Atypical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress

Abstract

Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of antioxidative proteins, including heme oxygenase-1 (HO-1). Nrf2 is sequestered in the cytoplasm by Keap1 under unstimulated conditions but translocates into the nucleus and transactivates the antioxidant responsive element (ARE) upon exposure to oxidative insults. It has recently been demonstrated that in vitro phosphorylation of Nrf2 on Ser40 by protein kinase C (PKC) facilitates the dissociation of Nrf2 from the Keap1 complex (Huang HC, Nguyen T, and Pickett CB. J Biol Chem 277: 42769–42774, 2002). The present study was designed to examine whether PKC is involved in oxidative stress-mediated nuclear translocation of Nrf2 in vivo and, if so, which PKC isoforms are involved. Induction of HO-1 gene expression by phorone, a glutathione depletor, and 4-hydroxy-2,3-nonenal (4-HNE), an end product of lipid peroxidation, was suppressed by a specific PKC inhibitor, Ro-31-8220, at concentrations that inhibit all isoforms in WI-38 cells. The induction of HO-1 was not affected by prolonged exposure of the cells to 12- O-tetradecanoylphorbol-13 acetate (TPA), suggesting that TPA-insensitive atypical PKC (aPKC) isoforms are involved. An immunocomplex kinase assay revealed that phorone and 4-HNE increased aPKCι activity. In COS-7 cells, 4-HNE induced nuclear translocation of the Nrf2-green fluorescent protein (GFP) fusion protein, but not the Nrf2(S40A)-GFP mutant. In the absence of oxidative insults, the Nrf2(S40E)-GFP mutant was distributed in the nucleus. The Nrf2-GFP accumulation in the nucleus was induced by coexpression of aPKCι, but not by a kinase inactive mutant aPKCι(K274W). The activity of an ARE-driven reporter was increased by coexpression of aPKCι, and this effect was eliminated by Ro-31-8220 in HepG2 cells. The reporter activity induced by 4-HNE was inhibited by coexpression of aPKCι(K274W). These results suggest that phosphorylation of Nrf2 Ser40 by aPKC(s) is involved in the nuclear translocation and ARE transactivation of Nrf2 by oxidative stress.

The chemistry of cell signaling by reactive oxygen and nitrogen species and 4-hydroxynonenal

“…In contrast to PKC-β, the novel PKC-δ activity was inhibited by 0.1 μM HNE but increased by 1-10 μM HNE (199,200) . Numazawa also reported that HNE-mediated HO-1 induction was completely abrogated by dominant-negative expression of atypical PKC-ι (201), suggesting that HNE may also activate PKC-ι.…”

Nuclear Factor–Erythroid-2–Related Factor 2 in Aging and Lung Fibrosis

“…74 Various isoforms of PKC and atypical PKC have been documented to be involved in phosphorylation of Nrf2. 15,75 Phosphatidylinositol 3-kinaseemediated phosphorylation of Nrf2 and its dissociation, nuclear translocation, and subsequent induction of antioxidants have been shown in human neuroblastoma cells. 76 In addition to the involvement of PKC and phosphatidylinositol 3-kinase in the activation of Nrf2, accumulating evidence suggests a role for mitogenactivated protein kinases 19,77,78 ; however, some reports suggest negative regulation of Nrf2 by these protein kinase pathways.…”

Self-regulatory role of 4-hydroxynonenal in signaling for stress-induced programmed cell death

“…It has been shown that Daxx interacts with HSF1 (the nuclear concentration of which is increased following HNE exposure) and upregulates the expression of HSF1-associated genes. Likewise, it is known that interaction of Daxx with other transcription factors such as ETS1, Pax 3, Pax5, smad4, and p53 also affects the expression of the associated genes [104][105][106][107]. Thus, HNE can influence a large set of fundamental cellular processes including proliferation, cell cycle events, and apoptosis and can modulate the expression of numerous genes.…”