This proposal focuses on the cyclin-dependent protein kinases or CDKs, a family of enzymes that controls the timing and coordination of eukaryotic cell cycle events. The structure and function of these proteins will be analyzed by several approaches, in the hope that a better understanding of these important enzymes will lead to improved knowledge of cell cycle control in both normal and neoplastic cells. Transient increases in CDK activity at specific cell cycle stages are believed to initiate key cell cycle events. The molecular mechanisms underlying these surges in CDK activity are remarkably complex: the activity of the CDK catalytic subunit is controlled by a variety of positive regulatory subunits (cyclins) and by phosphorylations at stimulatory and inhibitory sites. Experiments described in the first part of this proposal will provide a detailed understanding of the molecular mechanisms by which cyclins and phosphorylation control CDK activity. The proposed experiments are based on our recent determination of the crystal structure of human cyclin-dependent kinase-2 (CDK2), which was carried out as a collaboration with S.-H. Kim of U.C.Berkeley. The first specific aim of this proposal is to continue our collaboration to determine the crystal structure of cyclins and CDK/cyclin complexes in various states of phosphorylation. Second, we will use the information already obtained from the CDK2 structure to design and construct mutant CDK2 proteins that will reveal the role of specific residues and regions in CDK2 activity, substrate binding, and regulation by other subunits and regulatory molecules. Third, we will use a recently developed inducible promoter system to express various dominant negative and dominant positive CDK2 mutants in mammalian cells, in order to gain insight into the biological function of CDK2. Finally, homologous recombination methods will he employed to develop a cell line in which both CDK2 alleles are disrupted. This cell line will also contain a plasmid carrying the CDK2 cDNA under the control of a repressible promoter. By repressing the expression of CDK2 in this cell, we will be able to assess the role of CDK2 in cell cycle control. Our long term goal in these studies will he to use the information gained in these various structural and functional studies to design mutant proteins or other molecules that inhibit CDK2 activity or inhibit its interactions with regulatory proteins. These studies may eventually lead to the design of drugs that specifically inhibit CDK action (and therefore cell division) in cancer cells.
