| 2014 |
RIPK3 phosphorylates MLKL at T357 and S358; phosphorylated MLKL forms oligomers that bind phosphatidylinositol lipids and cardiolipin, enabling translocation from cytosol to plasma and intracellular membranes where it directly disrupts membrane integrity to cause necrotic cell death. |
In vitro phosphorylation assay, phospho-specific monoclonal antibody, lipid-binding assay, cell fractionation, live-cell imaging |
Molecular cell |
High |
24703947
|
| 2013 |
MLKL is a pseudokinase (catalytically inactive) that binds ATP but has no enzymatic activity; it comprises a four-helical bundle (4HB) domain tethered to a pseudokinase domain; RIPK3-mediated phosphorylation of the pseudoactive site is essential for necroptotic signaling; structure-guided mutation of the pseudoactive site causes constitutive, RIPK3-independent necroptosis, demonstrating a molecular switch mechanism. |
Crystal structure of MLKL, MLKL-deficient mouse generation, TNF-induced necroptosis rescue assay, structure-guided mutagenesis |
Immunity |
High |
24012422
|
| 2013 |
MLKL acts downstream of RIP3 kinase in TLR3/TRIF-driven programmed necrosis, independently of RIP1, placing MLKL as a required effector in the TRIF-RIP3-MLKL necroptosis axis. |
Genetic epistasis (MLKL knockdown/deficiency), RIP3 kinase inhibitors, cell death assays |
The Journal of biological chemistry |
Medium |
24019532
|
| 2016 |
MLKL forms cation channels permeable preferentially to Mg2+ over Ca2+ in the presence of Na+ and K+; the N-terminal domain (helices H1–H6) is sufficient for channel formation; helices H1, H2, H3, H5, and H6 are transmembrane segments while H4 is cytoplasmic; MLKL-induced membrane depolarization and cell death correlate with channel activity. |
Electrophysiology (patch-clamp), substituted cysteine accessibility method (SCAM), ion selectivity assays, cell death assays |
Cell research |
High |
27033670
|
| 2017 |
MLKL forms SDS-resistant, disulfide bond-dependent amyloid-like polymers during necroptosis; recombinant MLKL N-terminal domain forms amyloid-like fibers (~5 nm diameter) that bind Congo red; MLKL mutants unable to polymerize fail to induce necroptosis; necrosulfonamide conjugates cysteine 86 of human MLKL, blocking polymer formation and cell death. |
SDS-PAGE, proteinase K resistance assay, electron microscopy, Congo red binding, site-directed mutagenesis, chemical probe (necrosulfonamide) conjugation |
Proceedings of the National Academy of Sciences of the United States of America |
High |
28827318
|
| 2017 |
During necroptosis, MLKL-dependent Ca2+ influx and phosphatidylserine exposure on the outer plasma membrane leaflet precede membrane rupture; ESCRT-III machinery is recruited downstream of MLKL activation to form PS-exposing membrane 'bubbles' released from the cell surface, acting to delay/limit plasma membrane disruption and modulate necroptotic kinetics. |
Live-cell imaging (Ca2+ and PS reporters), flow cytometry, ESCRT-III genetic knockdown/knockout, cell death assays |
Cell |
High |
28388412
|
| 2017 |
MLKL associates with endosomes independently of RIPK3 (constitutive function) and controls endocytosed protein transport, receptor/ligand degradation, and extracellular vesicle generation; upon RIPK3 activation, enhanced MLKL–endosome association leads to MLKL binding to ESCRT proteins and flotillins and its release within extracellular vesicles. |
Co-immunoprecipitation with ESCRT proteins and flotillins, endosomal fractionation, extracellular vesicle isolation, MLKL-deficient cell comparison |
Immunity |
Medium |
28666573
|
| 2017 |
MLKL-induced NLRP3 inflammasome activation requires (i) the death effector four-helical bundle of MLKL, (ii) MLKL oligomerization and membrane association, and (iii) intracellular potassium efflux; this occurs cell-intrinsically before cell lysis; GSDMD is not required for MLKL-dependent IL-1β secretion during necroptosis. |
Domain deletion/mutation analysis, K+ efflux measurement, ASC speck imaging, NLRP3/caspase-1 genetic KO, GSDMD KO |
Proceedings of the National Academy of Sciences of the United States of America |
High |
28096356 28130493
|
| 2018 |
Inositol phosphate (IP) kinases IPMK and ITPK1 are essential for MLKL-dependent necroptosis; in IP kinase mutant cells, MLKL fails to oligomerize and localize to membranes despite proper RIPK3-mediated phosphorylation; highly phosphorylated IP products (not lowly phosphorylated precursors) displace the MLKL auto-inhibitory brace region, identifying metabolite control of MLKL activation. |
Genetic screen in human cells (CRISPR), IP kinase KO rescue, oligomerization assay, membrane localization assay, in vitro brace-displacement assay |
Molecular cell |
High |
29883610
|
| 2018 |
RIPK3 and MLKL continuously shuttle between nucleus and cytoplasm; during TNF-induced necroptosis, nuclear MLKL becomes phosphorylated and oligomerized; pharmacological inhibition of nuclear export retains RIPK3/MLKL in the nucleus, prevents cytosolic RIPK3/MLKL oligomerization, and reduces necroptotic cell death, indicating nuclear passage as a regulatory step for necrosome formation. |
Nuclear export inhibitor (leptomycin B), subcellular fractionation, immunofluorescence, cell death assays |
Communications biology |
Medium |
30271893
|
| 2018 |
MLKL activates PAD4-dependent NET formation in necroptotic neutrophils: MLKL translocates from cytoplasm to plasma membrane and stimulates downstream NADPH oxidase-independent ROS production, nuclear membrane breakdown, chromatin decondensation, histone hypercitrullination, and NET extrusion; PAD4 acts downstream of RIPK1/RIPK3/MLKL. |
Genetic KO of RIPK3 and MLKL, MLKL translocation imaging, ROS assay, PAD4 KO rescue, NET quantification |
Science signaling |
Medium |
30181240
|
| 2018 |
During peripheral nerve injury, MLKL is phosphorylated at serine 441 (not by RIP3) and targets the myelin sheath membrane of Schwann cells to promote myelin breakdown; Schwann cell-specific MLKL knockout delays myelin breakdown and reduces nerve regeneration; MLKL overexpression accelerates myelin breakdown. |
Schwann cell-specific conditional KO, MLKL overexpression, phospho-site mapping, myelin breakdown assay, axon regeneration assay |
Molecular cell |
High |
30344099
|
| 2019 |
TAM receptor tyrosine kinases (Tyro3, Axl, Mer) phosphorylate MLKL at Tyr376, promoting MLKL oligomerization (but not membrane translocation or RIPK3-mediated phosphorylation); pharmacological or genetic TAM inhibition potently inhibits necroptotic death and protects mice from systemic inflammatory response syndrome. |
TAM kinase inhibitors, TAM KO cells, phospho-Tyr376 MLKL detection, oligomerization assay, in vivo SIRS model |
Molecular cell |
High |
31230815
|
| 2019 |
MLKL directly binds to intracellular Listeria monocytogenes in the cytosol and inhibits their replication independently of host cell killing; RIPK3-MLKL pathway activation by Listeria leads to phospho-MLKL that does not cause cell death but suppresses bacterial replication. |
MLKL direct binding to bacteria (co-localization/pulldown), RIPK3/MLKL KO infection assay, in vivo Listeria dissemination in Ripk3-/- mice |
The Journal of cell biology |
Medium |
30975711
|
| 2020 |
Phosphorylated MLKL assembles into higher-order species at cytoplasmic necrosomes, then co-traffics with tight junction proteins to the cell periphery via Golgi-microtubule-actin-dependent mechanisms, and accumulates as micron-sized hotspots at the plasma membrane; this trafficking and plasma membrane accumulation are key checkpoints controlling necroptosis kinetics and threshold. |
Single-cell live imaging of endogenous MLKL, super-resolution microscopy, Golgi/microtubule/actin disruption, tight junction co-localization |
Nature communications |
High |
32561730
|
| 2020 |
MLKL regulates autophagic flux independently of RIPK3: MLKL translocates first to autophagosomes then to the plasma membrane upon palmitic acid treatment; MLKL deficiency (but not RIP3 deficiency) prevents autophagosome–lysosome fusion inhibition; MLKL overexpression blocks autophagy independently of stimulus. |
mRFP-GFP-LC3 reporter (autophagy flux assay), MLKL/RIP3 KO hepatocytes, MLKL overexpression, LC3-II/p62 Western blot |
Journal of hepatology |
Medium |
32220583
|
| 2020 |
A missense mutation in the MLKL brace region (D139V) confers constitutive, RIPK3-independent killing activity, demonstrating that the brace helix connecting the 4HB and pseudokinase domains is a critical regulatory element controlling MLKL auto-inhibition in vivo. |
Mouse knock-in model (MlklD139V), cell death assays in RIPK3-deficient background, histopathological analysis |
Nature communications |
High |
32561755
|
| 2021 |
Conformational interconversion of the MLKL pseudokinase domain occurs upon activation: after RIPK3-mediated phosphorylation, MLKL undergoes a large conformational change and disengages from RIPK3; crystal structures of the pseudokinase domain with Monobody-27 (binding the RIPK3-interface epitope only after disengagement) and Monobody-32 (constitutive brace-region binding) define two distinct pseudokinase conformations as key regulatory steps. |
Crystal structures of MLKL pseudokinase domain in complex with Monobodies, intracellular Monobody expression, necroptosis assays |
Nature communications |
High |
33850121
|
| 2021 |
Necroptosis-specific multi-mono-ubiquitylation of MLKL occurs after its activation and oligomerization; ubiquitylated MLKL accumulates at organellar/plasma membranes; ubiquitylation drives proteasome- and lysosome-dependent MLKL turnover; constitutive deubiquitylation of MLKL (via MLKL-DUB fusion) licenses MLKL auto-activation independent of necroptosis signaling, showing ubiquitylation restrains basal activated MLKL levels. |
MLKL-DUB fusion strategy, plasma membrane-targeted DUB expression, ubiquitin site mapping (MS), digitonin fractionation, proteasome/lysosome inhibitors |
The EMBO journal |
High |
34698396
|
| 2021 |
CAMK2/CaMKII phosphorylates MLKL in response to serum and amino acid starvation independently of RIPK3, promoting autophagic flux (autophagosome–lysosome fusion) and protecting cells from starvation-induced death; this is mechanistically distinct from the necroptotic RIPK3-MLKL pathway, which instead suppresses autophagic flux. |
CAMK2 inhibitor/siRNA, MLKL KO/siRNA, LC3 flux reporter (pHluorin-mKate2-LC3), co-immunoprecipitation |
Autophagy |
Medium |
34282994
|
| 2021 |
K63-linked polyubiquitin chains conjugated by the E3 ligase ITCH to distinct lysine residues in the MLKL N-terminal HeLo domain of phosphorylated MLKL redirect MLKL to endosomes (instead of plasma membrane), resulting in its release within extracellular vesicles and enhanced endosomal trafficking of intracellular bacteria to lysosomes. |
Site-specific ubiquitin chain characterization (MS), ITCH Co-IP/WW-domain interaction, MLKL lysine mutants, endosomal fractionation, bacterial trafficking assay |
Cell death and differentiation |
Medium |
34999730
|
| 2019 |
RIPK3-mediated phosphorylation of the MLKL pseudokinase domain drives pseudokinase domain dimerization as the initial oligomerization step; subsequent internal coiled-coil self-assembly forms the full MLKL oligomer; crystal structure of human MLKL reveals two inter-dimer interfaces; mutations destroying both interfaces prevent RIPK3-induced oligomerization and necroptosis; coiled-coil disruption prevents oligomerization but not dimerization. |
Crystal structure of human MLKL pseudokinase domain, site-directed mutagenesis of dimer interfaces and coiled-coil, necroptosis assays |
Cell death & disease |
High |
34158471
|
| 2023 |
RIPK3-mediated phosphorylation of the human MLKL activation loop drives pseudokinase domain dimerization, which nucleates elongated homotetramers via a central coiled coil formed by the ~80 Å brace helix; tetramerization is an essential prerequisite for release and reorganization of four-helix bundle domains for membrane permeabilization. |
Crystal structure, negative-stain electron microscopy, MLKL tetramerization-defective mutants, cell death assays |
Nature communications |
High |
37884510
|
| 2020 |
Hsp70 performs dual roles in necroptosis: it stabilizes MLKL protein under basal conditions (client protein function) and promotes MLKL N-terminal domain polymerization through its substrate-binding domain (SBD) during necroptosis; NBC1 covalently conjugates C574 and C603 of the Hsp70 SBD to block MLKL polymerization and cell death; SBD mutations at both cysteines abolish this pro-polymerization function. |
Biotin-NBC1 pulldown identifying Hsp70, in vitro Hsp70–MLKL-NTD interaction/polymerization assay, SBD mutagenesis, Hsp70 knockdown |
Proceedings of the National Academy of Sciences of the United States of America |
High |
32156734
|
| 2023 |
Activated MLKL translocates to the lysosomal membrane during necroptosis; MLKL polymerization induces lysosome clustering, fusion, and lysosomal membrane permeabilization (LMP), releasing cathepsin B (CTSB) into the cytosol; CTSB contributes to cell death downstream of MLKL, as CTSB inhibition or knockdown protects cells from necroptosis. |
MLKL-lysosome co-localization imaging, LMP assay, cathepsin activity measurement, CTSB inhibitor/siRNA rescue |
Cell death and differentiation |
Medium |
37996483
|
| 2025 |
Phospho-MLKL translocates to mitochondria and induces microtubule-dependent release of mitochondrial DNA (mtDNA) into the cytosol, which activates the cGAS-STING pathway to upregulate IFN-β expression; this represents a cell-autonomous inflammatory signaling mechanism downstream of MLKL activation. |
MLKL-mitochondria co-localization, mtDNA release assay, cGAS-STING pathway readout (IFN-β), microtubule disruption, in vivo IBD model with STING pathway interference |
Molecular cell |
High |
40614706
|
| 2019 |
Poxviral MLKL-like proteins function as dominant-negative mimics of host MLKL by sequestering RIPK3 via its kinase domain, thereby preventing MLKL engagement and phosphorylation and blocking necroptotic cell death. |
Expression of viral MLKL-like proteins in human and mouse cells, Co-IP with RIPK3, MLKL phosphorylation assay, cell death rescue assay |
Cell reports |
Medium |
31553902
|
| 2016 |
The MLKL 4HB (four-helix bundle) domain mediates membrane permeabilization and is evolutionarily conserved in this function across species; forced dimerization of the human MLKL 4HB domain overcomes a species-specific defect and triggers cell death; recombinant 4HB proteins from mouse, frog, human, and chicken all permeabilize liposomes, most effectively those mimicking plasma membrane composition. |
Recombinant protein liposome permeabilization assay, forced dimerization, cross-species domain expression, cell death assays |
Cell death and differentiation |
High |
26868910
|
| 2019 |
Activated MLKL attenuates autophagic flux by inhibiting autophagosome and/or autolysosome function upon translocation to intracellular membranes; this effect requires MLKL association with intracellular membranes and occurs independently of plasma membrane disruption. |
LC3B lipidation Western blot, autophagy flux assay, MLKL KO mouse dermal fibroblasts and HT-29 cells, MLKL intracellular membrane association |
Journal of cell science |
Medium |
30709919
|
| 2023 |
The E3 ligase Skp2 interacts with MLKL and promotes its ubiquitination-mediated proteasomal degradation in cisplatin-resistant NSCLC cells; Skp2 knockdown restores MLKL levels and sensitizes resistant cells to cisplatin. |
Co-immunoprecipitation (Skp2–MLKL), ubiquitination assay, Skp2 knockdown/OE, cisplatin sensitivity rescue |
Communications biology |
Medium |
37532777
|
| 2019 |
BRD4 forms a transcription complex with IRF1, P-TEFb, and RNA polymerase II to positively regulate MLKL transcription; BET inhibitors (JQ-1) downregulate MLKL expression by disrupting this complex, thereby inhibiting necroptosis. |
ChIP, Co-IP of transcription complex, BET inhibitor treatment, MLKL expression assay, cell death assays |
Cell death and differentiation |
Medium |
30644439
|
| 2022 |
In macrophages, LPS-induced MLKL phosphorylation and oligomerization drives translocation to intracellular phagosomes and lysosomes (not plasma membrane), and this MLKL activity is required for macrophage phagocytic capability; MLKL-deficient Kupffer cells phagocytose fewer bioparticles in vivo. |
Bone marrow transplant (myeloid vs. non-myeloid Mlkl-/-), phagocytosis assay, MLKL subcellular fractionation/immunofluorescence, in vivo bioparticle phagocytosis |
Hepatology (Baltimore, Md.) |
Medium |
35689613
|
| 2021 |
MLKL modulates insulin-stimulated PI(3,4,5)P3 production in liver cells, regulating hepatic insulin sensitivity independently of inflammation and independently of its canonical necroptosis-inducing function. |
MLKL KO mice (whole-body), in vitro hepatocyte insulin signaling (PI(3,4,5)P3 assay), inflammatory gene expression analysis (negative for inflammatory mechanism) |
Molecular metabolism |
Medium |
30837196
|
| 2022 |
TGFβ induces translocation of RIPK3 and MLKL to mitochondria, causing mitochondrial dysfunction and ROS production; mitochondrial ROS activates CaMKII, which phosphorylates Smad2/3 to drive extracellular matrix production; MLKL deficiency prevents this signaling axis and reduces kidney fibrosis. |
RIPK3/MLKL mitochondrial fractionation, ROS measurement, CaMKII phosphorylation, Smad2/3 phosphorylation, MLKL KO in CKD model |
Matrix biology : journal of the International Society for Matrix Biology |
Medium |
35964866
|
| 2021 |
MLKL activates downstream CaMKII in smooth muscle cells (SMCs) during RIPK3-mediated necroptosis; MLKL knockdown reduces CaMKII phosphorylation, whereas CaMKII knockdown does not affect MLKL phosphorylation, oligomerization, or trafficking, placing MLKL upstream of CaMKII in this pathway. |
siRNA knockdown of MLKL and CaMKII, phosphorylation Western blot, MLKL oligomerization assay, cell death assay |
Cells |
Medium |
34572045
|
| 2023 |
MLKL activity in pancreatic acinar cells controls CXCL10 secretion independently of RIPK3 and cell death; CXCL10 then drives macrophage M1 polarization; MLKL KO reduces M1 polarization and AP severity. |
Mlkl-/- and Ripk3-/- mice in cerulein AP model, CXCL10 measurement, macrophage polarization assay, in vivo CXCL10 neutralization |
Cell death & disease |
Medium |
36828808
|