INHIBITION OF NEURONAL CYCLIN-DEPENDENT KINASE-5 BY STAUROSPORINE ANDPURINE ANALOGS IS INDEPENDENT OF ACTIVATION BY MUNC-18

Neuronal cdk5 can phosphorylate certain lys-ser-pro (KSP) motifs of ne urofilaments and tau protein in the nervous system. We have immunoprec ipitated the cdk5 from rat brain using a polyclonal antibody raised ag ainst the C-terminus of cdk5. The immunoprecipitate has phosphorylated a KSPXK peptide analog of NF-H, as well as histone H1 and a bacterial ly expressed rat NF-H protein. The kinase activity was inhibited by st aurosporine, isopentanyladenine and olomoucine in a dose dependent man ner. Kinetic studies indicated Ki values of 39 nM, 38 mu M and 8 mu M, respectively for staurosporine, isopentanyladenine and olomoucine. Th e inhibition by staurosporine was non-competitive with respect to phos phoryl acceptor substrates. Western blot analysis of the immunoprecipi tate showed both cdk5 and p67 (Munc-18), a putative regulator molecule of the kinase. Addition of p67 fusion protein enhanced the kinase act ivity of the immunoprecipitate by 60% above the basal activity. P67 el evated Ki values for both staurosporine and olomoucine. The degree of inhibition at high concentrations of these inhibitors was unaltered by exogenous p67 indicating a lack of competitive interactions with p67. The high affinity of staurosporine for cdk5 suggests that cdk5 may be one of the targets for the neurotropic effect of staurosporine.