Inhibition of neuronal cyclin-dependent kinase-5 by staurosporine and purine analogs is independent of activation by munc-18
- 42 Downloads
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 μM and 8 μM, respectively for staurosporine, isopentanyladenine and olomoucine. The inhibition by staurosporine was non-competitive with respect to phosphoryl 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.
Key Wordscdk5 staurosporine phosphorylation p67 (Munc-18) neurofilament
Unable to display preview. Download preview PDF.
- 20.Gold, M. R., Sanghera, J. S., Stewart, J., and Pelech, S. L. 1992. Selective activation of p42 mitogen activated protein, MAP kinase in murine B lymphoma cell lines by membrane immunoglobulin cross linking. Evidence for protein kinase C independent and dependent mechanisms of activation. Biochem. J. 287:269–276.PubMedGoogle Scholar
- 23.Nakano, H., Kobayashi, E., Takahashi, I., Tamaoki, T., Kuzuu, Y., and Iba, H. 1987. Staurosporine inhibits tyrosine specific protein kinase activity of Rous sarcoma virus transforming protein P60. J. Antibiotics XL:706–708.Google Scholar
- 25.Rialet, V., and Meijer, L. 1991. A new screening test for antimitotic compounds using the universal M phase specific protein kinase, p34cdc2/cyclin Bcdc13, affinity immobilized on p13suc1-coated microtitration plates. Cancer Res. 11:1581–1590.Google Scholar
- 34.Aletta, J. M., Tsao, H., and Greene, L. A. 1990. How do neurites grow? Clues from NGF regulated cytoskeletal phosphoproteins. Pages 203–218, in L. A. Horrocks (ed) Tropic Factors and the Nervous System. Raven Press, New York.Google Scholar
- 35.Hisanaga, S., and Hirokawa, N. 1990. Dephosphorylation induced interaction of neurofilaments with microtubules. J. Biol. Chem. 266:21852–21858.Google Scholar
- 37.Eagles, P. A. M., Pant, H. C., and Gainer H. 1990. Neurofilaments. Pages 37–94,in Goldman R. D. and Steinert P. M. (eds.). Cellular and Molecular Biology of Intermediate Filaments. Plenum New York.Google Scholar