PROLIFERATIVE EFFECTS OF CDK4 AND CDK6 DYSREGULATION

描述：这项提议的总体目标是破译这一机制 CDK4/6的过表达可抑制P53和Rb介导的生长。在……里面 这种系统过度表达CDK4/6也导致了细胞的过度生产 细胞周期抑制物，p15，可能还有p16。通过以下方式了解该机制 P15的诱导发生是第一个特定的目标。私人侦探将 研究转录和转录后机制。此外, 不结合p15和p16的CDK4/6突变体将用于克隆 形成试验，以确定是否相互作用的激酶与 抑制剂对于推翻P53介导的生长抑制是必不可少的。这个 P21(p53的靶标)的作用也将通过p21缺失细胞来检测。 过表达温度敏感型突变体P53。在目标2中， CDK4/6与p15和p16的相关性也将作为 PRb被磷酸化从而失活的机制 这个系统。第三个特定目标将测试CDK4/6或突变体 不结合p15和p16的基因本身可以促进永生和 或啮齿动物细胞的转化。最后，PI将确定CDK4/6是否 P15/p16结合和水解三磷酸腺苷均有缺陷的突变体 可以作为显性的负癌基因发挥作用。这个实验将会有所帮助。 确定细胞周期中需要p16/p15功能的时间点 并且可以帮助识别衬底。

DESCRIPTION: The overall aim of this proposal is to decipher the mechanism by which overexpression of cdk4/6 overrides p53 and Rb mediated growth. In this system overexpression of cdk4/6 also leads to overproduction of the cell cycle inhibitors, p15 and possibly p16. Understanding the mechanism by which induction of p15 occurs is the first specific aim. The PI will examine transcriptional and post-transcriptional mechanisms. In addition, mutants of cdk4/6 that do not bind p15 and p16 will be used in colony formation assays to determine if the interaction of the kinases with the inhibitors is essential for overriding p53 mediated growth suppression. The role of p21 (a target of p53) will also be examined using p21 null cells overexpressing the temperature sensitive p53 mutant. In aim 2, the association of cdk4/6 with p15 and p16 will also be examined as the mechanism by which pRB becomes phosphorylated and thereby inactivated in this system. The third specific aim will test whether cdk4/6 or mutants that do not bind p15 and p16 can by themselves promote immortalization and or transformation of rodent cells. Lastly, the PI will determine if cdk4/6 mutants that are defective in both p15/p16 binding and in hydrolyzing ATP can function as dominant negative oncogenes. This experiment will help identify the point in the cell cycle at which p16/p15 function is required and may aid in identification of substrates.