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Dingbo Lin, Daniel L. Boyle, Dolores J. Takemoto; IGF-I-Induced Phosphorylation of Connexin 43 by PKCγ: Regulation of Gap Junctions in Rabbit Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(3):1160-1168. doi: 10.1167/iovs.02-0737.
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purpose. To determine the role of PKCγ in insulin-like growth factor (IGF)-I-induced phosphorylation of connexin (Cx)43 and control of gap junctions in lens epithelial cells.
methods. N/N1003A rabbit lens epithelial cells were used in the experiments. PKC translocation or in vivo Cx43 phosphorylation on serine was determined by Western blot analysis. Gap junction activity was measured by scrape-loading/dye-transfer assay. The number of cell surface gap junction plaques was detected by confocal microscopy. The interaction between PKCγ and Cx43 was determined by coimmunoprecipitation. In vitro Cx43 phosphorylation was assayed by PKC assay kit. Endogenous sn-1,2-diacylglycerol (DAG) was measured by detecting 32P-labeled phosphatidic acid.
results. IGF-I stimulated activation and translocation of PKCγ in a dose- and time-dependent manner, acidic FGF (aFGF) had no effect on translocation of PKCγ, and PKCα was not translocated by IGF-I at 25 ng/mL. PKCγ translocation resulted in coimmunoprecipitation with and phosphorylation of Cx43. IGF-I- or DAG-induced activation of PKCγ caused a decrease in gap junctions. IGF-I increased endogenous DAG. Exogenous CaCl2 and DAG stimulated PKCγ translocation. TMB-8, an internal calcium mobilization inhibitor, blocked CaCl2-induced PKCγ translocation; however, it had no effect on IGF-I- or DAG-induced translocation of PKCγ.
conclusions. PKCγ mediated IGF-I-induced decreases in gap junctional communication through interaction with and phosphorylation of Cx43. IGF-I caused an increase in DAG, and this increased translocation of PKCγ, whereas mobilization of calcium was not essential for IGF-I-stimulated translocation of PKCγ.
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