{"gene":"EYA2","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1999,"finding":"EYA2 physically interacts with DACH2 and SIX1 proteins, and these interactions synergistically regulate myogenic differentiation; EYA2-DACH2 and EYA2-SIX1 direct physical interactions were demonstrated by pulldown/co-immunoprecipitation assays.","method":"GST pulldown and co-immunoprecipitation assays; functional synergy assays in chick embryo electroporation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays plus functional epistasis in vivo, replicated across Drosophila and vertebrate systems in same study","pmids":["10617572"],"is_preprint":false},{"year":2000,"finding":"EYA2 is an intrinsically cytosolic protein that is translocated to the nucleus by SIX family homeodomain proteins; activated Gαz and Gαi2 interact with the C-terminal Eya consensus domain of EYA2, prevent its nuclear translocation, and inhibit SIX/EYA2-mediated transcription from MEF3/TATA promoter elements.","method":"Yeast two-hybrid, mammalian two-hybrid, GST pulldown, subcellular localization assays, reporter gene assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Y2H, mammalian 2-hybrid, GST pulldown, localization, reporter assay) in one study; domain mapping included","pmids":["10906137"],"is_preprint":false},{"year":2001,"finding":"PAX3 is necessary and sufficient to induce expression of SIX1 and its cofactor EYA2 prior to MyoD and myogenin induction during myogenesis; dominant-negative PAX3 abolishes SIX1 and EYA2 expression, placing PAX3 upstream of EYA2 in the myogenic transcriptional cascade.","method":"Gain-of-function and dominant-negative loss-of-function in pluripotent stem cells; RT-PCR expression analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean gain- and loss-of-function with defined molecular readout, single lab","pmids":["11262400"],"is_preprint":false},{"year":2004,"finding":"Gαi1, Gαi2, and Gαi3 (but not Gαo) interact with EYA2 in an activation-dependent manner; coexpression of activated Gαi family members recruits EYA2 to the plasma membrane, prevents nuclear translocation, abrogates EYA2/SIX4-mediated transcription, and EYA2 reciprocally relieves Gαi2-mediated inhibition of adenylyl cyclase.","method":"In vitro binding assays, co-immunoprecipitation, subcellular localization by microscopy, transcriptional reporter assays, adenylyl cyclase activity assay","journal":"Molecular pharmacology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding, localization, reporter, enzymatic assay) confirming bidirectional regulation, extending prior finding","pmids":["15308761"],"is_preprint":false},{"year":2006,"finding":"EYA1 and EYA2 act genetically upstream of PAX3 in hypaxial dermomyotome formation; SIX proteins recruit EYA proteins to MEF3 DNA sites to drive transcription during somitogenesis; compound Eya1/Eya2 double-knockout embryos lose PAX3 expression in ventrolateral dermomyotomes and lack hypaxial myogenic progenitors, phenocopying Six1/Six4 double knockouts.","method":"Genetic epistasis (double knockout mice), in situ hybridization, ChIP/reporter assays showing SIX-EYA binding to MEF3 sites","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with double-mutant rescue logic plus direct DNA-binding evidence, replicated across two gene pairs","pmids":["17098221"],"is_preprint":false},{"year":2009,"finding":"Crystal structure of the EYA domain (ED) of human EYA2 at 2.4-Å resolution reveals it belongs to the haloacid dehalogenase (HAD) family with an elongated helix-bundle motif; catalytic and SIX-interacting sites are on opposite faces, and the DACH-binding site lies between them, suggesting DACH acts as a transcriptional switch linking phosphatase activity to SIX binding; two reaction intermediates (acyl-phosphate and hydrolysis transition state) were captured.","method":"X-ray crystallography (2.4 Å), active-site intermediate capture","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mechanistic intermediate capture; detailed active-site and protein-interaction surface mapping","pmids":["19858093"],"is_preprint":false},{"year":2009,"finding":"EYA2 overexpression in transgenic mice prevents pressure-overload-induced adverse cardiac remodeling, preserves the PI3K/Akt/mTOR signaling cascade, and alters metabolic gene expression; EYA2 forms a complex with SIX1 during physiological hypertrophy.","method":"Cardiac-specific transgenic mice, pressure overload surgery, gene expression profiling, co-immunoprecipitation","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic mouse model with defined cardiac phenotype and signaling pathway readout; SIX1 complex confirmed by co-IP; single lab","pmids":["19272299"],"is_preprint":false},{"year":2011,"finding":"EYA2 directly binds to the mTOR promoter and activates mTOR expression; the EYA2-SIX1 complex binds the mTOR promoter synergistically; this pathway mediates physiological cardiac hypertrophy with preservation of mitochondrial integrity.","method":"Luciferase reporter assays, chromatin immunoprecipitation (ChIP), EMSA, transgenic mice, co-immunoprecipitation","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct DNA binding shown by ChIP and EMSA, reporter assay, and complex confirmed by co-IP, functional validation in transgenic mice; single lab","pmids":["22197309"],"is_preprint":false},{"year":2011,"finding":"EYA2 is required for SIX1 to induce TGF-β signaling, epithelial-mesenchymal transition, and cancer stem cell properties in breast cancer cells; knockdown of EYA2 reverses SIX1-mediated pro-metastatic characteristics, establishing EYA2 as a necessary co-factor for SIX1's oncogenic functions.","method":"siRNA knockdown in MCF7 cells, TGF-β signaling assays, EMT marker analysis, cancer stem cell assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with multiple defined molecular and cellular readouts; single lab","pmids":["21706047"],"is_preprint":false},{"year":2012,"finding":"A class of N-arylidenebenzohydrazide compounds selectively inhibits EYA2 phosphatase activity with IC50 values of 1.8–79 µM and does not significantly inhibit other cellular phosphatases; H2AX is confirmed as a known EYA2 substrate in this assay context.","method":"High-throughput screening, in vitro phosphatase assay (OMFP and malachite green), selectivity panel against other phosphatases","journal":"Journal of biomolecular screening","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with selectivity profiling; substrate confirmed; single lab","pmids":["22820394"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of the human SIX1-EYA2 complex at 2.0-Å resolution reveals SIX1 uses predominantly a single helix to interact with EYA2; substitution of a single amino acid in this helix disrupts SIX1-EYA2 interaction, SIX1-mediated EMT, and metastasis in mouse models; structure provides rationale for BOR syndrome mutations.","method":"X-ray crystallography (2.0 Å), site-directed mutagenesis, co-immunoprecipitation, in vivo mouse metastasis models","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure of the complex combined with mutagenesis and in vivo functional validation; multiple orthogonal methods","pmids":["23435380"],"is_preprint":false},{"year":2013,"finding":"CDK6 binds to and promotes the degradation of the EYA2 protein, suggesting CDK6 regulates EYA2 activity.","method":"Co-immunoprecipitation, protein degradation assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP and degradation assay, mechanistic detail is limited in abstract, single lab","pmids":["24196439"],"is_preprint":false},{"year":2014,"finding":"N-arylidenebenzohydrazide compounds are reversible, allosteric inhibitors of EYA2 phosphatase that do not bind the active site and do not require Mg2+ coordination; mutagenesis indicates binding occurs on the opposite face from the active site; these compounds inhibit EYA2 phosphatase-mediated cell migration selectively over EYA3.","method":"In vitro phosphatase assay, site-directed mutagenesis, reversibility assays, cell migration assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic characterization with mutagenesis defining allosteric mechanism plus cellular functional assay; single lab but multiple orthogonal methods","pmids":["24755226"],"is_preprint":false},{"year":2014,"finding":"miR-30a represses EYA2 expression by binding to the 3'-UTR of EYA2; EYA2 overexpression rescues the inhibition of breast cancer cell proliferation and migration caused by miR-30a, and EYA2 mediates G1/S cell cycle progression via regulation of cyclin A, cyclin D1, cyclin E, and c-Myc.","method":"3'-UTR reporter assay, siRNA knockdown, rescue overexpression, cell proliferation and migration assays, flow cytometry","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct 3'-UTR targeting confirmed by reporter assay; rescue experiment with EYA2 OE; multiple cellular readouts; single lab","pmids":["24508260"],"is_preprint":false},{"year":2017,"finding":"EYA2 interacts with SIX1 protein in astrocytoma cells; EYA2 positively regulates ERK activity and MMP9 expression; blockade of ERK signaling abolishes EYA2-induced MMP9 production and invasion; EYA2 fails to upregulate MMP9 expression when SIX1 is silenced, indicating EYA2 requires SIX1 for this function.","method":"Co-immunoprecipitation, siRNA knockdown, ERK inhibitor treatment, invasion assay, Western blot for MMP9","journal":"International journal of molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP confirming complex; epistasis via SIX1 knockdown; pathway inhibitor used; single lab","pmids":["28901379"],"is_preprint":false},{"year":2019,"finding":"Crystal structure of EYA2 ED in complex with allosteric inhibitor NCGC00249987 reveals it binds an induced pocket distant from the active site; inhibitor binding causes a conformational change unfavorable for Mg2+ binding, inhibiting tyrosine phosphatase activity; EYA2 tyrosine phosphatase activity is required for migration, invadopodia formation, and invasion of lung adenocarcinoma cells but not for growth or survival; EYA2 F290Y mutant abolishes compound binding, confirming on-target activity.","method":"X-ray crystallography, site-directed mutagenesis (F290Y), cell migration/invasion/invadopodia assays, genetic loss-of-function","journal":"Molecular cancer therapeutics","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of inhibitor-bound EYA2 plus mutagenesis confirming binding site plus genetic validation in cancer cells; multiple orthogonal methods","pmids":["31285279"],"is_preprint":false},{"year":2020,"finding":"EYA2 phosphatase activity regulates H2AX phosphorylation during blastema cell proliferation in axolotl limb regeneration; loss of eya2 (genetic ablation or pharmacological phosphatase inhibition) impairs cell cycle progression at G1/S and G2/M transitions and reduces regeneration rate.","method":"Eya2 mutant axolotl generation, pharmacological Eya2 phosphatase inhibition, phospho-H2AX immunostaining, cell cycle analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic and pharmacological perturbations with defined molecular readout (H2AX phosphorylation) and quantitative cell cycle phenotype; two independent approaches converge","pmids":["32142407"],"is_preprint":false},{"year":2020,"finding":"Small molecule NCGC00378430 (8430) reduces the SIX1/EYA2 protein-protein interaction; 8430 partially reverses SIX1-mediated transcriptional and metabolic profiles and reverses SIX1-induced TGF-β signaling and EMT; treatment significantly suppresses breast cancer metastasis in vivo without significantly altering primary tumor growth.","method":"Protein-protein interaction assay, transcriptomics, metabolomics, TGF-β signaling assays, EMT marker analysis, in vivo mouse metastasis model","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological disruption of SIX1-EYA2 complex with multiple molecular, transcriptomic, and in vivo phenotypic readouts; single lab but multiple orthogonal approaches","pmids":["32341035"],"is_preprint":false},{"year":2021,"finding":"EYA2 combines with DACH1 to transcriptionally regulate SOCS3 expression, suppressing JAK/STAT signaling; hepatocyte-specific deletion of EYA2 in mice promotes diethylnitrosamine-induced HCC development; EYA2(A510E) mutation leads to protein degradation via the unfolded protein response, weakening EYA2's tumor-suppressive function.","method":"Co-immunoprecipitation (EYA2-DACH1 complex), RNA sequencing, loss- and gain-of-function in cell lines, hepatocyte-specific knockout mice, unfolded protein response assays","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP demonstrating EYA2-DACH1 complex plus genetic KO mouse model plus mechanistic pathway (SOCS3/JAK/STAT) validated by multiple methods; single lab","pmids":["34044846"],"is_preprint":false},{"year":2021,"finding":"EYA2 localizes to centrosomes in glioblastoma stem cells (GSCs); EYA2 tyrosine phosphatase activity is essential for proper mitotic spindle assembly and survival of GSCs; genetic or pharmacological inhibition of EYA2 Tyr phosphatase causes mitotic catastrophe, cell cycle arrest, apoptosis, and loss of self-renewal in GSCs.","method":"Immunofluorescence localization to centrosomes, genetic EYA2 knockout/knockdown, pharmacological Tyr phosphatase inhibition, cell cycle analysis, apoptosis assays, in vivo mouse tumor model","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — novel centrosomal localization with functional consequence; convergent genetic and pharmacological evidence for phosphatase-dependent spindle assembly; in vivo validation","pmids":["34617969"],"is_preprint":false},{"year":2022,"finding":"FBXO7 binds and stabilizes EYA2, stimulating mesenchymal gene expression and suppressing IFN/chemokine/antigen-presentation pathways; ubiquitin ligase SCFFBXW7 antagonizes this by promoting EYA2 ubiquitin-mediated degradation; EYA2 Tyr phosphatase activity drives mesenchymal phenotypes and immune evasion; the AXL ligand GAS6 drives the FBXO7/EYA2 axis.","method":"Co-immunoprecipitation (FBXO7-EYA2), ubiquitination assays, genetic knockdown/knockout, transcriptomics, in vivo mouse tumor/immune infiltration assays, anti-PD-1 combination experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, ubiquitination assays, multiple genetic perturbations, in vivo immune phenotyping; multiple orthogonal methods in one study","pmids":["35182481"],"is_preprint":false},{"year":2023,"finding":"EYA2 regulates MYC expression and protein stability in Group 3 medulloblastoma; inhibition of EYA2 tyrosine phosphatase activity decreases MYC expression and global MYC transcriptional activity both in vitro and in vivo, reducing tumor growth.","method":"Genetic EYA2 knockdown, pharmacological Tyr phosphatase inhibition (NCGC00249987), MYC expression/stability assays, in vivo flank and intracranial tumor models","journal":"Neuro-oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — convergent genetic and pharmacological evidence for EYA2 phosphatase-dependent MYC regulation with in vivo validation; single lab","pmids":["37486991"],"is_preprint":false},{"year":2025,"finding":"Smurf2 ubiquitin ligase promotes EYA2 ubiquitination and degradation; Smurf2 knockdown suppresses EYA2 ubiquitination, elevates EYA2 protein levels, and inhibits mesangial cell proliferation and fibrosis under high glucose; EYA2 knockdown reverses the protective effects of Smurf2 knockdown, placing EYA2 downstream of Smurf2-mediated ubiquitination in diabetic nephropathy.","method":"Co-immunoprecipitation (Smurf2-EYA2), ubiquitination Western blot, siRNA knockdown, in vitro cell proliferation/fibrosis assays, in vivo diabetic nephropathy mouse model","journal":"Renal failure","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus ubiquitination assay plus epistasis rescue; single lab, recently published","pmids":["40556274"],"is_preprint":false}],"current_model":"EYA2 is a dual-function protein: a HAD-family tyrosine phosphatase (whose active site and allosteric regulatory pocket are structurally defined) and a transcriptional co-activator that is recruited to the nucleus by SIX family proteins, where it forms bipartite complexes with SIX1/SIX4 and DACH proteins to drive transcription from MEF3 sites regulating myogenesis, organogenesis, and oncogenic programs; its phosphatase activity dephosphorylates H2AX to promote DNA damage repair over apoptosis and is required for centrosome-dependent mitotic spindle assembly, cell migration/invasion, MYC stability, and mesenchymal/immune-evasion phenotypes, while its stability is governed by FBXO7 (stabilizing) and SCFFBXW7/Smurf2 (ubiquitin-mediated degradation), and its nuclear localization is negatively regulated by activated Gαi/Gαz proteins that sequester EYA2 at the plasma membrane."},"narrative":{"mechanistic_narrative":"EYA2 is a dual-function protein that operates both as a SIX-recruited transcriptional co-activator and as a HAD-family tyrosine phosphatase, integrating developmental and oncogenic transcriptional programs with phosphatase-dependent control of cell migration and mitosis [PMID:10617572, PMID:19858093]. Intrinsically cytosolic, EYA2 is translocated to the nucleus by SIX-family homeodomain proteins, where it forms bipartite complexes with SIX1/SIX4 and DACH proteins to drive transcription from MEF3 promoter elements; this complex governs PAX3-initiated myogenic and somitic programs during development [PMID:10906137, PMID:17098221]. Crystallographic analysis places the catalytic site and the SIX-interacting surface on opposite faces of the EYA domain with the DACH-binding site between them, and a high-resolution SIX1-EYA2 co-structure shows SIX1 engages EYA2 through a single helix whose single-residue disruption blocks SIX1-driven EMT and metastasis [PMID:19858093, PMID:23435380]. Nuclear access of EYA2 is negatively regulated by activated Gαi/Gαz proteins, which bind its C-terminal Eya domain and sequester it at the plasma membrane, while EYA2 reciprocally relieves Gαi2-mediated inhibition of adenylyl cyclase [PMID:10906137, PMID:15308761]. The intrinsic tyrosine phosphatase activity dephosphorylates H2AX and is required for cell cycle progression, mitotic spindle assembly at centrosomes, cell migration, invadopodia formation, and invasion, as established by allosteric inhibitors that bind a pocket distant from the active site and abrogate Mg2+ coordination [PMID:22820394, PMID:24755226, PMID:31285279, PMID:32142407, PMID:34617969]. Through this dual activity EYA2 drives oncogenic phenotypes — SIX1-dependent TGF-β signaling and EMT, ERK/MMP9-mediated invasion, MYC expression and stability, and mesenchymal/immune-evasion programs — and conversely acts as a tumor suppressor in liver by partnering DACH1 to induce SOCS3 and restrain JAK/STAT signaling [PMID:21706047, PMID:28901379, PMID:34044846, PMID:35182481, PMID:37486991]. EYA2 protein abundance is set by competing ubiquitin pathways: FBXO7 stabilizes it whereas SCFFBXW7 and Smurf2 promote its ubiquitin-mediated degradation [PMID:35182481, PMID:40556274].","teleology":[{"year":1999,"claim":"Established that EYA2 functions not in isolation but as a node in a SIX-EYA-DACH regulatory network controlling myogenic differentiation.","evidence":"GST pulldown and co-IP plus chick embryo electroporation epistasis showing EYA2-DACH2 and EYA2-SIX1 interactions","pmids":["10617572"],"confidence":"High","gaps":["Did not define the structural basis of the interactions","Catalytic activity of EYA2 not yet known"]},{"year":2000,"claim":"Resolved how EYA2 reaches its site of action and how it is restrained, showing it is cytosolic and SIX-imported while Gαz/Gαi2 block nuclear entry.","evidence":"Yeast and mammalian two-hybrid, GST pulldown, localization and MEF3/TATA reporter assays with domain mapping","pmids":["10906137"],"confidence":"High","gaps":["Mechanism by which SIX drives import not detailed","Physiological trigger for Gα-mediated sequestration unknown"]},{"year":2001,"claim":"Placed EYA2 within an ordered myogenic cascade by identifying PAX3 as a necessary and sufficient upstream inducer of SIX1 and EYA2.","evidence":"Gain- and dominant-negative loss-of-function in pluripotent stem cells with RT-PCR readout","pmids":["11262400"],"confidence":"Medium","gaps":["Direct vs indirect induction of EYA2 by PAX3 not resolved","Single lab, expression-level readout only"]},{"year":2004,"claim":"Extended Gα regulation to the Gαi family and demonstrated bidirectional crosstalk, with EYA2 relieving Gαi2 inhibition of adenylyl cyclase.","evidence":"In vitro binding, co-IP, localization microscopy, reporter and adenylyl cyclase activity assays","pmids":["15308761"],"confidence":"High","gaps":["In vivo relevance of EYA2-Gαi crosstalk untested","Whether phosphatase activity contributes to adenylyl cyclase modulation unknown"]},{"year":2006,"claim":"Revealed EYA2 as a developmental regulator acting genetically upstream of PAX3 in hypaxial myogenesis through SIX-MEF3 site engagement.","evidence":"Eya1/Eya2 double-knockout mice, in situ hybridization, ChIP/reporter MEF3 binding","pmids":["17098221"],"confidence":"High","gaps":["Functional redundancy with EYA1 prevents EYA2-specific dissection","Phosphatase contribution to this developmental role untested"]},{"year":2009,"claim":"Defined the structural and catalytic nature of EYA2, identifying it as a HAD-family phosphatase with spatially segregated catalytic, SIX, and DACH surfaces.","evidence":"2.4-Å X-ray crystallography of the EYA domain with reaction-intermediate capture","pmids":["19858093"],"confidence":"High","gaps":["Physiological substrates not identified in this study","Proposed DACH switch model not functionally tested here"]},{"year":2009,"claim":"Connected EYA2 to organ physiology by showing it protects against pathological cardiac remodeling via SIX1-complexed signaling.","evidence":"Cardiac-specific transgenic mice, pressure-overload surgery, expression profiling, co-IP","pmids":["19272299"],"confidence":"Medium","gaps":["Direct transcriptional targets not identified in this study","Phosphatase vs co-activator contribution not separated"]},{"year":2011,"claim":"Identified mTOR as a direct EYA2-SIX1 transcriptional target mediating physiological hypertrophy, providing a concrete promoter readout.","evidence":"Luciferase reporter, ChIP, EMSA, co-IP and transgenic mice","pmids":["22197309"],"confidence":"High","gaps":["Whether phosphatase activity is required for mTOR promoter activation unknown","Single lab"]},{"year":2011,"claim":"Established EYA2 as an obligatory co-factor for SIX1's oncogenic functions, linking it to TGF-β-driven EMT and cancer stem cell properties.","evidence":"siRNA knockdown in MCF7 cells with TGF-β, EMT marker, and stem-cell assays","pmids":["21706047"],"confidence":"Medium","gaps":["Did not separate phosphatase from co-activator requirement","Single cell line, single lab"]},{"year":2012,"claim":"Provided the first selective chemical probes of EYA2 phosphatase and confirmed H2AX as a substrate, enabling functional dissection of catalytic activity.","evidence":"HTS, in vitro phosphatase assays, selectivity panel","pmids":["22820394"],"confidence":"Medium","gaps":["Modest potency (IC50 1.8–79 µM)","Cellular target engagement not yet shown"]},{"year":2013,"claim":"Defined the SIX1-EYA2 interface at atomic resolution and proved a single interface residue controls EMT and metastasis, validating the complex as a therapeutic target.","evidence":"2.0-Å crystallography, site-directed mutagenesis, co-IP, in vivo metastasis models","pmids":["23435380"],"confidence":"High","gaps":["Did not address phosphatase-dependent functions","BOR mutation consequences inferred structurally"]},{"year":2013,"claim":"Suggested cell-cycle kinase control of EYA2 abundance by identifying CDK6-promoted degradation.","evidence":"Co-IP and protein degradation assays","pmids":["24196439"],"confidence":"Low","gaps":["Single co-IP and degradation assay without reciprocal or mechanistic validation","Ubiquitin pathway not defined"]},{"year":2014,"claim":"Demonstrated the inhibitors act allosterically rather than at the active site, separating EYA2 phosphatase-driven migration from EYA3 and defining a druggable regulatory pocket.","evidence":"In vitro phosphatase assay, mutagenesis, reversibility and migration assays","pmids":["24755226"],"confidence":"High","gaps":["Allosteric mechanism structurally undefined at this stage","Single lab"]},{"year":2014,"claim":"Placed EYA2 under post-transcriptional control by miR-30a and linked it to G1/S progression via cyclin and c-Myc regulation.","evidence":"3'-UTR reporter, knockdown, rescue overexpression, proliferation/migration and flow cytometry assays","pmids":["24508260"],"confidence":"Medium","gaps":["Mechanism of cyclin/c-Myc regulation not defined","Single lab"]},{"year":2017,"claim":"Showed EYA2 drives invasion through SIX1-dependent ERK activation and MMP9 induction in astrocytoma.","evidence":"Co-IP, siRNA knockdown, ERK inhibitor, invasion assay, MMP9 Western blot","pmids":["28901379"],"confidence":"Medium","gaps":["Direct vs indirect link between EYA2 and ERK unresolved","Single lab"]},{"year":2019,"claim":"Captured the inhibitor-bound EYA2 structure revealing an induced allosteric pocket and genetically confirmed phosphatase-dependent invasion in lung cancer.","evidence":"X-ray crystallography, F290Y mutagenesis, migration/invasion/invadopodia and loss-of-function assays","pmids":["31285279"],"confidence":"High","gaps":["Phosphatase substrate driving invasion not identified","Phosphatase dispensable for growth/survival in this model"]},{"year":2020,"claim":"Demonstrated in vivo that EYA2 phosphatase controls H2AX phosphorylation and cell cycle progression during regeneration, generalizing its catalytic role beyond cancer.","evidence":"Eya2 mutant axolotl, pharmacological inhibition, phospho-H2AX immunostaining, cell cycle analysis","pmids":["32142407"],"confidence":"High","gaps":["Whether H2AX is the direct in vivo substrate not formally proven","Co-activator contribution to regeneration untested"]},{"year":2020,"claim":"Validated pharmacological disruption of the SIX1/EYA2 complex as antimetastatic, showing it reverses EMT and TGF-β programs without affecting primary tumor growth.","evidence":"PPI assay, transcriptomics, metabolomics, EMT analysis, in vivo metastasis model","pmids":["32341035"],"confidence":"High","gaps":["Whether residual phosphatase activity persists with PPI disruption unknown","Single lab"]},{"year":2021,"claim":"Identified EYA2 as a context-dependent tumor suppressor in liver, acting with DACH1 to induce SOCS3 and dampen JAK/STAT signaling.","evidence":"Co-IP, RNA-seq, hepatocyte-specific KO mice, UPR assays of the A510E mutant","pmids":["34044846"],"confidence":"High","gaps":["Reconciliation of tumor-suppressive vs oncogenic roles across tissues unresolved","Role of phosphatase activity in SOCS3 regulation not defined"]},{"year":2021,"claim":"Revealed a centrosomal localization and a phosphatase-dependent role in mitotic spindle assembly essential for glioblastoma stem cell survival.","evidence":"Immunofluorescence, genetic KO/KD, pharmacological inhibition, cell cycle/apoptosis assays, in vivo tumor model","pmids":["34617969"],"confidence":"High","gaps":["Centrosomal substrate of EYA2 not identified","Link to H2AX dephosphorylation at centrosome unclear"]},{"year":2022,"claim":"Defined competing ubiquitin pathways controlling EYA2 stability and linked phosphatase-driven EYA2 to mesenchymal and immune-evasion programs downstream of GAS6/AXL.","evidence":"Reciprocal co-IP, ubiquitination assays, genetic perturbation, transcriptomics, in vivo immune phenotyping with anti-PD-1","pmids":["35182481"],"confidence":"High","gaps":["Direct phosphatase substrate driving immune evasion not identified","Interplay between FBXW7 and Smurf2 degradation not compared"]},{"year":2023,"claim":"Connected EYA2 phosphatase activity to MYC expression and stability, defining a targetable axis in Group 3 medulloblastoma.","evidence":"Genetic knockdown, pharmacological phosphatase inhibition, MYC expression/stability assays, in vivo tumor models","pmids":["37486991"],"confidence":"Medium","gaps":["Direct mechanism of MYC stabilization by EYA2 phosphatase not resolved","Single lab"]},{"year":2025,"claim":"Added Smurf2 as an EYA2 ubiquitin ligase relevant to diabetic nephropathy, with EYA2 placed downstream as the effector of fibrosis and proliferation.","evidence":"Co-IP, ubiquitination Western blot, siRNA knockdown, in vitro fibrosis assays, in vivo diabetic nephropathy model","pmids":["40556274"],"confidence":"Medium","gaps":["Smurf2 degron on EYA2 not mapped","Single lab, recently published"]},{"year":null,"claim":"The physiological tyrosine phosphatase substrates beyond H2AX and the basis for EYA2's opposite (oncogenic vs tumor-suppressive) roles across tissues remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct centrosomal or MYC-stabilizing phosphatase substrate identified","Switch between co-activator and phosphatase functions not mechanistically resolved","Tissue-specific determinants of oncogenic vs suppressive output unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,9,12,15,16,19]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[5,9,15]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[1,4,7,18]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[4,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,4]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[19]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,4,7,18]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,2,4]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[8,18,20,21]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[16,19,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,14,17,18]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[20,22]}],"complexes":["SIX1-EYA2-DACH transcriptional complex","SIX4-EYA2 complex","EYA2-DACH1 complex"],"partners":["SIX1","SIX4","DACH1","DACH2","FBXO7","FBXW7","SMURF2","GNAI2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O00167","full_name":"Protein phosphatase EYA2","aliases":["Eyes absent homolog 2"],"length_aa":538,"mass_kda":59.2,"function":"Functions both as protein phosphatase and as transcriptional coactivator for SIX1, and probably also for SIX2, SIX4 and SIX5 (PubMed:12500905, PubMed:23435380). Tyrosine phosphatase that dephosphorylates 'Tyr-142' of histone H2AX (H2AXY142ph) and promotes efficient DNA repair via the recruitment of DNA repair complexes containing MDC1. 'Tyr-142' phosphorylation of histone H2AX plays a central role in DNA repair and acts as a mark that distinguishes between apoptotic and repair responses to genotoxic stress (PubMed:19351884). Its function as histone phosphatase may contribute to its function in transcription regulation during organogenesis. 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EYA2-DACH2 and EYA2-SIX1 direct physical interactions were demonstrated by pulldown/co-immunoprecipitation assays.\",\n      \"method\": \"GST pulldown and co-immunoprecipitation assays; functional synergy assays in chick embryo electroporation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays plus functional epistasis in vivo, replicated across Drosophila and vertebrate systems in same study\",\n      \"pmids\": [\"10617572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"EYA2 is an intrinsically cytosolic protein that is translocated to the nucleus by SIX family homeodomain proteins; activated Gαz and Gαi2 interact with the C-terminal Eya consensus domain of EYA2, prevent its nuclear translocation, and inhibit SIX/EYA2-mediated transcription from MEF3/TATA promoter elements.\",\n      \"method\": \"Yeast two-hybrid, mammalian two-hybrid, GST pulldown, subcellular localization assays, reporter gene assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Y2H, mammalian 2-hybrid, GST pulldown, localization, reporter assay) in one study; domain mapping included\",\n      \"pmids\": [\"10906137\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PAX3 is necessary and sufficient to induce expression of SIX1 and its cofactor EYA2 prior to MyoD and myogenin induction during myogenesis; dominant-negative PAX3 abolishes SIX1 and EYA2 expression, placing PAX3 upstream of EYA2 in the myogenic transcriptional cascade.\",\n      \"method\": \"Gain-of-function and dominant-negative loss-of-function in pluripotent stem cells; RT-PCR expression analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean gain- and loss-of-function with defined molecular readout, single lab\",\n      \"pmids\": [\"11262400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Gαi1, Gαi2, and Gαi3 (but not Gαo) interact with EYA2 in an activation-dependent manner; coexpression of activated Gαi family members recruits EYA2 to the plasma membrane, prevents nuclear translocation, abrogates EYA2/SIX4-mediated transcription, and EYA2 reciprocally relieves Gαi2-mediated inhibition of adenylyl cyclase.\",\n      \"method\": \"In vitro binding assays, co-immunoprecipitation, subcellular localization by microscopy, transcriptional reporter assays, adenylyl cyclase activity assay\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding, localization, reporter, enzymatic assay) confirming bidirectional regulation, extending prior finding\",\n      \"pmids\": [\"15308761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"EYA1 and EYA2 act genetically upstream of PAX3 in hypaxial dermomyotome formation; SIX proteins recruit EYA proteins to MEF3 DNA sites to drive transcription during somitogenesis; compound Eya1/Eya2 double-knockout embryos lose PAX3 expression in ventrolateral dermomyotomes and lack hypaxial myogenic progenitors, phenocopying Six1/Six4 double knockouts.\",\n      \"method\": \"Genetic epistasis (double knockout mice), in situ hybridization, ChIP/reporter assays showing SIX-EYA binding to MEF3 sites\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with double-mutant rescue logic plus direct DNA-binding evidence, replicated across two gene pairs\",\n      \"pmids\": [\"17098221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Crystal structure of the EYA domain (ED) of human EYA2 at 2.4-Å resolution reveals it belongs to the haloacid dehalogenase (HAD) family with an elongated helix-bundle motif; catalytic and SIX-interacting sites are on opposite faces, and the DACH-binding site lies between them, suggesting DACH acts as a transcriptional switch linking phosphatase activity to SIX binding; two reaction intermediates (acyl-phosphate and hydrolysis transition state) were captured.\",\n      \"method\": \"X-ray crystallography (2.4 Å), active-site intermediate capture\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mechanistic intermediate capture; detailed active-site and protein-interaction surface mapping\",\n      \"pmids\": [\"19858093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EYA2 overexpression in transgenic mice prevents pressure-overload-induced adverse cardiac remodeling, preserves the PI3K/Akt/mTOR signaling cascade, and alters metabolic gene expression; EYA2 forms a complex with SIX1 during physiological hypertrophy.\",\n      \"method\": \"Cardiac-specific transgenic mice, pressure overload surgery, gene expression profiling, co-immunoprecipitation\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic mouse model with defined cardiac phenotype and signaling pathway readout; SIX1 complex confirmed by co-IP; single lab\",\n      \"pmids\": [\"19272299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EYA2 directly binds to the mTOR promoter and activates mTOR expression; the EYA2-SIX1 complex binds the mTOR promoter synergistically; this pathway mediates physiological cardiac hypertrophy with preservation of mitochondrial integrity.\",\n      \"method\": \"Luciferase reporter assays, chromatin immunoprecipitation (ChIP), EMSA, transgenic mice, co-immunoprecipitation\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct DNA binding shown by ChIP and EMSA, reporter assay, and complex confirmed by co-IP, functional validation in transgenic mice; single lab\",\n      \"pmids\": [\"22197309\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EYA2 is required for SIX1 to induce TGF-β signaling, epithelial-mesenchymal transition, and cancer stem cell properties in breast cancer cells; knockdown of EYA2 reverses SIX1-mediated pro-metastatic characteristics, establishing EYA2 as a necessary co-factor for SIX1's oncogenic functions.\",\n      \"method\": \"siRNA knockdown in MCF7 cells, TGF-β signaling assays, EMT marker analysis, cancer stem cell assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with multiple defined molecular and cellular readouts; single lab\",\n      \"pmids\": [\"21706047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"A class of N-arylidenebenzohydrazide compounds selectively inhibits EYA2 phosphatase activity with IC50 values of 1.8–79 µM and does not significantly inhibit other cellular phosphatases; H2AX is confirmed as a known EYA2 substrate in this assay context.\",\n      \"method\": \"High-throughput screening, in vitro phosphatase assay (OMFP and malachite green), selectivity panel against other phosphatases\",\n      \"journal\": \"Journal of biomolecular screening\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with selectivity profiling; substrate confirmed; single lab\",\n      \"pmids\": [\"22820394\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of the human SIX1-EYA2 complex at 2.0-Å resolution reveals SIX1 uses predominantly a single helix to interact with EYA2; substitution of a single amino acid in this helix disrupts SIX1-EYA2 interaction, SIX1-mediated EMT, and metastasis in mouse models; structure provides rationale for BOR syndrome mutations.\",\n      \"method\": \"X-ray crystallography (2.0 Å), site-directed mutagenesis, co-immunoprecipitation, in vivo mouse metastasis models\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure of the complex combined with mutagenesis and in vivo functional validation; multiple orthogonal methods\",\n      \"pmids\": [\"23435380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"CDK6 binds to and promotes the degradation of the EYA2 protein, suggesting CDK6 regulates EYA2 activity.\",\n      \"method\": \"Co-immunoprecipitation, protein degradation assays\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP and degradation assay, mechanistic detail is limited in abstract, single lab\",\n      \"pmids\": [\"24196439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"N-arylidenebenzohydrazide compounds are reversible, allosteric inhibitors of EYA2 phosphatase that do not bind the active site and do not require Mg2+ coordination; mutagenesis indicates binding occurs on the opposite face from the active site; these compounds inhibit EYA2 phosphatase-mediated cell migration selectively over EYA3.\",\n      \"method\": \"In vitro phosphatase assay, site-directed mutagenesis, reversibility assays, cell migration assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic characterization with mutagenesis defining allosteric mechanism plus cellular functional assay; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"24755226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"miR-30a represses EYA2 expression by binding to the 3'-UTR of EYA2; EYA2 overexpression rescues the inhibition of breast cancer cell proliferation and migration caused by miR-30a, and EYA2 mediates G1/S cell cycle progression via regulation of cyclin A, cyclin D1, cyclin E, and c-Myc.\",\n      \"method\": \"3'-UTR reporter assay, siRNA knockdown, rescue overexpression, cell proliferation and migration assays, flow cytometry\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct 3'-UTR targeting confirmed by reporter assay; rescue experiment with EYA2 OE; multiple cellular readouts; single lab\",\n      \"pmids\": [\"24508260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"EYA2 interacts with SIX1 protein in astrocytoma cells; EYA2 positively regulates ERK activity and MMP9 expression; blockade of ERK signaling abolishes EYA2-induced MMP9 production and invasion; EYA2 fails to upregulate MMP9 expression when SIX1 is silenced, indicating EYA2 requires SIX1 for this function.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, ERK inhibitor treatment, invasion assay, Western blot for MMP9\",\n      \"journal\": \"International journal of molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP confirming complex; epistasis via SIX1 knockdown; pathway inhibitor used; single lab\",\n      \"pmids\": [\"28901379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structure of EYA2 ED in complex with allosteric inhibitor NCGC00249987 reveals it binds an induced pocket distant from the active site; inhibitor binding causes a conformational change unfavorable for Mg2+ binding, inhibiting tyrosine phosphatase activity; EYA2 tyrosine phosphatase activity is required for migration, invadopodia formation, and invasion of lung adenocarcinoma cells but not for growth or survival; EYA2 F290Y mutant abolishes compound binding, confirming on-target activity.\",\n      \"method\": \"X-ray crystallography, site-directed mutagenesis (F290Y), cell migration/invasion/invadopodia assays, genetic loss-of-function\",\n      \"journal\": \"Molecular cancer therapeutics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of inhibitor-bound EYA2 plus mutagenesis confirming binding site plus genetic validation in cancer cells; multiple orthogonal methods\",\n      \"pmids\": [\"31285279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EYA2 phosphatase activity regulates H2AX phosphorylation during blastema cell proliferation in axolotl limb regeneration; loss of eya2 (genetic ablation or pharmacological phosphatase inhibition) impairs cell cycle progression at G1/S and G2/M transitions and reduces regeneration rate.\",\n      \"method\": \"Eya2 mutant axolotl generation, pharmacological Eya2 phosphatase inhibition, phospho-H2AX immunostaining, cell cycle analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic and pharmacological perturbations with defined molecular readout (H2AX phosphorylation) and quantitative cell cycle phenotype; two independent approaches converge\",\n      \"pmids\": [\"32142407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Small molecule NCGC00378430 (8430) reduces the SIX1/EYA2 protein-protein interaction; 8430 partially reverses SIX1-mediated transcriptional and metabolic profiles and reverses SIX1-induced TGF-β signaling and EMT; treatment significantly suppresses breast cancer metastasis in vivo without significantly altering primary tumor growth.\",\n      \"method\": \"Protein-protein interaction assay, transcriptomics, metabolomics, TGF-β signaling assays, EMT marker analysis, in vivo mouse metastasis model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological disruption of SIX1-EYA2 complex with multiple molecular, transcriptomic, and in vivo phenotypic readouts; single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"32341035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EYA2 combines with DACH1 to transcriptionally regulate SOCS3 expression, suppressing JAK/STAT signaling; hepatocyte-specific deletion of EYA2 in mice promotes diethylnitrosamine-induced HCC development; EYA2(A510E) mutation leads to protein degradation via the unfolded protein response, weakening EYA2's tumor-suppressive function.\",\n      \"method\": \"Co-immunoprecipitation (EYA2-DACH1 complex), RNA sequencing, loss- and gain-of-function in cell lines, hepatocyte-specific knockout mice, unfolded protein response assays\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP demonstrating EYA2-DACH1 complex plus genetic KO mouse model plus mechanistic pathway (SOCS3/JAK/STAT) validated by multiple methods; single lab\",\n      \"pmids\": [\"34044846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EYA2 localizes to centrosomes in glioblastoma stem cells (GSCs); EYA2 tyrosine phosphatase activity is essential for proper mitotic spindle assembly and survival of GSCs; genetic or pharmacological inhibition of EYA2 Tyr phosphatase causes mitotic catastrophe, cell cycle arrest, apoptosis, and loss of self-renewal in GSCs.\",\n      \"method\": \"Immunofluorescence localization to centrosomes, genetic EYA2 knockout/knockdown, pharmacological Tyr phosphatase inhibition, cell cycle analysis, apoptosis assays, in vivo mouse tumor model\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — novel centrosomal localization with functional consequence; convergent genetic and pharmacological evidence for phosphatase-dependent spindle assembly; in vivo validation\",\n      \"pmids\": [\"34617969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FBXO7 binds and stabilizes EYA2, stimulating mesenchymal gene expression and suppressing IFN/chemokine/antigen-presentation pathways; ubiquitin ligase SCFFBXW7 antagonizes this by promoting EYA2 ubiquitin-mediated degradation; EYA2 Tyr phosphatase activity drives mesenchymal phenotypes and immune evasion; the AXL ligand GAS6 drives the FBXO7/EYA2 axis.\",\n      \"method\": \"Co-immunoprecipitation (FBXO7-EYA2), ubiquitination assays, genetic knockdown/knockout, transcriptomics, in vivo mouse tumor/immune infiltration assays, anti-PD-1 combination experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, ubiquitination assays, multiple genetic perturbations, in vivo immune phenotyping; multiple orthogonal methods in one study\",\n      \"pmids\": [\"35182481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EYA2 regulates MYC expression and protein stability in Group 3 medulloblastoma; inhibition of EYA2 tyrosine phosphatase activity decreases MYC expression and global MYC transcriptional activity both in vitro and in vivo, reducing tumor growth.\",\n      \"method\": \"Genetic EYA2 knockdown, pharmacological Tyr phosphatase inhibition (NCGC00249987), MYC expression/stability assays, in vivo flank and intracranial tumor models\",\n      \"journal\": \"Neuro-oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — convergent genetic and pharmacological evidence for EYA2 phosphatase-dependent MYC regulation with in vivo validation; single lab\",\n      \"pmids\": [\"37486991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Smurf2 ubiquitin ligase promotes EYA2 ubiquitination and degradation; Smurf2 knockdown suppresses EYA2 ubiquitination, elevates EYA2 protein levels, and inhibits mesangial cell proliferation and fibrosis under high glucose; EYA2 knockdown reverses the protective effects of Smurf2 knockdown, placing EYA2 downstream of Smurf2-mediated ubiquitination in diabetic nephropathy.\",\n      \"method\": \"Co-immunoprecipitation (Smurf2-EYA2), ubiquitination Western blot, siRNA knockdown, in vitro cell proliferation/fibrosis assays, in vivo diabetic nephropathy mouse model\",\n      \"journal\": \"Renal failure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus ubiquitination assay plus epistasis rescue; single lab, recently published\",\n      \"pmids\": [\"40556274\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EYA2 is a dual-function protein: a HAD-family tyrosine phosphatase (whose active site and allosteric regulatory pocket are structurally defined) and a transcriptional co-activator that is recruited to the nucleus by SIX family proteins, where it forms bipartite complexes with SIX1/SIX4 and DACH proteins to drive transcription from MEF3 sites regulating myogenesis, organogenesis, and oncogenic programs; its phosphatase activity dephosphorylates H2AX to promote DNA damage repair over apoptosis and is required for centrosome-dependent mitotic spindle assembly, cell migration/invasion, MYC stability, and mesenchymal/immune-evasion phenotypes, while its stability is governed by FBXO7 (stabilizing) and SCFFBXW7/Smurf2 (ubiquitin-mediated degradation), and its nuclear localization is negatively regulated by activated Gαi/Gαz proteins that sequester EYA2 at the plasma membrane.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EYA2 is a dual-function protein that operates both as a SIX-recruited transcriptional co-activator and as a HAD-family tyrosine phosphatase, integrating developmental and oncogenic transcriptional programs with phosphatase-dependent control of cell migration and mitosis [#0, #5]. Intrinsically cytosolic, EYA2 is translocated to the nucleus by SIX-family homeodomain proteins, where it forms bipartite complexes with SIX1/SIX4 and DACH proteins to drive transcription from MEF3 promoter elements; this complex governs PAX3-initiated myogenic and somitic programs during development [#1, #4]. Crystallographic analysis places the catalytic site and the SIX-interacting surface on opposite faces of the EYA domain with the DACH-binding site between them, and a high-resolution SIX1-EYA2 co-structure shows SIX1 engages EYA2 through a single helix whose single-residue disruption blocks SIX1-driven EMT and metastasis [#5, #10]. Nuclear access of EYA2 is negatively regulated by activated Gαi/Gαz proteins, which bind its C-terminal Eya domain and sequester it at the plasma membrane, while EYA2 reciprocally relieves Gαi2-mediated inhibition of adenylyl cyclase [#1, #3]. The intrinsic tyrosine phosphatase activity dephosphorylates H2AX and is required for cell cycle progression, mitotic spindle assembly at centrosomes, cell migration, invadopodia formation, and invasion, as established by allosteric inhibitors that bind a pocket distant from the active site and abrogate Mg2+ coordination [#9, #12, #15, #16, #19]. Through this dual activity EYA2 drives oncogenic phenotypes — SIX1-dependent TGF-β signaling and EMT, ERK/MMP9-mediated invasion, MYC expression and stability, and mesenchymal/immune-evasion programs — and conversely acts as a tumor suppressor in liver by partnering DACH1 to induce SOCS3 and restrain JAK/STAT signaling [#8, #14, #18, #20, #21]. EYA2 protein abundance is set by competing ubiquitin pathways: FBXO7 stabilizes it whereas SCFFBXW7 and Smurf2 promote its ubiquitin-mediated degradation [#20, #22].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established that EYA2 functions not in isolation but as a node in a SIX-EYA-DACH regulatory network controlling myogenic differentiation.\",\n      \"evidence\": \"GST pulldown and co-IP plus chick embryo electroporation epistasis showing EYA2-DACH2 and EYA2-SIX1 interactions\",\n      \"pmids\": [\"10617572\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural basis of the interactions\", \"Catalytic activity of EYA2 not yet known\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Resolved how EYA2 reaches its site of action and how it is restrained, showing it is cytosolic and SIX-imported while Gαz/Gαi2 block nuclear entry.\",\n      \"evidence\": \"Yeast and mammalian two-hybrid, GST pulldown, localization and MEF3/TATA reporter assays with domain mapping\",\n      \"pmids\": [\"10906137\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which SIX drives import not detailed\", \"Physiological trigger for Gα-mediated sequestration unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Placed EYA2 within an ordered myogenic cascade by identifying PAX3 as a necessary and sufficient upstream inducer of SIX1 and EYA2.\",\n      \"evidence\": \"Gain- and dominant-negative loss-of-function in pluripotent stem cells with RT-PCR readout\",\n      \"pmids\": [\"11262400\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect induction of EYA2 by PAX3 not resolved\", \"Single lab, expression-level readout only\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Extended Gα regulation to the Gαi family and demonstrated bidirectional crosstalk, with EYA2 relieving Gαi2 inhibition of adenylyl cyclase.\",\n      \"evidence\": \"In vitro binding, co-IP, localization microscopy, reporter and adenylyl cyclase activity assays\",\n      \"pmids\": [\"15308761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of EYA2-Gαi crosstalk untested\", \"Whether phosphatase activity contributes to adenylyl cyclase modulation unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealed EYA2 as a developmental regulator acting genetically upstream of PAX3 in hypaxial myogenesis through SIX-MEF3 site engagement.\",\n      \"evidence\": \"Eya1/Eya2 double-knockout mice, in situ hybridization, ChIP/reporter MEF3 binding\",\n      \"pmids\": [\"17098221\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional redundancy with EYA1 prevents EYA2-specific dissection\", \"Phosphatase contribution to this developmental role untested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the structural and catalytic nature of EYA2, identifying it as a HAD-family phosphatase with spatially segregated catalytic, SIX, and DACH surfaces.\",\n      \"evidence\": \"2.4-Å X-ray crystallography of the EYA domain with reaction-intermediate capture\",\n      \"pmids\": [\"19858093\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates not identified in this study\", \"Proposed DACH switch model not functionally tested here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connected EYA2 to organ physiology by showing it protects against pathological cardiac remodeling via SIX1-complexed signaling.\",\n      \"evidence\": \"Cardiac-specific transgenic mice, pressure-overload surgery, expression profiling, co-IP\",\n      \"pmids\": [\"19272299\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transcriptional targets not identified in this study\", \"Phosphatase vs co-activator contribution not separated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified mTOR as a direct EYA2-SIX1 transcriptional target mediating physiological hypertrophy, providing a concrete promoter readout.\",\n      \"evidence\": \"Luciferase reporter, ChIP, EMSA, co-IP and transgenic mice\",\n      \"pmids\": [\"22197309\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether phosphatase activity is required for mTOR promoter activation unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established EYA2 as an obligatory co-factor for SIX1's oncogenic functions, linking it to TGF-β-driven EMT and cancer stem cell properties.\",\n      \"evidence\": \"siRNA knockdown in MCF7 cells with TGF-β, EMT marker, and stem-cell assays\",\n      \"pmids\": [\"21706047\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not separate phosphatase from co-activator requirement\", \"Single cell line, single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Provided the first selective chemical probes of EYA2 phosphatase and confirmed H2AX as a substrate, enabling functional dissection of catalytic activity.\",\n      \"evidence\": \"HTS, in vitro phosphatase assays, selectivity panel\",\n      \"pmids\": [\"22820394\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Modest potency (IC50 1.8–79 µM)\", \"Cellular target engagement not yet shown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined the SIX1-EYA2 interface at atomic resolution and proved a single interface residue controls EMT and metastasis, validating the complex as a therapeutic target.\",\n      \"evidence\": \"2.0-Å crystallography, site-directed mutagenesis, co-IP, in vivo metastasis models\",\n      \"pmids\": [\"23435380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address phosphatase-dependent functions\", \"BOR mutation consequences inferred structurally\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Suggested cell-cycle kinase control of EYA2 abundance by identifying CDK6-promoted degradation.\",\n      \"evidence\": \"Co-IP and protein degradation assays\",\n      \"pmids\": [\"24196439\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single co-IP and degradation assay without reciprocal or mechanistic validation\", \"Ubiquitin pathway not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated the inhibitors act allosterically rather than at the active site, separating EYA2 phosphatase-driven migration from EYA3 and defining a druggable regulatory pocket.\",\n      \"evidence\": \"In vitro phosphatase assay, mutagenesis, reversibility and migration assays\",\n      \"pmids\": [\"24755226\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Allosteric mechanism structurally undefined at this stage\", \"Single lab\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placed EYA2 under post-transcriptional control by miR-30a and linked it to G1/S progression via cyclin and c-Myc regulation.\",\n      \"evidence\": \"3'-UTR reporter, knockdown, rescue overexpression, proliferation/migration and flow cytometry assays\",\n      \"pmids\": [\"24508260\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of cyclin/c-Myc regulation not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed EYA2 drives invasion through SIX1-dependent ERK activation and MMP9 induction in astrocytoma.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, ERK inhibitor, invasion assay, MMP9 Western blot\",\n      \"pmids\": [\"28901379\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect link between EYA2 and ERK unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Captured the inhibitor-bound EYA2 structure revealing an induced allosteric pocket and genetically confirmed phosphatase-dependent invasion in lung cancer.\",\n      \"evidence\": \"X-ray crystallography, F290Y mutagenesis, migration/invasion/invadopodia and loss-of-function assays\",\n      \"pmids\": [\"31285279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase substrate driving invasion not identified\", \"Phosphatase dispensable for growth/survival in this model\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated in vivo that EYA2 phosphatase controls H2AX phosphorylation and cell cycle progression during regeneration, generalizing its catalytic role beyond cancer.\",\n      \"evidence\": \"Eya2 mutant axolotl, pharmacological inhibition, phospho-H2AX immunostaining, cell cycle analysis\",\n      \"pmids\": [\"32142407\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether H2AX is the direct in vivo substrate not formally proven\", \"Co-activator contribution to regeneration untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Validated pharmacological disruption of the SIX1/EYA2 complex as antimetastatic, showing it reverses EMT and TGF-β programs without affecting primary tumor growth.\",\n      \"evidence\": \"PPI assay, transcriptomics, metabolomics, EMT analysis, in vivo metastasis model\",\n      \"pmids\": [\"32341035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether residual phosphatase activity persists with PPI disruption unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified EYA2 as a context-dependent tumor suppressor in liver, acting with DACH1 to induce SOCS3 and dampen JAK/STAT signaling.\",\n      \"evidence\": \"Co-IP, RNA-seq, hepatocyte-specific KO mice, UPR assays of the A510E mutant\",\n      \"pmids\": [\"34044846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation of tumor-suppressive vs oncogenic roles across tissues unresolved\", \"Role of phosphatase activity in SOCS3 regulation not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed a centrosomal localization and a phosphatase-dependent role in mitotic spindle assembly essential for glioblastoma stem cell survival.\",\n      \"evidence\": \"Immunofluorescence, genetic KO/KD, pharmacological inhibition, cell cycle/apoptosis assays, in vivo tumor model\",\n      \"pmids\": [\"34617969\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Centrosomal substrate of EYA2 not identified\", \"Link to H2AX dephosphorylation at centrosome unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined competing ubiquitin pathways controlling EYA2 stability and linked phosphatase-driven EYA2 to mesenchymal and immune-evasion programs downstream of GAS6/AXL.\",\n      \"evidence\": \"Reciprocal co-IP, ubiquitination assays, genetic perturbation, transcriptomics, in vivo immune phenotyping with anti-PD-1\",\n      \"pmids\": [\"35182481\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phosphatase substrate driving immune evasion not identified\", \"Interplay between FBXW7 and Smurf2 degradation not compared\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected EYA2 phosphatase activity to MYC expression and stability, defining a targetable axis in Group 3 medulloblastoma.\",\n      \"evidence\": \"Genetic knockdown, pharmacological phosphatase inhibition, MYC expression/stability assays, in vivo tumor models\",\n      \"pmids\": [\"37486991\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct mechanism of MYC stabilization by EYA2 phosphatase not resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Added Smurf2 as an EYA2 ubiquitin ligase relevant to diabetic nephropathy, with EYA2 placed downstream as the effector of fibrosis and proliferation.\",\n      \"evidence\": \"Co-IP, ubiquitination Western blot, siRNA knockdown, in vitro fibrosis assays, in vivo diabetic nephropathy model\",\n      \"pmids\": [\"40556274\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Smurf2 degron on EYA2 not mapped\", \"Single lab, recently published\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The physiological tyrosine phosphatase substrates beyond H2AX and the basis for EYA2's opposite (oncogenic vs tumor-suppressive) roles across tissues remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct centrosomal or MYC-stabilizing phosphatase substrate identified\", \"Switch between co-activator and phosphatase functions not mechanistically resolved\", \"Tissue-specific determinants of oncogenic vs suppressive output unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 9, 12, 15, 16, 19]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [5, 9, 15]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 4, 7, 18]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [4, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 4, 7, 18]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 2, 4]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [8, 18, 20, 21]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [16, 19, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 14, 17, 18]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [20, 22]}\n    ],\n    \"complexes\": [\n      \"SIX1-EYA2-DACH transcriptional complex\",\n      \"SIX4-EYA2 complex\",\n      \"EYA2-DACH1 complex\"\n    ],\n    \"partners\": [\n      \"SIX1\",\n      \"SIX4\",\n      \"DACH1\",\n      \"DACH2\",\n      \"FBXO7\",\n      \"FBXW7\",\n      \"Smurf2\",\n      \"GNAI2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}