Abstract
Gap junctions are intercellular conduits that permit the passage of ions, small metabolites, and signaling molecules between cells. Connexin32 (Cx32) is a major gap junction protein in the liver and brain. Phosphorylation is integral to regulating connexin assembly, degradation, and electrical and metabolic coupling, as well as to interactions with molecular partners. Cx32 contains two intracellular tyrosine residues, and tyrosine phosphorylation of Cx32 has been detected after activation of the epidermal growth factor receptor; however, the specific tyrosine residue and the functional implication of this phosphorylation remain unknown. To address the limited available information on Cx32 regulation by tyrosine kinases, here we used the Cx32 C-terminal (CT) domain in an in vitro kinase-screening assay, which identified ephrin (Eph) receptor family members as tyrosine kinases that phosphorylate Cx32. We found that EphB1 and EphA1 phosphorylate the Cx32CT domain residue Tyr243 Unlike for Cx43, the tyrosine phosphorylation of the Cx32CT increased gap junction intercellular communication. We also demonstrated that T-cell protein-tyrosine phosphatase dephosphorylates pTyr243 The data presented above along with additional examples throughout the literature of gap junction regulation by kinases, indicate that one cannot extrapolate the effect of a kinase on one connexin to another.
Highlights
Gap junctions are intercellular conduits that permit the passage of ions, small metabolites, and signaling molecules between cells
To determine whether ephrin type-B receptor 1 (EphB1) and T-cell protein-tyrosine phosphatase (TC-PTP) affect the level of Cx32 pTyr243 in cells, HeLa cells stably expressing Cx32 WT were transfected with EphB1 and/or the TC-PTP catalytic domain, and the tyrosine phosphorylation level was detected on immunoprecipitated Cx32 (Fig. 4A)
A few studies have investigated the regulation of gap junction intercellular communication (GJIC) by Eph receptors and ephrins; none have looked at the Cx32 isoform, interestingly, their observations of blocking communication through gap junctions were all the same
Summary
Gap junctions are intercellular conduits that permit the passage of ions, small metabolites, and signaling molecules between cells. Gap junctions are intercellular channels that permit the free passage of ions, small metabolites, and signaling molecules between neighboring cells. They are important for a number of physiological processes, including cell growth, differentiation, and synchronization. Another study showed that GJIC is inhibited at ectopic ephrin boundaries and that ephrin-B1 physically interacts with Cx43 and influences its distribution [19] These studies suggest a mechanism by which Eph receptors and ligands mediate control of cell-to-cell communication through phosphorylation of the gap junction proteins. We identified that the Eph receptor isoforms EphB1 and EphA1 phosphorylate Cx32CT residue Tyr243, an event that increases GJIC. This study provides evidence that characterization of the same kinase for different connexin isoforms is important, as one cannot extrapolate the effect of a kinase on one connexin to another
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