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Analysis of degradation of cell cycle regulators Kip family

细胞周期调节因子Kip家族的降解分析

基本信息

批准号：
16390082
负责人：
KAMURA Takumi
金额：
$ 9.54万
依托单位：
Nagoya University (2005)Kyushu University (2004)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16390082/
关键词：
Cell Cycle Protein Degradation Ubiquitin p27 KPC

项目摘要

The cyclin-dependent kinase (CDK) inhibitor p27 is degraded at the G(0)-G(1) transition of the cell cycle by the ubiquitin-proteasome pathway in a Skp2-independent manner. We recently identified a novel ubiquitin ligase, KPC (Kip1 ubiquitylation-promoting complex), consisting of KPC1 and KPC2, which regulates the ubiquitin-dependent degradation of p27 at G(1) phase. We have now investigated the structural requirements for the interactions of KPC1 with KPC2 and p27. The NH(2)-terminal region of KPC1 was found to be responsible for binding to KPC2 and to p27. KPC1 mutants that lack this region failed to mediate polyubiquitylation of p27 in vitro and expression of one such mutant delayed p27 degradation in vivo. We also generated a series of deletion mutants of p27 and found that KPC failed to polyubiquitylate a p27 mutant that lacks the CDK inhibitory domain. Interestingly, the cyclin E.CDK2 complex prevented both the interaction of KPC with p27 as well as KPC-mediated polyubiquitylation of p27. A complex of cyclin E with a kinase-negative mutant of CDK2 also exhibited these inhibitory effects, suggesting that cyclin E.CDK2 competes with KPC1 for access to the CDK inhibitory domain of p27. These results suggest that free p27 is recognized by the NH(2)-terminal region of KPC1, which also associates with KPC2, and that p27 is then polyubiquitylated by the COOH-terminal RING-finger domain of KPC1.

细胞周期蛋白依赖性激酶(CDK)抑制物p27在细胞周期G(0)-G(1)转变时被泛素-蛋白酶体途径以Skp2不依赖的方式降解。我们最近发现了一种新的泛素连接酶KPC(Kip1泛素化促进复合体)，它由KPC1和KPC2组成，调节泛素依赖的p27在G(1)期的降解。我们现在已经研究了KPC1与KPC2和p27相互作用的结构要求。KPC1的NH(2)末端区域负责与KPC2和p27结合。缺乏该区域的KPC1突变体在体外未能介导p27的多泛素化，其中一个突变体的表达在体内延迟了p27的降解。我们还产生了p27的一系列缺失突变体，并发现KPC未能使缺乏CDK抑制结构域的p27突变体多泛素化。有趣的是，Cyclin E.CDK2复合体既阻止了KPC与p27的相互作用，也阻止了KPC介导的p27的多泛素化。细胞周期蛋白E与激酶阴性突变体CDK2的复合体也表现出这些抑制作用，提示细胞周期蛋白E.CDK2与KPC1竞争进入p27的CDK抑制域。这些结果表明，游离的p27是由KPC1的NH(2)末端区域识别的，该区域也与KPC2结合，然后p27被KPC1的COOH末端的环指结构域多泛素化。