Mechanism of Mitotic Checkpoint in Mammalian Cells

哺乳动物细胞有丝分裂检查点的机制

• 批准号: 7642532
• 负责人: Timothy Yen
• 金额: $ 38.39万
• 依托单位: RESEARCH INST OF FOX CHASE CAN CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-07 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7642532
• 关键词: 14-3-3 Proteins; Affect; Alleles; Antibodies; Autoantigens; Automobile Driving; Binding; Binding Proteins; Biochemical; Cell-Free System; Cells; Chromosome Segregation; Chromosomes; Clinic; Complex; Data; Defect; Development; Event; Exhibits; Health; Hela Cells; Human; In Vitro; Kinetochores; Lead; Light; Link; Mammalian Cell; Mechanics; Mitosis; Mitotic; Mitotic Checkpoint; Modification; Paclitaxel; Patients; Pharmaceutical Preparations; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Process; Proteins; RPS27 gene; Regulation; Reporting; Research; Role; Screening procedure; Signal Transduction; Sjogren' s Syndrome; Somatic Cell; Specific qualifier value; Staining method; Stains; Testing; To specify; Ubiquitin; Yeasts; anaphase-promoting complex; base; cancer cell; in vitro activity; in vivo; inhibitor/antagonist; mutant; prevent; public health relevance; tumor; ubiquitin ligase; ubiquitin-protein ligase; yeast two hybrid system

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.

描述（由申请人提供）：有丝分裂检查点由一组进化上保守的六种蛋白质指定，其功能是防止染色体未对齐的细胞过早退出有丝分裂。这些蛋白质检测错位染色体，然后在整个细胞中产生和转导抑制信号以阻止后期促进复合物/环体（APC/C）促进有丝分裂退出的机制是仍有待解决的主要问题。该提案的一个主要重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 的因素。要研究的因素之一是有丝分裂检查点复合物 (MCC)，这是一种由检查点蛋白 hBUBR1、hBUB3、Mad2 和 Cdc20 组成的复合物，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，我们还在 MCC 中发现了一个新的亚基。 Ro52 是一种存在于干燥综合征患者体内的自身抗原。 Ro52本身是一种E3泛素连接酶，可以通过修饰其一些亚基来调节MCC的功能。我们建议以hMps1和Ro52为重点来研究MCC的调控。我们将描述 hMps1 在有丝分裂中的调节方式以及 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 表现出泛素连接酶活性，并且 BubR1 是 MCC 内的底物之一。因此，我们计划测试 Ro52 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，从而影响体外和体内的功能。最近的研究支持有丝分裂检查点受泛素连接酶调节的可能性，这些研究表明泛素连接酶和解结合酶的拮抗作用调节检查点蛋白抑制 APC/C 的能力。公共健康相关性：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，因为该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。本主题还涉及了解癌细胞如何在临床常用药物（例如紫杉醇）的治疗中存活下来。