title: Stimulation of human neutrophils with formyl-methionyl-leucyl-phenylalanine induces tyrosine phosphorylation and activation of two distinct mitogen-activated protein-kinases.
abstract: Stimulation of human polymorphonuclear neutrophils with the chemotactic peptide FMLP induces an increase in the phosphorylation of several proteins on tyrosine residues. Immunoblotting of whole cell lysates with antiphosphotyrosine antibodies demonstrated this increase to be predominant in two proteins with apparent m.w. of 40,000 and 42,000, respectively. We identify these two proteins as members of the mitogen-activated-protein (MAP) kinase family. These results are based on comigration of the tyrosine phosphorylated proteins with two proteins of same apparent m.w., recognized by a series of antibodies raised against sequences contained within members of this family. This was further confirmed by immunoprecipitation with either an antiphosphotyrosine antibody or a MAP kinase-specific antibody. To assess whether neutrophil stimulation with FMLP increased MAP kinase activity, the cells were stimulated with various doses of FMLP and the phosphotransferase activity in cell lysates was measured against two identified substrates of MAP kinase: myelin basic protein and T-669, the epidermal growth factor receptor Thr 669 synthetic peptide modeled after the major site of phosphorylation of the receptor. A dose- and time-dependent increase in MAP kinase activity was detected in the lysates from FMLP-stimulated PMN, which closely correlated with the dose and time course of tyrosine phosphorylation of the 40- and 42-kDa proteins. Additionally, the MAP kinases in crude lysates were enriched by DEAE-cellulose chromatography and further resolved on a MonoQ column by fast performance liquid chromatography. The respective eluates contained the two proteins, each recognized by anti-MAP kinase antibodies and, after stimulation, each was phosphorylated on tyrosine residues and expressed T-669 phosphotransferase activity. Treatment with genistein, a tyrosine kinase inhibitor, reduced the tyrosine phosphorylation of the 40- and 42-kDa proteins in a dose-dependent manner, decreased the activation of the MAP kinases, and inhibited the production of superoxide anion by FMLP-stimulated neutrophils. Therefore, this study demonstrates that human polymorphonuclear neutrophils contain two distinct members of the MAP kinase family, antigenically related to sea star p44mpk and rat p43erk1 and that both MAP kinase isoforms are implicated in the cascade of protein phosphorylation induced by neutrophil stimulation with FMLP. Furthermore, these data suggest that tyrosine phosphorylation and activation of these two enzymes may play a regulatory role in the signal transduction pathway leading to the respiratory burst induced by FMLP.
authors.affiliation: Department of Pediatrics, Childrens Hospital Los Angeles, University of Southern California, School of Medicine 90027.
authors.forename: M
authors.initials: M
authors.lastname: Torres
authors.forename: F L
authors.initials: FL
authors.lastname: Hall
authors.forename: K
authors.initials: K
authors.lastname: O'Neill
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002451:Cell Compartmentation; D004789:Enzyme Activation; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D019833:Genistein; D006801:Humans; D007529:Isoflavones / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D016897:Respiratory Burst / Q000187:drug effects; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D007529:Isoflavones; D019000:Phosphotyrosine; D014443:Tyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019833:Genistein; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1