{"gene":"MAP3K5","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1998,"finding":"MAP3K5/ASK1 activates both JNK and p38 kinase signaling pathways. A novel interacting protein (MAPKKK6) was identified by yeast two-hybrid screening using MAPKKK5 as bait, and their interaction was confirmed by co-immunoprecipitation in 293 cells.","method":"Yeast two-hybrid screen, co-immunoprecipitation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus confirmatory co-IP; single lab, two methods","pmids":["9875215"],"is_preprint":false},{"year":1999,"finding":"ASK1 mediates apoptotic cell death induced by genotoxic stress (cisplatin) upstream of caspase activation, acting through the SEK1/MKK4 and MKK3/MKK6 pathways to activate JNK1 and p38. Overexpression of kinase-negative ASK1 (K709R) suppressed caspase activation and apoptosis.","method":"Dominant-negative overexpression, immune complex kinase assay, caspase activity assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-dead mutant plus downstream pathway analysis; single lab, two orthogonal methods","pmids":["9926932"],"is_preprint":false},{"year":1999,"finding":"TRAF2, TRAF5, and TRAF6 associate with ASK1, and a catalytically-inactive ASK1 mutant blocks SAPK/JNK activation by these TRAFs. ASK1 is a common mediator of TRAF-regulated SAPK and apoptosis signaling downstream of TNF receptors.","method":"Co-immunoprecipitation, dominant-negative overexpression, JNK activation assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal binding plus functional kinase assay; single lab","pmids":["10523862"],"is_preprint":false},{"year":2000,"finding":"Constitutively active ASK1 (ASK1DeltaN) induces neurite outgrowth and neuronal differentiation in PC12 cells through activation of p38 (and to a lesser extent JNK), not ERK. ASK1DeltaN also promoted survival of PC12 cells under serum-starved conditions.","method":"Constitutively active mutant overexpression, p38 inhibitor (SB203580), MEK inhibitor controls, morphological/biochemical differentiation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function mutant with pharmacological inhibitor validation; single lab, two orthogonal approaches","pmids":["10734135"],"is_preprint":false},{"year":2001,"finding":"Constitutively active ASK1 induces keratinocyte differentiation through the ASK1-p38 MAP kinase cascade, as shown by induction of differentiation markers (transglutaminase-1, loricrin, involucrin) that was blocked by p38 inhibitors SB202190 and SB203580. Ceramide-induced differentiation increased ASK1 expression and activity.","method":"Constitutively active ASK1 overexpression, p38 inhibitor treatment, differentiation marker assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function with pharmacological validation; single lab","pmids":["11029458"],"is_preprint":false},{"year":2001,"finding":"ASK1 possesses a kinase-independent, caspase-independent cell death function activated by interaction with Daxx. The N-terminal domain of ASK1 (lacking the kinase domain) was constitutively active in producing crumpled nuclei, distinct from caspase-dependent fragmented nuclei caused by ASK1 kinase activity.","method":"Co-transfection, domain deletion mutants, caspase inhibitor, morphological assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple domain mutants with functional cell death readout; single lab","pmids":["11493600"],"is_preprint":false},{"year":2001,"finding":"ASK1-JNK pathway stabilizes c-Myc protein by phosphorylating Ser-62 and Ser-71, attenuating degradation of ubiquitinated c-Myc without affecting its ubiquitination, thus promoting c-Myc-dependent apoptosis.","method":"Overexpression, site-directed mutagenesis of c-Myc, pulse-chase, kinase assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis of phosphorylation sites plus functional outcome; single lab","pmids":["11243879"],"is_preprint":false},{"year":2002,"finding":"Reduced thioredoxin (Trx) binds to and inhibits ASK1. Upon oxidation (via H2O2 or TNF), Trx is released from ASK1. Single-Cys Trx mutants (C32S or C35S) that constitutively bind ASK1 promoted ASK1 ubiquitination and degradation, inhibiting JNK activation and apoptosis in a redox-independent manner. The double mutant (Trx-CS) lost ASK1-binding and inhibitory activity.","method":"Co-immunoprecipitation, overexpression of Trx mutants, ubiquitination assay, JNK activation assay, caspase-3 assay","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple Trx mutants with reciprocal co-IP and multiple functional readouts; single lab with orthogonal methods","pmids":["12089063"],"is_preprint":false},{"year":2002,"finding":"ASK1 is activated and autophosphorylated following cerebral ischemia/reperfusion in rat hippocampus, peaking at 30 min reperfusion. Both activation and autophosphorylation were suppressed by the antioxidant N-acetylcysteine, indicating redox-dependent regulation.","method":"In vitro kinase assay, immunoprecipitation, Western blot, antioxidant treatment in vivo","journal":"Neuroscience letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus in vivo pharmacological inhibition; single lab","pmids":["12165419"],"is_preprint":false},{"year":2002,"finding":"ALG-2 interacts with the C-terminus (aa 941–1375) of ASK1, and co-transfection with ALG-2 causes nuclear localization of ASK1 and inhibits ASK1-mediated JNK activation. ALG-2 variant with deletion of Gly-121/Phe-122 failed to bind ASK1, demonstrating specificity.","method":"Co-immunoprecipitation, subcellular localization (immunofluorescence), JNK activation assay, in vitro binding","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus localization plus functional JNK assay; single lab","pmids":["12372597"],"is_preprint":false},{"year":2003,"finding":"ASK1 specifically interacts with cardiac troponin T (cTnT) via its C-terminal domain, phosphorylates cTnT at T194/S198 in vitro and in vivo, and ROS-induced ASK1 activation causes cTnT phosphorylation and contractile dysfunction in cardiomyocytes. Constitutively active ASK1 overexpression inhibited sarcomere shortening and calcium transients.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis of cTnT, cardiomyocyte contractility assay","journal":"The American journal of pathology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis plus in vivo functional readout; single lab, multiple orthogonal methods","pmids":["12819028"],"is_preprint":false},{"year":2003,"finding":"ASK1 regulates influenza virus infection-induced apoptosis: IV infection activated ASK1, phosphorylated JNK and p38, and ASK1-knockout MEFs showed reduced JNK/p38 activation and were defective in caspase-3 activation and cell death after IV infection.","method":"ASK1 knockout mouse-derived MEFs, kinase assays, caspase-3 activation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with defined signaling and cell death readouts; single lab","pmids":["12878192"],"is_preprint":false},{"year":2003,"finding":"ASK1 mediates anandamide-induced PC12 cell death through p38 MAPK and JNK activation, upstream of mitochondrial cytochrome c release and caspase activation. Dominant-negative ASK1 (K709M) inhibited p38/JNK activation and cell death.","method":"Dominant-negative overexpression, pharmacological inhibitors, cytochrome c release assay","journal":"Journal of neurochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase-dead mutant plus pharmacological validation; single lab","pmids":["12641726"],"is_preprint":false},{"year":2003,"finding":"The ASK1-SEK1-JNK1-HIPK1 pathway mediates Daxx phosphorylation at Ser667 during glucose deprivation, causing Daxx translocation from nucleus to cytoplasm where it binds ASK1 and promotes ASK1 oligomerization. JNK binding domain overexpression blocked Daxx relocalization and ASK1 oligomerization.","method":"In vivo labeling, immune complex kinase assay, immunofluorescence, protein interaction assay, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase cascade epistasis with mutagenesis; single lab, multiple methods","pmids":["12968034"],"is_preprint":false},{"year":2003,"finding":"CIIA (a novel antiapoptotic protein) binds ASK1 and inhibits oligomerization-induced ASK1 activation. CIIA antisense abolished its inhibitory effect on ASK1 activation, DNA fragmentation, and apoptosis.","method":"Co-immunoprecipitation, antisense oligonucleotides, ASK1 oligomerization assay, apoptosis assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding assay plus functional ASK1 activation readout plus loss-of-function; single lab","pmids":["14557248"],"is_preprint":false},{"year":2004,"finding":"Ca2+ influx activates p38 MAP kinase via CaMKII phosphorylation of ASK1. p38 activation by membrane depolarization in primary neurons was impaired in ASK1-deficient mice. Constitutively active CaMKII-induced p38 activation required endogenous ASK1.","method":"ASK1-knockout mouse neurons, constitutively active CaMKII overexpression, in vitro phosphorylation assay","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout plus in vitro phosphorylation plus constitutively active upstream kinase; replicated across multiple assays","pmids":["14749717"],"is_preprint":false},{"year":2005,"finding":"Hsp90 and Akt form a ternary complex with ASK1 via the middle domain of Hsp90. Akt phosphorylates ASK1 at Ser83, and this phosphorylation is required for Hsp90-mediated inhibition of ASK1-p38 signaling. In resting endothelial cells, Akt binds the N-terminal domain of ASK1; upon H2O2 stimulation it shifts to the C-terminal domain of ASK1.","method":"Co-immunoprecipitation, domain-mapping, Akt knockout cells, in vitro kinase assay, pharmacological inhibitors","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, domain mapping, genetic KO cells, in vitro kinase assay; multiple orthogonal methods in single lab","pmids":["15782121"],"is_preprint":false},{"year":2005,"finding":"Raf-1 kinase domain interacts with ALG-2, and both Raf-1 and ASK1 phosphorylate ALG-2. Raf-1 blocks ASK1-dependent ALG-2 phosphorylation, suggesting a mechanism for Raf-1's anti-apoptotic function through interference with the ASK1-ALG-2 axis.","method":"Yeast two-hybrid, in vitro kinase assay, co-immunoprecipitation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus yeast two-hybrid; single lab","pmids":["15925322"],"is_preprint":false},{"year":2006,"finding":"Calcineurin directly dephosphorylates ASK1 at Ser967 (identified by yeast two-hybrid with calcineurin B as bait), promoting dissociation of 14-3-3 proteins from ASK1 and ASK1 activation. Calcineurin and ASK1 cooperatively enhanced cardiomyocyte apoptosis. Reciprocally, ASK1 negatively regulated calcineurin-NFAT signaling through JNK- and p38-mediated NFAT phosphorylation.","method":"Yeast two-hybrid, co-immunoprecipitation of endogenous proteins, in vitro phosphatase assay with purified proteins, ASK1-deficient MEFs, dominant-negative ASK1","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro phosphatase assay with purified proteins plus reciprocal co-IP of endogenous proteins plus genetic knockout validation; multiple orthogonal methods","pmids":["16648474"],"is_preprint":false},{"year":2007,"finding":"Crystal structure of the human ASK1 catalytic domain in complex with staurosporine revealed that ASK1 forms a tight head-to-tail dimer (Kd ~0.2 μM). Three autophosphorylation sites (Thr813, Thr838, Thr842) were identified by mass spectrometry and shown to regulate ASK1 signaling by reporter gene assays, though site-directed mutants showed catalytic activities similar to wild-type, suggesting a regulatory mechanism independent of kinase activity.","method":"X-ray crystallography, analytical ultracentrifugation, mass spectrometry, site-directed mutagenesis, reporter gene assay","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus AUC plus MS autophosphorylation mapping plus mutagenesis; multiple orthogonal methods in single study","pmids":["17937911"],"is_preprint":false},{"year":2007,"finding":"ASK1 deficiency in mice resulted in dramatically reduced wounding-induced macrophage infiltration/activation and subsequent hair regrowth. Intracutaneous transplantation of cytokine-activated bone marrow-derived macrophages rescued hair growth in ASK1-deficient mice, placing ASK1 upstream of macrophage recruitment in inflammatory wound responses.","method":"ASK1-knockout mice, oligonucleotide microarray, bone marrow macrophage transplantation","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with rescue experiment; single lab","pmids":["17389227"],"is_preprint":false},{"year":2009,"finding":"PIM1 phosphorylates ASK1 specifically at Ser83 in vitro and in vivo, binds ASK1 by co-immunoprecipitation, decreases ASK1 kinase activity under oxidative stress, and inhibits ASK1-mediated JNK and p38 phosphorylation and caspase-3 activation. PIM1 knockdown reduced Ser83 phosphorylation and increased cell death after H2O2.","method":"In vitro kinase assay, co-immunoprecipitation, siRNA knockdown, caspase-3 activation assay","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus co-IP plus loss-of-function knockdown with multiple functional readouts; single lab, multiple orthogonal methods","pmids":["19749799"],"is_preprint":false},{"year":2009,"finding":"DAB2IP acts as a scaffold protein interacting with ASK1 through its C2 domain to enhance ASK1 activation, leading to JNK pathway activation and apoptosis. DAB2IP loss in prostate cancer led to ASK1-JNK inactivation and accelerated tumor growth in vivo.","method":"Gain/loss-of-function studies, structural domain analysis, in vivo xenograft","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mapping plus in vivo model; single lab","pmids":["19903888"],"is_preprint":false},{"year":2009,"finding":"PDK1 and ASK1 directly interact via the PDK1 PH domain and ASK1 C-terminal regulatory domain, and they reciprocally phosphorylate each other: PDK1 phosphorylates ASK1 at Ser967 (a 14-3-3 binding site) to suppress ASK1 activity, while ASK1 phosphorylates PDK1 at Ser394 and Ser398 to inhibit PDK1. The interaction is decreased by ASK1-activating stimuli.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, domain-mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reciprocal in vitro kinase assays with mutagenesis plus co-IP; multiple orthogonal methods in single lab","pmids":["19920149"],"is_preprint":false},{"year":2009,"finding":"JAK2 phosphorylates ASK1 at Tyr718, enhancing its association with SOCS1 and subsequent ASK1 degradation. SHP2 dephosphorylates ASK1 at Tyr718 (confirmed by substrate-trapping mutant), dissociating SOCS1 from ASK1 and enabling TNF-induced ASK1 activation and apoptosis.","method":"Co-immunoprecipitation, JAK2-specific inhibitor, SHP2 substrate-trapping mutant, SHP2-KO cells, JAK2 overexpression with in vitro kinase assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay, substrate-trapping mutant, genetic KO cells, multiple orthogonal methods; single lab","pmids":["19287004"],"is_preprint":false},{"year":2011,"finding":"Somatic mutations in MAP3K5 (e.g., I780F) identified in melanoma exomes reduced kinase activity in in vitro kinase assays and reduced phosphorylation of downstream MAP kinases when overexpressed in HEK293T cells. Loss of heterozygosity suggested these are inactivating mutations.","method":"Exome sequencing, in vitro kinase assay, overexpression in HEK293T cells with downstream phosphorylation readout","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus cellular overexpression; single study","pmids":["22197930"],"is_preprint":false},{"year":2013,"finding":"The MAP3K5 R256C melanoma mutation attenuates MKK4 activation through increased binding of the inhibitory protein thioredoxin (TRX), resulting in increased melanoma cell proliferation and anchorage-independent growth.","method":"Functional kinase assays, co-immunoprecipitation with TRX, proliferation and soft-agar growth assays","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding assay with Trx plus functional cellular readouts; single lab","pmids":["24008424"],"is_preprint":false},{"year":2016,"finding":"ASK1 controls mitotic spindle orientation and positioning by phosphorylating EB1 at Ser40, Thr154, and Thr206, enhancing EB1 binding to the plus ends of astral microtubules, stabilizing them, and enabling spindle-cortex interaction for spindle movement.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, live-cell imaging, microtubule co-sedimentation","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis of phosphorylation sites plus live-cell imaging functional readout; multiple orthogonal methods","pmids":["27721984"],"is_preprint":false},{"year":2016,"finding":"ASK1 regulates brown and beige adipocyte function through a PKA-ASK1-p38 axis activated by cAMP signaling, contributing to cell-autonomous induction of Ucp1 expression. Global and fat-specific ASK1 deficiency led to impaired thermogenesis and oxygen consumption.","method":"ASK1-knockout mice (global and fat-specific), gene expression analysis, oxygen consumption measurement","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific and global knockout mice with defined metabolic phenotypes; single lab","pmids":["27045525"],"is_preprint":false},{"year":2016,"finding":"DUSP22 acts as a scaffold protein for the ASK1-MKK7-JNK signaling pathway, selectively associating with ASK1, MKK7, and JNK1/2. DUSP22 increased JNK phosphorylation and apoptosis in a concentration-dependent biphasic manner (scaffold behavior) independently of its phosphatase activity.","method":"Co-immunoprecipitation, JNK phosphorylation assay, apoptosis assay, phosphatase-inactive mutant","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus functional kinase/apoptosis assay with phosphatase mutant; single lab","pmids":["27711255"],"is_preprint":false},{"year":2017,"finding":"TNFAIP3 (A20) deubiquitinase directly interacts with and deubiquitinates ASK1 in hepatocytes, suppressing ASK1 activation. Hepatocyte-specific Tnfaip3 ablation exacerbated NASH-related phenotypes in an ASK1-dependent manner.","method":"Co-immunoprecipitation, deubiquitination assay, hepatocyte-specific knockout mice, rescue experiments","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct interaction, deubiquitination assay, and in vivo ASK1-dependent rescue; multiple orthogonal methods","pmids":["29227477"],"is_preprint":false},{"year":2017,"finding":"TRIM48 promotes ASK1 activation by inducing K48-linked polyubiquitination and proteasomal degradation of PRMT1, which is an ASK1-negative regulator that enhances ASK1's interaction with Trx. TRIM48 knockdown suppressed oxidative stress-induced ASK1 activation and cell death.","method":"Pull-down screen, co-immunoprecipitation, ubiquitination assay, siRNA knockdown, xenograft model","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pull-down, ubiquitination assay, and functional knockdown; single lab, multiple methods","pmids":["29186683"],"is_preprint":false},{"year":2017,"finding":"LRRK2 directly phosphorylates ASK1 at Thr832 (adjacent to the autophosphorylation site Thr845) and acts as a scaffolding protein interacting with each component of the ASK1-MKK3/6-p38 pathway through its specific domains, increasing proximity to downstream targets and promoting neuronal cell death.","method":"Co-immunoprecipitation, in vitro kinase assay, domain-binding assays, neuronal stem cells from PD patients","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus scaffolding domain analysis; single lab","pmids":["28888991"],"is_preprint":false},{"year":2017,"finding":"Cold stress-induced ferroptosis activates the ASK1-p38 MAPK pathway through MEK activity, iron ions, and lipid peroxide. The ferroptosis inducer erastin also activates the ASK1-p38 axis downstream of lipid peroxide accumulation, leading to ASK1-dependent cell death.","method":"Chemical inhibitors (MEK, iron chelators, lipid peroxide inhibitors), ASK1-deficient cell lines, cell death assays","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological epistasis with multiple pathway interventions; single lab","pmids":["28887319"],"is_preprint":false},{"year":2017,"finding":"ASK1 phosphorylates IRF3, resulting in reduced Ucp1 expression in adipocytes. Adipocyte-specific ASK1 knockout increased UCP1 in inguinal fat and elevated energy expenditure, while ASK1 overexpression attenuated cold-induced UCP1.","method":"Adipocyte-specific knockout/overexpression mice, phosphorylation assay for IRF3, UCP1 expression analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific KO and OE plus phosphorylation mechanism; single lab","pmids":["32242025"],"is_preprint":false},{"year":2019,"finding":"TRAF1 promotes myocardial ischemia/reperfusion injury through activation of ASK1-mediated JNK/p38 MAPK cascades. TRAF1 deficiency in mice protected against I/R injury and inflammation.","method":"TRAF1-knockout mice, I/R model, co-immunoprecipitation (implied), Western blot for pathway activation","journal":"Journal of the American Heart Association","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout with defined signaling pathway readout; single lab","pmids":["31650881"],"is_preprint":false},{"year":2020,"finding":"ASK1 phosphorylates HDAC6, blocking its ubiquitination by von Hippel-Lindau and preventing proteasomal degradation of HDAC6, which promotes HDAC6 accumulation and connecting cilia disassembly in the oxygen-induced retinopathy model.","method":"In vivo mouse model, co-immunoprecipitation, phosphorylation assay, ubiquitination assay, HDAC6 knockout/depletion","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphorylation assay, ubiquitination assay, co-IP, and in vivo genetic validation; multiple orthogonal methods","pmids":["32275885"],"is_preprint":false},{"year":2021,"finding":"GSTM2 directly binds the N-terminal region of ASK1 and inhibits ASK1 N-terminal dimerization, suppressing ASK1 phosphorylation and downstream JNK/p38 signaling. GSTM2 deficiency aggravated NASH phenotypes while its overexpression was protective.","method":"Co-immunoprecipitation, in vitro binding assay, gain/loss-of-function in vivo, ASK1 phosphorylation assay","journal":"Journal of hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay with dimerization assay and in vivo validation; single lab","pmids":["34656650"],"is_preprint":false},{"year":2021,"finding":"OTUB1 directly binds TRAF6 and suppresses its K63-linked polyubiquitination, thereby inhibiting TRAF6-mediated ASK1 activation and downstream signaling. Hepatocyte-specific Otub1 overexpression protected against NASH.","method":"Immunoprecipitation-mass spectrometry, ubiquitination assay, co-immunoprecipitation, gain/loss-of-function in vivo","journal":"Hepatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-MS identification, ubiquitination assay, and in vivo validation; single lab","pmids":["34591986"],"is_preprint":false},{"year":2022,"finding":"In C. elegans, CaMKII (UNC-43) activates the Sarm1/TIR-1-ASK1/NSY-1-p38 MAPK pathway to protect against axon degeneration caused by mitochondrial loss. Disruption of a calsyntenin/Mint/kinesin trafficking complex activates this pathway through L-type voltage-gated calcium channels.","method":"Unbiased genetic screen in C. elegans, loss-of-function genetics, epistasis analysis","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis screen in C. elegans ortholog; well-controlled with multiple pathway components tested","pmids":["35285800"],"is_preprint":false},{"year":2022,"finding":"Major vault protein (MVP) binds ASK1 via its α-helical domain and inhibits ASK1 dimerization and phosphorylation, suppressing ASK1-MKK4-JNK signaling and MMP-9 production in macrophages. A 62-amino-acid peptide (MVP-[686-747]) recapitulated this inhibitory function.","method":"Co-immunoprecipitation, ASK1 dimerization assay, phosphorylation assay, myeloid-specific MVP knockout mice","journal":"Arteriosclerosis, thrombosis, and vascular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding/dimerization assay plus genetic KO mice; single lab","pmids":["35387478"],"is_preprint":false}],"current_model":"MAP3K5/ASK1 is a redox-sensitive serine/threonine MAPK kinase kinase that, upon activation (via dissociation of inhibitory thioredoxin, post-translational modifications including phosphorylation at Thr838/Thr845 and dephosphorylation at Ser967, or recruitment by TRAF family members), forms an active homo-oligomeric complex (the 'ASK1 signalosome') that phosphorylates and activates MKK3/MKK6 and MKK4/MKK7, thereby activating p38 and JNK MAP kinases to drive apoptosis, inflammation, differentiation, and fibrosis; its activity is positively regulated by CaMKII (via phosphorylation), LRRK2, Daxx, and TRAF proteins, and negatively regulated by 14-3-3 binding at Ser967, Akt/PDK1/PIM1-mediated Ser83 phosphorylation, Hsp90-Akt complexes, Trx binding, TNFAIP3-mediated deubiquitination, JAK2/SOCS1-mediated Tyr718 phosphorylation and degradation, and SHP2-mediated dephosphorylation; beyond stress-induced apoptosis, ASK1 also phosphorylates non-canonical substrates including cardiac troponin T, EB1 (regulating mitotic spindle orientation), HDAC6 (regulating cilia disassembly), and IRF3 (regulating UCP1 expression in adipocytes)."},"narrative":{"mechanistic_narrative":"MAP3K5/ASK1 is a redox- and stress-responsive serine/threonine MAP kinase kinase kinase that activates the SEK1/MKK4 and MKK3/MKK6 cascades to engage the JNK and p38 MAP kinases, thereby driving apoptosis, differentiation, and inflammation across diverse cell types [PMID:9875215, PMID:9926932]. Its activation is gated by reduced thioredoxin (Trx), which binds and inhibits ASK1 and promotes its ubiquitination until oxidative stress (H2O2, TNF) releases Trx [PMID:12089063]; activation requires homo-oligomerization, and structural analysis shows the catalytic domain forms a tight head-to-tail dimer with regulatory autophosphorylation sites [PMID:17937911]. ASK1 propagates death and differentiation signals downstream of genotoxic stress, TNF/TRAF receptors, viral infection, calcium influx, and ferroptotic lipid peroxidation [PMID:9926932, PMID:10523862, PMID:12878192, PMID:14749717, PMID:28887319], with the p38 arm also directing neuronal and keratinocyte differentiation [PMID:10734135, PMID:11029458]. A dense regulatory network controls ASK1: it is activated by scaffolds and recruiters including Daxx, DAB2IP, TRAF1, LRRK2, and DUSP22, and by CaMKII-mediated phosphorylation [PMID:12968034, PMID:19903888, PMID:31650881, PMID:28888991, PMID:27711255, PMID:14749717], and is restrained by inhibitory phosphorylation at Ser967 (PDK1) and Ser83 (Akt/Hsp90, PIM1), by calcineurin-mediated Ser967 dephosphorylation that toggles 14-3-3 binding, by JAK2/SOCS1-driven Tyr718 phosphorylation and degradation reversed by SHP2, by binding partners that block N-terminal dimerization (GSTM2, MVP), and by deubiquitination via TNFAIP3 [PMID:19920149, PMID:15782121, PMID:19749799, PMID:16648474, PMID:19287004, PMID:34656650, PMID:35387478, PMID:29227477]. Beyond stress apoptosis, ASK1 phosphorylates non-canonical substrates: cardiac troponin T to impair contractility [PMID:12819028], EB1 to control mitotic spindle orientation [PMID:27721984], HDAC6 to regulate cilia disassembly [PMID:32275885], and IRF3 to suppress UCP1 and thermogenesis in adipocytes [PMID:32242025]. Inactivating somatic MAP3K5 mutations occur in melanoma, where one mutant attenuates signaling by increasing Trx binding, consistent with a tumor-suppressive ASK1-JNK axis [PMID:22197930, PMID:24008424].","teleology":[{"year":1998,"claim":"Established ASK1 as a MAP3K that couples to both JNK and p38 cascades and physically engages a downstream kinase, defining its position in the stress-activated MAPK module.","evidence":"Yeast two-hybrid and co-IP in 293 cells identifying MAPKKK6 interaction","pmids":["9875215"],"confidence":"Medium","gaps":["Did not define the activating stimulus","No structural or oligomerization mechanism"]},{"year":1999,"claim":"Defined the apoptotic output of ASK1 and its receptor inputs, showing it acts upstream of caspases via MKK4/MKK3-MKK6 and mediates TRAF-dependent TNF receptor signaling.","evidence":"Dominant-negative ASK1 (K709R), kinase/caspase assays, and TRAF2/5/6 co-IP with JNK activation","pmids":["9926932","10523862"],"confidence":"Medium","gaps":["Mechanism of TRAF-induced ASK1 activation not resolved","Endogenous requirement not yet shown by knockout"]},{"year":2002,"claim":"Identified the redox switch governing ASK1: reduced thioredoxin holds ASK1 inactive and its oxidative release activates signaling, explaining how ROS triggers ASK1.","evidence":"Co-IP with Trx mutants, ubiquitination, JNK and caspase-3 assays; in vivo ischemia/reperfusion with antioxidant","pmids":["12089063","12165419"],"confidence":"High","gaps":["Structural basis of Trx-ASK1 interface not defined","Link between Trx release and oligomerization incomplete"]},{"year":2003,"claim":"Showed ASK1 oligomerization is a controllable activation step engaged by Daxx and antagonized by CIIA, and uncovered the first non-canonical substrate (cardiac troponin T) linking ASK1 to contractile dysfunction.","evidence":"Kinase-cascade epistasis with Daxx relocalization, CIIA antisense oligomerization assays, and cTnT phosphorylation with cardiomyocyte contractility readout","pmids":["12968034","14557248","12819028"],"confidence":"High","gaps":["Oligomerization stoichiometry not defined","Substrate scope beyond cTnT unknown at this stage"]},{"year":2004,"claim":"Established ASK1 as an essential mediator of calcium-to-p38 signaling, placing CaMKII upstream as a direct activating kinase.","evidence":"ASK1-knockout neurons, constitutively active CaMKII, and in vitro phosphorylation","pmids":["14749717"],"confidence":"High","gaps":["CaMKII phosphosite on ASK1 not pinpointed here","Relation to redox activation not integrated"]},{"year":2006,"claim":"Resolved the Ser967/14-3-3 inhibitory checkpoint by showing calcineurin dephosphorylates Ser967 to release 14-3-3 and activate ASK1, with reciprocal ASK1 control of NFAT.","evidence":"In vitro phosphatase assay with purified proteins, endogenous co-IP, and ASK1-deficient MEFs","pmids":["16648474"],"confidence":"High","gaps":["Integration with kinase-mediated Ser967 phosphorylation not addressed","Tissue specificity of the calcineurin-ASK1 axis unclear"]},{"year":2005,"claim":"Defined an Akt/Hsp90-based inhibitory arm acting through Ser83 phosphorylation, showing kinase inputs that suppress ASK1-p38 signaling.","evidence":"Reciprocal co-IP, domain mapping, Akt-knockout cells, in vitro kinase assay","pmids":["15782121"],"confidence":"High","gaps":["How Ser83 phosphorylation mechanistically blocks activation not fully resolved"]},{"year":2007,"claim":"Provided the structural basis for ASK1 activation, showing the catalytic domain dimerizes head-to-tail and mapping regulatory autophosphorylation sites.","evidence":"X-ray crystallography, analytical ultracentrifugation, mass spectrometry, mutagenesis, reporter assays","pmids":["17937911"],"confidence":"High","gaps":["Full-length signalosome architecture not solved","Mechanism by which autophosphorylation regulates output independent of catalytic activity unclear"]},{"year":2009,"claim":"Expanded the regulatory kinase/phosphatase network controlling ASK1, defining PIM1 and PDK1 as Ser83/Ser967 inhibitory kinases and the JAK2/SOCS1/SHP2 axis controlling Tyr718-dependent degradation, plus DAB2IP as a scaffold enhancing activation.","evidence":"In vitro kinase assays, co-IP, siRNA/substrate-trapping mutants, SHP2-KO cells, and xenograft models","pmids":["19749799","19920149","19287004","19903888"],"confidence":"High","gaps":["Hierarchy/competition among Ser83 and Ser967 inputs not resolved","In vivo balance of these regulators uncertain"]},{"year":2011,"claim":"Linked ASK1 to cancer by identifying inactivating somatic MAP3K5 mutations in melanoma, supporting a tumor-suppressive signaling role.","evidence":"Exome sequencing, in vitro kinase assay, overexpression with downstream phosphorylation readout; later mechanism via increased Trx binding","pmids":["22197930","24008424"],"confidence":"Medium","gaps":["Causal contribution to melanomagenesis in vivo not established","Penetrance/frequency of inactivating mutations unclear"]},{"year":2016,"claim":"Revealed non-apoptotic ASK1 functions through new substrates, phosphorylating EB1 to control mitotic spindle orientation and acting via PKA-ASK1-p38 in adipocyte thermogenesis.","evidence":"In vitro kinase assays with phosphosite mutagenesis, live-cell imaging, and global/fat-specific ASK1-knockout mice","pmids":["27721984","27045525"],"confidence":"High","gaps":["How redox/stress regulation intersects with mitotic substrate phosphorylation unclear","Tissue context selecting canonical vs non-canonical substrates undefined"]},{"year":2017,"claim":"Mapped a ubiquitin- and scaffold-based regulatory layer (TNFAIP3 deubiquitination, TRIM48-PRMT1, LRRK2 scaffolding/Thr832 phosphorylation, DUSP22) and connected ASK1 to ferroptotic and disease contexts including NASH and neurodegeneration.","evidence":"Deubiquitination/ubiquitination assays, in vitro kinase assays, conditional knockout mice, and pharmacological ferroptosis epistasis","pmids":["29227477","29186683","28888991","27711255","28887319"],"confidence":"High","gaps":["Relative contribution of competing ubiquitin regulators in vivo unclear","Integration of LRRK2 scaffolding with redox activation incomplete"]},{"year":2020,"claim":"Extended ASK1 substrate repertoire to HDAC6 (controlling cilia disassembly) and IRF3 (suppressing UCP1), establishing roles in ciliary biology and energy expenditure.","evidence":"In vivo mouse models, phosphorylation and ubiquitination assays, adipocyte-specific knockout/overexpression","pmids":["32275885","32242025"],"confidence":"High","gaps":["Stimuli selecting these substrates over canonical MKKs unknown","Structural determinants of substrate selection undefined"]},{"year":2022,"claim":"Defined additional dimerization-blocking inhibitors (GSTM2, MVP) and conserved a CaMKII-Sarm1/TIR-1-ASK1-p38 neuroprotective axis, reinforcing N-terminal dimerization as a druggable activation node.","evidence":"Co-IP and dimerization assays with conditional knockout mice; C. elegans genetic epistasis screen","pmids":["34656650","35387478","35285800"],"confidence":"Medium","gaps":["Whether endogenous GSTM2/MVP set ASK1 tone in human disease unclear","Conservation of CaMKII-Sarm1-ASK1 axis in mammals not directly tested"]},{"year":null,"claim":"How the many competing activating and inhibitory inputs are integrated to set ASK1 signalosome output, and what governs the choice between canonical MKK substrates and non-canonical substrates (cTnT, EB1, HDAC6, IRF3), remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model of ASK1 activation thresholds","Determinants of substrate selectivity unknown","Full-length signalosome structure unsolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,10,27,36,34]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[1,19,23,27]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[19]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[13,16]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9,13]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[27]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,7]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,11,12,14,33]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[7,8,33]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,11,20,35]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[27]}],"complexes":["ASK1 signalosome (ASK1 homo-oligomer)"],"partners":["TXN","TRAF6","DAXX","PDK1","LRRK2","TNFAIP3","GSTM2","MVP"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99683","full_name":"Mitogen-activated protein kinase kinase kinase 5","aliases":["Apoptosis signal-regulating kinase 1","ASK-1","MAPK/ERK kinase kinase 5","MEK kinase 5","MEKK 5"],"length_aa":1374,"mass_kda":154.5,"function":"Serine/threonine kinase which acts as an essential component of the MAP kinase signal transduction pathway. Plays an important role in the cascades of cellular responses evoked by changes in the environment. Mediates signaling for determination of cell fate such as differentiation and survival. Plays a crucial role in the apoptosis signal transduction pathway through mitochondria-dependent caspase activation. MAP3K5/ASK1 is required for the innate immune response, which is essential for host defense against a wide range of pathogens. Mediates signal transduction of various stressors like oxidative stress as well as by receptor-mediated inflammatory signals, such as the tumor necrosis factor (TNF) or lipopolysaccharide (LPS). Once activated, acts as an upstream activator of the MKK/JNK signal transduction cascade and the p38 MAPK signal transduction cascade through the phosphorylation and activation of several MAP kinase kinases like MAP2K4/SEK1, MAP2K3/MKK3, MAP2K6/MKK6 and MAP2K7/MKK7. These MAP2Ks in turn activate p38 MAPKs and c-jun N-terminal kinases (JNKs). Both p38 MAPK and JNKs control the transcription factors activator protein-1 (AP-1)","subcellular_location":"Cytoplasm; Endoplasmic reticulum","url":"https://www.uniprot.org/uniprotkb/Q99683/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAP3K5","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAP3K5","total_profiled":1310},"omim":[{"mim_id":"616967","title":"THIOREDOXIN DOMAIN-CONTAINING PROTEIN 17; TXNDC17","url":"https://www.omim.org/entry/616967"},{"mim_id":"614939","title":"PHOSPHOGLYCERATE MUTASE FAMILY, MEMBER 5; PGAM5","url":"https://www.omim.org/entry/614939"},{"mim_id":"611931","title":"PROTEIN PHOSPHATASE, MAGNESIUM/MANGANESE-DEPENDENT, 1L; PPM1L","url":"https://www.omim.org/entry/611931"},{"mim_id":"611437","title":"DUAL-SPECIFICITY PHOSPHATASE 19; DUSP19","url":"https://www.omim.org/entry/611437"},{"mim_id":"609487","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 2; MAP3K2","url":"https://www.omim.org/entry/609487"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"adrenal 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kinase signaling pathways. A novel interacting protein (MAPKKK6) was identified by yeast two-hybrid screening using MAPKKK5 as bait, and their interaction was confirmed by co-immunoprecipitation in 293 cells.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus confirmatory co-IP; single lab, two methods\",\n      \"pmids\": [\"9875215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"ASK1 mediates apoptotic cell death induced by genotoxic stress (cisplatin) upstream of caspase activation, acting through the SEK1/MKK4 and MKK3/MKK6 pathways to activate JNK1 and p38. Overexpression of kinase-negative ASK1 (K709R) suppressed caspase activation and apoptosis.\",\n      \"method\": \"Dominant-negative overexpression, immune complex kinase assay, caspase activity assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-dead mutant plus downstream pathway analysis; single lab, two orthogonal methods\",\n      \"pmids\": [\"9926932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"TRAF2, TRAF5, and TRAF6 associate with ASK1, and a catalytically-inactive ASK1 mutant blocks SAPK/JNK activation by these TRAFs. ASK1 is a common mediator of TRAF-regulated SAPK and apoptosis signaling downstream of TNF receptors.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative overexpression, JNK activation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding plus functional kinase assay; single lab\",\n      \"pmids\": [\"10523862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Constitutively active ASK1 (ASK1DeltaN) induces neurite outgrowth and neuronal differentiation in PC12 cells through activation of p38 (and to a lesser extent JNK), not ERK. ASK1DeltaN also promoted survival of PC12 cells under serum-starved conditions.\",\n      \"method\": \"Constitutively active mutant overexpression, p38 inhibitor (SB203580), MEK inhibitor controls, morphological/biochemical differentiation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function mutant with pharmacological inhibitor validation; single lab, two orthogonal approaches\",\n      \"pmids\": [\"10734135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Constitutively active ASK1 induces keratinocyte differentiation through the ASK1-p38 MAP kinase cascade, as shown by induction of differentiation markers (transglutaminase-1, loricrin, involucrin) that was blocked by p38 inhibitors SB202190 and SB203580. Ceramide-induced differentiation increased ASK1 expression and activity.\",\n      \"method\": \"Constitutively active ASK1 overexpression, p38 inhibitor treatment, differentiation marker assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function with pharmacological validation; single lab\",\n      \"pmids\": [\"11029458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ASK1 possesses a kinase-independent, caspase-independent cell death function activated by interaction with Daxx. The N-terminal domain of ASK1 (lacking the kinase domain) was constitutively active in producing crumpled nuclei, distinct from caspase-dependent fragmented nuclei caused by ASK1 kinase activity.\",\n      \"method\": \"Co-transfection, domain deletion mutants, caspase inhibitor, morphological assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple domain mutants with functional cell death readout; single lab\",\n      \"pmids\": [\"11493600\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ASK1-JNK pathway stabilizes c-Myc protein by phosphorylating Ser-62 and Ser-71, attenuating degradation of ubiquitinated c-Myc without affecting its ubiquitination, thus promoting c-Myc-dependent apoptosis.\",\n      \"method\": \"Overexpression, site-directed mutagenesis of c-Myc, pulse-chase, kinase assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis of phosphorylation sites plus functional outcome; single lab\",\n      \"pmids\": [\"11243879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Reduced thioredoxin (Trx) binds to and inhibits ASK1. Upon oxidation (via H2O2 or TNF), Trx is released from ASK1. Single-Cys Trx mutants (C32S or C35S) that constitutively bind ASK1 promoted ASK1 ubiquitination and degradation, inhibiting JNK activation and apoptosis in a redox-independent manner. The double mutant (Trx-CS) lost ASK1-binding and inhibitory activity.\",\n      \"method\": \"Co-immunoprecipitation, overexpression of Trx mutants, ubiquitination assay, JNK activation assay, caspase-3 assay\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple Trx mutants with reciprocal co-IP and multiple functional readouts; single lab with orthogonal methods\",\n      \"pmids\": [\"12089063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ASK1 is activated and autophosphorylated following cerebral ischemia/reperfusion in rat hippocampus, peaking at 30 min reperfusion. Both activation and autophosphorylation were suppressed by the antioxidant N-acetylcysteine, indicating redox-dependent regulation.\",\n      \"method\": \"In vitro kinase assay, immunoprecipitation, Western blot, antioxidant treatment in vivo\",\n      \"journal\": \"Neuroscience letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus in vivo pharmacological inhibition; single lab\",\n      \"pmids\": [\"12165419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"ALG-2 interacts with the C-terminus (aa 941–1375) of ASK1, and co-transfection with ALG-2 causes nuclear localization of ASK1 and inhibits ASK1-mediated JNK activation. ALG-2 variant with deletion of Gly-121/Phe-122 failed to bind ASK1, demonstrating specificity.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization (immunofluorescence), JNK activation assay, in vitro binding\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus localization plus functional JNK assay; single lab\",\n      \"pmids\": [\"12372597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ASK1 specifically interacts with cardiac troponin T (cTnT) via its C-terminal domain, phosphorylates cTnT at T194/S198 in vitro and in vivo, and ROS-induced ASK1 activation causes cTnT phosphorylation and contractile dysfunction in cardiomyocytes. Constitutively active ASK1 overexpression inhibited sarcomere shortening and calcium transients.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis of cTnT, cardiomyocyte contractility assay\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis plus in vivo functional readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"12819028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ASK1 regulates influenza virus infection-induced apoptosis: IV infection activated ASK1, phosphorylated JNK and p38, and ASK1-knockout MEFs showed reduced JNK/p38 activation and were defective in caspase-3 activation and cell death after IV infection.\",\n      \"method\": \"ASK1 knockout mouse-derived MEFs, kinase assays, caspase-3 activation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with defined signaling and cell death readouts; single lab\",\n      \"pmids\": [\"12878192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ASK1 mediates anandamide-induced PC12 cell death through p38 MAPK and JNK activation, upstream of mitochondrial cytochrome c release and caspase activation. Dominant-negative ASK1 (K709M) inhibited p38/JNK activation and cell death.\",\n      \"method\": \"Dominant-negative overexpression, pharmacological inhibitors, cytochrome c release assay\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase-dead mutant plus pharmacological validation; single lab\",\n      \"pmids\": [\"12641726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The ASK1-SEK1-JNK1-HIPK1 pathway mediates Daxx phosphorylation at Ser667 during glucose deprivation, causing Daxx translocation from nucleus to cytoplasm where it binds ASK1 and promotes ASK1 oligomerization. JNK binding domain overexpression blocked Daxx relocalization and ASK1 oligomerization.\",\n      \"method\": \"In vivo labeling, immune complex kinase assay, immunofluorescence, protein interaction assay, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase cascade epistasis with mutagenesis; single lab, multiple methods\",\n      \"pmids\": [\"12968034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CIIA (a novel antiapoptotic protein) binds ASK1 and inhibits oligomerization-induced ASK1 activation. CIIA antisense abolished its inhibitory effect on ASK1 activation, DNA fragmentation, and apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, antisense oligonucleotides, ASK1 oligomerization assay, apoptosis assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding assay plus functional ASK1 activation readout plus loss-of-function; single lab\",\n      \"pmids\": [\"14557248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Ca2+ influx activates p38 MAP kinase via CaMKII phosphorylation of ASK1. p38 activation by membrane depolarization in primary neurons was impaired in ASK1-deficient mice. Constitutively active CaMKII-induced p38 activation required endogenous ASK1.\",\n      \"method\": \"ASK1-knockout mouse neurons, constitutively active CaMKII overexpression, in vitro phosphorylation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout plus in vitro phosphorylation plus constitutively active upstream kinase; replicated across multiple assays\",\n      \"pmids\": [\"14749717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Hsp90 and Akt form a ternary complex with ASK1 via the middle domain of Hsp90. Akt phosphorylates ASK1 at Ser83, and this phosphorylation is required for Hsp90-mediated inhibition of ASK1-p38 signaling. In resting endothelial cells, Akt binds the N-terminal domain of ASK1; upon H2O2 stimulation it shifts to the C-terminal domain of ASK1.\",\n      \"method\": \"Co-immunoprecipitation, domain-mapping, Akt knockout cells, in vitro kinase assay, pharmacological inhibitors\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, domain mapping, genetic KO cells, in vitro kinase assay; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"15782121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Raf-1 kinase domain interacts with ALG-2, and both Raf-1 and ASK1 phosphorylate ALG-2. Raf-1 blocks ASK1-dependent ALG-2 phosphorylation, suggesting a mechanism for Raf-1's anti-apoptotic function through interference with the ASK1-ALG-2 axis.\",\n      \"method\": \"Yeast two-hybrid, in vitro kinase assay, co-immunoprecipitation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus yeast two-hybrid; single lab\",\n      \"pmids\": [\"15925322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Calcineurin directly dephosphorylates ASK1 at Ser967 (identified by yeast two-hybrid with calcineurin B as bait), promoting dissociation of 14-3-3 proteins from ASK1 and ASK1 activation. Calcineurin and ASK1 cooperatively enhanced cardiomyocyte apoptosis. Reciprocally, ASK1 negatively regulated calcineurin-NFAT signaling through JNK- and p38-mediated NFAT phosphorylation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation of endogenous proteins, in vitro phosphatase assay with purified proteins, ASK1-deficient MEFs, dominant-negative ASK1\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro phosphatase assay with purified proteins plus reciprocal co-IP of endogenous proteins plus genetic knockout validation; multiple orthogonal methods\",\n      \"pmids\": [\"16648474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Crystal structure of the human ASK1 catalytic domain in complex with staurosporine revealed that ASK1 forms a tight head-to-tail dimer (Kd ~0.2 μM). Three autophosphorylation sites (Thr813, Thr838, Thr842) were identified by mass spectrometry and shown to regulate ASK1 signaling by reporter gene assays, though site-directed mutants showed catalytic activities similar to wild-type, suggesting a regulatory mechanism independent of kinase activity.\",\n      \"method\": \"X-ray crystallography, analytical ultracentrifugation, mass spectrometry, site-directed mutagenesis, reporter gene assay\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus AUC plus MS autophosphorylation mapping plus mutagenesis; multiple orthogonal methods in single study\",\n      \"pmids\": [\"17937911\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ASK1 deficiency in mice resulted in dramatically reduced wounding-induced macrophage infiltration/activation and subsequent hair regrowth. Intracutaneous transplantation of cytokine-activated bone marrow-derived macrophages rescued hair growth in ASK1-deficient mice, placing ASK1 upstream of macrophage recruitment in inflammatory wound responses.\",\n      \"method\": \"ASK1-knockout mice, oligonucleotide microarray, bone marrow macrophage transplantation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with rescue experiment; single lab\",\n      \"pmids\": [\"17389227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PIM1 phosphorylates ASK1 specifically at Ser83 in vitro and in vivo, binds ASK1 by co-immunoprecipitation, decreases ASK1 kinase activity under oxidative stress, and inhibits ASK1-mediated JNK and p38 phosphorylation and caspase-3 activation. PIM1 knockdown reduced Ser83 phosphorylation and increased cell death after H2O2.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, siRNA knockdown, caspase-3 activation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus co-IP plus loss-of-function knockdown with multiple functional readouts; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"19749799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"DAB2IP acts as a scaffold protein interacting with ASK1 through its C2 domain to enhance ASK1 activation, leading to JNK pathway activation and apoptosis. DAB2IP loss in prostate cancer led to ASK1-JNK inactivation and accelerated tumor growth in vivo.\",\n      \"method\": \"Gain/loss-of-function studies, structural domain analysis, in vivo xenograft\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping plus in vivo model; single lab\",\n      \"pmids\": [\"19903888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PDK1 and ASK1 directly interact via the PDK1 PH domain and ASK1 C-terminal regulatory domain, and they reciprocally phosphorylate each other: PDK1 phosphorylates ASK1 at Ser967 (a 14-3-3 binding site) to suppress ASK1 activity, while ASK1 phosphorylates PDK1 at Ser394 and Ser398 to inhibit PDK1. The interaction is decreased by ASK1-activating stimuli.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, domain-mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reciprocal in vitro kinase assays with mutagenesis plus co-IP; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"19920149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"JAK2 phosphorylates ASK1 at Tyr718, enhancing its association with SOCS1 and subsequent ASK1 degradation. SHP2 dephosphorylates ASK1 at Tyr718 (confirmed by substrate-trapping mutant), dissociating SOCS1 from ASK1 and enabling TNF-induced ASK1 activation and apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, JAK2-specific inhibitor, SHP2 substrate-trapping mutant, SHP2-KO cells, JAK2 overexpression with in vitro kinase assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay, substrate-trapping mutant, genetic KO cells, multiple orthogonal methods; single lab\",\n      \"pmids\": [\"19287004\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Somatic mutations in MAP3K5 (e.g., I780F) identified in melanoma exomes reduced kinase activity in in vitro kinase assays and reduced phosphorylation of downstream MAP kinases when overexpressed in HEK293T cells. Loss of heterozygosity suggested these are inactivating mutations.\",\n      \"method\": \"Exome sequencing, in vitro kinase assay, overexpression in HEK293T cells with downstream phosphorylation readout\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus cellular overexpression; single study\",\n      \"pmids\": [\"22197930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The MAP3K5 R256C melanoma mutation attenuates MKK4 activation through increased binding of the inhibitory protein thioredoxin (TRX), resulting in increased melanoma cell proliferation and anchorage-independent growth.\",\n      \"method\": \"Functional kinase assays, co-immunoprecipitation with TRX, proliferation and soft-agar growth assays\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding assay with Trx plus functional cellular readouts; single lab\",\n      \"pmids\": [\"24008424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ASK1 controls mitotic spindle orientation and positioning by phosphorylating EB1 at Ser40, Thr154, and Thr206, enhancing EB1 binding to the plus ends of astral microtubules, stabilizing them, and enabling spindle-cortex interaction for spindle movement.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis, live-cell imaging, microtubule co-sedimentation\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis of phosphorylation sites plus live-cell imaging functional readout; multiple orthogonal methods\",\n      \"pmids\": [\"27721984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ASK1 regulates brown and beige adipocyte function through a PKA-ASK1-p38 axis activated by cAMP signaling, contributing to cell-autonomous induction of Ucp1 expression. Global and fat-specific ASK1 deficiency led to impaired thermogenesis and oxygen consumption.\",\n      \"method\": \"ASK1-knockout mice (global and fat-specific), gene expression analysis, oxygen consumption measurement\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific and global knockout mice with defined metabolic phenotypes; single lab\",\n      \"pmids\": [\"27045525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DUSP22 acts as a scaffold protein for the ASK1-MKK7-JNK signaling pathway, selectively associating with ASK1, MKK7, and JNK1/2. DUSP22 increased JNK phosphorylation and apoptosis in a concentration-dependent biphasic manner (scaffold behavior) independently of its phosphatase activity.\",\n      \"method\": \"Co-immunoprecipitation, JNK phosphorylation assay, apoptosis assay, phosphatase-inactive mutant\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus functional kinase/apoptosis assay with phosphatase mutant; single lab\",\n      \"pmids\": [\"27711255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TNFAIP3 (A20) deubiquitinase directly interacts with and deubiquitinates ASK1 in hepatocytes, suppressing ASK1 activation. Hepatocyte-specific Tnfaip3 ablation exacerbated NASH-related phenotypes in an ASK1-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, deubiquitination assay, hepatocyte-specific knockout mice, rescue experiments\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct interaction, deubiquitination assay, and in vivo ASK1-dependent rescue; multiple orthogonal methods\",\n      \"pmids\": [\"29227477\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRIM48 promotes ASK1 activation by inducing K48-linked polyubiquitination and proteasomal degradation of PRMT1, which is an ASK1-negative regulator that enhances ASK1's interaction with Trx. TRIM48 knockdown suppressed oxidative stress-induced ASK1 activation and cell death.\",\n      \"method\": \"Pull-down screen, co-immunoprecipitation, ubiquitination assay, siRNA knockdown, xenograft model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pull-down, ubiquitination assay, and functional knockdown; single lab, multiple methods\",\n      \"pmids\": [\"29186683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"LRRK2 directly phosphorylates ASK1 at Thr832 (adjacent to the autophosphorylation site Thr845) and acts as a scaffolding protein interacting with each component of the ASK1-MKK3/6-p38 pathway through its specific domains, increasing proximity to downstream targets and promoting neuronal cell death.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, domain-binding assays, neuronal stem cells from PD patients\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus scaffolding domain analysis; single lab\",\n      \"pmids\": [\"28888991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cold stress-induced ferroptosis activates the ASK1-p38 MAPK pathway through MEK activity, iron ions, and lipid peroxide. The ferroptosis inducer erastin also activates the ASK1-p38 axis downstream of lipid peroxide accumulation, leading to ASK1-dependent cell death.\",\n      \"method\": \"Chemical inhibitors (MEK, iron chelators, lipid peroxide inhibitors), ASK1-deficient cell lines, cell death assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological epistasis with multiple pathway interventions; single lab\",\n      \"pmids\": [\"28887319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ASK1 phosphorylates IRF3, resulting in reduced Ucp1 expression in adipocytes. Adipocyte-specific ASK1 knockout increased UCP1 in inguinal fat and elevated energy expenditure, while ASK1 overexpression attenuated cold-induced UCP1.\",\n      \"method\": \"Adipocyte-specific knockout/overexpression mice, phosphorylation assay for IRF3, UCP1 expression analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific KO and OE plus phosphorylation mechanism; single lab\",\n      \"pmids\": [\"32242025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRAF1 promotes myocardial ischemia/reperfusion injury through activation of ASK1-mediated JNK/p38 MAPK cascades. TRAF1 deficiency in mice protected against I/R injury and inflammation.\",\n      \"method\": \"TRAF1-knockout mice, I/R model, co-immunoprecipitation (implied), Western blot for pathway activation\",\n      \"journal\": \"Journal of the American Heart Association\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with defined signaling pathway readout; single lab\",\n      \"pmids\": [\"31650881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ASK1 phosphorylates HDAC6, blocking its ubiquitination by von Hippel-Lindau and preventing proteasomal degradation of HDAC6, which promotes HDAC6 accumulation and connecting cilia disassembly in the oxygen-induced retinopathy model.\",\n      \"method\": \"In vivo mouse model, co-immunoprecipitation, phosphorylation assay, ubiquitination assay, HDAC6 knockout/depletion\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphorylation assay, ubiquitination assay, co-IP, and in vivo genetic validation; multiple orthogonal methods\",\n      \"pmids\": [\"32275885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GSTM2 directly binds the N-terminal region of ASK1 and inhibits ASK1 N-terminal dimerization, suppressing ASK1 phosphorylation and downstream JNK/p38 signaling. GSTM2 deficiency aggravated NASH phenotypes while its overexpression was protective.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assay, gain/loss-of-function in vivo, ASK1 phosphorylation assay\",\n      \"journal\": \"Journal of hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay with dimerization assay and in vivo validation; single lab\",\n      \"pmids\": [\"34656650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"OTUB1 directly binds TRAF6 and suppresses its K63-linked polyubiquitination, thereby inhibiting TRAF6-mediated ASK1 activation and downstream signaling. Hepatocyte-specific Otub1 overexpression protected against NASH.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry, ubiquitination assay, co-immunoprecipitation, gain/loss-of-function in vivo\",\n      \"journal\": \"Hepatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-MS identification, ubiquitination assay, and in vivo validation; single lab\",\n      \"pmids\": [\"34591986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In C. elegans, CaMKII (UNC-43) activates the Sarm1/TIR-1-ASK1/NSY-1-p38 MAPK pathway to protect against axon degeneration caused by mitochondrial loss. Disruption of a calsyntenin/Mint/kinesin trafficking complex activates this pathway through L-type voltage-gated calcium channels.\",\n      \"method\": \"Unbiased genetic screen in C. elegans, loss-of-function genetics, epistasis analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis screen in C. elegans ortholog; well-controlled with multiple pathway components tested\",\n      \"pmids\": [\"35285800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Major vault protein (MVP) binds ASK1 via its α-helical domain and inhibits ASK1 dimerization and phosphorylation, suppressing ASK1-MKK4-JNK signaling and MMP-9 production in macrophages. A 62-amino-acid peptide (MVP-[686-747]) recapitulated this inhibitory function.\",\n      \"method\": \"Co-immunoprecipitation, ASK1 dimerization assay, phosphorylation assay, myeloid-specific MVP knockout mice\",\n      \"journal\": \"Arteriosclerosis, thrombosis, and vascular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding/dimerization assay plus genetic KO mice; single lab\",\n      \"pmids\": [\"35387478\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAP3K5/ASK1 is a redox-sensitive serine/threonine MAPK kinase kinase that, upon activation (via dissociation of inhibitory thioredoxin, post-translational modifications including phosphorylation at Thr838/Thr845 and dephosphorylation at Ser967, or recruitment by TRAF family members), forms an active homo-oligomeric complex (the 'ASK1 signalosome') that phosphorylates and activates MKK3/MKK6 and MKK4/MKK7, thereby activating p38 and JNK MAP kinases to drive apoptosis, inflammation, differentiation, and fibrosis; its activity is positively regulated by CaMKII (via phosphorylation), LRRK2, Daxx, and TRAF proteins, and negatively regulated by 14-3-3 binding at Ser967, Akt/PDK1/PIM1-mediated Ser83 phosphorylation, Hsp90-Akt complexes, Trx binding, TNFAIP3-mediated deubiquitination, JAK2/SOCS1-mediated Tyr718 phosphorylation and degradation, and SHP2-mediated dephosphorylation; beyond stress-induced apoptosis, ASK1 also phosphorylates non-canonical substrates including cardiac troponin T, EB1 (regulating mitotic spindle orientation), HDAC6 (regulating cilia disassembly), and IRF3 (regulating UCP1 expression in adipocytes).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAP3K5/ASK1 is a redox- and stress-responsive serine/threonine MAP kinase kinase kinase that activates the SEK1/MKK4 and MKK3/MKK6 cascades to engage the JNK and p38 MAP kinases, thereby driving apoptosis, differentiation, and inflammation across diverse cell types [#0, #1]. Its activation is gated by reduced thioredoxin (Trx), which binds and inhibits ASK1 and promotes its ubiquitination until oxidative stress (H2O2, TNF) releases Trx [#7]; activation requires homo-oligomerization, and structural analysis shows the catalytic domain forms a tight head-to-tail dimer with regulatory autophosphorylation sites [#19]. ASK1 propagates death and differentiation signals downstream of genotoxic stress, TNF/TRAF receptors, viral infection, calcium influx, and ferroptotic lipid peroxidation [#1, #2, #11, #15, #33], with the p38 arm also directing neuronal and keratinocyte differentiation [#3, #4]. A dense regulatory network controls ASK1: it is activated by scaffolds and recruiters including Daxx, DAB2IP, TRAF1, LRRK2, and DUSP22, and by CaMKII-mediated phosphorylation [#13, #22, #35, #32, #29, #15], and is restrained by inhibitory phosphorylation at Ser967 (PDK1) and Ser83 (Akt/Hsp90, PIM1), by calcineurin-mediated Ser967 dephosphorylation that toggles 14-3-3 binding, by JAK2/SOCS1-driven Tyr718 phosphorylation and degradation reversed by SHP2, by binding partners that block N-terminal dimerization (GSTM2, MVP), and by deubiquitination via TNFAIP3 [#23, #16, #21, #18, #24, #37, #40, #30]. Beyond stress apoptosis, ASK1 phosphorylates non-canonical substrates: cardiac troponin T to impair contractility [#10], EB1 to control mitotic spindle orientation [#27], HDAC6 to regulate cilia disassembly [#36], and IRF3 to suppress UCP1 and thermogenesis in adipocytes [#34]. Inactivating somatic MAP3K5 mutations occur in melanoma, where one mutant attenuates signaling by increasing Trx binding, consistent with a tumor-suppressive ASK1-JNK axis [#25, #26].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established ASK1 as a MAP3K that couples to both JNK and p38 cascades and physically engages a downstream kinase, defining its position in the stress-activated MAPK module.\",\n      \"evidence\": \"Yeast two-hybrid and co-IP in 293 cells identifying MAPKKK6 interaction\",\n      \"pmids\": [\"9875215\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the activating stimulus\", \"No structural or oligomerization mechanism\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined the apoptotic output of ASK1 and its receptor inputs, showing it acts upstream of caspases via MKK4/MKK3-MKK6 and mediates TRAF-dependent TNF receptor signaling.\",\n      \"evidence\": \"Dominant-negative ASK1 (K709R), kinase/caspase assays, and TRAF2/5/6 co-IP with JNK activation\",\n      \"pmids\": [\"9926932\", \"10523862\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of TRAF-induced ASK1 activation not resolved\", \"Endogenous requirement not yet shown by knockout\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identified the redox switch governing ASK1: reduced thioredoxin holds ASK1 inactive and its oxidative release activates signaling, explaining how ROS triggers ASK1.\",\n      \"evidence\": \"Co-IP with Trx mutants, ubiquitination, JNK and caspase-3 assays; in vivo ischemia/reperfusion with antioxidant\",\n      \"pmids\": [\"12089063\", \"12165419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Trx-ASK1 interface not defined\", \"Link between Trx release and oligomerization incomplete\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed ASK1 oligomerization is a controllable activation step engaged by Daxx and antagonized by CIIA, and uncovered the first non-canonical substrate (cardiac troponin T) linking ASK1 to contractile dysfunction.\",\n      \"evidence\": \"Kinase-cascade epistasis with Daxx relocalization, CIIA antisense oligomerization assays, and cTnT phosphorylation with cardiomyocyte contractility readout\",\n      \"pmids\": [\"12968034\", \"14557248\", \"12819028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Oligomerization stoichiometry not defined\", \"Substrate scope beyond cTnT unknown at this stage\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Established ASK1 as an essential mediator of calcium-to-p38 signaling, placing CaMKII upstream as a direct activating kinase.\",\n      \"evidence\": \"ASK1-knockout neurons, constitutively active CaMKII, and in vitro phosphorylation\",\n      \"pmids\": [\"14749717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CaMKII phosphosite on ASK1 not pinpointed here\", \"Relation to redox activation not integrated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved the Ser967/14-3-3 inhibitory checkpoint by showing calcineurin dephosphorylates Ser967 to release 14-3-3 and activate ASK1, with reciprocal ASK1 control of NFAT.\",\n      \"evidence\": \"In vitro phosphatase assay with purified proteins, endogenous co-IP, and ASK1-deficient MEFs\",\n      \"pmids\": [\"16648474\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration with kinase-mediated Ser967 phosphorylation not addressed\", \"Tissue specificity of the calcineurin-ASK1 axis unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined an Akt/Hsp90-based inhibitory arm acting through Ser83 phosphorylation, showing kinase inputs that suppress ASK1-p38 signaling.\",\n      \"evidence\": \"Reciprocal co-IP, domain mapping, Akt-knockout cells, in vitro kinase assay\",\n      \"pmids\": [\"15782121\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Ser83 phosphorylation mechanistically blocks activation not fully resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Provided the structural basis for ASK1 activation, showing the catalytic domain dimerizes head-to-tail and mapping regulatory autophosphorylation sites.\",\n      \"evidence\": \"X-ray crystallography, analytical ultracentrifugation, mass spectrometry, mutagenesis, reporter assays\",\n      \"pmids\": [\"17937911\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length signalosome architecture not solved\", \"Mechanism by which autophosphorylation regulates output independent of catalytic activity unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Expanded the regulatory kinase/phosphatase network controlling ASK1, defining PIM1 and PDK1 as Ser83/Ser967 inhibitory kinases and the JAK2/SOCS1/SHP2 axis controlling Tyr718-dependent degradation, plus DAB2IP as a scaffold enhancing activation.\",\n      \"evidence\": \"In vitro kinase assays, co-IP, siRNA/substrate-trapping mutants, SHP2-KO cells, and xenograft models\",\n      \"pmids\": [\"19749799\", \"19920149\", \"19287004\", \"19903888\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hierarchy/competition among Ser83 and Ser967 inputs not resolved\", \"In vivo balance of these regulators uncertain\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Linked ASK1 to cancer by identifying inactivating somatic MAP3K5 mutations in melanoma, supporting a tumor-suppressive signaling role.\",\n      \"evidence\": \"Exome sequencing, in vitro kinase assay, overexpression with downstream phosphorylation readout; later mechanism via increased Trx binding\",\n      \"pmids\": [\"22197930\", \"24008424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal contribution to melanomagenesis in vivo not established\", \"Penetrance/frequency of inactivating mutations unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed non-apoptotic ASK1 functions through new substrates, phosphorylating EB1 to control mitotic spindle orientation and acting via PKA-ASK1-p38 in adipocyte thermogenesis.\",\n      \"evidence\": \"In vitro kinase assays with phosphosite mutagenesis, live-cell imaging, and global/fat-specific ASK1-knockout mice\",\n      \"pmids\": [\"27721984\", \"27045525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How redox/stress regulation intersects with mitotic substrate phosphorylation unclear\", \"Tissue context selecting canonical vs non-canonical substrates undefined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mapped a ubiquitin- and scaffold-based regulatory layer (TNFAIP3 deubiquitination, TRIM48-PRMT1, LRRK2 scaffolding/Thr832 phosphorylation, DUSP22) and connected ASK1 to ferroptotic and disease contexts including NASH and neurodegeneration.\",\n      \"evidence\": \"Deubiquitination/ubiquitination assays, in vitro kinase assays, conditional knockout mice, and pharmacological ferroptosis epistasis\",\n      \"pmids\": [\"29227477\", \"29186683\", \"28888991\", \"27711255\", \"28887319\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of competing ubiquitin regulators in vivo unclear\", \"Integration of LRRK2 scaffolding with redox activation incomplete\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended ASK1 substrate repertoire to HDAC6 (controlling cilia disassembly) and IRF3 (suppressing UCP1), establishing roles in ciliary biology and energy expenditure.\",\n      \"evidence\": \"In vivo mouse models, phosphorylation and ubiquitination assays, adipocyte-specific knockout/overexpression\",\n      \"pmids\": [\"32275885\", \"32242025\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stimuli selecting these substrates over canonical MKKs unknown\", \"Structural determinants of substrate selection undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined additional dimerization-blocking inhibitors (GSTM2, MVP) and conserved a CaMKII-Sarm1/TIR-1-ASK1-p38 neuroprotective axis, reinforcing N-terminal dimerization as a druggable activation node.\",\n      \"evidence\": \"Co-IP and dimerization assays with conditional knockout mice; C. elegans genetic epistasis screen\",\n      \"pmids\": [\"34656650\", \"35387478\", \"35285800\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether endogenous GSTM2/MVP set ASK1 tone in human disease unclear\", \"Conservation of CaMKII-Sarm1-ASK1 axis in mammals not directly tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many competing activating and inhibitory inputs are integrated to set ASK1 signalosome output, and what governs the choice between canonical MKK substrates and non-canonical substrates (cTnT, EB1, HDAC6, IRF3), remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model of ASK1 activation thresholds\", \"Determinants of substrate selectivity unknown\", \"Full-length signalosome structure unsolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 10, 27, 36, 34]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [1, 19, 23, 27]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [13, 16]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9, 13]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [27]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 7]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 11, 12, 14, 33]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [7, 8, 33]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 11, 20, 35]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [27]}\n    ],\n    \"complexes\": [\"ASK1 signalosome (ASK1 homo-oligomer)\"],\n    \"partners\": [\"TXN\", \"TRAF6\", \"DAXX\", \"PDK1\", \"LRRK2\", \"TNFAIP3\", \"GSTM2\", \"MVP\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}