The CDK Activation Network of Fission Yeast

裂殖酵母的CDK激活网络

基本信息

批准号：
7525139
负责人：
ROBERT P FISHER
金额：
$ 24.84万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-06-01 至 2009-01-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7525139
关键词：
Animal Model Binding Biochemical Genetics Biological Models Bypass CDC2 Protein Kinase Cell Cycle Cell Proliferation Cell division Cells Chemicals Complex Condition Coupling Cyclin-Dependent Kinases Cyclins DNA Damage DNA Repair Defect Dependency Engineering Ensure Enzymes Eukaryota Eukaryotic Cell Event Fission Yeast Gene Expression Genes Genetic Genetic Transcription Genome Genomics Goals Growth Hand Homologous Gene Human In Vitro Laboratories Link Logic Maintenance Malignant Neoplasms Mammals Measures Mediating Medical Surveillance Messenger RNA Methyltransferase Modeling Normal tissue morphology Organism Orthologous Gene Pathway interactions Pattern Phosphorylation Phosphotransferases Play Polymerase Positive Transcriptional Elongation Factor B Process Proteins Public Health Quality Control RNA Polymerase II RNA chemical synthesis Refractory Research Resistance Role Saccharomyces cerevisiae Saccharomycetales Signal Transduction Testing Toxic effect Transcript Transcription Elongation Translating Work analog chemical genetics cyclin H design homologous recombination in vivo inhibitor/antagonist mRNA capping mutant neoplastic cell novel programs response small molecule tool transcription factor TFIIH tumor progression upstream kinase

项目摘要

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.

描述（由申请人提供）：拟议研究的长期目标是了解细胞周期蛋白依赖性激酶（CDK）和上游 CDK 激活激酶（CAK）网络如何协调细胞分裂与基因表达以及维持基因组完整性。一种模型真核生物，裂殖酵母裂殖酵母。在裂殖酵母中，与在多细胞生物中一样，一种激酶激活主要细胞周期 CDK (Cdk1) 以驱动细胞分裂，并磷酸化 RNA 聚合酶 II 以调节转录周期。这种酶 - Mcs6 与人类 Cdk7 同源 - 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，以产生成熟的信使 RNA (mRNA)，可以翻译成蛋白质。 S. pombe 还含有第二个 CAK，Csk1，它对于生存能力来说是可有可无的，但它会激活 Cdk1、Mcs6 和 Cdk9。缺乏 Csk1 的细胞存在生长缺陷，并且对 DNA 损伤剂和复制抑制剂高度敏感。因此，CAK-CDK 网络除了促进细胞增殖的典型功能外，还通过多种途径发出信号，并在基因表达和基因组监测中发挥关键的调节作用。我们将结合生化和遗传学方法来剖析裂殖酵母中 CAK-CDK 网络的功能和靶点。通过引入每个 CDK 的基因工程版本，该版本易于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。相同的操作还使我们能够鉴定目标激酶的蛋白质底物。具体目标是： 1. 研究 Mcs6 (Cdk7) 和 Cdk9 之间的协调，它们依次作用并以协调一致地控制选定基因的表达。我们将调查是否招募 Cdk9 或转录延伸复合物的活性取决于 Mcs6 的先前功能。 2. 研究 mRNA 转录（合成）与其成熟的耦合含有 Cdk9 和必需 mRNA 加工酶（mRNA 5'-cap）的复合物甲基转移酶Pcm1。 3. 鉴定CAKs和CDKs在DNA损伤反应中的新靶点和功能。公共健康相关性：CAK-CDK 网络的整合功能在人类细胞中是保守的，但在癌症中可能会受到破坏，其中细胞增殖控制受损、基因表达紊乱和基因组损伤都会导致肿瘤的发生和进展。如果我们要阻断或抑制该途径的功能以阻止肿瘤细胞的生长和分裂，则需要了解其多种功能以避免对正常组织的毒性。裂殖酵母中可用的强大遗传工具，以及人类途径功能和组织的基本保守性，使粟酒裂殖酵母成为进行这项工作的理想模型系统。