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Originally published In Press as doi:10.1074/jbc.M310492200 on February 25, 2004

J. Biol. Chem., Vol. 279, Issue 18, 19209-19216, April 30, 2004
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Cell Density Regulates Intracellular Localization of Aryl Hydrocarbon Receptor*

Togo Ikuta{ddagger}, Yasuhito Kobayashi§, and Kaname Kawajiri{ddagger}||

From the {ddagger}Research Institute and §Department of Pathology, Saitama Cancer Center, Saitama, 362-0806, Japan and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama 332-0012, Japan

Received for publication, September 22, 2003 , and in revised form, January 23, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that plays a role as an intracellular mediator of the xenobiotic signaling pathway. AhR contains signals for both nuclear localization and nuclear export (NES). The objective of this study was to demonstrate how AhR intracellular distribution was regulated physiologically in cells. We found that cell density, but not the cell cycle, influenced the subcellular distribution of AhR in a keratinocyte cell line, HaCaT: AhR was predominantly nuclear at sparse cell densities, both nuclear and cytoplasmic at subconfluence, and predominantly cytoplasmic at confluence. Stable transfectants of HaCaT carrying a reporter gene fused with xenobiotic responsive element showed an association between xenobiotic responsive element-mediated transcription and AhR relocalization. Leptomycin B promoted nuclear accumulation of AhR irrespective of cell density, suggesting that this alteration may be because of a change of the regulation of the nuclear export of AhR. We found that Ser-68 in the NES of AhR was phosphorylated after nuclear accumulation of activated AhR and the nuclear export of a chimeric GST-AhR-GFP fusion protein was suppressed by substitution of a serine residue (Ser-68) to aspartic acid, which mimics the negative charge of phosphorylation. This novel cell density-dependent AhR relocalization was affected by exposure to SB203580, okadaic acid, and low Ca2+ concentrations. These findings strongly suggest that cell density regulates the intracellular localization and function of AhR, because of modulation of nuclear export activity. The p38 MAPK-mediated phosphorylation of the NES and its dephosphorylation, regulated by cell-cell contact signals, may have pivotal roles in the novel AhR relocalization.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The aryl hydrocarbon receptor (AhR)1 is a ligand-activated transcription factor belonging to the basic-helix-loop-helix (bHLH)/PER-ARNT-SIM homology region (PAS) family. AhR is an intracellular mediator of the xenobiotic signaling pathway and remains in the cytoplasm as a complex with Hsp90 (1, 2), p23 (3, 4), and XAP2, also known as ARA9 or AIP (5-7). When environmental contaminants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and 3-methylcholanthrene (MC) bind to AhR, the receptor complex subsequently translocates into the nucleus, where AhR dissociates from Hsp90 to bind to the heterodimer partner AhR nuclear translocator (ARNT) (8-10). In the nucleus, AhR/ARNT binds to xenobiotic responsive elements (XRE) (11), which are enhancer DNA elements located in the 5'-flanking region of target genes. In addition to the genes for xenobiotic metabolizing enzymes such as CYP1A1 or glutathione S-transferase Ya, several genes such as p27Kip1 (12), AhR repressor (13), Bax (14), and DNA polymerase {kappa} (15) have also been identified as target genes for the AhR/ARNT system.

Analyses of AhR-deficient mice have shown that AhR is involved directly in chemical carcinogenesis caused by benzo-(a)pyrene (16), in teratogenesis such as cleft palate (17), hepatotoxicity, and immunosuppression because of thymic involution caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (18). Moreover, AhR may participate in the resolution of fetal vascular structures during development (19), and in the normal ovarian germ-cell dynamics of mice (20). It is also likely that AhR is involved indirectly in adverse biological responses because of endocrine disrupters in the environment by a molecular interaction of activated AhR and nuclear steroid hormone receptors such as the estrogen receptor (21, 22). However, despite an extensive study to elucidate the various roles of AhR, little is known of the physiological function of AhR because of a paucity of information of its endogenous ligands (23, 24).

We previously identified and characterized a nuclear localization signal (NLS) of AhR as amino acid residues 13-39 (25), the bipartite core that overlapped with domains of DNA or Hsp90 binding (26, 27). Moreover, we also showed that AhR has a leucine-rich nuclear export signal (NES) (28, 29) that is dependent on chromosome region maintenance 1 composed of amino acid residues 55-75 in helix 2 (25), the hydrophobic core involved in heterodimer formation with ARNT. Using these two signals, AhR shuttles between the cytoplasm and the nucleus of a cell (30), and the intracellular localization of AhR may be regulated by the masking and unmasking of NLS and NES with some interacting proteins. In general, the intracellular distribution of nucleo-cytoplasmic shuttling proteins such as transcription factors is also determined by the balance of nuclear import and export activity (31-34), which is specifically regulated by various physiological or environmental signals. Molecular modulations of phosphorylation or dephosphorylation (35), especially close to the NLS (36, 37) or NES (38, 39), by the network of signal transduction cascades, are frequently known to regulate the intracellular distribution of such proteins, resulting in spatial and temporal-specific gene regulation mediated by transcription factors.

To elucidate how the nuclear import and export of AhR is regulated physiologically in cells, we used a human keratinocyte cell line, HaCaT, which endogenously expresses both AhR and ARNT (40). Keratinocytes are a major component of the epidermis, which is the outermost layer of skin and protects the inner organs from various stimuli of xenochemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (41). The most prominent effect of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in humans is the development of chloracne, a persistent acneiform condition characterized by comedones and cysts of the skin caused by a disturbance of the normal differentiation process of keratinocytes (42). Because growth or differentiation of cultured keratinocytes is regulated in part by cell density, we examined the subcellular localization of AhR in HaCaT cells under various cell density conditions, and found that the cell density, but not the cell cycle, influenced not only the intracellular localization of AhR but also AhR transcriptional activation in HaCaT cells. It is likely that cell density may regulate the AhR relocalization partly by the p38 mitogen-activated protein kinase (MAPK)-dependent phosphorylation and dephosphorylation of the Ser-68 in the NES.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Cell lines used for this study were the human keratinocyte HaCaT kindly provided by Dr. N. Fusenig (43) and Madin-Darby bovine kidney cells. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum at 37 °C with 5% CO2 atmosphere. HaCaT cells were also cultured in DMEM in the presence of EGTA (4 mM), SB203580 (50 µM) (Calbiochem-Novabiochem Co., San Diego, CA), U0126 (10 µM) (Promega), and okadaic acid (200 nM) (Calbiochem-Novabiochem). To reduce the Ca2+ concentration, HaCaT cells were also placed in S-MEM (Invitrogen) supplemented with 10% fetal calf serum that was dialyzed against phosphate-buffered saline.

Plasmids—The XRE-driven pX4TK-Luc, containing four copies of the XRE fused with the TK promoter and luciferase, was kindly provided by Dr. J. Mimura (13). The XRE-TK fragment was subcloned into the HindIII site of pEGFP-1 (Clontech Co., Palo Alto, CA) to produce pX4TK-GFP. The construction of the GST-AhR-GFP fusion gene was carried out as described previously using GST-GFP2 vector (9). Replacement of amino acid residues was performed using a QuikChange site-directed mutagenesis kit (Stratagene) to generate AhR mutants, and each mutant was confirmed by sequencing.

Immunofluorescence of AhR in HaCaT Cells—HaCaT cells were cultured with sparse distribution, subconfluence or confluence on coverslips. Leptomycin B (LMB) (5 ng/ml) (Sigma) or MC (1 µM) (Wako Pure Chemical Industries, Ltd., Osaka) were added to the medium for 2 h before fixation. For immunofluorescence, an anti-AhR antibody (BIOMOL, Plymouth Meeting, PA) and an anti-rabbit IgG antibody coupled with fluorescein isothiocyanate were used for the primary and secondary antibodies, respectively. Incorporation of bromodeoxyuridine (BrdUrd) was carried out as described previously (44) using an anti-BrdUrd antibody (BIOSYS SA, Compiegne, France) and a goat antibody to rat IgG conjugated with rhodamine (Cappel, Durham, NC).

Preparation of Nuclear and Cytosolic Fractions—HaCaT cells on culture dishes were washed with phosphate-buffered saline and the cells were scraped off followed by centrifugation to collect the cells. The cell pellets were lysed in buffer (10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM NaCl, 10% glycerol, 1% Nonidet P-40) and incubated on ice for 10 min. The lysates were homogenized and centrifuged at 1,000 x g for 5 min. After centrifugation, the supernatants were pooled as the cytosolic fraction and the resulting pellets were resuspended in the lysis buffer as the nuclear fraction. The same protein concentrations of cytosolic and nuclear fractions were used for immunoblotting analysis.

Western Blotting—Samples for analysis by SDS-PAGE were lysed in SDS-lysis buffer, and the proteins were separated on appropriate concentrations of polyacrylamide gel followed by transfer to a nitrocellulose membrane. The probe antibodies used were as follows: anti-AhR antibodies, anti-ARNT antibodies (NOVUS, Littleton, CO), anti-CYP1A1 antibodies (Chemicon, Temecula, CA), anti-involucrine antibodies (Sigma), anti-phospho-p38 MAPK antibodies (Promega), and anti-AhR-(61-74)-pS68 antibodies, which was produced from chemically synthesized AhR-(61-74) containing phosphoserine 68 (Immuno-Biological Laboratories Co., Gunma, Japan). The membrane was incubated with secondary antibodies conjugated with alkaline phosphatase and visualized by an AP Conjugate Substrate Kit (Bio-Rad).

Cloning of Stable Transfectants—HaCaT cells were co-transfected with pSVneo and pX4TK-Luc or pX4TK-GFP by the Lipofectin method according to the manufacturer's instructions (Invitrogen). pGL3 basic (Promega) or pEGFP-1 plasmids were used for control experiments. After transfection, the cells were replated and incubated with a selection medium containing 0.8 mg/ml geneticin.

Luciferase Assay—Stable HaCaT transformants containing pX4TKLuc were seeded in a 12-well plate and cultured to the appropriate cell density. The luciferase activity was determined using a Luciferase Assay System (Promega) according to the manufacturer's instructions, and was adjusted to the protein content of each sample used. The protein concentrations of the lysates were determined by the BCA protein assay reagent (Pierce).

In Vitro Wound Healing Model—Stable HaCaT transformants containing pX4TK-GFP were cultured to confluence, partly scraped off, and then the scraped wound was allowed to heal in vitro (45). GFP expression was examined by fluorescent microscopy.

Microinjection Analysis—GST-AhR-(55-75)-GFP vector was introduced into Escherichia coli strain BL21, and the expressed fusion proteins were purified as described previously (9). The purified preparation of GST-AhR-(55-75)-GFP was injected into Madin-Darby bovine kidney cell nuclei along with Texas Red-labeled bovine serum albumin, which was co-injected to ensure a clean nuclear injection without leakage (25). After microinjection, the cells were incubated at 37 °C for 30 min before fixation with 4% formaldehyde. The injected protein was localized by fluorescence microscopy.

Immunohistochemistry of Mouse Livers—Male C57BL/6 mice received an intraperitoneal injection of MC (25 mg/kg of body weight) dissolved in corn oil each day for three successive days. Sections of frozen liver from MC-treated or corn oil-treated control mice were fixed with 4% paraformaldehyde for 10 min, and were blocked with goat serum. Colorimetric detection was performed by the protocol of the streptavidin method, using anti-AhR-(61-74)-pS68 antibody and anti-rabbit IgG antibody (Nichirei, Tokyo, Japan) as primary and secondary antibodies.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Density-dependent AhR Relocalization in HaCaT Cells—To elucidate how nuclear import and export of AhR is regulated physiologically in cells, we examined the subcellular localization of AhR in HaCaT cells under the conditions of sparse distribution, subconfluence or confluence (Fig. 1A, upper panels). HaCaT cells were seeded at 5 x 104 cells on a 3-cm culture dish with coverslip on day 0, and were cultured for the numbers of days indicated and then fixed. On day 2, when the cells were sparsely distributed, AhR was localized predominantly in the nucleus. On day 4, when the cells were maintained in subconfluence, the fluorescent signal of AhR was distributed equally both in the cytoplasm and the nucleus. However, when the cells were further grown to confluence on day 6, the immunostained AhR was localized predominantly in the cytoplasm. Using immunoblotting analysis, it was also shown that the relative concentration of AhR in the nucleus was gradually decreased in proportion to the cell density (Fig. 1B).



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  		FIG. 1.
The intracellular localization of AhR is influenced by the cell density of HaCaT cells. A, subcellular localization of AhR in HaCaT cells. HaCaT cells grown on coverslips were untreated (Control), treated with 5 ng/ml LMB or 1 µM MC for 2 h before fixation on days 2, 4, and 6. The distribution of AhR was observed by immunocytochemical staining with anti-AhR antibody followed by fluorescein isothiocyanate-labeled anti-rabbit IgG. Immunofluorescence was visualized and captured with fluorescence microscopy. B, subfractionation of HaCaT cells followed by immunoblot analysis of AhR. Cytosolic (C) and nuclear (N) fractions of HaCaT cells were isolated as described under "Experimental Procedures." AhR was detected by Western blot analysis using anti-AhR antibody in the same protein concentration of each fraction. C, effect of Ca2+ concentration on AhR localization. Confluent HaCaT cells were treated with either 4 mM EGTA (DMEM + EGTA) or calcium-deficient medium (S-MEM) for 2 h before fixation. AhR was visualized by immunofluorescence as described in A, and images were captured under a fluorescence microscope. D, AhR relocalization was not associated with the cell cycle. Asynchronous HaCaT cells with sparse distribution were treated with 50 µM BrdUrd for 5 min before fixation. AhR localization and incorporation of BrdUrd were analyzed by double staining with anti-AhR and anti-BrdUrd antibodies followed by incubation with fluorescein isothiocyanate-labeled anti-rabbit IgG (AhR) and rhodamine-conjugated anti-rat IgG (BrdUrd) secondary antibodies, respectively.

 
Next, we examined the effect of LMB, a specific inhibitor of nuclear export, on the intracellular localization of AhR under conditions of varying cell density (Fig. 1A, middle panels). Treatment with LMB for 2 h resulted in a promoted nuclear accumulation of AhR even with high cell density, suggesting that both nuclear import activity and some potential endogenous ligands for the AhR exist even when the HaCaT cells are confluent. These observations led us to consider that AhR in HaCaT cells alter its intracellular distribution according to cell density, and this alteration may be because of a change of the regulation of the nuclear export of AhR.

Because cell-to-cell communication of the HaCaT cells seems to be crucial in the novel AhR relocalization by cell density, we studied the effect of Ca2+ depression on the AhR distribution. When cells were grown to confluence on day 6 in DMEM followed by another 2-h incubation in the absence or presence of the Ca2+ chelator EGTA, or incubated with Ca2+-deficient S-MEM, a clear nuclear localization of AhR was observed in the cells, in which Ca2+-dependent cell-to-cell contact was disrupted by a low concentration of Ca2+ (Fig. 1C). The disrupted cell-cell interaction was confirmed by observation of subcellular localization of E-cadherin (data not shown). To study whether the AhR nuclear localization in sparse cell density was partly because of a distribution specific to the cell cycle, S-phase cells were identified among asynchronous HaCaT cell populations on day 2 by briefly labeling with BrdUrd. As shown in Fig. 1D, a predominant nuclear localization of AhR was not associated specifically with either BrdUrd positive (S-phase) or negative cells, indicating that AhR relocalization was dependent on the cell density but not on a particular event specific in the cell cycle.

AhR-mediated Transcription Is Regulated by Cell Density—To investigate whether or not altered intracellular localization of AhR because of cell density reflects AhR-mediated transcription, we established stable transfectants of HaCaT cells by introducing pX4TK-Luc, which contains four copies of the XRE fused with luciferase (Fig. 2A). Whereas the transfectant that contained the control plasmid TK-Luc did not show any activity, a transfectant containing pX4TK-Luc, Clone XRE.3, displayed luciferase activity even in the absence of exogenous ligand. The luciferase activity of XRE.3, however, was only doubled by MC treatment when compared with the activity of untreated cells. This may be explained because of the small effect of MC on the nuclear translocation of AhR in HaCaT cells (Fig. 1A, lower panels). Fig. 2B shows the XRE-driven luciferase activity in relation to the number of cultured XRE.3 cells. When XRE.3 was inoculated at 5 x 104 cells on a 3-cm culture dish on day 0, the cells were grown to reach subconfluence on day 4, and a maximal luciferase activity was observed as shown in Fig. 2B. When the cells were further grown to reach confluence on day 6, when AhR was predominantly distributed in the cytoplasm (Fig. 1A), the luciferase activity was reduced to basal levels. Another clone, XRE.11, yielded similar results (data not shown).



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  		FIG. 2.
XRE-mediated transcription is influenced by the cell density of HaCaT cells. A, isolation of stable transfectants carrying XRE-driven pX4TKLuc. A clone transfected with pGL3-basic and a clone designated XRE.3 carrying pX4TK-Luc were treated either with Me2SO (-) or 1 µM MC for 16 h. The luciferase activity is expressed as the mean ± S.D. of three experiments. B, XRE-mediated transcriptional activation in relation to cell density of HaCaT cells. Clone XRE.3 was seeded at 5 x 104 cells on a 3-cm culture dish. Both the number of cells in each dish (closed circle) and the luciferase activity (open circle) were shown in relation to the duration of cell culture. The luciferase activities are indicated as values normalized by the protein content of each sample. The results are given as the mean ± S.D. value of three experiments. C, cell density-dependent transcriptional activity mediated by XRE. XRE.3 cells (0.4-3.0 x 106 cells on each 3-cm dish) were cultured at 37 °C for 26 h, and the luciferase activities were assayed. D, reactivation of the luciferase activity by re-seeding. A confluent culture of XRE.3 cells in a 3-cm dish was trypsinized and re-plated to a 6-cm dish. After the indicated incubation, luciferase activity was assayed. E, immunoblot analysis of HaCaT cells derived from samples with different cell-density conditions: sparse (lane 1), subconfluent (lane 2), or confluent (lane 3). The nitrocellulose membrane was probed with anti-AhR, anti-ARNT, anti-CYP1A1, or anti-involucrine antibodies, respectively.

 
The decreased XRE-mediated transcription under high cell density was confirmed by an experiment in which different numbers of cells were inoculated on day 0 (Fig. 2C). When a large number of XRE.3 cells (1.5-3.0 x 106) were seeded in a dish and incubated for 26 h, the luciferase activity was decreased to 50% compared with when only a small number of cells (0.4 x 106) were inoculated. We also studied the effect of reseeding the confluent cells on the reactivation of luciferase (Fig. 2D). When XRE.3 cells in confluence on a 3-cm culture dish (day 6) were harvested with trypsin treatment followed by reseeding of 1.8 x 106 cells on a 6-cm culture dish on day 0, the luciferase activity increased.

Fig. 2E shows a representative profile of expression of the AhR/ARNT system in combination with a target gene product CYP1A1 under varying cell density. As shown in this figure, the expression levels of AhR or ARNT did not change under different conditions of cell density. Although CYP1A1 was expressed under sparse or subconfluent conditions, the down-regulation of CYP1A1 was observed in the confluent state, similar to the decrease of luciferase activity (Fig. 2B). On the contrary, the expression of involucrine, a specific differentiation marker of keratinocytes (45), increased gradually with increased cell density. Thus, it is likely that the altered XRE-mediated luciferase activity because of cell density resulted from a novel redistribution of AhR, but not from a different expression level of AhR.

XRE-mediated Transcription in a Wound-healing Model—To visualize the transcriptional activity of the AhR/ARNT system in cells, stable transfectants of HaCaT with a pX4TK-GFP expression plasmid containing XRE fused with GFP were introduced (Fig. 3A). Fig. 3B shows a representative profile of the XRE-mediated GFP expression of a transfectant, Clone 14, under various conditions of cell density. The GFP was expressed clearly either on days 1 or 3, when the cells were maintained at low density. In contrast, the green fluorescence gradually diminished after day 5 when the cells grew to confluence or superconfluence (days 7 or 9).



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  		FIG. 3.
XRE-mediated transcription in an in vitro wound healing model of HaCaT cells. A, isolation of stable transfectants carrying XRE-driven pX4TK-GFP. A representative expression profile of GFP transfected with pEGFP-1 control (vector) or pX4TK-GFP (Clone 14) are indicated. B, expression of GFP in relation to cell density. Clone 14 was seeded on coverslips and cultured for the various times indicated at 37 °C before fixation. C, expression of GFP in an in vitro wound healing model. A monolayer of Clone 14 with superconfluent cells (day 9) was injured in part by scraping followed by further incubation for 3 days. Images were captured under phase-contrast (a) and fluorescence microscopes (b).

 
To further investigate the relation between cell density-dependent AhR redistribution and transcriptional activity, we carried out an experiment using an in vitro wound-healing model, which requires coordinated activities of keratinocytes such as proliferation, migration, cell-to-cell adhesion, and differentiation (46). Whether or not the cell scraping resulted in a reactivation of GFP at the injury site, the adhering superconfluent monolayer of cells displaying no fluorescence (Fig. 3B, day 9) was scraped off in part and then the cells were further cultured for 3 days. Cells initiated migration from the edge to fuse both injured edges within 24 h after injury, but GFP reactivation was not initiated (data not shown). After 3 days of culture, however, the expression of GFP was evident among the cells immediately bordering the wound margin (Fig. 3C). These observations clearly suggest that XRE-mediated transcription was selectively activated at the wound edges, where the cells seem to be loosely associated. These results support the idea that XRE-mediated transcription is influenced by cell density and suggest that the AhR/ARNT system functions in this process.

Phosphorylation of Ser-68 within NES Inhibits Nuclear Export of AhR—Because cell density-dependent AhR relocalization may be because of a change of the regulation of the nuclear export of AhR, we examined which amino acid residues(s) of AhR-NES are important for the modulation of nuclear export activity. In addition to the functional NES located in the bHLH domain of AhR (25), a novel NES-like motif of AhR-(218-230) in the PAS domain was reported (47), but direct nuclear export activity of the sequence has not been shown. Fig. 4A shows a comparison of the functional NES core of AhR-(64-73) with the corresponding region of proteins having nuclear export activity. Among them, we developed great interest in Ser-68 or Ser-73, because some transcription factors possess a Ser or Thr in their NES, which is often phosphorylated in the activated or inactivated forms. An alanine substitution mimics the dephosphorylated form of the NES, whereas an aspartic acid substitution mimics the negative charge of the phosphorylated side chain (48). When injected into the nucleus of Madin-Darby bovine kidney cells, the wild type AhR-(55-75) fused with GST-GFP was found to be present in the cytoplasm and was excluded almost completely from the nucleus within 30 min. In contrast, coinjected Texas Red-labeled bovine serum albumin was localized in the nucleus. When injected into the nucleus, mutated AhR-(55-75) (S73D) retained nuclear export activity, whereas AhR-(55-75) (S68D) was unable to pass through the nuclear pore and remained in the nucleus (Fig. 4B), suggesting that phosphorylation of Ser-68 inhibits nuclear export activity.



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  		FIG. 4.
Phosphorylation of Ser-68 within NES inhibits nuclear export of AhR. A, comparison of the consensus NES of AhR with the corresponding region of proteins having nuclear export activity. B, nuclear export activity of wild-type (WT) AhR-(55-75) or mutants thereof having mutations at S68D or S73D. Purified GST-AhR-(55-75)-GFP proteins were microinjected into the nuclei of Madin-Darby bovine kidney cells with co-injected Texas Red-labeled bovine serum albumin. After incubation, the injected proteins were localized by fluorescence microscopy. C, phosphorylation at Ser-68 in the NES of AhR has occurred in the nucleus. a, specificity of anti-AhR-(61-74)-pS68 antibody. The chemically synthesized peptides of AhR-(61-74) containing phosphoserine 68 () or Ser-68 ({blacktriangleup}) were dotted to the nitrocellulose membrane followed by incubation with anti-AhR-(61-74)-pS68 antibody. The antibody used was specifically bound to the peptide of phosphorylated but not unphosphorylated AhR-(61-74). b, immunoblot analysis of HaCaT cells using anti-AhR antibody or anti-AhR-(61-74)-pS68 antibody. Cell extracts of HaCaT cells from confluent conditions cultured with DMEM (D), or from cells incubated with S-MEM for a further 10 h (S), were used. The nitrocellulose membrane was probed with anti-AhR antibody (BIOMOL) or with anti-AhR-(61-74)-pS68 antibody. c, immunohistochemical staining of AhR on frozen liver sections of MC-treated mice. Sections of frozen liver tissues from control corn oil-treated or MC-treated mice were incubated with anti-AhR-(61-74)-pS68 antibody. Colorimetric detection was performed by the streptavidin method. The liver sections were also stained with non-immunized rabbit IgG (data not shown). D, effect of SB203580 or U0126 on AhR localization. a, HaCaT cells in the subconfluent condition were treated with SB203580 (50 µM) or U0126 (10 µM) for 2 h, and AhR was visualized by immunofluorescence as described in the legend of Fig. 1. b, XRE-mediated reporter assay in the presence of SB203580. Confluent HaCaT cells cultured with DMEM were incubated with calcium-deficient medium (S-MEM) for another 5 h in the presence of U0126 or SB203580. XRE-mediated reporter assay was carried out as described in the legend to Fig. 2. c, analysis of p38 MAPK phosphorylation and CYP1A1 induction. Confluent HaCaT cells cultured with DMEM were incubated with S-MEM for the times designated. Cell lysates were analyzed by immunoblotting with antibodies to phospho-p38 MAPK and CYP1A1. E, effect of okadaic acid on AhR localization. HaCaT cells in the confluent condition were treated with okadaic acid (200 nM), and AhR was visualized by immunofluorescence as described in the legend to Fig. 1.

 
We produced specific antibodies against AhR-(61-74) containing phosphoserine 68 (anti-AhR-(61-74)-pS68 antibody) to test whether or not Ser-68 of the AhR was phosphorylated in the nucleus. Using in vitro binding of the antibody to phosphorylated or unphosphorylated AhR-(61-74) peptides, we confirmed that the anti-AhR-(61-74)-pS68 antibody specifically reacted with the phosphorylated peptide of AhR-(61-74) but not with the unphosphorylated AhR-(61-74) (Fig. 4C, a). When HaCaT cells were grown to confluence in DMEM, followed by another 10 h incubation with Ca2+-deficient S-MEM, a clear nuclear localization of AhR was observed as shown in Fig. 1C. After immunoblot analysis was carried out using anti-AhR antibody as a probe, a single band with adequate molecular size of AhR was observed in cell extracts from DMEM culture, whereas an additional band with slow migration (arrowhead) was observed from S-MEM. In contrast, when probed with anti-AhR-(61-74)-pS68 antibody, no band was observed in extract from DMEM, and only a single band with slow mobility shift (arrowhead) was observed in extract from S-MEM (Fig. 4C, b). One possible explanation for the large molecular weight of AhR detected in extracts from S-MEM is molecular modulation of phosphorylation. In addition, immunohistochemical staining of AhR was carried out on frozen liver sections prepared from corn oil-treated or MC-treated mice (Fig. 4C, c). In MC-treated livers, immunostaining of AhR by anti-AhR-(61-74)-pS68 antibody was clearly observed in the nuclei, whereas no positive staining was observed in the nuclei of cells from control corn oil-treated livers. These results led us to conclude that Ser-68 in the NES was phosphorylated in the nucleus.

A role for the p38 MAPK signaling pathway in suppressing the nuclear export of the p53 or estrogen receptor {alpha} has been described (38, 39). In this context, it is likely that Ser-68 within the NES of the AhR is the corresponding amino acid that is modified by the p38 MAPK system (Fig. 4A). If the molecular modulation of the AhR-NES by the activated p38 MAPK system is responsible for the cell density-dependent redistribution of AhR, exposure to an inhibitor of p38 MAPK would result in an altered subcellular localization of the endogenous AhR. To test this, cultured HaCaT cells in the subconfluent condition, in which the AhR distributes evenly in the cytoplasm and the nucleus, were treated briefly with a p38 MAPK inhibitor SB203580, and we observed a more cytoplasmic distribution of AhR. By contrast, when treated with U0126, a MAP kinase-ERK kinase (MEK) inhibitor of the ERK1/2 MAPK signal transduction cascade, the subcellular localization of AhR was not affected compared with that of Me2SO-treated controls (Fig. 4D, a). Nuclear retention of AhR mediated by activated p38 MAPK, but not ERK1/2, was also suggested by a reporter assay, as shown in Fig. 4D, b. In addition, we observed that the p38 MAPK was actually phosphorylated by the medium change from DMEM to calcium-deficient S-MEM after only 15 min with the HaCaT cells, followed by CYP1A1 induction (Fig. 4D, c). In contrast, when confluent HaCaT cells, in which AhR was distributed predominantly in the cytoplasm, were treated briefly with a protein serine/threonine phosphatase (PP) inhibitor, okadaic acid, a significant shift toward a more nuclear distribution of AhR was observed (Fig. 4E). Thus, we were able to alter the novel relocalization of AhR toward a more cytoplasmic (SB203580) or more nuclear (okadaic acid) distribution, suggesting that p38 MAPK-dependent phosphorylation of the AhR-NES and its dephosphorylation may be involved in the novel relocalization of AhR caused by cell density.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Because AhR contains both signals for nuclear import and export, it appears to show a different intracellular localization under particular physiological conditions such as proliferation, differentiation, development, and transformation, responsible for the physiological function of AhR. In the present study, we showed that AhR intracellular localization is regulated by cell density because of altered nuclear export activity by modulation of the NES. Also, our data showed an association between the AhR relocalization regulated by cell density and AhR-mediated transcriptional activity.

Using the keratinocyte cell line HaCaT, we found that the cell density, but not the cell cycle, influences the intracellular distribution of AhR, with a predominant nuclear localization of AhR in sparsely distributed cells, localization in both the nucleus and the cytoplasm in subconfluent cells, and predominant cytoplasmic localization in confluent cells (Fig. 1A). Studies in recent years have provided increasing evidence that nuclear localization of protein is regulated not only by nuclear import but also by export activity (49, 50). Because treatment with LMB promotes nuclear accumulation of AhR irrespective of cell density, high cell density may accelerate the nuclear export of AhR but not inhibit the nuclear import of AhR. Nuclear localization of activated AhR without MC may suggest the presence of an endogenous ligand or a ligand-independent activation mechanism in HaCaT cells. It has been known that the NES-dependent nuclear export of protein is primarily and precisely regulated by phosphorylation-dephosphorylation of the NES (38, 39). Actually, the nuclear export of a chimeric GST-AhR-(55-75)-GFP fusion protein was suppressed by substitution of a serine residue (Ser-68) to aspartic acid (Fig. 4B), which mimics the negative charge of phosphorylation. To show the phosphorylation of Ser-68 in the NES of AhR, we produced and used a polyclonal peptide antibody against AhR-(61-74) containing phosphoserine 68. We found that the Ser-68 was phosphorylated in confluent HaCaT cells cultured with DMEM followed by further incubation with S-MEM (Fig. 4C, b). A single band, which had a slower mobility shift than that of AhR because of possible molecular modulation by negative charge of phosphorylation, was detected by anti-AhR-(61-74)-pS68 antibody in cell extracts prepared from S-MEM, and this band was also recognized with a specific antibody to AhR (BIOMOL). In addition, an apparent phosphorylation of Ser-68 was also observed after nuclear accumulation of activated AhR in the nuclei of livers of MC-treated mice (Fig. 4C, c). The specificity of anti-AhR-(61-74)-pS68 antibody used was confirmed by specific binding with the phosphorylated peptide of AhR-(61-74), but not with unphosphorylated peptide, as shown in Fig. 4C, a. Thus, it is likely that the NES of AhR constitutes a phosphorylation-mediated regulatory module of nuclear export.

Although no consensus Ser-Pro motif feature shared by substrates for known MAPKs was observed, it should be emphasized that the p38 MAPK signaling pathway (51-53), which is an important mediator of the cellular response to environmental stressors such as UV radiation, heat shock, and proinflammatory cytokines, is involved in nuclear accumulation of environmental response gene products such as p53 (38) and estrogen receptor {alpha} (39). In this context, it is likely that Ser-68 within the NES of AhR is the corresponding amino acid that is modified by the p38 MAPK system (Fig. 4A), leading to a stress-dependent suppression of nuclear export activity. Then, we observed the effect of a p38-MAPK inhibitor SB203580 or U0126, an inhibitor of MEK in ERK-dependent MAPK, on cell density-dependent novel relocalization of AhR. We found an association between the p38-dependent, but not the ERK-dependent, signal transduction cascade and AhR relocalization (Fig. 4D). It is noteworthy that there was a large difference in activation of p38 MAPK between stimuli of chemicals such as MC and that of cell-cell contact signals. The rapid phosphorylation of p38 MAPK was observed in confluent HaCaT cells cultured with DMEM followed by only 15 min incubation with S-MEM (Fig. 4D, c), whereas MC activated p38 MAPK after a 16-24-h exposure to the cultured HepG2 cells (54). Although the physiological ligand of AhR is not yet known, nuclear import of AhR and subsequent inductive expression of CYP1A1 were observed at 2 h incubation of S-MEM, suggesting functional cross-talk between the activated p38 MAPK and AhR. Thus, it is likely that a p38 MAPK-dependent phosphorylation of AhR-NES may occur in the nucleus after the nuclear translocation of activated p38 MAPK (55, 56).

Although we have little information of which subfamilies of PP might participate in the dephosphorylation of AhR-NES to export from the nucleus, a PP inhibitor, okadaic acid, but not cyclosporin A (data not shown) an inhibitor of calcineurin, alters the redistribution of AhR, suggesting the involvement of regulated dephosphorylation by a PP in AhR relocalization (Fig. 4E). Two plausible hypotheses may explain the differential regulation of dephosphorylation under different cell densities. One of these is an altered PP activity in the nucleus with varying cell density, and the other is a different activity of PP inhibitors. A recent report showed that inhibitor-2 of type 1 PP (PP1) is concentrated in the nucleus of cells that are sparsely distributed, whereas cells growing at a high cell density exclude inhibitor-2 from the nucleus (57). Although redistribution of inhibitor-2 for PP1 may mediate for cell density-induced novel relocalization of AhR, a correlation between PP activity and cell density should be investigated.

The predominant cytoplasmic localization of endogenous AhR with high cell density was altered toward a more nuclear distribution by reducing the Ca2+ concentration (Fig. 1C), suggesting that cadherin-mediated signals derived from cell-cell contact may have pivotal roles in promoting the nuclear export of AhR. In this context, it is noteworthy that the suspension-induced increases in steady state levels of CYP1A1 mRNA and enzyme activity in keratinocytes are independent of xenobiotic AhR ligands (58). This may cause Hsp90 to be stripped from AhR leading to nuclear import of AhR (59). Alternatively, the decreased cell-cell contact in suspension culture of the keratinocytes may inhibit the AhR movement from the nucleus to the cytoplasm, just like cell density-dependent AhR relocalization. A proposed model for AhR relocalization is shown in Fig. 5.



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  		FIG. 5.
A proposed model for AhR relocalization. The intracellular localization of AhR is regulated not only by the presence or absence of endogenous or exogenous ligands but also by cell density. In the absence of ligands, the AhR is retained predominantly in the cytoplasm because of a steric hindrance masking of the NLS by Hsp90. Ligand binding stimulates the exposed NLS to bind importins for nuclear import. After a ligand-dependent nuclear translocation of the AhR, the AhR dissociates from Hsp90 to bind to the ARNT. In the nucleus, the AhR/ARNT heterodimer binds to the XRE to activate its target gene transcription. Exogenous ligands such as MC or endogenous ligands phosphorylate p38 resulting in the nuclear import of the stress-dependent MAPK. It is likely that activated p38 MAPK phosphorylates Ser-68 in the NES of the AhR resulting in nuclear accumulation of the receptor, which is because of a loss of nuclear export activity. The AhR is localized predominantly in the nucleus under low cell density, whereas it is distributed in the cytoplasm at high cell density. The cell density-dependent AhR relocalization may be explained by the balance of PP and PP inhibitor in the nucleus with varying cell density. The cadherin-mediated signal derived from cell-cell contact may have a pivotal role in promoting the nuclear export of AhR caused by dephosphorylation. The individual roles of NES in bHLH and PAS domains (47) and their interplay in nuclear export need further investigation.

 
Although a large number of proteins, including transcription factors, have been known to shuttle between the nucleus and the cytoplasm, there are only a few examples of cell densitydependent regulated proteins. The tumor suppressor proteins, von Hippel-Lindau (60) and adenomatous polyposis coli (48), both of which may control cell growth and transformation, are known as nucleocytoplasmic shuttling proteins, and their subcellular localization is regulated by cell density. These proteins also are localized predominantly in the nucleus with low cell density but in the cytoplasm with high cell density, just like AhR. However, it is interesting to note that there was a marked difference in regulation between cell density-dependent adenomatous polyposis coli and AhR relocalization. A novel redistribution of adenomatous polyposis coli was mediated by differential phosphorylation of casein kinase 2 and protein kinase A sites near the NLS for the adenomatous polyposis coli (48), whereas modulation of the NES was responsible for a novel relocalization of AhR.

The expression of AhR was specific to the cell type, organ/tissue, and the developmental stage, and intracellular localization of the protein was altered in a time-specific manner in mice embryos (61, 62), suggesting that the ligand-activated transcription factor may be important in normal embryonic development. Because the regulated intracellular localization of transcription factors can serve as a biological switch in response to various signals, a novel relocalization of AhR caused by cell density appears to be an essential event for the biological function of the AhR. Phosphorylation/dephosphorylation-dependent AhR movement in and of the nucleus provides a simple, reversible, and rapid means to control responses to different environmental or physiological conditions and extracellular signals in cells.


   FOOTNOTES
 
* This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, and Health Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Research Institute, Saitama Cancer Center, 818 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japan. Tel.: 81-48-722-1111 (ext. 4620); Fax: 81-48-722-1739; E-mail: kawajiri{at}cancer-c.pref.saitama.jp.

1 The abbreviations used are: AhR, aryl hydrocarbon receptor; ARNT, AhR nuclear translocator; NLS, nuclear localization signal; NES, nuclear export signal; bHLH, basic helix-loop-helix; PAS, PER-ARNT-SIM homology region; XRE, xenobiotic responsive element; MC, 3-methylcholanthrene; LMB, leptomycin B; GST, glutathione S-transferase; GFP, green fluorescent protein; MAPK, mitogen-activated protein kinase; PP, protein serine/threonine phosphatase; DMEM, Dulbecco's modified Eagle's medium; TK, thymidine kinase; BrdUrd, bromodeoxyuridine; ERK, extracellular signal-regulated kinase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Denis, M., Cuthill, S., Wikstrom, A. C., and Gustafsson, J. A. (1988) Biochem. Biophys. Res. Commun. 155, 801-807[CrossRef][Medline] [Order article via Infotrieve]
  2. Perdew, G. H. (1988) J. Biol. Chem. 263, 13802-13805[Abstract/Free Full Text]
  3. Nair, S. C., Toran, E. J., Rimerman, R. A., Hjermstad, S., Smithgall, T. E., and Smith, D. F. (1996) Cell Stress Chaperones 1, 237-250[CrossRef][Medline] [Order article via Infotrieve]
  4. Kazlauskas, A., Poellinger, L., and Pongratz, I. (1999) J. Biol. Chem. 274, 13519-13524[Abstract/Free Full Text]
  5. Carver, L. A., and Bradfield, C. A. (1997) J. Biol. Chem. 272, 11452-11456[Abstract/Free Full Text]
  6. Ma, Q., and Whitlock, J. P. (1997) J. Biol. Chem. 272, 8878-8884[Abstract/Free Full Text]
  7. Meyer, B. K., Pray-Grant, M. G., Vanden Heuvel, J. P., and Perdew, G. H. (1998) Mol. Cell. Biol. 18, 978-988[Abstract/Free Full Text]
  8. McGuire, J., Whitelaw, M. L., Pongratz, I., Gustafsson, J. A., and Poellinger, L. (1994) Mol. Cell. Biol. 14, 2438-2446[Abstract/Free Full Text]
  9. Eguchi, H., Ikuta, T., Tachibana, T., Yoneda, Y., and Kawajiri, K. (1997) J. Biol. Chem. 272, 17640-17647[Abstract/Free Full Text]
  10. Heid, S. E., Pollenz, R. S., and Swanson, H. I. (1999) Mol. Pharmacol. 57, 82-92
  11. Fujisawa-Sehara, A., Sogawa, K., Nishi, C., and Fujii-Kuriyama, Y. (1986) Nucleic Acids Res. 14, 1465-1477[Abstract/Free Full Text]
  12. Kolluri, S. K., Weiss, C., Koff, A., and Gottlicher, M. (1999) Genes Dev. 13, 1742-1753[Abstract/Free Full Text]
  13. Mimura, J., Ema, M., Sogawa, K., and Fujii-Kuriyama, Y. (1999) Genes Dev. 13, 20-25[Abstract/Free Full Text]
  14. Matikainen, T., Perez, G. I., Jurisicova, A., Pru, J., Schlezinger, J. J., Ryu, H-Y., Laine, J., Sakai, T., Korsmeyer, S., Casper, R. F., Sherr, D. H., and Tilly, J. L. (2001) Nat. Genet. 28, 355-359[CrossRef][Medline] [Order article via Infotrieve]
  15. Ogi, T., Mimura, J., Hikida, M., Fujimoto, H., Fujii-Kuriyama, Y., and Ohmori, H. (2001) Genes Cells 6, 943-953[Abstract]
  16. Shimizu, Y., Nakatsuru, Y., Ichinose, M., Takahashi, Y., Kume, H., Mimura, J., Fujii-Kuriyama, Y., and Ishikawa, T. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 779-782[Abstract/Free Full Text]
  17. Mimura, J., Yamashita, K., Nakamura, K., Morita, M., Takagi, N. T., Nakao, K., Ema, M., Sogawa, K., Yasuda, M., Katsuki, M., and Fujii-Kuriyama, Y. (1997) Genes Cells 2, 645-654[Abstract]
  18. Fernandez-Salguero, P. M., Hilbert, D. M., Rudikoff, S., Ward, J. M., and Gonzalez, F. J. (1996) Toxicol. Appl. Pharmacol. 140, 173-179[CrossRef][Medline] [Order article via Infotrieve]
  19. Lahvis, G., Lindell, S. L., Thomas, R. S., McCuskey, R. S., Murphy, C., Glover, E., Bentz, M., Southard, J., and Bradfield, C. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10442-10447[Abstract/Free Full Text]
  20. Robles, R., Morita, Y., Mann, K. K., Perez, G. I., Yang, S., Matikainen, T., Sherr, D. H., and Tilly, J. (2000) Endocrinology 141, 450-453[Abstract/Free Full Text]
  21. Klinge, C. M., Kaur, K., and Swanson, H. I. (2000) Arch. Biochem. Biophys. 373, 163-174[CrossRef][Medline] [Order article via Infotrieve]
  22. Ohtake, F., Takeyama, K., Matsumoto, T., Kitagawa, H., Yamamoto, Y., Nohara, K., Tohyama, C., Krust, A., Mimura, J., Chambon, P., Yanagisawa, J., Fujii-Kuriyama, Y., and Kato, S. (2003) Nature 423, 545-550[CrossRef][Medline] [Order article via Infotrieve]
  23. Adachi, J., Mori, Y., Matsui, S., Takigami, H., Fujino, J., Kitagawa, H., Miller, C. A., III, Kato, T., Saeki, K., and Matsuda, T. (2001) J. Biol. Chem. 276, 31475-31478[Abstract/Free Full Text]
  24. Song, J., Clagett-Dame, M., Peterson, R. E., Hahn, M. E., Westler, W. M., Sicinski, R. R., and DeLuca, H. F. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 14694-14699[Abstract/Free Full Text]
  25. Ikuta, T., Eguchi, H., Tachibana, T., Yoneda, Y., and Kawajiri, K. (1998) J. Biol. Chem. 273, 2895-2904[Abstract/Free Full Text]
  26. Lees, M. J., and Whitelaw, M. L. (1999) Mol. Cell. Biol. 19, 5811-5822[Abstract/Free Full Text]
  27. Kazlauskas, A., Sundstrom, S., Poellinger, L., and Pongratz, I. (2001) Mol. Cell. Biol. 21, 2594-2607[Abstract/Free Full Text]
  28. Fischer, U., Huber, J., Boelens, W., Mattaj, I. W., and Luhrmann, R. (1995) Cell 82, 475-483[CrossRef][Medline] [Order article via Infotrieve]
  29. Wen, W., Meinkkoth, J. L., Tsien, R. Y., and Taylor, S. S. (1995) Cell 82, 463-473[CrossRef][Medline] [Order article via Infotrieve]
  30. Ikuta, T., Tachibana, T., Watanabe, J., Yoshida, M., Yoneda, Y., and Kawajiri, K. (2000) J. Biochem. (Tokyo) 127, 503-509[Abstract/Free Full Text]
  31. Nigg, E. A. (1997) Nature 386, 779-787[CrossRef][Medline] [Order article via Infotrieve]
  32. Mattaj, I. W., and Englmeier, L. (1998) Annu. Rev. Biochem. 67, 265-306[CrossRef][Medline] [Order article via Infotrieve]
  33. Pennisi, E. (1998) Science 279, 1129-1131[Free Full Text]
  34. Hood, J. K., and Silver, P. A. (1999) Curr. Opin. Cell Biol. 11, 241-247[CrossRef][Medline] [Order article via Infotrieve]
  35. Jans, D. A., and Hubner, S. (1996) Physiol. Rev. 76, 651-685[Abstract/Free Full Text]
  36. Rihs, H. P., Jans, D. A., Fan, H., and Peters, R. (1991) EMBO J. 10, 633-639[Medline] [Order article via Infotrieve]
  37. Zhang, F., White, R. L., and Neufeld, K. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 12577-12582[Abstract/Free Full Text]
  38. Zhang, Y., and Xiong, Y. (2001) Science 292, 1910-1915[Abstract/Free Full Text]
  39. Lee, H., and Bai, W. (2002) Mol. Cell. Biol. 22, 5835-5845[Abstract/Free Full Text]
  40. Wanner, R., Brommer, S., Czarnetzki, B. M., and Rosenbach, T. (1995) Biochem. Biophys. Res. Commun. 209, 706-711[CrossRef][Medline] [Order article via Infotrieve]
  41. Sutter, T. R., Guzman, K., Dold, K. M., and Greenlee, W. F. (1991) Science 254, 415-418[Abstract/Free Full Text]
  42. Moses, M., and Prioleau, P. G. (1985) J. Am. Acad. Dermatol. 12, 497-506[Medline] [Order article via Infotrieve]
  43. Boukamp, P., Petrussevska, R. T., Breitkreutz, D., Hornung, J., Markham, A., and Fusenig, N. E. (1988) J. Cell Biol. 106, 761-771[Abstract/Free Full Text]
  44. Kimura, H., Nozaki, N., and Sugimoto, K. (1994) EMBO J. 13, 4311-4320[Medline] [Order article via Infotrieve]
  45. Michalik, L., Desvergne, B., Tan, N. S., Basu-Modak, S., Escher, P., Rieusset, J., Peters, J. M., Kaya, G., Gonzalez, F. J., Zakany, J., Metzger, D., Chambon, P., Duboule, D., and Wahli, W. (2001) J. Cell Biol. 154, 799-814[Abstract/Free Full Text]
  46. Martin, P. (1997) Science 276, 75-81[Abstract/Free Full Text]
  47. Berg, P., and Pongratz, I. (2001) J. Biol. Chem. 276, 43231-43238[Abstract/Free Full Text]
  48. Zhang, F., White, R. L., and Neufeld, K. L. (2001) Mol. Cell. Biol. 21, 8143-8156[Abstract/Free Full Text]
  49. Stommel, J. M., Marchenko, N. D., Jimenez, G. S., Moll, U., Hope, T. J., and Wahl, G. M. (1999) EMBO J. 18, 1660-1672[CrossRef][Medline] [Order article via Infotrieve]
  50. Johnson, C., Antwerp, A. V., and Hope, T. J. (1999) EMBO J. 18, 6682-6693[CrossRef][Medline] [Order article via Infotrieve]
  51. Han, J., Lee, J. D., Bibbs, L., and Ulevitch, R. J. (1994) Science 265, 808-811[Abstract/Free Full Text]
  52. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler, J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., and Young, P. R. (1994) Nature 372, 739-746[CrossRef][Medline] [Order article via Infotrieve]
  53. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., Zamanillo, D., Hunt, T., and Nebreda, A. R. (1994) Cell 78, 1027-1037[CrossRef][Medline] [Order article via Infotrieve]
  54. Kwon, Y-W., Ueda, S., Ueno, M., Yodoi, J., and Masutani, H. (2002) J. Biol. Chem. 277, 1837-1844[Abstract/Free Full Text]
  55. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) J. Biol. Chem. 270, 7420-7426[Abstract/Free Full Text]
  56. Seternes, O. M., Johansen, B., Hegge, B., Johannesen, M., Keyse, S. M., and Moens, U. (2002) Mol. Cell. Biol. 22, 6931-6945[Abstract/Free Full Text]
  57. Leach, C., Eto, M., and Brautigan, D. L. (2002) J. Cell Sci. 115, 3739-3745[Abstract/Free Full Text]
  58. Sladek, C. M., and Allen-Hoffmann, B. L. (1994) J. Biol. Chem. 269, 16067-16074[Abstract/Free Full Text]
  59. Pongratz. I., Mason, G. G., and Poellinger, L. (1992) J. Biol. Chem. 267, 13728-13734[Abstract/Free Full Text]
  60. Lee, S., Chen, D. Y., Humphrey, J. S., Gnarra, J. R., Linehan, W. M., and Klausner, R. D. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1770-1775[Abstract/Free Full Text]
  61. Abbott, B. D., Birnbaum, L. S., and Perdew, G. H. (1995) Dev. Dyn. 204, 133-143[Medline] [Order article via Infotrieve]
  62. Abbott, B. D., and Probst, M. R. (1995) Dev. Dyn. 204, 144-155[Medline] [Order article via Infotrieve]

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 All ASBMB Journals   Molecular and Cellular Proteomics 
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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
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