21. A biostatistician (E.J.O.) randomized each patientto either the active or placebo treatment group inblocks of six, stratified by functional class (28)severity.

22. The placebo consisted of 1.0-mi doses of 0.1 Macetic acid subjected to membrane filtration.

23. Three investigators (D.C., C.L., and K.L.S.) ob-tained the randomization and prepared medica-tion but did not have access to clinical data. Nounblinding occurred.

24. Conventional instruments were used to measureRA activity (16). Assistive devices were permittedfor walk times. The clinical investigator cared forthe patients during the trial and was responsiblefor safety monitoring. Laboratory safety assess-ment was performed immediately before random-ization and at 2, 4,8, and 12 weeks thereafter. Theassessment comprised a complete blood count,differential and platelet count, liver and renalfunction tests, prothrombin and partial thrombo-plastin times, urinalysis, and ESR. HLA typing wasperformed for alleles of the A, B, C, and DR/DQloci (36). Serum immunoglobulin M (IgM) rheuma-toid factor titers were determined by nephelome-try and lgG antibody titers to native type 11 colla-gen (expressed as -lo92) by enzyme-linked im-munosorbent assay (37) immediately before andat the end of collagen or placebo administration.

25. Before unblinding, decisions were made concern-ing the analysis of five subjects (8%) that failed tocomplete the study. One was noncompliant andwithdrew for personal reasons on day 40 after onlya baseline examination. This patient was excludedfrom analysis and had been randomized to colla-gen. Four discontinued their study medication be-fore the end of the 3-month treatment because ofworsening arthritis. They were assigned the worstscore in the sample for the remainder of the studyand included in the analyses. All four had beenrandomized to placebo. One protocol violationoccurred with a patient who increased the dailydose of prednisone from 5 mg to 10 mg just beforemonth 2. Because the patient continued to dopoorly and the 10-mg dose was consistent witheligibility requirements, the patient was included inthe analyses; the patient had been randomized tocollagen. No steroid injections or other problemswith compliance occurred.

26. Comparisons between collagen- and placebo-treated patients were performed with theWilcoxon rank-sum test for continuous measures(such as the number of swollen joints), the Fish-er's exact test for dichotomous measures (suchas narcotic usage), and the x2 trend test forfunctional class and patient and physician as-sessments. All measured end points such as thenumber of swollen joints were compared withentry values before testing; qualitative measures,such as patient and physician assessments andfunctional class, are presented and analyzedwithout adjustment for baseline responses. TheStudent's paired t test was used to assess wheth-er changes in the collagen group representedsignificant improvements over baseline values.Reported P values are two-sided.

27. Complete resolution is a more rigorous extensionof RA remission criteria (17), preformulated be-cause of the magnitude of improvement in somepatients in the initial trial, and is defined by thefollowing conditions: no swollen or tender joints,no morning stiffness or afternoon fatigue, absentarthritis on physician and patient appraisals, func-tional class I status, and normal ESR (<28 mm/hour) while off prednisone.

28. 0. Steinbrocker, C. H. Traeger, R. C. Batterman,J. Am. Med. Assoc. 140, 659 (1949).

29. Rather than assigning the placebo patients whowithdrew from the trial the worst observed value(25), they were given the value from their last visit.Because one of the four dropped out before the1-month follow-up, that patient was removed fromall analyses, reducing the sample size to 28collagen and 30 placebo patients. By this analy-sis, the number of tender joints, joint-tendernessindex, walk time, patient assessment of severe orvery severe disease, and analgesic use was

significantly (Ps 0.05) improved in the collagengroup compared with the placebo group.

30. H. J. Williams et al., Arthritis Rheum. 31, 702(1988).

31. Analysis of variance showed no significant inter-action between treatment effectiveness (as mea-sured by changes in the number of swollen joints,tender joints, or walk time) and any characteristicin Table 1.

32. R. Nussenblatt, personal communication.33. A. Friedman and H. L. Weiner, J. Immunol. 150,

4A (1993); H. L. Weiner etal., Ann. Rev. Immunol.,in press.

34. A. Al-Sabbagh, A. Miller, R. A. Sobel, H. L. Wein-

er, Neurology42 (suppl. 3), 346 (1992).35. S. M. Helfgott,R. A. Dynesius-Trentham, E. Brahn,

D. E. Trentham, J. Exp. Med. 162, 1531 (1985).36. G. M. Kammer and D. E. Trentham, Arthritis

Rheum. 27, 489 (1984).37. S. M. Helfgott etal., Lancet 337, 387 (1991).38. Supported by NIH grants M01 RR01032 and

AG00294 and by a grant from Autolmmune Inc.We thank E. Milford and C. B. Carpenter for HLAtyping. In accordance with disclosure guidelinesof the Harvard Medical School, D.A.H. and H.L.W.have a financial interest in Autolmmune Inc.

9 June 1993; accepted 23 August 1993

Tyrosine Phosphorylation of DNA Binding Proteinsby Multiple Cytokines

Andrew C. Larner, Michael David, Gerald M. Feldman,Ken-ichi Igarashi, Rebecca H. Hackett, Deborah S. A. Webb,Sharon M. Sweitzer, Emanuel F. Petricoin Ill, David S. Finbloom

Interferon-a (IFN-a) and IFN-y regulate gene expression by tyrosine phosphorylation of

several transcription factors that have the 91 -kilodalton (p91) protein of interferon-stimu-lated gene factor-3 (ISGF-3) as a common component. Interferon-activated protein com-

plexes bind enhancers present in the promoters of early response genes such as the

high-affinity Fc-y receptor gene (FcyRI). Treatment of human peripheral blood monocytes

or basophils with interleukin-3 (IL-3), IL-5, IL-10, or granulocyte-macrophage colony-stimulating factor (GM-CSF) activated DNA binding proteins that recognized the IFN-y

response region (GRR) located in the promoter of the FcVyRI gene. Although tyrosine

phosphorylation was required for the assembly of each of these GRR binding complexes,

only those formed as a result of treatment with IFN--y or IL-10 contained p91. Instead,

complexes activated by IL-3 or GM-CSF contained a tyrosine-phosphorylated protein of

80 kilodaltons. Induction of FcyRI RNA occurred only with IFN-y and IL-10, whereas

pretreatment of cells with GM-CSF or IL-3 inhibited IFN-y induction of FcyRI RNA. Thus,

several cytokines other than interferons can activate putative transcription factors by

tyrosine phosphorylation.

Nuclear or whole-cell extracts preparedfrom human monocytes incubated with ei-ther IFN-y or IFN-a contain a protein orproteins (FcRFy) that specifically recognizethe GRR in the promoter of the high-affinity immunoglobulin G Fc receptor gene(1-3). Within the FcRFy complex is a91-kD tyrosine-phosphorylated protein thatis a component of the ISGF-3 transcriptioncomplex, which causes IFN-a-stimulatedexpression of early response genes (2-4).Because the peripheral blood monocyte is acritical target cell for IFN-a, IFN-y, andother cytokines, experiments were done todetermine whether any cytokines otherthan the interferons might induce the for-mation of FcRFy. Whole-cell extracts wereprepared from monocytes incubated withvarious cytokines for 15 min at 37MC andanalyzed by electrophoretic mobility-shiftassays (EMSAs) with a 32P-labeled oligonu-cleotide corresponding to the GRR (Fig.1A) (5). Untreated cells showed no forma-

Division of Cytokine Biology, Center for BiologicsEvaluation and Research, Bethesda, MD 20892.

tion of FcRF^y, whereas extracts preparedfrom monocytes treated with IL-3 or GM-CSF contained GRR binding complexesthat migrated with a mobility different thanthat of the FcRFy (Fig. 1A) complex ob-served after IFN--y activation. In contrast,IL-10 activated the formation of a GRRbinding complex with a mobility similar tothat of FcRFy. Other cytokines that haveeffects on monocytes-IL-1, IL-2, IL-6,tumor necrosis factor (TNF), monocytecolony-stimulating factor (M-CSF), and li-popolysaccharide-showed no formation ofGRR binding complexes.

Binding of FcRFy and the complexesactivated by GM-CSF treatment of mono-cytes was inhibited by addition of excessunlabeled GRR (Fig. 1B), but not by addi-tion of an unlabeled oligonucleotide corre-sponding to the IFN--y activation sequence(GAS) within the promoter of the guanyl-ate-binding protein gene (Fig. 1B) (6). Thecomplexes induced by treatment of mono-cytes with IL-3 and IL-10 showed similarbinding specificities (7). When the GASoligonucleotide was used as a probe, only

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Fig. 1. Activation of GRR binding complexes by IFN-y,IL-3, IL-5, IL-10, and GM-CSF. (A) Electrophoretic mo-bility-shift assays with a 32P-labeled GRR probe andwhole-cell extracts. Human monocytes and basophilswere purified by leukaphoresis of normal volunteersfollowed by Ficoll-Hypaque sedimentation and counter-current centrifugal elutriation (1). Monocytes were thenincubated with recombinant human IFN-y (10 ng/ml;Genentech), recombinant human IL-i (100 ng/ml; Ot-suka), recombinant human IL-2 (1 gg/ml; Chiron), re-combinant human IL-3 (50 ng/ml; Amgen), recombinanthuman IL-5 (50 ng/ml; Genzyme), recombinant humanIL-6 (100 U/mI; gift of G. Tosato), recombinant humanIL-10 (50 ng/ml; Schering-Plough), recombinant humanGM-CSF (30 ng/ml; Schering-Plough), recombinant hu-man M-CSF (100 ng/ml; Genetics Institute), recombi-nant human TNF-a (10 ng/ml; Genentech), recombinanthuman TGF-P (10 ng/ml; Genzyme), or lipopolysaccha-ride (0.25 gg/ml; Sigma) for 15 min at 370C. Cell extracts were assayed forGRR binding proteins by EMSA with a 32P-labeled oligonucleotide probe[5'-AGCATGTTTCAAGGATTTGAGATGTAT1TCCCAG4AAAG-3' and itscomplement (1)] corresponding to the GRR of the human high-affinity Fcyreceptor gene. The arrows indicating the FcRFy complexes are intended

only for lane 2. Lanes 5, 7, 8, and 15 represent the shifted complexesactivated by the respective cytokine. (B) IFN-y- (lanes 1 to 3) andGM-CSF-induced (lanes 4 to 6) GRR binding complexes formed in thepresence of excess unlabeled GRR (lanes 2 and 5) or GAS (lanes 3 and6) (6).

extracts prepared from cells treated withIFN-y or IL-10 showed the IFN-.y activa-tion factor (GAF) transcription complex(7). The IFN-y-induced GAF complexcontains tyrosine-phosphorylated p91 (8).

Because the GM-CSF, IL-3, and theIL-5 receptor use a common , subunit fortransmembrane signal transduction (9), wedetermined whether IL-5 might also acti-vate the formation of a complex that boundto the GRR. Peripheral blood basophilswere treated with either IFN-y or IL-5 (Fig.lA). IL-5 treatment resulted in formationof a GRR binding complex that migratedmore slowly than FcRFy (Fig. lA). Unla-beled GRR, but not the GAS oligonucleo-tide, also competed for binding of the IL-5-inducible shift complex (7).

The ISGF-3, GAF, and FcRFy tran-scription complexes contain the tyrosine-phosphorylated protein p91 (2-4, 8, 10,1 1). To determine whether the complexesinduced by GM-CSF, IL-3, IL-5, or IL-10also contained p91, we did EMSAs aftertreatment of extracts with an antibody top91 (anti-p91) (Fig. 2A). Whereas theGRR binding complex present in extractsprepared from IFN--y- or IL-lO-treatedmonocytes was supershifted, those com-plexes activated by treatment with the oth-er cytokines were unchanged and thereforewere not recognized by anti-p91. An anti-body to the 113-kD component of ISGF-3did not form supershifted complexes withany of the GRR binding proteins.

IFN-y treatment ofmonocytes resulted inincreased concentrations ofFcyRI RNA (1).Ribonuclease protection assays were done todetermine whether any of the other cyto-kines that activated the formation of GRRbinding complexes might induce FcyRIRNA (Fig. 2B). Other than IFN-y, onlyIL-10 increased expression of Fc-yRI RNA.

Therefore, only those cytokines that activateGRR binding complexes that contain p91 arecapable of enhancing FcyRI RNA. BecauseGM-CSF and IL-3 did not induce FcyRIRNA, we pretreated monocytes with these

cytokines to determine whether they mightmodulate IFN--y-induced FcyRI RNA. Incu-bation of cells with IL-3 or GM-CSF beforeIFN--y treatment resulted in marked inhibi-tion of FcyRI RNA induction (Fig. 2C).

Flg. 2. Composition of the cytokine-induced DNA binding complexes.(A) Presence of p91 in extractsprepared from monocytes treatedwith IFN--y or IL-10. Extracts pre-pared from monocytes or basophilstreated with IFN-y (lanes 1 to 3),GM-CSF (lanes 4 to 6), IL-3 (lanes 7and 8), IL-10 (lanes 9 and 10), orIL-5 (lanes 11 and 12) were incu-bated for 1 hour at 40C either withantiserum to the 39 COOH-terminalamino acids unique to p91 (3)(lanes 2, 5, 8, 10, 12, and 14) orantiserum to the 113-kD protein ofISGF-3 (lanes 3, 6, and 15). Afterincubation with the antisera (diluted1 :100), EMSAs were done with theGRR probe. To demonstrate thespecificity of the antisera, we alsoincubated a nuclear extract pre-pared from IFN-a-treated humandiploid fibroblasts with the antibody(lanes 13 to 15). These extractswere then assayed for ISGF-3 for-mation with a 32P-labeled oligo-nucleotide corresponding to theIFN-stimulated response element(ISRE) of the ISG15 gene (11). (B)IFN-y- and IL-i0-induced expres-sion of the FcyRI RNA. Monocyteswere incubated without or with theindicated cytokines for 1 hour. Total RNA was extracted and hybridized with 32P antisense RNAprobes corresponding to FcyRI and to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (aninternal control) as described (1). The protected bands corresponding to FcyRI and GAPDH RNAsare indicated in the figure. Lane 1, control; lane 2, IFN-y; lane 3, GM-CSF; lane 4, IL-3; and lane 5,IL-10. (C) IL-3 and GMCSF inhibit IFN-y-induced FcyRI RNA expression. Monocytes wereuntreated (lane 1), treated with IFN-y alone for 1 hour (lane 2), or treated with either GMCSF (lane3) or IL-3 (lane 4) for 30 min before the addition of IFN-y for 1 hour. Total RNA was prepared andanalyzed for FcyRI and GAPDH RNA by ribonuclease protection assay.

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Assembly of the IFN-activated tran-scription complexes FcRFy, ISGF-3, andGAF requires tyrosine phosphorylation andcan be specifically disrupted by treatmentwith protein tyrosine phosphatase (PTPase)(4, 6, 11, 12). Similar experiments weredone to determine whether tyrosine phos-phorylation was required for DNA bindingby the complexes activated by IL-3, IL-5,IL-10, and GM-CSF. Whole cell extractswere incubated either without (at 4CC) orwith the addition of purified recombinantPTPase from Yersinia enterocolitca, in thepresence or absence of the PTPase inhibitororthovanadate at 30MC for 30 min. Sampleswere then assayed for the presence of GRRbinding complexes (Fig. 3). The integrityof each complex was sensitive to PTPaseregardless of the inducing cytokine. Vana-date inhibited the effects of the PTPase,confirming the specificity of the phospha-tase toward tyrosine (Fig. 3).

To further define which proteins withinthe DNA binding complexes were selec-tively tyrosine-phosphorylated in responseto cytokine treatment, we incubated mono-cytes with [32Plorthophosphate for 3 hoursand then with either IFN-y, GM-CSF,IL-3, or IL-10 for 15 min. Whole cellextracts were then prepared, and proteinswere precipitated either with anti-p91 (Fig.4A) or agarose beads coupled to the GRRoligonucleotide. Only extracts from mono-cytes treated with IFN-y or IL-10 contained32P-labeled p91 (Fig. 4A) (p91 migrateswith an apparent molecular size of 97 kDwhen it is phosphorylated). A protein of 84kD (the nature of which has not beendefined) immunoprecipitated with anti-

p91. Extracts from 32P-labeled monocytestreated with IFN-y, IL-3, or GM-CSF wereincubated with the GRR-agarose beads.Affinity-purified 32P-labeled proteins werethen resolved by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) as shown inFig. 4A. IL-3 and GM-CSF induced asso-ciation of a phosphorylated protein of 80kD with the GRR-agarose beads, whereasphosphorylated p91 was present only inextracts prepared from cells treated withIFN-y. Occasionally, a less prominentphosphorylated band of 97 kD was detectedin extracts of cells treated with GM-CSF orIL-3 (Fig. 4C). Labeling of monocytes with

[35Sjmethionine and [35S~cysteine revealedthat treatment with the same cytokinesinduced the binding of labeled proteins tothe GRR of the same molecular sizes asthose shown to be phosphorylated in thisexperiment (7).

For confirmation that the IFN-y- orIL-10-induced phosphorylation of p91 wason tyrosine, immunoprecipitates from IFN--y- or IL-10-treated monocytes were re-solved by SDS-PAGE, transferred to Immo-bilon membranes, and then probed withantibody to phosphotyrosine (Fig. 4B).Treatment of monocytes with either IFN-y-or IL-lO-induced tyrosine phosphorylation

Fig. 3. Effect of PTPase on the assembly of theGRR binding complexes. Extracts preparedfrom monocytes were incubated (40C) without(lanes 1, 4, 7, 10, and 13) or with purifiedrecombinant PTP (1 pg) at 300C for 30 minalone (lanes 2, 5, 8, 11, and 14) or in thepresence of 1 mM orthovanadate (lanes 3, 6, 9,12, and 15). The reaction mixtures were thenassayed for the presence of GRR binding com-plexes.

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of p91. However, the IL-10 immunoprecip-itate also contained a tyrosine phosphory-lated protein of 91 kD. This protein did notreact with an antibody that recognized bothp91 and its 84-kD spliced variant (7).

32P-labeled phosphoproteins bound toGRR-agarose were visualized by autoradiog-raphy and then incubated with Y. enteroco-litica PTPase (Fig. 4C) in the absence orpresence of orthovanadate. Yersiia PTPaseselectively hydrolyzed the 32P-labeled IL-3-and GM-CSF-induced 80-kD phosphopro-tein, whereas incubation of identical sam-ples in the presence of the PTPase andorthovanadate had no effect. A weakerband of 97 kD was also sensitive to PTPasetreatment. These results indicated that theproteins that associated with the GRR weretyrosine phosphorylated as a result of treat-ment with either IL-3 or GM-CSF.

Although it seems likely that IL-3, IL-5,IL-10, or GM-CSF induce the expression ofsets of early response genes that are respon-sible for their biological actions, no well-defined enhancers have been identified inthe promoters of cellular genes that arerapidly activated by these cytokines. IL-10and IEN-y activated the formation of aGRR binding complex that contained p91.The presence of p91 in these complexesappears to be important for the ultimateexpression of the FcyRI gene, because onlyIFN-y and IL-10 treatment resulted in in-creased expression of the RNA. The com-plexes activated by IL-3, IL-5, and GM-CSF do not contain either p91 or the113-kD protein of ISGF-3 (13-15), butrather a prominent phosphoprotein of 80kD. It appears that at least by their relativemigration in SDS-PAGE, none of theseproteins are identical to the 84-kD compo-nent of ISGF-3 that lacks the COOH-terminal 39 amino acids of p91 (15). Theassembly of these phosphoproteins intoDNA binding complexes appears to be ageneral mechanism by which growth factorscan modulate gene expression through ac-tivation of putative transcription factors bytyrosine phosphorylation.

REFERENCES AND NOTES

1. K. C. Wilson, D. S. Finbloom, Proc. Nati. Acad.Sci. U.S.A. 89,11964 (1992).

2. R. N. Pearse, R. Feinman, K. Shuai, J. E. DarnellJr., J. V. Ravetch, ibid. 90, 4314 (1993).

3. C. Perez, J. Wietzerbin, P. D. Benech, Mol. Cell.Biol. 13, 2182 (1993).

4. K. Igarashi, M. David, A. C. Larner, D. S. Fin-bloom, ibid., p. 3984.

5. Cells (107) were collected by centrifugation,washed with phosphate-buffered saline, and re-suspended in 200 gl of ice-cold extraction buffer[1 mM MgCI2, 20mM Hepes (pH 7.0), 10mM KCI,300 mM NaCI, 0.5 mM dithiothreitol, 0.1% TritonX-100, 200 FM phenylmethylsulfonyl fluoride, and20% glycerol]. The suspension was gently vor-texed for 10 s and allowed to incubate at 4°C for10 min. The mixture was centrifuged at 18,000gfor 10 min at 40C, and the supernatant was

transferred to a new tube. Protein concentrationswere determined and normalized by the additionof extraction buffer.

6. K. Igarashi, M. David, D. S. Finbloom, A. C.Lamer, Mol. Cell. Biol. 13,1634 (1993).

7. A. C. Larner, M. David, K. Igarashi, E. F. Petricoin,D. S. Finbloom, unpublished data.

8. K. Shuai, C. Schindler, V. R. Prezioso, J. E. DarnellJr., Science 258, 1808 (1992).

9. A. Miyajima, T. Kitamura, N. Harada, T. Yokoya, K.Arai, Annu. Rev. Immunol. 10, 295 (1992).

10. C. Schindler, K. Shuai, V. R. Prezioso, J. E. DarnellJr., Science 257, 809 (1992).

11. M. David, G. Romero, Z. Zhang, J. E. Dixon, A. C.

Larner, J. Biol. Chem. 268, 6593 (1993).12. X.-Y. Fu, Cell 70, 323 (1992).13. C. Schindler, T. Improta, R. Aebersold, J.

E. Darnell Jr., Proc. Nati. Acad. Sci. U.S.A. 89,7840 (1992).

14. X.-Y. Fu, D. S. Kessler, S. A. Veals, D. E. Levy, J.E. Darnell Jr., ibid. 87, 8555 (1990).

15. C. Schindler, X.-Y. Fu, T. Improta, R. Aebersold, J.E. Darnell Jr., ibid. 89, 7836 (1992).

16. M.D. was supported by a Schroedinger fellowshipfrom the Fonds zur Foerderung der wissenschaft-lichen Forschung, Austria.

2 July 1993; accepted 30 August 1993

Induction by EGF and Interferon-y of TyrosinePhosphorylated DNA Binding Proteins

in Mouse Liver Nuclei

Susan Ruff-Jamison, Katherine Chen, Stanley Cohen*Intraperitoneal injection of epidermal growth factor (EGF) into mice resulted in the ap-

pearance in liver nuclei of three tyrosine phosphorylated proteins (84, 91, and 92 kilo-daltons) within minutes after administration of EGF. Administration of interferon-y (IFN-y)resulted in the appearance in liver nuclei of two tyrosine phosphorylated proteins (84 and91 kilodaltons). The 84- and 91 -kilodalton proteins detected after either EGF or IFN-yadministration were identified as the IFN-y activation factors (GAF). Furthermore, gel shiftanalysis revealed that these GAF proteins, detected after either EGE or IFN-y adminis-tration, specifically bound to the sis-inducible element of the c-fos promoter. Thus, GAFproteins participate in nuclear signaling in both IFN-y and EGF pathways.

Epidermal growth factor elicits a wide rangeof physiological responses from a variety ofcell types (1-3). All of these responses arebelieved to be mediated by the membrane-spanning receptor for EGF (EGFR). Ligandbinding by the EGFR activates its intrinsictyrosine kinase activity and results in manycellular alterations, including the generationof signals that activate transcription (4-6)and the tyrosine phosphorylation of cyto-plasmic proteins (7-10). We have previouslyreported that the injection ofEGF into miceleads to increased tyrosine phosphorylationof a number of proteins in all organs exam-ined (11, 12).

To detect signaling proteins that mightbe involved in transcriptional regulation,we isolated liver nuclei and examined themfor the presence of tyrosine phosphorylatedproteins before and after the administrationof EGF (Fig. 1A). A tyrosine phosphorylat-ed protein of approximately 92 kD wasreadily apparent in nuclei 6 and 20 minafter the administration of EGF. The extentof tyrosine phosphorylation of the 92-kDprotein was diminished by 60 min and wasdetectable only in trace amounts in nucleifrom control mice. The 92-kD proteincould not be extracted from nuclei with

Department of Biochemistry, Vanderbilt UniversitySchool of Medicine, Nashville, TN 37232.

*To whom correspondence should be addressed.

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1.0% Triton X-100, indicating that theprotein was inside the nucleus and not acytoplasmic contaminant or a protein asso-ciated with the outer nuclear membrane(13). However, more than 90% of thisprotein could be extracted with 0.2 M NaCI(Fig. 1B). Because the 0.2 M NaCI extrac-tion procedure partially purified the 92-kDprotein from the nucleus, the material ex-tracted in this way was used in all subsequentprotein characterizations. EGF also inducedthe appearance of a cytoplasmic 92-kD tyro-sine phosphorylated protein (13).

Treatment of responsive cells with inter-feron-a (IFN-ct) induces the formation of atyrosine phosphorylated complex consistingof three protein subunits (IFN-stimulatedgene factor-3a, ISGF-3a) that is translo-cated into the nucleus and forms, with theaddition of a fourth subunit (ISGF-3-y), aDNA binding complex specific for the LEN-stimulated response element (ISRE) presentin promoters of IFN-a-responsive genes(14-18). Two of the components of theISGF-3a complex are a 91-kD protein andan 84-kD protein, the latter being identicalto the 91-kD species but lacking theCOOH-terminal 36 amino acids. Treat-ment of responsive cells with IFN--y inducesthe tyrosine phosphorylation of an LFN-yactivation factor (GAF), a DNA bindingprotein that binds to a sequence (IFN-,yactivation site, GAS) required for the tran-

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