The CAK Network in Metazoan Cell Cycle Regulation

描述（由申请人提供）：细胞周期蛋白依赖性激酶（CDKs）通过RNA聚合酶II （Pol II）控制细胞分裂和转录，并且本身受到严格调控。在后生动物中，细胞周期和转录途径共同的激活因子是cdk激活激酶（CAK）： Cdk7、细胞周期蛋白H和Mat1的复合物。Cdk7复合物在细胞分裂和基因表达中具有重要的功能：作为一个CAK，激活CDKs，驱动S期和有丝分裂；并且，作为转录因子TFIIH的一个组成部分，磷酸化Pol II（和其他蛋白质）。Cdk7及其靶标形成了一个网络，将基因表达与分裂细胞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如何完成其双重功能，以及它是否有助于协调基因表达模式与细胞分裂。我们已经定义了Cdk7在哺乳动物细胞周期控制和转录中的需求，通过一种新的化学遗传学方法：创建一种人类癌细胞系，其中Cdk7可以被小分子特异性抑制。Cdk7是激活Cdk2所必需的，主要CDK在S期活跃；以及Cdk1/细胞周期蛋白B的组装和激活，细胞周期蛋白B是有丝分裂的酶触发器。抑制Cdk7可抑制Pol - 1依赖性基因的特定亚群的表达。在体内操纵Cdk7活性的能力将使我们能够评估CAK-CDK途径作为人类疾病的治疗靶点。我们将采用化学遗传学方法来解决我们的具体目标：1)确定G1中Cdk7功能的靶点和时间。2)了解体内Cdk1/cyclin B组装依赖于CAK的机制。3)了解Cdk7是否在有丝分裂停止期间调节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，通过功能基因组学鉴定Cdk7应答基因，并通过生物化学鉴定其关键蛋白靶点和合作者。在癌症中，细胞增殖和基因信息的调控表达都受到干扰。一个叫做CDKs的调节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专门设计的CDK抑制剂敏感，来探测CDK的功能，并测试这些酶是否是抑制癌症治疗的潜在靶点。

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.