Inhibition of neuronal cyclin-dependent kinase-5 by staurosporine and purine analogs is independent of activation by munc-18 |
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Authors: | Veeranna K T Shetty N Amin P Grant R W Albers H C Pant |
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Institution: | (1) Dept. of Neurochemistry, NIMHANS, Bangalore, India;(2) Lab. of Neurochemistry (NINDS), NIH, Bldg. 36, Room 4D20, 20892-4130 Bethesda, MD |
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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 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. |
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Keywords: | cdk5 staurosporine phosphorylation p67 (Munc-18) neurofilament |
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