Kinetochore Function and Cell Cycle Progression

着丝粒功能和细胞周期进程

• 批准号: 6599020
• 负责人: KATSUMI KITAGAWA
• 金额: $ 27.6万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6599020
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; cell cycle; cell growth regulation; cell proliferation; centromere; chromosome movement; eukaryote; flow cytometry; high performance liquid chromatography; immunoprecipitation; mass spectrometry; matrix assisted laser desorption ionization; phosphorylation; polymerase chain reaction; protein binding; protein protein interaction; protein structure function; ubiquitin

DESCRIPTION (provided by applicant): Aneuploidy occurs in cancer cells when errors in chromosome segregation happen. Mutations leading to increased chromosome missegregation in certain cancers might be predisposing factors that accelerate tumorigenesis. By this model, the kinetochore and its regulatory system, which are essential for genome stability, are crucial in protecting against cancer development. The project's goal is to identify and characterize proteins required for mitotic chromosome segregation in eukaryotes, by using Saccharomyces cerevisiae as an experimental organism. Sgt1 and the core kinetochore protein, Skp1, promote assembly of the kinetochore core complex (CBF3) by activating Ctf13. Moreover, Skp1 and Sgt1 are part of the SCF complex (an E3 ubiquitin ligase). Specific Aim 1 is to investigate the biological function of Sgt1. This will be investigated by determining the time at which Sgt1 binds to CEN, and analyzing Sgt1's phosphorylation status. To test the hypothesis that Sgt1 and Skp1 are key components in the potential connection between kinetochore activation and ubiquitin-mediated degradation via the SCF complex, extragenic suppressor screens will be performed. Specific Aim 2 is to isolate and characterize proteins that interact with Sgt1. Immunoprecipitation mass spectrometry will be performed to isolate Sgt1 interactors. The function(s) of the isolated proteins will be characterized in genetics and biochemistry using the mutant strains that we will construct. Signaling from defective kinetochores to the mitotic checkpoint is thought to occur but is poorly understood. The principal investigator has shown that the spindle checkpoint protein Bub1 binds to Skp1 and that Bub1 associates with CEN DNA via Skp1. Therefore, Specific Aim 3 is to characterize the molecular interaction between kinetochores and the mitotic spindle checkpoint, especially the Bub1-Skp1 interaction. The contribution of the Bub1-Skp1 complex to the G2/M delays caused by mitotic defects, Bub1's Skp1 binding and CEN-associating domains, and additional Bub1 interactors will be analyzed. This work will reveal kinetochore functions of Skp1 and Sgt1 and novel kinetochore or cell-cycle regulators that can serve as entry points for further analysis of kinetochores in chromosome transmission and cell-cycle progression Achieving these aims will further elucidate mechanisms of cancer development.

描述（由申请人提供）：当染色体隔离中的误差发生时，癌细胞发生了非整倍性。突变导致某些癌症中染色体错误分类的增加可能是加速肿瘤发生的因素。通过这种模型，对于基因组稳定性至关重要的动力学及其调节系统对于防止癌症发展至关重要。该项目的目标是通过使用酿酒酵母作为实验生物，鉴定和表征真核生物中有丝分裂染色体所需的蛋白质。 SGT1和核心动物学蛋白SKP1通过激活CTF13促进动力学核心复合物（CBF3）的组装。此外，SKP1和SGT1是SCF复合物（E3泛素连接酶）的一部分。具体目的1是研究SGT1的生物学功能。这将通过确定SGT1与CEN结合的时间以及分析SGT1的磷酸化状态来研究。为了测试假说，即SGT1和SKP1是通过SCF复合物通过SCF复合物进行动力学激活与泛素介导的降解之间潜在连接的关键组成部分，将执行外基抑制器筛查。具体目的2是隔离和表征与SGT1相互作用的蛋白质。将执行免疫沉淀质谱法以分离SGT1相互作用。分离的蛋白质的功能将在遗传学和生物化学中使用我们要构建的突变菌株进行表征。 从有缺陷的动力学到有丝分裂检查点的信号传导被认为会发生，但知之甚少。主要研究者表明，主轴检查点蛋白BUB1与SKP1结合，并且BUB1通过SKP1与CEN DNA相关。因此，特定的目的3是表征动力学和有丝分裂主轴检查点之间的分子相互作用，尤其是BUB1-SKP1相互作用。将分析BUB1-SKP1复合物对有丝分裂缺陷，BUB1的SKP1结合和CEN缔解域以及其他BUB1相互作用的G2/M延迟的贡献。 这项工作将揭示SKP1和SGT1以及新型动力学或细胞周期调节剂的动力学功能，这些功能可以用作进一步分析染色体传递和细胞周期进展中的动物学的入口点，以实现这些目标，将进一步阐明癌症发展机制。