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Role of Tyrosine 441 of Interferon-γ Receptor Subunit 1 in SOCS-1-mediated Attenuation of STAT1 Activation

2004; Elsevier BV; Volume: 280; Issue: 3 Linguagem: Inglês

10.1074/jbc.m409863200

ISSN

1083-351X

Autores

Yulan Qing, Ana P. Costa‐Pereira, Diane Watling, George R. Stark,

Tópico(s)

interferon and immune responses

Resumo

Suppressor of cytokine signaling (SOCS)-1, the key negative regulator of interferon (IFN)-γ-dependent signaling, is induced in response to IFNγ. SOCS-1 binds to and inhibits the IFNγ receptor-associated kinase Janus-activated kinase (JAK) 2 and inhibits its function in vitro, but the mechanism by which SOCS-1 inhibits IFNγ-dependent signaling in vivo is not clear. Upon stimulation, mouse IFNγ receptor subunit 1 (IFNGR1) is phosphorylated on several cytoplasmic tyrosine residues, and Tyr419 is required for signal transducer and activator of transcription (STAT) 1 activation in mouse embryo fibroblasts. However, the functions of the other three cytoplasmic tyrosine residues are not known. Here we show that Tyr441 is required to attenuate STAT1 activation in response to IFNγ. Several tyrosine to phenylalanine mutants of IFNGR1, expressed at normal levels in stable pools of IFNGR1-null cells, were analyzed for the phosphorylation of STAT1 during a 48-h period, and antiviral activity in response to IFNγ was also measured. Stronger activation of STAT1 was observed in cells expressing all IFNGR1 variants mutated at Tyr441, and, consistently, stronger antiviral activity was also observed in these cells. Furthermore, constitutive overexpression of SOCS-1 inhibited IFNγ-dependent signaling only in cells expressing IFNGR1 variants that included the Tyr441 mutation. Mutation of Tyr441 also blocked the ability of SOCS-1 to bind to IFNGR1 and JAK2 in response to IFNγ and the normal down-regulation of STAT1 activation and antiviral activity. These results, together with data from the literature, suggest a model in which, in response to IFNγ, phosphorylation of Tyr441 creates a docking site for SOCS-1, which then binds to JAK2 within the receptor-JAK complex to partially inhibit JAK2 phosphorylation. Furthermore, the virtually complete blockade of STAT1 phosphorylation by overexpressed SOCS-1 in this experiment suggests that the binding of SOCS-1 to Tyr441 also blocks the access of STAT1 to Tyr419 and that this effect may be the principal mechanism of inhibition of downstream signaling. Suppressor of cytokine signaling (SOCS)-1, the key negative regulator of interferon (IFN)-γ-dependent signaling, is induced in response to IFNγ. SOCS-1 binds to and inhibits the IFNγ receptor-associated kinase Janus-activated kinase (JAK) 2 and inhibits its function in vitro, but the mechanism by which SOCS-1 inhibits IFNγ-dependent signaling in vivo is not clear. Upon stimulation, mouse IFNγ receptor subunit 1 (IFNGR1) is phosphorylated on several cytoplasmic tyrosine residues, and Tyr419 is required for signal transducer and activator of transcription (STAT) 1 activation in mouse embryo fibroblasts. However, the functions of the other three cytoplasmic tyrosine residues are not known. Here we show that Tyr441 is required to attenuate STAT1 activation in response to IFNγ. Several tyrosine to phenylalanine mutants of IFNGR1, expressed at normal levels in stable pools of IFNGR1-null cells, were analyzed for the phosphorylation of STAT1 during a 48-h period, and antiviral activity in response to IFNγ was also measured. Stronger activation of STAT1 was observed in cells expressing all IFNGR1 variants mutated at Tyr441, and, consistently, stronger antiviral activity was also observed in these cells. Furthermore, constitutive overexpression of SOCS-1 inhibited IFNγ-dependent signaling only in cells expressing IFNGR1 variants that included the Tyr441 mutation. Mutation of Tyr441 also blocked the ability of SOCS-1 to bind to IFNGR1 and JAK2 in response to IFNγ and the normal down-regulation of STAT1 activation and antiviral activity. These results, together with data from the literature, suggest a model in which, in response to IFNγ, phosphorylation of Tyr441 creates a docking site for SOCS-1, which then binds to JAK2 within the receptor-JAK complex to partially inhibit JAK2 phosphorylation. Furthermore, the virtually complete blockade of STAT1 phosphorylation by overexpressed SOCS-1 in this experiment suggests that the binding of SOCS-1 to Tyr441 also blocks the access of STAT1 to Tyr419 and that this effect may be the principal mechanism of inhibition of downstream signaling. Interferon (IFN) 1The abbreviations used are: IFN, interferon; JAK, Janus-activated kinase; STAT, signal transducer and activator of transcription; SOCS, suppressor of cytokine signaling; MEF, mouse embryo fibroblast; ISG, interferon-stimulated gene; SH, Src homology. -γ plays key roles in mediating antiviral and antigrowth responses and in modulating immune responses (1Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar). The major signal transduction pathway activated by IFNγ has been elucidated through both biochemical and genetic studies. The IFNγ receptor complex consists of two receptor subunits, IFNGR1 and IFNGR2, and the tyrosine kinases Janus-activated kinase (JAK) 1 and JAK2, which bind to IFNGR1 and IFNGR2, respectively. IFNγ induces the oligomerization of the receptor subunits, leading to the activation of JAK1 and JAK2, which then phosphorylate tyrosine residues within the cytoplasmic domain of IFNGR1. Signal transducer and activator of transcription (STAT) 1 is then recruited to the receptor complex and phosphorylated on Tyr701, allowing it to be released, form homodimers, translocate to the nucleus, and bind to γ-activated sequences to activate the transcription of interferon-stimulated genes (ISGs) (1Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3388) Google Scholar, 2Schindler C. Darnell Jr., J.E. Annu. Rev. Biochem. 1995; 64: 621-651Crossref PubMed Scopus (1657) Google Scholar, 3Bach E.A. Aguet M. Schreiber R.D. Annu. Rev. Immunol. 1997; 15: 563-591Crossref PubMed Scopus (879) Google Scholar). The activation of STAT1 by IFNγ is tightly controlled by several mechanisms (4Wormald S. Hilton D.J. J. Biol. Chem. 2004; 279: 821-824Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar). The SH2-containing phosphatase 2 binds to IFNGR1 and inhibits STAT1 activation without inhibiting the phosphorylation of IFNGR1 (5You M. Yu D.H. Feng G.S. Mol. Cell. Biol. 1999; 19: 2416-2424Crossref PubMed Google Scholar). Protein inhibitor of activated STAT 1 (PIAS-1) binds to STAT1 and prevents its association with target DNA (6Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10626-10631Crossref PubMed Scopus (636) Google Scholar). Both genetic and biochemical studies have shown that suppressor of cytokine signaling (SOCS)-1 is the most potent inhibitor of IFNγ signaling (7Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google Scholar). Mice lacking SOCS-1 develop a complex fatal neonatal disease (8Starr R. Metcalf D. Elefanty A.G. Brysha M. Willson T.A. Nicola N.A. Hilton D.J. Alexander W.S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14395-14399Crossref PubMed Scopus (380) Google Scholar, 9Marine J.C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J.N. Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 10Naka T. Matsumoto T. Narazaki M. Fujimoto M. Morita Y. Ohsawa Y. Saito H. Nagasawa T. Uchiyama Y. Kishimoto T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15577-15582Crossref PubMed Scopus (260) Google Scholar), and the mortality, which results from hypersensitivity to IFNγ, is largely prevented by administration of anti-IFNγ. In addition, premature death does not occur in mice lacking both SOCS-1 and IFNγ (9Marine J.C. Topham D.J. McKay C. Wang D. Parganas E. Stravopodis D. Yoshimura A. Ihle J.N. Cell. 1999; 98: 609-616Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 11Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. 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Chem. 1998; 273: 35056-35062Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar), and in vitro studies have shown that the SOCS-1 protein inhibits the kinase activity of JAK2 by binding directly to the active site loop domain (14Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (606) Google Scholar). SOCS-1 can also direct proteins to which it binds, including guanine nucleotide exchange factor VAV, insulin receptor substrates 1 and 2, and JAK2, to proteasome-mediated degradation (15Ungureanu D. Saharinen P. Junttila I. Hilton D.J. Silvennoinen O. Mol. Cell. Biol. 2002; 22: 3316-3326Crossref PubMed Scopus (213) Google Scholar, 16Kamizono S. Hanada T. Yasukawa H. Minoguchi S. Kato R. Minoguchi M. Hattori K. Hatakeyama S. Yada M. Morita S. Kitamura T. Kato H. Nakayama K. Yoshimura A. J. Biol. 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Abraham R.T. Curr. Opin. Immunol. 1995; 7: 320-326Crossref PubMed Scopus (47) Google Scholar). Receptor phosphorylation creates docking sites for downstream signaling components and mutation of specific tyrosine residues blocks signal transduction (24Mulcahy L. Semin. Oncol. 2001; 28: 19-23Crossref PubMed Google Scholar, 25Zhu T. Goh E.L. Graichen R. Ling L. Lobie P.E. Cell Signal. 2001; 13: 599-616Crossref PubMed Scopus (221) Google Scholar, 26Heldin C.H. Westermark B. Physiol. Rev. 1999; 79: 1283-1316Crossref PubMed Scopus (1996) Google Scholar, 27Jorissen R.N. Walker F. Pouliot N. Garrett T.P. Ward C.W. Burgess A.W. Exp. Cell Res. 2003; 284: 31-53Crossref PubMed Scopus (1274) Google Scholar, 28Klint P. Claesson-Welsh L. Front. Biosci. 1999; 4: D165-D177Crossref PubMed Google Scholar). The situation for IFNγ-dependent signaling has been well studied. Activation of JAK1 and JAK2 is mediated by trans- and autophosphorylation. Phosphorylated Tyr419 of IFNGR1 creates the docking site for STAT1 in mouse embryo fibroblasts (MEFs), and phosphorylation of Tyr440 of human IFNGR1 has a major role in activating STAT1 and mediating antiviral activity. Tyrosine to phenylalanine mutation of this motif impairs STAT1 activation as well as the expression of ISGs (29Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (376) Google Scholar, 30Strobl B. Arulampalam V. Is'harc H. Newman S.J. Schlaak J.F. Watling D. Costa-Pereira A.P. Schaper F. Behrmann I. Sheehan K.C. Schreiber R.D. Horn F. Heinrich P.C. Kerr I.M. EMBO J. 2001; 20: 5431-5442Crossref PubMed Scopus (32) Google Scholar). Tyrosine phosphorylation also provides the basis for negative regulation of receptor-dependent signaling (31Sasaki A. Yasukawa H. Shouda T. Kitamura T. Dikic I. Yoshimura A. J. Biol. 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Furthermore, Tyr759 is required for the binding of SH2-containing phosphatase 2 and SOCS-3 (36Nicholson S.E. De Souza D. Fabri L.J. Corbin J. Willson T.A. Zhang J.G. Silva A. Asimakis M. Farley A. Nash A.D. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6493-6498Crossref PubMed Scopus (398) Google Scholar, 37Schmitz J. Weissenbach M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: 12848-12856Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). SH2-containing phosphatase 2 mediates the activation of mitogen-activated protein kinase in pro-B cell lines (38Fukada T. Hibi M. Yamanaka Y. Takahashi-Tezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar) and negatively regulates STAT activation in gp130-dependent signaling (39Symes A. Stahl N. Reeves S.A. Farruggella T. Servidei T. Gearan T. Yancopoulos G. Fink J.S. Curr. Biol. 1997; 7: 697-700Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar); SOCS-3 is the key negative regulator of gp130-dependent signaling (40Yasukawa H. Ohishi M. Mori H. Murakami M. Chinen T. Aki D. Hanada T. Takeda K. Akira S. Hoshijima M. Hirano T. Chien K.R. Yoshimura A. Nat. Immunol. 2003; 4: 551-556Crossref PubMed Scopus (628) Google Scholar, 41Lang R. Pauleau A.L. Parganas E. Takahashi Y. Mages J. Ihle J.N. Rutschman R. Murray P.J. Nat. Immunol. 2003; 4: 546-550Crossref PubMed Scopus (372) Google Scholar, 42Croker B.A. Krebs D.L. Zhang J.G. Wormald S. Willson T.A. Stanley E.G. Robb L. Greenhalgh C.J. Forster I. Clausen B.E. Nicola N.A. Metcalf D. Hilton D.J. Roberts A.W. Alexander W.S. Nat. Immunol. 2003; 4: 540-545Crossref PubMed Scopus (667) Google Scholar). Mutation of Tyr759 to phenylalanine enhances signal transduction in response to gp130-linked cytokines, including interleukin-6, leukemia inhibitory factor, and oncostatin M (43Anhuf D. Weissenbach M. Schmitz J. Sobota R. Hermanns H.M. Radtke S. Linnemann S. Behrmann I. Heinrich P.C. Schaper F. J. Immunol. 2000; 165: 2535-2543Crossref PubMed Scopus (42) Google Scholar, 44Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (143) Google Scholar). Furthermore, mice expressing a gp130 mutant lacking Tyr759 have splenomegaly, lymphadenopathy, and an enhanced acute phase reaction (45Ohtani T. Ishihara K. Atsumi T. Nishida K. Kaneko Y. Miyata T. Itoh S. Narimatsu M. Maeda H. Fukada T. Itoh M. Okano H. Hibi M. Hirano T. Immunity. 2000; 12: 95-105Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Here, we investigate the involvement of specific tyrosine residues of IFNGR1 in IFNγ-dependent signaling and find that Tyr441 is required for attenuation. Tyrosine to phenylalanine mutation of this residue leads to stronger STAT1 activation and antiviral activity, and inhibition of signaling in response to SOCS-1 requires Tyr441, as does the IFNγ-dependent binding of SOCS-1 to IFNGR1 and JAK2. Constructs—Plasmids expressing murine IFNGR1 and the IFNGR1 mutant Y419F were kindly provided by Dr. Robert Schreiber (Washington University, St. Louis, MO). IFNGR1 and IFNGR1 Y419F cDNAs were subcloned into pBABEpuro3. Tyrosine to phenylalanine mutations of IFNGR1 were generated by PCR-splicing overlapping extension (46Qing Y. Stark G.R. J. Biol. Chem. 2004; 279: 41679-41685Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). The identity of each plasmid was confirmed by DNA sequencing. The plasmid encoding SOCS-1, kindly provided by Dr. Ke Shuai (University of California Los Angeles, Los Angeles, CA), was used to subclone this gene into pBluescript at the XbaI site and then into pLHCX at the HindIII and HpaI sites. Biological Reagents and Cell Culture—Recombinant murine IFNγ (PeproTech, Inc., Rocky Hill, NJ) was used at 1000 IU/ml. Bosc cells (American Type Culture Collection), wild-type MEFs, and IFNGR1-null MEFs (from Dr. Robert Schreiber) (47Woldman I. Varinou L. Ramsauer K. Rapp B. Decker T. J. Biol. Chem. 2001; 276: 45722-45728Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 μg/ml penicillin G, and 100 μg/ml streptomycin. Virus-infected cells were maintained in complete medium plus 2 μg/ml puromycin or 100 μg/ml hygromycin. Western Analyses—After treatment, cells at 80% confluence in 100-mm dishes were washed once with phosphate-buffered saline, and the cell pellets were lysed for 20 min at 4 °C in 100 μl of lysis buffer containing 1% Triton X-100, 50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 10% glycerol, 0.1 mm EDTA, 10 mm sodium fluoride, 1 mm sodium orthovanadate, 1 mm phenylmethanesulfonyl fluoride, 3 μg/ml aprotinin, 2 μg/ml pepstatin, and 1 μg/ml leupeptin. Cellular debris was pelleted by centrifugation at 16,000 × g at 4 °C for 10 min. Cell extracts were fractionated by electrophoresis in 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The following antibodies were used: anti-phospho-Tyr701 STAT1 (Upstate Biotechnology), anti-N-terminal STAT1 (Transduction Laboratories), anti-phospho-JAK2 (BIOSOURCE), anti-phospho-JAK1 (BIOSOURCE), anti-IFNGR1 (Santa Cruz Biotechnology), anti-FLAG (Sigma), and anti-actin (Neomarkers). Horseradish peroxidase-coupled goat anti-rabbit or goat anti-mouse immunoglobulin was used for visualization, using the enhanced chemiluminescence Western detection system (PerkinElmer Life Sciences). Northern Analyses—Total RNA was isolated by using the TRIzol reagent (Invitrogen). The RNA (15 μg) was denatured, separated by electrophoresis in a formaldehyde-1.2% agarose gel, and transferred to Hybond-N+ nylon membranes (Amersham Biosciences). Socs-3, ip-10, irf-1, and glyceraldehyde-3-phosphate dehydrogenase (gapdh) mRNAs were detected by using cDNAs labeled with [32P]dCTP (Amersham Biosciences) by nick-translation (Megaprime DNA Labeling System; Amersham Biosciences) and visualized by autoradiography. Antiviral Assays—Cells seeded into 96-well plates at 2 × 104 cells/well were incubated overnight at 37 °C and treated with serial 10-fold dilutions of IFNγ for 18 h. Where indicated, the cells were challenged for 20 h with encephalomyocarditis virus (0.5 plaque-forming unit/cell), fixed, and stained with Giemsa. Coimmunoprecipitation—After treatment, cells at 80% confluence in 150-mm dishes were washed once with phosphate-buffered saline, and cell pellets were lysed for 20 min at 4 °C in 1 ml of buffer containing 0.5% Triton X-100, 50 mm HEPES, pH 7.4, 150 mm NaCl, 15 mm MgCl2, 0.1 mm EGTA, 10 mm sodium fluoride, 1 mm sodium orthovanadate, 1 mm phenylmethanesulfonyl fluoride, 3 μg/ml aprotinin, 2 μg/ml pepstatin, and 1 μg/ml leupeptin. Cellular debris was pelleted by centrifugation at 16,000 × g at 4 °C for 10 min. Cell lysates were incubated with anti-FLAG antibody and protein G-Sepharose (Amersham Biosciences) overnight. Immunoprecipitates were washed four times with ice-cold lysis buffer and analyzed by the Western method. Tyrosine Residues of IFNGR1 Provide Negative Regulation of IFNγ-dependent Signaling—Mouse IFNGR1 has four cytoplasmic tyrosine residues, which are phosphorylated upon stimulation with IFNγ. Tyr419 is required for the activation of STAT1 (46Qing Y. Stark G.R. J. Biol. Chem. 2004; 279: 41679-41685Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 47Woldman I. Varinou L. Ramsauer K. Rapp B. Decker T. J. Biol. Chem. 2001; 276: 45722-45728Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), but the functions of the other tyrosines are not known. Previous studies have shown that Tyr759 of gp130, which is not needed for STAT3 activation, is required for the negative regulation of this process (36Nicholson S.E. De Souza D. Fabri L.J. Corbin J. Willson T.A. Zhang J.G. Silva A. Asimakis M. Farley A. Nash A.D. Metcalf D. Hilton D.J. Nicola N.A. Baca M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6493-6498Crossref PubMed Scopus (398) Google Scholar, 37Schmitz J. Weissenbach M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: 12848-12856Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar, 39Symes A. Stahl N. Reeves S.A. Farruggella T. Servidei T. Gearan T. Yancopoulos G. Fink J.S. Curr. Biol. 1997; 7: 697-700Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). To reveal whether a tyrosine residue of IFNGR1 other than Tyr419 is similarly required for the negative regulation of IFNγ-dependent signaling, IFNGR1-null cells expressing either wild-type or 3F/419Y IFNGR1 (three of the four cytoplasmic tyrosine residues were mutated to phenylalanines) were used to examine the phosphorylation on tyrosine of STAT1, which was stronger in cells expressing 3F/419Y than in cells expressing wild-type IFNGR1 (Fig. 1a). Because phosphorylated STAT1 is essential for the transcription of most IFNγ-induced genes, the expression of three ISGs was also examined. After treatment with IFNγ, the levels of irf-1, ip-10, and socs-3 mRNAs were increased by 2-fold or more in cells expressing 3F/419Y compared with cells expressing wild-type IFNGR1 (Fig. 1b). Therefore, STAT1 activation is negatively regulated by a tyrosine residue other than Tyr419 of IFNGR1.Fig. 1Tyrosine residues are involved in regulating STAT1 phosphorylation and ISG expression. a, cells expressing wild-type (WT) IFNGR1 or the 3F/419Y mutant were treated with IFNγ for 0.25, 0.5, 1, 6, or 16 h. Total cell lysates were analyzed by the Western method, using antibodies against phospho-STAT1 (pYSTAT1) and actin. b, cells expressing wild-type (WT) IFNGR1 or the 3F/419Y mutant were treated with IFNγ for 4 h. Total RNA was isolated and analyzed by the Northern method.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Tyr441 of IFNGR1 Is Required for Negative Regulation of STAT1 Activation in Response to IFNγ—To determine which tyrosine(s) might be required, several different tyrosine to phenylalanine mutants were generated (Fig. 2). In mutant 2F/441Y/419Y, STAT1 phosphorylation was attenuated normally, and the attenuation was abolished in all mutants that include Y441F (Fig. 3a). Note that the basal levels of STAT1 in these cells are very low. Detailed time courses with single tyrosine to phenylalanine mutants confirmed that Tyr441 is the primary residue that mediates negative regulation of STAT1 activation in response to IFNγ (Fig. 3b). To test whether Tyr441 is also involved in modulating the antiviral response, an assay was performed to assess the ability of IFNγ to protect cells from the cytopathic effects of encephalomyocarditis virus in cells expressing wild-type IFNGR1 or several mutants. Cells expressing the 3F/419Y, 2F/285Y/419Y, or 2F/370Y/419Y mutants of IFNGR1 were protected at much lower doses of IFNγ than were cells expressing wild-type IFNGR1 or the 2F/441Y/419Y mutant (Fig. 3c). These data provide additional evidence that Tyr441 of IFNGR1 mediates the negative regulation of IFNγ-dependent signaling, including antiviral activity. Inhibition of IFNγ-dependent Signaling by SOCS-1 Is Mediated by Tyr441—Previous studies identified SOCS-1 as a critical inhibitor of IFNγ-dependent signaling (7Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google Scholar); SOCS-1 was expressed constitutively in cells with wild-type IFNGR1 or the 3F/419Y, 2F/441Y/419Y, and 3Y/441F mutants, and responses to IFNγ were examined. SOCS-1 blocked STAT1 phosphorylation virtually completely, but only in cells expressing wild-type IFNGR1 or the 2F/441Y/419Y mutant, and not in cells expressing the 3F/419Y or 3Y/441F mutants (Fig. 4a). Constitutive overexpression of SOCS-1 attenuated the IFNγ-dependent phosphorylation of JAK2 by about 2-fold in cells expressing wild-type IFNGR1 or the 2F/441Y/419Y mutant, but not in cells expressing the 3F/419Y or 3Y/441F mutants, and the phosphorylation of JAK1 was intact (Fig. 4b). Protein levels of JAK1 and JAK2 did not change during the time course investigated (data not shown). Previous studies have shown that SOCS-1-null mice are more resistant to viral infection than are wild-type mice (11Alexander W.S. Starr R. Fenner J.E. Scott C.L. Handman E. Sprigg N.S. Corbin J.E. Cornish A.L. Darwiche R. Owczarek C.M. Kay T.W. Nicola N.A. Hertzog P.J. Metcalf D. Hilton D.J. Cell. 1999; 98: 597-608Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar) and, conversely, that overexpression of SOCS-1 completely blocks the antiviral activity of IFNγ (13Song M.M. Shuai K. J. Biol. Chem. 1998; 273: 35056-35062Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar). Constitutively overexpressed SOCS-1 abrogated antiviral responses in cells expressing wild-type IFNGR1 or the 2F/441Y/419Y mutant but did not inhibit this response in cells carrying the 3F/419Y or 3Y/441F mutants of IFNGR1. Consistent with these results, IFNγ protected cells expressing the 3F/419Y or 3Y/441F mutants of IFNGR1 at lower doses than cells with wild-type IFNGR1 or the 2F/441Y/419Y mutant (Fig. 4c). These results indicate that Tyr441 is required to mediate the inhibitory effect of SOCS-1 in IFNγ-dependent signaling. Tyr441 Is Required for the IFNγ-dependent Association of SOCS-1 and IFNGR1—The results cited above show that inhibition of IFNγ-dependent signaling by SOCS-1 requires Tyr441 of IFNGR1. Does SOCS-1 bind to IFNGR1, and, if so, does binding require Tyr441? Coimmunoprecipitation Western analysis showed that, in response to IFNγ, association was observed only in cells expressing wild-type IFNGR1 or the 2F/441Y/419Y mutant, and not in cells with the 3F/419Y or 3Y/441F mutants. In addition, association of SOCS-1 and JAK2 was also observed only in cells with wild-type IFNGR1 or the 2F/441Y/419Y mutant (Fig. 5). These data indicate that SOCS-1 is recruited to the IFNγ receptor complex in a ligand-dependent manner and that Tyr441 is required for this association. Contribution of Specific Tyrosine Residues of IFNGR1 to IFNγ-dependent Signaling—In response to IFNγ, the phosphorylation of Tyr419 of murine IFNGR1 is absolutely required for STAT1 activation (46Qing Y. Stark G.R. J. Biol. Chem. 2004; 279: 41679-41685Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar), the antiviral response (Fig. 3c), and the transcription of ISGs in reconstituted IFNGR1-null MEFs. 2Y. Qing and G. R. Stark, unpublished data. It is interesting that the requirement for the corresponding tyrosine residue of human IFNGR1, Tyr440, appears to be different: in primary human fibroblasts lacking IFNGR1 and reconstituted with the Y440F mutant, the expression of ISGs in response to IFNγ was substantial, although the antiviral effect was inhibited. 3A. P. Costa-Pereira, D. Watling, and I. M. Kerr, unpublished data. The hybrid murine fibroblast cell line SCC16-5, containing a single copy of human chromosome 21 encoding human IFNGR2, was used initially to characterize human IFNGR1 (49Janssen J.W. Collard J.G. Tulp A. Cox D. Millington-Ward A. Pearson P. Cytometry. 1986; 7: 411-417Crossref PubMed Scopus (11) Google Scholar, 50Jung V. Rashidbaigi A. Jones C. Tischfield J.A. Shows T.B. 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EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (376) Google Scholar) (data not shown). The functions of the other phosphorylated tyrosine residues of IFNGR1 have not been investigated until now. Here we show that an important function of one of these residues is attenuation of IFNγ-dependent responses, which are stronger in IFNGR1-null MEFs expressing the 3F/419Y mutant than in cells expressing wild-type IFNGR1 (Figs. 1 and 3c). Stronger STAT1 activation in response to IFNγ was also observed in SCC16-5 cells expressing the 4F/440Y mutant, in which four of the five tyrosines in the cytoplasmic domain of IFNGR1 were mutated to phenylalanines, than in cells with wild-type human IFNGR1 (data not shown). Moreover, Tyr441 is the primary residue that mediates this attenuation (Fig. 3). Therefore, IFNGR1 uses different tyrosine residues to provide activation and feedback inhibition signals. Mutant IFNGR1 constructs were made in a retroviral expression vector, and pools of IFNGR1-null cells were generated by infection, followed by selection with puromycin. By titering the viruses, relatively even expression of different mutant IFNGR1s was achieved, at levels matching that of IFNGR1 in wild-type cells (Fig. 2). We believe that it is advantageous to use stable pools of cells, allowing one to observe an average response and to avoid being misled by differences among different cell clones. Interaction of SOCS-1 and IFNGR1—Analyses of the mechanisms of the inhibitory effects of SOCS-1 have focused on the ability of SOCS-1 to bind to the active loop of JAKs, and interaction of SOCS-1 with the interleukin-2 receptor β chain has been shown not to be required for inhibitory effects (52Sporri B. Kovanen P.E. Sasaki A. Yoshimura A. Leonard W.J. Blood. 2001; 97: 221-226Crossref PubMed Scopus (105) Google Scholar). Our results show that, in IFNγ-dependent signaling, constitutive overexpression of SOCS-1 partially inhibited JAK2 phosphorylation (Fig. 4b), and this inhibition was observed only in cells retaining Tyr441 of IFNGR1. We were also able to show that SOCS-1 binds to IFNGR1 at Tyr441 in a ligand-dependent manner, presumably through an interaction between its SH2 domain and phospho-tyrosine 441, because tyrosine to phenylalanine mutation of Tyr441 abolishes the interaction (Fig. 5). These results, together with others in the literature, suggest that SOCS-1, like SOCS-3 (the most homologous member of the SOCS family) (53Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar), is likely to inhibit cytokine-dependent signaling though its interaction with a receptor (7Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google Scholar, 31Sasaki A. Yasukawa H. Shouda T. Kitamura T. Dikic I. Yoshimura A. J. Biol. Chem. 2000; 275: 29338-29347Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 34Hortner M. Nielsch U. Mayr L.M. Johnston J.A. Heinrich P.C. Haan S. J. Immunol. 2002; 169: 1219-1227Crossref PubMed Scopus (111) Google Scholar), although SOCS-1 does interact with and inhibit JAKs in vitro (14Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (606) Google Scholar, 48Sasaki A. Yasukawa H. Suzuki A. Kamizono S. Syoda T. Kinjyo I. Sasaki M. Johnston J.A. Yoshimura A. Genes Cells. 1999; 4: 339-351Crossref PubMed Scopus (311) Google Scholar). How SOCS-1 Inhibits IFNγ-dependent Signaling—Studies in vitro have shown that SOCS-1 inhibits the kinase activity of JAK2, probably by binding to the active site loop (14Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (606) Google Scholar, 53Starr R. Willson T.A. Viney E.M. Murray L.J. Rayner J.R. Jenkins B.J. Gonda T.J. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; 387: 917-921Crossref PubMed Scopus (1816) Google Scholar), and the binding of SOCS-1 may also target JAK2 for proteasome-dependent degradation (7Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google Scholar). Our results show that SOCS-1 binds to JAK2 only in cells expressing wild-type IFNGR1 or the 2F/441Y/419Y mutant and that mutation of Tyr441 completely abrogates the IFNγ-induced binding of SOCS-1 to IFNGR1 and JAK2 (Fig. 5). Consistently, in cells expressing the 3Y/441F or 3F/419Y mutant of IFNGR1, IFNγ-dependent signaling was prolonged, revealing that the interaction of SOCS-1 and Tyr441 of IFNGR1 is required for negative regulation. A likely scenario is that, in response to IFNγ, SOCS-1 expression is induced, and SOCS-1 is recruited to IFNGR1 through phospho-tyrosine 441, which brings it close enough to JAK2 to enable it to bind to the active site loop, thus inhibiting the kinase activity and possibly also catalyzing proteasome-mediated degradation of JAK2, leading to negative feedback of IFNγ-dependent signaling. However, the relatively small effect on JAK2 phosphorylation contrasts with the dramatic effect on STAT1 phosphorylation. Therefore, it seems likely that a second mechanism is more important in vivo, namely, the binding of SOCS-1 to Tyr441 of IFNGR1 blocks the access of STAT1 to Tyr419, thus preventing STAT1 activation. We thank Dr. Robert D. Schreiber for providing IFNGR1-null cells, Dr. Ian M. Kerr for critical reading of the manuscript, Dr. Mark Jackson for technical help with retroviral infections, and Drs. Anette van Boxel-Dezaire and Xudong Liao for helpful discussions.

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