TRAF4 directs oxidants to focal complexes

Endogenous oxidants participate in endothelial cell migration, suggesting that the enzymatic source of oxidants, like other proteins known to control cell migration, requires precise subcellular localization for spatial confinement of signaling effects. We previously found that the NADPH oxidase adapter p47 phox binds TRAF4, an orphan adapter whose congenital absence impairs ontogenic migration. Here, we report that both p47 phox and TRAF4 sequester within nascent focal complex‐like structures in lamellae of motile endothelial cells. We recovered the focal contact scaffold Hic‐5 as a TRAF4 binding partner from a yeast two‐hybrid screen, and knockdown of either protein, disruption of the complex through ectopic expression of truncation mutants, or oxidant scavenging blocked cell migration. An active mutant of TRAF4 increased GTP loading of the Rho GTPases Cdc42, Rac1, and RhoA, activated the NADPH oxidase downstream of the Cdc42/Rac1 effector PAK1, and oxidatively modified the Hic‐5‐associated focal contact tyrosine phosphatase, PTP‐PEST. Active TRAF4 stimulated robust membrane ruffling through Rac1, PAK1, the NADPH oxidase, and the tyrosine kinases c‐Src and Pyk2, while 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. Supported in part by the NHLBI, the NIGMS, the Veterans Administration, and the Robert Wood Johnson Foundation.