Portal de Periódicos da CAPES

Ubiquitin Makes Its Mark on Immune Regulation

2010; Cell Press; Volume: 33; Issue: 6 Linguagem: Inglês

10.1016/j.immuni.2010.12.007

1097-4180

Barbara A. Malynn, Averil Ma,

Immunotherapy and Immune Responses

Ubiquitination, the covalent attachment of ubiquitin molecules to proteins, is emerging as a widely utilized mechanism for rapidly regulating cell signaling. Recent studies indicate that ubiquitination plays potent roles in regulating a variety of signals in both innate and adaptive immune cells. Here, we will review recent studies of ubiquitin ligases, ubiquitin chain linkages, and ubiquitin binding proteins that highlight the diversity and specificity of ubiquitin dependent functions in immune cells. We will also review studies that shed light on how ubiquitination signals are integrated in cell-type-specific fashion to regulate the immune system in vivo. Ubiquitination, the covalent attachment of ubiquitin molecules to proteins, is emerging as a widely utilized mechanism for rapidly regulating cell signaling. Recent studies indicate that ubiquitination plays potent roles in regulating a variety of signals in both innate and adaptive immune cells. Here, we will review recent studies of ubiquitin ligases, ubiquitin chain linkages, and ubiquitin binding proteins that highlight the diversity and specificity of ubiquitin dependent functions in immune cells. We will also review studies that shed light on how ubiquitination signals are integrated in cell-type-specific fashion to regulate the immune system in vivo. The regulation of intracellular signals allows immune cells to integrate stimuli from their environment and to exhibit the dynamic plasticity characteristic of immune responses. The transduction of such signals requires rapid posttranslational modifications of proteins via processes such as phosphorylation or ubiquitination. While phosphorylation events are generally binary, comprising the presence or absence of a single phosphate group on selected amino acids of target proteins (e.g., serine, threonine, or tyrosine), ubiquitination events include the attachment of a variety of lengths and conformations of ubiquitin chains, mostly on lysine residues (Pickart and Fushman, 2004Pickart C.M. Fushman D. Polyubiquitin chains: Polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (790) Google Scholar). Understanding how ubiquitination events are regulated, and how they regulate a diverse array of cellular responses (Table 1), requires an understanding of the components of the ubiquitin system.Table 1E3 Ubiquitin Ligases Are Integral Mediators of Immune RegulationImmune Cell TypeE3 Ubiquitin LigaseFunctionReferencesT CellsTRAF6TCR signaling, central T cell toleranceKing et al., 2006King C.G. Kobayashi T. Cejas P.J. Kim T. Yoon K. Kim G.K. Chiffoleau E. Hickman S.P. Walsh P.T. Turka L.A. Choi Y. TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis.Nat. Med. 2006; 12: 1088-1092Crossref PubMed Scopus (160) Google Scholar, King et al., 2008King C.G. Buckler J.L. Kobayashi T. Hannah J.R. Bassett G. Kim T. Pearce E.L. Kim G.G. Turka L.A. Choi Y. Cutting edge: Requirement for TRAF6 in the induction of T cell anergy.J. Immunol. 2008; 180: 34-38PubMed Google Scholar, Lin and Mak, 2007Lin A.E. Mak T.W. The role of E3 ligases in autoimmunity and the regulation of autoreactive T cells.Curr. Opin. Immunol. 2007; 19: 665-673Crossref PubMed Scopus (55) Google Scholar, Cejas et al., 2010Cejas P.J. Walsh M.C. Pearce E.L. Han D. Harms G.M. Artis D. Turka L.A. Choi Y. TRAF6 inhibits Th17 differentiation and TGF-beta-mediated suppression of IL-2.Blood. 2010; 115: 4750-4757Crossref PubMed Scopus (51) Google ScholarCbl-BPeripheral T cell toleranceLin and Mak, 2007Lin A.E. Mak T.W. The role of E3 ligases in autoimmunity and the regulation of autoreactive T cells.Curr. Opin. Immunol. 2007; 19: 665-673Crossref PubMed Scopus (55) Google Scholar, Venuprasad, 2010Venuprasad K. Cbl-b and itch: Key regulators of peripheral T-cell tolerance.Cancer Res. 2010; 70: 3009-3012Crossref PubMed Scopus (26) Google ScholarITCH (AIP4)T cell tolerance; TH2 developmentVenuprasad, 2010Venuprasad K. Cbl-b and itch: Key regulators of peripheral T-cell tolerance.Cancer Res. 2010; 70: 3009-3012Crossref PubMed Scopus (26) Google ScholarGRAILT cell tolerance; Treg functionNurieva et al., 2010Nurieva R.I. Zheng S. Jin W. Chung Y. Zhang Y. Martinez G.J. Reynolds J.M. Wang S.L. Lin X. Sun S.C. et al.The E3 ubiquitin ligase GRAIL regulates T cell tolerance and regulatory T cell function by mediating T cell receptor-CD3 degradation.Immunity. 2010; 32: 670-680Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, Anandasabapathy et al., 2003Anandasabapathy N. Ford G.S. Bloom D. Holness C. Paragas V. Seroogy C. Skrenta H. Hollenhorst M. Fathman C.G. Soares L. GRAIL: An E3 ubiquitin ligase that inhibits cytokine gene transcription is expressed in anergic CD4+ T cells.Immunity. 2003; 18: 535-547Abstract Full Text Full Text PDF PubMed Scopus (226) Google ScholarSOCS proteinsT cell maturation, differentiation, and functionAlexander and Hilton, 2004Alexander W.S. Hilton D.J. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response.Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (582) Google Scholar, Palmer and Restifo, 2009Palmer D.C. Restifo N.P. Suppressors of cytokine signaling (SOCS) in T cell differentiation, maturation, and function.Trends Immunol. 2009; 30: 592-602Abstract Full Text Full Text PDF PubMed Scopus (174) Google ScholarB CellsTRAF2 and 3B cell developmentVallabhapurapu et al., 2008Vallabhapurapu S. Matsuzawa A. Zhang W. Tseng P.H. Keats J.J. Wang H. Vignali D.A. Bergsagel P.L. Karin M. Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling.Nat. Immunol. 2008; 9: 1364-1370Crossref PubMed Scopus (438) Google ScholarTRAF6CD40 signaling; B cell development and TD and TI responsesRowland et al., 2007Rowland S.L. Tremblay M.M. Ellison J.M. Stunz L.L. Bishop G.A. Hostager B.S. A novel mechanism for TNFR-associated factor 6-dependent CD40 signaling.J. Immunol. 2007; 179: 4645-4653PubMed Google Scholar, Kobayashi et al., 2009Kobayashi T. Kim T.S. Jacob A. Walsh M.C. Kadono Y. Fuentes-Pananá E. Yoshioka T. Yoshimura A. Yamamoto M. Kaisho T. et al.TRAF6 is required for generation of the B-1a B cell compartment as well as T cell-dependent and -independent humoral immune responses.PLoS ONE. 2009; 4: e4736Crossref PubMed Scopus (39) Google ScholarA20 (TNFAIP3)B cell tolerance; germinal center selection; CD40 signalingTavares et al., 2010Tavares R.M. Turer E.E. Liu C.L. Advincula R. Scapini P. Rhee L. Barrera J. Lowell C.A. Utz P.J. Malynn B.A. Ma A. The ubiquitin modifying enzyme A20 restricts B cell survival and prevents autoimmunity.Immunity. 2010; 33: 181-191Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, Chu et al., 2010Chu Y. Vahl J.C. Kumar D. Heger K. Bertossi A. Wójtowicz E. Soberon V. Schenten D. Mack B. Reutelshöfer M. et al.B cells lacking the tumor suppressor TNFAIP3/A20 display impaired differentiation, hyperactivation, cause inflammation and autoimmunity in aged mice.Blood. 2010; Google Scholarc-IAP-2GALT B cell homeostasis; noncanonical NF-κB signalingConze et al., 2010Conze D.B. Zhao Y. Ashwell J.D. Non-canonical NF-κB activation and abnormal B cell accumulation in mice expressing ubiquitin protein ligase-inactive c-IAP2.PLoS Biol. 2010; 8: e1000518Crossref PubMed Scopus (40) Google ScholarAct1B cell survival and toleranceQian et al., 2004Qian Y. Qin J. Cui G. Naramura M. Snow E.C. Ware C.F. Fairchild R.L. Omori S.A. Rickert R.C. Scott M. et al.Act1, a negative regulator in CD40- and BAFF-mediated B cell survival.Immunity. 2004; 21: 575-587Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, Qian et al., 2008Qian Y. Giltiay N. Xiao J. Wang Y. Tian J. Han S. Scott M. Carter R. Jorgensen T.N. Li X. Deficiency of Act1, a critical modulator of B cell function, leads to development of Sjögren's syndrome.Eur. J. Immunol. 2008; 38: 2219-2228Crossref PubMed Scopus (55) Google ScholarInnate CellsTRAF2 and 5TNF signalingMicheau and Tschopp, 2003Micheau O. Tschopp J. Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell. 2003; 114: 181-190Abstract Full Text Full Text PDF PubMed Scopus (1783) Google ScholarTRAF3TLR-mediated IRF3 signalingOganesyan et al., 2006Oganesyan G. Saha S.K. Guo B. He J.Q. Shahangian A. Zarnegar B. Perry A. Cheng G. Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response.Nature. 2006; 439: 208-211Crossref PubMed Scopus (677) Google ScholarTRAF6TLR signalingDeng et al., 2000Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1391) Google ScholarcIAPsTNF signalingBertrand 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. cIAP1 and cIAP2 facilitate cancer cell survival by functioning as E3 ligases that promote RIP1 ubiquitination.Mol. Cell. 2008; 30: 689-700Abstract Full Text Full Text PDF PubMed Scopus (766) Google ScholarITCHTNF signalingChang et al., 2006Chang L. Kamata H. Solinas G. Luo J.L. Maeda S. Venuprasad K. Liu Y.C. Karin M. The E3 ubiquitin ligase itch couples JNK activation to TNFalpha-induced cell death by inducing c-FLIP(L) turnover.Cell. 2006; 124: 601-613Abstract Full Text Full Text PDF PubMed Scopus (560) Google ScholarA20 (TNFAIP3)TNF, TLR, NLR signalingLee et al., 2000Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Failure to regulate TNF-induced NF-kappaB and cell death responses in A20-deficient mice.Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1133) Google Scholar, Boone et al., 2004Boone D.L. Turer E.E. Lee E.G. Ahmad R.C. Wheeler M.T. Tsui C. Hurley P. Chien M. Chai S. Hitotsumatsu O. et al.The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses.Nat. Immunol. 2004; 5: 1052-1060Crossref PubMed Scopus (855) Google Scholar, Turer et al., 2008Turer E.E. Tavares R.M. Mortier E. Hitotsumatsu O. Advincula R. Lee B. Shifrin N. Malynn B.A. Ma A. Homeostatic MyD88-dependent signals cause lethal inflamMation in the absence of A20.J. Exp. Med. 2008; 205: 451-464Crossref PubMed Scopus (218) Google Scholar, Hitotsumatsu et al., 2008Hitotsumatsu O. Ahmad R.C. Tavares R. Wang M. Philpott D. Turer E.E. Lee B.L. Shiffin N. Advincula R. Malynn B.A. et al.The ubiquitin-editing enzyme A20 restricts nucleotide-binding oligomerization domain containing 2-triggered signals.Immunity. 2008; 28: 381-390Abstract Full Text Full Text PDF PubMed Scopus (250) Google ScholarTRIM25RIG-I signaling; type I interferon productionGack et al., 2007Gack M.U. Shin Y.C. Joo C.H. Urano T. Liang C. Sun L. Takeuchi O. Akira S. Chen Z. Inoue S. Jung J.U. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity.Nature. 2007; 446: 916-920Crossref PubMed Scopus (1053) Google ScholarLUBACTNF, IL-1β signalingHaas 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.Recruitment of the linear ubiquitin chain assembly complex stabilizes the TNF-R1 signaling complex and is required for TNF-mediated gene induction.Mol. Cell. 2009; 36: 831-844Abstract Full Text Full Text PDF PubMed Scopus (505) 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.Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation.Nat. Cell Biol. 2009; 11: 123-132Crossref PubMed Scopus (674) Google Scholar Open table in a new tab Ubiquitination involves a three-step enzymatic reaction catalyzed by three different types of proteins, termed E1, E2, and E3 ubiquitin ligases (Pickart and Eddins, 2004Pickart C.M. Eddins M.J. Ubiquitin: Structures, functions, mechanisms.Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (925) Google Scholar). An E1 enzyme first "activates" ubiquitin by forming a thiol ester bond. Activated ubiquitin is transferred to an E2 ubiquitin-conjugating enzyme. The E2 enzyme-ubiquitin complex interacts with an E3 ubiquitin ligase that facilitates transfer of the ubiquitin to the epsilon-amino group of a lysine (K) on substrate proteins. Together with ubiquitin binding proteins (or sensors) and proteases that function as deubiquitinating enzymes (DUBs), E1, E2, and E3 ubiquitin ligase complexes constitute the core biochemical machinery for building, editing, and removing ubiquitin chains. While two known E1 enzymes "charge" or activate ubiquitin molecules for virtually all ubiquitination events in the mammalian proteome, diverse combinations of E2 and E3 ubiquitin ligases attach distinct types of ubiquitin chains to specific substrate proteins. Approximately 38 E2 enzymes are predicted to exist (Ye and Rape, 2009Ye Y. Rape M. Building ubiquitin chains: E2 enzymes at work.Nat. Rev. Mol. Cell Biol. 2009; 10: 755-764Crossref PubMed Scopus (609) Google Scholar). As there are many more E3 ubiquitin ligases (>600 predicted) than E2s, most E2s functionally interact with many E3 ubiquitin ligases. In addition, at least some E3s can bind to multiple E2s. For example, the E3 ubiquitin ligase complex of BRCA1 and BARD1 can interact with ten different E2s that display divergent functions; one E2 may mediate ubiquitin initiation and other E2s may mediate the elongation of various linkages depending on the E2 used (Christensen et al., 2007Christensen D.E. Brzovic P.S. Klevit R.E. E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages.Nat. Struct. Mol. Biol. 2007; 14: 941-948Crossref PubMed Scopus (248) Google Scholar, Christensen and Klevit, 2009Christensen D.E. Klevit R.E. Dynamic interactions of proteins in complex networks: Identifying the complete set of interacting E2s for functional investigation of E3-dependent protein ubiquitination.FEBS J. 2009; 276: 5381-5389Crossref PubMed Scopus (38) Google Scholar). Hence, a vast number of E2 and E3 combinations are available to specify the target proteins to be modified and the type of ubiquitin chains to be added. E2 enzymes play a major role in determining the length and linkage type of ubiquitin chains that are formed (Christensen and Klevit, 2009Christensen D.E. Klevit R.E. Dynamic interactions of proteins in complex networks: Identifying the complete set of interacting E2s for functional investigation of E3-dependent protein ubiquitination.FEBS J. 2009; 276: 5381-5389Crossref PubMed Scopus (38) Google Scholar, Ye and Rape, 2009Ye Y. Rape M. Building ubiquitin chains: E2 enzymes at work.Nat. Rev. Mol. Cell Biol. 2009; 10: 755-764Crossref PubMed Scopus (609) Google Scholar). For example, the E2 enzyme Ubc13 preferentially builds K63-linked ubiquitin chains that support mitogen-activated protein (MAP) kinase signal propagation (Yamamoto et al., 2006aYamamoto M. Okamoto T. Takeda K. Sato S. Sanjo H. Uematsu S. Saitoh T. Yamamoto N. Sakurai H. Ishii K.J. et al.Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling.Nat. Immunol. 2006; 7: 962-970Crossref PubMed Scopus (218) Google Scholar, Rodrigo-Brenni et al., 2010Rodrigo-Brenni M.C. Foster S.A. Morgan D.O. Catalysis of lysine 48-specific ubiquitin chain assembly by residues in E2 and ubiquitin.Mol. Cell. 2010; 39: 548-559Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, David et al., 2010David Y. Ziv T. Admon A. Navon A. The E2 ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines.J. Biol. Chem. 2010; 285: 8595-8604Crossref PubMed Scopus (109) Google Scholar). Ubc13 is required for interleukin-1 (IL-1) and lipopolysaccharide (LPS)-induced MAP kinase activation but appears less important for Nuclear Factor-κB (NF-κB) signaling from these ligands in macrophages and fibroblasts (Yamamoto et al., 2006aYamamoto M. Okamoto T. Takeda K. Sato S. Sanjo H. Uematsu S. Saitoh T. Yamamoto N. Sakurai H. Ishii K.J. et al.Key function for the Ubc13 E2 ubiquitin-conjugating enzyme in immune receptor signaling.Nat. Immunol. 2006; 7: 962-970Crossref PubMed Scopus (218) Google Scholar). Ubc13 also appears to be dispensable for tumor necrosis factor (TNF)-induced NF-κB signaling. In contrast, Ubc13 is important for T cell receptor (TCR)-induced NF-κB signaling in thymocytes (Yamamoto et al., 2006bYamamoto M. Sato S. Saitoh T. Sakurai H. Uematsu S. Kawai T. Ishii K.J. Takeuchi O. Akira S. Cutting edge: Pivotal function of Ubc13 in thymocyte TCR signaling.J. Immunol. 2006; 177: 7520-7524PubMed Google Scholar). It is possible that other E2 ligases, such as Ubc5, can support K63 ubiquitin-dependent signals, depending on the cell type and stimulus. The selectivity of E2s for certain subsets of E3 enzymes (and hence substrates) and the predilection of E2s to form particular ubiquitin chain linkages combine to render these enzymes important regulators as well as mediators of ubiquitination. E3 ubiquitin ligases confer substrate specificity to the ubiquitin reaction by binding to and mediating transfer of ubiquitin from E2 enzymes to target proteins such as signaling molecules. E3 ubiquitin ligases have been divided into two general types depending on the type of protein domain used to recognize substrates: really interesting new gene (RING) and homologous to E6-associated protein carboxyl terminus (HECT) E3 ubiquitin ligases. RING E3s make up the largest number by far, with over 600 predicted to be encoded in the human genome, while 28 HECT E3s are predicted to exist (Deshaies and Joazeiro, 2009Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1677) Google Scholar, Rotin and Kumar, 2009Rotin D. Kumar S. Physiological functions of the HECT family of ubiquitin ligases.Nat. Rev. Mol. Cell Biol. 2009; 10: 398-409Crossref PubMed Scopus (690) Google Scholar). RING and HECT E3s mediate substrate ubiquitination by different mechanisms. RING E3s use their RING finger domain to direct the transfer of ubiquitin from the activated E2-Ub to the substrate, whereas HECT E3s accept the ubiquitin from the E2-Ub to form a covalent thioester bond intermediate before transferring ubiquitin to the target protein. Because HECT E3s are charged with a ubiquitin while bound to their target protein, they may also help determine the specificity of linkage chain formation (Ye and Rape, 2009Ye Y. Rape M. Building ubiquitin chains: E2 enzymes at work.Nat. Rev. Mol. Cell Biol. 2009; 10: 755-764Crossref PubMed Scopus (609) Google Scholar). In addition, many RING E3s have ubiquitin binding domains (UBDs) that may serve to orient the acceptor ubiquitin molecule for attack and thereby influence the type of linkage formed. Indeed, a subset of E3 ubiquitin ligases, occasionally termed E4 ligases, may preferentially extend ubiquitin chains on ubiquitinated substrates. Hence, E3 enzymes mediate target recognition and can also contribute to linkage specificity. In addition to RING and HECT domain-containing E3 ubiquitin ligases, other protein motifs, such as plant homology domains (PHD), U box domains, and a subset of zinc fingers, have been implicated in E3 ubiquitin ligase activity. The U box, so designated by the domain found in the yeast ubiquitination factor UFD2, is a modified RING finger that lacks the canonical cysteine residues for zinc binding but can nevertheless mediate ubiquitin ligase activity (Aravind and Koonin, 2000Aravind L. Koonin E.V. The U box is a modified RING finger - a common domain in ubiquitination.Curr. Biol. 2000; 10: R132-R134Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, Hatakeyama et al., 2001Hatakeyama S. Yada M. Matsumoto M. Ishida N. Nakayama K.I. U box proteins as a new family of ubiquitin-protein ligases.J. Biol. Chem. 2001; 276: 33111-33120Crossref PubMed Scopus (449) Google Scholar). More recently, a zinc finger motif in the ubiquitin ligase A20 protein has been shown to mediate E3 ubiquitin ligase activity (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.De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling.Nature. 2004; 430: 694-699Crossref PubMed Scopus (1368) Google Scholar). Although all proteins bearing such motifs have not been tested for E3 ubiquitin ligase activity, it is likely that the number of bona fide E3 ubiquitin ligases will grow significantly. Moreover, E3 ubiquitin ligases typically possess the ability to ubiquitinate multiple substrates, suggesting that a significant portion of the mammalian proteome undergoes ubiquitination. Ubiquitination events in immune cells mediate diverse cell signals and cellular responses. Part of this diversity is due to the fact that ubiquitin molecules can be attached to proteins as monomers or as polymers (Figure 1). Monoubiquitination events regulate DNA repair, receptor endocytosis, vesicle sorting, and gene silencing (Sigismund et al., 2004Sigismund S. Polo S. Di Fiore P.P. Signaling through monoubiquitination.Curr. Top. Microbiol. Immunol. 2004; 286: 149-185Crossref PubMed Scopus (129) Google Scholar). Ubiquitination of DNA repair proteins can impact immune processes such as class switch recombination (Li et al., 2010Li L. Halaby M.J. Hakem A. Cardoso R. El Ghamrasni S. Harding S. Chan N. Bristow R. Sanchez O. Durocher D. Hakem R. Rnf8 deficiency impairs class switch recombination, spermatogenesis, and genomic integrity and predisposes for cancer.J. Exp. Med. 2010; 207: 983-997Crossref PubMed Scopus (87) Google Scholar, Santos et al., 2010Santos M.A. Huen M.S. Jankovic M. Chen H.T. López-Contreras A.J. Klein I.A. Wong N. Barbancho J.L. Fernandez-Capetillo O. Nussenzweig M.C. et al.Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8.J. Exp. Med. 2010; 207: 973-981Crossref PubMed Scopus (75) Google Scholar, Sun and Chen, 2004Sun L. Chen Z.J. The novel functions of ubiquitination in signaling.Curr. Opin. Cell Biol. 2004; 16: 119-126Crossref PubMed Scopus (359) Google Scholar). Monoubiquitination has also been implicated in persistent NF-κB signaling that has implications for human T cell leukemia virus-1 (HTLV-1) infection and signaling thresholds mediating positive and negative thymic selection (Carter et al., 2005Carter R.S. Pennington K.N. Arrate P. Oltz E.M. Ballard D.W. Site-specific monoubiquitination of IkappaB kinase IKKbeta regulates its phosphorylation and persistent activation.J. Biol. Chem. 2005; 280: 43272-43279Crossref PubMed Scopus (32) Google Scholar, Wada et al., 2009Wada K. Niida M. Tanaka M. Kamitani T. Ro52-mediated monoubiquitination of IKKbeta down-regulates NF-kappaB signalling.J. Biochem. 2009; 146: 821-832Crossref PubMed Scopus (53) Google Scholar, Wang et al., 2010Wang H. Holst J. Woo S.R. Guy C. Bettini M. Wang Y. Shafer A. Naramura M. Mingueneau M. Dragone L.L. et al.Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.EMBO J. 2010; 29: 1285-1298Crossref PubMed Scopus (33) Google Scholar). Polyubiquitin chains can be formed by using any one of the seven internal lysine residues (K6, K11, K27, K29, K33, K48, and K63) or the N-terminal amino group of ubiquitin (Komander, 2009Komander D. The emerging complexity of protein ubiquitination.Biochem. Soc. Trans. 2009; 37: 937-953Crossref PubMed Scopus (515) Google Scholar) to form distinct ubiquitin chain linkage types (Figure 1). Structural studies have revealed that different chain linkages adopt distinct conformations (Pickart and Fushman, 2004Pickart C.M. Fushman D. Polyubiquitin chains: Polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (790) Google Scholar, Fushman and Walker, 2010Fushman D. Walker O. Exploring the linkage dependence of polyubiquitin conformations using molecular modeling.J. Mol. Biol. 2010; 395: 803-814Crossref PubMed Scopus (45) Google Scholar). Hence, distinct chain types could be distinguished by ubiquitin-dependent proteins. K48-linked ubiquitin chains that are at least four ubiquitin molecules in length target misfolded or senescent proteins for recruitment to the proteasome for proteolytic degradation (Pickart and Fushman, 2004Pickart C.M. Fushman D. Polyubiquitin chains: Polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (790) Google Scholar). In the context of cell signaling, K48-linked polyubiquitin chains facilitate degradation of signaling proteins, including both agonists and inhibitors of signal transduction. Signaling proteins probably do not exhibit the same biochemical features as misfolded proteins. E3 ubiquitin ligases that target signaling proteins often recognize modifications to these proteins that occur during their activation, such as phosphorylated residues. For example, phosphorylation of the NF-κB inhibitor IkBα leads to its recognition by a Skp1-Cul1-F box E3 complex called SCFβTrCP that adds K48 ubiquitin chains to IkBα and promotes its degradation (Skaug et al., 2009Skaug B. Jiang X. Chen Z.J. The role of ubiquitin in NF-kappaB regulatory pathways.Annu. Rev. Biochem. 2009; 78: 769-796Crossref PubMed Scopus (388) Google Scholar). Hence, regulated degradation of signaling inhibitors supports the propagation of canonical NF-κB signals. By contrast, K48 ubiquitination of agonist signaling proteins limits the duration of signals. An example of this type of ubiquitin regulation is the negative feedback inhibition of cytokine signaling by suppressors of cytokine signaling (SOCS)-family proteins. SOCS proteins are E3 ubiquitin ligases that tag cytokine signaling proteins, such as Janus kinases (JAKs) and cytokine receptors, with ubiquitin, marking them for degradation (Alexander and Hilton, 2004Alexander W.S. Hilton D.J. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response.Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (582) Google Scholar). Regulated attachment of K48 ubiquitin chains to agonist signaling molecules has been increasingly recognized in restricting various immune signaling cascades. A major revelation in cell signaling biology was the discovery that ubiquitin chains assembled in certain conformations can target proteins for outcomes other than proteosomal degradation (Deng et al., 2000Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain.Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1391) Google Scholar, Pickart and Fushman, 2004Pickart C.M. Fushman D. Polyubiquitin chains: Polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (790) Google Scholar). Structural studies showed that K63 ubiquitin chains are more flexible than K48 chains, providing a biochemical basis for selective recognition of K63-ubiquitinated proteins (Pickart and Fushman, 2004Pickart C.M. Fushman D. Polyubiquitin chains: Polymeric protein signals.Curr. Opin. Chem. Biol. 2004; 8: 610-616Crossref PubMed Scopus (790) Google Scholar, Winget and Mayor, 2010Winget J.M. Mayor T. The diversity of ubiquitin recognition: Hot spots and varied specificity.Mol. Cell. 2010; 38: 627-635Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, Fushman and Walker, 2010Fushman D. Walker O. Exploring the linkage dependence of polyubiquitin conformations using molecular modeling.J. Mol. Biol. 2010; 395: 803-814Crossref PubMed Scopus (45) Google Scholar). A quantitative proteomics profile of polyubiquitin linkages in yeast showed that all the lysines in ubiquitin can form chains and, with the exception of lysine 63, can directly target proteins to the proteasome for degradation with varying efficiency (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. Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.Cell. 2009; 137: 133-145Abstract Full Text Full Text PDF PubMed Scopus (785) Google Scholar). While ubiquitin chains of distinct conformations had been defined in yeast, their importance in signaling pathways in metazoan organisms is now being more fully realized. We now highlight some of the recent discoveries that manifest how the diversity of ubiquitin signaling modalities impact immune cell signaling pathways. The presence of four ubiquitin-coding genes and the widespread use of ubiquitin modifications in multiple aspects of cell biology render strategies for the genetic manipulation of ubiquitin linkages difficult. Chen and colleagues devised a ubiquitin replacement strategy for testing the requirements for specific ubiquitin chain linkage in cells. Using an inducible system for the coordinated knockdown of endogenous ubiquitin and expression of mutant ubiquitin in model cell lines, they showed that IL-1-induced NF-κB signaling requires K63 ubiquitin chains. By contrast, TNF-induced NF-κB signaling can occur in the absence of K63 (Xu et al., 2009aXu M. Skaug B. Zeng W. Chen Z.J. A ubiquitin replacement strategy in human cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta.Mol. Cell. 2009; 36: 302-314Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). This strategy was also used to show that K63 ubiquitination is required for viral activation of interferon regulatory factor-3 (IRF3)