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. 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. tumor necrosis factor human immunodeficiency virus Jun N-terminal kinase untranslated region hemagglutinin green fluorescent protein glutathione S-transferase mitogen-activated protein kinase TNF receptor-associated factor 4 human umbilical vein endothelial cell The vascular endothelium is generally well supplied with oxygen and produces significant quantities of oxidants when stimulatedin vivo or in vitro (1Al-Mehdi A.B. Zhao G. Dodia C. Tozawa K. Costa K. Muzykantov V. Ross C. Blecha F. Dinauer M. Fisher A.B. Circ. Res. 1998; 83: 730-737Crossref PubMed Scopus (248) Google Scholar, 2Gu Y., Wu, R.F., Xu, Y.C. Flores S.C. Terada L.S. Virology. 2001; 286: 62-71Crossref PubMed Scopus (42) Google Scholar). 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 (3Bhunia A.K. Arai T. Bulkley G. Chatterjee S. J. Biol. Chem. 1998; 273: 34349-34357Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 4Abid M.R. Kachra Z. Spokes K.C. Aird W.C. FEBS Lett. 2000; 486: 252-256Crossref PubMed Scopus (182) Google Scholar, 5Deshpande S.S. Angkeow P. Huang J. Ozaki M. Irani K. FASEB J. 2000; 14: 1705-1714Crossref PubMed Scopus (205) Google Scholar). However, the relatively broad biochemical reactivity of these oxidants poses a potential problem for signal specificity. As an example, a number of studies now support the participation of oxidants in both proliferative (6Ushio-Fukai M. Zafari A.M. Fukui T. Ishizaka N. Griendling K.K. J. Biol. Chem. 1996; 271: 23317-23321Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar, 7Sundaresan M., Yu, Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2322) Google Scholar, 8Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1441) Google Scholar) and apoptotic (9Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2092) Google Scholar, 10Manna S.K. Zhang H.J. Yan T. Oberley L.W. Aggarwal B.B. J. Biol. Chem. 1998; 273: 13245-13254Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar) 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 (11Mohazzab K.M. Kaminski P.M. Wolin M.S. Am. J. Physiol. 1994; 266: H2568-H2572PubMed Google Scholar) thought to participate in a number of these signal pathways. Inhibitors of this oxidase suppress growth factor, TNFα,1 HIV-1 Tat, and shear cessation-induced signaling (2Gu Y., Wu, R.F., Xu, Y.C. Flores S.C. Terada L.S. Virology. 2001; 286: 62-71Crossref PubMed Scopus (42) Google Scholar, 4Abid M.R. Kachra Z. Spokes K.C. Aird W.C. FEBS Lett. 2000; 486: 252-256Crossref PubMed Scopus (182) Google Scholar, 12Gu Y., Xu, Y.C., Wu, R.F. Souza R.F. Nwariaku F.E. Terada L.S. Exp. Cell Res. 2002; 272: 62-74Crossref PubMed Scopus (68) Google Scholar, 13Wei Z. Costa K., Al- Mehdi A.B. Dodia C. Muzykantov V. Fisher A.B. Circ. Res. 1999; 85: 682-689Crossref PubMed Scopus (130) Google Scholar), and dominant negative Rac1 disrupts TNFα signaling (5Deshpande S.S. Angkeow P. Huang J. Ozaki M. Irani K. FASEB J. 2000; 14: 1705-1714Crossref PubMed Scopus (205) Google Scholar) in endothelial cells. Recently, both cytochrome subunits of the oxidase, p22 phox , and gp91 phox , were cloned from rat and human endothelial cells (14Bayraktutan U. Blayney L. Shah A.M. Arterio. Thromb. Vasc. Biol. 2000; 20: 1903-1911Crossref PubMed Scopus (209) Google Scholar, 15Gorlach A. Brandes R.P. Nguyen K. Amidi M. Dehghani F. Busse R. Circ. Res. 2000; 87: 26-32Crossref PubMed Scopus (543) Google Scholar). We subsequently cloned the oxidase adapter subunit p47 phox from HUVEC, demonstrating its participation in TNFα signaling (12Gu Y., Xu, Y.C., Wu, R.F. Souza R.F. Nwariaku F.E. Terada L.S. Exp. Cell Res. 2002; 272: 62-74Crossref PubMed Scopus (68) Google Scholar). 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 (12Gu Y., Xu, Y.C., Wu, R.F. Souza R.F. Nwariaku F.E. Terada L.S. Exp. Cell Res. 2002; 272: 62-74Crossref PubMed Scopus (68) Google Scholar). 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 oxidase-activating endothelial agonists TNFα and HIV-1 Tat. 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 −2), creating a newNcoI site. The NcoI-EcoRI fragment containing the coding region and 3′-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 theEcoRI 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 (16Yang Y.S. Yang M.C. Wang B. Weissler J.C. Am. J. Respir. Cell Mol. Biol. 2001; 24: 30-37Crossref PubMed Scopus (27) Google Scholar) to create pCINF-T4(CT). pGBKT7-p47-(1–205) was constructed by removing the C-terminalBamHI-BamHI fragment from pGBKT7-p47, and pGBKT7-p47-(205–390) was obtained by isolation of the N-terminalSalI-BamHI fragment of p47 phox and ligation into pGBKT7. pGBKT7-p47-(1–346) was derived by excision of aSmaI-SmaI segment from pGBKT7-p47. pGBKT7-p47-(1–298) was derived by complete restriction of pGBKT7-p47 with EcoRI, partial restriction with NarI, T4 polymerase end fill-in, gel purification, and blunt-end ligation to reseal the plasmid. pGBKT7-p47-(347–390) was obtained by PCR deletion of p47-(1–346) from pGBKT7-p47 and frame correction by NcoI restriction, end fill-in, and blunt-end resealing. GAL4-BD fusions for p47-(153–286), (299–345), and (299–390) were derived by PCR amplification of segments between EcoRI and SalI sites, with insertion of appropriate stop codons, followed by ligation into pGBKT7. GAL4-AD fusions with TRAF4-(266–307) and TRAF4-(308–470) were produced by PCR amplification of segments between EcoRI and XhoI sites and ligation into pGADT7 (CLONTECH). The coding region of p47 phox was PCR-amplified betweenEcoRI and SalI sites and ligated into pCIneo-FLAG to yield pCINF-p47, and between two EcoRI sites with ligation into pGEX-2TK to yield pGEX-p47. HA-JNK2 was derived by reversal of HA-JNK2(APF) mutant (gift from Dr. Lynn Heasley) back to wild type with PCR mutagenesis, and HA-JNK1 was a gift from Dr. Stephen Dreskin (17Dreskin S.C. Thomas G.W. Dale S.N. Heasley L.E. J. Immunol. 2001; 166: 5646-5653Crossref PubMed Scopus (87) Google Scholar). pGAD424-TRAF1 and pGAD424-TRAF2 were gifts from Dr. Preet Chaudry. The frame of the former was to The fusion was produced by PCR amplification of p47 phox between EcoRI sites and ligation into between the and TRAF4 C was constructed as previously (12Gu Y., Xu, Y.C., Wu, R.F. Souza R.F. Nwariaku F.E. Terada L.S. Exp. Cell Res. 2002; 272: 62-74Crossref PubMed Scopus (68) Google Scholar). All were by HUVEC were with and cells were and (12Gu Y., Xu, Y.C., Wu, R.F. Souza R.F. Nwariaku F.E. Terada L.S. Exp. Cell Res. 2002; 272: 62-74Crossref PubMed Scopus (68) Google Scholar), with and using a A human lung library cloned into was obtained (CLONTECH). endothelial cell library was constructed using from HUVEC was cloned into the EcoRI and XhoI sites of (Stratagene). The library was as and the library was in the yeast shuttle vector by excision using a (Stratagene). The was (CLONTECH) were with the bait vector pGBKT7-p47 and p47 phox was found to were then with the lung or endothelial cell libraries using yeast two and the control of that using in and were for using a were and single were for from were and for GAL4-AD and back into negative were then with yeast containing pGBKT7-p47 and A was to the HUVEC library using TRAF4 as bait of with pGBKT7-T4. cells with were in and for at 4 for and at at 4 was then performed using protein at were performed with the C terminus of TRAF4 or were as previously (12Gu Y., Xu, Y.C., Wu, R.F. Souza R.F. Nwariaku F.E. Terada L.S. Exp. Cell Res. 2002; 272: 62-74Crossref PubMed Scopus (68) Google Scholar), and cytosolic were recovered by of yeast with were for negative were with yeast with deletion were on for both and expression with a were with pGBKT7 and the control was with were by of a negative for were in vitro S. A. J.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). (Stratagene) were with pGEX-2TK or for at and the proteins were on of or was of binding Full-length TRAF4 was and in vitro from pCI-T4 using and of was to each binding In some was in vitro and from and directly to the binding with TRAF4. A mutation was in p47 phox to a potential site. The p47 phox was excised with and and ligated into and the expression was then subcloned into the (CLONTECH). The was then into cells and and was constructed to the (CLONTECH). expression of p47 phox was by at of of and was in of cells. JNK of cells was using a kinase using and (2Gu Y., Wu, R.F., Xu, Y.C. Flores S.C. Terada L.S. Virology. 2001; 286: 62-71Crossref PubMed Scopus (42) Google Scholar). of JNK was with using a To HUVEC HUVEC were at the with J.L. V. 1994; 17: Google Scholar). cells were with of each for each HUVEC were with HA-JNK1 or were with human TNFα or HIV-1 Tat, as a fusion as previously (2Gu Y., Wu, R.F., Xu, Y.C. Flores S.C. Terada L.S. Virology. 2001; 286: 62-71Crossref PubMed Scopus (42) Google Scholar). The JNK of was then We found that to the with ECV-304 cells (12Gu Y., Xu, Y.C., Wu, R.F. Souza R.F. Nwariaku F.E. Terada L.S. Exp. Cell Res. 2002; 272: 62-74Crossref PubMed Scopus (68) Google Scholar), p47 phox appears to constitutively with the cytoskeleton of with as well as in unstimulated HUVEC To binding partners for p47 phox , a lung library was for an screen of its of were and and were single were found to be for PCR amplification and restriction an containing an in-frame library this was and found to the C-terminal of TRAF4. To a were screened from the HUVEC and were found to were and two were subsequently found to be were by restriction of the C-terminal of TRAF4. of binding of p47 phox to and in cells, TRAF4 with the with a of the protein found in the the cytoskeletal of p47 phox (12Gu Y., Xu, Y.C., Wu, R.F. Souza R.F. Nwariaku F.E. Terada L.S. Exp. Cell Res. 2002; 272: 62-74Crossref PubMed Scopus (68) Google Scholar). TRAF4 was found to with TRAF4 was found to with using at In addition, TRAF4 with the C-terminal TRAF domain of TRAF4 suggesting this were to of p47 phox and TRAF4. The C terminus of p47 phox containing a an basic and a C-terminal was both and for interaction with TRAF4 the C terminus of p47 phox was by and the segment was by or to the of the protein N-terminal to The a interaction of p47 phox with vitro In addition, in with p47 phox for TRAF4 with a specific interaction of the C terminus of p47 phox with TRAF4. The of the TRAF4 library obtained to both the and the TRAF domain of TRAF4, an interaction of p47 phox with this The TRAF of a C-terminal by a and secondary these and of TRAF4 C. C. C. P. M.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus (205) Google Scholar). studies suggested that the and were each to p47 phox , whereas the TRAF domain p47 phox of p47 phox or TRAF4 alone in cells not JNK In contrast, overexpression of both p47 phox and TRAF4 increased JNK suggesting a as well as interaction between the two proteins of p47 phox and TRAF4 increased with an in oxidant To that interaction of p47 phox with the TRAF4 TRAF domain was for JNK activation, we this TRAF domain to the C terminus of p47 phox of the fusion protein increased JNK in HUVEC whereas overexpression of p47 phox or the TRAF4 TRAF domain alone This activation was decreased by the antioxidant N-acetyl To further phox in endogenous endothelial cell signaling pathways, we the of TRAF4 p47 phox binding domain overexpression on signaling by TNFα and HIV-1 Tat, two agonists that activate endothelial cell JNK p47 phox oxidant production (2Gu Y., Wu, R.F., Xu, Y.C. Flores S.C. Terada L.S. 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In endothelial cell p47 phox with TRAF4 a tail-to-tail Coexpression or association of these two proteins downstream activation of JNK, whereas disruption of this interaction JNK activation by the TNFα or HIV-1 Tat. TRAF4 may to a number of proteins We speculate that TRAF4 may p47 phox to signaling involved in cell as a of the of oxidants We the of