Molecular interactions between p27(Kip1) and p21(Cip1) and G1 cyclin/Cdk complexes

The importance of cyclin/cdk complexes for cell cycle progression has been apparent for a number of years. However, the mechanisms of and reasons for the interaction of these kinases with the Cip/Kip class of proteins have been difficult to determine. There is evidence that the Cip/Kip proteins can inhibit the catalytic activity of the kinases, redirect the substrate preferences of the kinases, and even promote the stable association of the kinases. Different conclusions are reached dependent on the kip/cyclin/cdk combination studied and the source material and purification methods of the components used. We have approached this problem by examining the binding of limiting amounts of two Cip/Kip proteins, p27Kip1 and p21 Cip1, with active and inactive cyclin E/cdk2 and cyclin D2/cdk4 complexes in vitro. We have found that one molecule of either p27 or p21 is sufficient to achieve complete inhibition of cyclin E/cdk2, but two molecules of each inhibitor are needed to inhibit cyclin D2/cdk4. Whereas the inhibitors can not associate with active cyclin E/cdk2 complexes, one molecule of either p27 or p21 activates cyclin D2/cdk4 activity. p27's activation of cyclin D2/cdk4 is to a higher degree, but shorter lived, than that of p21. Thus, if the functional differences exhibited by p27 and p21 at the biological level—p27s arrest in G1 to allow differentiation contrasted with p21's arrest in G1 to allow growth—are manifest at the molecular level, they are more likely to be attributable to differences in their interactions with cyclin D2/cdk4 than with cyclin E/cdk2. Ramifications of this idea are discussed and suggestions of other sources for the kips' different roles in cell cycle regulation are posited. Our results also suggest that the importance of the nonconserved α-helix between the kips' conserved LFG cyclin binding domain and WNFDF cdk binding region seems to be purely structural rather than dependent on a particular amino acid sequence.