The tyrosine phosphorylation of Munc18c induces a switch in binding specificity from syntaxin 4 to Doc2beta.
Glucose-stimulated insulin secretion is mediated by syntaxin 4-based SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes and the Sec1/Munc18 protein Munc18c. Our laboratory recently reported that Munc18c-syntaxin 4 complexes are further regulated by the competitive binding of the double C2 domain protein Doc2beta to Munc18c, although the underlying mechanism for this is unknown. Because the Doc2beta binding region of Munc18c contained residue Tyr-219 and this residue becomes phosphorylated in response to glucose stimulation, we hypothesized that the mechanism would involve Munc18c phosphorylation. Coimmunoprecipitation analyses using detergent lysates prepared from pervanadate-treated MIN6 beta cells revealed that the tyrosine phosphorylation of Munc18c corresponded to a 60% decrease in Munc18c-syntaxin 4 association with a coordinate 2-fold increase in Munc18c-Doc2beta binding. In vitro binding assays identified syntaxin 4 residues 118-194 as sufficient to confer its interaction with Munc18c; residues 118-194 contain the Hc alpha-helix and flexible linker region controlling transition of syntaxins between closed and open conformations. When overexpressed in MIN6 cells, this Hc-linker region functioned as a competitive inhibitor of endogenous syntaxin 4-Munc18c binding, increased syntaxin 4 binding to VAMP2, and significantly enhanced glucose-stimulated secretion. Molecular modeling of these new interactions yielded the predictions 1) that Tyr-219 of Munc18c remains buried under basal conditions in a conformation that is favorable for interaction with "closed" syntaxin 4 and 2) that stimulation leads to changes in syntaxin 4 contacts to facilitate exposure of Munc18c Tyr-219 for phosphorylation and Doc2beta binding.
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA.
Jenna L
JL
Jewell
Eunjin
E
Oh
Sara M
SM
Bennett
Samy O
SO
Meroueh
Debbie C
DC
Thurmond
D000818:Animals; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D005947:Glucose / Q000378:metabolism; D051379:Mice; D008968:Molecular Conformation; D051938:Munc18 Proteins / Q000737:chemistry* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D050765:Qa-SNARE Proteins / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D014443:Tyrosine / Q000737:chemistry*
D002135:Calcium-Binding Proteins; C490420:DOC2B protein, human; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D014443:Tyrosine; D005947:Glucose