The linear ubiquitin chain assembly complex (LUBAC) is a RING between RING (RBR) E3 ligase complex composed of two E3 ubiquitin ligases HOIL-1L (RBCK1) and HOIP (RNF31) and an adaptor protein SHARPIN (Kirisako T et al. 2006; Walczak H et al. 2012; Rittinger K & Ikeda F 2017; Carvajal AR et al. 2021; Fuseya Y & Iwai K 2021). The catalytic activity of LUBAC specifically generates Met1 (M1)-linked (also known as linear) polyubiquitin (polyUb) chains. The formation of mixed K63-/M1-linked heterotypic chains by LUBAC was also detected (Dittmar G & Winklhofer KF 2019; Carvajal AR et al. 2021). LUBAC regulates TNFα-induced TNFR1 signaling pathway through conjugating M1-linked polyUb to different components downstream of TNFR1 (Gerlach B et al. 2011; Haas TL et al. 2009). CASP8 and FADD-like apoptosis regulator (CFLAR, also known as cellular FLICE-like inhibitory protein (cFLIP)) has been identified as the LUBAC substrate (Tang Y et la. 2018). Co-immunoprecipitation analysis revealed that CFLAR interacts with the components of LUBAC in human cells. In vitro ubiquitination assay showed that LUBAC in combination with UBE1 (E1) and UBE2D3 (E2) conjugates M1-linked ubiquitination chains to CFLAR (cFLIP) (Tang Y et la. 2018). UBE2L3 was also reported to function as E2-conjugating enzyme for LUBAC (Lewis MJ et al. 2015; Fu B et al. 2014). UBE2L3, but not UBE2D3, associated with RBCK1 (HOIL-1L) subunit of the LUBAC complex to facilitate the assembly of linear pUb chains in human embryonic kidney 293T (HEK293T) and HeLa cells (Fu B et al. 2014). Overexpression of LUBAC components promoted linear polyUb of CFLAR and RIPK1 in HEK293T cells (Tang Y et la. 2018). Mutagenesis analysis showed that the catalytic activity of RNF31 (LUBAC) is critical for attachment of M1-linked polyUb chains to CFLAR. Further, LUBAC-mediated M1-linked polyubiquitination stabilized cFLIP by competing with K48–linked ubiquitination of cFLIP in HeLa cells. Site-directed mutagenesis identified K351 and K353 as the main M1-linked ubiquitination sites of CFLAR (cFLIP) (Tang Y et la. 2018).

CFLAR (cFLIP) is expressed in two major isoforms cFLIP long (FLIP(L)) and cFLIP short (FLIP(S)). While both cFLIP(L) and cFLIP(S) form heterodimers with procaspase-8, they differentially control caspase-8 activation. FLIP(L) interacts with procaspase-8 through both death effector domain (DED) and caspase-like domain (CLD). The procaspase-8 catalytic domain prefers heterodimerization with the CLD of FLIP(L) over homodimerization with catalytic domains of other procaspase-8 molecules (Boatright KM et al. 2004; Yu JW et al. 2009). The regulatory function of cFLIP(L) (CFLAR) has been found to differ depending on its expression levels. cFLIP(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 (Boatright KM et al. 2004; Okano H et al. 2003; Yerbes R et al. 2011; Hughes MA et al. 2016). Further, CFLAR was found to degrade faster in HOIP (RNF31)-deficient TNFα-induced HeLa cells (Tang Y et la. 2018). These data suggest that LUBAC-catalyzed M1-linked polyubiquitination of CFLAR (cFLIP(L)) stabilizes CFLAR (cFLIP) protecting cells from TNFα-induced cell death (Tang Y et la. 2018).

This Reactome event shows LUBAC-mediated M1-linked polyubiquitination of CFLAR (cFLIP) at K351 and K353 in the presence of Ub:UBE2L3.