Recruitment of the Linear Ubiquitin Chain Assembly Complex Stabilizes the TNF-R1 Signaling Complex and Is Required for TNF-Mediated Gene Induction

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-κB 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. © 2009 Elsevier Inc. All rights reserved

The linear ubiquitin chain assembly complex (LUBAC) forms part of the TNF-R1 signalling complex and is required for effective TNF-induced gene induction and prevents TNF-induced apoptosis

For antigen and various cytokine receptor-mediated signalling events, ubiquitination is a fundamental regulatory mechanism implicated in the different signalling pathways emanating from these receptors. Since the discovery of the importance of lysine 48-linked ubiquitin chains for NF-?B activation and the finding that the ubiquitin ligases TRAF2 and cIAP1/2 are recruited to the TNF-RSC, it became clear that the activation of TNF-induced signalling pathways depends strongly on the ubiquitin system. TNF plays a critical role in inflammatory processes and is involved in the regulation of immune responses. Depending on the cellular context, TNF initiates a complex cascade of signalling events that can result in induction of proinflammatory cytokines, cell proliferation, differentiation or cell death. Ligand-induced trimerisation of TNF-R1 leads to the formation of a multi-protein complex, the TNFR1 signalling complex (TNF-RSC). To be able to understand the multifaceted regulatory functions of the ubiquitin network and to comprehend the complex interplay of signalling pathways emanating from TNF-R1, the TNF-RSC and its composition need to be understood at the molecular level. Therefore we newly developed a modified tandem affinity purification (moTAP) procedure which allowed us to physically determine the molecular composition of the TNF-RSC in an unbiased manner. Using the moTAP approach, HOIL-1 and HOIP were identified as two novel, functional components of the native TNF-RSC. Together they were shown to form a linear ubiquitin chain assembly complex (LUBAC), catalysing the formation of linear head-to-tail ubiquitin chains. LUBAC mediates ubiquitination of NEMO with linear ubiquitin chains, required for efficient NF-?B activation following TNF stimulation. We show that the stimulation-dependent recruitment of LUBAC to the TNF-RSC is impaired in TRADD-, TRAF2-, and cIAP1/2-, but not in RIP1- and NEMO-deficient mouse embryonic fibroblast (MEF) cell lines. Furthermore, we demonstrate that the E3 ligase activity of cIAPs, but not of TRAF2, is required for HOIL-1 recruitment to the TNF-RSC. This result, together with the ability of HOIL-1 and HOIP to bind polyubiquitin chains of various linkage types, suggests that LUBAC is recruited to the TNF-RSC via cIAP-generated ubiquitin chains. LUBAC enhances NEMO interaction with the TNF-RSC, stabilises this protein complex, and is required for efficient TNF-induced activation of NF-?B and JNK, resulting in apoptosis inhibition. Finally, we show that the activity of LUBAC is required for stabilisation of the TNF-RSC, thereby adding a third form of ubiquitin linkage to the control of TNF signalling. The identification of HOIL-1 and HOIP as functional constituents of the TNF-RSC provides evidence that LUBAC is an important regulator at the apex of TNF-induced signalling cascades and increases the combinatorial complexity of ubiquitin modifications within this receptor complex. \uc2\ua9 2011 Springer Science+Business Media, LLC

Linear ubiquitination prevents inflammation and regulates immune signalling

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