Ca2+ and calmodulin-regulated endogenous tubulin kinase activity in presynaptic nerve terminal preparations.
Synaptosomal tubulin was shown to be the major substrate for a Ca2+-calmodulin regulated protein kinase in synaptosome soluble fractions as determined by two-dimensional gel electrophoresis and peptide mapping. Ca2+ activated this endogenous tubulin kinase system in presynaptic nerve terminal preparations. The Ca2+-dependent activation of the tubulin kinase system was mediated by the Ca2+ binding protein, calmodulin. Trifluoperazine, a known inhibitor of calmodulin, significantly blocked the calmodulin-stimulated [32P]phosphate incorporation into synaptic tubulin. This inhibition of endogenous tubulin phosphorylation could be reversed by addition of exogenous calmodulin to the reaction mixture. The concentrations of Ca2+ and calmodulin required to produce a half-maximal stimulation of the tubulin kinase were 0.8 microM and 0.3 microM respectively. Greater than 70% of soluble tubulin present in the nerve terminal was phosphorylated in less than 50 s by this kinase system. Evidence is presented indicating that the synaptic Ca2+-calmodulin tubulin kinase is a distinct enzyme system from the previously described cyclic AMP microtubule-associated kinase. The anticonvulsant phenytoin inhibited the Ca2+-calmodulin stimulated phosphorylation of tubulin, and alpha- and beta-tubulin were identified as major components of previously designated synaptic phosphoprotein bands of DPH-L and DPH-M. Existence of the kinase as a calmodulin-tubulin-kinase complex is suggested from kinetic studies. The Ca2+-calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. The activation of the tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of tubulin in the nerve terminal and may mediate some of the effects of Ca2+ on synaptic function.
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology*; D002540:Cerebral Cortex / Q000201:enzymology*; D000242:Cyclic AMP / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013574:Synaptosomes / Q000201:enzymology; D014268:Trifluoperazine / Q000494:pharmacology; D014404:Tubulin / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D014404:Tubulin; D014268:Trifluoperazine; D000242:Cyclic AMP; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium