The role of hybrid ubiquitin chains in the MyD88 and other innate immune signalling pathways

Abstract

The adaptor protein MyD88 is required for signal transmission by toll-like receptors and receptors of the interleukin-1 family of cytokines. MyD88 signalling triggers the formation of Lys63-linked and Met1-linked ubiquitin (K63-Ub, M1-Ub) chains within minutes. The K63-Ub chains, which are formed by the E3 ubiquitin ligases TRAF6, Pellino1 and Pellino2, activate TAK1, the master kinase that switches on mitogen-activated protein (MAP) kinase cascades and initiates activation of the canonical IκB kinase (IKK) complex. The M1-Ub chains, which are formed by the linear ubiquitin chain assembly complex (LUBAC), bind to the NEMO (NF-κB essential modulator) component of the IKK complex and are required for TAK1 to activate IKKs, but not MAP kinases. An essential E3 ligase-independent role of TRAF6 is to recruit LUBAC into the MyD88 signalling complex, where it recognises preformed K63-Ub chains attached to protein components of these complexes, such as IRAK1 (IL-1 receptor-associated kinase), producing ubiquitin chains containing both types of linkage, termed K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids, which is a feature of several innate immune signalling pathways, permits the co-recruitment of proteins that interact with either K63-Ub or M1-Ub chains. Two likely roles for K63/M1-Ub hybrids are to facilitate the TAK1-dependent activation of the IKK complex and to prevent the hyperactivation of these kinases by recruiting A20 and A20-binding inhibitor of NF-κB1 (ABIN1). These proteins restrict activation of the TAK1 and IKK complexes, probably by competing with them for binding to K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids may also regulate the rate at which the ubiquitin linkages in these chains are hydrolysed. The IKK-catalysed phosphorylation of some of its substrates permits their recognition by the E3 ligase SCF^βTRCP, leading to their Lys48-linked ubiquitylation and proteasomal degradation. Innate immune signalling is therefore controlled by the formation and destruction of three different types of ubiquitin linkage.

Figure 1

How the MyD88 pathway triggers the ubiquitylation of IRAK1. The interaction of IL-1 with its receptor leads to the formation of the Myddosome and the phosphorylation and activation of IRAK1. The interaction of IRAK1 with TRAF6 induces dimerisation of the RING domain of TRAF6, activating its E3 ligase function. IRAK1 also phosphorylates Pellino1 and Pellino2 converting them from inactive to active E3 ubiquitin ligases. In the presence of E1-activating enzyme and the E2-conjugating complex Ubc13-Uev1a, TRAF6 and Pellinos 1 and 2 both contribute to the formation of K63-Ub chains, which become attached to IRAK1 and other components of the Myddosome. One essential role of TRAF6 is to recruit LUBAC into the IL-1 signalling complex where it interacts with K63-Ub-substrates, such as IRAK1, to produce K63/M1-Ub-IRAK1. In the human cell lines we have studied, the expression of IRAK1 is essential for IL-1 signalling and the IRAK2 in these cells cannot compensate for the loss of IRAK1. Protein kinases are highlighted in red and E3 ligases in green

Figure 2

The formation of M1-Ub chains and their interaction with NEMO are required for TAK1 to initiate the activation of the canonical IKK complex. (a) IL-1 α induces the formation of M1-Ub chains in embryonic fibroblasts from wild-type (WT) mice, but not in knock-in mice expressing the E3 ligase-inactive HOIP[C879S] mutant. (b) The IL-1α-dependent, TAK1-catalysed phosphorylation of IKKβ at Ser177 is suppressed in embryonic fibroblasts from HOIP[C879S] knock-in mice that do not form M1-Ub chains, or in embryonic fibroblasts from NEMO[D311N] knock-in mice in which M1-Ub chains are formed, but are unable to interact with NEMO. Adapted from Emmerich et al. and Zhang et al.

Figure 3

The formation of K63/M1-Ub hybrids facilitates the activation of TAK1 and IKK complexes, and the phosphorylation of their substrates. The K63/M1-Ub hybrids produced by innate immune signalling pathways recruit the TAK1 and IKK complexes. The interaction of K63-Ub chains with TAB2 and TAB3 activates TAK1 allowing it to phosphorylate and activate MAP kinase kinases 4 and 7 (MKK4, MKK7), which activate the MAP kinases, termed JNK1 and JNK2. The binding of M1-Ub chains to NEMO induces a conformational change that permits TAK1 to phosphorylate IKKβ at Ser177. IKKβ completes the activation process by phosphorylating itself at Ser181. IKKβ can then activate transcription factors essential for inflammatory mediator production (NF-κB and IRF5). IKKβ also activates the Tpl2 kinase complex, which activates MEK1 and MEK2, the protein kinases that activate ERK1 and ERK2. Tpl2 additionally phosphorylates MKK3 and MKK6. MKK3 and MKK6 operate redundantly with MKK4 to phosphorylate and activate p38α in some cells. Protein kinases are highlighted in red, ubiquitin-binding proteins in purple and ubiquitin (Ub) molecules in blue. Further details are given in the text

Figure 4

ABIN1 restricts the activation of TAK1 and the canonical IKK complex. (a) ABIN1 binds to K63/M1-Ub hybrids and competes with the TAK1 and IKK complexes for binding to these ubiquitin chains. This suppresses the interaction of the TAK1 and IKK complexes with these ubiquitin chains, restricting their activation and the production of inflammatory mediators. (b) In dendritic cells and B cells from knock-in mice expressing the ubiquitin-binding-defective ABIN1[D485N] mutant, this restriction is removed, leading to hyperactivation of the TAK1 and IKK complexes and the overproduction of inflammatory mediators (red arrows). This leads to the spontaneous development of SLE when the ABIN1[D485N] mice are 4–5 months old. The colour key is as in Figures 1 and 3