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Originally published In Press as doi:10.1074/jbc.M602556200 on June 16, 2006

J. Biol. Chem., Vol. 281, Issue 33, 23870-23879, August 18, 2006
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Regulation of Nucleocytoplasmic Trafficking of Transcription Factor OREBP/TonEBP/NFAT5*

Edith H. Y. Tong{ddagger}§, Jin-Jun Guo{ddagger}§, Ai-Long Huang, Han Liu||, Chang-Deng Hu||, Stephen S. M. Chung{ddagger}**1, and Ben C. B. Ko{ddagger}§2

From the {ddagger}Department of Chemistry, §Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, and the **Department of Physiology, the University of Hong Kong, Hong Kong Special Administrative Region, China, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chong Qing, China, and the ||Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University School of Pharmacy, West Lafayette, Indiana 47907

Received for publication, March 20, 2006 , and in revised form, June 16, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels, including nucleocytoplasmic trafficking. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The exposure of mammalian cells to extracellular hypertonicity elicits a genetic program of adaptive cellular responses, which includes the synthesis and accumulation of organic osmolytes such as sorbitol, betaine, and myoinositol (1) to replace intracellular electrolytes that are otherwise deleterious to normal cellular function (2). Hypertonicity also induces the expression of heat shock protein 70 (HSP-70) (3) that acts as a molecular chaperone to protect cells from osmotic stress-induced apoptosis (4). The accumulation of organic osmolytes is brought about by the induction of a battery of genes, including aldose reductase (AR), betaine/{gamma}-aminobutyric acid transporter (BGT-1), and Na+-dependent myoinositol transporter (SMIT), which is responsible for the synthesis of sorbitol and the uptake of betaine and myoinositol, respectively. The hypertonic induction of these genes, including HSP-70, is controlled at the transcriptional level and is mediated by a common cisacting element known as the osmotic response element or the TonE (tonicity-responsive enhancer) (5-8).

The transcription factor that binds to the TonE/ORE, known as TonEBP3 or OREBP, was independently identified by a yeast one-hybrid assay (9) and its affinity purification (10). OREBP/TonEBP was also shown to be identical in sequence to NFAT5, a protein independently identified as a member of the NFAT family of transcription factors (11). However, unlike other members of the NFAT family, such as NFATc1-4 (12), OREBP/TonEBP lacks the calcineurin binding domain and is therefore not regulated by Ca2+ and calcineurin. It also does not form a cooperative complex with Fos and Jun for DNA binding. Therefore, OREBP/TonEBP is regarded as a distant member of the NFAT family (11).

OREBP/TonEBP is regulated at multiple levels. Hypertonicity induces its nuclear localization (10, 13, 14). This is accompanied by an increase in the level of OREBP/TonEBP mRNA andprotein(9,13). In addition, hypertonicity increases the phosphorylation of OREBP/TonEBP as well as the activity of its transactivation domain (14-16). Nevertheless, the identification of specific phosphorylation site(s) and the evidence directly showing whether phosphorylation is required for its transcriptional activation activity remain elusive (14).

It appears that the proteasome is involved in the hypertonicity-induced nuclear translocation of OREBP/TonEBP, as the prevention of its nuclear accumulation using a proteasome inhibitor blocked hypertonic induction of SMIT and BGT-1(17). Apparently the nuclear translocation is bi-directional, because hypotonicity reduces the nuclear staining of OREBP/TonEBP (13). Here we identify and characterize three protein motifs, including a nuclear export sequence (NES), a putative auxiliary export domain (AED), and a nuclear localization signal (NLS), that are responsible for OREBP/TonEBP nucleocytoplasmic shuttling in response to changes in extracellular tonicity. These results provide a better understanding of the mechanism of tonicity-regulated nucleocytoplasmic shuttling of OREBP/TonEBP.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Constructs—Human OREBP/TonEBP cDNA clone KIAA0827 was a gift from Dr. Takahiro Nagase (Kazusa DNA Research Institute, Japan). FLAG-OREBPWT, FLAG-OREBP1-581, FLAG-OREBP1-581{Delta}1-131, and FLAG-OREBP1-581{Delta}1-156 were derived by in-frame insertion of KIAA0827 cDNA corresponding to amino acid residues 1-1531, 1-581, 132-581, and 157-581 into NotI and BamHI restriction sites of pFLAG-CMV-2 mammalian expression vector (Sigma), respectively. FLAG-OREBP1-581{Delta}132-156 was derived by in-frame insertion of the cDNA fragment corresponding to amino acid residues 1-131 into the NotI restriction site of FLAG-OREBP1-581{Delta}1-156. FLAG-OREBP1-581L4A, FLAG-OREBP1-581RKR202-204AAA, and FLAG-OREBP1-581KRR213-215AAA were constructed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) using FLAG-OREBP1-581 as template. To construct the OREBP-GFP chimeras, OREBP/TonEBP cDNAs corresponding to amino acid residues 1-581 and 157-581 were cloned in-frame into pEGFP-N1 (Clontech), respectively. CRM1 cDNA (OriGene Technologies, Inc.) was cloned in-frame into Myc tag expression vector pCMV-Tag-3C (Stratagene).

A newly engineered fluorescent protein, Venus (18), was used for the bimolecular fluorescence complementation (BiFC) analysis. The cDNA encoding N-terminal residues 1-172 (VN173) and C-terminal residues 155-238 (VC155) of Venus were subcloned into pHA-CMV (Clontech) vectors to make BiFC cloning vectors. pHA-CRM1566-720VN was constructed as described (19). OREBP1-581VC and OREBP1-581L4AVC were derived by in-frame insertion of the OREBP/TonEBP mutants to VC155. All constructs were verified by DNA sequencing.

Cell Cultures and Transfection—HeLa cells (American Type Culture Collection, Manassas, VA) were maintained in minimal essential medium supplemented with 10% fetal bovine serum. For OREBP/TonEBP subcellular localization studies, cells grown in 6-well plates to 50% confluence were transfected with 0.3-0.5 µg of the plasmids expressing the proteins as indicated in each experiment using GeneJuice (Novagen) or Lipofectamine (Invitrogen) according to the manufacturers' instructions. Transfected cells were incubated for 16-24 h in complete growth medium before switching to medium with different osmolality. Hypotonic (250 mosmol/kg H2O), isotonic (300 mosmol/kg H2O), or hypertonic (450 mosmol/kg H2O) medium was prepared by supplementing NaCl to NaCl-deficient growth medium (5.4 mM KCl, 0.8 mM MgSO4, 1.8 mM CaCl2, 1 mM NaH2PO4, 25 mM NaHCO3, 5.5 mM glucose, 1x minimum Eagle's medium amino acids solution, and 1x vitamin solution (Invitrogen), 2 mM glutamine, 10% fetal bovine serum, pH 7.4) to the desired osmolality. Medium osmolality was measured by the Vapro® vapor pressure osmometer (Wescor). For nuclear export assays, cells were either left untreated or treated with 10 ng/ml leptomycin B (Sigma) for the indicated times and osmolality. For cycloheximide studies, cells were treated with 5 µg/ml cycloheximide for the indicated time.

Immunofluorescence, Green Fluorescence Microscopy, and BiFC Analysis—Cells were washed three times with PBS and fixed with 4% w/v paraformaldehyde for 15 min at 4 °C. The cells were permeabilized with absolute methanol for 2 min at room temperature. Primary antibodies against FLAG (Sigma), TonEBP (a kind gift from Prof. H. M. Kwon, University of Maryland), or Myc (Santa Cruz Biotechnology) were used. The fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibody (Chemicon International, Temecula, CA) or TRITC-conjugated anti-rabbit antibodies (Molecular Probes) were used as secondary antibodies. To visualize the nuclei, the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma). Expression of GFP fusion proteins was determined by green fluorescence microscopy.

For BiFC analysis, HeLa cells were transfected with plasmids encoding VN and VC fusion proteins. HA-ECFP vector was cotransfected as an internal control to measure the BiFC efficiency of fragments derived from Venus. Cells were incubated for 16 h before analysis. Yellow fluorescent protein emission of the VN155 and VC173 complexes was measured at 520 ± 10 nm during excitation at 490 ± 15 nm. CFP fluorescence was measured at 470 ± 15 nm during excitation at 436 ± 5 nm. The fluorescent images were captured using an Olympus IX71 fluorescence microscope fitted with a SPOT RT digital camera (Diagnostic Instrument, Inc., Melville, NY). The intensity of fluorescence of the individual cell was quantified using an automated intensity recognition feature of Metamorph II (Universal Imaging Corp., Downingtown, PA). The median of the Venus/CFP ratio was used to calculate the fold increase of BiFC efficiency as a better measure for highly skewed distribution (20).

Western Blotting Analysis—Expression of recombinant proteins was also confirmed by Western blotting analysis of total cell extracts. At 16 h after transfection, cells were washed twice with ice-cold PBS and collected in lysis buffer containing 20 mM Tris-Cl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM beta-glycerol phosphate, 1% Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitors. After being rotated at 4 °C for 20 min, the cell lysates were cleared by high speed centrifugation at 4 °C, and the supernatants were collected as total cell extracts. For immunoblotting, the total cell extracts (10 mg/lane) were resolved by reducing SDS-PAGE, electrotransferred to nitrocellulose membrane, and subsequently immunoblotted with anti-FLAG (Sigma), anti-HA (Roche Applied Science), or anti-{alpha}-tubulin (Sigma) antibodies as indicated. Anti-rabbit, anti-rat, or anti-mouse horseradish peroxidase-linked IgG (Amersham Biosciences) was used as secondary antibodies. TBS with 0.1% Tween 20 was used for washing the membranes. Blots were developed with ECLTM Western blotting reagents (Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
OREBP/TonEBP Undergoes Nucleocytoplasmic Trafficking in Response to Changes in Tonicity, but the C-terminal Transactivation Domain Is Not Required for This Process—OREBP/TonEBP consists of an N-terminal transactivation domain (AD1, amino acids 1-76) (14), a putative bipartite NLS (amino acids 199-216), a Rel-homologous DNA binding domain that shares significant homology with the NFAT family of transcription factors (amino acids 264-543) (10, 11), followed by an extended glutamine-rich transactivation domain at the C terminus, which can be further divided into multiple modulation and activation domains (AD2, amino acids 618-1476) (Fig. 1A) (14). Indirect immunofluorescence analysis with anti-OREBP/TonEBP antibodies showed that hypertonicity increases the nuclear staining of OREBP/TonEBP (9), whereas the staining was reduced substantially when cells were subjected to prolonged hypotonic treatment (18 h) (13), suggesting that OREBP/TonEBP activity may be regulated by nucleocytoplasmic trafficking. To examine the mechanism of OREBP/TonEBP nucleocytoplasmic trafficking, we sought to identify the protein domains that were responsible for its nuclear import and export. The structural organization of wild type OREBP/TonEBP and that of the deletion mutants created for this study are depicted (Fig. 1A). We transiently expressed FLAG-tagged wild type OREBP/TonEBP (FLAG-OREBPWT) cDNA in HeLa cells, and we determined its subcellular localization under different extracellular tonicity conditions. As shown in Fig. 1B, indirect immunofluorescence analysis using fluorescein-tagged FLAG antibodies showed that under isotonicity, a majority of the transfected cells (~50%) exhibited cytoplasmic fluorescence, whereas the remaining cells showed either pan-cellular (~30%) or nucleoplasmic (~20%) fluorescence. However, the fluorescence signal was found exclusively in the cytoplasm or in the nucleus when cells were challenged with hypotonicity or hypertonicity, respectively. The presence of cycloheximide did not alter the hypotonicity- or hypertonicity-induced subcellular localization of FLAG-OREBPWT, suggesting that the observed translocation of FLAG-OREBPWT was because of bona fide nucleocytoplasmic trafficking rather than de novo protein synthesis (Fig. 1C).

To determine whether the transactivation domain AD2 contains protein motif(s) responsible for nucleocytoplasmic trafficking, a mutant OREBP/TonEBP lacking AD2 (FLAG-OREBP1-581) was generated (Fig. 1A). Similar to the findings with the FLAG-OREBPWT protein, the majority of FLAG-OREBP1-581 was detected in the cytoplasm when cells were maintained in isotonic medium, whereas hypotonicity or hypertonicity led to cytoplasmic or nuclear localization, respectively (Fig. 1B). To demonstrate that this AD2-deficient OREBP/TonEBP can be subjected to nucleocytoplasmic trafficking similar to the exogenous protein in response to changes in tonicity, OREBP1-581 was fused to GFP (OREBP1-581-GFP), and the localization of the resultant fusion protein was assessed (Fig. 1A). OREBP1-581-GFP was found to undergo nucleocytoplasmic trafficking similar to that of FLAG-OREBPWT in response to the changes in tonicity. On the other hand, GFP alone was found in both the nucleus and the cytoplasm of transfected cells, and its localization did not alter with changes in tonicity (Fig. 1D). Western blot analysis showed that the FLAG-OREBPWT, FLAG-OREBP1-581, and OREBP1-581-GFP proteins were correctly expressed (Fig. 1E). Taken together, these data suggested that the first 581 amino acid residues of OREBP/TonEBP contain essential protein domain(s) required for nucleocytoplasmic trafficking.

A Nuclear Import Signal Is Responsible for OREBP/TonEBP Nuclear Import—Next we addressed whether the isotonic distribution of OREBP is also actively regulated, i.e. whether the resting localization represents a steady state of continuous import and export. Because the size of OREBP/TonEBP (>180 kDa) precludes a passive nuclear transport mechanism (21), we investigated whether it underwent active nucleocytoplasmic shuttling under isotonic conditions. Large proteins shuttle between the nucleus and the cytoplasm through nuclear pore complexes by virtue of their NLS and NES, which are recognized by specific import and export receptors, respectively (22). A motif scan of its amino acid sequence revealed that OREBP/TonEBP, similar to many other nuclearly targeted proteins, contains a consensus bipartite NLS (amino acids 199-216) where two clusters of basic amino acids, residues 202-204 (RKR) and residues 213-215 (KRR), were aligned in tandem (Fig. 2A) (23). To assess the importance of these two clusters of basic amino acids in the identified NLS sequence, we constructed two mutants of FLAG-OREBP1-581, FLAG-OREBP1-581RKR202-204AAA and FLAG-OREBP1-581KRR213-215AAA, where the first or second clusters of basic amino acids were mutated to alanines, respectively (Fig. 2A). The mutation at the first basic cluster completely abolished nuclear localization of OREBP/TonEBP when cells were treated with hypertonicity (Fig. 2B). In contrast, mutation of the second basic cluster had no effect on subcellular localization of the mutant protein (Fig. 2B). These results indicated that only the first basic cluster contributes to the nuclear import of OREBP/TonEBP, and therefore the identified NLS is a monopartite NLS.

A CRM-1-responsive Nuclear Export Signal Is Responsible for OREBP/TonEBP Nuclear Export under Isotonic Conditions—To examine the presence of functional NES in OREBP/TonEBP, HeLa cells were transfected with FLAG-OREBP1-581 or OREBP1-581-GFP and treated with leptomycin B (LMB) (24), a specific inhibitor of the nuclear export receptor exportin-1 (CRM1) (25, 26). As shown in Fig. 3A, under isotonic conditions in the absence of LMB, FLAG-OREBP1-581 and OREBP1-581-GFP proteins were detected in both the cytoplasmic and nuclear compartments within HeLa cells (isotonic, -LMB). The addition of LMB led to the nuclear retention of both proteins (Fig. 3A, isotonic, +LMB), suggesting that OREBP/TonEBP is subjected to active nuclear export in a CRM1-dependent manner. To examine if LMB also blocks hypotonicity-induced OREBP/TonEBP nuclear export, HeLa cells were first subjected to hypertonic conditions in the presence of LMB to induce nuclear translocation of the FLAG and GFP fusion proteins. This was followed by moving the cells to a hypotonic medium to induce nuclear export in the presence or absence of LMB. FLAG-OREBP1-581 and OREBP1-581-GFP were found to localize exclusively in the nucleus when cells were treated with hypertonic medium (data not shown) and localized entirely in the cytoplasm when switched to hypotonic medium in the absence of LMB (Fig. 3A, hypotonic, -LMB). Interestingly, LMB failed to prevent hypotonicity-induced nuclear export of FLAG-OREBP1-581 and OREBP1-581-GFP (Fig. 3A, hypotonic, +LMB).

The inhibition of the nuclear export of OREBP/TonEBP by LMB indicated that the protein may contain a NES. Canonical NESs contain a stretch of hydrophobic residues, predominantly leucine and isoleucine, separated by a short spacer of 1-4 amino acids (25). We searched for putative nuclear export sequences in OREBP/TonEBP using the NES prediction server, NetNES (27). Analysis of the human OREBP/TonEBP sequence revealed one core region containing four leucine residues (leucine 8, 9, 13, and 15) with a NES motif score of >0.5. Furthermore, analysis of this leucine-rich sequence revealed the presence of a NES domain (amino acid 8-15) similar to other well characterized NESs, including Rev, p53, c-ABL, and Ran-BP1 (Fig. 3B). To test the functional relevance of this putative NES, we generated an OREBP/TonEBP mutant, in which the four consensus leucine residues in the core NES were substituted with alanines (FLAG-OREBP1-581L4A) (Fig. 3C). Unlike FLAG-OREBP1-581, FLAG-OREBP1-581L4A was localized to the nucleus under isotonic conditions (Fig. 3D, isotonic), suggesting that the putative NES is a functional NES motif. However, similar to FLAG-OREBP1-581, FLAG-OREBP1-581L4A was subjected to hypotonicity-induced nuclear export (Fig. 3D, hypotonic). Nuclear export was not inhibited by the addition of LMB (data not shown). Moreover, cycloheximide did not alter the subcellular localization of FLAG-OREBP1-581L4A under hypotonic conditions (data not shown), and therefore the observed cytoplasmic signal was because of bona fide nuclear export. Collectively, these data suggested that nuclear export of OREBP/TonEBP under isotonic conditions is a NES-dependent process mediated by CRM1, whereas hypotonicity-induced nuclear export is mediated by the NES- and CRM1-independent process.


Figure 1
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  		FIGURE 1.
Subcellular localization of OREBP/TonEBP mutants. A, schematic illustration of OREBP/TonEBP and mutants. AD1 and AD2, putative transcriptional activation domains; DD, dimerization domain. B, representative fluorescence images of fixed HeLa cells expressing recombinant OREBP/TonEBP. Cells were transfected with the indicated OREBP expression plasmids. Subsequently, the cells were either treated with isotonic, hypotonic, or hypertonic medium for 90 min, respectively. FLAG-tagged recombinant protein was visualized after fixation with a FLAG antibody and a FITC-labeled secondary antibody, counterstained with DAPI, and analyzed by fluorescence microscopy. C, effect of cycloheximide on subcellular localization of recombinant OREBP/TonEBP. HeLa cells transfected with FLAG-OREBPWT were pretreated with cycloheximide (5 µg/ml) for 1 h. Subsequently, the cells were either treated with hypertonic or hypotonic medium in the presence of the drug for another 90 min. D, subcellular localization of OREBP1-581-GFP fusion protein under different osmolality. HeLa cells were transfected with OREBP1-581-GFP or GFP-expressing construct and were cultured in isotonic, hypotonic, or hypertonic medium for 90 min, respectively. GFP fluorescence was evaluated in live cells. E, expression of FLAG-tagged and GFP-tagged OREBP fusion proteins as determined by Western blotting. HeLa cells were transfected with plasmids encoding FLAG-OREBPWT, FLAG-OREBP1-581, and OREBP1-581-GFP, respectively. Cell lysates were loaded for the determination of protein expression using immunoblotting analysis with anti-FLAG (for FLAG-OREBPWT and FLAG-OREBP1-581), anti-OREBP (for OREBP1-581-GFP), and anti-tubulin antibodies. Images were examined with a x40 objective. Scale bar, 100 µm. Data shown are representative of at least three independent transfections for each construct.

 
Identification of a Protein Domain That Is Critical for Hypotonicity-induced Nuclear Export—To identify the functional domain responsible for hypotonicity-induced nuclear export, we generated deletion mutants of FLAG-OREBP1-581 (Fig. 4A) and determined the subcellular localization of these mutants under different tonicities. HeLa cells expressing these mutants were transferred to hypotonic, isotonic, or hypertonic medium, respectively, and the subcellular localization of the mutant proteins was recorded and scored quantitatively (Fig. 4B). Representative pictures of the mutant proteins under different culture conditions are shown in Fig. 4C. As expected, FLAG-OREBP1-581 was predominantly localized to the cytoplasm (98%) or to the nucleus (95%) under hypotonic or hypertonic conditions, respectively, whereas the majority of the FLAG signal was cytoplasmic (54%) and pan-cellular (28%) under isotonic conditions. However, FLAG-OREBP1-581{Delta}1-131 (with the first 131 amino acids including the canonical NES deleted) was predominantly localized to the nucleus (79%) under isotonic conditions, whereas its subcellular localization under hypotonic (95% cytoplasmic; 5% nuclear) and hypertonic conditions (3% cytoplasmic, 97% nuclear) was similar to that of FLAG-OREBP1-581. Similar to the FLAG-OREBP1-581L4A, cycloheximide did not alter the subcellular localization of FLAG-OREBP1-581{Delta}1-131 under hypotonic conditions (data not shown), and therefore the observed cytoplasmic signal was because of bona fide nuclear export. Remarkably, an OREBP/TonEBP mutant with the first 156 amino acids removed (FLAG-OREBP1-581{Delta}1-156) localized almost exclusively to the nucleus even in hypotonic medium (2% cytoplasmic, 89% nuclear), which suggested that amino acids residues 132-156 contained a protein domain that is necessary for the hypotonicity-induced nuclear export. Similarly, the GFP chimera of this mutant, OREBP1-581{Delta}1-156-GFP (Fig. 4A), also exhibited constitutive localization to the nucleus (Fig. 4D).


Figure 2
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  		FIGURE 2.
Mutational analysis of the NLS motif in OREBP/TonEBP. A, amino acid sequences of the putative bipartite nuclear localization signal and illustration of the point mutant FLAG-OREBP1-581RKR202-204AAA and FLAG-OREBP1-581KRR213-215AAA. Basic amino acid-enriched regions are underlined. The alanine substitution mutations are indicated in boldface. B, representative fluorescence images of fixed HeLa cells expressing FLAG-OREBP1-581, FLAG-OREBP1-581RKR202-204AAA, or FLAG-OREBP1-581KRR213-215AAA. Cells were transfected with the indicated OREBP expression plasmids. Subsequently, the cells were treated with hypertonic medium for 90 min. FLAG signal was visualized after fixation using a FLAG antibody and a FITC-labeled secondary antibody. The cells were counterstained with DAPI and analyzed by fluorescence microscopy. Images were examined with x40 objective. Scale bar, 100 µm.

 
To further examine the role of this protein domain in OREBP/TonEBP nuclear export, we generated mutant FLAG-OREBP1-581 {Delta}132-156 (Fig. 4A). Similar to the FLAG-OREBP1-581{Delta}1-156, this mutant protein was also constitutively localized to the nucleus under isotonic conditions and remained predominantly nuclear (2% cytoplasmic, 85% nuclear) when the cells were challenged with hypotonic medium (Fig. 4, B and C). Furthermore, deletion of this protein domain alone was sufficient to abrogate nucleocytoplasmic shuttling of OREBP/TonEBP, as this mutant was predominantly localized to the nucleus under isotonic conditions. Taken together these data demonstrated the presence of a putative auxiliary export domain (composed of amino acids 132-156, designated as AED) that is critical for the hypotonicity-induced nuclear export of OREBP/TonEBP. Intriguingly, despite the presence of NES, deletion of AED abrogated isotonic nucleocytoplasmic shuttling of OREBP/TonEBP, suggesting that AED plays a dominant role in the nuclear export process. However, the fusion of OREBP/TonEBP amino acids 132-156 to the N terminus of GFP did not alter its subcellular localization (data not shown), suggesting that AED alone might not be a functional NES, or it might only function as a NES in a protein context-dependent manner. Western blotting analysis showed that these mutants were correctly expressed (Fig. 4E).

CRM1 Targets NES but Not AED—Analysis of the AED amino acid sequences revealed no similarity to canonical NESs or other known nuclear export sequences, suggesting that it may contribute to nuclear export through a yet unknown mechanism. Although it is well established that CRM1 mediates nuclear export of proteins that contain canonical NESs, it has been shown that CRM1 also recognizes and exports proteins that contain atypical NESs (28, 29). Therefore, we sought to determine whether there was an in vivo interaction between NES and CRM1 and to establish whether AED was an unconventional substrate of CRM1. To achieve this, we used a BiFC assay to directly visualize the interaction of CRM1 with OREBP/TonEBP mutant proteins in living cells. This approach is based on the formation of a fluorescent protein complex when two fragments of a fluorescent protein are brought together by the physical interaction of the two test proteins bearing the fluorescent fragments (30). The in vivo interaction between CRM1 and ATF2 has been demonstrated recently by this approach (19). HeLa cells co-transfected with pHA-CRM1566-720VN and OREBP1-581VC exhibited nuclear fluorescence, indicating that the two proteins formed a complex in vivo (Fig. 5A). Substitution of the leucine residues of NES with alanines (OREBP1-581L4AVC) abolished the fluorescence complementation (Fig. 5A). The BiFC signal derived from OREBP1-581VC-pHA-CRM1566-720VN interaction was 3-fold higher than from the OREBP1-581L4AVCpHA-CRM1566-720VN interaction (Fig. 5B). Western blotting analysis showed that these mutants were correctly expressed (Fig. 5C). These data suggested that NES plays a major role in the interaction with Crm1.

We then investigated whether CRM1 overexpression would direct nuclear export of OREBP/TonEBP. As shown in Fig. 5D, the exogenous expression of CRM1 (Myc-CRM1) promotes the cytoplasmic localization of FLAG-OREBP1-581, suggesting that CRM1 directs the NES-containing OREBP/TonEBP to the cytoplasm. However, we found that CRM-1 overexpression had minimal effect on the nuclear localization of the OREBP/TonEBP mutant protein FLAG-OREBP1-581{Delta}1-131. This is consistent with the data obtained from the fluorescence complementation experiments, which showed that NES is required for CRM1-mediated nuclear export of OREBP/TonEBP, whereas AED does not interact with CRM1.


Figure 3
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  		FIGURE 3.
Identification of a CRM1-responsive NES in OREBP/TonEBP. A, effect of LMB (10 ng/ml) on the subcellular localization of the indicated OREBP mutants expressed in HeLa cells. Representative fluorescence images of fixed cells expressing FLAG-OREBP1-581 and live cells expressing OREBP1-581-GFP. For isotonic treatment, cells transfected with the indicated expression plasmids were cultured in isotonic medium with or without LMB for 5 h. For hypotonic treatment, cells were pretreated with hypertonic medium for 90 min to induce nuclear translocation of the fusion proteins with or without LMB. Subsequently, cells were cultured in hypotonic medium for another 90 min with or without LMB. FLAG-OREBP1-581 recombinant protein was visualized after fixation using a FLAG antibody and a FITC-labeled secondary antibody and was analyzed by fluorescence microscopy. OREBP1-581-GFP recombinant protein was visualized by green fluorescence microscopy in live cells. B, alignment of OREBP/TonEBP nuclear export signal with several characterized NES motifs. The conserved residues are highlighted. C, amino acid sequences of the OREBP/TonEBP NES and illustration of the point mutant. The mutated residues are in boldface. D, functional analysis of the NES. Representative fluorescence images of fixed HeLa cells. Cells expressing the indicated mutants were treated with hypotonic or isotonic medium for 90 min. Recombinant protein was visualized after fixation with a FLAG antibody and a FITC-labeled secondary antibody and was analyzed by fluorescence microscopy. Images were examined with x40 objective. Scale bar, 100 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
OREBP/TonEBP activity is regulated at multiple levels in response to changes in extracellular tonicity. For example, it has been shown that OREBP/TonEBP transactivation activity is regulated osmotically in a p38-, Fyn-, and cAMP-dependent protein kinase- and ATM-dependent manner (15, 16, 31, 32). More recently, the transactivation has been linked with NaCl-induced production of mitochondrial reactive oxygen species (33). Nevertheless, the precise molecular changes of the transcription factor associated with its activation remain poorly defined. In this study, we examined how changes in osmolality regulate nucleocytoplasmic trafficking of OREBP/TonEBP. We identified three protein motifs in the transcription factor that control its proper subcellular localization under different extracellular osmolality, one of which is the NLS motif. Alanine substitution of three basic residues in this motif completely eliminated the nuclear staining of OREBP/TonEBP, suggesting that this NLS plays an important role in nuclear import. Moreover, we identified a canonical NES in the extreme N terminus (with the amino acid sequence 5FISLLSADLDL15) that is primarily responsible for the nuclear export of OREBP/TonEBP specifically under isotonic conditions. We showed that mutational inactivation of NES or treatment with LMB both led to the nuclear localization of FLAG-OREBP1-581, suggesting that although the majority of the OREBP/TonEBP signals are found in the cytoplasm under isotonic conditions, the transcription factor undergoes active nucleocytoplasmic shuttling. More importantly, the finding that hypotonicity-induced nuclear export was abolished when amino acids 132-156 were removed from the OREBP/TonEBP protein suggested the presence of a protein domain (AED) important for the hypotonicity-induced nuclear export (with the amino acid sequence 132HPSTP-KRHTVLYISPPPEDLLDNSR156). Unlike the canonical NES motifs, AED does not contain the leucine signature or the hydrophobic patch (LXXLXXLXL) that is characteristic of a CRM1 binding domain (25). We further showed that AED does not belong to the family of atypical NES sequences that are subjected to CRM1-mediated and LMB-sensitive nuclear export, such as those found in Axin (34), Bach2 (35), snuportin1 (36), and equine infectious anemia virus Rev protein (28). Our conclusions are supported by the fact that the hypotonicity-induced nuclear export of FLAG-OREBP1-581 was not inhibited by LMB. Moreover, although the NES binding domain of CRM1 interacts with NES containing the OREBP/TonEBP protein, as evidenced by fluorescence complementation, it does not interact with the mutant protein that contains the AED.

Although AED alone did not drive the nuclear export of a fused heterologous protein, it is clear from our deletion analysis that AED has a role in the nuclear export of OREBP/TonEBP. This is supported by the fact that the NES-deficient OREBP/TonEBP mutant was subjected to hypotonicity-induced nuclear export, whereas the AED-deficient OREBP/TonEBP mutant is constitutively nuclear localized. We postulated that AED may contribute to the nuclear export of OREBP/TonEBP in two ways. First, it may function as a NLS interacting domain similar to that of NFAT1 (37). In the case of NFAT1, multiple phosphorylations of a serine-rich domain led to a conformational switch that masked the NLS, resulting in cytoplasmic retention of the transcription factor. In OREBP/TonEBP, hypotonicity might lead to post-translational modification(s) of the AED or nearby sequences, resulting in a similar conformational switch that masks the NLS and causes full exposure of the nuclear export domain(s), leading to nuclear export. Second, the conformational switch could subsequently induce a novel docking site for nuclear export factors (38). The recent identification of exportin 7 (39), a novel nuclear transport receptor that recognized folded motifs instead of short linear sequences, supports the feasibility of this model and may account for the fact that AED did not confer nuclear export function to its fusion protein. In such a case, AED may contribute to the correct folding of OREBP/TonEBP for nuclear export. Regardless of the mode of action, OREBP/TonEBP utilizes a dual nuclear export mechanism that is distinct from other nucleocytoplasmic shuttling proteins that rely exclusively on canonical NESs for nuclear export, such as histone deacetylase 4 (40), breast cancer susceptibility gene 1 (41), and p53 (42, 43). Although additional nuclear export pathways may also be present, an inactivation mutation of the NES in these transcription factors was sufficient to prevent nuclear exclusion. The existence of two nuclear export mechanisms may allow OREBP/TonEBP to be regulated by yet unidentified extracellular signals and to allow greater flexibility in the control of nucleocytoplasmic trafficking.


Figure 4
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  		FIGURE 4.
Mapping of the AED. A, schematic illustration of OREBP/TonEBP deletion and GFP fusion mutants. B, quantification of the subcellular localization of different OREBP/TonEBP mutants treated with hypotonic, isotonic, and hypertonic medium, respectively. For each condition >100 cells were counted. The data represented are the average of three independent experiments. C, representative fluorescence images of fixed HeLa cells expressing OREBP/TonEBP deletion mutants. Cells were treated with hypotonic, isotonic, and hypertonic medium, respectively. Recombinant protein was visualized with a FLAG antibody and a FITC-labeled secondary antibody and analyzed by fluorescence microscopy. D, representative fluorescence images of live HeLa cells expressing OREBP/TonEBP deletion mutant OREBP1-581{Delta}1-156-GFP. E, expression of FLAG-tagged and GFP-tagged OREBP fusion proteins as determined by Western blotting. HeLa cells were transfected with plasmids encoding FLAG-OREBP1-581{Delta}1-131, FLAG-OREBP1-581{Delta}1-156, FLAG-OREBP1-581{Delta}132-156, and OREBP1-581{Delta}1-156-GFP, respectively. Cell lysates were loaded for the determination of protein expression using immunoblotting analysis with anti-FLAG (for FLAG-OREBP1-581{Delta}1-131, FLAG-OREBP1-581{Delta}1-156, and FLAG-OREBP1-581{Delta}132-156), anti-OREBP (for OREBP1-581{Delta}1-156-GFP), and anti-tubulin antibodies. Images were examined with x40 objective. Scale bar, 100 µm.

 


Figure 5
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  		FIGURE 5.
In vivo interaction of NES with CRM1. A, BiFC analysis of interaction between CRM1 and OREBP/TonEBP. Representative fluorescence images of HeLa cells co-transfected with and expressing the indicated plasmids. At 16 h after transfection, cells were treated with hypertonic medium for 2 h and fixed. Fluorescence emission of the cells was imaged. B, quantitation of BiFC signal. The median of yellow fluorescent protein (YFP)/CFP ratios in each group was derived from more than 100 transfected cells. C, expression of VN173 and VC155 fusion proteins as determined by Western blotting. HeLa cells were transfected with plasmids encoding pHA-OREBP1-581VC and pHA-OREBP1-581L4AVC and pHA-CRM1-VN, respectively. Cell lysates were loaded for the determination of protein expression using immunoblotting analysis with anti-HA (for pHA-CRM1-VN), anti-OREBP (for OREBP1-581VC and OREBP1-581L4AVC), and anti-tubulin antibodies. D, effect of overexpressed CRM1 on subcellular localization of OREBP/TonEBP mutants. Myc-CRM1 expression plasmid was transfected into HeLa cells along with expression plasmids for FLAG-tagged OREBP/TonEBP mutants. Cells were treated with hypertonic medium for 2 h, fixed, and stained with anti-FLAG antibody to determine subcellular localization of OREBP/TonEBP mutant proteins (green) and anti-Myc antibody to detect recombinant CRM1 by indirect immunofluorescence microscopy (red). The cells were counterstained with DAPI to visualize nuclei. Images were examined with x40 objective. Scale bar, 100 µm.

 
OREBP/TonEBP coordinates the induction of a battery of osmoprotective genes in response to hypertonicity (44, 45). The coordinated induction of these genes is crucial for cell survival (4, 46). Because the kidney is the only organ where extreme hyperosmolality is regularly encountered because of the operation of the urine concentration mechanism, it is conceivable that prompt nuclear accumulation of OREBP/TonEBP, as mediated by NLS, is a key step in the timely induction of gene expression to ensure cellular protection. Similarly, during diuresis when hypotonic urine is produced, rapid nuclear exclusion of OREBP/TonEBP can effectively turn off OREBP/TonEBP-dependent gene transcription to avoid excess accumulation of the organic osmolytes that could otherwise be deleterious, as has been demonstrated to occur in other tissues such as the lens and the nerves (47-49). To date, OREBP/TonEBP is the only transcription factor known to undergo nucleocytoplasmic trafficking in response to hypertonicity as well as hypotonicity. Although several other transcription factors, including STAT1 and STAT3 (50), also undergo nuclear translocation in response to hypertonic stress, whether these transcription factors undergo hypotonicity-induced nuclear export remains to be investigated. It has been shown that STAT1 and STAT3 contain multiple CRM1-responsive, canonical nuclear export sequences (51-54). However, we failed to identify AED-like motifs in both STATs. It is therefore likely that the regulation of the hypotonicity-induced nuclear export of STATs, if it occurs, may be through a different mechanism from that of OREBP/TonEBP.

OREBP/TonEBP mRNA is widely distributed in many organs (9) and is highly expressed in the developing heart (55). Furthermore, OREBP/TonEBP-dependent gene expression and/or osmoprotective responses have also been identified in cell types that are non-renal in origin, such as T cells and Jurkat T cells (56, 57), neurons (58), keratinocytes (59), HepG2 cells (60), and HeLa cells (9), etc. Although the physiological function of OREBP/TonEBP remains to be elucidated in tissues where osmolality is maintained at fairly constant levels, a role in cell growth and metastasis has recently been suggested (61, 62). Interestingly, two of the three known alternatively spliced OREBP/TonEBP cDNAs identified in T cells (63) encode putative protein variants that are devoid of NES and are most probably constitutively localized to the nucleus. Consistent with this postulation, the presence of NES-deficient OREBP/TonEBP variants in non-renal cells could facilitate the initiation of transcriptional programs other than that of the osmoprotective genes under isotonic conditions. Taken together, these data also suggest the physiological significance of the presence of two distinctive domains in the regulation of nuclear export in OREBP/TonEBP.

It has been shown that hypertonicity-induced nuclear translocation of OREBP/TonEBP can be blocked by proteasome inhibitors (17) and cyclosporin A (64), although their modes of action remain elusive. More recently, the ataxia telangiectasia-mutated kinase (ATM) was shown to play a role in the process (65). However, the protein targets of the ATM inhibitors used are unclear, and the underlying mechanism(s) remain to be established. Here we identify motifs on the OREBP/TonEBP protein that are essential for its nucleocytoplasmic translocation. Our findings will assist future investigations into the molecular mechanisms underlying the operation of this important protein and help to identify the signaling pathways involved.


   FOOTNOTES
 
* This work was supported by the University Grant Committee of the Hong Kong Special Administrative Region Area of Excellence Scheme Grant AoE/P-10/01, by the University of Hong Kong Generic Drugs Research Program, and by Research Grant Council Grants HKU 7419/03 M and 7427/04 M (to B. C. B. K.). 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

1 To whom correspondence may be addressed. E-mail: smchung{at}hkucc.hku.hk.

2 To whom correspondence may be addressed. E-mail: cbko{at}hkucc.hku.hk.

3 The abbreviations used are: TonEBP, tonicity enhancer-binding protein; NES, nuclear export sequence; NLS, nuclear localization signal; OREBP, osmotic response element-binding protein; GFP, green fluorescent protein; DAPI, 4',6-diamidino-2-phenylindole; AED, auxiliary export domain; BiFC, bimolecular fluorescence complementation; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate; CFP, cyan fluorescent protein; ATM, ataxia telangiectasia-mutated kinase; LMB, leptomycin B. Back


   ACKNOWLEDGMENTS
 
We thank Prof. Anjana Rao, Prof. H. M. Kwon, Dr. D. Y. Jin, Dr. Andras Kapus, and Dr. Rory Watt for critical comments on the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S. Xu, C. C. L. Wong, E. H. Y. Tong, S. S. M. Chung, J. R. Yates III, Y. Yin, and B. C. B. Ko
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