Ca2+ and Calmodulin‐Dependent Phosphorylation of Endogenous Synaptic Vesicle Tubulin by a Vesicle‐Bound Calmodulin Kinase System

Abstract: Endogenous synaptic vesicle α‐and β‐tubulin were shown to be the major substrates for a Ca2+‐calmodulin‐regulated protein kinase system in enriched synaptic vesicle preparations from rat cortex as determined by two‐dimensional gel electrophoresis and peptide mapping. The activation of this endogenous tubulin kinase system was dependent on Ca2+ and the Ca2+ binding protein, calmodulin. Under maximally stimulated conditions, approximately 40% of the tubulin present in enriched synaptic vesicles was phosphorylated within less than 50 s by the vesicle Ca2+‐calmodulin kinase. Evidence is presented indicating that the Ca2+‐calmodulin tubulin kinase is an enzyme system distinct from previously described cyclic AMP protein kinases. α‐Tubulin and β‐tubulin were identified as major components of previously designated vesicle phosphorylation bands DPH‐L and DPH‐M. The Ca2+‐calmodulin tubulin kinase is very labile and specialized isolation procedures were necessary to retain activity. Ca2+‐activated synaptic vesicle tubulin phosphorylation correlated with vesicle neurotransmitter release. Depolarization‐dependent Ca2+ uptake in intact synaptosomes simultaneously stimulated the release of neurotransmitters and the phosphorylation of synaptic vesicle α‐and β‐tubulin. The results indicate that regulation of the synaptic vesicle tubulin kinase by Ca2+ and calmodulin may play a role in the functional utilization of synaptic vesicle tubulin and may mediate some of the effects of Ca2+ on vesicle function and neurosecretion.