REGULATION OF P27 AND ITS ROLE IN G1 PROGRESSION

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DESCRIPTION: This is a resubmission of an application to study the CDK inhibitor p27. p27 was originally identified and characterized by the PI while a post-doctoral fellow. This grant is based on the analysis of three models that might explain the normal role of p27. In one model, p27 levels provide a threshold of inhibitor in G1. To activate a G1 CDK, the levels of cyclin-CDK complexes must increase above the threshold of p27 in the cells. In the second model p27 is a scaffold on which cyclin and CDK polypeptides are assembled. The third model suggests that different specificities of p27 binding are achieved by various post-translational modifications. This application proposes to test various aspects of these models. In the first aim, the PI will test the properties of the threshold model. The timing of p27 protein and RNA expression will be studied in synchronized cell populations. Discrepancies of RNA and protein expression will be followed by determining the rates of synthesis and degradation of p27. Next, the regulation of association of p27 with cyclin-CDK complexes will be determined by analyzing timing of association, relative levels of p27 versus the cyclin-CDK proteins, and changes in post-translational modification. Then the regulation of p27 associated kinase activity will be determined. The kinases that account for this activity, the timing of activity versus assembly, and the range of substrates for p27 will be determined. Using this characterization for comparison, the second specific aim will analyze how p27 changes in different growth and differentiation systems. The PI proposes to study differences in protein synthesis and degradation rates, if p27-associated kinases still exist in non-proliferating cells, and whether p27 properties change in direct response to differentiating agents. Next analogous differentiation and growth arrest procedures will be performed with p27- null ES cells to determine if p27 is required for these changes. In the final aim, the PI will analyze cells for proteins that associate with p27 by immunoprecipitation and two-hybrid analysis. Previous work has shown that p27 can be isolated in a large molecular weight complex. The characterization of this complex will be done in both proliferating and differentiating cells.