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（CDK）通过RNA聚合酶II（Pol II）控制细胞分裂和转录，并且自身受到严格调控。多细胞动物中细胞周期和转录途径的共同激活剂是CDK激活激酶（CAK）：Cdk 7，细胞周期蛋白H和Mat 1的复合物。Cdk 7复合物在细胞分裂和基因表达中具有重要功能：作为CAK，激活驱动S期和有丝分裂的CDK;以及作为转录因子TFIIH的组分，磷酸化Pol II（和其他蛋白质）。cdk 7及其靶点形成了一个网络，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Cdk 7如何实现其双重功能，以及它是否有助于协调基因表达模式与细胞分裂。我们已经定义了在哺乳动物细胞的细胞周期控制和转录的Cdk 7的要求，通过一种新的，化学遗传学的方法：创建一个人癌细胞系，其中Cdk 7可以特异性地抑制小分子。Cdk 7是激活Cdk 2（S期主要的CDK活性）和组装和激活Cdk 1/细胞周期蛋白B（有丝分裂的酶促触发剂）所必需的。Cdk 7的抑制抑制Pol II依赖性基因的特定子集的表达。在体内操纵Cdk 7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。我们将采取化学遗传学方法，以解决我们的具体目标：1）确定Cdk 7在G1中的作用靶点和时间。2)了解Cdk 1/cyclin B组装体在体内对CAK依赖性的机制。3)探讨在有丝分裂停滞期间Cdk 7是否调节Cdk 1/细胞周期蛋白B的组装和活性。4)明确基因表达中Cdk 7活性的精确要求，通过功能基因组学鉴定Cdk 7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，细胞增殖和遗传信息的调节表达都受到干扰。一个名为CDK的调节酶网络协调这两个过程。我们采用了一种新的方法，用对专门设计的CDK抑制剂敏感的工程化人类癌细胞来探测CDK的功能，并测试这些酶是否是癌症治疗中抑制的潜在靶点。

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.