Cyclin-Dependent Kinase 5 (Cdk5): Preparation and Measurement of Kinase Activity

Cyclin-dependent kinase 5 (Cdk5) is a versatile protein kinase that plays a role in a variety of neuronal activities including neuronal migration during brain development, synaptic activities in mature neurons, and neuronal cell death in neurodegenerative diseases. However, the functions of Cdk5 in brains have not been elucidated fully and its extraneuronal functions are being explored. It is important to identify the functional substrates of Cdk5 and to elucidate the mechanism regulating its activity. Cdk5 is a catalytic subunit of an active kinase complex with the regulatory subunit, p35 or p39. Because the kinase activity of Cdk5 is regulated mainly by synthesis and degradation of the regulatory subunits, care is needed when detecting and measuring its kinase activity. We describe here how to prepare kinase-active Cdk5, to measure its kinase activity, and to distinguish Cdk5 phosphorylation from other types of kinase-dependent phosphorylation.