Limits...
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.

Show MeSH

Related in: MedlinePlus

TRAF4 associates with Hic-5. (A) In vitro–translated full-length Hic-5 was specifically pulled down by GST-TRAF4, not by GST or beads alone. Top panel is an autoradiogram of captured 35S-methionine–Hic-5; bottom panel is Coomassie stain of GST fusion input. (B) Phoenix-293 cells were transfected with TRAF4 with or without Hic-5 and immunoprecipitated with antibodies against Hic-5 (left), TRAF4 (right), or irrelevant antibodies. Immunoblots were sequentially probed for Hic-5 and TRAF4. Hic-5 appears as a doublet straddling the Ig heavy chain (HC). (C) Lysate from untransfected human lung microvascular endothelial cells was immunoprecipitated with antibodies for TRAF4 or Pyk2 and immunoblotted as shown. Endogenous Hic-5 coprecipitated with TRAF4 and both TRAF4 and Hic-5 coprecipitated with Pyk2. (D) HUVEC were infected with adenoviruses harboring lacZ, p47phox(wt), p47(W193R), or p47(S303D,S304D,S328D) (MOI = 100:1). 24 h later, lysate was immunoprecipitated for Pyk2 and immunoblotted for p47phox and Pyk2. Bottom panel shows total p47phox. (E) Partial colocalization of Hic5-GFP and DsRed-TRAF4 at focal structures within the membrane edge (arrows) is seen. (F) Immunofluorescent image showing the presence of endogenous Hic-5 (AlexaFluor633) in small- to medium-sized vinculin (AlexaFluor555) structures at the edge of a protrusion (arrows). (G) Immunofluorescent stain showing the simultaneous appearance of TRAF4 (AlexaFluor488), Hic-5 (AlexaFluor633), and vinculin (AlexaFluor555) in small (closed head arrow) to medium (open head arrow)-sized complexes at the periphery of a lamella. E–G are TIRF microscopy images. Bars (E and F), 10 μm; (G) 20 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171295&req=5

fig3: TRAF4 associates with Hic-5. (A) In vitro–translated full-length Hic-5 was specifically pulled down by GST-TRAF4, not by GST or beads alone. Top panel is an autoradiogram of captured 35S-methionine–Hic-5; bottom panel is Coomassie stain of GST fusion input. (B) Phoenix-293 cells were transfected with TRAF4 with or without Hic-5 and immunoprecipitated with antibodies against Hic-5 (left), TRAF4 (right), or irrelevant antibodies. Immunoblots were sequentially probed for Hic-5 and TRAF4. Hic-5 appears as a doublet straddling the Ig heavy chain (HC). (C) Lysate from untransfected human lung microvascular endothelial cells was immunoprecipitated with antibodies for TRAF4 or Pyk2 and immunoblotted as shown. Endogenous Hic-5 coprecipitated with TRAF4 and both TRAF4 and Hic-5 coprecipitated with Pyk2. (D) HUVEC were infected with adenoviruses harboring lacZ, p47phox(wt), p47(W193R), or p47(S303D,S304D,S328D) (MOI = 100:1). 24 h later, lysate was immunoprecipitated for Pyk2 and immunoblotted for p47phox and Pyk2. Bottom panel shows total p47phox. (E) Partial colocalization of Hic5-GFP and DsRed-TRAF4 at focal structures within the membrane edge (arrows) is seen. (F) Immunofluorescent image showing the presence of endogenous Hic-5 (AlexaFluor633) in small- to medium-sized vinculin (AlexaFluor555) structures at the edge of a protrusion (arrows). (G) Immunofluorescent stain showing the simultaneous appearance of TRAF4 (AlexaFluor488), Hic-5 (AlexaFluor633), and vinculin (AlexaFluor555) in small (closed head arrow) to medium (open head arrow)-sized complexes at the periphery of a lamella. E–G are TIRF microscopy images. Bars (E and F), 10 μm; (G) 20 μm.

Mentions: Using full-length TRAF4 as a Gal4-binding domain bait fusion in a yeast two-hybrid screen, two clones were retrieved from an endothelial cell Gal4 activation domain library (Xu et al., 2002) containing residues 45–444 of Hic-5, including the three LD motifs and four COOH-terminal LIM domains. Yeast mating studies demonstrated interactions between p47phox and TRAF4 and TRAF4 and Hic-5 but not between p47phox and Hic-5 (unpublished data). Binding of TRAF4 to Hic-5 was direct, as indicated by specific pull down of in vitro translated Hic-5 by GST-TRAF4 (Fig. 3 A). Ectopically expressed TRAF4 and Hic-5 cross-coimmunoprecipitated in Phoenix-293 cells (Fig. 3 B), and endogenous TRAF4 coimmunoprecipitated with endogenous Hic-5 in lung microvascular endothelial cells (Fig. 3 C).


Subcellular targeting of oxidants during endothelial cell migration.

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

TRAF4 associates with Hic-5. (A) In vitro–translated full-length Hic-5 was specifically pulled down by GST-TRAF4, not by GST or beads alone. Top panel is an autoradiogram of captured 35S-methionine–Hic-5; bottom panel is Coomassie stain of GST fusion input. (B) Phoenix-293 cells were transfected with TRAF4 with or without Hic-5 and immunoprecipitated with antibodies against Hic-5 (left), TRAF4 (right), or irrelevant antibodies. Immunoblots were sequentially probed for Hic-5 and TRAF4. Hic-5 appears as a doublet straddling the Ig heavy chain (HC). (C) Lysate from untransfected human lung microvascular endothelial cells was immunoprecipitated with antibodies for TRAF4 or Pyk2 and immunoblotted as shown. Endogenous Hic-5 coprecipitated with TRAF4 and both TRAF4 and Hic-5 coprecipitated with Pyk2. (D) HUVEC were infected with adenoviruses harboring lacZ, p47phox(wt), p47(W193R), or p47(S303D,S304D,S328D) (MOI = 100:1). 24 h later, lysate was immunoprecipitated for Pyk2 and immunoblotted for p47phox and Pyk2. Bottom panel shows total p47phox. (E) Partial colocalization of Hic5-GFP and DsRed-TRAF4 at focal structures within the membrane edge (arrows) is seen. (F) Immunofluorescent image showing the presence of endogenous Hic-5 (AlexaFluor633) in small- to medium-sized vinculin (AlexaFluor555) structures at the edge of a protrusion (arrows). (G) Immunofluorescent stain showing the simultaneous appearance of TRAF4 (AlexaFluor488), Hic-5 (AlexaFluor633), and vinculin (AlexaFluor555) in small (closed head arrow) to medium (open head arrow)-sized complexes at the periphery of a lamella. E–G are TIRF microscopy images. Bars (E and F), 10 μm; (G) 20 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171295&req=5

fig3: TRAF4 associates with Hic-5. (A) In vitro–translated full-length Hic-5 was specifically pulled down by GST-TRAF4, not by GST or beads alone. Top panel is an autoradiogram of captured 35S-methionine–Hic-5; bottom panel is Coomassie stain of GST fusion input. (B) Phoenix-293 cells were transfected with TRAF4 with or without Hic-5 and immunoprecipitated with antibodies against Hic-5 (left), TRAF4 (right), or irrelevant antibodies. Immunoblots were sequentially probed for Hic-5 and TRAF4. Hic-5 appears as a doublet straddling the Ig heavy chain (HC). (C) Lysate from untransfected human lung microvascular endothelial cells was immunoprecipitated with antibodies for TRAF4 or Pyk2 and immunoblotted as shown. Endogenous Hic-5 coprecipitated with TRAF4 and both TRAF4 and Hic-5 coprecipitated with Pyk2. (D) HUVEC were infected with adenoviruses harboring lacZ, p47phox(wt), p47(W193R), or p47(S303D,S304D,S328D) (MOI = 100:1). 24 h later, lysate was immunoprecipitated for Pyk2 and immunoblotted for p47phox and Pyk2. Bottom panel shows total p47phox. (E) Partial colocalization of Hic5-GFP and DsRed-TRAF4 at focal structures within the membrane edge (arrows) is seen. (F) Immunofluorescent image showing the presence of endogenous Hic-5 (AlexaFluor633) in small- to medium-sized vinculin (AlexaFluor555) structures at the edge of a protrusion (arrows). (G) Immunofluorescent stain showing the simultaneous appearance of TRAF4 (AlexaFluor488), Hic-5 (AlexaFluor633), and vinculin (AlexaFluor555) in small (closed head arrow) to medium (open head arrow)-sized complexes at the periphery of a lamella. E–G are TIRF microscopy images. Bars (E and F), 10 μm; (G) 20 μm.
Mentions: Using full-length TRAF4 as a Gal4-binding domain bait fusion in a yeast two-hybrid screen, two clones were retrieved from an endothelial cell Gal4 activation domain library (Xu et al., 2002) containing residues 45–444 of Hic-5, including the three LD motifs and four COOH-terminal LIM domains. Yeast mating studies demonstrated interactions between p47phox and TRAF4 and TRAF4 and Hic-5 but not between p47phox and Hic-5 (unpublished data). Binding of TRAF4 to Hic-5 was direct, as indicated by specific pull down of in vitro translated Hic-5 by GST-TRAF4 (Fig. 3 A). Ectopically expressed TRAF4 and Hic-5 cross-coimmunoprecipitated in Phoenix-293 cells (Fig. 3 B), and endogenous TRAF4 coimmunoprecipitated with endogenous Hic-5 in lung microvascular endothelial cells (Fig. 3 C).

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