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

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Abstract
Endogenous synaptic vesicle .alpha.- and .beta.-tubulin were the major substrates for a Ca2+-calmodulin-regulated protein kinase system in enriched synaptic vesicle preparations from rat cortex as determined by 2-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, .apprx. 40% of the tubulin present in enriched synaptic vesicles was phosphorylated within < 50 s by the vesicle Ca2+-calmodulin kinase. The Ca2+-calmodulin tubulin kinase is an enzyme system distinct from previously described cAMP protein kinases. .alpha.-Tubulin and .beta.-tubulin were identified as major components of previously designated vesicle phosphorylation bands DPH-L [dilantin-L] and DPH-M [dilantin-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 .alpha.- and .beta.-tubulin. Apparently, 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.