Rank K B, Evans D B, Sharma S K
Protein Science, Pharmacia and Upjohn, Michigan, 49007, Kalamazoo.
Biochem Biophys Res Commun. 2000 May 10;271(2):469-73. doi: 10.1006/bbrc.2000.2648.
It has been suggested that binding of p27 and p21 kinase inhibitory proteins (KIPs) to cyclin-dependent kinases (cdks) render them inaccessible to cdk-activating kinase (CAK), presumably by steric hindrance by the C-terminal residues. However, this common mechanism of inhibition is inconsistent with the known structural divergence in the p27 and p21 C-terminal domains. Therefore, we studied the direct binding of N-terminal minimal domain of p27 (amino acids 28-81) to cdk2/cyclin E. An unlabeled p27 minimal domain, mutated in the N-terminal LFG motif, was unable to compete with a labeled minimal domain for binding to cdk2/cyclin E. The p27 and its minimal domain inhibited CAK-mediated phosphorylation of cdk2/cyclin E. This inhibitory effect was significantly diminished with p27 minimal domain mutated in the LFG motif. A synthetic peptide, ACRRLFGPVDSE, from the N-terminal residues 17-28 of p21, was also a potent inhibitor of CAK-mediated cdk2/cyclin E phosphorylation. Taken together, these results show that anchoring of p27 or p21 KIPs to cyclin E via the N-terminal LFG-containing motif can block CAK access to its cdk2/cyclin E substrate.
有人提出，p27和p21激酶抑制蛋白（KIPs）与细胞周期蛋白依赖性激酶（cdks）结合后，可能由于C末端残基的空间位阻，使细胞周期蛋白依赖性激酶激活激酶（CAK）无法接近它们。然而，这种常见的抑制机制与p27和p21 C末端结构域已知的结构差异不一致。因此，我们研究了p27的N末端最小结构域（氨基酸28 - 81）与cdk2/细胞周期蛋白E的直接结合。在N末端LFG基序中发生突变的未标记p27最小结构域，无法与标记的最小结构域竞争结合cdk2/细胞周期蛋白E。p27及其最小结构域抑制了CAK介导的cdk2/细胞周期蛋白E的磷酸化。在LFG基序中发生突变的p27最小结构域，这种抑制作用显著减弱。来自p21 N末端残基17 - 28的合成肽ACRRLFGPVDSE，也是CAK介导的cdk2/细胞周期蛋白E磷酸化的有效抑制剂。综上所述，这些结果表明，p27或p21 KIPs通过含N末端LFG的基序锚定到细胞周期蛋白E上，可以阻止CAK接近其cdk2/细胞周期蛋白E底物。
