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Stat5B Shuttles Between Cytoplasm and Nucleus in a Cytokine-Dependent and -Independent Manner This information is current as of June 15, 2017. Rong Zeng, Yutaka Aoki, Minoru Yoshida, Ken-ichi Arai and Sumiko Watanabe J Immunol 2002; 168:4567-4575; ; doi: 10.4049/jimmunol.168.9.4567 http://www.jimmunol.org/content/168/9/4567 Subscription Permissions Email Alerts This article cites 61 articles, 33 of which you can access for free at: http://www.jimmunol.org/content/168/9/4567.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017 References Stat5B Shuttles Between Cytoplasm and Nucleus in a Cytokine-Dependent and -Independent Manner1 Rong Zeng,*‡ Yutaka Aoki,*‡ Minoru Yoshida,†‡ Ken-ichi Arai,*‡ and Sumiko Watanabe2* R oles of Janus kinases (Jaks)3 and Stats in cytokine signal transduction were first established in IFN signaling pathways (1). Although the cytokine receptor superfamily is not structurally related to the IFN receptor, the data indicated that all members of the cytokine receptor superfamily use Jaks and Stats (2). Furthermore, it is now well known that receptor tyrosine kinases such as epidermal growth factor or insulin-like growth factor activate the Jak and Stat pathways (3, 4). As observed initially in the IFN system, accumulating evidence suggests that Stats are involved in the activation of cytokine-specific genes (3, 5–7), and knock-out studies of various Stats strongly support this idea (8, 9). To date, Stats 1– 6 with similar structural features have been identified (10). All of these members have a DNA-binding domain located in the amino-terminal half, and linker and Src homology 2 (SH2) domains, followed by the transactivation domain, in their C-terminal half (2). A conserved tyrosine residue is located in the C-terminal region and phosphorylation of this residue plays an essential role in dimerization and nuclear translocation of Stats. Serine residues on the C-terminal side of this tyrosine are phosphorylated by extracellular signal-related kinase, p38 mitogen-activated protein kinase (MAPK), or c-Jun N-terminal kinase, and *Department of Molecular and Developmental Biology, Institute of Medical Science, and †Department of Biotechnology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Tokyo, Japan; and ‡Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Tokyo, Japan Received for publication January 16, 2002. Accepted for publication February 21, 2002. 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. 1 R.Z. is recipient of the Okazaki International Scholarship. 2 Address correspondence and reprint requests to Dr. Sumiko Watanabe, Department of Molecular and Development Biology, Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail address: [email protected] 3 Abbreviations used in this paper: Jak, Janus kinase; SH2, Src homology 2; MAPK, mitogen-activated protein kinase; NLS, nuclear localization signal; CRM1, chromosome region maintenance 1; NES, nuclear export signal; LMB, leptomycin B; c,  subunit; m, mouse; h, human; EGFP, enhanced green fluorescent protein; PI, propidium iodide. Copyright © 2002 by The American Association of Immunologists have been implicated in the transcriptional activity of Stat1 and Stat3 (11). Stats exist as monomers in the cytoplasm before receptor activation (1). Cytokine stimulation leads to Jak activation followed by phosphorylation of tyrosine residues in the cytoplasmic part of the cytokine receptor. Phosphotyrosine of the receptor recruits Stats through their SH2 domain, making Jak2 accessible to phosphorylate Stats. Activated Jaks, which phosphorylates the C-terminal tyrosine residue of Stats, leads to Stat dimer formation by the intermolecular interactions of the SH2 domain and the phosphorylated tyrosine (12). Once dimerized, Stats dissociate from the receptors and translocate to the nucleus in the dimer form where they bind to target DNA. Stats are thought to be translocated back to the cytoplasm after dephosphorylation, which means the regulation of translocation of Stats is important for the regulation of Stat activation (13). In contrast, the involvement of proteasomedependent or ubiquitin-associated degradation of Stats in the nucleus as a shut-off mechanism has also been proposed (14, 15). Stat proteins have a mass over the generally considered largest size for diffusion through the nuclear pore (16), and thus they are assumed to be actively transported into and of the nucleus. However, the mechanism regulating the subcellular distribution of Stats is not well understood. Stat1 dimers bind the nuclear import complex importin ␣,  (17), thus indicating the involvement of nuclear import machinery. Neither classical nuclear localization signals (NLS) nor regions critical for this process have been found in Stat1. Chromosome region maintenance 1 (CRM1)/exportin 1 was identified as the nuclear export receptor (18, 19). CRM1 interacts with Ras-like nuclear G protein GTPase, and this complex binds to the nuclear pore to translocate nuclear export signal (NES)-containing proteins (20). NES is a short stretch of amino acids composed of leucine (or hydrophobic amino acid) spaced by two to three lengths of amino acids. The functions of many signaling molecules and transcription factors, including protein kinase inhibitor, MAPK kinase, IB, p53, and NFAT, were found to be regulated by CRM1-dependent nuclear export (21). Leptomycin B (LMB), an antifungal antibiotic blocking the yeast CRM1 function (22), was recently shown to inhibit NES-dependent nuclear export 0022-1767/02/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017 In response to cytokine stimuli, Stats are phosphorylated and translocated to the nucleus to activate target genes. Then, most are dephosphorylated and returned to the cytoplasm. Using Ba/F3 cells, we found that the nuclear export of Stat5B by cytokine depletion was inhibited by leptomycin B (LMB), a specific inhibitor of nuclear export receptor chromosome region maintenance 1. Interestingly, LMB treatment in the absence of cytokine led to the accumulation of Stat5B in the nucleus, suggesting that Stat5B shuttles between the nucleus and the cytoplasm as a monomer without cytokine stimulation. This notion is supported by the observation that LMB-induced accumulation of Stat5B in the nucleus was also observed with Stat5B having a mutated tyrosine 699, which is essential for dimer formation. Using a series of mutant Stat5Bs, we identified a part of the coiled coil domain to be a critical region for monomer nuclear import and a more N-terminal region to be critical for the cytokine stimulation dependent import of Stat5B. Taken together, we propose a model in which Stat5B shuttles between the nucleus and cytoplasm by two different mechanisms, one being a factor-independent constitutive shuttling by monomeric form, and the other, a factor stimulationdependent one regulated by tyrosine phosphorylation and subsequent dimerization. The Journal of Immunology, 2002, 168: 4567– 4575. 4568 Materials and Methods Chemicals and cytokines FCS was purchased from Biocell Laboratories (Carson, CA), and RPMI 1640 and DMEM were from Nikken Biomedical Laboratories (Kyoto, Japan). Mouse (m) rIL-3 expressed in the silkworm Bombyx mori was purified as described (38). Human (h) GM-CSF and G418 were kind gifts from Schering-Plough (Madison, NJ). Anti-Stat5 Ab (C-17) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and anti-Stat5 mAb was obtained from Transduction Laboratories (Lexington, KY). Anti-phosphoStat5A/B Ab was obtained from Upstate Biotechnology (Lake Placid, NY). Anti-green fluorescent protein Ab (8362-1) came from Clontech Laboratories (Palo Alto, CA), and anti-FLAG Ab (F3165), from Sigma-Aldrich (St. Louis, MO). Cell lines and culture methods The mIL-3-dependent pro-B cell line, Ba/F3 (39), was maintained in RPMI 1640 medium containing 5% FCS, 0.25 ng/ml mIL-3, 100 U/ml penicillin, and 100 U/ml streptomycin. For cell washing or depletion, the same medium without mIL-3 was used. Various Ba/F3 cell clones expressing hGMCSFR␣ and hGM-CSFR (Ba/F-GMR) and Stat5B or its mutants were selected and maintained in the same type of medium with 500 g/ml G418. A monkey kidney epithelial cell line, COS7, was maintained in low glucose DMEM with 10% FCS, 100 U/ml penicillin, and 100 U/ml streptomycin. Serum depletion was done by washing COS7 cells with DMEM two times. Construction and expression of Stat5B mutants The mammalian expression vector pME18S used in this study contained the SR␣ promoter (40). pME-Stat5B-enhanced green fluorescent protein (EGFP) was generated by ligating together the EcoRI-SacI fragment of Stat5B, which contained aa 1–771, the NotI-EcoRI fragment of the pME18S vector, and the SacI-NotI fragment of pEGFP-1 (Clontech Laboratories, Palo Alto, CA). Stat5B-FLAG was constructed by replacing the NarI-NotI fragment of pME-Stat5B-EGFP with an NarI-NotI-digested PCR fragment amplified with primers, designed to create FLAG tag DYKDDDDK (eight amino acids) followed by a termination codon and NotI site. Sequences of the primers used are as follows: 5⬘-CTGACGGATTCGTGAAGCCACAGA TCA-3⬘ (sense) and 5⬘-CTAGTCTAGCGGCCGCTTGTCGTCGTCGTCC TTGTAGTCCATGGTGGCGACCGGTG-3⬘ (antisense). For NES mutants, pME-F570A/F574A-EGFP, ⫺M578A/V580A-EGFP, and ⫺L656A/ L660A-EGFP, all of which contained two amino acid substitutions in the conserved NES, were prepared. These point mutations were introduced by PCR mutagenesis and the XhoI (1730)-AgeI (2149)-digested PCR product containing these mutations at the NES site was used to replace the same fragment in the wild-type Stat5B. pME-I324A-EGFP, which contained one amino acid mutation at the NES1 site, was constructed by PCR mutagenesis, and the SapI-BstEII fragment was replaced with the corresponding fragment of the wild-type Stat5B. NES-EGFP was constructed by ligating fragments of the NES sequence, which had a start codon ATG at the N terminus, into pME-EGFP. Construction of F mutants was done as described (41). For a series of N-terminal deletion mutants, the XhoI site followed by ATG was placed at aa 85, 104, 138, 165 by PCR-based mutagenesis. XhoI and BstEII or XhoI and ApaLI fragments were then used to replace the same fragment of the wild-type Stat5B. All the PCR products and junctions of the ligated fragments were confirmed by sequencing, and the size of proteins was examined by the expression in COS7 cells followed by Western blotting using anti-Stat, GFP, or FLAG Abs. Subcellular localization of Stat5B Subcellular localization was examined either by biochemical cell fractionation or histochemical analysis. Biochemical cell fractionation of Ba/F3 cells was done as described (41). Briefly, cells were incubated in buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, 0.1 mM PMSF) for 15 min on ice, and Nonidet P-40 was added at the final concentration of 0.6%. The cells were mixed vigorously for 15 s, then centrifuged. Nuclear proteins were extracted as described (42). For histochemical analysis, Ba/F3 cells were harvested on glass slides (Matsunami, Kishiwada, Osaka) by use of Cytospin3 (Shandon, Pittsburgh, PA) and COS7 cells were cultured in LabTekII chamber slides (Nunc, Rochester, NY). These cells were fixed with 3.7% (volume/volume) formaldehyde in PBS for 15 min at room temperature. The fixed cells were then rinsed with PBS and permeabilized with 0.5% Triton X-100 in PBS for 15 min. In some cases, cells were incubated with the anti-FLAG M2 monoclonal Ab at 1 g/ml in PBS and 1% BSA followed by FITC-rabbit antimouse IgG Ab (Zymed Laboratories, South San Francisco, CA) at 15 g/ml in PBS. Then, propidium iodide (PI; 5 ng/ml) was applied to stain the nuclei of the cells. Slide glasses were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and analyzed with a laser scanning confocal imaging system (MRC-1024; Bio-Rad, Hercules, CA) or with a fluorescence microscope (BX50; Olympus, Tokyo, Japan). Images were processed by using Adobe Photoshop (Adobe Systems, Mountain View, CA). Each experiment was repeated at least twice, and images representing over 80% of the total cell population are shown in the figures otherwise indicated. Transient transfection and Western blotting Ba/F3 or COS7 cells were transfected with plasmid DNA by electroporation as described (30, 41). Briefly, cells in 200 l of OPTI-MEM (Life Technologies, Tokyo, Japan) were mixed with plasmid DNA and electroshocked with a gene pulser (Bio-Rad) at 960 F, 200 V for Ba/F3 cells, or at 100 V for COS7 cells. Two days after transfection COS7 cells were lysed and subjected to SDS-PAGE followed by Western blotting. For the luciferase assay, Ba/F-GMR cells were transfected with the -casein promoter luciferase plasmid (7) by electroporation. After a 12 h of culture in mIL-3 medium, the cells were depleted of mIL-3 for 5 h and restimulated with hGM-CSF (10 ng/ml) for 5 h, and then the luciferase activity was analyzed, as described (43). Results Nuclear export of Stat5B in Ba/F3 cells is LMB-sensitive Many proteins are exported from the nucleus via the CRM1 (exportin 1) system (21). CRM1 recognizes its target protein through a short stretch of amino acids composed of leucine (or hydrophobic amino acid) spaced by two to three lengths of amino acids named NES (44, 45). When we examined the sequence of Stat5B, we found at least three amino acid stretches that matched the consensus of NES; all of these sequences are highly conserved among other members of the Stat family. LMB is a specific inhibitor of the NES-dependent nuclear export receptor CRM1 (exportin 1) and can be used to block the nuclear export pathway (18, 24, 25). We Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017 by specific binding to the CRM1 (23–25). Quite recently, the inhibition by LMB of Stat1 nuclear export was reported (26, 27). The cytokines GM-CSF and IL-3 stimulate proliferation and differentiation as well as promote the survival of various hemopoietic cells (28). GM-CSFR and IL-3R consist of two subunits, ␣ and , both of which are members of the cytokine receptor superfamily (29). The ␣ subunit is specific to each cytokine, and the  subunit (c) is shared by GM-CSF, IL-3, and IL-5 receptors (29). GM-CSF and IL-3 induce tyrosine phosphorylation of c and various cellular proteins and activate expression of early response genes and cell proliferation in hemopoietic cells and in fibroblasts (30, 31). The c contains conserved box 1 and box 2 regions and eight tyrosine residues located in the cytoplasmic region (32). GMCSF activates Jak2 and Stat5 A and B in various hemopoietic cells (33, 34). Stat5A and Stat5B genes encode proteins that are ⬃95% identical in amino acid sequence (35). Although Stat5 is activated by various cytokines such as erythropoietin, IL-3/IL-5/GM-CSF, prolactin, growth hormone and thrombopoietin, Stat5A and/or Stat5B knockout mice show a role for physiological responses associated with growth hormone and with prolactin (36). As other Stats may play pivotal roles for cell differentiation and the function of various cells, including hemopoietic cells, these results suggest that Stat5A/B are obligate mediators of mammopoietic and lactogenic signaling rather than of cell proliferation (37). We analyzed the nuclear import and export of Stat5B through c signals in the mouse IL-3-dependent cell line Ba/F3 and the monkey kidney epithelial cell line COS7. In addition to the nuclear import in response to c signals, interestingly, we found that Stat5B shuttles between the nucleus and the cytoplasm as a nonphosphorylated form regardless of cytokine stimulation. This finding reveals a new and unique feature of Stat5B transport. NUCLEAR TRANSLOCATION OF STAT5B The Journal of Immunology 4569 Addition of LMB leads to the accumulation of Stat5B in the nucleus in the absence of cytokine stimulation FIGURE 1. LMB-sensitive nuclear export of Stat5 in Ba/F3 cells. A, Effects of LMB on disappearance of Stat5 from the nucleus were analyzed. Ba/F-GMR cells were depleted of cytokine and LMB (0, 2, or 11 ng/ml) was added simultaneously with stimulation by hGM-CSF (10 ng/ml) as indicated. After 30 min of stimulation, cells were depleted of GM-CSF and cultured in the depletion medium containing the same concentration of LMB. Nuclear proteins were extracted and run through SDS-PAGE followed by Western blotting by using anti-Stat5 Ab. B, Nucleocytoplasmic translocation of Stat5B fused with EGFP or FLAG at the C terminus. Ba/F3 cells stably expressing Stat5B-EGFP or transiently expressing Stat5BFLAG were depleted of mIL-3 for 5 h and then stimulated with mIL-3 (1 ng/ml) for 1 h. Then cells were depleted of mIL-3 in the presence or absence of LMB. Cells were harvested on glass slides and stained with PI (for Ba/F3-Stat5B-EGFP), or PI and anti-FLAG Ab conjugated with FITC (for Ba/F3-Stat5B-FLAG). Images of EGFP and FITC are shown. asked if LMB affects the nuclear export of Stat5B. We have been analyzing hGM-CSF receptor signaling in mIL-3 dependent Ba/F3 cells expressing the hGM-CSF receptor (Ba/F-GMR). Ba/F-GMR can survive and proliferate in the presence of either mIL-3 or hGM-CSF (30). Ba/F-GMR cells were depleted of mIL-3 for 5 h and then stimulated with hGM-CSF (10 ng/ml) in the presence or absence of LMB. Then, hGM-CSF was washed out, and cells were harvested at indicated time points. The same concentration of the LMB was present in the washing and depletion medium. Nuclear proteins were subjected to Western blotting using an anti-Stat5 Ab. In the absence of LMB, Stat5 accumulated in the nucleus by 30 min of GM-CSF stimulation (Fig. 1A, lanes 1 and 2) and then disappeared from the nucleus 2– 4 h after the depletion of hGMCSF (lanes 3 and 6). When LMB was added, the disappearance of Stat5 was inhibited in a dose-dependent manner (lanes 4 and 5) at 2 h after the depletion. Even at 4 h after depletion, the dose-dependent effects of LMB were still evident (lanes 7 and 8). These results indicate that Stat5 was translocated from the nucleus by a CRM1-dependent pathway. In the course of our experiments, we found that the addition of LMB in the absence of cytokine also resulted in accumulation of Stat5B in the nucleus. As shown in Fig. 1A, Stat5B disappeared from the nucleus in the cytoplasm 4 h after cytokine depletion in Ba/F3 cells. At that time point, we added LMB and examined its effect on the subcellular localization of Stat5B. As shown in Fig. 2A, Stat5B disappeared from the cytoplasm and accumulated in the nucleus within 15 min after the addition of LMB. When we transfected COS7 cells with Stat5B-EGFP, Stat5B was exclusively localized in the cytosol (Fig. 2A) because there is no stimulus that induces the nuclear translocation of Stat5B in COS7 cells. We then examined the effects of LMB on Stat5B subcellular localization in COS7 cells. Stat5B began to accumulate in the nucleus by LMB addition and only a trace amount was detectable in the cytoplasm after 60 min. These results indicate that Stat5B shuttles between the nucleus and the cytoplasm, even in the absence of cytokines. Because FCS contains trace amount of cytokines, we next did similar experiments using COS7 cells in the absence of FCS. As in the presence of FCS, Stat5B accumulated in nucleus after LMB treatment in the absence of FCS and cytokines, indicating that the accumulation of Stat5B was not caused by FCS. Tyrosine phosphorylation of Stat5B is not essential for factorindependent shuttling Because Stats are believed to form dimers after tyrosine phosphorylation induced by cytokines (12), it is speculated that Stats exist as monomers in the absence of cytokines. To determine whether the cytokine independent shuttling of Stat5B was conducted with Stat5B as monomers, we next constructed a tyrosine mutant of Stat5B. Tyrosine 699 of Stat5B, which is phosphorylated in response to IL-3 or GM-CSF and plays an essential role in dimerization (46), was replaced with phenylalanine and the mutant was fused with EGFP or FLAG at its C terminus (Fig. 2B). Ba/F3 or COS7 cells were transiently transfected with the F mutant constructs, and the effects of LMB on subcellular localization of Stat5B in the absence of cytokine were examined. As shown in Fig. 2B (upper panel), Stat5B-F-EGFP or Stat5B-F-FLAG accumulated in the nucleus by adding LMB in the absence of cytokines. Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017 Because there is another possibility, that Stat5 is activated by LMB and remains for a longer time in the nucleus, we examined whether the Stat5 was activated by the addition of LMB. Tyrosine phosphorylation, DNA binding, and -casein luciferase activation were not observed by the addition of LMB in the absence of cytokine (data not shown). These results thus indicate that LMB inhibits the nuclear export of Stat5 in Ba/F3 cells. For more detailed analysis of the nuclear shuttling of Stat5, we tagged mouse Stat5B with EGFP or FLAG at the C terminus (Fig. 1B). Subcellular localization of Stat5B-EGFP in stable clones of Ba/F3 cells and of transiently expressed Stat5B-FLAG in Ba/F3 cells was examined under a fluorescence microscope. By cytokine depletion, both fusion proteins accumulated in the cytoplasm, and then were translocated to the nucleus after cytokine stimulation. Although the C-terminal 15 amino acids of Stat5B were deleted at the junction between Stat5B and EGFP or FLAG, Stat5B-EGFP and Stat5B-FLAG both behaved in a similar manner as wild-type Stat5B. Nuclear export of these proteins was inhibited by LMB, and their tyrosine phosphorylation, DNA binding activity, and transactivation potential were all equivalent to those observed with wild-type Stat5B (data not shown). When we added cycloheximide to block de novo protein synthesis, essentially the same pattern was observed (data not shown). 4570 NUCLEAR TRANSLOCATION OF STAT5B In COS7 cells, the addition of LMB also resulted in accumulation of Stat5B in the nucleus. Taken together, these results suggest that cytokine-independent shuttling of Stat5B occurs with Stat5B in the monomer state. Different region required for factor-dependent and -independent imports of Stat5B To determine the region of Stat5B required for subcellular localization, we constructed a series of truncation mutants from the N terminus of Stat5B, as shown in Fig. 3A. These mutants were transiently expressed in COS7 cells, and the subcellular localization in the presence or absence of LMB (20 ng/ml, 60 min) was analyzed (Fig. 3A). All mutants accumulated in the cytoplasm in the absence of LMB, suggesting that the nuclear export of mutants ⌬N104, Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017 FIGURE 2. Stat5B shuttles between cytoplasm and nucleus in the absence of cytokines. A, Effects of LMB on factor-depleted Ba/F3 and COS7 cells. As schematically shown, Ba/FStat5B-EGFP were depleted of cytokine for 5 h. LMB (20 ng/ ml) was added and cells were harvested at the indicated time points. COS7 cells were transfected with Stat5B-EGFP and cultured for 2 days, then LMB was added and the subcellular localization of Stat5B-EGFP was examined at the indicated time points. In the FCS depletion experiments, cells were washed with DMEM 16 h before LMB addition and no FCS was contained in following steps. B, Effect of mutation of tyrosine 699 of Stat5B on factor-independent shuttling. Mutant Stat5B-EGFP or FLAG, which contained substitution of tyrosine 699 (Stat5B-FEGFP, Stat5B-F-FLAG) with phenylalanine, was introduced to Ba/F3 cells and COS7 cells, and the effects of LMB were examined as described in Fig. 1B. 138, and 165 occurred as in the wild type. When LMB was added, ⌬N104 and 138 translocated to the nucleus. In contrast, ⌬N165 remained in the cytoplasm in the presence of LMB, suggesting that the region between residues 138 and 165 is essential for monomer import of Stat5B. We next prepared stable clones of Ba/F3 cells expressing ⌬N104, 138, and 165, and examined the subcellular localization of the mutant Stat5B in these cells. Ba/F3 cell clones expressing one of these mutants were depleted of mIL-3 for 5 h, and then LMB was added and incubation was continued for 60 min. As shown in Fig. 3B (left column), all mutants accumulated in the cytosol after factor depletion, as expected. ⌬N104-EGFP and ⌬N138-EGFP accumulated in nucleus in the presence of LMB as did wild-type Stat5B (right column). Further deletion up to residue 165 (⌬N165-EGFP) abrogated the response to the LMB. The Journal of Immunology 4571 minal mutants can be tyrosine phosphorylated by mIL-3 stimulation (data not shown), thereby suggesting that the region up to residue 104 is essential for the step of dimer formation or subsequent nuclear translocation. A region containing SH2 is sufficient for nuclear export Taken together, as in COS7 cells, the region between residues 138 and 165 is essential for cytokine-independent import of Stat5B from the cytoplasm to the nucleus in Ba/F3 cells. Interestingly, when we examined the nuclear import of factor-induced dimers (center column, stimulated), none of the mutants could translocate to the nucleus in response to mIL-3 stimulation. All these N-ter- Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017 FIGURE 3. Role of the N-terminal region for subcellular localization of Stat5B in Ba/F3 and COS7 cells. A, Schematic representation of a series of N-terminal deletion mutants of Stat5B and subcellular localization of these mutants in COS7 cells. A series of N-terminal deletion mutants of Stat5B were transfected to COS7 cells and their subcellular localization, in the presence or absence of LMB, was examined. B, Ba/F3 cells stably expressing N-terminal truncation mutants of Stat5B were depleted of mIL-3 for 5 h then restimulated with mIL-3 or treated with LMB (20 ng/ml). Subcellular localization of Stat5B was examined. We next analyzed the region required for the nuclear export of Stat5B. Various mutants of Stat5B-EGFP containing a single or double amino acid substitution at putative NES sites were constructed (Fig. 4A). Stable lines of Ba/F3 cells expressing these mutants were established, and the subcellular localization was microscopically examined. Mutants I324A-EGFP and M578A/ V580A-EGFP accumulated in the cytoplasm after cytokine depletion (Fig. 4A). Both mutants translocated to the nucleus after stimulation, and the nuclear export was inhibited by LMB (data not shown). In contrast, F570A/F574A-EGFP and S656A/L660AEGFP were distributed both in the cytoplasm and nucleus, in the absence of cytokines. We then analyzed the transcriptional activation potential of these mutants. -casein is a target gene of Stat5, and the promoter region covering ⬃0.3 kb is sufficient for the induction by various stimulators including IL-3, GM-CSF (7). Each NES mutant was introduced into Ba/F-GMR with -caseinluciferase plasmids and the luciferase activity, in the presence or absence of hGM-CSF, was analyzed. The mutants I324A-EGFP and M578A/V580A-EGFP activated the -casein promoter as did the wild type, but the mutants F570A/F574A-EGFP and S656A/ L660A-EGFP could not activate -casein luciferase (data not shown). We constructed the same set of Stat5B mutants, but without EGFP, and essentially the same results were obtained in Ba/F3 cells (data not shown). These data suggest the possible involvement of either NES2 or NES3 in the nuclear export of Stat5B. To determine whether one of the isolated Stat5B NES1, 2, or 3 regions would promote EGFP to cytoplasm as a nuclear export signal, we constructed NES-EGFP in which each NES sequence was fused with the N terminus of EGFP and the constructs were introduced into COS7 cells. All the mutant fusion proteins were localized in the nucleus in the absence of cytokine, and the subcellular localization did not change in the presence of LMB (Fig. 4B). Therefore, it seems clear that none of the putative NES regions alone can support transport of EGFP to the cytoplasm. Based on these results, we next made deletion mutants from both the N and C termini of Stat5B. All the mutants were transiently expressed in COS7 cells, and their subcellular localization in the absence or presence of LMB was analyzed. We mutated tyrosine 699 of these mutants to exclude unexpected phosphorylation and possible following dimerization with the SH2 region of endogenous Stat5B. N-terminal deletion up to residue 578 (⌬N578) resulted in the loss of NES1 and NES2, and this mutant exclusively localized in the cytoplasm without LMB and accumulated in the nucleus with LMB treatment (Fig. 4C). The mutant ⌬N578 redistributed into the nucleus in the presence of LMB, and it may have been caused by the fact that EGFP tended to accumulate in the nucleus. To test this hypothesis, we made the same type of mutant using a FLAG tag. Our hypothesis was supported by the finding that ⌬N578-F-FLAG localized in the cytoplasm both in the absence and presence of LMB and prolonged LMB treatment resulted in the nuclear localization of this mutant (data not shown). We further deleted a part of Stat5B from the N terminus, and the mutant ⌬N675-F-EGFP localized in the nucleus in the presence and absence of LMB, suggesting that the region between 578 and 675 is essential for the export of Stat5B from the nucleus to the cytoplasm. To identify the minimum region for the export of Stat5B, we then deleted further from the C terminus of this mutant. Deletion from the C terminus up to residue 723 did not abrogate 4572 NUCLEAR TRANSLOCATION OF STAT5B Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017 FIGURE 4. Region containing SH2 and NES3 of Stat5B is sufficient to direct EGFP from nucleus to cytoplasm. A, Schematic representation of putative NES of Stat5B and other Stat family members and subcellular localization of mutants Stat5B-EGFP. Consensus sequences of NES and various Stat5BEGFP with mutation of putative NES are also shown. Ba/F3 cells stably expressing one of the Stat5B (1⬃771-EGFP) mutants containing mutations with consensus amino acids of putative NES sequence (I324A-EGFP, F570A/F574A-EGFP, M578A/V580A-EGFP, S656A/L660A-EGFP) were depleted of mIL-3 for 5 h. Subcellular localization was microscopically analyzed using EGFP fluorescence. B, Subcellular localization of EGFP fused with the putative NES sequence derived from STAT5B. NES-EGFP, schematically represented, was transfected to COS7 cells and subcellular localization, in the presence or absence of LMB, was examined. C, Deletion mutants Stat5B of N and/or C terminus fused with EGFP were transfected to COS7 cells and subcellular distribution in the presence or absence of LMB was examined. the nuclear export of this mutant (578⬃723-F-EGFP), but further deletion up to 611 (611⬃723-EGFP) disrupted the nuclear export. Taken together, the data indicate that the region between residues 578 and 723 is sufficient for the export of Stat5B from the nucleus to the cytoplasm. Because this region contains a putative NES motif (NES3) and this transport is LMB-sensitive, it seems likely that the region mediates the CRM1-dependent nuclear export of Stat5B. Discussion Cytokine stimulation-independent nuclear translocation of Stat5B Using LMB and various Stat5B mutants, we demonstrated evidence for new features of both the nuclear export and import of Stat5B. We found that two different types of nuclear import systems and the LMB-sensitive nuclear export are coupled to regulate The Journal of Immunology 4573 Statc required DIF signaling via sequences located in the N-terminal half of the Dd-Statc, suggesting that some factor-inducible cue, which cannot be explained by the previous tyrosine-SH2 dimerization model, is required. Because Stat5B translocates to the nucleus in response to LMB treatment even in the absence of FCS, such a factor-inducible mechanism of monomer translocation is less feasible. Dimerization-dependent nuclear localization Stat5B subcellular distribution. We found that Stat5B accumulated in the nucleus in the presence of LMB, even in the absence of cytokine stimulation. Because Stats are not tyrosine phosphorylated in the absence of cytokine stimulation, this nuclear translocation may have occurred with Stat5B in its monomer form. This notion was supported by the finding that the Stat5B mutant with tyrosine 699 replaced with phenylalanine could also translocate to the nucleus with LMB treatment. Because some mutants that could translocate to the nucleus cytokine independently could not translocate there by cytokine stimulation, it is likely that the region required for cytokine-independent and -dependent nuclear translocation differs. These findings led to the idea that two different mechanisms by which Stat5B is imported to the nucleus are present. Monomer and dimer translocation of a single molecule was noted in the case of MAP kinase (47). The size of MAPK (42 kDa) is small enough to pass through the nuclear pore and this monomer translocation is assumed to occur by passive diffusion. In contrast, the molecular mass of Stat5B is ⬃94 kDa, which is much larger than the maximal size for passive diffusion. Therefore, the cytokine-independent transport of Stat5B must be conducted by active transport. Physiological relevance of monomer shuttling of Stat5B Although there is no suggestion of monomer transport of Stat1, there are several findings indicating the role of monomer or nonphosphorylated Stat1 in the nucleus. Unphosphorylated Stat1 has been detected in the nuclei of rat liver cells (48). Stat1 mediates the constitutive transcription of many genes such as cpp32 or ich-1, and a mutant Stat1 in which the site of tyrosine phosphorylation was mutated supports expression of these genes and TNF-induced apoptosis (49). In the case of low molecular mass polypeptide 2 (LMP2), the same type of mutant Stat1 (Y701F), which does not form a SH2-phophotyrosine-dependent dimer, binds to the IFN-␥activated sequence element and supports low molecular mass polypeptide 2 expression (50). In this case, unphosphorylated Stat1 forms dimer through its N-terminal domain. These findings suggest the possibility that shuttling Stat5 mediates constitutive expression of target genes. Recently tyrosine phosphorylation independent nuclear translocation of a Dictyostelium Stat, Dd-Statc, was reported (51). Interestingly, nuclear translocation of the Dd- Involvement of NES and CRM1 for Stat5B nuclear exclusion LMB is a specific inhibitor of the nuclear export receptor CRM1 (24) and we found that the nuclear export of Stat5 was blocked by LMB. This effect of LMB on Stat1 nuclear export was reported earlier, based on two independent studies (26, 27). Both of these investigations and our results indicate multiple NES sequences within Stat family members and conservation of all these motifs among members of the Stat family. Nevertheless, all results suggest different regions as important motifs for nuclear export. We found that two different mutations disrupted the correct subcellular distribution of Stat5B. But in both cases, the mutant Stat5B was also distributed in some amount in the cytoplasm rather than exclusively accumulating in the nucleus in the absence of cytokines, hence, we could not conclude that the phenomenon means inhibition of nuclear export. In addition, these mutants could not be Downloaded from http://www.jimmunol.org/ by guest on June 15, 2017 FIGURE 5. Schematic representation of Stat5B cytoplasm/nucleus shuttling Tyrosine phosphorylation of Stats by Jak2 followed by nuclear translocation is a generally accepted model for all members of Stat family and thus much attention has been paid to the mechanism of the cytokine-dependent import. As is the case for other Stat members, only the tyrosine phosphorylated form of Stat5 was observed to translocate to the nucleus in response to IL-3 or GM-CSF stimulation in Ba/F or COS7 cells (data not shown). Because the mutant ⌬N104, which cannot translocate to the nucleus upon IL-3 stimulation, was tyrosine phosphorylated by IL-3 or GM-CSF stimulation (data not shown), we speculate that the mutant ⌬104 is defective in some steps such as dimer formation and nuclear translocation. Thus, it is probable that the N-terminal region plays an essential role in nuclear translocation of Stat5B. Proteins that translocate to the nucleus usually contain the NLS. The best known NLS consists of a single stretch of basic amino acids or a bipartite sequence of basic amino acids (21). There is no documentation of classical NLS within any member of the Stat family, and we also could not find any consensus NLS within Stat5A and B. The involvement of the N-terminal region or DNA-binding domain in the nuclear import of Stat1 and Stat5 has been reported (47, 52, 53). Interestingly, the Stat5B N-terminal coiled coil domain possesses leucine zipper-like motifs which have the potential to interact with other proteins. Therefore, we speculate that this region may mediate the interaction of Stat5B with the nuclear import machinery, probably through some other NLS-possessing adapter molecule. Indeed, the N-terminal region of Stat5B was reported to interact with other molecules such as Nmi or protein inhibitors of activated Stats (54, 55). It remains to be examined if they act as adapters for the nuclear import of Stats. Nuclear import in the dimer form has been analyzed in the case of Stat1 induced by IFN-␥ and the involvement of Ran in the nuclear import was evident (56). In this case, the interaction of IFN ␥ with NPI-1, but not with Rch1, has been reported (17), but the involvement of any basic cluster of amino acid stretch was not indicated, and the region responsible for the interaction was not defined. There is another report of identification of an essential region within the DNA-binding domain of Stat5B for nuclear translocation (57). A mutant of this region can be tyrosine phosphorylated by growth hormone stimulation followed by dimer formation. 4574 tyrosine phosphorylated. There are two different explanations for the lack of tyrosine phosphorylation of these mutants, one is that the mutants locate exclusively in the nucleus regardless of cytokine stimulation, and another is that a disrupted conformation of these mutants resulted in a lack of response to IL-3 stimulation. 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Studies on the three-dimensional structure of Stat1 and Stat3 (58, 59) revealed that Stats dimerize through their SH2 domain and amino acids surrounding the phosphorylated tyrosine residue. As the SH2 domain serve as a binding site of homodimerization, this region may be masked during dimer formation and unmasked after dephosphorylation of tyrosine 699 followed by monomerization in the nucleus. Although there is no information on the monomer structure of Stat5B, it is possible that some intra- or intermolecular masking mechanism is involved in the regulation of the subcellular localization of Stat5B. This notion is supported by reports that artificial dimerization of Stats without tyrosine phosphorylation triggered nuclear translocation of Stats (60, 61). We propose a model for the nucleocytoplasmic transport of Stat5B as shown in Fig. 5. In the absence of cytokine stimulation, the nonphosphorylated, monomeric form of Stat5B shuttles continuously between the nucleus and the cytoplasm. 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