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M. E. Barnett, R. McCulley, D. J. Takemoto; PKC Functions as a Regulator of Survival in Mammalian Lens. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5646.
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The primary purpose for this work is to investigate the potential regulatory and functional roles of PKCε in the mammalian lens.
Protein/protein interaction experiments were conducted using co-immunoprecipitation combined with Western blotting utilizing either whole lens homogenates or mitochondrial preparations. Protein/protein interactions were verified using immuno-histochemistry and confocal microscopy. Protein phosphorylation analysis was carried out with Western blotting. Enzymatic activities were measured with commercially available assays. Sections prepared for confocal, light, or electron microscopy were prepared from whole eyes or lenses that were fixed in 4% paraformaldehyde.
Western blotting demonstrated that both PKCγ as well as PKCε are expressed in the lens epithelial layers as well as in the differentiating fiber cells. They exist at lower amounts in the outer mature fibers and even less in the mature fibers. Co-immunoprecipitation of cytochrome c oxidase subunit IV (COIV) and PKCε from isolated lens mitochondria demonstrate that this interaction is enhanced in the absence of oxygen, and diminished in the presence of oxygen. The enhanced interaction is correlated with increased cytochrome c oxidase activity while the weakened interaction is associated with a loss of cytochrome c oxidase activity. Confocal microscopy results confirm that under normal hypoxic conditions in the lens, PKCε is localized to the mitochondria where it interacts with Tom-20 and COIV. Taken together, these results indicate that PKCε under normal hypoxic conditions in the lens interacts with mitochondrial components but in the presence of higher concentrations of oxygen, PKCε has a switch that allows it to respond differently to various stimuli.
Both pro-survival and pro-apoptotic roles have been cast for PKCε in various cell systems. In the hypoxic environment of the mammalian lens, co-immunoprecipitation and confocal experiments show that PKCε acts to preserve oxidative phosphorylation by binding to cytochrome c oxidase subunit IV, increasing its activity via phosphorylation and prolonging its half-life. This is likely related to its activity as an anti-apoptotic factor which may contribute to the needed stability of differentiating fibers as they transition to fiber cells. It is likely that PKCε is exerting other pro-survival effects upstream of cytochrome c oxidase in lens, contributing to the survival of differentiating fibers at multiple cellular locations.
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