Ferguson Michelle, Luciani M Gloria, Finlan Lee, Rankin Elaine M, Ibbotson Sally, Fersht Alan, Hupp Tedd R
Cancer Research UK Laboratories, Division of Pathology and Neurosciences, University of Dundee Dundee, Scotland, UK.
Cell Cycle. 2004 Jan;3(1):80-9.
Cyclin-dependent protein kinases play important roles in cell cycle progression and are attractive targets for the design of anti-proliferative drugs. Two distinct synthetic CDK1/2 inhibitors, Roscovitine and NU2058, are pharmacologically distinct in their ability to modify p53-dependent transcription and perturb cell cycle progression. Although such active-site CDK1/2 inhibitors comprise the most standard type of enzyme inhibitor, many protein kinases are proving to harbour high affinity docking sites that may provide a potentially novel interface for the design of kinase-inhibitors. We examined whether CDK2 has a docking site for its oligomeric substrate p53, whether small-peptide leads can be developed that inhibit CDK2 function, and whether such peptide-inhibitors are pharmacologically distinct from Roscovitine or NU2058. A docking site for CDK2 was identified in the tetramerization domain of p53 at a site that is distinct from the phospho-acceptor site. Peptides derived from the tetramerization domain of p53 block CDK2 phosphorylation and identification of critical CDK2 contacts in the tetramerization domain of p53 suggest that kinase docking does not require tetramerization of the substrate. Transient transfection assays were developed to show that the GFP-CDK2 docking site fusion protein (GFP-CIP) attenuates p53 activity in vivo and suppresses p21WAF1 induction which is similar to NU2058 but distinct from Roscovitine. A stable cell line with an inducible GFP-CIP gene attenuates p53 activity and induces significant cell death in a drug-resistant melanoma cell line, sensitizes cells to death induced by Doxorubicin, and suppresses cell growth in a colony formation assay. These data indicate that CDK2, in addition to cyclin A, can have a high affinity docking site for a substrate and highlights the possibility that CDK2 docking sites may represent effective targets for inhibitor design.
细胞周期蛋白依赖性蛋白激酶在细胞周期进程中发挥重要作用，是抗增殖药物设计的有吸引力的靶点。两种不同的合成CDK1/2抑制剂，Roscovitine和NU2058，在调节p53依赖性转录和扰乱细胞周期进程的能力上具有药理学差异。尽管这种活性位点CDK1/2抑制剂是最标准类型的酶抑制剂，但许多蛋白激酶被证明具有高亲和力对接位点，这可能为激酶抑制剂的设计提供一个潜在的新界面。我们研究了CDK2是否具有其寡聚底物p53的对接位点，是否可以开发抑制CDK2功能的小肽先导物，以及这种肽抑制剂在药理学上是否与Roscovitine或NU2058不同。在p53的四聚化结构域中确定了一个与磷酸化受体位点不同的CDK2对接位点。源自p53四聚化结构域的肽阻断CDK2磷酸化，并且在p53四聚化结构域中关键CDK2接触的鉴定表明激酶对接不需要底物的四聚化。开发了瞬时转染试验以表明绿色荧光蛋白 - CDK2对接位点融合蛋白（GFP - CIP）在体内减弱p53活性并抑制p21WAF1诱导，这与NU2058相似但与Roscovitine不同。具有可诱导GFP - CIP基因的稳定细胞系在耐药黑色素瘤细胞系中减弱p53活性并诱导显著的细胞死亡，使细胞对阿霉素诱导的死亡敏感，并在集落形成试验中抑制细胞生长。这些数据表明，除了细胞周期蛋白A之外，CDK2可以具有底物的高亲和力对接位点，并突出了CDK2对接位点可能代表抑制剂设计有效靶点的可能性。
