{"gene":"REPS2","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2003,"finding":"REPS2/POB1 overexpression in prostate cancer cell lines induced apoptosis within 48 h and inhibited signalling towards a TPA response element luciferase reporter, indicating REPS2 negatively regulates growth factor signalling through the Ral signalling pathway.","method":"Transient transfection overexpression, luciferase reporter assay, apoptosis assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single lab, two orthogonal functional readouts (apoptosis + reporter assay) but no direct biochemical mechanism resolved","pmids":["12771942"],"is_preprint":false},{"year":2004,"finding":"The EH domain of REPS2 directly binds the NPF-motif in the NF-κB subunit p65, as established by yeast two-hybrid, mammalian two-hybrid, and co-immunoprecipitation; this interaction is triggered by PMA stimulation, linking PMA-sensitive signalling pathways to REPS2-p65 interaction and NF-κB modulation.","method":"Yeast two-hybrid, mammalian two-hybrid, co-immunoprecipitation, crystal structure data-guided mutagenesis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal two-hybrid plus co-IP with mutagenesis support, single lab","pmids":["15184881"],"is_preprint":false},{"year":2008,"finding":"The central proline-rich domain of POB1/REPS2 contains two closely spaced binding motifs: one for 14-3-3 proteins and one for the SH3 domains of Amphiphysin II and Grb2. Ectopic expression of this domain exerts a dominant-negative effect on EGFR endocytosis (but not transferrin receptor endocytosis), and mutation of these motifs abolishes the inhibitory effect, indicating these interactions are functionally required for EGFR endocytosis. 14-3-3 is proposed to bridge EGFR and POB1/REPS2.","method":"Phage display, peptide arrays, bioinformatics, mutagenesis, co-immunoprecipitation, dominant-negative overexpression with EGFR endocytosis assay","journal":"BMC biochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (phage display, peptide array, co-IP, mutagenesis, functional endocytosis assay) in a single rigorous study","pmids":["18647389"],"is_preprint":false},{"year":2011,"finding":"REPS2 suppresses the ability of its binding partner RalBP1 to transport chemotherapeutic drugs (e.g., doxorubicin) out of the cell, establishing a role for REPS2 in modulating drug efflux via the RalBP1 complex.","method":"Review/summary citing prior experimental data (molecular interaction characterisation)","journal":"The international journal of biochemistry & cell biology","confidence":"Low","confidence_rationale":"Tier 4 / Weak — review article summary, no new primary experiment described in abstract","pmids":["21907823"],"is_preprint":false},{"year":2016,"finding":"miR-675-5p targets REPS2 (validated as a direct target), and REPS2 knockdown abrogates the G1 arrest, anti-proliferative, and anti-metastatic effects induced by miR-675-5p inhibition; REPS2 acts upstream in the RalBP1/RAC1/CDC42 signalling pathway.","method":"miRNA target validation, siRNA knockdown, cell cycle analysis, proliferation/invasion assays, in vivo xenograft","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct target validation plus epistasis experiment (REPS2 KD rescues miR-675-5p inhibition phenotype), single lab, multiple readouts","pmids":["27120794"],"is_preprint":false},{"year":2022,"finding":"LXR agonist T0901317 upregulates REPS2 at the transcriptional level via LXR binding to an LXRE in the REPS2 promoter (shown by promoter activity assay and ChIP); increased REPS2 expression inhibits EGF-mediated EGFR endocytosis and downstream AKT/NF-κB, p38MAPK, and ERK1/2 activation in HCC cells.","method":"Promoter activity assay, chromatin immunoprecipitation (ChIP), EGFR endocytosis assay, western blot for downstream signalling, REPS2 knockdown rescue","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus functional endocytosis and signalling assays, single lab, multiple orthogonal methods","pmids":["35995867"],"is_preprint":false},{"year":2022,"finding":"REPS2 knockdown in human lens epithelial cells activates FAK phosphorylation and Cdc42, promoting FGF-induced proliferation, EMT, ECM synthesis, and cytoskeletal reorganisation; pharmacological FAK inhibition (PF573228) abolishes these effects, placing REPS2 as a negative regulator upstream of the FAK/Cdc42 signalling axis.","method":"siRNA knockdown, G-protein pulldown, western blot, F-actin staining, FAK inhibitor rescue, wound healing and Transwell migration assays","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via pharmacological inhibitor rescue plus G-protein pulldown, single lab, multiple orthogonal readouts","pmids":["35690292"],"is_preprint":false},{"year":2025,"finding":"REPS2 promotes autophagy-lysosome-mediated degradation of β-catenin by facilitating interaction between β-catenin and p62 (SQSTM1); REPS2, p62, and β-catenin form a complex, leading to reduced Wnt signalling and attenuation of cancer cell stemness.","method":"CRISPR/Cas9 genome-wide screen, co-immunoprecipitation (p62–β-catenin–REPS2 complex), autophagy/lysosome inhibition assays, in vitro and in vivo functional assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide CRISPR screen identification plus co-IP complex validation and functional rescue, single lab","pmids":["40514427"],"is_preprint":false}],"current_model":"REPS2 is a scaffold protein containing EH domains, proline-rich regions, and a coiled-coil domain that acts as a negative regulator of growth factor receptor (particularly EGFR) endocytosis and downstream signalling: its EH domain binds the NPF-motif of NF-κB p65 to modulate NF-κB activity; its proline-rich region recruits 14-3-3, Amphiphysin II, and Grb2 to coordinate EGFR internalisation; it suppresses RalBP1/RAC1/CDC42 and FAK/Cdc42 signalling axes; and it promotes p62-mediated autophagy-lysosomal degradation of β-catenin to attenuate Wnt/cancer-stemness signalling, with its expression transcriptionally activated by LXR."},"narrative":{"mechanistic_narrative":"REPS2 (POB1) is a multidomain scaffold protein that acts as a negative regulator of growth factor receptor signalling, restraining proliferative, survival, and stemness programs across multiple cancer cell types [PMID:12771942, PMID:35995867, PMID:40514427]. Its central proline-rich domain carries adjacent binding motifs for 14-3-3 and for the SH3 domains of Amphiphysin II and Grb2, and engagement of these motifs is required for REPS2 to suppress EGFR (but not transferrin receptor) endocytosis, with 14-3-3 bridging REPS2 to EGFR [PMID:18647389]; by blocking EGFR internalisation REPS2 dampens downstream AKT/NF-κB, p38MAPK, and ERK1/2 signalling [PMID:35995867]. Its EH domain directly binds the NPF motif of the NF-κB subunit p65 in a PMA-inducible manner, providing a second route to NF-κB modulation [PMID:15184881]. REPS2 additionally constrains small-GTPase signalling, acting upstream of the RalBP1/RAC1/CDC42 axis and the FAK/Cdc42 axis, where its loss drives proliferation, EMT, and cytoskeletal reorganisation [PMID:27120794, PMID:35690292], and it promotes p62 (SQSTM1)-dependent autophagy-lysosomal degradation of β-catenin within a REPS2–p62–β-catenin complex to attenuate Wnt signalling and cancer stemness [PMID:40514427]. REPS2 expression is transcriptionally activated by LXR through an LXRE in its promoter [PMID:35995867]. No structural model of full-length REPS2 or its assembled signalling complexes has been characterized in the available corpus.","teleology":[{"year":2003,"claim":"Established REPS2 as a functional negative regulator of growth factor/Ral signalling rather than a passive structural protein, by linking its overexpression to a measurable cellular consequence.","evidence":"Transient overexpression in prostate cancer cells with apoptosis and TPA-response-element luciferase reporter readouts","pmids":["12771942"],"confidence":"Medium","gaps":["No direct biochemical mechanism resolved","Relies on overexpression rather than endogenous loss-of-function","Connection to specific receptor not defined"]},{"year":2004,"claim":"Defined a direct molecular link between REPS2 and NF-κB by showing its EH domain binds an NPF motif in p65, explaining how REPS2 can modulate an inflammatory/survival transcription factor.","evidence":"Yeast and mammalian two-hybrid, co-IP, and structure-guided mutagenesis with PMA stimulation","pmids":["15184881"],"confidence":"Medium","gaps":["Single lab","Functional consequence on NF-κB target genes not fully quantified","Stimulus dependence beyond PMA unexplored"]},{"year":2008,"claim":"Identified the receptor-selective mechanism of REPS2 in endocytosis, mapping the proline-rich motifs required for it to inhibit EGFR (but not transferrin receptor) internalisation and naming the bridging partners.","evidence":"Phage display, peptide arrays, mutagenesis, co-IP, and dominant-negative EGFR endocytosis assay","pmids":["18647389"],"confidence":"High","gaps":["14-3-3 bridging of EGFR proposed but not structurally resolved","Endogenous regulation versus dominant-negative fragment not distinguished","Spatial/temporal step of endocytosis affected not defined"]},{"year":2011,"claim":"Extended REPS2's RalBP1 partnership to a drug-resistance phenotype by implicating it in modulating RalBP1-mediated efflux of chemotherapeutics.","evidence":"Review summary citing prior molecular interaction data","pmids":["21907823"],"confidence":"Low","gaps":["Review without new primary experiment in abstract","Direct effect of REPS2 on efflux kinetics not shown here","Mechanism of RalBP1 inhibition unspecified"]},{"year":2016,"claim":"Placed REPS2 in a defined oncogenic regulatory circuit, showing it is a direct miR-675-5p target acting upstream of RalBP1/RAC1/CDC42 to control cell cycle, proliferation, and metastasis.","evidence":"miRNA target validation, siRNA knockdown rescue, cell cycle/proliferation/invasion assays, and xenografts","pmids":["27120794"],"confidence":"Medium","gaps":["Direct biochemical inhibition of RalBP1 by REPS2 not shown in this study","Single lab","Downstream RAC1/CDC42 activation not directly assayed"]},{"year":2022,"claim":"Connected REPS2 to a second small-GTPase axis (FAK/Cdc42) and to a transcriptional activator (LXR), establishing how REPS2 levels are set and how its loss promotes EMT and growth.","evidence":"siRNA knockdown with G-protein pulldown and FAK inhibitor rescue in lens epithelial cells; ChIP and promoter assays plus EGFR endocytosis/signalling readouts in HCC cells","pmids":["35690292","35995867"],"confidence":"Medium","gaps":["Whether FAK/Cdc42 regulation is direct or downstream of receptor endocytosis unclear","LXR-driven REPS2 induction tested in limited cell contexts","In vivo relevance of LXR–REPS2 axis not fully established"]},{"year":2025,"claim":"Revealed a degradative mechanism by which REPS2 suppresses Wnt/stemness signalling, showing it scaffolds β-catenin to p62 for autophagy-lysosomal turnover.","evidence":"Genome-wide CRISPR/Cas9 screen, co-IP of REPS2–p62–β-catenin complex, autophagy/lysosome inhibition, and in vitro/in vivo assays","pmids":["40514427"],"confidence":"Medium","gaps":["Domain of REPS2 mediating p62/β-catenin engagement not mapped","Single lab","Selectivity for β-catenin over other autophagy cargo unaddressed"]},{"year":null,"claim":"It remains unresolved how REPS2's distinct scaffolding activities (EGFR endocytosis, NF-κB, RalBP1/Cdc42 axes, and β-catenin degradation) are coordinated and which are direct versus secondary consequences of receptor regulation.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of full-length REPS2 or its complexes","No unified model linking endocytic and degradative roles","Physiological versus disease-context functions not separated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,5,6]}],"localization":[],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,5]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,6]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[7]}],"complexes":["REPS2–p62–β-catenin complex"],"partners":["RALBP1","YWHAB","BIN1","GRB2","RELA","SQSTM1","CTNNB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8NFH8","full_name":"RalBP1-associated Eps domain-containing protein 2","aliases":["Partner of RalBP1","RalBP1-interacting protein 2"],"length_aa":660,"mass_kda":71.5,"function":"Involved in ligand-dependent receptor mediated endocytosis of the EGF and insulin receptors as part of the Ral signaling pathway (PubMed:10393179, PubMed:12771942, PubMed:9422736). By controlling growth factor receptors endocytosis may regulate cell survival (PubMed:12771942). Through ASAP1 may regulate cell adhesion and migration (PubMed:12149250)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q8NFH8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/REPS2","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":[{"gene":"RALBP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/REPS2","total_profiled":1310},"omim":[{"mim_id":"607262","title":"EPSIN 1; EPN1","url":"https://www.omim.org/entry/607262"},{"mim_id":"302350","title":"NANCE-HORAN SYNDROME; NHS","url":"https://www.omim.org/entry/302350"},{"mim_id":"300317","title":"RALBP1-ASSOCIATED EPS DOMAIN-CONTAINING PROTEIN 2; REPS2","url":"https://www.omim.org/entry/300317"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/REPS2"},"hgnc":{"alias_symbol":["POB1"],"prev_symbol":[]},"alphafold":{"accession":"Q8NFH8","domains":[{"cath_id":"1.10.238.10","chopping":"28-48_70-141","consensus_level":"medium","plddt":84.0451,"start":28,"end":141},{"cath_id":"1.10.238.10","chopping":"281-366","consensus_level":"high","plddt":89.6371,"start":281,"end":366},{"cath_id":"1.20.5","chopping":"626-656","consensus_level":"medium","plddt":93.2787,"start":626,"end":656}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NFH8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NFH8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8NFH8-F1-predicted_aligned_error_v6.png","plddt_mean":58.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=REPS2","jax_strain_url":"https://www.jax.org/strain/search?query=REPS2"},"sequence":{"accession":"Q8NFH8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8NFH8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8NFH8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8NFH8"}},"corpus_meta":[{"pmid":"27120794","id":"PMC_27120794","title":"miR-675-5p enhances tumorigenesis and metastasis of esophageal squamous cell carcinoma by targeting REPS2.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27120794","citation_count":46,"is_preprint":false},{"pmid":"12771942","id":"PMC_12771942","title":"REPS2/POB1 is downregulated during human prostate cancer progression and inhibits growth factor signalling in prostate cancer cells.","date":"2003","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/12771942","citation_count":44,"is_preprint":false},{"pmid":"15184881","id":"PMC_15184881","title":"Identification of REPS2 as a putative modulator of NF-kappaB activity in prostate cancer cells.","date":"2004","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/15184881","citation_count":19,"is_preprint":false},{"pmid":"18647389","id":"PMC_18647389","title":"The central proline rich region of POB1/REPS2 plays a regulatory role in epidermal growth factor receptor endocytosis by binding to 14-3-3 and SH3 domain-containing proteins.","date":"2008","source":"BMC biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18647389","citation_count":12,"is_preprint":false},{"pmid":"21907823","id":"PMC_21907823","title":"Reps2: a cellular signaling and molecular trafficking nexus.","date":"2011","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/21907823","citation_count":11,"is_preprint":false},{"pmid":"35995867","id":"PMC_35995867","title":"Liver X receptor agonists exert antitumor effects against hepatocellular carcinoma via inducing REPS2 expression.","date":"2022","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/35995867","citation_count":11,"is_preprint":false},{"pmid":"23803043","id":"PMC_23803043","title":"Expression and clinical significance of REPS2 in human esophageal squamous cell carcinoma.","date":"2013","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/23803043","citation_count":10,"is_preprint":false},{"pmid":"35690292","id":"PMC_35690292","title":"REPS2 downregulation facilitates FGF-induced adhesion and migration in human lens epithelial cells through FAK/Cdc42 signaling and contributes to posterior capsule opacification.","date":"2022","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/35690292","citation_count":4,"is_preprint":false},{"pmid":"40514427","id":"PMC_40514427","title":"REPS2 attenuates cancer stemness through inhibiting Wnt signaling by autophagy mediated degradation of β-catenin.","date":"2025","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/40514427","citation_count":3,"is_preprint":false},{"pmid":"41996722","id":"PMC_41996722","title":"CRISPR/Cas9-mediated generation of a REPS2 knockout human embryonic stem cell line.","date":"2026","source":"Stem cell research","url":"https://pubmed.ncbi.nlm.nih.gov/41996722","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":6652,"output_tokens":2157,"usd":0.026156,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9106,"output_tokens":2930,"usd":0.05939,"stage2_stop_reason":"end_turn"},"total_usd":0.085546,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"REPS2/POB1 overexpression in prostate cancer cell lines induced apoptosis within 48 h and inhibited signalling towards a TPA response element luciferase reporter, indicating REPS2 negatively regulates growth factor signalling through the Ral signalling pathway.\",\n      \"method\": \"Transient transfection overexpression, luciferase reporter assay, apoptosis assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single lab, two orthogonal functional readouts (apoptosis + reporter assay) but no direct biochemical mechanism resolved\",\n      \"pmids\": [\"12771942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The EH domain of REPS2 directly binds the NPF-motif in the NF-κB subunit p65, as established by yeast two-hybrid, mammalian two-hybrid, and co-immunoprecipitation; this interaction is triggered by PMA stimulation, linking PMA-sensitive signalling pathways to REPS2-p65 interaction and NF-κB modulation.\",\n      \"method\": \"Yeast two-hybrid, mammalian two-hybrid, co-immunoprecipitation, crystal structure data-guided mutagenesis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal two-hybrid plus co-IP with mutagenesis support, single lab\",\n      \"pmids\": [\"15184881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The central proline-rich domain of POB1/REPS2 contains two closely spaced binding motifs: one for 14-3-3 proteins and one for the SH3 domains of Amphiphysin II and Grb2. Ectopic expression of this domain exerts a dominant-negative effect on EGFR endocytosis (but not transferrin receptor endocytosis), and mutation of these motifs abolishes the inhibitory effect, indicating these interactions are functionally required for EGFR endocytosis. 14-3-3 is proposed to bridge EGFR and POB1/REPS2.\",\n      \"method\": \"Phage display, peptide arrays, bioinformatics, mutagenesis, co-immunoprecipitation, dominant-negative overexpression with EGFR endocytosis assay\",\n      \"journal\": \"BMC biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (phage display, peptide array, co-IP, mutagenesis, functional endocytosis assay) in a single rigorous study\",\n      \"pmids\": [\"18647389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"REPS2 suppresses the ability of its binding partner RalBP1 to transport chemotherapeutic drugs (e.g., doxorubicin) out of the cell, establishing a role for REPS2 in modulating drug efflux via the RalBP1 complex.\",\n      \"method\": \"Review/summary citing prior experimental data (molecular interaction characterisation)\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — review article summary, no new primary experiment described in abstract\",\n      \"pmids\": [\"21907823\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"miR-675-5p targets REPS2 (validated as a direct target), and REPS2 knockdown abrogates the G1 arrest, anti-proliferative, and anti-metastatic effects induced by miR-675-5p inhibition; REPS2 acts upstream in the RalBP1/RAC1/CDC42 signalling pathway.\",\n      \"method\": \"miRNA target validation, siRNA knockdown, cell cycle analysis, proliferation/invasion assays, in vivo xenograft\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct target validation plus epistasis experiment (REPS2 KD rescues miR-675-5p inhibition phenotype), single lab, multiple readouts\",\n      \"pmids\": [\"27120794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LXR agonist T0901317 upregulates REPS2 at the transcriptional level via LXR binding to an LXRE in the REPS2 promoter (shown by promoter activity assay and ChIP); increased REPS2 expression inhibits EGF-mediated EGFR endocytosis and downstream AKT/NF-κB, p38MAPK, and ERK1/2 activation in HCC cells.\",\n      \"method\": \"Promoter activity assay, chromatin immunoprecipitation (ChIP), EGFR endocytosis assay, western blot for downstream signalling, REPS2 knockdown rescue\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus functional endocytosis and signalling assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35995867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"REPS2 knockdown in human lens epithelial cells activates FAK phosphorylation and Cdc42, promoting FGF-induced proliferation, EMT, ECM synthesis, and cytoskeletal reorganisation; pharmacological FAK inhibition (PF573228) abolishes these effects, placing REPS2 as a negative regulator upstream of the FAK/Cdc42 signalling axis.\",\n      \"method\": \"siRNA knockdown, G-protein pulldown, western blot, F-actin staining, FAK inhibitor rescue, wound healing and Transwell migration assays\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via pharmacological inhibitor rescue plus G-protein pulldown, single lab, multiple orthogonal readouts\",\n      \"pmids\": [\"35690292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"REPS2 promotes autophagy-lysosome-mediated degradation of β-catenin by facilitating interaction between β-catenin and p62 (SQSTM1); REPS2, p62, and β-catenin form a complex, leading to reduced Wnt signalling and attenuation of cancer cell stemness.\",\n      \"method\": \"CRISPR/Cas9 genome-wide screen, co-immunoprecipitation (p62–β-catenin–REPS2 complex), autophagy/lysosome inhibition assays, in vitro and in vivo functional assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide CRISPR screen identification plus co-IP complex validation and functional rescue, single lab\",\n      \"pmids\": [\"40514427\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"REPS2 is a scaffold protein containing EH domains, proline-rich regions, and a coiled-coil domain that acts as a negative regulator of growth factor receptor (particularly EGFR) endocytosis and downstream signalling: its EH domain binds the NPF-motif of NF-κB p65 to modulate NF-κB activity; its proline-rich region recruits 14-3-3, Amphiphysin II, and Grb2 to coordinate EGFR internalisation; it suppresses RalBP1/RAC1/CDC42 and FAK/Cdc42 signalling axes; and it promotes p62-mediated autophagy-lysosomal degradation of β-catenin to attenuate Wnt/cancer-stemness signalling, with its expression transcriptionally activated by LXR.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"REPS2 (POB1) is a multidomain scaffold protein that acts as a negative regulator of growth factor receptor signalling, restraining proliferative, survival, and stemness programs across multiple cancer cell types [#0, #5, #7]. Its central proline-rich domain carries adjacent binding motifs for 14-3-3 and for the SH3 domains of Amphiphysin II and Grb2, and engagement of these motifs is required for REPS2 to suppress EGFR (but not transferrin receptor) endocytosis, with 14-3-3 bridging REPS2 to EGFR [#2]; by blocking EGFR internalisation REPS2 dampens downstream AKT/NF-\\u03baB, p38MAPK, and ERK1/2 signalling [#5]. Its EH domain directly binds the NPF motif of the NF-\\u03baB subunit p65 in a PMA-inducible manner, providing a second route to NF-\\u03baB modulation [#1]. REPS2 additionally constrains small-GTPase signalling, acting upstream of the RalBP1/RAC1/CDC42 axis and the FAK/Cdc42 axis, where its loss drives proliferation, EMT, and cytoskeletal reorganisation [#4, #6], and it promotes p62 (SQSTM1)-dependent autophagy-lysosomal degradation of \\u03b2-catenin within a REPS2\\u2013p62\\u2013\\u03b2-catenin complex to attenuate Wnt signalling and cancer stemness [#7]. REPS2 expression is transcriptionally activated by LXR through an LXRE in its promoter [#5]. No structural model of full-length REPS2 or its assembled signalling complexes has been characterized in the available corpus.\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established REPS2 as a functional negative regulator of growth factor/Ral signalling rather than a passive structural protein, by linking its overexpression to a measurable cellular consequence.\",\n      \"evidence\": \"Transient overexpression in prostate cancer cells with apoptosis and TPA-response-element luciferase reporter readouts\",\n      \"pmids\": [\"12771942\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct biochemical mechanism resolved\", \"Relies on overexpression rather than endogenous loss-of-function\", \"Connection to specific receptor not defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined a direct molecular link between REPS2 and NF-\\u03baB by showing its EH domain binds an NPF motif in p65, explaining how REPS2 can modulate an inflammatory/survival transcription factor.\",\n      \"evidence\": \"Yeast and mammalian two-hybrid, co-IP, and structure-guided mutagenesis with PMA stimulation\",\n      \"pmids\": [\"15184881\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Functional consequence on NF-\\u03baB target genes not fully quantified\", \"Stimulus dependence beyond PMA unexplored\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified the receptor-selective mechanism of REPS2 in endocytosis, mapping the proline-rich motifs required for it to inhibit EGFR (but not transferrin receptor) internalisation and naming the bridging partners.\",\n      \"evidence\": \"Phage display, peptide arrays, mutagenesis, co-IP, and dominant-negative EGFR endocytosis assay\",\n      \"pmids\": [\"18647389\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"14-3-3 bridging of EGFR proposed but not structurally resolved\", \"Endogenous regulation versus dominant-negative fragment not distinguished\", \"Spatial/temporal step of endocytosis affected not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended REPS2's RalBP1 partnership to a drug-resistance phenotype by implicating it in modulating RalBP1-mediated efflux of chemotherapeutics.\",\n      \"evidence\": \"Review summary citing prior molecular interaction data\",\n      \"pmids\": [\"21907823\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Review without new primary experiment in abstract\", \"Direct effect of REPS2 on efflux kinetics not shown here\", \"Mechanism of RalBP1 inhibition unspecified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Placed REPS2 in a defined oncogenic regulatory circuit, showing it is a direct miR-675-5p target acting upstream of RalBP1/RAC1/CDC42 to control cell cycle, proliferation, and metastasis.\",\n      \"evidence\": \"miRNA target validation, siRNA knockdown rescue, cell cycle/proliferation/invasion assays, and xenografts\",\n      \"pmids\": [\"27120794\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical inhibition of RalBP1 by REPS2 not shown in this study\", \"Single lab\", \"Downstream RAC1/CDC42 activation not directly assayed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected REPS2 to a second small-GTPase axis (FAK/Cdc42) and to a transcriptional activator (LXR), establishing how REPS2 levels are set and how its loss promotes EMT and growth.\",\n      \"evidence\": \"siRNA knockdown with G-protein pulldown and FAK inhibitor rescue in lens epithelial cells; ChIP and promoter assays plus EGFR endocytosis/signalling readouts in HCC cells\",\n      \"pmids\": [\"35690292\", \"35995867\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether FAK/Cdc42 regulation is direct or downstream of receptor endocytosis unclear\", \"LXR-driven REPS2 induction tested in limited cell contexts\", \"In vivo relevance of LXR\\u2013REPS2 axis not fully established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a degradative mechanism by which REPS2 suppresses Wnt/stemness signalling, showing it scaffolds \\u03b2-catenin to p62 for autophagy-lysosomal turnover.\",\n      \"evidence\": \"Genome-wide CRISPR/Cas9 screen, co-IP of REPS2\\u2013p62\\u2013\\u03b2-catenin complex, autophagy/lysosome inhibition, and in vitro/in vivo assays\",\n      \"pmids\": [\"40514427\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Domain of REPS2 mediating p62/\\u03b2-catenin engagement not mapped\", \"Single lab\", \"Selectivity for \\u03b2-catenin over other autophagy cargo unaddressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how REPS2's distinct scaffolding activities (EGFR endocytosis, NF-\\u03baB, RalBP1/Cdc42 axes, and \\u03b2-catenin degradation) are coordinated and which are direct versus secondary consequences of receptor regulation.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of full-length REPS2 or its complexes\", \"No unified model linking endocytic and degradative roles\", \"Physiological versus disease-context functions not separated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 5, 6]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 6]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"REPS2\\u2013p62\\u2013\\u03b2-catenin complex\"],\n    \"partners\": [\"RALBP1\", \"YWHAB\", \"BIN1\", \"GRB2\", \"RELA\", \"SQSTM1\", \"CTNNB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}