Abstract 8711: Double Serine Phosphorylation of P47Phox At 303-304 is Critical for Acute Tnfα-Induced Ros Production but Not for Mapk Activation in Endothelial Cells
p47phox is a major regulatory subunit of Nox2 enzyme, which by generating superoxide plays important role in TNFα-induced endothelial dysfunction. The p47phox has several serines clustered within the carboxyl terminus, and their phosphorylation has been reported to be critical in Nox2 activation and endothelial reactive oxygen species (ROS) production in response to stimulation. To understand the mechanisms of p47phox phosphorylation in mediating acute TNFα redox-signaling, we replaced serines (303-4, 310, 315, 320, 328, 345, 348, 359, 370 and 379) to alanines and transfected mouse microvascular endothelial cells (SVEC4-10) with these constructs. Compared to vector controls, S303-4A (double mutation), S310A, S315A, S328A, S345A, S370A and S379A reduced significantly the TNFα (100 U/mL for 30 min)-induced ROS production (P<0.05), whereas only S328A, S345A, S370A and S379A (but not S303-4A, S310A and S315A) inhibited TNFα-induced ERK1/2 phosphorylation. The different effects of S303-304A on TNFα-induced ROS production and TNFα signaling were further investigated using coronary microvascular endothelial cells isolated from p47phox knockout mice. Compared to the cells transfected with a wild-type p47phox cDNA, S303-4A mutation significantly inhibited (~51+2%) TNFα-induced ROS production, but showed no significant effect on TNFα-induced MAPK activation, p47phox association with TRAF4, p47phox binding to p22phox and p47phox membrane translocation. Knockdown of TRAF4 with siRNA abolished TNFα-induced ERK1/2 phosphorylation in S303-4A transfected p47phoxknockout cells. In conclusion, phosphorylation of p47phox at different serines plays distinct roles in endothelial cell response to acute TNFα stimulation. Double serine phosphorylation (S303 and S304) of p47phox is crucial in acute TNFα-induced ROS production, but not involved in TNFα-signaling through TRAF4 and MAPK in endothelial cells.
- © 2011 by American Heart Association, Inc.