CHEMOTACTIC PEPTIDE-INDUCED ACTIVATION OF MEK-2, THE PREDOMINANT ISOFORM IN HUMAN NEUTROPHILS - INHIBITION BY WORTMANNIN

Exposure of neutrophils to a variety of agonists including chemoattrac tant peptides and cytokines induces degranulation and activation of th e oxidative burst which are required for bacterial killing, The signal ing pathways regulating these important functions are incompletely cha racterized, Mitogen-activated protein (MAP) kinases, which include the extracellular signal-regulated kinases (ERKs), are activated rapidly in neutrophils, suggesting that they may regulate cell activation, We found that neutrophils express two isoforms of MAP/ERK kinase (MEK), m ixed-function kinases that are responsible for phosphorylation and act ivation of ERK. Like MEK-1, MEK-2 was found to reside in the cytosol b oth before and after stimulation, Studies were undertaken to define th e relative abundance and functional contribution of MEK-1 and MEK-2 in neutrophils and to characterize the signaling pathways leading to the ir activation, Although the abundance of the two isoforms was similar, the activity of MEK-2 was at least 3-fold greater than that of MEK-1, A rise in cytosolic [Ca2+] was insufficient for MEK stimulation, and blunting the [Ca2+] change with intracellular chelators failed to prev ent receptor-mediated activation of either isoform, implying that cyto solic Ca2+ transients are not necessary. In contrast, both MEK-1 and M EK-2 were activated by exposure of cells to protein kinase C (PKC) ago nists. Conversely, PKC antagonists inhibited the chemotactic stimulati on of both isoforms, suggesting that PKC was required for their activa tion, Despite these similarities, clear differences were also found in the pathways leading to activation of the MEK isoforms, In particular , MEK-2 was considerably more sensitive than MEK-1 to the phosphatidyl inositol 3-kinase inhibitor wortmannin, Phosphorylation and activation of ERK-1 and ERK-2 were also reduced by this inhibitor, In summary, M EK-2 is stimulated in formyl-methionyl-leucyl-phenylalanine-treated ne utrophils, where it appears to be functionally the predominant isoform , The time course and inhibitor sensitivity of MEK-2 activation parall el those of several components of the microbicidal response, suggestin g a signaling role of the MEK-ERK pathway.