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

Abstract

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 can 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 α-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.