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 γ (PKCγ) acts as a primary oxidative stress sensor in the lens. PKCγ has a Zn-finger C1B stress switch domain, residues 101–150. Mutation, H101Y, in the C1B domain of PKCγ proteins causes a failure of the PKCγ 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 PKCγ 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 PKCγ C1B mutants had lower basal enzyme activities and were not activated by H2O2. Furthermore, the PKCγ mutations caused a failure of endogenous wild type PKCγ to be activated by H2O2. These PKCγ mutations abolished the effect of H2O2 on phosphorylation of Cx43 and Cx50 by H2O2 activation of PKCγ. The cells with PKCγ C1B mutations had more Cx43 and/or Cx50 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 μM, 3 h) activated a caspase-3 apoptotic pathway in the lens epithelial cells but was more severe in cells expressing PKCγ mutations. The presence of 18α-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 PKCγ C1B mutations on endogenous PKCγ 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 PKCγ 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 PKCγ.