CDK inhibitor regulation during the Xenopus cell cycle

描述(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什么不受控制地分裂至关重要。在许多癌细胞中，CDK抑制物等负性细胞周期调节因子不表达或以异常低的水平表达。已知的CIP/KIP类型的CDK抑制物受蛋白质周转调节，但它们的破坏如何在分子水平上与DNA复制启动或环境应激反应相协调仍不清楚。为了了解CDK抑制剂功能调控的潜在机制，我们以非洲爪蛙卵为材料，研究了CDK抑制剂蛋白水解酶与DNA复制启动的关系。我们的研究表明，被称为p27xicl(Xicl)的非洲爪哇属Cip/Kip型CDK抑制剂仅针对细胞核内的蛋白降解，其作用方式依赖于细胞周期、泛素化和蛋白酶体。我们最近的发现表明，Xic1蛋白降解在细胞周期中受到磷酸化和DNA复制检查点的调控。从机制上讲，我们已经证明了Xic1蛋白的降解严重依赖于与DNA复制机制的一个组成部分--增殖细胞核抗原(PCNA)的结合。这表明，在Xic1被泛素化和降解之前，它必须通过与增殖细胞核抗原的结合而被招募到起始位置。因此，我们的发现将Xic1的蛋白分解与DNA聚合酶的切换和DNA复制的开始联系在一起。在这些发现的基础上，我们假设Xic1的蛋白分解在细胞周期和细胞周期检查点受复制起始点的增殖细胞核抗原和DNA依赖的机制调控。我们的具体目标是：(1)阐明Xic1泛素化和降解的分子机制；(2)定义和表征在DNA损伤、DNA复制受阻和DNA修复后DNA合成重新开始过程中Xic1蛋白降解的调节；以及(3)研究Xic1磷酸化和去磷酸化如何在细胞周期中调节Xic1蛋白降解。

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.