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Inhibition of Gap Junction Activity through the Release of the C1B Domain of Protein Kinase Cγ (PKCγ) from 14-3-3

T. Nguyen,L. Takemoto,D. Takemoto

Published 2004 in Journal of Biological Chemistry

ABSTRACT

We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cγ (PKCγ)to the membrane and the phosphorylation of Cx43 by PKCγ 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 PKCγ-mediated phosphorylation of Cx43. PKCγ 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 PKCγ-C1B domain that are responsible for its interaction with 14-3-3ϵ. Two sites, C1B1 (residues 101–112) and C1B5 (residues 141–151), are located within the C1 domain of PKCγ. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3ϵ, resulting in the release of endogenous cellular PKCγ from 14-3-3ϵ, in vivo or in vitro, in activation of PKCγ enzyme activity, phosphorylation of PKCγ, in the subsequent translocation of PKCγ 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 μm 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 PKCγ are regulated by its binding to 14-3-3ϵ at the C1B domain of PKCγ. Synthetic peptides corresponding to these regions of PKCγ successfully competed for the binding of 14-3-3ϵ to endogenous PKCγ, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.

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