purpose. To demonstrate the interactions of PKCγ with caveolin (Cav)-1 and connexin (Cx) 43 in lipid rafts and its regulation of gap junctions.

methods. N/N1003A lens epithelial cells, bovine primary lens epithelial cells, and stably transfected N/N1003A lens epithelial cells were used. Coimmunoprecipitation and Western blot analysis were used to detect protein expression and their interactions. Cav-1–containing lipid rafts and redistribution of Cav-1, PKCγ, and Cx43 were analyzed by sucrose gradients and by consequent Western blot analysis. Cell surface gap junction Cx43 plaques were detected by confocal microscopy. PKCγ activity was measured with a PKC assay kit.

results. Cav-1 and-2 were found in N/N1003A and bovine primary lens epithelial cells. Cx43 was associated with Cav-1 in lipid rafts. Phorbol ester (TPA) and insulin-like growth factor (IGF)-1 recruited PKCγ into Cav-1–containing lipid rafts and stimulated the interactions of PKCγ with Cav-1 and Cx43. TPA and IGF-1 induced redistribution of Cav-1 and Cx43 from light-density fractions to higher density fractions on sucrose gradients. PKCγ redistributed with Cav-1–and Cx43-containing fractions on stimulation with TPA or IGF-1. Overexpression of PKCγ-enhanced green fluorescent protein (EGFP) increased the interaction of PKCγ-EGFP with Cav-1 and Cx43 and decreased gap junction Cx43 plaques without exogenous growth factors. Overexpression of a loss-of-function PKCγ mutant did not decrease gap junction Cx43 plaques or cause redistribution in lipid rafts, even though the PKCγ mutant still interacted with Cav-1 and Cx43.

conclusions. Activation of PKCγ by TPA or IGF-1 stimulated the interaction of PKCγ with Cav-1 and Cx43 in lipid rafts, causing Cx43, Cav-1, and PKCγ to redistribute within the lipid rafts, and this resulted in a decrease in gap junction plaques.