Analysis of degradation of cell cycle regulators Kip family

周期蛋白依赖性激酶（CDK）抑制剂p27在细胞周期的G(0)-G(1)过渡阶段通过泛素-蛋白酶体途径以不依赖skp2的方式降解。我们最近发现了一种新的泛素连接酶，KPC （Kip1泛素化促进复合物），由KPC1和KPC2组成，它在G(1)期调节p27的泛素依赖性降解。我们现在研究了KPC1与KPC2和p27相互作用的结构要求。发现KPC1的NH(2)末端区域负责与KPC2和p27结合。缺少该区域的KPC1突变体在体外不能介导p27的多泛素化，而其中一个突变体的表达延迟了p27在体内的降解。我们还生成了一系列p27的缺失突变体，并发现KPC未能多聚修饰缺乏CDK抑制结构域的p27突变体。有趣的是，细胞周期蛋白E.CDK2复合物既阻止了KPC与p27的相互作用，也阻止了KPC介导的p27多泛素化。细胞周期蛋白E与CDK2激酶阴性突变体的复合物也表现出这些抑制作用，这表明细胞周期蛋白E.CDK2与KPC1竞争进入p27的CDK抑制结构域。这些结果表明，游离p27可被KPC1的NH(2)末端区域识别，该区域也与KPC2结合，然后p27被KPC1的cooh末端RING-finger结构域多泛素化。

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.