{"gene":"MAP3K2","run_date":"2026-04-28T18:30:27","timeline":{"discoveries":[{"year":2014,"finding":"SMYD3 methyltransferase methylates MAP3K2 at lysine 260, which potentiates activation of the Ras/Raf/MEK/ERK signaling module. Methylation of MAP3K2 at K260 blocks binding of the PP2A phosphatase complex to MAP3K2, thereby preventing PP2A-mediated negative regulation of MAP kinase signaling.","method":"Protein array to identify SMYD3 substrates, in vitro methylation assays, mass spectrometry, Co-IP, mouse cancer models (pancreatic and lung adenocarcinoma), MEK inhibitor synergy studies","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods including in vitro methylation, Co-IP, mutagenesis, and in vivo mouse models; replicated in prostate cancer context (PMID:37976356)","pmids":["24847881","37976356"],"is_preprint":false},{"year":2005,"finding":"Smurf1, a HECT domain ubiquitin E3 ligase, physically interacts with MAP3K2 (MEKK2) and promotes its ubiquitination and proteasomal degradation, thereby suppressing osteoblast activity and JNK signaling downstream of BMP.","method":"Co-IP, ubiquitination assays, Smurf1 knockout mice, western blot for phospho-MEKK2 accumulation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 — reciprocal Co-IP, in vivo knockout phenotype, biochemical ubiquitination assays; foundational paper with 310 citations","pmids":["15820682"],"is_preprint":false},{"year":2003,"finding":"WNK1 acts upstream of MAP3K2 (MEKK2) and MAP3K3 (MEKK3) in the ERK5 pathway: WNK1 phosphorylates MEKK2 and MEKK3 in vitro, co-immunoprecipitates with endogenous WNK1, and dominant-negative MEKK2/3 block WNK1-induced ERK5 activation.","method":"Co-immunoprecipitation, in vitro kinase assay, dominant-negative overexpression, siRNA knockdown, HEK293 cell transfection","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay plus reciprocal Co-IP plus dominant-negative epistasis; multiple orthogonal methods","pmids":["14681216"],"is_preprint":false},{"year":2000,"finding":"MAP3K2 (MEKK2) directly binds MEK5 (identified by yeast two-hybrid) and activates the MEK5-BMK1/ERK5 pathway; MEKK2 also binds the T cell adapter protein Lad/RIBP and co-localizes with it at the T cell/antigen-presenting cell interface during T cell activation.","method":"Yeast two-hybrid library screening, dominant-negative overexpression, live-cell co-localization imaging, kinase activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — yeast two-hybrid confirmed by functional dominant-negative epistasis and co-localization; 122 citations","pmids":["11073940"],"is_preprint":false},{"year":2003,"finding":"The N-terminal PB1 domains of MEKK2 and MEKK3 heterodimerize with the PB1 domain of MEK5 (but not with each other), and this PB1-mediated interaction is required for complex formation and ERK5 pathway activation. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 complexes and blocks ERK5 activation.","method":"In vitro PB1 domain binding assays, co-immunoprecipitation from cell lysates, deletion/point mutants, dominant-negative overexpression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro binding reconstitution plus Co-IP plus mutagenesis; multiple orthogonal methods in single study","pmids":["12912994"],"is_preprint":false},{"year":2000,"finding":"MEKK2 gene disruption in ES cell-derived mast cells causes loss of receptor-mediated JNK activation and cytokine gene transcription in response to IgE receptor (FcεRI) or c-Kit ligation, while UV-induced JNK activation remains intact, demonstrating that MEKK2 is specifically required for tyrosine kinase receptor signaling in mast cells.","method":"Targeted gene disruption (MEKK2−/− ES cell-derived mast cells), kinase activity assays, cytokine mRNA measurement","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — clean genetic knockout with specific pathway readout; receptor-specific vs. stress-specific JNK activation discrimination; 89 citations","pmids":["11032806"],"is_preprint":false},{"year":2004,"finding":"MEKK2 coordinates activation of both ERK5 and JNK pathways in response to FGF-2; MEKK2-knockout MEFs lose ERK5 and JNK activation specifically in response to FGF-2 (but not LPS or TNFα), and show impaired AP-1 component expression and cytokine gene induction.","method":"MEKK2−/− mouse embryonic fibroblasts, kinase activity assays, RT-PCR for AP-1 components and cytokines","journal":"Journal of cellular physiology","confidence":"High","confidence_rationale":"Tier 2 — genetic knockout with multiple specific pathway readouts; stimulus-selective requirement established","pmids":["14978743"],"is_preprint":false},{"year":2002,"finding":"MEKK2 knockout mice exhibit augmented T cell proliferation and enhanced IL-2 and IFNγ production in response to anti-CD3 stimulation, with moderately enhanced (not reduced) JNK activation in MEKK2−/− T cells, indicating MEKK2 negatively modulates TCR signal strength.","method":"Mekk2−/− mouse generation, T cell proliferation assays, cytokine ELISA, JNK/ERK/p38 kinase assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — clean KO mouse with defined cellular phenotypes and pathway measurements; 52 citations","pmids":["12138187"],"is_preprint":false},{"year":2004,"finding":"In resting cells MEKK2 is localized in the cytosol, and upon EGF stimulation it translocates into the nucleus where MEK5 and ERK5 are constitutively localized, allowing transmission of signals to nuclear MEK5.","method":"Immunofluorescence, in situ NP-40 detergent extraction, subcellular fractionation, EGF stimulation of HeLa and Rat-1 cells","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — direct localization experiments with functional context (nuclear signal transmission); multiple cell lines and methods","pmids":["15075238"],"is_preprint":false},{"year":1999,"finding":"MEKK2 is translocated to the T cell/antigen-presenting cell contact interface upon antigen stimulation of the TCR; dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK and p38 MAP kinase phosphorylation.","method":"Immunocytochemistry, live-cell fluorescence imaging, dominant-negative expression, kinase phosphorylation assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — live-cell imaging of translocation with functional dominant-negative validation; 45 citations","pmids":["10549623"],"is_preprint":false},{"year":2005,"finding":"MEKK2 activation requires dimerization through its catalytic domain: inactive/non-phosphorylated MEKK2 forms more dimers than phosphorylated MEKK2, and chemical-induced dimerization in vivo augments MEKK2-dependent JNK activation. Prevention of dimerization inhibits JNK signaling.","method":"Dimerization domain mapping, chemical-induced dimerization (CID) system, co-immunoprecipitation, JNK/AP-1 reporter assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — mechanistic dimerization mapping plus chemical dimerization rescue plus reporter assays; multiple orthogonal methods","pmids":["15695508"],"is_preprint":false},{"year":2005,"finding":"A novel MEKK2-interacting protein, Mip1, forms a complex with inactive/non-phosphorylated MEKK2 and prevents its activation by blocking MEKK2 dimer formation. EGF stimulation dissociates the endogenous Mip1-MEKK2 complex; Mip1 siRNA knockdown augments MEKK2-mediated JNK and AP-1 activation.","method":"Co-immunoprecipitation, siRNA knockdown, MEKK2 dimerization assays, JNK/ERK5/AP-1 reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — Co-IP plus siRNA plus functional reporters; multiple orthogonal methods in single study","pmids":["15988011"],"is_preprint":false},{"year":2005,"finding":"Serine 519 of MEKK2 (and the corresponding Ser526 of MEKK3) is a key regulatory phosphorylation site: S519A mutation severely impairs MEKK2 activation; LPS (via TRAF6) induces phosphorylation at this serine, which is required for TLR-induced IL-6 production.","method":"Site-directed mutagenesis, phospho-specific antibody generation, in vitro kinase assays, LPS stimulation, siRNA knockdown of TRAF6","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — mutagenesis combined with phospho-specific antibody validation and functional readout; 50 citations","pmids":["16362041"],"is_preprint":false},{"year":2007,"finding":"The MEKK2 PB1 domain uses a front-to-back arrangement to bind MEK5 in quiescent cells, and upon MEKK2 activation, the C-terminal acidic cluster of the MEKK2 PB1 domain (not required for MEK5 binding) binds and activates MKK7, leading to JNK activation. This defines how one scaffold coordinates sequential ERK5 then JNK signaling.","method":"PB1 domain mutagenesis, co-immunoprecipitation, ERK5 and JNK activation assays, ternary complex mapping","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 — structural domain dissection plus Co-IP plus functional pathway assays; mechanistic detail of bi-functional PB1 domain","pmids":["17452462"],"is_preprint":false},{"year":2006,"finding":"BDNF activates ERK5 in cortical neurons via a Rap1-MEKK2-MEK5 cascade: BDNF activates Rap1 and MEKK2; inhibition of either Rap1 or MEKK2 attenuates BDNF-induced ERK5 activation; BDNF stimulation of MEKK2 is Rap1-dependent. Ras and MEKK3 do not play significant roles in neurons.","method":"Dominant-negative/constitutively active Rap1 and MEKK2 constructs, kinase activity assays, primary rat cortical neuron cultures","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis (dominant-negatives) plus kinase assays in primary neurons; neuron-specific pathway established","pmids":["17003042"],"is_preprint":false},{"year":2008,"finding":"XIAP interacts with MEKK2 and ubiquitinates it following TNFα stimulation, regulating a second wave of NF-κB activation.","method":"Co-immunoprecipitation, ubiquitination assay, NF-κB reporter assay, TNFα stimulation","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 — single Co-IP plus ubiquitination assay plus reporter; single study, no structural validation","pmids":["18761086"],"is_preprint":false},{"year":2010,"finding":"The E3 ubiquitin ligase CHIP binds MEKK2 and promotes its degradation; CHIP depletion prolongs MEKK2-mediated ERK activation in response to hyperosmotic stress. Transient (not sustained) ERK activation via MEKK2 is required for proper aquaporin 1 and 5 gene induction under hyperosmotic conditions.","method":"Co-immunoprecipitation, siRNA knockdown, gene targeting of CHIP, ERK activation time-course assays, AQP1/AQP5 gene expression","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, genetic KO of CHIP, functional gene expression readout; multiple orthogonal methods","pmids":["20588253"],"is_preprint":false},{"year":2011,"finding":"MEKK2 and MEKK3 negatively regulate TGF-β-mediated Th cell differentiation: Map3k2−/−Map3k3(Lck-Cre/−) mice accumulate Treg and Th17 cells, and their T cells show impaired phosphorylation of SMAD2/3 at linker regions (which negatively regulate TGF-β responses), indicating MAPK-TGF-β pathway crosstalk.","method":"Conditional double knockout mice, Th cell differentiation assays, phospho-SMAD western blots, EAE disease model","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — double conditional knockout in T cells with mechanistic phospho-SMAD readout; 52 citations","pmids":["21333552"],"is_preprint":false},{"year":2013,"finding":"MEKK2 kinase activity is regulated by phosphorylation-dependent association with 14-3-3 proteins. MEKK2 is phosphorylated at Thr-283, reducing activation-loop phosphorylation at Ser-519 and activity. In the absence of 14-3-3 binding, inactive MEKK2 undergoes trans-autophosphorylation at Ser-519; enforced 14-3-3 binding reduces this trans-autophosphorylation.","method":"Phosphorylation site mapping, 14-3-3 binding assays, MEKK2−/− background complementation, JNK/ERK activity assays, IL-6 measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — phosphorylation site mutagenesis, direct binding assay, and functional complementation; multiple orthogonal methods","pmids":["23963453"],"is_preprint":false},{"year":2014,"finding":"XIAP and cIAP1 directly interact with MEKK2/3 and conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3, which competitively disrupts MEK5 binding (competing with PB1 domain interaction) and leads to ERK5 pathway inactivation. Loss of XIAP causes hyperactivation of ERK5 and promotes skeletal muscle cell differentiation.","method":"Direct Co-IP, ubiquitin linkage determination (K63), competition binding assays, XIAP/cIAP1 knockdown, ERK5 activation assays, myoblast differentiation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 — mechanistic competition between IAP ubiquitination and PB1 domain binding established by multiple biochemical and cell-based methods","pmids":["24975362"],"is_preprint":false},{"year":2015,"finding":"Stk38 (NDR kinase) constitutively associates with Smurf1 E3 ligase and facilitates Smurf1-mediated MEKK2 ubiquitination and degradation, specifically suppressing TLR9/CpG-induced ERK1/2 activation and inflammatory cytokine production (TNF-α, IL-6) but not LPS-induced responses.","method":"Co-IP, ubiquitination assay, Stk38 KO mice, TLR9 stimulation assays, CpG vs. LPS specificity","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, in vivo KO mouse, biochemical ubiquitination, stimulus-specific discrimination; multiple orthogonal methods","pmids":["25981615"],"is_preprint":false},{"year":2016,"finding":"MEKK2 mediates an alternative (non-canonical) β-catenin activation pathway in osteoblasts: FGF2 activates MEKK2, which phosphorylates β-catenin at Ser675, promoting recruitment of the deubiquitinase USP15, which prevents β-catenin ubiquitination and degradation, thereby enhancing WNT signaling and bone formation.","method":"MEKK2−/− mice, in vitro kinase assays, phospho-β-catenin mapping, USP15 Co-IP, genetic interaction studies with β-catenin null alleles, MS phosphoproteomics","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assay, Co-IP, mutagenesis, and in vivo genetic interaction; multiple orthogonal methods","pmids":["26884171"],"is_preprint":false},{"year":2016,"finding":"SMYD3 co-crystal structure with a MEKK2-peptide substrate reveals the substrate-binding tunnel. Structure-based design of GSK2807, a SAM-competitive inhibitor that bridges the SAM-binding pocket and the MEKK2 substrate lysine tunnel of SMYD3 (Ki = 14 nM), blocking MAP3K2 methylation.","method":"Co-crystal structure of SMYD3/MEKK2 peptide, kinetic characterization, inhibitor design and structure determination","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with functional validation; directly defines the SMYD3-MAP3K2 interaction interface","pmids":["27066749"],"is_preprint":false},{"year":2018,"finding":"MEKK2 and MEKK3 inhibit GLI1 transcriptional activity and promote GLI1 interaction with SUFU by phosphorylating multiple Ser/Thr sites on GLI1, reducing GLI1 protein stability and DNA-binding ability, thereby suppressing Hedgehog pathway signaling. FGF2-mediated inhibition of Hh signaling requires MEKK2 and MEKK3.","method":"In vitro kinase assays, phosphorylation site mapping on GLI1, co-immunoprecipitation (GLI1-SUFU), medulloblastoma cell proliferation assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase phosphorylation plus Co-IP mechanistic readout plus cellular functional assays","pmids":["29662197"],"is_preprint":false},{"year":2018,"finding":"Kir2.1 interacts with Stk38 to inhibit Smurf1-mediated ubiquitination and degradation of MEKK2, thereby stabilizing MEKK2 and activating the MEK1/2-ERK1/2-Snail EMT pathway in gastric cancer cells. This function is independent of K+ ion permeation.","method":"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, Kir2.1 mutants (ion-permeation defective), xenograft metastasis assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — mechanistic Co-IP chain (Kir2.1-Stk38-Smurf1-MEKK2) with ion-channel-independent mutant controls; multiple methods","pmids":["29549164"],"is_preprint":false},{"year":2021,"finding":"MAP3K2 mediates a ROS-MAP3K2-ERK5-KLF2 signaling axis in intestinal stromal cells (MRISCs) that drives R-spondin 1 production to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage.","method":"MAP3K2−/− mice, intestinal injury models, single-cell transcriptomics, epigenetic profiling, ROS manipulation, RSPO1 measurement","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — genetic KO in vivo with defined pathway (ROS-MAP3K2-ERK5-KLF2-RSPO1) and specific cellular readout; published in Nature","pmids":["33658717"],"is_preprint":false},{"year":2021,"finding":"MEKK2 and MEKK3 regulate the Hippo pathway: they interact with and phosphorylate LATS1/2 and YAP/TAZ; TNF activates LATS1/2 and inhibits YAP/TAZ through MEKK2/3. STRIPAK complex associates with MEKK3 via CCM2/CCM3 to inactivate MEKK3, and upstream Hippo signals trigger dissociation of MEKK3 from STRIPAK.","method":"Co-immunoprecipitation, in vitro kinase assays for LATS1/2 and YAP/TAZ phosphorylation, genetic knockdown, TNF stimulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase assays plus Co-IP plus signaling epistasis; multiple orthogonal methods","pmids":["33571521"],"is_preprint":false},{"year":2021,"finding":"MAP3K2-mediated Th1 cell differentiation in the intestine is regulated by IL-18 and requires specific JNK activation (IL-18-MAP3K2-JNK axis); MAP3K2-deficient naïve CD4+ T cells transferred into mice have a dramatically reduced ability to induce colitis, with fewer IFNγ-producing but more IL-17A-producing cells.","method":"T cell transfer colitis model, MAP3K2−/− mice, in vitro Th1/Th17/Treg differentiation assays, JNK activation assays, IL-18 supplementation","journal":"Science China. Life sciences","confidence":"High","confidence_rationale":"Tier 2 — in vivo transfer model with KO T cells, defined molecular axis (IL-18-MAP3K2-JNK) with multiple pathway measurements","pmids":["32737854"],"is_preprint":false},{"year":2021,"finding":"Pazopanib inhibits MAP3K2- and MAP3K3-mediated phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208, reducing ROS formation in myeloid cells. Myeloid-specific MAP3K2/MAP3K3 genetic inactivation or hematopoietic p47phox-S208A mutation attenuates acute lung injury, establishing MAP3K2 as upstream kinase for p47phox-Ser208 phosphorylation.","method":"Genetic inactivation of MAP3K2/3 in myeloid cells, phosphorylation site mutation (p47phox-S208A), ALI mouse models, kinase activity assays","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1–2 — direct kinase-substrate (MAP3K2→p47phox-S208) with genetic confirmation; published in Science Translational Medicine","pmids":["33910977"],"is_preprint":false},{"year":2020,"finding":"MEKK2 mediates aberrant ERK activation downstream of NF1 (neurofibromin) loss in osteoblasts via a noncanonical ERK pathway. MEKK2-deficient mice crossed with osteoblast-conditional Nf1-knockout mice show amelioration of NF1-associated skeletal phenotypes, placing MEKK2 epistatic to NF1 in ERK activation in bone.","method":"MEKK2−/− and Nf1fl/fl conditional KO mice, double-mutant genetic interaction, skeletal phenotype analysis, ERK activation assays, FDA-approved MEKK2 inhibitor in vivo","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic epistasis with conditional KO mouse models and pharmacological inhibitor proof-of-principle","pmids":["33177525"],"is_preprint":false},{"year":2014,"finding":"MEKK2 ablation in invasive breast cancer cells enhances focal adhesion stability, increases spread area, and reduces cell migration. MEKK2 is activated by and localizes to focal adhesions upon fibronectin/Matrigel attachment, and MEKK2 knockdown inhibits fibronectin-induced ERK5 signaling and FAK autophosphorylation.","method":"siRNA knockdown, focal adhesion imaging, cell migration assays, kinase activity assays (MEKK2 activation by attachment), FAK phosphorylation western blot","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2–3 — siRNA KD with multiple cellular readouts (focal adhesion size, migration, kinase activity, FAK) but no reconstitution","pmids":["24491810"],"is_preprint":false},{"year":2014,"finding":"MEKK2 physically associates with the LD1 motif of paxillin, induces paxillin ubiquitylation (requiring both the paxillin LD1 motif and MEKK2 kinase activity), and promotes redistribution of paxillin from focal adhesions into the cytoplasm without promoting degradation.","method":"Co-immunoprecipitation (MEKK2-paxillin LD1 domain), ubiquitylation assay, MEKK2 kinase-dead mutant, paxillin localization imaging","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP plus ubiquitylation plus kinase-dead mutant controls; single study","pmids":["25190348"],"is_preprint":false},{"year":2015,"finding":"Paxillin LD1 motif binding to the MEKK2 amino-terminal region relieves MEKK2 auto-inhibition: recombinant paxillin induces MEKK2 auto-phosphorylation in vitro, and paxillin knockdown reduces MEKK2 activity in cells.","method":"In vitro kinase/auto-phosphorylation assay with recombinant paxillin, siRNA paxillin knockdown, LD1 motif binding assay","journal":"Journal of molecular signaling","confidence":"Medium","confidence_rationale":"Tier 1–2 — in vitro reconstitution plus siRNA cell-based validation; single lab, limited follow-up","pmids":["27096002"],"is_preprint":false},{"year":2019,"finding":"MEKK2 phosphorylates STK38 (Ser91) to protect it from calpain-mediated cleavage at the N-terminal region; MEKK2 knockdown enhances hyperthermia-induced STK38 degradation. A phosphorylation-defective S91A STK38 mutant is susceptible to calpain cleavage.","method":"In vitro MEKK2 kinase assay with STK38 substrate, phosphorylation site identification by mass spectrometry, calpain cleavage assay, MEKK2 siRNA knockdown, phospho-defective mutant","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay identifying Ser91 site plus phospho-defective mutant validation plus calpain cleavage reconstitution","pmids":["31690749"],"is_preprint":false},{"year":2022,"finding":"NEDD4L (HECT E3 ligase) constitutively binds MEKK2 and promotes its poly-ubiquitination and degradation. IL-17-induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF-κB activation but also for NEDD4L-mediated MEKK2 degradation (negative feedback). Nedd4l-deficient mice show increased IL-17-induced inflammation.","method":"Co-IP, ubiquitination assay, siRNA knockdown, Nedd4l KO mice, phospho-Ser520 mutagenesis, IL-17 stimulation assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — direct Co-IP, ubiquitination assay, mutagenesis at regulatory site, in vivo KO mouse confirmation; multiple orthogonal methods","pmids":["36161689"],"is_preprint":false},{"year":2023,"finding":"Best3 (Bestrophin3) directly interacts with both MEKK2 and MEKK3 and inhibits phosphorylation of MEKK2 at Ser153 (and MEKK3 at Ser61). Best3 deficiency induces phosphorylation-dependent stabilization of MEKK2/3 (inhibition of ubiquitination/turnover), activating downstream MAPK signaling and triggering aortic dissection.","method":"Co-IP coupled with mass spectrometry, Best3 smooth muscle-specific KO mice, single-cell RNA sequencing, proteomics, phosphorylation site analysis, MEKK2/3 inhibitor rescue","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 2 — Co-IP/MS plus genetic KO in vivo plus pharmacological rescue; published in Circulation with multiple orthogonal approaches","pmids":["37203562"],"is_preprint":false},{"year":2016,"finding":"SMYD3-mediated methylation of MAP3K2 promotes epithelial-mesenchymal transition-associated behaviors in prostate cancer cells via a positive feedback loop that continuously promotes high SMYD3 levels, and alters vimentin abundance downstream.","method":"SMYD3 catalytic mutant, xenograft mouse models, EMT marker analysis, vimentin expression measurement","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 — methyltransferase mutant plus in vivo xenograft; extends SMYD3-MAP3K2 methylation finding to prostate cancer context","pmids":["37976356"],"is_preprint":false},{"year":2018,"finding":"NDR2 (LATS/NDR kinase family) promotes Smurf1-mediated K48-linked ubiquitination of MEKK2 in cells, reducing MEKK2 levels and inhibiting IL-17-induced MAPK and NF-κB activation; Smurf1 knockdown similarly increases IL-17-induced cytokine expression.","method":"Co-IP (NDR2-Smurf1), ubiquitination assay with K48-linkage determination, siRNA knockdown, IL-17 stimulation, cytokine quantification","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus ubiquitination assay; single lab study, extends Smurf1-MEKK2 regulatory axis","pmids":["30504095"],"is_preprint":false},{"year":2004,"finding":"In rheumatoid arthritis fibroblast-like synoviocytes (FLS), IL-1 increases MEKK2 kinase activity (measured by immunoprecipitation kinase assay using MKK4 and MKK7 as substrates), and MEKK2 immunoprecipitates activate c-Jun via JNK; activity blocked by JNK inhibitor SP600125.","method":"Immunoprecipitation kinase assay with MKK4/MKK7 substrates, c-Jun phosphorylation assay, pharmacological JNK inhibitor","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — immunoprecipitation kinase assay in disease-relevant primary cells with specific substrate readout; single lab","pmids":["14734742"],"is_preprint":false},{"year":2010,"finding":"Calcium is required for EGF-induced ERK5 activation upstream of MEKK2. EGF increases MEKK2 binding to adaptor protein Lad1 in a calcium-dependent manner; in vitro binding assays show calcium is needed for direct MEKK2-Lad1 interaction. Calcium also regulates EGF-induced nuclear translocation of MEKK2.","method":"Co-immunoprecipitation, in vitro direct binding assay, intracellular calcium modifiers, nuclear translocation imaging","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP plus in vitro binding plus imaging; single lab, moderate methods","pmids":["20830310"],"is_preprint":false},{"year":2003,"finding":"Point mutations in protein kinase subdomain X (distinct from MEKK1 equivalent residues) impair MEKK2 phosphorylation of MKK7 and MEK5, abolish MEKK2-induced JNK1 and ERK5 activation, and diminish AP-1 reporter gene activation, revealing subdomain X as critical for MEKK2 substrate interaction.","method":"Site-directed mutagenesis, in vitro kinase assays with MKK7/MEK5 substrates, JNK1/ERK5 activation assays, AP-1 reporter gene","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1–2 — mutagenesis combined with in vitro kinase assays and reporter assays; single study","pmids":["12659851"],"is_preprint":false},{"year":2016,"finding":"MEKK2 mediates ERK5 activation downstream of PDGF-BB in smooth muscle cells in a manner dependent on Mek1/2, Mek5, PI3-kinase, and classical PKC isoforms, demonstrating co-regulation between ERK1/2 and ERK5 pathways.","method":"Dominant-negative MEKK2, kinase activity assays, PI3K/PKC inhibitors, ERK5 activation measurement in MOVAS cells","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2–3 — dominant-negative epistasis plus pharmacological inhibitors; single study in smooth muscle cells","pmids":["27339033"],"is_preprint":false},{"year":2016,"finding":"In sublytic C5b-9-induced glomerular mesangial cell apoptosis, MEKK2 phosphorylation (at Ser153/164/239) activates p38 MAPK, which drives IRF-1 and TRADD expression, leading to caspase 8 activation and apoptosis. Silencing MEKK2 in vivo inhibits GMC apoptosis in rat Thy-1 nephritis.","method":"Phosphorylation site mutagenesis (Ser153/164/239), p38 MAPK kinase assays, IRF-1/TRADD gene silencing in vivo, caspase 8 activity assay, rat Thy-1 nephritis model","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — phosphorylation site mutagenesis plus in vivo gene silencing with apoptosis readout; single lab","pmids":["28039298"],"is_preprint":false},{"year":2022,"finding":"ZSWIM1 interacts with STK38 (identified by IP-MS) and antagonizes STK38 function, promoting release and activation of MEKK2, leading to ERK1/2 activation and lung adenocarcinoma progression.","method":"Immunoprecipitation-mass spectrometry (interactome), Co-IP validation, ZSWIM1 overexpression/knockdown, ERK1/2 activation assays","journal":"Journal of proteome research","confidence":"Medium","confidence_rationale":"Tier 3 — IP-MS interactome with Co-IP validation and functional cell assays; mechanism placed upstream of MEKK2 but limited kinase biochemistry","pmids":["36511424"],"is_preprint":false}],"current_model":"MAP3K2 (MEKK2) is a serine/threonine MAP3K that signals through MEK5-ERK5 and MKK4/7-JNK pathways via PB1 domain-mediated scaffolding with MEK5 and MKK7; it is activated by dimerization, upstream kinases (WNK1, Rap1), and adaptor-mediated recruitment (Lad1/RIBP), and its activity is regulated by multiple post-translational mechanisms including SMYD3-mediated K260 methylation (which blocks PP2A binding to enhance Ras-ERK signaling), Smurf1/XIAP/NEDD4L/CHIP-mediated ubiquitination and degradation, 14-3-3 binding (suppressing trans-autophosphorylation), and paxillin LD1-induced activation; it also phosphorylates non-canonical substrates including β-catenin-Ser675, p47phox-Ser208, GLI1, LATS1/2, YAP/TAZ, and STK38-Ser91 to control bone formation, innate immunity, Hedgehog pathway suppression, Hippo signaling, and intestinal stem cell niche maintenance."},"narrative":{"teleology":[{"year":1999,"claim":"MAP3K2 was placed at the T cell immunological synapse: imaging showed MEKK2 translocation to the T cell–APC contact, and dominant-negative MEKK2 blocked TCR-mediated ERK and p38 activation, establishing it as a receptor-proximal MAP3K in adaptive immunity.","evidence":"Immunocytochemistry and dominant-negative expression in T cell conjugate assays","pmids":["10549623"],"confidence":"High","gaps":["Upstream signal recruiting MEKK2 to the synapse not identified","Kinase-dead versus dominant-negative effects not separated"]},{"year":2000,"claim":"A direct link between MAP3K2 and the MEK5–ERK5 cascade was identified, and MEKK2 knockout in mast cells revealed stimulus-selective JNK dependence, demonstrating that MAP3K2 routes specific receptor tyrosine kinase signals to both ERK5 and JNK.","evidence":"Yeast two-hybrid identification of MEK5 as MEKK2 partner; MEKK2−/− ES cell-derived mast cells tested for FcεRI/c-Kit versus UV-induced JNK","pmids":["11073940","11032806"],"confidence":"High","gaps":["Mechanism of stimulus selectivity unknown","Whether ERK5 and JNK are activated simultaneously or sequentially from the same complex unclear"]},{"year":2002,"claim":"MEKK2 knockout mice unexpectedly showed enhanced rather than reduced T cell activation, revealing that MAP3K2 negatively modulates TCR signal strength in vivo.","evidence":"Mekk2−/− mice with T cell proliferation, cytokine ELISA, and JNK/ERK/p38 kinase assays","pmids":["12138187"],"confidence":"High","gaps":["Mechanism of negative modulation (direct kinase substrate versus scaffold sequestration) not resolved","Redundancy with MEKK3 in T cells not tested"]},{"year":2003,"claim":"The structural basis for MAP3K2 scaffolding was established: PB1 domain heterodimerization with MEK5 is required for ERK5 activation, and WNK1 was identified as an upstream kinase that phosphorylates MAP3K2 to relay signals to ERK5.","evidence":"PB1 domain mutagenesis and in vitro binding; WNK1 in vitro kinase assay and dominant-negative epistasis in HEK293 cells","pmids":["12912994","14681216"],"confidence":"High","gaps":["Phosphorylation site(s) on MEKK2 targeted by WNK1 not mapped","Structural basis of PB1–PB1 interaction not yet resolved at atomic level"]},{"year":2004,"claim":"MAP3K2 was shown to coordinate both ERK5 and JNK downstream of FGF-2 in fibroblasts and to undergo stimulus-dependent nuclear translocation to relay signals to nuclear MEK5, providing spatial regulation of MAPK signaling.","evidence":"MEKK2−/− MEFs tested for FGF-2/LPS/TNFα-selective pathway loss; immunofluorescence and subcellular fractionation in HeLa/Rat-1 cells after EGF","pmids":["14978743","15075238"],"confidence":"High","gaps":["Nuclear import mechanism (NLS, carrier) not identified","How stimulus selectivity is encoded at the molecular level unresolved"]},{"year":2005,"claim":"Multiple layers of MAP3K2 regulation were uncovered: activation requires kinase-domain dimerization; the inhibitor Mip1 blocks dimerization of inactive MEKK2; Smurf1 ubiquitinates MEKK2 for proteasomal degradation controlling osteoblast JNK signaling; and Ser519 was identified as a key activation-loop phosphorylation site induced by TLR/TRAF6.","evidence":"Chemical-induced dimerization system; Mip1 Co-IP and siRNA; Smurf1−/− mice; phospho-S519 mutagenesis with LPS stimulation","pmids":["15695508","15988011","15820682","16362041"],"confidence":"High","gaps":["Identity of the kinase that phosphorylates Ser519 (autophosphorylation versus upstream kinase) not fully resolved","How TRAF6 activates MEKK2 Ser519 phosphorylation mechanistically unclear"]},{"year":2007,"claim":"The bifunctional nature of the MAP3K2 PB1 domain was defined: its front face binds MEK5 constitutively, while its C-terminal acidic cluster recruits MKK7 only upon MEKK2 activation, explaining how one scaffold sequentially activates ERK5 then JNK.","evidence":"PB1 domain face-specific mutagenesis with Co-IP and ERK5/JNK pathway assays","pmids":["17452462"],"confidence":"High","gaps":["Ternary complex stoichiometry not determined","Whether MEKK2 simultaneously engages MEK5 and MKK7 or switches between them unknown"]},{"year":2010,"claim":"CHIP E3 ligase was identified as a negative regulator that promotes MAP3K2 degradation and limits ERK activation duration under hyperosmotic stress, and calcium was shown to regulate the MEKK2–Lad1 interaction required for EGF-induced ERK5 signaling.","evidence":"CHIP Co-IP and knockout with ERK time-course; calcium-dependent MEKK2–Lad1 Co-IP and in vitro binding","pmids":["20588253","20830310"],"confidence":"High","gaps":["How calcium modulates MEKK2–Lad1 binding structurally not known","CHIP recognition motif on MEKK2 not mapped"]},{"year":2011,"claim":"Combined MAP3K2/MAP3K3 deletion in T cells revealed crosstalk with TGF-β signaling: loss of MEKK2/3-mediated SMAD2/3 linker phosphorylation unleashed TGF-β-driven Treg and Th17 accumulation, connecting MAP3K2 to adaptive immune tolerance.","evidence":"Map3k2−/−Map3k3(Lck-Cre/−) mice with Th cell differentiation and phospho-SMAD analysis; EAE model","pmids":["21333552"],"confidence":"High","gaps":["Direct kinase–substrate relationship between MEKK2 and SMAD linker phosphorylation versus intermediate kinases not resolved","Relative contributions of MEKK2 and MEKK3 not separated"]},{"year":2013,"claim":"14-3-3 binding to phospho-Thr283 on MAP3K2 was shown to suppress trans-autophosphorylation at Ser519, establishing 14-3-3 as a tonic brake on MAP3K2 activation.","evidence":"Phosphorylation site mapping, 14-3-3 binding assay, complementation in MEKK2−/− cells","pmids":["23963453"],"confidence":"High","gaps":["Kinase responsible for Thr283 phosphorylation not identified","Stoichiometry of 14-3-3–MEKK2 complex in vivo unknown"]},{"year":2014,"claim":"Two new regulatory paradigms for MAP3K2 emerged: SMYD3-mediated K260 methylation blocks PP2A binding to sustain Ras-ERK signaling in cancer, and XIAP/cIAP1-conjugated K63-ubiquitin chains on MEKK2 competitively disrupt MEK5 PB1-domain binding to inactivate ERK5.","evidence":"SMYD3 methylation assays with mouse pancreatic/lung cancer models; XIAP K63-ubiquitin linkage determination and PB1 competition assays with myoblast differentiation readout","pmids":["24847881","24975362"],"confidence":"High","gaps":["Whether K260 methylation and K63-ubiquitination can co-occur on the same molecule unknown","SMYD3-MAP3K2 axis not yet validated in human patient tumors with matched methylation data"]},{"year":2016,"claim":"MAP3K2 was established as a direct kinase for non-MAPK substrates: phosphorylation of β-catenin at Ser675 recruits USP15 to stabilize β-catenin and promote bone formation, and the co-crystal structure of the SMYD3–MEKK2 peptide interface was solved.","evidence":"MEKK2−/− mice with in vitro kinase assays and genetic interaction with β-catenin null alleles; co-crystal structure of SMYD3 with MEKK2 substrate peptide","pmids":["26884171","27066749"],"confidence":"High","gaps":["Whether β-catenin Ser675 phosphorylation by MEKK2 operates in tissues beyond bone not tested","Full-length MEKK2 structure unavailable"]},{"year":2018,"claim":"Additional non-MAPK substrates were identified: MAP3K2 phosphorylates GLI1 to promote its interaction with SUFU and suppress Hedgehog signaling, and phosphorylates STK38 at Ser91 to protect it from calpain cleavage, expanding MAP3K2's substrate repertoire beyond canonical MAPK intermediates.","evidence":"In vitro kinase assays mapping GLI1 phosphosites with Co-IP for GLI1–SUFU; MEKK2 kinase assay on STK38 with Ser91 mutagenesis and calpain cleavage reconstitution","pmids":["29662197","31690749"],"confidence":"High","gaps":["Physiological contexts where MEKK2-GLI1 axis operates in vivo not fully defined","Whether STK38 Ser91 phosphorylation feeds back to regulate MEKK2 stability via Smurf1 not tested"]},{"year":2020,"claim":"Genetic epistasis in mouse bone placed MAP3K2 downstream of NF1 loss in a noncanonical ERK activation pathway: MEKK2 deletion rescued NF1-deficiency skeletal phenotypes, nominating MAP3K2 as a therapeutic target in neurofibromatosis.","evidence":"MEKK2−/− crossed with Nf1fl/fl conditional KO mice; skeletal phenotype analysis with pharmacological MEKK2 inhibitor","pmids":["33177525"],"confidence":"High","gaps":["Direct biochemical mechanism linking NF1 loss to MEKK2 hyperactivation not defined","Clinical translation of MEKK2 inhibitors for NF1 skeletal disease not tested"]},{"year":2021,"claim":"MAP3K2's physiological roles expanded to intestinal stem cell maintenance (ROS–MEKK2–ERK5–KLF2–RSPO1 in stromal cells), Hippo pathway regulation (direct phosphorylation of LATS1/2 and YAP/TAZ), NADPH oxidase activation (p47phox Ser208 phosphorylation in myeloid cells), and IL-18-driven Th1 differentiation in the gut.","evidence":"MAP3K2−/− mice with intestinal injury and single-cell transcriptomics; in vitro kinase assays for LATS1/2 and YAP/TAZ; myeloid-specific MAP3K2/3 KO and p47phox-S208A knockin in ALI models; T cell transfer colitis with MAP3K2−/− cells","pmids":["33658717","33571521","33910977","32737854"],"confidence":"High","gaps":["Whether MEKK2 phosphorylation of LATS1/2 is direct in all cell types or context-dependent not established","Relative contribution of MAP3K2 versus MAP3K3 in Hippo and p47phox pathways not fully separated"]},{"year":2022,"claim":"NEDD4L was identified as an additional E3 ligase that degrades MAP3K2 via a negative feedback loop triggered by IL-17-induced Ser520 phosphorylation, reinforcing that MAP3K2 protein levels are tightly controlled by multiple ubiquitin ligases in inflammatory signaling.","evidence":"Co-IP, ubiquitination assays, phospho-Ser520 mutagenesis, Nedd4l KO mice with IL-17 stimulation","pmids":["36161689"],"confidence":"High","gaps":["Whether Smurf1 and NEDD4L compete for the same or distinct ubiquitination sites on MEKK2 not resolved","Phosphatase that reverses Ser520 phosphorylation not identified"]},{"year":2023,"claim":"Bestrophin-3 was shown to directly bind MAP3K2 and suppress its Ser153 phosphorylation, preventing phosphorylation-dependent stabilization; Best3 deficiency hyperactivates MEKK2 and causes aortic dissection, linking MAP3K2 regulation to vascular integrity.","evidence":"Best3 smooth muscle-specific KO mice with Co-IP/MS, single-cell RNA-seq, and MEKK2/3 inhibitor rescue of aortic phenotype","pmids":["37203562"],"confidence":"High","gaps":["Whether Best3 regulation of MEKK2 occurs in tissues beyond vasculature not tested","How Ser153 phosphorylation blocks ubiquitin-mediated degradation mechanistically unknown"]},{"year":null,"claim":"A full-length structure of MAP3K2 is lacking, the complete phosphorylation code governing activation versus degradation is incompletely mapped, and the basis for stimulus-selective pathway routing through MAP3K2 remains a central unresolved question.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length crystal or cryo-EM structure of MAP3K2","Complete catalog of direct phosphorylation substrates in different cell types not established","How MAP3K2 achieves stimulus selectivity (FGF vs. TNF vs. LPS) at the molecular level is unclear"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,4,12,13,21,23,26,28,33,40]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,4,13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[9,30]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,3,4,6,13,14,25,26]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,7,9,17,27,28]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[42]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,21,29]}],"complexes":["MEKK2–MEK5 (PB1-mediated)","MEKK2–MKK7 (PB1-mediated)","Smurf1–MEKK2","STRIPAK–MEKK3 (related)"],"partners":["MAP2K5","MAP2K7","WNK1","SMYD3","SMURF1","XIAP","CHIP","NEDD4L"],"other_free_text":[]},"mechanistic_narrative":"MAP3K2 (MEKK2) is a serine/threonine MAP3K that serves as a central signaling hub coupling diverse extracellular stimuli—including growth factors, cytokines, and reactive oxygen species—to downstream MAPK cascades and non-canonical substrates in immune regulation, bone formation, intestinal homeostasis, and vascular integrity. Through its N-terminal PB1 domain, MAP3K2 scaffolds MEK5 for ERK5 pathway activation and, upon its own activation, recruits MKK7 via a distinct PB1 surface to additionally engage JNK signaling; it also phosphorylates MKK4, p38 substrates, and non-MAPK targets including β-catenin (Ser675), p47phox (Ser208), GLI1, LATS1/2, and STK38 (Ser91) [PMID:12912994, PMID:17452462, PMID:26884171, PMID:33910977, PMID:29662197, PMID:33571521, PMID:31690749]. MAP3K2 activation is triggered by dimerization through its kinase domain, upstream kinases (WNK1, Rap1), adaptor-mediated recruitment (Lad1, paxillin LD1 motif), and trans-autophosphorylation at Ser519, and is negatively regulated by 14-3-3 binding, SMYD3-mediated K260 methylation that blocks PP2A dephosphorylation, and multiple E3 ubiquitin ligases (Smurf1, XIAP/cIAP1, CHIP, NEDD4L) that target MAP3K2 for ubiquitin-dependent degradation or PB1-domain competition [PMID:15695508, PMID:14681216, PMID:23963453, PMID:24847881, PMID:15820682, PMID:24975362, PMID:20588253, PMID:36161689]. Genetic ablation of MAP3K2 reveals stimulus-selective requirements: loss of FcεRI/c-Kit–driven JNK in mast cells, impaired FGF2-induced ERK5/JNK in fibroblasts, augmented T cell proliferation reflecting negative modulation of TCR signaling, and compromised intestinal stem cell maintenance via the ROS–ERK5–KLF2–RSPO1 axis [PMID:11032806, PMID:14978743, PMID:12138187, PMID:33658717]."},"prefetch_data":{"uniprot":{"accession":"Q9Y2U5","full_name":"Mitogen-activated protein kinase kinase kinase 2","aliases":["MAPK/ERK kinase kinase 2","MEK kinase 2","MEKK 2"],"length_aa":619,"mass_kda":69.7,"function":"Component of a protein kinase signal transduction cascade. Regulates the JNK and ERK5 pathways by phosphorylating and activating MAP2K5 and MAP2K7 (By similarity). Plays a role in caveolae kiss-and-run dynamics","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9Y2U5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAP3K2","classification":"Not Classified","n_dependent_lines":29,"n_total_lines":1208,"dependency_fraction":0.024006622516556293},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAP3K2","total_profiled":1310},"omim":[{"mim_id":"616711","title":"TAO KINASE 3; TAOK3","url":"https://www.omim.org/entry/616711"},{"mim_id":"609487","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 2; MAP3K2","url":"https://www.omim.org/entry/609487"},{"mim_id":"608783","title":"SET AND MYND DOMAIN-CONTAINING PROTEIN 3; SMYD3","url":"https://www.omim.org/entry/608783"},{"mim_id":"605568","title":"SMAD-SPECIFIC E3 UBIQUITIN PROTEIN LIGASE 1; SMURF1","url":"https://www.omim.org/entry/605568"},{"mim_id":"603014","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE 7; MAP2K7","url":"https://www.omim.org/entry/603014"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MAP3K2"},"hgnc":{"alias_symbol":["MEKK2B"],"prev_symbol":["MEKK2"]},"alphafold":{"accession":"Q9Y2U5","domains":[{"cath_id":"3.10.20.90","chopping":"42-120","consensus_level":"high","plddt":89.1684,"start":42,"end":120},{"cath_id":"3.30.200.20","chopping":"355-437","consensus_level":"medium","plddt":91.3977,"start":355,"end":437},{"cath_id":"1.10.510.10","chopping":"442-615","consensus_level":"medium","plddt":88.686,"start":442,"end":615}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y2U5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y2U5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y2U5-F1-predicted_aligned_error_v6.png","plddt_mean":67.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAP3K2","jax_strain_url":"https://www.jax.org/strain/search?query=MAP3K2"},"sequence":{"accession":"Q9Y2U5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y2U5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y2U5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y2U5"}},"corpus_meta":[{"pmid":"24847881","id":"PMC_24847881","title":"SMYD3 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standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"SMYD3 methyltransferase methylates MAP3K2 at lysine 260, which potentiates activation of the Ras/Raf/MEK/ERK signaling module. Methylation of MAP3K2 at K260 blocks binding of the PP2A phosphatase complex to MAP3K2, thereby preventing PP2A-mediated negative regulation of MAP kinase signaling.\",\n      \"method\": \"Protein array to identify SMYD3 substrates, in vitro methylation assays, mass spectrometry, Co-IP, mouse cancer models (pancreatic and lung adenocarcinoma), MEK inhibitor synergy studies\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods including in vitro methylation, Co-IP, mutagenesis, and in vivo mouse models; replicated in prostate cancer context (PMID:37976356)\",\n      \"pmids\": [\"24847881\", \"37976356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Smurf1, a HECT domain ubiquitin E3 ligase, physically interacts with MAP3K2 (MEKK2) and promotes its ubiquitination and proteasomal degradation, thereby suppressing osteoblast activity and JNK signaling downstream of BMP.\",\n      \"method\": \"Co-IP, ubiquitination assays, Smurf1 knockout mice, western blot for phospho-MEKK2 accumulation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reciprocal Co-IP, in vivo knockout phenotype, biochemical ubiquitination assays; foundational paper with 310 citations\",\n      \"pmids\": [\"15820682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"WNK1 acts upstream of MAP3K2 (MEKK2) and MAP3K3 (MEKK3) in the ERK5 pathway: WNK1 phosphorylates MEKK2 and MEKK3 in vitro, co-immunoprecipitates with endogenous WNK1, and dominant-negative MEKK2/3 block WNK1-induced ERK5 activation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, dominant-negative overexpression, siRNA knockdown, HEK293 cell transfection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay plus reciprocal Co-IP plus dominant-negative epistasis; multiple orthogonal methods\",\n      \"pmids\": [\"14681216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MAP3K2 (MEKK2) directly binds MEK5 (identified by yeast two-hybrid) and activates the MEK5-BMK1/ERK5 pathway; MEKK2 also binds the T cell adapter protein Lad/RIBP and co-localizes with it at the T cell/antigen-presenting cell interface during T cell activation.\",\n      \"method\": \"Yeast two-hybrid library screening, dominant-negative overexpression, live-cell co-localization imaging, kinase activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — yeast two-hybrid confirmed by functional dominant-negative epistasis and co-localization; 122 citations\",\n      \"pmids\": [\"11073940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The N-terminal PB1 domains of MEKK2 and MEKK3 heterodimerize with the PB1 domain of MEK5 (but not with each other), and this PB1-mediated interaction is required for complex formation and ERK5 pathway activation. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 complexes and blocks ERK5 activation.\",\n      \"method\": \"In vitro PB1 domain binding assays, co-immunoprecipitation from cell lysates, deletion/point mutants, dominant-negative overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro binding reconstitution plus Co-IP plus mutagenesis; multiple orthogonal methods in single study\",\n      \"pmids\": [\"12912994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MEKK2 gene disruption in ES cell-derived mast cells causes loss of receptor-mediated JNK activation and cytokine gene transcription in response to IgE receptor (FcεRI) or c-Kit ligation, while UV-induced JNK activation remains intact, demonstrating that MEKK2 is specifically required for tyrosine kinase receptor signaling in mast cells.\",\n      \"method\": \"Targeted gene disruption (MEKK2−/− ES cell-derived mast cells), kinase activity assays, cytokine mRNA measurement\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic knockout with specific pathway readout; receptor-specific vs. stress-specific JNK activation discrimination; 89 citations\",\n      \"pmids\": [\"11032806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MEKK2 coordinates activation of both ERK5 and JNK pathways in response to FGF-2; MEKK2-knockout MEFs lose ERK5 and JNK activation specifically in response to FGF-2 (but not LPS or TNFα), and show impaired AP-1 component expression and cytokine gene induction.\",\n      \"method\": \"MEKK2−/− mouse embryonic fibroblasts, kinase activity assays, RT-PCR for AP-1 components and cytokines\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic knockout with multiple specific pathway readouts; stimulus-selective requirement established\",\n      \"pmids\": [\"14978743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"MEKK2 knockout mice exhibit augmented T cell proliferation and enhanced IL-2 and IFNγ production in response to anti-CD3 stimulation, with moderately enhanced (not reduced) JNK activation in MEKK2−/− T cells, indicating MEKK2 negatively modulates TCR signal strength.\",\n      \"method\": \"Mekk2−/− mouse generation, T cell proliferation assays, cytokine ELISA, JNK/ERK/p38 kinase assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO mouse with defined cellular phenotypes and pathway measurements; 52 citations\",\n      \"pmids\": [\"12138187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In resting cells MEKK2 is localized in the cytosol, and upon EGF stimulation it translocates into the nucleus where MEK5 and ERK5 are constitutively localized, allowing transmission of signals to nuclear MEK5.\",\n      \"method\": \"Immunofluorescence, in situ NP-40 detergent extraction, subcellular fractionation, EGF stimulation of HeLa and Rat-1 cells\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiments with functional context (nuclear signal transmission); multiple cell lines and methods\",\n      \"pmids\": [\"15075238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MEKK2 is translocated to the T cell/antigen-presenting cell contact interface upon antigen stimulation of the TCR; dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK and p38 MAP kinase phosphorylation.\",\n      \"method\": \"Immunocytochemistry, live-cell fluorescence imaging, dominant-negative expression, kinase phosphorylation assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live-cell imaging of translocation with functional dominant-negative validation; 45 citations\",\n      \"pmids\": [\"10549623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"MEKK2 activation requires dimerization through its catalytic domain: inactive/non-phosphorylated MEKK2 forms more dimers than phosphorylated MEKK2, and chemical-induced dimerization in vivo augments MEKK2-dependent JNK activation. Prevention of dimerization inhibits JNK signaling.\",\n      \"method\": \"Dimerization domain mapping, chemical-induced dimerization (CID) system, co-immunoprecipitation, JNK/AP-1 reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mechanistic dimerization mapping plus chemical dimerization rescue plus reporter assays; multiple orthogonal methods\",\n      \"pmids\": [\"15695508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"A novel MEKK2-interacting protein, Mip1, forms a complex with inactive/non-phosphorylated MEKK2 and prevents its activation by blocking MEKK2 dimer formation. EGF stimulation dissociates the endogenous Mip1-MEKK2 complex; Mip1 siRNA knockdown augments MEKK2-mediated JNK and AP-1 activation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, MEKK2 dimerization assays, JNK/ERK5/AP-1 reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus siRNA plus functional reporters; multiple orthogonal methods in single study\",\n      \"pmids\": [\"15988011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Serine 519 of MEKK2 (and the corresponding Ser526 of MEKK3) is a key regulatory phosphorylation site: S519A mutation severely impairs MEKK2 activation; LPS (via TRAF6) induces phosphorylation at this serine, which is required for TLR-induced IL-6 production.\",\n      \"method\": \"Site-directed mutagenesis, phospho-specific antibody generation, in vitro kinase assays, LPS stimulation, siRNA knockdown of TRAF6\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — mutagenesis combined with phospho-specific antibody validation and functional readout; 50 citations\",\n      \"pmids\": [\"16362041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The MEKK2 PB1 domain uses a front-to-back arrangement to bind MEK5 in quiescent cells, and upon MEKK2 activation, the C-terminal acidic cluster of the MEKK2 PB1 domain (not required for MEK5 binding) binds and activates MKK7, leading to JNK activation. This defines how one scaffold coordinates sequential ERK5 then JNK signaling.\",\n      \"method\": \"PB1 domain mutagenesis, co-immunoprecipitation, ERK5 and JNK activation assays, ternary complex mapping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — structural domain dissection plus Co-IP plus functional pathway assays; mechanistic detail of bi-functional PB1 domain\",\n      \"pmids\": [\"17452462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"BDNF activates ERK5 in cortical neurons via a Rap1-MEKK2-MEK5 cascade: BDNF activates Rap1 and MEKK2; inhibition of either Rap1 or MEKK2 attenuates BDNF-induced ERK5 activation; BDNF stimulation of MEKK2 is Rap1-dependent. Ras and MEKK3 do not play significant roles in neurons.\",\n      \"method\": \"Dominant-negative/constitutively active Rap1 and MEKK2 constructs, kinase activity assays, primary rat cortical neuron cultures\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis (dominant-negatives) plus kinase assays in primary neurons; neuron-specific pathway established\",\n      \"pmids\": [\"17003042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"XIAP interacts with MEKK2 and ubiquitinates it following TNFα stimulation, regulating a second wave of NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, NF-κB reporter assay, TNFα stimulation\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP plus ubiquitination assay plus reporter; single study, no structural validation\",\n      \"pmids\": [\"18761086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The E3 ubiquitin ligase CHIP binds MEKK2 and promotes its degradation; CHIP depletion prolongs MEKK2-mediated ERK activation in response to hyperosmotic stress. Transient (not sustained) ERK activation via MEKK2 is required for proper aquaporin 1 and 5 gene induction under hyperosmotic conditions.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, gene targeting of CHIP, ERK activation time-course assays, AQP1/AQP5 gene expression\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, genetic KO of CHIP, functional gene expression readout; multiple orthogonal methods\",\n      \"pmids\": [\"20588253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MEKK2 and MEKK3 negatively regulate TGF-β-mediated Th cell differentiation: Map3k2−/−Map3k3(Lck-Cre/−) mice accumulate Treg and Th17 cells, and their T cells show impaired phosphorylation of SMAD2/3 at linker regions (which negatively regulate TGF-β responses), indicating MAPK-TGF-β pathway crosstalk.\",\n      \"method\": \"Conditional double knockout mice, Th cell differentiation assays, phospho-SMAD western blots, EAE disease model\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — double conditional knockout in T cells with mechanistic phospho-SMAD readout; 52 citations\",\n      \"pmids\": [\"21333552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MEKK2 kinase activity is regulated by phosphorylation-dependent association with 14-3-3 proteins. MEKK2 is phosphorylated at Thr-283, reducing activation-loop phosphorylation at Ser-519 and activity. In the absence of 14-3-3 binding, inactive MEKK2 undergoes trans-autophosphorylation at Ser-519; enforced 14-3-3 binding reduces this trans-autophosphorylation.\",\n      \"method\": \"Phosphorylation site mapping, 14-3-3 binding assays, MEKK2−/− background complementation, JNK/ERK activity assays, IL-6 measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — phosphorylation site mutagenesis, direct binding assay, and functional complementation; multiple orthogonal methods\",\n      \"pmids\": [\"23963453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"XIAP and cIAP1 directly interact with MEKK2/3 and conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3, which competitively disrupts MEK5 binding (competing with PB1 domain interaction) and leads to ERK5 pathway inactivation. Loss of XIAP causes hyperactivation of ERK5 and promotes skeletal muscle cell differentiation.\",\n      \"method\": \"Direct Co-IP, ubiquitin linkage determination (K63), competition binding assays, XIAP/cIAP1 knockdown, ERK5 activation assays, myoblast differentiation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — mechanistic competition between IAP ubiquitination and PB1 domain binding established by multiple biochemical and cell-based methods\",\n      \"pmids\": [\"24975362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Stk38 (NDR kinase) constitutively associates with Smurf1 E3 ligase and facilitates Smurf1-mediated MEKK2 ubiquitination and degradation, specifically suppressing TLR9/CpG-induced ERK1/2 activation and inflammatory cytokine production (TNF-α, IL-6) but not LPS-induced responses.\",\n      \"method\": \"Co-IP, ubiquitination assay, Stk38 KO mice, TLR9 stimulation assays, CpG vs. LPS specificity\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, in vivo KO mouse, biochemical ubiquitination, stimulus-specific discrimination; multiple orthogonal methods\",\n      \"pmids\": [\"25981615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MEKK2 mediates an alternative (non-canonical) β-catenin activation pathway in osteoblasts: FGF2 activates MEKK2, which phosphorylates β-catenin at Ser675, promoting recruitment of the deubiquitinase USP15, which prevents β-catenin ubiquitination and degradation, thereby enhancing WNT signaling and bone formation.\",\n      \"method\": \"MEKK2−/− mice, in vitro kinase assays, phospho-β-catenin mapping, USP15 Co-IP, genetic interaction studies with β-catenin null alleles, MS phosphoproteomics\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assay, Co-IP, mutagenesis, and in vivo genetic interaction; multiple orthogonal methods\",\n      \"pmids\": [\"26884171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SMYD3 co-crystal structure with a MEKK2-peptide substrate reveals the substrate-binding tunnel. Structure-based design of GSK2807, a SAM-competitive inhibitor that bridges the SAM-binding pocket and the MEKK2 substrate lysine tunnel of SMYD3 (Ki = 14 nM), blocking MAP3K2 methylation.\",\n      \"method\": \"Co-crystal structure of SMYD3/MEKK2 peptide, kinetic characterization, inhibitor design and structure determination\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation; directly defines the SMYD3-MAP3K2 interaction interface\",\n      \"pmids\": [\"27066749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MEKK2 and MEKK3 inhibit GLI1 transcriptional activity and promote GLI1 interaction with SUFU by phosphorylating multiple Ser/Thr sites on GLI1, reducing GLI1 protein stability and DNA-binding ability, thereby suppressing Hedgehog pathway signaling. FGF2-mediated inhibition of Hh signaling requires MEKK2 and MEKK3.\",\n      \"method\": \"In vitro kinase assays, phosphorylation site mapping on GLI1, co-immunoprecipitation (GLI1-SUFU), medulloblastoma cell proliferation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase phosphorylation plus Co-IP mechanistic readout plus cellular functional assays\",\n      \"pmids\": [\"29662197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Kir2.1 interacts with Stk38 to inhibit Smurf1-mediated ubiquitination and degradation of MEKK2, thereby stabilizing MEKK2 and activating the MEK1/2-ERK1/2-Snail EMT pathway in gastric cancer cells. This function is independent of K+ ion permeation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, siRNA knockdown, Kir2.1 mutants (ion-permeation defective), xenograft metastasis assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic Co-IP chain (Kir2.1-Stk38-Smurf1-MEKK2) with ion-channel-independent mutant controls; multiple methods\",\n      \"pmids\": [\"29549164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MAP3K2 mediates a ROS-MAP3K2-ERK5-KLF2 signaling axis in intestinal stromal cells (MRISCs) that drives R-spondin 1 production to maintain LGR5+ intestinal stem cells and protect against acute intestinal damage.\",\n      \"method\": \"MAP3K2−/− mice, intestinal injury models, single-cell transcriptomics, epigenetic profiling, ROS manipulation, RSPO1 measurement\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO in vivo with defined pathway (ROS-MAP3K2-ERK5-KLF2-RSPO1) and specific cellular readout; published in Nature\",\n      \"pmids\": [\"33658717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MEKK2 and MEKK3 regulate the Hippo pathway: they interact with and phosphorylate LATS1/2 and YAP/TAZ; TNF activates LATS1/2 and inhibits YAP/TAZ through MEKK2/3. STRIPAK complex associates with MEKK3 via CCM2/CCM3 to inactivate MEKK3, and upstream Hippo signals trigger dissociation of MEKK3 from STRIPAK.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assays for LATS1/2 and YAP/TAZ phosphorylation, genetic knockdown, TNF stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase assays plus Co-IP plus signaling epistasis; multiple orthogonal methods\",\n      \"pmids\": [\"33571521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MAP3K2-mediated Th1 cell differentiation in the intestine is regulated by IL-18 and requires specific JNK activation (IL-18-MAP3K2-JNK axis); MAP3K2-deficient naïve CD4+ T cells transferred into mice have a dramatically reduced ability to induce colitis, with fewer IFNγ-producing but more IL-17A-producing cells.\",\n      \"method\": \"T cell transfer colitis model, MAP3K2−/− mice, in vitro Th1/Th17/Treg differentiation assays, JNK activation assays, IL-18 supplementation\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo transfer model with KO T cells, defined molecular axis (IL-18-MAP3K2-JNK) with multiple pathway measurements\",\n      \"pmids\": [\"32737854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Pazopanib inhibits MAP3K2- and MAP3K3-mediated phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208, reducing ROS formation in myeloid cells. Myeloid-specific MAP3K2/MAP3K3 genetic inactivation or hematopoietic p47phox-S208A mutation attenuates acute lung injury, establishing MAP3K2 as upstream kinase for p47phox-Ser208 phosphorylation.\",\n      \"method\": \"Genetic inactivation of MAP3K2/3 in myeloid cells, phosphorylation site mutation (p47phox-S208A), ALI mouse models, kinase activity assays\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct kinase-substrate (MAP3K2→p47phox-S208) with genetic confirmation; published in Science Translational Medicine\",\n      \"pmids\": [\"33910977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MEKK2 mediates aberrant ERK activation downstream of NF1 (neurofibromin) loss in osteoblasts via a noncanonical ERK pathway. MEKK2-deficient mice crossed with osteoblast-conditional Nf1-knockout mice show amelioration of NF1-associated skeletal phenotypes, placing MEKK2 epistatic to NF1 in ERK activation in bone.\",\n      \"method\": \"MEKK2−/− and Nf1fl/fl conditional KO mice, double-mutant genetic interaction, skeletal phenotype analysis, ERK activation assays, FDA-approved MEKK2 inhibitor in vivo\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic epistasis with conditional KO mouse models and pharmacological inhibitor proof-of-principle\",\n      \"pmids\": [\"33177525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MEKK2 ablation in invasive breast cancer cells enhances focal adhesion stability, increases spread area, and reduces cell migration. MEKK2 is activated by and localizes to focal adhesions upon fibronectin/Matrigel attachment, and MEKK2 knockdown inhibits fibronectin-induced ERK5 signaling and FAK autophosphorylation.\",\n      \"method\": \"siRNA knockdown, focal adhesion imaging, cell migration assays, kinase activity assays (MEKK2 activation by attachment), FAK phosphorylation western blot\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — siRNA KD with multiple cellular readouts (focal adhesion size, migration, kinase activity, FAK) but no reconstitution\",\n      \"pmids\": [\"24491810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MEKK2 physically associates with the LD1 motif of paxillin, induces paxillin ubiquitylation (requiring both the paxillin LD1 motif and MEKK2 kinase activity), and promotes redistribution of paxillin from focal adhesions into the cytoplasm without promoting degradation.\",\n      \"method\": \"Co-immunoprecipitation (MEKK2-paxillin LD1 domain), ubiquitylation assay, MEKK2 kinase-dead mutant, paxillin localization imaging\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP plus ubiquitylation plus kinase-dead mutant controls; single study\",\n      \"pmids\": [\"25190348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Paxillin LD1 motif binding to the MEKK2 amino-terminal region relieves MEKK2 auto-inhibition: recombinant paxillin induces MEKK2 auto-phosphorylation in vitro, and paxillin knockdown reduces MEKK2 activity in cells.\",\n      \"method\": \"In vitro kinase/auto-phosphorylation assay with recombinant paxillin, siRNA paxillin knockdown, LD1 motif binding assay\",\n      \"journal\": \"Journal of molecular signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro reconstitution plus siRNA cell-based validation; single lab, limited follow-up\",\n      \"pmids\": [\"27096002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MEKK2 phosphorylates STK38 (Ser91) to protect it from calpain-mediated cleavage at the N-terminal region; MEKK2 knockdown enhances hyperthermia-induced STK38 degradation. A phosphorylation-defective S91A STK38 mutant is susceptible to calpain cleavage.\",\n      \"method\": \"In vitro MEKK2 kinase assay with STK38 substrate, phosphorylation site identification by mass spectrometry, calpain cleavage assay, MEKK2 siRNA knockdown, phospho-defective mutant\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay identifying Ser91 site plus phospho-defective mutant validation plus calpain cleavage reconstitution\",\n      \"pmids\": [\"31690749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4L (HECT E3 ligase) constitutively binds MEKK2 and promotes its poly-ubiquitination and degradation. IL-17-induced MEKK2 Ser520 phosphorylation is required not only for downstream p38 and NF-κB activation but also for NEDD4L-mediated MEKK2 degradation (negative feedback). Nedd4l-deficient mice show increased IL-17-induced inflammation.\",\n      \"method\": \"Co-IP, ubiquitination assay, siRNA knockdown, Nedd4l KO mice, phospho-Ser520 mutagenesis, IL-17 stimulation assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct Co-IP, ubiquitination assay, mutagenesis at regulatory site, in vivo KO mouse confirmation; multiple orthogonal methods\",\n      \"pmids\": [\"36161689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Best3 (Bestrophin3) directly interacts with both MEKK2 and MEKK3 and inhibits phosphorylation of MEKK2 at Ser153 (and MEKK3 at Ser61). Best3 deficiency induces phosphorylation-dependent stabilization of MEKK2/3 (inhibition of ubiquitination/turnover), activating downstream MAPK signaling and triggering aortic dissection.\",\n      \"method\": \"Co-IP coupled with mass spectrometry, Best3 smooth muscle-specific KO mice, single-cell RNA sequencing, proteomics, phosphorylation site analysis, MEKK2/3 inhibitor rescue\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP/MS plus genetic KO in vivo plus pharmacological rescue; published in Circulation with multiple orthogonal approaches\",\n      \"pmids\": [\"37203562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SMYD3-mediated methylation of MAP3K2 promotes epithelial-mesenchymal transition-associated behaviors in prostate cancer cells via a positive feedback loop that continuously promotes high SMYD3 levels, and alters vimentin abundance downstream.\",\n      \"method\": \"SMYD3 catalytic mutant, xenograft mouse models, EMT marker analysis, vimentin expression measurement\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — methyltransferase mutant plus in vivo xenograft; extends SMYD3-MAP3K2 methylation finding to prostate cancer context\",\n      \"pmids\": [\"37976356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NDR2 (LATS/NDR kinase family) promotes Smurf1-mediated K48-linked ubiquitination of MEKK2 in cells, reducing MEKK2 levels and inhibiting IL-17-induced MAPK and NF-κB activation; Smurf1 knockdown similarly increases IL-17-induced cytokine expression.\",\n      \"method\": \"Co-IP (NDR2-Smurf1), ubiquitination assay with K48-linkage determination, siRNA knockdown, IL-17 stimulation, cytokine quantification\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus ubiquitination assay; single lab study, extends Smurf1-MEKK2 regulatory axis\",\n      \"pmids\": [\"30504095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In rheumatoid arthritis fibroblast-like synoviocytes (FLS), IL-1 increases MEKK2 kinase activity (measured by immunoprecipitation kinase assay using MKK4 and MKK7 as substrates), and MEKK2 immunoprecipitates activate c-Jun via JNK; activity blocked by JNK inhibitor SP600125.\",\n      \"method\": \"Immunoprecipitation kinase assay with MKK4/MKK7 substrates, c-Jun phosphorylation assay, pharmacological JNK inhibitor\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — immunoprecipitation kinase assay in disease-relevant primary cells with specific substrate readout; single lab\",\n      \"pmids\": [\"14734742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Calcium is required for EGF-induced ERK5 activation upstream of MEKK2. EGF increases MEKK2 binding to adaptor protein Lad1 in a calcium-dependent manner; in vitro binding assays show calcium is needed for direct MEKK2-Lad1 interaction. Calcium also regulates EGF-induced nuclear translocation of MEKK2.\",\n      \"method\": \"Co-immunoprecipitation, in vitro direct binding assay, intracellular calcium modifiers, nuclear translocation imaging\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP plus in vitro binding plus imaging; single lab, moderate methods\",\n      \"pmids\": [\"20830310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Point mutations in protein kinase subdomain X (distinct from MEKK1 equivalent residues) impair MEKK2 phosphorylation of MKK7 and MEK5, abolish MEKK2-induced JNK1 and ERK5 activation, and diminish AP-1 reporter gene activation, revealing subdomain X as critical for MEKK2 substrate interaction.\",\n      \"method\": \"Site-directed mutagenesis, in vitro kinase assays with MKK7/MEK5 substrates, JNK1/ERK5 activation assays, AP-1 reporter gene\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — mutagenesis combined with in vitro kinase assays and reporter assays; single study\",\n      \"pmids\": [\"12659851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MEKK2 mediates ERK5 activation downstream of PDGF-BB in smooth muscle cells in a manner dependent on Mek1/2, Mek5, PI3-kinase, and classical PKC isoforms, demonstrating co-regulation between ERK1/2 and ERK5 pathways.\",\n      \"method\": \"Dominant-negative MEKK2, kinase activity assays, PI3K/PKC inhibitors, ERK5 activation measurement in MOVAS cells\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — dominant-negative epistasis plus pharmacological inhibitors; single study in smooth muscle cells\",\n      \"pmids\": [\"27339033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In sublytic C5b-9-induced glomerular mesangial cell apoptosis, MEKK2 phosphorylation (at Ser153/164/239) activates p38 MAPK, which drives IRF-1 and TRADD expression, leading to caspase 8 activation and apoptosis. Silencing MEKK2 in vivo inhibits GMC apoptosis in rat Thy-1 nephritis.\",\n      \"method\": \"Phosphorylation site mutagenesis (Ser153/164/239), p38 MAPK kinase assays, IRF-1/TRADD gene silencing in vivo, caspase 8 activity assay, rat Thy-1 nephritis model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phosphorylation site mutagenesis plus in vivo gene silencing with apoptosis readout; single lab\",\n      \"pmids\": [\"28039298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ZSWIM1 interacts with STK38 (identified by IP-MS) and antagonizes STK38 function, promoting release and activation of MEKK2, leading to ERK1/2 activation and lung adenocarcinoma progression.\",\n      \"method\": \"Immunoprecipitation-mass spectrometry (interactome), Co-IP validation, ZSWIM1 overexpression/knockdown, ERK1/2 activation assays\",\n      \"journal\": \"Journal of proteome research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — IP-MS interactome with Co-IP validation and functional cell assays; mechanism placed upstream of MEKK2 but limited kinase biochemistry\",\n      \"pmids\": [\"36511424\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAP3K2 (MEKK2) is a serine/threonine MAP3K that signals through MEK5-ERK5 and MKK4/7-JNK pathways via PB1 domain-mediated scaffolding with MEK5 and MKK7; it is activated by dimerization, upstream kinases (WNK1, Rap1), and adaptor-mediated recruitment (Lad1/RIBP), and its activity is regulated by multiple post-translational mechanisms including SMYD3-mediated K260 methylation (which blocks PP2A binding to enhance Ras-ERK signaling), Smurf1/XIAP/NEDD4L/CHIP-mediated ubiquitination and degradation, 14-3-3 binding (suppressing trans-autophosphorylation), and paxillin LD1-induced activation; it also phosphorylates non-canonical substrates including β-catenin-Ser675, p47phox-Ser208, GLI1, LATS1/2, YAP/TAZ, and STK38-Ser91 to control bone formation, innate immunity, Hedgehog pathway suppression, Hippo signaling, and intestinal stem cell niche maintenance.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MAP3K2 (MEKK2) is a serine/threonine MAP3K that serves as a central signaling hub coupling diverse extracellular stimuli—including growth factors, cytokines, and reactive oxygen species—to downstream MAPK cascades and non-canonical substrates in immune regulation, bone formation, intestinal homeostasis, and vascular integrity. Through its N-terminal PB1 domain, MAP3K2 scaffolds MEK5 for ERK5 pathway activation and, upon its own activation, recruits MKK7 via a distinct PB1 surface to additionally engage JNK signaling; it also phosphorylates MKK4, p38 substrates, and non-MAPK targets including β-catenin (Ser675), p47phox (Ser208), GLI1, LATS1/2, and STK38 (Ser91) [PMID:12912994, PMID:17452462, PMID:26884171, PMID:33910977, PMID:29662197, PMID:33571521, PMID:31690749]. MAP3K2 activation is triggered by dimerization through its kinase domain, upstream kinases (WNK1, Rap1), adaptor-mediated recruitment (Lad1, paxillin LD1 motif), and trans-autophosphorylation at Ser519, and is negatively regulated by 14-3-3 binding, SMYD3-mediated K260 methylation that blocks PP2A dephosphorylation, and multiple E3 ubiquitin ligases (Smurf1, XIAP/cIAP1, CHIP, NEDD4L) that target MAP3K2 for ubiquitin-dependent degradation or PB1-domain competition [PMID:15695508, PMID:14681216, PMID:23963453, PMID:24847881, PMID:15820682, PMID:24975362, PMID:20588253, PMID:36161689]. Genetic ablation of MAP3K2 reveals stimulus-selective requirements: loss of FcεRI/c-Kit–driven JNK in mast cells, impaired FGF2-induced ERK5/JNK in fibroblasts, augmented T cell proliferation reflecting negative modulation of TCR signaling, and compromised intestinal stem cell maintenance via the ROS–ERK5–KLF2–RSPO1 axis [PMID:11032806, PMID:14978743, PMID:12138187, PMID:33658717].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"MAP3K2 was placed at the T cell immunological synapse: imaging showed MEKK2 translocation to the T cell–APC contact, and dominant-negative MEKK2 blocked TCR-mediated ERK and p38 activation, establishing it as a receptor-proximal MAP3K in adaptive immunity.\",\n      \"evidence\": \"Immunocytochemistry and dominant-negative expression in T cell conjugate assays\",\n      \"pmids\": [\"10549623\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signal recruiting MEKK2 to the synapse not identified\", \"Kinase-dead versus dominant-negative effects not separated\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"A direct link between MAP3K2 and the MEK5–ERK5 cascade was identified, and MEKK2 knockout in mast cells revealed stimulus-selective JNK dependence, demonstrating that MAP3K2 routes specific receptor tyrosine kinase signals to both ERK5 and JNK.\",\n      \"evidence\": \"Yeast two-hybrid identification of MEK5 as MEKK2 partner; MEKK2−/− ES cell-derived mast cells tested for FcεRI/c-Kit versus UV-induced JNK\",\n      \"pmids\": [\"11073940\", \"11032806\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of stimulus selectivity unknown\", \"Whether ERK5 and JNK are activated simultaneously or sequentially from the same complex unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"MEKK2 knockout mice unexpectedly showed enhanced rather than reduced T cell activation, revealing that MAP3K2 negatively modulates TCR signal strength in vivo.\",\n      \"evidence\": \"Mekk2−/− mice with T cell proliferation, cytokine ELISA, and JNK/ERK/p38 kinase assays\",\n      \"pmids\": [\"12138187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of negative modulation (direct kinase substrate versus scaffold sequestration) not resolved\", \"Redundancy with MEKK3 in T cells not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The structural basis for MAP3K2 scaffolding was established: PB1 domain heterodimerization with MEK5 is required for ERK5 activation, and WNK1 was identified as an upstream kinase that phosphorylates MAP3K2 to relay signals to ERK5.\",\n      \"evidence\": \"PB1 domain mutagenesis and in vitro binding; WNK1 in vitro kinase assay and dominant-negative epistasis in HEK293 cells\",\n      \"pmids\": [\"12912994\", \"14681216\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphorylation site(s) on MEKK2 targeted by WNK1 not mapped\", \"Structural basis of PB1–PB1 interaction not yet resolved at atomic level\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"MAP3K2 was shown to coordinate both ERK5 and JNK downstream of FGF-2 in fibroblasts and to undergo stimulus-dependent nuclear translocation to relay signals to nuclear MEK5, providing spatial regulation of MAPK signaling.\",\n      \"evidence\": \"MEKK2−/− MEFs tested for FGF-2/LPS/TNFα-selective pathway loss; immunofluorescence and subcellular fractionation in HeLa/Rat-1 cells after EGF\",\n      \"pmids\": [\"14978743\", \"15075238\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear import mechanism (NLS, carrier) not identified\", \"How stimulus selectivity is encoded at the molecular level unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Multiple layers of MAP3K2 regulation were uncovered: activation requires kinase-domain dimerization; the inhibitor Mip1 blocks dimerization of inactive MEKK2; Smurf1 ubiquitinates MEKK2 for proteasomal degradation controlling osteoblast JNK signaling; and Ser519 was identified as a key activation-loop phosphorylation site induced by TLR/TRAF6.\",\n      \"evidence\": \"Chemical-induced dimerization system; Mip1 Co-IP and siRNA; Smurf1−/− mice; phospho-S519 mutagenesis with LPS stimulation\",\n      \"pmids\": [\"15695508\", \"15988011\", \"15820682\", \"16362041\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the kinase that phosphorylates Ser519 (autophosphorylation versus upstream kinase) not fully resolved\", \"How TRAF6 activates MEKK2 Ser519 phosphorylation mechanistically unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"The bifunctional nature of the MAP3K2 PB1 domain was defined: its front face binds MEK5 constitutively, while its C-terminal acidic cluster recruits MKK7 only upon MEKK2 activation, explaining how one scaffold sequentially activates ERK5 then JNK.\",\n      \"evidence\": \"PB1 domain face-specific mutagenesis with Co-IP and ERK5/JNK pathway assays\",\n      \"pmids\": [\"17452462\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ternary complex stoichiometry not determined\", \"Whether MEKK2 simultaneously engages MEK5 and MKK7 or switches between them unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"CHIP E3 ligase was identified as a negative regulator that promotes MAP3K2 degradation and limits ERK activation duration under hyperosmotic stress, and calcium was shown to regulate the MEKK2–Lad1 interaction required for EGF-induced ERK5 signaling.\",\n      \"evidence\": \"CHIP Co-IP and knockout with ERK time-course; calcium-dependent MEKK2–Lad1 Co-IP and in vitro binding\",\n      \"pmids\": [\"20588253\", \"20830310\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How calcium modulates MEKK2–Lad1 binding structurally not known\", \"CHIP recognition motif on MEKK2 not mapped\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Combined MAP3K2/MAP3K3 deletion in T cells revealed crosstalk with TGF-β signaling: loss of MEKK2/3-mediated SMAD2/3 linker phosphorylation unleashed TGF-β-driven Treg and Th17 accumulation, connecting MAP3K2 to adaptive immune tolerance.\",\n      \"evidence\": \"Map3k2−/−Map3k3(Lck-Cre/−) mice with Th cell differentiation and phospho-SMAD analysis; EAE model\",\n      \"pmids\": [\"21333552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct kinase–substrate relationship between MEKK2 and SMAD linker phosphorylation versus intermediate kinases not resolved\", \"Relative contributions of MEKK2 and MEKK3 not separated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"14-3-3 binding to phospho-Thr283 on MAP3K2 was shown to suppress trans-autophosphorylation at Ser519, establishing 14-3-3 as a tonic brake on MAP3K2 activation.\",\n      \"evidence\": \"Phosphorylation site mapping, 14-3-3 binding assay, complementation in MEKK2−/− cells\",\n      \"pmids\": [\"23963453\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for Thr283 phosphorylation not identified\", \"Stoichiometry of 14-3-3–MEKK2 complex in vivo unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Two new regulatory paradigms for MAP3K2 emerged: SMYD3-mediated K260 methylation blocks PP2A binding to sustain Ras-ERK signaling in cancer, and XIAP/cIAP1-conjugated K63-ubiquitin chains on MEKK2 competitively disrupt MEK5 PB1-domain binding to inactivate ERK5.\",\n      \"evidence\": \"SMYD3 methylation assays with mouse pancreatic/lung cancer models; XIAP K63-ubiquitin linkage determination and PB1 competition assays with myoblast differentiation readout\",\n      \"pmids\": [\"24847881\", \"24975362\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether K260 methylation and K63-ubiquitination can co-occur on the same molecule unknown\", \"SMYD3-MAP3K2 axis not yet validated in human patient tumors with matched methylation data\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"MAP3K2 was established as a direct kinase for non-MAPK substrates: phosphorylation of β-catenin at Ser675 recruits USP15 to stabilize β-catenin and promote bone formation, and the co-crystal structure of the SMYD3–MEKK2 peptide interface was solved.\",\n      \"evidence\": \"MEKK2−/− mice with in vitro kinase assays and genetic interaction with β-catenin null alleles; co-crystal structure of SMYD3 with MEKK2 substrate peptide\",\n      \"pmids\": [\"26884171\", \"27066749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether β-catenin Ser675 phosphorylation by MEKK2 operates in tissues beyond bone not tested\", \"Full-length MEKK2 structure unavailable\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Additional non-MAPK substrates were identified: MAP3K2 phosphorylates GLI1 to promote its interaction with SUFU and suppress Hedgehog signaling, and phosphorylates STK38 at Ser91 to protect it from calpain cleavage, expanding MAP3K2's substrate repertoire beyond canonical MAPK intermediates.\",\n      \"evidence\": \"In vitro kinase assays mapping GLI1 phosphosites with Co-IP for GLI1–SUFU; MEKK2 kinase assay on STK38 with Ser91 mutagenesis and calpain cleavage reconstitution\",\n      \"pmids\": [\"29662197\", \"31690749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where MEKK2-GLI1 axis operates in vivo not fully defined\", \"Whether STK38 Ser91 phosphorylation feeds back to regulate MEKK2 stability via Smurf1 not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Genetic epistasis in mouse bone placed MAP3K2 downstream of NF1 loss in a noncanonical ERK activation pathway: MEKK2 deletion rescued NF1-deficiency skeletal phenotypes, nominating MAP3K2 as a therapeutic target in neurofibromatosis.\",\n      \"evidence\": \"MEKK2−/− crossed with Nf1fl/fl conditional KO mice; skeletal phenotype analysis with pharmacological MEKK2 inhibitor\",\n      \"pmids\": [\"33177525\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical mechanism linking NF1 loss to MEKK2 hyperactivation not defined\", \"Clinical translation of MEKK2 inhibitors for NF1 skeletal disease not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"MAP3K2's physiological roles expanded to intestinal stem cell maintenance (ROS–MEKK2–ERK5–KLF2–RSPO1 in stromal cells), Hippo pathway regulation (direct phosphorylation of LATS1/2 and YAP/TAZ), NADPH oxidase activation (p47phox Ser208 phosphorylation in myeloid cells), and IL-18-driven Th1 differentiation in the gut.\",\n      \"evidence\": \"MAP3K2−/− mice with intestinal injury and single-cell transcriptomics; in vitro kinase assays for LATS1/2 and YAP/TAZ; myeloid-specific MAP3K2/3 KO and p47phox-S208A knockin in ALI models; T cell transfer colitis with MAP3K2−/− cells\",\n      \"pmids\": [\"33658717\", \"33571521\", \"33910977\", \"32737854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MEKK2 phosphorylation of LATS1/2 is direct in all cell types or context-dependent not established\", \"Relative contribution of MAP3K2 versus MAP3K3 in Hippo and p47phox pathways not fully separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"NEDD4L was identified as an additional E3 ligase that degrades MAP3K2 via a negative feedback loop triggered by IL-17-induced Ser520 phosphorylation, reinforcing that MAP3K2 protein levels are tightly controlled by multiple ubiquitin ligases in inflammatory signaling.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, phospho-Ser520 mutagenesis, Nedd4l KO mice with IL-17 stimulation\",\n      \"pmids\": [\"36161689\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Smurf1 and NEDD4L compete for the same or distinct ubiquitination sites on MEKK2 not resolved\", \"Phosphatase that reverses Ser520 phosphorylation not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Bestrophin-3 was shown to directly bind MAP3K2 and suppress its Ser153 phosphorylation, preventing phosphorylation-dependent stabilization; Best3 deficiency hyperactivates MEKK2 and causes aortic dissection, linking MAP3K2 regulation to vascular integrity.\",\n      \"evidence\": \"Best3 smooth muscle-specific KO mice with Co-IP/MS, single-cell RNA-seq, and MEKK2/3 inhibitor rescue of aortic phenotype\",\n      \"pmids\": [\"37203562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Best3 regulation of MEKK2 occurs in tissues beyond vasculature not tested\", \"How Ser153 phosphorylation blocks ubiquitin-mediated degradation mechanistically unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structure of MAP3K2 is lacking, the complete phosphorylation code governing activation versus degradation is incompletely mapped, and the basis for stimulus-selective pathway routing through MAP3K2 remains a central unresolved question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length crystal or cryo-EM structure of MAP3K2\", \"Complete catalog of direct phosphorylation substrates in different cell types not established\", \"How MAP3K2 achieves stimulus selectivity (FGF vs. TNF vs. LPS) at the molecular level is unclear\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 4, 12, 13, 21, 23, 26, 28, 33, 40]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 4, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [9, 30]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 3, 4, 6, 13, 14, 25, 26]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 7, 9, 17, 27, 28]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [42]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 21, 29]}\n    ],\n    \"complexes\": [\n      \"MEKK2–MEK5 (PB1-mediated)\",\n      \"MEKK2–MKK7 (PB1-mediated)\",\n      \"Smurf1–MEKK2\",\n      \"STRIPAK–MEKK3 (related)\"\n    ],\n    \"partners\": [\n      \"MAP2K5\",\n      \"MAP2K7\",\n      \"WNK1\",\n      \"SMYD3\",\n      \"SMURF1\",\n      \"XIAP\",\n      \"CHIP\",\n      \"NEDD4L\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}