THE JOURNAL OF CELL BIOLOGY JCB: REPORT © The Rockefeller University Press $8.00The Journal of Cell Biology, Vol. 170, No. 6, September 12, 2005 873–880http://www.jcb.org/cgi/doi/10.1083/jcb.200505040 JCB 873 Microtubule capture by CENP-E silences BubR1-dependent mitotic checkpoint signaling Yinghui Mao, 1 Arshad Desai, 1,2 and Don W. Cleveland 1,2 1 Ludwig Institute for Cancer Research and 2 Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093 he mitotic checkpoint is the major cell cycle controlmechanism for maintaining chromosome contentin multicellular organisms. Prevention of prema-ture onset of anaphase requires activation at unattachedkinetochores of the BubR1 kinase, which acts with othercomponents to generate a diffusible “stop anaphase” in-hibitor. Not only does direct binding of BubR1 to thecentromere-associated kinesin family member CENP-Eactivate its essential kinase, binding of a motorless frag-ment of CENP-E is shown here to constitutively activateBubR1 bound at kinetochores, producing checkpoint sig-T naling that is not silenced either by spindle microtubulecapture or the tension developed at those kinetochoresby other components. Using purified BubR1, microtu-bules, and CENP-E, microtubule capture by the CENP-Emotor domain is shown to silence BubR1 kinase activityin a ternary complex of BubR1–CENP-E–microtubule.Together, this reveals that CENP-E is the signal transduc-ing linker responsible for silencing BubR1-dependentmitotic checkpoint signaling through its capture at kinet-ochores of spindle microtubules. Introduction To assure accurate sister chromatid segregation during celldivision, eukaryotic cells have evolved a mitotic checkpointto prevent premature advance to anaphase before successfulattachment of every chromosome to microtubules of the mi-totic spindle. Chromosome instability leading to an abnormalchromosome number (aneuploidy) is associated with loss offunction of the mitotic checkpoint in some human cancers(Cahill et al., 1998; Hanks et al., 2004). Genetics in yeast ini-tially identified seven components of the mitotic checkpoint,Mad1-3 (Li and Murray, 1991), Bub1-3 (Hoyt et al., 1991),and Mps1 (Weiss and Winey, 1996). There are vertebrate ho-mologues of all of these except Bub2 (Chen et al., 1996,1998; Li and Benezra, 1996; Taylor and McKeon, 1997; Tay-lor et al., 1998; Abrieu et al., 2001). Additional essential con-tributors lacking yeast counterparts are known in metazoans.Without the kinetochore-associated microtubule motor proteinCENP-E, the checkpoint cannot be established or maintainedin vitro (Abrieu et al., 2000) or in mice (Putkey et al., 2002).Inhibition of ZW10 or Rod by mutation (Basto et al., 2000),antibody injection (Chan et al., 2000), or depletion withsiRNA (Kops et al., 2005) has revealed both to be requiredfor checkpoint signaling.As initially shown for Mad2 (Chen et al., 1996), all ofthese checkpoint proteins are recruited onto unattached kinet-ochores where they generate a diffusible signal to preventanaphase onset (for review see Cleveland et al., 2003). Beforespindle attachment, a Mad1–Mad2 complex is stably targetedto kinetochores. There it recruits additional Mad2 that is con-verted into a rapidly released, inhibitory form (Shah et al.,2004), including the possible assembly of a complex withother checkpoint proteins (Sudakin et al., 2001; Fang, 2002).The activated inhibitor(s) binds to and sequesters Cdc20, aspecificity factor required for an E3 ubiquitin ligase called theanaphase-promoting complex/cyclosome (APC/C) to recog-nize substrates (including securin and cyclin B) whose de-struction is required for advance to anaphase (Cleveland etal., 2003).Although the basic plan of the signaling cascade is estab-lished, how spindle microtubule binding to kinetochores si-lences mitotic checkpoint signaling is not known. However, thediscovery that CENP-E is associated with BubR1 (Chan et al.,1998; Yao et al., 2000) and stimulates its kinase activity (Maoet al., 2003) had implicated it in activation and maintenance ofmitotic checkpoint signaling in Xenopus egg extracts (Abrieuet al., 2000). This is true as well in the mammalian mitotic Correspondence to Don W. Cleveland: [email protected]. Mao’s present address is Department of Pathology, Columbia UniversityCollege of Physicians and Surgeons, New York, NY 10032.Abbreviations used in this paper: APC/C, anaphase-promoting complex/cyclo-some; CSF, cytostatic factor.The online version of this article contains supplemental material. on March 23, 2006 www.jcb.orgDownloaded from http://www.jcb.org/cgi/content/full/jcb.200505040/DC1Supplemental Material can be found at:JCB • VOLUME 170 • NUMBER 6 • 2005874 checkpoint where CENP-E was shown to be required for pre-vention of premature advance to anaphase in the presence ofunattached kinetochores in regenerating liver in mice (Putkeyet al., 2002). BubR1, the vertebrate homologue of yeast Mad3,has acquired a kinase domain in every species (including Dro-sophila , Xenopus , mice, and human) that also has a CENP-Ehomologue. BubR1 kinase activity is essential for the metazoanmitotic checkpoint and is directly stimulated by CENP-E bind-ing to it (Mao et al., 2003; Weaver et al., 2003).CENP-E is also one of the components directly responsi-ble for capture and stabilization of spindle microtubules by ki-netochores (Lombillo et al., 1995; McEwen et al., 2001; Putkeyet al., 2002). Full chromosome alignment is precluded by dis-ruption of CENP-E function in Xenopus egg extracts (Wood etal., 1997), in mammalian cultured cells (Yao et al., 2000;McEwen et al., 2001; Putkey et al., 2002), or in vivo in mice(Putkey et al., 2002). All of this has combined to make CENP-Ea candidate for a signal transducing linker responsible forsilencing BubR1-dependent checkpoint signaling at each ki-netochore through capture by its motor domain of spindlemicrotubules. We now test this model directly by eliminat-ing CENP-E–mediated microtubule capture in Xenopus eggextracts and mammalian-cultured cells, and by reconstruc-tion with purified components of silencing of BubR1 kinasethrough CENP-E–mediated microtubule capture. Results and discussion Motorless CENP-E tail chronically activates BubR1 kinase and mitotic checkpoint signaling in Xenopus egg extracts If CENP-E capture of spindle microtubules is required for mi-totic checkpoint signal silencing, replacement of CENP-E witha