Investigation into the molecular mechanisms underlying proteolysis of CDK inhibitor p27

我们已经研究了CDK抑制剂p27丰度调控的机制。p27在GO-G1过渡过程中被降解，在细胞增殖过程中其丰度仍然很低。SCF/Skp2泛素连接酶复合物被认为在p27降解中起关键作用。然而，对Skp2缺陷小鼠的分析显示，在Skp2-/-细胞中，GO-G1过渡时p27的降解正常进行，而在这些Skp2缺陷细胞中，S-G2期的p27蛋白水解受损。这些结果表明，在GO-G1过渡过程中，存在不依赖skp2的途径来控制p27的泛素化。利用体外泛素化实验，我们生化纯化了一个由两个蛋白组成的复合物，命名为KPC （Kip1泛素化促进复合物）1和KPC2。鉴于KPC1/2定位于细胞质中，p27的核输出似乎先于KPC1/2的泛素化。在哺乳动物细胞中，野生型KPC1/2的过表达促进了p27的降解，而显性阴性突变型KPC1/2的表达延缓了p27的降解。通过RNA干扰减少KPC1也抑制了p27的降解。这些数据表明，kpc1 /2泛素连接酶复合物在GO-G1过渡阶段控制p27的降解，而Skp2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。

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.