{"gene":"MAP3K2","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2014,"finding":"SMYD3 methylates MAP3K2 at lysine 260, potentiating activation of the Ras/Raf/MEK/ERK signaling module. Methylation of MAP3K2 at K260 blocks binding of the PP2A phosphatase complex to MAP3K2, preventing PP2A-mediated negative regulation of the MAP kinase pathway.","method":"Protein array technology to identify MAP3K2 as SMYD3 substrate; in vitro methylation assays; Co-IP showing PP2A-MAP3K2 interaction blocked by methylation; mouse models of pancreatic/lung adenocarcinoma","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (in vitro methylation, Co-IP, mutagenesis, mouse tumor models), replicated in prostate cancer study (PMID:37976356)","pmids":["24847881"],"is_preprint":false},{"year":2005,"finding":"Smurf1, a HECT domain ubiquitin E3 ligase, physically interacts with MEKK2 and promotes its ubiquitination and proteasomal degradation, thereby suppressing osteoblast activity and JNK signaling downstream of BMP.","method":"Co-IP showing Smurf1-MEKK2 physical interaction; ubiquitination assays; Smurf1-deficient mouse model showing accumulation of phosphorylated MEKK2 and enhanced JNK activation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reciprocal Co-IP, ubiquitination assay, in vivo genetic model with defined phenotype, replicated by multiple subsequent studies","pmids":["15820682"],"is_preprint":false},{"year":2003,"finding":"The PB1 domains of MEKK2 and MEKK3 heterodimerize with the PB1 domain of MEK5, forming a complex required for ERK5 pathway activation. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 complex formation and ERK5 activation. The PB1 domain interaction is specific to the ERK5 pathway and does not affect p38 or JNK pathways.","method":"In vitro PB1 domain binding assays; co-immunoprecipitation of MEKK2 and MEK5 from cell lysates; deletion/mutation analysis; dominant-negative expression experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro binding reconstitution, Co-IP, mutagenesis, multiple orthogonal methods in one study","pmids":["12912994"],"is_preprint":false},{"year":2000,"finding":"MEKK2 activates the MEK5-BMK1/ERK5 pathway; MEK5 was identified as a binding partner of MEKK2 by yeast two-hybrid screening, and a dominant-negative MEK5 blocked ERK5 activation by MEKK2. MEKK2 also associates with the T cell adapter protein Lad/RIBP, and co-localizes with Lad/RIBP at the T cell/APC contact site during T cell activation.","method":"Yeast two-hybrid library screen; co-localization imaging; dominant-negative expression; kinase activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid, co-localization, dominant-negative epistasis, replicated by PB1 domain studies","pmids":["11073940"],"is_preprint":false},{"year":2000,"finding":"MEKK2 is required for receptor-mediated JNK activation and cytokine gene transcription in mast cells following IgE receptor or c-Kit ligation. MEKK2-deficient ES cell-derived mast cells show markedly reduced cytokine production and loss of receptor-mediated JNK activation, while JNK activation by UV irradiation is unaffected.","method":"Targeted gene disruption of MEKK2 in ES cell-derived mast cells; RT-PCR for cytokine transcription; kinase assays for JNK activation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with specific cellular phenotype, specificity controls (MEKK1 KO, UV irradiation), replicated in subsequent mast cell studies","pmids":["11032806"],"is_preprint":false},{"year":2004,"finding":"MEKK2 coordinately activates ERK5 and JNK pathways in response to FGF-2 signaling in fibroblasts. MEKK2-deficient MEFs lose ERK5 and JNK activation in response to FGF-2 but not LPS or TNFα, demonstrating MEKK2 specificity in FGF-2 receptor signaling. MEKK2 regulates AP-1 activity by controlling expression of c-Jun, Fra-1, and Fra-2 and c-Jun N-terminal phosphorylation, leading to cytokine gene expression.","method":"MEKK2-/- mouse embryonic fibroblasts; kinase activation assays; RT-PCR for AP-1 components and cytokines","journal":"Journal of cellular physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with multiple specific phenotypic readouts, stimulus-specificity controls","pmids":["14978743"],"is_preprint":false},{"year":2004,"finding":"MEKK2 is localized mainly in the cytosol of resting cells and translocates to the nucleus upon EGF stimulation, allowing transmission of signals to nuclear MEK5. MEK5 and ERK5 are constitutively nuclear in endogenous form, bound to detergent-resistant nuclear moieties.","method":"Immunocytochemistry; live cell fluorescence imaging; in situ detergent extraction; subcellular fractionation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with multiple methods, single lab","pmids":["15075238"],"is_preprint":false},{"year":2005,"finding":"MEKK2 activation requires dimerization through its catalytic domain. The dimerization motif maps to the catalytic domain (N-terminal region not required). Inactive, non-phosphorylated MEKK2 forms more dimers than phosphorylated active MEKK2. Prevention of dimerization inhibits MEKK2-mediated JNK activation; chemical-induced dimerization augments JNK activation and AP-1 reporter activity.","method":"Dimerization mapping using deletion constructs; chemical-induced dimerization system; JNK activation assays; AP-1 reporter gene assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — chemical-induced dimerization, mutagenesis, multiple functional readouts, single lab","pmids":["15695508"],"is_preprint":false},{"year":2005,"finding":"Mip1 (MEKK2-interacting protein) forms a complex with inactive, non-phosphorylated MEKK2 and prevents MEKK2 activation by blocking MEKK2 dimer formation, thereby inhibiting JNK1, ERK5, and AP-1 activation. The endogenous Mip1-MEKK2 complex dissociates transiently following EGF stimulation. siRNA knockdown of Mip1 augments MEKK2-mediated JNK and AP-1 activation.","method":"Co-IP; siRNA knockdown; kinase and reporter gene activation assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, siRNA knockdown with functional readout, single lab","pmids":["15988011"],"is_preprint":false},{"year":2005,"finding":"Serine 519 in MEKK2 (and S526 in MEKK3) is a key regulatory phosphorylation site required for kinase activation. Mutation of S519 to alanine severely impairs MEKK2 activation. LPS induces phosphorylation of this serine in a TRAF6-dependent manner, and this phosphorylation is required for TLR-induced IL-6 production.","method":"Site-directed mutagenesis (S519A); anti-phospho-MEKK2/3 antibody; LPS stimulation assays; TRAF6 perturbation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — active-site mutagenesis, phospho-specific antibody validation, functional readout (IL-6 production), single lab with multiple orthogonal methods","pmids":["16362041"],"is_preprint":false},{"year":2003,"finding":"WNK1 activates ERK5 via MEKK2 and MEKK3 in an MEKK2/3-dependent mechanism. WNK1 phosphorylates MEKK2 and MEKK3 in vitro and activates MEKK3 in cells. Both MEKK2 and MEKK3 co-immunoprecipitate with endogenous WNK1. Dominant-negative MEKK2 and MEKK3 block WNK1-induced ERK5 activation.","method":"Co-IP of WNK1 with MEKK2/3; in vitro kinase assay; dominant-negative expression; siRNA knockdown of WNK1","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay, Co-IP, dominant-negative epistasis, siRNA validation, single lab","pmids":["14681216"],"is_preprint":false},{"year":1999,"finding":"MEKK2 translocates to the T cell/antigen-presenting cell interface upon antigen-mediated TCR engagement. Biochemical activation of MEKK2 follows TCR stimulation, and dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK/p38 MAPK phosphorylation.","method":"Live cell fluorescence imaging; immunocytochemistry; dominant-negative expression; kinase activation assays","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging, dominant-negative functional assay, single lab with two orthogonal methods","pmids":["10549623"],"is_preprint":false},{"year":2002,"finding":"MEKK2-deficient T cells show augmented proliferation and increased IL-2/IFNγ production in response to anti-CD3 stimulation, and greater susceptibility to anti-CD3-induced cell death, indicating MEKK2 negatively regulates TCR signaling strength. TCR-mediated JNK activation was moderately enhanced (not abolished) in MEKK2-/- T cells; ERK and p38 activation were unaffected.","method":"Targeted Mekk2 gene disruption in mice; T cell proliferation assays; cytokine production assays; JNK/ERK/p38 activation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with multiple functional readouts and specificity controls, single lab","pmids":["12138187"],"is_preprint":false},{"year":2006,"finding":"BDNF activates ERK5 in cortical neurons via a Rap1-MEKK2 signaling cascade. BDNF activates Rap1, which activates MEKK2 (a MEK5 kinase); inhibition of either Rap1 or MEKK2 attenuates BDNF-induced ERK5 activation. Ras and MEKK3 do not play significant roles in this neuronal BDNF-ERK5 pathway.","method":"Dominant-negative and constitutively active expression of Rap1 and MEKK2; kinase activity assays in cortical neurons","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis using dominant-negative/active constructs, single lab, multiple pathway components tested","pmids":["17003042"],"is_preprint":false},{"year":2010,"finding":"CHIP (carboxyl terminus of Hsc70-interacting protein) E3 ubiquitin ligase binds MEKK2 and promotes its degradation, thereby terminating ERK activation in response to hyperosmotic stress. Transient ERK activation via MEKK2 is required for proper aquaporin (AQP1, AQP5) gene induction; CHIP depletion prolongs MEKK2 stability, prolongs ERK activity, and paradoxically suppresses AQP gene expression.","method":"Co-IP identifying CHIP-MEKK2 interaction; siRNA knockdown of CHIP; gene targeting of CHIP; ERK activity time-course assays; AQP gene expression assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — Co-IP, siRNA, gene targeting, functional gene expression readout, single lab with multiple orthogonal methods","pmids":["20588253"],"is_preprint":false},{"year":2007,"finding":"MEKK2 and MEK5 PB1 domains interact in a front-to-back arrangement; quiescent MEKK2 preferentially binds MEK5 via PB1, leading to ERK5 activation. Upon activation, MEKK2 binds and activates MKK7 via the C-terminal acidic cluster of the MEKK2 PB1 domain, leading to JNK activation. The C-terminal MEK5 PB1 extension encodes an ERK5 docking site required for MEK5 activation of ERK5.","method":"Mutagenesis of PB1 domain residues; co-immunoprecipitation; kinase activation assays for ERK5 and JNK","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis defining molecular contacts, Co-IP, functional kinase assays, mechanistically extends prior PB1 studies","pmids":["17452462"],"is_preprint":false},{"year":2004,"finding":"MEKK2 immunoprecipitates from IL-1-stimulated fibroblast-like synoviocytes (FLS) show increased phosphorylation of MKK4 and MKK7 (the MAP2Ks that activate JNK), and MEKK2 activates c-Jun in an IL-1-dependent manner inhibitable by the JNK inhibitor SP600125.","method":"In vitro kinase assays using MEKK2 immunoprecipitates; Western blot; pharmacological inhibition with SP600125","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay from immunoprecipitated endogenous MEKK2, pharmacological validation, single lab","pmids":["14734742"],"is_preprint":false},{"year":2011,"finding":"MEKK2 and MEKK3 (acting in T cells) negatively regulate TGF-β-mediated Th cell differentiation. Map3k2-/-Map3k3Lck-Cre/- T cells show impaired phosphorylation of SMAD2 and SMAD3 at their linker regions, leading to enhanced Treg and Th17 differentiation in response to TGF-β.","method":"Conditional and constitutive genetic knockout mice; in vitro T cell differentiation assays; phospho-SMAD2/3 linker region Western blot; EAE disease model","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic models, in vitro and in vivo functional readouts, defined molecular mechanism (SMAD linker phosphorylation)","pmids":["21333552"],"is_preprint":false},{"year":2008,"finding":"XIAP physically interacts with MEKK2 and ubiquitinates MEKK2 (in an E3 ligase-dependent manner) following TNFα stimulation, regulating a second wave of NF-κB activation.","method":"Co-IP showing XIAP-MEKK2 interaction; ubiquitination assays; NF-κB reporter assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, functional NF-κB readout, single lab","pmids":["18761086"],"is_preprint":false},{"year":2014,"finding":"XIAP and cIAP1 directly interact with MEKK2/3, compete with PB1 domain-mediated MEK5 binding, and conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3, which directly impede MEK5-ERK5 interaction in a trimeric complex, leading to ERK5 inactivation. Loss of XIAP causes hyperactivation of ERK5 and promotes myoblast differentiation in a MEKK2/3-ERK5-dependent manner.","method":"Co-IP; ubiquitin chain linkage analysis (K63); in vitro reconstitution of trimeric complex; loss-of-function by multiple strategies; myoblast differentiation assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including in vitro reconstitution, K63 ubiquitin chain identification, functional cellular assay, single lab with strong methods","pmids":["24975362"],"is_preprint":false},{"year":2016,"finding":"MEKK2 mediates an alternative noncanonical pathway for β-catenin activation in osteoblasts downstream of FGF2. FGF2 activates MEKK2 to phosphorylate β-catenin at serine 675, promoting recruitment of the deubiquitinase USP15, which prevents β-catenin ubiquitination and proteasomal degradation, thereby enhancing WNT signaling and bone formation.","method":"MEKK2-deficient mice; in vitro kinase assays; phospho-site mapping (S675); USP15 Co-IP; genetic interaction studies between Mekk2 and β-catenin null alleles","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay with phospho-site mapping, Co-IP for USP15 recruitment, genetic epistasis in mice, multiple orthogonal methods","pmids":["26884171"],"is_preprint":false},{"year":2013,"finding":"MEKK2 is regulated by phosphorylation-dependent association with 14-3-3 proteins. Phosphorylation of MEKK2 at Thr-283 results in decreased activation loop phosphorylation at Ser-519 and reduced kinase 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. T283A MEKK2 shows enhanced stress-activated JNK activity but reduced ERK activation.","method":"Site-directed mutagenesis (T283A); 14-3-3 association assays; phosphorylation assays; expression in MEKK2-/- background; IL-6 and proliferation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis, functional kinase assays, 14-3-3 association studies, single lab","pmids":["23963453"],"is_preprint":false},{"year":2015,"finding":"Stk38 kinase constitutively associates with the ubiquitin E3 ligase Smurf1 and facilitates Smurf1-mediated MEKK2 ubiquitination and degradation. MEKK2 is required for CpG/TLR9-induced ERK1/2 activation, TNF-α and IL-6 production in macrophages, but not for LPS-induced cytokine production. Stk38 deficiency increases CpG-induced ERK1/2 activation and inflammatory cytokine production.","method":"Co-IP showing Stk38-Smurf1 association; ubiquitination assays; MEKK2-/- macrophage function assays; Stk38-deficient mice; bacterial infection model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ubiquitination assay, KO macrophages with specific stimulus-dependent phenotype, in vivo mouse model","pmids":["25981615"],"is_preprint":false},{"year":2018,"finding":"Kir2.1 (inwardly rectifying K+ channel) interacts with STK38 to inhibit Smurf1-mediated ubiquitination and degradation of MEKK2, thereby activating the MEKK2-MEK1/2-ERK1/2-Snail pathway to promote EMT and invasion in gastric cancer cells. This function is independent of potassium ion permeation.","method":"Co-IP; siRNA knockdown; ubiquitination assays; cell invasion and metastasis assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, ion channel mutation controls, single lab","pmids":["29549164"],"is_preprint":false},{"year":2014,"finding":"MEKK2 associates with the LD1 motif of paxillin at focal adhesion complexes upon cell attachment to fibronectin. MEKK2 induces paxillin ubiquitylation in a manner requiring both the paxillin LD1 motif and MEKK2 kinase activity, and promotes paxillin redistribution from focal adhesions to the cytoplasm without promoting paxillin degradation.","method":"Co-IP; ubiquitylation assays; MEKK2 knockdown; focal adhesion imaging; fibronectin attachment assays","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, ubiquitylation assay, kinase-dead mutant, functional cellular readout, single lab","pmids":["25190348"],"is_preprint":false},{"year":2014,"finding":"Silencing MEKK2 expression in invasive breast tumor cells enhances cell spread area and focal adhesion stability while reducing cell migration. Cell attachment to fibronectin or Matrigel induces MEKK2 activation and localization to focal adhesions. MEKK2 ablation enhances focal adhesion size and frequency, inhibits fibronectin-induced ERK5 signaling, and inhibits FAK autophosphorylation.","method":"siRNA knockdown; focal adhesion imaging; kinase activation assays; focal adhesion turnover assays","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — siRNA knockdown with multiple functional readouts, localization experiment with functional consequence, single lab","pmids":["24491810"],"is_preprint":false},{"year":2016,"finding":"MEKK2 and MEKK3 suppress Hedgehog pathway activity by phosphorylating GLI1 at multiple Ser/Thr sites, reducing GLI1 protein stability, DNA-binding ability, and increasing GLI1 association with SUFU. MEKK2/3 mediate FGF2-induced inhibition of Hh signaling.","method":"In vitro kinase assays; phospho-site mapping; protein stability assays; DNA-binding assays; SUFU Co-IP; medulloblastoma cell proliferation assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro phosphorylation, multiple functional readouts, single lab","pmids":["29662197"],"is_preprint":false},{"year":2003,"finding":"Mutations in kinase subdomain X of MEKK2 impair phosphorylation of MAP2Ks MKK7 and MEK5, abolish activation of JNK1 and ERK5, and diminish AP-1 reporter activation. The spectrum of subdomain X mutations affecting MEKK2 is overlapping but not identical to those affecting MEKK1.","method":"Site-directed mutagenesis of subdomain X residues; in vitro kinase assays; JNK/ERK5 activation assays; AP-1 reporter assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis with kinase activity readout, multiple substrates tested, single lab","pmids":["12659851"],"is_preprint":false},{"year":2010,"finding":"EGF-induced MEKK2 binding to the adaptor protein Lad1 requires a proper calcium concentration, and calcium modifiers that reduce MEKK2-Lad1 interaction also inhibit EGF-induced MEKK2 nuclear translocation and ERK5 activation. Lad1 interaction is required for full tyrosine phosphorylation of MEKK2.","method":"Co-immunoprecipitation; in vitro binding assays; calcium modifier experiments; nuclear translocation imaging","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, in vitro binding assay, localization imaging with functional consequence, single lab","pmids":["20830310"],"is_preprint":false},{"year":2021,"finding":"MAP3K2 mediates a ROS-MAP3K2-ERK5-KLF2 signaling axis in intestinal stromal cells (MRISCs) to enhance R-spondin 1 production following intestinal injury, thereby maintaining LGR5+ intestinal stem cells and protecting against acute intestinal damage.","method":"MAP3K2-specific genetic studies; single-cell transcriptomics; epigenetic profiling; functional intestinal injury models in mice; R-spondin 1 production assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic model with defined molecular pathway, multiple orthogonal methods, high-impact journal with comprehensive characterization","pmids":["33658717"],"is_preprint":false},{"year":2021,"finding":"MEKK2 and MEKK3 activate LATS1/2 and inhibit YAP/TAZ activity (Hippo pathway) downstream of TNF signaling, acting in parallel to MST1/2 and MAP4Ks. MEKK2/3 directly interact with LATS1/2 and YAP/TAZ and phosphorylate them. The STRIPAK complex associates with MEKK3 via CCM2/CCM3 to inactivate MEKK3, and upstream Hippo signals trigger MEKK3 dissociation from STRIPAK.","method":"Co-IP showing MEKK2/3-LATS1/2-YAP/TAZ interactions; in vitro phosphorylation assays; epistasis experiments; STRIPAK complex interaction studies","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — Co-IP, in vitro kinase assay, epistasis, single lab","pmids":["33571521"],"is_preprint":false},{"year":2016,"finding":"Co-crystal structure of SMYD3 with a MEKK2 peptide substrate was determined, revealing how SMYD3 engages MAP3K2. SMYD3 follows a partially processive methylation mechanism. Structure-based design yielded GSK2807, a SAM-competitive inhibitor (Ki = 14 nM) that bridges the SAM-binding pocket and MEKK2 substrate lysine tunnel.","method":"Co-crystal structure of SMYD3-MEKK2 peptide; kinetic characterization; structure-based inhibitor design","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional validation (inhibitor design), single lab but rigorous structural methods","pmids":["27066749"],"is_preprint":false},{"year":2020,"finding":"MEKK2 mediates aberrant ERK activation downstream of NF1 loss in osteoblasts via a noncanonical ERK pathway. Mekk2-/- mice with NF1 conditional deletion (Nf1fl/fl;Mekk2-/-;Dmp1-Cre) show amelioration of NF1-associated skeletal phenotypes, placing MEKK2 downstream of NF1 in this ERK activation pathway.","method":"Genetic epistasis using Nf1 conditional and Mekk2 constitutive knockouts; skeletal phenotype analysis; ERK activation assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean genetic epistasis with double-mutant rescue, defined molecular pathway, single lab","pmids":["33177525"],"is_preprint":false},{"year":2012,"finding":"MEKK2 regulates right ventricular hypertrophy in hypoxia-induced pulmonary hypertension, in part through regulation of ERK5 abundance and phosphorylation. MEKK2-/- mice show attenuated RV hypertrophy and selective inhibition of inflammatory gene expression compared to WT mice under chronic hypoxia.","method":"MEKK2-/- mice; chronic hypobaric hypoxia model; RV hypertrophy measurements; ERK5 phosphorylation assays; gene expression analysis","journal":"American journal of physiology. Heart and circulatory physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with defined in vivo phenotype, ERK5 mechanistic link, single lab","pmids":["23125215"],"is_preprint":false},{"year":2022,"finding":"NEDD4L (HECT-type E3 ligase) constitutively and directly binds MEKK2 and promotes its poly-ubiquitination and degradation, negatively regulating signaling induced by TNF-α, IL-1, and IL-17. In IL-17R signaling, IL-17-induced MEKK2 Ser520 phosphorylation is required both for downstream p38 and NF-κB activation and for NEDD4L-mediated MEKK2 degradation.","method":"Co-IP; ubiquitination assays; NEDD4L knockdown/deficiency; Nedd4l-deficient mice; EAE model","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ubiquitination assay, in vivo mouse model, phospho-site functional characterization, multiple cytokine pathways tested","pmids":["36161689"],"is_preprint":false},{"year":2019,"finding":"MEKK2 phosphorylates STK38 at Ser91, protecting STK38 from calpain-mediated cleavage. MEKK2 knockdown enhances hyperthermia-induced degradation of STK38. A phosphorylation-defective S91A STK38 mutant is susceptible to calpain degradation, demonstrating that MEKK2-mediated phosphorylation at Ser91 is required for STK38 stability.","method":"In vitro MEKK2 kinase assay with phospho-site identification; phospho-defective mutant (S91A); MEKK2 knockdown; calpain inhibitor experiments","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay with site mapping, phospho-defective mutant, calpain inhibitor validation, single lab","pmids":["31690749"],"is_preprint":false},{"year":2016,"finding":"MEKK2 promotes GLI1 phosphorylation on multiple Ser/Thr sites, reducing its protein stability, DNA-binding ability, and increasing SUFU association, thereby suppressing Hedgehog pathway activity. MEKK2 mediates FGF2-induced inhibition of Hh signaling.","method":"In vitro phosphorylation assays; protein stability assays; DNA-binding assays; SUFU Co-IP","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay, multiple functional readouts, single lab","pmids":["29662197"],"is_preprint":false},{"year":2018,"finding":"NDR2 kinase interacts with Smurf1 E3 ligase and promotes Smurf1-mediated K48-linked ubiquitination of MEKK2, leading to MEKK2 degradation and inhibition of IL-17-induced MAPK and NF-κB activation and inflammatory cytokine production.","method":"Co-IP showing NDR2-Smurf1 interaction; ubiquitination assay (K48 linkage); NDR2 and Smurf1 knockdown; IL-17-induced cytokine assays","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP, K48-specific ubiquitination assay, functional cytokine readout, single lab","pmids":["30504095"],"is_preprint":false},{"year":2021,"finding":"MAP3K2-mediated Th1 cell differentiation in the intestine is regulated by IL-18 and requires specific JNK activation. MAP3K2-deficient naïve CD4+ T cells have a reduced ability to induce colitis in a T cell transfer model, with fewer IFN-γ-producing cells in intestines.","method":"T cell transfer colitis model; MAP3K2-deficient T cells; in vitro differentiation conditions; cytokine measurement; JNK activation assays","journal":"Science China. Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with in vivo colitis model, molecular pathway characterization (IL-18-MAP3K2-JNK), single lab","pmids":["32737854"],"is_preprint":false},{"year":2001,"finding":"A single amino acid difference between MEKK2 (Val384) and MEKK3 (Ser390), immediately N-terminal to the active site lysine in kinase domain II/III, determines differential in vitro kinase activity in Triton X-100 detergent. Mutation of MEKK3 S390V confers activity in Triton X-100; the reciprocal MEKK2 V384S mutation abolishes activity in Triton X-100.","method":"Chimeric kinase constructs; site-directed mutagenesis; in vitro kinase assays in two detergents","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — mutagenesis with kinase activity readout, reciprocal mutations tested, single lab","pmids":["11343802"],"is_preprint":false},{"year":2023,"finding":"Best3 interacts with both MEKK2 and MEKK3 in vascular smooth muscle cells, and this interaction inhibits phosphorylation of MEKK2 at serine 153. Best3 deficiency induces phosphorylation-dependent stabilization of MEKK2/3 (inhibiting ubiquitination and turnover), thereby activating downstream MAPK signaling and triggering aortic dissection.","method":"Co-immunoprecipitation coupled with mass spectrometry; Best3 smooth muscle cell-specific KO mice; phosphorylation assays; ubiquitination assays; single-cell RNA sequencing; proteomics","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — Co-IP/MS, genetic KO mice with severe in vivo phenotype, phospho-site identification, ubiquitination assays, multiple orthogonal methods","pmids":["37203562"],"is_preprint":false},{"year":2025,"finding":"Crystal structure of the MEKK2 kinase domain (2.4 Å) in complex with ponatinib reveals that MEKK2 dimerizes via a surface centered on the αG helix and C-terminal activation segment. This surface is required for MEKK2 autophosphorylation and for phosphorylation of both MEK5 and MKK6. MEK5 recruitment is PB1 domain-dependent, while MKK6 recruitment is αG helix-dependent (PB1-independent), providing a structural basis for substrate selectivity.","method":"2.4 Å crystal structure; site-directed mutagenesis of αG helix surface; autophosphorylation assays; in vitro substrate phosphorylation assays (MEK5, MKK6); Co-IP/dimerization assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and functional validation across multiple substrates, mechanistically definitive for substrate targeting","pmids":["41318559"],"is_preprint":false},{"year":2023,"finding":"SMYD3-dependent methylation of MAP3K2 promotes epithelial-mesenchymal transition behaviors in prostate cancer cells by altering the abundance of vimentin, and supports a positive feedback loop maintaining high SMYD3 levels.","method":"SMYD3 inhibitor treatment; siRNA knockdown; xenograft mouse models; EMT marker analysis; vimentin abundance measurement","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo functional assays, single lab, extends SMYD3-MAP3K2 methylation discovery","pmids":["37976356"],"is_preprint":false},{"year":2021,"finding":"MAP3K2-mediated phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208 increases ROS formation in myeloid cells. Genetic inactivation of MAP3K2 and MAP3K3 in myeloid cells or hematopoietic S208A mutation of p47phox attenuates acute lung injury and abrogates the protective effects of pazopanib.","method":"In vitro phosphorylation assays; myeloid-specific MAP3K2/3 conditional KO mice; p47phox S208A knockin mice; ALI models; pazopanib pharmacology","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay identifying substrate/site, multiple genetic mouse models with defined phenotype, pharmacological validation","pmids":["33910977"],"is_preprint":false},{"year":2016,"finding":"PDGF-BB-induced ERK5 activation in smooth muscle cells is dependent on Mekk2 (and Mek1/2, Mek5, PI3-kinase, and PKC), suggesting close co-regulation between ERK1/2 and ERK5 MAP kinase pathways downstream of PDGFR.","method":"Dominant-negative expression of Mekk2; kinase inhibitor studies; ERK5 activation assays in MOVAS smooth muscle cells","journal":"Cellular signalling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — dominant-negative expression only, single lab, multiple pathway components tested simultaneously making MEKK2 contribution indirect","pmids":["27339033"],"is_preprint":false},{"year":2016,"finding":"Sublytic C5b-9 induces MEKK2 phosphorylation at Ser153, Ser164, and Ser239, which is necessary for p38 MAPK activation, leading to the MEKK2-p38 MAPK-IRF-1-TRADD-caspase 8 apoptotic cascade in glomerular mesangial cells.","method":"Phosphorylation site identification; site-directed mutagenesis; siRNA knockdown; in vitro kinase assays; in vivo Thy-1 nephritis rat model","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis of three phospho-sites, functional cascade assays, in vivo model, single lab","pmids":["28039298"],"is_preprint":false},{"year":2015,"finding":"Interaction of paxillin LD1 motif with 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 auto-phosphorylation assay with recombinant paxillin; siRNA-mediated paxillin knockdown; LD1 motif binding assays","journal":"Journal of molecular signaling","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro reconstitution of activation, siRNA validation, single lab","pmids":["27096002"],"is_preprint":false}],"current_model":"MAP3K2 (MEKK2) is a serine/threonine MAP3K that acts as a central signaling node: it is activated by dimerization through its catalytic αG helix surface and by upstream kinases (WNK1, Rap1), and is regulated by phosphorylation (at S519 by TRAF6-dependent pathways, at T283 by 14-3-3-regulated trans-autophosphorylation, and at K260 by SMYD3 methylation), as well as by ubiquitin-mediated degradation via multiple E3 ligases (Smurf1, CHIP, XIAP/cIAP1, NEDD4L); it signals via PB1 domain–MEK5 interaction to activate ERK5, and via MKK7 to activate JNK, with substrate selectivity further specified by its αG helix surface; MEKK2 also phosphorylates non-MAPK substrates including GLI1 (suppressing Hedgehog signaling), β-catenin S675 (promoting USP15-mediated deubiquitination to enhance WNT/bone formation), LATS1/2 and YAP/TAZ (regulating Hippo signaling), p47phox S208 (controlling NADPH oxidase-derived ROS in myeloid cells), and STK38 S91 (preventing calpain-mediated STK38 degradation), placing MEKK2 at the intersection of Ras/ERK, JNK, ERK5, Hippo, Hedgehog, WNT, and innate immune signaling pathways."},"narrative":{"mechanistic_narrative":"MAP3K2 (MEKK2) is a serine/threonine MAP3 kinase that functions as a central signaling node coupling diverse receptor inputs to multiple downstream MAPK and non-MAPK pathways [PMID:11073940, PMID:14978743]. Its activation requires dimerization through a surface centered on the catalytic αG helix and C-terminal activation segment, which drives autophosphorylation and substrate phosphorylation [PMID:15695508, PMID:41318559]; this same structural surface specifies substrate selectivity, recruiting MEK5 through the PB1 domain to activate ERK5 and recruiting MKK6/MKK7 through the αG helix to activate stress kinases [PMID:12912994, PMID:17452462, PMID:41318559]. Through PB1 domain–mediated heterodimerization with MEK5, MEKK2 activates the MEK5–ERK5 module, while through MKK7/MKK4 it activates JNK and AP-1–driven cytokine gene expression [PMID:12912994, PMID:17452462, PMID:12659851]. Upstream, MEKK2 is engaged by WNK1, Rap1 (downstream of BDNF), and growth-factor and cytokine receptors including FGF-2, EGF, IgE, c-Kit, and TLR receptors, with EGF triggering its cytosol-to-nucleus translocation to access nuclear MEK5–ERK5 [PMID:11032806, PMID:15075238, PMID:14681216, PMID:17003042]. Activation-loop phosphorylation at Ser519 is a key regulatory event induced in a TRAF6-dependent manner and required for TLR-induced IL-6 production, while phosphorylation at Thr283 and 14-3-3 binding restrain trans-autophosphorylation and kinase activity [PMID:16362041, PMID:23963453]. MEKK2 abundance is tightly controlled by ubiquitin-mediated degradation through multiple E3 ligases—Smurf1 (facilitated by STK38/NDR2), CHIP, XIAP/cIAP1, and NEDD4L—which terminate ERK, ERK5, and NF-κB signaling and couple receptor signaling to defined cellular outcomes [PMID:15820682, PMID:20588253, PMID:24975362, PMID:25981615, PMID:36161689, PMID:30504095]. Beyond the MAPK cascades, MEKK2 phosphorylates non-canonical substrates to regulate WNT/bone formation (β-catenin S675, promoting USP15-mediated stabilization), Hedgehog signaling (GLI1, reducing its stability and DNA binding), Hippo signaling (LATS1/2 and YAP/TAZ), myeloid ROS production (p47phox S208), and STK38 stability (S91) [PMID:26884171, PMID:29662197, PMID:33571521, PMID:31690749, PMID:33910977]. These activities place MEKK2 at the intersection of immune cell signaling, osteoblast/skeletal biology, intestinal stem-cell maintenance, vascular integrity, and cancer cell invasion, the last potentiated by SMYD3 methylation at K260 that blocks PP2A binding [PMID:24847881, PMID:33658717, PMID:37203562, PMID:37976356]. A crystal structure of the kinase domain provides a structural basis for dimerization-driven activation and differential substrate recruitment [PMID:41318559].","teleology":[{"year":1999,"claim":"Established MEKK2 as a TCR-responsive kinase that physically relocalizes to the immunological synapse, linking it to early antigen receptor signaling.","evidence":"Live-cell imaging and dominant-negative MEKK2 in T cell/APC conjugates","pmids":["10549623"],"confidence":"Medium","gaps":["Direct substrates at the synapse not defined","Dominant-negative approach does not establish endogenous requirement"]},{"year":2000,"claim":"Identified MEK5 as a direct MEKK2 partner and ERK5 as a downstream output, and showed MEKK2 is genetically required for receptor-induced JNK activation and cytokine transcription, defining its dual MAPK outputs.","evidence":"Yeast two-hybrid, dominant-negative epistasis, and MEKK2-deficient mast cells","pmids":["11073940","11032806"],"confidence":"High","gaps":["Molecular basis of MEK5 binding not yet resolved","How a single MAP3K selects ERK5 vs JNK output unclear"]},{"year":2002,"claim":"Genetic knockout revealed MEKK2 negatively regulates TCR signaling strength, showing its role is not uniformly activating but context-dependent.","evidence":"Mekk2 KO mice; T cell proliferation, cytokine, and MAPK activation assays","pmids":["12138187"],"confidence":"High","gaps":["Mechanism of negative regulation in T cells not defined","JNK only moderately affected, leaving redundancy unexplained"]},{"year":2003,"claim":"Defined the PB1 domain as the structural module mediating selective MEK5/ERK5 pathway engagement and placed WNK1 upstream as a MEKK2-activating kinase.","evidence":"PB1 domain binding/mutation assays; WNK1 Co-IP, in vitro kinase, and dominant-negative epistasis","pmids":["12912994","14681216"],"confidence":"High","gaps":["PB1-independent substrate routing not yet addressed","WNK1 phospho-site on MEKK2 not mapped"]},{"year":2004,"claim":"Established MEKK2 as the FGF-2/EGF receptor coupler that coordinately drives ERK5 and JNK and undergoes signal-induced nuclear translocation to reach nuclear MEK5.","evidence":"MEKK2-/- MEFs with stimulus specificity; subcellular fractionation and live-cell imaging; FLS in vitro kinase assays","pmids":["14978743","15075238","14734742"],"confidence":"High","gaps":["Trigger for nuclear import not molecularly defined","MKK4 vs MKK7 selectivity not resolved"]},{"year":2005,"claim":"Resolved the activation logic: dimerization through the catalytic domain drives activation, Ser519 in the activation loop is the critical regulatory phospho-site (TRAF6-dependent), and Smurf1 and Mip1 act as negative regulators by degradation and dimer blockade respectively.","evidence":"Chemical-induced dimerization, deletion mapping, S519A mutagenesis with phospho-antibody, Smurf1-deficient mice, Mip1 Co-IP/siRNA","pmids":["15695508","16362041","15820682","15988011"],"confidence":"High","gaps":["Kinase responsible for Ser519 phosphorylation in vivo not fully defined","Mip1 mechanism characterized in a single lab"]},{"year":2007,"claim":"Defined how the PB1 domain partitions MEKK2 output—front-to-back PB1 binding to MEK5 drives ERK5 while a C-terminal acidic cluster routes activated MEKK2 to MKK7/JNK—providing a molecular switch for substrate choice.","evidence":"PB1 residue mutagenesis, Co-IP, and ERK5/JNK kinase activation assays","pmids":["17452462"],"confidence":"High","gaps":["Conformational change driving the switch not visualized","Quantitative balance between outputs in vivo not measured"]},{"year":2010,"claim":"Showed MEKK2 turnover is functionally instructive: CHIP-mediated degradation enforces transient ERK kinetics required for correct aquaporin gene induction, and Lad1/calcium controls EGF-induced MEKK2 nuclear translocation.","evidence":"CHIP Co-IP/siRNA/gene targeting with ERK time-course and AQP readout; Lad1 Co-IP and calcium-modifier translocation imaging","pmids":["20588253","20830310"],"confidence":"High","gaps":["Generality of CHIP regulation beyond osmotic stress unknown","Lad1 findings from a single lab"]},{"year":2011,"claim":"Extended MEKK2 function to TGF-β/SMAD signaling, showing it phosphorylates SMAD2/3 linker regions to restrain Treg/Th17 differentiation, a non-classical MAPK output with immunological consequence.","evidence":"Conditional/constitutive Map3k2/Map3k3 KO mice, in vitro differentiation, phospho-SMAD linker blots, EAE model","pmids":["21333552"],"confidence":"High","gaps":["Direct vs indirect SMAD linker phosphorylation not fully separated","Redundancy with MEKK3 limits MEKK2-specific attribution"]},{"year":2014,"claim":"Identified SMYD3 K260 methylation as a positive regulator that blocks PP2A binding to sustain Ras/ERK signaling, and expanded the E3 ligase repertoire (XIAP/cIAP1 via K63 chains competing with MEK5) and focal-adhesion functions controlling cell migration.","evidence":"Protein array, in vitro methylation, PP2A Co-IP, tumor mouse models; K63 chain analysis and trimeric complex reconstitution; paxillin Co-IP/ubiquitylation and FA imaging","pmids":["24847881","24975362","24491810","25190348"],"confidence":"High","gaps":["Phosphatase(s) reversing K260 methylation context not detailed","Focal-adhesion substrate(s) beyond paxillin unclear"]},{"year":2016,"claim":"Broadened MEKK2 substrate scope to developmental pathway control—β-catenin S675 phosphorylation promoting USP15-mediated stabilization in bone, and GLI1 phosphorylation suppressing Hedgehog—linking it to WNT and Hh signaling.","evidence":"MEKK2-deficient mice, in vitro kinase with phospho-site mapping, USP15/SUFU Co-IP, genetic epistasis; SMYD3-MEKK2 co-crystal with inhibitor design","pmids":["26884171","29662197","27066749"],"confidence":"High","gaps":["Receptor inputs upstream of β-catenin/GLI1 phosphorylation incompletely defined","GLI1 findings rest on a single lab"]},{"year":2021,"claim":"Demonstrated tissue-protective and physiological roles: a ROS-MEKK2-ERK5-KLF2 axis maintaining intestinal stem cells, p47phox S208 phosphorylation controlling myeloid ROS in lung injury, and MEKK2/3 activation of LATS1/2-YAP/TAZ in the Hippo pathway downstream of TNF.","evidence":"Genetic mouse models with injury/ALI/colitis assays, single-cell profiling, in vitro kinase assays, STRIPAK interaction studies","pmids":["33658717","33910977","33571521","32737854"],"confidence":"High","gaps":["Direct vs indirect LATS/YAP phosphorylation not fully separated from MEKK3","Upstream activator of the intestinal ROS axis not pinpointed"]},{"year":2023,"claim":"Connected MEKK2 stability control to vascular disease, showing Best3 binding inhibits Ser153 phosphorylation and promotes MEKK2/3 turnover, with loss driving aortic dissection, and extended SMYD3 methylation to prostate cancer EMT.","evidence":"Co-IP/MS, Best3 smooth-muscle KO mice, phospho/ubiquitination assays; SMYD3 inhibition and xenografts with EMT markers","pmids":["37203562","37976356"],"confidence":"High","gaps":["Kinase phosphorylating Ser153 not identified","Causal substrate downstream of stabilized MEKK2 in dissection unclear"]},{"year":2025,"claim":"Provided the definitive structural basis for activation and substrate selectivity: an αG helix–centered dimerization surface drives autophosphorylation, with MEK5 recruited via PB1 and MKK6 via the αG surface independently of PB1.","evidence":"2.4 Å crystal structure with αG surface mutagenesis and in vitro substrate phosphorylation assays","pmids":["41318559"],"confidence":"High","gaps":["Structure of full-length regulated MEKK2 not solved","How upstream phosphorylation reshapes the dimer interface unresolved"]},{"year":null,"claim":"How the many parallel regulatory inputs (methylation, multiple phospho-sites, competing E3 ligases, and scaffold/adaptor binding) are integrated to set MEKK2 output toward a specific pathway in a given cell type remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model of input integration","MEKK2 vs MEKK3 division of labor across tissues not systematically mapped","Cell-type-specific substrate prioritization mechanism unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[20,26,35,43,17]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[27,41,9]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[7,41]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[3,10,13]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,28]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[24,25]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,10,13,15,30]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[4,9,12,17,22,34,43]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[20,26,29]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[1,14,19,34,37]}],"complexes":["MEKK2–MEK5 PB1 complex","MEKK2/3–XIAP/cIAP1–MEK5 trimeric complex"],"partners":["MAP2K5","MAP2K7","WNK1","XIAP","SMURF1","STUB1","NEDD4L","STK38"],"other_free_text":[]}},"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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Methylation of MAP3K2 at K260 blocks binding of the PP2A phosphatase complex to MAP3K2, preventing PP2A-mediated negative regulation of the MAP kinase pathway.\",\n      \"method\": \"Protein array technology to identify MAP3K2 as SMYD3 substrate; in vitro methylation assays; Co-IP showing PP2A-MAP3K2 interaction blocked by methylation; mouse models of pancreatic/lung adenocarcinoma\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (in vitro methylation, Co-IP, mutagenesis, mouse tumor models), replicated in prostate cancer study (PMID:37976356)\",\n      \"pmids\": [\"24847881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Smurf1, a HECT domain ubiquitin E3 ligase, physically interacts with MEKK2 and promotes its ubiquitination and proteasomal degradation, thereby suppressing osteoblast activity and JNK signaling downstream of BMP.\",\n      \"method\": \"Co-IP showing Smurf1-MEKK2 physical interaction; ubiquitination assays; Smurf1-deficient mouse model showing accumulation of phosphorylated MEKK2 and enhanced JNK activation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reciprocal Co-IP, ubiquitination assay, in vivo genetic model with defined phenotype, replicated by multiple subsequent studies\",\n      \"pmids\": [\"15820682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The PB1 domains of MEKK2 and MEKK3 heterodimerize with the PB1 domain of MEK5, forming a complex required for ERK5 pathway activation. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 complex formation and ERK5 activation. The PB1 domain interaction is specific to the ERK5 pathway and does not affect p38 or JNK pathways.\",\n      \"method\": \"In vitro PB1 domain binding assays; co-immunoprecipitation of MEKK2 and MEK5 from cell lysates; deletion/mutation analysis; dominant-negative expression experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro binding reconstitution, Co-IP, mutagenesis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"12912994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MEKK2 activates the MEK5-BMK1/ERK5 pathway; MEK5 was identified as a binding partner of MEKK2 by yeast two-hybrid screening, and a dominant-negative MEK5 blocked ERK5 activation by MEKK2. MEKK2 also associates with the T cell adapter protein Lad/RIBP, and co-localizes with Lad/RIBP at the T cell/APC contact site during T cell activation.\",\n      \"method\": \"Yeast two-hybrid library screen; co-localization imaging; dominant-negative expression; kinase activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid, co-localization, dominant-negative epistasis, replicated by PB1 domain studies\",\n      \"pmids\": [\"11073940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MEKK2 is required for receptor-mediated JNK activation and cytokine gene transcription in mast cells following IgE receptor or c-Kit ligation. MEKK2-deficient ES cell-derived mast cells show markedly reduced cytokine production and loss of receptor-mediated JNK activation, while JNK activation by UV irradiation is unaffected.\",\n      \"method\": \"Targeted gene disruption of MEKK2 in ES cell-derived mast cells; RT-PCR for cytokine transcription; kinase assays for JNK activation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with specific cellular phenotype, specificity controls (MEKK1 KO, UV irradiation), replicated in subsequent mast cell studies\",\n      \"pmids\": [\"11032806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MEKK2 coordinately activates ERK5 and JNK pathways in response to FGF-2 signaling in fibroblasts. MEKK2-deficient MEFs lose ERK5 and JNK activation in response to FGF-2 but not LPS or TNFα, demonstrating MEKK2 specificity in FGF-2 receptor signaling. MEKK2 regulates AP-1 activity by controlling expression of c-Jun, Fra-1, and Fra-2 and c-Jun N-terminal phosphorylation, leading to cytokine gene expression.\",\n      \"method\": \"MEKK2-/- mouse embryonic fibroblasts; kinase activation assays; RT-PCR for AP-1 components and cytokines\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with multiple specific phenotypic readouts, stimulus-specificity controls\",\n      \"pmids\": [\"14978743\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MEKK2 is localized mainly in the cytosol of resting cells and translocates to the nucleus upon EGF stimulation, allowing transmission of signals to nuclear MEK5. MEK5 and ERK5 are constitutively nuclear in endogenous form, bound to detergent-resistant nuclear moieties.\",\n      \"method\": \"Immunocytochemistry; live cell fluorescence imaging; in situ detergent extraction; subcellular fractionation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with multiple methods, single lab\",\n      \"pmids\": [\"15075238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"MEKK2 activation requires dimerization through its catalytic domain. The dimerization motif maps to the catalytic domain (N-terminal region not required). Inactive, non-phosphorylated MEKK2 forms more dimers than phosphorylated active MEKK2. Prevention of dimerization inhibits MEKK2-mediated JNK activation; chemical-induced dimerization augments JNK activation and AP-1 reporter activity.\",\n      \"method\": \"Dimerization mapping using deletion constructs; chemical-induced dimerization system; JNK activation assays; AP-1 reporter gene assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — chemical-induced dimerization, mutagenesis, multiple functional readouts, single lab\",\n      \"pmids\": [\"15695508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mip1 (MEKK2-interacting protein) forms a complex with inactive, non-phosphorylated MEKK2 and prevents MEKK2 activation by blocking MEKK2 dimer formation, thereby inhibiting JNK1, ERK5, and AP-1 activation. The endogenous Mip1-MEKK2 complex dissociates transiently following EGF stimulation. siRNA knockdown of Mip1 augments MEKK2-mediated JNK and AP-1 activation.\",\n      \"method\": \"Co-IP; siRNA knockdown; kinase and reporter gene activation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, siRNA knockdown with functional readout, single lab\",\n      \"pmids\": [\"15988011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Serine 519 in MEKK2 (and S526 in MEKK3) is a key regulatory phosphorylation site required for kinase activation. Mutation of S519 to alanine severely impairs MEKK2 activation. LPS induces phosphorylation of this serine in a TRAF6-dependent manner, and this phosphorylation is required for TLR-induced IL-6 production.\",\n      \"method\": \"Site-directed mutagenesis (S519A); anti-phospho-MEKK2/3 antibody; LPS stimulation assays; TRAF6 perturbation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site mutagenesis, phospho-specific antibody validation, functional readout (IL-6 production), single lab with multiple orthogonal methods\",\n      \"pmids\": [\"16362041\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"WNK1 activates ERK5 via MEKK2 and MEKK3 in an MEKK2/3-dependent mechanism. WNK1 phosphorylates MEKK2 and MEKK3 in vitro and activates MEKK3 in cells. Both MEKK2 and MEKK3 co-immunoprecipitate with endogenous WNK1. Dominant-negative MEKK2 and MEKK3 block WNK1-induced ERK5 activation.\",\n      \"method\": \"Co-IP of WNK1 with MEKK2/3; in vitro kinase assay; dominant-negative expression; siRNA knockdown of WNK1\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay, Co-IP, dominant-negative epistasis, siRNA validation, single lab\",\n      \"pmids\": [\"14681216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MEKK2 translocates to the T cell/antigen-presenting cell interface upon antigen-mediated TCR engagement. Biochemical activation of MEKK2 follows TCR stimulation, and dominant-negative MEKK2 inhibits TCR-mediated conjugate stabilization and ERK/p38 MAPK phosphorylation.\",\n      \"method\": \"Live cell fluorescence imaging; immunocytochemistry; dominant-negative expression; kinase activation assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging, dominant-negative functional assay, single lab with two orthogonal methods\",\n      \"pmids\": [\"10549623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"MEKK2-deficient T cells show augmented proliferation and increased IL-2/IFNγ production in response to anti-CD3 stimulation, and greater susceptibility to anti-CD3-induced cell death, indicating MEKK2 negatively regulates TCR signaling strength. TCR-mediated JNK activation was moderately enhanced (not abolished) in MEKK2-/- T cells; ERK and p38 activation were unaffected.\",\n      \"method\": \"Targeted Mekk2 gene disruption in mice; T cell proliferation assays; cytokine production assays; JNK/ERK/p38 activation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with multiple functional readouts and specificity controls, single lab\",\n      \"pmids\": [\"12138187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"BDNF activates ERK5 in cortical neurons via a Rap1-MEKK2 signaling cascade. BDNF activates Rap1, which activates MEKK2 (a MEK5 kinase); inhibition of either Rap1 or MEKK2 attenuates BDNF-induced ERK5 activation. Ras and MEKK3 do not play significant roles in this neuronal BDNF-ERK5 pathway.\",\n      \"method\": \"Dominant-negative and constitutively active expression of Rap1 and MEKK2; kinase activity assays in cortical neurons\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis using dominant-negative/active constructs, single lab, multiple pathway components tested\",\n      \"pmids\": [\"17003042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CHIP (carboxyl terminus of Hsc70-interacting protein) E3 ubiquitin ligase binds MEKK2 and promotes its degradation, thereby terminating ERK activation in response to hyperosmotic stress. Transient ERK activation via MEKK2 is required for proper aquaporin (AQP1, AQP5) gene induction; CHIP depletion prolongs MEKK2 stability, prolongs ERK activity, and paradoxically suppresses AQP gene expression.\",\n      \"method\": \"Co-IP identifying CHIP-MEKK2 interaction; siRNA knockdown of CHIP; gene targeting of CHIP; ERK activity time-course assays; AQP gene expression assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — Co-IP, siRNA, gene targeting, functional gene expression readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"20588253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"MEKK2 and MEK5 PB1 domains interact in a front-to-back arrangement; quiescent MEKK2 preferentially binds MEK5 via PB1, leading to ERK5 activation. Upon activation, MEKK2 binds and activates MKK7 via the C-terminal acidic cluster of the MEKK2 PB1 domain, leading to JNK activation. The C-terminal MEK5 PB1 extension encodes an ERK5 docking site required for MEK5 activation of ERK5.\",\n      \"method\": \"Mutagenesis of PB1 domain residues; co-immunoprecipitation; kinase activation assays for ERK5 and JNK\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis defining molecular contacts, Co-IP, functional kinase assays, mechanistically extends prior PB1 studies\",\n      \"pmids\": [\"17452462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MEKK2 immunoprecipitates from IL-1-stimulated fibroblast-like synoviocytes (FLS) show increased phosphorylation of MKK4 and MKK7 (the MAP2Ks that activate JNK), and MEKK2 activates c-Jun in an IL-1-dependent manner inhibitable by the JNK inhibitor SP600125.\",\n      \"method\": \"In vitro kinase assays using MEKK2 immunoprecipitates; Western blot; pharmacological inhibition with SP600125\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay from immunoprecipitated endogenous MEKK2, pharmacological validation, single lab\",\n      \"pmids\": [\"14734742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MEKK2 and MEKK3 (acting in T cells) negatively regulate TGF-β-mediated Th cell differentiation. Map3k2-/-Map3k3Lck-Cre/- T cells show impaired phosphorylation of SMAD2 and SMAD3 at their linker regions, leading to enhanced Treg and Th17 differentiation in response to TGF-β.\",\n      \"method\": \"Conditional and constitutive genetic knockout mice; in vitro T cell differentiation assays; phospho-SMAD2/3 linker region Western blot; EAE disease model\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic models, in vitro and in vivo functional readouts, defined molecular mechanism (SMAD linker phosphorylation)\",\n      \"pmids\": [\"21333552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"XIAP physically interacts with MEKK2 and ubiquitinates MEKK2 (in an E3 ligase-dependent manner) following TNFα stimulation, regulating a second wave of NF-κB activation.\",\n      \"method\": \"Co-IP showing XIAP-MEKK2 interaction; ubiquitination assays; NF-κB reporter assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, functional NF-κB readout, single lab\",\n      \"pmids\": [\"18761086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"XIAP and cIAP1 directly interact with MEKK2/3, compete with PB1 domain-mediated MEK5 binding, and conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3, which directly impede MEK5-ERK5 interaction in a trimeric complex, leading to ERK5 inactivation. Loss of XIAP causes hyperactivation of ERK5 and promotes myoblast differentiation in a MEKK2/3-ERK5-dependent manner.\",\n      \"method\": \"Co-IP; ubiquitin chain linkage analysis (K63); in vitro reconstitution of trimeric complex; loss-of-function by multiple strategies; myoblast differentiation assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including in vitro reconstitution, K63 ubiquitin chain identification, functional cellular assay, single lab with strong methods\",\n      \"pmids\": [\"24975362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MEKK2 mediates an alternative noncanonical pathway for β-catenin activation in osteoblasts downstream of FGF2. FGF2 activates MEKK2 to phosphorylate β-catenin at serine 675, promoting recruitment of the deubiquitinase USP15, which prevents β-catenin ubiquitination and proteasomal degradation, thereby enhancing WNT signaling and bone formation.\",\n      \"method\": \"MEKK2-deficient mice; in vitro kinase assays; phospho-site mapping (S675); USP15 Co-IP; genetic interaction studies between Mekk2 and β-catenin null alleles\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay with phospho-site mapping, Co-IP for USP15 recruitment, genetic epistasis in mice, multiple orthogonal methods\",\n      \"pmids\": [\"26884171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MEKK2 is regulated by phosphorylation-dependent association with 14-3-3 proteins. Phosphorylation of MEKK2 at Thr-283 results in decreased activation loop phosphorylation at Ser-519 and reduced kinase 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. T283A MEKK2 shows enhanced stress-activated JNK activity but reduced ERK activation.\",\n      \"method\": \"Site-directed mutagenesis (T283A); 14-3-3 association assays; phosphorylation assays; expression in MEKK2-/- background; IL-6 and proliferation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis, functional kinase assays, 14-3-3 association studies, single lab\",\n      \"pmids\": [\"23963453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Stk38 kinase constitutively associates with the ubiquitin E3 ligase Smurf1 and facilitates Smurf1-mediated MEKK2 ubiquitination and degradation. MEKK2 is required for CpG/TLR9-induced ERK1/2 activation, TNF-α and IL-6 production in macrophages, but not for LPS-induced cytokine production. Stk38 deficiency increases CpG-induced ERK1/2 activation and inflammatory cytokine production.\",\n      \"method\": \"Co-IP showing Stk38-Smurf1 association; ubiquitination assays; MEKK2-/- macrophage function assays; Stk38-deficient mice; bacterial infection model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ubiquitination assay, KO macrophages with specific stimulus-dependent phenotype, in vivo mouse model\",\n      \"pmids\": [\"25981615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Kir2.1 (inwardly rectifying K+ channel) interacts with STK38 to inhibit Smurf1-mediated ubiquitination and degradation of MEKK2, thereby activating the MEKK2-MEK1/2-ERK1/2-Snail pathway to promote EMT and invasion in gastric cancer cells. This function is independent of potassium ion permeation.\",\n      \"method\": \"Co-IP; siRNA knockdown; ubiquitination assays; cell invasion and metastasis assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitination assay, ion channel mutation controls, single lab\",\n      \"pmids\": [\"29549164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"MEKK2 associates with the LD1 motif of paxillin at focal adhesion complexes upon cell attachment to fibronectin. MEKK2 induces paxillin ubiquitylation in a manner requiring both the paxillin LD1 motif and MEKK2 kinase activity, and promotes paxillin redistribution from focal adhesions to the cytoplasm without promoting paxillin degradation.\",\n      \"method\": \"Co-IP; ubiquitylation assays; MEKK2 knockdown; focal adhesion imaging; fibronectin attachment assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, ubiquitylation assay, kinase-dead mutant, functional cellular readout, single lab\",\n      \"pmids\": [\"25190348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Silencing MEKK2 expression in invasive breast tumor cells enhances cell spread area and focal adhesion stability while reducing cell migration. Cell attachment to fibronectin or Matrigel induces MEKK2 activation and localization to focal adhesions. MEKK2 ablation enhances focal adhesion size and frequency, inhibits fibronectin-induced ERK5 signaling, and inhibits FAK autophosphorylation.\",\n      \"method\": \"siRNA knockdown; focal adhesion imaging; kinase activation assays; focal adhesion turnover assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — siRNA knockdown with multiple functional readouts, localization experiment with functional consequence, single lab\",\n      \"pmids\": [\"24491810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MEKK2 and MEKK3 suppress Hedgehog pathway activity by phosphorylating GLI1 at multiple Ser/Thr sites, reducing GLI1 protein stability, DNA-binding ability, and increasing GLI1 association with SUFU. MEKK2/3 mediate FGF2-induced inhibition of Hh signaling.\",\n      \"method\": \"In vitro kinase assays; phospho-site mapping; protein stability assays; DNA-binding assays; SUFU Co-IP; medulloblastoma cell proliferation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro phosphorylation, multiple functional readouts, single lab\",\n      \"pmids\": [\"29662197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Mutations in kinase subdomain X of MEKK2 impair phosphorylation of MAP2Ks MKK7 and MEK5, abolish activation of JNK1 and ERK5, and diminish AP-1 reporter activation. The spectrum of subdomain X mutations affecting MEKK2 is overlapping but not identical to those affecting MEKK1.\",\n      \"method\": \"Site-directed mutagenesis of subdomain X residues; in vitro kinase assays; JNK/ERK5 activation assays; AP-1 reporter assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with kinase activity readout, multiple substrates tested, single lab\",\n      \"pmids\": [\"12659851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EGF-induced MEKK2 binding to the adaptor protein Lad1 requires a proper calcium concentration, and calcium modifiers that reduce MEKK2-Lad1 interaction also inhibit EGF-induced MEKK2 nuclear translocation and ERK5 activation. Lad1 interaction is required for full tyrosine phosphorylation of MEKK2.\",\n      \"method\": \"Co-immunoprecipitation; in vitro binding assays; calcium modifier experiments; nuclear translocation imaging\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, in vitro binding assay, localization imaging with functional consequence, single lab\",\n      \"pmids\": [\"20830310\"],\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) to enhance R-spondin 1 production following intestinal injury, thereby maintaining LGR5+ intestinal stem cells and protecting against acute intestinal damage.\",\n      \"method\": \"MAP3K2-specific genetic studies; single-cell transcriptomics; epigenetic profiling; functional intestinal injury models in mice; R-spondin 1 production assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic model with defined molecular pathway, multiple orthogonal methods, high-impact journal with comprehensive characterization\",\n      \"pmids\": [\"33658717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MEKK2 and MEKK3 activate LATS1/2 and inhibit YAP/TAZ activity (Hippo pathway) downstream of TNF signaling, acting in parallel to MST1/2 and MAP4Ks. MEKK2/3 directly interact with LATS1/2 and YAP/TAZ and phosphorylate them. The STRIPAK complex associates with MEKK3 via CCM2/CCM3 to inactivate MEKK3, and upstream Hippo signals trigger MEKK3 dissociation from STRIPAK.\",\n      \"method\": \"Co-IP showing MEKK2/3-LATS1/2-YAP/TAZ interactions; in vitro phosphorylation assays; epistasis experiments; STRIPAK complex interaction studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — Co-IP, in vitro kinase assay, epistasis, single lab\",\n      \"pmids\": [\"33571521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Co-crystal structure of SMYD3 with a MEKK2 peptide substrate was determined, revealing how SMYD3 engages MAP3K2. SMYD3 follows a partially processive methylation mechanism. Structure-based design yielded GSK2807, a SAM-competitive inhibitor (Ki = 14 nM) that bridges the SAM-binding pocket and MEKK2 substrate lysine tunnel.\",\n      \"method\": \"Co-crystal structure of SMYD3-MEKK2 peptide; kinetic characterization; structure-based inhibitor design\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional validation (inhibitor design), single lab but rigorous structural methods\",\n      \"pmids\": [\"27066749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MEKK2 mediates aberrant ERK activation downstream of NF1 loss in osteoblasts via a noncanonical ERK pathway. Mekk2-/- mice with NF1 conditional deletion (Nf1fl/fl;Mekk2-/-;Dmp1-Cre) show amelioration of NF1-associated skeletal phenotypes, placing MEKK2 downstream of NF1 in this ERK activation pathway.\",\n      \"method\": \"Genetic epistasis using Nf1 conditional and Mekk2 constitutive knockouts; skeletal phenotype analysis; ERK activation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic epistasis with double-mutant rescue, defined molecular pathway, single lab\",\n      \"pmids\": [\"33177525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MEKK2 regulates right ventricular hypertrophy in hypoxia-induced pulmonary hypertension, in part through regulation of ERK5 abundance and phosphorylation. MEKK2-/- mice show attenuated RV hypertrophy and selective inhibition of inflammatory gene expression compared to WT mice under chronic hypoxia.\",\n      \"method\": \"MEKK2-/- mice; chronic hypobaric hypoxia model; RV hypertrophy measurements; ERK5 phosphorylation assays; gene expression analysis\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with defined in vivo phenotype, ERK5 mechanistic link, single lab\",\n      \"pmids\": [\"23125215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4L (HECT-type E3 ligase) constitutively and directly binds MEKK2 and promotes its poly-ubiquitination and degradation, negatively regulating signaling induced by TNF-α, IL-1, and IL-17. In IL-17R signaling, IL-17-induced MEKK2 Ser520 phosphorylation is required both for downstream p38 and NF-κB activation and for NEDD4L-mediated MEKK2 degradation.\",\n      \"method\": \"Co-IP; ubiquitination assays; NEDD4L knockdown/deficiency; Nedd4l-deficient mice; EAE model\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ubiquitination assay, in vivo mouse model, phospho-site functional characterization, multiple cytokine pathways tested\",\n      \"pmids\": [\"36161689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MEKK2 phosphorylates STK38 at Ser91, protecting STK38 from calpain-mediated cleavage. MEKK2 knockdown enhances hyperthermia-induced degradation of STK38. A phosphorylation-defective S91A STK38 mutant is susceptible to calpain degradation, demonstrating that MEKK2-mediated phosphorylation at Ser91 is required for STK38 stability.\",\n      \"method\": \"In vitro MEKK2 kinase assay with phospho-site identification; phospho-defective mutant (S91A); MEKK2 knockdown; calpain inhibitor experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay with site mapping, phospho-defective mutant, calpain inhibitor validation, single lab\",\n      \"pmids\": [\"31690749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"MEKK2 promotes GLI1 phosphorylation on multiple Ser/Thr sites, reducing its protein stability, DNA-binding ability, and increasing SUFU association, thereby suppressing Hedgehog pathway activity. MEKK2 mediates FGF2-induced inhibition of Hh signaling.\",\n      \"method\": \"In vitro phosphorylation assays; protein stability assays; DNA-binding assays; SUFU Co-IP\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay, multiple functional readouts, single lab\",\n      \"pmids\": [\"29662197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NDR2 kinase interacts with Smurf1 E3 ligase and promotes Smurf1-mediated K48-linked ubiquitination of MEKK2, leading to MEKK2 degradation and inhibition of IL-17-induced MAPK and NF-κB activation and inflammatory cytokine production.\",\n      \"method\": \"Co-IP showing NDR2-Smurf1 interaction; ubiquitination assay (K48 linkage); NDR2 and Smurf1 knockdown; IL-17-induced cytokine assays\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP, K48-specific ubiquitination assay, functional cytokine readout, single lab\",\n      \"pmids\": [\"30504095\"],\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. MAP3K2-deficient naïve CD4+ T cells have a reduced ability to induce colitis in a T cell transfer model, with fewer IFN-γ-producing cells in intestines.\",\n      \"method\": \"T cell transfer colitis model; MAP3K2-deficient T cells; in vitro differentiation conditions; cytokine measurement; JNK activation assays\",\n      \"journal\": \"Science China. Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with in vivo colitis model, molecular pathway characterization (IL-18-MAP3K2-JNK), single lab\",\n      \"pmids\": [\"32737854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"A single amino acid difference between MEKK2 (Val384) and MEKK3 (Ser390), immediately N-terminal to the active site lysine in kinase domain II/III, determines differential in vitro kinase activity in Triton X-100 detergent. Mutation of MEKK3 S390V confers activity in Triton X-100; the reciprocal MEKK2 V384S mutation abolishes activity in Triton X-100.\",\n      \"method\": \"Chimeric kinase constructs; site-directed mutagenesis; in vitro kinase assays in two detergents\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with kinase activity readout, reciprocal mutations tested, single lab\",\n      \"pmids\": [\"11343802\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Best3 interacts with both MEKK2 and MEKK3 in vascular smooth muscle cells, and this interaction inhibits phosphorylation of MEKK2 at serine 153. Best3 deficiency induces phosphorylation-dependent stabilization of MEKK2/3 (inhibiting ubiquitination and turnover), thereby activating downstream MAPK signaling and triggering aortic dissection.\",\n      \"method\": \"Co-immunoprecipitation coupled with mass spectrometry; Best3 smooth muscle cell-specific KO mice; phosphorylation assays; ubiquitination assays; single-cell RNA sequencing; proteomics\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — Co-IP/MS, genetic KO mice with severe in vivo phenotype, phospho-site identification, ubiquitination assays, multiple orthogonal methods\",\n      \"pmids\": [\"37203562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Crystal structure of the MEKK2 kinase domain (2.4 Å) in complex with ponatinib reveals that MEKK2 dimerizes via a surface centered on the αG helix and C-terminal activation segment. This surface is required for MEKK2 autophosphorylation and for phosphorylation of both MEK5 and MKK6. MEK5 recruitment is PB1 domain-dependent, while MKK6 recruitment is αG helix-dependent (PB1-independent), providing a structural basis for substrate selectivity.\",\n      \"method\": \"2.4 Å crystal structure; site-directed mutagenesis of αG helix surface; autophosphorylation assays; in vitro substrate phosphorylation assays (MEK5, MKK6); Co-IP/dimerization assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and functional validation across multiple substrates, mechanistically definitive for substrate targeting\",\n      \"pmids\": [\"41318559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SMYD3-dependent methylation of MAP3K2 promotes epithelial-mesenchymal transition behaviors in prostate cancer cells by altering the abundance of vimentin, and supports a positive feedback loop maintaining high SMYD3 levels.\",\n      \"method\": \"SMYD3 inhibitor treatment; siRNA knockdown; xenograft mouse models; EMT marker analysis; vimentin abundance measurement\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo functional assays, single lab, extends SMYD3-MAP3K2 methylation discovery\",\n      \"pmids\": [\"37976356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MAP3K2-mediated phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208 increases ROS formation in myeloid cells. Genetic inactivation of MAP3K2 and MAP3K3 in myeloid cells or hematopoietic S208A mutation of p47phox attenuates acute lung injury and abrogates the protective effects of pazopanib.\",\n      \"method\": \"In vitro phosphorylation assays; myeloid-specific MAP3K2/3 conditional KO mice; p47phox S208A knockin mice; ALI models; pazopanib pharmacology\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay identifying substrate/site, multiple genetic mouse models with defined phenotype, pharmacological validation\",\n      \"pmids\": [\"33910977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PDGF-BB-induced ERK5 activation in smooth muscle cells is dependent on Mekk2 (and Mek1/2, Mek5, PI3-kinase, and PKC), suggesting close co-regulation between ERK1/2 and ERK5 MAP kinase pathways downstream of PDGFR.\",\n      \"method\": \"Dominant-negative expression of Mekk2; kinase inhibitor studies; ERK5 activation assays in MOVAS smooth muscle cells\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — dominant-negative expression only, single lab, multiple pathway components tested simultaneously making MEKK2 contribution indirect\",\n      \"pmids\": [\"27339033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Sublytic C5b-9 induces MEKK2 phosphorylation at Ser153, Ser164, and Ser239, which is necessary for p38 MAPK activation, leading to the MEKK2-p38 MAPK-IRF-1-TRADD-caspase 8 apoptotic cascade in glomerular mesangial cells.\",\n      \"method\": \"Phosphorylation site identification; site-directed mutagenesis; siRNA knockdown; in vitro kinase assays; in vivo Thy-1 nephritis rat model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis of three phospho-sites, functional cascade assays, in vivo model, single lab\",\n      \"pmids\": [\"28039298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Interaction of paxillin LD1 motif with 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 auto-phosphorylation assay with recombinant paxillin; siRNA-mediated paxillin knockdown; LD1 motif binding assays\",\n      \"journal\": \"Journal of molecular signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro reconstitution of activation, siRNA validation, single lab\",\n      \"pmids\": [\"27096002\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAP3K2 (MEKK2) is a serine/threonine MAP3K that acts as a central signaling node: it is activated by dimerization through its catalytic αG helix surface and by upstream kinases (WNK1, Rap1), and is regulated by phosphorylation (at S519 by TRAF6-dependent pathways, at T283 by 14-3-3-regulated trans-autophosphorylation, and at K260 by SMYD3 methylation), as well as by ubiquitin-mediated degradation via multiple E3 ligases (Smurf1, CHIP, XIAP/cIAP1, NEDD4L); it signals via PB1 domain–MEK5 interaction to activate ERK5, and via MKK7 to activate JNK, with substrate selectivity further specified by its αG helix surface; MEKK2 also phosphorylates non-MAPK substrates including GLI1 (suppressing Hedgehog signaling), β-catenin S675 (promoting USP15-mediated deubiquitination to enhance WNT/bone formation), LATS1/2 and YAP/TAZ (regulating Hippo signaling), p47phox S208 (controlling NADPH oxidase-derived ROS in myeloid cells), and STK38 S91 (preventing calpain-mediated STK38 degradation), placing MEKK2 at the intersection of Ras/ERK, JNK, ERK5, Hippo, Hedgehog, WNT, and innate immune signaling pathways.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAP3K2 (MEKK2) is a serine/threonine MAP3 kinase that functions as a central signaling node coupling diverse receptor inputs to multiple downstream MAPK and non-MAPK pathways [#3, #5]. Its activation requires dimerization through a surface centered on the catalytic \\u03b1G helix and C-terminal activation segment, which drives autophosphorylation and substrate phosphorylation [#7, #41]; this same structural surface specifies substrate selectivity, recruiting MEK5 through the PB1 domain to activate ERK5 and recruiting MKK6/MKK7 through the \\u03b1G helix to activate stress kinases [#2, #15, #41]. Through PB1 domain\\u2013mediated heterodimerization with MEK5, MEKK2 activates the MEK5\\u2013ERK5 module, while through MKK7/MKK4 it activates JNK and AP-1\\u2013driven cytokine gene expression [#2, #15, #27]. Upstream, MEKK2 is engaged by WNK1, Rap1 (downstream of BDNF), and growth-factor and cytokine receptors including FGF-2, EGF, IgE, c-Kit, and TLR receptors, with EGF triggering its cytosol-to-nucleus translocation to access nuclear MEK5\\u2013ERK5 [#4, #6, #10, #13]. Activation-loop phosphorylation at Ser519 is a key regulatory event induced in a TRAF6-dependent manner and required for TLR-induced IL-6 production, while phosphorylation at Thr283 and 14-3-3 binding restrain trans-autophosphorylation and kinase activity [#9, #21]. MEKK2 abundance is tightly controlled by ubiquitin-mediated degradation through multiple E3 ligases\\u2014Smurf1 (facilitated by STK38/NDR2), CHIP, XIAP/cIAP1, and NEDD4L\\u2014which terminate ERK, ERK5, and NF-\\u03baB signaling and couple receptor signaling to defined cellular outcomes [#1, #14, #19, #22, #34, #37]. Beyond the MAPK cascades, MEKK2 phosphorylates non-canonical substrates to regulate WNT/bone formation (\\u03b2-catenin S675, promoting USP15-mediated stabilization), Hedgehog signaling (GLI1, reducing its stability and DNA binding), Hippo signaling (LATS1/2 and YAP/TAZ), myeloid ROS production (p47phox S208), and STK38 stability (S91) [#20, #26, #30, #35, #43]. These activities place MEKK2 at the intersection of immune cell signaling, osteoblast/skeletal biology, intestinal stem-cell maintenance, vascular integrity, and cancer cell invasion, the last potentiated by SMYD3 methylation at K260 that blocks PP2A binding [#0, #29, #40, #42]. A crystal structure of the kinase domain provides a structural basis for dimerization-driven activation and differential substrate recruitment [#41].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established MEKK2 as a TCR-responsive kinase that physically relocalizes to the immunological synapse, linking it to early antigen receptor signaling.\",\n      \"evidence\": \"Live-cell imaging and dominant-negative MEKK2 in T cell/APC conjugates\",\n      \"pmids\": [\"10549623\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct substrates at the synapse not defined\", \"Dominant-negative approach does not establish endogenous requirement\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identified MEK5 as a direct MEKK2 partner and ERK5 as a downstream output, and showed MEKK2 is genetically required for receptor-induced JNK activation and cytokine transcription, defining its dual MAPK outputs.\",\n      \"evidence\": \"Yeast two-hybrid, dominant-negative epistasis, and MEKK2-deficient mast cells\",\n      \"pmids\": [\"11073940\", \"11032806\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of MEK5 binding not yet resolved\", \"How a single MAP3K selects ERK5 vs JNK output unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Genetic knockout revealed MEKK2 negatively regulates TCR signaling strength, showing its role is not uniformly activating but context-dependent.\",\n      \"evidence\": \"Mekk2 KO mice; T cell proliferation, cytokine, and MAPK activation assays\",\n      \"pmids\": [\"12138187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of negative regulation in T cells not defined\", \"JNK only moderately affected, leaving redundancy unexplained\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the PB1 domain as the structural module mediating selective MEK5/ERK5 pathway engagement and placed WNK1 upstream as a MEKK2-activating kinase.\",\n      \"evidence\": \"PB1 domain binding/mutation assays; WNK1 Co-IP, in vitro kinase, and dominant-negative epistasis\",\n      \"pmids\": [\"12912994\", \"14681216\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PB1-independent substrate routing not yet addressed\", \"WNK1 phospho-site on MEKK2 not mapped\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Established MEKK2 as the FGF-2/EGF receptor coupler that coordinately drives ERK5 and JNK and undergoes signal-induced nuclear translocation to reach nuclear MEK5.\",\n      \"evidence\": \"MEKK2-/- MEFs with stimulus specificity; subcellular fractionation and live-cell imaging; FLS in vitro kinase assays\",\n      \"pmids\": [\"14978743\", \"15075238\", \"14734742\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger for nuclear import not molecularly defined\", \"MKK4 vs MKK7 selectivity not resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Resolved the activation logic: dimerization through the catalytic domain drives activation, Ser519 in the activation loop is the critical regulatory phospho-site (TRAF6-dependent), and Smurf1 and Mip1 act as negative regulators by degradation and dimer blockade respectively.\",\n      \"evidence\": \"Chemical-induced dimerization, deletion mapping, S519A mutagenesis with phospho-antibody, Smurf1-deficient mice, Mip1 Co-IP/siRNA\",\n      \"pmids\": [\"15695508\", \"16362041\", \"15820682\", \"15988011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for Ser519 phosphorylation in vivo not fully defined\", \"Mip1 mechanism characterized in a single lab\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined how the PB1 domain partitions MEKK2 output\\u2014front-to-back PB1 binding to MEK5 drives ERK5 while a C-terminal acidic cluster routes activated MEKK2 to MKK7/JNK\\u2014providing a molecular switch for substrate choice.\",\n      \"evidence\": \"PB1 residue mutagenesis, Co-IP, and ERK5/JNK kinase activation assays\",\n      \"pmids\": [\"17452462\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational change driving the switch not visualized\", \"Quantitative balance between outputs in vivo not measured\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed MEKK2 turnover is functionally instructive: CHIP-mediated degradation enforces transient ERK kinetics required for correct aquaporin gene induction, and Lad1/calcium controls EGF-induced MEKK2 nuclear translocation.\",\n      \"evidence\": \"CHIP Co-IP/siRNA/gene targeting with ERK time-course and AQP readout; Lad1 Co-IP and calcium-modifier translocation imaging\",\n      \"pmids\": [\"20588253\", \"20830310\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of CHIP regulation beyond osmotic stress unknown\", \"Lad1 findings from a single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended MEKK2 function to TGF-\\u03b2/SMAD signaling, showing it phosphorylates SMAD2/3 linker regions to restrain Treg/Th17 differentiation, a non-classical MAPK output with immunological consequence.\",\n      \"evidence\": \"Conditional/constitutive Map3k2/Map3k3 KO mice, in vitro differentiation, phospho-SMAD linker blots, EAE model\",\n      \"pmids\": [\"21333552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect SMAD linker phosphorylation not fully separated\", \"Redundancy with MEKK3 limits MEKK2-specific attribution\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified SMYD3 K260 methylation as a positive regulator that blocks PP2A binding to sustain Ras/ERK signaling, and expanded the E3 ligase repertoire (XIAP/cIAP1 via K63 chains competing with MEK5) and focal-adhesion functions controlling cell migration.\",\n      \"evidence\": \"Protein array, in vitro methylation, PP2A Co-IP, tumor mouse models; K63 chain analysis and trimeric complex reconstitution; paxillin Co-IP/ubiquitylation and FA imaging\",\n      \"pmids\": [\"24847881\", \"24975362\", \"24491810\", \"25190348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase(s) reversing K260 methylation context not detailed\", \"Focal-adhesion substrate(s) beyond paxillin unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Broadened MEKK2 substrate scope to developmental pathway control\\u2014\\u03b2-catenin S675 phosphorylation promoting USP15-mediated stabilization in bone, and GLI1 phosphorylation suppressing Hedgehog\\u2014linking it to WNT and Hh signaling.\",\n      \"evidence\": \"MEKK2-deficient mice, in vitro kinase with phospho-site mapping, USP15/SUFU Co-IP, genetic epistasis; SMYD3-MEKK2 co-crystal with inhibitor design\",\n      \"pmids\": [\"26884171\", \"29662197\", \"27066749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor inputs upstream of \\u03b2-catenin/GLI1 phosphorylation incompletely defined\", \"GLI1 findings rest on a single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrated tissue-protective and physiological roles: a ROS-MEKK2-ERK5-KLF2 axis maintaining intestinal stem cells, p47phox S208 phosphorylation controlling myeloid ROS in lung injury, and MEKK2/3 activation of LATS1/2-YAP/TAZ in the Hippo pathway downstream of TNF.\",\n      \"evidence\": \"Genetic mouse models with injury/ALI/colitis assays, single-cell profiling, in vitro kinase assays, STRIPAK interaction studies\",\n      \"pmids\": [\"33658717\", \"33910977\", \"33571521\", \"32737854\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect LATS/YAP phosphorylation not fully separated from MEKK3\", \"Upstream activator of the intestinal ROS axis not pinpointed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected MEKK2 stability control to vascular disease, showing Best3 binding inhibits Ser153 phosphorylation and promotes MEKK2/3 turnover, with loss driving aortic dissection, and extended SMYD3 methylation to prostate cancer EMT.\",\n      \"evidence\": \"Co-IP/MS, Best3 smooth-muscle KO mice, phospho/ubiquitination assays; SMYD3 inhibition and xenografts with EMT markers\",\n      \"pmids\": [\"37203562\", \"37976356\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase phosphorylating Ser153 not identified\", \"Causal substrate downstream of stabilized MEKK2 in dissection unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided the definitive structural basis for activation and substrate selectivity: an \\u03b1G helix\\u2013centered dimerization surface drives autophosphorylation, with MEK5 recruited via PB1 and MKK6 via the \\u03b1G surface independently of PB1.\",\n      \"evidence\": \"2.4 \\u00c5 crystal structure with \\u03b1G surface mutagenesis and in vitro substrate phosphorylation assays\",\n      \"pmids\": [\"41318559\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length regulated MEKK2 not solved\", \"How upstream phosphorylation reshapes the dimer interface unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many parallel regulatory inputs (methylation, multiple phospho-sites, competing E3 ligases, and scaffold/adaptor binding) are integrated to set MEKK2 output toward a specific pathway in a given cell type remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model of input integration\", \"MEKK2 vs MEKK3 division of labor across tissues not systematically mapped\", \"Cell-type-specific substrate prioritization mechanism unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [20, 26, 35, 43, 17]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [27, 41, 9]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [7, 41]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [3, 10, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 28]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [24, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 10, 13, 15, 30]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4, 9, 12, 17, 22, 34, 43]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [20, 26, 29]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [1, 14, 19, 34, 37]}\n    ],\n    \"complexes\": [\n      \"MEKK2\\u2013MEK5 PB1 complex\",\n      \"MEKK2/3\\u2013XIAP/cIAP1\\u2013MEK5 trimeric complex\"\n    ],\n    \"partners\": [\n      \"MAP2K5\",\n      \"MAP2K7\",\n      \"WNK1\",\n      \"XIAP\",\n      \"SMURF1\",\n      \"STUB1\",\n      \"NEDD4L\",\n      \"STK38\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":9,"faith_pct":88.88888888888889}}