Regulation of RIPK3- and RHIM-dependent Necroptosis by the Proteasome

Abstract

Receptor-interacting protein kinase 3 (RIPK3) is a serine/ threonine kinase with essential function in necroptosis. The activity of RIPK3 is controlled by phosphorylation. Once activated, RIPK3 phosphorylates and activates the downstream effector mixed lineage kinase domain-like (MLKL) to induce necroptosis. In certain situations, RIPK3 has also been shown to promote apoptosis or cytokine expression in a necroptosis and kinase-independent manner. The ubiquitin-proteasome system is the major pathway for selective degradation of cellular proteins and thus has a critical role in many cellular processes such as cell survival and cell death. Clinically, proteasome inhibition has shown promise as an anti-cancer agent. Here we show that the proteasome inhibitors MG132 and bortezomib activate the RIPK3-MLKL necroptotic pathway in mouse fibroblasts as well as human leukemia cells. Unlike necroptosis induced by classical TNF-like cytokines, necroptosis induced by proteasome inhibitors does not require caspase inhibition. However, an intact RIP homotypic interaction motif (RHIM) is essential. Surprisingly, when recruitment of MLKL to RIPK3 is restricted, proteasome inhibitors induced RIPK3-dependent apoptosis. Proteasome inhibition led to accumulation of K48-linked ubiquitinated RIPK3, which was partially reduced when Lys-264 was mutated. Taken together, these results reveal the ubiquitin-proteasome system as a novel regulatory mechanism for RIPK3-dependent necroptosis.Tissue homeostasis is maintained through dynamic balance between cell survival and cell death. A number of diseases such as cancer can be attributed to perturbation of this balance. Caspase-dependent apoptosis is a key cell death module with important functions in development and certain disease pathologies. In addition to apoptosis, recent evidence shows that a form of regulated necrosis, termed necroptosis, is significantly associated with a number of common clinical diseases such as viral infection, ischemia-reperfusion injury, myocardial infarction, atherosclerosis, and inflammatory bowel diseases (1, 2). Necroptosis is driven by the cytosolic serine/threonine kinase receptor-interacting protein kinase 3 (RIPK3) 2 (3). A wide variety of stimuli including tumor necrosis factor (TNF) superfamily death ligands have been reported to induce necroptosis (1). Ligation of TNF to TNF receptor (TNFR) 1 causes formation of receptor-associated complex I, which comprises of TNFR1-associated via death domain (TRADD), TNFR-associated factor 2 (TRAF2), cellular inhibitor of apoptosis 1 (cIAP1), cIAP2, linear ubiquitin chain assembly complex (LUBAC), and RIPK1. cIAPs and LUBAC promotes RIPK1 ubiquitination through Lys-11, Lys-63, and linear ubiquitin linkages, which promotes activation of NF-B and cell survival in part by limiting the formation of the cytosolic death-inducing signaling complex (DISC, aka complex IIa). Removal of the ubiquitin chains on RIPK1 by the de-ubiquitinase cylindromatosis (CYLD) promotes Complex IIa formation and apoptosis (4). When caspase 8 act...

Distinct Kinase-Independent Role of RIPK3 in CD11c + Mononuclear Phagocytes in Cytokine-Induced Tissue Repair

“…Moreover, Ripk3 ΔR/ΔR MEFs were resistant to apoptosis induced by the RIPK3 kinase inhibitor GSK’843 (Fig. S3H), which causes caspase 8 activation through RHIM-mediated formation of the ripoptosome (Mandal et al, 2014; Moriwaki and Chan, 2016). Furthermore, Ripk3 ΔR/ΔR BMDCs were resistant to LPS and CHX-induced caspase 3/8 activation and apoptosis (Fig.…”

Caspase‐8: regulating life and death

“…Chip −/− mice are viable but die within several weeks of birth due to intestinal inflammation and necroptotic cell death and are rescued by co-ablation of ripk3 (142). Inhibition of the proteasome induces RIPK3-mediated necroptosis dependent on the RHIM domain, but without the need for caspase-8 inhibition (143). TAK1, a kinase required for IKK activation, also regulates apoptotic and necroptotic signaling by activating RIPK1 and caspase-8 (144).…”

The Inflammatory Signal Adaptor RIPK3: Functions Beyond Necroptosis

“…Normally, this event does not lead to necroptosis as the unmasked RIPK3 is rapidly degraded by the proteasome. However, inhibition of the proteasome can trigger receptor-independent necroptosis (Moriwaki and Chan, 2016). Hence, the requirement for oligomerization-induced activation may serve to keep RIPK3 in check and to prevent inadvertent necroptosis during normal protein turnover.…”

“…A20, a unique ubiquitin editing protein that carries both deubiquitination and E3 ligase activities, was also reported to block necroptosis through stabilization of linear ubiquitin chains in TNFR1 complex and direct deubiquitination of RIPK3 (Draber et al, 2015; Onizawa et al, 2015; Wertz et al, 2015). However, the precise lysine residue mediating this effect is controversial (Moriwaki and Chan, 2016). As such, many regulators of the NF-κB pathway negatively control RIPK3-dependent necroptosis in NF-κB-dependent and independent manners.…”