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. 1999 Jan 1;13(1):76-86.
doi: 10.1101/gad.13.1.76.

Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain

K Itoh  1 , N Wakabayashi, Y Katoh, T Ishii, K Igarashi, J D Engel, M Yamamoto
Affiliations

Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain

K Itoh et al. Genes Dev. .

Abstract

Transcription factor Nrf2 is essential for the antioxidant responsive element (ARE)-mediated induction of phase II detoxifying and oxidative stress enzyme genes. Detailed analysis of differential Nrf2 activity displayed in transfected cell lines ultimately led to the identification of a new protein, which we named Keap1, that suppresses Nrf2 transcriptional activity by specific binding to its evolutionarily conserved amino-terminal regulatory domain. The closest homolog of Keap1 is a Drosophila actin-binding protein called Kelch, implying that Keap1 might be a Nrf2 cytoplasmic effector. We then showed that electrophilic agents antagonize Keap1 inhibition of Nrf2 activity in vivo, allowing Nrf2 to traverse from the cytoplasm to the nucleus and potentiate the ARE response. We postulate that Keap1 and Nrf2 constitute a crucial cellular sensor for oxidative stress, and together mediate a key step in the signaling pathway that leads to transcriptional activation by this novel Nrf2 nuclear shuttling mechanism. The activation of Nrf2 leads in turn to the induction of phase II enzyme and antioxidative stress genes in response to electrophiles and reactive oxygen species.

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Figures

Figure 1
Figure 1
Schematic representation of the regions conserved between chicken and human Nrf2 proteins. (A) Six conserved domains, designated Neh1–Neh6, are found between human and chicken Nrf2. Neh1 corresponds to the CNC region and bZip structure. (B) Sequence homology in Neh2. Amino acid residues conserved between at least two proteins are shaded. The 33 amino-terminal residues, including the hydrophobic region, are conserved among Nrf1, hNrf2, and cNrf2 (ECH); the next 40 residues of Neh2 are rich in hydrophilic residues and specifically conserved between cross-species Nrf2 molecules. The strikingly homologous region, containing hydrophilic residues, is boxed. (▾) Restriction enzyme sites.
Figure 2
Figure 2
Transcriptional activation activity of Gal4 DBD–Nrf2 fusion proteins. (A) Schematic presentation of the Gal4 DBD–Nrf2 fusion proteins. Gal4–Nrf2#5 contains both Gal4 DBD and full-length chicken Nrf2 (ECH). (B) Transactivation activity of Nrf2 chimeric proteins. Trans-activation activity of each fusion protein was determined by cotransfection assay into the quail fibroblast cell line QT6, utilizing a LUC reporter plasmid, which is transcriptionally directed by five Gal4-binding sites.Each column corresponds to the construct shown in A. LUC activity of the reporter plasmid in the absence of any effector plasmid was arbitrarily set at 100, and mean values of three independent experiments, each carried out in duplicate, are shown with the standard error of the mean (s.e.).
Figure 3
Figure 3
The Neh2 domain is required for negative regulation of Nrf2 activity in HD3 erythroblasts. (A) Schematic representation of the wild-type and Neh2 deletion mutant (M1) of cNrf2. (B,C) Transfection of incremental amounts of the wild-type and mutant Nrf2 expression plasmids into QT6 cells or HD3 erythroblasts, respectively. (█) Wild; (○) M1. The pRBGP2 reporter, containing triplicated MARE binding sites from the chicken β-globin enhancer, was transfected simultaneously. LUC activity with the wild-type Nrf2 at an effector/reporter ratio of 20 was arbitrarily set at 100% in B, and that of the mutant at an effector/reporter ratio of 24 was set at 100% in C. Mean values of three independent experiments, each carried out in duplicate, are shown with the s.e.
Figure 4
Figure 4
Overexpression of Gal4 DBD–Neh2 restores Nrf2-dependent activation in HD3 cells. The full-length cNrf2 expression plasmid (4 μg) was cotransfected into HD3 cells together with the pRBGP2 reporter (0.5 μg) in the presence of increasing concentrations of expression plasmids encoding either Gal4 DBD–Neh2 or Gal4 DBD. LUC activity in the absence of effector plasmids was arbitrarily set at 0.1. Mean values of three independent experiments, each carried out in duplicate, are shown with the s.e.. Note that the addition of Gal4 DBD–Neh2 releases Nrf2 activity from repression in HD3 cells. (Stippled bars) Gal 4–Neh2; (hatched bars) Gal4–DBD.
Figure 5
Figure 5
Two-hybrid screening of Keap1. (A) Structural homology of Keap1 with the Drosophila Kelch protein, which contains BTB and DGR domains. Boxed Gs indicate the DGR domain. (B) Keap1 shows high similarity to the human clone KIAA0132 (Nagase et al. 1995). Amino acid identities conserved between the two proteins are shaded.
Figure 5
Figure 5
Two-hybrid screening of Keap1. (A) Structural homology of Keap1 with the Drosophila Kelch protein, which contains BTB and DGR domains. Boxed Gs indicate the DGR domain. (B) Keap1 shows high similarity to the human clone KIAA0132 (Nagase et al. 1995). Amino acid identities conserved between the two proteins are shaded.
Figure 6
Figure 6
Interaction between the Neh2 domain of Nrf2 and the DGR domain of Keap1. (A) Schematic representations of the wild-type and mutant Gal4 DBD–Neh2 fusion proteins (B1 to B4) and the Gal4 AD–Keap1 fusion proteins (L1–L3). Construct B1 contains full length Neh2. B4 is the same construct as the Neh2 deletion mutant of Nrf2 in Fig. 3. B2 contains two amino acid substitutions in Neh2, whereas B3 lacks the amino-terminal 15 residues. L2 and L3 lack the DGR or BTB domain of Keap1, respectively. (B) Expression plasmids for various Gal4 DBD and Gal4 AD fusion proteins were introduced into the reporter yeast strain in the given combinations. The resulting transformants were tested for the His+ phenotype by spotting onto His− or His+ plates. (C) Results of the BIAcore interaction assay. Ligand (GST–Neh2, amino acids 1–73, shaded bars) or GST alone (open bars), and analyte (MBP–Keap1–DGR, amino acids 308–624) binding was tested at three different concentrations. The test and the control values, each carried out in triplicate, are shown with the s.e.
Figure 7
Figure 7
Keap1 repression of Nrf2 activity. (A) Increasing amounts of Keap1 expression plasmid were cotransfected along with a constant amount of the pRBGP2 reporter and the wild-type or Neh2 deletion mutant (M1) cNrf2 expression plasmids into (normally Nrf2 permissive) QT6 cells. (B) Increasing amounts of Keap1 expression plasmid were transfected into QT6 cells along with a constant concentration of the wild-type mNrf2 or a second cNrf2 mutant (missing only the conserved hydrophilic core; amino acids 33–73) expression plasmid and the pRBGP2 reporter gene. LUC activity in the absence of any effector plasmid was arbitrarily set at 100, and the mean values of three independent experiments, each carried out in duplicate, are shown with the s.e.
Figure 8
Figure 8
Electrophilic agents liberate Nrf2 from repression by Keap1. Both Nrf2 and Keap1 expression plasmids were transfected into QT6 fibroblasts. After 12 hr of culture, the cells were washed with fresh medium, and then increasing amounts of DEM (A) or catechol (D) were added to the replacement medium. The QT6 cells were cultured for another 36 hr. LUC reporter activity of cells transfected with only pRBGP2 reporter (B) or the reporter and the Nrf2 expression plasmid (C), and treated with DEM, are also shown as controls. Results of three independent experiments, each of which were carried out in duplicate, are shown with the s.e.
Figure 9
Figure 9
Subcellular localization of Keap1 and Neh2. (A,B) Subcellular localization of Keap1–GFP. Expression plasmids for GFP alone (A) or the Keap1–GFP fusion protein (B) were transfected into QT6 cells. (C,D) Subcellular localization of Neh2–GFP. The expression plasmid for Neh2 (mNrf2)–GFP fusion protein was transfected into 293T cells either alone (C) or in combination with a plasmid expressing Keap1 (D).
Figure 10
Figure 10
Subcellular localization of Nrf2. Nrf2 alone (A,B) or both Nrf2 and Keap1 (C,D) were force expressed in 293T cells. Nrf2 and Keap1 were also force-expressed in the presence of 100 μm DEM (E,F). (A,C,E) Localization of Nrf2 was detected using an anti-human Nrf2 antibody; (B,D,F) the same fields stained with propidium iodide. The arrowhead and arrow in C and D, respectively, show the characteristic perinuclear ring structure described in the text.
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