Ligation of TNF-α to TNF receptor 1 (TNFR1) results in the sequential formation of several signaling complexes (Walczak H 2011). The rapidly forming complex-I (TNFR1 signaling complex) is assembled at the receptor’s cytoplasmic tail and consists of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1 (receptor-interacting serine/threonine protein kinase 1), and E3 ubiquitin (Ub) ligases BIRC2, BIRC3 (cIAP1/2, cellular inhibitor of apoptosis) and LUBAC (linear ubiquitin chain assembly complex) (Micheau O and Tschopp J 2003; Yuan J et al. 2019). Within this complex, RIPK1 and other proteins are rapidly conjugated with Ub chains by various E3 ligases. The ubiquitination status of RIPK1 determines cell fate downstream of the TNFR1 signaling complex. The conjugation of K63-linked Ub chains by BIRC2/3 or Met1-linked Ub chains by LUBAC, have been shown to promote RIPK1-dependent pro-survival NF-kappa-B signaling while inhibiting RIPK1-mediated apoptosis and necroptosis (Micheau O and Tschopp J 2003; Yuan J et al. 2019). Deubiquitinating enzymes, such as Ub carboxyl-terminal hydrolase CYLD, remove Ub chains from RIPK1 leading to the formation of the cytosolic complex IIa, TRADD:TRAF2:RIPK1:FADD:caspase-8 (CASP8), which activates apoptosis. In addition, RIPK1 also interacts with RIPK3 and MLKL to form the cytosolic complex IIb, which activates necroptosis (Micheau O and Tschopp J 2003; Yuan J et al. 2019). In these cell death-inducing complexes, RIPK1 activity is also regulated by ubiquitination (Amin P et al. 2018; de Almagro M et al. 2015). Besides, a RING-type E3 Ub ligase mind bomb-2 (MIB2) is also recruited to the TNFR1 signaling complex-I, where it conjugates inhibitory Ub chains to RIPK1 (Feltham R et al. 2018; Nakabayashi O et al. 2021). Studies in TNF-α-stimulated epithelial cells including human breast carcinoma MDA-MB-231, fibrosarcoma HT1080, and renal adenocarcinoma 786-0 cell lines showed that endogenous MIB2 is recruited to the TNFR1 complex in a time-dependent manner, peaking at 15 min (Feltham R et al 2018). Similar results were obtained in HeLa cells (Nakabayashi O et al. 2021). Endogenous MIB2 co-immunoprecipitated with ubiquitylated RIPK1 and other components of complex-I such as TRADD, TNFR1, and SHARPIN in TNF-treated MDA-MB-231 and 786-0 cells (Feltham R et al. 2018). RIPK1 is thought to mediate the recruitment of MIB2 to the TNFR1 complex (Feltham R et al. 2018). Co-immunoprecipitation analysis showed interaction between MIB2 and RIPK1 upon co-expression of tagged proteins in human embryonic kidney 293T (HEK293T) cells (Feltham R et al. 2018). In vitro-synthesized MIB2 co-purified RIPK1 from cellular extract of MDA-MB-231 cells upon TNF/Smac mimetic-164/zVAD-fm-induced cell death. Mutagenesis studies using overexpressed proteins revealed that the N-terminal MZM region of MIB2, which contains two Mib-HERC2 domains and a ZZ-type zinc finger motif (ZZ domain), interacted with the linker region of RIPK1 in HEK293T cells (Feltham R et al. 2018). Further, mutant MIB2 carrying mutation of the coordinating Cys residue of the ZZ domain within the MZM region did not interact with RIPK1 (Feltham R et al. 2018). When compared to wild-type (wt) MIB2, mutant MIB2 lacking the functional ZZ domain required for RIPK1 binding, failed to suppress TNF-induced CASP8 activation and cell death in MIB2-deficient MDA-MB-231 cells reconstituted with either wt MIB2 or mutant MIB2 (Feltham R et al. 2018). Structural and biochemical studies of MIB1, a paralogue of MIB2, further support the essential role of the MZM region in a substrate recognition (McMillan BJ et al. 2015; Guo B et al. 2016). MIB1 also interacted with RIPK1 in overexpression experiments using HEK293T cells (not shown here). However, RIPK1 was less efficiently ubiquitinated by MIB1 than by MIB2 (Feltham R et al. 2018). Upon TNF stimulation, recruited MIB2 conjugates different types of polyUb chains to RIPK1 targeting multiple lysine (K) residues (Feltham R et al. 2018). MIB2-mediated ubiquitination of K377 is thought to suppress auto-phosphorylation of RIPK1 at S166. Auto-phosphorylated RIPK1 provides a platform to form downstream death-inducing signalling complexes-IIa and IIb, causing apoptosis or necroptosis respectively (Laurien L et al. 2020; Chen X et al. 2022). MIB2-mediated ubiquitination of K604 and K634 within the death-domain (DD) of RIPK1 is predicted to affect both DD-mediated homotypic as well as heterotypic interactions of RIPK1 (Feltham R et al. 2018). MIB2 deficiency enhanced TNF-induced phosphorylation of RIPK1 at S166 in HeLa cells (Nakabayashi O et al. 2021). Depletion of MIB2 sensitized 786-0 cells to TNF-Induced and RIPK1-dependent cell death (Feltham R et al. 2018). Further, MIB2 deficiency had no effect on TNF-α-induced NF-kappa-B signaling pathway in human colorectal carcinoma HCT116 cells, HeLa cells (Nakabayashi O et al. 2021), MDA-MB-231, 786-0 and HEK293T cells (Feltham R et al. 2018) suggesting that the E3 Ub ligase activity of MIB2 protects cells from TNF-induced cell death without affecting the NF-kappa-B-mediated signaling.

Besides RIPK1, MIB2 was found to interact with other components downstream of the TNFR1 signaling complex. MIB2-mediated K48-linked ubiquitination of CYLD targets CYLD for proteasomal degradation thus preventing CYLD-mediated deubiquitination of RIPK1 and RIPK1-dependent cell death (Uematsu A et al. 2019). In addition, MIB2 binds cellular FLICE-like inhibitory protein long (FLIP(L) encoded by the CFLAR gene) in a RIPK1-independent manner (Nakabayashi O et al. 2021). As a homolog of caspase-8 (CASP8), CFLAR controls the pro-apoptotic function of CASP8 within the complex-IIa, TRADD:FADD:TRAF2:RIPK1:CASP8. The MIB2:CFLAR interaction increased stability of CFLAR (FLIP(L)) thereby attenuating TNF-α–induced apoptosis (Nakabayashi O et al. 2021). Thus, MIB2 is thought to suppress both RIPK1 kinase activity-dependent and -independent cell death, through ubiquitination of RIPK1 and cFLIPL, respectively (Nakabayashi O et al. 2021). Together, these data suggest that the E3 ligase activity of MIB2 suppresses the TNF-induced cell death by attaching polyUb chains to RIPK1, CFLAR and CYLD (Feltham R et al. 2018; Uematsu A et al. 2019; Nakabayashi O et al. 2021).

This Reactome event describes MIB2 binding to RIPK1 within the TNF-α:TNFR1:TRADD:RIPK1:TRAF2 complex.