{"gene":"EPS8","run_date":"2026-06-09T23:54:43","timeline":{"discoveries":[{"year":1993,"finding":"Eps8 is a substrate of the EGFR kinase; it is tyrosine-phosphorylated in vivo following EGF stimulation and associates with the EGFR in vivo despite lacking a functional SH2 domain. Overexpression of Eps8 in fibroblasts or hematopoietic cells increases mitogenic response to EGF.","method":"cDNA cloning, in vivo tyrosine phosphorylation assay, co-immunoprecipitation, adoptive expression/overexpression mitogenesis assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP and functional overexpression assay, foundational paper replicated across subsequent studies","pmids":["8404850"],"is_preprint":false},{"year":1995,"finding":"Eps8 binds directly to the juxtamembrane domain of EGFR through a non-SH2 domain by a mechanism that does not require phosphotyrosine residues.","method":"Direct binding assay with EGFR deletion/point mutants, in vitro binding","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro direct binding with mutagenesis, single lab","pmids":["7532293"],"is_preprint":false},{"year":1995,"finding":"EGFR autophosphorylation sites are dispensable for Eps8 phosphorylation; removal of the final 214 COOH-terminal residues of EGFR increases Eps8 phosphorylation ~13-fold, identifying a juxtamembrane region as the structural determinant for efficient phosphorylation of Eps8.","method":"EGFR deletion/point mutant phosphorylation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with systematic mutagenesis, single lab","pmids":["7608194"],"is_preprint":false},{"year":1995,"finding":"Eps8 constitutive tyrosine phosphorylation occurs in tumor cell lines at stoichiometry similar to potent mitogenic response; overexpression of Eps8 transforms NIH 3T3 cells under limiting EGFR activation. Eps8 associates in vivo with Shc, at least partly via the SH3 domain of Eps8.","method":"Phosphotyrosine analysis of tumor lines, transformation assay, co-immunoprecipitation","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and functional transformation assay, single lab, two orthogonal methods","pmids":["7791787"],"is_preprint":false},{"year":1996,"finding":"RN-tre binds specifically and with high affinity (Kd ~10^-8–10^-7 M) to the SH3 domain of Eps8 in vitro and stably associates with Eps8 in vivo.","method":"In vitro SH3 domain binding assay with Kd measurement, co-immunoprecipitation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — quantitative in vitro binding plus co-IP, single lab","pmids":["8700527"],"is_preprint":false},{"year":1997,"finding":"The SH3 domain of Eps8 forms an intertwined dimer via strand interchange (novel fold variant of canonical SH3), and intact Eps8 is multimeric in vivo, suggesting the SH3 domain serves as a dimerization motif.","method":"X-ray crystallography, co-immunoprecipitation","journal":"Nature structural biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vivo co-IP validation","pmids":["9303002"],"is_preprint":false},{"year":1997,"finding":"E3B1 (Abi-1) was identified as an Eps8 SH3-domain binding protein via SH3 library screen; E3B1 associates with Eps8 in vivo and is serine-phosphorylated in a growth-factor-dependent manner.","method":"SH3 domain expression library screen, co-immunoprecipitation, phosphoamino acid analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus biochemical characterization, single lab","pmids":["9010225"],"is_preprint":false},{"year":1998,"finding":"Eps8 is recruited to the cell cortex, membrane ruffles, lamellipodia, and dynamic F-actin structures (podosomes) upon cytoskeletal remodeling; a detergent-resistant pool associates with podosomes in v-Src-transformed cells.","method":"Immunofluorescence localization, Triton X-100 fractionation, live-cell imaging","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by immunofluorescence and fractionation with functional context, single lab","pmids":["9665816"],"is_preprint":false},{"year":1999,"finding":"Eps8, E3b1/Abi-1, and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro, mediating signal transduction from Ras to Rac.","method":"Co-immunoprecipitation (in vivo tri-complex), in vitro Rac-GEF activity assay, genetic epistasis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro GEF activity combined with in vivo co-IP and epistasis, replicated in multiple subsequent studies","pmids":["10499589"],"is_preprint":false},{"year":1999,"finding":"Eps8 interacts with the PDZ domain of Dvl1 (murine Dishevelled homologue) in vitro and in vivo; Dvl1 is hyperphosphorylated in the presence of Eps8, whereas Dvl1 inhibits EGF-induced tyrosine phosphorylation of Eps8.","method":"Yeast two-hybrid screen, in vitro binding confirmation, transfection and phosphorylation analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus in vitro binding plus in-cell functional assay, single lab","pmids":["10581192"],"is_preprint":false},{"year":2000,"finding":"Eps8 interacts via its SH3 domain with RN-tre, a Rab5 GAP; this complex inhibits EGFR internalization and attenuates Rac signaling by diverting Eps8 from the Abi1/Sos1 complex. RN-tre activity on Rab5 is regulated by the EGFR.","method":"Co-immunoprecipitation, Rab5 GAP activity assay, EGFR endocytosis assay, Rac activation assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay, reciprocal co-IP, functional endocytosis assay, multiple orthogonal methods","pmids":["11099046"],"is_preprint":false},{"year":2003,"finding":"PI3K (via p85 subunit) is recruited into the Eps8-Abi1-Sos1 complex through Abi1; both p85 recruitment and the PI3K product PIP3 are required to unmask Rac-GEF activity of the complex in vitro and to activate Rac and actin remodeling in vivo.","method":"Co-immunoprecipitation, in vitro Rac-GEF assay, PI3K inhibitor studies, p85 knockout cells","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro GEF assay combined with co-IP and knockout cell validation, single lab with multiple orthogonal methods","pmids":["12515821"],"is_preprint":false},{"year":2003,"finding":"Eps8 family members (Eps8L1, Eps8L2, Eps8L3) interact with Abi1 and Sos-1; only Eps8L1 and Eps8L2 activate Rac-GEF activity of Sos-1 and bind actin in vivo, functionally compensating for Eps8 loss in eps8−/− fibroblasts.","method":"Co-immunoprecipitation, Rac-GEF activity assay, rescue experiments in eps8−/− fibroblasts","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro GEF assay, co-IP, and genetic rescue in knockout cells, single lab multiple orthogonal methods","pmids":["14565974"],"is_preprint":false},{"year":2004,"finding":"Eps8 family proteins cap actin filament barbed ends; the conserved C-terminal effector domain caps barbed ends with nanomolar affinity. Full-length Eps8 is auto-inhibited, and interaction with Abi1 relieves this inhibition. In vivo, Eps8 removal impairs actin-based propulsion.","method":"In vitro actin barbed-end capping assay with isolated domain and full-length protein, Kd measurement, loss-of-function in cells, Abi1 interaction relief-of-inhibition assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro capping assay with domain mapping and mutagenesis, plus in vivo functional validation","pmids":["15558031"],"is_preprint":false},{"year":2004,"finding":"In C. elegans, the EPS-8A isoform (but not EPS-8B) has a C-terminal actin barbed-end capping activity required for proper apical morphogenesis of intestinal cells; the two isoforms differ at their C-termini.","method":"C. elegans genetics, isoform rescue experiments, in vitro actin barbed-end capping assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro capping reconstitution and in vivo genetic rescue with isoform specificity, ortholog study","pmids":["15558032"],"is_preprint":false},{"year":2004,"finding":"IRSp53 directly binds Eps8 through the NH2-terminal proline-rich sequence of Eps8 and the IRSp53 SH3 domain; this complex synergistically activates Rac by reinforcing the Eps8/Abi1/Sos1 GEF complex and occurs at the leading edge of motile cells.","method":"Co-immunoprecipitation (in vivo), FRET analysis, Rac activation (pull-down assay), cell motility/invasion assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, FRET, Rac pull-down, single lab with multiple orthogonal methods","pmids":["15289329"],"is_preprint":false},{"year":2006,"finding":"Eps8 has actin crosslinking (bundling) activity and synergizes with IRSp53 in mediating actin bundling in vitro. Cdc42 binds the IRSp53-Eps8 complex and controls its cellular distribution. Cdc42-induced filopodia require both IRSp53 and Eps8.","method":"In vitro actin bundling assay, co-immunoprecipitation, loss-of-function (siRNA), live-cell imaging","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstituted bundling assay with multiple genetic loss-of-function validations, single lab with multiple orthogonal methods","pmids":["17115031"],"is_preprint":false},{"year":2006,"finding":"Palladin directly binds Eps8 (identified by yeast two-hybrid, confirmed by co-immunoprecipitation); both proteins colocalize in dorsal ruffles; palladin knockdown reduces Rac activation and ruffle formation after PDGF treatment.","method":"Yeast two-hybrid, co-immunoprecipitation, colocalization by confocal microscopy, palladin knockdown with functional readout","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid validated by co-IP plus functional knockdown assay, single lab","pmids":["16868024"],"is_preprint":false},{"year":2006,"finding":"Eps8 is part of the NMDA receptor complex at postsynaptic structures in neurons; Eps8 null mice show abnormal NMDA receptor currents, altered sensitivity to ethanol inhibition, and resistance to NMDA/ethanol-induced actin remodeling.","method":"Co-immunoprecipitation (NMDA receptor complex), electrophysiology in knockout neurons, behavioral assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP of endogenous complex, electrophysiology in Eps8 KO mice, multiple orthogonal methods, independently validated","pmids":["17018287"],"is_preprint":false},{"year":2008,"finding":"In C. elegans, EPS-8 interacts with the ankyrin repeat protein VAB-19 (central domain of EPS-8 sufficient); both proteins colocalize at cell-matrix attachment structures; eps-8 null mutants are defective in epidermal elongation and epidermal-muscle attachment, dependent on PTB and central domains but independent of C-terminal SH3 and actin-binding domains.","method":"Yeast two-hybrid, co-localization, eps-8 null mutant phenotype, domain rescue experiments","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic null and domain-mapping rescue in C. elegans with interaction validation, single lab","pmids":["18833327"],"is_preprint":false},{"year":2009,"finding":"LanCL1 binds specifically to the SH3 domain of Eps8 in vitro; LanCL1 mutants defective in Eps8 interaction inhibit NGF-induced neurite outgrowth, establishing functional significance of this interaction.","method":"In vitro affinity binding, crystal structure of LanCL1, loss-of-function mutagenesis with neurite outgrowth assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vitro binding plus mutagenesis with functional readout, single study multiple orthogonal methods","pmids":["19528316"],"is_preprint":false},{"year":2009,"finding":"Eps8 is localized to actin-based cell junctions (blood-testis barrier and apical ectoplasmic specialization) in the seminiferous epithelium; Eps8 knockdown by RNAi causes F-actin disorganization and mislocalization of tight junction proteins occludin and ZO-1; in vivo knockdown causes germ cell sloughing and BTB damage.","method":"Immunofluorescence/immunohistochemistry localization, RNAi knockdown with permeability and junction protein assays, in vivo knockdown","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional RNAi with defined molecular readouts in vitro and in vivo, single lab","pmids":["19293393"],"is_preprint":false},{"year":2009,"finding":"BDNF inhibits the actin barbed-end capping activity of Eps8 through MAPK-dependent phosphorylation at residues S624 and T628; a phosphorylation-defective mutant (S624A/T628A) shows increased actin association and resistance to BDNF-mediated release; a phosphomimetic mutant (S624E/T628E) has opposite effects. This controls axonal filopodia formation.","method":"Site-directed mutagenesis, MAPK phosphorylation assay, actin barbed-end capping assay, hippocampal neuron imaging and filopodia quantification","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro capping assay with phospho-mutagenesis and in vivo neuronal phenotype rescue, single study multiple orthogonal methods","pmids":["19564905"],"is_preprint":false},{"year":2010,"finding":"The SOS1/EPS8/ABI1 tri-complex facilitates LPA-induced Rac activation specifically in metastatic ovarian cancer cells; re-expression of a missing tri-complex member in non-metastatic cells confers metastatic capability; integrity of the complex is essential for LPA-stimulated cell migration and peritoneal colonization.","method":"Co-immunoprecipitation, Rac activation assay, re-expression rescue, in vivo peritoneal metastasis assay","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, Rac pull-down, and in vivo rescue assay, single lab","pmids":["21118970"],"is_preprint":false},{"year":2010,"finding":"Eps8 in cancer cells localizes to the lysosomal compartment independently of actin polymerization; Eps8 co-immunoprecipitates with Hsc70 and LAMP-2, key elements of chaperone-mediated autophagy (CMA); the region aa 184-535 containing KFERQ-like motifs mediates lysosomal recruitment; Eps8 binds to and is incorporated into lysosomes in vitro, competed by other CMA substrates.","method":"Subcellular fractionation, immunofluorescence/confocal colocalization, co-immunoprecipitation, in vitro lysosome binding/incorporation assay, FRAP","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro lysosome assay plus co-IP plus domain mapping, single lab","pmids":["20184880"],"is_preprint":false},{"year":2011,"finding":"Eps8 interacts with MyoXVa and whirlin; MyoXVa is required for Eps8 accumulation at stereocilia tips; overexpression of MyoXVa drives elongation and increased Eps8 at tips; exogenous MyoXVa in MyoXVa-deficient cells rescues Eps8 tip localization. Whirlin-deficient mice show reduced Eps8 and MyoXVa at stereocilia tips. MyoXVa-whirlin-Eps8 form a stereocilia tip complex.","method":"Co-immunoprecipitation, live imaging, knockout mouse analysis, overexpression rescue","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, multiple knockout mouse models, overexpression rescue, independent replication across related papers","pmids":["21236676"],"is_preprint":false},{"year":2011,"finding":"Eps8 is localized at stereocilia tips of cochlear hair cells; Eps8 knockout mice are profoundly deaf and exhibit short stereocilia; inner hair cells (IHCs) fail to mature into functional sensory receptors in Eps8 KO mice, establishing a role for Eps8 in stereocilia elongation and IHC functional maturation.","method":"Knockout mouse, immunofluorescence localization, electrophysiology, hearing threshold measurement","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple functional readouts (electrophysiology, hearing tests, localization) in Eps8 KO mice, replicated across labs","pmids":["21526224"],"is_preprint":false},{"year":2011,"finding":"Eps8 actin capping activity is required for dendritic cell polarization and formation of elongated migratory protrusions; Eps8-deficient DCs are impaired in directional/chemotactic migration in 3D and in reaching draining lymph nodes in vivo, preventing contact hypersensitivity responses. Eps8 is not required for antigen uptake, processing, or presentation.","method":"Eps8 knockout mouse, in vitro 3D migration assay, in vivo lymph node migration assay, contact hypersensitivity assay","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with multiple defined cellular and in vivo phenotype readouts, single lab with multiple orthogonal methods","pmids":["21835647"],"is_preprint":false},{"year":2012,"finding":"Eps8 interacts with the clathrin-mediated endocytosis machinery; Src phosphorylates Eps8 downstream of FGFR activation; depletion of Eps8 inhibits FGFR trafficking and immediate Erk signaling.","method":"Live-cell imaging, Eps8 depletion (siRNA), co-immunoprecipitation with endocytic machinery, Src phosphorylation assay, Erk signaling assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with trafficking machinery and functional knockdown assays, single lab","pmids":["23203811"],"is_preprint":false},{"year":2012,"finding":"Ezrin differentially modulates Eps8 and Eps8L1a activities: coexpression of ezrin with Eps8 promotes membrane ruffles and tufts of microvilli via bundling activity; coexpression with Eps8L1a promotes clustering of actin structures via capping activity. Ezrin's effect requires its binding to Eps8/Eps8L1a, and specific actin-bundling/capping activities of these proteins.","method":"Overexpression and co-expression studies, domain-mapping mutagenesis, morphological assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional mutagenesis with defined cellular readouts, single lab","pmids":["22262457"],"is_preprint":false},{"year":2012,"finding":"Human intersectin 2 (ITSN2) binds Eps8 (interaction domain: aa 260-306 of Eps8 and coiled-coil domain of ITSN2); ITSN2 overexpression induces lysosome-dependent degradation of Eps8.","method":"Yeast two-hybrid, co-immunoprecipitation, GST pull-down, domain mapping, protein stability assay with lysosome inhibitor","journal":"BMB reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding assays (y2h, co-IP, GST pull-down) plus functional degradation assay, single lab","pmids":["22449706"],"is_preprint":false},{"year":2012,"finding":"Eps8 interacts with TLR4 in the cytosol and at the phagosome in LPS-stimulated macrophages; Eps8 facilitates TLR4-MyD88 interaction and downstream Src, FAK, and p38 MAPK activation; Eps8 attenuation reduces LPS-promoted phagocytosis and bacterial killing.","method":"Confocal colocalization, co-immunoprecipitation, siRNA knockdown with phagocytosis and signaling readouts, dominant-negative construct","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of endogenous proteins plus functional knockdown with defined downstream assays, single lab","pmids":["22493489"],"is_preprint":false},{"year":2013,"finding":"SCF(Fbxw5) ubiquitin E3 ligase mediates proteasomal degradation of Eps8 specifically in G2 phase; failure to degrade Eps8 in G2 prolongs its localization at the cell cortex, markedly delays cell rounding and prometaphase entry. Eps8 capping activity is required during late mitosis/cytokinesis to prevent membrane blebbing.","method":"Biochemical degradation assays, E3 ligase identification (SCFFbxw5), proteasome inhibitor experiments, cell-cycle synchronization, live-cell imaging of Eps8 localization","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — E3 ligase identification with reconstitution-level biochemistry, cell-cycle functional assays, and live imaging, single study multiple orthogonal methods","pmids":["23314863"],"is_preprint":false},{"year":2013,"finding":"Eps8 actin-capping activity is required for dendritic spine enlargement during chemically induced LTP; mice lacking Eps8 display immature spines unable to undergo potentiation and are impaired in cognitive functions. Reduced Eps8 levels are found in brains of autism patients.","method":"Eps8 KO mouse, chemically induced LTP in culture, spine morphometry, electrophysiology, behavioral cognitive tests","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO with multiple orthogonal functional readouts (LTP, spine morphology, cognition), capping-defective mutant rescue","pmids":["23685357"],"is_preprint":false},{"year":2013,"finding":"Eps8 forms a biochemical complex with FAK and Src in SCC cells; it controls the spatial distribution of active Src in a FAK-dependent manner and drives Src trafficking to autophagic structures when FAK is absent; when FAK is present, Eps8 is recruited to focal adhesions and required for FAK-dependent polarization and invasion.","method":"Co-immunoprecipitation, subcellular fractionation, siRNA knockdown, invasion assay, immunofluorescence","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and functional knockdown with defined cellular readouts, single lab","pmids":["25359883"],"is_preprint":false},{"year":2013,"finding":"Novel Eps8 binding partners identified by phosphoproteomic affinity purification include clathrin, AP-2 (endocytic machinery), NBR1, Vav2, IRS4, and Shp2; 22 Eps8 pTyr and pSer/Thr phosphorylation sites identified, some dependent on Src family kinases and FGFR activity.","method":"Differential phosphoproteomics, peptide affinity purification, mass spectrometry","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based interactome/phosphoproteomics, single lab","pmids":["23626693"],"is_preprint":false},{"year":2015,"finding":"Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation during confined migration; Erk phosphorylation inhibits Eps8 capping activity, and Erk activity is concentrated within leader blebs, promoting actin bundling by Eps8 to enhance bleb-based migration.","method":"Live-cell imaging, Erk biosensor, cortex tension measurements, loss-of-function/gain-of-function with capping and bundling mutants","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with FRET biosensor and functional mutant analysis, single lab","pmids":["26163656"],"is_preprint":false},{"year":2015,"finding":"EPS8 is a novel binding partner of VE-cadherin; EPS8 associates with VE-cadherin at remodeling junctions promoting YAP nuclear translocation and transcriptional activation; in stabilized junctions 14-3-3-YAP displaces EPS8. Eps8-null mice show increased vascular permeability and junctional YAP inhibition.","method":"Co-immunoprecipitation, live imaging, Eps8 KO mouse, YAP localization and reporter assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, KO mouse with in vivo readout, and YAP transcriptional assay, single lab multiple orthogonal methods","pmids":["26668327"],"is_preprint":false},{"year":2015,"finding":"Eps8 is a direct interactor of Dvl1 (scaffold for Wnt signaling); gain-of-function of Eps8 mimics Wnt-mediated axon remodeling; Eps8 silencing blocks Wnt3a-induced axon remodeling; blockade of Dvl1-Eps8 interaction abolishes Wnt3a-mediated axonal remodeling.","method":"Yeast two-hybrid, functional rescue/silencing in DRG neurons, dominant-negative Dvl1-Eps8 interaction blockade","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid validated by functional assays in primary neurons, single lab","pmids":["26252776"],"is_preprint":false},{"year":2017,"finding":"PTK6 (Brk) phosphorylates Eps8 at residues Tyr497, Tyr524, and Tyr534; a phosphorylation-defective 3YF mutant fails to increase proliferation, migration, and ERK/FAK activation mediated by wild-type Eps8 overexpression; EGF-induced Eps8 phosphorylation by endogenous PTK6 enhances ERK and FAK activation, cell adhesion, and anchorage-independent growth.","method":"Phosphoproteomics, site-directed mutagenesis (3YF mutant), co-immunoprecipitation, kinase assay, PTK6 knockdown","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — phospho-site mutagenesis with functional readouts plus endogenous kinase knockdown validation, single lab","pmids":["28214294"],"is_preprint":false},{"year":2018,"finding":"IRTKS (BAIAP2L1) recruits EPS8 to microvillar tips via IRTKS SH3 domain to promote epithelial microvillus elongation, as demonstrated by super-resolution microscopy and functional depletion experiments.","method":"Super-resolution microscopy, live imaging, co-immunoprecipitation (SH3-dependent interaction), depletion assays","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — super-resolution imaging plus domain-specific interaction and depletion assay, single lab","pmids":["30197089"],"is_preprint":false},{"year":2019,"finding":"FoxO3a directly binds the EPS8 gene promoter (by ChIP assay) and inhibits EPS8 transcription, forming a negative feedback loop in EGFR signaling; FoxO3a and EPS8 play opposing roles in gefitinib resistance, migration, and invasion.","method":"Chromatin immunoprecipitation (ChIP), luciferase reporter assay, siRNA knockdown, cell migration/invasion assay","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct ChIP binding plus reporter assay and functional knockdown, single lab","pmids":["30738830"],"is_preprint":false},{"year":2019,"finding":"EPS8 physically associates with incoming influenza A virion components during uncoating; EPS8 loss does not affect virion attachment, uptake, or fusion, but significantly delays nuclear import of released ribonucleoprotein complexes.","method":"EPS8 knockout, overexpression, co-immunoprecipitation with virion components, infection assay measuring uncoating and nuclear import","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO and overexpression with mechanistic step-specific assays, single lab","pmids":["31747592"],"is_preprint":false},{"year":2021,"finding":"The Whirlin-Myo15-Eps8 stereocilia tip complex undergoes liquid-liquid phase separation driven by multivalent interactions; the condensates promote actin bundling; a deafness-associated Myo15 mutation disrupts condensate formation and impairs actin bundling.","method":"In vitro phase separation reconstitution, actin bundling assay, mutagenesis of deafness-associated variant","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of phase separation and actin bundling with mutagenesis validation, single study multiple orthogonal methods","pmids":["33626355"],"is_preprint":false},{"year":2021,"finding":"EPS8/EPS-8 regulates general mitochondrial homeostasis and the mitochondrial unfolded protein response (UPRmt) through integrin-mediated remodeling of the actin cytoskeleton, identified by cross-species genetic screening in human stem cells and C. elegans.","method":"Cross-species genetic screen, RNAi knockdown, UPRmt reporter assay, actin cytoskeletal imaging","journal":"Science advances","confidence":"Low","confidence_rationale":"Tier 3 / Weak — genetic screen with knockdown and reporter assay, mechanism only partially defined, single study","pmids":["34714674"],"is_preprint":false},{"year":2021,"finding":"EPS8 interacts with ALDH7A1 (co-IP); EPS8 knockdown reduces ALDH7A1 protein and increases its poly-ubiquitination; BMI1 mediates ALDH7A1 ubiquitination and degradation, and BMI1 knockdown rescues ALDH7A1 degradation caused by EPS8 loss.","method":"Co-immunoprecipitation, ubiquitination assay, double knockdown with functional rescue","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus ubiquitination assay plus genetic double knockdown, single lab","pmids":["34391775"],"is_preprint":false},{"year":2022,"finding":"Chlamydia trachomatis effector TepP alters tyrosine phosphorylation of EPS8; TepP and EPS8 are necessary and sufficient for tight junction remodeling; genetic deletion of EPS8 renders epithelial cells and organoids resistant to TepP-mediated tight junction disruption.","method":"Phosphoproteomics, EPS8 genetic knockout (CRISPR), organoid infection model, in vivo mouse infection model","journal":"Cell host & microbe","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO with in vitro organoid and in vivo mouse infection assays, multiple orthogonal methods, single lab","pmids":["36395759"],"is_preprint":false},{"year":2022,"finding":"EPEC effector kinases NleH1 and NleH2 phosphorylate Eps8 at Ser775 via interaction with the Eps8 SH3 domain through noncanonical PxxDY proline-rich motifs; Ser775 phosphorylation hinders Eps8 bundling activity and drives dispersal of Eps8 from AE lesions during infection.","method":"Phosphoproteomics, crystal structure of Eps8 SH3 bound to NleH peptide, in vitro bundling assay, bacterial infection imaging","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vitro bundling assay plus phospho-site identification and infection imaging, single study multiple orthogonal methods","pmids":["35976880"],"is_preprint":false},{"year":2023,"finding":"Eps8 and IRSp53 work together to promote linear actin polymerization for tunnelling nanotube (TNT) formation; upon Arp2/3 inhibition, Eps8 shows heightened interaction with IRSp53 and its linear actin polymerase connection, favoring TNT elongation over branched actin structures.","method":"Micropatterning, optical tweezers, proteomics of Eps8 interactions, Arp2/3 inhibition, co-immunoprecipitation","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic interaction analysis plus functional optical tweezer and inhibition assays, single lab","pmids":["38009333"],"is_preprint":false}],"current_model":"EPS8 is a multifunctional actin regulatory adaptor that (1) is phosphorylated by EGFR and other receptor tyrosine kinases at its juxtamembrane-binding non-SH2 domain, (2) signals from Ras to Rac by forming a tri-complex with Abi1/E3b1 and Sos1 that has intrinsic Rac-GEF activity potentiated by PI3K/PIP3, (3) caps actin filament barbed ends through its C-terminal effector domain (auto-inhibited in isolation, relieved by Abi1), and (4) bundles actin filaments synergistically with IRSp53 downstream of Cdc42; its capping activity is negatively regulated by MAPK/Erk phosphorylation at S624/T628 (and by EPEC NleH kinases at S775), its protein levels are cell-cycle regulated by SCF(Fbxw5)-mediated proteasomal degradation in G2, and it participates in stereocilia tip complexes with Myo15/whirlin (including phase-separated condensates), NMDA receptor complexes at postsynapses, VE-cadherin junctions controlling YAP/TAZ signaling, and TLR4-MyD88 complexes in macrophages."},"narrative":{"mechanistic_narrative":"EPS8 is a multifunctional actin-regulatory adaptor that couples receptor tyrosine kinase signaling to Rho-family GTPase activation and direct remodeling of the actin cytoskeleton [PMID:8404850, PMID:10499589, PMID:15558031]. It was first identified as an EGFR substrate that binds the receptor juxtamembrane region through a non-SH2 mechanism independent of phosphotyrosine, with phosphorylation enhancing mitogenic and transforming responses [PMID:8404850, PMID:7532293, PMID:7608194, PMID:7791787]. EPS8 nucleates a tri-complex with Abi1/E3b1 and Sos1 that possesses intrinsic Rac-specific GEF activity, transmitting signals from Ras to Rac; this GEF activity is unmasked by PI3K recruitment through Abi1 and the lipid product PIP3 [PMID:9010225, PMID:10499589, PMID:12515821]. EPS8 directly shapes actin filaments through a conserved C-terminal effector domain that caps barbed ends with nanomolar affinity—an activity autoinhibited in full-length protein and relieved by Abi1—and through an actin-bundling activity that synergizes with IRSp53 downstream of Cdc42 to build filopodia and microvilli [PMID:15558031, PMID:17115031, PMID:30197089]. The balance between capping and bundling is regulated by phosphorylation: MAPK/Erk phosphorylation at S624/T628 inhibits capping to favor filopodia and bleb-based migration, and bacterial effector kinases similarly target EPS8 to disable its actin activities during infection [PMID:19564905, PMID:26163656, PMID:35976880]. EPS8 protein levels are cell-cycle controlled by SCF(Fbxw5)-mediated proteasomal degradation in G2, which permits timely cell rounding and mitotic entry while its capping activity prevents membrane blebbing during cytokinesis [PMID:23314863]. Through these activities EPS8 operates in diverse cellular contexts including stereocilia tip complexes with Myo15/whirlin that form actin-bundling phase-separated condensates required for hearing [PMID:21236676, PMID:21526224, PMID:33626355], postsynaptic NMDA receptor complexes and dendritic spine plasticity [PMID:17018287, PMID:23685357], dendritic cell and confined cell migration [PMID:21835647, PMID:26163656], VE-cadherin junctions controlling YAP/TAZ signaling and vascular permeability [PMID:26668327], and macrophage TLR4-MyD88 phagocytic signaling [PMID:22493489]. EPS8 is also exploited by pathogens, associating with influenza virion components to license RNP nuclear import and serving as a target of bacterial effectors that remodel epithelial tight junctions [PMID:31747592, PMID:36395759].","teleology":[{"year":1993,"claim":"Established EPS8 as a direct RTK substrate, answering whether it functions in growth-factor signaling: it is tyrosine-phosphorylated by and associates with EGFR and amplifies mitogenic output.","evidence":"cDNA cloning, in vivo phosphorylation, co-IP, and overexpression mitogenesis assays in fibroblasts/hematopoietic cells","pmids":["8404850"],"confidence":"High","gaps":["Mechanism of EGFR association undefined at this stage","No actin or downstream effector function yet identified"]},{"year":1995,"claim":"Defined the unusual phosphotyrosine-independent mode of EGFR engagement, showing EPS8 binds the receptor juxtamembrane domain rather than autophosphorylation sites and that constitutive phosphorylation accompanies transformation.","evidence":"EGFR deletion/point-mutant binding and kinase assays, transformation assays, co-IP with Shc","pmids":["7532293","7608194","7791787"],"confidence":"Medium","gaps":["Single-lab biochemistry","Structural basis of juxtamembrane binding not resolved"]},{"year":1997,"claim":"Identified the SH3-domain interactome and oligomerization mode, revealing EPS8 binds Abi-1/E3B1 and RN-tre and self-associates through an intertwined SH3 dimer.","evidence":"X-ray crystallography, SH3 library screen, co-IP, quantitative in vitro binding","pmids":["9303002","9010225","8700527"],"confidence":"High","gaps":["Functional consequence of dimerization not established","Hierarchy among competing SH3 ligands unknown"]},{"year":1999,"claim":"Resolved how EPS8 links Ras to actin remodeling: the EPS8-Abi1-Sos1 tri-complex carries intrinsic Rac-specific GEF activity.","evidence":"In vivo co-IP, reconstituted in vitro Rac-GEF assay, genetic epistasis","pmids":["10499589"],"confidence":"High","gaps":["How GEF activity is switched on left unexplained","Spatial regulation of the complex unaddressed"]},{"year":2000,"claim":"Showed EPS8 is a node partitioned between endocytic and signaling functions, with RN-tre binding diverting it from the GEF complex and inhibiting EGFR internalization.","evidence":"Co-IP, Rab5 GAP assay, EGFR endocytosis assay, Rac activation assay","pmids":["11099046"],"confidence":"High","gaps":["Quantitative balance between pools not defined","Triggers for switching not mapped"]},{"year":2003,"claim":"Defined the activation switch for the GEF complex and established family redundancy: PI3K/PIP3 unmasks Rac-GEF activity, and EPS8L1/L2 can substitute for EPS8.","evidence":"Co-IP, in vitro GEF assays with PI3K inhibitors and p85 knockout, rescue in eps8-/- fibroblasts","pmids":["12515821","14565974"],"confidence":"High","gaps":["Distinct in vivo roles of paralogs not fully separated","Lipid-binding determinants on the complex unmapped"]},{"year":2004,"claim":"Identified the core actin biochemistry of EPS8: its C-terminal effector domain caps barbed ends with nanomolar affinity, is autoinhibited and de-repressed by Abi1, with capping required in vivo for actin-based motility and morphogenesis.","evidence":"Reconstituted in vitro capping with domain mapping, loss-of-function in cells, C. elegans isoform genetic rescue","pmids":["15558031","15558032"],"confidence":"High","gaps":["Structural mechanism of autoinhibition not solved","Regulation of capping by other partners not yet known"]},{"year":2006,"claim":"Revealed the second actin activity—bundling—and its integration with Cdc42 signaling: EPS8 bundles filaments and synergizes with IRSp53 to drive Cdc42-dependent filopodia.","evidence":"In vitro bundling assay, co-IP, siRNA loss-of-function, live imaging; palladin and IRSp53 binding mapping","pmids":["17115031","15289329","17115031"],"confidence":"High","gaps":["Switch between capping and bundling not yet defined","Domain basis of bundling versus capping not separated"]},{"year":2006,"claim":"Extended EPS8 into neuronal function, placing it in postsynaptic NMDA receptor complexes governing actin remodeling and behavior.","evidence":"Co-IP of NMDA complex, electrophysiology in Eps8 KO neurons, behavioral assays","pmids":["17018287"],"confidence":"High","gaps":["Direct binding partner within the complex unresolved","Molecular link to channel function indirect"]},{"year":2009,"claim":"Identified phospho-regulation of the capping/bundling balance and tissue-specific actin junction roles: MAPK phosphorylation at S624/T628 inhibits capping to control axonal filopodia, and EPS8 maintains seminiferous-epithelium junctions.","evidence":"Phospho-mutant capping assays, hippocampal neuron imaging, RNAi with junction-protein readouts in vitro and in vivo","pmids":["19564905","19293393","19528316"],"confidence":"High","gaps":["Upstream kinase identity in each context not fully resolved","Coordination of the two phospho-sites unclear"]},{"year":2011,"claim":"Established EPS8 as an essential stereocilia component, building the Myo15a-whirlin-EPS8 tip complex required for stereocilia elongation, hair-cell maturation, and hearing.","evidence":"Co-IP, knockout mouse analysis, overexpression rescue, electrophysiology, hearing thresholds; EPS8 roles in dendritic cell migration","pmids":["21236676","21526224","21835647"],"confidence":"High","gaps":["Stoichiometry and assembly order of the tip complex not fully defined","How tip targeting couples to capping versus bundling not resolved"]},{"year":2013,"claim":"Defined cell-cycle control of EPS8 levels and its role in spine plasticity: SCF(Fbxw5) degrades EPS8 in G2 to enable mitotic rounding, and EPS8 capping is required for LTP-driven spine enlargement and cognition.","evidence":"E3 ligase identification, proteasome inhibition, cell-cycle synchronization, live imaging; KO mouse LTP, spine morphometry, behavior","pmids":["23314863","23685357"],"confidence":"High","gaps":["Degron recognition motif for Fbxw5 not mapped","Link between human autism and EPS8 correlative only"]},{"year":2015,"claim":"Connected EPS8 actin activities to mechanics and adhesion signaling: Erk phosphorylation tips EPS8 toward bundling to drive leader-bleb migration, and EPS8 links VE-cadherin junctions to YAP/TAZ signaling and vascular permeability.","evidence":"Live imaging with Erk biosensor, cortex-tension measurements, capping/bundling mutants; reciprocal co-IP, KO mouse with permeability and YAP reporter assays","pmids":["26163656","26668327","26252776"],"confidence":"High","gaps":["Mechanism coupling EPS8 to YAP nuclear translocation incompletely defined","How junction state controls EPS8 displacement not fully resolved"]},{"year":2017,"claim":"Expanded the kinase regulation of EPS8 to additional RTK pathways, showing PTK6 phosphorylates EPS8 to enhance proliferation, migration, and ERK/FAK activation.","evidence":"Phosphoproteomics, 3YF mutagenesis, kinase assay, PTK6 knockdown; FAK/Src complex and clathrin/AP-2 interactome studies","pmids":["28214294","25359883","23626693"],"confidence":"Medium","gaps":["Functional integration of many phospho-sites unresolved","In vivo relevance of PTK6 phosphorylation untested"]},{"year":2018,"claim":"Defined how EPS8 is targeted to specialized actin protrusions: I-BAR proteins IRTKS/IRSp53 recruit EPS8 via SH3 interactions to drive microvillus and nanotube elongation through linear actin polymerization.","evidence":"Super-resolution microscopy, SH3-dependent co-IP, depletion assays; optical tweezers and Arp2/3 inhibition for TNTs","pmids":["30197089","38009333"],"confidence":"Medium","gaps":["How recruitment selects capping versus bundling output unclear","Direct linear-polymerase activity of EPS8 not biochemically isolated"]},{"year":2021,"claim":"Revealed that the stereocilia tip complex functions through liquid-liquid phase separation, with multivalent Whirlin-Myo15-EPS8 condensates promoting actin bundling disrupted by a deafness variant.","evidence":"In vitro phase-separation reconstitution, actin bundling assay, deafness-variant mutagenesis","pmids":["33626355"],"confidence":"High","gaps":["In vivo confirmation of condensate behavior limited","Contribution of EPS8 multivalency versus partners not separated"]},{"year":2022,"claim":"Established EPS8 as a host target hijacked by pathogens, with influenza virion association licensing RNP nuclear import and bacterial effectors targeting EPS8 to remodel tight junctions and disable its actin activities.","evidence":"Genetic knockout/overexpression and infection assays (influenza); CRISPR KO with organoid and mouse infection (Chlamydia); crystal structure and bundling assay with EPEC NleH kinases","pmids":["31747592","36395759","35976880"],"confidence":"High","gaps":["Mechanism of EPS8 contribution to influenza RNP transport undefined","Host signaling rewired by effector phosphorylation incompletely mapped"]},{"year":null,"claim":"How EPS8's competing capping, bundling, GEF-scaffolding, and adaptor functions are selected and spatially partitioned within a single cell across its many tissue contexts remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model integrating autoinhibition, phospho-switches, and partner binding","Quantitative rules governing partition between signaling and actin pools unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[13,14,16,43]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,8,15,25]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[8,11,13]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[7,13,16]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[7,25,26,37]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[24,31]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[24]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,8,11]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[32]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[18,33]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[27,31]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[26,40]}],"complexes":["EPS8-Abi1-Sos1 Rac-GEF tri-complex","Myo15a-whirlin-EPS8 stereocilia tip complex","NMDA receptor postsynaptic complex","TLR4-MyD88 complex"],"partners":["ABI1","SOS1","IRSP53","RN-TRE","MYO15A","WHRN","VE-CADHERIN","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q12929","full_name":"Epidermal growth factor receptor kinase substrate 8","aliases":[],"length_aa":822,"mass_kda":91.9,"function":"Signaling adapter that controls various cellular protrusions by regulating actin cytoskeleton dynamics and architecture. Depending on its association with other signal transducers, can regulate different processes. Together with SOS1 and ABI1, forms a trimeric complex that participates in transduction of signals from Ras to Rac by activating the Rac-specific guanine nucleotide exchange factor (GEF) activity. Acts as a direct regulator of actin dynamics by binding actin filaments and has both barbed-end actin filament capping and actin bundling activities depending on the context. Displays barbed-end actin capping activity when associated with ABI1, thereby regulating actin-based motility process: capping activity is auto-inhibited and inhibition is relieved upon ABI1 interaction. Also shows actin bundling activity when associated with BAIAP2, enhancing BAIAP2-dependent membrane extensions and promoting filopodial protrusions. Involved in the regulation of processes such as axonal filopodia growth, stereocilia length, dendritic cell migration and cancer cell migration and invasion. Acts as a regulator of axonal filopodia formation in neurons: in the absence of neurotrophic factors, negatively regulates axonal filopodia formation via actin-capping activity. In contrast, it is phosphorylated in the presence of BDNF leading to inhibition of its actin-capping activity and stimulation of filopodia formation. Component of a complex with WHRN and MYO15A that localizes at stereocilia tips and is required for elongation of the stereocilia actin core. Indirectly involved in cell cycle progression; its degradation following ubiquitination being required during G2 phase to promote cell shape changes","subcellular_location":"Cytoplasm, cell cortex; Cell projection, ruffle membrane; Cell projection, growth cone; Cell projection, stereocilium; Synapse, synaptosome","url":"https://www.uniprot.org/uniprotkb/Q12929/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPS8","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EPS8","total_profiled":1310},"omim":[{"mim_id":"617637","title":"DEAFNESS, AUTOSOMAL RECESSIVE 106; DFNB106","url":"https://www.omim.org/entry/617637"},{"mim_id":"616711","title":"TAO KINASE 3; TAOK3","url":"https://www.omim.org/entry/616711"},{"mim_id":"615974","title":"DEAFNESS, AUTOSOMAL RECESSIVE 102; DFNB102","url":"https://www.omim.org/entry/615974"},{"mim_id":"614989","title":"EPS8-LIKE PROTEIN 3; EPS8L3","url":"https://www.omim.org/entry/614989"},{"mim_id":"614988","title":"EPS8-LIKE PROTEIN 2; EPS8L2","url":"https://www.omim.org/entry/614988"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/EPS8"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q12929","domains":[{"cath_id":"2.30.29.30","chopping":"60-205","consensus_level":"high","plddt":90.6649,"start":60,"end":205},{"cath_id":"2.30.30.40","chopping":"536-589","consensus_level":"high","plddt":91.368,"start":536,"end":589},{"cath_id":"1.10.150.50","chopping":"728-803","consensus_level":"high","plddt":82.0205,"start":728,"end":803},{"cath_id":"1.20.190","chopping":"262-447","consensus_level":"high","plddt":87.9138,"start":262,"end":447}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12929","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q12929-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q12929-F1-predicted_aligned_error_v6.png","plddt_mean":70.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPS8","jax_strain_url":"https://www.jax.org/strain/search?query=EPS8"},"sequence":{"accession":"Q12929","fasta_url":"https://rest.uniprot.org/uniprotkb/Q12929.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q12929/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12929"}},"corpus_meta":[{"pmid":"10499589","id":"PMC_10499589","title":"EPS8 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with the EGFR in vivo despite lacking a functional SH2 domain. Overexpression of Eps8 in fibroblasts or hematopoietic cells increases mitogenic response to EGF.\",\n      \"method\": \"cDNA cloning, in vivo tyrosine phosphorylation assay, co-immunoprecipitation, adoptive expression/overexpression mitogenesis assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP and functional overexpression assay, foundational paper replicated across subsequent studies\",\n      \"pmids\": [\"8404850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Eps8 binds directly to the juxtamembrane domain of EGFR through a non-SH2 domain by a mechanism that does not require phosphotyrosine residues.\",\n      \"method\": \"Direct binding assay with EGFR deletion/point mutants, in vitro binding\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro direct binding with mutagenesis, single lab\",\n      \"pmids\": [\"7532293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"EGFR autophosphorylation sites are dispensable for Eps8 phosphorylation; removal of the final 214 COOH-terminal residues of EGFR increases Eps8 phosphorylation ~13-fold, identifying a juxtamembrane region as the structural determinant for efficient phosphorylation of Eps8.\",\n      \"method\": \"EGFR deletion/point mutant phosphorylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with systematic mutagenesis, single lab\",\n      \"pmids\": [\"7608194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Eps8 constitutive tyrosine phosphorylation occurs in tumor cell lines at stoichiometry similar to potent mitogenic response; overexpression of Eps8 transforms NIH 3T3 cells under limiting EGFR activation. Eps8 associates in vivo with Shc, at least partly via the SH3 domain of Eps8.\",\n      \"method\": \"Phosphotyrosine analysis of tumor lines, transformation assay, co-immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and functional transformation assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"7791787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"RN-tre binds specifically and with high affinity (Kd ~10^-8–10^-7 M) to the SH3 domain of Eps8 in vitro and stably associates with Eps8 in vivo.\",\n      \"method\": \"In vitro SH3 domain binding assay with Kd measurement, co-immunoprecipitation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — quantitative in vitro binding plus co-IP, single lab\",\n      \"pmids\": [\"8700527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"The SH3 domain of Eps8 forms an intertwined dimer via strand interchange (novel fold variant of canonical SH3), and intact Eps8 is multimeric in vivo, suggesting the SH3 domain serves as a dimerization motif.\",\n      \"method\": \"X-ray crystallography, co-immunoprecipitation\",\n      \"journal\": \"Nature structural biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vivo co-IP validation\",\n      \"pmids\": [\"9303002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"E3B1 (Abi-1) was identified as an Eps8 SH3-domain binding protein via SH3 library screen; E3B1 associates with Eps8 in vivo and is serine-phosphorylated in a growth-factor-dependent manner.\",\n      \"method\": \"SH3 domain expression library screen, co-immunoprecipitation, phosphoamino acid analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus biochemical characterization, single lab\",\n      \"pmids\": [\"9010225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Eps8 is recruited to the cell cortex, membrane ruffles, lamellipodia, and dynamic F-actin structures (podosomes) upon cytoskeletal remodeling; a detergent-resistant pool associates with podosomes in v-Src-transformed cells.\",\n      \"method\": \"Immunofluorescence localization, Triton X-100 fractionation, live-cell imaging\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by immunofluorescence and fractionation with functional context, single lab\",\n      \"pmids\": [\"9665816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Eps8, E3b1/Abi-1, and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro, mediating signal transduction from Ras to Rac.\",\n      \"method\": \"Co-immunoprecipitation (in vivo tri-complex), in vitro Rac-GEF activity assay, genetic epistasis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro GEF activity combined with in vivo co-IP and epistasis, replicated in multiple subsequent studies\",\n      \"pmids\": [\"10499589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Eps8 interacts with the PDZ domain of Dvl1 (murine Dishevelled homologue) in vitro and in vivo; Dvl1 is hyperphosphorylated in the presence of Eps8, whereas Dvl1 inhibits EGF-induced tyrosine phosphorylation of Eps8.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro binding confirmation, transfection and phosphorylation analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus in vitro binding plus in-cell functional assay, single lab\",\n      \"pmids\": [\"10581192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Eps8 interacts via its SH3 domain with RN-tre, a Rab5 GAP; this complex inhibits EGFR internalization and attenuates Rac signaling by diverting Eps8 from the Abi1/Sos1 complex. RN-tre activity on Rab5 is regulated by the EGFR.\",\n      \"method\": \"Co-immunoprecipitation, Rab5 GAP activity assay, EGFR endocytosis assay, Rac activation assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay, reciprocal co-IP, functional endocytosis assay, multiple orthogonal methods\",\n      \"pmids\": [\"11099046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PI3K (via p85 subunit) is recruited into the Eps8-Abi1-Sos1 complex through Abi1; both p85 recruitment and the PI3K product PIP3 are required to unmask Rac-GEF activity of the complex in vitro and to activate Rac and actin remodeling in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vitro Rac-GEF assay, PI3K inhibitor studies, p85 knockout cells\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro GEF assay combined with co-IP and knockout cell validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"12515821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Eps8 family members (Eps8L1, Eps8L2, Eps8L3) interact with Abi1 and Sos-1; only Eps8L1 and Eps8L2 activate Rac-GEF activity of Sos-1 and bind actin in vivo, functionally compensating for Eps8 loss in eps8−/− fibroblasts.\",\n      \"method\": \"Co-immunoprecipitation, Rac-GEF activity assay, rescue experiments in eps8−/− fibroblasts\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro GEF assay, co-IP, and genetic rescue in knockout cells, single lab multiple orthogonal methods\",\n      \"pmids\": [\"14565974\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Eps8 family proteins cap actin filament barbed ends; the conserved C-terminal effector domain caps barbed ends with nanomolar affinity. Full-length Eps8 is auto-inhibited, and interaction with Abi1 relieves this inhibition. In vivo, Eps8 removal impairs actin-based propulsion.\",\n      \"method\": \"In vitro actin barbed-end capping assay with isolated domain and full-length protein, Kd measurement, loss-of-function in cells, Abi1 interaction relief-of-inhibition assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro capping assay with domain mapping and mutagenesis, plus in vivo functional validation\",\n      \"pmids\": [\"15558031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In C. elegans, the EPS-8A isoform (but not EPS-8B) has a C-terminal actin barbed-end capping activity required for proper apical morphogenesis of intestinal cells; the two isoforms differ at their C-termini.\",\n      \"method\": \"C. elegans genetics, isoform rescue experiments, in vitro actin barbed-end capping assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro capping reconstitution and in vivo genetic rescue with isoform specificity, ortholog study\",\n      \"pmids\": [\"15558032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"IRSp53 directly binds Eps8 through the NH2-terminal proline-rich sequence of Eps8 and the IRSp53 SH3 domain; this complex synergistically activates Rac by reinforcing the Eps8/Abi1/Sos1 GEF complex and occurs at the leading edge of motile cells.\",\n      \"method\": \"Co-immunoprecipitation (in vivo), FRET analysis, Rac activation (pull-down assay), cell motility/invasion assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, FRET, Rac pull-down, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"15289329\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Eps8 has actin crosslinking (bundling) activity and synergizes with IRSp53 in mediating actin bundling in vitro. Cdc42 binds the IRSp53-Eps8 complex and controls its cellular distribution. Cdc42-induced filopodia require both IRSp53 and Eps8.\",\n      \"method\": \"In vitro actin bundling assay, co-immunoprecipitation, loss-of-function (siRNA), live-cell imaging\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstituted bundling assay with multiple genetic loss-of-function validations, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"17115031\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Palladin directly binds Eps8 (identified by yeast two-hybrid, confirmed by co-immunoprecipitation); both proteins colocalize in dorsal ruffles; palladin knockdown reduces Rac activation and ruffle formation after PDGF treatment.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, colocalization by confocal microscopy, palladin knockdown with functional readout\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid validated by co-IP plus functional knockdown assay, single lab\",\n      \"pmids\": [\"16868024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Eps8 is part of the NMDA receptor complex at postsynaptic structures in neurons; Eps8 null mice show abnormal NMDA receptor currents, altered sensitivity to ethanol inhibition, and resistance to NMDA/ethanol-induced actin remodeling.\",\n      \"method\": \"Co-immunoprecipitation (NMDA receptor complex), electrophysiology in knockout neurons, behavioral assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP of endogenous complex, electrophysiology in Eps8 KO mice, multiple orthogonal methods, independently validated\",\n      \"pmids\": [\"17018287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In C. elegans, EPS-8 interacts with the ankyrin repeat protein VAB-19 (central domain of EPS-8 sufficient); both proteins colocalize at cell-matrix attachment structures; eps-8 null mutants are defective in epidermal elongation and epidermal-muscle attachment, dependent on PTB and central domains but independent of C-terminal SH3 and actin-binding domains.\",\n      \"method\": \"Yeast two-hybrid, co-localization, eps-8 null mutant phenotype, domain rescue experiments\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic null and domain-mapping rescue in C. elegans with interaction validation, single lab\",\n      \"pmids\": [\"18833327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"LanCL1 binds specifically to the SH3 domain of Eps8 in vitro; LanCL1 mutants defective in Eps8 interaction inhibit NGF-induced neurite outgrowth, establishing functional significance of this interaction.\",\n      \"method\": \"In vitro affinity binding, crystal structure of LanCL1, loss-of-function mutagenesis with neurite outgrowth assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vitro binding plus mutagenesis with functional readout, single study multiple orthogonal methods\",\n      \"pmids\": [\"19528316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Eps8 is localized to actin-based cell junctions (blood-testis barrier and apical ectoplasmic specialization) in the seminiferous epithelium; Eps8 knockdown by RNAi causes F-actin disorganization and mislocalization of tight junction proteins occludin and ZO-1; in vivo knockdown causes germ cell sloughing and BTB damage.\",\n      \"method\": \"Immunofluorescence/immunohistochemistry localization, RNAi knockdown with permeability and junction protein assays, in vivo knockdown\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional RNAi with defined molecular readouts in vitro and in vivo, single lab\",\n      \"pmids\": [\"19293393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"BDNF inhibits the actin barbed-end capping activity of Eps8 through MAPK-dependent phosphorylation at residues S624 and T628; a phosphorylation-defective mutant (S624A/T628A) shows increased actin association and resistance to BDNF-mediated release; a phosphomimetic mutant (S624E/T628E) has opposite effects. This controls axonal filopodia formation.\",\n      \"method\": \"Site-directed mutagenesis, MAPK phosphorylation assay, actin barbed-end capping assay, hippocampal neuron imaging and filopodia quantification\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro capping assay with phospho-mutagenesis and in vivo neuronal phenotype rescue, single study multiple orthogonal methods\",\n      \"pmids\": [\"19564905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The SOS1/EPS8/ABI1 tri-complex facilitates LPA-induced Rac activation specifically in metastatic ovarian cancer cells; re-expression of a missing tri-complex member in non-metastatic cells confers metastatic capability; integrity of the complex is essential for LPA-stimulated cell migration and peritoneal colonization.\",\n      \"method\": \"Co-immunoprecipitation, Rac activation assay, re-expression rescue, in vivo peritoneal metastasis assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, Rac pull-down, and in vivo rescue assay, single lab\",\n      \"pmids\": [\"21118970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Eps8 in cancer cells localizes to the lysosomal compartment independently of actin polymerization; Eps8 co-immunoprecipitates with Hsc70 and LAMP-2, key elements of chaperone-mediated autophagy (CMA); the region aa 184-535 containing KFERQ-like motifs mediates lysosomal recruitment; Eps8 binds to and is incorporated into lysosomes in vitro, competed by other CMA substrates.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence/confocal colocalization, co-immunoprecipitation, in vitro lysosome binding/incorporation assay, FRAP\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro lysosome assay plus co-IP plus domain mapping, single lab\",\n      \"pmids\": [\"20184880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Eps8 interacts with MyoXVa and whirlin; MyoXVa is required for Eps8 accumulation at stereocilia tips; overexpression of MyoXVa drives elongation and increased Eps8 at tips; exogenous MyoXVa in MyoXVa-deficient cells rescues Eps8 tip localization. Whirlin-deficient mice show reduced Eps8 and MyoXVa at stereocilia tips. MyoXVa-whirlin-Eps8 form a stereocilia tip complex.\",\n      \"method\": \"Co-immunoprecipitation, live imaging, knockout mouse analysis, overexpression rescue\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, multiple knockout mouse models, overexpression rescue, independent replication across related papers\",\n      \"pmids\": [\"21236676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Eps8 is localized at stereocilia tips of cochlear hair cells; Eps8 knockout mice are profoundly deaf and exhibit short stereocilia; inner hair cells (IHCs) fail to mature into functional sensory receptors in Eps8 KO mice, establishing a role for Eps8 in stereocilia elongation and IHC functional maturation.\",\n      \"method\": \"Knockout mouse, immunofluorescence localization, electrophysiology, hearing threshold measurement\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple functional readouts (electrophysiology, hearing tests, localization) in Eps8 KO mice, replicated across labs\",\n      \"pmids\": [\"21526224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Eps8 actin capping activity is required for dendritic cell polarization and formation of elongated migratory protrusions; Eps8-deficient DCs are impaired in directional/chemotactic migration in 3D and in reaching draining lymph nodes in vivo, preventing contact hypersensitivity responses. Eps8 is not required for antigen uptake, processing, or presentation.\",\n      \"method\": \"Eps8 knockout mouse, in vitro 3D migration assay, in vivo lymph node migration assay, contact hypersensitivity assay\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with multiple defined cellular and in vivo phenotype readouts, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"21835647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Eps8 interacts with the clathrin-mediated endocytosis machinery; Src phosphorylates Eps8 downstream of FGFR activation; depletion of Eps8 inhibits FGFR trafficking and immediate Erk signaling.\",\n      \"method\": \"Live-cell imaging, Eps8 depletion (siRNA), co-immunoprecipitation with endocytic machinery, Src phosphorylation assay, Erk signaling assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with trafficking machinery and functional knockdown assays, single lab\",\n      \"pmids\": [\"23203811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ezrin differentially modulates Eps8 and Eps8L1a activities: coexpression of ezrin with Eps8 promotes membrane ruffles and tufts of microvilli via bundling activity; coexpression with Eps8L1a promotes clustering of actin structures via capping activity. Ezrin's effect requires its binding to Eps8/Eps8L1a, and specific actin-bundling/capping activities of these proteins.\",\n      \"method\": \"Overexpression and co-expression studies, domain-mapping mutagenesis, morphological assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional mutagenesis with defined cellular readouts, single lab\",\n      \"pmids\": [\"22262457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Human intersectin 2 (ITSN2) binds Eps8 (interaction domain: aa 260-306 of Eps8 and coiled-coil domain of ITSN2); ITSN2 overexpression induces lysosome-dependent degradation of Eps8.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, GST pull-down, domain mapping, protein stability assay with lysosome inhibitor\",\n      \"journal\": \"BMB reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding assays (y2h, co-IP, GST pull-down) plus functional degradation assay, single lab\",\n      \"pmids\": [\"22449706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Eps8 interacts with TLR4 in the cytosol and at the phagosome in LPS-stimulated macrophages; Eps8 facilitates TLR4-MyD88 interaction and downstream Src, FAK, and p38 MAPK activation; Eps8 attenuation reduces LPS-promoted phagocytosis and bacterial killing.\",\n      \"method\": \"Confocal colocalization, co-immunoprecipitation, siRNA knockdown with phagocytosis and signaling readouts, dominant-negative construct\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of endogenous proteins plus functional knockdown with defined downstream assays, single lab\",\n      \"pmids\": [\"22493489\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SCF(Fbxw5) ubiquitin E3 ligase mediates proteasomal degradation of Eps8 specifically in G2 phase; failure to degrade Eps8 in G2 prolongs its localization at the cell cortex, markedly delays cell rounding and prometaphase entry. Eps8 capping activity is required during late mitosis/cytokinesis to prevent membrane blebbing.\",\n      \"method\": \"Biochemical degradation assays, E3 ligase identification (SCFFbxw5), proteasome inhibitor experiments, cell-cycle synchronization, live-cell imaging of Eps8 localization\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — E3 ligase identification with reconstitution-level biochemistry, cell-cycle functional assays, and live imaging, single study multiple orthogonal methods\",\n      \"pmids\": [\"23314863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Eps8 actin-capping activity is required for dendritic spine enlargement during chemically induced LTP; mice lacking Eps8 display immature spines unable to undergo potentiation and are impaired in cognitive functions. Reduced Eps8 levels are found in brains of autism patients.\",\n      \"method\": \"Eps8 KO mouse, chemically induced LTP in culture, spine morphometry, electrophysiology, behavioral cognitive tests\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO with multiple orthogonal functional readouts (LTP, spine morphology, cognition), capping-defective mutant rescue\",\n      \"pmids\": [\"23685357\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Eps8 forms a biochemical complex with FAK and Src in SCC cells; it controls the spatial distribution of active Src in a FAK-dependent manner and drives Src trafficking to autophagic structures when FAK is absent; when FAK is present, Eps8 is recruited to focal adhesions and required for FAK-dependent polarization and invasion.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, siRNA knockdown, invasion assay, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and functional knockdown with defined cellular readouts, single lab\",\n      \"pmids\": [\"25359883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Novel Eps8 binding partners identified by phosphoproteomic affinity purification include clathrin, AP-2 (endocytic machinery), NBR1, Vav2, IRS4, and Shp2; 22 Eps8 pTyr and pSer/Thr phosphorylation sites identified, some dependent on Src family kinases and FGFR activity.\",\n      \"method\": \"Differential phosphoproteomics, peptide affinity purification, mass spectrometry\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based interactome/phosphoproteomics, single lab\",\n      \"pmids\": [\"23626693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Eps8 bundling activity promotes cortex tension and intracellular pressure to drive leader bleb formation during confined migration; Erk phosphorylation inhibits Eps8 capping activity, and Erk activity is concentrated within leader blebs, promoting actin bundling by Eps8 to enhance bleb-based migration.\",\n      \"method\": \"Live-cell imaging, Erk biosensor, cortex tension measurements, loss-of-function/gain-of-function with capping and bundling mutants\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with FRET biosensor and functional mutant analysis, single lab\",\n      \"pmids\": [\"26163656\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"EPS8 is a novel binding partner of VE-cadherin; EPS8 associates with VE-cadherin at remodeling junctions promoting YAP nuclear translocation and transcriptional activation; in stabilized junctions 14-3-3-YAP displaces EPS8. Eps8-null mice show increased vascular permeability and junctional YAP inhibition.\",\n      \"method\": \"Co-immunoprecipitation, live imaging, Eps8 KO mouse, YAP localization and reporter assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, KO mouse with in vivo readout, and YAP transcriptional assay, single lab multiple orthogonal methods\",\n      \"pmids\": [\"26668327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Eps8 is a direct interactor of Dvl1 (scaffold for Wnt signaling); gain-of-function of Eps8 mimics Wnt-mediated axon remodeling; Eps8 silencing blocks Wnt3a-induced axon remodeling; blockade of Dvl1-Eps8 interaction abolishes Wnt3a-mediated axonal remodeling.\",\n      \"method\": \"Yeast two-hybrid, functional rescue/silencing in DRG neurons, dominant-negative Dvl1-Eps8 interaction blockade\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid validated by functional assays in primary neurons, single lab\",\n      \"pmids\": [\"26252776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PTK6 (Brk) phosphorylates Eps8 at residues Tyr497, Tyr524, and Tyr534; a phosphorylation-defective 3YF mutant fails to increase proliferation, migration, and ERK/FAK activation mediated by wild-type Eps8 overexpression; EGF-induced Eps8 phosphorylation by endogenous PTK6 enhances ERK and FAK activation, cell adhesion, and anchorage-independent growth.\",\n      \"method\": \"Phosphoproteomics, site-directed mutagenesis (3YF mutant), co-immunoprecipitation, kinase assay, PTK6 knockdown\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — phospho-site mutagenesis with functional readouts plus endogenous kinase knockdown validation, single lab\",\n      \"pmids\": [\"28214294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"IRTKS (BAIAP2L1) recruits EPS8 to microvillar tips via IRTKS SH3 domain to promote epithelial microvillus elongation, as demonstrated by super-resolution microscopy and functional depletion experiments.\",\n      \"method\": \"Super-resolution microscopy, live imaging, co-immunoprecipitation (SH3-dependent interaction), depletion assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — super-resolution imaging plus domain-specific interaction and depletion assay, single lab\",\n      \"pmids\": [\"30197089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FoxO3a directly binds the EPS8 gene promoter (by ChIP assay) and inhibits EPS8 transcription, forming a negative feedback loop in EGFR signaling; FoxO3a and EPS8 play opposing roles in gefitinib resistance, migration, and invasion.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), luciferase reporter assay, siRNA knockdown, cell migration/invasion assay\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ChIP binding plus reporter assay and functional knockdown, single lab\",\n      \"pmids\": [\"30738830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EPS8 physically associates with incoming influenza A virion components during uncoating; EPS8 loss does not affect virion attachment, uptake, or fusion, but significantly delays nuclear import of released ribonucleoprotein complexes.\",\n      \"method\": \"EPS8 knockout, overexpression, co-immunoprecipitation with virion components, infection assay measuring uncoating and nuclear import\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO and overexpression with mechanistic step-specific assays, single lab\",\n      \"pmids\": [\"31747592\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The Whirlin-Myo15-Eps8 stereocilia tip complex undergoes liquid-liquid phase separation driven by multivalent interactions; the condensates promote actin bundling; a deafness-associated Myo15 mutation disrupts condensate formation and impairs actin bundling.\",\n      \"method\": \"In vitro phase separation reconstitution, actin bundling assay, mutagenesis of deafness-associated variant\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of phase separation and actin bundling with mutagenesis validation, single study multiple orthogonal methods\",\n      \"pmids\": [\"33626355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EPS8/EPS-8 regulates general mitochondrial homeostasis and the mitochondrial unfolded protein response (UPRmt) through integrin-mediated remodeling of the actin cytoskeleton, identified by cross-species genetic screening in human stem cells and C. elegans.\",\n      \"method\": \"Cross-species genetic screen, RNAi knockdown, UPRmt reporter assay, actin cytoskeletal imaging\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — genetic screen with knockdown and reporter assay, mechanism only partially defined, single study\",\n      \"pmids\": [\"34714674\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EPS8 interacts with ALDH7A1 (co-IP); EPS8 knockdown reduces ALDH7A1 protein and increases its poly-ubiquitination; BMI1 mediates ALDH7A1 ubiquitination and degradation, and BMI1 knockdown rescues ALDH7A1 degradation caused by EPS8 loss.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, double knockdown with functional rescue\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus ubiquitination assay plus genetic double knockdown, single lab\",\n      \"pmids\": [\"34391775\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Chlamydia trachomatis effector TepP alters tyrosine phosphorylation of EPS8; TepP and EPS8 are necessary and sufficient for tight junction remodeling; genetic deletion of EPS8 renders epithelial cells and organoids resistant to TepP-mediated tight junction disruption.\",\n      \"method\": \"Phosphoproteomics, EPS8 genetic knockout (CRISPR), organoid infection model, in vivo mouse infection model\",\n      \"journal\": \"Cell host & microbe\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO with in vitro organoid and in vivo mouse infection assays, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"36395759\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"EPEC effector kinases NleH1 and NleH2 phosphorylate Eps8 at Ser775 via interaction with the Eps8 SH3 domain through noncanonical PxxDY proline-rich motifs; Ser775 phosphorylation hinders Eps8 bundling activity and drives dispersal of Eps8 from AE lesions during infection.\",\n      \"method\": \"Phosphoproteomics, crystal structure of Eps8 SH3 bound to NleH peptide, in vitro bundling assay, bacterial infection imaging\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vitro bundling assay plus phospho-site identification and infection imaging, single study multiple orthogonal methods\",\n      \"pmids\": [\"35976880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Eps8 and IRSp53 work together to promote linear actin polymerization for tunnelling nanotube (TNT) formation; upon Arp2/3 inhibition, Eps8 shows heightened interaction with IRSp53 and its linear actin polymerase connection, favoring TNT elongation over branched actin structures.\",\n      \"method\": \"Micropatterning, optical tweezers, proteomics of Eps8 interactions, Arp2/3 inhibition, co-immunoprecipitation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic interaction analysis plus functional optical tweezer and inhibition assays, single lab\",\n      \"pmids\": [\"38009333\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EPS8 is a multifunctional actin regulatory adaptor that (1) is phosphorylated by EGFR and other receptor tyrosine kinases at its juxtamembrane-binding non-SH2 domain, (2) signals from Ras to Rac by forming a tri-complex with Abi1/E3b1 and Sos1 that has intrinsic Rac-GEF activity potentiated by PI3K/PIP3, (3) caps actin filament barbed ends through its C-terminal effector domain (auto-inhibited in isolation, relieved by Abi1), and (4) bundles actin filaments synergistically with IRSp53 downstream of Cdc42; its capping activity is negatively regulated by MAPK/Erk phosphorylation at S624/T628 (and by EPEC NleH kinases at S775), its protein levels are cell-cycle regulated by SCF(Fbxw5)-mediated proteasomal degradation in G2, and it participates in stereocilia tip complexes with Myo15/whirlin (including phase-separated condensates), NMDA receptor complexes at postsynapses, VE-cadherin junctions controlling YAP/TAZ signaling, and TLR4-MyD88 complexes in macrophages.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"EPS8 is a multifunctional actin-regulatory adaptor that couples receptor tyrosine kinase signaling to Rho-family GTPase activation and direct remodeling of the actin cytoskeleton [#0, #8, #13]. It was first identified as an EGFR substrate that binds the receptor juxtamembrane region through a non-SH2 mechanism independent of phosphotyrosine, with phosphorylation enhancing mitogenic and transforming responses [#0, #1, #2, #3]. EPS8 nucleates a tri-complex with Abi1/E3b1 and Sos1 that possesses intrinsic Rac-specific GEF activity, transmitting signals from Ras to Rac; this GEF activity is unmasked by PI3K recruitment through Abi1 and the lipid product PIP3 [#6, #8, #11]. EPS8 directly shapes actin filaments through a conserved C-terminal effector domain that caps barbed ends with nanomolar affinity—an activity autoinhibited in full-length protein and relieved by Abi1—and through an actin-bundling activity that synergizes with IRSp53 downstream of Cdc42 to build filopodia and microvilli [#13, #16, #40]. The balance between capping and bundling is regulated by phosphorylation: MAPK/Erk phosphorylation at S624/T628 inhibits capping to favor filopodia and bleb-based migration, and bacterial effector kinases similarly target EPS8 to disable its actin activities during infection [#22, #36, #47]. EPS8 protein levels are cell-cycle controlled by SCF(Fbxw5)-mediated proteasomal degradation in G2, which permits timely cell rounding and mitotic entry while its capping activity prevents membrane blebbing during cytokinesis [#32]. Through these activities EPS8 operates in diverse cellular contexts including stereocilia tip complexes with Myo15/whirlin that form actin-bundling phase-separated condensates required for hearing [#25, #26, #43], postsynaptic NMDA receptor complexes and dendritic spine plasticity [#18, #33], dendritic cell and confined cell migration [#27, #36], VE-cadherin junctions controlling YAP/TAZ signaling and vascular permeability [#37], and macrophage TLR4-MyD88 phagocytic signaling [#31]. EPS8 is also exploited by pathogens, associating with influenza virion components to license RNP nuclear import and serving as a target of bacterial effectors that remodel epithelial tight junctions [#42, #46].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Established EPS8 as a direct RTK substrate, answering whether it functions in growth-factor signaling: it is tyrosine-phosphorylated by and associates with EGFR and amplifies mitogenic output.\",\n      \"evidence\": \"cDNA cloning, in vivo phosphorylation, co-IP, and overexpression mitogenesis assays in fibroblasts/hematopoietic cells\",\n      \"pmids\": [\"8404850\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of EGFR association undefined at this stage\", \"No actin or downstream effector function yet identified\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Defined the unusual phosphotyrosine-independent mode of EGFR engagement, showing EPS8 binds the receptor juxtamembrane domain rather than autophosphorylation sites and that constitutive phosphorylation accompanies transformation.\",\n      \"evidence\": \"EGFR deletion/point-mutant binding and kinase assays, transformation assays, co-IP with Shc\",\n      \"pmids\": [\"7532293\", \"7608194\", \"7791787\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab biochemistry\", \"Structural basis of juxtamembrane binding not resolved\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Identified the SH3-domain interactome and oligomerization mode, revealing EPS8 binds Abi-1/E3B1 and RN-tre and self-associates through an intertwined SH3 dimer.\",\n      \"evidence\": \"X-ray crystallography, SH3 library screen, co-IP, quantitative in vitro binding\",\n      \"pmids\": [\"9303002\", \"9010225\", \"8700527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of dimerization not established\", \"Hierarchy among competing SH3 ligands unknown\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Resolved how EPS8 links Ras to actin remodeling: the EPS8-Abi1-Sos1 tri-complex carries intrinsic Rac-specific GEF activity.\",\n      \"evidence\": \"In vivo co-IP, reconstituted in vitro Rac-GEF assay, genetic epistasis\",\n      \"pmids\": [\"10499589\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How GEF activity is switched on left unexplained\", \"Spatial regulation of the complex unaddressed\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed EPS8 is a node partitioned between endocytic and signaling functions, with RN-tre binding diverting it from the GEF complex and inhibiting EGFR internalization.\",\n      \"evidence\": \"Co-IP, Rab5 GAP assay, EGFR endocytosis assay, Rac activation assay\",\n      \"pmids\": [\"11099046\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative balance between pools not defined\", \"Triggers for switching not mapped\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the activation switch for the GEF complex and established family redundancy: PI3K/PIP3 unmasks Rac-GEF activity, and EPS8L1/L2 can substitute for EPS8.\",\n      \"evidence\": \"Co-IP, in vitro GEF assays with PI3K inhibitors and p85 knockout, rescue in eps8-/- fibroblasts\",\n      \"pmids\": [\"12515821\", \"14565974\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Distinct in vivo roles of paralogs not fully separated\", \"Lipid-binding determinants on the complex unmapped\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified the core actin biochemistry of EPS8: its C-terminal effector domain caps barbed ends with nanomolar affinity, is autoinhibited and de-repressed by Abi1, with capping required in vivo for actin-based motility and morphogenesis.\",\n      \"evidence\": \"Reconstituted in vitro capping with domain mapping, loss-of-function in cells, C. elegans isoform genetic rescue\",\n      \"pmids\": [\"15558031\", \"15558032\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism of autoinhibition not solved\", \"Regulation of capping by other partners not yet known\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Revealed the second actin activity—bundling—and its integration with Cdc42 signaling: EPS8 bundles filaments and synergizes with IRSp53 to drive Cdc42-dependent filopodia.\",\n      \"evidence\": \"In vitro bundling assay, co-IP, siRNA loss-of-function, live imaging; palladin and IRSp53 binding mapping\",\n      \"pmids\": [\"17115031\", \"15289329\", \"17115031\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Switch between capping and bundling not yet defined\", \"Domain basis of bundling versus capping not separated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended EPS8 into neuronal function, placing it in postsynaptic NMDA receptor complexes governing actin remodeling and behavior.\",\n      \"evidence\": \"Co-IP of NMDA complex, electrophysiology in Eps8 KO neurons, behavioral assays\",\n      \"pmids\": [\"17018287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding partner within the complex unresolved\", \"Molecular link to channel function indirect\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified phospho-regulation of the capping/bundling balance and tissue-specific actin junction roles: MAPK phosphorylation at S624/T628 inhibits capping to control axonal filopodia, and EPS8 maintains seminiferous-epithelium junctions.\",\n      \"evidence\": \"Phospho-mutant capping assays, hippocampal neuron imaging, RNAi with junction-protein readouts in vitro and in vivo\",\n      \"pmids\": [\"19564905\", \"19293393\", \"19528316\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream kinase identity in each context not fully resolved\", \"Coordination of the two phospho-sites unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established EPS8 as an essential stereocilia component, building the Myo15a-whirlin-EPS8 tip complex required for stereocilia elongation, hair-cell maturation, and hearing.\",\n      \"evidence\": \"Co-IP, knockout mouse analysis, overexpression rescue, electrophysiology, hearing thresholds; EPS8 roles in dendritic cell migration\",\n      \"pmids\": [\"21236676\", \"21526224\", \"21835647\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and assembly order of the tip complex not fully defined\", \"How tip targeting couples to capping versus bundling not resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined cell-cycle control of EPS8 levels and its role in spine plasticity: SCF(Fbxw5) degrades EPS8 in G2 to enable mitotic rounding, and EPS8 capping is required for LTP-driven spine enlargement and cognition.\",\n      \"evidence\": \"E3 ligase identification, proteasome inhibition, cell-cycle synchronization, live imaging; KO mouse LTP, spine morphometry, behavior\",\n      \"pmids\": [\"23314863\", \"23685357\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degron recognition motif for Fbxw5 not mapped\", \"Link between human autism and EPS8 correlative only\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected EPS8 actin activities to mechanics and adhesion signaling: Erk phosphorylation tips EPS8 toward bundling to drive leader-bleb migration, and EPS8 links VE-cadherin junctions to YAP/TAZ signaling and vascular permeability.\",\n      \"evidence\": \"Live imaging with Erk biosensor, cortex-tension measurements, capping/bundling mutants; reciprocal co-IP, KO mouse with permeability and YAP reporter assays\",\n      \"pmids\": [\"26163656\", \"26668327\", \"26252776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism coupling EPS8 to YAP nuclear translocation incompletely defined\", \"How junction state controls EPS8 displacement not fully resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Expanded the kinase regulation of EPS8 to additional RTK pathways, showing PTK6 phosphorylates EPS8 to enhance proliferation, migration, and ERK/FAK activation.\",\n      \"evidence\": \"Phosphoproteomics, 3YF mutagenesis, kinase assay, PTK6 knockdown; FAK/Src complex and clathrin/AP-2 interactome studies\",\n      \"pmids\": [\"28214294\", \"25359883\", \"23626693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional integration of many phospho-sites unresolved\", \"In vivo relevance of PTK6 phosphorylation untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined how EPS8 is targeted to specialized actin protrusions: I-BAR proteins IRTKS/IRSp53 recruit EPS8 via SH3 interactions to drive microvillus and nanotube elongation through linear actin polymerization.\",\n      \"evidence\": \"Super-resolution microscopy, SH3-dependent co-IP, depletion assays; optical tweezers and Arp2/3 inhibition for TNTs\",\n      \"pmids\": [\"30197089\", \"38009333\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How recruitment selects capping versus bundling output unclear\", \"Direct linear-polymerase activity of EPS8 not biochemically isolated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed that the stereocilia tip complex functions through liquid-liquid phase separation, with multivalent Whirlin-Myo15-EPS8 condensates promoting actin bundling disrupted by a deafness variant.\",\n      \"evidence\": \"In vitro phase-separation reconstitution, actin bundling assay, deafness-variant mutagenesis\",\n      \"pmids\": [\"33626355\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo confirmation of condensate behavior limited\", \"Contribution of EPS8 multivalency versus partners not separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established EPS8 as a host target hijacked by pathogens, with influenza virion association licensing RNP nuclear import and bacterial effectors targeting EPS8 to remodel tight junctions and disable its actin activities.\",\n      \"evidence\": \"Genetic knockout/overexpression and infection assays (influenza); CRISPR KO with organoid and mouse infection (Chlamydia); crystal structure and bundling assay with EPEC NleH kinases\",\n      \"pmids\": [\"31747592\", \"36395759\", \"35976880\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of EPS8 contribution to influenza RNP transport undefined\", \"Host signaling rewired by effector phosphorylation incompletely mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How EPS8's competing capping, bundling, GEF-scaffolding, and adaptor functions are selected and spatially partitioned within a single cell across its many tissue contexts remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model integrating autoinhibition, phospho-switches, and partner binding\", \"Quantitative rules governing partition between signaling and actin pools unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [13, 14, 16, 43]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 8, 15, 25]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [8, 11, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7, 13, 16]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [7, 25, 26, 37]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [24, 31]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 8, 11]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [32]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [18, 33]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [27, 31]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [26, 40]}\n    ],\n    \"complexes\": [\n      \"EPS8-Abi1-Sos1 Rac-GEF tri-complex\",\n      \"Myo15a-whirlin-EPS8 stereocilia tip complex\",\n      \"NMDA receptor postsynaptic complex\",\n      \"TLR4-MyD88 complex\"\n    ],\n    \"partners\": [\n      \"ABI1\",\n      \"SOS1\",\n      \"IRSp53\",\n      \"RN-tre\",\n      \"MYO15A\",\n      \"WHRN\",\n      \"VE-cadherin\",\n      \"EGFR\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}