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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 2

Use of an antibody to phospho-Ser345 demonstrates that GM-CSF and TNF-α induce phosphorylation of p47phox on Ser345 in a concentration- and time-dependent manner.

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                  Use of an antibody to phospho-Ser345 demonstrates tha...
(A) Neutrophils (1 × 107 cells/ml) were incubated with various concentrations of GM-CSF for 20 minutes or were incubated with GM-CSF (12.5 ng/ml) for the times indicated. Cells were lysed, and proteins from 4 × 105 cells were analyzed with SDS-PAGE and immunoblotting with anti–phospho-Ser345 antibody (p-Ser345) or anti-p47phox antibody (p47phox). (B) Neutrophils (1 × 107 cells/ml) were incubated with various concentrations of TNF-α for 20 minutes or were incubated with TNF-α (10 ng/ml) for the times indicated. Cells were lysed, and proteins from 4 × 105 cells were analyzed with SDS-PAGE and immunoblotting with anti–phospho-Ser345 antibody or anti-p47phox antibody. Western blots from different experiments were scanned; phosphorylated and total p47phox were quantified by densitometry; and the intensity of phosphorylated p47phox was corrected for the amount of p47phox. Results are expressed as mean ± SEM (n = 3). *P < 0.05 compared with untreated neutrophils or time 0 minutes.

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

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