The tyrosine phosphorylation of Munc18c induces a switch in binding specificity from syntaxin 4 to Doc2β

Jenna L. Jewell, Eunjin Oh, Sara M. Bennett, Samy Meroueh, Debbie C. Thurmond

Research output: Contribution to journalArticle

35 Citations (Scopus)

Abstract

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 Doc2β to Munc18c, although the underlying mechanism for this is unknown. Because the Doc2β 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-Doc2β 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 α-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 Doc2β binding.

Original languageEnglish
Pages (from-to)21734-21746
Number of pages13
JournalJournal of Biological Chemistry
Volume283
Issue number31
DOIs
StatePublished - Aug 1 2008

Fingerprint

Qa-SNARE Proteins
Phosphorylation
Tyrosine
Switches
SNARE Proteins
Glucose
Conformations
Munc18 Proteins
Vesicle-Associated Membrane Protein 2
Syntaxin 1
Molecular modeling
Competitive Binding
Detergents
Assays
Proteins
Association reactions
Insulin

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

The tyrosine phosphorylation of Munc18c induces a switch in binding specificity from syntaxin 4 to Doc2β. / Jewell, Jenna L.; Oh, Eunjin; Bennett, Sara M.; Meroueh, Samy; Thurmond, Debbie C.

In: Journal of Biological Chemistry, Vol. 283, No. 31, 01.08.2008, p. 21734-21746.

Research output: Contribution to journalArticle

Jewell, Jenna L. ; Oh, Eunjin ; Bennett, Sara M. ; Meroueh, Samy ; Thurmond, Debbie C. / The tyrosine phosphorylation of Munc18c induces a switch in binding specificity from syntaxin 4 to Doc2β. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 31. pp. 21734-21746.
@article{a7ccb4334c7e4d41b2bdb079817d8ccd,
title = "The tyrosine phosphorylation of Munc18c induces a switch in binding specificity from syntaxin 4 to Doc2β",
abstract = "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 Doc2β to Munc18c, although the underlying mechanism for this is unknown. Because the Doc2β 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-Doc2β 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 α-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 Doc2β binding.",
author = "Jewell, {Jenna L.} and Eunjin Oh and Bennett, {Sara M.} and Samy Meroueh and Thurmond, {Debbie C.}",
year = "2008",
month = "8",
day = "1",
doi = "10.1074/jbc.M710445200",
language = "English",
volume = "283",
pages = "21734--21746",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "31",

}

TY - JOUR

T1 - The tyrosine phosphorylation of Munc18c induces a switch in binding specificity from syntaxin 4 to Doc2β

AU - Jewell, Jenna L.

AU - Oh, Eunjin

AU - Bennett, Sara M.

AU - Meroueh, Samy

AU - Thurmond, Debbie C.

PY - 2008/8/1

Y1 - 2008/8/1

N2 - 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 Doc2β to Munc18c, although the underlying mechanism for this is unknown. Because the Doc2β 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-Doc2β 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 α-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 Doc2β binding.

AB - 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 Doc2β to Munc18c, although the underlying mechanism for this is unknown. Because the Doc2β 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-Doc2β 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 α-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 Doc2β binding.

UR - http://www.scopus.com/inward/record.url?scp=52049101363&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=52049101363&partnerID=8YFLogxK

U2 - 10.1074/jbc.M710445200

DO - 10.1074/jbc.M710445200

M3 - Article

C2 - 18541526

AN - SCOPUS:52049101363

VL - 283

SP - 21734

EP - 21746

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 31

ER -