Molecular dissection of the interaction between p27 and Kip1 ubiquitylation-promoting complex, the ubiquitin ligase that regulates proteolysis of p27 in G1 phase
Molecular dissection of the interaction between p27 and Kip1 ubiquitylation-promoting complex, the ubiquitin ligase that regulates proteolysis of p27 in G1 phase
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DOI：
10.1074/jbc.m500866200
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发表时间：
2005-05-06
影响因子：
4.8
通讯作者：
Nakayama, KI
中科院分区：
文献类型：
--
作者：
Kotoshiba, S;Kamura, T;Nakayama, KI
细胞周期蛋白依赖性激酶（CDK）抑制剂p27在细胞周期的G（0）-G（1）转换时通过泛素-蛋白酶体途径以Skp 2非依赖性方式降解。我们最近发现了一种新的泛素连接酶KPC（Kip 1 ubiquitylation-promoting complex），它由KPC 1和KPC 2组成，调节G（1）期p27的泛素依赖性降解。我们现在已经研究了KPC 1与KPC 2和p27相互作用的结构要求。发现KPC 1的NH 2-末端区域负责与KPC 2和p27结合。缺乏该区域的KPC 1突变体未能在体外介导p27的多聚遍在化，并且一种此类突变体的表达延迟了体内p27的降解。我们还产生了一系列的p27缺失突变体，并发现KPC未能polyubiquitylate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复合物阻止了KPC与p27的相互作用以及KPC介导的p27的多泛素化。细胞周期蛋白E与CDK 2的激酶阴性突变体的复合物也表现出这些抑制作用，表明细胞周期蛋白E.CDK2与KPC 1竞争进入p27的CDK抑制结构域。这些结果表明，游离p27被KPC 1的NH 2-末端区域识别，该区域也与KPC 2相关，然后p27被KPC 1的COOH-末端环指结构域聚泛素化。
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 NH2-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 NH2-terminal region of KPC1, which also associates with KPC2, and that p27 is then polyubiquitylated by the COOH-terminal RING-finger domain of KPC1.