title: A 14-3-3 mode-1 binding motif initiates gap junction internalization during acute cardiac ischemia.
abstract: Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14-3-3 proteins can regulate protein trafficking, and a 14-3-3 mode-1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C-terminus. We hypothesized that Cx43(Ser373) phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14-3-3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14-3-3τ in cell lines increases gap junction plaque size at cell-cell borders. Cx43(S373A) mutation prevents Cx43/14-3-3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff-perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no-flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen-activated protein kinase has previously been implicated in Cx43 internalization. The Cx43(S373A) mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43(Ser373) phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.
authors.affiliation: Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, CA, 90048, USA.
authors.forename: James W
authors.initials: JW
authors.lastname: Smyth
authors.forename: Shan-Shan
authors.initials: SS
authors.lastname: Zhang
authors.forename: Jose M
authors.initials: JM
authors.lastname: Sanchez
authors.forename: Samy
authors.initials: S
authors.lastname: Lamouille
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Vogan
authors.forename: Geoffrey G
authors.initials: GG
authors.lastname: Hesketh
authors.forename: Tingting
authors.initials: T
authors.lastname: Hong
authors.forename: Gordon F
authors.initials: GF
authors.lastname: Tomaselli
authors.forename: Robin M
authors.initials: RM
authors.lastname: Shaw
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009154:Mutation; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D055550:Protein Stability; D021381:Protein Transport
chemical_list: D048948:14-3-3 Proteins; D018031:Connexin 43; D011493:Protein Kinase C; D020929:Mitogen-Activated Protein Kinase Kinases
keywords: 14-3-3; connexin; endocytosis; gap junction; ischemia