Mechanism of Mitotic Checkpoint in Mammalian Cells

描述(申请人提供)：有丝分裂检查点是由进化上保守的六种蛋白质组指定的，它们的功能是防止染色体不对齐的细胞过早退出有丝分裂。这些蛋白质检测染色体错位，然后在整个细胞内产生和转导抑制信号以阻止后期促进复合体/环体(APC/C)促进有丝分裂退出的机制是有待解决的主要问题。这项建议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(S)。为此，我们开发了一个体细胞无细胞系统，使我们能够确定和表征负责抑制APC/C的因素。在被研究的因素中，有丝分裂检查点复合体(MCC)是由检查点蛋白hBUBR1、hBUB3、MAD2和CDC20组成的复合体，它是迄今报道的最有效的APC/C抑制因子。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，我们还在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，可能通过修改MCC的一些亚基来调节MCC的功能。我们建议通过研究hMps1和Ro52来研究MCC的调控。我们将描述hMps1在有丝分裂中是如何调控的，以及hMps1在体内和体外对MCC组装的作用。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，BubR1是MCC内的底物之一。因此，我们计划测试Ro52是否对MCC中的泛素连接酶活性负责，以及这种修饰是否影响MCC的组成，从而在体外和体内发挥作用。最近的研究支持有丝分裂检查点受泛素连接酶调控的可能性，这些研究表明泛素连接酶和去结合酶的拮抗作用调节了检查点蛋白抑制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指定人类细胞中准确染色体分离的关键机械和调节事件。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，从而可能导致肿瘤形成或发育缺陷。这一主题也与理解癌细胞如何在临床上常用的药物如紫杉醇的治疗下存活有关。

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.