Inhibition of neuronal cyclin-dependent kinase-5 by staurosporine and purine analogs is independent of activation by Munc-18.
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.
Veeranna
Laboratory of Neurochemistry, NINDS, NIH, Bethesda, MD, USA 20892-4130, USA.
Shetty
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Grant
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Albers
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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
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