The binding of TNF-α to TNF receptor 1 (TNFR1) results in the sequential formation of several signaling complexes (Walczak H 2011). The rapidly forming complex-I (TNFR1 signaling complex) is assembled at the receptor’s cytoplasmic tail and consists of TNFR1, TRADD (TNFR1-associated death domain), TRAF2 (TNF receptor associated factor-2), RIPK1 (receptor-interacting serine/threonine protein kinase 1), and E3 ubiquitin (Ub) ligases BIRC2, BIRC3 (cIAP1/2, cellular inhibitor of apoptosis) and LUBAC (linear ubiquitin chain assembly complex) (Micheau O and Tschopp J 2003; Yuan J et al. 2019). Within this complex, RIPK1 and other proteins are rapidly conjugated with Ub chains by various E3 ligases (Micheau O and Tschopp J 2003; Yuan J et al. 2019). The ubiquitination status of RIPK1 determines cell fate downstream of the TNFR1 signaling complex (Yuan J et al. 2019). The conjugation of K63-linked Ub chains by BIRC2/3 or Met1-linked Ub chains by LUBAC, have been shown to promote RIPK1-dependent pro-survival NF-kappa-B signaling while inhibiting RIPK1 kinase-mediated apoptosis and necroptosis. In addition, RIPK1 also interacts with FADD/Caspase-8 or RIPK3/MLKL to form complex IIa or IIb, which activate apoptosis or necroptosis respectively (Yuan J et al. 2019). In these cell death-inducing complexes, RIPK1 function is also regulated by ubiquitination (Amin P et al. 2018; de Almagro MC et al. 2015). The protein stability of RIPK1 is negatively regulated by the carboxyl-terminus of HSC70-interacting protein (CHIP, also known as STIP1 homology and U-Box containing protein 1, STUB1) (Seo J et al. 2016). STUB1 (CHIP), as an E3 ligase, mediates K48-linked ubiquitination of RIPK1 at K571, K604, and K627 and targets it to lysosomal degradation (Seo J et al. 2016). Co-immunoprecipitation analysis using overexpressed proteins revealed interactions between STUB1 (CHIP) and RIPK1 in human embryonic kidney 293T (HEK293T) cells (Seo J et al. 2016). Interaction of endogenous STUB1 and RIPK1 proteins was detected in mouse fibroblast L929 cells. Direct interaction between recombinant proteins was confirmed by GST-pull-down assay (Seo J et al. 2016). Domain mapping revealed that the RHIM domain of RIPK1 interacts with the U-Box domain of STUB1 (Seo J et al. 2016). Similarly, STUB1 interacts with and ubiquitinates RIPK3 inducing lysosome-dependent destabilization of RIPK3 (Seo J et al. 2016). These data suggest that the E3 ligase activity of STUB1 negatively regulates TNF-α–induced cell death by conjugating K48-linked ubiquitin chains to RIPK1 and RIPK3, thereby promoting their degradation.

This Reactome event describes STUB1 (CHIP) binding to RIPK1.