Affinage

Showing AFDN — AF6 is a alias.

AFDN

Afadin · UniProt P55196

Length
1824 aa
Mass
206.8 kDa
Annotated
2026-06-09
69 papers in source corpus 39 papers cited in narrative 38 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 7/7 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

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).

Mechanistic history

Synthesis pass · year-by-year structured walk · 23 steps
  1. 1993 Medium

    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

    PMID:8242616

    Open questions at the time
    • No functional role for native AF-6 defined
    • Mechanism by which the fusion transforms cells unknown
  2. 1996 High

    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

    PMID:8557659

    Open questions at the time
    • Functional consequence of Ras binding at junctions not yet established
    • GTPase selectivity not fully mapped
  3. 1997 High

    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

    PMID:9348294

    Open questions at the time
    • Whether Ras-ZO-1 competition operates in vivo not shown
    • Downstream cytoskeletal effects undefined
  4. 1997 Medium

    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

    PMID:9349501

    Open questions at the time
    • Functional consequence of nuclear mislocalization not yet linked to transformation
    • Single-lab localization data
  5. 1998 High

    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

    PMID:9707552 PMID:9722616

    Open questions at the time
    • Physiological signals that trigger AF-6 ubiquitination unclear
    • Downstream effect of Eph phosphorylation on AF-6 function undefined
  6. 1999 High

    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

    PMID:10224125 PMID:10334923

    Open questions at the time
    • Cellular outcome of Rap1A vs Ras selectivity not resolved
    • M-Ras interaction rests on a single Co-IP (#10)
  7. 1999 High

    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

    PMID:10469590

    Open questions at the time
    • Molecular pathway connecting AF-6 loss to junctional collapse not dissected
    • Tissue-specific requirements not separable in null embryos
  8. 1999 High

    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

    PMID:9922461

    Open questions at the time
    • Functional synaptic consequence not yet shown
    • Link to cytoskeletal remodeling undefined
  9. 2000 High

    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

    PMID:10856295 PMID:10922060

    Open questions at the time
    • Functional impact of profilin recruitment on actin dynamics not measured
    • Rap1 binding does not perturb AF-6 localization, leaving its role at junctions unclear
  10. 2003 High

    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

    PMID:12590145 PMID:12808105 PMID:14504224 PMID:14507782

    Open questions at the time
    • How AF-6 integrates competing Ras/Rap1 inputs not fully resolved
    • Drosophila pathway position established but mammalian mechanism for ommatidial-type processes limited
  11. 2005 High

    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

    PMID:15684424 PMID:16051602 PMID:16301177

    Open questions at the time
    • How Rap1-GTP/GDP switching mechanically reshapes spines unresolved
    • Whether AF-6 sequesters Rap-GTP unproductively rests on correlation (#19)
  12. 2006 Medium

    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

    PMID:16882694 PMID:17183697

    Open questions at the time
    • Mammalian relevance of Drosophila multipathway repression not tested
    • Isoform-specific functions in vivo unmapped
  13. 2007 High

    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

    PMID:17013812 PMID:17473018 PMID:17491594 PMID:18052198

    Open questions at the time
    • Physiological trigger for s-afadin nuclear-body formation unclear
    • Whether allosteric PDZ regulation extends to non-Bcr ligands untested
  14. 2008 High

    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

    PMID:18550750

    Open questions at the time
    • How this integrates with the Rap1-driven spine pathway not reconciled
    • In vivo requirement not tested
  15. 2010 High

    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

    PMID:20395419

    Open questions at the time
    • Structural basis of RA1 self-association not yet defined here
    • Downstream effectors of fusion dimerization unknown
  16. 2012 High

    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

    PMID:22815517

    Open questions at the time
    • Mammalian Robo-AFDN relevance not addressed
    • Mechanism of Robo localization control undefined
  17. 2013 High

    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

    PMID:23361907 PMID:24695851

    Open questions at the time
    • How nuclear AF6 mechanistically elevates cytoplasmic RAS-GTP not fully resolved
    • Whether DOT1L and RAS effects are mechanistically coupled unclear
  18. 2013 High

    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

    PMID:23393160

    Open questions at the time
    • How a junctional scaffold acts at mitochondria mechanistically unclear
    • Physiological relevance to Parkinson disease not established in mammals
  19. 2015 Medium

    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

    PMID:26013125

    Open questions at the time
    • How AF6 normally restrains the Dvl2-FOXE1 complex undefined
    • Single-lab mechanism
  20. 2017 High

    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

    PMID:28848400 PMID:29062045

    Open questions at the time
    • Druggability of the dimerization interface in patients untested
    • Mechanism of AF-6 neuroprotection in PD models remains correlative
  21. 2019 High

    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

    PMID:31127058

    Open questions at the time
    • How AF6 modulates SHP2 catalytic activity mechanistically unclear
    • Connection to AF6's junctional scaffolding role unexplored
  22. 2023 High

    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

    PMID:37828052

    Open questions at the time
    • How AF6 recruits USP21 to RIPK1 not detailed
    • Relationship to AF6's other ubiquitination-regulated functions unexplored
  23. 2025 Medium

    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)

    PMID:bio_10.1101_2025.10.02.680170

    Open questions at the time
    • Preprint not yet peer-reviewed
    • Molecular mechanism of PAK4 recruitment by Afadin undefined

Open questions

Synthesis pass · forward-looking unresolved questions
  • 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.
  • 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

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0060090 molecular adaptor activity 4 GO:0098772 molecular function regulator activity 3 GO:0005198 structural molecule activity 2 GO:0008092 cytoskeletal protein binding 2
Localization
GO:0005634 nucleus 3 GO:0005829 cytosol 2 GO:0005856 cytoskeleton 2 GO:0005886 plasma membrane 2
Pathway
R-HSA-162582 Signal Transduction 4 R-HSA-1500931 Cell-Cell communication 3 R-HSA-1643685 Disease 3 R-HSA-5357801 Programmed Cell Death 1 R-HSA-9612973 Autophagy 1
Partners
BCREPHB2F11RRAP1ARASSRCTJP1
Complex memberships
adherens junctiontight junction

Evidence

Reading pass · 38 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1993 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. Molecular cloning, cDNA characterization Cancer research Medium 8242616
1996 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. GST affinity chromatography, recombinant protein binding assay The Journal of biological chemistry High 8557659
1997 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. Immunoelectron microscopy, in vitro binding assay, co-immunoprecipitation, overexpression of activated Ras The Journal of cell biology High 9348294
1997 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. Immunofluorescence, cell fractionation Oncogene Medium 9349501
1998 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. Protein purification, peptide sequencing, co-immunoprecipitation, in vitro binding, ubiquitination assay The Journal of cell biology High 9722616
1998 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. Yeast two-hybrid, mutational analysis, co-immunoprecipitation, phosphorylation assay in NIH 3T3 and NG108 cells Proceedings of the National Academy of Sciences of the United States of America High 9707552
1999 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. Site-directed mutagenesis, co-immunoprecipitation, reporter assay Biochemical and biophysical research communications High 10334923
1999 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. Biochemical binding assay, 31P NMR spectroscopy, kinetic analysis with GTP analogs The Journal of biological chemistry High 10224125
1999 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. Co-immunoprecipitation from brain lysates, co-transfection with GFP-AF-6, immunohistochemistry, in vitro phosphorylation The Journal of cell biology High 9922461
1999 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. Targeted gene knockout in mice, histology, immunofluorescence, electron microscopy Current biology : CB High 10469590
1999 M-Ras co-immunoprecipitates with AF-6 in mammalian cells, extending the range of Ras-subfamily GTPases that can interact with AF-6. Co-immunoprecipitation The Journal of biological chemistry Low 10446149
2000 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. Co-immunoprecipitation from endothelial cells, in vitro binding, co-localization by immunofluorescence, truncation mutants The Journal of biological chemistry High 10856295
2000 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. In vitro binding with GST-fusion proteins, co-immunoprecipitation in mammalian cells, yeast two-hybrid, co-localization Proceedings of the National Academy of Sciences of the United States of America High 10922060
2003 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. Yeast two-hybrid, co-localization, genetic epistasis in Drosophila, loss-of-function analysis Genetics High 14504224
2003 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. Co-immunoprecipitation, kinase assay, mutagenesis, co-localization, reporter assay for ERK pathway Molecular and cellular biology High 12808105
2003 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. Co-immunoprecipitation, in vitro binding with truncation mutants and point mutants, cell adhesion assay, immunostaining The Journal of biological chemistry High 12590145
2003 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. Drosophila genetics, epistasis analysis, loss-of-function and gain-of-function alleles Development (Cambridge, England) Medium 14507782
2005 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. NMR structure determination, BIACORE surface plasmon resonance, NMR chemical shift perturbation, molecular dynamics simulation The Journal of biological chemistry High 15684424
2005 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. Live imaging in cultured neurons, dominant-active and dominant-negative Rap1 constructs, NMDA receptor activation, spine morphology quantification Neuron High 16301177
2005 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. RNA interference knockdown, overexpression, integrin-mediated cell adhesion assay, Rap-GTP pull-down The Journal of biological chemistry Medium 16051602
2006 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. RNAi knockdown, isoform-specific expression, wound closure assay, co-immunoprecipitation, F-actin sedimentation Journal of cell science Medium 16882694
2006 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. Yeast two-hybrid, co-immunoprecipitation, Drosophila genetics, loss-of-function analysis PloS one Medium 17183697
2007 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. PDZ domain binding assay, mutagenesis, kinase assay, co-immunoprecipitation, co-localization, knockdown of AF-6 The EMBO journal High 17491594
2007 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. NMR structure determination, relaxation dispersion NMR, backbone dynamics analysis Protein science / Biochemistry High 17473018 18052198
2007 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. GFP-tagged isoform expression, live-cell imaging, cell cycle inhibition, transcription inhibition, growth factor treatment Journal of cellular physiology Medium 17013812
2008 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. Co-immunoprecipitation, co-localization, N-cadherin clustering assay, Rac1 pull-down, PAK phosphorylation, dominant-negative and siRNA knockdown The Journal of neuroscience High 18550750
2010 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. Retroviral transduction of truncation/point-mutant constructs, myeloid progenitor immortalization assay, mouse leukemia model, self-association assay Blood High 20395419
2011 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. Co-immunoprecipitation, GST pull-down, immunofluorescence co-localization in brain tissue The European journal of neuroscience Medium 22211808
2012 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. Co-immunoprecipitation from embryo lysates, genetic epistasis in Drosophila, immunostaining for Robo localization, mutant analysis The Journal of neuroscience High 22815517
2013 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. Gene-expression analysis, ChIP-seq, conditional Dot1l knockout mouse, small molecule inhibitor treatment Blood High 23361907
2013 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. Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, mitophagy assay, disease-mutant analysis, co-localization Human molecular genetics High 23393160
2014 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. Subcellular fractionation, immunofluorescence, RAS-GTP pull-down, siRNA knockdown, clonogenic assay Blood High 24695851
2015 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. siRNA knockdown, promoter ChIP assay, nuclear localization studies, in vitro migration/invasion assays, in vivo tumor model Nature communications Medium 26013125
2017 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. Transgenic Drosophila overexpression and RNAi, locomotion assay, mitochondrial morphology analysis, rotenone treatment model Frontiers in cellular neuroscience Medium 28848400
2017 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. Structural analysis, NMR/biochemical binding, proteomic interaction mapping (proximity biotinylation), mouse leukemia model with dimerization-disrupting mutants Nature communications High 29062045
2019 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. Liver-specific knockout mice, adenovirus overexpression, co-immunoprecipitation, SHP2 phosphatase activity assay, glucose tolerance test, insulin signaling (pAKT) measurement Diabetes High 31127058
2023 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. Co-immunoprecipitation, ubiquitination assay, hepatocyte-specific knockout mice, adenoviral overexpression, necroptosis assay, in vivo liver disease models Cell death & disease High 37828052
2025 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. Afdn knockout, PAK4 inhibition/knockout, targeted PAK4 expression in Afdn-KO cells, MDCK live imaging, Xenopus embryo live imaging, barrier function assay bioRxiv (preprint)preprint Medium bio_10.1101_2025.10.02.680170

Source papers

Stage 0 corpus · 69 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2000 Junctional adhesion molecule interacts with the PDZ domain-containing proteins AF-6 and ZO-1. The Journal of biological chemistry 338 10856295
1997 The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells. The Journal of cell biology 270 9348294
1993 Cloning of the ALL-1 fusion partner, the AF-6 gene, involved in acute myeloid leukemias with the t(6;11) chromosome translocation. Cancer research 245 8242616
1996 Identification of AF-6 and canoe as putative targets for Ras. The Journal of biological chemistry 184 8557659
1998 PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor. Proceedings of the National Academy of Sciences of the United States of America 176 9707552
2000 The junctional multidomain protein AF-6 is a binding partner of the Rap1A GTPase and associates with the actin cytoskeletal regulator profilin. Proceedings of the National Academy of Sciences of the United States of America 170 10922060
1999 The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain. The Journal of cell biology 164 9922461
1999 Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development. Current biology : CB 160 10469590
2013 Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l. Blood 141 23361907
1999 Thermodynamic and kinetic characterization of the interaction between the Ras binding domain of AF6 and members of the Ras subfamily. The Journal of biological chemistry 121 10224125
1999 M-Ras/R-Ras3, a transforming ras protein regulated by Sos1, GRF1, and p120 Ras GTPase-activating protein, interacts with the putative Ras effector AF6. The Journal of biological chemistry 102 10446149
1998 The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme. The Journal of cell biology 102 9722616
2003 The AF-6 homolog canoe acts as a Rap1 effector during dorsal closure of the Drosophila embryo. Genetics 99 14504224
2005 Activity-dependent dendritic spine structural plasticity is regulated by small GTPase Rap1 and its target AF-6. Neuron 97 16301177
2003 AF-6 controls integrin-mediated cell adhesion by regulating Rap1 activation through the specific recruitment of Rap1GTP and SPA-1. The Journal of biological chemistry 82 12590145
2008 Coordination of synaptic adhesion with dendritic spine remodeling by AF-6 and kalirin-7. The Journal of neuroscience : the official journal of the Society for Neuroscience 81 18550750
2003 The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain. Molecular and cellular biology 76 12808105
2003 Egfr signaling regulates ommatidial rotation and cell motility in the Drosophila eye via MAPK/Pnt signaling and the Ras effector Canoe/AF6. Development (Cambridge, England) 71 14507782
2013 Afadin/AF-6 and canoe: roles in cell adhesion and beyond. Progress in molecular biology and translational science 68 23481206
2014 Disrupted interaction between CFTR and AF-6/afadin aggravates malignant phenotypes of colon cancer. Biochimica et biophysica acta 60 24373847
2015 Loss of polarity protein AF6 promotes pancreatic cancer metastasis by inducing Snail expression. Nature communications 55 26013125
2011 Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth. Oncogene 51 21478912
1999 In vivo interaction of AF-6 with activated Ras and ZO-1. Biochemical and biophysical research communications 51 10334923
2006 Regulation of epithelial wound closure and intercellular adhesion by interaction of AF6 with actin cytoskeleton. Journal of cell science 46 16882694
2014 MLL-AF6 fusion oncogene sequesters AF6 into the nucleus to trigger RAS activation in myeloid leukemia. Blood 44 24695851
2011 The effector and scaffolding proteins AF6 and MUPP1 interact with connexin36 and localize at gap junctions that form electrical synapses in rodent brain. The European journal of neuroscience 41 22211808
2006 The PDZ protein Canoe/AF-6 links Ras-MAPK, Notch and Wingless/Wnt signaling pathways by directly interacting with Ras, Notch and Dishevelled. PloS one 40 17183697
2005 AF6 negatively regulates Rap1-induced cell adhesion. The Journal of biological chemistry 39 16051602
2002 RYK, a catalytically inactive receptor tyrosine kinase, associates with EphB2 and EphB3 but does not interact with AF-6. The Journal of biological chemistry 39 11956217
2010 Self-association mediated by the Ras association 1 domain of AF6 activates the oncogenic potential of MLL-AF6. Blood 37 20395419
1996 Analysis of the t(6;11)(q27;q23) in leukemia shows a consistent breakpoint in AF6 in three patients and in the ML-2 cell line. Genes, chromosomes & cancer 34 8703846
1997 Chimeric MLL products with a Ras binding cytoplasmic protein AF6 involved in t(6;11) (q27;q23) leukemia localize in the nucleus. Oncogene 33 9349501
2017 CLDN6 enhances chemoresistance to ADM via AF-6/ERKs pathway in TNBC cell line MDAMB231. Molecular and cellular biochemistry 31 29159771
1996 Fusion of the MLL gene with two different genes, AF-6 and AF-5alpha, by a complex translocation involving chromosomes 5, 6, 8 and 11 in infant leukemia. Oncogene 31 8950979
2005 Solution structure of AF-6 PDZ domain and its interaction with the C-terminal peptides from Neurexin and Bcr. The Journal of biological chemistry 30 15684424
2007 AF6/s-afadin is a dual residency protein and localizes to a novel subnuclear compartment. Journal of cellular physiology 28 17013812
2006 Expression of JAM-A, AF-6, PAR-3 and PAR-6 during the assembly and remodeling of RPE tight junctions. Brain research 28 16859655
2007 Regulation of c-Src by binding to the PDZ domain of AF-6. The EMBO journal 26 17491594
2000 Co-localization of FAM and AF-6, the mammalian homologues of Drosophila faf and canoe, in mouse eye development. Mechanisms of development 26 10704870
1998 Complete genomic structure DNA polymorphisms, and alternative splicing of the human AF-6 gene. DNA research : an international journal for rapid publication of reports on genes and genomes 26 9679199
2012 The actin-binding protein Canoe/AF-6 forms a complex with Robo and is required for Slit-Robo signaling during axon pathfinding at the CNS midline. The Journal of neuroscience : the official journal of the Society for Neuroscience 23 22815517
2018 The basic helix-loop-helix transcription factor SHARP1 is an oncogenic driver in MLL-AF6 acute myelogenous leukemia. Nature communications 22 29692408
2017 Evolution of AF6-RAS association and its implications in mixed-lineage leukemia. Nature communications 22 29062045
2015 Loss of AF-6/afadin induces cell invasion, suppresses the formation of glandular structures and might be a predictive marker of resistance to chemotherapy in endometrial cancer. BMC cancer 22 25879875
2013 AF-6 is a positive modulator of the PINK1/parkin pathway and is deficient in Parkinson's disease. Human molecular genetics 22 23393160
2020 Afadin (AF6) in cancer progression: A multidomain scaffold protein with complex and contradictory roles. BioEssays : news and reviews in molecular, cellular and developmental biology 21 33165933
1998 Identification of Ce-AF-6, a novel Caenorhabditis elegans protein, as a putative Ras effector. Gene 21 9931431
2007 Solution structure and backbone dynamics of the AF-6 PDZ domain/Bcr peptide complex. Protein science : a publication of the Protein Society 18 17473018
2007 Interesting structural and dynamical behaviors exhibited by the AF-6 PDZ domain upon Bcr peptide binding. Biochemistry 18 18052198
2017 AF-6 Protects Against Dopaminergic Dysfunction and Mitochondrial Abnormalities in Drosophila Models of Parkinson's Disease. Frontiers in cellular neuroscience 16 28848400
2019 Polarity Protein AF6 Controls Hepatic Glucose Homeostasis and Insulin Sensitivity by Modulating IRS1/AKT Insulin Pathway in an SHP2-Dependent Manner. Diabetes 14 31127058
1995 AF6 gene on chromosome band 6q27 maps distal to the minimal region of deletion in epithelial ovarian cancer. Genes, chromosomes & cancer 14 8589040
2004 A close correlation in the expression patterns of Af-6 and Usp9x in Sertoli and granulosa cells of mouse testis and ovary. Reproduction (Cambridge, England) 12 15509704
2002 The LIM domain protein Lmo2 binds to AF6, a translocation partner of the MLL oncogene. FEBS letters 12 12067721
2023 Polarity protein AF6 functions as a modulator of necroptosis by regulating ubiquitination of RIPK1 in liver diseases. Cell death & disease 11 37828052
2020 Tumour suppressive long non-coding RNA AFDN-DT inhibits gastric cancer invasion via transcriptional regulation. Journal of cellular and molecular medicine 9 31981317
2020 Thioridazine requires calcium influx to induce MLL-AF6-rearranged AML cell death. Blood advances 9 32931582
2024 Long non-coding RNA MLLT4 antisense RNA 1 induces autophagy to inhibit tumorigenesis of cervical cancer through modulating the myosin-9/ATG14 axis. Scientific reports 7 38493244
2002 Monitoring minimal residual disease in patients with MLL-AF6 fusion transcript-positive acute myeloid leukemia following allogeneic bone marrow transplantation. International journal of hematology 7 11999359
2014 Small-molecule inhibitors of AF6 PDZ-mediated protein-protein interactions. ChemMedChem 6 24668962
2007 Role of AF6 protein in cell-to-cell spread of Herpes simplex virus 1. FEBS letters 6 17967423
2022 Detection of a Cryptic KMT2A/AFDN Gene Fusion [ins(6;11)(q27;q23q23)] in a Pediatric Patient with Newly Diagnosed Acute Myeloid Leukemia. Laboratory medicine 5 34894139
2022 Multifunctional liposomal nanostructure-mediated siRNA/bortezomib co-delivery for SHARP1 knockdown in MLL-AF6 acute myeloid leukemia. Biomaterials advances 5 35577689
2022 Damaging Mutations in Contribute to Risk of Nonsyndromic Cleft Lip With or Without Cleft Palate. The Cleft palate-craniofacial journal : official publication of the American Cleft Palate-Craniofacial Association 5 36384317
2024 AFDN Deficiency Promotes Liver Tropism of Metastatic Colorectal Cancer. Cancer research 4 39047222
2026 AF6 knockout-induced upregulation of bile acid production promotes CXCL14-mediated antitumor immunity in HCC. Journal of hepatology 1 41577299
2025 Cryptic KMT2A::AFDN Fusion Due to AFDN Insertion into KMT2A in a Patient with Acute Monoblastic Leukemia. Genes 1 40149468
2025 Regulation of Metastasis and Growth of Colorectal Cancer via Targeting the PTEN Pathway: An Update on the Progress of LncRNA MLLT4-AS1. Recent patents on anti-cancer drug discovery 0 38835131
2025 AF6 regulates intestinal IgA via crosstalk between intestinal epithelial cells and immune cells in inflammatory bowel disease. iScience 0 40687779

Missed literature

Know a paper Affinage missed for AFDN? Flag it for the maintainers and the community.

No submissions yet.