EYA4 is a transcriptional coactivator and aspartate-based protein tyrosine phosphatase of the eyes-absent family. Its N-terminal transactivation region cooperates with SIX-family transcription factors, including SIX3, which recruits EYA4 to the nucleus and promotes target-gene expression. Its conserved C-terminal HAD-like catalytic domain hydrolyzes protein phosphotyrosine. In human cells, EYA4 associates with PLK1 at G2 centrosomes and promotes PLK1 activation and successful mitosis. EYA4 also occupies the nucleoplasm, and its H2AX-directed phosphatase activity supports an evolutionarily conserved role in DNA-repair regulation. EYA4 contributes to sensory-system development and maintenance; pathogenic variants cause autosomal dominant sensorineural hearing loss, sometimes with dilated cardiomyopathy. Alternative splicing generates distinct isoforms, and expression is enriched in heart and skeletal muscle.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0004725 protein tyrosine phosphatase activity | IBA GO_REF:0000033 | ACCEPT | Summary: Conserved EYA tyrosine-phosphatase activity has direct human EYA4 corroboration. Reason: Human EYA4 leaf PTN002468882 lies below the positive PTHR10190 IBD at PTN000022641; the ungapped alignment exactly matches displayed O95677 (639 residues), with no target-path NOT. Independently, PMID:38360978 assays purified human EYA4 residues 367–639 on PLK1 phosphotyrosine peptides and H2AX-pY142. This supports the inherited activity without equating the isolated domain with all five natural products. Supporting Evidence: |
| GO:0004725 protein tyrosine phosphatase activity | IEA GO_REF:0000120 | ACCEPT | Summary: The combined family and enzyme mappings match measured EYA4 chemistry. Reason: The actual O95677 InterPro match includes IPR028472 with GO:0004725, and UniProt links its phosphotyrosine-hydrolysis reaction to Rhea 10684 and EC 3.1.3.48. The official EC reaction and purified human EYA4-domain peptide assays in PMID:38360978 agree with this chemistry. The direct Rhea RDF request failed, so its mapping is traced through UniProt rather than claimed independently inspected. No autonomous serine/threonine activity is inferred. Supporting Evidence: |
| GO:0004725 protein tyrosine phosphatase activity | ISS GO_REF:0000024 | ACCEPT | Summary: The EYA1-based inference is independently supported on human EYA4. Reason: The current Q99502 donor record includes experimental tyrosine-phosphatase support, including PMID:19234442. Its Eya1/3 affinity preparations and human knockdown versus mouse rescue are distinct from EYA4. Crucially, PMID:38360978 directly measures the purified human EYA4 367–639 domain on phosphotyrosine peptides, so this judgment does not rest solely on paralog similarity or transfer EYA3-specific substrate comparisons to EYA4. |
| GO:0005515 protein binding | IPI PMID:19606496 EYA4, deleted in a case with middle interhemispheric variant... | MODIFY | Summary: SIX3 association supports the specific DNA-binding transcription factor binding activity. Reason: PMID:19606496 reports EYA4–SIX3 co-immunoprecipitation, SIX3-dependent nuclear recruitment and cooperative reporter activation. The experimentally supported UniProt CC:SUBUNIT statement independently curates that same functional interaction on human EYA4. These observations establish a more informative partner class than generic protein binding and support transcriptional coactivation, without requiring purified binary-affinity measurements. The literal WITH accession Q62233 denotes mouse Six3; the historical EYA4 construct and isoform remain unresolved because full Methods are unavailable. The source accession is preserved, and no human–human binary assay is asserted. Proposed replacements: DNA-binding transcription factor binding |
| GO:0005634 nucleus | IBA GO_REF:0000033 | MODIFY | Summary: Human immunofluorescence supports the more precise nucleoplasmic location. Reason: The positive nucleus IBD at PTN000022641 is valid on the independently traced EYA4 path. Current HPA004805 images in HeLa, Rh30 and U2OS show diffuse/granular nuclear signal, and HPA assigns approved nucleoplasm localization. Refine the broad compartment to GO:0005654, a current part_of descendant of nucleus. The complete HPA antigen exactly matches product 5; the other four products retain long matching segments. This polyclonal-antibody observation neither identifies the stained product nor establishes product-5-specific localization. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN000022641 · PTN000022641 SUPPORTS TRANSFER The positive GO:0005634 nucleus IBD is ancestral to the independently verified EYA4 leaf PTN002468882 in PTHR10190; its 639-residue MSA sequence exactly matches displayed human EYA4 and no target-path NOT assertion was found. Broad nuclear inheritance is supported. The nucleoplasm refinement uses independent human HPA observations and does not dispute this node placement or identify the stained natural product. Proposed replacements: nucleoplasm Supporting Evidence: HPA:ENSG00000112319 |
| GO:0005634 nucleus | IEA GO_REF:0000044 | MODIFY | Summary: The broad UniProt nucleus mapping can be refined using human HPA imaging. Reason: SL-0191 correctly maps nucleus and is present in the target UniProt record as an EYA1-based inference. Independent target HPA004805 immunofluorescence supports nucleoplasm in three human cell lines. Use GO:0005654 while retaining the original mapping provenance; approved antibody-level evidence is not a claim of exclusive nuclear localization or isoform-specific staining. Proposed replacements: nucleoplasm Supporting Evidence: HPA:ENSG00000112319 |
| GO:0005634 nucleus | ISS GO_REF:0000024 | MODIFY | Summary: Target-specific imaging resolves the EYA1-derived nuclear assertion more precisely. Reason: Q99502 is the recorded ISS donor, but human EYA4 HPA004805 images independently support nucleoplasm. The observed nuclear signal and current HPA assignment justify GO:0005654 without assuming every donor nuclear experiment used EYA4. Antigen overlap prevents assigning the observation independently to each of the five source products. Proposed replacements: nucleoplasm Supporting Evidence: HPA:ENSG00000112319 |
| GO:0005737 cytoplasm | IEA GO_REF:0000044 | KEEP AS NON CORE | Summary: Retain the broad cytoplasmic pool separately from the functional centrosomal pool. Reason: The recorded UniProt/EYA1-derived cytoplasm assertion is consistent with EYA4 nuclear recruitment by SIX3. The specific G2 centrosomal pool is independently supported by endogenous EYA4 localization and depletion in PMID:38360978 and is represented in the phosphatase core. The diffuse cytoplasmic distribution associated with SIX3-dependent nuclear recruitment is not reducible to the G2 centrosomal pool. Retain cytoplasm as non-core distribution evidence rather than imply that all extranuclear EYA4 is centrosomal. |
| GO:0005737 cytoplasm | ISS GO_REF:0000024 | KEEP AS NON CORE | Summary: Retain the broad cytoplasmic pool separately from the functional centrosomal pool. Reason: The recorded UniProt/EYA1-derived cytoplasm assertion is consistent with EYA4 nuclear recruitment by SIX3. The specific G2 centrosomal pool is independently supported by endogenous EYA4 localization and depletion in PMID:38360978 and is represented in the phosphatase core. The diffuse cytoplasmic distribution associated with SIX3-dependent nuclear recruitment is not reducible to the G2 centrosomal pool. Retain cytoplasm as non-core distribution evidence rather than imply that all extranuclear EYA4 is centrosomal. |
| GO:0007601 visual perception | TAS PMID:9887327 EYA4, a novel vertebrate gene related to Drosophila eyes abs... | UNDECIDED | Summary: The original TAS support for visual-stimulus processing remains unverified. Reason: The accessible PMID:9887327 abstract establishes EYA4 characterization and reports predominantly craniofacial, dermamyotome and limb expression in mouse embryos, with no pathogenic EYA4 variant found in the examined ODD cohort. Predominant expression does not exclude ocular expression, and a negative ODD screen does not refute visual-stimulus processing. Those findings therefore cannot establish either the positive visual-perception assertion or its contradiction. The original supporting passage remains unavailable; retain UNDECIDED without turning an abstract-only negative disease screen into evidence that the annotation is false. Supporting Evidence: |
| GO:0009653 anatomical structure morphogenesis | TAS PMID:9887327 EYA4, a novel vertebrate gene related to Drosophila eyes abs... | MODIFY | Summary: Refine broad anatomical morphogenesis to the independently supported embryonic context. Reason: Embryonic morphogenesis is an is_a descendant of anatomical structure morphogenesis and is independently supported for the EYA4 lineage by the accepted PTN002565156 IBD and mouse/zebrafish developmental evidence. Use GO:0048598 instead of retaining an unresolved broad assertion merely because the older TAS source is unavailable in full. This refinement does not claim that the 1999 paper itself performed the later experiments. Inner ear development is not an is_a descendant of anatomical morphogenesis and is not added to the replacement list. Proposed replacements: embryonic morphogenesis |
| GO:0030154 cell differentiation | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: Conserved developmental differentiation is retained as a contextual role. Reason: The target inherits the positive cell-differentiation IBD at PTN000022641. The current IBD includes zebrafish evidence absent from the imported WITH list; both versions remain distinct. Actual PMID:18799547 uses two zebrafish eya4 splice-blocking morpholinos, mismatch controls, hair-cell readouts and downstream atp1b2b rescue, supporting a developmental regulatory contribution. It does not establish a human EYA4 hair-cell assay or direct atp1b2b promoter binding. Retain the broad process as non-core relative to the molecular activity. Supporting Evidence: |
| GO:0045739 positive regulation of DNA repair | IBA GO_REF:0000033 | ACCEPT | Summary: A conserved H2AX phosphotyrosine-regulatory mechanism supports the repair inference. Reason: The positive DNA-repair-regulation IBD at PTN000022641 lies on the actual EYA4 target path and is grounded in EYA1/EYA3 descendant evidence. PMID:19234442 connects H2AX tyrosine state with repair-factor association, with Eya1/3 and peptide/extract experiments kept distinct. PMID:38360978 independently shows human EYA4-domain hydrolysis of the H2AX-pY142 peptide. Together these support the conserved regulatory capacity; neither the peptide assay nor the PLK1 mitosis experiments alone demonstrate a cellular EYA4 DNA-repair outcome. No target-specific loss was found that would overturn the ancestral placement. |
| GO:0048598 embryonic morphogenesis | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: The EYA4-clade developmental inference is retained with organism-specific scope. Reason: The actual target path passes through the embryonic-morphogenesis IBD at PTN002565156. Imported WITH lists mouse MGI:1337104; the current IBD additionally lists zebrafish ZDB-GENE-050327-93. PMID:18219393 uses mouse Eya4 deletion and identifies a palate-maturation delay at birth plus later middle-ear/eustachian-tube defects; its postnatal phenotype is not mislabeled as an isolated embryonic assay. PMID:18799547 directly examines zebrafish otic morphogenesis and regulatory rescue. These support a conserved developmental context, without transferring a complete human product-specific mechanism. |
| GO:0048839 inner ear development | IBA GO_REF:0000033 | KEEP AS NON CORE | Summary: The conserved inner-ear developmental role is supported independently of rat expression alone. Reason: The target descends from the positive PTN002565156 inner-ear IBD. The imported rat RGD:1307099 source traces to an IEP assertion from PMID:11159937; its operative original was not recovered here. The current IBD also contains zebrafish evidence. In the actually read PMID:18799547, eya4 depletion disrupts otic-vesicle epithelial pillars, semicircular-canal formation and sensory hair-cell development, and atp1b2b mRNA rescues substantial defects. Retain this as a non-core developmental inference; mouse middle-ear disease and human late-onset hearing loss are not substituted for a direct inner-ear developmental assay. |
| GO:0048839 inner ear development | IEA GO_REF:0000107 | KEEP AS NON CORE | Summary: The rat-derived developmental annotation is retained with an explicit source-access limit. Reason: Current rat A0A8I6AS60 corresponds to Eya4/RGD:1307099 and Ensembl ENSRNOP00000097961.2; its GO:0048839 assertion is IEP PMID:11159937. Only verified metadata and abstract were accessible for that paper, so no claim is made to have inspected its rat expression experiment or to infer participation from expression alone. Independent actual zebrafish PMID:18799547 and the target-path PTN002565156 IBD support the broader conserved inner-ear developmental role. Retain the original source context as non-core rather than reject an unread experiment or assign it to all human products. |
| GO:0005813 centrosome | IDA PMID:38360978 The Eyes Absent family members EYA4 and EYA1 promote PLK1 ac... | NEW | Summary: Endogenous EYA4 localizes to centrosomes in G2 human cells. Reason: PMID:38360978 Results and Figure 1G report endogenous EYA4 colocalization with PLK1 at centrosomes in G2-arrested HeLa cells, with EYA4-depletion controls. This independently observed functional pool supports a centrosome annotation; it neither replaces the broader cytoplasmic distribution nor assigns localization to every splice isoform. The original text and figure caption were inspected; no new figure-pixel assessment is claimed. Supporting Evidence: |
| GO:0003713 transcription coactivator activity | IDA PMID:19606496 EYA4, deleted in a case with middle interhemispheric variant... | NEW | Summary: EYA4 cooperates with SIX3 to enhance transcriptional reporter activity. Reason: The PMID:19606496 abstract explicitly reports EYA4–SIX3 co-immunoprecipitation, SIX3-dependent nuclear recruitment and cooperative reporter activation. The human EYA4 UniProt O95677 entry experimentally curates this functional interaction from the same paper. Together these support gene-level transcriptional coactivation, beyond localization or binding alone. IDA describes the reported primary reporter assay. The original Methods and historical construct sequences remain unavailable, and the source partner Q62233 is mouse Six3. This is not a claim of a newly verified purified human–human assay or equivalent activity of all five current EYA4 products. |
| GO:0071902 positive regulation of protein serine/threonine kinase activity | IMP PMID:38360978 The Eyes Absent family members EYA4 and EYA1 promote PLK1 ac... | NEW | Summary: EYA4 phosphatase activity promotes PLK1 kinase activity in human cells. Reason: PMID:38360978 Figure 2 and its Results show that EYA4 depletion reduces PLK1-pT210 relative to total PLK1 and reduces phosphorylation of the PLK1 substrates TCTP and CDC25C; the Cyclin B change is not significant. EYA4 catalytic and docking mutants alter PLK1 activation, and Figure 4C directly measures dephosphorylation of PLK1 phosphotyrosine peptides by the purified human EYA4 catalytic domain. EYA4 therefore performs the regulatory dephosphorylation step, while PLK1 performs the downstream serine/threonine phosphorylation. The claim is not based solely on necessity or a missing annotation in comparator genes. The nonsignificant cellular pY445 trend is not treated as site-specific proof, and no full-length isoform equivalence is asserted. The selected term is more specific than GO:0045860 and does not require demonstrating initiation from a completely inactive kinase. Supporting Evidence: |
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Download this section (compressed HTML)Q: Does endogenous EYA4 dephosphorylate H2AX-pY142 in human nuclei, and under which conditions does that reaction alter DNA-repair outcomes?
Q: Which EYA4 splice isoforms are expressed, catalytically active or SIX-dependent coactivators in relevant cells, and is the predicted nonsense-mediated decay of isoform3 observed?
Q: What is the quantitative contribution of endogenous EYA4 to PLK1-pY445 turnover and PLK1 activation during the G2-to-mitosis transition?
Q: GO:0071902 and its parent GO:0045860 remain non-obsolete but are marked as obsoletion candidates. Which future GO-CAM or replacement representation should retain the demonstrated EYA4 phosphatase step that increases PLK1 activity?
Experiment: Compare EYA4 depletion with rescue by sequence-verified full-length wild-type and catalytic-site mutant EYA4 at matched expression. Measure H2AX-pY142 loss, damage-site recruitment and repair/survival kinetics after a defined DNA-damage challenge. Include EYA1 perturbation separately to distinguish paralog contributions and quantify EYA4 nuclear abundance rather than assuming it from antibody staining.
Hypothesis: EYA4-dependent H2AX phosphotyrosine removal may provide a cellular repair-regulatory mechanism, but isolated-domain activity need not predict its contribution in nuclei.
Type: Cellular substrate attribution and repair outcome analysis
Experiment: Measure isoform-specific transcript junctions and discriminating peptides, including isoform3 abundance with and without nonsense-mediated-decay inhibition. Compare sequence-verified full-length proteins in matched-expression phosphotyrosine assays, SIX3-dependent reporter assays and nuclear/centrosomal localization experiments. Use independent endogenous tags or epitope-mapped antibodies, and distinguish isolated-domain chemistry from full-length isoform function.
Hypothesis: EYA4 splice isoforms may differ in abundance, phosphatase activity, SIX-dependent coactivation and localization; isoform3 may be depleted by nonsense-mediated decay.
Type: Product-resolved biochemical and localization comparison
Experiment: Quantify endogenous PLK1-pY445 with isotope-labeled phosphopeptide standards across synchronized G2 and mitotic entry after EYA4 depletion and wild-type or catalytic mutant rescue. Measure PLK1-pT210 and selected PLK1 substrate phosphorylation in parallel, controlling for total PLK1 abundance and cell-cycle composition. Treat Y445F as a functional perturbation rather than a direct measurement of physiological phosphosite occupancy, and distinguish EYA4 perturbation from pan-EYA inhibition.
Hypothesis: EYA4 promotes PLK1 activation partly through pY445 turnover, but the size and timing of the endogenous contribution remain uncertain.
Type: Time-resolved phosphosite quantification and kinase coupling
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