Subcellular targeting of oxidants during endothelial cell migration.
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 p47(phox) 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.
Wu
Ru Feng
RF
University of Texas Southwestern, Dallas, TX 75390, USA.
Xu
You Cheng
YC
Ma
Zhenyi
Z
Nwariaku
Fiemu E
FE
Sarosi
George A
GA
Terada
Lance S
LS
D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D042783:Endothelial Cells / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D009154:Mutation; D019255:NADPH Oxidases / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D017027:Protein Tyrosine Phosphatases / Q000502:physiology; D011554:Pseudopodia / Q000502:physiology; D053449:TNF Receptor-Associated Factor 4; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins / Q000235:genetics / Q000502:physiology*; D054462:p21-Activated Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D060588:LIM Domain Proteins; D016877:Oxidants; D010750:Phosphoproteins; C490351:TGFB1I1 protein, human; D053449:TNF Receptor-Associated Factor 4; C506367:TRAF4 protein, human; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C515903:PAK1 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; C516738:PTPN12 protein, human; D054594:Protein Tyrosine Phosphatase, Non-Receptor Type 12; D017027:Protein Tyrosine Phosphatases; D020830:rac1 GTP-Binding Protein