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J. Biol. Chem., Vol. 280, Issue 14, 13682-13693, April 8, 2005

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Oxidative Activation of Protein Kinase C through the C1 Domain

EFFECTS ON GAP JUNCTIONS^*

Dingbo Lin and Dolores J. Takemoto

From the Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

The accumulation of reactive oxygen species (ROS, for example H₂O₂) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169–180). Protein kinase C (PKC) is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H₂O₂. Mutations (H101Y, G118D, S119P, and G128D) in the PKC Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839–849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760–1765). This could be due to a failure of the mutant PKC proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKC by H₂O₂ and the resultant effects on gap junction activity. H₂O₂ stimulated PKC enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H₂O₂-stimulated PKC activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H₂O₂, suggesting a direct oxidation of PKC at the Cys-rich C1 domain. H₂O₂ induced the C1 domain of PKC to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H₂O₂ on PKC were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKC formed disulfide bonds in response to H₂O₂. H₂O₂-activated PKC was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKC phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H₂O₂ caused oxidation of the C1 domain, activation of the PKC, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.

Received for publication, July 9, 2004 , and in revised form, December 23, 2004.

^* This work was supported by National Institutes of Health Grant EY13421 (to D. J. T.) and by a grant from the National Organization for Rare Disorders (to D. L.). This is publication 04-018-J from the Kansas Agricultural Experiment Station. 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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