Apontic B-Protein binds O the O translational O repressor O Bruno B-Protein and O is O implicated O in O regulation O of O oskar B-Protein mRNA O translation O . O The O product O of O the O oskar B-Protein gene O directs O posterior O patterning O in O the O Drosophila O oocyte O , O where O it O must O be O deployed O specifically O at O the O posterior O pole O . O Proper O expression O relies O on O the O coordinated O localization O and O translational O control O of O the O oskar B-Protein mRNA O . O Translational O repression O prior O to O localization O of O the O transcript O is O mediated O , O in O part O , O by O the O Bruno B-Protein protein O , O which O binds O to O discrete O sites O in O the O 3 O ' O untranslated O region O of O the O oskar B-Protein mRNA O . O To O begin O to O understand O how O Bruno B-Protein acts O in O translational O repression O , O we O performed O a O yeast O two O - O hybrid O screen O to O identify O Bruno B-Protein - O interacting O proteins O . O One O interactor O , O described O here O , O is O the O product O of O the O apontic B-Protein gene O . O Coimmunoprecipitation O experiments O lend O biochemical O support O to O the O idea O that O Bruno B-Protein and O Apontic B-Protein proteins O physically O interact O in O Drosophila O . O Genetic O experiments O using O mutants O defective O in O apontic B-Protein and O bruno B-Protein reveal O a O functional O interaction O between O these O genes O . O Given O this O interaction O , O Apontic B-Protein is O likely O to O act O together O with O Bruno B-Protein in O translational O repression O of O oskar B-Protein mRNA O . O Interestingly O , O Apontic B-Protein , O like O Bruno B-Protein , O is O an O RNA O - O binding O protein O and O specifically O binds O certain O regions O of O the O oskar B-Protein mRNA O 3 O ' O untranslated O region O . O NuMA B-Protein is O well O known O for O its O role O in O the O formation O and O stabilization O of O the O mitotic O spindle O . O Accumulation O of O NuMA B-Protein has O been O shown O at O the O mitotic O spindle O poles O and O at O or O near O the O minus O ends O of O microtubules O . O Sequestration O of O endogenous O NuMA B-Protein by O microinjection O of O anti O - O NuMA B-Protein antibodies O into O mitotic O cultured O cells O was O shown O to O disrupt O the O bipolar O mitotic O spindles O . O The O spindle O association O activity O of O NuMA B-Protein was O mapped O to O its O COOH O - O terminal O part O ( O residues O 1538 O - O 2115 O ) O . O A O recent O study O showed O that O the O critical O sequences O for O spindle O pole O localization O are O contained O within O the O amino O acid O residues O 1750 O - O 1800 O of O NuMA B-Protein . O However O , O the O mechanism O by O which O NuMA B-Protein binds O to O microtubules O is O not O clear O . O NuMA B-Protein does O not O contain O a O microtubule O - O binding O region O of O kinesin B-Protein , O Map2 B-Protein , O or O tau B-Protein , O but O has O been O shown O to O directly O bind O to O tubulin B-Protein and O organize O microtubule O formation O through O the O distal O portion O of O its O COOH O - O terminal O domain O . O HIV O - O 1 O Nef B-Protein alters O the O expression O of O betaII O and O epsilon O isoforms O of O protein B-Family kinase I-Family C I-Family and O the O activation O of O the O long O terminal O repeat O promoter O in O human O astrocytoma O cells O . O In O the O human O immunodeficiency O virus O type O 1 O ( O HIV O - O 1 O ) O - O infected O brain O , O the O virus O does O not O replicate O in O astrocytes O , O but O a O synthesis O of O viral O regulatory O proteins O occurs O in O these O cells O , O leading O to O accumulation O of O Nef B-Protein . O As O an O approach O to O understand O the O effects O of O Nef B-Protein on O astrocyte O functional O activity O , O we O analyzed O whether O intracellular O Nef B-Protein interferes O with O the O expression O and O activation O of O the O enzyme O protein B-Family kinase I-Family C I-Family ( O PKC B-Family ) O , O which O is O an O important O regulator O of O astroglial O functions O and O HIV O - O 1 O replication O . O Astrocytoma O clones O ( O U251 O MG O ) O not O expressing O Nef B-Protein ( O Neo O ) O , O or O expressing O wild O - O type O Nef B-Protein ( O Bru O ) O or O nonmyristoylated O Nef B-Protein ( O TH O ) O were O used O to O monitor O the O expression O and O activation O of O 10 O PKC O isoforms O . O The O same O clones O were O used O to O evaluate O the O effect O of O Nef B-Protein on O the O viral O long O terminal O repeat O ( O LTR O ) O promoter O after O activation O of O PKC B-Family with O the O phorbol B-Chemical ester I-Chemical 12 I-Chemical - I-Chemical myristate I-Chemical 13 I-Chemical - I-Chemical acetate I-Chemical ( O PMA B-Chemical ) O . O PKC B-Family intracellular O distribution O and O activation O were O evaluated O by O Western O blot O analysis O of O cytosolic O and O membrane O fractions O of O control O and O Nef B-Protein - O expressing O clones O . O PMA B-Chemical - O induced O LTR O activation O was O analyzed O in O clones O transfected O with O a O plasmid O encoding O for O the O CAT B-Protein reporter O gene O controlled O by O the O LTR O promoter O , O by O using O an O enzyme O - O linked O immunosorbent O assay O to O measure O CAT B-Protein expression O . O Nef B-Protein selectively O downregulated O the O expression O and O activation O of O betaII B-Protein and O epsilon B-Protein PKC I-Protein isoforms O in O astrocytoma O cells O . O Such O downregulation O correlated O with O an O inhibition O of O LTR O activation O after O PMA B-Chemical stimulation O . O The O myristoylation O of O Nef B-Protein and O its O membrane O localization O were O essential O for O these O effects O . O These O results O suggest O that O Nef B-Protein may O alter O astrocytic O functions O by O interfering O with O PKC B-Family expression O and O activation O and O contribute O to O the O restriction O of O HIV O - O 1 O replication O in O astrocytes O . O Interaction O of O Alzheimer O ' O s O presenilin B-Protein - I-Protein 1 I-Protein and O presenilin B-Protein - I-Protein 2 I-Protein with O Bcl B-Protein - I-Protein X I-Protein ( I-Protein L I-Protein ) I-Protein . O A O potential O role O in O modulating O the O threshold O of O cell O death O . O The O familial O Alzheimer O ' O s O disease O gene O products O , O presenilin B-Protein - I-Protein 1 I-Protein and O presenilin B-Protein - I-Protein 2 I-Protein , O have O been O reported O to O be O functionally O involved O in O amyloid B-Protein precursor I-Protein protein I-Protein processing O , O notch B-Protein receptor I-Protein signaling O , O and O programmed O cell O death O or O apoptosis O . O However O , O the O molecular O mechanisms O by O which O presenilins B-Family regulate O these O processes O remain O unknown O . O With O regard O to O the O latter O , O we O describe O a O molecular O link O between O presenilins B-Family and O the O apoptotic O pathway O . O Bcl B-Protein - I-Protein X I-Protein ( I-Protein L I-Protein ) I-Protein , O an O anti O - O apoptotic O member O of O the O Bcl B-Family - I-Family 2 I-Family family O was O shown O to O interact O with O the O carboxyl O - O terminal O fragments O of O PS1 B-Protein and O PS2 B-Protein by O the O yeast O two O - O hybrid O system O . O In O vivo O interaction O analysis O revealed O that O both O PS2 B-Protein and O its O naturally O occurring O carboxyl O - O terminal O products O , O PS2short B-Protein and O PS2Ccas B-Protein , O associated O with O Bcl B-Protein - I-Protein X I-Protein ( I-Protein L I-Protein ) I-Protein , O whereas O the O caspase B-Protein - I-Protein 3 I-Protein - O generated O amino O - O terminal O PS2Ncas B-Protein fragment O did O not O . O This O interaction O was O corroborated O by O demonstrating O that O Bcl B-Protein - I-Protein X I-Protein ( I-Protein L I-Protein ) I-Protein and O PS2 B-Protein partially O co O - O localized O to O sites O of O the O vesicular O transport O system O . O Functional O analysis O revealed O that O presenilins B-Family can O influence O mitochondrial O - O dependent O apoptotic O activities O , O such O as O cytochrome B-Protein c I-Protein release O and O Bax B-Protein - O mediated O apoptosis O . O Together O , O these O data O support O a O possible O role O of O the O Alzheimer O ' O s O presenilins B-Family in O modulating O the O anti O - O apoptotic O effects O of O Bcl B-Protein - I-Protein X I-Protein ( I-Protein L I-Protein ) I-Protein . O Cell O cycle O - O regulated O attachment O of O the O ubiquitin B-Protein - O related O protein O SUMO B-Family to O the O yeast O septins B-Family . O SUMO B-Family is O a O ubiquitin B-Protein - O related O protein O that O functions O as O a O posttranslational O modification O on O other O proteins O . O SUMO B-Family conjugation O is O essential O for O viability O in O Saccharomyces O cerevisiae O and O is O required O for O entry O into O mitosis O . O We O have O found O that O SUMO B-Family is O attached O to O the O septins B-Family Cdc3 B-Protein , O Cdc11 B-Protein , O and O Shs1 B-Protein / O Sep7 B-Protein specifically O during O mitosis O , O with O conjugates O appearing O shortly O before O anaphase O onset O and O disappearing O abruptly O at O cytokinesis O . O Septins B-Family are O components O of O a O belt O of O 10 O - O nm O filaments O encircling O the O yeast O bud O neck O . O Intriguingly O , O only O septins B-Family on O the O mother O cell O side O of O the O bud O neck O are O sumoylated O . O We O have O identified O four O major O SUMO B-Family attachment O - O site O lysine O residues O in O Cdc3 B-Protein , O one O in O Cdc11 B-Protein , O and O two O in O Shs1 B-Protein , O all O within O the O consensus O sequence O ( O IVL O ) O KX O ( O ED O ) O . O Mutating O these O sites O eliminated O the O vast O majority O of O bud O neck O - O associated O SUMO B-Family , O as O well O as O the O bulk O of O total O SUMO B-Family conjugates O in O G O ( O 2 O ) O / O M O - O arrested O cells O , O indicating O that O sumoylated O septins B-Family are O the O most O abundant O SUMO B-Family conjugates O at O this O point O in O the O cell O cycle O . O This O mutant O has O a O striking O defect O in O disassembly O of O septin B-Family rings O , O resulting O in O accumulation O of O septin B-Family rings O marking O previous O division O sites O . O Thus O , O SUMO B-Family conjugation O plays O a O role O in O regulating O septin B-Family ring O dynamics O during O the O cell O cycle O . O Iontophoresis O of O lysophosphatidic B-Chemical acid I-Chemical into O rabbit O cornea O induces O HSV O - O 1 O reactivation O : O evidence O that O neuronal O signaling O changes O after O infection O . O PURPOSE O : O Lysophosphatidic B-Chemical acid I-Chemical induces O neurite O retraction O ; O it O is O also O present O in O tears O and O aqueous O humor O . O We O determined O whether O lysophosphatidic B-Chemical acid I-Chemical induces O HSV O - O 1 O reactivation O in O latently O infected O rabbits O and O whether O the O nerve O growth O associated O protein O GAP B-Protein - I-Protein 43 I-Protein undergoes O posttranslational O modification O during O the O course O of O HSV O - O 1 O infection O . O METHODS O : O Rabbits O were O infected O with O HSV O - O 1 O and O acute O infection O was O documented O by O slit O lamp O examination O . O Corneas O of O latently O infected O rabbits O were O treated O with O lysophosphatidic B-Chemical acid I-Chemical or O lysophosphatidylserine B-Chemical ( O structurally O similar O but O lacking O biological O potency O ) O . O For O application O to O the O cornea O , O these O compounds O were O impregnated O into O collagen B-Complex shields O , O applied O as O topical O drops O , O or O iontophoresed O . O In O another O experiment O , O corneas O of O latently O infected O rabbits O were O either O untreated O or O treated O iontophoretically O with O lysophosphatidic B-Chemical acid I-Chemical , O lysophosphatidylserine B-Chemical , O or O saline O . O Ocular O swabs O detected O shedding O of O infectious O virus O . O Western O blot O and O immunoprecipitation O identified O GAP B-Protein - I-Protein 43 I-Protein in O corneal O extracts O and O densitometry O of O silver B-Chemical - O stained O isoelectric O focusing O gels O measured O changes O in O GAP B-Protein - I-Protein 43 I-Protein isoform O abundance O . O RESULTS O : O Iontophoresis O of O lysophosphatidic B-Chemical acid I-Chemical induced O HSV O - O 1 O shedding O more O frequently O than O lysophosphatidylserine B-Chemical or O saline O . O Viral O shedding O induced O by O collagen B-Complex shield O and O topical O drop O administration O was O low O and O not O significantly O different O for O lysophosphatidic B-Chemical acid I-Chemical and O lysophosphatidylserine B-Chemical . O Five O discrete O GAP B-Protein - I-Protein 43 I-Protein isoforms O predominated O in O the O IEF O gels O . O Most O abundant O were O the O pI O 4 O . O 7 O band O in O uninfected O cornea O and O the O pI O 5 O . O 05 O band O in O latently O - O infected O cornea O . O Compared O to O latently O - O infected O cornea O , O there O was O no O significant O change O in O isoform O abundance O 1 O h O after O lysophosphatidic B-Chemical acid I-Chemical iontophoresis O , O but O 24 O and O 72 O h O later O , O the O pI O 5 O . O 05 O band O was O diminished O . O CONCLUSIONS O : O Lysophosphatidic B-Chemical acid I-Chemical can O induce O HSV O - O 1 O reactivation O and O changes O in O GAP B-Protein - I-Protein 43 I-Protein pI O suggest O that O posttranslational O modifications O , O possibly O related O to O phosphorylation O and O ADP O - O ribosylation O , O are O occurring O during O HSV O - O 1 O latency O and O after O LPA B-Chemical is O iontophoretically O applied O to O the O cornea O . O How O lysophosphatidic B-Chemical acid I-Chemical - O induced O signaling O , O HSV O - O 1 O reactivation O , O and O GAP B-Protein - I-Protein 43 I-Protein pI O are O related O remains O to O be O determined O . O Human B-Protein topoisomerase I-Protein IIalpha I-Protein and O IIbeta B-Protein interact O with O the O C O - O terminal O region O of O p53 B-Protein . O The O p53 B-Protein tumor O suppressor O protein O is O a O critical O regulator O of O cell O cycle O progression O and O apoptosis O following O exposure O of O cells O to O DNA O damaging O agents O such O as O ionizing O radiation O or O anticancer O drugs O . O An O important O group O of O anticancer O drugs O , O including O compounds O such O as O etoposide B-Chemical and O doxorubicin B-Chemical ( O Adriamycin B-Chemical ) O , O interacts O with O DNA B-Protein topoisomerase I-Protein II I-Protein ( O topo B-Protein II I-Protein ) O , O causing O the O accumulation O of O enzyme O - O DNA O adducts O that O ultimately O lead O to O double O - O strand O breaks O and O cell O death O via O apoptosis O . O Human B-Protein topo I-Protein IIbeta I-Protein has O previously O been O shown O to O interact O with O p53 B-Protein , O and O we O have O extended O this O analysis O to O show O that O both O topo B-Protein IIalpha I-Protein and O IIbeta B-Protein interact O with O p53 B-Protein in O vivo O and O in O vitro O . O Furthermore O , O we O show O that O the O regulatory O C O - O terminal O basic O region O of O p53 B-Protein ( O residues O 364 O - O 393 O ) O is O necessary O and O sufficient O for O interaction O with O DNA B-Protein topo I-Protein II I-Protein . I-Protein The O Saccharomyces O cerevisiae O Cdc14 B-Protein phosphatase O is O implicated O in O the O structural O organization O of O the O nucleolus O . O Cdc14 B-Protein , O a O dual O - O specificity O protein O phosphatase O , O has O been O previously O implicated O in O triggering O exit O from O mitosis O in O the O yeast O Saccharomyces O cerevisiae O . O Using O immunofluorescence O microscopy O and O immunogold O labeling O , O we O demonstrate O that O a O functional O HA B-Protein - O tagged O version O of O the O phosphatase O Cdc14 B-Protein localizes O to O the O nucleolus O . O Moreover O , O Cdc14 B-Protein - O HA B-Protein co O - O localized O with O the O nucleolar O NOP2 B-Protein and O GAR1 B-Protein proteins O . O By O immunofluorescence O , O Cdc14 B-Protein - O HA B-Protein was O found O in O the O nucleolus O during O most O of O the O mitotic O cell O cycle O , O except O during O anaphase O - O telophase O when O it O redistributed O along O the O mitotic O spindle O . O While O this O work O was O in O progress O , O the O same O pattern O of O Cdc14 B-Protein localization O was O described O by O others O ( O Visintin O et O al O , O Nature O 398 O ( O 1999 O ) O 818 O ) O . O Constitutive O overexpression O of O CDC14 B-Protein was O toxic O and O led O to O cell O cycle O arrest O of O cells O , O mainly O in O G1 O . O This O correlated O with O the O appearance O of O abnormal O nuclear O structures O . O A O genetic O search O for O suppressors O of O the O lethality O associated O with O CDC14 B-Protein overexpression O identified O YJL076W B-Protein . O Because O overproduction O of O Yj1076w B-Protein buffered O the O toxic O effect O of O Cdc14 B-Protein overproduction O , O this O suggested O that O it O might O be O a O substrate O of O Cdc14 B-Protein . O This O has O indeed O been O found O to O be O the O case O by O others O who O recently O described O Yj1076w B-Protein / O Netl B-Protein as O a O nucleolar O protein O that O physically O associates O with O Cdc14 B-Protein ( O Shou O et O al O , O Cell O 97 O ( O 1999 O ) O 233 O ) O . O The O present O data O confirm O several O recently O uncovered O aspects O of O the O regulation O of O Cdc14 B-Protein localization O and O activity O and O suggest O that O the O level O of O expression O of O CDC14 B-Protein influences O the O structural O organization O of O the O nucleolus O . O Nedd8 B-Protein modification O of O cul B-Protein - I-Protein 1 I-Protein activates O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex - O dependent O ubiquitination O of O IkappaBalpha B-Protein . O Regulation O of O NF B-Complex - I-Complex kappaB I-Complex occurs O through O phosphorylation O - O dependent O ubiquitination O of O IkappaBalpha B-Protein , O which O is O degraded O by O the O 26S B-Complex proteasome I-Complex . O Recent O studies O have O shown O that O ubiquitination O of O IkappaBalpha B-Protein is O carried O out O by O a O ubiquitin O - O ligase O enzyme O complex O called O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex . O Here O we O show O that O Nedd8 B-Protein modification O of O the O Cul B-Protein - I-Protein 1 I-Protein component O of O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex is O important O for O function O of O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex in O ubiquitination O of O IkappaBalpha B-Protein . O In O cells O , O Nedd8 B-Protein - O conjugated O Cul B-Protein - I-Protein 1 I-Protein was O complexed O with O two O substrates O of O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex , O phosphorylated O IkappaBalpha B-Protein and O beta B-Protein - I-Protein catenin I-Protein , O indicating O that O Nedd8 B-Protein - O Cul B-Protein - I-Protein 1 I-Protein conjugates O are O part O of O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex in O vivo O . O Although O only O a O minute O fraction O of O total O cellular O Cul B-Protein - I-Protein 1 I-Protein is O modified O by O Nedd8 B-Protein , O the O Cul B-Protein - I-Protein 1 I-Protein associated O with O ectopically O expressed O betaTrCP B-Protein was O highly O enriched O for O the O Nedd8 B-Protein - O conjugated O form O . O Moreover O , O optimal O ubiquitination O of O IkappaBalpha B-Protein required O Nedd8 B-Protein and O the O Nedd8 B-Protein - O conjugating O enzyme O , O Ubc12 B-Protein . O The O site O of O Nedd8 B-Protein ligation O to O Cul B-Protein - I-Protein 1 I-Protein is O essential O , O as O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex containing O a O K720R O mutant O of O Cul B-Protein - I-Protein 1 I-Protein only O weakly O supported O IkappaBalpha B-Protein ubiquitination O compared O to O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex containing O WT O Cul B-Protein - I-Protein 1 I-Protein , O suggesting O that O the O Nedd8 B-Protein ligation O of O Cul B-Protein - I-Protein 1 I-Protein affects O the O ubiquitination O activity O of O SCF B-Complex ( I-Complex beta I-Complex ( I-Complex TrCP I-Complex ) I-Complex ) I-Complex . O These O observations O provide O a O functional O link O between O the O highly O related O ubiquitin B-Protein and O Nedd8 B-Protein pathways O of O protein O modification O and O show O how O they O operate O together O to O selectively O target O the O signal O - O dependent O degradation O of O IkappaBalpha B-Protein . O v B-Protein - I-Protein Src I-Protein suppresses O SHPS B-Protein - I-Protein 1 I-Protein expression O via O the O Ras B-Family - O MAP B-Family kinase I-Family pathway O to O promote O the O oncogenic O growth O of O cells O . O We O investigated O the O effect O of O cell O transformation O by O v B-Protein - I-Protein src I-Protein on O the O expression O and O tyrosine O phosphorylation O of O SHPS B-Protein - I-Protein 1 I-Protein , O a O putative O docking O protein O for O SHP B-Protein - I-Protein 1 I-Protein and O SHP B-Protein - I-Protein 2 I-Protein . O We O found O that O transformation O by O v B-Protein - I-Protein src I-Protein virtually O inhibited O the O SHPS B-Protein - I-Protein 1 I-Protein expression O at O mRNA O level O . O While O nontransforming O Src B-Family kinases O including O c B-Protein - I-Protein Src I-Protein , O nonmyristoylated O forms O of O v B-Protein - I-Protein Src I-Protein had O no O inhibitory O effect O on O SHPS B-Protein - I-Protein 1 I-Protein expression O , O transforming O Src B-Family kinases O including O wild O - O type O v B-Protein - I-Protein Src I-Protein and O chimeric O mutant O of O c B-Protein - I-Protein Src I-Protein bearing O v B-Protein - I-Protein Src I-Protein SH3 O substantially O suppressed O the O SHPS B-Protein - I-Protein 1 I-Protein expression O . O In O cells O expressing O temperature O sensitive O mutant O of O v B-Protein - I-Protein Src I-Protein , O suppression O of O the O SHPS B-Protein - I-Protein 1 I-Protein expression O was O temperature O - O dependent O . O In O contrast O , O tyrosine O phosphorylation O of O SHPS B-Protein - I-Protein 1 I-Protein was O rather O activated O in O cells O expressing O c B-Protein - I-Protein Src I-Protein or O nonmyristoylated O forms O of O v B-Protein - I-Protein Src I-Protein . O SHPS B-Protein - I-Protein 1 I-Protein expression O in O SR3Y1 O was O restored O by O treatment O with O herbimycin B-Chemical A I-Chemical , O a O potent O inhibitor O of O tyrosine O kinase O , O or O by O the O expression O of O dominant O negative O form O of O Ras B-Family . O Contrary O , O active O form O of O Mekl B-Protein markedly O suppressed O SHPS B-Protein - I-Protein 1 I-Protein expression O . O Finally O , O overexpression O of O SHPS B-Protein - I-Protein 1 I-Protein in O SR3Y1 O led O to O the O drastic O reduction O of O anchorage O independent O growth O of O the O cells O . O Taken O together O , O our O results O suggest O that O the O suppression O of O SHPS B-Protein - I-Protein 1 I-Protein expression O is O a O pivotal O event O for O cell O transformation O by O v B-Protein - I-Protein src I-Protein , O and O the O Ras B-Family - O MAP B-Family kinase I-Family cascade O plays O a O critical O role O in O the O suppression O . O Functional O interactions O between O the O estrogen B-Protein receptor I-Protein and O DRIP205 B-Protein , O a O subunit O of O the O heteromeric O DRIP B-Complex coactivator O complex O . O Nuclear O receptors O regulate O transcription O in O direct O response O to O their O cognate O hormonal O ligands O . O Ligand O binding O leads O to O the O dissociation O of O corepressors O and O the O recruitment O of O coactivators O . O Many O of O these O factors O , O acting O in O large O complexes O , O have O emerged O as O potential O chromatin O remodelers O through O intrinsic O histone B-Family modifying O activities O . O In O addition O , O other O ligand O - O recruited O complexes O appear O to O act O more O directly O on O the O transcriptional O apparatus O . O The O DRIP B-Complex complex O is O a O 15 O - O subunit O complex O required O for O nuclear B-Family receptor I-Family transcriptional O activation O in O vitro O . O It O is O recruited O to O the O receptor O in O response O to O ligand O through O specific O interactions O of O one O subunit O , O DRIP205 B-Protein . O We O present O evidence O that O DRIP205 B-Protein interacts O with O another O member O of O the O steroid O receptor O subfamily O , O estrogen B-Family receptor I-Family ( O ER B-Family ) O . O This O interaction O occurs O in O an O agonist O - O stimulated O fashion O which O in O turn O is O inhibited O by O several O ER B-Family antagonists O . O In O vivo O , O a O fragment O of O DRIP205 B-Protein containing O only O its O receptor O interacting O region O acts O to O selectively O inhibit O ER B-Family ' O s O ability O to O activate O transcription O in O response O to O estradiol B-Chemical . O These O observations O suggest O a O key O role O for O the O DRIP B-Complex coactivator O complex O in O estrogen B-Chemical - O ER B-Family signaling O . O Akr1p B-Protein and O the O type B-Family I I-Family casein I-Family kinases O act O prior O to O the O ubiquitination O step O of O yeast O endocytosis O : O Akr1p B-Protein is O required O for O kinase O localization O to O the O plasma O membrane O . O Ubiquitination O of O the O plasma O membrane O - O localized O yeast O a B-Protein - I-Protein factor I-Protein receptor I-Protein ( O Ste3p B-Protein ) O triggers O a O rapid O , O ligand O - O independent O endocytosis O leading O to O its O vacuolar O degradation O . O This O report O identifies O two O mutants O that O block O uptake O by O blocking O ubiquitination O , O these O being O mutant O either O for O the O ankyrin O repeat O protein O Akr1p B-Protein or O for O the O redundant O type B-Family I I-Family casein I-Family kinases O Yck1p B-Protein and O Yck2p B-Protein . O While O no O obvious O defect O was O seen O for O wild O - O type O Ste3p B-Protein phosphorylation O in O akr1 B-Protein or O yck B-Family mutant O backgrounds O , O examination O of O the O Delta320 O - O 413 O Ste3p B-Protein deletion O mutant O phosphorylation O did O reveal O a O clear O defect O in O both O mutants O . O The O Delta320 O - O 413 O deletion O removes O 18 O Ser O - O Thr O residues O ( O possible O YCK B-Family - O independent O phosphorylation O sites O ) O yet O retains O the O 15 O Ser O - O Thr O residues O of O the O Ste3p B-Protein PEST O - O like O ubiquitination O - O endocytosis O signal O . O Two O other O phenotypes O link O akr1 B-Protein and O yck B-Family mutants O : O both O are O defective O in O phosphorylation O of O wild O - O type O alpha B-Protein - I-Protein factor I-Protein receptor I-Protein , O and O while O both O are O defective O for O Ste3p B-Protein constitutive O internalization O , O both O remain O partially O competent O for O the O Ste3p B-Protein ligand O - O dependent O uptake O mode O . O Yck1p B-Protein - O Yck2p B-Protein may O be O the O function O responsible O in O phosphorylation O of O the O PEST O - O like O ubiquitination O - O endocytosis O signal O . O Akr1p B-Protein appears O to O function O in O localizing O Yck1p B-Protein - O Yck2p B-Protein to O the O plasma O membrane O , O a O localization O that O depends O on O prenylation O of O C O - O terminal O dicysteinyl O motifs O . O In O akr1Delta B-Protein cells O , O Yck2p B-Protein is O mislocalized O , O showing O a O diffuse O cytoplasmic O localization O identical O to O that O seen O for O a O Yck2p B-Protein mutant O that O lacks O the O C O - O terminal O Cys O - O Cys O , O indicating O a O likely O Akr1p B-Protein requirement O for O the O lipid O modification O of O Yck2p B-Protein , O for O prenylation O , O or O possibly O for O palmitoylation O . O Heat B-Protein shock I-Protein protein I-Protein 70 I-Protein binds O to O human B-Protein apurinic I-Protein / I-Protein apyrimidinic I-Protein endonuclease I-Protein and O stimulates O endonuclease O activity O at O abasic O sites O . O The O interaction O of O human B-Protein heat I-Protein shock I-Protein protein I-Protein 70 I-Protein ( O HSP70 B-Protein ) O with O human B-Protein apurinic I-Protein / I-Protein apyrimidinic I-Protein endonuclease I-Protein ( O HAP1 B-Protein ) O was O demonstrated O by O coimmunoprecipitation O . O A O combination O of O HSP70 B-Protein and O HAP1 B-Protein also O caused O a O shift O in O the O electrophoretic O mobility O of O a O DNA O fragment O containing O an O apurinic O / O apyrimidinic O site O . O The O functional O consequence O of O the O HSP70 B-Protein / O HAP1 B-Protein interaction O was O a O 10 O - O 100 O - O fold O enhancement O of O endonuclease O activity O at O abasic O sites O . O The O physical O and O functional O interaction O between O HSP70 B-Protein and O HAP1 B-Protein did O not O require O the O addition O of O ATP O . O The O association O of O HSP70 B-Protein and O a O key O base O excision O repair O enzyme O suggests O a O role O for O heat O shock O proteins O in O promoting O base O excision O repair O . O These O findings O provide O a O possible O mechanism O by O which O HSP70 B-Protein protects O cells O against O oxidative O stress O . O JAK2 B-Protein activates O TFII B-Protein - I-Protein I I-Protein and O regulates O its O interaction O with O extracellular O signal O - O regulated O kinase O . O TFII B-Protein - I-Protein I I-Protein is O a O transcription O factor O that O shuttles O between O the O cytoplasm O and O nucleus O and O is O regulated O by O serine O and O tyrosine O phosphorylation O . O Tyrosine O phosphorylation O of O TFII B-Protein - I-Protein I I-Protein can O be O regulated O in O a O signal O - O dependent O manner O in O various O cell O types O . O In O B O lymphocytes O , O Bruton B-Protein ' I-Protein s I-Protein tyrosine I-Protein kinase I-Protein has O been O identified O as O a O TFII B-Protein - I-Protein I I-Protein tyrosine O kinase O . O Here O we O report O that O JAK2 B-Protein can O phosphorylate O and O regulate O TFII B-Protein - I-Protein I I-Protein in O nonlymphoid O cells O . O The O activity O of O TFII B-Protein - I-Protein I I-Protein on O the O c B-Protein - I-Protein fos I-Protein promoter O in O response O to O serum O can O be O abolished O by O dominant O negative O JAK2 B-Protein or O the O specific O JAK2 B-Protein kinase O inhibitor O AG490 B-Chemical . O Consistent O with O this O , O we O have O also O found O that O JAK2 B-Protein is O activated O by O serum O stimulation O of O fibroblasts O . O Tyrosine O 248 O of O TFII B-Protein - I-Protein I I-Protein is O phosphorylated O in O vivo O upon O serum O stimulation O or O JAK2 B-Protein overexpression O , O and O mutation O of O tyrosine O 248 O to O phenylalanine O inhibits O the O ability O of O JAK2 B-Protein to O phosphorylate O TFII B-Protein - I-Protein I I-Protein in O vitro O . O Tyrosine O 248 O of O TFII B-Protein - I-Protein I I-Protein is O required O for O its O interaction O with O and O phosphorylation O by O ERK B-Family and O its O in O vivo O activity O on O the O c B-Protein - I-Protein fos I-Protein promoter O . O These O results O indicate O that O the O interaction O between O TFII B-Protein - I-Protein I I-Protein and O ERK B-Family , O which O is O essential O for O its O activity O , O can O be O regulated O by O JAK2 B-Protein through O phosphorylation O of O TFII B-Protein - I-Protein I I-Protein at O tyrosine O 248 O . O Thus O , O like O the O STAT B-Family factors O , O TFII B-Protein - I-Protein I I-Protein is O a O direct O substrate O of O JAK2 B-Protein and O a O signal O - O dependent O transcription O factor O that O integrates O signals O from O both O tyrosine O kinase O and O mitogen O - O activated O protein O kinase O pathways O to O regulate O transcription O . O BAG B-Protein - I-Protein 1M I-Protein , I-Protein an O isoform O of O Bcl B-Protein - I-Protein 2 I-Protein - O interacting O protein O BAG B-Protein - I-Protein 1 I-Protein , O enhances O gene O expression O driven O by O CMV O promoter O . O BAG B-Protein - I-Protein 1M I-Protein , O one O of O the O isoforms O of O BAG B-Protein - I-Protein 1 I-Protein , O was O reported O to O bind O to O DNA O and O stimulate O general O transcription O when O cells O were O stressed O by O heat O shock O ( O Zeiner O , O M O . O , O et O al O . O , O Proc O . O Natl O . O Acad O . O Sci O . O USA O 96 O , O 10194 O - O 10199 O , O 1999 O ) O . O Here O we O show O that O BAG B-Protein - I-Protein 1M I-Protein binds O and O enhances O transcriptional O activity O of O Cytomegalovirus O ( O CMV O ) O early O gene O promoter O under O unstressed O conditions O . O This O activity O is O unique O to O BAG B-Protein - I-Protein 1M I-Protein in O that O other O isoforms O , O BAG B-Protein - I-Protein 1S I-Protein and O BAG B-Protein - I-Protein 1L I-Protein , O are O much O weaker O in O this O activity O , O although O all O of O the O isoforms O share O common O ubiquitin B-Protein - O like O domain O and O BAG O domain O interacting O with O Hsp70 B-Protein / O Hsc70 B-Protein . O Deletion O analysis O of O BAG B-Protein - I-Protein 1M I-Protein showed O that O C O - O terminal O BAG O domain O is O necessary O to O enhance O the O CMV O promoter O activity O , O suggesting O that O interaction O with O Hsp70 B-Protein / O Hsc70 B-Protein proteins O may O mediate O this O function O . O Another O mutation O in O N O - O terminus O , O BAG B-Protein - I-Protein 1M I-Protein K O ( O 2 O - O 4 O ) O A O , O lost O DNA O binding O capacity O and O majority O of O the O promoter O - O enhancing O activity O . O Our O study O demonstrates O that O both O N O - O terminal O DNA O binding O site O and O C O - O terminal O Hsp70 B-Protein / O Hsc70 B-Protein binding O site O of O BAG B-Protein - I-Protein 1M I-Protein play O an O important O role O in O enhancing O the O CMV O promoter O activity O . O Expression O , O cellular O distribution O and O protein O binding O of O the O glioma B-Protein amplified I-Protein sequence I-Protein ( O GAS41 B-Protein ) O , O a O highly O conserved O putative O transcription O factor O . O The O glioma B-Protein amplified I-Protein sequence I-Protein 41 I-Protein ( O GAS41 B-Protein ) O was O previously O isolated O by O microdissection O mediated O cDNA O capture O from O the O glioblastoma O multiforme O cell O line O TX3868 O and O shown O to O be O frequently O amplified O in O human O gliomas O . O We O determined O the O complete O cDNA O sequence O of O the O GAS41 B-Protein gene O , O demonstrated O that O the O GAS41 B-Protein protein O is O evolutionarily O conserved O , O specifically O at O the O N O - O terminus O , O and O identified O the O yeast O transcription O factor O tf2f O domain O within O the O GAS41 B-Protein sequence O . O A O human O multiple O - O tissue O Northern O blot O revealed O ubiquitous O expression O of O GAS41 B-Protein with O the O highest O expression O in O human O brain O . O After O generating O polyclonal O antibodies O we O found O GAS41 B-Protein protein O expression O in O the O nucleus O of O the O TX3868 O cell O line O by O Western O blot O analysis O and O immunofluorescence O microscopy O . O The O nuclear O localization O was O confirmed O for O several O human O tumors O including O gliomas O of O different O grades O of O malignancy O . O In O neuroblastoma O however O , O GAS41 B-Protein was O found O in O the O nucleoli O but O not O in O the O nucleoplasm O . O Yeast O two O - O hybrid O screening O of O the O TX3868 O cell O line O identified O the O nuclear B-Protein mitotic I-Protein apparatus I-Protein protein I-Protein ( O NuMA B-Protein ) O , O the O KIAA1009 B-Protein protein O , O and O prefoldin B-Protein subunit I-Protein 1 I-Protein ( O PFDN1 B-Protein ) O as O potential O interacting O partners O of O GAS41 B-Protein . O We O generated O a O polyclonal O antibody O against O the O KIAA1009 B-Protein protein O and O we O demonstrated O that O the O KIAA1009 B-Protein protein O is O a O nuclear O protein O , O which O appears O to O be O co O - O localized O with O the O GAS41 B-Protein protein O and O NuMA B-Protein . O A O novel O Rtg2p B-Protein activity O regulates O nitrogen B-Chemical catabolism O in O yeast O . O The O inactivity O of O Ure2p B-Protein , O caused O by O either O a O ure2 B-Protein mutation O or O the O presence O of O the O [ O URE3 B-Protein ] O prion O , O increases O DAL5 B-Protein transcription O and O thus O enables O Saccharomyces O cerevisiae O to O take O up O ureidosuccinate B-Chemical ( O USA B-Chemical + O ) O . O Rtg2p B-Protein regulates O transcription O of O glutamate B-Chemical - O repressible O genes O by O facilitation O of O the O nuclear O entry O of O the O Rtg1 B-Protein and O Rtg3 B-Protein proteins O . O We O find O that O rtg2 B-Protein Delta O cells O take O up O USA B-Chemical even O without O the O presence O of O [ O URE3 B-Protein ] O . O Thus O , O the O USA B-Chemical + O phenotype O of O rtg2 B-Protein Delta O strains O is O not O the O result O generation O of O the O [ O URE3 B-Protein ] O prion O but O is O a O regulatory O effect O . O Because O rtg1 B-Protein Delta O or O rtg3 B-Protein Delta O mutations O or O the O presence O of O glutamate B-Chemical do O not O produce O the O USA B-Chemical + O phenotype O , O this O is O a O novel O function O of O Rtg2p B-Protein . O The O USA B-Chemical + O phenotype O of O rtg2 B-Protein Delta O strains O depends O on O GLN3 B-Protein , O is O caused O by O overexpression O of O DAL5 B-Protein , O and O is O blocked O by O mks1 B-Protein Delta O , O but O not O by O overexpression O of O Ure2p B-Protein . O These O characteristics O suggest O that O Rtg2p B-Protein acts O in O the O upstream O part O of O the O nitrogen B-Chemical catabolism O regulation O pathway O . O Domain O structure O of O the O NRIF3 B-Protein family O of O coregulators O suggests O potential O dual O roles O in O transcriptional O regulation O . O The O identification O of O a O novel O coregulator O for O nuclear O hormone O receptors O , O designated O NRIF3 B-Protein , O was O recently O reported O ( O D O . O Li O et O al O . O , O Mol O . O Cell O . O Biol O . O 19 O : O 7191 O - O 7202 O , O 1999 O ) O . O Unlike O most O known O coactivators O , O NRIF3 B-Protein exhibits O a O distinct O receptor O specificity O in O interacting O with O and O potentiating O the O activity O of O only O TRs B-Protein and O RXRs B-Protein but O not O other O examined O nuclear O receptors O . O However O , O the O molecular O basis O underlying O such O specificity O is O unclear O . O In O this O report O , O we O extended O our O study O of O NRIF3 B-Protein - O receptor O interactions O . O Our O results O suggest O a O bivalent O interaction O model O , O where O a O single O NRIF3 B-Protein molecule O utilizes O both O the O C O - O terminal O LXXIL O ( O receptor O - O interacting O domain O 1 O [ O RID1 O ] O ) O and O the O N O - O terminal O LXXLL O ( O RID2 O ) O modules O to O cooperatively O interact O with O TR B-Protein or O RXR B-Protein ( O presumably O a O receptor O dimer O ) O , O with O the O spacing O between O RID1 O and O RID2 O playing O an O important O role O in O influencing O the O affinity O of O the O interactions O . O During O the O course O of O these O studies O , O we O also O uncovered O an O NRIF3 B-Protein - O NRIF3 B-Protein interaction O domain O . O Deletion O and O mutagenesis O analyses O mapped O the O dimerization O domain O to O a O region O in O the O middle O of O NRIF3 B-Protein ( O residues O 84 O to O 112 O ) O , O which O is O predicted O to O form O a O coiled O - O coil O structure O and O contains O a O putative O leucine O zipper O - O like O motif O . O By O using O Gal4 B-Protein fusion O constructs O , O we O identified O an O autonomous O transactivation O domain O ( O AD1 O ) O at O the O C O terminus O of O NRIF3 B-Protein . O Somewhat O surprisingly O , O full O - O length O NRIF3 B-Protein fused O to O the O DNA O - O binding O domain O of O Gal4 B-Protein was O found O to O repress O transcription O of O a O Gal4 B-Protein reporter O . O Further O analyses O mapped O a O novel O repression O domain O ( O RepD1 O ) O to O a O small O region O at O the O N O - O terminal O portion O of O NRIF3 B-Protein ( O residues O 20 O to O 50 O ) O . O The O NRIF3 B-Protein gene O encodes O at O least O two O additional O isoforms O due O to O alternative O splicing O . O These O two O isoforms O contain O the O same O RepD1 O region O as O NRIF3 B-Protein . O Consistent O with O this O , O Gal4 B-Protein fusions O of O these O two O isoforms O were O also O found O to O repress O transcription O . O Cotransfection O of O NRIF3 B-Protein or O its O two O isoforms O did O not O relieve O the O transrepression O function O mediated O by O their O corresponding O Gal4 B-Protein fusion O proteins O , O suggesting O that O the O repression O involves O a O mechanism O ( O s O ) O other O than O the O recruitment O of O a O titratable O corepressor O . O Interestingly O , O a O single O amino O acid O residue O change O of O a O potential O phosphorylation O site O in O RepD1 O ( O Ser O ( O 28 O ) O to O Ala O ) O abolishes O its O transrepression O function O , O suggesting O that O the O coregulatory O property O of O NRIF3 B-Protein ( O or O its O isoforms O ) O might O be O subjected O to O regulation O by O cellular O signaling O . O Taken O together O , O our O results O identify O NRIF3 B-Protein as O an O interesting O coregulator O that O possesses O both O transactivation O and O transrepression O domains O and O / O or O functions O . O Collectively O , O the O NRIF3 B-Protein family O of O coregulators O ( O which O includes O NRIF3 B-Protein and O its O other O isoforms O ) O may O play O dual O roles O in O mediating O both O positive O and O negative O regulatory O effects O on O gene O expression O . O Impairment O of O mineralocorticoid B-Protein receptor I-Protein ( O MR B-Protein ) O - O dependent O biological O response O by O oxidative O stress O and O aging O : O correlation O with O post O - O translational O modification O of O MR B-Protein and O decreased O ADP O - O ribosylatable O level O of O elongating B-Protein factor I-Protein 2 I-Protein in O kidney O cells O . O Acute O and O chronic O treatments O of O mice O with O the O glutathione B-Chemical - O depleting O agent O , O L B-Chemical - I-Chemical buthionine I-Chemical - I-Chemical ( I-Chemical SR I-Chemical ) I-Chemical - I-Chemical sulfoximine I-Chemical ( O BSO B-Chemical ) O , O impaired O the O mineralocorticoid B-Protein receptor I-Protein ( O MR B-Protein ) O - O dependent O biological O response O by O inhibiting O aldosterone B-Chemical binding O . O This O steroid B-Chemical - O binding O inhibition O was O fully O reversed O when O reducing O agents O were O added O to O kidney O cytosol O obtained O from O mice O treated O for O 5 O h O , O but O it O was O only O partially O reversed O in O cytosol O obtained O from O mice O treated O for O 10 O days O . O Although O the O oligomeric O structure O of O the O MR B-Protein - O hsp90 B-Protein heterocomplex O was O always O unaffected O , O a O decreased O amount O of O MR B-Protein protein O was O evidenced O after O the O long O term O treatment O . O Such O a O deleterious O effect O was O correlated O with O a O post O - O translational O modification O of O MR B-Protein , O as O demonstrated O by O an O increased O level O of O receptor O carbonylation O . O In O addition O , O a O failure O at O the O elongation O / O termination O step O was O also O observed O during O the O receptor O translation O process O in O a O reticulocyte O lysate O system O . O Thus O , O a O high O polyribosomes B-Complex / O monomers O ratio O and O both O increased O proteolysis O and O decreased O ADP O - O ribosylatable O concentration O of O elongation B-Protein factor I-Protein 2 I-Protein ( O EF B-Protein - I-Protein 2 I-Protein ) O were O shown O . O Importantly O , O similar O observations O were O also O performed O in O vivo O after O depletion O of O glutathione B-Chemical . O Notwithstanding O the O EF B-Protein - I-Protein 2 I-Protein functional O disruption O , O not O all O renal O proteins O were O equally O affected O as O the O MR B-Protein . O Interestingly O , O both O EF B-Protein - I-Protein 2 I-Protein and O MR B-Protein expressed O in O old O mice O were O similarly O affected O as O in O L B-Chemical - I-Chemical buthionine I-Chemical - I-Chemical ( I-Chemical SR I-Chemical ) I-Chemical - I-Chemical sulfoximine I-Chemical - O treated O young O mice O . O We O therefore O propose O that O a O dramatic O depletion O of O glutathione B-Chemical in O kidney O cells O mimics O the O cumulative O effect O of O aging O which O , O at O the O end O , O may O lead O to O a O renal O mineralocorticoid B-Chemical dysfunction O . O The O Khd1 B-Protein protein O , O which O has O three O KH O RNA O - O binding O motifs O , O is O required O for O proper O localization O of O ASH1 B-Protein mRNA O in O yeast O . O RNA O localization O is O a O widespread O mechanism O for O achieving O localized O protein O synthesis O . O In O Saccharomyces O cerevisiae O , O Ash1 B-Protein is O a O specific O repressor O of O transcription O that O localizes O asymmetrically O to O the O daughter O cell O nucleus O through O the O localization O of O ASH1 B-Protein mRNA O to O the O distal O tip O of O the O daughter O cell O . O This O localization O depends O on O the O actin B-Family cytoskeleton O and O five O She O proteins O , O one O of O which O is O a O type B-Family V I-Family myosin I-Family motor I-Family , O Myo4 B-Protein . O We O show O here O that O a O novel O RNA O - O binding O protein O , O Khd1 B-Protein ( O KH B-Protein - I-Protein domain I-Protein protein I-Protein 1 I-Protein ) O , O is O required O for O efficient O localization O of O ASH1 B-Protein mRNA O to O the O distal O tip O of O the O daughter O cell O . O Visualization O of O ASH1 B-Protein mRNA O in O vivo O using O GFP B-Protein - O tagged O RNA O demonstrated O that O Khd1 B-Protein associates O with O the O N O element O , O a O cis O - O acting O localization O sequence O within O the O ASH1 B-Protein mRNA O . O Co O - O immunoprecipitation O studies O also O indicated O that O Khd1 B-Protein associates O with O ASH1 B-Protein mRNA O through O the O N O element O . O A O khd1Delta B-Protein mutation O exacerbates O the O phenotype O of O a O weak O myo4 B-Protein mutation O , O whereas O overexpression O of O KHD1 B-Protein decreases O the O concentration O of O Ash1 B-Protein protein O and O restores O HO B-Protein expression O to O she B-Protein mutants O . O These O results O suggest O that O Khd1 B-Protein may O function O in O the O linkage O between O ASH1 B-Protein mRNA O localization O and O its O translation O . O Formation O of O the O approximately O 350 O - O kDa O Apg12 B-Protein - O Apg5 B-Protein . O Apg16 B-Protein multimeric O complex O , O mediated O by O Apg16 B-Protein oligomerization O , O is O essential O for O autophagy O in O yeast O . O Autophagy O , O responsible O for O the O delivery O of O cytoplasmic O components O to O the O lysosome O / O vacuole O for O degradation O , O is O the O major O degradative O pathway O in O eukaryotic O cells O . O This O process O requires O a O ubiquitin B-Protein - O like O protein O conjugation O system O , O in O which O Apg12 B-Protein is O covalently O bound O to O Apg5 B-Protein . O In O the O yeast O Saccharomyces O cerevisiae O , O the O Apg12 B-Protein - O Apg5 B-Protein conjugate O further O interacts O with O a O small O coiled O - O coil O protein O , O Apg16 B-Protein . O The O Apg12 B-Protein - O Apg5 B-Protein and O Apg16 B-Protein are O localized O in O the O cytosol O and O pre O - O autophagosomal O structures O and O play O an O essential O role O in O autophagosome O formation O . O Here O we O show O that O the O Apg12 B-Protein - O Apg5 B-Protein conjugate O and O Apg16 B-Protein form O a O approximately O 350 O - O kDa O complex O in O the O cytosol O . O Because O Apg16 B-Protein was O suggested O to O form O a O homo O - O oligomer O , O we O generated O an O in O vivo O system O that O allowed O us O to O control O the O oligomerization O state O of O Apg16 B-Protein . O With O this O system O , O we O demonstrated O that O formation O of O the O approximately O 350 O - O kDa O complex O and O autophagic O activity O depended O on O the O oligomerization O state O of O Apg16 B-Protein . O These O results O suggest O that O the O Apg12 B-Protein - O Apg5 B-Protein conjugate O and O Apg16 B-Protein form O a O multimeric O complex O mediated O by O the O Apg16 B-Protein homo O - O oligomer O , O and O formation O of O the O approximately O 350 O - O kDa O complex O is O required O for O autophagy O in O yeast O . O Paracrine O regulation O of O fat O cell O formation O in O bone O marrow O cultures O via O adiponectin B-Protein and O prostaglandins B-Chemical . O Adiponectin B-Protein , O an O adipocyte O - O derived O hormone O , O was O recently O shown O to O have O potential O therapeutic O applications O in O diabetes O and O obesity O because O of O its O influence O on O glucose B-Chemical and O lipid O metabolism O . O We O found O that O brown O fat O in O normal O human O bone O marrow O contains O this O protein O and O used O marrow O - O derived O preadipocyte O lines O and O long O - O term O cultures O to O explore O potential O roles O in O hematopoiesis O . O Recombinant O adiponectin B-Protein blocked O fat O cell O formation O in O long O - O term O bone O marrow O cultures O and O inhibited O the O differentiation O of O cloned O stromal O preadipocytes O . O Adiponectin B-Protein also O caused O elevated O expression O of O cyclooxygenase B-Protein - I-Protein 2 I-Protein ( O COX B-Protein - I-Protein 2 I-Protein ) O by O these O stromal O cells O and O induced O release O of O prostaglandin B-Chemical E I-Chemical ( I-Chemical 2 I-Chemical ) I-Chemical ( O PGE B-Chemical ( I-Chemical 2 I-Chemical ) I-Chemical ) O . O The O COX B-Protein - I-Protein 2 I-Protein inhibitor O Dup B-Chemical - I-Chemical 697 I-Chemical prevented O the O inhibitory O action O of O adiponectin B-Protein on O preadipocyte O differentiation O , O suggesting O involvement O of O stromal O cell O - O derived O prostanoids B-Chemical . O Furthermore O , O adiponectin B-Protein failed O to O block O fat O cell O generation O when O bone O marrow O cells O were O derived O from O B6 O , O 129S O ( O Ptgs2tm1Jed O ) O ( O COX B-Protein - I-Protein 2 I-Protein ( O + O / O - O ) O ) O mice O . O These O observations O show O that O preadipocytes O represent O direct O targets O for O adiponectin B-Protein action O , O establishing O a O paracrine O negative O feedback O loop O for O fat O regulation O . O They O also O link O adiponectin B-Protein to O the O COX B-Protein - I-Protein 2 I-Protein - O dependent O PGs B-Chemical that O are O critical O in O this O process O . O Site O - O directed O mutagenesis O of O platelet B-Protein glycoprotein I-Protein Ib I-Protein alpha I-Protein demonstrating O residues O involved O in O the O sulfation O of O tyrosines O 276 O , O 278 O , O and O 279 O . O The O interaction O between O platelet B-Protein glycoprotein I-Protein ( I-Protein GP I-Protein ) I-Protein Ib I-Protein alpha I-Protein and O von B-Protein Willebrand I-Protein factor I-Protein ( O VWF B-Protein ) O is O essential O for O initiation O of O hemostasis O . O The O sulfation O of O the O 3 O tyrosine O residues O 276 O , O 278 O , O and O 279 O in O GPIb B-Protein alpha I-Protein is O an O important O posttranslational O modification O that O seems O to O promote O the O interaction O with O VWF B-Protein . O The O environment O where O sulfation O of O tyrosines O occurs O has O been O proposed O to O contain O highly O acidic O residues O . O This O investigation O has O examined O the O highly O acidic O region O from O Asp249 O to O Asp287 O in O the O mature O GPIb B-Protein alpha I-Protein protein O . O Changes O to O most O of O the O carboxylic O acids O in O this O region O resulted O in O decreased O reactivity O to O VWF B-Protein . O Only O 3 O mutants O ( O Glu270Gln O , O Asp283Asn O , O Asp283Asn O / O Glu285Gln O / O Asp287Asn O ) O resulted O in O the O abolition O of O sulfation O . O Two O novel O mutations O were O also O created O . O First O , O a O deletion O of O the O 7 O amino O acids O from O Tyr276 O to O Glu282 O led O to O a O loss O of O sulfation O and O totally O abolished O VWF B-Protein binding O in O the O presence O of O botrocetin B-Chemical . O This O confirms O that O it O is O these O 3 O tyrosines O that O undergo O sulfation O and O that O this O region O is O crucial O for O botrocetin B-Chemical - O mediated O VWF B-Protein binding O . O The O second O mutation O involves O changing O the O lysine O residues O at O 253 O , O 258 O , O and O 262 O to O alanine O . O This O also O led O to O distinct O changes O in O VWF B-Protein binding O and O abolition O of O sulfation O . O The O postsynaptic O glutamate O receptor O subunit O DGluR B-Protein - I-Protein IIA I-Protein mediates O long O - O term O plasticity O in O Drosophila O . O The O developing O neuromuscular O junctions O ( O NMJs O ) O of O Drosophila O larvae O can O undergo O long O - O term O strengthening O of O signal O transmission O , O a O process O that O has O been O shown O recently O to O involve O local O subsynaptic O protein O synthesis O and O that O is O associated O with O an O elevated O synaptic O accumulation O of O the O postsynaptic O glutamate O receptor O subunit O DGluR B-Protein - I-Protein IIA I-Protein . O To O analyze O the O role O of O altered O postsynaptic O glutamate O receptor O expression O during O this O form O of O genetically O induced O junctional O plasticity O , O we O manipulated O the O expression O levels O of O two O so O far O - O described O postsynaptic O receptor O subunit O genes O , O dglur B-Protein - I-Protein IIA I-Protein and O dglur B-Protein - I-Protein IIB I-Protein , O in O wild O - O type O animals O and O plasticity O mutants O . O Here O we O show O that O elevated O synaptic O expression O of O DGluR B-Protein - I-Protein IIA I-Protein , O which O was O achieved O by O direct O transgenic O overexpression O , O by O genetically O increased O subsynaptic O protein O synthesis O , O or O by O a O reduced O dglur B-Protein - I-Protein IIB I-Protein gene O copy O number O , O results O in O an O increased O recruitment O of O active O zones O , O a O corresponding O enhancement O in O the O strength O of O junctional O signal O transmission O , O and O a O correlated O addition O of O boutons O to O the O NMJ O . O Ultrastructural O evidence O demonstrates O that O active O zones O appear O throughout O NMJs O at O a O typical O density O regardless O of O genotype O , O suggesting O that O the O space O requirements O of O active O zones O are O responsible O for O the O homogeneous O synapse O distribution O and O that O this O regulation O results O in O the O observed O growth O of O additional O boutons O at O strengthened O NMJs O . O These O phenotypes O were O suppressed O by O reduced O or O eliminated O DGluR B-Protein - I-Protein IIA I-Protein expression O , O which O resulted O from O either O a O reduced O dglur B-Protein - I-Protein IIA I-Protein gene O copy O number O or O transgenic O overexpression O of O DGluR B-Protein - I-Protein IIB I-Protein . O Our O results O demonstrate O that O persistent O alterations O of O neuronal O activity O and O subsynaptic O translation O result O in O an O elevated O synaptic O accumulation O of O DGluR B-Protein - I-Protein IIA I-Protein , O which O mediates O the O observed O functional O strengthening O and O morphological O growth O apparently O through O the O recruitment O of O additional O active O zones O . O CAND1 B-Protein binds O to O unneddylated O CUL1 B-Protein and O regulates O the O formation O of O SCF B-Complex ubiquitin I-Complex E3 I-Complex ligase I-Complex complex O . O The O SCF B-Complex ubiquitin I-Complex E3 I-Complex ligase I-Complex regulates O ubiquitin B-Protein - O dependent O proteolysis O of O many O regulatory O proteins O such O as O p27 B-Protein ( O Kip1 B-Protein ) O , O IkappaB B-Family , O and O beta B-Protein - I-Protein catenin I-Protein . O We O report O the O isolation O of O a O CUL1 B-Protein binding O protein O , O p120 B-Protein ( O CAND1 B-Protein ) O . O We O found O the O majority O of O CUL1 B-Protein is O in O a O complex O with O CAND1 B-Protein and O ROC1 B-Protein independent O of O SKP1 B-Protein and O F B-Family box I-Family protein O SKP2 B-Protein . O Both O in O vivo O and O in O vitro O , O CAND1 B-Protein prevents O the O binding O of O SKP1 B-Protein and O SKP2 B-Protein to O CUL1 B-Protein while O dissociation O of O CAND1 B-Protein from O CUL1 B-Protein promotes O the O reverse O reaction O . O Neddylation O of O CUL1 B-Protein or O the O presence O of O SKP1 B-Protein and O ATP O causes O CAND1 B-Protein dissociation O . O Our O data O suggest O that O CAND1 B-Protein regulates O the O formation O of O the O SCF B-Complex complex O , O and O its O dissociation O from O CUL1 B-Protein is O coupled O with O the O incorporation O of O F B-Family box I-Family proteins O into O the O SCF B-Complex complex O , O causing O their O destabilization O . O Genotoxic O stress O - O induced O activation O of O Plk3 B-Protein is O partly O mediated O by O Chk2 B-Protein . O Polo B-Protein - I-Protein like I-Protein kinase I-Protein 3 I-Protein ( O Plk3 B-Protein , O alternatively O termed O Prk B-Protein ) O is O involved O in O the O regulation O of O DNA O damage O checkpoint O as O well O as O in O M O - O phase O function O . O Plk3 B-Protein physically O interacts O with O p53 B-Protein and O phosphorylates O this O tumor O suppressor O protein O on O serine O - O 20 O , O suggesting O that O the O role O of O Plk3 B-Protein in O cell O cycle O progression O is O mediated O , O at O least O in O part O , O through O direct O regulation O of O p53 B-Protein . O Here O we O show O that O Plk3 B-Protein is O rapidly O activated O by O reactive O oxygen B-Chemical species O in O normal O diploid O fibroblast O cells O ( O WI O - O 38 O ) O , O correlating O with O a O subsequent O increase O in O p53 B-Protein protein O level O . O Plk3 B-Protein physically O interacts O with O Chk2 B-Protein and O the O interaction O is O enhanced O upon O DNA O damage O . O In O addition O , O Chk2 B-Protein immunoprecipitated O from O cell O lysates O of O Daudi O ( O which O expressed O little O Plk3 B-Protein ) O is O capable O of O stimulating O the O kinase O activity O of O purified O recombinant O Plk3 B-Protein in O vitro O , O and O this O stimulation O is O more O pronounced O when O Plk3 B-Protein is O supplemented O with O Chk2 B-Protein immunoprecipitated O from O Daudi O after O DNA O damage O . O Furthermore O , O ectopic O expression O Chk2 B-Protein activates O cellular O Plk3 B-Protein . O Together O , O our O studies O suggest O Chk2 B-Protein may O mediate O direct O activation O of O Plk3 B-Protein in O response O to O genotoxic O stresses O . O Modulation O of O p120E4F B-Protein transcriptional O activity O by O the O Gam1 B-Protein adenoviral O early O protein O . O Gam1 B-Protein , O an O early O adenoviral O CELO B-Family protein O , O is O required O for O viral O replication O . O Consistent O with O its O ability O to O inhibit O histone B-Family deacetylation O by O HDAC1 B-Protein , O Gam1 B-Protein activates O transcription O . O In O this O report O , O we O identify O the O cellular O transcription O factor O p120 B-Protein ( O E4F B-Protein ) O as O a O Gam1 B-Protein interaction O partner O . O p120 B-Protein ( O E4F B-Protein ) O is O a O low O - O abundance O transcription O factor O that O represses O the O adenovirus O E4 B-Protein promoter O . O Here O we O demonstrate O that O p120 B-Protein ( O E4F B-Protein ) O interacts O with O HDAC1 B-Protein in O vivo O and O in O vitro O , O and O that O E4F B-Protein - O mediated O transcriptional O repression O is O alleviated O by O the O HDAC B-Family inhibitor O trichostatin B-Chemical A I-Chemical or O by O overexpressing O Gam1 B-Protein . O A O mutant O E4 B-Protein promoter O unresponsive O to O E4F B-Protein - O mediated O transcriptional O repression O is O also O not O stimulated O by O Gam1 B-Protein . O Moreover O , O our O cofractionation O experiments O demonstrate O that O p120 B-Protein ( O E4F B-Protein ) O , O HDAC1 B-Protein and O Gam1 B-Protein may O be O concomitantly O present O in O protein O complexes O . O We O conclude O that O Gam1 B-Protein activates O E4 B-Protein - O dependent O transcription O possibly O by O inactivating O HDAC1 B-Protein . O Identification O and O characterization O of O a O nuclear B-Protein interacting I-Protein partner I-Protein of I-Protein anaplastic I-Protein lymphoma I-Protein kinase I-Protein ( O NIPA B-Protein ) O . O Anaplastic O large O - O cell O lymphoma O is O a O subtype O of O non O - O Hodgkin O lymphomas O characterized O by O the O expression O of O CD30 B-Protein . O More O than O half O of O these O lymphomas O carry O a O chromosomal O translocation O t O ( O 2 O ; O 5 O ) O leading O to O expression O of O the O oncogenic O tyrosine O kinase O nucleophosmin B-Protein - I-Protein anaplastic I-Protein lymphoma I-Protein kinase I-Protein ( O NPM B-Protein - I-Protein ALK I-Protein ) O . O NPM B-Protein - I-Protein ALK I-Protein is O capable O of O transforming O fibroblasts O and O lymphocytes O in O vitro O and O of O causing O lymphomas O in O mice O . O Previously O , O we O and O others O demonstrated O phospholipase B-Protein C I-Protein - I-Protein gamma I-Protein and O phosphatidylinositol B-Protein 3 I-Protein - I-Protein kinase I-Protein as O crucial O downstream O signaling O mediators O of O NPM B-Protein - I-Protein ALK I-Protein - O induced O oncogenicity O . O In O this O study O , O we O used O an O ALK B-Protein fusion O protein O as O bait O in O a O yeast O two O - O hybrid O screen O identifying O NIPA B-Protein ( O nuclear B-Protein interacting I-Protein partner I-Protein of I-Protein ALK I-Protein ) O as O a O novel O downstream O target O of O NPM B-Protein - I-Protein ALK I-Protein . O NIPA B-Protein encodes O a O 60 O - O kDa O protein O that O is O expressed O in O a O broad O range O of O human O tissues O and O contains O a O classical O nuclear O translocation O signal O in O its O C O terminus O , O which O directs O its O nuclear O localization O . O NIPA B-Protein interacts O with O NPM B-Protein - I-Protein ALK I-Protein and O other O ALK B-Protein fusions O in O a O tyrosine B-Protein kinase I-Protein - O dependent O manner O and O is O phosphorylated O in O NPM B-Protein - I-Protein ALK I-Protein - O expressing O cells O on O tyrosine O and O serine O residues O with O serine O 354 O as O a O major O phosphorylation O site O . O Overexpression O of O NIPA B-Protein in O Ba O / O F3 O cells O was O able O to O protect O from O apoptosis O induced O by O IL B-Protein - I-Protein 3 I-Protein withdrawal O . O Mutations O of O the O nuclear O translocation O signal O or O the O Ser O - O 354 O phosphorylation O site O impaired O the O antiapoptotic O function O of O NIPA B-Protein . O In O NPM B-Protein - I-Protein ALK I-Protein - O transformed O Ba O / O F3 O cells O , O apoptosis O triggered O by O wortmannin B-Chemical treatment O was O enhanced O by O overexpression O of O putative O dominant O - O negative O NIPA B-Protein mutants O . O These O results O implicate O an O antiapoptotic O role O for O NIPA B-Protein in O NPM B-Protein - I-Protein ALK I-Protein - O mediated O signaling O events O . O Molecular O cloning O , O expression O , O and O characterization O of O a O novel O class O of O synaptotagmin B-Family ( O Syt B-Protein XIV I-Protein ) O conserved O from O Drosophila O to O humans O . O Synaptotagmins B-Family ( O Syts B-Family ) O represent O a O large O family O of O putative O membrane O trafficking O proteins O found O in O various O species O from O different O phyla O . O In O this O study O , O I O identified O a O novel O class O of O Syt B-Family ( O named O Syt B-Protein XIV I-Protein ) O conserved O from O Drosophila O to O humans O and O its O highly O related O molecule O , O Strep14 B-Protein ( O Syt B-Protein XIV I-Protein - I-Protein related I-Protein protein I-Protein ) O . O Although O both O Syt B-Protein XIV I-Protein and O Strep14 B-Protein belong O to O the O C O - O terminal O - O type O ( O C O - O type O ) O tandem O C2 O protein O family O , O only O Syt B-Protein XIV I-Protein has O a O single O transmembrane O domain O at O the O N O - O terminus O and O a O putative O fatty O - O acylation O site O just O downstream O of O the O transmembrane O domain O . O Biochemical O analyses O have O indicated O that O Syt B-Protein XIV I-Protein is O a O Ca B-Chemical ( I-Chemical 2 I-Chemical + I-Chemical ) I-Chemical - O independent O Syt B-Family ( O e O . O g O . O , O Syts B-Protein VIII I-Protein , O XII B-Protein , O and O XIII B-Protein ) O and O that O , O like O other O Syt B-Family family O proteins O , O it O is O capable O of O forming O a O Ca B-Chemical ( I-Chemical 2 I-Chemical + I-Chemical ) I-Chemical - O independent O oligomer O . O Unlike O other O Syt B-Family isoforms O , O however O , O expression O of O Syt B-Protein XIV I-Protein and O Strep14 B-Protein mRNA O is O highly O restricted O to O mouse O heart O and O testis O and O absent O in O the O brain O , O where O most O other O Syts B-Family are O abundantly O expressed O , O suggesting O that O Syt B-Protein XIV I-Protein and O Strep14 B-Protein may O be O involved O in O membrane O trafficking O in O specific O tissues O outside O the O brain O . O I O also O identified O all O of O the O C O - O type O tandem O C2 O proteins O in O humans O , O the O mouse O , O the O fruit O fly O , O a O nematode O , O a O plant O , O and O a O yeast O and O discuss O the O molecular O evolution O of O the O C O - O type O tandem O C2 O protein O families O , O including O the O Syt B-Family family O , O the O Syt B-Family - I-Family like I-Family protein I-Family ( O Slp B-Family ) O family O , O and O the O Doc2 B-Family family O . O The O B B-Protein cell I-Protein - I-Protein specific I-Protein major I-Protein raft I-Protein protein I-Protein , O Raftlin B-Protein , O is O necessary O for O the O integrity O of O lipid O raft O and O BCR B-Family signal O transduction O . O Recent O evidence O indicates O that O membrane O microdomains O , O termed O lipid O rafts O , O have O a O role O in O B O - O cell O activation O as O platforms O for O B B-Family - I-Family cell I-Family antigen I-Family receptor I-Family ( O BCR B-Family ) O signal O initiation O . O To O gain O an O insight O into O the O possible O functioning O of O lipid O rafts O in O B O cells O , O we O applied O liquid O chromatography O electrospray O ionization O tandem O mass O spectrometry O ( O LC O - O ESI O - O MS O / O MS O ) O methodologies O to O the O identification O of O proteins O that O co O - O purified O with O lipid O rafts O of O Raji O cells O . O Among O these O raft O proteins O , O we O characterized O a O novel O protein O termed O Raftlin B-Protein ( O raft B-Protein - I-Protein linking I-Protein protein I-Protein ) O . O Like O the O Src B-Family family O kinase O , O Raftlin B-Protein is O localized O exclusively O in O lipid O rafts O by O fatty O acylation O of O N O - O terminal O Gly2 O and O Cys3 O , O and O is O co O - O localized O with O BCR B-Family before O and O after O BCR B-Family stimulation O . O Disruption O of O the O Raftlin B-Protein gene O in O the O DT40 O B O - O cell O line O resulted O in O a O marked O reduction O in O the O quantity O of O lipid O raft O components O , O including O Lyn B-Protein and O ganglioside O GM1 B-Chemical , O while O overexpression O of O Raftlin B-Protein increased O the O content O of O raft O protein O . O Moreover O , O BCR B-Family - O mediated O tyrosine O phosphorylation O and O calcium B-Chemical mobilization O were O impaired O by O the O lack O of O Raftlin B-Protein and O actually O potentiated O by O overexpression O of O Raftlin B-Protein . O These O data O suggest O that O Raftlin B-Protein plays O a O pivotal O role O in O the O formation O and O / O or O maintenance O of O lipid O rafts O , O therefore O regulating O BCR B-Family - O mediated O signaling O . O Omi B-Protein / O HtrA2 B-Protein catalytic O cleavage O of O inhibitor B-Family of I-Family apoptosis I-Family ( O IAP B-Family ) O irreversibly O inactivates O IAPs B-Family and O facilitates O caspase B-Family activity O in O apoptosis O . O Omi B-Protein / O HtrA2 B-Protein is O a O mitochondrial O serine O protease O that O is O released O into O the O cytosol O during O apoptosis O to O antagonize O inhibitors B-Family of I-Family apoptosis I-Family ( O IAPs B-Family ) O and O contribute O to O caspase B-Family - O independent O cell O death O . O Here O , O we O demonstrate O that O Omi B-Protein / O HtrA2 B-Protein directly O cleaves O various O IAPs B-Family in O vitro O , O and O the O cleavage O efficiency O is O determined O by O its O IAP B-Family - O binding O motif O , O AVPS O . O Cleavage O of O IAPs B-Family such O as O c B-Protein - I-Protein IAP1 I-Protein substantially O reduces O its O ability O to O inhibit O and O ubiquitylate O caspases B-Family . O In O contrast O to O the O stoichiometric O anti O - O IAP B-Family activity O by O Smac B-Protein / O DIABLO B-Protein , O Omi B-Protein / O HtrA2 B-Protein cleavage O of O c B-Protein - I-Protein IAP1 I-Protein is O catalytic O and O irreversible O , O thereby O more O efficiently O inactivating O IAPs B-Family and O promoting O caspase B-Family activity O . O Elimination O of O endogenous O Omi B-Protein by O RNA O interference O abolishes O c B-Protein - I-Protein IAP1 I-Protein cleavage O and O desensitizes O cells O to O apoptosis O induced O by O TRAIL B-Complex . O In O addition O , O overexpression O of O cleavage O - O site O mutant O c B-Protein - I-Protein IAP1 I-Protein makes O cells O more O resistant O to O TRAIL B-Complex - O induced O caspase B-Family activation O . O This O IAP B-Family cleavage O by O Omi B-Protein is O independent O of O caspase B-Family . O Taken O together O , O these O results O indicate O that O unlike O Smac B-Protein / O DIABLO B-Protein , O Omi B-Protein / O HtrA2 B-Protein ' O s O catalytic O cleavage O of O IAPs B-Family is O a O key O mechanism O for O it O to O irreversibly O inactivate O IAPs B-Family and O promote O apoptosis O . O Enhanced O gene O activation O by O Notch B-Family and O BMP B-Family signaling O cross O - O talk O . O The O signaling O systems O of O Notch B-Family and O bone B-Family morphogenetic I-Family protein I-Family ( O BMP B-Family ) O are O highly O conserved O from O flies O to O mammals O and O have O been O shown O to O be O important O in O the O development O of O multiple O organs O . O For O instance O , O in O the O fate O determination O of O mouse O neuroepithelial O cells O , O Notch B-Family signaling O plays O a O role O in O keeping O the O progenitors O from O differentiating O into O neurons O . O BMP B-Family is O also O known O to O inhibit O neuronal O differentiation O . O In O this O paper O , O we O show O that O BMP2 B-Protein enhances O Notch B-Family - O induced O transcriptional O activation O of O Hes B-Protein - I-Protein 5 I-Protein and O Hesr B-Protein - I-Protein 1 I-Protein in O mouse O neuroepithelial O cells O . O BMP2 B-Protein stimulation O , O in O addition O to O the O introduction O of O the O intracellular O domain O of O Notch B-Family ( O NIC O ) O , O resulted O in O enhanced O activation O of O the O Hes B-Protein - I-Protein 5 I-Protein gene O promoter O . O RBP B-Protein - I-Protein Jkappa I-Protein binding O to O its O target O sequence O is O important O not O only O for O Notch B-Family signaling O , O but O also O for O BMP2 B-Protein signaling O , O to O activate O the O Hes B-Protein - I-Protein 5 I-Protein gene O promoter O . O Smad1 B-Protein , O a O Smad B-Family species O that O is O activated O by O BMP2 B-Protein , O barely O interacted O with O NIC O , O but O did O form O a O complex O with O NIC O in O the O simultaneous O presence O of O the O coactivators O P B-Protein / I-Protein CAF I-Protein and O p300 B-Protein . O Recruitment O of O p300 B-Protein to O the O NIC O - O containing O complex O was O facilitated O by O activated O Smad1 B-Protein , O which O is O suggested O to O contribute O to O BMP2 B-Protein - O mediated O enhancement O of O Notch B-Protein - O induced O Hes B-Protein - I-Protein 5 I-Protein expression O . O These O data O suggest O a O novel O functional O cooperation O between O Notch B-Family signaling O and O BMP B-Family signaling O . O Origin O of O endogenous O DNA O abasic O sites O in O Saccharomyces O cerevisiae O . O Abasic O ( O AP O ) O sites O are O among O the O most O frequent O endogenous O lesions O in O DNA O and O present O a O strong O block O to O replication O . O In O Saccharomyces O cerevisiae O , O an O apn1 B-Protein apn2 B-Protein rad1 B-Protein triple O mutant O is O inviable O because O of O its O incapacity O to O repair O AP O sites O and O related O 3 O ' O - O blocked O single O - O strand O breaks O ( O M O . O Guillet O and O S O . O Boiteux O , O EMBO O J O . O 21 O : O 2833 O , O 2002 O ) O . O Here O , O we O investigated O the O origin O of O endogenous O AP O sites O in O yeast O . O Our O results O show O that O the O deletion O of O the O UNG1 B-Protein gene O encoding O the O uracil B-Protein DNA I-Protein glycosylase I-Protein suppresses O the O lethality O of O the O apn1 B-Protein apn2 B-Protein rad1 B-Protein mutant O . O In O contrast O , O inactivation O of O the O MAG1 B-Protein , O OGG1 B-Protein , O or O NTG1 B-Protein and O NTG2 B-Protein genes O encoding O DNA O glycosylases O involved O in O the O repair O of O alkylation O or O oxidation O damages O does O not O suppress O lethality O . O Although O viable O , O the O apn1 B-Protein apn2 B-Protein rad1 B-Protein ung1 B-Protein mutant O presents O growth O delay O due O to O a O G O ( O 2 O ) O / O M O checkpoint O . O These O results O point O to O uracil O as O a O critical O source O of O the O formation O of O endogenous O AP O sites O in O DNA O . O Uracil O can O arise O in O DNA O by O cytosine O deamination O or O by O the O incorporation O of O dUMP O during O replication O . O Here O , O we O show O that O the O overexpression O of O the O DUT1 B-Protein gene O encoding O the O dUTP B-Protein pyrophosphatase I-Protein ( O Dut1 B-Protein ) O suppresses O the O lethality O of O the O apn1 B-Protein apn2 B-Protein rad1 B-Protein mutant O . O Therefore O , O this O result O points O to O the O dUTP O pool O as O an O important O source O of O the O formation O of O endogenous O AP O sites O in O eukaryotes O . O A O novel O transmembrane O protein O recruits O numb B-Protein to O the O plasma O membrane O during O asymmetric O cell O division O . O Numb B-Protein , O an O evolutionarily O conserved O cell O fate O - O determining O factor O , O plays O a O pivotal O role O in O the O development O of O Drosophila O and O vertebrate O nervous O systems O . O Despite O lacking O a O transmembrane O segment O , O Numb B-Protein is O associated O with O the O cell O membrane O during O the O asymmetric O cell O division O of O Drosophila O neural O precursor O cells O and O is O selectively O partitioned O to O one O of O the O two O progeny O cells O from O a O binary O cell O division O . O Numb B-Protein contains O an O N O - O terminal O phosphotyrosine O - O binding O ( O PTB O ) O domain O that O is O essential O for O both O the O asymmetric O localization O and O the O fate O specification O function O of O Numb B-Protein . O We O report O here O the O isolation O and O characterization O of O a O novel O PTB O domain O - O binding O protein O , O NIP B-Protein ( O Numb B-Protein - I-Protein interacting I-Protein protein I-Protein ) O . O NIP B-Protein is O a O multipass O transmembrane O protein O that O contains O two O PTB O domain O - O binding O , O NXXF O motifs O required O for O the O interaction O with O Numb B-Protein . O In O dividing O Drosophila O neuroblasts O , O NIP B-Protein is O colocalized O to O the O cell O membrane O with O Numb B-Protein in O a O basal O cortical O crescent O . O Expression O of O NIP B-Protein in O Cos O - O 7 O cells O recruited O Numb B-Protein from O the O cytosol O to O the O plasma O membrane O . O This O recruitment O of O Numb B-Protein to O membrane O by O NIP B-Protein was O dependent O on O the O presence O of O at O least O one O NXXF O site O . O In O Drosophila O Schneider O 2 O cells O , O NIP B-Protein and O Numb B-Protein were O colocalized O at O the O plasma O membrane O . O Inhibition O of O NIP B-Protein expression O by O RNA O interference O released O Numb B-Protein to O the O cytosol O . O These O results O suggest O that O a O direct O protein O - O protein O interaction O between O NIP B-Protein and O Numb B-Protein is O necessary O and O sufficient O for O the O recruitment O of O Numb B-Protein to O the O plasma O membrane O . O Recruitment O of O Numb B-Protein to O a O basal O cortical O crescent O in O a O dividing O neuroblast O is O essential O for O Numb B-Protein to O function O as O an O intrinsic O cell O fate O determinant O . O AML1 B-Protein is O functionally O regulated O through O p300 B-Protein - O mediated O acetylation O on O specific O lysine O residues O . O AML1 B-Protein ( O RUNX1 B-Protein ) O is O one O of O the O most O frequently O disrupted O genes O in O human O leukemias O . O AML1 B-Protein encodes O transcription O factors O , O which O play O a O pivotal O role O in O hematopoietic O differentiation O , O and O their O inappropriate O expression O is O associated O with O leukemic O transformation O of O hematopoietic O cells O . O Previous O studies O demonstrated O that O the O transcription O cofactor O p300 B-Protein binds O to O the O C O - O terminal O region O of O AML1 B-Protein and O stimulates O AML1 B-Protein - O dependent O transcription O during O myeloid O cell O differentiation O . O Here O , O we O report O that O AML1 B-Protein is O specifically O acetylated O by O p300 B-Protein in O vitro O . O Mutagenesis O analyses O reveal O that O p300 B-Protein acetylates O AML1 B-Protein at O the O two O conserved O lysine O residues O ( O Lys O - O 24 O and O Lys O - O 43 O ) O . O AML1 B-Protein is O subject O to O acetylation O at O the O same O sites O in O vivo O , O and O p300 B-Protein - O mediated O acetylation O significantly O augments O the O DNA O binding O activity O of O AML1 B-Protein . O Disruption O of O these O two O lysines O severely O impairs O DNA O binding O of O AML1 B-Protein and O reduced O the O transcriptional O activity O and O the O transforming O potential O of O AML1 B-Protein . O Taken O together O , O these O data O indicate O that O acetylation O of O AML1 B-Protein through O p300 B-Protein is O a O critical O manner O of O posttranslational O modification O and O identify O a O novel O mechanism O for O regulating O the O function O of O AML1 B-Protein . O Structural O mechanism O of O the O bromodomain O of O the O coactivator O CBP B-Protein in O p53 B-Protein transcriptional O activation O . O Lysine O acetylation O of O the O tumor B-Protein suppressor I-Protein protein I-Protein p53 B-Protein in O response O to O a O wide O variety O of O cellular O stress O signals O is O required O for O its O activation O as O a O transcription O factor O that O regulates O cell O cycle O arrest O , O senescence O , O or O apoptosis O . O Here O , O we O report O that O the O conserved O bromo O - O domain O of O the O transcriptional O coactivator O CBP B-Protein ( O CREB B-Protein binding I-Protein protein I-Protein ) O binds O specifically O to O p53 B-Protein at O the O C O - O terminal O acetylated O lysine O 382 O . O This O bromodomain O / O acetyl O - O lysine O binding O is O responsible O for O p53 B-Protein acetylation O - O dependent O coactivator O recruitment O after O DNA O damage O , O a O step O essential O for O p53 B-Protein - O induced O transcriptional O activation O of O the O cyclin B-Family - I-Family dependent I-Family kinase I-Family inhibitor I-Family p21 B-Protein in O G1 O cell O cycle O arrest O . O We O further O present O the O three O - O dimensional O nuclear O magnetic O resonance O structure O of O the O CBP B-Protein bromodomain O in O complex O with O a O lysine O 382 O - O acetylated O p53 B-Protein peptide O . O Using O structural O and O biochemical O analyses O , O we O define O the O molecular O determinants O for O the O specificity O of O this O molecular O recognition O . O PIASy B-Protein - O mediated O repression O of O the O androgen B-Protein receptor I-Protein is O independent O of O sumoylation O . O PIASy B-Protein , O a O member O of O the O protein B-Family inhibitor I-Family of I-Family activated I-Family STAT I-Family ( O PIAS B-Family ) O family O , O represses O the O transcriptional O activity O of O the O androgen B-Protein receptor I-Protein ( O AR B-Protein ) O . O In O this O report O , O we O investigate O the O mechanism O of O PIASy B-Protein - O mediated O repression O of O AR B-Protein . O We O show O that O AR B-Protein binds O to O the O RING O - O finger O like O domain O of O PIASy B-Protein . O PIASy B-Protein contains O two O transcriptional O repression O domains O , O RD1 O and O RD2 O . O RD1 O , O but O not O RD2 O , O is O required O for O PIASy B-Protein - O mediated O repression O of O AR B-Protein . O We O show O that O the O RD1 O domain O binds O HDAC1 B-Protein and O HDAC2 B-Protein and O that O HDAC B-Family activity O is O required O for O PIASy B-Protein - O mediated O AR B-Protein repression O . O PIAS B-Family proteins O possess O small B-Family ubiquitin I-Family - I-Family related I-Family modifier I-Family ( O SUMO B-Family ) O E3 O ligase O activity O . O Conjugation O of O SUMO B-Protein - I-Protein 1 I-Protein to O AR B-Protein has O been O implicated O in O the O regulation O of O AR B-Protein activity O . O We O examine O if O the O SUMO B-Family ligase O activity O of O PIASy B-Protein is O required O for O PIASy B-Protein to O repress O AR B-Protein . O We O show O that O a O mutant O PIASy B-Protein , O defective O in O promoting O sumoylation O , O retains O the O ability O to O repress O AR B-Protein transcription O . O In O addition O , O mutation O of O all O the O known O sumoylation O acceptor O sites O of O AR B-Protein does O not O affect O the O transrepression O activity O of O PIASy B-Protein on O AR B-Protein . O Our O results O suggest O that O PIASy B-Protein may O repress O AR B-Protein by O recruiting O histone B-Family deacetylases I-Family , O independent O of O its O SUMO B-Family ligase O activity O . O Integrin O - O dependent O apposition O of O Drosophila O extraembryonic O membranes O promotes O morphogenesis O and O prevents O anoikis O . O BACKGROUND O : O Two O extraembryonic O tissues O form O early O in O Drosophila O development O . O One O , O the O amnioserosa O , O has O been O implicated O in O the O morphogenetic O processes O of O germ O band O retraction O and O dorsal O closure O . O The O developmental O role O of O the O other O , O the O yolk O sac O , O is O obscure O . O RESULTS O : O By O using O live O - O imaging O techniques O , O we O report O intimate O interactions O between O the O amnioserosa O and O the O yolk O sac O during O germ O band O retraction O and O dorsal O closure O . O These O tissue O interactions O fail O in O a O subset O of O myospheroid B-Protein ( O mys B-Protein : O betaPS B-Protein integrin I-Protein ) O mutant O embryos O , O leading O to O failure O of O germ O band O retraction O and O dorsal O closure O . O The O Drosophila O homolog O of O mammalian O basigin B-Protein ( O EMMPRIN B-Protein , O CD147 B-Protein ) O - O an O integrin O - O associated O transmembrane O glycoprotein O - O is O highly O enriched O in O the O extraembryonic O tissues O . O Strong O dominant O genetic O interactions O between O basigin B-Protein and O mys B-Protein mutations O cause O severe O defects O in O dorsal O closure O , O consistent O with O basigin B-Protein functioning O together O with O betaPS B-Protein integrin I-Protein in O extraembryonic O membrane O apposition O . O During O normal O development O , O JNK B-Family signaling O is O upregulated O in O the O amnioserosa O , O as O midgut O closure O disrupts O contact O with O the O yolk O sac O . O Subsequently O , O the O amnioserosal O epithelium O degenerates O in O a O process O that O is O independent O of O the O reaper B-Protein , O hid B-Protein , O and O grim B-Protein cell O death O genes O . O In O mys B-Protein mutants O that O fail O to O establish O contact O between O the O extraembryonic O membranes O , O the O amnioserosa O undergoes O premature O disintegration O and O death O . O CONCLUSIONS O : O Intimate O apposition O of O the O amnioserosa O and O yolk O sac O prevents O anoikis O of O the O amnioserosa O . O Survival O of O the O amnioserosa O is O essential O for O germ O band O retraction O and O dorsal O closure O . O We O hypothesize O that O during O normal O development O , O loss O of O integrin O - O dependent O contact O between O the O extraembryonic O tissues O results O in O JNK B-Family - O dependent O amnioserosal O disintegration O and O death O , O thus O representing O an O example O of O developmentally O programmed O anoikis O . O Disruption O of O the O Rb B-Protein - O - O Raf B-Protein - I-Protein 1 I-Protein interaction O inhibits O tumor O growth O and O angiogenesis O . O The O retinoblastoma B-Protein tumor I-Protein suppressor I-Protein protein I-Protein ( O Rb B-Protein ) O plays O a O vital O role O in O regulating O mammalian O cell O cycle O progression O and O inactivation O of O Rb B-Protein is O necessary O for O entry O into O S O phase O . O Rb B-Protein is O inactivated O by O phosphorylation O upon O growth O factor O stimulation O of O quiescent O cells O , O facilitating O the O transition O from O G O ( O 1 O ) O phase O to O S O phase O . O Although O the O signaling O events O after O growth O factor O stimulation O have O been O well O characterized O , O it O is O not O yet O clear O how O these O signals O contact O the O cell O cycle O machinery O . O We O had O found O previously O that O growth O factor O stimulation O of O quiescent O cells O lead O to O the O direct O binding O of O Raf B-Protein - I-Protein 1 I-Protein kinase I-Protein to O Rb B-Protein , O leading O to O its O inactivation O . O Here O we O show O that O the O Rb B-Protein - O Raf B-Protein - I-Protein 1 I-Protein interaction O occurs O prior O to O the O activation O of O cyclin B-Family and O / O or O cyclin B-Family - I-Family dependent I-Family kinases I-Family and O facilitates O normal O cell O cycle O progression O . O Raf B-Protein - I-Protein 1 I-Protein - O mediated O inactivation O of O Rb B-Protein is O independent O of O the O mitogen B-Family - I-Family activated I-Family protein I-Family kinase I-Family cascade O , O as O well O as O cyclin B-Family - I-Family dependent I-Family kinases I-Family . O Binding O of O Raf B-Protein - I-Protein 1 I-Protein seemed O to O correlate O with O the O dissociation O of O the O chromatin O remodeling O protein B-Protein Brg1 I-Protein from O Rb B-Protein . O Disruption O of O the O Rb B-Protein - O Raf B-Protein - I-Protein 1 I-Protein interaction O by O a O nine O - O amino O - O acid O peptide O inhibits O Rb B-Protein phosphorylation O , O cell O proliferation O , O and O vascular B-Family endothelial I-Family growth I-Family factor I-Family - O mediated O capillary O tubule O formation O . O Delivery O of O this O peptide O by O a O carrier O molecule O led O to O a O 79 O % O reduction O in O tumor O volume O and O a O 57 O % O reduction O in O microvessel O formation O in O nude O mice O . O It O appears O that O Raf B-Protein - I-Protein 1 I-Protein links O mitogenic O signaling O to O Rb B-Protein and O that O disruption O of O this O interaction O could O aid O in O controlling O proliferative O disorders O . O Cited1 B-Protein is O a O bifunctional O transcriptional O cofactor O that O regulates O early O nephronic O patterning O . O In O a O screen O to O identify O factors O that O regulate O the O conversion O of O mesenchyme O to O epithelium O during O the O early O stages O of O nephrogenesis O , O it O was O found O that O the O Smad4 B-Protein - O interacting O transcriptional O cofactor O , O Cited1 B-Protein , O is O expressed O in O the O condensed O cap O mesenchyme O surrounding O the O tip O of O the O ureteric O bud O ( O UB O ) O , O is O downregulated O after O differentiation O into O epithelia O , O and O has O the O capacity O to O block O UB O branching O and O epithelial O morphogenesis O in O cultured O metanephroi O . O Cited1 B-Protein represses O Wnt B-Family / O beta B-Protein - I-Protein catenin I-Protein but O activates O Smad4 B-Protein - O dependent O transcription O involved O in O TGF B-Family - I-Family beta I-Family and O Bmp B-Protein signaling O . O By O modifying O these O pathways O , O Cited1 B-Protein may O coordinate O cellular O differentiation O and O survival O signals O that O regulate O nephronic O patterning O in O the O metanephros O . O Requirement O of O Cul3 B-Protein for O axonal O arborization O and O dendritic O elaboration O in O Drosophila O mushroom O body O neurons O . O Cul3 B-Protein belongs O to O the O family O of O cullin B-Family proteins O , O which O function O as O scaffold O proteins O of O E3 O ubiquitin B-Protein ligase O complexes O . O Here O we O show O cell O - O autonomous O involvement O of O Cul3 B-Protein in O axonal O arborization O and O dendritic O elaboration O of O Drosophila O mushroom O body O neurons O . O Cul3 B-Protein mutant O neurons O are O defective O in O terminal O morphogenesis O of O neurites O . O Interestingly O , O mutant O axons O often O terminate O around O branching O points O . O In O addition O , O dendritic O elaboration O is O severely O affected O in O Cul3 B-Protein mutant O neurons O . O However O , O loss O of O Cul3 B-Protein function O does O not O affect O extension O of O the O axons O that O rarely O arborize O . O Function O of O cullin B-Family - O type O proteins O has O been O shown O to O require O covalent O attachment O of O Nedd8 B-Protein ( O neural O precursor O cell O - O expressed O developmentally O downregulated O ) O , O a O ubiquitin B-Protein - O like O protein O . O Consistent O with O this O notion O , O Cul3 B-Protein is O inactivated O by O a O mutation O in O its O conserved O neddylation O site O , O and O Nedd8 B-Protein mutant O neurons O exhibit O similar O neuronal O morphogenetic O defects O . O Together O , O Cul3 B-Protein plays O an O essential O role O in O both O axonal O arborization O and O proper O elaboration O of O dendrites O and O may O require O neddylation O for O its O proper O function O . O The O kinesinlike B-Protein protein I-Protein Subito I-Protein contributes O to O central O spindle O assembly O and O organization O of O the O meiotic O spindle O in O Drosophila O oocytes O . O In O the O oocytes O of O many O species O , O bipolar O spindles O form O in O the O absence O of O centrosomes O . O Drosophila O melanogaster O oocyte O chromosomes O have O a O major O role O in O nucleating O microtubules O , O which O precedes O the O bundling O and O assembly O of O these O microtubules O into O a O bipolar O spindle O . O Here O we O present O evidence O that O a O region O similar O to O the O anaphase O central O spindle O functions O to O organize O acentrosomal O spindles O . O Subito B-Protein mutants O are O characterized O by O the O formation O of O tripolar O or O monopolar O spindles O and O nondisjunction O of O homologous O chromosomes O at O meiosis O I O . O Subito B-Protein encodes O a O kinesinlike B-Family protein O and O associates O with O the O meiotic O central O spindle O , O consistent O with O its O classification O in O the O Kinesin B-Family 6 I-Family / O MKLP1 B-Protein family O . O This O class O of O proteins O is O known O to O be O required O for O cytokinesis O , O but O our O results O suggest O a O new O function O in O spindle O formation O . O The O meiotic O central O spindle O appears O during O prometaphase O and O includes O passenger O complex O proteins O such O as O AurB B-Protein and O Incenp B-Protein . O Unlike O mitotic O cells O , O the O passenger O proteins O do O not O associate O with O centromeres O before O anaphase O . O In O the O absence O of O Subito B-Protein , O central O spindle O formation O is O defective O and O AurB B-Protein and O Incenp B-Protein fail O to O properly O localize O . O We O propose O that O Subito B-Protein is O required O for O establishing O and O / O or O maintaining O the O central O spindle O in O Drosophila O oocytes O , O and O this O substitutes O for O the O role O of O centrosomes O in O organizing O the O bipolar O spindle O . O The O adaptor O protein O 3BP2 B-Protein binds O human O CD244 B-Protein and O links O this O receptor O to O Vav B-Protein signaling O , O ERK B-Family activation O , O and O NK O cell O killing O . O Adaptor O proteins O , O molecules O that O mediate O intermolecular O interactions O , O are O crucial O for O cellular O activation O . O The O adaptor O 3BP2 B-Protein has O been O shown O to O positively O regulate O NK O cell O - O mediated O cytotoxicity O . O In O this O study O we O present O evidence O for O a O physical O interaction O between O 3BP2 B-Protein and O the O CD244 B-Protein receptor O . O CD244 B-Protein , O a O member O of O the O CD150 B-Protein family O , O is O a O cell O surface O protein O expressed O on O NK O , O CD8 B-Protein + O T O , O and O myeloid O cells O . O CD244 B-Protein interacts O via O its O Src B-Protein homology O 2 O domain O with O the O X O - O linked O lymphoproliferative O disease O gene O product O signaling B-Protein lymphocytic I-Protein activation I-Protein molecule I-Protein - I-Protein associated I-Protein protein I-Protein ( O SAP B-Protein ) O / O SH2 B-Protein domain I-Protein protein I-Protein 1A I-Protein . O 3BP2 B-Protein interacts O with O human O but O not O murine O CD244 B-Protein . O CD244 B-Protein - O 3BP2 B-Protein interaction O was O direct O and O regulated O by O phosphorylation O , O as O shown O by O a O three O - O hybrid O analysis O in O yeast O and O NK O cells O . O Tyr337 O on O CD244 B-Protein , O part O of O a O consensus O motif O for O SAP B-Protein / O SH2 B-Protein domain I-Protein protein I-Protein 1A I-Protein binding O , O was O critical O for O the O 3BP2 B-Protein interaction O . O Although O mutation O of O Tyr337 O to O phenylalanine O abrogated O human O 3BP2 B-Protein binding O , O we O still O observed O SAP B-Protein association O , O indicating O that O this O motif O is O not O essential O for O SAP B-Protein recruitment O . O CD244 B-Protein ligation O induced O 3BP2 B-Protein phosphorylation O and O Vav B-Protein - I-Protein 1 I-Protein recruitment O . O Overexpression O of O 3BP2 B-Protein led O to O an O increase O in O the O magnitude O and O duration O of O ERK B-Family activation O , O after O CD244 B-Protein triggering O . O This O enhancement O was O concomitant O with O an O increase O in O cytotoxicity O due O to O CD244 B-Protein ligation O . O However O , O no O differences O in O IFN B-Protein - I-Protein gamma I-Protein secretion O were O found O when O normal O and O 3BP2 B-Protein - O transfected O cells O were O compared O . O These O results O indicate O that O CD244 B-Protein - O 3BP2 B-Protein association O regulates O cytolytic O function O but O not O IFN B-Protein - I-Protein gamma I-Protein release O , O reinforcing O the O hypothesis O that O , O in O humans O , O CD244 B-Protein - O mediated O cytotoxicity O and O IFN B-Protein - I-Protein gamma I-Protein release O involve O distinct O NK O pathways O . O Functional O interactions O of O stimulatory O and O inhibitory O GDP O / O GTP O exchange O proteins O and O their O common O substrate O small B-Family GTP I-Family - I-Family binding I-Family protein I-Family . O smg B-Protein GDS I-Protein and O rho B-Protein GDI I-Protein are O stimulatory O and O inhibitory O GDP O / O GTP O exchange O proteins O , O respectively O , O for O a O group O of O ras B-Family p21 I-Family - I-Family related I-Family small I-Family GTP I-Family - I-Family binding I-Family proteins I-Family ( O G B-Family proteins I-Family ) O . O rho B-Protein p21 I-Protein is O a O common O substrate O small O G B-Family protein I-Family for O both O GDP O / O GTP O exchange O proteins O . O We O examined O here O the O functional O interactions O of O these O GDP O / O GTP O exchange O proteins O with O rho B-Protein p21 I-Protein as O a O substrate O . O smg B-Protein GDS I-Protein and O rho B-Protein GDI I-Protein interacted O with O the O GDP B-Chemical - O bound O form O of O rho B-Protein p21 I-Protein and O thereby O stimulated O and O inhibited O , O respectively O , O the O dissociation O of O GDP B-Chemical . O The O inhibitory O effect O of O rho B-Protein GDI I-Protein was O much O stronger O than O the O stimulatory O effect O of O smg B-Protein GDS I-Protein . O The O GDP B-Chemical - O bound O form O of O rho B-Protein p21 I-Protein formed O a O complex O with O rho B-Protein GDI I-Protein but O not O with O smg B-Protein GDS I-Protein in O their O simultaneous O presence O . O Since O the O content O of O smg B-Protein GDS I-Protein was O generally O less O than O that O of O rho B-Protein GDI I-Protein in O cells O , O these O results O suggest O that O there O is O some O mechanism O to O release O the O inhibitory O action O of O rho B-Protein GDI I-Protein and O to O make O rho B-Protein p21 I-Protein sensitive O to O the O smg B-Protein GDS I-Protein action O during O the O conversion O of O rhoA B-Protein p21 I-Protein from O the O GDP B-Chemical - O bound O inactive O form O to O the O GTP B-Chemical - O bound O active O form O in O intact O cells O . O On O the O other O hand O , O rho B-Protein p21 I-Protein was O previously O shown O to O be O ADP O - O ribosylated O by O bacterial O ADP B-Family - I-Family ribosyltransferases I-Family , O named O C3 B-Protein and O EDIN B-Protein , O at O Asn41 O in O the O putative O effector O region O of O rho B-Protein p21 I-Protein . O This O ADP O - O ribosylation O was O inhibited O by O rho B-Protein GDI I-Protein much O more O efficiently O than O by O smg B-Protein GDS I-Protein . O These O results O suggest O that O rho B-Protein GDI I-Protein may O mask O the O putative O effector O region O of O rho B-Protein p21 I-Protein and O thereby O inhibit O its O interaction O with O the O target O protein O even O in O the O presence O of O smg B-Protein GDS I-Protein . O Thus O , O both O smg B-Protein GDS I-Protein and O rho B-Protein GDI I-Protein are O important O to O regulate O the O rho B-Protein p21 I-Protein activity O and O action O in O cooperation O with O each O other O . O In O the O standard O halo O assay O , O minimal O medium O with O allantoin B-Chemical ( O MMA B-Chemical ) O is O supplemented O with O 30 O mug O / O ml O leucine B-Chemical to O induce O PTR2 B-Protein expression O ( O MMA B-Chemical + O Leu B-Chemical medium O ) O . O Under O these O conditions O , O wild O - O type O strains O varied O substantially O in O sensitivity O to O Ala B-Chemical - I-Chemical Eth I-Chemical toxicity O ( O Figure O 7 O ) O . O S288c B-Protein was O the O most O sensitive O to O the O dipeptide O , O whereas O W303 B-Protein was O completely O resistant O . O In O the O absence O of O the O leucine B-Chemical inducer O , O only O the O clinical O isolates O YAT17 B-Protein and O YAT21 B-Protein were O sensitive O to O Ala B-Chemical - I-Chemical Eth I-Chemical ( O Figure O 7 O ) O . O Histone B-Protein deacetylase I-Protein 1 I-Protein - O mediated O histone B-Family modification O regulates O osteoblast O differentiation O . O Osteogenesis O is O a O complex O process O associated O with O dramatic O changes O in O gene O expression O . O To O elucidate O whether O modifications O in O chromatin O structure O are O involved O in O osteoblast O differentiation O , O we O examined O the O expression O levels O of O histone B-Family deacetylases I-Family ( O HDACs B-Family ) O and O the O degree O of O histone B-Family acetylation O at O the O promoter O regions O of O osteogenic O genes O . O During O osteogenesis O , O total O HDAC B-Family enzymatic O activity O was O decreased O with O significant O reduction O in O HDAC1 B-Protein expression O . O Consistently O , O recruitment O of O HDAC1 B-Protein to O the O promoters O of O osteoblast O marker O genes O , O including O osterix B-Protein and O osteocalcin B-Protein , O was O down O - O regulated O , O whereas O histone B-Protein H3 I-Protein and O H4 B-Protein were O hyperacetylated O at O those O promoters O during O osteoblast O differentiation O . O Moreover O , O suppression O of O HDAC B-Family activity O with O a O HDAC B-Family inhibitor O , O sodium O butyrate O , O accelerated O osteogenesis O by O inducing O osteoblast O marker O genes O including O osteopontin B-Protein and O alkaline B-Protein phosphatase I-Protein . O Consistently O , O knockdown O of O HDAC1 B-Protein by O the O short O interference O RNA O system O stimulated O osteoblast O differentiation O . O Taken O together O , O these O data O propose O that O down O - O regulation O of O HDAC1 B-Protein is O an O important O process O for O osteogenesis O . O The O proprotein B-Family convertase I-Family ( O PC B-Family ) O PCSK9 B-Protein is O inactivated O by O furin B-Protein and O / O or O PC5 B-Protein / O 6A B-Protein : O functional O consequences O of O natural O mutations O and O post O - O translational O modifications O . O PCSK9 B-Protein is O the O ninth O member O of O the O proprotein B-Family convertase I-Family ( O PC B-Family ) O family O . O Some O of O its O natural O mutations O have O been O genetically O associated O with O the O development O of O a O dominant O form O of O familial O hyper O - O or O hypocholesterolemia O . O The O exact O mechanism O of O action O of O PCSK9 B-Protein is O not O clear O , O although O it O is O known O to O enhance O the O intracellular O degradation O of O the O low B-Protein density I-Protein lipoprotein I-Protein ( I-Protein LDL I-Protein ) I-Protein receptor I-Protein in O acidic O compartments O , O likely O the O endosomes O / O lysosomes O . O We O analyzed O the O post O - O translational O modifications O of O PCSK9 B-Protein and O show O that O it O is O sulfated O within O its O prosegment O at O Tyr38 O . O We O also O examined O the O susceptibility O of O PCSK9 B-Protein to O proteolytic O cleavage O by O the O other O members O of O the O PC B-Family family O . O The O data O show O that O the O natural O gain O - O of O - O function O mutations O R218S O , O F216L O , O and O D374Y O associated O with O hypercholesterolemia O result O in O total O or O partial O loss O of O furin B-Protein / O PC5 B-Protein / O 6A B-Protein processing O at O the O motif O RFHR218 O downward O arrow O . O In O contrast O , O the O loss O - O of O - O function O mutations O A443T O and O C679X O lead O either O to O the O lack O of O trans O - O Golgi O network O / O recycling O endosome O localization O and O an O enhanced O susceptibility O to O furin B-Protein cleavage O ( O A443T O ) O or O to O the O inability O of O PCSK9 B-Protein to O exit O the O endoplasmic O reticulum O ( O C679X O ) O . O Furthermore O , O we O report O the O presence O of O both O native O and O furin B-Protein - O like O cleaved O forms O of O PCSK9 B-Protein in O circulating O human O plasma O . O Thus O , O we O propose O that O PCSK9 B-Protein levels O are O finely O regulated O by O the O basic O amino O acid O convertases O furin B-Protein and O PC5 B-Protein / O 6A B-Protein . O The O latter O may O reduce O the O lifetime O of O this O proteinase O and O its O ability O to O degrade O the O cell O - O surface O LDL B-Protein receptor I-Protein , O thereby O regulating O the O levels O of O circulating O LDL B-Chemical cholesterol I-Chemical . O Involvement O of O the O IkappaB B-Family kinase I-Family ( I-Family IKK I-Family ) I-Family - I-Family related I-Family kinases I-Family tank B-Protein - I-Protein binding I-Protein kinase I-Protein 1 I-Protein / O IKKi B-Protein and O cullin B-Family - I-Family based I-Family ubiquitin I-Family ligases I-Family in O IFN B-Protein regulatory I-Protein factor I-Protein - I-Protein 3 I-Protein degradation O . O Activation O of O the O innate O arm O of O the O immune O system O following O pathogen O infection O relies O on O the O recruitment O of O latent O transcription O factors O involved O in O the O induction O of O a O subset O of O genes O responsible O for O viral O clearance O . O One O of O these O transcription O factors O , O IFN B-Protein regulatory I-Protein factor I-Protein 3 I-Protein ( O IRF B-Protein - I-Protein 3 I-Protein ) O , O is O targeted O for O proteosomal O degradation O following O virus O infection O . O However O , O the O molecular O mechanisms O involved O in O this O process O are O still O unknown O . O In O this O study O , O we O show O that O polyubiquitination O of O IRF B-Protein - I-Protein 3 I-Protein increases O in O response O to O Sendai O virus O infection O . O Using O an O E1 B-Family temperature O - O sensitive O cell O line O , O we O demonstrate O that O polyubiquitination O is O required O for O the O observed O degradation O of O IRF B-Protein - I-Protein 3 I-Protein . O Inactivation O of O NEDD8 B-Protein - O activating O E1 B-Family enzyme O also O results O in O stabilization O of O IRF B-Protein - I-Protein 3 I-Protein suggesting O the O NEDDylation O also O plays O a O role O in O IRF B-Protein - I-Protein 3 I-Protein degradation O following O Sendai O virus O infection O . O In O agreement O with O this O observation O , O IRF B-Protein - I-Protein 3 I-Protein is O recruited O to O Cullin1 B-Protein following O virus O infection O and O overexpression O of O a O dominant O - O negative O mutant O of O Cullin1 B-Protein significantly O inhibits O the O degradation O of O IRF B-Protein - I-Protein 3 I-Protein observed O in O infected O cells O . O We O also O asked O whether O the O C O - O terminal O cluster O of O phosphoacceptor O sites O of O IRF B-Protein - I-Protein 3 I-Protein could O serve O as O a O destabilization O signal O and O we O therefore O measured O the O half O - O life O of O C O - O terminal O phosphomimetic O IRF B-Protein - I-Protein 3 I-Protein mutants O . O Interestingly O , O we O found O them O to O be O short O - O lived O in O contrast O to O wild O - O type O IRF B-Protein - I-Protein 3 I-Protein . O In O addition O , O no O degradation O of O IRF B-Protein - I-Protein 3 I-Protein was O observed O in O TBK1 B-Protein ( O - O / O - O ) O mouse O embryonic O fibroblasts O . O All O together O , O these O data O demonstrate O that O virus O infection O stimulates O a O host O cell O signaling O pathway O that O modulates O the O expression O level O of O IRF B-Protein - I-Protein 3 I-Protein through O its O C O - O terminal O phosphorylation O by O the O IkappaB B-Family kinase I-Family - I-Family related I-Family kinases I-Family followed O by O its O polyubiquitination O , O which O is O mediated O in O part O by O a O Cullin B-Family - I-Family based I-Family ubiquitin I-Family ligase I-Family . O Two O novel O members O of O the O ABLIM B-Family protein O family O , O ABLIM B-Protein - I-Protein 2 I-Protein and O - B-Protein 3 I-Protein , O associate O with O STARS B-Protein and O directly O bind O F B-Protein - I-Protein actin I-Protein . O In O addition O to O regulating O cell O motility O , O contractility O , O and O cytokinesis O , O the O actin B-Family cytoskeleton O plays O a O critical O role O in O the O regulation O of O transcription O and O gene O expression O . O We O have O previously O identified O a O novel O muscle O - O specific O actin B-Family - O binding O protein O , O STARS B-Protein ( O striated B-Protein muscle I-Protein activator I-Protein of I-Protein Rho I-Protein signaling I-Protein ) O , O which O directly O binds O actin B-Family and O stimulates O serum B-Protein - I-Protein response I-Protein factor I-Protein ( O SRF B-Protein ) O - O dependent O transcription O . O To O further O dissect O the O STARS B-Protein / O SRF B-Protein pathway O , O we O performed O a O yeast O two O - O hybrid O screen O of O a O skeletal O muscle O cDNA O library O using O STARS B-Protein as O bait O , O and O we O identified O two O novel O members O of O the O ABLIM B-Family protein O family O , O ABLIM B-Protein - I-Protein 2 I-Protein and O - B-Protein 3 I-Protein , O as O STARS B-Protein - O interacting O proteins O . O ABLIM B-Protein - I-Protein 1 I-Protein , O which O is O expressed O in O retina O , O brain O , O and O muscle O tissue O , O has O been O postulated O to O function O as O a O tumor O suppressor O . O ABLIM B-Protein - I-Protein 2 I-Protein and O - B-Protein 3 I-Protein display O distinct O tissue O - O specific O expression O patterns O with O the O highest O expression O levels O in O muscle O and O neuronal O tissue O . O Moreover O , O these O novel O ABLIM B-Family proteins O strongly O bind O F B-Protein - I-Protein actin I-Protein , O are O localized O to O actin B-Family stress O fibers O , O and O synergistically O enhance O STARS B-Protein - O dependent O activation O of O SRF B-Protein . O Conversely O , O knockdown O of O endogenous O ABLIM B-Family expression O utilizing O small O interfering O RNA O significantly O blunted O SRF B-Protein - O dependent O transcription O in O C2C12 O skeletal O muscle O cells O . O These O findings O suggest O that O the O members O of O the O novel O ABLIM B-Family protein O family O may O serve O as O a O scaffold O for O signaling O modules O of O the O actin O cytoskeleton O and O thereby O modulate O transcription O . O Inhibitor B-Protein of I-Protein growth I-Protein 4 I-Protein suppresses O cell O spreading O and O cell O migration O by O interacting O with O a O novel O binding O partner O , O liprin B-Protein alpha1 I-Protein . O Inhibitor B-Protein of I-Protein growth I-Protein 4 I-Protein ( O ING4 B-Protein ) O is O a O candidate O tumor O suppressor O that O plays O a O major O role O in O gene O regulation O , O cell O cycle O control O , O apoptosis O , O and O angiogenesis O . O ING4 B-Protein expression O is O down O - O regulated O in O glioblastoma O cells O and O head O and O neck O squamous O cell O carcinoma O . O Here O , O we O identified O liprin B-Protein alpha1 I-Protein / O PPFIA1 B-Protein , O a O cytoplasmic O protein O necessary O for O focal O adhesion O formation O and O axon O guidance O , O as O a O novel O interacting O protein O with O ING4 B-Protein . O ING4 B-Protein and O liprin B-Protein alpha1 I-Protein colocalized O at O lamellipodia O in O the O vicinity O of O vinculin B-Protein . O Overexpressed O ING4 B-Protein suppressed O cell O spreading O and O cell O migration O . O In O contrast O , O overexpressed O liprin B-Protein alpha1 I-Protein enhanced O cell O spreading O and O cell O migration O . O Knockdown O of O endogenous O ING4 B-Protein with O RNA O interference O induced O cell O motility O , O whereas O knockdown O of O endogenous O liprin B-Protein alpha1 I-Protein suppressed O cell O motility O . O ING4 B-Protein also O suppressed O cell O motility O that O was O enhanced O by O liprin B-Protein alpha1 I-Protein . O However O , O ING4 B-Protein did O not O further O suppress O cell O motility O when O liprin B-Protein alpha1 I-Protein was O suppressed O with O RNA O interference O , O suggesting O a O functional O and O mechanistic O interdependence O between O these O proteins O . O In O addition O to O its O nuclear O functions O , O cytoplasmic O ING4 B-Protein interacts O with O liprin B-Protein alpha1 I-Protein to O regulate O cell O migration O and O , O with O its O known O antiangiogenic O function O , O may O prevent O invasion O and O metastasis O . O SCABP8 B-Protein / O CBL10 B-Protein , O a O putative O calcium O sensor O , O interacts O with O the O protein O kinase O SOS2 B-Protein to O protect O Arabidopsis O shoots O from O salt B-Chemical stress O . O The O SOS O ( O for O Salt O Overly O Sensitive O ) O pathway O plays O essential O roles O in O conferring O salt B-Chemical tolerance O in O Arabidopsis O thaliana O . O Under O salt B-Chemical stress O , O the O calcium B-Chemical sensor O SOS3 B-Protein activates O the O kinase O SOS2 B-Protein that O positively O regulates O SOS1 B-Protein , O a O plasma O membrane O sodium B-Chemical / O proton B-Chemical antiporter O . O We O show O that O SOS3 B-Protein acts O primarily O in O roots O under O salt B-Chemical stress O . O By O contrast O , O the O SOS3 B-Protein homolog O SOS3 B-Protein - I-Protein LIKE I-Protein CALCIUM I-Protein BINDING I-Protein PROTEIN8 I-Protein ( O SCABP8 B-Protein ) O / O CALCINEURIN B-Protein B I-Protein - I-Protein LIKE10 I-Protein functions O mainly O in O the O shoot O response O to O salt B-Chemical toxicity O . O While O root O growth O is O reduced O in O sos3 B-Protein mutants O in O the O presence O of O NaCl B-Chemical , O the O salt B-Chemical sensitivity O of O scabp8 B-Protein is O more O prominent O in O shoot O tissues O . O SCABP8 B-Protein is O further O shown O to O bind O calcium B-Chemical , O interact O with O SOS2 B-Protein both O in O vitro O and O in O vivo O , O recruit O SOS2 B-Protein to O the O plasma O membrane O , O enhance O SOS2 B-Protein activity O in O a O calcium B-Chemical - O dependent O manner O , O and O activate O SOS1 B-Protein in O yeast O . O In O addition O , O sos3 B-Protein scabp8 B-Protein and O sos2 B-Protein scabp8 B-Protein display O a O phenotype O similar O to O sos2 B-Protein , O which O is O more O sensitive O to O salt B-Chemical than O either O sos3 B-Protein or O scabp8 B-Protein alone O . O Overexpression O of O SCABP8 B-Protein in O sos3 B-Protein partially O rescues O the O sos3 B-Protein salt B-Chemical - O sensitive O phenotype O . O However O , O overexpression O of O SOS3 B-Protein fails O to O complement O scabp8 B-Protein . O These O results O suggest O that O SCABP8 B-Protein and O SOS3 B-Protein are O only O partially O redundant O in O their O function O , O and O each O plays O additional O and O unique O roles O in O the O plant O salt B-Chemical stress O response O . O Increased O acylated O plasma O ghrelin B-Protein , O but O improved O lipid O profiles O 24 O - O h O after O consumption O of O carob O pulp O preparation O rich O in O dietary O fibre O and O polyphenols B-Chemical . O We O have O recently O shown O that O a O polyphenol B-Chemical - O rich O insoluble O dietary O fibre O preparation O from O carob O pulp O ( O Ceratonia O siliqua O L O ; O carob O fibre O ) O decreased O postprandial O acylated O ghrelin B-Protein , O TAG B-Chemical and O NEFA B-Chemical during O an O acute O liquid O meal O challenge O test O . O However O , O delayed O effects O of O carob O fibre O consumption O are O unknown O . O Therefore O , O a O randomized O controlled O crossover O study O in O nineteen O healthy O volunteers O consuming O foods O with O or O without O 50 O g O carob O fibre O was O conducted O . O On O the O subsequent O day O ( O day O 2 O ) O , O glucose B-Chemical , O TAG B-Chemical , O total O and O acylated O ghrelin B-Protein as O well O as O insulin B-Protein , O NEFA B-Chemical and O leptin B-Protein were O assessed O at O baseline O and O at O timed O intervals O for O 300 O min O after O ingestion O of O standardized O bread O . O Consumption O of O carob O fibre O - O enriched O foods O did O not O affect O fasting O concentrations O of O glucose B-Chemical , O TAG B-Chemical , O total O ghrelin B-Protein , O NEFA B-Chemical , O insulin B-Protein and O leptin B-Protein . O Fasting O acylated O ghrelin B-Protein was O increased O on O the O day O subsequent O to O carob O fibre O consumption O compared O with O control O ( O P O = O 0 O . O 046 O ) O . O After O consumption O of O the O standard O bread O on O day O 2 O , O glucose B-Chemical response O ( O P O = O 0 O . O 029 O ) O was O increased O , O and O TAG B-Chemical ( O P O = O 0 O . O 033 O ) O and O NEFA B-Chemical ( O P O < O 0 O . O 001 O ) O responses O were O decreased O compared O with O control O . O Postprandial O responses O of O total O and O acylated O ghrelin B-Protein , O insulin B-Protein and O leptin B-Protein on O day O 2 O were O unaffected O by O carob O fibre O consumption O the O previous O day O . O In O conclusion O , O an O increase O in O total O and O acylated O plasma O ghrelin B-Protein accompanied O by O enhanced O lipid O metabolism O after O carob O fibre O consumption O suggests O higher O lipid O utilization O and O suppressed O lipolysis O on O the O day O subsequent O to O carob O fibre O consumption O . O However O , O elevated O glucose B-Chemical levels O after O carob O fibre O consumption O need O to O be O addressed O in O future O studies O . O Herpes O simplex O virus O protein O UL11 B-Protein but O not O UL51 B-Protein is O associated O with O lipid O rafts O . O The O UL11 B-Protein and O UL51 B-Protein gene O products O of O herpes O simplex O virus O ( O HSV O ) O are O membrane O - O associated O tegument O proteins O that O are O incorporated O into O the O HSV O virion O . O UL11 B-Protein and O UL51 B-Protein are O conserved O throughout O the O herpesvirus O family O . O Both O UL11 B-Protein and O UL51 B-Protein , O either O singly O or O in O combination O , O are O involved O in O virion O envelopment O and O / O or O egress O . O Both O proteins O are O fatty O acylated O : O UL11 B-Protein is O both O acylated O by O myristoic O and O palmitoic O acids O and O UL51 B-Protein is O monoacylated O by O palmitoic O acid O . O Using O confocal O microscopy O and O sucrose O gradient O fractionations O in O transfected O or O HSV O - O infected O cells O , O we O found O that O HSV O - O 2 O UL11 B-Protein but O not O UL51 B-Protein was O associated O with O lipid O rafts O . O The O dual O acylation O of O UL11 B-Protein was O necessary O for O lipid O raft O association O , O as O mutations O in O the O myristoylation O or O palmitoylation O sites O prevented O lipid O raft O association O . O These O differences O in O lipid O raft O association O may O contribute O to O the O functional O differences O between O UL11 B-Protein and O UL51 B-Protein . O Altered O pattern O of O Cul B-Protein - I-Protein 1 I-Protein protein O expression O and O neddylation O in O human O lung O tumours O : O relationships O with O CAND1 B-Protein and O cyclin B-Protein E I-Protein protein O levels O . O The O Cul B-Protein - I-Protein 1 I-Protein protein O is O the O scaffold O element O of O SCF B-Complex complexes O that O are O involved O in O the O proteasomal O degradation O of O numerous O proteins O regulating O cell O cycle O progression O . O Owing O to O this O central O role O in O cell O growth O control O , O aberrant O expression O of O the O components O of O SCF B-Complex is O thought O to O play O a O role O during O tumourigenesis O . O Nothing O is O known O about O Cul B-Protein - I-Protein 1 I-Protein expression O in O human O tumours O . O In O this O study O , O we O have O analysed O its O status O in O a O series O of O 128 O human O lung O carcinomas O , O comprising O 50 O non O - O small O cell O lung O cancers O ( O NSCLCs O ; O 29 O squamous O cell O carcinomas O and O 21 O adenocarcinomas O ) O and O 78 O neuroendocrine O ( O NE O ) O lung O tumours O ( O 24 O typical O and O atypical O carcinoids O , O 19 O large O cell O NE O carcinomas O and O 35 O small O cell O lung O carcinomas O ) O , O using O immunohistochemistry O . O We O report O for O the O first O time O an O altered O pattern O of O Cul B-Protein - I-Protein 1 I-Protein expression O in O human O tumours O ; O indeed O , O we O show O that O Cul B-Protein - I-Protein 1 I-Protein expression O is O up O - O regulated O in O 40 O % O ( O 51 O / O 128 O ) O of O all O lung O tumours O as O compared O to O normal O lung O tissues O , O including O 34 O % O ( O 17 O / O 50 O ) O , O 75 O % O ( O 18 O / O 24 O ) O and O 30 O % O ( O 16 O / O 54 O ) O of O NSCLCs O , O carcinoids O and O high O grade O neuroendocrine O lung O carcinomas O , O respectively O . O Furthermore O , O we O demonstrate O that O high O levels O of O Cul B-Protein - I-Protein 1 I-Protein protein O are O associated O with O a O low O KI67 O proliferative O index O ( O p O = O 0 O . O 005 O ) O and O with O a O decrease O in O the O cyclin B-Protein E I-Protein oncoprotein O ( O p O = O 0 O . O 0003 O ) O , O one O of O the O major O targets O of O SCF B-Complex complexes O . O These O data O suggest O that O up O - O regulation O of O Cul B-Protein - I-Protein 1 I-Protein could O protect O cells O from O hyperproliferative O signals O through O cyclin B-Protein E I-Protein down O - O regulation O . O Cul B-Protein - I-Protein 1 I-Protein is O modified O by O neddylation O , O a O post O - O translational O modification O that O grafts O ubiquitin B-Protein - O like O Nedd8 B-Protein / O Rub1 B-Protein residues O and O controls O Cul B-Protein - I-Protein 1 I-Protein activity O . O We O also O provide O evidence O that O neddylated O forms O of O Cul B-Protein - I-Protein 1 I-Protein are O specifically O expressed O in O high O - O grade O NE O lung O tumours O and O are O associated O with O down O - O regulation O of O the O Cul B-Protein - I-Protein 1 I-Protein inhibitor O CAND1 B-Protein ( O p O = O 0 O . O 03 O ) O and O a O high O level O of O cyclin B-Protein E I-Protein ( O p O = O 0 O . O 0002 O ) O . O These O data O support O the O notion O that O alterations O in O the O Cul B-Protein - I-Protein 1 I-Protein neddylation O / O deneddylation O pathway O could O contribute O to O the O development O of O these O highly O aggressive O lung O tumours O . O Although O the O above O - O mentioned O simulation O must O be O regarded O as O very O preliminary O and O will O have O to O be O validated O by O more O advanced O computational O methods O and O perhaps O also O by O biophysical O measurements O , O it O generally O agrees O with O our O experimental O finding O that O mutations O of O residues O at O the O Trm6p B-Protein / O Trm61p B-Protein interface O do O not O disrupt O oligomerization O , O but O interfere O with O tRNA O binding O . O Thus O , O we O propose O that O the O presence O of O salt O bridges O in O the O yeast O m1A58 O Mtase O ( O Trm61p B-Protein - O E255 O and O Trm6p B-Protein - O R420 O , O and O between O Trm6p B-Protein - O E416 O and O Trm61p B-Protein - O R259 O ) O , O and O most O likely O also O in O bacterial O TrmI B-Protein Mtases O ( O e O . O g O . O E299 O - O R233 O in O Rv2118c B-Protein ) O , O serves O to O establish O the O structure O of O the O tRNA O - O binding O region O rather O than O to O promote O binding O of O subunits O to O each O other O . O We O hypothesize O that O the O mutations O reported O in O this O work O could O have O long O - O range O structural O effects O on O the O conformation O of O the O positively O charged O loop O ( O residues O 265 O - O 345 O ) O in O the O Trm61p B-Protein subunit O , O which O may O be O involved O in O tRNA O binding O . O By O identifying O amino O acids O involved O in O tRNA O binding O , O this O study O has O provided O a O foundation O on O which O further O studies O can O be O built O , O such O as O experiments O to O address O our O hypothesis O regarding O tRNA O binding O and O to O determine O how O the O yeast O m1A58 O Mtase O is O able O to O recognize O substrate O tRNAs O amongst O the O cellular O tRNA O pool O . O Tousled B-Family - I-Family like I-Family kinase I-Family in O a O microbial O eukaryote O regulates O spindle O assembly O and O S O - O phase O progression O by O interacting O with O Aurora B-Protein kinase I-Protein and O chromatin O assembly O factors O . O The O Tousled B-Family - I-Family like I-Family kinases I-Family are O an O evolutionarily O conserved O family O of O proteins O implicated O in O DNA O repair O , O DNA O replication O and O mitosis O in O metazoans O and O plants O . O Their O absence O from O the O yeasts O and O other O eukaryotic O ' O microbes O ' O suggests O a O specific O role O for O them O in O the O development O of O multicellular O organisms O . O In O this O study O , O two O closely O related O Tousled B-Family - I-Family like I-Family kinase I-Family homologs O , O TLK1 B-Protein and O TLK2 B-Protein , O were O identified O in O Trypanosoma O brucei O , O a O unicellular O protozoan O parasite O . O Only O TLK1 B-Protein plays O an O essential O role O in O cell O growth O , O and O a O deficiency O in O TLK1 B-Protein led O to O an O enrichment O of O S O - O phase O cells O , O defective O spindle O formation O and O aberrant O chromosome O segregation O . O Although O both O TLK B-Family proteins O localize O to O the O nucleus O , O only O TLK1 B-Protein also O concentrates O in O the O spindle O poles O during O mitosis O . O Both O TLK O proteins O are O phosphorylated O by O the O Aurora B-Protein kinase I-Protein ( O AUK1 B-Protein ) O , O and O both O can O autophosphorylate O and O phosphorylate O histone B-Protein H3 I-Protein and O the O chromatin O assembly O factors O Asf1A B-Protein and O Asf1B B-Protein in O vitro O , O but O only O TLK1 B-Protein is O autophosphorylated O and O capable O of O oligomerizing O and O interacting O with O AUK1 B-Protein , O Asf1A B-Protein and O Asf1B B-Protein in O vivo O . O These O discrepancies O between O the O two O TLK B-Family proteins O can O be O attributed O to O minor O differences O between O their O N O - O and O C O - O terminal O sequences O . O In O summary O , O TLK1 B-Protein cooperates O with O Aurora B-Protein kinase I-Protein to O regulate O spindle O assembly O and O chromosome O segregation O , O and O it O performs O a O role O in O DNA O replication O probably O by O regulating O histone B-Family modification O in O trypanosomes O . O Regulation O of O histone B-Family modification O and O cryptic O transcription O by O the O Bur1 B-Complex and O Paf1 B-Complex complexes O . O The O Bur1 B-Protein - O Bur2 B-Protein and O Paf1 B-Complex complexes O function O during O transcription O elongation O and O affect O histone B-Family modifications O . O Here O we O describe O new O roles O for O Bur1 B-Protein - O Bur2 B-Protein and O the O Paf1 B-Complex complex O . O We O find O that O histone B-Protein H3 I-Protein K36 O tri O - O methylation O requires O specific O components O of O the O Paf1 B-Complex complex O and O that O K36 O tri O - O methylation O is O more O strongly O affected O at O the O 5 O ' O ends O of O genes O in O paf1delta B-Protein and O bur2delta B-Protein strains O in O parallel O with O increased O acetylation O of O histones B-Protein H3 I-Protein and O H4 B-Protein . O Interestingly O , O the O 5 O ' O increase O in O histone B-Family acetylation O is O independent O of O K36 O methylation O , O and O therefore O is O mechanistically O distinct O from O the O methylation O - O driven O deacetylation O that O occurs O at O the O 3 O ' O ends O of O genes O . O Finally O , O Bur1 B-Protein - O Bur2 B-Protein and O the O Paf1 B-Complex complex O have O a O second O methylation O - O independent O function O , O since O bur2delta B-Protein set2delta B-Protein and O paf1delta B-Protein set2delta B-Protein double O mutants O display O enhanced O histone B-Family acetylation O at O the O 3 O ' O ends O of O genes O and O increased O cryptic O transcription O initiation O . O These O findings O identify O new O functions O for O the O Paf1 B-Complex and O Bur1 B-Protein - O Bur2 B-Protein complexes O , O provide O evidence O that O histone B-Family modifications O at O the O 5 O ' O and O 3 O ' O ends O of O coding O regions O are O regulated O by O distinct O mechanisms O , O and O reveal O that O the O Bur1 B-Protein - O Bur2 B-Protein and O Paf1 B-Complex complexes O repress O cryptic O transcription O through O a O Set2 B-Protein - O independent O pathway O . O Puf1p B-Protein acts O in O combination O with O other O yeast O Puf B-Family proteins O to O control O mRNA O stability O . O The O eukaryotic O Puf B-Family proteins O bind O 3 O ' O untranslated O region O ( O UTR O ) O sequence O elements O to O regulate O the O stability O and O translation O of O their O target O transcripts O , O and O such O regulatory O events O are O critical O for O cell O growth O and O development O . O Several O global O genome O analyses O have O identified O hundreds O of O potential O mRNA O targets O of O the O Saccharomyces O cerevisiae O Puf B-Family proteins O ; O however O , O only O three O mRNA O targets O for O these O proteins O have O been O characterized O thus O far O . O After O direct O testing O of O nearly O 40 O candidate O mRNAs O , O we O established O two O of O these O as O true O mRNA O targets O of O Puf B-Family - O mediated O decay O in O yeast O , O HXK1 B-Protein and O TIF1 B-Protein . O In O a O novel O finding O , O multiple O Puf B-Family proteins O , O including O Puf1p B-Protein , O regulate O both O of O these O mRNAs O in O combination O . O TIF1 B-Protein mRNA O decay O can O be O stimulated O individually O by O Puf1p B-Protein and O Puf5p B-Protein , O but O the O combination O of O both O proteins O is O required O for O full O regulation O . O This O Puf B-Family - O mediated O decay O requires O the O presence O of O two O UGUA O binding O sites O within O the O TIF1 B-Protein 3 O ' O UTR O , O with O one O site O regulated O by O Puf5p B-Protein and O the O other O by O both O Puf1p B-Protein and O Puf5p B-Protein . O Alteration O of O the O UGUA O site O in O the O tif1 B-Protein 3 O ' O UTR O to O more O closely O resemble O the O Puf3p B-Protein binding O site O broadens O the O specificity O to O include O regulation O by O Puf3p B-Protein . O The O stability O of O the O endogenously O transcribed O HXK1 B-Protein mRNA O , O cellular O levels O of O Hxk1 B-Protein protein O activity O , O and O HXK1 B-Protein 3 O ' O UTR O - O directed O decay O are O affected O by O Puf1p B-Protein and O Puf5p B-Protein as O well O as O Puf4p B-Protein . O Together O these O results O identify O the O first O mRNA O targets O of O Puf1p B-Protein - O mediated O decay O , O describe O similar O yet O distinct O combinatorial O control O of O two O new O target O mRNAs O by O the O yeast O Puf B-Family proteins O , O and O suggest O the O importance O of O direct O testing O to O evaluate O RNA O - O regulatory O mechanisms O . O Acetylation O in O nuclear B-Family receptor I-Family signaling O and O the O role O of O sirtuins B-Family . O It O has O been O known O since O the O early O 1970s O that O nuclear B-Family receptor I-Family complexes O bind O DNA O in O association O with O coregulatory O proteins O . O Characterization O of O these O nuclear B-Family receptor I-Family coregulators O has O revealed O diverse O enzymatic O activities O that O temporally O and O spatially O coordinate O nuclear B-Family receptor I-Family activity O within O the O context O of O local O chromatin O in O response O to O diverse O hormone O signals O . O Chromatin O - O modifying O proteins O , O which O dictate O the O higher O - O order O chromatin O structure O in O which O DNA O is O packaged O , O in O turn O orchestrate O orderly O recruitment O of O nuclear B-Family receptor I-Family complexes O . O Modifications O of O histones B-Protein include O acetylation O , O methylation O , O phosphorylation O , O ubiquitylation O , O sumoylation O , O ADP O ribosylation O , O deimination O , O and O proline O isomerization O . O At O this O time O , O we O understand O how O a O subset O of O these O modifications O regulates O nuclear B-Family receptor I-Family signaling O . O However O , O the O effects O , O particularly O of O acetylation O and O demethylation O , O are O profound O . O The O finding O that O nuclear B-Family receptors I-Family are O directly O acetylated O and O that O acetylation O in O turn O directly O regulates O contact O - O independent O growth O has O broad O therapeutic O implications O . O Studies O over O the O past O 7 O yr O have O led O to O the O understanding O that O nuclear B-Family receptor I-Family acetylation O is O a O conserved O function O , O regulating O diverse O nuclear B-Family receptor I-Family activity O . O Furthermore O , O we O now O know O that O acetylation O of O multiple O and O distinct O substrates O within O nuclear B-Family receptor I-Family signaling O pathways O , O form O an O acetylation O signaling O network O from O the O cell O surface O to O the O nucleus O . O The O finding O that O nicotinamide O adenine O dinucleotide O ( O NAD O ) O - O dependent O histone B-Family deacetylases I-Family , O the O sirtuins B-Family , O are O capable O of O deacetylating O nuclear B-Family receptors I-Family provides O a O new O level O of O complexity O in O the O control O of O nuclear B-Family receptor I-Family activity O in O which O local O intracellular O concentrations O of O NAD O may O regulate O nuclear B-Family receptor I-Family physiology O . O Structure O of O tumor O suppressor O p53 B-Protein and O its O intrinsically O disordered O N O - O terminal O transactivation O domain O . O Proteins O with O intrinsically O disordered O domains O are O implicated O in O a O vast O range O of O biological O processes O , O especially O in O cell O signaling O and O regulation O . O Having O solved O the O quaternary O structure O of O the O folded O domains O in O the O tumor O suppressor O p53 B-Protein by O a O multidisciplinary O approach O , O we O have O now O determined O the O average O ensemble O structure O of O the O intrinsically O disordered O N O - O terminal O transactivation O domain O ( O TAD O ) O by O using O residual O dipolar O couplings O ( O RDCs O ) O from O NMR O spectroscopy O and O small O - O angle O x O - O ray O scattering O ( O SAXS O ) O . O Remarkably O , O not O only O were O we O able O to O measure O RDCs O of O the O isolated O TAD O , O but O we O were O also O able O to O do O so O for O the O TAD O in O both O the O full O - O length O tetrameric O p53 B-Protein protein O and O in O its O complex O with O a O specific O DNA O response O element O . O We O determined O the O orientation O of O the O TAD O ensemble O relative O to O the O core O domain O , O found O that O the O TAD O was O stiffer O in O the O proline O - O rich O region O ( O residues O 64 O - O 92 O ) O , O which O has O a O tendency O to O adopt O a O polyproline O II O ( O PPII O ) O structure O , O and O projected O the O TAD O away O from O the O core O . O We O located O the O TAD O in O SAXS O experiments O on O a O complex O between O tetrameric O p53 B-Protein and O four O Taz2 B-Protein domains O that O bind O tightly O to O the O TAD O ( O residues O 1 O - O 57 O ) O and O acted O as O " O reporters O . O " O The O p53 B-Protein - O Taz2 B-Protein complex O was O an O extended O cross O - O shaped O structure O . O The O quality O of O the O SAXS O data O enabled O us O to O model O the O disordered O termini O and O the O folded O domains O in O the O complex O with O DNA O . O The O core O domains O enveloped O the O response O element O in O the O center O of O the O molecule O , O with O the O Taz2 B-Protein - O bound O TADs O projecting O outward O from O the O core O . O Constitutively O active O Rheb B-Protein induces O oncogenic O transformation O . O Rheb B-Protein ( O Ras B-Protein - I-Protein homolog I-Protein enriched I-Protein in I-Protein brain I-Protein ) O is O a O component O of O the O phosphatidylinositol B-Family 3 I-Family - I-Family kinase I-Family ( O PI3K B-Family ) O target B-Protein of I-Protein rapamycin I-Protein ( O TOR B-Protein ) O signaling O pathway O , O functioning O as O a O positive O regulator O of O TOR B-Protein . O Constitutively O active O mutants O of O Rheb B-Protein induce O oncogenic O transformation O in O cell O culture O . O The O transformed O cells O are O larger O and O contain O more O protein O than O their O normal O counterparts O . O They O show O constitutive O phosphorylation O of O the O ribosomal B-Family protein I-Family S6 I-Family kinase I-Family and O the O eukaryotic B-Protein initiation I-Protein factor I-Protein 4E I-Protein - I-Protein binding I-Protein protein I-Protein 1 I-Protein , O two O downstream O targets O of O TOR B-Protein . O The O TOR B-Protein - O specific O inhibitor O rapamycin B-Chemical strongly O interferes O with O transformation O induced O by O constitutively O active O Rheb B-Protein , O suggesting O that O TOR B-Protein activity O is O essential O for O the O oncogenic O effects O of O mutant O Rheb B-Protein . O Rheb B-Protein - O induced O transformation O is O also O dependent O on O a O C O - O terminal O farnesylation O signal O that O mediates O localization O to O a O cellular O membrane O . O An O engineered O N O - O terminal O myristylation O signal O can O substitute O for O the O farnesylation O . O Immunofluorescence O localizes O wild O - O type O and O mutant O Rheb B-Protein to O vesicular O structures O in O the O cytoplasm O , O overlapping O with O the O endoplasmic O reticulum O . O The O WW B-Protein domain I-Protein containing I-Protein E3 I-Protein ubiquitin I-Protein protein I-Protein ligase I-Protein 1 I-Protein upregulates O ErbB2 B-Protein and O EGFR B-Protein through O RING B-Protein finger I-Protein protein I-Protein 11 I-Protein . O The O WW B-Protein domain I-Protein containing I-Protein E3 I-Protein ubiquitin I-Protein protein I-Protein ligase I-Protein 1 I-Protein ( O WWP1 B-Protein ) O is O a O homologous O to O the O E6 B-Protein - O associated O protein O C O terminus O - O type O E3 O ligase O frequently O overexpressed O in O human O prostate O and O breast O cancers O due O to O gene O amplification O . O Previous O studies O suggest O that O WWP1 B-Protein promotes O cell O proliferation O and O survival O ; O however O , O the O mechanism O of O WWP1 B-Protein action O is O still O poorly O understood O . O Here O , O we O showed O that O WWP1 B-Protein upregulates O and O maintains O erythroblastic B-Protein leukemia I-Protein viral I-Protein oncogene I-Protein homolog I-Protein 2 I-Protein ( O ErbB2 B-Protein ) O and O epithelial B-Protein growth I-Protein factor I-Protein receptor I-Protein ( O EGFR B-Protein ) O in O multiple O cell O lines O . O WWP1 B-Protein depletion O dramatically O attenuates O the O EGF B-Protein - O induced O ERK B-Family phosphorylation O . O WWP1 B-Protein forms O a O protein O complex O with O RING B-Protein finger I-Protein protein I-Protein 11 I-Protein ( O RNF11 B-Protein ) O , O a O negative O regulator O of O ErbB2 B-Protein and O EGFR B-Protein . O The O protein O - O protein O interaction O is O through O the O first O and O third O WW O domains O of O WWP1 B-Protein and O the O PY O motif O of O RNF11 B-Protein . O Although O WWP1 B-Protein is O able O to O ubiquitinate O RNF11 B-Protein in O vitro O and O in O vivo O , O WWP1 B-Protein neither O targets O RNF11 B-Protein for O degradation O nor O changes O RNF11 B-Protein ' O s O cellular O localization O . O Importantly O , O inhibition O of O RNF11 B-Protein can O rescue O WWP1 B-Protein siRNA O - O induced O ErbB2 B-Protein and O EGFR B-Protein downregulation O and O growth O arrest O . O Finally O , O we O demonstrated O that O RNF11 B-Protein is O overexpressed O in O a O panel O of O prostate O and O breast O cancer O cell O lines O with O WWP1 B-Protein expression O . O These O findings O suggest O that O WWP1 B-Protein may O promote O cell O proliferation O and O survival O partially O through O suppressing O RNF11 B-Protein - O mediated O ErbB2 B-Protein and O EGFR B-Protein downregulation O . O Biochemical O characterization O of O plasma O - O derived O tissue B-Protein factor I-Protein pathway I-Protein inhibitor I-Protein : O post O - O translational O modification O of O free O , O full O - O length O form O with O particular O reference O to O the O sugar O chain O . O BACKGROUND O : O Tissue B-Protein factor I-Protein pathway I-Protein inhibitor I-Protein ( O TFPI B-Protein ) O is O a O physiological O protease O inhibitor O that O inhibits O the O initial O reactions O of O the O extrinsic O blood O coagulation O pathway O . O Most O TFPI B-Protein in O human O plasma O is O associated O with O lipoproteins O ; O however O , O the O most O functionally O active O form O is O thought O to O be O the O free O , O full O - O length O form O ( O f O - O pTFPI B-Protein ) O . O Cell O culture O derived O TFPI B-Protein and O recombinant O TFPI B-Protein ( O rTFPI O ) O exhibit O variations O in O their O respective O anticoagulant O activity O , O which O may O be O caused O by O post O - O translational O modifications O , O such O as O the O frequent O differences O in O sugar O chain O structures O among O recombinant O proteins O . O Sugar O chain O structures O in O rTFPI O expressed O in O Chinese O hamster O ovary O ( O CHO O ) O cells O have O been O reported O previously O , O but O those O of O plasma O TFPI B-Protein have O not O been O . O OBJECTIVES O : O To O purify O f O - O pTFPI B-Protein and O analyze O the O sugar O chain O structures O . O RESULTS O AND O CONCLUSION O : O f O - O pTFPI B-Protein was O purified O to O homogeneity O from O blood O plasma O using O a O combination O of O anion O - O exchange O , O heparin B-Chemical affinity O , O immunoaffinity O , O and O reversed O - O phase O chromatographies O , O resulting O in O a O yield O of O 76 O % O . O f O - O pTFPI B-Protein showed O a O partially O phosphorylated O glycoprotein O comprising O a O total O of O 276 O amino O acids O by O peptide O mapping O . O The O sugar O chain O structures O were O analyzed O by O two O - O dimensional O sugar O mapping O combined O with O exoglycosidase B-Chemical digestion O of O the O pyridylamino O sugar O chains O and O the O following O results O were O obtained O . O ( B-Chemical Sialyl I-Chemical ) I-Chemical Galbeta1 I-Chemical - I-Chemical 3GalNAc I-Chemical was O linked O to O Thr O ( O 175 O ) O , O partially O to O Thr O ( O 14 O ) O and O Ser O ( O 174 O ) O ; O sialyl O complex O - O type O sugar O chains O to O Asn O ( O 117 O ) O and O Asn O ( O 167 O ) O , O whereas O Asn O ( O 228 O ) O was O not O glycosylated O . O Neuraminidase O - O resistant O acidic O sugar O chains O including O sulfated O sugar O chains O were O not O observed O significantly O . O The O protease O inhibitory O activities O of O f O - O pTFPI B-Protein towards O activated O factor B-Protein ( I-Protein F I-Protein ) I-Protein X I-Protein and O tissue B-Complex factor I-Complex - I-Complex activated I-Complex FVII I-Complex complex O were O identical O to O those O of O full O - O length O rTFPI O expressed O in O CHO O cells O . O Subcellular O localization O directs O signaling O specificity O of O the O Cryptococcus O neoformans O Ras1 B-Protein protein O . O In O the O human O fungal O pathogen O Cryptococcus O neoformans O , O Ras B-Family signaling O mediates O sexual O differentiation O , O morphogenesis O , O and O pathogenesis O . O By O studying O Ras B-Family prenylation O and O palmitoylation O in O this O organism O , O we O have O found O that O the O subcellular O localization O of O this O protein O dictates O its O downstream O signaling O specificity O . O Inhibiting O C O . O neoformans O Ras1 B-Protein prenylation O results O in O the O defective O general O membrane O targeting O of O this O protein O and O the O loss O of O all O Ras B-Family function O . O In O contrast O , O palmitoylation O mediates O localization O of O Ras1 B-Protein to O the O plasma O membrane O and O is O required O for O normal O morphogenesis O and O survival O at O high O temperatures O . O However O , O palmitoylation O and O plasma O membrane O localization O are O not O required O for O Ras B-Family - O dependent O sexual O differentiation O . O Likely O as O a O result O of O its O effect O on O thermotolerance O , O Ras1 B-Protein palmitoylation O is O also O required O for O the O pathogenesis O of O C O . O neoformans O . O These O data O support O an O emerging O paradigm O of O compartmentalized O Ras B-Family signaling O . O However O , O our O studies O also O demonstrate O fundamental O differences O between O the O Ras B-Family pathways O in O different O organisms O that O emphasize O the O functional O flexibility O of O conserved O signaling O cascades O . O DDB1 B-Protein targets O Chk1 B-Protein to O the O Cul4 B-Protein E3 O ligase O complex O in O normal O cycling O cells O and O in O cells O experiencing O replication O stress O . O The O Chk1 B-Protein protein O kinase O preserves O genome O integrity O in O normal O proliferating O cells O and O in O cells O experiencing O replicative O and O genotoxic O stress O . O Chk1 B-Protein is O currently O being O targeted O in O anticancer O regimens O . O Here O , O we O identify O damaged B-Protein DNA I-Protein - I-Protein binding I-Protein protein I-Protein 1 I-Protein ( O DDB1 B-Protein ) O as O a O novel O Chk1 B-Protein - O interacting O protein O . O DDB1 B-Protein is O part O of O an O E3 O ligase O complex O that O includes O the O cullin B-Family proteins O Cul4A B-Protein and O Cul4B B-Protein . O We O report O that O Cul4A B-Protein / O DDB1 B-Protein negatively O regulates O Chk1 B-Protein stability O in O vivo O . O Chk1 B-Protein associates O with O Cul4A B-Protein / O DDB1 B-Protein during O an O unperturbed O cell O division O cycle O and O both O Chk1 B-Protein phosphorylation O and O replication O stress O enhanced O these O interactions O . O Cul4A B-Protein / O DDB1 B-Protein regulates O Chk1 B-Protein ubiquitination O in O vivo O and O Chk1 B-Protein is O directly O ubiquitinated O in O vitro O in O a O Cul4A B-Protein / O DDB1 B-Protein - O dependent O manner O . O Furthermore O , O Chk1 B-Protein is O stabilized O in O cells O deficient O for O Cul4A B-Protein / O DDB1 B-Protein . O This O study O shows O that O Chk1 B-Protein abundance O is O regulated O by O the O Cul4A B-Protein / O DDB1 B-Protein ubiquitin B-Protein ligase O during O an O unperturbed O cell O division O cycle O , O in O response O to O replicative O stress O and O on O heat B-Protein shock I-Protein protein I-Protein 90 I-Protein inhibition O , O and O that O deregulation O of O the O Chk1 B-Protein / O Cul4A B-Protein / O DDB1 B-Protein pathway O perturbs O the O ionizing O radiation O - O induced O G O ( O 2 O ) O checkpoint O . O Deletion O analysis O indicated O that O a O sizable O segment O , O residues O 1 O - O 383 O at O the O N O - O terminus O , O could O be O removed O , O along O with O a O shorter O segment O at O the O C O - O terminus O , O to O yield O a O core O region O that O is O active O in O V O ( O D O ) O J O recombination O in O vitro O and O in O cells O . O Despite O being O recognized O for O years O , O the O function O of O the O N O - O terminal O region O remains O mysterious O . O Its O existence O is O conserved O through O the O evolution O of O RAG1 B-Protein , O yet O it O clearly O is O not O needed O for O the O central O enzymatic O role O as O a O recombinase O . O Sequence O alignments O show O that O this O region O exhibits O a O greater O divergence O through O evolution O than O the O enzymatic O core O . O Notable O in O such O alignments O is O the O absolute O conservation O of O a O cluster O of O cystine O and O histidine O residues O recognized O as O a O special O zinc O - O binding O motif O termed O a O ' O RING O finger O ' O . O RING O structures O have O been O found O in O enzymes O ( O E3 O ligases O ) O that O help O modify O other O proteins O through O the O covalent O addition O of O small O modifier O proteins O . O In O biochemical O assays O , O the O RAG1 B-Protein N O - O terminus O can O act O as O an O E3 O ubiquitin B-Protein ligase O . O While O characterizing O the O structural O requirements O for O Fra B-Protein - O mediated O axon O attraction O , O we O observed O that O neuronal O expression O of O a O dominant O negative O form O of O Fra B-Protein ( O FraDeltaC B-Protein ) O leads O to O a O dose O - O dependent O " O commissureless B-Protein " O phenotype O . O Searching O for O candidate O genes O that O modify O this O phenotype O , O we O found O that O removing O one O copy O of O comm B-Protein enhances O the O midline O crossing O defects O caused O by O expressing O UASFraDeltaC B-Protein ( O fig O . O S1 O ) O , O suggesting O a O role O for O Fra B-Protein in O regulating O Comm B-Protein during O midline O guidance O . O Consistent O with O this O idea O , O removing O one O copy O of O comm B-Protein in O hypomorphic O fra B-Protein mutants O increases O the O commissural O defects O as O shown O by O thin O or O missing O commissures O in O many O segments O , O as O well O as O an O increased O frequency O of O non O - O crossing O defects O in O a O subset O of O commissural O neurons O : O the O eagle B-Protein neurons O ( O Fig O . O 1 O and O Table O S1 O ) O . O Similar O genetic O interactions O are O also O observed O using O additional O alleles O of O both O fra B-Protein and O comm B-Protein ( O fig O . O S2 O and O Table O S1 O ) O . O These O dose O - O dependent O genetic O interactions O suggest O that O fra B-Protein and O comm B-Protein may O function O in O the O same O pathway O to O control O commissural O axon O guidance O . O At O low O expression O levels O , O GFP B-Protein - O MinCC B-Protein / O MinD B-Protein localizes O to O the O Z O ring O without O disrupting O it O . O This O localization O is O dependent O upon O FtsZ B-Protein but O not O other O early O division O proteins O such O as O FtsA B-Protein , O ZipA B-Protein and O ZapA B-Protein , O suggesting O that O MinCC B-Protein / O MinD B-Protein interacts O with O FtsZ B-Protein directly O . O An O interaction O between O FtsZ B-Protein and O MinCC B-Protein / O MinD B-Protein was O observed O in O an O in O vitro O assay O , O which O strongly O supports O this O idea O . O In O this O paper O we O further O investigate O the O mechanism O by O which O MinCC B-Protein / O MinD B-Protein antagonizes O Z O ring O formation O . O We O isolated O FtsZ B-Protein mutants O that O are O resistant O to O MinCC B-Protein / O MinD B-Protein and O then O used O these O mutants O to O study O the O MinCC B-Protein / O MinD B-Protein - O FtsZ B-Protein interaction O and O the O basis O of O the O toxicity O associated O with O MinCC B-Protein / O MinD B-Protein . O We O have O previously O shown O that O forced O expression O of O Optix B-Protein in O the O developing O eye O leads O to O severe O alterations O in O eye O structure O . O This O activity O was O mapped O to O the O nonconserved O C O - O terminal O tail O as O the O replacement O of O the O So B-Protein CT O with O that O of O Optix B-Protein leads O to O the O same O degree O of O roughening O as O full O - O length O Optix B-Protein . O In O this O report O , O we O have O extended O this O early O observation O by O functionally O dissecting O the O C O - O terminal O segment O of O the O Optix B-Protein protein O and O ( O 1 O ) O demonstrated O that O while O the O CT O is O necessary O for O inhibiting O eye O development O , O it O also O requires O the O activity O of O the O protein O - O protein O interaction O and O DNA O binding O regions O ; O ( O 2 O ) O identified O that O regions O A O , O C O , O and O D O of O the O CT O are O required O for O blocking O retinal O formation O ; O ( O 3 O ) O shown O that O region O D O is O specifically O conserved O just O within O the O Drosophilids O ; O ( O 4 O ) O demonstrated O that O the O CT O is O required O for O the O ability O of O Optix B-Protein to O induce O ectopic O eyes O ; O ( O 5 O ) O shown O that O none O of O the O vertebrate O SIX B-Family proteins O are O capable O of O inducing O ectopic O eyes O in O flies O ; O and O ( O 6 O ) O demonstrated O that O in O addition O to O so B-Protein homologs O , O expression O of O DSix4 B-Protein and O Six4 B-Protein can O also O rescue O so1 B-Protein loss O - O of O - O function O mutants O . O Regulation O of O AMPA B-Complex receptor I-Complex extrasynaptic O insertion O by O 4 B-Protein . I-Protein 1N I-Protein , O phosphorylation O and O palmitoylation O . O The O insertion O of O AMPA B-Complex receptors I-Complex ( O AMPARs B-Complex ) O into O the O plasma O membrane O is O an O important O step O in O the O synaptic O delivery O of O AMPARs B-Complex during O the O expression O of O synaptic O plasticity O . O However O , O the O molecular O mechanisms O regulating O AMPAR B-Complex insertion O remain O elusive O . O By O directly O visualizing O individual O insertion O events O of O the O AMPAR B-Complex subunit O GluR1 B-Protein in O rodents O , O we O found O that O the O protein O 4 B-Protein . I-Protein 1N I-Protein was O required O for O activity O - O dependent O GluR1 B-Protein insertion O . O Protein B-Family kinase I-Family C I-Family ( O PKC B-Family ) O phosphorylation O of O the O serine O 816 O ( O S816 O ) O and O S818 O residues O of O GluR1 B-Protein enhanced O 4 B-Protein . I-Protein 1N I-Protein binding O to O GluR1 B-Protein and O facilitated O GluR1 B-Protein insertion O . O In O addition O , O palmitoylation O of O GluR1 B-Protein C811 O residue O modulated O PKC B-Family phosphorylation O and O GluR1 B-Protein insertion O . O Finally O , O disrupting O 4 B-Protein . I-Protein 1N I-Protein - O dependent O GluR1 B-Protein insertion O decreased O surface O expression O of O GluR1 B-Protein and O the O expression O of O long O - O term O potentiation O . O Our O study O uncovers O a O previously O unknown O mechanism O that O governs O activity O - O dependent O GluR1 B-Protein trafficking O , O reveals O an O interaction O between O AMPAR B-Complex palmitoylation O and O phosphorylation O , O and O underscores O the O functional O importance O of O 4 B-Protein . I-Protein 1N I-Protein in O AMPAR B-Complex trafficking O and O synaptic O plasticity O . O Characterization O of O subcellular O localization O of O duck O enteritis O virus O UL51 B-Protein protein O . O BACKGROUND O : O Knowledge O of O the O subcellular O localization O of O a O protein O can O provide O useful O insights O about O its O function O . O While O the O subcellular O localization O of O many O alphaherpesvirus O UL51 B-Protein proteins O has O been O well O characterized O , O little O is O known O about O where O duck O enteritis O virus O ( O DEV O ) O UL51 B-Protein protein O ( O pUL51 B-Protein ) O is O targeted O to O . O Thus O , O in O this O study O , O we O investigated O the O subcellular O localization O and O distribution O of O DEV O pUL51 B-Protein by O computer O aided O analysis O , O as O well O as O indirect O immunofluorescence O ( O IIF O ) O and O transmission O immunoelectron O microscopy O ( O TIEM O ) O approaches O in O DEV O - O infected O cells O . O RESULTS O : O The O DEV O UL51 B-Protein gene O product O was O identified O as O an O approximate O 34 O kDa O protein O in O DEV O - O infected O cells O analyzed O by O western O blotting O . O Computer O aided O analysis O suggested O that O DEV O pUL51 B-Protein is O not O targeted O to O the O mitochondrial O , O extra O - O cellular O or O nucleus O , O but O be O targeted O to O the O cytoplasmic O in O host O cells O , O more O specifically O , O palmitoylation O of O the O pUL51 B-Protein through O the O N O - O terminal O cysteine O at O position O 9 O makes O membrane O association O and O Golgi O localization O possible O . O Using O IIF O analysis O , O we O found O that O DEV O pUL51 B-Protein was O first O detected O in O a O juxtanuclear O region O of O DEV O - O infected O cells O at O 9 O h O postinfection O ( O p O . O i O . O ) O , O and O then O was O detected O widely O distributed O in O the O cytoplasm O and O especially O was O stronger O in O the O juxtanuclear O region O from O 12 O to O 60 O h O p O . O i O . O TIEM O analysis O revealed O that O DEV O pUL51 B-Protein was O mainly O associated O with O cytoplasmic O virions O and O also O with O some O membranous O structure O near O the O pUL51 B-Protein - O specific O immuno O - O labeling O intracellular O virion O in O the O cytoplasmic O vesicles O ; O moreover O , O the O pUL51 B-Protein efficiently O accumulated O in O the O Golgi O apparatus O at O first O , O and O then O was O sent O to O the O plasma O membrane O from O the O Golgi O by O some O unknown O mechanism O . O CONCLUSION O : O In O this O work O , O we O described O the O basic O characteristics O of O pUL51 B-Protein subcellular O localization O and O distribution O for O the O first O time O . O From O these O results O , O we O concluded O that O palmitoylation O at O the O N O - O terminal O cysteine O , O which O is O conserved O in O all O alphaherpesvirus O UL51 B-Protein homologs O , O is O required O for O its O membrane O association O and O Golgi O localization O , O and O the O pUL51 B-Protein mainly O localized O to O the O juxtanuclear O region O of O DEV O - O infected O cells O , O as O well O seemed O to O be O incorporated O into O mature O virions O as O a O component O of O the O tegument O . O The O research O will O provide O useful O clues O for O DEV O pUL51 B-Protein functional O analysis O , O and O will O be O usefull O for O further O understanding O the O localization O properties O of O alphaherpesvirus O UL51 B-Protein homologs O . O Palmitoylation O of O the O sphingosine B-Family 1 I-Family - I-Family phosphate I-Family receptor I-Family S1P I-Family is O involved O in O its O signaling O functions O and O internalization O . O The O lipid O mediator O sphingosine B-Chemical 1 I-Chemical - I-Chemical phosphate I-Chemical ( O S1P B-Chemical ) O regulates O several O cellular O processes O through O binding O to O its O receptors O ( O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein - O S1P B-Protein ( I-Protein 5 I-Protein ) I-Protein ) O , O which O are O heterotrimeric O G B-Family protein I-Family - I-Family coupled I-Family receptors I-Family . O Here O , O we O report O that O all O S1P B-Family receptors I-Family are O palmitoylated O . O In O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein , O three O Cys O residues O in O the O cytoplasmic O tail O are O palmitoylated O . O We O examined O the O roles O of O palmitoylation O of O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein using O model O cells O in O which O wild O - O type O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein or O a O non O - O palmitoylated O mutant O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein was O overproduced O . O Compared O with O wild O - O type O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein , O the O non O - O palmitoylated O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein exhibited O binding O affinity O similar O to O the O natural O ligand O S1P B-Chemical but O lower O to O the O synthetic O ligand O FTY720 B-Chemical phosphate I-Chemical ( O FTY720 B-Chemical - I-Chemical P I-Chemical ) O , O the O active O form O of O the O immunomodulator O FTY720 B-Chemical . O However O , O downstream O signaling O of O non O - O palmitoylated O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein was O similarly O affected O by O S1P B-Chemical and O FTY720 B-Chemical - I-Chemical P I-Chemical stimulation O . O Moreover O , O upon O stimulation O with O S1P B-Chemical , O internalization O of O the O mutant O non O - O palmitoylated O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein was O retarded O , O compared O with O that O of O the O wild O - O type O protein O . O This O effect O was O much O more O pronounced O with O FTY720 B-Chemical - I-Chemical P I-Chemical stimulation O . O Similar O differences O were O observed O for O the O phosphorylation O of O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein and O its O mutant O . O These O findings O may O provide O insights O into O the O molecular O mechanisms O of O the O pharmacological O effects O of O FTY720 B-Chemical . O Finally O , O palmitoylation O of O wild O - O type O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein increased O upon O treatment O with O S1P B-Chemical , O suggesting O that O S1P B-Protein ( I-Protein 1 I-Protein ) I-Protein undergoes O a O palmitoylation O / O depalmitoylation O cycle O after O stimulation O by O its O ligands O . O Dvl B-Protein is O a O critical O regulator O of O Wnt B-Family signaling O pathways O . O In O the O canonical O Wnt B-Family signaling O pathway O , O Wnt B-Family signal O is O passed O from O the O membrane O Wnt B-Family receptor O Fz B-Family to O Dvl B-Protein , O which O then O relays O the O signal O to O downstream O components O . O The O direct O recognition O between O Dvl B-Protein PDZ O domain O and O a O conserved O sequence O ( O KTXXXW O ) O in O Fz B-Family , O localized O two O residues O after O the O seventh O transmembrane O domain O , O is O the O key O interaction O in O the O pathway O . O Indeed O , O Dvl B-Protein PDZ O - O binding O peptides O as O well O as O small O molecules O targeting O the O Dvl B-Protein PDZ O domain O can O effectively O block O Wnt B-Family signaling O . O Here O we O show O that O sulindac B-Chemical and O sulindac B-Chemical sulfone I-Chemical bind O the O peptide O - O binding O site O of O the O Dvl B-Protein PDZ O domain O with O an O affinity O comparable O to O that O of O the O native O binding O partners O and O that O , O like O other O Dvl B-Protein PDZ O inhibitors O , O sulindac B-Chemical blocks O Wnt B-Family signaling O at O the O Dvl B-Protein level O in O Xenopus O . O Therefore O , O we O propose O that O sulindac B-Chemical exerts O its O chemoprotective O anticancer O effect O not O only O by O inhibiting O COX B-Protein enzymes O but O also O by O inhibiting O Wnt B-Family signaling O via O Dvl B-Protein . O Tyrosine O sulfation O : O an O increasingly O recognised O post O - O translational O modification O of O secreted O proteins O . O The O post O - O translational O sulfation O of O tyrosine O residues O occurs O in O numerous O secreted O and O integral O membrane O proteins O and O , O in O many O cases O , O plays O a O crucial O role O in O controlling O the O interactions O of O these O proteins O with O physiological O binding O partners O as O well O as O invading O pathogens O . O Recent O advances O in O our O understanding O of O protein O tyrosine O sulfation O have O come O about O owing O to O the O cloning O of O two O human O tyrosylprotein O sulfotransferases O ( O TPST B-Protein - I-Protein 1 I-Protein and O TPST B-Protein - I-Protein 2 I-Protein ) O , O the O development O of O novel O analytical O and O synthetic O methodologies O and O detailed O studies O of O proteins O and O peptides O containing O sulfotyrosine O residues O . O In O this O article O , O we O describe O the O TPST O enzymes O , O review O the O major O techniques O available O for O studying O the O presence O , O location O and O function O of O tyrosine O sulfation O in O proteins O and O discuss O the O biological O functions O and O biochemical O interactions O of O several O proteins O ( O or O protein O families O ) O in O which O tyrosine O sulfation O influences O the O protein O function O . O In O particular O , O we O describe O the O detailed O evidence O supporting O the O importance O of O tyrosine O sulfation O in O the O cellular O adhesion O function O of O P B-Protein - I-Protein selectin I-Protein glycoprotein I-Protein ligand I-Protein - I-Protein 1 I-Protein , O the O leukocyte O trafficking O and O pathogen O invasion O functions O of O chemokine O receptors O and O the O ligand O binding O and O activation O of O other O G B-Family - I-Family protein I-Family - I-Family coupled I-Family receptors I-Family by O complement O proteins O , O phospholipdis O and O glycoprotein O hormones O . O The O miRNA O pathway O involves O the O loading O of O a O miRNA O into O RISC B-Complex ( O miRISC B-Complex ) O , O and O the O active O association O of O miRISC B-Complex with O target O mRNAs O . O We O visualized O the O distribution O of O all O miRISC B-Complex complexes O ( O active O , O loaded O , O and O inactive O ) O in O Drosophila O cells O using O an O antibody O recognizing O the O Ago1 B-Protein protein O , O a O key O subunit O of O miRISC B-Complex . O Ago1 B-Protein was O detected O in O the O cytoplasm O of O cells O , O and O was O enriched O in O the O perinuclear O region O ( O Fig O . O 3f O ) O , O where O lysosomes O tend O to O localize O . O In O dHPS4 B-Protein mutant O cells O , O Ago1 B-Protein was O not O enriched O in O the O perinuclear O region O but O was O more O dispersed O throughout O the O cytoplasm O ( O Fig O . O 3g O ) O . O This O result O suggests O that O Ago1 B-Protein localization O correlates O with O trafficking O endosomes O . O As O further O evidence O , O cells O mutant O for O vps25 B-Protein and O the O ESCRT B-Complex regulator O myopic B-Protein showed O Ago1 B-Protein concentrated O around O large O cytoplasmic O vesicles O ( O Fig O . O 3h O - O i O ) O . O These O mutations O generate O cells O with O enlarged O early O endosomes O because O they O block O sorting O of O early O endosomes O into O MVBs O . O Hence O , O the O distribution O of O miRISC B-Complex appears O to O correlate O with O the O status O of O MVBs O ; O blocking O MVB O formation O concentrates O miRISC B-Complex in O early O endosomes O while O blocking O MVB O turnover O disperses O miRISC B-Complex from O lysosomes O . O Even O though O Ago1 B-Protein is O dispersed O in O dHPS4 B-Protein cells O , O it O is O still O associated O with O membranes O in O the O cytoplasm O ( O SFig O . O 3a O ) O . O We O separated O cytoplasmic O membrane O from O cytosol O by O OptiPrep O density O gradient O centrifugation O , O and O found O a O substantial O fraction O of O Ago1 B-Protein protein O associated O with O membrane O . O There O was O little O change O in O its O association O with O membrane O in O dHPS4 B-Protein mutant O preparations O . O A O similar O result O was O observed O with O Ago2 B-Protein protein O ( O SFig O . O 3a O ) O . O GSK B-Protein - I-Protein 3 I-Protein phosphorylates O delta B-Protein - I-Protein catenin I-Protein and O negatively O regulates O its O stability O via O ubiquitination O / O proteosome O - O mediated O proteolysis O . O Delta B-Protein - I-Protein catenin I-Protein was O first O identified O because O of O its O interaction O with O presenilin B-Protein - I-Protein 1 I-Protein , O and O its O aberrant O expression O has O been O reported O in O various O human O tumors O and O in O patients O with O Cri O - O du O - O Chat O syndrome O , O a O form O of O mental O retardation O . O However O , O the O mechanism O whereby O delta B-Protein - I-Protein catenin I-Protein is O regulated O in O cells O has O not O been O fully O elucidated O . O We O investigated O the O possibility O that O glycogen B-Protein - I-Protein synthase I-Protein kinase I-Protein - I-Protein 3 I-Protein ( O GSK B-Protein - I-Protein 3 I-Protein ) O phosphorylates O delta B-Protein - I-Protein catenin I-Protein and O thus O affects O its O stability O . O Initially O , O we O found O that O the O level O of O delta B-Protein - I-Protein catenin I-Protein was O greater O and O the O half O - O life O of O delta B-Protein - I-Protein catenin I-Protein was O longer O in O GSK B-Protein - I-Protein 3beta I-Protein ( O - O / O - O ) O fibroblasts O than O those O in O GSK B-Protein - I-Protein 3beta I-Protein ( O + O / O + O ) O fibroblasts O . O Furthermore O , O four O different O approaches O designed O to O specifically O inhibit O GSK B-Protein - I-Protein 3 I-Protein activity O , O i O . O e O . O GSK B-Protein - I-Protein 3 I-Protein - O specific O chemical O inhibitors O , O Wnt B-Family - O 3a O conditioned O media O , O small O interfering O RNAs O , O and O GSK B-Protein - I-Protein 3alpha I-Protein and O - B-Protein 3beta I-Protein kinase I-Protein dead O constructs O , O consistently O showed O that O the O levels O of O endogenous O delta B-Protein - I-Protein catenin I-Protein in O CWR22Rv O - O 1 O prostate O carcinoma O cells O and O primary O cortical O neurons O were O increased O by O inhibiting O GSK B-Protein - I-Protein 3 I-Protein activity O . O In O addition O , O it O was O found O that O both O GSK B-Protein - I-Protein 3alpha I-Protein and O - B-Protein 3beta I-Protein interact O with O and O phosphorylate O delta B-Protein - I-Protein catenin I-Protein . O The O phosphorylation O of O DeltaC207 O - O delta B-Protein - I-Protein catenin I-Protein ( O lacking O 207 O C O - O terminal O residues O ) O and O T1078A O delta B-Protein - I-Protein catenin I-Protein by O GSK B-Protein - I-Protein 3 I-Protein was O noticeably O reduced O compared O with O that O of O wild O type O delta B-Protein - I-Protein catenin I-Protein , O and O the O data O from O liquid O chromatography O - O tandem O mass O spectrometry O analyses O suggest O that O the O Thr O ( O 1078 O ) O residue O of O delta B-Protein - I-Protein catenin I-Protein is O one O of O the O GSK B-Protein - I-Protein 3 I-Protein phosphorylation O sites O . O Treatment O with O MG132 B-Chemical or O ALLN B-Chemical , O specific O inhibitors O of O proteosome B-Complex - O dependent O proteolysis O , O increased O delta B-Protein - I-Protein catenin I-Protein levels O and O caused O an O accumulation O of O ubiquitinated O delta B-Protein - I-Protein catenin I-Protein . O It O was O also O found O that O GSK B-Protein - I-Protein 3 I-Protein triggers O the O ubiquitination O of O delta B-Protein - I-Protein catenin I-Protein . O These O results O suggest O that O GSK B-Protein - I-Protein 3 I-Protein interacts O with O and O phosphorylates O delta B-Protein - I-Protein catenin I-Protein and O thereby O negatively O affects O its O stability O by O enabling O its O ubiquitination O / O proteosome B-Complex - O mediated O proteolysis O . O In O order O to O successfully O infect O human O cells O , O HIV O - O 1 O has O to O neutralize O cellular O restriction O factors O that O impede O its O replication O at O multiple O steps O . O HIV O - O 1 O Vpu B-Protein serves O this O goal O by O counteracting O a O blockade O imposed O by O the O newly O identified O protein O tetherin B-Protein . O Under O basal O conditions O , O tetherin B-Protein is O expressed O in O B O and O T O cells O , O plasmacytoid O dendritic O cells O and O myeloid O cells O . O In O addition O , O tetherin B-Protein expression O is O strongly O upregulated O in O many O cell O types O by O type O - O I O interferon O ( O IFN O ) O , O a O situation O typically O encountered O in O viral O infections O . O Tetherin B-Protein is O a O heavily O glycosylated O type O - O II O transmembrane O protein O with O an O unusual O topology O , O which O is O otherwise O only O found O in O mammals O in O a O minor O but O pathologically O important O topological O variant O of O the O prion O protein O . O Tetherin B-Protein is O indeed O linked O to O membranes O both O by O its O one O - O pass O transmembrane O domain O and O by O a O C O - O ter O GPI O anchor O . O This O anti O - O viral O factor O is O mostly O intracellular O , O but O it O is O also O localized O at O the O cell O surface O in O lipid O rafts O , O from O where O it O is O continually O recycled O to O the O trans O - O Golgi O network O . O In O cells O expressing O tetherin B-Protein , O HIV O - O 1 O viruses O deleted O for O the O Vpu B-Protein gene O can O bud O normally O but O remain O tethered O to O the O cell O surface O through O a O protein O bond O . O The O mechanistic O details O of O this O phenomenon O remain O to O be O clarified O . O A O hypothesis O , O that O still O awaits O confirmation O , O is O that O tetherin B-Protein itself O forms O the O protein O tether O between O the O cell O surface O and O the O virion O owing O to O its O ability O to O form O stable O dimers O . O The O affected O virions O are O then O endocytosed O and O probably O degraded O in O lysosomes O . O In O addition O to O inhibiting O HIV O - O 1 O , O tetherin B-Protein also O blocks O the O replication O of O numerous O retroviruses O , O as O well O as O other O non O - O related O enveloped O viruses O . O The O importance O of O this O restriction O in O the O cellular O antiviral O arsenal O is O underscored O by O the O apparent O positive O selection O that O tetherin B-Protein undergoes O , O which O is O the O hallmark O of O an O ongoing O molecular O fight O with O pathogens O . O Although O we O do O not O have O sufficient O data O to O model O the O replisome O , O the O electrostatic O surface O potential O of O Pol B-Protein gammaA I-Protein is O informative O . O As O expected O , O the O putative O DNA O - O binding O channel O is O lined O with O positively O charged O residues O but O the O opposite O surface O of O the O protein O presents O a O large O negatively O charged O region O near O the O exo O domain O and O the O tip O of O the O AID O subdomain O also O contains O four O sequential O glutamates O ( O 535EEEE538 O , O E O - O tract O ; O Fig O . O 5A O ) O . O The O human O mitochondrial O helicase O , O Twinkle B-Protein , O has O a O highly O positively O charged O C O - O terminal O region O that O could O contact O one O of O these O regions O . O If O the O interaction O is O through O the O negatively O charged O E O - O tract O in O the O replisome O , O Twinkle B-Protein would O be O positioned O in O a O location O close O to O that O of O the O 363RKK365 O and O 328RK329 O , O residues O important O in O Twinkle B-Protein - O dependent O replication O of O duplex O DNA O . O Validation O of O a O method O for O the O quantitation O of O ghrelin B-Protein and O unacylated O ghrelin B-Protein by O HPLC O . O An O HPLC O / O UV O method O was O first O optimized O for O the O separation O and O quantitation O of O human O acylated O and O unacylated O ( O or O des O - O acyl O ) O ghrelin B-Protein from O aqueous O solutions O . O This O method O was O validated O by O an O original O approach O using O accuracy O profiles O based O on O tolerance O intervals O for O the O total O error O measurement O . O The O concentration O range O that O achieved O adequate O accuracy O extended O from O 1 O . O 85 O to O 59 O . O 30microM O and O 1 O . O 93 O to O 61 O . O 60microM O for O acylated O and O unacylated O ghrelin B-Protein , O respectively O . O Then O , O optimal O temperature O , O pH O and O buffer O for O sample O storage O were O determined O . O Unacylated O ghrelin B-Protein was O found O to O be O stable O in O all O conditions O tested O . O At O 37 O degrees O C O acylated O ghrelin B-Protein was O stable O at O pH O 4 O but O unstable O at O pH O 7 O . O 4 O , O the O main O degradation O product O was O unacylated O ghrelin B-Protein . O Finally O , O this O validated O HPLC O / O UV O method O was O used O to O evaluate O the O binding O of O acylated O and O unacylated O ghrelin B-Protein to O liposomes O . O Activation O of O the O Drosophila O IMD B-Protein pathway O by O DAP O - O type O peptidoglycan B-Chemical ( O PGN B-Chemical ) O leads O to O the O robust O and O rapid O production O of O a O battery O of O antimicrobial O peptides O ( O AMPs O ) O and O other O immune O responsive O genes O . O Two O peptidoglycan B-Family recognition I-Family protein I-Family ( O PGRP B-Family ) O receptors O are O responsible O for O the O recognition O of O DAP O - O type O PGN B-Chemical , O the O cell O surface O receptor O PGRP B-Protein - I-Protein LC I-Protein and O the O cytosolic O receptor O PGRP B-Protein - I-Protein LE I-Protein . O DAP O - O type O PGN B-Chemical binding O causes O these O receptors O to O multimerize O or O cluster O triggering O signal O transduction O . O IMD B-Protein signaling O culminates O in O activation O of O the O NF B-Complex - I-Complex kappaB I-Complex precursor O Relish B-Protein and O transcriptional O induction O of O AMP O genes O . O Primase B-Protein directs O the O release O of O DnaC B-Protein from O DnaB B-Protein . O An O AAA B-Protein + I-Protein ATPase I-Protein , O DnaC B-Protein , O delivers O DnaB B-Protein helicase I-Protein at O the O E O . O coli O chromosomal O origin O by O a O poorly O understood O process O . O This O report O shows O that O mutant O proteins O bearing O alanine O substitutions O for O two O conserved O arginines O in O a O motif O named O box O VII O are O defective O in O DNA O replication O , O but O this O deficiency O does O not O arise O from O impaired O interactions O with O ATP O , O DnaB B-Protein , O or O single O - O stranded O DNA O . O Despite O their O ability O to O deliver O DnaB B-Protein to O the O chromosomal O origin O to O form O the O prepriming O complex O , O this O intermediate O is O inactive O . O Quantitative O analysis O of O the O prepriming O complex O suggests O that O the O DnaB B-Protein - O DnaC B-Protein complex O contains O three O DnaC B-Protein monomers O per O DnaB B-Protein hexamer O and O that O the O interaction O of O primase B-Protein with O DnaB B-Protein and O primer O formation O triggers O the O release O of O DnaC B-Protein , O but O not O the O mutants O , O from O DnaB B-Protein . O The O interaction O of O primase B-Protein with O DnaB B-Protein and O the O release O of O DnaC B-Protein mark O discrete O events O in O the O transition O from O initiation O to O the O elongation O stage O of O DNA O replication O . O Three O - O dimensional O organization O of O promyelocytic O leukemia O nuclear O bodies O . O Promyelocytic O leukemia O nuclear O bodies O ( O PML O - O NBs O ) O are O mobile O subnuclear O organelles O formed O by O PML B-Protein and O Sp100 B-Protein protein O . O They O have O been O reported O to O have O a O role O in O transcription O , O DNA O replication O and O repair O , O telomere O lengthening O , O cell O cycle O control O and O tumor O suppression O . O We O have O conducted O high O - O resolution O 4Pi O fluorescence O laser O - O scanning O microscopy O studies O complemented O with O correlative O electron O microscopy O and O investigations O of O the O accessibility O of O the O PML O - O NB O subcompartment O . O During O interphase O PML O - O NBs O adopt O a O spherical O organization O characterized O by O the O assembly O of O PML B-Protein and O Sp100 B-Protein proteins O into O patches O within O a O 50 O - O to O 100 O - O nm O - O thick O shell O . O This O spherical O shell O of O PML B-Protein and O Sp100 B-Protein imposes O little O constraint O to O the O exchange O of O components O between O the O PML O - O NB O interior O and O the O nucleoplasm O . O Post O - O translational O SUMO B-Family modifications O , O telomere O repeats O and O heterochromatin B-Protein protein I-Protein 1 I-Protein were O found O to O localize O in O characteristic O patterns O with O respect O to O PML B-Protein and O Sp100 B-Protein . O From O our O findings O , O we O derived O a O model O that O explains O how O the O three O - O dimensional O organization O of O PML O - O NBs O serves O to O concentrate O different O biological O activities O while O allowing O for O an O efficient O exchange O of O components O . O Negative O role O of O RIG B-Protein - I-Protein I I-Protein serine O 8 O phosphorylation O in O the O regulation O of O interferon B-Protein - I-Protein beta I-Protein production O . O RIG B-Protein - I-Protein I I-Protein ( O retinoic B-Protein acid I-Protein - I-Protein inducible I-Protein gene I-Protein I I-Protein ) O and O TRIM25 B-Protein ( O tripartite B-Protein motif I-Protein protein I-Protein 25 I-Protein ) O have O emerged O as O key O regulatory O factors O to O induce O interferon B-Protein ( O IFN B-Protein ) O - O mediated O innate O immune O responses O to O limit O viral O replication O . O Upon O recognition O of O viral O RNA O , O TRIM25 B-Protein E3 O ligase O binds O the O first O caspase B-Family recruitment O domain O ( O CARD O ) O of O RIG B-Protein - I-Protein I I-Protein and O subsequently O induces O lysine O 172 O ubiquitination O of O the O second O CARD O of O RIG B-Protein - I-Protein I I-Protein , O which O is O essential O for O the O interaction O with O downstream O MAVS B-Protein / O IPS B-Protein - I-Protein 1 I-Protein / O CARDIF B-Protein / O VISA B-Protein and O , O thereby O , O IFN B-Protein - I-Protein beta I-Protein mRNA O production O . O Although O ubiquitination O has O emerged O as O a O major O factor O involved O in O RIG B-Protein - I-Protein I I-Protein activation O , O the O potential O contribution O of O other O post O - O translational O modifications O , O such O as O phosphorylation O , O to O the O regulation O of O RIG B-Protein - I-Protein I I-Protein activity O has O not O been O addressed O . O Here O , O we O report O the O identification O of O serine O 8 O phosphorylation O at O the O first O CARD O of O RIG B-Protein - I-Protein I I-Protein as O a O negative O regulatory O mechanism O of O RIG B-Protein - I-Protein I I-Protein - O mediated O IFN B-Protein - I-Protein beta I-Protein production O . O Immunoblot O analysis O with O a O phosphospecific O antibody O showed O that O RIG B-Protein - I-Protein I I-Protein serine O 8 O phosphorylation O steady O - O state O levels O were O decreased O upon O stimulation O of O cells O with O IFN B-Protein - I-Protein beta I-Protein or O virus O infection O . O Substitution O of O serine O 8 O in O the O CARD O RIG B-Protein - I-Protein I I-Protein functional O domain O with O phosphomimetic O aspartate O or O glutamate O results O in O decreased O TRIM25 B-Protein binding O , O RIG B-Protein - I-Protein I I-Protein ubiquitination O , O MAVS B-Protein binding O , O and O downstream O signaling O . O Finally O , O sequence O comparison O reveals O that O only O primate O species O carry O serine O 8 O , O whereas O other O animal O species O carry O an O asparagine O , O indicating O that O serine O 8 O phosphorylation O may O represent O a O primate O - O specific O regulation O of O RIG B-Protein - I-Protein I I-Protein activation O . O Collectively O , O these O data O suggest O that O the O phosphorylation O of O RIG B-Protein - I-Protein I I-Protein serine O 8 O operates O as O a O negative O switch O of O RIG B-Protein - I-Protein I I-Protein activation O by O suppressing O TRIM25 B-Protein interaction O , O further O underscoring O the O importance O of O RIG B-Protein - I-Protein I I-Protein and O TRIM25 B-Protein connection O in O type O I O IFN B-Protein signal O transduction O . O Deltamba1 B-Protein / O Deltamdm38 B-Protein mitochondria O show O severe O defects O in O complexes B-Complex III I-Complex and O IV B-Complex of O the O respiratory O chain O . O ( O A O - O C O ) O . O Complex B-Complex III I-Complex , O complex B-Complex IV I-Complex , O and O malate B-Chemical dehydrogenase I-Chemical activities O were O measured O in O isolated O mitochondria O from O the O strains O indicated O . O SEs B-Protein were O calculated O from O three O independent O experiments O . O ( O D O ) O Mitochondrial O protein O complexes O were O resolved O by O BN O - O PAGE O , O transferred O to O PVDF O membranes O , O and O probed O with O antibodies O against O Rip1 B-Protein ( O complex B-Complex III I-Complex ) O , O Cox4 B-Protein ( O complex B-Complex IV I-Complex ) O , O Tim54 B-Protein ( O TIM22 B-Complex complex I-Complex ) O , O and O Atp5 B-Protein ( O FoF1 B-Complex - I-Complex ATPase I-Complex ) O . O Positions O of O molecular O - O weight O markers O in O kDa O are O indicated O . O ( O E O ) O Mitochondria O ( O 50 O mug O ) O of O the O indicated O strains O were O analyzed O by O Western O blotting O with O antibodies O against O the O indicated O proteins O . O Cyt B-Protein b I-Protein , O cytochrome B-Protein b I-Protein ; O Cytc1 B-Protein , O cytochrome B-Protein c1 I-Protein ; O F1alpha B-Protein , O alpha B-Protein subunit I-Protein of I-Protein the I-Protein FoF1 I-Protein - I-Protein ATPase I-Protein . O The O arrows O indicate O proteins O that O show O diminished O levels O in O the O double O mutant O . O An O S O - O acylation O switch O of O conserved O G O domain O cysteines O is O required O for O polarity O signaling O by O ROP B-Family GTPases I-Family . O Rho B-Family GTPases I-Family are O master O regulators O of O cell O polarity O . O For O their O function O , O Rhos B-Family must O associate O with O discrete O plasma O membrane O domains O . O Rho B-Family of I-Family Plants I-Family ( O ROPs B-Family ) O or O RACs B-Family comprise O a O single O family O . O Prenylation O and O S O - O acylation O of O hypervariable O domain O cysteines O of O Ras B-Family and O Rho B-Family GTPases I-Family are O required O for O their O function O ; O however O , O lipid O modifications O in O the O G O domain O have O never O been O reported O . O Reversible O S O - O acylation O involves O the O attachment O of O palmitate B-Chemical ( O C16 O : O 0 O ) O or O other O saturated O lipids O to O cysteines O through O a O thioester O linkage O and O was O implicated O in O the O regulation O of O signaling O . O Here O we O show O that O transient O S O - O acylation O of O Arabidopsis O AtROP6 B-Protein takes O place O on O two O conserved O G O domain O cysteine O residues O , O C21 O and O C156 O . O C21 O is O relatively O exposed O and O is O accessible O for O modification O , O but O C156 O is O not O , O implying O that O its O S O - O acylation O involves O a O conformational O change O . O Fluorescence O recovery O after O photobleaching O beam O - O size O analysis O shows O that O S O - O acylation O of O AtROP6 B-Protein regulates O its O membrane O - O association O dynamics O , O and O detergent O - O solubilization O studies O indicate O that O it O regulates O AtROP6 B-Protein association O with O lipid O rafts O . O Site O - O specific O acylation O - O deficient O AtROP6 B-Protein mutants O can O bind O and O hydrolyze O GTP B-Chemical but O display O compromised O effects O on O polar O cell O growth O , O endocytic O uptake O of O the O tracer O dye O FM4 B-Chemical - I-Chemical 64 I-Chemical , O and O distribution O of O reactive O oxygen B-Chemical species O . O These O data O reveal O an O S O - O acylation O switch O that O regulates O Rho B-Family signaling O . O Post O - O replication O repair O suppresses O duplication O - O mediated O genome O instability O . O RAD6 B-Protein is O known O to O suppress O duplication O - O mediated O gross O chromosomal O rearrangements O ( O GCRs O ) O but O not O single O - O copy O sequence O mediated O GCRs O . O Here O , O we O found O that O the O RAD6 B-Protein - O and O RAD18 B-Protein - O dependent O post O - O replication O repair O ( O PRR O ) O and O the O RAD5 B-Protein - O , O MMS2 B-Protein - O , O UBC13 B-Protein - O dependent O error O - O free O PRR O branch O acted O in O concert O with O the O replication O stress O checkpoint O to O suppress O duplication O - O mediated O GCRs O formed O by O homologous O recombination O ( O HR O ) O . O The O Rad5 B-Protein helicase O activity O , O but O not O its O RING O finger O , O was O required O to O prevent O duplication O - O mediated O GCRs O , O although O the O function O of O Rad5 B-Protein remained O dependent O upon O modification O of O PCNA B-Protein at O Lys164 O . O The O SRS2 B-Protein , O SGS1 B-Protein , O and O HCS1 B-Protein encoded O helicases O appeared O to O interact O with O Rad5 B-Protein , O and O epistasis O analysis O suggested O that O Srs2 B-Protein and O Hcs1 B-Protein act O upstream O of O Rad5 B-Protein . O In O contrast O , O Sgs1 B-Protein likely O functions O downstream O of O Rad5 B-Protein , O potentially O by O resolving O DNA O structures O formed O by O Rad5 B-Protein . O Our O analysis O is O consistent O with O models O in O which O PRR O prevents O replication O damage O from O becoming O double O strand O breaks O ( O DSBs O ) O and O / O or O regulates O the O activity O of O HR O on O DSBs O . O dHIP14 B-Protein - O dependent O palmitoylation O promotes O secretion O of O the O BMP O antagonist O Sog B-Protein . O Analysis O of O diverse O signaling O systems O has O revealed O that O one O important O level O of O control O is O regulation O of O membrane O trafficking O of O ligands O and O receptors O . O The O activities O of O some O ligands O are O also O regulated O by O whether O they O are O membrane O bound O or O secreted O . O In O Drosophila O , O several O morphogenetic O signals O that O play O critical O roles O in O development O have O been O found O to O be O subject O to O such O regulation O . O For O example O , O activity O of O the O Hedgehog B-Protein ( O Hh B-Protein ) O is O regulated O by O Raspberry B-Protein , O which O palmitoylates O Hh B-Protein . O Similarly O , O the O palmitoylases O Porcupine B-Protein and O Raspberry B-Protein increase O the O activities O of O Wingless B-Protein ( O Wg B-Protein ) O and O the O EGF B-Protein - O ligand O Spitz B-Protein ( O Spi B-Protein ) O , O respectively O . O In O contrast O to O its O vertebrate O homologues O , O which O have O typical O N O - O terminal O signal O sequences O , O the O precursor O form O of O Drosophila O Hh B-Protein contains O an O internal O type O - O II O secretory O signal O motif O . O The O Short B-Protein Gastrulation I-Protein ( O Sog B-Protein ) O protein O is O another O secreted O Drosophila O protein O that O contains O a O type O - O II O signal O and O differs O from O its O vertebrate O ortholog O Chordin B-Protein which O contains O a O standard O signal O peptide O . O In O this O study O , O we O examine O the O regulation O of O Sog B-Protein secretion O and O regulation O by O dHIP14 B-Protein , O the O ortholog O of O a O mammalian O palmitoylase O first O identified O as O Huntington O Interacting O Protein O ( O HIP O ) O . O We O show O that O dHIP14 B-Protein binds O to O Sog B-Protein and O that O Sog B-Protein is O palmitoylated O . O In O S2 O cells O , O dHIP14 B-Protein promotes O secretion O of O Sog B-Protein as O well O as O stabilizing O a O membrane O associated O form O of O Sog B-Protein . O We O examined O the O requirement O for O candidate O cysteine O residues O in O the O N O - O terminal O predicted O cytoplasmic O domain O of O Sog B-Protein and O find O that O Cys27 O , O one O of O two O adjacent O cysteines O ( O Cys27 O and O Cys28 O ) O , O is O essential O for O the O full O activity O of O dHIP14 B-Protein and O its O effect O on O Sog B-Protein . O Finally O , O we O find O that O dHIP14 B-Protein promotes O the O activity O of O Sog B-Protein in O vivo O . O These O studies O highlight O the O growing O importance O of O lipid O modification O in O regulating O signaling O at O the O level O of O ligand O production O and O localization O . O FAN1 B-Protein is O easily O identifiable O in O Dictyostelium O discoideum O , O which O has O orthologs O of O several O Fanconi O anemia O proteins O but O no O obvious O orthologs O are O apparent O in O Drosophila O or O Xenopus O . O Interestingly O , O an O S O . O pombe O ortholog O of O FAN1 B-Protein has O a O SAP O domain O , O TRP O domain O and O a O nuclease O domain O , O thus O it O is O predicted O to O also O function O in O DNA O repair O transactions O . O Fission O yeast O does O not O possess O the O classical O FA O proteins O except O for O an O ortholog O of O FANCM B-Protein , O Fml1 B-Protein , O which O promotes O Rad51 B-Protein - O dependent O gene O conversion O at O stalled O replication O forks O and O limits O crossing O over O during O mitotic O double O - O strand O break O repair O . O It O will O be O interesting O to O test O if O the O S O . O pombe O FAN1 B-Protein mutant O is O sensitive O to O crosslinking O agents O and O if O so O , O how O it O functions O without O the O other O proteins O present O in O human O cells O . O This O could O shed O light O on O alternative O pathways O for O crosslink O repair O in O human O cells O . O Interplay O of O palmitoylation O and O phosphorylation O in O the O trafficking O and O localization O of O phosphodiesterase B-Protein 10A I-Protein : O implications O for O the O treatment O of O schizophrenia O . O Phosphodiesterase B-Protein 10A I-Protein ( O PDE10A B-Protein ) O is O a O striatum O - O enriched O , O dual O - O specific O cyclic B-Family nucleotide I-Family phosphodiesterase I-Family that O has O gained O considerable O attention O as O a O potential O therapeutic O target O for O psychiatric O disorders O such O as O schizophrenia O . O As O such O , O a O PDE10A B-Protein - O selective O inhibitor O compound O , O MP B-Chemical - I-Chemical 10 I-Chemical , O has O recently O entered O clinical O testing O . O Since O little O is O known O about O the O cellular O regulation O of O PDE10A B-Protein , O we O sought O to O elucidate O the O mechanisms O that O govern O its O subcellular O localization O in O striatal O medium O spiny O neurons O . O Previous O reports O suggest O that O PDE10A B-Protein is O primarily O membrane O bound O and O is O transported O throughout O medium O spiny O neuron O axons O and O dendrites O . O Moreover O , O it O has O been O shown O in O PC12 O cells O that O the O localization O of O the O major O splice O form O , O PDE10A2 B-Protein , O may O be O regulated O by O protein B-Family kinase I-Family A I-Family phosphorylation O at O threonine O 16 O ( O Thr O - O 16 O ) O . O Using O an O antibody O that O specifically O recognizes O phosphorylated O Thr O - O 16 O ( O pThr O - O 16 O ) O of O PDE10A2 B-Protein , O we O provide O evidence O that O phosphorylation O at O Thr O - O 16 O is O critical O for O the O regulation O of O PDE10A B-Protein subcellular O localization O in O vivo O . O Furthermore O , O we O demonstrate O in O primary O mouse O striatal O neuron O cultures O that O PDE10A B-Protein membrane O association O and O transport O throughout O dendritic O processes O requires O palmitoylation O of O cysteine O 11 O ( O Cys O - O 11 O ) O of O PDE10A2 B-Protein , O likely O by O the O palmitoyl O acyltransferases O DHHC B-Protein - I-Protein 7 I-Protein and O - B-Protein 19 I-Protein . O Finally O , O we O show O that O Thr O - O 16 O phosphorylation O regulates O PDE10A B-Protein trafficking O and O localization O by O preventing O palmitoylation O of O Cys O - O 11 O rather O than O by O interfering O with O palmitate B-Chemical - O lipid O interactions O . O These O data O support O a O model O whereby O PDE10A B-Protein trafficking O and O localization O can O be O regulated O in O response O to O local O fluctuations O in O cAMP B-Chemical levels O . O Given O this O , O we O propose O that O excessive O striatal O dopamine B-Chemical release O , O as O occurs O in O schizophrenia O , O might O exert O differential O effects O on O the O regulation O of O PDE10A B-Protein localization O in O the O two O striatal O output O pathways O . O Protein O tyrosine O phosphatases O ( O PTPs O ) O remove O phosphate O groups O from O phosphorylated O tyrosine O residues O , O and O play O critical O roles O in O cell O communication O , O shape O , O motility O , O proliferation O and O differentiation O . O One O well O - O known O PTP O , O protein B-Protein tyrosine I-Protein phosphatase I-Protein receptor I-Protein type I-Protein C I-Protein ( O PTPRC B-Protein , O CD45 B-Protein ) O , O is O important O for O thymocyte O development O and O T O cell O activation O , O and O is O expressed O on O all O nucleated O cells O of O hemopoietic O origin O . O The O association O between O MBL B-Protein and O CD45 B-Protein in O immature O T O cells O influences O thymocyte O development O . O The O SpMyh1 B-Protein - O Chimera O construct O was O derived O from O pET11a O - O SpMyh1 B-Protein . O First O , O the O QuikChange O XL O Site O - O Directed O Mutagenesis O Kit O ( O Stratagene O ) O was O used O to O create O a O SalI O restriction O enzyme O cut O site O within O the O Spmyh1 B-Protein + O gene O using O primers O , O SpMyh B-Protein - O Sal O - O F O and O SpMyh B-Protein - O Sal O - O R O . O The O SalI O site O was O created O immediately O 3 O ' O to O the O segment O of O DNA O that O encodes O for O the O SpMyh1 B-Protein IDC O region O and O immediately O 5 O ' O to O the O segment O of O DNA O that O encodes O for O the O SpMyh1 B-Protein C O - O terminal O domain O . O The O pET11a O - O SpMyh1 B-Protein - O SalI O mutagenesis O product O was O digested O with O NdeI O and O SalI O and O the O digested O DNA O fragment O containing O the O pET11a O vector O and O the O DNA O encoding O for O the O C O - O terminal O domain O of O SpMyh1 B-Protein ( O pET11a O - O CTDSpMyh1 O ) O was O gel O purified O . O Simultaneously O , O PCR O was O completed O to O amplify O DNA O containing O a O 5 O ' O - O NdeI O cut O site O and O the O 5 O ' O - O end O of O the O Spmyh1 B-Protein + O gene O up O to O the O beginning O of O the O section O of O DNA O that O encodes O for O the O SpMyh1 B-Protein linker O region O with O primers O SpMyh B-Protein - O NdeI O and O SpMyh B-Protein - O SalI O . O The O SpMyh B-Protein - O SalI O primer O used O in O the O PCR O reaction O included O DNA O to O synthesize O the O specified O section O of O Spmyh1 B-Protein + O , O the O E O . O coli O MutY B-Protein linker O region O , O and O a O SalI O cut O site O . O This O PCR O product O was O digested O with O NdeI O and O SalI O and O ligated O into O the O NdeI O - O SalI O digested O pET11a O - O CTDSpMyh1 O . O Using O the O QuikChange O XL O Site O - O Directed O Mutagenesis O Kit O ( O Stratagene O ) O and O primers O , O SpCHIM O - O Sal O - O to O - O Nat O - O F O and O SpCHIM O - O Sal O - O to O - O Nat O - O R O , O mutagenesis O was O completed O again O to O remove O the O SalI O site O . O The O pET11a O - O SpMyh1 B-Protein - O Chimera O construct O was O used O as O a O template O for O subcloning O into O the O pLM303 O vector O . O BMK1 B-Protein is O activated O by O mitogens O and O oncogenic O signals O and O , O thus O , O is O strongly O implicated O in O tumorigenesis O . O We O found O that O BMK1 B-Protein interacted O with O promyelocytic B-Protein leukemia I-Protein protein I-Protein ( O PML B-Protein ) O , O and O inhibited O its O tumor O - O suppressor O function O through O phosphorylation O . O Furthermore O , O activated O BMK1 B-Protein notably O inhibited O PML B-Protein - O dependent O activation O of O p21 B-Protein . O To O further O investigate O the O BMK B-Protein - O mediated O inhibition O of O the O tumor O suppressor O activity O of O PML B-Protein in O tumor O cells O , O we O developed O a O small O - O molecule O inhibitor O of O the O kinase O activity O of O BMK1 B-Protein , O XMD8 B-Chemical - I-Chemical 92 I-Chemical . O Inhibition O of O BMK1 B-Protein by O XMD8 B-Chemical - I-Chemical 92 I-Chemical blocked O tumor O cell O proliferation O in O vitro O and O significantly O inhibited O tumor O growth O in O vivo O by O 95 O % O , O demonstrating O the O efficacy O and O tolerability O of O BMK1 B-Protein - O targeted O cancer O treatment O in O animals O . O Actin B-Family filament O associated O protein O mediates O c B-Protein - I-Protein Src I-Protein related O SRE B-Protein / O AP B-Complex - I-Complex 1 I-Complex transcriptional O activation O . O AFAP B-Protein is O an O adaptor O protein O involved O in O cytoskeletal O organization O and O intracellular O signaling O . O AFAP B-Protein binds O and O activates O c B-Protein - I-Protein Src I-Protein ; O however O , O the O downstream O signals O of O this O interaction O remain O unknown O . O Here O we O show O that O co O - O expression O of O AFAP B-Protein and O c B-Protein - I-Protein Src I-Protein induce O transcriptional O activation O of O SRE B-Protein and O AP B-Complex - I-Complex 1 I-Complex in O a O c B-Protein - I-Protein Src I-Protein activity O dependent O fashion O . O Structural O - O functional O studies O suggest O that O the O proline O - O rich O motif O in O the O N O - O terminus O of O AFAP B-Protein is O critical O for O c B-Protein - I-Protein Src I-Protein activation O , O and O subsequent O SRE B-Protein / O AP B-Complex - I-Complex 1 I-Complex transactivation O and O the O actin B-Family - O binding O domain O in O the O AFAP B-Protein C O - O terminus O is O negatively O involved O in O the O regulation O of O AFAP B-Protein / O c B-Protein - I-Protein Src I-Protein mediated O SRE B-Protein / O AP B-Complex - I-Complex 1 I-Complex transactivation O . O Selective O deletion O of O this O domain O enhances O transactivation O of O SRE B-Protein . O We O conclude O that O in O addition O to O its O role O in O the O regulation O of O cytoskeletal O structures O , O AFAP B-Protein may O also O be O involved O in O the O c B-Protein - I-Protein Src I-Protein related O transcriptional O activities O . O Saccharomyces O cerevisiae O protein O phosphatase O Ppz1 B-Protein and O protein O kinases O Sat4 B-Protein and O Hal5 B-Protein are O involved O in O the O control O of O subcellular O localization O of O Gln3 B-Protein by O likely O regulating O its O phosphorylation O state O . O A O Saccharomyces O cerevisiae O mutant O lacking O PPZ1 B-Protein , O encoding O a O serine B-Protein / I-Protein threonine I-Protein protein I-Protein phosphatase I-Protein ( O PPase B-Protein ) O , O is O caffeine B-Chemical - O sensitive O . O To O clarify O the O function O of O Ppz1 B-Protein in O resistance O to O caffeine B-Chemical , O we O attempted O systematically O to O identify O protein O kinase O ( O PKase O ) O whose O disruption O lead O to O suppression O of O caffeine B-Chemical sensitive O phenotype O of O the O incrementppz1 B-Protein disruptant O since O disruption O of O PPZ1 B-Protein might O cause O caffeine B-Chemical sensitivity O by O increasing O its O phosphorylated O substrates O and O we O presumed O that O disruption O of O genes O for O PKase O sharing O the O substrate O with O Ppz1 B-Protein could O restore O the O resistance O through O bypassing O necessity O for O dephosphorylation O of O substrates O . O Among O the O 102 O viable O pkase O disruptions O , O disruption O of O either O SAT4 B-Protein or O HAL5 B-Protein suppressed O the O caffeine B-Chemical sensitivity O phenotype O and O increased O expression O of O ENA1 B-Protein , O encoding O a O P B-Family - I-Family type I-Family ATPase I-Family of O the O incrementppz1 B-Protein disruptant O . O Because O increased O expression O of O ENA1 B-Protein in O the O incrementppz1 B-Protein disruptant O was O found O to O be O suppressed O by O disruption O of O GLN3 B-Protein , O localization O and O phosphorylation O of O Gln3 B-Protein in O the O incrementppz1 B-Protein disruptant O was O compared O to O that O in O the O incrementppz1incrementsat4 B-Protein and O incrementppz1incrementhal5 B-Protein double O disruptants O . O Gln3 B-Protein was O found O to O accumulate O in O the O nucleus O in O the O incrementppz1 B-Protein disruptant O , O and O this O nuclear O localization O was O abolished O by O disruption O of O either O SAT4 B-Protein or O HAL5 B-Protein . O Interestingly O , O the O level O of O Gln3 B-Protein phosphorylation O in O the O incrementppz1incrementsat4 B-Protein and O incrementppz1incrementhal5 B-Protein disruptants O decreased O relative O to O wild O type O independent O of O caffeine B-Chemical . O From O these O observations O , O we O conclude O that O Ppz1 B-Protein controls O Gln3 B-Protein localization O by O regulating O its O phosphorylation O state O in O combination O with O Sat4 B-Protein and O Hal5 B-Protein . O Feedback O regulation O of O Drosophila O BMP B-Family signaling O by O the O novel O extracellular O protein O larval B-Protein translucida I-Protein . O The O cellular O response O to O the O Drosophila O BMP B-Protein 2 I-Protein / O 4 B-Protein - O like O ligand O Decapentaplegic B-Protein ( O DPP B-Protein ) O serves O as O one O of O the O best O - O studied O models O for O understanding O the O long O - O range O control O of O tissue O growth O and O pattern O formation O during O animal O development O . O Nevertheless O , O fundamental O questions O remain O unanswered O regarding O extracellular O regulation O of O the O ligand O itself O , O as O well O as O the O nature O of O the O downstream O transcriptional O response O to O BMP B-Family pathway O activation O . O Here O , O we O report O the O identification O of O larval B-Protein translucida I-Protein ( O ltl B-Protein ) O , O a O novel O target O of O BMP B-Family activity O in O Drosophila O . O Both O gain O - O and O loss O - O of O - O function O analyses O implicate O LTL B-Protein , O a O leucine O - O rich O repeat O protein O , O in O the O regulation O of O wing O growth O and O vein O patterning O . O At O the O molecular O level O , O we O demonstrate O that O LTL B-Protein is O a O secreted O protein O that O antagonizes O BMP B-Family - O dependent O MAD B-Protein phosphorylation O , O indicating O that O it O regulates O DPP B-Protein / O BMP B-Family signaling O at O or O above O the O level O of O ligand O - O receptor O interactions O . O Furthermore O , O based O on O genetic O interactions O with O the O DPP B-Protein - O binding O protein O Crossveinless B-Protein 2 I-Protein and O biochemical O interactions O with O the O glypican B-Family Dally B-Protein - I-Protein like I-Protein , O we O propose O that O LTL B-Protein acts O in O the O extracellular O space O where O it O completes O a O novel O auto O - O regulatory O loop O that O modulates O BMP B-Family activity O . O As O is O the O case O with O bur2Delta B-Protein cells O , O deletion O of O DST1 B-Protein , O LEO1 B-Protein , O or O SPT4 B-Protein renders O cells O sensitive O to O 6 B-Chemical - I-Chemical AU I-Chemical , O thus O indicating O their O roles O in O regulating O transcription O . O To O determine O whether O U2 B-Protein snRNA I-Protein alleles O affect O the O 6 B-Chemical - I-Chemical AU I-Chemical sensitivity O of O these O strains O we O tested O the O growth O of O the O double O mutants O on O medium O containing O 6 B-Chemical - I-Chemical AU I-Chemical . O Alterations O in O the O U2 B-Protein snRNA I-Protein secondary O structure O failed O to O produce O significant O changes O in O the O 6 B-Chemical - I-Chemical AU I-Chemical sensitivity O of O these O strains O ( O data O not O shown O and O Table O 1 O ) O . O Taken O together O , O the O genetic O analyses O summarized O in O Table O 1 O suggest O that O neither O CUS2 B-Protein nor O the O U2 B-Protein snRNA O conformation O mutants O exhibit O functional O overlap O with O transcription O factors O that O interact O with O the O CDK B-Family complexes O . O Although O Sp3 B-Protein is O the O only O protein O in O the O Sp B-Family subfamily O that O can O either O positively O or O negatively O modulate O the O Sp1 B-Protein - O dependent O gene O expression O , O our O experiments O demonstrated O that O Sp3 B-Protein positively O regulates O the O exon O 1b O promoter O activity O in O SL2 O cells O . O Taken O together O , O these O results O establish O that O Sp1 B-Protein and O Sp3 B-Protein are O biologically O essential O regulators O of O the O SVCT2 B-Protein exon O 1b O gene O expression O along O with O YY1 B-Protein . O RR B-Protein is O regulated O by O an O intricate O allosteric O mechanism O . O The O two O allosteric O sites O of O RR B-Protein are O the O specificity O site O ( O S O - O site O ) O , O which O determines O substrate O preference O , O and O the O activity O site O ( O A O - O site O ) O , O which O stimulates O or O inhibits O RR B-Protein activity O depending O on O whether O ATP O or O dATP O is O bound O . O While O it O is O known O that O dATP O , O which O is O less O abundant O in O the O cell O than O ATP O , O has O a O 100 O - O fold O higher O affinity O for O RR B-Protein , O the O mechanism O for O this O is O unknown O . O Most O of O our O knowledge O on O RR B-Protein has O been O derived O from O studies O on O the O E O . O coli O and O other O prokaryotic O enzymes O , O which O exist O as O alpha2beta2 O hetero O - O tetramers O . O This O organization O failed O , O however O , O to O mechanistically O explain O how O dATP O inactivates O and O ATP O activates O the O enzyme O . O Recent O biochemical O studies O suggest O that O both O dATP O and O ATP O regulate O mouse O RR B-Protein ( O mRR B-Protein ) O by O altering O its O oligomeric O state O in O an O ATP O / O dATP O concentration O - O dependent O manner O . O The O Cooperman O group O proposed O that O dATP O induces O an O inactive O mRR1 B-Protein tetramer O , O while O ATP O induces O active O mRR1 B-Protein dimers O and O hexamers O . O Interestingly O , O in O a O previous O study O Thelander O and O co O - O workers O observed O dATP O and O ATP O induced O tetramers O for O calf O thymus O RR1 B-Protein . O In O contrast O , O the O Hofer O group O proposed O that O both O dATP O and O ATP O induce O mRR1 B-Protein hexamers O . O In O fact O , O the O cancer O drug O gemcitabine B-Chemical has O been O shown O to O inactivate O a O higher O order O oligomer O of O human O RR B-Protein . O Additionally O , O recent O reports O suggest O that O the O E O . O coli O RR1 B-Protein instead O of O forming O hexamers O forms O tetramers O . O However O , O the O structural O basis O for O dNTP O regulation O by O RR B-Protein oligomerization O is O not O known O . O This O paper O describes O the O discovery O , O through O blind O mutant O screens O , O of O yeast O Ctf18 B-Protein - O RFC B-Complex mutants O that O destabilize O triplet O repeats O . O Genetic O analysis O indicates O Ctf18 B-Protein - O RFC B-Complex likely O acts O through O replication O fork O stabilization O and O / O or O post O - O replication O repair O ( O PRR O ) O , O not O SCC O , O to O prevent O triplet O repeat O instability O , O chromosome O fragility O and O cell O cycle O delays O in O S O and O G2 O / O M O phases O . O Our O data O also O support O a O general O role O for O the O Ctf18 B-Protein - O RFC B-Complex complex O in O preventing O DNA O damage O , O a O role O which O becomes O more O crucial O in O the O presence O of O an O at O - O risk O sequence O such O as O an O expanded O trinucleotide O repeat O tract O . O We O next O measured O the O impact O of O malin B-Protein pathogenic O mutations O on O the O glycogenic O capacity O of O R5 B-Protein / I-Protein PTG I-Protein protein O . O The O cotransfection O of O laforin B-Protein with O wild O - O type O malin B-Protein led O to O a O marked O decrease O of O glycogen B-Chemical levels O in O HEK293 O cells O expressing O R5 B-Protein / I-Protein PTG I-Protein compared O to O cells O overexpressing O only O R5 B-Protein / I-Protein PTG I-Protein ( O Fig O . O 8 O ) O . O This O result O is O consistent O with O the O fact O that O the O laforin B-Protein - O malin B-Protein complex O downregulates O R5 B-Protein / I-Protein PTG I-Protein via O a O proteasomal O degradation O pathway O . O In O contrast O , O malinC46Y B-Protein , O malinP69A B-Protein , O malinD146N B-Protein , O and O malinL261P B-Protein mutants O all O failed O to O reduce O glycogen B-Chemical levels O . O As O illustrated O in O Fig O . O 8 O , O accumulation O of O glycogen B-Chemical levels O was O detected O in O cells O expressing O malin B-Protein mutants O but O not O in O cells O expressing O wild O - O type O malin B-Protein ( O p O < O 0 O . O 01 O ) O . O Interleukin B-Protein enhancer I-Protein - I-Protein binding I-Protein factor I-Protein 3 I-Protein functions O as O a O liver O receptor O homologue O - O 1 O co O - O activator O in O synergy O with O the O nuclear O receptor O co O - O activators O PRMT1 B-Protein and O PGC B-Protein - I-Protein 1alpha I-Protein . O LRH B-Protein - I-Protein 1 I-Protein ( O liver B-Protein receptor I-Protein homologue I-Protein - I-Protein 1 I-Protein ) O , O a O transcription O factor O and O member O of O the O nuclear O receptor O superfamily O , O regulates O the O expression O of O its O target O genes O , O which O are O involved O in O bile B-Chemical acid I-Chemical and O cholesterol B-Chemical homoeostasis O . O However O , O the O molecular O mechanisms O of O transcriptional O control O by O LRH B-Protein - I-Protein 1 I-Protein are O not O completely O understood O . O Previously O , O we O identified O Ku80 B-Protein and O Ku70 B-Protein as O LRH B-Protein - I-Protein 1 I-Protein - O binding O proteins O and O reported O that O they O function O as O co O - O repressors O . O In O the O present O study O , O we O identified O an O additional O LRH B-Protein - I-Protein 1 I-Protein - O binding O protein O , O ILF3 B-Protein ( O interleukin B-Protein enhancer I-Protein - I-Protein binding I-Protein factor I-Protein 3 I-Protein ) O . O ILF3 B-Protein formed O a O complex O with O LRH B-Protein - I-Protein 1 I-Protein and O the O other O two O nuclear O receptor O co O - O activators O PRMT1 B-Protein ( O protein B-Protein arginine I-Protein methyltransferase I-Protein 1 I-Protein ) O and O PGC B-Protein - I-Protein 1alpha I-Protein ( O peroxisome B-Protein proliferator I-Protein - I-Protein activated I-Protein receptor I-Protein gamma I-Protein co I-Protein - I-Protein activator I-Protein - I-Protein 1alpha I-Protein ) O . O We O demonstrated O that O ILF3 B-Protein , O PRMT1 B-Protein and O PGC B-Protein - I-Protein 1alpha I-Protein were O recruited O to O the O promoter O region O of O the O LRH B-Protein - I-Protein 1 I-Protein - O regulated O SHP B-Protein ( O small B-Protein heterodimer I-Protein partner I-Protein ) O gene O , O encoding O one O of O the O nuclear O receptors O . O ILF3 B-Protein enhanced O SHP B-Protein gene O expression O in O co O - O operation O with O PRMT1 B-Protein and O PGC B-Protein - I-Protein 1alpha I-Protein through O the O C O - O terminal O region O of O ILF3 B-Protein . O In O addition O , O we O found O that O the O small O interfering O RNA O - O mediated O down O - O regulation O of O ILF3 B-Protein expression O led O to O a O reduction O in O the O occupancy O of O PGC B-Protein - I-Protein 1alpha I-Protein at O the O SHP B-Protein promoter O and O SHP B-Protein expression O . O Taken O together O , O our O results O suggest O that O ILF3 B-Protein functions O as O a O novel O LRH B-Protein - I-Protein 1 I-Protein co O - O activator O by O acting O synergistically O with O PRMT1 B-Protein and O PGC B-Protein - I-Protein 1alpha I-Protein , O thereby O promoting O LRH B-Protein - I-Protein 1 I-Protein - O dependent O gene O expression O . O Our O results O suggest O a O two O - O step O model O for O PFN B-Protein delivery O of O Gzms B-Family in O which O PFN B-Protein first O forms O transient O pores O in O the O cell O membrane O that O trigger O the O target O cell O membrane O repair O response O leading O to O coendocytosis O of O Gzms B-Family and O PFN B-Protein . O PFN B-Protein then O forms O larger O , O more O stable O , O pores O in O the O endosomal O membrane O to O trigger O release O of O Gzms B-Family . O Although O the O experiments O presented O here O used O GzmB B-Protein , O we O obtained O similar O results O when O the O other O major O Gzm B-Family ( O GzmA B-Protein ) O was O substituted O ( O data O not O shown O ) O . O Therefore O we O expect O this O model O applies O to O all O the O Gzms B-Family . O This O model O , O which O suggests O that O PFN B-Protein can O form O at O least O two O types O of O pores O of O different O size O and O stability O , O is O supported O by O a O recent O study O that O measured O conductance O through O various O sorts O of O membranes O ( O planar O lipid O bilayers O and O unilamellar O vesicles O of O different O lipid O composition O and O size O ) O treated O with O PFN B-Protein . O There O was O a O good O deal O of O heterogeneity O in O PFN B-Protein pores O ; O in O particular O formation O of O small O , O highly O unstable O pores O preceded O the O development O of O more O stable O and O larger O pores O with O a O distribution O in O size O . O Heterogeneous O pore O formation O was O confirmed O by O cryoelectron O microscopy O . O We O therefore O hypothesize O that O the O rapid O membrane O repair O response O interferes O with O the O formation O of O larger O pores O on O the O plasma O membrane O , O but O that O PFN B-Protein multimerizes O into O larger O stable O pores O on O the O gigantosome O membrane O that O increase O in O size O within O 5 O - O 15 O min O after O adding O PFN B-Protein . O Based O on O the O kinetics O of O early O endosome O acidification O , O our O data O suggest O that O the O smaller O pores O form O within O the O gigantosome O membrane O almost O immediately O to O interfere O with O acidification O and O allow O PFN B-Protein to O remain O active O . O As O deleting O its O initiation O motif O abolished O geminin B-Protein degradation O , O we O used O this O substrate O to O identify O key O residues O required O for O promoting O initiation O . O We O found O that O mutation O of O charged O residues O ( O RE40 O ; O KRK50 O - O 52 O ; O HR53 O / O 54 O ) O to O alanine O interfered O with O the O APC O / O C O - O dependent O ubiquitination O and O degradation O of O geminin B-Protein ( O Figure O 2A O ; O Figure O S2A O ) O . O Assays O with O methylubiquitin B-Protein revealed O that O RE40 O / O 41 O , O KRK50 O - O 52 O , O and O HR53 O / O 54 O were O required O for O efficient O chain O initiation O ( O Figure O 2B O ) O . O Interestingly O , O changing O all O Lys O residues O to O arginine O did O not O strongly O affect O geminin B-Protein degradation O or O chain O initiation O , O showing O that O the O initiation O motif O has O functions O in O addition O to O providing O ubiquitin B-Protein acceptor O sites O . O As O expected O for O a O motif O controlling O the O degradation O of O a O key O cell O cycle O regulator O , O functionally O important O , O but O not O irrelevant O , O residues O are O highly O conserved O among O geminin B-Protein homologs O from O different O organisms O ( O Figure O S2B O ) O . O A O yeast O two O hybrid O screen O identifies O SPATA4 B-Protein as O a O TRAPP B-Complex interactor O . O The O TRAPP B-Complex vesicle O - O tethering O complex O consists O of O more O than O 10 O distinct O polypeptides O and O is O involved O in O protein O transport O . O Using O the O C2 B-Protein subunit O as O bait O we O identified O SPATA4 B-Protein , O a O spermatocyte O - O specific O protein O of O unknown O function O , O as O an O interacting O partner O in O a O yeast O two O hybrid O screen O . O Further O studies O indicate O SPATA4 B-Protein interacts O with O the O C2 B-Protein portion O of O the O TRAPP B-Complex complex O . O SPATA4 B-Protein fractionates O with O both O cytosolic O and O nuclear O fractions O suggesting O it O may O have O several O distinct O functions O . O SPATA4 B-Protein is O one O of O only O three O human O proteins O that O contain O a O DUF1042 O domain O and O we O show O that O C2 B-Protein does O not O interact O with O another O one O of O the O DUF1042 O domain O - O containing O proteins O . O Our O results O suggest O a O role O for O SPATA4 B-Protein in O membrane O traffic O and O a O specialized O function O for O TRAPP B-Complex in O spermatocytes O . O Fission O yeast O Swi5 B-Protein - O Sfr1 B-Protein protein O complex O , O an O activator O of O Rad51 B-Protein recombinase O , O forms O an O extremely O elongated O dogleg O - O shaped O structure O . O In O eukaryotes O , O DNA O strand O exchange O is O the O central O reaction O of O homologous O recombination O , O which O is O promoted O by O Rad51 B-Protein recombinases O forming O a O right O - O handed O nucleoprotein O filament O on O single O - O stranded O DNA O , O also O known O as O a O presynaptic O filament O . O Accessory O proteins O known O as O recombination O mediators O are O required O for O the O formation O of O the O active O presynaptic O filament O . O One O such O mediator O in O the O fission O yeast O Schizosaccharomyces O pombe O is O the O Swi5 B-Protein - O Sfr1 B-Protein complex O , O which O has O been O identified O as O an O activator O of O Rad51 B-Protein that O assists O in O presynaptic O filament O formation O and O stimulates O its O strand O exchange O reaction O . O Here O , O we O determined O the O 1 O : O 1 O binding O stoichiometry O between O the O two O subunits O of O the O Swi5 B-Protein - O Sfr1 B-Protein complex O using O analytical O ultracentrifugation O and O electrospray O ionization O mass O spectrometry O . O Small O - O angle O x O - O ray O scattering O experiments O revealed O that O the O Swi5 B-Protein - O Sfr1 B-Protein complex O displays O an O extremely O elongated O dogleg O - O shaped O structure O in O solution O , O which O is O consistent O with O its O exceptionally O high O frictional O ratio O ( O f O / O f O ( O 0 O ) O ) O of O 2 O . O 0 O + O / O - O 0 O . O 2 O obtained O by O analytical O ultracentrifugation O . O Furthermore O , O we O determined O a O rough O topology O of O the O complex O by O comparing O the O small O - O angle O x O - O ray O scattering O - O based O structures O of O the O Swi5 B-Protein - O Sfr1 B-Protein complex O and O four O Swi5 B-Protein - O Sfr1 B-Protein - O Fab O complexes O , O in O which O the O Fab O fragments O of O monoclonal O antibodies O were O specifically O bound O to O experimentally O determined O sites O of O Sfr1 B-Protein . O We O propose O a O model O for O how O the O Swi5 B-Protein - O Sfr1 B-Protein complex O binds O to O the O Rad51 B-Protein filament O , O in O which O the O Swi5 B-Protein - O Sfr1 B-Protein complex O fits O into O the O groove O of O the O Rad51 B-Protein filament O , O leading O to O an O active O and O stable O presynaptic O filament O . O Germline O inactivation O of O the O VHL O tumor O suppressor O gene O is O linked O with O development O of O von O Hippel O - O Lindau O ( O VHL O ) O disease O , O an O autosomal O dominantly O inherited O cancer O syndrome O . O VHL O patients O are O predisposed O to O develop O various O vascular O tumors O , O including O hemangioblastomas O of O the O retinas O and O central O nervous O system O , O clear O cell O RCC O , O pancreatic O cysts O and O adenocarcinomas O , O and O adrenal O pheochromocytomas O . O The O VHL O gene O is O also O inactivated O in O the O majority O of O patients O with O sporadic O clear O cell O RCC O . O The O VHL O gene O encodes O proteins O ( O pVHL B-Protein ) O of O 25 O and O 19 O kDa O through O use O of O alternative O translation O initiation O codons O , O and O both O isoforms O appear O to O possess O tumor O suppressor O activity O . O pVHL B-Protein has O alpha O and O beta O structural O domains O that O are O critical O to O its O function O as O the O substrate O recognition O component O of O a O cullin B-Complex - I-Complex RING I-Complex E3 I-Complex ubiquitin I-Complex ligase I-Complex ( O CRL B-Complex ) O . O The O N O - O terminal O beta O domain O is O associated O with O target O protein O recognition O , O while O the O alpha O domain O contains O a O SOCS B-Protein box O that O interacts O with O elongin B-Protein C I-Protein and O links O pVHL B-Protein to O the O ubiquitin B-Protein - O ligase O complex O containing O elongin B-Protein B I-Protein and O C B-Protein and O Cul2 B-Protein . O Perhaps O the O best O characterized O pVHL B-Protein CRL B-Complex targets O are O the O alpha O subunits O of O hypoxia B-Protein - I-Protein inducible I-Protein factor I-Protein ( O HIF B-Protein ) O . O Hydroxylation O of O conserved O prolines O on O HIFalpha B-Protein subunits O under O normoxic O ( O 21 O % O O2 B-Chemical ) O conditions O provides O a O substrate O recognition O motif O for O pVHL B-Protein polyubiquitylation O , O and O proteasomal O degradation O . O In O hypoxia O ( O 1 O % O O2 B-Chemical ) O prolyl O hydroxylase O activity O is O inhibited O , O unmodified O HIFalpha B-Protein subunits O are O stabilized O , O and O the O hypoxia O response O is O initiated O . O We O recently O demonstrated O that O pVHL B-Protein levels O are O suppressed O in O hypoxia O , O providing O an O additional O mechanism O for O HIFalpha B-Protein upregulation O in O hypoxia O . O A O sulfurtransferase B-Family is O essential O for O activity O of O formate O dehydrogenases O in O Escherichia O coli O . O l B-Family - I-Family Cysteine I-Family desulfurases I-Family provide O sulfur B-Chemical to O several O metabolic O pathways O in O the O form O of O persulfides O on O specific O cysteine O residues O of O an O acceptor O protein O for O the O eventual O incorporation O of O sulfur B-Chemical into O an O end O product O . O IscS B-Protein is O one O of O the O three O Escherichia O coli O l B-Family - I-Family cysteine I-Family desulfurases I-Family . O It O interacts O with O FdhD B-Protein , O a O protein O essential O for O the O activity O of O formate O dehydrogenases O ( O FDHs O ) O , O which O are O iron B-Chemical / O molybdenum B-Chemical / O selenium B-Chemical - O containing O enzymes O . O Here O , O we O address O the O role O played O by O this O interaction O in O the O activity O of O FDH B-Protein - I-Protein H I-Protein ( O FdhF B-Protein ) O in O E O . O coli O . O The O interaction O of O IscS B-Protein with O FdhD B-Protein results O in O a O sulfur B-Chemical transfer O between O IscS B-Protein and O FdhD B-Protein in O the O form O of O persulfides O . O Substitution O of O the O strictly O conserved O residue O Cys O - O 121 O of O FdhD B-Protein impairs O both O sulfur O transfer O from O IscS B-Protein to O FdhD B-Protein and O FdhF B-Protein activity O . O Furthermore O , O inactive O FdhF B-Protein produced O in O the O absence O of O FdhD B-Protein contains O both O metal O centers O , O albeit O the O molybdenum B-Chemical cofactor O is O at O a O reduced O level O . O Finally O , O FdhF B-Protein activity O is O sulfur B-Chemical - O dependent O , O as O it O shows O reversible O sensitivity O to O cyanide B-Chemical treatment O . O Conclusively O , O FdhD B-Protein is O a O sulfurtransferase B-Family between O IscS B-Protein and O FdhF B-Protein and O is O thereby O essential O to O yield O FDH O activity O . O Yeast O transcription O termination O factor O Rtt103 B-Protein functions O in O DNA O damage O response O . O YKu70 B-Protein / O YKu80 B-Protein is O a O heterodimer O that O is O essential O for O repair O of O DNA O double O strand O breaks O through O non O - O homologous O end O joining O pathway O in O the O yeast O Saccharomyces O cerevisiae O . O Yku70 B-Protein / O 80 B-Protein proteins O are O associated O with O telomeres O and O are O important O for O maintaining O the O integrity O of O telomeres O . O These O proteins O protect O telomeres O from O recombination O events O , O nuclease O attacks O , O support O the O formation O of O heterochromatin O at O telomeres O and O anchor O telomeres O to O the O nuclear O periphery O . O To O identify O components O in O molecular O networks O involved O in O the O multiple O functions O of O Yku70 B-Protein / O 80 B-Protein complex O , O we O performed O a O genetic O screen O for O suppressors O of O yku70 B-Protein deletion O . O One O of O the O suppressors O identified O was O RTT103 B-Protein , O which O encodes O a O protein O implicated O in O transcription O termination O . O We O show O that O rtt103Delta B-Protein are O sensitive O to O multiple O forms O of O genome O insults O and O that O RTT103 B-Protein is O essential O for O recovery O from O DNA O double O strand O breaks O in O the O chromosome O . O We O further O show O that O Rtt103 B-Protein associates O with O sites O of O DNA O breaks O and O hence O is O likely O to O play O a O direct O role O in O response O to O DNA O damage O . O To O compare O our O low O resolution O SAXS O data O with O the O crystal O structure O , O we O computed O the O theoretical O SAXS O curves O and O the O pair O - O distance O distribution O function O for O the O crystal O structures O of O hPPARgamma B-Protein DBD I-Protein - I-Protein LBD I-Protein monomer O and O hPPARgamma B-Protein / O hRXRalpha B-Protein DBD I-Protein - I-Protein LBD I-Protein heterodimer O ( O Figure O 5 O ) O . O The O crystallographic O models O do O not O fit O well O to O the O DAMs O derived O from O our O SAXS O experiments O . O The O profiles O of O the O distance O distribution O functions O p O ( O r O ) O corresponding O to O DAMs O and O generated O for O crystallography O structures O are O typical O for O elongated O particles O . O Nevertheless O , O the O Dmax O of O the O DAMs O are O larger O than O the O crystallographic O structure O , O which O indicates O that O the O protein O in O solution O is O more O elongated O than O in O the O crystal O . O All O dysc B-Protein alleles O were O recessive O ; O trans O - O heterozygotic O combinations O of O the O three O alleles O were O largely O arrhythmic O ; O and O heterozygosity O for O both O dysc B-Protein s168 O and O dysc B-Protein c03838 O in O combination O with O a O deficiency O removing O the O dysc B-Protein locus O also O resulted O in O arrhythmia O ( O Table O 1 O ) O . O In O addition O , O precise O excision O of O the O dysc B-Protein s168 O P O - O element O restored O wild O - O type O patterns O of O locomotion O ( O Table O 1 O ) O , O indicating O that O the O P O - O element O insertion O is O responsible O for O arrhythmicity O . O Finally O , O to O test O whether O transgenic O expression O of O dysc B-Protein could O restore O rhythmic O behavior O , O we O generated O flies O carrying O a O UAS B-Protein - O dysc B-Protein transgene O encoding O a O long O isoform O of O DYSC B-Protein . O Pan O - O neuronal O expression O of O the O UAS B-Protein - O dysc B-Protein transgene O in O dysc B-Protein mutants O was O sufficient O to O rescue O rhythmic O behavior O ( O Figure O 2E O and O Table O 2 O ) O . O Consistent O with O the O fact O that O dysc B-Protein s168 O homozygotes O , O which O express O normal O levels O of O the O short O isoform O , O are O arrhythmic O , O transgenic O expression O of O a O short O DYSC B-Protein isoform O did O not O restore O rhythmicity O in O dysc B-Protein c03838 O mutants O ( O Table O 2 O ; O see O Figure O S3 O for O expression O levels O of O long O and O short O dysc B-Protein transgenes O ) O . O Over O - O expression O of O either O the O long O or O short O isoforms O of O DYSC B-Protein in O a O wild O - O type O background O did O not O affect O circadian O rhythmicity O ( O Table O 2 O ) O . O These O results O comprehensively O demonstrate O that O DYSC B-Protein is O required O for O circadian O alterations O in O locomotor O activity O , O and O furthermore O indicate O that O correct O circadian O output O requires O DYSC B-Protein expression O in O the O nervous O system O . O Secondly O , O we O determined O the O molar O binding O ratio O of O GFP B-Protein - O Ligase B-Protein III I-Protein to O RFP B-Protein - O Xrcc1 B-Protein . O Xrcc1 B-Protein binds O in O a O molar O ratio O of O 0 O . O 61 O + O / O - O 0 O . O 14 O to O Ligase B-Protein III I-Protein but O did O not O bind O to O other O proteins O such O as O GFP B-Protein - O PBD B-Protein , O GFP B-Protein - O Dnmt1DeltaPBD B-Protein or O GFP B-Protein . O Previous O studies O demonstrated O that O DNA B-Protein Ligase I-Protein III I-Protein was O recruited O to O DNA O repair O sites O via O its O BRCT O domain O mediated O interaction O with O Xrcc1 B-Protein . O To O identify O the O putative O interacting O protein O , O we O used O an O unbiased O forward O genetic O strategy O and O discovered O SOL B-Protein - I-Protein 2 I-Protein , O a O CUB O - O domain O transmembrane O protein O that O is O the O homologue O of O the O vertebrate O Neto B-Family proteins O , O with O 2 O CUB O - O domains O and O a O LDLa O - O domain O . O As O predicted O , O we O found O that O s O - O SOL B-Protein - I-Protein 1 I-Protein function O was O dependent O on O SOL B-Protein - I-Protein 2 I-Protein and O that O SOL B-Protein - I-Protein 2 I-Protein associates O with O the O GLR B-Protein - I-Protein 1 I-Protein signaling O complex O . O We O show O that O surface O delivery O of O GLR B-Protein - I-Protein 1 I-Protein and O SOL B-Protein - I-Protein 1 I-Protein occurs O in O the O absence O of O SOL B-Protein - I-Protein 2 I-Protein ; O however O , O the O stability O or O function O of O the O complex O appears O compromised O in O sol B-Protein - I-Protein 2 I-Protein mutants O . O In O sol B-Protein - I-Protein 1 I-Protein mutants O , O the O remaining O components O of O the O GLR B-Protein - I-Protein 1 I-Protein complex O are O also O delivered O to O the O postsynaptic O membrane O , O indicating O that O SOL B-Protein - I-Protein 1 I-Protein does O not O have O an O essential O role O in O assembly O or O trafficking O of O the O signaling O complex O . O We O demonstrate O that O GLR B-Protein - I-Protein 1 I-Protein - O mediated O currents O depend O on O both O SOL B-Protein - I-Protein 1 I-Protein and O SOL B-Protein - I-Protein 2 I-Protein , O and O that O currents O in O sol B-Protein - I-Protein 1 I-Protein and O sol B-Protein - I-Protein 2 I-Protein mutants O can O be O rescued O in O adults O , O thus O demonstrating O an O ongoing O role O for O these O CUB O - O domain O proteins O in O synaptic O transmission O . O The O NDP52 B-Protein CLIR O was O disordered O in O crystals O of O apo O - O NDP52 B-Protein but O became O structured O in O the O 2 O . O 5 O Aa O complex O structure O of O NDP52 B-Protein bound O to O LC3C B-Protein ( O Figure O 4B O ) O . O Similar O to O other O LC3 B-Family - O LIR B-Protein complexes O and O to O SUMO B-Family - O SIM O interactions O , O the O CLIR O forms O a O short O beta O strand O ( O Figure O 4B O ) O that O fits O into O the O canonical O , O hydrophobic O LIR B-Protein binding O groove O of O LC3C B-Protein ( O Figures O 4C O and O 4D O ) O . O As O expected O from O the O NMR O data O and O mutational O analysis O ( O Figure O 3 O ) O , O the O interaction O is O driven O by O extensive O hydrophobic O interactions O of O Leu134 O , O Val135 O , O and O Val136 O . O We O also O wanted O to O determine O if O the O interaction O with O ABI B-Protein - I-Protein 1 I-Protein is O isoform O specific O with O respect O to O MIG B-Protein - I-Protein 10 I-Protein . O As O described O previously O , O the O three O predicted O splice O variants O of O mig B-Protein - I-Protein 10 I-Protein encode O isoforms O that O differ O only O in O their O N O - O termini O ( O Fig O . O 1 O ) O . O In O particular O , O the O N O - O terminus O of O MIG B-Protein - I-Protein 10A I-Protein contains O a O proline O - O rich O region O with O a O putative O SH3 O binding O site O that O is O missing O in O MIG B-Protein - I-Protein 10B I-Protein , O the O smallest O isoform O . O MIG B-Protein - I-Protein 10B I-Protein : O V5 O also O co O - O immunoprecipitated O with O wild O type O ABI B-Protein - I-Protein 1 I-Protein : O GFP B-Protein ( O Fig O . O 2E O ) O , O demonstrating O that O both O isoforms O are O capable O of O interaction O with O ABI B-Protein - I-Protein 1 I-Protein , O and O suggesting O that O the O N O - O termini O of O the O MIG B-Protein - I-Protein 10 I-Protein isoforms O are O not O essential O to O the O interaction O . O Domain O organization O of O Drosophila O melanogaster O GW182 B-Protein , O Hs O TNRC6C B-Protein and O the O corresponding O chimeric O proteins O . O ABD O , O AGO O - O binding O domain O ; O ABD2 O , O AGO O - O binding O domain O from O Caenorhabditis O elegans O AIN B-Protein - I-Protein 2 I-Protein ; O NED O , O N O - O terminal O effector O domain O ; O UBA O , O ubiquitin B-Protein associated O - O like O domain O ; O QQQ O , O region O rich O in O glutamine O ; O Mid O , O middle O region O containing O the O PAM2 O motif O ( O dark O blue O ) O , O which O divides O the O Mid O region O into O the O M1 O and O M2 O regions O ; O RRM O , O RNA O recognition O motif O ; O C O - O term O , O C O - O terminal O region O ; O SD O , O silencing O domain O . O The O position O of O the O conserved O CIM O - O 1 O , O CIM O - O 2 O and O P O - O GL O motifs O are O indicated O . O Amino O acid O positions O at O domain O boundaries O are O indicated O below O the O protein O outlines O . O Vertical O red O lines O indicate O the O positions O of O GW O repeats O . O Vertical O green O lines O indicate O the O positions O of O tryptophan O residues O in O the O M2 O region O that O are O involved O in O NOT1 B-Protein - O binding O . O Sequence O alignments O of O the O PAM2 O , O CIM O - O 1 O , O CIM O - O 2 O and O P O - O GL O motifs O and O the O amino O acids O mutated O in O this O study O are O shown O in O Supplementary O Figure O S7 O . O The O Newton O - O Raphson O algorithm O ( O Equation O 9 O ) O uses O known O values O of O PT O ( O i O ) O , O FPAT O ( O i O ) O and O RT O ( O i O ) O and O chosen O values O of O Ka1 O and O Ka2 O where O iterations O ( O i O ) O proceed O to O convergence O where O F1 O = O F2 O = O 0 O . O where O V O is O a O column O vector O with O rows O P O and O FPA B-Chemical , O and O F O is O a O column O vector O with O rows O F1 O and O F2 O . O We O next O followed O the O vesicular O movement O of O full O - O length O Serp B-Protein - O GFP B-Protein , O and O found O that O it O was O associated O with O the O Rab9 B-Protein - O positive O endosomal O membrane O , O and O some O of O the O Serp B-Protein - O GFP B-Protein was O concentrated O at O the O Rab9 B-Protein - O enriched O domains O ( O white O arrowheads O in O Fig O . O 5f O ) O . O Importantly O , O the O Serp B-Protein puncta O colocalized O with O small O GFP B-Protein - O Rab9 B-Protein vesicles O that O were O budding O from O larger O endosomes O ( O blue O arrowheads O in O Fig O . O 5f O ) O . O In O Arabidopsis O , O CBL1 B-Protein is O activated O by O various O abiotic O stresses O and O functions O as O a O positive O regulator O in O salt B-Chemical or O drought O stress O responses O . O It O was O also O described O as O a O negative O regulator O of O cold O response O . O By O contrast O , O VDAC1 B-Protein was O stable O under O salt B-Chemical , O cold O or O drought O stresses O in O Arabidopsis O during O 24 O h O detected O by O semi O - O quantitative O RT O - O PCR O , O whereas O its O expression O was O enhanced O in O mature O plants O after O 48 O h O under O cold O stress O examined O by O real O - O time O PCR O method O ( O Figure O 1b O ) O , O indicating O a O possible O role O in O cold O stress O response O . O Overexpression O of O VDAC1 B-Protein reduced O freezing O tolerance O , O whereas O vdac1 B-Protein mutant O plants O showed O superior O performance O to O WT O under O freezing O stress O , O suggesting O that O VDAC1 B-Protein functions O as O a O negative O regulator O in O cold O stress O response O . O This O is O consistent O with O the O role O of O CBL1 B-Protein in O cold O stress O response O . O CBL1 B-Protein was O shown O to O alter O the O expression O of O a O series O of O cold O - O induced O genes O when O subjected O to O cold O stress O . O To O test O if O VDAC1 B-Protein and O CBL1 B-Protein could O be O involved O in O the O regulation O of O a O common O set O of O genes O , O the O expression O of O genes O found O to O be O altered O by O CBL1 B-Protein under O cold O treatment O were O quantified O in O VDAC1 B-Protein overexpressing O and O vdac1 B-Protein mutant O lines O . O Among O these O genes O , O the O expression O of O DREB1A B-Protein , O a O gene O specifically O responsive O to O cold O , O was O inhibited O in O VDAC1 B-Protein - O overexpressing O plants O , O suggesting O that O VDAC1 B-Protein overexpression O exerted O negative O regulation O on O DREB1A B-Protein . O On O the O contrary O , O DREB1A B-Protein was O highly O induced O in O vdac1 B-Protein mutant O plants O ( O Figure O 2f O ) O . O These O results O indicate O that O VDAC1 B-Protein and O CBL1 B-Protein have O similar O effects O on O the O expression O of O DREB1A B-Protein . O The O vdac1 B-Protein mutant O also O showed O superior O performance O during O seed O germination O under O low O temperature O , O and O the O phenotypes O observed O for O cbl1 B-Protein resembled O those O of O vdac1 B-Protein ( O Figure O 4c O ) O . O To O determine O if O the O visual O phenotypes O of O the O mutants O at O 4 O degreesC O were O due O to O quicker O germination O , O but O not O faster O growth O , O seeds O were O germinated O at O 22 O degreesC O for O two O days O before O being O transferred O to O 4 O degreesC O . O The O mutants O did O not O present O any O obvious O morphological O differences O ( O data O not O shown O ) O . O These O results O demonstrated O that O both O VDAC1 B-Protein and O CBL1 B-Protein function O during O seed O germination O at O low O temperature O , O suggesting O a O biological O relevance O of O the O two O genes O . O Cohesin B-Complex is O highly O enriched O throughout O the O pericentromere O , O yet O the O Scc2 B-Protein / O 4 B-Protein cohesin B-Complex loader O shows O strong O enrichment O only O within O the O core O ~ O 125 O bp O centromere O ( O see O Figures O S1A O and O S1B O available O online O ) O . O Enrichment O of O Scc2 B-Protein at O centromeres O , O but O not O at O a O control O tRNA O site O , O requires O Ctf19 B-Complex complex O components O ( O Figures O S1C O and O S1D O ) O . O Scc2 B-Protein turns O over O rapidly O near O kinetochores O and O does O not O stably O associate O with O the O Ctf19 B-Complex complex O ( O Figures O S1E O , O S1F O , O and O Table O S3 O ) O . O Even O when O the O Ctf19 B-Complex complex O was O purified O from O cells O producing O a O version O of O Smc3 B-Protein ( O Smc3E1155Q B-Protein ) O blocked O at O an O early O step O in O cohesin B-Complex loading O , O virtually O the O entire O kinetochore O , O yet O only O very O few O peptides O of O cohesin B-Complex and O its O loader O , O were O recovered O ( O Figure O S1E O and O Table O S3 O ) O . O These O data O suggest O that O the O Ctf19 B-Complex complex O promotes O cohesin B-Complex enrichment O throughout O the O pericentromere O by O enabling O the O transient O association O of O the O Scc2 B-Protein / O 4 B-Protein cohesin B-Complex loader O with O centromeres O . O The O p21 B-Family - I-Family activated I-Family kinase I-Family Mbt B-Protein is O a O component O of O the O apical O protein O complex O in O central O brain O neuroblasts O and O controls O cell O proliferation O . O The O final O size O of O the O central O nervous O system O is O determined O by O precisely O controlled O generation O , O proliferation O and O death O of O neural O stem O cells O . O We O show O here O that O the O Drosophila O PAK B-Protein protein O Mushroom B-Protein bodies I-Protein tiny I-Protein ( O Mbt B-Protein ) O is O expressed O in O central O brain O progenitor O cells O ( O neuroblasts O ) O and O becomes O enriched O to O the O apical O cortex O of O neuroblasts O in O a O cell O cycle O - O and O Cdc42 B-Protein - O dependent O manner O . O Using O mushroom O body O neuroblasts O as O a O model O system O , O we O demonstrate O that O in O the O absence O of O Mbt B-Protein function O , O neuroblasts O and O their O progeny O are O correctly O specified O and O are O able O to O generate O different O neuron O subclasses O as O in O the O wild O type O , O but O are O impaired O in O their O proliferation O activity O throughout O development O . O In O general O , O loss O of O Mbt B-Protein function O does O not O interfere O with O establishment O or O maintenance O of O cell O polarity O , O orientation O of O the O mitotic O spindle O and O organization O of O the O actin O or O tubulin O cytoskeleton O in O central O brain O neuroblasts O . O However O , O we O show O that O mbt B-Protein mutant O neuroblasts O are O significantly O reduced O in O cell O size O during O different O stages O of O development O , O which O is O most O pronounced O for O mushroom O body O neuroblasts O . O This O phenotype O correlates O with O reduced O mitotic O activity O throughout O development O . O Additionally O , O postembryonic O neuroblasts O are O lost O prematurely O owing O to O apoptosis O . O Yet O , O preventing O apoptosis O did O not O rescue O the O loss O of O neurons O seen O in O the O adult O mushroom O body O of O mbt B-Protein mutants O . O From O these O results O , O we O conclude O that O Mbt B-Protein is O part O of O a O regulatory O network O that O is O required O for O neuroblast O growth O and O thereby O allows O proper O proliferation O of O neuroblasts O throughout O development O . O With O the O discovery O of O the O diverse O functions O of O RNA O transcripts O , O increasing O interest O has O been O placed O upon O the O role O of O alternative O splicing O in O tumor O progression O . O Although O neoplastic O tissues O have O been O reported O to O display O a O perturbation O in O mRNA O isoform O expression O compared O to O matched O normal O tissues O , O the O precise O role O of O this O altered O transcriptome O and O splicing O pattern O in O tumorigenesis O remains O uncertain O . O Previously O , O it O was O shown O that O the O metastasis O suppressor O Nm23 B-Protein - I-Protein H1 I-Protein regulates O changes O in O the O expression O of O RNA O post O - O transcriptional O modification O proteins O , O including O Gemin5 B-Protein , O a O component O of O the O spliceosome O . O Yet O , O a O direct O role O for O metastasis O modifiers O in O splicing O has O not O yet O been O reported O , O although O it O seems O that O many O factors O involved O in O splicing O have O a O variety O of O cellular O functions O . O Here O , O using O RNA O - O seq O in O combination O with O other O biochemical O methods O , O the O metastasis O modifier O RRP1B B-Protein has O been O demonstrated O to O directly O regulate O alternative O isoform O expression O . O This O suggests O that O alternative O splicing O , O and O the O diverse O isoforms O generated O from O that O process O , O may O play O a O role O in O the O regulation O of O various O factors O implicated O in O metastasis O . O While O HOT1 B-Protein localizes O to O a O subset O of O telomeres O together O with O shelterin B-Complex components O , O our O HOT1 B-Protein immunoprecipitation O experiments O failed O to O establish O any O putative O association O between O HOT1 B-Protein and O shelterin B-Complex complex O members O . O Likewise O , O we O could O not O retrieve O HOT1 B-Protein after O a O POT1 B-Protein - O IP O , O which O is O in O agreement O with O several O other O studies O that O searched O for O shelterin B-Complex - O associated O factors O either O by O immunoprecipitation O or O by O bimolecular O fluorescence O complementation O . O Our O comparison O of O the O cocrystal O structures O of O telomeric O DNA O with O the O HOT1 B-Protein , O TRF1 B-Protein and O TRF2 B-Protein homeodomains O indicates O that O while O HOT1 B-Protein is O shifted O ' O down O ' O in O 5 O ' O - O - O > O 3 O ' O direction O by O one O base O towards O the O following O telomeric O repeat O , O the O binding O sites O are O largely O overlapping O . O Hence O , O it O is O intriguing O how O these O proteins O coexist O at O telomeres O , O how O they O compete O for O binding O sites O and O whether O the O shelterin B-Complex proteins O are O found O interspersed O on O telomeres O or O whether O there O are O discrete O , O mutually O exclusive O patches O along O the O telomeric O tracts O . O This O might O further O contribute O to O answering O the O question O how O HOT1 B-Protein is O selectively O restricted O to O a O subset O of O telomeres O . O Synthesis O , O in O vitro O , O and O in O cell O studies O of O a O new O series O of O [ O indoline B-Chemical - I-Chemical 3 I-Chemical , I-Chemical 2 I-Chemical ' I-Chemical - I-Chemical thiazolidine I-Chemical ] O - O based O p53 B-Protein modulators O . O Analogues O of O the O previously O described O spiro B-Chemical [ I-Chemical imidazo I-Chemical [ I-Chemical 1 I-Chemical , I-Chemical 5 I-Chemical - I-Chemical c I-Chemical ] I-Chemical thiazole I-Chemical - I-Chemical 3 I-Chemical , I-Chemical 3 I-Chemical ' I-Chemical - I-Chemical indoline I-Chemical ] I-Chemical - I-Chemical 2 I-Chemical ' I-Chemical , I-Chemical 5 I-Chemical , I-Chemical 7 I-Chemical ( I-Chemical 6H I-Chemical , I-Chemical 7aH I-Chemical ) I-Chemical - I-Chemical trione I-Chemical p53 B-Protein modulators O were O prepared O to O explore O new O structural O requirements O at O the O thiazolidine B-Chemical domain O for O the O antiproliferative O activity O and O p53 B-Protein modulation O . O In O cell O , O antiproliferative O activity O was O evaluated O against O two O human O tumor O cell O lines O . O Derivative O 5 B-Chemical - I-Chemical bromo I-Chemical - I-Chemical 3 I-Chemical ' I-Chemical - I-Chemical ( I-Chemical cyclohexane I-Chemical carbonyl I-Chemical ) I-Chemical - I-Chemical 1 I-Chemical - I-Chemical methyl I-Chemical - I-Chemical 2 I-Chemical - I-Chemical oxospiro I-Chemical [ I-Chemical indoline I-Chemical - I-Chemical 3 I-Chemical , I-Chemical 2 I-Chemical ' I-Chemical - I-Chemical thiazolidine I-Chemical ] I-Chemical ( O 4n B-Chemical ) O emerged O as O the O most O potent O compound O of O this O series O , O inhibiting O in O vitro O 30 O % O of O p53 B-Protein - O MDM2 B-Protein interaction O at O 5 O muM O and O the O cell O growth O of O different O human O tumor O cells O at O nanomolar O concentrations O . O Docking O studies O confirmed O the O interactions O of O 4n B-Chemical with O the O well O - O known O Trp23 O and O Phe19 O clefts O , O explaining O the O reasons O for O its O binding O affinity O for O MDM2 B-Protein . O 4n B-Chemical at O 50 O nM O is O capable O of O inducing O the O accumulation O of O p53 B-Protein protein O , O inducing O significant O apoptotic O cell O death O without O affecting O the O cell O cycle O progression O . O Comparative O studies O using O nutlin B-Chemical in O the O same O cellular O system O confirm O the O potential O of O 4n B-Chemical as O a O tool O for O increasing O understanding O of O the O process O involved O in O the O nontranscriptional O proapoptotic O activities O of O p53 B-Protein . O Negative O regulation O of O RIG B-Protein - I-Protein I I-Protein - O mediated O antiviral O signaling O by O TRK B-Protein - I-Protein fused I-Protein gene I-Protein ( O TFG B-Protein ) O protein O . O RIG B-Protein - I-Protein I I-Protein ( O retinoic B-Protein acid I-Protein inducible I-Protein gene I-Protein I I-Protein ) O - O mediated O antiviral O signaling O serves O as O the O first O line O of O defense O against O viral O infection O . O Upon O detection O of O viral O RNA O , O RIG B-Protein - I-Protein I I-Protein undergoes O TRIM25 B-Protein ( O tripartite B-Protein motif I-Protein protein I-Protein 25 I-Protein ) O - O mediated O K63 O - O linked O ubiquitination O , O leading O to O type B-Family I I-Family interferon I-Family ( O IFN O ) O production O . O In O this O study O , O we O demonstrate O that O TRK B-Protein - I-Protein fused I-Protein gene I-Protein ( O TFG B-Protein ) O protein O , O previously O identified O as O a O TRIM25 B-Protein - O interacting O protein O , O binds O TRIM25 B-Protein upon O virus O infection O and O negatively O regulates O RIG B-Protein - I-Protein I I-Protein - O mediated O type B-Family - I-Family I I-Family IFN I-Family signaling O . O RIG B-Protein - I-Protein I I-Protein - O mediated O IFN O production O and O nuclear B-Complex factor I-Complex ( I-Complex NF I-Complex ) I-Complex - I-Complex kappaB I-Complex signaling O pathways O were O upregulated O by O the O suppression O of O TFG B-Protein expression O . O Furthermore O , O vesicular O stomatitis O virus O ( O VSV O ) O replication O was O significantly O inhibited O by O small O inhibitory O hairpin O RNA O ( O shRNA O ) O - O mediated O knockdown O of O TFG B-Protein , O supporting O the O suppressive O role O of O TFG B-Protein in O RIG B-Protein - I-Protein I I-Protein - O mediated O antiviral O signaling O . O Interestingly O , O suppression O of O TFG B-Protein expression O increased O not O only O RIG B-Protein - I-Protein I I-Protein - O mediated O signaling O but O also O MAVS B-Protein ( O mitochondrial B-Protein antiviral I-Protein signaling I-Protein protein I-Protein ) O - O induced O signaling O , O suggesting O that O TFG B-Protein plays O a O pivotal O role O in O negative O regulation O of O RNA O - O sensing O , O RIG B-Family - I-Family I I-Family - I-Family like I-Family receptor I-Family ( O RLR B-Family ) O family O signaling O pathways O . O The O Vps33 B-Protein family O of O SM B-Family proteins O , O unlike O the O Sec1 B-Family / I-Family Munc18 I-Family , O Sly1 B-Protein , O and O Vps45 B-Protein families O , O does O not O appear O to O interact O with O the O N O - O peptides O of O Qa O - O SNAREs B-Family . O The O structural O basis O for O this O key O distinction O among O SM B-Family protein O families O can O be O understood O by O superimposing O domain O 1 O of O Vps33 B-Protein upon O that O of O other O , O N O - O peptide O - O binding O SM B-Family proteins O . O This O analysis O reveals O that O the O binding O site O normally O occupied O by O the O N O - O peptide O ' O s O conserved O Arg O side O chain O ( O Fig O . O 5A O ) O is O , O uniquely O in O the O case O of O Vps33 B-Protein , O filled O by O Arg O - O 115 O of O the O SM B-Family protein O itself O ( O Fig O . O 5B O ) O . O The O positioning O of O the O Arg O - O 115 O side O chain O is O reinforced O by O a O salt O - O bridge O formed O with O Asp O - O 120 O . O Also O blocked O - O and O only O in O Vps33 B-Protein - O is O the O pocket O that O normally O accommodates O a O conserved O hydrophobic O residue O located O four O residues O C O - O terminal O to O the O N O - O peptide O Arg O residue O ( O Fig O . O 5C O , O D O ) O . O In O this O case O a O displacement O of O the O Vps33 B-Protein backbone O , O relative O to O other O SM B-Family proteins O , O causes O the O side O chain O of O Leu O - O 129 O to O occupy O the O binding O pocket O . O Thus O , O both O of O the O pockets O that O in O other O SM B-Family proteins O accommodate O conserved O Qa O - O SNARE B-Family N O - O peptide O residues O are O missing O in O Vps33 B-Protein . O Likewise O , O the O N O - O terminal O region O of O the O relevant O Qa O - O SNARE B-Family Vam3 B-Protein lacks O the O sequence O determinants O - O including O the O conserved O Arg O - O found O in O the O N O - O peptides O of O the O Qa O - O SNAREs B-Family that O bind O SM B-Family proteins O . O Chondrosarcoma O ( O CS O ) O is O the O second O most O common O primary O malignant O bone O tumour O . O When O such O tumours O arise O as O solitary O lesions O in O the O medullary O cavity O ( O central O CS O ) O or O more O rarely O in O the O periosteum O , O ~ O 50 O % O harbour O either O a O somatic O IDH1 B-Protein ( O isocitrate B-Protein dehydrogenase I-Protein 1 I-Protein ) O or O IDH2 B-Protein heterozygous O mutation O . O In O a O minority O of O individuals O , O these O tumours O are O multiple O , O and O affected O individuals O are O at O risk O of O developing O other O neoplasms O , O including O spindle O cell O haemangiomas O , O and O high O - O grade O gliomas O / O secondary O glioblastomas O , O among O others O . O In O this O setting O , O the O mosaic O distribution O of O tumours O is O caused O by O somatic O early O post O - O zygotic O mutations O of O IDH1 B-Protein and O IDH2 B-Protein ( O ref O . O ) O . O The O same O mutations O have O been O previously O identified O in O ~ O 70 O % O of O sporadic O high O - O grade O gliomas O and O secondary O glioblastomas O , O ~ O 10 O % O of O acute O myeloid O leukemias O ( O AMLs O ) O and O cholangiocarcinomas O ( O CCs O ) O , O and O much O less O commonly O in O other O neoplasms O . O The O mutant O ( O mt O ) O IDH B-Protein enzyme O catalyses O the O reduction O of O alpha B-Chemical - I-Chemical ketoglutarate I-Chemical ( O alpha B-Chemical - I-Chemical KG I-Chemical ) O to O D B-Chemical - I-Chemical 2 I-Chemical - I-Chemical hydroxyglutarate I-Chemical ( O 2 B-Chemical - I-Chemical HG I-Chemical ) O , O an O oncometabolite O affecting O the O activity O of O alpha B-Chemical - I-Chemical KG I-Chemical - O dependent O dioxygenases O : O these O events O affect O a O number O of O cellular O responses O , O and O have O been O shown O to O induce O CpG O island O DNA O hypermethylation O in O low O - O grade O gliomas O ( O LGGs O ) O , O CCs O and O AMLs O harbouring O IDH1 B-Protein and O IDH2 B-Protein mutations O . O The O TET O dioxygenases O are O responsible O for O the O conversion O of O 5 B-Chemical - I-Chemical methylcytosine I-Chemical to O 5 B-Chemical - I-Chemical hydroxymethylcytosine I-Chemical ( O 5hmC B-Chemical ) O , O an O intermediate O metabolite O in O the O recently O discovered O active O demethylation O pathway O , O and O it O is O possible O that O mt O IDH1 B-Protein enzyme O mediates O the O observed O hypermethylation O phenotype O through O inhibition O of O TET O by O 2 O - O HG O . O To O screen O for O correct O formation O and O localization O of O the O septum O , O we O stained O the O cells O with O Calcofluor B-Chemical , O together O with O DAPI B-Chemical to O visualize O the O nuclei O . O Figure O 3D O shows O the O fraction O of O normally O dividing O , O septated O cells O with O normal O septa O at O 25degreesC O ( O t O = O 0 O ) O , O and O at O times O after O transfer O to O the O restrictive O temperature O for O overexpression O of O three O of O the O mutants O . O At O t O = O 0 O nearly O all O septa O were O normal O . O After O 4 O hours O , O ~ O 30 O % O of O the O cells O with O ' O vector O only O ' O had O normal O septa O . O In O cells O over O - O expressing O wildtype O or O mutant O Cdc8p B-Protein , O after O 4 O hours O there O were O ~ O 50 O % O normal O septa O reflecting O the O residual O ts O phenotype O ( O multiple O , O poorly O organized O septa O and O abnormal O morphologies O ) O . O After O 17 O - O 24 O hours O , O almost O no O cdc8 B-Protein - O 27 O cells O were O normal O ; O most O had O > O 2 O nuclei O , O abnormal O and O multiple O septa O , O and O were O branched O . O By O 17 O hours O most O cells O expressing O wildtype O and O E104A O had O normal O septa O while O D16A O and O R121A O . O D131A O . O E138A O had O an O elevated O fraction O of O cells O with O an O abnormal O morphology O and O number O of O septa O ( O Figure O 3D O ) O . O Certain O other O mutants O divided O normally O based O on O nuclear O number O , O yet O had O a O higher O percentage O of O abnormalities O including O shape O , O position O or O number O of O septa O ( O E6A O , O D16A O , O E107A O . O R110A O , O for O example O ; O Table O S3 O in O File O S1 O ) O . O Two O mutants O ( O E82A O , O V114S O . O E117A O . O H118A O ) O were O so O variable O from O one O transformation O to O the O next O that O we O did O not O report O values O in O Table O S3 O in O File O S1 O . O In O general O , O however O , O overexpression O of O mutant O Cdc8p B-Protein had O poor O penetrance O ; O most O cells O were O morphologically O similar O to O wildtype O . O ( O A O ) O AZI1 B-Protein - O myc B-Protein protein O levels O are O reduced O in O mpk3 B-Protein mutants O . O Proteins O were O extracted O from O seedlings O of O 35S O : O : O AZI1 B-Protein and O from O 35S O : O : O AZI1 B-Protein / O mpk3 B-Protein ( O homozygous O crossings O of O line O 35S O : O : O AZI1 B-Protein _ O 11 O . O 8 O with O mpk3 B-Protein ) O . O AZI1 B-Protein - O myc B-Protein expression O was O detected O by O immunoblotting O with O an O antibody O directed O against O the O myc B-Protein epitope O tag O . O Protein O loading O was O visualized O by O subsequent O Coomassie B-Chemical Blue I-Chemical staining O of O the O membrane O ( O CBB B-Chemical ) O . O Homozygous O 35S O : O : O AZI1 B-Protein / O mpk3 B-Protein lines O ( O three O are O shown O ) O have O consistently O less O AZI1 B-Protein - O myc B-Protein protein O . O The O experiment O was O repeated O three O times O , O with O similar O results O . O The O ability O to O form O homodimers O is O essential O for O RDM1 B-Protein to O function O in O RNA O - O directed O DNA O methylation O . O RDM1 B-Protein ( O RNA B-Protein - I-Protein DIRECTED I-Protein DNA I-Protein METHYLATION1 I-Protein ) O is O a O small O plant O - O specific O protein O required O for O RNA O - O directed O DNA O methylation O ( O RdDM O ) O . O RDM1 B-Protein interacts O with O RNA B-Complex polymerase I-Complex II I-Complex ( O Pol B-Complex II I-Complex ) O , O ARGONAUTE4 B-Protein ( O AGO4 B-Protein ) O , O and O the O de O novo O DNA O methyltransferase O DOMAINS B-Protein REARRANGED I-Protein METHYLTRANSFERASE2 I-Protein ( O DRM2 B-Protein ) O and O binds O to O methylated O single O stranded O DNA O . O As O the O only O protein O identified O so O far O that O interacts O directly O with O DRM2 B-Protein , O RDM1 B-Protein plays O a O pivotal O role O in O the O RdDM O mechanism O by O linking O the O de O novo O DNA O methyltransferase O activity O to O AGO4 B-Protein , O which O binds O short O interfering O RNAs O ( O siRNAs O ) O that O presumably O base O - O pair O with O Pol B-Complex II I-Complex or O Pol B-Complex V I-Complex scaffold O transcripts O synthesized O at O target O loci O . O RDM1 B-Protein also O acts O together O with O the O chromatin O remodeler O DEFECTIVE B-Protein IN I-Protein RNA I-Protein - I-Protein DIRECTED I-Protein DNA I-Protein METHYLATION1 I-Protein ( O DRD1 B-Protein ) O and O the O structural O - O maintenance O - O of O - O chromosomes O solo O hinge O protein O DEFECTIVE B-Protein IN I-Protein MERISTEM I-Protein SILENCING3 I-Protein ( O DMS3 B-Protein ) O to O form O the O DDR B-Complex complex O , O which O facilitates O synthesis O of O Pol B-Complex V I-Complex scaffold O transcripts O . O The O manner O in O which O RDM1 B-Protein acts O in O both O the O DDR B-Complex complex O and O as O a O factor O bridging O DRM2 B-Protein and O AGO4 B-Protein remains O unclear O . O RDM1 B-Protein contains O no O known O protein O domains O but O a O prior O structural O analysis O suggested O distinct O regions O that O create O a O hydrophobic O pocket O and O promote O homodimer O formation O , O respectively O . O We O have O tested O several O mutated O forms O of O RDM1 B-Protein altered O in O the O predicted O pocket O and O dimerization O regions O for O their O ability O to O complement O defects O in O RdDM O and O transcriptional O gene O silencing O , O support O synthesis O of O Pol B-Complex V I-Complex transcripts O , O form O homodimers O , O and O interact O with O DMS3 B-Protein . O Our O results O indicate O that O the O ability O to O form O homodimers O is O essential O for O RDM1 B-Protein to O function O fully O in O the O RdDM O pathway O and O may O be O particularly O important O during O the O de O novo O methylation O step O . O Interestingly O , O when O we O stimulated O the O nerve O at O a O high O frequency O ( O 10 O Hz O ) O for O a O prolonged O period O ( O 10 O min O ) O , O mnb1 B-Protein showed O significantly O faster O rundown O than O controls O ( O Fig O . O 3e O - O f O ) O . O This O result O suggests O that O Mnb B-Protein promotes O efficient O synaptic O vesicle O recycling O during O conditions O of O high O activity O . O Presynaptic O overexpression O of O mnb B-Protein - O F O alone O did O not O affect O basal O synaptic O transmission O and O responses O to O high O frequency O stimulation O as O compared O to O the O control O , O but O restored O maintenance O of O synaptic O transmission O when O expressed O in O mnb1 B-Protein mutant O background O ( O Fig O . O 3a O - O f O ) O . O Together O , O these O data O indicate O neuronal O Mnb B-Protein is O required O for O rapid O synaptic O vesicle O recycling O . O The O PTK7 B-Protein - O related O transmembrane O proteins O off B-Protein - I-Protein track I-Protein and O off B-Protein - I-Protein track I-Protein 2 I-Protein are O co O - O receptors O for O Drosophila O Wnt2 B-Protein required O for O male O fertility O . O Wnt B-Family proteins O regulate O many O developmental O processes O and O are O required O for O tissue O homeostasis O in O adult O animals O . O The O cellular O responses O to O Wnts B-Family are O manifold O and O are O determined O by O the O respective O Wnt B-Family ligand O and O its O specific O receptor O complex O in O the O plasma O membrane O . O Wnt B-Family receptor O complexes O contain O a O member O of O the O Frizzled B-Family family O of O serpentine B-Protein receptors O and O a O co O - O receptor O , O which O commonly O is O a O single O - O pass O transmembrane O protein O . O Vertebrate O protein B-Protein tyrosine I-Protein kinase I-Protein 7 I-Protein ( O PTK7 B-Protein ) O was O identified O as O a O Wnt B-Family co O - O receptor O required O for O control O of O planar O cell O polarity O ( O PCP O ) O in O frogs O and O mice O . O We O found O that O flies O homozygous O for O a O complete O knock O - O out O of O the O Drosophila O PTK7 B-Protein homolog O off B-Protein track I-Protein ( O otk B-Protein ) O are O viable O and O fertile O and O do O not O show O PCP O phenotypes O . O We O discovered O an O otk B-Protein paralog O ( O otk2 B-Protein , O CG8964 B-Protein ) O , O which O is O co O - O expressed O with O otk B-Protein throughout O embryonic O and O larval O development O . O Otk B-Protein and O Otk2 B-Protein bind O to O each O other O and O form O complexes O with O Frizzled B-Family , O Frizzled2 B-Protein and O Wnt2 B-Protein , O pointing O to O a O function O as O Wnt B-Family co O - O receptors O . O Flies O lacking O both O otk B-Protein and O otk2 B-Protein are O viable O but O male O sterile O due O to O defective O morphogenesis O of O the O ejaculatory O duct O . O Overexpression O of O Otk B-Protein causes O female O sterility O due O to O malformation O of O the O oviduct O , O indicating O that O Otk B-Protein and O Otk2 B-Protein are O specifically O involved O in O the O sexually O dimorphic O development O of O the O genital O tract O . O Structural O efforts O have O attempted O to O elucidate O the O mechanistic O details O of O signal O transduction O spanning O several O domains O from O the O periplasmic O sensors O to O the O cytoplasmic O DHp O domain O , O and O numerous O structures O have O been O reported O . O Crystal O structures O are O now O available O for O multiple O domains O of O two O - O component O and O chemotaxis O systems O , O including O a O structure O of O the O periplasmic O sensor O domain O of O PhoQ B-Protein . O NMR O and O X O - O ray O structures O have O been O solved O for O HAMP B-Protein domains O as O well O as O TM O regions O . O Many O recent O multi O - O domain O crystal O structures O give O us O detailed O view O of O the O connections O between O cytoplasmic O domains O . O A O full O length O structure O of O an O engineered O , O cytoplasmic O two O - O component O sensor O ( O lacking O a O TM O domain O ) O was O determined O , O as O was O the O structure O of O the O cytoplasmic O region O of O VicK B-Protein , O from O Streptococcus O mutans O . O Despite O these O advances O , O several O competing O proposals O still O remain O for O a O mechanism O of O transmembrane O signaling O . O A O hallmark O property O of O circadian O rhythms O is O that O the O period O length O is O very O constant O over O a O wide O range O of O physiologically O relevant O temperatures O , O termed O temperature O compensation O . O To O investigate O whether O phosphorylation O of O dCLK B-Protein might O have O a O role O in O temperature O compensation O , O we O analyzed O behavioral O rhythms O at O three O standard O temperatures O ( O i O . O e O . O , O 18degrees O , O 25degrees O and O 29degreesC O ) O . O Although O we O noted O a O decrease O in O rhythmicity O for O dClk B-Protein - O 15A O ; O Clk O out O flies O at O 29degreesC O , O the O periods O were O quite O similar O over O the O temperature O range O tested O ( O Table O 2 O ) O , O suggesting O that O global O phosphorylation O of O dCLK B-Protein does O not O play O a O major O role O in O temperature O compensation O . O Emerging O optical O tools O allow O much O finer O control O and O observation O of O cellular O processes O using O light O , O an O actuator O that O can O be O delivered O immediately O and O with O subcellular O spatial O resolution O . O Optogenetic O actuators O allow O light O control O of O specific O processes O such O as O neuronal O firing O , O but O more O general O tools O have O also O been O developed O to O manipulate O protein O transcription O , O protein O - O protein O interactions O , O and O a O variety O of O other O cellular O biochemical O events O . O In O previous O work O , O we O developed O one O such O tool O , O a O system O using O the O Arabidopsis O flavoprotein B-Family cryptochrome B-Protein 2 I-Protein ( O CRY2 B-Protein ) O and O its O interacting O partner O CIB1 B-Protein to O inducibly O control O protein O interactions O with O light O . O While O these O proteins O do O not O interact O in O the O dark O , O blue O light O absorption O triggers O a O conformational O change O in O CRY2 B-Protein allowing O binding O to O CIB1 B-Protein that O is O reversible O after O several O minutes O ( O t O ( O 1 O / O 2 O ) O ~ O 5 O . O 5 O min O ) O . O When O attached O to O diverse O target O proteins O , O the O CRY2 B-Protein and O CIB1 B-Protein modules O allow O tight O light O regulation O of O target O protein O activity O . O A O HYL1 B-Protein promoter O region O ( O 1240 O bp O upstream O of O the O translation O start O site O ) O and O a O full O - O length O coding O sequence O ( O 1257 O bp O ) O were O amplified O from O Columbia O seedlings O . O Then O both O sequences O were O cloned O into O pCAMBIA1301 O binary O vectors O to O obtain O the O pHYL1 B-Protein : O : O HYL1 B-Protein constructs O . O Site O - O directed O mutagenesis O was O performed O , O and O the O primers O used O for O polymerase O chain O reactions O ( O PCRs O ) O are O listed O in O Supplementary O Table O S3 O . O The O cDNA O encoding O RCD1 B-Protein was O cloned O into O the O bait O vector O pGBT9 O and O the O Rap2 B-Protein . I-Protein 4a I-Protein cDNA O into O the O prey O vector O pACT2 O . O Double O transformants O of O the O yeast O strain O HF7c O showed O protein O - O protein O interaction O by O complementation O of O the O histidine O auxotrophy O of O the O yeast O strain O . O On O 20 O mM O 3 B-Chemical - I-Chemical AT I-Chemical , O which O increases O the O stringency O by O inhibiting O histidine O biosynthesis O , O the O double O transformant O grew O almost O as O good O as O the O commonly O used O positive O control O , O pVA O - O pTD1 O double O transformants O ( O Figure O 4 O left O ) O ( O Li O et O al O . O , O ) O . O The O pAct2 O - O Rap2 B-Protein . I-Protein 4a I-Protein - O empty O pGBT9 O double O transformants O ( O negative O control O ) O did O not O grow O . O Rap1GAP B-Protein is O a O member O of O a O family O of O GTPase B-Family - I-Family activating I-Family proteins I-Family ( O GAPs B-Family ) O that O specifically O stimulate O the O GTP B-Chemical hydrolysis O of O Rap1 B-Family GTPases I-Family . O Rap1 B-Protein is O one O of O the O Ras B-Family - I-Family like I-Family small I-Family GTPases I-Family that O are O critical O players O in O signaling O pathways O that O control O cell O growth O , O migration O , O and O differentiation O . O Rap1 B-Protein shuttles O between O an O inactive O GDP B-Chemical - O and O active O GTP B-Chemical - O bound O form O . O Activation O of O Rap1 B-Protein ( O Rap1 B-Protein - O GTP B-Chemical ) O is O mediated O by O guanine O nucleotide O exchange O factors O ( O GEFs O ) O , O including O C3G B-Protein , O PDZ B-Protein - I-Protein GEF I-Protein , O Epac B-Protein , O and O CalDAG B-Protein . O Inactivation O of O Rap1 B-Protein is O mediated O by O GTPase B-Family activating I-Family proteins I-Family ( O GAPs B-Family ) O , O including O Rap1GAP B-Protein and O Rap1GAP2 B-Protein , O SPA B-Protein - I-Protein 1 I-Protein / O SIPA1 B-Protein and O SIPA1L1 B-Protein / O SPAR B-Protein . O ( O A O ) O Hierarchical O clustering O of O No O - O 0 O and O slh1 B-Protein temperature O - O dependent O differential O gene O expression O . O Fold O - O change O values O of O 5611 O genes O ( O differentially O expressed O at O least O in O one O time O point O ) O are O shown O . O The O numbers O on O top O of O the O heat O map O indicate O the O time O ( O h O ) O after O temperature O shift O . O Black O , O red O and O green O colours O indicate O no O change O , O up O - O regulated O and O down O - O regulated O , O respectively O . O ( O B O ) O qRT O - O PCR O analysis O of O selected O RRS1SLH1 B-Protein - O regulated O genes O following O the O temperature O shift O ( O 28degreesC O to O 19degreesC O ) O in O 4 O week O - O old O No O - O 0 O and O slh1 B-Protein plants O . O Transcript O accumulation O is O presented O relative O to O No O - O 0 O before O temperature O shift O ( O 28degreesC O ) O . O Our O previous O proteomic O screens O identified O a O number O of O proteins O associated O with O AP B-Protein - I-Protein 3 I-Protein that O could O coordinate O both O the O interaction O of O SEPT6 B-Protein or O SEPT7 B-Protein with O F B-Protein - I-Protein actin I-Protein and O that O of O AP B-Protein - I-Protein 3 I-Protein with O membranes O or O could O regulate O a O switch O from O F B-Protein - I-Protein actin I-Protein to O microtubules O during O MVB O biogenesis O . O Their O function O can O now O be O tested O in O the O light O of O our O current O findings O . O Structure O of O the O Kti11 B-Protein / O Kti13 B-Protein heterodimer O and O its O double O role O in O modifications O of O tRNA O and O eukaryotic B-Protein elongation I-Protein factor I-Protein 2 I-Protein . O The O small O , O highly O conserved O Kti11 B-Protein alias O Dph3 B-Protein protein O encoded O by O the O Kluyveromyces B-Protein lactis I-Protein killer I-Protein toxin I-Protein insensitive I-Protein gene O KTI11 B-Protein / O DPH3 B-Protein is O involved O in O the O diphthamide O modification O of O eukaryotic B-Protein elongation I-Protein factor I-Protein 2 I-Protein and O , O together O with O Kti13 B-Protein , O in O Elongator O - O dependent O tRNA O wobble O base O modifications O , O thereby O affecting O the O speed O and O accuracy O of O protein O biosynthesis O through O two O distinct O mechanisms O . O We O have O solved O the O crystal O structures O of O Saccharomyces O cerevisiae O Kti13 B-Protein and O the O Kti11 B-Protein / O Kti13 B-Protein heterodimer O at O 2 O . O 4 O and O 2 O . O 9 O Aa O resolution O , O respectively O , O and O validated O interacting O residues O through O mutational O analysis O in O vitro O and O in O vivo O . O We O show O that O metal O coordination O by O Kti11 B-Protein and O its O heterodimerization O with O Kti13 B-Protein are O essential O for O both O translational O control O mechanisms O . O Our O structural O and O functional O analyses O identify O Kti13 B-Protein as O an O additional O component O of O the O diphthamide O modification O pathway O and O provide O insight O into O the O molecular O mechanisms O that O allow O the O Kti11 B-Protein / O Kti13 B-Protein heterodimer O to O coregulate O two O consecutive O steps O in O ribosomal O protein O synthesis O . O elp1Delta B-Protein yeast O strains O expressing O myc B-Protein - O tagged O ELP2 B-Protein and O either O HA B-Protein - O tagged O ELP3 B-Protein ( O A O ) O or O ELP5 B-Protein ( O B O ) O were O transformed O with O either O empty O vector O or O plasmids O expressing O the O indicated O ELP1 B-Protein alleles O ( O Table O S2 O ) O . O Elp2 B-Protein - O myc B-Protein was O immunoprecipitated O from O extracts O and O immunoprecipitates O were O examined O by O Western O blotting O with O anti O - O myc B-Protein and O anti O - O HA B-Protein antibodies O to O detect O co O - O immunoprecipitated O Elp1 B-Protein , O Elp3 B-Protein and O Elp5 B-Protein as O indicated O . O Note O that O Elp1 B-Protein runs O as O a O doublet O as O observed O previously O . O CDC28 B-Protein phosphorylates O Cac1p B-Protein and O regulates O the O association O of O chromatin B-Protein assembly I-Protein factor I-Protein I I-Protein with O chromatin O . O Chromatin B-Protein Assembly I-Protein Factor I-Protein I I-Protein ( O CAF B-Protein - I-Protein I I-Protein ) O plays O a O key O role O in O the O replication O - O coupled O assembly O of O nucleosomes O . O It O is O expected O that O its O function O is O linked O to O the O regulation O of O the O cell O cycle O , O but O little O detail O is O available O . O Current O models O suggest O that O CAF B-Protein - I-Protein I I-Protein is O recruited O to O replication O forks O and O to O chromatin O via O an O interaction O between O its O Cac1p B-Protein subunit O and O the O replication O sliding O clamp O , O PCNA B-Protein , O and O that O this O interaction O is O stimulated O by O the O kinase O CDC7 B-Protein . O Here O we O show O that O another O kinase O , O CDC28 B-Protein , O phosphorylates O Cac1p B-Protein on O serines O 94 O and O 515 O in O early O S O phase O and O regulates O its O association O with O chromatin O , O but O not O its O association O with O PCNA B-Protein . O Mutations O in O the O Cac1p B-Protein - O phosphorylation O sites O of O CDC28 B-Protein but O not O of O CDC7 B-Protein substantially O reduce O the O in O vivo O phosphorylation O of O Cac1p B-Protein . O However O , O mutations O in O the O putative O CDC7 B-Protein target O sites O on O Cac1p B-Protein reduce O its O stability O . O The O association O of O CAF B-Protein - I-Protein I I-Protein with O chromatin O is O impaired O in O a O cdc28 B-Protein - O 1 O mutant O and O to O a O lesser O extent O in O a O cdc7 B-Protein - O 1 O mutant O . O In O addition O , O mutations O in O the O Cac1p B-Protein - O phosphorylation O sites O by O both O CDC28 B-Protein and O CDC7 B-Protein reduce O gene O silencing O at O the O telomeres O . O We O propose O that O this O phosphorylation O represents O a O regulatory O step O in O the O recruitment O of O CAF B-Protein - I-Protein I I-Protein to O chromatin O in O early O S O phase O that O is O distinct O from O the O association O of O CAF B-Protein - I-Protein I I-Protein with O PCNA B-Protein . O Hence O , O we O implicate O CDC28 B-Protein in O the O regulation O of O chromatin O reassembly O during O DNA O replication O . O These O findings O provide O novel O mechanistic O insights O on O the O links O between O cell O - O cycle O regulation O , O DNA O replication O and O chromatin O reassembly O . O Concerted O and O differential O actions O of O two O enzymatic O domains O underlie O Rad5 B-Protein contributions O to O DNA O damage O tolerance O . O Many O genome O maintenance O factors O have O multiple O enzymatic O activities O . O In O most O cases O , O how O their O distinct O activities O functionally O relate O with O each O other O is O unclear O . O Here O we O examined O the O conserved O budding O yeast O Rad5 B-Protein protein O that O has O both O ubiquitin O ligase O and O DNA O helicase O activities O . O The O Rad5 B-Protein ubiquitin O ligase O activity O mediates O PCNA B-Protein poly O - O ubiquitination O and O subsequently O recombination O - O based O DNA O lesion O tolerance O . O Interestingly O , O the O ligase O domain O is O embedded O in O a O larger O helicase O domain O comprising O seven O consensus O motifs O . O How O features O of O the O helicase O domain O influence O ligase O function O is O controversial O . O To O clarify O this O issue O , O we O use O genetic O , O 2D O gel O and O biochemical O analyses O and O show O that O a O Rad5 B-Protein helicase O motif O important O for O ATP O binding O is O also O required O for O PCNA B-Protein poly O - O ubiquitination O and O recombination O - O based O lesion O tolerance O . O We O determine O that O this O requirement O is O due O to O a O previously O unrecognized O contribution O of O the O motif O to O the O PCNA B-Protein and O ubiquitination O enzyme O interaction O , O and O not O due O to O its O canonical O role O in O supporting O helicase O activity O . O We O further O show O that O Rad5 B-Protein ' O s O helicase O - O mediated O contribution O to O replication O stress O survival O is O separable O from O recombination O . O These O findings O delineate O how O two O Rad5 B-Protein enzymatic O domains O concertedly O influence O PCNA B-Protein modification O , O and O unveil O their O discrete O contributions O to O stress O tolerance O . O To O gain O new O functional O insights O into O Brc1 B-Protein we O carried O out O an O E O - O MAP O analysis O to O quantify O the O genetic O interactions O between O brc1Delta B-Protein and O a O S O . O pombe O gene O deletion O library O of O nonessential O genes O . O E O - O MAP O values O were O determined O with O a O simple O growth O phenotype O that O measures O negative O ( O aggravating O ) O interactions O , O such O as O synthetic O sick O / O lethal O ( O SSL O ) O interactions O , O as O well O as O positive O ( O alleviating O ) O interactions O in O which O the O double O mutant O is O healthier O than O would O be O expected O based O on O the O growth O of O the O two O single O mutants O . O An O SSL O interaction O often O identifies O proteins O that O function O in O distinct O but O parallel O pathways O , O whereas O a O positive O interaction O score O may O indicate O either O suppression O or O masking O effects O , O in O which O loss O of O one O gene O masks O the O effect O of O losing O another O , O as O seen O when O two O proteins O act O together O in O a O common O complex O or O pathway O . O The O RGMCND O - O BMP2 B-Protein complex O was O solved O by O molecular O replacement O in O PHASER O using O the O structure O of O the O disulfide O bonded O BMP2 B-Protein dimer O ( O PDB O 3BMP B-Complex ) O as O a O search O model O . O Extra O electron O density O for O two O molecules O of O RGMCND O in O the O asymmetric O unit O was O immediately O discernible O after O density O modification O in O PARROT O ( O Supplementary O Fig O . O 2a O , O b O ) O . O The O RGMC B-Protein polypeptide O chain O was O traced O using O iterative O rounds O of O BUCCANEER O , O manual O building O in O COOT O and O refinement O in O autoBUSTER O and O PHENIX O . O This O resulted O in O a O well O - O defined O model O for O the O RGMCND O - O BMP2 B-Protein complex O that O included O two O molecules O of O RGMC B-Protein ( O residues O Q36 O - O P129 O ) O bound O to O a O disulfide O linked O BMP2 B-Protein dimer O ( O residues O K293 O - O R396 O ) O ( O Supplementary O Fig O . O 2c O ) O . O The O RGMAND O - O BMP2 B-Protein and O RGMBND O - O BMP2 B-Protein complexes O were O solved O by O molecular O replacement O using O PHASER O with O the O RGMCND O - O BMP2 B-Protein complex O as O a O search O model O . O Molecular O replacement O with O PHASER O was O applied O to O solve O the O BMP2 B-Protein - O eRGMB O - O NEO1FN56 O structure O using O the O RGMBND O - O BMP2 B-Protein Form O 1 O ( O from O this O study O ) O and O the O NEO1FN56 O - O RGMB B-Protein ( O PDB O ID O . O 4BQ6 B-Complex ) O structures O . O The O complexes O were O refined O using O autoBUSTER O and O PHENIX O and O , O where O applicable O , O non O - O crystallographic O restraints O were O applied O . O For O the O BMP2 B-Protein - O eRGMB O - O NEO1FN56 O structure O target O weight O refinement O using O the O individual O high O resolution O structures O of O the O BMP2 B-Protein , O RGMBND O , O eRGMB O , O NEO1FN5 O and O NEO1FN6 O domains O as O targets O was O applied O . O Crystallographic O statistics O are O given O in O Table O 1 O . O Stereochemical O properties O were O assessed O by O MOLPROBITY O . O Superpositions O were O calculated O using O the O program O COOT O and O electrostatic O potentials O were O generated O using O APBS O as O implemented O in O PYMOL O . O Buried O surface O areas O of O protein O - O protein O interactions O were O calculated O using O the O PISA O webserver O for O a O probe O radius O of O 1 O . O 4 O Aa O . O The O first O crystal O structures O of O a O complete O ECF B-Complex transporter I-Complex revealed O the O subunits O associated O in O a O 1A B-Protein ' I-Protein : O 1A B-Protein : O 1T B-Protein : O 1S B-Protein quaternary O architecture O as O anticipated O from O solution O studies O on O ECF B-Complex transporters I-Complex from O L O . O lactis O . O The O nucleotide O - O free O EcfAA B-Protein ' I-Protein ATPase O subunits O were O observed O in O an O open O conformation O . O EcfT B-Protein displayed O a O novel O L O - O shaped O fold O in O which O a O parallel O bundle O of O five O TM O helices O is O roughly O perpendicular O to O three O cytoplasmic O helices O ( O CH1 O - O 3 O ) O . O The O S B-Protein subunit O in O each O structure O interacts O exclusively O with O EcfT B-Protein . O Unexpectedly O , O the O substrate O - O binding O site O is O facing O the O cytoplasm O , O as O if O the O entire O EcfS B-Protein subunit O has O been O " O toppled O " O from O an O extracellular O - O facing O , O substrate O - O capturing O orientation O . O As O a O result O , O these O crystal O structures O may O have O captured O a O post O - O translocation O state O in O which O nucleotides O had O dissociated O and O the O transport O substrate O was O released O into O the O cytoplasm O . O If O TMEM106B B-Protein associates O with O CHMP2B B-Protein , O what O is O its O role O in O the O ESCRT B-Complex - O involved O endolysosomal O pathway O ? O Because O TMEM106B B-Protein associates O with O CHMP2B B-Protein in O endosomal O pathways O and O functions O in O lysosomes O , O TMEM106B B-Protein might O be O involved O in O autophagy O with O reference O to O its O association O with O CHMP2B B-Protein . O In O order O to O investigate O this O question O , O we O examined O whether O the O expression O of O the O TMEM106B B-Protein variants O affected O autophagic O flux O . O In O order O to O measure O autophagic O flux O , O the O levels O of O microtubule B-Protein - I-Protein associated I-Protein protein I-Protein 1A I-Protein / I-Protein 1B I-Protein - I-Protein light I-Protein chain I-Protein 3 I-Protein ( I-Protein LC3 I-Protein ) I-Protein - I-Protein II I-Protein in O cells O expressing O T185 O or O S185 O in O the O presence O or O absence O of O the O lysosomotrophic O reagent O ammonium B-Chemical chloride I-Chemical ( O NH4Cl B-Chemical ) O were O quantified O in O a O western O blot O analysis O . O As O shown O in O Fig O . O 2e O - O f O , O cells O expressing O T185 O showed O a O slight O reduction O in O autophagic O flux O compared O to O cells O expressing O S185 O . O These O results O suggested O that O the O enhanced O sequestration O of O CHMP2B B-Protein might O reduce O autophagic O flux O . O Morpholino B-Protein oligonucleotides O , O mRNA O , O and O plasmids O were O injected O into O two O animal O dorsal O blastomeres O at O the O eight O - O cell O stage O for O observation O of O embryo O phenotypes O and O RT O - O PCR O analysis O , O into O four O animal O blastomeres O for O western O blot O analysis O , O or O into O two O ventral O blastomeres O at O the O four O - O cell O stage O for O RT O - O PCR O analysis O . O The O cytoplasmic O and O nuclear O fractions O were O prepared O as O described O with O modifications O 23 O . O Quantitative O RT O - O PCR O analysis O : O Total O RNA O was O prepared O using O TRIzol O ( O Invitrogen O , O Carlsbad O , O CA O , O USA O ) O from O the O injected O region O at O the O gastrula O or O neurula O stage O . O cDNA O synthesis O was O carried O out O using O Moloney O murine O leukemia O virus O reverse O transcriptase O ( O Invitrogen O ) O . O The O sequences O of O the O primer O pairs O were O previously O reported O 24 O , O 25 O , O 26 O and O as O follows O : O xWDR26 O : O Forward O 5 O ' O - O ATGGCAACCTGCTTGACTCC O - O 3 O ' O ; O Reverse O 5 O ' O - O ACAGTACCGTCGTCAGAAGC O - O 3 O ' O . O hWDR26 B-Protein : O Forward O 5 O ' O - O CCGGAACTCGCCTGCTTGTC O - O 3 O ' O ; O Reverse O 5 O ' O - O TGACATCCTCATCTGACTGG O - O 3 O ' O . O Xnr3 B-Protein : O Forward O 5 O ' O - O CTTCTGCACTAGATTCTG O - O 3 O ' O ; O Reverse O 5 O ' O - O CAGCTTCTGGCCAAGACT O - O 3 O ' O . O Xtwn B-Protein : O Forward O 5 O ' O - O AACCCAAGAAGGCGACACTATC O - O 3 O ' O ; O Reverse O 5 O ' O - O GTGCCGATGGTAGGAAATGATC O - O 3 O ' O . O Xenopus B-Protein embryonic I-Protein ornithine I-Protein decarboxylase I-Protein ( O xODC B-Protein ) O was O used O for O normalization O of O cDNA O samples O . O Molecular O mechanism O of O APC B-Complex / I-Complex C I-Complex activation O by O mitotic O phosphorylation O . O In O eukaryotes O , O the O anaphase B-Complex - I-Complex promoting I-Complex complex I-Complex ( O APC B-Complex / I-Complex C I-Complex , O also O known O as O the O cyclosome B-Complex ) O regulates O the O ubiquitin B-Protein - O dependent O proteolysis O of O specific O cell O - O cycle O proteins O to O coordinate O chromosome O segregation O in O mitosis O and O entry O into O the O G1 O phase O . O The O catalytic O activity O of O the O APC B-Complex / I-Complex C I-Complex and O its O ability O to O specify O the O destruction O of O particular O proteins O at O different O phases O of O the O cell O cycle O are O controlled O by O its O interaction O with O two O structurally O related O coactivator O subunits O , O Cdc20 B-Protein and O Cdh1 B-Protein . O Coactivators O recognize O substrate O degrons O , O and O enhance O the O affinity O of O the O APC B-Complex / I-Complex C I-Complex for O its O cognate O E2 B-Family ( O refs O 4 O - O 6 O ) O . O During O mitosis O , O cyclin B-Family - I-Family dependent I-Family kinase I-Family ( O Cdk B-Family ) O and O polo B-Protein - I-Protein like I-Protein kinase I-Protein ( O Plk B-Protein ) O control O Cdc20 B-Protein - O and O Cdh1 B-Protein - O mediated O activation O of O the O APC B-Complex / I-Complex C I-Complex . O Hyperphosphorylation O of O APC B-Complex / I-Complex C I-Complex subunits O , O notably O Apc1 B-Protein and O Apc3 B-Protein , O is O required O for O Cdc20 B-Protein to O activate O the O APC B-Complex / I-Complex C I-Complex , O whereas O phosphorylation O of O Cdh1 B-Protein prevents O its O association O with O the O APC B-Complex / I-Complex C I-Complex . O Since O both O coactivators O associate O with O the O APC B-Complex / I-Complex C I-Complex through O their O common O C O - O box O and O Ile O - O Arg O tail O motifs O , O the O mechanism O underlying O this O differential O regulation O is O unclear O , O as O is O the O role O of O specific O APC B-Complex / I-Complex C I-Complex phosphorylation O sites O . O Here O , O using O cryo O - O electron O microscopy O and O biochemical O analysis O , O we O define O the O molecular O basis O of O how O phosphorylation O of O human O APC B-Complex / I-Complex C I-Complex allows O for O its O control O by O Cdc20 B-Protein . O An O auto O - O inhibitory O segment O of O Apc1 B-Protein acts O as O a O molecular O switch O that O in O apo O unphosphorylated O APC B-Complex / I-Complex C I-Complex interacts O with O the O C O - O box O binding O site O and O obstructs O engagement O of O Cdc20 B-Protein . O Phosphorylation O of O the O auto O - O inhibitory O segment O displaces O it O from O the O C O - O box O - O binding O site O . O Efficient O phosphorylation O of O the O auto O - O inhibitory O segment O , O and O thus O relief O of O auto O - O inhibition O , O requires O the O recruitment O of O Cdk B-Family - O cyclin B-Family in O complex O with O a O Cdk B-Family regulatory O subunit O ( O Cks O ) O to O a O hyperphosphorylated O loop O of O Apc3 B-Protein . O We O also O find O that O the O small O - O molecule O inhibitor O , O tosyl B-Chemical - I-Chemical l I-Chemical - I-Chemical arginine I-Chemical methyl I-Chemical ester I-Chemical , O preferentially O suppresses O APC B-Complex / I-Complex C I-Complex ( O Cdc20 B-Protein ) O rather O than O APC B-Complex / I-Complex C I-Complex ( O Cdh1 B-Protein ) O , O and O interacts O with O the O binding O sites O of O both O the O C O - O box O and O Ile O - O Arg O tail O motifs O . O Our O results O reveal O the O mechanism O for O the O regulation O of O mitotic O APC B-Complex / I-Complex C I-Complex by O phosphorylation O and O provide O a O rationale O for O the O development O of O selective O inhibitors O of O this O state O . O Mutant O allele O of O rna14 B-Protein in O fission O yeast O affects O pre O - O mRNA O splicing O . O Spliceosome B-Complex and O 3 B-Complex ' I-Complex - I-Complex end I-Complex processing I-Complex complexes O are O necessary O for O the O precursor O mRNA O ( O pre O - O mRNA O ) O maturation O . O Spliceosome B-Complex complex O removes O noncoding O introns O , O while O 3 B-Complex ' I-Complex - I-Complex end I-Complex processing I-Complex involves O in O cleavage O and O addition O of O poly O ( O A O ) O tails O to O the O nascent O transcript O . O Rna14 B-Protein protein O in O budding O yeast O has O been O implicated O in O cleavage O and O polyadenylation O of O mRNA O in O the O nucleus O but O their O role O in O the O pre O - O mRNA O splicing O has O not O been O studied O . O Here O , O we O report O the O isolation O of O a O mutant O allele O of O rna14 B-Protein in O fission O yeast O , O Schizosaccharomyces O pombe O that O exhibits O reduction O in O protein O level O of O Chk1 B-Protein at O the O nonpermissive O temperature O , O primarily O due O to O the O defects O in O posttranscriptional O processing O . O Reverse O transcriptase O - O polymerase O chain O reaction O analysis O reveals O defective O splicing O of O the O chk1 B-Protein ( O + O ) O transcript O at O the O nonpermissive O temperature O . O Apart O from O chk1 B-Protein ( O + O ) O , O the O splicing O of O some O other O genes O were O also O found O to O be O defective O at O the O nonpermissive O temperature O suggesting O that O Rna14 B-Protein might O be O involved O in O pre O - O mRNA O splicing O . O Subsequently O , O genetic O interaction O of O Rna14 B-Protein with O prp1 B-Protein and O physical O interactions O with O Prp28 B-Protein suggest O that O the O Rna14 B-Protein might O be O part O of O a O larger O protein O complex O responsible O for O the O pre O - O mRNA O maturation O . O IKKepsilon B-Protein inhibits O PKC B-Family to O promote O Fascin B-Protein - O dependent O actin B-Family bundling O . O Signaling O molecules O have O pleiotropic O functions O and O are O activated O by O various O extracellular O stimuli O . O Protein B-Family kinase I-Family C I-Family ( O PKC B-Family ) O is O activated O by O diverse O receptors O , O and O its O dysregulation O is O associated O with O diseases O including O cancer O . O However O , O how O the O undesired O activation O of O PKC B-Family is O prevented O during O development O remains O poorly O understood O . O We O have O previously O shown O that O a O protein O kinase O , O IKKepsilon B-Protein , O is O active O at O the O growing O bristle O tip O and O regulates O actin B-Family bundle O organization O during O Drosophila O bristle O morphogenesis O . O Here O , O we O demonstrate O that O IKKepsilon B-Protein regulates O the O actin B-Family bundle O localization O of O a O dynamic O actin B-Family cross O - O linker O , O Fascin B-Protein . O IKKepsilon B-Protein inhibits O PKC B-Family , O thereby O protecting O Fascin B-Protein from O inhibitory O phosphorylation O . O Excess O PKC B-Family activation O is O responsible O for O the O actin B-Family bundle O defects O in O IKKepsilon B-Protein - O deficient O bristles O , O whereas O PKC B-Family is O dispensable O for O bristle O morphogenesis O in O wild O - O type O bristles O , O indicating O that O PKC B-Family is O repressed O by O IKKepsilon B-Protein in O wild O - O type O bristle O cells O . O These O results O suggest O that O IKKepsilon B-Protein prevents O excess O activation O of O PKC B-Family during O bristle O morphogenesis O . O Role O of O variant O prealbumin B-Protein in O the O pathogenesis O of O familial O amyloidotic O polyneuropathy O : O fate O of O normal O and O variant O prealbumin B-Protein in O the O circulation O . O According O to O recent O studies O on O protein O chemistry O and O genetic O engineering O , O replacement O of O the O Val30 O residue O of O prealbumin B-Protein by O methionine O is O believed O to O play O a O critical O role O in O the O formation O of O amyloid O deposit O and O the O pathogenesis O of O familial O amyloidotic O polyneuropathy O ( O FAP O ) O . O However O , O only O limited O information O is O available O concerning O the O behavior O of O prealbumin B-Protein in O the O circulation O . O To O obtain O the O molecular O insight O into O the O mechanism O of O amyloid O deposition O , O it O is O indispensable O to O know O the O fates O of O normal O and O variant O prealbumin B-Protein in O vivo O . O Thus O , O the O fates O of O prealbumin B-Protein samples O from O normal O and O FAP O patients O were O studied O in O normal O rats O as O well O as O in O animals O that O were O challenged O with O acute O inflammation O induced O by O turpentine B-Chemical . O The O effect O of O in O vitro O photooxidation O of O prealbumin B-Protein samples O on O their O behavior O was O also O examined O in O vivo O . O Kinetic O analysis O revealed O no O appreciable O difference O between O prealbumin B-Protein samples O from O normal O and O FAP O patients O . O These O results O suggest O that O factors O other O than O the O rate O of O transfer O of O the O variant O form O prealbumin B-Protein from O plasma O to O an O extravascular O compartment O may O play O a O critical O role O in O the O pathogenesis O of O amyloid O deposition O in O FAP O patients O . O An O Mcm10 B-Protein Mutant O Defective O in O ssDNA O Binding O Shows O Defects O in O DNA O Replication O Initiation O . O Mcm10 B-Protein is O an O essential O protein O that O functions O to O initiate O DNA O replication O after O the O formation O of O the O replication O fork O helicase O . O In O this O manuscript O , O we O identified O a O budding O yeast O Mcm10 B-Protein mutant O ( O Mcm10 B-Protein - O m2 O , O 3 O , O 4 O ) O that O is O defective O in O DNA O binding O in O vitro O . O Moreover O , O this O Mcm10 B-Protein - O m2 O , O 3 O , O 4 O mutant O does O not O stimulate O the O phosphorylation O of O Mcm2 B-Protein by O Dbf4 B-Protein - I-Protein dependent I-Protein kinase I-Protein ( O DDK B-Protein ) O in O vitro O . O When O we O expressed O wild O - O type O levels O of O mcm10 B-Protein - O m2 O , O 3 O , O 4 O in O budding O yeast O cells O , O we O observed O a O severe O growth O defect O and O a O substantially O decreased O DNA O replication O . O We O also O observed O a O substantially O reduced O replication O protein O A O - O chromatin O immunoprecipitation O signal O at O origins O of O replication O , O reduced O levels O of O DDK B-Protein - O phosphorylated O Mcm2 B-Protein , O and O diminished O Go B-Protein , O Ichi B-Protein , O Ni B-Protein , O and O San B-Protein ( O GINS O ) O association O with O Mcm2 B-Protein - O 7 O in O vivo O . O mcm5 B-Protein - O bob1 B-Protein bypasses O the O growth O defect O conferred O by O DDK B-Protein - O phosphodead O Mcm2 B-Protein in O budding O yeast O . O However O , O the O growth O defect O observed O by O expressing O mcm10 B-Protein - O m2 O , O 3 O , O 4 O is O not O bypassed O by O the O mcm5 B-Protein - O bob1 B-Protein mutation O . O Furthermore O , O origin O melting O and O GINS B-Protein association O with O Mcm2 B-Protein - O 7 O are O substantially O decreased O for O cells O expressing O mcm10 B-Protein - O m2 O , O 3 O , O 4 O in O the O mcm5 B-Protein - O bob1 B-Protein background O . O Thus O , O the O origin O melting O and O GINS B-Protein - O Mcm2 B-Protein - O 7 O interaction O defects O we O observed O for O mcm10 B-Protein - O m2 O , O 3 O , O 4 O are O not O explained O by O decreased O Mcm2 B-Protein phosphorylation O by O DDK B-Protein , O since O the O defects O persist O in O an O mcm5 B-Protein - O bob1 B-Protein background O . O These O data O suggest O that O DNA O binding O by O Mcm10 B-Protein is O essential O for O the O initiation O of O DNA O replication O . O Nuclear O / O cytoplasmic O distribution O and O phosphorylation O of O YAP B-Protein significantly O influence O its O stabilization O . O To O find O out O if O TNFAIP8 B-Protein could O stabilize O YAP B-Protein protein O , O we O treated O HCC O cells O with O protein O synthesis O inhibitor O cycloheximide B-Chemical ( O CHX B-Chemical ) O after O 40 O hours O of O transfection O . O Relative O protein O intensity O was O shown O in O Supplementary O Figure O S4A O . O We O observed O that O , O after O 6 O hours O of O CHX B-Chemical treatment O , O TNFAIP8 B-Protein overexpression O significantly O upregulated O total O YAP B-Protein protein O . O TNFAIP8 B-Protein siRNA O showed O the O opposite O effects O ( O Figure O 4C O & O Supplementary O Figure O S4A O ) O . O These O results O indicate O that O the O half O - O life O of O endogenous O YAP B-Protein increased O with O TNFAIP8 B-Protein transfection O and O decreased O after O siRNA O treatment O . O Then O we O performed O luciferase O reporter O assay O using O reporter O plasmid O with O 8xGTIIC O - O luciferase O . O The O activity O of O TEAD B-Protein luciferase I-Protein reporter O indicates O transcriptional O activity O of O YAP B-Protein . O Upregulation O of O TEAD B-Protein luciferase I-Protein activity O correlates O with O inhibition O of O Hippo B-Family signaling O pathway O . O As O shown O in O Figure O 4D O , O TNFAIP8 B-Protein positively O regulated O transcription O of O YAP B-Protein / O TEAD B-Protein . O Based O on O the O above O results O , O we O assumed O that O TNFAIP8 B-Protein could O stabilize O the O YAP B-Protein protein O and O thus O increase O its O steady O - O state O protein O level O and O nuclear O localization O , O leading O to O inhibition O of O Hippo B-Family signaling O . O Breast O cancer O tissue O microarray O ( O TMA O ) O of O 334 O cases O of O invasive O ductal O carcinomas O is O obtained O from O the O tissue O bank O at O the O Markey O Cancer O Center O ' O s O tissue O repository O at O our O institute O . O Tissue O samples O were O stained O with O anti O - O Dub3 B-Protein ( O Abcam O , O ab12991 O , O 1 O : O 100 O dilution O ) O and O anti O - O Snail1 B-Protein ( O Abcam O , O ab53519 O , O 1 O : O 250 O dilution O ) O antibodies O , O and O each O sample O was O scored O by O an O H O - O score O method O that O combines O the O values O of O immunoreaction O intensity O and O the O percentage O of O tumour O cell O staining O as O described O previously O . O Chi O - O square O analysis O was O used O to O analyse O the O relationship O between O Dub3 B-Protein and O Snail1 B-Protein expression O ; O statistical O significance O was O defined O as O P O < O 0 O . O 05 O . O In O vivo O characterization O of O the O poly O ( O ADP O - O ribosylation O ) O of O SV40 O chromatin O and O large B-Protein T I-Protein antigen I-Protein by O immunofractionation O . O We O have O confirmed O the O poly O ( O ADP O - O ribosylation O ) O of O large B-Protein T I-Protein antigen I-Protein of O SV40 O by O using O antibodies O to O both O large B-Protein T I-Protein antigen I-Protein and O poly B-Chemical ( I-Chemical ADP I-Chemical - I-Chemical ribose I-Chemical ) I-Chemical and O consequently O have O begun O to O characterize O how O this O post O - O translational O nuclear O modification O of O the O viral O protein O modulates O its O biological O functions O . O SV40 O minichromosomal O subpopulation O containing O replicative O intermediate O DNA O was O shown O to O have O a O significantly O higher O affinity O for O anti O - O poly B-Chemical ( I-Chemical ADP I-Chemical - I-Chemical Rib I-Chemical ) I-Chemical - I-Chemical Sepharose I-Chemical than O viral O chromatin O fractions O containing O mature O minichromosomal O DNA O . O An O anti O - O large B-Chemical T I-Chemical - I-Chemical Sepharose I-Chemical column O was O used O to O isolate O T B-Protein antigen I-Protein from O crude O extracts O by O two O different O approaches O : O ( O 1 O ) O large B-Protein T I-Protein antigen I-Protein was O labeled O with O [ O 35S O ] O methionine O in O vivo O and O the O infected O cell O extract O was O immunofractionated O to O isolate O large B-Protein T I-Protein antigen I-Protein and O ( O 2 O ) O large B-Protein T I-Protein antigen I-Protein from O infected O cell O extracts O was O immunofractionated O followed O by O immunostaining O . O Using O these O techniques O , O 1 O - O 10 O % O of O the O total O T B-Protein antigen I-Protein from O infected O cells O was O found O to O be O poly O ( O ADP O - O ribosylated O ) O . O Minichromosome O preparations O per O se O were O also O subjected O to O immunofractionation O on O anti O - O large B-Chemical T I-Chemical - I-Chemical Sepharose I-Chemical . O The O high O level O of O retention O of O poly O ( O ADP O - O ribosylated O ) O species O of O minichromosomes O on O this O matrix O suggested O that O this O post O - O translational O modification O of O viral O chromatin O may O be O related O to O those O steps O in O viral O replication O and O transcription O under O regulation O by O large B-Protein T I-Protein antigen I-Protein . O Functional O diversity O among O putative O E2 B-Family isozymes O in O the O mechanism O of O ubiquitin B-Protein - O histone B-Family ligation O . O The O covalent O ligation O of O the O 8 O . O 6 O - O kDa O protein O ubiquitin B-Protein to O histones B-Family within O transcriptionally O poised O regions O is O believed O to O participate O in O the O localized O regulation O of O chromatin O structure O . O This O unique O post O - O translational O modification O is O thought O to O be O distinct O from O similar O cytosolic O reactions O in O requiring O ubiquitin B-Family - I-Family activating I-Family enzyme I-Family ( O E1 B-Family ) O and O one O or O more O putative O ubiquitin B-Family carrier I-Family proteins I-Family ( O E2 B-Family ) O but O not O isopeptide O ligase O ( O E3 O ) O . O Apparently O homogeneous O preparations O of O the O E2 B-Family isozymes O were O tested O for O their O ability O to O catalyze O the O E3 O - O independent O conjugation O of O ubiquitin B-Protein to O linker O histone B-Protein H1 I-Protein and O core O histones O H2A B-Protein , O H2B B-Protein , O H3 B-Protein , O and O H4 B-Protein in O the O presence O of O catalytic O amounts O of O E1 B-Family . O Significant O rates O of O nonprocessive O core O histone B-Family monoubiquitination O were O catalyzed O by O the O E2 B-Family ( O 14kDa O ) O , O E2 B-Family ( O 20kDa O ) O , O and O E2 B-Family ( O 32kDa O ) O isozymes O but O not O by O either O E2 B-Family ( O 17kDa O ) O or O E2 B-Family ( O 24kDa O ) O . O The O former O three O E2 B-Family isozymes O also O supported O slow O rates O of O direct O multiple O ubiquitination O to O secondary O ligation O sites O on O the O histones B-Family . O Rate O studies O for O the O monoubiquitination O of O H2A B-Protein and O H2B B-Protein revealed O that O : O 1 O ) O E2 B-Family ( O 14kDa O ) O catalyzed O a O second O order O reaction O with O respect O to O histone B-Family concentration O ; O 2 O ) O E2 B-Family ( O 32kDa O ) O - O mediated O ligation O proceeded O by O hyperbolic O kinetics O , O yielding O Km O values O of O 2 O . O 8 O and O 12 O microM O for O H2A B-Protein and O H2B B-Protein , O respectively O ; O and O 3 O ) O E2 B-Family ( O 20kDa O ) O exhibited O complex O kinetics O composed O of O both O second O order O and O hyperbolic O pathways O , O the O latter O having O Km O values O of O 0 O . O 83 O and O 1 O . O 5 O microM O for O H2A B-Protein and O H2B B-Protein , O respectively O . O Pulse O - O chase O kinetics O suggested O that O both O ubiquitin B-Protein thiol O esters O formed O to O E2 B-Family ( O 20kDa O ) O were O catalytically O competent O in O H2A B-Protein ligation O . O The O active O E2 B-Family isozymes O also O catalyzed O the O processive O multiple O ubiquitination O of O calf O thymus O H1 O . O Other O rate O studies O determined O that O Kd O values O for O binding O of O the O active O E2 B-Family species O to O E1 B-Family ternary O complex O were O 0 O . O 1 O nM O for O E2 B-Family ( O 14kDa O ) O , O 0 O . O 4 O nM O for O E2 B-Family ( O 32kDa O ) O , O and O 3 O . O 6 O nM O for O E2 B-Family ( O 20kDa O ) O . O The O data O indicate O that O E2 B-Family ( O 20kDa O ) O and O E2 B-Family ( O 32kDa O ) O are O specific O but O mechanistically O distinct O ligation O enzymes O responsible O for O the O conjugation O of O ubiquitin B-Protein to O nucleosomal O proteins O . O In O contrast O to O other O cyclins B-Family involved O in O cell O cycle O progression O , O CycC B-Protein does O not O oscillate O . O Instead O , O Cdk8 B-Protein / O CycC B-Protein activity O appears O to O be O regulated O by O other O Mediator B-Complex subunits O . O In O line O with O a O model O previously O suggested O by O others O and O us O , O we O believe O that O Med12 B-Protein and O Med13 B-Protein keep O the O Cdk8 B-Protein / O CycC B-Protein pair O in O a O repressed O state O , O which O can O be O released O when O activators O and O / O or O signaling O pathways O induce O structural O changes O in O the O Mediator B-Complex complex O . O Loss O of O Med12 B-Protein and O / O or O Med13 B-Protein may O abolish O this O repressive O effect O , O which O explains O the O early O entry O into O mitosis O observed O in O med12Delta B-Protein and O med13Delta B-Protein cells O . O In O fact O , O loss O of O Med12 B-Protein and O Med13 B-Protein leads O to O the O release O of O CycC B-Protein and O Cdk8 B-Protein from O Mediator B-Complex . O We O reasoned O that O a O free O pool O of O CycC B-Protein and O Cdk8 B-Protein might O affect O cell O cycle O progression O by O interacting O with O alternative O cyclins B-Family and O Cdks B-Family . O In O support O of O this O idea O , O a O previous O study O of O human O CycC B-Protein showed O that O the O protein O can O interact O with O an O alternative O Cdk B-Family , O namely O CDK3 B-Protein . O To O address O this O possibility O experimentally O , O here O we O fused O CycC B-Protein to O Cdk8 B-Protein , O thereby O restricting O interaction O to O the O cognate O partner O and O analyzed O the O effects O of O med12 B-Protein + O and O med13 B-Protein + O deletion O on O cell O cycle O progression O . O As O demonstrated O here O , O the O effects O of O med12Deltamed13Delta B-Protein on O mitotic O progression O are O identical O in O the O Cdk8 B-Protein - O L O - O CycC B-Protein strain O to O the O effects O observed O in O wt O cells O , O strongly O suggesting O that O Cdk8 B-Protein and O CycC B-Protein act O in O concert O to O regulate O timing O of O mitosis O . O Loss O of O Med12 B-Protein and O Med13 B-Protein also O causes O early O entry O into O S O phase O . O Of O interest O , O this O effect O is O not O observed O in O CycC B-Protein - O L O - O Cdk8 B-Protein cells O . O The O data O suggest O that O eliminating O the O possibility O for O Cdk8 B-Protein and O CycC B-Protein to O interact O with O alternative O cyclins B-Family and O Cdks B-Family can O restrict O the O effects O of O med12 B-Protein + O and O med13 B-Protein + O deletions O on O cell O cycle O progression O . O Both O overproduction O of O Cdk8 B-Protein and O mutations O in O CycC B-Protein are O observed O in O human O tumors O . O It O is O tempting O to O speculate O that O these O mutagenic O changes O may O cause O the O formation O of O a O free O Cdk8 B-Protein pool O in O mammalian O cells O , O which O could O result O in O spontaneous O interactions O with O alternative O cyclins B-Family and O promote O S O - O phase O progression O . O Limited O information O is O available O on O the O role O of O Cdk8 B-Protein and O CycC B-Protein in O M O - O phase O progression O in O mammalian O cells O . O We O hope O that O our O findings O will O stimulate O others O to O elucidate O whether O the O effects O we O observe O in O fission O yeast O are O also O conserved O in O higher O organisms O . O 2 O ODs O of O cells O grown O in O SD O media O to O exponential O phase O ( O OD600 O = O 0 O . O 5 O - O 1 O ) O were O spheroplasted O by O treatment O with O 2 O . O 5 O U O per O OD O of O Zymolyase O ( O Zymo O Research O ; O Orange O , O CA O ) O . O The O total O RNA O was O extracted O using O the O NucleoSpin O RNA O extraction O kit O ( O Macherey O Nagel O ) O . O 1 O mug O of O resulting O RNAs O were O then O treated O with O DNase B-Protein I I-Protein ( O NEB B-Protein ) O and O subjected O to O reverse O transcriptionusing O Maxima O First O Strand O cDNA O synthesis O Kit O for O quantitative O PCR O with O reverse O transcription O ( O Thermo O Scientific O ) O . O Quantitative O real O time O PCR O was O performed O with O Maxima O SYBR O Green O qPCR O Master O Mix O ( O Thermo O Scientific O ) O in O a O Bio O - O Rad O CFX96 O Real O - O Time O PCR O system O using O OLE1 B-Protein and O ACT1 B-Protein specific O primers O . O The O OLE1 B-Protein mRNA O abundance O in O mutants O was O normalized O to O ACT1 B-Protein mRNA O levels O and O expressed O relative O to O OLE1 B-Protein expression O in O WT O using O the O Livak O method O . O Six O experiments O were O performed O and O the O relative O mRNA O levels O were O averaged O . O Nedd4 B-Protein - I-Protein 2 I-Protein mis O - O localization O in O PCK O kidney O is O associated O with O increased O apical O ENaC B-Protein and O enhanced O sodium B-Chemical reabsorption O . O ( O a O ) O Kidneys O from O 12 O - O week O old O strain O control O ( O Crj O : O CD O / O SD O ) O ( O Wt O ) O ; O left O ) O and O PCK O ( O right O ) O rats O immunostained O for O Nedd4 B-Protein - I-Protein 2 I-Protein ( O red O ) O , O Lamp1 B-Protein ( O green O ) O and O DAPI B-Chemical ( O blue O ) O . O Similar O patterns O were O observed O in O sections O from O three O different O mutants O , O and O abnormalities O were O restricted O to O collecting O ducts O . O Scale O bars O , O 20 O mum O . O ( O b O ) O Kidneys O from O 12 O - O week O old O strain O control O ( O Wt O ) O and O PCK O rats O immunostained O for O each O of O the O three O subunits O of O ENaC B-Protein ( O red O ) O , O DBA B-Protein ( O green O ) O and O DAPI B-Chemical ( O blue O ) O . O Scale O bars O , O 20 O mum O . O ( O c O ) O Primary O CD O cells O from O 12 O - O week O old O strain O control O and O PCK O rats O were O isolated O and O cultured O on O Transwell O filters O and O then O immunostained O for O each O of O the O three O ENaC B-Protein subunits O ( O red O ) O and O DAPI B-Chemical ( O blue O ) O . O Scale O bars O , O 10 O mum O . O ( O d O ) O Open O - O circuit O measurements O of O primary O CD O cells O from O three O 12 O - O week O old O strain O control O rats O ( O Wt1 O , O Wt2 O and O Wt3 O ) O and O three O PCK O rats O ( O PCK1 O , O PCK2 O , O PCK3 O ) O cultured O on O Transwell O filters O either O in O the O absence O or O presence O ( O " O + O Amil B-Chemical ) O of O 50 O muM O amiloride B-Chemical in O the O apical O side O . O Measurements O were O collected O from O at O least O 10 O independent O wells O per O animal O . O * O P O < O 0 O . O 05 O , O * O * O P O < O 0 O . O 01 O . O To O confirm O that O plasma O cBIN1 B-Protein - O MPs O are O of O cardiomyocyte O origin O , O we O took O advantage O of O mice O with O cardiac O - O specific O heterozygous O deletion O of O the O Bin1 B-Protein gene O ( O Bin1 B-Protein HT O ) O . O Bin1 B-Protein HT O mice O are O grossly O phenotypically O normal O with O normal O cardiac O contractile O function O at O young O adulthood O ( O 2 O - O 4 O months O ) O . O Yet O , O their O cardiomyocytes O lose O cBIN1 B-Protein - O microfolds O at O t O - O tubules O , O and O t O - O tubule O remodeling O occurs O with O this O loss O of O microfolds O within O the O t O - O tubule O membrane O . O Plasma O for O MP O acquisition O was O obtained O from O Bin1 B-Protein HT O mice O and O their O wild O - O type O ( O WT O ) O littermate O controls O . O Purified O MPs O were O fixed O and O colabeled O with O anti O - O cBIN1 B-Protein - O Alexa O 647 O and O annexin B-Protein V I-Protein - O Alexa O 488 O . O Plasma O MPs O were O loaded O in O TruCount O tubes O , O and O a O total O of O 20 O , O 000 O events O were O counted O . O Based O on O the O known O concentration O of O TruCount O reference O beads O , O the O plasma O concentration O of O MPs O could O be O determined O . O Both O the O total O MP O population O and O the O subpopulation O of O double O positive O in O cBIN1 B-Protein and O annexin B-Protein V I-Protein ( O cBIN1 B-Protein - O MPs O ) O were O quantified O and O compared O between O WT O and O Bin1 B-Protein HT O mice O . O As O indicated O in O Fig O 1D O , O although O total O plasma O MP O concentration O is O not O significantly O altered O in O Bin1 B-Protein HT O mice O ( O Fig O 1D O , O left O panel O ) O , O the O plasma O concentration O of O cBIN1 B-Protein - O MPs O ( O Fig O 1D O , O right O panel O ; O representative O flow O scatter O plots O against O IgG B-Complex isotype O control O are O in O S1C O Fig O ) O in O Bin1 B-Protein HT O mice O ( O 251 O . O 8 O + O / O - O 48 O . O 3 O MPs O / O ml O ) O is O significantly O decreased O by O 47 O % O from O WT O mice O ( O 470 O . O 4 O + O / O - O 67 O . O 8 O MPs O / O ml O ) O ( O p O < O 0 O . O 05 O ) O ( O Fig O 1D O ) O . O Consistent O with O less O cBIN1 B-Protein - O MPs O , O biochemical O immunoprecipitation O followed O by O western O blot O detection O further O confirmed O that O the O cBIN1 B-Protein protein O level O is O reduced O in O plasma O from O Bin1 B-Protein HT O mice O , O corresponding O to O a O similar O reduction O of O cBIN1 B-Protein protein O in O heart O lysates O from O the O same O mice O ( O Fig O 1E O ) O . O Taken O together O , O these O data O support O that O cBIN1 B-Protein in O plasma O is O present O in O MPs O of O cardiomyocyte O origin O . O Exomer B-Complex is O an O adaptor O complex O required O for O the O direct O transport O of O a O selected O number O of O cargoes O from O the O < O i O > O trans O < O / O i O > O - O Golgi O network O ( O TGN O ) O to O the O plasma O membrane O in O < O i O > O Saccharomyces O cerevisiae O < O / O i O > O However O , O exomer B-Complex mutants O are O highly O sensitive O to O increased O concentrations O of O alkali B-Chemical metal I-Chemical cations I-Chemical , O a O situation O that O remains O unexplained O by O the O lack O of O transport O of O any O known O cargoes O . O Here O we O identify O several O < O i O > O HAL B-Protein < O / O i O > O genes O that O act O as O multicopy O suppressors O of O this O sensitivity O and O are O connected O to O the O reduced O function O of O the O sodium O ATPase O Ena1 B-Protein . O Furthermore O , O we O find O that O Ena1 B-Protein is O dependent O on O exomer B-Complex function O . O Even O though O Ena1 B-Protein can O reach O the O plasma O membrane O independently O of O exomer B-Complex , O polarized O delivery O of O Ena1 B-Protein to O the O bud O requires O functional O exomer B-Complex . O Moreover O , O exomer B-Complex is O required O for O full O induction O of O Ena1 B-Protein expression O after O cationic O stress O by O facilitating O the O plasma O membrane O recruitment O of O the O molecular O machinery O involved O in O Rim101 B-Protein processing O and O activation O of O the O RIM101 B-Protein pathway O in O response O to O stress O . O Both O the O defective O localization O and O the O reduced O levels O of O Ena1 B-Protein contribute O to O the O sensitivity O of O exomer B-Complex mutants O to O alkali B-Chemical metal I-Chemical cations I-Chemical . O Our O work O thus O expands O the O spectrum O of O exomer B-Complex - O dependent O proteins O and O provides O a O link O to O a O more O general O role O of O exomer B-Complex in O TGN O organization O . O We O identified O slam B-Protein as O an O mRNA O with O noncoding O information O for O localization O and O translational O control O in O addition O to O its O coding O information O . O On O the O molecular O level O , O slam B-Protein is O special O in O that O mRNA O and O protein O associate O in O a O complex O as O demonstrated O by O co O - O immunoprecipitation O . O This O unconventional O relationship O of O slam B-Protein RNA O and O protein O may O be O important O for O the O tightly O restricted O subcellular O localization O and O strong O increase O in O protein O levels O at O the O FC O . O The O functional O interaction O of O slam B-Protein RNA O and O protein O constitutes O a O positive O feedback O loop O , O which O contributes O to O the O fast O increase O in O Slam B-Protein protein O levels O at O the O FC O . O In O an O established O model O of O metastatic O lung O cancer O , O H460 O cells O with O IGF2BP3 B-Protein overexpression O were O injected O into O the O tail O vein O of O nude O mice O . O Animals O were O sacrificed O at O day O 21 O and O the O lungs O were O resected O and O photographed O . O The O number O of O tumor O nodules O in O lungs O was O significantly O enhanced O in O IGF2BP3 B-Protein overexpressing O group O compared O with O control O group O ( O Figure O 4A O and O 4B O ) O . O In O agreement O with O the O subcutaneous O lung O cancer O model O , O IGF2BP3 B-Protein overexpressing O group O in O the O metastatic O lung O cancer O model O also O showed O reduced O survival O rate O ( O Figure O 4C O ) O . O To O support O this O , O we O also O analyzed O the O survival O rate O of O lung O cancer O patients O by O an O online O tool O " O Kaplan O - O Meier O Plotter O " O analysis O ( O http O : O / O / O kmplot O . O com O / O analysis O / O ) O . O As O shown O in O Figure O 4D O , O lung O cancer O patients O with O high O expression O of O IGF2BP3 B-Protein had O shorter O survival O rate O compared O to O those O with O low O expression O of O IGF2BP3 B-Protein . O Thus O , O both O in O vitro O and O in O vivo O assays O suggest O that O overexpression O of O IGF2BP3 B-Protein promotes O proliferation O , O metastasis O and O tumorigenicity O of O lung O cancer O cells O . O RAD4 B-Protein and O RAD23 B-Protein / O HMR B-Protein Contribute O to O Arabidopsis O UV O Tolerance O . O In O plants O , O exposure O to O solar O ultraviolet O ( O UV O ) O light O is O unavoidable O , O resulting O in O DNA O damage O . O Damaged O DNA O causes O mutations O , O replication O arrest O , O and O cell O death O , O thus O efficient O repair O of O the O damaged O DNA O is O essential O . O A O light O - O independent O DNA O repair O pathway O called O nucleotide O excision O repair O ( O NER O ) O is O conserved O throughout O evolution O . O For O example O , O the O damaged O DNA O - O binding O protein O Radiation B-Protein sensitive I-Protein 4 I-Protein ( O Rad4 B-Protein ) O in O Saccharomyces O cerevisiae O is O homologous O to O the O mammalian O NER O protein O Xeroderma B-Protein Pigmentosum I-Protein complementation I-Protein group I-Protein C I-Protein ( O XPC B-Protein ) O . O In O this O study O , O we O examined O the O role O of O the O Arabidopsis O thaliana O Rad4 B-Protein / O XPC B-Protein homologue O ( O AtRAD4 B-Protein ) O in O plant O UV O tolerance O by O generating O overexpression O lines O . O AtRAD4 B-Protein overexpression O , O both O with O and O without O an O N O - O terminal O yellow B-Protein fluorescent I-Protein protein I-Protein ( O YFP B-Protein ) O tag O , O resulted O in O increased O UV O tolerance O . O YFP B-Protein - O RAD4 B-Protein localized O to O the O nucleus O , O and O UV O treatment O did O not O alter O this O localization O . O We O also O used O yeast O two O - O hybrid O analysis O to O examine O the O interaction O of O AtRAD4 B-Protein with O Arabidopsis O RAD23 B-Protein and O found O that O RAD4 B-Protein interacted O with O RAD23B B-Protein as O well O as O with O the O structurally O similar O protein O HEMERA B-Protein ( O HMR B-Protein ) O . O In O addition O , O we O found O that O hmr B-Protein and O rad23 B-Protein mutants O exhibited O increased O UV O sensitivity O . O Thus O , O our O analysis O suggests O a O role O for O RAD4 B-Protein and O RAD23 B-Protein / O HMR B-Protein in O plant O UV O tolerance O . O Deubiquitylation O and O stabilization O of O p21 B-Protein by O USP11 B-Protein is O critical O for O cell O - O cycle O progression O and O DNA O damage O responses O . O p21WAF1 B-Protein / O CIP1 B-Protein is O a O broad O - O acting O cyclin B-Family - I-Family dependent I-Family kinase I-Family inhibitor O . O Its O stability O is O essential O for O proper O cell O - O cycle O progression O and O cell O fate O decision O . O Ubiquitylation O by O the O multiple O E3 O ubiquitin B-Protein ligase O complexes O is O the O major O regulatory O mechanism O of O p21 B-Protein , O which O induces O p21 B-Protein degradation O . O However O , O it O is O unclear O whether O ubiquitylated O p21 B-Protein can O be O recycled O . O In O this O study O , O we O report O USP11 B-Protein as O a O deubiquitylase O of O p21 B-Protein . O In O the O nucleus O , O USP11 B-Protein binds O to O p21 B-Protein , O catalyzes O the O removal O of O polyubiquitin O chains O conjugated O onto O p21 B-Protein , O and O stabilizes O p21 B-Protein protein O . O As O a O result O , O USP11 B-Protein reverses O p21 B-Protein polyubiquitylation O and O degradation O mediated O by O SCFSKP2 B-Protein , O CRL4CDT2 B-Protein , O and O APC B-Protein / O CCDC20 B-Protein in O a O cell O - O cycle O - O independent O manner O . O Loss O of O USP11 B-Protein causes O the O destabilization O of O p21 B-Protein and O induces O the O G1 O / O S O transition O in O unperturbed O cells O . O Furthermore O , O p21 B-Protein accumulation O mediated O by O DNA O damage O is O completely O abolished O in O cells O depleted O of O USP11 B-Protein , O which O results O in O abrogation O of O the O G2 O checkpoint O and O induction O of O apoptosis O . O Functionally O , O USP11 B-Protein - O mediated O stabilization O of O p21 B-Protein inhibits O cell O proliferation O and O tumorigenesis O in O vivo O . O These O findings O reveal O an O important O mechanism O by O which O p21 B-Protein can O be O stabilized O by O direct O deubiquitylation O , O and O they O pinpoint O a O crucial O role O of O the O USP11 B-Protein - O p21 B-Protein axis O in O regulating O cell O - O cycle O progression O and O DNA O damage O responses O . O The O human O receptor O for O T B-Protein - I-Protein cell I-Protein growth I-Protein factor I-Protein . O Evidence O for O variable O post O - O translational O processing O , O phosphorylation O , O sulfation O , O and O the O ability O of O precursor O forms O of O the O receptor O to O bind O T B-Protein - I-Protein cell I-Protein growth I-Protein factor I-Protein . O The O T B-Protein - I-Protein cell I-Protein growth I-Protein factor I-Protein ( I-Protein TCGF I-Protein ) I-Protein receptor I-Protein on O phytohemagglutinin B-Chemical - O activated O normal O peripheral O blood O T O - O cells O is O characterized O as O a O glycoprotein O with O an O apparent O Mr O = O 55 O , O 000 O that O contains O N O - O linked O and O O O - O linked O carbohydrate O with O only O approximately O 33 O , O 000 O daltons O of O peptide O structure O ( O p33 B-Protein ) O as O evaluated O by O sodium B-Chemical dodecyl I-Chemical sulfate I-Chemical - O polyacrylamide B-Chemical gel O electrophoresis O . O There O are O two O N O - O linked O glycosylated O intermediate O precursor O forms O ( O apparent O Mr O = O 35 O , O 000 O ( O p35 B-Protein ) O and O 37 O , O 000 O ( O p37 B-Protein ] O . O This O receptor O differs O from O the O TCGF B-Protein receptor I-Protein on O HUT O - O 102B2 O cells O ( O apparent O Mr O = O 50 O , O 000 O ) O because O of O differences O in O post O - O translational O processing O . O Experiments O with O the O carboxylic O ionophore O monensin B-Chemical demonstrate O blockade O of O the O transition O of O the O p35 B-Protein and O p37 B-Protein receptor O precursor O forms O to O the O mature O receptor O , O presumably O secondary O to O inhibition O of O Golgi O - O associated O receptor O processing O . O We O identify O the O primary O translation O product O of O TCGF B-Protein receptor I-Protein mRNA O as O intermediate O in O size O between O the O p33 B-Protein and O the O p35 B-Protein / O p37 B-Protein forms O . O We O further O demonstrate O that O the O p33 B-Protein , O p35 B-Protein , O and O p37 B-Protein precursor O forms O , O but O not O the O primary O translation O product O , O are O all O capable O of O binding O TCGF B-Protein . O Thus O , O the O removal O of O the O signal O peptide O and O / O or O conformational O changes O of O the O primary O translation O product O are O necessary O for O ligand O binding O ; O however O , O the O extensive O post O - O translational O modifications O are O not O . O Lastly O , O we O demonstrate O that O at O least O some O TCGF B-Protein receptors I-Protein are O phosphorylated O and O sulfated O , O and O that O TCGF B-Protein receptors I-Protein on O phytohemagglutinin B-Chemical - O activated O normal O T O - O cells O are O more O heavily O sulfated O than O those O on O HUT O - O 102B2 O cells O . O Pex30 B-Protein has O an O N O - O terminal O reticulon B-Family homology O domain O ( O RHD O ) O . O Reticulons B-Family and O reticulon B-Family - I-Family like I-Family proteins I-Family are O abundant O conserved O ER O - O shaping O membrane O proteins O that O stabilize O the O highly O curved O portions O of O the O ER O , O tubules O , O and O the O edges O of O ER O sheets O through O the O RHDs O forming O wedge O - O shaped O hydrophobic O hairpins O . O We O found O that O overexpression O of O the O RHD O domain O of O Pex30 B-Protein restores O ER O structure O in O S O . O cerevisiae O cells O lacking O the O reticulons B-Family . O Endogenously O expressed O Pex30 B-Protein is O in O ER O subdomains O in O tubules O and O the O edges O of O sheets O , O as O are O reticulons B-Family . O The O function O of O Pex30 B-Protein is O not O known O but O it O may O play O a O role O in O peroxisome O biogenesis O since O the O size O and O number O of O peroxisomes O is O altered O in O cells O lacking O Pex30 B-Protein . O Pex30 B-Protein has O also O been O suggested O to O reside O at O ER O - O peroxisome O contacts O . O The O screen O highlighted O 9 O loci O in O IKZF3 B-Protein ZF2 O whose O amino O acid O identities O were O critical O for O degradation O ( O Fig O . O 3 O , O B O and O C O , O and O fig O . O S3 O , O C O and O D O ) O . O These O loci O again O included O residues O that O maintain O the O tertiary O ZF O fold O ( O IKZF3 B-Protein C148 O , O C151 O , O F155 O , O L161 O , O H164 O , O H168 O ) O and O the O beta O - O hairpin O glycine O residue O ( O IKZF3 B-Protein G152 O ) O . O In O addition O , O the O screen O highlighted O two O non O - O structural O residues O ( O IKZF3 B-Protein Q147 O and O A153 O ) O that O varied O among O the O other O ZF O degrons O ( O Fig O . O 3A O ) O , O but O were O important O for O degradation O of O the O IKZF3 B-Protein ZF2 O reporter O ( O Fig O . O 3 O , O B O and O C O ) O . O Mutation O of O these O two O residues O impaired O degradation O in O validation O experiments O , O whereas O N149 O , O a O residue O that O was O not O highlighted O in O the O mutagenesis O screen O , O tolerated O mutation O ( O Fig O . O 3D O ) O . O These O data O show O that O in O addition O to O the O residues O maintaining O the O ZF O fold O , O non O - O structural O amino O acids O ( O IKZF3 B-Protein Q147 O , O G152 O and O A153 O ) O contribute O to O degron O specificity O . O The O above O experiments O suggested O , O that O the O CTR O plays O a O RENT B-Complex complex O - O independent O role O supporting O cellular O growth O , O presumably O by O interacting O with O the O 35S B-Protein rDNA O promoter O . O We O therefore O tested O if O the O CTR O harbors O the O Pol B-Protein I I-Protein transcription O stimulating O function O of O Net1 B-Protein . O Haploid O yeast O strains O carrying O a O NET1 B-Protein or O a O net1Deltactr B-Protein allele O and O co O - O expressing O MNHA B-Protein fusion O proteins O of O the O Pol B-Protein I I-Protein subunits O Rpa43 B-Protein or O Rpa190 B-Protein were O subjected O to O ChEC O and O ChIP O experiments O . O ChEC O analyses O revealed O that O Rpa43 B-Protein - O MNHA B-Protein and O Rpa190 B-Protein - O MNHA B-Protein cleavage O events O at O the O 35S B-Protein rDNA O promoter O region O and O at O the O 35S B-Protein rDNA O transcription O termination O site O upstream O of O the O RFB O were O reduced O in O net1Deltactr B-Protein strains O , O when O compared O to O cleavages O in O NET1 B-Protein strains O ( O Fig O 5A O compare O lanes O 1 O - O 4 O with O lanes O 5 O - O 8 O , O and O lanes O 17 O - O 20 O with O lanes O 21 O - O 24 O ) O . O This O pointed O to O a O lower O occupancy O of O Pol B-Protein I I-Protein molecules O within O the O 35S B-Protein rRNA O gene O region O . O ChIP O experiments O strongly O supported O this O finding O , O since O co O - O precipitation O of O 35S B-Protein rDNA O fragments O with O the O tagged O Pol B-Protein I I-Protein subunits O was O substantially O impaired O in O net1Deltactr B-Protein strains O when O compared O with O ChIP O in O NET1 B-Protein strains O ( O Fig O 5B O , O ChIP O of O fragments O 2 O - O 4 O ) O . O Importantly O , O ChEC O and O ChIP O experiments O indicated O that O expression O of O FLAGCTR O in O trans O could O fully O restore O Pol B-Protein I I-Protein association O with O the O 35S B-Protein rDNA O in O net1Deltactr B-Protein cells O to O wild O - O type O levels O , O in O good O correlation O with O the O re O - O establishment O of O normal O cell O growth O in O these O strains O ( O Fig O 5A O , O compare O lanes O 9 O - O 12 O , O and O 25 O - O 28 O with O lanes O 13 O - O 16 O , O and O 29 O - O 32 O , O respectively O ; O Fig O 5B O , O ChIP O of O fragments O 2 O - O 4 O ; O S3 O Table O , O doubling O times O ) O . O To O verify O that O Abf2 B-Protein and O Mhr1 B-Protein are O required O for O mtDNA O maintenance O , O we O analyzed O mtDNA O levels O relative O to O nuclear O DNA O using O quantitative O PCR O . O We O observed O that O the O mtDNA O level O in O incrementabf2 B-Protein cells O was O less O than O half O ( O 46 O . O 3 O + O / O - O 8 O . O 6 O % O ) O that O of O WT O cells O grown O in O YPGly O medium O ( O Fig O . O 6a O ) O . O Consistent O with O our O previous O results O , O a O large O proportion O ( O 83 O . O 9 O + O / O - O 15 O . O 3 O % O ) O of O mtDNA O content O was O retained O in O mhr1 B-Protein - O 1 O cells O grown O in O glycerol B-Chemical medium O , O while O we O observed O no O additive O effect O on O the O depletion O of O mtDNA O in O incrementabf2 B-Protein mhr1 B-Protein - O 1 O double O - O mutant O cells O ( O 51 O . O 4 O + O / O - O 8 O . O 8 O % O ) O , O suggesting O Abf2 B-Protein is O dispensable O for O Mhr1 B-Protein - O driven O mtDNA O replication O ( O Fig O . O 6a O ) O . O Purification O and O postsynthetic O modifications O of O Friend O erythroleukemic O cell O high O mobility O group O protein O HMG B-Protein - I-Protein I I-Protein . O We O have O previously O detected O and O purified O a O Friend O erythroleukemic O mouse O cell O nonhistone O chromatin O protein O having O extraction O and O acid O - O solubility O properties O like O the O low O molecular O weight O " O high B-Family mobility I-Family group I-Family " O ( O HMG B-Family ) O nuclear O proteins O . O We O show O here O that O the O electrophoretic O properties O and O the O amino O acid O composition O of O this O mouse O cell O " B-Family HMG I-Family - I-Family like I-Family " I-Family protein I-Family is O comparable O to O those O of O the O HMG B-Protein - I-Protein I I-Protein proteins O isolated O from O human O HeLa O S3 O cells O , O African O green O monkey O cells O , O Ehrlich O ascites O mouse O cells O , O and O rat O fibroblast O cells O . O Therefore O , O we O have O also O designated O the O Friend O erythroleukemic O mouse O cell O protein O as O HMG B-Protein - I-Protein I I-Protein . O In O common O with O the O other O HMG B-Family proteins O the O Friend O cell O HMG B-Protein - I-Protein I I-Protein protein O can O undergo O a O variety O of O post O - O translational O biochemical O modifications O including O acetylation O , O ADP O - O ribosylation O , O glycosylation O , O and O phosphorylation O . O Surprisingly O , O in O the O course O of O these O studies O we O found O that O in O vivo O radiolabeling O experiments O revealed O that O only O two O minor O HMG B-Protein - I-Protein 14 I-Protein subspecies O ( O and O / O or O possibly O a O minor O HMG B-Protein - I-Protein I I-Protein subspecies O ) O are O phosphorylated O whereas O HMG B-Protein - I-Protein 1 I-Protein , O - B-Protein 2 I-Protein , I-Protein - B-Protein 17 I-Protein , O and O the O major O HMG B-Protein - I-Protein 14 I-Protein are O not O heavily O phosphorylated O . O Reduced O but O accurate O translation O from O a O mutant O AUA O initiation O codon O in O the O mitochondrial O COX2 B-Protein mRNA O of O Saccharomyces O cerevisiae O . O We O have O changed O the O translation O initiation O codon O of O the O COX2 B-Protein mRNA O of O Saccharomyces O cerevisiae O from O AUG O to O AUA O , O generating O a O mutation O termed O cox2 B-Protein - O 10 O . O This O mutation O reduced O translation O of O the O COX2 B-Protein mRNA O at O least O five O - O fold O without O affecting O the O steady O - O state O level O of O the O mRNA O , O and O produced O a O leaky O nonrespiratory O growth O phenotype O . O To O address O the O question O of O whether O residual O translation O of O the O cox2 B-Protein - O 10 O mRNA O was O initiating O at O the O altered O initiation O codon O or O at O the O next O AUG O codon O downstream O ( O at O position O 14 O ) O , O we O took O advantage O of O the O fact O that O the O mature O coxII B-Protein protein O is O generated O from O the O electrophoretically O distinguishable O coxII B-Protein precursor O by O removal O of O the O amino O - O terminal O 15 O residues O , O and O that O this O processing O can O be O blocked O by O a O mutation O in O the O nuclear O gene O PET2858 B-Protein . O We O constructed O a O pet2858 B-Protein , O cox2 B-Protein - O 10 O double O mutant O strain O using O a O pet2858 B-Protein allele O from O our O mutant O collection O . O The O double O mutant O accumulated O low O levels O of O a O polypeptide O which O comigrated O with O the O coxII B-Protein precursor O protein O , O not O the O mature O species O , O providing O strong O evidence O that O residual O initiation O was O occurring O at O the O mutant O AUA O codon O . O Residual O translation O of O the O mutant O mRNA O required O the O COX2 B-Protein mRNA O - O specific O activator O PET111 B-Protein . O Furthermore O , O growth O of O cox2 B-Protein - O 10 O mutant O strains O was O sensitive O to O alterations O in O PET111 B-Protein gene O dosage O : O the O respiratory O - O defective O growth O phenotype O was O partially O suppressed O in O haploid O strains O containing O PET111 B-Protein on O a O high O - O copy O - O number O vector O , O but O became O more O severe O in O diploid O strains O containing O only O one O functional O copy O of O PET111 B-Protein . O Biosynthesis O of O GlyCAM B-Protein - I-Protein 1 I-Protein , O a O mucin B-Family - O like O ligand O for O L B-Protein - I-Protein selectin I-Protein . O L B-Protein - I-Protein selectin I-Protein , O a O member O of O the O selectin B-Family family O of O leukocyte O - O endothelial O adhesion O proteins O , O mediates O the O initial O attachment O of O lymphocytes O to O lymph O node O high O endothelial O venules O during O lymphocyte O recirculation O . O One O of O the O endothelial O - O associated O ligands O for O L B-Protein - I-Protein selectin I-Protein is O GlyCAM B-Protein - I-Protein 1 I-Protein , O a O mucin B-Family - O like O glycoprotein O , O which O presents O novel O sulfated O , O sialylated O and O fucosylated O O O - O glycans O . O In O order O to O understand O the O generation O of O these O glycans O , O we O have O examined O the O biosynthesis O of O GlyCAM B-Protein - I-Protein 1 I-Protein in O lymph O node O organ O culture O . O Using O peptide O - O specific O antibodies O , O lectins B-Family , O and O recombinant O L B-Protein - I-Protein selectin I-Protein , O we O detected O the O following O species O of O GlyCAM B-Protein - I-Protein 1 I-Protein : O unglycosylated O ( O < O 28 O kDa O ) O ; O modified O with O GalNAc O only O ( O 28 O - O 33 O kDa O ) O ; O modified O with O sialic O acid O , O fucose O , O and O sulfate O but O lacking O L B-Protein - I-Protein selectin I-Protein reactivity O ( O 40 O - O 50 O kDa O ) O ; O and O mature O ( O L B-Protein - I-Protein selectin I-Protein - O reactive O ) O ligand O ( O 50 O - O 60 O kDa O ) O . O Pulse O - O chase O labeling O at O 15 O degrees O C O suggested O that O GalNAc O is O added O in O a O pre O - O Golgi O compartment O . O Treatment O with O brefeldin B-Chemical A I-Chemical almost O completely O blocked O sulfation O , O indicating O that O this O modification O occurs O in O the O trans O - O Golgi O network O . O Two O distinct O sialylation O events O occurred O in O the O presence O of O brefeldin B-Chemical A I-Chemical , O while O fucosylation O was O partially O blocked O . O We O conclude O that O sialylation O precedes O both O fucosylation O and O sulfation O during O biosynthesis O . O This O ordering O will O help O to O identify O the O critical O acceptor O structures O recognized O by O lymph O node O glycosyltransferases O and O sulfotransferases O . O P B-Protein - I-Protein selectin I-Protein is O acylated O with O palmitic B-Chemical acid I-Chemical and O stearic B-Chemical acid I-Chemical at O cysteine O 766 O through O a O thioester O linkage O . O We O report O that O the O adhesion O receptor O P B-Protein - I-Protein selectin I-Protein can O be O metabolically O labeled O with O [ O 3H O ] O palmitic B-Chemical acid I-Chemical in O human O platelets O . O Analysis O of O alkaline O methanolysis O products O from O labeled O protein O demonstrated O that O the O radioactivity O associated O with O P B-Protein - I-Protein selectin I-Protein was O covalently O bound O palmitic B-Chemical acid I-Chemical . O [ O 3H O ] O Palmitic B-Chemical acid I-Chemical was O cleaved O by O hydroxylamine B-Chemical treatment O at O neutral O pH O and O by O reducing O agents O , O indicating O that O acylation O occurred O through O a O thioester O linkage O . O Both O stearic B-Chemical acid I-Chemical and O palmitic B-Chemical acid I-Chemical were O detected O by O gas O chromatography O - O mass O spectrometry O analysis O of O alkaline O hydrolysates O of O purified O P B-Protein - I-Protein selectin I-Protein . O Deletion O or O mutation O of O Cys766 O eliminated O [ O 3H O ] O palmitic B-Chemical acid I-Chemical labeling O of O P B-Protein - I-Protein selectin I-Protein in O transfected O COS O - O 7 O cells O . O We O conclude O that O the O cytoplasmic O domain O of O P B-Protein - I-Protein selectin I-Protein is O acylated O at O Cys766 O through O a O thioester O bond O . O Fatty O acid O acylation O may O regulate O intracellular O trafficking O or O other O functions O of O P B-Protein - I-Protein selectin I-Protein . O Terminal O marking O of O avian O triosephosphate B-Family isomerases I-Family by O deamidation O and O oxidation O . O Triosephosphate B-Family isomerase I-Family ( O TPI B-Family ) O provides O an O excellent O model O for O terminal O marking O and O protein O degradation O . O Mammalian O TPI B-Family is O terminally O modified O by O deamidation O at O Asn71 O - O Gly O , O resulting O in O unfolding O , O dissociation O , O and O proteolysis O . O In O contrast O , O chicken O TPI B-Family , O which O contains O a O lysine O at O position O 71 O , O is O terminally O modified O by O the O oxidation O of O Cys126 O . O Thus O , O both O deamidation O and O oxidation O initiate O degradation O of O TPI B-Family from O different O species O . O To O explore O the O terminal O marking O in O other O avians O , O we O have O purified O the O turkey O TPI B-Family to O homogeneity O and O determined O its O characteristics O . O Although O the O molecular O properties O of O the O turkey O and O chicken O TPI B-Family were O very O similar O , O their O tolerances O to O temperature O , O oxidants O , O and O alkaline O pH O were O very O different O . O For O example O , O chicken O TPI B-Family was O inactivated O 80 O % O in O either O 10 O mM O oxidized O glutathione B-Chemical or O H2O2 B-Chemical , O whereas O 120 O mM O GSSG B-Chemical had O no O effect O on O turkey O TPI B-Family , O and O > O 120 O mM O H2O2 B-Chemical was O needed O for O 80 O % O inactivation O . O Under O alkaline O conditions O that O cause O rapid O deamidation O of O the O mammalian O TPI B-Family , O neither O avian O enzyme O deamidated O . O Chicken O TPI B-Family , O however O , O aggregated O . O Aggregation O was O reversed O by O 2 B-Chemical - I-Chemical mercaptoethanol I-Chemical . O Under O prolonged O exposure O to O milder O conditions O the O turkey O enzyme O was O completely O inactivated O and O deamidated O at O Asn15 O - O Gly O . O Thus O , O there O are O marked O differences O in O the O susceptibility O of O these O two O avian O enzymes O to O oxidation O and O deamidation O , O and O their O terminal O marking O mechanisms O appear O to O be O different O . O Molecular O cloning O of O ras B-Family and O rap B-Family genes O from O Entamoeba O histolytica O . O To O better O understand O growth O regulation O in O the O protozoan O parasite O Entamoeba O histolytica O , O ameba O genes O homologous O to O the O ras B-Family oncogene O and O rap B-Family ( O Krev B-Protein - I-Protein 1 I-Protein ) O anti O - O oncogene O were O cloned O . O Two O putative O ameba O ras B-Family genes O ( O Ehras1 B-Protein and O Ehras2 B-Protein ) O were O identified O , O which O contain O 205 O and O 203 O amino O acid O ( O aa O ) O open O reading O frames O ( O ORFs O ) O , O respectively O . O The O Ehras1 B-Protein ORF O shows O an O 91 O % O positional O identity O with O that O of O Ehras2 B-Protein , O a O 55 O % O identity O with O Dictyostelium O discoideum O ( O Dd O ) O ras B-Family , O and O a O 47 O % O identity O with O human O ( O Hs O ) O ras B-Family . O Two O ameba O rap B-Family genes O ( O Ehrap1 B-Protein and O Ehrap2 B-Protein ) O were O identified O , O both O of O which O contain O 184 O - O aa O ORFs O . O The O Ehrap1 B-Protein ORF O shows O a O 93 O % O positional O identity O with O that O of O Ehrap2 B-Protein , O a O 60 O % O identity O with O Dd O rap B-Family , O a O 61 O % O identity O with O Hs O Krev B-Protein - I-Protein 1 I-Protein , O and O a O 45 O % O identity O with O that O of O Ehras1 B-Protein . O Conserved O aa O in O each O ameba O ras B-Family and O rap B-Family ORF O include O GTP O - O binding O sites O , O effector O site O , O site O of O ADP O - O ribosylation O by O Pseudomonas O exoenzyme B-Protein S I-Protein , O and O COOH O - O terminus O CAAX O . O As O all O Xs O = O Leu O or O Phe O , O ameba O ras B-Family and O rap B-Family proteins O may O be O gerenylgerenylated O and O not O farnesylated O . O Both O ras B-Family and O rap B-Family genes O are O transcribed O by O trophozoites O . O A O single O 21 O - O kDa O ameba O ras B-Family protein O reacts O with O the O rat O Y13 O - O 259 O anti O - O ras B-Family monoclonal O antibody O , O which O is O located O on O the O cytosolic O side O of O the O plasma O membrane O . O These O are O the O first O ras B-Family and O rap B-Family genes O identified O from O a O protozoan O parasite O . O Isoprenylation O of O large B-Protein hepatitis I-Protein delta I-Protein antigen I-Protein is O necessary O but O not O sufficient O for O hepatitis O delta O virus O assembly O . O Hepatitis O delta O virus O ( O HDV O ) O encodes O two O proteins O , O the O small B-Protein hepatitis I-Protein delta I-Protein antigen I-Protein ( O SHDAg B-Protein ) O and O large B-Protein hepatitis I-Protein delta I-Protein antigen I-Protein ( O LHDAg B-Protein ) O . O Both O proteins O are O identical O except O for O the O presence O of O additional O 19 O amino O acids O at O the O C O terminus O of O LHDAg B-Protein . O While O SHDAg B-Protein is O required O for O HDV O RNA O replication O , O LHDAg B-Protein inhibits O replication O and O is O required O together O with O hepatitis O B O surface O antigen O for O the O assembly O of O HDV O . O The O C O - O terminal O last O 4 O amino O acids O of O LHDAg B-Protein ( O Cys O - O Arg O - O Pro O - O Gln O ) O is O an O isoprenylation O motif O . O It O has O previously O been O shown O that O the O mutation O of O the O Cys O inhibited O the O assembly O of O HDV O . O In O order O to O discern O whether O this O effect O is O due O to O change O of O amino O acid O residue O or O abolition O of O isoprenylation O , O we O constructed O several O LHDAg B-Protein mutants O of O the O terminal O three O amino O acid O residues O and O tested O their O abilities O to O be O packaged O with O HBsAg B-Protein by O cotransfection O experiments O . O We O also O made O GST O - O fusion O proteins O of O these O mutants O and O tested O their O abilities O to O be O isoprenylated O in O rabbit O reticulocyte O lysate O system O . O We O found O that O some O , O but O not O all O , O of O the O substitutions O of O the O amino O acid O residues O other O than O the O Cys O also O inhibited O isoprenylation O and O that O the O status O of O isoprenylation O of O these O mutant O proteins O correlated O well O with O their O abilities O to O be O packaged O with O HBsAg B-Protein into O virions O . O This O result O indicates O that O isoprenylation O , O rather O than O the O primary O amino O acid O sequence O , O is O required O for O LHDAg B-Protein packaging O . O Furthermore O , O we O found O that O the O attachment O of O an O isoprenylation O motif O to O SHDAg B-Protein did O not O enable O it O to O be O packaged O with O HBsAg B-Protein and O that O the O deletions O of O any O 5 O amino O acids O in O the O last O 15 O amino O acids O ( O amino O acids O 196 O to O 210 O ) O unique O to O the O LHDAg B-Protein abolished O the O packaging O ability O . O In O contrast O , O the O deletion O of O 33 O amino O acids O ( O amino O acids O 163 O to O 195 O ) O upstream O of O the O last O C O - O terminal O 19 O amino O acids O of O LHDAg B-Protein did O not O interfere O with O its O packaging O ability O . O Therefore O , O we O conclude O that O the O 15 O amino O acids O upstream O of O the O isoprenylation O site O of O LHDAg B-Protein are O also O essential O for O HDV O assembly O , O and O a O large O portion O of O the O alleged O C O - O terminal O Pro O / O Gly O - O rich O region O ( O amino O acids O 146 O to O 195 O ) O is O not O required O for O the O assembly O process O . O The O mevalonate B-Chemical pathway O : O importance O in O mesangial O cell O biology O and O glomerular O disease O . O Products O of O intracellular O mevalonate B-Chemical metabolism O are O critical O for O the O growth O and O proliferation O of O eukaryotic O cells O . O These O products O include O cholesterol B-Chemical and O several O nonsterol B-Chemical isoprenoids I-Chemical . O The O isoprenoid B-Chemical farnesyl B-Chemical is O a O particularly O important O intermediate O in O the O mevalonate B-Chemical pathway O . O Farnesyl B-Chemical can O be O used O to O synthesize O cholesterol B-Chemical and O can O also O bind O covalently O to O several O low O molecular O mass O GTP B-Family - I-Family binding I-Family proteins I-Family such O as O p21 B-Protein ras I-Protein . O Farnesylated O p21 B-Protein ras I-Protein may O be O critical O for O mitogenic O signalling O stimulated O by O growth O factors O such O as O platelet B-Family - I-Family derived I-Family growth I-Family factor I-Family . O Inhibitors O of O the O enzyme O 3 B-Protein - I-Protein hydroxy I-Protein - I-Protein 3 I-Protein - I-Protein methylglutaryl I-Protein coenzyme I-Protein A I-Protein reductase I-Protein , O such O as O lovastatin B-Chemical and O compactin B-Chemical , O block O the O production O of O mevalonate B-Chemical and O its O metabolites O . O These O agents O have O been O shown O to O inhibit O proliferation O of O many O cell O types O . O Recently O we O demonstrated O that O lovastatin B-Chemical inhibited O proliferation O of O cultured O glomerular O mesangial O cells O . O Lovastatin B-Chemical inhibition O was O overcome O by O the O simultaneous O addition O of O either O mevalonate B-Chemical or O farnesol B-Chemical , O but O not O by O exogenous O low O density O lipoprotein O cholesterol B-Chemical . O These O results O suggested O that O farnesyl B-Chemical is O critical O for O mesangial O cell O proliferation O . O In O several O experimental O models O of O renal O disease O , O chronic O lovastatin B-Chemical administration O reduced O the O extent O of O glomerular O injury O . O The O beneficial O effects O of O lovastatin B-Chemical have O been O attributed O to O lowering O of O circulating O lipid O and O lipoprotein O levels O . O In O view O of O recent O data O , O however O , O it O is O possible O that O lovastatin B-Chemical may O act O to O reduce O glomerular O injury O , O at O least O in O part O , O through O a O direct O action O on O mesangial O cell O proliferation O . O Vitamin B-Protein K I-Protein - I-Protein dependent I-Protein carboxylase I-Protein activity O , O prothrombin B-Protein mRNA O , O and O prothrombin B-Protein production O in O two O cultured O rat O hepatoma O cell O lines O . O The O presence O of O under O - O gamma O - O carboxylated O forms O of O plasma O prothrombin B-Protein is O a O marker O for O human O primary O hepatocellular O carcinoma O . O A O rat O hepatoma O cell O line O ( O 7777 O ) O which O was O previously O shown O to O secrete O undercarboxylated O prothrombin B-Protein when O grown O as O a O solid O tumor O has O now O been O grown O in O monolayer O culture O . O This O cell O line O has O a O decreased O activity O of O the O microsomal O vitamin B-Protein K I-Protein - I-Protein dependent I-Protein carboxylase I-Protein when O compared O to O a O control O ( O H4IIEC3 O ) O hepatoma O line O , O does O not O increase O intracellular O prothrombin B-Protein concentrations O in O response O to O vitamin B-Chemical K I-Chemical depletion O , O and O secretes O undercarboxylated O prothrombin B-Protein even O when O grown O in O vitamin B-Chemical K I-Chemical supplemented O media O . O Prothrombin B-Protein gene O expression O in O the O 7777 O cell O line O , O as O measured O by O prothrombin B-Protein mRNA O levels O , O was O not O altered O in O the O 7777 O cell O line O . O This O cell O line O appears O to O be O a O model O for O assessing O the O cellular O alterations O responsible O for O undercarboxylated O prothrombin B-Protein excretion O by O human O hepatocellular O tumors O . O Multiple O palmitoylation O of O synaptotagmin B-Family and O the O t B-Family - I-Family SNARE I-Family SNAP B-Protein - I-Protein 25 I-Protein . O Synaptotagmin B-Family , O a O likely O calcium B-Chemical sensor O for O synaptic O transmission O , O and O SNAP B-Protein - I-Protein 25 I-Protein , O a O t B-Family - I-Family SNARE I-Family of O the O presynaptic O plasma O membrane O , O are O key O proteins O for O the O docking O and O fusion O of O synaptic O and O other O vesicles O . O We O report O that O both O synaptotagmin B-Family and O SNAP B-Protein - I-Protein 25 I-Protein are O palmitoylated O with O their O fatty O acids O attached O in O a O labile O thioester O - O type O bond O . O A O SNAP B-Protein - I-Protein 25 I-Protein mutant O with O deleted O palmitoylation O sites O is O found O exclusively O in O the O cytosol O after O cell O fractionation O , O whereas O the O palmitoylated O form O of O SNAP B-Protein - I-Protein 25 I-Protein is O membrane O - O bound O , O establishing O that O SNAP B-Protein - I-Protein 25 I-Protein is O membrane O - O anchored O via O covalently O linked O palmitate B-Chemical . O Purification O of O a O protein O palmitoyltransferase O that O acts O on O H B-Protein - I-Protein Ras I-Protein protein O and O on O a O C O - O terminal O N B-Protein - I-Protein Ras I-Protein peptide O . O Mammalian O H B-Protein - I-Protein Ras I-Protein and O N B-Protein - I-Protein Ras I-Protein are O GTP B-Family - I-Family binding I-Family proteins I-Family that O must O be O post O - O translationally O lipidated O to O function O as O molecular O switches O in O signal O transduction O cascades O controlling O cell O growth O and O differentiation O . O These O proteins O contain O a O C O - O terminal O farnesyl O - O cysteine O alpha O - O methyl O ester O and O palmitoyl O groups O attached O to O nearby O cysteines O . O Data O is O presented O showing O that O rat O liver O microsomes O contain O an O enzyme O that O transfers O the O palmitoyl O group O from O palmitoyl B-Chemical - I-Chemical coenzyme I-Chemical A I-Chemical to O cysteine O residues O of O H B-Protein - I-Protein Ras I-Protein protein O and O of O a O synthetic O peptide O having O the O structure O of O the O C O terminus O of O N B-Protein - I-Protein Ras I-Protein . O This O protein O palmitoyltransferase O ( O PPT B-Protein ) O was O solubilized O from O membranes O and O purified O 10 O , O 500 O - O fold O to O apparent O homogeneity O with O an O overall O yield O of O 10 O % O . O On O an O SDS B-Chemical gel O , O PPT B-Protein appears O as O two O proteins O of O molecular O masses O of O approximately O 30 O and O approximately O 33 O kDa O . O If O the O palmitoylation O sites O of O the O N B-Protein - I-Protein Ras I-Protein peptide O ( O the O non O - O farnesylated O cysteine O ) O or O H B-Protein - I-Protein Ras I-Protein protein O ( O cysteines O 181 O and O 184 O ) O are O changed O to O serine O , O palmitoylation O by O PPT B-Protein does O not O occur O . O Non O - O farnesylated O H B-Protein - I-Protein Ras I-Protein produced O in O bacteria O as O well O as O in O vitro O farnesylated O bacterial O H B-Protein - I-Protein Ras I-Protein are O not O substrates O for O PPT B-Protein nor O is O the O non O - O farnesylated O , O methylated O N B-Protein - I-Protein Ras I-Protein peptide O . O These O results O suggest O , O but O do O not O prove O , O that O farnesylation O and O possibly O C O - O terminal O methylation O are O prerequisites O for O Ras B-Family palmitoylation O . O PPT B-Protein shows O a O large O preference O for O palmitoyl B-Chemical - I-Chemical coenzyme I-Chemical A I-Chemical over O myristoyl B-Chemical - I-Chemical coenzyme I-Chemical as O the O acyl O donor O . O Values O of O Km O for O palmitoyl B-Chemical - I-Chemical CoA I-Chemical and O H B-Protein - I-Protein Ras I-Protein are O 4 O . O 3 O + O / O - O 1 O . O 2 O and O 0 O . O 8 O + O / O - O 0 O . O 3 O microM O , O respectively O . O PPT B-Protein is O the O first O protein O palmitoyltransferase O to O be O purified O , O and O the O availability O of O pure O enzyme O should O contribute O to O our O understanding O of O the O function O and O regulation O of O Ras B-Family palmitoylation O in O cells O . O Molecular O cloning O and O characterization O of O nlpH B-Protein , O encoding O a O novel O , O surface O - O exposed O , O polymorphic O , O plasmid O - O encoded O 33 O - O kilodalton O lipoprotein O of O Borrelia O afzelii O . O Borrelia O burgdorferi O sensu O lato O organisms O , O comprising O B O . O burgdorferi O sensu O stricto O , O Borrelia O afzelii O , O and O Borrelia O garinii O , O are O tick O - O borne O pathogens O causing O Lyme O borreliosis O in O humans O . O To O identify O putative O virulence O determinants O , O a O B O . O afzelii O DNA O library O was O screened O for O Congo B-Chemical red I-Chemical dye O binding O , O a O property O associated O with O virulence O in O pathogenic O bacteria O . O One O clone O was O found O to O carry O a O 663 O - O nucleotide O - O long O open O reading O frame O encoding O a O Congo B-Chemical red I-Chemical dye O - O binding O protein O with O a O calculated O molecular O mass O of O 25 O , O 660 O Da O . O The O amino O acid O sequence O deduced O from O its O nucleotide O sequence O was O found O to O include O a O consensus O bacterial O lipidation O site O present O at O residues O 15 O to O 18 O ( O Leu O - O Ser O - O Gly O - O Cys O ) O . O The O lipoprotein O nature O was O demonstrated O by O incorporation O of O radioactive O palmitate B-Chemical ; O hence O , O this O protein O has O been O termed O NlpH B-Protein , O for O new O lipoprotein O H O . O NlpH B-Protein is O located O on O the O surface O of O B O . O afzelii O , O and O the O nlpH B-Protein gene O is O found O on O a O circular O plasmid O . O The O nlpH B-Protein gene O is O also O found O in O B O . O burgdorferi O sensu O stricto O and O B O . O garinii O . O Immediately O upstream O of O nlpH B-Protein is O located O a O smaller O reading O frame O encoding O a O polypeptide O containing O the O casein B-Family kinase I-Family II I-Family phosphorylation O recognition O sequence O , O ( O Ser O / O Thr O ) O - O X O - O Y O - O ( O Glu O / O Asp O ) O , O repeated O 10 O times O . O Restricted O expression O of O a O novel O murine O atonal B-Protein - O related O bHLH B-Family protein O in O undifferentiated O neural O precursors O . O Tissue O - O specific O bHLH B-Family proteins O play O important O roles O in O the O specification O and O differentiation O of O neural O cell O lineages O in O invertebrate O and O vertebrate O organisms O . O Two O groups O of O bHLH B-Family proteins O , O atonal B-Protein and O achaete B-Protein - I-Protein scute I-Protein , O have O proneural O activities O in O Drosophila O , O and O the O mouse O achaete B-Protein - I-Protein scute I-Protein homolog O MASH1 B-Protein is O required O for O the O differentiation O of O several O neural O lineages O . O In O a O screen O for O proteins O interacting O with O MASH1 B-Protein , O we O have O isolated O a O novel O bHLH B-Family protein O related O to O atonal B-Protein , O named O MATH4A B-Protein , O which O is O broadly O expressed O in O neural O precursor O cells O in O the O mouse O embryonic O CNS O and O PNS O . O Interaction O assays O in O yeast O and O in O vitro O demonstrate O that O MATH4A B-Protein interacts O efficiently O with O both O MASH1 B-Protein and O the O ubiquitous O bHLH B-Family protein O E12 B-Protein . O MATH4A B-Protein - O E12 B-Protein heterodimers O , O but O not O MATH4A B-Protein - O MASH1 B-Protein , O bind O to O a O consensus O E O - O box O sequence O . O Math4A B-Protein expression O is O restricted O to O undifferentiated O neural O precursors O and O is O complementary O to O that O of O Mash1 B-Protein in O most O regions O of O the O nervous O system O . O In O particular O , O Math4A B-Protein is O transcribed O at O high O levels O in O the O cerebral O cortex O , O dorsal O thalamus O , O and O epibranchial O placodes O , O which O present O little O or O no O Mash1 B-Protein expression O . O However O , O expression O of O the O two O genes O shows O limited O overlap O in O certain O CNS O regions O ( O retina O , O preoptic O area O of O the O hypothalamus O , O midbrain O , O hindbrain O ) O . O Its O structure O and O expression O pattern O suggest O that O MATH4A B-Protein may O regulate O an O early O step O of O neural O development O , O either O as O a O partner O of O ubiquitous O bHLH B-Family proteins O or O associated O with O other O neural O - O specific O bHLH O proteins O . O Transcription B-Protein elongation I-Protein factor I-Protein S I-Protein - I-Protein II I-Protein is O not O required O for O transcription O - O coupled O repair O in O yeast O . O Two O different O subpathways O play O a O role O in O removal O of O UV O - O induced O cyclobutane O pyrimidine O dimers O ( O CPDs O ) O by O nucleotide O excision O repair O ( O NER O ) O . O The O relatively O slow O global O genome O repair O subpathway O operates O on O all O CPDs O irrespective O of O their O position O in O the O DNA O , O whereas O the O transcription O - O coupled O repair O subpathway O is O responsible O for O the O rapid O removal O of O CPDs O from O transcribed O strands O . O In O Saccharomyces O cerevisiae O , O the O RAD26 B-Protein gene O is O implicated O in O transcription O - O coupled O repair O . O However O , O transcription O - O coupled O repair O is O not O completely O absent O in O rad26 B-Protein mutants O , O and O therefore O other O gene O products O are O possibly O involved O in O this O subpathway O . O Based O on O in O vitro O experiments O with O purified O components O , O the O transcription B-Protein elongation I-Protein factor I-Protein S I-Protein - I-Protein II I-Protein appeared O to O be O a O candidate O for O a O function O in O transcription O - O coupled O repair O . O To O investigate O a O possible O role O of O S B-Protein - I-Protein II I-Protein in O transcription O - O coupled O repair O in O vivo O in O yeast O , O S B-Protein - I-Protein II I-Protein null O mutations O were O introduced O into O various O genetic O backgrounds O differing O in O NER O capacity O . O UV O sensitivity O was O not O altered O by O disruption O of O the O S B-Protein - I-Protein II I-Protein gene O in O a O RAD O + O ( O NER O proficient O ) O strain O , O or O in O rad26 B-Protein ( O impaired O in O efficient O transcription O - O coupled O repair O ) O , O rad7 B-Protein ( O lacking O global O genome O repair O ) O , O or O rad7 B-Protein rad26 B-Protein ( O lacking O global O genome O repair O , O but O having O residual O transcription O - O coupled O repair O capacity O ) O mutants O . O Moreover O , O S B-Protein - I-Protein II I-Protein did O not O influence O the O repair O rate O on O the O transcribed O strand O of O the O RPB2 B-Protein gene O , O either O in O repair O - O proficient O or O in O rad7 B-Protein rad26 B-Protein backgrounds O . O Hence O , O transcription O - O coupled O repair O is O fully O functional O in O yeast O cells O lacking O the O gene O encoding O S B-Protein - I-Protein II I-Protein . O Furthermore O , O S B-Protein - I-Protein II I-Protein is O not O required O for O the O Rad26 B-Protein - O independent O residual O transcription O - O coupled O repair O in O vivo O . O An O interesting O property O of O plakoglobin B-Protein is O that O it O binds O to O both O classical B-Family and O desmosomal B-Family cadherins I-Family . O The O observation O that O DP B-Protein - I-Protein NTP I-Protein binds O to O plakoglobin B-Protein and O clusters O desmosomal O cadherin B-Protein - O plakoglobin B-Protein complexes O implies O that O desmoplakin B-Protein binds O to O plakoglobin B-Protein that O is O associated O with O the O desmosomal O cadherin B-Protein cytoplasmic O domain O . O In O contrast O , O plakoglobin B-Protein that O is O associated O with O the O desmosomal O cadherin B-Protein cytoplasmic O domain O is O unable O to O bind O to O alpha B-Protein - I-Protein catenin I-Protein ( O Fig O . O 8 O ) O . O Recent O studies O have O demonstrated O that O the O alpha B-Protein - I-Protein catenin I-Protein and O Dsg1 B-Protein binding O sites O on O plakoglobin B-Protein overlap O in O the O amino O - O terminal O armadillo O repeats O of O plakoglobin B-Protein , O suggesting O that O Dsg1 B-Protein and O alpha B-Protein - I-Protein catenin I-Protein cannot O bind O to O the O same O plakoglobin B-Protein molecule O simultaneously O . O However O , O the O classical O cadherins O bind O to O the O central O armadillo O repeats O of O plakoglobin B-Protein , O leaving O the O amino O - O terminal O armadillo O repeats O of O plakoglobin B-Protein available O to O bind O alpha B-Protein - I-Protein catenin I-Protein . O In O the O present O study O , O deletion O mutants O of O plakoglobin B-Protein lacking O either O the O amino O - O or O carboxyl O - O terminal O domain O co O - O immunoprecipitated O with O DP B-Protein - I-Protein NTP I-Protein and O were O clustered O in O L O - O cells O co O - O expressing O DP B-Protein - I-Protein NTP I-Protein . O These O results O , O along O with O the O ability O of O the O amino O - O terminal O plakoglobin B-Protein deletion O to O interact O with O DP B-Protein - I-Protein NTP I-Protein in O the O two O hybrid O system O , O suggest O that O desmoplakin B-Protein may O bind O to O the O central O armadillo O repeats O of O plakoglobin B-Protein . O In O A431 O cells O , O the O expression O of O plakoglobin B-Protein deletion O mutants O lacking O the O amino O - O and O carboxyl O - O terminal O end O domains O promoted O the O assembly O of O elongated O and O fused O desmosomes O , O suggesting O that O the O central O armadillo O repeats O of O plakoglobin B-Protein can O promote O interactions O between O desmosomal O proteins O , O which O may O include O desmoplakin B-Protein . O Future O studies O will O be O directed O at O mapping O precisely O where O desmoplakin B-Protein binds O to O plakoglobin B-Protein in O relation O to O the O sites O on O plakoglobin B-Protein that O bind O to O the O desmosomal B-Protein cadherins I-Protein . O Such O studies O should O provide O insight O into O the O mechanisms O by O which O the O cadherin B-Protein cytoplasmic O tails O govern O the O domains O on O plakoglobin B-Protein that O are O available O for O interactions O with O either O alpha B-Protein - I-Protein catenin I-Protein or O desmoplakin B-Protein . O It O appears O that O the O removal O of O alpha B-Protein - I-Protein SNAP I-Protein stimulation O of O NSF B-Protein ATPase O activity O results O in O a O mutant O unable O to O stimulate O exocytosis O in O permeabilized O chromaffin O cells O . O It O is O probable O that O exogenously O added O alpha B-Protein - I-Protein SNAP I-Protein must O first O interact O with O an O endogenous O factor O , O probably O SNAREs B-Complex , O before O the O stimulation O of O NSF B-Protein . O Addition O of O an O alpha B-Protein - I-Protein SNAP I-Protein mutant O that O is O able O to O bind O but O not O stimulate O NSF B-Protein would O be O predicted O to O inhibit O the O action O of O exogenously O added O alpha B-Protein - I-Protein SNAP I-Protein in O chromaffin O cells O . O To O test O this O possibility O , O digitonin B-Chemical - O permeabilized O chromaffin O cells O were O preincubated O with O either O buffer O , O alpha B-Protein - I-Protein SNAP I-Protein ( O 1 O - O 160 O ) O ( O as O a O control O ) O , O alpha B-Protein - I-Protein SNAP I-Protein ( O 1 O - O 285 O ) O , O or O alpha B-Protein - I-Protein SNAP I-Protein ( O L294A O ) O for O 25 O min O and O stimulated O with O 10 O muM O Ca2 B-Chemical + I-Chemical for O 30 O min O with O or O without O 25 O mug O / O ml O alpha B-Protein - I-Protein SNAP I-Protein . O The O results O of O four O separate O experiments O were O averaged O , O and O the O results O were O expressed O as O a O percentage O of O the O 10 O muM O Ca2 B-Chemical + I-Chemical stimulation O . O When O preincubated O with O buffer O alone O , O alpha B-Protein - I-Protein SNAP I-Protein stimulated O exocytosis O above O control O levels O ( O Fig O . O 5 O B O ) O . O Preincubation O with O alpha B-Protein - I-Protein SNAP I-Protein ( O 1 O - O 160 O ) O had O no O inhibitory O effect O on O alpha B-Protein - I-Protein SNAP I-Protein action O ( O Fig O . O 5 O B O ) O , O which O would O be O predicted O because O such O a O large O truncation O would O abolish O the O ability O to O interact O with O SNAREs B-Complex . O However O , O addition O of O 25 O mug O / O ml O of O either O alpha B-Protein - I-Protein SNAP I-Protein ( O 1 O - O 285 O ) O or O alpha B-Protein - I-Protein SNAP I-Protein ( O L294A O ) O resulted O in O the O complete O inhibition O of O the O stimulatory O activity O of O exogenous O alpha B-Protein - I-Protein SNAP I-Protein . O The O inhibitory O effect O of O these O mutants O was O still O seen O , O even O when O an O additional O 10 O - O min O wash O step O was O added O between O incubation O of O mutant O proteins O and O the O stimulation O in O the O presence O of O alpha B-Protein - I-Protein SNAP I-Protein ( O data O not O shown O ) O . O It O should O be O noted O that O the O mutants O alpha B-Protein - I-Protein SNAP I-Protein ( O 1 O - O 285 O ) O and O alpha B-Protein - I-Protein SNAP I-Protein ( O L294A O ) O had O little O effect O on O endogenous O exocytosis O in O the O absence O of O added O alpha B-Protein - I-Protein SNAP I-Protein , O nor O did O they O do O so O even O up O to O concentrations O as O high O as O 150 O mug O / O ml O ( O data O not O shown O ) O . O POU O transcription O factors O Brn B-Protein - I-Protein 3a I-Protein and O Brn B-Protein - I-Protein 3b I-Protein interact O with O the O estrogen B-Protein receptor I-Protein and O differentially O regulate O transcriptional O activity O via O an O estrogen B-Chemical response O element O . O The O estrogen B-Protein receptor I-Protein ( O ER B-Protein ) O modulates O transcription O by O forming O complexes O with O other O proteins O and O then O binding O to O the O estrogen B-Chemical response O element O ( O ERE O ) O . O We O have O identified O a O novel O interaction O of O this O receptor O with O the O POU O transcription O factors O Brn B-Protein - I-Protein 3a I-Protein and O Brn B-Protein - I-Protein 3b I-Protein which O was O independent O of O ligand O binding O . O By O pull O - O down O assays O and O the O yeast O two O - O hybrid O system O , O the O POU O domain O of O Brn B-Protein - I-Protein 3a I-Protein and O Brn B-Protein - I-Protein 3b I-Protein was O shown O to O interact O with O the O DNA O - O binding O domain O of O the O ER B-Protein . O Brn B-Protein - I-Protein 3 I-Protein - O ER B-Protein interactions O also O affect O transcriptional O activity O of O an O ERE O - O containing O promoter O , O such O that O in O estradiol O - O stimulated O cells O , O Brn B-Protein - I-Protein 3b I-Protein strongly O activated O the O promoter O via O the O ERE O , O while O Brn B-Protein - I-Protein 3a I-Protein had O a O mild O inhibitory O effect O . O The O POU O domain O of O Brn B-Protein - I-Protein 3b I-Protein which O interacts O with O the O ER B-Protein was O sufficient O to O confer O this O activation O potential O , O and O the O change O of O a O single O amino O acid O in O the O first O helix O of O the O POU O homeodomain O of O Brn B-Protein - I-Protein 3a I-Protein to O its O equivalent O in O Brn B-Protein - I-Protein 3b I-Protein can O change O the O mild O repressive O effect O of O Brn B-Protein - I-Protein 3a I-Protein to O a O stimulatory O Brn B-Protein - I-Protein 3b I-Protein - O like O effect O . O These O observations O and O their O implications O for O transcriptional O regulation O by O the O ER B-Protein are O discussed O . O The O epidermal B-Protein growth I-Protein factor I-Protein receptor I-Protein associates O with O and O recruits O phosphatidylinositol B-Protein 3 I-Protein - I-Protein kinase I-Protein to O the O platelet B-Protein - I-Protein derived I-Protein growth I-Protein factor I-Protein beta I-Protein receptor I-Protein . O Receptor O tyrosine O kinases O are O classified O into O subfamilies O , O which O are O believed O to O function O independently O , O with O heterodimerization O occurring O only O within O the O same O subfamily O . O In O this O study O , O we O present O evidence O suggesting O a O direct O interaction O between O the O epidermal B-Protein growth I-Protein factor I-Protein ( I-Protein EGF I-Protein ) I-Protein receptor I-Protein ( O EGFR B-Protein ) O and O the O platelet B-Protein - I-Protein derived I-Protein growth I-Protein factor I-Protein beta I-Protein ( I-Protein PDGFbeta I-Protein ) I-Protein receptor I-Protein ( O PDGFbetaR B-Protein ) O , O members O of O different O receptor O tyrosine O kinase O subfamilies O . O We O find O that O the O addition O of O EGF B-Protein to O COS O - O 7 O cells O and O to O human O foreskin O Hs27 O fibroblasts O results O in O a O rapid O tyrosine O phosphorylation O of O the O PDGFbetaR B-Protein and O results O in O the O recruitment O of O phosphatidylinositol B-Protein 3 I-Protein - I-Protein kinase I-Protein to O the O PDGFbetaR B-Protein . O In O R1hER O cells O , O which O overexpress O the O EGFR B-Protein , O we O find O ligand O - O independent O tyrosine O phosphorylation O of O the O PDGFbetaR B-Protein and O the O constitutive O binding O of O a O substantial O amount O of O PI O - O 3 O kinase O activity O to O it O , O mimicking O the O effect O of O ligand O in O untransfected O cells O . O In O support O of O the O possibility O that O this O may O be O a O direct O interaction O , O we O show O that O the O two O receptors O can O be O coimmunoprecipitated O from O untransfected O Hs27 O fibroblasts O and O from O COS O - O 7 O cells O . O This O association O can O be O reconstituted O by O introducing O the O two O receptors O into O 293 O EBNA O cells O . O The O EGFR B-Protein / O PDGFbetaR B-Protein association O is O ligand O - O independent O in O all O cell O lines O tested O . O We O also O demonstrate O that O the O fraction O of O PDGFbetaR B-Protein bound O to O the O EGFR B-Protein in O R1hER O cells O undergoes O an O EGF B-Protein - O induced O mobility O shift O on O Western O blots O indicative O of O phosphorylation O . O Our O findings O indicate O that O direct O interactions O between O receptor O tyrosine O kinases O classified O under O different O subfamilies O may O be O more O widespread O than O previously O believed O . O Myoblast B-Protein city I-Protein , O the O Drosophila O homolog O of O DOCK180 B-Protein / O CED B-Protein - I-Protein 5 I-Protein , O is O required O in O a O Rac B-Family signaling O pathway O utilized O for O multiple O developmental O processes O . O The O Rac B-Family and O Cdc42 B-Protein GTPases O share O several O regulators O and O effectors O , O yet O perform O distinct O biological O functions O . O The O factors O determining O such O specificity O in O vivo O have O not O been O identified O . O In O a O mutational O screen O in O Drosophila O to O identify O Rac B-Family - O specific O signaling O components O , O we O isolated O 11 O alleles O of O myoblast B-Protein city I-Protein ( O mbc B-Protein ) O . O mbc B-Protein mutant O embryos O exhibit O defects O in O dorsal O closure O , O myogenesis O , O and O neural O development O . O DOCK180 B-Protein , O the O mammalian O homolog O of O Mbc B-Protein , O associates O with O Rac B-Family , O but O not O Cdc42 B-Protein , O in O a O nucleotide O - O independent O manner O . O These O results O suggest O that O Mbc B-Protein is O a O specific O upstream O regulator O of O Rac B-Family activity O that O mediates O several O morphogenetic O processes O in O Drosophila O embryogenesis O . O Crm1p B-Protein mediates O regulated O nuclear O export O of O a O yeast O AP B-Family - I-Family 1 I-Family - I-Family like I-Family transcription I-Family factor I-Family . I-Family The O yeast O AP B-Family - I-Family 1 I-Family - I-Family like I-Family transcription I-Family factor I-Family , O Yap1p B-Protein , O activates O genes O required O for O the O response O to O oxidative O stress O . O Yap1p B-Protein is O normally O cytoplasmic O and O inactive O , O but O will O activate O by O nuclear O translocation O if O cells O are O placed O in O an O oxidative O environment O . O Here O we O show O that O Yap1p B-Protein is O a O target O of O the O beta B-Family - I-Family karyopherin I-Family - I-Family like I-Family nuclear I-Family exporter I-Family , O Crm1p B-Protein . O Yap1p B-Protein is O constitutively O nuclear O in O a O crm1 B-Protein mutant O , O and O Crm1p B-Protein binds O to O a O nuclear O export O sequence O ( O NES O ) O - O like O sequence O in O Yap1p B-Protein in O the O presence O of O RanGTP B-Protein . O Recognition O of O Yap1p B-Protein by O Crm1p B-Protein is O inhibited O by O oxidation O , O and O this O inhibition O requires O at O least O one O of O the O three O cysteine O residues O flanking O the O NES O . O These O results O suggest O that O Yap1p B-Protein localization O is O largely O regulated O at O the O level O of O nuclear O export O , O and O that O the O oxidation O state O affects O the O accessibility O of O the O Yap1p B-Protein NES O to O Crm1p B-Protein directly O . O We O also O show O that O a O mutation O in O RanGAP B-Protein ( O rna1 B-Protein - I-Protein 1 I-Protein ) O is O synthetically O lethal O with O crm1 B-Protein mutants O . O Yap1p B-Protein export O is O inhibited O in O both O rna1 B-Protein - I-Protein 1 I-Protein and O prp20 B-Protein ( O RanGNRF B-Protein ) O mutant O strains O , O but O Yap1p B-Protein rapidly O accumulates O at O the O nuclear O periphery O after O shifting O rna1 B-Protein - I-Protein 1 I-Protein , O but O not O other O mutant O cells O to O the O non O - O permissive O temperature O . O Thus O , O disassembly O of O export O complexes O in O response O to O RanGTP B-Protein hydrolysis O may O be O required O for O release O of O substrate O from O a O terminal O binding O site O at O the O nuclear O pore O complex O ( O NPC O ) O . O hnRNP B-Protein H I-Protein is O a O component O of O a O splicing O enhancer O complex O that O activates O a O c B-Protein - I-Protein src I-Protein alternative O exon O in O neuronal O cells O . O The O regulation O of O the O c B-Protein - I-Protein src I-Protein N1 O exon O is O mediated O by O an O intronic O splicing O enhancer O downstream O of O the O N1 O 5 O ' O splice O site O . O Previous O experiments O showed O that O a O set O of O proteins O assembles O onto O the O most O conserved O core O of O this O enhancer O sequence O specifically O in O neuronal O WERI O - O 1 O cell O extracts O . O The O most O prominent O components O of O this O enhancer O complex O are O the O proteins O hnRNP B-Protein F I-Protein , O KSRP B-Protein , O and O an B-Protein unidentified I-Protein protein I-Protein of I-Protein 58 I-Protein kDa I-Protein ( O p58 B-Protein ) O . O This O p58 B-Protein protein O was O purified O from O the O WERI O - O 1 O cell O nuclear O extract O by O ammonium B-Chemical sulfate I-Chemical precipitation O , O Mono O Q O chromatography O , O and O immunoprecipitation O with O anti O - O Sm O antibody O Y12 O . O Peptide O sequence O analysis O of O purified O p58 B-Protein protein O identified O it O as O hnRNP B-Protein H I-Protein . O Immunoprecipitation O of O hnRNP B-Protein H I-Protein cross O - O linked O to O the O N1 O enhancer O RNA O , O as O well O as O gel O mobility O shift O analysis O of O the O enhancer O complex O in O the O presence O of O hnRNP B-Protein H I-Protein - O specific O antibodies O , O confirmed O that O hnRNP B-Protein H I-Protein is O a O protein O component O of O the O splicing O enhancer O complex O . O Immunoprecipitation O of O splicing O intermediates O from O in O vitro O splicing O reactions O with O anti O - O hnRNP B-Protein H I-Protein antibody O indicated O that O hnRNP B-Protein H I-Protein remains O bound O to O the O src B-Protein pre O - O mRNA O after O the O assembly O of O spliceosome O . O Partial O immunodepletion O of O hnRNP B-Protein H I-Protein from O the O nuclear O extract O partially O inactivated O the O splicing O of O the O N1 O exon O in O vitro O . O This O inhibition O of O splicing O can O be O restored O by O the O addition O of O recombinant O hnRNP B-Protein H I-Protein , O indicating O that O hnRNP B-Protein H I-Protein is O an O important O factor O for O N1 O splicing O . O Finally O , O in O vitro O binding O assays O demonstrate O that O hnRNP B-Protein H I-Protein can O interact O with O the O related O protein O hnRNP B-Protein F I-Protein , O suggesting O that O hnRNPs B-Protein H I-Protein and O F B-Protein may O exist O as O a O heterodimer O in O a O single O enhancer O complex O . O These O two O proteins O presumably O cooperate O with O each O other O and O with O other O enhancer O complex O proteins O to O direct O splicing O to O the O N1 O exon O upstream O . O