Dimerization of the amino terminal domain of p57Kip2 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 other hand, multiple inhibitor molecules are required to inhibit Cdks. In the present 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 complexes do not compete for the p57(Kip2) homodimers formation. However, a mutation in the cc-helix domain of p57(Kip2) (R33L) strongly reduced homodimer formation but did not modify interaction with cyclin D1-Cdk4 complexes. Also, increasing amounts of p57(Kip2) lead in vivo to a significant augmentation in the level of p57(Kip2) homodimerization associated with cyclin D1-Cdk4 complexes and to a marked inhibition of the cyclin D1-Cdk4 kinase activity. Altogether, these data suggest a model whereby p57(Kip2) associates with itself by using the NH2 domain to form a homodimeric species which interacts with and inhibits the cyclin D1-Cdk4 complexes.