Live Cell Imaging Reveals Continuous STAT6 Nuclear Trafficking Hui-Chen Chen and Nancy C. Reich This information is current as J Immunol 2010; 185:64-70; Prepublished online 24 May of September 24, 2021. 2010; doi: 10.4049/jimmunol.0903323 http://www.jimmunol.org/content/185/1/64 Downloaded from

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Live Cell Imaging Reveals Continuous STAT6 Nuclear Trafficking

Hui-Chen Chen and Nancy C. Reich

The STAT6 is essential for the development of protective immunity; however, the consequences of its activity can also contribute to the pathogenesis of autoimmune disease. Tyrosine phosphorylation is known to activate STAT6 in response to stimulation, but there is a gap in our understanding of the mechanisms by which it enters the nucleus. In this study, live cell imaging was used in conjunction with photobleaching techniques to demonstrate the continual nuclear import of STAT6, independent of tyrosine phosphorylation. The domain required for nuclear entry includes the coiled coil region of STAT6 and functions similarly before or after cytokine stimulation. The dynamic nuclear shuttling of STAT6 seems to be mediated by the classical importin-a–importin-b1 system. Although STAT6 is imported to the nucleus continually,

it accumulates in the nucleus following tyrosine phosphorylation as a result of its ability to bind DNA. These findings will Downloaded from impact diagnostic approaches and strategies to block the deleterious effects of STAT6 in autoimmunity. The Journal of Immunology, 2010, 185: 64–70.

eciphering the signaling events initiated by specific effects of STAT6 (12–14). Accurate cellular localization is key to is critical to understanding their biological the function of a transcription factor, but how the STAT6 protein effects. The STAT6 transcription factor was identified as gains access to the nucleus is not well understood. D http://www.jimmunol.org/ a DNA-binding factor activated in response to IL-4 (1–3). It is now Movement of in and out of the nucleus occurs by known to be required for the generation of Th2 lymphocytes, the passage through nuclear pore complexes that span the nuclear normal function of B lymphocytes, and protection against parasitic membrane (15). Typically, nuclear import of a large protein nematodes (4–7); however, collateral damage accompanies its depends on the presence of a nuclear localization signal (NLS). positive effects in the immune response. Hyperactivity of STAT6 The NLS is recognized by a karyopherin transport that predisposes lymphoproliferative disease and is responsible for facilitates import through the nuclear pore complex (16, 17). The diseases associated with Th2 cell pathologies, like asthma (8–10). classical import receptor consists of a dimer with two distinct Given the considerable evidence that STAT6 contributes to an subunits: an importin-a adapter that binds the NLS and effective immune response and plays a dominant role in asthmatic importin-b that binds importin-a and interacts with the nuclear by guest on September 24, 2021 lung pathology, understanding the mechanisms that regulate its pore complex. In the nucleus, importin-b binds Ran-GTP, lead- nuclear trafficking is essential for therapeutic intervention. ing to release of the NLS cargo. Current knowledge of the nu- STAT6 is a member of the family of signal transducers and clear trafficking of STAT factors has shown that their nuclear activators of transcription and is activated by tyrosine phosphor- import is regulated distinctly (18). For example, nuclear import ylation stimulated in response to Th2 cytokines IL-4 and -13 (11). of the STAT1 factor is conditional and dependent on its dimer- Following cytokine binding to cell-surface receptors, associated ization mediated by tyrosine phosphorylation (19). However, the Janus kinases phosphorylate STAT6 specifically on tyrosine 641. STAT3 transcription factor is imported continually to the nu- Tyrosine phosphorylation promotes the formation of STAT6 dimers cleus, independent of tyrosine phosphorylation (20). The STAT via reciprocal Src homology 2 (SH2) domain and phosphotyrosine molecules share a similar arrangement of functional motifs that interactions. The STAT6 dimer gains the ability to bind DNA tar- including an N terminus, coiled coil domain, DNA-binding do- gets, leading to new responsible for the biological main, SH2 domain, phosphorylated tyrosine, and carboxyl trans- activation domain. Following tyrosine phosphorylation and Department of Molecular Genetics and Microbiology, Stony Brook University, Stony dimerization, STAT1 gains the function of an NLS within its Brook, NY 11794 DNA-binding domain, whereas STAT3 has a constitutive NLS Received for publication October 15, 2009. Accepted for publication April 18, 2010. within the coiled coil domain, independent of tyrosine phosphor- This work was supported by National Institutes of Health Grant RO1CA122910 (to ylation. N.C.R.). To assess the dynamic movement of STAT6 we used live cell Address correspondence and reprint requests to Dr. Nancy C. Reich, Department of imaging with photobleaching techniques. We provide evidence Molecular Genetics and Microbiology, Stony Brook University, Nicolls Road, Life that STAT6 is imported continually into the nucleus, independent Sciences Building, Stony Brook, NY 11794. E-mail address: nancy.reich@stony brook.edu of tyrosine phosphorylation, and it seems to use the importin- The online version of this article contains supplemental material. a2importin-b1 system. In addition, a region required for NLS Abbreviations used in this paper: anti-pSTAT6, anti-STAT6 phosphotyrosine 641 Ab; function was found to map within the coiled coil domain. c, control; C, cytoplasm; dl, deletion; Fl, fluorescent; FLIP, fluorescence loss in Although nuclear import rates of STAT6 are similar before and photobleaching; FRAP, fluorescence recovery after photobleaching; G, GFP; hIL-4, after tyrosine phosphorylation, nuclear accumulation occurs after human IL-4; impb1, importin-b1; IP, immunoprecipitated; MBP, maltose-binding protein; N, nucleus; NLS, nuclear localization signal; PS, Ponceau S; S, STAT6; phosphorylation, and this is dependent on the DNA-binding abil- SH2, Src homology 2; siRNA, short interfering RNA; STAT6-V5, STAT6 tagged ity of STAT6. Live cell imaging has provided critical insight to the with the V5 epitope; WB, Western blot; wtSTAT6, wild type STAT6. spatial distribution of STAT6, which impacts its function as a tran- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 scription factor. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903323 The Journal of Immunology 65

Materials and Methods Axiovert 200M microscope using a heating insert coupled with the In- Cell cultures and reagents cubator S (Zeiss). During imaging, the cells were maintained at 37˚C and 5% CO2 using the Zeiss Tempcontrol 37-2 Digital and CTI Controller HeLa and Cos1 cells (American Type Culture Collection, Manassas, VA) 3700. The time-series images for photobleaching assays were taken with were cultured in DMEM with 8% FBS. Cells were treated with human the Zeiss LSM 510 META NLO two-photon laser-scanning microscope rIL-4 (R&D Systems, Minneapolis, MN) at 10 ng/ml. DNA transfections system using a 403 oil objective (Plan-Neofluar, numerical aperture 1.3, were carried out using TransIT-LT1 transfection reagent (Mirus, Madison, differential interference contrast microscopy objective). The excitation WI), according to the manufacturer’s instructions. Rabbit anti-STAT6 Ab wavelength used for GFP was 488 nm, and emission was detected with (Santa Cruz Biotechnology, Santa Cruz, CA), anti-STAT6 phosphotyrosine a 505-nm filter. For fluorescence recovery after photobleaching (FRAP) 641 Ab (anti-pSTAT6) (Cell Signaling Technology, Danvers, MA), and mu- analysis, a region in the nucleus was bleached at 100% power of an argon rine anti-GFP Ab (Roche Diagnostic Systems, Indianapolis, IN) were used laser at 488 nm for 70 s. For fluorescence loss in photobleaching (FLIP) for Western blotting at a 1:1000 dilution. HRP-conjugated anti-rabbit and analysis, a region in the nucleus or cytoplasm was bleached every 12 s at anti-mouse Ig were used as secondary Abs for Western blotting (1:5000). maximum laser intensity for 5 or 50 min. Images were acquired using LSM GFP Ab and murine IgG2b (MOPC-144) control Ab (Sigma-Aldrich, 510 META version 3.2 imaging software. The fluorescence intensity was St. Louis, MO) were used in EMSA, at 1 mg in 40-ml reactions. Two micro- quantified in the region of interest using LSM Imaging software and graph- grams of anti-V5 Ab (Invitrogen, Carlsbad, CA) was used for the in vitro ically depicted using Microsoft Excel (Microsoft, Redmond, WA). binding assays. Luciferase assay Plasmid constructs and protein purification HeLa cells were transfected with IL-4R–luciferase (22), Renilla luciferase Full-length STAT6 cDNA and deletion mutants created by PCR were cloned (Promega, Madison, WI), and STAT6-GFP wild type or mutant plasmids. into pEF1/V5-His (Invitrogen) or pMAL-c4X (New England Biolabs, Ips- Two days after transfection, cells were treated or not with 3 ng/ml

witch, MA) to generate V5 or maltose-binding protein (MBP) fusion pro- hIL-4 for 8 h prior to harvest. Dual-luciferase reporter assays were per- Downloaded from teins. A monomeric form of enhanced GFP was produced by mutating formed according to the manufacturer’s instructions (Promega, Madison, A206K, L221K, and F223R in the vector pEGFP-N1 (BD Clontech, Moun- WI). The luciferase results were normalized to Renilla luciferase values to tain View, CA), and it was used to generate GFP-tagged STAT6 proteins compensate for variations in transfection efficiency. (21). Site-directed mutagenesis was performed with targeted oligonucleoti- des and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). All con- In vitro importin binding assay structs were confirmed by DNA sequencing. Importin-a constructs lacking the importin-b1–binding domain were generated and purified, as reported The GST–importin-as lacking the aminoterminal importin-b–binding previously (20). MBP-STAT6(1–267) and MBP-STAT6(1–267 deletion [dl] domain were expressed in bacteria and purified by binding and elution http://www.jimmunol.org/ 136–140) proteins were prepared following the manufacturer’s instructions from glutathione beads (20). Cos1 cells expressing STAT6 tagged with (New England Biolabs). the V5 epitope (STAT6-V5) were lysed with buffer (280 mM NaCl, 50 mM Tris-HCl [pH 8.2], 5 mM EDTA, and 0.5% Nonidet P-40), and 500 mg Western blot protein lysate was used for each assay. STAT6 was captured with anti-V5 Ab, bound to protein G beads, and incubated with 15 mg purified GST– Two days after transfection, cells were serum starved for 24 h and were importin-as. Bound protein complexes were eluted with SDS sample treated or not with human IL-4 (hIL-4) for 30 min and lysed with cold lysis buffer and analyzed by Western blot with anti-V5 and anti-GST Abs. To buffer (50 mM Tris [pH 8], 5 mM EDTA, 0.5% Nonidet P-40, 280 mM NaCl, test importin binding to bacterially expressed STAT6, rGST–importin-as 1mMPMSF,13 protease inhibitor mixture [Sigma-Aldrich], 1 mM NaF, were incubated with bacterially expressed MBP-tagged STAT6 proteins and 1 mM sodium vanadate). Proteins were separated by 8% SDS-PAGE immobilized on amylase resin in column buffer (20 mM Tris-HCl, 200 and transferred to nitrocellulose membrane (Pierce, Rockford, IL). The mM NaCl, 1 mM EDTA, and 1 mM DTT) with 0.05% Nonidet P-40. by guest on September 24, 2021 proteins were detected by reacting with Abs to STAT6, STAT6 phosphotyr- Binding was detected by Western blot with anti-GST Ab, and the STAT6 osine, or GFP and detected using the enhanced chemiluminescence system protein was quantified by Ponceau S staining. or Odyssey Infrared Imaging System (LI-COR, Lincoln, NE). RNA interference EMSA Short interfering RNA (siRNA) duplexes specific for human importin-b1 Cells were lysed with hypotonic lysis buffer (15 mM HEPES [pH 7.9], 0.2 (Qiagen, Valencia, CA) or vimentin (control) were transfected with mM spermine, 0.5 mM spermidine, 2 mM potassium-EDTA, 80 mM KCl, 1% X-tremeGENE siRNA transfection reagent (Roche). Twenty-four hours glycerol, 0.0025% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 1 mM sodium 3 after siRNA transfection, cells were transfected with STAT6-GFP. Cellu- vanadate, 1 mM NaF, and 1 protease inhibitor mixture) to prepare cyto- lar localization of STAT6-GFP was observed after 24 h by fluorescence plasmic extracts. Nuclei were collected by centrifugation and extracted in microscopy.RNAextractionwasperformedwithSurePrepTrueTotal hypertonic buffer (20 mM HEPES [pH 7.9], 0.2 mM spermine, 0.5 mM RNA purification (Fisher Scientific, Pittsburgh, PA), and cDNA spermidine, 0.2 mM potassium-EDTA, 0.4 M NaCl, 10% glycerol, 1 mM 3 was synthesized with M-MLV reverse transcriptase (Promega). RT-PCR DTT, 1 mM PMSF, 1 mM sodium vanadate, 1 mM NaF, and 1 protease was performed with specific primers for importin-b1orGAPDHas inhibitor mixture). Nuclear and cytoplasmic extracts were combined for the an internal control. Image J software was used to estimate quantity DNA-binding reactions. Lysates were preincubated with Abs or 100-fold (freely available in the National Institutes of Health public domain). excess nonradiolabeled probe for 30 min at room temperature prior to in- Primer sequences for importin-b1were59-AATCCAGGAAACAGT- cubation with radiolabeled oligonucleotide probe for 30 min. The dsDNA CAGGTTGC-39 (forward) and 59-AGCACTGAGACCCTCAATCAG-39 oligonucleotide corresponding to 2407 to 2387 of the IL-4Ra gene (59- 9 9 9 (reverse) and for GAPDH were 5 -GGAGCCAAAAGGGTCATCAT- AGCTTCTTCATCTGAAAAGGG-3 )was5 end radiolabeled and used in CTC-39 (forward) and 59-AGTGGGTGTCGCTGTTGAGTC-39 (reverse). the binding reactions. Complexes were separated on nondenaturing acry- lamide gels and exposed to x-ray film for autoradiography. Confocal microscopy Results STAT6 nuclear import is independent of tyrosine Cells were plated on glass coverslips, transfected with STAT6constructs, and phosphorylation serum starved overnight. Cells were treated with or without hIL-4 for 30 min and fixed with 4% paraformaldehyde. GFP-tagged protein was observed with Fluorescence microscopy was used to visualize nuclear trafficking a Carl Zeiss LSM 5 laser-scanning microscope using a 403 oil objective of STAT6. STAT6 was tagged at its C terminus with GFP (STAT6- (Plan-Neofluar, numerical aperture 1.3, differential interference contrast mi- GFP) and expressed in cells that were serum starved and stimulated croscopy objective [Jena, Germany]). GFP was excited at 488 nm using an argon laser, and emission was collected using a 505 long-pass filter. Images or not with IL-4 for 30 min (Fig. 1A). The microscopic images were captured using Zeiss LSM 5 Pascal imaging software. revealed latent unphosphorylated STAT6-GFP in the cytoplasm and nucleus (Fig. 1Aa). This result indicated that tyrosine phos- Live cell imaging phorylation was not required for STAT6 nuclear import. Following HeLa cells were seeded on glass-bottom tissue culture dishes (Mattek, Ash- tyrosine phosphorylation in response to IL-4, STAT6-GFP accu- land, MA) and transfected. The dishes were mounted on a Zeiss inverted mulated dominantly in the nucleus (Fig. 1Ab). Results were similar 66 STAT6 NUCLEAR TRAFFICKING

FIGURE 2. Live cell imaging of STAT6 constitutive nuclear import. FIGURE 1. STAT6 nuclear import is independent of tyrosine phos- Nuclear FRAP assays were performed with cells expressing STAT6-GFP Downloaded from phorylation. A, HeLa cells were transfected with STAT6-GFP or STAT6 without IL-4 stimulation (upper row) or with IL-4 stimulation (middle row) 2 (RY)-GFP, serum starved, and left untreated ( ) or treated (+) with IL-4 or with cells expressing STAT6(RY)-GFP with IL-4 stimulation (lower for 30 min. Cellular localization of STAT6 was visualized by fluorescence row). The nucleus (N) was subjected to high-intensity laser to bleach the 3 microscopy (original magnification 100). B, EMSAs were performed fluorescent STAT6. Subsequent fluorescence recovery with time in the with the radiolabeled IL-4R target oligonucleotide and protein lysates from nucleus was monitored and quantified relative to a site in the cytoplasm cells, as treated in A. Effects of additions of Abs to the reaction to GFP (G), (C). The quantitative data of relative fluorescent (Fl) intensity with time STAT6 (S), control (c), or 100-fold excess unlabeled oligonucleotide are shown in the panels on the far right. Experiments are representative of http://www.jimmunol.org/ (DNA) are shown. C, Western blots of protein lysates were performed more than three independent studies. with anti-pSTAT6, anti-STAT6, or anti-GFP Abs. with endogenous STAT6 or V5-tagged STAT6 detected by immu- by 15 min and complete by 45 min (top panel). Following tyrosine nofluorescence (Supplemental Fig. 1A,1B). Analysis of endoge- phosphorylation in response to IL-4, nuclear fluorescence recov- nous STAT6 in primary lymphocytes also clearly showed ery also was half maximal by 15 min; however, within 30–45 min, unphosphorylated STAT6 present in nuclei prior to IL-4 treatment phosphorylated STAT6-GFP accumulated in the nucleus to and an increase in nuclear STAT6 following IL-4 treatment (Sup- a greater extent than in the cytoplasm. This result could reflect plemental Fig. 1C). To confirm STAT6 nuclear import was inde- more efficient import of the tyrosine phosphorylated form of pendent of tyrosine phosphorylation, the behavior of a STAT6 STAT6 or, alternatively, a decrease in STAT6 nuclear export. by guest on September 24, 2021 protein with a double mutation was evaluated. The tyrosine 641 The kinetics of nuclear accumulation of the STAT6(RY)-GFP mu- that is specifically phosphorylated in response to cytokine stimula- tant (bottom panel) were similar to that of unphosphorylated tion was substituted with phenylalanine (Y641F), and the critical STAT6 and confirm that nuclear import of STAT6 is continuous arginine562intheSH2domainthatfunctionstoformdimersca- and independent of tyrosine phosphorylation. pable of specific DNA binding was mutated to alanine (R562A). FLIP was used to address the basis of nuclear accumulation fol- Imaging results showed the double mutant, STAT6(RY)-GFP, was lowing tyrosine phosphorylation of STAT6 (Fig. 3). A high-intensity imported to the nucleus but did not accumulate following stimula- laser was continually directed to a small region in the cytoplasm of tionwithIL-4(Fig1Ac,1Ad). These data show that STAT6 nuclear cells expressing unphosphorylated STAT6-GFP or tyrosine phos- import is independent of tyrosine phosphorylation and that nuclear phorylated STAT6-GFP (+IL-4). STAT6 passing through the laser accumulation requires tyrosine phosphorylation. path of this small region is bleached, and the loss of fluorescence EMSAs and Western blotting were performed to ensure that correlates with STAT6 mobility. Fluorescence intensity rapidly de- STAT6-GFP was tyrosine phosphorylated accurately and capable of creased in the cytoplasm of cells expressing unphosphorylated binding DNA, whereas the STAT6(RY)-GFP lacked these abilities. STAT6-GFP or tyrosine phosphorylated STAT6-GFP, indicating The EMSAs showed that STAT6-GFP can bind a specific DNA tar- rapid movement through the cytoplasm. For unphosphorylated get only following tyrosine phosphorylation, and the STAT6(RY)- STAT6-GFP, this loss was followed by a loss of fluorescence in GFP lacks this ability (Fig. 1B). Western blotting with Abs that the nucleus that was nearly complete by 50 min. The loss of nuclear recognize phosphotyrosine 641 STAT6 confirmed that STAT6-GFP fluorescence indicates continual STAT6 export from the nucleus and is accurately tyrosine phosphorylated after IL-4 treatment, but passage through the laser path in the cytoplasm. In contrast, a differ- STAT6 (RY)-GFP is not phosphorylated (Fig. 1C). ent result was found for tyrosine phosphorylated STAT6-GFP (lower panel). Nuclear fluorescence of phosphorylated STAT6 did not de- Live cell imaging reveals STAT6 constitutive nuclear shuttling crease during the duration of the experiment. These results suggest The spatial and temporal dynamics of STAT6 were evaluated by that the nuclear accumulation that is evident after STAT6 tyrosine live cell imaging with nuclear FRAP (Fig. 2). Nuclei of cells phosphorylation is due to a decrease in nuclear export. expressing STAT6-GFP were subjected to a high-intensity laser to bleach fluorescence in this compartment (top panel). The recovery DNA binding retains STAT6 in the nucleus of fluorescence in the nucleus with time was monitored relative to Tyrosine phosphorylation activates STAT proteins by promoting a region of interest in the cytoplasm for STAT6 in unstimulated the formation of dimers that have the ability to bind specific DNA cells, STAT6 in IL-4–stimulated cells (+IL-4), or the STAT6(RY) target sites. To determine whether the increased nuclear accu- mutant in IL-4–stimulated cells (+IL-4). Fluorescence recovery in mulation of STAT6 seen following tyrosine phosphorylation was the nucleus of unphosphorylated STAT6-GFP was half maximal due to a gain in the ability to bind DNA, the behavior of a DNA- The Journal of Immunology 67

accurately tyrosine phosphorylated in response to IL-4, it did not bind target DNA sequences (Fig. 4A). Microscopic imaging indi- cated that STAT6(KR)was imported to the nucleus with and without IL-4 stimulation, but it did not accumulate in the nucleus in response to IL-4. This indicated that DNA binding contributes to nuclear accumulation following tyrosine phosphorylation. If DNA binding retains STAT6 in the nucleus, the mobility of tyrosine-phosphorylated STAT6 within the nucleus would be expected to be slower than unphosphorylated STAT6. A nuclear FLIP assay was used to investigate this possibility (Fig. 4B). A small region (region 1) in the nucleus of cells expressing STAT6- GFP, with or without IL-4 stimulation, was subjected to continu- FIGURE 3. Live cell imaging demonstrates decreased STAT6 nuclear ous laser bleaching for 5 min. The fluorescence intensity of region export following tyrosine phosphorylation. Cytoplasmic FLIP assays were performed with cells expressing STAT6-GFP not treated (upper row)or 1 was compared with a distinct region in the nucleus (region 2). If treated with IL-4 (lower row). A small region in the cytoplasm (C) was movement is rapid through the path of the laser, the fluorescence subjected to continuous high-intensity laser. Fluorescence loss was moni- intensity in region 2 will decrease similarly to region 1, along with tored with time in the cytoplasm and compared with the fluorescence loss the entire nucleus. This was the case for unphosphorylated STAT6. in a region of the nucleus (N). The quantitative data of relative fluorescent However, following tyrosine phosphorylation in response to IL-

(Fl) intensity with time are shown in the panels on the far right. Experi- 4, STAT6 showed significantly slower movement. The fluores- Downloaded from ments are representative of more than three independent studies. cence decrease in region 2 and the remainder of the nucleus was delayed considerably compared with region 1. The tyrosine- binding mutant was evaluated. A STAT6 DNA-binding mutant was phosphorylated DNA-binding mutant, STAT6(KR), showed the generated based on other STAT DNA-binding mutants (23). Lysines same rapid nuclear movement as unphosphorylated STAT6. To and arginines within aa 366–374 were substituted with alanines to establish that the DNA-binding mutant is not retained in the nu- generate STAT6(KR). Although the STAT6(KR) mutant was cleus following IL-4 stimulation, imaging with cytoplasmic FLIP http://www.jimmunol.org/ was used (Supplemental Fig. 2). The export kinetics of tyrosine- phosphorylated STAT6(KR) were similar to unphosphorylated wild type STAT6 (wtSTAT6). Together, the results support the premise that STAT6 accumulates in the nucleus only if it has a functional DNA-binding domain. Identification of amino acids in STAT6 that are required for nuclear import

Nuclear import of large molecules, such as STAT6, requires an by guest on September 24, 2021 amino acid sequence or structure that serves as an NLS. To identify amino acids that function to facilitate STAT6 nuclear import, a series of deletion mutations were generated, and the cellular localization of the truncated proteins was evaluated with or without IL-4 stimulation. Small proteins were tagged with two GFP mol- ecules to ensure that they did not passively diffuse into the nucleus; a diagram of some of the truncations is shown in Fig. 5. The cellular localization of these truncations indicated that a region in the coiled coil domain is needed for nuclear import. STAT6(1– 267) containing the N terminus and the coiled coil domain of STAT6 was imported to the nucleus. However, STAT6(268–847) containing the DNA-binding domain, SH2 domain, and transacti- vation domain remained in the cytoplasm with or without IL-4 stimulation. Deletions within the coiled coil domain identi- fied a region required for STAT6 nuclear import. STAT6(136–847) FIGURE 4. DNA binding promotes nuclear accumulation of tyrosine- was imported and accumulated in the nucleus following tyrosine phosphorylated STAT6. A, The DNA-binding mutant STAT6(KR)-GFP phosphorylation, whereas STAT6(141–847) remained in the cyto- was expressed in cells and examined by fluorescence microscopy before plasm with or without tyrosine phosphorylation. Western blotting (2) or after (+) treatment with IL-4 (original magnification 3100). West- with Abs to STAT6 phosphotyrosine 641 confirmed that the dele- ern blot (lower panels) was performed with anti-pSTAT6 or anti-GFP Abs. tions were accurately phosphorylated in response to IL-4 (Fig. 5). EMSA (right panel) was performed with the IL-4R target oligonucleotide The studies with STAT6 truncations identified a sequence be- and lysates from cells expressing STAT6-GFP (wt) or STAT6(KR)-GFP tween aa 136–140 (RLQHR) that is required for nuclear import. 2 (KR) without ( ) or with (+) IL-4 treatment. Abs to STAT6 (S), GFP (G), To determine the effect of a specific deletion or substitution of or MOPC control (c) were added to the binding reactions. B, Live cell these amino acids in otherwise full-length STAT6, we evaluated imaging was used with nuclear FLIP to evaluate STAT6-GFP and STAT6 the localization of two mutants linked to GFP (Fig. 6A). STAT6 dl (KR)-GFP mobility within the nucleus. A small region in the nucleus (region 1) was subjected to continuous high-intensity laser. Fluorescence 136–140 or STAT6 containing a substitution of 135–140 aa with loss was monitored with time in this region and a distinct region in the alanine residues (sub6A) were expressed in cells stimulated or not nucleus (region 2). Quantitative data of relative fluorescent (Fl) intensity with IL-4. The cellular localization of both mutants was restricted with time are shown in the panels on the far right. Experiments are rep- to the cytoplasm, indicating a deficiency in nuclear import. These resentative of more than three independent studies. mutants were accurately tyrosine phosphorylated in response to 68 STAT6 NUCLEAR TRAFFICKING

FIGURE 5. Identification of sequences required for STAT6 nuclear import. Top panel, linear diagram of STAT6 functional domains and corresponding deletion mutations. Numbers indicate amino acids. Bottom left, fluorescent images of STAT6-GFP deletion mutations expressed in serum-starved cells (2IL-4) or cells stim- ulated with IL-4 (+IL-4) (original magnification 3100). Bottom right, Western blot of protein lysates from cells performed with anti-pSTAT6, anti-STAT6, or anti-GFP Abs.

IL-4, indicating that the internal deletion and substitution did not whereas STAT6(dl136–140) and STAT6(KR) did not induce the disrupt STAT6 activation. To evaluate the influence of specific res- gene (Fig. 6B).

idues in this region, each amino acid was mutated in the context of Downloaded from Evidence supporting a role of importin-a/b1 in STAT6 nuclear full-length STAT6. However, the individual point mutants behaved import as wtSTAT6 (Supplemental Fig. 3). Together, these results indicate that aa 136–140 are required for STAT6 nuclear import, but they Active transport of large molecules through the nuclear pore may function within the context of a conformational NLS. complex usually requires facilitation by carrier proteins of the Transcriptional regulation is the primary function of STAT6, and karyopherin-b family. Importin-b1 is a primary karyopherin-b for this reason we evaluated the ability of STAT6 mutants to induce transporter that can bind directly to NLS-containing proteins or in- http://www.jimmunol.org/ gene expression. Mutants defective in nuclear localization, STAT6 directly via the family of importin-a adapters. Importin-a adapters (dl136–140), or DNA binding, STAT6(KR), were tested for their bind directly to the NLS. In vitro binding assays were performed to competence to induce the characterized promoter of the IL-4R evaluate whether one or more of the importin-as can recognize gene (22). Transient transfections clearly demonstrated the ability of wtSTAT6 to induce the IL-4R reporter in response to IL-4, by guest on September 24, 2021

FIGURE 7. STAT6 import is mediated by importin-a/b1 system. A, Immunoprecipitated (IP) STAT6-V5 expressed in COS1 cells was col- lected on protein G beads and incubated in vitro with bacterially expressed GST-importins. Western blots (WB) identified bound importins (anti-GST) and STAT6-V5 (anti-V5). Ten percent of importin input is shown in the FIGURE 6. Amino acids 136–140 are required for STAT6 nuclear im- bottom panel (anti-GST). B, Bacterial-expressed MBP-STAT6(1–267) and port. A, STAT6-GFP with an internal deletion of aa 135–140 (dl) or MBP-STAT6(1–267dl136–140) were immobilized on amylase resin and STAT6-GFP with a substitution of six alanine residues for aa 135–140 incubated with purified GST–importin-a1 or GST–importin-a3. The (sub6A) were expressed in cells that were serum starved (2) or stimulated binding was evaluated by Western blot with anti-GST Ab. Ponceau S with IL-4 (+). STAT6 localization was visualized microscopically (original (PS) staining showed that the same amount of STAT6 proteins were used. magnification 3100). Bottom panel displays Western blot of protein C, Fluorescent images of cells transfected with vimentin siRNA (control) lysates with anti-pSTAT6 or anti-GFP Abs. B, HeLa cells were transfected or importin-b1 siRNA (impb1) for 24 h followed by transfection with with IL-4R site-TKLuc reporter construct, Renilla luciferase vector, and STAT6-GFP. Cytoplasmic localization was seen in ∼10% of cells. Lower different STAT6-GFP constructs. Cells were treated or not with hIL-4 for 8 panel displays the effect of control vimentin siRNA or importin-b1 siRNA h before the dual-luciferase reporter assay. Luciferase result was normal- on endogenous importin-b1 mRNA or GAPDH mRNA as evaluated by ized to Renilla luciferase value. RT-PCR. The Journal of Immunology 69

STAT6 (Fig. 7A). STAT6-V5 was expressed in mammalian cells binding. STAT6 that completely lacks 135 aa from the N terminus is and immunoprecipitated from cell lysates with V5 Ab and protein imported to the nucleus, is tyrosine phosphorylated in response to G agarose beads. GST-tagged importin-as were expressed in IL-4, and can bind DNA (Fig. 5) (H.C. Chen and N.C. Reich, un- bacteria and added to the STAT6-V5 immunocomplexes collected published observations). on beads. Interaction of importins with STAT6 was detected by By studying the cellular localization of various STAT6 deletions, Western blot with Ab to GST. The results indicated that STAT6 is we identified a region within the coiled coil domain required for recognized primarily by importin-a3 and -a6. Similar results were STAT6 nuclear import (Fig. 5). STAT6(136–847) was imported to obtained with STAT6 isolated from untreated cells or IL-4– the nucleus constitutively, whereas STAT6(141–847) was not stimulated cells, indicating that binding is independent of tyrosine imported. Deletion or substitution of the small region between aa phosphorylation. Because importin-a6 is restricted to the testes, 135–140 eliminated the ability of otherwise full-length STAT6 to importin-a3 seems to be the primary import adapter (24, 25). be imported to the nucleus, although the proteins were still tyro- Because aa 136–140 in the coiled coil region of STAT6 are critical sine phosphorylated accurately (Fig. 6). The best-characterized for nuclear import, we determined whether this sequence was re- classical NLS sequences contain one or two stretches of basic quired for direct interaction with importin-a3. We expressed amino acids, particularly lysines (31). Although the sequence fragments of STAT6 tagged with MBP in bacteria corresponding to 135–140 (RRLQHR) contains arginine residues, site-directed mu- STAT6 1–267 aa or 1–267 containing the 136–140 deletion. MBP- tation of individual amino acids within this region was not suffi- STAT6(1–267) and MBP-STAT6(dl136–140) were incubated with cient to block nuclear import (Supplemental Fig. 3). This finding bacterially expressed GST–importin-a3 or GST–importin-a1as suggests that a noncanonical NLS may be functional within 136– a control and evaluated for binding (Fig. 7B). The results showed 267. Other STAT molecules seem to use noncanonical NLSs to Downloaded from that STAT6(1–267) can bind importin-a3 specifically, but the drive import, whether they are constitutive or conditional (18). deletion mutant cannot. These data suggest aa 136–140 are required Although the STATs do not display classical NLSs, they seem for STAT6 binding to importin-a3 and nuclear import in vivo. to use the importin-a–importin-b1 receptors. Importin-a5 binds to Given that the importin-a/b1 system may mediate STAT6 nuclear STAT1 when it is in the conformation of a tyrosine-phosphorylated import, we evaluated the effect of RNA interference on the inhibition dimer and facilitates its nuclear import (19, 32, 33), whereas

of expression of importin-b1 (Fig. 7C). siRNA duplexes corres- importin-a3and-a6 bind constitutively to STAT3 (20). In this http://www.jimmunol.org/ ponding to importin-b1 or to vimentin as a control were study, we found that importin-a3 and -a6 also bind constitutively transfected into cells with STAT6-GFP, and the localization of to STAT6; additionally, downmodulation of importin-b1byRNA STAT6-GFP was visualized microscopically. The behavior of interference notably reduces STAT6 nuclear import. The results STAT6-GFP was notably different in the cells treated with suggest that STAT6 is imported by importin-a–importin-b1re- importin-b1 siRNA. Approximately 10% of cells showed STAT6 ceptors (Fig. 7). It is challenging to determine specific importin-a restricted to the cytoplasmic compartment, often with punctate recognition of a particular NLS outside the framework of the native cytoplasmic fluorescence. Because the siRNA may not completely protein, because recognition depends on the NLS sequence, as well as inhibit importin-b1expressioninallcellsexpressingSTAT6-GFP,the the protein context (34). The crystal structure of STAT6 remains to be effect seems to be significant. To evaluate the effectiveness of the solved. However, the identity of the importin-a that binds a particular by guest on September 24, 2021 importin-b1 siRNA complexes, mRNA levels in cells treated with protein may be significant because the importin-a proteins display control or importin-b1 siRNAwere assayed by RT-PCR. The siRNA specific expression in tissues and during differentiation (25, 35). It to importin-b1 reduced endogenous mRNA by ∼70%. Together, the was reported that a Rac GTPase-activating protein is responsible results suggest that importin-a/importin-b1 may mediate STAT6 for nuclear import of activated STAT proteins and that the dominant nuclear import. negative N17Rac1 protein can block nuclear import of the STATs (36). For this reason, we tested the effect of N17 Rac1 on STAT6 nuclear import but did not detect any effect (Supplemental Fig. 4). Discussion Latent-unphosphorylated and tyrosine-phosphorylated STAT6 are Nuclear trafficking of STAT6 is integral to its function as a signal imported to the nucleus. The difference is that STAT6 accumulates in transducer and activator of transcription. By attaching a fluorescent the nucleus when it is tyrosine phosphorylated (Fig. 1). Live cell probe to STAT6 we were able to study its intracellular dynamics imaging withphotobleaching techniquesprovidesa more quantitative with microscopy in real time. The advantage of live cell imaging is and temporal measure of protein mobility and localization (37–39). that it avoids fixation techniques that can influence cellular ar- By using the technique of nuclear FRAP,the transport of STAT6-GFP chitecture. Cell fractionation has been used to evaluate cellular into the nucleus was observed to be similar for unphosphorylated or localization; however, the technique is limited in interpreting tyrosine-phosphorylated STAT6-GFP (Fig. 2). However, the average in vivo protein localization, particularly if the protein is actively fluorescence intensity of phosphorylated STAT6-GFP becomes sig- imported and exported from the nucleus. Our studies indicated that nificantly greater in the nucleus than in the cytoplasm. The nuclear STAT6 moves continually within the cytoplasm; additionally, it is accumulation is the consequence of decreased nuclear export. This transported continually into and out of the nucleus, independent of was demonstrated with cytoplasmic FLIP (Fig. 3). Repeated photo- tyrosine phosphorylation. bleaching of one small region in the cytoplasm resulted in the loss of Specific phosphorylation of tyrosine 641 promotes STAT6 di- total cytoplasmic fluorescence, independent of STAT6 phosphoryla- merization and its ability to bind DNA target sites. In addition to this tion. For unphosphorylated STAT6-GFP,this was followed by a grad- activating modification, other modifications have been reported that ual loss of fluorescent signal from the nucleus, indicating continuous include serine phosphorylation of the carboxyl transactivation do- export. In contrast, nuclear fluorescence of tyrosine-phosphorylated main, which may influence DNA binding (26–28), and acetylation, STAT6-GFP did not decrease during the experiment. Therefore, the which may contribute to induction of gene expression (12, 29). increase in STAT6 nuclear accumulation following tyrosine phos- Methylation of arginine 27 was reported to be required for STAT6 phorylation is a result of decreased nuclear export. tyrosine phosphorylation, nuclear translocation, and DNA binding The mechanism of STAT6 nuclear export remains to be de- (30). However, our studies indicate that arginine 27 is not necessary termined; nonetheless, it seems that DNA binding is responsible for for tyrosine phosphorylation, nuclear translocation, or DNA STAT6 nuclear accumulation. A STAT6 DNA-binding mutant was 70 STAT6 NUCLEAR TRAFFICKING shown to behave like unphosphorylated STAT6 and did not accu- 14. Hebenstreit, D., G. Wirnsberger, J. Horejs-Hoeck, and A. Duschl. 2006. Signaling mechanisms, interaction partners, and target genes of STAT6. mulate in the nucleus following phosphorylation (Fig. 4). 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