The CAK Network in Metazoan Cell Cycle Regulation

描述(申请人提供)：在真核生物中，细胞周期蛋白依赖性激酶(CDKs)控制着细胞分裂周期和RNA聚合酶II转录周期的关键步骤。CDK7在后生动物CDKs中是独一无二的，因为它在这两个周期中都发挥着重要的功能，作为主要的CDK激活激酶(CAK)和转录因子IIH(TFIIH)的组成部分。因此，CDK7是连接细胞分裂和基因表达的网络的中心元件--这种连接可能在癌症中被破坏。癌细胞的另一个标志是确保基因组复制和分离的保真度的监测机制功能障碍；真菌中的CDK7和相关酶参与了DNA损伤的修复和细胞周期反应。因此，了解CAK网络如何协调细胞分裂、生长和DNA损伤反应，对于了解这种控制是如何在癌症中失去的，以及在抗癌治疗中针对该途径都是至关重要的。然而，很难弄清楚CDK7是如何受到监管的，这恰恰是因为它有许多必需的功能。显然，CDK7的不同催化功能可以独立调节；拟议的工作旨在揭示调节因子，并发现CDK7作用的新靶点。第一个目的是通过对不同的含CDK7的复合体的酶学特性以及通过研究磷酸化在决定CDK7亚细胞定位中的作用，来识别影响CDK7的CAK功能的信号和信号分子。第二个目的是通过体内和体外对CDK7活性的经典和化学遗传操作来检验CDK7是关键细胞周期转变和/或特定转录程序的调节因子的假设。第三个目的是用经典的生化方法鉴定和表征调节CDK7的S磷酸化状态的酶，从而确定其稳定性和催化能力。最后，通过鉴定与酵母CAK网络的关键组成部分Csk1相关的一种新的哺乳动物CDK样激酶，将扩大对CAK网络的理解。哺乳动物的激酶可以替代酵母中的Csk1，在DNA损伤反应中发挥关键作用。它在暴露于DNA损伤剂的哺乳动物细胞中扮演着类似的角色，这一点将通过消融其功能来测试。

DESCRIPTION (provided by applicant): In eukaryotes, cyclin-dependent kinases (CDKs) control critical steps in both the cell division cycle and the transcription cycle of RNA polymerase II. CDK7 is unique among metazoan CDKs in that it performs essential functions in both cycles, as the major CDK-activating kinase (CAK) and as a component of transcription factor IIH (TFIIH). CDK7 is therefore the central element in a network linking cell division with gene expression--a connection that may be disrupted in cancer. Another hallmark of cancer cells is the dysfunction of surveillance mechanisms that insure fidelity of genome duplication and segregation; CDK7 and related enzymes in fungi have been implicated both in the repair of, and in the cell-cycle response to, DNA damage. Thus, understanding how the CAK network coordinates cell division, growth and the DNA damage response is crucial, both for understanding how that control is lost in cancer, and for targeting the pathway in anti-cancer therapy. It has been difficult to discern how CDK7 is regulated, however, precisely because of its many required functions. It is clear that the different catalytic functions of CDK7 can be independently regulated; the proposed work aims to uncover the regulators and to discover new targets for CDK7 action. The first aim is to identify signals and signaling molecules that affect the CAK function of CDK7, by enzymologic characterization of different CDK7-containing complexes and by investigating the role of phosphorylation in determining CDK7 subcellular localization. The second aim is to test the hypothesis that CDK7 is a regulator of key cell cycle transitions and/or specific transcriptional programs by both classical- and chemical-genetic manipulation of CDK7 activity in vivo and in vitro. The third aim is to identify and characterize the enzymes that modulate CDK7's phosphorylation state, and thereby its stability and catalytic power, by classical biochemical methods. Finally, understanding of the CAK network will be expanded by characterization of a novel mammalian CDK-like kinase related to Csk1, a key component of the yeast CAK network. The mammalian kinase can substitute for Csk1 in yeast to execute a critical function in the DNA damage response. That it plays a similar role in mammalian cells exposed to DNA-damaging agents will be tested by ablating its function.