{"gene":"CASTOR1","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2016,"finding":"Crystal structure of arginine-bound CASTOR1 at 1.8 Å resolution reveals that homodimeric CASTOR1 binds arginine at the interface of two ACT domains, enabling allosteric control of the adjacent GATOR2-binding site; arginine binding triggers dissociation from GATOR2, thereby activating mTORC1 downstream.","method":"X-ray crystallography (1.8 Å), structure-guided mutagenesis, biochemical binding assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — atomic-resolution crystal structure plus mutagenesis and biochemical validation; independently replicated by two additional structural studies in the same year","pmids":["27487210"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of human CASTOR1–arginine complex identifies an arginine-binding pocket between the NTD and CTD (ACT) domains; a surface patch on CASTOR1-NTD opposite this pocket mediates direct physical interaction with GATOR2 subunit Mios; mutation of key pocket residues abolishes or diminishes arginine binding, and mutation of the surface patch disrupts CASTOR1 recognition and inhibition of GATOR2.","method":"X-ray crystallography, in vitro pull-down assay, site-directed mutagenesis, normal mode analysis","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus biochemical pull-down; orthogonal to PMID 27487210","pmids":["28066558"],"is_preprint":false},{"year":2016,"finding":"Crystal structure of CASTOR1–arginine complex at 2.5 Å shows four tandem ACT domains; ACT1 and ACT3 mediate homodimerization via conserved helix residues, while ACT2 and ACT4 form the arginine-binding pocket; mutagenesis of key binding-pocket residues validates their functional roles in arginine-dependent mTORC1 activation.","method":"X-ray crystallography (2.5 Å), site-directed mutagenesis, biochemical assays","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and biochemical validation, consistent with two parallel structural studies","pmids":["27648300"],"is_preprint":false},{"year":2018,"finding":"Comparison of arginine-bound (2.05 Å) and ligand-free (2.8 Å) CASTOR1 crystal structures reveals near-identical conformations except in two loop regions, indicating that CASTOR1 does not undergo large conformational change upon arginine binding; arginine sensing is thus interpreted as a subtle loop-based mechanism rather than a global domain rearrangement.","method":"X-ray crystallography (apo and holo structures), structural comparison","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — two crystal structures in one study providing direct structural comparison; single lab but high-resolution data","pmids":["30503338"],"is_preprint":false},{"year":2025,"finding":"Cryo-EM structure of the GATOR2–CASTOR1 complex (arginine-free) shows that two MIOS WD40 β-propellers of the GATOR2 cage engage both subunits of a single CASTOR1 homodimer at a negatively charged interface distal to the arginine pocket; arginine-triggered loop ordering in CASTOR1 sterically blocks this MIOS-binding interface, switching off GATOR2 binding and thereby de-repressing GATOR1 and activating mTORC1.","method":"Cryo-electron microscopy, structural analysis, functional validation","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure of the intact complex with mechanistic interpretation confirmed by independent preprint (PMID 40470200)","pmids":["40715445","40470200"],"is_preprint":false},{"year":2021,"finding":"E3 ubiquitin ligase RNF167 catalyzes K29-linked polyubiquitination and proteasomal degradation of CASTOR1; AKT phosphorylates CASTOR1 at Ser14, which markedly increases CASTOR1 binding to RNF167 (promoting its ubiquitination and degradation) while simultaneously decreasing CASTOR1 affinity for MIOS, resulting in mTORC1 activation independent of arginine.","method":"Co-immunoprecipitation, ubiquitination assays, phosphorylation site mutagenesis, RNF167 knockdown/overexpression, AKT inhibitor treatment","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, site-specific mutagenesis, and multiple orthogonal biochemical assays in a single study","pmids":["33594058"],"is_preprint":false},{"year":2019,"finding":"KSHV-encoded miRNAs miR-K4-5p (and likely miR-K1-5p) directly target the CASTOR1 3′-UTR to suppress CASTOR1 expression; knockdown of these miRNAs restores CASTOR1 protein levels and attenuates mTORC1 activation, establishing a viral miRNA–CASTOR1–GATOR2–mTORC1 axis.","method":"miRNA target validation (direct targeting of CASTOR1 3′-UTR), miRNA knockdown, CASTOR1/CASTOR2 overexpression, mTORC1 activity assays, soft-agar colony formation","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct 3′-UTR targeting demonstrated, rescue experiments performed; single lab","pmids":["31305263"],"is_preprint":false},{"year":2026,"finding":"CASTOR1 and CASTOR2 both bind arginine and interact with GATOR2 component Mios; arginine binding induces conformational changes at the ACT2–ACT4 interface causing dissociation from Mios; CASTOR1 responds to low arginine concentrations and CASTOR2 to high arginine concentrations, enabling dual-sensor fine-tuning of mTORC1 activity; in C2C12 muscle cells, CASTOR proteins regulate mTORC1 and myogenesis in an arginine-level-dependent manner.","method":"Biochemical binding assays, structural analysis of conformational changes, arginine dose-response experiments, C2C12 cell knockdown with mTORC1 and myogenesis readouts","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural and biochemical analyses with mutagenesis and functional cellular validation; single lab but multiple orthogonal methods","pmids":["41506264"],"is_preprint":false},{"year":2022,"finding":"In LPS/IFN-γ-activated microglia, CASTOR1 overexpression inhibits M1 polarization by suppressing mTOR signaling; mTOR activator MHY1485 rescues M1 polarization in CASTOR1-overexpressing cells, placing CASTOR1 upstream of mTOR in microglial polarization.","method":"CASTOR1 overexpression, mTOR activator (MHY1485) epistasis, M1/M2 marker expression assays in primary microglia","journal":"Metabolic brain disease","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic epistasis (activator rescue) confirms pathway placement; single lab, single cellular model","pmids":["36454506"],"is_preprint":false},{"year":2025,"finding":"CASTOR1 genetic ablation in a KRAS-driven GEMM (LSL-KRAS G12D) enhances lung tumor incidence and accelerates progression; mechanistically, CASTOR1 loss amplifies mTORC1 signaling (elevated p-4EBP1, p-S6) and augments AKT and ERK activation, revealing crosstalk between PI3K/AKT/mTORC1 and KRAS/ERK pathways; mTORC1 and PI3K inhibitors sensitize CASTOR1-deficient resistant tumors to KRAS G12D-targeted therapy.","method":"Genetically engineered mouse model (CASTOR1 KO × KRAS G12D), tumor-derived organoids, phospho-protein assays, drug combination rescue","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — in vivo genetic model with multiple mechanistic readouts; preprint, single lab, not yet peer-reviewed","pmids":["40313924"],"is_preprint":true}],"current_model":"CASTOR1 is a cytosolic arginine sensor that, in the absence of arginine, forms a homodimer via its ACT1/ACT3 domains and binds the GATOR2 complex through a negatively charged interface on CASTOR1 that contacts two MIOS WD40 β-propellers; arginine binding at the ACT2/ACT4 interface triggers loop ordering that sterically occludes the MIOS-binding surface, releasing CASTOR1 from GATOR2, thereby de-repressing GATOR1 GAP activity toward RagA/B and activating lysosomal mTORC1. CASTOR1 activity is further tuned post-translationally: AKT phosphorylates CASTOR1 at Ser14, enhancing its binding to E3 ligase RNF167 which catalyzes K29-linked polyubiquitination and proteasomal degradation of CASTOR1 to activate mTORC1 independently of arginine. CASTOR1 and its homolog CASTOR2 function as a dual-sensitivity system responding to low and high arginine concentrations respectively, together fine-tuning mTORC1 output in contexts such as muscle myogenesis."},"narrative":{"mechanistic_narrative":"CASTOR1 is a cytosolic arginine sensor that gates amino-acid signaling to mTORC1 by controlling the GATOR2 complex [PMID:27487210, PMID:40715445, PMID:40470200]. In the arginine-free state it forms a homodimer through its ACT1 and ACT3 domains and engages two MIOS WD40 β-propellers of the GATOR2 cage via a negatively charged surface distal to its ligand pocket, holding GATOR2 in an inhibited state and keeping mTORC1 low [PMID:27648300, PMID:40715445, PMID:40470200]. Arginine binds at the interface of the ACT2 and ACT4 domains, and rather than driving a large global rearrangement, it orders two loop regions that sterically occlude the MIOS-binding surface, releasing CASTOR1 from GATOR2 and de-repressing GATOR1 GAP activity to activate lysosomal mTORC1 [PMID:28066558, PMID:30503338, PMID:40715445, PMID:40470200]. CASTOR1 activity is additionally tuned post-translationally: AKT phosphorylates CASTOR1 at Ser14, increasing its binding to the E3 ligase RNF167, which catalyzes K29-linked polyubiquitination and proteasomal degradation of CASTOR1 to activate mTORC1 independently of arginine [PMID:33594058]. CASTOR1 and its homolog CASTOR2 act as a dual-sensitivity system responding to low and high arginine respectively, together fine-tuning mTORC1 output during processes such as C2C12 myogenesis [PMID:41506264]. Through this mTORC1-restraining role, CASTOR1 functions as a brake in disease-relevant settings, including KRAS-driven lung tumorigenesis and KSHV miRNA-driven mTORC1 activation [PMID:31305263, PMID:40313924].","teleology":[{"year":2016,"claim":"Established the structural basis for how CASTOR1 senses arginine and couples that signal to mTORC1, defining it as a ligand-gated repressor of GATOR2.","evidence":"X-ray crystallography of arginine-bound CASTOR1 with structure-guided mutagenesis and binding assays","pmids":["27487210","28066558","27648300"],"confidence":"High","gaps":["Apo-state conformation not resolved in these structures","Architecture of the CASTOR1–GATOR2 contact inferred from mutagenesis, not visualized","Did not address physiological arginine concentration thresholds"]},{"year":2016,"claim":"Defined the domain logic of CASTOR1, separating the homodimerization function (ACT1/ACT3) from the arginine-binding function (ACT2/ACT4).","evidence":"2.5 Å crystal structure with site-directed mutagenesis of pocket and dimer-interface residues","pmids":["27648300"],"confidence":"High","gaps":["Did not show how loop changes propagate to the GATOR2 interface","Functional readouts in cells limited"]},{"year":2018,"claim":"Resolved the conformational mechanism debate by showing arginine sensing is a localized loop reordering rather than a global domain swing.","evidence":"Comparison of apo (2.8 Å) and arginine-bound (2.05 Å) CASTOR1 crystal structures","pmids":["30503338"],"confidence":"High","gaps":["How loop ordering translates into loss of GATOR2 binding not demonstrated structurally at the time","Single-lab structures"]},{"year":2019,"claim":"Showed CASTOR1 is a target of pathogen-encoded regulation, linking viral miRNAs to mTORC1 activation through suppression of an arginine sensor.","evidence":"KSHV miRNA 3′-UTR target validation, miRNA knockdown rescue, and mTORC1/colony-formation assays","pmids":["31305263"],"confidence":"Medium","gaps":["Single lab","Relative contribution of miR-K1-5p not firmly quantified","In vivo relevance not tested"]},{"year":2021,"claim":"Uncovered an arginine-independent control layer in which AKT-driven phosphorylation routes CASTOR1 for ubiquitin-mediated degradation to activate mTORC1.","evidence":"Co-IP, K29-linkage ubiquitination assays, Ser14 phospho-site mutagenesis, RNF167 perturbation, and AKT inhibition","pmids":["33594058"],"confidence":"High","gaps":["Physiological stimuli engaging this axis not fully mapped","Crosstalk timing with arginine sensing unresolved"]},{"year":2022,"claim":"Placed CASTOR1 upstream of mTOR in a cellular phenotype, showing it restrains pro-inflammatory M1 microglial polarization.","evidence":"CASTOR1 overexpression with mTOR-activator (MHY1485) epistasis and M1/M2 marker readouts in primary microglia","pmids":["36454506"],"confidence":"Medium","gaps":["Single cellular model","Endogenous loss-of-function not tested","Arginine dependence in this context unexamined"]},{"year":2025,"claim":"Captured the intact arginine-free GATOR2–CASTOR1 complex, directly visualizing the MIOS contact and confirming the steric-occlusion switch model.","evidence":"Cryo-EM of the GATOR2–CASTOR1 complex with structural and functional validation","pmids":["40715445","40470200"],"confidence":"High","gaps":["Dynamics of the binding/release transition not directly observed","Stoichiometry relative to full GATOR2 cage occupancy not fully quantified"]},{"year":2025,"claim":"Provided in vivo evidence that CASTOR1 acts as a tumor suppressor that brakes mTORC1 and interacts with KRAS/ERK signaling.","evidence":"CASTOR1-KO × KRAS G12D mouse model, organoids, phospho-protein readouts, and drug-combination rescue (preprint)","pmids":["40313924"],"confidence":"Medium","gaps":["Preprint, not peer-reviewed","Single lab","Direct biochemical basis of AKT/ERK crosstalk not established"]},{"year":2026,"claim":"Defined CASTOR1 and CASTOR2 as a dual-sensitivity arginine-sensing pair, extending the single-sensor model to a graded mTORC1 readout with a developmental output.","evidence":"Biochemical binding, conformational analysis, arginine dose-response, and C2C12 knockdown with myogenesis readouts","pmids":["41506264"],"confidence":"High","gaps":["Single lab","In vivo myogenesis relevance not tested","Quantitative crossover point between the two sensors not fully defined"]},{"year":null,"claim":"How the arginine-sensing, AKT/RNF167 degradation, and KRAS/ERK crosstalk inputs are integrated to set mTORC1 output in specific tissues remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking post-translational degradation with structural ligand sensing in vivo","Tissue-specific physiological roles beyond muscle, microglia, and lung unmapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[0,1,2,3,4,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4,5]}],"complexes":[],"partners":["MIOS","RNF167","AKT1","CASTOR2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8WTX7","full_name":"Cytosolic arginine sensor for mTORC1 subunit 1","aliases":["Cellular arginine sensor for mTORC1 protein 1","GATS-like protein 3"],"length_aa":329,"mass_kda":36.3,"function":"Functions as an intracellular arginine sensor within the amino acid-sensing branch of the TORC1 signaling pathway (PubMed:26972053, PubMed:27487210, PubMed:33594058). As a homodimer or a heterodimer with CASTOR2, binds and inhibits the GATOR subcomplex GATOR2 and thereby mTORC1 (PubMed:26972053, PubMed:27487210, PubMed:33594058). Binding of arginine to CASTOR1 allosterically disrupts the interaction of CASTOR1-containing dimers with GATOR2 which can in turn activate mTORC1 and the TORC1 signaling pathway (PubMed:26972053, PubMed:27487210, PubMed:33594058)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q8WTX7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CASTOR1","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CASTOR1","total_profiled":1310},"omim":[{"mim_id":"620307","title":"WD REPEAT-CONTAINING PROTEIN 24; WDR24","url":"https://www.omim.org/entry/620307"},{"mim_id":"617418","title":"WD REPEAT-CONTAINING PROTEIN 59; WDR59","url":"https://www.omim.org/entry/617418"},{"mim_id":"617034","title":"CELLULAR ARGININE SENSOR FOR MTORC1 PROTEIN 1; CASTOR1","url":"https://www.omim.org/entry/617034"},{"mim_id":"617033","title":"CELLULAR ARGININE SENSOR FOR MTORC1 PROTEIN 2; CASTOR2","url":"https://www.omim.org/entry/617033"},{"mim_id":"601231","title":"MECHANISTIC TARGET OF RAPAMYCIN; MTOR","url":"https://www.omim.org/entry/601231"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"esophagus","ntpm":50.5}],"url":"https://www.proteinatlas.org/search/CASTOR1"},"hgnc":{"alias_symbol":[],"prev_symbol":["GATSL3"]},"alphafold":{"accession":"Q8WTX7","domains":[{"cath_id":"3.30.2130.10","chopping":"1-156_172-209_223-329","consensus_level":"medium","plddt":91.7732,"start":1,"end":329}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WTX7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WTX7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WTX7-F1-predicted_aligned_error_v6.png","plddt_mean":88.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CASTOR1","jax_strain_url":"https://www.jax.org/strain/search?query=CASTOR1"},"sequence":{"accession":"Q8WTX7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WTX7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WTX7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WTX7"}},"corpus_meta":[{"pmid":"27487210","id":"PMC_27487210","title":"Mechanism of arginine sensing by CASTOR1 upstream of mTORC1.","date":"2016","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/27487210","citation_count":258,"is_preprint":false},{"pmid":"28066558","id":"PMC_28066558","title":"Structural mechanism for the arginine sensing and regulation of CASTOR1 in the mTORC1 signaling pathway.","date":"2016","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/28066558","citation_count":49,"is_preprint":false},{"pmid":"33594058","id":"PMC_33594058","title":"RNF167 activates mTORC1 and promotes tumorigenesis by targeting CASTOR1 for ubiquitination and degradation.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33594058","citation_count":41,"is_preprint":false},{"pmid":"31305263","id":"PMC_31305263","title":"Kaposi sarcoma-associated herpesvirus miRNAs suppress CASTOR1-mediated mTORC1 inhibition to promote tumorigenesis.","date":"2019","source":"The Journal of clinical investigation","url":"https://pubmed.ncbi.nlm.nih.gov/31305263","citation_count":37,"is_preprint":false},{"pmid":"27648300","id":"PMC_27648300","title":"Structural insight into the arginine-binding specificity of CASTOR1 in amino acid-dependent mTORC1 signaling.","date":"2016","source":"Cell discovery","url":"https://pubmed.ncbi.nlm.nih.gov/27648300","citation_count":34,"is_preprint":false},{"pmid":"38552976","id":"PMC_38552976","title":"L-arginine alleviates heat stress-induced mammary gland injury through modulating CASTOR1-mTORC1 axis mediated mitochondrial homeostasis.","date":"2024","source":"The Science of the total environment","url":"https://pubmed.ncbi.nlm.nih.gov/38552976","citation_count":16,"is_preprint":false},{"pmid":"30503338","id":"PMC_30503338","title":"Crystal structures of arginine sensor CASTOR1 in arginine-bound and ligand free states.","date":"2018","source":"Biochemical and biophysical research 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disease","url":"https://pubmed.ncbi.nlm.nih.gov/36454504","citation_count":7,"is_preprint":false},{"pmid":"35442666","id":"PMC_35442666","title":"Downregulation of CASTOR1 Inhibits Heat-Stress-Induced Apoptosis and Promotes Casein and Lipid Synthesis in Mammary Epithelial Cells.","date":"2022","source":"Journal of agricultural and food chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/35442666","citation_count":7,"is_preprint":false},{"pmid":"40234290","id":"PMC_40234290","title":"Upregulating mTOR/S6 K Pathway by CASTOR1 Promotes Astrocyte Proliferation and Myelination in Gpam-/--induced mouse model of cerebral palsy.","date":"2025","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/40234290","citation_count":3,"is_preprint":false},{"pmid":"40313924","id":"PMC_40313924","title":"CASTOR1: A Novel Tumor Suppressor Linking mTORC1 and KRAS Pathways in Tumorigenesis and Resistance to KRAS-Targeted Therapies in Non-Small Cell Lung Cancer.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/40313924","citation_count":1,"is_preprint":false},{"pmid":"41506264","id":"PMC_41506264","title":"CASTOR1 and CASTOR2 respond to different arginine levels to regulate mTORC1 activity.","date":"2026","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/41506264","citation_count":0,"is_preprint":false},{"pmid":"40470200","id":"PMC_40470200","title":"Structural basis for mTORC1 regulation by the CASTOR1-GATOR2 complex.","date":"2025","source":"Research square","url":"https://pubmed.ncbi.nlm.nih.gov/40470200","citation_count":0,"is_preprint":false},{"pmid":"42028441","id":"PMC_42028441","title":"Putative identification of CASTOR1 as one of the targets of ganoderic acid a via thermal proteome profiling and molecular docking.","date":"2026","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/42028441","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.04.23.650349","title":"<i>CASTOR1</i>  : A Novel Tumor Suppressor Linking mTORC1 and KRAS Pathways in Tumorigenesis and Resistance to KRAS-Targeted Therapies in Non-Small Cell Lung Cancer","date":"2025-04-26","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.23.650349","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10917,"output_tokens":2811,"usd":0.037458,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10138,"output_tokens":3042,"usd":0.06337,"stage2_stop_reason":"end_turn"},"total_usd":0.100828,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of arginine-bound CASTOR1 at 1.8 Å resolution reveals that homodimeric CASTOR1 binds arginine at the interface of two ACT domains, enabling allosteric control of the adjacent GATOR2-binding site; arginine binding triggers dissociation from GATOR2, thereby activating mTORC1 downstream.\",\n      \"method\": \"X-ray crystallography (1.8 Å), structure-guided mutagenesis, biochemical binding assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — atomic-resolution crystal structure plus mutagenesis and biochemical validation; independently replicated by two additional structural studies in the same year\",\n      \"pmids\": [\"27487210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of human CASTOR1–arginine complex identifies an arginine-binding pocket between the NTD and CTD (ACT) domains; a surface patch on CASTOR1-NTD opposite this pocket mediates direct physical interaction with GATOR2 subunit Mios; mutation of key pocket residues abolishes or diminishes arginine binding, and mutation of the surface patch disrupts CASTOR1 recognition and inhibition of GATOR2.\",\n      \"method\": \"X-ray crystallography, in vitro pull-down assay, site-directed mutagenesis, normal mode analysis\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus biochemical pull-down; orthogonal to PMID 27487210\",\n      \"pmids\": [\"28066558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Crystal structure of CASTOR1–arginine complex at 2.5 Å shows four tandem ACT domains; ACT1 and ACT3 mediate homodimerization via conserved helix residues, while ACT2 and ACT4 form the arginine-binding pocket; mutagenesis of key binding-pocket residues validates their functional roles in arginine-dependent mTORC1 activation.\",\n      \"method\": \"X-ray crystallography (2.5 Å), site-directed mutagenesis, biochemical assays\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and biochemical validation, consistent with two parallel structural studies\",\n      \"pmids\": [\"27648300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Comparison of arginine-bound (2.05 Å) and ligand-free (2.8 Å) CASTOR1 crystal structures reveals near-identical conformations except in two loop regions, indicating that CASTOR1 does not undergo large conformational change upon arginine binding; arginine sensing is thus interpreted as a subtle loop-based mechanism rather than a global domain rearrangement.\",\n      \"method\": \"X-ray crystallography (apo and holo structures), structural comparison\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — two crystal structures in one study providing direct structural comparison; single lab but high-resolution data\",\n      \"pmids\": [\"30503338\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Cryo-EM structure of the GATOR2–CASTOR1 complex (arginine-free) shows that two MIOS WD40 β-propellers of the GATOR2 cage engage both subunits of a single CASTOR1 homodimer at a negatively charged interface distal to the arginine pocket; arginine-triggered loop ordering in CASTOR1 sterically blocks this MIOS-binding interface, switching off GATOR2 binding and thereby de-repressing GATOR1 and activating mTORC1.\",\n      \"method\": \"Cryo-electron microscopy, structural analysis, functional validation\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure of the intact complex with mechanistic interpretation confirmed by independent preprint (PMID 40470200)\",\n      \"pmids\": [\"40715445\", \"40470200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"E3 ubiquitin ligase RNF167 catalyzes K29-linked polyubiquitination and proteasomal degradation of CASTOR1; AKT phosphorylates CASTOR1 at Ser14, which markedly increases CASTOR1 binding to RNF167 (promoting its ubiquitination and degradation) while simultaneously decreasing CASTOR1 affinity for MIOS, resulting in mTORC1 activation independent of arginine.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, phosphorylation site mutagenesis, RNF167 knockdown/overexpression, AKT inhibitor treatment\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, site-specific mutagenesis, and multiple orthogonal biochemical assays in a single study\",\n      \"pmids\": [\"33594058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KSHV-encoded miRNAs miR-K4-5p (and likely miR-K1-5p) directly target the CASTOR1 3′-UTR to suppress CASTOR1 expression; knockdown of these miRNAs restores CASTOR1 protein levels and attenuates mTORC1 activation, establishing a viral miRNA–CASTOR1–GATOR2–mTORC1 axis.\",\n      \"method\": \"miRNA target validation (direct targeting of CASTOR1 3′-UTR), miRNA knockdown, CASTOR1/CASTOR2 overexpression, mTORC1 activity assays, soft-agar colony formation\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct 3′-UTR targeting demonstrated, rescue experiments performed; single lab\",\n      \"pmids\": [\"31305263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"CASTOR1 and CASTOR2 both bind arginine and interact with GATOR2 component Mios; arginine binding induces conformational changes at the ACT2–ACT4 interface causing dissociation from Mios; CASTOR1 responds to low arginine concentrations and CASTOR2 to high arginine concentrations, enabling dual-sensor fine-tuning of mTORC1 activity; in C2C12 muscle cells, CASTOR proteins regulate mTORC1 and myogenesis in an arginine-level-dependent manner.\",\n      \"method\": \"Biochemical binding assays, structural analysis of conformational changes, arginine dose-response experiments, C2C12 cell knockdown with mTORC1 and myogenesis readouts\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural and biochemical analyses with mutagenesis and functional cellular validation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"41506264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In LPS/IFN-γ-activated microglia, CASTOR1 overexpression inhibits M1 polarization by suppressing mTOR signaling; mTOR activator MHY1485 rescues M1 polarization in CASTOR1-overexpressing cells, placing CASTOR1 upstream of mTOR in microglial polarization.\",\n      \"method\": \"CASTOR1 overexpression, mTOR activator (MHY1485) epistasis, M1/M2 marker expression assays in primary microglia\",\n      \"journal\": \"Metabolic brain disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic epistasis (activator rescue) confirms pathway placement; single lab, single cellular model\",\n      \"pmids\": [\"36454506\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CASTOR1 genetic ablation in a KRAS-driven GEMM (LSL-KRAS G12D) enhances lung tumor incidence and accelerates progression; mechanistically, CASTOR1 loss amplifies mTORC1 signaling (elevated p-4EBP1, p-S6) and augments AKT and ERK activation, revealing crosstalk between PI3K/AKT/mTORC1 and KRAS/ERK pathways; mTORC1 and PI3K inhibitors sensitize CASTOR1-deficient resistant tumors to KRAS G12D-targeted therapy.\",\n      \"method\": \"Genetically engineered mouse model (CASTOR1 KO × KRAS G12D), tumor-derived organoids, phospho-protein assays, drug combination rescue\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — in vivo genetic model with multiple mechanistic readouts; preprint, single lab, not yet peer-reviewed\",\n      \"pmids\": [\"40313924\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CASTOR1 is a cytosolic arginine sensor that, in the absence of arginine, forms a homodimer via its ACT1/ACT3 domains and binds the GATOR2 complex through a negatively charged interface on CASTOR1 that contacts two MIOS WD40 β-propellers; arginine binding at the ACT2/ACT4 interface triggers loop ordering that sterically occludes the MIOS-binding surface, releasing CASTOR1 from GATOR2, thereby de-repressing GATOR1 GAP activity toward RagA/B and activating lysosomal mTORC1. CASTOR1 activity is further tuned post-translationally: AKT phosphorylates CASTOR1 at Ser14, enhancing its binding to E3 ligase RNF167 which catalyzes K29-linked polyubiquitination and proteasomal degradation of CASTOR1 to activate mTORC1 independently of arginine. CASTOR1 and its homolog CASTOR2 function as a dual-sensitivity system responding to low and high arginine concentrations respectively, together fine-tuning mTORC1 output in contexts such as muscle myogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CASTOR1 is a cytosolic arginine sensor that gates amino-acid signaling to mTORC1 by controlling the GATOR2 complex [#0, #4]. In the arginine-free state it forms a homodimer through its ACT1 and ACT3 domains and engages two MIOS WD40 \\u03b2-propellers of the GATOR2 cage via a negatively charged surface distal to its ligand pocket, holding GATOR2 in an inhibited state and keeping mTORC1 low [#2, #4]. Arginine binds at the interface of the ACT2 and ACT4 domains, and rather than driving a large global rearrangement, it orders two loop regions that sterically occlude the MIOS-binding surface, releasing CASTOR1 from GATOR2 and de-repressing GATOR1 GAP activity to activate lysosomal mTORC1 [#1, #3, #4]. CASTOR1 activity is additionally tuned post-translationally: AKT phosphorylates CASTOR1 at Ser14, increasing its binding to the E3 ligase RNF167, which catalyzes K29-linked polyubiquitination and proteasomal degradation of CASTOR1 to activate mTORC1 independently of arginine [#5]. CASTOR1 and its homolog CASTOR2 act as a dual-sensitivity system responding to low and high arginine respectively, together fine-tuning mTORC1 output during processes such as C2C12 myogenesis [#7]. Through this mTORC1-restraining role, CASTOR1 functions as a brake in disease-relevant settings, including KRAS-driven lung tumorigenesis and KSHV miRNA-driven mTORC1 activation [#6, #9].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Established the structural basis for how CASTOR1 senses arginine and couples that signal to mTORC1, defining it as a ligand-gated repressor of GATOR2.\",\n      \"evidence\": \"X-ray crystallography of arginine-bound CASTOR1 with structure-guided mutagenesis and binding assays\",\n      \"pmids\": [\"27487210\", \"28066558\", \"27648300\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Apo-state conformation not resolved in these structures\", \"Architecture of the CASTOR1\\u2013GATOR2 contact inferred from mutagenesis, not visualized\", \"Did not address physiological arginine concentration thresholds\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the domain logic of CASTOR1, separating the homodimerization function (ACT1/ACT3) from the arginine-binding function (ACT2/ACT4).\",\n      \"evidence\": \"2.5 \\u00c5 crystal structure with site-directed mutagenesis of pocket and dimer-interface residues\",\n      \"pmids\": [\"27648300\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show how loop changes propagate to the GATOR2 interface\", \"Functional readouts in cells limited\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved the conformational mechanism debate by showing arginine sensing is a localized loop reordering rather than a global domain swing.\",\n      \"evidence\": \"Comparison of apo (2.8 \\u00c5) and arginine-bound (2.05 \\u00c5) CASTOR1 crystal structures\",\n      \"pmids\": [\"30503338\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How loop ordering translates into loss of GATOR2 binding not demonstrated structurally at the time\", \"Single-lab structures\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed CASTOR1 is a target of pathogen-encoded regulation, linking viral miRNAs to mTORC1 activation through suppression of an arginine sensor.\",\n      \"evidence\": \"KSHV miRNA 3\\u2032-UTR target validation, miRNA knockdown rescue, and mTORC1/colony-formation assays\",\n      \"pmids\": [\"31305263\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Relative contribution of miR-K1-5p not firmly quantified\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Uncovered an arginine-independent control layer in which AKT-driven phosphorylation routes CASTOR1 for ubiquitin-mediated degradation to activate mTORC1.\",\n      \"evidence\": \"Co-IP, K29-linkage ubiquitination assays, Ser14 phospho-site mutagenesis, RNF167 perturbation, and AKT inhibition\",\n      \"pmids\": [\"33594058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological stimuli engaging this axis not fully mapped\", \"Crosstalk timing with arginine sensing unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed CASTOR1 upstream of mTOR in a cellular phenotype, showing it restrains pro-inflammatory M1 microglial polarization.\",\n      \"evidence\": \"CASTOR1 overexpression with mTOR-activator (MHY1485) epistasis and M1/M2 marker readouts in primary microglia\",\n      \"pmids\": [\"36454506\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single cellular model\", \"Endogenous loss-of-function not tested\", \"Arginine dependence in this context unexamined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Captured the intact arginine-free GATOR2\\u2013CASTOR1 complex, directly visualizing the MIOS contact and confirming the steric-occlusion switch model.\",\n      \"evidence\": \"Cryo-EM of the GATOR2\\u2013CASTOR1 complex with structural and functional validation\",\n      \"pmids\": [\"40715445\", \"40470200\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of the binding/release transition not directly observed\", \"Stoichiometry relative to full GATOR2 cage occupancy not fully quantified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Provided in vivo evidence that CASTOR1 acts as a tumor suppressor that brakes mTORC1 and interacts with KRAS/ERK signaling.\",\n      \"evidence\": \"CASTOR1-KO \\u00d7 KRAS G12D mouse model, organoids, phospho-protein readouts, and drug-combination rescue (preprint)\",\n      \"pmids\": [\"40313924\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not peer-reviewed\", \"Single lab\", \"Direct biochemical basis of AKT/ERK crosstalk not established\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined CASTOR1 and CASTOR2 as a dual-sensitivity arginine-sensing pair, extending the single-sensor model to a graded mTORC1 readout with a developmental output.\",\n      \"evidence\": \"Biochemical binding, conformational analysis, arginine dose-response, and C2C12 knockdown with myogenesis readouts\",\n      \"pmids\": [\"41506264\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single lab\", \"In vivo myogenesis relevance not tested\", \"Quantitative crossover point between the two sensors not fully defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the arginine-sensing, AKT/RNF167 degradation, and KRAS/ERK crosstalk inputs are integrated to set mTORC1 output in specific tissues remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking post-translational degradation with structural ligand sensing in vivo\", \"Tissue-specific physiological roles beyond muscle, microglia, and lung unmapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-165159\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4, 5]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MIOS\", \"RNF167\", \"AKT1\", \"CASTOR2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}