The epsilon subtype of protein kinase C is required for cardiomyocyte connexin-43 phosphorylation.
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, PKCepsilon 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 PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.
Doble
B W
BW
Institute of Cardiovascular Sciences, University of Manitoba, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.
Ping
P
P
Kardami
E
E
D000470:Alkaloids; D000818:Animals; D053119:Benzophenanthridines; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010617:Phenanthridines / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D051744:Protein Kinase C-epsilon; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014018:Tissue Distribution
D000470:Alkaloids; D053119:Benzophenanthridines; D018031:Connexin 43; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010617:Phenanthridines; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; C016299:chelerythrine; C497257:Prkce protein, rat; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D020928:Mitogen-Activated Protein Kinases; D013755:Tetradecanoylphorbol Acetate