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.
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.
Authors: Jessica R Nealon; Stephen J Blanksby; Paul J Donaldson; Roger J W Truscott; Todd W Mitchell Journal: Invest Ophthalmol Vis Sci Date: 2011-02-09 Impact factor: 4.799
Authors: K Barth; M Gentsch; R Bläsche; A Pfüller; I Parshyna; R Koslowski; G Barth; M Kasper Journal: Histochem Cell Biol Date: 2004-11-20 Impact factor: 4.304
Authors: Anastasia F Thévenin; Tia J Kowal; John T Fong; Rachael M Kells; Charles G Fisher; Matthias M Falk Journal: Physiology (Bethesda) Date: 2013-03