Identification of a STAT4 Binding Site in the Interleukin-12 Receptor Required for Signaling (1999)

The specificity of the various STAT SH2 domains for different tyrosine-containing peptides enables cytokines to activate different signaling pathways and to induce distinct patterns of gene expression. We show that STAT4 has a unique peptide specificity and binds to the peptide sequence pYLPSNID (where pY represents phosphotyrosine). This motif is found at tyrosine residue 800 in the β2 subunit of the interleukin-12 receptor and is required for DNA binding and transcriptional activity of STAT4. Our data demonstrate that transfection of interleukin-12 receptor β1 and β2 subunits is sufficient for STAT4 activation but not for STAT1 or STAT3 activation. The specificity of the various STAT SH2 domains for different tyrosine-containing peptides enables cytokines to activate different signaling pathways and to induce distinct patterns of gene expression. We show that STAT4 has a unique peptide specificity and binds to the peptide sequence pYLPSNID (where pY represents phosphotyrosine). This motif is found at tyrosine residue 800 in the β2 subunit of the interleukin-12 receptor and is required for DNA binding and transcriptional activity of STAT4. Our data demonstrate that transfection of interleukin-12 receptor β1 and β2 subunits is sufficient for STAT4 activation but not for STAT1 or STAT3 activation. interleukin interferon IL-12 receptor cytomegalovirus electrophoretic mobility shift assay. IL-121 is a heterodimeric cytokine secreted from antigen presenting cells in response to bacteria and intracellular parasites and thus plays an important role in host defense against bacterial pathogens (1Trinchieri G. Annu. Rev. Immunol. 1995; 13: 251-276Crossref PubMed Scopus (2217) Google Scholar). IL-12 promotes the proliferation of T cells and NK cells and is required for the differentiation of T cells into the Th1 subset of T helper cells. Th1 cells are critical for cell-mediated immune responses, because they secrete IFN-γ, which enhances the activity of cytotoxic T cells and NK cells (1Trinchieri G. Annu. Rev. Immunol. 1995; 13: 251-276Crossref PubMed Scopus (2217) Google Scholar, 2Hsieh C. Macatonia S.E. Tripp C.S. Wolf S.F. O'Garra A. Murphy K.M. Science. 1993; 260: 547-549Crossref PubMed Scopus (2861) Google Scholar, 3Manetti R. Parronchi P. Guidizi M.G. Piccinni M.-P. Maggi E. Trinchieri G. Romagnani S. J. Exp. Med. 1993; 177: 1199-1204Crossref PubMed Scopus (1639) Google Scholar). In addition to these essential functions, Th1-dominated responses are associated with pathologic autoimmune and inflammatory conditions such as rheumatoid arthritis and inflammatory bowel disease (4Romagnani S. Clin. Immunol. Immunopathol. 1996; 80: 225-235Crossref PubMed Scopus (370) Google Scholar). Given this potential for immunopathology, it is important to understand the mechanisms that control the Th1 response to develop possible therapeutic interventions.IL-12 signals through the IL-12 receptor, which is composed of at least two subunits designated β1 and β2 (5Chua A.O. Chizzonite R. Desai B.B. Truitt T.P. Nunes P. Minetti L.J. Warrier R.J. Presky D.H. Levine J.F. Gately M.K. Gubler U. J. Immunol. 1994; 153: 128-136PubMed Google Scholar, 6Presky D.H. Yang H. Minetti L.J. Chua A.O. Nabavi N. Wu C.Y. Gately M.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14002-14007Crossref PubMed Scopus (574) Google Scholar). Both IL-12 receptor subunits are members of the hemapoietin receptor superfamily and have strong homology to the gp130 receptor (5Chua A.O. Chizzonite R. Desai B.B. Truitt T.P. Nunes P. Minetti L.J. Warrier R.J. Presky D.H. Levine J.F. Gately M.K. Gubler U. J. Immunol. 1994; 153: 128-136PubMed Google Scholar). The β1 receptor, although a low affinity binder of IL-12, is not capable of transducing an IL-12-mediated signal (6Presky D.H. Yang H. Minetti L.J. Chua A.O. Nabavi N. Wu C.Y. Gately M.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14002-14007Crossref PubMed Scopus (574) Google Scholar). A second subunit of the IL-12 receptor was subsequently identified that when coexpressed with the β1 subunit forms a high affinity receptor for IL-12 and confers IL-12 signaling (6Presky D.H. Yang H. Minetti L.J. Chua A.O. Nabavi N. Wu C.Y. Gately M.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14002-14007Crossref PubMed Scopus (574) Google Scholar). IL-12R β2 expression is differentially regulated in Th1versus Th2 cells (7Szabo S.J. Dighe A.S. Gubler U. Murphy K.M. J. Exp. Med. 1997; 185: 817-824Crossref PubMed Scopus (866) Google Scholar). Th1 cells but not Th2 cells express the β2 subunit of the IL-12 receptor (8Rogge L. Barberis-Maino L. Biffi M. Passini N. Presky D.H. Gubler U. Sinigaglia F. J. Exp. Med. 1997; 185: 825-831Crossref PubMed Scopus (673) Google Scholar). Following T cell activation, IL-12 and IFN-γ treatment induces β2 expression, whereas IL-4, a cytokine produced by Th2 cells, inhibits β2 expression resulting in loss of IL-12 signaling (7Szabo S.J. Dighe A.S. Gubler U. Murphy K.M. J. Exp. Med. 1997; 185: 817-824Crossref PubMed Scopus (866) Google Scholar, 8Rogge L. Barberis-Maino L. Biffi M. Passini N. Presky D.H. Gubler U. Sinigaglia F. J. Exp. Med. 1997; 185: 825-831Crossref PubMed Scopus (673) Google Scholar). Therefore, expression of the β2 subunit of the IL-12 receptor is a crucial determinant of Th1 versus Th2 development.Cytokine binding to its receptor leads to activation of JAK kinases that phosphorylate the receptor on tyrosines located in the intracellular domain. The phosphorylated regions are binding sites for signal transduction molecules called STATs that are rapidly recruited to the receptor and tyrosine phosphorylated by JAK kinases. Tyrosine phosphorylation of STAT proteins induces their dimerization and translocation to the nucleus where they bind to specific DNA sequences and regulate transcription. IL-12 stimulation results in TYK2 and JAK2 phosphorylation and interaction of TYK2 with the β1 subunit of the IL-12 receptor and interaction of JAK2 with the β2 subunit (9Bacon C.B. McVicar D.W. Ortaldo J.R. Rees R.C. O'Shea J.J. Johnston J.A. J. Exp. Med. 1995; 181: 399-404Crossref PubMed Scopus (291) Google Scholar, 10Zou J. Presky D.H. Wu C.-Y. Gubler U. J. Biol. Chem. 1997; 272: 6073-6077Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Unlike STAT4, whose expression is limited to lymphoid and spermatogonia cells, JAK2 and TYK2 are ubiquitously expressed (11Zhong Z. Wen Z. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4806-4810Crossref PubMed Scopus (338) Google Scholar, 12Yamamoto K. Quelle F.W. Thierfelder W.E. Kreider B.L. Gilbert D.J. Jenkins N.A. Copeland N.G. Silvennoinen O. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4342-4349Crossref PubMed Scopus (204) Google Scholar).In response to IL-12, STAT4 has been shown to be tyrosine phosphorylated and activated in Th1 lymphocytes and NK cells (9Bacon C.B. McVicar D.W. Ortaldo J.R. Rees R.C. O'Shea J.J. Johnston J.A. J. Exp. Med. 1995; 181: 399-404Crossref PubMed Scopus (291) Google Scholar, 13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google Scholar). In addition, it has been reported that STAT1, STAT3, and STAT5 can be activated in response to IL-12 (13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google Scholar, 14Yu C.-R. Lin J.-X. Fink D.W. Akira S. Bloom E.T. Yamauchi A. J. Immunol. 1996; 157: 126-137PubMed Google Scholar, 15Gollob J.A. Murphy E.A. Mahajan S. Schnipper C.P. Ritz J. Frank D.A. Blood. 91: PubMed Google by of their SH2 with specific on cytokine U. 1996; PubMed Scopus Google Scholar). the binding sites of the STAT proteins have been the tyrosine of STAT4 has not been The β2 subunit of the IL-12 receptor tyrosines in its whereas the β1 subunit tyrosine in its (5Chua A.O. Chizzonite R. Desai B.B. Truitt T.P. Nunes P. Minetti L.J. Warrier R.J. Presky D.H. Levine J.F. Gately M.K. Gubler U. J. Immunol. 1994; 153: 128-136PubMed Google Scholar, 6Presky D.H. Yang H. Minetti L.J. Chua A.O. Nabavi N. Wu C.Y. Gately M.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14002-14007Crossref PubMed Scopus (574) Google Scholar). Therefore, the of tyrosine phosphorylation in cytokine signaling on the tyrosines of the β2 subunit as possible sites for the STAT4 signaling in response to that STAT4 is activated through interaction with the tyrosine at 800 in the IL-12 receptor β2 STAT4 activation on interaction with the peptide sequence pYLPSNID (where pY represents thus for the specificity of the STAT4 SH2 for a unique binding in the IL-12 In addition, show that STAT1 STAT3 is activated through the IL-12 stimulation results in activation of STAT4 through interaction with the IL-12 receptor (13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google Scholar, Ortaldo J.R. Rees R.C. Johnston J.A. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). In response to IL-12 STAT4 is tyrosine phosphorylated and is for DNA binding and transcriptional activation of STAT4 is for the IL-12 response and is crucial for Th1 and IFN-γ as shown by STAT4 1996; PubMed Scopus Google Scholar, W.E. K. Tripp D.A. Ihle J.N. 1996; PubMed Scopus Google binding by STAT proteins has been shown to be specific and by the SH2 of the STAT proteins in R.M. PubMed Scopus Google Jr., J.E. Science. 1994; PubMed Scopus Google Scholar, and C. Darnell Jr., J.E. Annu. Rev. 1995; PubMed Scopus Google Scholar). STAT interaction with cytokine on the residue in the receptor and the to the that In this demonstrate that STAT4 is activated by with the IL-12 receptor and that the interaction of the STAT4 SH2 is specific for the peptide sequence the peptide at tyrosine 800 in the IL-12R β2 This tyrosine at 800 in the IL-12R β2 subunit is required for STAT4 DNA binding activity and transcriptional activation of the STAT4 is not capable of binding the peptide sequences at tyrosine or of the IL-12 β2 and these peptide sequences are not required for the activation of STAT4. The IL-12R peptide sequence pYLPSNID to be a specific binding for STAT4. STAT1, which has a SH2 the IL-12R pYLPSNID STAT1 and STAT3 are activated by a of cytokines C. Darnell Jr., J.E. Annu. Rev. 1995; PubMed Scopus Google Scholar, K. Darnell Jr., J.E. Science. 1993; PubMed Scopus Google Scholar). it has been reported that STAT1 and STAT3 are activated in response to (13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google reported that IL-12 tyrosine phosphorylation of STAT3 and DNA binding activity of STAT3 in addition to STAT4. that STAT3 was of a DNA that STAT4, that STAT3 and STAT4 C.-R. Lin J.-X. Fink D.W. Akira S. Bloom E.T. Yamauchi A. J. Immunol. 1996; 157: 126-137PubMed Google STAT4, and STAT3 treatment of NK cells with not the of STAT1, STAT3, or STAT4 with the IL-12 in STAT1 STAT3 was activated by IL-12 through the IL-12 receptor subunits conditions in which STAT4 was STAT1 and STAT3 be recruited to the IL-12 receptor through that as an and is not in the and in peptides have been shown to binding specificity N. Zhong Z. Darnell Jr., J.E. Science. 1995; PubMed Scopus Google Scholar). the sites for STAT3, for have a residue at the to of the tyrosine peptides in the IL-12 β2 receptor a in the they sequence to the STAT1 in the IFN-γ receptor, B.L. Schreiber R.D. J. 1994; 13: PubMed Scopus Google Scholar, Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This is with results that STAT1 and STAT3 are not activated by with the IL-12 specificity of the various STAT SH2 domains for different tyrosine-containing peptides results in activation of specific cytokine signaling pathways and the of distinct gene expression We that STAT4 has a unique peptide binding specificity the STAT and binds to the peptide sequence The unique specificity of STAT4 for its tyrosine-containing peptide sequence the distinct of IL-12 binding peptides from cytokine have the to STAT and STAT tyrosine J. U. M. Science. 1994; PubMed Scopus Google Scholar, B.L. Schreiber R.D. J. 1994; 13: PubMed Scopus Google Scholar, H. S. Schreiber R.D. C. J.J. J. 1996; PubMed Scopus Google Scholar). be possible to develop to the the thus STAT activation and to IL-12 signaling by the β1 and β2 subunits of the IL-12 receptor the of IL-12 receptor and In addition to activation of the resulting in STAT tyrosine IL-12 STAT4 phosphorylation by or C. Rees R.C. R. O'Shea J.J. J. Immunol. 1996; 157: Google Scholar, C. L. Ritz J. Blood. 1994; PubMed Google Scholar, C. M. Ritz J. Cell. Immunol. 1995; PubMed Scopus Google Scholar). be to which regions of the receptor are required for these signaling IL-121 is a heterodimeric cytokine secreted from antigen presenting cells in response to bacteria and intracellular parasites and thus plays an important role in host defense against bacterial pathogens (1Trinchieri G. Annu. Rev. Immunol. 1995; 13: 251-276Crossref PubMed Scopus (2217) Google Scholar). IL-12 promotes the proliferation of T cells and NK cells and is required for the differentiation of T cells into the Th1 subset of T helper cells. Th1 cells are critical for cell-mediated immune responses, because they secrete IFN-γ, which enhances the activity of cytotoxic T cells and NK cells (1Trinchieri G. Annu. Rev. Immunol. 1995; 13: 251-276Crossref PubMed Scopus (2217) Google Scholar, 2Hsieh C. Macatonia S.E. Tripp C.S. Wolf S.F. O'Garra A. Murphy K.M. Science. 1993; 260: 547-549Crossref PubMed Scopus (2861) Google Scholar, 3Manetti R. Parronchi P. Guidizi M.G. Piccinni M.-P. Maggi E. Trinchieri G. Romagnani S. J. Exp. Med. 1993; 177: 1199-1204Crossref PubMed Scopus (1639) Google Scholar). In addition to these essential functions, Th1-dominated responses are associated with pathologic autoimmune and inflammatory conditions such as rheumatoid arthritis and inflammatory bowel disease (4Romagnani S. Clin. Immunol. Immunopathol. 1996; 80: 225-235Crossref PubMed Scopus (370) Google Scholar). Given this potential for immunopathology, it is important to understand the mechanisms that control the Th1 response to develop possible therapeutic IL-12 signals through the IL-12 receptor, which is composed of at least two subunits designated β1 and β2 (5Chua A.O. Chizzonite R. Desai B.B. Truitt T.P. Nunes P. Minetti L.J. Warrier R.J. Presky D.H. Levine J.F. Gately M.K. Gubler U. J. Immunol. 1994; 153: 128-136PubMed Google Scholar, 6Presky D.H. Yang H. Minetti L.J. Chua A.O. Nabavi N. Wu C.Y. Gately M.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14002-14007Crossref PubMed Scopus (574) Google Scholar). Both IL-12 receptor subunits are members of the hemapoietin receptor superfamily and have strong homology to the gp130 receptor (5Chua A.O. Chizzonite R. Desai B.B. Truitt T.P. Nunes P. Minetti L.J. Warrier R.J. Presky D.H. Levine J.F. Gately M.K. Gubler U. J. Immunol. 1994; 153: 128-136PubMed Google Scholar). The β1 receptor, although a low affinity binder of IL-12, is not capable of transducing an IL-12-mediated signal (6Presky D.H. Yang H. Minetti L.J. Chua A.O. Nabavi N. Wu C.Y. Gately M.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14002-14007Crossref PubMed Scopus (574) Google Scholar). A second subunit of the IL-12 receptor was subsequently identified that when coexpressed with the β1 subunit forms a high affinity receptor for IL-12 and confers IL-12 signaling (6Presky D.H. Yang H. Minetti L.J. Chua A.O. Nabavi N. Wu C.Y. Gately M.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14002-14007Crossref PubMed Scopus (574) Google Scholar). IL-12R β2 expression is differentially regulated in Th1versus Th2 cells (7Szabo S.J. Dighe A.S. Gubler U. Murphy K.M. J. Exp. Med. 1997; 185: 817-824Crossref PubMed Scopus (866) Google Scholar). Th1 cells but not Th2 cells express the β2 subunit of the IL-12 receptor (8Rogge L. Barberis-Maino L. Biffi M. Passini N. Presky D.H. Gubler U. Sinigaglia F. J. Exp. Med. 1997; 185: 825-831Crossref PubMed Scopus (673) Google Scholar). Following T cell activation, IL-12 and IFN-γ treatment induces β2 expression, whereas IL-4, a cytokine produced by Th2 cells, inhibits β2 expression resulting in loss of IL-12 signaling (7Szabo S.J. Dighe A.S. Gubler U. Murphy K.M. J. Exp. Med. 1997; 185: 817-824Crossref PubMed Scopus (866) Google Scholar, 8Rogge L. Barberis-Maino L. Biffi M. Passini N. Presky D.H. Gubler U. Sinigaglia F. J. Exp. Med. 1997; 185: 825-831Crossref PubMed Scopus (673) Google Scholar). Therefore, expression of the β2 subunit of the IL-12 receptor is a crucial determinant of Th1 versus Th2 binding to its receptor leads to activation of JAK kinases that phosphorylate the receptor on tyrosines located in the intracellular domain. The phosphorylated regions are binding sites for signal transduction molecules called STATs that are rapidly recruited to the receptor and tyrosine phosphorylated by JAK kinases. Tyrosine phosphorylation of STAT proteins induces their dimerization and translocation to the nucleus where they bind to specific DNA sequences and regulate transcription. IL-12 stimulation results in TYK2 and JAK2 phosphorylation and interaction of TYK2 with the β1 subunit of the IL-12 receptor and interaction of JAK2 with the β2 subunit (9Bacon C.B. McVicar D.W. Ortaldo J.R. Rees R.C. O'Shea J.J. Johnston J.A. J. Exp. Med. 1995; 181: 399-404Crossref PubMed Scopus (291) Google Scholar, 10Zou J. Presky D.H. Wu C.-Y. Gubler U. J. Biol. Chem. 1997; 272: 6073-6077Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Unlike STAT4, whose expression is limited to lymphoid and spermatogonia cells, JAK2 and TYK2 are ubiquitously expressed (11Zhong Z. Wen Z. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4806-4810Crossref PubMed Scopus (338) Google Scholar, 12Yamamoto K. Quelle F.W. Thierfelder W.E. Kreider B.L. Gilbert D.J. Jenkins N.A. Copeland N.G. Silvennoinen O. Ihle J.N. Mol. Cell. Biol. 1994; 14: 4342-4349Crossref PubMed Scopus (204) Google Scholar). In response to IL-12, STAT4 has been shown to be tyrosine phosphorylated and activated in Th1 lymphocytes and NK cells (9Bacon C.B. McVicar D.W. Ortaldo J.R. Rees R.C. O'Shea J.J. Johnston J.A. J. Exp. Med. 1995; 181: 399-404Crossref PubMed Scopus (291) Google Scholar, 13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google Scholar). In addition, it has been reported that STAT1, STAT3, and STAT5 can be activated in response to IL-12 (13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google Scholar, 14Yu C.-R. Lin J.-X. Fink D.W. Akira S. Bloom E.T. Yamauchi A. J. Immunol. 1996; 157: 126-137PubMed Google Scholar, 15Gollob J.A. Murphy E.A. Mahajan S. Schnipper C.P. Ritz J. Frank D.A. Blood. 91: PubMed Google Scholar). STAT by of their SH2 with specific on cytokine U. 1996; PubMed Scopus Google Scholar). the binding sites of the STAT proteins have been the tyrosine of STAT4 has not been The β2 subunit of the IL-12 receptor tyrosines in its whereas the β1 subunit tyrosine in its (5Chua A.O. Chizzonite R. Desai B.B. Truitt T.P. Nunes P. Minetti L.J. Warrier R.J. Presky D.H. Levine J.F. Gately M.K. Gubler U. J. Immunol. 1994; 153: 128-136PubMed Google Scholar, 6Presky D.H. Yang H. Minetti L.J. Chua A.O. Nabavi N. Wu C.Y. Gately M.K. Gubler U. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14002-14007Crossref PubMed Scopus (574) Google Scholar). Therefore, the of tyrosine phosphorylation in cytokine signaling on the tyrosines of the β2 subunit as possible sites for the STAT4 signaling in response to We that STAT4 is activated through interaction with the tyrosine at 800 in the IL-12 receptor β2 STAT4 activation on interaction with the peptide sequence pYLPSNID (where pY represents thus for the specificity of the STAT4 SH2 for a unique binding in the IL-12 In addition, show that STAT1 STAT3 is activated through the IL-12 stimulation results in activation of STAT4 through interaction with the IL-12 receptor (13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google Scholar, Ortaldo J.R. Rees R.C. Johnston J.A. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). In response to IL-12 STAT4 is tyrosine phosphorylated and is for DNA binding and transcriptional activation of STAT4 is for the IL-12 response and is crucial for Th1 and IFN-γ as shown by STAT4 1996; PubMed Scopus Google Scholar, W.E. K. Tripp D.A. Ihle J.N. 1996; PubMed Scopus Google binding by STAT proteins has been shown to be specific and by the SH2 of the STAT proteins in R.M. PubMed Scopus Google Jr., J.E. Science. 1994; PubMed Scopus Google Scholar, and C. Darnell Jr., J.E. Annu. Rev. 1995; PubMed Scopus Google Scholar). STAT interaction with cytokine on the residue in the receptor and the to the that In this demonstrate that STAT4 is activated by with the IL-12 receptor and that the interaction of the STAT4 SH2 is specific for the peptide sequence the peptide at tyrosine 800 in the IL-12R β2 This tyrosine at 800 in the IL-12R β2 subunit is required for STAT4 DNA binding activity and transcriptional activation of the STAT4 is not capable of binding the peptide sequences at tyrosine or of the IL-12 β2 and these peptide sequences are not required for the activation of STAT4. The IL-12R peptide sequence pYLPSNID to be a specific binding for STAT4. STAT1, which has a SH2 the IL-12R pYLPSNID STAT1 and STAT3 are activated by a of cytokines C. Darnell Jr., J.E. Annu. Rev. 1995; PubMed Scopus Google Scholar, K. Darnell Jr., J.E. Science. 1993; PubMed Scopus Google Scholar). it has been reported that STAT1 and STAT3 are activated in response to (13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google reported that IL-12 tyrosine phosphorylation of STAT3 and DNA binding activity of STAT3 in addition to STAT4. that STAT3 was of a DNA that STAT4, that STAT3 and STAT4 C.-R. Lin J.-X. Fink D.W. Akira S. Bloom E.T. Yamauchi A. J. Immunol. 1996; 157: 126-137PubMed Google STAT4, and STAT3 treatment of NK cells with not the of STAT1, STAT3, or STAT4 with the IL-12 in STAT1 STAT3 was activated by IL-12 through the IL-12 receptor subunits conditions in which STAT4 was STAT1 and STAT3 be recruited to the IL-12 receptor through that as an and is not in the and in peptides have been shown to binding specificity N. Zhong Z. Darnell Jr., J.E. Science. 1995; PubMed Scopus Google Scholar). the sites for STAT3, for have a residue at the to of the tyrosine peptides in the IL-12 β2 receptor a in the they sequence to the STAT1 in the IFN-γ receptor, B.L. Schreiber R.D. J. 1994; 13: PubMed Scopus Google Scholar, Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This is with results that STAT1 and STAT3 are not activated by with the IL-12 specificity of the various STAT SH2 domains for different tyrosine-containing peptides results in activation of specific cytokine signaling pathways and the of distinct gene expression We that STAT4 has a unique peptide binding specificity the STAT and binds to the peptide sequence The unique specificity of STAT4 for its tyrosine-containing peptide sequence the distinct of IL-12 binding peptides from cytokine have the to STAT and STAT tyrosine J. U. M. Science. 1994; PubMed Scopus Google Scholar, B.L. Schreiber R.D. J. 1994; 13: PubMed Scopus Google Scholar, H. S. Schreiber R.D. C. J.J. J. 1996; PubMed Scopus Google Scholar). be possible to develop to the the thus STAT activation and to IL-12 signaling by the β1 and β2 subunits of the IL-12 receptor the of IL-12 receptor and In addition to activation of the resulting in STAT tyrosine IL-12 STAT4 phosphorylation by or C. Rees R.C. R. O'Shea J.J. J. Immunol. 1996; 157: Google Scholar, C. L. Ritz J. Blood. 1994; PubMed Google Scholar, C. M. Ritz J. Cell. Immunol. 1995; PubMed Scopus Google Scholar). be to which regions of the receptor are required for these signaling IL-12 stimulation results in activation of STAT4 through interaction with the IL-12 receptor (13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google Scholar, Ortaldo J.R. Rees R.C. Johnston J.A. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). In response to IL-12 STAT4 is tyrosine phosphorylated and is for DNA binding and transcriptional activation of STAT4 is for the IL-12 response and is crucial for Th1 and IFN-γ as shown by STAT4 1996; PubMed Scopus Google Scholar, W.E. K. Tripp D.A. Ihle J.N. 1996; PubMed Scopus Google Scholar). binding by STAT proteins has been shown to be specific and by the SH2 of the STAT proteins in R.M. PubMed Scopus Google Jr., J.E. Science. 1994; PubMed Scopus Google Scholar, and C. Darnell Jr., J.E. Annu. Rev. 1995; PubMed Scopus Google Scholar). STAT interaction with cytokine on the residue in the receptor and the to the that In this demonstrate that STAT4 is activated by with the IL-12 receptor and that the interaction of the STAT4 SH2 is specific for the peptide sequence the peptide at tyrosine 800 in the IL-12R β2 This tyrosine at 800 in the IL-12R β2 subunit is required for STAT4 DNA binding activity and transcriptional activation of the STAT4 is not capable of binding the peptide sequences at tyrosine or of the IL-12 β2 and these peptide sequences are not required for the activation of STAT4. The IL-12R peptide sequence pYLPSNID to be a specific binding for STAT4. STAT1, which has a SH2 the IL-12R pYLPSNID STAT1 and STAT3 are activated by a of cytokines C. Darnell Jr., J.E. Annu. Rev. 1995; PubMed Scopus Google Scholar, K. Darnell Jr., J.E. Science. 1993; PubMed Scopus Google Scholar). it has been reported that STAT1 and STAT3 are activated in response to (13Jacobson N.G. Szabo S.J. Weber-Nordt R.M. Zhong Z. Schreiber R.D. Murphy K.M. J. Exp. Med. 1995; 181: 1755-1762Crossref PubMed Scopus (578) Google reported that IL-12 tyrosine phosphorylation of STAT3 and DNA binding activity of STAT3 in addition to STAT4. that STAT3 was of a DNA that STAT4, that STAT3 and STAT4 C.-R. Lin J.-X. Fink D.W. Akira S. Bloom E.T. Yamauchi A. J. Immunol. 1996; 157: 126-137PubMed Google STAT4, and STAT3 treatment of NK cells with not the of STAT1, STAT3, or STAT4 with the IL-12 in STAT1 STAT3 was activated by IL-12 through the IL-12 receptor subunits conditions in which STAT4 was STAT1 and STAT3 be recruited to the IL-12 receptor through that as an and is not in cells. the and in peptides have been shown to binding specificity N. Zhong Z. Darnell Jr., J.E. Science. 1995; PubMed Scopus Google Scholar). the sites for STAT3, for have a residue at the to of the tyrosine peptides in the IL-12 β2 receptor a in the they sequence to the STAT1 in the IFN-γ receptor, B.L. Schreiber R.D. J. 1994; 13: PubMed Scopus Google Scholar, Schreiber R.D. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). This is with results that STAT1 and STAT3 are not activated by with the IL-12 The specificity of the various STAT SH2 domains for different tyrosine-containing peptides results in activation of specific cytokine signaling pathways and the of distinct gene expression We that STAT4 has a unique peptide binding specificity the STAT and binds to the peptide sequence The unique specificity of STAT4 for its tyrosine-containing peptide sequence the distinct of IL-12 STAT binding peptides from cytokine have the to STAT and STAT tyrosine J. U. M. Science. 1994; PubMed Scopus Google Scholar, B.L. Schreiber R.D. J. 1994; 13: PubMed Scopus Google Scholar, H. S. Schreiber R.D. C. J.J. J. 1996; PubMed Scopus Google Scholar). be possible to develop to the the thus STAT activation and The to IL-12 signaling by the β1 and β2 subunits of the IL-12 receptor the of IL-12 receptor and In addition to activation of the resulting in STAT tyrosine IL-12 STAT4 phosphorylation by or C. Rees R.C. R. O'Shea J.J. J. Immunol. 1996; 157: Google Scholar, C. L. Ritz J. Blood. 1994; PubMed Google Scholar, C. M. Ritz J. Cell. Immunol. 1995; PubMed Scopus Google Scholar). be to which regions of the receptor are required for these signaling We and for DNA for peptide and for the We and Lin Wu for and on the