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

Abstract

Protein Kinase C (PKC) isoforms have been identified as major cellular signaling proteins that act directly in response to oxidation conditions. In retina and lens two isoforms of PKC respond to changes in oxidative stress, PKCγ and PKCε, while only PKCε is found in heart. In heart the PKCε acts on connexin 43 to protect from hypoxia. The presence of both isoforms in the lens led to this study to determine if lens PKCε had unique targets. Both lens epithelial cells in culture and whole mouse lens were examined using PKC isoform-specific enzyme activity assays, coimmunoprecipitation, confocal microscopy, immunoblots, and light and electron microscopy. PKCε was found in lens epithelium and cortex but not in the nucleus of mouse lens. The PKCε isoform was activated in both epithelium and whole lens by 5% oxygen when compared to activity at 21% oxygen. In hypoxic conditions (5% oxygen) the PKCε co-immunoprecipitated with the mitochondrial cytochrome C oxidase IV subunit (CytCOx). Concomitant with this the CytCOx enzyme activity was elevated and increased co-localization of CytCOx with PCKε was observed using immunolabeling and confocal microscopy. In contrast, no hypoxia-induced activation of CytCOx was observed in lenses from the PKCε knockout mice. Lens from 6 week old PKCε knockout mice had a disorganized bow region which was filled with vacuoles indicating a possible loss of mitochondria but the size of the lens was not altered. Electron microscopy demonstrated that the nuclei of the PCKε knockout mice were abnormal in shape. Thus, PKCε is found to be activated by hypoxia and this results in the activation of the mitochondrial protein CytCOx. This could protect the lens from mitochondrial damage under the naturally hypoxic conditions observed in this tissue. Lens oxygen levels must remain low. Elevation of oxygen which occurs during vitreal detachment or liquification is associated with cataracts. We hypothesize that elevated oxygen could cause inhibition of PKCε resulting in a loss of mitochondrial protection.

Hypoxia-Regulated Activity of PKCε in the Lens

“…6A). Considering that hypoxia is the natural state of the lens 1–3 and our previous data that PKCε is activated in the fresh lens, 50 we confirmed the activation of PKCε by detecting phosphorylated PKCε on Ser-729 in the whole lens lysates (Figs. 6B, 6C).…”

“…Previously, we demonstrated that PKCε is a prerequisite for hypoxia-induced activation of the CytCOxIV. 50 The second possibility may result from an effect of PKCε on Cx43 which is absent in PKCε-KO mice.…”

“…We found that in the lens, like in the heart, hypoxia activates PKCε which is associated with CytCOxIV in the mitochondria. 50 We also demonstrated that hypoxia-induced activation of CytCOx in the lens is PKCε dependent: Hypoxia not only activated PKCε but also stimulated interaction with CytCOx, which was not observed in lenses from the PKCε knockout mice (KO). However, it is not certain what role PKCε plays in the control of lens homeostasis.…”

PI3K–AKT Pathway Protects Cardiomyocytes Against Hypoxia-Induced Apoptosis by MitoKATP-Mediated Mitochondrial Translocation of pAKT

“…CcO is a large transmembrane protein complex located on the inner mitochondrial [14]. Previous studies found that the protein kinase C could protect the lens from mitochondria damage under hypoxia through the activation of CcO enzyme activity [25]. In order to further clarify the mechanisms by which the PI3K-AKT pathway inhibits apoptosis, the activity of CcO was evaluated in cultured cardiomyocytes.…”

Canonical Bcl-2 Motifs of the Na⁺/K⁺ Pump Revealed by the BH3 Mimetic Chelerythrine: Early Signal Transducers of Apoptosis?

“…PKCs play important roles in lens development such as protection against hypoxia by PKCε of mitochondrial c oxidase IV subunits [4]. Inhibition of PKC, and hence phosphorylation of proteins involved in vital cellular functions, elicits apoptosis [5,6].…”