Live Cell Imaging Reveals Continuous STAT6 Nuclear Trafficking

Deciphering the signaling events initiated by specific cytokines is critical to understanding their biological effects. The STAT6 transcription factor was identified as a DNA-binding factor activated in response to IL-4 (1–3). It is now known to be required for the generation of Th2 lymphocytes, the normal function of B lymphocytes, and protection against parasitic nematodes (4–7); however, collateral damage accompanies its positive effects in the immune response. Hyperactivity of STAT6 predisposes lymphoproliferative disease and is responsible for diseases associated with Th2 cell pathologies, like asthma (8–10). Given the considerable evidence that STAT6 contributes to an effective immune response and plays a dominant role in asthmatic lung pathology, understanding the mechanisms that regulate its nuclear 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 phosphorylation stimulated in response to Th2 cytokines IL-4 and -13 (11). Following cytokine binding to cell-surface receptors, associated Janus kinases phosphorylate STAT6 specifically on tyrosine 641. Tyrosine phosphorylation promotes the formation of STAT6 dimers via reciprocal Src homology 2 (SH2) domain and phosphotyrosine interactions. The STAT6 dimer gains the ability to bind DNA targets, 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 nuclear membrane (15). Typically, nuclear import of a large protein depends on the presence of a nuclear localization signal (NLS). The NLS is recognized by a karyopherin transport receptor that facilitates import through the nuclear pore complex (16, 17). The classical import receptor consists of a dimer with two distinct subunits: an importin-α adapter that binds the NLS and importin-β that binds importin-α and interacts with the nuclear pore complex. In the nucleus, importin-β binds Ran-GTP, leading to release of the NLS cargo. Current knowledge of the nuclear trafficking of STAT factors has shown that their nuclear import is regulated distinctly (18). For example, nuclear import of the STAT1 factor is conditional and dependent on its dimerization mediated by tyrosine phosphorylation (19). However, the STAT3 transcription factor is imported continually to the nucleus, independent of tyrosine phosphorylation (20). The STAT molecules share a similar arrangement of functional motifs that including an N terminus, coiled coil domain, DNA-binding domain, SH2 domain, phosphorylated tyrosine, and carboxyl transactivation domain. Following tyrosine phosphorylation and dimerization, STAT1 gains the function of an NLS within its DNA-binding domain, whereas STAT3 has a constitutive NLS within the coiled coil domain, independent of tyrosine phosphorylation.

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-α−importin-β1 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 ability of STAT6. Live cell imaging has provided critical insight to the spatial distribution of STAT6, which impacts its function as a transcription factor.

HeLa and Cos1 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM with 8% FBS. Cells were treated with human rIL-4 (R&D Systems, Minneapolis, MN) at 10 ng/ml. DNA transfections were 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 phosphotyrosine 641 Ab (anti-pSTAT6) (Cell Signaling Technology, Danvers, MA), and murine anti-GFP Ab (Roche Diagnostic Systems, Indianapolis, IN) were used for Western blotting at a 1:1000 dilution. HRP-conjugated anti-rabbit and anti-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 μg in 40-μl reactions. Two micrograms of anti-V5 Ab (Invitrogen, Carlsbad, CA) was used for the in vitro binding assays.

Full-length STAT6 cDNA and deletion mutants created by PCR were cloned into pEF1/V5-His (Invitrogen) or pMAL-c4X (New England Biolabs, Ipswitch, MA) to generate V5 or maltose-binding protein (MBP) fusion proteins. A monomeric form of enhanced GFP was produced by mutating A206K, L221K, and F223R in the vector pEGFP-N1 (BD Clontech, Mountain View, CA), and it was used to generate GFP-tagged STAT6 proteins (21). Site-directed mutagenesis was performed with targeted oligonucleotides and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). All constructs were confirmed by DNA sequencing. Importin-α constructs lacking the importin-β1–binding domain were generated and purified, as reported previously (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).

Two days after transfection, cells were serum starved for 24 h and were treated or not with human IL-4 (hIL-4) for 30 min and lysed with cold lysis buffer (50 mM Tris [pH 8], 5 mM EDTA, 0.5% Nonidet P-40, 280 mM NaCl, 1 mM PMSF, 1× protease inhibitor mixture [Sigma-Aldrich], 1 mM NaF, and 1 mM sodium vanadate). Proteins were separated by 8% SDS-PAGE and transferred to nitrocellulose membrane (Pierce, Rockford, IL). The proteins were detected by reacting with Abs to STAT6, STAT6 phosphotyrosine, or GFP and detected using the enhanced chemiluminescence system or Odyssey Infrared Imaging System (LI-COR, Lincoln, NE).

Cells were lysed with hypotonic lysis buffer (15 mM HEPES [pH 7.9], 0.2 mM spermine, 0.5 mM spermidine, 2 mM potassium-EDTA, 80 mM KCl, 1% glycerol, 0.0025% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 1 mM sodium vanadate, 1 mM NaF, and 1× protease inhibitor mixture) to prepare cytoplasmic extracts. Nuclei were collected by centrifugation and extracted in hypertonic buffer (20 mM HEPES [pH 7.9], 0.2 mM spermine, 0.5 mM spermidine, 0.2 mM potassium-EDTA, 0.4 M NaCl, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 mM sodium vanadate, 1 mM NaF, and 1× protease inhibitor mixture). Nuclear and cytoplasmic extracts were combined for the DNA-binding reactions. Lysates were preincubated with Abs or 100-fold excess nonradiolabeled probe for 30 min at room temperature prior to incubation with radiolabeled oligonucleotide probe for 30 min. The dsDNA oligonucleotide corresponding to −407 to −387 of the IL-4Rα gene (5′-AGCTTCTTCATCTGAAAAGGG-3′) was 5′ end radiolabeled and used in the binding reactions. Complexes were separated on nondenaturing acrylamide gels and exposed to x-ray film for autoradiography.

Cells were plated on glass coverslips, transfected with STAT6 constructs, and 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 a Carl Zeiss LSM 5 laser-scanning microscope using a 40× oil objective (Plan-Neofluar, numerical aperture 1.3, differential interference contrast microscopy objective [Jena, Germany]). GFP was excited at 488 nm using an argon laser, and emission was collected using a 505 long-pass filter. Images were captured using Zeiss LSM 5 Pascal imaging software.

HeLa cells were seeded on glass-bottom tissue culture dishes (Mattek, Ashland, MA) and transfected. The dishes were mounted on a Zeiss inverted Axiovert 200M microscope using a heating insert coupled with the Incubator S (Zeiss). During imaging, the cells were maintained at 37°C and 5% CO₂ using the Zeiss Tempcontrol 37-2 Digital and CTI Controller 3700. The time-series images for photobleaching assays were taken with the Zeiss LSM 510 META NLO two-photon laser-scanning microscope system using a 40× oil objective (Plan-Neofluar, numerical aperture 1.3, differential interference contrast microscopy objective). The excitation wavelength used for GFP was 488 nm, and emission was detected with a 505-nm filter. For fluorescence recovery after photobleaching (FRAP) analysis, a region in the nucleus was bleached at 100% power of an argon laser 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 at maximum laser intensity for 5 or 50 min. Images were acquired using LSM 510 META version 3.2 imaging software. The fluorescence intensity was quantified in the region of interest using LSM Imaging software and graphically depicted using Microsoft Excel (Microsoft, Redmond, WA).

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/ml hIL-4 for 8 h prior to harvest. Dual-luciferase reporter assays were performed according to the manufacturer’s instructions (Promega, Madison, WI). The luciferase results were normalized to Renilla luciferase values to compensate for variations in transfection efficiency.

The GST–importin-αs lacking the aminoterminal importin-β–binding domain were expressed in bacteria and purified by binding and elution from glutathione beads (20). Cos1 cells expressing STAT6 tagged with 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 μg protein lysate was used for each assay. STAT6 was captured with anti-V5 Ab, bound to protein G beads, and incubated with 15 μg purified GST–importin-αs. Bound protein complexes were eluted with SDS sample buffer and analyzed by Western blot with anti-V5 and anti-GST Abs. To test importin binding to bacterially expressed STAT6, rGST–importin-αs were incubated with bacterially expressed MBP-tagged STAT6 proteins immobilized on amylase resin in column buffer (20 mM Tris-HCl, 200 mM 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 STAT6 protein was quantified by Ponceau S staining.

Short interfering RNA (siRNA) duplexes specific for human importin-β1 (Qiagen, Valencia, CA) or vimentin (control) were transfected with X-tremeGENE siRNA transfection reagent (Roche). Twenty-four hours after siRNA transfection, cells were transfected with STAT6-GFP. Cellular localization of STAT6-GFP was observed after 24 h by fluorescence microscopy. RNA extraction was performed with SurePrep TrueTotal RNA purification kit (Fisher Scientific, Pittsburgh, PA), and cDNA was synthesized with M-MLV reverse transcriptase (Promega). RT-PCR was performed with specific primers for importin-β1 or GAPDH as an internal control. Image J software was used to estimate quantity (freely available in the National Institutes of Health public domain). Primer sequences for importin-β1 were 5′-AATCCAGGAAACAGTCAGGTTGC-3′ (forward) and 5′-AGCACTGAGACCCTCAATCAG-3′ (reverse) and for GAPDH were 5′-GGAGCCAAAAGGGTCATCATCTC-3′ (forward) and 5′-AGTGGGTGTCGCTGTTGAGTC-3′ (reverse).

Fluorescence microscopy was used to visualize nuclear trafficking of STAT6. STAT6 was tagged at its C terminus with GFP (STAT6-GFP) and expressed in cells that were serum starved and stimulated or not with IL-4 for 30 min (Fig. 1A). The microscopic images revealed latent unphosphorylated STAT6-GFP in the cytoplasm and nucleus (Fig. 1Aa). This result indicated that tyrosine phosphorylation was not required for STAT6 nuclear import. Following tyrosine phosphorylation in response to IL-4, STAT6-GFP accumulated dominantly in the nucleus (Fig. 1Ab). Results were similar with endogenous STAT6 or V5-tagged STAT6 detected by immunofluorescence (Supplemental Fig. 1A, 1B). Analysis of endogenous STAT6 in primary lymphocytes also clearly showed unphosphorylated STAT6 present in nuclei prior to IL-4 treatment and an increase in nuclear STAT6 following IL-4 treatment (Supplemental Fig. 1C). To confirm STAT6 nuclear import was independent of tyrosine phosphorylation, the behavior of a STAT6 protein with a double mutation was evaluated. The tyrosine 641 that is specifically phosphorylated in response to cytokine stimulation was substituted with phenylalanine (Y641F), and the critical arginine 562 in the SH2 domain that functions to form dimers capable of specific DNA binding was mutated to alanine (R562A). Imaging results showed the double mutant, STAT6(RY)-GFP, was imported to the nucleus but did not accumulate following stimulation with IL-4 (Fig 1Ac, 1Ad). These data show that STAT6 nuclear import is independent of tyrosine phosphorylation and that nuclear accumulation requires tyrosine phosphorylation.

EMSAs and Western blotting were performed to ensure that STAT6-GFP was tyrosine phosphorylated accurately and capable of binding DNA, whereas the STAT6(RY)-GFP lacked these abilities. The EMSAs showed that STAT6-GFP can bind a specific DNA target only following tyrosine phosphorylation, and the STAT6(RY)-GFP lacks this ability (Fig. 1B). Western blotting with Abs that recognize phosphotyrosine 641 STAT6 confirmed that STAT6-GFP is accurately tyrosine phosphorylated after IL-4 treatment, but STAT6 (RY)-GFP is not phosphorylated (Fig. 1C).

The spatial and temporal dynamics of STAT6 were evaluated by live cell imaging with nuclear FRAP (Fig. 2). Nuclei of cells expressing STAT6-GFP were subjected to a high-intensity laser to bleach fluorescence in this compartment (top panel). The recovery of fluorescence in the nucleus with time was monitored relative to a region of interest in the cytoplasm for STAT6 in unstimulated cells, STAT6 in IL-4–stimulated cells (+IL-4), or the STAT6(RY) mutant in IL-4–stimulated cells (+IL-4). Fluorescence recovery in the nucleus of unphosphorylated STAT6-GFP was half maximal by 15 min and complete by 45 min (top panel). Following tyrosine phosphorylation in response to IL-4, nuclear fluorescence recovery also was half maximal by 15 min; however, within 30–45 min, phosphorylated STAT6-GFP accumulated in the nucleus to a greater extent than in the cytoplasm. This result could reflect more efficient import of the tyrosine phosphorylated form of STAT6 or, alternatively, a decrease in STAT6 nuclear export. The kinetics of nuclear accumulation of the STAT6(RY)-GFP mutant (bottom panel) were similar to that of unphosphorylated STAT6 and confirm that nuclear import of STAT6 is continuous and independent of tyrosine phosphorylation.

FLIP was used to address the basis of nuclear accumulation following tyrosine phosphorylation of STAT6 (Fig. 3). A high-intensity laser was continually directed to a small region in the cytoplasm of cells expressing unphosphorylated STAT6-GFP or tyrosine phosphorylated STAT6-GFP (+IL-4). STAT6 passing through the laser path of this small region is bleached, and the loss of fluorescence correlates with STAT6 mobility. Fluorescence intensity rapidly decreased in the cytoplasm of cells expressing unphosphorylated STAT6-GFP or tyrosine phosphorylated STAT6-GFP, indicating rapid movement through the cytoplasm. For unphosphorylated STAT6-GFP, this loss was followed by a loss of fluorescence in the nucleus that was nearly complete by 50 min. The loss of nuclear fluorescence indicates continual STAT6 export from the nucleus and passage through the laser path in the cytoplasm. In contrast, a different result was found for tyrosine phosphorylated STAT6-GFP (lower panel). Nuclear fluorescence of phosphorylated STAT6 did not decrease during the duration of the experiment. These results suggest that the nuclear accumulation that is evident after STAT6 tyrosine phosphorylation is due to a decrease in nuclear export.

Tyrosine phosphorylation activates STAT proteins by promoting the formation of dimers that have the ability to bind specific DNA target sites. To determine whether the increased nuclear accumulation of STAT6 seen following tyrosine phosphorylation was due to a gain in the ability to bind DNA, the behavior of a DNA-binding mutant was evaluated. A STAT6 DNA-binding mutant was generated based on other STAT DNA-binding mutants (23). Lysines and arginines within aa 366–374 were substituted with alanines to generate STAT6(KR). Although the STAT6(KR) mutant was accurately tyrosine phosphorylated in response to IL-4, it did not bind target DNA sequences (Fig. 4A). Microscopic imaging indicated 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 continuous laser bleaching for 5 min. The fluorescence intensity of region 1 was compared with a distinct region in the nucleus (region 2). If movement is rapid through the path of the laser, the fluorescence intensity in region 2 will decrease similarly to region 1, along with the entire nucleus. This was the case for unphosphorylated STAT6. However, following tyrosine phosphorylation in response to IL-4, STAT6 showed significantly slower movement. The fluorescence decrease in region 2 and the remainder of the nucleus was delayed considerably compared with region 1. The tyrosine-phosphorylated DNA-binding mutant, STAT6(KR), showed the same rapid nuclear movement as unphosphorylated STAT6. To establish that the DNA-binding mutant is not retained in the nucleus following IL-4 stimulation, imaging with cytoplasmic FLIP 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.

Nuclear import of large molecules, such as STAT6, requires an 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 molecules 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 transactivation domain remained in the cytoplasm with or without IL-4 stimulation. Deletions within the coiled coil domain identified a region required for STAT6 nuclear import. STAT6(136–847) was imported and accumulated in the nucleus following tyrosine phosphorylation, whereas STAT6(141–847) remained in the cytoplasm with or without tyrosine phosphorylation. Western blotting with Abs to STAT6 phosphotyrosine 641 confirmed that the deletions were accurately phosphorylated in response to IL-4 (Fig. 5).

The studies with STAT6 truncations identified a sequence between aa 136–140 (RLQHR) that is required for nuclear import. To determine the effect of a specific deletion or substitution of these amino acids in otherwise full-length STAT6, we evaluated the localization of two mutants linked to GFP (Fig. 6A). STAT6 dl 136–140 or STAT6 containing a substitution of 135–140 aa with alanine residues (sub6A) were expressed in cells stimulated or not with IL-4. The cellular localization of both mutants was restricted to the cytoplasm, indicating a deficiency in nuclear import. These mutants were accurately tyrosine phosphorylated in response to IL-4, indicating that the internal deletion and substitution did not disrupt STAT6 activation. To evaluate the influence of specific residues in this region, each amino acid was mutated in the context of full-length STAT6. However, the individual point mutants behaved as wtSTAT6 (Supplemental Fig. 3). Together, these results indicate that aa 136–140 are required for STAT6 nuclear import, but they may 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 induce gene expression. Mutants defective in nuclear localization, STAT6(dl136–140), or DNA binding, STAT6(KR), were tested for their competence to induce the characterized promoter of the IL-4R gene (22). Transient transfections clearly demonstrated the ability of wtSTAT6 to induce the IL-4R reporter in response to IL-4, whereas STAT6(dl136–140) and STAT6(KR) did not induce the gene (Fig. 6B).

Active transport of large molecules through the nuclear pore complex usually requires facilitation by carrier proteins of the karyopherin-β family. Importin-β1 is a primary karyopherin-β transporter that can bind directly to NLS-containing proteins or indirectly via the family of importin-α adapters. Importin-α adapters bind directly to the NLS. In vitro binding assays were performed to evaluate whether one or more of the importin-αs can recognize STAT6 (Fig. 7A). STAT6-V5 was expressed in mammalian cells and immunoprecipitated from cell lysates with V5 Ab and protein G agarose beads. GST-tagged importin-αs were expressed in bacteria and added to the STAT6-V5 immunocomplexes collected on beads. Interaction of importins with STAT6 was detected by Western blot with Ab to GST. The results indicated that STAT6 is recognized primarily by importin-α3 and -α6. Similar results were obtained with STAT6 isolated from untreated cells or IL-4–stimulated cells, indicating that binding is independent of tyrosine phosphorylation. Because importin-α6 is restricted to the testes, importin-α3 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 required for direct interaction with importin-α3. We expressed fragments of STAT6 tagged with MBP in bacteria corresponding to STAT6 1–267 aa or 1–267 containing the 136–140 deletion. MBP-STAT6(1–267) and MBP-STAT6(dl136–140) were incubated with bacterially expressed GST–importin-α3 or GST–importin-α1 as a control and evaluated for binding (Fig. 7B). The results showed that STAT6(1–267) can bind importin-α3 specifically, but the deletion mutant cannot. These data suggest aa 136–140 are required for STAT6 binding to importin-α3 and nuclear import in vivo.

Given that the importin-α/β1 system may mediate STAT6 nuclear import, we evaluated the effect of RNA interference on the inhibition of expression of importin-β1 (Fig. 7C). siRNA duplexes corresponding to importin-β1 or to vimentin as a control were transfected into cells with STAT6-GFP, and the localization of STAT6-GFP was visualized microscopically. The behavior of STAT6-GFP was notably different in the cells treated with importin-β1 siRNA. Approximately 10% of cells showed STAT6 restricted to the cytoplasmic compartment, often with punctate cytoplasmic fluorescence. Because the siRNA may not completely inhibit importin-β1 expression in all cells expressing STAT6-GFP, the effect seems to be significant. To evaluate the effectiveness of the importin-β1 siRNA complexes, mRNA levels in cells treated with control or importin-β1 siRNA were assayed by RT-PCR. The siRNA to importin-β1 reduced endogenous mRNA by ~70%. Together, the results suggest that importin-α/importin-β1 may mediate STAT6 nuclear import.

Nuclear trafficking of STAT6 is integral to its function as a signal transducer and activator of transcription. By attaching a fluorescent probe to STAT6 we were able to study its intracellular dynamics with microscopy in real time. The advantage of live cell imaging is that it avoids fixation techniques that can influence cellular architecture. Cell fractionation has been used to evaluate cellular localization; however, the technique is limited in interpreting in vivo protein localization, particularly if the protein is actively imported and exported from the nucleus. Our studies indicated that STAT6 moves continually within the cytoplasm; additionally, it is transported continually into and out of the nucleus, independent of tyrosine phosphorylation.

Specific phosphorylation of tyrosine 641 promotes STAT6 dimerization and its ability to bind DNA target sites. In addition to this activating modification, other modifications have been reported that include serine phosphorylation of the carboxyl transactivation domain, 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 STAT6 tyrosine phosphorylation, nuclear translocation, and DNA binding (30). However, our studies indicate that arginine 27 is not necessary for tyrosine phosphorylation, nuclear translocation, or DNA binding. STAT6 that completely lacks 135 aa from the N terminus is imported to the nucleus, is tyrosine phosphorylated in response to IL-4, and can bind DNA (Fig. 5) (H.C. Chen and N.C. Reich, unpublished observations).

By studying the cellular localization of various STAT6 deletions, we identified a region within the coiled coil domain required for STAT6 nuclear import (Fig. 5). STAT6(136–847) was imported to the nucleus constitutively, whereas STAT6(141–847) was not imported. Deletion or substitution of the small region between aa 135–140 eliminated the ability of otherwise full-length STAT6 to be imported to the nucleus, although the proteins were still tyrosine phosphorylated accurately (Fig. 6). The best-characterized classical NLS sequences contain one or two stretches of basic amino acids, particularly lysines (31). Although the sequence 135–140 (RRLQHR) contains arginine residues, site-directed mutation of individual amino acids within this region was not sufficient to block nuclear import (Supplemental Fig. 3). This finding suggests that a noncanonical NLS may be functional within 136–267. Other STAT molecules seem to use noncanonical NLSs to drive import, whether they are constitutive or conditional (18).

Although the STATs do not display classical NLSs, they seem to use the importin-α–importin-β1 receptors. Importin-α5 binds to STAT1 when it is in the conformation of a tyrosine-phosphorylated dimer and facilitates its nuclear import (19, 32, 33), whereas importin-α3 and -α6 bind constitutively to STAT3 (20). In this study, we found that importin-α3 and -α6 also bind constitutively to STAT6; additionally, downmodulation of importin-β1 by RNA interference notably reduces STAT6 nuclear import. The results suggest that STAT6 is imported by importin-α–importin-β1 receptors (Fig. 7). It is challenging to determine specific importin-α recognition of a particular NLS outside the framework of the native protein, because recognition depends on the NLS sequence, as well as the protein context (34). The crystal structure of STAT6 remains to be solved. However, the identity of the importin-α that binds a particular protein may be significant because the importin-α proteins display specific expression in tissues and during differentiation (25, 35). It was reported that a Rac GTPase-activating protein is responsible for nuclear import of activated STAT proteins and that the dominant 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).

Latent-unphosphorylated and tyrosine-phosphorylated STAT6 are imported to the nucleus. The difference is that STAT6 accumulates in the nucleus when it is tyrosine phosphorylated (Fig. 1). Live cell imaging with photobleaching techniques provides a more quantitative and temporal measure of protein mobility and localization (37–39). By using the technique of nuclear FRAP, the transport of STAT6-GFP into the nucleus was observed to be similar for unphosphorylated or tyrosine-phosphorylated STAT6-GFP (Fig. 2). However, the average fluorescence intensity of phosphorylated STAT6-GFP becomes significantly greater in the nucleus than in the cytoplasm. The nuclear accumulation is the consequence of decreased nuclear export. This was demonstrated with cytoplasmic FLIP (Fig. 3). Repeated photobleaching of one small region in the cytoplasm resulted in the loss of total cytoplasmic fluorescence, independent of STAT6 phosphorylation. For unphosphorylated STAT6-GFP, this was followed by a gradual loss of fluorescent signal from the nucleus, indicating continuous export. In contrast, nuclear fluorescence of tyrosine-phosphorylated STAT6-GFP did not decrease during the experiment. Therefore, the increase in STAT6 nuclear accumulation following tyrosine phosphorylation is a result of decreased nuclear export.

The mechanism of STAT6 nuclear export remains to be determined; nonetheless, it seems that DNA binding is responsible for STAT6 nuclear accumulation. A STAT6 DNA-binding mutant was shown to behave like unphosphorylated STAT6 and did not accumulate in the nucleus following phosphorylation (Fig. 4). In addition, nuclear FLIP analyses determined that DNA binding dramatically reduced STAT6 movement within the nucleus. These observations indicate that nuclear accumulation of tyrosine-phosphorylated STAT6 is due to retention by association with DNA. DNA binding may be a general cause for observed nuclear accumulation of STAT proteins (23, 38, 40, 41).

Accurate cellular localization is essential for the effective function of transcription factors, such as STAT6. The constitutive nuclear import and export of latent STAT6 may provide an advantage for the rapid response to cytokine-stimulated tyrosine phosphorylation, or it may enable an activating response to nuclear kinases. Alternatively, because there is precedence for the function of unphosphorylated STATs contributing to gene expression, unphosphorylated STAT6 may have an undiscovered function in the nucleus (42). Understanding the mechanisms that regulate STAT6 nuclear trafficking will support means to manipulate its activity in health and disease.

We 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.

Disclosures The authors have no financial conflicts of interest.