RIPK1-mediated regulated necrosis (Homo sapiens)

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16, 31, 6125, 30, 584329, 5110, 23, 365234, 4235, 534822, 32, 387, 46, 47, 551824501852446, 8, 125, 495, 9, 49182, 13, 20, 4539, 5014, 387, 12, 46, 4739, 5014, 3722, 38, 44, 5726264830, 58561721lysosomal lumencytosolDISC:procaspase-8:FLIP(L)K48pUb-XIAP p-S199,227, K48pUb-363-RIPK3 K48pUb-BIRC2 TNFRSF10B MLKL ATPPELI1 RIPK3(329-518)CFLAR(1-376)p-S166-RIPK1 FASLG(1-281) p-S-RIPK1:p-S199,227, K48pUb-363-RIPK3:PELI1p-S-RIPK1:p-S199,227-RIPK3 oligomer p-T357,S358-MLKL DISC:procaspase-8:FLIP(S)RIPK3 RIPK3:HSP90:CDC37UL36p-T357,S358-MLKL UBC(305-380) FLOT1:FLOT2TRADD UBA52(1-76) TNFRSF10A PI(3,4,5)P3 K48polyUbUBC(229-304) IP6 TRAF2 PI(4,5)P2 PDCD6IP RIPK1:RIPK3CASP8(1-479) TRADD:TRAF2:RIPK1:FADDUBB(77-152) p-S166-RIPK1:p-S199,227-RIPK3PI4P CRMA PI4P,PI(4,5)P2,PIP3RIPK1 CFLAR(1-480) Ub-K55,363-RIPK3 p-T357,S358-MLKL oligomer RPS27A(1-76) RIPK3 MLKLADPp-S166-RIPK1 O-glycosyl 3aTNFRSF10A HSV1 RIR1:RIPK1SPI-2 FADD NS1 3atetramer:(RIPK1:RIPK3) oligomerFLIP(S) STUB1 SDCBPp-S-RIPK1:p-S199,227-RIPK3 oligomer:4xMLKL:IP6,IP5, IP4RIPK3UL36 MLKL (RIPK1:RIPK3)oligomer:4xMLKLRIPK1 TRAF2 p-S-RIPK1:p-S199,227-RIPK3:PELI1viral serpinsactivecaspase-8:viralCRMA/SPI-2TRADD small moleculeinhibitors ofRIPK1, RIPK3I(1,3,4,6)P4FASLG(1-281) p-S-RIPK1:p-S199,227-RIPK3 oligomer UBC(77-152) RIPK1(325-671) p-T357,S358-MLKL oligomer (p-S-RIPK1:p-S199,227-RIPK3) oligomer:4xp-T357,S358-MLKLHSP90AA1 HSP90HSP90AA1 TRADD UDP-GlcNAcp-S-RIPK1:p-S199,227-RIPK3 oligomer FADD STUB1 RIR1OGlcNAc-T467-RIPK3RIPK1 CASP8(1-479)CASP8(385-479) p-S-RIPK1:p-S199,227-RIPK3 oligomer UBC(533-608) O-glycosyl 3a 3atetramer:(RIPK1:RIPK3) oligomerHSP90AA1 RIPK1 ATPFLOT2 ApoptosisCASP8(217-374) I(1,3,4,5,6)P5 ATPp-S199,227-RIPK3 RIPK3 TNFSF10 RIPK3(1-328)FLOT1 UBC(609-684) PELI1p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomerUbMLKL RIPK1(1-324)RIR1 PI4P TRAF2 CDC37 NS1:MLKLRIPK3 CASP8(385-479) K48polyUbRIPK3XIAP FLOT1 I(1,3,4,6)P4 STUB1:STUB1BIRC2,3,4p-S-RIPK1:p-S199,227-RIPK3 oligomer CASP8(217-374) CDC37 UBB(153-228) ADPSDCBP K48pUb-BIRC3 p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomer:PDCD6IP:SDCBPp-S-RIPK1:RIPK3RIPK1 TRADD MLKL:HSP90:CDC37RIR1 FADD TRADD CASP8(1-479) O-glycosyl 3a Ub-K55,363-RIPK3:STUB1MLKL p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomer:FLOT1:FLOT2RIPK3 FADD p-T357,S358-MLKL oligomer p-S-RIPK1:p-S199,227-RIPK3 oligomer TRAF2 RIPK1 O-glycosyl 3a p-S-RIPK1:p-S199,227-RIPK3 oligomerRIPK1 RIP1:RIP3:MLKLoligomer:PIPsUDPactive caspase-8p-S-RIPK1:p-S199,227-RIPK3 oligomer IP6CASP8(1-479) (p-S-RIPK1:p-S199,227-RIPK3) oligomer:4x(MLKL:ATP)FAS HSV1 RIR1:RIPK3FAS TRAF2 TNFRSF10B p-S166-RIPK1 p-S-RIPK1:p-S199,227-RIPK3 oligomer UBC(153-228) SPI-2 MLKL CASP8(1-479) NS1 dimerFLOT2 MLKL:UL36UBC(381-456) I(1,3,4,5,6)P5K48pUb- BIRC2,3,4Influenza InfectionFAS FASLG(1-281) ADPSARS-CoV-13a:(RIPK1:RIPK3)oligomerTNFRSF10B HSV1 RIR1:CASP8UBC(1-76) PI(4,5)P2 STUB1 BIRC3 FADD TNFSF10 BIRC2 p-S-RIPK1:p-S199,227-RIPK3 oligomer RIPK1:RIPK3:sorafenib, ponatinib, pazopanibCRMA NS1 p-S-RIPK1:p-S199,227-RIPK3 oligomer CDC37PELI1 DISC:procaspase-8p-S166-RIPK1 RIPK3:STUB1PI(3,4,5)P3 OGTTNFRSF10A p-S199,227-RIPK3 CDC37 RIR1 TRAF2:TRADD:RIPK1(325-671):FADDMLKLUBB(1-76) p-S-RIPK1:p-S199,227-RIPK3 oligomer TNFSF10 FLIP(S)RIPK1PDCD6IPUBC(457-532) p-T357,S358-MLKL oligomer ATP RIPK3 584122, 441440332227, 2840142222, 444026401, 6025, 5822, 44584022, 4448483, 4, 11, 15, 19...22


Description

Receptor-interacting serine/threonine-kinase protein 1 (RIPK1) and RIPK3-dependent necrosis is called necroptosis or programmed necrosis. The kinase activities of RIPK1 and RIPK3 are essential for the necroptotic cell death in human, mouse cell lines and genetic mice models (Cho YS et al. 2009; He S et al. 2009, 2011; Zhang DW et al. 2009; McQuade T et al. 2013; Newton et al. 2014). The initiation of necroptosis can be stimulated by the same death ligands that activate extrinsic apoptotic signaling pathway, such as tumor necrosis factor (TNF) alpha, Fas ligand (FasL), and TRAIL (TNF-related apoptosis-inducing ligand) or toll like receptors 3 and 4 ligands (Holler N et al. 2000; He S et al. 2009; Feoktistova M et al. 2011; Voigt S et al. 2014). In contrast to apoptosis, necroptosis represents a form of cell death that is optimally induced when caspases are inhibited (Holler N et al. 2000; Hopkins-Donaldson S et al. 2000; Sawai H 2014). Specific inhibitors of caspase-independent necrosis, necrostatins, have recently been identified (Degterev A et al. 2005, 2008). Necrostatins have been shown to inhibit the kinase activity of RIPK1 (Degterev A et al. 2008). Importantly, cell death of apoptotic morphology can be shifted to a necrotic phenotype when caspase 8 activity is compromised, otherwise active caspase 8 blocks necroptosis by the proteolytic cleavage of RIPK1 and RIPK3 (Kalai M et al. 2002; Degterev A et al. 2008; Lin Y et al. 1999; Feng S et al. 2007). When caspase activity is inhibited under certain pathophysiological conditions or by pharmacological agents, deubiquitinated RIPK1 is engaged in physical and functional interactions with the cognate kinase RIPK3 leading to formation of necrosome, a necroptosis-inducing complex consisting of RIPK1 and RIPK3 (Sawai H 2013; Moquin DM et al. 2013; Kalai M et al. 2002; Cho YS et al. 2009, He S et al. 2009, Zhang DW et al. 2009). Within the necrosome RIPK1 and RIPK3 bind to each other through their RIP homotypic interaction motif (RHIM) domains. The RHIMs can facilitate RIPK1:RIPK3 oligomerization, allowing them to form amyloid-like fibrillar structures (Li J et al. 2012; Mompean M et al. 2018). RIPK3 in turn interacts with mixed lineage kinase domain-like protein (MLKL) (Sun L et al. 2012; Zhao J et al. 2012; Murphy JM et al. 2013; Chen W et al. 2013). The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020). Mouse MLKL activation relies on transient engagement of RIPK3 to facilitate phosphorylation of the pseudokinase domain (Murphy JM et al. 2013; Petrie EJ et al. 2019a), while it appears that stable recruitment of human MLKL by necrosomal RIPK3 is an additional crucial step in human MLKL activation (Davies KA et al. 2018; Petrie EJ et al. 2018, 2019b). RIPK3-mediated phosphorylation is thought to initiate MLKL oligomerization, membrane translocation and membrane disruption (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2020; Samson AL et al. 2020). Studies in human cell lines suggest that upon induction of necroptosis MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014), but it is trafficking via a Golgi-microtubule-actin-dependent mechanism that facilitates plasma membrane translocation, where membrane disruption causes death (Samson AL et al. 2020). The mechanisms of necroptosis regulation and execution downstream of MLKL remain elusive. The precise oligomeric form of MLKL that mediates plasma membrane disruption has been highly debated (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014; Petrie EJ et al. 2017, 2018; Samson AL et al. 2020 ). However, microscopy data revealed that MLKL assembles into higher molecular weight species upon cytoplasmic necrosomes within human cells, and upon phosphorylation by RIPK3, MLKL is trafficked to the plasma membrane (Samson AL et al. 2020). At the plasma membrane, phospho-MLKL forms heterogeneous higher order assemblies, which are thought to permeabilize cells, leading to release of DAMPs to invoke inflammatory responses. While RIPK1, RIPK3 and MLKL are the core signaling components in the necroptosis pathway, many additional molecules have been proposed to positively and negatively tune the signaling pathway. Currently, this picture is evolving rapidly as new modulators continue to be discovered.

The Reactome module describes MLKL-mediated necroptotic events on the plasma membrane. View original pathway at Reactome.</div>

Comments

Reactome-Converter 
Pathway is converted from Reactome ID: 5213460
Reactome-version 
Reactome version: 75
Reactome Author 
Reactome Author: Shamovsky, Veronica

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Bibliography

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History

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CompareRevisionActionTimeUserComment
115106view19:00, 25 January 2021EgonwAnother empty reference.
115074view17:02, 25 January 2021ReactomeTeamReactome version 75
113586view08:12, 3 November 2020EgonwRemoved another empty reference (also a book?)
113557view13:09, 2 November 2020DeSlOntology Term : 'programmed cell death pathway' added !
113554view12:51, 2 November 2020DeSlOntology Term : 'cell death pathway' added !
113553view12:50, 2 November 2020DeSlOntology Term : 'regulatory pathway' added !
113552view12:49, 2 November 2020DeSlChanged layout for several complexes (included at least one small DataNode at top left corner, stretching the whole complex visually).
113543view12:02, 2 November 2020ReactomeTeamNew pathway

External references

DataNodes

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NameTypeDatabase referenceComment
(RIPK1:RIPK3)oligomer:4xMLKLComplexR-HSA-5218909 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:4x(MLKL:ATP)ComplexR-HSA-9687633 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:4xp-T357,S358-MLKLComplexR-HSA-5218902 (Reactome)
3a tetramer:(RIPK1:RIPK3) oligomerComplexR-HSA-9686334 (Reactome)
3a tetramer:(RIPK1:RIPK3) oligomerComplexR-HSA-9692649 (Reactome)
ADPMetaboliteCHEBI:456216 (ChEBI)
ATP MetaboliteCHEBI:30616 (ChEBI)
ATPMetaboliteCHEBI:30616 (ChEBI)
ApoptosisPathwayR-HSA-109581 (Reactome) Apoptosis is a distinct form of cell death that is functionally and morphologically different from necrosis. Nuclear chromatin condensation, cytoplasmic shrinking, dilated endoplasmic reticulum, and membrane blebbing characterize apoptosis in general. Mitochondria remain morphologically unchanged. In 1972 Kerr et al introduced the concept of apoptosis as a distinct form of "cell-death", and the mechanisms of various apoptotic pathways are still being revealed today.
The two principal pathways of apoptosis are (1) the Bcl-2 inhibitable or intrinsic pathway induced by various forms of stress like intracellular damage, developmental cues, and external stimuli and (2) the caspase 8/10 dependent or extrinsic pathway initiated by the engagement of death receptors
The caspase 8/10 dependent or extrinsic pathway is a death receptor mediated mechanism that results in the activation of caspase-8 and caspase-10. Activation of death receptors like Fas/CD95, TNFR1, and the TRAIL receptor is promoted by the TNF family of ligands including FASL (APO1L OR CD95L), TNF, LT-alpha, LT-beta, CD40L, LIGHT, RANKL, BLYS/BAFF, and APO2L/TRAIL. These ligands are released in response to microbial infection, or as part of the cellular, humoral immunity responses during the formation of lymphoid organs, activation of dendritic cells, stimulation or survival of T, B, and natural killer (NK) cells, cytotoxic response to viral infection or oncogenic transformation.
The Bcl-2 inhibitable or intrinsic pathway of apoptosis is a stress-inducible process, and acts through the activation of caspase-9 via Apaf-1 and cytochrome c. The rupture of the mitochondrial membrane, a rapid process involving some of the Bcl-2 family proteins, releases these molecules into the cytoplasm. Examples of cellular processes that may induce the intrinsic pathway in response to various damage signals include: auto reactivity in lymphocytes, cytokine deprivation, calcium flux or cellular damage by cytotoxic drugs like taxol, deprivation of nutrients like glucose and growth factors like EGF, anoikis, transactivation of target genes by tumor suppressors including p53.
In many non-immune cells, death signals initiated by the extrinsic pathway are amplified by connections to the intrinsic pathway. The connecting link appears to be the truncated BID (tBID) protein a proteolytic cleavage product mediated by caspase-8 or other enzymes.
BIRC2 ProteinQ13490 (Uniprot-TrEMBL)
BIRC2,3,4ComplexR-HSA-5357900 (Reactome)
BIRC3 ProteinQ13489 (Uniprot-TrEMBL)
CASP8(1-479) ProteinQ14790 (Uniprot-TrEMBL)
CASP8(1-479)ProteinQ14790 (Uniprot-TrEMBL)
CASP8(217-374) ProteinQ14790 (Uniprot-TrEMBL)
CASP8(385-479) ProteinQ14790 (Uniprot-TrEMBL)
CDC37 ProteinQ16543 (Uniprot-TrEMBL)
CDC37ComplexR-HSA-1225828 (Reactome)
CFLAR(1-376)ProteinO15519-1 (Uniprot-TrEMBL)
CFLAR(1-480) ProteinO15519-1 (Uniprot-TrEMBL)
CRMA ProteinP07385 (Uniprot-TrEMBL)
DISC:procaspase-8:FLIP(L)ComplexR-HSA-3371381 (Reactome)
DISC:procaspase-8:FLIP(S)ComplexR-HSA-3465352 (Reactome)
DISC:procaspase-8ComplexR-HSA-3465443 (Reactome)
FADD ProteinQ13158 (Uniprot-TrEMBL)
FAS ProteinP25445 (Uniprot-TrEMBL)
FASLG(1-281) ProteinP48023 (Uniprot-TrEMBL)
FLIP(S) ProteinO15519-2 (Uniprot-TrEMBL)
FLIP(S)ProteinO15519-2 (Uniprot-TrEMBL)
FLOT1 ProteinO75955 (Uniprot-TrEMBL)
FLOT1:FLOT2ComplexR-HSA-9688487 (Reactome)
FLOT2 ProteinQ14254 (Uniprot-TrEMBL)
HSP90AA1 ProteinP07900 (Uniprot-TrEMBL)
HSP90ComplexR-HSA-1221657 (Reactome)
HSV1 RIR1:CASP8ComplexR-HSA-9687483 (Reactome)
HSV1 RIR1:RIPK1ComplexR-HSA-9687467 (Reactome)
HSV1 RIR1:RIPK3ComplexR-HSA-9687468 (Reactome)
I(1,3,4,5,6)P5 MetaboliteCHEBI:16322 (ChEBI)
I(1,3,4,5,6)P5MetaboliteCHEBI:16322 (ChEBI)
I(1,3,4,6)P4 MetaboliteCHEBI:16155 (ChEBI)
I(1,3,4,6)P4MetaboliteCHEBI:16155 (ChEBI)
IP6 MetaboliteCHEBI:17401 (ChEBI)
IP6MetaboliteCHEBI:17401 (ChEBI)
Influenza InfectionPathwayR-HSA-168255 (Reactome) For centuries influenza epidemics have plagued man; with influenza probably being the disease described by Hippocrates in 412 BC. Today it remains a major cause of morbidity and mortality worldwide with large segments of the human population affected every year. Many animal species can be infected by influenza viruses, often with catastrophic consequences. An influenza pandemic is a continuing global level threat. The 1918 influenza pandemic is a modern example of how devastating such an event could be with an estimated 50 million deaths worldwide.

Influenza viruses belong to the family of Orthomyxoviridae; viruses with segmented RNA genomes that are negative sense and single-stranded (Baltimore 1971). Influenza virus strains are named according to their type (A, B, or C), the species from which the virus was isolated (omitted if human), location of isolate, the number of the isolate, the year of isolation, and in the case of influenza A viruses, the hemagglutinin (H) and neuraminidase (N) subtype. For example, the virus of H5N1 subtype isolated from chickens in Hong Kong in 1997 is: influenza A/chicken/Hong Kong/220/97(H5N1) virus. Currently 16 different hemagglutinin (H1 to H16) subtypes and 9 different neuraminidase (N1 to N9) subtypes are known for influenza A viruses. Most human disease is due to influenza viruses of the A type. The events of influenza infection have been annotated in Reactome primarily use protein and genome references to the Influenza A virus A/Puerto Rico/8/1934 H1N1 strain.


The influenza virus particle initially associates with a human host cell by binding to sialic acid receptors on the host cell surface. Sialic acids are found on many vertebrate cells and numerous viruses make use of this ubiquitous receptor. The bound virus is endocytosed by one of four distinct mechanisms. Once endocytosed the low endosomal pH sets in motion a number of steps that lead to viral membrane fusion mediated by the viral hemagglutinin (HA) protein, and the eventual release of the uncoated viral ribonucleoprotein complex into the cytosol of the host cell. The ribonucleoprotein complex is transported through the nuclear pore into the nucleus. Once in the nucleus, the incoming negative-sense viral RNA (vRNA) is transcribed into messenger RNA (mRNA) by a primer-dependent mechanism. Replication occurs via a two step process. A full-length complementary RNA (cRNA), a positive-sense copy of the vRNA, is first made and this in turn is used as a template to produce more vRNA. The viral proteins are expressed and processed and eventually assemble with vRNAs at what will become the budding sites on the host cell membrane. The viral protein and ribonucleoprotein complexes are assembled into complete viral particles and bud from the host cell, enveloped in the host cell's membrane.

Infection of a human host cell with influenza virus triggers an array of defensive host processes. This coevolution has driven the development of host processes that interfere with viral replication, notably the production of type I interferon. At the some time the virus counters these responses with the viral NS1 protein playing a central role in the viral response to the host cells defense.

K48pUb- BIRC2,3,4ComplexR-HSA-5675472 (Reactome)
K48pUb-BIRC2 ProteinQ13490 (Uniprot-TrEMBL)
K48pUb-BIRC3 ProteinQ13489 (Uniprot-TrEMBL)
K48pUb-XIAP ProteinP98170 (Uniprot-TrEMBL)
K48polyUbR-HSA-912740 (Reactome)
MLKL ProteinQ8NB16 (Uniprot-TrEMBL)
MLKL:HSP90:CDC37ComplexR-HSA-9698842 (Reactome)
MLKL:UL36ComplexR-HSA-9698671 (Reactome)
MLKLProteinQ8NB16 (Uniprot-TrEMBL)
NS1 ProteinP03496 (Uniprot-TrEMBL)
NS1 dimerComplexR-FLU-169143 (Reactome)
NS1:MLKLComplexR-HSA-9686346 (Reactome)
O-glycosyl 3a ProteinP59632 (Uniprot-TrEMBL)
O-glycosyl 3aProteinP59632 (Uniprot-TrEMBL)
OGTProteinO15294 (Uniprot-TrEMBL)
OGlcNAc-T467-RIPK3ProteinQ9Y572 (Uniprot-TrEMBL)
PDCD6IP ProteinQ8WUM4 (Uniprot-TrEMBL)
PDCD6IPProteinQ8WUM4 (Uniprot-TrEMBL)
PELI1 ProteinQ96FA3 (Uniprot-TrEMBL)
PELI1ProteinQ96FA3 (Uniprot-TrEMBL)
PI(3,4,5)P3 MetaboliteCHEBI:16618 (ChEBI)
PI(4,5)P2 MetaboliteCHEBI:18348 (ChEBI)
PI4P MetaboliteCHEBI:17526 (ChEBI)
PI4P,PI(4,5)P2,PIP3ComplexR-ALL-5620974 (Reactome)
RIP1:RIP3:MLKL oligomer:PIPsComplexR-HSA-5620984 (Reactome)
RIPK1 ProteinQ13546 (Uniprot-TrEMBL)
RIPK1(1-324)ProteinQ13546 (Uniprot-TrEMBL)
RIPK1(325-671) ProteinQ13546 (Uniprot-TrEMBL)
RIPK1:RIPK3:sorafenib, ponatinib, pazopanibComplexR-HSA-9693943 (Reactome)
RIPK1:RIPK3ComplexR-HSA-5218862 (Reactome)
RIPK1ProteinQ13546 (Uniprot-TrEMBL)
RIPK3 ProteinQ9Y572 (Uniprot-TrEMBL)
RIPK3(1-328)ProteinQ9Y572 (Uniprot-TrEMBL)
RIPK3(329-518)ProteinQ9Y572 (Uniprot-TrEMBL)
RIPK3:HSP90:CDC37ComplexR-HSA-9688479 (Reactome)
RIPK3:STUB1ComplexR-HSA-9688827 (Reactome)
RIPK3ProteinQ9Y572 (Uniprot-TrEMBL)
RIR1 ProteinP08543 (Uniprot-TrEMBL)
RIR1ProteinP08543 (Uniprot-TrEMBL)
RPS27A(1-76) ProteinP62979 (Uniprot-TrEMBL)
SARS-CoV-1

3a:(RIPK1:RIPK3)

oligomer
ComplexR-HSA-9686348 (Reactome)
SDCBP ProteinO00560 (Uniprot-TrEMBL)
SDCBPProteinO00560 (Uniprot-TrEMBL)
SPI-2 ProteinP15059 (Uniprot-TrEMBL)
STUB1 ProteinQ9UNE7 (Uniprot-TrEMBL)
STUB1:STUB1ComplexR-HSA-9688809 (Reactome) Structural studies suggest that STUB1 functions as homodimer (Zhang M et al. 2005)
TNFRSF10A ProteinO00220 (Uniprot-TrEMBL)
TNFRSF10B ProteinO14763 (Uniprot-TrEMBL)
TNFSF10 ProteinP50591 (Uniprot-TrEMBL)
TRADD ProteinQ15628 (Uniprot-TrEMBL)
TRADD:TRAF2:RIPK1:FADDComplexR-HSA-140977 (Reactome)
TRAF2 ProteinQ12933 (Uniprot-TrEMBL)
TRAF2:TRADD:RIPK1(325-671):FADDComplexR-HSA-5357809 (Reactome)
UBA52(1-76) ProteinP62987 (Uniprot-TrEMBL)
UBB(1-76) ProteinP0CG47 (Uniprot-TrEMBL)
UBB(153-228) ProteinP0CG47 (Uniprot-TrEMBL)
UBB(77-152) ProteinP0CG47 (Uniprot-TrEMBL)
UBC(1-76) ProteinP0CG48 (Uniprot-TrEMBL)
UBC(153-228) ProteinP0CG48 (Uniprot-TrEMBL)
UBC(229-304) ProteinP0CG48 (Uniprot-TrEMBL)
UBC(305-380) ProteinP0CG48 (Uniprot-TrEMBL)
UBC(381-456) ProteinP0CG48 (Uniprot-TrEMBL)
UBC(457-532) ProteinP0CG48 (Uniprot-TrEMBL)
UBC(533-608) ProteinP0CG48 (Uniprot-TrEMBL)
UBC(609-684) ProteinP0CG48 (Uniprot-TrEMBL)
UBC(77-152) ProteinP0CG48 (Uniprot-TrEMBL)
UDP-GlcNAcMetaboliteCHEBI:16264 (ChEBI)
UDPMetaboliteCHEBI:17659 (ChEBI)
UL36 ProteinP16767 (Uniprot-TrEMBL)
UL36ProteinP16767 (Uniprot-TrEMBL)
Ub-K55,363-RIPK3 ProteinQ9Y572 (Uniprot-TrEMBL)
Ub-K55,363-RIPK3:STUB1ComplexR-HSA-9688828 (Reactome)
UbComplexR-HSA-113595 (Reactome)
XIAP ProteinP98170 (Uniprot-TrEMBL)
active

caspase-8:viral

CRMA/SPI-2
ComplexR-HSA-2672221 (Reactome)
active caspase-8ComplexR-HSA-2562550 (Reactome)
p-S-RIPK1:RIPK3ComplexR-HSA-5218868 (Reactome)
p-S-RIPK1:p-S199,227, K48pUb-363-RIPK3:PELI1ComplexR-HSA-9686928 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomer R-HSA-5218908 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomer:4xMLKL:IP6,IP5, IP4ComplexR-HSA-9687620 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomerR-HSA-5218908 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:PELI1ComplexR-HSA-9686923 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomer:FLOT1:FLOT2ComplexR-HSA-9688468 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomer:PDCD6IP:SDCBPComplexR-HSA-9688894 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomerComplexR-HSA-5357794 (Reactome)
p-S166-RIPK1 ProteinQ13546 (Uniprot-TrEMBL)
p-S166-RIPK1:p-S199,227-RIPK3ComplexR-HSA-5218870 (Reactome)
p-S199,227, K48pUb-363-RIPK3 ProteinQ9Y572 (Uniprot-TrEMBL)
p-S199,227-RIPK3 ProteinQ9Y572 (Uniprot-TrEMBL)
p-T357,S358-MLKL ProteinQ8NB16 (Uniprot-TrEMBL)
p-T357,S358-MLKL oligomer R-HSA-5357857 (Reactome)
ponatinib
small molecule

inhibitors of

RIPK1, RIPK3
ComplexR-HSA-9694028 (Reactome)
viral serpinsComplexR-NUL-2672224 (Reactome)

Annotated Interactions

View all...
SourceTargetTypeDatabase referenceComment
(RIPK1:RIPK3)oligomer:4xMLKLArrowR-HSA-5218891 (Reactome)
(RIPK1:RIPK3)oligomer:4xMLKLR-HSA-5218906 (Reactome)
(RIPK1:RIPK3)oligomer:4xMLKLR-HSA-9687625 (Reactome)
(RIPK1:RIPK3)oligomer:4xMLKLmim-catalysisR-HSA-5218906 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:4x(MLKL:ATP)ArrowR-HSA-9687625 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:4x(MLKL:ATP)TBarR-HSA-5357927 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:4xp-T357,S358-MLKLArrowR-HSA-5218906 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:4xp-T357,S358-MLKLR-HSA-5357927 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:4xp-T357,S358-MLKLR-HSA-9687638 (Reactome)
3a tetramer:(RIPK1:RIPK3) oligomerArrowR-HSA-9686336 (Reactome)
3a tetramer:(RIPK1:RIPK3) oligomerArrowR-HSA-9686338 (Reactome)
3a tetramer:(RIPK1:RIPK3) oligomerR-HSA-9686338 (Reactome)
ADPArrowR-HSA-5213464 (Reactome)
ADPArrowR-HSA-5213466 (Reactome)
ADPArrowR-HSA-5218906 (Reactome)
ATPR-HSA-5213464 (Reactome)
ATPR-HSA-5213466 (Reactome)
ATPR-HSA-5218906 (Reactome)
ATPR-HSA-9687625 (Reactome)
ArrowR-HSA-5357927 (Reactome)
BIRC2,3,4R-HSA-5675470 (Reactome)
BIRC2,3,4TBarR-HSA-5213462 (Reactome)
BIRC2,3,4mim-catalysisR-HSA-5675470 (Reactome)
CASP8(1-479)R-HSA-9687458 (Reactome)
CDC37R-HSA-9688459 (Reactome)
CDC37R-HSA-9698844 (Reactome)
CFLAR(1-376)R-HSA-5675456 (Reactome)
DISC:procaspase-8:FLIP(L)ArrowR-HSA-5675456 (Reactome)
DISC:procaspase-8:FLIP(L)TBarR-HSA-5213462 (Reactome)
DISC:procaspase-8:FLIP(S)ArrowR-HSA-3465429 (Reactome)
DISC:procaspase-8:FLIP(S)ArrowR-HSA-5213462 (Reactome)
DISC:procaspase-8R-HSA-3465429 (Reactome)
DISC:procaspase-8R-HSA-5675456 (Reactome)
FLIP(S)R-HSA-3465429 (Reactome)
FLOT1:FLOT2R-HSA-9688456 (Reactome)
HSP90R-HSA-9688459 (Reactome)
HSP90R-HSA-9698844 (Reactome)
HSV1 RIR1:CASP8ArrowR-HSA-9687458 (Reactome)
HSV1 RIR1:RIPK1ArrowR-HSA-9687465 (Reactome)
HSV1 RIR1:RIPK3ArrowR-HSA-9687455 (Reactome)
I(1,3,4,5,6)P5R-HSA-9687638 (Reactome)
I(1,3,4,6)P4R-HSA-9687638 (Reactome)
IP6R-HSA-9687638 (Reactome)
K48pUb- BIRC2,3,4ArrowR-HSA-5675470 (Reactome)
K48polyUbR-HSA-5675470 (Reactome)
K48polyUbR-HSA-9686920 (Reactome)
MLKL:HSP90:CDC37ArrowR-HSA-9698844 (Reactome)
MLKL:UL36ArrowR-HSA-9698677 (Reactome)
MLKLR-HSA-5218891 (Reactome)
MLKLR-HSA-9686343 (Reactome)
MLKLR-HSA-9698677 (Reactome)
MLKLR-HSA-9698844 (Reactome)
NS1 dimerR-HSA-9686343 (Reactome)
NS1:MLKLArrowR-HSA-9686343 (Reactome)
O-glycosyl 3aR-HSA-9686345 (Reactome)
OGTmim-catalysisR-HSA-9687828 (Reactome)
OGlcNAc-T467-RIPK3ArrowR-HSA-9687828 (Reactome)
PDCD6IPR-HSA-9688832 (Reactome)
PELI1R-HSA-9686922 (Reactome)
PI4P,PI(4,5)P2,PIP3R-HSA-5620975 (Reactome)
R-HSA-2672196 (Reactome) SPI-2/CrmA (cytokine response modifier A) is a poxvirus gene product with homology to members of the serpin (serine protease inhibitor) superfamily. Cowpox virus-derived and vaccinia virus-derived CrmA cDNAs transfected into cells inhibit apoptosis induced by Fas-ligation and activation of TNFR1 (Tewari M and Dixit VM 1995; Miura M et al, 1995; Kettle S et al. 1997). Cowpox virus-derived CrmA was shown to selectively inhibit caspases in Fas-mediated apoptosis, showing the highest affinity for interleukin-1 beta-converting enzyme (ICE) and a similarly high affinity for caspase-8, Ki = 0.95 nM (Zhou Q et al. 1997).
R-HSA-3465429 (Reactome) The short form of cellular FLIP (FLIP(S) or c-FLIPS) has two death effector domains DEDs, which can bind to FADD and caspase-8 (CASP8). FLIP(S) protects the cells from apoptosis by inhibiting the processing of caspase-8 at the receptor level (Scaffidi C et al. 1999; Micheau O et al 2001).

FLIP(S) is a short-lived protein which is sensitive to ubiquitination and proteasomal degradation (Poukkula M et al. 2005). Protein kinase C (PKC)- mediated phosphorylation of FLIP(S) at Ser193 was shown to prolongs the half-life of FLIP(S) by inhibiting its polyubiquitination (Kaunisto A et al. 2009).

R-HSA-5213462 (Reactome) Necroptosis is a regulated form of necrotic cell death that is mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3 and mixed-lineage kinase domain-like protein (MLKL). The initiation of necroptosis can be stimulated by the same death ligands that activate apoptosis, such as tumor necrosis factor (TNF) alpha, Fas ligand (FasL), and TRAIL (TNF-related apoptosis-inducing ligand) or ligands for toll like receptors 3 (TLR3) and TLR4 (Holler N et al. 2000; He S et al. 2009; Feoktistova M et al. 2011; Voigt S et al. 2014). In contrast to apoptosis, however, necroptosis is optimally induced when caspases are inhibited (Holler N et al. 2000; Sawai H 2014). Otherwise active caspase 8 (CASP8) blocks necroptosis by the proteolytic cleavage of RIPK1 and RIPK3 (Kalai M et al. 2002; Degterev A et al. 2008; Feng S et al. 2007). When CASP8 activity is inhibited under certain pathophysiological conditions or by pharmacological agents, RIPK1 is engaged in physical and functional interactions with its homolog RIPK3 leading to formation of the necrosome, a cytosolic necroptosis-inducing complex consisting of RIPK1 and RIPK3 (Sawai H 2013; Moquin DM et al. 2013; Kalai M et al. 2002; He S et al. 2009, Zhang DW et al. 2009). RIPK3 was found to be essential for necroptosis (He S et al. 2009; Cho YC et al. 2009; Zhang DW et al. 2009). Embryonic fibroblasts from RIPK3 knockout mice were resistant to necrosis induced by TNF or during virus infection (He S et al. 2009; Cho YC et al. 2009). RIPK3-/- mice exhibited severely impaired vaccinia virus (VV)-induced tissue necrosis, inflammation, and control of viral replication (Cho YC et al. 2009). RIPK3 knockout animals were devoid of inflammation inflicted tissue damage in an acute pancreatitis model (He S et al. 2009). Further, RIPK3 knockdown in the human colorectal adenocarcinoma (HT-29) cell line, that stably expressed a shRNA targeting RIPK3, led to blockage of TNF-alpha, TRAIL or FAS-induced pronecrotic signaling pathway (He S et al. 2009). Knockdown of RIPK3 in human keratinocyte HaCaT cells blocked TLR3-mediated necroptosis without affecting the apoptotic response. Moreover, overexpression of RIPK3 in human epithelial carcinoma (HeLa) cells led to increased caspase-independent TLR3-induced cell death in the absence of inhibitors of apoptosis (IAPs) (Feoktistova M et al. 2011). Within the necrosome RIPK1 and RIPK3 bind to each other through their RIP homotypic interaction motif (RHIM) domains (Sun X et al. 2002; Li J et al. 2012; Mompean M et al. 2018). The RHIMs can facilitate RIPK1:RIPK3 oligomerization, allowing them to form amyloid-like fibrillar structures (Li J et al. 2012; Mompean M et al. 2018). RIPK1 serves as a scaffold to enable RIPK3 to assemble into homooligomers. Owing to the size and the toxicity arising from overexpressing RIPK1 and RIPK3 in cells, this has been problematic to study in detail. The underlying mechanism is still debated, but RIPK3 transphosphorylation is believed to be crucial for MLKL activation (Orozco S et al. 2014; Cook WD et al. 2014). Necroptosis is a tightly regulated process. The balance between caspase-dependent apoptosis and RIPK-dependent necroptosis was found to depend on the levels of CASP8 and cellular FADD-like interleukin-1 beta converting enzyme (FLICE)-inhibitory protein (cFLIP, encoded by the CFLAR gene) (Feoktistova M et al. 2011). cFLIP exists in two main isoforms: long cFLIP(L) and short cFLIP(S) forms. cFLIP(L) (CFLAR) prevented apoptosis and necroptosis, whereas FLIP(S) inhibited apoptosis but promoted necroptosis (Feoktistova M et al. 2011; Dillon CP et al. 2012). A blockage of CASP8 activity in the presence of viral FLIP-like protein was found to switch signaling to necrotic cell death (Sawai H 2013). Cell level of free active RIPK1 can be controlled by targeting RIPK1 for proteasomal degradation via K48-linked polyubiquitination mediated by baculoviral IAP repeat containing proteins BIRC2 and BIRC3 (also known as cellular inhibitor of apoptosis proteins cIAP1 and cIAP2) (Varfolomeev E et al, 2008; Bertrand MJM et al. 2008; Tenev T et al. 2011). The carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) was shown to negatively regulate necroptosis by ubiquitylation-mediated degradation of RIPK3 (Seo J et al. 2016). Further, O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) was found to prevent necroptosis by suppressing RIPK3 activity (Li X et al. 2019; Zhang B et al. 2019). During infection in human cells, herpes simplex virus (HSV)-1 and HSV-2 can modulate cell death pathways using the large subunit (R1) of viral ribonucleotide reductase (RIR1 or UL39). Viral RIR1 blocked necroptosis in infected human cells by interactions with RIPK1, RIPK3 and CASP8 (Guo H et al. 2015; Mocarski ES et al. 2015).

This Reactome event shows RHIM-dependent interaction of RIPK1 and RIPK3.

R-HSA-5213464 (Reactome) RIPK1 interaction with RIPK3 further potentiates their kinase activation through autophosphorylation and/or cross-phosphorylation (Cho YS et al. 2009). The kinase function of RIPK1 and RIPK3 is thought to stabilize RIPK1:RIPK3 association within the pronecrotic complex.

Reconstitution of RIPK1-deficient human Jurkat cells with mutated kinase-inactive RIPK1 or RIPK1 lacking the N-terminal serine/threonine kinase domain did not trigger FASL-induced necrotic cell death (Holler N et al. 2000). Similarly, mutations in the kinase domain and RIP homotypic interaction motif (RHIM) of RIPK1 also abolished the RIPK1-mediated rescue of tumor necrosis factor (TNF)/zVAD-fmk-induced regulated necrosis in RIPK1-deficient Jurkat cells (Cho YS et al. 2009). Furthermore, the results of structural and mutagenesis studies using necrostatins, which inhibit RIPK1 kinase activity by targeting the kinase domain, revealed that the N-terminal kinase domain of RIPK1 is required for propagating the pronecrotic signal (Degterev A et al. 2008; Cho YS et al. 2009; Xie T et al. 2013). Mass spectroscopy showed that human RIPK1 is phosphorylated within the kinase domain at multiple serine residues, such as Ser14/15, Ser20, Ser161 and Ser166, suggesting that the phosphorylation might regulate RIPK1 kinase activity (Degterev A et al. 2008). Using in vitro cellular systems, two independent studies reported that alanine substitution at Ser161 (S161A) leads to a reduction in RIPK1 kinase activity (Degterev A et al. 2008; McQuade T et al. 2013). RIPK1 autophosphorylation at Ser166 was found to modulate RIPK1 kinase activation (Laurien L et al. 2020). Studies with Ripk1 S166A/S166A knock-in mice revealed that abolishing phosphorylation at S166 prevented the development of RIPK1-mediated inflammatory conditions in vivo in four relevant mouse models of inflammation. Further, abolishing phosphorylation at S166 considerably inhibited RIPK1 kinase activity-dependent cell death downstream of tumor necrosis factor receptor 1 (TNFR1), toll-like receptor 3 (TLR3) and TLR4 in mouse cells isolated from Ripk1 S166A/S166A mice (Laurien L et al. 2020). Phosphorylation of S166 RIPK1 has been established as a biomarker of RIPK1 target engagement (Degterev A et al. 2008; Ofengeim D et al. 2015). The biological role of phosphorylation of individual serine residues in the kinase domain of RIPK1 remains to be further characterized (McQuade T et al. 2013).

RIPK1 is subjected to complex phosphorylation including several events possibly mediated by other kinases such as MAPK-activated protein kinase 2 (MK2) (Dondelinger Y et al. 2016; Jaco I et al. 2017; Delanghe T et al. 2020). S320 and S335 on human RIPK1 (S321 and S336 in mouse RIPK1) were identified as MK2 phosphorylation sites (Jaco I et al. 2017; Menon NB et al. 2017; Dondelinger Y et al. 2017). Transforming growth factor β-activated kinase 1 (TAK1) was also shown to phosphorylate RIPK1 along with TANK binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε) to prevent TNF-induced necroptosis or to dictate the multiple cell death pathways in mammalian cells (Lafont E et al. 2018; Xu D et al. 2018). In addition, IKKα/IKKβ is also able to phosphorylate RIPK1 in order to block RIPK1-dependent cell death in mouse models of infection and inflammation (Dondelinger Y et al. 2015, 2019). RIPK3 might also regulate RIPK1 phosphorylation in mammalian cells. For instance, RIPK3 was shown to directly phosphorylate RIPK1 when kinase-dead RIPK1 and RIPK3 were co-expressed in human embryonic kidney HEK293 cells, immunoprecipitated, and subjected to an in vitro kinase assay (Sun X et al. 2002; Cho et al. 2009). Importantly, mutation within RHIM motif of RIPK3 abrogated RIPK1 phosphorylation by RIPK3, suggesting that RIPK1 phosphorylation by RIPK3 is dependent on the formation of the RIPK1:RIPK3 complex (Sun X et al. 2002).

Several FDA-approved anticancer drugs, including sorafenib, pazopanib and ponatinib showed anti-necroptotic activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). RIPK1 has been identified as the main functional target of pazopanib, while sorafenib and ponatinib directly targeted both RIPK1 and RIPK3 (Fauster A et al. 2015; Najjar M et al. 2015; Martens S et al. 2017).

R-HSA-5213466 (Reactome) Necroptosis is a form of regulated necrotic cell death mediated by interaction of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 via a RIP homotypic interaction motif (RHIM) domain. RIPK1:RIPK3 complex formation further potentiates kinase activation through autophosphorylation and/or transphosphorylation, propagating the pronecrotic signal. RIPK1, RIPK3 and their kinase activities were shown to be essential for necroptosis (Degterev A et al. 2008; Cho YS et al. 2009). A RIPK3 kinase-dead mutant (K50A) was found to function as a dominant negative mutant, which blocked tumor necrosis factor alpha (TNFα)-induced necrotic pathway in human colorectal adenocarcinoma HT-29 cells (He S et al. 2009). Studies in mice expressing catalytically inactive RIPK3 showed that RIPK3 D161N stimulated RIPK1-dependent apoptosis and embryonic lethality in RIPK3 D161N homozygous mice, while K51A knock in mice developed into fertile and immunocompetent adults, suggesting that the kinase activity of RIPK3 determines whether cells die by necroptosis or caspase-8-dependent apoptosis (Mandal P et al. 2014; Newton K et al. 2014; Raju S et al. 2018). Further, differentially tagged constructs of RIPK3 kinase domain (KD) were found to form dimers after their co-expression in human embryonic kidney (HEK) 293T cells, and mutation of residues at the dimer interface impaired dimerization (Raju S et al. 2018). Phosphorylation on the serine residue 227 (S227) of human RIPK3 (S231 and S232 on mouse RIPK3) is thought to mediate recruitment and activation of mixed-lineage kinase domain-like (MLKL), a crucial downstream substrate of RIPK3 in the necrosis pathway (Sun et al. 2012; Chen et al. 2013). The phosphorylation occurs in the αG helix in the C-lobe of the RIPK3 kinase, not the activation loop (Petrie EJ et al. 2019;. Consequently it remains unclear why this would be an activating event and how this would lead to MLKL interaction Although RIPK1 activation is associated with phosphorylation of the RIPK3 activation loop, most studies, however, suggest that RIPK1 does not phosphorylate RIPK3 (Cho YS et al. 2009). Rather, it is thought that active RIPK1 serves as a scaffold to enable RIPK3 to assemble into homooligomers. The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020). The underlying mechanism is still debated, but the point is that RIPK3 transphosphorylation is crucial for MLKL activation (Cook WD et al. 2014; Orozco S et al. 2014; Mompean M et al. 2018).

FDA-approved anticancer drugs, including sorafenib and ponatinib, showed anti-necroptotic activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). These compounds are tyrosine kinase inhibitors (TKI) that directly targeted RIPK3 and RIPK1 and blocked their kinase activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). Pazopanib, another multi-targeting TKI, was shown to suppress necroptosis preferentially by targeting RIPK1 (Fauster A et al. 2015).

R-HSA-5218891 (Reactome) Exogenous stimuli provoke assembly of the receptor-interacting serine/threonine protein kinase RIPK1:RIPK3 oligomeric complex termed the necrosome, which acts as a platform for recruiting and activating mixed lineage kinase domain-like protein (MLKL), the terminal effector pseudokinase in the necroptotic signaling pathway (Sun L et al. 2012; Zhao J et al. 2012; reviewed by Murphy JM 2020). Mass spectrometry analysis identified MLKL as a necrosome component associated with RIPK3 in a HeLa cell line in which RIPK3 was expressed and caspase-8 was knocked down to induce necrosis in the presence of necrosulfonamide (NSA) (Sun L et al. 2012). NSA was found to specifically block TNFα-induced necroptosis downstream of RIPK3 activation in human colon cancer HT-29 cells, FADD null human T cell leukemia Jurkat cells and other RIPK3-expressing cells (Sun L et al. 2012). Short hairpin (sh) RNA-mediated genetic screens targeting human kinases, phosphatases, genes involved in protein ubiquination also identified MLKL as a key RIPK3 downstream component of TNFα-induced necroptosis in HT-29 cells (Zhao J et al. 2012). Further, MLKL knockout mice and cells derived from MLKL-deficient mice demonstrated the indispensable role of Mlkl in necroptosis (Wu J et al. 2013; Murphy JM et al. 2013). MLKL knockout in human myeloid leukaemia U937cells was shown to abrogate necroptosis, while induced expression of wild-type human MLKL in MLKL-/- U937 cells restored sensitivity to the necroptotic stimulus (Petrie EJ et al. 2018; Davies KA et al. 2020). Knockdown of MLKL by shRNA in HT-29 or gastric cancer MKN45 cells inhibited tumor necrosis factor alpha (TNFα)-induced necroptosis (Sun L et al. 2012; Zhao J et al. 2012; Wang H et al. 2014). The RIPK3 kinase activity is required for interaction and activation of MLKL in necroptosis as kinase-dead RIPK3 mutants were unable to bind MLKL or mediate TNF-induced necroptosis (Zhao J et al. 2012; Murphy JM et al. 2013; Chen W et al. 2013). The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020). The pseudokinase domain (psKD) of MLKL is known to engage the kinase domain (KD) of RIPK3, stably in the case of the human system (Sun L et al. 2012; Zhao J et al. 2012; Davies KA et al. 2018; Petrie EJ et al. 2018), but transiently in the mouse system (Murphy JM et al. 2013; Chen W et al. 2013; Petrie EJ et al. 2019a). Structural studies of the mouse MLKL pseudokinase domain in complex with the mouse RIPK3 kinase domain revealed juxtaposition of RIPK3 active site next to the pseudoactive site of mouse MLKL for phosphorylation of the latter’s activation loop (Xie T et al. 2013). The KD:psKD complex is governed by extensive lobe-to-lobe interaction interfaces, stabilized by hydrophobic and electrostatic interactions. The C-lobe interface is mediated by mouse RIPK3 autophosphorylated residues. It was observed that F373 of mouse MLKL projects from the αF-αG loop into a cavity adjacent to αG in RIPK3 (Xie T et al. 2013). The structure of the human RIPK3:MLKL complex has not been determined, but its modeling based on the mouse complex suggests that similar interaction may occur, governed by different electrostatic surface potentials (Petrie EJ et al. 2019b). Ala substitution of the equivalent human MLKL residue, F386, abrogated reconstitution of necroptotic signaling in MLKL-/- U937 cells, suggesting more broadly that this C-lobe:C-lobe interaction underpins RIPK3 engagement by MLKL (Petrie EJ et al. 2019b).

MLKL is composed of an amino-terminal four-helix bundle (4HB) domain, a two-helix “brace� region, and a carboxy-terminal pseudokinase domain (Murphy JM et al. 2013; Petrie EJ et al. 2018). The 4HB domain functions as the executioner domain by virtue of its membrane permeabilization activity (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Hildebrand JM et al. 2014; Su L et al. 2014; Wang H et al. 2014; Tanzer MC et al. 2016). The 4HB domain enables membrane translocation of MLKL and is responsible for the plasma membrane permeabilization that characterizes necroptotic cell death (Chen X et al. 2014; Cai Z et al. 2014; Dondelinger Y. et al. 2014; Hildebrand JM et al. 2014; Petrie EJ et al. 2020). The 4HB domain executioner function is regulated by the C-terminal pseudokinase domain, which serves as a receiver for upstream signals, such as activation loop phosphorylation by RIPK3 (Hildebrand JM et al. 2014; Sun L et al. 2012; Rodriguez DA et al. 2016; Petrie EJ et al. 2018). Studies using mouse:human MLKL chimeras showed that the first brace helix and the adjacent loop (that connect the 4HB to the pseudokinase domain) of MLKL mediate interdomain communication and oligomerisation upon RIPK3-mediated activation of MLKL (Davies KA et al. 2018). RIPK3-mediated phosphorylation is thought to trigger a conformational change within the pseudokinase of MLKL that promotes 4HB domain exposure, enabling MLKL to form oligomers, which are trafficked to the plasma membrane where cell permeabilization occurs (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2020; Samson AL et al. 2020). Even though the Reactome annotation shows that 4 molecules of MLKL bind to the RIPK1:RIPK3 oligomer, the exact stoichiometry of the binding and the oligomerization of MLKL has been highly debated (Chen X et al. 2014; Cai Z et al. 2014; Davies KA et al. 2018; Petrie EJ et al. 2018; Petrie EJ 2017). While trimers, tetramers, hexamers were reported in studies with the recombinant MLKL protein, single-cell imaging approaches revealed that endogenous human phosphorylated MLKL assembles into higher order species that are heterogeneous in MLKL stoichiometry (Samson AL et al. 2020). The mechanisms of necroptosis regulation and execution downstream of MLKL remain elusive.


R-HSA-5218905 (Reactome) Structural studies showed that activation of RIPK3 by RIPK1 involves the formation of a functional hetero-oligomeric amyloidal signaling complex that mediated programmed necrosis (Li J et al. 2012; Mompean M et al. 2018). The RIP homotypic interaction motifs (RHIMs) of RIPK1 and RIPK3 were found to mediate the assembly of these heterodimeric filamentous structures (Li J et al. 2012). RIPK1 was reported to control RIPK3 oligomerization in both postive and negative manners (Orozco S et al. 2014). RIPK3 recruitment to other RIPK3 protomers within this assembly may be favored by allosteric interactions between their kinase domains and activation by autophosphorylation of a site in the C-lobe of their kinase domains (Raju S et al. 2018). Presumably this autophosphorylation leads to an electrostatic repulsion or conformational change that disfavors RIPK3 hetero-oligomer formation to allow RIPK3 to preferentially self-associate within the necrosome complex. Owing to the size and the toxicity arising from overexpressing RIPK1 and RIPK3 in cells, this has been problematic to study in detail. The underlying mechanism is still debated, but RIPK3 transphosphorylation is believed to be crucial for MLKL activation (Orozco S et al. 2014; Cook WD et al. 2014).
R-HSA-5218906 (Reactome) Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) was shown to activate mixed lineage kinase domain-like protein (MLKL) by phosphorylation of the threonine 357 (T357) and serine 358 (S358) residues within the kinase-like domain in human MLKL and S345 in mouse MLKL (Sun L et al. 2012: Wang H et al. 2014; Murphy JM et al. 2013; Tanzer MC et al. 2015; Rodriguez DA et al. 2016). The precise mechanism of MLKL activation by RIPK3 is incompletely understood and may vary across species (Davies KA et al. 2020; reviewed by Murphy JM 2020). The pseudokinase domain (psKD) of MLKL is known to engage the kinase domain (KD) of RIPK3, stably in the case of the human system (Sun L et al. 2012; Davies KA et al. 2018; Petrie EJ et al. 2018, 2019a), but transiently in the mouse system (Tanzer MC et al. 2015; Rodriguez DA et al. 2016; Petrie EJ et al. 2019b). The kinase-dead RIPK3 mutants were unable to bind MLKL or mediate TNF-induced necroptosis in human and mouse cells (Sun L et al. 2012; Zhao J et al. 2012; Murphy JM et al. 2013; Chen W et al. 2013). Studies involving knockout of endogenous MLKL in human histiocytic lymphoma U937 and adenocarcinoma HT-29 cells support the idea that activation of MLKL relies on the RIPK3-mediated phosphorylation of T357 and S358 in human MLKL (Petrie EJ et al. 2018). While wild-type human MLKL could reconstitute the necroptotic signaling, both the T357E/S358E phosphomimic and the T357A/S358A phospho-ablating human MLKL constructst blocked necroptosis in MLKL-/- U937 and HT-29 cell lines in the presence of necroptosis stimuli (Petrie EJ et al. 2018). Furthermore, introduction of constructs harboring mutations within the human MLKL pseudoactive site, such as those observed in colon, lung, and endometrial carcinomas and melanoma specimens, into MLKL-/- U937 cells did not promote MLKL's killing activity, but rather delayed the kinetics of cell death following treatment with a necroptosis stimulus (Petrie EJ et al. 2018). Biophysical data suggest that defective MLKL variants are locked in a monomeric conformation, which hampers assembly into higher order oligomers that are responsible for cell death (Petrie EJ et al. 2018). Although wild-type human MLKL robustly bound human RIPK3 kinase domain, no binding was detected for the human MLKL T357E/S358E constructsts (Petrie et al. 2018). These data support the idea that human MLKL activation relies on recruitment to human RIPK3 in cells as a precursor to its activation (Petrie EJ et al. 2019). RIPK3-mediated phosphorylation of human MLKL is thought to trigger a conformational change within the pseudokinase of MLKL that promotes the N-terminal four-helix bundle (4HB) domain exposure, enabling MLKL to form higher order MLKL assemblies which are trafficked to the plasma membrane (Sun L et al. 2012; Wang H et al. 2014; Petrie EJ et al. 2017,2018, 2019; 2020; Samson AL et al. 2020). The phosphorylation of MLKL may induce disengagement of MLKL from RIPK3 followed by translocation to the the plasma membrane where cell permeabilization occurs (Davies KA et al. 2020; Murphy JM 2020). Important to note that the assembly of MLKL into higher order species and the translocation of MLKL oligomers to the plasma membrane are hallmarks of necroptosis (Davies KA et al. 2020; Petrie EJ et al. 2020; Samson AL et al. 2020). This Reactome event shows that 4 molecules of MLKL are bound to RIPK1:RIPK3 oligomer, however the exact stoichiometry of MLKL binding remains unclear (Chen X et al. 2014; Cai Z et al. 2014; Davies KA et al. 2018; Petrie EJ et al. 2018; reviewed by Petrie EJ 2017). Single-cell imaging approaches revealed that endogenous human MLKL assembles on necrosomes into higher order species that are heterogeneous in MLKL stoichiometry (Samson AL et al. 2020). The mechanisms of necroptosis regulation and execution downstream of MLKL remain elusive.
R-HSA-5357828 (Reactome) Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can be a part of cell death and survival signaling complexes. Whether RIPK1 functions in apoptosis, necroptosis or NFκB signaling is dependent on autocrine/paracrine signals, on the cellular context and tightly regulated by posttranslational modifications of RIP1 itself. The pro-survival function of RIPK1 is achieved by polyubiquitination which is required for recruitment of signaling molecules/complexes such as the IKK complex and the TAB2:TAK1 complex to mediate activation of NFκB signaling (Ea CK et al. 2006). CYLD-mediated deubiquitination of RIPK1 switches its pro-survival function to caspase-mediated pro-apoptotic signaling (Fujikura D et al. 2012; Moquin DM et al. 2013). Caspase-8 (CASP8) in human and rodent cells facilitates the cleavage of kinases RIPK1 and RIPK3 and prevents RIPK1/RIPK3-dependent necroptosis (Lin Y et al. 1999; Hopkins-Donaldson S et al. 2000; Newton K et al. 2019; Zhang X et al. 2019; Lalaoui N et al. 2020). CASP8-mediated cleavage of human RIPK1 after D324 (D325 in mice) separates the amino-terminal kinase domain from the carboxy-terminal part of the molecule preventing RIPK1 kinase activation through dimerization via the carboxy-terminal death domain and leads to the dissociation of the complex TRADD:TRAF2:RIP1:FADD:CASP8 (Lin Y et al. 1999; Meng H et al. 2018). The lack of CASP8 proteolytic activity in the presence of viral (e.g. CrmA and vICA) or pharmacological caspase inhibitors results in necroptosis induction via RIPK1 and RIPK3 (Tewari M & Dixit VM 1995; Fliss PM & Brune W 2012; Hopkins-Donaldson S et al. 2000). Cellular FLICE-like inhibitory protein (cFLIP), which is an NF-κB target gene, form heterodimer with procaspase-8 and inhibits activation of CASP8 within the the TRADD:TRAF2:RIP1:FADD:CASP8:FLIP complex (Yu JW et al. 2009; Pop C et al. 2011). The presence of cFLIP (long form) limits CASP8 to cleave CASP3/7 but allow cleavage of RIPK1 to cause the dissociation of the TRADD:TRAF2:RIP1:FADD:CASP8, thereby inhibiting both apoptosis and necroptosis (Boatright KM et al. 2004; Yu JW et al. 2009; Pop C et al. 2011; Feoktistova M et al. 2011). Mice that lack CASP8 or knock-in mice that express catalytically inactive CASP8 (C362A) die in a RIPK3- and MLKL-dependent manner during embryogenesis (Kaiser WJ et al. 2011; Newton K et al. 2019). Studies using mice that express RIPK1(D325A), in which the CASP8 cleavage site Asp325 had been mutated, further confirmed that cleavage of RIPK1 by CASP8 is a mechanism for dismantling death-inducing complexes for limiting aberrant cell death in response to stimuli (Newton K et al. 2019; Lalaoui N et al. 2020). Disrupted cleavage of RIPK1 variants with mutations at D324 by CASP8 in humans leads to an autoinflammatory response by promoting the activation of RIPK1 (Tao P et al. 2020; Lalaoui N et al. 2020).
R-HSA-5357927 (Reactome) Mixed lineage kinase domain-like protein (MLKL) was found to form oligomers that translocate to and mediate permeabilisation of plasma membrane (Hildebrand JM et al. 2014; Davies KA et al. 2018; Petrie EJ et al. 2018; Samson AL et al. 2020). The oligomerization of MLKL was observed in a variaty of human (colon adenocarcinoma HT-29, FADD-null Jurkat cells, leukemic monocyte lymphoma U937) and mouse cells upon necroptosis induced by (TNF+Smac mimetic+caspase inhibitor z-VAD-FMK) (Cai Z et al. 2014; Chen X et al. 2014; Davies KA et al. 2018; Petrie EJ et al. 2018). The precise oligomeric form of MLKL that mediates plasma membrane disruption has been highly debated (Chen X et al. 2014; Cai Z et al. 2014; Davies KA et al. 2018; Petrie EJ et al. 2018; reviewed by Petrie EJ 2017). Native mass spectrometry (MS) defined the human MLKL oligomer as a tetramer (Petrie EJ et al. 2018). Low-resolution techniques including cross-linking and deuterium exchange MS and small angle X-ray scattering (SAXS) showed that MLKL exists in equilibrium between a monomer and a daisy chain tetramer with the N-terminal four‑helix bundle (4HB) of one monomer binding to the pseudokinase domain (psKD) of another monomer (Petrie EJ et al. 2018). Cys-oxidation under nonreducing conditions and crosslinking analyses detected tetramers and octamers in L929 murine fibroblast and HEK293 cells undergoing TNF-mediated necroptosis, although the relationship of these disulfide crosslinks to MLKL’s killer function remains unknown (Huang D et al. 2017). While trimers, tetramers, hexamers were reported in studies with the recombinant MLKL protein (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014; Petrie EJ et al. 2018), single-cell imaging approaches revealed that endogenous human phosphorylated MLKL assembles on necrosomes into higher order species that are heterogeneous in MLKL stoichiometry (Samson AL et al. 2020). RIPK3-mediated phosphorylation of MLKL’s pseudokinase domain leads to MLKL switching from an inert to activated state, where exposure of 4HB ‘executioner’ domain leads to cell death (Hildebrand JM et al 2014; Petrie EJ et al. 2018). Following activation, toggling within the MLKL pseudokinase domain promotes 4HB domain disengagement from the pseudokinase domain αC helix and pseudocatalytic loop, to enable formation of a necroptosis-inducing tetramer (Petrie EJ et al. 2018). Despite lacking catalytic activity, the pseudokinase domain of MLKL has retained the ability to bind ATP (Murphy JM et al. 2013, 2014; Petrie EJ et al. 2018). The ATP binding has been shown to negatively regulate MLKL-mediated membrane permeabilization by destabilizing the MLKL tetramers and shifting the tetramer:monomer equilibrium toward the monomeric state (Petrie EJ et al. 2018). The two interdomain helices, termed the ‘brace’ helices, contribute to MLKL oligomerization by connecting phosphorylation of the pseudokinase domain to the release or activation of the 4HB domain executioner function to enable its participation in membrane localisation, permeabilization and cell death (Davies KA et al. 2018). In addition, the autoinhibited N-terminal 4HB of human MLKL is activated by inositol phosphate metabolites IP4, IP5 and IP6 produced by inositol phosphate multikinase (IPMK), inositol tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPPK) (Dovey CM et al. 2018; McNamara DE et al. 2019). These inositol phosphates promote MLKL-mediated necroptosis through directly binding 4HB domain of MLKL and dissociating its auto-inhibitory region (McNamara DE et al. 2019). Oligomers of MLKL translocate to membrane compartments (Cai Z et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014; Hildebrand JM et al. 2014; Davies KA et al. 2018; Petrie EJ et al. 2020; Samson AL et al. 2020). MLKL oligomerization and membrane translocation are hallmarks of the necroptosis pathway, which plays a crucial role in the host defense response against many pathogens (Upton JW et al. 2017). In response, pathogens have developed different strategies to target the host necroptosis machinery (Upton JW et al. 2017; Pearson JC et al. 2017; Petrie EJ et al. 2019; Gaba A et al. 2019).

Even though the stoichiometry of the MLKL oligomerization in the Reactome event depicts MLKL homotetramer, the endogenous MLKL was shown to assemble on necrosomes into higher order species that are heterogeneous in MLKL stoichiometry (Samson AL et al. 2020).

R-HSA-5620975 (Reactome) Activated by phosphorylation, mixed lineage kinase domain-like protein (MLKL) was found to translocate to the plasma membrane, where MLKL interacts with phosphatidylinositol phosphates (PIPs) via a patch of positively charged amino acids at the surface of a four-helical bundle domain (4HBD) located in its N-terminal region (Dondelinger Y et al. 2014; Wang H et al. 2014; Hildebrand JM et al. 2014; Su L et al. 2014; Quarato G et al. 2016). Interfering with the formation of PI(5)P or PI(4,5)P2 using PIP binders such as PIKfyve (P5i) efficiently inhibited TNF-induced necroptosis in both mouse L929 and the human FADD-null Jurkat cells (Dondelinger Y et al. 2014). In vitro liposome experiments revealed that MLKL induces leakage of PIP- or cardiolipin-containing liposomes suggesting that MLKL may have pore-forming capacities to mediate cell death by membrane's permeabilizing (Dondelinger Y et al. 2014; Wang H et al. 2014; Tanzer MC et al. 2016; Petrie EJ et al. 2018). Liposome permeabilization assays demonstrated that the N-terminal 4HB domain of MLKL compromised membrane integrity, and was more effective on liposomes whose composition resembled that of plasma membranes than on those mimicking mitochondrial membranes (Tanzer MC et al. 2016). One study has proposed the 4HB domain might reorganize in membranes to assemble into ion channels (Xia B et al. 2016); however, this remains to be fully explored in cellular contexts and structurally. Other studies implicated MLKL in engaging mitochondrial membranes to provoke mitochondrial fission or promote ion channel activity (Cai Z et al. 2014), although subsequent studies have discounted these possibilities (Murphy JM et al. 2013; Tait SW et al. 2013; Moujalled DM et al. 2014; Wang H et al. 2014; reviewd by Murphy JM 2020). Based on studies showing that the 4HB domain can permeabilize membranes in vitro (Dondelinger Y et al. 2014; Su L et al. 2014; Wang H et al. 2014; Tanzer MC et al. 2016; Petrie EJ et al. 2018), it is thought that MLKL kills cells via direct action on the plasma membrane (Murphy JM 2020).

Even in the Cai NCB 2013 paper this was not true of all cell types tested, despite this being written into folklore.

In terms of “pores� - this is a term that implies some sort of ordered structure. I prefer membrane perturbations or disruption as a term. If you look at Samson Nat Comm 2020, you can see these membrane structures are irregular in size and form. They also argue against channels as the destination for MLKL. Instead pMLKL coalesces with tight junction proteins in HT29 cells, although whether this is because of the membrane topology at this site or because of other accessory proteins being localized to these junctions, remains unknown.

Also Zargarian Plos Biol 2017. However, the idea of necroptotic bubbles was challenged in Samson Nat Comm 2020 because the bubbles could come and go at sites pMLKL did not accumulate and where membrane damage (“blowout�) did not occur

Various studies showed that the endosomal sorting complexes required for transport (ESCRT) pathway can remove phosphorylated MLKL-containing membrane vesicles from cells undergoing necroptosis, thereby attenuating the cell death process (Gong YN et al. 2017; Yoon S et al. 2017; Fan W et al. 2019). The ESCRT-associated proteins, programmed cell death 6-interacting protein (PDCD6IP or ALG-2-interacting protein X, ALIX) and syntenin-1 (SDCBP), were found to antagonize MLKL-mediated plasma membrane alteration (Fan W et al. 2019). In addition, flotillin-mediated endocytosis was proposed to suppress necroptosis by removing MLKL from the plasma membrane and redirecting it for lysosomal degradation (Fan W et al. 2019).

Studies in human cell lines suggest that upon induction of necroptosis MLKL shifts to the plasma membrane and membranous organelles such as mitochondria, lysosome, endosome and ER (Wang H et al. 2014), but it is trafficking via a Golgi-microtubule-actin-dependent mechanism that facilitates plasma membrane translocation, where membrane disruption causes death (Samson AL et al. 2020).


Based on studies showing that the 4HB domain can permeabilize membranes in vitro (Dondelinger et al. 2014; Su et al. 2014; Wang et al. 2014; Tanzer et al. 2016; Petrie et al. 2018), it is thought that MLKL kills cells via direct action on the plasma membrane.

R-HSA-5675456 (Reactome) The balance between caspase-dependent apoptosis and RIPK-dependent necroptosis was found to depend on the levels of caspase-8 (CASP8) and cellular FADD-like interleukin-1 beta converting enzyme (FLICE)-inhibitory protein (cFLIP, encoded by the CFLAR gene) (Feoktistova M et al. 2011; Hughes MA et al. 2016; reviewed in Tummers B & Green DR 2017). cFLIP exists in two main isoforms: long FLIP(L) and short FLIP(S) forms. Both FLIP(L) and FLIP(S) form heterodimers with procaspase-8, however they differentially regulate CASP8 activation (Feoktistova M et al. 2011; Dillon CP et al. 2012). The pseudoprotease FLIP(L) interacts with procaspase-8 through both death effector domains (DED) and caspase-like domain (CLD) that lacks catalytic activity due to absence of a cysteine residue in FLIP(L). The procaspase-8 catalytic domain prefers heterodimerization with the CLD of FLIP(L) over homodimerization with catalytic domains of other procaspase-8 molecules (Boatright KM et al. 2004; Yu JW et al. 2009). Heterodimerization to FLIP(L) rearranges the catalytic site of procaspase-8, producing a conformation that renders the heterodimer highly active even in the absence of proteolytic processing of either caspase-8 or cFLIPL (Micheau O et al. 2002; Yu JW et al. 2009; reviewed in Tummers B & Green DR 2017). The regulatory function of FLIP(L) has been found to differ depending on its expression levels. FLIP(L) was shown to inhibit death receptor (DR)-mediated apoptosis only when expressed at high levels, while low cell levels of FLIP(L) enhanced DR signaling to apoptosis (Boatright KM et al. 2004; Okano H et al. 2003; Yerbes R et al. 2011; Hughes MA et al. 2016). When FLIP(L) is expressed at high levels, the enzymatic activity of the FLIP(L):CASP8 heterodimer with procaspase-8 being an active unit is insufficient to generate active CASP8 heterotetramers for the apoptosis induction in mammalian cells. In contrary, the residual catalytic activity of FLIP(L):CASP8 is sufficient for RIPK1/RIPK3 cleavage, which inhibited the necroptotic cell death mode (Feoktistova M et al. 2011; Dillon CP et al. 2012; Oberst A et al. 2011).
R-HSA-5675470 (Reactome) Cellular inhibitor of apoptosis proteins (BIRC2, BIRC3 also known as cIAP1 and cIAP2) are E3 ubiquitin ligases that contribute to the cell survival by conjugating ubiquitin chains to components of cell death-activating platforms (such as ripoptosome, TNFR1- or toll like receptor 3 signaling complexes). BIRC2 or 3 can generate K11, K48- or K63-linked polyubiquitin chains depending on the cell content (Varfolomeev E et al, 2008; Bertrand MJM et al, 2008; Dynek JN et al. 2010). For instance, BIRC2/3-mediated K63-linked ubiquitination of RIPK1 enables activation of pro-survival NFkB signaling events downstream of TNFR1 complex, while cell level of free active RIPK1 can be controlled by targeting RIPK1 for proteasomal degradation via BIRC2/3-mediated K48-linked polyubiquitination (Varfolomeev E et al, 2008; Bertrand MJM et al. 2008; Tenev T et al. 2011; Darding M & Meier P 2012). Such BIRC2/3-mediated events prevent the formation of cell death signaling platforms. Following genotoxic stress, cytokine signaling-induced depletion of BIRCs, or Smac mimetics treatment, BIRC2, BIRC3, and XIAP levels rapidly decline and/or are inactivated (Bertrand MJM et al. 2008; Tenev T et al. 2011; Feoktistova M et al. 2011). BIRC2/3 antagonist are thought to induce self-mediated K48-linked ubiquitination followed by a proteasomal degradation. This allows accumulation of RIPK1 and formation of ripoptosome or other RIPK1-dependent death signaling platforms. In the absence of BIRCs the balance between caspase-dependent apoptosis and RIP-dependent necroptosis was found to depend on the levels of caspase-8, CFLAR (cFLIPL) and also FLIP(S) (Feoktistova M et al. 2011). CFLAR prevented apoptosis and necroptosis, whereas FLIPS inhibited apoptosis but promoted necroptosis (Feoktistova M et al. 2011; Dillon CP et al. 2012; Oberst A et al. 2001).
R-HSA-9686336 (Reactome) Receptor interacting serine/threonine protein kinase 3 (RIPK3) was found to induce oligomerization of severe acute respiratory syndrome-associated coronavirus type 1 (SARS-CoV-1) 3a (studied with the oligomerization-deficient viral 3a-flag C133A mutant) in human embryonic kidney 293 (HEK293) that do not express endogenous RIPK3 or MLKL, after co-transfection of viral 3a and RIPK3 (Yue Y et al. 2018). RIPK3-induced oligomerization of viral 3a helped drive necrotic cell death in RIPK3-expressing HEK293 and 5-Aza-2′-deoxycytidine (5-AD)-treated human alveolar epithelial A549 cells (Yue Y et al. 2018). The A549 cell line is resistant to the traditional necroptotic stimuli, but treatment with hypomethylating agents such as 5-AD induced RIPK3 expression (Yue Y et al. 2018). The results of the study suggest that SARS-Cov-1 3a does not induce cell death in the absence of RIPK3, but induces significant oligomerization-dependent death in the presence of endogenous RIPK3. (Yue Y et al. 2018). RIPK3 kinase activity was dispensable for the RIPK3-driven oligomerization of 3a (Yue Y et al. 2018). Further, a disulfide bond formation at cysteine-133 was found to mediate the oligomerization of 3a (Lu W et al. 2006) and the addition of DTT to cell lysates from HEK293 cells after co-transfection of viral 3a and RIPK3 completely erased the oligomerization 3a, confirming it is disulfide bond dependent (Yue Y et al. 2018). SARS-CoV-1 3a formed homodimer and homotetramer complexes in 3a-cDNA-transfected HEK 293 cells (Lu W et al. 2006). The tetrameric pattern is a very common feature of a protein involved in ion channel formation (Shi N et al. 2006).
R-HSA-9686338 (Reactome) Severe acute respiratory syndrome-associated coronavirus type 1 (SARS-CoV-1) open reading frame-3a has been implicated in host cell death pathways. Receptor interacting serine/threonine protein kinase 3 (RIPK3) was found to induce oligomerization of SARS-CoV-1 3a after co-transfection of viral 3a and RIPK3 in human embryonic kidney 293 (HEK293) that do not express endogenous RIPK3 or MLKL (Yue Y et al. 2018). Confocal imaging showed that co-expressed SARS-CoV-1 3a and RIPK3 co-localized with lysosomal-associated membrane protein 1 (LAMP1) in HeLa cells (Yue Y et al. 2018). Quantification of colocalization revealed that 3a likely targets RIPK3 to lysosomes. Further, lysosomal galectin puncta assay showed that SARS-CoV-1 3a caused lysosomal membrane permeablization. The SARS-CoV-1 3a-mediated release of cathepsins from lysosome resulted in impaired lysosomal degradation capacity in HeLa cells (Yue Y et al. 2018). Thus, RIPK3 is thought to induce oligomerization of SARS-CoV-1 3a, which facilitates membrane insertion and ion channel functionality of SARS-CoV-1 3a to promote virus release and inflammatory cell death (Yue Y et al. 2018).
R-HSA-9686343 (Reactome) Co-immunoprecipitation assays in influenza A virus (IAV)-infected human leukemia monocytic THP1 cell, as well as NS1-transfected human embryonic kidney (HEK293) cells showed that viral NS1 interacts with mixed-lineage kinase domain-like protein (MLKL) in an RNA-dependent manner (Gaba A et al. 2019). The second brace helix of MLKL is responsible for interacting with NS1 (Gaba A et al. 2019). The interaction of NS1 with MLKL is thought to increased MLKL membrane translocation. Moreover, the MLKL:NS1 interaction enhanced NLRP3 inflammasome activation and increases IL-1β processing and secretion (Gaba A et al. 2019).
R-HSA-9686345 (Reactome) Severe acute respiratory syndrome-associated coronavirus (SARS-CoV-1) 3a protein was shown to interact with receptor interacting protein kinase 3 (RIPK3) by immunoprecipitation analysis upon co-expression of viral 3a and RIPK3 in human embryonic kidney 293 (HEK293) cells (Yue Y et al. 2018). Mapping of the interaction between RIPK3 and 3a showed that the kinase domain of RIPK3 (1–326) interacted with SARS-CoV-1 3a, but that the RIP homotypic interaction motif (RHIM) containing C-terminus (327–518) interacted very weakly. Time-lapse confocal microscopy using Cherry-tagged RIP3 in HeLa cells expressing SARS-CoV-1 3a-GFP showed that expression of RIPK3 drives cell death in the presence of SARS 3a. Further, RIPK3-induced oligomerization of SARS-CoV-1 3a (studied with the oligomerization-deficient viral 3a-flag C133A mutant) helped drive necrotic cell death in RIPK3-expressing HEK293, HeLa and 5-Aza-2′-deoxycytidine (5-AD)-treated human alveolar epithelial A549 cells (Yue Y et al. 2018). The A549 cell line is resistant to the traditional necroptotic stimuli, but treatment with hypomethylating agents such as 5-AD induced RIPK3 expression (Yue Y et al. 2018). The results of the study suggest that SARS-Cov-1 3a does not induce cell death in the absence of RIPK3, but induces significant oligomerization-dependent death in the presence of endogenous RIPK3. (Yue Y et al. 2018).

During tumor necrosis factor (TNF)-induced necroptosis, RIPK3 and RIPK1 associate with each other through their RHIM domains into heteromeric RIPK1:RIPK3 complexes that further polymerize into filamentous β-amyloid structures promoting the activation of RIPK3 kinase (Cho Y et al. 2009; Li J et al. 2012). Functionally active RIPK3 activates mixed-lineage kinase domain-like pseudokinase (MLKL), the membrane-disrupting effector of programmed necrosis (Sun L et al. 2012; Murphy JM et al. 2013; Wang H et al. 2014). Other RHIM-containing proteins, such as the TLR3/TLR4 adaptor TRIF (also known as TICAM1) and the DNA sensor DAI/ZBP can form the necroptotic signaling platforms to support activation of RIPK3 and its interaction with MLKL (Kaiser W et al. 2013; Lin J et al. 2016).

This Reactome event shows a scaffolding role of RIPK3 bound to RIPK1 in supporting the formation of SARS-CoV-1 3a oligomers.

R-HSA-9686920 (Reactome) Receptor-interacting protein kinase-3 (RIP3 or RIPK3) interacts with pellino E3 ubiquitin protein ligase 1 (PELI1) through its forkhead-associated (FHA) domain to mediate K48-linked polyubiquitination of RIPK3 (Choi SW et al. 2018). Mass spectrometric analysis identified K363 of RIPK3 as the main target for PELI1-mediated ubiquitination. In vitro ubiquitination assay showed that PELI1 utilized the ubiquitin-conjugating enzyme UbcH5a to ubiquitinate RIPK3. Proteasome inhibitors (MG132 and BTZ), but not lysosome inhibitors, prevented PELI1-facilitated degradation of stably expressed RIPK3 in HeLa cells and endogenous RIPK3 in HT-29 cells, suggesting that PELI1 controls RIP3 protein destabilization via ubiquitylation-dependent proteasome-mediated degradation (Choi SW et al. 2018). Phosphorylation of RIPK3 at T182 was necessary for PELI1 recruitment and K48-linked polyubiquitination. PELI1-mediated RIPK3 degradation abolished the phosphorylation of mixed lineage kinase domain like pseudokinase (MLKL) and necroptotic cell death in human colorectal adenocarcinoma (HT29) cells, human neonatal epidermal keratinocytes (HEKn), and in lung tissues from PELI1 transgenic mice (Choi SW et al. 2018).


R-HSA-9686922 (Reactome) Pellino E3 ubiquitin protein ligase 1 (PELI1) was identified as a binding partner of receptor-interacting protein kinase-3 (RIPK3) in proteome microarrays (Choi SW et al. 2018). Endogenous PELI1 was found to interact with RIPK3 and RIPK1 in the necrosome complex upon necroptotic stimuli treatment in both human colorectal adenocarcinoma cells (HT-29) and mouse embryonic fibroblasts (MEF). PELI1:RIPK3 interaction was further studied by iImmunoprecipitation combined with site-directed mutagenesis assays using necroptosis-resistant RIPK3-deficient human embryonic kidney 293T (HEK293T) and human cervical cancer-derived HeLa cells, which were transfected with the epitope-tagged RIPK3 and PELI1 proteins. PELI1 was found to bind RIPK3 through its forkhead-associated (FHA) domain. Phosphorylation of RIPK3 at T182 was necessary for PELI1 recruitment (Choi SW et al. 2018).

MEFs lacking PELI1 have increased RIPK3 and MLKL phosphorylation, and necroptosis in response to necroptotic stimuli. Lung tissues from PELI1 transgenic mice showed a decrease in basal MLKL phosphorylation indicating that upregulated PELI1 may function to preferentially remove activated RIPK3 and reduce MLKL phosphorylation in vivo. In addition, PELI1 reduced endogenous RIPK3 in human lung adenocarcinoma (H2009) and human B lymphoblastoid (Raji) cell lines; conversely, knockdown of PELI1 in HT-29 and RIPK3 (ectopic)-expressing HeLa cell lines led to increased RIPK3 protein without affecting RIPK3 mRNA expression. Proteasome inhibitors (MG132 and BTZ), but not by lysosome inhibitors, prevented PELI1-facilitated degradation of stably expressed RIPK3 in HeLa cells and endogenous RIPK3 in HT-29 cells. In keratinocytes from toxic epidermal necrolysis (TEN) patients, PELI1 expression is low and inversely correlated with RIP3 protein, suggesting that reduction in PELI1 leads to upregulated RIP3 expression, thus contributing to disease progression. Thus, PELI1 is thought to control RIPK3 protein destabilization via ubiquitylation-dependent proteasome-mediated degradation (Choi SW et al. 2018).


R-HSA-9686930 (Reactome) Caspase-8 (CASP8) in human and rodent cells facilitates the cleavage of receptor-interacting protein kinases RIPK1 and RIPK3 and prevents RIPK1/RIPK3-dependent regulated necrosis (Lin Y et al. 1999; Hopkins-Donaldson S et al. 2000). These cleavage sites are identified to be Asp324 in RIPK1 and Asp328 in RIPK3 in humans (Lin Y et al. 1999; Feng S et al. 2007). The lack of CASP8 proteolytic activity in the presence of viral (e.g. CrmA and vICA) or pharmacological caspase inhibitors results in necroptosis induction via RIPK1 and RIPK3 (Tewari M & Dixit VM 1995; Fliss PM & Brune W 2012; Hopkins-Donaldson S et al. 2000).
R-HSA-9687455 (Reactome) During infection in human cells, herpes simplex virus (HSV)-1 and HSV-2 modulate cell death pathways using the large subunit (R1) of viral ribonucleotide reductase (RIR1 or UL39) (Dufour F et al. 2011; Guo H et al. 2015; Yu X et al. 2016; Ali M et al.2019). The N-terminal region of RIR1 protein carrying the RIP homotypic interaction motif (RHIM)-like element is sufficient for RHIM-dependent interaction with receptor�interacting protein kinase 1 (RIPK1) and receptor�interacting protein kinase 3 (RIPK3) thus inhibiting the interaction between RIPK1 and RIPK3 (Guo H et al. 2015; Yu X et al. 2015). An intact RHIM is required for the interaction between RIPK1 and RIPK3 that occurs downstream of tumour necrosis factor receptor 1 (TNFR1) activation during the programmed cell death response known as necroptosis (Sun X et al. 2002). In addition, the large carboxyl-terminal region of HSV RIR1 protein mediates the binding to caspase 8 (CASP8) (Dufour F et al. 2011; Guo H et al. 2015). HSV RIR1 is thought to block necroptosis in infected human cells by interactions with RIPK1, RIPK3 and CASP8 (Guo H et al. 2015; Mocarski ES et al. 2015).
R-HSA-9687458 (Reactome) During infection in human cells, herpes simplex virus 1 (HSV1) and HSV2 modulate cell death pathways using the large subunit (R1) of viral ribonucleotide reductase (RIR1 or UL39) proteins (Dufour F et al. 2011; Guo H et al. 2015; Yu X et al. 2016; Ali M et al. 2019). The HSV1 and HSV2 RIR1 proteins suppress death receptor-dependent apoptosis by interacting with death effector domains of caspase 8 (CASP8) via a conserved C-terminal ribonucleotide reductase (RNR) domain (Dufour F et al. 2011). The ability of HSV1 RIR1 and HSV2 RIR1 to bind CASP8 is integral to their suppression activity against necroptosis in human cells. Necroptosis complements apoptosis as a host defense pathway to stop virus infection and is mediated by the interaction between receptor�interacting protein kinase 1 (RIPK1) and RIPK3 that occurs downstream of tumor necrosis factor receptor 1 (TNFR1) activation during the programmed cell death response (Sun X et al. 2002). The N-terminal region of HSV1 and HSV2 RIR1 proteins carrying the RIP homotypic interaction motif (RHIM)-like element is sufficient for RHIM-dependent interaction with RIPK1 and RIPK3 thus inhibiting the interaction between RIPK1 and RIPK3 (Guo H et al. 2015; Yu X et al. 2015). HSV1 RIR1 and HSV2 RIR1 are thought to block the programmed cell death responses in infected human cells by interactions with RIPK1, RIPK3 and CASP8 (Guo H et al. 2015; Mocarski ES et al. 2015).
R-HSA-9687465 (Reactome) Necroptosis complements apoptosis as a host defense pathway to stop virus infection. During infection in human cells, herpes simplex virus (HSV)-1 and HSV-2 modulate cell death pathways using the large subunit (R1) of viral ribonucleotide reductase (RIR1 or UL39) (Dufour F et al. 2011; Guo H et al. 2015; Yu X et al. 2016; Ali M et al.2019). The N-terminal region of RIR1 protein carrying the RIP homotypic interaction motif (RHIM)-like element is sufficient for RHIM-dependent interaction with receptor�interacting protein kinase 1 (RIPK1) and receptor�interacting protein kinase 3 (RIPK3) thus inhibiting the interaction between RIPK1 and RIPK3 (Guo H et al. 2015; Yu X et al. 2015). An intact RHIM is required for the interaction between RIPK1 and RIPK3 that occurs downstream of tumour necrosis factor receptor 1 (TNFR1) activation during the programmed cell death response known as necroptosis (Sun X et al. 2002). In addition, the large carboxyl-terminal region of HSV RIR1 protein mediates the binding to caspase 8 (CASP8) (Dufour F et al. 2011; Guo H et al. 2015). HSV RIR1 is thought to block necroptosis in infected human cells by interactions with RIPK1, RIPK3 and CASP8 (Guo H et al. 2015; Mocarski ES et al. 2015).
R-HSA-9687625 (Reactome) Recombinant pseudokinase mixed lineage kinase domain-like (MLKL) exists in both monomeric form (∼30% of total) and tetrameric form (∼70% of total) in solution (Petrie EJ et al. 2018). Higher order oligomersof MLKL mediates necroptosis, an inflammatory form of programmed cell death executed through plasma membrane rupture (Wang H et al. 2014; Dondelinger Y et al. 2014; Tanzer MC et al. 2016; Petrie EJ et al. 2018; Samson AL et al. 2020). Biophysics, mass spectrometry (MS) and cellular assays revealed that the pseudokinase domain of human MLKL functions as a molecular switch in directing the transition of MLKL from a basal monomeric state to a pro-necroptotic tetramer (Petrie EJ et al. 2018). Despite lacking catalytic activity, the pseudokinase domain of MLKL has retained the ability to bind ATP (Murphy JM et al. 2013, 2014; Petrie EJ et al. 2018). The ATP binding has been shown to negatively regulate MLKL-mediated iposome permeabilization by destabilizing the MLKL tetramers and shifting the tetramer:monomer equilibrium toward the monomeric state (Petrie EJ et al. 2018). However, MLKL mutants with defective ATP-binding were still able to induce necroptosis when expressed in MLKL-/- HT-29 cells, suggesting that ATP-binding is most likely reflective of the ancestral function of the pseudokinase domain fold than regulatory in cells (Petrie EJ et al. 2018).
R-HSA-9687638 (Reactome) Metabolites of the inositol phosphate (IP) pathway I(1,3,4,6)P4, I(1,3,4,5,6)P5, and IP6 promote membrane permeabilization mediated by the pseudokinase mixed lineage kinase domain-like (MLKL) through directly binding the N‑terminal four-helical bundle (4HB) domain and dissociating its auto-inhibitory region (Dovey CM et al. 2018; McNamara DE et al. 2019). This is consistent with the findings that inositol polyphosphate kinases (IPK) IPMK and ITPK1 are essential regulators of MLKL-mediated necroptosis in a forward genetic screen performed with the human haploid cell line HAP1 (Dovey CM et al. 2018). Subsequent genetic deletion of IPK genes IPMK, ITPK1 and IPPK of the IP code metabolic pathway blocked MLKL-mediated necroptosis in human colon adenocarcinoma HT-29 cells (Dovey CM et al. 2018; McNamara DE et al. 2019). Activating IPs bind three sites on MLKL with affinity of 100-600 μM to destabilize contacts between the auto-inhibitory region and NED of MLKL. This liberates NED, promoting oligomerization and activation of MLKL (McNamara DE et al. 2019).
R-HSA-9687828 (Reactome) Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) plays an integral role in mediating a pro-inflammatory form of cell death, termed necroptosis. RIPK3-dependent signaling is tightly regulated by post-translational modifications, including proteolysis, phosphorylation and ubiquitylation. O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT), a key enzyme for protein O-GlcNAcylation, was found to limit RIPK3 kinase-mediated inflammation and necroptosis in lipopolysaccharide (LPS)-stimulated mouse bone marrow-derived macrophages (BMM) and human monocyte-like THP-1 cells (Li X et al. 2019). Genetic deletion of Ogt in myeloid cells markedly exacerbated cytokine storm and host mortality in experimental sepsis in mice (Li X et al. 2019). Further, impaired O-GlcNAc signaling in patients with liver cirrhosis and in mice with ethanol-induced liver injury exhibited a significant increase in the level of phosphorylated mixed lineage kinase domain-like (MLKL) (Zhang B et al. 2019). OGT utilizes uridine diphosphate (UDP)-GlcNAc to catalyze O-linked attachment of a single GlcNAc to serine or threonine residues in target proteins. Co-immunoprecipitation assay showed that RIPK3 directly interacts with OGT in human embryonic kidney 293T (HEK293T) cells that were transfected with the tagged RIPK3 and OGT proteins (Li X et al. 2019; Zhang B et al. 2019). Further, both RIPK3 O-GlcNAcylation and the association between RIPK3 and OGT increased upon LPS stimulation in mouse BMM cells, despite attenuated total protein O-GlcNAcylation, which suggests that OGT actively and specifically promotes RIPK3 O-GlcNAcylation in response to LPS. In addition, upon LPS challenge, Ogt-deficient BMMs produced significantly higher amounts of inflammatory mediators. Similarly, OGT-deficient human THP-1 cells increased cytokine production in response to TLR2 (Pam3Cys), TLR4 (LPS) or TLR9 (CpG) agonists, suggesting that OGT negatively regulates cytokine production both in mouse and human cells (Li X et al. 2019). Thiamet-G (TMG), that increased intracellular O-GlcNAc levels, effectively shortened the half-life of RIPK3 in human non-small cell lung carcinoma cell line derived from the lymph node (H1299) as compared with the vehicle control suggesting that O-GlcNAcylation of RIPK3 decreases its protein stability (Zhang B et al. 2019). Truncations of human RIPK3 in conjunction with mass spectrometry and subsequent site-directed mutagenesis pinpointed the site of O-GlcNAcylation as residue T467 within the RIP homotypic interaction motif (RHIM) domain of RIPK3 (Li X et al. 2019). Examination of a RIPK3 T467A mutant, which is resistant to O-GlcNAc modification, confirmed that this modification repressed LPS-induced RIPK3-mediated phosphorylation events, cytokine production, and necroptotic cell death in THP-1 and RIPK3-expressing HEK293T cells (Li X et al. 2019). It should be noted that T467 on human RIPK3 is only partially conserved among mammalian species, suggesting a possibility that additional functional O-GlcNAcylation site(s) could exist in other species (Li X et al. 2019). Further, the O-GlcNAcylation of RIPK3 diminished RIPK1:RIPK3 and RIPK3:RIPK3 RHIM interactions and downstream RIPK3 kinase activation in RIPK3-expressing HEK293T cells. These findings are supported by structural modeling showing that O-GlcNAc modification lies in close proximity to the conserved RHIM VQVG motif and likely perturbs RHIM-mediated protein interaction through steric hinderance. Collectively these data show that OGT targets residue T467 of RIPK3 for O-GlcNAcylation to prevent RIPK3 activation in human cells. These findings demonstrate an immuno-metabolic crosstalk linking the hexosamine biosynthesis pathway (HBP)-associated O-GlcNAc signaling and innate immune cell activation (Li X et al. 2019; Zhang B et al. 2019).

R-HSA-9688456 (Reactome) Upon regulated necrosis, receptor interacting protein kinase 3 (RIPK3) phosphorylates the mixed lineage kinase domain-like (MLKL) protein at Thr357 and Ser358 located in the activation loop of its pseudokinase domain. The RIPK3-mediated phosphorylation relieves the inhibitory effect of the pseudokinase domain of MLKL, thus allowing the activated MLKL to oligomerize and translocate from the cytosol to cell membranes to cause membrane disintegration. The flotillin-mediated endocytosis was found to antagonize MLKL-mediated plasma membrane alteration to sustain survival of the cell (Fan W et al. 2019). Protein cross-linking followed by affinity purification assays detected the lipid raft-associated proteins flotillin-1 (FLOT1) and flotillin-2 (FLOT2) in membrane-localized MLKL immunoprecipitates isolated from human colon cancer HT-29 cells expressing MLKL fused with a 3xFlag-HA (in which MLKL fused with a 3xFlag-HA (hemagglutinin) tagged to its C terminus was expressed while endogenous MLKL was knocked down (Fan W et al. 2019). Mass spectrometry and immunoblotting assay identified showed that MLKL was associated with FLOT1 and FLOT2 within extracellular vesicles (EVs) released from caspase inhibitor z-VAD-fmk-treated HT-29 cells, suggesting that RIPK3 activation triggered MLKL association with flotillins (Yoon S et al. 2017). Phosphorylated MLKL was eventually directed to lysosomes in HT-29 cells immunolabeled with phospho-MLKL and LAMP1 (lysosome-associated membrane protein 1) antibodies (Fan W et al. 2019). Bafilomycin A1 prevented the degradation of phosphorylated MLKL only in parental HT-29 cells but not in cells lacking either flotillin suggesting that flotillins mediated the relocalization of phospho-MLKL from cell membranes to lysosomes (Fan W et al. 2019). However, the other study reported that phosphorylated MLKL did not colocalize to a significant extent with LAMP-2-containing lysosomes, and inhibiting lysosomal degradation during the effector phase of necroptosis did not significantly alter the extent of necroptotic cell death (Samson AL et al. 2020). Binding between the N-terminal helix bundle of phospho-MLKL and a C-terminal region of flotillin-1 was required for flotillin-mediated endocytosis of phosphorylated MLKL (Fan W et al. 2019). In addition, mice with a double knockout of Flot1 and Flot2 showed accelerated death upon injection with TNFα plus z-VAD-fmk to induce systemic inflammatory response syndrome (SIRS). Moreover, all flotillin-null mice survived TNFα/z-VAD-fmk-induced SIRS when coinjected with the RIPK1 inhibitor RIPA-56, confirming that the accelerated death in flotillin-null mice resulted from enhanced necroptosis (Fan W et al. 2019). Thus, phosphorylated MLKL is removed from membranes through FLOT1, 2-mediated endocytosis followed by lysosomal degradation (Fan W et al. 2019).
R-HSA-9688459 (Reactome) Receptor-interacting serine/threonine protein kinase 3 (RIPK3 or RIP3) activation following the induction of necroptosis in human HT-29 colorectal adenocarcinoma cells requires the activity of a heat-shock protein 90 (HSP90) and cell division cycle 37 (CDC37) cochaperone complex (Li D et al. 2015). This complex physically associates with RIPK3. Chemical inhibitors of HSP90 efficiently block necroptosis by preventing RIP3 activation in HT-29. Cells with knocked down CDC37 were unable to respond to necroptosis stimuli (Li D et al. 2015). Further, geldanamycin, an inhibitor of HSP90, and siRNA/shRNA of HSP90α, protected cultured neurons from oxygen-glucose deprivation induced necroptosis by decreasing RIP3 expression (Wang Z et al. 2018). Geldanamycin was reported to nhibit programmed necrosis by suppressing RIPK3 protein expression (Cho YS et al. 2009).Moreover, the HSP90:CDC37 complex was found to interact and regulate the stability of mixed lineage kinase domain-like (MLKL), the downstream effector of the necroptotic signalling pathway (Bigenzahn JW et al. 2016; Jacobsen AV et al. 2016; Zhao XM et al. 2016).
R-HSA-9688831 (Reactome) STUB1 (CHIP) is a cochaperone E3 ligase containing three tandem repeats of tetratricopeptide (TPR) motifs and a C-terminal U-box domain separated by a charged coiled-coil region (Paul I & Ghosh MK 2014). STUB1 functions as a negative co-chaperone for the HSP90/HSP70 chaperone to regulate protein quality control by targeting unfolded or misfolded proteins for proteasomal degradation. STUB1 (CHIP) also targets many mature proteins for ubiquitination and degradation or degradation-independent regulation (Paul I & Ghosh MK 2014). Structural studies suggest that STUB1 functions as homodimer (Zhang M et al. 2005)

Receptor-interacting serine/threonine protein kinase 3 (RIPK3) functions as a key regulator of necroptosis. STUB1, as an E3 ligase, mediates ubiquitylation of RIPK3 at Lys55 and Lys363 and targets it to lysosomal degradation in human non-small cell lung carcinoma (H1299) cells (Seo J et al. 2016). Treatment with geldanamycin (an inhibitor of HSP90) induced the degradation of RIPK3 in mouse fibroblasts L929 cells even under STUB1-depleted conditions, suggesting that HSP90 might not be involved in the STUB1-mediated degradation of RIPK3 (Seo J et al. 2016). Further, RIP3 kinase activity was not required for its interaction with STUB1 in human embryonic kidney 293 (HEK293) cells, suggesting that STUB1-mediated regulation is independent of RIPK3 phosphorylation status (Choi SW et al. 2018). Moreover, Chip(-/-) mouse embryonic fibroblasts, CHIP-depleted L929 and human colorectal adenocarcinoma (HT-29) cells exhibited higher levels of RIPK3 expression, resulting in increased sensitivity to necroptosis induced by TNFα (Seo J et al. 2016). Supporting these findings, in vivo studies demonstrated that the inflammatory and lethal phenotypes of Chip−/− mice were rescued by crossing with Ripk3 knockout mice (Seo J et al. 2016). The ubiquitin E3 ligase function of STUB1 was also essential for the degradation of RIPK3 in mouse neuroblastoma N2a cell line. Ansiomycin, an inhibitor of protein synthesis, attenuated necroptosis by upregulating STUB1 in oxygen-glucose deprivation (OGD)-challenged N2a cells and primary cultured mouse hippocampal neurons (Tang MB et al. 2018). These data suggest that STUB1 (CHIP) can negatively regulate necroptosis by ubiquitylation-mediated degradation of RIPK3.

R-HSA-9688832 (Reactome) Upon necroptosis, receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates the mixed lineage kinase domain-like (MLKL) protein at Thr357 and Ser358 located in the activation loop of pseudokinase domain. The RIPK3-mediated phosphorylation relieves the inhibitory effect of the pseudokinase domain of MLKL, thus allowing the activated MLKL to oligomerize and translocate from the cytosol to cell membranes to cause membrane disintegration. Various studies showed that the endosomal sorting complexes required for transport (ESCRT) pathway can remove phosphorylated MLKL-containing membrane vesicles from cells undergoing necroptosis, thereby attenuating the cell death process (Gong YN et al. 2017; Yoon S et al. 2017; Fan W et al. 2019). The ESCRT-associated proteins, programmed cell death 6-interacting protein (PDCD6IP or ALG-2-interacting protein X, ALIX) and syntenin-1 (SDCBP), were found to mediate exocytosis of MLKL antagonizing MLKL-mediated plasma membrane alteration (Fan W et al. 2019). Protein cross-linking followed by affinity purification assays detected PDCD6IP and SDCBP in immunoprecipitates of membrane-localized MLKL isolated from human HT-29 colon cancer cells expressing a 3xFlag-HA (hemagglutinin)-tagged MLKL (Fan W et al. 2019). Knockdown of either PDCD6IP or SDCBP reduced the levels of phospho-MLKL in the exosome fractions collected from the culture medium of caspase inhibitor z-VAD-fmk-treated HT-29 cells, suggesting that phosphorylated MLKL was eventually removed from membranes through PDCD6IP:SDCBP-mediated exocytosis. Mass spectrometry and immunoblotting showed that MLKL was associated with some components of ESCRT system within extracellular vesicles (EVs) released from caspase inhibitor z-VAD-fmk-treated HT-29 cells (Yoon S et al. 2017). Further, PDCD6IP was shown to bind tumor susceptibility gene 101 (TSG101 also known as VPS23, vacuolar protein sorting 23), the ESCRT-I subunit protein (von Schwedler UK et al. 2003; Okumura M et al. 2009). Knockdown of TSG101 prevented the exocytosis of phosphorylated MLKL in HT-29 cells, further confirming that mediated exocytosis of phospho-MLKL depends on the ESCRT pathway (Fan W et al. 2019).

SDCBP (syntenin-1) interacts directly with PDCD6IP (ALIX) through three LYPX(n)L motifs located in its N-terminus and with the conserved cytoplasmic domains of the syndecans, via its PDZ domains (Baietti MF et al. 2012). Syndecans are a family of proteins that by virtue of their extracellular heparan sulfate chains interact with a plethora of signaling and adhesion molecules (Sarrazin S et al. 2011). Since PDCD6IP binds several ESCRT proteins, PDCD6IP:SDCBP adapts syndecans and syndecan cargo to the ESCRT budding machinery, playing a role in membrane budding and scission at the endosome and generating intraluminal vesicles (ILVs) that are released as exosomes when multivesicular endosomes fuse with the plasma membrane (Baietti MF et al. 2012 ). Exocytosis was proposed to counteract the effector phase of necroptosis via ESCRT-, PDCD6IP:SDCBP- or RAB27A/B-mediated expulsion of MLKL-containing bubbles to diminish the MLKL residing at the plasma membrane (Gong YN et al. 2017; Yoon S et al. 2017; Fan W et al. 2019). However, the other study suggests that membrane bubbling during necroptosis is an incredibly dynamic and heterogenous phenomenon with protrusions variously extending, retracting and shedding in a fashion seemingly independent of the primary sites of MLKL accumulation and membrane damage (Samson AL et al. 2020).

R-HSA-9688838 (Reactome) Receptor-interacting serine/threonine protein kinase 3 (RIPK3) functions as a key regulator of necroptosis.The protein stability of RIPK3 is negatively regulated by the C-terminus of HSC70-interacting protein (CHIP, also known as STIP1 homology and U-Box containing protein 1, STUB1) (Seo J et al. 2016). STUB1, as an E3 ligase, mediates ubiquitylation of RIPK3 at Lys55 and Lys363 and targerts it to lysosomal degradation. Coimmunoprecipitation analysis using overexpressed, endogenous or recombinant proteins revealed interactions between STUB1 (CHIP) and RIPK3 in human embryonic kidney 293T (HEK293T) cells (Seo J et al. 2016). Domain mapping revealed that the kinase domain of RIPK3 interacts with the tetratricopeptide repeat (TPR) region of STUB1 (Seo J et al. 2016). Treatment with geldanamycin (an inhibitor of HSP90) induced the degradation of RIPK3 in mouse fibroblasts L929 cells even under STUB1-depleted conditions, suggesting that HSP90 might not be involved in the STUB1-mediated degradation of RIPK3 (Seo J et al. 2016).
R-HSA-9693978 (Reactome) Several FDA-approved anticancer drugs, including sorafenib, pazopanib and ponatinib showed anti-necroptotic activity (Fauster A et al. 2015; Martens S et al. 2017; Fulda S 2018). Sorafenib was identified as an inhibitor of necroptosis using a high-content screening of FDA-approved drug libraries and small compounds (Martens S et al. 2017). Sorafenib has been demonstrated to block kinase activity of both receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3 by targeting their inactive states (Martens S et al. 2017). In pull-down experiments, biotinylated sorafenib has been found to directly interact with RIPK1, RIPK3 and MLKL (Martens S et al. 2017). Consequently, sorafenib rescued murine as well as human cell lines from TNFα-stimulated necroptosis (Martens S et al. 2017). Also, sorafenib can protect apoptosis-resistant acute myeloid leukemia (AML) cells from second mitochondria-derived activator of caspases (SMAC) mimetic-induced necroptosis, including primary, patient-derived AML blasts (Feldmann F et al. 2017). Importantly, sorafenib has been shown to provide protection in two in vivo models of necroptosis, that is in renal ischemia-reperfusion injury and in TNF-induced systemic inflammatory response syndrome (Martens S et al. 2017). Sorafenib was unable to inhibit necroptosis in mouse dermal fibroblasts (MDFs) (Hildebrand JM et al. 2014). In addition to sorafenib, a cellular screen with FDA-approved drugs identified pazopanib and ponatinib as necroptosis inhibitors that suppressed necroptosis in human cells at submicromolar EC50 concentrations (Fauster A et al. 2015). Both drugs inhibited necroptotic signaling triggered by various cell death receptors, whereas they did not interfere with apoptosis. Ponatinib and pazopanib abrogated phosphorylation of MLKL upon TNF-α-induced necroptosis in human adenocarcinoma HT-29 cells, suggesting that both agents target a component upstream of MLKL (Fauster A et al. 2015). RIPK1 has been identified as the main functional target of pazopanib, while ponatinib directly targeted both RIPK1 and RIPK3 (Fauster A et al. 2015; Najjar M et al. 2015).
R-HSA-9698677 (Reactome) Human cytomegalovirus (HCMV) protein pUL36 was found to bind mixed lineage kinase domain-like protein (MLKL) and target MLKL for degradation in HCMV-infected TERT-immortalized primary human fetal foreskin fibroblasts (HFFF-TERTs) (Fletcher-Etherington A et al. 2020). Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis in HFFF-TERTs. The same residue was also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, suggesting that pUL36 acts as a multifunctional inhibitor of both apoptotic and necroptotic cell death (Fletcher-Etherington A et al. 2020; Skaletskaya A et al. 2001).
R-HSA-9698844 (Reactome) The pseudokinase mixed lineage kinase domain-like (MLKL), is the terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by receptor interacting protein kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. Heat‑shock protein 90 (HSP90) and cell division cycle 37 (CDC37) cochaperone complex contributes to activation of MLKL (Jacobsen AV et al. 2016; Zhao XM et al. 2016; Bigenzahn JW et al. 2016). Whether HSP90 exerts its effects on MLKL folding, oligomerization, or translocation to membranes has not been precisely determined, although it is plausible HSP90 impacts each of these activation steps (Murphy JM 2020).
RIP1:RIP3:MLKL oligomer:PIPsArrowR-HSA-5620975 (Reactome)
RIPK1(1-324)ArrowR-HSA-5357828 (Reactome)
RIPK1:RIPK3:sorafenib, ponatinib, pazopanibArrowR-HSA-9693978 (Reactome)
RIPK1:RIPK3:sorafenib, ponatinib, pazopanibTBarR-HSA-5213464 (Reactome)
RIPK1:RIPK3:sorafenib, ponatinib, pazopanibTBarR-HSA-5213466 (Reactome)
RIPK1:RIPK3ArrowR-HSA-5213462 (Reactome)
RIPK1:RIPK3R-HSA-5213464 (Reactome)
RIPK1:RIPK3R-HSA-9693978 (Reactome)
RIPK1:RIPK3mim-catalysisR-HSA-5213464 (Reactome)
RIPK1:RIPK3mim-catalysisR-HSA-5213466 (Reactome)
RIPK1R-HSA-5213462 (Reactome)
RIPK1R-HSA-9687465 (Reactome)
RIPK3(1-328)ArrowR-HSA-9686930 (Reactome)
RIPK3(329-518)ArrowR-HSA-9686930 (Reactome)
RIPK3:HSP90:CDC37ArrowR-HSA-9688459 (Reactome)
RIPK3:STUB1ArrowR-HSA-9688838 (Reactome)
RIPK3:STUB1R-HSA-9688831 (Reactome)
RIPK3:STUB1mim-catalysisR-HSA-9688831 (Reactome)
RIPK3R-HSA-5213462 (Reactome)
RIPK3R-HSA-9686930 (Reactome)
RIPK3R-HSA-9687455 (Reactome)
RIPK3R-HSA-9687828 (Reactome)
RIPK3R-HSA-9688459 (Reactome)
RIPK3R-HSA-9688838 (Reactome)
RIR1R-HSA-9687455 (Reactome)
RIR1R-HSA-9687458 (Reactome)
RIR1R-HSA-9687465 (Reactome)
RIR1TBarR-HSA-5213462 (Reactome)
SARS-CoV-1

3a:(RIPK1:RIPK3)

oligomer
ArrowR-HSA-9686345 (Reactome)
SARS-CoV-1

3a:(RIPK1:RIPK3)

oligomer
R-HSA-9686336 (Reactome)
SDCBPR-HSA-9688832 (Reactome)
STUB1:STUB1R-HSA-9688838 (Reactome)
TBarR-HSA-5213462 (Reactome)
TBarR-HSA-5620975 (Reactome)
TRADD:TRAF2:RIPK1:FADDR-HSA-5357828 (Reactome)
TRAF2:TRADD:RIPK1(325-671):FADDArrowR-HSA-5357828 (Reactome)
UDP-GlcNAcR-HSA-9687828 (Reactome)
UDPArrowR-HSA-9687828 (Reactome)
UL36R-HSA-9698677 (Reactome)
Ub-K55,363-RIPK3:STUB1ArrowR-HSA-9688831 (Reactome)
UbR-HSA-9688831 (Reactome)
active

caspase-8:viral

CRMA/SPI-2
ArrowR-HSA-2672196 (Reactome)
active caspase-8R-HSA-2672196 (Reactome)
active caspase-8TBarR-HSA-5213462 (Reactome)
active caspase-8mim-catalysisR-HSA-5357828 (Reactome)
active caspase-8mim-catalysisR-HSA-9686930 (Reactome)
p-S-RIPK1:RIPK3ArrowR-HSA-5213464 (Reactome)
p-S-RIPK1:RIPK3R-HSA-5213466 (Reactome)
p-S-RIPK1:p-S199,227, K48pUb-363-RIPK3:PELI1ArrowR-HSA-9686920 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomer:4xMLKL:IP6,IP5, IP4ArrowR-HSA-5357927 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomer:4xMLKL:IP6,IP5, IP4ArrowR-HSA-9687638 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomerArrowR-HSA-5218905 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomerR-HSA-5218891 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomerR-HSA-9686345 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:PELI1ArrowR-HSA-9686922 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:PELI1R-HSA-9686920 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:PELI1mim-catalysisR-HSA-9686920 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomer:FLOT1:FLOT2ArrowR-HSA-9688456 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomer:FLOT1:FLOT2TBarR-HSA-5620975 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomer:PDCD6IP:SDCBPArrowR-HSA-9688832 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomerArrowR-HSA-5357927 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomerR-HSA-5620975 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomerR-HSA-9688456 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomerR-HSA-9688832 (Reactome)
p-S166-RIPK1:p-S199,227-RIPK3ArrowR-HSA-5213466 (Reactome)
p-S166-RIPK1:p-S199,227-RIPK3R-HSA-5218905 (Reactome)
p-S166-RIPK1:p-S199,227-RIPK3R-HSA-9686922 (Reactome)
small molecule

inhibitors of

RIPK1, RIPK3
R-HSA-9693978 (Reactome)
viral serpinsR-HSA-2672196 (Reactome)

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