Deciphering opsonized zymosan-induced phosphorylation of p47phox and NADPH oxidase activation in human neutrophils Available to Purchase

• • Serum-opsonized zymosan induces a rapid phosphorylation / dephosphorylation of p47phox on Ser304, Ser315, Ser320 and Ser328

• • The membrane receptors FcgR and CR3 induce the phosphorylation of p47phox involving Src/Syk, PI3K, PLC, PLD, Ca++ and PKCβ2

Neutrophils play a key role in innate immunity by killing microbes through phagocytosis and superoxide anion production by the phagocyte NADPH oxidase. The signaling pathways regulating NADPH oxidase activation in neutrophils have been extensively studied using soluble agonists, but are less understood during phagocytosis, a fundamental function of neutrophils. The aim of this study was to investigate the phosphorylation of the cytosolic NADPH oxidase protein p47phox in human neutrophils stimulated by serum-opsonized zymosan (OZ), which induces phagocytosis, using antibodies against phosphorylated sites. The results show that OZ induced rapid phosphorylation of p47phox on Ser304, Ser315, Ser320 and Ser328, followed by rapid dephosphorylation. Interestingly, despite the transient nature of p47phox phosphorylation, OZ-induced NADPH oxidase activity was sustained for a longer period of time in intact cells and in isolated membranes. OZ-induced p47phox phosphorylation at these sites was concentration dependent and preceded particle ingestion. IgG- and C3bi-opsonised zymosan similarly induced rapid phosphorylation and dephosphorylation of p47phox on Ser304, Ser315, Ser320 and Ser328, suggesting that FcγR and CR3 are involved in this process. Inhibitors of Src and Syk tyrosine kinases, PI3K, PLC, PLD, Ca++ and PKCβ2 inhibited OZ-induced phosphorylation of p47phox at these serine residues. These results suggest that: 1)-OZ-induced p47phox phosphorylation on Ser304, Ser315, Ser320 and Ser328 is required for the initiation of NADPH oxidase activation but not for its maintenance during phagocytosis; 2)-the membrane receptors FcγR and CR3 mediate this phosphorylation; and 3)-Src and Syk tyrosine kinases, PI3K, PLD, Ca++ and PKCβ2 control the phosphorylation of p47phox during phagocytosis.