IDENTIFICATION OF CDK4 SEQUENCES INVOLVED IN CYCLIN D1 AND P16 BINDING

Activation of CDK4 is regulated, in part, by its association with a D- type cyclin. Conversely, CDK4 activity is inhibited when it is bound t o the cyclin-dependent kinase inhibitor, p16(INK4A). TO investigate th e molecular basis of the interactions between CDK4 and cyclin D1 or p1 6(INK4A) we performed site-directed mutagenesis of CDK4. The interacti on was examined using in vitro translated wild type and mutant CDK4 pr oteins and bacterially expressed cyclin D1 and pig fusion proteins. As mutational analysis of CDC2 suggests that its cyclin binding domain i s primarily located near its amino terminus, the majority of the mutat ions constructed in CDK4 were located near its amino terminus. In addi tion, CDK4 residues homologous to CDCS sites involved in Suc1 binding were also mutated. Our analysis indicates that cyclin D1 and pie bindi ng sites are overlapping and located primarily near the amino terminus . All CDK4 mutations that resulted in decreased pig binding capability also diminished cyclin D1 binding. In contrast, amino-terminal sequen ces were identified, including the PSTAIRE region, that are important for cyclin D1 binding but are not involved in p16 binding.