Moskowitz N K, Borao F J, Dardashti O, Cohen H D, Germino F J
University of Medicine and Dentistry of New Jersey, Cancer Institute of New Jersey, New Brunswick 08901, USA.
Oncol Res. 1996;8(9):343-52.
The cyclin-dependent kinase (Cdk) inhibitor known as p21, which is transcriptionally regulated by p53, can induce G1 arrest when overexpressed and inhibit the kinase activity of a wide variety of cyclin-Cdk complexes. Previous studies have demonstrated that a portion of the conserved region of p21 (amino acids 46-78), which is homologous to similar regions in the related Cdk inhibitors p27 and p57, can bind to Cdk2, and that this region is essential for kinase inhibition. However, the site(s) on Cdk2 that are involved in p21 binding have not been identified. We therefore created mutant Cdk2 molecules with various N-terminal and C-terminal deletions and tested each for their ability to bind to p21 by the yeast two-hybrid and the double-tagging assays. None of the deletion mutants tested bound to p21 by either assay. We next tested whether p21 could bind to Cdk7, a component of the cyclin-activating kinase complex. By both the double-tagging and yeast two-hybrid assays, p21 failed to bind to this protein, consistent with previous reports. However, hybrid molecules consisting of the amino-terminal half of Cdk2 and the carboxy-terminal half of Cdk7 (Cdk2/Cdk7) could bind to p21 by both assays, whereas the Cdk7/Cdk2 hybrids could not. Furthermore, the yeast Cdc28 protein, which is 65% identical with Cdk2, failed to bind to p21 by both the yeast two-hybrid and double-tagging assays. Cdk2/Cdc28 hybrids but not Cdc28/Cdk2 hybrids could bind to p21. These results suggest that the amino-terminal half of Cdk2 is important for p21 binding, consistent with the recently published crystal-lographic data. Our data also suggest that the three-dimensional structure of Cdk2 is likely altered by creating deletion mutants from either the amino- or carboxy-terminal end of the protein. Finally, we have mutated the Cdc28/Cdk2 hybrid protein and isolated several mutants, which are able to bind to p21. This approach may be useful for identifying residues in Cdk2 and Cdc28 that affect their ability to bind to p21 and complement the crystallographic data.
细胞周期蛋白依赖性激酶（Cdk）抑制剂p21受p53转录调控，过表达时可诱导G1期阻滞，并抑制多种细胞周期蛋白-Cdk复合物的激酶活性。以往研究表明，p21保守区域的一部分（氨基酸46 - 78）与相关Cdk抑制剂p27和p57的类似区域同源，可与Cdk2结合，且该区域对激酶抑制至关重要。然而，尚未确定Cdk2上参与p21结合的位点。因此，我们构建了具有不同N端和C端缺失的突变型Cdk2分子，并通过酵母双杂交和双标签分析测试了它们与p21结合的能力。所测试的缺失突变体均未通过任何一种分析与p21结合。接下来，我们测试了p21是否能与细胞周期蛋白激活激酶复合物的一个组分Cdk7结合。通过双标签分析和酵母双杂交分析，p21均未能与该蛋白结合，这与之前的报道一致。然而，由Cdk2的氨基端一半和Cdk7的羧基端一半组成的杂合分子（Cdk2/Cdk7）在两种分析中均能与p21结合，而Cdk7/Cdk2杂合分子则不能。此外，与Cdk2有65%同源性的酵母Cdc28蛋白在酵母双杂交和双标签分析中均未能与p21结合。Cdk2/Cdc28杂合分子而非Cdc28/Cdk2杂合分子能与p21结合。这些结果表明，Cdk2的氨基端一半对p21结合很重要，这与最近发表的晶体学数据一致。我们的数据还表明，从蛋白的氨基端或羧基端创建缺失突变体可能会改变Cdk2的三维结构。最后，我们对Cdc28/Cdk2杂合蛋白进行了突变，并分离出了几个能够与p21结合的突变体。这种方法可能有助于确定Cdk2和Cdc28中影响它们与p21结合能力的残基，并补充晶体学数据。
