The epsilon subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation

Gap junctions (GJs), composed of connexins, are intercellular channels ensu ring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibro blast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by s timulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to pr otein kinase C (PKC) and mitogen-activated protein kinase, we first investi gated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhib itor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membr ane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKC epsilon displayed a d iscontinuous pattern of localization at intercellular contact sites and par tial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKC epsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKC epsilon. In immunoprecipitation experiments using specific anti-Cx43 a ntibodies, PKC epsilon but not PKC alpha coprecipitated with Cx43, FGF-2 in creased levels of coprecipitated PKC epsilon, suggesting increased associat ion between PKC epsilon and Cx43 on stimulation. Transient gene transfer an d overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKC epsilon decreased cardiomyocyte Cx43 phosphorylation signific antly. We conclude that PKC mediates the FGF-2-induced effects on cardiac G Js and that PKC epsilon likely interacts with and phosphorylates cardiac Cx 43 at sites of intercellular contact.