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. 2011 Nov;31(22):4500-12.
doi: 10.1128/MCB.05663-11. Epub 2011 Sep 12.

Dual regulation of the transcriptional activity of Nrf1 by β-TrCP- and Hrd1-dependent degradation mechanisms

Yoshiki Tsuchiya  1 , Tomoko Morita, Mehee Kim, Shun-ichiro Iemura, Tohru Natsume, Masayuki Yamamoto, Akira Kobayashi
Affiliations

Dual regulation of the transcriptional activity of Nrf1 by β-TrCP- and Hrd1-dependent degradation mechanisms

Yoshiki Tsuchiya et al. Mol Cell Biol. 2011 Nov.

Abstract

A growing body of evidence suggests that Nrf1 is an inducible transcription factor that maintains cellular homeostasis. Under physiological conditions, Nrf1 is targeted to the endoplasmic reticulum (ER), implying that it translocates into the nucleus in response to an activating signal. However, the molecular mechanisms by which the function of Nrf1 is modulated remain poorly understood. Here, we report that two distinct degradation mechanisms regulate Nrf1 activity and the expression of its target genes. In the nucleus, β-TrCP, an adaptor for the SCF (Skp1-Cul1-F-box protein) ubiquitin ligase, promotes the degradation of Nrf1 by catalyzing its polyubiquitination. This activity requires a DSGLS motif on Nrf1, which is similar to the canonical β-TrCP recognition motif. The short interfering RNA (siRNA)-mediated silencing of β-TrCP markedly augments the expression of Nrf1 target genes, such as the proteasome subunit PSMC4, indicating that β-TrCP represses Nrf1 activation. Meanwhile, in the cytoplasm, Nrf1 is degraded and suppressed by the ER-associated degradation (ERAD) ubiquitin ligase Hrd1 and valosin-containing protein (VCP) under normal conditions. We identified a cytoplasmic degradation motif on Nrf1 between the NHB1 and NHB2 domains that exhibited species conservation. Thus, these results clearly suggest that both β-TrCP- and Hrd1-dependent degradation mechanisms regulate the transcriptional activity of Nrf1 to maintain cellular homeostasis.

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Figures

Fig. 1.
Fig. 1.
The proteasomal degradation of the transcription factor Nrf1 in the cytoplasm and the nucleus. (A) MG132 stabilized the endogenous Nrf1 in MEFs. MEFs derived from wild-type (WT) and Nrf1 knockout (Nrf1 KO) mice were treated with MG132 (10 μM) for 8 h. Cytoplasmic (C) and nuclear (N) extracts from the cells were subjected to immunoblot analysis with rat monoclonal anti-Nrf1 antibody. Lamin B1 and α-tubulin were utilized as nuclear and cytoplasmic markers, respectively. (B) Schematic structures of the wild type and of the P1 and ΔbZip deletion mutants of Nrf1. The domain nomenclature was defined previously (40). The numbers denote the positions of the amino acid residues in the complete sequence of Nrf1. (C) The P1 and ΔbZip Nrf1 mutants predominantly localized in the nucleus and the cytoplasm, respectively, of COS7 cells. Nrf1 wild-type and mutants were stained with anti-Flag antibody, and the nuclei were stained with DAPI. A bar graph depicts the results of a quantitative analysis of the subcellular localization of the Nrf1 wild-type and mutants. More than 100 transfected cells were observed for the expression of each plasmid and classified into three different categories: C < N, nucleus-dominant staining; C = N, roughly equal distribution between the cytoplasmic and nuclear compartments; C > N, cytoplasm-dominant staining. (D) Both the P1 and ΔbZip Nrf1 mutants were degraded by the proteasome in COS7 cells. Cells were treated with MG132 (10 μM) for 8 h, and the expression levels of Nrf1 wild-type and deletion mutants were analyzed by immunoblot analysis with anti-Flag antibody. GFP was coexpressed as an internal control. α, anti.
Fig. 2.
Fig. 2.
The β-TrCP protein regulates the nuclear degradation of Nrf1. (A) HeLa cells were transfected with the 3×Flag-Nrf1 and GFP expression vectors at 24 h after two rounds of transfection with control (C), β-TrCP1 (1), β-TrCP2 (2), or β-TrCP1/2 (1/2) siRNA. At 24 h after the last transfection, whole-cell extracts were prepared and subjected to immunoblot analysis. Duplicate samples are presented. (B) The knockdown efficiency of each siRNA for β-TrCP1 and β-TrCP2 was determined by real-time quantitative PCR analysis. Duplicate samples are presented. (C) HeLa cells were transfected with siRNA against β-TrCP1 and β-TrCP2 (1/2), Keap1 (K), or the control (C) along with 3×Flag-tagged P1 and the ΔbZip Nrf1 mutant. Cells were left untreated or treated with MG132 (10 μM) for 8 h. Cell extracts were subjected to immunoblot analysis with the indicated antibodies. (D) Real-time quantitative PCR analysis revealed that siRNA directed against β-TrCP significantly reduced mRNA expression of both β-TrCP1 and β-TrCP2 but not Nrf1 in HeLa cells. The values were normalized with GAPDH and are presented as the means ± standard deviations (n = 3; *, P < 0.001). (E) Control (C), Keap1 (K), or β-TrCP1/2 (1/2) siRNA was transfected into HeLa cells in combination with the 3×Flag-Nrf1 vector or the Flag Nrf2 vector. Whole-cell extracts were subjected to immunoblot analysis with the indicated antibodies. (F) The siRNA directed against β-TrCP stabilized the endogenous Nrf1. β-TrCP (1/2) or the control (C) siRNA was twice transfected into HeLa cells. At 48 h after the second transfection, the cells were pretreated with MG132 (10 μM) for 6 h and treated with cycloheximide (CHX) (20 μg/ml). Whole-cell extracts were prepared for immunoblot analysis with anti-Nrf1 (H-285) antibody. The β-catenin protein is a canonical β-TrCP substrate, and α-tubulin is an internal control. The graph depicts the quantified band intensities of Nrf1. The values were normalized with α-tubulin and are presented as the means ± standard error (n = 3; *, P < 0.005). The values at 1 h after cycloheximide treatment were set to 1.
Fig. 3.
Fig. 3.
Colocalization and physical interaction of Nrf1 with β-TrCP2. (A) Colocalization of wild-type Nrf1 with β-TrCP2 in COS7 cells. The 3×Flag-Nrf1 and HA–β-TrCP2 expressed in COS7 cells were immunostained with the indicated antibodies. (B) Physical interaction of Nrf1 with β-TrCP2. Whole-cell extracts of COS7 cells expressing 3×Flag-Nrf1 and HA-tagged β-TrCP2 were subjected to immunoprecipitation (IP) with anti-Flag antibody, followed by immunoblot (IB) analysis with the indicated antibodies. (C) Schematic structures of β-TrCP2 deletion mutants. β-TrCP2 comprises the F-box and WD40 repeat domains. (D) The WD40 repeat domain of β-TrCP2 was required for its association with Nrf1. The 3×Flag-Nrf1 and the HA–β-TrCP2 wild type (WT) or ΔF-box (ΔF) or ΔWD40 (ΔWD) mutant were expressed into COS7 cells, and immunoprecipitation with anti-Flag antibody was performed, as described above. (E) Schematic structures of Nrf1 deletion mutants. (F) Identification of β-TrCP2-binding regions within Nrf1. Immunoprecipitation with anti-Flag antibody was performed, as described above. To ensure that the Nrf1 mutant proteins were expressed at similar levels, we transfected various amounts of plasmids, as follows: WT and M1, 1 μg; P2, P3, and M2, 0.3 μg; P4, P5, M3, and M4, 30 ng.
Fig. 4.
Fig. 4.
A region (residues 243 to 463) of Nrf1 is required for its β-TrCP-mediated degradation. (A) The Nrf1 mutants lacking amino acid residues 243 to 463 were not stabilized by the siRNA-mediated knockdown of β-TrCP. Control (C) or β-TrCP (1/2) siRNA was transfected into HeLa cells in combination with the indicated Nrf1 mutants. The expression levels of the mutants were analyzed, as described in the legend to Fig. 2C. (B) The deletion of residues 243 to 463 significantly stabilized Nrf1. HeLa cells were transfected with the expression plasmid for the wild type or the P3 or P4 mutant of 3×Flag-Nrf1. Next, the cells were treated with cycloheximide (CHX) (20 μg/ml), and the whole-cell extracts were prepared at the indicated time points. The assays were performed three times. Data normalized with cotransfected GFP are presented as the means ± standard errors (n = 3; *, P < 0.005; **, P < 0.001 versus wild type). (C) The β-TrCP-dependent polyubiquitination of Nrf1 in cultured cells. HeLa cells were transfected with the P3 Nrf1 mutant, HA-ubiquitin (Ub), and the Myc β-TrCP2 wild type (WT) or ΔF-box (ΔF) mutant. The P3 mutant was immunoprecipitated (IP) by anti-Flag antibody, and its ubiquitination was detected by immunoblot analysis with anti-HA antibody.
Fig. 5.
Fig. 5.
A DSGLS motif is required for the β-TrCP-mediated degradation of Nrf1. (A) Alignment of Nrf1 sequences among species and NF-E2-related factors around the highly conserved DSGLS motif, which is similar to the β-TrCP recognition motifs of the human YAP and erythropoietin receptor (EpoR). Shaded areas denote conserved serine residues that were replaced with alanine for subsequent experiments. (B) β-TrCP siRNA stabilized endogenous Nrf2. Transfection of siRNA and subsequent cycloheximide chase were performed, as described in the legend to Fig. 2F. (C) Alanine substitution of serine residues in the DSGLS motif (P3-SA) stabilized the P3 Nrf1 mutant. At 48 h after transfection of siRNA and Nrf1 mutant constructs, the cycloheximide chase experiment was conducted, as described in the legend to Fig. 4B. Assays were performed three times. The values were normalized with GFP and presented as the means ± standard errors (n = 3; *, P < 0.05 versus P3 with control siRNA). (D and E) A nuclear degradation mechanism repressed the transcriptional activity of Nrf1. COS7 cells were transfected with either P3 (WT) or P3-SA (SA) Nrf1 mutant plasmid in combination with a luciferase reporter containing a single ARE of NQO1 gene (D) or three tandem copies of ARE of PSMA4 (E). The levels of luciferase activity were normalized with the Renilla luciferase activity of an internal control pRL-TK, and results are presented as the means ± standard deviations (n = 3 [D] and n = 4 [E]; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (F) β-TrCP is involved in the regulation of the expression of Nrf1 target genes induced by proteasome inhibition. HeLa cells transfected twice with the indicated siRNA were left untreated (−) or were treated with (+) MG132 (1 μM) for 16 h, and then MG132-treated cells were incubated without MG132 for another 3 h (w.o.). The mRNA expression levels were determined by real-time quantitative PCR analysis. The values were normalized with 18S rRNA and are presented as the means ± standard deviations (n = 3; *, P < 0.05 versus control siRNA). (G and H) The siRNA-mediated knockdown of Nrf1. HeLa cells were twice transfected with control (C) or Nrf1 siRNA. At 24 h after the second transfection, Nrf1 knockdown efficiency was determined by immunoblot analysis with anti-Nrf1 (H-285) antibody (G) or by real-time quantitative PCR analysis (H). For immunoblot analysis, cells were treated with MG132 (10 μM) for 8 h. The values were normalized with 18S rRNA and are presented as the means ± standard deviations (n = 4; *, P < 0.001).
Fig. 6.
Fig. 6.
Hrd1 and VCP regulate the cytoplasmic degradation of Nrf1. (A) VCP or Hrd1 siRNA stabilized endogenous Nrf1 in HeLa cells. Transfection was carried out twice. The whole-cell extracts were prepared and analyzed by immunoblotting with anti-Nrf1 (H-285) antibody. α-Tubulin is an internal control. (B) Knockdown efficiency of ERAD-related E3 ligases. A knockdown experiment was conducted as described in the legend to Fig. 5H. The values were normalized with 18S rRNA and are presented as the means ± standard deviations (n = 4; *, P < 0.001). (C) Knockdown of VCP and Hrd1 inhibited Nrf1 degradation. Transfection of siRNA and subsequent cycloheximide chase were performed, as described in the legend to Fig. 2F. Data are presented as the means ± standard errors (n = 5; *, P < 0.05; **, P < 0.01 versus control siRNA). (D) Hrd1 is involved in the regulation of the expression of Nrf1 target genes induced by proteasome inhibition. The assays were performed according to the legend of Fig. 5F. Data are presented as the means ± standard deviations (n = 3; *, P < 0.05 versus control siRNA). (E) Schematic structures of deletion mutants derived from the ΔbZip Nrf1 mutant. (F and G) A region (residues 31 to 81) of Nrf1 is required for the cytoplasmic degradation of Nrf1. Cycloheximide chase experiments using the Nrf1 deletion mutants were performed according to the legend of Fig. 4B. Data are presented as the means ± standard errors (n = 7 [F]; n ≥ 3 [G]; *, P < 0.05; **, P < 0.005; ***, P < 0.001 versus wild-type Nrf1). (H) Amino acid residues (31 to 81) of mouse Nrf1 are highly conserved among species and in mouse Nrf3 (mNrf3). Shaded amino acid positions highlight amino acid identity (orange) or similarity (blue) between Nrf1 and Nrf3.
Fig. 7.
Fig. 7.
A model for the dual mechanisms of Nrf1 degradation in regulating the expression of Nrf1 target genes. Nrf1 is degraded via the ERAD pathway under physiological conditions. Upon activation, Nrf1 is stabilized and translocates into the nucleus. In the nucleus, β-TrCP-mediated degradation prevents inappropriate transcription of Nrf1 target genes such as proteasome subunit genes. This degradation may also facilitate the clearance of Nrf1 from the nucleus after the activation of Nrf1 is abolished. Ub, ubiquitin.
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