The CDK Activation Network of Fission Yeast

描述(申请人提供)：拟议研究的长期目标是了解细胞周期蛋白依赖性蛋白激酶(CDK)和上游CDK激活蛋白(CAK)的网络如何协调细胞分裂与基因表达和维持真核生物--裂殖酵母--裂殖酵母的基因组完整性。在分裂酵母中，就像在多细胞生物体中一样，一种激酶激活主要细胞周期CDK(CDK1)来驱动细胞分裂，并磷酸化RNA聚合酶II来调节转录周期。这种与人类CDK7同源的酶-Mcs6-与另一种CDK-CDK9一起工作，控制与细胞周期相关的基因表达程序，并协调RNA分子的合成及其加工，以产生可翻译成蛋白质的成熟信使RNA(MRNAs)。S.pombe还含有第二个CAK，Csk1，它对于生存是必不可少的，但它激活了CDK1、Mcs6和CDk9。缺乏Csk1的细胞有生长缺陷，对DNA损伤剂和复制抑制物高度敏感。因此，CAK-CDK网络通过多种途径传递信号，并在基因表达和基因组监测中发挥关键的调节作用，此外，它还具有促进细胞增殖的典型功能。我们将结合生化和遗传学的方法来剖析分裂酵母中CAK-CDK网络的功能和靶点。通过引入每个CDK的基因工程版本，它容易受到特殊设计的小分子的抑制，我们能够在体内关闭激酶活性，并测量对细胞增殖和基因表达的影响。同样的操作也使我们能够识别靶向激酶的蛋白底物。具体目标是： 1.研究Mcs6(CDK7)和CDK9之间的协调，它们依次作用于 协调控制精选基因的表达。我们将调查CDK9招聘或 转录延伸复合体的活性依赖于MCS6的先前功能。 2.研究mRNAs转录(合成)与其成熟过程的偶联 含有CDK9和必需的mRNA处理酶--mRNA5‘-帽的复合体 甲基转移酶Pcm1。 3.寻找CAKs和CDKs在DNA损伤反应中的新靶点和新功能。 与公共卫生相关：CAK-CDK网络的整合功能在人类细胞中是保守的，在癌症中可能被破坏，其中细胞增殖控制受损、基因表达紊乱和基因组损伤都有助于肿瘤的发生和发展。如果我们要阻断或抑制这一途径的功能，以阻止肿瘤细胞的生长和分裂，则需要了解其多重功能，以避免对正常组织的毒性。裂解酵母中强大的遗传工具，以及与人类途径功能和组织的基本保守，使S.pombe成为执行这项工作的理想模型系统。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand how a network of cyclindependent kinases (CDKs) and upstream CDK-activating kinases (CAKs) coordinates cell division with gene expression and the maintenance of genome integrity in a model eukaryotic organism, the fission yeast Schizosaccharomyces pombe. In fission yeast, as in multicellular organisms, one kinase activates the major cell-cycle CDK (Cdk1) to drive cell division and phosphorylates RNA polymerase II to regulate the transcription cycle. That enzyme-Mcs6, which is homologous to human Cdk7-works together with another CDK, Cdk9, to control gene expression programs linked to the cell cycle, and to coordinate synthesis of RNA molecules with their processing to produce mature messenger RNAs (mRNAs) that can be translated into proteins. S. pombe also contains a second CAK, Csk1, which is dispensable for viability, but which activates Cdk1, Mcs6 and Cdk9. Cells lacking Csk1 have growth defects and are hypersensitive to DNA-damaging agents and replication inhibitors. The CAK-CDK network therefore signals through multiple pathways, and plays critical regulatory roles in gene expression and genomic surveillance, in addition to its canonical function in promoting cell proliferation. We will combine biochemical and genetic approaches to dissect the functions and targets of the CAK-CDK network in fission yeast. By introducing a genetically engineered version of each CDK that is susceptible to inhibition by a specially designed small molecule, we are able to switch kinase activity off in vivo, and measure the consequences for cell proliferation and gene expression. The same manipulation also allows us to identify the protein substrates of the targeted kinase. The specific aims are: 1. To investigate coordination between Mcs6 (Cdk7) and Cdk9, which act sequentially and in concert to control expression of select genes. We will investigate whether Cdk9 recruitment or activity in transcription elongation complexes depends on prior function of Mcs6. 2. To investigate the coupling of transcription (synthesis) of mRNAs with their maturation by a complex containing Cdk9 and an essential mRNA-processing enzyme, the mRNA 5'-cap methyltransferase Pcm1. 3. To identify new targets and functions of CAKs and CDKs in the DNA damage response. PUBLIC HEALTH RELEVANCE: The integrating function of the CAK-CDK network is conserved in human cells, and potentially disrupted in cancer, where impaired control of cell proliferation, derangement of gene expression and damage to the genome all contribute to the initiation and progression of tumors. If we are to block or inhibit functions of this pathway in order to stop growth and division of tumor cells, an understanding of its multiple functions will be needed to avoid toxicity to normal tissue. The powerful genetic tools available in fission yeast, and the fundamental conservation of pathway function and organization with humans, make S. pombe the ideal model system in which to perform this work.