Oxidative Activation of Protein Kinase C gamma through the C1 Domain: Effects On Gap Junctions

The accumulation of reactive oxygen species (ROS, for example H sub(2)O sub(2)) 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) gamma is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H sub(2)O sub(2). Mutations (H101Y, G118D, S119P, and G128D) in the PKC gamma 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 gamma 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 gamma by H sub(2)O sub(2) and the resultant effects on gap junction activity. H sub(2)O sub(2) stimulated PKC gamma enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H sub(2)O sub(2)-stimulated PKC gamma activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H sub(2)O sub(2), suggesting a direct oxidation of PKC gamma at the Cys-rich C1 domain. H sub(2)O sub(2) induced the C1 domain of PKC gamma to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H sub(2)O sub(2) on PKC gamma were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKC gamma formed disulfide bonds in response to H sub(2)O sub(2). H sub(2)O sub(2)-activated PKC gamma was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKC gamma 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 H sub(2)O sub(2) caused oxidation of the C1 domain, activation of the PKC gamma , and inhibition of gap junctions. This inhibition