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A more recent version of this article appeared on October 1, 2006
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Submitted on April 24, 2006
Revised on July 18, 2006
Accepted on July 21, 2006
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020
Monitoring Editor: Yixian Zheng
Accurate chromosome segregation is controlled by the spindle checkpoint, which responses to the lack of microtubule-kinetochore attachment or of tension across sister kinetochores through phosphorylation of kinetochore proteins by the Mps1, Bub1, BubR1, Aurora B and Plk1/Plx1 kinases. The presence of the 3F3/2 phospho-epitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immuno-depletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phospho-epitope is dependent on the checkpoint activation, but the loading of the 3F3/2 substrate to kinetochores also requires the prior assembly of Mps1, Bub1 and BubR1 onto kinetochores. Interestingly, generation of the 3F3/2 epitope in checkpoint extracts requires the kinase activities of Mps1 and Bub1, but not that of BubR1. Furthermore, we demonstrate that checkpoint proteins in Xenopus extracts are assembled onto kinetochores in a highly ordered pathway consisting of three steps. Mps1 and Bub1 are loaded first, BubR1 and Plx1 s, followed by Mad1 and Mad2. The characterization of this ordered assembly pathway provides a framework for the biochemical mechanism of the checkpoint signaling and will aid the eventual identification of the 3F3/2 substrate.
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