{"gene":"MAPK6","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2004,"finding":"ERK3 specifically interacts with and phosphorylates MAPK-activated protein kinase 5 (MK5/PRAK), activating it both in vitro and in vivo. This interaction causes nuclear exclusion of both ERK3 and MK5. Endogenous MK5 activity is reduced by siRNA-mediated knockdown of ERK3 and in ERK3-/- mouse fibroblasts. Reciprocally, MK5 depletion causes dramatic reduction in endogenous ERK3 levels, suggesting MK5 acts as a chaperone for ERK3.","method":"Co-immunoprecipitation, in vitro kinase assay, siRNA knockdown, ERK3-/- mouse fibroblasts, PC12 cell differentiation model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro kinase assay, genetic KO, siRNA KD with defined phenotypic readouts, independently replicated in companion paper (PMID:15538386)","pmids":["15577943"],"is_preprint":false},{"year":2004,"finding":"ERK3 specifically interacts with MK5 in vitro and in vivo; ERK3 expression drives nuclear-cytoplasmic translocation and activation of MK5. Activation of MK5 is independent of ERK3 enzymatic activity but depends on MK5's own catalytic activity and a region in ERK3's C-terminal extension. Deletion of MK5 leads to strong reduction of ERK3 protein levels.","method":"Co-immunoprecipitation, mammalian cell expression, kinase-dead ERK3 mutants, MK5 knockout mice","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, kinase-dead mutant analysis, genetic KO, independently replicated alongside PMID:15577943","pmids":["15538386"],"is_preprint":false},{"year":1996,"finding":"ERK3 is constitutively localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells; the C-terminal 180 aa are not required for nuclear localization. Recombinant ERK3 autophosphorylates on Ser189 in vitro and in vivo; mutation of conserved catalytic Asp171 eliminates autophosphorylation. ERK3 does not phosphorylate typical MAP kinase substrates (myelin basic protein, etc.).","method":"Immunoblotting, subcellular fractionation, immunofluorescence, in vitro kinase assay, site-directed mutagenesis (D171A, S189)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis, direct localization experiment, replicated in multiple subsequent studies","pmids":["8621539"],"is_preprint":false},{"year":2003,"finding":"ERK3 undergoes CRM1-dependent nuclear export; treatment with leptomycin B causes nuclear accumulation, and ectopic CRM1 promotes cytoplasmic relocalization. CRM1 binds ERK3 in vitro. Forced nuclear or cytoplasmic localization of ERK3 attenuates its ability to induce cell cycle arrest in fibroblasts, indicating nucleocytoplasmic shuttling is required for its negative effect on cell cycle progression.","method":"Leptomycin B treatment, CRM1 overexpression, snurportin-1 overexpression, GST pulldown (CRM1 binds ERK3 in vitro), cell cycle analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct localization experiments with pharmacological and genetic tools, in vitro binding, functional consequence on cell cycle measured","pmids":["12915405"],"is_preprint":false},{"year":2008,"finding":"ERK3 (and ERK4) are phosphorylated on activation loop Ser189 in vivo by an upstream cellular kinase, detectable in resting cells. Activation loop phosphorylation stimulates intrinsic catalytic activity of ERK3 and is required for formation of stable active complexes with MK5, and for efficient cytoplasmic redistribution of ERK3/MK5 complexes.","method":"Phospho-specific antibodies, in vitro kinase assay, MK5 interaction assays, subcellular fractionation","journal":"Journal of cellular physiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — phospho-specific antibodies, in vitro kinase assays, multiple orthogonal methods in one study","pmids":["18720373"],"is_preprint":false},{"year":2010,"finding":"Group I p21-activated kinases (PAK1/2/3) phosphorylate ERK3 on activation loop Ser189 (and ERK4 on Ser186) both in vitro and in vivo. Activated Rac1 augments this phosphorylation; siRNA silencing of PAK1/2/3 abolishes Rac1-induced ERK3 Ser189 phosphorylation. PAK-mediated phosphorylation results in enzymatic activation of ERK3 and downstream activation of MK5, defining a PAK-ERK3-MK5 signaling pathway.","method":"Biochemical kinase purification, in vitro kinase assay, RNAi, phospho-specific antibodies, activated Rac1 expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — unbiased biochemical purification, in vitro reconstitution, RNAi validation, multiple orthogonal methods","pmids":["21177870"],"is_preprint":false},{"year":2011,"finding":"Recombinant PAK2 directly phosphorylates ERK3 at Ser189 in vitro (confirmed by protein microarray and solution-based kinase assay). Selective inhibition of class I PAK kinase activity in cells increases nuclear accumulation of ERK3, reduces Ser189 phosphorylation, and inhibits ERK3-PRAK complex formation.","method":"Protein microarray, in vitro kinase assay, phospho-specific antisera, PAK inhibitor treatment, nuclear/cytoplasmic fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution confirmed by two orthogonal methods; corroborates PMID:21177870","pmids":["21317288"],"is_preprint":false},{"year":2009,"finding":"ERK3 and ERK4 interact with MK5 through a novel FRIEDE motif in the L16 extension C-terminal to the CD domain; a single I→K substitution in FRIEDE completely abolishes binding, activation, and translocation of MK5 by both ERK3 and ERK4. The canonical CD domain is dispensable for this interaction. Activation loop phosphorylation of ERK3/ERK4 is required for MK5 binding, suggesting a phosphorylation-dependent switch mechanism.","method":"Peptide overlay assays, site-directed mutagenesis (FRIEDE motif), Co-immunoprecipitation, MK5 translocation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis defining interaction motif, multiple orthogonal binding and functional assays","pmids":["19473979"],"is_preprint":false},{"year":1996,"finding":"A novel nuclear/cytosolic protein kinase activity phosphorylates ERK3 at a single site, Ser189, and is distinct from MEK1/2. This kinase is inactivated by PP2A and does not phosphorylate ERK2 or ERK2 mutants bearing the ERK3 phospho-acceptor sequence, indicating high specificity for ERK3.","method":"Biochemical purification, in vitro kinase assay, PP2A treatment, ERK3/ERK2 chimeric mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis and biochemical fractionation; specificity established by negative controls","pmids":["8662649"],"is_preprint":false},{"year":2012,"finding":"ERK3 interacts with and phosphorylates SRC-3 (steroid receptor coactivator 3) at Ser857. This phosphorylation is essential for SRC-3 interaction with PEA3 transcription factor, which promotes MMP gene expression and proinvasive activity in lung cancer cells. Knockdown of ERK3 inhibits lung cancer cell invasion and tumor formation in xenograft mouse models.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis (S857A), gene knockdown, xenograft mouse model, MMP expression assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, co-IP, functional cell and in vivo xenograft validation","pmids":["22505454"],"is_preprint":false},{"year":2012,"finding":"The ERK3/MK5 signaling complex interacts with septin7 (Sept7) to form a ternary complex that can phosphorylate Borg proteins (regulators of Sept7). MK5 also interacts with and phosphorylates kalirin-7 (Kal7) in neurons. The ERK3/MK5 module stimulates Sept7-dependent dendrite development and spine formation in transfected primary neurons; MK5-deficient mice show impaired dendritic spine formation in hippocampal neurons.","method":"Large-scale interaction screens, Co-immunoprecipitation, in vitro kinase assay, MK5 knockout mice, primary neuron transfection","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, in vitro kinase assay, genetic KO with defined neuronal phenotype, multiple orthogonal methods","pmids":["22508986"],"is_preprint":false},{"year":2017,"finding":"DUSP2 (an inducible nuclear dual-specificity MAP kinase phosphatase) binds to ERK3 and ERK4 via the ERK3/ERK4 CD domain interacting with the DUSP2 KIM motif. This interaction is direct and results in dephosphorylation of ERK3/ERK4 activation loop, stabilization of DUSP2, and inhibition of MK5 activation downstream.","method":"Co-immunoprecipitation, in vitro phosphatase assay, domain mapping, MK5 activation assays, DUSP2 stabilization experiments","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — direct binding and dephosphorylation demonstrated in vitro, functional downstream consequence measured, multiple methods","pmids":["28252035"],"is_preprint":false},{"year":2010,"finding":"ERK3 is stoichiometrically hyperphosphorylated during mitotic entry and dephosphorylated at M→G1 transition. Cyclin B-Cdk1 phosphorylates four C-terminal ERK3 sites (Ser684, Ser688, Thr698, Ser705) in vitro and in vivo. Cdc14A and Cdc14B phosphatases bind ERK3 and reverse its C-terminal phosphorylation. Alanine substitution of the four phosphorylation sites markedly decreases ERK3 half-life in mitosis, linking Cdk1-mediated phosphorylation to ERK3 stabilization.","method":"In vitro kinase assay with purified cyclin B-Cdk1, mass spectrometry phosphosite identification, Cdc14 binding assay, protein half-life measurements (pulse-chase), site-directed mutagenesis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay, MS-based phosphosite identification, mutagenesis functional validation, phosphatase binding","pmids":["20236090"],"is_preprint":false},{"year":2007,"finding":"Human Cdc14A phosphatase interacts with ERK3 directly via ERK3's C-terminal domain (GST pulldown). Cdc14A can remove Cdk-mediated phosphorylation of ERK3 in vitro. Cdc14A forms a stable complex with ERK3 in human cells independent of phosphatase activity. Cdc14A upregulation leads to redistribution of ERK3 substrate MK5 from nucleus to cytoplasm and stabilizes the ERK3/cyclin D3 complex.","method":"Yeast two-hybrid screen, GST pulldown, Co-immunoprecipitation, in vitro phosphatase assay, subcellular localization analysis","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct in vitro binding and phosphatase assay, Co-IP in cells, but single lab and limited functional mechanistic depth","pmids":["18235225"],"is_preprint":false},{"year":2016,"finding":"ERK3 phosphorylates tyrosyl-DNA phosphodiesterase 2 (TDP2) at Ser60 and regulates TDP2's phosphodiesterase activity, thereby protecting lung cancer cells against Topoisomerase 2 inhibitor-induced DNA damage and growth inhibition.","method":"In vitro kinase assay, site-directed mutagenesis (S60), TDP2 phosphodiesterase activity assay, cell viability assays with Top2 inhibitors","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, enzymatic activity assay, functional cell-based validation; single lab","pmids":["26701725"],"is_preprint":false},{"year":2020,"finding":"β-adrenergic stimulation stabilizes ERK3, leading to formation of an ERK3/MK5 complex that drives lipolysis in adipocytes. The ERK3/MK5 pathway promotes expression of the lipolytic enzyme ATGL via the transcription factor FOXO1. Targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, elevates energy dissipation, and promotes a lean phenotype with improved diabetes markers.","method":"High-throughput screen, co-immunoprecipitation, adipocyte-specific ERK3 knockout mice, lipolysis assays, FOXO1 and ATGL expression analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — adipocyte-specific genetic KO with defined metabolic phenotype, co-IP, pathway analysis with multiple downstream readouts","pmids":["32139423"],"is_preprint":false},{"year":2021,"finding":"MAPK6 directly activates AKT by phosphorylating AKT at Ser473 independent of mTORC2. MAPK6 interacts with AKT through its C34 region and C-terminal tail. Inhibiting MAPK6 sensitizes cancer cells to mTOR kinase inhibitors.","method":"Co-immunoprecipitation (domain mapping), in vitro kinase assay (AKT phosphorylation at S473), mTOR inhibitor combination studies, cancer cell proliferation assays","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, in vitro kinase assay; single lab but multiple orthogonal methods","pmids":["34767444"],"is_preprint":false},{"year":2020,"finding":"ERK3 is necessary for production of interleukin-8 (IL-8) and is critical for AP-1 signaling through its interaction with and regulation of c-Jun protein. The secretome of ERK3-deficient cells is defective in chemotaxis of neutrophils and monocytes in vitro and in vivo.","method":"3D organoid system, ERK3 knockdown/knockout, secretome analysis, chemotaxis assays (in vitro and in vivo), c-Jun interaction/regulation experiments","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KD/KO with defined secretome and chemotaxis phenotypes, c-Jun interaction documented; single lab","pmids":["32314963"],"is_preprint":false},{"year":2023,"finding":"ERK3 directly acts as a guanine nucleotide exchange factor for CDC42 and phosphorylates ARP3 (subunit of ARP2/3 complex) at Ser418 to promote actin polymerization and filopodia formation. ERK3 protein bound directly to the purified ARP2/3 complex and augmented polymerization of actin in vitro. Depletion of ERK3 prevented CDC42/RAC1 activation, F-actin maintenance, filopodia formation, and epithelial cell migration.","method":"In vitro GEF assay (CDC42 nucleotide exchange), in vitro kinase assay (ARP3 phosphorylation at S418), in vitro actin polymerization assay with purified ARP2/3, ERK3 depletion with defined cytoskeletal phenotypes","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution (GEF assay, ARP3 kinase assay, actin polymerization), multiple orthogonal methods; single lab","pmids":["37057894"],"is_preprint":false},{"year":2022,"finding":"FBXW7 acts as an E3 ubiquitin ligase for ERK3, targeting it for ubiquitination-mediated proteasomal degradation. ERK3 binds FBXW7 through its C34D region, specifically at Thr417 and Thr421, which interact with the WD40 domain of FBXW7. T417A/T421A double mutation abrogates FBXW7-mediated ubiquitination. FBXW7 depletion restores ERK3 protein levels.","method":"Mammalian two-hybrid assay, co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, site-directed mutagenesis (T417A/T421A)","journal":"Experimental & molecular medicine","confidence":"High","confidence_rationale":"Tier 2 / Moderate — two-hybrid, Co-IP, ubiquitination assay, mutagenesis; multiple orthogonal methods in single lab","pmids":["35022544"],"is_preprint":false},{"year":2022,"finding":"EGLN3 catalyzes hydroxylation of ERK3, and this hydroxylation antagonizes chaperone-mediated autophagy (CMA)-mediated degradation of ERK3 by reducing ERK3 interaction with LAMP2A. ERK3 interacts with HSC70 and LAMP2A (core CMA components) and is degraded by the CMA-lysosome pathway; EGLN3-mediated hydroxylation blocks this.","method":"Co-immunoprecipitation (ERK3-HSC70 and ERK3-LAMP2A), hydroxylation assay, CMA-lysosome pathway assays, EGLN3 catalytically inactive knock-in mice","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of CMA components, enzymatic hydroxylation, genetic knock-in mice; mechanistic chain established but hydroxylation assay details limited in abstract","pmids":["35124697"],"is_preprint":false},{"year":2022,"finding":"ERK3 and MK5 act in a linear pathway to control postnatal myogenic differentiation: ERK3 kinase-inactive mice (Mapk6KD/KD) have impaired skeletal muscle growth and regeneration. MK5 directly phosphorylates FoxO3, promoting its degradation and reducing FoxO3 association with MyoD. FoxO3 depletion partially rescues premature differentiation caused by ERK3/MK5 inactivation.","method":"Mapk6KD/KD (kinase-dead knock-in) mice, MK5 KO mice, C2C12/primary myoblast differentiation assays, MK5 in vitro kinase assay (FoxO3 phosphorylation), MK5 inhibitor, FoxO3 rescue experiments","journal":"Journal of cellular physiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay for FoxO3 phosphorylation, genetic epistasis with KD knock-in and substrate rescue; multiple orthogonal methods","pmids":["35141958"],"is_preprint":false},{"year":2022,"finding":"Supervillin (SVIL) is a direct ERK3 substrate; ERK3 phosphorylates SVIL at Ser245 to regulate myosin II activation and cytokinesis completion. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild-type SVIL but not by non-phosphorylatable S245A mutant.","method":"Quantitative phosphoproteomics, in vitro kinase assay, site-directed mutagenesis (S245A), siRNA knockdown, cytokinesis and multinucleation assays","journal":"Journal of cellular physiology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — phosphoproteomics-guided substrate identification, in vitro kinase assay, mutagenesis rescue experiment, multiple orthogonal methods","pmids":["36576983"],"is_preprint":false},{"year":2001,"finding":"The C-terminal halves of ERK2 and ERK3 (catalytic domain) are primarily responsible for their distinct subcellular localizations in resting cells. The N-terminal folding domain of ERK2 is required for its activation by MEK1, interaction with MEK1, and nuclear accumulation; swapping in the ERK3 N-terminal domain abrogates these. ERK3 is thus constitutively nuclear through a mechanism residing in its C-terminal catalytic domain half, independent of MEK.","method":"ERK2/ERK3 chimeric protein constructs, subcellular localization assays, MEK1 interaction assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chimeric domain-swap constructs with localization readout; single lab","pmids":["11741894"],"is_preprint":false},{"year":2001,"finding":"ERK3 expression is upregulated at the mRNA and protein level by proteasome inhibitors in a p38 pathway-dependent manner. p38 pathway kinase inhibitors prevent proteasome-dependent ERK3 induction. ERK3 upregulation is independent of p53, Bcl2, and caspase-3. Ectopic ERK3 expression increases cellular resistance to proteasome inhibition.","method":"Proteasome inhibitor treatment (peptide-based inhibitors, lactacystin), p38 inhibitors, Western blot, ERK3 ectopic expression, cell viability assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological perturbation with multiple inhibitors; p38 pathway link established indirectly; single lab","pmids":["11148204"],"is_preprint":false},{"year":1996,"finding":"ERK3 activity is constitutively activated (~10-fold increase) in colon cancer cells stably transfected with PKC-beta1 or PKC-beta2, without a change in ERK3 protein level. TPA-induced downregulation of PKC activity reduces ERK3 activity, establishing a causal link. Activated ERK3 is found in nuclear and membrane fractions in PKC-beta transfectants.","method":"Stable transfection, immune complex kinase assay, in-gel kinase assay, TPA-mediated PKC downregulation, subcellular fractionation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple cell lines and rescue with TPA; indirect causal link via PKC; single lab","pmids":["8626698"],"is_preprint":false},{"year":2006,"finding":"ERK3/MAPK6 expression is upregulated by BRAF(V600E) signaling via MEK1/2; pharmacological inhibition of BRAF or MEK1/2, or RNAi-mediated BRAF knockdown, leads to rapid ERK3 protein degradation in melanoma cells.","method":"Conditional BRAF(V600E) expression system, MEK inhibitors, BRAF RNAi, Western blot, microarray","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological and genetic perturbation with protein stability readout; multiple approaches corroborating MEK→ERK3 regulation","pmids":["16964379"],"is_preprint":false},{"year":2006,"finding":"ERK3 associates with microtubule-associated protein MAP2 in pancreatic beta cells; PMA-induced ERK3 phosphorylation is accompanied by increased ERK3/MAP2 association and MAP2 phosphorylation. Antisense oligonucleotide-mediated ERK3 knockdown abolishes glucose-stimulated insulin secretion and PMA-induced insulin secretion, indicating ERK3 is required for stimulus-secretion coupling in beta cells.","method":"Co-immunoprecipitation (ERK3-MAP2), antisense oligonucleotide knockdown, insulin secretion assays, immunohistochemistry (beta cell-specific expression)","journal":"Molecular and cellular endocrinology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and functional knockdown; single lab, limited mechanistic depth on MAP2 phosphorylation consequence","pmids":["16597486"],"is_preprint":false},{"year":2010,"finding":"ERK3 localizes to spindle fibers and asters in mouse oocytes during meiosis I–II. Deletion of ERK3 by morpholino injection causes oocyte arrest at MI with impaired spindles, misaligned chromosomes, persistent BubR1 on kinetochores, disrupted kinetochore-microtubule attachments, and failure of homologous chromosome segregation.","method":"ERK3 morpholino microinjection in mouse oocytes, immunofluorescence (spindle localization, BubR1, α-tubulin), chromosome spreading, low-temperature kinetochore-MT stability assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — morpholino KD with defined meiotic phenotypes, direct localization by immunofluorescence; single lab","pmids":["20927325"],"is_preprint":false},{"year":2014,"finding":"ERK3 promotes endothelial cell migration, proliferation, and tube formation by upregulating SRC-3/SP1-mediated VEGFR2 expression. The mechanism involves ERK3-stimulated formation of a transcriptional complex containing SRC-3, SP-1, and CBP. ERK3 gene expression is upregulated by cytokines through c-Jun binding to the ERK3 gene promoter.","method":"Co-immunoprecipitation (SRC-3/SP-1/CBP complex), VEGFR2 promoter reporter assays, ERK3 knockdown, endothelial migration/proliferation/tube formation assays","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — transcriptional complex Co-IP, reporter assays, functional KD; single lab","pmids":["24585635"],"is_preprint":false},{"year":2018,"finding":"Activation loop phosphorylation (Ser189) is important for ERK3 kinase activity toward SRC-3: S189A mutation greatly decreases ERK3 kinase activity toward SRC-3 and reduces the ability to promote lung cancer cell migration, invasion, and MMP expression. A kinase-inactive ERK3 mutant still promotes invasion to a lesser extent, indicating both kinase-dependent and kinase-independent mechanisms contribute to ERK3-driven invasiveness.","method":"In vitro kinase assay (S189A and kinase-dead mutants vs. SRC-3 substrate), lung cancer cell migration/invasion assays, MMP expression analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with multiple mutants, correlated with cell-based functional readouts; builds on established ERK3-SRC3 substrate axis","pmids":["30166347"],"is_preprint":false},{"year":2025,"finding":"Intracellular acidification markedly increases ERK3 half-life, while alkalinization accelerates its degradation. This pH-dependent regulation is rapid, reversible, and cell-type consistent. A region in ERK3's C-terminus contains pH-sensing motifs responsible for this regulation.","method":"Intracellular pH manipulation, protein half-life measurements, quantitative proteomics, C-terminus deletion/mutant analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cell types, pharmacological pH manipulation, proteomics; pH-sensing motif defined but in abstract with limited mutagenesis detail","pmids":["41123996"],"is_preprint":false},{"year":2023,"finding":"ERK3 interacts with DGKζ (diacylglycerol kinase ζ) via its C34 domain; DGKζ binds to the N-terminal and C1 domains of ERK3. Co-overexpression of DGKζ and ERK3 completely blocks ERK3-promoted lung cancer cell migration; DGKζ requires the C34 domain of ERK3 to prevent ERK3-mediated migration.","method":"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay, co-localization, ERK3 domain deletion mutants, lung cancer cell migration assays","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP confirmed by in vitro binding and co-localization, domain mapping, functional migration assay; single lab","pmids":["37287450"],"is_preprint":false},{"year":2020,"finding":"ERK3 L290P/V cancer mutations enhance ERK3's cytoplasmic localization by increasing interaction with the nuclear export factor CRM1, thereby increasing migration/invasion-promoting capability without clear effects on intrinsic kinase activity.","method":"Site-directed mutagenesis (L290P, L290V), CRM1 co-immunoprecipitation, subcellular fractionation, migration/invasion assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and fractionation with multiple mutants; functional migration assay; single lab","pmids":["29101390"],"is_preprint":false},{"year":2023,"finding":"ERK3 interacts with Snail and enhances Snail protein stability by inhibiting the binding of FBXO11 (an E3 ubiquitin ligase) to Snail, thereby preventing Snail ubiquitination and degradation. ERK3 does not directly phosphorylate Snail.","method":"Co-immunoprecipitation, ubiquitination assay, protein stability assay, negative result for direct phosphorylation","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP showing competition for FBXO11 binding, ubiquitination assay; single lab; direct phosphorylation explicitly negative","pmids":["38201533"],"is_preprint":false},{"year":2025,"finding":"TRIM21 (ubiquitin ligase) binds MAPK6 and promotes its ubiquitin-proteasome degradation in endothelial cells exposed to disturbed shear stress. Endothelial MAPK6 regulates inflammation via the EGR1/CXCL12 axis; endothelium-specific MAPK6 knockout increases plaque area in ApoE-/- mice, reversible by CXCL12 neutralization.","method":"Co-immunoprecipitation (TRIM21-MAPK6), RNA-seq, proteomic analysis, endothelium-specific Mapk6 knockout mice, AAV-MAPK6 overexpression, CXCL12 neutralization rescue","journal":"Clinical and translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP identifying TRIM21, conditional KO mice with atherosclerosis phenotype, pathway rescue; single lab","pmids":["39763069"],"is_preprint":false},{"year":2015,"finding":"ERK3 localizes to the cell periphery of breast cancer cells during adhesion to collagen I. ERK3 overexpression reduces cell spread area and increases migration speed. Importantly, a kinase-inactive ERK3 mutant phenocopies wild-type ERK3 overexpression in reducing spread area and increasing migration, revealing a kinase-independent function.","method":"Live cell imaging, ERK3 overexpression (WT and kinase-dead), siRNA knockdown, morphological analysis during cell adhesion","journal":"Cell adhesion & migration","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — localization and kinase-dead mutant analysis; single lab, limited pathway placement","pmids":["26588708"],"is_preprint":false},{"year":2020,"finding":"The C-terminus tail of ERK3 is required for full kinase activity toward SRC-3 and for ERK3-promoted cancer cell migration/invasion. Septin 7, which interacts with ERK3 via its C-terminus tail, acts as a downstream effector for ERK3-induced cancer cell migration.","method":"C-terminus deletion mutants, in vitro kinase assay (SRC-3 substrate), septin 7 knockdown, migration/invasion assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — in vitro kinase assay with deletion mutants, functional migration assay with septin 7 KD; single lab","pmids":["32516969"],"is_preprint":false},{"year":1996,"finding":"ERK3 specifically co-elutes with B-Raf (but not c-Raf1) after anion exchange chromatography of rat hippocampal lysates; ERK3 is released from B-Raf immunoprecipitates upon ATP incubation, suggesting a specific association of ERK3 with B-Raf in rat hippocampus.","method":"Anion exchange chromatography, co-immunoprecipitation, ATP-dependent dissociation assay","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-fractionation and Co-IP experiment; single lab, not followed up mechanistically","pmids":["8954940"],"is_preprint":false},{"year":2014,"finding":"ERK3 expression is induced in CD4+ and CD8+ T cells following TCR stimulation, requiring ERK1/2 activation. ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. ERK3-deficient T cells show decreased proliferation and impaired cytokine secretion following stimulation with low-dose anti-CD3.","method":"ERK3-/- mice, T cell activation assays, ERK1/2 inhibitor treatment, co-immunoprecipitation (ERK3-MK5 in activated T cells), cytokine measurement","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined immune phenotype, ERK1/2 epistasis via inhibitor, Co-IP; single lab","pmids":["24475167"],"is_preprint":false},{"year":2018,"finding":"ERK3 regulates epithelial architecture in Xenopus embryos and human breast epithelial cells: ERK3 knockdown impairs adherens and tight-junction protein distribution and tight-junction barrier function. ERK3 is required for full activation of TFAP2A (AP-2α)-dependent transcription, and TFAP2A knockdown phenocopies ERK3 knockdown.","method":"Xenopus ERK3 knockdown (morpholino), human epithelial cell ERK3 KD, microarray gene expression, tight-junction barrier assay, TFAP2A luciferase reporter","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two model systems (Xenopus and human cells), barrier assay, reporter assay; single lab","pmids":["29674317"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of the ERK3 kinase domain was determined, revealing a distinct ATP binding pocket compared to ERK2, particularly in the A-loop, GC-loop, and αC-helix conformations. The structure also indicates a potential structural link toward MK5 interaction via the FHIEDE motif.","method":"X-ray crystallography (ERK3 kinase domain), small molecule inhibitor screening with biochemical and cellular assays (NanoBRET)","journal":"Bioorganic & medicinal chemistry letters","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure with inhibitor binding mode defined; functional connection to MK5 motif is structural inference; single lab","pmids":["32927028"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of the ERK3 kinase domain was determined providing molecular insights into its distinct ATP binding pocket relative to ERK2, explaining differences in inhibitor binding properties.","method":"X-ray crystallography (ERK3 kinase domain and CLK1 in complex with inhibitor CAF052), medium-scale small molecule screening","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure determined; functional mechanism inferred from structure alone; single lab","pmids":["33114754"],"is_preprint":false},{"year":2024,"finding":"ERK3 is expressed in atrial and ventricular cardiac fibroblasts (not myocytes). ERK3 co-immunoprecipitates with MK5 in heart lysates. ERK3 haploinsufficiency reduces cardiac hypertrophy and Col1a1 mRNA increase after transverse aortic constriction. ERK3 knockdown reduces TGF-β-induced collagen expression and impairs myofibroblast motility.","method":"Co-immunoprecipitation (heart lysates), ERK3+/- mice with TAC model, siRNA knockdown in primary cardiac fibroblasts, collagen assays, motility assays","journal":"Physiological reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic haploinsufficiency with defined cardiac phenotype, co-IP in tissue, siRNA in primary cells; single lab","pmids":["38872461"],"is_preprint":false}],"current_model":"ERK3 (MAPK6) is an atypical MAPK with constitutive nuclear/cytoplasmic distribution controlled by CRM1-dependent nuclear export and regulated by intracellular pH; it is activated by group I PAKs phosphorylating its unique SEG activation loop at Ser189, and it signals primarily through a direct interaction with MK5 (via the FRIEDE docking motif) to form a kinase-scaffold complex that drives MK5 activation, nuclear export, and downstream phosphorylation of substrates including FoxO3 (regulating myogenesis), FOXO1/ATGL (regulating lipolysis), supervillin/Ser245 (regulating cytokinesis), and CDC42/ARP3 (regulating actin dynamics); ERK3 also directly phosphorylates SRC-3/Ser857 to promote invasiveness, TDP2/Ser60 to enhance DNA damage repair, and AKT/Ser473 to promote tumor growth, while its protein stability is controlled by ubiquitin-mediated proteasomal degradation through FBXW7 and TRIM21, by CMA-lysosome pathway involving HSC70/LAMP2A counteracted by EGLN3 hydroxylation, and by Cdk1-mediated C-terminal phosphorylation stabilizing it in mitosis."},"narrative":{"mechanistic_narrative":"MAPK6 (ERK3) is an atypical mitogen-activated protein kinase that functions as a constitutively active signaling kinase whose biology is dominated by its activation loop, its regulated nucleocytoplasmic distribution, and its tightly controlled protein stability [PMID:8621539, PMID:12915405]. Unlike conventional MAP kinases, ERK3 carries a single phospho-acceptor (Ser189) in its SEG activation loop that is phosphorylated by a dedicated upstream activity rather than by MEK; group I PAKs (PAK1/2/3) directly phosphorylate Ser189 downstream of Rac1, stimulating intrinsic catalytic activity [PMID:21177870, PMID:21317288, PMID:8662649], while DUSP2 reverses this phosphorylation and damps signaling [PMID:28252035]. ERK3 shuttles between nucleus and cytoplasm via CRM1-dependent export, and this shuttling is required for its effects on cell cycle progression [PMID:12915405]. Its principal effector axis is a kinase-scaffold complex with MK5/PRAK formed through the non-canonical FRIEDE docking motif in the C-terminal L16 extension; complex formation depends on Ser189 phosphorylation and drives MK5 activation and co-redistribution to the cytoplasm, with ERK3 and MK5 mutually stabilizing one another [PMID:15577943, PMID:15538386, PMID:18720373, PMID:19473979]. Through this module ERK3 controls myogenic differentiation via MK5-mediated FoxO3 phosphorylation [PMID:35141958] and adipocyte lipolysis via a FOXO1/ATGL program [PMID:32139423]. Beyond MK5, ERK3 directly phosphorylates substrates governing the cytoskeleton and cell division — supervillin at Ser245 to enable cytokinesis [PMID:36576983], and ARP3 at Ser418 while acting as a CDC42 GEF to drive actin polymerization and filopodia formation [PMID:37057894] — and phosphorylates SRC-3 at Ser857 to promote MMP expression and cancer cell invasion [PMID:22505454, PMID:30166347]. ERK3 abundance is rate-limiting and is set by multiple degradative routes, including FBXW7- and TRIM21-mediated proteasomal degradation [PMID:35022544, PMID:39763069], chaperone-mediated autophagy through HSC70/LAMP2A that is antagonized by EGLN3 hydroxylation [PMID:35124697], and intracellular pH sensing through its C-terminus [PMID:41123996]; conversely, Cdk1 phosphorylation of four C-terminal sites stabilizes ERK3 during mitosis [PMID:20236090].","teleology":[{"year":1996,"claim":"Establishing ERK3 as an atypical MAP kinase required defining its activation site and constitutive localization, distinguishing it from classical ERKs.","evidence":"In vitro kinase assays with D171A/S189 mutants plus subcellular fractionation and immunofluorescence","pmids":["8621539"],"confidence":"High","gaps":["Did not identify the physiological upstream kinase for Ser189","No substrate beyond autophosphorylation identified at this stage"]},{"year":1996,"claim":"Biochemical purification showed Ser189 is phosphorylated by a specific cellular kinase distinct from MEK1/2, indicating ERK3 uses a non-canonical activation route.","evidence":"Biochemical purification, in vitro kinase assay, PP2A treatment, ERK3/ERK2 chimeras","pmids":["8662649"],"confidence":"High","gaps":["The kinase was not molecularly identified here","Physiological signals controlling this activity unknown at the time"]},{"year":2001,"claim":"Domain-swap experiments localized the determinant of ERK3's MEK-independent constitutive nuclear localization to its catalytic-domain half, explaining its divergence from ERK2.","evidence":"ERK2/ERK3 chimeric constructs with localization and MEK1 interaction readouts","pmids":["11741894"],"confidence":"Medium","gaps":["Specific export/import signals not mapped here","Single lab"]},{"year":2003,"claim":"Defining CRM1-dependent export established that ERK3 shuttling, not static localization, governs its cell-cycle effects.","evidence":"Leptomycin B, CRM1 overexpression, GST pulldown, cell cycle analysis","pmids":["12915405"],"confidence":"High","gaps":["Mechanism linking shuttling to cell cycle arrest not resolved","Direct nuclear export signal not mapped"]},{"year":2004,"claim":"Identification of MK5/PRAK as the direct ERK3 partner and substrate defined the core effector axis and revealed mutual protein stabilization.","evidence":"Reciprocal Co-IP, in vitro kinase assay, siRNA, ERK3-/- fibroblasts, MK5 KO mice (two companion papers)","pmids":["15577943","15538386"],"confidence":"High","gaps":["Physiological downstream substrates of activated MK5 not yet defined","Whether MK5 activation is strictly kinase-dependent vs scaffolding was contested between studies"]},{"year":2008,"claim":"Showing that activation-loop phosphorylation drives stable ERK3–MK5 complex formation and cytoplasmic redistribution linked Ser189 status to effector engagement.","evidence":"Phospho-specific antibodies, in vitro kinase assay, MK5 interaction and fractionation assays","pmids":["18720373"],"confidence":"High","gaps":["Did not identify the upstream kinase","Quantitative stoichiometry of the complex not determined"]},{"year":2009,"claim":"Defining the FRIEDE docking motif explained how ERK3/ERK4 recruit MK5 through a non-canonical mechanism independent of the canonical CD domain.","evidence":"Peptide overlay, FRIEDE mutagenesis, Co-IP, MK5 translocation assays","pmids":["19473979"],"confidence":"High","gaps":["Structural basis of FRIEDE–MK5 contact not solved here","Whether other partners use this motif unknown"]},{"year":2010,"claim":"Identifying group I PAKs as the Ser189 kinase placed ERK3 downstream of Rac1, completing a PAK–ERK3–MK5 pathway.","evidence":"Biochemical purification, in vitro kinase assay, RNAi of PAK1/2/3, phospho-specific antibodies, activated Rac1","pmids":["21177870","21317288"],"confidence":"High","gaps":["Upstream receptors/signals engaging Rac1-PAK toward ERK3 in physiological contexts not fully mapped","Whether other kinases contribute to Ser189 in vivo"]},{"year":2010,"claim":"Cdk1-mediated C-terminal phosphorylation, reversed by Cdc14 phosphatases, was shown to stabilize ERK3 during mitosis, revealing cell-cycle control of ERK3 abundance.","evidence":"In vitro kinase assay with cyclin B-Cdk1, MS phosphosite mapping, Cdc14 binding, pulse-chase, mutagenesis","pmids":["20236090","18235225"],"confidence":"High","gaps":["Functional consequence of mitotic ERK3 stabilization on substrates unclear","Cdc14 in vivo relevance limited"]},{"year":2012,"claim":"Discovery of SRC-3/Ser857 as a direct substrate connected ERK3 to a transcriptional program promoting invasion, extending its role beyond the MK5 axis.","evidence":"Co-IP, in vitro kinase assay, S857A mutagenesis, knockdown, xenograft, MMP assays","pmids":["22505454"],"confidence":"High","gaps":["Whether SRC-3 phosphorylation occurs in non-cancer contexts unknown","Relative contribution vs MK5 axis not quantified"]},{"year":2012,"claim":"Extending the ERK3/MK5 module to septin7 and neuronal targets linked it to cytoskeletal and dendritic spine development.","evidence":"Interaction screens, Co-IP, in vitro kinase assay, MK5 KO mice, primary neuron transfection","pmids":["22508986"],"confidence":"High","gaps":["Direct ERK3 substrate among these targets vs MK5-mediated effects not fully separated","In vivo ERK3-specific neuronal phenotype not tested"]},{"year":2017,"claim":"Identifying DUSP2 as a direct phosphatase that dephosphorylates the ERK3/ERK4 activation loop established negative regulation of the pathway.","evidence":"Co-IP, in vitro phosphatase assay, domain mapping, MK5 activation and DUSP2 stabilization assays","pmids":["28252035"],"confidence":"High","gaps":["Physiological signals inducing DUSP2 toward ERK3 not defined","Single study"]},{"year":2018,"claim":"Demonstrating Ser189-dependence of ERK3 activity toward SRC-3, alongside residual kinase-independent invasion, revealed dual mechanisms underlying ERK3-driven invasiveness.","evidence":"In vitro kinase assays with S189A and kinase-dead mutants, migration/invasion and MMP assays","pmids":["30166347"],"confidence":"High","gaps":["Molecular basis of the kinase-independent component undefined","Single lab"]},{"year":2022,"claim":"Linking the ERK3/MK5 module to FoxO3 phosphorylation in muscle and to FOXO1/ATGL in adipocytes mapped tissue-level physiological outputs of the pathway.","evidence":"Mapk6KD/KD and MK5 KO mice, adipocyte-specific ERK3 KO, in vitro kinase assay, lipolysis and differentiation assays","pmids":["35141958","32139423"],"confidence":"High","gaps":["Whether FoxO3/FOXO1 are direct ERK3 vs MK5 substrates fully delineated","Crosstalk between metabolic and myogenic outputs untested"]},{"year":2022,"claim":"Identification of supervillin/Ser245 as a direct substrate connected ERK3 kinase activity to cytokinesis completion and ploidy control.","evidence":"Phosphoproteomics, in vitro kinase assay, S245A mutagenesis rescue, cytokinesis/multinucleation assays","pmids":["36576983"],"confidence":"High","gaps":["Upstream signals timing ERK3 activity to cytokinesis unknown","In vivo relevance of multinucleation phenotype untested"]},{"year":2022,"claim":"Defining FBXW7-mediated proteasomal degradation and EGLN3-regulated chaperone-mediated autophagy established that ERK3 abundance is controlled by parallel degradative routes.","evidence":"Two-hybrid, Co-IP, ubiquitination assays, T417A/T421A mutagenesis, CMA-component Co-IP, EGLN3 knock-in mice","pmids":["35022544","35124697"],"confidence":"Medium","gaps":["Relative flux through proteasome vs CMA in different cell states unclear","Signals selecting one route over the other undefined"]},{"year":2023,"claim":"Showing ERK3 acts as a CDC42 GEF and phosphorylates ARP3/Ser418 directly coupled ERK3 to actin nucleation and filopodia formation, broadening its catalytic repertoire.","evidence":"In vitro GEF assay, ARP3 in vitro kinase assay, actin polymerization with purified ARP2/3, ERK3 depletion phenotypes","pmids":["37057894"],"confidence":"High","gaps":["Structural basis for GEF activity in a kinase fold undefined","Single lab"]},{"year":2025,"claim":"Demonstrating intracellular pH-dependent control of ERK3 half-life via C-terminal pH-sensing motifs added a metabolic/microenvironmental layer to ERK3 stability regulation.","evidence":"Intracellular pH manipulation, half-life measurements, quantitative proteomics, C-terminus mutants","pmids":["41123996"],"confidence":"Medium","gaps":["Molecular identity of pH-sensing residues only partially defined","Connection to the FBXW7/CMA degradation machinery not established"]},{"year":null,"claim":"How the multiple direct substrates (MK5, SRC-3, ARP3, supervillin, AKT, TDP2) and the kinase-independent functions are coordinated, and what receptor-proximal signals select among ERK3's degradative versus stabilizing pathways, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying model integrating direct substrates with the MK5 scaffold axis","Physiological triggers gating ERK3 stability vs degradation undefined","Kinase-independent mechanisms remain mechanistically opaque"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,9,18,22,30]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[2,5,18,22]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[2,41]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[18]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[18,22]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,3,23]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[18,28,36]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[25,36]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,7]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,12,22]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[12,19,20]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[9,30,33]}],"complexes":["ERK3–MK5 complex","ERK3/MK5/septin7 ternary complex"],"partners":["MK5","PAK1","PAK2","CRM1","DUSP2","FBXW7","SRC-3","TRIM21"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q16659","full_name":"Mitogen-activated protein kinase 6","aliases":["Extracellular signal-regulated kinase 3","ERK-3","MAP kinase isoform p97","p97-MAPK"],"length_aa":721,"mass_kda":82.7,"function":"Atypical MAPK protein. Phosphorylates microtubule-associated protein 2 (MAP2) and MAPKAPK5. The precise role of the complex formed with MAPKAPK5 is still unclear, but the complex follows a complex set of phosphorylation events: upon interaction with atypical MAPKAPK5, ERK3/MAPK6 is phosphorylated at Ser-189 and then mediates phosphorylation and activation of MAPKAPK5, which in turn phosphorylates ERK3/MAPK6. May promote entry in the cell cycle (By similarity)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q16659/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAPK6","classification":"Not Classified","n_dependent_lines":67,"n_total_lines":1208,"dependency_fraction":0.055463576158940396},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"MAP4","stoichiometry":0.2},{"gene":"MIF","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MAPK6","total_profiled":1310},"omim":[{"mim_id":"609949","title":"COMPLEMENT COMPONENT 5a RECEPTOR 2; C5AR2","url":"https://www.omim.org/entry/609949"},{"mim_id":"606784","title":"GLYCOGEN SYNTHASE KINASE 3-ALPHA; GSK3A","url":"https://www.omim.org/entry/606784"},{"mim_id":"606723","title":"MITOGEN-ACTIVATED PROTEIN KINASE-ACTIVATED PROTEIN KINASE 5; MAPKAPK5","url":"https://www.omim.org/entry/606723"},{"mim_id":"605554","title":"CD244 ANTIGEN; CD244","url":"https://www.omim.org/entry/605554"},{"mim_id":"605004","title":"GLYCOGEN SYNTHASE KINASE 3-BETA; GSK3B","url":"https://www.omim.org/entry/605004"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"},{"location":"Microtubules","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MAPK6"},"hgnc":{"alias_symbol":["ERK3","p97MAPK","HsT17250"],"prev_symbol":["PRKM6"]},"alphafold":{"accession":"Q16659","domains":[{"cath_id":"3.30.200.20","chopping":"13-110","consensus_level":"medium","plddt":81.8109,"start":13,"end":110},{"cath_id":"1.10.510.10","chopping":"114-333","consensus_level":"high","plddt":90.0202,"start":114,"end":333}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16659","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q16659-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q16659-F1-predicted_aligned_error_v6.png","plddt_mean":58.78},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAPK6","jax_strain_url":"https://www.jax.org/strain/search?query=MAPK6"},"sequence":{"accession":"Q16659","fasta_url":"https://rest.uniprot.org/uniprotkb/Q16659.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q16659/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16659"}},"corpus_meta":[{"pmid":"15577943","id":"PMC_15577943","title":"Activation 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This interaction causes nuclear exclusion of both ERK3 and MK5. Endogenous MK5 activity is reduced by siRNA-mediated knockdown of ERK3 and in ERK3-/- mouse fibroblasts. Reciprocally, MK5 depletion causes dramatic reduction in endogenous ERK3 levels, suggesting MK5 acts as a chaperone for ERK3.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, siRNA knockdown, ERK3-/- mouse fibroblasts, PC12 cell differentiation model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vitro kinase assay, genetic KO, siRNA KD with defined phenotypic readouts, independently replicated in companion paper (PMID:15538386)\",\n      \"pmids\": [\"15577943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ERK3 specifically interacts with MK5 in vitro and in vivo; ERK3 expression drives nuclear-cytoplasmic translocation and activation of MK5. Activation of MK5 is independent of ERK3 enzymatic activity but depends on MK5's own catalytic activity and a region in ERK3's C-terminal extension. Deletion of MK5 leads to strong reduction of ERK3 protein levels.\",\n      \"method\": \"Co-immunoprecipitation, mammalian cell expression, kinase-dead ERK3 mutants, MK5 knockout mice\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, kinase-dead mutant analysis, genetic KO, independently replicated alongside PMID:15577943\",\n      \"pmids\": [\"15538386\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"ERK3 is constitutively localized in the nucleus in exponentially growing, quiescent, and growth factor-stimulated cells; the C-terminal 180 aa are not required for nuclear localization. Recombinant ERK3 autophosphorylates on Ser189 in vitro and in vivo; mutation of conserved catalytic Asp171 eliminates autophosphorylation. ERK3 does not phosphorylate typical MAP kinase substrates (myelin basic protein, etc.).\",\n      \"method\": \"Immunoblotting, subcellular fractionation, immunofluorescence, in vitro kinase assay, site-directed mutagenesis (D171A, S189)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis, direct localization experiment, replicated in multiple subsequent studies\",\n      \"pmids\": [\"8621539\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ERK3 undergoes CRM1-dependent nuclear export; treatment with leptomycin B causes nuclear accumulation, and ectopic CRM1 promotes cytoplasmic relocalization. CRM1 binds ERK3 in vitro. Forced nuclear or cytoplasmic localization of ERK3 attenuates its ability to induce cell cycle arrest in fibroblasts, indicating nucleocytoplasmic shuttling is required for its negative effect on cell cycle progression.\",\n      \"method\": \"Leptomycin B treatment, CRM1 overexpression, snurportin-1 overexpression, GST pulldown (CRM1 binds ERK3 in vitro), cell cycle analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization experiments with pharmacological and genetic tools, in vitro binding, functional consequence on cell cycle measured\",\n      \"pmids\": [\"12915405\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ERK3 (and ERK4) are phosphorylated on activation loop Ser189 in vivo by an upstream cellular kinase, detectable in resting cells. Activation loop phosphorylation stimulates intrinsic catalytic activity of ERK3 and is required for formation of stable active complexes with MK5, and for efficient cytoplasmic redistribution of ERK3/MK5 complexes.\",\n      \"method\": \"Phospho-specific antibodies, in vitro kinase assay, MK5 interaction assays, subcellular fractionation\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-specific antibodies, in vitro kinase assays, multiple orthogonal methods in one study\",\n      \"pmids\": [\"18720373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Group I p21-activated kinases (PAK1/2/3) phosphorylate ERK3 on activation loop Ser189 (and ERK4 on Ser186) both in vitro and in vivo. Activated Rac1 augments this phosphorylation; siRNA silencing of PAK1/2/3 abolishes Rac1-induced ERK3 Ser189 phosphorylation. PAK-mediated phosphorylation results in enzymatic activation of ERK3 and downstream activation of MK5, defining a PAK-ERK3-MK5 signaling pathway.\",\n      \"method\": \"Biochemical kinase purification, in vitro kinase assay, RNAi, phospho-specific antibodies, activated Rac1 expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — unbiased biochemical purification, in vitro reconstitution, RNAi validation, multiple orthogonal methods\",\n      \"pmids\": [\"21177870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Recombinant PAK2 directly phosphorylates ERK3 at Ser189 in vitro (confirmed by protein microarray and solution-based kinase assay). Selective inhibition of class I PAK kinase activity in cells increases nuclear accumulation of ERK3, reduces Ser189 phosphorylation, and inhibits ERK3-PRAK complex formation.\",\n      \"method\": \"Protein microarray, in vitro kinase assay, phospho-specific antisera, PAK inhibitor treatment, nuclear/cytoplasmic fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution confirmed by two orthogonal methods; corroborates PMID:21177870\",\n      \"pmids\": [\"21317288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ERK3 and ERK4 interact with MK5 through a novel FRIEDE motif in the L16 extension C-terminal to the CD domain; a single I→K substitution in FRIEDE completely abolishes binding, activation, and translocation of MK5 by both ERK3 and ERK4. The canonical CD domain is dispensable for this interaction. Activation loop phosphorylation of ERK3/ERK4 is required for MK5 binding, suggesting a phosphorylation-dependent switch mechanism.\",\n      \"method\": \"Peptide overlay assays, site-directed mutagenesis (FRIEDE motif), Co-immunoprecipitation, MK5 translocation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis defining interaction motif, multiple orthogonal binding and functional assays\",\n      \"pmids\": [\"19473979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"A novel nuclear/cytosolic protein kinase activity phosphorylates ERK3 at a single site, Ser189, and is distinct from MEK1/2. This kinase is inactivated by PP2A and does not phosphorylate ERK2 or ERK2 mutants bearing the ERK3 phospho-acceptor sequence, indicating high specificity for ERK3.\",\n      \"method\": \"Biochemical purification, in vitro kinase assay, PP2A treatment, ERK3/ERK2 chimeric mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis and biochemical fractionation; specificity established by negative controls\",\n      \"pmids\": [\"8662649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ERK3 interacts with and phosphorylates SRC-3 (steroid receptor coactivator 3) at Ser857. This phosphorylation is essential for SRC-3 interaction with PEA3 transcription factor, which promotes MMP gene expression and proinvasive activity in lung cancer cells. Knockdown of ERK3 inhibits lung cancer cell invasion and tumor formation in xenograft mouse models.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis (S857A), gene knockdown, xenograft mouse model, MMP expression assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, co-IP, functional cell and in vivo xenograft validation\",\n      \"pmids\": [\"22505454\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The ERK3/MK5 signaling complex interacts with septin7 (Sept7) to form a ternary complex that can phosphorylate Borg proteins (regulators of Sept7). MK5 also interacts with and phosphorylates kalirin-7 (Kal7) in neurons. The ERK3/MK5 module stimulates Sept7-dependent dendrite development and spine formation in transfected primary neurons; MK5-deficient mice show impaired dendritic spine formation in hippocampal neurons.\",\n      \"method\": \"Large-scale interaction screens, Co-immunoprecipitation, in vitro kinase assay, MK5 knockout mice, primary neuron transfection\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, in vitro kinase assay, genetic KO with defined neuronal phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"22508986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DUSP2 (an inducible nuclear dual-specificity MAP kinase phosphatase) binds to ERK3 and ERK4 via the ERK3/ERK4 CD domain interacting with the DUSP2 KIM motif. This interaction is direct and results in dephosphorylation of ERK3/ERK4 activation loop, stabilization of DUSP2, and inhibition of MK5 activation downstream.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphatase assay, domain mapping, MK5 activation assays, DUSP2 stabilization experiments\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — direct binding and dephosphorylation demonstrated in vitro, functional downstream consequence measured, multiple methods\",\n      \"pmids\": [\"28252035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ERK3 is stoichiometrically hyperphosphorylated during mitotic entry and dephosphorylated at M→G1 transition. Cyclin B-Cdk1 phosphorylates four C-terminal ERK3 sites (Ser684, Ser688, Thr698, Ser705) in vitro and in vivo. Cdc14A and Cdc14B phosphatases bind ERK3 and reverse its C-terminal phosphorylation. Alanine substitution of the four phosphorylation sites markedly decreases ERK3 half-life in mitosis, linking Cdk1-mediated phosphorylation to ERK3 stabilization.\",\n      \"method\": \"In vitro kinase assay with purified cyclin B-Cdk1, mass spectrometry phosphosite identification, Cdc14 binding assay, protein half-life measurements (pulse-chase), site-directed mutagenesis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay, MS-based phosphosite identification, mutagenesis functional validation, phosphatase binding\",\n      \"pmids\": [\"20236090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Human Cdc14A phosphatase interacts with ERK3 directly via ERK3's C-terminal domain (GST pulldown). Cdc14A can remove Cdk-mediated phosphorylation of ERK3 in vitro. Cdc14A forms a stable complex with ERK3 in human cells independent of phosphatase activity. Cdc14A upregulation leads to redistribution of ERK3 substrate MK5 from nucleus to cytoplasm and stabilizes the ERK3/cyclin D3 complex.\",\n      \"method\": \"Yeast two-hybrid screen, GST pulldown, Co-immunoprecipitation, in vitro phosphatase assay, subcellular localization analysis\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct in vitro binding and phosphatase assay, Co-IP in cells, but single lab and limited functional mechanistic depth\",\n      \"pmids\": [\"18235225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ERK3 phosphorylates tyrosyl-DNA phosphodiesterase 2 (TDP2) at Ser60 and regulates TDP2's phosphodiesterase activity, thereby protecting lung cancer cells against Topoisomerase 2 inhibitor-induced DNA damage and growth inhibition.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (S60), TDP2 phosphodiesterase activity assay, cell viability assays with Top2 inhibitors\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with mutagenesis, enzymatic activity assay, functional cell-based validation; single lab\",\n      \"pmids\": [\"26701725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"β-adrenergic stimulation stabilizes ERK3, leading to formation of an ERK3/MK5 complex that drives lipolysis in adipocytes. The ERK3/MK5 pathway promotes expression of the lipolytic enzyme ATGL via the transcription factor FOXO1. Targeted deletion of ERK3 in mouse adipocytes inhibits lipolysis, elevates energy dissipation, and promotes a lean phenotype with improved diabetes markers.\",\n      \"method\": \"High-throughput screen, co-immunoprecipitation, adipocyte-specific ERK3 knockout mice, lipolysis assays, FOXO1 and ATGL expression analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — adipocyte-specific genetic KO with defined metabolic phenotype, co-IP, pathway analysis with multiple downstream readouts\",\n      \"pmids\": [\"32139423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MAPK6 directly activates AKT by phosphorylating AKT at Ser473 independent of mTORC2. MAPK6 interacts with AKT through its C34 region and C-terminal tail. Inhibiting MAPK6 sensitizes cancer cells to mTOR kinase inhibitors.\",\n      \"method\": \"Co-immunoprecipitation (domain mapping), in vitro kinase assay (AKT phosphorylation at S473), mTOR inhibitor combination studies, cancer cell proliferation assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, in vitro kinase assay; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"34767444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ERK3 is necessary for production of interleukin-8 (IL-8) and is critical for AP-1 signaling through its interaction with and regulation of c-Jun protein. The secretome of ERK3-deficient cells is defective in chemotaxis of neutrophils and monocytes in vitro and in vivo.\",\n      \"method\": \"3D organoid system, ERK3 knockdown/knockout, secretome analysis, chemotaxis assays (in vitro and in vivo), c-Jun interaction/regulation experiments\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KD/KO with defined secretome and chemotaxis phenotypes, c-Jun interaction documented; single lab\",\n      \"pmids\": [\"32314963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ERK3 directly acts as a guanine nucleotide exchange factor for CDC42 and phosphorylates ARP3 (subunit of ARP2/3 complex) at Ser418 to promote actin polymerization and filopodia formation. ERK3 protein bound directly to the purified ARP2/3 complex and augmented polymerization of actin in vitro. Depletion of ERK3 prevented CDC42/RAC1 activation, F-actin maintenance, filopodia formation, and epithelial cell migration.\",\n      \"method\": \"In vitro GEF assay (CDC42 nucleotide exchange), in vitro kinase assay (ARP3 phosphorylation at S418), in vitro actin polymerization assay with purified ARP2/3, ERK3 depletion with defined cytoskeletal phenotypes\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution (GEF assay, ARP3 kinase assay, actin polymerization), multiple orthogonal methods; single lab\",\n      \"pmids\": [\"37057894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBXW7 acts as an E3 ubiquitin ligase for ERK3, targeting it for ubiquitination-mediated proteasomal degradation. ERK3 binds FBXW7 through its C34D region, specifically at Thr417 and Thr421, which interact with the WD40 domain of FBXW7. T417A/T421A double mutation abrogates FBXW7-mediated ubiquitination. FBXW7 depletion restores ERK3 protein levels.\",\n      \"method\": \"Mammalian two-hybrid assay, co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, site-directed mutagenesis (T417A/T421A)\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two-hybrid, Co-IP, ubiquitination assay, mutagenesis; multiple orthogonal methods in single lab\",\n      \"pmids\": [\"35022544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EGLN3 catalyzes hydroxylation of ERK3, and this hydroxylation antagonizes chaperone-mediated autophagy (CMA)-mediated degradation of ERK3 by reducing ERK3 interaction with LAMP2A. ERK3 interacts with HSC70 and LAMP2A (core CMA components) and is degraded by the CMA-lysosome pathway; EGLN3-mediated hydroxylation blocks this.\",\n      \"method\": \"Co-immunoprecipitation (ERK3-HSC70 and ERK3-LAMP2A), hydroxylation assay, CMA-lysosome pathway assays, EGLN3 catalytically inactive knock-in mice\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of CMA components, enzymatic hydroxylation, genetic knock-in mice; mechanistic chain established but hydroxylation assay details limited in abstract\",\n      \"pmids\": [\"35124697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ERK3 and MK5 act in a linear pathway to control postnatal myogenic differentiation: ERK3 kinase-inactive mice (Mapk6KD/KD) have impaired skeletal muscle growth and regeneration. MK5 directly phosphorylates FoxO3, promoting its degradation and reducing FoxO3 association with MyoD. FoxO3 depletion partially rescues premature differentiation caused by ERK3/MK5 inactivation.\",\n      \"method\": \"Mapk6KD/KD (kinase-dead knock-in) mice, MK5 KO mice, C2C12/primary myoblast differentiation assays, MK5 in vitro kinase assay (FoxO3 phosphorylation), MK5 inhibitor, FoxO3 rescue experiments\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay for FoxO3 phosphorylation, genetic epistasis with KD knock-in and substrate rescue; multiple orthogonal methods\",\n      \"pmids\": [\"35141958\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Supervillin (SVIL) is a direct ERK3 substrate; ERK3 phosphorylates SVIL at Ser245 to regulate myosin II activation and cytokinesis completion. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild-type SVIL but not by non-phosphorylatable S245A mutant.\",\n      \"method\": \"Quantitative phosphoproteomics, in vitro kinase assay, site-directed mutagenesis (S245A), siRNA knockdown, cytokinesis and multinucleation assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — phosphoproteomics-guided substrate identification, in vitro kinase assay, mutagenesis rescue experiment, multiple orthogonal methods\",\n      \"pmids\": [\"36576983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The C-terminal halves of ERK2 and ERK3 (catalytic domain) are primarily responsible for their distinct subcellular localizations in resting cells. The N-terminal folding domain of ERK2 is required for its activation by MEK1, interaction with MEK1, and nuclear accumulation; swapping in the ERK3 N-terminal domain abrogates these. ERK3 is thus constitutively nuclear through a mechanism residing in its C-terminal catalytic domain half, independent of MEK.\",\n      \"method\": \"ERK2/ERK3 chimeric protein constructs, subcellular localization assays, MEK1 interaction assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chimeric domain-swap constructs with localization readout; single lab\",\n      \"pmids\": [\"11741894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"ERK3 expression is upregulated at the mRNA and protein level by proteasome inhibitors in a p38 pathway-dependent manner. p38 pathway kinase inhibitors prevent proteasome-dependent ERK3 induction. ERK3 upregulation is independent of p53, Bcl2, and caspase-3. Ectopic ERK3 expression increases cellular resistance to proteasome inhibition.\",\n      \"method\": \"Proteasome inhibitor treatment (peptide-based inhibitors, lactacystin), p38 inhibitors, Western blot, ERK3 ectopic expression, cell viability assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological perturbation with multiple inhibitors; p38 pathway link established indirectly; single lab\",\n      \"pmids\": [\"11148204\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"ERK3 activity is constitutively activated (~10-fold increase) in colon cancer cells stably transfected with PKC-beta1 or PKC-beta2, without a change in ERK3 protein level. TPA-induced downregulation of PKC activity reduces ERK3 activity, establishing a causal link. Activated ERK3 is found in nuclear and membrane fractions in PKC-beta transfectants.\",\n      \"method\": \"Stable transfection, immune complex kinase assay, in-gel kinase assay, TPA-mediated PKC downregulation, subcellular fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple cell lines and rescue with TPA; indirect causal link via PKC; single lab\",\n      \"pmids\": [\"8626698\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ERK3/MAPK6 expression is upregulated by BRAF(V600E) signaling via MEK1/2; pharmacological inhibition of BRAF or MEK1/2, or RNAi-mediated BRAF knockdown, leads to rapid ERK3 protein degradation in melanoma cells.\",\n      \"method\": \"Conditional BRAF(V600E) expression system, MEK inhibitors, BRAF RNAi, Western blot, microarray\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological and genetic perturbation with protein stability readout; multiple approaches corroborating MEK→ERK3 regulation\",\n      \"pmids\": [\"16964379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ERK3 associates with microtubule-associated protein MAP2 in pancreatic beta cells; PMA-induced ERK3 phosphorylation is accompanied by increased ERK3/MAP2 association and MAP2 phosphorylation. Antisense oligonucleotide-mediated ERK3 knockdown abolishes glucose-stimulated insulin secretion and PMA-induced insulin secretion, indicating ERK3 is required for stimulus-secretion coupling in beta cells.\",\n      \"method\": \"Co-immunoprecipitation (ERK3-MAP2), antisense oligonucleotide knockdown, insulin secretion assays, immunohistochemistry (beta cell-specific expression)\",\n      \"journal\": \"Molecular and cellular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and functional knockdown; single lab, limited mechanistic depth on MAP2 phosphorylation consequence\",\n      \"pmids\": [\"16597486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ERK3 localizes to spindle fibers and asters in mouse oocytes during meiosis I–II. Deletion of ERK3 by morpholino injection causes oocyte arrest at MI with impaired spindles, misaligned chromosomes, persistent BubR1 on kinetochores, disrupted kinetochore-microtubule attachments, and failure of homologous chromosome segregation.\",\n      \"method\": \"ERK3 morpholino microinjection in mouse oocytes, immunofluorescence (spindle localization, BubR1, α-tubulin), chromosome spreading, low-temperature kinetochore-MT stability assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — morpholino KD with defined meiotic phenotypes, direct localization by immunofluorescence; single lab\",\n      \"pmids\": [\"20927325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ERK3 promotes endothelial cell migration, proliferation, and tube formation by upregulating SRC-3/SP1-mediated VEGFR2 expression. The mechanism involves ERK3-stimulated formation of a transcriptional complex containing SRC-3, SP-1, and CBP. ERK3 gene expression is upregulated by cytokines through c-Jun binding to the ERK3 gene promoter.\",\n      \"method\": \"Co-immunoprecipitation (SRC-3/SP-1/CBP complex), VEGFR2 promoter reporter assays, ERK3 knockdown, endothelial migration/proliferation/tube formation assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — transcriptional complex Co-IP, reporter assays, functional KD; single lab\",\n      \"pmids\": [\"24585635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Activation loop phosphorylation (Ser189) is important for ERK3 kinase activity toward SRC-3: S189A mutation greatly decreases ERK3 kinase activity toward SRC-3 and reduces the ability to promote lung cancer cell migration, invasion, and MMP expression. A kinase-inactive ERK3 mutant still promotes invasion to a lesser extent, indicating both kinase-dependent and kinase-independent mechanisms contribute to ERK3-driven invasiveness.\",\n      \"method\": \"In vitro kinase assay (S189A and kinase-dead mutants vs. SRC-3 substrate), lung cancer cell migration/invasion assays, MMP expression analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with multiple mutants, correlated with cell-based functional readouts; builds on established ERK3-SRC3 substrate axis\",\n      \"pmids\": [\"30166347\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Intracellular acidification markedly increases ERK3 half-life, while alkalinization accelerates its degradation. This pH-dependent regulation is rapid, reversible, and cell-type consistent. A region in ERK3's C-terminus contains pH-sensing motifs responsible for this regulation.\",\n      \"method\": \"Intracellular pH manipulation, protein half-life measurements, quantitative proteomics, C-terminus deletion/mutant analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cell types, pharmacological pH manipulation, proteomics; pH-sensing motif defined but in abstract with limited mutagenesis detail\",\n      \"pmids\": [\"41123996\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ERK3 interacts with DGKζ (diacylglycerol kinase ζ) via its C34 domain; DGKζ binds to the N-terminal and C1 domains of ERK3. Co-overexpression of DGKζ and ERK3 completely blocks ERK3-promoted lung cancer cell migration; DGKζ requires the C34 domain of ERK3 to prevent ERK3-mediated migration.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vitro binding assay, co-localization, ERK3 domain deletion mutants, lung cancer cell migration assays\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP confirmed by in vitro binding and co-localization, domain mapping, functional migration assay; single lab\",\n      \"pmids\": [\"37287450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ERK3 L290P/V cancer mutations enhance ERK3's cytoplasmic localization by increasing interaction with the nuclear export factor CRM1, thereby increasing migration/invasion-promoting capability without clear effects on intrinsic kinase activity.\",\n      \"method\": \"Site-directed mutagenesis (L290P, L290V), CRM1 co-immunoprecipitation, subcellular fractionation, migration/invasion assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and fractionation with multiple mutants; functional migration assay; single lab\",\n      \"pmids\": [\"29101390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ERK3 interacts with Snail and enhances Snail protein stability by inhibiting the binding of FBXO11 (an E3 ubiquitin ligase) to Snail, thereby preventing Snail ubiquitination and degradation. ERK3 does not directly phosphorylate Snail.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, protein stability assay, negative result for direct phosphorylation\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP showing competition for FBXO11 binding, ubiquitination assay; single lab; direct phosphorylation explicitly negative\",\n      \"pmids\": [\"38201533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TRIM21 (ubiquitin ligase) binds MAPK6 and promotes its ubiquitin-proteasome degradation in endothelial cells exposed to disturbed shear stress. Endothelial MAPK6 regulates inflammation via the EGR1/CXCL12 axis; endothelium-specific MAPK6 knockout increases plaque area in ApoE-/- mice, reversible by CXCL12 neutralization.\",\n      \"method\": \"Co-immunoprecipitation (TRIM21-MAPK6), RNA-seq, proteomic analysis, endothelium-specific Mapk6 knockout mice, AAV-MAPK6 overexpression, CXCL12 neutralization rescue\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identifying TRIM21, conditional KO mice with atherosclerosis phenotype, pathway rescue; single lab\",\n      \"pmids\": [\"39763069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ERK3 localizes to the cell periphery of breast cancer cells during adhesion to collagen I. ERK3 overexpression reduces cell spread area and increases migration speed. Importantly, a kinase-inactive ERK3 mutant phenocopies wild-type ERK3 overexpression in reducing spread area and increasing migration, revealing a kinase-independent function.\",\n      \"method\": \"Live cell imaging, ERK3 overexpression (WT and kinase-dead), siRNA knockdown, morphological analysis during cell adhesion\",\n      \"journal\": \"Cell adhesion & migration\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — localization and kinase-dead mutant analysis; single lab, limited pathway placement\",\n      \"pmids\": [\"26588708\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The C-terminus tail of ERK3 is required for full kinase activity toward SRC-3 and for ERK3-promoted cancer cell migration/invasion. Septin 7, which interacts with ERK3 via its C-terminus tail, acts as a downstream effector for ERK3-induced cancer cell migration.\",\n      \"method\": \"C-terminus deletion mutants, in vitro kinase assay (SRC-3 substrate), septin 7 knockdown, migration/invasion assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — in vitro kinase assay with deletion mutants, functional migration assay with septin 7 KD; single lab\",\n      \"pmids\": [\"32516969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"ERK3 specifically co-elutes with B-Raf (but not c-Raf1) after anion exchange chromatography of rat hippocampal lysates; ERK3 is released from B-Raf immunoprecipitates upon ATP incubation, suggesting a specific association of ERK3 with B-Raf in rat hippocampus.\",\n      \"method\": \"Anion exchange chromatography, co-immunoprecipitation, ATP-dependent dissociation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-fractionation and Co-IP experiment; single lab, not followed up mechanistically\",\n      \"pmids\": [\"8954940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ERK3 expression is induced in CD4+ and CD8+ T cells following TCR stimulation, requiring ERK1/2 activation. ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. ERK3-deficient T cells show decreased proliferation and impaired cytokine secretion following stimulation with low-dose anti-CD3.\",\n      \"method\": \"ERK3-/- mice, T cell activation assays, ERK1/2 inhibitor treatment, co-immunoprecipitation (ERK3-MK5 in activated T cells), cytokine measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined immune phenotype, ERK1/2 epistasis via inhibitor, Co-IP; single lab\",\n      \"pmids\": [\"24475167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ERK3 regulates epithelial architecture in Xenopus embryos and human breast epithelial cells: ERK3 knockdown impairs adherens and tight-junction protein distribution and tight-junction barrier function. ERK3 is required for full activation of TFAP2A (AP-2α)-dependent transcription, and TFAP2A knockdown phenocopies ERK3 knockdown.\",\n      \"method\": \"Xenopus ERK3 knockdown (morpholino), human epithelial cell ERK3 KD, microarray gene expression, tight-junction barrier assay, TFAP2A luciferase reporter\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two model systems (Xenopus and human cells), barrier assay, reporter assay; single lab\",\n      \"pmids\": [\"29674317\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of the ERK3 kinase domain was determined, revealing a distinct ATP binding pocket compared to ERK2, particularly in the A-loop, GC-loop, and αC-helix conformations. The structure also indicates a potential structural link toward MK5 interaction via the FHIEDE motif.\",\n      \"method\": \"X-ray crystallography (ERK3 kinase domain), small molecule inhibitor screening with biochemical and cellular assays (NanoBRET)\",\n      \"journal\": \"Bioorganic & medicinal chemistry letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with inhibitor binding mode defined; functional connection to MK5 motif is structural inference; single lab\",\n      \"pmids\": [\"32927028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of the ERK3 kinase domain was determined providing molecular insights into its distinct ATP binding pocket relative to ERK2, explaining differences in inhibitor binding properties.\",\n      \"method\": \"X-ray crystallography (ERK3 kinase domain and CLK1 in complex with inhibitor CAF052), medium-scale small molecule screening\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure determined; functional mechanism inferred from structure alone; single lab\",\n      \"pmids\": [\"33114754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ERK3 is expressed in atrial and ventricular cardiac fibroblasts (not myocytes). ERK3 co-immunoprecipitates with MK5 in heart lysates. ERK3 haploinsufficiency reduces cardiac hypertrophy and Col1a1 mRNA increase after transverse aortic constriction. ERK3 knockdown reduces TGF-β-induced collagen expression and impairs myofibroblast motility.\",\n      \"method\": \"Co-immunoprecipitation (heart lysates), ERK3+/- mice with TAC model, siRNA knockdown in primary cardiac fibroblasts, collagen assays, motility assays\",\n      \"journal\": \"Physiological reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic haploinsufficiency with defined cardiac phenotype, co-IP in tissue, siRNA in primary cells; single lab\",\n      \"pmids\": [\"38872461\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ERK3 (MAPK6) is an atypical MAPK with constitutive nuclear/cytoplasmic distribution controlled by CRM1-dependent nuclear export and regulated by intracellular pH; it is activated by group I PAKs phosphorylating its unique SEG activation loop at Ser189, and it signals primarily through a direct interaction with MK5 (via the FRIEDE docking motif) to form a kinase-scaffold complex that drives MK5 activation, nuclear export, and downstream phosphorylation of substrates including FoxO3 (regulating myogenesis), FOXO1/ATGL (regulating lipolysis), supervillin/Ser245 (regulating cytokinesis), and CDC42/ARP3 (regulating actin dynamics); ERK3 also directly phosphorylates SRC-3/Ser857 to promote invasiveness, TDP2/Ser60 to enhance DNA damage repair, and AKT/Ser473 to promote tumor growth, while its protein stability is controlled by ubiquitin-mediated proteasomal degradation through FBXW7 and TRIM21, by CMA-lysosome pathway involving HSC70/LAMP2A counteracted by EGLN3 hydroxylation, and by Cdk1-mediated C-terminal phosphorylation stabilizing it in mitosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAPK6 (ERK3) is an atypical mitogen-activated protein kinase that functions as a constitutively active signaling kinase whose biology is dominated by its activation loop, its regulated nucleocytoplasmic distribution, and its tightly controlled protein stability [#2, #3]. Unlike conventional MAP kinases, ERK3 carries a single phospho-acceptor (Ser189) in its SEG activation loop that is phosphorylated by a dedicated upstream activity rather than by MEK; group I PAKs (PAK1/2/3) directly phosphorylate Ser189 downstream of Rac1, stimulating intrinsic catalytic activity [#5, #6, #8], while DUSP2 reverses this phosphorylation and damps signaling [#11]. ERK3 shuttles between nucleus and cytoplasm via CRM1-dependent export, and this shuttling is required for its effects on cell cycle progression [#3]. Its principal effector axis is a kinase-scaffold complex with MK5/PRAK formed through the non-canonical FRIEDE docking motif in the C-terminal L16 extension; complex formation depends on Ser189 phosphorylation and drives MK5 activation and co-redistribution to the cytoplasm, with ERK3 and MK5 mutually stabilizing one another [#0, #1, #4, #7]. Through this module ERK3 controls myogenic differentiation via MK5-mediated FoxO3 phosphorylation [#21] and adipocyte lipolysis via a FOXO1/ATGL program [#15]. Beyond MK5, ERK3 directly phosphorylates substrates governing the cytoskeleton and cell division — supervillin at Ser245 to enable cytokinesis [#22], and ARP3 at Ser418 while acting as a CDC42 GEF to drive actin polymerization and filopodia formation [#18] — and phosphorylates SRC-3 at Ser857 to promote MMP expression and cancer cell invasion [#9, #30]. ERK3 abundance is rate-limiting and is set by multiple degradative routes, including FBXW7- and TRIM21-mediated proteasomal degradation [#19, #35], chaperone-mediated autophagy through HSC70/LAMP2A that is antagonized by EGLN3 hydroxylation [#20], and intracellular pH sensing through its C-terminus [#31]; conversely, Cdk1 phosphorylation of four C-terminal sites stabilizes ERK3 during mitosis [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing ERK3 as an atypical MAP kinase required defining its activation site and constitutive localization, distinguishing it from classical ERKs.\",\n      \"evidence\": \"In vitro kinase assays with D171A/S189 mutants plus subcellular fractionation and immunofluorescence\",\n      \"pmids\": [\"8621539\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the physiological upstream kinase for Ser189\", \"No substrate beyond autophosphorylation identified at this stage\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Biochemical purification showed Ser189 is phosphorylated by a specific cellular kinase distinct from MEK1/2, indicating ERK3 uses a non-canonical activation route.\",\n      \"evidence\": \"Biochemical purification, in vitro kinase assay, PP2A treatment, ERK3/ERK2 chimeras\",\n      \"pmids\": [\"8662649\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The kinase was not molecularly identified here\", \"Physiological signals controlling this activity unknown at the time\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Domain-swap experiments localized the determinant of ERK3's MEK-independent constitutive nuclear localization to its catalytic-domain half, explaining its divergence from ERK2.\",\n      \"evidence\": \"ERK2/ERK3 chimeric constructs with localization and MEK1 interaction readouts\",\n      \"pmids\": [\"11741894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific export/import signals not mapped here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defining CRM1-dependent export established that ERK3 shuttling, not static localization, governs its cell-cycle effects.\",\n      \"evidence\": \"Leptomycin B, CRM1 overexpression, GST pulldown, cell cycle analysis\",\n      \"pmids\": [\"12915405\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking shuttling to cell cycle arrest not resolved\", \"Direct nuclear export signal not mapped\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of MK5/PRAK as the direct ERK3 partner and substrate defined the core effector axis and revealed mutual protein stabilization.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro kinase assay, siRNA, ERK3-/- fibroblasts, MK5 KO mice (two companion papers)\",\n      \"pmids\": [\"15577943\", \"15538386\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological downstream substrates of activated MK5 not yet defined\", \"Whether MK5 activation is strictly kinase-dependent vs scaffolding was contested between studies\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing that activation-loop phosphorylation drives stable ERK3–MK5 complex formation and cytoplasmic redistribution linked Ser189 status to effector engagement.\",\n      \"evidence\": \"Phospho-specific antibodies, in vitro kinase assay, MK5 interaction and fractionation assays\",\n      \"pmids\": [\"18720373\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the upstream kinase\", \"Quantitative stoichiometry of the complex not determined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defining the FRIEDE docking motif explained how ERK3/ERK4 recruit MK5 through a non-canonical mechanism independent of the canonical CD domain.\",\n      \"evidence\": \"Peptide overlay, FRIEDE mutagenesis, Co-IP, MK5 translocation assays\",\n      \"pmids\": [\"19473979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of FRIEDE–MK5 contact not solved here\", \"Whether other partners use this motif unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying group I PAKs as the Ser189 kinase placed ERK3 downstream of Rac1, completing a PAK–ERK3–MK5 pathway.\",\n      \"evidence\": \"Biochemical purification, in vitro kinase assay, RNAi of PAK1/2/3, phospho-specific antibodies, activated Rac1\",\n      \"pmids\": [\"21177870\", \"21317288\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream receptors/signals engaging Rac1-PAK toward ERK3 in physiological contexts not fully mapped\", \"Whether other kinases contribute to Ser189 in vivo\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Cdk1-mediated C-terminal phosphorylation, reversed by Cdc14 phosphatases, was shown to stabilize ERK3 during mitosis, revealing cell-cycle control of ERK3 abundance.\",\n      \"evidence\": \"In vitro kinase assay with cyclin B-Cdk1, MS phosphosite mapping, Cdc14 binding, pulse-chase, mutagenesis\",\n      \"pmids\": [\"20236090\", \"18235225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of mitotic ERK3 stabilization on substrates unclear\", \"Cdc14 in vivo relevance limited\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery of SRC-3/Ser857 as a direct substrate connected ERK3 to a transcriptional program promoting invasion, extending its role beyond the MK5 axis.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, S857A mutagenesis, knockdown, xenograft, MMP assays\",\n      \"pmids\": [\"22505454\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SRC-3 phosphorylation occurs in non-cancer contexts unknown\", \"Relative contribution vs MK5 axis not quantified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extending the ERK3/MK5 module to septin7 and neuronal targets linked it to cytoskeletal and dendritic spine development.\",\n      \"evidence\": \"Interaction screens, Co-IP, in vitro kinase assay, MK5 KO mice, primary neuron transfection\",\n      \"pmids\": [\"22508986\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ERK3 substrate among these targets vs MK5-mediated effects not fully separated\", \"In vivo ERK3-specific neuronal phenotype not tested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identifying DUSP2 as a direct phosphatase that dephosphorylates the ERK3/ERK4 activation loop established negative regulation of the pathway.\",\n      \"evidence\": \"Co-IP, in vitro phosphatase assay, domain mapping, MK5 activation and DUSP2 stabilization assays\",\n      \"pmids\": [\"28252035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals inducing DUSP2 toward ERK3 not defined\", \"Single study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating Ser189-dependence of ERK3 activity toward SRC-3, alongside residual kinase-independent invasion, revealed dual mechanisms underlying ERK3-driven invasiveness.\",\n      \"evidence\": \"In vitro kinase assays with S189A and kinase-dead mutants, migration/invasion and MMP assays\",\n      \"pmids\": [\"30166347\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of the kinase-independent component undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linking the ERK3/MK5 module to FoxO3 phosphorylation in muscle and to FOXO1/ATGL in adipocytes mapped tissue-level physiological outputs of the pathway.\",\n      \"evidence\": \"Mapk6KD/KD and MK5 KO mice, adipocyte-specific ERK3 KO, in vitro kinase assay, lipolysis and differentiation assays\",\n      \"pmids\": [\"35141958\", \"32139423\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether FoxO3/FOXO1 are direct ERK3 vs MK5 substrates fully delineated\", \"Crosstalk between metabolic and myogenic outputs untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identification of supervillin/Ser245 as a direct substrate connected ERK3 kinase activity to cytokinesis completion and ploidy control.\",\n      \"evidence\": \"Phosphoproteomics, in vitro kinase assay, S245A mutagenesis rescue, cytokinesis/multinucleation assays\",\n      \"pmids\": [\"36576983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals timing ERK3 activity to cytokinesis unknown\", \"In vivo relevance of multinucleation phenotype untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defining FBXW7-mediated proteasomal degradation and EGLN3-regulated chaperone-mediated autophagy established that ERK3 abundance is controlled by parallel degradative routes.\",\n      \"evidence\": \"Two-hybrid, Co-IP, ubiquitination assays, T417A/T421A mutagenesis, CMA-component Co-IP, EGLN3 knock-in mice\",\n      \"pmids\": [\"35022544\", \"35124697\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative flux through proteasome vs CMA in different cell states unclear\", \"Signals selecting one route over the other undefined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showing ERK3 acts as a CDC42 GEF and phosphorylates ARP3/Ser418 directly coupled ERK3 to actin nucleation and filopodia formation, broadening its catalytic repertoire.\",\n      \"evidence\": \"In vitro GEF assay, ARP3 in vitro kinase assay, actin polymerization with purified ARP2/3, ERK3 depletion phenotypes\",\n      \"pmids\": [\"37057894\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for GEF activity in a kinase fold undefined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrating intracellular pH-dependent control of ERK3 half-life via C-terminal pH-sensing motifs added a metabolic/microenvironmental layer to ERK3 stability regulation.\",\n      \"evidence\": \"Intracellular pH manipulation, half-life measurements, quantitative proteomics, C-terminus mutants\",\n      \"pmids\": [\"41123996\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular identity of pH-sensing residues only partially defined\", \"Connection to the FBXW7/CMA degradation machinery not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple direct substrates (MK5, SRC-3, ARP3, supervillin, AKT, TDP2) and the kinase-independent functions are coordinated, and what receptor-proximal signals select among ERK3's degradative versus stabilizing pathways, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying model integrating direct substrates with the MK5 scaffold axis\", \"Physiological triggers gating ERK3 stability vs degradation undefined\", \"Kinase-independent mechanisms remain mechanistically opaque\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 9, 18, 22, 30]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [2, 5, 18, 22]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [2, 41]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [18, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 3, 23]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [18, 28, 36]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [25, 36]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 7]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 12, 22]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [12, 19, 20]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [9, 30, 33]}\n    ],\n    \"complexes\": [\n      \"ERK3–MK5 complex\",\n      \"ERK3/MK5/septin7 ternary complex\"\n    ],\n    \"partners\": [\n      \"MK5\",\n      \"PAK1\",\n      \"PAK2\",\n      \"CRM1\",\n      \"DUSP2\",\n      \"FBXW7\",\n      \"SRC-3\",\n      \"TRIM21\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}