Chemotactic peptides induce phosphorylation and activation of MEK-1 in human neutrophils.

Extracellular signal-regulated kinase (Erk) (mitogen-activated protein (MAP) kinase) is rapidly activated when neutrophils are stimulated. Several isoforms of MAP/Erk kinase (MEK), a kinase capable of phosphorylating and activating Erk, have been identified, but their distribution and differential roles in leukocytes are unknown. We studied the effect of chemotactic stimulation on MEK-1, using isoform-specific antibodies. MEK-1 was found to be phosphorylated on serine and threonine residues in unstimulated human neutrophils. Stimulation by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) enhanced serine/threonine phosphorylation of MEK-1, while reducing its electrophoretic mobility. MEK-1 activity, measured as autophosphorylation or as phosphorylation of a glutathione S-transferase-Erk fusion protein, was undetectable in unstimulated cells but became evident after treatment with chemoattractant. Phosphorylation and activation of MEK-1 were rapid and transient, peaking after 1-2 min and returning to base line by 10 min. Experiments using electropermeabilized cells indicated that elevation of cytosolic Ca2+ is not required for activation of MEK-1 by fMLP. Moreover, MEK-1 was not stimulated by either platelet-activating factor or thapsigargin, which increase Ca2+ to levels comparable with those attained in chemoattractant-activated cells. In contrast, activation of MEK-1 was induced by phorbol esters, and the stimulatory effect of fMLP was blocked by an antagonist of protein kinase C. Stimulation of MEK-1 was also blocked by concentrations of erbstatin that prevent the fMLP-induced accumulation of tyrosine-phosphorylated proteins. The data suggest that MEK-1 is largely responsible for the activation of Erk in chemoattractant-stimulated neutrophils and that protein kinase C and/or tyrosine kinases mediate this effect, whereas elevated cytosolic Ca2+ is not essential.