Involvement of TRAF4 in Oxidative Activation of c-Jun N-terminal Kinase*

We previously found that the angiogenic factors TNFα and HIV-1 Tat activate an NAD(P)H oxidase in endothelial cells, which operates upstream of c-Jun N-terminal kinase (JNK), a MAPK involved in the determination of cell fate. To further understand oxidant-related signaling pathways, we screened lung and endothelial cell libraries for interaction partners of p47 phox and recovered the orphan adapter TNF receptor-associated factor 4 (TRAF4). Domain analysis suggested a tail-to-tail interaction between the C terminus of p47 phox and the conserved TRAF domain of TRAF4. In addition, TRAF4, like p47 phox , was recovered largely in the cytoskeleton/membrane fraction. Coexpression of p47 phox and TRAF4 increased oxidant production and JNK activation, whereas each alone had minimal effect. In addition, a fusion between p47 phox and the TRAF4 C terminus constitutively activated JNK, and this activation was decreased by the antioxidant N-acetyl cysteine. In contrast, overexpression of the p47 phox binding domain of TRAF4 blocked endothelial cell JNK activation by TNFα and HIV-1 Tat, suggesting an uncoupling of p47 phox from upstream signaling events. A secondary screen of endothelial cell proteins for TRAF4-interacting partners yielded a number of proteins known to control cell fate. We conclude that endothelial cell agonists such as TNFα and HIV-1 Tat initiate signals that enter basic signaling cassettes at the level of TRAF4 and an NAD(P)H oxidase. We speculate that endothelial cells may target endogenous oxidant production to specific sites critical to cytokine signaling as a mechanism for increasing signal specificity and decreasing toxicity of these reactive species.

The vascular endothelium is generally well supplied with oxygen and produces significant quantities of oxidants when stimulated in vivo or in vitro (1,2). As in other cell types, such tightly regulated oxidant bursts appear to transduce a variety of signals. Mechanical forces, growth factors, and cytokines stimulate oxidant production by endothelial cells, leading to migration, proliferation, apoptosis, or adhesion protein expression (3)(4)(5). However, the relatively broad biochemical reactivity of these oxidants poses a potential problem for signal specific-ity. As an example, a number of studies now support the participation of oxidants in both proliferative (6 -8) and apoptotic (9,10) pathways, depending on stimulus and context. The basis for the divergent responses to oxidants is not clear.
Endothelial cells possess an NAD(P)H oxidase (11) thought to participate in a number of these signal pathways. Inhibitors of this oxidase suppress growth factor, TNF␣, 1 HIV-1 Tat, and shear cessation-induced signaling (2,4,12,13), and dominant negative Rac1 disrupts TNF␣ signaling (5) in endothelial cells. Recently, both cytochrome subunits of the oxidase, p22 phox , and gp91 phox , were cloned from rat and human endothelial cells (14,15). We subsequently cloned the oxidase adapter subunit p47 phox from HUVEC, demonstrating its participation in TNF␣ signaling (12). Unexpectedly, endogenous p47 phox was found to be constitutively associated with the cytoskeleton of unstimulated ECV-304 cells, contrasting the free cytosolic location of p47 phox in unstimulated neutrophils. Because most signaling proteins are associated with the cytoskeleton at some point in their activation cycle, the strong association of p47 phox with the endothelial cytoskeleton suggested specific localization of the oxidase with cytoskeletally anchored signaling complexes. Indeed, cytoskeletal disruption derailed both oxidase activation and downstream signaling (12). Spatial targeting of the oxidase may therefore potentially confer signal specificity to these evanescent radicals.
To identify potential vicinal signaling elements associated with the endothelial NAD(P)H oxidase, we screened lung and HUVEC libraries for p47 phox -interacting proteins and recovered the orphan adapter TRAF4. This interaction appears to participate in downstream activation of JNK by the oxidaseactivating endothelial agonists TNF␣ and HIV-1 Tat.

EXPERIMENTAL PROCEDURES
Plasmid Construction-All PCR amplifications for subcloning or mutagenesis were performed with Pfu Turbo (Stratagene). The bait vector pGBKT7-p47 was created by a single base mutation of p47 phox (T to C at Ϫ2), creating a new NcoI site. The NcoI-EcoRI fragment containing the coding region and 3Ј-UTR of p47 phox was then subcloned into pGBKT7 (CLONTECH) in-frame with the GAL4-BD. Full-length TRAF4 was PCR-amplified from a HUVEC library (Stratagene) between the EcoRI and SalI sites. It was directly ligated into the expression vector pCI (Promega) to create pCI-T4 and into the yeast shuttle vector pGBKT7 to create pGBKT7-T4. The C-terminal TRAF domain of TRAF4 was excised from the library prey plasmid pACT2-T4 using EcoRI and PshAI and ligated into pCIneo-FLAG (16) to create pCINF-T4(CT). pGBKT7-p47-(1-205) was constructed by removing the C-terminal BamHI-BamHI fragment from pGBKT7-p47, and pGBKT7-p47-(205-390) was * This work was supported by the National Institutes of Health (R01-HL61897) and the Medical Research Service of the Department of Veterans Affairs. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
GAL4-AD Libraries-A commercial human lung library cloned into pACT2 was obtained (CLONTECH). An endothelial cell library was constructed using poly(A ϩ ) RNA from passage 4 -5 HUVEC (Clonetics). Oligo(dT)-primed cDNA was cloned directionally into the EcoRI and XhoI sites of lambda phage HybriZAP 2.1XR (Stratagene). The primary library (4.8 ϫ 10 6 plaque-forming units) was amplified once as lambda phage, and the library was dropped out in the yeast shuttle vector pAD-GAL4-2.1 by mass excision using a kit (Stratagene). The average insert size was 1.7 kb.
Yeast Two-hybrid Screening-Saccharomyces cerevisiae AH109 (CLONTECH) were stably transformed with the bait vector pGBKT7-p47 under tryptophan-deficiency selection, and full-length p47 phox was found to lack autonomous transactivation activity. Stable transformants were then secondarily transformed with the lung or endothelial cell libraries using lithium acetate. AH109 yeast contains two auxotrophic reporter genes (ADE2 and HIS3) and lacZ under the control of three distinct promoters. Therefore, colonies that survived selection using medium deficient in tryptophan (bait selection), leucine (library selection), adenine, and histidine (interaction selection) were tested for lacZ expression using a filter lift assay. Positive colonies were restreaked, and single clones were retested for auxotrophy and lacZ expression. Library plasmids from triple-positive clones were extracted and passaged through Escherichia coli with selection for GAL4-AD plasmids and stably transformed back into AH109. Autonomous transactivation negative clones were then mated with Y187 yeast containing pGBKT7-p47 and diploids tested for lacZ expression. A similar approach was used to rescreen the HUVEC library using full-length TRAF4 as bait after stable transformation of AH109 with pGBKT7-T4.
Yeast Mating-AH109 yeast stably transformed with GAL4-AD-TRAF4 constructs were tested first for autonomous transactivation; negative colonies were mated with Y187 yeast stably transformed with various GAL4-BD-p47 deletion constructs. Diploids were replated on medium selecting for both plasmids and tested for lacZ expression with a filter lift assay. Negative controls were Tyr-187 transformed with empty pGBKT7 and pGBKT7-lamin C; the positive control was AH109 transformed with holo-GAL4 (pCL1, CLONTECH). Positive interactions were identified by development of a blue color within 1 h, negative interactions remained white for Ͼ24 h.
GST Pull-down-Direct interactions were confirmed in vitro (18). BL21-RP E. coli (Stratagene) were transformed with either pGEX-2TK or pGEX-p47, induced for 3 h at 37°C, and the GST proteins were captured on glutathione-Sepharose (Amersham Biosciences, Inc.). Approximately 2 g of GST or GST-p47 was used per 200 l of binding reaction. Full-length TRAF4 was transcribed and translated in vitro from pCI-T4 (TNT Quick Coupled, Promega) using [ 35 S]methionine, and 5 l (3 Ci) of translation mix was added to each binding reaction. In some reactions, p47-(299 -390) was in vitro transcribed and translated from pGBKT7-p47-(299 -390) without isotope and added directly to the binding reaction simultaneously with labeled TRAF4.
Adenoviral Construction-A silent G360C mutation was introduced in p47 phox to eliminate a potential PI-CeuI site. The entire p47 phox cDNA was excised with XbaI and KpnI and ligated into pShuttle, and the expression cassette was then subcloned into the backbone pAdeno-X (CLONTECH). The linearized adenoviral DNA was then transfected into HEK-293 cells and replication-incompetent viruses harvested and titered. Ad-lacZ was similarly constructed according to the manufacturer's suggestions (CLONTECH). Robust expression of p47 phox was demonstrated by immunoblot at multiplicity of infections of 50 -200 (not shown), and lacZ expression was demonstrated in Ͼ95% of cells.
JNK Activity-JNK activity of Fx cells was assessed using a traditional immunoprecipitation kinase technique using anti-JNK1 (Santa Cruz Biotechnology, C-17) and GST-Jun (Santa Cruz Biotechnology) (2). Equivalent capture of JNK was assessed with immunoblot using a pan-specific anti-JNK (JNK-FL, Santa Cruz Biotechnology). To increase HUVEC transfection efficiency, passage 2-3 HUVEC (Clonetics) were synchronized at the G 1 -S transition with 3.5 mM thymidine overnight (19). Six hours after thymidine release, cells were electroporated with 10 g of each plasmid, keeping total DNA concentration constant for each experiment. HUVEC were cotransfected with either HA-JNK1 or HA-JNK2. Cells were stimulated with either human TNF␣ (100 ng/ml, Peprotech) or HIV-1 Tat, prepared as a GST fusion as previously described (2). The JNK activity of anti-HA immunoprecipitates was then assessed.

RESULTS
TRAF4 Interacts with p47 phox -We found first that similar to the situation with ECV-304 cells (12), p47 phox appears to constitutively associate with the actin cytoskeleton of HUVEC. p47-GFP colocalized with submembranous lateral actin bundles as well as actin microspikes in unstimulated HUVEC (Fig.  1). To find binding partners for p47 phox , a whole lung library was chosen for an initial screen because of its high representation of endothelium. 1.7 ϫ 10 5 transformants were screened, and 80 colonies survived initial auxotrophic selection and were restreaked. Of these, 23 single clones were found to be positive for lacZ expression. PCR amplification and HaeIII restriction digest pattern revealed nine independent clones. Only one clone represented an autonomous transactivation-negative interaction-positive clone containing an in-frame library insert; this clone was bidirectionally sequenced and found to encode the C-terminal 210 amino acid residues of TRAF4. To accomplish a more endothelial-specific screen, 1.9 ϫ 10 6 clones were screened from the HUVEC library, and only 10 colonies were found to survive auxotrophic selection. Of these, four were lacZ-positive and two were subsequently found to be true positives. These clones were identical by restriction digest analysis. Sequence of one revealed the C-terminal 287 residues of TRAF4. Yeast mating demonstrated lack of binding of p47 phox to full-length TRAF1 and 2 (not shown).
When overexpressed in Fx cells, TRAF4 preferentially remained with the detergent-insoluble fraction, with a smaller portion of the protein found in the detergent-soluble fraction (Fig. 2a), mimicking the cytoskeletal distribution of p47 phox (12). When coexpressed, TRAF4 was found to specifically coprecipitate with FLAG-p47 (Fig. 2b). TRAF4 was also found to coprecipitate with p47-GFP, using anti-GFP, though at reduced efficiency (not shown). In addition, full-length TRAF4 coprecipitated with the C-terminal TRAF domain of TRAF4 (Fig. 2c), suggesting self-association through this domain.
The C terminus of p47 phox Interacts with the TRAF Domain of TRAF4 -Two-hybrid interactions were used to determine interacting domains of p47 phox and TRAF4. The C terminus of p47 phox -(299 -390), containing a variant proline-rich (PR) sequence, an arginine-rich basic motif, and a C-terminal PR site, was both necessary and sufficient for interaction with TRAF4 (Fig. 3a). Interestingly, the extreme C terminus (347-390) of p47 phox was necessary but by itself insufficient, and the adjacent segment (299 -345) was also necessary but insufficient either by itself or attached to the rest of the protein N-terminal to it. The GST pull-down assay confirmed a direct interaction of p47 phox with full-length TRAF4 in vitro (Fig. 3b). In addition, in vitro-translated p47-(347-390) competed with full-length p47 phox for TRAF4 binding, consistent with a specific interaction of the C terminus of p47 phox with TRAF4.
The smaller of the TRAF4 library inserts obtained encoded residues 261-470, corresponding to both the 6th exon and the TRAF domain of TRAF4, indicating an interaction of p47 phox with this domain. The TRAF domains of TRAFs 1-6 contain a C-terminal ␤-sheet-predominant subdomain (TRAF-C) preceded by a shorter coiled-coil subdomain (TRAF-N). By homology and secondary structure prediction, these regions span residues 308 -470 and 267-307, respectively, of TRAF4 (20). Yeast mating studies suggested that the isolated TRAF-N and TRAF-C domains were each insufficient to bind p47 phox , whereas the entire TRAF domain bound p47 phox (Fig. 4).
TRAF4 Participates in JNK Activation-Overexpression of either p47 phox or full-length TRAF4 alone in Fx cells did not appreciably affect JNK activity. In contrast, overexpression of both p47 phox and TRAF4 increased JNK activity greater than 4-fold over control, suggesting a functional as well as physical interaction between the two proteins (Fig. 5a). Similarly, coexpression of p47 phox and TRAF4 increased DCF oxidation, consistent with an increase in oxidant production. To demonstrate that interaction of p47 phox with the TRAF4 TRAF domain was sufficient for JNK activation, we fused this TRAF domain to the C terminus of p47 phox . Overexpression of the fusion protein increased JNK activity in HUVEC (Fig. 5b), whereas overexpression of either p47 phox or the TRAF4 TRAF domain alone did not. This activation was decreased by the antioxidant Nacetyl cysteine (NAC).
To further implicate TRAF4-p47 phox interactions in endogenous endothelial cell signaling pathways, we investigated the effect of TRAF4 p47 phox binding domain overexpression on signaling by TNF␣ and HIV-1 Tat, two agonists that activate endothelial cell JNK through p47 phox -dependent oxidant pro-duction (2,12). Overexpression of this TRAF4 truncation consistently decreased activation of both HA-JNK1 and HA-JNK2 by TNF␣ and HIV-1 Tat in HUVEC (Fig. 6), whereas fulllength TRAF4 did not affect JNK activation (not shown).
Association of TRAF4 with Other Signaling Proteins-A secondary screen of the HUVEC library using full-length TRAF4 was performed on 5 ϫ 10 6 AH109 transformants. Of 92 His ϩ / Ade ϩ /LacZ ϩ clones, 73 were thought to be unique by PCR and digest pattern. These clones were isolated, passaged through E. coli, and 71 clones were confirmed by mating back to Y187/ pGBKT7-T4 and lacZ expression testing. These clones were all FIG. 2. Coprecipitation of TRAF4 and p47 phox . a, full-length TRAF4 (TRAF4(FL)) or the truncated TRAF domain of TRAF4 (TRAF4(CT)) were overexpressed in Fx cells, Triton X-100-fractionated, and immunoblotted with anti-TRAF4. Both full-length and C-terminal TRAF4 were recovered in both fractions, with a predominance in the detergent-insoluble fraction. b, Fx cells were cotransfected with pCINF-p47 (FLAG-p47) and/or pCI-T4 (untagged full-length TRAF4, TRAF4(FL)), immunoprecipitated with either anti-FLAG or irrelevant isotype antibody and immunoblotted for TRAF4 and then FLAG. TRAF4 specifically coprecipitated with FLAG-p47. c, Fx cells were cotransfected with pCINF-T4(CT) (FLAG-tagged C-terminal TRAF4) and/or pCI-T4 (untagged full-length TRAF4), immunoprecipitated with anti-FLAG or irrelevant antibody and immunoblotted with antisera recognizing the C terminus of TRAF4. Full-length TRAF4 (upper band) specifically coprecipitated with FLAG-T4(CT) (lower band).

DISCUSSION
TRAF4 is the least well understood of the traditional TRAF family members. Originally identified in a differential screen of metastatic breast cancer lymph nodes (21), it was subsequently shown to be widely expressed in normal human tissues, especially actively dividing epithelium (22). Its closest relative in both domain organization and amino acid sequence is the Drosophila adapter DTRAF1, which interacts with Misshapen (Msn), a MAP4K acting upstream of JNK (23). The biological function of TRAF4, however, is poorly understood, beyond its ability to prevent dimerization of the neurotrophin receptor p75 NTR (24) and to contribute to normal tracheal development in mice (25).
Full-length TRAF4 demonstrated a preference for the detergent-insoluble cell fraction, a distribution similar to that of p47 phox in endothelial cells (12). The constitutive association of p47 phox with the cytoskeleton demonstrated in ECV-304 cells in this prior study and in HUVEC in the present study stands in marked contrast to its behavior in neutrophils. In the latter cell type, p47 phox migrates from a cytosolic location to the cytoskeleton and membrane skeleton only upon stimulation (26,27). The constitutive association of p47 phox with the endothelial cytoskeleton suggests that the oxidase may exist in a preformed but inactive complex in endothelial cells. Abrupt cessation of flow, for instance, causes oxidant production by endothelial cell NADPH oxidase within 15 s (28). Although the basis for such constitutive cytoskeletal association is not known, there are clear differences in cytoskeletal structure between adherent endothelial cells and suspended neutrophils. Adhesiondependent reorganization of the actin cytoskeleton may create or expose p47 binding sites or initiate partial phosphorylation of p47 phox , resulting in cytoskeletal localization. Notably, adhesion primes neutrophils for a robust respiratory burst upon TNF␣ stimulation in a mechanism dependent upon actin polymerization (29). A similar mechanism may operate in adherent endothelial cells.
The isolated TRAF domain of TRAF4 also demonstrated a preference for the detergent-insoluble fraction. Because the TRAF4 TRAF domain also bound p47 phox , these data do not allow us to determine whether TRAF4 is primarily associated with the cytoskeleton or whether this localization arises secondarily from its association with p47 phox . The retrieval of ␣-actinin as a potential TRAF4-interacting partner (Table I) is consistent with the former possibility. This situation may be somewhat different from the localization of TRAF5, because the zinc finger motifs of this latter protein rather that its TRAF domain appear to confer detergent insolubility (30). It is equally plausible that TRAF4 and p47 phox each have independent cytoskeletal association domains.
The C-terminal tail of p47 phox from residues 299 -390 comprised the minimum TRAF4 binding domain we identified. This region encompasses a variant proline-rich site (299 -302), a basic region (314 -347), and a type II polyproline motif (362-368). In addition, this tail harbors three basic peptides corresponding to residues 305-319, 325-339, and 373-387, which independently block activation of the NADPH oxidase in vitro in a reconstituted phagocyte membrane system (31). GAL4-AD fusions containing either the variant proline-rich and basic regions or the C-terminal polyproline motif were each necessary but insufficient for binding TRAF4 in the two-hybrid assay, suggesting either a cooperative interaction or a tertiary structure requiring both halves of the C-terminal tail.
Our data also indicate an interaction of p47 phox specifically with the C-terminal TRAF domain of TRAF4, and suggest that both TRAF-N and TRAF-C domains are necessary for this interaction. The TRAF domain also appeared to be sufficient for TRAF4 self-association, consistent with prior studies suggesting homomultimerization of TRAF proteins through the TRAF domain (32,33). Besides self-association, the TRAF domains of TRAFs 2, 3, and 6 are also capable of binding downstream signaling elements such as cIAP-1/2, TRADD, receptorinteracting protein (RIP), NF-B-inducing kinase (NIK), MIP-3, and TTRAP (34 -39). Following this paradigm, the NAD(P)H oxidase may lie downstream of TRAF4. Consistent with this interpretation, we found that ectopic expression of both TRAF4 and p47 phox in Fx cells act cooperatively to stimulate activation of JNK. Further, forced association of p47 phox and the TRAF4 TRAF domain through genetic fusion caused activation of JNK in HUVEC. In the latter experiment, the C-terminal TRAF domain of the fusion protein may have associated with endogenous endothelial cell TRAF4, thus recruiting p47 phox to full-length TRAF4. Alternatively, the TRAF domain of TRAF4 may be sufficient to initiate distal signaling in the presence of p47 phox , a possibility we have not excluded.
In endothelial cells, TNF␣ and HIV-1 Tat are strong JNK activators that recognize distinct receptors yet appear to both signal through the NAD(P)H oxidase (2,12). Interestingly, we found that overexpression of the p47 phox binding TRAF4 TRAF domain interrupted signaling initiated by these two endothelial cell agonists in HUVEC. The isolated TRAF domains of TRAF2, 3, 5, and 6 have been demonstrated to block upstream signals originating from the TNF␣, LT␣/␤, and IL-1 receptors (37,40,41), but we are not aware of any reports in which the TRAF4 TRAF domain acts as a dominant negative for extracellular ligand-induced signaling. In addition, TRAF4 does not appear to mimic other TRAF members, as it does not, for instance, heterotrimerize with TRAF1, 2, or 3, and does not bind to TNFR1 or TNFR2 (22,32). These data raise the possibility that the ring and/or zinc fingers of TRAF4 may link p47 phox with upstream elements necessary for TNF␣-and Tatdependent signals, whereas the C-terminal TRAF domain may link p47 phox with distal events leading to JNK activation.
Both TNF␣ and HIV-1 Tat affect angiogenesis and vascular remodeling and therefore influence cell fate. Tat, for instance, triggers either proliferative or apoptotic pathways in endothelial cells (42,43), and TNF␣ initiates opposing death and survival pathways in endothelium (5). Notably, therefore, our screen of endothelial proteins interacting with TRAF4 recovered a number of proteins involved with proliferation and apoptosis. UBC9, for instance, binds to the death domains of TNFR1 and Fas and is thought to participate in apoptosis (44,45). Likewise, phospholipid scramblase facilitates phosphatidyl serine exposure during apoptosis (46) and NRAGE mediates nerve growth factor-induced apoptosis (47). In contrast, TARBP2 overexpression leads to cell transformation (48), and HCR has been linked with psoriasis, a skin condition marked by hyperproliferation and angiogenesis (49). Similarly, VRP was identified in a functional genetic screen of endothelial cells for angiogenic proteins (50).
The last two proteins retrieved, ArgBP2 and Hic-5, are both tyrosine kinase scaffolds. ArgBP2 binds the protooncogene c-Abl, which lies upstream of JNK (51). Interestingly, c-Abl is an oxidant-sensitive kinase, which upon activation by H 2 O 2 also mediates apoptosis (52). Hic-5 is a paralog of the cytoskeletal scaffold paxillin, which binds to and is a substrate for the related adhesion focal tyrosine kinase (RAFTK/Pyk2) (53). RAFTK lies upstream of JNK and mediates signals originating from both TNF␣ and VEGF, the latter sharing a receptor with HIV-1 Tat (54,55). Like c-Abl, RAFTK is activated by oxidants and can mediate either apoptosis or proliferation (54,56,57). Of interest, TRAF4 is robustly expressed in mature granulocytes (22), and RAFTK participates in TNF␣-induced activation of the NADPH oxidase in adherent neutrophils (58), raising the possibility of a similar functional and structural complex in myeloid cells.
In summary, endothelial cell p47 phox interacts with TRAF4 through a tail-to-tail interaction. Coexpression or forced association of these two proteins causes downstream activation of JNK, whereas disruption of this interaction blocks JNK activation by the extracellular ligands TNF␣ or HIV-1 Tat. TRAF4 itself may bind to a number of proteins mediating proliferation and/or apoptosis. We speculate that TRAF4 may tether p47 phox to signaling complexes involved in cell fate decisions as a way of focusing the transient effects of oxidants upon relevant proteins.