Phosphorylation of Nrf2 at Ser-40 by Protein Kinase C Regulates Antioxidant Response Element-mediated Transcription

Abstract

Nrf2, a basic leucine zipper transcription factor, is an essential activator of the coordinated transcription of genes encoding antioxidant enzymes and phase II detoxifying enzymes through the regulatory sequence termed antioxidant response element (ARE). Recently we reported evidence for the involvement of protein kinase C (PKC) in phosphorylating Nrf2 and triggering its nuclear translocation in response to oxidative stress. We show here that phosphorylation of purified rat Nrf2 by the catalytic subunit of PKC was blocked by a synthetic peptide mimicking one of the potential PKC sites. Accordingly, Nrf2 bearing a Ser to Ala mutation at amino acid 40 (S40A) could not be phosphorylated by PKC. The S40A mutation did not affect in vitro binding of Nrf2/ MafK to the ARE. However, it partially impaired Nrf2 activation of ARE-driven transcription in a reporter gene assay when Keap1 was overexpressed. In vitro transcribed/translated Keap1 could be coimmunoprecipitated with Nrf2. Phosphorylation of wild-type Nrf2 by PKC promoted its dissociation from Keap1, whereas the Nrf2-S40A mutant remained associated. These findings together with our prior studies suggest that the PKC-catalyzed phosphorylation of Nrf2 at Ser-40 is a critical signaling event leading to ARE-mediated cellular antioxidant response. The antioxidant response element (ARE)1 is a regulatory sequence involved in the coordinated transcriptional activation of genes coding for a number of antioxidant enzymes and phase II detoxifying enzymes (1-6). Reactive oxygen species and electrophiles are potent activators of genes containing an ARE, mediated by the basic leucine zipper (bZIP) transcription factor Nrf2 (NF-E2-related factor 2) (7-9). Accumulated evidence from studies of nrf2-null mice has established that Nrf2 is an essential ARE-binding factor involved in both constitutive and inducible gene expression via the ARE (9 -11). An important regulatory step leading to ARE activation is the oxidative stress-induced nuclear translocation of Nrf2, which normally appears to be sequestered in the cytoplasm by the cytoskeletonbinding Keap1 protein (12-14). However, the precise mechanism by which ARE-activating signals reach Nrf2 and cause dissociation of the putative inhibitory Nrf2-Keap1 complex remains unclear.Several protein kinase pathways have been implicated in transducing oxidative stress signals to gene expression mediated through the ARE. A number of reports have addressed a possible role for extracellular signal-regulated kinase (ERK1/2) in ARE activation. The findings have however remained controversial: ERK1/2 has been found to regulate the ARE positively in certain hepatoma cells (15-17) but negatively in others (18). Similarly, p38 MAP (mitogen-activated protein) kinase has also been shown to affect ARE activity, either positively (17,19,20) or negatively (16,21). More recently, phosphatidylinositol 3-kinase and its downstream target Akt/PKB (protein kinase B) have been linked to activation of the ARE in hepatoma (18, 19) and neuroblastoma (22) cell lin...

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“…Protein kinase C phosphorylates Nrf2 at Ser40, leading to Nrf2 dissociation from its endogenous inhibitor Kelch-like ECH associated protein 1 (Keap1), which results in nuclear translocation of Nrf2 in response to oxidative stress [54]. Therefore, we measured the levels of p-(Ser40)-Nrf2/Nrf2 in both cellular and nuclear extracts of striatal cells.…”

Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR‐32 neuroblastoma cells

“…It has been reported that the serine 40 residue (S40) of Nrf2, which is within the CD4 sequence, was phosphorylated by protein kinase C; phosphorylation at S40 was inducible by tBHQ and was necessary for the dissociation of Nrf2 from Keap1 and for the transcription activation activity of Nrf2 in a reporter assay in HepG2 cells [18,19]. We thus tested whether phosphorylation of S40 contributes to the slower migrating form of Nrf2.…”

“…Experiments employing specific inhibitors of the kinases implicated protein phosphorylation/dephosphorylation in the activation of Nrf2 [18,19]. However, due to the lack of direct biochemical evidence of phosphorylation of the Nrf2 protein, the role and mechanism of phosphorylation in Nrf2 regulation remain elusive.…”

Flunarizine induces Nrf2-mediated transcriptional activation of heme oxygenase-1 in protection of auditory cells from cisplatin

“…1 Transcriptional activation through ARE is mainly regulated by NF-E2-related factor 2 (Nrf2), a member of Cap'n'Collar (CNC) family of transcription factors that share a highly conserved basic region-leucine zipper (bZIP) structure. 2 Nrf2 is abundantly expressed in liver, intestine, lung and kidney, where detoxification reactions occur routinely. Recently, Nrf2 and its downstream proteins have been shown to be critically important regulators in protection of cells from oxidative stress-and chemicalinduced damages of liver and lung tissues.…”