Complete inhibition of Cdk/cyclin by one molecule of p21Cip1

Cell-cycle phase transitions are controlled by cyclin-dependent kinases (Cdks). Key to the regulation of these kinase activities are Cdk inhibitors, proteins that are induced in response to various antiproliferative signals but that can also oscillate during cell-cycle progression, leading to Cdk inactivation. A current dogma is that kinase complexes containing the prototype Cdk inhibitor p21 transit between active and inactive states, in that Cdk complexes associated with one p21 molecule remain active until they associate with additional p21 molecules. However, using a number of different techniques including analytical ultracentrifugation of purified p21/cyclin A/Cdk2 complexes we demonstrate unambiguously that a single p21 molecule is sufficient for kinase inhibition and that p21-saturated complexes contain only one stably bound inhibitor molecule. Even phosphorylated forms of p21 remain efficient inhibitors of Cdk activities. Therefore the level of Cdk inactivation by p21 is determined by the fraction of kinase complexed with the inhibitor and not by the stoichiometry of inhibitor bound to the kinase or the phosphorylation state of the Cdk inhibitor.