Calcium-dependent serine phosphorylation of synaptophysin.
The phosphorylation of synaptophysin, a major integral membrane protein of small synaptic vesicles, was found to be regulated in a Ca(2+)-dependent manner in rat cerebrocortical slices, synaptosome preparations, and highly purified synaptic vesicles isolated from rat forebrain. K(+)-induced depolarization of slices and synaptosomes prelabeled with 32P-orthophosphate produced a rapid, transient increase in serine phosphorylation of synaptophysin. In synaptosomes, the depolarization-dependent increase in synaptophysin phosphorylation required the presence of external Ca2+ in the incubation medium. The addition of Ca2+ plus calmodulin to purified synaptic vesicles resulted in a 4-fold increase in serine phosphorylation of synaptophysin, and this phosphorylation was antagonized by a peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II(. Purified rat forebrain CaM kinase II phosphorylated both purified synaptophysin and endogenous, vesicle-associated synaptophysin, and the resulting 2-dimensional chymotryptic phosphopeptide maps were similar to those derived from synaptophysin phosphorylated in cerebrocortical slices. These data demonstrate that Ca(2+)-dependent phosphorylation of synaptophysin, mediated by CaM kinase II, occurs under physiological conditions.
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021-6399.
J L
JL
Rubenstein
P
P
Greengard
A J
AJ
Czernik
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000494:pharmacology; D002540:Cerebral Cortex / Q000378:metabolism* / Q000502:physiology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D066298:In Vitro Techniques; D008297:Male; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism*; D010748:Phosphopeptides / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D016548:Prosencephalon / Q000378:metabolism / Q000502:physiology; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D016708:Synaptophysin / Q000302:isolation & purification / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology
D002147:Calmodulin; D010710:Phosphates; D010748:Phosphopeptides; D010761:Phosphorus Radioisotopes; D016708:Synaptophysin; D012694:Serine; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D011188:Potassium; D002118:Calcium