Purpose: To determine the effects of protein kinase Cgamma (PKCgamma) on phosphorylation of Cx43, the gap junction protein of lens epithelial cells, and on cell surface assembly/disassembly of Cx43-gap junction complexes.
Methods: Association and phosphorylation of Cx43 by PKCgamma was determined using co-immunoprecipitation and reaction with phosphoserine antisera. Activation of PKCgamma was with 200 nM phorbol ester for 30 to 60 min. Effects of specific PKC isoforms was determined after overexpression of either PKCalpha or PKCgamma for 24 h in N/N 1003A rabbit lens epithelial cells or in two retinal cell lines, WERI and Y79. Gap junction plaques were counted on the cell surface by immunolabeling of Cx43 using confocal microscopy.
Results: Co-immunoprecipitation of Cx43 with PKCgamma was observed only in cells over expressing PKCgamma and in cells activated with phorbol ester. Both overexpression and phorbol ester produced a rapid phosphorylation of Cx43 on serine. Cx43 cell surface gap junction plaques decreased in cells over expressing PKCgamma and in cells treated with phorbol ester. Similar results were observed using the retinal cell lines, WERI and Y79. The effect of PKCgamma overexpression was persistent for 7 days but total cell Cx43 was not decreased. Overexpression of PKCa resulted in an increase in cell surface gap junction plaques.
Conclusions: PKCgamma can be co-immunoprecipitated with Cx43 from lens epithelial cells using phorbol ester activation. PKCgamma phosphorylates Cx43 on serine and this causes disassembly and loss of gap junction Cx43 from the cell surface. Overexpression of PKCgamma confirmed that only this PKC isoform caused the loss of cell surface Cx43. Overexpression of PKCalpha, the other major lens PKC isoform, caused an increase in cell surface Cx43. The presence of PKCgamma and loss of surface Cx43 from two retinal cell lines, WERI and Y79, upon phorbol ester activation further suggests that activation of PKCgamma may be a common mechanism for control of cell surface Cx43.