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）：有丝分裂检查点是由六个蛋白质的进化保守的组指定的，其功能是防止具有未对准染色体的细胞过早退出有丝裂。这些蛋白检测到未对准的染色体，然后在整个细胞中产生和转导抑制信号的机制，以阻止促进促丝分裂出口的后期促进复合体/循环体（APC/C）的机制，是促进有丝分裂出口的基础的主要问题，是尚待解决的主要问题。该提案的主要重点是检查有丝分裂检查点抑制APC/C的生化机制。为此，我们开发了一个无体细胞系统，使我们能够识别和表征负责抑制APC/C的因素。在要研究的因素中，有丝分裂检查点复合物（MCC），该复合物由检查点蛋白HBUBR1，HBUB3，MAD2和CDC20组成，这是迄今报告的APC/C中最有效的抑制剂。我们发现MCC的形成与有丝分裂的进入和退出有关，并且严重取决于另一个检查点蛋白HMPS1激酶。此外，我们在MCC中确定了一个新的亚基。 RO52是一种自动抗原，存在于Sjogren综合征患者中。 RO52本身是一种E3泛素连接酶，可以通过修改其某些亚基来调节MCC的功能。我们建议通过关注HMPS1和RO52来研究MCC的调节。我们将表征HMPS1在有丝分裂中的调节以及HMPS1在体内和体外的贡献。初步数据表明，从有丝分裂细胞纯化的MCC表现出泛素连接酶活性，而BUBR1是MCC中的底物之一。因此，我们计划测试RO52是否负责MCC中这种泛素连接酶的活性，并且这种修饰是否影响MCC组成，从而在体外和体内发挥作用。最近的研究表明，有丝分裂检查点受泛素连接酶调节的可能性，该研究表明泛素连接酶的拮抗作用和Deconjugase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的染色体隔离的关键机械和调节事件。这与人类健康直接相关，因为此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。该主题还与了解癌细胞在诊所中通常使用的药物（例如紫杉醇）的治疗方法有关。