Antioxidant-induced modification of INrf2 cysteine 151 and PKC-δ-mediated phosphorylation of Nrf2 serine 40 are both required for stabilization and nuclear translocation of Nrf2 and increased drug resistance

Abstract

Antioxidants cause dissociation of nuclear factor erythroid 2-related factor 2 (Nrf2) from inhibitor of Nrf2 (INrf2) and so Nrf2:INrf2 can serve as a sensor of oxidative stress. Nrf2 translocates to the nucleus, binds to antioxidant response element (ARE) and activates defensive gene expression, which protects cells. Controversies exist regarding the role of antioxidant-induced modification of INrf2 cysteine 151 or protein kinase C (PKC)-mediated phosphorylation of Nrf2 serine 40 in the release of Nrf2 from INrf2. In addition, the PKC isoform that phosphorylates Nrf2S40 remains unknown. Here, we demonstrate that antioxidant-induced PKC-δ-mediated phosphorylation of Nrf2S40 leads to release of Nrf2 from INrf2. This was evident from specific chemical inhibitors of PKC isoenzymes in reporter assays, in vitro kinase assays with purified Nrf2 and PKC isoenzymes, in vivo analysis with dominant-negative mutants and siRNA against PKC isoforms, use of PKC-δ+/+ and PKC-δ–/– cells, and use of Nrf2S40 phospho-specific antibody. The studies also showed that antioxidant-induced INrf2C151 modification was insufficient for the dissociation of Nrf2 from INrf2. PKC-δ-mediated Nrf2S40 phosphorylation was also required. Nrf2 and mutant Nrf2S40A both bind to INrf2. However, antioxidant treatment led to release of Nrf2 but not Nrf2S40A from INrf2. In addition, Nrf2 and mutant Nrf2S40A both failed to dissociate from mutant INrf2C151A. Furthermore, antioxidant-induced ubiquitylation of INrf2 in PKC-δ+/+ and PKC-δ–/– cells occurred, but Nrf2 failed to be released in PKC-δ–/– cells. The antioxidant activation of Nrf2 reduced etoposide-mediated DNA fragmentation and promoted cell survival in PKC-δ+/+ but not in PKC-δ–/– cells. These data together demonstrate that both modification of INrf2C151 and PKC-δ-mediated phosphorylation of Nrf2S40 play crucial roles in Nrf2 release from INrf2, antioxidant induction of defensive gene expression, promoting cell survival, and increasing drug resistance.

Kinetic, Thermodynamic, and Structural Characterizations of the Association between Nrf2-DLGex Degron and Keap1

“…It has been reported that Ser40 within the DLGex motif is phosphorylated by protein kinase C and that this promotes the dissociation of Nrf2 from Keap1 (37)(38)(39)(40). However, in our analysis with an Nrf2 S40E mutant that mimics the phosphorylation of Ser40, Ser40 phosphorylation does not affect the interaction between the DLGex motif and the DC domain of Keap1.…”

“…It was reported that phosphorylation of Nrf2 Ser40 by protein kinase C promoted the dissociation of Nrf2 from Keap1 and the consequent activation of Nrf2 in response to oxidative stress (37)(38)(39)(40). Because Ser40 was included in Nrf2-DLGex, we were curious about whether the modification of Ser40 has an effect on the association between Nrf2-DLGex and Keap1-DC.…”

Src Subfamily Kinases Regulate Nuclear Export and Degradation of Transcription Factor Nrf2 to Switch Off Nrf2-mediated Antioxidant Activation of Cytoprotective Gene Expression

“…Another mechanism is that antioxidant-induced protein kinase C (PKC) phosphorylates Nrf2Ser-40, leading to dissociation of Nrf2 from INrf2 and nuclear localization of Nrf2 (5,6). Recently, it has been shown that the two mechanisms work in concert (7). Ultimately, Nrf2 translocates into the nucleus, binds with antioxidant-response elements (ARE) 2 in the promoter regions of cytoprotective genes, and coordinately activates gene expression.…”

Nrf2 stabilization prevents critical oxidative damage in Down syndrome cells

“…PKCδ levels were similar in NL and DS cells (Figure 3d). To evaluate whether Nrf2 phosphorylation in DS cells is dependent on PKCδ kinase activity (Niture et al, 2009), we used the PKCδ inhibitor Gö6983 and also Gö6976, an inhibitor that do not affect PKCδ but interfere PKCα and PKCβ isoforms instead (Gschwendt et al, 1996; Martiny‐Baron et al, 1993; Stempka et al, 1999). Treatment with Gö6983 prevented pNrf2 nuclear translocation in both basal conditions and after PQ treatment, while Gö6976 had no effect (Figure 3e).…”

“…Under oxidative conditions, the cysteine‐rich interacting domain in Keap1 changes its conformation, disabling its default association with Nrf2. As a result, Nrf2 accumulates in the cytoplasm and after phosphorylation by PKCδ, pNrf2ser40 translocates into the nucleus where it activates transcription of antioxidant response elements (ARE) (Niture et al, 2009; Villeneuve et al, 2010; Zhang & Hannink, 2003). …”