Experimental Cell Research 272, 45–55 (2002)doi:10.1006/excr.2001.5405, available online at http://www.idealibrary.com on

Cell Type-Specific and Tyrosine Phosphorylation-Independent NuclearPresence of STAT1 and STAT3

Thomas Meyer, Karsten Gavenis,1 and Uwe Vinkemeier2

Nachwuchsgruppe Zellulare Signalverarbeitung, Forschungsinstitut fur Molekulare Pharmakologie, and Freie Universitat,

Institut fur Kristallographie, Berlin, Germany

Tyrosine phosphorylation in response to cytokinestimulation of cells is believed to be required for thenuclear translocation of cytoplasmic STAT proteins(signal transducers and activators of transcription).In this study we examined the nucleocytoplasmic dis-tribution of STAT1 and STAT3 in transformed celllines and primary cells prior to stimulation with cyto-kines. It was found that both STAT1 and STAT3 areconstitutively nuclear in resting cells. Moreover, theextent of nuclear presence of both proteins differed ina cell type-specific mode as revealed by immunocyto-chemistry and confocal microscopy. We investigatedwhether varying degrees of tyrosine phosphorylationcould account for these differences. The results showthat depletion of type I interferons from culture me-dium with blocking antibodies did not influence theSTAT1 distribution in unstimulated cells. In addition,blocking tyrosine kinase activity with staurosporinealso did not influence the nucleocytoplasmic STAT1distribution in resting cells. Nuclear extracts fromunstimulated HeLa-S3 cells, which are demon-strated to be exceptionally high in the nuclear con-centration of STAT1, did not contain detectablequantities of tyrosine-phosphorylated STAT1. Inaddition, the nucleocytoplasmic distribution of aSTAT1 mutant which can no longer be phosphory-lated or dimerize did not differ from wild-type pro-tein. Thus, these data indicate that tyrosine phosphor-ylation of STATs does not constitute a mandatoryrequirement for the nuclear presence of these transcrip-tion factors. © 2001 Elsevier Science

INTRODUCTION

The STAT proteins are important regulators of amultitude of cellular processes, such as immune re-

1 Present address: Universitatsklinikum Aachen, Pauwelsstrabe30, 52074 Aachen, Germany.

2 To whom correspondence and reprint requests should be ad-dressed at Nachwuchsgruppe Zellulare Signalverarbeitung, For-schungsinstitut fur Molekulare Pharmakologie, Robert-Rossle-Str.10, 13125 Berlin, Germany. Fax: 49-30-94793-179. E-mail:

vinkemeier@fmp-berlin.de.

45

sponses, antiviral protection, and growth (reviewed in[1–3]). As their name implies, these factors performroles in the cytoplasm as well as in the nucleus. Inresponse to the binding of cytokines or growth factorsto their respective receptors the STAT proteins arephosphorylated on a single tyrosine residue [4]. Thisphosphorylation event triggers their dimerization viareciprocal phosphotyrosine–SH2 interactions [5–7].The dimerization transforms the STATs into DNA-binding proteins which recognize palindromic nonam-ers, termed GAS sites ([8], reviewed in [9]), and it isaccompanied by a rapid and transient nuclear accumu-lation [10, 11]. This sequence of events, usually termedSTAT activation, leads to the induction of immediateearly genes only a few minutes after cytokine bindingto the cell surface [12]. However, as is the case fornuclear accumulation, also the cytokine-stimulatedgene induction persists for only a few hours.

Recently, another and less well understood path totranscriptional activation by STATs has been discov-ered. As has been shown for STAT1, the STATs canalso participate in permanent transcriptional pro-grams, since STAT1 is required for the constitutiveexpression of caspase genes and the concomitantTNFa-induced onset of apoptosis [13, 14]. In unstimu-lated cells the STATs appear cytoplasmic, which leadsto the term “latent cytoplasmic transcription factors” todescribe their localization and function prior to cyto-kine stimulation. The rapid nuclear build-up of STATsas a consequence of cytokine or growth factor stimula-tion has been well described for various cell types ([1]and [3] and references therein), and a number of celltypes and conditions have been reported with elevatednuclear STATs due to constitutive tyrosine phosphor-ylation of these proteins [15, 16]. Comparatively littleis known about the regulation of their subcellular dis-tribution in unstimulated cells before the addition ofcytokines. There are only incidental reports on thedistribution in resting cells, and these do not elaborateon the underlying mechanism responsible for the ob-served nucleocytoplasmic distribution. We thereforeinitiated a study of this mechanism and performed a

comprehensive examination of the subcellular distri-

0014-4827/01 $35.00© 2001 Elsevier Science

All rights reserved.

46 MEYER, GAVENIS, AND VINKEMEIER

bution of STAT1 and STAT3 in various stable cell linesand extended these studies to different primary celltypes. Since tyrosine phosphorylation is usually con-sidered to be required for nuclear presence of STATs,we investigated the role of this modification in theprestimulation subcellular distribution of STAT1. Ourresults reveal constitutive, unique, and cell type-spe-cific levels of nuclear STAT1 and STAT3 prior to cyto-kine stimulation. Additionally, we find that this pro-cess is controlled entirely independent of tyrosinephosphorylation.

MATERIALS AND METHODS

Plasmid construction. The fusion protein of STAT1 to the N-terminus of the green fluorescent protein (GFP) has been described[11]. The mutations of arginine 602 to leucine and tyrosine 701 tophenylalanine were generated by site-directed mutagenesis usingthe Quick-Change kit according to the manufacturer’s protocol(Stratagene).

Cell culture, DNA transfections, and fluorescence microscopy.Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)with 10% fetal calf serum (GIBCO), 1% penicilline/streptomycin, and1% amphotericine (growth medium) to a confluence of approx. 70%.With cells grown in 12-well plates transient transfections using 0.8mg/plate of the different expression plasmids were performed by theLipofectamine method (GIBCO). Cells were left to recover for 24 h ingrowth medium. The influence of serum starvation on STAT1 nu-clear presence was tested with HeLa-S3 cells, which are shown to behigh in nuclear STAT1. For this purpose, cells were grown for 16 h inmedium without serum. To evaluate the effects of tyrosine phosphor-ylation on STAT1 distribution, cells were treated for 1 h with 500 nMstaurosporine (Sigma). The effectiveness of kinase inhibition wastested by subsequent stimulation of the cells with interferon-gamma(IFNg). Treatment with IFNg (5 ng/ml) was for 30 min. For the directdetection of GFP fusion proteins, cells were fixed in 3.7% paraform-aldehyde in PBS for 10 min at room temperature. Subsequently, thenuclei were stained for 3 min with 5 mg/ml Hoechst 33258 (Sigma)and the samples were mounted in fluorescence mounting medium(Dako). Fluorescence microscopy was performed using a Leica DMLB(Solms, Germany) microscope, equipped with A, N2.1, and I3 fluo-rescence filters. Images were taken with a Sensicam CCD cameraand were processed with the Axiovision (Zeiss, Gottingen) and AdobePhotoshop (Adobe Systems, Mountain View, CA) packages.

Detection of STAT proteins by immunocytochemistry. The follow-ing stable cell lines were used for immunocytochemistry: humancervix carcinoma cells HeLa-S3 (ATCC No. CCL-2), human embryo-nal kidney fibroblasts Hek 293 (ATCC No. CRL-1573), human hep-atoma cells HepG2 (ATCC No. HB-8065), monkey kidney cells COS7(ATCC No. CRL-1651), human urinary bladder carcinoma cellsECV304 (ECACC No. 92091712), human neuroblastoma cells SK-N-SH (ATCC No. HTB-11), human fibroblasts U3A (a gift from Dr. G.Stark, Cleveland). Primary cultures of human fibroblasts, smoothmuscle cells, chondrocytes, umbilical vein endothelial cells, keratino-cytes, nasal epithelial cells, and osteoblasts were purchased fromCell Lining (Berlin), grown in 16-well Lab-tec plates (Nunc) and wereused directly for immunocytochemical detection of STAT1. All stablecell lines grown on poly-L-lysine-coated coverslips were fixed withmethanol for 6 min (stable cell lines), or for 15 min (primary cells) at220°C. Thereafter, nuclei were stained with Hoechst dye and unspe-cific binding was blocked by incubation with 25% fetal calf serum inPBS for 30 min at room temperature. The samples were incubatedfor 1 h with affinity-purified polyclonal antibodies against STAT1a

and STAT3 (sc-345 and sc-482G, respectively; Santa Cruz Biotech-

nology) and visualized by a second incubation with Cy3-conjugatedsecondary antibodies (Jackson Research) for 1 h at room tempera-ture. Mounting and fluorescence microscopy were performed as de-scribed before.

Inhibition of IFN with anti-IFN antibodies. Neutralizing anti-human IFNa and IFNb antibodies (Strathmann Biotech, Hannover)were added to HeLa-S3 cells grown on poly-L-lysine-coated coverslipsat a final concentration of 10 mg/ml each for 16 h. In a parallel set ofexperiments cells were stimulated either with IFNa alone (Biomol;500 IU/ml) for 1 h or with IFNa which had been pretreated for 16 hat 4°C with 10 mg/ml of the appropriate antibody. Subsequently, cellswere fixed in methanol and STAT1 was detected by immunocyto-chemistry as described.

Western blotting and electrophoretic mobility-shift assay (EMSA).Cells grown on 6-cm dishes were lysed in 200 ml cytosolic extractionbuffer (10 mM KCl, 20 mM Hepes, 0.2% NP-40, 1 mM EDTA, 10%glycerol, 0.1 mM vanadate, 0.1 mM PMSF; 0.1 mM DTT, CompleteMini protease inhibitors (Roche), pH 7.4) for 20 min on ice. Theextracts were spun at 3000g at 4°C for 3 min and the supernatantswere used as cytosolic extracts for electrophoretic mobility-shift as-say and Western blotting. The pellets were washed twice in cytosolicextraction buffer (500 ml each time) and were then resuspended in100 ml nuclear extraction buffer (420 mM KCl, 20 mM Hepes, 0.1 mMvanadate, 20% glycerol, 1 mM EDTA, 0.1 mM PMSF, 1 mM DTT,complete protease inhibitors, pH 7.6) and left on ice for 30 min. Theextract were spun at 4°C at 15,000g for 20 min, and the supernatantswere used as nuclear extracts. Representation of equal cell numbersin cytosolic and nuclear extracts was assured by loading only half theamount of nuclear extract relative to the cytosolic extract. Whole-cellextracts were prepared in 200 ml whole-cell extraction buffer (50 mMTris, 0.5% NP-40, 0.2 mM EDTA, 2 mM EGTA, 10% glycerol, 10 mMglycerophosphate, 1 mM vanadate, 0.1 mM PMSF, 1 mM DTT,complete protease inhibitors, pH 8.0). Cells were lysed on ice for 20min before centrifugation (10 min; 15,000g). The resulting superna-tant was used as whole-cell extract. Proteins were separated by 8%SDS–PAGE and transferred to nitrocellulose by semi-dry blotting(Biorad). Molecular weights were determined by a prestained stan-dard (See Blue; Novex). Phosphorylated STAT1 was detected byincubation for 1 h with a polyclonal antibody specific for phospho-STAT1-Tyr701 (New England Biolabs) and developed with horserad-ish peroxidase-conjugated secondary antibodies (Dako) using en-hanced chemiluminescence (NEN). Subsequently, the blots werestripped off bound antibodies for 40 min at 50°C in 62.5 mM Tris–HCl, pH 6.8, 2% SDS, 0.7% (vol/vol) b-mercaptoethanol and werereprobed with a polyclonal STAT1-specific antibody (sc-345; SantaCruz). The cytoplasmic marker protein Eps15 was detected with apolyclonal antibody (sc-534; Santa Cruz). For EMSA analysis, 4 ml ofnuclear extract was incubated with 1 ng of 32P-labeled M67 probe[11] for 5 min at room temperature. The shift reactions were resolvedon 4% 29:1 acrylamid/bisacrylamid gels at 4°C as described previ-ously [11]. Binding activity was visualized with a phosphoimagingsystem (Storm 820, Molecular Dynamics).

Quantification of immunofluorescence intensities. STAT1 andSTAT3 were labeled by immunocytochemistry as described. Fluores-cence signals were detected and quantified by confocal microscopywith a laser scanning microscope (LSM 410; Zeiss), as described [17,18]. Briefly, for quantification of STAT protein content, an adherentmonolayer of cells was scanned in z direction, leading to a gallery ofconsecutive slices through the cells. The median slice (x/y image),being the slice with the best resolution of nucleus and cytoplasm, wastaken for fluorescence quantification. The outlines of both the cellmembrane (boundary of total cellular fluorescence) and the nuclearmembrane (boundary of nuclear fluorescence) were drawn on theimage and the sums of the fluorescence signals were determinedinside the marked areas with an image analysis software (KS400,Kontron Electronics). Unspecific background signals from areas

without cells were determined and subtracted from the specific sig-

47CONSTITUTIVELY NUCLEAR STATs

nals. The fluorescence intensities of 20 cells were determined foreach sample, and values for mean and standard deviation were

FIG. 1. Characterization of antibodies used for the immunocyto-chemical detection of STAT proteins. (A) SDS–PAGE profile of IFNg-stimulated HeLa-S3 whole-cell extracts stained by Coomassie bril-liant blue (indicated by C); and Western blots of the correspondingproteins incubated with affinity-purified anti-STAT1a antibodies (in-dicated as S1; note the double band representing unphosphorylated(lower band) and tyrosine-phosphorylated (upper band) STAT1a);anti-phospho-tyrosine STAT1 antibodies (indicated with S1P; notethe staining of tyrosine-phosphorylated STAT1a (upper band) andthe faster migrating tyrosine-phosphorylated STAT1b); or anti-STAT3 antibodies (indicated with S3). (B) U3A cells (U3A; A, B) andU3A cells stably expressing STAT1 (U3A-S; C, D) were stained withanti-STAT1 antibodies and Hoechst dye. Shown are the fluorescencemicrographs of the immunostained cells (A, C) and the positions ofthe nuclei (B, D).

calculated.

RESULTS

STAT1 and STAT3 Are Nuclear Prior to Stimulationwith Cytokines

To assess the cross-reactivity of the antibodies em-ployed for quantitative analysis, we treated HeLa-S3whole-cell extracts with the respective affinity-purifiedantibodies for STAT1a, tyrosine-phosphorylatedSTAT1a/b, and STAT3 (Fig. 1A). In addition, we im-munocytochemically stained U3A cells, which do notexpress STAT1 [19], with anti STAT1 antibodies, aswell as U3A cells stably transfected with a STAT1expression vector (Fig. 1B). These experiments revealthe high ratio of specific to unspecific binding and lowbackground levels of the antibodies used.

In order to accurately determine the steady-statedistribution of endogenous STAT1 and STAT3, we em-ployed confocal microscopy of various cell types labeledimmunofluorescently with the respective specific anti-bodies. An example of the data generation is shown inFig. 2A. A layer of adherent HeLa-S3 cells was scannedin the z direction in steps distanced 1.8 mm, producinga gallery of consecutive slices through the cell body.The median slice was taken for quantitative analysisas detailed above. A total of 13 different cell types, 7 ofwhich were primary cells of human origin, were in-cluded in the investigation. Generally, the cells werestained with little cell-to-cell variability. In less thanabout 5% of the cells the characteristic staining patterndeviated substantially. Labeling artifacts and dead ormitotic cells accounted for most of such cases. Thesecells were not included in the analysis. Notably, theprimary cells usually displayed a higher degree of het-erogeneity, which is reflected in the higher standarddeviations in the quantitative analysis.

We then investigated a number of established celllines and primary cell types for their subcellularSTAT1 distribution. It becomes immediately obviousfrom Fig. 2B that all unstimulated cells examined dis-played STAT1 immunoreactivity in the nucleus. How-ever, the degree of nuclear staining varied considerablybetween the different cell types. As the quantitativeanalysis (Table 1) revealed, nuclear STAT1 proteinaccounted for up to 43% of the total (HeLa-S3 cells),and was found to be as low as 19% in primary smoothmuscle cells. We found several cell lines with remark-ably high levels (.35%) of nuclear STAT1 in the ab-sence of cytokine stimulation. No striking differencesbetween established cell lines and primary cells couldbe detected (Fig. 2B and Table 1).

STAT1 and STAT3 Differ from One Another in TheirNucleocytoplasmic Distribution

Additionally, we focused on STAT3 distribution us-

ing immunocytochemically labeled cells. As was seen

48 MEYER, GAVENIS, AND VINKEMEIER

FIG. 2. Nucleocytoplasmic distribution of STAT1 in cultured mammalian stable cell lines and primary isolates. (A) Gallery of opticalslices spaced 1.8 mm (z disc) through a layer of HeLa-S3 cells immunocytochemically labeled with affinity-purified STAT1 antibodies. Theimage in the center (corresponding to the equator of the nucleus) was used for further quantitative analysis. (B) Confocal images of equatorialsections through stable cell lines (A–F) and primary cells (G–J). Shown are COS7 cells (A); Hek 293 cells (B); SK-N-SH cells (C); ECV cells(D); HeLa-S3 cells (E); HepG2 cells (F); nasal epithelial cells (G); human fibroblasts (H); keratinocytes (I); and chondrocytes (J). Staining wasperformed with STAT1 antibodies. (C) Comparison of the nucleocytoplasmic distribution of STAT1 and STAT3. Endogenous STAT1 andSTAT3 proteins were detected by immunocytochemistry using confocal microscopy. Shown are HepG2 cells (A, B); SK-N-SH cells (C, D); and

COS7 cells (E, F). Panels A, C, and E were labeled for STAT1; panels B, D, and F depict the localization of STAT3.

49CONSTITUTIVELY NUCLEAR STATs

for STAT1, also STAT3 can be readily detected in thenuclear compartment of unstimulated cells (Fig. 2C).Moreover, the investigated cell lines were distin-guished by the amount of nuclear STAT3 immunore-activity that each of them displayed (Table 1). Whenthe nuclear presence of both STATs in various stable

TABLE 1

Quantitative Analysis of Nuclear and Cytoplasmic STAT1and STAT3 Content in Various Cell Lines

STAT1(% nuclear)

STAT3(% nuclear)

HeLaUnstim 43.7 6 6.6 41.4 6 6.3Stim 67.8 6 4.9 ND

HekUnstim 41.9 6 6.5 42.2 6 10.7Stim 57.1 6 7.3 ND

Hep G2Unstim 33.4 6 6.1 50.4 6 7.4Stim 80.4 6 5.8 ND

COSUnstim 28.0 6 6.9 38.9 6 8.0Stim 74.5 6 10.8 ND

ECVUnstim 27.7 6 7.1 32.8 6 8.7Stim 75.9 6 8.1 ND

SK-N-SHUnstim 29.6 6 8.3 41.5 6 11.4Stim 62.8 6 8.8 ND

KERUnstim 25.3 6 6.2 NDStim 33.4 6 10.7 ND

CHONUnstim 36.7 6 8.1 NDStim 44.6 6 11.6 ND

NECUnstim 28.4 6 8.5 NDStim 55.9 6 12.0 ND

SMCUnstim 19.3 6 7.3 NDStim 46.6 6 9.2 ND

OBUnstim 20.8 6 10.5 NDStim 53.8 6 12.1 ND

FIBUnstim 25.0 6 9.0 NDStim 41.5 6 14.0 ND

VECUnstim 21.5 6 3.8 NDStim 48.8 6 7.5 ND

Note. Images as depicted in Fig. 1B were used for quantitativeanalysis of endogenous STAT1 and STAT3 immunoreactivity. Listedis the percentage of total cellular staining that was found in thenucleus before (unstim) and after treatment with IFNg for 30 min(stim). Fluorescence signals for wild-type STAT1 (WT) and thedimerization mutant R602L, Y701F (R602, Y701) were measured ornot determined (ND). The following abbreviations are used for pri-mary cells: KER, keratinocytes; CHON, chondrocytes; NEC, nasalepithelial cells; SMC, smooth muscle cells; OB, osteoblasts; FIB,fibroblasts; VEC, umbilical vein endothelial cells.

cell lines was compared (Fig. 2C and Table 1), each of

them showed a unique staining pattern. It was foundthat the two transcription factors in some cell linesshowed a very similar distribution (e.g., HeLa-S3),while in others a more disparate distribution was re-corded (e.g., HepG2). Thus, the presence of STAT1 andSTAT3 in the nuclei of resting cells is prevailing and ahallmark of individual cell types.

In addition to the analysis of untreated cells, wequantified the degree of nuclear accumulation ofSTAT1 after incubation with IFNg, which rapidly in-duced the well-known nuclear build-up (Fig. 3 andTable 1). While in all cells examined the nuclear pres-ence of STAT1 was markedly increased by treatmentwith IFNg, we also detected a surprisingly sizeablepool of residual cytoplasmic STAT1. The size of theSTAT1 pool that remained cytoplasmic varied consid-erably between different cell types, but it did not fallbelow 20% of the total STAT1.

Nucleocytoplasmic Distribution of STAT1 IsRegulated Independent of Tyrosine Phosphorylation

According to the current model describing howSTATs function, the nuclear import of STAT proteinsrequires their tyrosine phosphorylation and concomi-tant dimerization [20]. To investigate the role of ty-rosine modification in the constitutive nuclear pres-ence of STAT1, we employed immunofluorescencemicroscopy as well as biochemical analysis of nuclearand cytoplasmic cell extracts. Most experiments wereperformed in HeLa-S3 cells, which were found to con-tain nuclear STAT1 to conspicuously high levels (Table1). We initially serum-starved HeLa-S3 cells to sup-press any stimulatory influences stemming from bo-vine serum, but nucleocytoplasmic allocation of STAT1remained unaltered (not shown).

The most potent promoters of tyrosine phosphoryla-tion of STAT1 are type I and II interferons. While theexpression of type II IFN (IFNg) is limited to activatedT lymphocytes and natural killer cells during the cel-lular immune response [21], type I interferons (IFNaand b) appear to be secreted by a variety of cell lines[22]. To exclude any contributions to STAT1 phosphor-ylation by low-level autocrine type I IFN secretion intothe growth medium, we incubated the cells for 16 h inthe presence of blocking anti-IFNa and IFNb antibod-ies (Fig. 3A). Again, this treatment did not modify thenucleocytoplasmic distribution of STAT1. The effec-tiveness of this method to inhibit the action of IFNs isdemonstrated in panel D of Fig. 3A. Preincubation withneutralizing antibodies did entirely abrogate the ef-fects of IFNa on nuclear accumulation of STAT1. Thus,we conclude that IFN-induced tyrosine phosphoryla-tion can be excluded as the cause for the elevatednuclear presence of STAT1 in HeLa-S3 cells.

However, tyrosine phosphorylation of STAT1 can be

50 MEYER, GAVENIS, AND VINKEMEIER

induced by a plethora of cytokines and growth factors[23]. Therefore, we used two further approaches toexclude the possibility of tyrosine phosphorylation af-fecting constitutively nuclear STAT1. First, we treatedcells with the broad range kinase inhibitor staurospor-ine [24]. As expected, treatment of cells with this re-agent precluded STAT activation with subsequent nu-clear accumulation, presumably through blocking of

FIG. 3. Influence of type I IFN and tyrosine kinase activity on theI IFN activity with an equimolar mixture of anti-IFNa and b antibodfluorescence microscopic images of HeLa-S3 preparations stained witwere either left untreated (A) or treated with IFNa for 1 h (B). A 16-mg/ml each) is depicted in C. Cells exposed to IFNa that had been prthe effectiveness of IFN blocking. (B) Effects on the subcellular STstaurosporine. Fluorescence micrographs of STAT1 immunostainingcompared to identical samples that were pretreated with the tyrosineSTAT1 does not display immunoreactivity with antibodies directedShown are fluorescence micrographs of HeLa-S3 cells stained with pincubation of the cells with IFNg for 30 min.

JAK kinase activity (Fig. 3B). However, while IFNg-

induced nuclear accumulation of STAT1 was preventedby staurosporine, thus demonstrating its inhibitory ef-fect on tyrosine phosphorylation, this treatment on theother hand did not detectably diminish the amount ofnuclear STAT1.

In addition, we generated a mutant of STAT1 withsubstitution of tyrosine 701 to phenylalanine and adouble mutant with an additional substitution of argi-

bcellular localization of STAT1 in HeLa-S3 cells. (A) Blocking of typedoes not influence STAT1 nucleocytoplasmic distribution. Shown aretibodies against STAT1 and the corresponding Hoechst stains. Cellscubation in the presence of neutralizing anti-IFNa/b antibodies (10cubated with anti-IFNa/b antibodies (D) are shown to demonstratedistribution by unspecifically blocking tyrosine kinase activity by

HeLa-S3 cells before (A) and after 1 h treatment with IFNg (B) arenase inhibitor staurosporine (500 nM) for 1 h (C, D). (C) The nuclearainst STAT1 phosphotyrosine before stimulation of cells with IFN.photyrosine-specific anti-STAT1 antibodies before (A) and after (B)

suiesh anh inein

AT1inkiaghos

nine 602 to leucine. Tyrosine 701 is the sole site of

51CONSTITUTIVELY NUCLEAR STATs

tyrosine phosphorylation of STAT1 [4], and arginine602 is located at the bottom of the phosphotyrosinebinding pocket in the STAT1 SH2 domain [25]. Thus,the double mutant is incapable of phospho-tyrosine-mediated homo- or heterodimerization with endoge-nous wild-type STAT1 [5, 26, 27]. To visualize thesubcellular distribution of the mutants we generatedfusion proteins with the N terminus of GFP. As can beseen from a comparison of Figs. 2 and 3 with Fig. 4B forHeLa-S3 cells, and of Fig. 1B with Fig. 4F for U3Acells, the addition of the green fluorescent protein to

FIG. 4. Constitutive nuclear presence of unphosphorylated STfluorescence microscopical images of cells transiently expressing GFPand dimerization-deficient double mutant arginine 602 to leucine/tyrotreated with IFNg for 30 min before fixation. Depicted is the distribstained with Hoechst dye (Hoechst), and a merged image (Merge).

the C terminus of STAT1 did not alter the subcellular

STAT1 distribution. The STAT1-GFP fusion proteinsfaithfully reproduced the cell type-specific distributionof endogenous and untagged stably expressed STAT1in HeLa-S3 and U3A cells, respectively. In addition,upon stimulation with IFNg a strong nuclear accumu-lation could be induced only with wild-type STAT1-GFP (shown for HeLa-S3 cells in Fig. 4C). Importantly,the phosphorylation and dimerization incompetentdouble mutant did not differ from the cell type-specificnucleocytoplasmic distribution of wild-type STAT1 inresting cells (compare Figs. 1 and 4). As was seen with

1 in Hek 293, HeLa-S3, and U3A cells (as indicated). Shown areion proteins of either wild-type STAT1 (WT) or the phosphorylation-e 701 to phenylalanine (R602, Y701). Where indicated (1), cells weren of STAT1-GFP fusion proteins (STAT1), the localization of nuclei

ATfussin

utio

wild-type STAT1 in these cells, a substantial amount of

52 MEYER, GAVENIS, AND VINKEMEIER

the mutant R602L, Y701F resided in the nucleus priorto stimulation with IFNg, particularly in HeLa-S3 cells(Fig. 4D). The strong nuclear presence of unphosphor-ylated STAT1 in unstimulated HeLa-S3 cells was alsoevident with the single mutant tyrosine 701 to phenyl-alanine (not shown). Expectedly, addition of IFNg nei-ther induced nuclear accumulation of these STAT1mutants nor brought about any other detectablechange in their nucleocytoplasmic localization pattern(Fig. 4E).

In a further set of experiments we tried to directlydetect tyrosine phosphorylation of STAT1 by Westernblotting and probing nuclear extracts for DNA bindingactivity with gel-shift assays. We prepared nuclear andcytoplasmic extracts from HeLa-S3 cells before andafter stimulation with IFNg (Fig. 5A). To demonstratethe absence of cytoplasmic contamination in the nu-clear extracts, we examined the distribution of thecytosolic marker protein Eps15 [28, 29] by Westernblotting. As is shown in Fig. 5A, the nuclear extractswere virtually free of Eps15, which was present in highamounts in the cytoplasmic extracts. We then probedfor the presence of phosphorylated STAT1 in theseextracts by immunoblotting with an antibody specifi-cally recognizing tyrosine-phosphorylated STAT1. Thebinding reactivity of this antibody was entirely limitedto extracts derived from cells that had been stimulatedwith IFNg (Fig. 5A). Probing of an identical blot with aSTAT1-specific antibody revealed significant amountsof constitutively nuclear STAT1 also in unstimulatedcells. Remarkably, the total amount of nuclear STAT1did not drastically increase after cytokine stimulation(Fig. 5A). We also probed these extracts for STAT1DNA binding activity. Detection of DNA binding is anindirect, yet very sensitive, measure to trace low levelsof tyrosine phosphorylated and dimeric STATs. How-ever, gel-shift assays with the HeLa-S3 nuclear ex-tracts did not yield any evidence of constitutive ty-rosine phosphorylation prior to treatment with IFNg(Fig. 5B). Only after stimulation with the cytokine didDNA binding activity become apparent (Fig. 5B). Ex-pression of STAT1 fusion proteins in HeLa-S3 cells ledto the same result (Fig. 5B). No DNA binding activitywas detectable prior to stimulation with IFNg. Inter-estingly, stimulation with IFNg did not deplete thecytoplasm of STAT1 DNA binding activity (Fig. 5B,right panel). This finding is in line with the quantita-tive data in Table 1, which also show significant levelsof residual cytoplasmic STAT1 in IFNg-treated cells.

We also investigated extracts from U3A cells whichlack endogenous STAT1 for the presence of tyrosine-phosphorylated STAT1 prior to stimulation with IFNg.We used this cell line to exclude any influences onnuclear transport of the double mutant caused by in-teractions with the endogenous wild-type STAT1. Con-

firming our immunofluorescence data (Fig. 4), tran-

siently expressed wild-type and mutant STAT1 werefound with identical, albeit low, amounts in the nu-cleus. Despite this and in agreement with the resultsobtained with HeLa-S3 nuclear extracts, we could de-tect gel-shift activity in nuclear extracts from U3A cellsexpressing wild-type STAT1 only after IFNg stimula-tion (Fig. 5C). A direct evaluation of the expressionlevels and degree of tyrosine phosphorylation was per-formed by Western blotting. As is shown in Fig. 5D, wedetected significant amounts of unphosphorylatedwild-type STAT1 as well as the mutant protein innuclear extracts of unstimulated cells. Taken together,these results provide strong evidence that entry ofSTAT1 into the nuclei of resting cells is feasible despitethe absence of tyrosine phosphorylation.

DISCUSSION

The STAT proteins rapidly induce transcription ofgenes in response to cytokines. The responsive genesare characterized by the presence of STAT bindingsites in their promoter regions. In order to gain accessto these sites, the STATs need to be tyrosine phosphor-ylated, which was shown to induce their dimerizationand subsequent accumulation into the nucleus. Thenuclear translocation as well as the induction of cyto-kine-responsive genes is transient, and over the courseof several hours the initial distribution is restored. Thediscovery of constitutive functions of STAT1 in thenucleus, however, has been at odds with an exclusivelycytoplasmic localization of STATs in unstimulatedcells. Therefore, in the present study we aimed to char-acterize the nucleocytoplasmic localization of STAT1and STAT3. We employed several transformed celllines as well as a number of different primary cellswhich were included to get a more representative in-sight into the potential variability between differentcell types. For localization analysis, antibody stainingof the endogenous STAT proteins in combination withconfocal microscopy was used. In addition, we wantedto assess the role of tyrosine phosphorylation for thenuclear presence of STAT1. To this end, cell fraction-ations with cells expressing wild-type or mutantSTAT1 were performed. The resulting nuclear extractswere probed for STAT1 dimerization by gel-shift as-says and for tyrosine phosphorylation by Western blot-ting. The major findings of this study were as follows:(i) in all cells examined including primary isolates wedetected STAT1 and STAT3 in the nuclei of unstimu-lated cells; (ii) the prestimulation STAT levels vary celltype- and STAT-specifically; (iii) tyrosine phosphoryla-tion is not a prerequisite for nuclear entry and nuclearpresence of STAT proteins; (iv) nuclear entry and nu-clear accumulation are independent events.

Our results clearly demonstrated that STAT tran-

scription factors are constitutively nuclear. Some cells,

odcub

53CONSTITUTIVELY NUCLEAR STATs

such as HeLa-S3, showed an almost even nucleocyto-plasmic distribution in resting cells. We also used pri-

FIG. 5. Analysis of STAT1 tyrosine phosphorylation and gel-shifprotein content in nuclear and cytoplasmic extracts representing equIFNg for 30 min. The distribution of total STAT1a, tyrosine-phosphwas detected by Western blotting of nuclear and cytoplasmic extractsanalysis of cytoplasmic and nuclear extracts from untransfected HeLSTAT1-GFP (lanes 3–6). The extracts were incubated with radiolabelindicated (1), cells had been treated with IFNg for 30 min before eSTAT1 protein (bottom bar), of the STAT1-GFP homodimers (top baof the slots where the gel was loaded; (**) unspecific band. (C) Gel-shSTAT1, transiently expressing wild-type (lanes 1 and 2) and the pho(lanes 3 and 4) fused to GFP. Where indicated (1), cells had beenposition of the specific STAT1/DNA complex. (D) Western blot analyresolved on a denaturing polyacrylamide gel and probed with an antibP). Subsequently, the blot was stripped off bound antibodies and in

mary cells to exclude any artifacts introduced by cell

transformation. Yet, the primary cells did not substan-tially differ from the transformed stable lines and also

tivity in cytoplasmic and nuclear cell extracts. (A) Demonstration ofell numbers of HeLa-S3 cells before (2) and after (1) treatment withlated STAT1, and the cytoplasmic marker protein Eps15 (142 kDa)separate experiments with the appropriate antibodies. (B) Gel-shift3 cells (lanes 1 and 2) and from HeLa-S3 cells transiently expressingoligonucleotides containing a strong M67 STAT1 binding site. Wherection. Bars indicate the position of homodimers of the endogenousnd of heterodimers thereof (middle bar) bound to DNA. (*) Positionanalysis of nuclear extracts from U3A cells, which lack endogenousorylation- and dimerization-deficient mutant STAT1 R602L, Y701Fted with IFNg for 30 min before extraction. The bar indicates the

of STAT1 phosphorylation. The extracts used for EMSA in (C) werey specifically recognizing tyrosine-phosphorylated STAT1 (a-STAT1-ated with a STAT1-specific antibody (aSTAT1).

t acal coryin

a-Sedxtrar) aift

sphtreasis

contained relatively high amounts of nuclear STAT1

54 MEYER, GAVENIS, AND VINKEMEIER

and STAT3. Since our analysis by confocal microscopywas performed with the endogenous proteins, we canexclude a distorted subcellular localization as a conse-quence of transfection with potential overexpression.Given the constitutive nuclear function of STAT1 inapoptosis, it will be interesting to examine whether thedifferent levels of nuclear STAT1 influence caspasegene activity or the degree of susceptibility to apopto-sis-inducing agents such as tumor necrosis factor orIFNg [13].

We could exclude tyrosine phosphorylation as thecause of nuclear import of STAT1. No traces of phos-phorylation were detectable before stimulation withIFNg. Moreover, blocking of tyrosine kinases withstaurosporine or mutations of two critical residues,which prevent tyrosine phosphorylation and dimeriza-tion, were without influence on nucleocytoplasmic dis-tribution in resting cells. These findings agree with anearlier study that reported the nuclear presence of thephosphorylation incompetent STAT1 mutant Y701F inU3A cells [14]. We noted the absence of STAT1 immu-noreactivity or green fluorescence of the STAT1-GFPfusion proteins from the nucleoli of unstimulated cellsfor both wild-type and mutant STAT1 (Fig. 4). Thisbehavior is typical also for tyrosine-phosphorylatedSTAT1 after nuclear accumulation (Fig. 4 and Ref.[30]). Additionally, the permanent nuclear presenceseems to be a more general attribute of STAT proteins.So far a constitutive nuclear function has been identi-fied for STAT1 only. As has been demonstrated in thisreport, also STAT3 was found in the nucleus in theabsence of treatment with cytokines, thus hinting at acurrently unknown constitutive nuclear function forthe unphosphorylated protein. The results with themutant STAT derivatives also indicate that nuclearimport of STATs does not neccesarily lead to nuclearaccumulation (Fig. 4). Thus, the phosphorylatedSTATs do not differ from the unphosphorylated mole-cules in their ability to enter the nucleus, but mayrather display altered rates of nuclear import or ex-port, e.g., due to an inability to leave the nuclear com-partment.

No canonical import signal has been identified inSTAT proteins to date. However, it is known that thephosphorylated STAT1 requires the small GTPase Ranfor nuclear import [31], and associates with the b sub-unit of the nuclear pore targeting complex via theNPI-1 family of a subunits to enter the nucleus [32].The STAT binding domain of NPI-1 was shown to bedistinct from the conventional NLS binding domain,which consists of armadillo repeats in the N-terminalregion of NPI-1 [32]. The corresponding binding inter-face on STAT1 is unknown at present. Currently ex-periments are under way in our laboratory to investi-gate the role of the b subunit in nuclear import of

unphosphorylated STAT1.

Several groups recently described the existence ofnuclear export signals of the leucine-rich, CRM-1-de-pendent type in STAT1 [11, 33, 34]. Mutational knock-out of the NES function or the inhibition of nuclearexport by leptomycin B, which blocks CRM-1-depen-dent pathways, was without effects on the nucleocyto-plasmic distribution in resting cells [11, 33]. Thus, it isunclear at the moment how the nuclear presence ofSTAT1 is regulated in unstimulated cells. Nonetheless,it is modulated in a cell type-specific fashion. Differingrates of nuclear import and export, and/or the differ-ential expression of an anchoring substrate in the cy-toplasm, could account for these cell-specific differ-ences. An example for the latter mechanism insignaling to the nucleus by extracellular ligands isprovided by the Smad proteins Smad2 and Smad3. Inunstimulated cells, these transcription factors are lo-calized to the cytoplasm through interactions with theFYVE domain protein SARA [35]. Cytoplasmic pro-teins that interact with STATs have recently been dis-covered [36, 37] and may well fulfill comparable roles.As the presented study indicates for the STAT pro-teins, the control of the subcellular distribution ofthese transcriptional regulators entails many moreregulatory mechanisms than are currently known.

The authors thank G. Papsdorf and G. R. Stark for providing celllines, B. Oczko and M. Koroll for expert technical assistance inpreparing confocal images, and B. Wiesner for support in the quan-titative image analysis. We also thank P. Striedelmeyer and M. vanRossum for technical assistance and Doratha E. Drake for criticalreading of and comments on the manuscript. This work was sup-ported by a grant from the Bundesministerium fur Bildung undForschung (0311872 to U.V.).

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Received April 18, 2001Revised version received September 28, 2001Published online November 19, 2001