Subcellular targeting of oxidants during endothelial cell migration

Abstract

Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins controlling cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We found that the nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase adaptor p47phox and its binding partner TRAF4 were sequestered within nascent, focal complexlike structures in the lamellae of motile endothelial cells. TRAF4 directly associated with the focal contact scaffold Hic-5, and the knockdown of either protein, disruption of the complex, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 activated the NADPH oxidase downstream of the Rho GTPases and p21-activated kinase 1 (PAK1) and oxidatively modified the focal contact phosphatase PTP-PEST. The oxidase also functioned upstream of Rac1 activation, suggesting its participation in a positive feedback loop. Active TRAF4 initiated robust membrane ruffling through Rac1, PAK1, and the oxidase, whereas the knockdown of PTP-PEST increased ruffling independent of oxidase activation. Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.