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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 Rho family GTPases and PAK1. (A) HUVECs were transfected with empty vector, native TRAF4 (wt), or Myr-TRAF4. After 24 h, cells were lysed, and GTP-loaded Rho proteins were captured with GST-CRIB or GST–Rho-binding domain. Immunoblots for Cdc42, Rac1, and RhoA are shown in lysates and after capture. (B and C) HUVECs were transfected with the indicated vectors, infected with Ad-lacZ, Ad-RhoA(N19), or Ad-Rac1(N17) (MOI = 100:1), and RhoA, Rac1, and Cdc42 activity were assessed. Myr-TRAF4 activated Cdc42 and Rac1 upstream of RhoA. (D) HUVECs were transfected with the indicated plasmids and assessed for PAK1 activity by immunoprecipitation kinase. Phosphorylation of myelin basic protein (top) and immunoblot for captured PAK1 (bottom) are shown. Myr-TRAF4 but not wtTRAF4 activated PAK1.
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fig5: Myr-TRAF4 activates Rho family GTPases and PAK1. (A) HUVECs were transfected with empty vector, native TRAF4 (wt), or Myr-TRAF4. After 24 h, cells were lysed, and GTP-loaded Rho proteins were captured with GST-CRIB or GST–Rho-binding domain. Immunoblots for Cdc42, Rac1, and RhoA are shown in lysates and after capture. (B and C) HUVECs were transfected with the indicated vectors, infected with Ad-lacZ, Ad-RhoA(N19), or Ad-Rac1(N17) (MOI = 100:1), and RhoA, Rac1, and Cdc42 activity were assessed. Myr-TRAF4 activated Cdc42 and Rac1 upstream of RhoA. (D) HUVECs were transfected with the indicated plasmids and assessed for PAK1 activity by immunoprecipitation kinase. Phosphorylation of myelin basic protein (top) and immunoblot for captured PAK1 (bottom) are shown. Myr-TRAF4 but not wtTRAF4 activated PAK1.

Mentions: The Rho family of small GTPases associate avidly with raft domains upon activation (del Pozo et al., 2004) and control focal complex dynamics, migration, and NADPH oxidase activation. Using GST fusions of the PAK1 Cdc42–Rac1 interaction binding (CRIB) domain or the rhotekin Rho-binding domain in pull-down assays, we found that Myr-TRAF4 stimulated GTP loading of Cdc42, Rac1, and RhoA (Fig. 5 A). Furthermore, Rac1(N17) diminished Myr-TRAF4–induced RhoA activation, whereas RhoA(N19) had no effect on Rac1 or Cdc42 activation (Fig. 5, B and C). Thus, TRAF4 activates Cdc42 and Rac1 upstream of RhoA, which is consistent with the sequence of Rho GTPase activation during spontaneous focal contact formation after matrix attachment (Nobes and Hall, 1995). Confirming the activation of Rho GTPases in vivo, Myr-TRAF4 constitutively activated the Cdc42 and Rac1 effector PAK1 (Fig. 5 D). In some cells (presumably high transgene expressors), actin cytoskeletal collapse was noted (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200507004/DC1), which is a morphology resulting from the expression of active PAK1 mutants (Manser et al., 1997) or agonist-dependent PAK1 activation (Wu et al., 2004). Indeed, adenoviral delivery of kinase-dead PAK1(K298A) completely suppressed Myr-TRAF4–induced cytoskeletal remodeling (Fig. S4).


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 Rho family GTPases and PAK1. (A) HUVECs were transfected with empty vector, native TRAF4 (wt), or Myr-TRAF4. After 24 h, cells were lysed, and GTP-loaded Rho proteins were captured with GST-CRIB or GST–Rho-binding domain. Immunoblots for Cdc42, Rac1, and RhoA are shown in lysates and after capture. (B and C) HUVECs were transfected with the indicated vectors, infected with Ad-lacZ, Ad-RhoA(N19), or Ad-Rac1(N17) (MOI = 100:1), and RhoA, Rac1, and Cdc42 activity were assessed. Myr-TRAF4 activated Cdc42 and Rac1 upstream of RhoA. (D) HUVECs were transfected with the indicated plasmids and assessed for PAK1 activity by immunoprecipitation kinase. Phosphorylation of myelin basic protein (top) and immunoblot for captured PAK1 (bottom) are shown. Myr-TRAF4 but not wtTRAF4 activated PAK1.
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fig5: Myr-TRAF4 activates Rho family GTPases and PAK1. (A) HUVECs were transfected with empty vector, native TRAF4 (wt), or Myr-TRAF4. After 24 h, cells were lysed, and GTP-loaded Rho proteins were captured with GST-CRIB or GST–Rho-binding domain. Immunoblots for Cdc42, Rac1, and RhoA are shown in lysates and after capture. (B and C) HUVECs were transfected with the indicated vectors, infected with Ad-lacZ, Ad-RhoA(N19), or Ad-Rac1(N17) (MOI = 100:1), and RhoA, Rac1, and Cdc42 activity were assessed. Myr-TRAF4 activated Cdc42 and Rac1 upstream of RhoA. (D) HUVECs were transfected with the indicated plasmids and assessed for PAK1 activity by immunoprecipitation kinase. Phosphorylation of myelin basic protein (top) and immunoblot for captured PAK1 (bottom) are shown. Myr-TRAF4 but not wtTRAF4 activated PAK1.
Mentions: The Rho family of small GTPases associate avidly with raft domains upon activation (del Pozo et al., 2004) and control focal complex dynamics, migration, and NADPH oxidase activation. Using GST fusions of the PAK1 Cdc42–Rac1 interaction binding (CRIB) domain or the rhotekin Rho-binding domain in pull-down assays, we found that Myr-TRAF4 stimulated GTP loading of Cdc42, Rac1, and RhoA (Fig. 5 A). Furthermore, Rac1(N17) diminished Myr-TRAF4–induced RhoA activation, whereas RhoA(N19) had no effect on Rac1 or Cdc42 activation (Fig. 5, B and C). Thus, TRAF4 activates Cdc42 and Rac1 upstream of RhoA, which is consistent with the sequence of Rho GTPase activation during spontaneous focal contact formation after matrix attachment (Nobes and Hall, 1995). Confirming the activation of Rho GTPases in vivo, Myr-TRAF4 constitutively activated the Cdc42 and Rac1 effector PAK1 (Fig. 5 D). In some cells (presumably high transgene expressors), actin cytoskeletal collapse was noted (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200507004/DC1), which is a morphology resulting from the expression of active PAK1 mutants (Manser et al., 1997) or agonist-dependent PAK1 activation (Wu et al., 2004). Indeed, adenoviral delivery of kinase-dead PAK1(K298A) completely suppressed Myr-TRAF4–induced cytoskeletal remodeling (Fig. S4).

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