{"gene":"MAP3K10","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1997,"finding":"MLK2/MST (MAP3K10) directly phosphorylates and activates SEK1/MKK4/JNKK in vitro, establishing it as a MAP kinase kinase kinase that preferentially activates the JNK/SAPK pathway; recombinant MLK2 produced in bacteria phosphorylated SEK1 in an in vitro kinase assay.","method":"In vitro kinase assay with bacterially expressed recombinant MLK2; COS-1 cell overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro kinase assay with recombinant protein, replicated by activation data in cells, consistent with multiple subsequent studies","pmids":["9182538"],"is_preprint":false},{"year":1998,"finding":"MLK2 interacts with the GTP-bound (activated) forms of Rac and Cdc42 (preferring Rac) via its CRIB motif, as shown by yeast two-hybrid and in vitro dot-blot assays. MLK2 also interacts with members of the KIF3 kinesin superfamily motor proteins and with KAP3A (the targeting component of KIF3 complexes), and co-localizes with dually phosphorylated (active) JNK1/2 along microtubules in fibroblasts.","method":"Yeast two-hybrid, in vitro dot-blot, COS cell transfection, immunofluorescence co-localization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Y2H, in vitro binding, cellular co-localization), independently consistent with the known CRIB motif function","pmids":["9427749"],"is_preprint":false},{"year":1998,"finding":"Transfection of MLK2 into COS cells leads to activation of the JNK, ERK, and p38 MAP kinase cascades, with strongest and constitutive activation of JNK.","method":"COS cell transfection with kinase activity assays","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based overexpression with kinase activity readouts, single lab but consistent with biochemical data","pmids":["9427749"],"is_preprint":false},{"year":2000,"finding":"Normal huntingtin interacts with MLK2, and polyglutamine expansion of huntingtin disrupts this interaction. Expression of MLK2 induces JNK activation and apoptosis in HN33 neuronal cells; dominant-negative MLK2 attenuates apoptosis induced by polyglutamine-expanded huntingtin.","method":"Co-immunoprecipitation, transfection with dominant-negative constructs, JNK activity assay, apoptosis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional epistasis with dominant-negative, single lab","pmids":["10801775"],"is_preprint":false},{"year":2001,"finding":"Activated JNK2 phosphorylates MLK2 at multiple sites predominantly in its noncatalytic C-terminal region both in vitro and in vivo. The C-terminal domain of MLK2 is required for MLK2-induced apoptosis (N-terminal domain alone can activate JNK but is insufficient for apoptosis), identifying a feedback phosphorylation loop where JNK phosphorylates MLK2 to enable its apoptotic function.","method":"Phosphopeptide mapping, in vitro kinase assay with activated JNK2, cotransfection with dominant-negative JNK kinase, deletion mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with phosphopeptide mapping plus in vivo validation and deletion mutagenesis, single lab with multiple orthogonal methods","pmids":["11278395"],"is_preprint":false},{"year":2003,"finding":"MLK2 phosphorylates the NeuroD basic helix-loop-helix transcription factor and stimulates its transcriptional activity. Huntingtin and HAP1 interact with NeuroD and facilitate activation of NeuroD by MLK2, suggesting a scaffold function for Htt/HAP1 in MLK2-mediated NeuroD activation.","method":"Yeast two-hybrid screen, in vitro kinase assay, transcription reporter assays, co-immunoprecipitation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Y2H plus in vitro kinase assay and functional reporter, single lab with multiple methods","pmids":["12881483"],"is_preprint":false},{"year":2003,"finding":"PAK1 and MLK2 interact via their catalytic domains and PAK1 squelches MLK2-mediated JNK activation, suggesting that PAK1 recruits MLK2 to an activated receptor via the NCK adapter but cannot itself induce JNK cascade activation.","method":"Co-immunoprecipitation, overexpression/squelching assays, JNK activity assays","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus functional squelching assays, single lab, single paper","pmids":["12753919"],"is_preprint":false},{"year":2003,"finding":"Xenopus MLK2 (62% homology to mammalian MLK2) activates JNK in a SEK1/MKK4-dependent manner in COS7 cells. In vivo antisense inactivation and dominant-negative xMLK2 show that xMLK2 is required for normal cement gland development and pronephric tubule formation, establishing a tissue-restricted in vivo role for MLK2 in organogenesis.","method":"Antisense knockdown, dominant-negative overexpression, COS7 cell transfection with kinase assay, in situ hybridization","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative and antisense loss-of-function with specific developmental phenotypes, ortholog in Xenopus consistent with mammalian function","pmids":["12591241"],"is_preprint":false},{"year":2013,"finding":"RNAi-mediated depletion of MAP3K10 inhibits TGFβ-induced p38 MAPK phosphorylation in MEFs and HaCaT keratinocytes. Depletion of MAP3K10 from cells carrying a catalytically inactive MAP3K4 knock-in completely abolishes TGFβ-induced p38 phosphorylation, placing MAP3K10 and MAP3K4 as sufficient mediators of TGFβ→p38 signaling (and ruling out TAK1/MAP3K7 in this context).","method":"RNAi knockdown, catalytically inactive MAP3K4 knock-in cells, immunoblotting for p38 phosphorylation","journal":"Open biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis (RNAi + knock-in), negative control (TAK1 excluded), replicated in two cell types","pmids":["23760366"],"is_preprint":false},{"year":2020,"finding":"MAP3K10 interacts with and phosphorylates GlyRS (glycyl-tRNA synthetase) in bovine mammary epithelial cells, acting as an upstream kinase of GlyRS. This phosphorylation is stimulated by methionine through the GPR87-CDC42/Rac1-MAP3K10 signaling axis. Phosphorylated GlyRS then activates NFκB1.","method":"Co-immunoprecipitation, mass spectrometry, Western blotting, in vitro kinase assay, signaling pathway knockdown/inhibition","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vitro kinase assay and pathway manipulation, single lab, multiple orthogonal methods","pmids":["31954518"],"is_preprint":false},{"year":2012,"finding":"MAP3K10 overexpression in pancreatic cancer cells upregulates Hedgehog pathway components Gli-1 and Gli-2, promoting cell proliferation and decreasing gemcitabine sensitivity; MAP3K10 knockdown decreases proliferation and sensitizes cells to gemcitabine, but neither manipulation affects cell migration.","method":"Overexpression and shRNA knockdown, viability/proliferation assays, Western blotting for Gli-1/Gli-2","journal":"Cancer letters","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method set, no direct kinase-substrate experiment establishing the GLI connection","pmids":["23178452"],"is_preprint":false}],"current_model":"MAP3K10 (MLK2) is a dual-specificity MAPK kinase kinase that directly phosphorylates and activates SEK1/MKK4, preferentially activating the JNK/SAPK cascade (and also ERK and p38 to a lesser extent); it is activated by binding GTP-Rac/Cdc42 via its CRIB motif, co-localizes with active JNK along microtubules via interaction with KIF3 motor complexes, undergoes feedback phosphorylation by activated JNK2 in its C-terminal domain (required for apoptosis), participates in TGFβ-induced p38 activation alongside MAP3K4, phosphorylates non-canonical substrates including NeuroD and GlyRS, and is recruited to JNK-activating complexes through interactions with huntingtin, HAP1, NCK, and PAK1."},"narrative":{"mechanistic_narrative":"MAP3K10 (MLK2) is a mixed-lineage MAP kinase kinase kinase that nucleates activation of stress-responsive MAPK cascades, most prominently the JNK/SAPK pathway, by directly phosphorylating and activating SEK1/MKK4 [PMID:9182538]. Its output spans JNK, ERK, and p38, with strongest and constitutive activation of JNK upon expression [PMID:9427749]. Activation is controlled by small GTPase input: MLK2 binds GTP-loaded Rac and Cdc42 (preferring Rac) through its CRIB motif, links to KIF3 kinesin motor complexes and the KAP3A targeting subunit, and co-localizes with active JNK along microtubules [PMID:9427749]. A feedback loop refines its function — activated JNK2 phosphorylates MLK2 at multiple sites in its noncatalytic C-terminal region, and this C-terminal domain is required for MLK2-induced apoptosis even though the N-terminal catalytic domain alone suffices to activate JNK [PMID:11278395]. MLK2 is recruited into JNK-activating complexes through scaffolding and adaptor interactions, including normal huntingtin (an interaction disrupted by polyglutamine expansion), HAP1, and PAK1 acting via the NCK adapter [PMID:10801775, PMID:12753919]. Beyond canonical MAPK substrates, MLK2 phosphorylates the bHLH transcription factor NeuroD to stimulate its transcriptional activity in a huntingtin/HAP1-facilitated manner [PMID:12881483], and phosphorylates GlyRS downstream of a GPR87-CDC42/Rac1 axis to activate NFκB1 [PMID:31954518]. In TGFβ signaling, MAP3K10 together with MAP3K4 are sufficient mediators of TGFβ-induced p38 activation, with TAK1/MAP3K7 excluded in this context [PMID:23760366]. An in vivo developmental role is established in Xenopus, where MLK2 is required for cement gland and pronephric tubule formation [PMID:12591241].","teleology":[{"year":1997,"claim":"Established MLK2 as a bona fide MAP3K by demonstrating it directly phosphorylates and activates the JNK-pathway MAP2K SEK1/MKK4, placing it upstream in stress kinase signaling.","evidence":"In vitro kinase assay with bacterially expressed recombinant MLK2 plus COS-1 overexpression","pmids":["9182538"],"confidence":"High","gaps":["Did not define physiological upstream activators","Relative contribution to ERK/p38 versus JNK not resolved here"]},{"year":1998,"claim":"Defined how MLK2 is activated and spatially organized — GTP-Rac/Cdc42 binding via the CRIB motif and association with KIF3/KAP3A motors targeting it along microtubules with active JNK.","evidence":"Yeast two-hybrid, in vitro dot-blot, COS transfection, immunofluorescence co-localization","pmids":["9427749"],"confidence":"High","gaps":["Mechanistic consequence of microtubule localization for signaling output not established","Direct kinetic effect of GTPase binding on kinase activity not quantified"]},{"year":1998,"claim":"Showed MLK2 engages multiple MAPK cascades in cells, with preferential JNK activation, defining its signaling breadth.","evidence":"COS cell transfection with kinase activity assays","pmids":["9427749"],"confidence":"Medium","gaps":["Overexpression context may overstate ERK/p38 engagement","Endogenous selectivity untested"]},{"year":2000,"claim":"Linked MLK2 to neuronal apoptosis and disease, showing huntingtin binds MLK2, polyQ expansion disrupts this, and dominant-negative MLK2 blunts polyQ-huntingtin-induced death.","evidence":"Reciprocal Co-IP, dominant-negative transfection, JNK and apoptosis assays in HN33 neuronal cells","pmids":["10801775"],"confidence":"Medium","gaps":["Single-lab functional epistasis","Direct causal role in vivo not shown"]},{"year":2001,"claim":"Uncovered a JNK→MLK2 feedback loop, mapping JNK2 phosphorylation to the C-terminal region and showing this domain is required for the apoptotic, but not the JNK-activating, function of MLK2.","evidence":"Phosphopeptide mapping, in vitro kinase assay with activated JNK2, dominant-negative JNK cotransfection, deletion mutants","pmids":["11278395"],"confidence":"High","gaps":["Specific phosphosites and their individual contributions not resolved","Downstream effectors of C-terminal phosphorylation unknown"]},{"year":2003,"claim":"Expanded MLK2 substrate repertoire beyond MAP2Ks by showing it phosphorylates and activates the transcription factor NeuroD, with huntingtin/HAP1 serving as scaffold.","evidence":"Yeast two-hybrid, in vitro kinase assay, transcription reporter assays, Co-IP","pmids":["12881483"],"confidence":"Medium","gaps":["Phosphosites on NeuroD not mapped","Physiological/in vivo relevance unestablished"]},{"year":2003,"claim":"Identified PAK1 as a catalytic-domain partner that recruits MLK2 (via NCK) but squelches its JNK activation, indicating receptor-coupled regulation of MLK2 output.","evidence":"Co-IP, overexpression/squelching assays, JNK activity assays","pmids":["12753919"],"confidence":"Medium","gaps":["Single paper, single lab","Direct NCK-MLK2-PAK1 ternary complex at a receptor not demonstrated"]},{"year":2003,"claim":"Provided in vivo evidence for an organismal role, showing Xenopus MLK2 activates JNK via SEK1/MKK4 and is required for cement gland and pronephric tubule development.","evidence":"Antisense knockdown, dominant-negative overexpression, COS7 kinase assay, in situ hybridization","pmids":["12591241"],"confidence":"Medium","gaps":["Mammalian developmental role not directly demonstrated","Tissue-specific effectors unknown"]},{"year":2012,"claim":"Implicated MAP3K10 in pancreatic cancer cell proliferation and gemcitabine resistance through Hedgehog/Gli upregulation.","evidence":"Overexpression and shRNA knockdown, proliferation assays, Western blotting for Gli-1/Gli-2","pmids":["23178452"],"confidence":"Low","gaps":["No direct kinase-substrate experiment linking MAP3K10 to GLI","Mechanism connecting MAP3K10 to Hedgehog signaling unresolved"]},{"year":2013,"claim":"Placed MAP3K10 in the TGFβ→p38 pathway, showing it and MAP3K4 are sufficient mediators of TGFβ-induced p38 phosphorylation while excluding TAK1.","evidence":"RNAi knockdown, catalytically inactive MAP3K4 knock-in cells, p38 phospho-immunoblotting in MEFs and HaCaT cells","pmids":["23760366"],"confidence":"High","gaps":["Whether MAP3K10 acts on the same MAP2Ks as MAP3K4 not resolved","Direct substrate in the TGFβ context not identified"]},{"year":2020,"claim":"Defined a methionine-responsive signaling axis where GPR87-CDC42/Rac1 activates MAP3K10 to phosphorylate GlyRS, which in turn activates NFκB1.","evidence":"Co-IP, mass spectrometry, in vitro kinase assay, pathway knockdown/inhibition in bovine mammary epithelial cells","pmids":["31954518"],"confidence":"Medium","gaps":["GlyRS phosphosites not fully defined","Generalizability beyond mammary epithelial context untested"]},{"year":null,"claim":"How MAP3K10 selects among JNK, ERK, and p38 outputs and among canonical versus non-canonical substrates in a given cellular and developmental context remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of GTPase/scaffold-gated activation","Endogenous substrate selectivity not systematically mapped","Mammalian loss-of-function phenotype undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,4,5,9]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,5,9]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,4]}],"complexes":[],"partners":["MAP2K4","RAC1","CDC42","KAP3A","HTT","HAP1","PAK1","GARS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q02779","full_name":"Mitogen-activated protein kinase kinase kinase 10","aliases":["Mixed lineage kinase 2","Protein kinase MST"],"length_aa":954,"mass_kda":103.7,"function":"Activates the JUN N-terminal pathway","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q02779/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAP3K10","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAP3K10","total_profiled":1310},"omim":[{"mim_id":"614793","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 21; MAP3K21","url":"https://www.omim.org/entry/614793"},{"mim_id":"609479","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 20; MAP3K20","url":"https://www.omim.org/entry/609479"},{"mim_id":"600137","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 10; MAP3K10","url":"https://www.omim.org/entry/600137"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Actin filaments","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":73.5}],"url":"https://www.proteinatlas.org/search/MAP3K10"},"hgnc":{"alias_symbol":["MST","MEKK10"],"prev_symbol":["MLK2"]},"alphafold":{"accession":"Q02779","domains":[{"cath_id":"2.30.30.40","chopping":"21-79","consensus_level":"high","plddt":86.919,"start":21,"end":79},{"cath_id":"1.10.510.10","chopping":"185-200_219-225_267-438","consensus_level":"medium","plddt":91.7722,"start":185,"end":438}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q02779","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q02779-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q02779-F1-predicted_aligned_error_v6.png","plddt_mean":60.28},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAP3K10","jax_strain_url":"https://www.jax.org/strain/search?query=MAP3K10"},"sequence":{"accession":"Q02779","fasta_url":"https://rest.uniprot.org/uniprotkb/Q02779.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q02779/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q02779"}},"corpus_meta":[{"pmid":"9427749","id":"PMC_9427749","title":"The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.","date":"1998","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9427749","citation_count":228,"is_preprint":false},{"pmid":"9182538","id":"PMC_9182538","title":"MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates SEK1, an activator of c-Jun N-terminal kinase/stress-activated protein kinase.","date":"1997","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9182538","citation_count":168,"is_preprint":false},{"pmid":"23189122","id":"PMC_23189122","title":"Regulation of microRNA-155 in atherosclerotic inflammatory responses by targeting MAP3K10.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23189122","citation_count":113,"is_preprint":false},{"pmid":"10801775","id":"PMC_10801775","title":"Activation of MLK2-mediated signaling cascades by polyglutamine-expanded huntingtin.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10801775","citation_count":68,"is_preprint":false},{"pmid":"12881483","id":"PMC_12881483","title":"Stimulation of NeuroD activity by huntingtin and huntingtin-associated proteins HAP1 and MLK2.","date":"2003","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12881483","citation_count":61,"is_preprint":false},{"pmid":"9150946","id":"PMC_9150946","title":"Two-dimensional electrophoretic analysis of human breast carcinoma proteins: mapping of proteins that bind to the SH3 domain of mixed lineage kinase MLK2.","date":"1997","source":"Electrophoresis","url":"https://pubmed.ncbi.nlm.nih.gov/9150946","citation_count":41,"is_preprint":false},{"pmid":"23760366","id":"PMC_23760366","title":"The TGFβ-induced phosphorylation and activation of p38 mitogen-activated protein kinase is mediated by MAP3K4 and MAP3K10 but not TAK1.","date":"2013","source":"Open biology","url":"https://pubmed.ncbi.nlm.nih.gov/23760366","citation_count":23,"is_preprint":false},{"pmid":"18414056","id":"PMC_18414056","title":"Mice lacking both mixed-lineage kinase genes Mlk1 and Mlk2 retain a wild type phenotype.","date":"2008","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/18414056","citation_count":21,"is_preprint":false},{"pmid":"23178452","id":"PMC_23178452","title":"MAP3K10 promotes the proliferation and decreases the sensitivity of pancreatic cancer cells to gemcitabine by upregulating Gli-1 and Gli-2.","date":"2012","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/23178452","citation_count":21,"is_preprint":false},{"pmid":"11278395","id":"PMC_11278395","title":"Activated JNK phosphorylates the c-terminal domain of MLK2 that is required for MLK2-induced apoptosis.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11278395","citation_count":21,"is_preprint":false},{"pmid":"33995647","id":"PMC_33995647","title":"MiR-146b-3p regulates proliferation of pancreatic cancer cells with stem cell-like properties by targeting MAP3K10.","date":"2021","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/33995647","citation_count":20,"is_preprint":false},{"pmid":"12753919","id":"PMC_12753919","title":"PAK interacts with NCK and MLK2 to regulate the activation of jun N-terminal kinase.","date":"2003","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12753919","citation_count":15,"is_preprint":false},{"pmid":"31954518","id":"PMC_31954518","title":"Methionine stimulates GlyRS phosphorylation via the GPR87-CDC42/Rac1-MAP3K10 signaling pathway.","date":"2020","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/31954518","citation_count":14,"is_preprint":false},{"pmid":"12591241","id":"PMC_12591241","title":"A tissue restricted role for the Xenopus Jun N-terminal kinase kinase kinase MLK2 in cement gland and pronephric tubule differentiation.","date":"2003","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/12591241","citation_count":13,"is_preprint":false},{"pmid":"28214207","id":"PMC_28214207","title":"MicroRNA-155 targets MAP3K10 and regulates osteosarcoma cell growth.","date":"2017","source":"Pathology, research and practice","url":"https://pubmed.ncbi.nlm.nih.gov/28214207","citation_count":7,"is_preprint":false},{"pmid":"29431572","id":"PMC_29431572","title":"MLK1 and MLK2 integrate gibberellins and circadian clock signaling to modulate plant growth.","date":"2018","source":"Plant signaling & behavior","url":"https://pubmed.ncbi.nlm.nih.gov/29431572","citation_count":6,"is_preprint":false},{"pmid":"32323857","id":"PMC_32323857","title":"DNA methylation‑regulated miR‑155‑5p depresses sensitivity of esophageal carcinoma cells to radiation and multiple chemotherapeutic drugs via suppression of MAP3K10.","date":"2020","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/32323857","citation_count":6,"is_preprint":false},{"pmid":"36341729","id":"PMC_36341729","title":"DNA methylation of the MAP3K10 gene may participate in the development of intracranial aneurysm.","date":"2022","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/36341729","citation_count":4,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.28.684510","title":"Comparative genomic analyses of trans-ithmanian reef fishes reveals different molecular targets of environmental adaptation in different families.","date":"2025-10-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.28.684510","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.07.08.663677","title":"Microbial Scl1 Activates TGFβR1 receptor kinase signalling to Drive Fibrosis – Inflammation Axis in Alcohol-Associated Liver Disease","date":"2025-07-08","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.08.663677","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12172,"output_tokens":2817,"usd":0.039385,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10069,"output_tokens":3666,"usd":0.070998,"stage2_stop_reason":"end_turn"},"total_usd":0.110383,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"MLK2/MST (MAP3K10) directly phosphorylates and activates SEK1/MKK4/JNKK in vitro, establishing it as a MAP kinase kinase kinase that preferentially activates the JNK/SAPK pathway; recombinant MLK2 produced in bacteria phosphorylated SEK1 in an in vitro kinase assay.\",\n      \"method\": \"In vitro kinase assay with bacterially expressed recombinant MLK2; COS-1 cell overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro kinase assay with recombinant protein, replicated by activation data in cells, consistent with multiple subsequent studies\",\n      \"pmids\": [\"9182538\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"MLK2 interacts with the GTP-bound (activated) forms of Rac and Cdc42 (preferring Rac) via its CRIB motif, as shown by yeast two-hybrid and in vitro dot-blot assays. MLK2 also interacts with members of the KIF3 kinesin superfamily motor proteins and with KAP3A (the targeting component of KIF3 complexes), and co-localizes with dually phosphorylated (active) JNK1/2 along microtubules in fibroblasts.\",\n      \"method\": \"Yeast two-hybrid, in vitro dot-blot, COS cell transfection, immunofluorescence co-localization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Y2H, in vitro binding, cellular co-localization), independently consistent with the known CRIB motif function\",\n      \"pmids\": [\"9427749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Transfection of MLK2 into COS cells leads to activation of the JNK, ERK, and p38 MAP kinase cascades, with strongest and constitutive activation of JNK.\",\n      \"method\": \"COS cell transfection with kinase activity assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based overexpression with kinase activity readouts, single lab but consistent with biochemical data\",\n      \"pmids\": [\"9427749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Normal huntingtin interacts with MLK2, and polyglutamine expansion of huntingtin disrupts this interaction. Expression of MLK2 induces JNK activation and apoptosis in HN33 neuronal cells; dominant-negative MLK2 attenuates apoptosis induced by polyglutamine-expanded huntingtin.\",\n      \"method\": \"Co-immunoprecipitation, transfection with dominant-negative constructs, JNK activity assay, apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional epistasis with dominant-negative, single lab\",\n      \"pmids\": [\"10801775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Activated JNK2 phosphorylates MLK2 at multiple sites predominantly in its noncatalytic C-terminal region both in vitro and in vivo. The C-terminal domain of MLK2 is required for MLK2-induced apoptosis (N-terminal domain alone can activate JNK but is insufficient for apoptosis), identifying a feedback phosphorylation loop where JNK phosphorylates MLK2 to enable its apoptotic function.\",\n      \"method\": \"Phosphopeptide mapping, in vitro kinase assay with activated JNK2, cotransfection with dominant-negative JNK kinase, deletion mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with phosphopeptide mapping plus in vivo validation and deletion mutagenesis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"11278395\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"MLK2 phosphorylates the NeuroD basic helix-loop-helix transcription factor and stimulates its transcriptional activity. Huntingtin and HAP1 interact with NeuroD and facilitate activation of NeuroD by MLK2, suggesting a scaffold function for Htt/HAP1 in MLK2-mediated NeuroD activation.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro kinase assay, transcription reporter assays, co-immunoprecipitation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Y2H plus in vitro kinase assay and functional reporter, single lab with multiple methods\",\n      \"pmids\": [\"12881483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PAK1 and MLK2 interact via their catalytic domains and PAK1 squelches MLK2-mediated JNK activation, suggesting that PAK1 recruits MLK2 to an activated receptor via the NCK adapter but cannot itself induce JNK cascade activation.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/squelching assays, JNK activity assays\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus functional squelching assays, single lab, single paper\",\n      \"pmids\": [\"12753919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Xenopus MLK2 (62% homology to mammalian MLK2) activates JNK in a SEK1/MKK4-dependent manner in COS7 cells. In vivo antisense inactivation and dominant-negative xMLK2 show that xMLK2 is required for normal cement gland development and pronephric tubule formation, establishing a tissue-restricted in vivo role for MLK2 in organogenesis.\",\n      \"method\": \"Antisense knockdown, dominant-negative overexpression, COS7 cell transfection with kinase assay, in situ hybridization\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative and antisense loss-of-function with specific developmental phenotypes, ortholog in Xenopus consistent with mammalian function\",\n      \"pmids\": [\"12591241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RNAi-mediated depletion of MAP3K10 inhibits TGFβ-induced p38 MAPK phosphorylation in MEFs and HaCaT keratinocytes. Depletion of MAP3K10 from cells carrying a catalytically inactive MAP3K4 knock-in completely abolishes TGFβ-induced p38 phosphorylation, placing MAP3K10 and MAP3K4 as sufficient mediators of TGFβ→p38 signaling (and ruling out TAK1/MAP3K7 in this context).\",\n      \"method\": \"RNAi knockdown, catalytically inactive MAP3K4 knock-in cells, immunoblotting for p38 phosphorylation\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis (RNAi + knock-in), negative control (TAK1 excluded), replicated in two cell types\",\n      \"pmids\": [\"23760366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MAP3K10 interacts with and phosphorylates GlyRS (glycyl-tRNA synthetase) in bovine mammary epithelial cells, acting as an upstream kinase of GlyRS. This phosphorylation is stimulated by methionine through the GPR87-CDC42/Rac1-MAP3K10 signaling axis. Phosphorylated GlyRS then activates NFκB1.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, Western blotting, in vitro kinase assay, signaling pathway knockdown/inhibition\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vitro kinase assay and pathway manipulation, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"31954518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MAP3K10 overexpression in pancreatic cancer cells upregulates Hedgehog pathway components Gli-1 and Gli-2, promoting cell proliferation and decreasing gemcitabine sensitivity; MAP3K10 knockdown decreases proliferation and sensitizes cells to gemcitabine, but neither manipulation affects cell migration.\",\n      \"method\": \"Overexpression and shRNA knockdown, viability/proliferation assays, Western blotting for Gli-1/Gli-2\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method set, no direct kinase-substrate experiment establishing the GLI connection\",\n      \"pmids\": [\"23178452\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAP3K10 (MLK2) is a dual-specificity MAPK kinase kinase that directly phosphorylates and activates SEK1/MKK4, preferentially activating the JNK/SAPK cascade (and also ERK and p38 to a lesser extent); it is activated by binding GTP-Rac/Cdc42 via its CRIB motif, co-localizes with active JNK along microtubules via interaction with KIF3 motor complexes, undergoes feedback phosphorylation by activated JNK2 in its C-terminal domain (required for apoptosis), participates in TGFβ-induced p38 activation alongside MAP3K4, phosphorylates non-canonical substrates including NeuroD and GlyRS, and is recruited to JNK-activating complexes through interactions with huntingtin, HAP1, NCK, and PAK1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAP3K10 (MLK2) is a mixed-lineage MAP kinase kinase kinase that nucleates activation of stress-responsive MAPK cascades, most prominently the JNK/SAPK pathway, by directly phosphorylating and activating SEK1/MKK4 [#0]. Its output spans JNK, ERK, and p38, with strongest and constitutive activation of JNK upon expression [#2]. Activation is controlled by small GTPase input: MLK2 binds GTP-loaded Rac and Cdc42 (preferring Rac) through its CRIB motif, links to KIF3 kinesin motor complexes and the KAP3A targeting subunit, and co-localizes with active JNK along microtubules [#1]. A feedback loop refines its function — activated JNK2 phosphorylates MLK2 at multiple sites in its noncatalytic C-terminal region, and this C-terminal domain is required for MLK2-induced apoptosis even though the N-terminal catalytic domain alone suffices to activate JNK [#4]. MLK2 is recruited into JNK-activating complexes through scaffolding and adaptor interactions, including normal huntingtin (an interaction disrupted by polyglutamine expansion), HAP1, and PAK1 acting via the NCK adapter [#3, #6]. Beyond canonical MAPK substrates, MLK2 phosphorylates the bHLH transcription factor NeuroD to stimulate its transcriptional activity in a huntingtin/HAP1-facilitated manner [#5], and phosphorylates GlyRS downstream of a GPR87-CDC42/Rac1 axis to activate NF\\u03baB1 [#9]. In TGF\\u03b2 signaling, MAP3K10 together with MAP3K4 are sufficient mediators of TGF\\u03b2-induced p38 activation, with TAK1/MAP3K7 excluded in this context [#8]. An in vivo developmental role is established in Xenopus, where MLK2 is required for cement gland and pronephric tubule formation [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established MLK2 as a bona fide MAP3K by demonstrating it directly phosphorylates and activates the JNK-pathway MAP2K SEK1/MKK4, placing it upstream in stress kinase signaling.\",\n      \"evidence\": \"In vitro kinase assay with bacterially expressed recombinant MLK2 plus COS-1 overexpression\",\n      \"pmids\": [\"9182538\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define physiological upstream activators\", \"Relative contribution to ERK/p38 versus JNK not resolved here\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined how MLK2 is activated and spatially organized — GTP-Rac/Cdc42 binding via the CRIB motif and association with KIF3/KAP3A motors targeting it along microtubules with active JNK.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro dot-blot, COS transfection, immunofluorescence co-localization\",\n      \"pmids\": [\"9427749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic consequence of microtubule localization for signaling output not established\", \"Direct kinetic effect of GTPase binding on kinase activity not quantified\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Showed MLK2 engages multiple MAPK cascades in cells, with preferential JNK activation, defining its signaling breadth.\",\n      \"evidence\": \"COS cell transfection with kinase activity assays\",\n      \"pmids\": [\"9427749\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Overexpression context may overstate ERK/p38 engagement\", \"Endogenous selectivity untested\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Linked MLK2 to neuronal apoptosis and disease, showing huntingtin binds MLK2, polyQ expansion disrupts this, and dominant-negative MLK2 blunts polyQ-huntingtin-induced death.\",\n      \"evidence\": \"Reciprocal Co-IP, dominant-negative transfection, JNK and apoptosis assays in HN33 neuronal cells\",\n      \"pmids\": [\"10801775\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab functional epistasis\", \"Direct causal role in vivo not shown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Uncovered a JNK\\u2192MLK2 feedback loop, mapping JNK2 phosphorylation to the C-terminal region and showing this domain is required for the apoptotic, but not the JNK-activating, function of MLK2.\",\n      \"evidence\": \"Phosphopeptide mapping, in vitro kinase assay with activated JNK2, dominant-negative JNK cotransfection, deletion mutants\",\n      \"pmids\": [\"11278395\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphosites and their individual contributions not resolved\", \"Downstream effectors of C-terminal phosphorylation unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Expanded MLK2 substrate repertoire beyond MAP2Ks by showing it phosphorylates and activates the transcription factor NeuroD, with huntingtin/HAP1 serving as scaffold.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro kinase assay, transcription reporter assays, Co-IP\",\n      \"pmids\": [\"12881483\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphosites on NeuroD not mapped\", \"Physiological/in vivo relevance unestablished\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified PAK1 as a catalytic-domain partner that recruits MLK2 (via NCK) but squelches its JNK activation, indicating receptor-coupled regulation of MLK2 output.\",\n      \"evidence\": \"Co-IP, overexpression/squelching assays, JNK activity assays\",\n      \"pmids\": [\"12753919\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single paper, single lab\", \"Direct NCK-MLK2-PAK1 ternary complex at a receptor not demonstrated\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Provided in vivo evidence for an organismal role, showing Xenopus MLK2 activates JNK via SEK1/MKK4 and is required for cement gland and pronephric tubule development.\",\n      \"evidence\": \"Antisense knockdown, dominant-negative overexpression, COS7 kinase assay, in situ hybridization\",\n      \"pmids\": [\"12591241\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian developmental role not directly demonstrated\", \"Tissue-specific effectors unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Implicated MAP3K10 in pancreatic cancer cell proliferation and gemcitabine resistance through Hedgehog/Gli upregulation.\",\n      \"evidence\": \"Overexpression and shRNA knockdown, proliferation assays, Western blotting for Gli-1/Gli-2\",\n      \"pmids\": [\"23178452\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct kinase-substrate experiment linking MAP3K10 to GLI\", \"Mechanism connecting MAP3K10 to Hedgehog signaling unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed MAP3K10 in the TGF\\u03b2\\u2192p38 pathway, showing it and MAP3K4 are sufficient mediators of TGF\\u03b2-induced p38 phosphorylation while excluding TAK1.\",\n      \"evidence\": \"RNAi knockdown, catalytically inactive MAP3K4 knock-in cells, p38 phospho-immunoblotting in MEFs and HaCaT cells\",\n      \"pmids\": [\"23760366\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MAP3K10 acts on the same MAP2Ks as MAP3K4 not resolved\", \"Direct substrate in the TGF\\u03b2 context not identified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined a methionine-responsive signaling axis where GPR87-CDC42/Rac1 activates MAP3K10 to phosphorylate GlyRS, which in turn activates NF\\u03baB1.\",\n      \"evidence\": \"Co-IP, mass spectrometry, in vitro kinase assay, pathway knockdown/inhibition in bovine mammary epithelial cells\",\n      \"pmids\": [\"31954518\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GlyRS phosphosites not fully defined\", \"Generalizability beyond mammary epithelial context untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MAP3K10 selects among JNK, ERK, and p38 outputs and among canonical versus non-canonical substrates in a given cellular and developmental context remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of GTPase/scaffold-gated activation\", \"Endogenous substrate selectivity not systematically mapped\", \"Mammalian loss-of-function phenotype undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 4, 5, 9]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 5, 9]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MAP2K4\", \"RAC1\", \"CDC42\", \"KAP3A\", \"HTT\", \"HAP1\", \"PAK1\", \"GARS1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":8,"faith_total":8,"faith_pct":100.0}}