Proc. Natl. Acad. Sci. USAVol. 91, pp. 4776-4780, May 1994Biochemistry

Transcription factor ISGF-3 formation requires phosphorylatedStat9l protein, but Statll3 protein is phosphorylatedindependently of Stat9l protein

(ranrlpton/terfe/trosne phosphorylatlo)

TERESA IMPROTA*, CHRIS SCHINDLERt, CURT M. HORVATH*, IAN M. KERRt, G. R. STARK§,AND J. E. DARNELL, JR.*¶*Laboratory of Molecular Cell Biology, The Rockefeller University, New York, NY 10021; tDepartment of Medicine, Columbia University College ofPhysicians & Surgeons, New York, NY 10032; tImperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX,United Kingdom; and #Cleveland Clinic Foundation Research Institute, Cleveland, OH 44194

Contributed by J. E. Darnell, Jr., February 7, 1994

ABSTRACT Transcription factor ISGF-3 is a multipro-tein, interferon a-activated transcription complex constei ofa 48-kDa DNA-binding protein and two proteins termed Stats(for signal jransducers and activators of transcription) thatbecome phosphorylated on tyrosine in the cell cytoplasm, a113-kDa and either a 91- or 84-kDa polypeptide, the latter twoof which arise from dUifentilay spiced mRNAs. Using celllines lacking the Stat9l or StatS4 proteins, we show thatmutations in several dfferent sites in the 91-kDa protein blockthe interferon a-duced phosphorylation of the 91-kDa pro-tein and subseient ISGF-3 formation. Although correct ty-rosine phosphorylation on residue 690 of the Statll3 proteinoccurs Independent of the Stat91/84 protein, the Statll3phosphoprotein by itself moves to the cell nucleus much lessefficiently in the absence ofphosphorylated Stat91/84 protein.

The activation of transcription by interferon a (IFN-a) in-volves phosphorylation on tyrosine of three proteins-113,91, and 84 kDa in size (1-4). Together with a 48-kDaDNA-binding protein (4-6) these larger proteins composeISGF-3, the multiprotein transcriptional activator that bindsto a conserved 15-bp DNA element, the IFN stimulationresponse element (ISRE) (7-10). Because the phosphopro-teins are involved in both signal transduction and activationof transcription, we refer to them as Statll3, Stat9l, andStat84 (11). The Stat9l and Stat84 proteins are identical,except for 38 amino acids found at the carboxyl terminus ofthe Stat9l that are absent in the Stat84 (1). A series of celllines that were selected as deficient in the IFN-a responsepathway fall into several different complementation groups(12-14), one of which, the U3 group, lacks the Stat9l andStat84 mRNA and, therefore, the Stat9l and Stat84 proteins(13). Correction of the IFN-a response, as judged by IFN-a-dependent mRNA induction, was obtained by reconstitu-tion ofU3 cells with either the Stat91- or 84-kDa protein (13).Thus, either the Stat9l or the Stat84 protein is competenttogether with the Statll3 protein, the 48-kDa protein, and theappropriate deoxyoligonucleotide to form the transcriptionfactor ISGF-3 multiprotein complex. In this paper we use aU3 mutant cell line to explore further the role of Stat9l invarious IFN-a-dependent intracellular events.

MATERIALS AND METHODSCell Culture. Parental cells 2fTGH and U3 mutant cells

were grown and transfected, as described (12, 13), in Dul-becco's modified Eagle's medium/10%o heat-inactivated fetal

bovine serum. G418-resistant cells were selected and main-tained in medium containing G418 at 400 ug/ml. Selection inhypoxanthine/aminopterin/thymidine medium was done inthe presence of IFN-a at 500 international units/ml, asdescribed. Human recombinant IFNs were supplied by Hoff-mann-La Roche and Amgen (P. Sorter and D. Vapnek,respectively).

Plasnuds and Mutations. cDNA clones containing the cod-ing regions for the 91- and 84-kDa proteins were cloned intothe mammalian expression vector Rc/CMV (Invitrogen).The Y -- F plasmid contained the 91-kDa coding region withTyr70W changed to Phe. Plasmids A141 and A216 contained,respectively, 141- and 216-aa NH2-terminal deletions fromthe 91-kDa protein. The L20 plasmid contained a 20-aadeletion between aa 199 and 220 of the 91-kDa proteinleucine-rich region. All the deletions and point mutationswere obtained by standard PCR techniques (15). Sequenceswere verified by DNA sequencing (United States Biochem-ical).

Eletrophoretic Mobility-Shift Assay. This technique wasdone as described (7, 9).Immunoblotting and Immunoprecipitation. Immunoblots

and immunoprecipitations were done by standard methods(1-3, 16) on protein extracts obtained from 1 x 107 cellsuntreated or treated with IFN-a (500 international units/ml)and IFN-y (5 ng/ml), as indicated. Whole-cell lysates wereprecipitated with either anti-113 or anti-91T antibody (2, 3) atthe final dilution of 1:100. Tyrosine phosphorylation wasdetected by incubating filters for 2 hr with PY20 (Zymed) and4G10 antibodies (Upstate Biotechnology) dilution, 1:2000,respectively for the precipitated 91 or 113 proteins afterimmunoblots. Filters were then washed and incubated withsecondary antibody anti-mouse peroxidase conjugated andstained with ECL (Amersham).Phosphopeptide Mapping and Phospho Amino Acid Deter-

mination. Cell extracts from 32P-labeled cells were preparedand precipitated with antiserum as described (3, 11, 16).Proteolytic digestion and two-dimensional phosphopeptidemapping were also done as described (3, 11, 16). One-dimensional phosphopeptide analysis of p113 was done asfollows: the single IFN-a-induced p113 thermolysin-derivedphosphopeptide (3) was separated by a two-dimensionalanalysis. This phosphopeptide was eluted from the celluloseTLC plate by successive incubation with pH 3.5 buffer andlyophilized. The peptide (400-500 cpm) was then dissolved in200 kid of 0.05 mM NaHCO3 buffer, pH 8, and subjected totwo-dimensional analysis, as described (3).

Abbreviations: Stat, signal transducer and activator of transcription;IFN, interferon.ITo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The in vivo-labeled peptide was prepared, as described(11). Ten micrograms of synthetic peptide (indicated in Fig.1A) was phosphorylated in vitro in 10 id of LCK buffer (25mM Tris, pH 7.4/100 mM NaCI/10 mM MnCl2/and 0.2 mMdithiothreitol) with 100 pmol (2 x 104 dpm/pmol) of ATP([Ft32P]ATP) by the addition of 3 A1 of Ick protein (gift of R.Aebersold, University of British Columbia) for 30 min at300C. The basic peptide was subsequently separated byone-dimensional TLC and eluted as described above. Thepeptide was then digested in 200 4 of NaHCO3 buffer, pH 8(1000 cpm), with thermolysin at 0.1 mg/ml for 6-10.hr at37°C, lyophilized, and analyzed in two dimensions, as shownin Figs. 5 and 6.

RESULTSRequired Sites for IFN-a-Induced Stat9l Phosphorylation

and ISGF-3 Formation. To determine the residues in theStat91 required for IFN-a-induced phosphorylation and in-teraction with transcription Statl13 to form factor ISGF-3,we used the series of mutants shown in Fig. 1A. Stable celllines derived from U3 cells, all producing about the sameamount of Stat9l protein, or mutant Stat91.protein wereestablished for each mutation and deletion (Fig. 1B). ThecDNA constructs included (i) deletion of 141 or 216 aa fromthe NH2 terminus, (ii) deletion of20 internal amino acids thatremoved the heptad leucine repeats (2), (iii) mutation ofTyr701 to Phe, which removed the tyrosine residue that isphosphorylated after IFN-a and IFN-y treatment (11, 16),and (iv) ArgW2 -+ Leu mutation, which changed a crucialarginine in the phosphotyrosine-binding pocket of the Srchomology 2 domain (16, 17). Immediately after IFN-a treat-ment and phosphorylation of the cytoplasmic Statll3 andStat9l/84, the extracts of treated cells are competent, in thepresence of DNA and the 48-kDa-binding protein (9), toassemble the ISGF-3 protein-DNA complex. Also, U3 cellsreconstituted with either Stat9l or Stat84 acquire the capa-bility, like their parental cell line 2fTGH, of growing in theselective medium hypoxanthine/aminopterin/thymidine inthe presence ofIFN-a. We, therefore, tested the ability ofthe

2rrflH ) -* F A 141 \ 216

f ls(;ISF3

FIG. 2. ISGF-3 activity in cells expressing deletions and mutationofStat9l/84-kDa protein. Total extracts were prepared from parentalcell line 2fTGH and from the U3 cells permanently expressing 5'truncated 91-kDa proteins (A141 or A216) or the Tyr701--+ Phe mutantproteins. Cells were treated with IFN-a and with IFN--y for 16 hrfollowed by IFN-a for 30 min (30') or 3 hr (3h) (lanes y/a), asindicated; c, competition of the ISGF-3 band with 5Ox excess IFNstimulation response element oligonucleotide. Arrow, position ofISGF-3-complex migration on 4.5% polyacrylamide gel.

various Stat9l mutant cell lines to produce proteins capableof forming ISGF-3 (Fig. 2). None of the extracts from cellsbearing mutant proteins produced ISGF-3, except for thecells expressing the construct encoding Stat9l but lacking theNH2-terminal 141 aa (A141 cells), which produced a trace ofthe ISGF-3 complex (Fig. 2). Furthermore, none of the cell

A 2fTGH 91+ 84+ Y -sF 2fTGH L20 A216 A141- 4 +- -+ -- - -±+ - t+ -+t

113 KD -

91 KD -84 KD -

_-It _OR#w -' -

A CMV91 kD coding region

84 kD coding region141 aa 5' deletion

216 aa 5' deletion

YL region 20 aa deletion

Y-FY 701 point mutation

R-*LR 602 in SH2 domain

B+ +

91 KD -

84 KD

FIG. 1. (A) cDNAs constructs used for stable expression in U3cells. (B) Immunoblots probed with anti-Stat9l and 91T antibodiesshowing the level of mutated/deleted forms of Stat91/84 protein inthe cell lines expressing the cDNAs described above. Ten micro-grams of total protein obtained from crude cell extracts was loadedon SDS/7.5% polyacrylamide gel. A cell line bearing the Stat9lArgW -- Leu construct also produced equivalent amounts of protein(16). CMV, cytomegalovirus; SH2, Src homology region 2; KD, kDa.

B 2fTGH U3-+ -+

113 KD _ C

91 KD - 113

R6o2->L

a

IkD -

D 2tTGH L2C' A21 6 \141

1 l3kD-91kD -

FIG. 3. Phosphorylation in Statll3 and Stat9l in various cell linesdetected by anti-phosphotyrosine antibody. (A, B, C) Parental2fIGH, reconstituted U3 cells, and various mutants established inU3 cells; precipitation by anti-Statll3 antiserum followed by anti-phosphotyrosine 4G10 antibody blot. Cells were untreated, treatedwith IFN-a for 20 min, or treated with IFN-y for 15 min (+ or -

indicates IFN-a or no treatment; a or y indicates IFN-a or IFN-y,respectively). (D) Anti-Stat9l was used for precipitation, and the blotwas probed with PY20 antiphosphotyrosine antibody. Cells wereuntreated (-) and treated with IFN-a for 20 min (a) or with IFN-'yfor 15 min ('y); the same result was obtained with all other Stat9imutants in Fig. 1A. KD, kDa.

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4778 Biochemistry: Improta et al.

lines could survive in the selective hypoxanthine/aminopter-in/thymidine medium containing IFN-a, as could their parent2fTGH cells or Stat9l-reconstituted U3 cells. However, thecells containing the A141 mutant Stat9l were not promptlykilled in the selective medium, whereas cells totally lackingStat9l were promptly killed (T.I., unpublished results). Po-tentially, therefore, the survival ofa cell is the most sensitiveindicator of a minimal response in the IFN-a signal-transduction pathway.

Tests for phosphotyrosine in Statll3 or Stat9l were doneon each cell line after immunoprecipitation of extracts ofIFN-treated or untreated cells with either the Statll3 anti-body or the anti-Stat9l antibody followed by electrophoresisand immunoblotting with an antiphosphotyrosine antibody.The data for precipitation with the anti-113 antibody allowedtwo conclusions: (i) U3 cells lacking Stat9l phosphorylatedStatll3 normally in response to IFN-a. So did all of the cellsbearing Stat9l mutations (or U3 cells reconstituted withStat9l or Stat84). Thus, Stat9l was not required for Statll3phosphorylation. (ii) The other finding relates to interactionbetween Statll3 and Stat9l. Because the Statll3 antiserumprecipitates 35S-labeled Statll3 but does not precipitateStat91 in untreated cells, it is considered specific (1, 3). AfterIFN-a treatment, the Statll3 precipitates some Stat9l alongwith Statll3; probably because the two proteins interact, forexample, we now know that phospho Stat9l forms a ho-modimer after IFN-y-induced phosphorylation (18). ThisIEN-a-induced coprecipitation of Statll3 and Stat9l is dem-onstrated in Fig. 3A, lanes 2, 4, and 6. None of the mutantStat9l proteins coprecipitated, suggesting they did not forma Stat91-Stat113 complex. Finally we examined the phos-phorylation of mutant Stat9l proteins directly in anti-Stat9lprecipitates (Fig. 3D). The wild-type protein was clearlyphosphorylated, but none of the mutant Stat9l proteins listed

A c

in Fig. 1A were phosphorylated. Thus, not only did mutationin Tyr701 (the site known to be phosphorylated after IFN-yand IFN-a) block phosphorylation, but so did the Arg" -Leu (the Src homology 2 group mutant) and the collection ofNH2-terminal deletions and the leucine-heptad-region dele-tion. (Three mutants are shown in Fig. 3D, but all were testedand none were phosphorylated.)

Decreased Translocation or Nuclear Stability of Phosphor-ylated Statll3 Without Stat9l. Because phosphorylatedStat9l and Statll3 appear together in the nucleus (3) inIFN-a-treated wild-type cells, we determined whether theStatll3 could be translocated to the nucleus in the absence ofStat91/84. Nuclear translocation of Statll3 was demon-strated by immunofluorescence in U3 cells complementedwith Stat9l [as was earlier shown to be true ofwild-type cells(3)], but we could not detect a shift in immunofluorescence inU3 cells (data not shown). Because the immunofluorescencetechnique might not be sensitive to small amounts of trans-located Statll3, we fractionated IFN-a-treated U3 cells and2fTGH cells into nuclear and cytoplasmic samples and testedfor Statll3 by gel electrophoresis and immunoblotting ofproteins. By this technique the majority of the Statll3 in2fTGH cells was found in the nucleus after IFN-a treatment.In addition, a smaller amount of Statll3 was translocated inU3 cells and in the A141 cell line expressing the NH2-terminaltruncation described above (Fig. 4A). A time course of thenuclear appearance of the Statll3 in IFN-a-treated U3 cellsshowed a clear increase of nuclear Statll3 during IFN-atreatment. Note, however, that only about one-sixth as muchof the cytoplasmic sample relative to the nuclear sample wasused in the immunoblots of Fig. 4B, whereas an equal aliquotofnuclear and cytoplasmic sample was used in Fig. 4A. Thus,

2fTGH S Y' LJ391

N

2fTGH .A141' U3 2fTGH A141' U3

_-+-+-+ -±-+-++

113 KD- am* _91 KD-- q, __0ltie * x

4

j3 Y ..F S 7 ;fB N 01O 20 30 1h2h 3h

l13kD

0 1O 20 30' lh 2h 3h

113kD-- - -- - -s

FIG. 4. Nuclear translocation of 113-kDa (KD) protein. (A)Immunoblot of cytoplasmic (C) and nuclear (N) extracts preparedfrom 2fTGH, A141, and U3 cells untreated and treated with IFN-afor 1 hr, stained with anti-113 and anti-91T antibodies. Cells treated(+) or untreated (-) with IFN-a were fractionated into nuclear (N)and cytoplasmic (C) fractions, and equivalent aliquots were sub-jected to electrophoresis and immunoblotting with a mixture ofanti-Statll3 and anti-Stat91T antibodies. (B) U3 cells treated withIFN-a were fractionated into nuclear (N) and cytoplasmic -(C)samples. An amount of cytoplasmic extract equal to one-sixth asmany cells as that used for the nuclear sample was subjected toelectrophoresis and immunoblotting with the anti-113-kDa antise-rum. ', min; h, hr.

+ _ +

FIG. 5. Phosphopeptide and phospho amino acid analysis ofStatll3 protein from various cells labeled with 32P and treated withIFN-a. Statll3 was isolated by immunoprecipitation and gel elec-trophoresis and digested with thermolysin; phosphopeptides wereanalyzed by a two-dimensional procedure (3), as indicated. (Nodominant single peptides were present in extracts of cells not treatedwith IFN.) The single lEN-a-induced phosphopeptides were recov-ered, and individual phospho amino acids were determined after acidhydrolysis and chromatography: phosphoserine (S), phosphothreo-nine (T), and phosphotyrosine (Y) determinations for the recoveredprotein are shown in Insets.

C

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we conclude that Statll3 can, indeed, be moved to thenucleus in the absence of Stat9l, although inefficiently.

Phospborylation of Statll3 Without Stat9l. We next deter-mined whether the Statll3 phosphorylation that occurredwithout phosphorylated Stat9l protein (Fig. 3 A-C) was atthe correct tyrosine residue.

Cells labeled with 32P and treated or untreated with IFN-awere used to select labeled Statll3 (3, 11). Immunoprecipi-tation with antibody to Statll3 detected IFN-dependent32P-labeled 113-kDa protein in U3 cells, 2fTGH cells, and aswell a stable transfectant of U3 cells complemented with theStat9l protein (U3, 91+) and the Stat9l protein mutantcontaining Tyr70t -- Phe (data not shown).The 32P-labeled Statll3 protein was collected from the

various cell types, protein was cleaved to amino acids, and inevery case IFN-a-induced phosphotyrosine was observed inthe total protein from the 113-kDa band (data not shown).Samples of the 32P-labeled Statll3 were then subjected tothermolysin digestion, and two-dimensional phosphopeptidecomparisons were carried out (Fig. 5). From all cell samples(2fTGH cells, U3 cells, U3 plus Stat9l, and U3 plus Tyr701mutant), a single IFN-a-induced peptide was seen that mi-grated in the same position as that earlier described fromIFN-a-induced HeLa cells (Fig. 5 and ref. 3). The phospho-amino acid in this spot was confirmed to be phosphotyrosine(Fig. 5 Insets).The single phosphorylated peptide was proven to include

phosphotyrosine at position 690 by several analytical experi-

A

1'eptidie

StiltllI y y

Stat)1 Y Y

L

-Q E K YN L Q E

S R P K E A P E P M E L D G

ments (Fig. 6). (i) A small tryptic peptide was released fromStat9l, which upon Edman degradation yielded phosphoty-rosine in the first degradative step (Fig. 6B). Thus, thephosphotyrosine in Statll3 had argpnine or lysine (the targetsof trypsin) as its NHrterminal neighbor. (ii) The thermolysinpeptide (as in Fig. 5) migrated in one-dimensional electropho-resis as a very basic peptide, suggesting several basic residues(data not shown; see Fig. 6A). (iii)We knew the position ofthephosphotyrosine in Stat9l was Tyr70", and the homologousregion in Statll3 (Fig. 6A) contained a tyrosine next to lysineor arginine with several basic amino acids in the vicinity. Wetherefore synthesized a peptide (LQERRKY) and exposed itto labeled ATP and to lck protein, a tyrosine kinase (from R.Aebersold), and cleaved it with thermolysin. This mixtureyielded a labeled peptide migatng in the same position as theauthentic peptide, as determined by mixing the authenticpeptide with the lck-peptide reaction (Fig. 6C). (Only whenthe synthetic peptide was added did the LCK kinase producethe spot labeled.) Thus, we conclude that Tyr4"0 is the singletyrosine that becomes phosphorylated in Statll3 and that thissame phosphorylation can occur in cells lacking Stat9l.

DISCUSSIONThese results establish that the Tyr701 site in Stat9l and theArg0w site are required for IFN-a phosphorylation of Stat9l,as was earlier shown after IFN-y treatment (16). Moreover,the results show that the Stat9l phosphorylation is sensitiveto several other mutations (NH2-terminal deletions and re-

Q E R R - - K Y L K H R L 1 V V

R R - - K YI L

P K G T G Y I70

K H R L I V V

K T E L I S V

S N 1F Q D E

S E V H r R

B Edmancycle 0

cycle 1

cycle 2

cycle 3

C Synthetic Peptide

4_4oi. -i.

.q.

b

*i.

A

+

._L ~ ~ L

I.. ..

.-.. -Al_~l.-..n

Native Peptide

+

FIG. 6. Phosphorylation site in Statll3. (A) Amino acid sequences of Stat9l and Statll3 in region of tyrosine phosphorylation. Bar aboveindicates limits of synthesized peptide, and the arrow indicates the site of thermolysin cleavage. (B) 32P-labeled Statll3 was recovered fromIFN-a-treated cells and subjected to trypsin digestion and electrophoresis. The smallest peptide was recovered and subjected to Edmandegradation, showing release of orthophosphate in the first cycle. Arrow, point of application. (C) Two-dimensional analysis of thermolysin-derived phosphopeptides (done as for Fig. 5). The synthetic peptide indicated by the bar above A sequences was 32P-labeled in vitro by LCKkinase, producing the peptide labeled a (Left); spots labeled b and c were enzyme background and occurred whether or not the synthetic peptidewas added. Spot a was excised, as was a native peptide spot (Middle), and the two were rerun together (Right).

Bo th

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moval of the leucine heptad in the aa 199-220 region),implying that the functional Stat9l structure may be complexand easily inactivated as a kinase substrate. None of thenonphosphorylated Stat9l molecules could participate inISGF-3 formation, perhaps indicating that like the Stat9lhomodimer that forms in IFN-y-treated cells (18) the Stat91-113 interaction that occurs in IFN-a-treated cells might alsouse phosphotyrosine-Src homology 2 interaction.To study the phosphorylation of Statll3 meaningfully we

needed to know where that protein becomes phosphorylatedin response to IFN-a. We earlier showed that a singletyrosine phosphopeptide was observed after IFN-a, and wethen located a single tyrosine, Tyr690, in the Statll3 that is ina homologous state to the Tyr701 in Stat9l. We then showedthat Statll3 phosphorylation occurs on the correct Tyr6" sitein response to IFN-a, even in the total absence of the Stat91protein or in the presence of any of the nonphosphorylatedmutants. However, accumulation in the nucleus (either trans-location or nuclear stability) of the phospho-Statll3 wassubstantially diminished in the absence of Stat9l. Thus, itseems likely that each substrate protein is handled indepen-dently by the presumed receptor-JAK (Janus kinase) com-plex that is thought responsible for phosphorylating the Statprotein substrates (16, 19, 20). However, the Stat91-113interaction that normally occurs after IFN-a treatment facil-itates the transport or nuclear stability of the phosphorylatedStatll3.

This work was supported by National Institutes of Health GrantA132489 (J.E.D.).

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