of June 13, 2018.This information is current as

Activation Domain of Stat5trans- Genes Through the bcl-x and bcl-2,myc,Proliferation and Induction of the c-

The IL-2 Receptor Promotes Lymphocyte

Brad H. NelsonJames D. Lord, Bryan C. McIntosh, Philip D. Greenberg and

http://www.jimmunol.org/content/164/5/2533doi: 10.4049/jimmunol.164.5.2533

2000; 164:2533-2541; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/164/5/2533.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The IL-2 Receptor Promotes Lymphocyte Proliferation andInduction of the c-myc, bcl-2,and bcl-x Genes Through thetrans-Activation Domain of Stat51

James D. Lord,*†‡ Bryan C. McIntosh,* Philip D. Greenberg,†‡§ and Brad H. Nelson2*‡

Studies assessing the role of Stat5 in the IL-2 proliferative signal have produced contradictory, and thus inconclusive, results. Onefactor confounding many of these studies is the ability of IL-2R to deliver redundant mitogenic signals from different cytoplasmictyrosines on the IL-2R b-chain (IL-2Rb). Therefore, to assess the role of Stat5 in mitogenic signaling independent of any redun-dant signals, all cytoplasmic tyrosines were deleted from IL-2Rb except for Tyr510, the most potent Stat5-activating site. Thisdeletion mutant retained the ability to induce Stat5 activation and proliferation in the T cell line CTLL-2 and the pro-B cell lineBA/F3. A set of point mutations at or near Tyr510 that variably compromised Stat5 activation also compromised the proliferativesignal and revealed a quantitative correlation between the magnitude of Stat5 activation and proliferation. Proliferative signalingby a receptor mutant with a weak Stat5 activating site could be rescued by overexpression of wt Stat5a or b. Additionally, theability of this receptor mutant to induce c-myc, bcl-x, and bcl-2 was enhanced by overexpression of wt Stat5. By contrast,overexpression of a version of Stat5a lacking the C-terminaltrans-activation domain inhibited the induction of these genes and cellproliferation. Thus, Stat5 is a critical component of the proliferative signal from Tyr510 of the IL-2R and regulates expression ofboth mitogenic and survival genes through itstrans-activation domain. The Journal of Immunology,2000, 164: 2533–2541.

T he T cell mitogen IL-2 binds to a heterotrimeric receptorcomplex consisting of IL-2Ra, IL-2Rb, andgc chains (1),with the consequent heterodimerization of IL-2Rb andgc

inducing the proliferative signal (2, 3).gc contributes to prolifer-ative signaling by recruiting the tyrosine kinase Jak3(4–8), theactivation of which also requires a membrane-proximal S region ofIL-2Rb (9, 10). Additionally, at least one of three cytoplasmictyrosine phosphorylation sites (Tyr338, Tyr393, or Tyr510) distal tothe S region on IL-2Rb must be present for proliferation, suggest-ing that obligate mitogenic signaling molecules interact with thesephosphotyrosines (11, 12). Indeed, the adapter molecule Shc in-teracts with Tyr338 (11, 13) and can deliver a proliferative signal(14). The other two tyrosines, Tyr510 and Tyr393, provide dockingsites for the transcription factor Stat5 (11), which has led to thehypothesis that Stat5 mediates a proliferative signal parallel to theone involving Shc. However, Shc and Stat5 have very differentbiochemical properties, which raises the question of whether theycould indeed generate redundant proliferative signals from IL-2Rb. In addition, the fact that a single phosphotyrosine on a re-ceptor chain can interact with multiple signaling molecules, asexemplified by studies of the platelet-derived growth factor recep-tor (15), raises the possibility that a factor other than Stat5 medi-ates the proliferative signal from Tyr393 and/or Tyr510.

Stat5 refers to either of two highly homologous members of thelarger Stat family of signal-transducing activators of transcription,Stat5a and Stat5b (16–18). Stat activation conventionally com-mences with selective binding of a Stat SH2 domain to a phos-phorylated tyrosine motif on a ligated receptor (19–22), approxi-mating the Stat molecule with a receptor-associated, activatedJanus kinase which phosphorylates a key tyrosine residue on theStat molecule (23, 24). These Stat phosphotyrosines preferentiallybind to Stat SH2 domains (21), causing Stat molecules to dimerize,release from receptors, enter the nucleus, and bind palindromicDNA sequences (22) in certain promoters (25–27), inducing genetranscription via a C-terminaltrans-activation domain (TAD)3

(28–31). Some members of the Stat family also demonstrate lessconventional activities. Stat3, for example, serves as an adaptermolecule between PI3 kinase and type I IFN receptors (32). Fur-thermore, Stat1 and, in cooperation with the glucocorticoid recep-tor, Stat5 mediate the expression of certain genes through a TAD-independent mechanism (33–35). Thus, not all signaling functionsof Stat molecules involve the conventionaltrans-activationdomain.

A critical role for Stat5 in IL-2-mediated mitogenesis has beensuggested by the failure of TCR-stimulated T cells from mice lack-ing both the Stat5a and Stat5b genes to proliferate in response toIL-2 (36). However, the IL-2R can mediate proliferation in theabsence of detectable Stat5 activation through alternative mito-genic signaling molecules, such as Shc (11, 14). Indeed, IL-2Rbmutants that fail to activate Stat5 mediate proliferation in culturedcell lines (11, 37, 38), and primary T cells (39). Thus, the inabilityof Stat5-null T cells to proliferate may reflect a recently describedessential role for Stat5 in TCR signaling (40) rather than a requisiterole in IL-2 signaling.

This report assesses the role of Stat5 in signals derived from theIL-2R in cell lines that do not require TCR stimulation for prolif-eration. A receptor mutagenesis and rescue strategy was used to

*Virginia Mason Research Center, Seattle, WA 98101;†Fred Hutchinson CancerResearch Center, Seattle, WA 98104; and Departments of‡Immunology and§Med-icine, University of Washington, Seattle, WA 98195

Received for publication September 3, 1999. Accepted for publication December10, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grant GM57931 from the National Institutes of Health.J.D.L. was supported by a National Defense Science and Engineering Graduate fel-lowship from the U.S. Department of Defense and by a grant from the Poncin Schol-arship Fund.2 Address correspondence and reprint requests to Dr. Brad Nelson, Virginia MasonResearch Center, 1201 9th Avenue, Seattle, WA 98101-2795. 3 Abbreviations used in this paper: TAD,trans-activation domain; wt, wild type.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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study signaling by Stat5 in the absence of any redundant signalsfrom Shc or other molecules potentially associating with IL-2Rb.We demonstrate that Tyr510 of IL-2Rb mediates proliferationthrough the activation of Stat5 and that either the Stat5a or theStat5b isoform can transduce the mitogenic signal. Furthermore,proliferative signaling by Stat5 is critically dependent on its TAD,indicating that Stat5a mediates proliferation through its conven-tional role as a transcription factor. Indeed, IL-2-induced transcrip-tion of the proliferative and survival genes c-myc,bcl-2,andbcl-xis mediated by Stat5a through the TAD.

Materials and MethodsPlasmid construction

Expression vectors encoding the chimericag- and bb-chains (formerlydenoted GMa/2g and GMb/2b) under the control of the humanb-actinpromoter have been described previously (2, 9, 14, 41). Mutants ofbbwere generated by annealing sense and antisense oligonucleotides encod-ing novel C-terminal sequences forbb and/or premature stop codons, andcloning overhanging ends of these annealed primers between a uniqueAflIIsite in the cytoplasmic domain ofbb and a uniqueXbaI site immediately39 to the stop codon ofbb. For analyses in BA/FG cells, the full length,nonchimeric human IL-2Rb cDNA was cloned into the expression vectorused above, and the C terminus of this chain, between theAflII site and aunique 39ScaI site, was then replaced with the respective mutated se-quences. TheD713 mutant of Stat5a was generated by PCR with an anti-sense oligonucleotide encoding residues 708 to 712 of Stat5a followed bythe flag epitope, a stop codon, and anEcoRV site. This PCR product wasthen cut withXhoI andEcoRV enzymes and ligated between these samesites in a vector containing murine Stat5a, described previously (29).D713,as well as C-terminally flag epitope-tagged wild-type (wt) murine Stat5aand Stat5b, were next excised withEcoRI andHindIII and cloned betweenSalI andHindIII sites in theb-actin promoter-driven plasmid used abovefor receptor expression. All mutated Stat5a and IL-2Rb regions were se-quenced with the ABI Prism dye terminator cycle sequencing kit (Perkin-Elmer, Norwalk, CT).

Cell culture and transfection

The murine T cell line CTLL-2 was obtained from the American TypeCulture Collection (Manassas, VA) and maintained as described previously(14). BA/F3 pro-B cells were obtained from Immunex (Seattle, WA) andmaintained in RPMI supplemented with 10% FCS, 2 mML-glutamine,50U/ml penicillin, and 50 mg/ml streptomycin, as well as 10% WEHI3-conditioned medium as a source of murine IL-3. To grow BA/F3 cells inthe absence of Stat5 activation, the cells were transfected with a chimericG-CSFR/gp130 receptor chain described previously (42) and maintainedwith 100 ng/ml recombinant human G-CSF (Amgen, Thousand Oaks, CA)instead of IL-3. The resultant cell line is referred to hereafter as BA/FG.Linearized plasmids were introduced into cells by electroporation, andtransfectants were selected for resistance to G418 (Life Technologies,Gaithersburg, MD) in 96-well plates at limiting dilution to isolate inde-pendent subclones. Receptor expression was assessed by flow cytometrywith Abs to human GM-CSFRa or bc (Santa Cruz Biotechnology, SantaCruz, CA), or to IL-2Rb (PharMingen, San Diego, CA). Stat5 expressionwas assessed by Western blot with an Ab to Stat5 (Transduction Labs,Lexington, KY), or by flow cytometry of cells stained intracellularly withan Ab to the flag epitope (Sigma, St. Louis, MO). Subclones with compa-rable receptor and/or Stat5 expression were chosen for further analyses.

Proliferative assays

Thymidine incorporation assays were conducted in triplicate wells with 104

cells/well exposed to the indicated doses of GM-CSF, IL-2, G-CSF, or IL-3for 24 h, with [3H]thymidine (2.5mCi/well) present during the last 4 h.Cells were harvested onto glass fiber filters, and DNA synthesis was quan-tified by liquid scintillation counting. The data presented in Figs. 1–4 used100 ng/ml GM-CSF or 3000 U/ml IL-2, which were found to elicit a max-imal response from all functional receptor mutants in CTLL-2 or BA/F3cells, respectively.

Immunoprecipitaion and immunoblotting

Jak1 and Jak3 were immunoprecipitated and subjected to kinase assays asdescribed previously (9). Whole cell lysates were generated by boiling cellsin 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 50 mM DTT, and0.1% bromphenol blue. Lysates or immunoprecipitates were electropho-

resed on acrylamide gels and transferred to nitrocellulose. Nitrocelluloseblots were blocked with 0.1 M Tris base (pH 7.5), 0.9% sodium chloride,0.05% Tween 20 (TTBS) containing 1% BSA (for antiphosphotyrosineprobes) or 5% powdered skim milk (Carnation, Glendale, CA), and probedwith rabbit antisera recognizing Jak1 (Santa Cruz Biotechnology) or Jak3(Upstate Biotechnology, Lake Placid, NY) or murine Abs recognizingphosphotyrosine (4G10; Upstate Biotechnology) or Stat5 (TransductionLaboratories, Lexington, KY). Blots were then washed with TTBS, probedwith peroxidase-conjugated goat anti-rabbit or anti-mouse Abs (Life Tech-nologies), and washed again with TTBS. Bound Abs were detected byenhanced chemiluminescence (DuPont NEN, Boston, MA). Blots werestripped between probings with a 30-min, 50°C incubation in 62.6 mMTris-HCl (pH 6.7), 0.1 Mb-mercaptoethanol, and 2% SDS.

Electrophoretic mobility shift assays

Cells stimulated as indicated were washed once with buffer H (20 mMHEPES (pH 7.9), 1 mM EDTA, 0.1 mM EGTA, 2 mM magnesium chlo-ride, 1 mM sodiumo-vanadate, 20 mM sodium fluoride, 1 mM DTT, 0.1mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 1 mg/ml leupeptin)and lysed in buffer H plus 0.2% Nonidet P-40 at 0°C. Nuclei were pelletedby centrifugation and extracted with buffer K (buffer H plus 0.42 M sodiumchloride and 20% v/v glycerol). To generate a probe for Stat activity,incompletely overlapping oligonucleotides corresponding to the sense andantisense strands of the Stat-responsive DNA element from the FcgR1promoter were annealed, radiolabeled with [a-32P]dCTP by an end-fillingT4 polymerase reaction, and purified with a MicroSpin G-25 column (Phar-macia, Piscataway, NJ). The probe was added to nuclear extracts in 50 mMpotassium chloride, 10 mM HEPES (pH 7.9), 10% glycerol, 1 mM DTT,and 87.5 mg/ml dITP/dCTP at room temperature for 30 min in the presenceor absence of either an Ab recognizing the flag epitope (Sigma) or a com-bination of Abs recognizing Stat5a and Stat5b (Santa Cruz Biotechnology).Reaction mixtures were electrophoresed on a nonreducing 0.253 Tris-buffered EDTA-acrylamide gel which was then dried and subjected toautoradiography.

Northern blots

Cells stimulated as indicated were pelleted by centrifugation and flash-frozen in a dry ice-ethanol bath. RNA was harvested from thawed pelletswith the RNA Stat 60 kit (Tel Test, Friendswood, TX); denatured for 10min at 65°C in 20 mM MOPS, 5 mM sodium acetate, 0.5 mM EDTA, 2.4M formaldehyde, and 50% formamide; and run on a 1.2% agarose gel(containing 20 mM MOPS, 5 mM sodium acetate, 0.5 mM EDTA, and 1.1M formaldehyde) in 20 mM MOPS, 5 mM sodium acetate, and 0.5 mMEDTA at pH 7.0. RNA was passively transferred to Zetabind membranes(Cuno, Meriden, CT) with 103SSC (1.5 M sodium chloride, 0.15 Msodium citrate, pH 7.0), and UV cross-linked. Blots were prehybridized at43°C in hybridization buffer (1 M sodium phosphate (pH 7.1), 2 mMEDTA, 2% BSA, 10% SDS, 50% formamide, and 0.16 mg/ml yeast tRNAor herring sperm DNA). To generate nucleic acid probes, a 0.4-kbPstIfragment of murine c-myc, a 0.9-kbPstI fragment of murinebcl-2, a 1-kbEcoRI fragment of murinebcl-x, and a 1.2-kbPstI fragment of murineGAPDH cDNA were radiolabeled with [a-32P]dCTP using a random-primed labeling kit (Boehringer Mannheim, Indianapolis, IN) and purifiedwith Centri-Sep spin columns (Princeton Separations, Adelphia, NJ).Probes were boiled for 5 min and added to blots in hybridization buffer.After overnight incubation at 43°C, blots were washed 2–3 times with 23SSC, 0.1% SDS; once with 0.23SSC, 0.1% SDS at room temperature; and0–2 times with 0.23SSC, 0.1% SDS at 55°C before autoradiography.Blots were stripped between probings with a 2-min immersion in boilingwater.

ResultsProliferative signaling by Tyr510 of IL-2Rb correlatesquantitatively with Stat5 activation

Structure/function analyses of the cytoplasmic domain of IL-2Rbwere performed in the IL-2-dependent murine T cell line CTLL-2.To avoid activation of the endogenous IL-2R, we introduced apreviously described chimeric GM-CSF/IL-2 receptor consistingof two chains,ag andbb, containing the extracellular domains ofthe human GM-CSF receptora- andb-chains fused, respectively,to the transmembrane and intracellular regions ofgc and IL-2Rb.

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When coexpressed,ag andbb deliver in response to human GM-CSF a signal that is biochemically and physiologically indistin-guishable from that induced in the same cell by the wt IL-2 re-ceptor (2, 9, 14, 41). A CTLL-2 clone demonstrating stable highexpression ofag was generated and transfected with wt or mutatedderivatives ofbb (Figs. 1Aand 2B). Subclones of transfectantswere analyzed for receptor expression by flow cytometry (data notshown), and those expressing bothag andbb-chains at compara-

ble levels were chosen for further study. The wt version ofbbdelivered a proliferative signal in response to GM-CSF similar tothat mediated by the endogenous IL-2R. By contrast, a version ofbb from which all cytoplasmic tyrosines had been removed byintroduction of a premature stop codon at codon 325 (relative tothe human IL-2Rb sequence) (bbD325 (Fig. 1A)) failed to mediateproliferation (Fig. 1B), in accordance with prior reports (11, 12,14). However, bothbbwt and bbD325 induced phosphorylationand activation of Jak1 and Jak3 (Fig. 1C), confirming that Januskinase activation, whereas critical for Stat activation, is not suffi-cient for proliferation.

To investigate the role of Stat5 in proliferation, 13 amino acidsfrom the C terminus of IL-2Rb that encompass Tyr510, the mostefficient Stat5-activating tyrosine on IL-2Rb (11), were attached tothe C terminus of thebbD325 receptor (bbD325 1 Tyr510 (Fig.1A)). Like bbwt and the endogenous IL-2R, this receptor inducedstrong Stat5 activation (Fig. 1D), as well as cell proliferation (Fig.1B). Thymidine incorporation was 30–40% less than that inducedby bbwt or the endogenous IL-2R, likely due to the absence of aconcurrent proliferative signal mediated through Shc (11, 14).Nonetheless,bbD325 1 Tyr510 supported the long term prolifer-ation and survival of CTLL-2 cells in culture (Fig. 3,D andE; datanot shown). However, if Tyr510 was mutated to phenylalanine inthis receptor context (bbD325 1 Phe510 (Fig. 1A)), neither Stat5

FIGURE 2. Activation of Stat5 by Tyr510 of IL-2Rb shows a quantita-tive correlation with proliferation.A, Schematic comparison of the residuessurrounding Stat5-activating tyrosines (Y) (underlined) in the cytoplasmicdomains of various receptors. Conserved hydrophobic residues are in bold.B, Schematic depicting point mutations (bold face) introduced C-terminalto Tyr510 (underlined) inbbD3251 Tyr510. C, Stat5 activation in CTLL-2cells mediated by mutant chimeric receptors, assessed as per Fig. 1D.D,Thymidine incorporation in CTLL-2 cells mediated by the indicated chi-meric receptors, assessed and depicted as per Fig. 1B.

FIGURE 1. An IL-2 proliferative signal is mediated through a con-served 13-residue motif flanking Tyr510 (Y510).A, Schematic depicting wtand mutant versions of chimeric GM-CSF/IL-2Rb-chain (bb). TM, trans-membrane.B, Thymidine incorporation mediated by the indicated chimericreceptors.F, Individual CTLL-2 clones stimulated with 100 ng/ml humanGM-CSF; E, the same clones cultured without cytokine. Data for eachclone are expressed as a percentage of the thymidine incorporated afterstimulation with 100 U/ml human IL-2.C, bbD325 retains the ability toactivate Janus kinases. Representative CTLL-2 clones bearing the indicatedchimeric receptors were deprived of cytokine for 4 h and then stimulatedfor 10 min with 100 ng/ml human GM-CSF (G), 100 U/ml human IL-2 (2),or no cytokine (0). The indicated Janus kinases were then immunoprecipi-tated (IP) and either subjected to an in vitro autokinase assay or a Westernblot for phosphorylated tyrosines (pTyr). The phosphotyrosine Westernblots were then stripped and reprobed for Jak1 or Jak3, as indicated, toassess loading.D, bbD325 1 Tyr510 activates Stat5 through Tyr510. Rep-resentative CTLL-2 clones bearing the indicated chimeric receptors weredeprived of cytokine for 4 h and then stimulated for 10 min with 100 ng/mlhuman GM-CSF (G), 100 U/ml human IL-2 (2), or no cytokine (0). Nu-clear extracts were harvested and subjected to an electrophoretic mobilityshift assay using a Stat5-binding probe derived from the FcgR1 promoter.In lanes marked Ab, extracts from GM-CSF-stimulated cells were treatedwith anti-Stat5 Abs to specifically supershift Stat5 complexes. F510,Phe510.

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activation nor proliferation was induced (Fig. 1,B and D), indi-cating both events were dependent on Tyr510.

Like previous reports (11, 43), these data show a correlationbetween Stat5 activation and proliferation but do not exclude theparticipation of an undefined molecule interacting with Tyr510. In-deed, such a molecule has been previously proposed to explainhow Tyr510 mediates IL-2Ra expression (44). If a molecule otherthan Stat5 mediates the proliferative signal from Tyr510, specificdisruption of the interaction between Stat5 andbbD3251 Tyr510

should not eliminate mitogenesis. To test this possibility, pointmutations were introduced near Tyr510 in bbD325 1 Tyr510 tospecifically disrupt the consensus Stat5-binding motif withouteliminating Tyr510. Analysis of Stat5-binding sites in several cy-tokine receptors suggested that the SH2 domain of Stat5 prefer-entially associates with a phosphorylated tyrosine followed by hy-drophobic amino acids one and three positions C-terminal to thetyrosine (i.e., YLSL in the case of IL-2Rb) (Fig. 2A). These res-idues, in the context ofbbD325 1 Tyr510, were point mutatedindividually or concomitantly to arginine or glutamic acid to gen-erate four mutant receptors (Fig. 2B). Compared withbbD325 1Tyr510, these receptors demonstrated a spectrum of Stat5 activationpotencies (Fig. 2C) which correlated directly with proliferation(Fig. 2D).

IL-2 delivers a proliferative signal through Stat5a or Stat5b

Although designed to specifically disrupt Stat5 binding, these mu-tations near Tyr510 could also disrupt the binding of other putativemitogenic molecules. Therefore, to directly test whether Stat5 de-livers the proliferative signal from Tyr510, the effect of overex-pressing Stat5 on the marginal Stat5-activating potential ofbbD325 1 YRSL was assessed. By mass action, overexpressionof Stat5 was expected to overcome the affinity barrier betweenStat5 and Tyr510 created by the mutation of Leu511 to arginine. Aflag epitope-tagged version of murine Stat5a was cotransfectedwith bbD325 1 YRSL into CTLL-2 cells bearingag, and stablesubclones that coexpressedag, bb, and flag-Stat5a were identifiedby flow cytometry (data not shown). Expression levels of trans-fected Stat5a were estimated by Western blot to be 5- to 10-foldhigher than endogenous Stat5 (Fig. 3A). As intended, overexpres-sion of Stat5a restored the ability ofbbD325 1 YRSL to induceStat5 DNA-binding activity in response to GM-CSF to levels equalto or greater than that mediated bybbD325 1 Tyr510 (Fig. 3B).Rescue of Stat5 activation in turn restored the ability ofbbD3251YRSL to induce thymidine incorporation (Fig. 3C), and cell pro-liferation (Fig. 3D) and maintain cell viability (Fig. 3E). Thus, theproliferative signal from Tyr510 of IL-2Rb can be mediated byStat5a. Overexpression of Stat5a also enhanced the level of Stat5DNA-binding activity induced by the endogenous IL-2R (Fig. 3B)but had no observable effect on IL-2-mediated proliferation or vi-ability of CTLL-2 cells (Fig. 3,D andE). This suggests that theFIGURE 3. Overexpression of Stat5a or Stat5b rescues the ability of

bbD325 1 YRSL to mediate proliferation.A, Stat5 expression in trans-fected CTLL-2 cells. Whole cell lysates of CTLL-2 clones transfected witha vector encodingbbD325 1 YRSL either alone (0) or with a vectorencoding either Stat5a (6 lanes on left) or Stat5b (4 lanes on right) wereassessed for Stat5 content by Western blot using a Stat5-specific Ab. Theblot was stripped and reprobed with a Jak1-specific Ab to demonstrate evenloading. B, Stat5 overexpression rescues Stat5 activation bybbD325 1YRSL. Representative CTLL-2 clones expressingbbD325 1 Tyr510

(Y510), bbD325 1 YRSL (10), or bbD325 1 YRSL plus the indicatedStat5 isotype were assessed by electrophoreticmobility shift assay for theability to activate Stat5 as per Fig. 1D. C, Stat5 overexpression rescuesthymidine incorporation mediated bybbD325 1 YRSL. The ability ofmultiple CTLL-2 clones expressingbbD325 1 Tyr510 or bbD325 1YRSL with or without the indicated Stat5 isotype to induce thymidineincorporation was assessed and depicted as in Fig. 1B. D, Stat5 over-

expression rescues cell expansion mediated bybbD325 1 YRSL. Repre-sentative CTLL-2 clones expressing the indicated chimeric receptor/Stat5combinations were cultured for several days in the presence of 100 ng/mlhuman GM-CSF (GM), 100 U/ml human IL-2 or no cytokine (0). Every 2days, cells were stained with trypan blue and live cells were counted. Cellswere split as needed to maintain a density of,5 3 105 cells/ml. All clonesexpanded similarly in response to IL-2, and all died within 2 days in theabsence of cytokine (data not shown).E, Stat5 overexpression rescuesviability mediated bybbD325 1 YRSL. Representative CTLL-2 cloneswere cultured and counted as inD. Cells excluding and incorporatingtrypan blue were counted as live and dead, respectively. All clones re-mained comparably viable in the presence of IL-2 and died at a similar ratein the absence of cytokine (data not shown).

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degree of Stat5 activation normally induced by the IL-2R is satu-rating with respect to cell proliferation.

Functional differences have been described between the DNA-binding activities of Stat5a and Stat5b (45, 46). Additionally, micelacking either Stat5a or Stat5b genes have distinct phenotypic ab-normalities (26, 47–50). Thus, Stat5a and Stat5b may serve non-redundant functions. To determine whether Stat5b can also medi-ate a proliferative signal from Tyr510, a flag epitope-tagged versionof Stat5b was cotransfected withbbD325 1 YRSL, as above.Although we were unable to overexpress Stat5b to the levelsachieved with Stat5a (Fig. 3A), Stat5b overexpression neverthelessenhanced the ability ofbbD325 1 YRSL to activate Stat5 (Fig.3B) induce thymidine incorporation (Fig. 3C), support cell prolif-eration (Fig. 3D), and inhibit cell death (Fig. 3E).

The TAD of Stat5 is critical for Stat5 to mediate proliferation

Stat molecules conventionally require both a conserved tyrosinephosphorylation site and a C-terminal TAD to mediate transcrip-tion (23, 24, 29). However, the TAD of Stat5 has been reported tobe dispensable for some transcriptional events, such asb-caseininduction mediated by Stat5 in cooperation with the glucocorticoidreceptor (34, 35). Similarly, the TAD of Stat5 appears dispensablefor induction of c-mycby IL-3 (51) and for proliferation of thepromyeloid line 32D in response to the Stat5-activating cytokinesEpo and IL-3 (29).

To test the requirement for the TAD of Stat5 in IL-2R signaling,we assessed whether overexpression of a TAD-deficient version ofStat5 would enhance or inhibit the proliferative signal mediated byTyr510 of IL-2Rb. D713 is a naturally occurring isoform of Stat5athat lacks the TAD (Fig. 4A) but retains all other functional do-mains and therefore can become tyrosine phosphorylated and bindDNA in response to cytokine stimulation (29). If overexpressed inCTLL2 cells, D713 could potentially exert a dominant negativeblockade on some Stat5-mediated transcriptional events (29, 31,51) and impose undesirable selective pressure before analysis.Therefore, experiments withD713 were performed in a cytokine-dependent lymphocyte line that can grow in the absence of anyStat5 activity. The IL-3-dependent pro-B cell line BA/F3 wastransfected with a chimeric receptor containing the extracellulardomain of the human G-CSF receptor and the transmembrane andcytoplasmic domains of the gp130 receptor chain (42), to producea cell line termed BA/FG. gp130 activates Stat3, but not Stat5 (52),so this chimeric receptor promoted the proliferation of BA/F3-derived cells in response to human G-CSF (42) without activationof Stat5 (data not shown). BA/FG cells express an endogenousgcchain but do not respond to IL-2 due to the absence of IL-2Rb,which made it unnecessary to use the chimericag andbb receptorchains used in CTLL-2 cells. Instead, IL-2Rb mutants were testedusing the normal ectodomain of IL-2Rb. Stable transfectants ofBA/FG cells were generated that expressed the different IL-2Rbmutants at similar levels, as assessed by flow cytometry (data

FIGURE 4. Stat5-mediated proliferation requires the TAD of Stat5.A,Schematic depiction of wt or mutant versions of IL-2Rb and flag epitope-tagged Stat5a used in these studies.B, Stat5 DNA binding activity is en-hanced by wtStat5a orD713. Representative BA/FG clones bearing theindicated versions of IL-2Rb and Stat5a were deprived of cytokine for 4 hand then stimulated for 10 min with 3000 U/ml human IL-2 (2), 10%IL-3-conditioned media (3), or no cytokine (0). Nuclear extracts were thenharvested and assessed for Stat5 DNA-binding activity as in Fig. 1D. Lanesmarked Ab represent extracts from IL-2-stimulated cells treated with ananti-flag Ab to specifically supershift transfected Stat5.C, Thymidine in-corporation is enhanced by wtStat5a and inhibited byD713. Three BA/FGclones expressing each of the indicated combinations of IL-2Rb and/or

Stat5a variants were stimulated with 3000 U/ml human IL-2 (F), 10%IL-3-conditioned medium (Œ), or no cytokine (E). Data for each clone areexpressed as a percentage of the thymidine incorporated by that clone whenstimulated with 100 ng/ml human G-CSF.D, IL-2-mediated expansion ofBA/FG expressingD325 1 Tyr510 is inhibited byD713. A representativeBA/FG clone expressingD325 1 Tyr510 with or without D713 was cul-tured in the presence of 3000 U/ml human IL-2, 100 ng/ml human G-CSF,or no cytokine (0). Cell proliferation was assessed and depicted as in Fig.3D. Cells were split as needed to maintain a density of,106 cells/ml. Allclones proliferated similarly in response to G-CSF and died at a similar ratein the absence of cytokine (data not shown).

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not shown). Experiments were performed with a high dose ofIL-2 (3000 U/ml) sufficient to saturate IL-2Rb andgc irrespec-tive of IL-2Ra expression, in that the latter is dependent onStat5 activity (27).

We first confirmed that BA/FG cells, like CTLL2 T cells, weredependent on Stat5 to mediate the proliferative signal from Tyr510

of IL-2Rb. As expected, BA/FG expressingD325 1 Tyr510 acti-vated Stat5 and proliferated in response to IL-2, unless Tyr510

was point mutated to phenylalanine (D325 1 Phe510 (Fig. 4)).Moreover, BA/FG cells expressingD325 1 YRSL demonstrateddramatically impaired Stat5 activation and proliferation, and bothdefects could be rescued by overexpression of wtStat5a (Fig. 4,BandC).

To test the role of the Stat5 TAD in signaling, stable BA/FGclones coexpressingD325 1 Tyr510 and theD713 version ofStat5a were generated. Expression ofD713 did not appear to im-pose adverse selective pressure on BA/FG cells, inasmuch as 1)cells expressingD713 and wtStat5a were generated with similarcloning efficiencies, 2) the two versions of Stat5 were expressed atsimilar levels as revealed by flow cytometry (data not shown), and3) D713 had no effect on the proliferative response of BA/FG cellsto G-CSF (Fig. 4D). Similar to wtStat5a, overexpression ofD713dramatically enhanced Stat5 DNA-binding activity induced byD325 1 Tyr510, as well as by the endogenous IL-3 receptor (Fig.4B), confirming that the TAD is not required for receptor-mediatednuclear translocation or DNA binding by Stat5(29). Despite thecapacity to inducibly bind DNA,D713 severely reduced the abilityof either D325 1 Tyr510 or the IL-3 receptor to induce BA/FGproliferation (Fig. 4,C andD). Thus, the transcriptional activationdomain is essential for Stat5 to mediate proliferative signals fromthe Tyr510 site of IL-2Rb and the full length IL-3 receptor.

Stat5 mediates induction of c-myc,bcl-2, and bcl-x throughits TAD

The obligate role for the TAD in Stat5 mitogenic signaling sug-gests that Stat5 may mediate proliferation through induction ofgenes critical for cell cycle progression and/or survival. c-myc, aprotooncogene induced by IL-2 (53), is essential for T cells toenter S phase in response to stimulation (54).bcl-2andbcl-xL, alsoinduced by IL-2 (55), have been shown to support lymphocytesurvival (56, 57). Constitutive expression of both c-mycandbcl-2is sufficient to support factor-independent growth of the lymphoidcell line BAF/B03 (58).

To assess the role of Stat5 in c-myc,bcl-2, andbcl-x induction,Northern blot analyses were performed with the different BA/FGtransfectants described above. TheCISgene was used as an inter-nal control for Stat5 activity (26).D325 1 YRSL, which inducesminimal Stat5 activation, induced c-myc to a moderate level andonly weakly inducedCIS,bcl-2, andbcl-x in comparison with theendogenous IL-3 receptor (Fig. 5A). Overexpression of Stat5a withD325 1 YRSL enhanced induction not only ofCIS but also ofc-myc,bcl-2, andbcl-x (Fig. 5A), thereby implicating Stat5 in theregulation of all four genes. To test the role of the Stat5 TAD in theinduction of these genes, the effects ofD713 were assessed. Al-though expression ofD713 enhanced the ability ofD3251 Tyr510

to promote Stat5 DNA-binding activity (Fig. 4B), it prevented in-duction of theCIS gene by this receptor, as well as the IL-3 re-ceptor (Fig. 5B). Thus,D713 exerted dominant negative inhibitionof conventional Stat5 transcriptional activity, consistent with priorassessment of this construct (29). In so doing,D713 also inhibitedthe ability of D325 1 Tyr510, as well as the IL-3 receptor, toinduce c-myc,bcl-2, andbcl-x (Fig. 5B), indicating that Stat5 reg-ulates expression of these genes by a TAD-dependent mechanism.

To determine whether the induction of these genes by Stat5 is animmediate-early event or requires the de novo synthesis of inter-mediate transcription factors, the transcriptional consequences ofD325 1 Tyr510 signaling were assessed in the presence of the

FIGURE 5. Stat5 mediates c-myc,bcl-2,andbcl-x induction through itsTAD. A andB, BA/FG cells expressing the indicated versions of IL-2Rband Stat5a were deprived of cytokine for 8 h and then stimulated with 3000U/ml IL-2, 10% IL-3 conditioned medium, or 100 ng/ml G-CSF for theindicated times. RNA was harvested from cells and assessed by Northernblot with serial probing for expression ofCIS, c-myc,bcl-2, bcl-x, andGAPDH. C,The ability of BA/FG expressingD325 1 Tyr510 (Y510) toinduce c-myc, bcl-2andbcl-x with (1CHX) or without 20mg/ml cyclo-heximide administered 30 min before cytokine stimulation was assessed asin A andB.

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protein synthesis inhibitor cycloheximide. Although cyclohexi-mide enhanced the basal expression of genes in the absence ofcytokine as reported for c-myc(59, 60), IL-2 and IL-3 still inducedexpression of c-mycandbcl-x above this basal level (Fig. 5C). Bycontrast, induction ofbcl-2, which occurred with slower kineticsthan seen with c-mycandbcl-x (Fig. 5,A andB), was blocked bycycloheximide treatment (Fig. 5C). Thus, whereasbcl-2 inductionrequires de novo protein synthesis, c-mycandbcl-x are induced byStat5 in an immediate-early fashion, suggesting that Stat5 mayinteract directly with the promoters of these genes rather than viaan intermediary transcription factor.

DiscussionThis report investigates the mechanism of mitogenic signaling bya single cytoplasmic tyrosine (Tyr510) on IL-2Rb which has pre-viously been shown to interact with the transcription factors Stat5aand Stat5b (11, 43). By eliminating all other tyrosines from IL-2Rb and then introducing point mutations at or near Tyr510, wehave established a quantitative correlation between the level ofStat5 activation mediated by IL-2Rb and subsequent proliferationof the murine T cell line CTLL-2. Moreover, overexpression ofStat5a or Stat5b rescued the proliferative signal mediated by areceptor mutant with a low affinity Stat5-binding site, thereby di-rectly implicating Stat5a and Stat5b as mediators of mitogenic sig-naling in T cells. Proliferative signaling by Stat5a required a C-terminal TAD, which was similarly required for Stat5 to induceexpression of the c-myc, bcl-x,and bcl-2 genes, indicating thatStat5 mediates proliferation bytrans-activation of both promito-genic and antiapoptotic genes.

Previous studies of the role of Stat5 in proliferative signalinghave yielded somewhat contradictory results. Mui et al. (51) re-ported that a dominant-negative mutant of Stat5 significantly in-hibited the proliferation of the pro-B cell line BA/F3 in response toIL-3, another cytokine which activates Stat5. This occurred in theface of normal c-mycinduction, suggesting that Stat5 was criticalfor some, but not all, aspects of the proliferative signal. By con-trast, Wang et al. (29) reported that dominant-negative inhibitionof Stat5 activity in the promyeloid cell line 32D had no impact onproliferative responses to IL-3 and Epo. The residual proliferativeresponses seen in these two studies may have been mediated byone or more redundant proliferative pathways operating in parallelwith Stat5, such as the Shc pathway (14, 61). Indeed, the Stat5-binding sites on IL-2Rb (Tyr510 and Tyr393) are only critical forproliferation when Shc activity is abrogated by mutation of itsbinding site at Tyr338 (11). Consequently,D713 or other dominantnegative versions of Stat5 would not be expected to block prolif-eration mediated by full length IL-2Rb. For this reason, the ex-periments in the present study utilized a mutated version of IL-2Rb that lacked the Shc-binding site and contained only the Stat5activation site at Tyr510. By eliminating potentially redundantpathways, a definitive role for Stat5 in proliferative signaling wasrevealed.

Although both Stat5 and Shc are clearly able to mediate cellproliferation, the extent to which each contributes to the IL-2-induced proliferation of primary T cells remains controversial. Fu-jii et al. (39) reconstituted IL-2Rb-deficient mice with a mutatedversion of IL-2Rb lacking the ability to detectably activate Stat5,but containing an intact Shc-binding site. They found that thismutated receptor promoted a normal proliferative response to IL-2in primary T cells (39), consistent with prior studies in cell lines(11, 37, 38, 62). By contrast, Moriggl et al. reported that T cellsfrom mice lacking both the Stat5A and Stat5B genes show noproliferative response to IL-2 (36), which led them to conclude that

Stat5 is absolutely essential for IL-2-induced proliferation in pri-mary T cells. It is unclear why no Stat5-independent proliferativepathway was operational in T cells from the latter mice, as it ap-parently was in the cells studied by Fujii et al. (39). One possibleexplanation for the discrepant results is that the mutant form ofIL-2Rb used by Fujii et al. might have been able to activate Stat5to a physiologically relevant but experimentally undetectable level.However, this seems an unlikely explanation, in that we show herethat even detectable, low level Stat5 activity is insufficient for thehigh level of T cell proliferation observed by Fujii et al. (39) (Figs.2 and 3,B--E). An alternative explanation is that deletion of boththe Stat5a and Stat5b genes by Moriggl et al. had unanticipateddetrimental effects on the expression or function of one or moreessential components of the IL-2R complex, such as Jak3, render-ing the receptor globally dysfunctional in the total absence ofStat5. There is precedent for such a mechanism, because the basalactivity of Stat1 has recently been shown to be required for ex-pression of caspases, which in turn are required to sensitize cells toTNF-a-mediated apoptosis (33). Finally, it is also possible that thesevere proliferative defect seen in Stat5a/Stat5b-deficient T cells inpart reflects impaired signaling through the TCR, which has re-cently been show to induce transient activation of Stat5 (40).

The molecular mechanism by which Stat5 mediates proliferativesignaling has also remained obscure. A truncated version of Stat5,which was unable to dimerize and hence bind DNA, inhibited IL-3-mediated proliferation by a dominant negative mechanism (51),thus implicating Stat5 in the IL-3 proliferative signal. However, ina second study by Wang et al. (29), a less severely truncated ver-sion of Stat5 lacking only the TAD had no impact on the IL-3-proliferative signal. Together, these studies suggested that Stat5may mediate proliferative signaling by a TAD-independent mech-anism. Consistent with this notion, a version of Stat5 capable ofbinding DNA, but lacking its TAD, can nevertheless interact withthe glucocorticoid receptor to induce transcription of theb-caseingene (34, 35), thereby demonstrating that Stat5 is capable of TAD-independent transcriptional regulation. However, our results definea clear requirement for the C-terminal TAD of Stat5 for the pro-liferative response of lymphocytes to both IL-2 and IL-3. Thisindicates that, despite its capacity for TAD-independent signaling,Stat5 induces cell proliferation through the conventional TAD atits C terminus. The discrepancy between our findings and those ofWang et al. may reflect the fact that their cells were cultured withIL-3 in the face of constitutive dominant negative Stat5 expression,which would be expected to impose a strong selective pressure infavor of Stat5-independent signaling mechanisms. In the presentstudy, we avoided this potential problem by creating a cell line,BA/FG, that does not use Stat5 for growth and instead responds toa Stat3-dependent proliferative signal delivered by the gp130 re-ceptor subunit. This allowed for expression of the TAD-deficientform of Stat5 at a level sufficient for complete dominant negativeactivity, which likely accounts for the more pronounced impact onIL-2- and IL-3-mediated proliferation observed in this study com-pared with Wang et al. (29) and Mui et al. (51).

In addition to cell proliferation, Stat5 was also found to induceexpression of the growth-related genes c-myc, bcl-2, and bcl-xthrough a TAD-dependent mechanism (Fig. 5,A andB). Inductionof the c-mycand bcl-x genes by Stat5 did not require de novoprotein synthesis (Fig. 5C), suggesting that Stat5a interacts directlywith the promoters of these genes. Although no Stat5 binding siteshave yet been functionally defined in these promoters, other Statfamily members have been shown to regulate these genes. Stat1can interact with a TTCGGAGAA motif in the humanbcl-x pro-moter, and this site is involved in the regulation ofbcl-xexpression(63). Stat3 has also been proposed to regulatebcl-xexpression (64)

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and can associate with andtrans-activate the c-mycpromoter (65).However, the site in the c-mycpromoter with which Stat3 interactsis not appreciably bound by Stat5(65) (J.D.L., unpublished obser-vations), suggesting that Stat3 and Stat5 mediate c-myc inductionby binding distinct promoter sites.

In contrast to c-mycandbcl-x, induction ofbcl-2 by Stat5 ap-pears to be a delayed early event, because it occurs with slowerkinetics and requires de novo protein synthesis (Fig. 5). This sug-gests that Stat5 induces expression of one or more secondary tran-scription factors that in turn bind to andtrans-activate thebcl-2promoter. Such a model, in which Stat5 initiates a cascade of geneactivation events, would explain how a single transcription factorcould promote the diverse array of cellular changes that are nec-essary for cell cycle progression, including protooncogene induc-tion, up-regulation of cell metabolism, and activation of the cellcycle machinery.

Although theD3251 Tyr510 receptor was able to mediate Stat5activation at least as potently asbbwt and the endogenous wt IL-2receptor (Fig. 1D), it was;40% weaker in its ability to promotecell proliferation (Fig. 1B). This suggests that Stat5 is not sufficientto mediate all aspects of the IL-2 proliferative signal. Indeed, ac-tivation of theras/receptor-activated factor/mitogen-activated pro-tein kinase signaling pathway, culminating in the induction of c-fos, c-jun, and other genes, is mediated exclusively by Shc througha Stat5-independent mechanism (14, 66–68). Conversely, Stat5uniquely induces expression of theCIS andCD25 genes (26, 27,44) and mediates abcl-2-independent survival signal that cannotbe delivered through Shc (38). Perhaps STAT5 acts cooperativelywith Shc, or potentially other proliferative signals from the IL-2R,to deliver a signal of maximum mitogenic potential. In vivo, wherereceptor and/or cytokine concentrations may be limiting, synergybetween Stat5- and Shc-mediated mitogenic signals from theIL-2R may be essential for rapid T cell expansion, and hence thegeneration of an effective immune response to an invadingpathogen.

AcknowledgmentsWe thank David Hockenbery, Lee James, and Pierre Rollini for providingNorthern blot probes; James Ihle for providing Stat5 cDNAs; and MarkBenson, Laurel Hickock, Meghan Parsons, and Ben Wittenstein for tech-nical assistance.

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