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 (Walczak H 2011). 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). RIPK1 is thought to mediate the recruitment of MIB2 to the TNFR1 complex (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. Autophosphorylated RIPK1 provides a platform to form downstream death-inducing signaling complexes-IIa and IIb, causing apoptosis or necroptosis respectively (Laurien L et al. 2020; Chen X et al. 2022). Ubiquitination of RIPK1 at K377 is also mediated by BIRC2/3. Further, 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 TNFα-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 FLIP(L), 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-mediated polyubiquitination of RIPK1 at K377, K604 and K634 within the TNFα:TNFR1:TRADD:RIPK1:TRAF2 complex.