Cell Cycle Control by Cyclin-Dependent Kinases

描述(申请人提供)：这个项目将探索管理真核细胞分裂周期各个阶段进展的调控系统，重点是有丝分裂中染色体分离的控制。细胞周期的进程由一类重要的蛋白激酶控制，称为细胞周期蛋白依赖性蛋白激酶或CDK，其活性依赖于与细胞周期蛋白调节亚单位的联系。在拟议的研究中，将使用生化和分子遗传学方法对萌芽酵母酿酒酵母进行研究，以探讨CDK在整个细胞周期中控制进程的机制，特别是通过有丝分裂的机制。与前一个资助期一样，大部分拟议工作将侧重于识别和表征细胞中被CDK磷酸化的蛋白质底物。在第一个目标中，将使用创新的基于质谱学的方法来识别新的CDK靶点，然后将对选定的靶点进行详细分析，以评估它们在细胞周期中的功能以及CDK影响该功能的机制。第二个目标是研究CDK底物磷酸化状态的变化如何有助于控制后期复制染色体的分离和分离。初步研究表明，CDK底物Securin的光调节增强了中期到后期转换的开关性质，拟议的实验将通过开发新的方法来分析这种转换来解决这一可能性。其他实验将探索这样一种可能性，即后期重复DNA区域的有效分离依赖于特定CDK底物的去磷酸化。最后，第三个目标中的实验将针对一种名为CDC14的磷酸酶底物的鉴定和表征，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，从而增强我们对癌症等细胞周期控制缺陷的疾病的理解。与公共卫生相关：当一个细胞繁殖时，染色体首先被复制，然后被分离成一对子细胞。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量缺陷，其中任何一种都可能加速癌症进展或导致发育缺陷。拟议中的研究重点是被称为细胞周期蛋白依赖性激酶的酶，它是所有真核生物中细胞分裂的关键调节因素。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出现的。

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.