Regulation of synaptotagmin I phosphorylation by multiple protein kinases

Synaptotagmin I has been suggested to function as a low-affinity calcium se nsor for calcium-triggered exocytosis from neurons and neuroendocrine cells . We have studied the phosphorylation of synaptotagmin I by a variety of pr otein kinases in vitro and in intact preparations. Syntagl, the purified, r ecombinant, cytoplasmic domain of rat synaptotagmin I, was an effective sub strate in vitro for Ca2+/calmodulin-dependent protein kinase II (CaMKII), p rotein kinase C (PKC), and casein kinase II (caskII), Sequencing of tryptic phosphopeptides from syntagI revealed that CaMKII and PKC phosphorylated t he same residue, corresponding to Thr(112), whereas caskII phosphorylated t wo residues, corresponding to Thr(125) and Thr(128) Endogenous synaptotagmi n I was phosphorylated on purified synaptic vesicles by all three kinases, In contrast, no phosphorylation was observed on clathrin-coated vesicles, s uggesting that phosphorylation of synaptotagmin I in vivo occurs only at sp ecific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosom es and PC12 cells, K+-evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr( 112). The results suggest the possibility that the phosphorylation of synap totagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.