Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca2+-depleted human neutrophils

Abstract

Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.

Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella

“…Translocation of p47 phox to the membrane requires phosphorylation [55] of a number of serine sites located in its C-terminus, resulting in exposure of its SH3 domain, which binds directly to the p22 phox subunit of the flavocytochrome [56]. Neutrophils contain five of the eleven isoforms of PKC and there are several lines of evidence supporting a role for protein kinase C (PKC) a, bII, d and z in the phosphorylation of p47 phox [57] with a direct interaction between this substrate and the kinase been observed [58].…”

Tyrosine phosphorylation and subcellular redistribution of p125 ras guanosine triphosphatase‐activating protein in human neutrophils stimulated with FMLP

“…Proteins were electroblotted as previously described [22,33]. The blots were incubated overnight with 2 ~tg/ml anti-phosphotyrosine mAbs 4G10 or 2 Ixg/ml anti-125 GAP polyclonal antibody in blocking buffer [31].…”

Modulation of p47 PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

“…In this experiment, the cell-free assay was scaled down to 1 ⁄5 of the total volume while preserving the original concentrations, with the exception of the addition of 5 g of p47 PHOX and the addition of 1 Ci of [␥-32 P]ATP to the assay before activation with NADPH as noted . The identical mobilities of the phosphopeptides indicate that the Cterminal region of the protein (residues 279 -390) is phosphorylated (13,14,16). Fig.…”