Activation of MEKK by formyl-methionyl-leucyl-phenylalanine in human neutrophils. Mapping pathways for mitogen-activated protein kinase activation

• Published in: The Journal of biological chemistry Vol. 271; no. 52; pp. 33598 - 33606
• Main Authors: Avdi, N J, Winston, B W, Russel, M, Young, S K, Johnson, G L, Worthen, G S
• Format: Journal Article
• Language: English
• Published: United States 27-12-1996
• Subjects: Androstadienes - pharmacology; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Enzyme Activation; Enzyme Inhibitors - pharmacology; Flavonoids - pharmacology; Humans; Indoles - pharmacology; Maleimides - pharmacology; MAP Kinase Kinase Kinase 1; N-Formylmethionine Leucyl-Phenylalanine - pharmacology; Neutrophils - drug effects; Neutrophils - enzymology; Pertussis Toxin; Phosphatidylinositol 3-Kinases; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Protein Kinase C - metabolism; Protein-Serine-Threonine Kinases - metabolism; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-raf; Recombinant Proteins - metabolism; Virulence Factors, Bordetella - pharmacology; Wortmannin
• ISSN: 0021-9258
• DOI: 10.1074/jbc.271.52.33598

Mechanisms of neutrophil activation in response to chemoattractants remain incompletely understood. We have recently reported a Ras-mediated c-Raf pathway leading to the activation of mitogen-activated protein (MAP) kinase in human neutrophils stimulated with the chemoattractant formyl-Met-Leu-Phe (FMLP). However, concern that Raf activation may not fully account for the early FMLP-mediated human neutrophil responses prompted us to investigate the activation of MAP kinase/ERK kinase (MEK) by MEK kinase (MEKK). In cell lysates we identified protein species at 180, 160, 110, 72, and 54 kDa with a monoclonal antibody to MEKK. Activation of MEKK was determined on immunoprecipitates from FMLP-stimulated neutrophils by in vitro kinase assay, which utilized both MEK1 and MEK2 as substrates. It was rapid, detectable at 30 s and reaching a plateau at 5 min, and it was inhibited in a dose-dependent fashion by a specific phosphatidylinositol 3-kinase inhibitor, wortmannin. Partial inhibition by pertussis toxin was observed. We were unable to show inhibition of the MEKK response by GF 109203X, a protein kinase C-specific inhibitor. These data indicate that in neutrophils activation of MEKK in addition to Raf may underlie stimulation of MAP kinase and other MAP kinase homologues by FMLP.