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，了解细胞周期进程的调节对于确定癌细胞为何不受抑制地分裂至关重要。在许多癌细胞中，细胞周期负调控因子如CDK抑制剂不表达或以异常低的水平表达。已知Cip/Kip型CDK抑制剂受蛋白质周转调节，但其破坏如何在分子水平上与DNA复制起始或环境应激反应事件协调仍不清楚。为了了解调节CDK抑制剂功能的潜在机制，我们使用青蛙卵，非洲爪蟾，研究CDK抑制剂蛋白水解和DNA复制起始之间的关系。我们的研究表明，非洲爪蟾Cip/Kip型CDK抑制剂称为p27 xicl（Xicl）的蛋白水解的目标，只有在细胞核中，并在一定程度上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，Xic 1蛋白水解调节在细胞周期中的磷酸化和DNA复制检查点。从机制上讲，我们已经证明，Xic 1蛋白水解是严重依赖于结合到增殖细胞核抗原（PCNA），DNA复制机制的一个组成部分。 这表明Xic 1必须通过与PCNA结合被募集到起始位点，然后才能被靶向泛素化和降解。因此，我们的研究结果将Xic 1的蛋白水解与DNA聚合酶转换和进行性DNA复制的发生联系起来。基于这些研究结果，我们假设Xic 1蛋白水解在细胞周期和细胞周期检查点期间由PCNA和DNA依赖性机制在复制起始位点调节。我们的具体目标是：（1）阐明Xic 1泛素化和降解的分子机制;（2）定义和表征在DNA损伤、DNA复制阻断和DNA修复后DNA合成重新启动过程中Xic 1蛋白水解的调节;（3）表征在细胞周期中Xic 1磷酸化和去磷酸化如何调节Xic 1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.