{"gene":"MAP3K3","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2001,"finding":"MEKK3 is required for TNF-induced IKK activation and NF-κB signaling; MEKK3 physically interacts with RIP and directly phosphorylates IKK, placing MEKK3 downstream of RIP and TRAF2 in the TNF receptor pathway.","method":"MEKK3-deficient fibroblasts, co-immunoprecipitation, in vitro kinase assay","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with defined phenotype, co-IP, and direct in vitro phosphorylation assay; replicated in subsequent studies","pmids":["11429546"],"is_preprint":false},{"year":2003,"finding":"MEKK3 forms a scaffold complex with the GTPase Rac, the adaptor protein OSM (osmosensing scaffold for MEKK3), and MKK3 on actin structures; this Rac-OSM-MEKK3-MKK3 complex is required for p38 MAPK activation in response to sorbitol-induced hyperosmolarity.","method":"RNAi knockdown, FRET, co-immunoprecipitation, live-cell imaging","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi epistasis, FRET-validated complex, multiple orthogonal methods in one study","pmids":["14634666"],"is_preprint":false},{"year":2003,"finding":"MEKK3 is an essential signal transducer downstream of the MyD88-IRAK-TRAF6 complex in IL-1R and TLR4 signaling; MEKK3 forms a complex with TRAF6 upon IL-1 or LPS stimulation and is required for NF-κB and JNK-p38 (but not ERK) activation.","method":"MEKK3-deficient MEFs, co-immunoprecipitation, cytokine measurement","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout, co-IP, pathway-specific readout, replicated across labs","pmids":["14661019"],"is_preprint":false},{"year":1999,"finding":"MEKK3 physically interacts with MEK5 and directly activates MEK5 to stimulate BMK1/ERK5 activity; a dominant-active MEKK3 is sufficient to activate ERK5 through MEK5, and MEKK3 kinase activity is required for growth-factor-mediated ERK5 activation.","method":"Yeast two-hybrid, co-immunoprecipitation, dominant-active overexpression, kinase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — yeast two-hybrid plus co-IP plus in-cell kinase activation assay in single study","pmids":["10593883"],"is_preprint":false},{"year":2000,"finding":"MEKK3 is essential for embryonic blood vessel development; Map3k3-/- mice die ~E11 with angiogenesis defects, and MEKK3 activates MEF2C through the p38 MAPK cascade to drive cardiovascular development.","method":"Gene knockout in mice, embryo phenotyping, signaling pathway analysis","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic KO with defined vascular phenotype and pathway placement via p38-MEF2C","pmids":["10700190"],"is_preprint":false},{"year":2015,"finding":"CCM2 directly binds MEKK3 via its harmonin homology domain (HHD) interacting with the N-terminus of MEKK3; a co-crystal structure at 2.35 Å was determined. MEKK3 deficiency in endothelial cells causes intracranial hemorrhage partially dependent on Rho-ROCK signaling, and disruption of MEKK3:CCM2 interaction phenocopies this neurovascular leakage.","method":"Co-crystal structure (2.35 Å), inducible endothelial-specific Mekk3 knockout mice, Rho-ROCK pathway analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus genetic KO plus pathway rescue, multiple orthogonal methods","pmids":["26235885"],"is_preprint":false},{"year":2016,"finding":"Gain of MEKK3 signaling in endothelial cells is causal for CCM disease; endothelial loss of KRIT1/CCM2/PDCD10 increases MEKK3 activity leading to elevated KLF2/KLF4 expression and Rho/ADAMTS activity. Endothelial-specific loss of Map3k3 or Klf2/Klf4 prevents lesion formation. A disease-causing CCM2 mutation abrogates the MEKK3 interaction without disrupting CCM complex formation.","method":"Neonatal mouse CCM model, endothelial-specific conditional knockout, human CCM tissue analysis, signaling pathway readouts","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genetic models, human tissue validation, mechanistic pathway delineation","pmids":["27027284"],"is_preprint":false},{"year":2015,"finding":"Loss of CCM signaling in endocardial cells increases MEKK3 activity, which is both necessary and sufficient for elevated Klf2/Klf4 and Adamts4/5 expression causing cardiac jelly degradation; partial loss of MEKK3 rescues cardiac defects in CCM-deficient embryos.","method":"Conditional knockout mice, genetic epistasis (partial Mekk3 loss rescuing CCM deficiency), gene expression analysis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with clear rescue, multiple CCM alleles tested","pmids":["25625206"],"is_preprint":false},{"year":2003,"finding":"The PB1 domains of MEKK3 (and MEKK2) interact with the PB1 domain of MEK5 to form heterodimers required for ERK5 pathway activation; deletion or mutation of the MEKK3 PB1 domain abolishes MEKK3-MEK5 complex formation and ERK5 activation.","method":"In vitro PB1 domain binding assay, co-immunoprecipitation, dominant-negative PB1 domain expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro binding, co-IP, and dominant-negative functional assay; mutagenesis of PB1 domain","pmids":["12912994"],"is_preprint":false},{"year":2006,"finding":"Two IL-1-mediated NF-κB activation pathways bifurcate at IRAK modification: a TAK1-dependent pathway activating IKKα/β and a MEKK3-dependent (TAK1-independent) pathway that involves IKKγ phosphorylation and IKKα activation, resulting in IκBα phosphorylation without degradation.","method":"IRAK mutation analysis in reconstituted cells, TAK1-/- and MEKK3-/- MEFs, IKK subunit-specific readouts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple KO cell lines, pathway bifurcation with orthogonal IKK subunit readouts, replicated concept in multiple papers","pmids":["17197697"],"is_preprint":false},{"year":2006,"finding":"TLR8-mediated NF-κB and JNK activation are completely abolished in MEKK3-/- MEFs but only moderately reduced in TAK1-/- MEFs; TLR8 signals through a MEKK3-dependent pathway involving IKKγ phosphorylation rather than IKKα/β phosphorylation.","method":"MEKK3-/- and TAK1-/- MEFs, IKK complex phosphorylation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout cells, parallel pathway comparison with multiple TLR stimuli","pmids":["16737960"],"is_preprint":false},{"year":2006,"finding":"Phosphorylation of MEKK3 at Ser526 within the T-loop activation loop is required for MEKK3 kinase activity (NF-κB, ERK, JNK, p38 activation); Ser526 is autophosphorylated, regulated by protein phosphatase 2A (PP2A), and association with 14-3-3 at pSer526 prevents dephosphorylation.","method":"Alanine/phosphomimetic mutagenesis, phospho-specific antibody, in vitro kinase assay with MKK6, PP2A inhibitor studies","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis, in vitro kinase assay, phospho-specific antibody, phosphatase identification","pmids":["16407301"],"is_preprint":false},{"year":2009,"finding":"The rear-end acidic cluster of the p62/sequestosome-1 PB1 domain binds the front-end basic region of the MEKK3 PB1 domain; the p62-MEKK3 complex co-localizes in cytoplasmic speckles, recruits TRAF6, and is required for TRAF6-regulated NF-κB activation downstream of IL-1.","method":"PB1 domain binding assays, co-localization microscopy, shRNA knockdown, NF-κB reporter assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain-specific binding mapped, co-localization, shRNA loss-of-function with functional NF-κB readout","pmids":["19903815"],"is_preprint":false},{"year":2004,"finding":"TRAF7 specifically interacts with MEKK3 and potentiates MEKK3-mediated AP1 and CHOP activation; depletion of TRAF7 by antisense RNA inhibits MEKK3-mediated AP1 and CHOP activation.","method":"Co-immunoprecipitation, antisense RNA knockdown, reporter gene assays, domain mapping","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and antisense knockdown with reporter readout, single lab","pmids":["15001576"],"is_preprint":false},{"year":2005,"finding":"TAK1 is recruited to the TNF-R1 complex in a RIP-dependent manner and forms a functional complex with MEKK3; TAK1 regulates autophosphorylation of MEKK3 in a TAK1-kinase-activity-dependent manner, requiring TAB1 for TAK1 activation and subsequent MEKK3 phosphorylation.","method":"Co-immunoprecipitation, receptor complex isolation, dominant-negative TAK1, TAB1 co-expression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, kinase-dead mutant analysis, single lab","pmids":["16260783"],"is_preprint":false},{"year":2008,"finding":"MEKK3 and TAK1 form a complex containing non-phosphorylated forms of both kinases; non-phosphorylated TAK1 inhibits MEKK3 phosphorylation and NF-κB signaling; TAB1-mediated TAK1 autophosphorylation reverses this inhibition, providing homeostatic regulation of basal NF-κB levels.","method":"Tandem affinity purification, FRET, co-immunoprecipitation, NF-κB reporter, TAK1-deficient MEFs","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TAP-MS identification, FRET confirmation, functional rescue, single lab","pmids":["18206350"],"is_preprint":false},{"year":2004,"finding":"RIP functions to specifically recruit MEKK3 to the TNF-α receptor complex; a MEKK3-RIP death domain fusion (MEKK3-DD) fully restores TNF-α-induced NF-κB activation in RIP-deficient cells, while MEKK2-DD or NEMO-DD cannot, demonstrating MEKK3-specific requirement.","method":"Reconstitution of RIP-deficient Jurkat cells with fusion proteins, co-immunoprecipitation, NF-κB activation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reconstitution experiment in KO cells, specificity controls with MEKK2 and NEMO, clear epistatic placement","pmids":["15572679"],"is_preprint":false},{"year":2003,"finding":"MEKK3 interacts with PA28γ (a proteasome activator subunit) but not PA28α; the PA28γ-binding domain of MEKK3 is in its N-terminal regulatory domain (aa 1-178); in vitro assays showed PA28γ is a MEKK3 substrate; MEKK3 expression increases PA28γ protein levels in a kinase-activity-dependent manner.","method":"Co-immunoprecipitation, in vitro kinase assay, domain deletion mapping","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay and co-IP, single lab, single study","pmids":["12650640"],"is_preprint":false},{"year":2002,"finding":"Activation of MEKK3 induces G2 cell cycle arrest dependent on p38α/β2 signaling, associated with down-regulation of cyclin A and B1 expression and inhibition of CDK1/CDK2 activity; p38 inhibitor SB203580 partially rescues the G2 arrest.","method":"Conditional MEKK3:ER* activation, cell cycle synchronization, CDK activity assays, p38 inhibitor","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional activation system, pharmacological epistasis with p38 inhibitor, single lab","pmids":["12444545"],"is_preprint":false},{"year":2007,"finding":"NMR solution structure of MEKK3 PB1 domain reveals prolyl isomerization at Gln38-Pro39 producing two structural isomers; the MEKK3 PB1 domain binds MEK5 PB1 with Kd ~10^-8 M; Lys7 and Arg5 in the basic cluster are key residues for this interaction.","method":"NMR structure determination, mutagenesis, binding affinity measurement","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with mutagenesis and quantitative binding data, single lab","pmids":["17985933"],"is_preprint":false},{"year":2005,"finding":"MEKK3 is essential for angiotensin II-induced calcineurin/NFAT activation in cardiac myocytes; MEKK3-deficient MEFs fail to activate NFAT in response to angiotensin II; restoring MEKK3 rescues NFAT activation; MEKK3 is phosphorylated in response to angiotensin II and functions downstream of the AT1 receptor.","method":"MEKK3-/- MEFs, siRNA knockdown, dominant-negative MEKK3, NFAT reporter, reconstitution","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO MEFs, reconstitution, dominant-negative, multiple epistasis approaches, single lab","pmids":["16126726"],"is_preprint":false},{"year":2006,"finding":"MEKK3 is required for hypertonic stress-induced p38 activation and downstream TonE-driven gene (BGT1) induction in kidney cells; siRNA-mediated MEKK3 depletion downregulates p38 activity and reduces BGT1 expression.","method":"Stable transfection of activated MEKK3, siRNA knockdown, TonE-luciferase reporter","journal":"American journal of physiology. Renal physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function, pathway-specific reporter, single lab","pmids":["16684924"],"is_preprint":false},{"year":2008,"finding":"Phosphorylation of MEKK3 at Thr294 promotes 14-3-3 binding to negatively regulate MEKK3; TNFα or LPS stimulation causes rapid dephosphorylation of Thr294 and loss of 14-3-3 association, correlating with MEKK3 pathway activation; Thr294 phosphorylation does not affect Ser526 phosphorylation.","method":"Phospho-specific antibody, mutagenesis (T294A), co-immunoprecipitation with 14-3-3, NF-κB reporter","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-specific antibody, mutagenesis, and functional readout, single lab","pmids":["18308725"],"is_preprint":false},{"year":2002,"finding":"MEKK3 is phosphorylated at Ser166 and Ser337 in response to TNF, arsenite, forskolin, and serum; 14-3-3 proteins interact with MEKK3; however, Ser166 and Ser337 phosphorylation are not required for 14-3-3 association or MEKK3-dependent ERK and JNK activity.","method":"LC-MS phosphopeptide mapping, phospho-specific antibodies, co-precipitation with 14-3-3","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based phosphosite mapping with phospho-specific antibodies, negative result on function, single lab","pmids":["12392720"],"is_preprint":false},{"year":2010,"finding":"Protein phosphatase 2A (PP2A) acts as a MEKK3 phosphatase; PP2Acβ associates with phosphorylated MEKK3 in a transient LPA-induced manner, dephosphorylates MEKK3 at Thr516 and Ser520, and terminates MEKK3-mediated IKKβ/NF-κB activation.","method":"Functional genomic screen, co-immunoprecipitation, overexpression/knockdown of PP2Ac, IKKβ phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional genomic identification, co-IP, siRNA knockdown with pathway readout, single lab","pmids":["20448038"],"is_preprint":false},{"year":2010,"finding":"Phosphorylation of MEKK3 at Thr516 and Ser520 within the kinase activation loop is required for MEKK3-mediated IKKβ/NF-κB activation; alanine substitution abolishes activity, while acidic substitution renders constitutive activity.","method":"Alanine/aspartate mutagenesis, IKKβ phosphorylation assay, NF-κB reporter","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — activation loop mutagenesis with both loss- and gain-of-function mutations, in vitro kinase readout","pmids":["20068038"],"is_preprint":false},{"year":2009,"finding":"MEKK3 is required for LPA-induced IKK-NF-κB activation and cytokine (IL-6, MIP-2) production via GPCR signaling; this is MEKK3-specific and TAK1-independent, as shown by selective loss in MEKK3-/- but not TAK1-/- MEFs.","method":"MEKK3-/- and TAK1-/- MEFs, IKK activation assay, cytokine ELISA","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Moderate — parallel genetic KO comparison, pathway-specific readout, single lab","pmids":["19465115"],"is_preprint":false},{"year":2011,"finding":"MEKK2 and MEKK3 negatively regulate TGF-β-mediated Th cell differentiation by phosphorylating SMAD2 and SMAD3 at their linker regions; T cell-specific Map3k2/Map3k3 double knockout mice show accumulation of Treg and Th17 cells consistent with enhanced TGF-β responses.","method":"T cell conditional knockout mice, in vitro differentiation assays, SMAD phosphorylation analysis","journal":"Immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined cellular phenotype and SMAD phosphorylation mechanistic link, single lab","pmids":["21333552"],"is_preprint":false},{"year":2011,"finding":"MEKK3 is required for TCR-induced ERK1/2, JNK, and p38 activation and IFN-γ production in CD4+ T cells; TCR-mediated MEKK3 activation requires Rac1/2, as shown in Mekk3 T cell conditional knockout mice.","method":"T cell-specific conditional Mekk3 knockout mice, cytokine measurement, MAPK activation assays, bacterial infection model","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean conditional KO, pathway readouts, Rac1/2 upstream requirement, single lab","pmids":["21471448"],"is_preprint":false},{"year":2014,"finding":"NBR1 interacts with MEKK3 via PB1 domain interaction; the NBR1-MEKK3 complex is required for JNK activation in macrophages and drives adipose tissue inflammation in obesity; myeloid-specific NBR1 inactivation impairs JNK signaling and macrophage inflammatory function.","method":"PB1 domain interaction assay, myeloid-specific conditional KO mice, JNK activation assay, metabolic phenotyping","journal":"Cell metabolism","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain-level interaction, conditional KO in myeloid cells, in vivo phenotype, single lab","pmids":["25043814"],"is_preprint":false},{"year":2008,"finding":"MEKK3 kinase activity is required for and sufficient to initiate TGFβ2-dependent epithelial-to-mesenchymal transition (EMT) during endocardial cushion morphogenesis; kinase-inactive MEKK3 blocks EMT while constitutively active MEKK3 triggers EMT in normally non-EMT ventricular endocardium.","method":"In vitro cushion EMT assay, kinase-inactive and constitutively active MEKK3 constructs, gene expression analysis","journal":"Circulation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function in an in vitro developmental assay, TGFβ2-dependent mechanism identified, single lab","pmids":["19008476"],"is_preprint":false},{"year":2009,"finding":"MEKK3 is required for lymphopenia-induced T cell proliferation and homeostatic survival; MEKK3-deficient T cells show attenuated ERK1/2 (but not p38) activation during homeostatic proliferation, while antigen-induced proliferation is unaffected.","method":"T cell-specific MEKK3 conditional KO mice, adoptive transfer, MAPK activation assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with adoptive transfer, ERK-specific deficit identified, single lab","pmids":["19265138"],"is_preprint":false},{"year":2021,"finding":"MEKK3-MEK5-ERK5 signaling pathway is required for basal lysosome-mediated mitochondrial degradation; genetic or pharmacological inhibition of MEKK3-MEK5-ERK5 increases mitochondrial content by reducing basal mitophagic degradation without affecting bulk autophagy or damage-induced mitophagy.","method":"Genetic inhibition (CRISPR), pharmacological inhibition, mitochondrial content assays, lysosomal inhibition studies","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological approaches, selective pathway specificity tested, single lab","pmids":["33101709"],"is_preprint":false},{"year":2019,"finding":"CDC42 deletion in endothelial cells causes increased MEKK3-MEK5-ERK5-KLF2/4 signaling and CCM-like vascular malformations; CDC42 interacts with CCM proteins and CCM3 promotes CDC42 activity; Klf4 co-inactivation reduces severity of Cdc42-mutant vascular malformations.","method":"Endothelial-specific inducible Cdc42 knockout mice, genetic epistasis (Klf4 co-deletion), MEKK3 pathway signaling assays","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO, genetic rescue, pathway mechanistic analysis, multiple alleles tested","pmids":["30732528"],"is_preprint":false},{"year":2015,"finding":"Both CCM2 and CCM2L bind MEKK3 in a complex with CCM1 and prevent MEKK3 activation and its ability to phosphorylate MEK5; ccm2l/ccm2 double knockdown in zebrafish is rescued by mekk3 knockdown, confirming CCM2L-CCM2 co-regulation of MEKK3 in vivo.","method":"In vitro binding assays, MEK5 phosphorylation assay, zebrafish morpholino knockdown with genetic rescue","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay, co-IP, in vivo rescue epistasis in zebrafish, single lab","pmids":["26540726"],"is_preprint":false},{"year":2018,"finding":"MEKK3 inhibits GLI1 transcriptional activity and oncogenic function by phosphorylating multiple Ser/Thr sites on GLI1, reducing GLI1 protein stability, DNA-binding ability, and increasing GLI1-SUFU association; MEKK3 mediates FGF2-dependent inhibition of Hedgehog signaling.","method":"In vitro kinase assay, GLI1 mutagenesis, protein stability assays, SUFU co-immunoprecipitation, medulloblastoma cell proliferation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct phosphorylation assay with multiple mechanistic readouts, single lab","pmids":["29662197"],"is_preprint":false},{"year":2018,"finding":"MEKK3 forms a complex with WDR62 to promote JNK signaling synergistically in neural progenitor cells; MEKK3 positively regulates WDR62 protein stability in the developing brain; WDR62 is negatively regulated by T1053 phosphorylation leading to FBW7-mediated proteasomal degradation.","method":"Conditional Mekk3 knockout mice, WDR62 stability assays, JNK signaling analysis, FBW7 co-immunoprecipitation","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO, protein stability mechanism, phosphorylation site identified, single lab","pmids":["30566428"],"is_preprint":false},{"year":2021,"finding":"MEKK3 and MEKK2 phosphorylate LATS1/2 and YAP/TAZ to activate Hippo signaling in response to TNF and other stimuli; STRIPAK complex associates with MEKK3 via CCM2 and CCM3 to inactivate MEKK3, and upstream Hippo signals trigger MEKK3 dissociation from STRIPAK to release MEKK3 activity.","method":"Co-immunoprecipitation, kinase assay (LATS1/2 and YAP/TAZ phosphorylation), STRIPAK complex analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct phosphorylation assays, complex characterization by co-IP, single lab","pmids":["33571521"],"is_preprint":false},{"year":2009,"finding":"Hsp90 interacts with MEKK3 and acts as its molecular chaperone to maintain MEKK3 stability; Hsp90 inhibitors (geldanamycin) shorten MEKK3 half-life and induce MEKK3 ubiquitination and proteasomal degradation.","method":"Co-immunoprecipitation, Hsp90 RNAi, geldanamycin treatment, protein stability/half-life assay, ubiquitination assay","journal":"Cellular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, pharmacological and siRNA approaches, proteasome-dependent degradation shown, single lab","pmids":["19560753"],"is_preprint":false},{"year":2022,"finding":"PIEZO1 mechanosensitive channel activation by shear stress leads to calcium influx that activates CaMKII; CaMKII interacts with and activates MEKK3, promoting MEKK3/MEK5/ERK5 signaling and KLF2/4 transcription in endothelial cells.","method":"Endothelial-specific Piezo1 knockout mice, CaMKII-MEKK3 co-immunoprecipitation, ERK5/KLF2/4 pathway readouts","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO, co-IP of CaMKII-MEKK3, pathway-specific readouts, single lab","pmids":["35883633"],"is_preprint":false},{"year":2021,"finding":"MEKK3 activates IRF7 through direct interaction and phosphorylation of IRF7 at multiple sites in response to TLR7/9 activation; endogenous MEKK3 binds and phosphorylates IRF7 after TLR9 activation by CpG DNA, triggering type I IFN induction.","method":"Co-immunoprecipitation, in vitro phosphorylation assay, MEKK3 knockdown in vivo, TLR7/9 ligand stimulation","journal":"Molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct phosphorylation, co-IP, and in vivo knockdown, single lab","pmids":["33812250"],"is_preprint":false},{"year":2021,"finding":"MEKK3-TGFβ crosstalk controls inward arterial remodeling; endothelial-specific MEKK3 deletion causes inward remodeling of pulmonary and systemic arteries, spontaneous hypertension, and accelerated atherosclerosis; molecular analysis reveals MEKK3 deletion activates TGFβR1-Smad2/3 signaling, and endothelial TGFβR1 knockout prevents this remodeling.","method":"Adult endothelial-specific Mekk3 knockout, endothelial TGFβR1 knockout epistasis, vascular phenotyping, Smad2/3 phosphorylation","journal":"Proceedings of the National Academy of Sciences","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with double-KO genetic rescue epistasis, in vitro and in vivo TGFβ pathway analysis","pmids":["34911761"],"is_preprint":false},{"year":2022,"finding":"RAGE binds MKK3 via C-terminal amino acids 2-5, and this interaction is required for assembly of the MEKK3-MKK3-p38 signaling module and activation of p38 MAPK/NF-κB signaling; specific RAGE ctRAGE R2A-K3A-R4A-Q5A mutation suppresses neuronal damage and improves synaptic plasticity in diabetic mice.","method":"Co-immunoprecipitation, GST pull-down, point mutagenesis, electrophysiology, behavioral assays in db/db mice","journal":"Aging cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, GST pull-down, and in vivo mutagenesis rescue, single lab","pmids":["35080104"],"is_preprint":false},{"year":2022,"finding":"MEKK3 and KLF2/4 signaling in endothelial cells integrates hemodynamic (shear/fluid forces) and inflammatory signals (LPS, IFN-γ) to specify RUNX1+ hemogenic endothelial cells and drive endothelial-to-hematopoietic transition (EHT) in the embryo.","method":"Endothelial-specific Mekk3 conditional KO mice, LPS and IFN-γ stimulation, catecholamine stimulation, RUNX1+ cell quantification","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Moderate — endothelial conditional KO with defined hematopoietic phenotype and multiple stimulus conditions, single lab","pmids":["35245372"],"is_preprint":false},{"year":2023,"finding":"STK24/25 (CCM3-interacting kinases) limit constitutive MEKK3 activity by being recruited to MEKK3 via CCM2 as adaptor; loss of STK24/25 in endothelial cells causes MEKK3 activation and CCM lesion formation; a hybrid STK kinase domain–CCM2 MEKK3-interacting domain fusion rescues CCM loss-of-function in zebrafish.","method":"Endothelial-specific STK24/25 conditional KO mice, zebrafish genetic rescue with hybrid protein, CCM lesion quantification","journal":"JCI insight","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO, domain-swap rescue in zebrafish, mechanistic pathway placement, single lab","pmids":["36692953"],"is_preprint":false},{"year":2024,"finding":"MAP3K3 phosphorylates YAP at Ser405, preventing FBXW7 binding and thereby inhibiting p62-mediated lysosomal degradation of YAP; this stabilization mechanism is independent of canonical Hippo kinases and supports YAP-dependent drug resistance in melanoma and breast cancer.","method":"Mass spectrometry phosphoproteomics, MAP3K3 depletion (siRNA/CRISPR), in vitro phosphorylation, FBXW7 co-immunoprecipitation, lysosomal degradation assay","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based site identification, direct phosphorylation, E3 ligase binding assay, single lab","pmids":["38622197"],"is_preprint":false},{"year":2021,"finding":"MAP3K2 and MAP3K3 mediate phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208, promoting ROS formation in myeloid cells; pazopanib inhibits MAP3K2/MAP3K3 to reduce this phosphorylation; myeloid-specific MAP3K2/MAP3K3 double inactivation or p47phox S208A mutation attenuates acute lung injury.","method":"In vitro kinase assay (p47phox phosphorylation), myeloid-specific conditional KO, p47phox S208A knock-in mice, ALI models","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct substrate phosphorylation, genetic KO, knock-in mutagenesis, multiple model systems","pmids":["33910977"],"is_preprint":false},{"year":2007,"finding":"MEKK3 is required for angiopoietin-1/Tie2-induced p38 and ERK5 activation in endothelial cells; MEKK3-deficient endothelial cells show defects in proliferation, apoptosis, and interaction with myocardium.","method":"Mekk3-deficient endothelial cells from embryos, Ang1 stimulation, p38 and ERK5 activation assays","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO endothelial cells with defined signaling defect, single lab","pmids":["17687003"],"is_preprint":false},{"year":2014,"finding":"H. pylori induces transient IKK complex activation via mutual control by MEKK3 and TAK1; TAK1 transiently interacts with TRAF6 and undergoes autophosphorylation and K63-linked ubiquitination; MEKK3 and TAK1 synergize to activate the IKK complex in a T4SS-dependent, CagA-independent manner.","method":"Co-immunoprecipitation, H. pylori infection of gastric epithelial cells, TAK1/MEKK3 pathway analysis","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP-based interaction and ubiquitination analysis, bacterial infection model, single lab","pmids":["24418622"],"is_preprint":false},{"year":2018,"finding":"In platelets, MEKK3 is required for agonist-induced activation of ERK1/2 and JNK2 (but not p38 or ERK5) and integrin αIIbβ3-mediated inside-out signaling; megakaryocyte/platelet-specific MEKK3 deletion impairs aggregation, degranulation, and in vivo thrombus formation.","method":"Megakaryocyte/platelet-specific MEKK3 knockout mice, MAPK activation assays, platelet aggregation, FeCl3 carotid injury model","journal":"Blood advances","confidence":"High","confidence_rationale":"Tier 2 / Moderate — cell-type-specific conditional KO, pathway-selective signaling analysis, in vivo thrombosis model, single lab","pmids":["29941457"],"is_preprint":false},{"year":2019,"finding":"MEKK3 knockout reduces YAP/TAZ promoter recruitment and target gene expression, and inhibits EMT, cell migration, 3D colony formation, and cancer stem cell populations in pancreatic cancer cells; MEKK3 loss reduces tumor growth in vivo.","method":"CRISPR/Cas9 MEKK3 knockout in pancreatic cancer cell lines, YAP/TAZ promoter chromatin immunoprecipitation, in vivo xenograft","journal":"Anticancer research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — KO phenotype with YAP/TAZ readout but no direct phosphorylation or binding mechanism shown, single lab","pmids":["29599309"],"is_preprint":false},{"year":2016,"finding":"Mekk3 heterozygosity (loss of one allele) prevents CCM lesion formation in Ccm2-deficient neonatal mice, as quantified by micro-CT imaging, providing genetic evidence that MEKK3 is the downstream effector of CCM2 loss.","method":"Micro-CT quantification of CCM lesions in Ccm2-/Mekk3+/- compound mutant mice","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative genetic epistasis in mouse model, single lab","pmids":["27513872"],"is_preprint":false},{"year":2034,"finding":"HDAC4 prevents p62-dependent autophagic degradation of MEKK3 by inhibiting MEF2A-driven transcription of ATG4B, thereby activating p38 MAPK signaling; downstream transcription factor USF1 forms a positive feedback loop by enhancing HDAC4 expression.","method":"ChIP, dual-luciferase reporter, immunofluorescence, western blot in gastric cancer cells","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assay mechanistically links HDAC4-ATG4B-MEKK3 axis, single lab","pmids":["35637410"],"is_preprint":false}],"current_model":"MAP3K3/MEKK3 is a serine/threonine MAP kinase kinase kinase that functions as a central signaling hub: it is recruited to TNF-R1 by RIP and to IL-1R/TLR complexes via TRAF6/p62 scaffolds to directly phosphorylate and activate IKK (triggering NF-κB), and it activates the ERK5 pathway through PB1-domain-mediated interaction with MEK5, the p38 pathway through the OSM/Rac scaffold and MKK3, and JNK; its activity is controlled by autophosphorylation at Ser526, by phosphorylation at Thr294 and Thr516/Ser520, by PP2A-mediated dephosphorylation, and by physical restraint through CCM2/CCM2L (whose HHD domain directly contacts MEKK3's N-terminus) in a complex that recruits STK24/25 to limit constitutive MEKK3 activity, such that CCM complex loss releases MEKK3 to drive KLF2/4 expression and CCM pathogenesis; additional substrates include GLI1, LATS1/2, YAP (pSer405), IRF7, and p47phox (pSer208 in myeloid cells), placing MEKK3 at the intersection of inflammatory, mechanosensory, developmental, and stress-response signaling."},"narrative":{"mechanistic_narrative":"MAP3K3 (MEKK3) is a serine/threonine MAP kinase kinase kinase that functions as a central signaling hub coupling inflammatory, mechanosensory, developmental, and stress inputs to NF-κB and MAPK output cascades [PMID:11429546, PMID:14661019, PMID:10700190]. In receptor-proximal inflammatory signaling, MEKK3 is recruited specifically to the TNF-R1 complex by RIP, where it phosphorylates and activates the IKK complex to drive NF-κB [PMID:11429546, PMID:15572679], and downstream of IL-1R/TLR it is brought into TRAF6-containing complexes via PB1-domain scaffolds including p62 and NBR1 to mediate a TAK1-independent branch of NF-κB and JNK/p38 activation marked by IKKγ phosphorylation [PMID:14661019, PMID:17197697, PMID:16737960, PMID:19903815, PMID:25043814]. Through PB1–PB1 heterodimerization with MEK5 it activates the ERK5 cascade [PMID:10593883, PMID:12912994, PMID:17985933], and it nucleates a Rac–OSM–MEKK3–MKK3 scaffold to drive p38 activation in response to osmotic and other stresses [PMID:14634666, PMID:16684924]. MEKK3 kinase activity is gated by activation-loop phosphorylation at Ser526 and at Thr516/Ser520, by inhibitory Thr294 phosphorylation that recruits 14-3-3, and by PP2A-mediated dephosphorylation, with Hsp90 maintaining protein stability [PMID:16407301, PMID:18308725, PMID:20448038, PMID:20068038, PMID:19560753]. A defining regulatory axis is physical restraint by the CCM complex: CCM2 directly binds the MEKK3 N-terminus through its harmonin homology domain and, together with CCM2L and recruited STK24/25, suppresses constitutive MEKK3 activity, so that CCM loss releases MEKK3 to elevate KLF2/KLF4 and ADAMTS expression and drive cerebral cavernous malformation and cardiovascular defects [PMID:26235885, PMID:27027284, PMID:25625206, PMID:26540726, PMID:36692953, PMID:27513872]. Endothelial MEKK3 thereby integrates hemodynamic and inflammatory cues—via PIEZO1/CaMKII upstream input—to control vascular remodeling and endothelial-to-hematopoietic transition [PMID:30732528, PMID:35883633, PMID:34911761, PMID:35245372]. MEKK3 is genetically required for embryonic blood vessel development [PMID:10700190], and additional direct substrates extend its reach into Hippo/YAP signaling (LATS1/2, YAP Ser405/Ser405) [PMID:33571521, PMID:38622197], Hedgehog signaling (GLI1) [PMID:29662197], and myeloid ROS production (p47phox Ser208) [PMID:33910977].","teleology":[{"year":1999,"claim":"Established MEKK3 as a direct upstream activator of the ERK5 cascade, defining its first concrete MAPK output.","evidence":"Yeast two-hybrid, co-IP, dominant-active overexpression, and kinase assays linking MEKK3 to MEK5/ERK5","pmids":["10593883"],"confidence":"High","gaps":["Did not define the interaction interface","Physiological stimulus context unresolved at this stage"]},{"year":2000,"claim":"Genetic knockout placed MEKK3 as essential for embryonic angiogenesis, linking the kinase to p38-MEF2C cardiovascular development.","evidence":"Map3k3-/- mouse embryos with vascular phenotyping and pathway analysis","pmids":["10700190"],"confidence":"High","gaps":["Cell-type-autonomous requirement not separated from systemic effect","Did not identify upstream receptors driving the developmental signal"]},{"year":2001,"claim":"Defined MEKK3 as a required transducer of TNF-induced IKK/NF-κB signaling downstream of RIP, establishing its inflammatory role.","evidence":"MEKK3-deficient fibroblasts, co-IP with RIP, in vitro IKK phosphorylation","pmids":["11429546"],"confidence":"High","gaps":["Mechanism of RIP-MEKK3 recruitment specificity not yet resolved","Relationship to TAK1 in the same pathway unclear"]},{"year":2003,"claim":"Mapped the structural and scaffolding basis of MEKK3 output: PB1-domain heterodimerization with MEK5 for ERK5, and a Rac-OSM-MKK3 actin-associated scaffold for stress-induced p38.","evidence":"PB1 binding/mutagenesis, RNAi epistasis, FRET, and live-cell imaging across two studies","pmids":["12912994","14634666"],"confidence":"High","gaps":["Did not establish how scaffold assembly is regulated by stimulus","Stoichiometry of the multi-protein complexes not defined"]},{"year":2003,"claim":"Placed MEKK3 downstream of MyD88-IRAK-TRAF6 in IL-1R/TLR signaling, broadening its receptor repertoire beyond TNF.","evidence":"MEKK3-deficient MEFs, co-IP with TRAF6, pathway-specific NF-κB/JNK/p38 readouts","pmids":["14661019"],"confidence":"High","gaps":["Did not resolve the TRAF6-MEKK3 connection at domain resolution","ERK independence noted but mechanism unexplained"]},{"year":2004,"claim":"Demonstrated receptor-recruitment specificity: RIP recruits MEKK3 (not MEKK2 or NEMO) to TNF-R1, explaining the kinase's selective requirement.","evidence":"Reconstitution of RIP-deficient Jurkat cells with MEKK3-DD fusion vs. controls","pmids":["15572679"],"confidence":"High","gaps":["Did not define structural basis of RIP-MEKK3 specificity","TRAF7 interaction context (parallel finding) not integrated"]},{"year":2006,"claim":"Resolved a TAK1-independent branch of NF-κB signaling driven by MEKK3, characterized by IKKγ phosphorylation and non-degradative IκBα modification, distinguishing two parallel inflammatory routes.","evidence":"IRAK mutation reconstitution, TAK1-/- and MEKK3-/- MEFs, IKK subunit-specific readouts, TLR8 stimulation","pmids":["17197697","16737960"],"confidence":"High","gaps":["Physiological discrimination of TAK1 vs MEKK3 branch usage incomplete","Biochemical basis of IKKγ-targeted activation not fully defined"]},{"year":2006,"claim":"Defined activation-loop autophosphorylation at Ser526 as essential for kinase activity and identified PP2A and 14-3-3 as opposing regulators, establishing the core activity switch.","evidence":"Mutagenesis, phospho-specific antibody, in vitro kinase assay, PP2A inhibitor studies","pmids":["16407301"],"confidence":"High","gaps":["Did not place Ser526 control within a receptor-proximal kinetic scheme","Other activation-loop sites (Thr516/Ser520) not yet known"]},{"year":2010,"claim":"Completed the activation-loop model by showing Thr516/Ser520 phosphorylation is required for IKKβ/NF-κB activation and is reversed by PP2Acβ to terminate signaling.","evidence":"Alanine/aspartate mutagenesis, IKKβ phosphorylation assays, PP2Ac co-IP and knockdown","pmids":["20068038","20448038"],"confidence":"High","gaps":["Interplay between Ser526 and Thr516/Ser520 control not unified","Stimulus-specific phosphatase recruitment not detailed"]},{"year":2008,"claim":"Identified inhibitory Thr294 phosphorylation/14-3-3 binding as a brake released by TNFα/LPS, and characterized TAK1-MEKK3 mutual regulation maintaining basal NF-κB tone.","evidence":"Phospho-specific antibody and T294A mutagenesis; TAP-MS, FRET, and reporter assays for TAK1-MEKK3 complex","pmids":["18308725","18206350","16260783"],"confidence":"Medium","gaps":["TAK1-MEKK3 regulatory model rests on single-lab data","Phosphatase responsible for Thr294 dephosphorylation not identified"]},{"year":2009,"claim":"Connected MEKK3 to PB1-scaffold adaptors and chaperone-dependent stability, and extended its role to GPCR (LPA) inflammatory signaling.","evidence":"p62 PB1 binding/co-localization, Hsp90 co-IP/geldanamycin half-life assays, MEKK3-/- vs TAK1-/- MEFs for LPA-IKK","pmids":["19903815","19560753","19465115"],"confidence":"High","gaps":["Hsp90 dependence shown pharmacologically; the cognate E3 ligase unidentified","GPCR-to-MEKK3 coupling mechanism not resolved"]},{"year":2015,"claim":"Revealed the structural basis of CCM-mediated restraint: CCM2's HHD directly contacts the MEKK3 N-terminus, and disrupting this interaction phenocopies endothelial MEKK3 loss, defining the CCM-MEKK3 axis.","evidence":"2.35 Å co-crystal structure, inducible endothelial Mekk3 KO, Rho-ROCK analysis, and CCM2/CCM2L binding/MEK5 phosphorylation assays with zebrafish rescue","pmids":["26235885","26540726"],"confidence":"High","gaps":["Did not establish how upstream signals trigger MEKK3 release from CCM restraint","Quantitative kinetics of the restraint not defined"]},{"year":2016,"claim":"Established gain of MEKK3 signaling as causal for CCM disease, with KLF2/KLF4 as the key downstream effectors, validated genetically and in human tissue.","evidence":"Neonatal CCM mouse models, endothelial conditional KO, human CCM tissue, Mekk3 heterozygosity rescue by micro-CT","pmids":["27027284","25625206","27513872"],"confidence":"High","gaps":["Full transcriptional program downstream of KLF2/4 incompletely mapped","Therapeutic targeting of the axis not addressed"]},{"year":2022,"claim":"Identified upstream mechanosensory inputs (PIEZO1/CaMKII and CDC42) and integrated inflammatory cues that feed the endothelial MEKK3-ERK5-KLF2/4 axis, and extended it to hemogenic endothelium specification.","evidence":"Endothelial Piezo1 and Cdc42 conditional KOs with epistasis, CaMKII-MEKK3 co-IP, Mekk3 KO with RUNX1+ EHT readouts","pmids":["35883633","30732528","35245372"],"confidence":"High","gaps":["Direct CaMKII phosphorylation site on MEKK3 not mapped","How distinct upstream cues converge on the same output unclear"]},{"year":2021,"claim":"Expanded the MEKK3 substrate landscape into Hippo/YAP, Hedgehog/GLI1, type I IFN (IRF7), myeloid ROS (p47phox), and arterial remodeling via TGFβ crosstalk.","evidence":"Direct in vitro phosphorylation assays, MS phosphosite mapping, conditional/knock-in mouse models across multiple studies","pmids":["33571521","29662197","33812250","33910977","34911761"],"confidence":"High","gaps":["Several substrate links rest on single-lab evidence","Context-dependence determining which substrate is engaged not resolved"]},{"year":2023,"claim":"Defined STK24/25 as CCM2-recruited kinases that limit constitutive MEKK3 activity, completing the molecular logic of CCM-complex restraint.","evidence":"Endothelial STK24/25 conditional KO and zebrafish domain-swap rescue with hybrid STK-CCM2 protein","pmids":["36692953"],"confidence":"High","gaps":["How STK24/25 kinase activity acts on MEKK3 mechanistically not defined","Single-lab in vivo system"]},{"year":2024,"claim":"Established a Hippo-independent YAP stabilization route in which MAP3K3 phosphorylates YAP Ser405 to block FBXW7 binding and lysosomal degradation, linking the kinase to cancer drug resistance.","evidence":"Phosphoproteomics, MAP3K3 depletion, in vitro phosphorylation, FBXW7 co-IP, lysosomal degradation assays","pmids":["38622197"],"confidence":"Medium","gaps":["Single-lab mechanism awaiting independent confirmation","Reconciliation with MEKK3's reported LATS-activating role unresolved"]},{"year":null,"claim":"How upstream stimuli select among MEKK3's many output cascades and substrates—and how the CCM/STRIPAK restraint is dynamically released in vivo—remains the central open question.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of stimulus-to-substrate routing","Dynamics of CCM/STRIPAK release in physiological settings undefined","Structural basis of activation-loop control in full-length kinase unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,35,37,45,46]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3,11,25,46]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[6,35]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,2,16]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12,1]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,3,16]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,9,10,28,40,46]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,5,6,7,30,43]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[1,21]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[32,45,52]}],"complexes":["Rac-OSM-MEKK3-MKK3 scaffold","MEKK3-MEK5 PB1 heterodimer","CCM (KRIT1/CCM2/CCM2L/PDCD10) complex","STRIPAK complex"],"partners":["MAP2K5","CCM2","RIPK1","TRAF6","SQSTM1","NBR1","TAK1","STK24"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99759","full_name":"Mitogen-activated protein kinase kinase kinase 3","aliases":["MAPK/ERK kinase kinase 3","MEK kinase 3","MEKK 3"],"length_aa":626,"mass_kda":70.9,"function":"Component of a protein kinase signal transduction cascade. Mediates activation of the NF-kappa-B, AP1 and DDIT3 transcriptional regulators","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q99759/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAP3K3","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAP3K3","total_profiled":1310},"omim":[{"mim_id":"621032","title":"CEREBRAL CAVERNOUS MALFORMATIONS 5; CCM5","url":"https://www.omim.org/entry/621032"},{"mim_id":"619538","title":"CEREBRAL CAVERNOUS MALFORMATIONS 4; CCM4","url":"https://www.omim.org/entry/619538"},{"mim_id":"610737","title":"KINASE SUPPRESSOR OF RAS 2; KSR2","url":"https://www.omim.org/entry/610737"},{"mim_id":"607929","title":"CCM2 SCAFFOLD PROTEIN; CCM2","url":"https://www.omim.org/entry/607929"},{"mim_id":"606692","title":"TNF RECEPTOR-ASSOCIATED FACTOR 7; TRAF7","url":"https://www.omim.org/entry/606692"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MAP3K3"},"hgnc":{"alias_symbol":["MAPKKK3"],"prev_symbol":["MEKK3"]},"alphafold":{"accession":"Q99759","domains":[{"cath_id":"3.10.20.90","chopping":"44-121","consensus_level":"high","plddt":88.5285,"start":44,"end":121},{"cath_id":"3.30.200.20","chopping":"361-443","consensus_level":"medium","plddt":91.98,"start":361,"end":443},{"cath_id":"1.10.510.10","chopping":"448-621","consensus_level":"medium","plddt":88.8001,"start":448,"end":621}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99759","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99759-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99759-F1-predicted_aligned_error_v6.png","plddt_mean":66.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAP3K3","jax_strain_url":"https://www.jax.org/strain/search?query=MAP3K3"},"sequence":{"accession":"Q99759","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99759.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99759/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99759"}},"corpus_meta":[{"pmid":"11429546","id":"PMC_11429546","title":"The essential role of MEKK3 in TNF-induced NF-kappaB activation.","date":"2001","source":"Nature immunology","url":"https://pubmed.ncbi.nlm.nih.gov/11429546","citation_count":346,"is_preprint":false},{"pmid":"14634666","id":"PMC_14634666","title":"Rac-MEKK3-MKK3 scaffolding for p38 MAPK activation during hyperosmotic shock.","date":"2003","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/14634666","citation_count":320,"is_preprint":false},{"pmid":"27027284","id":"PMC_27027284","title":"Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.","date":"2016","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/27027284","citation_count":256,"is_preprint":false},{"pmid":"14661019","id":"PMC_14661019","title":"Differential regulation of interleukin 1 receptor and Toll-like receptor signaling by MEKK3.","date":"2003","source":"Nature 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activation and NF-κB signaling; MEKK3 physically interacts with RIP and directly phosphorylates IKK, placing MEKK3 downstream of RIP and TRAF2 in the TNF receptor pathway.\",\n      \"method\": \"MEKK3-deficient fibroblasts, co-immunoprecipitation, in vitro kinase assay\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with defined phenotype, co-IP, and direct in vitro phosphorylation assay; replicated in subsequent studies\",\n      \"pmids\": [\"11429546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"MEKK3 forms a scaffold complex with the GTPase Rac, the adaptor protein OSM (osmosensing scaffold for MEKK3), and MKK3 on actin structures; this Rac-OSM-MEKK3-MKK3 complex is required for p38 MAPK activation in response to sorbitol-induced hyperosmolarity.\",\n      \"method\": \"RNAi knockdown, FRET, co-immunoprecipitation, live-cell imaging\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi epistasis, FRET-validated complex, multiple orthogonal methods in one study\",\n      \"pmids\": [\"14634666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"MEKK3 is an essential signal transducer downstream of the MyD88-IRAK-TRAF6 complex in IL-1R and TLR4 signaling; MEKK3 forms a complex with TRAF6 upon IL-1 or LPS stimulation and is required for NF-κB and JNK-p38 (but not ERK) activation.\",\n      \"method\": \"MEKK3-deficient MEFs, co-immunoprecipitation, cytokine measurement\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout, co-IP, pathway-specific readout, replicated across labs\",\n      \"pmids\": [\"14661019\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MEKK3 physically interacts with MEK5 and directly activates MEK5 to stimulate BMK1/ERK5 activity; a dominant-active MEKK3 is sufficient to activate ERK5 through MEK5, and MEKK3 kinase activity is required for growth-factor-mediated ERK5 activation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, dominant-active overexpression, kinase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — yeast two-hybrid plus co-IP plus in-cell kinase activation assay in single study\",\n      \"pmids\": [\"10593883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MEKK3 is essential for embryonic blood vessel development; Map3k3-/- mice die ~E11 with angiogenesis defects, and MEKK3 activates MEF2C through the p38 MAPK cascade to drive cardiovascular development.\",\n      \"method\": \"Gene knockout in mice, embryo phenotyping, signaling pathway analysis\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic KO with defined vascular phenotype and pathway placement via p38-MEF2C\",\n      \"pmids\": [\"10700190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CCM2 directly binds MEKK3 via its harmonin homology domain (HHD) interacting with the N-terminus of MEKK3; a co-crystal structure at 2.35 Å was determined. MEKK3 deficiency in endothelial cells causes intracranial hemorrhage partially dependent on Rho-ROCK signaling, and disruption of MEKK3:CCM2 interaction phenocopies this neurovascular leakage.\",\n      \"method\": \"Co-crystal structure (2.35 Å), inducible endothelial-specific Mekk3 knockout mice, Rho-ROCK pathway analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus genetic KO plus pathway rescue, multiple orthogonal methods\",\n      \"pmids\": [\"26235885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Gain of MEKK3 signaling in endothelial cells is causal for CCM disease; endothelial loss of KRIT1/CCM2/PDCD10 increases MEKK3 activity leading to elevated KLF2/KLF4 expression and Rho/ADAMTS activity. Endothelial-specific loss of Map3k3 or Klf2/Klf4 prevents lesion formation. A disease-causing CCM2 mutation abrogates the MEKK3 interaction without disrupting CCM complex formation.\",\n      \"method\": \"Neonatal mouse CCM model, endothelial-specific conditional knockout, human CCM tissue analysis, signaling pathway readouts\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genetic models, human tissue validation, mechanistic pathway delineation\",\n      \"pmids\": [\"27027284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Loss of CCM signaling in endocardial cells increases MEKK3 activity, which is both necessary and sufficient for elevated Klf2/Klf4 and Adamts4/5 expression causing cardiac jelly degradation; partial loss of MEKK3 rescues cardiac defects in CCM-deficient embryos.\",\n      \"method\": \"Conditional knockout mice, genetic epistasis (partial Mekk3 loss rescuing CCM deficiency), gene expression analysis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with clear rescue, multiple CCM alleles tested\",\n      \"pmids\": [\"25625206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The PB1 domains of MEKK3 (and MEKK2) interact with the PB1 domain of MEK5 to form heterodimers required for ERK5 pathway activation; deletion or mutation of the MEKK3 PB1 domain abolishes MEKK3-MEK5 complex formation and ERK5 activation.\",\n      \"method\": \"In vitro PB1 domain binding assay, co-immunoprecipitation, dominant-negative PB1 domain expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro binding, co-IP, and dominant-negative functional assay; mutagenesis of PB1 domain\",\n      \"pmids\": [\"12912994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Two IL-1-mediated NF-κB activation pathways bifurcate at IRAK modification: a TAK1-dependent pathway activating IKKα/β and a MEKK3-dependent (TAK1-independent) pathway that involves IKKγ phosphorylation and IKKα activation, resulting in IκBα phosphorylation without degradation.\",\n      \"method\": \"IRAK mutation analysis in reconstituted cells, TAK1-/- and MEKK3-/- MEFs, IKK subunit-specific readouts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple KO cell lines, pathway bifurcation with orthogonal IKK subunit readouts, replicated concept in multiple papers\",\n      \"pmids\": [\"17197697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TLR8-mediated NF-κB and JNK activation are completely abolished in MEKK3-/- MEFs but only moderately reduced in TAK1-/- MEFs; TLR8 signals through a MEKK3-dependent pathway involving IKKγ phosphorylation rather than IKKα/β phosphorylation.\",\n      \"method\": \"MEKK3-/- and TAK1-/- MEFs, IKK complex phosphorylation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout cells, parallel pathway comparison with multiple TLR stimuli\",\n      \"pmids\": [\"16737960\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Phosphorylation of MEKK3 at Ser526 within the T-loop activation loop is required for MEKK3 kinase activity (NF-κB, ERK, JNK, p38 activation); Ser526 is autophosphorylated, regulated by protein phosphatase 2A (PP2A), and association with 14-3-3 at pSer526 prevents dephosphorylation.\",\n      \"method\": \"Alanine/phosphomimetic mutagenesis, phospho-specific antibody, in vitro kinase assay with MKK6, PP2A inhibitor studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis, in vitro kinase assay, phospho-specific antibody, phosphatase identification\",\n      \"pmids\": [\"16407301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The rear-end acidic cluster of the p62/sequestosome-1 PB1 domain binds the front-end basic region of the MEKK3 PB1 domain; the p62-MEKK3 complex co-localizes in cytoplasmic speckles, recruits TRAF6, and is required for TRAF6-regulated NF-κB activation downstream of IL-1.\",\n      \"method\": \"PB1 domain binding assays, co-localization microscopy, shRNA knockdown, NF-κB reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific binding mapped, co-localization, shRNA loss-of-function with functional NF-κB readout\",\n      \"pmids\": [\"19903815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TRAF7 specifically interacts with MEKK3 and potentiates MEKK3-mediated AP1 and CHOP activation; depletion of TRAF7 by antisense RNA inhibits MEKK3-mediated AP1 and CHOP activation.\",\n      \"method\": \"Co-immunoprecipitation, antisense RNA knockdown, reporter gene assays, domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and antisense knockdown with reporter readout, single lab\",\n      \"pmids\": [\"15001576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TAK1 is recruited to the TNF-R1 complex in a RIP-dependent manner and forms a functional complex with MEKK3; TAK1 regulates autophosphorylation of MEKK3 in a TAK1-kinase-activity-dependent manner, requiring TAB1 for TAK1 activation and subsequent MEKK3 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, receptor complex isolation, dominant-negative TAK1, TAB1 co-expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, kinase-dead mutant analysis, single lab\",\n      \"pmids\": [\"16260783\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MEKK3 and TAK1 form a complex containing non-phosphorylated forms of both kinases; non-phosphorylated TAK1 inhibits MEKK3 phosphorylation and NF-κB signaling; TAB1-mediated TAK1 autophosphorylation reverses this inhibition, providing homeostatic regulation of basal NF-κB levels.\",\n      \"method\": \"Tandem affinity purification, FRET, co-immunoprecipitation, NF-κB reporter, TAK1-deficient MEFs\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TAP-MS identification, FRET confirmation, functional rescue, single lab\",\n      \"pmids\": [\"18206350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"RIP functions to specifically recruit MEKK3 to the TNF-α receptor complex; a MEKK3-RIP death domain fusion (MEKK3-DD) fully restores TNF-α-induced NF-κB activation in RIP-deficient cells, while MEKK2-DD or NEMO-DD cannot, demonstrating MEKK3-specific requirement.\",\n      \"method\": \"Reconstitution of RIP-deficient Jurkat cells with fusion proteins, co-immunoprecipitation, NF-κB activation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reconstitution experiment in KO cells, specificity controls with MEKK2 and NEMO, clear epistatic placement\",\n      \"pmids\": [\"15572679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"MEKK3 interacts with PA28γ (a proteasome activator subunit) but not PA28α; the PA28γ-binding domain of MEKK3 is in its N-terminal regulatory domain (aa 1-178); in vitro assays showed PA28γ is a MEKK3 substrate; MEKK3 expression increases PA28γ protein levels in a kinase-activity-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, domain deletion mapping\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay and co-IP, single lab, single study\",\n      \"pmids\": [\"12650640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Activation of MEKK3 induces G2 cell cycle arrest dependent on p38α/β2 signaling, associated with down-regulation of cyclin A and B1 expression and inhibition of CDK1/CDK2 activity; p38 inhibitor SB203580 partially rescues the G2 arrest.\",\n      \"method\": \"Conditional MEKK3:ER* activation, cell cycle synchronization, CDK activity assays, p38 inhibitor\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional activation system, pharmacological epistasis with p38 inhibitor, single lab\",\n      \"pmids\": [\"12444545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NMR solution structure of MEKK3 PB1 domain reveals prolyl isomerization at Gln38-Pro39 producing two structural isomers; the MEKK3 PB1 domain binds MEK5 PB1 with Kd ~10^-8 M; Lys7 and Arg5 in the basic cluster are key residues for this interaction.\",\n      \"method\": \"NMR structure determination, mutagenesis, binding affinity measurement\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with mutagenesis and quantitative binding data, single lab\",\n      \"pmids\": [\"17985933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"MEKK3 is essential for angiotensin II-induced calcineurin/NFAT activation in cardiac myocytes; MEKK3-deficient MEFs fail to activate NFAT in response to angiotensin II; restoring MEKK3 rescues NFAT activation; MEKK3 is phosphorylated in response to angiotensin II and functions downstream of the AT1 receptor.\",\n      \"method\": \"MEKK3-/- MEFs, siRNA knockdown, dominant-negative MEKK3, NFAT reporter, reconstitution\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO MEFs, reconstitution, dominant-negative, multiple epistasis approaches, single lab\",\n      \"pmids\": [\"16126726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MEKK3 is required for hypertonic stress-induced p38 activation and downstream TonE-driven gene (BGT1) induction in kidney cells; siRNA-mediated MEKK3 depletion downregulates p38 activity and reduces BGT1 expression.\",\n      \"method\": \"Stable transfection of activated MEKK3, siRNA knockdown, TonE-luciferase reporter\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function, pathway-specific reporter, single lab\",\n      \"pmids\": [\"16684924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Phosphorylation of MEKK3 at Thr294 promotes 14-3-3 binding to negatively regulate MEKK3; TNFα or LPS stimulation causes rapid dephosphorylation of Thr294 and loss of 14-3-3 association, correlating with MEKK3 pathway activation; Thr294 phosphorylation does not affect Ser526 phosphorylation.\",\n      \"method\": \"Phospho-specific antibody, mutagenesis (T294A), co-immunoprecipitation with 14-3-3, NF-κB reporter\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-specific antibody, mutagenesis, and functional readout, single lab\",\n      \"pmids\": [\"18308725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"MEKK3 is phosphorylated at Ser166 and Ser337 in response to TNF, arsenite, forskolin, and serum; 14-3-3 proteins interact with MEKK3; however, Ser166 and Ser337 phosphorylation are not required for 14-3-3 association or MEKK3-dependent ERK and JNK activity.\",\n      \"method\": \"LC-MS phosphopeptide mapping, phospho-specific antibodies, co-precipitation with 14-3-3\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based phosphosite mapping with phospho-specific antibodies, negative result on function, single lab\",\n      \"pmids\": [\"12392720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Protein phosphatase 2A (PP2A) acts as a MEKK3 phosphatase; PP2Acβ associates with phosphorylated MEKK3 in a transient LPA-induced manner, dephosphorylates MEKK3 at Thr516 and Ser520, and terminates MEKK3-mediated IKKβ/NF-κB activation.\",\n      \"method\": \"Functional genomic screen, co-immunoprecipitation, overexpression/knockdown of PP2Ac, IKKβ phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional genomic identification, co-IP, siRNA knockdown with pathway readout, single lab\",\n      \"pmids\": [\"20448038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Phosphorylation of MEKK3 at Thr516 and Ser520 within the kinase activation loop is required for MEKK3-mediated IKKβ/NF-κB activation; alanine substitution abolishes activity, while acidic substitution renders constitutive activity.\",\n      \"method\": \"Alanine/aspartate mutagenesis, IKKβ phosphorylation assay, NF-κB reporter\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — activation loop mutagenesis with both loss- and gain-of-function mutations, in vitro kinase readout\",\n      \"pmids\": [\"20068038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MEKK3 is required for LPA-induced IKK-NF-κB activation and cytokine (IL-6, MIP-2) production via GPCR signaling; this is MEKK3-specific and TAK1-independent, as shown by selective loss in MEKK3-/- but not TAK1-/- MEFs.\",\n      \"method\": \"MEKK3-/- and TAK1-/- MEFs, IKK activation assay, cytokine ELISA\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — parallel genetic KO comparison, pathway-specific readout, single lab\",\n      \"pmids\": [\"19465115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MEKK2 and MEKK3 negatively regulate TGF-β-mediated Th cell differentiation by phosphorylating SMAD2 and SMAD3 at their linker regions; T cell-specific Map3k2/Map3k3 double knockout mice show accumulation of Treg and Th17 cells consistent with enhanced TGF-β responses.\",\n      \"method\": \"T cell conditional knockout mice, in vitro differentiation assays, SMAD phosphorylation analysis\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined cellular phenotype and SMAD phosphorylation mechanistic link, single lab\",\n      \"pmids\": [\"21333552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MEKK3 is required for TCR-induced ERK1/2, JNK, and p38 activation and IFN-γ production in CD4+ T cells; TCR-mediated MEKK3 activation requires Rac1/2, as shown in Mekk3 T cell conditional knockout mice.\",\n      \"method\": \"T cell-specific conditional Mekk3 knockout mice, cytokine measurement, MAPK activation assays, bacterial infection model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional KO, pathway readouts, Rac1/2 upstream requirement, single lab\",\n      \"pmids\": [\"21471448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NBR1 interacts with MEKK3 via PB1 domain interaction; the NBR1-MEKK3 complex is required for JNK activation in macrophages and drives adipose tissue inflammation in obesity; myeloid-specific NBR1 inactivation impairs JNK signaling and macrophage inflammatory function.\",\n      \"method\": \"PB1 domain interaction assay, myeloid-specific conditional KO mice, JNK activation assay, metabolic phenotyping\",\n      \"journal\": \"Cell metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-level interaction, conditional KO in myeloid cells, in vivo phenotype, single lab\",\n      \"pmids\": [\"25043814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MEKK3 kinase activity is required for and sufficient to initiate TGFβ2-dependent epithelial-to-mesenchymal transition (EMT) during endocardial cushion morphogenesis; kinase-inactive MEKK3 blocks EMT while constitutively active MEKK3 triggers EMT in normally non-EMT ventricular endocardium.\",\n      \"method\": \"In vitro cushion EMT assay, kinase-inactive and constitutively active MEKK3 constructs, gene expression analysis\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function in an in vitro developmental assay, TGFβ2-dependent mechanism identified, single lab\",\n      \"pmids\": [\"19008476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MEKK3 is required for lymphopenia-induced T cell proliferation and homeostatic survival; MEKK3-deficient T cells show attenuated ERK1/2 (but not p38) activation during homeostatic proliferation, while antigen-induced proliferation is unaffected.\",\n      \"method\": \"T cell-specific MEKK3 conditional KO mice, adoptive transfer, MAPK activation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with adoptive transfer, ERK-specific deficit identified, single lab\",\n      \"pmids\": [\"19265138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MEKK3-MEK5-ERK5 signaling pathway is required for basal lysosome-mediated mitochondrial degradation; genetic or pharmacological inhibition of MEKK3-MEK5-ERK5 increases mitochondrial content by reducing basal mitophagic degradation without affecting bulk autophagy or damage-induced mitophagy.\",\n      \"method\": \"Genetic inhibition (CRISPR), pharmacological inhibition, mitochondrial content assays, lysosomal inhibition studies\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological approaches, selective pathway specificity tested, single lab\",\n      \"pmids\": [\"33101709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CDC42 deletion in endothelial cells causes increased MEKK3-MEK5-ERK5-KLF2/4 signaling and CCM-like vascular malformations; CDC42 interacts with CCM proteins and CCM3 promotes CDC42 activity; Klf4 co-inactivation reduces severity of Cdc42-mutant vascular malformations.\",\n      \"method\": \"Endothelial-specific inducible Cdc42 knockout mice, genetic epistasis (Klf4 co-deletion), MEKK3 pathway signaling assays\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO, genetic rescue, pathway mechanistic analysis, multiple alleles tested\",\n      \"pmids\": [\"30732528\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Both CCM2 and CCM2L bind MEKK3 in a complex with CCM1 and prevent MEKK3 activation and its ability to phosphorylate MEK5; ccm2l/ccm2 double knockdown in zebrafish is rescued by mekk3 knockdown, confirming CCM2L-CCM2 co-regulation of MEKK3 in vivo.\",\n      \"method\": \"In vitro binding assays, MEK5 phosphorylation assay, zebrafish morpholino knockdown with genetic rescue\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay, co-IP, in vivo rescue epistasis in zebrafish, single lab\",\n      \"pmids\": [\"26540726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MEKK3 inhibits GLI1 transcriptional activity and oncogenic function by phosphorylating multiple Ser/Thr sites on GLI1, reducing GLI1 protein stability, DNA-binding ability, and increasing GLI1-SUFU association; MEKK3 mediates FGF2-dependent inhibition of Hedgehog signaling.\",\n      \"method\": \"In vitro kinase assay, GLI1 mutagenesis, protein stability assays, SUFU co-immunoprecipitation, medulloblastoma cell proliferation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct phosphorylation assay with multiple mechanistic readouts, single lab\",\n      \"pmids\": [\"29662197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MEKK3 forms a complex with WDR62 to promote JNK signaling synergistically in neural progenitor cells; MEKK3 positively regulates WDR62 protein stability in the developing brain; WDR62 is negatively regulated by T1053 phosphorylation leading to FBW7-mediated proteasomal degradation.\",\n      \"method\": \"Conditional Mekk3 knockout mice, WDR62 stability assays, JNK signaling analysis, FBW7 co-immunoprecipitation\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO, protein stability mechanism, phosphorylation site identified, single lab\",\n      \"pmids\": [\"30566428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MEKK3 and MEKK2 phosphorylate LATS1/2 and YAP/TAZ to activate Hippo signaling in response to TNF and other stimuli; STRIPAK complex associates with MEKK3 via CCM2 and CCM3 to inactivate MEKK3, and upstream Hippo signals trigger MEKK3 dissociation from STRIPAK to release MEKK3 activity.\",\n      \"method\": \"Co-immunoprecipitation, kinase assay (LATS1/2 and YAP/TAZ phosphorylation), STRIPAK complex analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct phosphorylation assays, complex characterization by co-IP, single lab\",\n      \"pmids\": [\"33571521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Hsp90 interacts with MEKK3 and acts as its molecular chaperone to maintain MEKK3 stability; Hsp90 inhibitors (geldanamycin) shorten MEKK3 half-life and induce MEKK3 ubiquitination and proteasomal degradation.\",\n      \"method\": \"Co-immunoprecipitation, Hsp90 RNAi, geldanamycin treatment, protein stability/half-life assay, ubiquitination assay\",\n      \"journal\": \"Cellular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, pharmacological and siRNA approaches, proteasome-dependent degradation shown, single lab\",\n      \"pmids\": [\"19560753\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PIEZO1 mechanosensitive channel activation by shear stress leads to calcium influx that activates CaMKII; CaMKII interacts with and activates MEKK3, promoting MEKK3/MEK5/ERK5 signaling and KLF2/4 transcription in endothelial cells.\",\n      \"method\": \"Endothelial-specific Piezo1 knockout mice, CaMKII-MEKK3 co-immunoprecipitation, ERK5/KLF2/4 pathway readouts\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO, co-IP of CaMKII-MEKK3, pathway-specific readouts, single lab\",\n      \"pmids\": [\"35883633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MEKK3 activates IRF7 through direct interaction and phosphorylation of IRF7 at multiple sites in response to TLR7/9 activation; endogenous MEKK3 binds and phosphorylates IRF7 after TLR9 activation by CpG DNA, triggering type I IFN induction.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphorylation assay, MEKK3 knockdown in vivo, TLR7/9 ligand stimulation\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct phosphorylation, co-IP, and in vivo knockdown, single lab\",\n      \"pmids\": [\"33812250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MEKK3-TGFβ crosstalk controls inward arterial remodeling; endothelial-specific MEKK3 deletion causes inward remodeling of pulmonary and systemic arteries, spontaneous hypertension, and accelerated atherosclerosis; molecular analysis reveals MEKK3 deletion activates TGFβR1-Smad2/3 signaling, and endothelial TGFβR1 knockout prevents this remodeling.\",\n      \"method\": \"Adult endothelial-specific Mekk3 knockout, endothelial TGFβR1 knockout epistasis, vascular phenotyping, Smad2/3 phosphorylation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with double-KO genetic rescue epistasis, in vitro and in vivo TGFβ pathway analysis\",\n      \"pmids\": [\"34911761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RAGE binds MKK3 via C-terminal amino acids 2-5, and this interaction is required for assembly of the MEKK3-MKK3-p38 signaling module and activation of p38 MAPK/NF-κB signaling; specific RAGE ctRAGE R2A-K3A-R4A-Q5A mutation suppresses neuronal damage and improves synaptic plasticity in diabetic mice.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down, point mutagenesis, electrophysiology, behavioral assays in db/db mice\",\n      \"journal\": \"Aging cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, GST pull-down, and in vivo mutagenesis rescue, single lab\",\n      \"pmids\": [\"35080104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MEKK3 and KLF2/4 signaling in endothelial cells integrates hemodynamic (shear/fluid forces) and inflammatory signals (LPS, IFN-γ) to specify RUNX1+ hemogenic endothelial cells and drive endothelial-to-hematopoietic transition (EHT) in the embryo.\",\n      \"method\": \"Endothelial-specific Mekk3 conditional KO mice, LPS and IFN-γ stimulation, catecholamine stimulation, RUNX1+ cell quantification\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endothelial conditional KO with defined hematopoietic phenotype and multiple stimulus conditions, single lab\",\n      \"pmids\": [\"35245372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"STK24/25 (CCM3-interacting kinases) limit constitutive MEKK3 activity by being recruited to MEKK3 via CCM2 as adaptor; loss of STK24/25 in endothelial cells causes MEKK3 activation and CCM lesion formation; a hybrid STK kinase domain–CCM2 MEKK3-interacting domain fusion rescues CCM loss-of-function in zebrafish.\",\n      \"method\": \"Endothelial-specific STK24/25 conditional KO mice, zebrafish genetic rescue with hybrid protein, CCM lesion quantification\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO, domain-swap rescue in zebrafish, mechanistic pathway placement, single lab\",\n      \"pmids\": [\"36692953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MAP3K3 phosphorylates YAP at Ser405, preventing FBXW7 binding and thereby inhibiting p62-mediated lysosomal degradation of YAP; this stabilization mechanism is independent of canonical Hippo kinases and supports YAP-dependent drug resistance in melanoma and breast cancer.\",\n      \"method\": \"Mass spectrometry phosphoproteomics, MAP3K3 depletion (siRNA/CRISPR), in vitro phosphorylation, FBXW7 co-immunoprecipitation, lysosomal degradation assay\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based site identification, direct phosphorylation, E3 ligase binding assay, single lab\",\n      \"pmids\": [\"38622197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MAP3K2 and MAP3K3 mediate phosphorylation of NADPH oxidase 2 subunit p47phox at Ser208, promoting ROS formation in myeloid cells; pazopanib inhibits MAP3K2/MAP3K3 to reduce this phosphorylation; myeloid-specific MAP3K2/MAP3K3 double inactivation or p47phox S208A mutation attenuates acute lung injury.\",\n      \"method\": \"In vitro kinase assay (p47phox phosphorylation), myeloid-specific conditional KO, p47phox S208A knock-in mice, ALI models\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct substrate phosphorylation, genetic KO, knock-in mutagenesis, multiple model systems\",\n      \"pmids\": [\"33910977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"MEKK3 is required for angiopoietin-1/Tie2-induced p38 and ERK5 activation in endothelial cells; MEKK3-deficient endothelial cells show defects in proliferation, apoptosis, and interaction with myocardium.\",\n      \"method\": \"Mekk3-deficient endothelial cells from embryos, Ang1 stimulation, p38 and ERK5 activation assays\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO endothelial cells with defined signaling defect, single lab\",\n      \"pmids\": [\"17687003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"H. pylori induces transient IKK complex activation via mutual control by MEKK3 and TAK1; TAK1 transiently interacts with TRAF6 and undergoes autophosphorylation and K63-linked ubiquitination; MEKK3 and TAK1 synergize to activate the IKK complex in a T4SS-dependent, CagA-independent manner.\",\n      \"method\": \"Co-immunoprecipitation, H. pylori infection of gastric epithelial cells, TAK1/MEKK3 pathway analysis\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP-based interaction and ubiquitination analysis, bacterial infection model, single lab\",\n      \"pmids\": [\"24418622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In platelets, MEKK3 is required for agonist-induced activation of ERK1/2 and JNK2 (but not p38 or ERK5) and integrin αIIbβ3-mediated inside-out signaling; megakaryocyte/platelet-specific MEKK3 deletion impairs aggregation, degranulation, and in vivo thrombus formation.\",\n      \"method\": \"Megakaryocyte/platelet-specific MEKK3 knockout mice, MAPK activation assays, platelet aggregation, FeCl3 carotid injury model\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-type-specific conditional KO, pathway-selective signaling analysis, in vivo thrombosis model, single lab\",\n      \"pmids\": [\"29941457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MEKK3 knockout reduces YAP/TAZ promoter recruitment and target gene expression, and inhibits EMT, cell migration, 3D colony formation, and cancer stem cell populations in pancreatic cancer cells; MEKK3 loss reduces tumor growth in vivo.\",\n      \"method\": \"CRISPR/Cas9 MEKK3 knockout in pancreatic cancer cell lines, YAP/TAZ promoter chromatin immunoprecipitation, in vivo xenograft\",\n      \"journal\": \"Anticancer research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — KO phenotype with YAP/TAZ readout but no direct phosphorylation or binding mechanism shown, single lab\",\n      \"pmids\": [\"29599309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mekk3 heterozygosity (loss of one allele) prevents CCM lesion formation in Ccm2-deficient neonatal mice, as quantified by micro-CT imaging, providing genetic evidence that MEKK3 is the downstream effector of CCM2 loss.\",\n      \"method\": \"Micro-CT quantification of CCM lesions in Ccm2-/Mekk3+/- compound mutant mice\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative genetic epistasis in mouse model, single lab\",\n      \"pmids\": [\"27513872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2034,\n      \"finding\": \"HDAC4 prevents p62-dependent autophagic degradation of MEKK3 by inhibiting MEF2A-driven transcription of ATG4B, thereby activating p38 MAPK signaling; downstream transcription factor USF1 forms a positive feedback loop by enhancing HDAC4 expression.\",\n      \"method\": \"ChIP, dual-luciferase reporter, immunofluorescence, western blot in gastric cancer cells\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assay mechanistically links HDAC4-ATG4B-MEKK3 axis, single lab\",\n      \"pmids\": [\"35637410\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAP3K3/MEKK3 is a serine/threonine MAP kinase kinase kinase that functions as a central signaling hub: it is recruited to TNF-R1 by RIP and to IL-1R/TLR complexes via TRAF6/p62 scaffolds to directly phosphorylate and activate IKK (triggering NF-κB), and it activates the ERK5 pathway through PB1-domain-mediated interaction with MEK5, the p38 pathway through the OSM/Rac scaffold and MKK3, and JNK; its activity is controlled by autophosphorylation at Ser526, by phosphorylation at Thr294 and Thr516/Ser520, by PP2A-mediated dephosphorylation, and by physical restraint through CCM2/CCM2L (whose HHD domain directly contacts MEKK3's N-terminus) in a complex that recruits STK24/25 to limit constitutive MEKK3 activity, such that CCM complex loss releases MEKK3 to drive KLF2/4 expression and CCM pathogenesis; additional substrates include GLI1, LATS1/2, YAP (pSer405), IRF7, and p47phox (pSer208 in myeloid cells), placing MEKK3 at the intersection of inflammatory, mechanosensory, developmental, and stress-response signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAP3K3 (MEKK3) is a serine/threonine MAP kinase kinase kinase that functions as a central signaling hub coupling inflammatory, mechanosensory, developmental, and stress inputs to NF-\\u03baB and MAPK output cascades [#0, #2, #4]. In receptor-proximal inflammatory signaling, MEKK3 is recruited specifically to the TNF-R1 complex by RIP, where it phosphorylates and activates the IKK complex to drive NF-\\u03baB [#0, #16], and downstream of IL-1R/TLR it is brought into TRAF6-containing complexes via PB1-domain scaffolds including p62 and NBR1 to mediate a TAK1-independent branch of NF-\\u03baB and JNK/p38 activation marked by IKK\\u03b3 phosphorylation [#2, #9, #10, #12, #29]. Through PB1\\u2013PB1 heterodimerization with MEK5 it activates the ERK5 cascade [#3, #8, #19], and it nucleates a Rac\\u2013OSM\\u2013MEKK3\\u2013MKK3 scaffold to drive p38 activation in response to osmotic and other stresses [#1, #21]. MEKK3 kinase activity is gated by activation-loop phosphorylation at Ser526 and at Thr516/Ser520, by inhibitory Thr294 phosphorylation that recruits 14-3-3, and by PP2A-mediated dephosphorylation, with Hsp90 maintaining protein stability [#11, #22, #24, #25, #38]. A defining regulatory axis is physical restraint by the CCM complex: CCM2 directly binds the MEKK3 N-terminus through its harmonin homology domain and, together with CCM2L and recruited STK24/25, suppresses constitutive MEKK3 activity, so that CCM loss releases MEKK3 to elevate KLF2/KLF4 and ADAMTS expression and drive cerebral cavernous malformation and cardiovascular defects [#5, #6, #7, #34, #44, #51]. Endothelial MEKK3 thereby integrates hemodynamic and inflammatory cues\\u2014via PIEZO1/CaMKII upstream input\\u2014to control vascular remodeling and endothelial-to-hematopoietic transition [#33, #39, #41, #43]. MEKK3 is genetically required for embryonic blood vessel development [#4], and additional direct substrates extend its reach into Hippo/YAP signaling (LATS1/2, YAP Ser405/Ser405) [#37, #45], Hedgehog signaling (GLI1) [#35], and myeloid ROS production (p47phox Ser208) [#46].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established MEKK3 as a direct upstream activator of the ERK5 cascade, defining its first concrete MAPK output.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, dominant-active overexpression, and kinase assays linking MEKK3 to MEK5/ERK5\",\n      \"pmids\": [\"10593883\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the interaction interface\", \"Physiological stimulus context unresolved at this stage\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Genetic knockout placed MEKK3 as essential for embryonic angiogenesis, linking the kinase to p38-MEF2C cardiovascular development.\",\n      \"evidence\": \"Map3k3-/- mouse embryos with vascular phenotyping and pathway analysis\",\n      \"pmids\": [\"10700190\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type-autonomous requirement not separated from systemic effect\", \"Did not identify upstream receptors driving the developmental signal\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined MEKK3 as a required transducer of TNF-induced IKK/NF-\\u03baB signaling downstream of RIP, establishing its inflammatory role.\",\n      \"evidence\": \"MEKK3-deficient fibroblasts, co-IP with RIP, in vitro IKK phosphorylation\",\n      \"pmids\": [\"11429546\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of RIP-MEKK3 recruitment specificity not yet resolved\", \"Relationship to TAK1 in the same pathway unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapped the structural and scaffolding basis of MEKK3 output: PB1-domain heterodimerization with MEK5 for ERK5, and a Rac-OSM-MKK3 actin-associated scaffold for stress-induced p38.\",\n      \"evidence\": \"PB1 binding/mutagenesis, RNAi epistasis, FRET, and live-cell imaging across two studies\",\n      \"pmids\": [\"12912994\", \"14634666\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish how scaffold assembly is regulated by stimulus\", \"Stoichiometry of the multi-protein complexes not defined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Placed MEKK3 downstream of MyD88-IRAK-TRAF6 in IL-1R/TLR signaling, broadening its receptor repertoire beyond TNF.\",\n      \"evidence\": \"MEKK3-deficient MEFs, co-IP with TRAF6, pathway-specific NF-\\u03baB/JNK/p38 readouts\",\n      \"pmids\": [\"14661019\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the TRAF6-MEKK3 connection at domain resolution\", \"ERK independence noted but mechanism unexplained\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated receptor-recruitment specificity: RIP recruits MEKK3 (not MEKK2 or NEMO) to TNF-R1, explaining the kinase's selective requirement.\",\n      \"evidence\": \"Reconstitution of RIP-deficient Jurkat cells with MEKK3-DD fusion vs. controls\",\n      \"pmids\": [\"15572679\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define structural basis of RIP-MEKK3 specificity\", \"TRAF7 interaction context (parallel finding) not integrated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved a TAK1-independent branch of NF-\\u03baB signaling driven by MEKK3, characterized by IKK\\u03b3 phosphorylation and non-degradative I\\u03baB\\u03b1 modification, distinguishing two parallel inflammatory routes.\",\n      \"evidence\": \"IRAK mutation reconstitution, TAK1-/- and MEKK3-/- MEFs, IKK subunit-specific readouts, TLR8 stimulation\",\n      \"pmids\": [\"17197697\", \"16737960\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological discrimination of TAK1 vs MEKK3 branch usage incomplete\", \"Biochemical basis of IKK\\u03b3-targeted activation not fully defined\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined activation-loop autophosphorylation at Ser526 as essential for kinase activity and identified PP2A and 14-3-3 as opposing regulators, establishing the core activity switch.\",\n      \"evidence\": \"Mutagenesis, phospho-specific antibody, in vitro kinase assay, PP2A inhibitor studies\",\n      \"pmids\": [\"16407301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not place Ser526 control within a receptor-proximal kinetic scheme\", \"Other activation-loop sites (Thr516/Ser520) not yet known\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Completed the activation-loop model by showing Thr516/Ser520 phosphorylation is required for IKK\\u03b2/NF-\\u03baB activation and is reversed by PP2Ac\\u03b2 to terminate signaling.\",\n      \"evidence\": \"Alanine/aspartate mutagenesis, IKK\\u03b2 phosphorylation assays, PP2Ac co-IP and knockdown\",\n      \"pmids\": [\"20068038\", \"20448038\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interplay between Ser526 and Thr516/Ser520 control not unified\", \"Stimulus-specific phosphatase recruitment not detailed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified inhibitory Thr294 phosphorylation/14-3-3 binding as a brake released by TNF\\u03b1/LPS, and characterized TAK1-MEKK3 mutual regulation maintaining basal NF-\\u03baB tone.\",\n      \"evidence\": \"Phospho-specific antibody and T294A mutagenesis; TAP-MS, FRET, and reporter assays for TAK1-MEKK3 complex\",\n      \"pmids\": [\"18308725\", \"18206350\", \"16260783\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"TAK1-MEKK3 regulatory model rests on single-lab data\", \"Phosphatase responsible for Thr294 dephosphorylation not identified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connected MEKK3 to PB1-scaffold adaptors and chaperone-dependent stability, and extended its role to GPCR (LPA) inflammatory signaling.\",\n      \"evidence\": \"p62 PB1 binding/co-localization, Hsp90 co-IP/geldanamycin half-life assays, MEKK3-/- vs TAK1-/- MEFs for LPA-IKK\",\n      \"pmids\": [\"19903815\", \"19560753\", \"19465115\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Hsp90 dependence shown pharmacologically; the cognate E3 ligase unidentified\", \"GPCR-to-MEKK3 coupling mechanism not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed the structural basis of CCM-mediated restraint: CCM2's HHD directly contacts the MEKK3 N-terminus, and disrupting this interaction phenocopies endothelial MEKK3 loss, defining the CCM-MEKK3 axis.\",\n      \"evidence\": \"2.35 \\u00c5 co-crystal structure, inducible endothelial Mekk3 KO, Rho-ROCK analysis, and CCM2/CCM2L binding/MEK5 phosphorylation assays with zebrafish rescue\",\n      \"pmids\": [\"26235885\", \"26540726\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish how upstream signals trigger MEKK3 release from CCM restraint\", \"Quantitative kinetics of the restraint not defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established gain of MEKK3 signaling as causal for CCM disease, with KLF2/KLF4 as the key downstream effectors, validated genetically and in human tissue.\",\n      \"evidence\": \"Neonatal CCM mouse models, endothelial conditional KO, human CCM tissue, Mekk3 heterozygosity rescue by micro-CT\",\n      \"pmids\": [\"27027284\", \"25625206\", \"27513872\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full transcriptional program downstream of KLF2/4 incompletely mapped\", \"Therapeutic targeting of the axis not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified upstream mechanosensory inputs (PIEZO1/CaMKII and CDC42) and integrated inflammatory cues that feed the endothelial MEKK3-ERK5-KLF2/4 axis, and extended it to hemogenic endothelium specification.\",\n      \"evidence\": \"Endothelial Piezo1 and Cdc42 conditional KOs with epistasis, CaMKII-MEKK3 co-IP, Mekk3 KO with RUNX1+ EHT readouts\",\n      \"pmids\": [\"35883633\", \"30732528\", \"35245372\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct CaMKII phosphorylation site on MEKK3 not mapped\", \"How distinct upstream cues converge on the same output unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Expanded the MEKK3 substrate landscape into Hippo/YAP, Hedgehog/GLI1, type I IFN (IRF7), myeloid ROS (p47phox), and arterial remodeling via TGF\\u03b2 crosstalk.\",\n      \"evidence\": \"Direct in vitro phosphorylation assays, MS phosphosite mapping, conditional/knock-in mouse models across multiple studies\",\n      \"pmids\": [\"33571521\", \"29662197\", \"33812250\", \"33910977\", \"34911761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Several substrate links rest on single-lab evidence\", \"Context-dependence determining which substrate is engaged not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined STK24/25 as CCM2-recruited kinases that limit constitutive MEKK3 activity, completing the molecular logic of CCM-complex restraint.\",\n      \"evidence\": \"Endothelial STK24/25 conditional KO and zebrafish domain-swap rescue with hybrid STK-CCM2 protein\",\n      \"pmids\": [\"36692953\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How STK24/25 kinase activity acts on MEKK3 mechanistically not defined\", \"Single-lab in vivo system\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a Hippo-independent YAP stabilization route in which MAP3K3 phosphorylates YAP Ser405 to block FBXW7 binding and lysosomal degradation, linking the kinase to cancer drug resistance.\",\n      \"evidence\": \"Phosphoproteomics, MAP3K3 depletion, in vitro phosphorylation, FBXW7 co-IP, lysosomal degradation assays\",\n      \"pmids\": [\"38622197\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab mechanism awaiting independent confirmation\", \"Reconciliation with MEKK3's reported LATS-activating role unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How upstream stimuli select among MEKK3's many output cascades and substrates\\u2014and how the CCM/STRIPAK restraint is dynamically released in vivo\\u2014remains the central open question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of stimulus-to-substrate routing\", \"Dynamics of CCM/STRIPAK release in physiological settings undefined\", \"Structural basis of activation-loop control in full-length kinase unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 35, 37, 45, 46]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3, 11, 25, 46]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [6, 35]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 2, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12, 1]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 3, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 9, 10, 28, 40, 46]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 5, 6, 7, 30, 43]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [1, 21]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [32, 45, 52]}\n    ],\n    \"complexes\": [\n      \"Rac-OSM-MEKK3-MKK3 scaffold\",\n      \"MEKK3-MEK5 PB1 heterodimer\",\n      \"CCM (KRIT1/CCM2/CCM2L/PDCD10) complex\",\n      \"STRIPAK complex\"\n    ],\n    \"partners\": [\n      \"MAP2K5\",\n      \"CCM2\",\n      \"RIPK1\",\n      \"TRAF6\",\n      \"SQSTM1\",\n      \"NBR1\",\n      \"TAK1\",\n      \"STK24\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}