Cross Talk between IRAK4 and the NADPH Oxidase.

The ability of human neutrophils to combat bacterial and fungal infections depends on their production of reactive oxygen species. The enzymatic complex NADPH oxidase which is responsible for the generation of superoxide anion, is essential for the microbicidal activity of neutrophils, since patients with chronic granulomatous disease, whose NADPH oxidase is inactive, suffer recurrent infections. The exposure of neutrophils to lipopolysaccharide (LPS) amplifies their oxidative response to formylated-peptides (priming). However, the relationship between the signaling downstream of the toll-like receptor 4 after LPS stimulation and the activation of the oxidase has not been elucidated. The phosphorylation of the NADPH oxidase cytosolic factor p47phox is an essential step during the oxidase activation. Here, we test the hypothesis that Interleukin-1 receptor-associated kinase-4 (IRAK4) regulates the NADPH oxidase through phosphorylation of p47phox. We first show that p47phox is a substrate for IRAK4. We showed that IRAK4-phosphorylated p47phox could be subsequently phosphorylated by PKC suggesting that they phosphorylate p47phox at different residues. IRAK4 phosphorylated p47phox to a similar extent as PKC, however, while p47phox was phosphorylated by PKC only in serines, IRAK4 phosphorylated p47phox also in threonines as determined by two-dimensional electrophoresis. Importantly, IRAK4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system (significantly different from the unphosphorylated control, p<0.05). Furthermore, cophosphorylation of p47phox by IRAK4 and PKC significantly potentiated the activity of the NADPH oxidase when compared to PKC phosphorylation alone (p<0.01). Finally, we identified, by mass spectrometry, the residues in p47phox that are targets for IRAK4 phosphorylation. We found that IRAK4 phosphorylates p47phox at a threonine rich domain which possibly constitutes a novel regulatory domain.