Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1
Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1
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DOI：
10.1021/bi001524e
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发表时间：
2000-11-14
期刊：
影响因子：
2.9
通讯作者：
Lumb, KJ
中科院分区：
文献类型：
--
作者：
Adkins, JN;Lumb, KJ
真核细胞周期的进展受磷酸化的调节，而磷酸化是由细胞周期蛋白依赖的蛋白激酶催化的。细胞周期蛋白依赖性激酶通过多种机制进行调节，包括p21的负性调节(CAP20、Cip1、Sdi1和WAF1)。已经提出需要多个p21分子来抑制细胞周期蛋白依赖性激酶，这样p21就可以作为细胞周期蛋白依赖性蛋白激酶活性的敏感缓冲区，或者作为细胞周期蛋白和细胞周期蛋白依赖性蛋白激酶形成的复合体的组装因子。使用纯化的全长已知浓度的蛋白质(由吸光度确定)和已知活性的细胞周期蛋白A-CDK2(用星形孢子素校准)，我们发现p21和细胞周期蛋白A-CDK2的摩尔比为1：1，在二元细胞周期蛋白A-CDK2复合体中和在增殖细胞核抗原(PCNA)存在的情况下都能抑制CDK2的活性。我们的结果表明，p21抑制细胞周期蛋白A-CDK2的机制不涉及多个结合的p21分子。
Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.