Human Spinocerebellar Ataxia Mutations in the C1B Domain of PKC Cause Altered Cell Gap Junctional Communication in Lens Epithelial Cells

Abstract

Abstract: : Purpose: Missense mutations in the C1B domain of PKCγ cause Spinocerebellar ataxia (SCA–14) in humans. The purpose of this study is to determine how these mutations alter cell gap junctional communication in lens epithelial cells. Methods: Mutations (H101Y, S119P, or G128D) were introduced into the wild type PKC γ:EGFP vector by site–directed mutagenesis. These mutations were transfected into 80 % confluent N/N1003A lens epithelial cells, and stably transfected cells were selected in G418–containing DMEM media. Activation of these mutants by phorbol–12 myristate 13 acetate (TPA) or H₂O₂ was determined by PKCγ enzyme activity assay and translocation of these EGFP fusion proteins was measured by confocal microscopy and Western blot. Direct oxidation and disulfide bond formation within PKCγ were determined using off–diagonal 2–dimensional SDS–PAGE and Western blot. Cell surface Cx43 gap junction plaques were immuno–labeled and counted by confocal microscopy. Results: PKCγH101Y and G128D had basal enzyme activity similar or identical to the wild type but were not activated by TPA or H₂O₂. PKCγS119P had lower basal enzyme activity than the wild type and was also not activated by TPA or H₂O₂. PKCγ activation by TPA or H₂O₂ resulted from direct oxidation and disulfide bond formation in –SH groups of Cys residues in the C1 domain, and the mutations in the C1B domain (H101Y, S119P, or G128D) caused abolishment of these effects, suggesting that oxidative stress–activation of PKCγ is through the C1B domain. Overexpression of the PKCγ mutants resulted in a lower response of cell surface Cx43 gap junction plaques which are normally disassembled by TPA or H₂O₂–activation of the wild type PKCγ. Conclusions: Inhibition of gap junctions by PKC γ could provide a protection to cells against oxidative stress, whereas, ataxia mutations could cause an improper defense response to oxidative stress. Support: EY13421 to DJT, and a grant from the National Organization for Rare Diseases (NORD) to DL

Keywords: stress response • gap junctions/coupling • signal transduction