IGF-I-induced phosphorylation of connexin 43 by PKCgamma: regulation of gap junctions in rabbit lens epithelial cells.

Abstract

To determine the role of PKCgamma in insulin-like growth factor (IGF)-I-induced phosphorylation of connexin (Cx)43 and control of gap junctions in lens epithelial cells. 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 PKCgamma 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 (32)P-labeled phosphatidic acid. IGF-I stimulated activation and translocation of PKCgamma in a dose- and time-dependent manner, acidic FGF (aFGF) had no effect on translocation of PKCgamma, and PKCalpha was not translocated by IGF-I at 25 ng/mL. PKCgamma translocation resulted in coimmunoprecipitation with and phosphorylation of Cx43. IGF-I- or DAG-induced activation of PKCgamma caused a decrease in gap junctions. IGF-I increased endogenous DAG. Exogenous CaCl(2) and DAG stimulated PKCgamma translocation. TMB-8, an internal calcium mobilization inhibitor, blocked CaCl(2)-induced PKCgamma translocation; however, it had no effect on IGF-I- or DAG-induced translocation of PKCgamma. PKCgamma 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 PKCgamma, whereas mobilization of calcium was not essential for IGF-I-stimulated translocation of PKCgamma.