title: Cross-talk between IRAK-4 and the NADPH oxidase.
abstract: Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.
authors.affiliation: Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Pacquelet
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Johnson
authors.forename: Beverly A
authors.initials: BA
authors.lastname: Ellis
authors.forename: Agnieszka A
authors.initials: AA
authors.lastname: Brzezinska
authors.forename: William S
authors.initials: WS
authors.lastname: Lane
authors.forename: Daniela B
authors.initials: DB
authors.lastname: Munafo
authors.forename: Sergio D
authors.initials: SD
authors.lastname: Catz
mesh_terms: D000595:Amino Acid Sequence; D004789:Enzyme Activation; D006801:Humans; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D019255:NADPH Oxidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D010766:Phosphorylation; D015854:Up-Regulation; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
chemical_list: D008070:Lipopolysaccharides; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C507773:IRAK4 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases; D048051:p38 Mitogen-Activated Protein Kinases