The Stimulus-induced Tyrosine Phosphorylation of Munc18c Facilitates Vesicle Exocytosis

Eunjin Oh,Debbie C Thurmond

doi:10.1074/jbc.m601581200

Abstract

Stimulus-induced tyrosine phosphorylation of Munc18c was investigated as a potential regulatory mechanism by which the Munc18c-Syntaxin 4 complex can be dissociated in response to divergent stimuli in multiple cell types. Use of [(32)P]orthophosphate incorporation, pervanadate treatment, and phosphotyrosine-specific antibodies demonstrated that Munc18c underwent tyrosine phosphorylation. Phosphorylation was apparent under basal conditions, but levels were significantly increased within 5 min of glucose stimulation in MIN6 beta cells. Tyrosine phosphorylation of Munc18c was also detected in 3T3L1 adipocytes and increased with insulin stimulation, suggesting that this may be a conserved mechanism. Syntaxin 4 binding to Munc18c decreased as Munc18c phosphorylation levels increased in pervanadate-treated cells, suggesting that phosphorylation dissociates the Munc18c-Syntaxin 4 complex. Munc18c phosphorylation was localized to the N-terminal 255 residues. Mutagenesis of one residue in this region, Y219F, significantly increased the affinity of Munc18c for Syntaxin 4, whereas mutation of three other candidate sites was without effect. Moreover, Munc18c-Y219F expression in MIN6 cells functionally inhibited glucose-stimulated SNARE complex formation and insulin granule exocytosis. These data support a novel and conserved mechanism for the dissociation of Munc18c-Syntaxin 4 complexes in a stimulus-dependent manner to facilitate the increase in Syntaxin 4-VAMP2 association and to promote vesicle/granule fusion.

Highlights

Led to the general concept that most types of regulated vesicle fusion occur by a common mechanism
Tyrosine Phosphorylation of Munc18c insulin-stimulated GLUT4 vesicle translocation rapidly and coordinately maintain whole body euglycemia, we propose that commonalities in post-translational modifications may provide the basis for a conserved mechanism by which the Munc18c-Syntaxin 4 complex regulates exocytosis
Using insulin-stimulated GLUT4 vesicle translocation as our model system, we have shown previously that Munc18c-Syntaxin 4 complexes are important for the fusion step of vesicle exocytosis [25], the mechanism for this has remained unknown

Summary

Introduction

Led to the general concept that most types of regulated vesicle fusion occur by a common mechanism. Immunoprecipitation of labeled lysates using anti-Syntaxin 4 antibody failed to show any incorporation of phosphate, indicating that Syntaxin 4 is not phosphorylated in beta cells (data not shown) and that phosphorylation is specific to Munc18c in this complex. Tyrosine Phosphorylation of Munc18c Leads to Its Dissociation from Syntaxin 4: A Conserved Mechanism—To determine whether the tyrosine phosphorylation of Munc18c alters its ability to interact with Syntaxin 4, we treated MIN6 cells with increasing concentrations of pervanadate and prepared lysates for immunoprecipitation with anti-Munc18c antibody.

Results

Conclusion