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

Abstract

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 and Takemoto (2005) (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 H 2 O 2 -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 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 cellto-cell in lens epithelial cells may be through open gap junctions. The control of gap junctions requires PKCγ.

Protein kinase C epsilon activates lens mitochondrial cytochrome c oxidase subunit IV during hypoxia

“…Because gap junctions are used in cell-tocell communication pathways, PKCγout mice display learning deficits, insensitivity to pain, do not develop tolerance to alcohol like normal mice ( Abeliovich, et al, 1993), and are more sensitive to hydrogen peroxide induced cataract formation (Lin, 2006). It is thought that these deficits are partially a result of the improper control of gap junctions due to loss of PKCγ (Lin, et al, 2007).…”

Drug target identification at the crossroad of neuronal apoptosis and survival

“…Among potential deregulated therapeutic targets, Hspa5 (i), Limk2 (ii), Mcl1 (iii), and Gja8 were all found to be upregulated by Igf1 and Pacap. Inhibition of Gja8 by 18alpha-glycyrrhetinic acid [35] and activation of Hspa5 by salubrinal, 2DG, and NE-100 [36,37] have already been shown to support cell survival. Similarly, treatment with LIMKi and A-1210477, two selective inhibitors of Limk2 and Mcl1, respectively, prevents apoptosis in cancer [38,39].…”

Protection from ataxia-linked apoptosis by gap junction inhibitors

“…PKCγ SCA-14 mutant plasmid construction and transfection C-terminal EGFP-tagged PKCγ SCA14 mutants (H101Y, S119P, or G128D) were generated previously [18]. In addition, PKCγ SCA14 mutant cDNA was cloned into the pCMVHA vector (Clontech, California) to generate N-terminal HA-tagged PKCγ SCA14 mutants by introducing 5'EcoRI and 3'KpnI sites.…”

“…We have previously demonstrated that lens epithelial cells with PKCγ SCA14 mutants (H101Y, S119P, or G128D) lack PKCγ enzyme activity even when endogenous PKCγ is present [18]. This dominant negative effect on endogenous PKCγ has been linked to failure of control of gap junctions and this causes cells to be more susceptible to H 2 O 2 -induced, caspase-3-dependent cell apoptosis [18].…”

“…This dominant negative effect on endogenous PKCγ has been linked to failure of control of gap junctions and this causes cells to be more susceptible to H 2 O 2 -induced, caspase-3-dependent cell apoptosis [18]. In the current study, we used the hippocampal HT22 cells which has endogenous PKCγ, thus, the overexpression of the SCA14 mutant PKCγ should more directly reflect SCA14 patients who are heterozygous.…”