Aurora B Couples Chromosome Alignment with Anaphase by Targeting Bubr1, Mad2, and Cenp-E to Kinetochores

The Journal of Cell Biology

JCB

T phenotypes areduetoinhibitionof Aurora B, not Aurora A paclitaxel. RNA interferenceexperimentssuggestthatthese Indeed, indicating thatthespindlecheckpoint iscompromised. ZM447439-treated cellsexitmitosiswithnormalkinetics, all fail.Despitethepresenceofmalorientedchromosomes, ever, chromosome alignment,segregation,andcytokinesis enter mitosisnormally, andassemblebipolarspindles.How- Cells treatedwithZM447439progressthroughinterphase, Claire Ditchﬁeld, Trevor Johnson, and Cenp-Etokinetochores with anaphaseby targetingBubR1,Mad2, Aurora Bcoupleschromosome alignment Downloaded from ing syntelicorientations,possiblybymonitoringtensionat yeast, Ipl1ensuresaccuratechromosomesegregationbyresolv- Prigent, 1999;Adamsetal.,2001a;Nigg,2001).Inbudding roles inmitosis(BischoffandPlowman,1999;Giet orientation errorsarepoorlyunderstood. and Hardwick,2002),themechanismsthatdetectresolve chromosomes correctlybi-orient(forreviewseeMusacchio mechanism, thespindlecheckpoint,delaysanaphaseuntilall 1998). Althoughitiswellestablishedthatasurveillance some malorientation(Nicklas,1997;RiederandSalmon, stochastic process,itiserrorproneandcanresultinchromo- opposite spindlepoles.Becausekinetochoreattachmentisa sister kinetochoresattachtomicrotubulesemanatingfrom Accurate chromosomesegregationinmitosisrequiresthat 2 1 Published Online:28April,2003|SuppInfo: Introduction http://www.jcb.org/cgi/doi/10.1083/jcb.200208091 The Journal ofCellBiology, Volume 161, Number 2,April 28,2003267–280  ZM447439 Key words: mitosis;spindlecheckpoint;chemical biology;aneuploidy; 5763. E-mail:[email protected] Manchester M139PT,UK.Tel.: 44-161-275-5100. Fax:44-161-275- University ofManchester,2.205 StopfordBuilding,OxfordRd., Address correspondenceto The onlineversionofthisarticleincludessupplementalmaterial.

Cancer andInfection Research Area, AstraZeneca Pharmaceuticals, Mereside, Cheshire SK104TG, UK School ofBiological Sciences,University ofManchester, Manchester M139PT, UK

TheRockefeller University Press, 0021-9525/2003/04/267/14$8.00 Article

The Ipl1/Aurorafamilyofproteinkinasesplaysmultiple

ZM447439, anovel selective Aurora kinaseinhibitor. roles inmitosisandcytokinesis. Here,wedescribe he Aurora/Ipl1 familyofproteinkinasesplays multiple

ZM447439 prevents mitoticarrestafterexposureto

jcb.rupress.org 2 Andrew Mortlock, 1 Victoria L.Johnson, Stephen S.Taylor,SchoolofBiological Sciences, on May11,2019 http://doi.org/10.1083/jcb.200208091 2 Nicholas Keen, 1 Anthony Tighe, 2 andStephen S. Taylor 1 Rebecca Ellston, chores onlyattachasinglemicrotubule. Therefore,ifthe of tensionatcentromeres.However, buddingyeastkineto- required forspindlecheckpoint activationinresponsetoloss tension despitemicrotubuleattachment, arguingthatIpl1is kinetochores inthesecellslack sisters,theyfailtocomeunder Ipl1-dependent manner(BigginsandMurray,2001).Because their DNA.Insuchmutants,anaphaseispreventedinan chromatid cohesionorentermitosiswithoutreplicating comes fromanalyzingbuddingyeastmutantsthatlacksister suggestingthatIpl1monitorstension atcentromeres evidence Ipl1 isauniversalfeatureoftheAurorakinasefamily.Further whether thekinetochore–SPBresolvingactivityexhibitedby during mitosis(Nicklas,1997),andtherefore,itisunclear 2002). Inhighereukaryotes,syntelicorientationsarerare both kinetochoresattachedtotheoldSPB(Tanakaetal., SPB duplication,buddingyeastcellsthenentermitosiswith pole body(SPB)inG1.Becausecentromeresreplicatebefore in thatcentromeresconnecttotheunduplicatedspindle et al.,1999).However,buddingyeastareatypical (Biggins may regulatekinetochore–microtubuleinteractionsdirectly neto tubules (Tanakaetal.,2002).Ipl1phosphorylatestheki- centromeres anddestabilizinginappropriatelyboundmicro- chromosome alignmentwithanaphaseonset. checkpoint proteinstokinetochores, Aurora Bcouples ment. Together, theseresults suggestthatby targeting point function,butisalsorequiredforchromosome align- we show thatBubR1isnotonlyrequiredforspindlecheck- for phosphorylationofBubR1onentryintomitosis.Finally, centromeric tension. Aurora Bkinaseactivity isalsorequired of BubR1tometaphasekinetochores afterareductionin inhibition of Aurora Bkinaseactivity prevents therebinding nents BubR1,Mad2,andCenp-Eisdiminished.Furthermore, kinetochore localizationofthespindlecheckpoint compo- or someotherkinase.Intheabsenceof Aurora Bfunction, chore componentNdc10invitro,suggestingthatit 2 Carolyn Haworth, 1 2 267 The Journal of Cell Biology 268 Aurora Bproteincomplexes. due toreducedAuroraBkinaseactivityorthedisruptionof and therefore,itisnotclearwhetherthesephenotypesare rora Bcanitselfaffectcelldivision(Tatsukaetal.,1998), are complicatedbythefactthatexpressionofwild-typeAu- tions (Murata-HoriandWang,2002).Theseexperiments ment duetothefailureofkinetochore–microtubuleinterac- dicates thatAuroraBK109Rpreventschromosomealign- al., 1998;Teradaet1998).However,anotherreportin- ity isnotrequiredforchromosomesegregation(Tatsukaet failed toundergocytokinesis,suggestingthatAuroraBactiv- cells expressingAuroraBK109Rcompletedmitosis,but these studieshaveyieldedconflictingresults.Intwocases, ically expressingmutantsinmammaliancells.However, role ofAuroraBkinaseactivityhasbeenaddressedbyectop- and Prigent,1999;Adamsetal.,2001a;Nigg,2001).The tion, andcytokinesis(BischoffPlowman,1999;Giet cluding histoneH3phosphorylation,chromosomesegrega- inner centromereprotein,affectsmultiplemitoticeventsin- ams etal.,2001a;Nigg,2001).InhibitionofAuroraB,an (Bischoff andPlowman,1999;GietPrigent,Ad- quired forbipolarspindleformationinavarietyofsystems Aurora AandClocalizetospindlepoles,isre- conditions wherereplicationandcohesionarenormal. there isaclearneedtoanalyzeAurorakinasefunctionunder chromatid cohesion(Morishitaetal.,2001).Therefore, tion ofthefissionyeastAurorakinase,Ark1,requiressister tions arecomplicatedbythefactthatcentromericlocaliza- that Ipl1monitorstensionatcentromeres,theseinterpreta- the analysisofreplicationandcohesionmutantssuggests binding sites(Tanakaetal.,2002).Furthermore,although simply asecondaryconsequenceofexposingmicrotubule the apparentroleofIpl1incheckpointactivationmaybe primary roleofIpl1istodestabilizeboundmicrotubules, tion ofAurora kinaseactivitydoesnotprevent progression Aurora kinase activity inhumancells.Weshow thatinhibi- ZM447439 asaresearchtool, wedirectlyaddresstheroleof thekinaseactivity ofAuroraAandB.Using inhibits activity. Here,wedescribeZM447439, whichselectively have generatedsmallmolecule inhibitorsofAurorakinase netic instability.Todevelop novelanti-cancerdrugs,we either byprovidingagrowth advantage orbypromotingge- vated Aurorakinaseactivitymaypromotetumorevolution mors innudemice(Bischoffetal.,1998).Therefore,ele- pressing AuroraA,butnotakinasemutant,readilyformtu- al., 2001b;Meraldiet2002).Inaddition,cellsoverex- al., 1998;TatsukaetZhouAdams et tion, centrosomeabnormalities,andaneuploidy(Bischoff et ectopic overexpressioninculturedcellsleadstotransforma- phenotypes arecurrentlylacking. picture isconfusing,andmolecularexplanationsforthese many roleshavebeenattributedtoAuroraB,theemerging microtubule attachment,notjusttension.Thus,although merization wasinhibited,suggestingthatAuroraBmonitors strains, mitoticexitalsooccurredwhenmicrotubulepoly- lio etal.,2002).However,incontrasttoIpl1deficient tent witharoleforAuroraBinthespindlecheckpoint(Kal- anti-Aurora Bantibodiesexitmitosisprematurely,consis- Higher eukaryotesexpresstwoormoreAurorakinases. Aurora AandBareoverexpressedinhumantumors, The JournalofCellBiology

Volume 161,Number2,2003 Xenopus cellsinjectedwith teins, ZM447439inhibitedAuroraAandBwithIC A). Ininvitrokinaseassaysusingpurifiedrecombinantpro- methoxy-7-(3-(1-morpholino)propoxy)quinazoline; Fig.1 division, apanelofhumancelllineswastreatedwith2 serine 10.TodeterminewhetherZM447439inhibitscell division andinhibitphosphorylationofhistoneH3on we predictedthatanAurorainhibitorshouldpreventcell hibited byZM447439.Basedondatafrommodelsystems, the majorityofotherproteinkinasesassayedwerenotin- ues of110and130nM,respectively(Fig.1B).Incontrast, To identifynovelAurorainhibitors, ZM447439, anovelinhibitorofAuroraAandB Aurora A. that thesephenotypesareduetoinhibitionofAuroraB,not Elbashiretal., 2001),wedemonstrate terference (RNAi;* alignment andspindlecheckpointfunction.UsingRNAin- rora kinaseactivityisrequiredforcorrectchromosome tion, orkinetochore–microtubuleinteractions.Rather,Au- through interphase,mitoticentry,bipolarspindleforma- *Abbreviation used inthispaper:RNAi,RNAinterference. cells expressingadominant-negative p53mutantendoredu- 2001). Consistently,inthepresence ofZM447439,U2OS aberrant mitosisand/orcytokinesis (Andreassenetal., p53-dependent post-mitoticcheckpoint thatoccursafteran due toZM447439,butwas rather duetoactivationofthe arrest exhibitedbytheA549andHMEcellswasnotdirectly idly lostviability.Thisraisesthepossibilitythat4N/8N functional p53response,continuedDNAsynthesisandrap- tents (Fig.1C).Incontrast,HeLacells,whichlack a tually allthecellsarrestedwitheither4Nor8NDNAcon- doreduplicated inthepresenceofZM447439,after48hvir- Although asignificantfractionofA549andHMEcellsen- in theabsenceofp53function ZM447439-induced endoreduplicationisenhanced affect chromosomecondensation. the lackofhistoneH3phosphorylationdidnotappearto sistent withpreviousobservations(Adamsetal.,2001c), does inhibitmitoticphosphorylationofhistoneH3.Con- H3 wasnotdetectable,demonstratingthatZM447439 ever, afterbriefexposurestoZM447439,phosphohistone stained positiveforphosphohistoneH3(Fig.1D).How- body (Hsuetal.,2000).Inuntreatedcells,chromosomes cells werestainedwithananti-phosphohistoneH3anti- tone H3onserine10,untreatedandZM447439-treated mine whetherZM447439inhibitsphosphorylationofhis- that ZM447439completelyinhibitscelldivision.Todeter- cells withDNAcontentsgreaterthan4N,demonstrating the linesanalyzedthenendoreduplicated,accumulating majority ofcellsinallthelineshad4NDNAcontents.All ZM447439 forupto96h(Fig.1C).After18h,thevast produce ZM447439(4-(4-( substrate. Oneinhibitoridentifiedwasfurthermodifiedto recombinant humanAuroraAagainstanartificialpeptide were screenedfortheabilitytoinhibitkinaseactivityof Results N -benzoylamino)anilino)-6- 250,000 compounds 50 val- M The Journal of Cell Biology ZM447439. MCF7 cellswereselectedfor this experiment, potentialofcellsafter a72-hexposureto ony-forming for whytheHeLacellslost viability, weanalyzedthecol- progression inthepresence ofZM447439mayaccount ZM447439-induced division failure.Totestifcellcycle strating thatp53doesrestrain cellcycleprogressionafter cient parentalU2OScellshad 8NDNAcontents,demon- contents (Fig.2A).Incontrast, only38%ofthep53-profi- plicated efficientlysuchthatby48h,78%had8NDNA Figure 1. times indicatedinhours.(D)MitoticDLD-1cellsstainedforphosphohistone H3(green)andDNA(red). ZM447439 againstapanelofproteinkinases.(C)DNAcontenthistograms ofHeLa,A549,andHMEcellstreatedwithZM447439for the ZM447439 inhibitsAuroraAandB.

(A)ChemicalstructureofZM447439.(B)TableshowingtheIC proliferating (Fig.2B).Consistently, at1.25and2.5 ZM447439 gaverisetomore coloniesthanthosethatwere gens. Cellsthatweregrowth-arrested duringexposureto as theycanbearrestedinG0bytreatmentwithanti-estro- ZM447439 leads tolossofviability,the p53-deficient Although continued cellcycleprogressionin thepresenceof the growth-arrestedcellswas largelyunaffected(Fig.2C). was reducedtobelow40%,whereas thecloningefficiencyof ZM447439, thecloningefficiency oftheproliferatingcells Aurora Bregulatesthespindlecheckpoint| 50 values( Ditchfieldetal. M) of 269 M The Journal of Cell Biology 270 ZM447439-induced tetraploidization. p53-independent mechanismsmayalsoaffect cellfateafter HeLa cells.Thereasonforthis isunclear,butitsuggeststhat U2OS cellsdidnotappearto loseviabilityasrapidlythe ZM447439. Figure 2. represents themeanandSDfrom threeindependentexperiments. treated witharangeofZM447439 concentrations.Eachvalue (C) BargraphquantitatingthecloningefficiencyofMCF-7cells assayed fortheabilitytoformcoloniesinabsenceofZM447439. MCF-7 cellswereexposedtoZM447439for72h,andthen for thetimesindicatedinhours.(B)Growth-arrestedorproliferating or without(p53 The JournalofCellBiology p53 restrainsendoreduplicationinthepresenceof (A)DNAcontenthistogramsofU20Scellswith(p53 ) afunctionalp53responsetreatedwithZM447439

Volume 161,Number2,2003

) similar response(unpublisheddata). mally, butfailtodivide.A549andHMEcellsexhibiteda ence ofZM447439,HeLacellsenterandexitmitosisnor- similar tothecontrolculture(Fig.3B).Thus,inpres- which themitoticindexincreasedanddecreasedwasvery phase (Fig.3,AandC).Significantly,thekineticswith graded cyclinB1normally,andthenenteredasecondS ZM447439 progressedthroughSphase,failedtodivide,de- els fortheremainderofexperiment.Cellsreleasedinto mitosis, andthenremainedarrestedwithhighcyclinB1lev- mitosis (Fig.3,BandC).Nocodazole-treatedcellsentered at 10handcyclinB1levelsdecreasedasthecellscompleted than 2N(Fig.3A).Consistently,themitoticindexpeaked such thatby18h,themajorityhadDNAcontentsgreater control cellsdividedby12hthenenteredasecondSphase DNA contenthistogramsshowthatthevastmajorityof mine DNAcontent,mitoticindex,andcyclinB1levels. various timesafterG1/S,thecellswereharvestedtodeter- toxin thatpreventsmicrotubulepolymerization(Fig.3).At plemented witheitherZM447439ornocodazole,aspindle release fromaG1/Sblockintofreshmediumormediasup- tween thesetwopossibilities,weanalyzedHeLacellsafter dividing andthenenterasecondSphase.Todistinguishbe- tosis, oralternatively,thecellsenterandexitmitosiswithout ther thecellsre-replicatetheirgenomeswithoutenteringmi- ditional Sphaseswithoutdividingraisestwopossibilities:ei- The observationthatZM447439-treatedcellscanenterad- ZM447439-treated cellsentermitosis,butfailtodivide were observed in 12cases.Eightofthesecould betracedto 19 controlcells, fiberscontainingmultiple microtubules thin sectionsanalyzedbyelectron microscopy(Fig.4D).In deed, kinetochoresandkinetochore fiberswereobservedin kinetochore–microtubule interactions weretakingplace.In- netochores wereorientedtoward thespindle,suggestingthat length ofthespindle(Fig.4A). Intheselattercases,theki- than aligningatthespindleequator,linedupalong mosomes eithersplayedoutthroughoutthecellor,rather mal inthepresenceofZM447439.Inparticular,chro- segregation. Chromosomealignmentalsoappearedabnor- cells enterandexitmitosiswithoutundergoingchromosome accumulate inmitosis,confirmingthatZM447439-treated Cells treatedwithZM447439andnocodazoledidhowever ZM447439 alonedidnotaccumulateinmitosis(Fig.4C). (Fig. 4B).Despitethelackofanaphases,cellstreatedwith dicating thatZM447439inhibitschromosomesegregation metaphase andanaphasespindleswasmarkedlyreduced,in- prophase appearednormal.However,theproportionof bipolar spindleswereobservedandtheproportionofcellsin apparent (Fig.4,AandB).InZM447439-treatedcultures, rosettes, andmetaphaseanaphasespindleswerereadily in ZM447439-treatedcultures.Incontrols,prometaphase chromosome segregation,weanalyzedspindlemorphology ing cytokinesis.TodeterminewhetherZM447439prevents fails, oralternatively,chromosomesegregationprevent- mosome segregationtakesplacenormallybutcytokinesis mitosis butfailtodivideraisestwopossibilities:eitherchro- The observationthatZM447439-treatedcellsenterandexit ZM447439 inhibitschromosomealignment The Journal of Cell Biology effect ofZM447439 oncellsafterreleasefrom anocodazole checkpoint function. Totestthispossibility, weanalyzedthe (Fig. 3B),suggestingthat ZM447439 maycompromise ZM447439-treated cellsexit mitosiswithnormalkinetics checkpoint,andthuscausemitoticarrest. However, dle chromosome alignmentdefects shouldactivatethespin- The observationsdescribed abovepresentaparadox: ZM447439 compromisesspindlecheckpointfunction block tochromosomesegregation. cytokinesis ispreventedasasecondaryconsequenceofthe whether ZM447439directlyinhibitscytokinesisor prevent lossofsisterchromatidcohesion.Itisunclear (unpublished data),suggestingthatZM447439doesnot following mitosis.Diplochromosomeswereneverobserved ZM447439, andthenanalyzedchromosomespreadsinthe we allowedcellstopassthroughmitosisinthepresenceof whether ZM447439preventssisterchromatidseparation, hibit chromosomealignmentandsegregation.Todetermine bipolar spindleformation.However,ZM447439doesin- does notpreventkinetochore–microtubuleinteractionsor and/or microtubulebindingcapacity,ZM447439clearly the possibilitythatZM447439effectskinetochorestructure were tracedtokinetochores.Althoughwecannotruleout ing multiplemicrotubuleswereobservedin15cases,andsix kinetochores. In20ZM447439-treatedcells,fiberscontain- to thecontrols.Tubulinisusedasaloadingcontrol. similarkineticswith similarkineticstothecontrols.(C)ImmunoblotsshowingthatcyclinB1levelsriseandfallinZM447439-treatedcells (B) Graphplottingmitoticindex,asdeterminedbyMPM-2reactivity,againsttimeshowingthatZM447439-treatedcellsenterand exitmitosis histograms showingthatZM447439-treatedcellsfailtodivide,butre-replicatetheirgenomeswithsimilarkineticsthecontrol cells. G1/S. ThedatainCarefromanindependentexperimentwherethemitoticindexpeaked12hafterrelease(A)DNAcontent three independentexperiments.ThedatainAandBarefromthesameexperimentwheremitoticindexpeaked10hafterrelease from harvested atthetimesindicatedinhours,andthenanalyzedbyflowcytometryimmunoblotting.Thedatashownarerepresentative of Figure 3. ZM447439-treated cellsentermitosis,butfailtodivide.

HeLacellswerereleasedfromG1/Sintovariousdrugcombinations, haps ZM447439 onlycompromisesthecheckpoint when der allcircumstances. Toexplainthis,wereasoned thatper- cating thatZM447439doesnot overridethecheckpointun- and nocodazoleaccumulated cells inmitosis(Fig.4C),indi- spindle checkpoint. consistent withthenotionthat ZM447439overridesthe after 1h.Thus,ZM447439-treatedcellsrapidlyexitmitosis, controls (Fig.5B).Consistently,cyclinB1wasundetectable treated cellswerestillinmitosis,comparedwith67%ofthe fell extremelyrapidly:after1h,only4%oftheZM447439- C). Strikingly,themitoticindexofZM447439-treatedcells h butremainedhighinthepresenceof nocodazole(Fig.5 2 B). Consistently,incontrolcells,cyclinB1levelsfellafter of nocodazolethecellsremainedarrestedinmitosis(Fig. 5 15% 2hafterrelease.Incontrast,inthecontinuedpresence index ofthecontrolcellswasinitiallyhigh,butthenfellto treated cellsfailedtodivide(unpublisheddata).Themitotic ZM447439, nocodazole,andZM447439plusnocodazole- mosome segregationanddivided(Fig.5A).Incontrast, release, themajorityofcontrolcellshadcompletedchro- DNA content,mitoticindex,andcyclinB1levels.2hafter Atvarioustimesthecellswereharvested todetermine A–C). were thenreplatedinvariousdrugcombinations(Fig.5, lective detachment,washedtoremovethenocodazole,and block. Nocodazole-arrestedmitoticcellswereisolatedbyse- However, asynchronouscultures treatedwithZM447439 Aurora Bregulatesthespindlecheckpoint| Ditchfieldetal. 271 The Journal of Cell Biology 272 kinetochore fibers. in whichatleast1,000cellswerecounted.(D)Electronmicrographs ofZM447439-treatedHeLacellsshowingkinetochores(arrows) and ZM447439 plusnocodazole(bluetriangles).ValuesinBandCrepresent themeanandSEMderivedfromthreeindependentexperiments or of DLD-1culturesatthetimesindicatedaftertreatmentwitheither ZM447439alone(reddiamonds),nocodazole(yellowsquares), DLD-1 cellstoZM447439for1h,showingthatchromosomealignment andsegregationareinhibited.(C)Graphshowingthemitotic index for 1h.(B)Graphquantitatingthenumberofprophase(P),prometaphase (PM),metaphase(M),andanaphase(A)cellsafterexposure of (ACA, green),andDNA(red).BottompanelsshowexamplesofabnormalprometaphasesfrequentlyobservedaftertreatmentwithZM4 treated cultureremainedlow at 5 D).Incontrast,themitotic indexoftheZM447439- ZM447439, themitoticindex reached the activates thecheckpointbystabilizingmicrotubules.In totic cellsinresponsetopaclitaxel,aspindletoxinthat alyzed theeffectofZM447439onaccumulationmi- microtubules areallowedtopolymerize.Totestthis,wean- 4. Figure paclitaxel-treated culturesoverarangeofZM447439 con- effect, wedetermined themitoticindexofnocodazole- and ZM447439 reachedonly12%. Toconfirmthisdifferential totic indexoftheculture treated withpaclitaxelplus The JournalofCellBiology presence ofpaclitaxel,nocodazole, ornocodazoleplus ZM447439 preventschromosomealignmentandsegregation.

Volume 161,Number2,2003 7%. Significantly,themi- 25% after6h(Fig.

(A)MitoticDLD-1cellsstainedfortubulin,kinetochores/centromeres prolonged mitotic arrest,ZM447439canalso compromise nocodazole alone (Fig.5B).Thissuggeststhat perhapsafter plus ZM447439exitmitosis fasterthancellsreleasedinto 12-h nocodazoleblock,mitotic cellsreleasedintonocodazole to culturestreatedwithnocodazole alone(Fig.4C),aftera and nocodazoleaccumulatemitotic cellsinamannersimilar point whenmicrotubulesare allowed topolymerize. that ZM447439canefficientlyoverridethespindlecheck- on checkpointfunction.Thus,theseobservationsconfirm the pharmacologicaleffectsnocodazoleandpaclitaxelhave centrations. Fig.5EclearlyshowsthatZM447439resolves Although asynchronouscultures treatedwithZM447439 47439 The Journal of Cell Biology that ZM447439compromisesnocodazole-inducedcheckpointactivationafterprolongedmitoticarrest. or nocodazoleplusZM447439(whitebars),andthemitoticindexwasdeterminedbymeasuringMPM-2reactivityafter4h.Bargraph shows (blackbar) no drug(Control,greencircles).(F)MitoticHeLacellsharvestedaftera2-or12-hnocodazoleblockwerereplatedin ZM447439 alone(ZM,reddiamonds);nocodazoleplus(NZ,bluetriangles);paclitaxel(TZ,greensquares); and which atleast1,000cellswerecounted.Drugcombinationsusednocodazolealone(Noc,yellowsquares);paclitaxel(Tax); with respecttospindlecheckpointfunction.ValuesinDandErepresentthemeanSEMderivedfromthreeindependentexperiments in mitotic arrest.(E)Linegraphplottingtheindexafter8h,showingthatZM447439resolveseffectsofnocodazoleand paclitaxel microscopy. (D)Bargraphplottingthemitoticindexafter6h,showingthatZM447439compromisespaclitaxel-butnotnocodazole-induced ZM447439-treated cells.(DandE)DLD-1cellsweretreatedwithdrugcombinationsthenfixedanalyzedbyfluorescence showing thatZM447439acceleratesmitoticexit.(C)ImmunoblotscyclinB1andphosphohistoneH3levelsfallrapidly in content histogramsshowingthatthecontrolcellsdivideafter1h.(B)Graphplottingmitoticindex,asdeterminedbyMPM-2 reactivity, 12-h nocodazoleblock,replatedinvariousdrugscombinations,andthenharvestedatthetimesindicatedhoursanalyzed. (A)DNA mosome alignment andcheckpointfunction, weanalyzed To gaininsight intohowZM447439compromises chro- Mad2, andCenp-E ZM447439 inhibitskinetochore localizationofBubR1, codazole aloneornocodazoleplusZM447439.Although 12-h nocodazoleblock,thenreplatedfor4hineitherno- To testthis,mitoticHeLacellswereharvestedaftera2-or the checkpointinducedbymicrotubuledepolymerization. Figure 5. but failtoarrestwhenmicrotubules arestabilized(Fig.5E). dergo mitoticarrestwhenmicrotubules aredepolymerized, ysis isthatintheshortterm,ZM447439-treated cellsdoun- lymerization. However,whatisclearlyevidentfromthisanal- compromise checkpointarrestinducedbymicrotubuledepo- rested. Thus,afterprolongedmitoticarrest,ZM447439can rested inthepresenceofZM447439andnocodazole,only 20% ofthecellsisolatedaftera12-hblockremainedar- 80% ofthecellsharvestedaftera2-hblockremainedar- ZM447439 compromisesspindlecheckpointfunction.

(A–C)MitoticHeLacellswereisolatedbyselectivedetachmentaftera

Mad2 to S1, andTableSI).ZM447439 reducedkinetochore-bound Fig. presence andabsenceofmicrotubule toxins(Fig.7B, duced kinetochore-associated BubR1to Quantitation ofpixelintensitiesshowsthatZM447439re- http: Fig. S1,availableat Cenp-E, andMad2(Fig.7A ZM447439 didreducekinetochoreboundBubR1, ever, Aurora BandSurvivintocentromeres(Fig.6A).How- spindle poles(unpublisheddata)orthelocalizationof ZM447439 didnotpreventlocalizationofAuroraAto Mad2, andthekinesin-relatedmotorproteinCenp-E. vivin, thespindlecheckpointcomponentsBubR1and its effectonthelocalizationofAuroraA,B,Sur- plus paclitaxel, respectively.However,in the presenceof and 23%incells treatedwithZM447439 and ZM447439 (Fig. S1C).Kinetochore-bound Cenp-Ewasreducedto28 in thepresenceofnocodazole andpaclitaxel,respectively //www.jcb.org/cgi/content/full/jcb.200208091/DC1). Aurora Bregulatesthespindlecheckpoint| 10% inprometaphasecellsand to30and43% 10%, bothinthe Ditchfieldetal. 273 The Journal of Cell Biology 274 greater thanthe meannocodazoleonlyvalue). ZM447439 and nocodazole(0%haveaBubR1/ACA ratio netochores hadnormalBubR1 levelsinthepresenceof cellstreatedwithnocodazoleonly).Incontrast, noki- in ACA ratiogreaterthanthe mean valueforkinetochores ber retainednormalCenp-E levels(30%haveaCenp-E/ Cenp-E atthemajorityofkinetochores, asignificantnum- dazole, althoughZM447439reducedkinetochore-bound histograms (Fig.S2)showsthatinthepresenceofnoco- 59%. Interestingly,inspectionofthefluorescenceintensity nocodazole andZM447439,Cenp-Ewasonlyreducedto (red), andDNA(blue). K106R stainedtodetectAuroraB(green),Myc-taggedproteins expressing eitherMyc-taggedAuroraBor of AuroraBsiRNAduplexes.(C)TransientlytransfectedHeLacells (blue) after(A)exposuretoZM447439for1hor(B)transfection cells stainedtodetectAuroraB(green),Survivin(red),andDNA centromeres. Figure 6. The JournalofCellBiology ZM447439 doesnotpreventlocalizationofAuroraBto ImmunofluorescenceimagesofprometaphaseDLD-1

Volume 161,Number2,2003

totic cellswereharvested whether ZM447439preventsBubR1phosphorylation,mi- (Chan etal.,1999;Taylor2001).Todetermine to 3.86 from thepaclitaxel-treatedandcontrolcells(P mean interkinetochoredistancewasreducedfrom0.89 (Fig. 7CandTableSII).Afterpaclitaxeltreatment,the ter a40-mintreatmentwitheitherZM447439orpaclitaxel and interkinetochoredistanceatmetaphasekinetochoresaf- after lossoftension,wemeasuredBubR1signalintensity hibits thereassociationofBubR1withalignedkinetochores etal.,2001).TodeterminewhetherZM447439in- (Skoufias the lossoftensioninducedbylowlevelsvinblastine 2001), itisrecruitedbacktometaphasekinetochoresafter chromosome alignment(Chanetal.,1999;Taylor 0.20 nation forthese differencesisthatinaddition toitscatalytic tance wasmarkedly reduced(Fig.8E).One possibleexpla- interactions. Accordingly,the meaninterkinetochoredis- 8 D),suggestingtheabsence ofkinetochore–microtubule chromosomes werefrequently adjacenttothespindle(Fig. codazole aswellpaclitaxel (Fig.8B).Furthermore,the RNAi compromisedcheckpoint arrestinresponsetono- those inducedbyZM447439.Inparticular,Aurora B The AuroraBRNAiphenotypesappearmoreseverethan the endogenousprotein Overexpression ofAuroraBK106Rmislocalizes inhibition ofAuroraB,notAorsomeotherkinase. ZM447439-induced phenotypesdescribedabovearedueto C andFig.S3).Thus,theseobservationsindicatethatthe netochore localizationofBubR1,Cenp-E,andMad2(Fig. 8 B). RepressionofAuroraB(butnotA)inhibitedki- accumulation ofmitoticcellsafterspindledamage(Fig. 8 bust spindlecheckpoints,repressionofAuroraBreducedthe A). AlthoughcontrolandAuroraARNAicultureshadro- other kinase,werepressedAuroraAandBbyRNAi(Fig.8 ZM447439 phenotypesmightbeduetoinhibitionofan- these functions,andtoruleoutthepossibilitythat point function.TodeterminewhichAuroraisrequiredfor required forchromosomealignmentandspindlecheck- is The ZM447439datasuggestthatAurorakinaseactivity localization ofBubR1,Cenp-E,andMad2 Repression ofAuroraBinhibitskinetochore BubR1/ACA fluorescenceratioincreasedfrom2.23 creased to1.11 did notinhibitBubR1(Fig.7E). ZM447439. AlthoughZM447439inhibitedAuroraA,it sayed forkinaseactivityinthepresenceandabsenceof hibits BubR1directly,immunoprecipitateswereas- (Fig. 7D).ToruleoutthepossibilitythatZM447439in- tectable, eitherinthepresenceorabsenceofnocodazole ZM447439, thephosphorylatedformofBubR1wasnotde- block intovariousdrugcombinations.Inthepresenceof 0.55 ZM447439 reducedthemeaninterkinetochoredistanceto

BubR1 isphosphorylatedinresponsetospindledamage Although kinetochore-boundBubR1diminishesafter

m to0.58 0.14

2.41(P

m, theBubR1/ACAfluorescenceratiode-

00.88

0.001).Incontrast,althoughexposureto

0.15 m, whichisstatisticallydifferent 9.5 hafterreleasefromaG1/S m (P 0.001),andthemean 0.001). 1.21 The Journal of Cell Biology rora Bwasnot apparentatcentromeres(Fig. 6C).Thus,in pressing moderate tohighlevelsoftheK106R mutant,Au- and Wang[2002]).However, inprometaphasecellsex- rat K109Rmutantdescribed previouslyinMurata-Hori (note thathumanAuroraBK106R mutantisequivalentto the AuroraBK106Rmutant alsolocalizedtocentromeres (unpublished data).Inaddition, atlowlevelsofexpression, prometaphase (Fig.6C)andmidbodiesaftercelldivision ogen tant. AftertransienttransfectionsofHeLacells,theex- tromeres afteroverexpressionoftheAuroraBkinasemu- Therefore, wetestedwhetherAuroraBlocalizedtocen- phenotype thatismoreconsistentwithourRNAidata. teractions (Murata-HoriandWang,2002),resultingin a of AuroraBK109Rabolishedkinetochore–microtubulein- phenotype similartoZM447439.However,overexpression pression ofanAuroraBkinasemutantshouldproduce a RNAi (Fig.6B).Iftheaboveargumentisvalid,ectopicex- ZM447439 treatment(Fig.6A),itdoesnotafterAuroraB tently, althoughSurvivinlocalizestocentromeresafter than simplyinhibitingAuroraBkinaseactivity.Consis- this complexmayhavemoreextensiveconsequencesrather tosis andcytokinesis(Adamsetal.,2001a).Disruptionof form acomplexthatisrequiredformultipleaspectsofmi- tromeres. Significantly,AuroraB,INCENP,andSurvivin role, AuroraBmayalsoplayastructuralroleatcen- Figure 7. value representsthemeanandSEMderivedfromthreeindependentexperiments. (E) BubR1wasaffinitypurifiedformnocodazole-arrestedmitoticHeLacellsandassayedforkinaseactivityinthepresenceZM447439. Each the drugsindicatedwereanalyzedbyimmunoblotting.InpresenceofZM447439,phosphorylatedformBubR1isnotdetectable. The ovalsencompassatleast75%ofthedatapoints.(D)MitoticHeLacellscollectedbyselectivedetachmentafterreleasefrom G1/Sinto position. or cellsexposedtoZM447439(reddiamonds)paclitaxel(bluesquares)for40min.ACAfociwereuseddeterminekinetochore different cells.(C)PlotofBubR1signalintensityversusinterkinetochoredistanceatmetaphasekinetochoresinuntreatedcells (greencircles) signal by (B) BargraphplottingthefluorescenceratioofBubR1toACAsignalundervariousconditions,showingthatZM447439reduces BubR1 mitotic DLD-1cellstreatedfor1hwiththedrugsindicatedandthenstainedtodetectkinetochores/centromeres(ACA),BubR1, and DNA. ous wild-typeAuroraBlocalizedtocentromeresin ZM447439 inhibitskinetochorelocalizationandphosphorylationofBubR1. 10-fold. ValuesrepresentthemeanandSEMofatleast26differentkinetochore/centromerepairsanalyzedinthree ization oftheendogenousproteintocentromeres. overexpression ofanAuroraBkinasemutantpreventslocal- contrast totreatmentwiththeAurorakinaseinhibitor, tached tothe spindle, themeaninterkinetochore distance (Fig. 9A).Although thesechromosomesappear tobeat- length ofthespindlerather thanatthemetaphaseplate peared abnormalwiththechromosomes alignedalongthe prometaphase cellsinBubR1 RNAiculturesoftenap- posure tospindletoxins(unpublished data).Strikingly, RNAi culturesdidnotaccumulate mitoticcellsonex- S4).Furthermore,BubR1 lagging chromosomes(Fig. phases wasreducedandtheanaphasesfrequentlydisplayed part BubR1 compromisedspindlecheckpointfunction.In injection experiments(Chanetal.,1999),repressionof hibited chromosomealignment.Consistentwithantibody used RNAitodeterminewhetherrepressionofBubR1in- at leastinpart,viaitsaffectonBubR1.Totestthis,we ing correctchromosomealignmentmightalsobemediated, we reasonedthattherequirementforAuroraBinpromot- BubR1 bindsCenp-E(Chanetal.,1999;Yao2000), least inpart,bytargetingBubR1tokinetochores.Because rora Bkinaseactivityregulatesthespindlecheckpoint,at Our observationsareconsistentwiththenotionthatAu- BubR1 isrequiredforchromosomealignment icular, inasynchronouscultures,thenumberofmeta- Aurora Bregulatesthespindlecheckpoint| (A)Projectionsofdeconvolvedimagestacksshowing

Ditchfieldetal. 275 The Journal of Cell Biology 276 metaphases roseto BubR1 isindeedrequiredforchromosomealignment. the accumulationofcellsinmetaphase,suggestingthat spindle checkpointactivation,repressionofBubR1reduces metaphase–anaphase transitionisblockeddownstreamof metaphases reachedonly agents andexperimental researchtoolsdepends ontheselec- The usefulness ofproteinkinaseinhibitors astherapeutic kinase activity ZM447439 isanovelspecific inhibitorofAurora mained roughlyconstantat S4). Incontrolcultures,theproportionofprometaphasesre- almost zeroinbothcontrolandBubR1RNAicultures(Fig. timecourse,thenumberofprophasesandanaphasesfellto 3-h some inhibitorMG132topreventanaphaseonset.Overa treated controlandBubR1RNAicultureswiththeproteo- simply duetothecellsprematurelyenteringanaphase,we tent withareductioninpullingforces. was reducedcomparedwithcontrolcells(Fig.9B),consis- of interkinetochoredistanceincontrolandAuroraB–repressedcells. of siRNAstargetingAuroraA(top)andB(bottom).(D)ExamplesspindlemorphologyincontrolRNAicultures. (E)Plot in whichatleast1,000cellswerecounted.(C)Projecteddeconvolvedimagestacksofnocodazole-treatedmitoticDLD-1after transfection experimentsexposed tospindletoxins,andthemitoticindexwasmeasuredovertime.ValuesrepresentmeanSEMfromthreeindependent to represseitherAuroraAorB.(A)ImmunoblotsofHeLacelllysatesshowingrepressionand(B)TransfectedDLD-1 cellswere Figure 8. prometaphases rosefrom However, inBubR1-repressedcultures,theproportionof MG132 preventingthemetaphase–anaphasetransition. Discussion To ruleoutthepossibilitythatthisalignmentdefectwas The JournalofCellBiology Repression ofAuroraBpreventskinetochorelocalizationBubR1. 66% (Fig.9C),consistentwith

32 to 44% (Fig.9B).Thus,whenthe Volume 161,Number2,2003 25% andtheproportionof 49%, andthenumberof in vitrowithIC tivity oftheinhibitor.ZM447439inhibitsAuroraAandB Aurora Aisrequired forbipolarspindleformation in tion ofAurora B, notAuroraAoranyother proteinkinase. dence thatthephenotypesdescribed hereareduetoinhibi- not tested,theRNAidata(Fig. 8)providecompellingevi- possibility thatZM447439 inhibits otherproteinkinases C, inasimilarmanner.Although wecannotruleoutthe ZM447439 probablyinhibits AuroraB,andpossibly rora kinases(BischoffandPlowman,1999)suggeststhat in otherkinases.ThesequencehomologybetweentheAu- ATP bindingpocketandanadjacentcleftthatisnotpresent communication). Thisshowsthattheinhibitoroccupies Richard Pauptit,AstraZenecaPharmaceuticals,personal complexed witharelatedinhibitor(AndrewPanniferand of ZM447439comesfromthecrystalstructureAurora A 1999). Insightintothemechanismunderlyingselectivity progression(Moasseretal.,1999;Wright G2/M ZM447439, selectiveSRCandMEK1inhibitorsdelay to inhibitionoftheseenzymesbecause,incontrast range, thephenotypesdescribedhereareunlikelytobedue cated inmitoticprogression,wereinhibitedthe1 CDK1 andPLK1,wereinhibitedwithIC verse structuraltypes,11,includingthemitotickinases when testedagainstapanelof14otherproteinkinasesdi- M. AlthoughMEK1andSRC,whichhavebeenimpli- HeLaandDLD-1cellsweretransfectedwithsiRNAduplexes 50 valuesinthe100nMrange.Incontrast,

values Dro- 10 M The Journal of Cell Biology bipolarity inhumansomaticcells. suggesting thatperhapsAuroraAisnotrequiredforspindle when AuroraAisrepressedbyRNAi(unpublisheddata), ence ofZM447439.However,bipolarspindlesalsoform trosome maturation,bipolarspindlesdoforminthepres- though wehavenottestedtheeffectofZM447439oncen- rect chromosome alignment.Sucharole for AuroraBis tions, butrather regulatestheseinteractions to promotecor- tivity isnotrequiredforkinetochore–microtubule interac- presence ofZM447439,suggesting thatAuroraBkinaseac- cellular processes.Indeed,kinetochore fibersforminthe fulness ofsmallmoleculeinhibitors indissectingcomplex those inducedbyZM447439, ourdatademonstratetheuse- pression andoverexpressionappearmoreextensivethan processes. Becausethephenotypesderivedfromproteinre- time, therequirementforAuroraBkinaseactivityinthese hibitor, wehavebeenabletodirectlyaddress,forthefirst in humancells.Byusinganovelselectiveproteinkinasein- checkpoint functionandmetaphasechromosomealignment vations showthatAuroraBisrequiredforbothspindle lio etal.,2002;Murata-HoriandWang,2002),ourobser- Consistent withpreviousreports(Adamsetal.,2001c;Kal- alignment andthespindlecheckpoint Aurora Bkinaseactivityregulateschromosome sophila Figure 9. maturation in Values representthemeanandSEMderivedfromthreeindependentexperimentsinwhichatleast100mitoticcellswerecounted. position. (C)Proportionofmitoticcellsexhibitingeitherprometaphaseormetaphasechromosomealignmentaftera1-hexposuretoMG132. kinetochorewith chromosomesalignedalongthelengthofspindle.(B)PlotinterkinetochoredistancesmeasuredusingBub1focitodetermine aligned alongthelengthofspindleratherthanmetaphaseplate.Bargraphshowsthat were thenstainedtodetectBub1(green),tubulin(red),andDNA(blue).(A)Examplesofabnormalprometaphasecellswithchromosomes embryosand Repression ofBubR1preventschromosomealignment. C. elegans Xenopus embryos(Hannaketal.,2001).Al- eggextractsandcentrosome DLD-1cellsweretransfectedwithsiRNAduplexestorepressBubR1and B kinaseactivity. Ifthisisthecase,thenAurora Bkinaseac- of multiplepathways, oneofwhichdoesnot requireAurora tive” interpretationisthatthe checkpointmustbecomposed qualitative innature,theother quantitative.The“qualita- ble explanationsforthisobservation, oneofwhichis taxel-induced mitoticarrest. Below, weconsidertwopossi- tial effectthatZM447439has onnocodazole-andpacli- quired forcheckpointfunction. least, AuroraBkinaseactivitydoesappeartobedirectlyre- netochores isseverelyreduced.Thus,inhumancells at ZM447439, localizationofbothBubR1andMad2toki- tubules. However,inthepresenceofnocodazoleand should localizetokinetochoresthatlackboundmicro- absence ofAuroraBkinaseactivity,BubR1andMad2 2002)? Ifthiswerethecase,wewouldpredictthatin kinetochores, ashasbeenarguedforIpl1(Tanakaetal., tion isasecondaryconsequenceofgeneratingunattached alignment, isitpossiblethatitsroleincheckpointactiva- alignment. BecauseAuroraBalsopromoteschromosome tion (BigginsandMurray,2001)aswellchromosome (Tanaka etal.,2002). microtubule interactionstoensurecorrectbi-orientation the spindle,butratherresolvesinappropriatekinetochore– Ipl1 isnotrequiredfortheattachmentofchromosomesto entirely consistentwiththeroleIpl1playsinbuddingyeast: The moststrikingobservationinthisworkisthedifferen- Ipl1 hasalsobeenimplicatedinspindlecheckpointfunc- Aurora Bregulatesthespindlecheckpoint| 40% ofprometaphasesappearabnormal Ditchfieldetal.

277

The Journal of Cell Biology 278 ZM447439. In addition,cyclingcellsrapidly loseviability to continuecell cycleprogressioninthe presenceof functional p53response,p53-deficient cellsaremorelikely thermore, wehaveshown that relativetocellswitha nase activityincellswitha small moleculeinhibitor.Fur- tions showthatitispossibleto selectivelyinhibitAuroraki- 1998; Zhouetal.,Adams etal.,2001a).Ourobserva- oncogenic potential(Bischoffetal.,1998;Tatsuka The Aurorasrepresentanewfamilyofproteinkinaseswith Aurora inhibitorsaspotentialtherapeuticagents chore-associated BubR1andMad2(by of tension.AlthoughZM447439clearlyreduceskineto- simply targetsBubR1andMad2tokinetochoresirrespective at centromeres. rather activatesthecheckpointinresponsetolossoftension rectly monitorkinetochore–microtubuleinteractions,but yeast (BigginsandMurray,2001),AuroraBdoesnotdi- the spindle.Thiswouldindicatethat,likeIpl1inbudding chores cancapturemicrotubules,butnotcorrectlyalignon destruction, butisessentialunderconditionswherekineto- loss ofkinetochore–microtubuleinteractionsand/orspindle tivity isnotessentialforcheckpointactivationinresponseto molecular level. monitor chromosomealignmentareinterweavedatthe function indicatesthatthemechanismswhichregulateand that BubR1playsadualroleincongressionandcheckpoint and Koch,1969).Furthermore,ourobservationshowing that tensionstabilizesmicrotubuleattachment(Nicklas checkpoint function.Indeed,itwasshownmanyyearsago tension andattachmentarenotseparableeventsintermsof 2001; Shannonetal.,2002).However,itispossiblethat to alignedkinetochoresafterlossoftension(Skoufiasetal., 1998), andinparticularBubR1,butnotMad2,isrecruited are clearlysensitivetochangesintension(Watersetal., ment (Riederetal.,1995).Yetmammaliankinetochores malian somaticcellsonlymonitorsmicrotubuleattach- compelling evidencetoarguethatthecheckpointinmam- to thelackoftension(BigginsandMurray,2001),thereis suggests thatIpl1doesactivatethecheckpointinresponse Hardwick, 2002).Althoughevidencefrombuddingyeast tachment, orbothremainsunsolved(Musacchioand the spindlecheckpointmonitorstension,microtubuleat- tinguish betweenthesetwopossibilities.Indeed,whether mitotic arrestinthepresenceofnocodazole. sufficient toactivatetheresidualBubR1andthusmaintain Yao etal.,2000),perhapsthiselevatedlevelofCenp-Eis to activatethecheckpointviaBubR1(Chanetal.,1999; with ZM447439pluspaclitaxel.BecauseCenp-Eisthought ZM447439 andnocodazolecomparedwithcellstreated Cenp-E isalmosttwofoldhigherincellstreatedwith ZM447439 andpaclitaxel.Interestingly,kinetochore-bound may explainwhycellscannotarrestinthepresenceof cient totheninactivatetheremainingboundprotein,this interactions.Ifmicrotubuleoccupancyissuffi- microtubule to sustainmitoticarrestintheabsenceofkinetochore– spectively), perhapstheresidualboundproteinissufficient An alternative“quantitative”explanationisthatAuroraB At present,itisdifficulttoimaginehowonecoulddis- The JournalofCellBiology

Volume 161,Number2,2003 90 and 70%, re- Materials andmethods assayed forincorporationof the productswerecapturedonP30nitrocellulosefilters(Whatman)and min. Reactionswerestoppedbyadditionof20%phosphoricacid,and Ci/mmol; AmershamBiosciences),andwasthenincubatedatRTfor60 substrate (Biotinyl-Ahx-tetra(LRRWSLG);Bachem),10 mM KCl,2.5NaF,0.6DTT,6.25MnCl added toareactioncocktailcontaining25mMTris-HCl,pH7.5,12.5 Flow (AmershamBiosciences).1ngpurifiedrecombinantenzymewas was purifiedbyionexchangechromatographyusingCMSepharoseFast purified byaffinitychromatographyusingNi-NTAagarose,andAuroraB GIBCO BRL)accordingtothemanufacturer’sinstructions.AuroraAwas tagged fusionproteinsusingabaculovirusexpressionsystem(FastBac™; Recombinant AuroraAandBwereexpressedasNH In vitrokinaseassays develop novelanti-canceragents. ing tumorcells,andthereforeopenupnewopportunitiesto rora kinaseinhibitorsmaybeselectivelytoxictoproliferat- tain viability.Together,theseobservationssuggestthatAu- in thepresenceofZM447439,whereasnondividingcellsre- MnCl) andwere then incubatedatRTfor1hinkinase buffersupple- (10 mMTris,pH7.5, 5mMKCl,1NaF,0.24 DTT,and2.5mM lished data).ForIPkinaseassays,beads wereequilibratedinkinasebuffer lines stablyexpressingMyc-tagged hMad2orhSurvivinORFs(unpublished data).ForlocalizationofMad2 andSurvivin,weusedDLD-1cell (Transduction Laboratories)orasheep anti–humanAuroraBpAb(unpub- (9E10). AuroraBwasdetectedusing eithertheanti-AIM-1mousemAb ACA); BubR1(SBR1.1);Bub1(4B12); AuroraA(RAA.1);andMyc-tag state Biotechnology);tubulin(TAT1); centromere/kinetochores(human the following:phosphohistoneH3(UpstateBiotechnology);cyclinB1(Up- done asdescribedpreviously(Tayloretal.,2001)usingantibodiesagainst Immunofluorescence, immunoblotting,andimmunoprecipitationswere all Antibody techniques ZM447439-treated platescomparedwithDMSO-treatedcontrols. and counted.Thecloningefficiencyrepresentsthenumberofcolonieson ZM447439. After10d,thecolonieswerefixed,stainedwithcrystalviolet, plated ineachwellofa6-wellplatecompletemediawithout centrations for72h.Thecellswereharvested,washed,and 182780 at1 (HyClone), andwerethentreatedwithorwithouttheanti-estrogenICI MCF7 cellswereplatedinphenolredfreeDMEplus5%strippedserum previously (TaylorandMcKeon,1997).Todeterminecloningefficiency, HeLa cellsatG1/Susingadoublethymidineblockweredoneasdescribed DNA contentandmitoticindexmeasurementssynchronizationofTA- Cell cycleanalysisandcloningassays then freshlydilutedinmedia.TheIC for upto9moinindividualaliquotsavoidfreeze-thawcycles,andwas M. ZM447439wasdissolvedinDMSOat10mMandstored biochem) wasdissolvedinDMSOandusedatafinalconcentrationof20 dine wereusedasdescribedpreviously(Tayloretal.,2001).MG132(Cal- were culturedinMEGM(Clonetics).Nocodazole,paclitaxel,andthymi- mary epithelialcellsexpressinghTERT(CLONTECHLaboratories,Inc.) described previously(Tayloretal.,2001).Nontransformedhumanmam- Beatson InstituteforCancerResearch,Glasgow,UK)wereallculturedas pCMVp53 143A,andparentalU2OScells(bothfromKarenVousden, HeLa cells(TaylorandMcKeon,1997),U2OSstablytransfectedwith A549, MCF-7,andDLD-1cells(AmericanTypeCultureCollection),TA- Cell culture 5 eca Pharmaceuticals,Cheshire,UK). ity. FurtherdetailsareavailableonrequestfromNicholasKeen(AstraZen- concentration ofZM447439,whichgave50%inhibitionenzymeactiv- enzyme andnocompoundcontrolvalueswereusedtodeterminethe the cellularATPconcentrationis nM) weredeterminedattheKmforATP(seeabove).However,because drug-free culturestoaccountforthesolvent. of 2 ZM447439 isanATPcompetitor,weusedataconcentration M ATPforAuroraB,and0.2 M inallthecellassaysunlessstatedotherwise.DMSOwasaddedto M for48h.ZM447439wasthenaddedattheindicatedcon- 33 P withaBetaplate™counter(Wallac).No Ci 50 valuesforAuroraAandB( [ 33 200-fold higher,andbecause P]ATP (specificactivity 2, M forAuroraAor 10 2 -terminal His M peptide 400 cells 2,500 20 100

C 6

The Journal of Cell Biology manufacturer’s instructions.Inbrief,10 were transfectedusingOligofectAMINE™(Invitrogen)accordingtothe CACAAUUGA-3 as indicatedtodeterminekinetochoreposition. used tomeasureinterkinetochoredistancesusingeitherACAorBub1foci count foranyvariationsinstainingorimageacquisition.SoftWoRxwas tracted, andthevalueswerethennormalizedagainstACAsignaltoac- imaging software(AppliedPrecision).Backgroundreadingsweresub- In brief,kinetochorefluorescencevaluesweredeterminedusingsoftWoRx quantitation wereperformedasdescribedpreviously(Tayloretal.,2001). scintillation counting.Deconvolutionmicroscopyandpixelintensity five washesin0.5%phosphoricacid,boundradiolabelwasquantitatedby phoric acid,andwerethenspottedontoP30filtermat(Whatman).After (LRRWSLG) peptidesubstrate.Reactionswerestoppedwith20%phos- Adams, R.R.,H.Maiato, W.C.Earnshaw,andM.Carmena. 2001c.Essentialroles Adams, R.R.,D.M.Eckley,P.Vagnarelli, S.P.Wheatley,D.L.Gerloff,A.M. Adams, R.R.,M.Carmena,andW.C.Earnshaw. 2001a.Chromosomalpassengers References Accepted: 24February2003 Revised: 24February2003 Submitted: 15August2002 Council (BBSRC).S.S.TaylorisaBBSRCDavidPhillipsResearchFellow. search UKandTheBiotechnologyBiologicalSciencesResearch work wouldnothavebeenpossible. colleagues atAstraZeneca,especiallyStephenGreen,withoutwhomthis Hagan andReneMedemaforcommentsonthemanuscript;allour cluding GordonTurner,SarahHoltandClaireBullock,forreagents;Iain for assistancewithelectronmicroscopy;membersoftheTaylorlab,in- We aregratefultoKarenVousdenforp53 www.jcb.org/cgi/content/full/jcb.200208091/DC1 tance andBubR1binding.Onlinesupplementalmaterialavailableathttp:// experiment examiningtheeffectofZM447439oninterkinetochoredis- RNAi onthespindlecheckpoint;and(3)quantitationofdatafrom Aurora BRNAionCenp-EandMad2localization;(2)theeffectofBubR1 The supplementalfiguresandtablesshow(1)theeffectsofZM447439 Online supplementalmaterial cence asdescribedabove. fixed with1%formaldehydeinPBSandprocessedforimmunofluores- calcium phosphatekit(Promega).24haftertransfection,thecellswere then transfectedintoTA-HeLacellsoncoverslipsusingtheProFection K106R, andsequenced,allafterstandardprocedures.PlasmidDNAwas cloned intopcDNA-3Myc(TaylorandMcKeon,1997),mutatedtocreate The humanAuroraBORF(Bischoffetal.,1998)wasamplifiedbyPCR, Transient transfections AAGCACAAAAGCUUGUCUCCA-3 siRNA duplexes(DharmaconResearch)designedtorepressAuroraA(5 RNAi examined onanelectronmicroscope(Tecnai12BioTWIN;FEICompany). 70-nm sectionswerecut,stainedwithuranylacetateandleadcitrate,then 16 hat4 at RT.Afterwashes,sampleswerestainedenblocin1%uranylacetatefor RT, andwerethenpelletedpost-fixedin1%osmiumtetroxidefor1h Cells werefixedwith2.5%glutaraldehydeinphosphatebufferfor2hat Electron microscopy mented with2.5 lyzed 48–72haftertransfection. tion ofcompletemedia.24hlater,thecellswerereplatedandthenana- siRNA/lipid complexeswerethenaddedtocellsfor4hfollowedbyaddi- fectAMINE™ weredilutedinmedia,mixed,andincubatedfor20min. 24-well plate24hbeforetransfection.siRNAduplexesandOligo- C. DitchfieldisfundedbyAstraZeneca,andV.L.JohnsonCancerRe- of somes andisoverexpressedintumourcells. man INCENPcolocalizeswiththeAurora-B/AIRK2 kinaseonchromo- Mackay, P.A.Svingen,S.H.Kaufmann, andW.C.Earnshaw.2001b.Hu- and the(aurora)ABCsofmitosis. Drosophila C, dehydratedwithacetone,andthenembeddedinSpurr’sresin. ), andBubR1(5 M ATP,5 innercentromereprotein (INCENP)andauroraBinhistone Ci [ -AACGGGCAUUUGAAUAUGAAA-3 32 ), AuroraB(5 Trends CellBiol. P]ATP, and3 5 cellswereseededinwellsofa Chromosoma. / U2OScells;SamNewby 11:49–54. -AACGCGGCACUU- M biotinyl-Ahx-tetra 110:65–74. - ) ® Shannon, K.B.,J.C.Canman,andE.D.Salmon.2002.Mad2BubR1function Rieder, C.L.,R.W.Cole,A.Khodjakov,andG.Sluder.1995.Thecheckpointde- Rieder, C.L.,andE.D.Salmon.1998.Thevertebratecellkinetochoreit’sroles Tanaka, T.U.,N.Rachidi, C.Janke,G.Pereira,M.Galova, E.Schiebel,M.J. Skoufias, D.A.,P.R.Andreassen,F.B. Lacroix, L.Wilson,andR.L.Margolis. Nigg, E.A.2001.Mitotickinasesasregulatorsofcelldivisionanditscheckpoints. Nicklas, R.B.,andC.A.Koch.1969.Chromosomemicromanipulation.3.Spindle Nicklas, R.B.1997.Howcellsgettherightchromosomes. Musacchio, A.,andK.G.Hardwick.2002.Thespindlecheckpoint:structuralin- Murata-Hori, M.,andY.Wang.2002.ThekinaseactivityofAuroraBisrequired Morishita, J.,T.Matsusaka,G.Goshima,Nakamura,H.Tatebe,andM. Moasser, M.M.,M.Srethapakdi,K.S.Sachar,A.J.Kraker,andN.Rosen.1999. Meraldi, P.,R.Honda,andE.A.Nigg.2002.Aurora-Aoverexpressionrevealstet- Kallio, M.J.,M.L.McCleland,P.T.Stukenberg,andG.J.Gorbsky.2002.Inhibi- Hsu, J.Y.,Z.W.Sun,X.Li,M.Reuben,K.Tatchell,D.K.Bishop,J.M.Grushcow, Hannak, E.,M.Kirkham,A.A.Hyman,andK.Oegema.2001.Aurora-Akinaseis Giet, R.,andC.Prigent.1999.Aurora/Ipl1p-relatedkinases,anewoncogenicfam- Elbashir, S.M.,J.Harborth,W.Lendeckel,A.Yalcin,K.Weber,andT.Tuschl. Chan, G.K.,S.A.Jablonski,V.Sudakin,J.C.Hittle,andT.J.Yen.1999.Human Bischoff, J.R.,L.Anderson,Y.Zhu,K.Mossie,Ng,B.Souza,Schryver,P. Bischoff, J.R.,andG.D.Plowman.1999.TheAurora/Ipl1pkinasefamily:regulators Biggins, S.,F.F.Severin,N.Bhalla,I.Sassoon,A.A.Hyman,andA.W.Murray. Biggins, S.,andA.W.Murray.2001.ThebuddingyeastproteinkinaseIpl1/Au- Andreassen, P.R.,O.D.Lohez,F.B.Lacroix,andR.L.Margolis.2001.Tetraploid Cell. in asinglecheckpointpathwaythatresponds toalossoftension. 941–948. an inhibitorysignalproducedbyunattachedkinetochores. laying anaphaseinresponsetochromosomemonoorientationismediatedby during mitosis. Stark, andK.Nasmyth. 2002.EvidencethattheIpl1-Sli15 (Aurora kinase- 4492–4497. ment andkinetochore-tensioncheckpoints. 2001. Mammalianmad2andbub1/bubR1 recognizedistinctspindle-attach- Nat. Rev.Mol.CellBiol. Biol. fiber tensionandthereorientationofmal-orientedchromosomes. sights intodynamicsignalling. 894–899. for kinetochore-microtubuleinteractionsduringmitosis. Genes Cells. loss ofcohesionisrequiredforcondensation,spindleelongationandrepair. Yanagida. 2001.Bir1/Cut17movingfromchromosometospindleuponthe totic arrest. Inhibition ofSrckinasesbyaselectivetyrosinekinaseinhibitorcausesmi- cells. raploidization asamajorroutetocentrosomeamplificationinp53 12:900–905. dle checkpoint,andperturbsmicrotubuledynamicsinmitosis. tion ofauroraBkinaseblockschromosomesegregation,overridesthespin- in buddingyeastandnematodes. of histoneH3isgovernedbyIpl1/aurorakinaseandGlc7/PP1phosphatase C.J. Brame,J.A.Caldwell,D.F.Hunt,etal.2000.Mitoticphosphorylation 155:1109–1116. required forcentrosomematurationin ily ofmitoticserine-threoninekinases. tured mammaliancells. 2001. Duplexesof21-nucleotideRNAsmediateRNAinterferenceincul- kinetochores andbindsthecyclosome/APC. BUBR1 isamitoticcheckpointkinasethatmonitorsCENP-Efunctionsat cers. sophila Flanagan, F.Clairvoyant,C.Ginther,etal.1998.Ahomologueof of chromosomesegregationandcytokinesis. kinetochores inbuddingyeast. 1999. TheconservedproteinkinaseIpl1regulatesmicrotubulebindingto Dev. rora allowstheabsenceoftensiontoactivatespindlecheckpoint. G1. state inducesp53-dependentarrestofnontransformedmammaliancellsin junction, andchromosomesegregation. H3 phosphorylation,metaphasechromosomealignment,kinetochoredis- Aurora Bregulatesthespindlecheckpoint| 13:3706–3719. Mol. Biol.Cell. 43:40–50. 15:3118–3129. EMBO J. EMBO J. aurorakinaseisoncogenicandamplifiedinhumancolorectalcan- 6:743–763. Cancer Res. 17:3052–3065. 21:483–492. Trends CellBiol. 12:1315–1328. 59:6145–6152. Nature. 2:21–32. Nat. Rev.Mol.CellBiol. Genes Dev. 411:494–498. 8:310–318. Cell. 102:279–291. J. CellSci. J. CellBiol. Caenorhabditis elegans 13:532–544. Trends CellBiol. Proc. Natl.Acad.Sci.USA. J. CellBiol. 112:3591–3601. 153:865–880. Science. Ditchfieldetal. 3:731–741. 146:941–954. 9:454–459. J. CellBiol. 275:632–637. Curr. Biol. . J. CellBiol. Curr. Biol. Mol. Biol. J. Cell Genes 130: Dro- 12: 279 98: / The Journal of Cell Biology 280 Taylor, S.S.,andF.McKeon.1997.KinetochorelocalizationofmurineBub1isre- Tatsuka, M.,H.Katayama,T.Ota,Tanaka,S.Odashima,F.Suzuki,andY. Terada, Y.,M.Tatsuka,F.Suzuki,Y.Yasuda,S.Fujita,andOtsu.1998. Taylor, S.S.,D.Hussein,Y.Wang,S.Elderkin,andC.J.Morrow.2001.Kineto- The JournalofCellBiology age. quired fornormalmitotictimingandcheckpointresponsetospindledam- in humancancercells. sion oftheaurora-andIpl1-likemidbody-associatedproteinmitotickinase Terada. 1998.Multinuclearityandincreasedploidycausedbyoverexpres- chore-spindle poleconnections. INCENP) complexpromoteschromosomebi-orientationbyalteringkineto- AIM-1: amammalianmidbody-associatedproteinrequiredforcytokinesis. man cells. nents Bub1andBubR1aredifferentiallyregulatedbyspindleeventsinhu- chore localisationandphosphorylationofthemitoticcheckpointcompo- Cell. 89:727–735. J. CellSci. 114:4385–4395. Cancer Res. |

Volume 161,Number2,2003 Cell. 58:4811–4816. 108:317–329. Yao, X.,A.Abrieu,Y.Zheng,K.F.Sullivan,andD.W.Cleveland.2000.CENP-E Wright, J.H.,E.Munar,D.R.Jameson,P.R.Andreassen,R.L.Margolis,R.Seger, Waters, J.C.,R.H.Chen,A.W.Murray,andE.D.Salmon.1998.Localizationof Zhou, H.,J.Kuang,L.Zhong,W.L.Kuo,J.W.Gray,A.Sahin,B.R.Brinkley,and and themitoticcheckpoint. forms alinkbetweenattachmentofspindlemicrotubulestokinetochores Proc. Natl.Acad.Sci.USA. quired fortheG(2)/Mtransitionofcellcycleinmammalianfibroblasts. and E.G.Krebs.1999.Mitogen-activatedproteinkinaseactivityisre- Cell Biol. Mad2 tokinetochoresdependsonmicrotubuleattachment,nottension. EMBO J. amplification, aneuploidyandtransformation. S. Sen.1998.TumouramplifiedkinaseSTK15/BTAKinducescentrosome 141:1181–1191. 17:667–676. 96:11335–11340. Nat. CellBiol. 2:484–491. Nat. Genet. 20:189–193. J.