Cell Cycle Control by Cyclin-Dependent Kinases

描述（由申请人提供）：本项目将探索真核细胞分裂周期各阶段的调控系统，重点研究有丝分裂过程中染色体分离的控制。细胞周期进程是由一类重要的蛋白激酶控制的，称为周期蛋白依赖性激酶或Cdks，其活性取决于与周期蛋白调节亚基的关联。在拟议的研究中，生化和分子遗传学方法将用于出芽酵母酿酒酵母，以解决Cdks通过细胞周期控制进程的机制，特别是通过有丝分裂。与之前的资助期一样，大部分拟议的工作将集中在细胞中被Cdks磷酸化的蛋白质底物的鉴定和表征上。在第一个目标中，将使用基于质谱的创新方法来识别新的Cdk靶标，之后将详细分析选定的靶标，以评估它们在细胞周期中的功能以及Cdks影响该功能的机制。在第二个目标中提出的工作是针对Cdk底物磷酸化状态的变化如何帮助控制后期复制染色体的分离和分离的研究。初步研究表明，Cdk底物之一securin的磷酸化调控增强了中期到后期转变的开关特性，提出的实验将通过开发分析这种转变的新方法来解决这种可能性。其他实验将探索重复DNA区域在后期的有效分离取决于特定Cdk底物的去磷酸化的可能性。最后，第三个目标中的实验将针对磷酸酶Cdc14的底物的鉴定和表征，该酶已知在有丝分裂后期使许多Cdk底物去磷酸化。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，从而增强我们对细胞周期控制有缺陷的疾病（如癌症）的理解。公共卫生相关性：当细胞繁殖时，染色体首先复制，然后分离成一对子细胞。这些过程中的错误可能导致不受控制的细胞增殖、遗传损伤或染色体数目缺陷，其中任何一种都可能加速癌症进展或导致发育缺陷。拟议的研究集中在称为周期蛋白依赖激酶的酶上，它是所有真核生物细胞分裂的关键调节因子。这些研究将有助于更好地理解细胞分裂和染色体分离错误是如何在人类疾病中产生的。

DESCRIPTION (provided by applicant): This project will explore the regulatory system that governs progression through the stages of the eukaryotic cell division cycle, with an emphasis on the control of chromosome segregation in mitosis. Cell-cycle progression is governed by an important class of protein kinases called the cyclin-dependent kinases or Cdks, whose activity depends on association with cyclin regulatory subunits. In the proposed studies, biochemical and molecular genetic approaches will be used in the budding yeast Saccharomyces cerevisiae to address the mechanisms by which Cdks control progression through the cell cycle in general and through mitosis in particular. As in the previous funding period, much of the proposed work will focus on the identification and characterization of the protein substrates that are phosphorylated by Cdks in the cell. In the first aim, innovative mass-spectrometry-based approaches will be used to identify new Cdk targets, after which selected targets will be analyzed in detail to assess their function in the cell cycle and the mechanisms by which Cdks influence that function. The work proposed in the second aim is directed toward the study of how changes in the phosphorylation state of Cdk substrates help govern the separation and segregation of the duplicated chromosomes in anaphase. Preliminary studies suggest that the phosphoregulation of one Cdk substrate, securin, enhances the switchlike properties of the metaphase-to-anaphase transition, and the proposed experiments will address this possibility through the development of novel methods to analyze this transition. Other experiments will pursue the possibility that the efficient segregation of repetitive DNA regions in anaphase depends on the dephosphorylation of specific Cdk substrates. Finally, the experiments in the third aim will be directed toward the identification and characterization of substrates for a phosphatase, Cdc14, that is known to dephosphorylate many Cdk substrates in late mitosis. The information gained from these studies will provide important new insights into the control of cell-cycle progression and thereby enhance our understanding of diseases, such as cancer, in which cell-cycle control is defective. PUBLIC HEALTH RELEVANCE: When a cell reproduces, the chromosomes are first duplicated and then segregated into a pair of daughter cells. Errors in these processes can result in uncontrolled cell proliferation, genetic damage or defects in chromosome number, any of which can accelerate cancer progression or cause developmental defects. The proposed studies focus on enzymes called the cyclin-dependent kinases, which are key regulators of cell division in all eukaryotes. These studies will lead to a better understanding of how errors in cell division and chromosome segregation can arise in human disease.