title: Protein kinase C gamma 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 gamma (PKCgamma) acts as a primary oxidative stress sensor in the lens. PKCgamma has a Zn-finger C1B stress switch domain, residues 101-150. Mutation, H101Y, in the C1B domain of PKCgamma proteins causes a failure of the PKCgamma 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 PKCgamma 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 PKCgamma C1B mutants had lower basal enzyme activities and were not activated by H(2)O(2). Furthermore, the PKCgamma mutations caused a failure of endogenous wild type PKCgamma to be activated by H(2)O(2). These PKCgamma mutations abolished the effect of H(2)O(2) on phosphorylation of Cx43 and Cx50 by H(2)O(2) activation of PKCgamma. The cells with PKCgamma C1B mutations had more Cx43 and/or Cx50 gap junction plaques which were not decreased by H(2)O(2). Since open gap junctions could have a bystander effect this could cause apoptosis to occur. H(2)O(2) (100 microM, 3 h) activated a caspase-3 apoptotic pathway in the lens epithelial cells but was more severe in cells expressing PKCgamma mutations. The presence of 18alpha-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 PKCgamma C1B mutations on endogenous PKCgamma 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 PKCgamma 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 PKCgamma.
authors.affiliation: Department of Biochemistry, Kansas State University, 141 Chalmers Hall, Manhattan, KS 66506, USA.
authors.forename: Dingbo
authors.initials: D
authors.lastname: Lin
authors.forename: Denton
authors.initials: D
authors.lastname: Shanks
authors.forename: Om
authors.initials: O
authors.lastname: Prakash
authors.forename: Dolores J
authors.initials: DJ
authors.lastname: Takemoto
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology*; D053148:Caspase 3 / Q000235:genetics*; D002460:Cell Line; D018031:Connexin 43 / Q000032:analysis; D017630:Connexins / Q000032:analysis; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D005136:Eye Proteins / Q000032:analysis; D017629:Gap Junctions / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006034:Glycyrrhetinic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007908:Lens, Crystalline / Q000187:drug effects / Q000502:physiology*; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D011817:Rabbits; D000099264:Connexin 50
chemical_list: D053148:Caspase 3; D018031:Connexin 43; D017630:Connexins; D005136:Eye Proteins; D006034:Glycyrrhetinic Acid; D006861:Hydrogen Peroxide; D016877:Oxidants; D011493:Protein Kinase C; D000099264:Connexin 50; C119129:18alpha-glycyrrhetinic acid; C087857:protein kinase C gamma