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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 induces marked membrane ruffling through Rac1, PAK1, and the NADPH oxidase. (A) HUVECs were cotransfected with either empty vector or Myr-TRAF4 and either actin-GFP or DsRed-p47. Myr-TRAF4–induced ruffling is seen on multiple edges of cotransfected cells (middle). Ruffles contained DsRed-p47 (right). (B and C) HUVECs were cotransfected with actin-GFP and either empty vector (first bar) or Myr-TRAF4 and additionally with one of the indicated plasmids. PAK1(K298A) and p67(V204A) were expressed via adenoviral transduction. 100 μM MnTMPyP was added for 1 h. Inhibition of PAK1, the NADPH oxidase, Pyk2, or Src decreased the percentage of cells ruffling in response to Myr-TRAF4. *, P < 0.05 compared with vector control; †, P < 0.05 compared with Myr-TRAF4 alone. (D) HUVECs were transfected with siRNA against PTP-PEST. Immunoblot shows protein levels after 48 h. PTP-PEST knockdown increased the percentage of ruffling cells. *, P < 0.05 compared with control siRNA. Expression of p67(V204A) had no effect on the ruffling rate in PTP-PEST knockdown cells. Examples are shown below. Error bars represent SEM. Bars, 20 μm.
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fig8: Myr-TRAF4 induces marked membrane ruffling through Rac1, PAK1, and the NADPH oxidase. (A) HUVECs were cotransfected with either empty vector or Myr-TRAF4 and either actin-GFP or DsRed-p47. Myr-TRAF4–induced ruffling is seen on multiple edges of cotransfected cells (middle). Ruffles contained DsRed-p47 (right). (B and C) HUVECs were cotransfected with actin-GFP and either empty vector (first bar) or Myr-TRAF4 and additionally with one of the indicated plasmids. PAK1(K298A) and p67(V204A) were expressed via adenoviral transduction. 100 μM MnTMPyP was added for 1 h. Inhibition of PAK1, the NADPH oxidase, Pyk2, or Src decreased the percentage of cells ruffling in response to Myr-TRAF4. *, P < 0.05 compared with vector control; †, P < 0.05 compared with Myr-TRAF4 alone. (D) HUVECs were transfected with siRNA against PTP-PEST. Immunoblot shows protein levels after 48 h. PTP-PEST knockdown increased the percentage of ruffling cells. *, P < 0.05 compared with control siRNA. Expression of p67(V204A) had no effect on the ruffling rate in PTP-PEST knockdown cells. Examples are shown below. Error bars represent SEM. Bars, 20 μm.

Mentions: The expression of Myr-TRAF4 by endothelial cells caused dramatic membrane ruffling on multiple edges with a concentration of DsRed-p47 within ruffles, suggesting diffuse constitutive activation of Rac1 and PAK1 and high focal complex turnover (Fig. 8 A). Extensive lamella formation was occasionally seen in conjunction with ruffling (not depicted). As expected, Rac1(N17) diminished ruffling to basal levels (Fig. 8 B). In addition, kinase-inactive PAK1(K298A) and wild-type PID (but not the inactive PID[LF] mutant peptide) also blocked Myr-TRAF4–induced ruffling (Fig. 8 B). Finally, both MnTMPyP and p67(V204A) reduced Myr-TRAF4–induced ruffling to basal levels (Fig. 8 C). Thus, by morphological as well biochemical criteria, Rac1, PAK1, and the NADPH oxidase act downstream of TRAF4.


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 induces marked membrane ruffling through Rac1, PAK1, and the NADPH oxidase. (A) HUVECs were cotransfected with either empty vector or Myr-TRAF4 and either actin-GFP or DsRed-p47. Myr-TRAF4–induced ruffling is seen on multiple edges of cotransfected cells (middle). Ruffles contained DsRed-p47 (right). (B and C) HUVECs were cotransfected with actin-GFP and either empty vector (first bar) or Myr-TRAF4 and additionally with one of the indicated plasmids. PAK1(K298A) and p67(V204A) were expressed via adenoviral transduction. 100 μM MnTMPyP was added for 1 h. Inhibition of PAK1, the NADPH oxidase, Pyk2, or Src decreased the percentage of cells ruffling in response to Myr-TRAF4. *, P < 0.05 compared with vector control; †, P < 0.05 compared with Myr-TRAF4 alone. (D) HUVECs were transfected with siRNA against PTP-PEST. Immunoblot shows protein levels after 48 h. PTP-PEST knockdown increased the percentage of ruffling cells. *, P < 0.05 compared with control siRNA. Expression of p67(V204A) had no effect on the ruffling rate in PTP-PEST knockdown cells. Examples are shown below. Error bars represent SEM. Bars, 20 μm.
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fig8: Myr-TRAF4 induces marked membrane ruffling through Rac1, PAK1, and the NADPH oxidase. (A) HUVECs were cotransfected with either empty vector or Myr-TRAF4 and either actin-GFP or DsRed-p47. Myr-TRAF4–induced ruffling is seen on multiple edges of cotransfected cells (middle). Ruffles contained DsRed-p47 (right). (B and C) HUVECs were cotransfected with actin-GFP and either empty vector (first bar) or Myr-TRAF4 and additionally with one of the indicated plasmids. PAK1(K298A) and p67(V204A) were expressed via adenoviral transduction. 100 μM MnTMPyP was added for 1 h. Inhibition of PAK1, the NADPH oxidase, Pyk2, or Src decreased the percentage of cells ruffling in response to Myr-TRAF4. *, P < 0.05 compared with vector control; †, P < 0.05 compared with Myr-TRAF4 alone. (D) HUVECs were transfected with siRNA against PTP-PEST. Immunoblot shows protein levels after 48 h. PTP-PEST knockdown increased the percentage of ruffling cells. *, P < 0.05 compared with control siRNA. Expression of p67(V204A) had no effect on the ruffling rate in PTP-PEST knockdown cells. Examples are shown below. Error bars represent SEM. Bars, 20 μm.
Mentions: The expression of Myr-TRAF4 by endothelial cells caused dramatic membrane ruffling on multiple edges with a concentration of DsRed-p47 within ruffles, suggesting diffuse constitutive activation of Rac1 and PAK1 and high focal complex turnover (Fig. 8 A). Extensive lamella formation was occasionally seen in conjunction with ruffling (not depicted). As expected, Rac1(N17) diminished ruffling to basal levels (Fig. 8 B). In addition, kinase-inactive PAK1(K298A) and wild-type PID (but not the inactive PID[LF] mutant peptide) also blocked Myr-TRAF4–induced ruffling (Fig. 8 B). Finally, both MnTMPyP and p67(V204A) reduced Myr-TRAF4–induced ruffling to basal levels (Fig. 8 C). Thus, by morphological as well biochemical criteria, Rac1, PAK1, and the NADPH oxidase act downstream of TRAF4.

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