Phosphorylation of Munc-18/n-Sec1/rbSec1 by Protein Kinase C

doi:10.1074/jbc.271.13.7265

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 (*)

From the (1)Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Japan, the
(2)Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444, Japan, the
(3)Department of Molecular Genetics, Howard Hughes Medical Institute, University of Texas Southwestern Medical School, Dallas, Texas 75235, and the
(4)Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, California 94305

^¶To whom correspondence should be addressed:
Dept. of Molecular Biology and Biochemistry, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan.
Tel.: 81-6-879-3410; Fax: 81-6-879-3419; ytakai{at}molbio.med.osaka-u.ac.jp.

↵^§ Present address: Takai Biotimer Project, Exploratory Research for Advanced Technology (ERATO), 2-2-10 Murotani, Nishi-ku, Kobe 651-22, Japan.

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 Ca-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 Ca- 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 and Ser. 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.

Footnotes

↵* This investigation at Osaka University was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Science, Sports, and Culture, Japan(1995), by grants-in-aid for Abnormalities in Hormone Receptor Mechanisms and for Aging and Health from the Ministry of Health and Welfare, Japan(1995), and by a grant from the Uehara Memorial Foundation(1995). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SNARE

SNAP receptor

SNAP

soluble NSF attachment protein

NSF

N-ethylmaleimide-sensitive fusion protein

SNAP-25

synaptosomal-associated protein of 25 KDa

t-SNARE

target SNARE

VAMP

vesicle-associated membrane protein

v-SNARE

vesicle SNARE

PKC

protein kinase C

GST

glutathione S-transferase

PKA

cyclic AMP-dependent protein kinase

CaMKII

calmodulin-dependent protein kinase II

PS

phosphatidylserine

DTT

dithiothreitol

TPA

12-O-tetradecanoylphorbol-13-acetate

PAGE

polyacrylamide gel electrophoresis.
- Received November 30, 1995.
- Revision received January 15, 1996.