PHOSPHORYLATION OF MUNC-18 N-SEC1/RBSEC1 BY PROTEIN-KINASE-C - ITS IMPLICATION IN REGULATING THE INTERACTION OF MUNC-18/N-SEC1/RBSEC1 WITH SYNTAXIN/

Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inh ibits the association of vesicle-associated membrane protein (VAMP)/sy naptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) wi th syntaxin. Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmalei mide-sensitive fusion protein attachment protein (SNAP) receptors esse ntial for docking and/or fusion of synaptic vesicles with the presynap tic plasma membrane. Genetic analyses in yeast, Caenorhabditis elegans , and Drosophila suggest that Munc-18 is essential for vesicle transpo rt. On the other hand, protein kinase C (PKC) stimulates Ca2+-dependen t exocytosis in various types of secretory cells. However, the modes o f action of Munc-18 and PKC in vesicle transport have not been clarifi ed. Here, we show that recombinant Munc-18 is phosphorylated by conven tional PKC in a Ca2+- and phospholipid-dependent manner in a cell-free system. About 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18. The major phosphorylation sites are Ser(306) and Ser(313) . The Munc-18 complexed with syntaxin is not phosphorylated. The PKC-c atalyzed phosphorylation of Munc-18 inhibits its interaction with synt axin. These results suggest that the PKC-catalyzed phosphorylation of Munc-18 plays an important role in regulating the interaction of Munc- 18 with syntaxin and thereby the docking and/or the fusion of synaptic vesicles with the presynaptic plasma membrane.