3 years ago

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

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

Publisher URL: http://doi.org/10.1091/mbc.E16-07-0496

DOI: 10.1091/mbc.E16-07-0496

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