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STAT6 Nuclear TraffickingLive Cell Imaging Reveals Continuous

Hui-Chen Chen and Nancy C. Reich

http://www.jimmunol.org/content/185/1/64doi: 10.4049/jimmunol.09033232010;

2010; 185:64-70; Prepublished online 24 MayJ Immunol 

MaterialSupplementary

3.DC1http://www.jimmunol.org/content/suppl/2010/05/21/jimmunol.090332

Referenceshttp://www.jimmunol.org/content/185/1/64.full#ref-list-1

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The Journal of Immunology

Live Cell Imaging Reveals Continuous STAT6 NuclearTrafficking

Hui-Chen Chen and Nancy C. Reich

The STAT6 transcription factor 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 cytokine 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 protein 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

impact diagnostic approaches and strategies to block the deleterious effects of STAT6 in autoimmunity. The Journal of

Immunology, 2010, 185: 64–70.

Deciphering the signaling events initiated by specificcytokines is critical to understanding their biologicaleffects. The STAT6 transcription factor was identified as

a DNA-binding factor activated in response to IL-4 (1–3). It is nowknown to be required for the generation of Th2 lymphocytes, thenormal function of B lymphocytes, and protection against parasiticnematodes (4–7); however, collateral damage accompanies itspositive effects in the immune response. Hyperactivity of STAT6predisposes lymphoproliferative disease and is responsible fordiseases associated with Th2 cell pathologies, like asthma (8–10).Given the considerable evidence that STAT6 contributes to aneffective immune response and plays a dominant role in asthmaticlung pathology, understanding the mechanisms that regulate itsnuclear trafficking is essential for therapeutic intervention.STAT6 is a member of the family of signal transducers and

activators of transcription and is activated by tyrosine phosphor-ylation stimulated in response to Th2 cytokines IL-4 and -13 (11).Following cytokine binding to cell-surface receptors, associatedJanus kinases phosphorylate STAT6 specifically on tyrosine 641.Tyrosine phosphorylation promotes the formation of STAT6 dimersvia reciprocal Src homology 2 (SH2) domain and phosphotyrosineinteractions. The STAT6 dimer gains the ability to bind DNA tar-gets, leading to new gene expression responsible for the biological

effects of STAT6 (12–14). Accurate cellular localization is key to

the function of a transcription factor, but how the STAT6 protein

gains access to the nucleus is not well understood.Movement of proteins in and out of the nucleus occurs by

passage through nuclear pore complexes that span the nuclearmembrane (15). Typically, nuclear import of a large proteindepends on the presence of a nuclear localization signal (NLS).The NLS is recognized by a karyopherin transport receptor thatfacilitates import through the nuclear pore complex (16, 17). Theclassical import receptor consists of a dimer with two distinctsubunits: an importin-a adapter that binds the NLS andimportin-b that binds importin-a and interacts with the nuclearpore complex. In the nucleus, importin-b binds Ran-GTP, lead-ing to release of the NLS cargo. Current knowledge of the nu-clear trafficking of STAT factors has shown that their nuclearimport is regulated distinctly (18). For example, nuclear importof the STAT1 factor is conditional and dependent on its dimer-ization mediated by tyrosine phosphorylation (19). However, theSTAT3 transcription factor is imported continually to the nu-cleus, independent of tyrosine phosphorylation (20). The STATmolecules share a similar arrangement of functional motifs thatincluding an N terminus, coiled coil domain, DNA-binding do-main, SH2 domain, phosphorylated tyrosine, and carboxyl trans-activation domain. Following tyrosine phosphorylation anddimerization, STAT1 gains the function of an NLS within itsDNA-binding domain, whereas STAT3 has a constitutive NLSwithin the coiled coil domain, independent of tyrosine phosphor-ylation.To assess the dynamic movement of STAT6 we used live cell

imaging with photobleaching techniques. We provide evidence

that STAT6 is imported continually into the nucleus, independent

of tyrosine phosphorylation, and it seems to use the importin-

a2importin-b1 system. In addition, a region required for NLS

function was found to map within the coiled coil domain.

Although nuclear import rates of STAT6 are similar before and

after tyrosine phosphorylation, nuclear accumulation occurs after

phosphorylation, and this is dependent on the DNA-binding abil-

ity of STAT6. Live cell imaging has provided critical insight to the

spatial distribution of STAT6, which impacts its function as a tran-

scription factor.

Department of Molecular Genetics and Microbiology, Stony Brook University, StonyBrook, NY 11794

Received for publication October 15, 2009. Accepted for publication April 18, 2010.

This work was supported by National Institutes of Health Grant RO1CA122910 (toN.C.R.).

Address correspondence and reprint requests to Dr. Nancy C. Reich, Department ofMolecular Genetics and Microbiology, Stony Brook University, Nicolls Road, LifeSciences Building, Stony Brook, NY 11794. E-mail address: nancy.reich@stonybrook.edu

The online version of this article contains supplemental material.

Abbreviations used in this paper: anti-pSTAT6, anti-STAT6 phosphotyrosine 641 Ab;c, control; C, cytoplasm; dl, deletion; Fl, fluorescent; FLIP, fluorescence loss inphotobleaching; FRAP, fluorescence recovery after photobleaching; G, GFP; hIL-4,human IL-4; impb1, importin-b1; IP, immunoprecipitated; MBP, maltose-bindingprotein; N, nucleus; NLS, nuclear localization signal; PS, Ponceau S; S, STAT6;SH2, Src homology 2; siRNA, short interfering RNA; STAT6-V5, STAT6 taggedwith the V5 epitope; WB, Western blot; wtSTAT6, wild type STAT6.

Copyright� 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903323

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Materials and MethodsCell cultures and reagents

HeLa and Cos1 cells (American Type Culture Collection, Manassas, VA)were cultured in DMEM with 8% FBS. Cells were treated with humanrIL-4 (R&D Systems, Minneapolis, MN) at 10 ng/ml. DNA transfectionswere carried out using TransIT-LT1 transfection reagent (Mirus, Madison,WI), according to the manufacturer’s instructions. Rabbit anti-STAT6 Ab(Santa Cruz Biotechnology, Santa Cruz, CA), anti-STAT6 phosphotyrosine641 Ab (anti-pSTAT6) (Cell Signaling Technology, Danvers, MA), and mu-rine anti-GFPAb (Roche Diagnostic Systems, Indianapolis, IN) were usedfor Western blotting at a 1:1000 dilution. HRP-conjugated anti-rabbit andanti-mouse Ig were used as secondary Abs for Western blotting (1:5000).GFP Ab and murine IgG2b (MOPC-144) control Ab (Sigma-Aldrich,St. Louis, MO) were used in EMSA, at 1 mg in 40-ml reactions. Two micro-grams of anti-V5 Ab (Invitrogen, Carlsbad, CA) was used for the in vitrobinding assays.

Plasmid constructs and protein purification

Full-length STAT6 cDNA and deletion mutants created by PCR were clonedinto pEF1/V5-His (Invitrogen) or pMAL-c4X (New England Biolabs, Ips-witch, MA) to generate V5 or maltose-binding protein (MBP) fusion pro-teins. A monomeric form of enhanced GFP was produced by mutatingA206K, L221K, and F223R in the vector pEGFP-N1 (BD Clontech, Moun-tain View, CA), and it was used to generate GFP-tagged STAT6 proteins(21). Site-directed mutagenesis was performed with targeted oligonucleoti-des and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). All con-structs were confirmed by DNA sequencing. Importin-a constructs lackingthe importin-b1–binding domain were generated and purified, as reportedpreviously (20). MBP-STAT6(1–267) and MBP-STAT6(1–267 deletion [dl]136–140) proteins were prepared following the manufacturer’s instructions(New England Biolabs).

Western blot

Two days after transfection, cells were serum starved for 24 h and weretreated or not with human IL-4 (hIL-4) for 30 min and lysed with cold lysisbuffer (50 mMTris [pH 8], 5 mMEDTA, 0.5%Nonidet P-40, 280 mMNaCl,1 mM PMSF, 13 protease inhibitor mixture [Sigma-Aldrich], 1 mM NaF,and 1 mM sodium vanadate). Proteins were separated by 8% SDS-PAGEand transferred to nitrocellulose membrane (Pierce, Rockford, IL). Theproteins were detected by reacting with Abs to STAT6, STAT6 phosphotyr-osine, or GFP and detected using the enhanced chemiluminescence systemor Odyssey Infrared Imaging System (LI-COR, Lincoln, NE).

EMSA

Cells were lysed with hypotonic lysis buffer (15 mM HEPES [pH 7.9], 0.2mM spermine, 0.5mMspermidine, 2mMpotassium-EDTA, 80mMKCl, 1%glycerol, 0.0025% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 1 mM sodiumvanadate, 1 mM NaF, and 13 protease inhibitor mixture) to prepare cyto-plasmic extracts. Nuclei were collected by centrifugation and extracted inhypertonic buffer (20 mM HEPES [pH 7.9], 0.2 mM spermine, 0.5 mMspermidine, 0.2 mM potassium-EDTA, 0.4 M NaCl, 10% glycerol, 1 mMDTT, 1 mM PMSF, 1 mM sodium vanadate, 1 mM NaF, and 13 proteaseinhibitor mixture). Nuclear and cytoplasmic extracts were combined for theDNA-binding reactions. Lysates were preincubated with Abs or 100-foldexcess nonradiolabeled probe for 30 min at room temperature prior to in-cubation with radiolabeled oligonucleotide probe for 30 min. The dsDNAoligonucleotide corresponding to 2407 to 2387 of the IL-4Ra gene (59-AGCTTCTTCATCTGAAAAGGG-39) was 59 end radiolabeled and used inthe binding reactions. Complexes were separated on nondenaturing acry-lamide gels and exposed to x-ray film for autoradiography.

Confocal microscopy

Cellswere plated on glass coverslips, transfectedwith STAT6 constructs, andserum starved overnight. Cells were treated with or without hIL-4 for 30minandfixedwith 4%paraformaldehyde.GFP-taggedproteinwas observedwitha Carl Zeiss LSM 5 laser-scanning microscope using a 403 oil objective(Plan-Neofluar, numerical aperture 1.3, differential interference contrast mi-croscopy objective [Jena, Germany]). GFP was excited at 488 nm using anargon laser, and emission was collected using a 505 long-pass filter. Imageswere captured using Zeiss LSM 5 Pascal imaging software.

Live cell imaging

HeLa cells were seeded on glass-bottom tissue culture dishes (Mattek, Ash-land, MA) and transfected. The dishes were mounted on a Zeiss inverted

Axiovert 200M microscope using a heating insert coupled with the In-cubator S (Zeiss). During imaging, the cells were maintained at 37˚Cand 5% CO2 using the Zeiss Tempcontrol 37-2 Digital and CTI Controller3700. The time-series images for photobleaching assays were taken withthe Zeiss LSM 510 META NLO two-photon laser-scanning microscopesystem using a 403 oil objective (Plan-Neofluar, numerical aperture 1.3,differential interference contrast microscopy objective). The excitationwavelength used for GFP was 488 nm, and emission was detected witha 505-nm filter. For fluorescence recovery after photobleaching (FRAP)analysis, a region in the nucleus was bleached at 100% power of an argonlaser at 488 nm for 70 s. For fluorescence loss in photobleaching (FLIP)analysis, a region in the nucleus or cytoplasm was bleached every 12 s atmaximum laser intensity for 5 or 50 min. Images were acquired using LSM510 META version 3.2 imaging software. The fluorescence intensity wasquantified in the region of interest using LSM Imaging software and graph-ically depicted using Microsoft Excel (Microsoft, Redmond, WA).

Luciferase assay

HeLa cells were transfected with IL-4R–luciferase (22), Renilla luciferase(Promega, Madison, WI), and STAT6-GFP wild type or mutant plasmids.Two days after transfection, cells were treated or not with 3 ng/mlhIL-4 for 8 h prior to harvest. Dual-luciferase reporter assays were per-formed according to the manufacturer’s instructions (Promega, Madison,WI). The luciferase results were normalized to Renilla luciferase values tocompensate for variations in transfection efficiency.

In vitro importin binding assay

The GST–importin-as lacking the aminoterminal importin-b–bindingdomain were expressed in bacteria and purified by binding and elutionfrom glutathione beads (20). Cos1 cells expressing STAT6 tagged withthe V5 epitope (STAT6-V5) were lysed with buffer (280 mM NaCl, 50mM Tris-HCl [pH 8.2], 5 mM EDTA, and 0.5% Nonidet P-40), and 500 mgprotein lysate was used for each assay. STAT6 was captured with anti-V5Ab, bound to protein G beads, and incubated with 15 mg purified GST–importin-as. Bound protein complexes were eluted with SDS samplebuffer and analyzed by Western blot with anti-V5 and anti-GST Abs. Totest importin binding to bacterially expressed STAT6, rGST–importin-aswere incubated with bacterially expressed MBP-tagged STAT6 proteinsimmobilized on amylase resin in column buffer (20 mM Tris-HCl, 200mM NaCl, 1 mM EDTA, and 1 mM DTT) with 0.05% Nonidet P-40.Binding was detected by Western blot with anti-GST Ab, and the STAT6protein was quantified by Ponceau S staining.

RNA interference

Short interfering RNA (siRNA) duplexes specific for human importin-b1(Qiagen, Valencia, CA) or vimentin (control) were transfected withX-tremeGENE siRNA transfection reagent (Roche). Twenty-four hoursafter siRNA transfection, cells were transfected with STAT6-GFP. Cellu-lar localization of STAT6-GFP was observed after 24 h by fluorescencemicroscopy. RNA extraction was performed with SurePrep TrueTotalRNA purification kit (Fisher Scientific, Pittsburgh, PA), and cDNAwas synthesized with M-MLV reverse transcriptase (Promega). RT-PCRwas performed with specific primers for importin-b1 or GAPDH asan internal control. Image J software was used to estimate quantity(freely available in the National Institutes of Health public domain).Primer sequences for importin-b1 were 59-AATCCAGGAAACAGT-CAGGTTGC-39 (forward) and 59-AGCACTGAGACCCTCAATCAG-39(reverse) and for GAPDH were 59-GGAGCCAAAAGGGTCATCAT-CTC-39 (forward) and 59-AGTGGGTGTCGCTGTTGAGTC-39 (reverse).

ResultsSTAT6 nuclear import is independent of tyrosinephosphorylation

Fluorescence microscopy was used to visualize nuclear traffickingof STAT6. STAT6 was tagged at its C terminus with GFP (STAT6-GFP) and expressed in cells that were serum starved and stimulatedor not with IL-4 for 30 min (Fig. 1A). The microscopic imagesrevealed latent unphosphorylated STAT6-GFP in the cytoplasmand nucleus (Fig. 1Aa). This result indicated that tyrosine phos-phorylation was not required for STAT6 nuclear import. Followingtyrosine phosphorylation in response to IL-4, STAT6-GFP accu-mulated dominantly in the nucleus (Fig. 1Ab). Results were similar

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with endogenous STAT6 or V5-tagged STAT6 detected by immu-nofluorescence (Supplemental Fig. 1A, 1B). Analysis of endoge-nous STAT6 in primary lymphocytes also clearly showedunphosphorylated STAT6 present in nuclei prior to IL-4 treatmentand an increase in nuclear STAT6 following IL-4 treatment (Sup-plemental Fig. 1C). To confirm STAT6 nuclear import was inde-pendent of tyrosine phosphorylation, the behavior of a STAT6protein with a double mutation was evaluated. The tyrosine 641that is specifically phosphorylated in response to cytokine stimula-tion was substituted with phenylalanine (Y641F), and the criticalarginine 562 in the SH2 domain that functions to form dimers ca-pable of specific DNA binding was mutated to alanine (R562A).Imaging results showed the double mutant, STAT6(RY)-GFP, wasimported to the nucleus but did not accumulate following stimula-tion with IL-4 (Fig 1Ac, 1Ad). These data show that STAT6 nuclearimport is independent of tyrosine phosphorylation and that nuclearaccumulation requires tyrosine phosphorylation.EMSAs and Western blotting were performed to ensure that

STAT6-GFP was tyrosine phosphorylated accurately and capable ofbinding DNA, whereas the STAT6(RY)-GFP lacked these abilities.The EMSAs showed that STAT6-GFP can bind a specific DNA tar-get only following tyrosine phosphorylation, and the STAT6(RY)-GFP lacks this ability (Fig. 1B). Western blotting with Abs thatrecognize phosphotyrosine 641 STAT6 confirmed that STAT6-GFPis accurately tyrosine phosphorylated after IL-4 treatment, butSTAT6 (RY)-GFP is not phosphorylated (Fig. 1C).

Live cell imaging reveals STAT6 constitutive nuclear shuttling

The spatial and temporal dynamics of STAT6 were evaluated bylive cell imaging with nuclear FRAP (Fig. 2). Nuclei of cellsexpressing STAT6-GFP were subjected to a high-intensity laser tobleach fluorescence in this compartment (top panel). The recoveryof fluorescence in the nucleus with time was monitored relative toa region of interest in the cytoplasm for STAT6 in unstimulatedcells, STAT6 in IL-4–stimulated cells (+IL-4), or the STAT6(RY)mutant in IL-4–stimulated cells (+IL-4). Fluorescence recovery inthe nucleus of unphosphorylated STAT6-GFP was half maximal

by 15 min and complete by 45 min (top panel). Following tyrosinephosphorylation in response to IL-4, nuclear fluorescence recov-ery also was half maximal by 15 min; however, within 30–45 min,phosphorylated STAT6-GFP accumulated in the nucleus toa greater extent than in the cytoplasm. This result could reflectmore efficient import of the tyrosine phosphorylated form ofSTAT6 or, alternatively, a decrease in STAT6 nuclear export.The kinetics of nuclear accumulation of the STAT6(RY)-GFP mu-tant (bottom panel) were similar to that of unphosphorylatedSTAT6 and confirm that nuclear import of STAT6 is continuousand independent of tyrosine phosphorylation.FLIP was used to address the basis of nuclear accumulation fol-

lowing tyrosine phosphorylation of STAT6 (Fig. 3). A high-intensitylaser was continually directed to a small region in the cytoplasm ofcells expressing unphosphorylated STAT6-GFP or tyrosine phos-phorylated STAT6-GFP (+IL-4). STAT6 passing through the laserpath of this small region is bleached, and the loss of fluorescencecorrelates with STAT6 mobility. Fluorescence intensity rapidly de-creased in the cytoplasm of cells expressing unphosphorylatedSTAT6-GFP or tyrosine phosphorylated STAT6-GFP, indicatingrapid movement through the cytoplasm. For unphosphorylatedSTAT6-GFP, this loss was followed by a loss of fluorescence inthe nucleus that was nearly complete by 50 min. The loss of nuclearfluorescence indicates continual STAT6 export from the nucleus andpassage through the laser path in the cytoplasm. In contrast, a differ-ent result was found for tyrosine phosphorylated STAT6-GFP (lowerpanel). Nuclear fluorescence of phosphorylated STAT6 did not de-crease during the duration of the experiment. These results suggestthat the nuclear accumulation that is evident after STAT6 tyrosinephosphorylation is due to a decrease in nuclear export.

DNA binding retains STAT6 in the nucleus

Tyrosine phosphorylation activates STAT proteins by promotingthe formation of dimers that have the ability to bind specific DNAtarget sites. To determine whether the increased nuclear accu-mulation of STAT6 seen following tyrosine phosphorylation wasdue to a gain in the ability to bind DNA, the behavior of a DNA-

FIGURE 1. STAT6 nuclear import is independent of tyrosine phos-

phorylation. A, HeLa cells were transfected with STAT6-GFP or STAT6

(RY)-GFP, serum starved, and left untreated (2) or treated (+) with IL-4

for 30 min. Cellular localization of STAT6 was visualized by fluorescence

microscopy (original magnification 3100). B, EMSAs were performed

with the radiolabeled IL-4R target oligonucleotide and protein lysates from

cells, as treated in A. Effects of additions of Abs to the reaction to GFP (G),

STAT6 (S), control (c), or 100-fold excess unlabeled oligonucleotide

(DNA) are shown. C, Western blots of protein lysates were performed

with anti-pSTAT6, anti-STAT6, or anti-GFP Abs.

FIGURE 2. Live cell imaging of STAT6 constitutive nuclear import.

Nuclear FRAP assays were performed with cells expressing STAT6-GFP

without IL-4 stimulation (upper row) or with IL-4 stimulation (middle row)

or with cells expressing STAT6(RY)-GFP with IL-4 stimulation (lower

row). The nucleus (N) was subjected to high-intensity laser to bleach the

fluorescent STAT6. Subsequent fluorescence recovery with time in the

nucleus was monitored and quantified relative to a site in the cytoplasm

(C). The quantitative data of relative fluorescent (Fl) intensity with time

are shown in the panels on the far right. Experiments are representative of

more than three independent studies.

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binding mutant was evaluated. A STAT6 DNA-binding mutant wasgenerated based on other STATDNA-bindingmutants (23). Lysinesand arginines within aa 366–374 were substituted with alanines togenerate STAT6(KR). Although the STAT6(KR) mutant was

accurately tyrosine phosphorylated in response to IL-4, it did notbind target DNA sequences (Fig. 4A). Microscopic imaging indi-cated that STAT6(KR)was imported to the nucleuswith andwithoutIL-4 stimulation, but it did not accumulate in the nucleus in responseto IL-4. This indicated that DNA binding contributes to nuclearaccumulation following tyrosine phosphorylation.If DNA binding retains STAT6 in the nucleus, the mobility of

tyrosine-phosphorylated STAT6 within the nucleus would beexpected to be slower than unphosphorylated STAT6. A nuclearFLIP assay was used to investigate this possibility (Fig. 4B). Asmall region (region 1) in the nucleus of cells expressing STAT6-GFP, with or without IL-4 stimulation, was subjected to continu-ous laser bleaching for 5 min. The fluorescence intensity of region1 was compared with a distinct region in the nucleus (region 2). Ifmovement is rapid through the path of the laser, the fluorescenceintensity in region 2 will decrease similarly to region 1, along withthe entire nucleus. This was the case for unphosphorylated STAT6.However, following tyrosine phosphorylation in response to IL-4, STAT6 showed significantly slower movement. The fluores-cence decrease in region 2 and the remainder of the nucleuswas delayed considerably compared with region 1. The tyrosine-phosphorylated DNA-binding mutant, STAT6(KR), showed thesame rapid nuclear movement as unphosphorylated STAT6. Toestablish that the DNA-binding mutant is not retained in the nu-cleus following IL-4 stimulation, imaging with cytoplasmic FLIPwas used (Supplemental Fig. 2). The export kinetics of tyrosine-phosphorylated STAT6(KR) were similar to unphosphorylatedwild type STAT6 (wtSTAT6). Together, the results support thepremise that STAT6 accumulates in the nucleus only if it hasa functional DNA-binding domain.

Identification of amino acids in STAT6 that are required fornuclear import

Nuclear import of large molecules, such as STAT6, requires anamino acid sequence or structure that serves as an NLS. To identifyamino acids that function to facilitate STAT6 nuclear import,a series of deletion mutations were generated, and the cellularlocalization of the truncated proteins was evaluated with or withoutIL-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. Thecellular localization of these truncations indicated that a regionin the coiled coil domain is needed for nuclear import. STAT6(1–267) containing the N terminus and the coiled coil domain ofSTAT6 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 withoutIL-4 stimulation. Deletions within the coiled coil domain identi-fied a region required for STAT6 nuclear import. STAT6(136–847)was imported and accumulated in the nucleus following tyrosinephosphorylation, whereas STAT6(141–847) remained in the cyto-plasm with or without tyrosine phosphorylation. Western blottingwith Abs to STAT6 phosphotyrosine 641 confirmed that the dele-tions were accurately phosphorylated in response to IL-4 (Fig. 5).The studies with STAT6 truncations identified a sequence be-

tween aa 136–140 (RLQHR) that is required for nuclear import.To determine the effect of a specific deletion or substitution ofthese amino acids in otherwise full-length STAT6, we evaluatedthe localization of two mutants linked to GFP (Fig. 6A). STAT6 dl136–140 or STAT6 containing a substitution of 135–140 aa withalanine residues (sub6A) were expressed in cells stimulated or notwith IL-4. The cellular localization of both mutants was restrictedto the cytoplasm, indicating a deficiency in nuclear import. Thesemutants were accurately tyrosine phosphorylated in response to

FIGURE 3. Live cell imaging demonstrates decreased STAT6 nuclear

export following tyrosine phosphorylation. Cytoplasmic FLIP assays were

performed with cells expressing STAT6-GFP not treated (upper row) or

treated with IL-4 (lower row). A small region in the cytoplasm (C) was

subjected to continuous high-intensity laser. Fluorescence loss was moni-

tored with time in the cytoplasm and compared with the fluorescence loss

in a region of the nucleus (N). The quantitative data of relative fluorescent

(Fl) intensity with time are shown in the panels on the far right. Experi-

ments are representative of more than three independent studies.

FIGURE 4. DNA binding promotes nuclear accumulation of tyrosine-

phosphorylated STAT6. A, The DNA-binding mutant STAT6(KR)-GFP

was expressed in cells and examined by fluorescence microscopy before

(2) or after (+) treatment with IL-4 (original magnification 3100). West-

ern blot (lower panels) was performed with anti-pSTAT6 or anti-GFPAbs.

EMSA (right panel) was performed with the IL-4R target oligonucleotide

and lysates from cells expressing STAT6-GFP (wt) or STAT6(KR)-GFP

(KR) without (2) or with (+) IL-4 treatment. Abs to STAT6 (S), GFP (G),

or MOPC control (c) were added to the binding reactions. B, Live cell

imaging was used with nuclear FLIP to evaluate STAT6-GFP and STAT6

(KR)-GFP mobility within the nucleus. A small region in the nucleus

(region 1) was subjected to continuous high-intensity laser. Fluorescence

loss was monitored with time in this region and a distinct region in the

nucleus (region 2). Quantitative data of relative fluorescent (Fl) intensity

with time are shown in the panels on the far right. Experiments are rep-

resentative of more than three independent studies.

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IL-4, indicating that the internal deletion and substitution did notdisrupt STAT6 activation. To evaluate the influence of specific res-idues in this region, each amino acid was mutated in the context offull-length STAT6. However, the individual point mutants behavedas wtSTAT6 (Supplemental Fig. 3). Together, these results indicatethat aa 136–140 are required for STAT6 nuclear import, but theymay function within the context of a conformational NLS.Transcriptional regulation is the primary function of STAT6, and

for this reason we evaluated the ability of STAT6 mutants to inducegene expression. Mutants defective in nuclear localization, STAT6(dl136–140), or DNA binding, STAT6(KR), were tested for theircompetence to induce the characterized promoter of the IL-4Rgene (22). Transient transfections clearly demonstrated the abilityof wtSTAT6 to induce the IL-4R reporter in response to IL-4,

whereas STAT6(dl136–140) and STAT6(KR) did not induce thegene (Fig. 6B).

Evidence supporting a role of importin-a/b1 in STAT6 nuclearimport

Active transport of large molecules through the nuclear porecomplex usually requires facilitation by carrier proteins of thekaryopherin-b family. Importin-b1 is a primary karyopherin-btransporter that can bind directly to NLS-containing proteins or in-directly via the family of importin-a adapters. Importin-a adaptersbind directly to the NLS. In vitro binding assays were performed toevaluate whether one or more of the importin-as can recognize

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 magnification3100).

Bottom right, Western blot of protein lysates from cells

performed with anti-pSTAT6, anti-STAT6, or anti-GFP

Abs.

FIGURE 6. Amino acids 136–140 are required for STAT6 nuclear im-

port. A, STAT6-GFP with an internal deletion of aa 135–140 (dl) or

STAT6-GFP with a substitution of six alanine residues for aa 135–140

(sub6A) were expressed in cells that were serum starved (2) or stimulated

with IL-4 (+). STAT6 localization was visualized microscopically (original

magnification 3100). Bottom panel displays Western blot of protein

lysates with anti-pSTAT6 or anti-GFP Abs. B, HeLa cells were transfected

with IL-4R site-TKLuc reporter construct, Renilla luciferase vector, and

different STAT6-GFP constructs. Cells were treated or not with hIL-4 for 8

h before the dual-luciferase reporter assay. Luciferase result was normal-

ized to Renilla luciferase value.

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

bottom panel (anti-GST). B, Bacterial-expressed MBP-STAT6(1–267) and

MBP-STAT6(1–267dl136–140) were immobilized on amylase resin and

incubated with purified GST–importin-a1 or GST–importin-a3. The

binding was evaluated by Western blot with anti-GST Ab. Ponceau S

(PS) staining showed that the same amount of STAT6 proteins were used.

C, Fluorescent images of cells transfected with vimentin siRNA (control)

or importin-b1 siRNA (impb1) for 24 h followed by transfection with

STAT6-GFP. Cytoplasmic localization was seen in ∼10% of cells. Lower

panel displays the effect of control vimentin siRNA or importin-b1 siRNA

on endogenous importin-b1 mRNA or GAPDH mRNA as evaluated by

RT-PCR.

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STAT6 (Fig. 7A). STAT6-V5 was expressed in mammalian cellsand immunoprecipitated from cell lysates with V5 Ab and proteinG agarose beads. GST-tagged importin-as were expressed inbacteria and added to the STAT6-V5 immunocomplexes collectedon beads. Interaction of importins with STAT6 was detected byWestern blot with Ab to GST. The results indicated that STAT6 isrecognized primarily by importin-a3 and -a6. Similar results wereobtained with STAT6 isolated from untreated cells or IL-4–stimulated cells, indicating that binding is independent of tyrosinephosphorylation. Because importin-a6 is restricted to the testes,importin-a3 seems to be the primary import adapter (24, 25).Because aa 136–140 in the coiled coil region of STAT6 are critical

for nuclear import, we determined whether this sequence was re-quired for direct interaction with importin-a3. We expressedfragments of STAT6 tagged with MBP in bacteria corresponding toSTAT6 1–267 aa or 1–267 containing the 136–140 deletion. MBP-STAT6(1–267) and MBP-STAT6(dl136–140) were incubated withbacterially expressed GST–importin-a3 or GST–importin-a1 asa control and evaluated for binding (Fig. 7B). The results showedthat STAT6(1–267) can bind importin-a3 specifically, but thedeletion mutant cannot. These data suggest aa 136–140 are requiredfor STAT6 binding to importin-a3 and nuclear import in vivo.Given that the importin-a/b1 systemmaymediate STAT6 nuclear

import, we evaluated the effect of RNA interference on the inhibitionof expression of importin-b1 (Fig. 7C). siRNA duplexes corres-ponding to importin-b1 or to vimentin as a control weretransfected into cells with STAT6-GFP, and the localization ofSTAT6-GFP was visualized microscopically. The behavior ofSTAT6-GFP was notably different in the cells treated withimportin-b1 siRNA. Approximately 10% of cells showed STAT6restricted to the cytoplasmic compartment, often with punctatecytoplasmic fluorescence. Because the siRNA may not completelyinhibit importin-b1 expression in all cells expressingSTAT6-GFP, theeffect seems to be significant. To evaluate the effectiveness of theimportin-b1 siRNA complexes, mRNA levels in cells treated withcontrol or importin-b1 siRNAwere assayed by RT-PCR. The siRNAto importin-b1 reduced endogenous mRNA by ∼70%. Together, theresults suggest that importin-a/importin-b1 may mediate STAT6nuclear import.

DiscussionNuclear trafficking of STAT6 is integral to its function as a signaltransducer and activator of transcription. By attaching a fluorescentprobe to STAT6 we were able to study its intracellular dynamicswith microscopy in real time. The advantage of live cell imaging isthat it avoids fixation techniques that can influence cellular ar-chitecture. Cell fractionation has been used to evaluate cellularlocalization; however, the technique is limited in interpretingin vivo protein localization, particularly if the protein is activelyimported and exported from the nucleus. Our studies indicated thatSTAT6 moves continually within the cytoplasm; additionally, it istransported continually into and out of the nucleus, independent oftyrosine phosphorylation.Specific phosphorylation of tyrosine 641 promotes STAT6 di-

merization and its ability to bind DNA target sites. In addition to thisactivating modification, other modifications have been reported thatinclude serine phosphorylation of the carboxyl transactivation do-main, which may influence DNA binding (26–28), and acetylation,which may contribute to induction of gene expression (12, 29).Methylation of arginine 27 was reported to be required for STAT6tyrosine phosphorylation, nuclear translocation, and DNA binding(30). However, our studies indicate that arginine 27 is not necessaryfor tyrosine phosphorylation, nuclear translocation, or DNA

binding. STAT6 that completely lacks 135 aa from the N terminus isimported to the nucleus, is tyrosine phosphorylated in response toIL-4, and can bind DNA (Fig. 5) (H.C. Chen and N.C. Reich, un-published observations).By studying the cellular localization of various STAT6 deletions,

we identified a region within the coiled coil domain required forSTAT6 nuclear import (Fig. 5). STAT6(136–847) was imported tothe nucleus constitutively, whereas STAT6(141–847) was notimported. Deletion or substitution of the small region between aa135–140 eliminated the ability of otherwise full-length STAT6 tobe imported to the nucleus, although the proteins were still tyro-sine phosphorylated accurately (Fig. 6). The best-characterizedclassical NLS sequences contain one or two stretches of basicamino acids, particularly lysines (31). Although the sequence135–140 (RRLQHR) contains arginine residues, site-directed mu-tation of individual amino acids within this region was not suffi-cient to block nuclear import (Supplemental Fig. 3). This findingsuggests that a noncanonical NLS may be functional within 136–267. Other STAT molecules seem to use noncanonical NLSs todrive import, whether they are constitutive or conditional (18).Although the STATs do not display classical NLSs, they seem

to use the importin-a–importin-b1 receptors. Importin-a5 binds toSTAT1 when it is in the conformation of a tyrosine-phosphorylateddimer and facilitates its nuclear import (19, 32, 33), whereasimportin-a3 and -a6 bind constitutively to STAT3 (20). In thisstudy, we found that importin-a3 and -a6 also bind constitutivelyto STAT6; additionally, downmodulation of importin-b1 by RNAinterference notably reduces STAT6 nuclear import. The resultssuggest that STAT6 is imported by importin-a–importin-b1 re-ceptors (Fig. 7). It is challenging to determine specific importin-arecognition of a particular NLS outside the framework of the nativeprotein, because recognition depends on the NLS sequence, as well asthe protein context (34). The crystal structure of STAT6 remains to besolved. However, the identity of the importin-a that binds a particularprotein may be significant because the importin-a proteins displayspecific expression in tissues and during differentiation (25, 35). Itwas reported that a Rac GTPase-activating protein is responsiblefor nuclear import of activated STAT proteins and that the dominantnegative N17Rac1 protein can block nuclear import of the STATs(36). For this reason, we tested the effect of N17 Rac1 on STAT6nuclear import but did not detect any effect (Supplemental Fig. 4).Latent-unphosphorylated and tyrosine-phosphorylated STAT6 are

imported to the nucleus. The difference is that STAT6 accumulates inthe nucleus when it is tyrosine phosphorylated (Fig. 1). Live cellimagingwithphotobleaching techniquesprovidesamorequantitativeand temporal measure of protein mobility and localization (37–39).By using the technique of nuclear FRAP, the transport of STAT6-GFPinto the nucleus was observed to be similar for unphosphorylated ortyrosine-phosphorylated STAT6-GFP (Fig. 2). However, the averagefluorescence intensity of phosphorylated STAT6-GFP becomes sig-nificantly greater in the nucleus than in the cytoplasm. The nuclearaccumulation is the consequence of decreased nuclear export. Thiswas demonstrated with cytoplasmic FLIP (Fig. 3). Repeated photo-bleaching of one small region in the cytoplasm resulted in the loss oftotal cytoplasmic fluorescence, independent of STAT6 phosphoryla-tion. For unphosphorylated STAT6-GFP, thiswas followed by a grad-ual loss of fluorescent signal from the nucleus, indicating continuousexport. In contrast, nuclear fluorescence of tyrosine-phosphorylatedSTAT6-GFP did not decrease during the experiment. Therefore, theincrease in STAT6 nuclear accumulation following tyrosine phos-phorylation is a result of decreased nuclear export.The mechanism of STAT6 nuclear export remains to be de-

termined; nonetheless, it seems that DNA binding is responsible forSTAT6 nuclear accumulation. A STAT6 DNA-binding mutant was

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shown to behave like unphosphorylated STAT6 and did not accu-mulate in the nucleus following phosphorylation (Fig. 4). In ad-dition, nuclear FLIP analyses determined that DNA bindingdramatically reduced STAT6 movement within the nucleus. Theseobservations indicate that nuclear accumulation of tyrosine-phosphorylated STAT6 is due to retention by association withDNA. DNA binding may be a general cause for observed nuclearaccumulation of STAT proteins (23, 38, 40, 41).Accurate cellular localization is essential for the effective

function of transcription factors, such as STAT6. The constitutivenuclear import and export of latent STAT6 may provide an ad-vantage for the rapid response to cytokine-stimulated tyrosinephosphorylation, or it may enable an activating response to nuclearkinases. Alternatively, because there is precedence for the functionof unphosphorylated STATs contributing to gene expression,unphosphorylated STAT6 may have an undiscovered function inthe nucleus (42). Understanding the mechanisms that regulateSTAT6 nuclear trafficking will support means to manipulate itsactivity in health and disease.

AcknowledgmentsWe thank the current and past members of the laboratory for their support,

particularly Janaki Iyer, Velasco Cimica, and Sarah Van Scoy. We express

our thanks to Dr. Guo-Wei Tian and Dr. Vitaly Citovsky for their support

with imaging experiments. Many thanks to Dr. Nick Carpino and Dr. Martha

Furie for their helpful support.

DisclosuresThe authors have no financial conflicts of interest.

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