Chen J, Saha P, Kornbluth S, Dynlacht B D, Dutta A
Department of Pathology, Division of Molecular Oncology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Cell Biol. 1996 Sep;16(9):4673-82. doi: 10.1128/MCB.16.9.4673.
The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex.
细胞周期蛋白依赖性激酶（Cdk）抑制剂p21由肿瘤抑制因子p53诱导产生，是DNA损伤细胞中G1-S期阻滞所必需的。我们报告称，p21中有两个细胞周期蛋白结合基序，即Cy1和Cy2，它们可独立于Cdk2与细胞周期蛋白相互作用。p21的细胞周期蛋白结合基序在体外对细胞周期蛋白-Cdk激酶的最佳抑制以及在体内对生长抑制都是必需的。仅包含Cy1或Cy2基序的肽在体外可部分抑制细胞周期蛋白-Cdk激酶活性，并抑制非洲爪蟾卵提取物中的DNA复制。一种识别p21的Cy1位点的单克隆抗体可特异性破坏细胞提取物中p21与细胞周期蛋白E-Cdk2以及细胞周期蛋白D1-Cdk4的结合。综上所述，这些观察结果表明，p21的细胞周期蛋白结合基序对于激酶抑制以及细胞中p21-细胞周期蛋白-Cdk复合物的形成很重要。最后，我们表明，如果仅与p21的细胞周期蛋白结合基序相关联，细胞周期蛋白-Cdk复合物会部分激活，这就解释了在体内如何发现p21与活性细胞周期蛋白-Cdk复合物相关联。Cy序列可能是Cdk抑制剂或底物用于在细胞周期蛋白-Cdk复合物中与细胞周期蛋白相互作用的通用基序。
