Purpose: Fiber cell gap junction proteins connexin 46 (Cx46) and connexin 50 (Cx50) play distinct roles in the avascular lens. The purpose of this study was to determine how protein kinase Cgamma (PKCgamma) differentially regulates phosphorylation of Cx46 and Cx50 in oxidatively stressed lenses.
Methods: Sprague Dawley rats (six week old) were used in the experiments. PKCgamma enzyme activity was analyzed by use of the PepTag assay kit. Phosphorylation of caveolin-1, Cx46, and Cx50 was determined by immunoblotting. Lipid rafts were isolated by continuous sucrose gradient centrifugation. Lipid raft-localization of PKCgamma, Cx46, or Cx50 was demonstrated by immunoblotting. Association of caveolin-1 with PKCgamma, Cx46, or Cx50 was revealed by co-immunoprecipitation.
Results: H2O2 (100 microM) stimulated PKCgammaactivation in rat whole lens. Activated PKCgamma was recruited into caveolin-1 (Cav-1) containing lipid rafts and this activation enhanced the coimmunoprecipitation of Cav-1, Cx46, and Cx50 with PKCgamma. Both Cx50 and Cx46 were associated with Cav-1 in lipid rafts. H2O2 significantly induced threonine (Thr) phosphorylation of Cx46 and Cx50, and serine (Ser) phosphorylation of Cx50. However, There was only a small stimulation of Cx46 phosphorylation at Ser by H2O2, as Cx46 was already phosphorylated.
Conclusions: Activation of PKCgamma by H2O2 stimulated differential Ser phosphorylation of Cx50 versus Cx46, within lipid rafts. This suggests that Cx50 and Cx46 may have different functions in lens.