Journal of Cell Science ervrlTt(rdl ta. 95.TI3 subiquitously depending is of varies TRIM32 level domain the 1995). activation but tissues, al., the mammalian et for in expressed (Fridell partner Tat binding are retroviral a TRIM32, as particular identified unknown. in largely is proteins, signaling processes, cellular these cell by various how regulated in development, al., implication and their et whether and differentiation, genes species (Nisole TRIM many of response prevalence in the proliferation, Despite antiviral 2011). Hatakeyama, and 2005; cell apoptosis oncogenesis, members, as been family processes biological also TRIM diverse such in have Several implicated homologs are species. TRIM32, and including various members, in TRIM identified protein–protein encodes 70 genome mediate than human The to more 2001). thought al., et to are (Reymond specific interactions and are which TRIMs domain, (NHL) individual LIN-41 the and and (PHD), HT2A and ryanodine homeodomain NCL-1, al., plant and additional the SP1a by domain, et the (SPRY) followed receptor including (Reymond B- usually domains, two domain is C-terminal or motif variable coiled-coil one RBCC finger, predicted The as RING 2001). a known a also and contain are they boxes, that as proteins proteins TRIM of RBCC of member family a is expanding (TRIM32) the 32 protein motif-containing Tripartite Introduction connection novel a the reveal of that results formation mutant the words: Our TRIM32 stimulating Key by growth. a events whereas cell cell network, of facilitate variety microtubule to a the complex. modulate unavailable could signaling from which was 14-3-3–TRIM32 away pathways, and pool phosphorylation and cytoplasmic multimerization disrupting ubiquitylation of distinct underwent between TRIM32 consequence a dimerized a 14-3-3 Consequently, part, in dimerization. bind in sequestered its is, cannot affecting was 14-3-3 without of 14-3-3 protein-kinase- self-association effect on to higher-order and inhibitory dependent undergo bound the autoregulatory various TRIM32 is to that interaction potential TRIM32 phosphorylated found to 14-3-3–TRIM32 of are We to The propensity Ser651. contributes which the TRIM32. bind at free bodies, ligase) TRIM32 proteins soluble of cytoplasmic of 14-3-3 ubiquitin-protein phosphorylation TRIM32-containing concentration A-catalyzed that the of E3 reduce biochemistry, formation can an and that the mechanisms proteomics (TRIM32, and quantitative autoubiquitylation 32 using TRIM32 protein prevent report, motif-containing we Here tripartite diseases. of expression Deregulated Summary 10.1242/jcs.122069 doi: 2014–2026 126, ß Science Cell of Journal 2013 January 28 Accepted ( correspondence for *Author 5 4 3 2 1 Ichimura Tohru formation and body autoubiquitylation cytoplasmic TRIM32 repress soluble to of ligase pool ubiquitin a sequester proteins 14-3-3 2014 ohaiIsobe Toshiaki eateto iceity okioUiest rdaeSho fMdcn,Spoo okio0083,Japan 060-8638, Hokkaido Sapporo, Medicine, of School Japan Graduate 160-8402, Japan University Tokyo 650-0017, Hokkaido Shinjuku, Kobe Biochemistry, University, Medicine, of Medical of Department Tokyo School Japan Neurophysiology, Graduate 192-0397, of University Tokyo Department Japan Kobe Hachioji, 239-8686, Microbiology, University, Kanagawa of Metropolitan Yokosuka, Division Tokyo Academy, Chemistry, Defense of National Department Chemistry, Applied of Department 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. RM2i nRC-H-yeTI ebroriginally member TRIM RBCC-NHL-type an is TRIM32 hshrlto,Uiutlto,PA 433 TRIM32 14-3-3, PKA, Ubiquitylation, Phosphorylation, 2 1,2, n am Hachiya Naomi and [email protected] ,Mst Taoka Masato *, ) 2 koShoji Ikuo , 4 3 ioiKato Hiroki , rpryi htTI3 a uobqiyainatvt in al., activity (here et Albor activity 2005; autoubiquitylation al., ligase et (Kudryashova has ubiquitin-protein transubiquitylation) called Another conventional TRIM32 2007). to more that al., a addition and et is CBs (Campbell between property fraction proteins Diaz-Griffero the cytoplasmic 2005; exchange al., diffuse or et 2006) (Song al., proteins et TRIM level free proper soluble the maintain of aggresome that as compartments TRIM19 function storage is may with or CBs functions precursors that particularly TRIM32 suggested studies, in have Locke TRIM5, CBs recent and 2006; of understood, al., role fully the et not Although (Albor 2009). (CBs) cells al., in et bodies expressed cytoplasmic transiently insoluble when called autoregulatory forms and puncta potential 2011). self-associates often al., two high-molecular-mass it et that has is (Kudryashova TRIM32 One properties. cells TRIMs, in many degradation Like TRIM32, its destabilize mutation dystrophy to to shown missense leading muscular recently was limb-girdle common (LGMD2H), phenotypes 2H causes the type directly neurogenic 2009); Alzheimer’s which and al., D489N, of myopathic lobe Moreover, et several 2006). occipital (Kudryashova al., the display et in cells mice (Yokota cancer and high patients skin Abnormally 2004) al., 2008). disease human et al., al., in Frosk et et demonstrated 1995; (Horn Kano been al., 2004; has et al., expression (Fridell et conditions Horn and 2002; type cell on 4 ooouSato Tomonobu , 5 hgtuuHatakeyama Shigetsugu , eerhArticle Research Trim32 5 -null , Journal of Cell Science 05]wr aee nmdu otiig[ containing medium in labeled were 2005)] bqii–rtaoesse Abre l,20;Kn tal., induce et the can overexpression Kano TRIM32 through that 2006; shown apoptosis also al., was prevent It et transforming 2008). and (Albor proliferation, system motility for ubiquitin–proteasome cell signal cell a and promote as activity, serve TRIM32 can also that reported may recently overexpression was it It their although trafficking. and regulate cells, localization to in ligases E3 levels by own used is Autoubiquitylation 2006). ta. 08.Tu,P1M el amncoa C2cl line cell PC12 monoclonal [a 14-3-3 MEF-fused cells the expressing PC12Mh Ichimura stably Thus, 2005; 2008). proteomics al., signaling tag, al., et purification et published (Ichimura PKA affinity (myc-TEV-FLAG) tandem previously in MEF the called 14-3-3 a with combined of employed technology interactions we novel pathways, identify cells To in in pathways interactions signaling 14-3-3 PKA of potential screening of proteomic Quantitative a regulation provide Results PKA- PKA/14-3-3-dependent and a the as 14-3-3 TRIM32. Nedd4-2, for of including mechanism interactor ligases ubiquitin regulated to addition other in 14-3-3- TRIM32, mass-spectrometry- the six identify results using of Our ligases repertoire quantification. ubiquitin based wider E3 of a control demonstrated this assited have In stability, localization. we activity, intracellular study their and modulate interactions to response conformation, that and in 14-3-3 molecules proteins The phosphorylation signaling eukaryotic and 2006). protein enzymes of of al., variety family et a diverse al., bind Nagaki ligase a et 2005; constitute (Bhalla al., 14-3-3 proteins E3 of et protein HECT-type Ichimura the a recruiting 2005; role Nedd4-2, by ligase, regulates ubiquitin or have direct negatively (PKA) others A kinase kinase and SGK1 protein the by We phosphorylation few regulation. that relatively demonstrated demonstrated functional documented, in well phosphorylation have is ligases studies E3 by recognition differentiation growth, is cell function. normal concentration and of TRIM32 maintenance cellular the for of crucial control proper that suggest microRNA the of let7 functions activating by differentiation neuronal lhuhteiprac fpopoyaini substrate in phosphorylation of importance the Although Shabr ta. 09.Tgte,teefnig strongly findings these Together, 2009). al., et (Schwamborn g sfr Ihmr tal., et (Ichimura isoform 13 ]y and C]Lys 6% aebe eotdt id1--s(e al ) the 1), Table only to (see limited been 14-3-3s has proteins association bind their 51 to in the PKA reported 14-3-3 of of involvement been 33 forskolin-regulated have although Interestingly, (65%) candidate 1). (Table as targets the in dataset highly proteins) (19 a showed exclusion 200-protein least or at clearly proteins) range displayed (32 In that proteins enrichment narrow proteins twofold 51 200 1C). and selected (Fig. We normal the profiles. variable have of proteins that several reproducible proteins forskolin responses, to many 200 the response to examined the the contrast of we (ratio) for interactions, log identified stimulation mean the the the obtain of of of To two distribution 1B). view least independent Fig. at global S1; five from Table a recovered material with (supplementary were 14-3- tests (62%) with five times 200 interact which that five liquid We of proteins 3, 324 analysis two-dimensional identified 1A). and (Fig. this preparations by out spectrometry quantified carried mass the and chromatography–tandem with and identified purification immunoaffinity specifically then multistep tag-based associated MEF using that MEF- expressed and partners cells, non-stimulated an unlabeled, with interacting from mixed lysate was of cells amount stimulated equal from Lysate activator. PKA a ieitrcos CaMKK interactors: five ugse htms,i o l,o h neatosslce nthis our in PKA. selected of of interactions downstream the located of utility strongly were all, not screen and the if interactions most, demonstrated novel that suggested identify results to cell-type procedure These reflecting proteomic S1B, probably panel). Fig. material HEK293, (supplementary right in interaction in this not for reconstituted specificity was but association cells TFEB, (supplementary PC12 For cells S1A). HEK293 in Fig. forskolin material of cPKA absence of of the coexpression that in for by even except reconstituted tested, successfully RNF20). be were to (SMURF1, TFEB, associations signaling the in of PKA all implicated nor Intriguingly, those binding signaling (TRIM32, and 14-3-3 signaling TFEB), PKA neither PKA AKT1S1, in in WDR20, interactions TPD52L1, their 14-3-3 for for unknown but targets the on known (CaMKK based using proteins criteria: 11 assay chose We three co-transfection (cPKA). PKA a of subunit catalytic performed we interactions, [ 13 odrcl sestecnrbto fPAt h observed the to PKA of contribution the assess directly To ]r n tmltdfr2 iue ih50 with minutes 20 for stimulated and C]Arg yarrows. by oaihial.Tectf ons( plotted points proteins cutoff identified The reproducibly logarithmically. ( the analysis. of number each the change and in interaction total) proteins proteins identified (200 uniquely analyses consistently of two proteins least of at number in the identified shows figure The analyses. spectrometry. mass tandem ( MS/MS, two-dimensional; chromatography; 2D, liquid pathways. LC, signaling PKA in interactions signaling PKA in analysis interactome pathways. 14-3-3 Quantitative 1. Fig. B hrceiaino 433itrcosietfe rmfive from identified interactors 14-3-3 of Characterization ) a ed-,T,TR2,kontresfr14-3-3 for targets known TORC2), TH, Nedd4-2, , euaino RM2b 4332015 14-3-3 by TRIM32 of Regulation ( g A taeyfrietfigadqatfig14-3-3 quantifying and identifying for Strategy ) eeprfe rmtecmie ellysates cell combined the from purified were a ed-,PEA HadTORC2. and TH PDE3A, Nedd4-2, , 6 -odcag)aeindicated are change) 2-fold C Relative ) m forskolin, M Journal of Cell Science N1Srn/henn-rti iaeWK .4Yes Yes 2.04 WNK1 kinase Serine/threonine-protein 0.25 Yes No No Yes 2.21 No Yes dynamics microtubule of Regulator 0.30 WNK1 0.27 0.18 2.39 mKIAA0226 0.37 to TRIM32 ubiquitin-protein similar PREDICTED: ligase E3 FAM82A2 No voltage-gated channel, Potassium No KIAA0226 2.58 4 Yes deacetylase binding promoter Histone c-myc TRIM32 (proline-rich) Yes to Similar 1 substrate 2.54 AKT1 1 Yes protein 2.59 1 interacting protein-like CASK factor KCNC1 exchange nucleotide Rho-guanine 3.16 Cordon-bleu 1 RNF20 ubiquitin-protein ligase protein E3 binding Yes HDAC4 KARP-1 0.36 cytomatrix Yes (presynaptic AKT1S1 MYCBP Bassoon Yes transcription regulated coactivator 2 CREB Need4-2 ubiquitin-protein ligase E3 4 member CASKIN1 2.80 family domain COBLL1 TBC1 to Yes Similar 0.45 DNA RGNEF No to 0.43 similar PREDICTED: RNF20 Yes MYCBP2 ubiquitin-protein 3.36 shootin-1 ligase E3 KAB 18 Cyclin-dependentNEDD4L CRTC2 kinase 3.34 1 BSN TBC1D4 gene expressed 3.37 preferentially Aortic FLJ22471 KIAA1598 Yes No pombe) (S. homolog containing, Yes Yes MYCBP2 domain 1 homology Pleckstrin export RNA hypothetical protein PREDICTED: CDK18 4.31 Yes 3.47 ZNRF2 ubiquitin-protein ligase 4.32 E3 3.85 EB factor SPEG PLEKHA6 transcription 3.74 FAM83E SMURF1 ligase protein ubiquitin E3 RAE1 Yes ZNRF2 band family protein membrane Wiskott-Aldrich protein Signal-induced Erythrocyte proliferation-associated syndrome 1 protein 1 GYF 4.48 interacting TFEB GRB10 1 SMURF1 D52-like 20 protein domain Tumor containing, repeat domain homology Pleckstrin WD Yes SIPA1L3 EPB41L3 WASF2 D52 protein Tumor GIGYF1 proteins Downregulated PLEKHA7 9.78 Yes TPD52L1 No factor MitochondrialWDR20 fission Yes 7.21 spectrin-associated regulated Calmodulin 15.01 TPD52 10.37 Patatin-like phospholipase domain CAMSAP2 MFF serine/threonine associated Microtubule hydroxylase Tyrosine 2 PNPLA7 D52-like protein Tumor MAST1 3A Phosphodiesterase TPD52L2 TH A Cytospin PDE3A Calcium/calmodulin-dependent protein SPECCIL CAMKK1 proteins Upregulated atral xrse n uiid14-3-3 using assay purified pull-down inherited We 14-3-3 and a TRIM32. analysis. several in expressed further interaction in for bacterially the Because interaction implicated examined 2A). this initially (Fig. is other selected six we Nedd4-2 Nedd4-2, with diseases, including like together ligases screen TRIM32, the E3 in ubiquitin partner identified binding was 14-3-3 a TRIM32 as TRIM32 of Confirmation Ratio description Protein homolog Human 2016 sn l ee amla 433ioom ( isoforms 14-3-3 assay mammalian by co-precipitation seven a phosphorylated in all when interaction using the TRIM32 assessed next to We suggesting directly no PKA. 3), binds (lane 4); 2) PKA 14-3-3 and lane of 1 absence that 2B, the (lanes in (Fig. alone GST–TRIM32 moiety PKA with GST or the by with phosphorylated detected was was complex it which in a vrg ai ffl hnei 433bnigwt osoi tmlto oprdt basal. to compared stimulation forskolin with binding 14-3-3 in change fold of ratio Average ora fCl cec 2 (9) 126 Science Cell of Journal g emn,Cr5 Chr segment, 6 member A family 3 4.1-like 2 member family, protein 7 containing 1 kinase alpha 1, kinase kinase rti 3 protein 2 isoform protein 1 member subfamily, Shaw-related protein on oteGTTI3 ne conditions under GST–TRIM32 the to bound al .Smayo osoi-epnie1-- idn agt dniidb LC-MS/MS by identified targets binding 14-3-3 forskolin-responsive of Summary 1. Table g g , n GST-fused and b , 58 e P1Rgltro osnetasrps105 Yes 0.50 1 transcripts nonsense of Regulator UPF1 Yes 15.83 .1Yes No 2.11 2.15 No No 0.12 2.23 Yes 2.29 helicase-like RecQ syndrome, Bloom No Yes BLM 2.66 0.29 No HECTD1 ligase ubiquitin-protein E3 3.26 No 0.43 HECTD1 Yes mixed-lineage Myeloid/lymphoid or 3.63 1 MLLT4 homolog slit to Similar No activating GTPase LOC652055 Rho PREDICTED: 4.34 ARHGAP23 No No 6.78 6.80 c , e a , f , idn ua ooo rti ecito Ratio description Protein homolog Human Binding h , s ) C2clswt A-433atbde,adthe and antibodies, a the in with PAN-14-3-3 detected blotting readily was TRIM32. western TRIM32 naive with by antibody. TRIM32-specific analyzed and from were cells immunoprecipitated immunocomplexes 14-3-3s were PC12 endogenous the 14-3-3s Thus, investigated isoforms then Endogenous between panel). 14-3-3 We many cells. bottom HEK293 with interaction in interacting 2C, co-expressed of (Fig. into when capable incorporation antibodies is phospho- Phosphate using TRIM32 substrate 2011). confirmed was al., conditions PKA et Locke these Ryu 2008; under al., TRIM32 of 2009; study et the al., (Kano in used localization et widely and line cell isoforms interaction a 14-3-3 TRIM32 cPKA 2C), of (Fig. these cells coexpression all HEK293 by in of stimulated were interaction FLAG–TRIM32 The with myc. with tagged protein) 4 to, translocated leukemia; 23 protein 3 like 7 member A family 2 member (TORC2) .7No Yes 0.37 0.41 Yes Yes 0.44 0.44 Yes Yes 0.47 0.50 .8Yes 0.28 a Binding Journal of Cell Science RM2i C2cls el eemitie sdsrbdpeiul Ngk ta. 06,icbtdi eu-remdu o 6hus n hnstimu then and hours, 16 for a medium stripped serum-free ( also in left. incubated was 2006), the blot al., at middle et indicated (Nagaki The 50 previously th are panel). described with kDa, and (middle as minutes in beads, maintained anti-FLAG 20 markers, were anti-FLAG–Sepharose monoclonal Cells size with and cells. Molecular (IP) PC12 panel) panel). in immunoprecipitated (upper TRIM32 (bottom was anti-myc substrate FLAG–TRIM32 monoclonal anti-phospho-PKA expressed with with the WB analyzed hours, by 24 analyzed After were combinations. precipitates indicated the at cells u assetoer aa hs eut ugse that suggested results These data. with consistent the spectrometry 4), increased (lane mass stimulation precipitate forskolin the our note, in Of abundance 3). TRIM32 lane immunoprecipitate control 2D, the (Fig. not but immunoprecipitate 14-3-3 ( panel). (middle phosphorylation monoc and monitor ( anti-PAN-14-3-3 to with cells. substrate WB non-stimulated (5 by in FLAG–TRIM32 analyzed (5 level then of the cPKA the were and mutants in of immunoprecipitates constructs shown The change TRIM32 band anti-FLAG. fold indicated with TRIM32 ( as the immunoprecipitated anti-FLAG. each with expressed were of transfected and mutants were intensity densitometry truncation cells The its by HEK293 and panel). quantified 14-3-3. (lower also binding anti-PAN14-3-3 was for or 4) responsible domain panel) and (upper 3 anti-TRIM32 (lanes with panel WB upper by analyzed were immunocomplexes ( panel). (lower anti-GST and panel) (5 FLAG–TRIM32 (upper cells. 14-3-3 HEK293 anti-PAN with WB by analyzed partner. interacting 14-3-3 ( PKA-regulated a ( as partners. TRIM32 binding of Identification 2. Fig. , 0.5 m ah a nuae ih14-3-3 with incubated was each) g F feto h RM2S7Ao 61 uaino 433bnig h xeietwscnutda nE xetta 40 n 61 point S651A and S470A that except E, in as conducted was experiment The binding. 14-3-3 on mutation S651A or S470A TRIM32 the of Effect ) B ) m osoi sidctd fe Pwt niPN1-- (2 14-3-3 anti-PAN with IP After indicated. as forskolin M nvitro In soito f1-- ihpopoyae RM2a sesdb etr ltig(B.Prfe S–RM2o S alone GST or GST–TRIM32 Purified (WB). blotting western by assessed as TRIM32 phosphorylated with 14-3-3 of association m ah eeue nta ftetucto uat.Tebto ltwsas tipdadrpoe ihanti-phospho-PKA with reprobed and stripped also was blot bottom The mutants. truncation the of instead used were each) g m ) n myc-14-3-3 and g), g (1 m )i h bec rpeec fPA ulddw ihguahoeSpaoe n h rcpttswere precipitates the and glutathione–Sepharose, with down pulled PKA, of presence or absence the in g) G g osre eunemtf o 433bniglctdi h H oan fkonTRIM32s. known of domains NHL the in located binding 14-3-3 for motifs sequence Conserved ) , b , c , e , f , h or s (5 m )pamd eetasetdtgte iho ihu PA(5 cPKA without or with together transfected were plasmids g) m K ciain ute vdneta RM2i 14-3-3- a myc–14-3-3 is using and assay TRIM32 colocalization upon a FLAG–TRIM32 that with 14-3-3s obtained evidence was with partner interacting complex Further a activation. in PKA resides TRIM32 endogenous C ,lns3ad4 rpemueIG(2 IgG preimmune or 4) and 3 lanes g, K-nue neato fTI3 n amla 433ioom in isoforms 14-3-3 mammalian and TRIM32 of interaction PKA-induced ) ( A 3uiutnpoenlgssietfe sfrklnrsosv 14-3-3- forskolin-responsive as identified ligases ubiquitin-protein E3 ) euaino RM2b 4332017 14-3-3 by TRIM32 of Regulation D soito fedgnu 433and 14-3-3 endogenous of Association ) m m g ah,adteepesdTRIM32 expressed the and each), g ,lns1ad2,the 2), and 1 lanes g, oehrwt PA The cPKA. with together E nlsso h TRIM32 the of Analysis ) m )it HEK293 into g) ae for lated lonal e nd Journal of Cell Science f1--–RM2cmlxfrain emntrdteeffect the monitored we formation, significance 3A, complex functional (Fig. 14-3-3–TRIM32 TRIM5 the investigate of hollow To of 2007). al., and reminiscent et (Campbell spherical panels), right approximately and size middle are in vary microscopy but CBs Fluorescence these shape, that panel). we revealed left microscopy TRIM32 3A, electron FLAG-tagged and (Fig. cells the expressed cultured 2011), transiently in when al., Locke CBs et formed 2006; spontaneously al., Ryu generated et 2009; (Albor al., constructs et TRIM32 other many Like TRIM32- an of CBs not formation containing the does affects in binding al., which 14-3-3 et I, Coblitz manner 2005; binds mode al., et than (Ganguly probably cPKA-dependent position rather 2006). that in III 14-3-3 Pro a mode require Thus, via still in TRIM32 could assay. P653A mutant 14-3-3s for immunoprecipitation necessary the 2F,G). not because (Fig. was bind site 14-3-3 residue Pro binding with this 14-3-3 interaction that I however, mode found, consensus We a the contains fits S( also Ser651 of that sequence at downstream with TRIM32 residues PKA The two by interacts proline domain. phosphorylated NHL specifically is its 14-3-3 ligase within the Thus, when preferentially site. TRIM32 (lower antibody antibody the this substrate that suggesting recognizes PKA 6), the lane a of the panel, caused middle binding also 2F, S470A S651A of Fig. the Interestingly, loss 4). whereas complete (lane 6), in effect lane no panel, shown had top mutant (lower As mutant by S651A above. disrupted completely the assay was described an 14-3-3s endogenous in with and that binding interaction (S470A 14-3-3 to for Ala of mutants with similar mutants these residue point tested Ser and two either S651A) prepared replaced thus that We TRIM32 2G). (Fig. various sequence from III homologs TRIM32 although in organisms, conserved these highly domain importantly, NHL are 2F,G); the motifs (Fig. within Ser651 and occur Ser470 indeed revealed encompassing TRIM32 motifs human sequence of such sequence that acid amino the of Analysis S/T( where Rxx[S/T( motifs Rxxx[S/T( sequence I), consensus the in residue These binding. necessary 10). 14-3-3 is and observed domain the 2 NHL for lanes C-terminal sufficient C- panels, and the C-140, N-361 lower with that the not 2E, suggested but (Fig. results as cPKA, C-365 well of and presence as 265 the TRIM32 C- in 14-3-3s or only full-length endogenous fragment N- expressed of the the N-361; co-precipitation in with specific either C-365, revealed truncated HEK293 cells C-265, was from immunoprecipitation which Subsequent (C-140, domain. of terminal FLAG- each deletions four 2E), the generated Fig. characterize TRIM32 we the further 14-3-3, called To with tagged segment panel). TRIM32 40-residue of upper the association 2E, (Fig. of repeat repeats domain NHL six NHL unique al., includes the et carries that (Reymond C-terminus its domain contrast, RBCC In 2001). tripartite the III contains mode TRIM32 via TRIM32 binds panels). 14-3-3 left indeed 3B, proteins Fig. two (see the cells that in myc–14-3-3 colocalize confirming similar, and generally FLAG–TRIM32 were of patterns immunostaining 2018 433poen eonz hshrltdsrn rthreonine or serine phosphorylated a recognize proteins 14-3-3 ora fCl cec 2 (9) 126 Science Cell of Journal P spopoSrTradxi n mn acid. amino any is x and phospho-Ser/Thr is ) P ]P(oeI) r[S/T( or II), (mode )]xP ai rerio Danio RM2cnan nytemode the only contains TRIM32 P P 61(r63,aresidue a (Pro653), )651 ]xCO md III), (mode )]xx-COOH a cnann CBs -containing P ]P(mode )]xP g eosrt h bevdrl o 433i iigcls CFP– cells. living in 14-3-3 to TRIM32 for to role (CFP) observed protein the fluorescent demonstrate through cyan formation tethering CB protein of step initial TRIM32. the phosphorylated at that with suggested role association observations a right These plays refractory 14-3-3. 14-3-3 3C, be of (Fig. may accessibility formed, the CBs once to CBs, TRIM32 the that pre-existing suggesting panels), the with colocalize h 61 uatee ntepeec f14-3-3 of middle presence the with and in seen even the top was mutant microtubules compare S651A with from the S3, association Because Fig. away panels). material 14- without cytoplasm (supplementary with expressed 14-3-3 TRIM32 TRIM32 CB the 3-3 whereas TRIM32-bound of to network, throughout microtubule 14-3-3 association expected, of the distributed As contribution analyzed microtubules. the we panels). two of characterize bottom formation, the addition 3D, further (Fig. prevented that CBs To agent, found TRIM32 larger we TRIM5 destabilizing of this, formation microtubule (Campbell or with a CBs Consistent larger nocodazole, TRIM19 2007). form al., e.g. to et microtubules TRIMs, along CBs transported diffuse data. middle other immunofluorescence 3D, be the (Fig. containing with CBs to consistent visible panels), observed no two with also cytoplasm the was top throughout 3D, 14-3-3 CFP–TRIM32 (Fig. when signals, CBs CFP transfected, larger Importantly, form faint panels). to with coalesced along two CBs formed smaller CBs transfection. the after small and hours cells, 4 these within In detected initially was TRIM32 RM2rdsrbto iia ota bevdupon observed 14-3-3 post-expressed that Moreover, 3C). (Fig. to not did similar 14-3-3 of assessed. coexpression redistribution was 14-3-3 formation of CB pre-expression TRIM32 that on cPKA, indicated 14-3-3 experiment and this This TRIM32 of with influence the transfection and after or before expressed sfrsas on RM2i PAdpnetmanner cPKA-dependent a in these TRIM32 that 2C). fact bound (Fig. the with also line in isoforms isoforms S4), 14-3-3 Fig. mammalian a material seven Similar microtubule all (supplementary CBs. in with into in obtained pool incorporation were the cytoplasmic results to defect is prior separate 14-3-3 general TRIM32 a with complex that into a wild-type suggested sequestered to with results specifically results these due our Collectively, not that integrity. probable were is TRIM32 it panels), (bottom etrdsetterl o 433i hspoes 14-3-3 process, this in 2009), To 14-3-3 S2). al., for Fig. role et material the (supplementary Schwamborn dissect change better 2008; a through such al., altered TRIM32 cause et not bind did (Kano that This domain proteins NHL CBs. other 3B). its a two (Fig. of Abi2, to coexpression TRIM32 and because CBs fraction AGO1 relevant from of appeared cytoplasmic localization distribution TRIM32 S651A) formation diffuse not (but the more TRIM32 of on shift myc-14-3-3 binding of Coexpression 14-3-3 of om(hlae l,20;Ihmr ta. 05.T examine we To activity, 2005). TRIM32 al., affects et similarly Ichimura binding to 2005; 14-3-3 PKA al., inactive whether enzymatically or et an (Bhalla SGK1 in form ligase by phosphorylated stable phosphorylated a the forms maintain Nedd4-2 14-3-3 that with shown previously complex have others and We transubiquitylation autoubiquitylation TRIM32 TRIM32 and both prevents binding 14-3-3 ielpemcocp a hnpromdwt fusion a with performed then was microscopy Time-lapse g a ofndt B oae ln microtubules along located CBs to confined was g hra otepeso ih14-3-3 with post-expression whereas , g n PAidcdadramatic a induced cPKA and g n PAwr co- were cPKA and g n cPKA and g g g a also was i not did g a caused are , was g Journal of Cell Science mgn.Rpeettv mgso el rmtreidpneteprmnsaeshown. are experiments independent three from cells of 10 images of Representative presence imaging. the in CFP–TRIM32 or xeiet eecnutda nB xetta myc-14-3-3 that ( except cPKA. B, and in as conducted were experiments RM2(etpnl)o h 61 uat(ih aes eetasetdit E23cls fe 4hus el eesandwt oylnlrabbit polyclonal with stained were cells hours, 24 After ( cells. HEK293 (red). into anti-myc transfected mouse were panels) monoclonal (right and mutant (green) S651A the or panels) (left TRIM32 el.HK9 el eetasetytasetdwt LGTI3,adTI3 oaiainwsaaye yimnfursec ab n electron and (a,b) immunofluorescence by analyzed was localization TRIM32 and FLAG–TRIM32, with ( transfected (c,d). CBs. transiently microscopy TRIM32-containing were of cells formation HEK293 the cells. prevents 14-3-3 3. Fig. D ielpeiaigwt E23clsepesn F–RM2(o w aes,CPTI3,cK n myc-14-3-3 and cPKA CFP–TRIM32, panels), two (top CFP–TRIM32 expressing cells HEK293 with imaging Time-lapse ) B muoloecnedul aeigo RM2adisS5Amtn bt re)ad14-3-3 and green) (both mutant S651A its and TRIM32 of labeling double Immunofluorescence ) m /lncdzl bto w aes.Clswr mgda -oritras ooaoewsadd6husbefore hours 6 added was Nocodazole intervals. 1-hour at imaged were Cells panels). two (bottom nocodazole g/ml C feto myc-14-3-3 of Effect ) g a xrse 4husbfr lf aes ratr(ih ae)taseto ihFLAG–TRIM32 with transfection panel) (right after or panels) (left before hours 24 expressed was ( A g muoloecneadeeto irsoyiae fTI3 B nHEK293 in CBs TRIM32 of images microscopy electron and Immunofluorescence ) r-xrsinadps-xrsino uclua itiuino RM2 The TRIM32. of distribution subcellular on post-expression and pre-expression euaino RM2b 4332019 14-3-3 by TRIM32 of Regulation g rd.Myc–14-3-3 (red). g g mdl w panels), two (middle PAadFLAG– and cPKA , anti-FLAG Journal of Cell Science fehne IGE ciiis(eti ta. 02 etle al., et Pertel 2002; al., et (Kentsis activities expression E3 the RING and enhanced TRIM- 2011) that of of al., reported et formation been (Ganser-Pornillos has the CBs also in containing to It implicated 5A). is Fig. prerequisite interaction al., (see higher-order 2011) a et al., et (Ganser-Pornillos is Li 2011; oligomerization dimerization higher-order and efficient high- self-association, and been self- (dimerization) have TRIM32 low-order order self-association proteins; on TRIM for binding activity, for 14-3-3 modes described 14-3-3 ligase discrete which of ubiquitin Two effect E3 by association. the and mechanism investigated formation we CB molecular both the represses understand To but homodimerization self-association not higher-order TRIM32 blocks 14-3-3 this confirmed We FLAG–14-3-3 panels). with lower observation exogenous 4D, of (Fig. protein levels FLAG–Abi2 steady-state of the coexpression decrease Indeed, FLAG–14-3-3 upper 2). not 4D, lane did (Fig. with control TRIM32 compare positive 3–5, a lanes as panel, used was of that ubiquitylation FLAG–Abi2 strong despite phosphorylating conditions under nonphosphorylating TRIM32 or by ubiquitylated not tested was thus isoform We 2002). al., FLAG–14-3-3 et (Urano whether (TRIM25) Efp for ( substrate 14-3-3 a Notably, 5–6). (lanes constructs control with 14- of coexpression upon level 3-3 basal the almost wild- to of using decreased ubiquitylation ubiquitylation Abi2 TRIM32-catalyzed of TRIM32. level (non-tagged) its determined type and model a CB as FLAG–Abi2 a the known used [a we protein from end, this isoforms To results transubiquitylation. mediated 14-3-3 with Similar seven S4). consistent Fig. cells. material all 2–8), (supplementary assay formation with lanes in formation observed 4B, (Fig. complex autoubiquitylation was 14-3-3–TRIM32 suppression TRIM32 that suppresses suggested indicating These was 11–12), autoubiquitylation. results TRIM32 5–6, 14-3-3 autoubiquitylation decreased lanes binding in 4A 14-3-3 TRIM32 (Fig. deficient that controls of was these in that reduction detected mutant other Little the S651A and binding. TRIM32 2005), al., the et was (Ichimura Nedd4-2 14-3-3 14-3-3 including of of targets interaction mutant of the point specificity V180D disrupts the the specifically assess was to used one were finding; mutants lanes this with point compare Two 10, 9). and and 4 (lanes 3 alone cPKA not but cPKA 14-3-3 and of autoubiquitylated presence of of the intensity in heavily significantly smear 2 the expressed decreased that FLAG–TRIM32 lanes a however, found, the was 4A, by We (Fig. 8). 2011), demonstrated and HA–ubiquitin and of as al., species cells, active et high-molecular-mass the Ryu biologically in 2009; autoubiquitylated al., was et al., (Albor et FLAG–TRIM32 findings Locke previous with Materials 2006; also Consistent (see Methods). 2005) and al., et (Ichimura ubiquitin hemagglutinin (HA)-tagged involving assay ubiquitylation in-cell an performed 2020 hnamr eea fetsc sezm–usrt competition. enzyme–substrate as such effect general of more rather a TRIM32 inhibition phosphorylated than a to binding reflects 14-3-3-mediated 14-3-3 of anti-FLAG TRIM32 effect specific observed of with transubiquitylation the blotting and autoubiquitylation western Thus, by 4E). (Fig. analyzed anti-HA– by and separated first Sepharose were proteins HA-ubiquitylated total enx etdwehr1-- ol loafc TRIM32- affect also could 14-3-3 whether tested next We g n PA(i.4,ln ,cmaewt ae –)btnot but 2–3) lanes with compare 4, lane 4C, (Fig. cPKA and ora fCl cec 2 (9) 126 Science Cell of Journal g swl sedgnu 433poen,ulk the unlike proteins, 14-3-3 endogenous as well as nvivo in g usrt fTI3 Kn ta. 2008)] al., et (Kano TRIM32 of substrate a bqiyae n on htthis that found and ubiquitylated was g swl sFLAG–14-3-3 as well as g ihavreyof variety a with s sfr)is isoform) g which , s after g o igepoenitrcinhss ayeffects. explain affecting TRIM- many may so this of has 2011), interaction al., formation protein et single the Li a 2002; how 2011; al., both without al., et (Kentsis et for activity self-association Ganser-Pornillos ligase important ubiquitin and higher-order CBs be containing to that multimerization TRIM32 appears disrupts Given specifically homodimerization. binding 14-3-3 higher-order that results These homodimerization. suggested TRIM32 affect not does binding y–61 loc-rcpttdwt LGS5Ai the in FLAG–S651A myc-14-3-3 with of of amount co-precipitated 3). presence comparable also lane a FLAG–TRIM32 FLAG–S651A, out myc–S651A panel, and carried top myc–S651A was the co-immunoprecipitation with 5C, the (Fig. when in FLAG–TRIM32 Furthermore, 14-3-3 expressed with the recovered complex though even were immunocomplex myc–TRIM32 ujce oc-muorcptto odtc h TRIM32 was the extract resulting detect the to and co-immunoprecipitation were myc X-100, to with FLAG- Triton tagged subjected containing construct with same extracts TRIM32 the prepared cell including and widely HEK293 TRIM32 TRIMs is 2009). tagged many al., which et homodimers, of (Locke TRIM characterization for for source extract used potential cell a detergent-solubilized a as used We homodimerization. complex cells. 14-3-3–TRIM32 in self-association that higher-order TRIM32 suggested disrupts formation results These panel). value S 5; 14-3- with bound not to or bound complexes mutant 3 S651A centrifugation self-association its formed and TRIM32 sucrose-density-gradient in-cell of the used compare directly We 2011). nedfr ies motnl,we h aeco- same the containing when extracts Importantly, with myc-14-3-3 performed coexpressed dimers. was could the immunoprecipitation TRIM32s form 2), tagged with lane these myc–TRIM32 panel, indeed that (top precipitated confirming 5C effectively Fig. FLAG–TRIM32, FLAG in to seen be antibody can As homodimer. sue ht1-- ih lomdlt ellrlvl of 14-3-3 of levels we amounts cellular – increasing TRIM32 modulate containing TRIM32 with lysates also together cell soluble HEK293 might Thus, of TRIM32. 14-3-3 soluble level that the assumed reduce the autoregulatory and that potential autoubiquitylation – mechanisms indicated CBs TRIM32 data TRIM32-containing our of both active probably formation Because the prevents (higher- 2005). constitute 14-3-3 (most al., may forms that et 5A) (Song insoluble soluble Fig. fraction see than protein CBs; the or rather both oligomers states, in dimers) order cytoplasm and insoluble the monomers in and exist proteins soluble TRIM many cells NIH3T3 Although ability of its growth and TRIM32 accelerate soluble to of level the modulates 14-3-3 opee,btnttoecnann 61,wr almost were ( S651A, fractions TRIM32 containing lower-density wild-type the those in the recovered not however, completely but cells, 14-3-3 When in complexes, panel). the coexpressed in second mutant 5B, were S651A (Fig. the high-molecular-mass assay with same of obtained was consisted profile that value sedimenting tube (S the at complexes of as well as bottom 1 fraction the in predominantly detected were complexes n nlzdb etr ltig ned nrae 14-3-3 soluble increased the in Indeed, TRIM32 of blotting. level elevated western an centrifugation promoted by by expression fractions analyzed insoluble and and soluble into separated g enx xmndwehr1-- idn fet TRIM32 affects binding 14-3-3 whether examined next We ssoni i.5,tewl-ye(o-agd TRIM32 (non-tagged) wild-type the 5B, Fig. in shown As . , S hr ae,cmaewt h otmfourth bottom the with compare panel, third 8S, . 6;tppnl.Asmlrbtmore but similar A panel). top 16S; g g n PA loteulaonsof amounts equal almost cPKA, and n PA(ae4.Tu,14-3-3 Thus, 4). (lane cPKA and g n PAwere cPKA and g g n cPKA and , omda formed fraction Journal of Cell Science uato 61 RM2mtn nta fterrespective their of instead mutant TRIM32 S651A or 4 mutant and 4 lanes used we with when detected 14-3-3 not or insoluble was cPKA the elevation This in 6A). TRIM32 (Fig. in fraction decrease concomitant a with fraction (10 lysates Cell (E). anti-FLAG or (D) anti-HA monoclonal with WB by analyzed then was 3-3 transubiquitylation. TRIM32 and autoubiquitylation TRIM32 (5 plasmids inhibits indicated binding 14-3-3 4. Fig. eemn rti ees(oe aes.Smlrrslswr bandfra es he needn experiments. independent three least at for obtained were results Similar panels). (lower levels protein bar wer ( determine with results anti-FLAG indicated Similar with are immunoprecipitation (C). to species FLAG–Abi2 subjected Ubiquitylated and were forms. lysates (A,B) ( ubiquitylated and TRI FLAG–TRIM32 experiments. X-100, their precipitated independent indicate Triton the three detect arrows detect least to The to at panels) form). anti-FLAG for (upper monoclonal (monoubiquitylated obtained anti-HA with arrowheads (WB) monoclonal and blotting with forms) western or (polyubiquitylated by panels) followed (lower SDS-PAGE 7.5% Abi2 by and analyzed were proteins precipitated The g ln Fg B ae – n 1 and 1–3 lanes 6B, (Fig. alone m 9 rwe etasetdteV8D14-3-3 V180D the transfected we when or ) ah eelsdwt %SS n h xrse LGTI3 AB rFA–b2()wsimnpeiiae ihanti-FLAG. with immunoprecipitated was (C) FLAG–Abi2 or (A,B) FLAG–TRIM32 expressed the and SDS, 1% with lysed were each) g D , E E23clstasetyc-rnfce ihteidctdcntut (5 constructs indicated the with co-transfected transiently cells HEK293 ) 9 –3 9 compare , g D upre ytm-oreeprmnswt h protein was the the of finding with half About CB The experiments 6C). (Fig. and 4A,C). cycloheximide time-course Fig. 5 inhibitor autoubiquitylation synthesis 6, 3B; by from (Fig. and data supported assays 5 the lanes formation with 6B, (Fig. consistent constructs wild-type rat-A( anti-HA or ) m fpoenec)tetda nDwr loaaye yW to WB by analyzed also were D in as treated each) protein of g E olwdb 0 D-AE bqiyaino FLAG–14- of Ubiquitylation SDS-PAGE. 10% by followed ) ( euaino RM2b 4332021 14-3-3 by TRIM32 of Regulation A–C E23clstasetyc-rnfce ihthe with co-transfected transiently cells HEK293 ) m ah eelsdwt 1% with lysed were each) g 9 n 6 and s M32 e 9 ), Journal of Cell Science aaae(16) es loo eyrgns 73) S 48) n g ht yoye(.S.( (1.9S). lysozyme white egg and (4.8S), BSA (7.3S), (5 dehydrogenase plasmids alcohol yeast (11.6S), catalase fe omlzn h xrse rti mut eeaaye yW ihteidctdatbde.Teatrs eoe oobqiyae TRIM32/ monoubiquitylated denotes asterisk The antibodies. indicated (lowe the lysates with and WB panels) by (upper immunoprecipitates analyzed the were in amounts proteins protein FLAG-tagged expressed and myc- the included of normalizing proteins amounts after Marker The experiment. experiment. each (IP) immunoprecipitation in each protein of amount total the of percentage a as plotted were bands (5 constructs 100,000 indicated the with transfected neatoso RMpoen.Temdli ae ntepooa yGne-onlo ta.(asrPrilse l,21) ( 2011). al., et (Ganser-Pornillos al. et Ganser-Pornillos by proposal the on based is model The proteins. TRIM of interactions hshrlto n 433bnigaeipratfcosfor Ser651 factors important PKA-mediated are cell that cannot intrinsic TRIM32. binding yet the regulating 14-3-3 concluded elicit 6) ligase and not lane We could phosphorylation 4C, ubiquitin-protein 6), Fig. activity. lane 12; E3 growth 2F, and (Fig. has 6 14-3-3 lanes bind which Thus, 4A, Mock). (Fig. mutant, of under activity that growth to S651A cell similar NIH3T3 the rate on (growth effect conditions did stimulatory S651A parallel that NIH3T3 any however, (WT) of show observed, TRIM32 We not rate of Mock. growth with expression the compared with upon as agreement 2008), significantly In al., increased 14-3-3. (Kano et and cells (Kano PKA naturally to data is TRIM32 addition It previous in express 6D). 2008), (Fig. cells al., points et mutant NIH3T3 infection, time various retroviral that at using reported FLAG–S651A numbers (Mock) cell vector counted empty and (WT), an that or lines (S651A) FLAG–TRIM32 cell NIH3T3 stable expressed produced the we 14-3-3. in interaction, with TRIM32 interaction alterations in to to owing increase protein due observed the of was 48-hour the half-life a relative concentration was that after TRIM32 suggested even mutant, pool soluble results S651A soluble These the the of in chase. not retained half-life was but a TRIM32 TRIM32, with the soluble detected, from of co- disappeared level cells 14-3-3 in cells whereas with chase, of the transfected hours 10–12 in within fraction alone soluble expressed TRIM32 homodimerization. not but self-association higher-order TRIM32 prevents formation complex 14-3-3–TRIM32 5. Fig. 2022 oeaieteptnilbooia eeac fte14-3-3– the of relevance biological potential the examine To g 8hus,fatoae rmtebto,adaaye ySSPG olwdb etr ltig(B ihat-RM2 h neste fgel of intensities The anti-TRIM32. with (WB) blotting western by followed SDS-PAGE by analyzed and bottom, the from fractionated hours), 18 , ora fCl cec 2 (9) 126 Science Cell of Journal m ah eelsdwt %Tio -0 n ujce opeiiainwt niFA fe omlzn eeso xrse RM2poen for proteins TRIM32 expressed of levels normalizing after anti-FLAG with precipitation to subjected and X-100 Triton 1% with lysed were each) g g n PA lwrdces nthe in decrease slower a cPKA, and . 8hus Notably, hours. 48 m ah eehmgnzdi B p .)adsbetdt urs est-rdetcnrfgto (5–20%, centrifugation density-gradient sucrose to subjected and 7.4) (pH PBS in homogenized were each) g . 0 of 60% oaiet aiu uclua oain,icuigtecytoplasm, proteins the including identified locations, the subcellular addition, various transport, in to In metabolism, localize etc. involved signaling, expression, are cell gene proteins of growth, to aspects respect identified with different newly novel many The were interactions signaling. 46 the PKA For proteins, 1). 51 (Table 14-3-3 these activation of of coordinated PKA upon pathways targets bidirectional, altered signaling significantly binding PKA were the 51 in show identify interactome 14-3-3 and we the study 14-3- of response present a the In that suggest Discussion tissues, physiologically normal results is association cells. in this These in that significant even notion the 6). support exists which and complex PKA-dependent magnesium 3:phosphoTRIM32 formation 3 complex that and the mediating indicating ATP (lanes in involved homogenates, cAMP, is the of phosphorylation to addition association this by Importantly, chloride 2 4). (lanes stimulated and IgG 1 lanes was control with with compare not from 5, but and 14-3-3 homogenates with tissue these co-immunoprecipitated both 6E). clearly 14-3-3 (Fig. was brain native and TRIM32 muscle with skeletal physiological associated tested mouse from we is the immunopurified TRIM32, TRIM32 with validate 14-3-3 endogenous of To whether interaction proteins. the of recombinant importance cultured with or obtained were cells above described tissues data normal interaction from The co-precipitate TRIM32 and 14-3-3 C E23clstasetytasetdwt h indicated the with transfected transiently cells HEK293 ) shrci coli Escherichia ( A B E23clstasetyco- transiently cells HEK293 ) oe fintermolecular of model A ) aatsds 1S,bovine (16S), galactosidase g hs interactions whose panels) r S651A. Journal of Cell Science lutaino h rpsdrl o 433i euaino TRIM32. of regulation a in densitometry 14-3-3 by for quantified role also proposed was the panel of upper illustration the in shown band TRIM32 05m) T 05m)admgeimclrd 1 M eeaddt h ooeae n nuae t32 at incubated and homogenates the to added were mM) (10 chloride magnesium and mM) W (0.5 by ATP followed mM), SDS-PAGE (0.5 non-reducing to subjected were immunoprecipitates (4 IgG preimmune mean the represents i.6 433TI3 neato sipratfrTI3 regulation. TRIM32 for important is interaction 14-3-3/TRIM32 6. 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(10 cycloheximide with incubated were hours 24 after and each) g ..o he xeiet.( experiments. three of s.d. eut eenraie o1a ie0frec ex each for 0 time at 1 to normalized were Results ce ihteidctdepeso lsis(5 plasmids expression indicated the with ected E RM2i oimnprfe ih1-- rmm from 14-3-3 with co-immunopurified is TRIM32 ) 6 6 g 10 h eann noul eltwsslblzdi SDS- in solubilized was pellet insoluble remaining The . 4 el nsxwl ltsadhretdfrdtriaino h ubro el tteidctdtms ahvalue Each times. indicated the at cells of number the of determination for harvested and plates six-well in cells g sidctd ( indicated. as tnX10slblzdmueseea uce(ae –)adbanetat lns46.The 4–6). 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(lower anti-PAN14-3-3 or panel) (upper anti-TRIM32 with B B 433TI3 ope omto lvtstelvlo RM2i h detergent- the in TRIM32 of level the elevates formation complex 14-3-3–TRIM32 ) m eietadepesda ecnae.( percentages. as expressed and periment oul rtiswr xrce tteidctdtmsa nAadBand B and A in as times indicated the at extracted were proteins Soluble ah eelsdwt %Tio -0,adaslbepoenfraction protein soluble a and X-100, Triton 1% with lysed were each) g ueseea uceadban niPN1-- (4 14-3-3 Anti-PAN brain. and muscle skeletal ouse euaino RM2b 4332023 14-3-3 by TRIM32 of Regulation AEsml ufr h ellstswr nlzdby analyzed were lysates cell The buffer. sample PAGE ˚ o 0mnts(ae n ) h nest feach of intensity The 6). and 3 (lanes minutes 20 for C K9 el eec-rnfce ihthe with co-transfected were cells EK293 D 433bnigi eesr for necessary is binding 14-3-3 ) F Schematic ) u cAMP ous m 1A)orthe )or g) Journal of Cell Science 433bnigmyidc ofrainlcag rms the mask or change conformational that a assume induce somewhat We may 2011). binding with al., 14-3-3 (domain et although (Ganser-Pornillos self- domain efficiency form arrays, lower to SPRY capacity hexagonal the the truncated retains association-based TRIM32) a lacks of that NHL that is to finding equivalent TRIM5-21R finding recent This of provide the 2002). dimer and al., given et relevant al. (Kentsis particularly for the et functions critical be complement TRIM Kentsis may cellular this self-assembly results TRIM by that our evidences that suggested additional Thus, model and 3–5). and (Figs scaffolding activity ligase formation dimerization ubiquitin CB TRIM32 self- affecting both higher-order repressed interaction without TRIM32 reactions 14-3-3 disrupted that association enzymatic showed we specifically study, specific present model the partner interaction control In of 2002). ‘scaffolding’ or al., their assembly et the (Kentsis amplify the to support and presented could intrinsic proteins TRIM al. oligomers all be TRIM et of where should features Kentsis characteristics common form functions. to be these propensity to members, and appear Ganser-Pornillos oligomerization complexes 2005; Because al., large 2011). et in al., (Song complexes et formation large form CB and and activity PKA. with enzymatic interaction conjunction whose TRIM32 a proteins represent NHL-domain-binding inhibits co-immunoprecipitation may of 14-3-3 by class Thus, analyzed novel S5). TRIM32 as Fig. of material AGO1 (supplementary interaction and the Abi2 phosphorylated affect the with with significantly 14-3-3 toward not of domain did association pointed TRIM32 NHL that Furthermore, and found 2003). the indeed al., shaft of et 2), we in (Edwards (Fig. loop propeller side bottom occurred Ser651 C-terminal the electronegative the at interaction outside in TRIM32 this exposed located of is because phosphorylation which unique to be this response here From reported to 14-3-3 2003). with appears al., TRIM32 of et interaction the (Edwards perspective, the proteins face that ligand-binding NHL-containing proposed the been represent of may has surface it top and electropositive bottom electronegative, the more shaft. whereas much electropositive central is mostly a is around top radially the Importantly, a arrayed forms repeats, domain NHL the NHL individual only the lacks 2003), that al., six-bladed et family tumor (Edwards RBCC-NHL brain domain the of RING of domain al., NHL member et the a Schwamborn of (Brat), structure 2008; crystal al., the et In (Kano 2009). AGO1 domain with NHL and its Abi2 protein– shown including through been substrates tryptophan-rich the various has with a TRIM32 in interact indeed to is located and domain Ser651, module, interaction NHL at protein PKA The by domain. phosphorylated NHL is ligase the functions. and cell 3-3 of and range 14 wide between a link in direct PKA provides a further al., for in and evidence identified et proposal experimental genetic complexes (Gelperin the protein complements 14-3-3) the by study of of our caused diversity homolog lethality the Thus, the (yeast 1995). suppress Bmh partially of can depletion PKA, of of 3-3 subunit al., et and overexpression Roberts 14 1995; Indeed, al., between 1997). et links (Gelperin functional pathways signaling many PKA pointed be and have may yeast there with that studies out genetic Previous and membrane. reticulum cell endoplasmic mitochondria, cytoskeleton, nucleus, 2024 B a elc h blt fTI rtist self-associate to proteins TRIM of ability the reflect may CBs when directly TRIM32 binds 14-3-3 that indicate results Our ora fCl cec 2 (9) 126 Science Cell of Journal b poelr ihtebae,cnitn ftesix the of consisting blades, the with -propeller, TPK1 noigtecatalytic the encoding , n rtisi epnet hshrlto,although phosphorylation, and (Vincenz information to target other certain and requires results of response these unfolding From it promote enzymes proteins. and in may mature cases even itself proteins of phosphorylation most proteins other activities the in with modulate and can because interactions it its cell because unique for these 14-3-3 be by phosphorylation of regulation to however, formation viewpoint, appears These during this 2004). From separately, structures. either al., proteins, or 14- shock that heat et with cooperatively suggest interplay functional results, (Stoecklin have present may 3-3 our granules with together stress al., observations, 2010; et and (Okada al., bodies processing 2011) et participates of family formation/maintenance (Hwang 14-3-3 the the in granules Importantly, 2010). stress al., et CBs, and/or Johnston and TRIM formation including bodies, the foci, in processing cytoplasmic roles various important of play maintenance Hsp70 and the in Hsp90 domain NHL the arrays. of higher-order role TRIM32 Future the of mechanism and Ser651. assembly molecular interaction detailed 14-3-3 at the the phosphorylation of clarify complex will by once studies dimers triggered structural TRIM32 is of required is formation interaction that TRIM32 higher-order of parts for other possibly and domain NHL euaoymcaimmyas aea motn maton impact important this an phosphorylation, have several to also with response may mechanism associated in regulatory – proteins but processes misfolded native only pathological or not molecular – denatured the proteins many at also binds functions 14-3-3 TRIM32 signaling Because understanding control level. toward to may step that important here muscular and an pathways describe and we limb-girdle substrates first mechanism the regulatory in is TRIM32 The occurring 2H. specific have type mechanisms dystrophy efforts identify the many to decade, elucidate past made PKA- the a been through In TRIM32 and mechanism. Nedd4-2 dependent of types. regulation cell the other both to for as site) well as another for these (or in kinase(s) Ser651 binding protein 14-3-3 phosphorylate support additional also exclude could that cannot could TRIM32 We however, 14-3-3, 6D). possibility, viral (Fig. bind growth to the our cell unable NIH3T3 by 14-3-3 yet facilitate mutant, active not supported S651A enzymatically the is as is that which model showed which reversible, This (Dougherty experiment, dephosphorylation transduction 2004). be self- by Morrison, reversed the and be should This modulating can 6F). interactions by mechanism (Fig. state interactions regulatory of soluble protein–protein equilibrium the soluble–insoluble association-based the toward and blocking drives soluble and TRIM32 and autoubiquitylation a by formation of in proteasome. upstream CB TRIM32 stability complex the latent phosphorylated via functionally TRIM32 traps degradation 14-3-3 promote thus Thus, to and may 14-3-3 autoubiquitylation bind it ligase TRIM32 must represses activity, 14-3-3 Although TRIM32 formation. to CB (Locke however, Ser651; repress at levels phosphorylated, TRIM32 expression soluble phosphorylates be its PKA on 2009). al., depending et formation, autoubiquitylation CB self-association-based and by soluble of reduced concentration in is the cytoplasm and TRIM32 states, the to insoluble in Similar and distributed soluble 6F). is both (Fig. TRIM32 TRIM32 proteins, 14- for TRIM of role many control potential a PKA-dependent propose in we 2003) 3-3 al., et Chen 1996; Dixit, onigeiec niae htmlclrcaeossc as such chaperons molecular that indicates evidence Mounting ncnlso,w rps ht1-- sacmo cofactor common a is 14-3-3 that propose we conclusion, In Journal of Cell Science ucae rmUsaeadKdk epciey oylnlat-LGwas anti-FLAG Sigma. from Polyclonal purchased was respectively. were anti-tubulin Kodak, Monoclonal anti-FLAG Rockland. and and substrate from anti-HA Upstate purchased Monoclonal anti-phospho(Ser/Thr)-PKA from Signaling. Cell and purchased from Cruz anti- anti-myc purchased Santa were antibody, from Polyclonal PAN-14-3-3 purchased Anti-14-3-3 The Laboratories. were Siomi. anti-myc Haruhiko monoclonal Biotechnology. and Dr FLAG–AGO1 for anti-GST by plasmid TRIM32, The provided [(Itagaki templates. as TH kindly pcDNA3] and in pcDNA3], was myc–TH in for myc–Nedd4-2 1999) for al., 2005) et al., et [(Ichimura Nedd4-2 h amla xrsinpamdcryn LGtge RM2was TRIM32 FLAG-tagged 5 carrying oligonucleotides using plasmid GCAGCTTC-3 PCR by expression generated mammalian The Materials Methods aggregation-prone and Materials especially targets, targets. 14-3-3-binding other rznmueseea uceadbanwr ooeie n1 oue flysis 100,000 of at volumes centrifuged 10 were in homogenates homogenized the were and brain brain buffer, and and muscle muscle skeletal skeletal mouse mouse Frozen from 14-3-3 of Immunopurification previously (2–4 described cells as HEK293 2005). conducted al., were et (Ichimura analyses Co-immunoprecipitation assay pull-down and Co-immunoprecipitation expressing stably 2005) al., al., et et ( (Ichimura (Ichimura cells 14-3-3 previously PC12Mh MEF-fused of described dishes as 15-cm out Six 2008). carried was proteomics Quantitative proteomics Quantitative reported the in or pCMV-Tag2B), CaMKK pCMV-Tag2B), FLAG–TORC2 of in in for in FLAG–TFEB plasmids FLAG–WDR20 Takemori, for using for (Funakosi, (for shown PCR Yo pCMV-SPORT6 by (Toyobo, Dr Roth made plasmids pME18SFL3 by Richard were pCMV-Tag2B), or myc– provided Drs The FLAG–RNF20) (for (kindly from (for Koya TORC2/pSport6 Reinberg Abnova. gifts Daisuke Danny EGFP–TPD52L1), kind and 2006). from (for Smurf1), were al., Byrne et S1 Jennifer targeted obtained (Nagaki AKT1S1–HA), Fig. brain PCR-based was material porcine from supplementary of TRIM32 isolated was 14-3-3 protocol PKA Human The of standard subunit 2002). catalytic the GST–14-3-3 al., of to et cleavage (Ichimura according was mutagenesis construct created CFP-TRIM32 were The 2005). al., Eco the et ligating by (Ichimura created described been have poii,ad1 and aprotinin, ihbfe ,adtepeiiae opee eeaaye y1%SDS-PAGE 10% by analyzed blotting. were complexes western precipitated by the followed and A, buffer with LGtgvco CVTgB(nirgn,tre CVTI3.The pCMV-TRIM32. termed myc–14-3-3 (Invitrogen), for plasmids pCMV-Tag2B was expression fragment vector PCR The FLAG-tag template. as with Funakosi) digested (pOTB7, cDNA TRIM32 human 0mnts ye n3m flssbfe 5 MTi-C,p .,10mM 150 7.5, pH Tris-HCl, Na mM mM 1 [50 EGTA, buffer mM 10 lysis NaF, 50 mM of 100 with ml glycerol, 3 (w/v) stimulated 10% in were NaCl, lysed cells minutes, labeled 20 The days. 12 ihepeso lsis(ah5 (each plasmids expression with Ihmr ta. 05.Tercvrdpoen eedgse ihtysnadthen and trypsin with digested were proteins expressed recovered the The 2005). to al., et bound (Ichimura Proteins cells. PC12Mh MEF-14-3-3 non-stimulated unlabeled, from Sikw ta. 05.Frqatfcto,vle eecretdb TMfran for STEM [ by corrected 81% were of values programs efficiency quantification, software incorporation For STEM 2005). and al., Science) et (Matrix (Shinkawa MASCOT mass the chromatography–tandem using liquid spectrometry two-dimensional by quantified and identified oue(0m EE,p .,1 Mmgeimaeae n . MATP) 0.25 mM 0.1 of and absence acetate, magnesium or mM presence 10 7.5, the pH in HEPES, mM (50 volume )Tio -0,5m ZnCl mM 5 X-100, Triton v) ahfgr.FrGTpl-oneprmns S–RM2o S ln (each alone GST or in GST–TRIM32 indicated experiments, antibodies pull-down specific , SDS- the GST 10% using For by blotting figure. separated western each then (w/v) by were from analyzed 10% Proteins released and cRaf1). NaCl, PAGE was human mM of 14-3-3 [LSQRQRSTS( 150 250–265 bound phosphopeptide residues synthetic 7.5, the on mM pH and 1 X-100], Tris-HCl, with FLAG– beads Triton mM the (w/v) [50 expressed 0.1% washed A the and (Sigma), buffer hours, glycerol 24 with anti-FLAG–Sepharose times After with five cPKA. immunoprecipitated and was myc–14-3-3 TRIM32 without or with ute nuae o 0mntsa 4 at and minutes reaction 60 the for to incubated added were further (Pharmacia) beads glutathione–agarose incubation, 0.5 Ist fpCPC Cotc) h eeinadpitmtnso TRIM32 of mutants point and deletion The (Clontech). pECFP-C1 of site RI m )wsicbtdwt 14-3-3 with incubated was g) g eete uiidfo h obndlstsb h E method MEF the by lysates combined the from purified then were Bam 9 m /lluetn n obndwt neulvlm ftelysate the of volume equal an with combined and leupeptin] g/ml n 5 and Iand HI g a ioom eemtblclylbldwt [ with labeled metabolically were -isoform) Bam (ciuae l,20)frmyc–CaMKK for 2008) al., et [(Ichimura 9 g -CGGAATTCCTATGGGGTGGAATATCTTC-3 Eco Ngk ta. 06 ihFco BoRd.The (Bio-Rad). X Factor with 2006) al., et (Nagaki HI/ g 2 Eco Iadte netdit h lnn ieo the of site cloning the into inserted then and RI Mpeymtysloy loie 10 fluoride, phenylmethylsulfonyl mM 2 , 13 a ucae rmImmuno-Biological from purchased was Ifamn fpM-RM2it the into pCMV-TRIM32 of fragment RI m ]y n 0 [ 70% and C]Lys g )ecdn LGTI3 rismutants, its or FLAG–TRIM32 encoding g) g ˚ m (1 .Tebaswr hnwse ietimes five washed then were beads The C. ftectltcsbnto K.After PKA. of subunit catalytic the of l n t 10 uato FLAG–14-3-3 or mutant V180D its and m )fr2 iue t30 at minutes 20 for g) 9 6 -CGGGATCCATGGCTGCAGCA- 10 6 eetasetytransfected transiently were ) g 13 C]Arg. m rti a rprdby prepared was protein osoi Sga for (Sigma) forskolin M g o 0mntsa 4 at minutes 30 for P TNH,based )TPNVHA, 13 ˚ a Cin25 ]y/r for C]Lys/Arg 3 npcDNA3], in VO 4 %(w/ 1% , m 9 m final l Bgl g/ml and ˚ II/ C. g o 0mntsa 4 at 4 with minutes incubated 60 were mg) (1.5 for lysates cleared The n ltdwt D-AEsml ufr(lacking buffer sample SDS-PAGE with eluted and PS+]adfxdwt 0 omlni 0 B()fr3 iue t25 at minutes 30 for PBS(+) 70% in formalin 10% with fixed 0.5 and (each [PBS(+)] plasmids expression indicated the Instruments, Precision with World transfected (FluoroDish, plates were 3.5-cm Inc.) on grown cells microscopy HEK293 electron and microscopy Immunofluorescence uhr eeivle ndt icsin n rprn h final the preparing and discussions All manuscript. manuscript. data the of the in cells. version wrote involved NIH3T3 and stable were experiments of authors evaluated T.I. generation N.H. and the and in H.K. analyses. T.I. contributed I.S., immunocytochemical S.H. M.T., and and experiments. spectrometry T.S. the mass of with most helped out carried T.I. contributions Author experiments. Y. some and doing Nishijima in K. thank Uchida, help also C. for Y. We Yoshida, and Komata cells. J. Reinberg PC12 Kakiuchi, D. K. for Koya, Yamada, Greene D. S. L. Byrne, and J. plasmids, Roth, for R. Takemori Siomi, H. thank We Acknowledgements at seeded were cells Infected 2008). 2008). al., al., et et (Kano packaging (Kano previously Plat-E 1 pMX-puro by described produced S651A with as retroviruses with and cells constructed infected (WT) were were fibroblasts wild-type NIH3T3 (S651A) FLAG–TRIM32 mutant for point vectors expression counting Retrovirus cell and infection Retroviral (2–4 cells HEK293 gradient density Sucrose al., et (Ichimura previously described (2–4 as cells HEK293 performed 2005). was assay ubiquitylation The assay ubiquitylation In-cell ubra aiu times. various at number itcnlg)wsadd n yae eefrhricbtdfr6 iue at minutes 60 for incubated further were lysates 4 and added, was Biotechnology) rti)wr oddo oascoedniygain 52% /)adcentrifuged and w/v) (5–20%, gradient density 100,000 sucrose a at to on loaded were protein) 5 (each 4husuigLpfcaie20 Ivtoe) rnfce el eerinsed were CaCl cells g/l 0.1 Transfected containing (Invitrogen). (PBS) saline 2000 phosphate-buffered Lipofectamine with using hours 24 lsis(ah5 (each plasmids ihaTMH10(iah t)adsandwt UE OLCN8000 COOLSCAN cells SUPER and a myc–14-3-3-expressing citrate with and collected scanned lead FLAG–TRIM32-expressing and were and Ltd) Counting images (Hitachi acetate (Nikon). microscopy H7100 Electron uranyl TEM (Nisshin grids. a with resin with copper 812-based stained 150-mesh 7.2), room with Quetol (TAAB on (pH were with dehydrated at mounted sections then embedded hour buffer glutaraldehyde finally were 1 Ultrathin and Cells for phosphate 2.5% EM). 100%, buffer. tetroxide to phosphate sodium osmium with up sodium 1% ethanol M M with temperature 0.1 postfixed 0.1 in and room temperature in then PBS, with at and Ltd) washed PBS hour Equipment 1 with Laboratories for Inc.). times Precision, four (Applied fixed system washed microscope room Vision were at Delta hour a cells 1 using for stained visualized Probes) The (Molecular subsequently antibodies with anti- temperature. (see secondary incubated and (red) anti-mouse were antibodies cells or temperature primary Alexa-Fluor-594-conjugated fetal the rabbit specific and PBS, room 10% with with (green) times with at temperature four Alexa-Fluor-488-conjugated incubated washing minutes room After 30 then at legends). hour figure PBS, for 1 with PBS for times incubated in four serum washed bovine were cells fixed oeta 0 fteFA–RM2pstv el eeas oiiefrmyc- for positive also that were and cells 20–40% FLAG–TRIM32-positive expression. was the 14-3-3 expression of 90% FLAG–TRIM32 than of more efficiency the that revealed sapretg fttlaonso rti nec experiment. each in protein expressed of were amounts results 10% was total the to of image and percentage subjected gel (ATTO) a The densitometer and as anti-TRIM32. E-Graph acid, an with trichloroacetic with blotting quantified 10% western by with followed precipitated SDS-PAGE tubes, the of .%o 0 D-AEfloe ywsenbotn ihmncoa anti-HA monoclonal with by blotting anti- analyzed western The were with by proteins anti-FLAG. (Roche). followed bound incubated or and SDS-PAGE matrix A, 10% and buffer affinity or with buffer anti-HA 7.5% times five lysis with washed were of or beads (Sigma) volumes beads 9 FLAG–Sepharose with diluted subsequently 6 ˚ .Imnpeiiae 433cmlxswr ahdfv ie ihbfe A buffer with times five washed were complexes 14-3-3 Immunoprecipitated C. o lcrnmcocp,HK9 el rnfce ihFA–RM2were FLAG–TRIM32 with transfected cells HEK293 microscopy, electron For 10 4 el e eli i-elpae n avse o eemnto fcell of determination for harvested and plates six-well in well per cells m )fr3 or n ooeie nPS h ooeae (50 homogenates The PBS. in homogenized and hours 36 for g) g o 8husa 4 at hours 18 for euaino RM2b 4332025 14-3-3 by TRIM32 of Regulation 6 m )fr4 or n ye n1 D.Smlswere Samples SDS. 1% in lysed and hours 48 for g) 10 ˚ 6 .ImblzdpoenAGSpaoe(at Cruz (Santa A/G–Sepharose protein Immobilized C. eetasetdwt h niae xrsinplasmids expression indicated the with transfected were ) 6 10 6 ˚ .Tesmlswr rcintdfo h bottom the from fractionated were samples The C. eec-rnfce ihteidctdexpression indicated the with co-transfected were ) b m -mercaptoethanol). fat-A 433IgG 14-3-3 anti-PAN of g 2 n . / MgCl g/l 0.1 and m ˚ .The C. )for g) m g 2 Journal of Cell Science on .J,Abr . i,Y,E-iai . adrek .E,Bbok M., Babcock, E., G. Vanderbeek, S., El-Hizawi, Y., Liu, A., Albor, J., and E. K. Horn, Matsumoto, K., S. Irie, Hatakeyama, P., Roseboom, G., K., J. Nelson, J., Sodroski, Weigle, O., D., Gelperin, Pornillos, V., Chandrasekaran, K., B. Ganser-Pornillos, agl,S,Wle,J . o . hmna,P,Mlax .adKlein, and B. Malpaux, P., Chemineau, A., Ho, L., J. Weller, S., Ganguly, rs,P,Wie,T,Nln . uh,T,Geneg .R,Mra,K,Fujiwara, K., Morgan, R., C. Greenberg, T., Sudha, E., Nylen, T., Weiler, P., Frosk, rdl,R . adn,L . oed .P n uln .R. B. Cullen, and P. H. K. Bogerd, S., A. L. Aggarwal, Harding, A., and R. P. Fridell, R. Wharton, D., B. Wilkinson, A., T. Edwards, K. D. Morrison, and K. and M. M. Dougherty, Stremlau, S., Welikala, B., Song, H., Javanbakht, X., Li, F., Diaz-Griffero, olt,B,W,M,Siao .adL,M. Li, and S. Shikano, M., Wu, B., Coblitz, hn .K,FradzFnz . cvd,S . a,Y . atr .D., M. Kaytor, C., Y. Lam, F., S. Acevedo, P., Fernandez-Funez, K., H. Chen, apel .M,Ddig .P,Yp .W,W,X,GlosMnbu,S., Gallois-Montbrun, X., Wu, W., M. Yap, P., M. Dodding, M., E. Campbell, ciua . aaua . aaoo . oiaa . ak,M,Kakiuchi, M., Taoka, Y., Tominaga, K., Sasamoto, H., Yamamura, T., Ichimura, hla . Daidie V., Bhalla, ciua . aaiaTuua . tgk,C,Toa . aao . Natsume, T., Hayano, M., Taoka, C., Itagaki, A., Wakamiya-Tsuruta, T., Ichimura, lo,A,E-iai . on .J,Leeih . rs,P,Woean .and K. Wrogemann, P., Frosk, M., Laederich, J., E. Horn, S., El-Hizawi, A., Albor, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.122069/-/DC1 online available the material Supplementary and Foundation; Project, Memorial Pharma System Mitsubishi Suhara Foundation. the Proteomics the Sumitomo Frontier; foundation; the Integrated Genome Research the on Research S.H.]; and Pioneer 24112006 Culture, T.I.; to Education, to for 21570213 of Grants-in-Aid 24390065 numbers through Ministry [grant Japan Research the of Scientific Technology by and supported Science Sports, was work This Funding 2026 wn,C . ol . aa,D,Le . i,S,U,M,Ko,K .adSong, and S. K. Kwon, M., Um, S., Kim, Y., Lee, D., Rajan, J., Holl, Y., C. Hwang, odn .T,Hnig,H,Lzn,G,Wibr,W .e al. et C. W. Weinberg, G., Lozano, H., Hennings, T., G. Bowden, cerevisiae. Saccharomyces in signaling S. Lemmon, M. Yeager, and protein. TRIM5alpha I. W. Sundquist, c.USA Sci. phosphoserine-205. by mediated N-acetyltransferase arylalkylamine C. D. soitdwt uaini RM2 uaieE-bqii-iaegene. E3-ubiquitin-ligase putative a TRIM32, Genet. in K. Hum. mutation Wrogemann, with and associated M. T. fanvlhmnzn igrpoenta pcfclyitrcswt h activation the with interacts specifically that proteins. protein Tat finger lentiviral zinc of human domain novel a of complex. repressor translation tumor-Pumilio brain 2508-2513. the of Model Sci. TRIM5. factor restriction J. Sodroski, eetieadfnto f1-- proteins. 14-3-3 of function and repertoire neato fAtpopoyae txn1wt 433mdae neurodegeneration al. 1. et mediates type J. 14-3-3 ataxia Botas, with T., spinocerebellar H. in ataxin-1 Orr, Akt-phosphorylated M., E. of Skoulakis, Interaction A., Aitken, H., M. Fernandez, ai,M . ty,J .adHp,T J. structures. T. dynamic Hope, highly and P. are J. Stoye, H., M. Malim, ouaeteepeso feihla a hnesb phosphorylation-dependent by channels ligase. Na+ ubiquitin Nedd4-2 epithelial with of interaction T. Isobe, expression and the S. Shimada, modulate N., Takahashi, T., Shinkawa, K., euae iae1rgltsuiutnlgs erlpeusrcell-expressed, precursor neural ligase ubiquitin D. regulates Pearce, 1 and O. kinase Staub, regulated D., J. Stockand, eeomnal onrgltdpoen42b nuigitrcinwt 14-3-3. with interaction inducing by 4-2 Endocrinol. Mol. protein down-regulated developmentally n h 433protein. 14-3-3 the and 2 T. Isobe, and T. iaemttdi ibgrl uclrdsrpytp H rmtsPiasy promotes 2H, type NFkappaB. through dystrophy apoptosis Chem. muscular keratinocyte Biol. UVB-induced J. limb-girdle regulates and in degradation mutated ligase M. Kulesz-Martin, sit h odn fTRIM5alpha. of E3- folding the and assists anti-apoptotic has B. carcinogenesis skin properties. with ligase associated ubiquitin Trim32 protein 21) s7 neat ihtertoia etito atrTI5lh and TRIM5alpha factor restriction retroviral the with interacts Hsp70 (2010). 117 20) eaoi ytei:1---eedn ciainadihbto of inhibition and activation 14-3-3-dependent synthesis: Melatonin (2005). 1875-1884. , 102 ora fCl cec 2 (9) 126 Science Cell of Journal 1222-1227. , 70 19) 433poen:ptnilrlsi eiua rnpr n Ras and transport vesicular in roles potential proteins: 14-3-3 (1995). ,D,L,H,Po .C,LGag,L . ag . adwle A., Vandewalle, J., Wang, P., L. LaGrange, C., A. Pao, H., Li, D., ´, 21) RMpoen n cancer. and proteins TRIM (2011). 20) ai unvradpluiutlto fteretroviral the of polyubiquitylation and turnover Rapid (2006). 281 663-672. , 20) hshrlto-eedn neato fknsnlgtchain light kinesin of interaction Phosphorylation-dependent (2002). 19 25850-25866. , 3073-3084. , 20) h neato fPaywt rm2 nE3-ubiquitin an Trim32, with Piasy of interaction The (2006). rc al cd c.USA Sci. Acad. Natl. Proc. Biochemistry Virology Carcinogenesis o.Bo.Cell Biol. Mol. 20) ibgrl uclrdsrpytp 2H type dystrophy muscular Limb-girdle (2002). Cell Virology 349 .Bo.Chem. Biol. J. 113 21) eaoa sebyo restricting a of assembly Hexagonal (2011). 20) nokn h oeo 14-3-3. of code the Unlocking (2004). 300-315. , rc al cd c.USA Sci. Acad. Natl. Proc. 41 457-468. , 5566-5572. , .Bo.Chem. Biol. J. ESLett. FEBS 25 209 20) -emnlbnig nexpanded an binding: C-terminal (2006). 20) RM lh yolsi bodies cytoplasmic alpha TRIM5 (2007). 157-167. , 18 347-357. , 20) eu-adglucocorticoid- and Serum- (2005). 2102-2111. , 108 a.Rv Cancer Rev. Nat. 285 534-539. , 7827-7837. , 580 280 1531-1535. , 13187-13194. , 20) 433proteins 14-3-3 (2005). 19) Identification (1995). 92 rc al Acad. Natl. Proc. ee Dev. Genes 11 11539-11543. , 20) RING (2004). 792-804. , (2003). (2003). m J. Am. .Cell J. 17 , ooa . iha . ktu . ai . iaci . aaoo . Kosaka, T., Yamamoto, T., Miyauchi, Y., Tani, H., Akatsu, M., Mishra, T., Yokota, M. V. Dixit, and C. Vincenz, rn,T,Sio . ski . uia . oo,T,Mrmtu . Ouchi, M., Muramatsu, T., Hosoi, M., Fujita, T., Tsukui, T., Saito, T., Urano, teki,G,Sub,T,Kdrh,N,Wx . ib,W . lcwl,T .and K. T. Blackwell, F., W. Rigby, S., Wax, N., Kedersha, T., Stubbs, G., Stoecklin, aai . aaua . hmd,S,Sio . iaaa . ak,M,Isobe, M., Taoka, S., Hisanaga, T., Saito, S., Shimada, H., Yamamura, K., Nagaki, og . izGifr,F,Pr,D . oes . telu .adSodroski, and M. Stremlau, T., Rogers, H., D. Park, F., Diaz-Griffero, B., Song, ok,M,Tnly .L,Bno,M .adBae .J. D. Blake, and A. M. Benson, L., C. Tinsley, M., Locke, hnaa . ak,M,Ymuh,Y,Ihmr,T,Kj,H,Tkhsi .and N. Takahashi, H., Kaji, T., Ichimura, Y., Yamauchi, M., Taoka, A. T., J. Shinkawa, Knoblich, and E. Berezikov, C., J. S., Schwamborn, Y. Seo, H., J. Kim, S., J. Maeng, Y., C. Hwang, W., K. Lee, Y., Lee, S., Y. Ryu, Mo L., R. Roberts, i . en,D . ign .M n orsi J. Sodroski, and M. A. Fiegen, F., D. Yeung, X., Li, L. K. Borden, and E. R. Gordon, A., Kentsis, emn,A,Mrn,G,Fnoz,A,Mra . ar,S,Lz,L,Rgnli D., Riganelli, L., Luzi, S., Cairo, G., Merla, A., Fantozzi, G., Meroni, A., Reymond, Zu D., Morger, S., Hausmann, T., Pertel, urahv,E,Sry,A,Mkooa . ann .C n pne,M J. M. Spencer, and C. S. Cannon, E., Mokhonova, A., Struyk, E., Kudryashova, J. M. Spencer, and A. L. Havton, J., Wu, E., Kudryashova, J. M. Spencer, and I. Kramerova, D., Kudryashov, E., Kudryashova, S. Hatakeyama, and S. G. Fukuda, Hutvagner, N., and Miyajima, S., R. Kano, Hay, A., Sobala, C., M. Geoffroy, M., Johnston, Omata, T., Horigome, N., Nomura, T., Natsume, M., Taoka, T., Isobe, C., H. Itagaki, Tokumitsu, and K. Goto, Y., Hozumi, M., Taoka, T., Ichimura, kd,N,Ybt,N,Szk,H,Aln . rn .adNjm,H. Nojima, and M. Oren, Y., Aylon, H., Suzuki, N., Yabuta, N., Okada, ioe . ty,J .adSaı and P. J. Stoye, S., Nisole, nlssi lhie’ ies patients. disease K. Alzheimer’s Heese, in and analysis T. Sawada, Y., Nagai, K., pcfcmne n ucinbt scaeoeadaatrmolecules. adapter and chaperone as 271 both function and manner specific growth. tumour S. Inoue, and Y. rnl soito n R-RAdecay. ARE-mRNA and association granule P. Anderson, fteitrcinbtenteuiutnE iaeNd42adeihla Na+ epithelial and Nedd4-2 ligase E3 ubiquitin the between channels. interaction the of T. Ichimura, and T. niervrlactivity. antiretroviral J. bqii iaefrdysbindin. for ligase ubiquitin .Poem Res. Proteome J. neural T. mouse Isobe, in self-renewal prevents and microRNAs progenitors. activates TRIM32 protein (XIAP). apoptosis of inhibitor X-linked 25729-25738. against S. activity K. ligase Kwon, (TNF factor and necrosis B. tumor Song, H., K. You, re soito ftersrcinfco RMapaadohrtiatt motif tripartite other and TRIM5alpha factor restriction the proteins. (TRIM) of association order A/AKcsaesgaigdrn suoyhldvlpeti .cerevisiae. S. in development pseudohyphal during Cell signaling cascade RAS/MAPK al. et S. 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H. ¨sch, .Cl Sci. Cell J. Nature 20) f agt 433sgafrpoelssadpooe breast promotes and proteolysis for sigma 14-3-3 targets Efp (2002). 136 4 .Bo.Chem. Biol. J. 1826-1831. , 913-925. , 20) 433Mdae hshrlto-eedn inhibition phosphorylation-dependent Mediates 14-3-3 (2006). a.Rv Microbiol. Rev. Nat. Virology 45 417 6733-6740. , a b A. ¨b, 871-875. , 19) 433poen soit ihA0i nisoform- an in A20 with associate proteins 14-3-3 (1996). 124 -nue ppoi i t IGdmi-eedn E3 domain-dependent RING its via apoptosis )-induced u.Ml Genet. Mol. Hum. 21 MOJ. EMBO u.Ml Genet. Mol. Hum. 18 57-67. , 1462-1469. , 343 286 1353-1367. , 20) RMfml rtis ervrlrestriction retroviral proteins: family TRIM (2005). 201-211. , 27959-27970. , gr . ura . acn,J,Reinhard, J., Lascano, J., Guerra, S., ¨ger, Nature 20 u.J ln Invest. Clin. J. Eur. 21) RM2poensniie el to cells sensitizes protein TRIM32 (2011). 20) ri ieseii eeexpression gene site-specific Brain (2006). 2140-2151. , 3 20) oto fbohmclreactions biochemical of Control (2002). MOJ. EMBO 799-808. , 19) 433poen r seta for essential are proteins 14-3-3 (1997). 472 20 .Ml Biol. Mol. J. 20) h rpriemtffamily motif tripartite The (2001). 21) RM sa naeimmune innate an is TRIM5 (2011). 18 3925-3932. , 21) eemnnso h higher the of Determinants (2011). 361-365. , 2344-2358. , rc al cd c.USA Sci. Acad. Natl. Proc. ESLett. FEBS 23 1313-1324. , 19) Stimulus-coupled (1999). 20) RM2i nE3 an is TRIM32 (2009). 20) h TRIM-NHL The (2009). Biochemistry 20) eiinyo the of Deficiency (2009). 20) rpriemotif Tripartite (2008). 354 36 820-830. , .Bo.Chem. Biol. J. 413-424. , 582 20) Trim32 (2005). 20) 14-3-3 (2008). 661-665. , .Bo.Chem. Biol. J. 38 21) A (2011). 15673- , (2010). 286 99 , ,