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Interleukin-4 (IL-4) Induces Phosphatidylinositol 3-Kinase (p85) Dephosphorylation

IMPLICATIONS FOR THE ROLE OF SHP-1 IN THE IL-4-INDUCED SIGNALS IN HUMAN B CELLS*
  • Farhad Imani
    Correspondence
    To whom correspondence should be addressed
    Affiliations
    From the Division of Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Asthma and Allergy Center, Baltimore, Maryland 21224
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  • Kelly J. Rager
    Affiliations
    From the Division of Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Asthma and Allergy Center, Baltimore, Maryland 21224
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  • Branimir Catipovic
    Affiliations
    From the Division of Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Asthma and Allergy Center, Baltimore, Maryland 21224
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  • David G. Marsh
    Affiliations
    From the Division of Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Asthma and Allergy Center, Baltimore, Maryland 21224
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  • Author Footnotes
    * This work was supported by grants from the American Lung Association and The Johns Hopkins University (to F. I.) and a National Institutes of Health Grants AI0059 (to D. G. M.). 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.
    2 F. Imani, K. J. Rager, and D. G. Marsh, manuscript in preparation.
Open AccessPublished:March 21, 1997DOI:https://doi.org/10.1074/jbc.272.12.7927
      Interleukin 4 (IL-4) is a potent cytokine produced by T cells and to a lesser extent by tumor-associated natural killer cells, basophils, and mast cells. IL-4 treatment of T cells and macrophages leads to augmentation of their cytotoxic activity. In human B cells, IL-4 is a potent stimulator of Ig class switching from IgM to IgE. The diverse biological responses induced by IL-4 are mediated through a high affinity receptor complex (IL-4R). Although a wealth of information has accumulated regarding IL-4R, the exact mechanisms of IL-4R-mediated signaling pathways in human B cells are not well defined. In an attempt to characterize the IL-4-induced signals in human B cells, we have found that IL-4 treatment induced rapid dephosphorylation of the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase. To identify the protein-tyrosine phosphatase involved in the IL-4-mediated dephosphorylation, we performed Western blot analysis using monoclonal antibodies specific to protein-tyrosine phosphatases. Upon IL-4 treatment, SHP-1 was specifically translocated to the cellular membrane fraction. Furthermore, immunoprecipitation studies revealed that SHP-1 could be specifically coimmunoprecipitated with the IL-4R as well as with phosphatidylinositol 3-kinase (p85). Collectively, our observations suggest that in addition to protein phosphorylation, protein tyrosine dephosphorylation may play a role in the IL-4-induced signaling pathways.

      INTRODUCTION

      IL-4
      The abbreviations used are: IL4-R
      interleukin-4 receptor
      PI
      phosphatidylinositol
      mAb
      monoclonal antibody
      TPCK
      L-1-tosylamido-2-phenylethyl chloromethyl ketone
      PAGE
      polyacrylamide gel electrophoresis.
      is a potent cytokine with pleiotropic effects on many cell types. The biological effects of IL-4 include induction of IgE class switching, induction of proliferation in T and B cells, and up-regulation of CD23 and major histocompatibility complex class II molecules on human B cells (
      • Lundgren M.
      • Persson U.
      • Larsson P.
      • Magnusson C.
      • Smith C.I.E.
      • Hammarstrom L.
      • Severinson E.
      ,
      • Grabstein K.
      • Eisenman J.
      • Mochizuki D.
      • Shanebeck K.
      • Conlon P.
      • Hopp T.
      • March C.
      • Gillis S.
      ,
      • Defrance T.
      • Aubry J.P.
      • Rousset F.
      • Vanbervliet B.
      • Bonnefoy J.-Y.
      • Arai N.
      • Takebe Y.
      • Yokota T.
      • Lee F.
      • Arai K.
      • de Vries J.E.
      • Bancherau J.
      ,
      • Noelle R.
      • Krammer P.H.
      • Ohara J.
      • Uhr J.W.
      • Vitetta E.S.
      ).
      The IL-4 receptor complex is present on many hematopoietic and non-hematopoietic cell lines (
      • Lowenthal J.W.
      • Castle B.E.
      • Christiansen J.
      • Schreuers J.
      • Rennick D.
      • Arai N.
      • Hoy P.
      • Takebe U.
      • Howard M.
      ). Although many members of the cytokine receptor family, including the IL-4 receptor (IL-4R), lack protein kinase consensus domains, ligand binding to these receptors results in tyrosine phosphorylation as well as dephosphorylation and subsequent biological responses (
      • Keegan A.D.
      • Nelms K.
      • Wang L.-M.
      • Pierce J.H.
      • Paul W.E.
      ,
      • Mire-Sluis A.R.
      • Thorpe R.
      ).
      Early studies of Morla et al (
      • Morla A.O.
      • Schreus J.
      • Miyajima A.
      • Wang J.Y.
      ) reveal IL-4-induced tyrosine phosphorylation of proteins of 110 and 170 kDa in a murine mast cell line, IC 2.9. Subsequently, Wang et al. (
      • Wang L.-M.
      • Keegan A.D.
      • Li W.
      • Lienhard G.E.
      • Pacini S.
      • Gutkind J.S.
      • Myers M.G.
      • Sun X.-J.
      • White M.F.
      • Aaronson S.A.
      • Paul W.E.
      ) reported IL-4-induced tyrosine phosphorylation of a 170-kDa polypeptide, termed 4PS in murine myeloid cell lines. Keegan et al. (
      • Keegan A.D.
      • Nelms K.
      • White M.
      • Wang L.-M.
      • Pierce J.H.
      • Paul W.E.
      ) reported that 4PS may be antigenically and functionally similar to the insulin receptor substrate-1. Additional evidence of IL-4-induced signal transduction events was demonstrated by the IL-4-induced association of the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) with the phosphorylated form of 4PS; however, p85 itself was not phosphorylated (
      • Wang L.-M.
      • Keegan A.D.
      • Paul W.E.
      • Heidaran M.A.
      • Gutkind J.S.
      • Pierce J.H.
      ).
      Further studies have shown that IL-4 treatment induces the association of IL-4R with the γ chain of the IL-2 receptor complex (
      • Kondo M.
      • Takeshita T.
      • Ishii N.
      • Nakamura M.
      • Watanabe S.
      • Arai K.-I.
      • Sugamura K.
      ) that participates in IL-4-mediated signaling by γ chain-associated JAK kinases (
      • Yin T.
      • Tsang M.L.-S.
      • Yang Y.-C.
      ,
      • Witthuhn B.A.
      • Silvennoinen O.
      • Lai K.S.
      • Cwik C.
      • Liu E.T.
      • Ihle J.N.
      ). However, experiments by He and Malek (
      • He Y.-W.
      • Malek T.R.
      ) provide evidence for the presence of two distinct IL-4R-mediated signaling events, γ chain-dependent and -independent, providing an explanation for the pleiotropic effects exerted by IL-4. Also, Hou et al. (
      • Hou J.
      • Schindler U.
      • Henzel W.J.
      • Ho T.C.
      • Brasseur M.
      • McKnight S.L.
      ) reported that IL-4 could activate a tyrosine-phosphorylated DNA-binding protein termed IL-4-Stat (Stat-6) that is involved in IL-4-mediated transcription. Recently, Stat-6 knockout mice have been developed, and these mice are deficient in biological responses to IL-4 treatment (
      • Shimoda K.
      • Deursen J.V.
      • Sangster M.Y.
      • Sarawar S.R.
      • Carson R.T.
      • Tripp R.A.
      • Chu C.
      • Quelle F.W.
      • Nosaka T.
      • Vignali D.A.A.
      • Doherty P.C.
      • Grosweld G.
      • Paul W.E.
      • Ihle J.N.
      ).
      In this report we provide evidence for IL-4-induced protein tyrosine dephosphorylation of the 85-kDa subunit of the PI 3-kinase (p85). Also, we report the association of SHP-1, previously known as PTP-1C, with the IL-4R and PI 3-kinase (p85). It therefore appears that the IL-4-induced protein tyrosine dephosphorylation may play a role in the IL-4-induced signals, leading to diverse biological responses.

      DISCUSSION

      We have used the highly IL-4-sensitive subtype (2G6.4C10) of the human Burkitt's lymphoma B cell line Ramos. These cells are IgM+ and respond to IL-4 treatment by up-regulation of CD23 (
      • Siegal J.P.
      • Mostowski H.S.
      ). In this paper we report the IL-4-induced tyrosine dephosphorylation of an 85-kDa polypeptide in Ramos cells.
      This polypeptide appears to be identical to the 85-kDa regulatory subunit of PI 3-kinase (p85). Immunoprecipitation studies revealed that IL-4 treatment induced tyrosine dephosphorylation of PI 3-kinase (p85). Interestingly, an IL-4-induced tyrosine-dephosphorylated polypeptide at approximately 170 kDa was also coimmunoprecipitated with anti-PI 3-kinase (p85) (Fig. 2B, middle panel). At present the identity of the 170-kDa polypeptide (p170) is unknown, but it is possible that this molecule may represent insulin receptor substrate-2.
      Also, IL-4 treatment of Ramos cells resulted in specific translocation of SHP-1 to a cellular membrane fraction. Zhao et al. (
      • Zhao Z.
      • Shen S.-H.
      • Fischer E.H.
      ) reported SHP-1 phosphorylation and translocation to the cellular membrane by phorbol ester treatment of a human promyelocytic leukemia cell line. Furthermore, by performing immunoprecipitation studies we showed that SHP-1 could specifically associate with IL-4R as well as with PI 3-kinase (p85), suggesting a role for this phosphatase in IL-4-induced signaling pathways.
      The biological responses induced by IL-4 are transmitted by the high affinity IL-4R present in low abundance (102-103 copies/cell) on many hematopoietic and non-hematopoietic cells (
      • Park L.S.
      • Friend D.
      • Grabstein K.
      • Urdal D.L.
      ). Cross-linking and immunoprecipitation studies of cell surface radioiodinated cells showed that IL-4 treatment induces the association of IL-4R and the γ chain shared by the IL-2, IL-5, IL-7, and IL-9 receptors (
      • Kondo M.
      • Takeshita T.
      • Ishii N.
      • Nakamura M.
      • Watanabe S.
      • Arai K.-I.
      • Sugamura K.
      ). Since the IL-4R chain does not possess any protein kinase activity, IL-4-induced signals are thought to be mediated through IL-4R-associated protein kinase(s).
      Further evidence of IL-4-induced signal transduction was demonstrated by the observation that the 85-kDa subunit (p85) of PI 3-kinase was associated with the phosphorylated form of insulin receptor substrate-2 (4PS), suggesting that this interaction may be critical to IL-4-induced activation (
      • Wang L.-M.
      • Keegan A.D.
      • Paul W.E.
      • Heidaran M.A.
      • Gutkind J.S.
      • Pierce J.H.
      ). Hou et al. (
      • Hou J.
      • Schindler U.
      • Henzel W.J.
      • Ho T.C.
      • Brasseur M.
      • McKnight S.L.
      ) revealed the presence of an IL-4-activated transcription factor termed Stat-6. Stat-6 is phosphorylated upon IL-4 treatment, and the phosphorylated form of Stat-6 is translocated to the nucleus where it can bind and activate the transcription of IL-4-responsive genes.
      In contrast to the reported IL-4-induced protein phosphorylation events, in vivo phosphorylation studies by Mire-Sluis and Thorpe (
      • Mire-Sluis A.R.
      • Thorpe R.
      ) in human TF-1 cells showed the IL-4-induced dephosphorylation of an 80-kDa phosphotyrosine polypeptide. Pretreatment of cells with TGF-β, a known down-regulator of IL-4-induced IgE class switching (
      • Gauchat J.-F.
      • Aversa G.
      • Gascan H.
      • de Vries J.E.
      ,
      • Armitage R.J.
      • Macduff B.M.
      • Spriggs M.K.
      • Farnslow W.C.
      ), blocked this dephosphorylation (
      • Mire-Sluis A.R.
      • Thorpe R.
      ). Although it is not yet determined, it is possible that p80 (
      • Mire-Sluis A.R.
      • Thorpe R.
      ) is the same polypeptide as p85, and the difference in the molecular weight is due to different electrophoresis conditions and molecular weight markers.
      The discrepancy in the reports of the IL-4-induced signals may represent the differences between human and mouse systems. Alternatively, these differences in the IL-4-induced signals may be due to the different cell types used in the experiments.
      In our experiments, we have detected rapid protein tyrosine dephosphorylation upon IL-4 treatment. At this point we do not know the exact physiologic relevance of the IL-4-induced PI 3-kinase (p85) dephosphorylation. But based on previous reports showing tyrosine phosphorylation of PI 3-kinase (p85) by growth factors such as platelet-derived growth factor and insulin (
      • Kavanaugh W.M.
      • Turck C.W.
      • Klippel A.
      • Williams L.T.
      ,
      • Hayashi H.
      • Kamohara S.
      • Nishioka Y.
      • Kanai F.
      • Miyake N.
      • Fukui Y.
      • Shibasaki F.
      • Takenawa T.
      • Ebina Y.
      ), it is possible that the IL-4-induced inhibition of leukemic and normal B cell proliferation (
      • Pandrau D.
      • Saeland S.
      • Duvert V.
      • Manel A.-M.
      • Zabot M.-T.
      • Philippe N.
      • Banchereau J.
      ) may be due to the IL-4-induced PI 3-kinase (p85) dephosphorylation. Alternatively, based on our preliminary results showing that Na3VO4, a potent protein-tyrosine phosphatase inhibitor, could block the IL-4-induced IgE germline transcript expression,
      F. Imani, K. J. Rager, and D. G. Marsh, manuscript in preparation.
      it is tempting to speculate that protein tyrosine dephosphorylation may be necessary for the IL-4-induced signaling that leads to IgE class switching in human B cells. Further experiments are necessary to determine the exact role of PI 3-kinase (p85) dephosphorylation in the IL-4-induced signaling events.

      Acknowledgments

      We gratefully acknowledge Drs. David Proud and Stephen Desiderio (The Johns Hopkins University) for helpful suggestions and critical review of this manuscript.

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