Regulation of Nucleocytoplasmic Trafficking of Transcription Factor OREBP/TonEBP/NFAT5
2006; Elsevier BV; Volume: 281; Issue: 33 Linguagem: Inglês
10.1074/jbc.m602556200
ISSN1083-351X
AutoresEdith H.Y. Tong, Jinjun Guo, Ailong Huang, Han Liu, Chang‐Deng Hu, Stephen S. Chung, Ben C.B. Ko,
Tópico(s)Nuclear Receptors and Signaling
ResumoThe 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. 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. 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 (1Burg M.B. Kwon E.D. Kultz D. Annu. Rev. Physiol. 1997; 59: 437-455Crossref PubMed Scopus (328) Google Scholar) to replace intracellular electrolytes that are otherwise deleterious to normal cellular function (2Haussinger D. Biochem. J. 1996; 313: 697-710Crossref PubMed Scopus (500) Google Scholar). Hypertonicity also induces the expression of heat shock protein 70 (HSP-70) (3Neuhofer W. Beck F.X. Annu. Rev. Physiol. 2005; 67: 531-555Crossref PubMed Scopus (99) Google Scholar) that acts as a molecular chaperone to protect cells from osmotic stress-induced apoptosis (4Shim E.H. Kim J.I. Bang E.S. Heo J.S. Lee J.S. Kim E.Y. Lee J.E. Park W.Y. Kim S.H. Kim H.S. Smithies O. Jang J.J. Jin D.I. Seo J.S. EMBO Rep. 2002; 3: 857-861Crossref PubMed Scopus (81) Google Scholar). The accumulation of organic osmolytes is brought about by the induction of a battery of genes, including aldose reductase (AR), betaine/γ-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) (5Woo S.K. Lee S.D. Na K.Y. Park W.K. Kwon H.M. Mol. Cell. Biol. 2002; 22: 5753-5760Crossref PubMed Scopus (175) Google Scholar, 6Ko B.C. Ruepp B. Bohren K.M. Gabbay K.H. Chung S.S. J. Biol. Chem. 1997; 272: 16431-16437Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 7Takenaka M. Preston A.S. Kwon H.M. Handler J.S. J. Biol. Chem. 1994; 269: 29379-29381Abstract Full Text PDF PubMed Google Scholar, 8Rim J.S. Atta M.G. Dahl S.C. Berry G.T. Handler J.S. Kwon H.M. J. Biol. Chem. 1998; 273: 20615-20621Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The transcription factor that binds to the TonE/ORE, known as TonEBP 3The 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. or OREBP, was independently identified by a yeast one-hybrid assay (9Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (472) Google Scholar) and its affinity purification (10Ko B.C. Turck C.W. Lee K.W. Yang Y. Chung S.S. Biochem. Biophys. Res. Commun. 2000; 270: 52-61Crossref PubMed Scopus (120) Google Scholar). 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 (11Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (318) Google Scholar). However, unlike other members of the NFAT family, such as NFATc1-4 (12Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1557) Google Scholar), 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 (11Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (318) Google Scholar). OREBP/TonEBP is regulated at multiple levels. Hypertonicity induces its nuclear localization (10Ko B.C. Turck C.W. Lee K.W. Yang Y. Chung S.S. Biochem. Biophys. Res. Commun. 2000; 270: 52-61Crossref PubMed Scopus (120) Google Scholar, 13Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Am. J. Physiol. 2000; 278: F1006-F1012Crossref PubMed Google Scholar, 14Lee S.D. Colla E. Sheen M.R. Na K.Y. Kwon H.M. J. Biol. Chem. 2003; 278: 47571-47577Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This is accompanied by an increase in the level of OREBP/TonEBP mRNA andprotein(9Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (472) Google Scholar,13Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Am. J. Physiol. 2000; 278: F1006-F1012Crossref PubMed Google Scholar). In addition, hypertonicity increases the phosphorylation of OREBP/TonEBP as well as the activity of its transactivation domain (14Lee S.D. Colla E. Sheen M.R. Na K.Y. Kwon H.M. J. Biol. Chem. 2003; 278: 47571-47577Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 15Ferraris J.D. Williams C.K. Persaud P. Zhang Z. Chen Y. Burg M.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 739-744Crossref PubMed Scopus (120) Google Scholar, 16Ko B.C. Lam A.K. Kapus A. Fan L. Chung S.K. Chung S.S. J. Biol. Chem. 2002; 277: 46085-46092Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Nevertheless, the identification of specific phosphorylation site(s) and the evidence directly showing whether phosphorylation is required for its transcriptional activation activity remain elusive (14Lee S.D. Colla E. Sheen M.R. Na K.Y. Kwon H.M. J. Biol. Chem. 2003; 278: 47571-47577Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). 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(17Woo S.K. Maouyo D. Handler J.S. Kwon H.M. Am. J. Physiol. 2000; 278: C323-C330Crossref PubMed Google Scholar). Apparently the nuclear translocation is bi-directional, because hypotonicity reduces the nuclear staining of OREBP/TonEBP (13Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Am. J. Physiol. 2000; 278: F1006-F1012Crossref PubMed Google Scholar). 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. 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-OREBP1-581Δ1-131, and FLAG-OREBP1-581Δ1-156 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-OREBP1-581Δ132-156 was derived by in-frame insertion of the cDNA fragment corresponding to amino acid residues 1-131 into the NotI restriction site of FLAG-OREBP1-581Δ1-156. 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 (18Nagai T. Ibata K. Park E.S. Kubota M. Mikoshiba K. Miyawaki A. Nat. Biotechnol. 2002; 20: 87-90Crossref PubMed Scopus (2184) Google Scholar), 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 (19Liu H. Deng X. Shyu Y.J. Li J.J. Taparowsky E.J. Hu C.D. EMBO J. 2006; 25: 1058-1069Crossref PubMed Scopus (89) Google Scholar). 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, 1× minimum Eagle's medium amino acids solution, and 1× 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 (20Shyu Y.J. Liu H. Deng X. Hu C.D. BioTechniques. 2006; 40: 61-66Crossref PubMed Scopus (318) Google Scholar). 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 β-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-α-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 ECL™ Western blotting reagents (Amersham Biosciences). 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) (14Lee S.D. Colla E. Sheen M.R. Na K.Y. Kwon H.M. J. Biol. Chem. 2003; 278: 47571-47577Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), 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) (10Ko B.C. Turck C.W. Lee K.W. Yang Y. Chung S.S. Biochem. Biophys. Res. Commun. 2000; 270: 52-61Crossref PubMed Scopus (120) Google Scholar, 11Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (318) Google Scholar), 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) (14Lee S.D. Colla E. Sheen M.R. Na K.Y. Kwon H.M. J. Biol. Chem. 2003; 278: 47571-47577Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Indirect immunofluorescence analysis with anti-OREBP/TonEBP antibodies showed that hypertonicity increases the nuclear staining of OREBP/TonEBP (9Miyakawa H. Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2538-2542Crossref PubMed Scopus (472) Google Scholar), whereas the staining was reduced substantially when cells were subjected to prolonged hypotonic treatment (18 h) (13Woo S.K. Dahl S.C. Handler J.S. Kwon H.M. Am. J. Physiol. 2000; 278: F1006-F1012Crossref PubMed Google Scholar), 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 (21Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1009) Google Scholar), 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 (22Suntharalingam M. Wente S.R. Dev. Cell. 2003; 4: 775-789Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). 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) (23Dingwall C. Laskey R.A. Trends Biochem. Sci. 1991; 16: 478-481Abstract Full Text PDF PubMed Scopus (1711) Google Scholar). 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) (24Kudo N. Matsumori N. Taoka H. Fujiwara D. Schreiner E.P. Wolff B. Yoshida M. Horinouchi S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9112-9117Crossref PubMed Scopus (855) Google Scholar), a specific inhibitor of the nuclear export receptor exportin-1 (CRM1) (25Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1741) Google Scholar, 26Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Crossref PubMed Scopus (623) Google Scholar). 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 (25Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1741) Google Scholar). We searched for putative nuclear export sequences in OREBP/TonEBP using the NES prediction server, NetNES (27la Cour T. Kiemer L. Molgaard A. Gupta R. Skriver K. Brunak S. Protein Eng. Des. Sel. 2004; 17: 527-536Crossref PubMed Scopus (635) Google Scholar). 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. 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-OREBP1-581Δ1-131 (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-OREBP1-581Δ1-131 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-OREBP1-581Δ1-156) 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, OREBP1-581Δ1-156-GFP (Fig. 4A), also exhibited constitutive localization to the nucleus (Fig. 4D). To further examine the role of this protein domain in OREBP/TonEBP nuclear export, we generated mutant FLAG-OREBP1-581 Δ132-156 (Fig. 4A). Similar to the FLAG-OREBP1-581Δ1-156, 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 1
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