Ligation of TNF-α to TNF receptor 1 (TNFR1) induces the formation of the TNFR1 signaling complex composed of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1 (receptor-interacting serin/threonine protein kinase 1), and E3 ubiquitin ligases BIRC2, BIRC3 (cIAP1/2, cellular inhibitor of apoptosis) and LUBAC (linear ubiquitin chain assembly complex) (Micheau O and Tschopp J 2003). The conjugation of K63-linked ubiquitin (Ub) chains by BIRC2/3 to RIPK1 allows further recruitment and activation of the TAK1 (also known as MAP3K7) complex and IκB kinase (IKK) complex which in turn drives activation of NF-kappa-B (Ea CK et al. 2006; Haas et al. 2009). The catalytic activity of LUBAC, composed of HOIL-1L, HOIP, and SHARPIN, specifically generates Met1-linked (also known as linear) polyUb chains (Kirisako T et al. 2006; Walczak H et al. 2012; Rittinger K & Ikeda F 2017; Fuseya Y & Iwai K 2021). UBE2L3 functions as E2-conjugating enzyme for LUBAC (Lewis MJ et al. 2015; Fu B et al. 2014). LUBAC was shown to catalyze Met1-linked ubiquitination of RIPK1 at K627 upon co-expression of Flag-RIPK1 with Myc-tagged components of LUBAC (HOIP, HOIL1, and SHARPIN) in human embryonic kidney 293T (HEK293T) cells (Tu H et al. 2021). Linear ubiquitination of mouse Ripk1 on K612 (K627 in human) inhibited TNF-α–induced apoptosis and necroptosis in mouse embryonic fibroblast (MEF) cells. Mutagenesis analysis further confirmed the importance of the K627 (K612 in mouse) ubiquitination site of RIPK1 in the TNFR1 signaling pathway (Li X et al. 2020). Further, LUBAC is known to ubiquitinate IKBKG (NEMO), a regulatory component of the IKK complex. LUBAC-mediated IKBKG ubiquitination enhanced IKBKG interaction with the TNFR1 signaling complex and stabilized this protein complex to promote activation of NF-kappa-B (Haas TL et al. 2009). Importantly, deletion of the LUBAC component SHARPIN in mice or mutation of HOIL-1 in humans, lead to hyperinflammatory phenotypes, indicating key roles of LUBAC and linear Ub chains in the response to infection and inflammation (Gerlach B et al. 2011; Ikeda F et al. 2011; Tokunaga F et al. 2011; Boisson B et al. 2012). The data suggest that LUBAC-mediated linear ubiquitylation of RIPK1 limits TNF-α–induced cell death.

This Reactome event describes LUBAC-mediated Met1-linked polyubiquitination (M1polyUb) of RIPK1 at K627 within the TNFR1 signaling complex.