Interferon-α Engages the Insulin Receptor Substrate-1 to Associate with the Phosphatidylinositol 3′-Kinase

Interferon-α (IFNα) induces rapid tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), a docking protein with multiple tyrosine phosphorylation sites that bind to the Src homology 2 (SH2) domains of various signaling proteins. During IFNα stimulation, the p85 regulatory subunit of the phosphatidylinositol 3′-kinase binds via its SH2 domains to tyrosine-phosphorylated IRS-1, and phosphatidylinositol 3′-kinase activity is detected in association with IRS-1. Thus, IFNα responses occur by activation of the IRS signaling system, which it shares with insulin, insulin-like growth factor-1, and interleukin-4. Interferon-α (IFNα) induces rapid tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1), a docking protein with multiple tyrosine phosphorylation sites that bind to the Src homology 2 (SH2) domains of various signaling proteins. During IFNα stimulation, the p85 regulatory subunit of the phosphatidylinositol 3′-kinase binds via its SH2 domains to tyrosine-phosphorylated IRS-1, and phosphatidylinositol 3′-kinase activity is detected in association with IRS-1. Thus, IFNα responses occur by activation of the IRS signaling system, which it shares with insulin, insulin-like growth factor-1, and interleukin-4. INTRODUCTIONThe Type I interferons (IFNα, IFNβ, and IFNω)1( 1The abbreviations used are: IFNsinterferonsISGF3interferon-stimulated gene factor 3IRS-1insulin receptor substrate-1SH2Src homology 2PI 3′-kinasephosphatidylinositol 3′-kinasePAGEpolyacrylamide gel electrophoresisIGF-1insulin-like growth factor-1ILinterleukinGSTglutathione S-transferasePBSphosphate-buffered saline. )exert a variety of biological effects on normal and neoplastic cells that include antiviral and antiproliferative activities(1Petska S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1593) Google Scholar). Immediately after IFNα stimulation, several signaling proteins in the receptor complex become tyrosine-phosphorylated, including the α and β subunits of the Type I IFN receptor (2Platanias L.C. Colamonici O.R. J. Biol. Chem. 1992; 267: 24053-24057Abstract Full Text PDF PubMed Google Scholar, 3Platanias L.C. Uddin S. Colamonici O.R. J. Biol. Chem. 1994; 269: 17761-17764Abstract Full Text PDF PubMed Google Scholar) and the Tyk-2 and Jak-1 tyrosine kinases(4Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (637) Google Scholar, 5Shuai K. Ziemiecki A. Wilks A.F. Harpur A.G. Sadowski H.B. Gilman M.Z. Darnell J.E. Nature. 1993; 366: 580-583Crossref PubMed Scopus (407) Google Scholar, 6Silvennoinen O. Ihle J.N. Schlessinger J. Levy D.E. Nature. 1993; 366: 583-585Crossref PubMed Scopus (287) Google Scholar, 7Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar). Both kinases associate with components of the Type I IFN receptor(7Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar, 8Novick D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (576) Google Scholar), and their activation early in the IFNα signaling cascade is presumed to regulate tyrosine phosphorylation of various downstream signaling molecules. Expression of Jak-1 and Tyk-2 rescues an IFNα response in certain insensitive cell lines, suggesting that these tyrosine kinases or related members of the Janus family are essential for IFNα action(4Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (637) Google Scholar, 9Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (701) Google Scholar).Several proteins are substrates for IFNα-dependent tyrosine kinase activity. In response to IFNα treatment of cells, the Stat-2, Stat-1α, and Stat-1β components of the transcriptional activator ISGF3α are rapidly phosphorylated on tyrosine and associate with a 48-kDa protein (ISGF3γ) to form an active complex(10Fu X.-Y. Cell. 1992; 70: 323-335Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 11Schindler C. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (714) Google Scholar, 12Gutch M.J. Daly C. Reich N.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 8: 11411-11415Crossref Scopus (64) Google Scholar). This complex translocates to the nucleus and initiates gene transcription during binding to interferon-stimulated response elements (10-13). In addition, the vav proto-oncogene product (p95vav) is tyrosine-phosphorylated during IFNα stimulation (14Platanias L.C. Sweet M.E. J. Biol. Chem. 1994; 269: 3143-3146Abstract Full Text PDF PubMed Google Scholar); however, its precise role in the signal transduction of IFNα remains to be determined. The involvement of multiple pathways in IFNα signaling is consistent with its pleiotropic biological effects on cells and tissues.Many growth factor receptors, including those for epidermal growth factor and platelet-derived growth factor, associate directly through their autophosphorylation sites with a common set of signaling proteins that contain SH2 domains, including the phosphatidylinositol (PI) 3′-kinase, Grb-2, SH-PTP2, phospholipase Cγ, and Ras-GAP(15Pawson T. Gish G.D. Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (792) Google Scholar). The receptors for insulin, IGF-1, and IL-4, however, do not strongly associate with most SH2 proteins known to be involved in their signaling pathways(16Myers M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-294Abstract Full Text PDF PubMed Scopus (288) Google Scholar). Instead, they stimulate tyrosine phosphorylation of docking proteins in the IRS signaling family, notably IRS-1, which binds directly to various SH2 proteins(16Myers M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-294Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 17Sun X.-J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1274) Google Scholar, 18Wang L.-M. Keegan A.D. Li W. Lienhard G.E. Pacini S. Gutkind J.S. Sun X.-J. White M.F. Aaronson S.A. Paul W.E. Pierce J.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4032-4036Crossref PubMed Scopus (166) Google Scholar, 19Wang L.-M. Myers M.G. Sun X.-J. Aaronson S.A. White M.F. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (365) Google Scholar). We report that IFNα also engages the IRS signaling system, as evidenced by the rapid IFNα-dependent tyrosine phosphorylation of IRS-1 in several hematopoietic cell lines. We also demonstrate that the p85 regulatory subunit of the PI 3′-kinase binds via its SH2 domains the IFNα-induced phosphorylated form of IRS-1, and that PI 3′-kinase activity can be detected in association with IRS-1.EXPERIMENTAL PROCEDURESCells and ReagentsThe U-266 (human myeloma), Daudi (lymphoblastoid), and MOLT-4 (acute T-cell leukemia) cell lines were grown in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, Inc.) or 10% (v/v) defined calf serum (Hyclone Laboratories) and antibiotics. Human recombinant IFNα2 was provided by Hoffmann-La Roche and Schering-Plough. The antiphosphotyrosine monoclonal antibody (4G-10) and the monoclonal antibody against the regulatory subunit of PI 3′-kinase were obtained from Upstate Biotechnology (Lake Placid, NY) and were used for immunoblotting. Nonimmune rabbit IgG was obtained from Sigma. The polyclonal αIRS-1PH antibody was raised against a GST fusion protein of a region of rat IRS-1 (amino acid residues 1-135) containing its pleckstrin homology domain. An antibody (αIRS-1CT) against a synthetic peptide (TYASINFQKQPEDRQ), corresponding to a sequence present in the C terminus of IRS-1, was raised in rabbits. The polyclonal antibodies against recombinant rat IRS-1 and the p85 regulatory subunit of PI 3′-kinase have been described elsewhwere (20Backer J.M. Myers M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar) and were used for immunoprecipitations. A polyclonal antibody against Stat-2 (p113, 186-199) has been described elsewhere(21Colamonici O.R. Domanski P. Krolewski J.J. Fu X.-Y. Reich N.C. Pfeffer L.M. Sweet M.E. Platanias L.C. J. Biol. Chem. 1994; 269: 5660-5665Abstract Full Text PDF PubMed Google Scholar).Immunoprecipitations and ImmunoblottingCells were stimulated with the indicated amounts of IFNα or insulin for the indicated periods of time. After stimulation, the cells were rapidly centrifuged and lysed in a phosphorylation lysis buffer (0.5-1% Triton X-100, 150 mM NaCl, 200 μM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM EDTA, 50 mM Hepes, 1.5 mM magnesium chloride, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml aprotinin). Cell lysates were immunoprecipitated with the indicated antibodies and, after five washes with phosphorylation lysis buffer containing 0.1% Triton X-100, were analyzed by SDS-PAGE and transferred onto nitrocellulose (Schleicher and Schuell) or polyvinylidene difluoride (Immobilon) filters, and the residual binding sites on the filters were blocked by incubating with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20, 10% bovine serum albumin) for 1-3 h at room temperature or overnight at 4°C. The filters were subsequently incubated with the antiphosphotyrosine monoclonal antibody and developed using an enhanced chemiluminesence (ECL) kit following the manufacturer's procedure (Amersham).Preparation of Glutathione S-Transferase Fusion ProteinsGlutathione S-transferase fusion proteins containing the nSH2 and cSH2 domains of PI 3′-kinase (22Myers Jr., M.G. Backer J.M. Sun X.-J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar) were purified from transformed Escherichia coli bacteria which were induced with 1 mM isopropyl-1-thio-β-D-galactopyranoside as described previously(23Uddin S. Katzav S. White M.F. Platanias L.C. J. Biol. Chem. 1995; 270: 7712-7716Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Briefly, after 2 h of additional growth, bacteria were lysed by sonication in phosphate-buffered saline (PBS), pH 7.0. Lysed bacteria were spun for 25 min at 14,000 rpm at 4°C, and the supernatant was immobilized on glutathione-Sepharose beads (Pharmacia) and used for binding assays.Assays for PI 3′-Kinase ActivityPI 3′-kinase activity assays on αIRS-1PH immunoprecipitates obtained from IFNα- or insulin-stimulated cells were performed as described previously(24Backer J.M. Schroeder G. Kahn R.C. Myers Jr., M.G. Wilden P.A. Cahill D.A. White M.F. J. Biol. Chem. 1992; 267: 1367-1374Abstract Full Text PDF PubMed Google Scholar). Briefly, after IFNα or insulin stimulation, serum-starved Daudi cells were washed once with ice-cold PBS and twice with 20 mM Tris, pH 7.5, containing 137 mM NaCl, 1 mM MgCl2, and 100 μM Na3VO4 (Buffer A). The cells were lysed in 1 ml of Buffer A containing 1% Nonidet P-40, 10% glycerol, and 0.35 mg/ml phenylmethylsulfonyl fluoride, and insoluble material was removed by centrifugation. Cell lysates were immunoprecipitated with the indicated antibodies, and immunoprecipitates were washed in PBS with 1% Nonidet P-40 and 100 μM Na3VO4 (three times), 100 mM Tris, pH 7.5, containing 500 mM LiCl2 and 100 μM Na3VO4 (three times), and 10 mM Tris, pH 7.5, containing 100 mM NaCl, 1 mM EDTA, and 100 μM Na3VO4 (twice). The pellets were resuspended in 50 μl of 10 mM Tris, pH 7.5, containing 100 mM NaCl and 1 mM EDTA. 10 μl of 100 mM MnCl2 and 10 μl of phosphatidylinositol (2 μg/μl) sonicated in 10 mM Tris, pH 7.5, 1 mM EGTA were added to each pellet. The reaction was started by the addition of 10 μl of 440 μM ATP containing 30 μCi of 32PATP. After incubation for 10 min at 22°C, the reaction was stopped by the addition of 20 μl of 8 N HCl and 160 μl of CHCl3:methanol (1:1). The samples were and the was removed and to a gel which was with 1% were developed in and and on a performed in which the of tyrosine phosphorylation induced by IFNα or insulin in hematopoietic cells were We that IFNα and insulin stimulate tyrosine phosphorylation of a common protein in U-266 cells The of protein was to the of the insulin receptor X.-J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1274) Google Scholar), suggesting that IFNα an IRS signaling protein to certain IRS-1 is involved in IFNα lysates from or stimulated U-266 or Daudi cells were immunoprecipitated with polyclonal antibodies against IRS-1 and with an antiphosphotyrosine tyrosine phosphorylation of a corresponding to IRS-1, was detected in U-266 not in Daudi IFNα and insulin strongly stimulated tyrosine phosphorylation of IRS-1 in cell lines A and and not The IFNα-induced phosphorylation of IRS-1 was rapid and 1 min of treatment of Daudi or MOLT-4 cells and after min was also a response with the phosphorylation of proteins phosphorylation of IRS-1 in response to IFNα and serum-starved U-266 cells were with IFNα for min at as and cell lysates were immunoprecipitated with or normal rabbit serum as Daudi cells were incubated in the or of IFNα or insulin for min at Cell lysates were immunoprecipitated with or a polyclonal antibody against rat IRS-1 as of tyrosine phosphorylation of IRS-1. were stimulated with IFNα for the indicated and cell lysates were immunoprecipitated with or an antibody against recombinant rat IRS-1 as serum-starved U-266 cells were for 30 min at with the indicated amounts of and cell lysates were immunoprecipitated with or with a polyclonal antibody against Stat-2 tyrosine IRS-1 binds to several SH2 including the regulatory subunit of the PI J.M. Myers M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar). IRS-1 binds p85 during IFNα stimulation, immunoprecipitates obtained with an antibody from Daudi cell lysates were with antiphosphotyrosine IFNα or insulin treatment of the cells, a tyrosine-phosphorylated protein was detected in the This with IRS-1 immunoprecipitated directly with αIRS-1PH immunoprecipitates after IFNα or insulin stimulation The tyrosine-phosphorylated form of IRS-1 from IFNα- or insulin-stimulated cells also to a GST fusion protein containing the SH2 of p85 and were obtained using a GST fusion protein containing the SH2 of Uddin and L. C. IFNα the association of IRS-1 with the PI 3′-kinase, and most the SH2 domains in Jr., M.G. Backer J.M. Sun X.-J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google of IRS-1 with the p85 regulatory subunit of PI 3′-kinase in Daudi antiphosphotyrosine were incubated for min at in the or of IFNα or insulin as and cell lysates were immunoprecipitated with normal or as cells were incubated in the or of IFNα or insulin for min at as and cell lysates were immunoprecipitated with normal or as indicated and with a monoclonal antibody against cells were stimulated with IFNα or insulin for min at as and cell lysates were incubated for h at with GST or a GST fusion protein containing the SH2 of of which were to glutathione-Sepharose proteins were by SDS-PAGE and with cells were incubated for min with IFNα as and cell lysates were to GST or a fusion proteins were analyzed by SDS-PAGE and with PI 3′-kinase to an role in various biological responses and is by growth and L.C. C. B. A. S. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). activation by insulin during association with tyrosine-phosphorylated J.M. Myers M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar). PI 3′-kinase activity is detected in association with IRS-1 during IFNα stimulation, PI 3′-kinase assays were on stimulation, a of PI 3′-kinase activity was detected in the which was to the activity detected in immunoprecipitates with rabbit serum IFNα or insulin stimulated the association of PI 3′-kinase activity with IRS-1 that was immunoprecipitated with αIRS-1PH is that the PI 3′-kinase is by IFNα during its association with of PI 3′-kinase activity with IRS-1 during IFNα or insulin stimulation in Daudi cells were for min in the or of IFNα or insulin as cell lysates were immunoprecipitated with αIRS-1PH or and immunoprecipitates were in for PI 3′-kinase activity. The in the the and the the phosphorylated phosphatidylinositol were as the activation S.E. for has been in of IFNα signaling the and The a for the of which of containing the response J.E. Kerr I.M. Stark G.R. Science. 1994; PubMed Scopus Google Scholar). The used by IFNα to regulate signaling however, is that IFNα tyrosine phosphorylation of IRS-1 the of a for the of additional SH2 signaling proteins during IFNα be to SH2 proteins by IRS-1 in response to as as in response to and which also its phosphorylation on tyrosine a role in the signal transduction of insulin and and its signaling to be essential for the effects of these L.-M. Myers M.G. Sun X.-J. Aaronson S.A. White M.F. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (365) Google Scholar). has that IRS-1 also be involved in growth signaling J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). The protein tyrosine phosphorylation sites in various M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-294Abstract Full Text PDF PubMed Scopus (288) Google Scholar). tyrosine residues a role as substrates for tyrosine kinases and as docking sites for downstream SH2 proteins. tyrosine residues in IRS-1 phosphorylation by the insulin including residues and which are in and bind to p85 which the PI J.M. Myers M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar). are also phosphorylated by the insulin including which binds Grb-2, and and which bind X.-J. Myers Jr., M.G. Miralpeix M. White M.F. Cell. Biol. 1993; PubMed Google Scholar). to the of IRS-1 that are tyrosine-phosphorylated in response to IFNα on the by which the signaling is through the common of protein by and growth that the insulin receptor tyrosine kinase the phosphorylation of IRS the receptors for and IFNα Janus family tyrosine kinases to however, that IRS-1 as a for Janus kinases during IFNα stimulation remains to be receptors the Janus family of tyrosine most of do not IRS-1. The of the elements for are the receptors for insulin, IGF-1, and contain a common acid sequence which to to the of these receptors with A.D. K. White M. L.-M. Pierce J.H. Paul W.E. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar); however, sequence is not in the components of the Type I IFN D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (576) Google Scholar, G. G. Cell. Full Text PDF PubMed Scopus Google Scholar), suggesting that a be involved or subunit remains to be of the IRS signaling a common signaling for insulin, IGF-1, IL-4, and J. 1993; Google Scholar) that the transcriptional activation of gene in and related cell lines. the of is the of an IFNα and through the common of proteins in the IRS signaling is insulin and also IFNα in these and as INTRODUCTIONThe Type I interferons (IFNα, IFNβ, and IFNω)1( 1The abbreviations used are: IFNsinterferonsISGF3interferon-stimulated gene factor 3IRS-1insulin receptor substrate-1SH2Src homology 2PI 3′-kinasephosphatidylinositol 3′-kinasePAGEpolyacrylamide gel electrophoresisIGF-1insulin-like growth factor-1ILinterleukinGSTglutathione S-transferasePBSphosphate-buffered saline. )exert a variety of biological effects on normal and neoplastic cells that include antiviral and antiproliferative activities(1Petska S. Langer J.A. Zoon K.C. Samuel C.E. Annu. Rev. Biochem. 1987; 56: 727-777Crossref PubMed Scopus (1593) Google Scholar). Immediately after IFNα stimulation, several signaling proteins in the receptor complex become tyrosine-phosphorylated, including the α and β subunits of the Type I IFN receptor (2Platanias L.C. Colamonici O.R. J. Biol. Chem. 1992; 267: 24053-24057Abstract Full Text PDF PubMed Google Scholar, 3Platanias L.C. Uddin S. Colamonici O.R. J. Biol. Chem. 1994; 269: 17761-17764Abstract Full Text PDF PubMed Google Scholar) and the Tyk-2 and Jak-1 tyrosine kinases(4Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (637) Google Scholar, 5Shuai K. Ziemiecki A. Wilks A.F. Harpur A.G. Sadowski H.B. Gilman M.Z. Darnell J.E. Nature. 1993; 366: 580-583Crossref PubMed Scopus (407) Google Scholar, 6Silvennoinen O. Ihle J.N. Schlessinger J. Levy D.E. Nature. 1993; 366: 583-585Crossref PubMed Scopus (287) Google Scholar, 7Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar). Both kinases associate with components of the Type I IFN receptor(7Colamonici O.R. Uyttendaele H. Domanski P. Yan H. Krolewski J.J. J. Biol. Chem. 1994; 269: 3518-3522Abstract Full Text PDF PubMed Google Scholar, 8Novick D. Cohen B. Rubinstein M. Cell. 1994; 77: 391-400Abstract Full Text PDF PubMed Scopus (576) Google Scholar), and their activation early in the IFNα signaling cascade is presumed to regulate tyrosine phosphorylation of various downstream signaling molecules. Expression of Jak-1 and Tyk-2 rescues an IFNα response in certain insensitive cell lines, suggesting that these tyrosine kinases or related members of the Janus family are essential for IFNα action(4Müller M. Briscoe J. Laxton C. Guschin D. Ziemiecki A. Silvennoinen O. Harpur A.G. Barbieri G. Witthuhn B.A. Schindler C. Pellegrini S. Wilks A.F. Ihle J.N. Stark G.R. Kerr I.M. Nature. 1993; 366: 129-135Crossref PubMed Scopus (637) Google Scholar, 9Velazquez L. Fellous M. Stark G.R. Pellegrini S. Cell. 1992; 70: 313-322Abstract Full Text PDF PubMed Scopus (701) Google Scholar).Several proteins are substrates for IFNα-dependent tyrosine kinase activity. In response to IFNα treatment of cells, the Stat-2, Stat-1α, and Stat-1β components of the transcriptional activator ISGF3α are rapidly phosphorylated on tyrosine and associate with a 48-kDa protein (ISGF3γ) to form an active complex(10Fu X.-Y. Cell. 1992; 70: 323-335Abstract Full Text PDF PubMed Scopus (304) Google Scholar, 11Schindler C. Shuai K. Prezioso V.R. Darnell Jr., J.E. Science. 1992; 257: 809-813Crossref PubMed Scopus (714) Google Scholar, 12Gutch M.J. Daly C. Reich N.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 8: 11411-11415Crossref Scopus (64) Google Scholar). This complex translocates to the nucleus and initiates gene transcription during binding to interferon-stimulated response elements (10-13). In addition, the vav proto-oncogene product (p95vav) is tyrosine-phosphorylated during IFNα stimulation (14Platanias L.C. Sweet M.E. J. Biol. Chem. 1994; 269: 3143-3146Abstract Full Text PDF PubMed Google Scholar); however, its precise role in the signal transduction of IFNα remains to be determined. The involvement of multiple pathways in IFNα signaling is consistent with its pleiotropic biological effects on cells and tissues.Many growth factor receptors, including those for epidermal growth factor and platelet-derived growth factor, associate directly through their autophosphorylation sites with a common set of signaling proteins that contain SH2 domains, including the phosphatidylinositol (PI) 3′-kinase, Grb-2, SH-PTP2, phospholipase Cγ, and Ras-GAP(15Pawson T. Gish G.D. Cell. 1992; 71: 359-362Abstract Full Text PDF PubMed Scopus (792) Google Scholar). The receptors for insulin, IGF-1, and IL-4, however, do not strongly associate with most SH2 proteins known to be involved in their signaling pathways(16Myers M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-294Abstract Full Text PDF PubMed Scopus (288) Google Scholar). Instead, they stimulate tyrosine phosphorylation of docking proteins in the IRS signaling family, notably IRS-1, which binds directly to various SH2 proteins(16Myers M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-294Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 17Sun X.-J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1274) Google Scholar, 18Wang L.-M. Keegan A.D. Li W. Lienhard G.E. Pacini S. Gutkind J.S. Sun X.-J. White M.F. Aaronson S.A. Paul W.E. Pierce J.H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4032-4036Crossref PubMed Scopus (166) Google Scholar, 19Wang L.-M. Myers M.G. Sun X.-J. Aaronson S.A. White M.F. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (365) Google Scholar). We report that IFNα also engages the IRS signaling system, as evidenced by the rapid IFNα-dependent tyrosine phosphorylation of IRS-1 in several hematopoietic cell lines. We also demonstrate that the p85 regulatory subunit of the PI 3′-kinase binds via its SH2 domains the IFNα-induced phosphorylated form of IRS-1, and that PI 3′-kinase activity can be detected in association with IRS-1.