The CAK Network in Metazoan Cell Cycle Regulation

描述(申请人提供)：细胞周期蛋白依赖性蛋白激酶(CDK)由RNA聚合酶II(POL II)控制细胞分裂和转录，并且自身受到严格的调控。后生动物细胞周期和转录途径的共同激活物是CDK激活激酶(CAK)：由CDK7、细胞周期蛋白H和Mat1组成的复合体。CDK7复合体在细胞分裂和基因表达中具有重要的功能：作为一种细胞周期蛋白，激活CDKs，驱动S期和有丝分裂；作为转录因子TFIIH的一部分，磷酸化POL II(和其他蛋白质)。CDK7及其靶点形成了一个网络，将基因表达与细胞分裂中的基因组复制和分离联系起来。我们的长期目标是了解CDK7是如何完成其双重功能的，以及它是否起到协调基因表达模式和细胞分裂的作用。我们已经通过一种新的化学遗传学方法定义了在哺乳动物细胞中对CDK7的细胞周期控制和转录的要求：创建一种人类癌细胞系，其中CDK7可以被小分子特异性地抑制。CDK7是激活CDK2和组装和激活CDK1/Cyclin B所必需的，CDK2是S期的主要CDK，CDK1/Cyclin B是有丝分裂的酶触发因子。抑制CDK7抑制特定的Pol11依赖基因亚集的表达。在体内操纵CDK7活性的能力将使我们能够评估CAK-CDK通路作为人类疾病治疗靶点的作用。我们将采用化学遗传学的方法来解决我们的特定目标：1)确定CDK7在G1中功能的靶点和时间。2)了解体内CDK1/Cyclin B组装对细胞周期蛋白依赖性的作用机制(S)。3)探讨在有丝分裂停滞过程中，CDK7是否调节CDK1/Cyclin B的组装和活性。4)明确CDK7活性在基因表达中的精确要求，通过功能基因组学确定CDK7反应基因，并通过生化确定其关键的蛋白质靶点和合作者。细胞增殖和基因信息的调节表达在癌症中都受到干扰。一个称为CDK的调控酶网络协调这两个过程。我们采用了一种新的方法，利用工程化的人类癌细胞对特殊设计的CDK抑制剂敏感，来探索CDK的功能，并测试这些酶是否是癌症治疗中潜在的抑制靶点。

DESCRIPTION (provided by applicant): Cyclin-dependent kinases (CDKs) control cell division and transcription by RNA polymerase II (Pol II), and are themselves stringently regulated. An activator common to both cell-cycle and transcription pathways in metazoans is the CDK-activating kinase (CAK): a complex of Cdk7, cyclin H and Mat1. The Cdk7 complex has essential functions in cell division and gene expression: as a CAK, to activate CDKs that drive S phase and mitosis; and, as a component of the transcription factor TFIIH, to phosphorylate Pol II (and other proteins). Cdk7 and its targets form a network that links gene expression with genome duplication and segregation in dividing cells. Our long-term goal is to understand how Cdk7 accomplishes its dual functions, and whether it serves to coordinate patterns of gene expression with cell division. We have defined requirements for Cdk7 in cell-cycle control and transcription in mammalian cells, by a novel, chemical-genetic approach: the creation of a human cancer cell line in which Cdk7 can be specifically inhibited with small molecules. Cdk7 is required for the activation of Cdk2, the major CDK active in S phase; and for the assembly and activation of Cdk1/cyclin B, the enzymatic trigger of mitosis. Inhibition of Cdk7 represses expression of a specific subset of Pol ll-dependent genes. The ability to manipulate Cdk7 activity in vivo will allow us to evaluate the CAK-CDK pathway as a therapeutic target in human disease. We will take a chemical genetic approach, to address our Specific Aims: 1) To determine the targets and timing of Cdk7 function in G1. 2) To understand the mechanism(s) enforcing dependency of Cdk1/cyclin B assembly on CAK in vivo. 3) To ask if Cdk7 regulates Cdk1/cyclin B assembly and activity during a mitotic arrest. 4) To define precise requirements for Cdk7 activity in gene expression, through functional genomics to identify Cdk7-responsive genes, and through biochemistry to identify its critical protein targets and collaborators. Cell proliferation and the regulated expression of genetic information are both disturbed in cancer. A network of regulatory enzymes called CDKs coordinates both processes. We take a novel approach, with engineered human cancer cells sensitive to specifically designed CDK inhibitors, to probe the functions of CDKs, and to test whether these enzymes are potential targets for inhibition in the treatment of cancer.