Regulated Necrosis (Homo sapiens)

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5, 9, 113, 3218, 197, 2914, 28810, 17, 20, 3016, 21, 22, 257, 18282, 7, 2916, 22, 25, 264, 12, 263, 32cytosolTRAF2 p-S-RIPK1:p-S199,227-RIPK3 oligomer TNFRSF10A K48pUb-XIAP RIP1:RIP3:MLKLoligomer:PIPsCASP8(1-479) CASP8(385-479) ATPPIPsCFLAR(1-376)TRAF2 FASLG(1-281) DISC:procaspase-8:FLIP(L)TNFRSF10B SPI-2 TRADD p-S199,227-RIPK3 CRMA FASLG(1-281) (p-S-RIPK1:p-S199,227-RIPK3) oligomer:3xp-T357,S358-MLKLp-S-RIPK1 DISC:procaspase-8:FLIP(S)TRAF2:TRADD:RIP1(325-671)CASP8(385-479) PI4P MLKLp-T357,S358-MLKL oligomer FADD TNFRSF10B p-S-RIPK1:p-S199,227-RIPK3 oligomer p-S-RIPK1:p-S199,227-RIPK3ATPADPp-S-RIPK1:p-S199,227-RIPK3 oligomer TNFRSF10A RIPK3 RIPK1:RIPK3PI(4,5)P2 RIPK1 TNFSF10 CASP8(217-374) CFLAR(1-480) FLIP(S)PI4P p-S-RIPK1 TRAF2:TRADD:RIPK1TRADD FAS RIPK1(1-324)PI(3,4,5)P3 BIRC2,3,4TRADD TRADD K48polyUbFADD RIPK1 PI(3,4,5)P3 p-S-RIPK1:p-S199,227-RIPK3 oligomerCASP8(217-374) TRADD RIPK3 RIPK3RIPK1(325-671) FLIP(S) active caspase-8RIPK1 BIRC2 ATPTNFRSF10B K48pUb-BIRC3 TNFRSF10A activecaspase-8:viralCRMA/SPI-2p-T357,S358-MLKL oligomer PI(4,5)P2 FAS K48pUb-BIRC2 TRAF2 RIPK1CASP8(1-479) FASLG(1-281) p-S-RIPK1:RIPK3SPI-2 TRAF2 FADD MLKL (RIPK1:RIPK3)oligomer:3xMLKLCRMA p-T357,S358-MLKL RIPK1 TNFSF10 DISC:procaspase-8TRAF2 TNFSF10 CASP8(1-479) p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomerBIRC3 viral serpinsApoptosisADPp-S-RIPK1:p-S199,227-RIPK3 oligomer ADPXIAP FAS K48pUb- BIRC2,3,4RIPK1 14, 2818292929151, 6, 23, 27, 31...13, 242815281518


Necrosis has traditionally been considered as a passive, unregulated cell death. However, accumulating evidence suggests that necrosis, like apoptosis, can be executed by genetically controlled and highly regulated cellular process that is morphologically characterized by a loss of cell membrane integrity, intracellular organelles and/or the entire cell swelling (oncosis) (Rello S et al. 2005; Galluzzi L et al. 2007; Berghe TV et al. 2014). The morphological hallmarks of the nectotic death have been associated with different forms of programmed cell death including (but not limited to) parthanatos, necroptosis, glutamate-induced oxytosis, ferroptosis, inflammasome-mediated necrosis etc. Each of them can be triggered under certain pathophysiological conditions. For example UV, ROS or alkylating agents may induce poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation (parthanatos), while tumor necrosis factor (TNF) or toll like receptor ligands (LPS and dsRNA) can trigger necrosome-mediated necroptosis. The initiation events, e.g., PARP1 hyperactivation, necrosome formation, activation of NADPH oxidases, in turn trigger one or several common intracellular signals such as NAD+ and ATP-depletion, enhanced Ca2+ influx, dysregulation of the redox status, increased production of reactive oxygen species (ROS) and the activity of phospholipases. These signals affect cellular organelles and membranes leading to osmotic swelling, massive energy depletion, lipid peroxidation and the loss of lysosomal membrane integrity. Regulated or programmed necrosis eventually leads to cell lysis and release of cytoplasmic content into the extracellular region that is often associated with a tissue damage resulting in an intense inflammatory response.

The Reactome module describes necroptosis as the most characterized form of regulated necrosis. The molecular mechanisms behind the other types of regulated necrosis as well as interconnectivity among them need further studies. View original pathway at:Reactome.</div>


Pathway is converted from Reactome ID: 5218859
Reactome version: 66
Reactome Author 
Reactome Author: Shamovsky, Veronica

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Ontology Terms



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  14. He S, Wang L, Miao L, Wang T, Du F, Zhao L, Wang X.; ''Receptor interacting protein kinase-3 determines cellular necrotic response to TNF-alpha.''; PubMed Europe PMC
  15. Chinnaiyan AM, O'Rourke K, Tewari M, Dixit VM.; ''FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis.''; PubMed Europe PMC
  16. Pop C, Oberst A, Drag M, Van Raam BJ, Riedl SJ, Green DR, Salvesen GS.; ''FLIP(L) induces caspase 8 activity in the absence of interdomain caspase 8 cleavage and alters substrate specificity.''; PubMed Europe PMC
  17. Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS.; ''Target protease specificity of the viral serpin CrmA. Analysis of five caspases.''; PubMed Europe PMC
  18. Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, Wang X.; ''Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.''; PubMed Europe PMC
  19. Zhao J, Jitkaew S, Cai Z, Choksi S, Li Q, Luo J, Liu ZG.; ''Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis.''; PubMed Europe PMC
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  22. Scaffidi C, Schmitz I, Krammer PH, Peter ME.; ''The role of c-FLIP in modulation of CD95-induced apoptosis.''; PubMed Europe PMC
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  33. MacFarlane M, Williams AC.; ''Apoptosis and disease: a life or death decision.''; PubMed Europe PMC
  34. Kerr JF.; ''History of the events leading to the formulation of the apoptosis concept.''; PubMed Europe PMC


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101551view11:41, 1 November 2018ReactomeTeamreactome version 66
101087view21:24, 31 October 2018ReactomeTeamreactome version 65
100616view19:59, 31 October 2018ReactomeTeamreactome version 64
100167view16:44, 31 October 2018ReactomeTeamreactome version 63
99717view15:11, 31 October 2018ReactomeTeamreactome version 62 (2nd attempt)
99293view12:46, 31 October 2018ReactomeTeamreactome version 62
93852view13:40, 16 August 2017ReactomeTeamreactome version 61
93412view11:22, 9 August 2017ReactomeTeamreactome version 61
88132view12:50, 26 July 2016RyanmillerOntology Term : 'programmed cell death pathway' added !
88131view12:50, 26 July 2016RyanmillerOntology Term : 'regulatory pathway' added !
86501view09:19, 11 July 2016ReactomeTeamreactome version 56
83381view11:04, 18 November 2015ReactomeTeamVersion54
81559view13:06, 21 August 2015ReactomeTeamNew pathway

External references


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NameTypeDatabase referenceComment
(RIPK1:RIPK3)oligomer:3xMLKLComplexR-HSA-5218909 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:3xp-T357,S358-MLKLComplexR-HSA-5218902 (Reactome)
ADPMetaboliteCHEBI:16761 (ChEBI)
ATPMetaboliteCHEBI:15422 (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(217-374) ProteinQ14790 (Uniprot-TrEMBL)
CASP8(385-479) ProteinQ14790 (Uniprot-TrEMBL)
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)
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)
MLKLProteinQ8NB16 (Uniprot-TrEMBL)
PI(3,4,5)P3 MetaboliteCHEBI:16618 (ChEBI)
PI(4,5)P2 MetaboliteCHEBI:18348 (ChEBI)
PI4P MetaboliteCHEBI:17526 (ChEBI)
PIPsComplexR-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:RIPK3ComplexR-HSA-5218862 (Reactome)
RIPK1ProteinQ13546 (Uniprot-TrEMBL)
RIPK3 ProteinQ9Y572 (Uniprot-TrEMBL)
RIPK3ProteinQ9Y572 (Uniprot-TrEMBL)
SPI-2 ProteinP15059 (Uniprot-TrEMBL)
TNFRSF10A ProteinO00220 (Uniprot-TrEMBL)
TNFRSF10B ProteinO14763 (Uniprot-TrEMBL)
TNFSF10 ProteinP50591 (Uniprot-TrEMBL)
TRADD ProteinQ15628 (Uniprot-TrEMBL)
TRAF2 ProteinQ12933 (Uniprot-TrEMBL)
TRAF2:TRADD:RIP1(325-671)ComplexR-HSA-5357809 (Reactome)
TRAF2:TRADD:RIPK1ComplexR-HSA-140935 (Reactome)
XIAP ProteinP98170 (Uniprot-TrEMBL)


ComplexR-HSA-2672221 (Reactome)
active caspase-8ComplexR-HSA-2562550 (Reactome)
p-S-RIPK1 ProteinQ13546 (Uniprot-TrEMBL)
p-S-RIPK1:RIPK3ComplexR-HSA-5218868 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomer R-HSA-5218908 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3 oligomerR-HSA-5218908 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3:p-T357,S358-MLKL oligomerComplexR-HSA-5357794 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3ComplexR-HSA-5218870 (Reactome)
p-S199,227-RIPK3 ProteinQ9Y572 (Uniprot-TrEMBL)
p-T357,S358-MLKL ProteinQ8NB16 (Uniprot-TrEMBL)
p-T357,S358-MLKL oligomer R-HSA-5357857 (Reactome)
viral serpinsComplexR-NUL-2672224 (Reactome)

Annotated Interactions

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SourceTargetTypeDatabase referenceComment
(RIPK1:RIPK3)oligomer:3xMLKLArrowR-HSA-5218891 (Reactome)
(RIPK1:RIPK3)oligomer:3xMLKLR-HSA-5218906 (Reactome)
(RIPK1:RIPK3)oligomer:3xMLKLmim-catalysisR-HSA-5218906 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:3xp-T357,S358-MLKLArrowR-HSA-5218906 (Reactome)
(p-S-RIPK1:p-S199,227-RIPK3) oligomer:3xp-T357,S358-MLKLR-HSA-5357927 (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)
BIRC2,3,4R-HSA-5675470 (Reactome)
BIRC2,3,4TBarR-HSA-5213462 (Reactome)
BIRC2,3,4mim-catalysisR-HSA-5675470 (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)
K48pUb- BIRC2,3,4ArrowR-HSA-5675470 (Reactome)
K48polyUbR-HSA-5675470 (Reactome)
MLKLR-HSA-5218891 (Reactome)
PIPsR-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 inhibits 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-lives of FLIP(S) by inhibiting its polyubiquitination (Kaunisto A et al. 2009).

R-HSA-5213462 (Reactome) When caspase-8 (CASP8) activity is inhibited, receptor-interacting protein 1 and 3 (RIPK1 and RIPK3) form a complex also known as the necrosome (Sun X et al. 2002; Li J et al. 2012). The RIP homotypic interaction motifs (RHIMs) of RIPK1 and RIPK3 mediate their interaction.

RIPK3 was found to be essential for the regulated necrosis. RIPK3 knockdown in human colorectal adenocarcinoma (HT-29) cell line, that stably expressed a shRNA targeting RIPK3, lead 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 cell line HeLa led to increased caspase-independent TLR3-induced cell death in the absence of IAPs (Feoktistova M et al. 2011).

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 pronectrotic 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 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 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). However, site-directed mutagenesis revealed that alanine substitution of individual serine residues in the kinase domain of RIP1 had little effects on RIP1 kinase activity and TNF-induced programmed necrosis (McQuade T et al. 2013). At the same time, Ser89 was identified as an inhibitory phospho-acceptor site that may reduce RIP1 kinase activity to limit RIP1-dependent programmed necrosis (McQuade T et al. 2013). Thus, the biological role of phosphorylation on the serine residues in the kinase domain of RIPK1 remain to be further characterized.

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. 2001; 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. 2001).?

R-HSA-5213466 (Reactome) RIPK1:RIPK3 complex formation further potentiates kinase activation through autophosphorylation and/or cross-phosphorylation, propagating the pronecrotic signal. RIPK1, RIPK3 and their kinase activities were shown to be essential for regulated necrosis (Degterev A et al. 2008; Cho YS et al. 2009). RIPK3 kinase-dead mutant (K50A) was found to function as a dominant negative mutant, which blocked TNF-alpha induced necrotic pathway in human colorectal adenocarcinoma HT-29 cells (He S et al. 2009). Mutation of the RIP homotypic interaction motif (RHIM) of RIP3 abrogated RIP1 phosphorylation by RIPK3, suggesting that RIPK1 phosphorylation by RIP3 is dependent on the formation of a RIP:RIP3 complex (Sun X et al. 2001).

Phosphorylation on Ser227 is thought to mediate recruitment and activation of mixed-lineage kinase domain-like (MLKL), a crucial downstream substrate of RIP3 in the necrosis pathway (Sun et al. 2012; Chen et al. 2013).

R-HSA-5218891 (Reactome) Mixed lineage kinase domain-like protein (MLKL) was identified as an essential checkpoint for programmed necrosis. Knockdown of MLKL by shRNA in human colon adenocarcinoma (HT29) or gastric cancer MKN45 cells inhibited TNF alpha-induced necrosis (Sun L et al. 2012; Zhao J et al. 2012; Wang H et al. 2014). Treatment with the MLKL chemical inhibitor necrosulfonamide (NSA) also inhibited TNF-induced necrotic cell death in a variety of human cell lines, e.g., HT29 , FADD-null T cell leukemia Jurkat cells, pancreatic cancer Panc-1 cells (Sun L et al. 2012). As in humans, knockdown of MLKL or inhibition by NSA blocks TNF-induced necroptosis in mouse L929 fibrosarcoma cells (Sun L et al. 2012; Zhao J et al. 2012; Remijsen Q et al. 2014).

MLKL was found to interact with receptor interacting protein kinase 3 (RIPK3) when co-expressed in human embryonic kidney 293 (HEK293) cells (Zhao J et al. 2012). The C-terminal kinase-like domain of MLKL is responsible for association with RIPK3 (Sun L et al. 2012). MLKL-bound necrosome complex was reported to translocate to cell membrane systems (Wang Z et al. 2012; Chen X et al. 2014; Cai Z et al. 2014; Wang H et al. 2014). Translocation of MLKL to lipid rafts of the plasma membrane is thought to induce membrane permeability with subsequent loss of ionic homeostasis, which increases osmotic pressure, eventually leading to membrane rupture (Cai Z et al. 2014; Dondelinger Y et al. 2014).

Even though the Reactome annotation shows that 3 molecules of MLKL bind to the RIPK1:RIPK3 oligomer, the exact stoichiometry of the binding and the oligomerization of MLKL itself remains unclear (Chen X et al. 2014; Cai Z et al. 2014).

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). 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).
R-HSA-5218906 (Reactome) RIPK3 was shown to activate mixed lineage kinase domain-like protein (MLKL) by phosphorylation of the threonine 357 and serine 358 residues within the kinase-like domain (Sun L et al. 2012: Wang H et al. 2014). Knocking down of RIPK3 expression in human colon adenocarcinoma (HT29) cell line blocked MLKL phosphorylation (Wang H et al. 2014). These phosphorylation events are critical for necrosis (Sun L et al. 2012; Cai Z et al. 2014; Wang H et al. 2014). Phosphorylation of MLKL is thought to induce a monomer-to-oligomer transition followed by translocation to the membrane-containing compartments (Cai Z et al. 2014; Wang H et al. 2014).

The Reactome event shows that 3 molecules of MLKL are bound to RIPK1:RIPK3 oligomer however the exact stoichiometry of MLKL binding remains unclear.

R-HSA-5357828 (Reactome) Receptor-interacting protein 1 (RIP1 or RIPK1) can be a part of cell death and survival signaling complexes. Whether RIP1 functions in apoptosis, necroptosis or NFkB signaling is dependent on autocrine/paracrine signals, on the cellular context and tightly regulated posttranslational modifications of RIP1 itself. Pro-survival function of RIP1 is achieved by K63-polyubiquitination which is required for recruitment of signaling molecules/complexes such as the IKK complex and the TAB2:TAK1 complex to mediate activation of NFkB 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 regulated necrosis (Lin Y et al. 1999; Hopkins-Donaldson S et al. 2000). 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-5357927 (Reactome) Mixed lineage kinase domain-like protein (MLKL) was found to form oligomers (detected as homotrimers) when it was overexpressed in human embryonic kidney 293 (HEK293) cells (Cai Z et al. 2014). The oligomerization of MLKL was also observed in multiple human (colon adenocarcinoma HT29, FADD-null Jurkat cells, leukemic monocyte lymphoma U937) and mouse cells upon (TNF+Smac mimetic+caspase inhibitor z-VAD-FMK)-induced necroptosis (Cai Z et al. 2014; Chen X et al. 2014). Whether MLKL forms trimers, tetramers or higher molecular structures, remains to be clarified (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014). The oligomerization of MLKL was blocked by reducing agents such as dithiothreitol or beta-mercaptoethanol, suggesting that oligomeric forms are probably stabilized by disulphide bonds (Cai Z et al. 2014; Wang H et al. 2014). Oligomeric MLKL was not detected in cell lysates of HEK293 cells that were transfected with kinase-dead RIPK3 and MLKL (Cai Z et al. 2014). Mutations in phosphorylation sites of MLKL also blocked MLKL oligomerization (Cai Z et al. 2014; Wang H et al. 2014). Moreover, gel filtration chromatography analysis of recombinant wild-type or phosphorylation site mutant MLKL proteins is in agreement with immunoblotting data, confirming that RIPK3-mediated phosphorylation of MLKL promotes oligomerization of MLKL (Wang H et al. 2014). Oligomers of MLKL translocate to membrane compartments(Cai Z et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014).

Even though the stoichiometry of the MLKL oligomerization in the Reactome event depicts MLKL homotrimer, it remains unclear whether MLKL forms trimers, tetramers or higher molecular structure (Cai Z et al. 2014; Chen X et al. 2014; Dondelinger Y et al. 2014; Wang H et al. 2014).

R-HSA-5620975 (Reactome) Activated by phosphorylation mixed lineage kinase domain-like protein (MLKL) was found to translocate to lipid rafts of 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). Interfering with the formation of PI(5)P or PI(4,5)P2 using PIP inhibitors (such as PIKfyve (P5i)) efficiently inhibited TNFalpha-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 has induced leakage of PIP- or cardiolipin-containing liposomes suggesting that MLKL may have pore-forming capacities to mediate cell death by permeabilizing PIP- or cardiolipin-containing membranes (Dondelinger Y et al. 2014; Wang H et al. 2014). Other study showed that translocation of MLKL to plasma membrane following necroptosis induction in human colon adenocarcinoma HT29 and FADD-null Jurkat cells is associated with Ca2+ influx. MLKL-mediated calcium influx was completely blocked in MLKL-knockdown by shRNA HT29 cells and upon treatment with MLKL chemical inhibitor necrosulfonamide (NSA) (Cai Z et al. 2014). Thus, proposed MLKL functions are (1) binding to and creating pores on the plasma membrane surface and/or (2) regulating ion influx through channels and disturbing the osmotic homeostasis of the cell. Both scenarios lead to the typical cell swelling (“oncosis�) associated with necroptosis.
R-HSA-5675456 (Reactome) Heterodimerization with CFLAR is able to activate CASP8 and -10 because CFLAR uses the same dimer interface between the catalytic domains (Boatright et al. 2004; Dohrman et al. 2005; Micheau et al. 2002; Yu et al. 2009). However, the enzymatic activity of CFLAR:CASP8 heterodimers is insufficient to generate active CASP8 heterotetramers for the apoptosis induction in mammalian cells. In contrary, the residual catalitic activity of CASP8:CFLAR is sufficient for RIP1/RIP3 cleavage, which inhibited the necroptotic cell death mode (Feoktistova M et al. 2011; Dillon CP et al. 2012; Oberst A et al. 2001).
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).
RIP1:RIP3:MLKL oligomer:PIPsArrowR-HSA-5620975 (Reactome)
RIPK1(1-324)ArrowR-HSA-5357828 (Reactome)
RIPK1:RIPK3ArrowR-HSA-5213462 (Reactome)
RIPK1:RIPK3R-HSA-5213464 (Reactome)
RIPK1:RIPK3mim-catalysisR-HSA-5213464 (Reactome)
RIPK1:RIPK3mim-catalysisR-HSA-5213466 (Reactome)
RIPK1R-HSA-5213462 (Reactome)
RIPK3R-HSA-5213462 (Reactome)
TRAF2:TRADD:RIP1(325-671)ArrowR-HSA-5357828 (Reactome)
TRAF2:TRADD:RIPK1R-HSA-5357828 (Reactome)


ArrowR-HSA-2672196 (Reactome)
active caspase-8R-HSA-2672196 (Reactome)
active caspase-8TBarR-HSA-5213462 (Reactome)
active caspase-8mim-catalysisR-HSA-5357828 (Reactome)
p-S-RIPK1:RIPK3ArrowR-HSA-5213464 (Reactome)
p-S-RIPK1:RIPK3R-HSA-5213466 (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: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-RIPK3ArrowR-HSA-5213466 (Reactome)
p-S-RIPK1:p-S199,227-RIPK3R-HSA-5218905 (Reactome)
viral serpinsR-HSA-2672196 (Reactome)

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