PKC-gamma phosphorylation of connexin 46 in the lens cortex

Purpose: To identify the role of PKC-gamma in control of and phosphorylatio n of connexin 46 (Cx46) in the lens cortex. Methods: The association between PKC-gamma and Cx46 was determined by co-im munoprecipitation from whole lens. Phosphorylation of Cx46 and activity of PKC-gamma were determined using Western blots, PKC activity assays, and inh ibition of PKC activity by addition of isoform-specific PKC pseudosubstrate inhibitors. Results: Co-localization of PKC-gamma and Cx46 was observed in the bow regi ons and cortical regions of rat lens. PKC-gamma was not observed in the nuc lear region and Cx46 was not observed in the epithelial layer. PKC-alpha wa s not found in lens cortex or nuclear regions. PKC-gamma could be co-immuno precipitated with Cx46 from lens cortical regions. Cx46 was phosphorylated on both serine and threonine. No tyrosine phosphorylation was observed. The PKC-gamma specific pseudosubstrate inhibitor caused a 73% inhibition of se rine phosphorylation on Cx46 at 1 muM, and, 36% inhibition of threonine pho sphorylation at the same concentration. Inhibition of phosphorylation of Cx 46 with PKC-alpha pseudosubstrate inhibitor was not observed. Conclusions: PKC-gamma may phosphorylate Cx46, primarily on serine in whole lens. A role for PKC-gamma in control of lens cortical gap junctions is su ggested.