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.
