Nrf2 Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species

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1 Advan. Enzyme Regul. 46 (2006) Nrf2 Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species Makoto Kobayashi, Masayuki Yamamoto JST-ERATO Environmental Response Project, Center for Tsukuba Advanced Research Alliance, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba , Japan Introduction Xenobiotics are chemical substances that are foreign to biological systems and include naturally occurring compounds, drugs, and chemicals. A simple model for understanding the metabolic transformation or detoxication of xenobiotics in animals is division of the process into two consecutive reactions. The phase 1 reaction is mediated by cytochrome P450 monooxygenase systems, such as CYP1A1 and CYP1A2, which modify xenobiotics through oxidation and reduction. The products of phase 1 reactions are often electrophilic and highly reactive, thereby resulting in the harmful modification of DNA and proteins. Phase 2 enzymes promote the conjugation of phase 1 products with various hydrophilic moieties, such as glutathione and glucuronic acid. Xenobiotics often serve as ligands for Abbreviations: ARE, Antioxidant-responsive element; BHA, Butylated hydroxyanisole; BTB, Broad complextramtrack-bric-a-brac; bzip, Basic leucine zipper; CBP, CREB binding protein; C/EBPb, CCAAT/enhancer binding protein b; CNC, Cap n collar; Cox2, Cyclooxygenase 2; Cul3, Cullin 3; DexMes, dexamethasone 21- mesylate; DGR, Double glycine repeat; 15d-PGJ 2, 15-deoxy-D 12,14 -prostaglandin J 2 ; EpRE, Electrophileresponsive element; ERK, Extracellular signal-regulated kinases; EST, Expression sequence tagged; FAC1, Fetal ALZ-50 reactive clone 1; ggcs, g-glutamylcysteine synthetase; GSTs, Glutathione S-transferases; HO-1, Heme oxygenase-1; IAB, N-iodoacetyl-N-biotinylhexylenediamine; IVR, Intervening region; Keap1, Kelch-like ECH associating protein 1; NES, Nuclear export signal; NFkB, Nuclear factor-kb; NLS, Nuclear localizing signal; Neh, Nrf2-ECH homology; Nrf2, NF-E2-related protein 2; NQO-1, NAD(P)H:quinone oxidoreductase; PERK, PKR-like endoplasmic reticulum kinase; PKC, Protein kinase C; PPARg; Peroxisome proliferator-activated receptor g; ROS, Reactive oxygen species; sirna, Small interfering RNA; SNPs, Single nucleotide polymorphisms; tbhq, Tert-butylhydroquinone; UGTs, UDP Glucuronosyl transferases Corresponding author. addresses: masi@tara.tsukuba.ac.jp, geoweber@iupui.edu (M. Yamamoto) /$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi: /j.advenzreg

2 114 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) key transcriptional activators of phase 1 enzyme genes, while phase 2 enzyme genes are often induced by phase 1 metabolites (Primiano et al., 1997; Mimura and Fujii-Kuriyama, 2003). Characterization of the regulatory regions of phase 2 enzyme genes, such as those for NAD(P)H:quinone oxidoreductase (NQO-1), glutathione S-transferases (GSTs), and UDP-glucuronosyl transferases (UGTs), revealed that electrophiles transcriptionally activate the expression of these genes through the antioxidant-responsive element (ARE; Rushmore and Pickett, 1990) or electrophile-responsive element (EpRE; Friling et al., 1990). After several years of extensive searching for ARE/EpRE (hereafter ARE for simplicity) binding transcription factors, we discovered NF-E2-related protein 2 (Nrf2), the major transactivating factor for ARE-dependent gene transcription (Itoh et al., 1997). Analyses of Nrf2-null mutant mice revealed that Nrf2 not only regulates the electrophile-induced expression of phase 2 enzyme genes, but is also critical in the regulation of various antioxidant genes in response to the oxidative stress (Ishii et al., 2000) and inflammation (Itoh et al., 2004). The Nrf2 molecule and/or nrf2 gene have been identified in human, mouse, rat, chicken, and zebrafish, suggesting that this transcription factor exists in virtually all vertebrates. Gene knockdown analysis revealed that Nrf2 is required for the induction of GST and NQO-1 genes in zebrafish, as well as in mammals, an observation indicating a high-level of conservation in the Nrf2 system (Kobayashi et al., 2002; Suzuki et al., 2005). Nrf2 can be activated by endogenous products of oxidative stress or other stresses generated inside the body, such as 4-hydroxynonenal (Numazawa et al., 2003; Ishii et al., 2004), oxidized low-density lipoproteins (Ishii et al., 2004; Anwar et al., 2005), heme (Kim et al, 2001; Alam et al., 2003; Nakaso et al., 2003), and nitric oxide (Kang et al, 2002a; Buckley et al., 2003; Dhakshinamoorthy and Porter, 2004). In addition, 15-deoxy- D 12,14 -prostaglandin J 2 (15d-PGJ 2 ; Itoh et al., 2004; Levonen et al., 2004; Zhang et al., 2004b), keratinocyte growth factor (Braun et al., 2002), and fibroblast growth factor (Vargas et al., 2005) were also found to induce Nrf2-dependent gene expression. As these agents function as signaling molecules in many systems, the Nrf2 system has emerged as one of the central components of cellular defense networks. Since the importance and historical aspects of phase 2 gene regulation in the protection against electrophilic and oxidative stresses have already been excellently summarized by Talalay and colleagues (Talalay et al., 2003; Holtzclaw et al., 2004), we will focus on topics relating to Nrf2 and its partner Keap1 in this review. Brief history of the CNC and Maf family transcription factors Nrf2 is a basic region-leucine zipper (bzip)-type transcription factor (Moi et al., 1994; Itoh et al., 1995) and is structurally related to the p45 subunit of NF-E2 (nuclear factorerythroid 2). NF-E2 was originally identified as an erythroid-restricted DNA-binding activity that recognizes the specific DNA sequence (A/G)TGA(G/C)TCAGCA (NF-E2 binding motif) containing a 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE; TGA(G/C)TCA). NF-E2 was the second factor found among transcription factors that have specificity for erythroid genes, as the factor now known as GATA-1 was initially termed NF-E1. The NF-E2 binding motif is frequently found in the regulatory regions of globin and other erythroid genes and the functional importance of this motif for the

3 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) expression of these target genes has been demonstrated (Reitman and Felsenfeld, 1988; Mignotte et al., 1989; Ney et al., 1990; Strauss et al., 1992). Purification of the NF-E2 binding activity from human erythroleukemia cells revealed that NF-E2 is a heterodimeric protein consisting of a large p45 and small p18 subunit (Ney et al., 1993). It was shown by cdna cloning that p45 contains a Cap n collar (CNC)-type bzip domain (Andrews et al., 1993), which is highly conserved in the Drosophila transcription factor CNC (Mohler et al., 1991). The p45 subunit does not form a homodimer, but utilizes its CNC-bZip domain for heterodimerizing with p18 (Andrews et al., 1993). The p18 subunit was identified as MafK, one of the small Maf transcription factors that are cellular products of the Maf proto-oncogene family (Igarashi et al., 1994). The small Maf proteins MafF, MafG, and MafK possess a bzip domain and either homodimerize or heterodimerize with each other, but lack a canonical transactivation domain (Fujiwara et al., 1993; Kataoka et al., 1995). All three small Maf proteins can form heterodimers with p45, and the p45-small Maf heterodimers bind to the NF-E2 motif (Igarashi et al., 1994). In the heterodimeric association of mouse p45 and MafK, the small Maf protein confers DNA-binding activity to p45, while p45 activates transcription via its transactivation domain (Igarashi et al., 1995). The expression of p45 is restricted to hematopoietic progenitors and erythroid, megakaryocytic, and mast cells, which suggests that p45 is the major transcriptional activator in these lineages. Normal erythropoiesis occurs in p45-null mutant mice, but there is a high mortality due to severe hemorrhage resulting from the absence of circulating platelets (Shivdasani and Orkin, 1995; Shivdasani et al., 1995). On the contrary, small Maf family proteins are expressed in various hematopoietic and non-hematopoietic lineages (Fujiwara et al., 1993; Igarashi et al., 1994). Simple disruption of mafk or maff genes showed no apparent phenotype (Kotkow and Orkin, 1996; Shavit et al., 1998; Onodera et al., 1999), whereas mafg gene knockout mice displayed both behavioral abnormalities and impaired platelet formation (Shavit et al., 1998). The latter phenotype is related to that of the p45-null mouse, suggesting that MafG is the essential partner molecule of p45 in megakaryopoiesis. Each small Maf gene exhibits an overlapping but distinct tissue distribution pattern during development (Motohashi et al., 1996), which may be the reason why disruption of MafG alone resulted in the specific phenotypes (Shavit et al., 1998). However, the remarkable similarity in amino acid sequences of the three small Maf proteins suggests functional redundancy. Indeed, in vitro analyses revealed that MafF, MafG, and MafK are functionally interchangeable (Igarashi et al., 1994; Kataoka et al., 1995). Transgenic overexpression of MafK in vivo rescued MafG-null mutant mice from neuronal and megakaryocytic disorders (Motohashi et al., 2000). Consistent with the contention that the small Mafs share a functional redundancy, small Maf compound mutant lines of mice displayed more severe phenotypes than the MafG single mutant mice (Onodera et al., 2000; Katsuoka et al., 2003, 2005b; Motohashi et al., 2004). The data suggest that the gene dosage or expression level of small Mafs may also be an important determinant in the target gene expression. Supporting this notion, both elevation and reduction in the abundance of small Maf protein were found to severely affect megakaryopoiesis (Motohashi et al., 2000). Transgenic overexpression of MafK in mice severely affects the proliferation and function of T cells (Yoh et al., 2001b). The expression of small Maf proteins in non-hematopoietic tissues suggests the existence of heterodimeric partners additional to p45 and in fact five NF-E2-related proteins have

4 116 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) been cloned and characterized (Fig. 1). These factors have a common CNC-type bzip domain and exploit one of the small Mafs as an obligatory heterodimeric partner molecule for binding to the NF-E2 motif (Oyake et al., 1996; Toki et al., 1997; Kobayashi et al., 1999). A probe containing the tandem NF-E2 sites of the b-globin locus control region was used to isolate Nrf1 from an expression library (Chan et al., 1993; Caterina et al., 1994). Human Nrf2 was identified in the screen for molecules interacting with the NF-E2 binding motif (Moi et al., 1994), while ECH (erythroid-derived CNC homology protein) was independently isolated in the search for the p45 homologue in chicken (Itoh et al., 1995). The fourth member of the CNC family proteins, Nrf3, was isolated as an expression sequence tagged (EST) clone encoding an amino acid sequence homologous to that of Nrf1 (Kobayashi et al., 1999). Bach1 and Bach2 were discovered by yeast two-hybrid screening in the search for proteins that interact with MafK (Oyake et al., 1996). Unlike other NF-E2-related proteins, the Bach factors possess a BTB (broad complex-tramtrack-brica-brac) domain (Fig. 1). Nrf1, Nrf2, and Nrf3 are expressed in a wide range of tissues, with each factor showing a distinct, but overlapping expression profile. Nrf1-null mutant mice die of anemia in the late stage of gestation. Since Nrf1-null embryonic stem cells contribute to the development of adult blood cells in chimeric mice, defective erythropoiesis is a non-cell autonomous process (Farmer et al., 1997; Chan et al., 1998). On the other hand, Nrf2-null mutant mice do not display any significant abnormality in hematopoiesis (Chan et al., 1996; Itoh et al., 1997). To test the occurrence of compensation among members of the CNC family in the erythroid lineage, mutant mice compound for p45 and Nrf2 were generated and examined (Kuroha et al., 1998; Martin et al., 1998). The concomitant lack of p45 and Nrf2 did not Neh5 Neh6 Neh3 p45 NF-E 2 Neh2 Nrf1 Nrf2 Neh4 Nrf3 Bach1 Bach2 BTB CNC-bZIP Fig. 1. Structural properties of CNC protein family members. Six Neh domains are conserved among vertebrate Nrf2, while some of them are included in other CNC proteins. Among them, Neh4 is the only domain specific to Nrf2. Nrf1 also contains a Neh2-like motif, an interaction site for Keap1, but the precise function of Nrf1 Neh2 is not known. None of the Neh domains exist in the Bach proteins.

5 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) exacerbate the erythroid and megakaryocytic dysfunctions observed in the p45-null mice, suggesting that Nrf2 is dispensable for normal hematopoiesis. Nrf3-null mice developed normally and no obvious phenotype was observed (Kobayashi et al., 2004b; Derjuga et al., 2004). While Bach1 is expressed ubiquitously, its expression is high in hematopoietic organs, such as bone marrow and fetal liver (Igarashi et al., 1998). Bach2 is most abundant in the brain and B cell lineage (Oyake et al., 1996; Muto et al., 1998). Bach1-null mutant mice are viable and fertile (Sun et al., 2002). Genetic ablation of Bach2 in mice revealed that Bach2 is required for the class switch recombination of immunoglobulin and somatic hypermutation (Muto et al., 2004). Nrf2 activates cytoprotective genes under the regulation of the ARE Transcriptional activation of phase 2 enzyme genes by antioxidants and/or electrophiles is mediated by a cis-acting element called the ARE. The ARE has been detected in the promoter or upstream promoter regions of the genes encoding GST-Ya (rat and mouse; Friling et al., 1990; Rushmore et al., 1990), GST-P (rat; Okuda et al., 1989), and NQO-1 (rat and human; Favreau and Pickett, 1991; Li and Jaiswal, 1992). The mouse heme oxygenase-1 (HO-1) gene has two enhancers, both harboring multiple AREs (Prestera et al., 1995). AREs have also been identified in the regulatory regions of human g-glutamylcysteine synthetase (ggcs) heavy subunit (Mulcahy and Gipp, 1995), mouse GST-M1 and M3 (Reinhart and Pearson, 1993), and mouse ferritin light chain (Wasserman and Fahl, 1997). These ARE motifs match a common (A/G)TGACnnnGC sequence originally identified by mutagenesis of the rat GST-Ya ARE (Rushmore et al., 1991) and show high homology to the functional ARE sequence proposed by Wasserman and Fahl (1997). While extensive analyses were carried out to identify transcription factors transactivating through the ARE, it took a long time for the identification of transcription factors binding and transducing signals from the ARE in vivo. Retrospectively, this seems to be due to the fact that (i) a single transcription factor neither binds nor activates transcription through the ARE, (ii) AP-1 has been suggested to mediate the response based on the similarity of its TRE-binding motif to the ARE sequence (Friling et al., 1992; Li and Jaiswal, 1992), and (iii) reporter co-transfection analysis and gel retardation assays have been the major approaches and did not give rise to a clear resolution. Based of the similarity of the ARE and NF-E2 binding motif sequences, it was hypothesized that some of the CNC-small Maf heterodimers may regulate gene expression through the ARE. Nrf2 was thought to be the most probable CNC factor, as it is highly expressed in metabolic organs. Therefore, to test this hypothesis, Nrf2-null mutant mice were generated and examined. Following dietary administration of the phenolic antioxidant butylated hydroxyanisole (BHA) to Nrf2-null mutant mice, the expressions of GSTs and NQO-1 were examined in the liver and intestine (Itoh et al., 1997). Whereas BHA induced the gene expression of four GST subunits (Ya1, Ya3, Yp, and Yb) and NQO-1 in wild-type mice, the magnitude of the inductions were much lower in the Nrf2- null mutant mice. In the Nrf2-null mice, oxidant-induced expression of HO-1 and ggcs heavy subunit was also markedly reduced (Ishii et al., 2000; McMahon et al., 2001). Reduction in both the inducible expression and basal level expression of some AREregulated genes was observed (Chanas et al., 2002). Consistent with these results, it was

6 118 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) also reported that Nrf1 and Nrf2 elevate ARE-reporter expression in cotransfection transactivation experiments (Venugopal and Jaiswal, 1996). These results thus indicate that Nrf2 is an important activator of phase 2 and antioxidant enzyme genes. Nrf2 is the major regulator of cytoprotective gene expression through the ARE Since the other CNC family members bind to the ARE in vitro, it would be interesting to examine whether they can also regulate gene expression through the ARE in vivo. The contribution of Nrf1 and Nrf3 to the regulation of cytoprotective gene expression has been analyzed using gene-disrupted lines of mice. As mentioned above, Nrf1-deficient mice die during development (Farmer et al., 1997; Chan et al., 1998), but analyses of Nrf1 single and Nrf1 Nrf2 compound mutant cells suggest that Nrf1 contributes to the regulation of the basal expression level of certain cytoprotective enzyme genes, but not to their inducible expression (Kwong et al., 1999; Leung et al., 2003). Recently, liver-specific Nrf1-deficient mice were generated using the Cre-loxP system to bypass embryonic lethality. In these mice, a reduced basal level of expression of GST-P and some of the GST-Ms was observed (Xu et al., 2005), demonstrating the participation, albeit limited contribution, of Nrf1 in their cytoprotective gene expression. As mentioned above, ablation of the nrf3 gene revealed no obvious phenotypical differences (Kobayashi et al., 2004b; Derjuga et al., 2004). It also seems unlikely that Nrf3 acts as a major regulator of phase 2 enzyme genes, since Nrf3 is not expressed in tissues important for the electrophilic response, such as liver and intestine (Kobayashi et al., 1999; Derjuga et al., 2004). By the same token, p45 NF-E2 does not appear to be the major regulator (Shivdasani and Orkin, 1995; Shivdasani et al., 1995). Bach proteins have been shown to act as transcriptional repressors (see below; Muto et al., 1998; Yoshida et al., 1999). Despite all these observations, the possibility remains and seems plausible that Nrf1, Nrf3, p45, Bach1, and Bach2 may play crucial roles in the oxidative stress response in specific tissues. Nrf2 activity is finely tuned by the other ARE-binding transcription factors It is possible that other members of the CNC family or other bzip-type transcription factors inhibit the transactivation of Nrf2-small Maf heterodimers by competing for the ARE. Important transcription factors in this regard are Bach1 and Bach2, since Bach1 antagonizes Nrf2 during HO-1 gene expression (Sun et al., 2002). Oxidative stress induces the nuclear accumulation of Bach2 and reduces the expression of the ARE reporter gene (Muto et al., 2002). Small Maf proteins have also been shown to act as transcriptional repressors (Igarashi et al., 1994; Kataoka et al., 1995). Repression is brought about by their lack of a transactivation domain, such that small Maf homodimers tend to inhibit the binding of activating heterodimers to the ARE. Indeed, inhibition of Nrf2 activity by the expression of an excess amount of small Mafs has been demonstrated in various experiments utilizing cultured cells (Marini et al., 1997; Alam et al., 1999; Wild et al., 1999; Dhakshinamoorthy and Jaiswal, 2000; Nguyen et al., 2000) and in zebrafish embryos (Takagi et al., 2004). Similarly, overexpression of Nrf3 decreased the expression of an ARE-driven reporter and endogenous NQO-1 genes in Hep-G2 cells. The expression of these genes was recovered by the application of small interfering RNA (sirna) specific to Nrf3, suggesting that Nrf3

7 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) can behave as a negative regulator of cytoprotective gene expression (Sankaranarayanan and Jaiswal, 2004). Overexpression of c-fos, Fra1, or c-maf has also been reported to negatively regulate the expression of an ARE-driven reporter gene, probably through competitive binding to the ARE. These observations give rise to an interesting insight into the roles played by AP-1 family factors and large Maf proteins during the oxidative or electrophilic stress response; these factors may negatively regulate Nrf2 activity in vivo (Venugopal and Jaiswal, 1996; Dhakshinamoorthy and Jaiswal, 2002). Indeed, the basal expression level of cytoprotective genes is increased in c-fos-null mutant mice (Wilkinson et al., 1998). Similarly, it was reported that TGFb down-regulates g-gcs heavy chain gene expression by inducing Fra1-c-Jun heterodimer binding to the ARE (Jardine et al., 2002) or by inducing ATF3 expression (Bakin et al., 2005). Since Nrf2 is the most potent transcriptional activator among CNC proteins (Toki et al., 1997; Kobayashi et al., 1999,2004b; Katoh et al., 2001), Nrf2 may enhance cytoprotective gene expression even if the other transcription factors are present and occupy ARE. Thus, available lines of evidence support the contention that Nrf2 is the major activity inducing the expression of phase 2 and antioxidant enzyme genes, whereas the basal and inducible expression of these genes, and perhaps the timing of the induction and its termination, is finely tuned by the other ARE-binding transcription factors. Nrf2 regulon: orchestrated regulation of cytoprotective genes The induction of all known ARE-regulated genes is under the control of Nrf2. This led us to isolate unidentified ARE-regulated genes by screening for genes that are induced by phase 2 inducers in wild-type mice but not in Nrf2-null mice. Surprisingly, many genes encoding detoxifying, antioxidant, and glutathione-biosynthesis enzymes were found to be regulated by Nrf2, such as UGT1A6, UGT1A7, aflatoxin B1 aldehyde reductase, microsomal epoxide hydrolase, g-gcs light subunit, ubiquitin/pkc-z-interacting protein A170, peroxiredoxin 1, the heavy chain of ferritin, catalase, glutathione peroxidase, glutathione reductase, superoxide dismutase, thioredoxin, thioredoxin reductase, carboxylesterase, aldehyde oxidase, aldehyde dehydrogenases, and 1-Cys peroxiredoxin (Chan and Kan, 1999; Chan and Kwong, 2000; Ishii et al., 2000; Kim et al., 2001; Kwak et al., 2001; Cho et al., 2002a,2005; Lee et al., 2003a; Pietsch et al., 2003; Iida et al., 2004; Rangasamy et al., 2005). Nrf2 also regulates genes encoding metabolic enzymes that are directly or indirectly involved in detoxifying and anti-oxidative stress response processes and include glucose-6-phosphate dehydrogenase, UDP-glucose dehydrogenase, malic enzyme, the cysteine/glutamate exchange transporter xct, and the multi-drug resistance-associated protein 1/ATP-binding cassette transporter C (Sasaki et al., 2002; Thimmulappa et al., 2002; Hayashi et al., 2003; Rangasamy et al., 2004; Cho et al., 2005). These results suggest the existence of an orchestrated regulation by Nrf2 in the cellular protection against environmental stresses. Therefore, it seems reasonable to consider ARE-regulated genes as constituting Nrf2 regulon in the coordinated expression of genes related to detoxifying/antioxidant enzymes, as is the case for the soxrs and oxyr regulons in Escherichia coli (Pomposiello and Demple, 2001). Indeed, we found functional AREs in the regulatory regions of these newly identified genes, suggesting that Nrf2 directly governs a large number of cytoprotective genes via the ARE (Hudson and Kavanagh, 2000; Kim et al., 2001; Erickson et al., 2002; Sasaki et al., 2002; Solis et al., 2002;

8 120 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) Hayashi et al., 2003; Pietsch et al., 2003; Banning et al., 2005; Nishinaka and Yabe- Nishimura, 2005). To identify Nrf2-target genes systematically, microarray-based surveys have also been conducted (Li et al., 2002,2004b; Thimmulappa et al., 2002; Kwak et al., 2003b; Lee et al., 2003a, b; Kraft et al., 2004; Cho et al., 2005). The results suggest that Nrf2 may regulate more than 200 genes; not only genes related to detoxifying and antioxidant enzymes, but also genes that are classified into different categories. The expression of a number of 26S proteasome subunits is induced in an Nrf2-dependent manner by antioxidants (Kwak et al., 2003b). Especially, a reporter co-transfection assay and chromatin immunoprecipitation assay of the PSMB5 subunit gene revealed that the gene is a direct target of Nrf2 (Kwak et al., 2003a). Induction of the 26S proteasome may provide an efficient means for cells to survive in various stress conditions that collectively enhance the likelihood of chronic diseases. Heat shock proteins are also inducible by the Nrf2-dependent pathway (Kwak et al., 2003b). Since the accumulation of unfolded polypeptides following oxidative stress disturbs normal cellular function and triggers apoptosis, chaperone proteins, together with the proteasome system, seem to play essential roles in responding to stress by repairing and removing damaged proteins. CD36, a gene encoding the scavenger receptor that mediates the uptake of oxidized low-density lipoproteins, is also a target of Nrf2 in vascular smooth muscle cells (Ishii et al., 2004), implying that Nrf2 is an important component in the pathway preventing the development of atherosclerosis. In certain tissues or cells, the level of Nrf2 transcription itself is basically unchanged before and after treatment with phase 2 inducers. On the contrary, in keratinocytes, the nrf2 gene appears to be auto-regulated through an ARE sequence (Kwak et al., 2002). Oxidative stress induces the expression of small Maf proteins (Crawford et al., 1996; Suzuki et al., 2001; Moran et al., 2002; Takagi et al., 2004; Katsuoka et al., 2005b) and a functional ARE was recently identified in the mouse mafg gene (Katsuoka et al., 2005a). Nrf3 expression was upregulated in the skin of the Nrf2-null mouse (Braun et al., 2002). Since Nrf3 and small Maf homodimers suppress Nrf2-mediated gene expression, their gene induction may result in negative feedback regulation of phase 2 enzyme genes. Disruption of Nrf2 makes mice susceptible to various environmental stresses Nrf2-null mutant mice provide a valuable means for the study of cytoprotective gene expression in vivo. Nrf2 regulates the coordinated and global expression of the genes for phase 2 and anti-oxidative stress enzymes, such that a loss in the contribution of Nrf2 severely impairs the detoxification machinery. Therefore, although Nrf2-null mutant mice do not display a significant phenotype per se, these animals carry an enhanced susceptibility to environmental stresses. Indeed, without Nrf2, the induction of cytoprotective enzymes is insufficient, impinging upon the cells an increased susceptibility towards toxic xenobiotics, such as acetaminophen, butylated hydroxytoluene, diesel exhaust, and cigarette smoke (Aoki et al., 2001; Chan et al., 2001; Enomoto et al., 2001; Rangasamy et al., 2004). The activity of Nrf2 is also required for protection against the oxidative tissue damage induced by acute pulmonary injury, hyperoxia, and ultraviolet (UV) light (Chan and Kan, 1999; Cho et al., 2002a; Gao and Talalay, 2004; Hirota et al., 2005).

9 Elimination of Nrf2 reduces both the constitutive and inducible expression of cytoprotective genes, thereby enhancing the sensitivity of neurons and astrocytes to oxidative stress (Lee et al., 2003a, b; Calkins et al., 2005). Administration of mitochondrial complex II inhibitors to mice causes brain lesions. Disruption of Nrf2 expression increases the size of the lesions, while transplantation of Nrf2-overexpressing astrocytes reduces it (Calkins et al., 2005; Shih et al., 2005). These studies thus demonstrate that Nrf2 is fundamental to the defense against reactive oxygen species (ROS) and imply that Nrf2 deficiency is involved in the pathogenesis of lung, neural, and other chronic diseases. The redox status of wild-type and Nrf2-null mutant mice was measured using a combination of real-time electron paramagnetic resonance imaging and spin probe kinetic analysis and revealed that Nrf2 acts to reduce the ROS level in vivo (Hirayama et al., 2003). Nrf2-null mice also form a higher level of DNA adducts and show an elevated incidence of cancer following exposure to carcinogens such as aflatoxin B1, diesel particulate matter, benzo[a]pyrene, and N-nitroso compounds (Aoki et al., 2001; Kwak et al., 2001; Ramos- Gomez et al., 2003; Iida et al., 2004). The effects of cancer chemopreventive agents, such as oltipraz and sulforaphane, are abolished in Nrf2-deficient mice (Kwak et al., 2001; Ramos- Gomez et al., 2001, 2003; Fahey et al., 2002; Iida et al., 2004), indicating that an array of enzymes regulated by Nrf2 is critical for cancer chemoprevention. Nrf2 and diseases M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) Chronic inflammation, a process associated with elevated levels of various cytokines and ROS, has been regarded as a leading contributor to carcinogenesis. Recently, Nrf2 target genes were found to play anti-inflammatory roles. Using carrageenan-induced pleurisy as a model system for acute inflammation, it was found that the accumulation of neutrophils during inflammation persists and that macrophage recruitment is delayed in the Nrf2-null mutant mice (Itoh et al., 2004). Increased inflammation by nrf2-gene disruption has also been observed in wounded mouse skin (Braun et al., 2002), as well as in the lungs of cigarette smoke-exposed mice and ovalbumin-challenged mice (Rangasamy et al., 2004, 2005; Ishii et al., 2005; Iizuka et al., 2005). The expression of inducible nitric oxide (ino) synthase is suppressed by the anti-inflammatory agent triterpenoid and was abrogated by Nrf2 disruption in mouse embryonic fibroblasts (Dinkova-Kostova et al., 2005b). Laminar shear stress (Hosoya et al., 2005) and anti-rheumatic gold-containing compounds (Kataoka et al., 2001; Chen et al., 2003) activate phase 2 enzyme genes in an Nrf2- dependent manner. These broad observations demonstrate that the Nrf2 ARE pathway modulates inflammation by orchestrating the recruitment of inflammatory cells and regulating the expression of genes related to inflammation. Interestingly, wild-type mice pretreated with a cyclooxygenase 2 (Cox2) inhibitor displayed an increased neutrophil invasion and delayed recruitment of macrophages, an outcome similar to nrf2 gene disruption. In contrast, concomitant administration of 15d- PGJ 2 eliminated the effect of the Cox2 inhibitor (Itoh et al., 2004). Importantly, neither the Cox2 inhibitor nor 15d-PGJ 2 affected the Nrf2-null mutant mice. These findings strongly support the contention that Nrf2 is involved in regulation of the inflammatory process downstream of 15d-PGJ 2, an endogenous regulator of the inflammatory response. Nrf2-decificent cells are prone to apoptosis upon induction by H 2 O 2, nitric oxide, stressed endoplasmic reticulum, and glucose depletion (Cullinan et al., 2003; Cullinan and Diehl, 2004; Dhakshinamoorthy and Porter, 2004; Kraft et al., 2004; Lee et al., 2004).

10 122 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) Similarly, the neurons of Nrf2-null mice are sensitive to mitochondrial toxin-induced apoptosis (Lee et al., 2003a, b; Calkins et al., 2005; Shih et al., 2005). Disruption of Nrf2 increased the number of apoptotic cells following UV irradiation in dermal fibroblasts (Hirota et al., 2005). Overexpression of Nrf2 was shown to protect the cells from Fas-induced apoptosis (Kotlo et al., 2003). Nrf2 has been shown to regulate the sensitivity of cells to Fas-inducible apoptosis by affecting intracellular glutathione levels (Morito et al., 2003). These broad observations demonstrate the function of Nrf2 in cell survival, which may be mediated, at least in part, by inhibition of the FAS-dependent apoptosis pathway. Nrf2-null mutant mice developed normally under standard laboratory conditions (Itoh et al., 1997). However, female Nrf2-null mice displayed a short lifespan and developed severe lupus-like autoimmune nephritis (Yoh et al., 2001a; Li et al., 2004b), indicating that Nrf2 is one of the genes determining susceptibility to autoimmune diseases. Suh et al. (2004) reported an age-associated decline in glutathione levels in rat liver, which may be caused by dysregulation of Nrf2-dependent gene transcription. Thus, Nrf2-null mutant mice may also be susceptible to aging. Susceptibility of Nrf2-null mice to various stresses suggests that dysfunction of Nrf2 may be involved in the pathogenesis of human diseases. Polymorphisms and single nucleotide polymorphisms (SNPs) have already been found in the human NRF2 gene (Yamamoto et al., 2004). The frequency of these polymorphisms was examined in two groups of patients, those with systemic lupus erythematosis and those with chronic obstructive pulmonary disease. While a close connection between the risk of these two diseases and NRF2 polymorphisms was not established in that study, the result does not exclude the importance of examining the link between NRF2 polymorphisms and other oxidative stress-related disorders. Indeed, a correlation between mouse strain-specific variation in susceptibility to hyperoxia and a polymorphic T to C substitution in the nrf2 gene has been identified (Cho et al., 2002b). Suppression of Nrf2 activity by Keap1 in unstressed basal conditions Extensive domain structure-function analyses have been conducted and the function of domains/regions homologous between human Nrf2 and chicken ECH, which are referred to as Neh (Nrf2-ECH homology) domains (Itoh et al., 1997), have been characterized. Six Neh domains have been identified (Fig. 2). An important observation in the domain structure-function analyses is that deletion of the N-terminal Neh2 domain enhances the transcriptional activity of Nrf2 (Itoh et al., 1999). This observation suggests that the domain may recruit a negative regulator of Nrf2. This repressor was subsequently identified in a yeast two-hybrid screen and named Keap1 (Kelch-like ECH associating protein 1; Itoh et al., 1999). Keap1 possess two characteristic domains, the BTB domain and the DGR (double glycine repeat; Prag and Adams, 2003) domain. Keap1 has three additional domains/ regions: the N-terminal region (NTR), the intervening region (IVR), and the C-terminal region (CTR; see Fig. 2). Keap1 interacts with Nrf2 by exploiting the DGR domain (Itoh et al., 1999). Upon determination of the dissociation constant for the Keap1 Neh2 interaction, a tight association of the two molecules was identified (Itoh et al., 1999; Eggler et al., 2005). Moreover, the interaction between Nrf2 and Keap1 was proven in the following genetic experiment (Wakabayashi et al., 2003). While Keap1-null mutant mice

11 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) Nrf2 Neh2 Neh4 Neh5 Neh6 CNC-bZip (Neh1) Neh3 DLG S40 ETGE K44,K50,K52,K53,K56,K64,K68 NLS C506 NES Keap1 NTR BTB IVR DGR CTR S104 C151 C257 C297 C613 Cul3 binding? C273 C288 NES Fig. 2. The functional domains of Nrf2 and Keap1. [Nrf2] Keap1 binds to Neh2 through the ETGE and DLG motifs. Seven lysine residues between these two motifs are important for ubiquitination. Neh4 and Neh5 form the transcriptional activation domains to which CBP interacts. Neh6 and Neh2 are important for the degradation of Nrf2. Neh1 contains a bzip structure, which is critical for DNA binding and dimerization with small Mafs. A functional NLS and NES are also localized in Neh1. [Keap1] The DGR provides the binding surface for Nrf2 and actin. Cul3 interacts with the IVR and/or BTB domains. The BTB is also important for Keap1 dimerization. The IVR contains a NES. died within 3 weeks after birth due to hyperkeratosis in the esophagus and forestomach, the mice were rescued from the lethality by the simultaneous knockout of Nrf2. Knockdown of Keap1 by sirna in human cells has also been performed and upregulation of the ARE-driven genes has been confirmed (Devling et al., 2005). These results strongly argue that Keap1 acts as the principle regulator of Nrf2. Since Keap1 localizes mainly in the cytoplasm, it has been postulated that Keap1 associates with Nrf2 and tethers it in the cytoplasm under basal unstressed conditions, but upon the addition of electrophiles, Nrf2 is released from Keap1 and translocates into nucleus to bind to the ARE and activate gene transcription (Itoh et al., 1999). Indeed, in some cell lines, shifting of the intracellular localization of Nrf2 was detected before and after electrophile treatment (Huang et al., 2000; Morimitsu et al., 2002). A native electrophoretic mobility shift assay conducted with mouse Keap1 and Neh2 proteins showed that the addition of sulforaphane disrupts the Keap1 Neh2 complex (Dinkova- Kostova et al., 2002). This simple liberation model has been advanced to a more realistic one due to the extensive study and rapid progress of this topic. In most of the cell lines, it is difficult to detect Nrf2 in the cytoplasm if inducers are not present, suggesting that Nrf2 is rapidly degraded in unstressed conditions. Indeed, Nrf2 is a rapidly turned over protein, with the half time of Nrf2 degradation being less than 20 min (Itoh et al., 2003) or min (Alam et al., 2003; McMahon et al., 2003, Stewart et al., 2003; Zhang and Hannink, 2003) after exposure to cycloheximide. Importantly, this degradation was inhibited by the addition of proteasome inhibitors (Itoh et al., 2003; McMahon et al., 2003; Zhang and Hannink, 2003; Stewart et al., 2003; Nguyen et al., 2003), and ubiquitinated Nrf2 was observed when tagged-ubiquitin was expressed and/or cells were treated with proteasome inhibitors (Sekhar et al., 2002b; McMahon et al., 2003; Nguyen et al., 2003; Stewart et al., 2003;

12 124 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) Zhang and Hannink, 2003). These results suggest that Nrf2 is degraded continuously through the proteasomal pathway. Treatment of cells with various phase 2 inducers stops this rapid degradation of Nrf2 (Alam et al., 2003; Itoh et al., 2003; McMahon et al., 2003; Nguyen et al., 2003; Stewart et al., 2003; Zhang and Hannink, 2003). Several lines of evidence argue that the main pathway of Nrf2 degradation is Keap1 dependent. For instance, in Keap1-null mutant macrophages, Nrf2 accumulated to the same extent as or even to a higher level than in electrophile-treated wild-type macrophages (Itoh et al., 2003). Accumulation of Nrf2 was also observed when Keap1 was knocked-down by sirna in 293 T cells (Cullinan et al., 2004; Furukawa and Xiong, 2005). It has also been noted, however, that the Keap1- independent degradation pathway mediated by the Neh6 domain may also exist (Kobayashi et al., 2004a; McMahon et al., 2004; see Fig. 2). Keap1 functions as an adaptor for Cullin 3 (Cul3)-based E3 ligase to regulate the stability of Nrf2 (Cullinan et al., 2004; Kobayashi et al., 2004a; Zhang et al., 2004a; Furukawa and Xiong, 2005). Cul3 is a scaffold protein that forms the E3 ligase complex with Roc1/Rbx1/Hrt1 and recruits a cognate E2 enzyme. The functional relationship between Cul3 and Nrf2 degradation was confirmed by the fact that sirnadependent silencing of Cul3 expression leads to Nrf2 accumulation in the cytoplasm (Cullinan et al., 2004; Furukawa and Xiong, 2005). For the interaction of Keap1 with Cul3 to occur, the NTR of Cul3 and the IVR and/or BTB domains of Keap1 are required (Cullinan et al., 2004; Kobayashi et al., 2004b; Zhang et al., 2004a; Furukawa and Xiong, Fig. 3. A model for the electrophile-induced activation of Nrf2. Cul3 interacts with the BTB and/or IVR of Keap1 and ubiquitinates lysine residues in Nrf2. Dimerization of Keap1 is important for Nrf2 ubiquitination. Electrophiles and oxidants may disrupt this ubiquitination system in two ways: (i) they directly modify the reactive cysteine residues in Keap1 and disrupt the Keap1 Cul3 or Keap1 Keap1 interaction; or (ii) they activate PKC or other kinases by an unknown mechanism to phosphorylate Ser40 in Neh2 and disturb the Keap1 Nrf2 interaction.

13 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) ; see Figs. 2 and 3). Deletion of the IVR domain reduced the activity of Keap1 to repress Nrf2 transactivation activity, which resulted in stabilization of the Nrf2 protein and activation of its target genes (Kang et al., 2004; Kobayashi et al., 2004a). Mutations of Keap1 at the interface predicted for the Cul3 interaction in the BTB domain led to down regulation of Neh2 ubiquitination as well as Keap1 activity (Zhang et al., 2004a; Furukawa and Xiong, 2005). In conclusion, currently available data suggest that Keap1 supports ubiquitination of Nrf2 and its degradation as a member of the Cul3 E3 ligase complex. In many cases, the electrophile-induced nuclear accumulation of Nrf2 is abolished by the concomitant treatment of cells with cycloheximide, which inhibits the de novo synthesis of Nrf2. This observation supports the contention that Nrf2 activity is mainly regulated by its protein stability rather than by its active liberation from Keap1. In this regard, it is interesting to note that the stress response factors IkB, HIF-1a, and Nrf2 share a common feature that is repressed by the ubiquitin proteasome system under normal unstressed conditions exploiting the Cul-type E3 ligases Cul1, Cul2, and Cul3, respectively. The Neh2 domain acts as a degron The Neh2 domain is responsible for the Keap1-dependent degradation of Nrf2. Analyses of various fusion proteins revealed that Neh2 constitutes a redox-sensitive degron (Itoh et al., 2003; McMahon et al., 2003, 2004; Zhang et al., 2004a; Katoh et al., 2005). Through a yeast reverse two-hybrid screen, the ETGE motif was identified as a major Keap1-interacting site in the Neh2 domain (Kobayashi et al., 2002; Figs. 2 and 3). Nrf2 or Neh2-fusion proteins became stable when the ETGE motif was mutated (McMahon et al., 2004; Kobayashi et al., 2004a; Katoh et al., 2005; Furukawa and Xiong, 2005), indicating that the interaction between Nrf2 and Keap1 through the ETGE motif is critical for Nrf2 degradation. Ubiquitination of Neh2-fusion proteins has been observed and is dependent on Keap1 (McMahon et al., 2004; Zhang et al., 2004a; Katoh et al., 2005). In the presence of Keap1, seven lysine residues in the Neh2 domain were shown to be ubiquitinated (Zhang et al., 2004a) (Fig. 2). It has been suggested that ubiquitination of these lysine residues is critical for the degradation of Nrf2. Indeed, the Nrf2 protein was stabilized when all seven lysines were substituted by arginines (Zhang et al., 2004a). Recently, we identified another Keap1 binding site in the Neh2 domain. In our search for a region responsible for Nrf2 degradation through deletion construct analysis, we found that the NTR of the Neh2 domain, in which the ETGE motif is absent, is important for its instability (Katoh et al., 2005). Upon detailed sequence comparison among Nrf2 proteins, we identified the extensively conserved sequence LxxQDxDLG and referred to this sequence as the DLG motif (Fig. 2). Mutations in the DLG motif reduced the degradation of Nrf2 and Neh2-fusion proteins (McMahon et al., 2004; Katoh et al., 2005), suggesting the importance of this motif for the interaction with Keap1 and/or ubiquitination of Nrf2. We found from biophysical studies using nuclear magnetic resonance and isothermal calorimetry that a peptide containing the DLG motif interacts directly with Keap1, although more weakly than the ETGE motif (Tong et al., 2006). The DLG motif serves as a second Keap1-binding site in Nrf2, which may facilitate Nrf2 ubiquitination (Fig. 3).

14 126 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) Activation of Nrf2: escape from Keap1-dependent degradation Identification of the sensor molecule that detects phase 2 inducers and transduces their signals to Nrf2 has been a hot topic. Inducers of phase 2 enzyme genes have been classified into nine structurally diverse chemical groups (Dinkova-Kostova et al., 2001). A property these inducers have in common is the ability to modify sulfhydryl groups by alkylation, oxidation, or reduction. This observation suggests that cells possess a primary sensor for phase 2 inducers that is equipped with highly reactive cysteine residues. Keap1 contains 25 cysteine residues, several of which are reactive, that are conserved between human and mouse, implying that Keap1 may be a direct target of phase 2 inducers (Itoh et al., 1999; Dinkova-Kostova et al., 2002). The electrophile dexamethasone 21-mesylate (DexMes) was found to directly modify, in vitro, five cysteine residues of mouse Keap1, Cys257, Cys273, Cys288, Cys297, and Cys613 (Dinkova-Kostova et al., 2002; Fig. 2). These cysteine residues, except Cys613, exist in the IVR domain and mutations of these IVR cysteines to alanine disrupted the binding of DexMes to Keap1 almost completely, suggesting that the cysteine residues in the IVR are highly reactive to electrophiles (Dinkova-Kostova et al., 2005a). Further analysis of the cysteine residues in human Keap1 showed that N-iodoacetyl-N-biotinylhexylenediamine and biotinylated iodoacetamide modify residues Cys196, Cys226, Cys241, Cys257, Cys288, and Cys319 (Hong et al., 2005) and residues Cys151, Cys257, Cys273, Cys288, Cys297, Cys319, and Cys613 (Eggler et al., 2005), respectively. Differences in the profiles of these three reports might have arisen from the diversity of the inducers or experimental conditions. These cysteine residues are highly reactive to electrophiles, even in the presence of excess amounts of reducing agents. Other electrophiles, such as 15d-PGJ 2, 15-A 2t -isoprostane, and 4-hydroxy-2-nonenal, were also shown to bind directly to Keap1 in cultured cells and to activate the Nrf2 Keap1 system (Itoh et al., 2004; Levonen et al., 2004; Hosoya et al., 2005). When 7 cysteine residues in the IVR were mutated to alanine, 15d-PGJ 2 lost its ability to bind to Keap1. Since these cysteine residues bind directly to electrophiles in vitro, it was hypothesized that one or more cysteine residues in the IVR may be target sites for electrophiles in vivo. Studies using a Keap1 molecule whose cysteine residues were mutated to alanine or serine in a cell culture system unveiled that at least three cysteine residues, Cys151, Cys273 and Cys288, are crucial for its activity (Zhang and Hannink, 2003; Levonen et al., 2004; Wakabayashi et al., 2004). Cys273 and Cys288 in the IVR are crucial for the repressive activity of Keap1 (Zhang and Hannink, 2003; Levonen et al., 2004; Wakabayashi et al., 2004; Fig. 2). Keap1 proteins in which Cys273 or Cys288 were mutated to alanine or serine residues showed a decrease in Keap1-dependent ubiquitination and an increase in the stability of Nrf2 (Zhang and Hannink, 2003; Wakabayashi et al., 2004). In contrast, mutations of the other cysteine residues in the IVR had practically no effect on the activity of Nrf2 or on the response to electrophiles (Zhang and Hannink, 2003; Wakabayashi et al., 2004). Since the IVR is important for the interaction with Cul3 (Kobayashi et al., 2004a), these results suggest that these two cysteine residues play key roles in Keap1 Cul3 binding. One attractive hypothesis is that the electrophile-induced modification of these cysteine residues affects the Keap1 Cul3 interaction. However, mutations in both C273A and C288A did not affect the binding activity of Keap1 to Cul3 (Kobayashi et al., 2004a), so further analysis is necessary regarding this hypothesis.

15 M. Kobayashi, M. Yamamoto / Advan. Enzyme Regul. 46 (2006) Recently, Dinkova-Kostova et al. (2005a) showed that Keap1 is a zinc-containing protein. Alanine substitutions in both Cys273 and Cys288 reduced the binding affinity between Keap1 and zinc to 1/20, suggesting that these two cysteine residues participate in the binding to zinc. Interestingly, the addition of phase 2 inducers such as H 2 O 2 or sulforaphane released zinc from Keap1. These results thus suggest that Keap1 with a zinc atom may form a suitable structure for the ubiquitination of Nrf2, while phase 2 inducers lead to zinc release. Modification of Cys273 and Cys288 may cause a profound conformational change in Keap1 that makes the protein unable to support ubiquitination. Cys151 in the BTB domain may also play a critical role in electrophile signaling (Fig. 2). When Cys151 was mutated to serine, electrophile-induced down-regulation of Nrf2 ubiquitination and degradation was significantly reduced (Zhang and Hannink, 2003; Zhang et al., 2004a). A simple explanation for this observation is that electrophile-induced modification of Cys151 prevents the Cul3 complex from ubiquitinating Nrf2. Since a C151S mutation showed almost no effect on the ubiquitination and degradation of Nrf2 in normal conditions (Zhang and Hannink, 2003), Cys151 itself is not essential for the binding of Keap1 to Cul3 or for the activity of the Keap1 Cul3 complex in the ubiquitination of Nrf2. The results imply that modification of Cys151 by electrophiles may lead to a conformational change in Keap1 which ceases its activity for Nrf2 ubiquitination. The BTB domain acts to dimerize Keap1 (Zipper and Mulcahy, 2002). The selfassociation of Keap1 was demonstrated to be important for its activity in repressing Nrf2. A point mutation at Ser104 in the BTB domain decreased the association of Keap1 with both Nrf2 and Keap1 itself (Fig. 2), suggesting that Keap1 dimerization is important for Keap1 to interact with Nrf2. Efficient binding of Keap1 and Neh2 in a 2:1 stoichiometry supports this idea (Wakabayashi et al., 2004; Dinkova-Kostova et al., 2005a; Eggler et al., 2005). Interestingly, the concomitant expression of C273A and C288A mutants led to the substantial restoration of repressor activity (Wakabayashi et al., 2004). This observation supports the view that Keap1 acts as a dimer and further suggests that the simultaneous presence of Cys273 on one molecule of the dimer and Cys288 on the other is compatible with the repressor activity. It is interesting to note that Nrf2 contains two Keap1 binding sites in the Neh2 domain, the ETGE motif (Kobayashi et al., 2002) and the DLG motif (Katoh et al., 2005). Lysine residues targeted for ubiquitination are localized between these two sites (Zhang et al., 2004a; see Fig. 2). Taking into account the importance of Keap1 dimerization, we propose the model that the ETGE and DLG motifs in a single Nrf2 molecule interact with the DGR domains in two different Keap1 molecules (Tong et al., 2006; see Fig. 3). Recently, Keap1 itself was shown to be ubiquitinated by the Cul3 complex (Hong et al., 2005; Zhang et al., 2005). This ubiquitination was markedly increased in cells exposed to some phase 2 inducers, such as tert-butylhydroquinone (tbhq) and N-iodoacetyl-Nbiotinylhexylenediamine. Since multi-ubiquitination of Keap1 appears to require Lys63 of ubiquitin (Zhang et al., 2005) and not Lys48 that acts as a signal for substrate recognition and degradation by the 26S proteasome (Dubiel and Gordon, 1999), Keap1 ubiquitination may participate in a process other than protein degradation by the proteasome. Mutations of Keap1 at the interface predicted for Cul3 interaction in the BTB domain led not only to down-regulation of Neh2 ubiquitination, but also to up-regulation of Keap1 ubiquitination (Zhang et al., 2004a). Zhang et al. (2005) have speculated that electrophiles may trigger a switching of Cul3-dependent ubiquitination from Nrf2 to Keap1. A curious point to this hypothesis is that the ubiquitination of Keap1 was induced by tbhq, but not by