Ca2+ and calmodulin-regulated endogenous tubulin kinase activity in presynaptic nerve terminal preparations

Abstract

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 μM and 0.3 μM 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 α- and β-tubulin were identified as major components of previously designated synaptic phosphoprotein bands 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.