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D. Lin, J. Zhou, P.S. Zelenka, D.L. Boyle, D.J. Takemoto; Caveolin Regulation of Lens Epithelial Cell Gap Junctions through PKC . Invest. Ophthalmol. Vis. Sci. 2003;44(13):427.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Caveolins, principle structural proteins of the caveolae, mediate transmembrane signaling through direct interaction with diverse signaling molecules. Cell-cell gap junctional communications are regulated by environmental stimuli in the lens epithelial cells. Phorbol ester and growth factors can regulate lens gap junctions by activation of PKC γ, which in turn cause phosphorylation of connexin 43 and disassembly of gap junctions. This study demonstrates interactions of caveolin-1 with PKCγ and connexin43, and its regulation in gap junctions in response to growth factors. Methods: N/N 1003A lens epithelial cells (LECs), primary bovine LECs, and the stably transfected N/N 1003A LECs overexpressing PKCγ:GFP and point mutants as fusion proteins were used for the experiments. Cell surface gap junction Cx43 plaques, and colocalization of Cx43 and caveolin-1, were detected by confocal microscopy. Co-immunoprecipitation was performed to determine the in vivo protein-protein interactions. Redistribution of caveolin, PKCγ and Cx43 in response to growth factors was analyzed by isolation of detergent-resistant membranes on sucrose gradients and by consequent Western blotting. Results: Caveolin1 and 2 but not caveolin 3, was found in N/N1003A and primary bovine lens epithelial cells. TPA and IGF-I stimulated the interactions between caveolin 1 and PKCγ. However Cx43 was always associated with caveolin 1. TPA and IGF-I induced redistribution of caveolin1 and Cx43 from light density fractions to higher density fractions, indicating movement out of caveolae "lipid rafts". PKCγ::GFP fusion proteins overexpressed in N/N 1003A cells translocated to plasma membranes in the presence of TPA or IGF-I. Overexpression of PKCγ increased the interaction between PKCγ:GFP fusion protein and caveolin 1 or Cx43, and decreased gap junctional Cx43 plaques in N/N 1003A LEC without exogenous growth factors. However, overexpression of loss-of-function PKCγ mutants(activation loop or phosphorylation site mutations) did not decrease gap junctions, even though PKCγ still interacted with caveolin 1 and Cx43. Conclusions: Activation of PKCγ by TPA or IGF-I stimulated the interaction of PKCγ with caveolin1 and Cx43, both of which were initially colocalized in detergent resistant lipid rafts. This activation of PKCγ caused Cx43, caveolin 1 and PKCγ to localize out of caveolae. Use of loss-of-function PKCγ mutants suggests that PKCγ activity is required for gap junctions movement out of caveolae "lipid rafts".
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