| 1999 |
RIP3 (RIPK3) is a novel kinase containing an N-terminal kinase domain homologous to RIP and RIP2, but with a unique C-terminus. RIP3 binds RIP1 through its unique C-terminal segment, is recruited to the TNF receptor-1 signaling complex, and attenuates RIP1- and TNFR1-induced NF-κB activation. Overexpression induces apoptosis via selective binding to large prodomain initiator caspases. |
Co-immunoprecipitation, overexpression, NF-κB reporter assays |
The Journal of biological chemistry |
Medium |
10358032
|
| 2010 |
Murine cytomegalovirus (MCMV) infection induces RIP3-dependent necrosis. RIP3 kinase activity and RHIM-dependent interactions control virus-associated necrosis; this death proceeds independently of RIP1, distinguishing it from TNFα-induced necroptosis. The viral M45-encoded inhibitor vIRA targets RIP3 and disrupts RIP3-RIP1 interactions, suppressing both TNF-induced and virus-induced necroptosis. |
Genetic knockout (RIP3-deficient mice), viral infection models, co-immunoprecipitation, in vivo attenuation studies |
Cell host & microbe |
High |
20413098
|
| 2011 |
Genetic deficiency of RIP3 (RIPK3) prevents spontaneous epithelial cell necrosis, Paneth cell loss, and colitis in FADD-deficient intestinal epithelial cell-specific knockout mice, demonstrating that RIP3-dependent programmed necrosis drives intestinal inflammation when FADD-mediated apoptotic signaling is absent. |
Genetic epistasis (double-knockout mice: FADD IEC-KO × RIP3 KO), histopathology |
Nature |
High |
21804564
|
| 2012 |
Quantitative phosphoproteomic analysis (SILAC) comparing RIP3+/+ and RIP3−/− cells identified a potential RIP3 phosphorylation motif and a broad set of RIP3-regulated phosphoproteins spanning cell cycle, metabolism, and development, suggesting RIP3 kinase activity controls phosphorylation of substrates beyond direct necroptotic effectors. |
Quantitative phosphoproteomics (SILAC + IMAC-HILIC + nanoLC-MS/MS) in macrophages and MEFs |
Molecular & cellular proteomics |
Medium |
22942356
|
| 2013 |
Phosphorylation of RIP3 at S204 creates a phosphomimetic (S204D) that drives programmed necrosis independently of RIP1 and necrostatin-1. Mutation of RIP1 S89 to alanine enhances RIP1 kinase activity and necrosis without affecting necrosome formation, identifying S89 as an inhibitory phosphosite on RIP1 upstream of RIP3. |
Site-directed mutagenesis, cell death assays, siRNA knockdown, kinase activity assays |
The Biochemical journal |
Medium |
24059293
|
| 2014 |
RIP3 dimerization leads to RIP3 intramolecular autophosphorylation; phosphorylation of one RIP3 molecule within a dimer is sufficient for MLKL recruitment and necroptosis induction. RIP1-RIP3 heterodimers alone cannot trigger necroptosis without recruitment of additional RIP3; RIP3-RIP3 homo-interaction is the critical event for necroptosis execution. |
Inducible dimerization systems (chemical-inducible FKBP/rapamycin), phosphorylation assays, MLKL recruitment assays, cell death assays |
Cell death and differentiation |
High |
24902902
|
| 2014 |
RIPK1 both activates RIPK3 (via RHIM-dependent oligomerization in response to TNF signaling) and negatively regulates spontaneous RIPK3 oligomerization in the cytosol. Chemically enforced RIPK3 oligomerization triggers necroptosis independent of RHIM domain, TNF, or RIPK1 kinase activity. Caspase-8 controls propagation/stability of RIPK3 dimers seeded by RHIM interactions. |
Inducible dimerization/oligomerization constructs, RIPK1 KO cells, chemical inhibition of RIPK1, cell death assays |
Cell death and differentiation |
High |
24902904
|
| 2014 |
Three selective RIP3 kinase inhibitors inhibit necroptosis but, at higher concentrations, induce apoptosis via RHIM-driven recruitment of RIP1 to form a RIP3-FADD-caspase-8-cFLIP complex, independent of pronecrotic kinase activities and MLKL. A kinase-dead D161N RIP3 mutant spontaneously induces apoptosis, while K51A does not — RIP3 D161N/D161N mice die perinatally but RIP3 K51A/K51A mice are viable. |
Small-molecule inhibitors, kinase-dead mutants, RHIM mutagenesis, caspase-8 complex co-immunoprecipitation, knock-in mice |
Molecular cell |
High |
25459880
|
| 2015 |
In the absence of IAPs, LPS triggers RIPK3 to activate caspase-8 and promote apoptosis and NLRP3-caspase-1 activation independently of RIPK3 kinase activity and MLKL. When both IAPs and caspase-8 are absent, RIPK3 kinase activity and MLKL are required for TLR-induced NLRP3 activation. TRIF and IAPs regulate RIPK3 and MLKL ubiquitylation. |
Genetic KO models (IAP-deficient, caspase-8 KO, MLKL KO, RIPK3 KO), LPS treatment, inflammasome activation assays, in vivo arthritis model |
Nature communications |
High |
25693118
|
| 2016 |
RIP3 triggers myocardial necroptosis through activation of CaMKII (via phosphorylation or oxidation), rather than through RIP1 and MLKL, leading to mitochondrial permeability transition pore (mPTP) opening. RIP3 deficiency or CaMKII inhibition ameliorates ischemia-reperfusion and doxorubicin-induced myocardial necroptosis and heart failure in mice. |
RIP3 KO mice, CaMKII inhibition (genetic and pharmacologic), cardiac ischemia-reperfusion models, co-immunoprecipitation, phosphorylation assays |
Nature medicine |
High |
26726877
|
| 2016 |
Proteasome inhibitors (MG132, bortezomib) activate RIPK3-MLKL necroptotic pathway in a RHIM-dependent but caspase-inhibition-independent manner. K48-linked ubiquitination of RIPK3 (partially at K264) accumulates upon proteasome inhibition. When MLKL recruitment to RIPK3 is restricted under proteasome inhibition, RIPK3-dependent apoptosis is induced instead. |
Proteasome inhibitor treatment, RHIM mutagenesis, K264R ubiquitination mutants, cell death assays, Western blot |
The Journal of biological chemistry |
Medium |
26786097
|
| 2016 |
RIPK1 and RIPK3 kinase activities promote sustained activation of Erk, cFos, and NF-κB to drive pro-inflammatory gene expression in macrophages stimulated by LPS, independently of cell death functions. This regulation requires the adaptor TRIF and proceeds cell-autonomously. |
Kinase inhibitors, genetic KO, LPS stimulation of primary macrophages, cytokine/signaling assays, in vivo LPS challenge |
Immunity |
Medium |
27396959
|
| 2017 |
RIPK3 restricts West Nile virus pathogenesis in the CNS independently of necroptotic cell death: Ripk3−/− mice show enhanced mortality with suppressed neuronal chemokine expression and decreased CNS recruitment of T lymphocytes and inflammatory myeloid cells, while Mlkl−/− mice are unaffected, demonstrating a death-independent neuroinflammatory signaling role for RIPK3. |
Ripk3−/− and Mlkl−/− mouse models, WNV infection, flow cytometry, chemokine profiling |
Cell |
High |
28366204
|
| 2017 |
RIPK1 and RIPK3 kinase activities direct IFN-β synthesis induced by LPS via necrosome-like aggregates that facilitate canonical TRIF-dependent signaling downstream of TLR4, independently of MLKL and necroptosis. |
Genetic KO and kinase inhibitors, macrophage LPS stimulation, IFN-β ELISA, necrosome formation assays |
Journal of immunology |
Medium |
28461567
|
| 2018 |
PELI1 E3 ubiquitin ligase selectively targets kinase-active RIP3 for degradation: phosphorylation of RIP3 on T182 leads to interaction with the FHA domain of PELI1, which then mediates K48-linked polyubiquitination of RIP3 on K363, promoting proteasomal degradation and preventing cell death from RIP3 hyperactivation. |
Co-immunoprecipitation, ubiquitination assays, mutagenesis (T182, K363), in vitro kinase assays, cell death assays |
Molecular cell |
High |
29883609
|
| 2018 |
RIPK3 promotes kidney fibrosis through AKT-dependent activation of ATP citrate lyase (ACL), independently of MLKL-dependent necroptosis. RIPK3-deficient mice are protected from UUO- and adenine diet-induced fibrosis while MLKL-deficient mice are not. Genetic or chemical RIPK3 inhibition suppresses TGF-β1-induced AKT and ACL phosphorylation in fibroblasts. |
RIPK3 KO and MLKL KO mouse models, UUO/adenine diet fibrosis models, AKT/ACL phosphorylation assays, fibroblast TGF-β1 stimulation |
JCI insight |
High |
29415885
|
| 2018 |
RIPK3 kinase domain homodimerizes through a surface structurally similar to RAF family members; mutation of the dimer interface residues impairs dimerization and necroptosis. Kinase domain dimerization drives cis-autophosphorylation of RIPK3 and is required for necroptosis. The D161N mutation enhances this noncatalytic allosteric dimerization function, explaining its apoptosis-inducing activity. |
Structural analysis, dimerization interface mutagenesis, co-immunoprecipitation, cell death assays, RIPK3 D161N heterozygous × RIPK1 KO mice |
Science signaling |
High |
30131368
|
| 2018 |
RIPK3 deficiency in TAMs reduces ROS and inhibits caspase-1-mediated cleavage of PPARγ, enabling PPARγ activation and fatty acid oxidation, thus driving M2 polarization and immunosuppression in the tumor microenvironment of hepatocellular carcinoma. |
RIPK3 KO macrophages, ROS assays, caspase-1 activity assays, PPARγ cleavage assays, macrophage polarization assays |
Cancer immunology research |
Medium |
32122992
|
| 2019 |
Neuronal ZBP1 and RIPK3 activation during Zika virus infection restricts viral replication by upregulating the enzyme IRG1 and producing the metabolite itaconate, which inhibits succinate dehydrogenase. This immunometabolic pathway operates independently of necroptotic cell death. |
ZBP1 KO and RIPK3 KO mouse models, Zika infection, metabolomics, IRG1 expression assays, succinate dehydrogenase activity assays |
Immunity |
High |
30635240
|
| 2019 |
CK1γ1 and CK1γ3 promote TNF-induced necroptosis by forming a complex with the necrosome (RIPK1, RIPK3, MLKL) and phosphorylating RIPK3, affecting its activity. Autophosphorylated CK1γ3 (S344/345) is present in the necrosome. CK1γ is cleaved by caspase-8 during apoptosis, analogous to RIPK1. |
Gain-of-function kinase screen (546 kinases), Co-IP, in vitro kinase assays with purified proteins, siRNA knockdown, cell death assays, CK1γ inhibitor in vivo |
Cell death & disease |
Medium |
31801942
|
| 2020 |
TRIM25 E3 ubiquitin ligase directly interacts with RIP3 through its SPRY domain and mediates K48-linked polyubiquitination of RIP3 at K501, promoting proteasomal degradation. The RING domain of TRIM25 is required for this ubiquitination. TRIM25 deficiency inhibits RIP3 ubiquitination and promotes TNF-induced cell necrosis. |
Co-immunoprecipitation, ubiquitination assays, TRIM25 KO cells, mutagenesis (K501R), domain-deletion analysis |
Cell death and differentiation |
Medium |
33953350
|
| 2021 |
ZBP1 induces K63- and M1-linked ubiquitin chains on RIPK1 and ZBP1 itself through cIAP1 and LUBAC, using RIPK1 and RIPK3 as scaffolds (not through their kinase activity) to promote TAK1- and IKK-mediated inflammatory cytokine production, independently of cell death. ZBP1 associates with RIPK1, RIPK3, cIAP1, and LUBAC in HT29 cells. |
Co-immunoprecipitation, ubiquitin chain-type analysis, kinase inhibitors, caspase inhibitors, cytokine assays in HT29 cells |
EMBO reports |
Medium |
36268590
|
| 2021 |
A class of orthopoxvirus proteins (vIRD — viral inducer of RIPK3 degradation) binds host SKP1-Cullin1-F-box (SCF) machinery and RIPK3, triggering ubiquitination and proteasome-mediated degradation of RIPK3 to inhibit necroptosis and regulate virus-induced inflammation. |
siRNA screen, Co-immunoprecipitation, ubiquitination assays, RIPK3 KO and MLKL KO mice, viral replication assays in vivo |
Immunity |
High |
33444549
|
| 2021 |
Phosphorylated MLKL disengages from RIPK3 following necroptotic stimulation through a conformational transition of the MLKL pseudokinase domain. Monobody-27 binds MLKL via an epitope overlapping the RIPK3 binding site that is only exposed after pMLKL disengages from RIPK3, identifying MLKL disengagement from RIPK3 as a key regulatory step in necroptosis. |
Crystal structures of MLKL pseudokinase domain with Monobodies, cell-based necroptosis assays, phospho-MLKL pulldowns |
Nature communications |
High |
33850121
|
| 2022 |
RIPK3 phosphorylation at T224 and S227 (C-lobe) are synergistic, crucial sites for stable interaction with MLKL and promotion of necroptosis in human cells. Activation loop phosphorylation at S164/T165 negatively regulates RIPK3 kinase activity. Stable recruitment of MLKL by phospho-T224/S227 RIPK3 to the necrosome is an essential checkpoint independent of and prior to MLKL phosphorylation. |
Phosphoproteomics of RIPK3 in HT29 cells, mutagenesis of phosphosites, kinase-inactive RIPK3 controls, cell death assays |
Cell death & disease |
High |
35739084
|
| 2022 |
Super-resolution microscopy reveals that cellular necrosomes are mosaic structures comprising alternating RIP1 and RIP3 oligomeric domains. Small mosaics are round; large functional mosaics are rod-shaped. RIP3 oligomers of tetramer size or above serve as domains for MLKL oligomerization driven by phospho-RIP3. RIP1 autophosphorylation controls ordered RIP1 oligomerization and is required for RIP1-initiated RIP3 homo-oligomerization in correct configuration for functional rod-shaped mosaic formation. |
Super-resolution microscopy (STORM/PALM), inducible dimer systems, RIP1 autophosphorylation mutants, MLKL recruitment assays |
Nature cell biology |
High |
35256774
|
| 2022 |
RIPK3 deficiency restores mitochondrial biogenesis, bioenergetics, and function in NAFLD, and is accompanied by upregulation of antioxidant systems and alterations in lipid droplet architecture (smaller, more numerous LDs). RIPK3 deficiency upregulates LD-associated proteins PLIN1 and PLIN5. |
Ripk3−/− mice on CDAA diet, CRISPR-Cas9 Ripk3-null hepatocytes, mitochondrial function assays, lipid droplet imaging, proteomics |
Hepatology |
Medium |
36029129
|
| 2022 |
RIPK3 cleavage by caspase-8 at D333 is dispensable for blocking necroptosis during development (Ripk3D333A/D333A mice are viable), but caspase-8-mediated cleavage of RIPK3 restricts NLRP3 inflammasome activation, pyroptosis, and IL-1β secretion when IAPs are limiting. |
Knock-in mice (Ripk3D333A/D333A), cell death assays, NLRP3 inflammasome activation assays, IL-1β secretion |
Cell death and differentiation |
High |
38514849
|
| 2022 |
RIPK3-MLKL signaling drives STING pathway amplification: RIPK3 inhibits autophagic degradation of STING, while MLKL (when its pro-necroptotic membrane activity is blocked) binds activated STING and is secreted extracellularly to restrict TBK1 and IRF3 recruitment, thereby bidirectionally regulating STING signaling. |
RIPK3 KO HT29 cells, autophagy flux assays, STING co-immunoprecipitation with MLKL, cell death assays, in vivo sepsis model |
Clinical and translational medicine |
Medium |
37475188
|
| 2023 |
OASL undergoes liquid-liquid phase condensation upon viral infection and scaffolds RIPK3 and ZBP1 via protein-protein interactions, providing spatial segregation for RIPK3 nucleation. This OASL-driven liquid platform facilitates amyloid-like fibril formation and autophosphorylation of RIPK3, leading to MLKL phosphorylation and necroptosis. Oasl1-deficient mice show severely impaired necroptosis and uncontrolled viral dissemination. |
Phase condensation assays, co-immunoprecipitation, RIPK3 fibril formation assays, MLKL phosphorylation, Oasl1 KO mice with viral infection |
Nature cell biology |
High |
36604592
|
| 2023 |
PRMT1 methyltransferase methylates RIP3 at R486 (human) / R479 (mouse), and this methylation inhibits the RIP1-RIP3 interaction and suppresses necrosome complex formation, thereby blocking RIP3 phosphorylation and necroptosis activation. |
Co-immunoprecipitation, in vitro methylation assays, methylation-deficient RIP3 mutant, RIP3-RIP1 interaction assays, necroptosis readouts, patient sample analysis with di-methylation-specific antibody |
Cell death & disease |
Medium |
37005412
|
| 2023 |
p55γ promotes RIP3 proteasomal degradation in a ubiquitin-dependent manner, with MG53 functioning as the E3 ligase mediating p55γ-induced RIP3 degradation. HIF1α transcriptionally regulates p55γ expression. This p55γ-MG53-RIP3 axis underlies ischemic preconditioning-induced suppression of myocardial necroptosis. |
Co-immunoprecipitation, pulldown assays, mass spectrometry, ChIP assays, p55γ transgenic and KO mice, cardiac I/R model |
Cardiovascular research |
Medium |
37527538
|
| 2024 |
ZBP1 senses mitochondrial Z-form DNA induced by diquat poisoning in endothelial cells and interacts with RIPK3, leading to RIPK3-dependent necroptosis and ferroptosis. RIPK3 has a dual role: it phosphorylates MLKL (necroptosis) and also phosphorylates FSP1 to inhibit its enzymatic activity (promoting ferroptosis). Specific deletion of Zbp1 or Ripk3 in endothelial cells simultaneously inhibits both death pathways. |
Endothelial cell-specific Zbp1 and Ripk3 KO mice, Z-DNA detection, MLKL phosphorylation assays, FSP1 phosphorylation and activity assays, organ damage readouts |
Cell death and differentiation |
High |
38493248
|
| 2024 |
RIPK3 induces mitochondrial fission in diabetic podocytes via MLKL-PGAM5-Drp1 signaling: RIPK3 activates MLKL, which engages PGAM5 to promote phosphorylation and mitochondrial translocation of Drp1 (S616), causing mitochondrial fragmentation and dysfunction. Ripk3 KO mice are protected from DKD-associated albuminuria and podocyte loss. |
Ripk3 KO mice (DKD model), RIPK3 overexpression in podocytes, MLKL/PGAM5/Drp1 phosphorylation assays, mitochondrial imaging, single-cell RNA-seq |
Metabolism: clinical and experimental |
Medium |
39089491
|
| 2016 |
RIP3 nuclear translocation and formation of a RIP3-AIF complex, followed by nuclear translocation of this complex, is critical for ischemic neuronal DNA degradation and programmed necrosis after global cerebral ischemia/reperfusion injury in rats. |
Necrostatin-1 treatment, immunoprecipitation, immunofluorescence co-localization, western blot in rat I/R model |
Scientific reports |
Low |
27377128
|
| 2022 |
RIPK3-MLKL signaling activates mitochondrial CaMKII which drives phosphorylation of Smad2/3 and subsequent production of extracellular matrix proteins (αSMA, collagen 1α1) in response to TGF-β during chronic kidney disease. TGF-β induces translocation of RIPK3 and MLKL to mitochondria, causing mitochondrial dysfunction and ROS production that activates CaMKII. |
RIPK3 KO and MLKL KD experiments, mitochondrial fractionation, CaMKII inhibition, Smad2/3 phosphorylation assays, UUO model |
Matrix biology |
Medium |
35964866
|