A Tangled Web of Ubiquitin Chains: Breaking News in TNF-R1 Signaling
2009; Elsevier BV; Volume: 36; Issue: 5 Linguagem: Inglês
10.1016/j.molcel.2009.11.029
ISSN1097-4164
AutoresKatiuscia Bianchi, Pascal Meier,
Tópico(s)interferon and immune responses
ResumoA flurry of recent revelations is challenging the current dogma on how ubiquitin-dependent processes culminate in the activation of NF-κB by TNF. Here, we integrate these findings into a model for TNF-R1 signaling—and underscore the importance of individual components, including linear ubiquitin chains—which allows for the remarkable versatility of the ubiquitin system. A flurry of recent revelations is challenging the current dogma on how ubiquitin-dependent processes culminate in the activation of NF-κB by TNF. Here, we integrate these findings into a model for TNF-R1 signaling—and underscore the importance of individual components, including linear ubiquitin chains—which allows for the remarkable versatility of the ubiquitin system. The flow of molecular information frequently relies on the formation of transient protein complexes. Importantly, the assembly of such signaling hubs is controlled via posttranslational modification, such as the addition of small functional groups (e.g., phosphates) or entire proteins (e.g., Ubiquitin [Ub]). The conjugated adduct, thereby, forms part of a new docking site for proteins with specialized binding motifs that help to build reversible, short-lived signaling centers. While in the early years conjugation of Ub was often regarded as a tag for “garbage disposal” (Hershko et al., 2000Hershko A. Ciechanover A. Varshavsky A. Nat. Med. 2000; 6: 1073-1081Crossref PubMed Scopus (544) Google Scholar), it is now recognized as a sophisticated adduct influencing a legion of highly dynamic biological processes such as endocytic trafficking, NF-κB signaling, gene expression, DNA repair, and apoptosis (Chen and Sun, 2009Chen Z.J. Sun L.J. Mol. Cell. 2009; 33: 275-286Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar). Ub is conjugated either as a single moiety or as chains of variable length (Komander, 2009Komander D. Biochem. Soc. Trans. 2009; 37: 937-953Crossref PubMed Scopus (534) Google Scholar). Further complexity is provided by different linkage types, as Ub moieties can be conjugated to one another via different lysine (K) residues within Ub (Figure 1). At least eight different types of Ub chains exist that exert distinct effects on cellular processes (Xu et al., 2009bXu P. Duong D.M. Seyfried N.T. Cheng D. Xie Y. Robert J. Rush J. Hochstrasser M. Finley D. Peng J. Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). A flurry of recent studies have uncovered an ever-expanding complexity of Ub-conjugating, deubiquitylating enzymes and Ub receptors that build up, destroy, and recognize the Ub message, affecting signaling pathways through degradative and nondegradative means. Activation of NF-κB has, thereby, served as a productive playing field for the identification of new paradigms of Ub-mediated signaling. Early models postulated that activation of NF-κB by TNFα requires the formation of two differently linked polyUb chains—K63-linked chains for activation of the IκB-activating kinase (IKK), and K48-linked chains for targeting the NF-κB inhibitor IκB for degradation (Terzic et al., 2007Terzic J. Marinovic-Terzic I. Ikeda F. Dikic I. Biochem. Soc. Trans. 2007; 35: 942-945Crossref PubMed Scopus (25) Google Scholar). However, closer examination of “older” data, combined with more recent experimental evidence, suggest that the mechanism through which Ub mediates NF-κB activation differs from what was proposed previously. Here we re-evaluate older data and integrate recent findings regarding the mechanisms though which TNFα triggers Ub-mediated activation of NF-κB. The conjugation of Ub requires a stepwise process that involves Ub-activating enzymes (E1), Ub-conjugating enzymes (E2), and Ub protein ligases (E3) (Dikic et al., 2009Dikic I. Wakatsuki S. Walters K.J. Nat. Rev. Mol. Cell Biol. 2009; 10: 659-671Crossref PubMed Scopus (573) Google Scholar). E3s confer substrate specificity and enable the formation of an isopeptide linkage between the carboxyl terminus of Ub (glycine [G]76) and the amino group of a reactive side chain of the substrate. Ubiquitylation, like other modifications such as acetylation, methylation, and glycosylation, most frequently occurs on K because this residue harbours one of the most reactive ɛ-amino groups. However, the side chain of other amino acids, such as serine, threonine, and/or cysteine, can also function as Ub acceptors (Tait et al., 2007Tait S.W. de Vries E. Maas C. Keller A.M. D'Santos C.S. Borst J. J. Cell Biol. 2007; 179: 1453-1466Crossref PubMed Scopus (79) Google Scholar). Importantly, Ub itself has seven K residues, all of which can serve as acceptor for further attachment of Ub, generating polymeric chains of Ub. In addition to K-mediated polyubiquitylation, Ub can also be attached to the extreme amino terminus of Ub, creating head-to-tail-linked linear Ub chains in which the carboxy-terminal α-carboxyl group of G76 of one Ub is conjugated to the α-amino group of methionine of another (Kirisako et al., 2006Kirisako T. Kamei K. Murata S. Kato M. Fukumoto H. Kanie M. Sano S. Tokunaga F. Tanaka K. Iwai K. EMBO J. 2006; 25: 4877-4887Crossref PubMed Scopus (512) Google Scholar). Differently linked polyUb chains adopt distinct overall structures as the seven K of Ub are positioned at different surfaces of the Ub fold (Figure 1). K48-linked polyUb chains adopt a kinked topology while the ones of K63-linked and linear Ub resemble “beads on a string” (Komander, 2009Komander D. Biochem. Soc. Trans. 2009; 37: 937-953Crossref PubMed Scopus (534) Google Scholar). Recent evidence indicates that K6-, K11-, K27-, K29-, K33- and K48-linked modifications promote degradation through recognition by the 26S proteasome (Xu et al., 2009bXu P. Duong D.M. Seyfried N.T. Cheng D. Xie Y. Robert J. Rush J. Hochstrasser M. Finley D. Peng J. Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar), and thus regulate protein levels. This ensures normal protein turnover as well as targeted downregulation of signaling molecules. In contrast, mono-Ub, K63-, and linear linkages contribute to a variety of nondegradative signaling processes (Hoeller et al., 2006Hoeller D. Hecker C.M. Dikic I. Nat. Rev. Cancer. 2006; 6: 776-788Crossref PubMed Scopus (316) Google Scholar, Tokunaga et al., 2009Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. et al.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (693) Google Scholar). Most frequently, mono-Ub regulates protein activity by controlling cellular localization, whereas K63-linked and linear chains play more scaffolding roles in signal-transduction events. For example, K63-linked polyUb chains are formed upon stimulation of cytokine receptors and are required to activate downstream kinases. At the center of the remarkable diversity of the Ub signal lays the ability of the E3:E2 system to conjugate different types of Ub chains. E3s determine which substrate is ubiquitylated, as they bind to both E2 and substrate, bringing the E2 in position for Ub transfer. While the E3 determines which substrate is ubiquitylated, it is generally thought that the E2 provides the ultimate decision on which type of chain is formed. This view is predominantly based on the observation that the heterodimeric E2 Ubc13/Uev1A is specialized in generating K63-linked Ub chains (Hofmann and Pickart, 1999Hofmann R.M. Pickart C.M. Cell. 1999; 96: 645-653Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar). However, other E2s, such as UbcH5 and UbcH10, are more promiscuous and can promote a surprisingly diverse array of products, even synthesizing chains of mixed linkages (Jin et al., 2008Jin L. Williamson A. Banerjee S. Philipp I. Rape M. Cell. 2008; 133: 653-665Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, Kirkpatrick et al., 2006Kirkpatrick D.S. Hathaway N.A. Hanna J. Elsasser S. Rush J. Finley D. King R.W. Gygi S.P. Nat. Cell Biol. 2006; 8: 700-710Crossref PubMed Scopus (338) Google Scholar). Conjugation of polyUb chains of a particular topology may actually require the cooperative action of two different classes of E2s (Rodrigo-Brenni and Morgan, 2007Rodrigo-Brenni M.C. Morgan D.O. Cell. 2007; 130: 127-139Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). For example, Ubc13/Uev1a is very efficient in forming K63-linked chains; however, such chains are not attached to any substrates (Windheim et al., 2008Windheim M. Peggie M. Cohen P. Biochem. J. 2008; 409: 723-729Crossref PubMed Scopus (84) Google Scholar). While Ubc13/Uev1a performs poorly in attaching the first Ub moiety onto target substrate, UbcH5 is highly efficient in target ubiquitylation. However, although UbcH5 readily conjugates the first Ub to K residues in the substrate, it subsequently lacks specificity for any particular type of chain elongation. Therefore, conjugation of K63-linked chains on target substrates appears to be the result of a division of labor among chain initiating (UbcH5) and chain-elongating E2s (Ubc13/Uev1a) (Ye and Rape, 2009Ye Y. Rape M. Nat. Rev. Mol. Cell Biol. 2009; 10: 755-764Crossref PubMed Scopus (628) Google Scholar). Intriguingly, E3 ligases with RING domains can be active with more than one E2, which begs the question how the correct E2 is selected for the right occasion. For example, RING fingers of IAPs reportedly bind to Ubc5A, Ubc5C, Ubc7, Ubc8, and Ubc13/Uev1A (Vaux and Silke, 2005Vaux D.L. Silke J. Nat. Rev. Mol. Cell Biol. 2005; 6: 287-297Crossref PubMed Scopus (515) Google Scholar), while other RING E3s can even interact with 10 different E2s (Christensen and Klevit, 2009Christensen D.E. Klevit R.E. FEBS J. 2009; 276: 5381-5389Crossref PubMed Scopus (38) Google Scholar). There are approximately 30 E2s identified in the human genome; however, currently little is known how, and under what conditions, a particular E3 selects its E2 to promote a specific type of Ub modification. Although ubiquitylation may influence protein function directly, the most common mode of regulation by Ub conjugation involves specific Ub receptors that recognize ubiquitylated proteins and link them to downstream signaling processes (Dikic et al., 2009Dikic I. Wakatsuki S. Walters K.J. Nat. Rev. Mol. Cell Biol. 2009; 10: 659-671Crossref PubMed Scopus (573) Google Scholar). Ub receptors carry small Ub-binding domains (UBDs) through which they bind to the Ub modification via low-affinity, noncovalent interactions. Currently, more that 20 different types of UBDs are known that detect overlapping as well as distinct Ub modifications. Ub receptors that selectively recognize K48-linked polyUb chains, such as the proteasome subunit Rpn13 (Husnjak et al., 2008Husnjak K. Elsasser S. Zhang N. Chen X. Randles L. Shi Y. Hofmann K. Walters K.J. Finley D. Dikic I. Nature. 2008; 453: 481-488Crossref PubMed Scopus (459) Google Scholar), recruit modified proteins to the proteasome for degradation. In contrast, Ub receptors that bind to mono-Ub, K63 linkages, or linear Ub allow Ub-dependent association with signaling molecules (Hoeller et al., 2006Hoeller D. Hecker C.M. Dikic I. Nat. Rev. Cancer. 2006; 6: 776-788Crossref PubMed Scopus (316) Google Scholar). In particular, K63-linked Ub chains, and their respective Ub receptors, are critical for TNFα-mediated NF-κB activation and cell survival (Xu et al., 2009bXu P. Duong D.M. Seyfried N.T. Cheng D. Xie Y. Robert J. Rush J. Hochstrasser M. Finley D. Peng J. Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). Early models postulated that binding of trimeric TNFα to its cognate receptor, TNF-R1, triggers the recruitment of the adaptor protein TRADD; the E3 ligases TRAF2, TRAF5, cIAP1, and cIAP2; and the protein kinase RIP1. This membrane-localized complex is also referred to as “complex I” (Micheau and Tschopp, 2003Micheau O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar). While RIP1's kinase activity is not required for signaling to NF-κB, RIP1 itself and the covalent attachment of poly-Ub chains to RIP1K377 reportedly is essential. These chains are believed to be linked via K63 of Ub, based on experiments with Ub mutants (Ea et al., 2006Ea C.K. Deng L. Xia Z.P. Pineda G. Chen Z.J. Mol. Cell. 2006; 22: 245-257Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar, Li et al., 2006Li H. Kobayashi M. Blonska M. You Y. Lin X. J. Biol. Chem. 2006; 281: 13636-13643Crossref PubMed Scopus (208) Google Scholar, Wertz et al., 2004Wertz I.E. O'Rourke K.M. Zhou H. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. et al.Nature. 2004; 430: 694-699Crossref PubMed Scopus (1394) Google Scholar), and an anti-K63-selective antibody (Newton et al., 2008Newton K. Matsumoto M.L. Wertz I.E. Kirkpatrick D.S. Lill J.R. Tan J. Dugger D. Gordon N. Sidhu S.S. Fellouse F.A. et al.Cell. 2008; 134: 668-678Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Consistently, expression of a dominant-negative form of Ubc13 (Ubc13C87A) and siRNA-mediated knockdown of Ubc13/Uev1a resulted in defective NF-κB activation in human embryonic kidney 293T cells (Deng et al., 2000Deng 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 (1414) Google Scholar, Habelhah et al., 2004Habelhah H. Takahashi S. Cho S.G. Kadoya T. Watanabe T. Ronai Z. EMBO J. 2004; 23: 322-332Crossref PubMed Scopus (185) Google Scholar). The conjugation of K63-linked polyUb chains to RIP1 is thought to be mediated by TRAF2 (Wertz et al., 2004Wertz I.E. O'Rourke K.M. Zhou H. Eby M. Aravind L. Seshagiri S. Wu P. Wiesmann C. Baker R. Boone D.L. et al.Nature. 2004; 430: 694-699Crossref PubMed Scopus (1394) Google Scholar), which belongs to the RING finger family of E3 ligases. The Ub chains on RIP1 then bind and recruit the TAK/TAB (TAK1/TAB2/TAB3) and IKK (IKKα/β/γ[NEMO]) kinase complexes via the Ub receptors TAB2 and NEMO, respectively (Ea et al., 2006Ea C.K. Deng L. Xia Z.P. Pineda G. Chen Z.J. Mol. Cell. 2006; 22: 245-257Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar, Kanayama et al., 2004Kanayama 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 (662) Google Scholar, Li et al., 2006Li H. Kobayashi M. Blonska M. You Y. Lin X. J. Biol. Chem. 2006; 281: 13636-13643Crossref PubMed Scopus (208) Google Scholar, Wu et al., 2006Wu C.J. Conze D.B. Li T. Srinivasula S.M. Ashwell J.D. Nat. Cell Biol. 2006; 8: 398-406Crossref PubMed Scopus (493) Google Scholar). TAB2 carries one zinc-finger-type UBD, whereas NEMO harbors two distinct UBDs, a coiled-coil-type UBD, also called UBAN domain, and a C-terminal zinc-finger-type UBD. Both zinc-finger-type UBDs of TAB2 and NEMO confer selectivity for K63-linked polyUb chains (Komander et al., 2009Komander D. Reyes-Turcu F. Licchesi J.D. Odenwaelder P. Wilkinson K.D. Barford D. EMBO Rep. 2009; 10: 466-473Crossref PubMed Scopus (424) Google Scholar, Laplantine et al., 2009Laplantine E. Fontan E. Chiaravalli J. Lopez T. Lakisic G. Veron M. Agou F. Israel A. EMBO J. 2009; 28: 2885-2895Crossref PubMed Scopus (143) Google Scholar, Lo et al., 2009Lo Y.C. Lin S.C. Rospigliosi C.C. Conze D.B. Wu C.J. Ashwell J.D. Eliezer D. Wu H. Mol. Cell. 2009; 33: 602-615Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, Yoshikawa et al., 2009Yoshikawa A. Sato Y. Yamashita M. Mimura H. Yamagata A. Fukai S. FEBS Lett. 2009; 583: 3317-3322Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Once hooked to K63-linked polyubiquitylated RIP1, TAK1 activates IKKβ, most likely due to proximity-induced phosphorylation. Activated IKKβ subsequently phosphorylates IκB, which targets it for SCFbTrCP-mediated K48-linked polyubiquitylation and degradation (Xu et al., 2009bXu P. Duong D.M. Seyfried N.T. Cheng D. Xie Y. Robert J. Rush J. Hochstrasser M. Finley D. Peng J. Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (799) Google Scholar). This liberates NF-κB, which translocates to the nucleus where it drives target gene expression. TNF-R1 signaling also results in the formation of a cytoplasmic “complex II,” which mediates apoptosis, particularly in the absence of NF-κB activation (Micheau and Tschopp, 2003Micheau O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar). In contrast to complex I, complex II lacks TNF-R1 and instead includes the caspase-adaptor FADD and caspase-8. Although a large body of work supports the above-mentioned model, closer examination of older data, combined with new experimental results, suggests a surprisingly different scenario. Several converging lines of evidence have challenged the “standard” model at numerous points. The notion that TRAF2, together with Ubc13 forms K63-linked chains required for TNF-induced NF-κB activation, is unlikely to be correct for several reasons. First, conditional, homozygous inactivation of the E2-conjugating enzyme ubc13 does not affect TNFα-induced IκBα degradation, or stress kinase activation in mouse embryonic fibroblasts (MEFs) (Yamamoto et al., 2006Yamamoto M. Okamoto T. Takeda K. Sato S. Sanjo H. Uematsu S. Saitoh T. Yamamoto N. Sakurai H. Ishii K.J. et al.Nat. Immunol. 2006; 7: 962-970Crossref PubMed Scopus (222) Google Scholar). Since Ubc13 is critical for conjugating K63-linked chains to target proteins, this finding suggests that, at least in MEFs, Ubc13 is not required for TNFα-mediated activation of NF-κB. In fact, the knockout data strongly suggest that although Ubc13 deficiency slightly affects IκBα degradation in some cell types, Ubc13 is mostly dispensable for NF-κB activation stimulated by a variety of Toll-like receptors (TLR), B cell receptors (BCR), CD40, interleukin 1 receptor (IL-1R), and TNF-R1, under physiological conditions. Instead, Ubc13 is required for MAP kinase activation induced by TLR, IL-1R, BCR, and CD40, but not TNF-R1. Nevertheless, as with all knockout studies, it is formally possible that other E2 family members, such as Ubc5, which also mediates K63-linked polyUb chain synthesis (Duncan et al., 2006Duncan L.M. Piper S. Dodd R.B. Saville M.K. Sanderson C.M. Luzio J.P. Lehner P.J. EMBO J. 2006; 25: 1635-1645Crossref PubMed Scopus (205) Google Scholar, Varfolomeev et al., 2008Varfolomeev E. Goncharov T. Fedorova A.V. Dynek J.N. Zobel K. Deshayes K. Fairbrother W.J. Vucic D. J. Biol. Chem. 2008; 283: 24295-24299Crossref PubMed Scopus (415) Google Scholar), compensate for the loss of Ubc13 in some cell types. Second, although TRAF2/TRAF5 are required for TNFα-mediated NF-κB activation, their RING fingers, and hence their activity as E3 ligases, are dispensable (Vince et al., 2009Vince J.E. Pantaki D. Feltham R. Mace P.D. Cordier S.M. Schmukle A.C. Davidson A.J. Callus B.A. Wong W.W. Gentle I.E. et al.J Biol Chem. 2009; (in press. Published online October 8, 2009)https://doi.org/10.1074/jbc.M109.072256Crossref PubMed Scopus (168) Google Scholar). Third, TRAF2, and most likely TRAF5, have structural differences to TRAF6 that make it unlikely that they bind to either Ubc13, or other related E2s (Yin et al., 2009Yin Q. Lamothe B. Darnay B.G. Wu H. Biochemistry. 2009; 48: 10558-10567Crossref PubMed Scopus (83) Google Scholar). These results suggest that TRAF2 and Ubc13 are neither the E3 nor the E2 for TNF-R1-mediated activation of NF-κB. Instead, it seems that TRAF2 acts rather as an adaptor for cIAPs, and that these serve as the E3 ligases for TNF-R1 signaling. This view was already suggested by recent reports showing that loss of cIAPs abrogated TNF-R1 induced activation of NF-κB (Wu et al., 2007Wu H. Tschopp J. Lin S.C. Cell. 2007; 131: 655-658Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Moreover, TRAF2 mutants that cannot bind cIAPs fail to reconstitute TRAF2/5 double-knockout cells, while a cIAP-binding-competent, but RING-finger-defective TRAF2 mutant rescues TNFα-mediated activation of NF-κB (Haas et al., 2009Haas T.L. Emmerich C.H. Gerlach B. Schmukle A.C. Cordier S.M. Rieser E. Feltham R. Vince J. Warnken U. Wenger T. et al.Mol Cell. 2009; 36 (this issue): 831-844Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, Vince et al., 2009Vince J.E. Pantaki D. Feltham R. Mace P.D. Cordier S.M. Schmukle A.C. Davidson A.J. Callus B.A. Wong W.W. Gentle I.E. et al.J Biol Chem. 2009; (in press. Published online October 8, 2009)https://doi.org/10.1074/jbc.M109.072256Crossref PubMed Scopus (168) Google Scholar). Consistent with the notion that cIAPs are critical for TNFα signaling, loss of cIAP1/2 completely abrogates ubiquitylation of RIP1 and NF-κB activation, even though recruitment of TRAF2 to complex I is normal. Although the E3 activities of cIAPs are essential, it is currently unclear which type(s) of chain(s) is (or are) synthesized by cIAPs. Clearly, cIAPs can bind to several E2s, including UbcH5 and Ubc13/Uev1A, and reportedly promote the synthesis of both K48- and K63-linked chains (Bertrand et al., 2008Bertrand M.J. Milutinovic S. Dickson K.M. Ho W.C. Boudreault A. Durkin J. Gillard J.W. Jaquith J.B. Morris S.J. Barker P.A. Mol. Cell. 2008; 30: 689-700Abstract Full Text Full Text PDF PubMed Scopus (794) Google Scholar, Varfolomeev et al., 2008Varfolomeev E. Goncharov T. Fedorova A.V. Dynek J.N. Zobel K. Deshayes K. Fairbrother W.J. Vucic D. J. Biol. Chem. 2008; 283: 24295-24299Crossref PubMed Scopus (415) Google Scholar, Yang and Du, 2004Yang Q.H. Du C. J. Biol. Chem. 2004; 279: 16963-16970Crossref PubMed Scopus (166) Google Scholar). Whether cIAPs also promote the synthesis of other types of chains remains to be determined. Although the knockout data ruled out the involvement of Ubc13 in TNF-R1-signaling, it was formally possible that Ubc5, and its ability to promote K63-linked chains, compensates for the loss of Ubc13. However, this view has now been challenged since new data suggest that K63-linked chains are not involved in TNF-R1 signaling. Using a Ub-replacement strategy, Chen and colleagues found that neither the K63 of Ub nor the catalytic activity of Ubc13 are required for TNFα signaling (Xu et al., 2009aXu M. Skaug B. Zeng W. Chen Z.J. Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), independently supporting the Ubc13 knockout results. Instead, they report that TNFα-mediated activation of IKK requires Ubc5 and the synthesis of non-K63-linked chains. The identity of those non-K63-linked chains is still uncertain but recent data suggest that linear Ub chains maybe particularly important for TNFα-induced activation of NF-κB. The first data supporting this view was the discoveries that linear ubiquitylation of NEMO (Tokunaga et al., 2009Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. et al.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (693) Google Scholar), and the binding of the UBAN domain of NEMO to linear Ub chains (Rahighi et al., 2009Rahighi S. Ikeda F. Kawasaki M. Akutsu M. Suzuki N. Kato R. Kensche T. Uejima T. Bloor S. Komander D. et al.Cell. 2009; 136: 1098-1109Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar) were required for proper NF-κB activation by TNFα. Interestingly, NEMO's UBAN domain (Figure 2), which was suspected to be a K63-binding motif, displays a 100× greater affinity to linear chains than to K63-linked chains (Rahighi et al., 2009Rahighi S. Ikeda F. Kawasaki M. Akutsu M. Suzuki N. Kato R. Kensche T. Uejima T. Bloor S. Komander D. et al.Cell. 2009; 136: 1098-1109Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Moreover, NEMO mutants that fail to bind to linear chains also fail to reconstitute NEMO knockout cells. Interestingly, similar mutations are also found in patients that suffer from X-linked ectodermal dysplasia and immunodeficiency that is caused by impaird NF-κB signaling. The linear Ub chain assembly complex (LUBAC) is currently the only E3 ligase that promotes the synthesis of head-to-tail-linked linear chains. Intriguingly, LUBAC can use multiple E2s—including UbcH5, E2-25, and UbcH7—to generate head-to-tail Ub conjugates (Kirisako et al., 2006Kirisako T. Kamei K. Murata S. Kato M. Fukumoto H. Kanie M. Sano S. Tokunaga F. Tanaka K. Iwai K. EMBO J. 2006; 25: 4877-4887Crossref PubMed Scopus (512) Google Scholar). Moreover, LUBAC cannot generate polyUb chains with N-terminally tagged wild-type Ub, indicating that it exclusively produces linear chains. This also suggests that LUBAC and not the associated E2 is responsible for determining the linkage type. Stimulation of TNF-R1 results in the polyubiquitylation of RIP1, and Ub-dependent assembly of TAK/TAB and IKK. Since LUBAC does not polyubiquitylate RIP1, and is only known to bind to NEMO—a downstream signaling component whose recruitment to complex I is Ub dependent, it is not immediately apparent how LUBAC, and linear ubiquitylation of NEMO, might contribute to TNF-R1-signaling (Tokunaga et al., 2009Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. et al.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (693) Google Scholar). In this issue of Molecular Cell, new findings from Haas et al. now add flesh to the bones of TNF-R1 signaling. Using an elegant tandem-affinity purification strategy, coupled to tandem-mass spectrometric analysis, Walczak and colleagues identified the “full” complement of the native TNF-R1 signaling complex (Haas et al., 2009Haas T.L. Emmerich C.H. Gerlach B. Schmukle A.C. Cordier S.M. Rieser E. Feltham R. Vince J. Warnken U. Wenger T. et al.Mol Cell. 2009; 36 (this issue): 831-844Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). This yielded TNF-R1, TRADD, TRAF2, cIAP2, RIP1, Ub, ABIN1, IKKα, IKKβ, NEMO, TAK1, TAB1, and TAB2, plus two previously unrecognized components: the heme-oxidised IRP1 Ub ligase-1 (HOIL-1) and the HOIL-1-interacting protein (HOIP), which together constitute LUBAC (Kirisako et al., 2006Kirisako T. Kamei K. Murata S. Kato M. Fukumoto H. Kanie M. Sano S. Tokunaga F. Tanaka K. Iwai K. EMBO J. 2006; 25: 4877-4887Crossref PubMed Scopus (512) Google Scholar). Like other components of the TNF-R1-signaling complex, LUBAC is recruited to TNF-R1 in a ligand-dependent manner. Using a plethora of knockout cell lines, they unambiguously show that LUBAC recruitment requires TRADD, TRAF2, and cIAP1/2. Intriguingly, although LUBAC reportedly binds to NEMO, this interaction was not required for its recruitment since recruitment of LUBAC to the TNF-R1-signaling complex was unaffected in NEMO−/− cells. Instead, the E3 ligase activity of cIAPs, but not the one of TRAF2, was necessary for LUBAC recruitment. This suggests that recruitment of LUBAC is Ub dependent. LUBAC is composed of the two RING-in-between-RING proteins HOIL-1 and HOIP (Kirisako et al., 2006Kirisako T. Kamei K. Murata S. Kato M. Fukumoto H. Kanie M. Sano S. Tokunaga F. Tanaka K. Iwai K. EMBO J. 2006; 25: 4877-4887Crossref PubMed Scopus (512) Google Scholar). In addition to the double RINGs both HOIL-1 and HOIP also carry Npl4-type zinc finger (NZF) domains that are crucial for NF-κB activation (Tokunaga et al., 2009Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. et al.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (693) Google Scholar). Using copurification assays with HOIL-1, HOIP, and polyUb chains, Haas et al. now find that these NZF domains enable HOIL-1 and HOIP to bind to polyUb chains. The NZF domains of HOIP seem to be rather nonselective as they bind to K48-, K63-, as well as linear-Ub chains. This suggests that LUBAC is recruited to TNF-R1 via Ub chains that are attached to components of the TNF-R1 signaling complex. Although RIP1 is the most prominent target of cIAP-mediated ubiquitylation, RIP1 was neither required for LUBAC recruitment in MEFs nor essential for it in cancer cells (HeLa). Clearly, RIP1 is not the only protein that is modified by Ub in a TNFα-dependent manner. TRADD, TNF-R1, and most likely also cIAPs also undergo extensive posttranslational modifications in complex I (Micheau and Tschopp, 2003Micheau O. Tschopp J. Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar). Therefore, in the absence of RIP1, Ub chains on TRADD, TNF-R1, or cIAPs may recruit LUBAC into the TNF-R1 signaling complex. Importantly, Haas et al. found that recruitment of LUBAC results in the stabilization of the TNF-R1-signaling complex (Haas et al., 2009Haas T.L. Emmerich C.H. Gerlach B. Schmukle A.C. Cordier S.M. Rieser E. Feltham R. Vince J. Warnken U. Wenger T. et al.Mol Cell. 2009; 36 (this issue): 831-844Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Enhanced retention of cIAP1, TRAF2, RIP1, and TAK1 was dependent on LUBAC's E3 ligase activity. This suggests that linear ubiquitylation protects complex I from falling apart. In this context, it is interesting to mention that in addition to NEMO (Rahighi et al., 2009Rahighi S. Ikeda F. Kawasaki M. Akutsu M. Suzuki N. Kato R. Kensche T. Uejima T. Bloor S. Komander D. et al.Cell. 2009; 136: 1098-1109Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar), other components of the TNF-R1-signaling complex can also bind to linear Ub chains (Figure 2), such as cIAPs with their recently identified UBA domain (Gyrd-Hansen et al., 2008Gyrd-Hansen M. Darding M. Miasari M. Santoro M.M. Zender L. Xue W. Tenev T. da Fonseca P.C. Zvelebil M. Bujnicki J.M. et al.Nat. Cell Biol. 2008; 10: 1309-1317Crossref PubMed Scopus (183) Google Scholar), and ABINs via their UBAN domain (Komander et al., 2009Komander D. Reyes-Turcu F. Licchesi J.D. Odenwaelder P. Wilkinson K.D. Barford D. EMBO Rep. 2009; 10: 466-473Crossref PubMed Scopus (424) Google Scholar). LUBAC-mediated conjugation of linear Ub chains might, therefore, act as additional scaffolds for the retention of cIAPs, NEMO, and ABINs. The underlying mechanism for enhanced stability of the TNF-R1-signaling complex is, however, still unclear. It is interesting to mention that most deubiquitylating enzymes (DUBs), except CYLD, do not cleave linear Ub chains, or process them less efficiently than other chain types (Komander et al., 2009Komander D. Reyes-Turcu F. Licchesi J.D. Odenwaelder P. Wilkinson K.D. Barford D. EMBO Rep. 2009; 10: 466-473Crossref PubMed Scopus (424) Google Scholar). Therefore, the increased stability of complex I might simply be due to linear chains being more refractory to DUB-mediated disassembly. The “new” working model proposes that activation of TNF-R1 stimulates recruitment of the E3 ligase cIAP1 via TRAF2 and TRADD (Figure 3). cIAP1 then ubiquitylates several components of complex I, including RIP1 and cIAP1 itself, which causes Ub-dependent recruitment of LUBAC, TAK1/TAB2/TAB3, and NEMO/IKKα/IKKβ. Once recruited LUBAC further ubiquitylates NEMO, and most likely also other components of complex I. Since several components of this complex harbor UBDs that lock onto linear Ub chains, linear ubiquitylation stabilizes complex I, and supports additional recruitment, retention, ubiquitylation, and activation of NEMO/IKKα/IKKβ. The studies of Walczak and Chen (Haas et al., 2009Haas T.L. Emmerich C.H. Gerlach B. Schmukle A.C. Cordier S.M. Rieser E. Feltham R. Vince J. Warnken U. Wenger T. et al.Mol Cell. 2009; 36 (this issue): 831-844Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, Xu et al., 2009aXu M. Skaug B. Zeng W. Chen Z.J. Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) highlight a number of unsolved issues. First, it still remains unclear which type of linkage is established by cIAPs under physiological conditions. It is also not clear which component(s) of the TNF-R1-signaling complex actually serve(s) as target(s) for cIAP-mediated conjugation of Ub. Second, the contribution of K63-linked chains in TNF-R1 signaling has yet to be fully resolved. Although the Ub replacement strategy by Chen and colleagues leaves little room for K63-linked Ub chains (Xu et al., 2009aXu M. Skaug B. Zeng W. Chen Z.J. Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), there is a large body of literature that suggests otherwise. It is likely that Ub-replacement strategy and RNAi-mediated knockdown of endogenous Ub does not completely eliminate the synthesis of K63-linked chains. It is currently not clear how much, or how little, K63-linked polyubiquitylation is sufficient for signaling. Hence, low level of K63-linked polyubiquitylation may well be enough for TNF-R1-mediated activation of NF-κB, while it is not sufficient for IL-1R signaling. Importantly, the different signaling pathways also rely on different E3s (Chen and Sun, 2009Chen Z.J. Sun L.J. Mol. Cell. 2009; 33: 275-286Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar), which are likely to have distinct catalytic potentials. Therefore, the observed difference between TNF-R1 and IL-1R may simply reflect differences between cIAPs and TRAF6. Third, a detailed re-examination of the interactions between Ub receptors of the TNF-R1-signaling complex and different chain types need to be undertaken. This is crucial since it recently surfaced that the apparent K63 selectivity of some UBDs is actually due to avid interactions that are artificially promoted in the dimeric GST fusions used to classify the domains (Sims et al., 2009Sims J.J. Haririnia A. Dickinson B.C. Fushman D. Cohen R.E. Nat. Struct. Mol. Biol. 2009; 16: 883-889Crossref PubMed Scopus (67) Google Scholar). Despite all the potential avidity problems, it is clear that the Ub-receptor TAB2 does not bind to K48-linked chains or linear Ub, but exclusively binds to K63-linked chains (Komander et al., 2009Komander D. Reyes-Turcu F. Licchesi J.D. Odenwaelder P. Wilkinson K.D. Barford D. EMBO Rep. 2009; 10: 466-473Crossref PubMed Scopus (424) Google Scholar). Given the notion that K63 of Ub and the catalytic activity of Ubc13 might not be required for TNFα signaling, it will be important to resolve how TAB2, and other K63-binding proteins function as Ub receptors in TNF signaling. Finally, it will be vital to establish how chain selectivity is achieved. Clearly, E3s can use different E2s to mediate distinct types of Ub modifications; however, it remains enigmatic how, and under what conditions, a particular E3 selects its E2 to promote a specific type of modification (Brzovic and Klevit, 2006Brzovic P.S. Klevit R.E. Cell Cycle. 2006; 5: 2867-2873Crossref PubMed Scopus (66) Google Scholar). This issue is of fundamental importance to our understanding of the remarkable versatility and specificity within the Ub system. The discovery of LUBAC as a component of TNF-R1 complex I, and the ability of linear Ub chains to stabilize Ub-dependent protein complexes (Haas et al., 2009Haas T.L. Emmerich C.H. Gerlach B. Schmukle A.C. Cordier S.M. Rieser E. Feltham R. Vince J. Warnken U. Wenger T. et al.Mol Cell. 2009; 36 (this issue): 831-844Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, Tokunaga et al., 2009Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. et al.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (693) Google Scholar) almost certainly has wider implications. Ub-dependent signaling complexes not only occur downstream of TNF-R1, but are assembled for the regulation of various other cellular processes (Chen and Sun, 2009Chen Z.J. Sun L.J. Mol. Cell. 2009; 33: 275-286Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar). Since LUBAC displays a strong affinity for polyubiquitylated complexes, it is likely that LUBAC, and its ability to promote the conjugation of linear Ub chains, plays important roles in many other Ub-dependent signal transduction events. For what is known, LUBAC is also important for IL-1β-signaling (Tokunaga et al., 2009Tokunaga F. Sakata S. Saeki Y. Satomi Y. Kirisako T. Kamei K. Nakagawa T. Kato M. Murata S. Yamaoka S. et al.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (693) Google Scholar), a pathway that relies on TRAF6-, Ubc13/Uev1a, and Ub for signal transduction. Undoubtedly, future work will establish the requirement of LUBAC for other Ub-dependent signaling events. The notion that mutations in NEMO, which abrogate specific recognition of linear Ub chains (Rahighi et al., 2009Rahighi S. Ikeda F. Kawasaki M. Akutsu M. Suzuki N. Kato R. Kensche T. Uejima T. Bloor S. Komander D. et al.Cell. 2009; 136: 1098-1109Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar), are frequently found in X-linked ectodermal dysplasia, strongly suggests that loss of LUBAC's activity may contribute to immunodeficiency. Conversely, gain of LUBAC activity may well contribute to parainflammation and tumor formation.
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