Assay for activity of mammalian cyclin D-dependent kinases CDK4 and CDK6

This chapter discusses the assay for activity of mammalian cyclin D-dependent kinases CDK4 and CDK6. In mammalian cells, progression through the G1 phase is primarily controlled by two cyclin-dependent kinases, CDK4-cyclin D or CDK6-cyclin D and CDK2-cyclin E. The temporal activation and inactivation of CDK kinase activity during the cell cycle are multistep processes that are tightly regulated by several complex mechanisms including cyclin binding and activation, subunit phosphorylations, ubiquitin-mediated cyclin destruction, and inhibition by the binding of a CDK inhibitor. The analysis of cyclin D-dependent CDK4 and CDK6 kinase activity, however, has technically been more difficult. This chapter describes detailed protocols for the preparation and measurement of active CDK4 and CDK6 complexes from three different sources: bacterial, insect, and mammalian cells.