Fotedar R, Fitzgerald P, Rousselle T, Cannella D, Dorée M, Messier H, Fotedar A
Institut de Biologie Structurale J-P Ebel, Grenoble, France.
Oncogene. 1996 May 16;12(10):2155-64.
Cyclin dependent kinases regulate the progression of eukaryotic cells through the cell cycle. p21Cip1/Waf1/Sdi1 is an inhibitor of cdk-cyclin kinase activity, and has been shown to form complexes with cdk-cyclins and with PCNA, an accessory protein of DNA polymerase delta. The kinase inhibitory domain maps to the N-terminus (1-82) and contains the cdk2 binding site (28-82). We have generated a panel of deletion mutants of p21. A functional characterization of p21 mutants in the N-terminal domain reveals that cyclins bind to this domain independently of cdk2. Correlating with these results we find that p21 can associate with cyclin-cdk kinases in two functionally distinct forms, one in which the kinase activity is inhibited and the other in which the kinase is still active. The cdk2 and cyclin binding sites on p21 are both required to inhibit kinase activity. The second type of interaction, in which an active cyclin-cdk complex only interacts with p21 either via the cyclin or the cdk2 binding site but not through both, does not lead to inhibition of cyclin kinase activity. These results thus provide a basis for understanding the mechanism by which p21, and perhaps other cdk-cyclin kinase inhibitory proteins, suppress kinase activity.
细胞周期蛋白依赖性激酶调控真核细胞通过细胞周期的进程。p21Cip1/Waf1/Sdi1是一种细胞周期蛋白依赖性激酶活性的抑制剂，已被证明能与细胞周期蛋白依赖性激酶以及增殖细胞核抗原（DNA聚合酶δ的一种辅助蛋白）形成复合物。激酶抑制结构域定位于N端（1 - 82），并包含cdk2结合位点（28 - 82）。我们构建了一组p21的缺失突变体。对p21 N端结构域突变体的功能表征显示，细胞周期蛋白与该结构域的结合不依赖于cdk2。与这些结果相关的是，我们发现p21能以两种功能不同的形式与细胞周期蛋白 - cdk激酶结合，一种形式中激酶活性被抑制，另一种形式中激酶仍具有活性。p21上的cdk2和细胞周期蛋白结合位点对于抑制激酶活性都是必需的。第二种相互作用类型，即活性细胞周期蛋白 - cdk复合物仅通过细胞周期蛋白或cdk2结合位点与p21相互作用，但不是同时通过两者，不会导致细胞周期蛋白激酶活性的抑制。因此，这些结果为理解p21以及可能其他细胞周期蛋白依赖性激酶抑制蛋白抑制激酶活性的机制提供了基础。
