Regulation anf Function of Clp 1p Protein Phosphatase

Clp 1p 蛋白磷酸酶的调节和功能

• 批准号: 6675622
• 负责人: DANNEL MCCOLLUM
• 金额: $ 28.37万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-19 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6675622
• 关键词: cell cycle; cell growth regulation; cell nucleus; cyclin dependent kinase; enzyme activity; fungal proteins; gene mutation; genetic screening; molecular cloning; phenotype; phosphoprotein phosphatase; phosphorylation; protein localization; protein protein interaction; protein transport; site directed mutagenesis

DESCRIPTION (provided by applicant): Cyclin dependent kinases (Cdks) are the key regulators of cell cycle transitions in eukaryotic cells. Precise coordination of each cell cycle step with the others is essential for cells to correctly transmit an intact genome to each daughter cell. Failure to do so can lead to polyploidy, genomic instability and cancer. Recently work has shown that the Cdc 14-family of protein phosphatases may be important regulators of Cdk activity and play a crucial role in coordinating late mitotic events to maintain genomic stability. We have found that the fission yeast S. pombe Cdc 14 ortholog Clp 1p functions to block further rounds of cell cycle progression if cytokinesis is delayed. Clp 1p carries out this function by regulating Cdk activity and a conserved signaling pathway termed the SIN. Deletion of clp1 makes cells unable to restrain cell cycle progression if cytokinesis is delayed resulting in polyploid cells. The long-term goal of this project is to understand how Clp1p regulates Cdk activity, and how Clp1p is regulated to ensure that mitosis is properly coordinated with cytokinesis. My specific aims are: (1) To determine how release of Clp1p from the nucleus in early mitosis is regulated and how in late mitosis the SIN functions to keep Clp1p out of the nucleus, as well as the significance of this regulation for Clp1p function. (2) To characterize the role of Clp1p phosphorylation in regulating Clp1p activity and/or localization by (A) determining the effect of phosphorylation of on Clp1p phosphatase activity, (B) mapping and mutagenesis of Clp1p phosphorylation sites followed by characterization of the phenotype of Clp1p phosphorylation site mutants. (3) To identify (A) Clp1p interacting protein(s) at different cell cycle stages, as well as (B) Clp1p regulated processes using biochemical and genetic analysis. (4) To test the model that Clp1p regulates Cdk activity by antagonizing a self-activating mechanism by which Cdk1p promotes its own activity by phosphorylating and activating its activator Cdc25p and inhibiting its inhibitor Wee 1p.

描述(申请人提供)：细胞周期蛋白依赖性蛋白激酶(CDK)是真核细胞中细胞周期转变的关键调节因子。每个细胞周期步骤与其他步骤的精确协调对于细胞正确地将完整的基因组传递到每个子细胞是至关重要的。如果做不到这一点，可能会导致多倍体、基因组不稳定和癌症。最近的研究表明，CDC14蛋白磷酸酶家族可能是CDK活性的重要调节因子，并在协调晚期有丝分裂事件以维持基因组稳定方面发挥关键作用。我们已经发现，如果胞质分裂延迟，分裂酵母S.pombe CDC14同源CLP 1p可以阻止进一步的细胞周期进展。CLP-1P通过调节CDK活性和一条保守的信号通路SIN来实现这一功能。如果胞质分裂延迟导致多倍体细胞，CLP1的缺失使细胞不能抑制细胞周期的进展。这个项目的长期目标是了解Clp1p如何调节CDK活性，以及Clp1p是如何调节以确保有丝分裂与细胞质分裂适当协调的。我的具体目标是：(1)确定Clp1p在有丝分裂早期是如何从细胞核中释放出来的，在有丝分裂晚期SIN是如何将Clp1p排除在细胞核之外的，以及这种调节对Clp1p功能的意义。(2)通过(A)确定Clp1p磷酸化对Clp1p磷酸酶活性的影响，(B)Clp1p磷酸化位点的定位和突变，以及Clp1p磷酸化位点突变体的表型鉴定，来表征Clp1p磷酸化在调节Clp1p活性和/或定位中的作用。(3)通过生物化学和遗传学分析鉴定(A)Clp1p相互作用蛋白(S)，以及(B)Clp1p调控的过程。(4)验证Clp1p通过磷酸化并激活其激活剂CDc25p并抑制其抑制物Wee 1p而促进自身活性的自激活机制，从而验证Clp1p调节CDK活性的模型。