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J. Biol. Chem., Vol. 279, Issue 50, 52714-52725, December 10, 2004

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

IDENTIFICATION OF PKC-BINDING SITES^*

Thu Annelise Nguyen, Larry J. Takemoto, and Dolores J. Takemoto¶

From the Department of Biochemistry and Division of Biology, Kansas State University, Manhattan, Kansas 66506

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

Received for publication, March 18, 2004 , and in revised form, September 29, 2004.

^* This work was supported by National Institutes of Health Grants EY13421 (to D. J. T.) and EY02932 (to L. J. T.). This is Publication Number 04-304-J from the Kansas Agriculture Experiment Station, patent pending. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, 103 Willard Hall, Kansas State University, Manhattan, KS 66506. Tel.: 785-532-7009; Fax: 785-532-7278; E-mail: dtak{at}ksu.edu.

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