Regulation of synaptotagmin I phosphorylation by multiple protein kinases.
Synaptotagmin I has been suggested to function as a low-affinity calcium sensor for calcium-triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. SyntagI, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagI revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas caskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin-coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+-evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.
Hilfiker
S
S
Laboratory of Molecular and Cellular Neuroscience, Rockefeller University, New York, New York 10021, USA.
Pieribone
V A
VA
Nordstedt
C
C
Greengard
P
P
Czernik
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D047390:Casein Kinase II; D002454:Cell Differentiation; D002966:Clathrin / Q000494:pharmacology; D017124:Conserved Sequence; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D016716:PC12 Cells; D010449:Peptide Mapping; D019606:Phosphoamino Acids / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D013574:Synaptosomes / Q000378:metabolism; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D002966:Clathrin; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D019606:Phosphoamino Acids; C494310:SYT1 protein, human; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D011494:Protein Kinases; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases