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Regulation of Gap Junction Activity through Caveolin-1–Containing Lipid Rafts
1From the Department of Biochemistry, Kansas State University, Manhattan, Kansas; and the 2Laboratory of Molecular and Developmental Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland.
PURPOSE. To demonstrate the interactions of PKC
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, PKC, and Cx43 were analyzed by sucrose gradients and by consequent Western blot analysis. Cell surface gap junction Cx43 plaques were detected by confocal microscopy. PKC 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 PKC into Cav-1–containing lipid rafts and stimulated the interactions of PKC 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. PKC redistributed with Cav-1– and Cx43-containing fractions on stimulation with TPA or IGF-1. Overexpression of PKC-enhanced green fluorescent protein (EGFP) increased the interaction of PKC-EGFP with Cav-1 and Cx43 and decreased gap junction Cx43 plaques without exogenous growth factors. Overexpression of a loss-of-function PKC mutant did not decrease gap junction Cx43 plaques or cause redistribution in lipid rafts, even though the PKC mutant still interacted with Cav-1 and Cx43.
CONCLUSIONS. Activation of PKC by TPA or IGF-1 stimulated the interaction of PKC with Cav-1 and Cx43 in lipid rafts, causing Cx43, Cav-1, and PKC to redistribute within the lipid rafts, and this resulted in a decrease in gap junction plaques.
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