All cyclin-dependent kinases (CDKs) involved in eukaryotic cell cycle control require phosphorylation at a conserved threonine (or serine) residue within the activation- or T-loop to attain full enzymatic activity. The enzyme responsible for this activating phosphorylation, the CDK-activating kinase (CAK), is therefore essential for proliferation of all eukaryotic cells. We describe methods to assess the T-loop phosphorylation state of the major mammalian CDKs in vivo; to measure the levels of CAK activity in cell-free extracts; and to analyze the abundance, subunit composition, and phosphorylation state of CAK complexes in metazoan cells. When derangement of normal CDK regulation is suspected as a cause of disturbed cell cycle progression, the combination of these methodologies can ascertain whether a primary CAK defect is the explanation.
