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Genes to Cells (2007) 12, 1163-1178. doi:10.1111/j.1365-2443.2007.01118.x
© 2007 Blackwell Publishing or its licensors
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Subcellular localization and cytoplasmic complex status of endogenous Keap1

 Yoriko Watai1,2, Akira Kobayashi1,3,*, Hiroko Nagase1, Mio Mizukami4, Justina McEvoy5, Jeffrey D. Singer5, Ken Itoh1,4,6 and Masayuki Yamamoto1,3,4,*

1 Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan
2 Yokohama Laboratory, Summit Pharmaceuticals International Corporation, Yokohama, Japan
3 Department of Medical Biochemistry, Tohoku University Graduate School of Medicine, Sendai, Japan
4 Environmental Response Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, University of Tsukuba, Tsukuba, Japan
5 Department of Molecular Biology, Cell Biology and Biochemistry, and the Center for Genomics and Proteomics, Brown University, Providence, RI, USA
6 Center for Advanced Medical Research, Hirosaki University School of Medicine, Hirosaki, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Keap1 acts as a sensor for oxidative/electrophilic stress, an adaptor for Cullin-3-based ubiquitin ligase, and a regulator of Nrf2 activity through the interaction with Nrf2 Neh2 domain. However, the mechanism(s) of Nrf2 migration into the nucleus in response to stress remains largely unknown due to the lack of a reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new monoclonal antibody for the detection of endogenous Keap1 molecules. Immunocytochemical analysis of mouse embryonic fibroblasts with the antibody revealed that under normal, unstressed condition, Keap1 is localized primarily in the cytoplasm with minimal amount in the nucleus and endoplasmic reticulum. This subcellular localization profile of Keap1 appears unchanged after treatment of cells with diethyl maleate, an electrophile, and/or Leptomycin B, a nuclear export inhibitor. Subcellular fractionation analysis of mouse liver cells showed similar results. No substantial change in the subcellular distribution profile could be observed in cells isolated from butylated hydroxyanisole-treated mice. Analyses of sucrose density gradient centrifugation of mouse liver cells indicated that Keap1 appears to form multiprotein complexes in the cytoplasm. These results demonstrate that endogenous Keap1 remains mostly in the cytoplasm, and electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
The Nrf2–Keap1 system is one of major regulatory pathways regulating the cellular defense against oxidative/electrophilic stress. A basic region leucine zipper transcription factor Nrf2 mediates the inducible expression of phase 2 drug-metabolizing enzymes and antioxidant response proteins, such as NAD(P)H-quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST) and hemoxygenase 1 (HO-1), via a cis-acting DNA element, electrophile/antioxidant responsive elements (EpRE/ARE; reviewed in Motohashi & Yamamoto 2004; Dinkova-Kostova et al. 2005). Molecular and biochemical analyses, as well as mouse in vivo study, have been conducted in order to elucidate the mechanisms regulating the Nrf2 activity. The latter study demonstrated that Nrf2-deficient mice are susceptible to a variety of xenobiotic or oxidative insults (reviewed in Kobayashi & Yamamoto 2006). The Nrf2-null mice are prone to forestomach tumors when provoked with benzo(a)pyrene compared to wild-type mice (Ramos-Gomez et al. 2001), indicating that Nrf2 plays crucial roles in cancer chemoprevention.

We discovered Keap1, which represses Nrf2 in the cytoplasm under unstressed conditions (Itoh et al. 1999). Keap1 possesses three functional domains, the Broad complex, Tramtrack and Bric-a-Brac (BTB) domain, intervening region (IVR) and double glycine repeat (DGR)/Kelch domain (Itoh et al. 1999), and belongs to the BTB–Kelch protein family (reviewed in Adams et al. 2000). Keap1 binds to Nrf2 via the DGR domain and C-terminal region, which is collectively named as Keap1-DC domain. Upon exposure to oxidative/electrophilic stress, Nrf2 is liberated from Keap1 repression, accumulates in the nucleus and activates cytoprotective genes. We and another group recently identified somatic mutations of KEAP1 gene in tissues and cultured cells derived from human lung cancer patients (Padmanabhan et al. 2006; Singh et al. 2006). Although the physiological relevance between somatic KEAP1 mutations and lung carcinogenesis remains to be elucidated, these mutations seem to impair the ability of Keap1 to repress Nrf2, thereby allowing the constitutive activation of its downstream target genes and resulting in cytoprotection of cancer cells.

Keap1 directly interacts with the Neh2 (Nrf2-ECH homology 2) domain of Nrf2 via the Keap1-DC domain (Itoh et al. 1999). An important observation here is that Keap1 promotes rapid degradation of Nrf2 through the ubiquitin–proteasome pathway (Itoh et al. 2003; McMahon et al. 2003, 2004; Nguyen et al. 2003; Stewart et al. 2003). Keap1 serves as an adaptor for Cullin-3 (Cul3)-based E3 ubiquitin ligase, promoting poly-ubiquitin conjugation to Nrf2 (Cullinan et al. 2004; Kobayashi et al. 2004; Zhang et al. 2004; Furukawa & Xiong 2005). Based on these and other lines of data, we recently proposed a "two-site molecular recognition model" for the interaction of Nrf2 Neh2 domain and Keap1-DC domain (Tong et al. 2006a). In this model, DLG and ETGE motifs in Neh2 independently associate with the Keap1-DC domain; one Nrf2 molecule associates with two Keap1 molecules through the DLG and ETGE motifs, and this agrees with the finding that Keap1 forms a homodimer (Zipper & Mulcahy 2002).

Keap1 is a cysteine-rich protein that allows for oxidative and electrophilic sensing. Cys151 in the BTB domain or Cys273 and Cys288 in the IVR are such targets of electrophilic modification (Dinkova-Kostova et al. 2002; Zhang & Hannink 2003; Wakabayashi et al. 2004). These studies indicate that Cys151 appears to be required for the oxidative stress-mediated activation of Nrf2, whereas Cys273 and Cys288 are required for the repression activity of Keap1 (Zhang & Hannink 2003; Wakabayashi et al. 2004).

We and other groups recently demonstrated that the electrophilic stress does not disrupt the association of Keap1 and Nrf2, but rather it represses Keap1-mediated ubiquitination of Nrf2 (Zhang et al. 2004; Eggler et al. 2005; Kobayashi et al. 2006). After exposure to the electrophilic stress, newly synthesized Nrf2 molecules translocate into the nucleus bypassing the Keap1 "gate" of repression. As for the mechanisms underlying this de-repression process, we have proposed a Hinge and Latch model (Tong et al. 2006a, b), in which conformational changes in Keap1 molecule brought on by oxidative/electrophilic modification of the cysteine residues abrogates the ubiquitination activity of Keap1–Cul3 complex, thereby disrupting the repression of Nrf2 by Keap1.

Aside from the Hinge and Latch model, four additional mechanisms/models have been proposed. First, CAND1, an interference factor of Cul1 and its adaptor interaction, was reported to also disrupt the Keap1–Cul3 complex to stabilize Nrf2 (Lo & Hannink 2006). Second, it has been reported that oxidants and electrophiles promote degradation of Keap1 (Hong et al. 2005; Zhang et al. 2005). In this model, oxidative/electrophilic stress causes self-ubiquitination and degradation of Keap1, allowing for Nrf2 nuclear accumulation. A third mechanism suggests that oxidative/electrophilic stress affect subcellular localization of Keap1 molecules (Karapetian et al. 2005; Nguyen et al. 2005; Velichkova & Hasson 2005). Here, Keap1 harbors a nuclear export signal (NES) and shuttles between the nucleus and cytoplasm in an exportin/Crm1-dependent manner. Oxidative/electrophilic stress impairs the NES function of Keap1, causing nuclear accumulation of Keap1 and Nrf2. Finally, one study reported that Nrf2 possesses a redox-sensitive NES, and this NES directly senses oxidative stress to promote nuclear accumulation of Nrf2 (Li et al. 2006).

While the physiological validity of these four mechanisms, and our Hinge and Latch model is yet to be established, one critical issue is the lack of data examining subcellular localization of endogenous Keap1 molecules and its dynamic changes in vivo, as most studies were conducted in vitro in artificial over-expression experiments. This is, at least in part, due to the lack of reliable antibody for the detection of endogenous Keap1 molecule. Here, we report the generation of a new Keap1 antibody that effectively detects endogenous Keap1 and the evaluation of the molecule's subcellular localization in response to electrophilic stress utilizing the generated antibody. The results show endogenous Keap1 to be mainly localized in the cytoplasm with a small amount in the nucleus and endoplasmic reticulum (ER). The subcellular localization of endogenous Keap1 appears unchanged upon treatment of cells with electrophiles or Leptomycin B (LMB), indicating that oxidative/electrophilic stress provokes nuclear accumulation of Nrf2 without altering the cytoplasmic localization of Keap1. These results thus provide basic information related to subcellular localization and dynamics of Keap1, which is useful to execute further experiments to support or exclude the working models summarized above.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Generation of monoclonal antibody recognizing endogenous Keap1 protein

To examine subcellular distribution of endogenous Keap1, we generated anti-Keap1 monoclonal antibodies by immunizing rats with purified mouse Keap1 protein. We have selected one clone (no. 144) in our initial screening. Immunoblot experiments showed that the antibody recognized Keap1 plus a cross-reactive material (CRM), as the latter band was detected in both wild-type and Keap1-deficient mouse embryonic fibroblasts (MEF-WT and MEF-K0 cells, respectively; data not shown). To eliminate the CRM, protein G column-purified antibody was adsorbed with MEF-K0 cell extracts. Immunoblot experiments with this pre-cleared antibody recognized Keap1 alone and not the CRM; a band showing the Keap1 molecular size was observed in cell extracts of MEF-WT cell (Fig. 1A, lane 1), while the band was not detected in cell extracts of MEF-K0 cell (lane 2).


Figure 1
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  		Figure 1  Generation of monoclonal antibody for endogenous Keap1 protein. (A) Immunoblot analysis of cell extracts from mouse embryonic fibroblast (MEF) cells. Protein-G column-purified antibody was adsorbed with cell extracts from MEF cells of Keap1-null mice (MEF-K0) to eliminate cross-reactive material. The adsorbed Keap1 (ad-Keap1) antibody specifically recognized endogenous Keap1 protein in MEF-WT cells (lane 1) but not in MEF-K0 cells (lane 2). The immunoblot was also subjected to {alpha}-Tubulin as the loading control. (B) Immunofluorescent staining shows that Keap1 mainly localizes in the perinuclear region (left, MEF-WT). Right panel shows immunostaining of MEF-K0 cells as a negative control.

 
We also exploited the adsorbed fraction of this antibody in immunocytochemical staining of MEF cells. Fluorescence signal derived from the Keap1–antibody complex was observed in MEF-WT cells, but not in MEF-K0 cells (Fig. 1B). Based on these results, we conclude that the anti-Keap1 antibody we generated is appropriate for both immunoblot and immunocytostaining analyses for the detection of endogenous Keap1 molecule. We refer to the adsorbed antibody as ad-Keap1 antibody in this paper.

Endogenous Keap1 localizes mostly in the cytoplasm

To elucidate subcellular localization of endogenous Keap1 protein, we performed an immunocytological staining experiment of MEF-WT cells with the ad-Keap1 antibody. The results showed that Keap1 localizes predominantly in the perinuclear region of cytoplasm (Fig. 1B, left panel).

We then performed double immunocytostaining of MEF cells using ad-Keap1 antibody together with anti-organelle marker antibodies, which included those against Calreticulin (ER), Golgin130 (Golgi) or Mitochondrial complex II (mitochondria). We also stained actin with Alexa546-conjugated phalloidin, which specifically recognizes actin filaments (F-actin) or anti-actin antibody. The fluorescence images were captured by confocal microscopy. We found that Keap1 co-localizes with ER (Fig. 2A), but not with Golgi apparatus or mitochondria (Fig. 2B and C, respectively). The anti-actin antibody, which recognizes both globular (G) and filamentous (F) actin, stained the perinuclear region where endogenous Keap1 localizes (Fig. 2D), showing agreement with our previous observation that Keap1 co-localizes with actin filaments in the cytoplasm of MEF cells derived from Keap1-over-expressing transgenic mice (Kang et al. 2004). In contrast, we could not find clear-cut co-localization of endogenous Keap1 with actin filaments visualized by the phalloidin staining (Fig. 2E), suggesting that the relationship between Keap1 and the actin cytoskeleton as a scaffold is not straightforward and requires further examination.


Figure 2
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  		Figure 2  Endogenous Keap1 largely localizes in the cytoplasm. MEF-WT cells were co-immunostained with ad-Keap1 antibody (green) and various organelle antibodies (red); Calreticulin (A; ER), Golgin130 (B; Golgi), Mitochondrial protein complex (C; mitochondria), actin (D; G-/F-actin) and Alexa546-conjugated phalloidin (E; F-actin). Right panels show merged and magnified fluorescent images (bottom and top, respectively). Nuclei were stained with Hoechst33342 (bottom right, blue).

 
Subcellular localization of endogenous Keap1 does not change by diethyl maleate (DEM) or LMB

To examine Keap1 localization changes in response to electrophilic stress, MEF-WT cells were treated with DEM (final concentration 100 µM) for 4 h, followed by immunostaining with the ad-Keap1 antibody. We found that the DEM treatment did not influence the subcellular localization of endogenous Keap1 (Fig. 3A, upper panels), although it promoted nuclear accumulation of Nrf2 (bottom panels). The results were reproducible in the time course study of 0.5-, 1-, 2-, 6- and 12-h DEM treatment. Similarly, the Keap1 localization was not affected by the treatment of 100 µM tert-butylhydroquinone (tBHQ) for 4 h (data not shown). Previous study had showed the nuclear accumulation of endogenous Keap1 in NIH3T3 cells (Velichkova & Hasson 2005). We performed similar experiment in NIH3T3 cells with 16 h of serum starvation followed by serum supplementation to promote actin polymerization, followed by the treatment of cells with DEM for 2 h. Under our experimental conditions, we were unable to see nuclear accumulation of endogenous Keap1 (Fig. 3B).


Figure 3
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  		Figure 3  Diethyl maleate (DEM) does not affect subcellular localization of endogenous Keap1. (A) MEF-WT cells were treated with or without DEM (100 µM) for 4 h and immunostained with ad-Keap1 or anti-Nrf2 antibody. (B) NIH-3T3 cells were serum starved for 16 h followed by serum supplementation with DEM (100 µM) for 2 h.

 
We next examined whether Keap1 shuttles between the cytoplasm and nucleus in a nuclear export factor Crm1-dependent manner. NIH3T3 cells were treated with an inhibitor of Crm1, LMB (final concentration, 20 ng/mL), for 4 h and subjected to immunocytostaining with ad-Keap1 antibody. GFP-Bach2 expression plasmid was used as positive control, since Bach2 shuttles between the nucleus and cytoplasm in a Crm1-dependent manner and LMB treatment has been shown to promote nuclear accumulation of GFP-Bach2 (Hoshino et al. 2000). NIH3T3 cells transfected with GFP-Bach2 were treated with LMB for 4 h, and subcellular localization of Keap1 and GFP-Bach2 was monitored by immunocytostaining with ad-Keap1 antibody and green fluorescence, respectively. Although LMB treatment clearly caused nuclear accumulation of GFP-Bach2 (Fig. 4A and B), nuclear accumulation of endogenous Keap1 could not be observed in the GFP-Bach2 transfected cells or control cells (left and right panels, respectively). Similar results were seen in MEF-WT cells not only at 4 h (Fig. 4C) but also at 2 and 8 h (data not shown) after the LMB treatment.


Figure 4
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  		Figure 4  Leptomycin B (LMB) treatment does not affect Keap1 localization. (A, B) NIH3T3 cells were treated with or without LMB (20 ng/mL) for 4 h, and immunostained with ad-Keap1 antibody. Bach2-GFP plasmid was transfected as a positive control to monitor Crm1 inhibition. (C) MEF-WT cells were similarly treated with or without LMB. Cells in the right-most panel was co-treated with LMB and DEM (100 µM) for 4 h. All nuclei were stained with Hoechst33342.

 
Biochemical subcellular fractionation analysis of endogenous Keap1

To confirm our findings in immunocytochemical analysis, immunoblot experiments were performed. Cytoplasmic and nuclear extracts were prepared from equal number of MEF cells treated with DEM and subjected to immunoblot analysis with the ad-Keap1 antibody. We verified the minimum presence of cross-contamination between the two fractions with anti-{alpha}-Tubulin and anti-LaminB antibodies for cytoplasm and nucleus, respectively (Fig. 5, two bottom panels). While DEM treatment induced nuclear accumulation of Nrf2 (Fig. 5A, lanes 2 and 4), the treatment did not stimulate nuclear localization of Keap1. LMB treatment in the absence or presence of DEM increased the nuclear level of Keap1, although the effect was marginal (lanes 5–8). Important observation here is that even in the presence of LMB, DEM did not increase the nuclear level of Keap1. Densitometry analysis indicates that the increase in nuclear Keap1 was less than 20% in the presence of both DEM and LMB (Fig. 5B). This suggests that endogenous Keap1 is stably localized in the cytoplasm even in the presence of oxidative/electrophilic stress. Therefore, we conclude that the nuclear transfer and accumulation of endogenous Keap1 is not the major mechanism for the Nrf2 activation in response to electrophiles.


Figure 5
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  		Figure 5  Biochemical subcellular fractionation analysis of endogenous Keap1. (A) MEF-WT cells were treated with 100 µM DEM (D), 20 ng/mL LMB (L), or DEM and LMB (DL) for 4 h. Cytoplasmic (C) and nuclear extracts (N) prepared from equal number of the cells were used for immunoblot analyses with ad-Keap1 and anti-Nrf2 antibodies. (B) Anti-LaminB and anti-{alpha}-Tubulin antibodies were used as internal controls for nuclear and cytoplasmic extracts, respectively. Each bar represents the relative value of non-treated cells (–) set as 100%. Bar graph represents mean ± SD of % intensity of the Keap1 band (n = 3).

 
Subcellular localization of Keap1 in mouse liver cells

To further examine the Keap1 subcellular localization, we performed organelle fractionation of mouse liver and assessed the expression of Keap1. We isolated the following organelle fractions using standard fractionation method with isotonic sucrose; nuclei (N), mitochondria (M), smooth ER (sER) and rough ER (rER), Golgi (G) and cytoplasm (C). These organelle extracts were prepared and subjected to immunoblot analyses with the ad-Keap1 antibody (each fraction, 40 µg protein). Table 1 shows the recovery percentages of five organelle fractions from total homogenates. Compared to the previous reports with rat liver (such as Touster et al. 1970), the data showed comparable yields, indicating that the fractionation reflects the intrinsic population of these organelles in mouse liver cells. Immunoblot experiments show that Keap1 is mainly present in the cytoplasm fraction, but also in sER and rER fractions and nuclear fraction at lesser extent (Fig. 6A). Immunopositive Keap1 bands were quantified with densitometry, and subcellular distribution ratio of Keap1 was calculated. We estimated that Keap1 distribution ratio in the cytoplasm, ER and nucleus are 81.4% ± 13.1%, 13.7% ± 10.4% and 4.9% ± 3.2%, respectively. Asterisks indicate a CRM band, which we could not completely eliminate by adsorption. We envisage that this CRM is very rich in liver homogenates.


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  		Table 1  Yield of organelle fractions from mouse liver homogenates
 

Figure 6
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  		Figure 6  Subcellular localization of Keap1 in mouse liver cells. Organelle fractionation of mouse liver was performed by sucrose density gradient centrifugation. Organelle extracts of wild-type mouse liver (WT) were subjected to immunoblot analyses using ad-Keap1, anti-Nrf2 and anti-Cul3 antibodies. Anti-actin, Calreticulin, Golgin130, LaminB and Mitochondrial protein complex (Mito complex) antibodies were used as controls. Asterisk (*) indicates cross-reactive material. Similar experiments were performed using extracts from (B) BHA-treated (400 mg/kg body weight, 6 h) wild-type mice or (C) hepatocyte-specific Keap1-deficient mice (CKO), respectively.

 
To exclude cross-contamination among these fractions, we performed immunoblot analyses with antibodies against actin, LaminB, Calreticulin, Mito complex and Golgin130. The results demonstrate that the cross-contamination of the fractions is minimal. Since Keap1 forms E3 ubiquitin ligase complex with Cul3, we also examined co-localization of Keap1 with Cul3. Supporting our contention, Cul3 was detected in organelle fractions where Keap1 is expressed. On the contrary, Nrf2 was barely detected in these same fractions. This is most likely due to the proteasomal degradation pathway under homeostatic conditions.

To examine whether the electrophilic stress affects organelle localization of Keap1 in vivo or not, mice were orally administrated with an electrophile, butylated hydroxyanisole (BHA). Six hours after administration, liver homogenates were prepared from the mice and subjected to subcellular fractionation followed by immunoblot analysis with the ad-Keap1 antibody (Fig. 6B). Whereas BHA treatment promoted nuclear accumulation of Nrf2, this treatment did not affect localization of Keap1 in the cytoplasm, sER, rER or nucleus. Same experiments were performed on liver-tissue-specific Keap1-deficient mice as a negative control (Fig. 6C). Thus, results of immunocytological and immunoblot analyses utilizing newly prepared ad-Keap1 antibody demonstrate that the majority of Keap1 molecule is localized in the cytoplasm, and electrophilic stress induces nuclear accumulation of Nrf2 without altering the Keap1 subcellular localization.

Characterization of Keap1 complex in cytoplasm of mouse liver cells

We then investigated whether electrophilic stress affects cytoplasmic complex formation of Keap1. We monitored effects of BHA on the molecular size of Keap1-based complex in mouse liver cells. To this end, cytoplasmic extracts from mice treated with BHA were prepared and subjected to 5%–25% sucrose density gradient centrifugation, followed by immunoblot analyses with anti-Keap1, anti-Nrf2 and anti-Cul3 antibodies. As a control, we also performed similar experiment using purified Keap1 recombinant protein.

In this assay, purified Keap1 protein showed a fraction curve with approximately 180-kDa peak (Fig. 7A and B, broken line). Since calculated molecular weight of Keap1 is approximately 70 kDa, we considered that this peak coincides with a Keap1 homodimer as reported previously (McMahon et al. 2006). Under unstressed conditions, cytoplasmic Keap1 showed a broad peak curve rather than the symmetric curve of purified protein (the mass ranges from 140 to 300 kDa, Fig. 7A and B, arrows and open circles, respectively). From the data, we hypothesize that Keap1 is likely to form multiple complexes including the cytoplasmic homodimer.


Figure 7
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  		Figure 7  Characterization of Keap1 complex in cytoplasm of mouse liver cells. (A) Cytoplasmic extracts of mouse liver [wild-type (WT), BHA-treated mice (BHA) and hepatocyte-specific Keap1-deficient mice (CKO)] or purified recombinant Keap1 protein were fractionated by 5%–25% sucrose density gradient centrifugation. Fractions were subjected to immunoblot analyses with ad-Keap1, anti-Nrf2 and anti-Cul3 antibodies. Asterisk (*) indicates cross-reactive material; arrows indicate immunopositive signals of Keap1. For calibration of molecular size, ovalbumin (48.1 kDa), aldolase (158 kDa), catalase (232 kDa) and thyroglobin (669 kDa) were utilized. (B) Quantitated fraction curves of Keap1 (left) and Nrf2 (right). Open circles and squares indicate the fraction of liver cytoplasmic extracts of WT and BHA-treated mice, respectively. (n = 3) Broken line in the left graph represents the fraction curve of purified recombinant Keap1 protein.

 
Since barely detectable amount of Nrf2 exists in the cytoplasm under homeostatic conditions, we performed signal enhancement with biotin-streptavidin immunoblot analysis against anti-Nrf2 antibody and found that Nrf2 showed similar fractionation curve to that of Keap1. However, we could not detect Nrf2 protein in a similar experiment using Keap1-deficient mice (Fig. 7A, CKO), suggesting that cytoplasmic Nrf2 interacts with Keap1 and forms a protein complex. Without the presence of Keap1, Nrf2 seems to translocate rapidly into the nucleus. On the contrary, Cul3 was observed in fractions of much higher molecular size complex, but it co-existed with Keap1 and Nrf2 in the fraction around 300 kDa. This observation implies a possible formation of Keap1–Nrf2–Cul3 complex in the cytoplasm. We also examined effects of BHA treatment on these complexes in the liver cytoplasm. This treatment actually enhanced nuclear accumulation of Nrf2 (Fig. 6B). We also observed mild alteration of fractionation curves for Keap1 (Fig. 7B, open squares) within limitation of the resolution of this experiment.

Biochemical and genetic linkages between Nrf2, Keap1 and Cul3

In order to detect formation of Keap1–Nrf2–Cul3 complex within the cells, we carried out a series of immunoprecipitation experiments with anti-Nrf2 antibody (Fig. 8A). 293T cells were treated with MG132 to stabilize Nrf2 in the cytoplasm, and cell extracts were subjected to immunoprecipitation and immunoblot analyses. We observed that endogenous Keap1 was co-immunoprecipitated with endogenous Nrf2 (lane 3). On the contrary, whereas formation of the Keap1–Cul3 complex was previously reported (Furukawa & Xiong 2005), we could not co-precipitate the endogenous Cul3 with endogenous Keap1 in our experimental conditions using the anti-Nrf2 and anti-Keap1 antibodies (data not shown).


Figure 8
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  		Figure 8  Formation of Keap1–Nrf2–Cul3 complex. (A) Immunoprecipitation analysis shows the endogenous association of Keap1 and Nrf2. 293T cells were treated with MG132 for 12 h, and cell extracts were prepared and subjected to immunoprecipitation with anti-Nrf2 antibody. Immune complexes separated by SDS-PAGE were visualized by immunoblot analysis with anti-Nrf2 (SantaCruz, H-300), Keap1 and {alpha}-Tubulin. (B) Genetic linkage between Nrf2 and Cul3. MEF cells derived from Cul3 conditional knockout mouse embryos were used in this study (flox/flox, McEvoy et al. 2007). MEF cells were infected with adenovirus carrying Cre recombinase expression vector (Cre). As negative controls, adenovirus carrying LacZ or empty vector was utilized (LacZ and wt, respectively). The cells were simultaneously treated with tBHQ or MG132. Whole cell extracts were subjected to immunoblot analyses with anti-Nrf2, anti-Cul3 and anti-{alpha}-Tubulin antibodies.

 
As an alternative approach to verify the formation of Keap1–Nrf2–Cul3 complex, we executed a gene disruption experiment to ascertain the linkage between endogenous Nrf2 and Cul3. The rationale here is that since Cul3 forms a complex with Keap1 and does not directly interact with Nrf2 (Kobayashi et al. 2006), genetic evidence supporting the linkage between Nrf2 and Cul3 further strengthens the notion of Keap1–Nrf2–Cul3 complex within the cells. To this end, MEF cells derived from Cul3 conditional knockout mouse embryos (McEvoy et al. 2007) were infected with adenovirus expressing Cre recombinase (Cre). As a negative control, we utilized adenovirus carrying LacZ expression vector (LacZ) or empty vector (wt). Cell extracts were subjected to immunoblot analyses with anti-Nrf2, anti-Cul3 and {alpha}-Tubulin antibodies. While infection of adenovirus Cre decreased Cul3 protein amount (Fig. 8B, second panel, lane 4), it significantly stabilized Nrf2 protein (top panel, lane 4). This Cul3 deletion did not affect Keap1 protein levels (data not shown). Both tBHQ treatment and MG132 treatment stabilized Nrf2 protein more strongly than the Cul3 deletion (lanes 5–8). The reason why the Cul3 deletion does not fully stabilize the Nrf2 protein in the Cre-treated MEF cells is not clear at present. We speculate that it may be due to the presence of residual Cul3 protein or the presence of compensation by the other Cullin factors. Nonetheless, these results indicate that the Cul3 deletion did stabilize the Nrf2 protein in MEF cells. This genetic linkage between Nrf2 and Cul3 further supports our contention that Cul3 regulates the Nrf2 turnover through forming the Keap1–Nrf2–Cul3 complex.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we generated a new monoclonal anti-Keap1 antibody that can detect endogenous Keap1 and examined subcellular localization of endogenous Keap1 in cultured cells and mouse livers. We have confirmed authenticity of the antibody by immunoblot analysis utilizing the Keap1-deficient MEF cells. Our results demonstrated that endogenous Keap1 is mainly localized in the cytoplasm and this Keap1 localization is not influenced by the oxidative/electrophilic stress. These results support our contention that oxidative/electrophilic stress provokes nuclear accumulation of Nrf2 without altering cytoplasmic localization of Keap1.

We and other groups have shown that activation and nuclear accumulation of Nrf2 is a process of derepression of Nrf2 from Keap1 repression or Keap1-mediated proteasomal degradation (Zhang et al. 2004; Eggler et al. 2005; Kobayashi et al. 2006). Upon inactivation of Keap1 by oxidants or electrophiles, de novo synthesized Nrf2 proteins bypass the cytoplasmic Keap1–proteasome destruction gate and accumulate in the nucleus (as summarized in Fig. 9). In the Hinge and Latch model, electrophilic modifications of Keap1 cysteine residues provoke conformational changes and disrupt the Nrf2 binding to Keap1 (Tong et al. 2006b). The present results further support this model. Our results also conform the CAND1 model (Lo & Hannink 2006). In contrast, our results do not provide support for the hypothesis that majority of Keap1 may shuttle between the nucleus and cytoplasm in response to oxidative/electrophilic stress. We believe that this is most likely due to the differences in experimental conditions; while we are detecting endogenous Keap1, preceding experiments were looking at transfected Keap1 (Karapetian et al. 2005; Nguyen et al. 2005; Velichkova & Hasson 2005).


Figure 9
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  		Figure 9  Schematic diagram of cellular response against oxidative and electrophilic stresses. This study provides evidence for the cytoplasmic localization of Keap1 in both unstressed and stressed conditions. Under unstressed condition, Nrf2 is sequestered in the cytoplasm by Keap1 homodimer and rapidly degraded via the ubiquitin–proteasome system. Upon exposure to oxidants or electrophiles, Keap1 is inactivated but stays in the cytoplasm. In this situation, de novo synthesized Nrf2 protein can bypass the cytoplasmic Keap1–proteasome destruction gate ("Keap1 gate" in the figure) and accumulate in the nucleus. The present results support the model that electrophilic modifications of Keap1 cysteine residues provoke conformational changes of Keap1 and disrupt the Nrf2 binding to Keap1.

 
We previously reported that Keap1 requires actin filaments as a scaffold, based on cytological and functional analyses utilizing artificially over-expressed Keap1 protein and actin depolymerizing agents, respectively (Kang et al. 2004). Supporting this notion, Drosophila homolog of Keap1, Kelch, has been reported to be an actin binding protein (Xue & Cooley 1993; Robinson & Cooley 1997). Our present immunocytological experiments using anti-actin antibody, which recognizes both G- and F-actin, also showed co-localization of Keap1 and actin in the perinuclear region. In contrast, we found that the actin filaments visualized by phalloidin staining do not show clear-cut co-localization with the endogenous Keap1. The latter observation suggests that the association of Keap1 and actin cytoskeleton or cytoplasmic scaffold may not be direct or straightforward. Molecular basis for the perinuclear localization of Keap1 remains to be clarified.

We speculate that Keap1 molecules may need certain scaffolding during their stay in the cytoplasm, especially in the perinuclear region. While endogenous Keap1 molecules predominantly stay on the scaffold, some of the over-expressed Keap1 molecules may fail to bind to the scaffold and thus possibly shuttling between the nucleus and cytoplasm in a Crm1-dependent manner. Supporting this speculation, our present results showed the presence of endogenous Keap1 in the nucleus, albeit this amount is far less than that reported in the Keap1 over-expression experiments (Karapetian et al. 2005; Nguyen et al. 2005; Velichkova & Hasson 2005). There may also be a small amount of endogenous Keap1 protein unbound to the scaffold and these may be exhibiting nuclear–cytoplasmic shuttling characteristic. Our present study also demonstrates that Keap1 is stably localized in the cytoplasm even in the presence of electrophiles. This excludes the possibility of nuclear–cytoplasmic shuttling activity contributing to the electrophilic stress-mediated nuclear translocation and accumulation of Nrf2.

We found that the majority of the Keap1 is localized in the perinuclear region of the cytoplasm. This localization may allow Keap1 to effectively entrap de novo synthesized Nrf2 protein enroutes to the nucleus. In contrast, although mitochondria are well known as the major source of reactive oxygen species, Keap1 could not be identified in the organelle. On the contrary, we found that approximately 14% of Keap1 localizes in the ER. Phase 1 enzymes that initially metabolize xenobiotics are usually localized on the cytoplasmic surface of ER (Guengerich 1990), suggesting that Keap1 localized at this position may easily access the highly reactive phase 1 metabolites. In the organelle fractionation experiments with mouse liver, BHA treatment appeared not to affect the ER localization of Keap1, suggesting that ER localization is not essential for the stress response regulation by Keap1. pVHL, a repressor for hypoxia response transcription factors (e.g. HIFs), has also been reported to localize in ER (Schoenfeld et al. 2001). This observation is interesting as pVHL works to degrade HIFs in the cytoplasm through the formation of an E3 ubiquitin ligase complex with Cul2. The ER localization of Keap1 and pVHL may have some functional significance. However, critical roles that ER-localized Keap1 may play remain to be elucidated.

In a transfection over-expression assay, self-ubiquitination and degradation of Keap1 was found to be one of the ways to activate Nrf2 in response to electrophilic stress (Zhang et al. 2005). In contrast, immunoblot and immunocytological analyses in this study showed that DEM and BHA treatment of MEF cells and mouse, respectively, does not reduce endogenous Keap1 protein level. Our preliminary experiments indicate that endogenous Keap1 protein is relatively stable with a half-life of approximately 6 h in MEF cells under unstressed conditions and this half-life was not affected by electrophile treatment (A. Kobayashi et al., unpublished observation).

Sucrose density gradient experiments with mouse liver cells showed that Keap1 forms protein multiple complexes ranging from 140 to 300 kDa in the cytoplasm. Consistent with this observation, Keap1 homodimer exists in the range of 141 to 155 kDa in Cos7 cells (McMahon et al. 2006). Nrf2 exists in similarly ranged fractions as Keap1, but the peak position is located at a slightly higher-molecular-weight position than that of Keap1. While Cul3 co-exists with Keap1 in the fractions around 300 kDa, it mainly distributes in much higher-molecular-weight regions than those of Keap1 and Nrf2 range. Although the resolution of our density gradient centrifugation experiments has limitation to detect detailed complex exchange and this analysis is still preliminary, these observations further support the presence of the Keap1–Nrf2–Cul3 complex in the cells.

In summary, with the use of new monoclonal Keap1 antibody that specifically recognizes endogenous Keap1, we found that Keap1 localizes primarily in the cytoplasm, while a small amount of Keap1 is also localized in the nucleus and ER. This subcellular localization profile is reproducible in a biochemical subcellular fractionation analysis in mouse liver. The subcellular localization profile of Keap1 appears to remain similar upon treatment of cells or animals with electrophiles. Therefore, our results demonstrate that the majority of endogenous Keap1 localizes in the cytoplasm and that electrophiles promote nuclear accumulation of Nrf2 without altering the subcellular localization of Keap1.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Generation of rat monoclonal antibody against Keap1

Monoclonal antibodies against mouse Keap1 were raised by immunizing rats with purified recombinant protein expressed in Escherichia coli following the procedure as described (Dinkova-Kostova et al. 2002). After selecting hybridoma cells expressing antibodies, one positive clone for Keap1 (no. 144) was injected into mouse abdominal cavity to produce ascites, and the resultant antibody was purified from ascites using protein G column (Pierce).

Cell culture

Wild-type (MEF-WT) and Keap1 knockout (MEF-K0) mouse embryonic fibroblast cell lines were established as described previously (Wakabayashi et al. 2003). These cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; Sigma) supplemented with 10% heat-inactivated fetal bovine serum and 5% CO2 at 37 °C. In chemical challenge experiments, cells were treated with 100 µM dietylmaleate (DEM, Wako) or tert-butylhydroquinone (tBHQ, Sigma) as an electrophile or 20 ng/mL of Leptomycin B (LMB, Sigma) as a nuclear export inhibitor.

Antibodies and plasmids

Anti-Cul3, anti-actin, anti-Calreticulin and anti-Golgin130 antibodies were purchased from BD Biosciences. Anti-LaminB and anti-{alpha}-Tubulin antibodies were purchased from Sigma and an antibody against Mitochondrial complex II protein was from Mitosciences. We used anti-Nrf2 antibodies of newly prepared one (K. Itoh et al., unpublished observations) and commercially available ones (Santa Cruz, C-20 and H-300). Anti-mouse and rat IgG antibodies conjugated to horseradish peroxidase (HRP) were from Zymed, and biotinylated anti-rat IgG was from Vector Laboratories. Alexa546-conjugated anti-mouse IgG antibody, Alexa546-conjugated phalloidin and Alexa488-conjugated streptavidin were purchased from Molecular Probes. Bach2-GFP plasmid was kindly provided by Dr K. Igarashi.

Mice

Mice were housed in stainless steel cages in an animal room kept at 24 °C maintained in a 12-h light : dark cycle. All mice were kept in the specific-pathogen-free conditions, and were treated according to the regulations of The Standards for Human Care and Use of Laboratory Animals of the University of Tsukuba and Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan.

Organelle fractionation of mouse liver homogenates

Organelle fractionation was performed following the method as described previously (Touster et al. 1970; Fleischer & Kervina 1974) with slight modification. Livers from 24-h-fasted ICR female mouse treated with or without 400 mg/kg body weight butylated hydroxyanisole (BHA, Sigma; LD10) for 6 h were used. Hepatocyte-specific Keap1 knockout mice (CKO, Okawa et al. 2006) were used as the Keap1-deficient liver. Approximately 1 g of fresh liver was sliced into small pieces and washed with ice-cold phosphate-buffered saline (PBS) twice to remove blood, and homogenized with nine volumes of 0.25 M sucrose in 10 mM HEPES (pH 7.5) with Dounce homogenizer on ice. The homogenate was filtered through gauze and successively centrifuged at 600 g for 10 min, 8000 g for 10 min and 105 000 g for 60 min at 4 °C. The resuspended extract from the sediment was purified to the organelle fractions of nuclei, mitochondria, and smooth and rough endoplasmic reticulum (sER and rER). The final supernatant of 105 000 g centrifugation was used as the cytoplasmic fraction. Golgi apparatus was isolated according to the reported method (Hamilton et al. 1991). Yields (%) of purified organelle fractions were calculated as the percentage of protein amounts of each fraction in that of total homogenate (Table 1).

Sucrose density gradient centrifugation of mouse liver cytoplasm

Cytoplasmic fraction (500 µL) prepared as above was loaded onto 5%–25% sucrose gradient solution (25 mL) and centrifuged at 100 000 g for 24 h at 4 °C. After centrifugation, 1-mL fractions were collected from the top, and each fraction was subjected to immunoblot analysis using anti-Keap1, anti-Nrf2 and anti-Cul3 antibodies. Purified Keap1 protein expressed by E. coli was provided by Dr Tong (Tong et al. 2006a) as a Keap1 protein standard. Calibration of molecular size was done under the similar condition by using size marker proteins (Amersham), ovalbumin (48.1 kDa), aldolase (158 kDa), catalase (232 kDa) and thyroglobin (669 kDa).

Immunoblot analyses

Cell extracts from MEF-WT and MEF-K0 cell lines were prepared using the cell extraction kit (Active Motif) according to the manufacturer's instructions. Cell extracts and organelle fractions from mouse livers were solubilized in 2x Laemmli sample buffer [125 mM Tris–HCl (pH 6.8), 10% 2-mercaptoethanol, 4% sodium dodecyl sulfate, 10% sucrose and bromophenol blue; Laemmli 1970)] and boiled for 5 min. About 40 µg protein of each fraction was separated on 8% SDS-PAGE. In the case of cytoplasmic extracts, the fractions were 10-fold concentrated by trichloroacetic acid precipitation. Thirty microliters of samples were loaded onto 8% SDS-PAGE. After electrophoresis, proteins were electrotransferred to PVDF membranes (Millipore). The membranes were blocked with 3% non-fat milk in PBS-T (0.05% Tween 20 in PBS), treated with primary antibodies and then HRP-conjugated secondary antibody. Protein signals were developed with ECL Plus reagents (Amersham). For quantification, densitometric scannings of the bands of purpose were quantitated by NIH image (version 1.34r).

Immunofluorescent staining

Cells grown on chamber slides were washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.05% Triton X-100 for 20 min and washed 3 times with PBS. After blocking with 1% BSA and 5% rabbit serum in PBS for 1 h, cells were treated with the anti-Keap1 antibody (200-fold dilution, described as follows) or anti-Nrf2 antibody (100-fold dilution) at 4 °C. To eliminate nonspecific reaction of anti-Keap1 antibody, the antibody was pre-incubated with whole cell extracts of MEF-K0 cells for 1 h. After centrifugation at 5000 g for 5 min, the supernatants were used for experiments as the adsorbed-Keap1 antibody (ad-Keap1 antibody).

In double immunocytochemical staining experiment using the ad-Keap1 antibody, the cells were simultaneously treated with each 500-fold diluted antibody against organelle marker proteins, Calreticulin (ER), Golgin130 (Golgi) or Mitochondria complex (mitochondria). Alexa546-conjugated phalloidin (100-fold dilution) and anti-actin antibody (500-fold dilution) were used for actin filaments (F-actin) and total actin (G-actin and F-actin) staining, respectively. The cells were washed 3 times with PBS, and treated with secondary antibodies; biotinylated anti-rat IgG antibody (200-fold dilution) for anti-Keap1 or anti-Nrf2 antibody, and Alexa546-conjugated anti-mouse IgG antibody (500-fold dilution) for anti-organelle marker protein antibodies. The cells were then washed and treated with 1000-fold diluted mixtures of Alexa488-conjugated streptavidin and Hoechst33342 (Dojindo) for 1 h. After rinsing with PBS, antifade fluorescent mounting medium (DAKO) was dropped on each slide and cover glass was mounted. To monitor Crm1 inhibition, Bach2-GFP plasmids were transfected by Lipofectamine 2000 (Invitrogen) in NIH3T3 cells as a positive control. Fluorescence in the cells was acquired using confocal laser scanning microscopy (LSM510 Meta, Zeiss).

Immunoprecipitation

293T cells were treated with MG132 (final 2 µM) for 12 h and lysed with the NP40 buffer [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 50 mM NaF, 0.5% NP-40, 1 mM DTT, Protease inhibitor (Roche) and 10 µM MG132)]. The cell extracts were reacted with anti-Nrf2 antibody (SantaCruz, C-20) or normal rabbit IgG for 5 h. After centrifugation, immunocomplexes in the supernatants were precipitated with ProteinG sepharose (Amersham), solubilized with the Laemmli sample buffer and separated on 8% SDS-PAGE. We performed immunoblot analysis by utilizing monoclonal rat anti-Keap1 and anti-Nrf2 (SantaCruz, H-300) antibodies.

Cul3 gene deletion experiment

MEF cells derived from Cul3 conditional targeting mice (flox/flox) were established as described (McEvoy et al. 2007). To delete Cul3 gene, MEF cells cultured in DMEM supplemented with 5% FCS were infected with adenovirus carrying a Cre recombinase expression vector (AxCANCre, Takara) at M.O.I. 10 for 12 h. As negative controls, adenovirus carrying LacZ or empty vector was utilized (AxCAiLacZ and AxCAwt, respectively). After addition of FCS to the final concentration of 10%, the cells were cultured for 42 h and then treated with tBHQ (final 100 µM) or MG132 (a proteasome inhibitor, final 2 µM) for further 5 h. Whole cell extracts were prepared from the cells with 2x Laemmli sample buffer and subjected to immunoblot analyses with anti-Nrf2, Cul3 and {alpha}-Tubulin antibodies.


   Acknowledgements
 
We are grateful to Drs Kazuhiko Igarashi for a plasmid, Hiromi Okawa and Keiko Taguchi for Keap1 CKO mice, and Kit Tong for Keap1 recombinant protein. We also thank members of Yamamoto's laboratory for experimental advice and discussion. This work was supported by grants from JST-ERATO (M.Y.), the Ministry of Education, Culture, Sports, Science and Technology of Japan (A.K. and M.Y.) and Inamori Foundation (A.K.).


   Footnotes
 
Communicated by: Shunsuke Ishii

* Correspondence: E-mail: masi{at}tara.tsukuba.ac.jp,akirak{at}mail.tains.tohoku.ac.jp


   References
Top
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 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 6 March 2007
Accepted: 11 July 2007




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