PROLIFERATIVE EFFECTS OF CDK4 AND CDK6 DYSREGULATION

描述：本提案的总体目标是破译该机制 cdk 4/6的过度表达通过这种方式克服p53和Rb介导的生长。 在 该系统中CDK 4/6的过度表达也导致了 细胞周期抑制剂，p15和可能的p16。 通过以下方式了解机制 p15的诱导发生是第一个具体目标。 PI将 研究转录和转录后机制。 此外，本发明还提供了一种方法， 将不结合p15和p16的CDK 4/6突变体用于菌落中 形成测定，以确定激酶与蛋白质的相互作用， 抑制剂对于克服p53介导的生长抑制是必需的。 的 p21（p53的靶点）的作用也将使用p21空细胞来检查 过表达温度敏感性p53突变体。 在目标2中， cdk 4/6与p15和p16的关联也将被检查， pRB磷酸化从而失活的机制， 这系统. 第三个具体目标将测试cdk 4/6或突变体是否 不结合p15和p16的蛋白本身可以促进永生化， 或转化啮齿动物细胞。 最后，PI将确定cdk 4/6是否 在p15/p16结合和水解ATP方面都有缺陷的突变体 可以作为显性负性癌基因发挥作用。 这个实验将有助于 确定细胞周期中需要p16/p15功能的点 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.