CDK-activating kinases: detection and activity measurements

Methods Mol Biol. 2005;296:279-90. doi: 10.1385/1-59259-857-9:279.

Abstract

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.

MeSH terms

  • Animals
  • Antibodies, Monoclonal
  • Binding Sites
  • Cell Cycle / physiology
  • Cyclin-Dependent Kinases / analysis*
  • Cyclin-Dependent Kinases / chemistry
  • Cyclin-Dependent Kinases / immunology
  • Cyclin-Dependent Kinases / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Electrophoretic Mobility Shift Assay
  • Humans
  • Immunoblotting
  • Immunoprecipitation
  • In Vitro Techniques
  • Mice
  • Phosphorylation

Substances

  • Antibodies, Monoclonal
  • Cyclin-Dependent Kinases
  • cyclin-dependent kinase-activating kinase