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 heter odimer of Cdk5 and an essential 25-kDa regulatory subunit (Lew, J,, an d Wang, J, H, (1995) Trends Biochem, Sci, 20, 33-37), The regulatory s ubunit is an N-terminal truncated derivative of a 35-kDa protein expre ssed specifically in brain, hence the name neuronal Cdk5 activator, p2 5/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, t hree forms of Cdk5 have been detected in bovine brain: a monomeric Cdk 5 that can be activated by bacterially expressed GST-p21(nck5a), a het erodimer of Cdk5 and p25(nck5a) that displays high kinase activity, an d a Cdk5 . p35(nck5a) complex that is inactive and refractory to GST-p 21(nck5a) activation, Analysis of the Cdk5 . p35(nck5a) complex by gel filtration chromatography indicated that the complex was part of a ma cromolecular structure with a molecular mass of similar to 670 kDa, Wh en the macromolecular complex was subjected to gel filtration chromato graphy in the presences of 10% ethylene glycol, the fractions containi ng 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 inh ibitory factor that dissociated from the complex in 10% ethylene glyco l.