Mechanism of Mitotic Checkpoint in Mammalian Cells

描述（由申请人提供）：有丝分裂检查点由一组进化上保守的6种蛋白质指定，其功能是防止染色体未对齐的细胞过早退出有丝分裂。这些蛋白检测染色体错位，然后在整个细胞中产生并释放抑制信号以阻断后期促进复合物/细胞核小体（APC/C）促进有丝分裂退出的机制是仍有待解决的主要问题。该建议的一个主要焦点是检查有丝分裂检查点抑制APC/C的生化机制。为了实现这一目标，我们开发了一种无体细胞系统，使我们能够识别和表征负责抑制APC/C的因子。在待研究的因子中，有丝分裂检查点复合物（MCC）是由检查点蛋白hBUBR 1、hBUB 3、Mad 2和Cdc 20组成的复合物，其是迄今报道的APC/C的最有效的抑制剂。我们发现MCC的形成与有丝分裂的进入和退出有关，并且严重依赖于另一种检查点蛋白hMps 1激酶。此外，我们在MCC中鉴定了一个新的亚基。Ro 52是一种自身抗原，存在于干燥综合征患者中。Ro 52本身是一种E3泛素连接酶，可以通过修饰MCC的一些亚基来调节MCC的功能。我们建议通过关注hMps 1和Ro 52来研究MCC的调控。我们将描述hMps 1在有丝分裂中的调控方式以及hMps 1在体内和体外对MCC组装的贡献。初步数据表明，MCC纯化有丝分裂细胞表现出泛素连接酶活性和BubR 1的MCC内的底物之一。因此，我们计划测试Ro 52是否负责MCC中的这种泛素连接酶活性，以及这种修饰是否影响MCC的组成，从而在体外和体内发挥功能。有丝分裂检查点受泛素连接酶调节的可能性得到最近研究的支持，这些研究表明泛素连接酶和去偶联酶的拮抗作用调节检查点蛋白抑制APC/C的能力。公共卫生相关性：我们实验室的广泛目标是了解在人类细胞中指定准确染色体分离的关键机械和调控事件。这与人类健康直接相关，因为这一过程中的缺陷会导致染色体不平衡，从而导致肿瘤形成或发育缺陷。这个主题也与理解癌细胞如何在临床常用的紫杉醇等药物治疗中存活有关。

DESCRIPTION (provided by applicant): The mitotic checkpoint is specified by an evolutionarily conserved group of six proteins whose functions are to prevent cells with unaligned chromosomes from prematurely exiting mitosis. The mechanisms by which these proteins detect misaligned chromosomes, and then generate and transduce an inhibitory signal throughout the cell to block the Anaphase Promoting Complex/cyclosome (APC/C) from promoting mitotic exit are major questions that remain to be solved. A major focus of this proposal is to examine the biochemical mechanism(s) by which the mitotic checkpoint inhibits the APC/C. To accomplish this, we have developed a somatic cell-free system that allows us to identify and characterize factors responsible for inhibiting the APC/C. Amongst the factors to be studied is the Mitotic Checkpoint Complex (MCC), a complex consisting of checkpoint proteins hBUBR1, hBUB3, Mad2 and Cdc20, that is the most potent inhibitor of the APC/C reported to date. We discovered that formation of the MCC is linked to mitotic entry and exit and is critically dependent on another checkpoint protein, hMps1 kinase. In addition, we identified a new subunit in the MCC. Ro52 is an autoantigen that is present in patients with Sjogren's syndrome. Ro52 is itself an E3 ubiquitin ligase that may regulate the function of the MCC by modifying some of its subunits. We propose to study the regulation of MCC by focusing on hMps1 and Ro52. We will characterize how hMps1 is regulated in mitosis and how hMps1 contributes to MCC assembly in vivo and in vitro. Preliminary data show that MCC purified from mitotic cells exhibits ubiquitin ligase activity and that BubR1 is one of the substrates within the MCC. We therefore plan to test if Ro52 is responsible for this ubiquitin ligase activity in MCC and whether this modification affects MCC composition and thus function in vitro and in vivo. The possibility that the mitotic checkpoint is regulated by a ubiquitin ligase is supported by recent studies that showed the antagonistic actions of ubiquitin ligase and deconjugase regulates the ability of checkpoint proteins to inhibit the APC/C. PUBLIC HEALTH RELEVANCE: The broad objective of our lab is to understand the key mechanical and regulatory events that specify accurate chromosome segregation in human cells. This is directly relevant to human health as defects in this process results in chromosome imbalance that can lead to tumor formation or developmental defects. This topic is also relevant to understanding how cancer cells survive treatment with drugs such as paclitaxel that are commonly used in the clinic.