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. 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Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google were in with and cells were in complete 2 cells at in were with and the cell were for at in of 1 1 2 and 1 was by at at for Cell were by in and to The were STAT1 STAT1 and was used for the was by the The was by in a and to and were by with by DNA and by at 2 were at and with of IFNγ for the cells were for with and with cells at in were with and cell were for at in 1 of 1 1 2 and 1 was by at at for Cell were with and protein were four with and analyzed by the Tyrosine of IFNGR1 of IFNγ-dependent IFNGR1 has four cytoplasmic tyrosine residues, which are phosphorylated with IFNγ. Tyr419 is required for the activation of STAT1 Y. G.R. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, I. L. K. B. T. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), but the functions of the other are not known. studies have shown that of which is not for is required for the negative regulation of this D. Corbin J. Willson T.A. Zhang J.G. A. M. A. Metcalf D. Hilton D.J. Nicola N.A. M. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, J. M. Haan S. Heinrich P.C. Schaper F. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A. N. S.A. T. T. T. G. Curr. Biol. 1997; 7: Full Text Full Text PDF PubMed Scopus Google Scholar). a tyrosine of IFNGR1 other than Tyr419 is required for the negative regulation of IFNγ-dependent signaling, IFNGR1-null cells expressing wild-type IFNGR1 of the four cytoplasmic tyrosine residues were mutated to were used to the phosphorylation on tyrosine of which was stronger in cells expressing than in cells expressing wild-type IFNGR1 phosphorylated STAT1 is for the transcription of most the expression of three ISGs was also with IFNγ, the levels of and were by in cells expressing with cells expressing wild-type IFNGR1 STAT1 activation is by a tyrosine other than Tyr419 of residues are in STAT1 phosphorylation and cells expressing wild-type IFNGR1 the were with IFNγ for cell were analyzed by the and cells expressing wild-type IFNGR1 the were with IFNγ for was and analyzed by the Tyr441 of IFNGR1 for of STAT1 Activation in to which be several tyrosine to phenylalanine mutants were In STAT1 phosphorylation was and the was in all mutants that that the levels of STAT1 in these cells are with tyrosine to phenylalanine mutants that Tyr441 is the that negative regulation of STAT1 activation in response to IFNγ Tyr441 is also in modulating the antiviral was to the ability of IFNγ to cells from the of in cells expressing wild-type IFNGR1 several expressing the mutants of IFNGR1 were at much of IFNγ than were cells expressing wild-type IFNGR1 the These data that Tyr441 of IFNGR1 the negative regulation of IFNγ-dependent signaling, including antiviral activity. of IFNγ-dependent by SOCS-1 by studies SOCS-1 as a inhibitor of IFNγ-dependent signaling (7Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google SOCS-1 was expressed in cells with wild-type IFNGR1 the and and responses to IFNγ were SOCS-1 blocked STAT1 phosphorylation virtually but only in cells expressing wild-type IFNGR1 the and not in cells expressing the mutants overexpression of SOCS-1 the IFNγ-dependent phosphorylation of JAK2 by in cells expressing wild-type IFNGR1 the but not in cells expressing the and the phosphorylation of JAK1 was Protein levels of JAK1 and JAK2 not during the not studies have shown that mice are to 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 and, that overexpression of SOCS-1 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). overexpressed SOCS-1 antiviral responses in cells expressing wild-type IFNGR1 the but not inhibit this response in cells the mutants of IFNGR1. with these results, IFNγ cells expressing the mutants of IFNGR1 at than cells with wild-type IFNGR1 the These results that Tyr441 is required to the effect of SOCS-1 in IFNγ-dependent signaling. Tyr441 for the IFNγ-dependent of SOCS-1 and results show that inhibition of IFNγ-dependent signaling by SOCS-1 Tyr441 of IFNGR1. SOCS-1 bind to IFNGR1, and, does binding in response to IFNγ, association was observed only in cells expressing wild-type IFNGR1 the and not in cells with the In addition, association of SOCS-1 and JAK2 was also observed only in cells with wild-type IFNGR1 the These data that SOCS-1 is recruited to the IFNγ receptor complex in a and that Tyr441 is required for this of Tyrosine of IFNGR1 to IFNγ-dependent response to IFNγ, the phosphorylation of Tyr419 of IFNGR1 is required for STAT1 activation Y. G.R. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), the antiviral response and the transcription of ISGs in IFNGR1-null and G. R. is that the for the tyrosine of human IFNGR1, to be in human fibroblasts lacking IFNGR1 and with the the expression of ISGs in response to IFNγ was the antiviral effect was P. D. and I. M. The cell a of human human IFNGR2, was used to human IFNGR1 J.G. A. D. A. P. 7: PubMed Scopus Google Scholar, V. A. C. J.A. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The expression of mutants of IFNGR1 in these cells that Tyr440 is required for STAT1 activation and antiviral activity (29Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (376) Google Scholar, M.A. Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). These results, suggest that the of as well as the responses to IFNγ. the of human IFNGR1 was expressed in cells, the of mouse IFNGR1 was expressed in IFNGR1-null tyrosine phosphorylation of IFNGR1 in response to IFNγ was observed (29Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (376) Google not The functions of the other phosphorylated tyrosine residues of IFNGR1 have not been Here we show that function of of these residues is of IFNγ-dependent which are stronger in IFNGR1-null expressing the than in cells expressing wild-type IFNGR1 1 and Stronger STAT1 activation in response to IFNγ was also observed in cells expressing the in which four of the in the cytoplasmic domain of IFNGR1 were mutated to than in cells with wild-type human IFNGR1 not Tyr441 is the that this IFNGR1 tyrosine residues to activation and inhibition IFNGR1 were in a expression and pools of IFNGR1-null cells were by by with the expression of was at levels that of IFNGR1 in wild-type cells that it is to stable pools of cells, allowing to response and to by cell of SOCS-1 and of the mechanisms of the of SOCS-1 have on the ability of SOCS-1 to bind to the active loop of and of SOCS-1 with the receptor has been shown not to be required for B. P.E. Sasaki A. Yoshimura A. 2001; PubMed Scopus Google Scholar). results show in IFNγ-dependent signaling, constitutive overexpression of SOCS-1 partially inhibited JAK2 phosphorylation and this inhibition was observed only in cells Tyr441 of IFNGR1. were also to show that SOCS-1 binds to IFNGR1 at Tyr441 in a through its domain and tyrosine to phenylalanine mutation of Tyr441 the These results, together with in the literature, suggest that SOCS-1, most of the R. Willson T.A. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; PubMed Scopus Google Scholar), is to inhibit signaling its with a receptor (7Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google Scholar, 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), SOCS-1 does with and inhibit 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, A. Yasukawa H. A. S. T. I. Sasaki M. Johnston J.A. Yoshimura A. 1999; 4: PubMed Scopus Google Scholar). SOCS-1 IFNγ-dependent in vitro have shown that SOCS-1 inhibits the kinase activity of JAK2, 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, R. Willson T.A. Alexander W.S. Metcalf D. Nicola N.A. Hilton D.J. Nature. 1997; PubMed Scopus Google Scholar), and the binding of SOCS-1 may also target JAK2 for degradation (7Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (608) Google Scholar). results show that SOCS-1 binds to JAK2 only in cells expressing wild-type IFNGR1 the and that mutation of Tyr441 the binding of SOCS-1 to IFNGR1 and JAK2 in cells expressing the of IFNGR1, IFNγ-dependent signaling was that the of SOCS-1 and Tyr441 of IFNGR1 is required for negative is in response to IFNγ, SOCS-1 expression is and SOCS-1 is recruited to IFNGR1 through which it to JAK2 to it to bind to the active site inhibiting the kinase activity and also proteasome-mediated degradation of JAK2, leading to negative of IFNγ-dependent signaling. However, the effect on JAK2 phosphorylation with the effect on STAT1 phosphorylation. it that a mechanism is in the binding of SOCS-1 to Tyr441 of IFNGR1 blocks the access of STAT1 to STAT1 D. Schreiber for IFNGR1-null cells, M. Kerr for of the for with and and Liao for
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