Yam C H, Ng R W, Siu W Y, Lau A W, Poon R Y
Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
Mol Cell Biol. 1999 Jan;19(1):635-45. doi: 10.1128/MCB.19.1.635.
Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21(Cip1/WAF1) bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G1/S cell cycle arrest.
细胞周期蛋白A-Cdk2复合物在S期与Skp1和Skp2结合，但Skp1和Skp2的功能尚不清楚。Skp1与Skp2等F-box蛋白一起，是泛素连接酶E3复合物的一部分，该复合物将许多细胞周期调节因子作为泛素化介导的蛋白水解的靶点。在本研究中，我们研究了Skp1和Skp2对细胞周期蛋白A-Cdk2活性的潜在调节作用。我们发现Skp2在体外可抑制细胞周期蛋白A-Cdk2的激酶活性，其方式包括直接抑制细胞周期蛋白A-Cdk2以及抑制细胞周期蛋白依赖性激酶（CDK）激活激酶磷酸化对Cdk2的激活作用。Skp2仅降低Cdk2的激酶活性，而不降低Cdc2或Cdk5的激酶活性。Skp2在丝氨酸76位点被细胞周期蛋白A-Cdk2磷酸化，但Skp2的非磷酸化突变体仍可抑制细胞周期蛋白A-Cdk2对组蛋白H1的激酶活性。Skp2的F盒是其与Skp1结合所必需的，Skp2的N端和C端区域均参与与细胞周期蛋白A-Cdk2的结合。此外，Skp2与CDK抑制剂p21（Cip1/WAF1）以互斥方式结合细胞周期蛋白A-Cdk2。在哺乳动物细胞中过表达Skp而不是Skp1会导致G1/S期细胞周期停滞。
