{"gene":"EPHB6","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2001,"finding":"EphB6 is catalytically inactive due to alterations in conserved kinase domain residues, yet undergoes inducible tyrosine phosphorylation upon ephrin-B1 stimulation via transphosphorylation by the active kinase EphB1; EphB1 and EphB6 form a stable heterocomplex, and this transphosphorylation requires EphB1 catalytic activity and is ligand-dependent.","method":"Co-immunoprecipitation, overexpression of kinase-active/inactive EphB1 mutants, phosphotyrosine immunoblotting in transfected cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, kinase-dead mutant controls, ligand-dependence confirmed; moderate evidence from single lab with multiple orthogonal methods","pmids":["11713248"],"is_preprint":false},{"year":2001,"finding":"EphB6 constitutively associates with the proto-oncogene c-Cbl, requiring a functional phosphotyrosine-binding domain on Cbl but not the RING finger ubiquitin-transfer domain.","method":"Co-immunoprecipitation, domain-deletion mutants of c-Cbl","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with domain mutant controls; single lab","pmids":["11713248"],"is_preprint":false},{"year":2001,"finding":"Cross-linking of EphB6 together with CD3 in Jurkat T cells triggers general protein tyrosine phosphorylation, alters lymphokine secretion, inhibits proliferation, and induces Fas-mediated apoptosis; EphB6 associates with Cbl, and cross-linking causes Cbl dephosphorylation and dissociation from SHP-1.","method":"Antibody cross-linking, phosphotyrosine immunoblotting, Co-IP, apoptosis assays in Jurkat cells","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — functional readouts with Co-IP; single lab, multiple methods","pmids":["11466354"],"is_preprint":false},{"year":2003,"finding":"EphB6 overexpression in Jurkat T cells and stimulation with ephrin-B1 selectively inhibits TCR-mediated JNK activation but not the MAPK/ERK pathway; this occurs by preventing TCR-induced activation of the small GTPase Rac1, which is required to initiate the JNK cascade. EphB6 also blocks anti-CD3-induced IL-2 secretion and CD25 expression in a ligand-dependent manner, and inhibits TCR-mediated apoptosis in murine thymocytes.","method":"Overexpression and dominant-negative EphB6 in Jurkat cells, Rac1 activation assay, JNK kinase assay, cytokine ELISA, thymocyte apoptosis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — dominant-negative rescue experiment, Rac1 activation assay, multiple cellular readouts; moderate evidence from single lab","pmids":["12517763"],"is_preprint":false},{"year":2004,"finding":"EphB6-deficient mice show compromised in vitro and in vivo T cell responses (lymphokine secretion, proliferation, delayed-type hypersensitivity, experimental autoimmune encephalitis) while humoral responses remain normal; mechanistically, loss of EphB6 reduces ZAP-70 activation, LAT phosphorylation, PLCγ1-SLP-76 association, and p44/42 MAPK activation downstream of the TCR. EphB6 migrates to aggregated TCRs and lipid rafts after TCR activation.","method":"EphB6 knockout mice, T cell proliferation and cytokine assays, EAE model, TCR raft fractionation, phosphoflow/immunoblot for ZAP-70, LAT, PLCγ1, MAPK","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — KO mice with defined signaling phenotype plus localization to TCR rafts; strong evidence with multiple orthogonal methods","pmids":["15599401"],"is_preprint":false},{"year":2000,"finding":"Ephrin-B2 (but not ephrin-B1) is a high-affinity saturable ligand for EphB6; EphB6 binds transmembrane ephrin ligands (not GPI-anchored), and ephrin-B2 is expressed in human and mouse thymus where EphB6 is the dominant ephrin-B2-binding receptor on adult thymocytes.","method":"Soluble EphB6-Fc fusion protein binding to transfected COS cells, phosphatidylinositol-specific phospholipase C treatment, binding competition, RT-PCR, EphB6 KO thymocyte binding assay","journal":"FEBS letters / Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 — direct binding assay with saturation kinetics, confirmed with KO thymocytes; replicated across two papers","pmids":["10648835","12379224"],"is_preprint":false},{"year":2005,"finding":"EphB6 exerts biphasic effects on cell adhesion and migration: low concentrations of ephrin-B2 promote adhesion and migration, while high concentrations induce repulsion and inhibit migration. The cytoplasmic domain is required for the negative (repulsive) effects. EphB6 is constitutively associated with the Src-family kinase Fyn, and high ephrin-B2 concentrations induce tyrosine phosphorylation of EphB6 through Src-family kinase activity, acting as a molecular switch.","method":"Cell adhesion and migration assays with ephrin-B2 dose-response, cytoplasmic-domain truncation mutant, Co-IP with Fyn, Src kinase inhibitors, phosphotyrosine immunoblotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — truncation mutant phenotype, Co-IP of Fyn, dose-response functional assays; multiple orthogonal methods in single study","pmids":["15955811"],"is_preprint":false},{"year":2010,"finding":"EphB6 restoration in invasive breast cancer cells promotes actin-dependent cell spreading and attachment and blocks invasiveness; EphB6 stimulation induces its tyrosine phosphorylation mediated by the EphB4 receptor (transphosphorylation), accompanied by EphB6-c-Cbl interaction and c-Cbl partner Abl kinase phosphorylation. Cbl silencing suppresses Abl phosphorylation, cell adhesion, and the ability of EphB6 to inhibit invasiveness.","method":"EphB6 re-expression in MDA-MB-231 cells, siRNA knockdown of Cbl, invasion assays, Co-IP, phosphotyrosine immunoblotting, actin staining","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — siRNA rescue experiment, Co-IP, multiple functional assays; moderate evidence from single lab with orthogonal methods","pmids":["20086179"],"is_preprint":false},{"year":2010,"finding":"EPHB6 expression is silenced by promoter DNA hypermethylation in non-small cell lung cancer; restoration of EPHB6 expression increases cell adhesion, decreases migration, and almost entirely abolishes metastasis formation in NOD/SCID mice. 5-aza-2'-deoxycytidine treatment re-induces EPHB6 expression.","method":"Methylation-specific PCR, bisulfite sequencing, 5-azadC demethylation, EPHB6 re-expression, in vitro adhesion/migration assays, in vivo NOD/SCID metastasis model","journal":"Clinical cancer research","confidence":"High","confidence_rationale":"Tier 2 — epigenetic mechanism with functional rescue in vivo; multiple methods in single study","pmids":["20371680"],"is_preprint":false},{"year":2009,"finding":"EphB6 loss correlates with increased invasiveness in breast cancer cell lines; EphB6 re-expression in MDA-MB-231, MDA-MB-435, and BT549 cells decreases in vitro invasiveness, mediated by decreased MMP7 and MMP19 transcript levels and increased TIMP2 expression.","method":"EphB6 expression construct transfection, in vitro invasion assay, RT-PCR for MMP7, MMP19, TIMP2","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — loss-of-function/gain-of-function with defined molecular mechanism; single lab","pmids":["19234485"],"is_preprint":false},{"year":2012,"finding":"EphB6 and its ephrin-B ligands are expressed on vascular smooth muscle cells (VSMC); reverse signaling through ephrin-B ligands (not forward signaling through EphB6) reduces VSMC contraction via GRIP1 adaptor protein. Loss of EphB6 in castrated male mice increases small artery contractility, RhoA activation, and constitutive myosin light chain phosphorylation, leading to elevated blood pressure.","method":"EphB6 KO mice, ex vivo arterial contractility assay, RhoA activation assay, myosin light chain phosphorylation, in vitro VSMC cross-linking experiments, 24-h urine catecholamine measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — KO mouse phenotype with mechanistic dissection of forward vs reverse signaling and GRIP1 adaptor; multiple orthogonal methods","pmids":["22223652"],"is_preprint":false},{"year":2018,"finding":"EPHB6 interacts with the GRB2 adapter protein and activates the RAS-ERK pathway to elevate OCT4 expression, thereby promoting expansion of tumor-initiating cells (TICs) in triple-negative breast cancer. Suppression of ERK or OCT4 blocks EPHB6-induced proliferation. EPHB6 also partially suppresses epithelial-mesenchymal transition and sensitizes TICs to DNA-damaging chemotherapy.","method":"Co-IP of EPHB6-GRB2, ERK inhibition, OCT4 knockdown, xenograft tumor models, flow cytometry for TIC markers","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, pharmacological inhibition, in vivo xenograft; multiple orthogonal methods in single study","pmids":["29700392"],"is_preprint":false},{"year":2016,"finding":"EPHB6 induces fragmentation of the mitochondrial network in triple-negative breast cancer cells via activation of the ERK-DRP1 pathway, increasing organelle fission frequency and rendering cells more susceptible to DR5-mediated apoptosis; DRP1 activity is essential to both the mitochondrial fragmentation and the pro-apoptotic response.","method":"EPHB6 overexpression, ERK inhibition, DRP1 dominant-negative/siRNA, mitochondrial morphology imaging, DR5 agonist apoptosis assay, DRP1 phosphorylation immunoblot","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 2 — genetic and pharmacological inhibition of pathway components with functional rescue; multiple orthogonal methods","pmids":["27788485"],"is_preprint":false},{"year":2020,"finding":"EphB6 deletion in mice induces autism-like behavior and disturbs gut microbiota; the disturbed microbiota leads to vitamin B6 and dopamine deficits. Gut microbiota transplant from EphB6-deficient mice transfers autism-like behavior to antibiotic-treated wild-type mice, and wild-type microbiota transplant rescues behavior in EphB6-KO mice. At the cellular level, gut microbiota-mediated vitamin B6 deficiency regulates the excitation/inhibition balance in the medial prefrontal cortex.","method":"EphB6 KO mice, fecal microbiota transplantation, behavioral assays, metabolomics (vitamin B6, dopamine), electrophysiology for E/I balance","journal":"Microbiome","confidence":"High","confidence_rationale":"Tier 2 — bidirectional microbiota transplant with mechanistic metabolic and electrophysiological readouts in KO model","pmids":["32819434"],"is_preprint":false},{"year":2020,"finding":"CLDN1 upregulates EPHB6 and enhances its activation, resulting in suppression of ERK1/2 signaling via a CLDN1-EPHB6-ERK1/2-SLUG feedback axis that represses metastasis, drug resistance, and cancer stemness in lung adenocarcinoma.","method":"Immunoprecipitation, immunoblotting for ERK1/2 and SLUG, migration assay, sphere assay, aldefluor assay, xenograft experiments, methylation-specific PCR","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and multiple functional assays; single lab, EPHB6 placed downstream of CLDN1 and upstream of ERK","pmids":["32754286"],"is_preprint":false},{"year":2019,"finding":"EPHB6 mutation promotes paclitaxel resistance through interaction with EPHA2, which activates JNK-mediated cadherin 11 (CDH11) expression and RhoA/FAK-dependent cell adhesion-mediated drug resistance (CAM-DR); targeted inhibition of EPHA2 or CDH11 reverses the acquired resistance.","method":"EPHB6 mutation-expression constructs, JNK inhibition, EPHA2/CDH11 siRNA, RhoA/FAK phosphorylation immunoblot, drug resistance assays","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — pathway dissection with genetic knockdown and pharmacological inhibition; single lab","pmids":["31160603"],"is_preprint":false},{"year":2017,"finding":"Enzalutamide-mediated androgen receptor (AR) signaling decreases EPHB6 expression by binding to an androgen-response element (ARE) on the EPHB6 promoter, leading to JNK/MMP9 pathway activation and increased CRPC cell invasion; JNK inhibition reverses this enhanced invasion.","method":"ChIP for AR on EPHB6 promoter ARE, luciferase reporter assay, siRNA, invasion assays, JNK phosphorylation immunoblot","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP and reporter assay establish direct transcriptional mechanism; single lab","pmids":["28826721"],"is_preprint":false},{"year":2023,"finding":"EPHB6 interacts with EGFR (identified by BioID proximity proteomics and confirmed by co-immunoprecipitation) and modulates EGFR signaling to enhance cancer cell proliferation and tumor development.","method":"BioID proximity proteomics, Co-IP, genetic interaction screening, xenograft tumor assays","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — BioID plus Co-IP plus functional in vivo assay; single lab","pmids":["37392382"],"is_preprint":false},{"year":2021,"finding":"EphB6 promotes survival of disseminated dormant breast cancer cells in the lung by modulating TFEB transcriptional activity and lysosome formation; direct coculture with alveolar type I-like lung epithelial cells and in vivo lung dissemination drive lysosomal accumulation and EphB6 induction; EphB6 also promotes proliferation of surrounding lung parenchymal cells.","method":"Lung organotypic system, in vivo dissemination assay, TFEB reporter assay, lysosomal staining, EphB6 knockdown","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo dissemination model plus in vitro mechanistic assays; single lab","pmids":["33802447"],"is_preprint":false},{"year":2000,"finding":"Transfection of EPHB6 cDNA into neuroblastoma cell lines (IMR5 and SY5Y) that express little endogenous EPHB6 inhibits clonogenicity in culture and suppresses tumorigenicity in mouse xenograft models, establishing EPHB6 as a functional suppressor of malignant neuroblastoma phenotype.","method":"EPHB6 cDNA transfection, colony formation assay, mouse xenograft tumor model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 — gain-of-function with in vitro and in vivo readouts; single lab","pmids":["10984508"],"is_preprint":false},{"year":2000,"finding":"EphB6 is expressed specifically on T-cell lineage hematopoietic cells (CD2+CD7+CD3+CD4+ or CD8+ lymphocytes) and is prominently expressed in CD4+CD8+ double-positive thymocytes, with expression level decreasing with T-cell maturation, as determined by monoclonal antibody staining and flow cytometry.","method":"Monoclonal antibody generation, flow cytometry, immunophenotyping of human peripheral blood, bone marrow, and thymocytes","journal":"Growth factors","confidence":"Medium","confidence_rationale":"Tier 2 — direct protein-level localization by flow cytometry with lineage markers; single lab","pmids":["10831073"],"is_preprint":false}],"current_model":"EPHB6 is an intrinsically kinase-inactive Eph receptor tyrosine kinase that signals through transphosphorylation by active EphB-family partners (EphB1, EphB4), constitutively associates with c-Cbl and the Src-family kinase Fyn, and activates downstream pathways—including RAS-ERK, Abl, and Rac1/JNK modulation—to regulate T cell activation, cell adhesion and migration (biphasically, depending on ephrin-B2 concentration), vascular smooth muscle contractility via ephrin-B reverse signaling through GRIP1, mitochondrial dynamics via ERK-DRP1, and tumor suppression in multiple cancer contexts, while its promoter is frequently silenced by DNA hypermethylation in cancers."},"narrative":{"teleology":[{"year":2000,"claim":"Identifying EPHB6 as a T-lineage receptor with tumor-suppressive activity established its initial functional identity, addressing what cell types express it and whether it has growth-regulatory consequences.","evidence":"Flow cytometric immunophenotyping of human thymocytes/T cells; EPHB6 cDNA re-expression in neuroblastoma lines with colony formation and xenograft assays","pmids":["10831073","10984508","10648835"],"confidence":"Medium","gaps":["Mechanism of tumor suppression not identified","Only two neuroblastoma lines tested","No downstream signaling pathway defined"]},{"year":2001,"claim":"Demonstrating that EphB6 is catalytically dead yet becomes tyrosine-phosphorylated via EphB1 transphosphorylation in a ligand-dependent heterocomplex resolved how a kinase-dead receptor could initiate signaling, establishing the transphosphorylation paradigm central to all subsequent EPHB6 biology.","evidence":"Co-IP of EphB1–EphB6, kinase-dead EphB1 mutant controls, phosphotyrosine immunoblotting with ephrin-B1 stimulation; c-Cbl domain-deletion mutant Co-IPs","pmids":["11713248","11466354"],"confidence":"High","gaps":["Structural basis of the EphB1–EphB6 heterocomplex unknown","Whether EphB6 can be transphosphorylated by other Eph kinases (beyond EphB4, shown later) not tested"]},{"year":2003,"claim":"Defining EPHB6's selective inhibition of Rac1–JNK but not ERK downstream of the TCR revealed a pathway-specific modulatory role rather than a global signal amplifier, answering how EphB6 shapes T cell fate decisions.","evidence":"Overexpression and dominant-negative EphB6 in Jurkat cells; Rac1 activation pull-down, JNK kinase assay, IL-2 ELISA, thymocyte apoptosis assay","pmids":["12517763"],"confidence":"High","gaps":["Direct molecular link between EphB6 and Rac1 GEF/GAP not identified","Dominant-negative approach may have off-target effects on other Eph receptors"]},{"year":2004,"claim":"The EphB6-knockout mouse demonstrated that EphB6 is non-redundantly required for T cell responses in vivo, revealing its role in proximal TCR signaling (ZAP-70, LAT, MAPK) and its migration to lipid rafts upon TCR engagement.","evidence":"EphB6 KO mice; T cell proliferation/cytokine assays, EAE model, raft fractionation, phospho-immunoblot for ZAP-70/LAT/MAPK","pmids":["15599401"],"confidence":"High","gaps":["How a kinase-dead receptor facilitates ZAP-70 activation mechanistically is unclear","Contribution of reverse signaling through ephrin-B ligands on APCs not dissected"]},{"year":2005,"claim":"A biphasic dose–response to ephrin-B2 uncovered EPHB6 as a molecular switch: constitutive Fyn association supports pro-adhesive signaling at low ligand, while high-ligand Src-family-kinase-mediated phosphorylation triggers repulsion, resolving the paradox of EphB6 promoting both adhesion and repulsion.","evidence":"Dose-response adhesion/migration assays, cytoplasmic-domain truncation, Co-IP of Fyn, Src inhibitor treatment","pmids":["15955811"],"confidence":"High","gaps":["Downstream effectors mediating the attractive versus repulsive arms not fully identified","Whether Fyn directly phosphorylates EphB6 or acts indirectly not resolved"]},{"year":2009,"claim":"Linking EphB6 re-expression to reduced MMP7/MMP19 and increased TIMP2 provided a molecular mechanism for its anti-invasive activity in breast cancer, extending the tumor-suppressor paradigm beyond neuroblastoma.","evidence":"EphB6 expression construct in MDA-MB-231/MDA-MB-435/BT549; invasion assay, RT-PCR for MMPs and TIMP2","pmids":["19234485"],"confidence":"Medium","gaps":["Transcriptional mechanism connecting EphB6 to MMP/TIMP expression not defined","No in vivo validation of MMP changes"]},{"year":2010,"claim":"Two discoveries solidified EphB6's biological scope: (1) EphB4 transphosphorylates EphB6 to activate c-Cbl–Abl signaling that suppresses breast cancer invasion, and (2) EphB6 loss in vascular smooth muscle increases RhoA-mediated contractility via disrupted ephrin-B reverse signaling through GRIP1, causing hypertension in castrated male mice.","evidence":"EphB6 re-expression in MDA-MB-231, Cbl siRNA, Co-IP, invasion assays; EphB6 KO mice, arterial contractility, RhoA assay, MLC phosphorylation","pmids":["20086179","20371680","22223652"],"confidence":"High","gaps":["Identity of the Abl substrate that mediates adhesion not determined","Sex-hormone dependence of the vascular phenotype not mechanistically explained","How GRIP1 suppresses RhoA not fully delineated"]},{"year":2016,"claim":"Establishing that EPHB6 fragments mitochondria via ERK–DRP1 and thereby sensitizes cancer cells to DR5-mediated apoptosis revealed a novel organelle-level effector mechanism for a kinase-dead receptor.","evidence":"EPHB6 overexpression, ERK inhibition, DRP1 dominant-negative/siRNA, mitochondrial imaging, DR5 agonist apoptosis assay","pmids":["27788485"],"confidence":"High","gaps":["Whether mitochondrial fragmentation is relevant in non-cancer cell types unknown","Link between EPHB6 and DRP1 phosphorylation may involve intermediate kinases not mapped"]},{"year":2018,"claim":"The identification of a GRB2–RAS–ERK–OCT4 axis downstream of EPHB6 explained how the same receptor can promote tumor-initiating cell expansion in triple-negative breast cancer while simultaneously suppressing EMT, clarifying its context-dependent oncogenic versus suppressive roles.","evidence":"Co-IP of EPHB6–GRB2, ERK inhibition, OCT4 knockdown, xenograft models, flow cytometry for TIC markers","pmids":["29700392"],"confidence":"High","gaps":["Mechanism by which EPHB6 simultaneously activates ERK (pro-TIC) yet suppresses EMT is not resolved","Whether GRB2 interaction is direct or via transphosphorylation intermediates unclear"]},{"year":2019,"claim":"Demonstrating that EPHB6 mutations activate EPHA2–JNK–CDH11 and RhoA/FAK to drive paclitaxel resistance identified a gain-of-function mechanism for mutant EPHB6, distinct from loss-of-expression silencing.","evidence":"EPHB6 mutation constructs, JNK inhibition, EPHA2/CDH11 siRNA, drug resistance assays","pmids":["31160603"],"confidence":"Medium","gaps":["Specific mutations tested represent a limited panel; structural basis for EPHA2 interaction not defined","Single study without independent replication"]},{"year":2020,"claim":"Two 2020 studies expanded EPHB6 function to gut–brain and lung cancer axes: EphB6 KO mice develop autism-like behavior via gut-microbiota-mediated vitamin B6/dopamine deficits, and CLDN1 upregulates EPHB6 to suppress ERK1/2–SLUG–driven lung cancer stemness and metastasis.","evidence":"Bidirectional fecal microbiota transplant, metabolomics, electrophysiology in KO mice; CLDN1–EPHB6 Co-IP, xenograft, methylation-specific PCR in lung adenocarcinoma","pmids":["32819434","32754286"],"confidence":"High","gaps":["Mechanism linking EphB6 loss to gut microbiota dysbiosis is unknown","Whether the CLDN1–EPHB6 interaction is direct remains uncertain"]},{"year":2023,"claim":"Proximity proteomics identified EGFR as an EPHB6 interactor that functionally cooperates to enhance cancer cell proliferation, expanding the repertoire of receptor tyrosine kinases that EPHB6 cross-talks with beyond the Eph family.","evidence":"BioID proximity proteomics, Co-IP of EPHB6–EGFR, xenograft tumor assays","pmids":["37392382"],"confidence":"Medium","gaps":["Nature of the EPHB6–EGFR interaction (direct or scaffolded) not resolved","Whether EGFR transphosphorylates EPHB6 (like EphB1/B4) not tested","Single study without independent confirmation"]},{"year":null,"claim":"Key unresolved questions include the structural basis of the EphB6 heterocomplex with active Eph kinases, how a kinase-dead receptor is recruited to and activates TCR-proximal signaling, the mechanism connecting EphB6 loss to gut microbiota remodeling, and the rules governing EPHB6's context-dependent pro-tumorigenic versus tumor-suppressive activity.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal structure or cryo-EM structure of EphB6 or its heterocomplexes","No reconstituted signaling system with purified components","Context-dependent ERK activation versus suppression not mechanistically reconciled"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,6,7,11]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,4,14]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,6,20]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,3,4,20]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[7,8,9,19]}],"complexes":[],"partners":["EPHB1","EPHB4","CBL","FYN","GRB2","EPHA2","EGFR","EFNB2"],"other_free_text":[]},"mechanistic_narrative":"EPHB6 is a catalytically inactive Eph receptor tyrosine kinase that functions as a signaling scaffold in T cell activation, cell adhesion and migration, vascular tone regulation, and tumor suppression. Although devoid of intrinsic kinase activity, EPHB6 undergoes ligand-dependent tyrosine phosphorylation through transphosphorylation by active family members EphB1 and EphB4, and constitutively associates with the adaptor c-Cbl and the Src-family kinase Fyn, enabling downstream engagement of GRB2–RAS–ERK, Abl, and Rac1/JNK pathways [PMID:11713248, PMID:15955811, PMID:20086179, PMID:29700392]. In T cells, EPHB6 localizes to TCR–lipid-raft aggregates and is required for ZAP-70, LAT, and MAPK activation; EphB6-knockout mice exhibit impaired T cell responses and, separately, autism-like behavior linked to gut-microbiota-mediated vitamin B6 and dopamine deficits [PMID:15599401, PMID:32819434]. EPHB6 acts as a tumor and metastasis suppressor in neuroblastoma, breast cancer, and lung cancer—where its promoter is frequently silenced by DNA hypermethylation—by promoting cell adhesion, restricting MMP expression, modulating mitochondrial fission via ERK–DRP1, and sensitizing cells to apoptosis [PMID:10984508, PMID:20371680, PMID:19234485, PMID:27788485]."},"prefetch_data":{"uniprot":{"accession":"O15197","full_name":"Ephrin type-B receptor 6","aliases":["HEP","Tyrosine-protein kinase-defective receptor EPH-6"],"length_aa":1021,"mass_kda":110.7,"function":"Kinase-defective receptor for members of the ephrin-B family. Binds to ephrin-B1 and ephrin-B2. Modulates cell adhesion and migration by exerting both positive and negative effects upon stimulation with ephrin-B2. Inhibits JNK activation, T-cell receptor-induced IL-2 secretion and CD25 expression upon stimulation with ephrin-B2","subcellular_location":"Secreted","url":"https://www.uniprot.org/uniprotkb/O15197/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/EPHB6","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/EPHB6","total_profiled":1310},"omim":[{"mim_id":"602757","title":"EPHRIN RECEPTOR EphB6; EPHB6","url":"https://www.omim.org/entry/602757"},{"mim_id":"300035","title":"EPHRIN B1; EFNB1","url":"https://www.omim.org/entry/300035"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nuclear speckles","reliability":"Uncertain"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":69.8},{"tissue":"skin 1","ntpm":79.7}],"url":"https://www.proteinatlas.org/search/EPHB6"},"hgnc":{"alias_symbol":["HEP"],"prev_symbol":[]},"alphafold":{"accession":"O15197","domains":[{"cath_id":"2.60.120.260","chopping":"33-166_195-232","consensus_level":"high","plddt":85.1317,"start":33,"end":232},{"cath_id":"2.60.40.1770","chopping":"234-272_280-302","consensus_level":"medium","plddt":81.835,"start":234,"end":302},{"cath_id":"2.60.40.10","chopping":"374-414_424-484","consensus_level":"medium","plddt":87.8439,"start":374,"end":484},{"cath_id":"2.60.40.10","chopping":"496-579","consensus_level":"high","plddt":89.8535,"start":496,"end":579},{"cath_id":"3.30.200.20","chopping":"649-751","consensus_level":"high","plddt":86.7389,"start":649,"end":751},{"cath_id":"1.10.510.10","chopping":"752-929","consensus_level":"medium","plddt":88.1249,"start":752,"end":929},{"cath_id":"1.10.150.50","chopping":"944-1021","consensus_level":"medium","plddt":77.4015,"start":944,"end":1021}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O15197","model_url":"https://alphafold.ebi.ac.uk/files/AF-O15197-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O15197-F1-predicted_aligned_error_v6.png","plddt_mean":80.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=EPHB6","jax_strain_url":"https://www.jax.org/strain/search?query=EPHB6"},"sequence":{"accession":"O15197","fasta_url":"https://rest.uniprot.org/uniprotkb/O15197.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O15197/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O15197"}},"corpus_meta":[{"pmid":"2842359","id":"PMC_2842359","title":"Inhibition 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by the active kinase EphB1; EphB1 and EphB6 form a stable heterocomplex, and this transphosphorylation requires EphB1 catalytic activity and is ligand-dependent.\",\n      \"method\": \"Co-immunoprecipitation, overexpression of kinase-active/inactive EphB1 mutants, phosphotyrosine immunoblotting in transfected cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, kinase-dead mutant controls, ligand-dependence confirmed; moderate evidence from single lab with multiple orthogonal methods\",\n      \"pmids\": [\"11713248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"EphB6 constitutively associates with the proto-oncogene c-Cbl, requiring a functional phosphotyrosine-binding domain on Cbl but not the RING finger ubiquitin-transfer domain.\",\n      \"method\": \"Co-immunoprecipitation, domain-deletion mutants of c-Cbl\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with domain mutant controls; single lab\",\n      \"pmids\": [\"11713248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cross-linking of EphB6 together with CD3 in Jurkat T cells triggers general protein tyrosine phosphorylation, alters lymphokine secretion, inhibits proliferation, and induces Fas-mediated apoptosis; EphB6 associates with Cbl, and cross-linking causes Cbl dephosphorylation and dissociation from SHP-1.\",\n      \"method\": \"Antibody cross-linking, phosphotyrosine immunoblotting, Co-IP, apoptosis assays in Jurkat cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional readouts with Co-IP; single lab, multiple methods\",\n      \"pmids\": [\"11466354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EphB6 overexpression in Jurkat T cells and stimulation with ephrin-B1 selectively inhibits TCR-mediated JNK activation but not the MAPK/ERK pathway; this occurs by preventing TCR-induced activation of the small GTPase Rac1, which is required to initiate the JNK cascade. EphB6 also blocks anti-CD3-induced IL-2 secretion and CD25 expression in a ligand-dependent manner, and inhibits TCR-mediated apoptosis in murine thymocytes.\",\n      \"method\": \"Overexpression and dominant-negative EphB6 in Jurkat cells, Rac1 activation assay, JNK kinase assay, cytokine ELISA, thymocyte apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — dominant-negative rescue experiment, Rac1 activation assay, multiple cellular readouts; moderate evidence from single lab\",\n      \"pmids\": [\"12517763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"EphB6-deficient mice show compromised in vitro and in vivo T cell responses (lymphokine secretion, proliferation, delayed-type hypersensitivity, experimental autoimmune encephalitis) while humoral responses remain normal; mechanistically, loss of EphB6 reduces ZAP-70 activation, LAT phosphorylation, PLCγ1-SLP-76 association, and p44/42 MAPK activation downstream of the TCR. EphB6 migrates to aggregated TCRs and lipid rafts after TCR activation.\",\n      \"method\": \"EphB6 knockout mice, T cell proliferation and cytokine assays, EAE model, TCR raft fractionation, phosphoflow/immunoblot for ZAP-70, LAT, PLCγ1, MAPK\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mice with defined signaling phenotype plus localization to TCR rafts; strong evidence with multiple orthogonal methods\",\n      \"pmids\": [\"15599401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Ephrin-B2 (but not ephrin-B1) is a high-affinity saturable ligand for EphB6; EphB6 binds transmembrane ephrin ligands (not GPI-anchored), and ephrin-B2 is expressed in human and mouse thymus where EphB6 is the dominant ephrin-B2-binding receptor on adult thymocytes.\",\n      \"method\": \"Soluble EphB6-Fc fusion protein binding to transfected COS cells, phosphatidylinositol-specific phospholipase C treatment, binding competition, RT-PCR, EphB6 KO thymocyte binding assay\",\n      \"journal\": \"FEBS letters / Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct binding assay with saturation kinetics, confirmed with KO thymocytes; replicated across two papers\",\n      \"pmids\": [\"10648835\", \"12379224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"EphB6 exerts biphasic effects on cell adhesion and migration: low concentrations of ephrin-B2 promote adhesion and migration, while high concentrations induce repulsion and inhibit migration. The cytoplasmic domain is required for the negative (repulsive) effects. EphB6 is constitutively associated with the Src-family kinase Fyn, and high ephrin-B2 concentrations induce tyrosine phosphorylation of EphB6 through Src-family kinase activity, acting as a molecular switch.\",\n      \"method\": \"Cell adhesion and migration assays with ephrin-B2 dose-response, cytoplasmic-domain truncation mutant, Co-IP with Fyn, Src kinase inhibitors, phosphotyrosine immunoblotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — truncation mutant phenotype, Co-IP of Fyn, dose-response functional assays; multiple orthogonal methods in single study\",\n      \"pmids\": [\"15955811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EphB6 restoration in invasive breast cancer cells promotes actin-dependent cell spreading and attachment and blocks invasiveness; EphB6 stimulation induces its tyrosine phosphorylation mediated by the EphB4 receptor (transphosphorylation), accompanied by EphB6-c-Cbl interaction and c-Cbl partner Abl kinase phosphorylation. Cbl silencing suppresses Abl phosphorylation, cell adhesion, and the ability of EphB6 to inhibit invasiveness.\",\n      \"method\": \"EphB6 re-expression in MDA-MB-231 cells, siRNA knockdown of Cbl, invasion assays, Co-IP, phosphotyrosine immunoblotting, actin staining\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — siRNA rescue experiment, Co-IP, multiple functional assays; moderate evidence from single lab with orthogonal methods\",\n      \"pmids\": [\"20086179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"EPHB6 expression is silenced by promoter DNA hypermethylation in non-small cell lung cancer; restoration of EPHB6 expression increases cell adhesion, decreases migration, and almost entirely abolishes metastasis formation in NOD/SCID mice. 5-aza-2'-deoxycytidine treatment re-induces EPHB6 expression.\",\n      \"method\": \"Methylation-specific PCR, bisulfite sequencing, 5-azadC demethylation, EPHB6 re-expression, in vitro adhesion/migration assays, in vivo NOD/SCID metastasis model\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epigenetic mechanism with functional rescue in vivo; multiple methods in single study\",\n      \"pmids\": [\"20371680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"EphB6 loss correlates with increased invasiveness in breast cancer cell lines; EphB6 re-expression in MDA-MB-231, MDA-MB-435, and BT549 cells decreases in vitro invasiveness, mediated by decreased MMP7 and MMP19 transcript levels and increased TIMP2 expression.\",\n      \"method\": \"EphB6 expression construct transfection, in vitro invasion assay, RT-PCR for MMP7, MMP19, TIMP2\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function/gain-of-function with defined molecular mechanism; single lab\",\n      \"pmids\": [\"19234485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EphB6 and its ephrin-B ligands are expressed on vascular smooth muscle cells (VSMC); reverse signaling through ephrin-B ligands (not forward signaling through EphB6) reduces VSMC contraction via GRIP1 adaptor protein. Loss of EphB6 in castrated male mice increases small artery contractility, RhoA activation, and constitutive myosin light chain phosphorylation, leading to elevated blood pressure.\",\n      \"method\": \"EphB6 KO mice, ex vivo arterial contractility assay, RhoA activation assay, myosin light chain phosphorylation, in vitro VSMC cross-linking experiments, 24-h urine catecholamine measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse phenotype with mechanistic dissection of forward vs reverse signaling and GRIP1 adaptor; multiple orthogonal methods\",\n      \"pmids\": [\"22223652\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"EPHB6 interacts with the GRB2 adapter protein and activates the RAS-ERK pathway to elevate OCT4 expression, thereby promoting expansion of tumor-initiating cells (TICs) in triple-negative breast cancer. Suppression of ERK or OCT4 blocks EPHB6-induced proliferation. EPHB6 also partially suppresses epithelial-mesenchymal transition and sensitizes TICs to DNA-damaging chemotherapy.\",\n      \"method\": \"Co-IP of EPHB6-GRB2, ERK inhibition, OCT4 knockdown, xenograft tumor models, flow cytometry for TIC markers\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, pharmacological inhibition, in vivo xenograft; multiple orthogonal methods in single study\",\n      \"pmids\": [\"29700392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EPHB6 induces fragmentation of the mitochondrial network in triple-negative breast cancer cells via activation of the ERK-DRP1 pathway, increasing organelle fission frequency and rendering cells more susceptible to DR5-mediated apoptosis; DRP1 activity is essential to both the mitochondrial fragmentation and the pro-apoptotic response.\",\n      \"method\": \"EPHB6 overexpression, ERK inhibition, DRP1 dominant-negative/siRNA, mitochondrial morphology imaging, DR5 agonist apoptosis assay, DRP1 phosphorylation immunoblot\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological inhibition of pathway components with functional rescue; multiple orthogonal methods\",\n      \"pmids\": [\"27788485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"EphB6 deletion in mice induces autism-like behavior and disturbs gut microbiota; the disturbed microbiota leads to vitamin B6 and dopamine deficits. Gut microbiota transplant from EphB6-deficient mice transfers autism-like behavior to antibiotic-treated wild-type mice, and wild-type microbiota transplant rescues behavior in EphB6-KO mice. At the cellular level, gut microbiota-mediated vitamin B6 deficiency regulates the excitation/inhibition balance in the medial prefrontal cortex.\",\n      \"method\": \"EphB6 KO mice, fecal microbiota transplantation, behavioral assays, metabolomics (vitamin B6, dopamine), electrophysiology for E/I balance\",\n      \"journal\": \"Microbiome\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional microbiota transplant with mechanistic metabolic and electrophysiological readouts in KO model\",\n      \"pmids\": [\"32819434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CLDN1 upregulates EPHB6 and enhances its activation, resulting in suppression of ERK1/2 signaling via a CLDN1-EPHB6-ERK1/2-SLUG feedback axis that represses metastasis, drug resistance, and cancer stemness in lung adenocarcinoma.\",\n      \"method\": \"Immunoprecipitation, immunoblotting for ERK1/2 and SLUG, migration assay, sphere assay, aldefluor assay, xenograft experiments, methylation-specific PCR\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and multiple functional assays; single lab, EPHB6 placed downstream of CLDN1 and upstream of ERK\",\n      \"pmids\": [\"32754286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"EPHB6 mutation promotes paclitaxel resistance through interaction with EPHA2, which activates JNK-mediated cadherin 11 (CDH11) expression and RhoA/FAK-dependent cell adhesion-mediated drug resistance (CAM-DR); targeted inhibition of EPHA2 or CDH11 reverses the acquired resistance.\",\n      \"method\": \"EPHB6 mutation-expression constructs, JNK inhibition, EPHA2/CDH11 siRNA, RhoA/FAK phosphorylation immunoblot, drug resistance assays\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pathway dissection with genetic knockdown and pharmacological inhibition; single lab\",\n      \"pmids\": [\"31160603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Enzalutamide-mediated androgen receptor (AR) signaling decreases EPHB6 expression by binding to an androgen-response element (ARE) on the EPHB6 promoter, leading to JNK/MMP9 pathway activation and increased CRPC cell invasion; JNK inhibition reverses this enhanced invasion.\",\n      \"method\": \"ChIP for AR on EPHB6 promoter ARE, luciferase reporter assay, siRNA, invasion assays, JNK phosphorylation immunoblot\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP and reporter assay establish direct transcriptional mechanism; single lab\",\n      \"pmids\": [\"28826721\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EPHB6 interacts with EGFR (identified by BioID proximity proteomics and confirmed by co-immunoprecipitation) and modulates EGFR signaling to enhance cancer cell proliferation and tumor development.\",\n      \"method\": \"BioID proximity proteomics, Co-IP, genetic interaction screening, xenograft tumor assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — BioID plus Co-IP plus functional in vivo assay; single lab\",\n      \"pmids\": [\"37392382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"EphB6 promotes survival of disseminated dormant breast cancer cells in the lung by modulating TFEB transcriptional activity and lysosome formation; direct coculture with alveolar type I-like lung epithelial cells and in vivo lung dissemination drive lysosomal accumulation and EphB6 induction; EphB6 also promotes proliferation of surrounding lung parenchymal cells.\",\n      \"method\": \"Lung organotypic system, in vivo dissemination assay, TFEB reporter assay, lysosomal staining, EphB6 knockdown\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo dissemination model plus in vitro mechanistic assays; single lab\",\n      \"pmids\": [\"33802447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Transfection of EPHB6 cDNA into neuroblastoma cell lines (IMR5 and SY5Y) that express little endogenous EPHB6 inhibits clonogenicity in culture and suppresses tumorigenicity in mouse xenograft models, establishing EPHB6 as a functional suppressor of malignant neuroblastoma phenotype.\",\n      \"method\": \"EPHB6 cDNA transfection, colony formation assay, mouse xenograft tumor model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function with in vitro and in vivo readouts; single lab\",\n      \"pmids\": [\"10984508\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"EphB6 is expressed specifically on T-cell lineage hematopoietic cells (CD2+CD7+CD3+CD4+ or CD8+ lymphocytes) and is prominently expressed in CD4+CD8+ double-positive thymocytes, with expression level decreasing with T-cell maturation, as determined by monoclonal antibody staining and flow cytometry.\",\n      \"method\": \"Monoclonal antibody generation, flow cytometry, immunophenotyping of human peripheral blood, bone marrow, and thymocytes\",\n      \"journal\": \"Growth factors\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct protein-level localization by flow cytometry with lineage markers; single lab\",\n      \"pmids\": [\"10831073\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"EPHB6 is an intrinsically kinase-inactive Eph receptor tyrosine kinase that signals through transphosphorylation by active EphB-family partners (EphB1, EphB4), constitutively associates with c-Cbl and the Src-family kinase Fyn, and activates downstream pathways—including RAS-ERK, Abl, and Rac1/JNK modulation—to regulate T cell activation, cell adhesion and migration (biphasically, depending on ephrin-B2 concentration), vascular smooth muscle contractility via ephrin-B reverse signaling through GRIP1, mitochondrial dynamics via ERK-DRP1, and tumor suppression in multiple cancer contexts, while its promoter is frequently silenced by DNA hypermethylation in cancers.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"EPHB6 is a catalytically inactive Eph receptor tyrosine kinase that functions as a signaling scaffold in T cell activation, cell adhesion and migration, vascular tone regulation, and tumor suppression. Although devoid of intrinsic kinase activity, EPHB6 undergoes ligand-dependent tyrosine phosphorylation through transphosphorylation by active family members EphB1 and EphB4, and constitutively associates with the adaptor c-Cbl and the Src-family kinase Fyn, enabling downstream engagement of GRB2–RAS–ERK, Abl, and Rac1/JNK pathways [PMID:11713248, PMID:15955811, PMID:20086179, PMID:29700392]. In T cells, EPHB6 localizes to TCR–lipid-raft aggregates and is required for ZAP-70, LAT, and MAPK activation; EphB6-knockout mice exhibit impaired T cell responses and, separately, autism-like behavior linked to gut-microbiota-mediated vitamin B6 and dopamine deficits [PMID:15599401, PMID:32819434]. EPHB6 acts as a tumor and metastasis suppressor in neuroblastoma, breast cancer, and lung cancer—where its promoter is frequently silenced by DNA hypermethylation—by promoting cell adhesion, restricting MMP expression, modulating mitochondrial fission via ERK–DRP1, and sensitizing cells to apoptosis [PMID:10984508, PMID:20371680, PMID:19234485, PMID:27788485].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying EPHB6 as a T-lineage receptor with tumor-suppressive activity established its initial functional identity, addressing what cell types express it and whether it has growth-regulatory consequences.\",\n      \"evidence\": \"Flow cytometric immunophenotyping of human thymocytes/T cells; EPHB6 cDNA re-expression in neuroblastoma lines with colony formation and xenograft assays\",\n      \"pmids\": [\"10831073\", \"10984508\", \"10648835\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of tumor suppression not identified\", \"Only two neuroblastoma lines tested\", \"No downstream signaling pathway defined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that EphB6 is catalytically dead yet becomes tyrosine-phosphorylated via EphB1 transphosphorylation in a ligand-dependent heterocomplex resolved how a kinase-dead receptor could initiate signaling, establishing the transphosphorylation paradigm central to all subsequent EPHB6 biology.\",\n      \"evidence\": \"Co-IP of EphB1–EphB6, kinase-dead EphB1 mutant controls, phosphotyrosine immunoblotting with ephrin-B1 stimulation; c-Cbl domain-deletion mutant Co-IPs\",\n      \"pmids\": [\"11713248\", \"11466354\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the EphB1–EphB6 heterocomplex unknown\", \"Whether EphB6 can be transphosphorylated by other Eph kinases (beyond EphB4, shown later) not tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defining EPHB6's selective inhibition of Rac1–JNK but not ERK downstream of the TCR revealed a pathway-specific modulatory role rather than a global signal amplifier, answering how EphB6 shapes T cell fate decisions.\",\n      \"evidence\": \"Overexpression and dominant-negative EphB6 in Jurkat cells; Rac1 activation pull-down, JNK kinase assay, IL-2 ELISA, thymocyte apoptosis assay\",\n      \"pmids\": [\"12517763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular link between EphB6 and Rac1 GEF/GAP not identified\", \"Dominant-negative approach may have off-target effects on other Eph receptors\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The EphB6-knockout mouse demonstrated that EphB6 is non-redundantly required for T cell responses in vivo, revealing its role in proximal TCR signaling (ZAP-70, LAT, MAPK) and its migration to lipid rafts upon TCR engagement.\",\n      \"evidence\": \"EphB6 KO mice; T cell proliferation/cytokine assays, EAE model, raft fractionation, phospho-immunoblot for ZAP-70/LAT/MAPK\",\n      \"pmids\": [\"15599401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a kinase-dead receptor facilitates ZAP-70 activation mechanistically is unclear\", \"Contribution of reverse signaling through ephrin-B ligands on APCs not dissected\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"A biphasic dose–response to ephrin-B2 uncovered EPHB6 as a molecular switch: constitutive Fyn association supports pro-adhesive signaling at low ligand, while high-ligand Src-family-kinase-mediated phosphorylation triggers repulsion, resolving the paradox of EphB6 promoting both adhesion and repulsion.\",\n      \"evidence\": \"Dose-response adhesion/migration assays, cytoplasmic-domain truncation, Co-IP of Fyn, Src inhibitor treatment\",\n      \"pmids\": [\"15955811\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream effectors mediating the attractive versus repulsive arms not fully identified\", \"Whether Fyn directly phosphorylates EphB6 or acts indirectly not resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linking EphB6 re-expression to reduced MMP7/MMP19 and increased TIMP2 provided a molecular mechanism for its anti-invasive activity in breast cancer, extending the tumor-suppressor paradigm beyond neuroblastoma.\",\n      \"evidence\": \"EphB6 expression construct in MDA-MB-231/MDA-MB-435/BT549; invasion assay, RT-PCR for MMPs and TIMP2\",\n      \"pmids\": [\"19234485\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional mechanism connecting EphB6 to MMP/TIMP expression not defined\", \"No in vivo validation of MMP changes\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Two discoveries solidified EphB6's biological scope: (1) EphB4 transphosphorylates EphB6 to activate c-Cbl–Abl signaling that suppresses breast cancer invasion, and (2) EphB6 loss in vascular smooth muscle increases RhoA-mediated contractility via disrupted ephrin-B reverse signaling through GRIP1, causing hypertension in castrated male mice.\",\n      \"evidence\": \"EphB6 re-expression in MDA-MB-231, Cbl siRNA, Co-IP, invasion assays; EphB6 KO mice, arterial contractility, RhoA assay, MLC phosphorylation\",\n      \"pmids\": [\"20086179\", \"20371680\", \"22223652\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the Abl substrate that mediates adhesion not determined\", \"Sex-hormone dependence of the vascular phenotype not mechanistically explained\", \"How GRIP1 suppresses RhoA not fully delineated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Establishing that EPHB6 fragments mitochondria via ERK–DRP1 and thereby sensitizes cancer cells to DR5-mediated apoptosis revealed a novel organelle-level effector mechanism for a kinase-dead receptor.\",\n      \"evidence\": \"EPHB6 overexpression, ERK inhibition, DRP1 dominant-negative/siRNA, mitochondrial imaging, DR5 agonist apoptosis assay\",\n      \"pmids\": [\"27788485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether mitochondrial fragmentation is relevant in non-cancer cell types unknown\", \"Link between EPHB6 and DRP1 phosphorylation may involve intermediate kinases not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"The identification of a GRB2–RAS–ERK–OCT4 axis downstream of EPHB6 explained how the same receptor can promote tumor-initiating cell expansion in triple-negative breast cancer while simultaneously suppressing EMT, clarifying its context-dependent oncogenic versus suppressive roles.\",\n      \"evidence\": \"Co-IP of EPHB6–GRB2, ERK inhibition, OCT4 knockdown, xenograft models, flow cytometry for TIC markers\",\n      \"pmids\": [\"29700392\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which EPHB6 simultaneously activates ERK (pro-TIC) yet suppresses EMT is not resolved\", \"Whether GRB2 interaction is direct or via transphosphorylation intermediates unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrating that EPHB6 mutations activate EPHA2–JNK–CDH11 and RhoA/FAK to drive paclitaxel resistance identified a gain-of-function mechanism for mutant EPHB6, distinct from loss-of-expression silencing.\",\n      \"evidence\": \"EPHB6 mutation constructs, JNK inhibition, EPHA2/CDH11 siRNA, drug resistance assays\",\n      \"pmids\": [\"31160603\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific mutations tested represent a limited panel; structural basis for EPHA2 interaction not defined\", \"Single study without independent replication\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Two 2020 studies expanded EPHB6 function to gut–brain and lung cancer axes: EphB6 KO mice develop autism-like behavior via gut-microbiota-mediated vitamin B6/dopamine deficits, and CLDN1 upregulates EPHB6 to suppress ERK1/2–SLUG–driven lung cancer stemness and metastasis.\",\n      \"evidence\": \"Bidirectional fecal microbiota transplant, metabolomics, electrophysiology in KO mice; CLDN1–EPHB6 Co-IP, xenograft, methylation-specific PCR in lung adenocarcinoma\",\n      \"pmids\": [\"32819434\", \"32754286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking EphB6 loss to gut microbiota dysbiosis is unknown\", \"Whether the CLDN1–EPHB6 interaction is direct remains uncertain\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Proximity proteomics identified EGFR as an EPHB6 interactor that functionally cooperates to enhance cancer cell proliferation, expanding the repertoire of receptor tyrosine kinases that EPHB6 cross-talks with beyond the Eph family.\",\n      \"evidence\": \"BioID proximity proteomics, Co-IP of EPHB6–EGFR, xenograft tumor assays\",\n      \"pmids\": [\"37392382\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Nature of the EPHB6–EGFR interaction (direct or scaffolded) not resolved\", \"Whether EGFR transphosphorylates EPHB6 (like EphB1/B4) not tested\", \"Single study without independent confirmation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis of the EphB6 heterocomplex with active Eph kinases, how a kinase-dead receptor is recruited to and activates TCR-proximal signaling, the mechanism connecting EphB6 loss to gut microbiota remodeling, and the rules governing EPHB6's context-dependent pro-tumorigenic versus tumor-suppressive activity.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal structure or cryo-EM structure of EphB6 or its heterocomplexes\", \"No reconstituted signaling system with purified components\", \"Context-dependent ERK activation versus suppression not mechanistically reconciled\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 6, 7, 11]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 4, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 6, 20]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [0, 3, 4, 6, 11, 14]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 3, 4, 20]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [7, 8, 9, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"EPHB1\",\n      \"EPHB4\",\n      \"CBL\",\n      \"FYN\",\n      \"GRB2\",\n      \"EPHA2\",\n      \"EGFR\",\n      \"EFNB2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}