{"gene":"AFDN","run_date":"2026-06-09T22:02:42","timeline":{"discoveries":[{"year":1993,"finding":"AF-6 (AFDN) was identified as the fusion partner of the ALL-1/MLL gene in the t(6;11)(q27;q23) acute myeloid leukemia translocation; the AF-6 protein contains a GLGF/PDZ motif, suggesting a role at cell-cell junctions.","method":"Molecular cloning, cDNA characterization","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — initial cloning with domain identification, single lab, foundational molecular characterization","pmids":["8242616"],"is_preprint":false},{"year":1996,"finding":"AF-6 and its Drosophila homolog Canoe were identified as direct binding targets of activated (GTP-bound) Ras; the N-terminal domain of AF-6 specifically binds GTPγS-Ras but not GDP-Ras or Ras with an effector-domain mutation.","method":"GST affinity chromatography, recombinant protein binding assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical reconstitution with GTP/GDP specificity controls and effector mutant controls, replicated in subsequent studies","pmids":["8557659"],"is_preprint":false},{"year":1997,"finding":"AF-6 localizes to tight junctions in polarized MDCK epithelial cells, co-distributing with ZO-1 at tight junctions as shown by immunoelectron microscopy; native and recombinant AF-6 directly interact with ZO-1 in vitro via the Ras-binding domain of AF-6; this interaction is inhibited by activated Ras.","method":"Immunoelectron microscopy, in vitro binding assay, co-immunoprecipitation, overexpression of activated Ras","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (immunoelectron microscopy, in vitro binding, Co-IP), replicated across labs","pmids":["9348294"],"is_preprint":false},{"year":1997,"finding":"MLL-AF6 chimeric fusion protein localizes to the nucleus despite AF-6 itself being cytoplasmic; the nuclear localization is conferred by the AT-hook motif-containing N-terminal region of MLL.","method":"Immunofluorescence, cell fractionation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal localization methods, single lab","pmids":["9349501"],"is_preprint":false},{"year":1998,"finding":"AF-6 is a substrate of the FAM deubiquitinating enzyme; AF-6 is ubiquitinated in intact cells, and FAM (the mammalian homolog of Drosophila fat facets) prevents ubiquitination of AF-6. FAM and AF-6 interact in vivo and in vitro and co-localize at cell-cell contacts.","method":"Protein purification, peptide sequencing, co-immunoprecipitation, in vitro binding, ubiquitination assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical purification, co-IP, in vitro binding, and direct ubiquitination assay; multiple orthogonal methods","pmids":["9722616"],"is_preprint":false},{"year":1998,"finding":"The PDZ domain of AF-6 interacts with C-terminal sequences of Eph receptor tyrosine kinases (EphB3, EphA7, EphB2, EphB5, EphB6), neurexins, and Notch ligand Jagged; interaction of full-length AF-6 with EphB3 depends on kinase activity of EphB3; endogenous AF-6 is phosphorylated by EphB2 and EphB3 in a ligand-dependent manner.","method":"Yeast two-hybrid, mutational analysis, co-immunoprecipitation, phosphorylation assay in NIH 3T3 and NG108 cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — yeast two-hybrid with mutagenesis, co-IP, and in-cell phosphorylation assay; multiple orthogonal methods","pmids":["9707552"],"is_preprint":false},{"year":1999,"finding":"The N-terminal Ras-association (RA) domain of AF-6 mediates interaction with activated Ras in vivo; a single amino acid mutation in this domain abolishes Ras binding; overexpression of the RA domain inhibits Ras-dependent c-fos promoter stimulation; AF-6 is co-immunoprecipitated with ZO-1 from Rat1 cells.","method":"Site-directed mutagenesis, co-immunoprecipitation, reporter assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis combined with in vivo co-IP and functional reporter assay, consistent with multiple prior studies","pmids":["10334923"],"is_preprint":false},{"year":1999,"finding":"AF-6 binds Ras with thermodynamic and kinetic parameters similar to Raf and RalGEF; AF-6 binding stabilizes one of two major Ras conformational states (detected by 31P NMR); among Ras-related GTPases, AF-6 binds Rap1A with the highest affinity; AF-6 inhibits nucleotide dissociation and GAP activity but not intrinsic GTPase activity of Ras.","method":"Biochemical binding assay, 31P NMR spectroscopy, kinetic analysis with GTP analogs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — rigorous biophysical characterization with multiple methods (NMR, kinetics, biochemistry) in a single study","pmids":["10224125"],"is_preprint":false},{"year":1999,"finding":"AF-6 and Canoe (Drosophila homolog) cluster with Eph receptors at cell-cell contact sites; AF-6 forms a complex with endogenous Eph receptors in whole rat brain lysates, co-localizes with Eph receptors at postsynaptic membrane sites of excitatory synapses in hippocampus, and is a substrate for Eph receptor kinases.","method":"Co-immunoprecipitation from brain lysates, co-transfection with GFP-AF-6, immunohistochemistry, in vitro phosphorylation","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP from native tissue, co-localization, and phosphorylation assay; multiple orthogonal methods","pmids":["9922461"],"is_preprint":false},{"year":1999,"finding":"Loss of AF-6 in mouse embryos disrupts epithelial cell-cell junctions and cell polarity: homozygous null embryos show reduced apical junction length, basolateral gaps, and loss of neuroepithelial polarity by 7.5 dpc, leading to embryonic lethality by 10 dpc.","method":"Targeted gene knockout in mice, histology, immunofluorescence, electron microscopy","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with clear developmental phenotype, multiple morphological readouts, landmark study replicated in concept by subsequent work","pmids":["10469590"],"is_preprint":false},{"year":1999,"finding":"M-Ras co-immunoprecipitates with AF-6 in mammalian cells, extending the range of Ras-subfamily GTPases that can interact with AF-6.","method":"Co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP experiment, single lab, no functional follow-up on the AF-6 interaction","pmids":["10446149"],"is_preprint":false},{"year":2000,"finding":"The AF-6 PDZ domain interacts with the C-terminal PDZ-binding motif of junctional adhesion molecule (JAM); AF-6 and ZO-1 can both be co-precipitated with JAM from endothelial cell extracts; loss of the JAM C-terminus disrupts its co-distribution with AF-6 at cell contacts.","method":"Co-immunoprecipitation from endothelial cells, in vitro binding, co-localization by immunofluorescence, truncation mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal in vitro and in vivo binding with truncation mutants and functional localization readout","pmids":["10856295"],"is_preprint":false},{"year":2000,"finding":"The first Ras-binding domain of AF-6 mediates interaction with Ras and preferentially binds Rap1A over oncogenic Ras GTPases; AF-6 interacts with Rap1 in vivo in mammalian cells; Rap1A does not perturb AF-6 localization at cell-cell junctions. AF-6 binds profilin, an actin polymerization regulator, making AF-6 the only junctional component known to interact with profilin.","method":"In vitro binding with GST-fusion proteins, co-immunoprecipitation in mammalian cells, yeast two-hybrid, co-localization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (in vitro binding, Co-IP, yeast two-hybrid), two distinct binding partners characterized","pmids":["10922060"],"is_preprint":false},{"year":2003,"finding":"Canoe (Drosophila AF-6 homolog) acts as an effector of Rap1 in vivo during dorsal closure; Cno binds activated Rap1 in a two-hybrid assay, the two co-localize at adherens junctions, and genetic epistasis shows Rap1 acts upstream of Cno in the same pathway. Cno has a Rap1-independent function in JNK pathway activation.","method":"Yeast two-hybrid, co-localization, genetic epistasis in Drosophila, loss-of-function analysis","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid binding plus genetic epistasis in Drosophila with multiple alleles; Rap1-independent role also defined","pmids":["14504224"],"is_preprint":false},{"year":2003,"finding":"The Bcr protein kinase is a ligand for the PDZ domain of AF-6; Bcr phosphorylates AF-6 and phosphorylation enables efficient Bcr binding to AF-6's PDZ domain; Bcr, AF-6, and Ras form a trimeric complex; Bcr increases AF-6's affinity for Ras and a phosphorylation-site mutant of AF-6 shows reduced Ras binding; Bcr (but not PDZ-binding-deficient Bcr mutants) interferes with Ras-Raf/MEK/ERK signaling.","method":"Co-immunoprecipitation, kinase assay, mutagenesis, co-localization, reporter assay for ERK pathway","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — kinase assay with mutagenesis, reciprocal co-IP, and functional pathway readout; multiple orthogonal methods","pmids":["12808105"],"is_preprint":false},{"year":2003,"finding":"AF-6 controls integrin-mediated cell adhesion by scaffolding both SPA-1 (a Rap1 GAP) and Rap1-GTP via distinct domains: SPA-1 binds the PDZ domain of AF-6 via an internal PDZ-ligand motif; Rap1V12 binds the N-terminal domain; AF-6 overexpression inhibits Rap1-GTP levels and β1 integrin-mediated adhesion in a SPA-1-dependent context.","method":"Co-immunoprecipitation, in vitro binding with truncation mutants and point mutants, cell adhesion assay, immunostaining","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple binding assays with deletion/point mutants plus functional cell adhesion readout, single lab but multiple orthogonal methods","pmids":["12590145"],"is_preprint":false},{"year":2003,"finding":"Canoe/AF-6 acts downstream of Egfr/Ras signaling to regulate ommatidial rotation in the Drosophila eye; genetic epistasis places Canoe downstream of Egfr and Ras in a pathway linking Egfr to cytoskeletal elements (cadherins and myosin II) during this rotation process.","method":"Drosophila genetics, epistasis analysis, loss-of-function and gain-of-function alleles","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in Drosophila, single lab, pathway position defined but mechanistic details limited","pmids":["14507782"],"is_preprint":false},{"year":2005,"finding":"NMR solution structure of the AF-6 PDZ domain was determined; the domain has a unique hydrophilic residue (Gln70) at alphaB1, making its binding groove distinct from canonical class II PDZ domains; the AF-6 PDZ domain binds the C-terminal peptide of Neurexin (class II motif) with Kd ~408 nM and Bcr C-terminal peptide (class I motif) with Kd ~2230 nM.","method":"NMR structure determination, BIACORE surface plasmon resonance, NMR chemical shift perturbation, molecular dynamics simulation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with quantitative binding measurements by SPR and chemical shift perturbation, multiple orthogonal methods","pmids":["15684424"],"is_preprint":false},{"year":2005,"finding":"Activated Rap1 recruits AF-6 to the plasma membrane and induces dendritic spine neck elongation; inactive Rap1 dissociates AF-6 from membrane and induces spine enlargement; Rap1 bimodally regulates spine morphology through AF-6 following NMDA receptor activation in cortical neurons.","method":"Live imaging in cultured neurons, dominant-active and dominant-negative Rap1 constructs, NMDA receptor activation, spine morphology quantification","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple constructs, live imaging, functional spine morphology readout, receptor activation, single lab but multiple orthogonal approaches","pmids":["16301177"],"is_preprint":false},{"year":2005,"finding":"AF6 negatively regulates Rap1-induced integrin-mediated cell adhesion: AF6 knockdown enhanced Rap1-induced adhesion, while overexpression inhibited it; AF6-mediated inhibition correlated with increased Rap-GTP levels, suggesting AF6 sequesters Rap-GTP in an unproductive complex.","method":"RNA interference knockdown, overexpression, integrin-mediated cell adhesion assay, Rap-GTP pull-down","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi and overexpression with functional adhesion readout, single lab","pmids":["16051602"],"is_preprint":false},{"year":2006,"finding":"AF6 isoform 3 (AF6i3), which has an additional C-terminal F-actin-binding site, stabilizes E-cadherin-dependent intercellular adhesion by linking the E-cadherin/catenin complex to F-actin; knockdown of AF6i3 impairs E-cadherin association with F-actin and p120-catenin and increases cell migration directionality.","method":"RNAi knockdown, isoform-specific expression, wound closure assay, co-immunoprecipitation, F-actin sedimentation","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — isoform-specific knockdown with functional readout and biochemical evidence, single lab","pmids":["16882694"],"is_preprint":false},{"year":2006,"finding":"Canoe/AF-6 physically interacts with Ras, Notch, and Dishevelled (Dsh) in Drosophila; Cno represses Wingless/Wnt, Ras-MAPK, and Notch signaling through these direct interactions, acting as a modulator of signaling cross-communication.","method":"Yeast two-hybrid, co-immunoprecipitation, Drosophila genetics, loss-of-function analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding partners confirmed by two-hybrid and co-IP, functional repression shown genetically, single lab","pmids":["17183697"],"is_preprint":false},{"year":2007,"finding":"The AF-6 PDZ domain binds the C-terminus of c-Src; PDZ-mediated binding of c-Src to AF-6 interferes with phosphorylation of c-Src at Tyr527 by CSK and reduces c-Src autophosphorylation at Tyr416, resulting in a moderately activated c-Src; AF-6 recruits c-Src to cell-cell contact sites.","method":"PDZ domain binding assay, mutagenesis, kinase assay, co-immunoprecipitation, co-localization, knockdown of AF-6","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assays, mutagenesis of PDZ-binding residue, co-IP, and cell localization; multiple orthogonal methods","pmids":["17491594"],"is_preprint":false},{"year":2007,"finding":"Solution structure of the AF-6 PDZ domain in complex with a Bcr C-terminal peptide was determined by NMR; the complex exhibits a noncanonical PDZ/peptide binding mode; phosphorylation of AF-6 by Bcr kinase induces a conformational change making the PDZ domain accessible for efficient Bcr binding; millisecond dynamics mediate allosteric signal transmission through the PDZ domain.","method":"NMR structure determination, relaxation dispersion NMR, backbone dynamics analysis","journal":"Protein science / Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with dynamic analysis and allosteric mechanism defined by multiple NMR methods","pmids":["17473018","18052198"],"is_preprint":false},{"year":2007,"finding":"The short isoform of AF-6 (s-afadin) is a dual-residency protein that can localize to either plasma membrane or nucleus, forming nuclear bodies in a cell-cycle-dependent and transcription-dependent manner; the long isoform (l-afadin) cannot localize to the nucleus; nuclear body formation is regulated by growth factor receptor signaling and cytoplasmic tyrosine kinases.","method":"GFP-tagged isoform expression, live-cell imaging, cell cycle inhibition, transcription inhibition, growth factor treatment","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — isoform-specific expression with multiple pharmacological perturbations, single lab","pmids":["17013812"],"is_preprint":false},{"year":2008,"finding":"AF-6 scaffolds N-cadherin and the Rac1-GEF kalirin-7 at synapses; N-cadherin clustering promotes AF-6/kalirin-7 interaction and recruitment, increasing Rac1 at spines and PAK phosphorylation; N-cadherin-dependent spine enlargement requires AF-6 and kalirin-7; disruption of N-cadherin uncouples AF-6/kalirin-7 causing thin, Rac1-poor spines.","method":"Co-immunoprecipitation, co-localization, N-cadherin clustering assay, Rac1 pull-down, PAK phosphorylation, dominant-negative and siRNA knockdown","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, functional rescue, RNAi, dominant-negative, and biochemical pathway measurements; multiple orthogonal methods","pmids":["18550750"],"is_preprint":false},{"year":2010,"finding":"The RA1 domain of AF-6 is the minimal region sufficient for MLL-AF6-mediated myeloid progenitor immortalization in vitro and leukemogenesis in vivo; the RA1 domain mediates self-association of MLL-AF6, and this self-association (not Ras-binding per se) is the dominant oncogenic mechanism, as Ras-binding point mutations do not abrogate oncogenesis.","method":"Retroviral transduction of truncation/point-mutant constructs, myeloid progenitor immortalization assay, mouse leukemia model, self-association assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic domain mapping with multiple mutants, in vitro and in vivo functional readouts, single lab","pmids":["20395419"],"is_preprint":false},{"year":2011,"finding":"AF6 and MUPP1 associate with connexin36 (Cx36) at neuronal gap junctions in rodent brain; this interaction requires the C-terminal PDZ-binding motif of Cx36 for binding the single PDZ domain of AF-6; AF-6 and MUPP1 co-localize with Cx36 in many brain areas.","method":"Co-immunoprecipitation, GST pull-down, immunofluorescence co-localization in brain tissue","journal":"The European journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and pull-down from native tissue with domain-binding confirmation, single lab","pmids":["22211808"],"is_preprint":false},{"year":2012,"finding":"Canoe (Drosophila AF-6) forms a complex with Robo receptor in vivo and is required for Slit-Robo axon guidance signaling at the CNS midline; in cno mutants, Robo is mislocalized from growth cone filopodia, and the commissureless phenotype (excess Robo on all neurons) is suppressed in comm, cno double mutants, placing Cno as a positive regulator of Robo localization and signaling.","method":"Co-immunoprecipitation from embryo lysates, genetic epistasis in Drosophila, immunostaining for Robo localization, mutant analysis","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP from native tissue plus genetic epistasis with multiple allele combinations, functional axon guidance phenotype","pmids":["22815517"],"is_preprint":false},{"year":2013,"finding":"MLL-AF6 leukemia requires continued DOT1L H3K79 methyltransferase activity; MLL-AF6 target genes show markedly elevated H3K79 dimethylation; conditional knockout of Dot1l inhibits MLL-AF6 leukemogenesis; MLL-AF6-transformed cells are sensitive to the DOT1L inhibitor EPZ0004777, which reduces proliferation and causes cell cycle arrest.","method":"Gene-expression analysis, ChIP-seq, conditional Dot1l knockout mouse, small molecule inhibitor treatment","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO plus ChIP-seq and pharmacological inhibition with functional readouts, multiple orthogonal methods","pmids":["23361907"],"is_preprint":false},{"year":2013,"finding":"AF-6 is a novel parkin-interacting protein; parkin interacts with AF-6 via its C-terminus with the AF-6 PDZ region, leading to AF-6 ubiquitination and proteasomal degradation; mitochondrial AF-6 is not degraded by parkin but co-localizes with parkin and enhances PINK1/parkin-mediated mitophagy; parkin/PINK1 disease mutants are insensitive to AF-6 stimulation.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, mitophagy assay, disease-mutant analysis, co-localization","journal":"Human molecular genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, ubiquitination assay, functional mitophagy readout, disease-mutant mechanistic dissection; multiple orthogonal methods","pmids":["23393160"],"is_preprint":false},{"year":2014,"finding":"In MLL-AF6-rearranged AML cells, AF6 is sequestered in the nucleus (in contrast to cytoplasmic localization in healthy cells), leading to aberrant activation of RAS-GTP; silencing MLL-AF6 restores cytoplasmic AF6 localization and reduces RAS-GTP levels; co-silencing of MLL-AF6 and AF6 reverses the RAS-GTP reduction, confirming that nuclear sequestration of AF6 by MLL-AF6 drives RAS pathway activation.","method":"Subcellular fractionation, immunofluorescence, RAS-GTP pull-down, siRNA knockdown, clonogenic assay","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including fractionation, RAS-GTP pull-down, siRNA rescue experiments; mechanistic pathway clearly established","pmids":["24695851"],"is_preprint":false},{"year":2015,"finding":"AF6 depletion in pancreatic cancer cells upregulates Snail protein expression through increased formation of a Dvl2-FOXE1 complex on the Snail promoter; this requires nuclear localization of AF6; AF6 loss promotes cell proliferation and metastasis via this Dvl2-FOXE1-Snail axis.","method":"siRNA knockdown, promoter ChIP assay, nuclear localization studies, in vitro migration/invasion assays, in vivo tumor model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP assay on Snail promoter with mechanistic pathway mapping, single lab","pmids":["26013125"],"is_preprint":false},{"year":2017,"finding":"AF-6 overexpression in Drosophila parkin null and pink1 null flies rescues mitochondrial pathology and locomotion deficits; AF-6 overexpression also ameliorates pathological phenotypes in LRRK2-G2019S flies; silencing endogenous AF-6 aggravates LRRK2 mutant fly phenotypes; AF-6 overexpression protects dopaminergic neurons against rotenone.","method":"Transgenic Drosophila overexpression and RNAi, locomotion assay, mitochondrial morphology analysis, rotenone treatment model","journal":"Frontiers in cellular neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic rescue in multiple Drosophila PD models, single lab, functional phenotype readouts","pmids":["28848400"],"is_preprint":false},{"year":2017,"finding":"AF6 employs a non-canonical, evolutionarily conserved alpha-helix to bind RAS (distinct from other effectors); in all patients with MLL-AF6 translocations, the fusion protein lacks exactly this helix, exposing hydrophobic residues that drive dimerization; dimerization (oligomerization) is the dominant oncogenic mechanism for MLL-AF6, and inhibiting dimerization abrogates leukemogenesis in mice.","method":"Structural analysis, NMR/biochemical binding, proteomic interaction mapping (proximity biotinylation), mouse leukemia model with dimerization-disrupting mutants","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — structural evidence plus proteomic data plus in vivo mouse model, multiple orthogonal methods","pmids":["29062045"],"is_preprint":false},{"year":2019,"finding":"AF6 directly regulates IRS1/AKT insulin signaling by interacting with SHP2 and modulating SHP2 tyrosine phosphatase activity; liver-specific AF6 knockout mice show enhanced insulin sensitivity and liver glycogen storage, while AF6 overexpression causes insulin resistance; AF6-SHP2 interaction is required for this effect.","method":"Liver-specific knockout mice, adenovirus overexpression, co-immunoprecipitation, SHP2 phosphatase activity assay, glucose tolerance test, insulin signaling (pAKT) measurement","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific KO plus overexpression plus enzymatic activity assay plus co-IP; multiple orthogonal methods with in vivo confirmation","pmids":["31127058"],"is_preprint":false},{"year":2023,"finding":"AF6 promotes necroptosis by interacting with the intermediate domain of RIPK1 and regulating RIPK1 ubiquitination via the deubiquitylase USP21; hepatocyte-specific AF6 deletion protects against TNFα-induced necroptosis in NASH and SIRS mouse models; overexpression of AF6 accelerates TNFα-induced necroptotic mortality.","method":"Co-immunoprecipitation, ubiquitination assay, hepatocyte-specific knockout mice, adenoviral overexpression, necroptosis assay, in vivo liver disease models","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP defining domain interaction, ubiquitination assay, tissue-specific KO, and in vivo disease models; multiple orthogonal methods","pmids":["37828052"],"is_preprint":false},{"year":2025,"finding":"PAK4 recruitment to multicellular vertices requires the scaffolding protein Afadin (AFDN); Afdn-KO cells exhibit severe junctional defects and reduced barrier function; artificial targeting of PAK4 to junctions in Afdn-KO cells partially restores junctional continuity and barrier function, demonstrating that Afadin acts upstream of PAK4 in vertex remodeling.","method":"Afdn knockout, PAK4 inhibition/knockout, targeted PAK4 expression in Afdn-KO cells, MDCK live imaging, Xenopus embryo live imaging, barrier function assay","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic rescue experiment with functional readout, multiple model systems, preprint not yet peer-reviewed","pmids":["bio_10.1101_2025.10.02.680170"],"is_preprint":true}],"current_model":"AFDN (AF-6/afadin) is a multidomain scaffold protein that localizes to adherens and tight junctions where it directly binds activated Ras and Rap1 GTPases (via its RA1 domain), the tight junction protein ZO-1, Eph receptor tyrosine kinases, JAM, profilin, and c-Src (via its PDZ domain), thereby linking cell-surface adhesion complexes to the actin cytoskeleton and regulating Ras/Rap1 signaling, integrin-mediated adhesion, dendritic spine morphology, and epithelial polarity; AF-6 is itself regulated post-translationally by FAM/USP9X deubiquitination, Bcr kinase phosphorylation, and Eph-receptor-dependent phosphorylation, and in MLL-AF6 leukemia the fusion protein sequesters AF-6 nuclear localization to aberrantly activate RAS-GTP while the RA1-domain-driven dimerization of the fusion protein is the dominant oncogenic mechanism."},"narrative":{"mechanistic_narrative":"AFDN (AF-6/afadin) is a multidomain junctional scaffold that couples cell-surface adhesion receptors and small-GTPase signaling to the actin cytoskeleton, thereby controlling epithelial junction integrity, cell polarity, and adhesion [PMID:9348294, PMID:10469590]. Through its N-terminal Ras-association (RA1) domain it directly and selectively binds GTP-loaded Ras and, with highest affinity, Rap1A, stabilizing a defined Ras conformer and inhibiting nucleotide dissociation and GAP-stimulated hydrolysis [PMID:8557659, PMID:10224125, PMID:10922060]; this RA1-Ras interaction is mutually exclusive with AF-6 binding to the tight-junction protein ZO-1 [PMID:9348294, PMID:10334923]. Its single PDZ domain — structurally distinguished by a noncanonical binding groove — engages the C-termini of Eph receptor tyrosine kinases, JAM, neurexin, connexin36, Bcr kinase, and c-Src, integrating receptor and kinase inputs at cell contacts [PMID:9707552, PMID:10856295, PMID:15684424, PMID:17491594, PMID:22211808]. AF-6 acts as a Rap1 effector that bimodally remodels dendritic spines and scaffolds the Rac1-GEF kalirin-7 downstream of N-cadherin to drive spine enlargement, and it organizes integrin-mediated adhesion by simultaneously binding Rap1-GTP and the Rap1-GAP SPA-1 [PMID:12590145, PMID:16301177, PMID:18550750]. AF-6 stability and activity are set post-translationally by FAM/USP9X-mediated deubiquitination, Bcr-dependent phosphorylation that allosterically opens the PDZ domain, and Eph-receptor phosphorylation [PMID:9722616, PMID:9707552, PMID:17473018, PMID:18052198]. Beyond junctions, AF-6 modulates insulin signaling via SHP2 [PMID:31127058], promotes RIPK1-dependent necroptosis [PMID:37828052], and enhances PINK1/parkin mitophagy [PMID:23393160]. AFDN is recurrently fused to MLL in t(6;11) leukemia; the MLL-AF6 fusion retains the RA1 domain, and RA1-driven self-association/dimerization — rather than Ras binding per se — is the dominant oncogenic mechanism, with the fusion sequestering AF-6 in the nucleus to aberrantly elevate RAS-GTP and requiring continued DOT1L H3K79 methylation [PMID:20395419, PMID:23361907, PMID:24695851, PMID:29062045].","teleology":[{"year":1993,"claim":"Established AFDN's existence and disease relevance by identifying it as the MLL/ALL-1 fusion partner in t(6;11) leukemia and recognizing its PDZ/GLGF motif, hinting at a junctional function.","evidence":"Molecular cloning and cDNA characterization of the translocation breakpoint","pmids":["8242616"],"confidence":"Medium","gaps":["No functional role for native AF-6 defined","Mechanism by which the fusion transforms cells unknown"]},{"year":1996,"claim":"Defined AF-6 as a bona fide Ras effector by showing its N-terminal domain binds GTP-bound but not GDP-bound or effector-mutant Ras, linking adhesion scaffolding to Ras signaling.","evidence":"GST affinity chromatography with recombinant proteins and nucleotide/effector-mutant controls","pmids":["8557659"],"confidence":"High","gaps":["Functional consequence of Ras binding at junctions not yet established","GTPase selectivity not fully mapped"]},{"year":1997,"claim":"Placed AF-6 at tight junctions in complex with ZO-1 and showed activated Ras competes for the same domain, suggesting Ras toggles AF-6 between adhesion and signaling roles.","evidence":"Immunoelectron microscopy, in vitro binding, and Co-IP in MDCK cells with activated-Ras overexpression","pmids":["9348294"],"confidence":"High","gaps":["Whether Ras-ZO-1 competition operates in vivo not shown","Downstream cytoskeletal effects undefined"]},{"year":1997,"claim":"Explained how the MLL-AF6 fusion gains nuclear access despite cytoplasmic native AF-6, attributing nuclear localization to the MLL AT-hook region.","evidence":"Immunofluorescence and cell fractionation of the chimeric protein","pmids":["9349501"],"confidence":"Medium","gaps":["Functional consequence of nuclear mislocalization not yet linked to transformation","Single-lab localization data"]},{"year":1998,"claim":"Identified two post-translational control points — FAM/USP9X deubiquitination and Eph-receptor-dependent phosphorylation via the PDZ domain — establishing AF-6 as a regulated receptor adaptor.","evidence":"Protein purification, ubiquitination assays, yeast two-hybrid, Co-IP, and in-cell phosphorylation assays","pmids":["9722616","9707552"],"confidence":"High","gaps":["Physiological signals that trigger AF-6 ubiquitination unclear","Downstream effect of Eph phosphorylation on AF-6 function undefined"]},{"year":1999,"claim":"Quantitatively characterized AF-6's GTPase engagement (Raf-like affinity for Ras, highest affinity for Rap1A, conformer stabilization, inhibition of nucleotide exchange/GAP) and showed RA-domain mutation abolishes Ras binding and disrupts Ras-driven transcription.","evidence":"31P NMR, kinetic analysis, GST binding, site-directed mutagenesis, and c-fos reporter assays","pmids":["10224125","10334923"],"confidence":"High","gaps":["Cellular outcome of Rap1A vs Ras selectivity not resolved","M-Ras interaction rests on a single Co-IP (#10)"]},{"year":1999,"claim":"Demonstrated AF-6 is essential for epithelial junction organization and polarity in vivo, defining its developmental role through an embryonic-lethal knockout.","evidence":"Targeted mouse knockout with histology, immunofluorescence, and electron microscopy","pmids":["10469590"],"confidence":"High","gaps":["Molecular pathway connecting AF-6 loss to junctional collapse not dissected","Tissue-specific requirements not separable in null embryos"]},{"year":1999,"claim":"Connected AF-6 to Eph receptor signaling at synapses, showing native complex formation, postsynaptic co-localization, and phosphorylation by Eph kinases.","evidence":"Co-IP from brain lysates, immunohistochemistry, and in vitro phosphorylation","pmids":["9922461"],"confidence":"High","gaps":["Functional synaptic consequence not yet shown","Link to cytoskeletal remodeling undefined"]},{"year":2000,"claim":"Expanded the PDZ interactome to JAM and showed AF-6 bridges JAM, ZO-1, and profilin, identifying AF-6 as a direct junction-to-actin link via profilin.","evidence":"Reciprocal in vitro/in vivo binding, truncation mutants, yeast two-hybrid, and co-localization in endothelial cells","pmids":["10856295","10922060"],"confidence":"High","gaps":["Functional impact of profilin recruitment on actin dynamics not measured","Rap1 binding does not perturb AF-6 localization, leaving its role at junctions unclear"]},{"year":2003,"claim":"Resolved AF-6 as a Rap1 effector and signaling cross-talk hub: it scaffolds SPA-1 and Rap1 to control integrin adhesion, forms a Bcr-AF-6-Ras trimeric complex modulating ERK signaling, and acts downstream of Rap1/Egfr/Ras in Drosophila morphogenesis.","evidence":"Co-IP, kinase assays, mutagenesis, adhesion and reporter assays, and Drosophila genetic epistasis","pmids":["12590145","12808105","14504224","14507782"],"confidence":"High","gaps":["How AF-6 integrates competing Ras/Rap1 inputs not fully resolved","Drosophila pathway position established but mammalian mechanism for ommatidial-type processes limited"]},{"year":2005,"claim":"Defined the structural basis of AF-6 PDZ specificity and established AF-6 as the effector through which Rap1 bimodally controls dendritic spine morphology.","evidence":"NMR structure with SPR binding measurements; live neuronal imaging with dominant Rap1 constructs and NMDA activation; RNAi/overexpression adhesion assays","pmids":["15684424","16301177","16051602"],"confidence":"High","gaps":["How Rap1-GTP/GDP switching mechanically reshapes spines unresolved","Whether AF-6 sequesters Rap-GTP unproductively rests on correlation (#19)"]},{"year":2006,"claim":"Showed an isoform with an extra F-actin-binding site (AF6i3) stabilizes E-cadherin/catenin–actin coupling, and that Drosophila Canoe represses Wnt, Ras-MAPK, and Notch signaling, casting AF-6 as a multipathway modulator.","evidence":"Isoform-specific RNAi, F-actin sedimentation, wound assays; yeast two-hybrid, Co-IP, and Drosophila loss-of-function","pmids":["16882694","17183697"],"confidence":"Medium","gaps":["Mammalian relevance of Drosophila multipathway repression not tested","Isoform-specific functions in vivo unmapped"]},{"year":2007,"claim":"Established that AF-6 PDZ binding activates c-Src by blocking CSK-mediated inhibitory phosphorylation, and revealed Bcr-phosphorylation-driven allosteric opening of the PDZ domain plus a nuclear-competent short isoform.","evidence":"NMR complex structure with relaxation-dispersion dynamics; PDZ binding/kinase assays with mutagenesis; GFP isoform imaging","pmids":["17491594","17473018","18052198","17013812"],"confidence":"High","gaps":["Physiological trigger for s-afadin nuclear-body formation unclear","Whether allosteric PDZ regulation extends to non-Bcr ligands untested"]},{"year":2008,"claim":"Defined a synaptic mechanism in which N-cadherin clustering recruits the AF-6/kalirin-7 module to raise Rac1 activity and drive spine enlargement.","evidence":"Co-IP, N-cadherin clustering, Rac1 pull-down, PAK phosphorylation, and siRNA/dominant-negative rescue","pmids":["18550750"],"confidence":"High","gaps":["How this integrates with the Rap1-driven spine pathway not reconciled","In vivo requirement not tested"]},{"year":2010,"claim":"Pinpointed the RA1 domain as the minimal element for MLL-AF6 transformation and showed self-association, not Ras binding, drives leukemogenesis.","evidence":"Truncation/point-mutant retroviral transduction, immortalization assays, and a mouse leukemia model","pmids":["20395419"],"confidence":"High","gaps":["Structural basis of RA1 self-association not yet defined here","Downstream effectors of fusion dimerization unknown"]},{"year":2012,"claim":"Extended AF-6/Canoe function to axon guidance by showing it complexes with Robo and is required for proper Robo localization and Slit-Robo signaling at the midline.","evidence":"Co-IP from embryo lysates, Robo immunostaining, and genetic epistasis in Drosophila","pmids":["22815517"],"confidence":"High","gaps":["Mammalian Robo-AFDN relevance not addressed","Mechanism of Robo localization control undefined"]},{"year":2013,"claim":"Mechanistically linked the MLL-AF6 fusion to RAS pathway hyperactivation via nuclear sequestration of AF6 and established dependence on DOT1L H3K79 methyltransferase activity as a therapeutic vulnerability.","evidence":"Subcellular fractionation, RAS-GTP pull-down, siRNA rescue; ChIP-seq, conditional Dot1l knockout, and DOT1L inhibitor treatment","pmids":["24695851","23361907"],"confidence":"High","gaps":["How nuclear AF6 mechanistically elevates cytoplasmic RAS-GTP not fully resolved","Whether DOT1L and RAS effects are mechanistically coupled unclear"]},{"year":2013,"claim":"Implicated AF-6 in mitochondrial quality control by showing parkin-mediated degradation of AF-6 and AF-6 enhancement of PINK1/parkin mitophagy.","evidence":"Co-IP, ubiquitination/proteasome assays, mitophagy assay, and disease-mutant analysis","pmids":["23393160"],"confidence":"High","gaps":["How a junctional scaffold acts at mitochondria mechanistically unclear","Physiological relevance to Parkinson disease not established in mammals"]},{"year":2015,"claim":"Defined a tumor-suppressive role for AF6 in pancreatic cancer, where its loss permits a nuclear Dvl2-FOXE1-Snail axis driving proliferation and metastasis.","evidence":"siRNA knockdown, promoter ChIP, migration/invasion assays, and in vivo tumor model","pmids":["26013125"],"confidence":"Medium","gaps":["How AF6 normally restrains the Dvl2-FOXE1 complex undefined","Single-lab mechanism"]},{"year":2017,"claim":"Solved the structural rationale for MLL-AF6 oncogenesis: the fusion lacks a noncanonical Ras-binding alpha-helix, exposing hydrophobic residues that drive dimerization, the dominant transforming activity that can be therapeutically disrupted.","evidence":"Structural/NMR analysis, proximity-biotinylation proteomics, and mouse models with dimerization-disrupting mutants; Drosophila PD-model rescue separately implicated AF-6 in mitochondrial protection","pmids":["29062045","28848400"],"confidence":"High","gaps":["Druggability of the dimerization interface in patients untested","Mechanism of AF-6 neuroprotection in PD models remains correlative"]},{"year":2019,"claim":"Identified a metabolic role for AF6 as a regulator of insulin signaling through SHP2, with liver AF6 levels setting insulin sensitivity in vivo.","evidence":"Liver-specific knockout and overexpression, Co-IP, SHP2 phosphatase assays, and glucose/insulin signaling readouts","pmids":["31127058"],"confidence":"High","gaps":["How AF6 modulates SHP2 catalytic activity mechanistically unclear","Connection to AF6's junctional scaffolding role unexplored"]},{"year":2023,"claim":"Revealed AF6 as a pro-necroptotic scaffold that binds RIPK1 and tunes its ubiquitination via USP21, linking AF6 to inflammatory cell death in liver disease.","evidence":"Co-IP defining domain interaction, ubiquitination assays, hepatocyte-specific knockout, and in vivo NASH/SIRS models","pmids":["37828052"],"confidence":"High","gaps":["How AF6 recruits USP21 to RIPK1 not detailed","Relationship to AF6's other ubiquitination-regulated functions unexplored"]},{"year":2025,"claim":"Positioned Afadin upstream of PAK4 in junctional vertex remodeling, with PAK4 mistargeting rescue restoring barrier function in Afadin-null cells.","evidence":"Afdn knockout, PAK4 inhibition/targeting, and live imaging in MDCK and Xenopus with barrier assays (preprint)","pmids":["bio_10.1101_2025.10.02.680170"],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Molecular mechanism of PAK4 recruitment by Afadin undefined"]},{"year":null,"claim":"How AF-6's many context-specific roles — junctional/cytoskeletal scaffolding, GTPase regulation, mitophagy, insulin signaling, and necroptosis — are coordinated by a single multidomain protein, and which interactions are dominant in each tissue, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model integrating junctional vs metabolic vs cell-death functions","Isoform-specific contributions to each role incompletely mapped","Therapeutic targeting of the MLL-AF6 dimerization interface unproven in humans"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[2,11,15,25]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,22,35]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[12,20]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[2,9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,18]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,31]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[24,31,32]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[12,20]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,7,12,14]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell 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Nectin- and actin-filament-binding protein that connects nectin to the actin cytoskeleton (PubMed:11024295). May play a key role in the organization of epithelial structures of the embryonic ectoderm (By similarity). Essential for the organization of adherens junctions (PubMed:30463011)","subcellular_location":"Cell junction, adherens junction","url":"https://www.uniprot.org/uniprotkb/P55196/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AFDN","classification":"Not Classified","n_dependent_lines":39,"n_total_lines":1208,"dependency_fraction":0.03228476821192053},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/AFDN","total_profiled":1310},"omim":[{"mim_id":"609607","title":"NECTIN CELL ADHESION MOLECULE 4; NECTIN4","url":"https://www.omim.org/entry/609607"},{"mim_id":"600798","title":"NECTIN CELL ADHESION MOLECULE 2; NECTIN2","url":"https://www.omim.org/entry/600798"},{"mim_id":"600644","title":"NECTIN CELL ADHESION MOLECULE 1; NECTIN1","url":"https://www.omim.org/entry/600644"},{"mim_id":"159559","title":"AFADIN; AFDN","url":"https://www.omim.org/entry/159559"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Plasma membrane","reliability":"Enhanced"},{"location":"Cell Junctions","reliability":"Enhanced"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AFDN"},"hgnc":{"alias_symbol":["AF-6","AF6"],"prev_symbol":["MLLT4"]},"alphafold":{"accession":"P55196","domains":[{"cath_id":"3.10.20.90","chopping":"2-134_196-207","consensus_level":"medium","plddt":77.3086,"start":2,"end":207},{"cath_id":"3.10.20.90","chopping":"243-346","consensus_level":"high","plddt":82.0787,"start":243,"end":346},{"cath_id":"2.60.200.20","chopping":"417-504","consensus_level":"medium","plddt":87.8683,"start":417,"end":504},{"cath_id":"-","chopping":"599-780_946-997","consensus_level":"medium","plddt":87.7307,"start":599,"end":997},{"cath_id":"-","chopping":"793-936","consensus_level":"medium","plddt":92.1258,"start":793,"end":936},{"cath_id":"2.30.42.10","chopping":"1010-1105","consensus_level":"high","plddt":87.1506,"start":1010,"end":1105},{"cath_id":"1.10.287","chopping":"1529-1543_1553-1575","consensus_level":"medium","plddt":84.6063,"start":1529,"end":1575}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P55196","model_url":"https://alphafold.ebi.ac.uk/files/AF-P55196-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P55196-F1-predicted_aligned_error_v6.png","plddt_mean":63.22},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AFDN","jax_strain_url":"https://www.jax.org/strain/search?query=AFDN"},"sequence":{"accession":"P55196","fasta_url":"https://rest.uniprot.org/uniprotkb/P55196.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P55196/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P55196"}},"corpus_meta":[{"pmid":"10856295","id":"PMC_10856295","title":"Junctional 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research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — initial cloning with domain identification, single lab, foundational molecular characterization\",\n      \"pmids\": [\"8242616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"AF-6 and its Drosophila homolog Canoe were identified as direct binding targets of activated (GTP-bound) Ras; the N-terminal domain of AF-6 specifically binds GTPγS-Ras but not GDP-Ras or Ras with an effector-domain mutation.\",\n      \"method\": \"GST affinity chromatography, recombinant protein binding assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical reconstitution with GTP/GDP specificity controls and effector mutant controls, replicated in subsequent studies\",\n      \"pmids\": [\"8557659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"AF-6 localizes to tight junctions in polarized MDCK epithelial cells, co-distributing with ZO-1 at tight junctions as shown by immunoelectron microscopy; native and recombinant AF-6 directly interact with ZO-1 in vitro via the Ras-binding domain of AF-6; this interaction is inhibited by activated Ras.\",\n      \"method\": \"Immunoelectron microscopy, in vitro binding assay, co-immunoprecipitation, overexpression of activated Ras\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (immunoelectron microscopy, in vitro binding, Co-IP), replicated across labs\",\n      \"pmids\": [\"9348294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"MLL-AF6 chimeric fusion protein localizes to the nucleus despite AF-6 itself being cytoplasmic; the nuclear localization is conferred by the AT-hook motif-containing N-terminal region of MLL.\",\n      \"method\": \"Immunofluorescence, cell fractionation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal localization methods, single lab\",\n      \"pmids\": [\"9349501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"AF-6 is a substrate of the FAM deubiquitinating enzyme; AF-6 is ubiquitinated in intact cells, and FAM (the mammalian homolog of Drosophila fat facets) prevents ubiquitination of AF-6. FAM and AF-6 interact in vivo and in vitro and co-localize at cell-cell contacts.\",\n      \"method\": \"Protein purification, peptide sequencing, co-immunoprecipitation, in vitro binding, ubiquitination assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical purification, co-IP, in vitro binding, and direct ubiquitination assay; multiple orthogonal methods\",\n      \"pmids\": [\"9722616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The PDZ domain of AF-6 interacts with C-terminal sequences of Eph receptor tyrosine kinases (EphB3, EphA7, EphB2, EphB5, EphB6), neurexins, and Notch ligand Jagged; interaction of full-length AF-6 with EphB3 depends on kinase activity of EphB3; endogenous AF-6 is phosphorylated by EphB2 and EphB3 in a ligand-dependent manner.\",\n      \"method\": \"Yeast two-hybrid, mutational analysis, co-immunoprecipitation, phosphorylation assay in NIH 3T3 and NG108 cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — yeast two-hybrid with mutagenesis, co-IP, and in-cell phosphorylation assay; multiple orthogonal methods\",\n      \"pmids\": [\"9707552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The N-terminal Ras-association (RA) domain of AF-6 mediates interaction with activated Ras in vivo; a single amino acid mutation in this domain abolishes Ras binding; overexpression of the RA domain inhibits Ras-dependent c-fos promoter stimulation; AF-6 is co-immunoprecipitated with ZO-1 from Rat1 cells.\",\n      \"method\": \"Site-directed mutagenesis, co-immunoprecipitation, reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis combined with in vivo co-IP and functional reporter assay, consistent with multiple prior studies\",\n      \"pmids\": [\"10334923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"AF-6 binds Ras with thermodynamic and kinetic parameters similar to Raf and RalGEF; AF-6 binding stabilizes one of two major Ras conformational states (detected by 31P NMR); among Ras-related GTPases, AF-6 binds Rap1A with the highest affinity; AF-6 inhibits nucleotide dissociation and GAP activity but not intrinsic GTPase activity of Ras.\",\n      \"method\": \"Biochemical binding assay, 31P NMR spectroscopy, kinetic analysis with GTP analogs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — rigorous biophysical characterization with multiple methods (NMR, kinetics, biochemistry) in a single study\",\n      \"pmids\": [\"10224125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"AF-6 and Canoe (Drosophila homolog) cluster with Eph receptors at cell-cell contact sites; AF-6 forms a complex with endogenous Eph receptors in whole rat brain lysates, co-localizes with Eph receptors at postsynaptic membrane sites of excitatory synapses in hippocampus, and is a substrate for Eph receptor kinases.\",\n      \"method\": \"Co-immunoprecipitation from brain lysates, co-transfection with GFP-AF-6, immunohistochemistry, in vitro phosphorylation\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP from native tissue, co-localization, and phosphorylation assay; multiple orthogonal methods\",\n      \"pmids\": [\"9922461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Loss of AF-6 in mouse embryos disrupts epithelial cell-cell junctions and cell polarity: homozygous null embryos show reduced apical junction length, basolateral gaps, and loss of neuroepithelial polarity by 7.5 dpc, leading to embryonic lethality by 10 dpc.\",\n      \"method\": \"Targeted gene knockout in mice, histology, immunofluorescence, electron microscopy\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with clear developmental phenotype, multiple morphological readouts, landmark study replicated in concept by subsequent work\",\n      \"pmids\": [\"10469590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"M-Ras co-immunoprecipitates with AF-6 in mammalian cells, extending the range of Ras-subfamily GTPases that can interact with AF-6.\",\n      \"method\": \"Co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP experiment, single lab, no functional follow-up on the AF-6 interaction\",\n      \"pmids\": [\"10446149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The AF-6 PDZ domain interacts with the C-terminal PDZ-binding motif of junctional adhesion molecule (JAM); AF-6 and ZO-1 can both be co-precipitated with JAM from endothelial cell extracts; loss of the JAM C-terminus disrupts its co-distribution with AF-6 at cell contacts.\",\n      \"method\": \"Co-immunoprecipitation from endothelial cells, in vitro binding, co-localization by immunofluorescence, truncation mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal in vitro and in vivo binding with truncation mutants and functional localization readout\",\n      \"pmids\": [\"10856295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The first Ras-binding domain of AF-6 mediates interaction with Ras and preferentially binds Rap1A over oncogenic Ras GTPases; AF-6 interacts with Rap1 in vivo in mammalian cells; Rap1A does not perturb AF-6 localization at cell-cell junctions. AF-6 binds profilin, an actin polymerization regulator, making AF-6 the only junctional component known to interact with profilin.\",\n      \"method\": \"In vitro binding with GST-fusion proteins, co-immunoprecipitation in mammalian cells, yeast two-hybrid, co-localization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (in vitro binding, Co-IP, yeast two-hybrid), two distinct binding partners characterized\",\n      \"pmids\": [\"10922060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Canoe (Drosophila AF-6 homolog) acts as an effector of Rap1 in vivo during dorsal closure; Cno binds activated Rap1 in a two-hybrid assay, the two co-localize at adherens junctions, and genetic epistasis shows Rap1 acts upstream of Cno in the same pathway. Cno has a Rap1-independent function in JNK pathway activation.\",\n      \"method\": \"Yeast two-hybrid, co-localization, genetic epistasis in Drosophila, loss-of-function analysis\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid binding plus genetic epistasis in Drosophila with multiple alleles; Rap1-independent role also defined\",\n      \"pmids\": [\"14504224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"The Bcr protein kinase is a ligand for the PDZ domain of AF-6; Bcr phosphorylates AF-6 and phosphorylation enables efficient Bcr binding to AF-6's PDZ domain; Bcr, AF-6, and Ras form a trimeric complex; Bcr increases AF-6's affinity for Ras and a phosphorylation-site mutant of AF-6 shows reduced Ras binding; Bcr (but not PDZ-binding-deficient Bcr mutants) interferes with Ras-Raf/MEK/ERK signaling.\",\n      \"method\": \"Co-immunoprecipitation, kinase assay, mutagenesis, co-localization, reporter assay for ERK pathway\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — kinase assay with mutagenesis, reciprocal co-IP, and functional pathway readout; multiple orthogonal methods\",\n      \"pmids\": [\"12808105\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"AF-6 controls integrin-mediated cell adhesion by scaffolding both SPA-1 (a Rap1 GAP) and Rap1-GTP via distinct domains: SPA-1 binds the PDZ domain of AF-6 via an internal PDZ-ligand motif; Rap1V12 binds the N-terminal domain; AF-6 overexpression inhibits Rap1-GTP levels and β1 integrin-mediated adhesion in a SPA-1-dependent context.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding with truncation mutants and point mutants, cell adhesion assay, immunostaining\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple binding assays with deletion/point mutants plus functional cell adhesion readout, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12590145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Canoe/AF-6 acts downstream of Egfr/Ras signaling to regulate ommatidial rotation in the Drosophila eye; genetic epistasis places Canoe downstream of Egfr and Ras in a pathway linking Egfr to cytoskeletal elements (cadherins and myosin II) during this rotation process.\",\n      \"method\": \"Drosophila genetics, epistasis analysis, loss-of-function and gain-of-function alleles\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in Drosophila, single lab, pathway position defined but mechanistic details limited\",\n      \"pmids\": [\"14507782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NMR solution structure of the AF-6 PDZ domain was determined; the domain has a unique hydrophilic residue (Gln70) at alphaB1, making its binding groove distinct from canonical class II PDZ domains; the AF-6 PDZ domain binds the C-terminal peptide of Neurexin (class II motif) with Kd ~408 nM and Bcr C-terminal peptide (class I motif) with Kd ~2230 nM.\",\n      \"method\": \"NMR structure determination, BIACORE surface plasmon resonance, NMR chemical shift perturbation, molecular dynamics simulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with quantitative binding measurements by SPR and chemical shift perturbation, multiple orthogonal methods\",\n      \"pmids\": [\"15684424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Activated Rap1 recruits AF-6 to the plasma membrane and induces dendritic spine neck elongation; inactive Rap1 dissociates AF-6 from membrane and induces spine enlargement; Rap1 bimodally regulates spine morphology through AF-6 following NMDA receptor activation in cortical neurons.\",\n      \"method\": \"Live imaging in cultured neurons, dominant-active and dominant-negative Rap1 constructs, NMDA receptor activation, spine morphology quantification\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple constructs, live imaging, functional spine morphology readout, receptor activation, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"16301177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AF6 negatively regulates Rap1-induced integrin-mediated cell adhesion: AF6 knockdown enhanced Rap1-induced adhesion, while overexpression inhibited it; AF6-mediated inhibition correlated with increased Rap-GTP levels, suggesting AF6 sequesters Rap-GTP in an unproductive complex.\",\n      \"method\": \"RNA interference knockdown, overexpression, integrin-mediated cell adhesion assay, Rap-GTP pull-down\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi and overexpression with functional adhesion readout, single lab\",\n      \"pmids\": [\"16051602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"AF6 isoform 3 (AF6i3), which has an additional C-terminal F-actin-binding site, stabilizes E-cadherin-dependent intercellular adhesion by linking the E-cadherin/catenin complex to F-actin; knockdown of AF6i3 impairs E-cadherin association with F-actin and p120-catenin and increases cell migration directionality.\",\n      \"method\": \"RNAi knockdown, isoform-specific expression, wound closure assay, co-immunoprecipitation, F-actin sedimentation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isoform-specific knockdown with functional readout and biochemical evidence, single lab\",\n      \"pmids\": [\"16882694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Canoe/AF-6 physically interacts with Ras, Notch, and Dishevelled (Dsh) in Drosophila; Cno represses Wingless/Wnt, Ras-MAPK, and Notch signaling through these direct interactions, acting as a modulator of signaling cross-communication.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, Drosophila genetics, loss-of-function analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding partners confirmed by two-hybrid and co-IP, functional repression shown genetically, single lab\",\n      \"pmids\": [\"17183697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The AF-6 PDZ domain binds the C-terminus of c-Src; PDZ-mediated binding of c-Src to AF-6 interferes with phosphorylation of c-Src at Tyr527 by CSK and reduces c-Src autophosphorylation at Tyr416, resulting in a moderately activated c-Src; AF-6 recruits c-Src to cell-cell contact sites.\",\n      \"method\": \"PDZ domain binding assay, mutagenesis, kinase assay, co-immunoprecipitation, co-localization, knockdown of AF-6\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assays, mutagenesis of PDZ-binding residue, co-IP, and cell localization; multiple orthogonal methods\",\n      \"pmids\": [\"17491594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Solution structure of the AF-6 PDZ domain in complex with a Bcr C-terminal peptide was determined by NMR; the complex exhibits a noncanonical PDZ/peptide binding mode; phosphorylation of AF-6 by Bcr kinase induces a conformational change making the PDZ domain accessible for efficient Bcr binding; millisecond dynamics mediate allosteric signal transmission through the PDZ domain.\",\n      \"method\": \"NMR structure determination, relaxation dispersion NMR, backbone dynamics analysis\",\n      \"journal\": \"Protein science / Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with dynamic analysis and allosteric mechanism defined by multiple NMR methods\",\n      \"pmids\": [\"17473018\", \"18052198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The short isoform of AF-6 (s-afadin) is a dual-residency protein that can localize to either plasma membrane or nucleus, forming nuclear bodies in a cell-cycle-dependent and transcription-dependent manner; the long isoform (l-afadin) cannot localize to the nucleus; nuclear body formation is regulated by growth factor receptor signaling and cytoplasmic tyrosine kinases.\",\n      \"method\": \"GFP-tagged isoform expression, live-cell imaging, cell cycle inhibition, transcription inhibition, growth factor treatment\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isoform-specific expression with multiple pharmacological perturbations, single lab\",\n      \"pmids\": [\"17013812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"AF-6 scaffolds N-cadherin and the Rac1-GEF kalirin-7 at synapses; N-cadherin clustering promotes AF-6/kalirin-7 interaction and recruitment, increasing Rac1 at spines and PAK phosphorylation; N-cadherin-dependent spine enlargement requires AF-6 and kalirin-7; disruption of N-cadherin uncouples AF-6/kalirin-7 causing thin, Rac1-poor spines.\",\n      \"method\": \"Co-immunoprecipitation, co-localization, N-cadherin clustering assay, Rac1 pull-down, PAK phosphorylation, dominant-negative and siRNA knockdown\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, functional rescue, RNAi, dominant-negative, and biochemical pathway measurements; multiple orthogonal methods\",\n      \"pmids\": [\"18550750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The RA1 domain of AF-6 is the minimal region sufficient for MLL-AF6-mediated myeloid progenitor immortalization in vitro and leukemogenesis in vivo; the RA1 domain mediates self-association of MLL-AF6, and this self-association (not Ras-binding per se) is the dominant oncogenic mechanism, as Ras-binding point mutations do not abrogate oncogenesis.\",\n      \"method\": \"Retroviral transduction of truncation/point-mutant constructs, myeloid progenitor immortalization assay, mouse leukemia model, self-association assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic domain mapping with multiple mutants, in vitro and in vivo functional readouts, single lab\",\n      \"pmids\": [\"20395419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AF6 and MUPP1 associate with connexin36 (Cx36) at neuronal gap junctions in rodent brain; this interaction requires the C-terminal PDZ-binding motif of Cx36 for binding the single PDZ domain of AF-6; AF-6 and MUPP1 co-localize with Cx36 in many brain areas.\",\n      \"method\": \"Co-immunoprecipitation, GST pull-down, immunofluorescence co-localization in brain tissue\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and pull-down from native tissue with domain-binding confirmation, single lab\",\n      \"pmids\": [\"22211808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Canoe (Drosophila AF-6) forms a complex with Robo receptor in vivo and is required for Slit-Robo axon guidance signaling at the CNS midline; in cno mutants, Robo is mislocalized from growth cone filopodia, and the commissureless phenotype (excess Robo on all neurons) is suppressed in comm, cno double mutants, placing Cno as a positive regulator of Robo localization and signaling.\",\n      \"method\": \"Co-immunoprecipitation from embryo lysates, genetic epistasis in Drosophila, immunostaining for Robo localization, mutant analysis\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP from native tissue plus genetic epistasis with multiple allele combinations, functional axon guidance phenotype\",\n      \"pmids\": [\"22815517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MLL-AF6 leukemia requires continued DOT1L H3K79 methyltransferase activity; MLL-AF6 target genes show markedly elevated H3K79 dimethylation; conditional knockout of Dot1l inhibits MLL-AF6 leukemogenesis; MLL-AF6-transformed cells are sensitive to the DOT1L inhibitor EPZ0004777, which reduces proliferation and causes cell cycle arrest.\",\n      \"method\": \"Gene-expression analysis, ChIP-seq, conditional Dot1l knockout mouse, small molecule inhibitor treatment\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO plus ChIP-seq and pharmacological inhibition with functional readouts, multiple orthogonal methods\",\n      \"pmids\": [\"23361907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"AF-6 is a novel parkin-interacting protein; parkin interacts with AF-6 via its C-terminus with the AF-6 PDZ region, leading to AF-6 ubiquitination and proteasomal degradation; mitochondrial AF-6 is not degraded by parkin but co-localizes with parkin and enhances PINK1/parkin-mediated mitophagy; parkin/PINK1 disease mutants are insensitive to AF-6 stimulation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, mitophagy assay, disease-mutant analysis, co-localization\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, ubiquitination assay, functional mitophagy readout, disease-mutant mechanistic dissection; multiple orthogonal methods\",\n      \"pmids\": [\"23393160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In MLL-AF6-rearranged AML cells, AF6 is sequestered in the nucleus (in contrast to cytoplasmic localization in healthy cells), leading to aberrant activation of RAS-GTP; silencing MLL-AF6 restores cytoplasmic AF6 localization and reduces RAS-GTP levels; co-silencing of MLL-AF6 and AF6 reverses the RAS-GTP reduction, confirming that nuclear sequestration of AF6 by MLL-AF6 drives RAS pathway activation.\",\n      \"method\": \"Subcellular fractionation, immunofluorescence, RAS-GTP pull-down, siRNA knockdown, clonogenic assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including fractionation, RAS-GTP pull-down, siRNA rescue experiments; mechanistic pathway clearly established\",\n      \"pmids\": [\"24695851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"AF6 depletion in pancreatic cancer cells upregulates Snail protein expression through increased formation of a Dvl2-FOXE1 complex on the Snail promoter; this requires nuclear localization of AF6; AF6 loss promotes cell proliferation and metastasis via this Dvl2-FOXE1-Snail axis.\",\n      \"method\": \"siRNA knockdown, promoter ChIP assay, nuclear localization studies, in vitro migration/invasion assays, in vivo tumor model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP assay on Snail promoter with mechanistic pathway mapping, single lab\",\n      \"pmids\": [\"26013125\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AF-6 overexpression in Drosophila parkin null and pink1 null flies rescues mitochondrial pathology and locomotion deficits; AF-6 overexpression also ameliorates pathological phenotypes in LRRK2-G2019S flies; silencing endogenous AF-6 aggravates LRRK2 mutant fly phenotypes; AF-6 overexpression protects dopaminergic neurons against rotenone.\",\n      \"method\": \"Transgenic Drosophila overexpression and RNAi, locomotion assay, mitochondrial morphology analysis, rotenone treatment model\",\n      \"journal\": \"Frontiers in cellular neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue in multiple Drosophila PD models, single lab, functional phenotype readouts\",\n      \"pmids\": [\"28848400\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AF6 employs a non-canonical, evolutionarily conserved alpha-helix to bind RAS (distinct from other effectors); in all patients with MLL-AF6 translocations, the fusion protein lacks exactly this helix, exposing hydrophobic residues that drive dimerization; dimerization (oligomerization) is the dominant oncogenic mechanism for MLL-AF6, and inhibiting dimerization abrogates leukemogenesis in mice.\",\n      \"method\": \"Structural analysis, NMR/biochemical binding, proteomic interaction mapping (proximity biotinylation), mouse leukemia model with dimerization-disrupting mutants\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — structural evidence plus proteomic data plus in vivo mouse model, multiple orthogonal methods\",\n      \"pmids\": [\"29062045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"AF6 directly regulates IRS1/AKT insulin signaling by interacting with SHP2 and modulating SHP2 tyrosine phosphatase activity; liver-specific AF6 knockout mice show enhanced insulin sensitivity and liver glycogen storage, while AF6 overexpression causes insulin resistance; AF6-SHP2 interaction is required for this effect.\",\n      \"method\": \"Liver-specific knockout mice, adenovirus overexpression, co-immunoprecipitation, SHP2 phosphatase activity assay, glucose tolerance test, insulin signaling (pAKT) measurement\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific KO plus overexpression plus enzymatic activity assay plus co-IP; multiple orthogonal methods with in vivo confirmation\",\n      \"pmids\": [\"31127058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AF6 promotes necroptosis by interacting with the intermediate domain of RIPK1 and regulating RIPK1 ubiquitination via the deubiquitylase USP21; hepatocyte-specific AF6 deletion protects against TNFα-induced necroptosis in NASH and SIRS mouse models; overexpression of AF6 accelerates TNFα-induced necroptotic mortality.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, hepatocyte-specific knockout mice, adenoviral overexpression, necroptosis assay, in vivo liver disease models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP defining domain interaction, ubiquitination assay, tissue-specific KO, and in vivo disease models; multiple orthogonal methods\",\n      \"pmids\": [\"37828052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PAK4 recruitment to multicellular vertices requires the scaffolding protein Afadin (AFDN); Afdn-KO cells exhibit severe junctional defects and reduced barrier function; artificial targeting of PAK4 to junctions in Afdn-KO cells partially restores junctional continuity and barrier function, demonstrating that Afadin acts upstream of PAK4 in vertex remodeling.\",\n      \"method\": \"Afdn knockout, PAK4 inhibition/knockout, targeted PAK4 expression in Afdn-KO cells, MDCK live imaging, Xenopus embryo live imaging, barrier function assay\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue experiment with functional readout, multiple model systems, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.10.02.680170\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"AFDN (AF-6/afadin) is a multidomain scaffold protein that localizes to adherens and tight junctions where it directly binds activated Ras and Rap1 GTPases (via its RA1 domain), the tight junction protein ZO-1, Eph receptor tyrosine kinases, JAM, profilin, and c-Src (via its PDZ domain), thereby linking cell-surface adhesion complexes to the actin cytoskeleton and regulating Ras/Rap1 signaling, integrin-mediated adhesion, dendritic spine morphology, and epithelial polarity; AF-6 is itself regulated post-translationally by FAM/USP9X deubiquitination, Bcr kinase phosphorylation, and Eph-receptor-dependent phosphorylation, and in MLL-AF6 leukemia the fusion protein sequesters AF-6 nuclear localization to aberrantly activate RAS-GTP while the RA1-domain-driven dimerization of the fusion protein is the dominant oncogenic mechanism.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AFDN (AF-6/afadin) is a multidomain junctional scaffold that couples cell-surface adhesion receptors and small-GTPase signaling to the actin cytoskeleton, thereby controlling epithelial junction integrity, cell polarity, and adhesion [#2, #9]. Through its N-terminal Ras-association (RA1) domain it directly and selectively binds GTP-loaded Ras and, with highest affinity, Rap1A, stabilizing a defined Ras conformer and inhibiting nucleotide dissociation and GAP-stimulated hydrolysis [#1, #7, #12]; this RA1-Ras interaction is mutually exclusive with AF-6 binding to the tight-junction protein ZO-1 [#2, #6]. Its single PDZ domain — structurally distinguished by a noncanonical binding groove — engages the C-termini of Eph receptor tyrosine kinases, JAM, neurexin, connexin36, Bcr kinase, and c-Src, integrating receptor and kinase inputs at cell contacts [#5, #11, #17, #22, #27]. AF-6 acts as a Rap1 effector that bimodally remodels dendritic spines and scaffolds the Rac1-GEF kalirin-7 downstream of N-cadherin to drive spine enlargement, and it organizes integrin-mediated adhesion by simultaneously binding Rap1-GTP and the Rap1-GAP SPA-1 [#15, #18, #25]. AF-6 stability and activity are set post-translationally by FAM/USP9X-mediated deubiquitination, Bcr-dependent phosphorylation that allosterically opens the PDZ domain, and Eph-receptor phosphorylation [#4, #5, #23]. Beyond junctions, AF-6 modulates insulin signaling via SHP2 [#35], promotes RIPK1-dependent necroptosis [#36], and enhances PINK1/parkin mitophagy [#30]. AFDN is recurrently fused to MLL in t(6;11) leukemia; the MLL-AF6 fusion retains the RA1 domain, and RA1-driven self-association/dimerization — rather than Ras binding per se — is the dominant oncogenic mechanism, with the fusion sequestering AF-6 in the nucleus to aberrantly elevate RAS-GTP and requiring continued DOT1L H3K79 methylation [#26, #29, #31, #34].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Established AFDN's existence and disease relevance by identifying it as the MLL/ALL-1 fusion partner in t(6;11) leukemia and recognizing its PDZ/GLGF motif, hinting at a junctional function.\",\n      \"evidence\": \"Molecular cloning and cDNA characterization of the translocation breakpoint\",\n      \"pmids\": [\"8242616\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional role for native AF-6 defined\", \"Mechanism by which the fusion transforms cells unknown\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Defined AF-6 as a bona fide Ras effector by showing its N-terminal domain binds GTP-bound but not GDP-bound or effector-mutant Ras, linking adhesion scaffolding to Ras signaling.\",\n      \"evidence\": \"GST affinity chromatography with recombinant proteins and nucleotide/effector-mutant controls\",\n      \"pmids\": [\"8557659\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Ras binding at junctions not yet established\", \"GTPase selectivity not fully mapped\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Placed AF-6 at tight junctions in complex with ZO-1 and showed activated Ras competes for the same domain, suggesting Ras toggles AF-6 between adhesion and signaling roles.\",\n      \"evidence\": \"Immunoelectron microscopy, in vitro binding, and Co-IP in MDCK cells with activated-Ras overexpression\",\n      \"pmids\": [\"9348294\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Ras-ZO-1 competition operates in vivo not shown\", \"Downstream cytoskeletal effects undefined\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Explained how the MLL-AF6 fusion gains nuclear access despite cytoplasmic native AF-6, attributing nuclear localization to the MLL AT-hook region.\",\n      \"evidence\": \"Immunofluorescence and cell fractionation of the chimeric protein\",\n      \"pmids\": [\"9349501\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of nuclear mislocalization not yet linked to transformation\", \"Single-lab localization data\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identified two post-translational control points — FAM/USP9X deubiquitination and Eph-receptor-dependent phosphorylation via the PDZ domain — establishing AF-6 as a regulated receptor adaptor.\",\n      \"evidence\": \"Protein purification, ubiquitination assays, yeast two-hybrid, Co-IP, and in-cell phosphorylation assays\",\n      \"pmids\": [\"9722616\", \"9707552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals that trigger AF-6 ubiquitination unclear\", \"Downstream effect of Eph phosphorylation on AF-6 function undefined\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Quantitatively characterized AF-6's GTPase engagement (Raf-like affinity for Ras, highest affinity for Rap1A, conformer stabilization, inhibition of nucleotide exchange/GAP) and showed RA-domain mutation abolishes Ras binding and disrupts Ras-driven transcription.\",\n      \"evidence\": \"31P NMR, kinetic analysis, GST binding, site-directed mutagenesis, and c-fos reporter assays\",\n      \"pmids\": [\"10224125\", \"10334923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular outcome of Rap1A vs Ras selectivity not resolved\", \"M-Ras interaction rests on a single Co-IP (#10)\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Demonstrated AF-6 is essential for epithelial junction organization and polarity in vivo, defining its developmental role through an embryonic-lethal knockout.\",\n      \"evidence\": \"Targeted mouse knockout with histology, immunofluorescence, and electron microscopy\",\n      \"pmids\": [\"10469590\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular pathway connecting AF-6 loss to junctional collapse not dissected\", \"Tissue-specific requirements not separable in null embryos\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Connected AF-6 to Eph receptor signaling at synapses, showing native complex formation, postsynaptic co-localization, and phosphorylation by Eph kinases.\",\n      \"evidence\": \"Co-IP from brain lysates, immunohistochemistry, and in vitro phosphorylation\",\n      \"pmids\": [\"9922461\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional synaptic consequence not yet shown\", \"Link to cytoskeletal remodeling undefined\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Expanded the PDZ interactome to JAM and showed AF-6 bridges JAM, ZO-1, and profilin, identifying AF-6 as a direct junction-to-actin link via profilin.\",\n      \"evidence\": \"Reciprocal in vitro/in vivo binding, truncation mutants, yeast two-hybrid, and co-localization in endothelial cells\",\n      \"pmids\": [\"10856295\", \"10922060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional impact of profilin recruitment on actin dynamics not measured\", \"Rap1 binding does not perturb AF-6 localization, leaving its role at junctions unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Resolved AF-6 as a Rap1 effector and signaling cross-talk hub: it scaffolds SPA-1 and Rap1 to control integrin adhesion, forms a Bcr-AF-6-Ras trimeric complex modulating ERK signaling, and acts downstream of Rap1/Egfr/Ras in Drosophila morphogenesis.\",\n      \"evidence\": \"Co-IP, kinase assays, mutagenesis, adhesion and reporter assays, and Drosophila genetic epistasis\",\n      \"pmids\": [\"12590145\", \"12808105\", \"14504224\", \"14507782\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How AF-6 integrates competing Ras/Rap1 inputs not fully resolved\", \"Drosophila pathway position established but mammalian mechanism for ommatidial-type processes limited\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined the structural basis of AF-6 PDZ specificity and established AF-6 as the effector through which Rap1 bimodally controls dendritic spine morphology.\",\n      \"evidence\": \"NMR structure with SPR binding measurements; live neuronal imaging with dominant Rap1 constructs and NMDA activation; RNAi/overexpression adhesion assays\",\n      \"pmids\": [\"15684424\", \"16301177\", \"16051602\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Rap1-GTP/GDP switching mechanically reshapes spines unresolved\", \"Whether AF-6 sequesters Rap-GTP unproductively rests on correlation (#19)\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed an isoform with an extra F-actin-binding site (AF6i3) stabilizes E-cadherin/catenin–actin coupling, and that Drosophila Canoe represses Wnt, Ras-MAPK, and Notch signaling, casting AF-6 as a multipathway modulator.\",\n      \"evidence\": \"Isoform-specific RNAi, F-actin sedimentation, wound assays; yeast two-hybrid, Co-IP, and Drosophila loss-of-function\",\n      \"pmids\": [\"16882694\", \"17183697\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mammalian relevance of Drosophila multipathway repression not tested\", \"Isoform-specific functions in vivo unmapped\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that AF-6 PDZ binding activates c-Src by blocking CSK-mediated inhibitory phosphorylation, and revealed Bcr-phosphorylation-driven allosteric opening of the PDZ domain plus a nuclear-competent short isoform.\",\n      \"evidence\": \"NMR complex structure with relaxation-dispersion dynamics; PDZ binding/kinase assays with mutagenesis; GFP isoform imaging\",\n      \"pmids\": [\"17491594\", \"17473018\", \"18052198\", \"17013812\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological trigger for s-afadin nuclear-body formation unclear\", \"Whether allosteric PDZ regulation extends to non-Bcr ligands untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined a synaptic mechanism in which N-cadherin clustering recruits the AF-6/kalirin-7 module to raise Rac1 activity and drive spine enlargement.\",\n      \"evidence\": \"Co-IP, N-cadherin clustering, Rac1 pull-down, PAK phosphorylation, and siRNA/dominant-negative rescue\",\n      \"pmids\": [\"18550750\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How this integrates with the Rap1-driven spine pathway not reconciled\", \"In vivo requirement not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Pinpointed the RA1 domain as the minimal element for MLL-AF6 transformation and showed self-association, not Ras binding, drives leukemogenesis.\",\n      \"evidence\": \"Truncation/point-mutant retroviral transduction, immortalization assays, and a mouse leukemia model\",\n      \"pmids\": [\"20395419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of RA1 self-association not yet defined here\", \"Downstream effectors of fusion dimerization unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended AF-6/Canoe function to axon guidance by showing it complexes with Robo and is required for proper Robo localization and Slit-Robo signaling at the midline.\",\n      \"evidence\": \"Co-IP from embryo lysates, Robo immunostaining, and genetic epistasis in Drosophila\",\n      \"pmids\": [\"22815517\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian Robo-AFDN relevance not addressed\", \"Mechanism of Robo localization control undefined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mechanistically linked the MLL-AF6 fusion to RAS pathway hyperactivation via nuclear sequestration of AF6 and established dependence on DOT1L H3K79 methyltransferase activity as a therapeutic vulnerability.\",\n      \"evidence\": \"Subcellular fractionation, RAS-GTP pull-down, siRNA rescue; ChIP-seq, conditional Dot1l knockout, and DOT1L inhibitor treatment\",\n      \"pmids\": [\"24695851\", \"23361907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How nuclear AF6 mechanistically elevates cytoplasmic RAS-GTP not fully resolved\", \"Whether DOT1L and RAS effects are mechanistically coupled unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Implicated AF-6 in mitochondrial quality control by showing parkin-mediated degradation of AF-6 and AF-6 enhancement of PINK1/parkin mitophagy.\",\n      \"evidence\": \"Co-IP, ubiquitination/proteasome assays, mitophagy assay, and disease-mutant analysis\",\n      \"pmids\": [\"23393160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a junctional scaffold acts at mitochondria mechanistically unclear\", \"Physiological relevance to Parkinson disease not established in mammals\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined a tumor-suppressive role for AF6 in pancreatic cancer, where its loss permits a nuclear Dvl2-FOXE1-Snail axis driving proliferation and metastasis.\",\n      \"evidence\": \"siRNA knockdown, promoter ChIP, migration/invasion assays, and in vivo tumor model\",\n      \"pmids\": [\"26013125\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How AF6 normally restrains the Dvl2-FOXE1 complex undefined\", \"Single-lab mechanism\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Solved the structural rationale for MLL-AF6 oncogenesis: the fusion lacks a noncanonical Ras-binding alpha-helix, exposing hydrophobic residues that drive dimerization, the dominant transforming activity that can be therapeutically disrupted.\",\n      \"evidence\": \"Structural/NMR analysis, proximity-biotinylation proteomics, and mouse models with dimerization-disrupting mutants; Drosophila PD-model rescue separately implicated AF-6 in mitochondrial protection\",\n      \"pmids\": [\"29062045\", \"28848400\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Druggability of the dimerization interface in patients untested\", \"Mechanism of AF-6 neuroprotection in PD models remains correlative\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified a metabolic role for AF6 as a regulator of insulin signaling through SHP2, with liver AF6 levels setting insulin sensitivity in vivo.\",\n      \"evidence\": \"Liver-specific knockout and overexpression, Co-IP, SHP2 phosphatase assays, and glucose/insulin signaling readouts\",\n      \"pmids\": [\"31127058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How AF6 modulates SHP2 catalytic activity mechanistically unclear\", \"Connection to AF6's junctional scaffolding role unexplored\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed AF6 as a pro-necroptotic scaffold that binds RIPK1 and tunes its ubiquitination via USP21, linking AF6 to inflammatory cell death in liver disease.\",\n      \"evidence\": \"Co-IP defining domain interaction, ubiquitination assays, hepatocyte-specific knockout, and in vivo NASH/SIRS models\",\n      \"pmids\": [\"37828052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How AF6 recruits USP21 to RIPK1 not detailed\", \"Relationship to AF6's other ubiquitination-regulated functions unexplored\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Positioned Afadin upstream of PAK4 in junctional vertex remodeling, with PAK4 mistargeting rescue restoring barrier function in Afadin-null cells.\",\n      \"evidence\": \"Afdn knockout, PAK4 inhibition/targeting, and live imaging in MDCK and Xenopus with barrier assays (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.10.02.680170\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"Molecular mechanism of PAK4 recruitment by Afadin undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AF-6's many context-specific roles — junctional/cytoskeletal scaffolding, GTPase regulation, mitophagy, insulin signaling, and necroptosis — are coordinated by a single multidomain protein, and which interactions are dominant in each tissue, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model integrating junctional vs metabolic vs cell-death functions\", \"Isoform-specific contributions to each role incompletely mapped\", \"Therapeutic targeting of the MLL-AF6 dimerization interface unproven in humans\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [2, 11, 15, 25]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 22, 35]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [12, 20]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [2, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 18]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 31]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [24, 31, 32]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [12, 20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 7, 12, 14]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [2, 9, 11]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [26, 31, 32]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [36]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [30]}\n    ],\n    \"complexes\": [\"adherens junction\", \"tight junction\"],\n    \"partners\": [\"RAS\", \"RAP1A\", \"TJP1\", \"F11R\", \"SRC\", \"BCR\", \"EPHB2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}