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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 oxidatively modifies the focal contact phosphatase PTP-PEST through the NADPH oxidase. (A) Phoenix-293 cells were transfected with either empty vector or Myr-TRAF4. In top panels, cells were cotransfected with Flag–PTP-PEST. p67(V204A) was adenovirally transduced. Lysates were labeled with 5-IAF, and the indicated PTPs were immunoprecipitated and immunoblotted for fluorescein to detect 5-IAF labeling and for the respective PTPs to assess capture. Myr-TRAF4 decreased 5-IAF labeling of PTP-PEST, indicating oxidative modification of the active site cysteine. 5-IAF labeling of MKP-1 and SHP-2 was unaffected. Error bars represent SEM. (B) Immunofluorescent images were obtained with TIRF microscopy showing colocalization of p47phox, TRAF4, and Hic-5 (AlexaFluor488 or 633) with phosphotyrosinylated proteins (AlexaFluor555) within peripheral dots at the edges of protrusions. In bottom panels, endogenous TRAF or Hic-5 were knocked down in HUVECs as indicated. Bars, 10 μm. (C) HUVECs were transfected with the indicated plasmids and infected with the indicated Ad viruses. 200 μM MnTMPyP was present for 3 h before lysis. Rac1 GTP loading was assessed by binding to GST-CRIB. Both MnTMPyP and p67(V204A) blocked Myr-TRAF4–induced Rac1 activation.
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fig7: Myr-TRAF4 oxidatively modifies the focal contact phosphatase PTP-PEST through the NADPH oxidase. (A) Phoenix-293 cells were transfected with either empty vector or Myr-TRAF4. In top panels, cells were cotransfected with Flag–PTP-PEST. p67(V204A) was adenovirally transduced. Lysates were labeled with 5-IAF, and the indicated PTPs were immunoprecipitated and immunoblotted for fluorescein to detect 5-IAF labeling and for the respective PTPs to assess capture. Myr-TRAF4 decreased 5-IAF labeling of PTP-PEST, indicating oxidative modification of the active site cysteine. 5-IAF labeling of MKP-1 and SHP-2 was unaffected. Error bars represent SEM. (B) Immunofluorescent images were obtained with TIRF microscopy showing colocalization of p47phox, TRAF4, and Hic-5 (AlexaFluor488 or 633) with phosphotyrosinylated proteins (AlexaFluor555) within peripheral dots at the edges of protrusions. In bottom panels, endogenous TRAF or Hic-5 were knocked down in HUVECs as indicated. Bars, 10 μm. (C) HUVECs were transfected with the indicated plasmids and infected with the indicated Ad viruses. 200 μM MnTMPyP was present for 3 h before lysis. Rac1 GTP loading was assessed by binding to GST-CRIB. Both MnTMPyP and p67(V204A) blocked Myr-TRAF4–induced Rac1 activation.

Mentions: A prominent and perhaps central mechanism by which oxidants exert their effects on signaling pathways is through oxidative modification of the conserved catalytic cysteine of protein tyrosine phosphatases, rendering them inactive (Meng et al., 2002). PTP-PEST represents a potential oxidant target because it binds Hic-5, targets nascent focal contacts, blocks integrin-dependent Rac1 activation, and modulates cell migration (Angers-Loustau et al., 1999; Nishiya et al., 1999; Sastry et al., 2002). Therefore, we examined oxidative inactivation of PTP-PEST by assessing its covalent labeling with 5′-iodoacetamidofluorescein (5-IAF), which is known to selectively react with the acidic catalytic cysteine thiol of active PTPs (Wu et al., 1998). We found that Myr-TRAF4 increased oxidative modification (decreased 5-IAF labeling) of the acidic cysteine of coexpressed PTP-PEST in Phoenix-293 cells (Fig. 7 A). The expression of p67(V204A) completely prevented this oxidative modification, which is consistent with the predominant effect of the NADPH oxidase on PTP-PEST.


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 oxidatively modifies the focal contact phosphatase PTP-PEST through the NADPH oxidase. (A) Phoenix-293 cells were transfected with either empty vector or Myr-TRAF4. In top panels, cells were cotransfected with Flag–PTP-PEST. p67(V204A) was adenovirally transduced. Lysates were labeled with 5-IAF, and the indicated PTPs were immunoprecipitated and immunoblotted for fluorescein to detect 5-IAF labeling and for the respective PTPs to assess capture. Myr-TRAF4 decreased 5-IAF labeling of PTP-PEST, indicating oxidative modification of the active site cysteine. 5-IAF labeling of MKP-1 and SHP-2 was unaffected. Error bars represent SEM. (B) Immunofluorescent images were obtained with TIRF microscopy showing colocalization of p47phox, TRAF4, and Hic-5 (AlexaFluor488 or 633) with phosphotyrosinylated proteins (AlexaFluor555) within peripheral dots at the edges of protrusions. In bottom panels, endogenous TRAF or Hic-5 were knocked down in HUVECs as indicated. Bars, 10 μm. (C) HUVECs were transfected with the indicated plasmids and infected with the indicated Ad viruses. 200 μM MnTMPyP was present for 3 h before lysis. Rac1 GTP loading was assessed by binding to GST-CRIB. Both MnTMPyP and p67(V204A) blocked Myr-TRAF4–induced Rac1 activation.
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Related In: Results  -  Collection

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fig7: Myr-TRAF4 oxidatively modifies the focal contact phosphatase PTP-PEST through the NADPH oxidase. (A) Phoenix-293 cells were transfected with either empty vector or Myr-TRAF4. In top panels, cells were cotransfected with Flag–PTP-PEST. p67(V204A) was adenovirally transduced. Lysates were labeled with 5-IAF, and the indicated PTPs were immunoprecipitated and immunoblotted for fluorescein to detect 5-IAF labeling and for the respective PTPs to assess capture. Myr-TRAF4 decreased 5-IAF labeling of PTP-PEST, indicating oxidative modification of the active site cysteine. 5-IAF labeling of MKP-1 and SHP-2 was unaffected. Error bars represent SEM. (B) Immunofluorescent images were obtained with TIRF microscopy showing colocalization of p47phox, TRAF4, and Hic-5 (AlexaFluor488 or 633) with phosphotyrosinylated proteins (AlexaFluor555) within peripheral dots at the edges of protrusions. In bottom panels, endogenous TRAF or Hic-5 were knocked down in HUVECs as indicated. Bars, 10 μm. (C) HUVECs were transfected with the indicated plasmids and infected with the indicated Ad viruses. 200 μM MnTMPyP was present for 3 h before lysis. Rac1 GTP loading was assessed by binding to GST-CRIB. Both MnTMPyP and p67(V204A) blocked Myr-TRAF4–induced Rac1 activation.
Mentions: A prominent and perhaps central mechanism by which oxidants exert their effects on signaling pathways is through oxidative modification of the conserved catalytic cysteine of protein tyrosine phosphatases, rendering them inactive (Meng et al., 2002). PTP-PEST represents a potential oxidant target because it binds Hic-5, targets nascent focal contacts, blocks integrin-dependent Rac1 activation, and modulates cell migration (Angers-Loustau et al., 1999; Nishiya et al., 1999; Sastry et al., 2002). Therefore, we examined oxidative inactivation of PTP-PEST by assessing its covalent labeling with 5′-iodoacetamidofluorescein (5-IAF), which is known to selectively react with the acidic catalytic cysteine thiol of active PTPs (Wu et al., 1998). We found that Myr-TRAF4 increased oxidative modification (decreased 5-IAF labeling) of the acidic cysteine of coexpressed PTP-PEST in Phoenix-293 cells (Fig. 7 A). The expression of p67(V204A) completely prevented this oxidative modification, which is consistent with the predominant effect of the NADPH oxidase on PTP-PEST.

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