Tyrosine Kinase Phosphorylation in Human Neutrophil Activation by PAF and other Agonists

  • Julian Gomez-Cambronero
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)


Protein kinases specific for tyrosine (PTK) were initially detected in oncogenic products of viral tumors (pp60v-src ) and in some growth factor receptors (1). Until recently, protein phosphorylation on tyrosine residues was only linked to cellular growth and transformation (1). Over the past few years, however, a number of laboratories have reported the presence of tyrosine kinase activities in non-proliferative tissues and cells (e.g. neurons, platelets and leukocytes). Tyrosine-specific kinases, such as the products of the proto-oncogenes c-src, chck, c fes/fps and c-fgr, are known to be present in white blood cells, including neutrophils (2–5), and some are expressed at a high level. Both particulate extracts and cytosolic fractions from neutrophils contain tyrosine kinase and phosphotyrosine phosphatase activities (6,7). Moreover, the CD45 antigen, a glycoprotein of the plasma membrane of all leukocytes, possesses tyrosine phosphatase activity that might regulate signal transduction in those cells (8). Using antibodies to phosphotyrosine (aPTyr) in Western blots, it has been demonstrated that tyrosine-specific protein phosphorylati on occurs when human or rabbit neutrophils are stimulated with a variety of agonists, including platelet-activating factor (PAF), human cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF), the chemotactic peptide fMet-Leu-Phe, phorbol esters (PMA) and the calcium ionophore A23187 (9–17). A summary of the potency of those agents, as it relates to phosphorylation, is given in Table I. It has also been reported that the addition of GTPγS or vanadate to electropermeabilized neutrophils triggers rapid phosphorylation of tyrosine residues of several protein substrates (18,19).


HL60 Cell Tyrosine Phosphorylation Human Neutrophil Pertussis Toxin Calcium Ionophore A23187 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    T. Hunter and J.A. Carpenter, Protein tyrosine kinases, Ann. Rev. Biochem. 54: 897–930 (1985).PubMedCrossRefGoogle Scholar
  2. 2.
    C.E. Gee, J. Griffin, L. Sastre, L.J. Miller, T.A. Springer, H. PiwnicaWorms and T.M. Roberts, Differentiation of myeloid cells is accompanied by increased levels of pp60c-src protein and kinase activity, Proc. Natl. Acad. Sci. USA 83: 5131–5135 (1986).PubMedCrossRefGoogle Scholar
  3. 3.
    S.F. Ziegler, C.B. Wilson and R.M. Perlmutter, Augmented expression of a myeloid-specific protein tyrosine kinases gene (hck) after macrophage activation, J. Exp. Med. 168: 1801–1810 (1988).PubMedCrossRefGoogle Scholar
  4. 4.
    G. Yu, T.E. Smithgall and R. I. Glazer, K562 leukemia cells transfected with human c-fes gene acquire the ability to undergo myeloid differentiation, J. Biol. Chem. 264: 10276–10281 (1989).PubMedGoogle Scholar
  5. 5.
    J.S. Gutkind and K.C. Robbins, Translocation of the FGR protein-tyrosine kinase as a consequence on neutrophil activation, Proc. Natl. Acad. Sci. USA 86: 8783–8787 (1989).PubMedCrossRefGoogle Scholar
  6. 6.
    A.S. Kraft and R.L. Berkow, Tyrosine kinase and phosphotyrosine phosphatase activity in human promyelocytic leukemia cells and human polymorphonuclear leucocytes, Blood, 70: 356–362 (1987).PubMedGoogle Scholar
  7. 7.
    R.L. Berkow, R.W. Dodson and A.S. Kraft, Human neutrophils contain distinct cytosolic and particulate tyrosine kinase acitivities: Possible role in neutrophil activation, Biochem. Biophys. Acta, 997: 292–301 (1989).CrossRefGoogle Scholar
  8. 8.
    L. Harvath, J.A. Balke, N.P. Christiansen, A.A. Russell and K.M. Skubitz, Selected antibodies to leukocyte common antigen (CD45) inhibit human neutrophil chemotaxis, J. Immunol 146: 949–957 (1991).PubMedGoogle Scholar
  9. 9.
    J. Gomez-Cambronero, M. Yamazaki, F. Metwally, T.F.P. Molski, V.A. Bonak, C.-K. Huang, E.L. Becker and R.I. Sha’afi, Granulocyte-macrophage colony-stimulating factor and human neutrophils: Role of guanine nucleotide regulatory proteins, Proc. Natl. Acad. Sci. USA, 86: 3569–3573 (1989).PubMedCrossRefGoogle Scholar
  10. 10.
    J. Gomez-Cambronero, C.-K. Huang, V.A. Bonak, E. Wang, J.E. Casnellie, T. Shiraishi and R.I. Sha’afi, Tyrosine phosphorylation in human neutrophils, Biochem. Biophys. Res. Commun 162: 1478–14856 (1989).CrossRefGoogle Scholar
  11. 11.
    J. Gomez-Cambronero, E. Wang, G. Johnson, C.-K. Huang and R.I. Sha’afi, Platelet-activating factor induces tyrosine phosphorylation in human neutrophils, J. Biol. Chem. 266: 6240–6245 (1991).PubMedGoogle Scholar
  12. 12.
    R.L. Berkow and R.W. Dodson, Tyrosine-specific protein phosphorylation during activation of human neutrophils, Blood, 75: 2445–2452 (1990).PubMedGoogle Scholar
  13. 13.
    C.-K. Huang, V. Bonak, G.R. Laramee and J.E. Casnellie, Protein Tyrosine phosphorylation in rabbit peritoneal neutrophils, Biochem. J. 269: 431–436 (1990).PubMedGoogle Scholar
  14. 14.
    C.-K. Huang, G.R. Laramee and J.E. Casnellie, Chemotactic factor induced tyrosine phosphorylation of membrane associated proteins in rabbit peritoneal neutrophils, Biochem. Biophys. Res. Commun, 151: 794–801 (1988).CrossRefGoogle Scholar
  15. 15.
    P.H. Naccache, C. Gilbert, A.C. Caon, M. Gaundry, C.-K. Huang, V.A. Bonak, K. Umezawa and S.R. McColl, Selective inhibition of human neutrophil functional responsiveness by Erbstatin, an inhibitor of tyrosine protein kinase, Blood. 76: 2098–2104 (1990).PubMedGoogle Scholar
  16. 16.
    P.E. Nasmith, G.B. Mills and S. Grinstein, Guanine nucleotides induce tyrosine phosphorylation and activation of the respiratory burst in neutrophils, Biochem. J. 257: 893–897 (1989).PubMedGoogle Scholar
  17. 17.
    T.C. Wright, M.J. Karnovsky and J.M. Robinson, Tyrosine phosphorylation of the 43 kD and 41 kD proteins occurs during PMN activation, J. Cell Biol. 107: 57a (1988).Google Scholar
  18. 18.
    P.E. Nasmith, G.B. Mills and S. Grinstein, Guanine nucleotides induce tyrosine phosphorylation and activation of the respiratory burst in neutrophils, Biochem. J. 257: 893–897 (1989).PubMedGoogle Scholar
  19. 19.
    S. Gristein, W. Furuya, D.J. Lu and G.B. Mills, Vanadate stimulates oxygen consumption and tyrosine phosphorylation in electropermeabilized human neutrophils, J. Biol. Chem. 265: 318–327 (1990).Google Scholar
  20. 20.
    T. G. Tessner, J.T. O’Flaherty and R.L. Wykle, Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites, J. Biol. Chem. 264: 4794–4799 (1989).PubMedGoogle Scholar
  21. 21.
    J.T. O’Flaherty, J.R. Suries, J. Redman, D. Jacobson, C. Piantadosi and R.L. Wykle, Binding and metabolism of platelet-activating factor by human neutrophils, J. Clin Invest. 78: 381–388 (1986).PubMedCrossRefGoogle Scholar
  22. 22.
    F.J. Valone and F.J. Goetzl,. Specific binding by human polymorphonuclear leukocytees of the immunological mediator 1O-hexadecyl-octadecyl-2-acetyl-sn-glycero-3-phosphocholine, Immunology 48: 141–149 (1983).PubMedGoogle Scholar
  23. 23.
    R.I. Sha’afi and T.F.P. Molski, Activation of the neutrophil, Prog., Allergy 42:1–64 (1988).Google Scholar
  24. 24.
    A. Dhar, A.K. Paul and S.D. Shukla, Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with Genistein and monoclonal antibody to phosphotyrosine,.Mol. Pharmacol. 37: 519–525 (1990).Google Scholar
  25. 25.
    K.B. Glasser, R. Asmis and E.A. Dennis, Bacterial lipopolysaccharide priming of P388D1 macrophage-like cells for enhanced arachidonic acid metabolism, J. Biol. Chem. 265: 8658–8664 (1990).Google Scholar
  26. 26.
    A.O. Morla, J. Scheurs, A. Miyajima and J.Y.J. Wang, hematopoietic growth factors activate the tyrosine phosphorylation of distinct sets of proteins in interleukin-3-dependent murine cell lines, Molec. Cell Biol 8: 2214 (1988).Google Scholar
  27. 27.
    J.P.M. Evans, A.R. Mire-Sluis, A.V. Hoffbrand and R.G. Wickremasinghe, Binding of G-CSF, GM-CSF, tumor necrosis factor-a and y-interferon to cell surface receptors on human myeloid leukemia cells triggers rapid tyrosine and serine phosphorylation of a 75-kD protein, Blood 75: 88–95 (1990).Google Scholar
  28. 28.
    P.H. Sorensen, A.L. Mui, S.C. Murthy and G. Krystal, Interleukin-3, GM-CSF and TPA induce distinct phosphorylation events in an interleukin-3-dependent multipotential cell line, Blood. 73: 406–418 (1989).Google Scholar
  29. 29.
    D.P. Gearing, J.A. King, N.M. Gough and N.A. Nicola, Expression cloning of a receptor for human granuclocyte-macrophage colony-stimulating factor, EMBO J. 8: 3667–3676 (1989).PubMedGoogle Scholar
  30. 30.
    Y. Yarden, Growth factor receptor tyrosine kinases, Ann. Rev. Biochem. 57: 443–478 (1988).PubMedCrossRefGoogle Scholar
  31. 31.
    G. Carpenter and S. Cohen, Epidermal Growth Factor, J. Biol. Chem. 265: 7709–7712 (1990).PubMedGoogle Scholar
  32. 32.
    J. Nath, A. Powledge and D.G. Wright, Studies of signal transduction in the respiratory burst-associated stimulation of fMet-Leu-Pheinduced tubulin tyrosinolation and PMA-induced posttranslational incorporation of tyrosine into multiple proteins in activated neutrophile and HL-60 cells, J. Biol Cehm. 264: 848–855 (1989).Google Scholar
  33. 33.
    P.H. Naccache, M.M. Molski, M. Volpi, E.L. Becker and R.I. Sha’afi, Unique inhibitory profile of PAF induced by calcium mobilization, polyphosphoinositide turnover and granule enzyme secretion in rabbit neutrophil towards pertussis toxin and phorbol ester, Biochem. Biophys. Res. Commun. 130: 677–684 (1985).CrossRefGoogle Scholar
  34. 34.
    A.J. Rossomando, D.M. Payne, M.J. Weber and T.W. Sturgill, Evidence that pp42, a major tyrosine kinase target protein, is a mitogenactivated serine/threoinine protein kinase, Proc. Natl. Acad. Sci. USA 86: 6940–6943 (1989).PubMedCrossRefGoogle Scholar
  35. 35.
    L.B. Ray and T.W. Sturgill, Rapid stimulation by insulin of a serine/threonine kinase in 3T3–L1 adipocytes that phosphorylates microtubule-associted protein 2 in vitro. Proc. Natl. Acad. Sci. USA 84: 1502–1506 (1987).PubMedCrossRefGoogle Scholar
  36. 36.
    K.F. Balazovich and E.L. McEwen, Purification and characterization of a soluble 42 kilodalton protein kinase from human neutrophils, L Cell Biol. I II, 49a (abst.) (1990).Google Scholar
  37. 37.
    M. Katan and P.J. Parker, Oncogenes and cell control, Nature 32: 203 (1988).CrossRefGoogle Scholar
  38. 38.
    J.R. Downing, C.W. Rettenmier and C.J. Shen, Ligand-induced tyrosine kinase activity of the colony-stimulating factor 1 receptor in a murine macrophage cell line, Mol. Cell. Biol. 8: 1795–1799 (1988).Google Scholar
  39. 39.
    A. Veillette, M.A. Bookman, E.M. Horak, L.E. Samelson and J.B. Bolen, Signal transduction through the CD45 receptor involves the activation of the internal membrane tyrosine-protein kinase p561ck, Nature 338: 257–259 (1989).PubMedCrossRefGoogle Scholar
  40. 40.
    M.L. Stahl, C.R. Ferenz, K.L. Kelleher, R.W. Kriz and J.L. Knopf,. Sequence similarity of Phospholipase C with the non-catalytic region of src, Nature 332: 269–272 (1988).PubMedCrossRefGoogle Scholar
  41. 41.
    B.J. Mayer, M. Hamaggchi and H. Hanafusa, A novel viral oncogene with structural similarity to Phospholipase C, Nature 332: 272–275 (1988).PubMedCrossRefGoogle Scholar
  42. 42.
    M. Katan and P.J. Parker, Oncogenes and cell control, Nature 32: 203 (1988).CrossRefGoogle Scholar
  43. 43.
    T.L. Leto, K.J. Lomax, B.D. Volpp, H. Nunoi, J.M. Sechler, W.M. Nauseef, R.A. Clark, J.I. Gallin and H.L. Malech, Cloning a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src, Science 248: 727–730 (1990).PubMedCrossRefGoogle Scholar
  44. W.M. Nauseef, B.D. Volpp, S. McCormick, K.G. Leidal and R.A. Clark, Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components, J. Biol Chem. 266:5911–5917 (1991).Google Scholar
  45. 45.
    S. Trudel, G.P. Downey, S. Grinstein and M.R. Paquet, Activation of permeabilized HL60 cells by vanadate. Evidence for divergent signalling pathways, Biochem J. 269: 127–131 (1990).PubMedGoogle Scholar
  46. 46.
    S. Trudel, M.R. Paquet and S. Grinstein, Mechanism of vanadateinduced activation of tyrosine phosphorylation and of the respiratory burst in HL60 cells. Role of reduced oxygen metabolites, Biochem. J. 276: 611–619 (1991).Google Scholar
  47. 47.
    J. Gomez-Cambronero and R.I. Sha’afi, Granulocyte-macrophage colony-stimulating factor and the neutrophil: mechanism of action. In: Cell-cell interaction in the release of inflammatory mediators. Ed. P. Wong and C.N. Serhan, Plenum Press, 1991.Google Scholar
  48. 48.
    J. Gomez-Cambronero, M. Durstin, T.F.P. Molski, P.H. Naccache and R.I. Sha’afi, Calcium is nesessary but not sufficient for the platelet-activating factor release in human neutrophils stimulated by physiological stimuli. Role of G proteins. J. Biol. Chem. 264: 21699–21704 (1989).Google Scholar

Copyright information

© Springer Science+Business Media New York 1991

Authors and Affiliations

  • Julian Gomez-Cambronero
    • 1
  1. 1.Department of PhysiologyUniversity of Connecticut Health CenterFarmingtonUSA

Personalised recommendations