The Kinase Activity of Rip1 Is Not Required for Tumor Necrosis Factor-α-induced IκB Kinase or p38 MAP Kinase Activation or for the Ubiquitination of Rip1 by Traf2

The death domain kinase Rip1 is recruited to the tumor necrosis factor receptor type 1 and mediates the IκB kinase and p38 MAP kinase pathways. In response to tumor necrosis factor-α (TNF-α), we find Rip1 phosphorylated and ubiquitinated, suggesting that Rip1 phosphorylation may stimulate its ubiquitination. To address the contribution of the kinase activity of Rip1 to its ubiquitination and to TNF-α signaling, we introduced wild type Rip1 and a kinase-inactive form of Rip1, Rip1D138N, into rip1-/- murine embryonic fibroblast cells by retroviral infection. TNF-α-induced ubiquitination of Rip1 is observed in Rip1D138N cells, supporting the argument that Rip1 autophosphorylation is not required for Rip1 ubiquitination. TNF-α-induced Ikk and p38 MAP kinase activation is normal, and the Rip1D138N cells are resistant to TNF-α-induced cell death, indicating that the kinase activity of Rip1 is not required to mediate its antiapoptotic functions. In the absence of Traf2, TNF-α-induced ubiquitination of Rip1 is impaired, suggesting that Traf2 may be the E3 ubiquitin ligase responsible for the TNF-α-dependent, ubiquitination of Rip1. Finally, recruitment of the ubiquitinated Tak1 complex is dependent on the presence of Rip1, suggesting that Rip1 ubiquitination rather than its phosphorylation is critical in signaling. The death domain kinase Rip1 is recruited to the tumor necrosis factor receptor type 1 and mediates the IκB kinase and p38 MAP kinase pathways. In response to tumor necrosis factor-α (TNF-α), we find Rip1 phosphorylated and ubiquitinated, suggesting that Rip1 phosphorylation may stimulate its ubiquitination. To address the contribution of the kinase activity of Rip1 to its ubiquitination and to TNF-α signaling, we introduced wild type Rip1 and a kinase-inactive form of Rip1, Rip1D138N, into rip1-/- murine embryonic fibroblast cells by retroviral infection. TNF-α-induced ubiquitination of Rip1 is observed in Rip1D138N cells, supporting the argument that Rip1 autophosphorylation is not required for Rip1 ubiquitination. TNF-α-induced Ikk and p38 MAP kinase activation is normal, and the Rip1D138N cells are resistant to TNF-α-induced cell death, indicating that the kinase activity of Rip1 is not required to mediate its antiapoptotic functions. In the absence of Traf2, TNF-α-induced ubiquitination of Rip1 is impaired, suggesting that Traf2 may be the E3 ubiquitin ligase responsible for the TNF-α-dependent, ubiquitination of Rip1. Finally, recruitment of the ubiquitinated Tak1 complex is dependent on the presence of Rip1, suggesting that Rip1 ubiquitination rather than its phosphorylation is critical in signaling. The proinflammatory cytokine tumor necrosis factor-α (TNF-α) 1The abbreviations used are: TNF, tumor necrosis factor; Tnfr1, TNF receptor type 1; mTNF, murine TNF; MAP, mitogen-activated protein; MEFs, murine embryonic fibroblasts; IL, interleukin; GFP, green fluorescent protein; Ab, antibody; IKK, IκB kinase; Mkk, MAP kinase kinase; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; IRES, internal ribosome entry site; MSCV, murine stem cell virus. is a major mediator of apoptosis as well as inflammation and immunity. It has also been implicated in the pathogenesis of several human diseases including inflammatory bowel disease, arthritis, sepsis, diabetes, and cancer (1Feldmann M. Maini R.N. Annu. Rev. Immunol. 2001; 19: 163-196Crossref PubMed Scopus (1158) Google Scholar). TNF-α activates the transcription factors NFκB and activator protein-1 and exerts its effects by binding to two receptors, the tumor necrosis factor receptor type 1 (Tnfr1 or p55) and the Tnfr type 2 (Tnfr2 or p75) (2Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-962Abstract Full Text PDF PubMed Scopus (1841) Google Scholar). Signaling from the activated Tnfr1 is mediated by the adapter proteins Traf2 and the death domain serine/threonine kinase Rip1 (3Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1754) Google Scholar, 4Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (989) Google Scholar, 5Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1740) Google Scholar); yet, how Rip1 and/or Traf2 mediates activation of the downstream kinases remains unclear. Ubiquitination has been implicated in the regulation of the NFκB pathway as well as in many other biological processes. Ubiquitin is a 76-amino acid protein that is highly conserved and, like phosphorylation, is essential for the degradation of proteins that respond to changes or stresses in the micoenvironment (6Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1264) Google Scholar). Phosphorylation can stimulate or inhibit ubiquitination by affecting either the target protein or the ubiquitin enzymes. Typically, ubiquitin forms a linkage with proteins via one or more lysine residues. In vivo, Lys-48 polyubiquitin chains lead to the recognition and degradation of proteins by a proteasome. In contrast, Lys-63 linkages are not associated with the proteasomal degradation but have been implicated in other biological processes, including activation of the IκB kinase by the E3 ubiquitin ligase Traf6 (7Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1526) Google Scholar, 8Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1660) Google Scholar). On TNF-α treatment, we found that Rip1 undergoes autophosphorylation and ubiquitination, suggesting that one or both of these posttranslational modifications may be important in Ikk or Mkk-6 activation. Studies in transfected cells suggest that the kinase activity of Rip1 is not required for TNF-α-induced Ikk or p38 MAP kinase activation (9Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (472) Google Scholar, 10Yuasa T. Ohno S. Kehrl J.H. Kyriakis J.M. J. Biol. Chem. 1998; 273: 22681-22692Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar); however, transfection of kinase-inactive Rip1 results in its oligomerization with Traf proteins and the subsequent activation of these pathways (5Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1740) Google Scholar, 10Yuasa T. Ohno S. Kehrl J.H. Kyriakis J.M. J. Biol. Chem. 1998; 273: 22681-22692Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 11Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (873) Google Scholar). To determine whether Rip1 autophosphorylation triggers its ubiquitination and to test whether Rip1 ubiquitination contributes to MAP kinase activation, we introduced wild type rip1 and a kinase-inactive rip1 into rip1-deficient murine embryonic fibroblasts (MEFs) and examined TNF-α-induced ubiquitination of Rip1 as well as activation of Ikk and p38 MAP kinase. In response to TNF-α, both Rip1 and the kinase-inactive form of Rip1 are ubiquitinated, although phosphorylated Rip1 may be a preferred E3 substrate. In addition, TNF-α-induced activation of Ikk and p38 MAP is not affected in cells expressing an inactive Rip1 kinase, demonstrating that Rip1 autophosphorylation is not required for signaling. Although it is recruited to the Tnfr1, Rip1 ubiquitination is not observed when Traf-deficient cells are stimulated with TNF-α. Taken together, these studies suggest that TNF-α-induced Ikk and Mkk-6 activation is mediated by the ubiquitination of Rip1 and the subsequent recruitment of a ubiquitinated Tak1 complex. Preparation of Murine Embryonic Fibroblasts—Targeted disruption of the rip1 loci has been described previously (12Kelliher M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (928) Google Scholar). Embryonic day-14 wild type rip1+/- and rip1-/- embryos were equilibrated overnight in trypsin at 4° C. Trypsin was activated by incubating at 37 °C for 15 min. The embryos were then dissociated in Dulbecco's modified Eagle's medium (BioWhittaker) containing 10% fetal calf serum. MEFs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone), 1% penicillin/streptomycin, and 1% l-glutamine (Invitrogen). Experiments were performed on cells between passage 2 and passage 5. Immortalized wild type and rip1-/- 3T3 cell lines were prepared according to established protocols. traf2-deficient and traf2/traf5-/- MEFs were generously provided by Dr. Wen-chen Yeh (13Yeh W.C. Shahinian A. Speiser D. Kraunus J. Billia F. Wakeham A. de la Pompa J.L. Ferrick D. Hum B. Iscove N. Ohashi P. Rothe M. Goeddel D.V. Mak T.W. Immunity. 1997; 7: 715-725Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar) (Toronto University, Toronto, Ontario, Canada) and Dr. H. Nakano (Tokyo, Japan), respectively. Plasmids and Constructs—The full-length RIP-Myc, ΔKIN-Myc, and ΔDD-Myc constructs and RIP-KD have been described previously (9Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (472) Google Scholar, 11Stanger B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (873) Google Scholar). The HA-ubiquitin construct was provided by Dr. Zhijan James Chen (University of Texas, Southwestern, Dallas). In Vitro Kinase Assay—Wild type or rip1-/- cells infected with rip1 or rip1D138N retrovirus were plated at 1 × 106 cells/10-cm plate. The cells were left untreated or treated with 10 ng/ml TNF-α for the time periods indicated. Cells were washed once with cold phosphate-buffered saline, and an in vitro kinase assay was performed as described previously (14Lee T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google Scholar). Interleukin-6 Production Assays—Wild type, rip1-/-, or rip1-/- cells infected with rip1 or rip1D138N retrovirus were plated at 3 × 104 cells/well on 24-well plates. The cells were left untreated or treated with 10 ng/ml TNF-α for 20 h. The supernatants were collected, and the levels of IL-6 produced from each cell line were assayed by an OptEIA mouse IL-6 enzyme-linked immunosorbent assay kit (BD Biosciences). Co-immunoprecipitation and Western Blot Analysis—For all transfection studies, 293T cells were plated at 3 × 105 cells/plate on 6-well plates. The transfected cells were lysed in 0.2 ml E1A buffer (50 mm HEPES (pH 7.6), 250 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA), immunoprecipitated with α-RIP Ab (BD Biosciences), and immunoblotted with α-HA Ab. Expression of transfected constructs was confirmed by immunoblotting with an α-HA Ab. Rip1 proteins were detected by immunoblotting with an α-RIP1 antibody. To determine whether Rip1 ubiquitination occurs at the Tnfr1, wild type cells or cells expressing kinase-inactive Rip1 were left untreated or treated with mTNF-α for the time periods indicated. Cells were lysed in 0.5 ml of endogenous lysis buffer (0.5 m Tris (pH 7.6), 0.5 m NaCl, 0.1 m EDTA, 1% Triton X-100, 0.5 m NaF, and 0.5 m sodium pyrophosphate) supplemented with a protease mixture inhibitor (Roche Applied Science), immunoprecipitated with α-TNFR1 antibody, and then immunoblotted with α-RIP1, α-TAK1, or α-TRAF2 antibody (Santa Cruz Biotechnology). In other experiments, cells were lysed in radioimmune precipitation assay buffer and immunoprecipitated with α-RIP Ab, and ubiquitinated Rip1 proteins were detected by immunoblotting with an anti-ubiquitin antibody (Boston Biochem). To determine p38-α-MAP kinase activity, 40 μg of total protein was immunoblotted with an α-phosphop38 antibody (Cell Signaling Technology). To determine the relative expression levels of p38-α MAP kinase or Ikk, wild type rip1-/- or rip1-/- infected with rip1 or rip1D138N retroviruses were lysed, and 30 μg of total protein was immunoblotted with α-p38α Ab (Santa Cruz Biotechnology) or with α-IKK-α antibody (Santa Cruz Biotechnology). Cell Viability Assays—Briefly, 5 × 105 cells were plated overnight on 60-mm tissue culture dishes. Cells were then treated with 10 ng/ml mTNF and 250 ng/ml cycloheximide for 20 h. Cells were then collected and assayed either for cell viability by trypan blue staining or for cell death by annexin V/phosphatidylinositol staining on triplicate cultures. Death Domain Kinase Rip1 and on Tnfr1 and studies have implicated the Rip1 kinase in TNF-α H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (989) Google Scholar, A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (472) Google Scholar, 10Yuasa T. Ohno S. Kehrl J.H. Kyriakis J.M. J. Biol. Chem. 1998; 273: 22681-22692Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (928) Google Scholar, T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google the by Rip1 mediates the NFκB or p38 MAP kinase to TNF unclear. phosphorylation and, more ubiquitination have been implicated in the activation of the complex. To the of Rip1 in the of these wild type and rip1-deficient MEFs were left or treated with TNF-α for and 10 and Rip1 protein was detected by In the wild type cells, we observed the presence of two forms of Rip1. On TNF-α we observed of a form of Rip1. The presence of form was when the cell were treated with with the that Tnfr1 activation Rip1 phosphorylation and The of Rip1 phosphorylation with at 5 TNF-α suggest is an in the TNF-α response and may to signaling. To whether Rip1 undergoes autophosphorylation or is phosphorylated by kinase, we infected rip1-/- MEFs with a kinase-inactive of Rip1 or with retroviral Cells expressing kinase-inactive Rip1 to TNF-α-induced phosphorylation that Tnfr1 activation Rip1 In to phosphorylation and to studies M.A. J. C. Immunity. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. D. Immunity. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P. Goeddel D.V. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google the Rip1 kinase is when cells are stimulated with TNF-α Rip1 ubiquitination in response to TNF-α is by as that suggesting that Rip1 ubiquitination occurs once the activated Tnfr1 is recruited into Kinase of Rip1 to TNF-α-induced Ikk or p38 MAP Kinase or TNF-α-induced IL-6 death domain kinase Rip1 undergoes two of posttranslational modifications in response to TNF-α, but the of phosphorylated or ubiquitinated Rip1 in TNF are unclear. have previously that TNF-α-induced Ikk and p38 MAP kinase activation are both in rip1-deficient MEFs (12Kelliher M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (928) Google Scholar, T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google Scholar). To the of the kinase activity of Rip1 in TNF-α-induced Ikk and p38 MAP kinase activation, we infected rip1-/- MEFs with retroviruses that wild type a kinase-inactive or with the retroviral Cell were prepared from wild type MEFs and from the infected rip1-/- MEFs, and the Rip1 protein expression levels were rip1-/- MEFs with wild type rip1 or kinase-inactive rip1 levels of Rip1 protein to of wild type MEFs demonstrating that Rip1 and oligomerization are not in the infected type, rip1-/-, and rip1-/- MEFs infected with wild type or with the rip1D138N retrovirus were left untreated or were treated with mTNF-α for and min. To TNF-α-induced Ikk activation, cell were immunoprecipitated with antibody, and an in vitro kinase assay was performed as the substrate. TNF-α-induced Ikk activation was not observed in cells Rip1 2 and M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (928) Google and NFκB activity was in rip1-/- cells with wild type Rip1 or with a kinase-inactive of Rip1 To TNF-α-induced p38 MAP kinase activity, cell were with a p38 MAP kinase antibody. TNF-α-induced p38 MAP kinase activation was not observed in rip1-/- MEFs described in T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google Scholar); however, p38 MAP kinase activity was observed in wild type MEFs and in rip1-/- MEFs infected with the rip1 retrovirus as well as the MEFs expressing the kinase-inactive of Rip1. The NFκB and p38 MAP kinase pathways have been implicated in cytokine A. S. Lee P. EMBO J. 1996; 15: PubMed Scopus Google Scholar, J. S. M. J. R.J. Davis R.J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google and TNF-α-induced IL-6 is in cells (14Lee T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google Scholar). To test whether the kinase activity of Rip1 contributes to TNF-α-induced cytokine we examined IL-6 in response to TNF-α in the of wild type cells with TNF-α in of or TNF-α-induced IL-6 was observed in rip1-/- cells and T.H. Huang Q. Oikemus S. Shank J. Ventura J.J. Cusson N. Vaillancourt R.R. Su B. Davis R.J. Kelliher M.A. Mol. Cell. Biol. 2003; 23: 8377-8385Crossref PubMed Scopus (76) Google Scholar). TNF-α-induced IL-6 was in the rip1-/- MEFs infected with wild type rip1 or rip1D138N retroviruses Although the cells a of IL-6 a in IL-6 was observed when wild type MEFs or the MEFs were treated with TNF-α. with the p38 MAP kinase and NFκB to TNF observed in rip1D138N cells, these suggest that the Rip1 kinase activity not to TNF-α-induced p38 MAP kinase or Ikk activation. Kinase of Rip1 to the to transfection studies implicated the kinase activity of Rip1 in apoptosis and necrosis B.Z. Leder P. Lee T.H. Kim E. Seed B. Cell. 1995; 81: 513-523Abstract Full Text PDF PubMed Scopus (873) Google Scholar, N. M. A. S. J.L. P. Seed B. J. Nat. Immunol. 2000; PubMed Scopus Google Scholar). To whether the Rip1 kinase cell death in cells, we treated wild type and rip1-/- MEFs with TNF-α and cycloheximide and the a rip1 cells to TNF-α-induced cell death, with the NFκB response and M.A. Grimm S. Ishida Y. Kuo F. Stanger B.Z. Leder P. Immunity. 1998; 8: 297-303Abstract Full Text Full Text PDF PubMed Scopus (928) Google Scholar). The of rip1 into these cells is to of the cells and with the that of the cells are and Rip1 Cells expressing a kinase-inactive Rip1 resistant to TNF-α-induced cell death, of activation of indicating that kinase activity of Rip1 not to the antiapoptotic response to TNF-α Rip1 Phosphorylation for the TNF-α-induced ubiquitination of the inhibitor phosphorylation recognition by the complex A. A. Davis M. S. A.M. M. F. Y. Nature. 1998; PubMed Scopus Google Scholar). To test whether Rip1 phosphorylation the protein for ubiquitination, cells were left untreated or stimulated with TNF-α for 30 or and immunoprecipitated with an α-RIP antibody, and then ubiquitinated Rip1 proteins were detected by immunoblotting with an anti-ubiquitin antibody Rip1 was by TNF-α and Rip1 was also detected in cells expressing a kinase-inactive Rip1D138N TNF-α-induced ubiquitination of Rip1 and Rip1D138N was when the cells were with indicating that Rip1 ubiquitination may with the kinase-inactive Rip1 is in response to TNF-α, that Rip1 phosphorylation is not required for TNF-α-induced ubiquitination. The that the ubiquitination of kinase-inactive Rip1D138N may be in response to TNF-α, indicating that kinase-inactive Rip1D138N may be in its to be recruited to the To test wild type and kinase-inactive Rip1D138N cells were treated with TNF-α, immunoprecipitated with an Ab and then immunoblotted with an antibody. observed a of at 10 with the activation of TNF-α-induced however, Rip1 ubiquitination is Rip1 was not detected at the Tnfr1 at time with the Rip1 ubiquitination was also observed in cells expressing a kinase-inactive Rip1D138N indicating that the kinase-inactive Rip1 is recruited to the Tnfr1 and undergoes ubiquitination in response to TNF-α. IκB Rip1 is phosphorylated in response to TNF-α, but Rip1 phosphorylation is not required for its ubiquitination at the Traf2 Rip1 at the Rip1 ubiquitination has been observed by M.A. J. C. Immunity. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, A. D. Immunity. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, P. Goeddel D.V. Mol. Cell. Full Text Full Text PDF PubMed Scopus Google the of Rip1 phosphorylation in its ubiquitination has not been has the E3 ubiquitin ligase for Rip1 been Traf2 and Traf6 have been to as E3 ubiquitin and to (7Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1526) Google Scholar, 8Wang C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1660) Google Scholar). TNF-α has been to stimulate Traf2 and oligomerization Kehrl J.H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). To that Traf proteins Rip1 in response to TNF-α, we examined Rip1 ubiquitination in and traf2-deficient cell lines were left untreated or stimulated with mTNF for 10 or 20 and the proteins were immunoprecipitated with an α-TNFR1 antibody. with α-RIP antibody detected Rip1 in wild type MEFs treated with TNF-α for 10 min. contrast, Rip1 was not detected in or in cells treated with TNF-α To more that Rip1 is not detected in cells, we stimulated wild type and or cells with TNF-α and immunoprecipitated the with the α-RIP antibody by immunoblotting with an anti-ubiquitin antibody. Rip1 protein was detected in wild type cells but not in or cells and Taken together, these suggest that Traf2 may be the E3 ubiquitin ligase that TNF-α signaling, by Rip1. In the of Rip1 a Tak1 to the studies to a for the Rip1 kinase in TNF-α signaling, but suggest that ubiquitinated Rip1 may other ubiquitinated proteins of the Ikk complex or To test whether ubiquitinated Rip1 the Tak1 kinase to be activated by ubiquitination, to the Tnfr1 C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1660) Google we treated wild type cells and rip1-/- cells with TNF-α for and 20 and immunoprecipitated the observed the recruitment of a ubiquitinated Tak1 complex in wild type cells stimulated with TNF-α for 5 In contrast, in the absence of Rip1, TNF-α to recruitment of the Tak1 complex suggesting that Rip1 and ubiquitinated Rip1 are required for Tak1 recruitment to the Traf2 however, is by an absence of Rip1 and is recruited to the Tnfr1 when rip1-/- MEFs are stimulated with TNF-α that the death domain kinase Rip1 undergoes autophosphorylation and ubiquitination as as TNF-α Rip1 autophosphorylation occurs the and to Rip1 ubiquitination at the posttranslational modifications are of observed for and suggest that Rip1 autophosphorylation may its however, cells that a kinase-inactive Rip1 TNF-α-induced ubiquitination and respond to TNF-α. that Rip1 autophosphorylation is not required for its recruitment to the activated Tnfr1 or for its ubiquitination in Rip1 is required for the recruitment of a ubiquitinated Tak1 complex to the activated Taken together, these studies suggest that Rip1 ubiquitination rather than its phosphorylation to Ikk and Mkk-6 activation. that the E3 ubiquitin ligase Traf2 may be responsible for the ubiquitination of Rip1. Rip1 ubiquitination in response to TNF-α is not observed in cells Traf2 or Traf 5 Although both proteins E3 ubiquitin ligase activity has been for Traf2 and Traf6 C. Deng L. Hong M. Akkaraju G.R. Inoue J. Chen Z.J. Nature. 2001; 412: 346-351Crossref PubMed Scopus (1660) Google Scholar, Kehrl J.H. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). suggest that Traf2 may TNF-α by Rip1 and other of the Tnfr1 TNF-α-induced ubiquitination of Rip1 is suggesting that Rip1 ubiquitination may be by as E. E. T. H. A. Nature. 2003; PubMed Scopus Google Scholar, A. C. A. D. Nature. 2003; PubMed Scopus Google Scholar). can NFκB activation by Rip1 or Traf2 A. C. A. D. Nature. 2003; PubMed Scopus Google suggesting that Rip1 may be a in Although Rip1 autophosphorylation is on TNF and the Rip1 serine/threonine kinase is recruited to the Tnfr1 on the kinase activity of Rip1 not to in TNF-α signaling, is it required for or of the TNF-α Cells expressing a kinase-inactive Rip1 respond to TNF-α and the NFκB and p38 MAP kinase with to wild type that the kinase activity of Rip1 not the TNF-α pathway but may mediate from other receptor pathways. Rip1 has been implicated in the response E. V. F. Kelliher M. J. Nat. Immunol. PubMed Google Scholar). the kinase activity of Rip1 may mediate

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