Loading of the 3F3/2 antigen onto kinetochores is dependent on the ordered assembly of the spindle checkpoint proteins.
Accurate chromosome segregation is controlled by the spindle checkpoint, which responds 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 phosphoepitope on kinetochores, generated by Plk1/Plx1-mediated phosphorylation of an unknown protein, correlates with the activation of the tension-sensitive checkpoint pathway. Using immunodepletion approach and a rephosphorylation assay in Xenopus extracts, we report here that not only the formation of the 3F3/2 phosphoepitope is dependent on the checkpoint activation but also the loading of the 3F3/2 substrate to kinetochores 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, and BubR1 and Plx1 second, 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 in the eventual identification of the 3F3/2 substrate.
Wong
Oi Kwan
OK
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.
Fang
Guowei
G
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D000939:Epitopes / Q000096:biosynthesis; D005455:Fluorescent Antibody Technique; D018386:Kinetochores / Q000378:metabolism* / Q000648:ultrastructure; D008954:Models, Biological; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction; D008941:Spindle Apparatus / Q000502:physiology*; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000097869:Polo-Like Kinase 1
D018797:Cell Cycle Proteins; D000939:Epitopes; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D029867:Xenopus Proteins; C090464:Bub1 spindle checkpoint protein; C101085:Plk1 protein, Xenopus; D000097869:Polo-Like Kinase 1; C514980:RPS27 protein, Xenopus