Ubiquitination of RIP Is Required for Tumor Necrosis Factor α-induced NF-κB Activation

Abstract

Stimulation of cells with tumor necrosis factor (TNFα) triggers a recruitment of various signaling molecules, such as RIP, to the TNFαreceptor 1 complex, leading to activation of NF-κB. Previous studies indicate that RIP plays an essential role for TNFα-induced NF-κB activation, but the molecular mechanism by which RIP mediates TNFαsignals to activate NF-κB is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to be determined whether the ubiquitination of RIP is required for TNFα-induced NF-κB activation. In this study, we have identified Lys377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-κB activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNFαreceptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNFα-induced NF-κB activation. Stimulation of cells with tumor necrosis factor (TNFα) triggers a recruitment of various signaling molecules, such as RIP, to the TNFαreceptor 1 complex, leading to activation of NF-κB. Previous studies indicate that RIP plays an essential role for TNFα-induced NF-κB activation, but the molecular mechanism by which RIP mediates TNFαsignals to activate NF-κB is not fully defined. Earlier studies suggest that RIP undergoes a ligand-dependent ubiquitination. However, it remains to be determined whether the ubiquitination of RIP is required for TNFα-induced NF-κB activation. In this study, we have identified Lys377 of RIP as the functional ubiquitination site, because mutating this residue to arginine completely abolished RIP-mediated NF-κB activation. The K377R mutation of RIP cannot undergo ligand-dependent ubiquitination and fails to recruit its downstream signaling components into the TNFαreceptor 1 complex. Together, our studies provide the first genetic evidence that the ubiquitination of RIP is required for TNFα-induced NF-κB activation. NF-κB is a homo- or heterodimeric transcription factor that controls the expression of various genes involved in inflammatory, apoptotic, and immune responses. In resting cells, the activity of NF-κB is controlled through its cytoplasmic sequestration by a family of inhibitors, IκB (Inhibitor of NF-κB) (1Baldwin A.J. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5592) Google Scholar). In response to extracellular stimuli, IκB proteins are phosphorylated by the IκB kinase (IKK) 2The abbreviations used are: IKK, IκB kinase; TNF, tumor necrosis factor; TNFR, TNF receptor; TAK1, tumor growth factor β-activated kinase 1; MEKK3, mitogen-activated protein kinase kinase kinase 3; E1, ubiquitin-activating enzyme; E2, ubiquitin conjugation enzyme; E3, ubiquitin ligase; HEK, human embryonic kidney; WT, wild type; FITC, fluorescein isothiocyanate; CTxB, cholera toxin B. complex, then ubiquitinated, and rapidly degraded, which leads to the nuclear localization and activation of NF-κB (2Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Crossref PubMed Scopus (4106) Google Scholar). One of the most potent NF-κB activators is the proinflammatory cytokine, tumor necrosis factor (TNF)α. TNFα functions through the TNF receptors, mainly TNFR1, on the cell surface. The binding of TNFα to TNFR1 causes trimerization of the receptor and recruitment of the adaptor protein TRADD (TNF receptor-associated death domain) (3Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1749) Google Scholar). TRADD further recruits TARF2 (TNF receptor-associated factor 2) (4Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar), FADD (Fas-associated death domain) (4Hsu H. Shu H.B. Pan M.G. Goeddel D.V. Cell. 1996; 84: 299-308Abstract Full Text Full Text PDF PubMed Scopus (1738) Google Scholar), and RIP (receptor-interacting protein) (5Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar, 6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar). RIP is a serine/threonine kinase that plays an essential role in TNFα-induced NF-κB activation (6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar, 7Kelliher 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 (926) Google Scholar). It contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain. Earlier studies indicate that the RIP kinase domain is dispensable for the activation of NF-κB (6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar, 8Lee T.H. Shank J. Cusson N. Kelliher M.A. J. Biol. Chem. 2004; 279: 33185-33191Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), whereas its death domain is required for the association with the upstream signaling component TRADD (5Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar). The intermediate domain is required for RIP-mediated NF-κB activation (6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar) and plays an important role for interacting with its downstream signaling components such as NEMO, the regulatory subunit of the IKK complex, and other molecules (9Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Therefore, RIP likely functions as an adaptor molecule to mediate the TNFα signaling cascade. Although genetic studies have demonstrated that TAK1 (tumor growth factor β-activated kinase 1) and MEKK3 are involved in mediating TNFα-induced NF-κB activation downstream of RIP (10Blonska M. Shambharkar P.B. Kobayashi M. Zhang D. Sakurai H. Su B. Lin X. J. Biol. Chem. 2005; 280: 43056-43063Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 11Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 12Morlon A. Munnich A. Smahi A. Hum. Mol. Genet. 2005; 14: 3751-3757Crossref PubMed Scopus (77) Google Scholar, 13Shim J.H. Xiao C. Paschal A.E. Bailey S.T. Rao P. Hayden M.S. Lee K.Y. Bussey C. Steckel M. Tanaka N. Yamada G. Akira S. Matsumoto K. Ghosh S. Genes Dev. 2005; 19: 2668-2681Crossref PubMed Scopus (597) Google Scholar, 14Yang J. Lin Y. Guo Z. Cheng J. Huang J. Deng L. Liao W. Chen Z. Liu Z. Su B. Nat. Immunol. 2001; 2: 620-624Crossref PubMed Scopus (352) Google Scholar, 15Sato S. Sanjo H. Takeda K. Ninomiya-Tsuji J. Yamamoto M. Kawai T. Matsumoto K. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 1087-1095Crossref PubMed Scopus (767) Google Scholar), the mechanism by which RIP activates these downstream kinases is not fully defined. Our recent studies show that RIP fusion proteins, which contain the death domain of RIP and MEKK3 or TAK1, can effectively rescue TNFα-induced NF-κB activation in RIP-deficient cells (10Blonska M. Shambharkar P.B. Kobayashi M. Zhang D. Sakurai H. Su B. Lin X. J. Biol. Chem. 2005; 280: 43056-43063Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 16Blonska M. You Y. Geleziunas R. Lin X. Mol. Cell Biol. 2004; 24: 10757-10765Crossref PubMed Scopus (39) Google Scholar), suggesting that one of the roles of RIP is to recruit these downstream kinases to the TNFR1 complex. Ubiquitination has been shown to play various roles in the regulation of NF-κB signaling pathways (17Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1026) Google Scholar) as well as many other biological processes. Ubiquitin is a 76-amino-acid protein that is highly conserved among different species and is covalently attached to lysine residues of target proteins (18Haglund K. Dikic I. EMBO J. 2005; 24: 3353-3359Crossref PubMed Scopus (605) Google Scholar). This process is catalyzed in three steps. First, ubiquitin is activated in an ATP-dependent reaction by an ubiquitin activation enzyme (E1). Then, the activated ubiquitin is transferred to an ubiquitin-conjugation enzyme (E2). Subsequently, the ubiquitin is attached to the target protein through an isopeptide bond between the C-terminal of ubiquitin and the [cepsilon]-amino group of the lysine residue in the target protein in the presence of ubiquitin ligase (E3) (17Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1026) Google Scholar). Ubiquitin itself contains several lysine residues, in which Lys48 or Lys63 could be linked to another ubiquitin to form polyubiquitin chain on target proteins. In vivo, Lys48-linked polyubiquitin chains lead to the recognition and degradation of proteins by the proteasome complex. In contrast, Lys63-linked polyubiquitination does not undergo the proteasome degradation but has been implicated in other biological processes (17Chen Z.J. Nat. Cell Biol. 2005; 7: 758-765Crossref PubMed Scopus (1026) Google Scholar). Both forms of polyubiquitination play important roles in TNFα-induced NF-κB activation. IκB is ubiquitinated through Lys48-linked polyubiquitination and degraded in a proteasome-dependent manner, whereas Lys63-linked polyubiquitination has been suggested to be involved in the modification of NEMO (11Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 19Zhou H. Wertz I. O'Rourke K. Ultsch M. Seshagiri S. Eby M. Xiao W. Dixit V.M. Nature. 2004; 427: 167-171Crossref PubMed Scopus (453) Google Scholar), which plays an important role in the activation of those proteins. It has been shown that RIP is also ubiquitinated following TNFα stimulation (8Lee T.H. Shank J. Cusson N. Kelliher M.A. J. Biol. Chem. 2004; 279: 33185-33191Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 11Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 20Legler D.F. Micheau O. Doucey M.A. Tschopp J. Bron C. Immunity. 2003; 18: 655-664Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). The ubiquitination of RIP may occur in both Lys63 and Lys48 linkages (21Wertz I.E. O'Rourke K.M. Zhou H. Eby M. L. Seshagiri S. P. C. R. A. Dixit V.M. Nature. 2004; PubMed Scopus Google Scholar). It has been suggested that RIP is ubiquitinated by through the Lys63 following TNFα stimulation (21Wertz I.E. O'Rourke K.M. Zhou H. Eby M. L. Seshagiri S. P. C. R. A. Dixit V.M. Nature. 2004; PubMed Scopus Google Scholar). This ubiquitination may provide a on RIP for its association with a protein (11Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). In contrast, RIP may also be ubiquitinated by through a Lys48 (21Wertz I.E. O'Rourke K.M. Zhou H. Eby M. L. Seshagiri S. P. C. R. A. Dixit V.M. Nature. 2004; PubMed Scopus Google Scholar), which is suggested to play an important role for the of TNFα-induced NF-κB activation and degradation of RIP K. R. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the is whether the ubiquitination of RIP is required for TNFα-induced NF-κB activation. In this study, we have a of RIP and RIP-deficient cells with these Our indicate that Lys377 of RIP is the functional ubiquitination and is required for the recruitment of downstream signaling components to the TNFR1 complex, leading to NF-κB activation. Together, our provide both and genetic evidence that the ubiquitination of RIP is required for TNFα-induced NF-κB activation. and for and TAK1 ubiquitin NEMO and Cell human TNFα The and of RIP by the into of and by the into of RIP the expression by RIP into the and in a which contains a of and a protein downstream of the Cell and cells by B. Seed (6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar). and cells in with and embryonic cells in with and the in and of cells by a or into 1 The cells for and then with for The to and for the presence of RIP proteins cells the of of Cell cells in 1 The cells in with of different forms of RIP with of and of the to of used to the by to cells in for the between and and in for The of the determined by the or of the expression of RIP proteins in the and with different and in a 1 1 1 1 and a The cell to and or with The with and with the on and transferred to with by and by the to the cells in with and with the genes the cell and with the NF-κB determined by of to or its cells following various in of 1 and and on for The and the cytoplasmic proteins The nuclear then in of 1 1 and for the and the nuclear proteins in the of nuclear determined by the protein as the The nuclear with a or as a for on a and by Cell cells with different forms of RIP into and with or TNFα for then and for cell by or for cell death by on cells or cell or used to the of TNFR1 and recruitment of signaling components into the 1 cells with cholera toxin for then and with TNFR1 for by show the of TNFR1, cells with for by cells to in a with for and with in for cells and by controls to TNFR1 and cells and with as by a biological Previous studies indicate that RIP undergoes a ubiquitination (8Lee T.H. Shank J. Cusson N. Kelliher M.A. J. Biol. Chem. 2004; 279: 33185-33191Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 11Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 20Legler D.F. Micheau O. Doucey M.A. Tschopp J. Bron C. Immunity. 2003; 18: 655-664Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). whether the ubiquitination of RIP is required for NF-κB activation, we to the ubiquitination of RIP is of three the N-terminal kinase domain an intermediate domain and the C-terminal death domain we a of RIP that different The expression these into cells with a the to to activate NF-κB. cell a in the with (5Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar, 6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar), of the intermediate domain and the death domain of RIP could NF-κB activation In contrast, of the kinase domain of RIP to activate NF-κB which domain of RIP contains the ubiquitination of RIP, the and of RIP the cells, to and with shown in both and the intermediate domain of RIP ubiquitinated, whereas the kinase domain and the death domain not indicate that the intermediate domain of RIP contains the ubiquitination which lysine in the intermediate domain of RIP is the functional ubiquitination site, we the of the intermediate domain of and that residues and are conserved among different species of which be the ubiquitination in the intermediate domain of Therefore, we first a of RIP intermediate domain in which residues or both and to Although of these as well as wild RIP intermediate domain, can NF-κB activation the RIP intermediate domain with Lys377 to the most for NF-κB activation the ubiquitination for the K377R the RIP with Lys377 to the most for NF-κB activation not Together, these suggest that Lys377 may be the ubiquitination in the intermediate domain of RIP and plays an important role for RIP-mediated NF-κB activation. that Lys377 of RIP plays a role for TNFα-induced NF-κB activation, we RIP-deficient cells with wild RIP and its in which residues in the intermediate domain to shown in of these of RIP are we TNFα-induced NF-κB activation by and determined the of of that expression of not as well as wild of RIP could effectively TNFα-induced NF-κB activation in cells In contrast, forms of RIP, lysine and to TNFα-induced NF-κB activation in cells The in Lys377 for TNFα because NF-κB activation in these cell with these in RIP-deficient cells or but in RIP-deficient cells or following TNFα stimulation Together, these indicate that lysine of RIP plays a role for mediating TNFα-induced NF-κB activation. TNFα activates both and The of cell or death following TNFα is on the of these Earlier studies indicate that the TNFα-induced is on NF-κB activation, which the expression of several or or proteins cells TNFα-induced TNFα stimulation cannot activate NF-κB in RIP-deficient cells, these cells are to TNFα-induced (6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar, 8Lee T.H. Shank J. Cusson N. Kelliher M.A. J. Biol. Chem. 2004; 279: 33185-33191Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). whether the form of RIP can rescue cells TNFα-induced cells with different of RIP with TNFα for the cell TNFα are to and used as the TNFα of in RIP and cells, whereas the expression of or cells TNFα-induced This is highly because not in these cells Together, these further the that Lys377 of RIP is important for TNFα-induced NF-κB activation. further whether the functional of the is because of the of ubiquitination following TNFα RIP ubiquitination in RIP-deficient cells or shown in the ubiquitinated form of RIP can be in cells with TNFα for whereas in cells cannot be ubiquitinated following TNFα and are with these TNFα effectively of in but not in cells and that Lys377 in the intermediate domain of RIP is the functional ubiquitination for TNFα-induced NF-κB activation. Earlier studies suggest that RIP may provide to recruit downstream signaling components such as TAK1 and NEMO (11Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, A. A. Lin Y. Y. Kelliher M. Liu Z. Immunity. 2000; 12: Full Text Full Text PDF PubMed Scopus Google Scholar), and our recent also suggest that the role of RIP is to recruit TAK1 to the TNFR1 leading to NF-κB activation (10Blonska M. Shambharkar P.B. Kobayashi M. Zhang D. Sakurai H. Su B. Lin X. J. Biol. Chem. 2005; 280: 43056-43063Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Therefore, we that the of TNFα-induced NF-κB activation in the RIP-deficient cells is because cannot effectively with TAK1 or this or into cells The RIP proteins the cells, to and with with and other of RIP, the between and TAK1 However, it also could with the NEMO suggest that the association of RIP with TAK1 is mainly on the ubiquitination of whereas the association of RIP with the IKK may be of Lys377 ubiquitination. The the and has been suggested to in the activation of NF-κB in the TNFα signaling It has been shown that TNFR1 and downstream signaling components are to the following TNFα and this recruitment may be required for NF-κB activation. the recruitment of signaling components to TNFR1 the stimulation of TNF receptor we the localization of signaling components cells first with which with the show the localization of TNFR1, cells with or TNFR1 by the with In cells, and TNFR1 are on the cell TNFR1, TNFR1 recruitment into Therefore, we can whether signaling components are into the TNFR1 by with following the stimulation of further whether Lys377 of RIP plays an essential role for the recruitment of downstream signaling components to TNFR1 complex, we RIP-deficient cells with or and in the cells not The cells also with and with or TNFR1 in cells, TNFR1 with following the stimulation of TNFR1 in both and cells the localization of signaling these cells with or In the cells, RIP, TAK1, and in the RIP TAK1, and to the to on the in the cells In contrast, the recruitment of TAK1 in the cells the recruitment of TNFR1, RIP and in these cells and Together, our indicate that the ubiquitination of Lys377 in RIP is required for the recruitment of its downstream signaling components to the TNFR1 in RIP is an essential signaling component for the NF-κB activation in TNFα signaling (6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar, 7Kelliher 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 (926) Google Scholar) and is ubiquitinated following TNFα stimulation (8Lee T.H. Shank J. Cusson N. Kelliher M.A. J. Biol. Chem. 2004; 279: 33185-33191Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 11Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar, 20Legler D.F. Micheau O. Doucey M.A. Tschopp J. Bron C. Immunity. 2003; 18: 655-664Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), which has been suggested to play an important role in the NF-κB activation. In this study, we have the role of RIP ubiquitination by the ubiquitination and TNFα-induced NF-κB activation in the RIP-deficient cells with various RIP Our indicate that Lys377 in the intermediate domain of RIP is the functional ubiquitination site, because expression of the of RIP, in which Lys377 with an arginine cannot the TNFα-induced NF-κB activation in RIP-deficient This is likely because this RIP fails to recruit the downstream signaling components into the TNFR1 complex. Together, our studies that ubiquitination of RIP is required for TNFα-induced NF-κB activation. RIP is of an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain. with studies (5Hsu H. Huang J. Shu H.B. Baichwal V. Goeddel D.V. Immunity. 1996; 4: 387-396Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar, 6Ting A.T. Pimentel-Muinos F.X. Seed B. EMBO J. 1996; 15: 6189-6196Crossref PubMed Scopus (471) Google Scholar), we that the kinase domain of RIP cannot activate NF-κB in it is not ubiquitinated In contrast, the intermediate domain is ubiquitinated and can also activate the that ubiquitination of RIP is important for the NF-κB activation. the death domain, which is required for the recruitment of RIP to the upstream component does not contain an ubiquitination site, it can activate NF-κB. One for the death NF-κB activation is that the death domain may lead to NF-κB activation Lin J.H. T. S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) by the with this we have that RIP forms through its death domain not The intermediate domain of RIP contains several lysine residues, lysine and which are conserved among different species However, the mutation on Lys377 in the intermediate domain leads to the for ubiquitination that Lys377 may be the ubiquitination in the intermediate domain. Although the NF-κB activation by of in with wild intermediate domain or other it activity to activate NF-κB This activity is likely because of the that other residues in the intermediate domain may the of Lys377 is in RIP-deficient cells are completely in TNFα-induced NF-κB activation, whereas on and not have for the NF-κB activation. with the functional TNFα-induced ubiquitination is also for It has been that may as an ligase for the Lys63-linked polyubiquitination of RIP, and this Lys63-linked polyubiquitination of RIP has been suggested to provide a for the recruitment of (11Kanayama A. Seth R.B. Sun L. Ea C.K. Hong M. Shaito A. Chiu Y.H. Deng L. Chen Z.J. Mol. Cell. 2004; 15: 535-548Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). Our studies indicate that the mutation of Lys377 the between RIP and TAK1 suggesting that the ubiquitination is likely involved in this Lys63-linked ubiquitination and is required for the association of with this fails to recruit TAK1 to the TNFα receptor following the stimulation of TNFR1 However, we have not been to that Lys377 undergoes the Lys63-linked polyubiquitination in vivo, because the of ubiquitin a of Lys63-linked It has been suggested that functions as a enzyme to the Lys63-linked polyubiquitination of RIP and then functions as an ligase to further RIP through the Lys48-linked This Lys48-linked polyubiquitination may play a role in the of TNFα-induced NF-κB activation by RIP for degradation (21Wertz I.E. O'Rourke K.M. Zhou H. Eby M. L. Seshagiri S. P. C. R. A. Dixit V.M. Nature. 2004; PubMed Scopus Google Scholar). the mutation of Lys377 does not the of RIP not we that the Lys48-linked polyubiquitination of RIP is through a in cells, it could be ubiquitinated not It remains to be determined whether this ubiquitination of RIP is Lys48-linked polyubiquitination and is the functional of ubiquitination in It has been shown that the can be to the TNFα receptor through a but mechanism A. A. Lin Y. Y. Kelliher M. Liu Z. Immunity. 2000; 12: Full Text Full Text PDF PubMed Scopus Google Scholar). However, it has also been shown that NEMO can with the intermediate domain of RIP (9Zhang S.Q. Kovalenko A. Cantarella G. Wallach D. Immunity. 2000; 12: 301-311Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Although the association of with the IKK is can with NEMO Therefore, these suggest that the association of RIP with IKK may not be completely on the ubiquitination of and suggest that RIP functions as an adaptor molecule to TAK1 and IKK by the and process following TNFα In this complex, TAK1 the of or leading to NF-κB activation. of signaling components to the is required for the activation of NF-κB following the of TNFR1 D.F. Micheau O. Doucey M.A. Tschopp J. Bron C. Immunity. 2003; 18: 655-664Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, G. J. 2004; 24: PubMed Scopus Google Scholar). with these we have that TNFR1 and other signaling components are with (10Blonska M. Shambharkar P.B. Kobayashi M. Zhang D. Sakurai H. Su B. Lin X. J. Biol. Chem. 2005; 280: 43056-43063Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Although and wild RIP effectively to following the stimulation of TNFR1, the recruitment of TAK1 to in RIP-deficient cells Together, our indicate that ubiquitination of RIP is essential for the recruitment of TAK1 to the TNFR1 in the following the stimulation of TNFR1 In our provide the genetic and evidence that the ubiquitination of RIP is required for the TNFα-induced NF-κB activation through TAK1 to the TNFR1 complex. we have that Lys377 in the intermediate domain of RIP is the functional ubiquitination and Seed for