title: Inhibition of neuronal cyclin-dependent kinase-5 by staurosporine and purine analogs is independent of activation by Munc-18.
abstract: Neuronal cdk5 can phosphorylate certain lys-ser-pro (KSP) motifs of neurofilaments and tau protein in the nervous system. We have immunoprecipitated the cdk5 from rat brain using a polyclonal antibody raised against the C-terminus of cdk5. The immunoprecipitate has phosphorylated a KSPXK peptide analog of NF-H, as well as histone H1 and a bacterially expressed rat NF-H protein. The kinase activity was inhibited by staurosporine, isopentanyladenine and olomoucine in a dose dependent manner. Kinetic studies indicated Ki values of 39 nM, 38 microM and 8 microM, respectively for staurosporine, isopentanyladenine and olomoucine. The inhibition by staurosporine was non-competitive with respect to phosphoryl acceptor acceptor substrates. Western blot analysis of the immunoprecipitate showed both cdk5 and p67 (Munc-18), a putative regulator molecule of the kinase. Addition of p67 fusion protein enhanced the kinase activity of the immunoprecipitate by 60% above the basal activity. P67 elevated 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.
authors.affiliation: Laboratory of Neurochemistry, NINDS, NIH, Bethesda, MD, USA 20892-4130, USA.
authors.lastname: Veeranna
authors.forename: K T
authors.initials: KT
authors.lastname: Shetty
authors.forename: N
authors.initials: N
authors.lastname: Amin
authors.forename: P
authors.initials: P
authors.lastname: Grant
authors.forename: R W
authors.initials: RW
authors.lastname: Albers
authors.forename: H C
authors.initials: HC
authors.lastname: Pant
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D009842:Oligopeptides; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011687:Purines / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D006657:Histones; D016900:Neurofilament Proteins; D009842:Oligopeptides; D011687:Purines; D011994:Recombinant Proteins; C051774:neurofilament protein H; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases; D019311:Staurosporine