Cyclin-dependent kinase inhibitor regulation during the Xenopus cell cycle

描述（由申请人提供）：我们的长期目标是了解细胞周期蛋白依赖性激酶（CDK）抑制剂如何调节脊椎动物的细胞分裂。细胞分裂是生物体中最基本的过程之一，理解细胞周期进程的调节对于确定为什么癌细胞不受控制地分裂至关重要。在许多癌细胞中，负细胞周期调节因子如CDK抑制剂不表达或表达异常低水平。已知Cip/ kip型CDK抑制剂受蛋白质周转调节，但它们的破坏如何在分子水平上与DNA复制起始事件或环境应激反应协调尚不清楚。为了了解CDK抑制剂功能的潜在调节机制，我们利用非洲爪蟾（Xenopus laevis）蛙卵研究了CDK抑制剂蛋白水解与DNA复制起始之间的关系。我们的研究表明，Xenopus Cip/ kip型CDK抑制剂p27 xicl （xicl）仅在细胞核中靶向蛋白水解，其方式依赖于细胞周期阶段、泛素化和蛋白酶体。我们最近的研究结果表明，Xic1蛋白水解在细胞周期中通过磷酸化和DNA复制检查点受到调节。从机制上讲，我们已经证明Xic1蛋白水解严重依赖于与增殖细胞核抗原（PCNA）的结合，PCNA是DNA复制机制的一个组成部分。这表明Xic1必须通过与PCNA的结合被招募到起始位点，才能成为泛素化和降解的目标。因此，我们的研究结果将Xic1的蛋白水解与DNA聚合酶转换和DNA复制过程的开始联系起来。基于这些发现，我们假设Xic1蛋白水解在细胞周期和细胞周期检查点受到PCNA和DNA依赖机制在复制起始位点的调节。我们的具体目标是：(1)阐明Xic1泛素化和降解的分子机制；(2)定义和表征Xic1蛋白水解在DNA损伤响应、DNA复制阻断和DNA修复后DNA合成重启过程中的调控；(3)表征细胞周期中Xic1磷酸化和去磷酸化如何调控Xic1蛋白水解。

DESCRIPTION (provided by applicant): Our long-term goal is to understand how cyclin-dependent kinase (CDK) inhibitors regulate cell division in the vertebrate. Cell division is one of the most fundamental processes in living organisms and understanding the regulation of cell cycle progression is critical for determining why cancer cells divide unchecked. In many cancer cells, negative cell cycle regulators such as CDK inhibitors are not expressed or are expressed at abnormally low levels. CDK inhibitors of the Cip/Kip-type are known to be regulated by protein turnover, but how their destruction is coordinated at the molecular level with the events of DNA replication initiation or environmental stress responses is still unclear. To understand the underlying mechanisms regulating CDK inhibitor function, we use eggs from the frog, Xenopus laevis, to study the relationship between CDK inhibitor proteolysis and DNA replication initiation. Our studies have indicated that the Xenopus Cip/Kip-type CDK inhibitor called p27 xicl (Xicl) is targeted for proteolysis only in the nucleus and in a manner dependent upon cell cycle phase, ubiquitination, and the proteasome. Our recent findings suggest that Xic1 proteolysis is regulated during the cell cycle by phosphorylation and during a DNA replication checkpoint. Mechanistically, we have demonstrated that Xic1 proteolysis is critically dependent upon binding to Proliferating Cell Nuclear Antigen (PCNA), a component of the DNA replication machinery. This suggests that Xic1 must be recruited to a site of initiation through its binding to PCNA before it is targeted for ubiquitination and degradation. Our findings thus link the proteolysis of Xic1 to DNA polymerase switching and the onset of processive DNA replication. On the basis of these findings, we hypothesize that Xic1 proteolysis is regulated during the cell cycle and during a cell cycle checkpoint by a PCNA- and DNA dependent mechanism at sites of replication initiation. Our specific aims are: (1) To elucidate the molecular mechanism of Xic1 ubiquitination and degradation; (2) To define and characterize the regulation of Xic1 proteolysis during a response to DNA damage, a block to DNA replication, and the restart of DNA synthesis following DNA repair; and (3) To characterize how Xic1 phosphorylation and dephosphorylation regulates Xic1 proteolysis during the cell cycle.