title: The stimulus-induced tyrosine phosphorylation of Munc18c facilitates vesicle exocytosis.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Center for Diabetes Research, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, IN 46202, USA.
authors.forename: Eunjin
authors.initials: E
authors.lastname: Oh
authors.forename: Debbie C
authors.initials: DC
authors.lastname: Thurmond
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology*; D005947:Glucose / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D051938:Munc18 Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016296:Mutagenesis; D010766:Phosphorylation; D050765:Qa-SNARE Proteins / Q000378:metabolism; D011817:Rabbits; D014443:Tyrosine / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism
chemical_list: D000906:Antibodies; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D050988:Vesicle-Associated Membrane Protein 2; C494808:vesicle-associated membrane protein 2, mouse; D014443:Tyrosine; D005947:Glucose