The Mitogen-Activated Protein Kinase Extracellular Signal-Regulated Kinase 1/2 Pathway Is Involved in formyl-Methionyl-Leucyl-Phenylalanine-Induced p47phox Phosphorylation in Human Neutrophils

Abstract

Phosphorylation of p47 phagocyte oxidase, (p47phox), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47phox is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47phox phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47phox. However, PD98059 weakly affected PMA-induced p47phox phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47phox induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47phox phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47phox phosphorylation either directly or indirectly via other kinases. In PMA-induced p47phox phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47phox phosphorylation. However, in fMLP-induced p47phox phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47phox when tested alone, and exerted additive inhibitory effects on p47phox phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47phox. Furthermore, they strongly suggest that p47phox is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.