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Subcellular targeting of oxidants during endothelial cell migration.

Wu RF, Xu YC, Ma Z, Nwariaku FE, Sarosi GA, Terada LS - J. Cell Biol. (2005)

Bottom Line: 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.Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Southwestern, Dallas, TX 75390, USA.

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 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.

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Myr-TRAF4 activates the NADPH oxidase through PAK1. (A) Phoenix-293 cells overexpressing wt-p47phox were transfected with the indicated plasmids. Whole cell lysates were immunoblotted for p47phox (pS328) and reprobed for total p47phox, demonstrating phosphorylation of p47(S328) by Myr-TRAF4. (B) HUVECs were cotransfected with DsRed and one of the indicated plasmids and were infected with Ad-lacZ or Ad-p67(V204A). DCF fluorescence of adherent DsRed-expressing cells was quantified by image analysis as described in Oxidant production. Myr-TRAF4–induced oxidant production was blocked by p67(V204A). (C) HUVECs were cotransfected with DsRed and the indicated plasmids, and oxidant production of DsRed-expressing cells was measured without manipulation. The PAK inhibitory domain (PID(wt)) but not its nonbinding mutant (PID(LF)) decreased Myr-TRAF4–induced oxidant production. (B and C) *, P < 0.05 compared with control; †, P < 0.05 compared with Myr-TRAF4 alone. Error bars represent SEM.
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fig6: Myr-TRAF4 activates the NADPH oxidase through PAK1. (A) Phoenix-293 cells overexpressing wt-p47phox were transfected with the indicated plasmids. Whole cell lysates were immunoblotted for p47phox (pS328) and reprobed for total p47phox, demonstrating phosphorylation of p47(S328) by Myr-TRAF4. (B) HUVECs were cotransfected with DsRed and one of the indicated plasmids and were infected with Ad-lacZ or Ad-p67(V204A). DCF fluorescence of adherent DsRed-expressing cells was quantified by image analysis as described in Oxidant production. Myr-TRAF4–induced oxidant production was blocked by p67(V204A). (C) HUVECs were cotransfected with DsRed and the indicated plasmids, and oxidant production of DsRed-expressing cells was measured without manipulation. The PAK inhibitory domain (PID(wt)) but not its nonbinding mutant (PID(LF)) decreased Myr-TRAF4–induced oxidant production. (B and C) *, P < 0.05 compared with control; †, P < 0.05 compared with Myr-TRAF4 alone. Error bars represent SEM.

Mentions: PAK1 not only targets focal complexes (Manser et al., 1998; Zhao et al., 2000) and becomes active within membrane rafts (Krautkramer et al., 2004), but it has been shown to act upstream of the NADPH oxidase in endothelial cells (Wu et al., 2003, 2004). An early event in oxidase activation is the serine phosphorylation of p47phox, which unmasks binding domains (Faust et al., 1995). To assess NADPH oxidase activation, we developed antisera against p47phox phosphorylated on S328, a key phosphorylation site (Ago et al., 1999), and found that the expression of Myr-TRAF4 in Phoenix-293 cells increased p47phox S328 phosphorylation (Fig. 6 A). Accordingly, Myr-TRAF4 increased oxidant production in HUVECs, and this oxidant production was decreased by the NADPH oxidase–specific transdominant antagonist p67(V204A) (Fig. 6 B). Furthermore, oxidant production was completely blocked by the PAK inhibitory domain (PID), which binds and inhibits the PAK kinase domain (Fig. 6 C). In contrast, the PID harboring an equivalent of the PAK1(L107F) mutant, which was predicted to disrupt this interaction (Frost et al., 1998), had no significant effect. Thus, Myr-TRAF4 activates Rac1 and increases oxidant production through PAK1-dependent NADPH oxidase activation.


Subcellular targeting of oxidants during endothelial cell migration.

Wu RF, Xu YC, Ma Z, Nwariaku FE, Sarosi GA, Terada LS - J. Cell Biol. (2005)

Myr-TRAF4 activates the NADPH oxidase through PAK1. (A) Phoenix-293 cells overexpressing wt-p47phox were transfected with the indicated plasmids. Whole cell lysates were immunoblotted for p47phox (pS328) and reprobed for total p47phox, demonstrating phosphorylation of p47(S328) by Myr-TRAF4. (B) HUVECs were cotransfected with DsRed and one of the indicated plasmids and were infected with Ad-lacZ or Ad-p67(V204A). DCF fluorescence of adherent DsRed-expressing cells was quantified by image analysis as described in Oxidant production. Myr-TRAF4–induced oxidant production was blocked by p67(V204A). (C) HUVECs were cotransfected with DsRed and the indicated plasmids, and oxidant production of DsRed-expressing cells was measured without manipulation. The PAK inhibitory domain (PID(wt)) but not its nonbinding mutant (PID(LF)) decreased Myr-TRAF4–induced oxidant production. (B and C) *, P < 0.05 compared with control; †, P < 0.05 compared with Myr-TRAF4 alone. Error bars represent SEM.
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Related In: Results  -  Collection

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fig6: Myr-TRAF4 activates the NADPH oxidase through PAK1. (A) Phoenix-293 cells overexpressing wt-p47phox were transfected with the indicated plasmids. Whole cell lysates were immunoblotted for p47phox (pS328) and reprobed for total p47phox, demonstrating phosphorylation of p47(S328) by Myr-TRAF4. (B) HUVECs were cotransfected with DsRed and one of the indicated plasmids and were infected with Ad-lacZ or Ad-p67(V204A). DCF fluorescence of adherent DsRed-expressing cells was quantified by image analysis as described in Oxidant production. Myr-TRAF4–induced oxidant production was blocked by p67(V204A). (C) HUVECs were cotransfected with DsRed and the indicated plasmids, and oxidant production of DsRed-expressing cells was measured without manipulation. The PAK inhibitory domain (PID(wt)) but not its nonbinding mutant (PID(LF)) decreased Myr-TRAF4–induced oxidant production. (B and C) *, P < 0.05 compared with control; †, P < 0.05 compared with Myr-TRAF4 alone. Error bars represent SEM.
Mentions: PAK1 not only targets focal complexes (Manser et al., 1998; Zhao et al., 2000) and becomes active within membrane rafts (Krautkramer et al., 2004), but it has been shown to act upstream of the NADPH oxidase in endothelial cells (Wu et al., 2003, 2004). An early event in oxidase activation is the serine phosphorylation of p47phox, which unmasks binding domains (Faust et al., 1995). To assess NADPH oxidase activation, we developed antisera against p47phox phosphorylated on S328, a key phosphorylation site (Ago et al., 1999), and found that the expression of Myr-TRAF4 in Phoenix-293 cells increased p47phox S328 phosphorylation (Fig. 6 A). Accordingly, Myr-TRAF4 increased oxidant production in HUVECs, and this oxidant production was decreased by the NADPH oxidase–specific transdominant antagonist p67(V204A) (Fig. 6 B). Furthermore, oxidant production was completely blocked by the PAK inhibitory domain (PID), which binds and inhibits the PAK kinase domain (Fig. 6 C). In contrast, the PID harboring an equivalent of the PAK1(L107F) mutant, which was predicted to disrupt this interaction (Frost et al., 1998), had no significant effect. Thus, Myr-TRAF4 activates Rac1 and increases oxidant production through PAK1-dependent NADPH oxidase activation.

Bottom Line: 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.Our data suggest that TRAF4 specifies a molecular address within focal complexes that is targeted for oxidative modification during cell migration.

View Article: PubMed Central - PubMed

Affiliation: University of Texas Southwestern, Dallas, TX 75390, USA.

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 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.

Show MeSH
Related in: MedlinePlus