Mechanism of the Spindle Assembly Checkpoint Control

主轴装配检查点控制机制

• 批准号: 6710093
• 负责人: GUOWEI FANG
• 金额: $ 26.05万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6710093
• 关键词: Xenopus; biological signal transduction; cell cycle; cell cycle proteins; centromere; chromosome movement; enzyme activity; flow cytometry; fluorescence resonance energy transfer; immunoprecipitation; mass spectrometry; microtubules; mitotic spindle apparatus; phosphorylation; protein kinase; protein protein interaction; protein structure function; proteolysis; reversed phase chromatography; tissue /cell culture; western blottings

DESCRIPTION (provided by applicant): This proposal concerns the molecular mechanism of the spindle assembly checkpoint in the mammalian system. This mitotic checkpoint ensures the fidelity of chromosome segregation; inactivation of this checkpoint causes mis-segregation of chromosomes and aneuploidy, leading to tumorigenesis. The checkpoint mechanism prevents premature anaphase initiation by inhibiting the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase that controls sister chromatid separation. The activity of APC at metaphase is controlled by its activator CDC20 and by its inhibitor MAD2, a checkpoint protein. We propose experiments here to address the function of two checkpoint proteins, MAD2 and BUBR1. Our long-term goal is to identify all the components in the mitotic checkpoint pathway, to understand the biochemistry of each signaling step and to reconstitute the signaling pathway in vitro using purified metaphase chromosomes. 1. To detect a checkpoint-dependent chance in MAD2 structure. We will develop a mammalian cell-free checkpoint system using purified metaphase chromosomes and determine the biochemical basis of the checkpoint signaling by the MAD2 protein. 2. To define the role of BUBR1 in control of CDC20 activity. CDC20 is a substrate of the checkpoint kinase BUBR1. We will analyze the biochemical effect and physiological function of phosphorylation of CDC20 by BUBR1, both in vitro and in vivo. 3. To investigate the regulation of BUBR1 by checkpoint pathway. BUBR 1 is activated by the checkpoint pathway. We will study the molecular mechanism that regulates BUBR1 kinase activity in response to checkpoint activation. Results from proposed studies will provide a molecular pathway for the mitotic checkpoint control in mammalian cells. Since the checkpoint pathway is inactivated in several types of cancer, a better understanding of the biochemical pathway for the checkpoint signaling is not only essential to our understanding of the basic workings of the cell cycle machinery and regulated proteolysis in all eukaryotic cells, but also central for the development of novel strategies for cancer diagnosis and treatment.

描述（由申请人提供）：本提案涉及分子 在哺乳动物系统中的纺锤体组装检查点的机制。这 有丝分裂检查点确保染色体分离的保真度;失活 该检查点的缺失导致染色体的错误分离和非整倍性， 导致肿瘤发生。检查点机制防止过早的后期 通过抑制后期促进复合物（APC）/细胞周期体启动， 控制姐妹染色单体分离的泛素连接酶。APC的活动 在中期由其激活剂CDC 20和其抑制剂MAD 2控制， 检查点蛋白我们在这里提出实验来解决两个功能 检查点蛋白，MAD 2和BUBR 1。我们的长期目标是确定所有 有丝分裂检查点途径中的组分，以了解 每个信号传导步骤，并在体外使用 纯化的中期染色体。 1.检测MAD 2结构中的检查点相关机会。我们将开发一个 使用纯化的中期染色体的哺乳动物无细胞检查点系统， 通过MAD 2确定检查点信号传导的生物化学基础 蛋白 2.明确BUBR 1在调控CDC 20活性中的作用。CDC 20是一种 检查点激酶BUBR 1的底物。我们将分析 BUBR 1对CDC 20磷酸化作用及其生理功能， 体外和体内。 3.探讨BUBR 1通过检查点途径的调控。BUBR 1是 由检查点路径激活。我们将研究 调节BUBR 1激酶活性以响应检查点激活。 从拟议的研究结果将提供一个有丝分裂的分子途径， 哺乳动物细胞中的检查点控制。由于检查点路径是 在几种类型的癌症中失活，更好地了解 检查点信号传导的生化途径不仅对我们的 了解细胞周期机制的基本工作原理， 蛋白质水解在所有真核细胞中，但也是核心的发展， 癌症诊断和治疗的新策略。