CDK-activating kinases: detection and activity measurements.
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.
Larochelle
Stéphane
S
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Fisher
Robert P
RP
D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002453:Cell Cycle / Q000502:physiology; D018844:Cyclin-Dependent Kinases / Q000032:analysis* / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D051379:Mice; D010766:Phosphorylation; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D000911:Antibodies, Monoclonal; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase