Catalytically Active TYK2 Is Essential for Interferon-β-mediated Phosphorylation of STAT3 and Interferon-α Receptor-1 (IFNAR-1) but Not for Activation of Phosphoinositol 3-Kinase

TYK2, a Janus kinase, plays both structural and catalytic roles in type I interferon (IFN) signaling. We recently reported (Rani, M. R. S., Gauzzi, C., Pellegrini, S., Fish, E., Wei, T., and Ransohoff, R. M. (1999) J. Biol. Chem. 274, 1891–1897) that catalytically active TYK2 was necessary for IFN-β to induce the β-R1 gene. We now report IFN-β-mediated activation of STATs and other components in U1 (TYK2-null) cell lines that were complemented with kinase-negative (U1.KR930) or wild-type TYK2 (U1.wt). We found that IFN-β induced phosphorylation on tyrosine of STAT3 in U1.wt cells but not in U1.KR930 cells, whereas STAT1 and STAT2 were activated in both cell lines. Additionally, IFN-β-mediated phosphorylation of interferon-α receptor-1 (IFNAR-1) was defective in IFN-β treated U1.KR930 cells, but evident in U1.wt cells. In U1A-derived cells, the p85/p110 phosphoinositol 3-kinase isoform was associated with IFNAR-1 but not STAT3, and the association was ligand-independent. Further, IFN-β treatment stimulated IFNAR-1-associated phosphoinositol kinase activity equally in either U1.wt or U1.KR930 cells. Our results indicate that catalytically active TYK2 is required for IFN-β-mediated tyrosine phosphorylation of STAT3 and IFNAR-1 in intact cells. TYK2, a Janus kinase, plays both structural and catalytic roles in type I interferon (IFN) signaling. We recently reported (Rani, M. R. S., Gauzzi, C., Pellegrini, S., Fish, E., Wei, T., and Ransohoff, R. M. (1999) J. Biol. Chem. 274, 1891–1897) that catalytically active TYK2 was necessary for IFN-β to induce the β-R1 gene. We now report IFN-β-mediated activation of STATs and other components in U1 (TYK2-null) cell lines that were complemented with kinase-negative (U1.KR930) or wild-type TYK2 (U1.wt). We found that IFN-β induced phosphorylation on tyrosine of STAT3 in U1.wt cells but not in U1.KR930 cells, whereas STAT1 and STAT2 were activated in both cell lines. Additionally, IFN-β-mediated phosphorylation of interferon-α receptor-1 (IFNAR-1) was defective in IFN-β treated U1.KR930 cells, but evident in U1.wt cells. In U1A-derived cells, the p85/p110 phosphoinositol 3-kinase isoform was associated with IFNAR-1 but not STAT3, and the association was ligand-independent. Further, IFN-β treatment stimulated IFNAR-1-associated phosphoinositol kinase activity equally in either U1.wt or U1.KR930 cells. Our results indicate that catalytically active TYK2 is required for IFN-β-mediated tyrosine phosphorylation of STAT3 and IFNAR-1 in intact cells. interferon phosphatidylinositol 3-kinase genomic DNA affinity chromatography polyvinylidene difluoride interferon-α receptor polyacrylamide gel electrophoresis The role(s) of the tyrosine kinase TYK2 in the type I IFN1 signaling pathway has been demonstrated through studies carried out in the TYK2-minus cell line U1A (1Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (714) Google Scholar, 2Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (317) Google Scholar). U1A cells are completely refractory to IFN-α, yet retain a partial responsiveness to IFN-β (2Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (317) Google Scholar), suggesting that IFN-β, but not IFN-α, activates both TYK2-dependent and -independent signaling pathways (3Uze G. Lutfalla G. Mogensen K.E. J. Interferon Cytokine Res. 1995; 15: 3-26Crossref PubMed Scopus (232) Google Scholar). Reconstitution of U1A cells with wild-type or mutant forms of TYK2 has revealed a surprising diversity of structural and catalytic functions for TYK2 in the type I IFN receptor (IFNAR-1/2) complex. For example, the N region (residues 1–591) of TYK2 was required for stable cytoplasmic accumulation of IFNAR-1 protein (4Richter M.F. Dumenil G. Uze G. Fellous M. Pellegrini S. J. Biol. Chem. 1998; 273: 24723-24729Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Individual Janus kinase homology domains within the N region were specifically implicated in IFNAR-1/TYK2 interaction and signaling in response to IFN-α (4Richter M.F. Dumenil G. Uze G. Fellous M. Pellegrini S. J. Biol. Chem. 1998; 273: 24723-24729Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Interestingly, the TYK2 kinase domain was found to be dispensable for some aspects of IFN-α/β signaling, such as the IFN-dependent induction of many classical IFN-stimulated genes (5Velazquez L. Mogensen K.E. Barbieri G. Fellous M. Uze G. Pellegrini S. J. Biol. Chem. 1995; 270: 3327-3334Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 6Gauzzi M.C. Velazquez L. McKendry R. Mogensen K.E. Fellous M. Pellegrini S. J. Biol. Chem. 1996; 271: 20494-20500Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Recent studies using alanine substitutions on the extracellular domain of IFNAR-2 has revealed the type I IFNs interacted differently with the two receptor subunits IFNAR-1 and IFNAR-2 (7Lewerenz M. Mogensen E. Uze G. J. Mol. Biol. 1998; 282: 585-599Crossref PubMed Scopus (77) Google Scholar). TYK2 has been implicated in the direct phosphorylation of IFNAR-1 in vitro (8Colamonici O. Yan H. Domanski P. Handa R. Smalley D. Mullersman J. Witte M. Krishnan K. Krolewski J. Mol. Cell. Biol. 1994; 14: 8133-8142Crossref PubMed Google Scholar, 9Yan H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (160) Google Scholar), and we (10Rani M.R.S. Gauzzi C. Pellegrini S. Fish E. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) recently reported that catalytically functional TYK2 was required for induction of the β-R1/I-TAC gene by IFN-β. In addition to the above functions, TYK2 may also play a role in the recruitment of other signaling molecules to the IFNAR1/2 complex (11Yang C.-H. Shi W. Basu L. Murti A. Constantinescu S.N. Blatt L. Croze E Mullersman J.E. Pfeffer L.M. J. Biol. Chem. 1996; 271: 8057-8061Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). STAT3, which has been reported to “dock” on phosphorylated IFNAR-1, has also been described as an adaptor for coupling PI3K to the IFN pathway in Daudi cells (12Pfeffer L.M. Mullersman J.E. Pfeffer S.R. Murti A. Shi W. Yang C.H. Science. 1997; 276: 1418-1420Crossref PubMed Scopus (237) Google Scholar). Overexpression of STAT3 restored antiviral and antiproliferative responses in an IFN-resistant Daudi cell line (13Yang C.H. Murti A. Pfeffer L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5568-5572Crossref PubMed Scopus (119) Google Scholar). Although TYK2 is believed to mediate IFNAR-1 phosphorylation, which in turn is considered essential for recruiting STAT3 to the IFNAR-1/2 complex, the role of TYK2 in STAT3 activation has not been examined directly. PI3K designates a family of enzymes that phosphorylate the D3 position of phosphatidylinositol. PI3K consists of a 110-kDa catalytic subunit (p110) that associates with an 85-kDa regulatory subunit (p85). Ligand-dependent interactions between the SH2 domains of the p85 subunit and the phosphotyrosine containing YXXM motif present on several cytokine/growth factor receptors have been reported (14Fry D.W. Kraker A.J. McMichael A. Ambroso L.A. Nelson J.M. Leopold W.R. Connors R.W. Bridges A.J. Science. 1994; 265: 1093-1095Crossref PubMed Scopus (815) Google Scholar). The phosphorylated lipid products of this enzymatic reaction may act as second messengers to activate protein kinases such as the Akt gene product (15Exton J.H. Biochim. Biophys. Acta. 1994; 1212: 26-42Crossref PubMed Scopus (924) Google Scholar) or certain forms of protein kinase C (16Coffer P. Lutticken C. van Puijenbroek A. Klop-de Jong M. Horn F. Kruijer W. Oncogene. 1995; 10: 985-994PubMed Google Scholar). p85/p110 PI3K exhibits enhanced lipid kinase and protein serine kinase activity in type I IFN-treated cells. A major substrate for PI3K activity in the IFN pathway appears to be insulin receptor substrate-1 as evidenced by the detection of IFN-α-dependent serine phosphorylation of insulin receptor substrate-1 in U-266 cells and inhibition of such phosphorylation by the semi-selective PI3K inhibitor wortmannin (17Uddin S. Fish E.N. Sher D.A. Gardziola C. White M.F. Platanias L.C. J. Immunol. 1997; 158: 2390-2397PubMed Google Scholar). In these experiments no interactions between p85 and TYK2, JAK1, IFNAR-1, or IFNAR-2c were detected suggesting PI3K did not interact with IFN-α signaling components upstream of insulin receptor substrate-1 (17Uddin S. Fish E.N. Sher D.A. Gardziola C. White M.F. Platanias L.C. J. Immunol. 1997; 158: 2390-2397PubMed Google Scholar). To date, the role of serine/lipid kinases such as PI3K in the biological response to IFN remains poorly understood. In this report we demonstrate that catalytically active TYK2 is essential for IFN-β-dependent phosphorylation of STAT3 and IFNAR-1 in U1A-derived cell lines. We found PI3K to co-immunoprecipitate with IFNAR-1, but not STAT3. Furthermore, IFNAR-1-associated PI3K activity was markedly elevated by IFN-β in the presence or absence of catalytically active TYK2, consistent with the observation that PI3K association with the IFNAR-1 receptor component was phosphotyrosine-independent. Thus, it appears that in these cells TYK2 and PI3K activation are not interdependent, suggesting that these molecules have distinct downstream signaling functions. Human fibrosarcoma 2fTGH cells, mutant U1A, and derivative cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (2Pellegrini S. John J. Shearer M. Kerr I.M. Stark G.R. Mol. Cell. Biol. 1989; 9: 4605-4612Crossref PubMed Scopus (317) Google Scholar). U1.wt and U1.KR930 cells described earlier (10Rani M.R.S. Gauzzi C. Pellegrini S. Fish E. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) were maintained in Dulbecco's modified Eagles medium with 10% calf serum with 250 μg/ml of hygromycin and 450 μg/ml of G418 (5Velazquez L. Mogensen K.E. Barbieri G. Fellous M. Uze G. Pellegrini S. J. Biol. Chem. 1995; 270: 3327-3334Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Purified recombinant IFN-α2 (1 × 105 units/ml) was obtained from Wellcome Research Laboratories (Kent, United Kingdom), and recombinant IFN-β-1b (2 × 108 units/mg protein) was from Berlex Biosciences (Richmond, CA). IFNs were used at a final concentration of 1,000 units/ml unless stated otherwise. Cells were treated with 15,000 units/ml of IFN for 15 min, and nuclear extracts were prepared as described previously (18Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Briefly, cells were washed twice with ice-cold phosphate-buffered saline that contained 1 mm Na3VO4and 5 mm NaF and once with hypotonic buffer. Cells were lysed in hypotonic buffer containing 0.2% Triton X-100 and nuclear extracts collected by differential centrifugation. GDAC has been previously described (18Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). 50 μg of nuclear extract was incubated for 20 min with 25 μg of poly(dI-dC) (Amersham Pharmacia Biotech) in binding buffer as described previously (18Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). This mixture (200 μl) was incubated for 2 h with 100 μl of bovine genomic DNA-cellulose (Sigma). The DNA-binding proteins were eluted in high salt buffer, concentrated, and resolved by SDS-PAGE for analysis in Western immunoblotting experiments (18Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Cells were treated with recombinant IFN-β at 37 °C for 10–15 min before preparation of cell extracts. The antibodies used were: anti-STAT1 and anti-STAT2 (Transduction Laboratories, Lexington, KY), anti-STAT3 (D. Levy, NY University), anti-p85 (Upstate Biotechnology, Inc., Lake Placid, NY). STAT proteins were immunoprecipitated from extracts (19Han Y. Rogers N. Ransohoff R. J. Interferon Cytokine Res. 1999; 19: 731-740Crossref PubMed Scopus (17) Google Scholar, 20Han Y. Watling D. Rogers N.C. Stark G.R. Mol. Endocrinol. 1997; 11: 1180-1188Crossref PubMed Scopus (38) Google Scholar), separated by SDS-polyacrylamide gel electrophoresis, and adsorbed to polyvinylidene difluoride (PVDF) membranes (Stratagene, La Jolla, CA). Incubation with primary antibody was for 2 h at room temperature. Blots were washed thoroughly followed by incubation with secondary antibody for 1 h at room temperature. Immunoreactive bands were visualized with the ECL Western blotting system (Amersham Pharmacia Biotech). Tyrosine phosphorylation was monitored by Western blotting using anti-phosphotyrosine monoclonal antibodies PY20, (Transduction Laboratories) and 4G10 (Upstate Biotechnology, Inc.). Cells at 70–80% confluency in 150-mm-diameter culture dishes were serum-starved (0% fetal bovine serum) for 4 h to suppress endogenous PI3K activity (21Reddy S.A. Huang J.H. Liao W.S. J. Biol. Chem. 1997; 272: 29167-29173Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar) and treated with IFN-β for varying times. Cells were lysed with buffer containing 1% Nonidet P-40, 50 mm HEPES (pH 7.5), 10% glycerol, 150 mm NaCl, 1.5 mmMgCl2, 1 mm EGTA, 50 mm NaF, 1 mm Na3VO4, 14 mm2-mercaptoethanol, 0.2 mm phenylmethylsulfonyl fluoride, 10 μg/ml of aprotinin, leupeptin, and pepstatin. Protein concentrations in cell extracts, determined by Bradford reactions, were equalized for each assay. For immunoprecipitation, anti-IFNAR-1 antibodies (22Croze E. Russell-Harde D. Wagner T.C. Pu H. Pfeffer L.M. Perez H.D. J. Biol. Chem. 1996; 271: 33165-33168Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) were precoupled to protein A-agarose beads for 1 h at 4 °C with rotation, and immunoprecipitation was done at 4 °C for 3 h. The immunoprecipitates were washed with lysis buffer 4 to 5 times. The kinase assay in 50 μl contained 10 μg of phosphatidylinositol (Sigma), 200 mm Tris, pH 7.5, 100 mm NaCl, 0.5 mm EGTA, 10 mm MgCl2, and 10 μm ATP and γ-32PATP (specific activity 6,000 Ci/mmol, 10 μCi/sample). After incubation for 30 min at 37 °C, an equal volume of water was added, and the reaction was stopped with 300 μl of MeOH:CHCl3 (2:1). After the sample was mixed, 100 μl of water and 200 μl of CHCl3was added. After vortexing, the organic phase was collected, dried, and resuspended in 25 μl CHCl3:MeOH (1:1) and spotted on thin-layer chromatography plates. The plates were developed in CHCl3:MeOH:4N NH4OH:water (45:30:3:5), dried, and exposed to X-O-mat film (Eastman Kodak). The signal was quantitated using a PhosphorImager (Molecular Dynamics, Sunnyville, CA). The availability of sibling cell lines equal of catalytically active or (U1.KR930) TYK2 M.C. Velazquez L. McKendry R. Mogensen K.E. Fellous M. Pellegrini S. J. Biol. Chem. 1996; 271: 20494-20500Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) an to the for catalytic TYK2 in STAT This was using a that activated STATs for DNA binding (18Ghislain J.J. Fish E.N. J. Biol. Chem. 1996; 271: 12408-12413Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). U1.wt and U1.KR930 cells activated STAT1 and STAT2 IFN-α not or treatment 1 GDAC to STAT3 from of IFN-treated U1.KR930 cells 1 The activation of STAT1 and STAT2 was in the U1.KR930 cells with U1.wt cells as determined by using as activation of equally in both U1.wt and U1.KR930 cells not of tyrosine on and was by immunoprecipitation Western blotting STAT1 and STAT2 were activated in U1.wt and U1.KR930 cells in response to IFN-β with results obtained by STAT1 and STAT2 phosphorylation was by in the U1.KR930 cells with U1.wt cells as determined by with 2 activation of STAT3 was in U1.KR930 cells treated with IFN-β 2 of STAT3 protein were present in both the cell lines. The absence of activated STAT3 from DNA-binding was by using with an for the The is a in the that and of STAT1 and STAT3 Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). STAT3 containing DNA-binding were detected in the extracts from U1.KR930 cells not with the by indicate that STAT3 activation kinase of of type I IFN to receptor tyrosine phosphorylation of the receptor subunits IFNAR-1 and In vitro studies revealed that TYK2 and IFNAR-1 H. Krishnan K. Greenlund A.C. Gupta S. Lim J.T.E. Schreiber R.D. Schindler C.W. Krolewski J.J. EMBO J. 1996; 15: 1064-1074Crossref PubMed Scopus (160) Google Scholar, H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). STAT3 has also been to with IFNAR-1 in a tyrosine (11Yang C.-H. Shi W. Basu L. Murti A. Constantinescu S.N. Blatt L. Croze E Mullersman J.E. Pfeffer L.M. J. Biol. Chem. 1996; 271: 8057-8061Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). To the phosphorylation of the IFN-α receptor subunits in the presence and absence of catalytically active TYK2, cell from IFN-treated U1.wt and U1.KR930 cells were immunoprecipitated with antibodies to IFNAR-2c and IFNAR-1 and to Western assay. in IFNAR-2c was phosphorylated in response to either IFN-β or IFN-α in both cell lines. IFNAR-1 immunoprecipitates from IFN-treated U1.wt cells, but not U1.KR930 cells, contained IFNAR-1 We that catalytically active TYK2 is essential for tyrosine phosphorylation of IFNAR-1 in intact U1A-derived cells. The absence of STAT3 and IFNAR-1 phosphorylation in U1.KR930 cells an to IFN-β signaling to PI3K in the absence of these activated PI3K has been to be activated by IFN-α (17Uddin S. Fish E.N. Sher D.A. Gardziola C. White M.F. Platanias L.C. J. Immunol. 1997; 158: 2390-2397PubMed Google Scholar, S. L. White M.F. Platanias L.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). using Daudi cells that STAT3 act as an adaptor to PI3K to the IFN pathway (12Pfeffer L.M. Mullersman J.E. Pfeffer S.R. Murti A. Shi W. Yang C.H. Science. 1997; 276: 1418-1420Crossref PubMed Scopus (237) Google Scholar). In that tyrosine phosphorylation of STAT3 was to be essential for association with In experiments using IFN-treated cells no association of p85 with IFNAR-1, or IFNAR-2c (17Uddin S. Fish E.N. Sher D.A. Gardziola C. White M.F. Platanias L.C. J. Immunol. 1997; 158: 2390-2397PubMed Google Scholar). In cells, we found PI3K to co-immunoprecipitate with IFNAR-1 in the presence or absence of IFN-β in both U1.wt and U1.KR930 cells phosphatidylinositol kinase activity was detected in anti-IFNAR-1 of the presence of catalytically active TYK2 4 A in lipid kinase activity was in both cell lines. We also examined the role of catalytically active TYK2 in association of STAT3 with PI3K in the presence and absence of IFN-β. The p85 regulatory subunit of PI3K to with anti-STAT3 in 4 and to with from STAT3 not Further, STAT3 tyrosine phosphorylation did not induce PI3K association in U1.wt cells. lipid kinase activity was not detected in STAT3 immunoprecipitates by in vitro kinase assay. We that activated STAT3 not mediate adaptor for recruitment of PI3K to the IFN-α/β receptor in U1A-derived cells. We the of type I IFN receptor signaling components and TYK2 kinase by fibrosarcoma cells that structural of the type I IFN receptor but the kinase of We found that catalytically active TYK2 is essential for IFNAR-1 phosphorylation by type I In vitro studies that IFNAR-1 was a substrate for reported demonstrate that catalytically active TYK2 is required for IFN-β-mediated phosphorylation of IFNAR-1 in intact cells. phosphorylation of IFNAR-2 by IFNs was not on kinase activity of Further, the absence of IFNAR-1 phosphorylation in U1.KR930 cells, we did induction of IFN-stimulated genes the were for the wild-type cells (10Rani M.R.S. Gauzzi C. Pellegrini S. Fish E. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). analysis of type I IFNs L. Pfeffer L. M. D. Yang C.H. Murti A. Pellegrini S. S. Uze G. Mogensen K. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and studies of the of IFNAR-2 (7Lewerenz M. Mogensen E. Uze G. J. Mol. Biol. 1998; 282: 585-599Crossref PubMed Scopus (77) Google Scholar) have also that not a direct interaction of with active TYK2 was essential for STAT3 phosphorylation by type I Our that to phosphorylate IFNAR-1 STAT3 phosphorylation in U1.KR930 cells. The for STAT1 and STAT2 to the factor in response to type I IFN is S. S.A. Kerr I.M. J.E. Stark G.R. Mol. Cell. Biol. 1995; 15: PubMed Google Scholar, S.A. S. Kerr I.M. Stark G.R. J.E. Mol. Cell. Biol. 1996; PubMed Scopus Google Scholar, White J.M. Greenlund A.C. D. K. R. M. Schreiber R.D. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. R. M.C. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The role of STAT3 in the response to IFN is STAT3 was as a factor that responses to and J.E. Science. 1994; PubMed Scopus Google Scholar). STAT3 is activated by a of and and are not K. K. Shi W. T. M. N. T. S. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). Recent studies that STAT3 associates with the IFNAR-1 of the type I IFN receptor in a tyrosine IFN-α addition (11Yang C.-H. Shi W. Basu L. Murti A. Constantinescu S.N. Blatt L. Croze E Mullersman J.E. Pfeffer L.M. J. Biol. Chem. 1996; 271: 8057-8061Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). of STAT3 in a Daudi cell line to antiviral and antiproliferative of IFNs restored an (13Yang C.H. Murti A. Pfeffer L.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5568-5572Crossref PubMed Scopus (119) Google Scholar). In U1.wt and U1.KR930 cells antiviral R. S. and R. M. Ransohoff, in responses to IFN-β were not W. M. R. S. and R. M. Ransohoff, STAT3 activation may be required for induction of genes that mediate antiproliferative of type I IFNs in certain cell the role of STAT3 in IFN signaling and may be obtained through the of genes activated STAT3. PI3K induced by IFN-β in U1.KR930 cells, not to be for IFN-β-mediated antiproliferative in the absence of activated STAT3. induction by IFN-β appears to PI3K have been reported (12Pfeffer L.M. Mullersman J.E. Pfeffer S.R. Murti A. Shi W. Yang C.H. Science. 1997; 276: 1418-1420Crossref PubMed Scopus (237) Google Scholar). M. R. S. and R. M. Ransohoff, Our results of the by which IFN-β activates PI3K in U1A-derived cell lines. active is to be required for PI3K activation in response to IFN-α Rogers N.C. Watling D. J.M. S. G. Pellegrini S. White M.F. Kerr I.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). Our studies indicate TYK2 kinase activity is not required for activation of We also found that p85 did not with anti-STAT3 with from STAT3 not we a pathway for IFN-β-mediated PI3K through a association between p85 and In these cells, PI3K activation by IFN-β equally in the presence or absence of catalytically active This that TYK2 and signal downstream in through an kinase The of PI3K activation by IFN-β to be through experiments in which both TYK2 and signaling are We Stark for