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• W2149690803 abstract "Activation of JAK tyrosine kinases is an essential step in cell signaling by multiple hormones, cytokines, and growth factors, including growth hormone (GH) and interferon-γ. Previously, we identified SH2-Bβ as a potent activator of JAK2 (Rui, L., and Carter-Su, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7172–7177). Here, we investigated whether the activation of JAK2 by SH2-Bβ is specific to JAK2 and SH2-Bβ or extends to other JAKs or other members of the SH2-Bβ family. When SH2-Bβ was overexpressed with JAK1 or JAK3, SH2-Bβ failed to increase their activity. However, SH2-Bβ bound to both and was tyrosyl-phosphorylated by JAK1. In contrast to SH2-Bβ, APS decreased tyrosyl phosphorylation of GH-stimulated JAK2 as well as Stat5B, a substrate of JAK2. APS also decreased tyrosyl phosphorylation of JAK1, but did not affect the activity or tyrosyl phosphorylation of JAK3. Overexpressed APS bound to and was tyrosyl-phosphorylated by all three JAKs. Consistent with these data, in 3T3-F442A adipocytes, endogenous APS was tyrosyl-phosphorylated in response to GH and interferon-γ. These results suggest that 1) SH2-Bβ specifically activates JAK2, 2) APS negatively regulates both JAK2 and JAK1, and 3) both SH2-Bβ and APS may serve as adapter proteins for all three JAKs independent of any role they have in JAK activity. Activation of JAK tyrosine kinases is an essential step in cell signaling by multiple hormones, cytokines, and growth factors, including growth hormone (GH) and interferon-γ. Previously, we identified SH2-Bβ as a potent activator of JAK2 (Rui, L., and Carter-Su, C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7172–7177). Here, we investigated whether the activation of JAK2 by SH2-Bβ is specific to JAK2 and SH2-Bβ or extends to other JAKs or other members of the SH2-Bβ family. When SH2-Bβ was overexpressed with JAK1 or JAK3, SH2-Bβ failed to increase their activity. However, SH2-Bβ bound to both and was tyrosyl-phosphorylated by JAK1. In contrast to SH2-Bβ, APS decreased tyrosyl phosphorylation of GH-stimulated JAK2 as well as Stat5B, a substrate of JAK2. APS also decreased tyrosyl phosphorylation of JAK1, but did not affect the activity or tyrosyl phosphorylation of JAK3. Overexpressed APS bound to and was tyrosyl-phosphorylated by all three JAKs. Consistent with these data, in 3T3-F442A adipocytes, endogenous APS was tyrosyl-phosphorylated in response to GH and interferon-γ. These results suggest that 1) SH2-Bβ specifically activates JAK2, 2) APS negatively regulates both JAK2 and JAK1, and 3) both SH2-Bβ and APS may serve as adapter proteins for all three JAKs independent of any role they have in JAK activity. Janus tyrosine kinase growth hormone interferon interleukin signal transducers and activators of transcription platelet-derived growth factor anti-JAK antibody anti-Myc antibody anti-phosphotyrosine antibody anti-APS antibody green fluorescent protein growth hormone receptor Dulbecco's modified Eagle's medium mitogen-activated protein kinases extracellular signal-regulated kinase suppressor of cytokine signaling erythropoietin The Janus family tyrosine kinases, consisting of JAK1,1 JAK2, JAK3, and Tyk2, bind to members of the cytokine family of receptors and are activated upon ligand binding to these receptors. This family of receptors consists of more than 20 different proteins that are known to bind to at least 25 different ligands, including growth hormone (GH), prolactin, leptin, interferon (IFN)-α, IFN-β, IFN-γ, and most interleukins (1Ihle J.N. Adv. Immunol. 1995; 60: 1-35Crossref PubMed Google Scholar, 2Smit L.S. Meyer D.J. Argetsinger L.S. Schwartz J. Carter-Su C. Kostyo J.L. Handbook of Physiology. V. Oxford University Press, New York1999: 445-480Google Scholar). Of these 25 ligands, more than two-thirds activate JAK2. JAK1 and Tyk2, like JAK2, are ubiquitously expressed and, in general, are activated by a similar, although more limited set of ligands compared with JAK2. In contrast, JAK3 is predominantly expressed in hematopoietic cells and is activated by a different set of ligands, including interleukin (IL)-2, IL-4, and IL-7, which are not potent activators of JAK2 (2Smit L.S. Meyer D.J. Argetsinger L.S. Schwartz J. Carter-Su C. Kostyo J.L. Handbook of Physiology. V. Oxford University Press, New York1999: 445-480Google Scholar, 3Aringer M. Cheng A. Nelson J.W. Chen M. Sudarshan C. Zhou Y.J. O'Shea J.J. Life Sci. 1999; 64: 2173-2186Crossref PubMed Scopus (64) Google Scholar). Knockout studies have revealed specific and vital roles for JAK kinases. Mice deficient in JAK2 die by day 12 of embryogenesis from a lack of erythropoiesis (4Parganas E. Wang D. Stravopodis D. Topham D.J. Marine J.C. Teglund S. Vanin E.F. Bodner S. Colamonici O.R. van Deursen J.M. Grosveld G. Ihle J.N. Cell. 1998; 93: 385-395Abstract Full Text Full Text PDF PubMed Scopus (912) Google Scholar, 5Neubauer H. Cumano A. Muller M. Wu H. Huffstadt U. Pfeffer K. Cell. 1998; 93: 397-409Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar). JAK1-deficient mice are smaller than their littermates, fail to nurse, and die within 1 day of birth (6Rodig S.J. Meraz M.A. White J.M. Lampe P.A. Riley J.K. Arthur C.D. King K.L. Sheehan K.C. Yin L. Pennica D. Johnson Jr., E.M. Schreiber R.D. Cell. 1998; 93: 373-383Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). In contrast to JAK1- or JAK2-deficient mice, JAK3 knockout mice survive and develop normally in pathogen-free conditions. However, these mice exhibit severe defects in lymphoid development (7Nosaka T. van Deursen J.M. Tripp R.A. Thierfelder W.E. Witthuhn B.A. McMickle A.P. Doherty P.C. Grosveld G.C. Ihle J.N. Science. 1995; 270: 800-802Crossref PubMed Scopus (576) Google Scholar, 8Thomis D.C. Gurniak C.B. Tivol E. Sharpe A.H. Berg L.J. Science. 1995; 270: 794-797Crossref PubMed Scopus (477) Google Scholar, 9Park S.Y. Saijo K. Takahashi T. Osawa M. Arase H. Hirayama N. Miyake K. Nakauchi H. Shirasawa T. Saito T. Immunity. 1995; 3: 771-782Abstract Full Text PDF PubMed Scopus (446) Google Scholar). Mice lacking Tyk2 develop normally and exhibit no major abnormalities in fertility or blood cell development. However, Tyk2-deficient mice have reduced responses to specific cytokines, including IFN-α/β, IL-12, and IFN-γ (10Karaghiosoff M. Neubauer H. Lassnig C. Kovarik P. Schindler H. Pircher H. McCoy B. Bogdan C. Decker T. Brem G. Pfeffer K. Muller M. Immunity. 2000; 13: 549-560Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). Taken together, studies using cellular models as well as analysis of knockout mice show that activation of JAKs is critical for such diverse responses as growth, lactation, nerve cell differentiation, hematopoiesis, and immune responses (3Aringer M. Cheng A. Nelson J.W. Chen M. Sudarshan C. Zhou Y.J. O'Shea J.J. Life Sci. 1999; 64: 2173-2186Crossref PubMed Scopus (64) Google Scholar, 10Karaghiosoff M. Neubauer H. Lassnig C. Kovarik P. Schindler H. Pircher H. McCoy B. Bogdan C. Decker T. Brem G. Pfeffer K. Muller M. Immunity. 2000; 13: 549-560Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 11Kishimoto T. Taga T. Akira S. Cell. 1994; 76: 253-262Abstract Full Text PDF PubMed Scopus (1250) Google Scholar, 12Patterson P.H. Nawa H. Cell. 1993; 72: 123-137Abstract Full Text PDF PubMed Scopus (300) Google Scholar). Upon ligand binding to cytokine receptors, JAKs phosphorylate themselves and their associated receptors, thereby providing multiple binding sites for signaling proteins containing SH2 or other phosphotyrosine-binding domains. Signaling proteins that bind to receptor·JAK complexes and undergo tyrosyl phosphorylation include STATs (13Darnell Jr., J.E. Science. 1997; 277: 1630-1635Crossref PubMed Scopus (3401) Google Scholar), Shc (14VanderKuur J. Allevato G. Billestrup N. Norstedt G. Carter-Su C. J. Biol. Chem. 1995; 270: 7587-7593Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), insulin receptor substrates (15Argetsinger L.S. Hsu G.W. Myers Jr., M.G. Billestrup N. Norstedt G. White M.F. Carter-Su C. J. Biol. Chem. 1995; 270: 14685-14692Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 16Argetsinger L.S. Billestrup N. Norstedt G. White M.F. Carter-Su C. J. Biol. Chem. 1996; 271: 29415-29421Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), and focal adhesion kinase (17Zhu T. Goh E.L. Lobie P.E. J. Biol. Chem. 1998; 273: 10682-10689Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Although activation of JAKs by cytokine receptor ligands is generally rapid and transient, constitutive activation of JAKs has been observed in a variety of cancers, indicating that regulation of JAKs is critical for controlling cell growth and proliferation. For example, leukemic cells from patients with acute lymphoblastic leukemia (18Lacronique V. Boureux A. Valle V.D. Poirel H. Quang C.T. Mauchauffe M. Berthou C. Lessard M. Berger R. Ghysdael J. Bernard O.A. Science. 1997; 278: 1309-1312Crossref PubMed Scopus (682) Google Scholar) were shown to have constitutively active JAK2, and specific inhibition of JAK2 in cells derived from an acute lymphoblastic leukemia patient blocked cell growth by inducing apoptosis (19Meydan N. Grunberger T. Dadi H. Shahar M. Arpaia E. Lapidot Z. Leeder J.S. Freedman M. Cohen A. Gazit A. Levitzki A. Roifman C.M. Nature. 1996; 379: 645-648Crossref PubMed Scopus (848) Google Scholar). Similarly, JAK1 and JAK3 were found to be constitutively active in cells from patients suffering from adult T cell leukemia/lymphoma caused by human T cell leukemia/lymphotrophic virus type I (20Takemoto S. Mulloy J.C. Cereseto A. Migone T.S. Patel B.K. Matsuoka M. Yamaguchi K. Takatsuki K. Kamihira S. White J.D. Leonard W.J. Waldmann T. Franchini G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13897-13902Crossref PubMed Scopus (235) Google Scholar). Furthermore, specific inhibition of JAKs inhibits the growth of multiple breast cancer cell lines and induces apoptosis in MDA-MB-468 breast cancer cells (21Garcia R. Bowman T.L. Niu G. Yu H. Minton S. Muro-Cacho C.A. Cox C.E. Falcone R. Fairclough R. Parsons S. Laudano A. Gazit A. Levitzki A. Kraker A. Jove R. Oncogene. 2001; 20: 2499-2513Crossref PubMed Scopus (662) Google Scholar). The critical role of JAKs in so many normal physiological responses and their potential role in some cancers make it vitally important to obtain a better understanding of the mechanisms by which JAKs are regulated. Recently, our laboratory identified the SH2 domain-containing protein SH2-Bβ (22Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar) as a potent activator of JAK2 (23Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar). Addition of GH stimulates the phosphorylation of JAK2, leading to the association of SH2-Bβ via its SH2 domain to one or more phosphorylated tyrosines in JAK2. This latter interaction substantially activates JAK2, thereby increasing the phosphorylation of JAK2 as well as of downstream targets of JAK2 such as Stat5B (23Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar). SH2-B is a member of a family of adapter proteins, which also includes APS and Lnk (24Osborne M.A. Dalton S. Kochan J.P. Bio/Technology. 1995; 13: 1474-1478Crossref PubMed Scopus (140) Google Scholar, 25Yokouchi M. Suzuki R. Masuhara M. Komiya S. Inoue A. Yoshimura A. Oncogene. 1997; 15: 7-15Crossref PubMed Scopus (108) Google Scholar, 26Huang X. Li Y. Tanaka K. Moore K.G. Hayashi J.I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11618-11622Crossref PubMed Scopus (138) Google Scholar) (Fig. 1). All three contain a pleckstrin homology domain in their amino termini and an SH2 domain near their C termini. The various isoforms of SH2-B (α, β, γ, and δ) also contain at least three proline-rich regions (22Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar, 27Nelms K. O'Neill T.J. Li S. Hubbard S.R. Gustafson T.A. Paul W.E. Mamm. Genome. 1999; 10: 1160-1167Crossref PubMed Scopus (68) Google Scholar,28Riedel H. Wang J. Hansen H. Yousaf N. J. Biochem. (Tokyo). 1997; 122: 1105-1113Crossref PubMed Scopus (66) Google Scholar). Although we have shown SH2-Bβ to be a positive regulator of JAK2 (23Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar), studies by others suggest that APS and Lnk may be negative regulators of some signaling pathways. Lnk has been shown to play a pivotal role in the regulation of B cell production, as Lnk knockout mice show overproduction of pre-B cells in the spleen and pro-B cells in bone marrow (29Takaki S. Sauer K. Iritani B.M. Chien S. Ebihara Y. Tsuji K. Takatsu K. Perlmutter R.M. Immunity. 2000; 13: 599-609Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Overexpression of APS suppresses proliferation of NIH-3T3 cells as stimulated by platelet-derived growth factor (PDGF) (30Yokouchi M. Wakioka T. Sakamoto H. Yasukawa H. Ohtsuka S. Sasaki A. Ohtsubo M. Valius M. Inoue A. Komiya S. Yoshimura A. Oncogene. 1999; 18: 759-767Crossref PubMed Scopus (68) Google Scholar). To date, the effects of APS and Lnk on the kinase activity and tyrosyl phosphorylation of JAKs have not been examined. Furthermore, SH2-Bβ has not been examined as a regulator of JAKs other than JAK2. Because of the importance of the cytokine receptor family of ligands and the remarkable ability of SH2-Bβ to activate JAK2, we examined whether the activating ability of SH2-Bβ is specific to JAK2 or extends to other members of the JAK family of tyrosine kinases. We also examined whether the ability of SH2-Bβ to activate JAK2 is shared by APS. Finally, we examined whether SH2-Bβ or APS binds to JAK1, JAK2, or JAK3 and/or is tyrosyl-phosphorylated by any of these JAKs, thereby implicating SH2-Bβ or APS as a signaling molecule for these JAKs. The stocks of COS-7, 293T, and 3T3-F442A cells were provided by Drs. M. D. Uhler (University of Michigan, Ann Arbor, MI), O. A. MacDougald (University of Michigan), and H. Green (Harvard University, Cambridge, MA), respectively. Aprotinin, leupeptin, and Triton X-100 were from Roche Molecular Biochemicals. Recombinant protein A-agarose was from Repligen. The enhanced chemiluminescence detection system (ECL) was from Amersham Biosciences, Inc. Anti-JAK2 antiserum (αJAK2) was raised in rabbits against a synthetic peptide corresponding to amino acids 758–766 of murine JAK2 (31Silvennoinen O. Witthuhn B. Quelle F.W. Cleveland J.L. Yi T. Ihle J.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8429-8433Crossref PubMed Scopus (437) Google Scholar, 32Argetsinger L.S. Campbell G.S. Yang X. Witthuhn B.A. Silvennoinen O. Ihle J.N. Carter-Su C. Cell. 1993; 74: 237-244Abstract Full Text PDF PubMed Scopus (827) Google Scholar) and was used at dilutions of 1:500 for immunoprecipitation and 1:15,000 for immunoblotting. Antibody against the Myc tag (αMyc; 9E10) was from Santa Cruz Biotechnology and was used for immunoblotting at a dilution of 1:10,000. For immunoprecipitation, αMyc was used at a dilution of 1:100 with rabbit anti-mouse IgG (1:100) (Upstate Biotechnology, Inc.). Monoclonal anti-phosphotyrosine antibody (αPY; clone 4G10; Upstate Biotechnology, Inc.) was used at a dilution of 1:7500 for immunoblotting. Antibody against murine JAK1 (αJAK1) was kindly provided by Dr. A. C. Larner (Learner Research Institute, Cleveland Clinic, Cleveland, OH) (33David M. Chen H.E. Goelz S. Larner A.C. Neel B.G. Mol. Cell. Biol. 1995; 15: 7050-7058Crossref PubMed Scopus (318) Google Scholar) and was used for immunoprecipitation at a dilution of 1:300. Polyclonal rabbit αJAK1 (Pharmingen) was used for immunoblotting at a dilution of 1:5000. αJAK3 was raised in rabbits against a synthetic peptide corresponding to amino acids 1104–1124 of human JAK3 (34Johnston J.A. Kawamura M. Kirken R.A. Chen Y.-Q. Blake T.B. Shibuya K. Ortaldo J.R. McVicar D.W. O'Shea J.J. Nature. 1994; 370: 151-153Crossref PubMed Scopus (510) Google Scholar) and was used at dilutions of 1:300 for immunoprecipitation and 1:3000 for immunoblotting. Anti-green fluorescent protein (GFP) antibody was from CLONTECH and was used at a dilution of 1:5000 for immunoblotting. Polyclonal rabbit anti-phospho-Stat5B antibody was from Zymed Laboratories Inc. and was used at a dilution of 1 μg/ml for immunoblotting. Anti-APS antibody (αAPS) was kindly provided by Dr. D. D. Ginty (Johns Hopkins University School of Medicine, Baltimore, MD) (35Qian X. Riccio A. Zhang Y. Ginty D.D. Neuron. 1998; 21: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and was used at dilutions of 1:100 for immunoprecipitation and 1:1000 for immunoblotting. The cDNA for wild-type murine JAK2 was provided by Drs. J. N. Ihle and B. A. Witthuhn (St. Jude Children's Research Hospital, Memphis, TN) (31Silvennoinen O. Witthuhn B. Quelle F.W. Cleveland J.L. Yi T. Ihle J.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8429-8433Crossref PubMed Scopus (437) Google Scholar). The cDNA for human JAK3 was described previously (36Russell S.M. Johnston J.A. Noguchi M. Kawamura M. Bacon C.M. Friedmann M. Berg M. McVicar D.W. Witthuhn B.A. Silvennoinen O. Goldman A.S. Schmalstieg F.C. Ihle J.N. OShea J.J. Leonard W.J. Science. 1994; 266: 1042-1045Crossref PubMed Scopus (592) Google Scholar, 37Zhou Y.J. Hanson E.P. Chen Y.-Q. Magnuson K. Chen M. Swann P.G. Wange R.L. Changelian P.S. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13850-13855Crossref PubMed Scopus (99) Google Scholar). The cDNA for murine JAK1 was provided by Drs. X. Yang and C. L. Cepko (Harvard Medical School, Boston MA) (38Yang X. Chung D. Cepko C.L. J. Neurosci. 1993; 13: 3006-3017Crossref PubMed Google Scholar). cDNA encoding murine JAK1 with a Myc tag at its C terminus was kindly provided by Dr. R. D. Schreiber (Washington University, St. Louis, MO). Construction of the vector encoding SH2-Bβ with a Myc tag at its N terminus has been described previously (23Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar). cDNA encoding Myc-tagged rat APS was kindly provided by Dr. D. D. Ginty (35Qian X. Riccio A. Zhang Y. Ginty D.D. Neuron. 1998; 21: 1017-1029Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). cDNA encoding GFP-tagged Stat5B was constructed as described (39Herrington J. Rui L. Luo G. Yu-Lee L.-y. Carter-Su C. J. Biol. Chem. 1999; 274: 5138-5145Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). cDNA encoding the rat GH receptor (GHR) was provided by Dr. G. Norstedt (Karolinska Institute, Stockholm, Sweden) (40Emtner M. Mathews L.S. Norstedt G. Mol. Endocrinol. 1990; 4: 2014-2020Crossref PubMed Scopus (43) Google Scholar). COS-7, 293T, and 3T3-F442A cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml amphotericin (supplemented DMEM), and 10% fetal calf serum (COS-7 cells) or 8% calf serum (293T and 3T3-F442A cells). Cells were transiently transfected using calcium phosphate precipitation (41Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4824) Google Scholar). Transfected cells were assayed 36–48 h after transfection. Before stimulating transfected cells with hormone, cells were incubated overnight in serum-free medium containing 1% bovine serum albumin and treated with ligands at 37 °C. 3T3-F442A fibroblasts were differentiated to adipocytes by treating confluent cells for 48 h with 2 μg/ml insulin, 0.25 μm dexamethasone, 0.5 mm methylisobutylxanthine, and 10% fetal calf serum in supplemented DMEM for 48 h. Cells were treated for an additional 48 h with supplemented DMEM containing 10% fetal calf serum and 1 μg/ml insulin (42Sumantran V.N. Tsai M.L. Schwartz J. Endocrinology. 1992; 130: 2016-2024Crossref PubMed Scopus (28) Google Scholar). Adipocytes were maintained in supplemented DMEM plus 10% fetal calf serum. Before assays, cells were incubated overnight in serum-free medium containing 1% bovine serum albumin and treated with ligands at 37 °C. Immunoprecipitations and immunoblotting were performed as described (43Rui L. Gunter D.R. Herrington J. Carter-Su C. Mol. Cell. Biol. 2000; 20: 3168-3177Crossref PubMed Scopus (51) Google Scholar). Thirty (293T cells) to 48 (COS-7 cells) h after transfection, cells were rinsed three times with 10 mm sodium phosphate (pH 7.4), 150 mm NaCl, and 1 mmNa3VO4. Cells were then solubilized in lysis buffer (50 mm Tris (pH 7.5), 0.1% Triton X-100, 150 mm NaCl, 2 mm EGTA, 1 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin) and centrifuged at 14,000 × g for 10 min at 4 °C. The supernatant (cell lysate) was incubated with the indicated antibody on ice for 2 h. The immune complexes were collected on protein A-agarose (14-μl packed volume) for 1 h at 4 °C. The beads were washed three times with wash buffer (50 mm Tris (pH 7.5), 0.1% Triton X-100, 150 mm NaCl, and 2 mmEGTA) and boiled for 5 min in a 80:20 mixture of lysis buffer and SDS-PAGE sample buffer (250 mm Tris-HCl (pH 6.8), 10% SDS, 10% β-mercaptoethanol, 40% glycerol, and 0.01% bromphenol blue). The solubilized proteins were separated by SDS-PAGE (7.5 or 5–12% gradient), followed by immunoblotting with the indicated antibody and visualization with the ECL detection system. In vitro kinase assays were performed as described previously (43Rui L. Gunter D.R. Herrington J. Carter-Su C. Mol. Cell. Biol. 2000; 20: 3168-3177Crossref PubMed Scopus (51) Google Scholar). JAKs were immunoprecipitated with the appropriate αJAK using protein A-agarose. Bound proteins were washed twice with lysis buffer and twice with kinase buffer (50 mm HEPES (pH 7.6), 5 mmMnCl2, 0.5 mm dithiothreitol, 100 mm NaCl, and 1 mmNa3VO4). Immunoprecipitates were incubated at 30 °C for 30 min in 50 μl of kinase buffer containing [γ-32P]ATP, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. Immunoprecipitates were washed once with 500 μl of kinase buffer supplemented with 10 mm EDTA, followed by three washes with 500 μl of lysis buffer. Proteins were eluted by boiling in a 4:1 mixture of lysis buffer and SDS-PAGE sample buffer. Proteins were then resolved by SDS-PAGE (7.5 or 5–12% gradient), transferred to nitrocellulose membrane, and visualized by autoradiography, followed by immunoblotting with the appropriate αJAK. We have shown previously that SH2-Bβ is a potent activator of JAK2 (23Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar). To determine whether the activation of JAK2 by SH2-Bβ is specific to JAK2 or shared by other members of the Janus family of tyrosine kinases, we examined whether SH2-Bβ could also activate JAK1. Like JAK2, JAK1 is widely expressed and is activated by an overlapping set of ligands, including GH, leukemia inhibitory factor, and granulocyte colony-stimulating factor (2Smit L.S. Meyer D.J. Argetsinger L.S. Schwartz J. Carter-Su C. Kostyo J.L. Handbook of Physiology. V. Oxford University Press, New York1999: 445-480Google Scholar). We also examined whether SH2-Bβ could activate JAK3, which is activated by ligands that are not potent activators of JAK2 such as IL-7 (3Aringer M. Cheng A. Nelson J.W. Chen M. Sudarshan C. Zhou Y.J. O'Shea J.J. Life Sci. 1999; 64: 2173-2186Crossref PubMed Scopus (64) Google Scholar). Initially, we assessed the kinase activity of JAK3 using an in vitro kinase assay. For comparison, the experiment was performed concurrently with JAK2. cDNA encoding the appropriate JAK was transfected alone or with cDNA encoding Myc-tagged SH2-Bβ in COS-7 cells. The expressed JAK was immunoprecipitated with the appropriate αJAK and incubated with [γ-32P]ATP. The immunoprecipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose.32P-Labeled proteins were visualized by autoradiography, and the amount of immunoprecipitated JAK was determined by immunoblotting with the appropriate αJAK. As reported previously (23Rui L. Carter-Su C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7172-7177Crossref PubMed Scopus (120) Google Scholar), JAK2 was constitutively active when overexpressed in COS-7 cells (Fig. 2 A, lane 1,upper panel). Furthermore, its in vitro kinase activity was substantially increased by the coexpression of SH2-Bβ (lane 2, upper panel). JAK3 was also constitutively active when overexpressed in COS-7 cells (lane 3, upper panel). However, in contrast to JAK2, JAK3 was not activated by coexpression with SH2-Bβ (lane 4,upper panel). Similar results were obtained when JAK2 and JAK3 were expressed in 293T cells (data not shown). We next examined the ability of SH2-Bβ to increase the in vitro kinase activity of JAK1. cDNA encoding JAK1 (or JAK2 for comparison) was transfected into 293T cells alone or with cDNA encoding Myc-tagged SH2-Bβ. 293T cells were used to overexpress JAK1 because we had difficulty overexpressing JAK1 in COS-7 cells. Kinase assays with JAK1 were performed with [γ-32P]ATP as described for JAK3. However, JAK1 incorporated only a small amount of [γ-32P]ATP in vitro compared with JAK2 and JAK3, raising the possibility that our anti-JAK1 antibodies interfere with the in vitro kinase assay. We therefore probed JAK1 (and JAK2) with αPY to examine the effects of overexpressed SH2-Bβ on the tyrosyl phosphorylation of JAK1 (and JAK2). The tyrosyl phosphorylation of JAKs is thought to reflect their autophosphorylation. SH2-Bβ increased the tyrosyl phosphorylation of JAK2 (Fig. 2 B, lane 2, upper panel) consistent with increased activity of JAK2. However, SH2-Bβ had no reproducible effect on the tyrosyl phosphorylation of JAK1 (lane 4, upper panel), suggesting that SH2-Bβ has no effect on the activity of JAK1. SH2-Bβ binds to JAK2 (Fig. 3 A) as assessed by coprecipitation experiments using overexpressed or endogenous JAK2 and SH2-Bβ (22Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar). Furthermore, we have shown that overexpressed JAK2 tyrosyl-phosphorylates overexpressed SH2-Bβ both in vivoand in vitro and that endogenous SH2-Bβ is phosphorylated on tyrosines in 3T3-F442A cells in response to GH and IFN-γ, two ligands that activate JAK2 (22Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar). This tyrosyl phosphorylation suggests that SH2-Bβ may also function as an adapter protein for ligands that activate JAK2 by recruiting proteins that bind phosphotyrosines to JAK2·SH2-Bβ complexes. To determine whether SH2-Bβ might also serve as a signaling protein for ligands that activate JAK1 and JAK3, even though SH2-Bβ does not appear to activate these JAKs, we examined whether either JAK1 or JAK3 associates with and/or phosphorylates SH2-Bβ. Myc-tagged SH2-Bβ was transiently overexpressed with JAK1 or JAK2 in 293T cells or with JAK3 in COS-7 cells. Myc-SH2-Bβ was immunoprecipitated with αMyc, and precipitated proteins were blotted with the appropriate αJAK. As reported previously (22Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar), SH2-Bβ coprecipitated with JAK2 (Fig.3 A, lane 2, upper panel). It also coprecipitated with JAK1 (Fig. 3 B, lane 2,upper panel) 2In Fig. 2 B, murine JAK1 cDNA with a C-terminal Myc tag (provided by Dr. R. D. Schreiber) was used. In Fig. 3 B, murine JAK1 cDNA lacking a Myc tag (provided by Drs. X. Yang and C. L. Cepko) was used to avoid directly precipitating JAK1 with αMyc. The bottom band obtained using the latter cDNA (Fig. 3 B, lane 2, upper panel; and lane 4) is thought to represent either a degradation product of JAK1 or a truncated form of JAK1 due to a second transcriptional start site encoded in the cDNA. and JAK3 (Fig. 3 C, lane 2, upper panel), suggesting that SH2-Bβ forms a complex with all three of these JAKs. To determine whether SH2-Bβ is tyrosyl-phosphorylated when overexpressed with JAK1, JAK2, or JAK3, the blots were reprobed with αPY. As shown in Fig. 3 A (lane 4), SH2-Bβ was strongly phosphorylated on tyrosines when coexpressed with JAK2. Interestingly, although SH2-Bβ did not seem to stimulate JAK1, SH2-Bβ was phosphorylated on tyrosines in cells overexpressing JAK1 (Fig. 3 B, lane 4). No phosphorylation of SH2-Bβ was detected when cells were transfected with SH2-Bβ alone (data not shown), as reported previously (22Rui L. Mathews L.S. Hotta K. Gustafson T.A. Carter-Su C. Mol. Cell. Biol. 1997; 17: 6633-6644Crossref PubMed Google Scholar), consistent with SH2-Bβ being phosphorylated on tyrosines by JAK1. In contrast, no tyrosyl phosphorylation of SH2-Bβ was detected in cells overexpressing JAK3 (Fig. 3 C, lane 4). Thus, the ability of SH2-Bβ to activate appears to be specific to JAK2. However, SH2-Bβ appears to bind to both JAK1 and JAK3 and to serve as a substrate of JAK1. These data suggest that SH2-Bβ may serve as an adapter protein for ligands that activate JAK1 as well as for ligands that activate JAK2 via binding of signaling molecules to phosphorylated tyrosines in SH2-Bβ and possibly to other binding motifs within SH2-Bβ. Although signaling proteins that bind preferentially to phosphorylated tyrosines would not be expected to be recruited to SH2-Bβ complexed to JAK3, SH2-Bβ may still serve as a signaling molecule for ligands that activate JAK3 by recruiting molecules that bind other motifs (e.g. pleckstrin homology and proline-rich regions) within SH2-Bβ to JAK3·SH2-Bβ complexes. Because SH2-Bβ activates some but not all JAKs and is tyro" @default.
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• W2149690803 date "2002-03-01" @default.
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• W2149690803 title "SH2-B Family Members Differentially Regulate JAK Family Tyrosine Kinases" @default.
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• W2149690803 doi "https://doi.org/10.1074/jbc.m109165200" @default.
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