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Investigation into the molecular mechanisms underlying proteolysis of CDK inhibitor p27

CDK抑制剂p27蛋白水解的分子机制研究

基本信息

批准号：
12213097
负责人：
NAKAYAMA Keiichi
金额：
$ 35.97万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12213097/
关键词：
Cell Cycle CDK inhibitor p27 ubiquitin SCF complex Skp2 KPC タンパク分解ユビキチンリガーゼ CRM1 ユビキチン化 G0-G1移行期ノックアウトマウス RING finger サイクリンE p27Kip1

项目摘要

We have studied the mechamism underlying the regulation of the abundance of the CDK inhibitor p27. p27 is degraded at the GO-G1 transition, and its abundance remains low during cell proliferation. The SCF/Skp2 ubiquitin ligase complex has been thought to play a critical role in p27 degradation. However, analysis of Skp2-deficient mice revealed that degradation of p27 at the GO-G1 transition proceeds normally in Skp2-/-cells, whereas p27 proteolysis during S-G2 phases is impaired in these Skp2-deficient cells. These results suggested the presence of Skp2-independent pathway to control ubiquitination of p27 at the GO-G1 transition. Using in vitro ubiquitylation assay, we biochemically purified a complex consisting of two proteins, designated KPC (Kip1 ubiquitylation Promoting Complex)1 and KPC2. Given that KPC1/2 are localized in the cytoplasm, nuclear export of p27 seems to precede the ubiquitination by KPC1/2. Overexpression of wild-type KPC1/2 in mammalian cells promotes the degradation of p27, whereas expression of dominant-negative mutant KPC 1/2 delayed the degradation. Depletion of KPC1 by RNA interference also inhibited p27 degradation. These data suggest that KPC 1/2 ubiquitin ligase complex controls the degradation of p27 at the GO-G1 transition, whereas the major function of Skp2 may be the regulation of progression from G2 to M phase by mediating the degradation of p27.

我们研究了CDK抑制剂p27丰度调节的潜在机制。p27在GO-G1转换时降解，并且其丰度在细胞增殖期间保持较低。SCF/Skp 2泛素连接酶复合物被认为在p27降解中起关键作用。然而，对Skp 2缺陷小鼠的分析显示，在Skp 2-/-细胞中，GO-G1转换时p27的降解正常进行，而在这些Skp 2缺陷细胞中，S-G2期期间p27蛋白水解受损。这些结果表明存在Skp 2非依赖性途径来控制p27在GO-G1转换时的泛素化。使用体外泛素化测定，我们生化纯化的复合物由两种蛋白质，命名为KPC（Kip 1泛素化促进复合物）1和KPC 2。鉴于KPC 1/2定位于细胞质中，p27的核输出似乎先于KPC 1/2的泛素化。野生型KPC 1/2在哺乳动物细胞中的过表达促进p27的降解，而显性阴性突变体KPC 1/2的表达延迟了降解。通过RNA干扰去除KPC 1也抑制p27降解。这些数据表明，KPC 1/2泛素连接酶复合物控制p27在GO-G1转换时的降解，而Skp 2的主要功能可能是通过介导p27的降解来调节从G2到M期的进展。