The ε Subtype of Protein Kinase C Is Required for Cardiomyocyte Connexin-43 Phosphorylation

Abstract

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.

Exercise Preconditioning-Induced Early and Late Phase of Cardioprotection Is Associated With Protein Kinase C Epsilon Translocation

“…From these results, which indicated that p-PKCε Ser729 was key to induced cardioprotection, it was found that p-PKCε Ser729 could phosphorylate cytoplasmic membrane proteins such as CX43 and that the phosphorylation of CX43 could decrease cardiomyocyte GJ permeability, prevent the spreading of injury between conjoined cells and finally enhance resistance to ischemic injury. 30 Phosphorylation by PKCε opens mitoKATP channels, preserving mitochondrial function and generating local ROS, 31 which can further activate PKCε in a positive feedback mechanism and confer cardioprotection. 19 A significant quantitative difference in PKCε and p-PKCε Ser729 was observed between the early and late phases of EP.…”

The effects of connexin phosphorylation on gap junctional communication

“…PKCα and ε were found to associate with Cx43 in cardiomycytes (Bowling et al, 2001). Fibroblast growth factor-2, which decreases cardiomyocyte gap junctional permeability and increases Cx43 phosphorylation, increased colocalization of PKCε with Cx43 (Doble, Ping, & Kardami, 2000). Thus, it appears that several members of the conventional and novel PKC families influence gap junctional communication.…”

Intercellular Connections in the Heart: The Intercalated Disc

“…Conversely, phosphorylation of Ser-365 by protein kinase A (PKA) promotes gap junction assembly and communication (Burghardt et al, 1995;Solan et al, 2007;TenBroek et al, 2001). Although several isozymes of protein kinase C (PKC) phosphorylate connexin-43 in diverse cell types and tissues, PKC is the only isoform that phosphorylates it at the ID (Bowling et al, 2001;Doble et al, 2000;Lampe et al, 2000;Lin et al, 2003;Saez et al, 1997). Consistent with this, PKC suppresses gap junction communication in the ischemic heart through phosphorylation of connexin-43 at Ser-368 (Ek-Vitorin et al, 2006;Hund et al, 2007;Hund et al, 2008).…”