Correspondence to Lance S. Terada: Lance.Terada{at}med.va.gov
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Introduction |
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Focal complex dynamics, in turn, appear to be coordinated by the Rho family of small GTPases. Whereas Rac1 and Cdc42 promote focal complex formation within lamellipodia and filopodia, RhoA facilitates the maturation of focal complexes into stable focal adhesions (Nobes and Hall, 1995; Rottner et al., 1999). To fully polarize these events in the motile cell so that meaningful locomotion can occur, positive feedback loops are initiated, resulting in the rapid amplification of Rho GTPase and p21-activated kinase 1 (PAK1) activation at the leading edge and, thus, asymmetric formation of a lamellipodium (Li et al., 2003). The mechanism by which the small GTPases control focal contact behavior is incompletely understood, but it involves the initiation of protein tyrosine phophorylation events leading to intense tyrosine phosphorylation of focal contact proteins. Rac1 and RhoA, for instance, direct the tyrosine kinase Src to lamellipodia and focal adhesions, respectively, to cause remodeling of these integrin-containing structures (Timpson et al., 2001; Webb et al., 2004). Src, in turn, facilitates full activation of the related focal adhesion tyrosine kinase (Pyk2), and both tyrosine kinases are required for the phosphorylation of a number of focal complex proteins. Opposing these effects, the protein tyrosine phosphatase PTP-PEST is also directed to nascent integrin clusters, where it dephosphorylates and thus inactivates Pyk2 (Lyons et al., 2001; Sastry et al., 2002). Furthermore, PTP-PEST suppresses Rac1 activation at the leading edge, diminishing the polarization of migrating cells (Sastry et al., 2002). Although PTP-PEST is thought to target focal contacts through direct binding to paxillin and the paxillin paralogue Hic-5, the mechanism by which its activity is regulated is unclear.
Besides controlling focal complexes and lamellipodia at the leading edge of cells, Rac1 is necessary for activation of the NADPH oxidase. Recently, this oxidase was shown to mediate endothelial cell migration in a Rac1-dependent manner (Moldovan et al., 2000; Ushio-Fukai et al., 2002). Oxidant activity was noted to localize to membrane ruffles (Moldovan et al., 2000), suggesting that distinct functions of Rac1oxidase activation and control of lamellipodial dynamicsmay be spatially and functionally coordinated at the leading edge of migrating cells. Such physical targeting of a tightly regulated oxidant source would present a logical explanation for the observed specificity of oxidant signaling in response to migratory stimuli, circumventing the rapid diffusion and limited target specificity of oxidant molecules themselves. Therefore, we sought to identify molecular targeting devices that may specify the site of oxidant production and, thereby, focus their effects.
We previously reported that the orphan adaptor TRAF4 directly binds the NADPH oxidase subunit p47phox and controls activation of the c-Jun NH2-terminal kinase (JNK) MAPK (Xu et al., 2002). Notably, mice lacking TRAF4 display defective branchial somite and neural tube closure, and flies lacking the orthologous dTRAF1 fail to activate JNK and complete dorsal closure; both phenotypes suggest abnormal migration (Regnier et al., 2002; Cha et al., 2003). In this study, we found that both TRAF4 and p47phox target focal complexes in association with Hic-5 and initiate Rho GTPase signaling. TRAF4-directed oxidant production selectively targets the redox-sensitive phosphatase PTP-PEST and may participate in a positive feedback cycle that facilitates Rac1 activation.
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Results |
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TRAF4Hic-5 interactions control cell migration
Because focal complex formation is critical to cell migration, we next asked whether TRAF4Hic-5 interactions were important for such migration. First, we found that short inhibitory RNA (siRNA)mediated knockdown of either endogenous TRAF4 or Hic-5 diminished HUVEC migration across fibronectin-coated filters in response to a VEGF gradient (Fig. 4 A). Furthermore, the superoxide dismutase mimetic MnTMPyP also inhibited endothelial cell migration, which is consistent with a role for oxidants in migration. Next, to determine relevant binding domains, truncations of each protein were expressed in vivo to preserve zinc finger structures. COOH-terminal truncations of Hic-5, which resulted in serial loss of the four COOH-terminal LIM domains and three LD motifs, revealed that only full-length Hic-5 coprecipitated with TRAF4 (Fig. 4 B), indicating that the COOH-terminal LIM domain 4 (residues 388444) was necessary for TRAF4 binding. In addition, the Hic-5 LIM domain 4 in isolation did not coprecipitate with TRAF4, whereas the tandem LIM domains 3 and 4 (L3,4) did (Fig. 4 C). Thus, LIM domains 3 and 4 are each necessary but insufficient for TRAF4 binding. Conversely, full-length Hic-5 coprecipitated the COOH-terminal TRAF domain of TRAF4 (TRAF4-C) but not the NH2-terminal remainder containing the ring and zinc fingers (Fig. 4 D). Thus Hic-5(L3,4) binds the COOH terminus of TRAF4.
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Myr-TRAF4 activates Rho GTPases, PAK1, and the NADPH oxidase
An active mutant of TRAF4 was created through NH2-terminal fusion with the c-Src myristoylation motif (Myr-TRAF4). Myr-TRAF4 was isolated from detergent-resistant low density membrane fractions, which is consistent with raft localization, and also from the insoluble pellet fraction, which is consistent with cytoskeletal association (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200507004/DC1). Surprisingly, both endogenous TRAF4 and overexpressed native TRAF4 also quantitatively partitioned with raft fractions, although cytoskeletal association was weak. Raft localization of TRAF4 is consistent with the site of integrin activation upon matrix attachment (Lacalle et al., 2002; Labrecque et al., 2003).
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 Cdc42Rac1 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-TRAF4induced 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-TRAF4induced cytoskeletal remodeling (Fig. S4).
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Although our data indicate that TRAF4 activates a Rac1/PAK1/NADPH oxidase cascade, leading to oxidative inactivation of PTP-PEST, the latter phosphatase in its active form has been shown to decrease attachment-dependent Rac1 activation, possibly feeding back upstream in this cascade (Sastry et al., 2002). Were this the case, Rac1-dependent oxidant production would be expected to further increase Rac1 activity, thereby engaging in a positive feedback cycle. To test this hypothesis, we examined the effect of the NADPH oxidase on its upstream activator, Rac1. Indeed, both MnTMPyP and p67(V204A) completely suppressed Myr-TRAF4induced Rac1 GTP loading (Fig. 7 D), placing the oxidase both upstream and downstream of TRAF4-dependent Rac1 activation.
TRAF4-dependent membrane ruffling proceeds through Rac1, PAK1, and the NADPH oxidase
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-TRAF4induced ruffling (Fig. 8 B). Finally, both MnTMPyP and p67(V204A) reduced Myr-TRAF4induced ruffling to basal levels (Fig. 8 C). Thus, by morphological as well biochemical criteria, Rac1, PAK1, and the NADPH oxidase act downstream of TRAF4.
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PTP-PEST antagonizes the effects of the tyrosine kinase Pyk2 (Lyons et al., 2001). The latter protein, which is associated with the p47phoxTRAF4Hic-5 complex (Fig. 3 C), acts as a scaffold for another focal complex tyrosine kinase, Src, and facilitates Src activation (Park et al., 2004). Src, in turn, mediates focal complex turnover much like active PAK1 (Manser et al., 1997; Zhao et al., 2000; Webb et al., 2004). Accordingly, we found that kinase-dead mutants of both Pyk2 and Src also caused Myr-TRAF4transfected endothelial cells to lose membrane ruffles and become quiescent (Fig. 8 C), which is consistent with the focusing of TRAF4's effects on focal complex tyrosine phosphorylation events related to PTP-PEST.
Finally, as many of the proteins implicated in TRAF4 signaling (including TRAF4 itself, Rac1, PAK1, the NADPH oxidase cytochrome subunits, integrins, and Src) congregate within membrane rafts, it was not surprising to find that the disruption of raft structure with either filipin (not depicted) or methyl-ß cyclodextrin (Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200507004/DC1) effectively diminished Myr-TRAF4induced ruffling and lamellipodium formation. Although not specific for the TRAF4 complex, these data support the concept that TRAF4 operates within an expected microdomain to participate in Rac1-dependent integrin signaling.
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Discussion |
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Our data suggest that during cell migration, TRAF4 may tether p47phox to nascent lamellar focal complexes to direct oxidant production to these structures and bias the local environment toward tyrosine phosphorylation events and Rho GTPase activation. We provide microscopic, biochemical, and functional evidence for such targeting. First, we found that both chromophore fusions of TRAF4 and p47phox as well as the endogenous proteins are targeted to leading edge focal complexes, which are integrin-associated structures concentrated with tyrosine-phosphorylated proteins. This physical targeting of an NADPH oxidase subunit to such structures is consistent with the influence of integrin ligation on oxidase activation; for instance, either the detachment or reattachment of cells to the matrix induces NADPH oxidasedependent oxidant production (Kheradmand et al., 1998; Chiarugi et al., 2003). The appearance of TRAF4 in membrane rafts is also consistent with its functional interaction with integrin structures and the NADPH oxidase. Matrix attachment, for instance, directs ß1 and ß3 integrins into raft domains (Lacalle et al., 2002; Labrecque et al., 2003); in addition, the NADPH oxidase membraneassociated cytochrome b558 (gp91phox and p22phox), like TRAF4, is constitutively associated with lipid rafts (Vilhardt and Van Deurs, 2004). The further appearance of Src, Rac1, and RhoA in rafts after integrin ligation also suggests the importance of rafts as organizing structures involved in focal complex dynamics.
Second, we found evidence that a functional complex is formed along with the focal contact protein Hic-5. TRAF4 binds Hic-5 and forms a complex with the focal adhesion kinase Pyk2, a redox-sensitive kinase previously shown to mediate VEGF-induced endothelial cell migration (Avraham et al., 2003). Accordingly, knockdown of either TRAF4, Hic-5, or binding truncations of either protein disrupt VEGF-mediated chemotaxis and sheet migration into wounds. The association of TRAF4 with Hic-5 suggests a mechanism of specifying oxidase activation to integrin complexes and potentially places such a complex in proximity to other oxidant-related, Hic-5associated proteins. These include PAK1, which associates with Hic-5 through paxillin-kinase linker (Turner et al., 1999) and both associates with p47phox and acts upstream of its phosphorylation (Wu et al., 2003, 2004); Pyk2, which binds to the NH2 terminus of Hic-5 (Matsuya et al., 1998) and requires endogenous oxidants for integrin-dependent mechanosensory activation (Tai et al., 2002); and Src, which binds Pyk2 (Matsuya et al., 1998; Park et al., 2004), phosphorylates Hic-5 (Matsuya et al., 1998; Ishino et al., 2000), and becomes active upon integrin ligation via oxidant production (Chiarugi et al., 2003). Besides acting up or downstream of endogenous oxidants, all three of these kinases increase focal complex turnover and mediate ruffle formation as well as cell migration (Manser et al., 1997; Kiosses et al., 1999; Timpson et al., 2001; Okigaki et al., 2003; Webb et al., 2004).
Third, we found that a myristoylated form of TRAF4 activates Rho GTPases and the effector kinase PAK1, increases p47phox phosphorylation and oxidant production, and causes intense membrane ruffling, which is a morphologic correlate of Rac1 hyperactivation. Our data further suggest that oxidants facilitate focal complex signaling through the targeted inactivation of PTP-PEST. This phosphatase binds Hic-5 and translocates to nascent focal complexlike structures (Angers-Loustau et al., 1999; Nishiya et al., 1999; Sastry et al., 2002), where it can gainsay both Pyk2 and Src function (Angers-Loustau et al., 1999; Lyons et al., 2001). Notably, active PTP-PEST decreases Rac1 activity and membrane ruffling and is thought to participate in focal contact turnover (Angers-Loustau et al., 1999; Sastry et al., 2002; Cousin and Alfandari, 2004). Consistent with the combined observations that PTP-PEST may act both up and downstream of Rac1, we found that the NADPH oxidase may act similarly, thus participating in a possible positive feedback loop. Such loops are thought to be critical in sensing and amplifying shallow chemoattractant gradients and fully polarizing the motile cell (for review see Ridley et al., 2003). Interestingly, these loops involve PI3K, PAK1, and Rho GTPases such as Rac1, which are all capable of acting upstream of the NADPH oxidase and are all rapidly mobilized to the leading edge.
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Materials and methods |
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Microscopy
HUVECs were grown in EGM-2 media (Clonetics), electroporated 67 h after release from thymidine-induced cell cycle arrest, and plated on fibronectin-coated chambered coverslips (Lab-Tek). Because endothelial edge ruffles do not readily withstand fixation/permeabilization, cells were examined live with sequential acquisition of red and green channels. Confocal microscopy was performed using a LSM 410 laser scanning system (Axiovert S100TV; Carl Zeiss MicroImaging, Inc.). Images were acquired with either a 63x/1.2 or a 100x/1.3 objective using LSM software (Carl Zeiss MicroImaging, Inc.). Ruffling was quantified in actin-GFPcotransfected cells by examining all cells in at least four high power fields per chamber in at least four chambers. TIRF microscopy was performed on live cells using a fluorescent inverted microscope system (TE2000-U; Nikon). Images were acquired at 37°C with a 60x/1.45 oil immersion objective (Nikon) using Metamorph software (Molecular Devices).
Immunofluorescence
HUVEC plated on fibronectin-coated chambered coverslips were washed, fixed with 2% HCHO, and permeabilized with 0.2% Triton X-100. After subsequent washing, cells were blocked at 25°C for 30 min with either 10% normal goat serum or 5% BSA. Primary antibodies used were Hic-5 (1:50; Santa Cruz Biotechnology, Inc.), TRAF4 (1:50; Santa Cruz Biotechnology, Inc.), p47phox (1:50; Santa Cruz Biotechnology, Inc.), vinculin (1:100; clone Vin-11-5; Sigma-Aldrich), and phosphotyrosine (1:200; clone PT-66; Sigma-Aldrich); these antibodies were incubated with cells at 25°C for 1 h in either 5% goat serum or 2% BSA. Corresponding secondary antibodies conjugated to AlexaFluor488, 555, or 633 were obtained from Invitrogen. Cells were stained with each secondary antibody separately with extensive washing in between. Cells were observed with TIRF microscopy as described above using 488-, 543-, or 633-nm excitation lines. Corresponding controls without primary antibodies were confirmed to be negative, and no bleed-through to other channels was observed with any of the chromophores.
Yeast two-hybrid screening
A HUVEC library was previously constructed in lambda phage HybriZAP 2.1 XR (Xu et al., 2002), and the library was dropped out in the yeast shuttle vector pAD-GAL4-2.1 by mass excision. Saccharomyces cerevisiae AH109 (CLONTECH Laboratories, Inc.) were sequentially transformed with the bait vector pGBKT7-TRAF4 and with the endothelial library using lithium acetate. Candidate prey were identified with adenine and histidine auxotrophic selection and lacZ expression. Single yeast clones were retested for auxotrophy and lacZ expression, and library plasmids were passaged through Escherichia coli and confirmed with standard yeast mating techniques. Interactions were assessed using yeast mating techniques, with the assessment of lacZ expression by diploids. In vitro pull down was performed as previously described (Xu et al., 2002).
Coimmunoprecipitation
Phoenix-293 cells electroporated with indicated vectors were extracted in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF) for 30 min on ice, sonicated for 5 s, and centrifuged at 10,000 g for 20 min at 4°C. Immunoprecipitation was then performed using protein A or protein Gagarose (GE Healthcare) conjugates. Antibodies used are as follows: TRAF4 (Santa Cruz Biotechnology, Inc.), Flag (Sigma-Aldrich), Hic-5 and Pyk2 (BD Transduction), and p47phox (Upstate Biotechnology and gift of Bernard Babior, Scripps Research Institute, La Jolla, CA). In some experiments, human lung microvascular endothelial cells or HUVECs (both from Clonetics) were used.
Protein knockdown
RNA corresponding to both strands of human Hic-5 (803821), TRAF4 (175193), and PTP-PEST (15841602; coding region numbering) tailed with dTdT were synthesized by Dharmacon. A sequence from firefly luciferase was used as a control siRNA in all experiments. 10 nM duplex siRNA was transfected into HUVEC using 30 µl TransIT-TKO (Mirus) per 100-mm dish. Protein expression was assessed, and experiments were initiated after 48 h.
Migration assays
The bottom of 24-well transwell filters (0.8 µm; Costar) was coated with fibronectin (Sigma-Aldrich). HUVECs were cotransfected with pEGFP (CLONTECH Laboratories, Inc.) and one of the indicated vectors. 24 h after transfection, cells were plated onto filters in growth factordeficient EBM2 media (Clonetics) with 2% FBS. The bottom chamber contained the same media plus 100 ng/ml VEGF (PeproTech). Cells were allowed to migrate for 16 h, and then cells remaining on the top surface were removed with a cotton swab and GFP-positive cells were counted. After counting, cells were swabbed off the bottom surface, and the filters were reexamined to confirm the removal of remaining cells. Migration into wounds was examined by plating pEGFP-cotransfected HUVECs on fibronectin-coated etched coverslips (Bellco). At least five wounded fields per coverslip were analyzed on six coverslips per condition, and identical fields were photographed under phase and epifluorescence at 0, 18, 24, and 48 h. Phase-contrast and GFP images were overlaid and analyzed using Metamorph software.
Raft fractionation
Detergent-insoluble low density fractions were isolated as described previously (Lee et al., 2002). Five 100-mm dishes per condition were pooled, washed twice with PBS, and resuspended in 1 ml MES-buffered saline (MBS; 25 mM MES, pH 6.5, 150 mM NaCl, and 5 mM EDTA) with 1% Triton X-100 and protease inhibitors. Cells were disrupted with 10 passes through a 25-gauge needle and diluted with 1 ml 80% sucrose in MBS. Lysate was overlaid with 2 ml of 30% and 1 ml of 5% sucrose in MBS and centrifuged at 200,000 g for 18 h at 4°C with a rotor (SW55Ti; Beckman Coulter). 0.4-ml fractions were removed from the top, acetone precipitated twice, washed in acetone, and resuspended in Laemmli buffer. Antibodies for caveolin-1, Rac1, TfR, and TRAF4 were obtained from Santa Cruz Biotechnology, Inc.
Rho family GTP loading
The human PAK1 CRIB domain (residues 51135) was recovered by PCR from a HUVEC library and ligated into pGEX-2TK, and the Rho-binding domain of mouse rhotekin (residues 789) was recovered by RT-PCR from mouse endothelial cells and ligated into pGEX-2TK. GST fusion proteins were then produced in BL21-RP bacteria and captured on glutathione S-transferaseSepharose beads (GE Healthcare). Active GTP-loaded Rho proteins from the 10,000-g supernatant of HUVEC lysate were pulled down by the respective fusions and immunoblotted with antisera for Rac1 (BD Transduction), Cdc42 (Santa Cruz Biotechnology, Inc.), or RhoA (Santa Cruz Biotechnology, Inc.).
PAK1 activity
PAK1 activation was assessed by an immunoprecipitation kinase assay (Wu et al., 2004). In brief, HUVECs were extracted in cold lysis buffer, sonicated briefly, centrifuged, and immunoprecipitated with rabbit anti-PAK1 (Santa Cruz Biotechnology, Inc.). The kinase reaction was performed using 5 µg myelin basic protein as a substrate in the presence of -[32P]ATP at 30°C for 30 min. Two thirds of each sample was subjected to autoradiography, and one third was subjected to immunoblotting to assess the capture of PAK1.
p47phox phosphorylation
Rabbit polyclonal antisera was raised by immunization with a keyhole limpet hemocyaninconjugated peptide YRRN(pS)VRFLQQRR containing a phosphoserine corresponding to S328 of human p47phox. The antisera specifically recognized p47phox phosphorylated in vivo by phorbol ester stimulation. Phoenix-293 cells overexpressing p47phox were transfected with pCIN, Myr-TRAF4, or TRAF4, and whole cell lysates were serially immunoblotted for p47phox(pS328) and total p47phox (Upstate Biotechnology).
Oxidant production
HUVECs were cotransfected with pDsRed-C2 and the test vectors, plated on fibronectin-coated slides, and subsequently infected with adenovirus (multiplicity of infection [MOI] = 100:1). After 24 h, cells were washed twice with warm HBSS and incubated with 10 µM dichlorodihydrofluorescein diacetate (DCF) for 40 min. Cells were washed twice in HBSS, and epifluorescent green and red channel images were acquired with cells in an adherent state to avoid perturbation of integrin activity. DCF fluorescence of DsRed-expressing cells was quantified in 16-bit resolution using Metamorph software. The mean of at least 20 cells per chamber in 10 chambers was quantified for each condition.
5-IAF labeling
Oxidation of protein tyrosine phosphatase acidic cysteine residues was assessed as described previously (Wu et al., 1998). Phoenix-293 cells were cotransfected with FlagPTP-PEST and either empty vector or Myr-TRAF4. After lysis, extracts were labeled with 5 µM 5-IAF (Invitrogen) at 4°C for 60 min, immunoprecipitated with anti-Flag, and immunoblotted with rabbit antifluorescein (Invitrogen). MKP-1 and SHP-2 (SH-PTP2) were similarly immunoprecipitated (Santa Cruz Biotechnology, Inc.) and subjected to immunoblot with antifluorescein.
Online supplemental material
Fig. S1 shows that TRAF4-GFP colocalizes with DsRed-zyxin in focal complexlike structures. Fig. S2 shows that TRAF4-GFP colocalizes with DsRed-zyxin in TIRF microscopy. Fig. S3 shows that TRAF4 concentrates in low density detergent-insoluble membrane fractions. Fig. S4 shows examples of Myr-TRAF4induced PAK1-dependent cytoskeletal collapse. Fig. S5 shows that Raft disruption decreases Myr-TRAF4induced ruffling. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200507004/DC1.
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Acknowledgments |
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This work was supported by the National Heart, Lung, and Blood Institute (grants R01-HL61897 and R01-HL67256), the National Institute of General Medical Sciences (grant R01-GM67674), the Veterans Administration, and the Robert Wood Johnson Foundation (grant to F.E. Nwariaku).
Submitted: 1 July 2005
Accepted: 3 November 2005
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