G2 Role For Cdk2 in Human Cells

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保守，确保主要事件以正确的顺序发生，并且延迟到下一阶段的进展，直到缺陷可以纠正。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DNA合成起始所必需的，而Cdk1 （Cdc2）是有丝分裂起始所必需的。越来越多的证据表明，Cdk2通过促进Cdk1的激活起着另一个作用。在初步研究中，我们建立了一个简单的实验系统来更好地定义Cdk2的作用。通过诱导表达野生型和显性阴性形式的酶（分别为Cdk2-wt和Cdk2-dn），生成了稳定的U2-OS细胞克隆。Cdk2-wt对细胞分裂周期没有明显的影响，而Cdk2-dn主要在G2期阻滞细胞：这些细胞含有复制的DNA，未凝聚的染色体，低水平的Cdk1激酶活性和高水平的酪氨酸磷酸化Cdk1，一种无活性的酶。这些发现巩固了Cdk2是激活Cdk1所必需的证据。进一步的研究发现了Cdk2-dn对Cdk1激活因子Plk1、Cdc25C和Cdc25B以及Cdk1抑制剂Wee1的特异性作用。我们现在报道，干扰Cdk2激活亚基之一的细胞周期蛋白A的表达，在正常的人成纤维细胞中施加S和G2/M延迟，并定性地再现了在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起主要作用。我们提出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水平的机制及其对有丝分裂进入的影响，3)确定Cdk2介导Cdc25C和Cdc25B激活的机制，以及4)确定Cdk2介导Wee1磷酸化的机制。我们将通过系统的生化分析在复制细胞中抑制和消耗相关因素的影响来实现这些目标。这项工作将定义连接细胞复制的两个主要阶段的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，以响应DNA损伤。我们认为，在癌症中经常发生的Cdk2的失调，可能通过失调这些功能导致遗传不稳定和肿瘤进展。

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.