Protein kinase Cgamma regulation of gap junction activity through caveolin-1-containing lipid rafts

Invest Ophthalmol Vis Sci. 2003 Dec;44(12):5259-68. doi: 10.1167/iovs.03-0296.


Purpose: To demonstrate the interactions of PKCgamma 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, PKCgamma, and Cx43 were analyzed by sucrose gradients and by consequent Western blot analysis. Cell surface gap junction Cx43 plaques were detected by confocal microscopy. PKCgamma 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 PKCgamma into Cav-1-containing lipid rafts and stimulated the interactions of PKCgamma 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. PKCgamma redistributed with Cav-1- and Cx43-containing fractions on stimulation with TPA or IGF-1. Overexpression of PKCgamma-enhanced green fluorescent protein (EGFP) increased the interaction of PKCgamma-EGFP with Cav-1 and Cx43 and decreased gap junction Cx43 plaques without exogenous growth factors. Overexpression of a loss-of-function PKCgamma mutant did not decrease gap junction Cx43 plaques or cause redistribution in lipid rafts, even though the PKCgamma mutant still interacted with Cav-1 and Cx43.

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

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cattle
  • Caveolin 1
  • Caveolins / metabolism*
  • Cells, Cultured
  • Centrifugation, Density Gradient
  • Connexin 43 / metabolism
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism*
  • Gap Junctions / metabolism*
  • Green Fluorescent Proteins
  • Insulin-Like Growth Factor I / pharmacology
  • Isoenzymes / metabolism
  • Lens, Crystalline / drug effects
  • Lens, Crystalline / metabolism*
  • Lipid Metabolism*
  • Luminescent Proteins
  • Microscopy, Confocal
  • Mutagenesis, Site-Directed
  • Protein Kinase C / metabolism*
  • Rabbits
  • Tetradecanoylphorbol Acetate / pharmacology
  • Transfection


  • Caveolin 1
  • Caveolins
  • Connexin 43
  • Isoenzymes
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Insulin-Like Growth Factor I
  • protein kinase C gamma
  • Protein Kinase C
  • Tetradecanoylphorbol Acetate