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.