Mechanisms of Spindle Assembly Checkpoint Silencing

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DESCRIPTION (provided by applicant): Errors in chromosome segregation drive chromosomal instability and lead to developmental defects and oncogenesis. Faithful chromosome segregation is enforced by the spindle assembly checkpoint (SAC). The SAC prevents mitotic progression until all sister chromosomes form bipolar attachments to the mitotic spindle. Loss of SAC activity confers chromosomal instability and the generation of aneuploidy in cell culture and animal models. Although chromosomal instability is hallmark of cancer, evidence for mutation or loss of the SAC machinery is rare. Whether the mechanisms that mediate silencing/recovery from SAC activation may be deregulated in cancer are unknown, largely because these mechanisms are poorly characterized. p31Comet drives progression beyond metaphase by binding and inhibiting the SAC effector Mad2 largely by preventing Mad2 dimerization. However, p31Comet and Mad2 interact constitutively. Thus, how a window of SAC activity is created while Mad2 is interacting with its antagonist p31Comet is a key unanswered question in understanding SAC function. We have discovered that phosphorylation of p31Comet reduces its affinity for Mad2 during mitosis. We propose that p31Comet phosphorylation weakens p31Comet-Mad2 binding to promote Mad2 dimerization and SAC activity. We have identified the prominent phosphorylation site. We propose to modulate the phosphorylation state of this residue to 1) Test the role of its phosphorylation for SAC activation and 2) define the effects of its phosphorylation on the affinity of p31Comet for Mad2 in vitro and in vivo. We will then determine the spatio-temporal regulation of phosphorylation and identify the targeting kinase. The results of these studies will elucidate an unexplored mechanism controlling SAC activity. Gaining this knowledge can improve our understanding of SAC function in cancer and may provide the rationale for novel anti-mitotic therapeutic strategies.