Kinetochore activation of the spindle checkpoint by the kinase Mps1

Thesis (Ph.D.)--University of Washington, 2015Chromosome segregation is essential for faithful cell division and missegregation is associated with birth defects and cancer. Missegregation is prevented by the spindle checkpoint, which monitors attachments between microtubules and kinetochores prior to anaphase. Kinetochores are protein complexes that mediate microtubule-chromosome attachments and also signal the checkpoint by generating a soluble inhibitor of an ubiquitin-ligase that is necessary for anaphase onset. Checkpoint signaling is accomplished through localizing checkpoint proteins to kinetochores and requires activity of the conserved kinetochore kinase Mps1. These checkpoint proteins include Bub1 and Bub3, which perform additional functions to support chromosome segregation, as well as the checkpoint proteins Mad1, Mad2, and Mps1. Kinetochore localization of Mad1 and Mad2 is strictly correlated with checkpoint signaling. How these checkpoint proteins localize to kinetochores and the function of Mps1 activity was unknown. Here, I utilized purified kinetochore particles to address the role of Mps1 in checkpoint function. I first determined that Mps1 kinase activity was the dominant activity on kinetochores in vitro, even though the kinase Bub1 and phosphatases also copurify. I was then able to reconstitute Bub1 and Bub3 binding to kinetochores in vitro and demonstrated that this binding was dependent upon Mps1 phosphorylation of the core kinetochore protein Spc105 on conserved MELT-like motifs. Similarly, I was able to reconstitute the downstream Mad1 kinetochore binding event with native kinetochores as well as with recombinant proteins. Translational fusion experiments indicated that a domain of Bub1 is sufficient for Mad1 kinetochore binding, and this binding required Mps1 activity towards Bub1. Mps1 overexpression arrested cells independently of Spc105 MELT phosphorylation but not Bub1 phosphorylation, highlighting the significance of Bub1 phosphorylation in checkpoint signaling. Together, this data reveals the deterministic role for Mps1 kinase activity in kinetochore checkpoint activation and clarifies the steps that must be taken to inactivate the checkpoint upon microtubule attachment to kinetochores