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

J Clin Invest. 2006 Jul;116(7):2033-43. doi: 10.1172/JCI27544. Epub 2006 Jun 15.


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-alpha prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem mass spectrometry to show that GM-CSF and TNF-alpha 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-alpha-induced phosphorylation of Ser345. Transfection of HL-60 cells with a mutated p47phox (S345A) inhibited GM-CSF- and TNF-alpha-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-alpha-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arthritis, Rheumatoid / immunology
  • Cell Line
  • Enzyme Activation
  • Enzyme Inhibitors / metabolism
  • Genistein / metabolism
  • Granulocyte-Macrophage Colony-Stimulating Factor / metabolism
  • Humans
  • Inflammation / metabolism*
  • Mitogen-Activated Protein Kinases / antagonists & inhibitors
  • Mitogen-Activated Protein Kinases / metabolism*
  • Molecular Sequence Data
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • Neutrophils / enzymology*
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / metabolism
  • Phosphorylation
  • Reactive Oxygen Species / metabolism
  • Serine / metabolism*
  • Synovial Fluid / cytology
  • Tumor Necrosis Factor-alpha / metabolism


  • Enzyme Inhibitors
  • Peptides
  • Reactive Oxygen Species
  • Tumor Necrosis Factor-alpha
  • Serine
  • Granulocyte-Macrophage Colony-Stimulating Factor
  • Genistein
  • NADPH Oxidases
  • neutrophil cytosolic factor 1
  • Mitogen-Activated Protein Kinases