Trafficking of the transcription factor Nrf2 to promyelocytic leukemia-nuclear bodies: implications for degradation of NRF2 in the nucleus

Abstract

Ubiquitylation of Nrf2 by the Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase complex targets Nrf2 for proteasomal degradation in the cytoplasm and is an extensively studied mechanism for regulating the cellular level of Nrf2. Although mechanistic details are lacking, reports abound that Nrf2 can also be degraded in the nucleus. Here, we demonstrate that Nrf2 is a target for sumoylation by both SUMO-1 and SUMO-2. HepG2 cells treated with As2O3, which enhances attachment of SUMO-2/3 to target proteins, increased SUMO-2/3-modification (polysumoylation) of Nrf2. We show that Nrf2 traffics, in part, to promyelocytic leukemia-nuclear bodies (PML-NBs). Cell fractions harboring key components of PML-NBs did not contain biologically active Keap1 but contained modified Nrf2 as well as RING finger protein 4 (RNF4), a poly-SUMO-specific E3 ubiquitin ligase. Overexpression of wild-type RNF4, but not the catalytically inactive mutant, decreased the steady-state levels of Nrf2, measured in the PML-NB-enriched cell fraction. The proteasome inhibitor MG-132 interfered with this decrease, resulting in elevated levels of polysumoylated Nrf2 that was also ubiquitylated. Wild-type RNF4 accelerated the half-life (t½) of Nrf2, measured in PML-NB-enriched cell fractions. These results suggest that RNF4 mediates polyubiquitylation of polysumoylated Nrf2, leading to its subsequent degradation in PML-NBs. Overall, this work identifies Nrf2 as a target for sumoylation and provides a novel mechanism for its degradation in the nucleus, independent of Keap1.

Keywords: Degradation of Nrf2; Fluorescence Resonance Energy Transfer (FRET); Promyelocytic Leukemia-Nuclear Bodies; Protein Degradation; RNF4; Sumoylation; Trafficking; Transcription Factors.

FIGURE 1.

SENP1 abolishes MG-132-induced formation of GFP-Nrf2-containing nuclear aggregates. HepG2 cells grown in MEM were transfected with 1.5 μg of pEGFP-Nrf2. Some cultures were also transfected with 1 μg of pFLAG (vector) (columns 1 and 2) or 1 μg of expression plasmid for SENP1 (column 3) or SENP1mt (column 4). After 24 h, some cultures (lanes 2–4) were treated for 4 h with MG-132 (final concentration, 10 μm). All cells were then harvested and fixed as described under “Experimental Procedures.” This figure is representative of four experiments. Scale bars, 5 μm. Representative Western blot of SENP1 is shown. GAPDH, loading control. Anti-SENP1 antibody (catalog no. 2887-1) was obtained from Epitomics Inc. The number of GFP-Nrf2-containing nuclear aggregates for each treatment group (n = 23–41 cells) is shown in the histogram. Values plotted are means ± S.E. One-way analysis of variance was performed with Tukey multiple comparison test. *** Statistically different (p < 0.0001).

FIGURE 2.

Nuclear body-enriched preparation contains Nrf2 and components of PML-NBs. Whole cell lysate (W), prepared from HepG2 cell cultures treated with As₂O₃ (2 μm) for 4 h, was separated into cytoplasmic (C) and partially purified nuclear pellet (18). Beginning with the partially purified nuclear pellet, nuclear body-enriched fraction (NB) was prepared according to the protocol described by He et al. (23) as modified by Vertegaal et al. (24). The final pellet was suspended in 100 μl of buffer containing equal volumes of Tris-HCl solution (pH 6.8) and 2× SDS sample buffer. The cytoplasmic, whole cell lysate, and NB fractions were then blotted for the proteins indicated in A and B. *, modified (sumoylated) PML protein.

FIGURE 3.

GFP-Nrf2 co-localizes with endogenous PML protein and wild-type RFP-SUMO. HepG2 cells grown in MEM on coverslips to 50–80% confluence were co-transfected with 2 μg of pEGFP-Nrf2 with 0.5 μg of expression plasmid for RFP-SUMO-1 or RFP-SUMO-2 followed by immunostaining for endogenous PML protein, as described under “Experimental Procedures.” A, RFP-SUMO-1; B, RFP-SUMO-2; inset in panel 5 is an enlargement of the boxed area within panel 5. Panels 7–9 are representative images of cells from parallel experiments showing co-localization as in panel 5. Yellow arrows point to yellow dots representing co-localization of GFP-Nrf2 with RFP-SUMO; white arrows point to white dots representing co-localization of GFP-Nrf2 with RFP-SUMO and endogenous PML. Scale bars, 10 μm.

FIGURE 4.

Nonconjugatable SUMO does not co-localize with GFP-Nrf2 or with endogenous PML protein. HepG2 cells grown as in Fig. 3 were co-transfected with 2 μg of pEGFP-Nrf2 with 0.5 μg of expression plasmid for RFP-SUMO-1ΔGG (A and C) or RFP-SUMO-2ΔGG (B and D); the cells in C and D were immunostained for endogenous PML protein, as described under “Experimental Procedures.” Scale bars, 10 μm.

FIGURE 5.

FRET analysis of SUMO-modified Nrf2 in PML-NBs. HepG2 cells were transfected with pEGFP-Nrf2 (1 μg) along with expression plasmid for RFP-SUMO (0.5 μg). The cells were harvested and fixed as described under “Experimental Procedures.” Individual spots containing PML, GFP-Nrf2, and RFP-SUMO were subjected to FRET analysis. Donor (GFP) and acceptor (RFP) images were acquired with a Nikon A1R laser-scanning confocal microscope, as described under “Experimental Procedures.” A and B, the arrows indicate locations of pre- and post-bleaching. The differential interference contrast (DIC) images are shown in panels 7 and 8. A, FRET analysis of interaction of GFP-Nrf2 with RFP-SUMO-1. B, FRET analysis of interaction of GFP-Nrf2 with RFP-SUMO-2. C, lack of FRET signals between GFP-Nrf2 and nonconjugatable SUMO-1 (RFP-SUMO-1ΔGG). D, quantification of the FRET data. For each treatment, 10–20 cells were imaged. FRET efficiency (%) was calculated from the following equation: FRET efficiency (%) = ((GFP_after − GFP_before)/GFP_after) × 100 (20). Values plotted are means ± S.E. for n = 17–52. Scale bars, 10 μm.

FIGURE 6.

Sumoylation of Nrf2 in vitro. Using GST-Nrf2 fusion protein (purified as described under “Experimental Procedures”) as substrate (0.5 μg), the assays were performed with in vitro sumoylation assay kits (Active Motif), with p53 supplied in the kit as a positive control. The GST-Nrf2 fusion protein as well as products of the reaction were separated on 7% SDS-PAGE and analyzed by Western blotting using the indicated antibodies. A, representative Western blot of the purified GST-Nrf2 fusion protein. B, SUMO-1-modified p53. C and D, SUMO-1-conjugated (C) and SUMO-2-conjugated (D) Nrf2 detected with anti-Nrf2 antibody are shown in the upper panels. Lower panels show the same membranes blotted with anti-SUMO-1 and anti SUMO-2/3 antibody, respectively.

FIGURE 7.

Desumoylase SENP1 disrupts association of RFP-SUMO-1 with GFP-Nrf2 and with GFP-PML-I. HepG2 cells grown in MEM were transfected with 0.5 μg of expression plasmid for RFP-SUMO-1 along with 2 μg of pEGFP-Nrf2 (A–D) or expression plasmid for GFP-PML-I (2 μg) (E–H). Some cells were also transfected with 1 μg of pFLAG (vector) (A and E) or 1 μg of expression plasmid for SENP1 (B and F) or SENP1mt (C and G). After 24 h, all cells were harvested and processed for fluorescence microscope imaging as described under “Experimental Procedures.” Yellow dots in nuclei were counted in 34–36 cells per treatment group in A–D and in 44–47 cells per treatment group in E–H, and expressed as number of dots/nucleus for each group. Values plotted (D and H) are means ± S.E. Statistical analysis was performed with one-way analysis of variance with Tukey multiple comparison test. **, statistically different (p < 0.001). ***, statistically different (p < 0.0001). Scale bars, 5 μm.

FIGURE 8.

Endogenous SUMO-2/3 is conjugated to endogenous Nrf2 and to endogenous PML. HepG2 cells were treated with As₂O₃ (2 μm in B or 5 μm in A and C) for various times. Whole cell lysates were subjected to Co-IP assay. Nonimmune serum (IgG) was used as control. A–C, endogenous Nrf2 associates with endogenous SUMO-2/3. A, IP, Nrf2; WB, SUMO-2/3. B, input control. IP, Nrf2; WB, Nrf2. C (reverse Co-IP assay): IP, SUMO-2/3; WB, Nrf2. D–F, endogenous PML associates with endogenous (lanes 1-3) as well as with exogenous (lanes 4-6) SUMO-2/3. D, Western blots of PML performed with 30 μg of whole cell lysate protein. Loading control, β-actin. E, input assay. IP, PML; WB, PML. F, IP, PML; WB, SUMO-2/3. The data in A–F are representative of results from three separate experiments.

FIGURE 9.

Distribution of Nrf2, Keap1, RNF4, and the PML-NB-marker protein SP100 in detergent-fractionated samples of HepG2 cells. HepG2 cells were grown in T-25 flasks. Some cultures were treated with As₂O₃ (2 μm) or MG-132 (10 μm). All cells were washed and scraped into ice-cold lysis buffer containing 1% Triton X-100. The lysate was centrifuged (14,000 × g) to separate DS fraction (supernatant) from DI fraction (pellet). The DI fraction was suspended in 500 μl of buffer containing equal volumes of Tris-HCl solution (pH 6.8) and 2× SDS sample buffer. All fractions were then resolved on an SDS-polyacrylamide gel (8%) and immunoblotted for SP100 (A), SUMO-1 (B), Keap1 (C), Nrf2 (D), and RNF4 (E). HMW, high molecular weight.

FIGURE 10.

Polysumoylated Nrf2 is ubiquitylated by RNF4 resulting in decreased steady-state levels of Nrf2. Where indicated, cells were transfected with 2 μg of vector (pBOS-EFI-H2BGFP) alone or plasmid encoding wild-type RNF4-YFP or mutant RNF4-CS1-YFP. After 48 h, the cells were incubated with or without MG-132 (10 μm) and/or As₂O₃ (5 μm) for 4 h and then harvested. When used together, MG-132 was added 30 min prior to the addition of As₂O₃. Whole cell lysates were immunoprecipitated with anti-Nrf2 antibody (A–C and F–H) or anti-GFP antibody (D and E) and Western blotted as indicated. A–G, nonimmune serum (IgG) was used as control. A and B, Co-IP assays to assess ubiquitylated (A) or sumoylated (B) endogenous Nrf2. C, input control. D and E, Co-IP assays demonstrating interaction of RNF4 with Nrf2. RNF4-YFP was detected with anti-GFP antibody. D, input control. E, interaction of RNF4 with Nrf2. RNF4-YFP. F, wild-type RNF4, but not its mutant, enhances ubiquitylation of polysumoylated Nrf2. G, ubiquitylated Nrf2 is also sumoylated. H, steady-state levels of Nrf2 in cells transfected with plasmid encoding wild-type RNF4-YFP. Cells were treated with As₂O₃ (2 μm) for up to 8 h with or without MG-132 for 8.5 h, lysed, and then Western blotted for Nrf2. The blots were quantified densitometrically using UN-SCAN-IT software (Silk Scientific, Inc., Orem, UT). Values plotted are means for 2–3 experiments. The data are presented as percentage plots, taking the values for no treatment with As₂O₃ (controls) as 100%.

FIGURE 11.

Analysis of nuclear body-enriched preparation reveals that RNF4 induces degradation of Nrf2 in arsenic trioxide-treated cells. Cells were grown and treated as in the legend to Fig. 10. A, whole cell lysate (W) prepared from cultures treated with As₂O₃ (2 μm) for 4 h was separated into cytoplasmic (C) and nuclear body-enriched (NB) fractions as in Fig. 2. RNF4-YFP was detected with anti-GFP antibody (Invitrogen). Keap1 was detected with anti-Keap1 antibody (ab31973; Abcam). B, expressed levels of wild-type RNF4 (RNF4wt-YFP) and mutant RNF4 (RNF4-CS1-YFP) (right panel) and steady-state levels of endogenous Nrf2 (left panel) in the NB fraction. C, RNF4-mediated degradation of endogenous Nrf2, measured in nuclear body-enriched fraction of cells transfected with plasmid encoding wild-type RNF4-YFP. Forty eight hours after transfection, HepG2 cells were incubated with As₂O₃ (2 μm) and cycloheximide (CHX) (50 μg/μl) and then harvested at the indicated time points thereafter. Nuclear body-enriched fractions (NB fractions) were then immunoblotted for Nrf2. Representative blots are shown. The blots were quantified densitometrically using UN-SCAN-IT software (Silk Scientific, Inc., Orem, UT). The values plotted are means ± S.E. for five experiments. The data are presented as percentage plots, taking the values for no treatment with cycloheximide (zero time) as 100%. Open circles, treatment with empty vector; closed squares, treatment with RNF4wt-YFP.

FIGURE 12.

Nrf2-dependent gene transcription is abrogated by RNF4. HepG2 cells were co-transfected with 0.2 μg each of HO-1-ARE-luc reporter gene construct and heterologous Nrf2 (pCI-Nrf2) as inducer (19, 22), with or without plasmid pCMV6-RNF4-myc-DDK (OriGene Technologies) expressing RNF4. When needed, empty vector pCI-Neo (0.2 μg) or pCMV6-myc-DDK (0.2 μg) was also transfected. The total amount of DNA in each well was 0.6 μg. The electrophile tBHQ (20 μm) was added 24 h after transfection, and promoter activity was measured 8 h later, as described (19, 22). A, Nrf2-dependent gene promoter activity is inhibited by RNF4. B, tBHQ-enhanced Nrf2-dependent gene transcription is inhibited by RNF4. Values plotted are means ± S.E. for duplicate assays from 3 to 4 different experiments. *, statistically different (p < 0.05).

FIGURE 13.

Schematic depicting post-translational modifications of Nrf2 after its separation from Keap1. For simplicity, phosphorylation (, –86) has been deliberately omitted. Whether sumoylation of Nrf2 precedes its binding to the ARE or occurs after this event has not been determined. Ac, acetyl; O-Ac-ADP-Rib, O-acetyl-ADP-ribose; S, SUMO; U, ubiquitin.