Journal
CIRCULATION RESEARCH
Volume 86, Issue 3, Pages 293-301Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.RES.86.3.293
Keywords
cardiomyocyte gap junction; protein kinase C epsilon; fibroblast growth factor-2; phosphorylation
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Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating 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 protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated 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 inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKC epsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial 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 antibodies, PKC epsilon but not PKC alpha coprecipitated with Cx43, FGF-2 increased levels of coprecipitated PKC epsilon, suggesting increased association between PKC epsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKC epsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKC epsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.
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