The role of Thr160 phosphorylation of Cdk2 in substrate recognition

Full activation of cyclin-dependent kinases (Cdks) requires binding to a cyclin and phosphorylation on an activating site equivalent to Thr160 in Cdk2 by the Cdk-activating kinase. Much is known about the effects of cyclin binding, but the role of the activating phosphorylation is less well understood. We have characterized the effects of Thr160 phosphorylation of Cdk2 on its interactions with substrates, particularly with the P + 3 position. We find that an ionic interaction participates in the recognition of the P + 3 position of the substrate and confirms an observation from structural studies indicating that a key element of this recognition is an interaction between the lysine at the P + 3 position and the Thr160 phosphate of Cdk2. The major effect of disrupting the lysine-phosphate interaction was on k(cat) values rather than K-m values, suggesting that the energy from this interaction is used to align the substrate for efficient catalysis. A lack of effect of Thr160 phosphorylation on the ATPase activity of Cdk2 supported this interpretation.