Centromere-Associated Protein-E Is Essential for the Mammalian Mitotic Checkpoint to Prevent Aneuploidy Due to Single Chromosome

The Journal of Cell Biology

JCB

Introduction Beth A.A. Weaver, Zahid Q.Bonday, Frances R.Putkey, J.P.L. Geert Kops, AlainD.Silk, andDon W. Cleveland Spindles due tosinglechromosome loss mammalian mitoticcheckpoint toprevent aneuploidy Centromere-associated protein-E isessentialforthe control pathway inwhich unattached kinetochores prevent implicated inthemitoticcheckpoint, themajorcellcycle directly bindstoBubR1,akinetochore-associated kinase domain notfoundinthebuddingyeastprotein(Cahill et the vertebrateMad3homologue,BubR1,containsakinase of eachhavebeenidentifiedinhighereukaryotes,although BUB1 point: as essentialforthekinetochore-dependentmitoticcheck- tubules ofthemitoticspindle. polar attachmentsthroughtheirkinetochorestothemicro- anaphase untilallchromosomeshavemadeproductive,bi- the mitoticcheckpoint,whichpreventstransitionto cer cells.Maintenanceofploidyisensuredthroughaction condition knownasaneuploidy,whichisahallmarkofcan- tion ofcellswithaDNAcontentgreaterorlessthan2N, chromosomes duringmitoticdivisionsleadstotheproduc- ble offspring(forreviewseeCohen,2002).Lossorgainof in theproductionofgametesthatareunabletoproducevia- even asinglechromosomeduringmeiosismostoftenresults and equalsegregationofgeneticinformation.Lossorgain Successful cellularpropagationrequiresfaithfulreplication anaphase, resultinginaneuploidy in25%ofdivisions in kinetochores depletedofCENP-Ecannotblock entryinto anaphase onset.Here,weshow thatsingleunattached C La Jolla, CA92093 Ludwig Institute forCancerResearch ofCellularandMolecular andDepartment Medicine, University ofCalifornia,San Diego,  Key words: kinetochore;mitosis;cellcycle;LENP-E;BubR1 Tel.: (858)534-7811.Fax:534-7659. email:[email protected] Research, 3080CMM-East,9500Gilman Drive,LaJolla,CA92093-0670. Address correspondencetoDonW.Cleveland, LudwigInstituteforCancer http://www.jcb.org/cgi/doi/10.1083/jcb.200303167 The Journal ofCellBiology

TheRockefeller University Press, 0021-9525

Article Six geneshavebeenidentifiedin

and

MAD1

stable microtubulecaptureatkinetochores. It also sential mitotickinesinthatisrequiredforefﬁcient, entromere-associated protein-E(CENP-E)isanes-

gene

BUB3

MPS1 ,

MAD2 (Hoytetal.,1991),andthe (WeissandWiney,1996).Homologues , Volume 162, Number 4,August 18,2003 551–563 and MAD3 (LiandMurray,1991), /2003/08/551/13 $8.00 Saccharomyces cerevisiae Mono Polar et al.,2003). is requiredforadvancetoanaphase(forreviewseeCleveland prevents theubiquitinationofsubstrateswhosedestruction the anaphasepromotingcomplex/cyclosome(APC/C),and it inhibitstheCdc20-activatedformofaubiquitinligase, produced bythesekinetochoreshasnotbeenidentified,but 1994, 1995;LiandNicklas,1995).Thesignalorsignals radiation andmicromanipulationexperiments(Riederetal., progression toanaphase,asdemonstratedbyclassiclaserir- chores. Evenasinglekinetochoreissufficienttoprevent chromosome missegregation. tumorigenesis inmice,presumablybyenhancingtherateof (Michel etal.,2001)orBub3(Babu2003)promote ploidy incellculture.HeterozygousmutationsMad2 produces lethalityinmiceandrapidacquisitionofaneu- of Mad2(Doblesetal.,2000)orBub3(Babu2003) advance toanaphaseduringeverymitosis.Geneinactivation mammals itisanessentialmechanismthatservestocontrol of itsactivationinresponsetospindledamageyeast, al., 2001).Althoughinitiallynameda“checkpoint”because al., 1998;ChanetTaylorKaplan hepatocytes invivo primary mousefibroblasts invitroand95%ofregenerating centromere-associated protein-E;MEF, mouseembryonicfibroblast. recombinase; APC/C,anaphase-promoting complex/cyclosome;CENP-E, Abbreviations usedinthispaper:AdCre, adenovirusexpressingtheCre an essentialamplifier ofabasalmitoticcheckpoint signal. and forstimulatingBubR1kinaseactivity, implicatingitas binding partnerBubR1toeach unattached kinetochore Thus, CENP-Eisrequiredforenhancingrecruitmentofits stimulates thekinaseactivity ofpurified BubR1invitro. activity remainsatbasallevels. CENP-Ebindstoanddirectly BubR1 arerecruitedtokinetochores andBubR1kinase The checkpointsignalisgeneratedbyunattachedkineto- . Without CENP-E, diminishedlevels of 551 The Journal of Cell Biology 552 to afailureofmetaphasealignmentchromosomesin in multiplecontexts,asinhibitionofCENP-Efunctionleads required forcompletechromosomealignmentatmetaphase tubule capturebykinetochores(Putkeyetal.,2002).Thisis prometaphase throughanaphaseA,CENP-Estabilizesmicro- phase (Brownetal.,1994).Localizedatkinetochoresfrom in G2,isusedthroughoutmitosis,anddegradedtelo- Results normally cyclingmammaliancells. used totesttheroleofCENP-Einmitoticcheckpoint in primarycellsvitroandregeneratinglivervivoisnow sister chromatidsseparateduringthisarrest. somes, althoughthesereportsconflictaboutwhetherornot al., 2000)arrestinmitosiswithmanymisalignedchromo- pleted ofCENP-Ewithantisenseoligonucleotides(Yaoet bodies (Schaaretal.,1997;McEwen2001)orde- 2000). Incontrast,HeLacellsinjectedwithCENP-Eanti- to arrestafterimmunodepletionofCENP-E(Abrieuetal., longer recruitMad1andMad2totheirkinetochoresfail ter nocodazole-inducedmicrotubuledepolymerization)no ( exon intheCENP-E gene(identifiedhere to beexon4) inserted intointronic sequencesoneither side ofanearly nition sequences(loxPsites) fortheCrerecombinasewere conditional allelesofCENP-E (Putkeyetal.,2002).Recog- is essentialusinggenetargeting inmicetoproducenullor (Ashar etal.,2000).Recently, we havereportedthatCENP-E and aterminalCAAXbox that maydirectfarnesylation phorylation sites,asecondmicrotubule-bindingdomain, nm longcoiledcoil),threecdc2–cyclinBconsensusphos- like motordomain,acentraldomain(predictedtobe220 287 kD(2474aminoacids;Fig.1A),includingakinesin- and methods).The7,425-basecDNAencodesaproteinof CENP-E cDNAwasidentifiedandsequenced(seeMaterials chromosome 4(Testaetal.,1994).Afull-lengthmurine ously identifiedlocationofhumanCENP-Eon chromosome 3(Fig.1A),whichissyntenictotheprevi- The geneencodingmurineCENP-Eislocatedonmouse primary mousefibroblasts the singlefunctionalmurineCENP-Egenein Efficient Crerecombinase–mediateddisruptionof pus pus signaling havecometosharplydivergentconclusions. CENP-E affectskinetochore-dependentmitoticcheckpoint derlying kinetochore-boundcheckpointcomponents. tioned toserveasasensorlinkingmicrotubulecaptureun- of eachkinetochore(Yaoetal.,1997),itisappropriatelyposi- 2000) andextendsatleast50nmawayfromtheoutersurface the checkpointkinaseBubR1(Chanetal.,1998;Yao al., 2002).BecauseCENP-Ealsocoimmunoprecipitateswith al., 1997;Yaoet2000;McEwen2001;Putkey formed humanandmousecellsgrowninculture(Schaaret gaster To extendtheseearlierefforts,selectivegeneinactivation Centromere-associated protein-E(CENP-E)isalarge Efforts todeterminewhetherabsenceorinhibitionof 300 kD),essential,kinesin-likeproteinthataccumulates

extractsinwhichallkinetochoresareunattached The JournalofCellBiology laevis embryos(Yuceletal.,2000),andprimaryortrans- extracts(Woodetal.,1997),

Volume 162,Number4,2003

Drosophila

melano-

Xeno-

(af-

was anaccumulationofmitoticCENP-E a transientpreanaphasedelaywasindeedobserved,asthere mitosis afterCENP-Egenedisruption.Consistentwiththis, tained mitoticcheckpointarrest,forcingaccumulationin attached kinetochoreswouldbeexpectedtogenerateasus- left mitoticcheckpointsignalgenerationintact,theseun- spindle microtubules(Putkeyetal.,2002).IflossofCENP-E the misalignedchromosomesatpolesareunattachedto pseudo metaphasehaveshownthatbothkinetochoreson cells hereafterdescribedasCENP-E quent experimentswereundertakenatthistimepointin gene andproteinby48hafteradditionofAdCre,subse- (Fig. 1F).InlightofthequantitativelossCENP-E (CENP-E 2.4 relative tocontrolcellstreated withAdCre(5.0 type (CENP-E genotyped byPCRtoidentifyMEFsthatwereofthewild CENP-E metaphase (24%asopposedto0%CENP-E modest increaseinthemitotic indexofCENP-E chromosomes, inspectionof live cellsrevealedonlyavery and telophase(Fig.2C).However, despitechronicpolar relative reductioninfractions ofmitoticcellsinanaphase allele reached used todeterminethatexcisionoftheconditionalCENP-E (AdCre; AntonandGraham,1995).Real-timePCRwas cation-defective adenovirusexpressingtheCrerecombinase from d14embryoswereobtainedbymatingCENP-E key etal.,2002;Fig.1B). cates alloftheknownfunctionaldomainsCENP-E(Put- duces aprematurestopcodonataminoacid82andtrun- created byCre-mediatedexcisionofexon4,whichintro- fine as“pseudometaphase.”MostoftheCENP-E pole (Fig.1E,arrow;Fig.2A,arrows),aconditionwede- aligned hadatleastonechromosomejuxtaposedtoaspindle mice withCENP-E create theconditionalallele(loxP).Thenull( of mitoticCENP-E Yucel etal.,2000;McEwen2001),ahighpercentage (Schaar etal.,1997;WoodYao2000; In agreementwithpreviousexperimentsinothersystems checkpoint despiteunattachedkinetochores CENP-E–deleted fibroblastsdonotsustainamitotic munofluorescence in gle-cell level,CENP-Ewascompletelyundetectablebyim- 16-fold comparedwiththewildtype(Fig.1D).Atsin- measured byquantitativeimmunoblotting,werediminished addition ofAdCre(Fig.1C).CENP-Eproteinlevels,as with AdCre(5.0 fewer (Fig.2B). mosomes; 55%hadonlyoneortwoand90%five pseudo metaphasecontainedonlyoneorafewpolarchro- somes. Intwoindependentexperiments,69( addition ofAdCre(Fig.1E),evenafter10 CENP-E–null cells(CENP-E

Primary mouseembryonicfibroblasts(MEFs)derived Previous EMperformedinCENP-E

0.3%;Fig.2D) ortoCENP-E loxP/ / cellswiththemajorityoftheirchromosomes ). TheCENP-E 90% (87 / ) orwereCENP-E–conditional/null / 0.3versus2.8 /loxP fibroblastshavemisalignedchromo- 85% ofCENP-E mice.Individualembryoswere 6%, loxP/ / ) byinfectionwitharepli- n cellswereconvertedto

/ . loxP/ 4) within48hafter loxP/ 0.2%;Fig.2 E). /

/ MEFs untreated

fibroblastsin cells48hafter cells inpseudo overexposure / 4) percentof

cells) anda 0.3versus / /

cells in MEFs ) was / The Journal of Cell Biology that appearabnormallycloseto thespindlepoles(whitearrow).Tubulin,red;DNAstainedwith DAPI,blue.Bar,2.5 stop codonataa82(outof2,474).(C)Real-timePCRanalysis asingleexperimentindicatesthatexcisionoftheCENP-Egen (exon P,showninorange),duetoloxPsites(denotedbytriangles) incorporatedintotheadjacentintrons,whichintroducesa direct farnesylation.(B)ConversionoftheconditionalCENP-Eallele tothenullallelebyCrerecombinase–mediatedexcisiono region andassociatedexons;(blue)thecarboxy-terminalglobular domainandassociatedexons;CAAX,theterminalCAAXmotifth ATP-binding consensussiteofthemotordomainandisselectively deleted(seeB);(green)thediscontinuous cdc2–cyclin Bconsensusphosphorylationsites.(Red)Thekinesin-like motorregionandassociatedexons;(orange)exon4encode that producesa2,474-aaprotein.Apparentfunctionaldomains are labeled.KT,kinetochore;MT,microtubule;purplecirclesden of themurineCENP-Egene,mRNA,andprotein.The46exons theCENP-Egenespan61.5kbandencodea7,425-nucleotidemRNA Figure 1. times longerthantheCENP-Eimage intheCENP-E CENP-E (E) Immunofluorescencedetection ofCENP-E(green)inmitotic (lane 8)ascomparedwithcontrol cells(lane3).Lane1isa16-folddilutionoflane3.Coomassie stainisshownasaloading fluorescence detectionofCENP-E (red)inmitoticCENP-E 90% by48hafteradditionofAdCre. (D)ImmunoblotshowingthatCENP-Eproteinlevelsarediminished / cells.DAPI,blue. Murine CENP-Eisefficientlyremovedafterrecombinase-mediated excisionofthesinglemurineCENP-Egene. / cells.CENP-Eisstillundetectable atkinetochores(markedbyBubR1,green)in / orCENP-E /

CENP-E isessentialtopreventaneuploidyinmammals| orCENP-E / cells.TheCENP-Eimageinthe

cells.CENP-E / MEFsacquiremisalignedchromosomes 16-fold inCENP-E -helical coiled-coilstalk / cellswasexposed10 Weaveretal. m. (F)Immuno- (A)Schematic premature e reaches f exon4 control. / cells at may s the ote 553 The Journal of Cell Biology 554 CENP-E cells aredepictedbybluebarsand Cre confirmedthatCENP-E (A) ImmunofluorescenceofCENP-E presence ofunattachedkinetochores. a robustcellcyclearrestdespitethe allele ofCENP-E(CENP-E MEFs withoneconditionalandnull (E) Quantitationofthemitoticindex of CENP-E.Arrowsdenotemitoticcells. exhibit markedmitoticarrestduetoloss visualized byHoechst33258donot (D) LivecellswhoseDNAhasbeen or morechromosomesareatthepoles. aligned atthemetaphaseplate,butone which themajorityofchromosomesare Pseudo metaphasecellsarethosein scored withcertainty. Thosethathavetheir kinetochoresat whose armsare directedawayfromthespindle couldbe move tothepolesduringanaphase, onlypolarchromosomes Fig. 3A,inset).However,because thechromosomemasses sister chromatidsatoneorboth poles(Fig.3,A-C,arrows; phase revealedthat13%(12 of 90)suchcellshadpaired cells (leftcolumn)andCENP-E chromosomes. Examination of CENP-E whether cellsenteredanaphasedespitethepresenceofpolar captured andalignedbeforethecellsenteredanaphase,or question ofwhetherthesechromosomeswereeventually MEFs andtheabsenceofasustainedmitoticarrestraised The highfrequencyofpolarchromosomesinCENP-E from thelossorgainofone(orafew)chromosome(s) CENP-E isessentialtopreventaneuploidyresulting FACS Figure 2. point. (F)FACS 1,000 MEFswerecountedateachtime with AdCrefor0,40,and48h.Atleast does notinducerobustmitoticarrest. AdCre, indicatingthatlossofCENP-E iodide atvarioustimesafteradditionof (right column)stainedwithpropidium have occurredifasustainedcheckpointsignalwasgenerated. tent neveraccumulatedtohighlevels(Fig.2F),aswould in variousstagesofmitosis.CENP-E Graph showingthepercentageofcells somes perpseudometaphasecell.(C) showing thenumberofpolarchromo- DAPI, blue;tubulin,red.(B)Histogram Arrows denotepolarchromosomes. cells (48hafteradditionofAdCre). The JournalofCellBiology ® profilestakenatvarioustimesafteradditionofAd- / cellsaredepictedbyredbars. CENP-E ® profilesofCENP-E / MEFsdonotmount loxP/

| ) treated

loxP/ Volume 162,Number4,2003 / cells cellswitha4NDNAcon- / / /

/ MEFsinana- / E Fig. 5 the polebuttheirarmsdirectedintospindle(see timate. EachCENP-E quency of13%isprobablyanapproximatetwofoldunderes- mass adjacenttothepole.Forthisreason,observedfre- the polarchromosomes inCENP-E (Fig. 3C)anaphase B,appearedtoproceed normally. Thus, sequent mitoticsteps,including early(Fig.3B)andlate tion ofthecontinuedpresence ofpolarchromatidpairs,sub- was insufficienttopreventanaphase onset.Withtheexcep- mitotic checkpointsignal.However, anyinhibitorproduced gested thateachkinetochore wasattemptingtogeneratea seen inanaphasewerepairedsisterchromatids,andsug- a double-dotpatternconfirmedthatthepolarchromosomes Mad1 andMad2checkpointproteins(unpublisheddata)in cells orin63anaphasefigures. observed inCENP-E somes hadonlyoneortwo.Nopolarchromosomeswere The continuedlocalizationofBub1(Fig.3,AandB)or , atrightinpanel3)areobscuredbythechromosome / / cells,eitherin anaphasecellwithpolarchromo-

cellsdonot cause

100 metaphase The Journal of Cell Biology in CENP-E proach, weexamined regeneratinghepatocytes inCENP-E functional CENP-EgeneinCENPE (yellow arrows)invivoafterAdCre-mediateddeletionofthesingle ceeded through(E)anaphaseinthepresenceofpolarchromosomes (D andE)Regeneratinghepatocytesthathaveentered(D)pro- double dotpatternofBub1stainingonthepairedsisterchromatids. ment ofthepolarchromosomeatleftspindlepoleshowing DAPI, blue;tubulin,red.Bars,2.5 erly biorientedandalignedwillbemissegregated.Bub1,green; of one(BandC)ortwo(A)polarchromosomesthatwereneverprop- that haveenteredandproceededthroughanaphaseinthepresence (blue bars)andCENP-E livers fromfour differentanimalsafterdamage tothoselivers leted theonefunctionalCENP-E genein combinase (usingtailveininjection ofAdCre)successfullyde- Recently, wehavereportedthat administrationoftheCrere- an invivomitoticcheckpoint intheabsenceofCENP-E One orafewunattachedkinetochoresdonotsustain high frequency. sustained mitoticarrestandareultimatelymissegregated at vivo. presence ofoneorafewpolarchromosomesbothinvitroand Figure 3. numbers werenormalizedtoreflectthe70%excisionrate. the numberofpolarchromosomesperanaphasefigureinCENP-E (A–C)ImmunofluorescenceimagesofprimaryCENP-E Absence ofCENP-Ecausescellstoenteranaphaseinthe loxP/ mice(Putkeyetal.,2002). Using thisap- / (redbars)hepatocytesinvivo.CENP-E lox P/ m. TheinsetinAisanenlarge- mice.(F)Histogramsshowing 70% oflivercells / MEFs / /

CENP-E isessentialtopreventaneuploidyinmammals| of CENP-Eareinsufficienttoprevententryintoanaphase. vivo example,oneorafewunattachedkinetochoresdepleted tubule captureintheabsenceofCENP-E.Thus,this that werereleasedduringanaphaseduetotheunstablemicro- and appearedtorepresentpreviouslyattachedchromosomes somes, butmostofthoseweredistributedalongspindlefibers D–F). Someofthesehepatocytesalsohadlaggingchromo- two polarchromosomes,and85%hadfiveorfewer(Fig.3, CENP-E. Totestthis,CENP-E point canmaintainanarrestinthesecellstheabsenceof and invivoraisedthequestionofwhethermitoticcheck- ciently recruited tokinetochoresintheabsence ofCENP-E. Thus, atleast three essentialcheckpointproteins areineffi- 3.2-fold, respectively)ofall thesecheckpointcomponents. and C)recruitedtwo-tofourfold lowerlevels(2.1-,3.7-,and E (Fig. 4A).Whenallkinetochoreswereunattached,CENP- cells after16hofdrug-inducedmicrotubuledisassembly ing achroniccheckpointresponseinmajorityofcycling initiated andsustainedcheckpointsignalingefficiently,yield- with Hoechst-stainedDNA.Asexpected,wild-typeMEFs E than inwild-typecellsbecausethe4NDNApeakofCENP- the arrestinabsenceofCENP-Eappearstobelessrobust under conditionswhenallkinetochoresaresendingasignal, pression ofmicrotubuledynamics(Fig.4A).However,even tion ofeithermicrotubule-disruptingdrug(Fig.4A). 4 A).AsimilarmitoticarrestinbothCENP-E cells accumulatedwith4NDNAafter16hoftreatment(Fig. scored eitherbyFACS cemid toinhibitspindleassembly.Themitoticindexwasthen were incubatedwiththemicrotubule-depolymerizingagentcol- That murineCENP-E the CENP-E left). AsintheMEFs,despitetheirpresencemajorityof cells fromwild-typeanimalshadsuchchromosomes(Fig.3F, chromosomes (Fig.3,D–F,right),whereasveryfewanaphase ures inhepatocytes.95%ofanaphasefiguresexhibitedpolar CENP-E ledtopseudometaphaseandaberrantanaphasefig- was inducedbyexposuretocarbontetrachloride.Absenceof E tubule disassemblywithcolcemid. ComparedwithCENP- BubR1, Mad1,andMad2after rapidlyforcingspindlemicro- cence wasusedtodeterminelevelsofkinetochore-bound dent kinetochore-derivedsignal,quantitativeimmunofluores- components, therebyamplifyingabasalCENP-E–indepen- test ifCENP-Eenhancedrecruitmentofknowncheckpoint such awayastogenerateaninhibitorofCdc20-APC/C.To cruit checkpointproteinsandmodifyoneormoreofthemin To generateacheckpointsignal,eachkinetochoremustre- unattached kinetochores Mad1, andMad2toattachednewly CENP-E isrequiredforefficientrecruitmentofBubR1, when allkinetochoresareunattached CENP-E somes percell.55%ofCENP-E CENP-E / / / cellsissomewhatsmalleratboth8and16hafteraddi- cellsalsoshowedasustainedarrest,with60%ofcycling cells,kinetochoresinmitotic CENP-E / / cellssustainthemitoticcheckpoint cellswasalsoobservedaftertaxol-inducedsup- / hepatocytes,therewerefewpolarchromo- ® orbydirectvisualizationoflivecells / cellslosechromosomesinvitro / / hepatocyteshadoneor andCENP-E / Weaveretal. cells(Fig.4,B / / MEFs and 555

The Journal of Cell Biology 556 Mad2. MetaphasekinetochoresinCENP-E phase kinetochoreswerediminishedinBubR1,Mad1,and a210 01)04 01)N AN AN 2.62(0.40) NA NA 1.88(0.31) 2.00 (0.14) NA 0.52(0.08) NA NA NA 0.03(0.01) NA 0.33(0.05) 0.03(0.01) NA NA NA 0.17(0.10) NA 0.36(0.04) 0.37(0.08) 0.13(0.02) 0.48(0.14) 0.41(0.04) 0.53(0.13) 0.35 (0.05) 1.00(0.10) 0.95(0.09) 1.00(0.18) 0.52(0.06) 1.00(0.16) 1.00(0.13) Mad2 Mad1 Bub1 BubR1 Error barsrepresentstandarderrors.**,P of thenormalizedintegratedintensitieskinetochoresignalsinB. that CENP-EisrequiredformaximalkinetochoretargetingofBubR1,Mad1,andMad2(green).DNAshowninblue.Bar,2.5 Relative toCENP-E–containingcells,CENP-E were alsodeterminedintheabsenceofspindleinhibitors. their kinetochores. fluorescence ofCENP-E microtubule depolymerizationwithcolcemidorstabilizationtaxol.75%oftheseprimarycellsarecycling.(B) errors areinparentheses. Values representnormalized, integratedintensitiesofkinetochore-associated BubR1,Bub1, Mad1,andMad2,asdetailedinMater Table I. E decrease inanaphaseandtelophasefigurescyclingCENP- The increaseinpseudometaphasefigurescoupledwiththe kinetochores intheabsenceofCENP-E Specific reductioninBubR1recruitmenttopolar and CENP-E Figure 4. largely undetectableduringmetaphaseinbothCENP-E diminished inBubR1andMad1,whereasMad2levelswere sence ofCENP-E(Fig.5E ished onprometaphaseormetaphasekinetochoresintheab- (Jablonski etal.,1998;unpublisheddata),wasnotdimin- Bub1, whichisrecruitedtokinetochoresbeforeCENP-E CENP-E genotype Kinetochore levelsofBub1,BubR1,Mad1,andMad2 / The JournalofCellBiology cells(Fig.2C)suggestedthatthepolarchromosomes Diminished kinetochorerecruitmentofBubR1intheabsence CENP-E Microtubule depolymerizationcausesCENP-E / cells(Fig.5,A–D (A)FACS / cells(leftcolumn)andCENP-E / rmtpaeMtpaePed eaaindPseudometapolar Pseudometa aligned Metaphase Prometaphase

| ®

profileofpropidiumiodide–stainedCENP-E Volume 162,Number4,2003 ; TableI). / ; TableI).However, 0.001. / cellswerealso / prometa- / / / cells(rightcolumn)aftera30-mintreatmentwith200ng/mlcolcemid,showing cellstoarrestinmitosiswithreducedamountsofBubR1,Mad1,andMad2on / tense thanonalignedchromosomesinCENP-E on thesekinetochoreswere,average, of detection(Fig.5,B CENP-E BubR1 andMad1signalsonmanyalignedkinetochoresin pletely silencecheckpointsignaling.Consistentwiththis, the spindlewithnormalkineticswouldbeexpectedtocom- During suchadelay,alignedchromosomesthatattachedto in thesecellstransientlydelayedprogressionintoanaphase. contexts duringmitoticdelay(RiederandPalazzo,1992; normal prometaphase,aphenomenonobservedinmany to recruitcheckpointproteinslevelshigherthanduring a be expectednotonlytocontinuecheckpointsignaling,but chromosomes inCENP-E and C chromosomes inCENP-E phase cells,andfour-tosixfoldlessintensethanonaligned 800 kinetochoresfrom / Conversely, theunattachedkinetochoresonpolar / cells(leftcolumn)andCENP-E ; TableI). / pseudometaphasecellsdroppedbelowthelevel / andC 10 cellswerequantitatedforeachbar. / / / pseudometaphasecellswould metaphasecells(Fig.5,B ). BubR1andMad1signals / cells(rightcolumn)after ials andmethods.Standard / m. (C)Quantitation 10-fold lessin- Immuno- / meta- / The Journal of Cell Biology CENP-E–depleted kinetochores. Thus, thereisaspecificdefectinrecruitmentofBubR1to prometaphase kinetochoresinwildtypecells(Fig.6G). of BubR1(B Kinetochore proteinsareshowningreen.DNA,blue;tubulin,red.Bar,2.5 and metaphasecells.KinetochoresignalsonalignedchromosomesinCENP-E were unchangedrelativetoCENP-E In contrast,BubR1levelsonthosesamepolarkinetochores CENP-E et al.,2001) whereas, aswehaveshown here,primary et al.,1997;Yao2000;Harborth etal.,2001;McEwen CENP-E–depleted HeLacells arecheckpointarrested(Schaar number ofmisalignedchromosomes but arrestbecausetheyhave alarge amounts ofBubR1totheir kinetochores, HeLa cellsdepletedofCENP-Ealsorecruitreduced prometaphase kinetochoresinCENP-E these proteinsrisingtwo-tosixfoldovertheirlevelson continued torecruitMad1,Mad2,andBub1,withlevelsof with this,unattachedkinetochoresonpolarchromosomes Thrower etal.,1996;Hoffman2001).Consistent (A andA Figure 5. kinetochores inCENP-E Table I)andtwo-tothreefoldhigherthanonprometaphase chores inCENP-E (Fig. 6,AandF).Bothpolarprometaphasekineto- chores from2to19differentcellswerequantitatedforeachbar.*,P ), BubR1(BandB CENP-E isrequiredforefficientkinetochoretargetingofBubR1,Mad1,andMad2. /

), Mad1(C MEFs (inthe absence ofspindlepoisons) are / ), Mad2(D cellsrecruitedhalftheBubR1of ), Mad1(CandC / cells(Fig.5,C–E ), andBub1(E / prometaphaselevels / cells(Fig.6,B–F; ), Mad2(DandD ) signalsatkinetochoresinCENP-E ; Fig.6G). ), andBub1(EE

0.05;**,P CENP-E isessentialtopreventaneuploidyinmammals| orescence revealedthat,likeCENP-E depleted ofCENP-EusingRNAi.Quantitativeimmunoflu- not. Toclarifytheseparadoxicalfindings,HeLacellswere (Chan etal.,1998) andbycoimmunoprecipitation frommi- BubR1, ashas beenshownbyayeasttwo-hybrid assay Mammalian CENP-Einteracts withthecheckpointkinase CENP-E stimulatesmammalian BubR1kinaseactivity netochores inCENP-E of CENP-Esendareducedcheckpointsignal,similartoki- (Fig. 7,AandB).Thus,kinetochoresinHeLacellsdepleted 7 A)recruitonlyhalfasmuchBubR1totheirkinetochores treated HeLacellswithundetectablelevelsofCENP-E(Fig. cells invitroorvivoisnot. number ofunattachedchromosomes inCENP-E cient forasustainedcheckpoint arrest,whereasthesmall number ofunattachedkinetochoresintheHeLacellsissuffi- checkpoint signalinbothMEFsandHeLacells,butthelarge E–depleted kinetochoressendaweakerBubR1-dependent average of7.75percell(Fig.7C).Thus,unattachedCENP- HeLa cellscontained1–25misalignedchromosomes,withan polar chromosomeswithonebeingthemostcommon, CENP-E–depleted HeLacells.AlthoughtheMEFshad1–10 the MEFs,thereweremanymorepolarchromosomesin m. (B / pseudometaphasecellsarealsoshown.20–1,750kineto- –E 0.001.Errorbarsrepresentstandarderror. ) Quantitationofthenormalizedintegratedintensity / ) inCENP-E (bluebars)andCENP-E (A–E / / MEFs.However,comparedwith ) KinetochorelocalizationofCENP-E (A–E)andCENP-E / (redbars)prometaphase /

MEFs, colcemid- Weaveretal. / (A –E / mouse ) cells. 557 The Journal of Cell Biology 558 CENP-E andGSTalone.Newly madeproteinswere[ baculoviruses expressingCENP-E andGSTHis-BubR1or a highaffinitycomplex,insect cellswereinfectedwith lane 6). CENP-E inprimaryMEFs(Fig.8A,comparelane3with totic stimulationofBubR1kinaseactivityisdependenton with andwithoutCENP-E(Fig.8A,bottom).Thus,mi- comparable levelsofBubR1wereprecipitatedfromcells of CENP-E(Fig.8A,toppanel,lane5vs.6),although nase activityduringmitosiswasnotobservedintheabsence BubR1 (Chanetal.,1999).However,elevatedki- lane 2vs.3),aswaspreviouslydemonstratedforhuman elevated inwild-typecellsenrichedmitosis(Fig.8A,top, This revealedthatmurineBubR1kinaseactivityissharply for kinaseactivity,usinghistoneH1asaninvitrosubstrate. when itwascoexpressed withGSTHis-BubR1 (Fig.8B, with glutathione beads.CENP-Ewasefficiently recovered labeled withmethionineand wererecoveredbyincubation kinetochores from tated fromCENP-E fected bythelossofCENP-E.BubR1wasimmunoprecipi- interaction ledustotestwhetherBubR1kinaseactivityisaf- totic cellextracts(Chanetal.,1998;Yao2000).This of BubR1,Bub1,Mad1,andMad2signalsatkinetochores.PrometaphasekinetochoresinCENP-E point proteins,green,exceptinB,whichMad1isgreenandBubR1red.(FG)Comparisonofthenormalizedintegrat Insets showhighermagnificationimagesofdesignatedkinetochores.Arrowsindicatepolarchromosomes.DNA,blue;tubulin,red; cells (right)havehigherlevelsofBub1(C),Mad1(BandD),Mad2(E),butnotBubR1(AB)thanprometaphasekinetocho Figure 6. cells (bluebarsinG)arecomparedwithkinetochoresofpolarchromosomesCENP-E To verifythatCENP-Eand BubR1dodirectlybindin The JournalofCellBiology Kinetochores onpolarchromosomesexhibitaspecificdefectinrecruitingBubR1. 12 differentcellswerequantifiedforeachbar.*,P /

or CENP-E

Volume 162,Number4,2003 /

cells andwasassayed 35 S]- indeed fromBubR1 andnotduetoacontaminating kinase. that theactivity detectedinpurifiedwild-type BubR1was absence ofCENP-E(Fig.8D, lane1and2),indicating tectable forthisKD-BubR1mutant eitherinthepresenceor parallel (Fig.8C,lane2).Only tracekinaseactivitywasde- referred toasKD-BubR1), was expressedandpurifiedin that waspredictedtodisrupt kinase activity(K795R—tobe 6). BubR1withapointmutation intheATP-bindingdomain BubR1 kinaseactivity(Fig.8D,comparelane5and BubR1-binding partner,hCdc20,didnotaffectGSTHis- the GSTHis-BubR1kinaseinasmuchasadditionofanother lane 5with7).CENP-Ebindingselectivelystimulated addition ofCENP-E(Fig.8D,comparelane3with4; with BubR1byitself,thiswasstimulatedaboutfivefold strate. Althoughabasallevelofkinaseactivitywasassociated sence ofaddedCENP-E,againusinghistoneH1asasub- of GSTHis-BubR1wasthenmeasuredinthepresenceorab- were expressedandpurifiedfrominsectcells.Kinaseactivity C, lane1)andhumanCENP-E(312kD;Fig.83) ity, recombinanthumanGSTHis-BubR1(110kD;Fig.8 bottom). top), butasexpecteddidnotbindtoGSTalone(Fig.8B, 0.05;**,P To testifCENP-EdirectlystimulatesBubR1kinaseactiv- 0.001.Errorbarsrepresentstandarderrors. / pseudometaphasecells(redbars).Atleast53 (A–E)Polarkinetochoresinpseudometaphase / cells(stripedbarsinF)orCENP-E ed intensity check- res (left). /

The Journal of Cell Biology phosphorylation ofkinasecompetent BubR1. 10). Thus,CENP-Edirectlystimulatesintramolecularauto- by theadditionalcoexpressionofCENP-E(Fig.8E,lane GSTHis-BubR1, whosekinaseactivityhadbeenstimulated when coexpressedwithGSTHis-BubR1(Fig.8E,lane8)or BubR1 wasnotmodifiedtoamoreslowlymigratingform ing withCENP-E(Fig.8E,lane9).Furthermore,His-KD- for His-KD-BubR1,thoughitwasstillcapableofinteract- lane 4vs.3).Thisshiftinmigrationwasnotobserved was dependentonthecontinuedpresenceofATP(Fig.8E, grating speciesofBubR1(Fig.8E,lane3and6)that CENP-E resultedintheproductionofamoreslowlymi- However, coexpressionofwild-typeGSTHis-BubR1with lane 5forGSTHis-BubR1;7His-KD-BubR1). as singlebandswhenexpressedalone(Fig.8E,lane2and tivity. BothGSTHis-BubR1andHis-KD-BubR1migrated The purifiedBubR1swerethentestedforautokinaseac- GSH-Sepharose (Fig.8E,left)orNi-NTAright). ATP andthephosphataseinhibitorokadaicacidusing of CENP-E.Theproteinswerepurifiedinthepresence KD-BubR1 wereexpressedbythemselvesorinthepresence CENP-E enhancesthisactivity,GSTHis-BubR1andHis- totic checkpoint signaling.Now,wehave shownthat (Putkey etal., 2002), therebycontributing to silencingmi- and microtubulesstabilize theinteractionbetweenthem CENP-E haspreviouslybeen shown tobindkinetochores Discussion To testifBubR1phosphorylatesitselfandwhether

CENP-E isessentialtopreventaneuploidyinmammals| rate invitro(Babuetal.,2003), butCENP-E primary MEFsdonotgenerate aneuploidyatasignificant cient topreventprogressionanaphase.Second,wild-type 1995), asingleunattachedkinetochorehasbeenfoundsuffi- cromanipulation (Riederetal.,1994,1995;LiandNicklas, direct testshavebeenfeasibleusinglaserirradiationormi- few kinetochores).First,ineachofthetwosystemswhich phase inthepresenceofoneunattachedkinetochore(or a a fewchromosomes)thatwouldarisefromadvancetoana- signaling tosuppressthemitoticlossofonechromosome(or posal thatCENP-Eisessentialforenhancingcheckpoint duce acheckpointsignalthatisabletosustainmitoticarrest. of insufficientstrengthforoneorafewkinetochorestopro- when largenumbersofkinetochoresareunattached,butis mitotic checkpointthatissufficientforlong-termarrest (Fig. 8).ThesimplestviewisthatCENP-Eamplifiesabasal tokinase) activityofBubR1invitroandprimaryMEFs In addition,CENP-Edirectlystimulatesthekinase(andau- partner BubR1tokinetochoresinHeLacellsandMEFs. find thatCENP-Estimulatesrecruitmentofitsbinding chores, andisthusbifunctionalincheckpointsignaling.We mitotic checkpointsignalgenerationatindividualkineto- CENP-E isalsorequiredinvitroandvivoformaximal MEFs missegregateoneortwo chromosomesin in CENP-E rate ofchromosome loss(Babuetal.,2003) tothatseen by lossofonecopytheBub3 geneproducesasimilar chores signaling).Third,weakening ofcheckpointsignaling divisions (andenteranaphase withonlyoneorafewkineto- Several linesofevidenceofferstrongsupportforourpro- / depleted ofCENP-EbyRNAi(greenbars). CENP-E–depleted kinetochores(redbars). kinetochores (bluebars)recruitmoreBubR1than cells (top)andprimaryMEFs(bottom).Control intensity ofBubR1signalsatkinetochoresinHeLa blue. (B)Quantitationofthenormalizedintegrated 30-min treatmentwith200ng/mlcolcemid.DNA, HeLa cellstreatedwithcontrolRNAi(top)aftera recruit lessBubR1(red)totheirkinetochoresthan cells depletedofCENP-E(green)byRNAi(bottom) and havemanymisalignedchromosomes. reduced amountsofBubR1totheirkinetochores Figure 7. cell inCENP-E misaligned chromosomesperpseudometaphase dard error.(C)Histogramshowingthenumberof each bar.**,P kinetochores from MEFs.Fourth, CENP-E–deletedbutnot HeLa cellsdepletedofCENP-Erecruit / MEFs(redbars)andinHeLacells 0.001.Errorbarsrepresentstan- 10 cellswerequantitatedfor Weaveretal. / (A)HeLa primary 25% of 800 559 The Journal of Cell Biology 560 at least two chromosomesin unattached. Thisresultsinan effectiveloss/gainofoneor prevent anaphaseonsetifonly oneorafewkinetochoresare kinetochores isweakenedsuch thatthecheckpointcannot out CENP-E,thecheckpointsignalgeneratedbyindividual at individualkinetochoresisdependentonCENP-E.With- off, theintensityofmitoticcheckpointsignalgenerated ing asasimplemolecularswitchthattogglesbetweenonand not besustainedafterattachmentofmostkinetochores. checkpoint responsethatinitiallydelaysanaphasebutcan- chromosomes duringmostmitoses.Thisisconsistentwith a CENP-E cipitated fromCENP-E stimulated byCENP-E. Figure 8. mutants in mammalian cells (Chan et al., 1999) have im-mutants inmammalian cells(Chanetal., 1999) have 2002; Maoet al., 2003)orexpressionofdominant BubR1 phase onset. the thresholdrequiredtopreventpremature inhibitors, whicharenolongersufficienttomeet chore producesfewermoleculesofCdc20-APC/C with reducedkinaseactivity(nostar).Eachkineto- of Mad1(yellow),Mad2(blue),andBubR1(purple) unattached kinetochoresrecruitreducedamounts attached. IntheabsenceofCENP-E(right), to delayanaphaseonsetuntilithasbecome APC/C inhibitors,whichpermiteachkinetochore kinetochores assemblelargequantitiesofCdc20- stimulated byCENP-E(purplestar).Theunattached (blue), andBubR1(purple)whosekinaseactivityis amounts ofBub1(red),Mad1(yellow),Mad2 (green, left)unattachedkinetochoresrecruitlarge in checkpointsignaling.InthepresenceofCENP-E ATP. (F)ModelofCENP-EasanactivatorBubR1 purified inthepresenceofokadaicacidand2mM (right). Proteinsinalllanesexcept1and4were using GSH-Sepharose(left)orNi-NTAagarose singly orincombinationHi5cellsandpurified His-KD-hBubR1, andhCENP-Ethatwereexpressed strate. (E)CoomassiestainsofGSTHis-hBubR1, hCENP-E (lane2).HistoneH1wasusedasasub- (lane 4andlane7),BubR1kinase-dead BubR1 (lane 3andlane5),KD-hBubR1byitself1), vitro kinaseactivityofpurifiedhBubR1byitself pressed ininsectcellsusingbaculovirus.(D)In (KD-BubR1), CENP-E,andCdc20thatwereex- recombinant humanBubR1,kinase-deadBubR1 loaded inlane1.(C)Coomassiestainofpurified to GSTalone(lane2,bottom).25%ofinputwas hBubR1 boundtoGSHbeads(lane2,top),butnot (B) PurifiedhCENP-EinteractswithpurifiedGST- of themicrotubule-depolymerizingagentcolcemid. enriched inmitosisbya16-htreatmentwith50ng/ml BubR1 wasimmunoprecipitatedfromcells from randomlycyclingcells;lane3and6, lane 2and5,BubR1wasimmunoprecipitated (bottom). Lane1andlane4,beadsalonecontrol; levels ofBubR1wereconfirmedbyimmunoblot using histoneH1asasubstrate(top).Equivalent MEFs (lanes4–6),andwasassayedforkinaseactivity With allofthisinmind,weproposethat,insteadact- Immunodepletion ofBubR1from The JournalofCellBiology hCdc20(lane6),BubR1 95% ofdivisionsinhepatocytes invivo(Fig.3). Mammalian BubR1kinaseactivityis / hepatocytesinvivomissegregateoneortwo (A)BubR1wasimmunopre- / (lanes1–3)andCENP-E 25% ofcelldivisionsinvitro andin

Volume 162,Number4,2003 hCENP-E Xenopus ana-

/ extracts(Chen, yields amaximum of ence ofCENP-E, whosecellcycle–dependent accumulation (Tang etal.,2001).Kinaseactivity correlateswiththepres- but isnotrequiredforBubR1 toinhibitCdc20-APC/C nopus checkpoint inmammaliancells (Chanetal.,1999)and ization andkinaseactivity. Mad1 andMad2throughitsdirectactiononBubR1local- indirectly affectskinetochoretargetingandactivationof (Chen, 2002;Maoetal.,2003),wepredictthatCENP-E be essentialforkinetochorelocalizationofMad1andMad2 this essentialBubR1role.Becausehasbeenshownto itself), ourevidencesupportsaCENP-Edependencyin diminished intheabsenceofCENP-E(otherthan is BubR1 istheprimarycheckpointproteinwhosefunction plicated BubR1asanessentialcheckpointprotein.As BubR1 kinaseactivityisapparently essentialforthe extracts(Maoetal.,2003; see followingparagraph), 5,000 molecules duringmammalian Xe- The Journal of Cell Biology chronically activated checkpointwithout obvious Mad2 ochore component, Hec1,hasbeenreported toyielda al., 2000).Ontheotherhand, diminutionofanotherkinet- despite prominentMad2binding atkinetochores(Chanet and ZW10,whoseinhibition yieldsaninactivecheckpoint This issimilartothesituation withtwootherproteins,Rod CENP-E produceaneuploid progenyathighfrequency. sufficient topreventcellcycle advance,andcellslacking hibitory signalsentbytheseMad2-richkinetochoresisin- netochores inCENP-E–deletedcells,thestrengthofin- we findthatdespitethepresenceofMad2atunattachedki- measure ofongoingcheckpointsignalgeneration.However, and itspresenceatkinetochoreshasoftenbeentakenas a the downstreameffectormoleculeofmitoticcheckpoint, these reasons,Mad2hasfrequentlybeeninterpretedtobe Fang etal.,1998;KallioLuo2000).For shown tobindandinhibitCdc20-APC/C(Lietal.,1997; unattached kinetochores(Howelletal.,2000)andhasbeen the presenceofCENP-E,Mad2rapidlycyclesonandoff chores, butthisisinsufficienttopreventanaphaseentry.In bustly recruitedtooneorafewpolar,unattachedkineto- CENP-E, theknownCdc20-APC/CinhibitorMad2isro- unattached kinetochore. stimulated catalyticroletoamplifysignalgenerationper checkpoint becameessential,BubR1acquiredaCENP-E– ing evolutionofmorecomplexeukaryotesinwhichthe logue ispresentaswell.Thus,itseemsplausiblethatdur- homologue containsakinasedomain,CENP-Ehomo- Xenopus necessary forthecheckpointbecause does notrequirekinetochorebinding. kinase byCENP-Einvitrodemonstratesthatsuchactivity at kinetochores,althoughthedirectstimulationofBubR1 that CENP-EstimulationoftheBubR1kinaseisprimarily tle correspondinglysolubleCENP-E,itismostplausible al., 2001),andmostofwhichissoluble.Therefore,withlit- totic levelhasbeenestimatedtobe50timeshigher(Tanget (Brown etal.,1994).ThiscontrastswithBubR1,whosemi- mitosis, almostallofwhicharekinetochoreassociated visiae ever, inallspeciesforwhichthedataareavailable( not essentialforthecheckpointintheseorganisms.How- main, clearlydemonstratingthatBubR1kinaseactivityis the homologuein BubR1 inbothbuddingandfissionyeasts(Mad3),aswell concerning BubR1kinaseactivity.Thehomologueof offers anattractiveexplanationforinitialconundrum tation ofCENP-E–stimulatedBubR1kinaseactivityalso of thediffusiblecheckpointinhibitor(s).Suchaninterpre- active BubR1atkinetochoresparticipatesinthegeneration tivity (Maoetal.,2003).Therefore,wepredictthatkinase- BubR1 needstobekinaseactiverestorecheckpointac- periments hasshownthatonlyasmallproportionof subsequent seriesofimmunodepletionandreadditionex- present inthoseexperiments(seeFig.3ofChen,2002).A However, kinetochore-boundwild-typeBubR1wasstill after additionofkinase-inactivemutants(Chen,2002). pleted ofBubR1displayedefficientcheckpointsignaling One surprisingfeatureofthisworkisthat,without One reporthasarguedthatBubR1kinaseactivityisun- , Schizosaccharomyces pombe , Arabidopsis Caenorhabditis elegans , mice,andhuman),whentheBubR1 , C. elegans Xenopus , lacksakinasedo- , extractsde- Drosophila S. cere- ,

CENP-E isessentialtopreventaneuploidyinmammals| of aneuploidy. to preventanaphaseonsetandtheconsequentdevelopment BubR1 togenerateasufficientlyrobustcheckpointsignal depleted ofCENP-Edonotrecruitandactivateenough netochore (orinsomecasesafewunattachedkinetochores) arrest. Thus,ineachofthesesystems,oneunattachedki- unattached kinetochoresissufficienttosustaincheckpoint erates adiminishedsignal,itappearsthatthesumofthese (averaging thus yieldingalargernumberofmisalignedchromosomes on CENP-Eforcaptureandalignmentofchromosomes, However, HeLacellsexhibitamuchstrongerdependence in vivo(Fig.3,D–F),andHeLacells7,AB). (Fig. 3throughFig.6,and8A),murinehepatocytes Xenopus the checkpointresponseperkinetochoreisdiminishedin ual kinetochoreinallcontexts.IntheabsenceofCENP-E, tial formaximalmitoticcheckpointsignalingperindivid- checkpoint activation. Mad2 atkinetochoresisnotafaithfulreporterforsuccessful analyses, itnowseemsclearthatthedetectablepresenceof Though Mad2intensitywasnotquantifiedineitherofthese bound tokinetochores(Martin-Lluesmaetal.,2002). mM Pipes,1EGTA,MgSO luted 1:200. Secondary antibodies(JacksonImmunoResearchLaboratories)weredi- et al.,2001)1:250,Bub11:200,andXMad2(Waters1998)1:60. BubR1 antibody(Tayloretal.,2001)1:200,theMad1(Campbell three-dimensional polygonssurroundingeachkinetochore. netochore fluorescence wasquantifiedusingtheintegrated intensityof ures weregeneratedbyprojecting the sumofopticalsections.Ki- ble imagesweretakenatthesame exposure timesonthesameday.Fig- images wereprocessedusingDeltaVision SoftWoRx™software.Compara- TE200; Nikon)base.Opticalsections weretakenat0.2- convolution microscopesystembuilt onaninvertedmicroscope(model Deconvolution imageswerecollected usingaDeltaVisionwide-fieldde- Deconvolution microscopyand quantitation Cells werewashedwith37 Immunofluorescence blotting wasmadequantitativebyrunningserialdilutionsofcontrolextract. et al.,1996)diluted1:200in5%milk1 was testedbyimmunoblottingusingtheHpxanti-CENP-Eantibody(Brown by real-timePCR(Putkeyetal.,2002).ReductioninCENP-Eproteinlevels Diego, LaJolla,CA)atamultiplicityofinfection10.Excisionwastested Cre recombinase(agiftfromKennethChien,UniversityofCalifornia,San al., 2002).Forexcision,cellswereinfectedwithanadenovirusencoding MEFs werepreparedfromd14embryosasdescribedpreviously(Putkeyet MEF preparation,culture,andexcision The full-lengthmurineCENP-EcDNAwasclonedfroman8.5-d Library screening Materials andmethods 1:200, theDM1 bodies. TheHpxantibodytoCENP-E(Brownetal.,1996)wasdiluted FBS, and200mMglycineinPBSwasusedtoblockcellsdiluteanti- (Tousimis ResearchCorporation)for10min.0.1%TritonX-100,2.5% After extraction,cellswerewashedagainandfixedin4%formaldehyde tracted beforefixationwith0.5%TritonX-100inMTSBat37 independent sequences. cDNA libraries.Allregionsofthewereconfirmedbytwoormore ESTs, RT-PCRproductsfromEScellRNA,andPCRassorted fragments asprobes.Additionalsequenceswereobtainedfrommurine mouse cDNAlibraryusingpreviouslyobtained5 Finally, oureffortshereindicatethatCENP-Eisessen- extracts(Abrieuetal.,2000),primarymousecells 7) percell.Thougheachkinetochorestillgen- antibodyto C microtubulestabilizingbuffer(MTSB;100 -tubulin (Bloseetal.,1984)1:1,000,the5F9 4 , and30%glycerol)thenex- TBSplus0.1%Tween20.ECL and3 Weaveretal. m intervalsand murinecDNA C for5min. gt10 561 The Journal of Cell Biology 562 with pS-hCENP-E and pCMV-CD20inaratioof10:1. 48haftertransfec- melkamp etal.,2002) tomakepS-hCENP-E.HeLacells weretransfected T4 polynucleotidekinase,andligated toHindIII/BglII-cutpSUPER(Brum- ACCTCTCTTGAAGGTTCACCCTTCTCTCTGCGGG-3 to it.AfterSDS-PAGE,thegelwassubjected bated withglutathionebeadstopurifyGST-BubR1andanyCENP-Ebound Ni-NTA beadstopurifytotalCENP-EandBubR1.Theotherhalfwasincu- in eachcasewasdividedintotwoaliquots.Onehalfincubatedwith mM NaCl,and0.1%TritonX-100plusproteaseinhibitors.Thesupernatant plate). Thecellswerelysedinbuffercontaining50mMTris-Cl,pH7.0,100 3 AAGGGTGAACCTTCAAGAGAGGTTCACCCTTCTCTCTGCTTTTTGGAAA- histone H1)togetherwith5 expression). Hi5cells( into pFBandhCENP-EpFB-HTb(both6xHisvectorsforbaculovirus setts InstituteofTechnology,Cambridge,MA).GST-BubR1wasrecloned tors forbaculovirusexpressionweregiftsfromDr.PeterSorger(Massachu- Tris-Cl, pH7.0,250mMNaCl,2MgCl virus. After42–48hofinfection,cellswereharvestedandlysedin50mM Triton X-100,2mMMgCl cold PBSonicebeforebeinglysedin50mMTris7.4,100NaCl,1% for 16hwithorwithout50ng/mlcolcemidandwerewashed2 PAGE followedbyCoomassiestaining. ing GSH-SepharoseorNi-NTAagarose.ProteinsweresubjectedtoSDS- fied inPBSthepresenceorabsenceofokadaicacidand2mMATPus- alone, hCENP-Eorcombinationsofviruses.Theproteinswerepuri- singly ortogetherinthepresenceof 5 proach. TheCENP-EsiRNAsusedweredescribedinHarborthetal.(2001). RNAi wasperformedinHeLacellsusingsiRNAsoraplasmid-basedap- RNAi Hi5 cellswereinfectedwithGST-His Autokinase assay Hi5 cellswereinfectedwithHis GST pulldownassay teins ( Phosphorylation reactionscontainedpurifiedhBubR1/KD-hBubR1pro- Phosphorylation reactions Cdc20, hBubR1,andBubR1kinase-dead(K795R)inpFB-NHis Protein expressionandpurification Adherent andnonadherentCENP-E Immunoprecipitation andkinaseassay PBS beforebeingresuspendedin200 Adherent andnonadherentcellswerecollectedwashed3 FACS buffer. Alleluatesweredialyzedagainstlysisbufferwith150mMNaCl. After washing,proteinswereelutedwith500mMimidazoleinthesame statin asproteaseinhibitors.ThesupernatantwasincubatedwithNi-NTA. vitogen). Alternatively,theoligonucleotide 5 RNAs withOligofectAMINE™,asrecommended bythemanufacturer(In- blasts. Thereactionwasthenperformedat30 buffer wasaddedtotheBubR1immunoprecipitatesfrommousefibro- mM sodiumorthovanadate.Cellextractswerespunfor10minat4 ml leupeptin,pepstatin,andchymostatin,10mMsodiumfluoride,20 ( CellQuest™ software(BDBiosciences). on acytometer(FACSort™;BectonDickinson)byDr.FrankFurnariusing bated inthedarkfor resuspended in20 cold 100%ethanolwhilebeingvortexedmildly.Cellswerestoredat4 Tris-Cl, pH7.4,50 munofluorescence section)at4 bind Gbeads(AmershamBiosciences) full speedinamicrofugeandthesupernatantwasincubatedwithGamma- Cl, pH7.4,50 min ina40- lysis buffer,andphosphorylationreactionswereperformedat30 -mercaptoethanol containingPMSFandleupeptin,pepstatin,chymo- g/ml histoneH1)togetherwith5 wasannealedto5 40 ng)ina40- 10 The JournalofCellBiology ® 4 analysis 25 ng)inthepresenceorabsenceofhCENP-E( cellswereseededinto6-wellplates l reactioncontaininghistoneH1kinasebuffer(20mMTris- M coldATP,10mMMgCl l reactioncontaininghistoneH1kinasebuffer(20mM M coldATP,10mMMgCl g/ml propidiumiodideand40 30 minbeforebeingsorted(20,000events/sample) 3 -AGCTTTTCCAAAAAGCAGAGAGAAGGGTGA- 2 , 5mMEDTAwith400 10 Ci of

7 C for2–3h.Pelletswerewashed3 | cells)wereinfectedwiththeappropriate

6 Volume 162,Number4,2003 -hCENP-E andHis [ Ci of 32 / 35 P]ATP persample. andCENP-E 6 S-labeled methionine(50 -hBubR1 alone,His l coldPBSandfixedwith800 the5F9BubR1antibody(seeIm- 2 2 [ , 10mMEGTA,and750 , 0.3%TritonX-100,and5mM 32 P]ATP persample.Thesame C for30min. 35 - GATCCCCGCAGAGAG- 20 hbeforetransfectionof S-labeled fluorography. 2 , 10mMEGTA,and750 M pefablock,100 6 / -GST-BubR1 viruses , phosphorylatedby MEFsweregrown g/ml RNase,incu- 20 ng)orCdc20 6 -KD-hBubR1 10 -HA vec- C for30 incold Ci per with with g/ml C at C, g/ l Chen, R.H.2002.BubR1isessentialfor kinetochorelocalizationofotherspindle Chan, G.K.,S.A.Jablonski,D.A.Starr, M.L.Goldberg,andT.J.Yen.2000.Hu- and replatedontopoly- umn inamagneticstand,elutedbyremovingthecolumnfrommagnet, microbeads (MiltenyiBiotec)for20minonice,recoveredanMScol- for 20minonice,washedwithPBS,andincubatedgoatanti–mouse tion, cellpelletswereincubatedwithmouseanti-CD20(DakoCytomation) Chan, G.K.,S.A.Jablonski,V.Sudakin, J.C.Hittle,andT.J.Yen.1999.Human Chan, G.K.,B.T.Schaar,andT.J.Yen.1998.Characterizationofthekinetochore Campbell, M.S.,G.K.Chan,andT.J.Yen.2001.Mitoticcheckpointproteins Cahill, D.P.,C.Lengauer,J.Yu,G.J.Riggins,J.K.Willson,S.D.Markowitz, Brummelkamp, T.R.,R.Bernards,andAgami.2002.Asystemforstableexpres- cells werefixedforimmunofluorescence. Accepted: 19June2003 Submitted: 28March2003 We thankDr.FrankFurnariforassistancewiththeFACS Brown, K.D.,K.W.Wood,andD.W.Cleveland.1996.Thekinesin-likeprotein Brown, K.D.,R.M.Coulson,T.J.Yen,andD.W.Cleveland.1994.Cyclin-likeac- Blose, S.H.,D.I.Meltzer,andJ.R.Feramisco.1984.10-nmfilamentsareinduced Babu, J.R.,K.B.Jeganathan,D.J.Baker,X.Wu,N.Kang-Decker,andJ.M.van Ashar, H.R.,L.James,K.Gray,D.Carr,S.Black,Armstrong,W.R.Bishop, Anton, M.,andF.L.Graham.1995.Site-specificrecombinationmediatedbyan Abrieu, A.,J.A.Kahana,K.W.Wood,andD.W.Cleveland.2000.CENP-Easan References Institute forCancerResearch. ety. SalarysupportforD.W.ClevelandwasprovidedbytheLudwig tive. G.J.KopswassupportedbyafellowshipfromtheDutchCancerSoci- day wassupportedbyapostdoctoralfellowshipfromtheTobaccoInitia- and theLudwigInstituteforCancerResearch(T32CA67754).Z.Q.Bon- ported byapredoctoraltraininggrantfromtheNationalCancerInstitute Health toD.W.Cleveland(R37GM25913).B.A.A.Weaverhasbeensup- Mullen foruseofandassistancewiththedeconvolutionmicroscope. for usefuldiscussions.WealsothankDr.JamesFeramiscoandSteveMc- Maddox forhelpfulcommentsonthemanuscript;andDr.AnjonAudhya Manchester, UK)forthegiftofBubR1antibody;Dr.Paul Chapel Hill,NC)fortheMad2antibody;Dr.StephenTaylor(Universityof Dr. BonnieHowellandE.D.Salmon(UniversityofNorthCarolina, Yen (FoxChaseCancerCenter,Philadelphia,PA)fortheMad1antibody; This workhasbeensupportbyagrantfromtheNationalInstitutesof 158:487–496. checkpoint proteins anditsphosphorylationrequiresMad1. chores. man Zw10andRODaremitoticcheckpoint proteinsthatbindtokineto- kinetochores andbindsthecyclosome/APC. 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