Critical role for the 310 helix region of p57Kip2 in cyclin-dependent kinase 2 inhibition and growth suppression
Critical role for the 310 helix region of p57Kip2 in cyclin-dependent kinase 2 inhibition and growth suppression
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DOI：
10.1074/jbc.273.26.16544
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发表时间：
1998-06-26
影响因子：
4.8
通讯作者：
Nakanishi, M
中科院分区：
文献类型：
--
作者：
Hashimoto, Y;Kohri, K;Nakanishi, M
虽然细胞周期蛋白A/细胞周期蛋白依赖性激酶2（Cdk 2）/p27的晶体结构分析（Russo，A.一、杰弗里，警察，模式，A. K.，Massague，J.，和Pavletich，N. P.（1996）Nature 382，325-331）已经提出Cip/Kip家族的Cdk抑制剂中的3（10）螺旋区可能参与细胞周期蛋白/Cdk活性的抑制，但没有生物化学证据支持该假设。在本研究中，我们证明了p57的细胞周期蛋白和Cdk结合结构域是必要的，但本身并不足以抑制细胞周期蛋白A/Cdk 2和细胞周期蛋白E/Cdk 2，并且该蛋白的3（10）螺旋区域对于抑制这些复合物是不可缺少的。相反，p21和p27的3（10）螺旋区是不需要的，和细胞周期蛋白和Cdk结合域单独足以抑制所有细胞周期蛋白/Cdk复合物检查。定点诱变鉴定了p57的3（10）螺旋区域内的苯丙氨酸79和酪氨酸80作为激酶抑制的关键残基，支持了3（10）螺旋结合Cdk 2催化裂缝深处的结构证据，模拟ATP。p57分子的3（10）螺旋区域内的突变完全消除了在体内将细胞周期阻滞在G（1）的能力。这些结果表明，该区域特异性地被p57用于选择性地抑制细胞周期蛋白A或E/Cdk 2活性。因此，3（10）螺旋基序可能赋予一个特定的调节机制，p57差异调节Cdk 2和Cdk 4的活动。
Although crystal structural analysis of cyclin A/cyclin-dependent kinase 2 (Cdk2)/p27 (Russo, A. A., Jeffrey, P. D., Pattern, A. K., Massague, J., and Pavletich, N. P. (1996) Nature 382, 325-331) has suggested that the 3(10) helix region in Cdk inhibitors of the Cip/Kip family may be involved in the inhibition of cyclin/Cdk activities, there is no biochemical evidence supporting this hypothesis. In the present study, we demonstrated that cyclin and Cdk binding domains of p57 were necessary but were not sufficient in themselves for the inhibition of cyclin A/Cdk2 and cyclin E/Cdk2, and that the 3(10) helix region of this protein is indispensable for the inhibition of these complexes. In contrast, the 3(10) helix regions of p21 and p27 were not required, and cyclin- and Cdk-binding domains alone were sufficient for the inhibition of all cyclin/Cdk complexes examined. Site-directed mutagenesis identified phenylalanine 79 and tyrosine 80 within the 3(10) helix region of p57 as crucial residues for kinase inhibition, supporting the structural evidence that the 3(10) helix binds deep inside the catalytic cleft of Cdk2, mimicking ATP. Mutations within the 3(10) helix region of the p57 molecule completely abolished the ability to arrest the cell cycle at G(1) in vivo. These results indicate that this region is specifically utilized by p57 in selectively inhibiting cyclin A or E/Cdk2 activities. Thus the 3(10) helix motif may confer a specific regulatory mechanism by which p57 differentially regulates Cdk2 and Cdk4 activities.