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

Objectives-To define the mechanism of p47 phox phosphorylation in regulating endothelial cell response to tumor necrosis factor-α (TNFα) stimulation. Methods and 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. Conclusion-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. © 2012 American Heart Association, Inc.