Protein kinase Cgamma regulation of gap junction activity through caveolin-1-containing lipid rafts.
OBJECTIVE
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.
Lin
Dingbo
D
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.
Zhou
Jianzheng
J
Zelenka
Peggy S
PS
Takemoto
Dolores J
DJ
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D018031:Connexin 43 / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007908:Lens, Crystalline / Q000187:drug effects / Q000378:metabolism*; D050356:Lipid Metabolism; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D051242:Caveolin 1; D022461:Caveolins; D018031:Connexin 43; D007527:Isoenzymes; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D007334:Insulin-Like Growth Factor I; C087857:protein kinase C gamma; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate