Affinage

EYA3

Protein phosphatase EYA3 · UniProt Q99504

Length
573 aa
Mass
62.7 kDa
Annotated
2026-06-09
28 papers in source corpus 19 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: tie faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

EYA3 is a dual-function protein that operates both as a transcriptional co-activator in developmental and oncogenic programs and as a tyrosine phosphatase that controls DNA damage repair, cytoskeletal dynamics, and cell survival (PMID:31515519, PMID:29440662, PMID:19008232). As a co-activator, EYA3 partners with SIX-family transcription factors and additional cofactors at target promoters: it synergizes with Six1 (enhanced by Tef and Hlf) to drive photoperiodic TSHβ induction (PMID:21129973, PMID:20434341), joins Ski and Six1 at the MEF3 element to activate myogenin during muscle differentiation (PMID:19008232), and under hypoxia assembles with SIX5 and the acetyltransferase p300 to transactivate EGFR, VEGFD, and multiple MMPs in colorectal cancer (PMID:35957720); isoform choice driven by RBFOX2-dependent splicing of exon 7 redirects EYA3 between SIX4 and ZBTB1 partners during myogenesis (PMID:38026174). Its intrinsic catalytic activity is a tyrosine phosphatase that dephosphorylates H2AX-pY142 to promote DNA repair and cell survival and WDR1 to remodel the actin cytoskeleton, with Src-mediated tyrosine phosphorylation and EYA3 autodephosphorylation tuning its localization and proliferative output (PMID:29440662, PMID:31847183, PMID:33649104). EYA3's apparent Ser/Thr phosphatase activity is not intrinsic but arises from direct recruitment of the PP2A-B55α holoenzyme through an extended N-terminal peptide defined by cryo-EM, redirecting PP2A-B55α to dephosphorylate c-Myc pT58 and thereby stabilize Myc (PMID:29535359, PMID:40247147, PMID:40414499, PMID:39975004). Through this Myc-stabilizing axis and NF-κB/CCL2 signaling, EYA3 upregulates PD-L1, suppresses CD8+ T cell and NK cell anti-tumor responses, and promotes breast cancer growth and metastasis (PMID:29757193, PMID:40333987, PMID:39211066). A missense EYA3 variant and zebrafish knockdown link the gene to oculo-auriculo-vertebral spectrum craniofacial development (PMID:33475861).

Mechanistic history

Synthesis pass · year-by-year structured walk · 14 steps
  1. 2008 Medium

    Established EYA3 as a component of a sequence-specific transcriptional activator complex, answering how it contributes to developmental gene programs.

    Evidence ChIP, reciprocal Co-IP, and reporter assays showing Ski-Six1-Eya3 binding to the Myog MEF3 site during myoblast differentiation

    PMID:19008232

    Open questions at the time
    • Direct DNA contact by EYA3 itself not demonstrated
    • Role of phosphatase activity in this transcriptional function unaddressed
  2. 2010 Medium

    Placed Eya3 upstream of TSHβ in the photoperiodic response, defining a physiological context for its co-activator function with Six1.

    Evidence Genome-wide expression analysis and transcriptional reporter assays with acute light stimulation in mice, replicated in sheep

    PMID:20434341 PMID:21129973

    Open questions at the time
    • Molecular mechanism of light-induced Eya3 induction not resolved
    • Whether catalytic activity is required for TSHβ activation unknown
  3. 2012 Medium

    Showed how EYA3 is post-transcriptionally controlled in cancer and linked it to DNA repair-mediated chemoresistance.

    Evidence miR-708 3'-UTR luciferase reporter, EYA3 knockdown, and DNA damage/chemosensitivity assays in Ewing sarcoma

    PMID:22723308

    Open questions at the time
    • Direct EYA3 substrate in the repair pathway not identified here
    • Phosphatase requirement not tested
  4. 2018 High

    Resolved the long-standing puzzle of EYA Ser/Thr phosphatase activity by showing it is borrowed from PP2A-B55α and redirected to stabilize c-Myc via pT58 dephosphorylation.

    Evidence Co-IP, in vitro phosphatase assays, mutagenesis, and breast cancer xenograft metastasis model

    PMID:29535359

    Open questions at the time
    • Structural basis of recruitment not yet defined at this stage
    • Generality across substrates beyond c-Myc unclear
  5. 2018 Medium

    Defined EYA3 substrate specificity and regulation of its tyrosine phosphatase, identifying WDR1 as a substrate and Src as the upstream kinase controlling localization.

    Evidence Phosphotyrosine peptide microarray, in vitro phosphatase assays, subcellular fractionation, and actin imaging

    PMID:29440662

    Open questions at the time
    • WDR1 dephosphorylation site not mapped
    • Single-lab finding without reciprocal validation
  6. 2018 High

    Demonstrated that EYA3-directed Myc stabilization drives immune evasion via PD-L1, linking the phosphatase axis to anti-tumor immunity.

    Evidence Knockdown, phosphatase assays, CD8+ T cell depletion, and PD-L1 rescue in immune-competent mice

    PMID:29757193

    Open questions at the time
    • Mechanism linking Myc to PD-L1 transcription not fully detailed
    • Tissue scope beyond model unclear
  7. 2018 High

    Validated EYA3 tyrosine phosphatase activity as a druggable driver of cell survival under DNA damage in a non-cancer disease setting.

    Evidence Phosphatase-dead transgenic mouse and pharmacological inhibition in rat pulmonary hypertension model

    PMID:31515519

    Open questions at the time
    • Relevant substrate(s) in pulmonary arterial smooth muscle not pinpointed
    • Selectivity of inhibitor for EYA3 vs paralogs unaddressed
  8. 2019 Medium

    Mapped Src phosphorylation sites on EYA3 and tied specific tyrosines to its proliferative function, refining the autodephosphorylation regulatory loop.

    Evidence Native and bottom-up mass spectrometry, site-directed mutagenesis, and cell cycle analysis in HEK293T cells

    PMID:31847183

    Open questions at the time
    • Downstream effectors of proliferative tyrosines not identified
    • In vivo relevance of autodephosphorylation kinetics untested
  9. 2021 Medium

    Confirmed H2AX-pY142 as an in vivo EYA3 substrate and linked tyrosine phosphatase activity to angiogenesis and repair-driven tumor survival.

    Evidence Genetic knockdown, benzarone inhibition, xenograft and patient-derived xenograft with H2AX-pY142 readout

    PMID:33649104

    Open questions at the time
    • Mechanism connecting H2AX dephosphorylation to VEGFA regulation unclear
    • Single-lab finding
  10. 2021 Medium

    Connected EYA3 to a human developmental disorder, showing a stabilizing missense variant and a craniofacial phenotype on knockdown.

    Evidence Protein stability and H2AFX dephosphorylation assays, exome sequencing, and zebrafish morphant analysis

    PMID:33475861

    Open questions at the time
    • Causality of the variant beyond a single family not established
    • Mechanism linking increased half-life to phenotype unknown
  11. 2022 Medium

    Identified a hypoxia-induced EYA3-SIX5-p300 transcriptional complex driving pro-tumorigenic target genes in colorectal cancer.

    Evidence Co-IP/MS, ChIP for promoter occupancy, xenograft, and benzarone treatment

    PMID:35957720

    Open questions at the time
    • Whether phosphatase activity is required for complex function not separated
    • Single-lab finding
  12. 2023 Medium

    Showed that RBFOX2-dependent alternative splicing of EYA3 dictates which transcription-factor partner it engages during myogenesis.

    Evidence MS-based interactome proteomics, transcriptomics, and knockdown with alternative splicing analysis

    PMID:38026174

    Open questions at the time
    • Functional consequences of SIX4 vs ZBTB1 partnering on specific genes not fully resolved
    • Single-lab finding
  13. 2025 High

    Provided the structural basis for EYA3 recruitment of PP2A-B55α and demonstrated that recruitment dictates phosphosite-selective dephosphorylation.

    Evidence Cryo-EM, NMR structures and NMR-based dephosphorylation assays of PP2A:B55 with Eya3, plus a competitive B55i peptide that elevates Myc pT58 in TNBC cells

    PMID:39975004 PMID:40247147 PMID:40414499

    Open questions at the time
    • Whether other Eya-family members redirect PP2A to distinct substrates in cells untested
    • Therapeutic feasibility of disrupting the interaction in vivo unestablished
  14. 2025 High

    Extended EYA3's pro-metastatic role to innate immunity, showing NF-κB/CCL2-mediated suppression of NK cells in the pre-metastatic niche.

    Evidence Knockdown with NF-κB pathway rescue, in vitro NK activation assays, and in vivo pre-metastatic niche analysis in TNBC

    PMID:39211066 PMID:40333987

    Open questions at the time
    • Direct mechanism by which EYA3 activates NF-κB not defined
    • Relationship between NF-κB and the Myc/PD-L1 axis unresolved

Open questions

Synthesis pass · forward-looking unresolved questions
  • How EYA3 integrates its transcriptional co-activator and dual phosphatase activities within a single cell, and whether these functions are coordinately or independently deployed across tissues, remains unresolved.
  • No unified model linking catalytic and co-activator roles
  • Substrate repertoire of the tyrosine phosphatase incompletely mapped
  • Mechanism coupling EYA3 to mTORC1/autophagy not biochemically defined

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0016787 hydrolase activity 3 GO:0098772 molecular function regulator activity 3 GO:0140096 catalytic activity, acting on a protein 3 GO:0140110 transcription regulator activity 3
Localization
GO:0005634 nucleus 1 GO:0005856 cytoskeleton 1
Pathway
R-HSA-1266738 Developmental Biology 3 R-HSA-1643685 Disease 3 R-HSA-73894 DNA Repair 3 R-HSA-74160 Gene expression (Transcription) 3 R-HSA-168256 Immune System 2
Complex memberships
EYA3-SIX5-p300PP2A-B55α (recruited holoenzyme)Ski-Six1-Eya3

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
2018 The Ser/Thr phosphatase activity of Eya3 is not intrinsic but arises from its direct interaction with the PP2A-B55α holoenzyme. Eya3 alters PP2A regulation of c-Myc by enabling PP2A-B55α to dephosphorylate pT58 on c-Myc, increasing c-Myc stability—in direct contrast to PP2A-B56α-mediated dephosphorylation of pS62 which destabilizes c-Myc. Eya3 and PP2A-B55α promote metastasis in a xenograft model of breast cancer. Co-immunoprecipitation, in vitro phosphatase assays, mutagenesis, xenograft model Nature communications High 29535359
2025 Cryo-EM and NMR structures of PP2A:B55 with Eya3 reveal that Eya3 binds B55α through an extended peptide in the N-terminal domain. Eya3 and PP2A substrates share a common set of interaction pockets on B55α but with distinct binding mechanisms. The core B55 recruitment motif in Eya3 is conserved across the Eya family. NMR-based dephosphorylation assays demonstrated that B55 recruitment by Eya3 directs selective dephosphorylation of specific phosphosites. Cryo-electron microscopy, NMR spectroscopy, NMR-based dephosphorylation assays Nature structural & molecular biology High 40247147
2025 Cryo-EM structures of PP2A-B55α bound with Eya3 show that Eya3 binds B55α through an extended peptide in the Eya3 N-terminal domain. A competitive inhibitory peptide (B55i) disrupts the B55α–Eya3 interaction in vitro; when expressed in TNBC cells, B55i increases Myc pT58 and decreases Myc protein levels, confirming functional relevance of the Eya3–PP2A-B55α interaction for Myc stability. Cryo-electron microscopy, in vitro binding/inhibition assays, cell-based Myc phosphorylation/protein level assays The Journal of biological chemistry High 39975004 40414499
2018 Eya3 utilizes its Thr phosphatase activity (via PP2A-B55α) to dephosphorylate Myc at pT58, producing stabilized Myc, which is required for Eya3-mediated upregulation of PD-L1. Eya3 loss decreases tumor growth in immune-competent mice, increases CD8+ T cell infiltration, and CD8+ T cell depletion reverses the effects of Eya3 knockdown. Knockdown experiments, phosphatase assays, CD8+ T cell depletion, in vivo tumor growth assays, rescue experiments (PD-L1 re-expression) The Journal of clinical investigation High 29757193
2025 EYA3 up-regulates NF-κB signaling to enhance CCL2 expression, which suppresses cytotoxic NK cell activation and infiltration into the pre-metastatic niche (PMN), thereby promoting TNBC metastasis. Restoration of NF-κB signaling downstream of Eya3 knockdown rescues metastasis without restoring primary tumor growth, isolating EYA3/NF-κB effects to the metastatic site. Knockdown experiments, NF-κB pathway rescue, in vitro NK cell activation assays, in vivo PMN analysis Science advances High 39211066 40333987
2018 EYA3 tyrosine phosphatase activity promotes the survival of pulmonary arterial smooth muscle cells under DNA-damaging conditions. Transgenic mice with an inactivating mutation in the EYA3 tyrosine phosphatase domain are significantly protected from vascular remodeling. Pharmacological inhibition of EYA3 tyrosine phosphatase substantially reverses vascular remodeling in a rat model of pulmonary hypertension. Transgenic mouse with phosphatase-dead EYA3 mutation, pharmacological inhibition, rat disease model Nature communications High 31515519
2018 WDR1 is a substrate of EYA3 tyrosine phosphatase but not EYA1. Src kinase phosphorylates tyrosine residues in EYA3, and EYA3 can autodephosphorylate these residues. Src phosphorylation of EYA3 controls its subcellular localization (nuclear and cytoskeletal). EYA3-mediated dephosphorylation of WDR1 induces major changes in cellular actin cytoskeleton organization. Phosphotyrosine peptide microarray, in vitro phosphatase assays, subcellular fractionation/localization, actin cytoskeleton imaging Scientific reports Medium 29440662
2019 Src kinase phosphorylates EYA3 at 13 tyrosine residues with different phosphorylation and autodephosphorylation kinetics. Residues Y77, Y96, and Y237 are involved in cell proliferation; mutation of these three residues abolishes the pro-proliferative effect of EYA3 overexpression. EYA3 controls its own phosphorylation state through autodephosphorylation. Native and bottom-up mass spectrometry, site-directed mutagenesis, cell cycle analysis in HEK293T cells International journal of molecular sciences Medium 31847183
2012 EYA3 is regulated by the EWS/FLI1 fusion protein via repression of miR-708, which targets the EYA3 3'-UTR, rather than through direct promoter binding. EYA3 knockdown in Ewing sarcoma cells sensitizes them to DNA-damaging chemotherapeutics and impairs DNA damage repair, implicating EYA3 as a mediator of chemoresistance. miRNA luciferase reporter assay, EYA3 knockdown, DNA damage/repair assays, chemotherapy sensitivity assays Molecular cancer research : MCR Medium 22723308
2021 EYA3 tyrosine phosphatase activity regulates VEGFA levels in Ewing sarcoma tumors and promotes DNA damage repair and cell survival. Pharmacological inhibition of EYA3 tyrosine phosphatase (benzarone) inhibits tumor growth and angiogenesis. Elevated EYA3 substrate H2AX-pY142 upon EYA3 loss confirms H2AX-pY142 as an EYA3 substrate in vivo. Genetic knockdown and pharmacological inhibition, xenograft tumor model, patient-derived xenograft, H2AX-pY142 substrate measurement Molecular cancer therapeutics Medium 33649104
2010 Eya3 and its partner Six1 synergistically activate TSHβ expression in the pars tuberalis upon long-day photoperiod stimulation, and this activation is further enhanced by Tef and Hlf. Eya3 expression is acutely induced by late-night light stimulation and precedes TSHβ induction, placing Eya3 upstream of TSHβ in the photoperiodic pathway. Genome-wide expression analysis, transcriptional reporter assays, acute light stimulation experiments in CBA/N mice Current biology : CB Medium 20434341 21129973
2008 Ski directly interacts with Six1 and Eya3 via the Dachshund homology domain of Ski to form a complex that binds the MEF3 site on the Myog regulatory region and activates myogenin (Myog) transcription during muscle terminal differentiation. This interaction is required for Ski-mediated promotion of myoblast differentiation. Chromatin immunoprecipitation (ChIP), transcriptional reporter assays, retroviral overexpression/knockdown, co-immunoprecipitation The Journal of biological chemistry Medium 19008232
2022 Hypoxia-induced EYA3 couples with SIX5 and the histone acetyltransferase p300 to form a complex that transactivates EGFR, VEGFD, and multiple MMPs (MMP3, MMP7, MMP8, MMP21, MMP26) by binding their promoters in colorectal cancer. Disruption of the EYA3-SIX5-p300 complex decreases expression of these targets and inhibits CRC cell growth. Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation (ChIP), tumor xenograft model, benzarone inhibitor treatment Annals of translational medicine Medium 35957720
2023 RBFOX2 regulates alternative splicing of EYA3 exon 7, generating tissue-specific isoforms. Different EYA3 isoforms differentially partner with either SIX4 or ZBTB1 transcription factors to dictate gene expression during myogenesis. EYA3 expression is required for myoblast proliferation and differentiation. Mass spectrometry-based proteomics, genome-wide transcriptomics, knockdown experiments, alternative splicing analysis iScience Medium 38026174
2021 A missense variant in EYA3 (p.Asn358Ser) increases the half-life of the mutated protein without affecting its ability to dephosphorylate H2AFX following DNA damage repair pathway induction. Knockdown of eya3 in zebrafish embryos produces craniofacial abnormalities consistent with oculo-auriculo-vertebral spectrum. Cellular protein stability assay, H2AFX dephosphorylation assay, zebrafish knockdown morphant analysis, exome sequencing Human genetics Medium 33475861
2024 EYA3 promotes gastric cancer cell proliferation by activating the mTORC1 signaling pathway and inhibiting autophagy. EYA3 silencing reduces cell proliferation in vitro and slows tumor growth in vivo in a xenograft model. Gene silencing, gene set enrichment analysis, in vitro proliferation assays, xenograft tumor model Scientific reports Low 39550476

Source papers

Stage 0 corpus · 28 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2010 Acute induction of Eya3 by late-night light stimulation triggers TSHβ expression in photoperiodism. Current biology : CB 97 21129973
2012 EWS/FLI1 regulates EYA3 in Ewing sarcoma via modulation of miRNA-708, resulting in increased cell survival and chemoresistance. Molecular cancer research : MCR 78 22723308
2018 Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression. Nature communications 74 29535359
2010 Identification of Eya3 and TAC1 as long-day signals in the sheep pituitary. Current biology : CB 69 20434341
2018 Eya3 promotes breast tumor-associated immune suppression via threonine phosphatase-mediated PD-L1 upregulation. The Journal of clinical investigation 41 29757193
2008 Ski regulates muscle terminal differentiation by transcriptional activation of Myog in a complex with Six1 and Eya3. The Journal of biological chemistry 36 19008232
2008 Pleiotropic effects in Eya3 knockout mice. BMC developmental biology 33 19102749
2019 The EYA3 tyrosine phosphatase activity promotes pulmonary vascular remodeling in pulmonary arterial hypertension. Nature communications 27 31515519
2021 A recurrent missense variant in EYA3 gene is associated with oculo-auriculo-vertebral spectrum. Human genetics 21 33475861
2018 WDR1 is a novel EYA3 substrate and its dephosphorylation induces modifications of the cellular actin cytoskeleton. Scientific reports 20 29440662
2021 CircGNG4 Promotes the Progression of Prostate Cancer by Sponging miR-223 to Enhance EYA3/c-myc Expression. Frontiers in cell and developmental biology 19 34395419
2021 Targeting EYA3 in Ewing Sarcoma Retards Tumor Growth and Angiogenesis. Molecular cancer therapeutics 15 33649104
2022 Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression. Annals of translational medicine 11 35957720
2025 Cryo-EM structures of PP2A:B55 with p107 and Eya3 define substrate recruitment. Nature structural & molecular biology 7 40247147
2023 RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis. iScience 7 38026174
2019 Analysis of EYA3 Phosphorylation by Src Kinase Identifies Residues Involved in Cell Proliferation. International journal of molecular sciences 7 31847183
2021 Neuroendocrine regulation of reproduction in Atlantic cod (Gadus morhua): Evidence of Eya3 as an integrator of photoperiodic cues and nutritional regulation to initiate sexual maturation. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 6 34089890
2022 Inhibitors of EYA3 Protein in Ewing Sarcoma. Asian Pacific journal of cancer prevention : APJCP 5 35633536
2025 EYA3 regulation of NF-κB and CCL2 suppresses cytotoxic NK cells in the premetastatic niche to promote TNBC metastasis. Science advances 4 40333987
2022 Cytogenomic Characterization of a Novel de novo Balanced Reciprocal Translocation t(1;12) by Genome Sequencing Leading to Fusion Gene Formation of EYA3/EFCAB4b. Molecular syndromology 3 36588754
2025 Loss of circular EYA3 attenuates formaldehyde-induced cardiomyocyte pyroptosis and congenital heart defects by regulating Smad5 stability. The Science of the total environment 2 40435734
2024 An EYA3/NF-κB/CCL2 signaling axis suppresses cytotoxic NK cells in the pre-metastatic niche to promote triple negative breast cancer metastasis. bioRxiv : the preprint server for biology 2 39211066
2020 Expression analysis of DIO2, EYA3, KISS1 and GPR54 genes in year-round estrous and seasonally estrous rams. Archives animal breeding 2 33473370
2018 An Immunosuppressive Role for Eya3 in TNBC. Cancer discovery 2 29884726
2025 HOTAIR Promotes Spiral Ganglion Neuron Proliferation via miR-211-5p/EYA3 Regulation. Critical reviews in immunology 1 40743761
2024 EYA3 promotes the tumorigenesis of gastric cancer through activation of the mTORC1 signaling pathway and inhibition of autophagy. Scientific reports 1 39550476
2025 Cryo-EM structures reveal the PP2A-B55α and Eya3 interaction that can be disrupted by a peptide inhibitor. bioRxiv : the preprint server for biology 0 39975004
2025 Cryo-EM structures reveal the PP2A-B55α and Eya3 interaction that can be disrupted by a peptide inhibitor. The Journal of biological chemistry 0 40414499

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