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

• Published in: The Journal of biological chemistry Vol. 271; no. 13; pp. 7265 - 7268
• Main Authors: Fujita, Y, Sasaki, T, Fukui, K, Kotani, H, Kimura, T, Hata, Y, Südhof, T C, Scheller, R H, Takai, Y
• Format: Journal Article
• Language: English
• Published: United States 29.03.1996
• Subjects: Amino Acid Sequence; Animals; Caenorhabditis elegans; Caenorhabditis elegans - genetics; Caenorhabditis elegans - metabolism; Cell Fusion; Cell Line; Cell Membrane - metabolism; Drosophila; Drosophila - genetics; Drosophila - metabolism; Homeostasis; Membrane Proteins - metabolism; Molecular Sequence Data; Munc18 Proteins; Nerve Tissue Proteins - chemistry; Nerve Tissue Proteins - metabolism; Peptide Fragments - chemistry; Phosphorylation; Protein Kinase C - metabolism; Qa-SNARE Proteins; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Synaptic Vesicles - metabolism; Transfection; Vesicular Transport Proteins
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.13.7265

Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inhibits the association of vesicle-associated membrane protein (VAMP)/synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) with syntaxin. Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors essential for docking and/or fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic analyses in yeast, Caenorhabditis elegans, and Drosophila suggest that Munc-18 is essential for vesicle transport. On the other hand, protein kinase C (PKC) stimulates Ca2+-dependent exocytosis in various types of secretory cells. However, the modes of action of Munc-18 and PKC in vesicle transport have not been clarified. Here, we show that recombinant Munc-18 is phosphorylated by conventional 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 Ser306 and Ser313. The Munc-18 complexed with syntaxin is not phosphorylated. The PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin. 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.