G2 Role For Cdk2 in Human Cells

Cdk2 在人体细胞中的 G2 作用

• 批准号: 7367502
• 负责人: GREGORY H. ENDERS
• 金额: $ 14.54万
• 依托单位: FOX CHASE CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367502
• 关键词: DNA damage; RNA interference; cell cycle; cyclin dependent kinase; enzyme activity; enzyme inhibitors; neoplastic growth; phosphoprotein phosphatase; phosphorylation; protein kinase; protein tyrosine kinase; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): The human cell division cycle is governed by elegant regulatory pathways, many of them conserved throughout eukaryotes, which ensure that major events occur in the proper order and that progression to the next phase is delayed until defects can be corrected. Previous work indicates that Cyclin dependent kinase 2 (Cdk2) is required for initiation of DNA synthesis in human cells and that Cdk1 (Cdc2) is required for initiation of mitosis. Mounting evidence suggests that Cdk2 plays another role by fostering Cdk1 activation. In preliminary studies, a facile experimental system was established to better define the roles of Cdk2. Stable U2-OS cell clones were generated with inducible expression of wild type and dominant-negative forms of the enzyme (Cdk2-wt and Cdk2-dn, respectively). Cdk2-wt had no apparent effect on the cell division cycle, whereas Cdk2-dn primarily arrested cells in G2 phase: these cells contained replicated DNA, uncondensed chromosomes, low levels of Cdk1 kinase activity, and high levels of tyrosine-phosphorylated Cdk1, an inactive form of the enzyme. These findings solidified the evidence that Cdk2 is needed for activation of Cdk1. Further investigation identified specific effects of Cdk2-dn on the Cdk1 activators Plk1, Cdc25C, and Cdc25B and the Cdk1 inhibitor Wee1. We now report that interfering with expression of cyclin A, one of the Cdk2 activating subunits, imposed S and G2/M delays in normal human fibroblasts and qualitatively reproduced the major biochemical effects observed with Cdk2-dn induction in U2-OS cells. We therefore hypothesize that cyclin A/Cdk2 complexes play a major role in mitotic entry by antagonizing tyrosine phosphorylation of Cdk1. We propose to explore this hypothesis through four interrelated specific aims: 1) To delineate the S and G2 phase Cdk complexes that foster Cdk1 activation, 2) To determine the mechanism by which Cdk2 increases Plk1 levels and its impact on mitotic entry, 3) To determine the mechanism by which Cdk2 mediates activation of Cdc25C and Cdc25B, and 4) To determine the mechanism by which Cdk2 mediates phosphorylation of Wee1. We will accomplish these aims through systematic biochemical analysis of the effects of inhibition and depletion of the relevant factors in replicating cells. The proposed work will define functions of Cdk complexes that link the two major phases of cell replication. These functions appear to be subject to physiologic regulation by checkpoint pathways that inhibit entry into mitosis in response to DNA damage. We believe that deregulation of Cdk2, a frequent event in cancer, may contribute to genetic instability and neoplastic progression through deregulation of these functions.

描述（由申请人提供）：人类细胞分裂周期由优雅的调控途径控制，其中许多在真核生物中是保守的，这确保了主要事件以适当的顺序发生，并且延迟了进入下一阶段的进程，直到缺陷得到纠正。以前的工作表明，细胞周期蛋白依赖性激酶2（Cdk 2）是必需的启动DNA合成在人类细胞和Cdk 1（Cdc 2）是必需的启动有丝分裂。越来越多的证据表明，Cdk 2通过促进Cdk 1激活发挥另一种作用。在初步研究中，建立了一个简单的实验系统，以更好地定义Cdk 2的作用。用野生型和显性阴性形式的酶（分别为Cdk 2-wt和Cdk 2-dn）的诱导表达产生稳定的U2-OS细胞克隆。Cdk 2-wt对细胞分裂周期没有明显的影响，而Cdk 2-dn主要在G2期逮捕细胞：这些细胞含有复制的DNA，未浓缩的染色体，低水平的Cdk 1激酶活性，和高水平的酪氨酸磷酸化的Cdk 1，一种失活形式的酶。这些发现巩固了Cdk 2是激活Cdk 1所必需的证据。 进一步的研究确定了Cdk 2-dn对Cdk 1激活剂Plk 1、Cdc 25 C和Cdc 25 B以及Cdk 1抑制剂Wee 1的特异性作用。我们现在报告说，干扰细胞周期蛋白A的表达，Cdk 2激活亚基之一，施加S和G2/M延迟在正常人成纤维细胞和定性再现与Cdk 2-DN诱导在U2-OS细胞中观察到的主要生化效应。因此，我们推测，细胞周期蛋白A/Cdk 2复合物发挥了重要作用，在有丝分裂进入拮抗酪氨酸磷酸化的Cdk 1。我们建议通过四个相互关联的具体目标来探索这一假设：1）描述促进Cdk 1活化的S和G2期Cdk复合物，2）确定Cdk 2增加Plk 1水平的机制及其对有丝分裂进入的影响，3）确定Cdk 2介导Cdc 25 C和Cdc 25 B活化的机制，（4）研究Cdk 2介导Wee 1磷酸化的机制。我们将通过对复制细胞中相关因子的抑制和消耗的影响进行系统的生化分析来实现这些目标。拟议的工作将定义连接细胞复制两个主要阶段的Cdk复合物的功能。这些功能似乎受到生理调节的检查点途径，抑制进入有丝分裂响应DNA损伤。我们认为，Cdk 2的失调，在癌症中的常见事件，可能有助于遗传不稳定性和肿瘤的进展，通过这些功能的失调。