Prothymosin-α mediates nuclear import of the INrf2/Cul3·Rbx1 complex to degrade nuclear Nrf2.

Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for Cul3-Rbx1 mediated degradation of Nrf2. A majority of the INrf2/Cul3-Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that Cul3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-α both bind to the DGR region of INrf2. However, it is prothymosin-α and not Nrf2 that mediates nuclear import of INrf2/Cul3-Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/Cul3-Rbx1 complex. The INrf2/Cul3-Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2 resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α mediated nuclear import of INrf2/Cul3-Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression. Introduction Nrf2:INrf2 serve as sensor of chemical and radiation induced oxidative and electrophilic stress (1-3). Nrf2 is a nuclear transcription factor that regulates expression of several defensive genes including detoxifying enzymes and antioxidant genes (1-3). The induction of these enzymes is important for neutralizing the cellular stresses, providing cellular protection and survival (1-3). Nrf2 resides predominantly in the cytoplasm where it interacts with actin-associated cytosolic protein, INrf2 (inhibitor of Nrf2) or Keap1 (Kelch-like ECH-associated protein 1) (4-6). INrf2 functions as a substrate adaptor protein for a Cul3-Rbx1-dependent E3 ubiquitin ligase complex to ubiquitinate and degrade Nrf2 thus maintaining a steadystate levels of Nrf2 (7). The exposure to oxidative/electrophilic stress leads to dissociation of Nrf2 from INrf2. Nrf2 is stabilized, translocates into the nucleus, and activates the transcription of several defensive genes. Recently, the mechanisms by which Nrf2 is released from INrf2 under stress have been actively investigated. One mechanism is that cysteine thiol groups of INrf2 were shown to function as sensors for oxidative stress which are modified by the chemical inducers, causing formation of disulfide bonds between cysteines of two INrf2 peptides. This results in conformational change that renders INrf2 unable to bind to Nrf2 (8-10). On the other hand, antioxidantinduced protein kinase C mediated phosphorylation of serine40 in Nrf2 leads to 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M808084200 The latest version is at JBC Papers in Press. Published on March 11, 2009 as Manuscript M808084200 Copyright 2009 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from dissociation of Nrf2 from INrf2 (11-12). It is possible that two mechanisms work in concert or independently of each other. However, the evidence to support either is lacking. Interestingly we have recently demonstrated that an autoregulatory loop between Nrf2 and INrf2 controls their abundance inside the cells (13). In other words, Nrf2 induces INrf2 gene expression for its own degradation. Several reports suggest that persistent accumulation of Nrf2 in the nucleus is harmful. INrf2-null mice demonstrated persistent accumulation of Nrf2 in the nucleus that led to postnatal death from malnutrition resulting from hyperkeratosis in the esophagus and forestomach (14). Reversed phenotype of INrf2 deficiency by breeding to Nrf2-null mice suggested tightly-regulated negative feedback might be essential for cell survival (15). The recent systemic analysis of INrf2 genomic locus in human lung cancer patients and cell lines showed that deletion, insertion, and missense mutations in functionally important domains of INrf2 results in reduction of INrf2 affinity for Nrf2 and elevated expression of cytoprotective genes (16-17). Taken together, unrestrained activation of Nrf2 in cells increases a risk of adverse effects including tumorigenesis. On the other hand, stress-induced activation of the Nrf2 pathway in normal cells is tightly regulated and confers cytoprotection against oxidative and electrophilic stress and carcinogens. Therefore, it appears that cells contain mechanisms that auto-regulate cellular abundance of Nrf2. A majority of the INrf2 is present in the cytosol (7). However, 10-15% of INrf2 is also localized inside the nucleus (7). Procite analysis revealed that INrf2 does not contain a defined nuclear import signal and the mechanism of nuclear import of INrf2 and its function inside the nucleus remain unknown. In this report we investigated the mechanism of nuclear import of INrf2 and its role in Nrf2 degradation inside the nucleus. The studies demonstrated that the DGR region of INrf2 and prothymosin-α (PTMα) are required for nuclear import of INrf2. Studies also demonstrated that INrf2 is not transported alone but in complex with Cul3 and Rbx1. Interestingly, PTMα containing a nuclear localization signal binds to the DGR region of INrf2 and transports whole complex of INrf2/Cul3-Rbx1 inside the nucleus. The complex inside the nucleus releases PTMα, binds to Nrf2, and triggers Nrf2 degradation. This is presumably to rapidly switch off the activation of Nrf2 downstream gene expression. Materials and Methods Cell Cultures— Mouse hepatoma (Hepa-1) cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin (40 units/ml), and streptomycin (40 μg/ml). The cells were grown in monolayer in an incubator at 37 °C in 95% air and 5% CO2. Plasmid Constructs— Mammalian expression vector pEGFP-N1 was purchased from Clontech (CA USA). This vector upon transfection in cells expresses green fluorescence protein (GFP). The vector was designated as 1X-GFP. We modified this vector by cloning another coding sequence of GFP. The modified vector was designated as 2XGFP. A Fulllength mouse INrf2 coding sequence (1872 bp) was cloned into 1X GFP as well as 2X GFP vector. The primer sequences used for amplification of full-length INrf2, different deletion domains and mutations of INrf2 are shown in Supplement Table 1. Two leucine residues (L308 and L310) present between functional nuclear export signals 2 (NES2) of INrf2 ware mutated to alanine by using specific mutant primers and site directed mutagenesis kit (Invitrogen). cDNA of prothymosin-α (PTMα) was purchased from OriGene (Rockville, MD) and a full-length coding sequence was amplified by PCR and subcloned into pcDNA3.1/V5-His/Topo vector by TA cloning. The construction of pGL2B-NQO1-ARE, pcDNA-Flag-INrf2, pcDNA-Flag-Nrf2, pcDNA-INrf2-V5 and HAUB has been described previously (15). All 2 by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from site directed mutations and deletions mutations were confirmed by DNA sequencing. In vitro transcription/translationIn vitro transcription/translation of the plasmids encoding domains/deletions of INrf2-GFP and PTMα–V5 constructs were performed using the TNT-coupled rabbit reticulocyte lysate system (Promega). 0.2 μg of plasmid DNA was incubated with 25 μl of TNTcoupled rabbit reticulocyte lysate supplied with 40 μM of L-methionine at 30°C for 90 min. The plasmid encoding luciferase provided in the kit was used as a control for the transcription/translation reaction. After the coupled transcription/translation, the proteins were checked for their correct size by SDS-PAGE followed by immunoblotting. All of the in vitro transcribed/translated proteins gave the expected size bands. Generation of stable Flp-In T-REx HEK293 cells expressing tetracycline-inducible Flag INrf2, Flag-ΔDGR-INrf2 and Flag-DGRINrf2— Full-length INrf2, ΔDGR-INrf2 and DGR-INrf2 regions were amplified by PCR using specific sets of primers (Supplement Table 1) and cloned into modified pcDNA5/FRT/TO (Flag) X2-(His) 8 vector. Flp-In T-REx HEK-293 cells were purchased from Invitrogen and cotransfected separately with 0.1 μg of FlagINrf2-pcDNA5/FRT/TO, Flag-ΔDGR INrf2pcDNA5/FRT/TO and Flag-DGRINrf2pcDNA5/FRT/TO along with 0.9 μg pOG44 plasmids (Invitrogen) by Effectene (Qiagen, Valencia, CA) method, as described by the manufacturer’s instructions. Forty eight hours after transfection, the cells were grown in medium containing 100 μg/ml hygromycin B and 30 μg/ml blasticidin (Invitrogen). The 293/FRT/TO cells stably expressing tetracycline-inducible N-terminal Flag-tagged INrf2, Flag-tagged ΔDGR-INrf2 and Flag-tagged DGR-INrf2 were selected. The stably selected cells were grown and treated with 0.5 μg/ml tetracycline (Sigma) for varying periods of time to follow the overexpression of Flag-tagged INrf2, Flagtagged ΔDGR INrf2 and Flag tagged DGR INrf2 proteins. Subcellular fractionation and Western blotting— Hepa-1 cells, seeded in 100-mm plates and treated or transfected as displayed in the figures, were washed twice with ice-cold phosphate-buffered saline, trypsinized, and centrifuged at 1500 rpm for 5 min. For making whole cell lysates, the cells were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.2 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholic acid, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Sodium orthovanadate supplemented with protease inhibitor mixture (Roche Applied Science). Cytoplasmic and nuclear cell lysates were separated by using the Active Motif nuclear extract kit (Active Motif, Carlsbad, CA) by following the manufacturer's protocol. The protein concentration was determined using the protein assay reagent (Bio-Rad). 60 to 80 micrograms of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 3% non fat dry milk and incubated with anti-INrf2 (E-20) (1:1000), anti-Nrf2 (H-300) (1:500), anti Prothymosin α (H-50) (1:1000) and anti fetal alz-50 clone 1 (FAC1) (1:1000) antibodies, all purchased from Santa Cruz Biotechnology (CA). Anti Cul3 and anti Rbx1 antibodies were purchased from Cell Signaling (Boston, MA). AntiFlag-HRP (1:10000), anti-HA-HRP (1:10000) and anti-actin antibodies were obtained from Sigma. Anti-GFP and anti-V5 HRP antibodies obtained from Invitrogen. The membranes were washed three times with TBST and immunoreactive bands were visualized using a chemiluminescence’s system ECL (Amersham). The intensity of protein bands after Western blotting were quantitated by using QuantityOne 4.6.3 Image software (ChemiDoc XRS, Bio-Rad) and normalized against proper loading controls. To confirm the purity of nuclearcytoplasmic fractionation, the membranes were reprobed with cytoplasm-specific, antilactate dehydrogenase (Chemicon) and nuclear specific, anti-lamin B antibodies (Santa Cruz Biotechnology). In related experiments, the cells were treated with 50 to 100 μM t-BHQ, 10 ng/ml LMB and 20 μM 3 by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from genestein or DMSO as a vehicle for different time intervals. Immunoprecipitation1 mg of whole cell lysate or nuclear/cytoplasmic extract were equilibrated in RIPA buffer, pre-cleaned with protein-AGplus-agarose (Santacruz Biotechnology) and incubated with respective antibodies (1 μg) at 4oC for overnight. Immune-complexes were collected by addition of protein AG-agarose again after centrifugation. The immunecomplexes were washed three times with RIPA buffer containing 0.25% NP-40, and proteins were resolved by 10% reducing SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blocked with 3% non fat dry milk and incubated with their respective primary and secondary antibodies. Immunoreactive bands were visualized using a chemiluminescence’s system ECL (Amersham). Transient transfection and luciferase assay— Hepa-1 cells were plated in 100 mm plates at a density of 1x10 cells/plate 24h prior to transfection. In the related experiments, the cells were transfected with 1 μg of the indicated plasmids using Effectene transfection reagent (Qiagen) according to the manufacturer’s instructions. After 36h of transfection, cells were harvested and cellular specific protein regulation was examined by Western blotting. For luciferase reporter assay, Hepa-1 cells were grown in monolayer cultures in 12-well plates in DMEM mediumα supplemented with 10% fetal bovine serum. Then cells were co-transfected with 0.1 μg of reporter construct human NQO1ARE-Luc and 10 times less quantities of firefly Renilla luciferase encoded by plasmid pRL-TK. Renilla luciferase was used as the internal control in each transfections. After 24h of transfection, the cells were washed with 1X phosphate-buffered saline and lysed in 1X Passive lysis buffer from the DualLuciferase® reporter assay system kit (Promega, Madison, WI). The luciferase activity was measured using the procedures described previously (15) and plotted. siRNA interference assay—Control, Nrf2 and INrf2 siRNA purchased from Dharmacon were used to inhibit Nrf2 and INrf2 proteins respectively by a procedure described previously (10). Hepa-1 cells were transfected with 5, 25 and 50 nM of control or Nrf2 or INrf2 siRNA using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. Thirty two hours after transfection, cells were harvested and localization of INrf2, Cul3, RBX1 was analyzed by Western blotting by probing the membranes with INrf2, Nrf2 and Cul3 or RBX1 antibodies. Fac1 and PTMα siRNA was also purchased from Ambion (CA) and effect of these siRNA on localization of INrf2 was analyzed. Effect of PTMα siRNA on localization of INrf2 and NQO1 ARE luciferase activity was carried out after control siRNA or PTMα siRNA transfections. Immunofluorescence— Hepa-1 cells were grown in Lab-Tek II chamber slides in DMEM supplemented with 10% fetal bovine serum. In related experiments, cells were transfected with INrf2-GFP, INrf2-ΔDGRGFP or INrf2 DGR 2X GFP for 32h. After fixing with 2% formaldehyde, cells were stained with DAPI and directly observed under fluorescence microscope. Effect of PTMα siRNA on localization of INrf2 was performed by transient transfection of PTMα siRNA using the procedures described above. Twenty-four hours later, cells were fixed with 2% formaldehyde at 4°C for 10 min and permeabilized by 0.25% Triton X100. Cells were washed twice with PBS and incubated with 1:1000 dilution of goat INrf2 primary antibodies in 2% BSA at 4°C for 12h. Then cells were washed twice with PBS and incubated with FITC conjugated second antibody for 2 h at room temperature. Cells were washed and stained with DAPI and mounted. Effect of PTMα overexpression on localization of INrf2-V5 was also visualized by immunofluorescence. Hepa-1 cells were transfected with INrf2-V5 alone or in combination with pcDNA-PTMα. Twentyfour hours later, cells were treated with 50 μM t-BHQ for 2h, and cells were fixed and 4 by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from permeabilized by 0.25% TritonX-100. After washing, cells were incubated with 1:1000 dilution of FITC conjugated anti-V5 tag antibodies for 12 h. Cells were washed twice with PBS, stained with DAPI and mounted. After immunostaining, cells were observed under Nikon fluorescence microscope and photographs were captured. The cytoplasmic and nuclear FITC fluorescence intensities were quantified from 10 different cells (N=10) by using NIS-Elements BR2.30 SP4 software (Nikon). The green fluorescence intensity of the images were quantified by using Nikon Elements Advanced Research Software (Melville, NY). The entire green region of nuclear and cytosolic compartment was first marked and delineated, and the average fluorescence intensity of the green channel was measured. The experiments were repeated three times. Isolation and purification of INrf2/Cul3Rbx1/PTMα/Nrf2 complex— Human embryonic kidney FRT-HEK293 cells stably expressing Flag-INrf2 were treated with 0.5 μg/ml of tetracycline for 24hour followed by treatment with DMSO or tBHQ (50 μM) for 1 and 2h. Cells were harvested and cytoplasmic and nuclear extracts prepared by using Active Motif Kit. 5 mg of cytoplasmic and nuclear extracts were mixed with anti-Flag agarose beads (200 μl) at 4°C for 8 h, beads washed three times with RIPA buffer and bound protein were eluted with 100 μl of 1X Flag peptide (Sigma). The native protein complex was electrophoresed by 6% Native gel and stained with Coomassie Brilliant Blue. The same native complex was immunoblotted with anti INrf2, Nrf2, PTMα, Cul3 and Rbx1 antibodies. Ubiquitination assay—Hepa-1 cells were transfected with control or PTMα siRNA (50 nM) for 10h followed by co-transfection with Flag-INrf2 (0.5 μg), Nrf2-V5 (1.0 μg) and HA-Ub (0.2 μg). Nuclear and cytoplasmic extracts were prepared using active motif kit. To check the effect of PTMα siRNA on localization of INrf2 and PTMα protein, 60 μg of the cytoplasmic and nuclear extracts were immunoblotted with anti-Flag, anti-V5 and anti-PTMα antibodies. To check Nrf2 ubiquitination in cytoplasmic and nuclear compartments, one mg of cytoplasmic and nuclear lysates were immunoprecipitated with anti-V5 antibody (Invitrogen). Immunecomplexes were resolved by 10% SDSPAGE followed by immunoblotting with antiHA-HRP antibody. Statistical Analyses— Statistical analysis of the data from luciferase assays and immunoblotting band intensities were performed by using the SPSS-16 software. Error bars indicate standard error of the means (s.e.m) of triplicate samples and presented in figures. Statistical analysis was performed by one way ANOVA, followed by the Tukey-Kramer’s post hoc test for multiple comparisons. Significance P values were also calculated and presented.