Oxidative Activation of Protein Kinase Cγ through the C1 Domain

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) γ 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 PKCγ 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 PKCγ 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 PKCγ by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCγ enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCγ activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCγ at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCγ to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCγ were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCγ formed disulfide bonds in response to H2O2. H2O2-activated PKCγ was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCγ 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 PKCγ, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.