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A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites
Pham My-Chan Dang, … , Marie-Anne Gougerot-Pocidalo, Jamel El-Benna
Pham My-Chan Dang, … , Marie-Anne Gougerot-Pocidalo, Jamel El-Benna
Published July 3, 2006
Citation Information: J Clin Invest. 2006;116(7):2033-2043. https://doi.org/10.1172/JCI27544.
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Research Article Immunology

A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites

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Abstract

Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-α prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem mass spectrometry to show that GM-CSF and TNF-α induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM-CSF–induced phosphorylation of Ser345, while p38 MAPK inhibitor abrogated TNF-α–induced phosphorylation of Ser345. Transfection of HL-60 cells with a mutated p47phox (S345A) inhibited GM-CSF– and TNF-α–induced priming of ROS production. This event was also inhibited in neutrophils by a cell-permeable peptide containing a TAT-p47phox-Ser345 sequence. Furthermore, ROS generation, p47phox-Ser345 phosphorylation, and ERK1/2 and p38 MAPK phosphorylation were increased in synovial neutrophils from rheumatoid arthritis (RA) patients, and TAT-Ser345 peptide inhibited ROS production by these primed neutrophils. This study therefore identifies convergent MAPK pathways on Ser345 that are involved in GM-CSF– and TNF-α–induced priming of neutrophils and are activated in RA. Inhibition of the point of convergence of these pathways might serve as a novel antiinflammatory strategy.

Authors

Pham My-Chan Dang, Allan Stensballe, Tarek Boussetta, Houssam Raad, Cedric Dewas, Yolande Kroviarski, Gilles Hayem, Ole N. Jensen, Marie-Anne Gougerot-Pocidalo, Jamel El-Benna

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Figure 1

Mass spectrometry analysis of GM-CSF–induced p47phox phosphorylation: Ser345 was phosphorylated in primed human neutrophils.

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                  Mass spectrometry analysis of GM-CSF–induced p47phox
...
(A) LC-MS/MS analysis of an aliquot of the p47phox tryptic peptide mixture (gradient: 0–38% acetonitrile in 35 minutes). The 2 upper panels depict the base peak chromatograms of electrospray ionization MS/MS experiments performed during the analysis in data-dependent acquisition mode. The lower panel represents the base peak chromatogram of the MS survey scans only. The arrow indicates the elution time of the phosphopeptide that was sequenced by MS/MS. (B) Identification of the phosphorylated peptide QARPGPQ[pS]PGSPLEEER (amino acids 338–354). The MS/MS spectrum displayed a near-complete y-ion series that reflects the amino acid sequence and the position of the phosphate group at Ser345. The elimination of phosphoric acid from the phosphoserine residue during MS/MS generated a dehydroalanine residue at the position corresponding to Ser345 (s; residue mass, 69 Da). The spectrum also demonstrated that the Ser348 residue (S) was not phosphorylated (residue mass, 87 Da).

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ISSN: 0021-9738 (print), 1558-8238 (online)

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