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). RIPK1 functions as a key regulator of both cell survival and cell death (reviewed in Ju E et al. 2022). Enzymatically inactive polyUb-bound RIPK1 serves as a scaffold in the membrane-bound TNFR1 signaling complex (complex I) contributing to activation of mitogen-activated protein kinase (MAPK) and NF-kappa-B signaling pathways, which regulate expression of pro-survival and inflammatory genes. Deubiquitination of RIPK1 negatively regulates TNFα-induced pro-survival responses while promoting RIPK1-mediated cell death (reviewed in Ju E et al. 2022). As a deubiquitinating enzyme, CYLD removes K63-linked and Met1-linked polyUb chains from RIPK1 and other substrates downstream of TNFR1, thus enhancing cytotoxic potential of RIPK1 (Uematsu A et al. 2019). Upon co-expression of tagged CYLD and MIB2 in human embryonic kidney 293T (HEK293T) cells, MIB2 directly binds to and catalyzes K48-linked polyubiquitination of CYLD thus targeting CYLD for proteasomal degradation (Rajan N et al. 2014; Uematsu A et al. 2019). Immunoaffinity purification using antibodies that detect ubiquitin remnant motif (K-Epsilon-GG) on modified Lys residues coupled with quantitative mass spectrometry analysis revealed that MIB2 ubiquitinates CYLD at K338 and K530 upon co-expression in HEK293T cells. Mutagenesis analysis confirmed that MIB2-dependent ubiquitination of CYLD occurs at K338 and K530 (Uematsu A et al. 2019). MIB2-mediated degradation of CYLD is thought to prevent CYLD-mediated deubiquitination of RIPK1 suppressing RIPK1-dependent cell death (Uematsu A et al. 2019). Ub-conjugating Enzyme E2D 2 (UBE2D2) facilitated MIB2-mediated ubiquitination of CYLD in in vitro assay. Besides CYLD, MIB2 directly binds and ubiquitinates RIPK1 within the TNFα:TNFR1 signaling complex I inhibiting TNFα-induced cell death (Feltham R et al. 2018). These data suggest that the E3 ligase activity of MIB2 suppresses the TNF-induced cell death by attaching K48-linked polyUb chains to CYLD (Feltham R et al. 2018; Uematsu A et al. 2019; Nakabayashi O et al. 2021)

This Reactome event describes MIB2-mediated K48-linked ubiquitination of CYLD in the presence of UBE2D2.