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 re main 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 no t fully account for the early FMLP-mediated human neutrophil responses prompted us to investigate the activation of MAP kinase/ERK kinase (M EK) by MEK kinase (MEKK). In cell lysates we identified protein specie s at 180, 160, 110, 72, and 54 kDa with a monoclonal antibody to MEKK, Activation of MEKK was determined on immunoprecipitates from FMLP-sti mulated 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. Par tial inhibition by pertussis toxin was observed. We were unable to sho w inhibition of the MEKK response by GF 109203X, a protein kinase C-sp ecific inhibitor. These data indicate that in neutrophils activation o f MEKK in addition to Raf may under-lie stimulation of MAP kinase and other MAP kinase homologues by FMLP.