Abstract
Connexin43 (Cx43) assembly and degradation, the regulation of electrical and metabolic coupling, as well as modulating the interaction with other proteins, involve phosphorylation. Here, we identified and characterized the biological significance of a novel tyrosine kinase that phosphorylates Cx43, tyrosine kinase 2 (Tyk2). Activation of Tyk2 led to a decrease in Cx43 gap junction communication by increasing the turnover rate of Cx43 from the plasma membrane. Tyk2 directly phosphorylated Cx43 residues Tyr-247 and Tyr-265, leading to indirect phosphorylation on residues Ser-279/Ser-282 (MAPK) and Ser-368 (PKC). Although this phosphorylation pattern is similar to what has been observed following Src activation, the response caused by Tyk2 occurred when Src was inactive in NRK cells. Knockdown of Tyk2 at the permissive temperature (active v-Src) in LA-25 cells decreased Cx43 phosphorylation, indicating that although activation of Tyk2 and v-Src leads to phosphorylation of the same Cx43CT residues, they are not identical in level at each site. Additionally, angiotensin II activation of Tyk2 increased the intracellular protein level of Cx43 via STAT3. These findings indicate that, like Src, Tyk2 can also inhibit gap junction communication by phosphorylating Cx43.
Highlights
Cx43 channels can be regulated by a variety of molecules and physiological conditions (e.g. Ca2ϩ, pH, and intercellular voltage), including through phosphorylation [2, 3]
Src has been the only non-receptor tyrosine kinase to directly phosphorylate Cx43 leading to an inhibition of GJIC and subsequent gap junction disassembly [59, 60]
265 and lead to indirect phosphorylation on residues Ser-368 and Ser-279/Ser-282. This phosphorylation pattern is similar to what has been observed following Src activation [51], importantly, the response caused by tyrosine kinase 2 (Tyk2) occurred when Src was inactive in Normal Rat Kidney (NRK) cells
Summary
Tyk Directly Interacts with and Phosphorylates the Cx43CT Domain—Src is the only known tyrosine kinase to both directly phosphorylate the Cx43CT domain (pTyr-247 and pTyr-265). An in vitro tyrosine phosphorylation screen performed by Eurofins Scientific (KinaseProfiler) found that Tyk phosphorylated purified Cx43CT236–382 (Fig. 1A). To confirm the interaction between Cx43 and Tyk, purified GST-tagged Cx43CT236–382 was immobilized on glutathione-Sepharose beads, and lysate from MDA-MB-231 cells that express Tyk (but not Cx43) was used in a pulldown assay (Fig. 1B). Identifying the Cx43CT Tyrosine Residue(s) Phosphorylated by Tyk2—Purified Cx43CT236–382 was incubated in vitro with active Tyk (p-Tyk, Life Technologies) as described previously [49, 50], and phosphorylation was confirmed using an anti-phosphotyrosine antibody (Fig. 2B). To confirm that tyrosine phosphorylation can occur at residues other than Tyr-247 and Tyr-265, the in vitro kinase assay was performed using the Cx43CT236–382 (Y247,265F) (2YF) construct, and phosphorylation was detected with an anti-phosphotyrosine antibody (Fig. 2B). The data indicate that a majority of Tyk phosphorylation occurs on Tyr-247 and Tyr265 (of note, Tyr-247 and Tyr-265 phospho-specific antibodies will be used to further characterize these sites), other tyrosine sites (i.e. Tyr-267 and Tyr-313) can be phosphorylated by Tyk in vitro
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