Receptor-interacting protein 1 (RIPK1) polyubiquitination is required for the recruitment of the downstream signaling complexes such as the IkB kinase (IKK) complex in the response to TNFR1 stimulation (Ea CK et al. 2006; Blackwell K et al. 2013). RIPK1 is polyubiquitinated at Lys377 (K377, equivalent to K376 of mouse RIPK1) (Ea CK et al. 2006). A point mutation of RIPK1 at Lys377 (K377R) was found to abolish its polyubiquitination and prevent the recruitment and activation of IKK and the TGF-β activated kinase 1 (TAK1) complex (Ea CK et al. 2006). TNFα-induced recruitment of the IKK complex to TNFR1 is completely impaired, recruitment of TAK1 is severely reduced, and recruitment of the LUBAC E3 ligase complex, is also reduced in human RIPK1-deficient Jukart T-cells (Blackwell K et al. 2013). In vivo studies showed that RIPK1 K376R mutation activated cell death resulting in embryonic lethality in mice (Tang Y et al. 2019; Zhang X et al. 2019; Kist M et al. 2021). These data suggest that K63-linked ubiquitylation of RIPK1 at K377 prevents TNF-induced cell death.
Several E3 ligases are involved in TNF-α signaling to initiate an immediate and effective host response to infection or injury. Among them are anti-apoptotic regulators BIRC2 and BIRC3, also known as inhibitor of apoptosis proteins (cIAP1/2). BIRC2/3 were found to constitutively associate with TRAF2 and via TRAF2 they were recruited to the TNFR1 signaling complex (Samuel T et al. 2006; Bertrand et al, 2008; Varfolomeev E et al, 2008). BIRC2/3 can directly ubiquitinate RIPK1 within the TNFR1 receptor complex allowing it to bind to the TAB2:TAK1 complex, a process reversed by the deubiquitinase CYLD and A20 (Bertrand et al, 2008; Varfolomeev et al, 2008; Moquin DM et al. 2013; Shembade N et al. 2010; Wertz IE et al. 2004). In conjunction with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, BIRC2/3 was shown to mediate polymerization of both K63-linked and linear Met1-linked chains on RIPK1 (Varfolomeev E et al, 2008; Bertrand et al, 2008; Blackwell K et al. 2013). TRAF2 promotes BIRC-mediated linear and K63-linked ubiquitination of RIP1 (Blackwell K et al. 2013). K11-linked polyubiquitination of RIPK1 may also depend on BIRC2 and BIRC3 (Dynek JN et al. 2010).