Advertisement

Erythropoietin Induces the Tyrosine Phosphorylation of Insulin Receptor Substrate-2

AN ALTERNATE PATHWAY FOR ERYTHROPOIETIN-INDUCED PHOSPHATIDYLINOSITOL 3-KINASE ACTIVATION*
  • Frédérique Verdier
    Footnotes
    Affiliations
    Institut Cochin de Génétique Moléculaire, INSERM U363, Université René Descartes, 27 rue du Faubourg Saint Jacques, F75014 Paris
    Search for articles by this author
  • Stany Chrétien
    Affiliations
    Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, F75015 Paris
    Search for articles by this author
  • Claudine Billat
    Affiliations
    Laboratoire de Biochimie, CNRS UPRES-A6021, UFR Sciences Exactes et Naturelles, Université de Reims Champagne-Ardenne, F51687 Reims Cedex 2, France
    Search for articles by this author
  • Sylvie Gisselbrecht
    Affiliations
    Institut Cochin de Génétique Moléculaire, INSERM U363, Université René Descartes, 27 rue du Faubourg Saint Jacques, F75014 Paris
    Search for articles by this author
  • Catherine Lacombe
    Affiliations
    Institut Cochin de Génétique Moléculaire, INSERM U363, Université René Descartes, 27 rue du Faubourg Saint Jacques, F75014 Paris
    Search for articles by this author
  • Patrick Mayeux
    Correspondence
    To whom correspondence should be addressed: Inst. Cochin de Génétique Moléculaire, INSERM U363, Hôpital Cochin, 27 rue du Faubourg Saint Jacques, F75014 Paris, France. Tel.: 33-1-46-33-14-09; Fax: 33-1-46-33-92-97;
    Affiliations
    Institut Cochin de Génétique Moléculaire, INSERM U363, Université René Descartes, 27 rue du Faubourg Saint Jacques, F75014 Paris
    Search for articles by this author
  • Author Footnotes
    * This work was supported in part by Contract 1373 from the Association pour la Recherche sur le Cancer and by grants from the Ligue Nationale Contre le Cancer and from the Comité de la Manche and the Comité de la Marne of the Ligue Nationale Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Supported by Glaxo Wellcome.
Open AccessPublished:October 17, 1997DOI:https://doi.org/10.1074/jbc.272.42.26173
      In this report, we demonstrate that insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine following treatment of UT-7 cells with erythropoietin. We have investigated the expression of IRS-1 and IRS-2 in several cell lines with erythroid and/or megakaryocytic features, and we observed that IRS-2 was expressed in all cell lines tested. In contrast, we did not detect the expression of IRS-1 in these cells. In response to erythropoietin, IRS-2 was immediately phosphorylated on tyrosine, with maximal phosphorylation between 1 and 5 min. Tyrosine-phosphorylated IRS-2 was associated with phosphatidylinositol 3-kinase and with a 140-kDa protein that comigrated with the phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase, SHIP. Moreover, IRS-2 was constitutively associated with the erythropoietin receptor. We did not observe the association of IRS-2 with JAK2, Grb2, or PTP1D. Using BaF3 cells transfected with mutated erythropoietin receptors, we demonstrate that neither the tyrosine residues of the intracellular domain nor the last 109 amino acids of the erythropoietin receptor are required for erythropoietin-induced IRS-2 tyrosine phosphorylation. Altogether, our results indicate that erythropoietin-induced IRS-2 tyrosine phosphorylation could account for the previously reported activation of phosphatidylinositol 3-kinase mediated by erythropoietin receptors mutated in the phosphatidylinositol 3-kinase-binding site (Damen, J., Cutler, R. L., Jiao, H., Yi, T., and Krystal, G. (1995) J. Biol. Chem. 270, 23402–23406; Gobert, S., Porteu, F., Pallu, S., Muller, O., Sabbah, M., Dusanter-Fourt, I., Courtois, G., Lacombe, C., Gisselbrecht, S., and Mayeux, P. (1995)Blood 86, 598–606).
      Insulin receptor substrate-1 (IRS-1)
      The abbreviations used are: IRS, insulin receptor substrate; IGF, insulin-like growth factor; PTB domain, phosphotyrosine-binding domain; PI, phosphatidylinositol; IL, interleukin; Epo, erythropoietin; SCF, stem cell factor; GST, glutathione S-transferase; GM-CSF, granulocyte-macrophage colony-stimulating factor.
      1The abbreviations used are: IRS, insulin receptor substrate; IGF, insulin-like growth factor; PTB domain, phosphotyrosine-binding domain; PI, phosphatidylinositol; IL, interleukin; Epo, erythropoietin; SCF, stem cell factor; GST, glutathione S-transferase; GM-CSF, granulocyte-macrophage colony-stimulating factor.
      is a major substrate of the IGF-1 and insulin receptors (
      • White M.F.
      • Kahn C.R.
      ). IRS-1 is a hydrophilic protein with a theoretical molecular mass of 131 kDa that migrates between 160 and 185 kDa on SDS-polyacrylamide gel electrophoresis partially because of a high serine phosphorylation state (
      • Sun X.J.
      • Rothenberg P.
      • Kahn C.R.
      • Backer J.M.
      • Araki E.
      • Wilden P.A.
      • Cahill D.A.
      • Goldstein B.J.
      • White M.F.
      ,
      • Sun X.J.
      • Miralpeix M.
      • Myers Jr., M.G.
      • Glasheen E.M.
      • Backer J.M.
      • Kahn C.R.
      • White M.F.
      ). IRS-1 contains a pleckstrin homology domain, a PTB domain, and at least 20 potential tyrosine phosphorylation sites including nine YXXM motifs that are consensus binding sites for the SH2 domains of the regulatory subunit (p85) of PI 3-kinase (
      • Songyang Z.
      • Shoelson S.E.
      • Chaudhuri M.
      • Gish G.
      • Pawson T.
      • Haser W.G.
      • King F.
      • Roberts T.
      • Ratnofsky S.
      • Lechleider R.J.
      • Neel B.G.
      • Birge R.B.
      • Fajardo J.E.
      • Chou M.M.
      • Hanafusa H.
      • Schaffhausen B.
      • Cantley L.C.
      ). IRS-1 binds to the tyrosine-phosphorylated IGF-1 and insulin receptors through its PTB domain and becomes a docking protein for signaling proteins such as PI 3-kinase, Grb2, PTP1D, and Nck after tyrosine phosphorylation (
      • White M.F.
      ). Moreover, a recent report shows that the pleckstrin homology domain could also be involved in the association of IRS-1 with the insulin receptor (
      • Yenush L.
      • Makati K.J.
      • Smith-Hall J.
      • Ishibashi O.
      • Myers Jr., M.G.
      • White M.F.
      ). More recently, a second IRS protein was identified that was designated IRS-2 (
      • Sun X.J.
      • Wang L.M.
      • Zhang Y.
      • Yenush L.
      • Myers Jr., M.G.
      • Glasheen E.
      • Lane W.S.
      • Pierce J.H.
      • White M.F.
      ). This protein corresponds to the previously identified 4PS protein (for IL-4-inducedphosphotyrosine substrate) (
      • Wang L.M.
      • Keegan A.D.
      • Paul W.E.
      • Heidaran M.A.
      • Gutkind J.S.
      • Pierce J.H.
      ,
      • Wang L.M.
      • Keegan A.D.
      • Li W.
      • Lienhard G.E.
      • Pacini S.
      • Gutkind J.S.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.F.
      • Aaronson S.A.
      • Paul W.E.
      • Pierce J.H.
      ). IRS-2 exhibits a high structural similarity to IRS-1, with a strong conservation of the PTB and pleckstrin homology domains. Moreover, tyrosine phosphorylation sites shown to bind PI 3-kinase, Grb2, and PTP1D in IRS-1 are also conserved in IRS-2.
      Not only insulin and IGF-1 receptors, but also several cytokine and interferon receptors can induce tyrosine phosphorylation of IRS-1 or IRS-2 (
      • Wang L.M.
      • Keegan A.D.
      • Li W.
      • Lienhard G.E.
      • Pacini S.
      • Gutkind J.S.
      • Myers Jr., M.G.
      • Sun X.J.
      • White M.F.
      • Aaronson S.A.
      • Paul W.E.
      • Pierce J.H.
      ,
      • Wang L.M.
      • Myers Jr., M.G.
      • Sun X.J.
      • Aaronson S.A.
      • White M.
      • Pierce J.H.
      ,
      • Souza S.C.
      • Frick G.P.
      • Yip R.
      • Lobo R.B.
      • Tai L.-R.
      • Goodman H.M.
      ,
      • Argetsinger L.S.
      • Hsu G.W.
      • Myers Jr., M.G.
      • Billestrup N.
      • White M.F.
      • Carter-Su C.
      ,
      • Johnston J.A.
      • Wang L.-M.
      • Hanson E.P.
      • Sun X.J.
      • White M.F.
      • Oakes S.A.
      • Pierce J.H.
      • O'Shea J.J.
      ,
      • Ridderstrale M.
      • Degerman E.
      • Tornqvist H.
      ,
      • Argetsinger L.S.
      • Norstedt G.
      • Billestrup N.
      • White M.F.
      • Carter-Su C.
      ,
      • Platanias L.C.
      • Uddin S.
      • Yetter A.
      • Sun X.-J.
      • White M.F.
      ,
      • Berlanga J.J.
      • Gualillo O.
      • Buteau H.
      • Applanat M.
      • Kelly P.A.
      • Edery M.
      ,
      • Welham M.J.
      • Bone H.
      • Levings M.
      • Learmonth L.
      • Wang L.M.
      • Leslie K.B.
      • Pierce J.H.
      • Schrader J.W.
      ). IRS-1 and IRS-2 activation by IL-4 is well documented. Indeed, it has been shown that IRS-1 and IRS-2 bind to a peptidic sequence of the activated IL-4 receptor with a typical NPXY PTB domain-binding motif (
      • Keegan A.D.
      • Nelms K.
      • White M.
      • Wang L.M.
      • Pierce J.H.
      • Paul W.E.
      ), and IRS-1 or IRS-2 expression appears to be required for IL-4-induced mitogenesis (
      • Wang L.M.
      • Myers Jr., M.G.
      • Sun X.J.
      • Aaronson S.A.
      • White M.
      • Pierce J.H.
      ). However, other cytokine receptors that induce the tyrosine phosphorylation of IRS-1 and IRS-2 do not possess typical PTB domain-binding motifs, and the mechanism of IRS-1 and IRS-2 activation by these receptors remains unknown.
      The erythropoietin (Epo) receptor also belongs to the cytokine receptor family (
      • D'Andrea A.D.
      • Lodish H.F.
      • Wong G.G.
      ,
      • Bazan J.F.
      ). Epo binding to its receptor activates the receptor-associated JAK2 tyrosine kinase (
      • Witthuhn B.
      • Quelle F.W.
      • Silvennoinen O.
      • Yi T.
      • Tang B.
      • Muira O.
      • Ihle J.N.
      ) and induces the tyrosine phosphorylation of the Epo receptor (
      • Miura O.
      • D'Andrea A.
      • Kabat D.
      • Ihle J.N.
      ,
      • Damen J.
      • Mui A.L.F.
      • Hughes P.
      • Humphries K.R.
      • Krystal G.
      ,
      • Dusanter-Fourt I.
      • Casadevall N.
      • Lacombe C.
      • Muller O.
      • Billat C.
      • Fischer S.
      • Mayeux P.
      ,
      • Yoshimura A.
      • Lodish H.F.
      ) and other proteins. Several intracellular signaling pathways are subsequently activated, including mitogen-activated protein kinases (
      • Miura Y.
      • Muira O.
      • Ihle J.N.
      • Aoki N.
      ,
      • Gobert S.
      • Duprez V.
      • Lacombe C.
      • Gisselbrecht S.
      • Mayeux P.
      ), STAT5 (
      • Gouilleux F.
      • Pallard C.
      • Dusanter-Fourt I.
      • Wakao H.
      • Haldosen L.-A.
      • Norstedt G.
      • Levy D.
      • Groner B.
      ,
      • Wakao H.
      • Harada N.
      • Kitamura T.
      • Mui A.L.F.
      • Miyajima A.
      ), and PI 3-kinase (
      • Damen J.E.
      • Mui A.L.F.
      • Puil L.
      • Pawson T.
      • Krystal G.
      ,
      • He T.C.
      • Zhuang H.
      • Jiang N.
      • Waterfield M.D.
      • Wojchowski D.M.
      ,
      • Mayeux P.
      • Dusanter-Fourt I.
      • Muller O.
      • Mauduit P.
      • Sabbah M.
      • Drucker B.
      • Vainchenker W.
      • Fischer S.
      • Lacombe C.
      • Gisselbrecht S.
      ,
      • Miura O.
      • Nakamura N.
      • Ihle J.N.
      • Aoki N.
      ). PI 3-kinase was shown to associate with phosphorylated Tyr479 of the Epo receptor (
      • Damen J.E.
      • Cutler R.L.
      • Jiao H.
      • Yi T.
      • Krystal G.
      ), and removal of this tyrosine residue abrogates PI 3-kinase association with the Epo receptor (
      • Miura O.
      • Nakamura N.
      • Ihle J.N.
      • Aoki N.
      ,
      • Damen J.E.
      • Cutler R.L.
      • Jiao H.
      • Yi T.
      • Krystal G.
      ,
      • Gobert S.
      • Porteu F.
      • Pallu S.
      • Muller O.
      • Sabbah M.
      • Dusanter-Fourt I.
      • Courtois G.
      • Lacombe C.
      • Gisselbrecht S.
      • Mayeux P.
      ). However, binding of PI 3-kinase to the Epo receptor does not appear to be required for Epo-induced PI 3-kinase activation since Epo receptors devoid of Tyr479 still activate PI 3-kinase (
      • Damen J.E.
      • Cutler R.L.
      • Jiao H.
      • Yi T.
      • Krystal G.
      ,
      • Gobert S.
      • Porteu F.
      • Pallu S.
      • Muller O.
      • Sabbah M.
      • Dusanter-Fourt I.
      • Courtois G.
      • Lacombe C.
      • Gisselbrecht S.
      • Mayeux P.
      ). These results strongly suggest that Epo could activate PI 3-kinase by several mechanisms. However, these alternate pathways for Epo-induced PI 3-kinase activation have not been identified up to date. We have previously shown that one of these mechanisms required only the first 127 amino acids of the Epo receptor intracellular domain and was not dependent on the phosphorylation of the single tyrosine residue (Tyr343) present in this region (
      • Gobert S.
      • Porteu F.
      • Pallu S.
      • Muller O.
      • Sabbah M.
      • Dusanter-Fourt I.
      • Courtois G.
      • Lacombe C.
      • Gisselbrecht S.
      • Mayeux P.
      ).
      In this report, we show that erythropoietin induces the tyrosine phosphorylation of IRS-2. Epo-induced IRS-2 tyrosine phosphorylation does not require the tyrosine residues of the intracellular domain of the Epo receptor, and IRS-2 appears to be constitutively associated with the Epo receptor. After Epo-induced tyrosine phosphorylation, IRS-2 associates with PI 3-kinase and with a tyrosine-phosphorylated protein comigrating with SHIP (p140). In contrast, we did not detect the association of Grb2 with IRS-2 in Epo-stimulated cells. Thus, our results strongly suggest that IRS-2 binding could be an alternate mechanism for Epo-induced PI 3-kinase activation.

      DISCUSSION

      Expression of IRS-1 and IRS-2 in the hematopoietic system is relatively poorly documented. Both IRS-1 and IRS-2 were reported to be expressed and activated by IL-4 and IL-2 in human T lymphoblasts (
      • Johnston J.A.
      • Wang L.-M.
      • Hanson E.P.
      • Sun X.J.
      • White M.F.
      • Oakes S.A.
      • Pierce J.H.
      • O'Shea J.J.
      ). In contrast, mast cells (
      • Welham M.J.
      • Bone H.
      • Levings M.
      • Learmonth L.
      • Wang L.M.
      • Leslie K.B.
      • Pierce J.H.
      • Schrader J.W.
      ) or the murine myeloid progenitor cell line 32D (
      • Wang L.M.
      • Myers Jr., M.G.
      • Sun X.J.
      • Aaronson S.A.
      • White M.
      • Pierce J.H.
      ) expressed neither IRS-1 nor IRS-2. Expression of IRS-2, but not IRS-1, was also detected in other IL-3-dependent myeloid progenitor cell lines such as FDCP-1 and FDCP-2 (
      • Wang L.M.
      • Myers Jr., M.G.
      • Sun X.J.
      • Aaronson S.A.
      • White M.
      • Pierce J.H.
      ), murine macrophages, and murine B and T lymphocytes (
      • Welham M.J.
      • Bone H.
      • Levings M.
      • Learmonth L.
      • Wang L.M.
      • Leslie K.B.
      • Pierce J.H.
      • Schrader J.W.
      ). Here, we studied the expression of these proteins in cells with erythroid and megakaryocytic characteristics. UT-7 cells express markers from different differentiation lineages depending on the stimulatory cytokines. Indeed, it has been shown that erythroid or megakaryocytic differentiation markers are expressed in UT-7 cells stimulated by Epo (
      • Hermine O.
      • Mayeux P.
      • Titeux M.
      • Mitjavila M.T.
      • Casadevall N.
      • Guichard J.
      • Komatsu N.
      • Suda T.
      • Miura Y.
      • Vainchenker W.
      • Breton-Gorius J.
      ) or by thrombopoietin (
      • Porteu F.
      • Rouyez M.C.
      • Cocault L.
      • Benit L.
      • Charon M.
      • Picard F.
      • Gisselbrecht S.
      • Souyri M.
      • Dusanter-Fourt I.
      ), respectively. TF-1 and HCD57 cells seem to be strictly committed in the erythroid differentiation pathway, whereas Mo7E cells exhibit megakaryocytic characteristics (
      • Ruscetti S.K.
      • Janesh N.
      • Chakraborti A.
      • Sawyer S.T.
      • Hankins H.D.
      ,
      • Avanzi G.C.
      • Lista P.
      • Giovinazzo B.
      • Miniero R.
      • Saglio G.
      • Benetton G.
      • Coda R.
      • Cattoretti G.
      • Pegoraro L.
      ,
      • Kitamura T.
      • Tange T.
      • Terasawa T.
      • Chiba S.
      • Kuwaki T.
      • Miyagawa K.
      • Piao Y.F.
      • Miyazono K.
      • Urabe A.
      • Takaku F.
      ,
      • Chrétien S.
      • Varlet P.
      • Verdier F.
      • Gobert S.
      • Cartron J.-P.
      • Gisselbrecht S.
      • Mayeux P.
      • Lacombe C.
      ). T3Cl2 cells are Friend virus-transformed cells that correspond to erythroid cells blocked at the colony-forming unit erythroid/proerythroblast stage (
      • Ikawa Y.
      • Aida M.
      • Inoue Y.
      ). All these cell lines express IRS-2, whereas we did not detect IRS-1 expression in any hematopoietic cell line tested. Thus, our results confirm that hematopoietic cells express IRS-2 rather than IRS-1 and extend this observation to cells of the erythroid lineage.
      We observed that Epo stimulation of UT-7 cells induced the rapid tyrosine phosphorylation of IRS-2 since maximal tyrosyl phosphorylation was detected between 1 and 10 min and decreased after this time. This kinetics was slightly different from that reported for growth hormone or prolactin, which maximally induced the tyrosine phosphorylation of IRS-1 and IRS-2 after 10–20 min (
      • Argetsinger L.S.
      • Hsu G.W.
      • Myers Jr., M.G.
      • Billestrup N.
      • White M.F.
      • Carter-Su C.
      ,
      • Argetsinger L.S.
      • Norstedt G.
      • Billestrup N.
      • White M.F.
      • Carter-Su C.
      ,
      • Berlanga J.J.
      • Gualillo O.
      • Buteau H.
      • Applanat M.
      • Kelly P.A.
      • Edery M.
      ). The time course of Epo-induced IRS-2 tyrosine phosphorylation in UT-7 cells closely paralleled the kinetics of JAK2 activation in these cells (data not shown) and was superimposable to the activation kinetics of other intracellular signaling pathways such as PI 3-kinase (
      • Mayeux P.
      • Dusanter-Fourt I.
      • Muller O.
      • Mauduit P.
      • Sabbah M.
      • Drucker B.
      • Vainchenker W.
      • Fischer S.
      • Lacombe C.
      • Gisselbrecht S.
      ) and mitogen-activated protein kinases (
      • Gobert S.
      • Duprez V.
      • Lacombe C.
      • Gisselbrecht S.
      • Mayeux P.
      ). The rapid IRS-2 phosphorylation and its association with the Epo receptor (see below) strongly suggest that Epo-induced IRS-2 tyrosine phosphorylation is a direct event of Epo receptor activation.
      Although UT-7 cells are sensitive to GM-CSF and SCF, we did not detect the tyrosine phosphorylation of IRS-2 in UT-7 cells stimulated with these cytokines. Using another cell line, Welham et al. (
      • Welham M.J.
      • Bone H.
      • Levings M.
      • Learmonth L.
      • Wang L.M.
      • Leslie K.B.
      • Pierce J.H.
      • Schrader J.W.
      ) previously reported the stimulation of IRS-2 tyrosine phosphorylation by IL-3 and GM-CSF. This discrepancy could be due to the low number of high affinity GM-CSF receptors expressed in UT-7 cells. Indeed, we detected only a few hundred high affinity receptors for GM-CSF, whereas these cells express ∼7000 Epo receptors (
      • Hermine O.
      • Mayeux P.
      • Titeux M.
      • Mitjavila M.T.
      • Casadevall N.
      • Guichard J.
      • Komatsu N.
      • Suda T.
      • Miura Y.
      • Vainchenker W.
      • Breton-Gorius J.
      ). Moreover, as shown in Fig. 2 B, JAK2 activation was also much less efficient using GM-CSF than Epo in these cells. In contrast, the inability of SCF to induce IRS-2 tyrosine phosphorylation is not due to a low number of SCF receptors since these cells express ∼35,000 receptors for this cytokine.
      P. Mayeux, unpublished results.
      It should be noted that CSF-1, whose receptor shares strong similarity with c-Kit, also does not induce the tyrosine phosphorylation of IRS-2 (
      • Welham M.J.
      • Bone H.
      • Levings M.
      • Learmonth L.
      • Wang L.M.
      • Leslie K.B.
      • Pierce J.H.
      • Schrader J.W.
      ).
      Many cytokines whose receptors belong either to subclass 1 or 2 (interferon receptors) have now been reported to induce the tyrosine phosphorylation of IRS-1 and/or IRS-2, suggesting that this relay could be a common signaling pathway for this class of receptors. All these receptors mediate intracellular signaling through the activation of JAK kinases (reviewed in Ref.
      • Ihle J.N.
      ). Moreover, mutated ZERO receptors that essentially conserved few receptor sequences downstream of the region required for JAK2 activation can efficiently mediate Epo-induced IRS-2 activation (Fig. 8). The corresponding region of the growth hormone receptor was previously shown to be responsible for IRS-1 and IRS-2 activation (
      • Argetsinger L.S.
      • Hsu G.W.
      • Myers Jr., M.G.
      • Billestrup N.
      • White M.F.
      • Carter-Su C.
      ,
      • Argetsinger L.S.
      • Norstedt G.
      • Billestrup N.
      • White M.F.
      • Carter-Su C.
      ). Thus, an attractive hypothesis would be that IRS-1 and IRS-2 proteins could directly interact with the JAK kinases, although JAK2 does not contain NPXY motifs (
      • Silvennoinen O.
      • Witthuhn B.A.
      • Quelle F.W.
      • Cleveland J.L.
      • Yi T.
      • Ihle J.N.
      ). However, our results do not sustain this hypothesis. Indeed, we did not detect JAK2 in IRS-2 immunoprecipitates (Fig. 3), and no IRS-2 protein was detected in anti-JAK2 immunoprecipitates (data not shown). In contrast, we observed the constitutive association of IRS-2 with the Epo receptor in both Epo-stimulated and unstimulated UT-7 cells, strongly suggesting the direct association of IRS-2 with the Epo receptor. The Epo receptor is not tyrosine-phosphorylated in unstimulated UT-7 cells, and it does not contain an NPXY PTB domain-binding sequence. Moreover, the tyrosine residues of the Epo receptor intracellular domain are not necessary for Epo-induced IRS-2 tyrosine phosphorylation (Fig. 8). Taken together, these results indicate that the association between IRS-2 and the Epo receptor probably does not involve the IRS-2 PTB domain.
      Although the role of IRS-1 and IRS-2 in normal erythropoiesis was never directly addressed, some reports have evidenced a role in erythropoiesis for IGF-1, which mainly uses IRS-1 and IRS-2 for intracellular signaling. Indeed, IGF-1 has been shown to stimulate erythroid colony formation in vitro even in the absence of Epo (
      • Correa P.N.
      • Axelrad A.A.
      ). Moreover, erythroid progenitors from polycythemia vera patients have been shown to be hypersensitive to IGF-1, leading to Epo independence (
      • Correa P.N.
      • Eskinazi D.
      • Axelrad A.A.
      ). Thus, signaling through IRS-1 and IRS-2 could sustain the proliferation of erythroid progenitors. Our results show that PI 3-kinase seems to be the main target of IRS-2 in Epo-stimulated cells. Previously published data suggest that PI 3-kinase seems to be involved in the control of Epo-induced cell proliferation. Indeed, the PI 3-kinase inhibitor wortmannin inhibits Epo-induced proliferation of Epo receptor-transfected DA3 cells (
      • Damen J.E.
      • Cutler R.L.
      • Jiao H.
      • Yi T.
      • Krystal G.
      ) and of UT-7 cells.
      S. Gobert and P. Mayeux, unpublished results.
      PI 3-kinase activation by Epo can be achieved using several mechanisms. One of these mechanisms involves the binding of PI 3-kinase to the tyrosine-phosphorylated Epo receptor (
      • Damen J.E.
      • Mui A.L.F.
      • Puil L.
      • Pawson T.
      • Krystal G.
      ,
      • He T.C.
      • Zhuang H.
      • Jiang N.
      • Waterfield M.D.
      • Wojchowski D.M.
      ,
      • Mayeux P.
      • Dusanter-Fourt I.
      • Muller O.
      • Mauduit P.
      • Sabbah M.
      • Drucker B.
      • Vainchenker W.
      • Fischer S.
      • Lacombe C.
      • Gisselbrecht S.
      ,
      • Miura O.
      • Nakamura N.
      • Ihle J.N.
      • Aoki N.
      ). However, Epo receptors without tyrosine residues in the intracellular domain are able to mediate Epo-induced PI 3-kinase activation, although PI 3-kinase association with the Epo receptor is not detected (
      • Damen J.E.
      • Cutler R.L.
      • Jiao H.
      • Yi T.
      • Krystal G.
      ,
      • Gobert S.
      • Porteu F.
      • Pallu S.
      • Muller O.
      • Sabbah M.
      • Dusanter-Fourt I.
      • Courtois G.
      • Lacombe C.
      • Gisselbrecht S.
      • Mayeux P.
      ), demonstrating the presence of alternate pathways for Epo-induced PI 3-kinase activation. Our results show that IRS-2 could be one of these pathways and could be responsible for Epo-induced PI 3-kinase activation in cells expressing Epo receptors devoid of intracellular tyrosine residues such as BaF3 cells transfected with the ZERO Epo receptor mutant. Interestingly, a region of the Epo receptor conserved in the ZERO Epo receptor mutant and located between the JAK2-binding sequence and amino acid 329 was previously reported to be required for Epo-induced mitogenesis in the context of a truncated (
      • He T.-C.
      • Jiang N.
      • Zhuang H.
      • Quelle D.E.
      • Wojchowski D.M.
      ), but not a full-length (
      • Hilton C.J.
      • Berridge M.V.
      ), Epo receptor. This result suggests that an intracellular relay could be activated by this region of the receptor, but that other signaling pathways activated by Epo receptor sequences located downstream could substitute for this signal. Our results show that PI 3-kinase could be this relay since it could be activated through the C-terminal part of the Epo receptor by direct binding to the Epo receptor and through the region of the Epo receptor close to the transmembrane domain by using IRS-2. Although under experimental conditions these pathways appear to be redundant, it should be kept in mind that normal erythroid progenitors express lower levels of Epo receptors that the cell lines used as experimental models and that they have to respond in vivo to low Epo concentrations. Under these conditions, PI 3-kinase activation by these different pathways could be additive and required to allow a cellular response to a low level of Epo stimulation. One of the signaling relays downstream of PI 3-kinase was recently shown to be the serine/threonine kinase AKT (see Ref.
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      for review). Interestingly, AKT is activated by PI 3,4-bisphosphate, but not by PI 3,4,5-trisphosphate (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Klippel A.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ). One way to produce PI 3,4-bisphosphate is the dephosphorylation of the main product of PI 3-kinase, PI 3,4,5-trisphosphate, which could be performed by the PI-3,4,5-trisphosphate 5-phosphatase, SHIP. Our results show that a 140-kDa protein that most likely corresponds to SHIP also associates with IRS-2. Thus, the same protein complex appears to contain both PI 3-kinase and SHIP, and these associations could increase the efficiency of PI 3,4-bisphosphate production.

      Acknowledgments

      We thank Boehringer Mannheim and Kirin Brewery Co. for the generous provision of recombinant Epo and recombinant SCF, respectively. The excellent technical work of Paule Varlet and Odile Muller is gratefully acknowledged.

      REFERENCES

        • White M.F.
        • Kahn C.R.
        J. Biol. Chem. 1994; 269: 1-4
        • Sun 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: 73-77
        • Sun X.J.
        • Miralpeix M.
        • Myers Jr., M.G.
        • Glasheen E.M.
        • Backer J.M.
        • Kahn C.R.
        • White M.F.
        J. Biol. Chem. 1992; 267: 22662-22672
        • Songyang Z.
        • Shoelson S.E.
        • Chaudhuri M.
        • Gish G.
        • Pawson T.
        • Haser W.G.
        • King F.
        • Roberts T.
        • Ratnofsky S.
        • Lechleider R.J.
        • Neel B.G.
        • Birge R.B.
        • Fajardo J.E.
        • Chou M.M.
        • Hanafusa H.
        • Schaffhausen B.
        • Cantley L.C.
        Cell. 1993; 72: 767-778
        • White M.F.
        Curr. Opin. Genet. & Dev. 1994; 4: 47-54
        • Yenush L.
        • Makati K.J.
        • Smith-Hall J.
        • Ishibashi O.
        • Myers Jr., M.G.
        • White M.F.
        J. Biol. Chem. 1996; 271: 24300-24306
        • Sun X.J.
        • Wang L.M.
        • Zhang Y.
        • Yenush L.
        • Myers Jr., M.G.
        • Glasheen E.
        • Lane W.S.
        • Pierce J.H.
        • White M.F.
        Nature. 1995; 377: 173-177
        • Wang L.M.
        • Keegan A.D.
        • Paul W.E.
        • Heidaran M.A.
        • Gutkind J.S.
        • Pierce J.H.
        EMBO J. 1992; 11: 4899-4908
        • Wang L.M.
        • Keegan A.D.
        • Li W.
        • Lienhard G.E.
        • Pacini S.
        • Gutkind J.S.
        • Myers Jr., M.G.
        • Sun X.J.
        • White M.F.
        • Aaronson S.A.
        • Paul W.E.
        • Pierce J.H.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4032-4036
        • Wang L.M.
        • Myers Jr., M.G.
        • Sun X.J.
        • Aaronson S.A.
        • White M.
        • Pierce J.H.
        Science. 1993; 261: 1591-1594
        • Souza S.C.
        • Frick G.P.
        • Yip R.
        • Lobo R.B.
        • Tai L.-R.
        • Goodman H.M.
        J. Biol. Chem. 1994; 269: 30085-30088
        • Argetsinger L.S.
        • Hsu G.W.
        • Myers Jr., M.G.
        • Billestrup N.
        • White M.F.
        • Carter-Su C.
        J. Biol. Chem. 1995; 270: 14685-14692
        • Johnston J.A.
        • Wang L.-M.
        • Hanson E.P.
        • Sun X.J.
        • White M.F.
        • Oakes S.A.
        • Pierce J.H.
        • O'Shea J.J.
        J. Biol. Chem. 1995; 270: 28527-28530
        • Ridderstrale M.
        • Degerman E.
        • Tornqvist H.
        J. Biol. Chem. 1995; 270: 3471-3474
        • Argetsinger L.S.
        • Norstedt G.
        • Billestrup N.
        • White M.F.
        • Carter-Su C.
        J. Biol. Chem. 1996; 271: 29415-29421
        • Platanias L.C.
        • Uddin S.
        • Yetter A.
        • Sun X.-J.
        • White M.F.
        J. Biol. Chem. 1996; 271: 278-282
        • Berlanga J.J.
        • Gualillo O.
        • Buteau H.
        • Applanat M.
        • Kelly P.A.
        • Edery M.
        J. Biol. Chem. 1997; 272: 2050-2052
        • Welham M.J.
        • Bone H.
        • Levings M.
        • Learmonth L.
        • Wang L.M.
        • Leslie K.B.
        • Pierce J.H.
        • Schrader J.W.
        J. Biol. Chem. 1997; 272: 1377-1381
        • Keegan A.D.
        • Nelms K.
        • White M.
        • Wang L.M.
        • Pierce J.H.
        • Paul W.E.
        Cell. 1994; 76: 811-820
        • D'Andrea A.D.
        • Lodish H.F.
        • Wong G.G.
        Cell. 1989; 57: 277-285
        • Bazan J.F.
        Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938
        • Witthuhn B.
        • Quelle F.W.
        • Silvennoinen O.
        • Yi T.
        • Tang B.
        • Muira O.
        • Ihle J.N.
        Cell. 1993; 74: 227-236
        • Miura O.
        • D'Andrea A.
        • Kabat D.
        • Ihle J.N.
        Mol. Cell. Biol. 1991; 11: 4895-4902
        • Damen J.
        • Mui A.L.F.
        • Hughes P.
        • Humphries K.R.
        • Krystal G.
        Blood. 1992; 80: 1923-1932
        • Dusanter-Fourt I.
        • Casadevall N.
        • Lacombe C.
        • Muller O.
        • Billat C.
        • Fischer S.
        • Mayeux P.
        J. Biol. Chem. 1992; 267: 10670-10675
        • Yoshimura A.
        • Lodish H.F.
        Mol. Cell. Biol. 1992; 12: 706-715
        • Miura Y.
        • Muira O.
        • Ihle J.N.
        • Aoki N.
        J. Biol. Chem. 1994; 269: 29962-29969
        • Gobert S.
        • Duprez V.
        • Lacombe C.
        • Gisselbrecht S.
        • Mayeux P.
        Eur. J. Biochem. 1995; 234: 75-83
        • Gouilleux F.
        • Pallard C.
        • Dusanter-Fourt I.
        • Wakao H.
        • Haldosen L.-A.
        • Norstedt G.
        • Levy D.
        • Groner B.
        EMBO J. 1995; 14: 2005-2013
        • Wakao H.
        • Harada N.
        • Kitamura T.
        • Mui A.L.F.
        • Miyajima A.
        EMBO J. 1995; 14: 2527-2535
        • Damen J.E.
        • Mui A.L.F.
        • Puil L.
        • Pawson T.
        • Krystal G.
        Blood. 1993; 81: 3204-3210
        • He T.C.
        • Zhuang H.
        • Jiang N.
        • Waterfield M.D.
        • Wojchowski D.M.
        Blood. 1993; 82: 3530-3538
        • Mayeux P.
        • Dusanter-Fourt I.
        • Muller O.
        • Mauduit P.
        • Sabbah M.
        • Drucker B.
        • Vainchenker W.
        • Fischer S.
        • Lacombe C.
        • Gisselbrecht S.
        Eur. J. Biochem. 1993; 216: 821-828
        • Miura O.
        • Nakamura N.
        • Ihle J.N.
        • Aoki N.
        J. Biol. Chem. 1994; 269: 614-620
        • Damen J.E.
        • Cutler R.L.
        • Jiao H.
        • Yi T.
        • Krystal G.
        J. Biol. Chem. 1995; 270: 23402-23408
        • Gobert S.
        • Porteu F.
        • Pallu S.
        • Muller O.
        • Sabbah M.
        • Dusanter-Fourt I.
        • Courtois G.
        • Lacombe C.
        • Gisselbrecht S.
        • Mayeux P.
        Blood. 1995; 86: 598-606
        • Komatsu N.
        • Nakauchi H.
        • Miwa A.
        • Ishihara T.
        • Eguchi M.
        • Moroi M.
        • Okada M.
        • Sato Y.
        • Wada H.
        • Yamata Y.
        • Suda T.
        • Miura Y.
        Cancer Res. 1991; 51: 341-345
        • Ruscetti S.K.
        • Janesh N.
        • Chakraborti A.
        • Sawyer S.T.
        • Hankins H.D.
        J. Virol. 1990; 63: 1057-1062
        • Avanzi G.C.
        • Lista P.
        • Giovinazzo B.
        • Miniero R.
        • Saglio G.
        • Benetton G.
        • Coda R.
        • Cattoretti G.
        • Pegoraro L.
        Br. J. Haematol. 1988; 69: 359-366
        • Kitamura T.
        • Tojo A.
        • Kuwaki T.
        • Chiba S.
        • Miyazono K.
        • Urabe A.
        • Takaku F.
        Blood. 1989; 73: 373-380
        • Kitamura T.
        • Tange T.
        • Terasawa T.
        • Chiba S.
        • Kuwaki T.
        • Miyagawa K.
        • Piao Y.F.
        • Miyazono K.
        • Urabe A.
        • Takaku F.
        J. Cell. Physiol. 1989; 140: 323-334
        • Palacios R.
        • Steinmetz M.
        Cell. 1985; 41: 727-734
        • Dexter T.M.
        • Garland D.S.
        • Scolnick E.
        • Metcalf D.
        J. Exp. Med. 1980; 152: 1036-1047
        • Damen J.E.
        • Liu L.
        • Cutler R.L.
        • Krystal G.
        Blood. 1993; 82: 2296-2303
        • Damen J.A.
        • Liu L.
        • Rosten P.
        • Humphries R.K.
        • Jefferson A.B.
        • Majerus P.W.
        • Krystal G.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1689-1693
        • Lioubin M.N.
        • Algate P.A.
        • Tsai S.
        • Carlberg K.
        • Aebersold R.
        • Rohrschneider L.R.
        Genes Dev. 1996; 10: 1084-1095
        • Ware M.D.
        • Rosten P.
        • Damen J.A.
        • Liu L.
        • Humphries R.K.
        • Krystal G.
        Blood. 1996; 88: 2833-2840
        • Drachman J.G.
        • Kaushansky K.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2350-2355
        • Hermine O.
        • Mayeux P.
        • Titeux M.
        • Mitjavila M.T.
        • Casadevall N.
        • Guichard J.
        • Komatsu N.
        • Suda T.
        • Miura Y.
        • Vainchenker W.
        • Breton-Gorius J.
        Blood. 1992; 80: 3060-3069
        • Porteu F.
        • Rouyez M.C.
        • Cocault L.
        • Benit L.
        • Charon M.
        • Picard F.
        • Gisselbrecht S.
        • Souyri M.
        • Dusanter-Fourt I.
        Mol. Cell. Biol. 1996; 16: 2473-2482
        • Chrétien S.
        • Varlet P.
        • Verdier F.
        • Gobert S.
        • Cartron J.-P.
        • Gisselbrecht S.
        • Mayeux P.
        • Lacombe C.
        EMBO J. 1996; 15: 4174-4181
        • Ikawa Y.
        • Aida M.
        • Inoue Y.
        Gann. 1976; 67: 767-770
        • Ihle J.N.
        Nature. 1995; 377: 591-594
        • Silvennoinen O.
        • Witthuhn B.A.
        • Quelle F.W.
        • Cleveland J.L.
        • Yi T.
        • Ihle J.N.
        Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8429-8433
        • Correa P.N.
        • Axelrad A.A.
        Blood. 1991; 78: 2823-2833
        • Correa P.N.
        • Eskinazi D.
        • Axelrad A.A.
        Blood. 1994; 83: 99-112
        • He T.-C.
        • Jiang N.
        • Zhuang H.
        • Quelle D.E.
        • Wojchowski D.M.
        J. Biol. Chem. 1994; 269: 18291-18294
        • Hilton C.J.
        • Berridge M.V.
        Growth Factors. 1995; 121: 263-276
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        Cell. 1997; 88: 435-437
        • Franke T.F.
        • Kaplan D.R.
        • Cantley L.C.
        • Toker A.
        Science. 1997; 275: 685-688
        • Klippel A.
        • Kavanaugh W.M.
        • Pot D.
        • Williams L.T.
        Mol. Cell. Biol. 1997; 17: 338-344