Divergent Effects of p47 ^phox Phosphorylation at S303-4 or S379 on Tumor Necrosis Factor-α Signaling via TRAF4 and MAPK in Endothelial Cells

<jats:sec> <jats:title>Objectives—</jats:title> <jats:p> To define the mechanism of p47 <jats:sup>phox</jats:sup> phosphorylation in regulating endothelial cell response to tumor necrosis factor-α (TNFα) stimulation. </jats:p> </jats:sec> <jats:sec> <jats:title>Methods and Results—</jats:title> <jats:p> 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 <jats:sub>2</jats:sub> <jats:sup>.−</jats:sup> 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 <jats:sub>2</jats:sub> <jats:sup>.−</jats:sup> 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 <jats:sub>2</jats:sub> <jats:sup>.−</jats:sup> production. However, phosphorylation of S303-4 was not required for TNFα-induced p47 <jats:sup>phox</jats:sup> membrane translocation and binding to TNF receptor–associated factor 4, ERK1/2 activation, and subsequent vascular cell adhesion molecule-1 expression. Knockout of p47 <jats:sup>phox</jats:sup> 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. </jats:p> </jats:sec> <jats:sec> <jats:title>Conclusion—</jats:title> <jats:p> Phosphorylation of p47 <jats:sup>phox</jats:sup> at different serine sites plays distinct roles in endothelial cell response to TNFα stimulation. Double serine (S303-4) phosphorylation is crucial for acute O <jats:sub>2</jats:sub> <jats:sup>.−</jats:sup> production, but is not involved in TNFα signaling through TNF receptor–associated factor 4 and ERK1/2. p47 <jats:sup>phox</jats:sup> requires serine phosphorylation at distinct sites to support specific signaling events in response to TNFα. </jats:p> </jats:sec>