*2.2. Cellular Functions of LUBAC*

### 2.2.1. LUBAC in the Inflammatory Cytokine-Induced canonical NF-κB Activation Pathway

LUBAC principally participates in the regulation of the canonical NF-κB signaling pathway in various mammalian cells. NF-κB is a master transcription factor for the biological defense system, and is composed of homo- or hetero-dimers of Rel-homology domain-containing proteins, such as p65 (RelA), RelB, c-Rel, p105/p50, and p100/p52. NF-κB expression leads to the transcription of target genes in the inflammatory and immune responses [33]. LUBAC regulates the NF-κB activation pathways induced by proinflammatory cytokines, such as TNF-α and IL-1β, various pathogen-associated molecular patterns (PAMPs), T cell receptor (TCR) agonists, genotoxic stress, and NOD2-mediated inflammasome activation [14,15]. However, LUBAC is not involved in either the B cell receptor (BCR)-mediated pathway or the noncanonical NF-κB pathway [12,34].

Upon stimulation of cells with TNF-<sup>α</sup>, LUBAC is recruited to the TNF receptor (TNFR) through binding to K63-linked polyubiquitin chains, which are antecedently generated by c-IAP-1/2, TRAF2, and TRAF5, and functions as a member of the TNFR signaling complex I [35–37]. LUBAC then conjugates linear ubiquitin chains to NEMO, RIP1, and FADD (Figure 2) [11,38,39]. The linear ubiquitin chain functions as a scaffold to recruit canonical IκB kinase (IKK) molecules, which are composed of the kinase subunits of IKKα and IKKβ, and a regulatory subunit of NEMO. Importantly, NEMO contains a high-affinity linear ubiquitin binding site that accumulates multiple IKK molecules on the linear ubiquitin chain. The *trans*-phosphorylation of the IKK molecules principally leads to the activation of IKKβ, which subsequently phosphorylates the inhibitory protein of NF-κB, IκBα. Interestingly, the conjugation of two linearly linked molecules of ubiquitin (linear diubiquitin) to NEMO sufficiently induces IKK activation [40]. The phosphorylated IκBα is ubiquitinated by the E3 complex SCFβ-TrCP for the K48-ubiquitination-mediated proteasomal degradation of IκBα. After liberation from IκBα, the canonical NF-κB transcription factors, predominantly composed of homo- or hetero-dimers of p65 (RelA) and/or p50, translocate into the nucleus and activate NF-κB target genes (Figure 2) [32]. Upon TNF-α stimulation, mammalian Ste20-like kinase (MST1, also called STK4) is recruited to TNFR in a TRAF2-dependent manner and phosphorylates Ser1066 in the LDD domain of HOIP, which attenuates the E3 activity of LUBAC [41]. Recently, *Parkin-coregulated gene* (PACRG) was identified as a functional replacement of SHARPIN in TNF signaling in human and mouse cells [42]. Therefore, multiple factors regulate the LUBAC-mediated NF-κB activation pathway.

**Figure 2.** LUBAC-mediated regulation of the TNF-α-induced canonical NF-κB activation pathway and extrinsic apoptosis pathway.

Although IL-1β is another prominent proinflammatory cytokine that activates the canonical NF-κB activation pathway, both K63- and M1-linked ubiquitinations are required for the formation of the NEMO-containing punctate structure upon IL-1β stimulation [43]. Importantly, the K63/M1-hybrid ubiquitin chain can become conjugated to interleukin 1 receptor-associated kinase 1 (IRAK1) and IRAK4 [44]. Furthermore, HOIL-1L conjugates oxyester-bond monoubiquitin to its own Ser/Thr residues, as well as those in SHARPIN, IRAK1/2, and MyD88 in human keratinocyte HaCaT cells and mouse bone marrow-derived macrophages [23]. Thus, the E3 activity of HOIL-1L regulates the Myddosome components upon innate immune responses. These results indicate the differences in the LUBAC functions between the TNF-<sup>α</sup>- and IL-1β-mediated canonical NF-κB activation pathways.
