title: Divergent effects of p47(phox) phosphorylation at S303-4 or S379 on tumor necrosis factor-α signaling via TRAF4 and MAPK in endothelial cells.
abstract: OBJECTIVE
  To define the mechanism of p47(phox) phosphorylation in regulating endothelial cell response to tumor necrosis factor-α (TNFα) stimulation.
  
  
  RESULTS
  We replaced 11 serines (303-4, 310, 315, 320, 328, 345, 348, 359, 370, and 379) with alanines and investigated their effects on TNFα (100 U/mL, 30 minutes)-induced acute O(2)(.-) production and mitogen-activated protein kinase phosphorylation in endothelial cells. Seven constructs, S303-4A (double), S310A, S315A, S328A, S345A, S370A, and S379A, significantly reduced the O(2)(.-) production, and 4 of them (S328A, S345A, S370A, and S379A) also inhibited TNFα-induced extracellular-signal-regulated kinase (ERK) 1/2 phosphorylation. Blocking the phosphorylation of S303-4 and S379 inhibited most effectively TNFα-induced O(2)(.-) production. However, phosphorylation of S303-4 was not required for TNFα-induced p47(phox) membrane translocation and binding to TNF receptor-associated factor 4, ERK1/2 activation, and subsequent vascular cell adhesion molecule-1 expression. Knockout of p47(phox) or knockdown of TNF receptor-associated factor 4 using siRNA abolished TNFα-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 activation significantly reduced the TNFα-induced vascular cell adhesion molecule-1 expression.
  
  
  CONCLUSIONS
  Phosphorylation of p47(phox) at different serine sites plays distinct roles in endothelial cell response to TNFα stimulation. Double serine (S303-4) phosphorylation is crucial for acute O(2)(.-) production, but is not involved in TNFα signaling through TNF receptor-associated factor 4 and ERK1/2. p47(phox) requires serine phosphorylation at distinct sites to support specific signaling events in response to TNFα.
authors.affiliation: Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
authors.forename: Lei
authors.initials: L
authors.lastname: Teng
authors.forename: Lampson M
authors.initials: LM
authors.lastname: Fan
authors.forename: Daniel
authors.initials: D
authors.lastname: Meijles
authors.forename: Jian-Mei
authors.initials: JM
authors.lastname: Li
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D042783:Endothelial Cells / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D020935:MAP Kinase Signaling System; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D034622:RNA Interference; D012694:Serine; D013481:Superoxides / Q000378:metabolism; D053449:TNF Receptor-Associated Factor 4 / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D053449:TNF Receptor-Associated Factor 4; C506368:Traf4 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D013481:Superoxides; D012694:Serine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases