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The Stat1 Binding Motif of the Interferon-γ Receptor Is Sufficient to Mediate Stat5 Activation and Its Repression by SOCS3

2001; Elsevier BV; Volume: 276; Issue: 49 Linguagem: Inglês

10.1074/jbc.m105320200

ISSN

1083-351X

Autores

Irina Woldman, Louisa Varinou, Katrin Ramsauer, B Rapp, Thomas Decker,

Tópico(s)

NF-κB Signaling Pathways

Resumo

Signal transduction via the interferon-γ (IFN-γ) receptor requires the tyrosine phosphorylation of signal transducers and activators of transcription (Stats). Whereas tyrosine phosphorylation of Stat1 occurs in all cells, activation of Stat5 by IFN-γ is cell type-restricted. Here we investigated the mechanism of Stat5 activation by the IFN-γ receptor. In transfection assays both Stat5 isoforms, Stat5a and Stat5b, were phosphorylated on tyrosine in response to IFN-γ. Stat5 activation required the presence of tyrosine 420 (Tyr-420) in the murine IFNGR1 receptor chain, which also serves as the Stat1 binding site. Moreover, a peptide including Tyr-440, the Stat1 binding site of the human IFNGR1 chain, conferred the ability upon a synthetic receptor to activate Stat5. Suppressor of cytokine signaling 3 (SOCS3) inhibited the activation of Stat5 by the IFN-γ receptor, and the Tyr-440-containing peptide stretch was sufficient for repression. SOCS3 expression had little effect on the activity of Jak kinases not associated with cytokine receptors. In IFN-γ-treated, Stat1-deficient fibroblasts Stat5 was inefficient in inducing transcription of a Stat-dependent reporter gene, suggesting it does not per se make a major contribution to the expression of IFN-γ-responsive genes. Signal transduction via the interferon-γ (IFN-γ) receptor requires the tyrosine phosphorylation of signal transducers and activators of transcription (Stats). Whereas tyrosine phosphorylation of Stat1 occurs in all cells, activation of Stat5 by IFN-γ is cell type-restricted. Here we investigated the mechanism of Stat5 activation by the IFN-γ receptor. In transfection assays both Stat5 isoforms, Stat5a and Stat5b, were phosphorylated on tyrosine in response to IFN-γ. Stat5 activation required the presence of tyrosine 420 (Tyr-420) in the murine IFNGR1 receptor chain, which also serves as the Stat1 binding site. Moreover, a peptide including Tyr-440, the Stat1 binding site of the human IFNGR1 chain, conferred the ability upon a synthetic receptor to activate Stat5. Suppressor of cytokine signaling 3 (SOCS3) inhibited the activation of Stat5 by the IFN-γ receptor, and the Tyr-440-containing peptide stretch was sufficient for repression. SOCS3 expression had little effect on the activity of Jak kinases not associated with cytokine receptors. In IFN-γ-treated, Stat1-deficient fibroblasts Stat5 was inefficient in inducing transcription of a Stat-dependent reporter gene, suggesting it does not per se make a major contribution to the expression of IFN-γ-responsive genes. interferon INF-γ receptor erythropoietin Epo receptor γ-interferon activation site Janus kinase suppressor of cytokine signaling signal transducer and activator of transcription granulocyte-macrophage colony-stimulating factor receptors electrophoretic mobility shift assay wild type Interferon-γ (IFN-γ)1 is a cytokine with pleiotropic effects on both innate and adaptive immune responses. It activates macrophages for enhanced antibacterial performance and contributes to tumor surveillance and antiviral immunity (1Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3361) Google Scholar). The IFN-γR is composed of two different chains, IFNGR1 and IFNGR2, that bind, respectively, the Jak1 and Jak2 tyrosine kinases (2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar). Binding of IFN-γ to its receptor stimulates the activity of the Jak kinases, which phosphorylate Tyr-440 in the IFNGR1 receptor chain (3Farrar M.A. Schreiber R.D. Annu. Rev. Immunol. 1993; 11: 571-611Crossref PubMed Scopus (1248) Google Scholar, 4Bach E.A. Aguet M. Schreiber R.D. Annu. Rev. Immunol. 1997; 15: 563-591Crossref PubMed Scopus (858) Google Scholar, 5Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (373) Google Scholar, 6Greenlund A.C. Morales M.O. Viviano B.L. Yan H. Krolewski J. Schreiber R.D. Immunity. 1995; 2: 677-687Abstract Full Text PDF PubMed Scopus (248) Google Scholar). The phosphorylated tyrosine serves as a docking site for signal transducer and activator of transcription 1 (Stat1), an SH2 domain-containing transcription factor. Tyrosine phosphorylation of the receptor-associated Stat1 by the Jaks is followed by the translocation of the dimerized protein to the cell nucleus where it binds to GAS promoter sequences and induces the transcription of IFN-γ-responsive genes (7Decker T. Kovarik P. Meinke A. J. Interferon Cytokine. Res. 1997; 17: 121-134Crossref PubMed Scopus (334) Google Scholar, 8Decker T. Kovarik P. Cell. Mol. Life Sci. 1999; 55: 1535-1546Crossref PubMed Scopus (91) Google Scholar). The essential character of Stat1 in the biology of IFN-γ was demonstrated by targeted disruption of its gene in mice, which resulted in severely compromised innate immunity and adverse effects on the regulation of Th cell generation and function (9Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar, 10Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar), reviewed in Levy (11Levy D.E. Cell. Mol. Life Sci. 1999; 55: 1559-1567Crossref PubMed Scopus (67) Google Scholar).The ability of the IFN-γR to activate Stats is not restricted to Stat1. Stat3 or Stat5 tyrosine phosphorylation has been reported to occur in a number of cell types upon treatment with IFN-γ (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar, 13Caldenhoven E. Buitenhuis M. van Dijk T.B. Raaijmakers J.A. Lammers J.W. Koenderman L. de Groot R.P. J. Leukocyte Biol. 1999; 65: 391-396Crossref PubMed Scopus (49) Google Scholar). In the case of Stat5, preferential activation of only one of two Stat5 isoforms, Stat5a, was noted (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar). The molecular basis for Stat5 activation by the IFN-γR is not known, and there are at present no explanations for isoform selectivity or cell type specificity of its activation. Moreover, the biological implications of deploying Stat5 in response to IFN-γ are unclear. Mice with a targeted disruption of the Stat5a and/or Stat5b isoforms have not been analyzed for defects in IFN responses, and these investigations might be hampered by the fact that such animals develop defective T cell immunity that obscures any effects of Stat5 deficiency on the IFN-γ response (14Liu X. Robinson G.W. Wagner K.U. Garrett L. Wynshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (909) Google Scholar, 15Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (822) Google Scholar, 16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 17Moriggl R. Topham D.J. Teglund S. Sexl V. McKay C. Wang D. Hoffmeyer A. vanDeursen J. Sangster M.Y. Bunting K.D. Grosveld G.C. Ihle J.N. Immunity. 1999; 10: 249-259Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar).The suppressors of cytokine signaling (SOCS) genes are induced by activated Stats, and SOCS exert feedback inhibition on cytokine receptor signaling (reviewed in Refs. 18Hilton D.J. Cell. Mol. Life Sci. 1999; 55: 1568-1577Crossref PubMed Scopus (188) Google Scholar, 19Chen X.P. Losman J.A. Rothman P. Immunity. 2000; 13: 287-290Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 20Yasukawa H. Sasaki A. Yoshimura A. Annu. Rev. Immunol. 2000; 18: 143-164Crossref PubMed Scopus (509) Google Scholar). Different members of the SOCS family appear to apply different modes of repressing Stat activation. For example, SOCS1 (or Jab) efficiently binds to the phosphorylated activation loop of Jaks and obstructs their ability to bind and phosphorylate substrates (21Yasukawa 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 (598) Google Scholar). By contrast, the ability of SOCS3 to inhibit Jaks directly is controversial (22Nicholson S.E. Willson T.A. Farley A. Starr R. Zhang J.G. Baca M. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. EMBO J. 1999; 18: 375-385Crossref PubMed Scopus (365) Google Scholar, 23Sasaki 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 (300) Google Scholar). It appears that SOCS3 is a more potent inhibitor when bound to a cytokine receptor chain either by competing for Stat binding or through an increased inhibitory activity on Jak activity (24Cohney S.J. Sanden D. Cacalano N.A. Yoshimura A. Mui A. Migone T.S. Johnston J.A. Mol. Cell. Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (207) Google Scholar, 25Aman M.J. Migone T.S. Sasaki A. Ascherman D.P. Zhu M. Soldaini E. Imada K. Miyajima A. Yoshimura A. Leonard W.J. J. Biol. Chem. 1999; 274: 30266-30272Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 26Sasaki 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 (266) Google Scholar, 27Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). SOCS3 inhibits Stat5 activation in a number of cytokine responses, and it also inhibits tyrosine phosphorylation of Stat1 by the IFN-γ receptor (28Song M.M. Shuai K. J. Biol. Chem. 1998; 273: 35056-35062Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 29Stoiber D. Kovarik P. Cohney S. Johnston J.A. Steinlein P. Decker T. J. Immunol. 1999; 163: 2640-2647PubMed Google Scholar). The ability of SOCS3 to inhibit Stat5 activation in the context of IFN responses has not been investigated.Our study addresses the mechanism of Stat5 activation by the IFN-γR and of its inhibition by SOCS3. We report a crucial role of the peptide motif containing Tyr-440 (or Tyr-420 in the murine receptor) for both processes.DISCUSSIONThe binding of cytokines to their receptors frequently causes the activation of several different Stats. Despite this, studies in gene-targeted mice suggest that only one of these mediates the predominant biological effects of the activating cytokine (11Levy D.E. Cell. Mol. Life Sci. 1999; 55: 1559-1567Crossref PubMed Scopus (67) Google Scholar). IFN-γ causes tyrosine phosphorylation of Stat1 and, in a cell-type-restricted manner, that of Stat3 and Stat5. Stat1 deficiency alone abolishes the major immunological functions of IFN-γ like the activation of macrophages or the regulation of the adaptive immune response (9Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar, 10Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar). However, IFN-γ is a pleiotropic cytokine exerting a plethora of effects on the activation, growth, and differentiation of cells (46Trinchieri G. Perussia B. Immunol. Today. 1985; 6: 131-136Abstract Full Text PDF PubMed Scopus (574) Google Scholar,47Nacy C.A. Meltzer M.S. Curr. Opin. Immunol. 1991; 3: 330-335Crossref PubMed Scopus (59) Google Scholar). Importantly, some of the biological effects of IFN-γ, like those on the growth and differentiation of hematopoietic cells, may be redundant with other cytokines, whereas others, like the activation of macrophages, are not. Thus, one might argue that effects of Stat5 or Stat3 deficiency on the IFN-γ response are not readily overt because other cytokines compensate. Moreover, the absence of Stat3/5 in an IFN-γ response might cause only minor alterations that are hard to detect, particularly if analyzed against a background of more severe phenotypes caused by tissue-restricted Stat3 ablation (48Akira S. Oncogene. 2000; 19: 2607-2611Crossref PubMed Scopus (301) Google Scholar) or Stat5a/b deficiency (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 17Moriggl R. Topham D.J. Teglund S. Sexl V. McKay C. Wang D. Hoffmeyer A. vanDeursen J. Sangster M.Y. Bunting K.D. Grosveld G.C. Ihle J.N. Immunity. 1999; 10: 249-259Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar).In this paper we show that Stat5 activation by the IFN-γ receptor results from a promiscuity of the amino acids surrounding Tyr-420 and Tyr-440 of, respectively, the murine and human IFNGR1 chains for Stat1 and Stat5 association. Mutation of Tyr-420 strongly reduces Stat5 activation. Vice versa, addition of the Tyr-440-containing peptide to a synthetic receptor strongly stimulates the ability of that receptor to activate both Stat1 and Stat5. By contrast, the addition of the EpoR peptide containing Tyr-343, the major Stat5 binding site, specifically resulted in Stat5 but not Stat1 activation. Therefore, not all Stat5 binding sites are intrinsically promiscuous for Stat1 binding. On the other hand, receptor binding sites may be shared between Stats, as in the case of gp130, where Stat1 and Stat3 can bind to the same phosphorylated tyrosine (49Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1730) Google Scholar), or the interleukin-9 receptor, where a single tyrosine residue was shown to cause activation of Stats 1, 3, and 5 (50Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (165) Google Scholar). Some ability to activate Stat5 was retained by IFN-γ receptors containing the Y420F mutant of the IFNGR1 chain or by the ΔBR synthetic receptor without a Stat binding site. Our previous studies suggest this may be because of a direct interaction between the Jak kinase domain and Stat5 (51Barahmand-Pour F. Meinke A. Groner B. Decker T. J. Biol. Chem. 1998; 273: 12567-12575Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Our studies further show that the IFN-γ receptor has no intrinsic ability to distinguish between the Stat5a and Stat5b isoforms. This possibility was suggested by our earlier finding in hematopoietic progenitor cells where IFN-γ treatment resulted in the tyrosine phosphorylation of Stat5a but not of Stat5b despite expression of both proteins (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar). One possible explanation is that Stat5 activation by the IFN-γR is determined by threshold levels of expression that may in some cells be exceeded by Stat5a but not Stat5b. Alternatively, ancillary proteins may form complexes with Stat5a in some cells and create increased affinity for the IFN-γR complex.Compared with Stat1, Stat5, activated by the IFN-γR, displayed very little ability to support transcription of a transfected reporter gene. Similar observations were made with Stat5 in the case of GM-CSF or Epo-treated cells (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar, 52Gouilleux F. Pallard C. Dusanter Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (332) Google Scholar). The exact contribution of Stat5 to the Epo response in vivo is still controversial (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 53Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar), but the protein clearly affects the generation of monocytes/macrophages in response to GM-CSF (35Kieslinger M. Woldman I. Moriggl R. Hofmann J. Marine J.C. Ihle J.N. Beug H. Decker T. Genes Dev. 2000; 14: 232-244PubMed Google Scholar). Consistent with studies on its transactivation domain (54Moriggl R. Berchtold S. Friedrich K. Standke G.J. Kammer W. Heim M. Wissler M. Stocklin E. Gouilleux F. Groner B. Mol. Cell. Biol. 1997; 17: 3663-3678Crossref PubMed Scopus (111) Google Scholar), Stat5 may not per se be a potent transcription factor but, rather, may require interaction with other DNA-binding proteins to stimulate gene expression. Another important activity of Stat5 may lie in its ability to mediate gene repression. This assumption emerged from both studies in cultured cells (55Luo G. Yu-Lee L. J. Biol. Chem. 1997; 272: 26841-26849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and the analysis of knock-out mice (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar), which suggested a role for Stat5 in suppressing the expression of the testosterone 15α-hydroxylase gene in male mice.SOCS3 inhibited both Stat1 and Stat5 tyrosine phosphorylation by the synthetic Tyr-440 receptor with similar efficiency, suggesting that the mechanism of inhibition may be the same in both cases. In agreement with the results by others (22Nicholson S.E. Willson T.A. Farley A. Starr R. Zhang J.G. Baca M. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. EMBO J. 1999; 18: 375-385Crossref PubMed Scopus (365) Google Scholar), our findings suggest that SOCS3 inhibition of Stat1 or Stat5 activation cannot be explained entirely by the effect of the protein on Jak kinases. The gp130 Jak binding site of the synthetic receptor was shown to associate with Jak1, Jak2, and Tyk2 (56Lutticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pellegrini S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (704) Google Scholar, 57Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J.N. Yancopoulos G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (842) Google Scholar). Cytoplasmic Jak2 and Tyk2 could not be efficiently inhibited by even high amounts of co-transfected SOCS3. Surprisingly, Jak1 was inhibited by low amounts of SOCS3, but inhibition was lost with increasing SOCS3 expression. We have no explanation for this dose effect, but comparison to the dose response of Stat inhibition by the complete synthetic receptor rules out the possibility that our results are entirely due to a direct interaction between SOCS3 and Jak1. We propose that Jak inhibition by SOCS3 might be more efficient when Jaks are associated with receptors. Possibly receptor chains stabilize the Jak-SOCS complex, as has been suggested for the interleukin-2 receptor β-chain (24Cohney S.J. Sanden D. Cacalano N.A. Yoshimura A. Mui A. Migone T.S. Johnston J.A. Mol. Cell. Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (207) Google Scholar) and/or increase the ability of SOCS3 to inhibit Jaks (26Sasaki 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 (266) Google Scholar). Alternatively or additionally, SOCS3 might itself bind to the phosphorylated Tyr-440 of the synthetic receptor via its SH2 domain and compete for Stat binding. This mode of action has been suggested in the case of Epo receptor Tyr-401, which also associates with Stat5 (26Sasaki 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 (266) Google Scholar), and for the insulin receptor (27Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar).In conclusion, our studies show that promiscuity of a receptor binding site is the basis of Stat5 activation by IFN-γ and that this phosphorylated tyrosine suffices to mediate inhibition by SOCS3 in the case of both Stat1 and Stat5. We suggest that tissue-restricted Stat5 activation by the IFN-γR is determined by threshold levels of expression, but this assumption needs to be confirmed in future studies. Interferon-γ (IFN-γ)1 is a cytokine with pleiotropic effects on both innate and adaptive immune responses. It activates macrophages for enhanced antibacterial performance and contributes to tumor surveillance and antiviral immunity (1Stark G.R. Kerr I.M. Williams B.R. Silverman R.H. Schreiber R.D. Annu. Rev. Biochem. 1998; 67: 227-264Crossref PubMed Scopus (3361) Google Scholar). The IFN-γR is composed of two different chains, IFNGR1 and IFNGR2, that bind, respectively, the Jak1 and Jak2 tyrosine kinases (2Darnell Jr., J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (4950) Google Scholar). Binding of IFN-γ to its receptor stimulates the activity of the Jak kinases, which phosphorylate Tyr-440 in the IFNGR1 receptor chain (3Farrar M.A. Schreiber R.D. Annu. Rev. Immunol. 1993; 11: 571-611Crossref PubMed Scopus (1248) Google Scholar, 4Bach E.A. Aguet M. Schreiber R.D. Annu. Rev. Immunol. 1997; 15: 563-591Crossref PubMed Scopus (858) Google Scholar, 5Greenlund A.C. Farrar M.A. Viviano B.L. Schreiber R.D. EMBO J. 1994; 13: 1591-1600Crossref PubMed Scopus (373) Google Scholar, 6Greenlund A.C. Morales M.O. Viviano B.L. Yan H. Krolewski J. Schreiber R.D. Immunity. 1995; 2: 677-687Abstract Full Text PDF PubMed Scopus (248) Google Scholar). The phosphorylated tyrosine serves as a docking site for signal transducer and activator of transcription 1 (Stat1), an SH2 domain-containing transcription factor. Tyrosine phosphorylation of the receptor-associated Stat1 by the Jaks is followed by the translocation of the dimerized protein to the cell nucleus where it binds to GAS promoter sequences and induces the transcription of IFN-γ-responsive genes (7Decker T. Kovarik P. Meinke A. J. Interferon Cytokine. Res. 1997; 17: 121-134Crossref PubMed Scopus (334) Google Scholar, 8Decker T. Kovarik P. Cell. Mol. Life Sci. 1999; 55: 1535-1546Crossref PubMed Scopus (91) Google Scholar). The essential character of Stat1 in the biology of IFN-γ was demonstrated by targeted disruption of its gene in mice, which resulted in severely compromised innate immunity and adverse effects on the regulation of Th cell generation and function (9Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar, 10Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar), reviewed in Levy (11Levy D.E. Cell. Mol. Life Sci. 1999; 55: 1559-1567Crossref PubMed Scopus (67) Google Scholar). The ability of the IFN-γR to activate Stats is not restricted to Stat1. Stat3 or Stat5 tyrosine phosphorylation has been reported to occur in a number of cell types upon treatment with IFN-γ (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar, 13Caldenhoven E. Buitenhuis M. van Dijk T.B. Raaijmakers J.A. Lammers J.W. Koenderman L. de Groot R.P. J. Leukocyte Biol. 1999; 65: 391-396Crossref PubMed Scopus (49) Google Scholar). In the case of Stat5, preferential activation of only one of two Stat5 isoforms, Stat5a, was noted (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar). The molecular basis for Stat5 activation by the IFN-γR is not known, and there are at present no explanations for isoform selectivity or cell type specificity of its activation. Moreover, the biological implications of deploying Stat5 in response to IFN-γ are unclear. Mice with a targeted disruption of the Stat5a and/or Stat5b isoforms have not been analyzed for defects in IFN responses, and these investigations might be hampered by the fact that such animals develop defective T cell immunity that obscures any effects of Stat5 deficiency on the IFN-γ response (14Liu X. Robinson G.W. Wagner K.U. Garrett L. Wynshaw-Boris A. Hennighausen L. Genes Dev. 1997; 11: 179-186Crossref PubMed Scopus (909) Google Scholar, 15Udy G.B. Towers R.P. Snell R.G. Wilkins R.J. Park S.H. Ram P.A. Waxman D.J. Davey H.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7239-7244Crossref PubMed Scopus (822) Google Scholar, 16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 17Moriggl R. Topham D.J. Teglund S. Sexl V. McKay C. Wang D. Hoffmeyer A. vanDeursen J. Sangster M.Y. Bunting K.D. Grosveld G.C. Ihle J.N. Immunity. 1999; 10: 249-259Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). The suppressors of cytokine signaling (SOCS) genes are induced by activated Stats, and SOCS exert feedback inhibition on cytokine receptor signaling (reviewed in Refs. 18Hilton D.J. Cell. Mol. Life Sci. 1999; 55: 1568-1577Crossref PubMed Scopus (188) Google Scholar, 19Chen X.P. Losman J.A. Rothman P. Immunity. 2000; 13: 287-290Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 20Yasukawa H. Sasaki A. Yoshimura A. Annu. Rev. Immunol. 2000; 18: 143-164Crossref PubMed Scopus (509) Google Scholar). Different members of the SOCS family appear to apply different modes of repressing Stat activation. For example, SOCS1 (or Jab) efficiently binds to the phosphorylated activation loop of Jaks and obstructs their ability to bind and phosphorylate substrates (21Yasukawa 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 (598) Google Scholar). By contrast, the ability of SOCS3 to inhibit Jaks directly is controversial (22Nicholson S.E. Willson T.A. Farley A. Starr R. Zhang J.G. Baca M. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. EMBO J. 1999; 18: 375-385Crossref PubMed Scopus (365) Google Scholar, 23Sasaki 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 (300) Google Scholar). It appears that SOCS3 is a more potent inhibitor when bound to a cytokine receptor chain either by competing for Stat binding or through an increased inhibitory activity on Jak activity (24Cohney S.J. Sanden D. Cacalano N.A. Yoshimura A. Mui A. Migone T.S. Johnston J.A. Mol. Cell. Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (207) Google Scholar, 25Aman M.J. Migone T.S. Sasaki A. Ascherman D.P. Zhu M. Soldaini E. Imada K. Miyajima A. Yoshimura A. Leonard W.J. J. Biol. Chem. 1999; 274: 30266-30272Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 26Sasaki 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 (266) Google Scholar, 27Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). SOCS3 inhibits Stat5 activation in a number of cytokine responses, and it also inhibits tyrosine phosphorylation of Stat1 by the IFN-γ receptor (28Song M.M. Shuai K. J. Biol. Chem. 1998; 273: 35056-35062Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 29Stoiber D. Kovarik P. Cohney S. Johnston J.A. Steinlein P. Decker T. J. Immunol. 1999; 163: 2640-2647PubMed Google Scholar). The ability of SOCS3 to inhibit Stat5 activation in the context of IFN responses has not been investigated. Our study addresses the mechanism of Stat5 activation by the IFN-γR and of its inhibition by SOCS3. We report a crucial role of the peptide motif containing Tyr-440 (or Tyr-420 in the murine receptor) for both processes. DISCUSSIONThe binding of cytokines to their receptors frequently causes the activation of several different Stats. Despite this, studies in gene-targeted mice suggest that only one of these mediates the predominant biological effects of the activating cytokine (11Levy D.E. Cell. Mol. Life Sci. 1999; 55: 1559-1567Crossref PubMed Scopus (67) Google Scholar). IFN-γ causes tyrosine phosphorylation of Stat1 and, in a cell-type-restricted manner, that of Stat3 and Stat5. Stat1 deficiency alone abolishes the major immunological functions of IFN-γ like the activation of macrophages or the regulation of the adaptive immune response (9Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar, 10Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar). However, IFN-γ is a pleiotropic cytokine exerting a plethora of effects on the activation, growth, and differentiation of cells (46Trinchieri G. Perussia B. Immunol. Today. 1985; 6: 131-136Abstract Full Text PDF PubMed Scopus (574) Google Scholar,47Nacy C.A. Meltzer M.S. Curr. Opin. Immunol. 1991; 3: 330-335Crossref PubMed Scopus (59) Google Scholar). Importantly, some of the biological effects of IFN-γ, like those on the growth and differentiation of hematopoietic cells, may be redundant with other cytokines, whereas others, like the activation of macrophages, are not. Thus, one might argue that effects of Stat5 or Stat3 deficiency on the IFN-γ response are not readily overt because other cytokines compensate. Moreover, the absence of Stat3/5 in an IFN-γ response might cause only minor alterations that are hard to detect, particularly if analyzed against a background of more severe phenotypes caused by tissue-restricted Stat3 ablation (48Akira S. Oncogene. 2000; 19: 2607-2611Crossref PubMed Scopus (301) Google Scholar) or Stat5a/b deficiency (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 17Moriggl R. Topham D.J. Teglund S. Sexl V. McKay C. Wang D. Hoffmeyer A. vanDeursen J. Sangster M.Y. Bunting K.D. Grosveld G.C. Ihle J.N. Immunity. 1999; 10: 249-259Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar).In this paper we show that Stat5 activation by the IFN-γ receptor results from a promiscuity of the amino acids surrounding Tyr-420 and Tyr-440 of, respectively, the murine and human IFNGR1 chains for Stat1 and Stat5 association. Mutation of Tyr-420 strongly reduces Stat5 activation. Vice versa, addition of the Tyr-440-containing peptide to a synthetic receptor strongly stimulates the ability of that receptor to activate both Stat1 and Stat5. By contrast, the addition of the EpoR peptide containing Tyr-343, the major Stat5 binding site, specifically resulted in Stat5 but not Stat1 activation. Therefore, not all Stat5 binding sites are intrinsically promiscuous for Stat1 binding. On the other hand, receptor binding sites may be shared between Stats, as in the case of gp130, where Stat1 and Stat3 can bind to the same phosphorylated tyrosine (49Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1730) Google Scholar), or the interleukin-9 receptor, where a single tyrosine residue was shown to cause activation of Stats 1, 3, and 5 (50Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (165) Google Scholar). Some ability to activate Stat5 was retained by IFN-γ receptors containing the Y420F mutant of the IFNGR1 chain or by the ΔBR synthetic receptor without a Stat binding site. Our previous studies suggest this may be because of a direct interaction between the Jak kinase domain and Stat5 (51Barahmand-Pour F. Meinke A. Groner B. Decker T. J. Biol. Chem. 1998; 273: 12567-12575Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Our studies further show that the IFN-γ receptor has no intrinsic ability to distinguish between the Stat5a and Stat5b isoforms. This possibility was suggested by our earlier finding in hematopoietic progenitor cells where IFN-γ treatment resulted in the tyrosine phosphorylation of Stat5a but not of Stat5b despite expression of both proteins (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar). One possible explanation is that Stat5 activation by the IFN-γR is determined by threshold levels of expression that may in some cells be exceeded by Stat5a but not Stat5b. Alternatively, ancillary proteins may form complexes with Stat5a in some cells and create increased affinity for the IFN-γR complex.Compared with Stat1, Stat5, activated by the IFN-γR, displayed very little ability to support transcription of a transfected reporter gene. Similar observations were made with Stat5 in the case of GM-CSF or Epo-treated cells (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar, 52Gouilleux F. Pallard C. Dusanter Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (332) Google Scholar). The exact contribution of Stat5 to the Epo response in vivo is still controversial (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 53Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar), but the protein clearly affects the generation of monocytes/macrophages in response to GM-CSF (35Kieslinger M. Woldman I. Moriggl R. Hofmann J. Marine J.C. Ihle J.N. Beug H. Decker T. Genes Dev. 2000; 14: 232-244PubMed Google Scholar). Consistent with studies on its transactivation domain (54Moriggl R. Berchtold S. Friedrich K. Standke G.J. Kammer W. Heim M. Wissler M. Stocklin E. Gouilleux F. Groner B. Mol. Cell. Biol. 1997; 17: 3663-3678Crossref PubMed Scopus (111) Google Scholar), Stat5 may not per se be a potent transcription factor but, rather, may require interaction with other DNA-binding proteins to stimulate gene expression. Another important activity of Stat5 may lie in its ability to mediate gene repression. This assumption emerged from both studies in cultured cells (55Luo G. Yu-Lee L. J. Biol. Chem. 1997; 272: 26841-26849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and the analysis of knock-out mice (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar), which suggested a role for Stat5 in suppressing the expression of the testosterone 15α-hydroxylase gene in male mice.SOCS3 inhibited both Stat1 and Stat5 tyrosine phosphorylation by the synthetic Tyr-440 receptor with similar efficiency, suggesting that the mechanism of inhibition may be the same in both cases. In agreement with the results by others (22Nicholson S.E. Willson T.A. Farley A. Starr R. Zhang J.G. Baca M. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. EMBO J. 1999; 18: 375-385Crossref PubMed Scopus (365) Google Scholar), our findings suggest that SOCS3 inhibition of Stat1 or Stat5 activation cannot be explained entirely by the effect of the protein on Jak kinases. The gp130 Jak binding site of the synthetic receptor was shown to associate with Jak1, Jak2, and Tyk2 (56Lutticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pellegrini S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (704) Google Scholar, 57Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J.N. Yancopoulos G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (842) Google Scholar). Cytoplasmic Jak2 and Tyk2 could not be efficiently inhibited by even high amounts of co-transfected SOCS3. Surprisingly, Jak1 was inhibited by low amounts of SOCS3, but inhibition was lost with increasing SOCS3 expression. We have no explanation for this dose effect, but comparison to the dose response of Stat inhibition by the complete synthetic receptor rules out the possibility that our results are entirely due to a direct interaction between SOCS3 and Jak1. We propose that Jak inhibition by SOCS3 might be more efficient when Jaks are associated with receptors. Possibly receptor chains stabilize the Jak-SOCS complex, as has been suggested for the interleukin-2 receptor β-chain (24Cohney S.J. Sanden D. Cacalano N.A. Yoshimura A. Mui A. Migone T.S. Johnston J.A. Mol. Cell. Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (207) Google Scholar) and/or increase the ability of SOCS3 to inhibit Jaks (26Sasaki 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 (266) Google Scholar). Alternatively or additionally, SOCS3 might itself bind to the phosphorylated Tyr-440 of the synthetic receptor via its SH2 domain and compete for Stat binding. This mode of action has been suggested in the case of Epo receptor Tyr-401, which also associates with Stat5 (26Sasaki 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 (266) Google Scholar), and for the insulin receptor (27Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar).In conclusion, our studies show that promiscuity of a receptor binding site is the basis of Stat5 activation by IFN-γ and that this phosphorylated tyrosine suffices to mediate inhibition by SOCS3 in the case of both Stat1 and Stat5. We suggest that tissue-restricted Stat5 activation by the IFN-γR is determined by threshold levels of expression, but this assumption needs to be confirmed in future studies. The binding of cytokines to their receptors frequently causes the activation of several different Stats. Despite this, studies in gene-targeted mice suggest that only one of these mediates the predominant biological effects of the activating cytokine (11Levy D.E. Cell. Mol. Life Sci. 1999; 55: 1559-1567Crossref PubMed Scopus (67) Google Scholar). IFN-γ causes tyrosine phosphorylation of Stat1 and, in a cell-type-restricted manner, that of Stat3 and Stat5. Stat1 deficiency alone abolishes the major immunological functions of IFN-γ like the activation of macrophages or the regulation of the adaptive immune response (9Meraz M.A. White J.M. Sheehan K.C. Bach E.A. Rodig S.J. Dighe A.S. Kaplan D.H. Riley J.K. Greenlund A.C. Campbell D. Carver Moore K. DuBois R.N. Clark R. Aguet M. Schreiber R.D. Cell. 1996; 84: 431-442Abstract Full Text Full Text PDF PubMed Scopus (1383) Google Scholar, 10Durbin J.E. Hackenmiller R. Simon M.C. Levy D.E. Cell. 1996; 84: 443-450Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar). However, IFN-γ is a pleiotropic cytokine exerting a plethora of effects on the activation, growth, and differentiation of cells (46Trinchieri G. Perussia B. Immunol. Today. 1985; 6: 131-136Abstract Full Text PDF PubMed Scopus (574) Google Scholar,47Nacy C.A. Meltzer M.S. Curr. Opin. Immunol. 1991; 3: 330-335Crossref PubMed Scopus (59) Google Scholar). Importantly, some of the biological effects of IFN-γ, like those on the growth and differentiation of hematopoietic cells, may be redundant with other cytokines, whereas others, like the activation of macrophages, are not. Thus, one might argue that effects of Stat5 or Stat3 deficiency on the IFN-γ response are not readily overt because other cytokines compensate. Moreover, the absence of Stat3/5 in an IFN-γ response might cause only minor alterations that are hard to detect, particularly if analyzed against a background of more severe phenotypes caused by tissue-restricted Stat3 ablation (48Akira S. Oncogene. 2000; 19: 2607-2611Crossref PubMed Scopus (301) Google Scholar) or Stat5a/b deficiency (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 17Moriggl R. Topham D.J. Teglund S. Sexl V. McKay C. Wang D. Hoffmeyer A. vanDeursen J. Sangster M.Y. Bunting K.D. Grosveld G.C. Ihle J.N. Immunity. 1999; 10: 249-259Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar). In this paper we show that Stat5 activation by the IFN-γ receptor results from a promiscuity of the amino acids surrounding Tyr-420 and Tyr-440 of, respectively, the murine and human IFNGR1 chains for Stat1 and Stat5 association. Mutation of Tyr-420 strongly reduces Stat5 activation. Vice versa, addition of the Tyr-440-containing peptide to a synthetic receptor strongly stimulates the ability of that receptor to activate both Stat1 and Stat5. By contrast, the addition of the EpoR peptide containing Tyr-343, the major Stat5 binding site, specifically resulted in Stat5 but not Stat1 activation. Therefore, not all Stat5 binding sites are intrinsically promiscuous for Stat1 binding. On the other hand, receptor binding sites may be shared between Stats, as in the case of gp130, where Stat1 and Stat3 can bind to the same phosphorylated tyrosine (49Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1730) Google Scholar), or the interleukin-9 receptor, where a single tyrosine residue was shown to cause activation of Stats 1, 3, and 5 (50Demoulin J.B. Uyttenhove C. Van Roost E. DeLestre B. Donckers D. Van Snick J. Renauld J.C. Mol. Cell. Biol. 1996; 16: 4710-4716Crossref PubMed Scopus (165) Google Scholar). Some ability to activate Stat5 was retained by IFN-γ receptors containing the Y420F mutant of the IFNGR1 chain or by the ΔBR synthetic receptor without a Stat binding site. Our previous studies suggest this may be because of a direct interaction between the Jak kinase domain and Stat5 (51Barahmand-Pour F. Meinke A. Groner B. Decker T. J. Biol. Chem. 1998; 273: 12567-12575Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Our studies further show that the IFN-γ receptor has no intrinsic ability to distinguish between the Stat5a and Stat5b isoforms. This possibility was suggested by our earlier finding in hematopoietic progenitor cells where IFN-γ treatment resulted in the tyrosine phosphorylation of Stat5a but not of Stat5b despite expression of both proteins (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar). One possible explanation is that Stat5 activation by the IFN-γR is determined by threshold levels of expression that may in some cells be exceeded by Stat5a but not Stat5b. Alternatively, ancillary proteins may form complexes with Stat5a in some cells and create increased affinity for the IFN-γR complex. Compared with Stat1, Stat5, activated by the IFN-γR, displayed very little ability to support transcription of a transfected reporter gene. Similar observations were made with Stat5 in the case of GM-CSF or Epo-treated cells (12Meinke A. Barahmand-pour F. Wöhrl S. Stoiber D. Decker T. Mol. Cell. Biol. 1996; 16: 6937-6944Crossref PubMed Scopus (155) Google Scholar, 52Gouilleux F. Pallard C. Dusanter Fourt I. Wakao H. Haldosen L.A. Norstedt G. Levy D. Groner B. EMBO J. 1995; 14: 2005-2013Crossref PubMed Scopus (332) Google Scholar). The exact contribution of Stat5 to the Epo response in vivo is still controversial (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar, 53Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (613) Google Scholar), but the protein clearly affects the generation of monocytes/macrophages in response to GM-CSF (35Kieslinger M. Woldman I. Moriggl R. Hofmann J. Marine J.C. Ihle J.N. Beug H. Decker T. Genes Dev. 2000; 14: 232-244PubMed Google Scholar). Consistent with studies on its transactivation domain (54Moriggl R. Berchtold S. Friedrich K. Standke G.J. Kammer W. Heim M. Wissler M. Stocklin E. Gouilleux F. Groner B. Mol. Cell. Biol. 1997; 17: 3663-3678Crossref PubMed Scopus (111) Google Scholar), Stat5 may not per se be a potent transcription factor but, rather, may require interaction with other DNA-binding proteins to stimulate gene expression. Another important activity of Stat5 may lie in its ability to mediate gene repression. This assumption emerged from both studies in cultured cells (55Luo G. Yu-Lee L. J. Biol. Chem. 1997; 272: 26841-26849Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) and the analysis of knock-out mice (16Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1068) Google Scholar), which suggested a role for Stat5 in suppressing the expression of the testosterone 15α-hydroxylase gene in male mice. SOCS3 inhibited both Stat1 and Stat5 tyrosine phosphorylation by the synthetic Tyr-440 receptor with similar efficiency, suggesting that the mechanism of inhibition may be the same in both cases. In agreement with the results by others (22Nicholson S.E. Willson T.A. Farley A. Starr R. Zhang J.G. Baca M. Alexander W.S. Metcalf D. Hilton D.J. Nicola N.A. EMBO J. 1999; 18: 375-385Crossref PubMed Scopus (365) Google Scholar), our findings suggest that SOCS3 inhibition of Stat1 or Stat5 activation cannot be explained entirely by the effect of the protein on Jak kinases. The gp130 Jak binding site of the synthetic receptor was shown to associate with Jak1, Jak2, and Tyk2 (56Lutticken C. Wegenka U.M. Yuan J. Buschmann J. Schindler C. Ziemiecki A. Harpur A.G. Wilks A.F. Yasukawa K. Taga T. Kishimoto T. Barbieri G. Pellegrini S. Sendtner M. Heinrich P.C. Horn F. Science. 1994; 263: 89-92Crossref PubMed Scopus (704) Google Scholar, 57Stahl N. Boulton T.G. Farruggella T. Ip N.Y. Davis S. Witthuhn B.A. Quelle F.W. Silvennoinen O. Barbieri G. Pellegrini S. Ihle J.N. Yancopoulos G.D. Science. 1994; 263: 92-95Crossref PubMed Scopus (842) Google Scholar). Cytoplasmic Jak2 and Tyk2 could not be efficiently inhibited by even high amounts of co-transfected SOCS3. Surprisingly, Jak1 was inhibited by low amounts of SOCS3, but inhibition was lost with increasing SOCS3 expression. We have no explanation for this dose effect, but comparison to the dose response of Stat inhibition by the complete synthetic receptor rules out the possibility that our results are entirely due to a direct interaction between SOCS3 and Jak1. We propose that Jak inhibition by SOCS3 might be more efficient when Jaks are associated with receptors. Possibly receptor chains stabilize the Jak-SOCS complex, as has been suggested for the interleukin-2 receptor β-chain (24Cohney S.J. Sanden D. Cacalano N.A. Yoshimura A. Mui A. Migone T.S. Johnston J.A. Mol. Cell. Biol. 1999; 19: 4980-4988Crossref PubMed Scopus (207) Google Scholar) and/or increase the ability of SOCS3 to inhibit Jaks (26Sasaki 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 (266) Google Scholar). Alternatively or additionally, SOCS3 might itself bind to the phosphorylated Tyr-440 of the synthetic receptor via its SH2 domain and compete for Stat binding. This mode of action has been suggested in the case of Epo receptor Tyr-401, which also associates with Stat5 (26Sasaki 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 (266) Google Scholar), and for the insulin receptor (27Emanuelli B. Peraldi P. Filloux C. Sawka-Verhelle D. Hilton D. Van Obberghen E. J. Biol. Chem. 2000; 275: 15985-15991Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). In conclusion, our studies show that promiscuity of a receptor binding site is the basis of Stat5 activation by IFN-γ and that this phosphorylated tyrosine suffices to mediate inhibition by SOCS3 in the case of both Stat1 and Stat5. We suggest that tissue-restricted Stat5 activation by the IFN-γR is determined by threshold levels of expression, but this assumption needs to be confirmed in future studies. We thank Robert Schreiber, Silvio Hemmi and Michel Aguet for cDNAs encoding wt or mutant IFN-γR chains. We also thank Peter Heinrich and Friedemann Horn for providing plasmids encoding synthetic cytokine receptors. We thank Manuela Baccarini and Pavel Kovarik for reading and providing critical comments on this manuscript.

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