MOLECULAR AND CELLULAR BIOLOGY,0270-7306/97/$04.0010

Sept. 1997, p. 5307–5316 Vol. 17, No. 9

Copyright © 1997, American Society for Microbiology

Functional Differences between Stat3a and Stat3bTIMOTHY S. SCHAEFER, LAURA K. SANDERS, OHKMAE K. PARK, AND DANIEL NATHANS*

Howard Hughes Medical Institute and Department of Molecular Biology and Genetics,Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Received 25 November 1996/Returned for modification 23 January 1997/Accepted 28 May 1997

Stat3b is a short form of Stat3 that differs from the longer form (Stat3a) by the replacement of theC-terminal 55 amino acid residues of Stat3a by 7 residues specific to Stat3b. In COS cells transfected withStat3 expression plasmids, both Stat3a and Stat3b were activated for DNA binding and transcription by thesame set of growth factors and cytokines and both, when activated, formed homodimers and heterodimers withStat1. Only Stat3b was active in the absence of added cytokine or growth factor. Activation of each form,including constitutive activation of Stat3b, was correlated with the phosphorylation of tyrosine 705. ActivatedStat3b in transfected COS cells was more stable and had greater DNA-binding activity than activated Stat3a.However, relative to DNA-binding activity, Stat3a showed greater transcriptional activity than Stat3b. Amutant of Stat3a lacking its highly acidic C-terminal 48 amino acids had properties indistinguishable fromStat3b. We conclude that Stat3a and Stat3b have significantly different properties due to the presence orabsence of the acidic C-terminal tail of Stat3a rather than the C-terminal sequence peculiar to Stat3b. Inaddition to its effect on transcription, we speculate that the acidic tail may destabilize the active dimeric formof Stat3a, resulting in lower DNA-binding activity of the Y705-phosphorylated form compared to Stat3b andin more rapid dephosphorylation.

Growth factors and cytokines cause rapid changes in geneexpression mediated by the activation of latent transcriptionfactors. Activation of transcription factors occurs by diversepathways, including a cascade of protein phosphorylation re-actions leading to the activation of nuclear transcription fac-tors and a more direct phosphorylation pathway resulting inactivation of latent cytoplasmic transcription factors that moveto the nucleus. An example of the latter is the pathway involv-ing the activation of Stat proteins by a cytokine receptor-associated tyrosine protein kinase of the JAK family (reviewedin references 14, 15, and 25). In this pathway, initially charac-terized for genes responsive to interferons (7), the interactionof cytokines with their cognate receptors results in dimeriza-tion of receptor chains, association of JAK with a cytoplasmicdomain of the receptor, and specific tyrosine phosphorylationof Stat transcription factors. Tyrosine-phosphorylated Stat pro-teins form homodimers and heterodimers via their SH2 do-mains and rapidly move from the cytosol to the nucleus, wherethey bind to response elements in DNA and contribute to geneactivation. In the case of receptors with cytoplasmic proteinkinase domains, such as those for EGF and PDGF, activationof Stat is thought to occur by direct phosphorylation or via anassociated JAK protein(s) (21, 30, 34).

At present seven mammalian Stat genes have been identified(Stat1, Stat2, Stat3, Stat4, Stat5a, Stat5b, and Stat6) (1, 3, 13,20, 32, 35, 38, 39). Stat1, Stat3 and Stat5 encode multiple formsgenerated by alternative splicing of pre-mRNA, and in somecases these have distinct transcriptional properties (4, 11, 24,33). Given the numerous exons found in Stat genes so faranalyzed (36), it is likely that other isoforms remain to bediscovered. One of the important determinants of the speci-ficity of a given cytokine or growth factor is the array of Statproteins that it activates in a responsive cell. In turn, this is

dependent on the expression of Stat genes, the pattern of RNAsplicing, and the affinity of each Stat SH2 domain for specificphosphotyrosine-containing regions of the cytokine or growthfactor receptor (12, 28). Other important determinants of spec-ificity are likely to be the relative stabilities of Stat homodimersand heterodimers formed from the activated pool of Stat pro-teins and cooperativity with other transcription regulators thatare specific for a given Stat isoform or heterodimer. Examplesof such cooperativity are those between Stat3b and Jun (24),between Stat1 and Sp1 (17), and between Stat5 and the glu-cocorticoid receptor (27).

In this study, we explored the functional differences betweenStat3a and Stat3b, a short form of Stat3 that is missing 55C-terminal amino acid residues present in Stat3a and has 7amino acid residues at its C terminus specific for Stat3b (24).Using cells transfected with plasmids expressing Stat3a orStat3b, we compared the responses of these proteins to variouscytokines and growth factors; the site of phosphorylation re-sponsible for their activation, including constitutive activationof Stat3b; their DNA-binding and transcriptional activities;and the stability of their active forms.

MATERIALS AND METHODS

Construction of plasmids. Plasmids that express Stat3a or Stat3b and thechloramphenicol acetyltransferase (CAT) reporter plasmid containing a segmentof the rat a2-macroglobulin and thymidine kinase (TK) promoters have beendescribed previously (24). Plasmids in which tyrosine 705 was mutated to phe-nylalanine (3aY705F and 3bY705F) were made by the EXsite system (Strat-agene). Plasmids D48 and D55 were made by PCR amplification in which the 39primer introduced a stop codon after amino acid 724 or 717, respectively. Theexpression plasmids encoding c-Jun or b-galactosidase (pYN3214-lacZ) wereprovided by Yusaku Nakabeppu (Kyushu University, Fukuoka, Japan).

Purification of Stat3a and Stat3b proteins. Tyrosine 705-phosphorylatedStat3 proteins with N-terminal oligohistidine were purified by Ni21- chelatechromotography from Baculovirus-infected Sf9 cells coexpressing JAK1 andJAK2, as previously described (22). Sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) with Coomassie blue staining indicated that theproteins were purified to near homogeneity (22). In the preparations used in theexperiment in Fig. 4, Stat3a and Stat3b preparations were tyrosine phosphory-lated to approximately the same extent, as determined by Western blotting witha Stat3-specific monoclonal antibody (Transduction Laboratories) or anti-phos-photyrosine antibody (4G10; Upstate Biotechnology).

* Corresponding author. Mailing address: Howard Hughes MedicalInstitute and Department of Molecular Biology and Genetics, JohnsHopkins University School of Medicine, 725 N. Wolfe St., Baltimore,MD 21205. Phone: (410) 955-8445. Fax: (410) 955-9124.

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Cell culture. COS-7 cells were maintained in Dulbecco’s modified Eagle’smedium (DMEM) plus 10% fetal bovine serum (DMEM-10) at 37°C under 5%CO2. For preparation of nuclear extracts for electrophoretic mobility shift assays(EMSA) or transcription experiments, cells were plated at a density of 1.1 3 106

per 10-cm dish or 3 3 105 per 5-cm dish the day prior to transfection. At 2 hbefore transfection, the cells were fed with fresh DMEM-10. The cells weretransfected by the calcium phosphate method (10), and the amounts of plasmidused are indicated in the figure legends. After 16 h, the cells were washed twiceand fed with DMEM containing no serum. At 40 h posttransfection, the cellswere stimulated with human epidermal growth factor (EGF) (100 ng/ml) (Up-state Biotechnology, New York, N.Y.) or other cytokines for the times indicatedin the figure legends. CAT assays were performed as described previously (29).CAT activity was normalized to b-galactosidase activity resulting from a cotrans-fected b-galactosidase reporter plasmid. The amount of input Stat DNA and thetimes of growth factor and cytokine stimulation vary among experiments asindicated, resulting in variations in percent CAT conversion. COS-7 cells used inthe study comparing the stability of Stat3 homodimers were transfected as aboveand stimulated with EGF for 20 min prior to the addition of the EGF receptorkinase inhibitor PD157655 (0.2 mM) (kindly provided by D. Fry, Parke-Davis,Ann Arbor, Mich.). Nuclear extracts were prepared at the indicated times afterinhibitor treatment. Cells used to prepare whole-cell extracts were plated andtransfected identically to those used to prepare nuclear extracts and harvested ina buffer consisting of 100 mM NaCl, 30 mM Tris (pH 7.6), 1% Nonidet P-40, 30mM NaF, 1 mM EDTA, 1 mM Na3VO4, 1 mM molybdate, 0.5 mM phenylmeth-ylsulfonyl fluoride, 10 mg of leupeptin per ml, and 5 mg of pepstatin A per ml.The lysate was incubated on ice for 15 min and centrifuged for 10 min in amicrocentrifuge. The supernatant was transferred to a new tube, and the proteinconcentration was determined by a detergent-compatible protein assay (Bio-Rad).

EMSAs. Nuclear extracts were prepared by the method of Andrews and Faller(2) with the inclusion of Na3VO4 (1 mM), 10 mg of leupeptin per ml, and 5 mgof pepstatin A per ml. The reactions were performed for 15 min at roomtemperature in 24 ml of buffer consisting of 10 mM HEPES (pH 7.8), 50 mMKCl, 1 mM EDTA, 5 mM MgCl2, 10% glycerol, 5 mM dithiothreitol, 1 mg ofbovine serum albumin per ml, 10 mg of leupeptin per ml, 5 mg of pepstatin A perml, 0.5 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4 with 1 mg ofpoly(dI-dC) and 0.3 ng of 32P-labeled high-affinity sis-inducible element (hSIE)(Santa Cruz). Protein-DNA complexes were separated by electrophoresis on 4%polyacrylamide gels.

Western analysis. Whole-cell or nuclear extracts were analyzed for proteinexpression by SDS-PAGE. Protein samples were separated on 10% gels, trans-ferred to nitrocellulose, and visualized with enhanced chemiluminescence re-agents (Amersham). The monoclonal antibody (S21320) recognizing both formsof Stat3 as well as the mutant Stat3 proteins was obtained from TransductionLaboratories. The polyclonal antibody that specifically recognizes Stat3 Y705was obtained from New England Biolabs. The anti-phosphotyrosine antibody4G10 was obtained from Upstate Biotechnology.

DNaseI footprinting. A 298-bp HindIII-XhoI fragment from the rat a2-mac-roglobulin CAT reporter plasmid containing both Jun- and interleukin-6 (IL-6)(Stat3)-responsive elements was used in the footprinting reactions (24). Approx-imately 3 mg of plasmid DNA was linearized with HindIII and treated with DNApolymerase I Klenow fragment in the presence of [a-32P]dATP, [a-32P]dCTP,and [a-32P]dGTP. The DNA was then cleaved with XhoI and purified by PAGE.Binding of purified activated Stat proteins was performed in 100 ml of EMSAbinding buffer [without poly(dI-dC)] containing approximately 5 3 105 cpm ofprobe. After a 22-min incubation at 22°C, CaCl2 was added to a final concen-tration of 2.5 mM; this was followed immediately by a 2-min incubation withDNaseI (0.015 U/reaction; Promega). The samples were then extracted withphenol-chloroform and ethanol precipitated. The samples were Cerenkovcounted prior to electrophoresis on sequencing gels and autoradiography.

Preparation of Stat3a- and Stat3b-specific antibodies. To generate Stat3b-specific antiserum, a synthetic peptide corresponding to the carboxy-terminal 11amino acids of Stat3b, the C-terminal 7 amino acids of which are unique to 3b,plus an additional 3 alanines (AAAICVTPFIDAVWK) was coupled to keyholelimpet hemocyanin for immunization. To generate Stat3a-specific antibodies, aprotein fusion between glutathione-S-transferase and amino acids 715 to 771 ofStat3a was made. The coupled peptide and glutathione-S-transferase fusionprotein were used to immunize New Zealand White rabbits. Immunoglobulin Gwas purified from immune sera by protein A affinity chromtography (Pierce).

Immunoprecipitation and immunoblot analysis. Whole-cell extracts (1 mg)from COS-7 cells transfected with expression plasmids for Stat3a, Stat3b, ortheir Y705F mutant forms were precleared with 50 ml of a 50% slurry of proteinA/G-agarose beads (Santa Cruz) for 1 h at 4°C. Following centrifugation, thesupernatant was transferred to a new tube and the proteins were immunopre-cipitated for approximately 16 h at 4°C with the Stat3a- or Stat3b-specificantibodies. Then 75 ml of a 50% slurry of protein A/G-agarose (Santa Cruz)beads was added, and the mixture was allowed to incubate for an additional 60min at 4°C. The beads were washed three times with 0.75 ml of ice-cold phos-phate-buffered saline. Immune complexes were separated on SDS–10% poly-acrylamide gels, transferred to nitrocellulose, and detected as previously re-ported (24). The blots were first probed with 4G10 monoclonal antibody. Theblots were then stripped in buffer (62.5 mM Tris-HCl [pH 7.6], 2% SDS, 100 mM

b-mercaptoethanol) at 50°C for 30 min and reprobed with an anti-Stat3 N-terminal monoclonal antibody.

RESULTS

Activation of Stat3a and Stat3b by cytokines and growthfactors. Stat3 is known to be activated by EGF, IL-6, gammainterferon, IL-10, IL-11, oncostatin M, leukemia inhibitory fac-tor, ciliary neurotropic factor, granulocyte colony-stimulatingfactor, IL-12, IL-2, IL-15, growth hormone, platelet-derivedgrowth factor and CSF-1 in cells with cognate receptors (14,15, 25). To compare the activation of Stat3a and Stat3b byselected cytokines and growth factors, we transfected COS-7cells with either a Stat3a or Stat3b expression plasmid andexamined the DNA-binding activity of nuclear proteins by us-ing a Stat-specific DNA oligonucleotide corresponding to thehSIE (Fig. 1). In cells transfected with Stat3a or Stat3b plas-mid, the two isoforms were generally present at similar con-centrations or the level of Stat3a exceeded that of Stat3b (24)(see Fig. 3B and 5). In the absence of ligand, nuclear extractsof cells transfected with the plasmid vector showed no oligo-nucleotide-binding activity, and after stimulation by ligand,they formed a complex corresponding to endogenous Stat1

FIG. 1. DNA binding to the hSIE by nuclear extracts of cytokine- or growthfactor-treated COS-7 cells that had been transfected with Stat3a or Stat3bexpression plasmids or vector. COS-7 cells were transfected with Stat3 expressionplasmids (12.5 mg/5-cm dish) or vector as described in Materials and Methods. At40 h after transfection, including a 24-h incubation in serumless medium, thecells were treated with the indicated ligand for 15 min. Nuclear extracts werethen prepared, 3 mg of extract was incubated with [32P]hSIE, and the complexwas detected by PAGE. All the factors used were human. Oncostatin M (OnM)(20 ng/ml; Genzyme), EGF (100 ng/ml; Upstate Biochemicals), transforminggrowth factor a (TGF-a), (200 ng/ml; Gibco/BRL), and alpha interferon (IFN-a)(3 3 103 U; Collaborative Biomedical Products) were used. 2, nuclear extractfrom uninduced cells. Complexes noted as A*, A, B, and C are Stat3b ho-modimer-oligonucleotide, Stat3a homodimer-oligonucleotide, Stat3-Stat1 het-erodimer-oligonucleotide, and Stat1 homodimer-oligonucleotide, respectively,based on supershift experiments and prior reports or correspondence to com-plexes formed by purified Stat proteins (22). The experiment with vector alonewas performed separately, with a different batch of cells and a different prepa-ration of labelled probe. It can therefore be compared only qualitatively to theother results.

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homodimer-oligonucleotide (SIF-C [31]) and generally a smallamount of endogenous Stat1/Stat3 heterodimer-oligonucleo-tide complex (SIF-B [24]; for an example, see Fig. 3B). In theabsence of ligand, only extracts of Stat3b-transfected cellsshowed a Stat-oligonucleotide complex (mostly SIF-A*, Stat3bhomodimer complex), as previously reported (24). (The iden-tities of the various complexes were verified by use of Stat3 orStat1 antisera and/or purified Stat proteins.) Of the ligandstested, EGF, transforming growth factor a, alpha interferon,and oncostatin M showed clear activation of Stat3, and in allinstances both Stat3a and Stat3b were activated (Fig. 1). Theseexperiments confirm that Stat3b, but not Stat3a, is partiallyactivated in the absence of added ligand and indicate thatStat3a and Stat3b are activated in COS-7 cells by the samesubset of ligands among those tested. Both isoforms formDNA-binding heterodimers with Stat1. We also examined var-ious ligands for stimulation of transcription from a Stat3-re-sponsive a2-macroglobulin-TK promoter (16). In each in-stance, the results paralleled DNA binding; i.e., only ligandsthat activated Stat3 for binding to the hSIE stimulated tran-scription (data not shown).

Evidence that constitutively active Stat3b is phosphorylatedon tyrosine 705. It has been shown with Stat1 that cytokine-

induced phosphorylation of a single tyrosine residue (Y701)located distal to the SH2 domain is necessary and sufficient fordimer formation and nuclear translocation (26). The Stat3isoforms have a tyrosine residue located at a position (Y705)similar to that of Stat1. The capacity of Stat3b for DNA bind-ing and transcriptional activation in the absence of addedgrowth factor or cytokine suggested that some of the b isoformis constitutively phosphorylated on this residue. To determinewhether constitutively active Stat3b and EGF-activated Stat3aand Stat3b are phosphorylated on tyrosine 705, we assessedthe tyrosine phosphorylation status of the Stat3 isoforms fromcells transfected with plasmids encoding Stat3a, Stat3b, ormutants in which tyrosine 705 had been changed to phenylal-anine (3aY705F, 3bY705F) (Fig. 2). Wild-type Stat3b wastyrosine phosphorylated prior to treatment with EGF, but mu-tant 3bY705F was not (Fig. 2A). The tyrosine phosphorylationlevels of both isoforms increased after EGF treatment. In laterexperiments, we found that constitutively expressed and EGF-activated Stat3 isoforms reacted with antiserum specific for aStat3-phosphorylated Y705 peptide (see Fig. 3B and 5). Un-expectedly, tyrosine phosphorylation of 3bY705F was also in-creased by EGF, as was tyrosine phosphorylation 3aY705F toa lesser extent. However, neither 3aY705F nor 3bY705F was

FIG. 2. Constitutive Y705 phosphorylation of Stat3b. (A) COS-7 cells were transfected with 25 mg per 10-cm dish of plasmid encoding Stat3a or Stat3b or theirY705F mutant forms, as described in Materials and Methods. At 40 h after transfection, including 24 h in serumless medium, the cells were stimulated (1) or notstimulated (2) with 100 ng of EGF per ml for 15 min, as indicated. Whole-cell extracts containing 1 mg of protein were incubated with Stat3a- or Stat3b-specificantibody, and the immunoprecipitated (IP) protein was electrophoresed and immunoblotted with an anti-phosphotyrosine (a-pTyr) antibody. The blots were thenreprobed with a monoclonal antibody (mAb) that recognizes an N-terminal epitope common to both Stat3 isoforms. (B) Effect of the Y705F mutation of Stat3a andStat3b on DNA binding. COS-7 cells were transfected with the plasmids shown (25 mg per 10-cm dish) as described above and where indicated (1) treated with humanrecombinant EGF (100ng/ml) for 15 min. Vec, vector. (C) Effect of the Y705F mutation of Stat3a and Stat3b on transcriptional activity. COS-7 cells in 5-cm disheswere transfected with 5 mg of the indicated Stat expression plasmid, 5 mg of reporter plasmid (a2-macroglobulin–TK CAT) (23), and 2 mg of pYN3214-lacZ as describedin Materials and Methods. At 40 h after transfection, including a 24-h incubation in serumless medium, the cells were treated with human recombinant EGF (100 ng/ml)and harvested for measurement of CAT and b-galactosidase activity 8 h thereafter. Unstimulated samples were harvested at the same time as the 8-h-stimulatedsamples. The values shown are the mean values, corrected for transfection efficiency, for duplicate dishes, and the variation from the mean is noted. The results shownare representative of two independent experiments.

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activated by EGF as assessed by hSIE binding of extracts oftransfected COS-7 cells or transcriptional activity (Fig. 2B andC). Similarly, the mutant Stat3b showed no constitutive DNA-binding or transcriptional activity. We conclude that activationof both Stat3a and Stat3b by EGF, like activation of othermembers of the Stat family, as well as constitutive activation ofStat3b, requires phosphorylation of a specific tyrosine residuedistal to the SH2 domain. A similar conclusion was reached bymeasuring the activation of purified Stat3 proteins by EGFreceptor kinase in vitro (22).

DNA-binding activities of phosphorylated Stat3 isoforms.Since Stat3 isoforms have the same DNA-binding domain, theywould be expected to have similar DNA-binding activities.However, the results shown in Fig. 2 suggested the possibilitythat cellular EGF-activated Stat3b was more active than acti-vated Stat3a for DNA binding, as was reported for the purifiedproteins (22). To assess any difference more quantitatively, wemeasured hSIE-binding activity, Stat3 protein (by using amonoclonal antibody that recognizes an epitope common toboth isoforms), and Y705 phosphorylation of Stat3 (by usingan antiserum specific for a Stat3 Y705 peptide) in the sameextracts of COS-7 cells transfected with increasing amounts ofStat3 expression plasmids. In addition, to determine whetherany differences were due to simple deletion of C-terminal

sequence from Stat3a, we used plasmids expressing two trun-cated forms of Stat3a (Fig. 3A) as well as wild-type Stat3a andStat3b. One truncated construct (D48) lacks most of the neg-atively charged C-terminal 48 amino acids of Stat3a (Fig. 3A),resulting in a molecule with the same number of amino acidresidues as Stat3b. The other (D55) ends at the amino acidresidue where Stat3a and Stat3b diverge in sequence.

In a comparison of Stat3a and Stat3b, Fig. 3B shows that ateach level of input plasmid, a greater fraction of Stat3b than ofStat3a, is phosphorylated, explaining in part the greater DNAbinding by Stat3b per unit of Stat protein. For example, com-paring the autoradiographic results with 10 mg of Stat3a and 10mg of Stat3b plasmids, one sees that relative to the amount ofStat3 protein, phosphorylated Stat3b exceeds phosphorylatedStat3a; i.e., more Stat3b molecules than Stat3a molecules arephosphorylated (Fig. 3B). Data obtained from densitometermeasurements indicate that the ratio of Y705-phosphorylatedStat3 to total Stat3 is about three- to fourfold greater forStat3b than for Stat3a. In addition, the DNA-binding activityof Y705-phosphorylated Stat3b is higher than that of Y705-phosphorylated Stat3a. This can be seen most clearly by com-paring the autoradiographic results when 25 mg of Stat3a plas-mid and 5 mg of Stat3b plasmid were used in the transfectionexperiments. Even though the level of phosphorylated Stat3a

FIG. 3. (A) C-terminal amino acids of Stat3 proteins. The unique 7 amino acids of Stat3b are in boldface type. Also depicted are the termini of the D48 and D55truncation mutants. Charged amino acids are indicated. The serine residue located in a consensus mitogen-activated protein kinase sequence of Stat3a is shadowed.(B) DNA-binding activities of Stat3 isoforms and C-terminal deletion mutants. COS-7 cells in 10-cm dishes were transfected with the indicated amount of Stat3a,Stat3b, D48, or D55 expression plasmid as described in Materials and Methods. Vector plasmid was included such that each dish was transfected with a total of 25 mgof DNA. At 40 h after transfection, including a 24-h incubation in serumless medium, the cells were treated with EGF (100 ng/ml) for 15 min before being harvestedand analyzed. The top panel shows EMSA analysis of SIF complexes formed with the hSIE by 10 mg of nuclear extract. The middle panel shows immunoblot analysisof 20 mg of nuclear extract with polyclonal antibody specific for Stat3 Y705-phosphorylated peptide. The bottom panel shows immunoblot analysis of total Stat3 proteinin 20 mg of nuclear extract with a monoclonal antibody directed against an N-terminal epitope common to the Stat3 proteins. To the right are extracts from cellstransfected with 25 mg of Stat3aY705F, Stat3bY705F, or vector plasmids. Note the endogenous Stat3a protein and the SIF-B complex formed by endogenous Stat1and Stat3a in the vector control.

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exceeds that of Stat3b, the amount of oligonucleotide boundby Stat3b is greater. The data obtained from PhosphorImagerand densitometer measurements indicate that the ratio ofDNA-binding activity to the Y705-phosphorylated form forStat3b is about 10 to 20 times that for Stat3a. This differenceis not likely to be due to interacting cellular proteins, sincesimilar results were obtained with purified proteins preparedfrom Sf9 cells expressing a Stat3 isoform and JAK kinase, byusing either the hSIE (22) or the Stat3 element of the a2-macroglobulin promoter (Fig. 4). As seen in Fig. 4, DNAcontaining the a2-macrogobulin Stat3-binding site shows amarked difference in DNase protection per nanogram of phos-phorylated Stat3a and Stat3b.

Figure 3B also shows the results with the truncated forms ofStat3a. The D48 construct behaved like Stat3b. It had a phos-phorylation level and DNA-binding activity per unit of Y705-phosphorylated form similar to those of Stat3b. Like Stat3b,D48 also showed partial activation in the absence of addedgrowth factor (see Fig. 5 and 8). The D55 construct showedproperties intermediate between those of Stat3a and Stat3b.

We conclude that the functional differences between Stat3aand Stat3b described above are attributable primarily to thepresence or absence of the C-terminal segment in Stat3a.

The results shown in Fig. 3B are also relevant to the effect ofthe intracellular concentrations of different Stat proteins onthe type of dimers formed in response to cytokines or growthfactors. Since Stat3 and endogenous Stat1 were both activatedby EGF, Stat1-Stat3 heterodimer oligonucleotide complexeswere present and the relative amounts of homodimer andheterodimer complexes were greatly affected by the level ofStat3. Since different Stat dimers are likely to have specifictranscriptional properties, the level of each Stat protein acti-

FIG. 4. Comparison of the DNA-binding activities of purified Stat3a andStat3b by DNase I footprinting. Stat proteins were purified from Sf9 cells ex-pressing both an oligohistidine-tagged Stat isoform and JAK1 and JAK2, asdescribed previously (22). Each isoform was Y705-phosphorylated to approxi-mately the same extent, as judged by the results of immunoblotting the proteinswith anti-phosphotyrosine and anti-Stat3 monoclonal antibodies. The DNA usedcontains the a2-macroglobulin Stat3 (TTCTGGGAA)- and Jun (TGACTCT)-binding sites. The experiment was performed as described in Materials andMethods with the indicated amounts of Stat protein. Note the protection of theStat3 site.

FIG. 5. Stability of the active form of Stat3 proteins in transfected COS cells.COS-7 cells were transfected with Stat3a, Stat3b, D48, or D55 expression plas-mids (12.5 mg per 5-cm dish) as described in Materials and Methods. At 40 hafter transfection, including a 24-h incubation in serumless medium, the cellswere treated with 100 ng of EGF per ml for 20 min, at which time the EGFreceptor kinase inhibitor PD157655 (0.2 mM) was added, and the cells wereharvested at the times indicated. The left-hand lane (U) was from cells that werenot treated with EGF, and the right-hand lane (C) was from cells that weretreated with EGF for 80 min in the absence of inhibitor. The top panel in eachset shows EMSA analysis of hSIE SIF complexes formed with 2.5 mg of nuclearextract. The middle panel shows immunoblot analysis with antibody specific forY705-phosphorylated Stat3 protein in 10 mg of nuclear extract. The bottom panelshows immunoblot analysis with anti-Stat3 monoclonal antibody in 10 mg ofnuclear extract.

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vatable by a given cytokine or mixture of cytokines would be animportant determinant of the cellular responses to ligands.

Stability of EGF-activated Stat proteins in transfected cells.The higher fraction of phosphorylated Stat3b than Stat3afound in EGF-stimulated cells suggested that activated Stat3bmay be more stable than activated Stat3a. To assess the sta-bility of activated Stat proteins, COS-7 cells were transfectedwith Stat3a, Stat3b, D48, or D55 expression plasmids; incu-bated in serum-containing medium for 16 h; and then incu-bated in serum-free medium for 24 h. The cells were thentreated with EGF for 20 min, and PD157655, an inhibitor ofEGF receptor kinase, was added to prevent or greatly reducecontinuing Stat activation. (In experiments not shown,

PD157655 was found to inhibit the activation of Stat3a andStat3b in transfected COS cells in response to EGF but not inresponse to oncostatin M. However, the inhibitor did not affectthe level of constitutively activated Stat3b.) Nuclear extractswere prepared at various times and analyzed for binding to thehSIE oligonucleotide, the level of Y705-phosphorylated Stat3,and the level of total Stat protein, as described in the legend toFig. 5. The data were then quantitated, correcting for consti-tutive phosphorylation and oligonucleotide-binding activity,and normalized to the total Stat3 protein in each sample tocorrect for variation in transfection efficiency and plasmid ex-pression. We thus assumed that many hours after transfectionthere would be little change in the level of constitutively acti-vated Stat over the course of the experiment. Comparison ofthe decay of DNA-binding activity and Y705 phosphorylationrelative to total Stat3 protein revealed considerable differencein stability between the activated forms of Stat3a and Stat3b(Fig. 6). In all cases, there was good agreement between therate of loss of Y705 phosphorylation and DNA binding. Thehalf-life of active Stat3a was approximately 10 min comparedto greater than 1 h for Stat3b. The half-life of active D48 wassimilar to that of Stat3b, and the half-life of active D55 was alsosimilar to or somewhat shorter than that of Stat3b, but the datapoints were more scattered than with the other Stat proteins.We conclude that the active forms of Stat3b, D48, and D55 aremore stable than the active form of Stat3a. This could be dueto differences in rates of dephosphorylation of Y705 or turn-over of the proteins. The rapidity of the decay of active Stat3aleads us to favor the first possibility.

Transcriptional activities of Stat3 isoforms. We next com-pared the transcriptional activities of Stat3a, Stat3b, D48, andD55. For this purpose, COS-7 cells transfected with a Statexpression plasmid, the a2-macroglobulin–TK CAT plasmid,and a b-galactosidase plasmid to normalize for transfectionefficiency were assessed for CAT activity in the absence ofgrowth factor 6.5 or 8 h after stimulation with EGF (Fig. 7).Both Stat3b and D48 showed constitutive activity, whereas theothers did not, and EGF stimulated transcription by each Stat

FIG. 6. Changes in EGF-inducible SIE-binding activity and phosphorylatedY705 as a function of time after inhibition of EGF receptor kinase. For each timepoint and the unstimulated sample (U) shown in Fig. 5, DNA binding wasquantitated with a PhosphorImager (SIF-A for Stat3a and SIF-A* for Stat3b,D48, and D55) and the level of Y705-phosphorylated and total Stat3 species wereassessed by densitometric measurement of the respective immunoblots. SIEbinding and Stat3 Y705-phosphorylated protein were each then normalized tototal Stat3 species (Stat3a, Stat3b, D48, or D55) in the sample, and similarlynormalized constitutive binding activity or constitutive Y705 phosphorylationwas subtracted to give EGF-inducible SIE binding or StatY705 phosphorylation,the ordinates shown in the graphs. (A) EGF-inducible SIE binding normalized tototal Stat3 species and expressed as a percentage of the zero time value. (B)EGF-inducible Stat3 Y705 phosphorylation normalized to total Stat3 species andexpressed as a percentage of the zero time value.

FIG. 7. Transcriptional activation by Stat3a, Stat3b, D48, and D55 in trans-fected COS cells. COS-7 cells in 5-cm dishes were transfected with 5 mg of theindicated Stat expression plasmid (or vector plasmid), 5 mg of a2-macroglobu-lin–TK CAT plasmid (23), and 2 mg of pYN3214-lacZ, as described in Materialsand Methods. At 40 h after transfection, including a 24-h incubation in serumlessmedium, the cells were treated with EGF (100 ng/ml) for 6.5 h and analyzed forCAT and b-galactosidase activities. 2EGF samples were harvested at the sametime as the stimulated samples. The values shown are the mean values for twodishes, and the variation from the mean is indicated. The results shown arerepresentative of three independent experiments.

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except D55, which was inactive. We then determined the rela-tive transcriptional activities of the active forms of Stat3a,Stat3b, and D48 by measuring the CAT activity and binding ofnuclear extract to the hSIE oligonucleotide (Fig. 8). As can beseen by the accumulation of CAT at the later time points andthe DNA-binding activities at each time point, relative to DNAbinding the transcriptional activity of Stat3a exceeded that ofStat3b and D48. Whereas the accumulated levels of CAT weresimilar, DNA binding by Stat3b and D48 was several timesgreater than that by Stat3a. We conclude that the difference intranscriptional activities of the Stat3 isoforms is due to thepresence or absence of the C-terminal acidic activation domainfound in Stat3a. The difference between Stat3b (and D48) andD55 suggests that Stat3b contains a weak activation domainconferred by its specific C-terminal sequence.

We previously reported that Stat3b (but not Stat3a) andc-Jun cooperatively activate an a2-macroglobulin–TK pro-moter with binding sites for Stat3 and Jun (24). Since these

experiments were done with cells in the absence of addedcytokines or growth factors and having shown above thatStat3a requires such ligands to activate its transcriptional ac-tivity, we wanted to establish whether EGF-activated Stat3acould cooperatively activate transcription with c-Jun. In thepresence of EGF, cells that expressed both Stat3a and c-Junactivated the reporter gene to levels that were comparable tothe additive activation by Stat3a or c-Jun alone (Fig. 9A),whereas coexpression of Stat3b and c-Jun in similarly treatedcells resulted in synergistic activation of the reporter gene (Fig.9B).

DISCUSSION

The findings in our comparison of the activities of Stat3aand Stat3b can be summarized as follows: (i) both forms wereactivated for DNA binding and transcription by the same set ofcytokines and growth factors among those tested; (ii) when

FIG. 8. Transcriptional activation relative to DNA binding by Stat3a, Stat3b, and D48. COS-7 cells in 5-cm dishes were transfected in triplicate with 2 mg of theindicated Stat expression plasmid, 5 mg of a2-macroglobulin–TK CAT, and 2 mg pYN3214-lacZ, as described in Materials and Methods. At 40 h after transfection,including a 24-h incubation in serumless medium, the cells were treated with EGF (100 ng/ml). At the indicated times, one dish was harvested for nuclear extract andanalyzed for SIE binding and the other two were processed for CAT and b-galactosidase activities. The values shown are the mean values for two dishes, and thevariation from the mean is indicated. The results shown are representative of two independent experiments.

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activated, both formed homodimers and heterodimers withStat1; (iii) activation of each form, including constitutive acti-vation of Stat3b, correlated with phosphorylation of tyrosine705; (iv) the activated form of Stat3b had considerably greaterspecific DNA-binding activity than activated Stat3a; (v) thestability of the activated form of Stat3b in transfected cells wasgreater than that of the activated form of Stat3a; and (vi)relative to DNA-binding activity, Stat3a was transcriptionallymore active than Stat3b.

Since Stat3a and Stat3b differ only at their C termini, it isnot surprising that they were activated by the same set ofcytokines and growth factors. The ligand specificity of Statproteins presumably depends on their SH2 domains (12, 28)and on the context of the phosphorylatable tyrosine residue,both of which are identical in the Stat3 isoforms. In any case,our results indicate that the C-terminal 55 amino acids ofStat3a are not involved in this specificity.

The formation of activated Stat3a and Statb homodimersand heterodimers with Stat1 is of interest in relation to thediversity of gene activation and cellular responses followingcytokine and growth factor stimulation of cells. Many ligandslead to activation of multiple Stat families (14, 15, 25) and theformation of various Stat homodimers and heterodimers, asillustrated by the activation of Stat3 and Stat1 by EGF andoncostatin M. If individual dimers have unique transcriptionalactivities in a given cellular context, the relative amounts ofeach dimeric form would influence the specificity of gene ac-tivation. The relative amounts of various Stat dimers woulddepend on the concentrations of individual Stat proteins in thecell (as illustrated in Fig. 3B), the degree of activation ofdifferent Stat proteins by ligands, and the concentrations ofdiverse ligands acting on a cell. Hence, the network of differentfamilies and isoforms of Stat proteins present a rich substratefor mediating specific responses to mixtures of ligands that acell encounters in vivo, as previously noted (6).

A striking difference between Stat3a and Stat3b reportedpreviously (24) is the constitutive activity of Stat3b. This con-stitutive activity of Stat3b is not unique to transfected COS-7

cells, since we found similar results with transfected F9 terato-carcinoma, U3A (18), NIH 3T3, and HepG2 cell lines and Sf9cells infected with Stat3-recombinant baculovirus. Our exper-iments show that constitutive activity was correlated with thephosphorylation of Y705, since a Y705F mutant of Stat3b wasinactive and an antiserum specific for a Stat3 Y705 peptidereacted with the constitutively active form. Although we can-not exclude the possibility that “constitutive” activity is due tothe presence of an undefined cytokine, the experiments werecarried out with cells washed and held for 24 h in protein-freemedium. Another possibility is that activated Stat3b, beingmore stable than Stat3a (as indicated by the results in Fig. 5),persisted at a low level long after the cells were exposed toserum. In any case, the presence of constitutive or residualactivity of Stat3b in a cell could modify the response of the cellto cytokines. An unexpected finding of the phosphorylationexperiments was the detection of tyrosine phosphorylation incells transfected with Stat3 Y705F mutants, suggesting thatwild-type Stat3b and, to a lesser extent Stat3a, could be phos-phorylated on a tyrosine(s) other than Y705 in response toEGF, although it should be noted that the EGF concentrationused in these experiments was high. A similar result was re-ported for Y705FStat3 phosphorylation by EGF receptor ki-nase in vitro (22).

Stat3b, unlike the C-terminally truncated forms of other Statproteins, has 7 amino acid residues at its C terminus that arenot present in Stat3a. However, the experiments with trun-cated forms of Stat3a indicate that all of the functional differ-ences we detected between Stat3a and Stat3b are due to thepresence or absence of the C-terminal acidic tail of Stat3a andnot to the C-terminal amino acids specific to Stat3b, since themutant D48, missing the C-terminal 48 amino acid residues ofStat3a, was indistinguishable from Stat3b in stability, DNAbinding, and transcriptional activation. Since the D55 mutantwas not transcriptionally active, we infer that the specific tail ofStat3b confers weak transcriptional activity equivalent to thatof the tail of D48. Although the transcriptional differencesbetween Stat3 isoforms are consistent with the known effects of

FIG. 9. Cooperative transcriptional activity of c-Jun and Stat3b versus Stat3a in EGF-stimulated transfected COS cells. COS-7 cells in 5-cm dishes were transfectedwith 5 mg of Stat3 expression plasmid, 0.5 mg of c-Jun expression plasmid (where indicated), 5 mg of a2-macroglobulin–TK CAT, and 2 mg of pYN3214-lacZ, asdescribed in Materials and Methods. At 40 h posttransfection, including 24 h in serumless DMEM, the cells were treated with EGF (100 ng/ml) and harvested for CATand b-galactosidase activities at the times shown. The values shown are the mean values for two dishes, and the variation from the mean is indicated. The results shownare representative of two independent experiments. (A) Stat3a; (B) Stat3b.

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acidic activation domains, it is not clear how the acidic Cterminus influences the stability and specific DNA-binding ac-tivities of Y705-phosphorylated Stat3a in transfected cells.Since Y705-phosphorylated Stat3b (whether activation oc-curred in JAK-expressing Sf9 cells or by in vitro activation byEGF receptor kinase [Fig. 4] [22]) showed approximately 20-to 50-fold-greater activity for binding to a Stat3 DNA site thandid Stat3a, the difference is not likely to be due to the presenceor absence of other proteins in the nuclear extract-oligonucle-otide complex. One possible explanation for the DNA-bindingdifferences is that the Stat3b dimer (the DNA-binding form) ismore stable than the Stat3a dimer, i.e., that the negativelycharged C terminus of Stat3a destabilizes the active dimericform. Another possibility is that the acidic tail of Stat3a re-duces its affinity for DNA. On the assumption that dephos-phorylation of activated Stat occurs primarily as monomer, adifference in dimer stability is also one plausible explanationfor the difference observed in the stabilities of the active formsin COS cells. (Because of the short half-life of Y705-phosphor-ylated Stat3a—approximately 10 min—it seems more likelythat the decay measured is related to dephosphorylation ratherthan protein turnover, but this remains unresolved.)

With regard to the transcriptional activity of Stat3b, it hasbeen reported not to activate a response element from theintercellular cell adhesion molecule (ICAM) promoter intransfected COS1 cells and to inhibit activation of this pro-moter by Stat3a (4). In our hands, Stat3b was active withpromoter elements derived from the c-Fos gene and showedcooperativity with c-Jun comparable to that observed with thea2-macroglobulin promoter elements. It is clear that the tran-scriptional activity of Stat3b is promoter dependent and pos-sibly dependent on cooperative interactions with other tran-scription factors.

All of the comparisons of Stat3a and Stat3b reported herewere carried out with cells that overexpress a Stat3 transgeneor with purified Stat proteins. The physiological significance ofthe functional differences we found remains to be determined.We do know that Stat3b mRNA is a minor component of Stat3mRNA in mouse liver (24) and in a number of other tissuesthat we have examined (brain, lung, heart, ovary, and spleen).Furthermore, the presence of the Stat3b isoform has beenobserved in bone marrow and in several acute myeloid leuke-mia cell lines, and in some tumor-derived cells there is anincrease in the level of activated Stat3b (reference 5 and our un-published observations). Similarly, constitutively active Stat3-related proteins are also seen in peripheral blood cells fromacute leukemia patients (11). Increased levels of constitutivelyactive Stat3b could play a role in neoplasia similar to that ofconstitutively active forms of growth factor receptors (8, 9). Inthis regard, we note that DNA-binding Stat proteins present incells transformed by human T-cell leukemia virus type 1 (19) orby the Src oncoprotein are immunologically related to Stat3(37).

ACKNOWLEDGMENTS

We thank D. Fry (Parke-Davis, Ann Arbor, Mich.) for generouslyproviding the EGF kinase inhibitor PD157655, Y. Nakabeppu (KyushuUniversity, Fukuoka, Japan) for expression plasmids, and WenlanWang for thoughtful discussions.

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