Regulation of the levels of cyclins is not the only mechanism that controls CDK activity. Activating and inhibitory phosphorylation events on the CDK subunit itself are essential to control cyclin-CDK activity. Phosphorylation of a threonine residue near the active site of the enzyme is required for CDK activity. This phosphorylation is mediated by the CDK-activating kinase (CAK). In some organisms, cyclin binding is a prerequisite for CAK phosphorylation, whereas in others this phosphorylation event occurs prior to cyclin binding. Although the sequence of assembling active CDKs differs among organisms, it is clear that CAK phosphorylation of CDK is not a rate-limiting step in CDK activation. CAK activity is constant throughout the cell cycle and phosphorylates the CDK as soon as a cyclin-CDK complex is formed.

Two inhibitory phosphorylations on CDK also play a critical role in controlling CDK activity. In contrast to the CAK-induced activating phosphorylation, these inhibitory phosphorylations are regulated. A highly conserved tyrosine (Y15 in human CDKs) and an adjacent threonine (T14 in humans) are subject to regulated phosphorylation. Both residues are situated in the ATP-binding pocket of the CDK, and their phosphorylation most likely interferes with positioning of ATP in the pocket. Changes in the phosphorylation of these sites are essential for the regulation of mitotic CDKs and have also been implicated in the control of G₁/S and S phase CDKs. As we will see in Section 19.5, a highly conserved kinase called Wee1 brings about this inhibitory phosphorylation, and a highly conserved phosphatase called Cdc25 mediates dephosphorylation.