Báo cáo khoa học: TNFR1-induced activation of the classical NF-jB pathway Harald Wajant1 and Peter Scheurich2 pptx

MINIREVIEW TNFR1-induced activation of the classical NF-jB pathway Harald Wajant1 and Peter Scheurich2 Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wurzburg, Germany ă Institute of Cell Biology and Immunology, University of Stuttgart, Germany Keywords apoptosis; caspase 8; IKK necrosis; NF-jB, NEMO; RIP1; TNF; TRADD; TRAF2 Correspondence H Wajant, Division of Molecular Internal Medicine, Medical Clinic and Polyclinic II, University Hospital Wurzburg, Rontgenring ă ¨ 11, 97070 Wurzburg, Germany ¨ Fax: 49 931 201 71070 Tel: 49 931 201 71000 E-mail: harald.wajant@mail.uni-wuerzburg.de (Received 12 October 2010, revised December 2010, accepted 11 December 2010) The molecular mechanisms underlying activation of the IjB kinase (IKK) complex are presumably best understood in the context of tumor necrosis factor (TNF) receptor-1 (TNFR1) signaling In fact, it seems that most, if not all, proteins relevant for this process have been identified and extensive biochemical and genetic data are available for the role of these factors in TNF-induced IKK activation There is evidence that protein modification– independent assembly of a core TNFR1 signaling complex containing TNFR1-associated death domain, receptor interacting kinase 1, TNF receptor-associated factor and cellular inhibitor of apoptosis protein and starts a chain of nondegrading ubiquitination events that culminate in the recruitment and activation of IKK complex-stimulating kinases and the IKK complex itself Here, we sum up the known details of TNFR1induced IKK activation, address arising contradictions and discuss possible explanations resolving the apparent discrepancies doi:10.1111/j.1742-4658.2011.08015.x The NF-jB system The nuclear factor of kappa B (NF-jB) proteins are dimeric transcription factors composed of five different subunits, namely p65 (RelA), RelB, cRel, p50 and p52 In unstimulated cells, the NF-jB transcription dimers are retained in the cytoplasm in an inactive state by masking of their nuclear localization sequence [1,2] Two different structural modes of the blockade of the nuclear localization sequence can be distinguished (Fig 1) In the first case, a NF-jB dimer interacts intermolecularly with an inhibitor of kappa B (IjB) protein under the formation of an inactive ternary complex In the second case, blockade of the nuclear localization sequence is achieved by intramolecular binding of an inhibitory domain This is possible because the NF-jB subunits p50 and p52 are initially produced as large precursor proteins of 105 and 100 kDa, respectively, carrying IjB protein-like inhibitory domains in their C-terminal parts [1,2] The two mechanisms of NF-jB dimer inhibition correspond to the existence of two different NF-jB-activating pathways called the classical and the alternative NF-jB pathway (Fig 1) The classical NF-jB pathway is initiated by activation of tumor necrosis factor (TNF) receptor-associated factor (TRAF) adapter proteins and the subsequent stimulation of the IjB kinase (IKK) complex, which among others also contains the related kinases IKK1 and IKK2 and the structural ⁄ regulatory component NF-jB essential modulator Abbreviations cIAP, cellular inhibitor of apoptosis protein; HOIL-1, heme-oxidized IRP1 ubiquitin ligase; IjB, inhibitor of kappa B; IKK, IjB kinase; IL, interleukin; LUBAC, linear ubiquitin chain assembly complex; MEF, murine embryonal fibroblast; NEMO, NF-jB essential modulator; NF-jB, nuclear factor of kappa B; PK, protein kinase; RIP1, receptor-interacting kinase 1; RNF11, RING finger protein 11; S1P, sphingosine1-phosphate; TAX1BP1, Tax1 binding protein; TNF, tumor necrosis factor; TRADD, TNFR1-associated via death domain; TRAF2, TNF receptor-associated factor 862 FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS H Wajant and P Scheurich TNFR1 induced NF-jB signaling - - Fig Activation of classical and alternative NF-jB signaling The classical NF-jB pathway can be activated by a broad range of stimuli, including most ligands of the TNF family, IL-1, a variety of pathogen associated molecular patterns (e.g lipopolysaccharide) and physical stress (e.g UV irradiation) Activation of the classical NF-jB pathway involves stimulation of the kinase activity of the IKK complex and proteolytic degradation of IjB proteins The alternative NF-jB pathway is activated by a limited subgroup of TNF ligands and involves activation of NIK-mediated stimulation of IKK1 and conversion of p100-containing NF-jB complexes into p52-containing NF-jB complexes by proteolytic processing of p100 to p52 (NEMO) [1,2] The activated IKK complex phosphorylates IjB proteins, thereby triggering their proteasomal degradation As a consequence, the NF-jB dimers are released in the cytoplasm and can now translocate into the nucleus Activation of the alternative NF-jB pathway is independent of IKK2 and NEMO and requires degradation of TRAF proteins and subsequent activation of IKK1 [1,2] IKK1 phosphorylates p100 and thereby triggers its processing to p52 This event results in the conversion of p100-inhibited NF-jB complexes into p52-containing NF-jB dimers capable of translocating into the nucleus [1,2] Different NF-jB dimers regulate different target genes The two NF-jB pathways have, therefore, many nonredundant functions Although the classical pathway mainly acts in innate immunity, the alternative pathway is of central relevance for the organogenesis of lymphoid tissue The TNF-induced core signaling complex of TNFR1 Ligand-induced reorganization of preassembled receptor complexes enables TNFR1 to recruit the adapter protein TNF receptor-associated protein with a death domain (TRADD) and the serine–threonine kinase receptor-interacting protein (RIP1) [3] TRADD and RIP1 contain a C-terminal death domain which mediates binding to the death domain of TNFR1 There are contradictory reports claiming competitive, but also cooperative, effects in the recruitment of TRADD and RIP1 to TNFR1, but in sum, it is generally accepted that RIP1 and TRADD are capable of interacting independently with TNFR1 [4–6] In addition, TRADD and RIP1 also interact strongly with each other by virtue of their death domains, which could be of importance for the assembly ⁄ integrity of a cytosolic, TRADD and RIP1-containing complex that is formed upon TNFR1 stimulation (for more details see the accompanying minireview by O’Donnell and Ting [7]) Upon association with ligated TNFR1, TRADD further recruits the adapter protein TRAF2 via its N-terminal TRAF-binding domain (Fig 2) TRAF2 consists of an N-terminal RING domain followed by five zinc fingers and a C-terminal TRAF domain, which mediates homotrimerization and interaction with TRADD [8] TRAF2 forms stable homotrimeric mushroom-shaped complexes capable of interacting with one TRADD molecule with each of its protomers The interaction of TRAF2 and TRADD is much stronger compared with TRAF2 binding to those members of the TNF receptor family that directly interact with TRAF proteins, such as, for example, TNFR2 Although a similar surface at the edge of the FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 863 TNFR1 induced NF-jB signaling H Wajant and P Scheurich mushroom cap of a TRAF2 complex mediates interaction with TRADD as well as TRAF2-binding TNF receptors, the actual molecular contacts enabling these interactions are entirely different [9] TNF-induced recruitment of TRAF2 to TNFR1 is abrogated in TRADD-deficient murine embryonal fibroblasts (MEFs) [10–12], but there is still significant TRAF2 recruitment in TRADD-deficient macrophages [12] Because RIP1 is known to directly interact with TRAF2 and is highly expressed in macrophages, it was suggested that RIP1 might contribute to TRAF2 recruitment to TNFR1, thereby compensating to some extent for TRADD deficiency in this regard [12] Nevertheless, in the absence of RIP1, TRADD is fully sufficient to recruit TRAF2 into the TNFR1 signaling complex TRAF2 forms complexes with cellular inhibitor of apoptosis protein (cIAP)-1 and cIAP2 with high efficacy and therefore typically all these proteins are part of the TNF-induced TNFR1 signaling complex [13] TRADD, RIP1, TRAF2 and cIAPs are crucially involved in TNFR1-induced NF-jB signaling It is evident from studies with TRADD-, TRAF2- and RIP1-deficient mice that all these molecules play important roles in TNFR1-induced activation of the classical NFjB pathway, but are not absolutely indispensable Although TNFR1-induced phosphorylation and degradation of IjBa are almost completely abolished in TRADD-deficient MEFs, these hallmarks of classical NF-jB signaling are only attenuated in TRADD-deficient macrophages [11,12] These findings correspond well to the differential capability of TNFR1 in these cells to recruit TRAF2 in a TRADDindependent fashion (see above) The available genetic evidence also argues for a cell type-specific dependency of TNFR1-induced activation of the classical NF-jB pathway from RIP1 (Table 1) No significant signs of TNF-induced NF-jB signaling were found in a human Fig Formation of the NF-jB-stimulating TNFR1 signaling complex In a first step, binding of TNF to TNFR1 triggers recruitment of the death domain-containing proteins RIP1 and TRADD In a second step, there is recruitment of TRAF2–cIAP1 ⁄ complexes to TNFR1-bound TRADD At this point, there is no evidence that protein modifications, such as phosphorylation or ubiquitination, play a role in the assembly of the TNFR1 signaling complex The TRAF2-associated E3 ligases cIAP1 and cIAP2 (and possibly also TRAF2) now modify RIP1, TRAF2 and themselves with K63-linked ubiquitin chains This creates docking sites for the LUBAC complex, an E3 ligase capable of forming linear polyubiquitin chains The LUBAC complex ubiquitinates NEMO, a subunit of the IKK complex, which by help of its IKK2 subunit also interacts with TRADD-bound TRAF2 In parallel, the TAK1-TAB complex interacts with K63-ubiquitin modified RIP1 by use of the K63-ubiquitin binding TAB subunit TAK1 become activated and then phosphorylates and activates IKK2 which in turn now phosphorylates IjBa, marking it for K48-ubiquitination and proteasomal degradation 864 FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS H Wajant and P Scheurich TNFR1 induced NF-jB signaling Table Effect of receptor-interacting kinase (RIP1) deficiency on tumor necrosis factor (TNF)-induced activation of the classical NF-jB pathway IKK, IjB kinase; IL, interleukin; MEF, murine embryonal fibroblast; NF-jB, nuclear factor of kappa B Model RIP1 def Jurkat RIP1 def Jurkat Abelson-transformed B-cells RIP1 def MEFs RIP1 def MEFs RIP1 def MEFs Observed effects on TNF-induced activation of the classical NF-jB pathway No TNF-induced activation of a NF-jB-regulated reporter gene Complete inhibition of TNF-induced IjBa phosphorylation (western blot) and DNA-binding of NF-jB (EMSA) Completely inhibited TNF-induced DNA-binding of NF-jB (EMSA) No or at best traces (< 10% of wild-type) of TNF-induced IKK activation and IL-6 induction Minor (10–30% of wild-type) but significant TNF-induced IKK activation Moderately attenuated TNF-stimulated degradation of IjBa and only modestly reduced induction (residual induction 70–80% of wild-type) of NF-jB-regulated target genes in primary and SV40 transformed MEFs Ref [68] [39] [69] [14] [4] [70] mutant cell line lacking RIP1 and in Abelson murine leukemia virus-transfomed pre-B-cell lines derived from RIP1 knockout mice (Table 1) By contrast, for MEFs derived from RIP1 knockout mice, almost unaffected TNF-induced NF-jB signaling has been reported (Table 1) Remarkably, reconstitution experiments with kinase-dead mutants suggest that the kinase activity of RIP1 is dispensable for its role in TNFR1-induced NF-jB activation [14] Reports describing variable contributions of RIP1 to TNFR1-mediated NF-jB activation correspond well with the observation that the C-terminal subunit of the heterodimeric oncoprotein MUC1 can substitute for RIP1 in TNFR1-induced activation of the classical NF-jB pathway MUC1 is initially produced as a typ I transmembrane polypeptide which is rapidly autoproteolytically cleaved in its sea urchin sperm protein, enterokinase and agrin (SEA) module near the membrane The resulting N-terminal (MUC1-N) and C-terminal (MUC1-C) fragments tightly interact by noncovalent interaction In MCF-10A cells, MUC1-C has been shown to be recruited to TNFR1 in a TRADD- and TRAF2-dependent manner after TNF stimulation, whereas RIP1 was found to be dispensable for this interaction [15] Furthermore, molecular knockdown of TRADD, TRAF2, TAK1 or TAB 2, all known to be of relevance in TNFR1-induced and RIP1-mediated NF-jB activation, also resulted in downregulation of TNF-induced and MUC1-mediated NF-jB signaling in MCF-10A cells These data are in accordance with the hypothesis that MUC1-C is capable of substituting for RIP1 in TNFR1-mediated activation of the NF-jB signaling pathway As expected, knockdown of MUC1-C also strongly reduced the TNF-induced NF-jB response [15] MEFs derived from TRAF2-deficient mice showed only a slightly reduced efficiency of TNF-induced NF-jB activation, whereas MEFs derived from TRAF2–TRAF5 double-deficient mice were severely impaired in this regard [16] Accordingly, TRAF2 and TRAF5 were proposed to have at least partially redundant functions in TNFR1-induced NF-jB activation, although recruitment of TRAF5 into the TNFR1 signaling complex has not been demonstarted to date Notably, various studies analyzing MEFs deficient for TRAF2, TRAF5 or both molecules revealed quite contradictory effects on TNF-induced NF-jB signaling (Table 2) Because disagreements have been also reported in cells of the same genetic background, they might reflect differences in the cultivation conditions or cell culture-related adaptation ⁄ selection processes In any case, these discrepancies illustrate the problems and pitfalls in making generalizations based only on studies of MEFs In line with this, initial analyses of cIAP1- and cIAP2-deficient MEFs gave no evidence for a role of these molecules in TNFR1 signaling [17,18] However, follow-up studies demonstrated impaired TNF-induced IjBa degradation in MEFs when expression of both cIAP proteins had been downregulated by RNA interference [19–21] At a first glance, the NF-jB activation-promoting function of cIAP1 and -2 observed in the context of TNFR1 signaling dissents from other reports showing NF-jB activation after depletion of cIAP1 and -2 with second mitochondria-derived activator of caspase (SMAC) mimetics [22,23] However, cIAP1 and -2 are not only involved in TNFR1-induced activation of the IKK complex, but are also responsible for the constitutive degradation of the MAP3K NF-jB-inducing kinase (NIK) [24,25], which phosphorylates and thereby activates IKK1 to trigger limited processing of p100 Accordingly, cIAP1 and -2 are negative regulators of the alternative NF-jB pathway and SMAC mimetics have consequently been identified as strong inducers of p100 processing The still puzzling observation that SMAC mimetics also activate the classical NF-jB pathway might be related to the fact that the IjB FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 865 TNFR1 induced NF-jB signaling H Wajant and P Scheurich Table Effect of TNF receptor-associated (TRAF)2 and TRAF5 deficiency on tumor necrosis factor (TNF)-induced activation of the classical NF-jB pathway cIAP, cellular inhibitor of apoptosis protein; IKK, IjB kinase; IL, interleukin; MEF, murine embryonal fibroblast; NF-jB, nuclear factor of kappa B FLIP, FLICE-inhibitory protein Effect on TNF-induced activation of the classical NF-jB pathway Cell type )⁄) TRAF2 MEFs TRAF2) ⁄ ) MEFs TRAF2) ⁄ ) MEFs TRAF2) ⁄ ) MEFs TRAF2) ⁄ ) MEFs TRAF2) ⁄ ) Bone marrow macrophages TRAF5) ⁄ ) MEFs TRAF5) ⁄ ) Bone marrow macrophages TRAF2) ⁄ ) TRAF5) ⁄ ) MEFs TRAF2) ⁄ ) TRAF5) ⁄ ) MEFs TRAF2) ⁄ ) TRAF5) ⁄ ) MEFs TRAF2) ⁄ ) TRAF5) ⁄ ) MEFs IKK activity IjBa phosphorylation Attenuated, basal activity enhanced IjBa degradation NF-jB target Genes Minor inhibition Delayed, basal activity normal Attenuated Enhanced and prolonged Strongly reduced Poorly affected Unchanged Reduced Ref [71] Normal DNA binding (EMSA) [16] A20 and IL-6 strongly reduced Higher basal P-p65, but poorly inducible Strongly reduced p-Ser 536 Normal DNA binding (EMSA) [29] [72] NF-jB reporter gene fully blocked [73] [74] Normal Basally increased, but TNF-induced maximal effect comparable with wild-type Delayed and attenuated, basal activity normal [16] Normal DNA binding (EMSA) [75] Strong, but not complete inhibition Delayed and weak FLIP and cIAP1 un-changed But IL-6, IP10 and ICAM-1 reduced Reduced DNA binding (EMSA) Residual IjBa phosphorylation [71] [16] [35] Basally increased, but TNF-induced maximal effect comparable with wild-type Higher basal P-p65 but poor increase by TNF domain of one of the subunits of a p100 homodimer can inhibit p65 ⁄ p50 NF-jB dimers which are typically regulated by the classical pathway [26] Together, in this special case activation of the alternative NF-jB pathway and p100 processing result in the release of a ‘classical’ NF-jB dimer in a fashion independent of [76] Strongly reduced P-Ser 536 TRAF2) ⁄ ) TRAF5) ⁄ ) MEFs 866 Nuclear translocation and phosphorylation of p65 [51] IKK complex activity and IjB degradation Furthermore, TNF production itself is regulated by NF-jBs and SMAC mimetics thus induce production of TNF and TNF-mediated apoptosis in some cell types but upregulation of TNF may also lead to enhanced NF-jB activation [22,23] FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS H Wajant and P Scheurich TNFR1 induced NF-jB signaling Table Ubiquitination of components of the NF-jB-stimulating TNF receptor-associated factor (TNFR1) signaling complex LUBAC, linear ubiquitin chain assembly complex; NEMO, NF-jB essential modulator; NF-jB, nuclear factor of kappa B; RIP1, receptor-interacting kinase 1; S1P, shingosine-1-phosphate; TRAF2, TNF receptor-associated factor Modified component Modified residue(s) TRAF2 – – K-31 – – K-377 – – cIAP1 cIAP2 RIP1 TAK1 NEMO K-158 K-285, K309 E3 ligase cIAP1 – – – – cIAP1 and cIAP2 TRAF2–S1P – LUBAC Ubiquitin linkage type Ref – K63 K63 – – – K48, K63 K63 [77] [30] [29] [19,22] [19,22] [38,39] [19,21] [36] K63 Linear head to tail [31] [37] TNFR1-induced activation of classical NF-jB signaling is associated with recruitment of MAP3K and the IKK complex to ubiquitinated components of the TNFR1 core signaling complex Based on co-immunoprecipitation experiments showing TNF-induced recruitment of the IKK complex to TNFR1 in wild-type and RIP1-deficient cells, but defective recruitment in TRAF2-deficient cells, an initial simple model of TNFR1-induced NF-jB activation was proposed [4] According to this model, TRAF2 is responsible for the recruitment of the IKK complex to the TNFR1 core signaling complex where RIP1 activates the kinase subunits of the IKK complex Early on, it became evident that TNFR1, TRAF2 and particularly RIP1 undergo ubiquitination in the TNFR1 core signaling complex (Table 3) Ubiquitination of TNFR1 occurs after TNF-induced translocation into lipid rafts, but the functional consequences and the E3 ligases involved are still obscure [27] The multiubiquitin chains found in TNFR1-associated TRAF2 and RIP1 are not linked via K48, typically marking the modified proteins for proteasomal degradation, but are linked via K63 or linearly by headto-tail conjugation These latter two ubiquitination forms are now known to represent recognition sites for ubiquitin binding proteins in the absence of degradation [28–30] In fact, the IKK complex and the IKK-activating TAK1–TAB 2–TAB complex recruit to the ubiquitinated TNFR1 core signaling complex by means of their respective ubiquitin-binding subunits NEMO and TAB NEMO also becomes multiubiqui- tinated in course of TNFR1-induced NF-jB activation in a TRAF2- and RIP1-dependent manner (Table 3) Ubiquitination of ubiquitin binding proteins ⁄ protein complexes is not unusual and may serve to facilitate formation of stable supramolecular protein complexes of ubiquitinated proteins and ubiquitin binding proteins There is further evidence that TAK1 is activated by K63 multiubiquitination and then phosphorylates IKK1 and IKK2 in their activation loops to trigger IjBa phosphorylation and proteasomal degradation of IjBa [31] An important role of TAK1, TAB and TAB in TNFR1-induced activation of the classical NF-jB is evident from analyses of knockout mice and RNA interference experiments (Table 4) As observed for other components of the TNFR1 signaling complex, however, these molecules are not absolutely obligate for TNF-induced NF-jB signaling MEKK3 is another MAP3K family that has been implicated in TNFR1-mediated IKK activation MEKK3) ⁄ ) MEFs display strongly impaired TNF-induced NF-jB activation and biochemical studies further showed that this kinase interacts with RIP1 and TRAF2 [32] MEKK3 can phosphorylate IKKs, but also stimulates TAK1 Whether TAK1 and MEKK3 act in a redundant manner in TNFR1-induced activation of the IKK complex or whether these two molecules cooperate in this regard is currently unclear There is further evidence that MEKK2 is also involved in TNFR1-induced IKK activation Notably, MEKK2 and MEKK3 target different NF-jB complexes after TNF stimulation [33] TNF induces formation of an early complex composed of MEKK3, IjBa and the IKK complex, but in addition also the independent and delayed formation of a complex consisting of MEKK2, IjBb and again the IKK complex [33] Correspondingly, MEKK3 deficiency primarily reduces early NF-jB activation by TNF, whereas MEKK2 downregulation by RNA interference results in downregulation of delayed NF-jB activity Combined inhibition of MEKK2 and MEKK3 in turn results in an almost complete inhibition of TNF-induced phosphorylation and degradation of IjBa ⁄ b [33] Nevertheless, there is no detailed knowledge of the interplay of MEKK2, MEKK3 and TAK1 in TNFR1-induced NF-jB signaling and, in particular, it is unclear whether ubiquitination of MEKK3 or MEKK2 plays a role E3 ligases involved in TNFR1-induced activation of the classical NF-jB pathway Several E3 ligases have been implicated in the regulation of TNF-induced NF-jB signaling A fraction of FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 867 TNFR1 induced NF-jB signaling H Wajant and P Scheurich Table Effects of knockout ⁄ knockdown of components of the TNF receptor-associated factor (TNFR1) signaling complex on TNFR1induced activation of the classical NF-jB pathway cIAP, cellular inhibitor of apoptosis protein; HOIL-1, heme-oxidized IRP1 ubiquitin ligase; IKK, IjB kinase; LUBAC, linear ubiquitin chain assembly complex; MEF, murine embryonal fibroblast; NEMO, NF-jB essential modulator; NF-jB, nuclear factor of kappa B; PKC, protein kinase C; RIP1, receptor-interacting kinase 1; TRADD, TNFR1-associated death domain Target Effect of knockout ⁄ knockdown on TNF-induced NF-jB signaling Ref TRADD MEFs: strongly reduced phosphorylation and degradation of IjBa; no TNFR1-associated RIP1 polyubiquitination in MEFs Macrophages: only partial reduction of phosphorylation and degradation of IjBa See Table See Table cIAP1 KO MEFs: normal TNF-induced NF-jB signaling and constitutive enhanced cIAP2 expression cIAP2 KO MEFs: normal TNF-induced NF-jB signaling cIAP2 KO MEFs + cIAP1 siRNA: no IjBa degradation cIAP1 siRNA in C2C12 cells: no IjBa degradation cIAP1 or cIAP2 siRNA in MEFs or hepatocytes: normal IjBa degradation cIAP1 and cIAP2 siRNA in MEFs or hepatocytes: no IjBa degradation HOIL-1 KO MEFs: Reduced phosphorylation and degradation of IjBa and reduced gene induction HOIL-1 and ⁄ or HOIP siRNA in HeLa: Reduced phosphorylation and degradation of IjBa and reduced gene induction; normal recruitment of TRADD, but reduced recruitment of TRAF2, RIP1, TAK1 and NEMO MEFs: delayed TAK1 activation, but reduced NF-jB–DNA binding and attenuated phosphorylation and degradation of IjBa MEFs: almost complete inhibition of phosphorylation and degradation of IjBa and reporter gene activity; reduced p65 phosphorylation on S536 MEKK2 KO MEFs: Delayed phase of NF-jB -DNA binding is blocked MEKK3 KO MEFs: inhibited interaction of IjBa, but not IjBb containing NF-jB complexes to the IKK complex; NF-jB -DNA binding, phosphorylation and degradation of IjBa are delayed and almost completely reduced MEKK2-MEKK3 DKO MEFs: complete inhibition of NF-jB -DNA binding, phosphorylation and degradation of IjBa Primary embryonal fibroblasts: Reduced reporter gene synthesis and DNA binding activity but grossly normal IKK activation Lung cells: Strongly reduced IKK activation and DNA binding activity MEFs: TRAF2 phophorylation, IKK activation, interaction of TRAF2 with the IKK complex and expression of NF-jB -regulated gene are reduced MEFs + PKCd siRNA: phosphorylation, IKK activation, interaction of TRAF2 with the IKK complex and expression of NF-jB -regulated gene almost completely blocked MEFs: Sustained phosphorylation and degradation of IjBa and enhanced production of NF-jB -regulated genes MEFs: Prolonged NF-jB -DNA binding, sustained phosphorylation and degradation of IjBa and enhanced production of NF-jB -regulated genes MEFs: Prolonged NF-jB -DNA binding, sustained phosphorylation and degradation of IjBa and enhanced production of NF-jB -regulated genes; enhanced K63 ubiquitination and reduced K63 ubiquitination of RIP1 siRNA: Prolonged NF-jB -DNA binding, sustained phosphorylation and degradation of IjBa, enhanced production of NF-jB-regulated genes and enhanced RIP1 ubiquitination [10–12] TRAFs RIP1 cIAPs LUBAC TAB TAK1 MEKKs PKCf PKCe A20 TAX1BP1 Itch RNF11 these ligases modifies components of the TNFR1 signaling complex by K48-linked multiubiquitin chains and thus prompt their proteasomal degradation These E3 ligases are discussed in the paragraph dedicated to the termination of TNF-induced NF-jB signaling (see below) However, the RING finger domain-containing molecules TRAF2, cIAP1 and cIAP2 themselves posses E3 ubiquitin ligase activity Together with the linear ubiquitin chain assembly complex (LUBAC), consisting of the heme-oxidized IRP1 ubiquitin ligase (HOIL-1) and HOIL-1-interacting protein HOIP, there are four E3 ligases capable of modifying components of the TNFR1 signaling complex with ‘nondegrading’ 868 [17–20] [34,37] [66] [31] [32,33] [48] [29] [78] [57] [58] [59] regulatory multiubiquitin chains, either linearly linked or via lysine residues distinct from K48 of ubiquitin, especially K63 TRAF2 tightly interacts constitutively by use of the N-terminal part of the TRAF domain with the BIR domains of cIAP1 and cIAP2 [13] As discussed above, upon TNF stimulation, TRAF2 and the associated cIAPs are recruited to TNFR1bound TRADD with the help of the C-terminal half of the TRAF domain of TRAF2 Thus, the E3 ligases TRAF2, cIAP1 and cIAP2 enter the TNFR1 signaling complex essentially independent of the presence of ubiquitin chains By contrast, reconstitution experiments of TRAF2 or cIAP knockout cells with the FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS H Wajant and P Scheurich corresponding E3 ligase activity-defective mutants revealed that recruitment of LUBAC is indirect and dependent on cIAP- but not TRAF2-mediated ubiquitination events [34] Indeed, the HOIP subunit of LUBAC in particular interacts with K48-, K63- and linearly linked ubiquitin chains [34] The hypothesis that cIAP1 ⁄ 2-mediated ubiquitination of one or more components of the TNFR1 core signaling complex is crucially involved in recruitment and activation of the IKK complex is in good agreement with functional and biochemical data Cells with absent or downregulated cIAP1 and -2 showed no RIP1 ubiquitination, no recruitment of LUBAC and no significant IjBa degradation [34] TRAF2 itself is also a reasonable substrate for ubiquitination in vitro and becomes modified with K63-linked polyubiquitin chains within the TNFR1 signaling complex [29] K63-ubiquitination of TRAF2 primarily occurs at lysine 31 and is dependent on TNF-induced phosphorylation of TRAF2 on T117 by protein kinase (PK)Cd and PKCe [29] Reconstitution experiments with TRAF2 knockout MEFs and TRAF2 mutants that are defective in T117 phosporylation and ubiquitination of K31, together with analysis of PKCe knockout MEFs with knockdown of PKCd, revealed further evidence of a crucial role of K63-ubiquitination of the RING domain of TRAF2 for recruitment of the IKK complex into the TNFR1signaling complex and activation of the NF-jB pathway [29] However, there is also strong evidence that K63-ubiquitination of TRAF2, and therefore its RING domain, is not involved in TNFR1-mediated NF-jB activation Together, reconstitution experiments in MEFs derived from TRAF2 ⁄ double-deficient mice with TRAF2 mutants revealed that the capability of this molecule to interact with cIAPs is necessary to restore TNF-induced NF-jB activation, whereas its RING ⁄ E3 ligase domain is dispensable The function of TRAF2 as a bona fide E3 ligase is also controversial Some studies making use of transient expression experiments reported TRAF2 autoubiquitination that was dependent on the RING domain of TRAF2 and the dimeric Ubc13–Uev1A conjugating enzyme complex [28,30] By contrast, elucidation of the structure of the RING domain and the first zinc finger of TRAF2 revealed an unfavorable interface for interaction with Ubc13 and Ubc-related E2 proteins, which in this report, also correlated with a lack of TRAF2 autoubiquitination activity [35] However, these discrepancies may become resolved in view of a recent study by Alvarez et al [36] identifying sphingosine-1phosphate (S1P) as a cofactor for the E3 ligase activity of TRAF2 using in vitro RIP1 ubiquitinations assays TNFR1 induced NF-jB signaling with Ubc13–Uev1a as E2 component Future studies must now clarify whether binding of S1P to TRAF2 induces, for example, a structural change enabling TRAF2–Ubc13 interaction In conclusion, TRAF2 likely represents an authentic E3 ligase, but the relevance of this activity for the role of TRAF2 in TNFR1-induced NF-jB signaling remains unresolved Although recruitment of LUBAC is dependent on initial cIAP1 ⁄ 2-mediated ubiquitination of components of the TNFR1 core signaling complex, there is evidence that LUBAC increases overall ubiquitination, thereby improving recruitment of the IKK complex In line with these arguments, there is reduced interaction of NEMO with the TNFR1 signaling complex in cells with LUBAC knockdown [34] Additional consequences are reduced recruitment of TRAF2, RIP1 and TAK1, despite normal TNF–TNFR1 interaction Conversely, in cells with ectopic expression of LUBAC, a prolonged and increased formation of the TNFR1 core signaling complex was observed Thus, the role of LUBAC in TNF-induced NF-jB activation seems not to be restricted to recruitment of NEMO and the IKK complex, but may also involve stabilization of the TNFR1 signaling complex as a whole In accordance with the proposed supporting and stabilizing nature of the LUBAC, NF-jB activation was significantly reduced but not fully absent in HOIL-1deficient MEFs and LUBAC knockdown cells [34–37] Notably, ubiquitination of RIP1 in the TNFR1 signaling complex seems to be independent of LUBAC activity, emphasizing results from other studies which have identified cIAP1 ⁄ as the major E3 ligases of RIP1 in TNFR1 signaling [19] RIP1 ubiquitination and its relevance for TNFR1-induced NF-jB activation RIP1 is the most strongly ubiquitinated component of the TNFR1 signaling complex RIP1 can be modified with K63-linked multiubiquitin chains, mediating the recruitment of various ubiquitin-binding proteins involved in TNF signaling, but also with K48-linked ubiquitin chains that prompt proteasomal degradation TNF-induced K63 ubiquitination of RIP1 occurs preferentially at lysine 377 [38–40] and is dependent on TRAF2 and cIAPs, whereby the latter seems to be the essential E3 ligases Reconstitution experiments in RIP1-deficient cells with a RIP1 mutant carrying a defective K63 ubiquitination acceptor site (RIP1– K377R), suggest that TNFR1-associated ubiquitinated RIP1 serves as a recruitment platform for the binding of a complex containing the ubiquitin-binding proteins TAB and TAB 3, and the TAB ⁄ 3-interacting FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 869 TNFR1 induced NF-jB signaling H Wajant and P Scheurich MAP3 kinase, TAK1 [14,38] Remarkbly, the affinity of TAB for K63-linked ubiquitin chains is much higher than for linear ubiquitin chains [41] TNFinduced recruitment of the TAB ⁄ 3–TAK1 complex is followed by K63-linked polyubiquitination of TAK1 at K158 [31] The latter has been be achieved with purified TRAF2 in in vitro assays and reconstitution experiments with TAK1) ⁄ ) MEFs and a TAK1– K158R mutant, further arguing for a crucial role of this event in TNFR1-induced activation of the classical NF-jB pathway [31] Ubiquitinated TNFR1-bound RIP1 can also interact with the IKK complex [38,40] Whereas TRAF2 binding to the IKK complex relies on interaction with the leucin zipper motif of IKK1 or IKK2, ubiquitinated RIP1 interacts with the NEMO subunit of the IKK complex [38,40,42] The relative contribution of these two interactions to recruitment of the IKK complex within the TNFR1 signaling core complex is currently unclear One study reported an only slight reduction in TNF-induced recruitment of IKK1 and IKK2 to TNFR1 in NEMO-deficient MEFs [42], whereas others found no recruitment of these kinases in NEMO-deficient MEFs [40] or NEMO-deficient Jurkat cells [38] In addition, the unexpected observation has been reported that NEMO interacts better with LUBAC-generated head-to-tail linked linear ubiquitin chains than with K63-multiubiquitin chains [34,37] It is tempting to speculate that interaction of nonubiquitinated NEMO with K63-polyubiquitinated RIP1 initially stabilizes the interaction of the IKK complex with TRAF2, whereas after LUBAC-catalyzed modification of NEMO with linearly linked ubiquitin chains, NEMO and the IKK complex are stabilized in the TNFR1 signaling complex via interaction with the ubiquitin-binding domains of LUBAC Despite the convincing biochemical evidence for an important role of K63 ubiquitination of RIP1, TAK1 and NEMO in TNF-induced NF-jB activation, this concept is challenged by recent findings First, TNFinduced IjBa degradation and p65 nuclear translocation have been reported to be unchanged in MEFs deficient for Ubc13 [43], a part of the Ubc13–Uev1A heteromeric E2 complex catalyzing the K63 ubiquitination of RIP1 on K377 [38] Second, inducible replacement of endogenous ubiquitin with K63R mutants in U2SO cells showed no effect on TNF-induced IKK activation and IjBa degradation, but abrogated interleukin (IL)-1-induced NF-jB signaling, underscoring the feasibility of this approach [44] However, the latter study is based on a knockdown strategy of endogenous ubiquitin and concomitant expression of the K63R ubiquitin mutant Because inhibition of endogenous ubiquitin expression by knockdown approaches is 870 unavoidably incomplete, it can not entirely be ruled out that residual expression of minute amounts of endogenous ubiquitin remain present, being sufficient to allow K63-linked ubiquitination of RIP1 in the TNFR1 signaling complex In fact, the strongly reduced expression of endogenous ubiquitin shown by Xu et al [44] is demonstrated using total cell lysates, but RIP1 ubiquitination is at best detectable in immunoprecipitates of TNFR1 Furthermore, Ubc13 could be substituted in its E2 ligase activity of RIP1 by UbcH5 Thus, the controversial data regarding the role of RIP1 in general, and RIP1 ubiquitination in particular, in TNR1-mediated NF-jB activation is likely to be related to the use of different cell types, insufficient sensitivity of functional analyses and underestimated experimental pitfalls In any case, additional studies are required to resolve the contradictions arising from the available literature Regulation of TNF-induced NF-jB activity by modification of NF-jB subunits IKK-induced IjBa degradation and translocation of the NF-jB subunits into the nucleus is not sufficient to ensure full transcriptional activity In addition, the latter requires post-translational modifications of the NF-jB subunits themselfes to achieve high DNA-binding capacity and strong transcriptional activity [1,2] The regulatory mechanisms directly targeting the NF-jB subunits are typically of relevance for various NF-jB inducers and not specific for TNFR1 signaling We therefore address only briefly some aspects involved in the regulation of the activity of p65-containing NF-jB dimers, representing the major NF-jB targets of TNFR1-induced signaling Phosphorylation of p65 on serine residues 276, 311, 529, 536 and 576 has been implicated in the regulation of TNF-induced NF-jB signaling The catalytic subunit of PKA (PKAc) was the first kinase identified as a regulator of p65 activity by serine 276 phosphorylation [45] PKAc is associated with NF-jB–IjBa ⁄ b complexes and concomitantly released upon degradation of IjBa ⁄ b Serine 276 phosphorylation can also be stimulated by TNF-induced activation of MSK1 (mitogen- and stress-activated kinase-1) via the p38 and ERK pathways [46] MEFs derived from MSK1–MSK2 DKO mice showed normal DNA-binding of p65 in response to TNF, but impaired transcription of a subset of NF-jB-regulated genes [46] Phosphorylation of serine 276 enables recruitment of the cAMP-responsive element-binding protein (CREB)-binding protein (CBP) and p300 These transcriptional coactivators interact FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS H Wajant and P Scheurich with histone acetyltransfereases and mediate acetylation of p65 leading to enhanced transcriptional activity [1] TNF-induced phosphorylation of serine 311 of p65 has been assigned to the activation of PKCf [47] Similar to serine 276, this phosphorylation is not required for IKK activation, IjBa degradation and DNA-binding of p65 in embryonal fibroblasts, but has been rather implicated in CREB-binding protein recruitment and transactivation [47,48] Noteworthy, in the lung of PKCf-knockout mice there was in addition a defect in TNF-stimulated IKK activation and IjBa degradation [48] Phosphorylation of serine 529 of p65 is mediated by casein kinase II, but is prevented in nonstimulated cells by the interaction with IjBa [49] Thus, similar to PKAc-mediated phosphorylation of serine 276 of p65, phosphorylation on serine 529 by casein kinase II occurs in the cytoplasm after TNF-induced degradation of IjBa Again, p65 phosphorylation on serine 529 increases the transcriptional activity of p65-containing NF-jB dimers, but is not required for their nuclear translocation p65 can also be phosphorylated by IKKs in response to TNF on serine 536 and on S468 by IKK2 [50–52] TNF-induced IKK-mediated phosphorylation of p65 is strongly reduced in the absence of TRAF2 ⁄ or TAK1 [51], suggesting that p65 is an additional substrate for the IKK complex Noteworthy, in detail there are differences in the mechanisms of IKK-mediated phosphorylation of p65 and IjBa First, the protein Rap1 has been identified recently as a cofactor improving association of the IKK complex with p65 and was shown to facilitate phosphorylation of the latter, but appeared irrelevant for IjBa phosphorylation [53] Second, IKK1 seems dispensable for TNF-induced phosphorylation and degradation of IjBa in MEFs, whereas both IjB kinases were required for p65 transactivation Furthermore, IKK1, but not IKK2 or NEMO, corecruit with p65 and CREB-binding protein to promoters of NF-jB-regulated genes in TNF stimulated cells [54,55] Besides acting as a p65 kinase, promoter-bound IKK1 also phosphorylates histone H3 on serine 10 to trigger its subsequent acetylation on lysine 14 [54,55] Chromatin-bound IKK1 further phosphorylates silencing mediator of retinoid and thyoid hormone action, inducing the redistribution of histone deactylase-3-containing silencing mediator of retinoid and thyoid hormone action repressor complexes into the cytosol [56] Whereas phosphorylation of S536 contributes to TNFinduced NF-jB activation via the aforementioned mechanisms, phosphorylation of S468 rather elicits attenuating effects [52] TNFR1 induced NF-jB signaling Termination of TNFR1-induced NF-jB activation The mechanisms by which TNFR1-mediated activation of the classical NF-jB pathway is shut down are less well understood than the initiating events, but it is evident that a variety of mechanisms contribute to this task There exist general mechanisms targeting steps in the pathway downstream of IKK activation which will not be addressed here, but there are also upstream acting mechanisms regulating the activity of the NF-jBstimulating TNFR1 signaling complex Multiubiquitination of RIP1 on K63 in the course of TNFR1 signaling can be antagonized by A20, a protein containing two ubiquitin-editing domains with different specificities An N-terminal de-ubiquitination domain is capable removing K63-linked ubiquitin chains from RIP1, whereas a C-terminal ubiquitin ligase domain polyubiquitinates RIP1 with K48-linked ubiquitin chains to trigger its proteasomal degradation There is increasing evidence that A20 acts as part of a multiprotein complex in RIP1 deubiquitination that is formed 15–30 post TNFR1 stimulation In addition to A20 and its substrate RIP1 (or TRAF6 in LPS signaling), this ubiquitin-editing A20 complex also includes Tax1-binding protein (TAX1BP1), RING finger protein 11 (RNF11) and Itch [57–59] Accordingly, TNF-induced interaction of RIP1 with A20 is inhibited in MEFs deficient for TAX1BP1 or Itch and in RNF11 knockdown cells [57–59] Moreover, TNF-induced NF-jB activity and multiubiquitination of RIP1, detected in RIP1 immunoprecipitates after 15–30 (TAX1BP1, Itch11, RNF11) or in TNFR1 immunoprecipitates after 5–25 (A20), were enhanced in MEFs deficient for A20, TAX1BP1 or Itch and in RNF11 knockdown cells [57–60] In accordance with the initial idea that the ubiquitin-editing A20 complex removes K63-linked ubiquitin from RIP1 to subsequently mark it by K48 ubiquitination for proteasomal degradation, RIP1 is predominantly K48 ubiquitin linked in RIP1 immunoprecipitates of TNF-stimulated wild-type MEFs, but K63 multiubiquitinated in Itch-deficient MEFs [58] Moreover, upon inhibition of protein synthesis, TNF triggers degradation of RIP1 in wild-type, but not Itch- and TAX1BP1-deficient MEFs [58] The gene encoding A20 is regulated by NF-jB and thus not or only poorly expressed in most cells, but readily inducible by TNFR1 It is therefore tempting to speculate that A20 is particularly important for desensitization of cellular NF-jB responsiveness towards persistent TNF stimulation In fact, there is experimental evidence that inducible, but also constitutively FEBS Journal 278 (2011) 862–876 ª 2011 The Authors Journal compilation ª 2011 FEBS 871 TNFR1 induced NF-jB signaling H Wajant and P Scheurich expressed, A20 has only minor effects on early TNFinduced activation of NF-jB (< 0.5 h), whereas the later phase (> h) of NF-jB activity is significantly attenuated [61] Furthermore, MEFs derived from A20-deficient mice show prolonged and enhanced TNF signaling, which is in good accordance with the deadly chronic inflammation observed in A20-deficient mice A20 can also interact with ABIN proteins, a class of NF-jB-inhibitory proteins containing a special type of ubiquitin-binding domain also found in NEMO The relation ⁄ relevance of A20 interaction with ABIN proteins regarding the inhibitory effect of the ubiquin editing complex of A20, Itch, RNF11 and TAX1BP on TNF-induced NF-jB activation, however, is unclear Analysis of ABIN1-deficient MEFs revealed an, at best, moderate inhibitory effect on TNF-induced NF-jB activation [62], but it can not be ruled out that other A20-interacting ABIN proteins, like ABIN-2 or -3, compensate for ABIN-1 deficiency The A20-containing complex is presumably not the only regulatory factor targeting quality and quantity of RIP1 modification in the context of TNFR1 signaling There is evidence that deubiquitination by USP21 and stimulation of proteasomal degradation by the TRIAD3a E3 ligase and the endosome associated E3 ligase CARP-2 limit the amplitude of TNF-induced NF-jB signaling [63–65] Together, K63-polyubiquination not only paves the road for the recruitment of stimulating factors to the TNF1 signaling complex, but also allows recruitment of inhibitory proteins Thus, TAB 2-deficient MEFs show the expected reduced TNF-induced activation of IKKs, but also revealed prolonged activation of TAK1 which could be traced back to inhibition of recruitment of the TAK1 inactivating serine ⁄ threonine protein phosphatase-6 [66] Furthermore, the NEMO homolog optineurin, interacting with K63-polyubiquitinated RIP1, also negatively regulates TNFR1-induced NF-jB signaling by competition with NEMO for RIP1 binding [67] Conclusions and perspectives The genetic and biochemical findings highlighted in this review definitely teach us that there is not simply a linear chain of events connecting TNFR1 with stimulation of the IKK complex and activation of the classical NF-jB pathway Instead, we gain more and more evidence that recruitment, modification and activation and inactivation of the E3 ligases and kinases involved in this process are reciprocally and intimately linked As a consequence, it is difficult to experimentally separate an individual aspect of TNFR1-induced NF-jB signaling even when knockout (knockdown) 872 cells are used which are reconstituted with point mutations of the deleted protein lacking only one or a subset of its properties A comprehensive understanding of the molecular mechanisms involved in TNFR1induced activation of the IKK complex and the classical NF-jB pathway might thus become possible only with the help of mathematical modeling The development of a detailed and quantitative model of TNFR1induced NF-jB activation, however, is complicated by the high degree of functional redundancy of the signaling molecules involved Thus, the lack of a particular signaling protein (including its replacement by a functional defective mutant) often affects a biochemically defined step in TNFR1 signaling, but not in an all or nothing way Moreover, TNFR1-induced activation of the classical NF-jB pathway, in general, and activation and inactivation of the IKK complex, in particular, is highly 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the molecular mechanisms involved in TNFR1induced activation of the IKK complex and the classical NF-jB pathway might thus become possible only with the help of mathematical