G2 Role For Cdk2 in Human Cells

Cdk2 在人体细胞中的 G2 作用

• 批准号: 6878990
• 负责人: GREGORY H. ENDERS
• 金额: $ 29.32万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 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/6878990
• 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）：人类细胞分裂周期受到优雅的调控途径的控制，其中许多途径在真核生物中是保守的，这确保了主要事件以正确的顺序发生，并且延迟到下一阶段的进展，直到缺陷得到纠正。先前的研究表明，细胞周期蛋白依赖性激酶 2 (Cdk2) 是人类细胞中 DNA 合成的启动所必需的，而有丝分裂的启动则需要 Cdk1 (Cdc2)。越来越多的证据表明，Cdk2 通过促进 Cdk1 激活发挥另一个作用。在初步研究中，建立了一个简单的实验系统以更好地定义 Cdk2 的作用。通过诱导表达野生型和显性失活形式的酶（分别为 Cdk2-wt 和 Cdk2-dn），生成稳定的 U2-OS 细胞克隆。 Cdk2-wt 对细胞分裂周期没有明显影响，而 Cdk2-dn 主要将细胞阻滞在 G2 期：这些细胞含有复制的 DNA、未浓缩的染色体、低水平的 Cdk1 激酶活性和高水平的酪氨酸磷酸化 Cdk1（一种酶的非活性形式）。这些发现证实了 Cdk2 是 Cdk1 激活所必需的证据。 进一步的研究确定了 Cdk2-dn 对 Cdk1 激活剂 Plk1、Cdc25C 和 Cdc25B 以及 Cdk1 抑制剂 Wee1 的特定作用。我们现在报道，干扰细胞周期蛋白 A（Cdk2 激活亚基之一）的表达，会在正常人成纤维细胞中施加 S 和 G2/M 延迟，并定性地再现 U2-OS 细胞中 Cdk2-dn 诱导所观察到的主要生化效应。因此，我们假设细胞周期蛋白 A/Cdk2 复合物通过拮抗 Cdk1 的酪氨酸磷酸化在有丝分裂进入中发挥主要作用。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) 描绘促进 Cdk1 激活的 S 期和 G2 期 Cdk 复合物，2) 确定 Cdk2 增加 Plk1 水平及其对有丝分裂进入的影响的机制，3) 确定 Cdk2 介导 Cdc25C 和 Cdc25B 激活的机制，4) 确定 Cdk2 介导的机制 Wee1 的磷酸化。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。这些功能似乎受到检查点途径的生理调节，这些检查点途径抑制 DNA 损伤而进入有丝分裂。我们认为，Cdk2 的失调是癌症中的常见事件，可能通过这些功能的失调导致遗传不稳定和肿瘤进展。