Nuclear Export of Glucocorticoid Receptor is Enhanced by c-Jun N-Terminal Kinase-Mediated Phosphorylation

M. Itoh1, M. Adachi1, H. Yasui, M. Takekawa, H. Tanaka and K. Imai

First Department of Internal Medicine (M.I., M.A., H.Y., K.I.), Sapporo Medical University School of Medicine, Sapporo, 060-8543, Japan; and Division of Molecular Cell Signaling (M.T.), and Division of Clinical Immunology (H.T.), Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

Address all correspondence and requests for reprints to: Masaaki Adachi, First Department of Internal Medicine, Sapporo Medical University School of Medicine, S1 W16, Chuo-ku, Sapporo, 060-8543 Japan. E-mail: .


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The c-Jun N-terminal kinase (JNK) phosphorylates the glucocorticoid receptor (GR) and inhibits GR-mediated transcription. However, the biological effect of the GR phosphorylation remains unknown. Here we demonstrate that activated JNK phosphorylates human GR at Ser226 and enhances its nuclear export after withdrawal of a ligand for GR, dexamethasone. At 1 h after dexamethasone withdrawal, green fluorescent protein-GR molecules were mostly retained at the nucleus, whereas UV exposure enhanced its nuclear export, and approximately 30–40% of cells revealed distinct nuclear export. JNK overexpression alone mimics UV exposure and enhanced GR export accompanied by inhibition of GR-mediated transcription. However, mutation of the Ser226 JNK phosphorylation site in GR abrogated UV-mediated enhancement of GR nuclear export. Furthermore, overexpression of a dominant negative SEK1 mutant also abrogated the effects of UV exposure on GR export. Taken together, these findings suggest that JNK-mediated phosphorylation of the GR-Ser226 enhances GR nuclear export and may contribute to termination of GR-mediated transcription.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE GLUCOCORTICOID RECEPTOR (GR) is a cytoplasmic, hormone-dependent transcription factor that mediates a variety of biological responses including gluconeogenesis, antiinflammation, antiproliferation, and immunosuppression (1, 2, 3). Although GR is expressed ubiquitously, it up-regulates transcription of distinct sets of genes in a cell type-specific manner. GR is a nucleocytoplasmic shuttling protein (4, 5). In most unstimulated cells GR molecules are localized predominantly in the cytoplasm. Upon hormone binding, GR molecules homodimerize and are rapidly imported into the nucleus (6). In the nucleus the ligand-bound GRs bind to glucocorticoid response elements (GREs) and increase the transcription of target genes (7, 8, 9)

Consistent with the broad functions of GR-mediated signals, GR-mediated transcription is regulated both positively and negatively by phosphorylation. To date, GR has been reported to be phosphorylated at four major sites (Thr171, Ser224, Ser232, and Ser246 in rat GR) in its N-terminal transcriptional regulatory region (10). MAPK phosphorylates rat GR at Thr171 and Ser246, whereas cyclin-dependent kinase (Cdk) phosphorylates it at Ser224 and Ser232. Interestingly, MAPK and Cdk-mediated phosphorylation of GR have opposing effects on GR-mediated transcription, i.e. MAPK negatively regulates, whereas Cdk positively regulates, GR-mediated transcription (10). In addition, phosphorylation of Thr171 by glycogen synthase kinase-3 inhibits GR-mediated transcription (11). Furthermore, it has been reported that Ser246 is directly phosphorylated by c-Jun N-terminal kinase (JNK), but not by p38, and that JNK phosphorylation of GR directly inhibits GR-mediated activation of transcription (12).

JNK is a member of the MAPK family that includes ERK and p38 MAPK (13, 14). MAPKs recognize and phosphorylate a similar consensus sequence (nonpolar-X-Ser/Thr-Pro), but are differently activated by distinct stimuli (15). JNK is activated by a variety of stress conditions such as UV radiation and osmotic shock as well as by inflammatory cytokines; TNF{alpha}, IL-1, and the lipopolysaccharide of Gram-negative bacteria (13, 16, 17, 18). Inflammatory stimuli and glucocorticoids clearly exert opposing inflammatory responses because it has been shown that glucocorticoids strongly inhibit inflammatory responses (3). Similarly, JNK and GR modulate inflammation in an opposing manner. Because prolonged and extensive inflammation often causes organ damage or hematological disorder, tight regulation of these opposing signals is therefore crucial for homeostasis. After external stimulation, activated JNK phosphorylates transcription factors, such as activating transcription factor 2, Elk-1, and c-Jun, within their activation domains, thereby enhancing their transcriptional activity (19). In contrast, JNK phosphorylation of GR inactivates the GR transcriptional activity. This implies that JNK simultaneously activates inflammatory signals while inactivating GR-mediated antiinflammatory signals, and thus appears to be a crucial molecule in the regulation of inflammatory responses.

Although clearly of biological importance, the molecular mechanism(s) by which JNK negatively regulates GR-mediated signals remains unclear. One potential mechanism for JNK inhibition of the GR signal is that JNK phosphorylation of GR might modulate ligand-dependent nuclear-cytoplasmic shuttling of GR. To determine whether this might be the case, we generated a mutant form of human GR in which the GR-JNK phosphorylation site (S226) was mutated to alanine (GRS226A). We then examined the effect of this mutation on nuclear import and export of GR using green fluorescence protein (GFP)-GR hybrids. We show that Ser226 on human GR is the major site for JNK phosphorylation and that activation of JNK can accelerate GR nuclear export. JNK activation could neither enhance nuclear export of the mutant GR (GRS226A) nor inhibit GRS226A-mediated transcriptional activation. Thus, the inhibitory effect of JNK on GR-mediated signals requires phosphorylation of Ser226. The subsequent early nuclear export of GR may provide a mechanism to explain the inhibitory effects of JNK, at least in part, on GR-mediated transcriptional events.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
UV Radiation Enhances Nuclear Export of GR
It has been demonstrated that JNK activation by external stimuli inhibits GR-mediated biological responses. However, the molecular mechanism by which the JNK activation affects the responses remains obscure. To explore this, we investigated the effect of UV radiation, one of the most prominent activators of the JNK pathway (18), on the intracellular distribution of GFP-tagged GR (GFP-GR) after treatment with 1 µM of the GR ligand dexamethasone (DEX). It has previously been demonstrated that GFP-GR has a similar intracellular distribution and transcriptional activity as the unmodified GR (6). When COS-7 cells were exposed to 40-J/m2 UV radiation, JNK was highly phosphorylated at 0.5 h after exposure (Fig. 1AGo). GFP-GR was mostly nuclear imported within 15 min after treatment with 1 µM DEX regardless of UV radiation (data not shown). When cells are continuously stimulated by DEX, it is difficult to evaluate how JNK affects the intracellular localization of GR (data not shown). However, the effect of JNK activation was clearly observed after DEX withdrawal. Most GFP-GR signals were still retained in the nucleus in untreated COS-7 transfectants, whereas significant portions of the signals were clearly exported to the cytoplasm in UV-treated cells at 1 h after DEX withdrawal (Fig. 1BGo). At 4 h after DEX withdrawal, nuclear GFP-GR signals became very faint in UV-treated cells. Statistical analysis of more than 100 transfectants revealed that approximately 80% of the UV-treated cells showed distinct nuclear export of GR, whereas only 30% of the untreated cells did at 4 h after DEX withdrawal (Fig. 1BGo). This strongly suggests that UV-induced JNK activation may augment nuclear export of GR.



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Figure 1. Effect of JNK Activation on GR-Mediated Transcription

A, COS-7 cells were serum deprived for 14 h and then treated with (+) or without (-) UV (40 J/m2) and harvested at 0.5 h after radiation. The phosphorylation status of endogenous JNK was analyzed by immunoblotting with an antibody specific to phosphorylated JNK. Equivalent levels of JNK were loaded in each lane as determined by blotting with an anti-JNK antibody. B, COS-7 cells were transiently transfected with pCMX-GFP-GR, exposed to UV (20 J/m2), and then incubated with 1 µM DEX for 1 h. After withdrawal of DEX, the transfectants were further incubated and fixed at 1 or 4 h after DEX withdrawal. Intracellular distribution of GFP-GR was investigated with confocal microscopy. Quantitative analysis of the effect of UV radiation on nuclear export of GFP-GR is shown (right panel).

 
Ser226 Is the Major Phosphorylation Site of JNK in Human GR
It has been reported previously that JNK phosphorylates rat GR mainly at Ser246, thereby inhibiting GR-mediated transcriptional activity (12). The amino acid sequences of the JNK phosphorylation site and its adjacent segments are well conserved among several species (Ser246 in rat GR corresponds to Ser226 in human GR) (Fig. 2AGo). To investigate the importance of the Ser226 site in GR for JNK phosphorylation and GR-mediated biological responses, we generated a mutant form of human GR with a single serine-to-alanine substitution at Ser226 (GRS226A). We first investigated whether this site is a major phosphorylation site for JNK. For this purpose we generated glutathione-S-transferase (GST)-GR wild type (wt) (77–262), GST-GRS226A (77–262), GST-c-Jun (as a positive control), and GST (as a negative control) for use as exogenous substrates in an in vitro kinase assay with activated JNK. To prepare activated JNK, 293 cells were transfected with a hemagglutinin (HA)-JNK expression plasmid, and stimulated with osmotic stress (0.5 M sorbitol). As shown in Fig. 2BGo, HA-JNK was strongly activated by osmotic stress. Using the HA-JNK precipitates from transfectants treated with or without osmotic stress, an in vitro kinase assay revealed that the GST-GRwt was strongly phosphorylated by activated JNK (Fig. 2CGo). However JNK-mediated phosphorylation of GR was completely abolished when GST-GRS226A was used as a substrate, indicating that the single amino acid substitution at Ser226 on human GR abrogated JNK phosphorylation. We thus conclude that Ser226 is the major site in human GR for JNK phosphorylation, and that the GRS226A mutant cannot be phosphorylated by active JNK. This mutant therefore can be used in experiments to determine the role of JNK phosphorylation of GR in GR-mediated biological responses.



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Figure 2. Phosphorylation of Human GR by JNK

A, Alignment of the putative JNK phosphorylation site of human GR (S226) with the defined JNK phosphorylation site of rat GR (S246). B, HA-JNK-transfected 293 cells were serum deprived for 24 h and then stimulated with (+) or without (-) 0.5 M sorbitol for 15 min. JNK activation was evaluated by immunoblots using an antibody specific to phosphorylated JNK (p-JNK). Equal levels of JNK protein were loaded in each lane (JNK). C, HA-JNK1 immunoprecipitates from cells treated with or without 0.5 M sorbitol were incubated with GRwt-GST(77–262), GRS226A-GST(77–262), a specific JNK exogenous substrate (c-Jun-GST), and a negative control GST alone (GST). Phosphorylation of these substrates by JNK was evaluated by an in vitro kinase assay, subsequent gel electrophoresis, and autoradiography (upper panel). Similar amounts of the substrate proteins were incubated with the HA-JNK1 immmunoprecipitates (lower panel). Molecular mass markers are shown in kilodaltons.

 
When COS-7 cells were transfected with CMX-GFP-GRwt or -GRS226A cDNA, these GFP-GRs were abundantly expressed (Fig. 3AGo). The GFP-GRwt was localized mainly in the cytoplasm in unstimulated cells, which is similar to the distribution of endogenous GR in many cell types. Similarly, GFP-GRS226A was localized mostly in the cytoplasm in unstimulated COS-7 transfectants, indicating that its cellular distribution is similar to that of endogenous GR (Fig. 3BGo). When COS-7 transfectants were stimulated with DEX for 0.5 h, GFP-GRwt and GFP-GRS226A were both exclusively localized in the nuclei. We next investigated whether these GFP-tagged GRs are biologically active. When these GFP-GRwt or GFP-GRS226A proteins were overexpressed in COS-7 cells, the transfectant became responsive to DEX. Importantly, these GFP-tagged GRs have similar transcriptional activity compared with untagged GRwt assessed by a luciferase reporter assay (Fig. 3CGo). Thus, these data strongly suggest that our GFP-GRs are biologically active and their behavior is not altered by its modification or mutation.



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Figure 3. Human GFP-GRwt and GFP-GRS226A

A, COS-7 cells were transfected with either control GFP-vehicle, GFP-GRwt, or GFP-GRS226A as indicated. The cells were harvested 36 h after transfection and subjected to immunoblot analysis using an anti-GR antibody. The anti-Hsc70 antibody was used as a control for protein loading. B, The transfectants described in panel A were treated with (+) or without (-) 1 µM DEX for 0.5 h and fixed. Intracellular distribution of GFP-GRs was evaluated with confocal laser scanning. C, Control vehicle pCMX, pCMX-GRwt, pCMX-GFP-GRwt, or pCMX-GFP-GRS226A expression vectors were transfected into COS-7 reporter cells. Transfectants were stimulated with or without 2.5 µM DEX at 24 h after transfection. Cells were harvested at 12 h after DEX stimulation for measurement of luciferase activity. The columns display the mean ±SD of data from three separate experiments.

 
UV Radiation Enhances Nuclear Export of GFP-GRwt, But Not GFP-GRS226A
To elucidate whether JNK activation affects nuclear localization of GR after DEX withdrawal, intracellular distribution of both GFP-GRs were investigated. In the presence of 1 µM DEX, GFP-GRs remained in the nuclei at 0.5 h after DEX treatment in both transfectants. After withdrawal of DEX, both proteins slowly returned to the cytoplasm, but the export of the GFP-GRS226A was slower than that of the GFP-GRwt. At 1 h after DEX withdrawal, most of the GFP-GRwt and GRS226A transfectants still showed a distinct nuclear localization (Fig. 4AGo). However, exposure to UV radiation revealed the difference in the nuclear export of GRwt and GRS226A. When the transfectants were radiated by UV, approximately 34% of the cells revealed distinct cytoplasmic localization of GFP-GRwt at 1 h after DEX withdrawal. In contrast, UV radiation did not enhance the nuclear export of the GFP-GRS226A (Fig. 4AGo). Similar results were obtained when transfectants were cultured in fetal calf serum-containing medium (data not shown).



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Figure 4. Effect of UV Radiation on GR Nuclear Export and Subcellular Fractionation of GRs

A, COS-7 cells were transfected with GFP-GRwt or GFP-GRS226A. Sixteen hours after transfection, the transfectants were serum-deprived for 16 h and then treated with (+) or without (-) UV radiation before incubation with 1 µM DEX for 1 h. After withdrawal of DEX, the transfectants were incubated for an additional 0.5 to 2 h in media alone. Intracellular distribution of GFP-GRs was evaluated by confocal laser scanning and representative data are shown. Quantitative analysis of the cellular distribution of GFP-GRs is shown (lower panel). Nuclear export was determined as described in Materials and Methods. These experiments were repeated at least three times with similar results. B, COS-7 cells were transfected with GFP-GRwt or GFP-GRS226A. The transfectants were treated 24 h after transfection with (+) or without (-) UV radiation before incubation with 1 µM DEX for 1 h. After DEX withdrawal the transfectants were further incubated with 10 µg/ml CHX for 2 h. GR expression levels in total cell lysates were evaluated using a polyclonal anti-GR-specific antibody. The anti-Hsc70 antibody was used as a protein loading control. C, After stimulation with (+) or without (-) UV (20 J/m2) radiation before incubation with 1 µM DEX for 1 h, transfectants were extensively washed and further incubated with 10 µg/ml CHX for 2 h. Both nuclear and cytoplasmic fractions were collected. Recovery of GFP-GR was evaluated by immunoblotting using a polyclonal anti-GR-specific antibody. Hsc70 and lamin B are shown as indicators of protein loading. D, Quantitative analysis of subcellular fractionation described in panel C. GFP-GRwt and GFP-GRS226A signals were measured by NIH image densitometric analysis and normalized to Hsc70 or lamin B protein on the same blots. These experiments were repeated at least three times with similar results.

 
After ligand, stimulated GR that has been translocated to the nucleus may encounter different fates including either nuclear export, degradation, or recycling (20). To exclude the possibility that the disappearance of GR proteins from the nucleus was caused by degradation of GR, we monitored the concentration of the GR proteins after DEX withdrawal. For this purpose, COS-7 cells, transfected with either GRwt or GRS226A cDNAs, were incubated with 1 µM DEX and 10 µg/ml cycloheximide (CHX) for 1 h and were then deprived of DEX for 2 h. Both GRwt and GRS226A expression levels remained unchanged for at least 2 h after DEX withdrawal (Fig. 4BGo). Thus the disappearance of the GR proteins from the nucleus is more likely to be due to their nuclear export than to protein degradation, although we cannot completely exclude the possibility that their varied expression levels among the transfectants may affect intracellular distribution of GRs.

We further evaluated the nuclear export of the GR constructs by subcellular fractionation. The amount of GRwt protein recovered from the cytoplasmic fraction was increased 1.4-fold at 2 h after DEX withdrawal and was further increased (~1.8-fold) by UV radiation (Fig. 4Go, C and D). In addition, the recovery of GRwt from the nuclear fractions was decreased to 0.7-fold by UV radiation. In contrast, recovery of GRS226A from the cytoplasmic and nuclear fractions was unchanged after DEX withdrawal and/or UV radiation. Thus, subcellular fractionation confirmed our observations that UV radiation clearly augments the nuclear export of GRwt, but not that of the GRS226A mutant.

Nuclear Export of GR Is Enhanced by JNK Activation
As observed above, UV radiation enhances the nuclear export of GRwt, but not GRS226A. Thus, JNK activation may be responsible for the early nuclear export of GR. To explore this, we investigated whether overexpression of a dominant negative (DN)-SEK1 mutant might affect UV exposure-induced early nuclear export of GRwt. Gene transfer-mediated DN-SEK1 expression can suppress JNK activation induced by osmotic stress as shown in Fig. 5AGo.



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Figure 5. Effect of DN-SEK1 Overexpression on Nuclear Export of GRwt

A, DN-SEK or its vehicle-transfected COS-7 cells were serum deprived for 14 h, then treated with (+) or without (-) 0.5 M sorbitol for 15 min, and harvested at 30 min after the stress. The phosphorylation status of endogenous JNK was evaluated as described in Fig. 1. BGo, COS-7 cells were transfected with GFP-GRwt or GFP-GRS226A and DN-SEK1 or its vehicle as indicated and incubated for 16 h. The cells were serum deprived for 16 h, and then exposed (+) or unexposed (-) to UV radiation (20 J/m2) before incubation with 1 µM DEX for 1 h. After fixation at the indicated minute after DEX withdrawal, intracellular distribution of GFP-GRs was evaluated with confocal laser scanning. Quantitative analysis of the cellular distribution of GFP-GRs is shown (right panels).

 
As observed above, GFP-GRwt COS-7 cotransfectants exposed to UV showed the enhanced GR nuclear export, and approximately 40% showed a cytoplasmic localization at 1 h after DEX withdrawal. In contrast, the early nuclear export was not observed in COS-7 cotransfectants expressing DN-SEK1 mutant (Fig. 5BGo). The suppressive effect of DN-SEK1 mutant on early nuclear export of GFP-GRwt was similarly observed in the transfectants stimulated by osmotic stress (data not shown). However, DN-SEK1 mutant did not affect GR-export in GFP-GRS226A cotransfectants (Fig. 5BGo). We therefore conclude that JNK activation enhances GR nuclear export and that this event requires phosphorylation of Ser226 of GR by JNK.

GR-Mediated Transcription Was Not Affected by JNK Activation in COS-7 Cells Expressing GR Mutated at Ser226
As observed above, nuclear export of GR is enhanced by JNK activation. Because JNK activation inhibits GR-mediated transcriptional activation, JNK-mediated early nuclear export of GR may contribute to the inhibitory effect. COS-7 cells are a useful model system for these experiments because COS-7 cells have no functional GR, and gene transfer-mediated GR expression renders COS-7 cells responsive to DEX (Fig. 6AGo). We investigated the effect of the GRS226A mutation on GR-mediated transcription in these COS-7 reporter cells. The cells were transfected with either a GFP-GRwt or a GFP-GRS226A mutant expression plasmid, and DEX-induced GR-mediated transcription was monitored. There was no significant difference in DEX-induced GR-mediated transcription between GRwt- and GRS226A-transfected COS-7 cells (Fig. 6BGo). However, a large difference was observed in DEX-induced GR-mediated transcription of the transfected cells after UV radiation. UV radiation greatly reduced DEX-induced GR-mediated transcription in GRwt COS-7 transfectants. In contrast, UV irradiation was unable to suppress DEX-induced GR transcription in the GRS226A COS-7 transfectants (Fig. 6BGo). Thus, UV-induced suppression of GR-mediated transcription appears to depend upon JNK- mediated phosphorylation of GR at Ser226. To confirm that the UV effect was due to enhanced JNK activity, we investigated whether overexpression of JNK alone could suppress DEX-induced GR transcription. For this purpose COS-7 cells were transfected with GFP-GRwt or GFP-GRS226A plasmids together with either a HA-JNK1 expression vector or the vehicle alone and serum deprived for 24 h. Cotransfection of HA-JNK1 resulted in reduced GFP-GRwt-mediated transcription but had no effect on GFP-GRS226A-mediated transcription (Fig. 6CGo). Similar results were obtained in HeLa transfectants expressing HA-JNK1 with GFP-GRwt or GFP-GRS226A (data not shown). Thus, augmented JNK activity alone was able to suppress GR-mediated transcription depending upon phosphorylation at Ser226, indicating that the UV effect was mostly mediated by enhanced JNK activity.



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Figure 6. Effect of JNK Activation on GRwt and GRS226A-Mediated Transcription

A, COS-7 reporter cells were transiently transfected with either GFP-GRwt or its vehicle (Mock). Expressed human GR protein was detected by immunoblotting with an antibody specific to GR, and Hsc70 blotting was an indicator of protein loading (upper panels). Transfectants were treated with (+) or without (-) 1 µM DEX for 24 h, and transcriptional activity of the exogenous GR (solid bars) was assessed. B, GRwt (solid bars) or GRS226A (open bars) were transfected into COS-7 reporter cells. Similar amounts of GR and GRS226A proteins were expressed in the transfectants (upper panels). Transfectants were serum deprived for 2 h and exposed (+) or unexposed (-) to UV radiation (20 J/m2) before stimulation with (+) or without (-) 1 µM DEX for 1 h. Cells were harvested at 6 h after DEX withdrawal for measurement of luciferase activity. C, COS-7 reporter cells were transiently transfected with GRwt (solid bars) or GRS226A (open bars) together with a HA-JNK1 expression vector (+) or its vehicle (-). JNK1 and GR proteins were detected by immunoblotting with antibodies specific to JNK and GR, respectively (upper panels). Transfectants were serum deprived for 2 h before stimulation with (+) or without (-) 1 µM DEX for 1 h. Cells were harvested at 8 h after withdrawal of DEX for assessment of luciferase activity. The columns display the mean ± SD of data from three separate experiments.

 
Early Nuclear Export of GR Is Sensitive to Leptomycin B (LMB)
The exact mechanism by which GR nuclear export occurs is unclear at present. We therefore proceeded to characterize the mechanism by which JNK phosphorylation might modulate GR nuclear export. As described above, GR slowly returns to the cytoplasm after DEX withdrawal, taking about 4–8 h to display distinct cytoplasmic accumulation in most cells (see Fig. 1Go). In UV-exposed cells, nuclear export of GR was visible from as early as 30 min following DEX withdrawal. Because GR is a large molecule, it is unlikely that diffusion is responsible for the early nuclear export of GR, and thus an active transport mechanism is suggested. In fact, we found that cold temperature practically abolished UV-induced early nuclear export of GR (data not shown). GR possesses a leucine-rich segment with limited homology to a nuclear export signal (NES), but after DEX withdrawal, its nuclear export appears not to be sensitive to the LMB (21, 22), which directly binds to exportin 1/CRM1 and inhibits nuclear export of various signaling molecules (23). We thus investigated whether this active transport involves exportin 1/CRMI by determination of the effect of LMB on the early nuclear export of GR. GR nuclear export was monitored in the presence or absence of LMB after activation of the JNK pathway in COS-7 cells by UV radiation. In the absence of LMB, nuclear export of GR was strongly enhanced by UV radiation. However, LMB treatment significantly suppressed the early nuclear export of GR (Fig. 7Go). These observations strongly suggest that exportin 1/CRM1 plays a pivotal role in the stress-induced early nuclear export of GR.



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Figure 7. Effect of LMB on Early GR Nuclear Export

GRwt-transfected COS-7 cells were incubated in the presence (+) or absence (-) of 1 nM LMB for 1 h and then treated with or without UV radiation (20 J/m2) before addition of 1 µM DEX. After incubation for 1 h, DEX was then withdrawn and the cells were further incubated in the DEX-free medium containing LMB for 1 h. The cells were fixed with methanol, and the intracellular distribution of GFP-GRwt was investigated by confocal laser scanning. These experiments were repeated at least three times with similar results. Representative data of cells fixed at 60 min after DEX withdrawal are shown. As a control study, cells were fixed without LMB/UV treatment. Quantitative analysis of the sensitivity of GR nuclear export to LMB when treated with UV radiation is shown (right panel). Nuclear export was determined as described in Materials and Methods.

 
Similar Effect of JNK Activation on GR Nuclear Export in HeLa Cells
To explore whether JNK activation affects GR nuclear export in other cells, we investigated the effect of UV on the GR export in HeLa cells. When the cells were transfected with GFP-GRwt or GFP-GRS226A, and stimulated with DEX for 30 min, the GRs were both exclusively localized in the nucleus (data not shown). After withdrawal of DEX, both proteins slowly returned to the cytoplasm, and their nuclear export was not significantly different. As observed in COS-7 cells, UV radiation induced early nuclear export of GRwt; however, it resulted in limited effect in GRS226A transfectants (Fig. 8AGo). At 60 min after DEX withdrawal following UV radiation, approximately 28% of the GRwt transfectants, but only 8% of the GFP-GRS226A transfectants, showed a distinct cytoplasmic localization. In addition, we also investigated the effect of LMB on early nuclear export of GR. As observed in COS-7 GRwt-transfectants, UV-induced early nuclear export was substantially inhibited in the presence of LMB (Fig. 8BGo). Furthermore, the inhibitory effect of LMB was similarly observed in HeLa GRwt-transfectants exposed to 0.5 M sorbitol. Thus, the JNK-mediated early nuclear export of GR appears to be generally observed, and the export is sensitive to LMB regardless of the external stimulus employed (sorbitol or UV).



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Figure 8. Effect of UV Radiation on GR Nuclear Export in HeLa Cells and Its Sensitivity to LMB

A, HeLa cells were transiently transfected with CMX-GFP-GRwt or -GRS226A. Twenty-four hours after transfection, the cells were serum deprived for 12 h and then exposed (+) or unexposed (-) to UV radiation (20 J/m2) before incubation with 1 µM DEX for 1 h. After withdrawal of DEX, the transfectants were incubated for an additional 30–60 min in media alone and fixed. Intracellular distribution of GFP-GRs was evaluated by confocal laser scanning. Quantitative analysis of the effect of UV radiation on the nuclear export of GRs is shown (right panel). B, GRwt-transfected HeLa cells were incubated in the presence (+) or absence (-) of 1 nM LMB for 1 h and then treated with or without osmotic stress (0.5 M sorbitol) or UV (20 J/m2) radiation before addition of 1 µM DEX. After incubation for 1 h, DEX was then withdrawn and the cells were further incubated in the DEX-free medium containing LMB for 1 h. The cells were fixed and the intracellular distribution of GFP-GRwt was investigated by confocal laser scanning. These experiments were repeated at least three times with similar results. Representative data of cells fixed at 30 min after DEX withdrawal are shown. Quantitative analysis of the sensitivity of GR nuclear export to LMB when treated with osmotic stress or UV radiation is shown (right panel). Nuclear export was determined as described in Materials and Methods.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this paper we have demonstrated that JNK phosphorylates human GR at Ser226 and enhances its nuclear export after DEX withdrawal. UV irradiation, which activates the JNK pathway, and JNK1 overexpression enhance nuclear export of GRwt, but not that of GRS226A. In addition, overexpression of DN-SEK1, which is situated upstream of JNK1, inhibits early nuclear export. Thus enhanced nuclear export of GR appears to be the mechanism by which JNK phosphorylates GR at Ser226. Considering recent findings that nuclear export of transcription factors accounts for termination of factor-dependent expression of their target genes (24, 25, 26), these data suggest that the early nuclear export of GR is responsible for JNK-mediated inhibition of GR transcriptions. However, our data are not sufficient to confirm the hypothesis, because it is also conceivable that JNK may affect GR-mediated signals in many different ways, such as GR-DNA interaction, protein synthesis, and composition of GR complexes. Because JNK overexpression clearly inhibits GR-mediated signals, and the inhibition was mostly abrogated in the COS-7 transfectants expressing GRS226A mutant, GR phosphorylation appears to be crucial for the mechanism(s). In this regard, our findings illuminate early nuclear export of GR induced by JNK-mediated phosphorylation, but it should be further clarified how and where the GR phosphorylation at Ser226 affects the GR-mediated signaling pathways.

Although nuclear export of GR has been described, the precise molecular mechanism has not been established. Controversial results have emerged regarding the sensitivity of GR nuclear export to the exportin 1/CRM1 inhibitor LMB (21, 22, 27). A recent report suggests that nuclear export of GR is unlikely to be mediated by the exportin 1/CRM1 nuclear export pathway because GR export is insensitive to LMB (21). More recently, mutational analysis has revealed that a 15-amino acid sequence within the GR-DNA binding domain confers nuclear export (22). However, our preliminary data clearly demonstrate that at least the phosphorylation-dependent early nuclear export of GR is sensitive to LMB (Figs. 6Go and 7Go). This may indicate that there are two distinct mechanisms for the nuclear export of GR. The JNK-mediated early export is mediated by the exportin 1/CRM1-dependent system. However, this system is dispensable for the slow export of GR, which may be mediated by an export system(s) other than the exportin 1/CRM1 pathway. In fact, the previous report described the effect of LMB on only the slow nuclear export of GR, i.e. the exportation of GR at 4–8 h following DEX withdrawal (21), and our preliminary experiments also showed only a limited effect of LMB on the slow nuclear export of GR (data not shown).

It is interesting to note that the GR phosphorylation site Ser226 and its juxtaposed amino acid sequences are highly conserved between human and rat (see Fig. 2Go). Considering that both Ser226 (human) and Ser246 (rat) are major phosphorylation sites for JNK, the inhibitory effect of JNK on GR-mediated transcription is likely to be conserved throughout evolution. An examination of the sequences flanking the JNK phosphorylation sites suggest one potential mechanism by which this might occur. These flanking sequences include a stretch of leucine-rich sequences as previously pointed out (12, 21). It has been previously determined that one class of NES, which contributes to rapid and energy-dependent nuclear export, has hydrophobic and leucine-enriched sequences (28). Thus, it is likely that this segment in GR functions as an NES. It could be envisaged that phosphorylation of Ser226 might unmask the NES-like structure of GR, thereby enhancing its interaction with exportin 1/CRM1, thereby enhancing GR export. It is also notable that a small segment within the GR-DNA binding domain is suggested to be crucial for slow GR nuclear export after DEX withdrawal (22). These findings support our suggestion that JNK-mediated early nuclear export and DEX withdrawal-mediated slow export of GR are performed by two different mechanisms.

Regulation of the nuclear-cytoplasmic shuttling of GR by JNK is similar to the mechanism by which JNK negatively regulates the NFAT (nuclear factor of activated T cells) group of transcription factors (29, 30). NFAT is retained in the cytoplasm of quiescent cells, but once the cells are activated, NFAT is dephosphorylated by calcineurin and translocated to the nucleus (31). JNK activation opposes the nuclear accumulation of NFAT4 and induces its nuclear export (32). Thus, nuclear export of NFAT4 is mainly regulated by JNK-dependent phosphorylation. In the case of GR, nuclear accumulation is triggered by ligand binding, and its export is caused by withdrawal of ligand. Cytoplasmic distribution of GR can be distinctly observed in most cells at 4–8 h after DEX withdrawal, and its complete export from the nucleus takes more than 12 h (21). However, our data show that JNK activation strongly enhances the GR nuclear export within 1–2 h in significant populations. In this regard, JNK-mediated phosphorylation appears to modulate ligand-dependent nuclear-cytoplasmic shuttling of GR. To our knowledge, this modulation system is quite unique. In addition, we think that this regulation may reflect biological importance of GR-mediated signals. Considering that JNK is activated in response to exposure to environmental stresses and inflammatory signals (13, 16, 17, 18), whereas GR has potent antistress and antiinflammatory effects in a variety of cell types, they modulate conflicting signals. Our data therefore illuminate a cross-talk between JNK- and GR-mediated signals.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
Human GR cDNA was subcloned into the pCMX-GFP vector as described previously (33). GR, mutated at the JNK phosphorylation site Ser226 (GRS226A), was generated by replacing Ser226 with an Ala codon using a PCR mutagenesis method (34, 35). PCR was performed using the following oligonucleotide primers: 5'-CTGTTTGCTTGCTCCTCTGG-3' and 5'-CCAGAGGAGCAAGCAAACAG-3'. GRS226A cDNA was subcloned into the pCMX-GFP vector. The same constructs were also subcloned into the pCMX vector and used for luciferase assays. N-terminal domains (BglII-BglII-fragments) of both the wild-type and the GRS226A mutant were subcloned into the prokaryote expression pGEX4T-2 vector (Pharmacia Biotech, Piscataway, NJ). The mammalian expression plasmid pSR{alpha}-HA-JNK1 and pMT-HA-SEK1 (a dominant negative mutant; DN-SEK1) were described previously (36). The reporter plasmid GRE was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA).

Tissue Culture and Transient Transfection
HeLa and COS-7 cells were cultured in DMEM containing 10% fetal calf serum and transiently transfected using the Effectene Transfection Reagent (QIAGEN, Chatsworth, CA) according to the manufacturer‘s protocol. The cells were incubated with 1 µM water-soluble DEX (Sigma) 18–24 h after transfection and further incubated as indicated in the text. For the enhancement of JNK activation, cells were exposed to UV-C (20 or 40 J/m2) exposure or incubated in 0.5 M sorbitol for 15 min.

Western Blotting
After washing with ice-cold PBS, cells were lysed by the addition of 50 µl of lysis buffer (140 mM NaCl; 2 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; 1% Nonidet P-40; and 50 mM Tris-HCl, pH 7.2). Total cell lysates were collected and their protein concentration was evaluated using a protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). The lysates (80 µg/lane) were separated by 10–15% SDS-PAGE gels and then electrophoretically transferred to polyvinylidine difluoride membranes (Millipore Corp., Bedford, MA) at 18 V for 70 min. The membranes were blocked in 8% BSA overnight at 4 C followed by washing with washing buffer [140 mM NaCl, 25 mM Tris-HCl (pH 7.8), and 0.05% Tween 20]. The membranes were then incubated with primary antibodies overnight at 4 C, and thereafter incubated with the corresponding peroxidase-linked secondary antibody (Amersham Pharmacia Biotech, Arlington Heights, IL) or MBL for 1 h at room temperature. Signals were developed by a standard enhanced chemiluminescence method following the manufacturer’s protocol (Amersham Pharmacia Biotech).

Subcellular Fractionation
To prepare cytoplasmic and nuclear fractions, COS-7 transfectants were collected, washed, and suspended in cold hypotonic buffer (10 mM HEPES, pH 7.5; 1.5 mM MgCl2; 10 mM KCl; 1 mM phenylmethylsulfonyl fluoride; 0.5 mM dithiothreitol). The cells were lysed with a Dounce homogenizer, and nuclei were collected by centrifugation two times for 5 min at 5000 rpm in Eppendorf tubes (Eppendorf North America, Inc., Madison, WI). The supernatant was saved as the cytosolic fraction. Nuclei were lysed by addition of the lysis buffer and the lysate was saved as the nuclear fraction. These fractions were subjected to SDS-PAGE and immunoblotting.

In Vitro Kinase Reactions
The N-terminal domains (BglII-BglII fragments) of GR and GRS226A cDNAs were subcloned into the GEX 4T-2 prokaryote expression vector (Pharmacia Biotech, Piscataway, NJ). GST, GST-c-jun (full-length), GST-GR (77–262), and GST-GRS226A (77–262) were expressed in Escherichia coli BL21 and purified using glutathione-Sepharose beads according to the manufacturer‘s protocol. To define the JNK phosphorylation site within GR, HA-tagged JNK1 cDNA was transiently transfected into HeLa cells. The cells were activated by osmotic stress (0.5 M sorbitol for 15 min) and immediately lysed and immunoprecipitated with the anti-HA monoclonal antibody 12CA5 (Roche Molecular Biochemicals) for 2 h at 4 C. Immunocomplexes were recovered with the aid of Protein A-Sepharose beads, washed three times with lysis buffer and twice with kinase buffer [40 mM HEPES (pH 7.4), 3 mM MnCl2, 10 mM MgCl2, and 1 mM dithiothreitol], and resuspended in 30 µl of the kinase buffer containing 1 µg of the substrate protein (GST, GST-c-jun, GST-GR, or GST-GRS226A). The kinase reaction was initiated by the addition of 10 µCi of [{gamma}-32P] ATP (Amersham Pharmacia Biotech) and 5 µM cold ATP. After 10 min incubation at 25 C, the reactions were terminated by the addition of sodium dodecyl sulfate loading buffer, and the resultant samples were subjected to SDS-PAGE analysis.

Determination of GR-Mediated Transcription
GR-mediated transcription was measured using a dual reporter luciferase assay system. Briefly, COS-7 or HeLa cells were transfected with a pRL-TK reporter plasmid (Promega Corp., Madison, WI) and a luciferase reporter plasmid driven by the GRE motif (CLONTECH Laboratories, Inc.) using the Effectene Transfection Reagent. These cells are referred to as COS-7 or HeLa reporter cells. GR-mediated transcription, generated by cotransfected GR constructs, was measured as luciferase activity using a luminometer (Mini Lumat LB 9506) and normalized to Renilla luciferase activity. To detect the effect of the GRS226A mutation on GR-mediated transcription, either the pCMX-GR or -GRS226A expression plasmids were transfected into the COS-7 reporter cells. Eighteen hours after transfection, transfectants were stimulated with or without 1 µM DEX for 1 h. To exclude the effect of serum, some transfectants were serum starved for 2 h before stimulation with DEX. Cells were harvested at 8 h following withdrawal of DEX for assessment of luciferase activity. To evaluate the effect of UV radiation, transfectants were simultaneously stimulated with or without exposure to UV (20 J/m2). For the determination of the effect of JNK activation on GR-mediated transcription, the reporter cells were transfected with either a GRwt or a GRS226A expression plasmid together with HA-JNK or its vehicle. Similar experiments were performed using HeLa reporter cells. To investigate whether JNK activation is required for stress-mediated suppression of GR-transcription, the HeLa reporter cells were transfected with a DN-SEK1 mutant expression plasmid.

Confocal Laser Scanning
COS-7 and HeLa cells were transiently transfected with either pEGFP-GR or pEGFP-GRS226A using the Effectene Transfection Reagent and treated with 1 µM DEX for 5–60 min. To investigate the effect of JNK activation on intracellular distribution of GR, cells were exposed to UV (20 J/m2) or 0.5 M sorbitol (15 min). To evaluate intracellular distribution of GR, we selected transfectants expressing GFP-GRs moderately, which exhibited a major population at 24–36 h after transfection. Intracellular localization of GFP-GR was classified into two groups: 1) nuclear, defined as exclusively nuclear or nuclear exceeding cytoplasmic localization; and 2) cytoplasmic/nuclear export, defined as either equivalent nuclear and cytoplasmic localization or cytoplasmic exceeding nuclear localization. The nuclear export of GFP-GR was determined by counting at least 100 cells per experimental condition. Confocal imaging was performed using an MRC-1024 laser scanner (Bio-Rad Laboratories, Inc.).

Antibodies and Reagents
The anti-GR, -JNK, -GFP, -Lamin B, and -HSC70 antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antiphospho-JNK kinase antibody was from New England Biolabs, Inc. (Beverly, MA). The anti-HA monoclonal antibody 12CA5 was from Roche Molecular Biochemicals. Sorbitol, CHX, and LMB were from Sigma.


    ACKNOWLEDGMENTS
 
We are very grateful to Dr. Michel Karin for providing us JNK and c-jun cDNAs.


    FOOTNOTES
 
This work was supported by a Grants-in-Aid for Cancer Research and for Scientific Reserach from the Ministry of Education, Science, Sports and Culture of Japan.

1 M.I. and M.A. contributed equally to this work. Back

Abbreviations: Cdk, Cyclin-dependent kinase; CHX, cycloheximide; DEX, dexamethasone; DN, dominant negative; GFP, green fluorescent protein; GR, glucocorticoid receptor; GRE, glucocorticoid response element; GST, glutathione-S-transferase; HA, hemagglutinin; JNK, c-Jun N-terminal kinase; LMB, leptomycin B; NES, nuclear export signal; NFAT, nuclear factor of activated T cells; wt, wild-type.

Received for publication April 17, 2002. Accepted for publication June 12, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Yamamoto KR 1985 Steroid receptor regulated transcription of specific genes and gene networks. Annu Rev Genet 19:209–252[CrossRef][Medline]
  2. Angeli A, Masera RG, Sartori ML, Fortunati N, Racca S, Dovio A, Staurenghi A, Frairia R 1999 Modulation by cytokines of glucocorticoid action. Ann NY Acad Sci 876:210–220[Abstract/Free Full Text]
  3. Cato AC, Wade E 1996 Molecular mechanisms of anti-inflammatory action of glucocorticoids. Bioessays 18:371–378[Medline]
  4. Madan AP, DeFranco DB 1993 Bidirectional transport of glucocorticoid receptors across the nuclear envelope. Proc Natl Acad Sci USA 90:3588–3592[Abstract]
  5. Hache RJ, Tse R, Reich T, Savory JG, Lefebvre YA 1999 Nucleocytoplasmic trafficking of steroid-free glucocorticoid receptor. J Biol Chem 274:1432–1439[Abstract/Free Full Text]
  6. Htun H, Barsony J, Renyi I, Gould DL, Hager GL 1996 Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera. Proc Natl Acad Sci USA 93:4845–4850[Abstract/Free Full Text]
  7. Slater EP, Hesse H, Beato M 1994 Regulation of transcription by steroid hormones. Ann NY Acad Sci 733:103–112[Medline]
  8. Deroo BJ, Archer TK 2001 Glucocorticoid receptor- mediated chromatin remodeling in vivo. Oncogene 20:3039–3046[CrossRef][Medline]
  9. Herrlich P 2001 Cross-talk between glucocorticoid and AP-1. Oncogene 20:2465–2475[CrossRef][Medline]
  10. Krstic MD, Rogatsky I, Yamamoto KR, Garabedian MJ 1997 Mitogen-activated and cyclin-dependent protein kinases selectively and differentially modulate transcriptional enhancement by the glucocorticoid receptor. Mol Cell Biol 17:3947–3954[Abstract]
  11. Rogatsky I, Waase CL, Garabedian MJ 1998 Phosphorylation and inhibition of rat glucocorticoid receptor transcriptional activation by glycogen synthase kinase-3 (GSK-3). J Biol Chem 273:14315–14321[Abstract/Free Full Text]
  12. Rogatsky I, Logan SK, Garabedian MJ 1998 Antagonism of glucocorticoid receptor transcriptional activation by the c-Jun N-terminal kinase. Proc Natl Acad Sci USA 95:2050–2055[Abstract/Free Full Text]
  13. Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin M, Davis RJ 1994 JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell 76:1025–1037[Medline]
  14. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, Woodgett JR 1994 The stress-activated protein kinase subfamily of c-Jun kinases. Nature 369:156–160[CrossRef][Medline]
  15. Minden A, Lin A, McMahon M, Lange-Carter C, Derijard B, Davis RJ, Johnson GL, Karin M 1994 Differential activation of ERK and JNK mitogen-activated protain kinases by Raf-1 and MEKK. Science 266:1719–1723[Medline]
  16. Hibi M, Lin A, Smeal T, Minden A, Karin M 1993 Identification of an oncoprotein-and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev 7:2135–2148[Abstract]
  17. Westwick JK, Weitzel C, Minden A, Karin M, Brenner DA 1994 Tumor necrosis factor {alpha} stimulates AP-1 activity through prolonged activation of the c-Jun kinase. J Biol Chem 269:26396–26401[Abstract/Free Full Text]
  18. Sluss HK, Barrett T, Derijard B, Davis RJ 1994 Signal transduction by tumor necrosis factor mediated by JNK protein kinases. Mol Cell Biol 14:8376–8384[Abstract]
  19. Whitmarsh AJ, Davis RJ 1996 Transcription factor AP-1 regulation by mitogen-activated protein kinase signal transduction pathways. J Mol Med 74:589–607[CrossRef][Medline]
  20. Yang J, Liu J, DeFranco DB 1997 Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export. J Cell Biol 137:523–538[Abstract/Free Full Text]
  21. Liu J, DeFranco DB 2000 Protracted nuclear export of glucocorticoid receptor limits its turnover and does not require the exportin1/CRAM1-directed nuclear export pathway. Mol Endocrinol 14:40–51[Abstract/Free Full Text]
  22. Black BE, Holaska JM, Rastinejad F, Paschal BM 2001 DNA binding domains in diverse nuclear receptors function as nuclear export signals. Curr Biol 11:1749–1758[CrossRef][Medline]
  23. Kudo N, Wolff B, Sekimoto T, Schreiner EP, Yoneda Y, Yanagida M, Horinouchi S, Yoshida M 1998 Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. Exp Cell Res 242:540–547[CrossRef][Medline]
  24. Freedman DA, Levine AJ 1998 Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6. Mol Cell Biol 18:7288–7293[Abstract/Free Full Text]
  25. Yan C, Lee LH, Davis LI 1998 Crm1p mediates regulated nuclear export of a yeast AP-1-like transcription factor. EMBO J 17:7416–7429[Abstract/Free Full Text]
  26. Johnson C, Van Antwerp D, Hope TJ 1999 An N-terminal nuclear export signal is required for the nucleocytoplasmic shuttling of I{kappa}B{alpha}. EMBO J 18:6682–6693[Abstract/Free Full Text]
  27. Savory JG, Hsu B, Laquian IR, Griffin W, Reich T, Hache RJ, Lefebvre YA 1999 Discrimination between NL1-and NL2-mediated nuclear localization of the glucocorticoid receptor. Mol Cell Biol 19:1025–1037[Abstract/Free Full Text]
  28. Gerace L 1995 Nuclear export signals and the fast track to the cytoplasm. Cell 82:341–344[Medline]
  29. Hoey T, Sun YL, Williamson K, Xu X 1995 Isolation of two new members of the NF-AT gene family and functional characterization of the NF-AT proteins. Immunity 2:461–472[Medline]
  30. Masuda ES, Naito Y, Tokumitsu H, Campbell D, Saito F, Hannum C, Arai K, Arai N 1995 NFATx, a novel member of the nuclear factor of activated T cells family that is expressed predominantly in the thymus. Mol Cell Biol 15:2697–2706[Abstract]
  31. Beals CR, Clipstone NA, Ho SN, Crabtree GR 1997 Nuclear localization of NF-Atc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction. Genes Dev 11:824–834[Abstract]
  32. Chow CW, Rincon M, Cavanagh J, Dickens M, Davis RJ 1997 Nuclear accumulation of NFAT4 opposed by the JNK signal transduction pathway. Science 278:1638–1641[Abstract/Free Full Text]
  33. Okamoto K, Tanaka H, Ogawa H, Makino Y, Eguchi H, Hayashi S, Yoshikawa N, Pellinger L, Umesono K, Makino I 1999 Redox-dependent regulation of nuclear import of the glucocorticoid receptor. J Biol Chem 274:10363–10371[Abstract/Free Full Text]
  34. Kadowaki H, Kadowaki T, Wondisford FE, Taylor SL 1989 Use of polymerase chain reaction catalysed by Taq DNA polymerase for site-specific mutagenesis. Gene 76:161–166[CrossRef][Medline]
  35. Vallette F, Mege E, Reiss A, Milton A 1989 Construction of mutant and chimeric genes using the polymerase chain reaction. Nucleic Acids Res 17:723–733[Abstract]
  36. Takekawa M, Saito H 1998 A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95:521–530[Medline]