Ca2+ and calmodulin-dependent phosphorylation of endogenous synaptic vesicle tubulin by a vesicle-bound calmodulin kinase system.
Endogenous synaptic vesicle alpha- and beta-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. alpha-Tubulin and beta-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 alpha- and beta-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.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013572:Synaptic Vesicles / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D018377:Neurotransmitter Agents; D047428:Protein Kinase Inhibitors; D014404:Tubulin; D000242:Cyclic AMP; D011494:Protein Kinases; D008274:Magnesium; D002118:Calcium