Mechanism of Mitotic Checkpoint in Mammalian Cells

描述（由申请人提供）：有丝分裂检查点是由一组进化上保守的六种蛋白质指定的，其功能是防止染色体未对齐的细胞过早退出有丝分裂。这些蛋白检测错配染色体的机制，然后在整个细胞中产生和转导抑制信号，阻止后期促进复合体/环体（APC/C）促进有丝分裂退出，这是仍有待解决的主要问题。本提案的一个主要焦点是研究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抑制APC/C的因素。待研究的因子之一是有丝分裂检查点复合体（MCC），这是一种由检查点蛋白hBUBR1、hBUB3、Mad2和Cdc20组成的复合体，是迄今为止报道的最有效的APC/C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hMps1激酶。此外，我们在MCC中发现了一个新的亚基。Ro52是一种自身抗原，存在于干燥综合征患者中。Ro52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。我们建议以hMps1和Ro52为重点研究MCC的调控。我们将描述hMps1如何在有丝分裂中被调节，以及hMps1如何在体内和体外促进MCC组装。初步数据表明，从有丝分裂细胞中纯化的MCC具有泛素连接酶活性，而BubR1是MCC内的底物之一。因此，我们计划测试Ro52是否负责MCC中泛素连接酶的活性，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。最近的研究表明，泛素连接酶和去偶联酶的拮抗作用调节了检查点蛋白抑制APC/C的能力，这支持了有丝分裂检查点受泛素连接酶调节的可能性。公共卫生相关性：我们实验室的广泛目标是了解人类细胞中精确的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失衡，从而导致肿瘤形成或发育缺陷。这个主题也与了解癌细胞如何在临床常用的紫杉醇等药物治疗下存活有关。

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.