The E3 ubiquitin (Ub) ligase activity of mind bomb 2 (MIB2) protein controls the tumor necrosis factor alpha (TNFα)-induced pathway by targeting several downstream components including receptor-interacting serine/threonine protein kinase 1 (RIPK1), Ub carboxyl-terminal hydrolase CYLD and 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). 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. The CFLAR gene is expressed in several isoforms including full length FLIP(L) and short form FLIP(S) (Yerbes R et al. 2011; Hughes MA et al. 2016). While both FLIP(L) and FLIP(S) form heterodimers with proCASP8, they differentially control CASP8 activation. The regulatory function of FLIP(L) has been found to differ depending on its expression levels. FLIP(L) was shown to inhibit death receptor (DR)-mediated apoptosis only when expressed at high levels, while low cell levels of FLIP(L) enhanced DR signaling to apoptosis (Yerbes R et al. 2011; Hughes MA et al. 2016). The FLIP(S) protein blocks DR-dependent CASP8 activation. MIB2 was found to bind FLIP(L), but not short form, of CFLAR upon co-expression of tagged proteins in human embryonic kidney 293T (HEK293T) cells (Nakabayashi O et al. 2021). Immunoblotting assay using polyUb-specific antibodies recognized K48- and K63- polyUb chains anchored to CFLAR (FLIP(L)) upon co-expression of tagged CFLAR, MIB2 and Ub proteins in HEK293T cells. Similar results were obtained for endogenous FLIP(L) in TNFα-stimulated human cervical cancer HeLa cells. Both K48- and K63- polyUb chains were reduced in MIB2-deficient HeLa cells (Nakabayashi O et al. 2021). In vitro ubiquitination assay showed that MIB2 attached both K48- and K63-linked polyUb chains to CFLAR (FLIP(L)) in the presence of Ub-conjugating enzymes E2D (UBE2D1 or UBE2D2), while UBE2N:UBE2V1 catalyzed conjugation of K63-linkedUb chains (Nakabayashi O et al. 2021). 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, suggesting that MIB2 regulates FLIP(L) by attaching non-degradable Ub chains formed by K63 or cooperation between K48 and K63 linkage types, but not those containing K48-linkage alone (Nakabayashi O et al. 2021). MIB2 was found to target CFLAR at multiple lysine residues including several sites (i.g., K460, K462, K473, and K474) 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 association with CASP8, thereby suppressing CASP8 activation and TNFα-induced apoptosis (Nakabayashi O et al. 2021).

This Reactome event shows MIB2-mediated K63-linked ubiquitination of CFLAR (FLIP(L)) in the presence of UBE2D proteins.