Regulation of connexin43 by the tyrosine kinase Tyk2

Abstract

The identification of serine and tyrosine kinases that phosphorylate Connexin43 (Cx43) have been instrumental towards understanding the regulation of gap junction intercellular communication (GJIC). In this study, we performed an in vitro tyrosine phosphorylation screen using the purified carboxyl terminal domain (CT) of Cx43 and identified phosphorylation by tyrosine kinase 2 (Tyk2). This interaction was confirmed by using a GST‐tagged pull down assay involving the Cx43CT and MDA‐MB‐231 cell lysate (expresses Tyk2) as well as a co‐immunoprecipitation assay in Tyk2 transfected NRK cells (expresses Cx43). Both mass spectrometry analysis of the Cx43CT phosphorylated by Tyk2 in vitro and immunoblotting using phospho‐specific antibodies in constitutively active Tyk2 transfected NRK cells show that Tyk2 increased the level of Cx43 pY247 and pY265. The phosphorylation on Cx43 caused by Tyk2 is independent of Src which is known to phosphorylate Cx43 directly on Y247 and Y265. Immunofluorescence results combined with other molecular biological assays suggested that Tyk2 decreases the half‐life of the Cx43 gap junction plaque. Based upon the correlation between increased Angiotensin II (Ang II) levels leading to increased activation of Tyk2 and down regulation of ventricular Cx43 GJIC, we found that transfection of wild type Tyk2 in NRK cells enhanced Ang II induced tyrosine and serine phosphorylation of Cx43 (S279/282, S368, Y265, Y247), which explains the down regulation of Cx43 GJIC. Altogether, we identified a novel tyrosine kinase for Cx43 that leads to decreased GJIC and may be involved in Ang II mediated gap junction remodeling.