title: 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.
abstract: 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.
authors.affiliation: Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Fujita
authors.forename: T
authors.initials: T
authors.lastname: Sasaki
authors.forename: K
authors.initials: K
authors.lastname: Fukui
authors.forename: H
authors.initials: H
authors.lastname: Kotani
authors.forename: T
authors.initials: T
authors.lastname: Kimura
authors.forename: Y
authors.initials: Y
authors.lastname: Hata
authors.forename: T C
authors.initials: TC
authors.lastname: Südhof
authors.forename: R H
authors.initials: RH
authors.lastname: Scheller
authors.forename: Y
authors.initials: Y
authors.lastname: Takai
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism; D002459:Cell Fusion; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D006706:Homeostasis; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013572:Synaptic Vesicles / Q000378:metabolism*; D014162:Transfection; D033921:Vesicular Transport Proteins
chemical_list: D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D050765:Qa-SNARE Proteins; D011993:Recombinant Fusion Proteins; C497931:SEC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D011493:Protein Kinase C