{"gene":"KPTN","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":1999,"finding":"Kaptin (2E4/KPTN) is a novel actin-binding protein that binds to filamentous (F)-actin, as demonstrated by F-actin affinity chromatography, and is eluted from F-actin columns by ATP, indicating ATP-sensitive actin association. It localizes to the leading edge of platelets and lamellipodia of motile fibroblasts, and to the tips of elongating stereocilia of the inner ear, consistent with a role in actin polymerization dynamics.","method":"F-actin affinity chromatography, immunofluorescence localization in platelets, fibroblasts, and inner ear sensory epithelium","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical binding assay (F-actin affinity chromatography) plus multiple cell-type localization experiments in a single study; not independently replicated with mutagenesis","pmids":["10099934"],"is_preprint":false},{"year":2000,"finding":"KPTN (2E4/kaptin) localizes beyond the barbed ends of actin filaments at the tips of stereocilia, as shown by double-label immunofluorescence with F-actin markers, indicating it acts at the barbed-end actin polymerization site in stereocilia.","method":"Double-label immunofluorescence in inner ear sensory epithelium","journal":"Annals of human genetics","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single localization method (immunofluorescence), single study, no functional validation","pmids":["11409409"],"is_preprint":false},{"year":2013,"finding":"Endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures in primary neuronal cell cultures, and this association is lost upon introduction of disease-causing KPTN mutations identified in families with macrocephaly and neurodevelopmental delay, establishing that actin association is required for normal neuromorphogenesis.","method":"Immunofluorescence of endogenous kaptin and GFP-tagged kaptin in primary neuronal cultures, mutant constructs with patient-derived mutations","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization in primary neurons with functional mutation analysis; single lab, no reconstitution","pmids":["24239382"],"is_preprint":false},{"year":2023,"finding":"KPTN is a component of the mTOR regulatory complex KICSTOR (comprising KPTN, ITFG2, C12orf66, and SZT2), and loss of KPTN in mouse knockout and human iPSC models leads to increased mTORC1 signaling, which is rapamycin-sensitive, placing KPTN as a negative regulator of mTORC1 upstream of the lysosomal signaling axis.","method":"Mouse Kptn knockout model (biochemical and transcriptional analysis), human iPSC differentiation model, rapamycin treatment rescue experiment","journal":"Brain : a journal of neurology","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent model systems (mouse KO and human iPSC), biochemical mTOR pathway readouts, pharmacological rescue with rapamycin, consistent with prior KICSTOR complex data","pmids":["37437211"],"is_preprint":false},{"year":2024,"finding":"OTUD3 is a deubiquitinase for KPTN that interacts with KPTN via its OTU domain. KPTN is ubiquitinated at lysine residue K49, and this ubiquitination is a non-degradative, function-regulating modification. OTUD3-mediated deubiquitination of KPTN suppresses mTORC1 signaling and promotes GATOR1 lysosomal localization in a KPTN-dependent manner.","method":"In vivo ubiquitination assay, Co-immunoprecipitation (OTUD3–KPTN interaction), CRISPR/Cas9 OTUD3 knockout, immunofluorescence for GATOR1 lysosomal localization, cell proliferation assay, NMR","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo ubiquitination assay with domain-mapping, CRISPR KO, and functional mTOR readouts; single lab","pmids":["38288086"],"is_preprint":false},{"year":2025,"finding":"FBXO2 directly interacts with KPTN via its F-box-associated domain and promotes K48- and K63-linked polyubiquitination of KPTN at lysine residues K49, K67, K262, and K265. This ubiquitination disrupts KPTN's interaction with ITFG2 and SZT2 (other KICSTOR components), while enhancing its interaction with C12orf66, thereby impairing KICSTOR's ability to recruit the GATOR1 complex (DEPDC5, NPRL2, NPRL3) to the lysosomal surface and thus activating mTORC1 signaling.","method":"Co-immunoprecipitation (FBXO2–KPTN interaction, KPTN–ITFG2/SZT2/C12orf66), in vitro ubiquitination assay with linkage-type determination, domain-mapping using F-box-associated domain mutants, lysosomal co-localization assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — multiple orthogonal methods (Co-IP, in vitro ubiquitination with linkage specificity, domain mutagenesis, complex disruption assays, lysosomal recruitment assay) in a single rigorous study","pmids":["41401028"],"is_preprint":false},{"year":2025,"finding":"CRISPR/Cas9 Kptn knockout in N2a cells in vitro induces mTOR activation (elevated pS6) and mTOR-dependent multi-cell aggregate formation within 24–48 hours of plating, which is abolished by rapamycin treatment, establishing that KPTN loss leads to mTOR-dependent cellular aggregation phenotypes relevant to cortical dyslamination.","method":"CRISPR/Cas9 knockout in N2a cells, Western blotting for pS6, timelapse live-cell imaging, rapamycin treatment rescue, LC-MS/MS proteomics","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct KO with pharmacological rescue and multiple orthogonal readouts; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.11.02.685388"],"is_preprint":true},{"year":2026,"finding":"CRISPR/Cas9 Kptn knockout in vitro induces mTOR activation and an mTOR-dependent increase in cell size. Focal in utero electroporation-based Kptn knockout in the mouse cortex results in heterotopic neurons in the subcortical white matter, demonstrating a cell-autonomous role for KPTN in cortical neuronal positioning dependent on mTOR regulation.","method":"CRISPR/Cas9 knockout in vitro (cell size assay), in utero electroporation for focal cortical Kptn KO, histological analysis in Kptn-/- mice, rapamycin treatment of Kptn-/- mice","journal":"Annals of neurology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro KO and in vivo focal KO with defined cellular phenotypes, pharmacological rescue; single study","pmids":["41696790"],"is_preprint":false}],"current_model":"KPTN (kaptin) is an actin-binding protein and essential component of the KICSTOR complex (with ITFG2, C12orf66, and SZT2) that negatively regulates mTORC1 signaling by enabling GATOR1 complex recruitment to the lysosomal surface in response to amino acid availability; its ubiquitination by FBXO2 (at K49, K67, K262, K265) disrupts KICSTOR integrity and activates mTORC1, while OTUD3-mediated deubiquitination at K49 suppresses mTORC1 and promotes GATOR1 lysosomal localization, and loss-of-function mutations cause rapamycin-sensitive mTOR hyperactivation leading to macrocephaly, cortical dyslamination, and neurodevelopmental deficits."},"narrative":{"mechanistic_narrative":"KPTN (kaptin) is an actin-binding protein that functions as an essential subunit of the lysosomal KICSTOR complex, where it serves as a negative regulator of mTORC1 signaling controlling neuronal growth and cortical positioning [PMID:37437211, PMID:41401028]. It was first defined biochemically as an F-actin-binding protein that localizes to dynamic actin structures including platelet leading edges, fibroblast lamellipodia, and the barbed-end actin-polymerization sites at stereocilia tips, with actin association required for normal neuromorphogenesis [PMID:10099934, PMID:24239382]. Within KICSTOR (KPTN, ITFG2, C12orf66, SZT2), KPTN enables recruitment of the GATOR1 complex (DEPDC5, NPRL2, NPRL3) to the lysosomal surface, thereby restraining mTORC1; loss of KPTN produces rapamycin-sensitive mTORC1 hyperactivation [PMID:37437211, PMID:41401028]. KICSTOR integrity is set by competing ubiquitination events: FBXO2 binds KPTN through its F-box-associated domain and catalyzes K48/K63-linked polyubiquitination at K49, K67, K262, and K265, disrupting KPTN's interactions with ITFG2 and SZT2 and impairing GATOR1 recruitment to activate mTORC1, while OTUD3 reverses non-degradative ubiquitination at K49 to suppress mTORC1 and promote GATOR1 lysosomal localization [PMID:38288086, PMID:41401028]. Consistent with this regulatory role, KPTN loss-of-function causes mTOR-dependent phenotypes including increased cell size, cellular aggregation, and heterotopic neurons in the cortex, underlying a neurodevelopmental disorder with macrocephaly and cortical dyslamination [PMID:24239382, PMID:41696790].","teleology":[{"year":1999,"claim":"Established the first molecular identity of KPTN as an actin-binding protein, defining where it acts in the cell before any signaling role was known.","evidence":"F-actin affinity chromatography with ATP elution plus immunofluorescence in platelets, fibroblasts, and inner ear epithelium","pmids":["10099934"],"confidence":"Medium","gaps":["No mutagenesis to map the actin-binding interface","Functional consequence of actin binding not tested","No link to a signaling pathway"]},{"year":2000,"claim":"Refined KPTN's actin localization to the barbed-end polymerization site at stereocilia tips, implying a role at sites of active filament elongation.","evidence":"Double-label immunofluorescence with F-actin markers in inner ear sensory epithelium","pmids":["11409409"],"confidence":"Low","gaps":["Single localization method, no functional validation","Mechanism of barbed-end targeting unknown"]},{"year":2013,"claim":"Connected KPTN actin association to human disease, showing patient mutations abolish actin localization and impair neuromorphogenesis.","evidence":"Immunofluorescence of endogenous and GFP-tagged kaptin in primary neurons with patient-derived mutant constructs","pmids":["24239382"],"confidence":"Medium","gaps":["Did not establish the downstream signaling mechanism","Single lab, no reconstitution","Causal chain from actin mislocalization to neuronal phenotype unresolved"]},{"year":2023,"claim":"Placed KPTN within the KICSTOR complex as a negative regulator of mTORC1, reframing the disease as an mTOR signalopathy.","evidence":"Mouse Kptn knockout and human iPSC models with mTORC1 biochemical readouts and rapamycin rescue","pmids":["37437211"],"confidence":"High","gaps":["Did not resolve how KPTN loss is sensed at the lysosome","Relationship between actin function and mTOR role unclear"]},{"year":2024,"claim":"Identified post-translational control of KPTN, showing OTUD3 deubiquitinates KPTN at K49 to suppress mTORC1 and promote GATOR1 lysosomal localization.","evidence":"In vivo ubiquitination assay, Co-IP, OTUD3 CRISPR knockout, GATOR1 localization imaging, NMR","pmids":["38288086"],"confidence":"Medium","gaps":["The opposing ubiquitin ligase was not identified in this study","Single lab","Stoichiometry and dynamics of the modification unresolved"]},{"year":2025,"claim":"Identified FBXO2 as the ligase that ubiquitinates KPTN and resolved the mechanism by which this modification dismantles KICSTOR to activate mTORC1.","evidence":"Co-IP, in vitro ubiquitination with linkage typing, F-box-associated domain mutagenesis, KICSTOR subunit interaction and lysosomal recruitment assays","pmids":["41401028"],"confidence":"High","gaps":["Upstream signals controlling FBXO2 activity not defined","Interplay between FBXO2 and OTUD3 not quantified","In vivo relevance of specific lysine sites not tested"]},{"year":2025,"claim":"Demonstrated that acute KPTN loss drives mTOR-dependent cellular aggregation, linking the signaling defect to a phenotype relevant to cortical dyslamination.","evidence":"CRISPR knockout in N2a cells, pS6 Western blot, live-cell imaging, rapamycin rescue, LC-MS/MS (preprint)","pmids":["bio_10.1101_2025.11.02.685388"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Aggregation mechanism downstream of mTOR not defined","In vitro cell line system"]},{"year":2026,"claim":"Established a cell-autonomous, mTOR-dependent role for KPTN in cortical neuronal positioning in vivo.","evidence":"In utero electroporation focal Kptn KO in mouse cortex, histology, cell size assays, rapamycin treatment of Kptn-/- mice","pmids":["41696790"],"confidence":"Medium","gaps":["Molecular link between mTOR hyperactivation and migration defect unresolved","Single study","Contribution of actin-binding function to migration not separated from mTOR role"]},{"year":null,"claim":"It remains unresolved how KPTN's original actin-binding function mechanistically relates to its KICSTOR/mTORC1 regulatory role, and what physiological signals coordinate FBXO2 and OTUD3 to set KICSTOR integrity.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No experiment unifies the actin and mTOR functions","Upstream regulators of the ubiquitination switch unknown","No structural model of KPTN within KICSTOR"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,5]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,2]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[4,5]}],"pathway":[],"complexes":["KICSTOR"],"partners":["ITFG2","SZT2","C12ORF66","FBXO2","OTUD3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y664","full_name":"KICSTOR complex protein kaptin","aliases":["Actin-associated protein 2E4"],"length_aa":436,"mass_kda":48.1,"function":"As part of the KICSTOR complex functions in the amino acid-sensing branch of the TORC1 signaling pathway. Recruits, in an amino acid-independent manner, the GATOR1 complex to the lysosomal membranes and allows its interaction with GATOR2 and the RAG GTPases. Functions upstream of the RAG GTPases and is required to negatively regulate mTORC1 signaling in absence of amino acids. In absence of the KICSTOR complex mTORC1 is constitutively localized to the lysosome and activated. The KICSTOR complex is also probably involved in the regulation of mTORC1 by glucose","subcellular_location":"Lysosome membrane; Cell projection, lamellipodium; Cell projection, stereocilium","url":"https://www.uniprot.org/uniprotkb/Q9Y664/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/KPTN","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/KPTN","total_profiled":1310},"omim":[{"mim_id":"621100","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL RECESSIVE 83; MRT83","url":"https://www.omim.org/entry/621100"},{"mim_id":"617421","title":"INTEGRIN-ALPHA FG-GAP REPEAT-CONTAINING PROTEIN 2; ITFG2","url":"https://www.omim.org/entry/617421"},{"mim_id":"617420","title":"KICSTOR SUBUNIT 2; KICS2","url":"https://www.omim.org/entry/617420"},{"mim_id":"615637","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL RECESSIVE 41; MRT41","url":"https://www.omim.org/entry/615637"},{"mim_id":"615620","title":"KAPTIN; KPTN","url":"https://www.omim.org/entry/615620"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/KPTN"},"hgnc":{"alias_symbol":["2E4","KICS4"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y664","domains":[{"cath_id":"2.130.10.10","chopping":"195-261_271-362","consensus_level":"medium","plddt":94.2719,"start":195,"end":362}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y664","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y664-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y664-F1-predicted_aligned_error_v6.png","plddt_mean":88.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=KPTN","jax_strain_url":"https://www.jax.org/strain/search?query=KPTN"},"sequence":{"accession":"Q9Y664","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y664.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y664/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y664"}},"corpus_meta":[{"pmid":"24239382","id":"PMC_24239382","title":"Mutations in KPTN cause macrocephaly, neurodevelopmental delay, and seizures.","date":"2013","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24239382","citation_count":47,"is_preprint":false},{"pmid":"25847626","id":"PMC_25847626","title":"Novel homozygous mutation in KPTN gene causing a familial intellectual disability-macrocephaly syndrome.","date":"2015","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/25847626","citation_count":33,"is_preprint":false},{"pmid":"25596549","id":"PMC_25596549","title":"The mAb against adipocyte fatty acid-binding protein 2E4 attenuates the inflammation in the mouse model of high-fat diet-induced obesity via toll-like receptor 4 pathway.","date":"2015","source":"Molecular and cellular endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/25596549","citation_count":26,"is_preprint":false},{"pmid":"10099934","id":"PMC_10099934","title":"2E4 (kaptin): a novel actin-associated protein from human blood platelets found in lamellipodia and the tips of the stereocilia of the inner ear.","date":"1999","source":"European journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/10099934","citation_count":26,"is_preprint":false},{"pmid":"11409409","id":"PMC_11409409","title":"2E4/Kaptin (KPTN)--a candidate gene for the hearing loss locus, DFNA4.","date":"2000","source":"Annals of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11409409","citation_count":19,"is_preprint":false},{"pmid":"37437211","id":"PMC_37437211","title":"Models of KPTN-related disorder implicate mTOR signalling in cognitive and overgrowth phenotypes.","date":"2023","source":"Brain : a journal of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/37437211","citation_count":10,"is_preprint":false},{"pmid":"32358097","id":"PMC_32358097","title":"Pathogenic variants in KPTN gene identified by clinical whole-genome sequencing.","date":"2020","source":"Cold Spring Harbor molecular case studies","url":"https://pubmed.ncbi.nlm.nih.gov/32358097","citation_count":9,"is_preprint":false},{"pmid":"31999056","id":"PMC_31999056","title":"KPTN gene homozygous variant-related syndrome in the northeast of Brazil: A case report.","date":"2020","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/31999056","citation_count":7,"is_preprint":false},{"pmid":"29795597","id":"PMC_29795597","title":"Antibacterial Effect of (2E,2E)-4,4-Trisulfanediylbis(but-2-enoic acid) against Staphylococcus aureus.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29795597","citation_count":6,"is_preprint":false},{"pmid":"38288086","id":"PMC_38288086","title":"OTUD3 suppresses the mTORC1 signaling by deubiquitinating KPTN.","date":"2024","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/38288086","citation_count":5,"is_preprint":false},{"pmid":"36703628","id":"PMC_36703628","title":"Case report: KPTN gene-related syndrome associated with a spectrum of neurodevelopmental anomalies including severe epilepsy.","date":"2023","source":"Frontiers in neurology","url":"https://pubmed.ncbi.nlm.nih.gov/36703628","citation_count":4,"is_preprint":false},{"pmid":"31695071","id":"PMC_31695071","title":"Structural Features of a Conformation-dependent Antigen Epitope on ORFV-B2L Recognized by the 2E4 mAb.","date":"2019","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/31695071","citation_count":3,"is_preprint":false},{"pmid":"37311648","id":"PMC_37311648","title":"Nonsense variant in a consanguineous family expands the phenotype of KPTN gene-related syndrome to include hearing impairment.","date":"2023","source":"Clinical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/37311648","citation_count":2,"is_preprint":false},{"pmid":"41401028","id":"PMC_41401028","title":"FBXO2-mediated KPTN ubiquitination promotes amino acid-dependent mTORC1 signaling and tumor growth.","date":"2025","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/41401028","citation_count":1,"is_preprint":false},{"pmid":"41696790","id":"PMC_41696790","title":"The Clinical Spectrum and Neurodevelopmental Pathogenesis of KPTN-Related Disorder in a Mouse Model.","date":"2026","source":"Annals of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/41696790","citation_count":0,"is_preprint":false},{"pmid":"41357736","id":"PMC_41357736","title":"Kaptin-Actin Binding Protein (KPTN)-Related Disorder: A Case Report of Two Siblings Harboring a Novel KPTN Mutation.","date":"2025","source":"Cureus","url":"https://pubmed.ncbi.nlm.nih.gov/41357736","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.02.685388","title":"mTOR pathway gene knockout results in mTOR-dependent cellular aggregation","date":"2025-11-04","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.02.685388","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9948,"output_tokens":2409,"usd":0.032989,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9421,"output_tokens":2881,"usd":0.059565,"stage2_stop_reason":"end_turn"},"total_usd":0.092554,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"Kaptin (2E4/KPTN) is a novel actin-binding protein that binds to filamentous (F)-actin, as demonstrated by F-actin affinity chromatography, and is eluted from F-actin columns by ATP, indicating ATP-sensitive actin association. It localizes to the leading edge of platelets and lamellipodia of motile fibroblasts, and to the tips of elongating stereocilia of the inner ear, consistent with a role in actin polymerization dynamics.\",\n      \"method\": \"F-actin affinity chromatography, immunofluorescence localization in platelets, fibroblasts, and inner ear sensory epithelium\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical binding assay (F-actin affinity chromatography) plus multiple cell-type localization experiments in a single study; not independently replicated with mutagenesis\",\n      \"pmids\": [\"10099934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"KPTN (2E4/kaptin) localizes beyond the barbed ends of actin filaments at the tips of stereocilia, as shown by double-label immunofluorescence with F-actin markers, indicating it acts at the barbed-end actin polymerization site in stereocilia.\",\n      \"method\": \"Double-label immunofluorescence in inner ear sensory epithelium\",\n      \"journal\": \"Annals of human genetics\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single localization method (immunofluorescence), single study, no functional validation\",\n      \"pmids\": [\"11409409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures in primary neuronal cell cultures, and this association is lost upon introduction of disease-causing KPTN mutations identified in families with macrocephaly and neurodevelopmental delay, establishing that actin association is required for normal neuromorphogenesis.\",\n      \"method\": \"Immunofluorescence of endogenous kaptin and GFP-tagged kaptin in primary neuronal cultures, mutant constructs with patient-derived mutations\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization in primary neurons with functional mutation analysis; single lab, no reconstitution\",\n      \"pmids\": [\"24239382\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"KPTN is a component of the mTOR regulatory complex KICSTOR (comprising KPTN, ITFG2, C12orf66, and SZT2), and loss of KPTN in mouse knockout and human iPSC models leads to increased mTORC1 signaling, which is rapamycin-sensitive, placing KPTN as a negative regulator of mTORC1 upstream of the lysosomal signaling axis.\",\n      \"method\": \"Mouse Kptn knockout model (biochemical and transcriptional analysis), human iPSC differentiation model, rapamycin treatment rescue experiment\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent model systems (mouse KO and human iPSC), biochemical mTOR pathway readouts, pharmacological rescue with rapamycin, consistent with prior KICSTOR complex data\",\n      \"pmids\": [\"37437211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"OTUD3 is a deubiquitinase for KPTN that interacts with KPTN via its OTU domain. KPTN is ubiquitinated at lysine residue K49, and this ubiquitination is a non-degradative, function-regulating modification. OTUD3-mediated deubiquitination of KPTN suppresses mTORC1 signaling and promotes GATOR1 lysosomal localization in a KPTN-dependent manner.\",\n      \"method\": \"In vivo ubiquitination assay, Co-immunoprecipitation (OTUD3–KPTN interaction), CRISPR/Cas9 OTUD3 knockout, immunofluorescence for GATOR1 lysosomal localization, cell proliferation assay, NMR\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo ubiquitination assay with domain-mapping, CRISPR KO, and functional mTOR readouts; single lab\",\n      \"pmids\": [\"38288086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXO2 directly interacts with KPTN via its F-box-associated domain and promotes K48- and K63-linked polyubiquitination of KPTN at lysine residues K49, K67, K262, and K265. This ubiquitination disrupts KPTN's interaction with ITFG2 and SZT2 (other KICSTOR components), while enhancing its interaction with C12orf66, thereby impairing KICSTOR's ability to recruit the GATOR1 complex (DEPDC5, NPRL2, NPRL3) to the lysosomal surface and thus activating mTORC1 signaling.\",\n      \"method\": \"Co-immunoprecipitation (FBXO2–KPTN interaction, KPTN–ITFG2/SZT2/C12orf66), in vitro ubiquitination assay with linkage-type determination, domain-mapping using F-box-associated domain mutants, lysosomal co-localization assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple orthogonal methods (Co-IP, in vitro ubiquitination with linkage specificity, domain mutagenesis, complex disruption assays, lysosomal recruitment assay) in a single rigorous study\",\n      \"pmids\": [\"41401028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CRISPR/Cas9 Kptn knockout in N2a cells in vitro induces mTOR activation (elevated pS6) and mTOR-dependent multi-cell aggregate formation within 24–48 hours of plating, which is abolished by rapamycin treatment, establishing that KPTN loss leads to mTOR-dependent cellular aggregation phenotypes relevant to cortical dyslamination.\",\n      \"method\": \"CRISPR/Cas9 knockout in N2a cells, Western blotting for pS6, timelapse live-cell imaging, rapamycin treatment rescue, LC-MS/MS proteomics\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct KO with pharmacological rescue and multiple orthogonal readouts; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.11.02.685388\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CRISPR/Cas9 Kptn knockout in vitro induces mTOR activation and an mTOR-dependent increase in cell size. Focal in utero electroporation-based Kptn knockout in the mouse cortex results in heterotopic neurons in the subcortical white matter, demonstrating a cell-autonomous role for KPTN in cortical neuronal positioning dependent on mTOR regulation.\",\n      \"method\": \"CRISPR/Cas9 knockout in vitro (cell size assay), in utero electroporation for focal cortical Kptn KO, histological analysis in Kptn-/- mice, rapamycin treatment of Kptn-/- mice\",\n      \"journal\": \"Annals of neurology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro KO and in vivo focal KO with defined cellular phenotypes, pharmacological rescue; single study\",\n      \"pmids\": [\"41696790\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"KPTN (kaptin) is an actin-binding protein and essential component of the KICSTOR complex (with ITFG2, C12orf66, and SZT2) that negatively regulates mTORC1 signaling by enabling GATOR1 complex recruitment to the lysosomal surface in response to amino acid availability; its ubiquitination by FBXO2 (at K49, K67, K262, K265) disrupts KICSTOR integrity and activates mTORC1, while OTUD3-mediated deubiquitination at K49 suppresses mTORC1 and promotes GATOR1 lysosomal localization, and loss-of-function mutations cause rapamycin-sensitive mTOR hyperactivation leading to macrocephaly, cortical dyslamination, and neurodevelopmental deficits.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"KPTN (kaptin) is an actin-binding protein that functions as an essential subunit of the lysosomal KICSTOR complex, where it serves as a negative regulator of mTORC1 signaling controlling neuronal growth and cortical positioning [#3, #5]. It was first defined biochemically as an F-actin-binding protein that localizes to dynamic actin structures including platelet leading edges, fibroblast lamellipodia, and the barbed-end actin-polymerization sites at stereocilia tips, with actin association required for normal neuromorphogenesis [#0, #2]. Within KICSTOR (KPTN, ITFG2, C12orf66, SZT2), KPTN enables recruitment of the GATOR1 complex (DEPDC5, NPRL2, NPRL3) to the lysosomal surface, thereby restraining mTORC1; loss of KPTN produces rapamycin-sensitive mTORC1 hyperactivation [#3, #5]. KICSTOR integrity is set by competing ubiquitination events: FBXO2 binds KPTN through its F-box-associated domain and catalyzes K48/K63-linked polyubiquitination at K49, K67, K262, and K265, disrupting KPTN's interactions with ITFG2 and SZT2 and impairing GATOR1 recruitment to activate mTORC1, while OTUD3 reverses non-degradative ubiquitination at K49 to suppress mTORC1 and promote GATOR1 lysosomal localization [#4, #5]. Consistent with this regulatory role, KPTN loss-of-function causes mTOR-dependent phenotypes including increased cell size, cellular aggregation, and heterotopic neurons in the cortex, underlying a neurodevelopmental disorder with macrocephaly and cortical dyslamination [#2, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the first molecular identity of KPTN as an actin-binding protein, defining where it acts in the cell before any signaling role was known.\",\n      \"evidence\": \"F-actin affinity chromatography with ATP elution plus immunofluorescence in platelets, fibroblasts, and inner ear epithelium\",\n      \"pmids\": [\"10099934\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mutagenesis to map the actin-binding interface\", \"Functional consequence of actin binding not tested\", \"No link to a signaling pathway\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Refined KPTN's actin localization to the barbed-end polymerization site at stereocilia tips, implying a role at sites of active filament elongation.\",\n      \"evidence\": \"Double-label immunofluorescence with F-actin markers in inner ear sensory epithelium\",\n      \"pmids\": [\"11409409\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single localization method, no functional validation\", \"Mechanism of barbed-end targeting unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Connected KPTN actin association to human disease, showing patient mutations abolish actin localization and impair neuromorphogenesis.\",\n      \"evidence\": \"Immunofluorescence of endogenous and GFP-tagged kaptin in primary neurons with patient-derived mutant constructs\",\n      \"pmids\": [\"24239382\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish the downstream signaling mechanism\", \"Single lab, no reconstitution\", \"Causal chain from actin mislocalization to neuronal phenotype unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Placed KPTN within the KICSTOR complex as a negative regulator of mTORC1, reframing the disease as an mTOR signalopathy.\",\n      \"evidence\": \"Mouse Kptn knockout and human iPSC models with mTORC1 biochemical readouts and rapamycin rescue\",\n      \"pmids\": [\"37437211\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how KPTN loss is sensed at the lysosome\", \"Relationship between actin function and mTOR role unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified post-translational control of KPTN, showing OTUD3 deubiquitinates KPTN at K49 to suppress mTORC1 and promote GATOR1 lysosomal localization.\",\n      \"evidence\": \"In vivo ubiquitination assay, Co-IP, OTUD3 CRISPR knockout, GATOR1 localization imaging, NMR\",\n      \"pmids\": [\"38288086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The opposing ubiquitin ligase was not identified in this study\", \"Single lab\", \"Stoichiometry and dynamics of the modification unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified FBXO2 as the ligase that ubiquitinates KPTN and resolved the mechanism by which this modification dismantles KICSTOR to activate mTORC1.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination with linkage typing, F-box-associated domain mutagenesis, KICSTOR subunit interaction and lysosomal recruitment assays\",\n      \"pmids\": [\"41401028\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals controlling FBXO2 activity not defined\", \"Interplay between FBXO2 and OTUD3 not quantified\", \"In vivo relevance of specific lysine sites not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that acute KPTN loss drives mTOR-dependent cellular aggregation, linking the signaling defect to a phenotype relevant to cortical dyslamination.\",\n      \"evidence\": \"CRISPR knockout in N2a cells, pS6 Western blot, live-cell imaging, rapamycin rescue, LC-MS/MS (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.11.02.685388\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Aggregation mechanism downstream of mTOR not defined\", \"In vitro cell line system\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Established a cell-autonomous, mTOR-dependent role for KPTN in cortical neuronal positioning in vivo.\",\n      \"evidence\": \"In utero electroporation focal Kptn KO in mouse cortex, histology, cell size assays, rapamycin treatment of Kptn-/- mice\",\n      \"pmids\": [\"41696790\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between mTOR hyperactivation and migration defect unresolved\", \"Single study\", \"Contribution of actin-binding function to migration not separated from mTOR role\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how KPTN's original actin-binding function mechanistically relates to its KICSTOR/mTORC1 regulatory role, and what physiological signals coordinate FBXO2 and OTUD3 to set KICSTOR integrity.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experiment unifies the actin and mTOR functions\", \"Upstream regulators of the ubiquitination switch unknown\", \"No structural model of KPTN within KICSTOR\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [3, 5]}\n    ],\n    \"complexes\": [\"KICSTOR\"],\n    \"partners\": [\"ITFG2\", \"SZT2\", \"C12orf66\", \"FBXO2\", \"OTUD3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}