Activation of MEKK by formyl-methionyl-leucyl-phenylalanine in human neutrophils. Mapping pathways for mitogen-activated protein kinase activation.
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.
Avdi
N J
NJ
Department of Medicine, University of Colorado School of Medicine, Denver, Colorado 80206, USA.
Winston
B W
BW
Russel
M
M
Young
S K
SK
Johnson
G L
GL
Worthen
G S
GS
D000730:Androstadienes / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D048728:MAP Kinase Kinase Kinase 1; D008301:Maleimides / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology*; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D048728:MAP Kinase Kinase Kinase 1; D008301:Maleimides; D009240:N-Formylmethionine Leucyl-Phenylalanine; D037342:Pertussis Toxin; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D019908:Proto-Oncogene Proteins c-raf; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000077191:Wortmannin; C482576:MAP3K1 protein, human; C070515:bisindolylmaleimide I; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one