Regulatory Enzymes and Systems in Cell Cycle Control

 描述(申请人提供)：建议的工作探索控制真核细胞分裂周期的调控系统，重点是在正确的时间和正确的顺序触发细胞周期事件的机制，从而导致细胞的健壮和准确的复制。细胞周期控制系统由主要的调控酶组成，包括细胞周期蛋白依赖的蛋白激酶和称为后期促进复合体或APC的泛素连接酶。在拟议的研究中，这些酶及其生物学功能将使用生化、细胞学和计算方法在萌芽酵母酿酒酵母中进行分析。大部分工作将集中在APC激活和底物结合的机制上，这取决于APC与结合底物的限速激活子亚单位的瞬时结合。将鉴定和表征一种以前未知的调节激活剂与APC结合的活性，并将开发单分子方法来分析APC-激活剂相互作用。计算模型将被用来探索APC-底物相互作用的调制如何导致有丝分裂期间APC靶标的有序泛素化。生化方法将被用来剖析APC磷酸化对激活剂功能的影响，并探索激活剂在纺锤体组装检查点的调节。这项拟议的工作还包括通过蛋白质磷酸化调节细胞周期进程的研究，重点是通过特定的磷酸酶控制晚期有丝分裂事件。从这些研究中获得的信息将为控制细胞周期进程提供重要的新见解，从而增强我们对癌症等疾病的理解，在这些疾病中，细胞周期控制或染色体分离是有缺陷的。这些研究还将阐明蛋白质泛素化和磷酸化的一般机制，这些调节修饰在细胞生物学和人类疾病中具有重要意义。

 DESCRIPTION (provided by applicant): The proposed work explores the regulatory system that controls the eukaryotic cell division cycle, with an emphasis on the mechanisms that trigger cell-cycle events at the correct time and in the correct order, resulting in robust and accurate reproduction of the cell. The cell-cycle control system is composed of master regulatory enzymes that include the cyclin-dependent protein kinases and a ubiquitin ligase called the anaphase-promoting complex or APC. In the proposed studies, these enzymes and their biological functions will be analyzed using biochemical, cytological, and computational approaches in the budding yeast Saccharomyces cerevisiae. Much of the work will focus on mechanisms of APC activation and substrate binding, which depend on transient association of the APC with a rate-limiting activator subunit that binds the substrate. A previously unknown activity that regulates binding of activator to the APC will be identified and characterized, and single-molecule methods will be developed for analysis of APC-activator interactions. Computational modeling will be used to explore how the modulation of APC-substrate interactions leads to the ordered ubiquitination of APC targets during mitosis. Biochemical approaches will be used to dissect the effects of APC phosphorylation on activator function and to explore activator regulation in the spindle assembly checkpoint. The proposed work also includes studies of the regulation of cell-cycle progression by protein phosphorylation, with an emphasis on the control of late mitotic events by a specific phosphatase. The information gained from these studies will provide important new insights into the control of cell-cycle progression, and will thereby enhance our understanding of diseases, such as cancer, in which cell-cycle control or chromosome segregation is defective. These studies will also illuminate general mechanisms of protein ubiquitination and phosphorylation, regulatory modifications of major importance throughout cell biology and human disease.