RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity

Mandal, Pratyusha; Berger, Scott B.; Pillay, Sureshnee; Moriwaki, Kazuo; Huang, Chunzi; Guo, Hongyan; Lich, John D.; Finger, Joshua N.; Kasparcova, Viera; Votta, Bart; Ouellette, Michael T.; King, Bryan W.; Wisnoski, David D.; Lakdawala, Ami S.; DeMartino, Michael P.; Casillas, Linda; Haile, Pamela A.; Sehon, Clark A.; Marquis, Robert W.; Upton, Jason W.; Daley‐Bauer, Lisa P.; Roback, Linda; Ramia, Nancy; Dovey, Cole M.; Carette, Jan E.; Chan, Francis Ka Ming; Bertin, John; Gough, Peter J.; Mocarski, Edward S.; Kaiser, William J.

doi:10.1016/j.molcel.2014.10.021

Cited by 484 publications

(565 citation statements)

References 41 publications

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“…As recently reported (25), high concentration of the RIPK3 kinase inhibitor GSK'843 induced activation of caspase 3 and 8 in wild type RIPK3 and D143N mutant-expressing cells (Fig. 2C, lanes 4 and 12).…”

Section: B Lane 12 and C Lane 10)supporting

confidence: 86%

“…This is in contrast to treatment with the RIPK3 kinase inhibitor GSK'843, which induced strong RIPK1-RIPK3-FADD interaction as previously reported (Fig. 4A, lane 3) (25). Ripoptosome assembly required an intact RHIM, since tetraalanine RIPK3 RHIM mutant abolished GSKЈ843-induced ripoptosome assembly (Fig.…”

Section: B Lane 12 and C Lane 10)supporting

confidence: 61%

“…In addition, RIPK3 can also induce apoptosis in a kinase activity-independent manner (24,25). To determine the cell death mechanism induced by MG132, we first examined the cell death kinetics.…”

Section: Proteasome Inhibition Causes Ripk3-and Rhim-dependentmentioning

confidence: 99%

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

Moriwaki

Chan

2016

Journal of Biological Chemistry

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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...

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Section: B Lane 12 and C Lane 10)supporting

confidence: 86%

Section: B Lane 12 and C Lane 10)supporting

confidence: 61%

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

Moriwaki

Chan

2016

Journal of Biological Chemistry

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“…Curiously, elevated doses of two newly described RIPK3 inhibitors (GSK'843 and GSK'872) were able to fully block necroptosis but, at the same time, both induced apoptosis. 27,34 Similar results were seen in cells that expressed a kinase-dead RIPK3 (D161N), 34 or under conditions in which MLKL was silenced; 35 in all of these cases, kinase-inhibited RIPK3 mediates the recruitment of a complex comprised of RIPK1-FADD-FLIP, which recruits and activates caspase-8. In this study, we described a new molecule, GW'39B, which blocks RIPK3-mediated MLKL phosphorylation both in murine and human cells, thus inhibiting MLKL oligomerization.…”

Section: Discussionmentioning

confidence: 53%

Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis

et al. 2015

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Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the 'necrosome'), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.

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“…Interestingly, in contrast to Mlkl − / − MDFs, Ripk3 − / − MDFs were largely protected from IFNγ/SM-induced killing (Figure 3a), suggesting a necroptosis-independent role for RIPK3 that is not wholly unprecedented. 41,42 Both Ripk3 − / − Casp8 − / − and Mlkl − / − Casp8 − / − MDFs were completely resistant to IFNγ/ SM-induced cell death, suggesting that IFNγ/SM treatment causes a caspase-8-dependent apoptosis ( Figure 3a). Inhibition of necroptosis using the MLKL inhibitor, compound 1, 43 and the RIPK1 inhibitor, Nec-1 (necrostatin-1), 44 had no impact on the sensitivity to IFNγ/SM killing ( Figure 3b), but provided some protection when combined with the caspase inhibitor, QVD ( Figure 3c).…”

Section: Resultsmentioning

confidence: 99%

Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways

et al. 2017

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Peptido-mimetic inhibitor of apoptosis protein (IAP) antagonists (Smac mimetics (SMs)) can kill tumour cells by depleting endogenous IAPs and thereby inducing tumour necrosis factor (TNF) production. We found that interferon-γ (IFNγ) synergises with SMs to kill cancer cells independently of TNF − and other cell death receptor signalling pathways. Surprisingly, CRISPR/Cas9 HT29 cells doubly deficient for caspase-8 and the necroptotic pathway mediators RIPK3 or MLKL were still sensitive to IFNγ/SMinduced killing. Triple CRISPR/Cas9-knockout HT29 cells lacking caspase-10 in addition to caspase-8 and RIPK3 or MLKL were resistant to IFNγ/SM killing. Caspase-8 and RIPK1 deficiency was, however, sufficient to protect cells from IFNγ/SM-induced cell death, implying a role for RIPK1 in the activation of caspase-10. These data show that RIPK1 and caspase-10 mediate cell death in HT29 cells when caspase-8-mediated apoptosis and necroptosis are blocked and help to clarify how SMs operate as chemotherapeutic agents.

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RIP3 Induces Apoptosis Independent of Pronecrotic Kinase Activity

Cited by 484 publications

References 41 publications

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

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

Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis

Combination of IAP antagonist and IFNγ activates novel caspase-10- and RIPK1-dependent cell death pathways

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