Cell Cycle control by cyclin-dependent kinases

超出空间 提供了 细胞周期蛋白依赖性激酶（Cdks）驱动真核细胞周期的事件。cdk被认为是通过 磷酸化细胞中大量的蛋白质底物，但这些底物中很少被磷酸化。 鉴定在拟议的研究中，一种新的生化策略将用于执行大规模搜索 在芽殖酵母和脊椎动物中的Cdk底物。 我们的初步研究已经鉴定了芽殖酵母中约200种Cdkl底物， 在所提出的工作的第一个目的中，将详细分析这些衬底的子集。其他初步 研究表明，相关细胞周期蛋白亚基的特性对细胞周期蛋白的表达速率有着深远的影响， Cdkl磷酸化许多酵母底物，并且所提出的研究的第二个目的将是探索 这种细胞周期蛋白特异性的分子基础及其在细胞中的生理功能。 还将制定方法，系统地鉴定和表征大量的 脊椎动物细胞中的Cdk底物。最后，将采用“化学遗传”方法构建小鼠 和小鼠细胞系，其中Cdk1或Cdk2可以以高特异性被抑制，允许进行严格的分析 它们在正常细胞生物学和肿瘤中的功能。 这些研究有望为Cdks的基本机制提供广泛的新见解， 驱动细胞周期事件。这些见解是朝着更好地理解 染色体分离缺陷和其他细胞周期缺陷，这些缺陷是癌细胞的特征。

EXCEED THE SPACE PROVIDED. Cyclin-dependent kinases (Cdks) drive the events of the eukaryotic cell cycle. Cdks are thought to act by phosphorylating a large number of protein substrates in the cell, but few of these substrates have been identified. In the proposed studies, a novel biochemical strategy will be used to perform large scale searches for Cdk substrates in budding yeast and vertebrates. Our preliminary studies have led to the identification of about 200 Cdkl substrates in budding yeast, and in the first aim of the proposed work a subset of these substrates will be analyzed in detail. Other preliminary work has revealed that the identity of the associated cyclin subunit has a profound effect on the rate at which Cdkl phosphorylates many yeast substrates, and the second aim of the proposed studies will be to explore the molecular basis of this cyclin specificity and its physiological function in the cell. Methods will also be developed for the systematic identification and characterization of large numbers of Cdk substrates in vertebrate cells. Finally, a 'chemical genetic' approach will be employed to construct mice and mouse cell lines in which Cdkl or Cdk2 can be inhibited with high specificity, allowing rigorous analysis of their function in normal cell biology and in tumors. These studies promise to provide a wide range of new insights into the basic mechanisms by which Cdks drive cell-cycle events. Such insights are an important step toward a better understanding of the chromosome segregation defects and other cell-cycle defects that characterize the cancer cell.