{"gene":"KANK1","run_date":"2026-04-28T18:06:54","timeline":{"discoveries":[{"year":2016,"finding":"KANK1 directly binds to talin rod domain R7 via its conserved KN domain, recruiting cortical microtubule stabilizing complexes (containing CLASPs, KIF21A, LL5β, and liprins) to focal adhesions. A single point mutation in talin disrupting KANK1 binding abolishes this recruitment without affecting talin's adhesion function.","method":"Structural studies (crystal structure), Co-IP, single point mutagenesis, fluorescence microscopy","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis plus cell biological validation in one study","pmids":["27410476"],"is_preprint":false},{"year":2008,"finding":"KANK1 is an Akt substrate downstream of PI3K; Akt-mediated phosphorylation of KANK1 promotes its binding to 14-3-3 proteins, which inhibits KANK1's suppression of RhoA activity, actin stress fiber formation, and insulin-induced cell migration.","method":"Kinase assay, co-immunoprecipitation, overexpression/co-expression in NIH3T3 cells, RhoA activity assay (GST-rhotekin pulldown), migration assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro kinase substrate identification combined with multiple orthogonal functional assays","pmids":["18458160"],"is_preprint":false},{"year":2009,"finding":"KANK1 binds IRSp53 and specifically inhibits the IRSp53–Rac1 interaction, suppressing lamellipodia formation without affecting filopodia (Cdc42-dependent), thereby negatively regulating actin remodeling and cell spreading.","method":"Co-immunoprecipitation, GST pulldown, RNAi knockdown, overexpression, microscopy","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal pulldown/Co-IP plus RNAi epistasis with multiple phenotypic readouts","pmids":["19171758"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of the KANK1 ankyrin repeat domain (ANKRD) in complex with a KIF21A peptide revealed that target recognition involves combinatorial use of two interfaces; mutations at either interface disrupt the KANK1–KIF21A interaction and prevent KIF21A recruitment to focal adhesions.","method":"X-ray crystallography (high-resolution crystal structure), mutagenesis, biochemical binding assays, immunofluorescence localization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis and functional cellular validation","pmids":["29217769"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of the KANK1·KIF21A complex at 2.1 Å showed that a five-helix-bundle-capping domain immediately preceding the ANK repeats forms a supramodule with the ANK repeats to bind a conserved KIF21A peptide; cancer-associated missense mutations at this interface destabilize the complex.","method":"X-ray crystallography, biochemical assays, mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — atomic resolution structure with biochemical and mutagenic validation","pmids":["29158259"],"is_preprint":false},{"year":2017,"finding":"The KANK2 ankyrin domain binds the same ~22 amino acid KIF21A peptide as KANK1; both complex structures show KIF21A adopting helical conformations upon binding to two distinct pockets of the ankyrin domain.","method":"X-ray crystallography, site-directed mutagenesis, biochemical binding assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — dual crystal structures with mutagenesis","pmids":["29183992"],"is_preprint":false},{"year":2019,"finding":"The talin R7–KANK1 KN domain complex can withstand physiological forces (up to 10 pN) for seconds to minutes under shear-force geometry; mechanical stretching promotes KANK1 localization to the periphery of focal adhesions rather than the center.","method":"Magnetic tweezers single-molecule force spectroscopy, cell biology (live cell imaging, TIRF)","journal":"Nano letters","confidence":"High","confidence_rationale":"Tier 1 — single-molecule mechanical measurements combined with cell biological validation","pmids":["31389241"],"is_preprint":false},{"year":2009,"finding":"KANK1 interacts with the third and fourth coiled-coil domains of KIF21A via its ankyrin-repeat domain; KIF21A controls the subcellular distribution of KANK1, with KIF21A knockdown causing KANK1 to remain cytosolic, and the CFEOM1-associated KIF21A R954W mutation significantly enhancing KANK1 translocation to the membrane fraction.","method":"Co-immunoprecipitation, subcellular fractionation, siRNA knockdown, Western blotting","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP and fractionation; single lab","pmids":["19559006"],"is_preprint":false},{"year":2011,"finding":"KANK1 physically and functionally associates with BIG1 (a guanine nucleotide-exchange factor for ARFs); depletion of either BIG1 or KANK1 similarly disrupts directed cell migration and Golgi/MTOC orientation toward the wound edge, indicating both function in maintaining cell polarity during migration.","method":"Reciprocal co-immunoprecipitation, siRNA depletion, wound-healing migration assay, Golgi/MTOC orientation imaging","journal":"PNAS","confidence":"Medium","confidence_rationale":"Tier 2–3 — reciprocal IP plus functional migration assay; moderate mechanistic depth","pmids":["22084092"],"is_preprint":false},{"year":2006,"finding":"KANK1 contains functional nuclear localization signals (NLS1, NLS2) and nuclear export signals (NES1–NES3); nuclear export is CRM1-dependent (blocked by leptomycin B); KANK1 binds β-catenin and its nuclear import correlates with activation of β-catenin-dependent transcription (TOPFLASH reporter).","method":"NLS/NES mutagenesis, leptomycin B treatment, TOPFLASH reporter assay, co-immunoprecipitation, immunofluorescence","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenesis of transport signals plus functional reporter assay and Co-IP, single lab","pmids":["16968744"],"is_preprint":false},{"year":2017,"finding":"KANK1 knockdown causes centrosomal amplification and cytokinesis failure via hyperactivation of RhoA; KANK1 interacts with Daam1 (a RhoA activator), and excess Daam1 upon KANK1 loss hyperactivates RhoA, elevating Aurora-A activity and leading to abnormal centrosome numbers and multinucleate cells.","method":"RNAi knockdown, co-immunoprecipitation (KANK1–Daam1), RhoA activity assay, centrosome counting, Aurora-A activity measurement","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP plus functional pathway epistasis; single lab","pmids":["28284839"],"is_preprint":false},{"year":2015,"finding":"In rat glomeruli and cultured human podocytes, KANK2 interacts with ARHGDIA (a RHO GTPase regulator); KANK2 knockdown increases active GTP-bound RHOA and decreases podocyte migration; RNAi of the Drosophila KANK homolog disrupts slit diaphragm and lacuna channel structures in nephrocytes.","method":"Co-immunoprecipitation, RhoA activity assay (GTP-pulldown), RNAi in Drosophila nephrocytes, zebrafish kank2 morpholino knockdown, immunofluorescence co-localization","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and activity assay plus in vivo model validation; primarily KANK2 but in context of KANK family mechanism","pmids":["25961457"],"is_preprint":false},{"year":2011,"finding":"The KANK1-PDGFRβ fusion protein (from t(5;9) translocation) constitutively activates STAT5 and ERK1/2 in a JAK2-independent manner; three N-terminal coiled-coil domains of KANK1 are required for KANK1-PDGFRβ-induced oligomerization (homotrimers and heavier oligomers), signaling, and hematopoietic cell growth.","method":"Retroviral transduction of Ba/F3 cells and CD34+ progenitors, JAK inhibitor treatment, phosphorylation assays, mutagenesis of coiled-coil domains, gel filtration oligomerization analysis","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 — domain mutagenesis with functional signaling readouts in two cell systems","pmids":["21685469"],"is_preprint":false},{"year":2023,"finding":"KANK1 structures in complex with talin R7 and liprin-β were determined; KANK1 undergoes liquid-liquid phase separation (LLPS) important for its localization at the focal adhesion edge; KANK1 scaffolds the FA core and associated proteins to modulate FA shape in response to mechanical force.","method":"X-ray crystallography, biochemical assays, LLPS assay, cell biological analysis (immunofluorescence, live-cell imaging)","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 — multiple crystal structures combined with LLPS demonstration and functional cellular analysis","pmids":["37874676"],"is_preprint":false},{"year":2023,"finding":"The talin-KANK1 complex structure revealed a novel β-hairpin motif in the KN region of KANK1 that stabilizes its α-helical talin-binding interface with high affinity; actomyosin forces on talin exclude KANK1 from the FA center while retaining it at the adhesion periphery (adhesion belt).","method":"Non-covalent crystallographic chaperone approach (crystal structure), site-directed mutagenesis, myosin inhibitor treatment, constitutively active vinculin expression, immunofluorescence","journal":"Open biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with mutagenesis and mechanically perturbable cell biology","pmids":["37339751"],"is_preprint":false},{"year":2014,"finding":"Drosophila Kank (ortholog of human KANK1) directly binds EB1, and this interaction is essential for Kank localization to microtubule plus ends in cultured cells; in late embryos Kank accumulates at muscle-tendon attachment sites.","method":"Direct binding assay, immunofluorescence, deletion mutant analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2–3 — direct binding assay with localization phenotype in Drosophila ortholog","pmids":["25203404"],"is_preprint":false},{"year":2021,"finding":"TRAIP promotes polyubiquitination and proteasomal degradation of KANK1, leading to downregulation of IGFBP3 and activation of the AKT pathway, thereby enhancing osteosarcoma cell invasion and proliferation.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, KANK1 overexpression/knockdown, AKT pathway readouts","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 — ubiquitination assay plus functional pathway epistasis; single lab","pmids":["34349117"],"is_preprint":false},{"year":2022,"finding":"In C2C12 myoblasts, KANK1 depletion increases F-actin accumulation and promotes nuclear localization of YAP1 by reducing YAP1 phosphorylation in the cytoplasm, activating YAP1 target genes and promoting proliferation while inhibiting myogenic differentiation.","method":"siRNA knockdown, F-actin staining (phalloidin), YAP1 phosphorylation/localization by immunofluorescence and western blot, myogenic marker expression, myotube formation assay","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2–3 — clean KD with defined cellular phenotypes and pathway readout; single lab","pmids":["35805114"],"is_preprint":false},{"year":2017,"finding":"KANK1 restoration in MPNST cells induces apoptosis and inhibits growth via upregulation of CXXC5; knockdown of CXXC5 diminishes KANK1-induced apoptosis, placing CXXC5 downstream of KANK1 in apoptosis signaling.","method":"Overexpression and knockdown in cell lines, xenograft assay, RNA-seq, CXXC5 siRNA epistasis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2–3 — genetic epistasis via siRNA rescue; supported by transcriptomic identification of target","pmids":["28067315"],"is_preprint":false},{"year":2025,"finding":"KANK1 is locally enriched at the β-cell capillary interface and forms a complex linking talin (focal adhesion) to liprin-β1 (which anchors liprin-α1); KANK1 knockdown disrupts liprin-α1 subcellular localization, reduces glucose-induced insulin secretion, and misdirects insulin granule fusion away from the capillary interface.","method":"siRNA knockdown, co-immunoprecipitation, immunofluorescence localization, live-cell TIRF imaging of granule fusion, insulin secretion assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional secretion assay and live imaging; single lab but multiple orthogonal methods","pmids":["41380968"],"is_preprint":false},{"year":2023,"finding":"The KIF21A–KANK1 interaction is critical for dendritic spine morphogenesis and synaptic plasticity in neurons; knockdown of KIF21A or KANK1 inhibits spine morphogenesis and dendritic branching, rescued only by wild-type proteins but not by binding-deficient mutants (disrupting KIF21A–KANK1 or KANK1–talin1 interfaces).","method":"siRNA knockdown in neurons, rescue with binding-deficient mutants, LTP recording, cognitive behavioral assay","journal":"Neural regeneration research","confidence":"Medium","confidence_rationale":"Tier 2 — mutagenic rescue experiment plus in vivo LTP and behavior; single lab","pmids":["38767486"],"is_preprint":false},{"year":2024,"finding":"KANK1 localizes to the basal side of BM-attached epithelial tumor cells; upon BM contact loss, KANK1 relocates to cell-cell junctions where it competes with Scribble for NOS1AP binding, thereby reducing Scribble's ability to activate the Hippo pathway, leading to TAZ nuclear accumulation and tumor cell survival.","method":"In vivo PyMT mammary tumor model, KANK1 knockout, co-immunoprecipitation (KANK1–NOS1AP, Scribble–NOS1AP competition), TAZ/YAP nuclear localization assay, Hippo pathway reporters","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — in vivo genetic model plus Co-IP competition assay and signaling pathway readouts; multiple orthogonal methods","pmids":["39613731"],"is_preprint":false},{"year":2025,"finding":"The hub protein LC8 binds multiple weak motifs in the intrinsically disordered linker L2 of KANK1 cooperatively, converting this ~600 aa disordered region into an elongated rod-like assembly (~35–50 nm) long enough to bridge the membrane–microtubule gap at focal adhesions.","method":"In-cell assays, biochemical binding assays, AlphaFold multivalent assembly prediction, electron microscopy structural analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1–2 — EM structure plus biochemistry, but preprint not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"KANK1 is a scaffold adaptor protein that localizes at the periphery of focal adhesions (FA belt) via a direct, mechanosensitive interaction between its KN domain and talin rod domain R7, where it recruits cortical microtubule stabilizing complexes (via KIF21A, LL5β, CLASPs, and liprins) and links FA cores to adjacent liprin scaffolds; KANK1 also suppresses RhoA and Rac1 activity (through 14-3-3/Akt-regulated inhibition and by blocking IRSp53–Rac1 interaction), regulates cell polarity and migration, undergoes nucleo-cytoplasmic shuttling to modulate β-catenin signaling, and at the tissue level orients insulin secretion toward capillaries in β-cells and competes with Scribble for NOS1AP binding to modulate Hippo/TAZ signaling."},"narrative":{"teleology":[{"year":2006,"claim":"Establishing that KANK1 is not solely cytoplasmic resolved how it could influence transcription: it possesses functional NLS and NES sequences, shuttles between nucleus and cytoplasm in a CRM1-dependent manner, binds β-catenin, and activates β-catenin-dependent transcription.","evidence":"NLS/NES mutagenesis, leptomycin B treatment, TOPFLASH reporter assay, Co-IP in cultured cells","pmids":["16968744"],"confidence":"Medium","gaps":["Endogenous nuclear KANK1 levels not quantified under physiological conditions","β-catenin target genes regulated by KANK1 not identified","Relationship between nuclear shuttling and adhesion-site functions unclear"]},{"year":2008,"claim":"Identifying KANK1 as an Akt substrate explained how growth factor signaling tunes its cytoskeletal function: PI3K/Akt phosphorylation promotes 14-3-3 binding, which inactivates KANK1's RhoA-suppressive activity and permits stress fiber formation and cell migration.","evidence":"In vitro kinase assay, Co-IP, RhoA-GTP pulldown, migration assay in NIH3T3 cells","pmids":["18458160"],"confidence":"High","gaps":["Phosphorylation site(s) not mapped to specific serines at that time","Whether 14-3-3 binding also affects KANK1 localization or other interactions unknown"]},{"year":2009,"claim":"Demonstrating that KANK1 inhibits Rac1 via blocking IRSp53–Rac1 interaction, selectively suppressing lamellipodia but not filopodia, established KANK1 as a dual negative regulator of both RhoA and Rac1 branches of Rho-family signaling.","evidence":"Co-IP, GST pulldown, RNAi knockdown, cell spreading and lamellipodia assays","pmids":["19171758"],"confidence":"High","gaps":["Whether RhoA and Rac1 suppression are coordinated or independent remains unclear","Structural basis of KANK1–IRSp53 interaction not determined"]},{"year":2009,"claim":"Identifying KIF21A as a KANK1 interactor via the ankyrin repeat domain, and showing that KIF21A controls KANK1 subcellular distribution, provided the first link between KANK1 and microtubule motor function.","evidence":"Co-IP, subcellular fractionation, siRNA knockdown in cultured cells","pmids":["19559006"],"confidence":"Medium","gaps":["Interaction mapped only by Co-IP and fractionation, no structural detail at that time","Functional consequence for microtubule dynamics not tested"]},{"year":2011,"claim":"The discovery that KANK1 associates with BIG1 and that both are required for MTOC/Golgi reorientation during wound healing established KANK1 as a polarity regulator acting through the ARF-GEF pathway.","evidence":"Reciprocal Co-IP, siRNA depletion, wound-healing migration assay, Golgi/MTOC orientation imaging","pmids":["22084092"],"confidence":"Medium","gaps":["Whether KANK1–BIG1 interaction is direct or within a larger complex not resolved","ARF-dependent trafficking cargo regulated by this pathway not identified"]},{"year":2016,"claim":"The crystal structure of the KANK1 KN domain bound to talin R7, combined with a single point mutation that abolished recruitment without affecting adhesion, provided the definitive molecular mechanism by which KANK1 couples focal adhesions to cortical microtubule stabilizing complexes containing CLASPs, KIF21A, LL5β, and liprins.","evidence":"X-ray crystallography, Co-IP, single-point mutagenesis, fluorescence microscopy","pmids":["27410476"],"confidence":"High","gaps":["How KANK1 discriminates between different focal adhesion subpopulations unclear","Stoichiometry of the KANK1–talin complex at adhesions not determined"]},{"year":2017,"claim":"High-resolution structures of the KANK1 ankyrin repeat–KIF21A complex revealed a dual-interface supramodule recognition mechanism and showed that cancer-associated mutations destabilize this interaction, defining the structural basis for microtubule effector recruitment.","evidence":"X-ray crystallography at 2.1 Å, site-directed mutagenesis, biochemical binding assays","pmids":["29158259","29217769","29183992"],"confidence":"High","gaps":["Whether cancer-associated mutations affect KANK1 function in vivo not tested","How KIF21A recruitment alters microtubule dynamics at focal adhesions not directly measured"]},{"year":2017,"claim":"Demonstrating that KANK1 loss causes centrosome amplification and cytokinesis failure through Daam1-mediated RhoA hyperactivation expanded KANK1's role from adhesion scaffold to a regulator of cell division fidelity.","evidence":"RNAi knockdown, Co-IP of KANK1–Daam1, RhoA activity assay, centrosome counting, Aurora-A activity measurement","pmids":["28284839"],"confidence":"Medium","gaps":["Whether KANK1–Daam1 interaction is direct not confirmed","Single cell line study; generalizability unclear"]},{"year":2019,"claim":"Single-molecule force spectroscopy revealed that the talin R7–KANK1 KN complex withstands physiological forces up to 10 pN, explaining why KANK1 is mechanically excluded from the high-tension FA center and confined to the peripheral adhesion belt.","evidence":"Magnetic tweezers single-molecule force spectroscopy, TIRF live-cell imaging","pmids":["31389241"],"confidence":"High","gaps":["Force thresholds in cells not directly measured","Whether other adhesion proteins modulate force sensitivity of the complex unknown"]},{"year":2022,"claim":"Linking KANK1 depletion to YAP1 nuclear localization and impaired myogenic differentiation revealed that KANK1's actin-regulatory function feeds into the Hippo/YAP mechanotransduction axis.","evidence":"siRNA knockdown in C2C12 myoblasts, YAP1 phosphorylation and localization assays, myogenic differentiation markers","pmids":["35805114"],"confidence":"Medium","gaps":["Whether YAP activation is direct or secondary to F-actin changes not resolved","Single cell type tested"]},{"year":2023,"claim":"Structures of KANK1 with both talin R7 and liprin-β, combined with LLPS demonstration, established that KANK1 undergoes phase separation at the adhesion belt to organize a scaffold bridging FA cores to adjacent liprin assemblies and modulate FA shape.","evidence":"X-ray crystallography, LLPS assays, live-cell imaging, immunofluorescence","pmids":["37874676","37339751"],"confidence":"High","gaps":["LLPS regulation in vivo not characterized","Whether phase separation is required or merely correlative for function not fully tested"]},{"year":2023,"claim":"Showing that the KIF21A–KANK1–talin1 axis is required for dendritic spine morphogenesis and LTP extended KANK1's scaffolding function to neuronal synaptic plasticity.","evidence":"siRNA knockdown in neurons, rescue with binding-deficient mutants, LTP electrophysiology, behavioral assays","pmids":["38767486"],"confidence":"Medium","gaps":["Neuronal cell type specificity not explored","Downstream signaling from KANK1 loss in neurons not identified"]},{"year":2024,"claim":"Demonstrating that KANK1 relocates to cell–cell junctions upon basement membrane detachment, where it competes with Scribble for NOS1AP binding to suppress Hippo pathway activation, revealed a context-dependent signaling role that promotes TAZ-driven tumor cell survival.","evidence":"In vivo PyMT mammary tumor model, KANK1 knockout, Co-IP competition assay, TAZ localization and Hippo reporters","pmids":["39613731"],"confidence":"High","gaps":["Whether KANK1–NOS1AP competition occurs in non-tumor contexts unknown","Structural basis of KANK1–NOS1AP interaction not determined"]},{"year":2025,"claim":"Identifying KANK1 enrichment at the β-cell capillary interface and showing it directs insulin granule fusion toward capillaries via a talin–KANK1–liprin scaffold demonstrated a physiological secretory polarity role for KANK1.","evidence":"siRNA knockdown, Co-IP, TIRF imaging of granule fusion, insulin secretion assay in β-cells","pmids":["41380968"],"confidence":"Medium","gaps":["In vivo validation in animal models not yet performed","Whether other KANK family members compensate in β-cells not tested"]},{"year":null,"claim":"Key unresolved questions include: how KANK1 LLPS is regulated by post-translational modifications and mechanical signals, the full structural basis of the elongated KANK1 rod bridging focal adhesions to microtubules, and whether KANK1's nuclear, adhesion, and junctional functions are coordinated or independently regulated.","evidence":"","pmids":[],"confidence":"Low","gaps":["Full-length KANK1 structure not available","Coordination between nuclear shuttling, adhesion scaffolding, and junctional signaling roles unknown","Post-translational modification map beyond Akt phosphorylation incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3,4,13,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,10,21]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,6,15]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,6,13,14,15]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,7]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[7,21]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,2,10,21]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[0,6,13,14]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[10]}],"complexes":["Cortical microtubule stabilizing complex (CMSC)","Talin-KANK1-liprin scaffold"],"partners":["TLN1","KIF21A","PPFIA1","PPFIBP1","IRSP53","DAAM1","NOS1AP","ARFGEF1"],"other_free_text":[]},"mechanistic_narrative":"KANK1 is a mechanosensitive scaffold protein that links focal adhesions to cortical microtubule stabilizing complexes and modulates Rho-family GTPase signaling to coordinate cell polarity, migration, and adhesion dynamics. Its N-terminal KN domain binds directly to talin rod domain R7 with a force-resistant interaction that confines KANK1 to the focal adhesion periphery under actomyosin tension, while its C-terminal ankyrin repeat domain recruits KIF21A via a structurally defined dual-interface supramodule, thereby tethering microtubule plus-end machinery (CLASPs, LL5β, liprins) to adhesion sites [PMID:27410476, PMID:29158259, PMID:37874676, PMID:31389241]. KANK1 suppresses RhoA activity — regulated by Akt-mediated phosphorylation and 14-3-3 binding — and inhibits Rac1-driven lamellipodia by blocking the IRSp53–Rac1 interaction, thereby restraining actin remodeling and cell spreading [PMID:18458160, PMID:19171758]. KANK1 undergoes liquid–liquid phase separation at the adhesion belt and can undergo CRM1-dependent nucleo-cytoplasmic shuttling to activate β-catenin-dependent transcription; at cell–cell junctions it competes with Scribble for NOS1AP binding, suppressing Hippo pathway activation and promoting TAZ nuclear accumulation [PMID:37874676, PMID:16968744, PMID:39613731]."},"prefetch_data":{"uniprot":{"accession":"Q14678","full_name":"KN motif and ankyrin repeat domain-containing protein 1","aliases":["Ankyrin repeat domain-containing protein 15","Kidney ankyrin repeat-containing protein"],"length_aa":1352,"mass_kda":147.3,"function":"Adapter protein that links structural and signaling protein complexes positioned to guide microtubule and actin cytoskeleton dynamics during cell morphogenesis (PubMed:22084092, PubMed:24120883). At focal adhesions (FAs) rims, organizes cortical microtubule stabilizing complexes (CMSCs) and directly interacts with major FA component TLN1, forming macromolecular assemblies positioned to control microtubule-actin crosstalk at the cell edge (PubMed:24120883, PubMed:27410476). Recruits KIF21A in CMSCs at axonal growth cones and regulates axon guidance by suppressing microtubule growth without inducing microtubule disassembly once it reaches the cell cortex (PubMed:24120883). Interacts with ARFGEF1 and participates in establishing microtubule-organizing center (MTOC) orientation and directed cell movement in wound healing (PubMed:22084092). Regulates actin stress fiber formation and cell migration by inhibiting RHOA activation in response to growth factors; this function involves phosphorylation through PI3K/Akt signaling and may depend on the competitive interaction with 14-3-3 adapter proteins to sequester them from active complexes (PubMed:18458160, PubMed:25961457). Inhibits the formation of lamellipodia but not of filopodia; this function may depend on the competitive interaction with BAIAP2 to block its association with activated RAC1. Inhibits fibronectin-mediated cell spreading; this function is partially mediated by BAIAP2 (PubMed:19171758). In the nucleus, is involved in beta-catenin-dependent activation of transcription (PubMed:16968744). During cell division, may regulate DAAM1-dependent RHOA activation that signals centrosome maturation and chromosomal segregation. May also be involved in contractile ring formation during cytokinesis (By similarity). Potential tumor suppressor for renal cell carcinoma (Probable)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q14678/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KANK1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DYNLL1","stoichiometry":0.2},{"gene":"DYNLL2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/KANK1","total_profiled":1310},"omim":[{"mim_id":"614612","title":"KN MOTIF- AND ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 4; KANK4","url":"https://www.omim.org/entry/614612"},{"mim_id":"612900","title":"CEREBRAL PALSY, SPASTIC QUADRIPLEGIC, 2; CPSQ2","url":"https://www.omim.org/entry/612900"},{"mim_id":"607704","title":"KN MOTIF- AND ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 1; KANK1","url":"https://www.omim.org/entry/607704"},{"mim_id":"256300","title":"NEPHROTIC SYNDROME, TYPE 1; NPHS1","url":"https://www.omim.org/entry/256300"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/KANK1"},"hgnc":{"alias_symbol":["KIAA0172","KANK"],"prev_symbol":["ANKRD15"]},"alphafold":{"accession":"Q14678","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14678","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14678-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14678-F1-predicted_aligned_error_v6.png","plddt_mean":53.97},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KANK1","jax_strain_url":"https://www.jax.org/strain/search?query=KANK1"},"sequence":{"accession":"Q14678","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14678.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14678/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14678"}},"corpus_meta":[{"pmid":"25961457","id":"PMC_25961457","title":"KANK deficiency leads to podocyte dysfunction and nephrotic syndrome.","date":"2015","source":"The Journal of clinical 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A single point mutation in talin disrupting KANK1 binding abolishes this recruitment without affecting talin's adhesion function.\",\n      \"method\": \"Structural studies (crystal structure), Co-IP, single point mutagenesis, fluorescence microscopy\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis plus cell biological validation in one study\",\n      \"pmids\": [\"27410476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"KANK1 is an Akt substrate downstream of PI3K; Akt-mediated phosphorylation of KANK1 promotes its binding to 14-3-3 proteins, which inhibits KANK1's suppression of RhoA activity, actin stress fiber formation, and insulin-induced cell migration.\",\n      \"method\": \"Kinase assay, co-immunoprecipitation, overexpression/co-expression in NIH3T3 cells, RhoA activity assay (GST-rhotekin pulldown), migration assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro kinase substrate identification combined with multiple orthogonal functional assays\",\n      \"pmids\": [\"18458160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KANK1 binds IRSp53 and specifically inhibits the IRSp53–Rac1 interaction, suppressing lamellipodia formation without affecting filopodia (Cdc42-dependent), thereby negatively regulating actin remodeling and cell spreading.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, RNAi knockdown, overexpression, microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal pulldown/Co-IP plus RNAi epistasis with multiple phenotypic readouts\",\n      \"pmids\": [\"19171758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of the KANK1 ankyrin repeat domain (ANKRD) in complex with a KIF21A peptide revealed that target recognition involves combinatorial use of two interfaces; mutations at either interface disrupt the KANK1–KIF21A interaction and prevent KIF21A recruitment to focal adhesions.\",\n      \"method\": \"X-ray crystallography (high-resolution crystal structure), mutagenesis, biochemical binding assays, immunofluorescence localization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis and functional cellular validation\",\n      \"pmids\": [\"29217769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of the KANK1·KIF21A complex at 2.1 Å showed that a five-helix-bundle-capping domain immediately preceding the ANK repeats forms a supramodule with the ANK repeats to bind a conserved KIF21A peptide; cancer-associated missense mutations at this interface destabilize the complex.\",\n      \"method\": \"X-ray crystallography, biochemical assays, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — atomic resolution structure with biochemical and mutagenic validation\",\n      \"pmids\": [\"29158259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The KANK2 ankyrin domain binds the same ~22 amino acid KIF21A peptide as KANK1; both complex structures show KIF21A adopting helical conformations upon binding to two distinct pockets of the ankyrin domain.\",\n      \"method\": \"X-ray crystallography, site-directed mutagenesis, biochemical binding assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — dual crystal structures with mutagenesis\",\n      \"pmids\": [\"29183992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The talin R7–KANK1 KN domain complex can withstand physiological forces (up to 10 pN) for seconds to minutes under shear-force geometry; mechanical stretching promotes KANK1 localization to the periphery of focal adhesions rather than the center.\",\n      \"method\": \"Magnetic tweezers single-molecule force spectroscopy, cell biology (live cell imaging, TIRF)\",\n      \"journal\": \"Nano letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — single-molecule mechanical measurements combined with cell biological validation\",\n      \"pmids\": [\"31389241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"KANK1 interacts with the third and fourth coiled-coil domains of KIF21A via its ankyrin-repeat domain; KIF21A controls the subcellular distribution of KANK1, with KIF21A knockdown causing KANK1 to remain cytosolic, and the CFEOM1-associated KIF21A R954W mutation significantly enhancing KANK1 translocation to the membrane fraction.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, siRNA knockdown, Western blotting\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP and fractionation; single lab\",\n      \"pmids\": [\"19559006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KANK1 physically and functionally associates with BIG1 (a guanine nucleotide-exchange factor for ARFs); depletion of either BIG1 or KANK1 similarly disrupts directed cell migration and Golgi/MTOC orientation toward the wound edge, indicating both function in maintaining cell polarity during migration.\",\n      \"method\": \"Reciprocal co-immunoprecipitation, siRNA depletion, wound-healing migration assay, Golgi/MTOC orientation imaging\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — reciprocal IP plus functional migration assay; moderate mechanistic depth\",\n      \"pmids\": [\"22084092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"KANK1 contains functional nuclear localization signals (NLS1, NLS2) and nuclear export signals (NES1–NES3); nuclear export is CRM1-dependent (blocked by leptomycin B); KANK1 binds β-catenin and its nuclear import correlates with activation of β-catenin-dependent transcription (TOPFLASH reporter).\",\n      \"method\": \"NLS/NES mutagenesis, leptomycin B treatment, TOPFLASH reporter assay, co-immunoprecipitation, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenesis of transport signals plus functional reporter assay and Co-IP, single lab\",\n      \"pmids\": [\"16968744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"KANK1 knockdown causes centrosomal amplification and cytokinesis failure via hyperactivation of RhoA; KANK1 interacts with Daam1 (a RhoA activator), and excess Daam1 upon KANK1 loss hyperactivates RhoA, elevating Aurora-A activity and leading to abnormal centrosome numbers and multinucleate cells.\",\n      \"method\": \"RNAi knockdown, co-immunoprecipitation (KANK1–Daam1), RhoA activity assay, centrosome counting, Aurora-A activity measurement\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP plus functional pathway epistasis; single lab\",\n      \"pmids\": [\"28284839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In rat glomeruli and cultured human podocytes, KANK2 interacts with ARHGDIA (a RHO GTPase regulator); KANK2 knockdown increases active GTP-bound RHOA and decreases podocyte migration; RNAi of the Drosophila KANK homolog disrupts slit diaphragm and lacuna channel structures in nephrocytes.\",\n      \"method\": \"Co-immunoprecipitation, RhoA activity assay (GTP-pulldown), RNAi in Drosophila nephrocytes, zebrafish kank2 morpholino knockdown, immunofluorescence co-localization\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and activity assay plus in vivo model validation; primarily KANK2 but in context of KANK family mechanism\",\n      \"pmids\": [\"25961457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The KANK1-PDGFRβ fusion protein (from t(5;9) translocation) constitutively activates STAT5 and ERK1/2 in a JAK2-independent manner; three N-terminal coiled-coil domains of KANK1 are required for KANK1-PDGFRβ-induced oligomerization (homotrimers and heavier oligomers), signaling, and hematopoietic cell growth.\",\n      \"method\": \"Retroviral transduction of Ba/F3 cells and CD34+ progenitors, JAK inhibitor treatment, phosphorylation assays, mutagenesis of coiled-coil domains, gel filtration oligomerization analysis\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — domain mutagenesis with functional signaling readouts in two cell systems\",\n      \"pmids\": [\"21685469\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"KANK1 structures in complex with talin R7 and liprin-β were determined; KANK1 undergoes liquid-liquid phase separation (LLPS) important for its localization at the focal adhesion edge; KANK1 scaffolds the FA core and associated proteins to modulate FA shape in response to mechanical force.\",\n      \"method\": \"X-ray crystallography, biochemical assays, LLPS assay, cell biological analysis (immunofluorescence, live-cell imaging)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple crystal structures combined with LLPS demonstration and functional cellular analysis\",\n      \"pmids\": [\"37874676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The talin-KANK1 complex structure revealed a novel β-hairpin motif in the KN region of KANK1 that stabilizes its α-helical talin-binding interface with high affinity; actomyosin forces on talin exclude KANK1 from the FA center while retaining it at the adhesion periphery (adhesion belt).\",\n      \"method\": \"Non-covalent crystallographic chaperone approach (crystal structure), site-directed mutagenesis, myosin inhibitor treatment, constitutively active vinculin expression, immunofluorescence\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with mutagenesis and mechanically perturbable cell biology\",\n      \"pmids\": [\"37339751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila Kank (ortholog of human KANK1) directly binds EB1, and this interaction is essential for Kank localization to microtubule plus ends in cultured cells; in late embryos Kank accumulates at muscle-tendon attachment sites.\",\n      \"method\": \"Direct binding assay, immunofluorescence, deletion mutant analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — direct binding assay with localization phenotype in Drosophila ortholog\",\n      \"pmids\": [\"25203404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRAIP promotes polyubiquitination and proteasomal degradation of KANK1, leading to downregulation of IGFBP3 and activation of the AKT pathway, thereby enhancing osteosarcoma cell invasion and proliferation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, KANK1 overexpression/knockdown, AKT pathway readouts\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — ubiquitination assay plus functional pathway epistasis; single lab\",\n      \"pmids\": [\"34349117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In C2C12 myoblasts, KANK1 depletion increases F-actin accumulation and promotes nuclear localization of YAP1 by reducing YAP1 phosphorylation in the cytoplasm, activating YAP1 target genes and promoting proliferation while inhibiting myogenic differentiation.\",\n      \"method\": \"siRNA knockdown, F-actin staining (phalloidin), YAP1 phosphorylation/localization by immunofluorescence and western blot, myogenic marker expression, myotube formation assay\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — clean KD with defined cellular phenotypes and pathway readout; single lab\",\n      \"pmids\": [\"35805114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"KANK1 restoration in MPNST cells induces apoptosis and inhibits growth via upregulation of CXXC5; knockdown of CXXC5 diminishes KANK1-induced apoptosis, placing CXXC5 downstream of KANK1 in apoptosis signaling.\",\n      \"method\": \"Overexpression and knockdown in cell lines, xenograft assay, RNA-seq, CXXC5 siRNA epistasis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — genetic epistasis via siRNA rescue; supported by transcriptomic identification of target\",\n      \"pmids\": [\"28067315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KANK1 is locally enriched at the β-cell capillary interface and forms a complex linking talin (focal adhesion) to liprin-β1 (which anchors liprin-α1); KANK1 knockdown disrupts liprin-α1 subcellular localization, reduces glucose-induced insulin secretion, and misdirects insulin granule fusion away from the capillary interface.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, immunofluorescence localization, live-cell TIRF imaging of granule fusion, insulin secretion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional secretion assay and live imaging; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"41380968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The KIF21A–KANK1 interaction is critical for dendritic spine morphogenesis and synaptic plasticity in neurons; knockdown of KIF21A or KANK1 inhibits spine morphogenesis and dendritic branching, rescued only by wild-type proteins but not by binding-deficient mutants (disrupting KIF21A–KANK1 or KANK1–talin1 interfaces).\",\n      \"method\": \"siRNA knockdown in neurons, rescue with binding-deficient mutants, LTP recording, cognitive behavioral assay\",\n      \"journal\": \"Neural regeneration research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mutagenic rescue experiment plus in vivo LTP and behavior; single lab\",\n      \"pmids\": [\"38767486\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KANK1 localizes to the basal side of BM-attached epithelial tumor cells; upon BM contact loss, KANK1 relocates to cell-cell junctions where it competes with Scribble for NOS1AP binding, thereby reducing Scribble's ability to activate the Hippo pathway, leading to TAZ nuclear accumulation and tumor cell survival.\",\n      \"method\": \"In vivo PyMT mammary tumor model, KANK1 knockout, co-immunoprecipitation (KANK1–NOS1AP, Scribble–NOS1AP competition), TAZ/YAP nuclear localization assay, Hippo pathway reporters\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo genetic model plus Co-IP competition assay and signaling pathway readouts; multiple orthogonal methods\",\n      \"pmids\": [\"39613731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The hub protein LC8 binds multiple weak motifs in the intrinsically disordered linker L2 of KANK1 cooperatively, converting this ~600 aa disordered region into an elongated rod-like assembly (~35–50 nm) long enough to bridge the membrane–microtubule gap at focal adhesions.\",\n      \"method\": \"In-cell assays, biochemical binding assays, AlphaFold multivalent assembly prediction, electron microscopy structural analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 — EM structure plus biochemistry, but preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"KANK1 is a scaffold adaptor protein that localizes at the periphery of focal adhesions (FA belt) via a direct, mechanosensitive interaction between its KN domain and talin rod domain R7, where it recruits cortical microtubule stabilizing complexes (via KIF21A, LL5β, CLASPs, and liprins) and links FA cores to adjacent liprin scaffolds; KANK1 also suppresses RhoA and Rac1 activity (through 14-3-3/Akt-regulated inhibition and by blocking IRSp53–Rac1 interaction), regulates cell polarity and migration, undergoes nucleo-cytoplasmic shuttling to modulate β-catenin signaling, and at the tissue level orients insulin secretion toward capillaries in β-cells and competes with Scribble for NOS1AP binding to modulate Hippo/TAZ signaling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"KANK1 is a mechanosensitive scaffold protein that links focal adhesions to cortical microtubule stabilizing complexes and modulates Rho-family GTPase signaling to coordinate cell polarity, migration, and adhesion dynamics. Its N-terminal KN domain binds directly to talin rod domain R7 with a force-resistant interaction that confines KANK1 to the focal adhesion periphery under actomyosin tension, while its C-terminal ankyrin repeat domain recruits KIF21A via a structurally defined dual-interface supramodule, thereby tethering microtubule plus-end machinery (CLASPs, LL5β, liprins) to adhesion sites [PMID:27410476, PMID:29158259, PMID:37874676, PMID:31389241]. KANK1 suppresses RhoA activity — regulated by Akt-mediated phosphorylation and 14-3-3 binding — and inhibits Rac1-driven lamellipodia by blocking the IRSp53–Rac1 interaction, thereby restraining actin remodeling and cell spreading [PMID:18458160, PMID:19171758]. KANK1 undergoes liquid–liquid phase separation at the adhesion belt and can undergo CRM1-dependent nucleo-cytoplasmic shuttling to activate β-catenin-dependent transcription; at cell–cell junctions it competes with Scribble for NOS1AP binding, suppressing Hippo pathway activation and promoting TAZ nuclear accumulation [PMID:37874676, PMID:16968744, PMID:39613731].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing that KANK1 is not solely cytoplasmic resolved how it could influence transcription: it possesses functional NLS and NES sequences, shuttles between nucleus and cytoplasm in a CRM1-dependent manner, binds β-catenin, and activates β-catenin-dependent transcription.\",\n      \"evidence\": \"NLS/NES mutagenesis, leptomycin B treatment, TOPFLASH reporter assay, Co-IP in cultured cells\",\n      \"pmids\": [\"16968744\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous nuclear KANK1 levels not quantified under physiological conditions\", \"β-catenin target genes regulated by KANK1 not identified\", \"Relationship between nuclear shuttling and adhesion-site functions unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identifying KANK1 as an Akt substrate explained how growth factor signaling tunes its cytoskeletal function: PI3K/Akt phosphorylation promotes 14-3-3 binding, which inactivates KANK1's RhoA-suppressive activity and permits stress fiber formation and cell migration.\",\n      \"evidence\": \"In vitro kinase assay, Co-IP, RhoA-GTP pulldown, migration assay in NIH3T3 cells\",\n      \"pmids\": [\"18458160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphorylation site(s) not mapped to specific serines at that time\", \"Whether 14-3-3 binding also affects KANK1 localization or other interactions unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrating that KANK1 inhibits Rac1 via blocking IRSp53–Rac1 interaction, selectively suppressing lamellipodia but not filopodia, established KANK1 as a dual negative regulator of both RhoA and Rac1 branches of Rho-family signaling.\",\n      \"evidence\": \"Co-IP, GST pulldown, RNAi knockdown, cell spreading and lamellipodia assays\",\n      \"pmids\": [\"19171758\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RhoA and Rac1 suppression are coordinated or independent remains unclear\", \"Structural basis of KANK1–IRSp53 interaction not determined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying KIF21A as a KANK1 interactor via the ankyrin repeat domain, and showing that KIF21A controls KANK1 subcellular distribution, provided the first link between KANK1 and microtubule motor function.\",\n      \"evidence\": \"Co-IP, subcellular fractionation, siRNA knockdown in cultured cells\",\n      \"pmids\": [\"19559006\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interaction mapped only by Co-IP and fractionation, no structural detail at that time\", \"Functional consequence for microtubule dynamics not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"The discovery that KANK1 associates with BIG1 and that both are required for MTOC/Golgi reorientation during wound healing established KANK1 as a polarity regulator acting through the ARF-GEF pathway.\",\n      \"evidence\": \"Reciprocal Co-IP, siRNA depletion, wound-healing migration assay, Golgi/MTOC orientation imaging\",\n      \"pmids\": [\"22084092\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether KANK1–BIG1 interaction is direct or within a larger complex not resolved\", \"ARF-dependent trafficking cargo regulated by this pathway not identified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The crystal structure of the KANK1 KN domain bound to talin R7, combined with a single point mutation that abolished recruitment without affecting adhesion, provided the definitive molecular mechanism by which KANK1 couples focal adhesions to cortical microtubule stabilizing complexes containing CLASPs, KIF21A, LL5β, and liprins.\",\n      \"evidence\": \"X-ray crystallography, Co-IP, single-point mutagenesis, fluorescence microscopy\",\n      \"pmids\": [\"27410476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How KANK1 discriminates between different focal adhesion subpopulations unclear\", \"Stoichiometry of the KANK1–talin complex at adhesions not determined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"High-resolution structures of the KANK1 ankyrin repeat–KIF21A complex revealed a dual-interface supramodule recognition mechanism and showed that cancer-associated mutations destabilize this interaction, defining the structural basis for microtubule effector recruitment.\",\n      \"evidence\": \"X-ray crystallography at 2.1 Å, site-directed mutagenesis, biochemical binding assays\",\n      \"pmids\": [\"29158259\", \"29217769\", \"29183992\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cancer-associated mutations affect KANK1 function in vivo not tested\", \"How KIF21A recruitment alters microtubule dynamics at focal adhesions not directly measured\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrating that KANK1 loss causes centrosome amplification and cytokinesis failure through Daam1-mediated RhoA hyperactivation expanded KANK1's role from adhesion scaffold to a regulator of cell division fidelity.\",\n      \"evidence\": \"RNAi knockdown, Co-IP of KANK1–Daam1, RhoA activity assay, centrosome counting, Aurora-A activity measurement\",\n      \"pmids\": [\"28284839\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether KANK1–Daam1 interaction is direct not confirmed\", \"Single cell line study; generalizability unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Single-molecule force spectroscopy revealed that the talin R7–KANK1 KN complex withstands physiological forces up to 10 pN, explaining why KANK1 is mechanically excluded from the high-tension FA center and confined to the peripheral adhesion belt.\",\n      \"evidence\": \"Magnetic tweezers single-molecule force spectroscopy, TIRF live-cell imaging\",\n      \"pmids\": [\"31389241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Force thresholds in cells not directly measured\", \"Whether other adhesion proteins modulate force sensitivity of the complex unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linking KANK1 depletion to YAP1 nuclear localization and impaired myogenic differentiation revealed that KANK1's actin-regulatory function feeds into the Hippo/YAP mechanotransduction axis.\",\n      \"evidence\": \"siRNA knockdown in C2C12 myoblasts, YAP1 phosphorylation and localization assays, myogenic differentiation markers\",\n      \"pmids\": [\"35805114\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether YAP activation is direct or secondary to F-actin changes not resolved\", \"Single cell type tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Structures of KANK1 with both talin R7 and liprin-β, combined with LLPS demonstration, established that KANK1 undergoes phase separation at the adhesion belt to organize a scaffold bridging FA cores to adjacent liprin assemblies and modulate FA shape.\",\n      \"evidence\": \"X-ray crystallography, LLPS assays, live-cell imaging, immunofluorescence\",\n      \"pmids\": [\"37874676\", \"37339751\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"LLPS regulation in vivo not characterized\", \"Whether phase separation is required or merely correlative for function not fully tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showing that the KIF21A–KANK1–talin1 axis is required for dendritic spine morphogenesis and LTP extended KANK1's scaffolding function to neuronal synaptic plasticity.\",\n      \"evidence\": \"siRNA knockdown in neurons, rescue with binding-deficient mutants, LTP electrophysiology, behavioral assays\",\n      \"pmids\": [\"38767486\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Neuronal cell type specificity not explored\", \"Downstream signaling from KANK1 loss in neurons not identified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrating that KANK1 relocates to cell–cell junctions upon basement membrane detachment, where it competes with Scribble for NOS1AP binding to suppress Hippo pathway activation, revealed a context-dependent signaling role that promotes TAZ-driven tumor cell survival.\",\n      \"evidence\": \"In vivo PyMT mammary tumor model, KANK1 knockout, Co-IP competition assay, TAZ localization and Hippo reporters\",\n      \"pmids\": [\"39613731\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether KANK1–NOS1AP competition occurs in non-tumor contexts unknown\", \"Structural basis of KANK1–NOS1AP interaction not determined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying KANK1 enrichment at the β-cell capillary interface and showing it directs insulin granule fusion toward capillaries via a talin–KANK1–liprin scaffold demonstrated a physiological secretory polarity role for KANK1.\",\n      \"evidence\": \"siRNA knockdown, Co-IP, TIRF imaging of granule fusion, insulin secretion assay in β-cells\",\n      \"pmids\": [\"41380968\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo validation in animal models not yet performed\", \"Whether other KANK family members compensate in β-cells not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: how KANK1 LLPS is regulated by post-translational modifications and mechanical signals, the full structural basis of the elongated KANK1 rod bridging focal adhesions to microtubules, and whether KANK1's nuclear, adhesion, and junctional functions are coordinated or independently regulated.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Full-length KANK1 structure not available\", \"Coordination between nuclear shuttling, adhesion scaffolding, and junctional signaling roles unknown\", \"Post-translational modification map beyond Akt phosphorylation incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3, 4, 13, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 10, 21]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 6, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 6, 13, 14, 15]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 7]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [7, 21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 2, 10, 21]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [0, 6, 13, 14]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"complexes\": [\n      \"Cortical microtubule stabilizing complex (CMSC)\",\n      \"Talin-KANK1-liprin scaffold\"\n    ],\n    \"partners\": [\n      \"TLN1\",\n      \"KIF21A\",\n      \"PPFIA1\",\n      \"PPFIBP1\",\n      \"IRSp53\",\n      \"DAAM1\",\n      \"NOS1AP\",\n      \"ARFGEF1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}