Protein kinase C γ mutations in the C1B domain cause caspase-3-linked apoptosis in lens epithelial cells through gap junctions

Failure to control oxidative stress is closely related to aging and to a diverse range of human diseases. We have reported that protein kinase C gamma (PKC gamma) acts as a primary oxidative stress sensor in the lens. PKC gamma has a Zn-finger C1B stress switch domain, residues 101-150. Mutation, H101Y, in the C1B domain of PKC gamma proteins causes a failure of the PKC gamma oxidative stress response [Lin, D., Takemoto, D.J., 2005. Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions. J. Biol. Chem. 280, 13682-13693]. Some human neurodegenerative spinocerebellar ataxia type 14 are caused by mutations in the PKC gamma C1B domain. In the current study we have investigated the effects of these mutations on lens epithelial cell responses to oxidative stress. The results demonstrate that PKC gamma C1B mutants had lower basal enzyme activities and were not activated by H2O2. Furthermore, the PKC gamma mutations caused a failure of endogenous wild type PKC gamma to be activated by H2O2. These PKC gamma mutations abolished the effect of H2O2 on phosphorylation of Cx43 and Cx50 by H2O2 activation of PKC gamma. The cells with PKC gamma C1B mutations had more Cx43 and/orCx50 gap junction plaques which were not decreased by H2O2. Since open gap junctions could have a bystander effect this could cause apoptosis to occur. H2O2 (100 mu M, 3 h) activated a caspase-3 apoptotic pathway in the lens epithelial cells but was more severe in cells expressing PKC gamma mutations. The presence of I 18 alpha-glycyrrhetinic acid (AGA), an inhibitor of gap junctions, decreased Cx43 and Cx50 protein levels and gap junction plaque number. This reduction in gap junctions by AGA resulted in inhibition of H2O2-induced apoptosis. Our results demonstrate that there is a dominant negative effect of PKC gamma C1B mutations on endogenous PKC gamma which results in loss of control of gap junctions. Modeled structures suggest that the severity of C1B mutation effects may be related to the extent of loss of C1B structure. Mutations in the C1B domain of PKC gamma result in increased apoptosis in lens epithelial cells. This can be prevented by a gap junction inhibitor. Thus, propagation of apoptosis from cell-to-cell in lens epithelial cells may be through open gap junctions. The control of gap junctions requires PKCy. (c) 2007 Elsevier Ltd. All rights reserved.