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Targeted redox inhibition of protein phosphatase 1 by Nox4 regulates eIF2α‐mediated stress signaling
(2016)
Phosphorylation of translation initiation factor 2α (eIF2α) attenuates global protein synthesis but enhances translation of activating transcription factor 4 (ATF4) and is a crucial evolutionarily conserved adaptive pathway during cellular stresses. The serine–threonine protein phosphatase 1 (PP1) deactivates this pathway whereas prolonging eIF2α phosphorylation enhances cell survival. Here, we show that the reactive oxygen species‐generating NADPH oxidase‐4 (Nox4) is induced downstream of ATF4, binds to a PP1‐targeting subunit GADD34 at the endoplasmic reticulum, and inhibits PP1 activity to increase eIF2α phosphorylation and ATF4 levels. Other PP1 targets distant from the endoplasmic reticulum are unaffected, indicating a spatially confined inhibition of the phosphatase. PP1 inhibition involves metal center oxidation rather than the thiol oxidation that underlies redox inhibition of protein tyrosine phosphatases. We show that this Nox4‐regulated pathway robustly enhances cell survival and has a physiologic role in heart ischemia–reperfusion and acute kidney injury. This work uncovers a novel redox signaling pathway, involving Nox4–GADD34 interaction and a targeted oxidative inactivation of the PP1 metal center, that sustains eIF2α phosphorylation to protect tissues under stress.
NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.
