Nuclear factor-erythroid 2 p45 (NF-E2 p45)-related factor 2 (Nrf2) is a master regulator of redox homoeostasis that allows cells to adapt to oxidative stress and also promotes cell proliferation. In this review, we describe the molecular mechanisms by which oxidants/electrophilic agents and growth factors increase Nrf2 activity. In the former case, oxidants/electrophiles increase the stability of Nrf2 by antagonizing the ability of Kelch-like ECH-associated protein 1 (Keap1) to target the transcription factor for proteasomal degradation via the cullin-3 (Cul3)–RING ubiquitin ligase CRLKeap1. In the latter case, we speculate that growth factors increase the stability of Nrf2 by stimulating phosphoinositide 3-kinase (PI3K)−protein kinase B (PKB)/Akt signalling, which in turn results in inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3) and in doing so prevents the formation of a DSGIS motif-containing phosphodegron in Nrf2 that is recognized by the β-transducin repeat-containing protein (β-TrCP) Cul1-based E3 ubiquitin ligase complex SCFβ-TrCP. We present data showing that in the absence of Keap1, the electrophile tert-butyl hydroquinone (tBHQ) can stimulate Nrf2 activity and induce the Nrf2-target gene NAD(P)H:quinone oxidoreductase-1 (NQO1), whilst simultaneously causing inhibitory phosphorylation of GSK-3β at Ser9. Together, these observations suggest that tBHQ can suppress the ability of SCFβ-TrCP to target Nrf2 for proteasomal degradation by increasing PI3K−PKB/Akt signalling. We also propose a scheme that explains how other protein kinases that inhibit GSK-3 could stimulate induction of Nrf2-target genes by preventing formation of the DSGIS motif-containing phosphodegron in Nrf2.

Background

Four cap’n’collar (CNC) basic-region leucine zipper (bZIP) transcription factors have been described in mammalian species, designated nuclear factor-erythroid 2 p45 (NF-E2 p45) and NF-E2 p45-related factor 1 (Nrf1), Nrf2 and Nrf3 [Nrf1, Nrf2 and Nrf3 have also been called NF-E2 p45 like 1 (Nfe2l1), Nfe2l2 and Nfe2l3, respectively; 1]. They bind their cognate DNA sequences in the regulatory regions of target genes as heterodimers with the small musculoaponeurotic fibrosarcoma (Maf) bZIP proteins, MafF, MafG and MafK [24]. Expression of the NF-E2 p45 and Nrf3 transcription factors is restricted primarily to haematopoietic tissue and placenta, whereas Nrf1 and Nrf2 are widely expressed across mammalian tissues [57]. Each of the CNC-bZIP transcription factors is functionally distinct, as evidenced by the markedly different phenotypes observed upon their knockout in the mouse [8,9].

Since its discovery in 1994 as a protein that binds a DNA sequence consisting of a tandem repeat of the activating protein 1 (AP1) site present in the β-globin gene locus control region [10], the work of Masayuki Yamamoto, Tom Kensler and many others has revealed that Nrf2 plays a central role in cancer chemoprevention and to be a master regulator of redox homoeostasis [1115]. More recently it has become apparent that Nrf2 can markedly influence intermediary metabolism by controlling carbon flux through the pentose phosphate pathway, inhibiting lipid synthesis, increasing β-oxidation of fatty acids and supporting mitochondrial respiration [1619].

Stimulation of Nrf2 activity by both oxidants/electrophiles and growth factors

A cardinal feature of Nrf2 is that its activity and hence the expression of its target genes, is maintained at low levels under normal homoeostatic conditions but increases rapidly in response to redox and electrophilic stressors as well as by stimulation by growth factors. The diverse biological effects of Nrf2 are exerted through its ability to mediate induction of genes that contain in their promoter regions an antioxidant response element {ARE [The ARE has also been designated the electrophile response element (EpRE)], 5′-A/GTGAC/GNNNGCA/G-3′} [20] upon exposure to a wide spectrum of oxidants and soft electrophiles (see Figure 1 for some examples) [2123] or stimulation by epidermal growth factor [24,25], fibroblast growth factor [26], insulin [27,28], insulin-like growth factor [29], keratinocyte growth factor [30,31], nerve growth factor [32], platelet-derived growth factor [33] or supply of glucose [34]. To date, approximately 250 genes that contain ARE sequences have been reported in mice and humans [3539]. Many of the ARE-containing Nrf2-target genes are involved in: (1) maintaining the glutathione and thioredoxin antioxidant defence systems within the cell; (2) the detoxification of soft electrophiles and oxidants that could compromise cellular redox status and cellular function by modifying protein thiols; (3) the repair of damaged tissue. Thus, in the presence of electrophiles or growth factors, Nrf2 increases expression of genes encoding proteins and enzymes that contribute to glutathione-based antioxidant defence (i.e. the cystine/glutamate transporter, glutamate-cysteine ligase catalytic and modifier subunits, glutaredoxin, glutathione peroxidase and glutathione reductase), thioredoxin-based antioxidant defence (i.e., peroxiredoxin, sulfiredoxin, thioredoxin and thioredoxin reductase), drug-metabolism [i.e., aldo-keto reductases, glutathione S-transferases and NAD(P)H:quinone oxidoreductase-1 (NQO1)], drug efflux pumps (i.e. multidrug resistance-associated proteins) and cytoprotective proteins associated with haem and iron metabolism [(i.e. biliverdin reductase, ferrochelatase, ferritin heavy and light and haem oxygenase-1 (HMOX1)]. Induction of the above members of the ARE-gene battery helps suppress the levels of reactive oxygen species and/or electrophiles in the cell and prevent oxidative stress [40,41].

Chemical structures of electrophilic agents that induce Nrf2-target genes

Figure 1
Chemical structures of electrophilic agents that induce Nrf2-target genes

The structures shown are of tBHQ, SFN, PEITC, D3T, PDTC, CDDO and bardoxolone methyl.

Figure 1
Chemical structures of electrophilic agents that induce Nrf2-target genes

The structures shown are of tBHQ, SFN, PEITC, D3T, PDTC, CDDO and bardoxolone methyl.

In addition to the well-documented role of Nrf2 in orchestrating adaptation to oxidative stress, increasing evidence indicates that Nrf2 also contributes to cell growth and the repair of damaged tissue [4245]. In this case, activation of Nrf2 is presumed to facilitate cell growth by: (1) increasing nutrient availability; (2) augmenting the levels of NADPH, ATP and metabolic intermediates required for synthesis of macromolecules; (3) increasing production of signalling molecules; (4) limiting inflammation. Thus, activation of Nrf2 results in the up-regulation of genes encoding transporters (i.e. the glucose transporter GLUT1, cystine/glutamate transporter SLC7A11, glycine transporter SLC6A9 and fatty acid translocase CD36) that are involved in the cellular uptake of glucose, amino acids and fatty acids. Also, activation of Nrf2 results in the up-regulation of enzymes involved in: carbohydrate metabolism (i.e. glucose-6-phosphate 1-dehydrogenase, isocitrate dehydrogenase 1, malic enzyme 1, 6-phosphogluconate dehydrogenase, transaldolase and transketolase isoform 1) that regenerate NADPH; the β-oxidation of fatty acids (i.e. acetyl-CoA thioesterase, acetyl-CoA oxidase, carboxylesterase 1, and stearoyl-CoA desaturase) that produce ATP; purine nucleotide biosynthesis (i.e. phosphoribosyl pyrophosphate amidotransferase and methylenetetrahydrofolate dehydrogenase, (2) that aid DNA synthesis. Moreover, Nrf2 also regulates directly Notch1 and augmenter of liver regeneration (ALR), both of which contribute to tissue repair [43,45]. Taken together, the findings outlined above provide an important insight into the biochemical pathways by which activation of Nrf2 stimulates cell growth.

Molecular basis for the dual regulation of Nrf2 by cullin-based ubiquitin ligases

A key feature of Nrf2 biology is that like other stress-responsive transcription factors it is a highly unstable protein because under normal homoeostatic conditions it is continuously targeted for proteasomal degradation [46,47]. The Nrf2 protein can be subdivided into seven regions, called Nrf2−ECH homology (Neh) domains 1–7. The intrinsic instability of Nrf2 can be attributed primarily to Neh2 and Neh6 as they contain several destruction motifs, whereas Neh1, Neh3, Neh4 and Neh5 are required for transactivation activity [41].

The first major repressor of Nrf2 to be discovered was Kelch-like ECH-associated protein-1 (Keap1) and it was identified using the Neh2 domain of Nrf2 as bait in a yeast two-hybrid screen [48]. It was initially thought that Keap1 sequesters Nrf2 in the cytoplasm and that upon treatment with electrophiles the CNC-bZIP transcription factor is released by Keap1 to become free to translocate to the nucleus [48,49]. Several models were advanced to explain the nuclear accumulation of Nrf2 following treatment with stressors: firstly, it was proposed that the release of Nrf2 from Keap1 required phosphorylation of Ser40 in the Neh2 domain of Nrf2 by protein kinase C (PKC) [5052]; secondly, it was demonstrated that reactive cysteine residues in a domain of Keap1 called the intervening region (IVR) serve as sensors for electrophiles and it was proposed that the chemical modification of these cysteine residues triggered a conformational change in Keap1 that allowed release of Nrf2 [53]. Subsequently, several groups recognized that Keap1 targets Nrf2 for proteasomal degradation [54,55] and several groups showed in close succession that Keap1 is a substrate adaptor for the cullin-3 (Cul3)–RING box protein (Rbx1) E3 ubiquitin ligase, which is abbreviated as CRL [5659]. The mammalian Keap1 protein comprises 624 amino acids and contains at least four separate cysteine-based stress sensors in the IVR and elsewhere that are triggered by reactive oxygen/nitrogen species and electrophiles [41]. It is a dimeric protein with two Kelch-repeat domains that each binds Nrf2 through either DLG or ETGE motifs in the Neh2 domain and it seems improbable that Keap1 releases Nrf2 in a non-ubiquitylated state [6062]. Indeed, the binding between Keap1 and Nrf2 is tighter when electrophilic agents modify the ubiquitin ligase substrate adaptor [63]. It seems likely that upon stress, newly translated Nrf2 is able to bypass Keap1 [64], possibly because the latter protein is saturated with Nrf2 that is not ubiquitylated and degraded and the modified Keap1 is ultimately degraded by autophagy [65].

The second major repressor of Nrf2 to be discovered was β-transducin repeat-containing protein (β-TrCP). Prior to the discovery, it was recognized that mutant Nrf2 lacking the high-affinity ETGE Keap1-interaction motif (i.e. Nrf2ETGE-V5) was not particularly stable even though CRLKeap1 could not target the mutant protein for degradation. Systematic deletion mapping across the Nrf2ETGE-V5 transcription factor identified amino acids bordering the N- and C-terminal boundaries of the Neh6 domain (i.e. residues 329–339 and 363–379 of mouse Nrf2) that conferred instability on the protein [66]. The N- and C-terminal Neh6 regions contain the sequences DSGIS and DSAPGS, respectively, that resemble the consensus binding sequence for β-TrCP, a substrate adaptor for the S-phase kinase-associated protein-1 (Skp1)−Cul1−F-box protein (SCF) ubiquitin ligase that is responsible for turnover of inhibitor of κB, subunit α (IκBα) and β-catenin; the β-TrCP consensus site has been reported to be DSGϕXS [67,68]. Although neither the DSGIS motif nor the DSAPGS motif conforms exactly to the β-TrCP consensus site, both have been found to recruit β-TrCP and support ubiquitylation of Nrf2 by SCFβ-TrCP [69,70]. Unlike the DLG and ETGE motifs in the Neh2 domain that are both required for CRLKeap1 ubiquitylation of Nrf2, the DSGIS and DSAPGS motifs in the Neh6 domain function independently of each other [70]. The reason for this is uncertain, as both Keap1 and β-TrCP are dimeric substrate adaptors, but one possibility is that whereas the lysine ubiquitin acceptor residues for CRLKeap1 lie between the DLG and ETGE motifs in Neh2, the lysine ubiquitin acceptor residues for SCFβ-TrCP may lie outwith the region encompassed by the DSGIS and DSAPGS motifs.

Besides Keap1 and β-TrCP, Zhang and colleagues [71] have shown that Nrf2 is repressed by the E3 ubiquitin ligase Hrd1 (also called synoviolin). Hrd1 is activated in response to endoplasmic reticulum stress and it is therefore questionable whether is contributes to the repression of Nrf2 under normal homoeostatic conditions. Importantly, however, activation of Hrd1 leads to loss of Nrf2 activity in cirrhotic liver and therapeutic targeting of Hrd1 in a murine model suppresses liver cirrhosis [71].

Glycogen synthase kinase-3 allows regulation of Nrf2 by growth factors

Many proteins that are ubiquitylated by SCFβ-TrCP are themselves phosphorylated by glycogen synthase kinase-3 (GSK-3), including IκBα, β-catenin, Gli3 and securin [67,68,72,73]. Salazar et al. [74] first reported that GSK-3 inhibits the expression of Nrf2-target genes and is able to phosphorylate the transcription factor. Evidence has subsequently been provided that GSK-3 phosphorylates serine residues within the DSGIS motif [75,76] and that this promotes ubiquitylation of Nrf2 by SCFβ-TrCP [69,70]. By contrast, the DSAPGS degron does not appear to be influenced by GSK-3 [70].

Unlike most kinases, GSK-3 is active in the cell under resting conditions, but is inhibited by phosphorylation of an N-terminal serine residue (i.e. Ser9 and Ser21 in GSK-3β and GSK-3α respectively) by protein kinase B (PKB)/Akt, and in turn PKB/Akt lies downstream of phosphoinositide 3-kinase (PI3K) [77]. As PI3K is closely associated with growth factor receptors and is intimately involved in cell growth and differentiation [7880], it seems distinctly possible that the ability of epidermal growth factor, fibroblast growth factor, insulin, insulin-like growth factor, keratinocyte growth factor, nerve growth factor and platelet-derived growth factor to activate Nrf2 occurs through stimulation of the PI3K−PKB/Akt pathway and entails inhibition of GSK-3 and loss of repression by β-TrCP.

Regulation of Nrf2 by mitogen-activated protein kinases

Various cancer chemopreventive agents that induce Nrf2-target genes stimulate mitogen-activated protein kinase (MAPK) signalling, including extracellular signal-regulated protein kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 kinases. Specifically, it has been reported that tert-butyl hydroquinone (tBHQ) activates ERK and p38MAPK [8184], sulforaphane (SFN) activates ERK and can suppress activation of p38MAPK by aninomycin [82,85], phenethyl isothiocyanate (PEITC) activates ERK and JNK [86,87], dithiole-3-thione (D3T) activates ERK [88] and pyrrolidine dithiocarbamate (PDTC) activates ERK [89,90]; see Figure 1 for structures. In the above studies, the use of kinase inhibitors and dominant-negative mutants to blunt gene induction suggest that ERK and JNK positively regulate Nrf2 activity. In marked contrast, p38MAPK has been reported to both positively and negatively regulate Nrf2 [84,9194] and this may reflect cell-specific differences in p38MAPK signalling pathways or response to chemicals.

Many of the investigators who examined the effects of MAPKs on Nrf2 activity considered that their activation resulted in phosphorylation of Nrf2 and that this event controlled translocation of the transcription factor from the cytoplasm to the nucleus. There are however at least three problems with this conclusion: firstly, it assumes the only mechanism of Nrf2 regulation is subcellular compartmentalization and does not consider the impact of MAPK on transcriptional activation of the gene encoding Nrf2 (i.e. NFE2L2) [95]; secondly, it does not consider the impact of MAPK on protein stability nor does it explain how knockdown or knockout of Keap1 is sufficient to induce Nrf2-target genes [96,97]; thirdly, mutation of putative MAPK phosphorylation sites in Nrf2 have little impact on the activity of the transcription factor [90,98]. The accumulated evidence suggests that both ERK and JNK positively regulate Nrf2 activity, but that their effects are probably indirect. Most, but not all studies, suggest that p38MAPK inhibits Nrf2 and again this appears to be indirect.

Regulation of Nrf2 by phosphatidylinositol-3-kinase

Over the years, it has been found consistently that inhibition of PI3K by Wortmannin or LY294002 blunts Nrf2-mediated induction of ARE-driven genes. Johnson and colleagues [99] first used LY294002 to diminish induction of NQO1 by tBHQ in IMR-32 human neuroblastoma cells and they subsequently used microarray analysis of gene induction by tBHQ in IMR-32 cells and primary astrocytes from Nrf2+/+ and Nrf2−/− mice to link PI3K to the induction of Nrf2-target genes [100,101]. Around the same time, Cuadrado and colleagues [32] showed that stimulation of PI3K affected Nrf2 indirectly by demonstrating first that activation of PI3K by nerve growth factor required activation of PKB/Akt in order for HMOX1 to be induced and secondly that Nrf2 was required for HMOX1 induction [102]. Thereafter, Salazar et al. [74] proposed that GSK-3 provides the link between activation of the PI3K−PKB/Akt pathway and stimulation of Nrf2-mediated gene induction in that they reported activation of PKB/Akt resulted in inhibitory phosphorylation of GSK-3, and that this resulted in failure by GSK-3 to phosphorylate Nrf2.

Regulation of Nrf2 by glycogen synthase kinase-3

As activation of PI3K and PKB/Akt cause inhibition of GSK-3α and GSK-3β by phosphorylation of their Ser21 and Ser9 residues, we hypothesized that chemopreventive agents might up-regulate Nrf2 by activating PI3K and PKB/Akt, thereby preventing formation of the DSGIS-containing phosphodegron in the Neh6 domain of Nrf2. To test this idea, we treated Keap1−/− mouse embryonic fibroblast (MEF) cells with tBHQ or SFN. As expected, we found that under normal conditions the Nrf2-target genes Nqo1 and Hmox1 were overexpressed in the Keap1−/− MEFs, relative to Keap1+/+ MEFs. However, both Nqo1 and Hmox1 could be further induced by tBHQ, but not by SFN, in the mutant MEFs (Figure 2). Most importantly, induction of Nqo1 and Hmox1 by tBHQ in the Keap1−/− MEFs was accompanied by inhibitory phosphorylation of GSK-3β at Ser9 and by activating phosphorylation of PKB/Akt at Ser473. Also of note, the PI3K inhibitor LY294002 markedly reduced Nrf2 protein levels as well as the basal expression of Nqo1 and Hmox1 in Keap1−/− MEFs, and blocked induction of Nqo1 and Hmox1 by tBHQ. These data suggest that PI3K positively regulates Nrf2 and support the hypothesis that tBHQ, but not SFN, activates PI3K and PKB/Akt.

Keap1-independent induction of Nrf2-target genes by tBHQ but not by SFN

Figure 2
Keap1-independent induction of Nrf2-target genes by tBHQ but not by SFN

Duplicate sets of 60-mm petridishes of wild-type and Keap1−/− MEFs, plated in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS, were grown in DMEM containing low serum (0.1% FBS) for 16 h. Thereafter, they were transferred to fresh medium containing 0.2% FBS and treated with tBHQ, SFN or vehicle control for various time intervals. In some instances, the fibroblasts were pre-treated for 30 min with 10 μM LY294002 immediately prior to transfer to fresh medium and treatment with tBHQ or SFN. (A) Whole cell lysates were prepared from Keap1−/− MEFs that had been treated for 18 h with tBHQ, SFN or DMSO vehicle control. Thereafter, Nqo1 enzyme activity was measured in the 10000 g supernatant by the ‘Prochaska’ bioassay method [103]. Data obtained from the MEFs that had been pretreated with LY294002 are shown in solid bars. Results that were significantly higher than the DMSO vehicle control with P values of 0.01–0.001 or <0.001 are indicated with double (**) or triple (***) asterisk signs respectively; ns, not significant. (B) Nqo1 mRNA levels were measured in Keap1−/− MEFs that had been treated for 12 h with tBHQ, SFN or DMSO. Data from MEFs that had been pretreated with 10 μM LY294002 prior to treatment with tBHQ or SFN are shown in solid bars. (C) MEFs from Keap1−/− and Keap1+/+ (WT) mice (lanes 1–6 and lanes 7–12, respectively) were treated with tBHQ, SFN or DMSO for 2 h, either with or without pretreatment with LY294002. Whole cell lysates were probed with antibodies specific for Nrf2, Nqo1, Hmox1, phospho-Ser473 Akt (i.e. activated PKB/Akt), total Akt, phospho-Ser9 GSK-3β (i.e. inactive GSK-3β), total GSK-3 and Gapdh.

Figure 2
Keap1-independent induction of Nrf2-target genes by tBHQ but not by SFN

Duplicate sets of 60-mm petridishes of wild-type and Keap1−/− MEFs, plated in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS, were grown in DMEM containing low serum (0.1% FBS) for 16 h. Thereafter, they were transferred to fresh medium containing 0.2% FBS and treated with tBHQ, SFN or vehicle control for various time intervals. In some instances, the fibroblasts were pre-treated for 30 min with 10 μM LY294002 immediately prior to transfer to fresh medium and treatment with tBHQ or SFN. (A) Whole cell lysates were prepared from Keap1−/− MEFs that had been treated for 18 h with tBHQ, SFN or DMSO vehicle control. Thereafter, Nqo1 enzyme activity was measured in the 10000 g supernatant by the ‘Prochaska’ bioassay method [103]. Data obtained from the MEFs that had been pretreated with LY294002 are shown in solid bars. Results that were significantly higher than the DMSO vehicle control with P values of 0.01–0.001 or <0.001 are indicated with double (**) or triple (***) asterisk signs respectively; ns, not significant. (B) Nqo1 mRNA levels were measured in Keap1−/− MEFs that had been treated for 12 h with tBHQ, SFN or DMSO. Data from MEFs that had been pretreated with 10 μM LY294002 prior to treatment with tBHQ or SFN are shown in solid bars. (C) MEFs from Keap1−/− and Keap1+/+ (WT) mice (lanes 1–6 and lanes 7–12, respectively) were treated with tBHQ, SFN or DMSO for 2 h, either with or without pretreatment with LY294002. Whole cell lysates were probed with antibodies specific for Nrf2, Nqo1, Hmox1, phospho-Ser473 Akt (i.e. activated PKB/Akt), total Akt, phospho-Ser9 GSK-3β (i.e. inactive GSK-3β), total GSK-3 and Gapdh.

Although it has not been clearly established how tBHQ stimulates PI3K, Sporn and colleagues [104] have shown that the semi-synthetic triterpenoid CDDO-imidazolide (CDDO-Im), which is a potent inducer of Nrf2-target genes, can activate signalling downstream of PI3K by adducting to the catalytic Cys124 residue of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) [104], the enzyme that cleaves the product of the PI3K reaction, phosphatidylinositol (3,4,5)-trisphosphate (PI3,4,5P3 or PIP3). Importantly, all cysteine-dependent protein tyrosine phosphatases are redox sensitive [105]. We therefore propose that tBHQ either modifies Cys124 in PTEN or another protein tyrosine phosphatase that impinges on PI3K signalling. We envisage that tBHQ and CDDO-Im increase PI3,4,5P3 levels by inhibiting PTEN and this causes activation of PDK1, which in turn activates PKB/Akt resulting in inhibition of GSK-3 and failure to produce the DSGIS-containing phosphodegron.

Besides influencing the stability of Nrf2 by catalysing formation of the DSGIS-containing phosphodegron, it has been reported that GSK-3 indirectly controls the subcellular localization of Nrf2 because it lies upstream of Src non-receptor tyrosine kinases that phosphorylate mouse Nrf2 at Tyr568 and human Nrf2 at Tyr576. In this case, treatment of cells with H2O2 has been reported to result in phosphorylation of Nrf2 at Tyr568/Tyr576 by Fyn, Src, Yes and Fgr, which triggered nuclear export and degradation of the transcription factor [106,107]. Further work is required to establish the precise pathway by which H2O2 activates the Src kinases and the putative involvement of GSK-3 in the process.

Concluding comments

Nrf2 plays an indispensible role in maintaining redox homoeostasis and it has become clear in recent years that it also controls cell growth. It is well known that Nrf2 activity is increased upon treatment of cells with electrophilic agents because such chemicals antagonize the ability of Keap1 to direct the transcription factor to ubiquitylation by Cul3−Rbx1. It is however much less well appreciated that Nrf2 activity is controlled by growth factors. The body of literature we have reviewed suggests that GSK-3 plays a pivotal role in the regulation of Nrf2 by growth factors because it catalyses formation of the DSGIS-containing phosphodegron that is recognized by SCFβ-TrCP. We have emphasized the ability of the PI3K−PKB/Akt pathway to inhibit GSK-3. However, as shown in Figure 3, other kinases besides PKB/Akt can inhibit GSK-3α/β by catalysing phosphorylation of their Ser21/9 residues, such as mTOR−p70S6K, ERK−p90RSK, p38MAPK and PKC signalling pathways [77], suggesting other mechanisms by which Nrf2 can be regulated. It is intriguing that the kinases that potentially inhibit GSK-3 include ERK and PKC, which have been considered to be positive regulators of Nrf2 [50,83,84,90]. It is also notable that GSK-3 has a strong preference for substrates that have already been phosphorylated by a ‘priming’ kinase at a serine or threonine residue that is situated four or five residues to the C-terminal side of the amino acid that it phosphorylates [108]. It is neither known whether Nrf2 has to be ‘primed’ before it can be modified by GSK-3, nor is it known whether induction of such a ‘priming’ kinase might serve as an alternative means of regulating Nrf2. Our preliminary investigations with peptide-based mini-protein assays suggest priming greatly enhances phosphorylation of Nrf2 by GSK-3. It is becoming clear that the regulation of Nrf2 stability and function is far more complex than just control by Keap1 and the input of multiple growth factor signalling pathways opens up an exciting new chapter in Nrf2 research and its association with human disease.

Regulation of Nrf2 by phosphorylation of its Neh6 domain

Figure 3
Regulation of Nrf2 by phosphorylation of its Neh6 domain

Evidence indicates that GSK-3 negatively controls Nrf2 by phosphorylating the DSGIS motif in its Neh6 domain and promoting degradation via the actions of SCFβ-TrCP. As indicated in (1), shown on mid right-hand side, phosphorylation of most validated substrates by GSK-3 requires ‘priming’ (i.e. pre-phosphorylation) by an alternative kinase. Therefore, Nrf2 degradation could be regulated by control of ‘priming’. As indicated in (2), shown on mid left-hand side, GSK-3α/β is itself inhibited by phosphorylation of an N-terminal Ser21/9 residue. The main kinases reported to date to catalyse inhibitory phosphorylation of GSK-3 are members of the AGC class of kinases (including PKB/Akt, PKC and p70S6K). Moreover, p90RSK and p38MAPK have also been reported to inhibit GSK-3. Therefore, as indicated in (3), shown in upper half, activation of PKB/Akt, p70S6K, p90RSK, p38MAPK and PKC could inhibit GSK-3 and reduce Neh-6 phosphorylation and Nrf2 degradation.

Figure 3
Regulation of Nrf2 by phosphorylation of its Neh6 domain

Evidence indicates that GSK-3 negatively controls Nrf2 by phosphorylating the DSGIS motif in its Neh6 domain and promoting degradation via the actions of SCFβ-TrCP. As indicated in (1), shown on mid right-hand side, phosphorylation of most validated substrates by GSK-3 requires ‘priming’ (i.e. pre-phosphorylation) by an alternative kinase. Therefore, Nrf2 degradation could be regulated by control of ‘priming’. As indicated in (2), shown on mid left-hand side, GSK-3α/β is itself inhibited by phosphorylation of an N-terminal Ser21/9 residue. The main kinases reported to date to catalyse inhibitory phosphorylation of GSK-3 are members of the AGC class of kinases (including PKB/Akt, PKC and p70S6K). Moreover, p90RSK and p38MAPK have also been reported to inhibit GSK-3. Therefore, as indicated in (3), shown in upper half, activation of PKB/Akt, p70S6K, p90RSK, p38MAPK and PKC could inhibit GSK-3 and reduce Neh-6 phosphorylation and Nrf2 degradation.

We are very grateful to Professor Masayuki Yamamoto for supplying Keap1-null MEFs and to Professor Antonio Cuadrado for extremely helpful discussions.

Funding

This work was supported by the Cancer Research UK [grant number C4909/A13786].

Abbreviations

     
  • β-TrCP

    β-transducin repeat-containing protein

  •  
  • ARE

    antioxidant response element

  •  
  • bZIP

    basic-region leucine zipper

  •  
  • CDDO-Im

    2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide

  •  
  • CNC

    cap’n’collar

  •  
  • CRL

    cullin-RING ubiquitin ligase

  •  
  • Cul

    cullin

  •  
  • D3T

    dithiole-3-thione

  •  
  • ECH

    erythroid cell-derived protein with CNC homology

  •  
  • ERK

    extracellular signal-regulated protein kinase

  •  
  • GSK-3

    glycogen synthase kinase-3

  •  
  • HMOX1

    haem oxygenase-1

  •  
  • Hrd1

    Synoviolin

  •  
  • IκBα

    inhibitor of κB, subunit α

  •  
  • IVR

    intervening region

  •  
  • JNK

    c-Jun N-terminal kinase

  •  
  • Keap1

    Kelch-like ECH-associated protein 1

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • MAPK

    mitogen-activated protein kinase

  •  
  • Maf

    musculoaponeurotic fibrosarcoma

  •  
  • mTOR

    mechanistic target of rapamycin

  •  
  • Neh

    Nrf2−ECH homology

  •  
  • Nrf2

    NF-E2 p45-related factor 2

  •  
  • NF-E2 p45

    nuclear factor-erythroid 2 p45

  •  
  • NQO1

    NAD(P)H:quinone oxidoreductase-1

  •  
  • PDTC

    pyrrolidine dithiocarbamate

  •  
  • PEITC

    phenethyl isothiocyanate

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PKB

    protein kinase B

  •  
  • PKC

    protein kinase C

  •  
  • PTEN

    phosphatase and tensin homologue

  •  
  • Rbx1

    RING box protein

  •  
  • SCF

    Skp1-Cdc53/Cul1-F-box protein

  •  
  • SFN

    sulforaphane

  •  
  • tBHQ

    tert-butyl hydroquinone

The Keap1/Nrf2 Pathway in Health and Disease: Held at Robinson College, Cambridge, UK, 6–8 Jan 2015.

References

References
1
Amoutzias
G.D.
Veron
A.S.
Weiner
J.
III
Robinson-Rechavi
M.
Bornberg-Bauer
E.
Oliver
S.G.
Robertson
D.L.
One billion years of bZIP transcription factor evolution: conservation and change in dimerization and DNA-binding site specificity
Mol. Biol. Evol.
2007
, vol. 
24
 (pg. 
827
-
835
)
[PubMed]
2
Igarashi
K.
Kataoka
K.
Itoh
K.
Hayashi
N.
Nishizawa
M.
Yamamoto
M.
Regulation of transcription by dimerization of erythroid factor NF-E2 p45 with small Maf proteins
Nature
1994
, vol. 
367
 (pg. 
568
-
572
)
[PubMed]
3
Newman
J.R.
Keating
A.E.
Comprehensive identification of human bZIP interactions with coiled-coil arrays
Science
2003
, vol. 
300
 (pg. 
2097
-
2101
)
[PubMed]
4
Katsuoka
F.
Motohashi
H.
Ishii
T.
Aburatani
H.
Engel
J.D.
Yamamoto
M.
Genetic evidence that small maf proteins are essential for the activation of antioxidant response element-dependent genes
Mol. Cell. Biol.
2005
, vol. 
25
 (pg. 
8044
-
8051
)
[PubMed]
5
Andrews
N.C.
Erdjument-Bromage
H.
Davidson
M.B.
Tempst
P.
Orkin
S.H.
Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein
Nature
1993
, vol. 
362
 (pg. 
722
-
728
)
[PubMed]
6
Kobayashi
A.
Ito
E.
Toki
T.
Kogame
K.
Takahashi
S.
Igarashi
K.
Hayashi
N.
Yamamoto
M.
Molecular cloning and functional characterization of a new cap’n’ collar family transcription factor Nrf3
J. Biol. Chem.
1999
, vol. 
274
 (pg. 
6443
-
6452
)
[PubMed]
7
McMahon
M.
Itoh
K.
Yamamoto
M.
Chanas
S.A.
Henderson
C.J.
McLellan
L.I.
Wolf
C.R.
Cavin
C.
Hayes
J.D.
The cap’n’collar basic leucine zipper transcription factor Nrf2 (NF-E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes
Cancer Res.
2001
, vol. 
61
 (pg. 
3299
-
3307
)
[PubMed]
8
Sykiotis
G.P.
Bohmann
D.
Stress-activated cap’n’collar transcription factors in aging and human disease
Sci. Signal.
2010
, vol. 
3
 pg. 
re3
 
[PubMed]
9
Chevillard
G.
Blank
V.
NFE2L3 (NRF3): the Cinderella of the cap’n’collar transcription factors
Cell. Mol. Life Sci.
2011
, vol. 
68
 (pg. 
3337
-
3348
)
[PubMed]
10
Moi
P.
Chan
K.
Asunis
I.
Cao
A.
Kan
Y.W.
Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region
Proc. Natl. Acad. Sci. U.S.A.
1994
, vol. 
91
 (pg. 
9926
-
9930
)
[PubMed]
11
Ramos-Gomez
M.
Kwak
M.K.
Dolan
P.M.
Itoh
K.
Yamamoto
M.
Talalay
P
Kensler
T.W.
Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice
Proc. Natl. Acad. Sci. U.S.A.
2001
, vol. 
98
 (pg. 
3410
-
3415
)
[PubMed]
12
Chanas
S.A.
Jiang
Q.
McMahon
M.
McWalter
G.K.
McLellan
L.I.
Elcombe
C.R.
Henderson
C.J.
Wolf
C.R.
Moffat
G.J.
Itoh
K.
, et al. 
Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gsta1, Gsta2, Gstm1, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice
Biochem. J.
2002
, vol. 
365
 (pg. 
405
-
416
)
[PubMed]
13
Motohashi
H.
Yamamoto
M.
Nrf2-Keap1 defines a physiologically important stress response mechanism
Trends Mol. Med.
2004
, vol. 
10
 (pg. 
549
-
557
)
[PubMed]
14
Clements
C.M.
McNally
R.S.
Conti
B.J.
Mak
T.W.
Ting
J.P.
DJ-1, a cancer- and Parkinson's disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2
Proc. Natl. Acad. Sci. U.S.A.
2006
, vol. 
103
 (pg. 
15091
-
15096
)
[PubMed]
15
Kensler
T.W.
Wakabayashi
N.
Biswal
S.
Cell survival responses to environmental stresses via the Keap1-Nrf2-ARE pathway
Annu. Rev. Pharmacol. Toxicol.
2007
, vol. 
47
 (pg. 
89
-
116
)
[PubMed]
16
Mitsuishi
Y.
Taguchi
K.
Kawatani
Y.
Shibata
T.
Nukiwa
T.
Aburatani
H.
Yamamoto
M.
Motohashi
H.
Nrf2 redirects glucose and glutamine into anabolic pathways in metabolic reprogramming
Cancer Cell
2012
, vol. 
22
 (pg. 
66
-
79
)
[PubMed]
17
Holmström
K.M.
Baird
L.
Zhang
Y.
Hargreaves
I.
Chalasani
A.
Land
J.M.
Stanyer
L.
Yamamoto
M.
Dinkova-Kostova
A.T.
Abramov
A.Y.
Nrf2 impacts cellular bioenergetics by controlling substrate availability for mitochondrial respiration
Biol. Open
2013
, vol. 
2
 (pg. 
761
-
770
)
[PubMed]
18
Ludtmann
M.H.
Angelova
P.R.
Zhang
Y.
Abramov
A.Y.
Dinkova-Kostova
A.T.
Nrf2 affects the efficiency of mitochondrial fatty acid oxidation
Biochem. J.
2014
, vol. 
457
 (pg. 
415
-
424
)
[PubMed]
19
Meakin
P.J.
Chowdhry
S.
Sharma
R.S.
Ashford
F.B.
Walsh
S.V.
McCrimmon
R.J.
Dinkova-Kostova
A.T.
Dillon
J.F.
Hayes
J.D.
Ashford
M.L.
Susceptibility of Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a high-fat diet is associated with oxidative stress, perturbation of the unfolded protein response, and disturbance in the expression of metabolic enzymes but not with insulin resistance
Mol. Cell. Biol.
2014
, vol. 
34
 (pg. 
3305
-
3320
)
[PubMed]
20
Nioi
P.
McMahon
M.
Itoh
K.
Yamamoto
M.
Hayes
J.D.
Identification of a novel Nrf2-regulated antioxidant response element (ARE) in the mouse NAD(P)H:quinone oxidoreductase 1 gene: reassessment of the ARE consensus sequence
Biochem. J.
2003
, vol. 
374
 (pg. 
337
-
348
)
[PubMed]
21
Prestera
T.
Holtzclaw
W.D.
Zhang
Y.
Talalay
P.
Chemical and molecular regulation of enzymes that detoxify carcinogens
Proc. Natl. Acad. Sci. U.S.A.
1993
, vol. 
90
 (pg. 
2965
-
2969
)
[PubMed]
22
Dinkova-Kostova
A.T.
Fahey
J.W.
Talalay
P.
Chemical structures of inducers of nicotinamide quinone oxidoreductase 1 (NQO1)
Methods Enzymol.
2004
, vol. 
382
 (pg. 
423
-
448
)
[PubMed]
23
Hayes
J.D.
McMahon
M.
Chowdhry
S.
Dinkova-Kostova
A.T.
Cancer chemoprevention mechanisms mediated through the Keap1-Nrf2 pathway
Antioxid. Redox Signal.
2010
, vol. 
13
 (pg. 
1713
-
1748
)
[PubMed]
24
Papaiahgari
S.
Zhang
Q.
Kleeberger
S.R.
Cho
H.Y.
Reddy
S.P.
Hyperoxia stimulates an Nrf2-ARE transcriptional response via ROS-EGFR-PI3K-Akt/ERK MAP kinase signaling in pulmonary epithelial cells
Antioxid. Redox Signal.
2006
, vol. 
8
 (pg. 
43
-
52
)
[PubMed]
25
Yamadori
T.
Ishii
Y.
Homma
S.
Morishima
Y.
Kurishima
K.
Itoh
K.
Yamamoto
M.
Minami
Y.
Noguchi
M.
Hizawa
N.
Molecular mechanisms for the regulation of Nrf2-mediated cell proliferation in non-small-cell lung cancers
Oncogene
2012
, vol. 
31
 (pg. 
4768
-
4777
)
[PubMed]
26
Vargas
M.R.
Pehar
M.
Cassina
P.
Martínez-Palma
L.
Thompson
J.A.
Beckman
J.S.
Barbeito
L.
Fibroblast growth factor-1 induces heme oxygenase-1 via nuclear factor erythroid 2-related factor 2 (Nrf2) in spinal cord astrocytes: consequences for motor neuron survival
J. Biol. Chem.
2005
, vol. 
280
 (pg. 
25571
-
25579
)
[PubMed]
27
Harrison
E.M.
McNally
S.J.
Devey
L.
Garden
O.J.
Ross
J.A.
Wigmore
S.J.
Insulin induces heme oxygenase-1 through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in renal cells
FEBS J.
2006
, vol. 
273
 (pg. 
2345
-
2356
)
[PubMed]
28
Geraldes
P.
Yagi
K.
Ohshiro
Y.
He
Z.
Maeno
Y.
Yamamoto-Hiraoka
J.
Rask-Madsen
C.
Chung
S.W.
Perrella
M.A.
King
G.L.
Selective regulation of heme oxygenase-1 expression and function by insulin through IRS1/phosphoinositide 3-kinase/Akt-2 pathway
J. Biol. Chem.
2008
, vol. 
283
 (pg. 
34327
-
34336
)
[PubMed]
29
Kim
Y.
Li
E.
Park
S.
Insulin-like growth factor-1 inhibits 6-hydroxydopamine-mediated endoplasmic reticulum stress-induced apoptosis via regulation of heme oxygenase-1 and Nrf2 expression in PC12 cells
Int. J. Neurosci.
2012
, vol. 
122
 (pg. 
641
-
649
)
[PubMed]
30
Braun
S.
Hanselmann
C.
Gassmann
M.G.
auf dem Keller
U.
Born-Berclaz
C.
Chan
K.
Kan
Y.W.
Werner
S.
Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound
Mol. Cell. Biol.
2002
, vol. 
22
 (pg. 
5492
-
5505
)
[PubMed]
31
Chowdhury
I.
Fisher
A.B.
Christofidou-Solomidou
M.
Gao
L.
Tao
J.Q.
Sorokina
E.M.
Lien
Y.C.
Bates
S.R.
Feinstein
S.I.
Keratinocyte growth factor and glucocorticoid induction of human peroxiredoxin 6 gene expression occur by independent mechanisms that are synergistic
Antioxid. Redox Signal.
2014
, vol. 
20
 (pg. 
391
-
402
)
[PubMed]
32
Salinas
M.
Diaz
R.
Abraham
N.G.
Ruiz de Galarreta
C.M.
Cuadrado
A.
Nerve growth factor protects against 6-hydroxydopamine-induced oxidative stress by increasing expression of heme oxygenase-1 in a phosphatidylinositol 3-kinase-dependent manner
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
13898
-
13904
)
[PubMed]
33
Tohidnezhad
M.
Wruck
C.J.
Slowik
A.
Kweider
N.
Beckmann
R.
Bayer
A.
Houben
A.
Brandenburg
L.O.
Varoga
D.
Sönmez
T.T.
, et al. 
Role of platelet-released growth factors in detoxification of reactive oxygen species in osteoblasts
Bone
2014
, vol. 
65
 (pg. 
9
-
17
)
[PubMed]
34
Heiss
E.H.
Schachner
D.
Zimmermann
K.
Dirsch
V.M.
Glucose availability is a decisive factor for Nrf2-mediated gene expression
Redox Biol.
2013
, vol. 
1
 (pg. 
359
-
365
)
[PubMed]
35
Kwak
M.K.
Wakabayashi
N.
Itoh
K.
Motohashi
H.
Yamamoto
M.
Kensler
T.W.
Modulation of gene expression by cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway. Identification of novel gene clusters for cell survival
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
8135
-
8145
)
[PubMed]
36
MacLeod
A.K.
McMahon
M.
Plummer
S.M.
Higgins
L.G.
Penning
T.M.
Igarashi
K.
Hayes
J.D.
Characterization of the cancer chemopreventive NRF2-dependent gene battery in human keratinocytes: demonstration that the KEAP1-NRF2 pathway, and not the BACH1-NRF2 pathway, controls cytoprotection against electrophiles as well as redox-cycling compounds
Carcinogenesis
2009
, vol. 
30
 (pg. 
1571
-
1580
)
[PubMed]
37
Agyeman
A.S.
Chaerkady
R.
Shaw
P.G.
Davidson
N.E.
Visvanathan
K.
Pandey
A.
Kensler
T.W.
Transcriptomic and proteomic profiling of KEAP1 disrupted and sulforaphane-treated human breast epithelial cells reveals common expression profiles
Breast Cancer Res. Treat.
2012
, vol. 
132
 (pg. 
175
-
187
)
[PubMed]
38
Chorley
B.N.
Campbell
M.R.
Wang
X.
Karaca
M.
Sambandan
D.
Bangura
F.
Xue
P.
Pi
J.
Kleeberger
S.R.
Bell
D.A.
Identification of novel NRF2-regulated genes by ChIP-Seq: influence on retinoid X receptor alpha
Nucleic Acids Res.
2012
, vol. 
40
 (pg. 
7416
-
7429
)
[PubMed]
39
Hirotsu
Y.
Katsuoka
F.
Funayama
R.
Nagashima
T.
Nishida
Y.
Nakayama
K.
Engel
J.D.
Yamamoto
M.
Nrf2-MafG heterodimers contribute globally to antioxidant and metabolic networks
Nucleic Acids Res.
2012
, vol. 
40
 (pg. 
10228
-
10239
)
[PubMed]
40
Higgins
L.G.
Kelleher
M.O.
Eggleston
I.M.
Itoh
K.
Yamamoto
M.
Hayes
J.D.
Transcription factor Nrf2 mediates an adaptive response to sulforaphane that protects fibroblasts in vitro against the cytotoxic effects of electrophiles, peroxides and redox-cycling agents
Toxicol. Appl. Pharmacol.
2009
, vol. 
237
 (pg. 
267
-
280
)
[PubMed]
41
Hayes
J.D.
Dinkova-Kostova
A.T.
The Nrf2 regulatory network provides an interface between redox and intermediary metabolism
Trends Biochem. Sci.
2014
, vol. 
39
 (pg. 
199
-
218
)
[PubMed]
42
Beyer
T.A.
Xu
W.
Teupser
D.
auf dem Keller
U.
Bugnon
P.
Hildt
E.
Thiery
J.
Kan
Y.W.
Werner
S.
Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance
EMBO J.
2008
, vol. 
27
 (pg. 
212
-
223
)
[PubMed]
43
Wakabayashi
N.
Shin
S.
Slocum
S.L.
Agoston
E.S.
Wakabayashi
J.
Kwak
M.K.
Misra
V.
Biswal
S.
Yamamoto
M.
Kensler
T.W.
Regulation of notch1 signaling by nrf2: implications for tissue regeneration
Sci. Signal.
2010
, vol. 
3
 pg. 
ra52
 
[PubMed]
44
Dayoub
R.
Vogel
A.
Schuett
J.
Lupke
M.
Spieker
S.M.
Kettern
N.
Hildt
E.
Melter
M.
Weiss
T.S.
Nrf2 activates augmenter of liver regeneration (ALR) via antioxidant response element and links oxidative stress to liver regeneration
Mol. Med.
2013
, vol. 
19
 (pg. 
237
-
244
)
[PubMed]
45
Al-Sawaf
O.
Fragoulis
A.
Rosen
C.
Keimes
N.
Liehn
E.A.
Hölzle
F.
Kan
Y.W.
Pufe
T.
Sönmez
T.T.
Wruck
C.J.
Nrf2 augments skeletal muscle regeneration after ischaemia-reperfusion injury
J. Pathol.
2014
, vol. 
234
 (pg. 
538
-
547
)
[PubMed]
46
Nguyen
T.
Sherratt
P.J.
Huang
H.C.
Yang
C.S.
Pickett
C.B.
Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
4536
-
4541
)
[PubMed]
47
Stewart
D.
Killeen
E.
Naquin
R.
Alam
S.
Alam
J.
Degradation of transcription factor Nrf2 via the ubiquitin-proteasome pathway and stabilization by cadmium
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
2396
-
2402
)
[PubMed]
48
Itoh
K.
Wakabayashi
N.
Katoh
Y.
Ishii
T.
Igarashi
K.
Engel
J.D.
Yamamoto
M.
Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain
Genes Dev.
1999
, vol. 
13
 (pg. 
76
-
86
)
[PubMed]
49
Zipper
L.M.
Mulcahy
R.T.
The Keap1 BTB/POZ dimerization function is required to sequester Nrf2 in cytoplasm
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
36544
-
36552
)
[PubMed]
50
Huang
H.C.
Nguyen
T.
Pickett
C.B.
Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
42769
-
42774
)
[PubMed]
51
Numazawa
S.
Ishikawa
M.
Yoshida
A.
Tanaka
S.
Yoshida
T.
Atypical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress
Am. J. Physiol. Cell. Physiol.
2003
, vol. 
285
 (pg. 
C334
-
C342
)
[PubMed]
52
Bloom
D.A.
Jaiswal
A.K.
Phosphorylation of Nrf2 at Ser40 by protein kinase C in response to antioxidants leads to the release of Nrf2 from INrf2, but is not required for Nrf2 stabilization/accumulation in the nucleus and transcriptional activation of antioxidant response element-mediated NAD(P)H:quinone oxidoreductase-1 gene expression
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
44675
-
44682
)
[PubMed]
53
Dinkova-Kostova
A.T.
Holtzclaw
W.D.
Cole
R.N.
Itoh
K.
Wakabayashi
N.
Katoh
Y.
Yamamoto
M.
Talalay
P.
Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants
Proc. Natl. Acad. Sci. U.S.A.
2002
, vol. 
99
 (pg. 
11908
-
11913
)
[PubMed]
54
McMahon
M.
Itoh
K.
Yamamoto
M.
Hayes
J.D.
Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
21592
-
21600
)
[PubMed]
55
Itoh
K.
Wakabayashi
N.
Katoh
Y.
Ishii
T.
O’Connor
T.
Yamamoto
M.
Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles
Genes Cells
2003
, vol. 
8
 (pg. 
379
-
391
)
[PubMed]
56
Kobayashi
A.
Kang
M.I.
Okawa
H.
Ohtsuji
M.
Zenke
Y.
Chiba
T.
Igarashi
K.
Yamamoto
M.
Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2
Mol. Cell. Biol.
2004
, vol. 
24
 (pg. 
7130
-
7139
)
[PubMed]
57
Cullinan
S.B.
Gordan
J.D.
Jin
J.
Harper
J.W.
Diehl
J.A.
The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase
Mol. Cell. Biol.
2004
, vol. 
24
 (pg. 
8477
-
8486
)
[PubMed]
58
Zhang
D.D.
Lo
S.C.
Cross
J.V.
Templeton
D.J.
Hannink
M.
Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex
Mol. Cell. Biol.
2004
, vol. 
24
 (pg. 
10941
-
10953
)
[PubMed]
59
Furukawa
M.
Xiong
Y.
BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the cullin 3-Roc1 ligase
Mol. Cell. Biol.
2005
, vol. 
25
 (pg. 
162
-
171
)
[PubMed]
60
Eggler
A.L.
Liu
G.
Pezzuto
J.M.
van Breemen
R.B.
Mesecar
A.D.
Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2
Proc. Natl. Acad. Sci. U.S.A.
2005
, vol. 
102
 (pg. 
10070
-
10075
)
[PubMed]
61
Tong
K.I.
Katoh
Y.
Kusunoki
H.
Itoh
K.
Tanaka
T.
Yamamoto
M.
Keap1 recruits Neh2 through binding to ETGE and DLG motifs: characterization of the two-site molecular recognition model
Mol. Cell. Biol.
2006
, vol. 
26
 (pg. 
2887
-
2900
)
[PubMed]
62
McMahon
M.
Thomas
N.
Itoh
K.
Yamamoto
M.
Hayes
J.D.
Dimerization of substrate adaptors can facilitate cullin-mediated ubiquitylation of proteins by a “tethering” mechanism: a two-site interaction model for the Nrf2-Keap1 complex
J. Biol. Chem.
2006
, vol. 
281
 (pg. 
24756
-
24768
)
[PubMed]
63
Baird
L.
Llères
D.
Swift
S.
Dinkova-Kostova
A.T.
Regulatory flexibility in the Nrf2-mediated stress response is conferred by conformational cycling of the Keap1-Nrf2 protein complex
Proc. Natl. Acad. Sci. U.S.A.
2013
, vol. 
110
 (pg. 
15259
-
15264
)
[PubMed]
64
Kobayashi
A.
Kang
M.I.
Watai
Y.
Tong
K.I.
Shibata
T.
Uchida
K.
Yamamoto
M.
Oxidative and electrophilic stresses activate Nrf2 through inhibition of ubiquitination activity of Keap1
Mol. Cell. Biol.
2006
, vol. 
26
 (pg. 
221
-
219
)
[PubMed]
65
Taguchi
K.
Fujikawa
N.
Komatsu
M.
Ishii
T.
Unno
M.
Akaike
T.
Motohashi
H.
Yamamoto
M.
Keap1 degradation by autophagy for the maintenance of redox homeostasis
Proc. Natl. Acad. Sci. U.S.A.
2012
, vol. 
109
 (pg. 
13561
-
13566
)
[PubMed]
66
McMahon
M.
Thomas
N.
Itoh
K.
Yamamoto
M.
Hayes
J.D.
Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
31556
-
31567
)
[PubMed]
67
Aberle
H.
Bauer
A.
Stappert
J.
Kispert
A.
Kemler
R.
beta-catenin is a target for the ubiquitin-proteasome pathway
EMBO J.
1997
, vol. 
16
 (pg. 
3797
-
3804
)
[PubMed]
68
Winston
J.T.
Strack
P.
Beer-Romero
P.
Chu
C.Y.
Elledge
S.J.
Harper
J.W.
The SCFbeta-TRCP-ubiquitin ligase complex associates specifically with phosphorylated destruction motifs in IkappaBalpha and beta-catenin and stimulates IkappaBalpha ubiquitination in vitro
Genes Dev.
1999
, vol. 
13
 (pg. 
270
-
283
)
[PubMed]
69
Rada
P.
Rojo
A.I.
Chowdhry
S.
McMahon
M.
Hayes
J.D.
Cuadrado
A.
SCF/{beta}-TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a Keap1-independent manner
Mol. Cell. Biol.
2011
, vol. 
31
 (pg. 
1121
-
1133
)
[PubMed]
70
Chowdhry
S.
Zhang
Y.
McMahon
M.
Sutherland
C.
Cuadrado
A.
Hayes
J.D.
Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity
Oncogene
2013
, vol. 
32
 (pg. 
3765
-
3781
)
[PubMed]
71
Wu
T.
Zhao
F.
Gao
B.
Tan
C.
Yagishita
N.
Nakajima
T.
Wong
P.K.
Chapman
E.
Fang
D.
Zhang
D.D.
Hrd1 suppresses Nrf2-mediated cellular protection during liver cirrhosis
Genes Dev.
2014
, vol. 
28
 (pg. 
708
-
722
)
[PubMed]
72
Tempé
D.
Casas
M.
Karaz
S.
Blanchet-Tournier
M.F.
Concordet
J.P.
Multisite protein kinase A and glycogen synthase kinase 3beta phosphorylation leads to Gli3 ubiquitination by SCFbetaTrCP
Mol. Cell. Biol.
2006
, vol. 
26
 (pg. 
4316
-
4326
)
[PubMed]
73
Limón-Mortés
M.C.
Mora-Santos
M.
Espina
A.
Pintor-Toro
J.A.
López-Román
A.
Tortolero
M.
Romero
F.
UV-induced degradation of securin is mediated by SKP1-CUL1-beta TrCP E3 ubiquitin ligase
J. Cell Sci.
2008
, vol. 
121
 (pg. 
1825
-
1831
)
[PubMed]
74
Salazar
M.
Rojo
A.I.
Velasco
D.
de Sagarra
R.M.
Cuadrado
A.
Glycogen synthase kinase-3beta inhibits the xenobiotic and antioxidant cell response by direct phosphorylation and nuclear exclusion of the transcription factor Nrf2
J. Biol. Chem.
2006
, vol. 
281
 (pg. 
14841
-
14851
)
[PubMed]
75
Rada
P.
Rojo
A.I.
Evrard-Todeschi
N.
Innamorato
N.G.
Cotte
A.
Jaworski
T.
Tobón-Velasco
J.C.
Devijver
H.
García-Mayoral
M.F.
Van Leuven
F.
, et al. 
Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/β-TrCP axis
Mol. Cell. Biol.
2012
, vol. 
32
 (pg. 
3486
-
3499
)
[PubMed]
76
Rojo
A.I.
Rada
P.
Mendiola
M.
Ortega-Molina
A.
Wojdyla
K.
Rogowska-Wrzesinska
A.
Hardisson
D.
Serrano
M.
Cuadrado
A.
The PTEN/NRF2 Axis Promotes Human Carcinogenesis
Antioxid. Redox Signal.
2014
, vol. 
21
 (pg. 
2498
-
2514
)
[PubMed]
77
Kaidanovich-Beilin
O.
Woodgett
J.R.
GSK-3: Functional Insights from Cell Biology and Animal Models
Front. Mol. Neurosci.
2011
, vol. 
4
 pg. 
40
 
[PubMed]
78
Liu
P.
Cheng
H.
Roberts
T.M.
Zhao
J.J.
Targeting the phosphoinositide 3-kinase pathway in cancer
Nat. Rev. Drug Discov.
2009
, vol. 
8
 (pg. 
627
-
644
)
[PubMed]
79
Vanhaesebroeck
B.
Guillermet-Guibert
J.
Graupera
M.
Bilanges
B.
The emerging mechanisms of isoform-specific PI3K signalling
Nat. Rev. Mol. Cell. Biol.
2010
, vol. 
11
 (pg. 
329
-
341
)
[PubMed]
80
Thorpe
L.M.
Yuzugullu
H.
Zhao
J.J.
PI3K in cancer: divergent roles of isoforms, modes of activation and therapeutic targeting
Nat. Rev. Cancer
2014
, vol. 
15
 (pg. 
7
-
24
)
81
Yu
R.
Tan
T.H.
Kong
A.N.
Butylated hydroxyanisole and its metabolite tert-butylhydroquinone differentially regulate mitogen-activated protein kinases. The role of oxidative stress in the activation of mitogen-activated protein kinases by phenolic antioxidants
J. Biol. Chem.
1997
, vol. 
272
 (pg. 
28962
-
28970
)
[PubMed]
82
Yu
R.
Lei
W.
Mandlekar
S.
Weber
M.J.
Der
C.J.
Wu
J.
Kong
A.N.
Role of a mitogen-activated protein kinase pathway in the induction of phase II detoxifying enzymes by chemicals
J. Biol. Chem.
1999
, vol. 
274
 (pg. 
27545
-
27552
)
[PubMed]
83
Yu
R.
Chen
C.
Mo
Y.Y.
Hebbar
V.
Owuor
E.D.
Tan
T.H.
Kong
A.N.
Activation of mitogen-activated protein kinase pathways induces antioxidant response element-mediated gene expression via a Nrf2-dependent mechanism
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
39907
-
39913
)
[PubMed]
84
Yu
R.
Mandlekar
S.
Lei
W.
Fahl
W.E.
Tan
T.H.
Kong
A.N.
p38 mitogen-activated protein kinase negatively regulates the induction of phase II drug-metabolizing enzymes that detoxify carcinogens
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
2322
-
2327
)
[PubMed]
85
Keum
Y.S.
Yu
S.
Chang
P.P.
Yuan
X.
Kim
J.H.
Xu
C.
Han
J.
Agarwal
A.
Kong
A.N.
Mechanism of action of sulforaphane: inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response element-mediated heme oxygenase-1 in human hepatoma HepG2 cells
Cancer Res.
2006
, vol. 
66
 (pg. 
8804
-
8813
)
[PubMed]
86
Keum
Y.S.
Owuor
E.D.
Kim
B.R.
Hu
R.
Kong
A.N.
Involvement of Nrf2 and JNK1 in the activation of antioxidant responsive element (ARE) by chemopreventive agent phenethyl isothiocyanate (PEITC)
Pharm. Res.
2003
, vol. 
20
 (pg. 
1351
-
1356
)
[PubMed]
87
Xu
C.
Yuan
X.
Pan
Z.
Shen
G.
Kim
J.H.
Yu
S.
Khor
T.O.
Li
W.
Ma
J.
Kong
A.N.
Mechanism of action of isothiocyanates: the induction of ARE-regulated genes is associated with activation of ERK and JNK and the phosphorylation and nuclear translocation of Nrf2
Mol. Cancer Ther.
2006
, vol. 
5
 (pg. 
1918
-
1926
)
[PubMed]
88
Manandhar
S.
Cho
J.M.
Kim
J.A.
Kensler
T.W.
Kwak
M.K.
Induction of Nrf2-regulated genes by 3H-1, 2-dithiole-3-thione through the ERK signaling pathway in murine keratinocytes
Eur. J. Pharmacol.
2007
, vol. 
577
 (pg. 
17
-
27
)
[PubMed]
89
Zipper
L.M.
Mulcahy
R.T.
Inhibition of ERK and p38 MAP kinases inhibits binding of Nrf2 and induction of GCS genes
Biochem. Biophys. Res. Commun.
2000
, vol. 
278
 (pg. 
484
-
492
)
[PubMed]
90
Zipper
L.M.
Mulcahy
R.T.
Erk activation is required for Nrf2 nuclear localization during pyrrolidine dithiocarbamate induction of glutamate cysteine ligase modulatory gene expression in HepG2 cells
Toxicol. Sci.
2003
, vol. 
73
 (pg. 
124
-
134
)
[PubMed]
91
Alam
J.
Wicks
C.
Stewart
D.
Gong
P.
Touchard
C.
Otterbein
S.
Choi
A.M.
Burow
M.E.
Tou
J.
Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
27694
-
27702
)
[PubMed]
92
Keum
Y.S.
Yu
S.
Chang
P.P.
Yuan
X.
Kim
J.H.
Xu
C.
Han
J.
Agarwal
A.
Kong
A.N.
Mechanism of action of sulforaphane: inhibition of p38 mitogen-activated protein kinase isoforms contributing to the induction of antioxidant response element-mediated heme oxygenase-1 in human hepatoma HepG2 cells
Cancer Res.
2006
, vol. 
66
 (pg. 
8804
-
8813
)
[PubMed]
93
Naidu
S.
Vijayan
V.
Santoso
S.
Kietzmann
T.
Immenschuh
S.
Inhibition and genetic deficiency of p38 MAPK up-regulates heme oxygenase-1 gene expression via Nrf2
J. Immunol.
2009
, vol. 
182
 (pg. 
7048
-
7057
)
[PubMed]
94
Tsai
C.W.
Lin
C.Y.
Wang
Y.J.
Carnosic acid induces the NAD(P)H: quinone oxidoreductase 1 expression in rat clone 9 cells through the p38/nuclear factor erythroid-2 related factor 2 pathway
J. Nutr.
2011
, vol. 
141
 (pg. 
2119
-
2125
)
[PubMed]
95
DeNicola
G.M.
Karreth
F.A.
Humpton
T.J.
Gopinathan
A.
Wei
C.
Frese
K.
Mangal
D.
Yu
K.H.
Yeo
C.J.
Calhoun
E.S.
, et al. 
Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis
Nature
2011
, vol. 
475
 (pg. 
106
-
109
)
[PubMed]
96
Wakabayashi
N.
Itoh
K.
Wakabayashi
J.
Motohashi
H.
Noda
S.
Takahashi
S.
Imakado
S.
Kotsuji
T.
Otsuka
F.
Roop
D.R.
, et al. 
Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation
Nat. Genet.
2003
, vol. 
35
 (pg. 
238
-
245
)
[PubMed]
97
Devling
T.W.
Lindsay
C.D.
McLellan
L. I.
McMahon
M.
Hayes
J.D.
Utility of siRNA against Keap1 as a strategy to stimulate a cancer chemopreventive phenotype
Proc. Natl. Acad. Sci. U.S.A.
2005
, vol. 
102
 (pg. 
7280
-
7285A
)
[PubMed]
98
Sun
Z.
Huang
Z.
Zhang
D.D.
Phosphorylation of Nrf2 at multiple sites by MAP kinases has a limited contribution in modulating the Nrf2-dependent antioxidant response
PLoS One
2009
, vol. 
4
 pg. 
e6588
 
[PubMed]
99
Lee
J.M.
Hanson
J.M.
Chu
W.A.
Johnson
J.A.
Phosphatidylinositol 3-kinase, not extracellular signal-regulated kinase, regulates activation of the antioxidant-responsive element in IMR-32 human neuroblastoma cells
J. Biol. Chem.
2001
, vol. 
276
 (pg. 
20011
-
20016
)
[PubMed]
100
Li
J.
Lee
J.M.
Johnson
J.A.
Microarray analysis reveals an antioxidant responsive element-driven gene set involved in conferring protection from an oxidative stress-induced apoptosis in IMR-32 cells
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
388
-
394
)
[PubMed]
101
Lee
J.M.
Calkins
M.J.
Chan
K.
Kan
Y.W.
Johnson
J.A.
Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
12029
-
12038
)
[PubMed]
102
Martin
D.
Rojo
A.I.
Salinas
M.
Diaz
R.
Gallardo
G.
Alam
J.
De Galarreta
C.M.
Cuadrado
A.
Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol
J. Biol. Chem.
2004
, vol. 
279
 (pg. 
8919
-
8929
)
[PubMed]
103
Fahey
J.W.
Dinkova-Kostova
A.T.
Stephenson
K.K.
Talalay
P.
The “Prochaska” microtiter plate bioassay for inducers of NQO1
Methods Enzymol.
2004
, vol. 
382
 (pg. 
243
-
258
)
[PubMed]
104
Pitha-Rowe
I.
Liby
K.
Royce
D.
Sporn
M.
Synthetic triterpenoids attenuate cytotoxic retinal injury: cross-talk between Nrf2 and PI3K/AKT signaling through inhibition of the lipid phosphatase PTEN
Invest. Ophthalmol. Vis. Sci.
2009
, vol. 
50
 (pg. 
5339
-
5347
)
[PubMed]
105
Ross
S.H.
Lindsay
Y.
Safrany
S.T.
Lorenzo
O.
Villa
F.
Toth
R.
Clague
M.J.
Downes
C.P.
Leslie
N.R.
Differential redox regulation within the PTP superfamily
Cell Signal.
2007
, vol. 
19
 (pg. 
1521
-
1530
)
[PubMed]
106
Jain
A.K.
Jaiswal
A.K.
GSK-3beta acts upstream of Fyn kinase in regulation of nuclear export and degradation of NF-E2 related factor 2
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
16502
-
16510
)
[PubMed]
107
Niture
S.K.
Jain
A.K.
Shelton
P.M.
Jaiswal
A.K.
Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression
J. Biol. Chem.
2011
, vol. 
286
 (pg. 
28821
-
28832
)
[PubMed]
108
Cohen
P.
Frame
S.
The renaissance of GSK3
Nat. Rev. Mol. Cell. Biol.
2001
, vol. 
2
 (pg. 
769
-
776
)
[PubMed]