In the last funding period we obtained important findings suggesting that each of the four important proteins gankyrin, IkappaBalpha, Tax, and SEI-1 interacts with cyclin-dependent kinase 4 (CDK4), with potentially different mechanisms. The main thrust of the next granting period is to understand the structural and biochemical mechanisms and biological functions of the interactions between CDK4 and three constitutively expressed human proteins, gankyrin, IkappaBalpha, and SEI-1, while the viral protein Tax is not included in this revised proposal. Three major approaches will be used: (a) use of NMR to determine solution structures; (b) use of rational mutagenesis combined with activity assays to dissect the structural basis of specificity; (c) use of fluorescence-based binding assay to determine the Kd of gankyrin, IkappaBalpha, and SEI-1 with CDK4. In Specific Aim 1, we plan to use site-directed mutagenesis to identify key residues of gankyrin important for its binding to CDK4, and the Kd values of gankyrin and mutants will be evaluated using a fluorescence-based assay. Subsequently, a set of CDK4 mutants will be generated through rational design and will be used to map out the residues of CDK4 involved in the binding of gankyrin. Furthermore, a gankyrin/CDK4 structural model will be generated using both NMR and mutagenesis data, hi collaboration with C. Weghorst, we will characterize gankyrin mutants identified from cancer tissues. In Specific Aim 2, we plan to solve the solution structure of the CDK4-binding domain of IkappaBalpha (residues 1-206), which is also an ankyrin-repeat protein. The same mutagenesis studies as described for gankyrin-CDK4 interactions will be used to map out the interaction between IkappaBalpha and CDK4. These results will allow comparison between gankyrin, IkappaBalpha, and the INK4 family of tumor suppressors p16 and p18. In Specific Aim 3, we plan to determine the structures of SEI-1 and/or its truncated forms possessing different activities, and use site-directed mutagenesis to dissect the residues important for different activities, and to identify CDK4 residues important for binding of SEI-1 and different functional domains of SEI-1.