Phosphorylation regulates connexin43/ZO-1 binding and release, an important step in gap junction turnover

• DOI: 10.1091/mbc.e16-07-0496
• Publication Date: 2017-10-12T01:00:32Z
• Authors: Anastasia F. Thév...; Rachel A. Margraf; Charles G. Fisher; Rachael M. Kells-...; Matthias M. Falk
• Keywords: 0301 basic medicine; Binding Sites; Gap Junctions; Articles; Connexins; Endocytosis; Madin Darby Canine Kidney Cells; 03 medical and health sciences; Dogs; Connexin 43; Proteolysis; Zonula Occludens-1 Protein; Animals; Humans; Phosphorylation; HeLa Cells; Protein Binding

To investigate whether connexin phosphorylation regulates the known role of zonula occludens-1 protein (ZO-1) in gap junction (GJ) function, we generated and analyzed a series phosphomimetic phosphorylation-dead mutants by mutating conserved regulatory serine (S) residues 255, 279/282, 365, 368, 373 located C-terminal domain connexin43 (Cx43) into glutamic acid (E) or alanine (A) residues. All were translated stable, full-length proteins assembled GJs when expressed HeLa Madin-Darby canine kidney epithelial cells. However, with S exchanged at positions exhibited significantly altered ZO-1 interaction profile, while 255 279/282 did not. Unlike wild-type Cx43, which binding is restricted to periphery GJ plaques, S365A, S365E, S368A, S368E, S373A bound throughout S373E mutant not bind all. Inability disengage from correlated increased plaque size half-life, maintaining channels an open, functional state. Quantitative clathrin-binding analyses revealed no significant alterations efficiency, suggesting that inability prevented maturation nonfunctional/endocytic channels, rather than interfering endocytosis directly. Collectively, our results indicate channel accrual, disengagement critical for closure transitioning endocytosis. Intriguingly, these transitional binding/release channel-aging steps are mediated hierarchical phosphorylation/dephosphorylation events S373, S365, S368, well-known Cx43 Akt, kinase A, C sites vicinity site.