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Published as doi: 10.1096/fj.07-8965com.
 (The FASEB Journal. 2008;22:391-400.)
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(The FASEB Journal. 2008;22:391-400.)
© 2008 FASEB

Constitutive nuclear import of latent and activated STAT5a by its coiled coil domain

Janaki Iyer and Nancy C. Reich1

Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA

1Correspondence: Dept. of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11794, USA. E-mail: nreich{at}notes.cc.sunysb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Signal transducer and activator of transcription 5a (STAT5a) is a critical transcription factor for a number of physiological processes including hematopoiesis and mammary gland development. Cytokines such as growth hormone, prolactin, erythropoietin, and interleukin-2 stimulate the activation of STAT5a by tyrosine phosphorylation. Tyrosine phosphorylation confers a conformational change and the ability to bind specific target DNA. To execute its function as a signaling molecule and transcription factor, accurate cellular localization of STAT5a is essential. This study explores the nuclear trafficking of STAT5a both before phosphorylation and after tyrosine phosphorylation. With the use of live cell imaging we demonstrate the continuous shuttling of STAT5a in and out of the nucleus. Evaluation of a series of mutations and deletions identifies a region within the coiled coil domain of STAT5a that is critical for nuclear import of both unphosphorylated and tyrosine-phosphorylated forms. The mechanism that regulates transport of STAT5a through nuclear pore complexes into the nucleus is therefore independent of tyrosine phosphorylation. However, after tyrosine phosphorylation, STAT5a accumulates in the nucleus because of its retention by DNA binding. These findings should provide a foundation for further studies that involve targeting the activity of STAT5a.—Iyer, J., Reich, N. C. Constitutive nuclear import of latent and activated STAT5a by its coiled coil domain.


Key Words: transcription factor • nuclear trafficking • tyrosine phosphorylation • live cell imaging


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE SIGNAL TRANSDUCERS and activators of transcription (STAT) family of transcription factors have the ability both to sense environmental cues and to directly transmit those cues to regulate specific gene expression in the nucleus (1 2 3 4 5) . In mammals the family comprises seven members, and their signaling pathways play essential roles in proliferation, immunity, and development. STAT5a is a critical signaling mediator of mammary gland development and hematopoiesis (6 7 8 9 10 11) . It is activated in response to a number of hormones including growth hormone, prolactin, interleukin-2, erythropoietin, and epidermal growth factor that bind to cell surface receptors associated with Janus tyrosine kinases (JAKs) or receptors that possess intrinsic tyrosine kinase activity (12 , 13) . Recent findings have also linked the activity of STAT5a to cell survival, proliferation, and cancer (14 15 16 17 18 19 20) .

To fulfill its role as a transcription factor, STAT5a needs to gain entry into the nucleus. Efficient nuclear localization of macromolecules such as the STATs requires facilitated, energy-dependent transport through nuclear pore complexes (NPCs) that span the nuclear envelope (21 22 23 24) . This transport depends on the ability of the STAT to interact directly with proteins of the NPC or to bind to transport carriers that interact with the NPC. In either case, a specific amino acid sequence or structure of the STAT must serve to function as a nuclear localization signal (NLS) that binds to the transporter (4) .

The STAT family members are activated by tyrosine phosphorylation of a conserved tyrosine residue within the carboxyl portion of the molecule. This phosphorylation causes a conformational change that results in STAT dimerization via reciprocal phosphotyrosine and Src homology 2 (SH2) domain interactions (1 , 25 26 27) . For all of the STATs, this phosphorylation-dependent dimerization results in the gain of the ability of the STAT dimer to bind specific DNA targets. Although the STATs share this property, their ability to enter the nucleus differs and can be either constitutive or dependent on tyrosine phosphorylation. Studies with the founder member of the STAT family, STAT1, identified a conformational NLS within the DNA binding domain that is conditional and dependent on STAT1 dimerization via tyrosine phosphorylation (28 29 30) . However, another STAT, STAT3, does not require tyrosine phosphorylation or dimerization for nuclear import (31) . Because of the critical role that STAT5a plays in both development and disease, we directed our studies on its nuclear transport process.

We provide evidence that unphosphorylated STAT5a is continually imported into the nucleus, and tyrosine-phosphorylated STAT5a is retained in the nucleus. Therefore, the process of nuclear import does not change after tyrosine phosphorylation. The domain of STAT5a crucial for nuclear import is the same for both unphosphorylated and tyrosine-phosphorylated STAT5a and resides within the coiled coil domain amino-terminal to the DNA binding domain. Retention of phosphorylated STAT5a in the nucleus requires DNA binding. Understanding the mechanisms that regulate the nuclear import of STAT5a should provide knowledge needed to stimulate or inhibit its activity in clinical intervention.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
COS1, HeLa, and STAT1-deficient cell line U3A (a gift from Dr. George R. Stark, Cleveland Clinic Foundation Research Institute, Cleveland, OH, USA) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 8% FBS. Recombinant human growth hormone (PBL Biomedical Laboratories, New Brunswick, NJ, USA), human epidermal growth factor (Sigma-Aldrich Corp., St. Louis, MO, USA) and leptomycin B (gifted from Barbara Wolff-Winiski, Novartis Research Institute, Vienna, Austria) were used at 1 ng/ml, 10 ng/ml, and 10 nM, respectively. DNA transfections were performed using FuGENE 6 (Roche Diagnostics Corp., Indianapolis, IN, USA) according to the manufacturer’s instructions. Antiphosphotyrosine STAT5 (Cell Signaling Technology, Beverly, MA, USA) and anti-STAT5a (L-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used at a 1:1000 dilution for Western blotting. Anti-green fluorescent protein (GFP) conjugated to IRDye 88 (Rockland Immunochemicals, Gilbertsville, PA, USA) was used at a 1:5000 dilution for Western blots. Anti-V5 (Invitrogen, Grand Island NY, USA) was used at a dilution of 1:200 for immunofluorescence. Anti-GFP (Roche Diagnostics Corp.), murine anti-STAT1 (32) , anti-STAT3 (C-20; Santa Cruz Biotechnology) anti-STAT5 (C-17; Santa Cruz Biotechnology), or control (MOPC-141; Sigma-Aldrich Corp.) were used at 1 µg for electrophoretic mobility shift assays.

Plasmid constructs
Full-length human STAT5a cDNA was amplified by polymerase chain reaction and cloned into the vector pEGFP-N1 (Clontech, Mountain View, CA, USA) or pEF1/V5-His (Invitrogen) to generate enhanced green fluorescent protein (EGFP)-tagged STAT5a or V5 epitope-tagged STAT5a. cDNAs of various deletion mutants of STAT5a were also cloned into pEGFP-N1 to create GFP fusion proteins. Site-directed mutagenesis was performed using a QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). The plasmid encoding rat growth hormone receptor was a kind gift from Dr. Christin Carter-Su (University of Michigan, Ann Arbor, MI, USA).

Western blot
Cells were lysed in 50 mM Tris (pH 8.0), 5 mM EDTA, 0.5% Nonidet P-40, 280 mM NaCl, 1mM PMSF, 1x protease inhibitor cocktail (Sigma-Aldrich Corp.), 1mM NaF, and 1mM sodium vanadate at 4°C for 30 min. The cells were centrifuged at 12,000 g for 10 min, and the supernatants obtained were boiled in sodium dodecyl sulfate sample buffer. The cell lysates were separated on 8% SDS-PAGE and transferred to nitrocellulose membrane (Pierce Biotechnology, Rockford, IL, USA). The membrane was then reacted with antibodies to STAT5 phosphotyrosine, STAT5a, or GFP. Images were detected using the Odyssey infrared imaging system (Li-COR Biosciences, Lincoln, NE, USA).

Electrophoretic mobility shift assay
Cellular lysates were generated by combining cytosol from hypotonic lysis [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 dithiothreitol (DTT), 1 mM PMSF, 1 mM sodium vanadate, 1 mM NaF, and 1x protease inhibitor cocktail] with high salt extracts from nuclei [20 mM Hepes (pH 7.9), 0.2 mM spermine, 0.5 mM spermidine, 1 mM DTT, 0.2 mM potassium-EGTA, 0.4 M NaCl, 10% glycerol, 1 mM PMSF, 1 mM sodium vanadate, 1 mM NaF, and 1x protease inhibitor cocktail]. Lysates were incubated with radiolabeled double-stranded (ds) DNA oligonucleotide representing the pRL response element of the β-casein gene promoter (5'-AGATTTCTAGGAATTCAA-3') for 30 min (33 , 34) . Specific antibodies (1 µg), or 100-fold excess nonradiolabeled dsDNA oligonucleotide was added to lysates before radiolabeled DNA. Reactions were separated on nondenaturing gels and exposed to X-ray film for autoradiography

Confocal microscopy
Cells were seeded on glass coverslips, transfected, serum starved, and evaluated after 48 h of transfection. Cells were washed with 1x PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were observed with a Zeiss LSM 5 laser scanning microscope using a x40 oil objective [Plan-Neofluar, numerical aperture 1.3, differential interference contrast microscopy objective (DIC)]. 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 and processed and presented using Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Images shown are representative of >90% of the cell population.

Live cell imaging
Cells expressing STAT5a-GFP or GFP were plated on glass-bottom tissue culture dishes (Mattek Corporation, Ashland, MA, USA). The time series images for all photobleaching techniques were performed using a Zeiss LSA 510 META NLO two-photon laser scanning microscope system. The culture plates were mounted on a Zeiss inverted Axiovert 200M microscope using a heating insert coupled with the Incubator S (Zeiss). The cells were maintained at 37°C and 5% CO2 for the entire length of the experiment using the Zeiss Tempcontrol 37-2 Digital and CTI Controller 3700. All analyses were performed using a x40 oil objective (Plan-Neofluar, numerical aperture 1.3, DIC objective). GFP was excited with an argon laser at 488 nm, and emission was collected using a 505-nm long pass filter. For performing fluorescence recovery after photobleaching (FRAP) in the nucleus, a region of interest in the nucleus was bleached at 100% power of an argon laser at 488 nm for 70 s. For fluorescence recovery after photobleaching (FLIP) analysis, a region of interest in the cell was bleached every 12 s at maximum laser intensity for 10–75 min, depending on the protein expressed in cells. Images were acquired using LSM 510 Meta version 3.2 imaging software. The images were processed and presented using Adobe Photoshop. The fluorescence intensity of the bleached area and other regions on the test cell and a neighboring control cell was quantified automatically using the mean region of interest (ROI) function of the LSM Imaging software and graphically depicted using Microsoft Excel.

Immunofluorescence
Cells were seeded on glass coverslips, transfected with STAT5a-V5, serum-starved and evaluated after 48 h of transfection. Cells were washed with 1x PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then permeabilized with 0.2% Triton X-100 for 5 min and then blocked with 3% BSA in PBS for 60 min at room temperature. STAT5a was detected with anti-V5 antibody and a rhodamine-conjugated secondary antibody. Immunofluorescence was observed under a Zeiss LSM 5 laser scanning microscope using a x40 oil objective (Plan-Neofluar, numerical aperture 1.3, DIC objective). Images were captured using Zeiss LSM 5 Pascal imaging software and processed and presented using Adobe Photoshop. Images shown are representative of ~90% of the cell population.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
STAT5a nuclear import is independent of tyrosine phosphorylation
To evaluate the cellular localization of STAT5a, we used fluorescence microscopy to visualize STAT5a linked to enhanced GFP (STAT5a-GFP). Results are shown for STAT5a-GFP expressed in HeLa cells both before and after stimulation with human growth hormone (GH) (Fig. 1 A). The images indicate that STAT5a has a significant presence in the nucleus before tyrosine phosphorylation in response to GH. After GH stimulation and tyrosine phosphorylation, STAT5a is seen to accumulate in the nucleus. Various other cells were evaluated with similar results including U3A cells that lack endogenous STAT1, and other epitope tags of STAT5a were found to show the same behavior (Supplemental Fig. 1). These results indicate that STAT5a is imported into the nucleus both before and after tyrosine phosphorylation.


Figure 1
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  		Figure 1. Characterization of STAT5a-GFP. HeLa cells expressing GH receptor and STAT5a-GFP were serum deprived and untreated or treated with GH for 30 min. A) Fluorescence microscopy of latent unphosphorylated STAT5a in serum-deprived cells expressing STAT5a-GFP (–) or in cells stimulated with GH (+). B) Western blots of cell lysates were performed with antibodies to phosphotyrosine 694 STAT5 ({alpha}-STAT5-pY), GFP ({alpha}-GFP), or STAT5a ({alpha}-STAT5a). C) An EMSA was performed with cell lysates and a radiolabeled DNA fragment corresponding to the β-casein gene prolactin response element. Additions to the binding reactions include control (c) antibody (Ab), antibody to GFP (G), or 100-fold excess unlabeled specific DNA.

To ensure that STAT5a-GFP retains the functional characteristics of STAT5a, we evaluated tyrosine phosphorylation and DNA binding ability after GH stimulation of cells. Western blotting was performed with cell lysates and antibodies that detect specific phosphorylation of STAT5a tyrosine 694. Phosphorylation was clearly detected only in response to addition of GH (Fig. 1B ). The DNA binding ability of the phosphorylated proteins was determined by performing an electrophoretic mobility shift assay (EMSA) with the β-casein gene response element (33) . In response to GH, STAT5a DNA-binding complexes were identified and verified by the inclusion of specific antibodies (Fig. 1C ).

STAT5a nuclear import and export are constitutive
To assess the spatial-temporal dynamics of unphosphorylated STAT5a, we used live-cell imaging with laser microscopy. Cells expressing STAT5a-GFP were subjected to nuclear FRAP. With this technique the entire nucleus of the cell was subjected to photobleaching, resulting in the loss of nuclear fluorescence (Fig. 2 A, top panel, ROI 1). The recovery of STAT5a fluorescence in the nucleus was then monitored with time. Nuclear import of unphosphorylated STAT5a was clearly evident, with complete recovery of nuclear fluorescence by ~75 min. Because STAT5a is ~92 kDa, it cannot passively diffuse into the nucleus but must be actively transported. To compare the nuclear import of STAT5a with that of a small protein that can passively diffuse through the nuclear pore complex, we evaluated the movement of GFP (Fig. 2A , lower panel, ROI 1). The nucleus of a cell expressing GFP was subjected to photobleaching, resulting in the loss of fluorescence. The recovery of fluorescence intensity in the nucleus was monitored over time, and we observed that very quickly by ~3 min the recovery of fluorescence intensity in the nucleus was complete.


Figure 2
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  		Figure 2. Nuclear FRAP of STAT5a-GFP and GFP with live cell imaging. A) A HeLa cell expressing STAT5a-GFP was subjected to nuclear photobleaching within ROI 1) (top panel). Subsequent recovery of nuclear fluorescence is shown with time (arrow). A cell expressing GFP was similarly subjected to nuclear FRAP within ROI 1 (bottom panel, arrow). B) Quantitative measurement of relative fluorescence intensity in a nucleus that was bleached (ROI 1) relative to the site in cytoplasm of a photobleached cell (ROI 2) or nucleus of an adjacent cell (ROI 3) shown in A. Images are representative of more than five experiments.

A graphic depiction of the kinetic results is shown in Fig. 2B . The fluorescence intensity of ROI 1 in the nucleus that was photobleached is compared with a control region in the cytoplasm of the same cell (ROI 2) or the nucleus of an adjacent cell (ROI 3) with time. The greater relative speed with which GFP redistributed into the nucleus in comparison with STAT5a is consistent with its ability to passively diffuse into the nucleus. Results were similar with other cell lines such as the human fibrosarcoma HT1080 (unpublished observations). To evaluate the export of STAT5a out of the nucleus, we used the technique of cytoplasmic FLIP. A small region (ROI 1) in the cytoplasm of a cell expressing STAT5a-GFP was subjected to a continuous high-intensity laser beam, resulting in the effective bleaching of any fluorescent STAT5a-GFP molecules passing through that region. Within ~10 min the cytoplasmic fluorescence was lost, but nuclear fluorescence remained (Fig. 3 A, arrow in top panel). This result indicates rapid movement of STAT5a molecules within the cytoplasm. With continued photobleaching in the cytoplasm at ROI 1, nuclear fluorescence intensity decreased and was lost by ~75 min. The loss of nuclear fluorescence indicates that STAT5a is continuously exported out of the nucleus. A similar cytoplasmic FLIP analysis was performed in cells expressing GFP (Fig. 3 , bottom panel). Because GFP can pass between nuclear and cytoplasmic compartments by diffusion, the substantial loss in both cytoplasmic and nuclear fluorescence by 10 min was not unexpected (Fig. 3A , bottom panel). Quantitation of the results is depicted graphically in Fig. 3B .


Figure 3
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  		Figure 3. Cytoplasmic FLIP of STAT5a-GFP and GFP with live cell imaging. A) ROI 1 in the cytoplasm of a cell expressing STAT5a-GFP was subjected to continuous high-intensity laser pulses (top panel, arrow). A similar cytoplasmic FLIP was performed with cells expressing GFP (bottom panel, arrow). B) Quantitative measurement of relative fluorescence in the cytoplasmic focal point of a bleached cell (ROI 1) relative to a site in the nucleus (ROI 2) of a bleached cell or a nuclear site in an adjacent control cell (ROI 3) shown in A. Images are representative of more than five experiments.

The results obtained with the nuclear FRAP and cytoplasmic FLIP live cell imaging studies demonstrate that STAT5a-GFP constitutively shuttles between the nucleus and cytoplasm of living cells. The movement of STAT5a in and out of the nucleus is considerably slower than that of GFP, and it is independent of tyrosine phosphorylation.

A region in the coiled coil domain of STAT5a is critical for nuclear import
Because the size of STAT5a requires it to be actively transported to the nucleus, it is likely that STAT5a contains a motif that functions as an NLS. Many classic NLS motifs contain one or two stretches of basic amino acids (35) . Scanning the primary sequence of STAT5a for hallmarks of a classic NLS identified several candidate sequences; however, after mutational analyses, none were found to effect nuclear import. A recent publication reported the requirement of a region in the DNA binding domain of STAT5a amino acids (aa) 341–365 (VLKTQTKFAATVRLLVGGKLNVHMN) for nuclear import, and so we tested the localization of STAT5a with an internal deletion of aa 341–365 (36) . Although static images showed STAT5a only in the cytoplasm, the addition of leptomycin B, an inhibitor of the export transporter CRM1, showed its localization in the nucleus (Supplemental Fig. 2). This result indicates that the aa 341–365 deletion mutant is imported to the nucleus and is exported effectively. To identify sequences needed for nuclear import a logical approach was then used to evaluate the cellular localization of a set of STAT5a deletion mutants. The deletion mutants were tagged with GFP or with two tandem copies of GFP to prevent small fragments from entering the nucleus by passive diffusion (Fig. 4 A).


Figure 4
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  		Figure 4. Identification of a region of STAT5a required for nuclear import. A) Diagram of STAT5a functional motifs including the coiled coil (CC), DNA binding domain (DBD), SH2 domain, transcriptional activation domain (TAD), and specific phosphorylated tyrosine (Y694). Linear depiction of deletion mutations with encoding amino acid residues or amino acid residues removed ({Delta}) linked to GFP or two copies of GFP in tandem (GFP-GFP). B) Fluorescence microscopy of STAT5a deletion constructs. C) Location of aa 142–149 in a ribbon diagram of the STAT5a crystal structure (Protein Data Bank ID code 1Y1U).

Cellular localization of the unphosphorylated STAT5a mutants was evaluated by fluorescence microscopy and is shown in Fig. 4B . STAT5a containing the amino terminus and the coiled coil domain (aa 1–330) showed a clear nuclear presence, even greater than that of the wild type (wt) protein. In comparison, expression of a STAT5a mutant containing the DNA binding domain, SH2 domain, and transcriptional activation domain (aa 331–794) showed only cytoplasmic localization. This result suggested that a region critical for nuclear import of STAT5a was present within aa 1–330. Analysis of additional constructs indicated that STAT5a containing aa 145–794 was imported into the nucleus, but the STAT5a deletion, aa 150–794, was limited to the cytoplasm (with or without leptomycin B) (unpublished observations).

Because there was a distinct cellular localization of aa 145–794 in comparison with aa 150–794, the behavior of an internal deletion of aa 142–149 from otherwise full-length STAT5a ({Delta}142–149) was examined. Localization of this internal deletion mutant was found to be restricted to the cytoplasm, indicating that the deleted region was required for nuclear import of unphosphorylated STAT5a (Fig. 4B ). Localization was not affected by addition of leptomycin B (Supplemental Fig. 2). The position of the aa 142–149 region within a ribbon diagram of the STAT5a crystal structure is shown in Fig. 4C (37) . This sequence within the coiled coil domain is on the outer surface of the STAT5a molecule and is thereby available for interaction with transporter molecules.

Dynamic redistribution of STAT5a after tyrosine phosphorylation
Before tyrosine phosphorylation, STAT5a is present in both the nucleus and cytoplasm. However, after tyrosine phosphorylation STAT5a clearly accumulates in the nucleus (Fig. 1A ). Because the amino acid sequence 142–149 in the coiled coil domain of STAT5a was essential for nuclear import of the unphosphorylated form, we tested the requirement of this sequence for nuclear accumulation after tyrosine phosphorylation. Cells expressing wt STAT5-GFP or STAT5a ({Delta}142–149)-GFP were untreated or treated with human epidermal growth factor (EGF), and cellular localization was visualized microscopically (Fig. 5 A). Wt STAT5a was distributed in the nucleus and cytoplasm before hormone addition and accumulated in the nucleus after EGF treatment. In contrast, the STAT5a ({Delta}142–149) protein resided in the cytoplasm and did not localize to the nucleus either before or after EGF stimulation. To ensure that the STAT5a ({Delta}142–149) protein was phosphorylated in response to EGF, Western blot analysis with a specific antibody to the STAT5 phosphotyrosine was performed (Fig. 5B ). The results indicate that STAT5a ({Delta}142–149) is specifically phosphorylated after EGF stimulation. In addition, because tyrosine phosphorylation confers a conformational change that enables the STAT proteins to bind DNA, we tested the ability of the phosphorylated STAT5a ({Delta}142–149) internal deletion to bind DNA. Lysates were prepared from cells untreated or stimulated with EGF and expressing STAT5a ({Delta}142–149) protein, and DNA-binding reactions were prepared with a radiolabeled DNA oligonucleotide containing the STAT5 binding site within the β-casein promoter (Fig. 5C ). STAT5a ({Delta}142–149) clearly gained the ability to specifically bind DNA after tyrosine phosphorylation. Specific antibodies added to the binding reactions identified STAT5a ({Delta}142–149)-GFP in the DNA-binding complexes and produced supershift complexes. From these results, it appears that the inability of this mutant to localize to the nucleus is not due to defective phosphorylation or DNA binding. The aa 142–149 region of the coiled coil domain is therefore necessary for nuclear import of both unphosphorylated and tyrosine-phosphorylated STAT5a (Figs. 4A and 5A) . We propose that this mutant protein is not recognized by the nuclear transport machinery and hence is not imported into the nucleus either in an unphosphorylated form or a tyrosine-phosphorylated form.


Figure 5
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  		Figure 5. Identification of the STAT5a domain required for nuclear import. A) Fluorescence microscopy of COS1 cells expressing wt STAT5a-GFP (WT) or the internal deletion STAT5a ({Delta}142–149)-GFP without treatment (–) or stimulated with EGF for 45 min. B) Western blot analysis of protein lysates from cells treated in A performed with antibodies to phosphotyrosine 694 STAT5 ({alpha}-STAT5-pY), GFP ({alpha}-GFP), or STAT5a ({alpha}-STAT5a). C) An EMSA with lysates containing STAT5a ({Delta}142–149)-GFP from untreated cells (–) or cells treated with EGF (+). Specific antibody (Ab) to STAT1 (1), STAT3 (3), STAT5a (5a), STAT5 (5), GFP (G), or a control antibody (c) was added to the DNA-binding reactions. As a specific competitor 100-fold excess unlabeled DNA was added (DNA). The mobility of STAT5a-DNA complexes and the antibody-mediated supershift is noted. The mobility of endogenous STAT1 is also indicated.

DNA binding promotes nuclear accumulation of STAT5a
Tyrosine phosphorylation is not necessary for nuclear import, but we tested whether it is required to retain STAT5a in the nucleus after hormone stimulation. Two mutations were introduced in STAT5a to eliminate tyrosine phosphorylation and dimerization with phosphorylated STAT5 or other endogenous STATs. The critical tyrosine residue that is phosphorylated was mutated to phenylalanine (Y694F) and the critical arginine residue in the SH2 domain that mediates phosphotyrosine-SH2 domain interactions was mutated to alanine (R618A). This created the STAT5a-RY mutant that cannot associate with other STATs via reciprocal phosphotyrosine and SH2 domains. The distribution of the STAT5a-RY-GFP mutant before GH stimulation was similar to unphosphorylated STAT5a (Fig. 6 A). In addition, nuclear FRAP of the double mutant STAT5a-RY-GFP was also evaluated, and the rate of nuclear import was similar to that obtained with wt STAT5a-GFP (Supplemental Fig. 3). These results further strengthen the concept that STAT5a import is independent of tyrosine phosphorylation. Although STAT5a-RY-GFP is imported to the nucleus, it did not accumulate in the nucleus after cellular stimulation with GH treatment. The failure of STAT5a-RY-GFP to accumulate in the nucleus as wt STAT5a in response to GH indicates that accumulation above baseline requires tyrosine phosphorylation and/or DNA binding. The lack of tyrosine phosphorylation and DNA binding ability of STAT5a-RY-GFP was shown by Western blot and EMSA analyses (Fig. 6B, C ).


Figure 6
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  		Figure 6. DNA binding required for nuclear accumulation of tyrosine-phosphorylated STAT5a. A) Fluorescence microscopy of HeLa cells expressing the GH receptor, STAT5a-GFP with the SH2 and tyrosine 694 mutation (RY), or STAT5a-GFP with basic residues 422–430 replaced with alanine (KR). Cells were starved (–) or treated with GH (+). B) Western blots of lysates from cells expressing STAT5a wt or RY or KR mutations untreated (–) or treated with GH (+) performed with antibodies to phosphotyrosine 694 STAT5 ({alpha}-STAT5-pY), GFP ({alpha}-GFP), or STAT5a ({alpha}-STAT5a). C) Electrophoretic mobility shift assays with lysates from cells treated as in B. Control (c) antibody (Ab) or antibody to GFP (G) was added to the DNA-binding reactions. Specific DNA competition (DNA) was performed with 100-fold excess unlabeled oligonucleotide.

Tyrosine phosphorylation of STAT5 triggers the formation of dimers via reciprocal phosphotyrosine and SH2 domain interactions, and this confers the ability of STAT5 to bind DNA. To determine the requirement of DNA binding for nuclear import or for nuclear retention of STAT5a, we evaluated the distribution of a STAT5a mutant that has impaired DNA binding ability. A mutant that we originally generated to evaluate nuclear import was found to lack DNA binding. The basic amino acids in the DNA binding domain of STAT5a (422-KRIKRADRR-430) were replaced with alanine to generate STAT5a-KR-GFP. Before GH treatment, STAT5a-KR-GFP was found in the nucleus as well as in the cytoplasm, showing no evidence for a defect in nuclear import (Fig. 6A ). However, after tyrosine phosphorylation with GH treatment, STAT5a-KR-GFP did not show the nuclear accumulation that is seen with phosphorylated wt STAT5a. STAT5a-KR-GFP was accurately tyrosine phosphorylated in response to GH, as detected by Western blotting using specific phosphotyrosine STAT5 antibodies (Fig. 6B ). The DNA-binding ability of STAT5a-KR-GFP was also tested, and the results of an EMSA analysis revealed that this mutant was unable to bind DNA, even though it was phosphorylated efficiently after GH treatment (Fig. 6C ). Thus, the inability of phosphorylated STAT5a-KR-GFP to accumulate in the nucleus is correlated with a defect in DNA binding and not a defect in nuclear import. From these studies we can infer that the nuclear accumulation of phosphorylated STAT5a requires the ability to bind DNA for retention in the nucleus.

If DNA binding is responsible for nuclear retention after tyrosine phosphorylation of STAT5a, we would predict that the mobility of phosphorylated STAT5a in the nucleus would be slower than that of unphosphorylated STAT5a. This hypothesis was tested by comparing the rates of movement of unphosphorylated and phosphorylated wt STAT5a in the nucleus using the FLIP imaging technique. An intense laser was directed to a small region of the nucleus in cells expressing unphosphorylated STAT5a-GFP before GH stimulation (Fig. 7 A, top panel, ROI 1). Fluorescent molecules moving through the path of the laser will be bleached, and the loss of fluorescence will correlate with protein mobility in the nucleus. The fluorescence intensity of unphosphorylated STAT5a was lost throughout the nucleus in ~2 min, indicative of rapid movement of unphosphorylated STAT5a in the nucleus (ROI 2). After GH treatment a similar nuclear FLIP analysis was performed with cells expressing tyrosine-phosphorylated STAT5a-GFP (middle panel). The pattern of fluorescence loss in nuclei expressing tyrosine-phosphorylated STAT5a-GFP was significantly different. The small region of the nucleus exposed to the laser rapidly lost fluorescence intensity, whereas other areas of the nucleus retained fluorescence for a significant period of time. This result suggested that the phosphorylated STAT5a has slower mobility in the nucleus as a result of DNA binding.


Figure 7
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  		Figure 7. Live cell imaging with nuclear FLIP reveals slowed movement of STAT5a-GFP capable of binding to DNA. A) Nuclear FLIP in a cell expressing unphosphorylated STAT5a-GFP (top panel, arrow), tyrosine-phosphorylated wt STAT5a-GFP (middle panel, arrow), or tyrosine-phosphorylated DNA-binding mutant STAT5a-KR-GFP (bottom panel, arrow). B) Quantitative measure of fluorescence intensity at a focal point of the laser (ROI 1) compared with a distant site in nucleus (ROI 2) for images presented in A. Images are representative of more than five experiments.

To evaluate more directly whether the mobility of tyrosine-phosphorylated STAT5a decreased as a result of DNA binding, we tested the behavior of a tyrosine-phosphorylated molecule that cannot bind DNA. If our hypothesis was correct, the DNA binding mutant STAT5a-KR should move rapidly in the nucleus even after tyrosine phosphorylation. The results of a nuclear FLIP of STAT5a-KR are shown in the bottom panels of Fig. 7A . STAT5a-KR bleaches rapidly in the entire nucleus indicating rapid movement in the absence of DNA binding. A graphic quantitation of the results is provided in Fig. 7B .


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Seven mammalian genes encode STAT proteins that share a similar arrangement of functional domains and a similar means of activation by tyrosine phosphorylation. However,the different STATs respond distinctly to extracellular hormones and intracellular kinases, and they elicit distinct physiological responses by regulating expression of specific sets of genes. As signaling molecules and transcription factors, they must have the capability of moving between the cytoplasm and the nucleus, and this property appears distinctly regulated for individual STATs. The localization of STAT1 and STAT2 is predominantly cytoplasmic before ligand stimulation, whereas STAT3 is prominent in the nucleus (4) . In some cases a static image may be misleading as the STAT molecule can continuously shuttle in and out of the nucleus (31 , 38 39 40 41) .

Two closely related STAT genes, STAT5a and STAT5b, exhibit partial functional redundancy but clearly also have distinct roles, as revealed by defects of single and double gene knockouts in mice. Although both genes play critical roles in hematopoiesis, survival, and proliferation, STAT5a knockout mice have impaired mammary gland development whereas STAT5b knockout mice have sexually dimorphic growth retardation (6 , 42) . Previous reports on the cellular location of STAT5 proteins are mixed with respect to the presence of STAT5 in the nucleus before hormone stimulation (36 , 40 , 43 , 44) . Additionally, evidence has suggested the involvement of microtubules in the nuclear translocation of STAT5b but not STAT5a (45) . Some of the differences reported may be due to immunofluorescence techniques and antibody cross-reactivity or to interpretations of static images. In this report we make a distinction between nuclear import and nuclear accumulation or retention. In Fig. 1 it is evident that unphosphorylated STAT5a is present in the nucleus as well as in the cytoplasm. However, after tyrosine phosphorylation STAT5a clearly accumulates in the nucleus, and we provide evidence that the accumulation is due to retention via DNA binding.

By using live cell imaging and the nuclear FRAP technique, the temporal import of unphosphorylated STAT5a to the nucleus can be visualized (Fig. 2) . The constitutive nuclear import of unphosphorylated STAT5a indicates that STAT5a possesses a sequence or structure that functions as a constitutive NLS. This constitutive nuclear entry is distinct from the behavior of STAT1, which is regulated primarily by an inducible NLS that becomes accessible after tyrosine phosphorylation and a conformational change of the dimer (28 , 46 , 47) . Although nuclear import of STAT5a is constitutive, the NLS function of STAT5a does not appear as overriding as that of STAT3, whose nuclear appearance does not change with hormone stimulation (31) .

After tyrosine phosphorylation, STAT5a clearly accumulates in the nucleus. This accumulation could result from STAT5a binding to DNA and subsequent retention of STAT5a in the nucleus and/or to a conformational change that masks the function of a nuclear export signal. Tyrosine phosphorylation is not required for constitutive nuclear import, but it is required for nuclear accumulation (RY mutation) (Fig. 6) . To distinguish between the requirement of tyrosine phosphorylation and DNA binding, we generated a DNA binding mutation (KR). The DNA binding mutation maintained the ability to enter the nucleus constitutively but did not show significant nuclear accumulation after tyrosine phosphorylation. Therefore, although interaction of phosphotyrosine-SH2 domains induced a conformational change, it was not sufficient for nuclear retention. If retention of tyrosine-phosphorylated STAT5a in the nucleus results from tethering to DNA, we would predict that the movement of STAT5a in the nucleus would be significantly reduced. Live cell imaging with a nuclear FLIP technique confirmed this prediction (Fig. 7) . Unphosphorylated STAT5a moves rapidly within the nucleus as photobleaching at a single focal point in the nucleus leads to rapid bleaching of the entire nucleus. In comparison, phosphorylated STAT5a with the ability to bind DNA has significantly reduced movement in the nucleus as evident by the occurrence of photobleaching primarily at the focal point of the laser beam. In additional, the STAT5a-KR DNA binding mutant did not show reduced mobility in the nucleus after tyrosine phosphorylation. Therefore, the accumulation of STAT5a in the nucleus apparently results from retention due to binding of DNA.

A static image of unphosphorylated STAT5a shows it to be distributed both in the nucleus and the cytoplasm (Fig. 1) . To evaluate the dynamic movement of STAT5a we used live cell imaging with continuous FLIP at a focal point in the cytoplasm (Fig. 3) . The resultant rapid photobleaching of the entire cytoplasm indicated rapid movement of STAT5a within the cytoplasm. Continued FLIP in the cytoplasm also led to photobleaching of the nucleus, indicating that STAT5a was exported from the nucleus and was photobleached at a point in the cytoplasm. Unphosphorylated STAT5a is therefore continuously imported to the nucleus and exported from the nucleus. After tyrosine phosphorylation, nuclear export decreases as shown with a constitutively active form of STAT5a (Supplemental Fig. 4).

One of the common exportin transporters is CRM1, and its activity can be inhibited with the antibiotic leptomycin B (48) . A previous report indicated nuclear shuttling of STAT5b, which could be inhibited by leptomycin B (44) ; however, we did not detect an effect of leptomycin B on localization of unphosphorylated STAT5a (Supplemental Fig. 2). The mechanism of nuclear export of unphosphorylated STAT5a remains to be determined.

To identify the region of STAT5a that functions in constitutive nuclear import we evaluated the localization of deletion mutations. Amino-terminal deletions revealed the requirement of a region in the coiled coil domain including residues 145–150 (QTFEEL) (Fig. 4) . This sequence is conserved in STAT5 from various species (Supplemental Fig. 5). Our studies demonstrate that an internal deletion of aa 142–149 in otherwise full-length STAT5a lacks the ability to enter the nucleus either before or after tyrosine phosphorylation, in the presence or absence of leptomycin B. It should be noted that STAT5a ({Delta}142–149) is not tyrosine-phosphorylated in response to GH, but is phosphorylated in response to EGF. This finding indicates the deleted region may be required for interaction with the GH receptor or JAKs, and future studies are needed to explain substrate targeting. However, it is not a concern of the current study that is directed at STAT5a postphosphorylation events, and therefore we used EGF to phosphorylate STAT5a ({Delta}142–149). The lack of nuclear import of this mutant supports the concept that STAT5a possesses a single constitutive NLS within the coiled coil domain that functions similarly in unphosphorylated and tyrosine-phosphorylated conformations. This amino acid sequence is exposed to solvent in the crystal structure of unphosphorylated STAT5a and therefore has the potential of interacting with proteins that mediate nuclear import (37) .

A recent study indicated that male germ cell RacGAP binding to aa 341–365 of STAT5a was required for STAT5a nuclear import (36) . However, our evaluation of STAT5a with an internal deletion of aa 341–365 indicated that this region is not required for import to the nucleus. The STAT5a deletion was found to be present in the nucleus when cells were treated with the export inhibitor leptomycin B (Supplemental Fig. 2). Hence this region in the DNA-binding domain of STAT5a does not appear to be critical for nuclear import. The same study reported that tyrosine-phosphorylated STAT5a can bind to the transport adapters importin-{alpha}1 and importin-{alpha}5. Our preliminary experiments indicate that phosphorylated STAT5a can bind to importin-{alpha}5 but not to importin-{alpha}1 (unpublished observations).

The requirement of a region in the coiled coil domain of STAT5a for nuclear import is distinct from the region in STAT3 required for nuclear import (31 , 41 , 49) . The STAT5a sequence lies upstream of the region in STAT3 that is critical for constitutive nuclear import (31) . The dynamic shuttling of STAT5a in and out of the nucleus may allow STAT5a to respond to tyrosine kinases in the cytoplasm and/or to tyrosine kinases in the nucleus such as Abelson tyrosine kinase (50) . It is also possible that the unphosphorylated form of STAT5a has a function in the nucleus yet to be determined (51 , 52) . Future insight into the mechanisms of STAT5a shuttling and functional consequences will support the design of therapeutic agents in various pathologic conditions.


   ACKNOWLEDGMENTS
 
We thank Dr. Guo-Wei Tian for his technical assistance while performing the live cell imaging experiments and Dr. Vitaly Citovsky for his support with confocal microscopy. We also thank the current and past members of the laboratory for their support, particularly Sarah Van Scoy, Gregg Banninger, and Ling Liu. These studies were supported by grants from the U.S. National Institutes of Health (PO1CA2814 and RO1CA122910).

Received for publication May 10, 2007. Accepted for publication August 9, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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