Interaction of cyclin-dependent kinase 5 (Cdk5) and neuronal Cdk5 activator in bovine brain

Neuronal cdc2-like kinase (Nclk) purified from bovine brain is a heterodimer of Cdk5 and an essential 25-kDa regulatory subunit (Lew, J,, and Wang, J, H, (1995) Trends Biochem, Sci, 20, 33-37), The regulatory subunit is an N-terminal truncated derivative of a 35-kDa protein expressed specifically in brain, hence the name neuronal Cdk5 activator, p25/p35(nck5a), In this study, we probe the relationship between the two different forms of Nck5a and their interaction with and activation of Cdk5 in bovine brain extract, Using protein fraction ation procedures in combination with Western blot analysis and protein kinase assay, three forms of Cdk5 have been detected in bovine brain: a monomeric Cdk5 that can be activated by bacterially expressed GST-p21(nck5a), a heterodimer of Cdk5 and p25(nck5a) that displays high kinase activity, and a Cdk5 . p35(nck5a) complex that is inactive and refractory to GST-p21(nck5a) activation, Analysis of the Cdk5 . p35(nck5a) complex by gel filtration chromatography indicated that the complex was part of a macromolecular structure with a molecular mass of similar to 670 kDa, When the macromolecular complex was subjected to gel filtration chromatography in the presences of 10% ethylene glycol, the fractions containing both p35(nck5a) and Cdk5, although eluting at the same position as control, displayed high kinase activity, The result is compatible with the suggestion that the macromolecular complex contained a kinase inhibitory factor that dissociated from the complex in 10% ethylene glycol.