Signal transduction of the human granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors involves tyrosine phosphorylation of a common set of cytoplasmic proteins.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) exert multiple effects on the proliferation, differentiation, and function of myeloid lineage cells through their interaction with specific cell-surface receptors. There is a considerable degree of overlap in the biological effects of these two growth factors, but little is known about the mechanisms of postreceptor signal transduction. We have investigated the effects of GM-CSF and IL-3 on protein tyrosine-kinase activity in a human cell line, MO7E, which proliferates in response to either factor. Tyrosine-kinase activity was detected using immunoblotting with a monoclonal antibody (MoAb) specific for phosphotyrosine. GM-CSF and IL-3 were found to induce a nearly identical pattern of protein tyrosine phosphorylation using both one- and two-dimensional gel electrophoresis. Tyrosine phosphorylation of two cytosolic proteins in particular was increased more than 10-fold, a 93-Kd protein (pp93) and a 70-Kd protein (pp70). Tyrosine phosphorylation of pp93 and pp70 was observed within 1 minute, reached a maximum at 5 to 15 minutes, and gradually decreased thereafter. Other proteins of 150, 125, 63, 55, 42, and 36 Kd were also phosphorylated on tyrosine in response to both GM-CSF and IL-3, although to a lesser degree. Tyrosine phosphorylation was dependent on the concentration of GM-CSF over the range of 0.1 to 10 ng/mL and on IL-3 over the range of 1 to 30 ng/mL. Stimulation of MO7E cells with 12-0-tetradecanoyl-phorbol-13-acetate (TPA) or cytokines such as G-CSF, M-CSF, interleukin-1 (IL-1), interleukin-4 (IL-4), interleukin-6 (IL-6), interferon gamma, tumor necrosis factor (TNF), or transforming growth factor-beta (TGF-beta) did not induce tyrosine phosphorylation of pp93 or pp70, suggesting that these two phosphoproteins are specific for GM-CSF-or IL-3-induced activation. The extent and duration of phosphorylation of all the substrates were increased by pretreatment of cells with vanadate, an inhibitor of protein-tyrosine phosphatases. Importantly, culture of MO7E cells with vanadate (up to 10 mumol/L) resulted in a dose-dependent increase in GM-CSF-or IL-3-induced proliferation of up to 1.8-fold. These results suggest that tyrosine phosphorylation may be important for GM-CSF and IL-3 receptor-mediated signal transduction and that cell proliferation may be, at least partially, regulated by a balance between CSF-induced protein-tyrosine kinase activity and protein-tyrosine phosphatase activity.

[1]  M. Moran,et al.  Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases , 1990, Nature.

[2]  K. Arai,et al.  Cloning of an interleukin-3 receptor gene: a member of a distinct receptor gene family. , 1990, Science.

[3]  S. Cannistra,et al.  Regulation of surface expression of the granulocyte/macrophage colony-stimulating factor receptor in normal human myeloid cells. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[4]  A. Hoffbrand,et al.  Binding of G-CSF, GM-CSF, tumor necrosis factor-alpha, and gamma-interferon to cell surface receptors on human myeloid leukemia cells triggers rapid tyrosine and serine phosphorylation of a 75-Kd protein. , 1990, Blood.

[5]  D. Gearing,et al.  Expression cloning of a receptor for human granulocyte‐macrophage colony‐stimulating factor. , 1989, The EMBO journal.

[6]  P. Johnson,et al.  Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[7]  T. Sturgill,et al.  Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[8]  T. Mustelin,et al.  Rapid activation of the T-cell tyrosine protein kinase pp56lck by the CD45 phosphotyrosine phosphatase. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[9]  T. Hunter,et al.  Phospholipase C-γ is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro , 1989, Cell.

[10]  A. Zilberstein,et al.  EGF induces tyrosine phosphorylation of phospholipase C-II: A potential mechanism for EGF receptor signaling , 1989, Cell.

[11]  K. Miyazono,et al.  Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line. , 1989, Biochemical and biophysical research communications.

[12]  G. Swarup,et al.  Purification and characterization of a protein-phosphotyrosine phosphatase from rat spleen which dephosphorylates and inactivates a tyrosine-specific protein kinase. , 1989, The Journal of biological chemistry.

[13]  T. Molski,et al.  Granulocyte-macrophage colony-stimulating factor and human neutrophils: role of guanine nucleotide regulatory proteins. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[14]  R. Mooney,et al.  Phosphorylation of the insulin receptor in permeabilized adipocytes is coupled to a rapid dephosphorylation reaction. , 1989, The Journal of biological chemistry.

[15]  D. Friend,et al.  Heterogeneity in human interleukin-3 receptors. A subclass that binds human granulocyte/macrophage colony stimulating factor. , 1989, The Journal of biological chemistry.

[16]  T. Hunter,et al.  Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. , 1989, Cell.

[17]  S. Murthy,et al.  Interleukin-3, GM-CSF, and TPA induce distinct phosphorylation events in an interleukin 3-dependent multipotential cell line. , 1989, Blood.

[18]  J. Ihle,et al.  Stimulation of factor-dependent myeloid cell lines with interleukin 3 induces tyrosine phosphorylation of several cellular substrates. , 1988, The Journal of biological chemistry.

[19]  D. Brautigan,et al.  Protein phosphotyrosine phosphatase purified from the particulate fraction of human placenta dephosphorylates insulin and growth-factor receptors. , 1988, The Biochemical journal.

[20]  D. Morrison,et al.  Signal transduction from membrane to cytoplasm: growth factors and membrane-bound oncogene products increase Raf-1 phosphorylation and associated protein kinase activity. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[21]  J. Ihle,et al.  Interleukin 3 binds to a 140-kDa phosphotyrosine-containing cell surface protein. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[22]  W. Liu,et al.  Identification and subcellular localization of proteins that are rapidly phosphorylated in tyrosine in response to colony-stimulating factor 1. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[23]  M. Vadas,et al.  Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series. , 1988, Blood.

[24]  S. Clark,et al.  Specific binding, internalization, and degradation of human recombinant interleukin‐3 by cells of the acute myelogenous, leukemia line, KG‐1 , 1988, Journal of cellular physiology.

[25]  D. Ferris,et al.  Interleukin 3 stimulation of tyrosine kinase activity in FDC-P1 cells. , 1988, Biochemical and biophysical research communications.

[26]  G. Cattoretti,et al.  Selective growth response to IL‐3 of a human leukaemic cell line with megakaryoblastic features , 1988, British journal of haematology.

[27]  N. Tonks,et al.  Characterization of the major protein-tyrosine-phosphatases of human placenta. , 1988, The Journal of biological chemistry.

[28]  J. Wang,et al.  Hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-3-dependent murine cell lines , 1988, Molecular and cellular biology.

[29]  A. D. de Weck,et al.  Leukotriene production in human neutrophils primed by recombinant human granulocyte/macrophage colony-stimulating factor and stimulated with the complement component C5A and FMLP as second signals , 1988, The Journal of experimental medicine.

[30]  J. Schlessinger,et al.  Signal transduction through the EGF receptor transfected in IL-3-dependent hematopoietic cells. , 1988, Science.

[31]  J. Dipersio,et al.  Characterization of the human granulocyte-macrophage colony-stimulating factor receptor. , 1988, The Journal of biological chemistry.

[32]  A. Ullrich,et al.  Growth factor receptor tyrosine kinases. , 1988, Annual review of biochemistry.

[33]  K. Arai,et al.  Interleukin 3‐specific tyrosine phosphorylation of a membrane glycoprotein of Mr 150,000 in multi‐factor‐dependent myeloid cell lines. , 1987, The EMBO journal.

[34]  J. Griffin,et al.  Effects of recombinant human granulocyte and macrophage colony-stimulating factors on signal transduction pathways in human granulocytes. , 1987, Journal of immunology.

[35]  M. White,et al.  Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity , 1987, Cell.

[36]  R. Kamen,et al.  The human hematopoietic colony-stimulating factors. , 1987, Science.

[37]  J. Ihle,et al.  The v-fms oncogene induces factor-independent growth and transformation of the interleukin-3-dependent myeloid cell line FDC-P1. , 1987, Molecular and cellular biology.

[38]  T. Sturgill,et al.  Rapid stimulation by insulin of a serine/threonine kinase in 3T3-L1 adipocytes that phosphorylates microtubule-associated protein 2 in vitro. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Y. Furukawa,et al.  Establishment and characterization of four human monocytoid leukemia cell lines (JOSK-I, -S, -M and -K) with capabilities of monocyte-macrophage lineage differentiation and constitutive production of interleukin 1. , 1986, Cancer Research.

[40]  D. Friend,et al.  Characterization of the cell surface receptor for granulocyte-macrophage colony-stimulating factor. , 1986, The Journal of biological chemistry.

[41]  M. Tomonaga,et al.  High-affinity binding of granulocyte-macrophage colony-stimulating factor to normal and leukemic human myeloid cells. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[42]  Charles J. Sherr,et al.  The c-fms proto-oncogene product is related to the receptor for the mononuclear phagocyte growth factor, CSF 1 , 1985, Cell.

[43]  A. Burgess,et al.  Specific binding of radioiodinated granulocyte‐macrophage colony‐stimulating factor to hemopoietic cells. , 1985, EMBO Journal.

[44]  D. Golde,et al.  Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator , 1985, Nature.

[45]  T. Hunter,et al.  Protein-tyrosine kinases. , 1985, Annual review of biochemistry.

[46]  T. Mosmann Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.