FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2091245777> ?p ?o ?g. }

• W2091245777 endingPage "40748" @default.
• W2091245777 startingPage "40742" @default.
• W2091245777 abstract "The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr861 of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr861 is essential for activation of ERK1/2 and for full activation of the α2-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines. The common use of the cytokine receptor gp130 has served as an explanation for the extremely redundant biological activities exerted by interleukin (IL)-6-type cytokines. Indeed, hardly any differences in signal transduction initiated by these cytokines are known. In the present study, we demonstrate that oncostatin M (OSM), but not IL-6 or leukemia inhibitory factor, induces tyrosine phosphorylation of the Shc isoforms p52 and p66 and their association with Grb2. Concomitantly, OSM turns out to be a stronger activator of ERK1/2 MAPKs. Shc is recruited to the OSM receptor (OSMR), but not to gp130. Binding involves Tyr861 of the OSMR, located within a consensus binding sequence for the Shc PTB domain. Moreover, Tyr861 is essential for activation of ERK1/2 and for full activation of the α2-macroglobulin promoter, but not for an exclusively STAT-responsive promoter. This study therefore provides evidence for qualitative differential signaling mechanisms exerted by IL-6-type cytokines. oncostatin M interleukin leukemia inhibitory factor LIF receptor OSM receptor tissue inhibitors of metalloproteinases signal transducers and activators of transcription mitogen-activated protein kinase Src homology 2 α2-macroglobulin soluble IL-6 receptor IL-5 receptor extracellular signal-regulated kinase The 28-kDa protein oncostatin M (OSM)1 belongs to the family of interleukin (IL)-6-type cytokines, which additionally comprises IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, cardiotrophin-1, and the recently described novel neurotrophin-1/B-cell stimulatory factor-3 (1Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1715) Google Scholar, 2Senaldi G. Varnum B.C. Sarmiento U. Starnes C. Lile J. Scully S. Guo J. Elliott G. McNinch J. Shaklee C.L. Freeman D. Manu F. Simonet W.S. Boone T. Chang M.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11458-11463Crossref PubMed Scopus (196) Google Scholar). These cytokines play an important role in hematopoiesis, inflammation, the acute phase response, bone, and heart development as well as neurogenesis. Their redundant effects can be attributed to the shared use of the common signal transducing receptor chain glycoprotein (gp) 130. gp130 is homodimerized by IL-6 and IL-11 upon binding to their ligand-specific α-receptors. The other cytokines of this family trigger the heterodimerization of gp130 with the LIF receptor (LIFR) or the OSM-specific receptor (OSMR). Whereas human OSM has the capability to signal both via gp130-LIFR and gp130-OSMR heterodimers, murine OSM solely utilizes the gp130-OSMR heterodimer for signal transduction (1Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1715) Google Scholar, 3Ichihara M. Hara T. Kim H. Murate T. Miyajima A. Blood. 1997; 90: 165-173Crossref PubMed Google Scholar). OSM is involved in various biological responses. It supports the growth of AIDS-associated Kaposi's sarcoma cells (4Miles S.A. Martinez-Maza O. Rezai A. Magpantay L. Kishimoto T. Nakamura S. Radka S.F. Linsley P.S. Science. 1992; 255: 1432-1434Crossref PubMed Scopus (237) Google Scholar, 5Nair B.C. DeVico A.L. Nakamura S. Copeland T.D. Chen Y. Patel A. O'Neil T. Oroszlan S. Gallo R.C. Sarngadharan M.G. Science. 1992; 255: 1430-1432Crossref PubMed Scopus (209) Google Scholar), whereas it leads to growth inhibition of various solid tumors (6Grant S.L. Begley C.G. Mol. Med. Today. 1999; 5: 406-412Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Due to its ability to induce TIMP-1 and TIMP-3, profibrotic properties have been attributed to this cytokine (7Richards C.D. Shoyab M. Brown T.J. Gauldie J. J. Immunol. 1993; 150: 5596-5603PubMed Google Scholar, 8Gatsios P. Haubeck H.D. Van de Leur E. Frisch W. Apte S.S. Greiling H. Heinrich P.C. Graeve L. Eur. J. Biochem. 1996; 241: 56-63Crossref PubMed Scopus (54) Google Scholar, 9Li W.Q. Zafarullah M. J. Immunol. 1998; 161: 5000-5007PubMed Google Scholar). Indeed, transgenic mice expressing OSM in islet β-cells develop severe fibrosis (10Bamber B. Reife R.A. Haugen H.S. Clegg C.H. J. Mol. Med. 1998; 76: 61-69Crossref PubMed Scopus (46) Google Scholar). In addition, OSM is suggested to play a role in the wound healing process and in attenuation of the inflammatory response (11Wallace P.M. MacMaster J.F. Rouleau K.A. Brown T.J. Loy J.K. Donaldson K.L. Wahl A.F. J. Immunol. 1999; 162: 5547-5555PubMed Google Scholar). Compared with other IL-6-type cytokines, OSM often induces stronger effects e.g.with regard to STAT and MAPK activation, induction of protease inhibitors, or growth inhibition (11Wallace P.M. MacMaster J.F. Rouleau K.A. Brown T.J. Loy J.K. Donaldson K.L. Wahl A.F. J. Immunol. 1999; 162: 5547-5555PubMed Google Scholar, 12Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar, 13Liu J. Spence M.J. Wallace P.M. Forcier K. Hellstrom I. Vestal R.E. Cell Growth Differ. 1997; 8: 667-676PubMed Google Scholar, 14Kortylewski M. Heinrich P.C. Mackiewicz A. Schniertshauer U. Klingmüller U. Nakajima K. Hirano T. Horn F. Behrmann I. Oncogene. 1999; 18: 3742-3753Crossref PubMed Scopus (123) Google Scholar, 15Thoma B. Bird T.A. Friend D.J. Gearing D.P. Dower S.K. J. Biol. Chem. 1994; 269: 6215-6222Abstract Full Text PDF PubMed Google Scholar). The molecular basis for this phenomenon is not known. Concerning the functional properties of the three signal transducing receptor subunits, much progress has been achieved in elucidating the signaling events initiated by the gp130 receptor chain (reviewed in Ref. 1Heinrich P.C. Behrmann I. Müller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1715) Google Scholar). gp130 associates with tyrosine kinases of the Janus family (Jak1, Jak2, and Tyk2). Upon ligand binding and receptor dimerization, Jaks are activated and in turn phosphorylate gp130 on several cytoplasmic tyrosine residues, which then provide docking sites for SH2 domain-containing molecules, such as transcription factors of the STAT family (STAT3 and STAT1). Upon phosphorylation, the STATs translocate as dimers into the nucleus, where they bind to promoter regions of their specific response genes. Additionally, the tyrosine phosphatase SHP-2 becomes recruited to gp130 via tyrosine residue 759 (16Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (862) Google Scholar). SHP-2 has been implicated in the down-regulation of gp130-mediated signals (17Symes A. Stahl N. Reeves S.A. Farruggella T. Servidei T. Gearan T. Yancopoulos G. Fink J.S. Curr. Biol. 1997; 7: 697-700Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 18Kim H. Hawley T.S. Hawley R.G. Baumann H. Mol. Cell. Biol. 1998; 18: 1525-1533Crossref PubMed Scopus (104) Google Scholar, 19Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (140) Google Scholar). In addition, SHP-2 serves as an adapter molecule linking cytokine receptors like gp130 to the Ras/Raf/MAPK pathway (20Fukada T. Hibi M. Yamanaka Y. Takahashi-Tezuka M. Fujitani Y. Yamaguchi T. Nakajima K. Hirano T. Immunity. 1996; 5: 449-460Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). In the present study, we addressed the question whether the OSMR contributes distinguishably to signal transduction. We demonstrate that in contrast to gp130, the human OSMR does not recruit SHP-2 but utilizes another protein implicated in activation of the Ras/Raf/MAPK pathway, Shc. This protein exists in three different isoforms of 46 kDa (p46), 52 kDa (p52), and 66 kDa (p66); p46 and p52 are produced by using alternative translation initiation sites of the same transcript, whereas p66 results from a differentially spliced message (21Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 22Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1128) Google Scholar, 23Migliaccio E. Mele S. Salcini A.E. Pelicci G. Lai K.M. Superti-Furga G. Pawson T. Di Fiore P.P. Lanfrancone L. Pelicci P.G. EMBO J. 1997; 16: 706-716Crossref PubMed Scopus (360) Google Scholar, 24Okada S. Kao A.W. Ceresa B.P. Blaikie P. Margolis B. Pessin J.E. J. Biol. Chem. 1997; 272: 28042-28049Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The 46- and 52-kDa isoforms are expressed ubiquitously, whereas the 66-kDa isoform is predominantly found in cells of epithelial origin. Shc contains an SH2 and a PTB domain, separated by a proline/glycine-rich collagen homology domain (CH1). The p66 isoform contains a further CH domain (CH2) at the N terminus (23Migliaccio E. Mele S. Salcini A.E. Pelicci G. Lai K.M. Superti-Furga G. Pawson T. Di Fiore P.P. Lanfrancone L. Pelicci P.G. EMBO J. 1997; 16: 706-716Crossref PubMed Scopus (360) Google Scholar, 24Okada S. Kao A.W. Ceresa B.P. Blaikie P. Margolis B. Pessin J.E. J. Biol. Chem. 1997; 272: 28042-28049Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The SH2 and the PTB domain of Shc are involved in phosphorylation-dependent association with membrane receptors upon stimulation of cells with a number of growth factors and cytokines (21Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar). Moreover, the PTB domain can interact with acidic phospholipids, thereby possibly mediating membrane association (25Ravichandran K.S. Zhou M.M. Pratt J.C. Harlan J.E. Walk S.F. Fesik S.W. Burakoff S.J. Mol. Cell. Biol. 1997; 17: 5540-5549Crossref PubMed Google Scholar). Shc can be phosphorylated at three different tyrosine residues, Tyr239, Tyr240, and Tyr317 (26Salcini A.E. McGlade J. Pelicci G. Nicoletti I. Pawson T. Pelicci P.G. Oncogene. 1994; 9: 2827-2836PubMed Google Scholar, 27Gotoh N. Tojo A. Shibuya M. EMBO J. 1996; 15: 6197-6204Crossref PubMed Scopus (115) Google Scholar, 28van der Geer P. Wiley S. Gish G.D. Pawson T. Curr. Biol. 1996; 6: 1435-1444Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Grb2/Sos binds to Shc phosphorylated at Tyr317, which then leads to the activation of the Ras/Raf/MAPK pathway (26Salcini A.E. McGlade J. Pelicci G. Nicoletti I. Pawson T. Pelicci P.G. Oncogene. 1994; 9: 2827-2836PubMed Google Scholar). However, Shc is also involved in mediation of Ras-independent signals (29Gotoh N. Toyoda M. Shibuya M. Mol. Cell. Biol. 1997; 17: 1824-1831Crossref PubMed Scopus (139) Google Scholar, 30Owen-Lynch P.J. Wong A.K. Whetton A.D. J. Biol. Chem. 1995; 270: 5956-5962Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Other signaling molecules able to associate with Shc include phosphatidylinositol 4,5-bisphosphate (31Zhou M.M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar, 32Sato K. Yamamoto H. Otsuki T. Aoto M. Tokmakov A.A. Hayashi F. Fukami Y. FEBS Lett. 1997; 410: 136-140Crossref PubMed Scopus (21) Google Scholar) and the inositol 5-phosphatases SHIP-1 and SHIP-2 (33Damen J.E. Liu L. Rosten P. Humphries R.K. Jefferson A.B. Majerus P.W. Krystal G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693Crossref PubMed Scopus (560) Google Scholar, 34Wisniewski D. Strife A. Swendeman S. Erdjument-Bromage H. Geromanos S. Kavanaugh W.M. Tempst P. Clarkson B. Blood. 1999; 93: 2707-2720Crossref PubMed Google Scholar). Shc proteins have been implicated in processes like endocytosis (35Okabayashi Y. Sugimoto Y. Totty N.F. Hsuan J. Kido Y. Sakaguchi K. Gout I. Waterfield M.D. Kasuga M. J. Biol. Chem. 1996; 271: 5265-5269Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), cell migration (36Pelicci G. Giordano S. Zhen Z. Salcini A.E. Lanfrancone L. Bardelli A. Panayotou G. Waterfield M.D. Ponzetto C. Pelicci P.G. Comoglio P.M. Oncogene. 1995; 10: 1631-1638PubMed Google Scholar, 37Collins L.R. Ricketts W.A. Yeh L. Cheresh D. J. Cell Biol. 1999; 147: 1561-1568Crossref PubMed Scopus (39) Google Scholar, 38Gu J. Tamura M. Pankov R. Danen E.H. Takino T. Matsumoto K. Yamada K.M. J. Cell Biol. 1999; 146: 389-403Crossref PubMed Scopus (371) Google Scholar), cell survival (27Gotoh N. Tojo A. Shibuya M. EMBO J. 1996; 15: 6197-6204Crossref PubMed Scopus (115) Google Scholar, 39Wary K.K. Mainiero F. Isakoff S.J. Marcantonio E.E. Giancotti F.G. Cell. 1996; 87: 733-743Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar), or mitogenesis (40McGlade J. Cheng A. Pelicci G. Pelicci P.G. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8869-8873Crossref PubMed Scopus (237) Google Scholar, 41Lanfrancone L. Pelicci G. Brizzi M.F. Aronica M.G. Casciari C. Giuli S. Pegoraro L. Pawson T. Pelicci P.G. Oncogene. 1995; 10: 907-917PubMed Google Scholar, 42Stevenson L.E. Ravichandran K.S. Frackelton Jr., A.R. Cell Growth Differ. 1999; 10: 61-71PubMed Google Scholar, 43Palmer H.J. Tuzon C.T. Paulson K.E. J. Biol. Chem. 1999; 274: 11424-11430Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Moreover, p66 Shc is part of a signal transduction pathway that regulates stress apoptotic responses and life span in mammals (44Migliaccio E. Giorgio M. Mele S. Pelicci G. Reboldi P. Pandolfi P.P. Lanfrancone L. Pelicci P.G. Nature. 1999; 402: 309-313Crossref PubMed Scopus (1451) Google Scholar). In the present study, we show a clear difference in signal transduction between gp130 and the OSMR for which so far mainly shared functions have been reported (12Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar, 45Auguste P. Guillet C. Fourcin M. Olivier C. Veziers J. Pouplard-Barthelaix A. Gascan H. J. Biol. Chem. 1997; 272: 15760-15764Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 46Kuropatwinski K.K. De Imus C. Gearing D. Baumann H. Mosley B. J. Biol. Chem. 1997; 272: 15135-15144Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). We provide comparative data showing that upon receptor activation Shc binds to the OSMR (and not to gp130), whereas SHP-2 only binds to gp130 (and not to the OSMR). We locate the site of Shc/OSMR interaction to tyrosine residue 861 of the OSMR. Point mutation of this residue abrogates ERK activation and reduces the induction of an α2M-promoter-driven reporter gene. This finding promises to contribute to the understanding of specific characteristics that have been attributed to OSM. Simian monkey kidney cells (COS-7) were maintained in Dulbecco's modified Eagle's medium; human hepatoma cells (HepG2) were maintained in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal calf serum, 100 mg/liter streptomycin, and 60 mg/liter penicillin. The human melanoma cell line A375 was maintained in RPMI medium supplemented with 5% fetal calf serum, 100 mg/liter streptomycin, and 60 mg/liter penicillin. Recombinant human IL-5 and human OSM were obtained from Cell Concepts (Umkirch, Germany), recombinant human LIF was a kind gift from Dr. N. Nicola (Walter and Elisa Hall Institute, Melbourne, Australia), and recombinant IL-6 was prepared as described (47Arcone R. Pucci P. Zappacosta F. Fontaine V. Malorni A. Marino G. Ciliberto G. Eur. J. Biochem. 1991; 198: 541-547Crossref PubMed Scopus (129) Google Scholar). The specific activity was 2 × 106 B cell stimulatory factor-2 units/mg of protein. Soluble human IL-6 receptor (sIL-6R) was prepared in insect cells as described previously (48Weiergräber O. Hemmann U. Küster A. Müller-Newen G. Schneider J. Rose-John S. Kurschat P. Brakenhoff J.P. Hart M.H. Stabel S. Heinrich P.C. Eur. J. Biochem. 1995; 234: 661-669Crossref PubMed Scopus (85) Google Scholar). Approximately 1.5 × 107COS-7 cells were transiently transfected with 10–20 μg of plasmid DNA using a modified DEAE-dextran method as described (12Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar). HepG2 cells were transfected with 14 μg of plasmid DNA using the calcium phosphate method as described (19Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (140) Google Scholar). The construction of the IL-5Rβ chimeras β/gp130, β/gp130-B1/2, β/OSMR, β/OSMRΔ1, and β/OSMRΔ3 has been described previously (12Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar, 49Behrmann I. Janzen C. Gerhartz C. Schmitz-Van de Leur H. Hermanns H. Heesel B. Graeve L. Horn F. Tavernier J. Heinrich P.C. J. Biol. Chem. 1997; 272: 5269-5274Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). α/gp130YFFFFF was constructed by exchanging the cDNA encoding the extracellular part of gp130YFFFFF (50Schmitz J. Dahmen H. Grimm C. Gendo C. Müller-Newen G. Heinrich P.C. Schaper F. J. Immunol. 2000; 164: 848-854Crossref PubMed Scopus (73) Google Scholar) with the cDNA encoding the extracellular part of the IL-5Rα chain (49Behrmann I. Janzen C. Gerhartz C. Schmitz-Van de Leur H. Hermanns H. Heesel B. Graeve L. Horn F. Tavernier J. Heinrich P.C. J. Biol. Chem. 1997; 272: 5269-5274Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Two further C-terminal deletion mutants, β/OSMRΔ4 and β/OSMRΔ5, were generated by polymerase chain reaction using an antisense oligonucleotide incorporating an in-frame termination codon followed by the recognition site for BamHI. They retain 105 and 75 amino acids of the OSMR cytoplasmic tail, respectively. The point mutated constructs containing the amino acid substitution Y861F were generated by polymerase chain reaction using the respective mutated oligonucleotides with either the cDNA for β/OSMR or β/OSMRΔ1 as a template. The resulting products were cloned into theEcoRI/BamHI-digested expression plasmid pSVL-IL-5Rβ/OSMR, thereby generating the constructs encoding β/OSMRY861F and β/OSMRΔ1Y861F. The integrity of all constructs was verified by DNA sequence analyses using an ABI PRISM 310 Genetic Analyzer (PerkinElmer Life Sciences). For transfection of HepG2 cells,XhoI/BamHI fragments comprising the cDNA encoding the various receptor constructs were inserted intoXhoI/BglII-digested pCAGGS expression vector (51Nakajima K. Yamanaka Y. Nakae K. Kojima H. Ichiba M. Kiuchi N. Kitaoka T. Fukada T. Hibi M. Hirano T. EMBO J. 1996; 15: 3651-3658Crossref PubMed Scopus (518) Google Scholar). Expression plasmids for Jak1 and Jak1K907E were kindly provided by Dr. I. M. Kerr (Imperial Cancer Research Fund, London). A375 and HepG2 cells were stimulated for the indicated periods of time with 20–200 ng/ml IL-6, 100–500 ng/ml LIF, or 10–100 ng/ml OSM. Immediately after stimulation, cells were lysed in Triton lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10 mm NaF, 1 mmNa3VO4, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, and 5 μg/ml leupeptin), scraped off the dish, and left on ice for 30 min. For coimmunoprecipitations, COS-7 cells were stimulated for 20–30 min with 10 ng/ml IL-5 and lysed in BRIJ97 lysis buffer (as Triton lysis buffer but with 1% BRIJ97). Lysates were centrifuged with 14,000 rpm for 10 min at 4 °C, and the supernatants were incubated with either α-Shc or α-IL-5Rβ (S-16) antibody. After an overnight incubation at 4 °C, immune complexes were collected on protein A-Sepharose during a 60-min incubation, washed twice with washing buffer (as lysis buffer, but with only 0.1% BRIJ97), and boiled for 5 min in Laemmli buffer at 95 °C. Proteins were separated by SDS-PAGE in 7.5, 10, or 12.5% gels, followed by electroblotting onto a polyvinylidene difluoride membrane (PALL, Dreieich, Germany). Western blot analysis was conducted using the indicated antibodies and the enhanced chemiluminescence kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Before reprobing, blots were stripped in 2% SDS, 100 mm β-mercaptoethanol in 62.5 mm Tris-HCl (pH 6.7) for 20 min at 75 °C. The polyclonal antiserum against Shc and the monoclonal α-STAT3 antibody were obtained from Transduction Laboratories (Lexington, KY); monoclonal α-IL5Rβ (S-16), polyclonal α-IL5Rβ (N-20), polyclonal α-Grb2 (C-23), polyclonal α-SHP-2 (C-18), polyclonal α-ERK1 (C-16-G), and α-ERK2 (C-14-G) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); and the monoclonal α-Tyr(P) antibody (4G10) was from Upstate Biotechnology, Inc. (Lake Placid, NY). The phosphospecific antibody against activated ERK1/2 was purchased from Promega (Madison, WI); phosphospecific polyclonal α-STAT3(Y705) was from New England Biolabs (Beverly, MA); and horseradish peroxidase-conjugated secondary antibodies were from Dako (Hamburg, Germany). The polyclonal antiserum against Jak1 was a kind gift from Dr. A. Ziemiecki (University of Bern, Switzerland). pGL3α2M-215Luc contains the promoter region, −215 to +8, of the rat α2-macroglobulin gene upstream of the luciferase-encoding sequence of plasmid pGL3 (Promega, Madison, WI). The SIE-tk-Luc construct contains three copies of the STAT consensus binding sequence from the c-fos promoter upstream of a thymidine kinase minimal promoter (52Coqueret O. Gascan H. J. Biol. Chem. 2000; 275: 18794-18800Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and was kindly provided by Dr. H. Gascan (INSERM, Angers, France). HepG2 cells were transfected with 6 μg of luciferase reporter construct, 2 μg of β-galactosidase control plasmid pCH110 (Amersham Pharmacia Biotech), and 3 μg of each receptor expression vector using the calcium phosphate transfection method (12Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar). Twenty-four hours after transfection, cells were treated for 18 h with 10 ng/ml IL-5. Luciferase assays were performed using the Promega luciferase assay system. The IL-6-type cytokines IL-6, LIF, and OSM transduce their signals either via formation of a gp130 homodimer, a gp130-LIFR heterodimer, or a gp130-OSMR heterodimer. HepG2 hepatoma cells express all three signal-transducing receptor chains. We stimulated these cells for different periods of time with either LIF, OSM, or IL-6 in the presence of sIL-6R, which is known to act agonistically (53Taga T. Hibi M. Hirata Y. Yamasaki K. Yasukawa K. Matsuda T. Hirano T. Kishimoto T. Cell. 1989; 58: 573-581Abstract Full Text PDF PubMed Scopus (1183) Google Scholar, 54Mackiewicz A. Schooltink H. Heinrich P.C. Rose-John S. J. Immunol. 1992; 149: 2021-2027PubMed Google Scholar). Interestingly, a phosphorylation of the p66 and p52 isoforms of Shc occurs only after stimulation with OSM but not after treatment with IL-6 or LIF as demonstrated upon immunoprecipitation of the Shc proteins from lysates. The phosphorylation was transient with a maximum reached after 5 min, which continued for 30 min. Then the proteins become dephosphorylated again (Fig. 1 A, upper right panel). The p46 isoform of Shc could not be detected, although equally well expressed (data not shown), due to comigration with the Ig heavy chain. As shown in Fig. 1 A(upper right panel), several additional tyrosine-phosphorylated proteins of high molecular mass were coimmunoprecipitated with Shc. One protein might represent the wild-type OSMR. Since there are no antibodies available for Western blot analysis, we are presently unable to verify this prediction. The phosphoproteins could not be identified as Sos, ErbB2, or members of the Janus kinase family (data not shown), which have either been implicated in signal transduction downstream of Shc or coprecipitated with Shc (21Bonfini L. Migliaccio E. Pelicci G. Lanfrancone L. Pelicci P.G. Trends Biochem. Sci. 1996; 21: 257-261Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 55Qiu Y. Ravi L. Kung H.J. Nature. 1998; 393: 83-85Crossref PubMed Scopus (265) Google Scholar, 56Giordano V. De Falco G. Chiari R. Quinto I. Pelicci P.G. Bartholomew L. Delmastro P. Gadina M. Scala G. J. Immunol. 1997; 158: 4097-4103PubMed Google Scholar). Future studies will aim at the identification of the coimmunoprecipitated proteins. Several reports have demonstrated that Shc is able to bind Grb2 after tyrosine phosphorylation of Tyr317, thereby linking cytokine or growth factor receptors to the activation of the Ras/Raf/MAPK pathway. Indeed, we could demonstrate a transient coimmunoprecipitation of Grb2 with Shc that parallels Shc phosphorylation (Fig. 1 A, lower right panel). The lack of Shc phosphorylation in response to IL-6 and LIF was not due to limited amounts of cytokines applied, since no Shc phosphorylation was detectable after increasing the concentration of IL-6 up to 200 ng/ml and of LIF up to 500 ng/ml (data not shown). In contrast, an OSM concentration of 10 ng/ml was sufficient to induce Shc phosphorylation (not shown). However, all three cytokines induced tyrosine phosphorylation of STAT3, indicating that the Jak/STAT pathway is activated by IL-6, LIF, and OSM (Fig. 1 B, top panel). Analyzing the activation of the Ras/Raf/MAPK pathway as an additional signaling route activated by IL-6-type cytokines showed that OSM has a much higher potential to stimulate ERK1/2 phosphorylation compared with IL-6 or LIF (Fig. 1 B,third panel). This suggests that Shc may play a key role in bridging the gp130-OSMR complex to the Ras/Raf/MAPK pathway. Human OSM is able to transduce signals via gp130-LIFR and gp130-OSMR complexes. The experiments performed in HepG2 cells showed that stimulation with OSM but not with LIF mediates Shc activation, indicating the involvement of the OSM-specific receptor in phosphorylation of Shc. To verify this assumption, we took advantage of A375 melanoma cells expressing only gp130 and the OSMR, but not the LIFR (45Auguste P. Guillet C. Fourcin M. Olivier C. Veziers J. Pouplard-Barthelaix A. Gascan H. J. Biol. Chem. 1997; 272: 15760-15764Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 57Mosley B. De Imus C. Friend D. Boiani N. Thoma B. Park L.S. Cosman D. J. Biol. Chem. 1996; 271: 32635-32643Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). Therefore, all signals occurring after OSM stimulation must be transduced via the gp130-OSMR heterodimer. Indeed, Shc p66 and p52 isoforms were also phosphorylated in A375 cells in response to OSM but not to IL-6 (Fig. 2, upper right panel). We conclude that the phosphorylation of Shc can be attributed to the OSM-specific receptor. Since Shc phosphorylation turned out to be an OSMR-dependent event, we investigated whether the OSMR is able to recruit Shc. The studies were performed in COS-7 cells, since these cells can be transfected efficiently and yield high levels of heterologously expressed proteins. To analyze mutated receptors independently of endogenous receptors, we took advantage of receptor chimeras that have been described previously (12Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar, 49Behrmann I. Janzen C. Gerhartz C. Schmitz-Van de Leur H. Hermanns H. Heesel B. Graeve L. Horn F. Tavernier J. Heinrich P.C. J. Biol. Chem. 1997; 272: 5269-5274Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar): the transmembrane and intracellular parts of the OSMR or gp130, respectively, were fused to the extracellular region of the IL-5 receptor β-chain (Fig. 3). Moreover, this approach enabled us to use the same precipitating antibody (α-IL5Rβ) for a comparative side-by-side analysis of OSMR and gp130. We overexpressed a Janus kinase (Jak1) along with the IL-5Rβ chimeras in COS-7 cells. As demonstrated by others (58Pratt J.C. Weiss M. Sieff C.A. Shoelson S.E. Burakoff S.J. Ravichandran K.S. J. Biol. Chem. 1996; 271: 12137-12140Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), thereby a stimulation-independent receptor phosphorylation can be achieved (not shown). After lysis of the cells and immunoprecipitation of the receptor chimeras, we observed binding of Shc p66 and p52 to chimeras containing the intracellular part of the OSMR but not to chimeras containing gp130 cytoplasmic sequences (Fig. 4, upper left panel) or LIFR sequences (not shown). In contrast, SHP-2 bound exclusively to β/" @default.
• W2091245777 created "2016-06-24" @default.
• W2091245777 creator A5008200603 @default.
• W2091245777 creator A5018291433 @default.
• W2091245777 creator A5031238347 @default.
• W2091245777 creator A5038296391 @default.
• W2091245777 creator A5038571222 @default.
• W2091245777 date "2000-12-01" @default.
• W2091245777 modified "2023-10-13" @default.
• W2091245777 title "Non-redundant Signal Transduction of Interleukin-6-type Cytokines" @default.
• W2091245777 cites W1488722235 @default.
• W2091245777 cites W1496109170 @default.
• W2091245777 cites W1500857648 @default.
• W2091245777 cites W1568956696 @default.
• W2091245777 cites W1571617103 @default.
• W2091245777 cites W1596383188 @default.
• W2091245777 cites W1607864895 @default.
• W2091245777 cites W1644491781 @default.
• W2091245777 cites W1779691448 @default.
• W2091245777 cites W1782056969 @default.
• W2091245777 cites W1782126971 @default.
• W2091245777 cites W1939561089 @default.
• W2091245777 cites W1966129049 @default.
• W2091245777 cites W1971977518 @default.
• W2091245777 cites W1974968368 @default.
• W2091245777 cites W1975118026 @default.
• W2091245777 cites W1976434580 @default.
• W2091245777 cites W1983076846 @default.
• W2091245777 cites W1986758681 @default.
• W2091245777 cites W1988917700 @default.
• W2091245777 cites W1991765224 @default.
• W2091245777 cites W1996548067 @default.
• W2091245777 cites W1997503905 @default.
• W2091245777 cites W2006147172 @default.
• W2091245777 cites W2008008296 @default.
• W2091245777 cites W2018570422 @default.
• W2091245777 cites W2019948850 @default.
• W2091245777 cites W2021297146 @default.
• W2091245777 cites W2024834727 @default.
• W2091245777 cites W2025621343 @default.
• W2091245777 cites W2030624761 @default.
• W2091245777 cites W2031491552 @default.
• W2091245777 cites W2040258408 @default.
• W2091245777 cites W2044500153 @default.
• W2091245777 cites W2050562554 @default.
• W2091245777 cites W2054873490 @default.
• W2091245777 cites W2056550812 @default.
• W2091245777 cites W2058924545 @default.
• W2091245777 cites W2063821969 @default.
• W2091245777 cites W2069785923 @default.
• W2091245777 cites W2070054628 @default.
• W2091245777 cites W2072373148 @default.
• W2091245777 cites W2072947383 @default.
• W2091245777 cites W2075916030 @default.
• W2091245777 cites W2079252365 @default.
• W2091245777 cites W2083663211 @default.
• W2091245777 cites W2094020416 @default.
• W2091245777 cites W2098494612 @default.
• W2091245777 cites W2098573045 @default.
• W2091245777 cites W2106938542 @default.
• W2091245777 cites W2119074033 @default.
• W2091245777 cites W2127553886 @default.
• W2091245777 cites W2132246987 @default.
• W2091245777 cites W2149468827 @default.
• W2091245777 cites W2152295044 @default.
• W2091245777 cites W2159904509 @default.
• W2091245777 cites W2165209574 @default.
• W2091245777 cites W2168275907 @default.
• W2091245777 cites W2170064797 @default.
• W2091245777 cites W2171073129 @default.
• W2091245777 cites W2278462616 @default.
• W2091245777 cites W4245804658 @default.
• W2091245777 cites W4248469839 @default.
• W2091245777 cites W4250884557 @default.
• W2091245777 cites W4313333818 @default.
• W2091245777 cites W57220067 @default.
• W2091245777 doi "https://doi.org/10.1074/jbc.m005408200" @default.
• W2091245777 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11016927" @default.
• W2091245777 hasPublicationYear "2000" @default.
• W2091245777 type Work @default.
• W2091245777 sameAs 2091245777 @default.
• W2091245777 citedByCount "74" @default.
• W2091245777 countsByYear W20912457772012 @default.
• W2091245777 countsByYear W20912457772013 @default.
• W2091245777 countsByYear W20912457772014 @default.
• W2091245777 countsByYear W20912457772015 @default.
• W2091245777 countsByYear W20912457772016 @default.
• W2091245777 countsByYear W20912457772017 @default.
• W2091245777 countsByYear W20912457772018 @default.
• W2091245777 countsByYear W20912457772019 @default.
• W2091245777 countsByYear W20912457772020 @default.
• W2091245777 countsByYear W20912457772021 @default.
• W2091245777 countsByYear W20912457772022 @default.
• W2091245777 countsByYear W20912457772023 @default.
• W2091245777 crossrefType "journal-article" @default.
• W2091245777 hasAuthorship W2091245777A5008200603 @default.
• W2091245777 hasAuthorship W2091245777A5018291433 @default.
• W2091245777 hasAuthorship W2091245777A5031238347 @default.

• next