Dimerization of the amino terminal domain of p57(Kip2) inhibits cyclin D1-Cdk4 kinase activity

Previous studies have led to the proposal that a single molecule of Cki can associate with the cyclin/Cdk complex to repress its activity. On the othe r hand, multiple inhibitor molecules are required to inhibit Cdks. In the p resent work, by using differently tagged p57(Kip2) proteins we demonstrate that p57(Kip2),,, bind to itself in vitro and in vivo. Mutational deletion analysis showed that the NH2 terminal domain of p57(Kip2) is necessary and sufficient to dimerization, Using an in vitro competition/association assay , we demonstrate that cyclin D1 alone, Cdk4 alone and/or cyclin D1/Cdk4 com plexes do not compete for the p57(Kip2) homodimers formation. However, a mu tation in the cc-helix domain of p57(Kip2) (R33L) strongly reduced homodime r formation but did not modify interaction with cyclin D1-Cdk4 complexes. A lso, increasing amounts of p57(Kip2) lead in vivo to a significant augmenta tion in the level of p57(Kip2) homodimerization associated with cyclin D1-C dk4 complexes and to a marked inhibition of the cyclin D1-Cdk4 kinase activ ity. Altogether, these data suggest a model whereby p57(Kip2) associates wi th itself by using the NH2 domain to form a homodimeric species which inter acts with and inhibits the cyclin D1-Cdk4 complexes.