3796 Author Correction

Tumor suppressor p16INK4a inhibits cancer cell growth by downregulating eEF1A2 through a direct interaction Mee-Hyun Lee1,2,*, Bu Young Choi3,*, Yong-Yeon Cho1,4, Sung-Young Lee1,2, Zunnan Huang1,`, Joydeb Kumar Kundu5, Myoung Ok Kim1,6, Dong Joon Kim1,§, Ann M. Bode1, Young-Joon Surh2," and Zigang Dong1,2," 1The Hormel Institute, University of Minnesota, MN 55912, USA 2WCU Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology and Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, South Korea 3Pharmaceutical Science and Engineering, School of Convergence Bioscience and Technology, University of SeoWon, Chungju, Chungbuk, 361-742, South Korea 4College of Pharmacy, The Catholic University of Korea, Bucheon, Gyeonggi-do, South Korea 5College of Pharmacy, Keimyung University, Daegu, South Korea 6School of Animal Science, KyungPook National University, Sangju, South Korea *These authors contributed equally to this work `Present address: China-America Cancer Research Institute, Guangdong Medical College, Dongguan, Guangdong 523808, People’s Republic of China §Present address: Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea "Authors for correspondence ([email protected]; [email protected])

Journal of Cell Science 126, 3796 2013. Published by The Company of Biologists Ltd doi: 10.1242/jcs.137521

There was an error published in J. Cell Sci. 126, 1744-1752.

The affiliations of Zunnan Huang, Joydeb Kumar Kundu and Myoung Ok Kim were given incorrectly. The correct affiliations are as listed above.

We apologise for this error. Journal of Cell Science nterglto fteekroi elcce oee,temcaim neligteat-rlfrtv fet fp16 of effects anti-proliferative the underlying mechanisms the However, cycle. cell eukaryotic the of regulation the in ydwrgltn E12truhadrc interaction direct a through eEF1A2 downregulating by utemr,sprsino rti ytei yepeso fp16 of expression by synthesis of suppression Furthermore, fPamc,SolNtoa nvriy eu,SuhKorea South Seoul, University, National Seoul Pharmacy, of odbKmrKundu Kumar Joydeb 2 a oano E12 coi xrsino p16 of expression Ectopic eEF1A2. of domain aa) 327 neato fp16 of interaction ylnDcmlxb p16 CDK4/ the by of complex regulation The D of 1993). inhibitor al., cyclin specific et a (Serrano Cdk6 as and acting Cdk4 by partially, least at proliferation, utsbadBka,20) h uo upesrprotein suppressor tumor The 2002). Bakkar, p16 critical Gali- and are 2005; Garrett, Chk2) and Muhtasib (Collins and progression pathways cycle (Chk1 cell of kinases signaling regulators Cdk check-point (Cdks), Intracellular and kinases inhibitors, cyclin-dependent 1974). to cyclins, commit various (Pardee, and the cancer comprising after factors of division growth of mid-G1, One of in cell hallmark independent 1994). occurs become Kastan, a cells check-point, and which restriction is (Swanton, Hartwell an the checkpoints 1994; checkpoints, separation al., ensuring these et and by of (Hartwell Failure stability replication 2004). genetic cell- maintain Two phases. authentic I (M) tightly checkpoints mitosis gap and a phases: (G2) cycle different 2 through gap four (S), occurs comprises synthesis (G1), which division cycle, cell cell regulated Eukaryotic inappropriate involves division. cells cell transformed of progression Malignant Introduction words: Key p16 protein suppressor tumor The Summary " work § this to equally ` contributed authors *These 5 4 3 2 1 Lee Mee-Hyun p16 suppressor Tumor 1744 xliigteat-rlfrtv fet fp16 of effects anti-proliferative the explaining obnto fp16 of combination iagDong Zigang ees 98 hr n oet,19) oee,subcellular However, 1999). Roberts, and p16 Sherr of localization 1998; Peters, n ulu Eaglue l,20) hiadcolleagues and Choi 2004). al., et (Evangelou nucleus and o:10.1242/jcs.113613 doi: 1744–1752 126, Science Cell of Journal 2013 January 21 Accepted irijcinof Microinjection enflyeuiae.Uigyattohbi cenn,w dniidteekroi lnainfco eF12a oe interacting novel a as (eEF)1A2 factor elongation eukaryotic the identified we p16 screening, of two-hybrid partner yeast Using elucidated. fully been rsn drs:CiaAeiaCne eerhIsiue undn eia olg,Dnga,Gagog530,Pol’ eulco China of Republic People’s 523808, Guangdong Dongguan, College, Medical Guangdong Institute, Research Cancer China-America address: Present rsn drs:MdclGnmc eerhCne,KraRsac nttt fBocec n itcnlg,Deen ot Korea South Daejeon, Biotechnology, and Bioscience of Institute Research Korea Center, Research Genomics Medical address: Present uhr o orsodne( correspondence for Authors colo nmlSine ynPo ainlUiest,Snj,SuhKorea South Korea Sangju, South University, Gyeonggi-do, National Bucheon, KyungPook Korea, Science, of Animal University Korea of Catholic South School College The 445-970, and Pharmacy, Gyeonggi-do Technology of and City, College Science Hwasung Convergence Labs, of Research School C&C Graduate Sciences, Biopharmaceutical USA and 55912, Medicine Molecular MN of Minnesota, Department of WCU University Institute, Hormel The 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. INK4a asscl yl retadihbt uo cell tumor inhibits and arrest cycle cell causes p16 INK4a INK4a 1, " 1,2, INK4a INK4a p16 . nipoieaieefcs E12 vra acr rnltoa activity Translational cancer, Ovarian eEF1A2, effects, Anti-proliferative , eEF1A2 INK4a ,B on Choi Young Bu *, INK4a [email protected] a endcmne nbt h cytoplasm the both in documented been has iheFA,adsbeun oneuaino h xrsinadfnto feFA sanvlmechanism novel a is eEF1A2 of function and expression the of downregulation subsequent and eEF1A2, with INK4a n opoioeFA eutdi ute euto ntasainlatvt.W ocueta the that conclude We activity. translational in reduction further a in resulted morpholino-eEF1A2 and 2, RAit h yols of cytoplasm the into mRNA ` stogtt ucina nocgn ncnes h p16 The cancers. in oncogene an as function to thought is yugO Kim Ok Myoung , cusi h ulu Ra and (Ruas nucleus the in occurs INK4a ; samme fteIK aiyo ylndpnetkns Ck niios hc r involved are which inhibitors, (Cdk) kinase cyclin-dependent of family INK4 the of member a is [email protected] 3, ,Yn-enCho Yong-Yeon *, INK4a 1,5 . ogJo Kim Joon Dong , ) INK4a INK4a Xenopus erae h xrsino E12adihbtdcne elgrowth. cell cancer inhibited and eEF1A2 of expression the decreased 1,4 mro upesdtelcfrs RAtasain hra the whereas , mRNA luciferase the suppressed embryos niiscne elgrowth cell cancer inhibits ugYugLee Sung-Young , eosrtdta yolsi p16 cytoplasmic that demonstrated eltasomto yitrcigwt -u -emnlkinase p16 cytoplasmic N-terminal Thus, c-Jun 2005). al., with et interacting (Choi (JNK)-1 by transformation cell cdhmlg n lyn iia oei rti synthesis, protein and ( in location 6q14 chromosomal role extensively their in amino in similar differ a 98% isoforms of been two playing sharing these isoforms and Despite two have homology of unexplained. acid existence remains eEF1A2, the still of eEF1A two necessity and as the exists investigated, which the eEF1A1 eEF1A, a catalyzes mammalian in isoforms, of Although and roles 1985). the 1 biological Moldave, of 2002) the four 2002; A-site factor (Ejiri, of Ejiri, the manner elongation to one GTP-dependent 2002; aminoacyl-tRNA is eukaryotic of Proud, family, binding in the protein and alpha G constitute implicated (Browne 1 the White- that factor of been elongation subunits 2009; member Eukaryotic a have 2009). Hinnebusch, (eEF1A), al., factors and et Gilbertson (Sonenberg (eEF) (eIF) initiation carcinogenesis elongation translation and Ruggero eukaryotic 2004; and Several al., cells et 2003). Ruggero of Pandolfi, 2007; transformation Stokoe, malignant and with (Bilanges associated is machinery cells. in roles functional other eetsuisdmntae htdrglto ftranslational of deregulation that demonstrated studies Recent INK4a 1,§ n .Bode M. Ann , xvivo ex EEF1A2 INK4a a eiidb uieaerpre activity. reporter luciferase by verified was 1,2 rti neatdwt l u h 2(250– D2 the but all with interacted protein n2q3 Ln ta. 96 n their and 1996) al., et (Lund 20q13) in unnHuang Zunnan , 1 on-onSurh Young-Joon , INK4a upesdUV-induced suppressed eerhArticle Research 1 , INK4a INK4a 2, " and a play may aenot have EEF1A1 Journal of Cell Science Results cells. cancer ovarian in eEF1A2 downregulating by function es w-yrdsreigo ua eacDNA-GAL4 HeLa human a of screening two-hybrid yeast a lnainfco 1A2 factor elongation h rsn td,w eosrt oe ehns whereby mechanism In novel 2008). a al., demonstrate et in tissue p16 we (Pinke levels study, tumors of ovarian expression present primary the high results all in showed of the 30% abundance tumors about protein as addition, ovarian eEF1A2 primary cells of In 500 assessment and (ES-2) mice. the carcinoma for potential nude microarray ovarian oncogenic in of provides xenografts with growth eEF1A2 the of (NIH3T3) 2010; accelerates overexpression al., that fibroblasts ectopic et reported The mouse 2002). Li colleagues al., 2009; and in et Surh, Anand overexpressed 2002; is 2005). and al., eEF1A2 al., et Lee Anand et 2007; 2007; Tomlinson al., al., et et (Amiri identified Kulkarni oncogene been putative has in and a protein involved cells as be the of to functions in reported non-translational protein been role other has of defined 1994), eEF1A2 pathways process, its al., play translation Beyond or in et cells roles. Lee involved nonproliferating for non-translational 1993; be expressed preferentially are al., to that et is synthesis suggested Knudsen is eEF1A2 1998; cardiomyocytes eEF1A2 cycle al., neurons, cell Because the et al., from as deviated et (Kahns 1993). permanently such Lee have that 1992; cells, myocytes, al., al., and certain et in et Lee 1993; specifically Lee al., (Khalyfa et nonproliferation Knudsen 1995; of are state 2001; that a al., muscle, in et skeletal locked and only cells heart is present of brain, isoform is composed as eEF1A2 such eEF1A1 tissues, whereas The those eEF1A tissues, in distribution. all of almost tissue isoforms in their two expressed in The eEF1A1 While differ preference. GTP, 1998). also than opposite al., GDP the et for affinity (Kahns displays stronger GTP a and exhibits GDP eEF1A2 for affinity binding nodrt dniypoen htitrc ihp16 with interact that identify to order In p16 suppressor Tumor INK4a neat iheFA n xrsistmrsuppressor tumor its exerts and eEF1A2 with interacts nvitro in INK4a 0 foaintmr (Anand tumors ovarian of ,30% neat iheukaryote with interacts and xvivo ex INK4a ,weperformed xrsinpamdlbayuigafl-eghp16 full-length a using library plasmid expression i.S) nadto,ec E12famn a rdcdby produced was with fragment eEF1A2 interacted each aa) material addition, (77–156 supplementary In 2A; p16 p16 S2). (Fig. full-length eEF1A1 Fig. and with fusion not aa) but GST (1–80 eEF1A2, The p16 eEF1A2. protein, of domain binding utemr,edgnu p16 endogenous Furthermore, eind lsisecdn h orsodn rget of fragments corresponding the eEF1A2 encoding Plasmids designed. oNtria 5wr rninl otasetdwt pcDNAp- with co-transfected transiently 16 were V5 encoding N-terminal to Plasmids assay. immunoprecipitation sda oiiecnrlt hwtep16 the show to control was positive CDK4 a Serum-induced as 1C). used (Fig. eEF1A2 serum-induced between interaction The 1A). (Fig. cells p16 in VP16-eEF1A2 el,it amla vectors. mammalian into cells, oeua aso h rti s5 D.Frfrhrsuy ecloned we 1392 study, predicted further For The of kDa. acids. 53 amino is the consists protein 463 further the of clone of mass for protein molecular eEF1A2 a S1) clone encoding full-length Fig. the nucleotides, selected The material We (supplementary investigation. ‘bait’. eEF1A2 the as encoding domain DNA-binding GAL4 eosrt ietitrcinbtenp16 between interaction direct a demonstrate neat iheFA nmmaincells. mammalian in eEF1A2 with interacts )cls(i.1) hs eut niaeta p16 that indicate (PA- results cancer These 1D). ovarian (Fig. human cells in 1) eEF1A2 by immunoprecipitated aiu eeincntut fp16 of constructs deletion various muoltaayi eeldta p16 that revealed and beads. lysate analysis 4B whole a Sepharose Immunoblot using GST–p16-conjugated assay or pull-down GST-conjugated- GST a performed we eutdi h opeiiaino p16 of co-precipitation the in resulted hs eut niae htGAL4-p16 pVP-eEF1A2. that containing indicated domain VP16 binding results assay GAL4 and These the two-hybrid pM-BD-p16 with mammalian transfected containing the cells performed COS-7 using we system, cell ocnimteitrcinbtenp16 between interaction the confirm To oietf h neatn oan fp16 of domains interacting the identify To INK4a E12a oeta agto p16 of target potential a as eEF1A2 INK4a eEF1A2 eec-rnfce ihp16 with co-transfected were a muorcpttdwt p16 a with immunoprecipitated was 2huswt rwtot1 ea oiesrm(+/ serum bovine additional fetal 1% an without for or incubated with were hours cells 12 PA-1 hours) (24 serum-starved ( blotting. western by detected were nt omlzdt eaiecnrl(vrg ) ( %). (average control luminescence negative relative a in to change normalized the units assay. indicates two-hybrid activity mammalian luciferase the The using determined was pVP-eEF1A2 muorcptt a ujce oimnbo nlssuigan antibody. using polyclonal analysis rabbit immunoblot eEF1A2 to subjected was immunoprecipitate GST–p16 eEF1A2. V5-tagged ( against by antibody polyclonal detected a were with immunoprecipitates immunoblotting the in proteins eEF1A2 ellstswr muorcpttdwt p16 a with cells. immunoprecipitated PA-1 were from lysates co-immunoprecipitated Cell were proteins eEF1A2 and p16 a as identified is eEF1A2 1. Fig. ( uldw sa a sdt ofr h neato between interaction the confirm to used was assay pull-down A C) h neato fpoen xrse rmpM-BD-p16 from expressed proteins of interaction The A) noCS7cls muorcptto fV5-eEF1A2 of Immunoprecipitation cells. COS-7 into eecntutdadue oietf h p16 the identify to used and constructed were n E12wsfrhrcnimdb co- a by confirmed further was eEF1A2 and ee hc a xrce rmttlmN fHeLa of mRNA total from extracted was which , INK4a n E12o D4i hl ellsts n proteins and lysates, cell whole in CDK4 or eEF1A2 and INK4a n E12 h p16 The eEF1A2. and hl ellstspeae from prepared lysates cell Whole D) INK4a INK4a INK4a INK4a oolnlatbd.The antibody. monoclonal INK4a INK4a INK4a itrcigprotein. -interacting INK4a n E12were eEF1A2 and INK4a a loco- also was INK4a INK4a INK4a INK4a n E12i a in eEF1A2 and 2 INK4a neatdwith interacted with interacted INK4a eEF1A2 B Fg B.To 1B). (Fig. eu) p16 serum). nioyadthe and antibody n eEF1A2, and n eEF1A2, and O- cells COS-7 ) fusedtothe INK4a interaction. physically INK4a protein 1745 INK4a fused INK4a and - Journal of Cell Science 1746 LGtge E12(5–2 a i o idt p16 to bind not did aa) However, (250–327 proteins. aa) eEF1A2 (328–463 FLAG-tagged eEF1A2 or aa) (1–249 eEF1A2 iheulaonso S–1 n S,GTp6fl-egh(–5 a,GTp61(–0a)adGTp62(716a)(,tp.()FA-agdfull- FLAG-tagged (B) GST–p16 top). of (A, amounts aa) equal (77–156 with GST–p16D2 incubated and were aa) (top) (1–80 fragments GST–p16D1 deletion aa), various (1–156 and GST–p16-full-length eEF1A2 GST, length and GST–p16 of amounts equal with r2 np16 in Arg24 eetdb uoaigah A rwsenbotn B.( (B). blotting western or (A) autoradiography by detected ewe h eiusfo h orhrpa fp16 of (supplementary bonds repeat hydrogen fourth eEF1A2 the the from addition, residues in In the S3A,B). between Glu403 Fig. S1; with Table material bond hydrogen one ewe S–E12adwl-yep16 wild-type and GST–eEF1A2 between h is,scn n hr nyi eet fp16 of repeats ankyrin third and second first, the between networks bond 6 a,btntt E12(5–2 a Fg 2C). (Fig. aa) (250–327 eEF1A2 to not but aa), 463 i.2 dniiaino h neatn oan feFA n p16 and eEF1A2 of domains interacting the of Identification 2. Fig. p16 between binding The 2B). (Fig. protein, down fusion pulled were GST lysates p16 Sepharose-4B-bead-conjugated cell the the and using cells, 293 in transfection hw hr p16 where shown rmtepoenpoendcigeprmn ugse that suggested experiment docking protein–protein p16 the from i neatwt ahidvda 1 uat(2Ao 11) ersnaiebosaesonadterslso he elctosaeqatfe ntegraph. the in quantified are replications three of results the and shown ( are expression blots eEF1A2 Representative in R131E). decrease or significant (R24A a mutant is p16 There individual each with interact did p16 xetdt ecuili h idn fp16 of binding the in crucial be to expected DC.Temdlo h p16 the of algorithm docking model transform-based The Fourier ZDOCK. Fast the by predicted vsa oeua yais Hmhe ta. 96.( 1996). al., et (Humphrey dynamics) molecular (visual rmdmi feFA ol locnrbt otesaiiyof stability the to p16 contribute also would the eEF1A2 of I domain from ntepeitdp16 predicted the In INK4a INK4a INK4a INK4a eut fteGTpl-onasysoe that showed assay pull-down GST the of Results . neatdwt ullnt E12 n FLAG-tagged and eEF1A2, full-length with interacted ora fCl cec 2 (8) 126 Science Cell of Journal on oeFA 129a)adeFA (328– eEF1A2 and aa) (1–249 eEF1A2 to bound eFA ope splmnaymtra Table material (supplementary complex –eEF1A2 INK4a INK4a si rneadeFA nca.The cyan. in eEF1A2 and orange in is om w yrgnbnswt l34and Glu374 with bonds hydrogen two forms INK4a b srnsi oanIIo E12and eEF1A2 of III domain in -strands eFA ope,tehydrogen- the complex, /eEF1A2 INK4a INK4a INK4a eFA ope obtained complex –eEF1A2 rmtn 2AR3Ep16 R24A/R131E mutant or n E12wsalso was eEF1A2 and INK4a P h mn cd r2 n r11o p16 of Arg131 and Arg24 acids amino The D) , rdce oe ftep16 the of model Predicted C) .5 nwl-yep16 wild-type in 0.05) INK4a iheEF1A2. with a hlcsaedana prl n the and spirals as drawn are -helices INK4a n those and INK4a INK4a are INK4a . ( A, E12ddntitrc ihtedul uatp6(2AR3E,but (R24A/R131E), p16 mutant double the with interact not did eEF1A2 . INK4a B lnainfco nppiecaneogto uigprotein during elongation p16 chain protein antiproliferative peptide the in synthesis, factor elongation 4 a.T ofr hsie,w osrce p16 a constructed we idea, this confirm To aa). 249 ocnrtos(,10 0,40ad80n)o recombinant of nM) 800 and 400 200, p16 100, (0, possibility, this concentrations explore To vitro eEF1A2. in of activity translational the 1 i.SCD.Ag3 np16 in Arg131 S3C,D). Fig. S1; R4)adAg3 R3E irpe h neato of interaction the disrupted (R131E) Arg131 Arg24 of p16 mutation and Double Arg131. and (R24A) Arg24 at mutations with neato iheFA oanI/III. domain eEF1A2 with interaction niae htAg4adAg3 fp16 of Arg131 and Arg24 that indicated ete xmndtefntoa infcneo h interaction the of significance p16 functional between the examined then We p16 eepromduiglcfrs DAa h eotrgn and gene reporter the as cDNA luciferase using performed were ihGu1 neFA.Teehdoe od ol explain would bonds hydrogen These eEF1A2. the in Glu217 with oeo eEF1A2 of role p16 ullnt E11adeFA rtiswr yteie n incubated and synthesized were proteins eEF1A2 and eEF1A1 Full-length ) oprdt h obemtn 2AR3Ep16 R24A/R131E mutant double the to compared INK4a INK4a INK4a INK4a nvitro in INK4a rti ytei a sesdi h rsneo varying of presence the in assessed was synthesis protein The . n E12cmlx rs-ydsee iwo h ope is complex the of view stereo cross-eyed A complex. eEF1A2 and neato iheFA 3843a)adeFA (1– eEF1A2 and aa) (328–463 eEF1A2 with interaction niistasainlactivity translational inhibits iheFA sepce Fg D.Ti result This 2D). (Fig. expected as eEF1A2 with INK4a b and INK4a srnsa ros hs iue eegnrtduigVMD using generated were figures These arrows. as -strands nvitro in INK4a on rtiswr nlzdb 5 D-AEand SDS-PAGE 15% by analyzed were proteins Bound . n E12 eas E12fntosa an as functions eEF1A2 Because eEF1A2. and xvivo ex neatwt E12 eut fapl-onassay pull-down a of Results eEF1A2. with interact rncito n rnlto experiments translation and transcription xeietlfnig eadn the regarding findings experimental INK4a om w yrgnbonds hydrogen two forms INK4a nvitro in INK4a INK4a r motn for important are ih attenuate might . and INK4a nvivo in protein : Journal of Cell Science RA flcfrs ln rwt uieaeplus luciferase with or alone luciferase of mRNAs When activity. translational rnlto sa hwdasgiiatdces nprotein p16 in recombinant decrease of the significant addition of the a luciferase with Results showed the synthesis autoradiograpy. assay by and detected translation assay were translation activity and transcription h xrsino E12wsdcesdwt neto of injection with decreased was eEF1A2 of Moreover, 3C). expression (Fig. mRNA the luciferase of translation the inhibits rnlto sa oepoeteefc fp16 of an effect performed the explore we of to Therefore, identification assay 1982). translation the (Richter mechanisms al., for translation protein et method of study sensitive the and mRNAs very a constitutes 3A,B). (Fig. uieaetasainlatvt a infcnl erae nthe p16 in of decreased presence significantly was activity translational luciferase hwntcal hne ntesnhssof synthesis not the did control, in negative changes a with as noticeable GST performed of show experiments concentrations Analogous same 3A,B). the (Fig. nM) 800 h ir-neto fmNsinto mRNAs of micro-injection The INK4a ugsigta h xrsino p16 of expression the that suggesting , Xenopus mro eeijce with injected were embryos Xenopus INK4a nvv Xenopus vivo in INK4a 35 p16 S-luciferase neEF1A2 on INK4a embryos nvitro in 40or (400 INK4a the , Fg F.T lrf h feto p16 of effect the clarify To 3F). (Fig. (5 MO-eEF1A2 E12 eut niae htteepeso feFA was 10 eEF1A2 or endogenous of (5 of expression MO-eEF1A2 the by translation that decreased indicated the Results inhibited eEF1A2. specifically control, ciiyo eEF1A2 of activity uieae( g plus ng) (1 luciferase netdwt uieae(0 g ln,lcfrs plus luciferase alone, pg) (500 luciferase with injected activity, translational etr ltrslssoe that showed results We blot 3D). Western (Fig. into untreated by (MO-eEF1A2) left injecting siRNA or eEF1A2 morpholino-based alone a optimized luciferase with injected hs aasgetta p16 that suggest data These ru,btntteM-oto dt o hw) showed of presence shown), the not inhibition in this (data decreased and activity further MO-control luciferase was the the of MO-eEF1A2 inhibition not plus moderate luciferase but The group, MO-eEF1A2. plus luciferase 10p) uieaeplus luciferase pg), (100 E12a oeta agto p16 of target potential a as eEF1A2 rti fe neto flcfrs,lcfrs plus luciferase luciferase, of injection after protein p16 n bec fp16 of absence and ( of into injection MO-eEF1A2 after or protein MO-control luciferase translated the of activity analysis. blot western to MO-control subjected or were MO-eEF1A2 of concentrations indicated oto ovrf qa rti odn.( loading. protein equal verify to control needn ape n aaaesona means as shown are using data times and three samples least independent at repeated were experiments into mRNAs MO-eEF1A2 plus translated eiuoyesse.( system. reticulocyte noeoseFA n xgnu p16 exogenous and eEF1A2 endogenous uieaeplus luciferase E12adp16 and eEF1A2 i.3 p16 3. Fig. es he ie sn needn ape n aaare data and means samples as independent shown using times three least omsi le(B tiig h oe ae hw the ( shows panel lower (means The quantification by staining. confirmed (CB) was Blue loading Coomassie detected lane and Equal SDS-PAGE autoradiography. 15% by by analyzed were produced ln ri obnto with combination luciferase in of or mRNAs alone synthetic with injected were Embryos into E12 h xrsino E11wsntchanged. not was eEF1A1 ( of expression The eEF1A2. h p16 The eEF1A2. 0,o 0 MGTo GST–p16 or 200, GST 100, nM 0, 800 adding or by 400, determined was protein luciferase G uieaeatvt in activity Luciferase C) E Xenopus m yae from Lysates ) h uieaeatvt ftetasae luciferase translated the of activity luciferase The ) INK4a )in M) Xenopus nvitro in INK4a fe neto fp16 of injection after ( p16 35 A Xenopus INK4a p16 -e-aee uieaepoen.Teproteins The proteins. luciferase S-Met-labeled h uieaeatvt fan of activity luciferase The ) Xenopus mro tdfeetconcentrations. different at embryos rti fetvl erse h xrsinof expression the repressed effectively protein mrowseaie ywsenblotting. western by examined was embryo INK4a p16 and INK4a 6 INK4a niistetasainlatvt of activity translational the inhibits Xenopus INK4a INK4a INK4a ..( s.d. nvivo in 50p)cmae oembryos to compared pg) (500 B erae h uieaeactivity luciferase the decreased 6 eeue o h eeto of detection the for used were lsM-E12(2.5 MO-eEF1A2 plus uoaigaho the of Autoradiograph ) l xeiet eerpae at repeated were experiments All . lsM-E12 rluciferase or MO-eEF1A2, plus h bnac feFA and eEF1A2 of abundance The D) .. ftreexperiments. three of s.d.) MO-eEF1A2 mro eeseparately were embryos Xenopus niie h translational the inhibited m mro netdwt the with injected embryos ;Fg E.Ijcinof Injection 3E). Fig. M; . p16 INK4a Xenopus INK4a Xenopus INK4a mro ntepresence the in embryos p16 INK4a n uieaemRNA luciferase and nioisagainst Antibodies . b INK4a noarabbit a into INK4a INK4a u o MO- not but , F mro.All embryos. Atnsre sa as served -Actin nvitro in h luciferase The ) embryos. neEF1A2 on respectively. , Fg 3G). (Fig. nvitro in p16 translated p16 m 6 ) or M), 1747 INK4a INK4a s.d. , Journal of Cell Science 1748 a ofre yuigtesal rnfce p16 transfected stably the using by confirmed was E12wsdcesdi iedpnetmne.Moreover, of amounts manner. varying with time-dependent cells CHO-KI a of co-transfection in decreased was eEF1A2 ytmi eacls(i.4) fe eoa fdoxycycline, of removal After 4A). (Fig. cells p16 HeLa in system ee feFA Fg 4F). (Fig. eEF1A2 of level el ihwild-type with cells E12oeepesn vra acrcells cancer ovarian eEF1A2-overexpressing bptwy Rb pathway, Rb xrsino E12wsdcesdwt increasing with decreased was eEF1A2 of concentrations of expression n pcDNA-p16 and rwhwssgiiatyrtre ytepeec fp16 of presence the by retarded significantly was growth 10o 0 g i.5) nadto,P- el stably cells PA-1 addition, In 5A). 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( A–D Rb eEF1A2 2 F The ) / The ) INK4a 2 Journal of Cell Science n VA8cells. OVCAR8 and tiigo F A1 G KV n H VA8clstasetdwt ihrcnrlsRAo E12s-N.Aleprmnswr eetda es three least at repeated were experiments All means sh-RNA. as eEF1A2 ( shown or cells. control-shRNA are OVCAR8 either data with (E) and transfected and cells samples SKOV3, OVCAR8 independent (H) (D) using and PA-1, times SKOV3 (C) (G) PA-1, in (F) sh-control, of by staining not but sh-eEF1A2, with oecln omto oprdt E11expression eEF1A2 eEF1A1 of expression to The S5). compared Fig. formation material induced (supplementary colony expression eEF1A2 eEF1A1 that of more indicated properties Results oncogenic (supplementary assay eEF1A2. the 2012) growth and compare cell al., to types anchorage-independent performed et cell An was Scaggiante S4). cancer Fig. 2004; many material al., in et ubiquitously (Joseph expressed are protein p16 of Implications 5. 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(Fig. INK4a .( INK4a B Anchorage- ) INK4a 1749 INK4a but Journal of Cell Science 1750 neatn rtis r eddt eemn h specific with 2008) other the al., and determine et eEF1A2 (Panasyuk to relationship of p16 needed this interruption for are mechanism direct proteins, as interacting further However, such S6). Fig. studies, material (supplementary changed not of level mRNA mro ihcmiain of combinations with embryos /I.Ee huhrslsidct o idn fiiybetween affinity binding low a p16 indicate results though Even I/III. E12i h rsneo p16 of presence the in eEF1A2 3D,E). 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Fig. material (supplementary ubcosmdfe al’ eim(MM,RM-60adftlbovine (MEM), fetal medium and essential RPMI-1640 Minimum (DMEM), MO). medium Louis, Eagle’s (St. modified was Dulbecco’s Sigma from bromide] procured [3-(4,5-dimethyl-thiazolyl-2)-2,5-diphephenyl-tetrazolium MTT reagents and Chemicals Methods and Materials of level mRNA the examined we eEF1A2 cells, OVCAR8 and SKOV3 in p16 by inhibition the to activity due translational probably downregulatory was eEF1A2 the of eEF1A2, to only binds binding strong a prevent p16 might with of eEF1A2 cavity interaction binding to smaller compared the homology, eEF1A1 sequence high a exhibit hwd o h rnlto ciiyrglto feFA by we eEF1A2 as of regulation significant, activity quite translation eEF1A2, become p16 the of might For activity effect showed. the regulatory affects the cooperatively bond hydrogen RA n OeFA n h eut ofre that confirmed results the and MO-eEF1A2 p16 and mRNA, hc antataoe feFA n E11wr nthe in work eEF1A1 downregulation p16 heterodimer, and by a eEF1A2 eEF1A2 as of translation If of phase alone. eEF1A1, elongation functional act a cannot of exclusion which the with activity translational ad h oneuaino h rnltoa ciiyo eEF1A2 other of the activity p16 translational On the by shown). of not downregulation the (data hand, degradation and ubiquitination neato ewe p16 between interaction p16 and eEF1A2 rabbit with sequence coincides the Because eEF1A2 3A,B). human (Fig. frame of time limited a in system iesoa tutr feFA Fg C,dmi Ii located is p16 therefore II and domain I/III 2C), domain (Fig. behind eEF1A2 of structure dimensional Mre ta. 04.Orcmuainldcigmodel p16 docking hydrogen between computational 12 formed that Our indicates are S1) 2004). bonds Table material al., (supplementary et (Marles h xc ehns xliigtedcesdepeso of expression decreased the explaining mechanism exact The INK4a INK4a INKa INK4a INK4a INK4a , . the answered, be to remain questions many Although . an used we , euae E12atvt Fg ) pcfcly we Specifically, 3). 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Porter, M., Wernick, G., Amann, S., Murthy, N., M. Anand, J. Lee, and E. D. Pinke, G., Kulkarni, S., Jeganathan, F., Noei, A., Amiri, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.113613/-/DC1 online available material Supplementary months. 12 after Y.J.S.]. release to for R31-2008-000-10103-0 PMC World in number the Deposited to [grant of Korea CA081064, University of Ministry Republic Class Foundation, R37 Technology, Research and CA120388, National Science National Education the numbers and Z.D.]; [grant Foundation to Hormel ES016548 Health The of by supported Institutes was work This Funding ru eepoe n ooeie n15 per in embryos luciferase homogenized five or a and Four using pooled instructions. measured were manufacturer’s were and group to legends activities according figure Luciferase system the hours. in assay stage described 7–8 two-cell as or for morpholinos one- and incubated the mRNA at with Embryos injected NJ). were Brunswick, New Corp., Mfg. Chemical lcrpopae 1 glycerophosphate, caao,P,Dso-lkd,D., Dasiou-Plakida, P., Zacharatos, I., Peros, J., Bramis, K., Evangelou, Z. Seymour, Weng, M., and Schutte, R. S., Chen, M. Redston, T., L. Costa, da A., S. Hahn, C., Caldas, jr,S. Ejiri, G. C. Proud, and J. G. Browne, D. Stokoe, and B. Bilanges, xeiet eerpae tlattretmsuigidpnetsmls All and samples. (luciferase independent cDNAs by using the produced times of were microinjection Each three for least used mRNAs at synthetic repeated were experiments emn)adte sdfrwsenblotting. western for used then and Germany) 9 AGGGCTTTTCCTC(eeol,Pioah R.Control OR). Philomath, (GeneTools, -ATGTGTGTCTTCTCTTTCCCCATCC rvnscl rnfraintruhihbto fcJnpopoyainadAP-1 and phosphorylation c-Jun of Z. inhibition Dong, through activity. and transformation M. cell A. prevents Bode, Y., W. Ma, algorithm. al. et J. cancer. D. ovarian Demetrick, in oncogene J., putative Diebold, a is W., EEF1A2 J. factor elongation Gray, C., Collins, and invasion remodeling, actin Akt-dependent stimulates migration. and Akt activates eEF1A2 aoeools . Asimacopoulo N., Kalogeropoulos, pancreatic in gene (MTS1) p16 E. S. the Kern, of and deletions J. adenocarcinoma. C. homozygous Yeo, and H., R. mutations Hruban, somatic L., C. Weinstein, B., A. udigt icfne rti 1ascae ula localization. nuclear R1-associated Biochem. protein finger zinc to bundling inhibitors. kinase checkpoint cycle cell and docking. protein-protein cells. mammalian strategies. intervention therapeutic and tumors 301-305. E12a oeta agto p16 of target potential a as eEF1A2 ytei fcpe RA sn an using mRNAs capped of synthesis 9 -CCTCTTACCTCAGTTACAATTTATA-3 20) onihigfntoso oyetd lnainfco :fo actin from 1: factor elongation polypeptide of functions Moonlighting (2002). a.Src.Ml Biol. Mol. Struct. Nat. 1-21. 66, Proteins Oncogene a.Genet. Nat. u.J Biochem. J. Eur. 6 80-87. 52, 3027-3040. 26, rtiaeihbtrccti Clice,Darmstadt, (Calbiochem, cocktail inhibitor proteinase 20) oe hp opeetrt crn ucinfor function scoring complementarity shape novel A (2003). Proteins 20) ehnsso rnltoa eeuaini human in deregulation translational of Mechanisms (2007). 20) agtn h eldvso yl ncne:CDK cancer: in cycle division cell the Targeting (2005). 8 3 6 VO 27-32. , 699-707. 12, ...o tlattreidpnetexperiments. independent three least at of s.e.m. 20) DC:a nta-tg protein-docking initial-stage an ZDOCK: (2003). 20) euaino etd-hi lnainin elongation peptide-chain of Regulation (2002). 51 4 otiigo MPS,1 mM 15 PMSF, mM 1 of containing ) 269 p16 397-408. , ,P . its . aio,E and E. Marinos, C., Kittas, J., P. s, 5360-5368. , INK4a 20) h uo upesrp16(INK4a) suppressor tumor The (2005). Xenopus ur pn Pharmacol. Opin. Curr. a ierzdadue o the for used and linearized was ) m nvitro in Oncogene flssbfe e mro All embryo. per buffer lysis of l 9 eeTos a sda a as used was Tools) Gene ; E12(OeFA)was (MO-eEF1A2) eEF1A2 rncito i (Ambion, kit transcription ts n a and t-test 5973-5990. 26, nvitro in INK4a isi Biotechnol. Biosci. 19) Frequent (1994). P 20) Protein (2002). 5 a.Genet. Nat. 366-373. , vleo less of -value transcription. 1751 (2007). 31 b in - , Journal of Cell Science 1752 aiMhai,H n akr N. Bakkar, and H. Gali-Muhtasib, e,S,Wlri,L .adWn,E. Wang, and A. L. Wolfraim, S., Lee, E. Wang, and S. Liu, M., A. Francoeur, S., Lee, J. Y. Surh, and H. M. Lee, A., M. Andrade-Navarro, S., Jeganathan, A., Amiri, A., D. Turbin, G., Kulkarni, A. Berns, and A. Loonstra, J., W. Mooi, C., H. K. Quon, Leffers, P., Krimpenfort, and F. B. Clark, J., Wang, Frydenberg, and S. M., Xu, S. J., Knudsen, Pan, E., Petroulakis, E., Chen, D., Bourbeau, and J. A., Cavallius, Khalyfa, F., B. Clark, R., C. Knudsen, P., Ong, Kristensen, and A., Z. Lund, B. S., Yuan, Kahns, X., Y. Lei, S., Othumpangat, M., C. O’Kernick, P., Joseph, K. Schulten, and A. Dalke, B. W., Garvik, Humphrey, and L. Kadyk, T., Weinert, L., Hartwell, B. M. Kastan, and H. L. Hartwell, ud . nde,S . isn,H,Cak .adTmeu,N. Tommerup, and B. Clark, H., Vissing, M., S. Knudsen, A., Lund, Abbott, L., A. Kovalchuk, J., H. Newbery, A., Zingone, M., D. Shin, F., C. Qi, Z., Li, E. Wang, and A. Duttaroy, A., LeBlanc, S., Lee, E. Wang, and K. D. Ann, S., Lee, n rset o uuedu development. drug carcinomas. future lung for cell prospects and non-small in study A artifact. cells an tumor not Histochem. in and (CKI) inhibitor specific kinase is cyclin-dependent ‘‘nuclear’’ p16(INK4A) the of G. V. Gorgoulis, lnainfco- lh uigrtdevelopment. rat during alpha factor-1 alpha -1 elongation the of member a S1, family. of gene muscle and heart, brain, mammalian M. J. Sci. Lee, and cancer. breast G. in Treat. outcome D. Res. favorable predicts Huntsman, eEF1A2 factor D., elongation T. Wu, mice. in melanoma metastatic to 86. susceptibility confers p16Ink4a of elongation-factor human of variant novel isolation a isoforms: encoding alpha. alpha cDNA 1 factor-1 a elongation of of characterisation expression and the in variation dependent mice. wasted and normal in expression protein E. protein. the of characterization and cDNA the C. W. Merrick, lines. cell and tissues M. T. Graph. Mol. J. cancer. and integrity, genomic 1821-1828. sineto ua lnainfco apagns E1 ast chromosome to maps 20q13.3. EEF1A to : EEF1A2 1alpha and factor 6q14 elongation human of Assignment plasmacytomas. mouse in signaling e10755. AKT and JAK/STAT 3rd activates C., and H. Morse, and M. C. its and alpha neurons. factor-1 in elongation S1, translation of gene, expression sister the pathway. in alteration evolutionary dependent possible gene alpha a factor-1 elongation of mammalian Commun. the of analysis member second family: the S1, for coding 20) hrceiaino lnainfco-A(E1-)adeEF1A-2/S1 and (eEF1A-1) factor-1A elongation of Characterization (2001). 1171 20) xrsinpoieo uaytctasainfcosi ua cancer human in factors translation eukaryotic of profile Expression (2004). u.J Biochem. J. Eur. 87-93. , 203 ora fCl cec 2 (8) 126 Science Cell of Journal 102 79 .Bo.Chem. Biol. J. 1371-1377. , 5-10. , 31-41. , 14 19) h lnainfco - sfr rmrbi:coigof cloning rabbit: from isoform A-2 1 factor elongation The (1998). 20) lcrnmcocp vdneta yolsi localization cytoplasmic that evidence microscopy Electron (2004). 33-38. , o.Carcinog. Mol. 215 20) E12a uaieoncogene. putative a as eEF1A2 (2009). x.Cl Res. Cell Exp. 267 21) e12pooe elgot,ihbt apoptosis inhibits growth, cell promotes Eef1a2 (2010). 549-554. , odSrn ab yp un.Biol. Quant. Symp. Harb. Spring Cold Genomics 24064-24068. , 19) lnn fhmnadmuebancDNAs brain mouse and human of Cloning (1994). 19) elccecnrladcancer. and control cycle Cell (1994). 20) ouaigcl yl:cretapplications current cycle: cell Modulating (2002). 40 171-179. , 19) ifrnilepeso fS and S1 of expression Differential (1993). 359-361. 36, 19) M:vsa oeua dynamics. molecular visual VMD: (1996). 219 ur acrDu Targets Drug Cancer Curr. 589-597. , uli cd Res. Acids Nucleic .Bo.Chem. Biol. J. 19) iseseii xrsinin expression Tissue-specific (1992). .Bo.Chem. Biol. J. 19) emnldifferentiation- Terminal (1995). 20) xrsino protein of Expression (2007). 19) elccecheckpoints, cycle Cell (1994). ice.Bohs Res. Biophys. Biochem. 276 268 1884-1890. 26, 22915-22922. , n.N .Acad. Y. N. Ann. Nature 19) Tissue- (1993). 24453-24459. , ratCancer Breast 259-263. 59, LSONE PLoS Science 20) Loss (2001). 2 309-336. , 413 Biotech. (1996). 83- , 266 5 , , cgine . aa,B,Bnn . rsi . enr,C,Fra . Cristiano, R., Farra, C., Zennaro, M., Grassi, S., Bonin, B., Dapas, B., Scaggiante, Migliaccio, A., Lamberti, A., Sickmann, and P., R. C. Zahedi, C., Cordon-Cardo, Scheuermann, P., C., Sanges, Londei, W., Xu, L., Ma, L., Montanaro, D., Ruggero, ht-ibrsn . ut,D .adVeklJhsn C. Voelkel-Johnson, and T. D. Kurtz, S., Miller, White-Gilbertson, A., Larionov, J., Jackson, R., N. Wray, J., H. Newbery, A., V. Tomlinson, G. A. Hinnebusch, and N. Sonenberg, ors .C,Bro,P . ebr,H . otos .J n bot .M. C. Abbott, and J. D. Porteous, J., H. Newbery, N., P. Barlow, M. C., J. D. Roberts, Soares, and J. C. Sherr, J. C. Sherr, ern,M,Hno,G .adBah D. Beach, and J. G. Hannon, M., Serrano, D. L. Smith, and J. W. Wasserman, D., J. Richter, ugr,D n adli .P. P. Pandolfi, and D. Ruggero, G. Peters, and M. Ruas, M. J. Lee, and G. D. Huntsman, T., Francetic, E., S. Kalloger, E., D. Pinke, B. V. A. Pardee, A. El’skaya, and B. Negrutskii, V., Filonenko, I., Nemazanyy, G., Panasyuk, Z. Weng, K. and Moldave, R. Chen, R., Anderson, K., Wiehe, B., Pierce, J., Mintseris, wno,C. Swanton, als .A,Dhs,S,Hye,J,Ades .J n aisn .R. A. Davidson, and J. B. Andrews, J., Haynes, S., Dahesh, A., J. Marles, xrsino E112gnsi ua rsaecne el:teptnilof potential the cells: cancer prostate al. progression. and human et transformation prostate in for C. hallmark a genes Giansante, as EEF1A2 EEF1A1/2 F., of and Zanconati, expression 1A S., Siracusano, factor L., elongation cells. translation eukaryotic eukaryotic in al. stability of et its J. regulate phosphorylation Reinders, S., the Zappavigna, M., mediate Marra, A., Baljuls, N., lymphomagenesis. in c-Myc with cooperates P. P. Pandolfi, E12i oeta norti hti vrxrse ntotid fbreast of two-thirds in overexpressed is M. that C. oncoprotein Abbott, potential and tumours. a M. is J. eEF1A2 Dixon, R., W. lseso eunevrainadptnildfeecsi phosphorylation. in differences potential surface and distinct two variation reveal ONE eEF1A2 sequence and of eEF1A1 human clusters of models Structural (2009). progression. G1-phase of regulators oto asn pcfcihbto fcci D/CDK4. cyclin of inhibition specific causing control 166-173. Cancer relatives. maturation. oocyte Xenopus during 167. synthesis protein increased cancer. ovarian in eEF1A2 factor elongation Oncol. of significance prognostic The USA Sci. increased Acad. Natl. displays molecules. Proc. signalling eEF1A different Biol. factor with Cell interact Biochem. translation to J. mammalian ability Int. and of phosphorylation isoform tyrosine A2 (2008). prediction. complex protein into potentials 511-520. pair statistical Integrating rti ytei ncl yln n cancer. and cycling cell in synthesis protein targets. biological and mechanisms eukaryotes: rti-rti neato fiiypasacuilrl ncnrligteSho1p- the controlling in role yeast. crucial in pathway a transduction plays signal mediated affinity interaction Protein-protein 4 e6315. , 108 3 179-192. , M Cancer BMC ici.Bohs Acta Biophys. Biochim. 20) asn n4/r:‘pr’ 1-ulmice. p16-null ‘‘pure’’ Ink4a/Arf: Parsing (2001). 18) uaytcpoensynthesis. protein Eukaryotic (1985). 20) elccetree therapies. targeted Cell-cycle (2004). 561-568. , 17) etito on o oto fnra nmlcl proliferation. cell animal normal of control for point restriction A (1974). 20) h rnlto atreF4 rmtstmrfrainand formation tumor promotes eIF-4E factor translation The (2004). 5 19) h 1IKaCK2 uo upesradits and suppressor tumor p16INK4a/CDKN2A The (1998). 113. , 63-71. 40, 71 1286-1290. , 20) osterbsm rnlt cancer? translate ribosome the Does (2003). F115-F177. 1378, 19) D niios oiieadnegative and positive inhibitors: CDK (1999). ee Dev. Genes 20) euaino rnlto ntainin initiation translation of Regulation (2009). elDahDis. Death Cell 19) e euaoymtfi cell-cycle in motif regulatory new A (1993). o.Oncol. Mol. a.Med. Nat. 20) rnlto lnainfactor elongation Translation (2005). o.Cell Mol. Cell nu e.Biochem. Rev. Annu. 1501-1512. 13, actOncol. Lancet 136 Nature 18) h ehns for mechanism The (1982). 10 731-745. , 3 3 813-823. 14, e276. , 484-486. , 402-408. , Cell 21) iscigthe Dissecting (2012). 366 5 20) h oeof role The (2009). e.Biol. Dev. 27-36. , 21) a kinases Raf (2012). 106 704-707. , r .Cancer J. Br. 531-534. , 1109-1149. 54, Proteins a.Rev. Nat. Gynecol. 159- 89, (2008). (2007). (2004). PLoS 106 69 , ,