{"gene":"MAP2K5","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1995,"finding":"MEK5 was isolated as a novel MEK family member that does not phosphorylate ERK1, ERK2, ERK3, JNK/SAPK, or p38/HOG1, and is not highly phosphorylated by Raf-1, c-Mos, or MEKK1, indicating it lies in a distinct MAP kinase pathway. Alternative splicing produces a 50-kDa alpha isoform (expressed in liver and brain, particulate/associated with actin cytoskeleton-like domain) and a 40-kDa beta isoform (ubiquitous, primarily cytosolic), with the N-terminal 23-aa exon of MEK5alpha directing differential subcellular localization.","method":"PCR cloning, in vitro kinase assays, subcellular fractionation, Northern/Western blotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assays with multiple substrates and upstream kinases, fractionation for localization; foundational characterization paper replicated by subsequent work","pmids":["7499418"],"is_preprint":false},{"year":1999,"finding":"MEKK3 physically interacts with MEK5 (identified by yeast two-hybrid and confirmed by co-immunoprecipitation in mammalian cells), and constitutively active MEKK3 stimulates BMK1/ERK5 activity through MEK5. Dominant-active MEKK3 is required for growth factor-mediated activation of endogenous BMK1, placing MEKK3 as a direct upstream kinase of MEK5 in the BMK1/ERK5 pathway.","method":"Yeast two-hybrid, co-immunoprecipitation, dominant-active/dominant-negative overexpression, kinase activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus reciprocal co-IP plus functional epistasis with dominant mutants; replicated by subsequent PB1 domain studies","pmids":["10593883"],"is_preprint":false},{"year":1999,"finding":"The ERK5/MEK5 pathway is required for Raf-dependent cellular transformation. Constitutively active MEK5 (MEK5DD) synergizes with Raf to transform NIH 3T3 cells. Endogenous Raf-1 binds specifically to endogenous ERK5 (but not ERK2 or SAPK), suggesting regulatory protein-protein interactions contribute to Ras/Raf activation of ERK5.","method":"Co-immunoprecipitation of endogenous proteins, NIH 3T3 transformation assays, dominant-active MEK5 overexpression, epistasis analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — endogenous co-IP plus functional transformation assay with constitutively active MEK5; multiple orthogonal methods in one study","pmids":["10531364"],"is_preprint":false},{"year":2000,"finding":"MEKK2 binds MEK5 via yeast two-hybrid and co-immunoprecipitation, and MEKK2 expression stimulates BMK1/ERK5 activity. MEKK2 activates BMK1/ERK5 to a greater extent than MEKK3 and with higher apparent affinity for MEK5. Dominant-negative MEK5 blocks MEKK2-induced BMK1/ERK5 activation without affecting JNK, demonstrating MEK5 is a specific downstream effector of MEKK2. In D10 T cells, dominant-negative MEKK2 (but not MEKK3) inhibits BMK1/ERK5, showing cell-type-specific usage of upstream kinases.","method":"Yeast two-hybrid, co-immunoprecipitation, dominant-negative/constitutively active overexpression, kinase activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus co-IP plus functional epistasis with dominant mutants; independently corroborated by MEKK3 study","pmids":["11073940"],"is_preprint":false},{"year":2001,"finding":"MEK5 induces eccentric cardiac hypertrophy with serial sarcomere assembly in cardiomyocytes in vitro. Constitutively active MEK5 causes elongated cardiomyocyte morphology, and dominant-negative MEK5 specifically blocks LIF-induced cardiomyocyte elongation and fetal gene expression without blocking other LIF hypertrophic responses. Cardiac-specific transgenic expression of activated MEK5 in mice produces eccentric cardiac hypertrophy progressing to dilated cardiomyopathy and sudden death.","method":"Adenoviral overexpression of constitutively active and dominant-negative MEK5 in cardiomyocytes; cardiac-specific transgenic mice; morphological and gene expression analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro loss/gain-of-function plus in vivo transgenic mouse model, multiple orthogonal phenotypic readouts","pmids":["11387209"],"is_preprint":false},{"year":2001,"finding":"Atypical protein kinase C isoforms (zetaPKC and lambda/iotaPKC) interact with MEK5 in an EGF-inducible manner, and this interaction is required and sufficient for MEK5 activation in response to EGF. The aPKC-MEK5 interaction depends on the aPKC interaction domain (acidic amino acid stretch) present in MEK5. aPKC activation of MEK5 leads to MEF2C-element-dependent Jun promoter activation.","method":"Co-immunoprecipitation, dominant-negative/constitutively active overexpression, luciferase reporter assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional reporter assay, single lab, two orthogonal methods","pmids":["11158308"],"is_preprint":false},{"year":2003,"finding":"The MEK5-ERK5 pathway phosphorylates and stabilizes c-Fos and Fra-1. Phosphorylation of c-Fos by ERK5 (and downstream kinases) occurs at sites distinct from ERK1/2-dependent sites, and MEK5-ERK5 pathway activation markedly increases c-Fos transactivation activity. The C-terminal half of ERK5 is required for maximal transactivation of c-Fos and Fra-1.","method":"Constitutively active MEK5 overexpression, phosphorylation analysis, mutagenesis of ERK5, luciferase/transactivation assays","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional mutagenesis plus reporter assays, single lab, two orthogonal methods","pmids":["12622723"],"is_preprint":false},{"year":2003,"finding":"PB1 domains of MEKK2 and MEKK3 bind the PB1 domain of MEK5 but do not significantly homo- or heterodimerize with each other in vitro. Co-immunoprecipitation confirms MEKK2-MEK5 complex formation in vivo. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 interaction. Expression of free MEKK2 or MEKK3 PB1 domains inhibits ERK5 activation specifically (not p38 or JNK), demonstrating that PB1 domain-mediated association is required for MEK5 activation by MEKK2/3.","method":"In vitro PB1 domain binding assays, co-immunoprecipitation, PB1 deletion/mutation analysis, kinase activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro binding assays plus co-IP plus mutagenesis plus functional pathway inhibition; multiple orthogonal methods","pmids":["12912994"],"is_preprint":false},{"year":2003,"finding":"MEK5 overexpression in prostate cancer cells stimulates proliferation, motility, invasion, and selectively increases MMP-9 (but not MMP-2) mRNA expression. Luciferase and EMSA assays show MEK5 activates the MMP-9 promoter via AP-1 (not NF-κB) binding sites, and MEK5 enhances AP-1 transcriptional activity approximately two-fold.","method":"MEK5 transfection, proliferation/invasion assays, RT-PCR, luciferase reporter assays, EMSA","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function overexpression with multiple functional assays plus reporter/EMSA, single lab","pmids":["12618764"],"is_preprint":false},{"year":2004,"finding":"MEK5 and ERK5 are localized in the nucleus of both resting and EGF-stimulated HeLa and Rat-1 cells (endogenous proteins), where they are bound to detergent-resistant nuclear moieties. Exogenous overexpressed ERK5 localizes to the cytosol, revealing saturation of nuclear anchors by endogenous ERK5. Upon EGF stimulation, MEK5 is released from nuclear anchors but remains nuclear, while MEKK2 translocates from cytosol to nucleus, enabling signal transmission to nuclear MEK5.","method":"Immunofluorescence microscopy of endogenous proteins, in situ detergent (NP-40) extraction fractionation, EGF stimulation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct subcellular localization by immunofluorescence plus fractionation with NP-40 extraction, single lab, two orthogonal methods","pmids":["15075238"],"is_preprint":false},{"year":2004,"finding":"NMR structure of the PKCiota PB1 domain reveals a ubiquitin fold with an OPCA motif forming an acidic surface that interacts with the basic surface of target PB1 domains (including MEK5). Mutational analysis of the PKCiota-ZIP/p62 PB1-PB1 interaction confirms the acidic surface is critical for binding. The PKCiota PB1 domain can interact with targets via either its acidic (OPCA) surface or a conserved lysine on the opposite face.","method":"NMR structure determination, mutagenesis, PB1 domain interaction analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — NMR structure with mutagenesis for the PKCiota PB1 domain; MEK5 mentioned as interacting partner but direct MEK5 mutagenesis confirmation not described in abstract","pmids":["15143057"],"is_preprint":false},{"year":2005,"finding":"Targeted deletion of mek5 in mice results in embryonic lethality at ~E10.5 with abnormal cardiac development, decreased proliferation, and increased apoptosis in heart, head, and dorsal regions. MEK5-deficient mouse embryonic fibroblasts show impaired ERK5 activation and reduced MEF2 transcriptional activity, and are sensitized to sorbitol-induced caspase-3 activity. MEK5 is established in vivo as the essential activator of ERK5 and a required regulator of cell survival.","method":"Targeted gene knockout in mice, phenotypic analysis, MEF2 transcriptional reporter assays, caspase-3 activity assays in MEFs","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with multiple orthogonal phenotypic and molecular readouts; in vivo validation","pmids":["15601854"],"is_preprint":false},{"year":2005,"finding":"MEK5-ERK5 pathway activation is responsible for biliary epithelial cell hyperproliferation in PCK rats (Caroli's disease model). MEK5 is overexpressed in PCK rat biliary epithelial cells, leading to increased ERK5 phosphorylation. siRNA knockdown of MEK5 significantly inhibits the abnormal biliary cell proliferation, while MEK1/2 inhibitors (PD98059, U0126) are less effective.","method":"siRNA knockdown of MEK5, Western blotting for ERK5 phosphorylation, cell proliferation assays, pharmacological inhibitor comparison","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with functional proliferation readout, single lab, two orthogonal methods (genetic + pharmacological)","pmids":["15631999"],"is_preprint":false},{"year":2005,"finding":"MEK5-ERK5 pathway is required for neural differentiation in Xenopus embryos. Knockdown of Xenopus MEK5 or ERK5 with antisense morpholino oligonucleotides reduces head structure and inhibits neural differentiation. Forced activation of MEK5-ERK5 alone is sufficient to induce neural differentiation. MEK5-ERK5 is necessary for SoxD neuralizing activity and sufficient for expression of the proneural gene Xngnr1, placing MEK5-ERK5 downstream of SoxD and upstream of Xngnr1.","method":"Antisense morpholino knockdown in Xenopus embryos, constitutively active MEK5-ERK5 overexpression, genetic epistasis analysis","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function (morpholino) plus gain-of-function plus epistasis in intact vertebrate embryo; multiple orthogonal approaches","pmids":["16179948"],"is_preprint":false},{"year":2007,"finding":"MEKK2 and MEK5 PB1 domains interact in a front-to-back arrangement: basic residues on the MEKK2 PB1 front bind to acidic clusters on the MEK5 PB1 back. A 34-amino-acid C-terminal extension of the MEK5 PB1 domain constitutes an ERK5 docking site required for MEK5 activation of ERK5. In quiescent cells, MEKK2 preferentially binds MEK5; upon MEKK2 activation, it switches to bind MKK7 via its PB1 acidic cluster, leading to JNK activation.","method":"PB1 domain mutagenesis, co-immunoprecipitation, kinase activation assays, domain-binding specificity analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — detailed mutagenesis of PB1 interaction surfaces plus co-IP plus functional kinase assays; mechanistic detail replicated across multiple studies","pmids":["17452462"],"is_preprint":false},{"year":2007,"finding":"NMR solution structure of MEKK3 PB1 domain reveals a ubiquitin fold with prolyl isomerization at Gln38-Pro39 producing two structural conformers. MEKK3 PB1 binds MEK5 PB1 with Kd ~10^-8 M. Mutagenesis identifies Lys7 and Arg5 in the MEKK3 PB1 basic cluster as critical residues for interaction with MEK5 PB1.","method":"NMR structure determination, backbone dynamics, mutagenesis, binding affinity measurement","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure determination with mutagenesis and quantitative binding measurement; single lab but rigorous structural methods","pmids":["17985933"],"is_preprint":false},{"year":2008,"finding":"Constitutively active MEK5alpha (CA-MEK5alpha) inhibits ERK5 SUMOylation independently of MEK5 kinase activity but dependently on MEK5-ERK5 physical association. H2O2 and high glucose induce ERK5 SUMOylation (mediated by E2 conjugase Ubc9 and E3 ligase PIAS1), which represses ERK5 transcriptional activity. CA-MEK5alpha transgenic mice show protection against DM-exacerbated LV dysfunction and apoptosis after MI by preventing ERK5 SUMOylation.","method":"Kinase-dead MEK5 mutants, SUMOylation assays, siRNA knockdown of PIAS1, CA-MEK5alpha transgenic mice, cardiac function measurement post-MI","journal":"Circulation research","confidence":"High","confidence_rationale":"Tier 2 / Strong — kinase-dead mutagenesis separates kinase activity from protein-protein interaction, multiple orthogonal approaches (mutagenesis, siRNA, transgenic mice), single lab","pmids":["18467627"],"is_preprint":false},{"year":2008,"finding":"MEK5/ERK5-mediated EMT in TNF-alpha-resistant MCF-7 breast cancer cells involves upregulation of vimentin, GSTP1, CKB and EMT regulators SNAI2/ZEB1/N-cadherin, and downregulation of keratins and E-cadherin. RNA interference targeting ERK5 reverses MEK5-mediated EMT gene expression, establishing ERK5 as required downstream effector of MEK5 in this context.","method":"2D gel proteomics, LC-MS/MS, RT-PCR, immunofluorescence, shRNA knockdown of ERK5","journal":"Breast cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — shRNA epistasis to show ERK5 dependence, multiple molecular readouts, single lab","pmids":["19087274"],"is_preprint":false},{"year":2008,"finding":"MEK5-selective inhibitor BIX02188 completely reverses flow-mediated inhibition of TNF-induced JNK activation in endothelial cells, while MEK1 inhibitor PD184352 (at ERK1/2-blocking concentrations) has no effect. This establishes MEK5-BMK1 (ERK5) as the required pathway mediating atheroprotective flow inhibition of JNK, distinct from MEK1-ERK1/2.","method":"Pharmacological inhibitor (BIX02188 vs PD184352) comparison, shear stress application, JNK activity assays in endothelial cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — selective inhibitor comparison demonstrating pathway specificity, single lab, functional cellular readout","pmids":["18358237"],"is_preprint":false},{"year":2008,"finding":"BIX02188 and BIX02189 are selective inhibitors of MEK5 catalytic function that block ERK5 phosphorylation without affecting ERK1/2 phosphorylation in sorbitol-stimulated HeLa cells, and inhibit MEF2C transcriptional activation downstream of MEK5/ERK5.","method":"In vitro kinase assay with purified MEK5 enzyme, cellular ERK5/ERK1/2 phosphorylation assays, MEF2C trans-reporter assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzyme assay with purified MEK5 plus cellular selectivity assays plus reporter assay; multiple orthogonal methods","pmids":["18834865"],"is_preprint":false},{"year":2009,"finding":"Constitutive activation of MEK5/ERK5 signaling in endothelial cells strongly inhibits cell migration and increases focal contact number/size with altered actin organization, due to decreased expression of p130Cas, a key regulator of directed cell migration. This identifies MEK5/ERK5 as a regulator of endothelial cell migration and focal contact turnover.","method":"Retroviral constitutively active MEK5 overexpression, migration assays, Western blotting for p130Cas, fluorescence microscopy of focal contacts and actin","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — constitutively active kinase with defined molecular target (p130Cas) and functional migration readout, single lab","pmids":["19605361"],"is_preprint":false},{"year":2009,"finding":"MEK5 acts as a downstream mediator of VEGF and antagonizes Epac/Rap1 anti-angiogenic signaling by inducing Id1 and suppressing TSP1 expression in endothelial cells.","method":"Overexpression studies, Western blotting, in vivo angiogenesis assays, epistasis analysis","journal":"Blood","confidence":"Low","confidence_rationale":"Tier 3 / Weak — overexpression with molecular readouts, single lab, limited mechanistic detail on MEK5 specifically in abstract","pmids":["19710505"],"is_preprint":false},{"year":2009,"finding":"MEK5 and ERK5 are required for the pro-myogenic actions of IGF-2 in C2 myoblasts. ERK5 translocates from cytoplasm to nucleus upon activation by upstream MEK5, while dominant-negative ERK5AEF-GFP (phospho-acceptor mutant) remains cytoplasmic. Constitutively active MEK5 rescues IGF-2 knockdown myogenic defects; dominant-negative MEK5 blocks IGF-2-induced myogenesis.","method":"GFP-tagged ERK5 live imaging for localization, constitutively active/dominant-negative MEK5 rescue/block experiments, antisense IGF-2 knockdown, kinase activity assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — GFP localization assay plus epistasis with dominant mutants plus rescue experiment, single lab, multiple orthogonal methods","pmids":["19654213"],"is_preprint":false},{"year":2011,"finding":"MEK5-ERK5 pathway has a non-redundant role in thymocyte apoptosis: dominant-negative MEK5 inhibits and constitutively active MEK5 promotes thymocyte apoptosis. ERK5 activity correlates with Nur77 family member levels (not Bim), and MEK5 has no function in positive selection. This is distinct from ERK1/2 function in T cell development.","method":"Retroviral expression of dominant-negative or constitutively active MEK5 in primary thymocytes, apoptosis assays, Nur77/Bim expression analysis","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with specific apoptosis readouts, identification of Nur77 as downstream correlate, single lab","pmids":["18548009"],"is_preprint":false},{"year":2011,"finding":"MEK5 activation by laminar shear stress in human dermal microvascular endothelial cells activates ERK5, which in turn induces KLF4 expression (ERK5-dependent). MEK5/CA-transduced HDMECs show increased KLF4, thrombomodulin, eNOS, and ICAM-1 expression, and reduced TNF responsiveness partly mediated by KLF4. siRNA knockdown of ERK5 or KLF4 confirms the MEK5→ERK5→KLF4 pathway.","method":"Laminar shear stress application, retroviral MEK5/CA overexpression, siRNA knockdown of ERK5 and KLF4, Western blotting, microarrays, FACS","journal":"Microcirculation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function plus siRNA epistasis defining the MEK5→ERK5→KLF4 axis, single lab, multiple methods","pmids":["21166929"],"is_preprint":false},{"year":2014,"finding":"XIAP directly interacts with MEKK2/3 and competes with PB1 domain-mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3, which directly impedes MEK5-ERK5 interaction in a trimeric complex, leading to ERK5 inactivation. Loss of XIAP or cIAP1 causes hyperactivation of ERK5, and loss of XIAP promotes MEK5-ERK5-MEKK2/3-dependent differentiation of human primary skeletal myoblasts.","method":"Co-immunoprecipitation, ubiquitination assays, K63-linkage-specific ubiquitin analysis, XIAP/cIAP1 siRNA/knockout, myoblast differentiation assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP plus biochemical ubiquitination assays plus genetic loss-of-function with functional differentiation readout; multiple orthogonal methods","pmids":["24975362"],"is_preprint":false},{"year":2017,"finding":"YAP promotes myogenic differentiation via the MEK5-ERK5 pathway through activation of the Abl/Src/MEKK3/MEK5/ERK5 kinase cascade. YAP co-immunoprecipitates with MEKK3 and ERK5. MEKK3 contains a PPGY motif (aa 178-181) that interacts with YAP; site-directed mutagenesis of MEKK3 Y181F inhibits MEK5/ERK5 activation and myogenic differentiation.","method":"Co-immunoprecipitation, site-directed mutagenesis, stable cell line overexpression, pharmacological inhibitors of c-Abl/Src/MEK5, Western blotting, myogenesis assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus mutagenesis of specific PPGY motif plus pharmacological validation, single lab","pmids":["28356344"],"is_preprint":false},{"year":2018,"finding":"MAP2K5 variants A321T and M367T (located in the kinase domain) identified in familial non-medullary thyroid carcinoma patients constitutively phosphorylate ERK5 at Ser731+Thr733 or Ser496, promote ERK5 nuclear translocation, alter downstream target gene expression, and induce thyroid epithelial cell malignant transformation.","method":"Whole exome/target sequencing, functional overexpression of MAP2K5 variants, Western blotting for ERK5 phosphorylation, nuclear translocation assays, gene expression analysis, transformation assays","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain-of-function overexpression of specific point mutants with phosphorylation and localization readouts, single lab","pmids":["30132833"],"is_preprint":false},{"year":2018,"finding":"Upon acute KRAS suppression in PDAC, an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC activates ERK5 (via MEK5), which phosphorylates MYC at S62, preventing MYC proteasomal degradation. Concurrent inhibition of ERK1/2 and ERK5 synergistically causes MYC loss and suppresses PDAC growth.","method":"Kinome-wide proteomics, high-throughput screen for MYC degradation, pharmacological ERK1/2 and ERK5 inhibition, MYC phosphorylation assays","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — kinome-wide proteomics plus functional screen plus pharmacological validation plus defined phosphorylation site on MYC; multiple orthogonal methods","pmids":["30423298"],"is_preprint":false},{"year":2019,"finding":"Resistance to ERK inhibitor SCH772984 in BRAF V600E melanoma involves stimulation of the IGF1R-MEK5-ERK5 signaling pathway, which counteracts inhibition of ERK1/2 activation. IGF1R inhibition with linsitinib blocks ERK5 activation in SCH-resistant cells and decreases their growth in 3D spheroid assays and in NSG mice.","method":"Drug resistance cell line generation, Western blotting for pathway activation, pharmacological inhibition of IGF1R, 3D spheroid assays, xenograft mouse models","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological epistasis with in vivo validation, single lab, multiple readouts","pmids":["30833419"],"is_preprint":false},{"year":2019,"finding":"MEK5 knockdown by RNAi sensitizes prostate cancer cells to ionizing radiation and etoposide by impairing phosphorylation of the catalytic subunit of DNA-PK at Ser2056, delaying resolution of γH2AX and 53BP1 foci, and compromising nonhomologous end joining (NHEJ) DNA repair. MEK5 silencing combined with irradiation strongly inhibits tumor growth in mouse xenografts.","method":"RNAi knockdown, clonogenic survival assays, γH2AX/53BP1 foci analysis, DNA-PK phosphorylation Western blots, NHEJ cell-based assay, xenograft mouse model","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockdown with multiple orthogonal molecular readouts (DNA-PK phosphorylation, foci resolution, NHEJ assay) plus in vivo validation","pmids":["31980741"],"is_preprint":false},{"year":2020,"finding":"MEK5-ERK5 axis promotes SCLC cell survival and expansion in vitro and in vivo, and controls lipid metabolism including the mevalonate/cholesterol synthesis pathway. Loss of MEK5/ERK5 sensitizes SCLC cells to pharmacological mevalonate pathway inhibition by statins.","method":"MEK5/ERK5 knockdown/overexpression, transcriptomics, lipidomics, pharmacological statin combination, in vivo tumor growth assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown plus metabolomics/transcriptomics plus pharmacological synergy, single lab","pmids":["31969375"],"is_preprint":false},{"year":2020,"finding":"The MEKK3-MEK5-ERK5 kinase cascade is required for basal mitochondrial degradation (mitophagy) through lysosome-mediated pathways, independent of exogenous mitochondrial damage, bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis.","method":"Genetic and pharmacological inhibition of MEKK3-MEK5-ERK5 pathway, mitochondrial content quantification, lysosome-mediated degradation assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — both genetic and pharmacological inhibition with specific functional readouts distinguishing basal from damage-induced mitophagy, single lab","pmids":["33101709"],"is_preprint":false},{"year":2020,"finding":"MEK5/ERK5 signaling mediates IL-4-induced M2 macrophage differentiation through regulation of c-Myc expression. Pharmacological MEK5 inhibition reduces M2 markers (Arg-1, Ym-1, Fizz-1) and M2 chemokines/cytokines. Myeloid-specific Erk5 knockout (LysMcre/Erk5f/f mice) confirms ERK5 requirement. ERK5 regulates M2 differentiation independently of STAT3/STAT6 phosphorylation.","method":"Pharmacological MEK5/ERK5 inhibition, myeloid-specific conditional Erk5 knockout mice, Western blotting for STAT3/STAT6, qPCR for M2 markers","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological plus conditional genetic knockout in vivo, with pathway specificity (STAT3/STAT6 independence shown), multiple methods","pmids":["32745297"],"is_preprint":false},{"year":2012,"finding":"REST transcription factor promotes primordial germ cell (PGC) survival in part by regulating Mek5 expression. Mek5 is a downstream REST-target candidate gene that is downregulated in REST-deficient PGCs. A Mek5 mutation, like the REST mutation, increases PGC apoptosis, placing Mek5 in the REST-dependent PGC survival pathway.","method":"PGC-specific conditional REST mutation, Mek5 mutation analysis, apoptosis assays in embryos, gene expression analysis","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional genetic mutations with parallel phenotypic analysis establishing pathway relationship, single lab","pmids":["23022299"],"is_preprint":false},{"year":2019,"finding":"MEK5 inhibition by BIX02188/BIX02189 activates the autophagy-lysosome pathway (ALP) in an mTOR- and ERK5-independent manner, reducing p62 levels and increasing LC3-II/LC3-I ratio. MEK5 inhibition alleviates TDP-43 mislocalization and cell death in neuronal cells, identifying MEK5 as a novel autophagy modulator acting through a non-canonical pathway.","method":"MEK5 inhibitor treatment, mTOR pathway analysis, LC3-II/LC3-I ratio, p62 Western blotting, TDP-43 localization in neuronal cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition with multiple autophagy markers plus ERK5-independence control, single lab","pmids":["31005259"],"is_preprint":false},{"year":2023,"finding":"MEK5-ERK5-STAT3 pathway is required for maintaining glioma stem cell (GSC) stemness and tumorigenicity. ERK5 silencing in GSCs suppresses self-renewal and GBM growth concomitant with downregulation of STAT3 phosphorylation; STAT3 introduction counteracts the GSC phenotypes caused by ERK5 silencing, placing STAT3 downstream of ERK5 in this pathway.","method":"shRNA knockdown, constitutively active MEK5/ERK5 overexpression, STAT3 rescue experiment, RNA sequencing, in vivo GBM growth assays","journal":"Cancer research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function plus rescue experiment defining ERK5→STAT3 axis, single lab, multiple methods","pmids":["36968222"],"is_preprint":false},{"year":2025,"finding":"Aberrant MEK5 splicing mediated by the splicing factor RBM39 is required for multiple myeloma cell survival. Full-length MEK5 maintains MM cell survival, whereas aberrant MEK5 isoforms with exon loss are non-functional and prone to proteasomal degradation. Targeting RBM39 or MEK5 synergistically increases bortezomib cytotoxicity in MM cells via inhibition of p65 (NF-κB).","method":"RBM39 knockdown/indisulam-mediated degradation, splice isoform analysis, overexpression of MEK5 isoforms, bortezomib combination cytotoxicity assays, p65 Western blotting","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic and pharmacological RBM39 depletion with defined MEK5 splicing consequence and functional rescue, single lab","pmids":["40048740"],"is_preprint":false},{"year":2025,"finding":"MEK5/ERK5 co-inhibition with MEK inhibitor causes sustained G1 cell cycle arrest in NRAS-mutant melanoma by suppressing Cyclin D1 and E2F-mediated gene expression. Forced expression of Cyclin D1 and CDK4 restores cell cycle progression in MEKi/ERK5i-treated cells, identifying Cyclin D/CDK4 as the key downstream target of combined MEK/ERK5 inhibition.","method":"Transcriptome analysis, pharmacological MEKi/ERK5i combination, constitutive Cyclin D1/CDK4 overexpression rescue, cell cycle FACS analysis","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological combination plus genetic rescue experiment identifying Cyclin D/CDK4 as downstream effector, single lab","pmids":["41053077"],"is_preprint":false},{"year":2025,"finding":"MEK5-ERK5 pathway activates Hedgehog-GLI signaling: ERK5 silencing reduces GLI1 and GLI2 protein levels and transcriptional activity in melanoma cells, while constitutively active MEK5DD increases GLI1/GLI2 levels and GLI transcriptional activity. ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation.","method":"shRNA ERK5 knockdown, constitutively active MEK5DD overexpression, GLI luciferase reporter assay, pharmacological MEK5/ERK5 inhibitors, 3D spheroid assays","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal genetic gain- and loss-of-function with luciferase reporter validation, single lab","pmids":["39998753"],"is_preprint":false}],"current_model":"MAP2K5 (MEK5) is a dual-specificity MAP kinase kinase that functions as the obligate, specific upstream activator of ERK5 (BMK1); it is activated by MEKK2 and MEKK3 through PB1 domain-mediated heterodimerization and direct phosphorylation, and also by atypical PKC isoforms downstream of EGF; it is negatively regulated by XIAP/cIAP1-mediated K63-ubiquitination of MEKK2/3 that disrupts the MEKK3-MEK5-ERK5 trimeric complex; MEK5 physically associates with ERK5 and prevents its SUMOylation (independently of kinase activity) to maintain ERK5 transcriptional activity; the pathway controls downstream transcription factors including MEF2C, c-Fos/Fra-1, KLF4, Nur77, c-Myc, STAT3, and GLI1/2; and it is physiologically required for cardiac sarcomere assembly, embryonic survival, neural differentiation, myogenesis, thymocyte apoptosis, endothelial mechanosensing, M2 macrophage polarization, basal mitophagy, and NHEJ DNA repair, while being co-opted in multiple cancers as a drug-resistance and pro-metastatic pathway."},"narrative":{"mechanistic_narrative":"MAP2K5 (MEK5) is a dual-specificity MAP kinase kinase that defines a distinct MAP kinase pathway, functioning as the obligate and specific upstream activator of ERK5/BMK1 without acting on ERK1/2, JNK, or p38 [PMID:7499418, PMID:15601854]. It is recruited and activated by the upstream kinases MEKK2 and MEKK3 through PB1 domain-mediated, front-to-back heterodimerization in which a basic cluster on the MEKK PB1 face engages an acidic surface on the MEK5 PB1 domain, an interaction required for ERK5 activation; a C-terminal extension of the MEK5 PB1 domain serves as the ERK5 docking site, organizing a MEKK3-MEK5-ERK5 trimeric module [PMID:12912994, PMID:17452462, PMID:17985933]. MEK5 is alternatively activated by atypical PKC isoforms downstream of EGF and integrates growth-factor, shear-stress, and YAP/Abl/Src inputs into the cascade [PMID:11158308, PMID:21166929, PMID:28356344]. Beyond its catalytic role, MEK5 physically binds ERK5 and prevents its repressive SUMOylation independently of kinase activity, thereby maintaining ERK5 transcriptional output [PMID:18467627]. The pathway is restrained by XIAP/cIAP1, which conjugate K63-linked ubiquitin to MEKK2/3 to disrupt the trimeric complex [PMID:24975362]. Through ERK5, MEK5 controls transcriptional programs governing MEF2C, c-Fos/Fra-1, KLF4, Nur77, c-Myc, STAT3, and GLI1/2 [PMID:12622723, PMID:15601854, PMID:18548009, PMID:21166929, PMID:30423298, PMID:36968222, PMID:39998753], and is physiologically required for cardiac development and sarcomere assembly, embryonic survival, neural differentiation, myogenesis, thymocyte apoptosis, endothelial mechanosensing, M2 macrophage polarization, basal mitophagy, and NHEJ DNA repair [PMID:11387209, PMID:15601854, PMID:16179948, PMID:19654213, PMID:18548009, PMID:31980741, PMID:33101709, PMID:32745297]. The pathway is co-opted across cancers as a proliferative, pro-metastatic, and drug-resistance route, and kinase-domain MAP2K5 variants (A321T, M367T) are linked to familial non-medullary thyroid carcinoma [PMID:30132833, PMID:30423298, PMID:30833419].","teleology":[{"year":1995,"claim":"Established that MEK5 is a MEK-family kinase belonging to a distinct, previously uncharacterized MAP kinase pathway rather than the ERK1/2, JNK, or p38 modules, and that splicing generates isoforms with different localization.","evidence":"PCR cloning, in vitro kinase assays against multiple MAPK substrates and upstream kinases, subcellular fractionation","pmids":["7499418"],"confidence":"High","gaps":["Downstream substrate (ERK5) not yet identified","Physiological upstream activators unknown","Functional significance of alpha/beta isoform localization untested in vivo"]},{"year":1999,"claim":"Identified MEKK2 and MEKK3 as direct upstream kinases of MEK5 and placed the module downstream of growth-factor and Ras/Raf signaling, defining the MEKK→MEK5→ERK5 cascade and its transforming potential.","evidence":"Yeast two-hybrid, reciprocal co-IP, dominant-active/negative epistasis, endogenous co-IP, NIH 3T3 transformation assays","pmids":["10593883","11073940","10531364"],"confidence":"High","gaps":["Structural basis of MEKK-MEK5 binding not yet resolved","Cell-type determinants of MEKK2-vs-MEKK3 usage incompletely defined","Mechanism of Raf-ERK5 association unclear"]},{"year":2001,"claim":"Demonstrated alternative upstream input via atypical PKC downstream of EGF and established a physiological output in cardiac sarcomere assembly and hypertrophy.","evidence":"Co-IP, dominant mutants, luciferase reporters; adenoviral and cardiac-specific transgenic mouse models","pmids":["11158308","11387209"],"confidence":"Medium","gaps":["aPKC phosphorylation sites on MEK5 not mapped","Direct vs indirect cardiac transcriptional targets not fully defined"]},{"year":2003,"claim":"Resolved the PB1 domain interaction code governing MEK5 recruitment and identified the first transcriptional effectors (c-Fos/Fra-1, AP-1) of the pathway, linking it to invasion and MMP-9.","evidence":"In vitro PB1 binding assays, co-IP, PB1 mutagenesis, transactivation/luciferase reporters, EMSA","pmids":["12912994","12622723","12618764"],"confidence":"High","gaps":["Direct ERK5 substrates among transcription factors not all distinguished from indirect effects","Quantitative PB1 binding parameters not yet measured"]},{"year":2004,"claim":"Defined the subcellular logic of signaling, showing endogenous MEK5 and ERK5 are nuclear-anchored and that MEKK2 nuclear translocation transmits the EGF signal, and provided NMR-level structure of a PB1 partner surface.","evidence":"Immunofluorescence of endogenous proteins, NP-40 in situ fractionation; NMR structure of PKCiota PB1 with mutagenesis","pmids":["15075238","15143057"],"confidence":"Medium","gaps":["Identity of nuclear anchoring moieties unknown","Direct MEK5 PB1 mutagenesis not tested in the structural study"]},{"year":2005,"claim":"Genetically validated MEK5 in vivo as the essential ERK5 activator and a required regulator of proliferation, survival, and cardiac development, and extended its role to neural differentiation.","evidence":"Mek5 knockout mice (E10.5 lethality), MEFs with MEF2 reporters and caspase assays; Xenopus morpholino knockdown and gain-of-function epistasis","pmids":["15601854","16179948"],"confidence":"High","gaps":["Tissue-autonomous vs systemic requirements not separated due to embryonic lethality","Direct ERK5 substrates driving survival not identified"]},{"year":2007,"claim":"Defined the front-to-back PB1 binding geometry, the MEK5 PB1 C-terminal ERK5 docking site, and a competitive switch in MEKK2 partner choice, providing the quantitative structural mechanism of cascade assembly.","evidence":"PB1 mutagenesis, co-IP, kinase assays; NMR structure of MEKK3 PB1 with Kd measurement (~10^-8 M)","pmids":["17452462","17985933"],"confidence":"High","gaps":["Full MEKK-MEK5-ERK5 ternary structure not solved","Regulation of the MEKK2 MKK7-vs-MEK5 switch in physiological settings unclear"]},{"year":2008,"claim":"Uncovered a kinase-independent function of MEK5 in protecting ERK5 from repressive SUMOylation, and defined pathway-specific roles in EMT and endothelial mechanosensing using selective inhibitors.","evidence":"Kinase-dead mutants, SUMOylation and PIAS1 siRNA assays, CA-MEK5alpha transgenic mice; selective MEK5 inhibitors BIX02188/BIX02189; proteomics and ERK5 shRNA epistasis","pmids":["18467627","19087274","18358237","18834865"],"confidence":"High","gaps":["Mechanism by which MEK5 binding blocks Ubc9/PIAS1 access not structurally defined","EMT transcriptional targets not directly tied to ERK5 phosphorylation events"]},{"year":2011,"claim":"Defined non-redundant tissue-specific roles in thymocyte apoptosis (via Nur77) and endothelial KLF4 induction, distinguishing MEK5/ERK5 from ERK1/2 outputs.","evidence":"Retroviral dominant/active MEK5 in thymocytes with Nur77/Bim analysis; shear stress with ERK5/KLF4 siRNA epistasis","pmids":["18548009","21166929"],"confidence":"Medium","gaps":["Direct ERK5 substrate linking to Nur77 induction not identified","Whether KLF4 is a direct vs indirect ERK5 target unresolved"]},{"year":2014,"claim":"Identified the negative regulatory mechanism whereby XIAP/cIAP1-mediated K63-ubiquitination of MEKK2/3 disrupts the trimeric MEK5 complex, defining how the cascade is restrained during differentiation.","evidence":"Reciprocal co-IP, K63-linkage ubiquitination assays, XIAP/cIAP1 loss-of-function, myoblast differentiation assays","pmids":["24975362"],"confidence":"High","gaps":["Deubiquitinases reversing this mark not identified","Signals controlling XIAP engagement of MEKK2/3 unknown"]},{"year":2017,"claim":"Connected upstream YAP/Abl/Src signaling to the cascade through a MEKK3 PPGY motif, defining how mechanical/Hippo inputs feed MEK5 activation during myogenesis.","evidence":"Co-IP, MEKK3 Y181F mutagenesis, pharmacological inhibition of c-Abl/Src/MEK5, myogenesis assays","pmids":["28356344"],"confidence":"Medium","gaps":["Direct vs scaffold role of YAP at the MEKK3 PB1 module unclear","In vivo requirement of the PPGY interaction not tested"]},{"year":2018,"claim":"Established MAP2K5 mutations as drivers of familial thyroid carcinoma and defined an ERK1/2-suppression-induced feedforward route in which MEK5/ERK5 stabilizes MYC, explaining pathway co-option in cancer and drug resistance.","evidence":"Exome sequencing and functional expression of MAP2K5 variants; kinome proteomics, MYC degradation screen, pharmacological ERK1/2+ERK5 inhibition with S62-MYC phosphorylation readout","pmids":["30132833","30423298"],"confidence":"High","gaps":["Whether ERK5 directly phosphorylates MYC vs via a downstream kinase not fully resolved","Generality of MAP2K5 germline variants beyond studied families unknown"]},{"year":2020,"claim":"Broadened MEK5's physiological repertoire to basal mitophagy, M2 macrophage polarization (via c-Myc, STAT3/STAT6-independent), and lipid/mevalonate metabolism in cancer, showing pathway outputs extend beyond classical proliferation control.","evidence":"Genetic and pharmacological pathway inhibition, conditional Erk5 knockout mice, transcriptomics/lipidomics, statin synergy","pmids":["33101709","32745297","31969375"],"confidence":"High","gaps":["Direct effectors linking ERK5 to mitophagy machinery not identified","Mechanism of c-Myc regulation in M2 macrophages not fully mapped"]},{"year":2019,"claim":"Revealed MEK5 functions in genome maintenance and a non-canonical autophagy role: it supports NHEJ via DNA-PK Ser2056 phosphorylation, while its inhibition activates the autophagy-lysosome pathway ERK5- and mTOR-independently.","evidence":"RNAi knockdown, gammaH2AX/53BP1 foci, DNA-PK phospho-Westerns, NHEJ assay, xenografts; MEK5 inhibitor with LC3-II/p62 and TDP-43 readouts","pmids":["31980741","31005259"],"confidence":"High","gaps":["Whether MEK5 acts on DNA-PK directly or through ERK5 not established","Molecular target of the ERK5-independent autophagy effect unknown"]},{"year":2025,"claim":"Extended cancer-relevant outputs to Hedgehog-GLI activation, Cyclin D/CDK4-dependent cell-cycle control under combined inhibition, and RBM39-dependent MEK5 splicing required for myeloma survival, reinforcing MEK5 as a therapeutic node.","evidence":"Reciprocal ERK5/MEK5DD gain/loss-of-function, GLI and cell-cycle rescue experiments, RBM39 depletion with splice-isoform and bortezomib synergy assays","pmids":["39998753","41053077","40048740"],"confidence":"Medium","gaps":["Direct ERK5 substrates linking to GLI and Cyclin D1 not defined","Mechanism by which RBM39 controls functional MEK5 splicing not fully mapped"]},{"year":null,"claim":"The complete catalogue of direct ERK5 substrates downstream of MEK5, the structural basis of the kinase-independent anti-SUMOylation function, and which physiological outputs depend on catalytic versus scaffolding activity of MEK5 remain to be resolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No ternary MEKK-MEK5-ERK5 structure","Direct vs ERK5-mediated effects on DNA-PK, MYC, and transcription factors unresolved","Catalytic vs scaffold contributions to in vivo phenotypes not dissected"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,7,11,16,19]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,19,27]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[16]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9,22,27]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,9]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,3,7,14]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[6,11,24,36]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[11,13,22,25]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[27,28,29,31]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[30]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[32,35]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[23,33]}],"complexes":["MEKK3-MEK5-ERK5 trimeric module"],"partners":["MAPK7","MAP3K2","MAP3K3","PRKCI","PRKCZ","XIAP","YAP1","RAF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13163","full_name":"Dual specificity mitogen-activated protein kinase kinase 5","aliases":["MAPK/ERK kinase 5","MEK 5"],"length_aa":448,"mass_kda":50.1,"function":"Acts as a scaffold for the formation of a ternary MAP3K2/MAP3K3-MAP3K5-MAPK7 signaling complex. Activation of this pathway appears to play a critical role in protecting cells from stress-induced apoptosis, neuronal survival and cardiac development and angiogenesis. As part of the MAPK/ERK signaling pathway, acts as a negative regulator of apoptosis in cardiomyocytes via promotion of STUB1/CHIP-mediated ubiquitination and degradation of ICER-type isoforms of CREM (By similarity)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q13163/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MAP2K5","classification":"Not Classified","n_dependent_lines":18,"n_total_lines":1208,"dependency_fraction":0.014900662251655629},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MAP2K5","total_profiled":1310},"omim":[{"mim_id":"612853","title":"RESTLESS LEGS SYNDROME, SUSCEPTIBILITY TO, 7; RLS7","url":"https://www.omim.org/entry/612853"},{"mim_id":"611122","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 28; ANKRD28","url":"https://www.omim.org/entry/611122"},{"mim_id":"609487","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE 2; MAP3K2","url":"https://www.omim.org/entry/609487"},{"mim_id":"602520","title":"MITOGEN-ACTIVATED PROTEIN KINASE KINASE 5; MAP2K5","url":"https://www.omim.org/entry/602520"},{"mim_id":"602016","title":"KLF TRANSCRIPTION FACTOR 2; KLF2","url":"https://www.omim.org/entry/602016"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"},{"location":"Primary cilium","reliability":"Approved"},{"location":"Nucleoli","reliability":"Additional"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MAP2K5"},"hgnc":{"alias_symbol":["MEK5","MAPKK5","HsT17454"],"prev_symbol":["PRKMK5"]},"alphafold":{"accession":"Q13163","domains":[{"cath_id":"3.10.20.90","chopping":"16-108","consensus_level":"high","plddt":85.0494,"start":16,"end":108},{"cath_id":"3.30.200.20","chopping":"157-243","consensus_level":"medium","plddt":86.0823,"start":157,"end":243},{"cath_id":"1.10.510.10","chopping":"248-448","consensus_level":"medium","plddt":87.3719,"start":248,"end":448}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13163","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13163-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13163-F1-predicted_aligned_error_v6.png","plddt_mean":78.5},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MAP2K5","jax_strain_url":"https://www.jax.org/strain/search?query=MAP2K5"},"sequence":{"accession":"Q13163","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13163.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13163/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13163"}},"corpus_meta":[{"pmid":"11387209","id":"PMC_11387209","title":"Activated 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Alternative splicing produces a 50-kDa alpha isoform (expressed in liver and brain, particulate/associated with actin cytoskeleton-like domain) and a 40-kDa beta isoform (ubiquitous, primarily cytosolic), with the N-terminal 23-aa exon of MEK5alpha directing differential subcellular localization.\",\n      \"method\": \"PCR cloning, in vitro kinase assays, subcellular fractionation, Northern/Western blotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assays with multiple substrates and upstream kinases, fractionation for localization; foundational characterization paper replicated by subsequent work\",\n      \"pmids\": [\"7499418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"MEKK3 physically interacts with MEK5 (identified by yeast two-hybrid and confirmed by co-immunoprecipitation in mammalian cells), and constitutively active MEKK3 stimulates BMK1/ERK5 activity through MEK5. Dominant-active MEKK3 is required for growth factor-mediated activation of endogenous BMK1, placing MEKK3 as a direct upstream kinase of MEK5 in the BMK1/ERK5 pathway.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, dominant-active/dominant-negative overexpression, kinase activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus reciprocal co-IP plus functional epistasis with dominant mutants; replicated by subsequent PB1 domain studies\",\n      \"pmids\": [\"10593883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The ERK5/MEK5 pathway is required for Raf-dependent cellular transformation. Constitutively active MEK5 (MEK5DD) synergizes with Raf to transform NIH 3T3 cells. Endogenous Raf-1 binds specifically to endogenous ERK5 (but not ERK2 or SAPK), suggesting regulatory protein-protein interactions contribute to Ras/Raf activation of ERK5.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, NIH 3T3 transformation assays, dominant-active MEK5 overexpression, epistasis analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — endogenous co-IP plus functional transformation assay with constitutively active MEK5; multiple orthogonal methods in one study\",\n      \"pmids\": [\"10531364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"MEKK2 binds MEK5 via yeast two-hybrid and co-immunoprecipitation, and MEKK2 expression stimulates BMK1/ERK5 activity. MEKK2 activates BMK1/ERK5 to a greater extent than MEKK3 and with higher apparent affinity for MEK5. Dominant-negative MEK5 blocks MEKK2-induced BMK1/ERK5 activation without affecting JNK, demonstrating MEK5 is a specific downstream effector of MEKK2. In D10 T cells, dominant-negative MEKK2 (but not MEKK3) inhibits BMK1/ERK5, showing cell-type-specific usage of upstream kinases.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, dominant-negative/constitutively active overexpression, kinase activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus co-IP plus functional epistasis with dominant mutants; independently corroborated by MEKK3 study\",\n      \"pmids\": [\"11073940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"MEK5 induces eccentric cardiac hypertrophy with serial sarcomere assembly in cardiomyocytes in vitro. Constitutively active MEK5 causes elongated cardiomyocyte morphology, and dominant-negative MEK5 specifically blocks LIF-induced cardiomyocyte elongation and fetal gene expression without blocking other LIF hypertrophic responses. Cardiac-specific transgenic expression of activated MEK5 in mice produces eccentric cardiac hypertrophy progressing to dilated cardiomyopathy and sudden death.\",\n      \"method\": \"Adenoviral overexpression of constitutively active and dominant-negative MEK5 in cardiomyocytes; cardiac-specific transgenic mice; morphological and gene expression analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro loss/gain-of-function plus in vivo transgenic mouse model, multiple orthogonal phenotypic readouts\",\n      \"pmids\": [\"11387209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Atypical protein kinase C isoforms (zetaPKC and lambda/iotaPKC) interact with MEK5 in an EGF-inducible manner, and this interaction is required and sufficient for MEK5 activation in response to EGF. The aPKC-MEK5 interaction depends on the aPKC interaction domain (acidic amino acid stretch) present in MEK5. aPKC activation of MEK5 leads to MEF2C-element-dependent Jun promoter activation.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative/constitutively active overexpression, luciferase reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional reporter assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"11158308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The MEK5-ERK5 pathway phosphorylates and stabilizes c-Fos and Fra-1. Phosphorylation of c-Fos by ERK5 (and downstream kinases) occurs at sites distinct from ERK1/2-dependent sites, and MEK5-ERK5 pathway activation markedly increases c-Fos transactivation activity. The C-terminal half of ERK5 is required for maximal transactivation of c-Fos and Fra-1.\",\n      \"method\": \"Constitutively active MEK5 overexpression, phosphorylation analysis, mutagenesis of ERK5, luciferase/transactivation assays\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional mutagenesis plus reporter assays, single lab, two orthogonal methods\",\n      \"pmids\": [\"12622723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PB1 domains of MEKK2 and MEKK3 bind the PB1 domain of MEK5 but do not significantly homo- or heterodimerize with each other in vitro. Co-immunoprecipitation confirms MEKK2-MEK5 complex formation in vivo. Deletion or mutation of the MEKK2 PB1 domain abolishes MEKK2-MEK5 interaction. Expression of free MEKK2 or MEKK3 PB1 domains inhibits ERK5 activation specifically (not p38 or JNK), demonstrating that PB1 domain-mediated association is required for MEK5 activation by MEKK2/3.\",\n      \"method\": \"In vitro PB1 domain binding assays, co-immunoprecipitation, PB1 deletion/mutation analysis, kinase activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro binding assays plus co-IP plus mutagenesis plus functional pathway inhibition; multiple orthogonal methods\",\n      \"pmids\": [\"12912994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"MEK5 overexpression in prostate cancer cells stimulates proliferation, motility, invasion, and selectively increases MMP-9 (but not MMP-2) mRNA expression. Luciferase and EMSA assays show MEK5 activates the MMP-9 promoter via AP-1 (not NF-κB) binding sites, and MEK5 enhances AP-1 transcriptional activity approximately two-fold.\",\n      \"method\": \"MEK5 transfection, proliferation/invasion assays, RT-PCR, luciferase reporter assays, EMSA\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function overexpression with multiple functional assays plus reporter/EMSA, single lab\",\n      \"pmids\": [\"12618764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"MEK5 and ERK5 are localized in the nucleus of both resting and EGF-stimulated HeLa and Rat-1 cells (endogenous proteins), where they are bound to detergent-resistant nuclear moieties. Exogenous overexpressed ERK5 localizes to the cytosol, revealing saturation of nuclear anchors by endogenous ERK5. Upon EGF stimulation, MEK5 is released from nuclear anchors but remains nuclear, while MEKK2 translocates from cytosol to nucleus, enabling signal transmission to nuclear MEK5.\",\n      \"method\": \"Immunofluorescence microscopy of endogenous proteins, in situ detergent (NP-40) extraction fractionation, EGF stimulation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct subcellular localization by immunofluorescence plus fractionation with NP-40 extraction, single lab, two orthogonal methods\",\n      \"pmids\": [\"15075238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"NMR structure of the PKCiota PB1 domain reveals a ubiquitin fold with an OPCA motif forming an acidic surface that interacts with the basic surface of target PB1 domains (including MEK5). Mutational analysis of the PKCiota-ZIP/p62 PB1-PB1 interaction confirms the acidic surface is critical for binding. The PKCiota PB1 domain can interact with targets via either its acidic (OPCA) surface or a conserved lysine on the opposite face.\",\n      \"method\": \"NMR structure determination, mutagenesis, PB1 domain interaction analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with mutagenesis for the PKCiota PB1 domain; MEK5 mentioned as interacting partner but direct MEK5 mutagenesis confirmation not described in abstract\",\n      \"pmids\": [\"15143057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Targeted deletion of mek5 in mice results in embryonic lethality at ~E10.5 with abnormal cardiac development, decreased proliferation, and increased apoptosis in heart, head, and dorsal regions. MEK5-deficient mouse embryonic fibroblasts show impaired ERK5 activation and reduced MEF2 transcriptional activity, and are sensitized to sorbitol-induced caspase-3 activity. MEK5 is established in vivo as the essential activator of ERK5 and a required regulator of cell survival.\",\n      \"method\": \"Targeted gene knockout in mice, phenotypic analysis, MEF2 transcriptional reporter assays, caspase-3 activity assays in MEFs\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with multiple orthogonal phenotypic and molecular readouts; in vivo validation\",\n      \"pmids\": [\"15601854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"MEK5-ERK5 pathway activation is responsible for biliary epithelial cell hyperproliferation in PCK rats (Caroli's disease model). MEK5 is overexpressed in PCK rat biliary epithelial cells, leading to increased ERK5 phosphorylation. siRNA knockdown of MEK5 significantly inhibits the abnormal biliary cell proliferation, while MEK1/2 inhibitors (PD98059, U0126) are less effective.\",\n      \"method\": \"siRNA knockdown of MEK5, Western blotting for ERK5 phosphorylation, cell proliferation assays, pharmacological inhibitor comparison\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with functional proliferation readout, single lab, two orthogonal methods (genetic + pharmacological)\",\n      \"pmids\": [\"15631999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"MEK5-ERK5 pathway is required for neural differentiation in Xenopus embryos. Knockdown of Xenopus MEK5 or ERK5 with antisense morpholino oligonucleotides reduces head structure and inhibits neural differentiation. Forced activation of MEK5-ERK5 alone is sufficient to induce neural differentiation. MEK5-ERK5 is necessary for SoxD neuralizing activity and sufficient for expression of the proneural gene Xngnr1, placing MEK5-ERK5 downstream of SoxD and upstream of Xngnr1.\",\n      \"method\": \"Antisense morpholino knockdown in Xenopus embryos, constitutively active MEK5-ERK5 overexpression, genetic epistasis analysis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function (morpholino) plus gain-of-function plus epistasis in intact vertebrate embryo; multiple orthogonal approaches\",\n      \"pmids\": [\"16179948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"MEKK2 and MEK5 PB1 domains interact in a front-to-back arrangement: basic residues on the MEKK2 PB1 front bind to acidic clusters on the MEK5 PB1 back. A 34-amino-acid C-terminal extension of the MEK5 PB1 domain constitutes an ERK5 docking site required for MEK5 activation of ERK5. In quiescent cells, MEKK2 preferentially binds MEK5; upon MEKK2 activation, it switches to bind MKK7 via its PB1 acidic cluster, leading to JNK activation.\",\n      \"method\": \"PB1 domain mutagenesis, co-immunoprecipitation, kinase activation assays, domain-binding specificity analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — detailed mutagenesis of PB1 interaction surfaces plus co-IP plus functional kinase assays; mechanistic detail replicated across multiple studies\",\n      \"pmids\": [\"17452462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"NMR solution structure of MEKK3 PB1 domain reveals a ubiquitin fold with prolyl isomerization at Gln38-Pro39 producing two structural conformers. MEKK3 PB1 binds MEK5 PB1 with Kd ~10^-8 M. Mutagenesis identifies Lys7 and Arg5 in the MEKK3 PB1 basic cluster as critical residues for interaction with MEK5 PB1.\",\n      \"method\": \"NMR structure determination, backbone dynamics, mutagenesis, binding affinity measurement\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure determination with mutagenesis and quantitative binding measurement; single lab but rigorous structural methods\",\n      \"pmids\": [\"17985933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Constitutively active MEK5alpha (CA-MEK5alpha) inhibits ERK5 SUMOylation independently of MEK5 kinase activity but dependently on MEK5-ERK5 physical association. H2O2 and high glucose induce ERK5 SUMOylation (mediated by E2 conjugase Ubc9 and E3 ligase PIAS1), which represses ERK5 transcriptional activity. CA-MEK5alpha transgenic mice show protection against DM-exacerbated LV dysfunction and apoptosis after MI by preventing ERK5 SUMOylation.\",\n      \"method\": \"Kinase-dead MEK5 mutants, SUMOylation assays, siRNA knockdown of PIAS1, CA-MEK5alpha transgenic mice, cardiac function measurement post-MI\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — kinase-dead mutagenesis separates kinase activity from protein-protein interaction, multiple orthogonal approaches (mutagenesis, siRNA, transgenic mice), single lab\",\n      \"pmids\": [\"18467627\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MEK5/ERK5-mediated EMT in TNF-alpha-resistant MCF-7 breast cancer cells involves upregulation of vimentin, GSTP1, CKB and EMT regulators SNAI2/ZEB1/N-cadherin, and downregulation of keratins and E-cadherin. RNA interference targeting ERK5 reverses MEK5-mediated EMT gene expression, establishing ERK5 as required downstream effector of MEK5 in this context.\",\n      \"method\": \"2D gel proteomics, LC-MS/MS, RT-PCR, immunofluorescence, shRNA knockdown of ERK5\",\n      \"journal\": \"Breast cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — shRNA epistasis to show ERK5 dependence, multiple molecular readouts, single lab\",\n      \"pmids\": [\"19087274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"MEK5-selective inhibitor BIX02188 completely reverses flow-mediated inhibition of TNF-induced JNK activation in endothelial cells, while MEK1 inhibitor PD184352 (at ERK1/2-blocking concentrations) has no effect. This establishes MEK5-BMK1 (ERK5) as the required pathway mediating atheroprotective flow inhibition of JNK, distinct from MEK1-ERK1/2.\",\n      \"method\": \"Pharmacological inhibitor (BIX02188 vs PD184352) comparison, shear stress application, JNK activity assays in endothelial cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — selective inhibitor comparison demonstrating pathway specificity, single lab, functional cellular readout\",\n      \"pmids\": [\"18358237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BIX02188 and BIX02189 are selective inhibitors of MEK5 catalytic function that block ERK5 phosphorylation without affecting ERK1/2 phosphorylation in sorbitol-stimulated HeLa cells, and inhibit MEF2C transcriptional activation downstream of MEK5/ERK5.\",\n      \"method\": \"In vitro kinase assay with purified MEK5 enzyme, cellular ERK5/ERK1/2 phosphorylation assays, MEF2C trans-reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzyme assay with purified MEK5 plus cellular selectivity assays plus reporter assay; multiple orthogonal methods\",\n      \"pmids\": [\"18834865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Constitutive activation of MEK5/ERK5 signaling in endothelial cells strongly inhibits cell migration and increases focal contact number/size with altered actin organization, due to decreased expression of p130Cas, a key regulator of directed cell migration. This identifies MEK5/ERK5 as a regulator of endothelial cell migration and focal contact turnover.\",\n      \"method\": \"Retroviral constitutively active MEK5 overexpression, migration assays, Western blotting for p130Cas, fluorescence microscopy of focal contacts and actin\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — constitutively active kinase with defined molecular target (p130Cas) and functional migration readout, single lab\",\n      \"pmids\": [\"19605361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MEK5 acts as a downstream mediator of VEGF and antagonizes Epac/Rap1 anti-angiogenic signaling by inducing Id1 and suppressing TSP1 expression in endothelial cells.\",\n      \"method\": \"Overexpression studies, Western blotting, in vivo angiogenesis assays, epistasis analysis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — overexpression with molecular readouts, single lab, limited mechanistic detail on MEK5 specifically in abstract\",\n      \"pmids\": [\"19710505\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MEK5 and ERK5 are required for the pro-myogenic actions of IGF-2 in C2 myoblasts. ERK5 translocates from cytoplasm to nucleus upon activation by upstream MEK5, while dominant-negative ERK5AEF-GFP (phospho-acceptor mutant) remains cytoplasmic. Constitutively active MEK5 rescues IGF-2 knockdown myogenic defects; dominant-negative MEK5 blocks IGF-2-induced myogenesis.\",\n      \"method\": \"GFP-tagged ERK5 live imaging for localization, constitutively active/dominant-negative MEK5 rescue/block experiments, antisense IGF-2 knockdown, kinase activity assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — GFP localization assay plus epistasis with dominant mutants plus rescue experiment, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"19654213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MEK5-ERK5 pathway has a non-redundant role in thymocyte apoptosis: dominant-negative MEK5 inhibits and constitutively active MEK5 promotes thymocyte apoptosis. ERK5 activity correlates with Nur77 family member levels (not Bim), and MEK5 has no function in positive selection. This is distinct from ERK1/2 function in T cell development.\",\n      \"method\": \"Retroviral expression of dominant-negative or constitutively active MEK5 in primary thymocytes, apoptosis assays, Nur77/Bim expression analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with specific apoptosis readouts, identification of Nur77 as downstream correlate, single lab\",\n      \"pmids\": [\"18548009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MEK5 activation by laminar shear stress in human dermal microvascular endothelial cells activates ERK5, which in turn induces KLF4 expression (ERK5-dependent). MEK5/CA-transduced HDMECs show increased KLF4, thrombomodulin, eNOS, and ICAM-1 expression, and reduced TNF responsiveness partly mediated by KLF4. siRNA knockdown of ERK5 or KLF4 confirms the MEK5→ERK5→KLF4 pathway.\",\n      \"method\": \"Laminar shear stress application, retroviral MEK5/CA overexpression, siRNA knockdown of ERK5 and KLF4, Western blotting, microarrays, FACS\",\n      \"journal\": \"Microcirculation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function plus siRNA epistasis defining the MEK5→ERK5→KLF4 axis, single lab, multiple methods\",\n      \"pmids\": [\"21166929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"XIAP directly interacts with MEKK2/3 and competes with PB1 domain-mediated binding to MEK5. XIAP and cIAP1 conjugate predominantly K63-linked ubiquitin chains to MEKK2 and MEKK3, which directly impedes MEK5-ERK5 interaction in a trimeric complex, leading to ERK5 inactivation. Loss of XIAP or cIAP1 causes hyperactivation of ERK5, and loss of XIAP promotes MEK5-ERK5-MEKK2/3-dependent differentiation of human primary skeletal myoblasts.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, K63-linkage-specific ubiquitin analysis, XIAP/cIAP1 siRNA/knockout, myoblast differentiation assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP plus biochemical ubiquitination assays plus genetic loss-of-function with functional differentiation readout; multiple orthogonal methods\",\n      \"pmids\": [\"24975362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"YAP promotes myogenic differentiation via the MEK5-ERK5 pathway through activation of the Abl/Src/MEKK3/MEK5/ERK5 kinase cascade. YAP co-immunoprecipitates with MEKK3 and ERK5. MEKK3 contains a PPGY motif (aa 178-181) that interacts with YAP; site-directed mutagenesis of MEKK3 Y181F inhibits MEK5/ERK5 activation and myogenic differentiation.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis, stable cell line overexpression, pharmacological inhibitors of c-Abl/Src/MEK5, Western blotting, myogenesis assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus mutagenesis of specific PPGY motif plus pharmacological validation, single lab\",\n      \"pmids\": [\"28356344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MAP2K5 variants A321T and M367T (located in the kinase domain) identified in familial non-medullary thyroid carcinoma patients constitutively phosphorylate ERK5 at Ser731+Thr733 or Ser496, promote ERK5 nuclear translocation, alter downstream target gene expression, and induce thyroid epithelial cell malignant transformation.\",\n      \"method\": \"Whole exome/target sequencing, functional overexpression of MAP2K5 variants, Western blotting for ERK5 phosphorylation, nuclear translocation assays, gene expression analysis, transformation assays\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain-of-function overexpression of specific point mutants with phosphorylation and localization readouts, single lab\",\n      \"pmids\": [\"30132833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Upon acute KRAS suppression in PDAC, an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC activates ERK5 (via MEK5), which phosphorylates MYC at S62, preventing MYC proteasomal degradation. Concurrent inhibition of ERK1/2 and ERK5 synergistically causes MYC loss and suppresses PDAC growth.\",\n      \"method\": \"Kinome-wide proteomics, high-throughput screen for MYC degradation, pharmacological ERK1/2 and ERK5 inhibition, MYC phosphorylation assays\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — kinome-wide proteomics plus functional screen plus pharmacological validation plus defined phosphorylation site on MYC; multiple orthogonal methods\",\n      \"pmids\": [\"30423298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Resistance to ERK inhibitor SCH772984 in BRAF V600E melanoma involves stimulation of the IGF1R-MEK5-ERK5 signaling pathway, which counteracts inhibition of ERK1/2 activation. IGF1R inhibition with linsitinib blocks ERK5 activation in SCH-resistant cells and decreases their growth in 3D spheroid assays and in NSG mice.\",\n      \"method\": \"Drug resistance cell line generation, Western blotting for pathway activation, pharmacological inhibition of IGF1R, 3D spheroid assays, xenograft mouse models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological epistasis with in vivo validation, single lab, multiple readouts\",\n      \"pmids\": [\"30833419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MEK5 knockdown by RNAi sensitizes prostate cancer cells to ionizing radiation and etoposide by impairing phosphorylation of the catalytic subunit of DNA-PK at Ser2056, delaying resolution of γH2AX and 53BP1 foci, and compromising nonhomologous end joining (NHEJ) DNA repair. MEK5 silencing combined with irradiation strongly inhibits tumor growth in mouse xenografts.\",\n      \"method\": \"RNAi knockdown, clonogenic survival assays, γH2AX/53BP1 foci analysis, DNA-PK phosphorylation Western blots, NHEJ cell-based assay, xenograft mouse model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockdown with multiple orthogonal molecular readouts (DNA-PK phosphorylation, foci resolution, NHEJ assay) plus in vivo validation\",\n      \"pmids\": [\"31980741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MEK5-ERK5 axis promotes SCLC cell survival and expansion in vitro and in vivo, and controls lipid metabolism including the mevalonate/cholesterol synthesis pathway. Loss of MEK5/ERK5 sensitizes SCLC cells to pharmacological mevalonate pathway inhibition by statins.\",\n      \"method\": \"MEK5/ERK5 knockdown/overexpression, transcriptomics, lipidomics, pharmacological statin combination, in vivo tumor growth assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown plus metabolomics/transcriptomics plus pharmacological synergy, single lab\",\n      \"pmids\": [\"31969375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The MEKK3-MEK5-ERK5 kinase cascade is required for basal mitochondrial degradation (mitophagy) through lysosome-mediated pathways, independent of exogenous mitochondrial damage, bulk autophagy, damage-induced mitophagy, or restraint of mitochondrial biogenesis.\",\n      \"method\": \"Genetic and pharmacological inhibition of MEKK3-MEK5-ERK5 pathway, mitochondrial content quantification, lysosome-mediated degradation assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — both genetic and pharmacological inhibition with specific functional readouts distinguishing basal from damage-induced mitophagy, single lab\",\n      \"pmids\": [\"33101709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MEK5/ERK5 signaling mediates IL-4-induced M2 macrophage differentiation through regulation of c-Myc expression. Pharmacological MEK5 inhibition reduces M2 markers (Arg-1, Ym-1, Fizz-1) and M2 chemokines/cytokines. Myeloid-specific Erk5 knockout (LysMcre/Erk5f/f mice) confirms ERK5 requirement. ERK5 regulates M2 differentiation independently of STAT3/STAT6 phosphorylation.\",\n      \"method\": \"Pharmacological MEK5/ERK5 inhibition, myeloid-specific conditional Erk5 knockout mice, Western blotting for STAT3/STAT6, qPCR for M2 markers\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological plus conditional genetic knockout in vivo, with pathway specificity (STAT3/STAT6 independence shown), multiple methods\",\n      \"pmids\": [\"32745297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"REST transcription factor promotes primordial germ cell (PGC) survival in part by regulating Mek5 expression. Mek5 is a downstream REST-target candidate gene that is downregulated in REST-deficient PGCs. A Mek5 mutation, like the REST mutation, increases PGC apoptosis, placing Mek5 in the REST-dependent PGC survival pathway.\",\n      \"method\": \"PGC-specific conditional REST mutation, Mek5 mutation analysis, apoptosis assays in embryos, gene expression analysis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional genetic mutations with parallel phenotypic analysis establishing pathway relationship, single lab\",\n      \"pmids\": [\"23022299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MEK5 inhibition by BIX02188/BIX02189 activates the autophagy-lysosome pathway (ALP) in an mTOR- and ERK5-independent manner, reducing p62 levels and increasing LC3-II/LC3-I ratio. MEK5 inhibition alleviates TDP-43 mislocalization and cell death in neuronal cells, identifying MEK5 as a novel autophagy modulator acting through a non-canonical pathway.\",\n      \"method\": \"MEK5 inhibitor treatment, mTOR pathway analysis, LC3-II/LC3-I ratio, p62 Western blotting, TDP-43 localization in neuronal cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition with multiple autophagy markers plus ERK5-independence control, single lab\",\n      \"pmids\": [\"31005259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MEK5-ERK5-STAT3 pathway is required for maintaining glioma stem cell (GSC) stemness and tumorigenicity. ERK5 silencing in GSCs suppresses self-renewal and GBM growth concomitant with downregulation of STAT3 phosphorylation; STAT3 introduction counteracts the GSC phenotypes caused by ERK5 silencing, placing STAT3 downstream of ERK5 in this pathway.\",\n      \"method\": \"shRNA knockdown, constitutively active MEK5/ERK5 overexpression, STAT3 rescue experiment, RNA sequencing, in vivo GBM growth assays\",\n      \"journal\": \"Cancer research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function plus rescue experiment defining ERK5→STAT3 axis, single lab, multiple methods\",\n      \"pmids\": [\"36968222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Aberrant MEK5 splicing mediated by the splicing factor RBM39 is required for multiple myeloma cell survival. Full-length MEK5 maintains MM cell survival, whereas aberrant MEK5 isoforms with exon loss are non-functional and prone to proteasomal degradation. Targeting RBM39 or MEK5 synergistically increases bortezomib cytotoxicity in MM cells via inhibition of p65 (NF-κB).\",\n      \"method\": \"RBM39 knockdown/indisulam-mediated degradation, splice isoform analysis, overexpression of MEK5 isoforms, bortezomib combination cytotoxicity assays, p65 Western blotting\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic and pharmacological RBM39 depletion with defined MEK5 splicing consequence and functional rescue, single lab\",\n      \"pmids\": [\"40048740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MEK5/ERK5 co-inhibition with MEK inhibitor causes sustained G1 cell cycle arrest in NRAS-mutant melanoma by suppressing Cyclin D1 and E2F-mediated gene expression. Forced expression of Cyclin D1 and CDK4 restores cell cycle progression in MEKi/ERK5i-treated cells, identifying Cyclin D/CDK4 as the key downstream target of combined MEK/ERK5 inhibition.\",\n      \"method\": \"Transcriptome analysis, pharmacological MEKi/ERK5i combination, constitutive Cyclin D1/CDK4 overexpression rescue, cell cycle FACS analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological combination plus genetic rescue experiment identifying Cyclin D/CDK4 as downstream effector, single lab\",\n      \"pmids\": [\"41053077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MEK5-ERK5 pathway activates Hedgehog-GLI signaling: ERK5 silencing reduces GLI1 and GLI2 protein levels and transcriptional activity in melanoma cells, while constitutively active MEK5DD increases GLI1/GLI2 levels and GLI transcriptional activity. ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation.\",\n      \"method\": \"shRNA ERK5 knockdown, constitutively active MEK5DD overexpression, GLI luciferase reporter assay, pharmacological MEK5/ERK5 inhibitors, 3D spheroid assays\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal genetic gain- and loss-of-function with luciferase reporter validation, single lab\",\n      \"pmids\": [\"39998753\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAP2K5 (MEK5) is a dual-specificity MAP kinase kinase that functions as the obligate, specific upstream activator of ERK5 (BMK1); it is activated by MEKK2 and MEKK3 through PB1 domain-mediated heterodimerization and direct phosphorylation, and also by atypical PKC isoforms downstream of EGF; it is negatively regulated by XIAP/cIAP1-mediated K63-ubiquitination of MEKK2/3 that disrupts the MEKK3-MEK5-ERK5 trimeric complex; MEK5 physically associates with ERK5 and prevents its SUMOylation (independently of kinase activity) to maintain ERK5 transcriptional activity; the pathway controls downstream transcription factors including MEF2C, c-Fos/Fra-1, KLF4, Nur77, c-Myc, STAT3, and GLI1/2; and it is physiologically required for cardiac sarcomere assembly, embryonic survival, neural differentiation, myogenesis, thymocyte apoptosis, endothelial mechanosensing, M2 macrophage polarization, basal mitophagy, and NHEJ DNA repair, while being co-opted in multiple cancers as a drug-resistance and pro-metastatic pathway.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAP2K5 (MEK5) is a dual-specificity MAP kinase kinase that defines a distinct MAP kinase pathway, functioning as the obligate and specific upstream activator of ERK5/BMK1 without acting on ERK1/2, JNK, or p38 [#0, #11]. It is recruited and activated by the upstream kinases MEKK2 and MEKK3 through PB1 domain-mediated, front-to-back heterodimerization in which a basic cluster on the MEKK PB1 face engages an acidic surface on the MEK5 PB1 domain, an interaction required for ERK5 activation; a C-terminal extension of the MEK5 PB1 domain serves as the ERK5 docking site, organizing a MEKK3-MEK5-ERK5 trimeric module [#7, #14, #15]. MEK5 is alternatively activated by atypical PKC isoforms downstream of EGF and integrates growth-factor, shear-stress, and YAP/Abl/Src inputs into the cascade [#5, #24, #26]. Beyond its catalytic role, MEK5 physically binds ERK5 and prevents its repressive SUMOylation independently of kinase activity, thereby maintaining ERK5 transcriptional output [#16]. The pathway is restrained by XIAP/cIAP1, which conjugate K63-linked ubiquitin to MEKK2/3 to disrupt the trimeric complex [#25]. Through ERK5, MEK5 controls transcriptional programs governing MEF2C, c-Fos/Fra-1, KLF4, Nur77, c-Myc, STAT3, and GLI1/2 [#6, #11, #23, #24, #28, #36, #39], and is physiologically required for cardiac development and sarcomere assembly, embryonic survival, neural differentiation, myogenesis, thymocyte apoptosis, endothelial mechanosensing, M2 macrophage polarization, basal mitophagy, and NHEJ DNA repair [#4, #11, #13, #22, #23, #30, #32, #33]. The pathway is co-opted across cancers as a proliferative, pro-metastatic, and drug-resistance route, and kinase-domain MAP2K5 variants (A321T, M367T) are linked to familial non-medullary thyroid carcinoma [#27, #28, #29].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established that MEK5 is a MEK-family kinase belonging to a distinct, previously uncharacterized MAP kinase pathway rather than the ERK1/2, JNK, or p38 modules, and that splicing generates isoforms with different localization.\",\n      \"evidence\": \"PCR cloning, in vitro kinase assays against multiple MAPK substrates and upstream kinases, subcellular fractionation\",\n      \"pmids\": [\"7499418\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream substrate (ERK5) not yet identified\", \"Physiological upstream activators unknown\", \"Functional significance of alpha/beta isoform localization untested in vivo\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified MEKK2 and MEKK3 as direct upstream kinases of MEK5 and placed the module downstream of growth-factor and Ras/Raf signaling, defining the MEKK→MEK5→ERK5 cascade and its transforming potential.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, dominant-active/negative epistasis, endogenous co-IP, NIH 3T3 transformation assays\",\n      \"pmids\": [\"10593883\", \"11073940\", \"10531364\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of MEKK-MEK5 binding not yet resolved\", \"Cell-type determinants of MEKK2-vs-MEKK3 usage incompletely defined\", \"Mechanism of Raf-ERK5 association unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrated alternative upstream input via atypical PKC downstream of EGF and established a physiological output in cardiac sarcomere assembly and hypertrophy.\",\n      \"evidence\": \"Co-IP, dominant mutants, luciferase reporters; adenoviral and cardiac-specific transgenic mouse models\",\n      \"pmids\": [\"11158308\", \"11387209\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"aPKC phosphorylation sites on MEK5 not mapped\", \"Direct vs indirect cardiac transcriptional targets not fully defined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Resolved the PB1 domain interaction code governing MEK5 recruitment and identified the first transcriptional effectors (c-Fos/Fra-1, AP-1) of the pathway, linking it to invasion and MMP-9.\",\n      \"evidence\": \"In vitro PB1 binding assays, co-IP, PB1 mutagenesis, transactivation/luciferase reporters, EMSA\",\n      \"pmids\": [\"12912994\", \"12622723\", \"12618764\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ERK5 substrates among transcription factors not all distinguished from indirect effects\", \"Quantitative PB1 binding parameters not yet measured\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the subcellular logic of signaling, showing endogenous MEK5 and ERK5 are nuclear-anchored and that MEKK2 nuclear translocation transmits the EGF signal, and provided NMR-level structure of a PB1 partner surface.\",\n      \"evidence\": \"Immunofluorescence of endogenous proteins, NP-40 in situ fractionation; NMR structure of PKCiota PB1 with mutagenesis\",\n      \"pmids\": [\"15075238\", \"15143057\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of nuclear anchoring moieties unknown\", \"Direct MEK5 PB1 mutagenesis not tested in the structural study\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Genetically validated MEK5 in vivo as the essential ERK5 activator and a required regulator of proliferation, survival, and cardiac development, and extended its role to neural differentiation.\",\n      \"evidence\": \"Mek5 knockout mice (E10.5 lethality), MEFs with MEF2 reporters and caspase assays; Xenopus morpholino knockdown and gain-of-function epistasis\",\n      \"pmids\": [\"15601854\", \"16179948\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-autonomous vs systemic requirements not separated due to embryonic lethality\", \"Direct ERK5 substrates driving survival not identified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the front-to-back PB1 binding geometry, the MEK5 PB1 C-terminal ERK5 docking site, and a competitive switch in MEKK2 partner choice, providing the quantitative structural mechanism of cascade assembly.\",\n      \"evidence\": \"PB1 mutagenesis, co-IP, kinase assays; NMR structure of MEKK3 PB1 with Kd measurement (~10^-8 M)\",\n      \"pmids\": [\"17452462\", \"17985933\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full MEKK-MEK5-ERK5 ternary structure not solved\", \"Regulation of the MEKK2 MKK7-vs-MEK5 switch in physiological settings unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Uncovered a kinase-independent function of MEK5 in protecting ERK5 from repressive SUMOylation, and defined pathway-specific roles in EMT and endothelial mechanosensing using selective inhibitors.\",\n      \"evidence\": \"Kinase-dead mutants, SUMOylation and PIAS1 siRNA assays, CA-MEK5alpha transgenic mice; selective MEK5 inhibitors BIX02188/BIX02189; proteomics and ERK5 shRNA epistasis\",\n      \"pmids\": [\"18467627\", \"19087274\", \"18358237\", \"18834865\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which MEK5 binding blocks Ubc9/PIAS1 access not structurally defined\", \"EMT transcriptional targets not directly tied to ERK5 phosphorylation events\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined non-redundant tissue-specific roles in thymocyte apoptosis (via Nur77) and endothelial KLF4 induction, distinguishing MEK5/ERK5 from ERK1/2 outputs.\",\n      \"evidence\": \"Retroviral dominant/active MEK5 in thymocytes with Nur77/Bim analysis; shear stress with ERK5/KLF4 siRNA epistasis\",\n      \"pmids\": [\"18548009\", \"21166929\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ERK5 substrate linking to Nur77 induction not identified\", \"Whether KLF4 is a direct vs indirect ERK5 target unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified the negative regulatory mechanism whereby XIAP/cIAP1-mediated K63-ubiquitination of MEKK2/3 disrupts the trimeric MEK5 complex, defining how the cascade is restrained during differentiation.\",\n      \"evidence\": \"Reciprocal co-IP, K63-linkage ubiquitination assays, XIAP/cIAP1 loss-of-function, myoblast differentiation assays\",\n      \"pmids\": [\"24975362\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deubiquitinases reversing this mark not identified\", \"Signals controlling XIAP engagement of MEKK2/3 unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Connected upstream YAP/Abl/Src signaling to the cascade through a MEKK3 PPGY motif, defining how mechanical/Hippo inputs feed MEK5 activation during myogenesis.\",\n      \"evidence\": \"Co-IP, MEKK3 Y181F mutagenesis, pharmacological inhibition of c-Abl/Src/MEK5, myogenesis assays\",\n      \"pmids\": [\"28356344\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs scaffold role of YAP at the MEKK3 PB1 module unclear\", \"In vivo requirement of the PPGY interaction not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established MAP2K5 mutations as drivers of familial thyroid carcinoma and defined an ERK1/2-suppression-induced feedforward route in which MEK5/ERK5 stabilizes MYC, explaining pathway co-option in cancer and drug resistance.\",\n      \"evidence\": \"Exome sequencing and functional expression of MAP2K5 variants; kinome proteomics, MYC degradation screen, pharmacological ERK1/2+ERK5 inhibition with S62-MYC phosphorylation readout\",\n      \"pmids\": [\"30132833\", \"30423298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ERK5 directly phosphorylates MYC vs via a downstream kinase not fully resolved\", \"Generality of MAP2K5 germline variants beyond studied families unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Broadened MEK5's physiological repertoire to basal mitophagy, M2 macrophage polarization (via c-Myc, STAT3/STAT6-independent), and lipid/mevalonate metabolism in cancer, showing pathway outputs extend beyond classical proliferation control.\",\n      \"evidence\": \"Genetic and pharmacological pathway inhibition, conditional Erk5 knockout mice, transcriptomics/lipidomics, statin synergy\",\n      \"pmids\": [\"33101709\", \"32745297\", \"31969375\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct effectors linking ERK5 to mitophagy machinery not identified\", \"Mechanism of c-Myc regulation in M2 macrophages not fully mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed MEK5 functions in genome maintenance and a non-canonical autophagy role: it supports NHEJ via DNA-PK Ser2056 phosphorylation, while its inhibition activates the autophagy-lysosome pathway ERK5- and mTOR-independently.\",\n      \"evidence\": \"RNAi knockdown, gammaH2AX/53BP1 foci, DNA-PK phospho-Westerns, NHEJ assay, xenografts; MEK5 inhibitor with LC3-II/p62 and TDP-43 readouts\",\n      \"pmids\": [\"31980741\", \"31005259\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MEK5 acts on DNA-PK directly or through ERK5 not established\", \"Molecular target of the ERK5-independent autophagy effect unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended cancer-relevant outputs to Hedgehog-GLI activation, Cyclin D/CDK4-dependent cell-cycle control under combined inhibition, and RBM39-dependent MEK5 splicing required for myeloma survival, reinforcing MEK5 as a therapeutic node.\",\n      \"evidence\": \"Reciprocal ERK5/MEK5DD gain/loss-of-function, GLI and cell-cycle rescue experiments, RBM39 depletion with splice-isoform and bortezomib synergy assays\",\n      \"pmids\": [\"39998753\", \"41053077\", \"40048740\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ERK5 substrates linking to GLI and Cyclin D1 not defined\", \"Mechanism by which RBM39 controls functional MEK5 splicing not fully mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The complete catalogue of direct ERK5 substrates downstream of MEK5, the structural basis of the kinase-independent anti-SUMOylation function, and which physiological outputs depend on catalytic versus scaffolding activity of MEK5 remain to be resolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ternary MEKK-MEK5-ERK5 structure\", \"Direct vs ERK5-mediated effects on DNA-PK, MYC, and transcription factors unresolved\", \"Catalytic vs scaffold contributions to in vivo phenotypes not dissected\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 7, 11, 16, 19]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 19, 27]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9, 22, 27]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 9]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 3, 7, 14]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [6, 11, 24, 36]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [11, 13, 22, 25]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [27, 28, 29, 31]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [30]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [32, 35]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [23, 33]}\n    ],\n    \"complexes\": [\"MEKK3-MEK5-ERK5 trimeric module\"],\n    \"partners\": [\"MAPK7\", \"MAP3K2\", \"MAP3K3\", \"PRKCI\", \"PRKCZ\", \"XIAP\", \"YAP1\", \"RAF1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}