Mechanisms of Spindle Assembly Checkpoint Silencing

描述（由申请方提供）：染色体分离错误导致染色体不稳定，并导致发育缺陷和肿瘤发生。纺锤体组装检查点（SAC）可以确保染色体的可靠分离。SAC阻止有丝分裂进程，直到所有姐妹染色体形成双极附着到有丝分裂纺锤体。SAC活性的丧失导致细胞培养物和动物模型中染色体不稳定和非整倍性的产生。虽然染色体不稳定是癌症的标志，但SAC机制突变或缺失的证据很少。介导SAC激活的沉默/恢复的机制是否可能在癌症中失调尚不清楚，主要是因为这些机制的特征很差。p31 Comet通过结合和抑制SAC效应子Mad 2（主要是通过阻止Mad 2二聚化）来驱动中期以后的进展。然而，p31 Comet和Mad 2组成型相互作用。因此，当Mad 2与其拮抗剂p31彗星相互作用时，SAC活性的窗口是如何创建的，这是理解SAC功能的关键未回答的问题。我们已经发现在有丝分裂过程中p31 Comet的磷酸化降低了其对Mad 2的亲和力。我们认为p31 Comet磷酸化减弱p31 Comet-Mad 2结合，促进Mad 2二聚化和SAC活性。我们已经确定了突出的磷酸化位点。我们建议调节该残基的磷酸化状态以1）测试其磷酸化对SAC活化的作用和2）确定其磷酸化对p31 Comet对Mad 2的体外和体内亲和力的影响。然后，我们将确定磷酸化的时空调节，并确定靶向激酶。这些研究的结果将阐明一个未探索的机制控制SAC活动。获得这些知识可以提高我们对SAC在癌症中功能的理解，并可能为新型抗有丝分裂治疗策略提供理论基础。

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.