The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other
The mitotic checkpoint is the major cell cycle control mechanism for maintaining chromosome content in multicellular organisms. Prevention of premature onset of anaphase requires activation at unattached kinetochores of the BubR1 kinase, which acts with other components to generate a diffusible “stop anaphase” inhibitor. Not only does direct binding of BubR1 to the centromere-associated kinesin family member CENP-E activate its essential kinase, binding of a motorless fragment of CENP-E is shown here to constitutively activate BubR1 bound at kinetochores, producing checkpoint signaling that is not silenced either by spindle microtubule capture or the tension developed at those kinetochores by other components. Using purified BubR1, microtubules, and CENP-E, microtubule capture by the CENP-E motor domain is shown to silence BubR1 kinase activity in a ternary complex of BubR1–CENP-E–microtubule. Together, this reveals that CENP-E is the signal transducing linker responsible for silencing BubR1-dependent mitotic checkpoint signaling through its capture at kinetochores of spindle microtubules.
To assure accurate sister chromatid segregation during cell division, eukaryotic cells have evolved a mitotic checkpoint to prevent premature advance to anaphase before successful attachment of every chromosome to microtubules of the mitotic spindle. Chromosome instability leading to an abnormal chromosome number (aneuploidy) is associated with loss of function of the mitotic checkpoint in some human cancers (Cahill et al., 1998; Hanks et al., 2004). Genetics in yeast initially 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 homologues of all of these except Bub2 (Chen et al., 1996, 1998; Li and Benezra, 1996; Taylor and McKeon, 1997; Taylor et al., 1998; Abrieu et al., 2001). Additional essential contributors lacking yeast counterparts are known in metazoans. Without the kinetochore-associated microtubule motor protein CENP-E, the checkpoint cannot be established or maintained in 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 with siRNA (Kops et al., 2005) has revealed both to be required for checkpoint signaling.
As initially shown for Mad2 (Chen et al., 1996), all of these checkpoint proteins are recruited onto unattached kinetochores where they generate a diffusible signal to prevent anaphase onset (for review see Cleveland et al., 2003). Before spindle attachment, a Mad1–Mad2 complex is stably targeted to kinetochores. There it recruits additional Mad2 that is converted into a rapidly released, inhibitory form (Shah et al., 2004), including the possible assembly of a complex with other checkpoint proteins (Sudakin et al., 2001; Fang, 2002). The activated inhibitor(s) binds to and sequesters Cdc20, a specificity factor required for an E3 ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) to recognize substrates (including securin and cyclin B) whose destruction is required for advance to anaphase (Cleveland et al., 2003).
Although the basic plan of the signaling cascade is established, how spindle microtubule binding to kinetochores silences mitotic checkpoint signaling is not known. However, the discovery that CENP-E is associated with BubR1 (Chan et al., 1998; Yao et al., 2000) and stimulates its kinase activity (Mao et al., 2003) had implicated it in activation and maintenance of mitotic checkpoint signaling in Xenopus egg extracts (Abrieu et al., 2000). This is true as well in the mammalian mitotic checkpoint where CENP-E was shown to be required for prevention of premature advance to anaphase in the presence of unattached kinetochores in regenerating liver in mice 