OBJECTIVE: 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. 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.
OBJECTIVE: 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. 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: Daniel N Meijles; Sanghamitra Sahoo; Imad Al Ghouleh; Jefferson H Amaral; Raquel Bienes-Martinez; Heather E Knupp; Shireen Attaran; John C Sembrat; Seyed M Nouraie; Mauricio M Rojas; Enrico M Novelli; Mark T Gladwin; Jeffrey S Isenberg; Eugenia Cifuentes-Pagano; Patrick J Pagano Journal: Sci Signal Date: 2017-10-17 Impact factor: 8.192
Authors: Daniel N Meijles; Lampson M Fan; Maziah M Ghazaly; Brendan Howlin; Martin Krönke; Gavin Brooks; Jian-Mei Li Journal: Circulation Date: 2016-05-09 Impact factor: 29.690