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

Abstract

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.