Mind bomb 1 (MIB1) and MIB2 belong to RING-type E3 ubiquitin (Ub) ligases. Both MIB1 and MIB2 are known to regulate NOTCH signaling through targeting the NOTCH ligands Delta and Jagged for ubiquitination and internalization (reviewed in Dutta D et al 2022). MIB1 and MIB2 have been also implicated in regulation of NF-kappa-B-mediated inflammatory responses, interferon induction and cell death (Zhang L & Gallagher PJ 2009; Stempin CC et al. 2011; Li S et al 2011; Ye JC et al. 2014; Feltham R et al. 2018; Uematsu A et al. 2019). MIB2 has been implicated in regulation of the tumor necrosis factor alpha (TNFα)-induced pathway. Ligation of TNFα to TNF receptor 1 (TNFR1) induces the sequential formation of several signaling complexes to promote either cell survival (complex I) or cell death (complex IIa,b) (Micheau O and Tschopp J 2003; Walczak H 2011; Yuan J et al. 2019). The assembly of these complexes is tightly regulated by proteolysis, ubiquitination and phosphorylation of receptor-interacting serine/threonine protein kinase 1 (RIPK1) and other components of the TNFα signaling pathway (reviewed in Yuan J et al. 2019; Varfolomeev E & Vucic D 2022). The E3 Ub ligase activity of MIB2 controls the TNFα-induced pathway by targeting several components downstream of TNFα:TNFR1, including RIPK1, Ub carboxyl-terminal hydrolase CYLD and cellular FLICE-like inhibitory protein long (FLIP(L) encoded by the CFLAR gene) (Feltham R et al. 2018; Uematsu A et al. 2019; Nakabayashi O et al. 2021). The CFLAR gene is expressed in several isoforms including full length FLIP(L) and short form FLIP(S). MIB2 was found to bind long form, but not short form, of CFLAR (Nakabayashi O et al. 2021). Co-immunoprecipitation (Co-IP) assay identified MIB2:CFLAR interaction upon co-expression of tagged proteins in human embryonic kidney 293T (HEK293T) cells (Nakabayashi O et al. 2021). Co-IP also detected interaction between endogenous CFLAR (FLIP(L)) and MIB2 in human cervical cancer HeLa cells (Nakabayashi O et al. 2021). Immunofluorescence assay revealed that MIB2 co-localized with CFLAR (FLIP(L)) upon co-expression in HEK293T cells. As a homolog of caspase-8 (CASP8), CFLAR controls the pro-apoptotic function of CASP8 within the cytosolic complex-IIa, TRADD:FADD:TRAF2:RIPK1:CASP8. At high expression levels, FLIP(L) functions as an anti-apoptotic molecule, while low expression levels of FLIP(L) promote CASP8 activation. The MIB2:CFLAR interaction was shown to increase stability of CFLAR (FLIP(L)) in TNFα-stimulated HeLa cells and in human colorectal carcinoma HCT116 cells (Nakabayashi O et al. 2021). MIB2 was found to target CFLAR at multiple lysine residues including several sites in the C-terminal domain of CFLAR (FLIP(L)), which is responsible for dimerization with CASP8. MIB2-dependent ubiquitination of CFLAR is thought to affect a proper dimer formation with CASP8, thereby suppressing CASP8 activation and TNFα-induced apoptosis (Nakabayashi O et al. 2021). Similar to MIB2, MIB1 was shown to regulate the TNFα:TNFR1 signaling pathway by binding CFLAR (both long and short forms) upon co-expression of tagged proteins in HEK293 cells. The MIB1:CFLAR interaction decreased the association between CFLAR and CASP8 (Zhang L & Gallagher PJ 2009). However, unlike MIB2, the E3 ligase activity of MIB1 was shown to promote activation of CASP8 and initiation of cell death (Zhang L & Gallagher PJ 2009). More research is needed to clarify MIB1/2-mediated regulation of CFLAR downstream of TNFα:TNFR1.

The data suggest that the E3 ligase activity of MIB2 suppresses the TNF-induced RIPK1 kinase-dependent cell death by attaching polyUb chains to RIPK1 and CYLD, while ubiquitylation of FLIP(L) by MIB2 controls TNFa signaling pathway in a RIPK1 kinase activity-independent manner (Feltham R et al. 2018; Uematsu A et al. 2019; Nakabayashi O et al. 2021)

This Reactome event describes binding of MIB2 to CFLAR (FLIP(L)).