Abstract
We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma)to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565-572; Lin, D., Boyle, D. L., and Takemoto, D. J. (2003) Investig. Ophthalmol. Vis. Sci. 44, 1160-1168). Gap junction activity in lens epithelial cells is regulated by PKCgamma-mediated phosphorylation of Cx43. PKCgamma activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKCgamma-C1B domain that are responsible for its interaction with 14-3-3epsilon. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKCgamma. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3epsilon, resulting in the release of endogenous cellular PKCgamma from 14-3-3epsilon, in vivo or in vitro, in activation of PKCgamma enzyme activity, phosphorylation of PKCgamma, in the subsequent translocation of PKCgamma to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 microM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKCgamma are regulated by its binding to 14-3-3epsilon at the C1B domain of PKCgamma. Synthetic peptides corresponding to these regions of PKCgamma successfully competed for the binding of 14-3-3epsilon to endogenous PKCgamma, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.
Highlights
Proteins continue to generate interest because of their roles in signal transduction pathways that control cell cycle checkpoints, MAP kinase activation, and apoptosis (4 – 6)
LEDGF Increases DAG Levels, PKC␥ Enzyme Activity, and PKC␥ Autophosphorylation—We have shown previously that PKC␥ is a major regulator of gap junction assembly/disassembly when both PKC␣ and -␥ are activated in lens epithelial cells [34] and that LEDGF can cause the translocation of PKC␥, not PKC␣, from the cytosol to the membrane
The results demonstrate that 100 M peptides for 2 h can compete endogenous PKC␥ from binding to endogenous 14-3-3⑀. 14-3-3⑀ was co-immunoprecipitated with Raf-1, and its association was not affected by the presence of C1B1 or C1B5 peptides (Fig. 3C)
Summary
Proteins continue to generate interest because of their roles in signal transduction pathways that control cell cycle checkpoints, MAP kinase activation, and apoptosis (4 – 6). We have shown that growth factors such as IGF-I or LEDGF can induce PKC␥ translocation to membranes, interactions of the PKC␥ with the gap junction protein, connexin 43, and this results in the inhibition of gap junction activity in lens epithelial cells [1, 2]. Western blots from cells, which were treated with 10 ng/ml LEDGF for 15 min or 25 ng/ml IGF-I for 15 min, showed a decrease in co-immunoprecipitation of PKC␥ and 14-3-3⑀ compared with control without treatment (Fig. 2A).
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