Etude du mécanisme d’activation de la NADPH oxydase phagocytaire : analyse de la dynamique de phosphorylation de la p47phox dans le neutrophile activé

Abstract

Activation of the phagocytic NADPH oxidase is known to require absolutly phosphorylation of its subunit, p47phox on several serine residus (Ser303-379) in its C terminus. We developped in this study specific antibodies raised against 8 major phosphorylated human p47phox sites (Ser304, Ser315, Ser320, Ser328, Ser345, Ser359, Ser370 and Ser379) to stringently study this process during activation. The use of these antibodies reveals new findings about p47phox phosphorylation in fMLF and PMA stimulated neutrophils and allowed us to monitor, for the first time, this process in vivo. p47phox is phosphorylated on Ser304, 315, 320 and 328 in a time and dose-dependent manner in fMLF and PMA-stimulated human neutrophils, but Ser345 is not. fMLF-induced phosphorylation on all of these sites is very rapid and transient and is inhibited by genistein, wortmannin and GF109203X. Whereas PMA-induced phosphorylation on these sites is more sustained and is inhibited only by GF109203X. p47phox shows a phosphorylation on serine 359, 370 and 379 in non stimulated cells which is not modified by any activator or inhibitor used. Proteins phosphorylated on serine 304, 315, 320, 328, 345, 359, 370 and 379 are present in the cytosol of fMLF and PMA-stimulated neutrophils. Whereas p47phox present in membranes and granuls of fMLF-stimulated neutrophils is phosphorylated on serine 315, 320, 328 and 370 with serine 304 being slightly phosphorylated in granuls. In membranes and granuls of PMA-stimulated neutrophils, p47phox is phosphorylated on serine 304, 315, 320, 328 and 345. p47phox is also phosphorylated at least on serine 304, 315, 320, 328 and 345 in EBV-transformed B lymphocytes stimulated by pansorbine or PMA. Mutation of p47phox phosphorylation sites to alanine S(303-370)A abolishes ROS production by these cells. During differentiation of HL60 cells, basal phosphorylation of the expressing p47phox on serine 359 and 370 starts on the 3rd day than increases until the last day of cell differentiation, whereas phosphorylation of serine 379 appears on the first day and is more stable. In vivo studies in a carrageenan-induced pleurisy rat model show that Ser304, 320 and 328 are transiently phosphorylated, while Ser315 phosphorylation is sustained. Thus, these antibodies allowed to capture the spatial and temporal phosphorylation of p47phox and to monitor this process in vivo. Phosphorylation of Ser315 may be a good marker to monitor p47phox phophorylation in vivo under a variety of physiological conditions.