Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1β was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.

TNF is a key inflammatory cytokine. Using a modified tandem affinity purification approach, we identified HOIL-1 and HOIP as functional components of the native TNF-R1 signaling complex (TNF-RSC). Together, they were shown to form a linear ubiquitin chain assembly complex (LUBAC) and to ubiquitylate NEMO. We show that LUBAC binds to ubiquitin chains of different linkage types and that its recruitment to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2- but not in RIP1- or NEMO-deficient MEFs. Furthermore, the E3 ligase activity of cIAPs, but not TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. LUBAC enhances NEMO interaction with the TNF-RSC, stabilizes this protein complex, and is required for efficient TNF-induced activation of NF-kappaB and JNK, resulting in apoptosis inhibition. Finally, we demonstrate that sustained stability of the TNF-RSC requires LUBAC's enzymatic activity, thereby adding a third form of ubiquitin linkage to the triggering of TNF signaling by the TNF-RSC.

Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-Binducing kinase stability and suppress constitutive noncanonical NF-B activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-B activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-B signaling and that efficient activation of NF-B by TNFR1 requires the recruitment of cIAP1/2 by TRAF2. The inhibitor of apoptosis (IAP)7 family is composed of baculoviral IAP repeat-containing proteins, several of which also bear a RING domain that is capable of acting as a ubiquitin E3 ligase (1). cIAP1 and cIAP2 (cIAP1/2) are two RING-containing IAPs whose amplification or genetic mutation has been associated with cancers and may promote tumor cell survival (2-8). These highly conserved IAPs were initially identified as components of a TRAF2-containing complex bound to the cytoplasmic domain of TNFR2 (9), and they have subsequently been implicated in the regulation of signaling by several more receptors of the TNF superfamily (10 -21). Although the BIR1 domain of cIAP1/2 mediates binding to TRAF2 (17,22,23), the complementary binding region of TRAF2 is unknown, and the relative roles of TRAF2 and cIAP1/2 E3 ligase activity in TNF superfamily signaling remain unclear.Genetic deletion of TRAF2 in the mouse results in early postnatal lethality that is caused by increased NF-B-mediated TNF production and increased cellular sensitivity to TNF killing (12,24,25). Most TRAF family members, including TRAF2, bear RING E3 ubiquitin ligase domains that are believed to catalyze 27). One of the proposed targets of TRAF2 RING E3 ligase activity is RIPK1 (28, 29), which is modified with Lys-63-linked polyubiquitin chains upon TNFR1 activation. This modified TNFR1-complexed RIPK1 has been proposed to serve as a platform for the recruitment and/or activation of TAB2-TAB3-TAK1 and NEMO-IKK␣-IKK␤ kinase complexes (30 -32), which target the NF-B inhibitor IB for Lys-48-linked polyubiquitin-mediated proteasomal degradation, to initiate canonical p65-dependent NF-B gene transcription.As in the case of TRAF2, genetic deletion of cIAP1, or IAP antagonist-induced loss of cIAP1/2, also sensitizes cells to TNF killing (16,19,(33)(34)(35)(36). Also like TRAF2, both ...