{"gene":"TTI1","run_date":"2026-06-10T10:51:56","timeline":{"discoveries":[{"year":2010,"finding":"TTI1 (KIAA0406) constitutively interacts with mTOR in both mTORC1 and mTORC2, and also binds Tel2. Knockdown of TTI1 suppresses phosphorylation of mTORC1 substrates (S6K1, 4E-BP1) and the mTORC2 substrate Akt, induces autophagy, and causes disassembly of mTORC1 and mTORC2. TTI1 interacts with and stabilizes all six PIKK family members (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP).","method":"Co-immunoprecipitation, siRNA knockdown, immunoprecipitation and size-exclusion chromatography, phosphorylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, size-exclusion chromatography, multiple orthogonal methods (knockdown + signaling assays + complex assembly), foundational study replicated by subsequent papers","pmids":["20427287"],"is_preprint":false},{"year":2013,"finding":"Tel2 and TTI1 are targeted for degradation within mTORC1 by the SCF-Fbxo9 ubiquitin ligase complex. This process is primed by CK2, which translocates to the cytoplasm and mediates mTORC1-specific phosphorylation of Tel2/TTI1 upon growth factor deprivation, leading to mTORC1 inactivation.","method":"Ubiquitin ligase substrate identification, co-immunoprecipitation, CK2 phosphorylation assays, siRNA knockdown, cell fractionation","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, phosphorylation assays, fractionation, degradation assays) in a single rigorous study identifying the E3 ligase and kinase","pmids":["23263282"],"is_preprint":false},{"year":2014,"finding":"CK2 phosphorylates the TTT complex (Tel2, TTI1, TTI2), and this phosphorylation is enhanced by IP7 (generated by IP6K2), which binds CK2 as an allosteric activator. CK2-mediated phosphorylation of TTT stabilizes DNA-PKcs and ATM, promoting p53 phosphorylation at serine 15 and apoptotic cell death.","method":"In vitro kinase assays, IP7-binding assays, co-immunoprecipitation, pharmacological inhibition, cell death assays with genetic manipulation of IP6K2","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay with defined biochemical mechanism, multiple orthogonal methods, single lab","pmids":["24657168"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of the human TTT complex (TELO2-TTI1-TTI2) at 4.2 Å resolution reveals that all three proteins form elongated helical repeat (HEAT-repeat/α-solenoid) structures. TTI1 provides a central platform: TELO2 binds to the central region of TTI1 and TTI2 binds to its C-terminal end. The TELO2 C-terminal domain is required for interaction with TTI1 and recruitment of ATM. The N- and C-terminal segments of TTI1 recognize the FAT domain and N-terminal HEAT repeats of ATM, respectively. TELO2 CTD and TTI1 N- and C-terminal segments are required for cell survival after ionizing radiation.","method":"Cryo-EM structure determination, deletion/domain mapping, co-immunoprecipitation, cell survival assays after ionizing radiation","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with functional domain mapping and cellular validation, single lab","pmids":["34838521"],"is_preprint":false},{"year":2021,"finding":"Cryo-EM structure of the human R2TP-TTT complex reveals that the HEAT-repeat TTT complex binds the kinase domain of TOR (without blocking its activity) and delivers TOR to the R2TP chaperone. Additionally, TTT regulates R2TP by inhibiting RUVBL1-RUVBL2 ATPase activity and modulating the conformation and interactions of PIH1D1 and RPAP3 components of R2TP.","method":"Cryo-EM structure determination, biochemical ATPase assays, pull-down assays, mass spectrometry","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus biochemical reconstitution assays, independently corroborated by companion structural study (PMID:34838521)","pmids":["34233195"],"is_preprint":false},{"year":2019,"finding":"In fission yeast, destabilization of the TTT complex (via a tel2 mutation that weakens Tel2-Tti1 and Tel2-Tti2 interactions) nearly completely eliminates Rad3 (ATR ortholog)-mediated phosphosignaling specifically in the DNA replication checkpoint, while only moderately reducing DNA damage checkpoint signaling. The tel2 mutation also causes telomere shortening.","method":"Genetic screen, yeast genetics, co-immunoprecipitation, phosphorylation assays, telomere length analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic epistasis via point mutation with checkpoint signaling readout, single lab, fission yeast model","pmids":["31332096"],"is_preprint":false},{"year":2021,"finding":"In S. cerevisiae, single-residue substitutions in Tti1 suppress the essential requirement for Sis1 (J-domain protein/Hsp70 cochaperone). Upon Sis1 depletion, PIKK protein levels (Mec1, Tra1, Tor2, Tor1) decrease, indicating Sis1 functions as an Hsp70 cochaperone for PIKK folding/maintenance. Tti1 overexpression can rescue growth independently of the other TTT subunits (Tel2, Tti2), suggesting Tti1 can function outside the complex.","method":"Genetic suppressor analysis, yeast genetics, protein level measurement by western blot, rapamycin sensitivity assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple readouts (suppressor screen, protein levels, drug sensitivity), single lab, yeast model","pmids":["34935410"],"is_preprint":false},{"year":2023,"finding":"Bi-allelic loss-of-function variants in TTI1 in humans impair TTT complex assembly and reduce mTOR pathway activity in patient-derived HEK293T cells, fibroblasts, and lymphoblastoid cells. Rapamycin treatment partially improves mTOR pathway activity in these cells, indicating mTOR signaling dysregulation underlies the phenotype.","method":"Patient cell functional studies (HEK293T, fibroblasts, lymphoblastoid cells), western blot for TTT complex and mTOR substrates, rapamycin treatment rescue","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional validation in patient-derived cells with multiple orthogonal methods, but single lab","pmids":["36724785"],"is_preprint":false},{"year":2025,"finding":"TTI1 knockdown enhances sensitivity of rectal cancer cells to irradiation, while TTI1 overexpression promotes radioresistance. TTI1 activates the ATM signaling pathway to enhance DNA damage repair following irradiation. Blocking ATM signaling sensitizes RC tissue to irradiation.","method":"siRNA knockdown, overexpression, colony formation assay, western blot, comet assay, flow cytometry, xenograft assay, organoid and PDX models","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function with defined pathway (ATM) and specific phenotypic readout (radiosensitivity, DNA repair), single lab","pmids":["40514657"],"is_preprint":false},{"year":2026,"finding":"Under hypoxic conditions, VEGFR2 regulates erythroid differentiation of CD34+ hematopoietic stem cells through a TTI1-mTORC1 signaling axis. TTI1 siRNA knockdown under hypoxia impedes erythropoiesis, phenocopying mTOR inhibition with rapamycin and VEGFR2 neutralization.","method":"Proteomics, siRNA knockdown, VEGFR2 neutralizing antibody, rapamycin treatment, erythroid differentiation assays in CD34+ cells","journal":"Journal of cellular physiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, proteomics-guided knockdown with phenotypic readout but limited mechanistic detail on TTI1's direct role","pmids":["42152561"],"is_preprint":false}],"current_model":"TTI1 forms the heterotrimeric TTT (TELO2-TTI1-TTI2) co-chaperone complex with an elongated α-solenoid/HEAT-repeat structure in which TTI1 serves as the central scaffold; TTT binds the kinase domain of TOR/mTOR and all PIKK family members to stabilize and deliver them to the R2TP-HSP90 chaperone machinery, while also being regulated by CK2-mediated phosphorylation (enhanced by inositol pyrophosphate IP7) and ubiquitin-mediated degradation via SCF-Fbxo9 to control mTOR complex assembly and activity in response to growth factor availability."},"narrative":{"mechanistic_narrative":"TTI1 is the central scaffolding subunit of the heterotrimeric TTT (TELO2-TTI1-TTI2) co-chaperone complex that stabilizes and regulates the assembly of phosphatidylinositol-3-kinase-related kinases (PIKKs), most prominently mTOR [PMID:20427287, PMID:34838521]. TTI1 constitutively associates with mTOR in both mTORC1 and mTORC2 and binds and stabilizes all six PIKK family members (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP); its loss collapses mTOR complex integrity, suppresses phosphorylation of mTORC1 (S6K1, 4E-BP1) and mTORC2 (Akt) substrates, and induces autophagy [PMID:20427287]. Structurally, TTI1 adopts an elongated HEAT-repeat/α-solenoid that serves as the platform onto which TELO2 binds centrally and TTI2 binds at the C-terminus, while the N- and C-terminal segments of TTI1 engage the FAT domain and N-terminal HEAT repeats of substrate PIKKs such as ATM [PMID:34838521]. The TTT complex docks the kinase domain of TOR without blocking catalysis and delivers it to the R2TP-HSP90 chaperone machinery, where it also inhibits RUVBL1-RUVBL2 ATPase activity and remodels PIH1D1/RPAP3 [PMID:34233195]. TTT-mediated PIKK stabilization is dynamically controlled: CK2 phosphorylates the complex—an event enhanced by the inositol pyrophosphate IP7—to stabilize DNA-PKcs and ATM and promote p53-Ser15 phosphorylation [PMID:24657168], while growth-factor deprivation primes CK2-dependent phosphorylation that targets TELO2/TTI1 for SCF-Fbxo9-mediated degradation and mTORC1 inactivation [PMID:23263282]. Bi-allelic loss-of-function variants in TTI1 impair TTT assembly and reduce mTOR pathway activity in patient-derived cells, defining a human disorder of mTOR signaling that is partially corrected by rapamycin [PMID:36724785].","teleology":[{"year":2010,"claim":"Established TTI1 as a constitutive component of both mTOR complexes and a general stabilizer of PIKK kinases, answering what physical role this uncharacterized protein plays in mTOR signaling.","evidence":"Co-IP, siRNA knockdown with signaling assays, and size-exclusion chromatography in human cells","pmids":["20427287"],"confidence":"High","gaps":["Did not resolve the structural basis of PIKK recognition","Did not define how TTT delivers clients to downstream chaperones"]},{"year":2013,"claim":"Identified the degradation arm that turns TTT/mTORC1 off, showing how growth-factor status is transduced into TTI1 turnover.","evidence":"SCF-Fbxo9 substrate identification, CK2 phosphorylation assays, cell fractionation and degradation assays in human cells","pmids":["23263282"],"confidence":"High","gaps":["Did not map the specific phosphodegron residues","Did not address whether mTORC2-associated TTI1 is similarly regulated"]},{"year":2014,"claim":"Connected inositol pyrophosphate signaling to TTT regulation, showing CK2 phosphorylation of the complex stabilizes DNA-PKcs/ATM and feeds into p53-dependent apoptosis.","evidence":"In vitro kinase and IP7-binding assays, Co-IP, and cell death assays with IP6K2 manipulation","pmids":["24657168"],"confidence":"High","gaps":["Did not define the TTT phosphosites or their structural consequences","Did not reconcile stabilizing CK2 phosphorylation here with degradation-priming CK2 phosphorylation"]},{"year":2019,"claim":"Used yeast genetics to show TTT integrity is differentially required across PIKK functions, with ATR/Rad3 replication-checkpoint signaling especially dependent on the complex.","evidence":"tel2 point mutation weakening TTT interactions, Co-IP, phosphosignaling and telomere length analysis in fission yeast","pmids":["31332096"],"confidence":"Medium","gaps":["Single yeast model; human TTI1 contribution to ATR signaling not directly tested","Mechanism of checkpoint-specific dependence unresolved"]},{"year":2021,"claim":"Resolved the architecture of TTT and its engagement with both PIKK clients and the R2TP-HSP90 machinery, defining TTI1 as the central α-solenoid scaffold and showing how clients are delivered without inhibiting kinase activity.","evidence":"Cryo-EM of human TTT and R2TP-TTT, domain-mapping deletions, ATPase and pull-down assays, and ionizing-radiation survival assays","pmids":["34838521","34233195"],"confidence":"High","gaps":["Static structures do not capture the dynamics of client loading/handoff","Does not explain client selectivity among the six PIKKs"]},{"year":2021,"claim":"Genetic suppressor work in budding yeast placed TTI1/TTT downstream of Hsp70 cochaperone activity for PIKK maintenance and showed Tti1 can act partly outside the complex.","evidence":"Tti1 suppressor substitutions, Sis1 depletion with PIKK protein-level measurement, rapamycin sensitivity in S. cerevisiae","pmids":["34935410"],"confidence":"Medium","gaps":["Mechanism of Tti1 complex-independent function unknown","Relevance of Sis1/Hsp70 link to human TTI1 untested"]},{"year":2023,"claim":"Established TTI1 as a human disease gene, showing bi-allelic loss-of-function impairs TTT assembly and mTOR activity, partially rescuable by rapamycin.","evidence":"Patient-derived HEK293T, fibroblast and lymphoblastoid cell studies with western blot and rapamycin rescue","pmids":["36724785"],"confidence":"Medium","gaps":["Single lab; genotype-phenotype spectrum not fully defined","Tissue-specific consequences of mTOR dysregulation not established"]},{"year":2025,"claim":"Demonstrated a therapeutically relevant role for TTI1 in tumor radioresistance through ATM-pathway-driven DNA damage repair.","evidence":"Knockdown/overexpression with colony formation, comet assay, flow cytometry, xenograft, organoid and PDX rectal cancer models","pmids":["40514657"],"confidence":"Medium","gaps":["Whether the effect requires intact TTT or direct ATM stabilization not dissected","Single cancer context"]},{"year":2026,"claim":"Implicated TTI1 in a VEGFR2-mTORC1 axis controlling hypoxic erythroid differentiation of hematopoietic stem cells.","evidence":"Proteomics-guided siRNA knockdown, VEGFR2 neutralization and rapamycin in CD34+ erythroid differentiation assays","pmids":["42152561"],"confidence":"Low","gaps":["Limited mechanistic detail on TTI1's direct role in the axis","Correlative phenocopy rather than direct interaction mapping"]},{"year":null,"claim":"How TTT achieves selectivity among its six PIKK clients, and how the opposing CK2-driven stabilizing and degradation-priming phosphorylation events are coordinated in vivo, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No residue-level map distinguishing stabilizing from degradative TTI1 phosphorylation","Dynamics of client handoff to R2TP-HSP90 not captured"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,4]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,4]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[2,5,8]}],"complexes":["TTT (TELO2-TTI1-TTI2) complex","mTORC1","mTORC2","R2TP-TTT"],"partners":["TELO2","TTI2","MTOR","ATM","RUVBL1","RUVBL2","PIH1D1","RPAP3"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43156","full_name":"TELO2-interacting protein 1 homolog","aliases":["Protein SMG10"],"length_aa":1089,"mass_kda":122.1,"function":"Regulator of the DNA damage response (DDR). Part of the TTT complex that is required to stabilize protein levels of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family proteins. The TTT complex is involved in the cellular resistance to DNA damage stresses, like ionizing radiation (IR), ultraviolet (UV) and mitomycin C (MMC). Together with the TTT complex and HSP90 may participate in the proper folding of newly synthesized PIKKs. Promotes assembly, stabilizes and maintains the activity of mTORC1 and mTORC2 complexes, which regulate cell growth and survival in response to nutrient and hormonal signals","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O43156/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/TTI1","classification":"Common Essential","n_dependent_lines":1145,"n_total_lines":1208,"dependency_fraction":0.9478476821192053},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"FKBP5","stoichiometry":0.2},{"gene":"POLR2E","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/TTI1","total_profiled":1310},"omim":[{"mim_id":"620445","title":"NEURODEVELOPMENTAL DISORDER WITH MICROCEPHALY AND MOVEMENT ABNORMALITIES; NEDMIM","url":"https://www.omim.org/entry/620445"},{"mim_id":"616954","title":"YOU-HOOVER-FONG SYNDROME; YHFS","url":"https://www.omim.org/entry/616954"},{"mim_id":"615541","title":"INTELLECTUAL DEVELOPMENTAL DISORDER, AUTOSOMAL RECESSIVE 39; MRT39","url":"https://www.omim.org/entry/615541"},{"mim_id":"614426","title":"TELO2-INTERACTING PROTEIN 2; TTI2","url":"https://www.omim.org/entry/614426"},{"mim_id":"614425","title":"TELO2-INTERACTING PROTEIN 1; TTI1","url":"https://www.omim.org/entry/614425"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TTI1"},"hgnc":{"alias_symbol":["smg-10"],"prev_symbol":["KIAA0406"]},"alphafold":{"accession":"O43156","domains":[{"cath_id":"-","chopping":"194-367","consensus_level":"medium","plddt":84.4153,"start":194,"end":367},{"cath_id":"-","chopping":"400-451_475-580_617-684","consensus_level":"medium","plddt":85.8945,"start":400,"end":684},{"cath_id":"1.25.40","chopping":"692-772_858-899","consensus_level":"medium","plddt":88.7598,"start":692,"end":899}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43156","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43156-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43156-F1-predicted_aligned_error_v6.png","plddt_mean":81.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TTI1","jax_strain_url":"https://www.jax.org/strain/search?query=TTI1"},"sequence":{"accession":"O43156","fasta_url":"https://rest.uniprot.org/uniprotkb/O43156.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43156/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43156"}},"corpus_meta":[{"pmid":"20427287","id":"PMC_20427287","title":"Tti1 and Tel2 are critical factors in mammalian target of rapamycin complex assembly.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20427287","citation_count":206,"is_preprint":false},{"pmid":"24657168","id":"PMC_24657168","title":"Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/24657168","citation_count":105,"is_preprint":false},{"pmid":"23263282","id":"PMC_23263282","title":"SCFFbxo9 and CK2 direct the cellular response to growth factor withdrawal via Tel2/Tti1 degradation and promote survival in multiple myeloma.","date":"2013","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/23263282","citation_count":83,"is_preprint":false},{"pmid":"34233195","id":"PMC_34233195","title":"Structure of the TELO2-TTI1-TTI2 complex and its function in TOR recruitment to the R2TP chaperone.","date":"2021","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/34233195","citation_count":29,"is_preprint":false},{"pmid":"34838521","id":"PMC_34838521","title":"Structure of the Human TELO2-TTI1-TTI2 Complex.","date":"2021","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/34838521","citation_count":11,"is_preprint":false},{"pmid":"36403197","id":"PMC_36403197","title":"TTI1 promotes non-small-cell lung cancer progression by regulating the mTOR signaling pathway.","date":"2022","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/36403197","citation_count":11,"is_preprint":false},{"pmid":"31332096","id":"PMC_31332096","title":"A tel2 Mutation That Destabilizes the Tel2-Tti1-Tti2 Complex Eliminates Rad3ATR Kinase Signaling in the DNA Replication Checkpoint and Leads to Telomere Shortening in Fission Yeast.","date":"2019","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/31332096","citation_count":10,"is_preprint":false},{"pmid":"34935410","id":"PMC_34935410","title":"Essentiality of Sis1, a J-domain protein Hsp70 cochaperone, can be overcome by Tti1, a specialized PIKK chaperone.","date":"2021","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/34935410","citation_count":8,"is_preprint":false},{"pmid":"35116463","id":"PMC_35116463","title":"A role of TTI1 in the colorectal cancer by promoting proliferation.","date":"2021","source":"Translational cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/35116463","citation_count":7,"is_preprint":false},{"pmid":"37175973","id":"PMC_37175973","title":"TTT (Tel2-Tti1-Tti2) Complex, the Co-Chaperone of PIKKs and a Potential Target for Cancer Chemotherapy.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37175973","citation_count":6,"is_preprint":false},{"pmid":"36724785","id":"PMC_36724785","title":"Bi-allelic TTI1 variants cause an autosomal-recessive neurodevelopmental disorder with microcephaly.","date":"2023","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/36724785","citation_count":5,"is_preprint":false},{"pmid":"38501918","id":"PMC_38501918","title":"ALKBH5 promotes hepatocellular carcinoma cell proliferation, migration and invasion by regulating TTI1 expression.","date":"2024","source":"Biomolecules & biomedicine","url":"https://pubmed.ncbi.nlm.nih.gov/38501918","citation_count":3,"is_preprint":false},{"pmid":"40514657","id":"PMC_40514657","title":"TTI1 contributes to radioresistance by activating ATM pathway in rectal cancer.","date":"2025","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40514657","citation_count":1,"is_preprint":false},{"pmid":"42152561","id":"PMC_42152561","title":"Unveiling the VEGFR2-TTI1-mTORC1 Axis: A Crucial Pathway in Hypoxia-Induced Erythropoiesis.","date":"2026","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/42152561","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9214,"output_tokens":2823,"usd":0.034993,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10100,"output_tokens":3409,"usd":0.067863,"stage2_stop_reason":"end_turn"},"total_usd":0.102856,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"TTI1 (KIAA0406) constitutively interacts with mTOR in both mTORC1 and mTORC2, and also binds Tel2. Knockdown of TTI1 suppresses phosphorylation of mTORC1 substrates (S6K1, 4E-BP1) and the mTORC2 substrate Akt, induces autophagy, and causes disassembly of mTORC1 and mTORC2. TTI1 interacts with and stabilizes all six PIKK family members (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP).\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunoprecipitation and size-exclusion chromatography, phosphorylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, size-exclusion chromatography, multiple orthogonal methods (knockdown + signaling assays + complex assembly), foundational study replicated by subsequent papers\",\n      \"pmids\": [\"20427287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Tel2 and TTI1 are targeted for degradation within mTORC1 by the SCF-Fbxo9 ubiquitin ligase complex. This process is primed by CK2, which translocates to the cytoplasm and mediates mTORC1-specific phosphorylation of Tel2/TTI1 upon growth factor deprivation, leading to mTORC1 inactivation.\",\n      \"method\": \"Ubiquitin ligase substrate identification, co-immunoprecipitation, CK2 phosphorylation assays, siRNA knockdown, cell fractionation\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, phosphorylation assays, fractionation, degradation assays) in a single rigorous study identifying the E3 ligase and kinase\",\n      \"pmids\": [\"23263282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"CK2 phosphorylates the TTT complex (Tel2, TTI1, TTI2), and this phosphorylation is enhanced by IP7 (generated by IP6K2), which binds CK2 as an allosteric activator. CK2-mediated phosphorylation of TTT stabilizes DNA-PKcs and ATM, promoting p53 phosphorylation at serine 15 and apoptotic cell death.\",\n      \"method\": \"In vitro kinase assays, IP7-binding assays, co-immunoprecipitation, pharmacological inhibition, cell death assays with genetic manipulation of IP6K2\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay with defined biochemical mechanism, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"24657168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of the human TTT complex (TELO2-TTI1-TTI2) at 4.2 Å resolution reveals that all three proteins form elongated helical repeat (HEAT-repeat/α-solenoid) structures. TTI1 provides a central platform: TELO2 binds to the central region of TTI1 and TTI2 binds to its C-terminal end. The TELO2 C-terminal domain is required for interaction with TTI1 and recruitment of ATM. The N- and C-terminal segments of TTI1 recognize the FAT domain and N-terminal HEAT repeats of ATM, respectively. TELO2 CTD and TTI1 N- and C-terminal segments are required for cell survival after ionizing radiation.\",\n      \"method\": \"Cryo-EM structure determination, deletion/domain mapping, co-immunoprecipitation, cell survival assays after ionizing radiation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with functional domain mapping and cellular validation, single lab\",\n      \"pmids\": [\"34838521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cryo-EM structure of the human R2TP-TTT complex reveals that the HEAT-repeat TTT complex binds the kinase domain of TOR (without blocking its activity) and delivers TOR to the R2TP chaperone. Additionally, TTT regulates R2TP by inhibiting RUVBL1-RUVBL2 ATPase activity and modulating the conformation and interactions of PIH1D1 and RPAP3 components of R2TP.\",\n      \"method\": \"Cryo-EM structure determination, biochemical ATPase assays, pull-down assays, mass spectrometry\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus biochemical reconstitution assays, independently corroborated by companion structural study (PMID:34838521)\",\n      \"pmids\": [\"34233195\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"In fission yeast, destabilization of the TTT complex (via a tel2 mutation that weakens Tel2-Tti1 and Tel2-Tti2 interactions) nearly completely eliminates Rad3 (ATR ortholog)-mediated phosphosignaling specifically in the DNA replication checkpoint, while only moderately reducing DNA damage checkpoint signaling. The tel2 mutation also causes telomere shortening.\",\n      \"method\": \"Genetic screen, yeast genetics, co-immunoprecipitation, phosphorylation assays, telomere length analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic epistasis via point mutation with checkpoint signaling readout, single lab, fission yeast model\",\n      \"pmids\": [\"31332096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In S. cerevisiae, single-residue substitutions in Tti1 suppress the essential requirement for Sis1 (J-domain protein/Hsp70 cochaperone). Upon Sis1 depletion, PIKK protein levels (Mec1, Tra1, Tor2, Tor1) decrease, indicating Sis1 functions as an Hsp70 cochaperone for PIKK folding/maintenance. Tti1 overexpression can rescue growth independently of the other TTT subunits (Tel2, Tti2), suggesting Tti1 can function outside the complex.\",\n      \"method\": \"Genetic suppressor analysis, yeast genetics, protein level measurement by western blot, rapamycin sensitivity assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple readouts (suppressor screen, protein levels, drug sensitivity), single lab, yeast model\",\n      \"pmids\": [\"34935410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Bi-allelic loss-of-function variants in TTI1 in humans impair TTT complex assembly and reduce mTOR pathway activity in patient-derived HEK293T cells, fibroblasts, and lymphoblastoid cells. Rapamycin treatment partially improves mTOR pathway activity in these cells, indicating mTOR signaling dysregulation underlies the phenotype.\",\n      \"method\": \"Patient cell functional studies (HEK293T, fibroblasts, lymphoblastoid cells), western blot for TTT complex and mTOR substrates, rapamycin treatment rescue\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional validation in patient-derived cells with multiple orthogonal methods, but single lab\",\n      \"pmids\": [\"36724785\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TTI1 knockdown enhances sensitivity of rectal cancer cells to irradiation, while TTI1 overexpression promotes radioresistance. TTI1 activates the ATM signaling pathway to enhance DNA damage repair following irradiation. Blocking ATM signaling sensitizes RC tissue to irradiation.\",\n      \"method\": \"siRNA knockdown, overexpression, colony formation assay, western blot, comet assay, flow cytometry, xenograft assay, organoid and PDX models\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function with defined pathway (ATM) and specific phenotypic readout (radiosensitivity, DNA repair), single lab\",\n      \"pmids\": [\"40514657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Under hypoxic conditions, VEGFR2 regulates erythroid differentiation of CD34+ hematopoietic stem cells through a TTI1-mTORC1 signaling axis. TTI1 siRNA knockdown under hypoxia impedes erythropoiesis, phenocopying mTOR inhibition with rapamycin and VEGFR2 neutralization.\",\n      \"method\": \"Proteomics, siRNA knockdown, VEGFR2 neutralizing antibody, rapamycin treatment, erythroid differentiation assays in CD34+ cells\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, proteomics-guided knockdown with phenotypic readout but limited mechanistic detail on TTI1's direct role\",\n      \"pmids\": [\"42152561\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TTI1 forms the heterotrimeric TTT (TELO2-TTI1-TTI2) co-chaperone complex with an elongated α-solenoid/HEAT-repeat structure in which TTI1 serves as the central scaffold; TTT binds the kinase domain of TOR/mTOR and all PIKK family members to stabilize and deliver them to the R2TP-HSP90 chaperone machinery, while also being regulated by CK2-mediated phosphorylation (enhanced by inositol pyrophosphate IP7) and ubiquitin-mediated degradation via SCF-Fbxo9 to control mTOR complex assembly and activity in response to growth factor availability.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TTI1 is the central scaffolding subunit of the heterotrimeric TTT (TELO2-TTI1-TTI2) co-chaperone complex that stabilizes and regulates the assembly of phosphatidylinositol-3-kinase-related kinases (PIKKs), most prominently mTOR [#0, #3]. TTI1 constitutively associates with mTOR in both mTORC1 and mTORC2 and binds and stabilizes all six PIKK family members (mTOR, ATM, ATR, DNA-PKcs, SMG-1, TRRAP); its loss collapses mTOR complex integrity, suppresses phosphorylation of mTORC1 (S6K1, 4E-BP1) and mTORC2 (Akt) substrates, and induces autophagy [#0]. Structurally, TTI1 adopts an elongated HEAT-repeat/α-solenoid that serves as the platform onto which TELO2 binds centrally and TTI2 binds at the C-terminus, while the N- and C-terminal segments of TTI1 engage the FAT domain and N-terminal HEAT repeats of substrate PIKKs such as ATM [#3]. The TTT complex docks the kinase domain of TOR without blocking catalysis and delivers it to the R2TP-HSP90 chaperone machinery, where it also inhibits RUVBL1-RUVBL2 ATPase activity and remodels PIH1D1/RPAP3 [#4]. TTT-mediated PIKK stabilization is dynamically controlled: CK2 phosphorylates the complex—an event enhanced by the inositol pyrophosphate IP7—to stabilize DNA-PKcs and ATM and promote p53-Ser15 phosphorylation [#2], while growth-factor deprivation primes CK2-dependent phosphorylation that targets TELO2/TTI1 for SCF-Fbxo9-mediated degradation and mTORC1 inactivation [#1]. Bi-allelic loss-of-function variants in TTI1 impair TTT assembly and reduce mTOR pathway activity in patient-derived cells, defining a human disorder of mTOR signaling that is partially corrected by rapamycin [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established TTI1 as a constitutive component of both mTOR complexes and a general stabilizer of PIKK kinases, answering what physical role this uncharacterized protein plays in mTOR signaling.\",\n      \"evidence\": \"Co-IP, siRNA knockdown with signaling assays, and size-exclusion chromatography in human cells\",\n      \"pmids\": [\"20427287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of PIKK recognition\", \"Did not define how TTT delivers clients to downstream chaperones\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified the degradation arm that turns TTT/mTORC1 off, showing how growth-factor status is transduced into TTI1 turnover.\",\n      \"evidence\": \"SCF-Fbxo9 substrate identification, CK2 phosphorylation assays, cell fractionation and degradation assays in human cells\",\n      \"pmids\": [\"23263282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not map the specific phosphodegron residues\", \"Did not address whether mTORC2-associated TTI1 is similarly regulated\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected inositol pyrophosphate signaling to TTT regulation, showing CK2 phosphorylation of the complex stabilizes DNA-PKcs/ATM and feeds into p53-dependent apoptosis.\",\n      \"evidence\": \"In vitro kinase and IP7-binding assays, Co-IP, and cell death assays with IP6K2 manipulation\",\n      \"pmids\": [\"24657168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the TTT phosphosites or their structural consequences\", \"Did not reconcile stabilizing CK2 phosphorylation here with degradation-priming CK2 phosphorylation\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Used yeast genetics to show TTT integrity is differentially required across PIKK functions, with ATR/Rad3 replication-checkpoint signaling especially dependent on the complex.\",\n      \"evidence\": \"tel2 point mutation weakening TTT interactions, Co-IP, phosphosignaling and telomere length analysis in fission yeast\",\n      \"pmids\": [\"31332096\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single yeast model; human TTI1 contribution to ATR signaling not directly tested\", \"Mechanism of checkpoint-specific dependence unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the architecture of TTT and its engagement with both PIKK clients and the R2TP-HSP90 machinery, defining TTI1 as the central α-solenoid scaffold and showing how clients are delivered without inhibiting kinase activity.\",\n      \"evidence\": \"Cryo-EM of human TTT and R2TP-TTT, domain-mapping deletions, ATPase and pull-down assays, and ionizing-radiation survival assays\",\n      \"pmids\": [\"34838521\", \"34233195\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Static structures do not capture the dynamics of client loading/handoff\", \"Does not explain client selectivity among the six PIKKs\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Genetic suppressor work in budding yeast placed TTI1/TTT downstream of Hsp70 cochaperone activity for PIKK maintenance and showed Tti1 can act partly outside the complex.\",\n      \"evidence\": \"Tti1 suppressor substitutions, Sis1 depletion with PIKK protein-level measurement, rapamycin sensitivity in S. cerevisiae\",\n      \"pmids\": [\"34935410\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of Tti1 complex-independent function unknown\", \"Relevance of Sis1/Hsp70 link to human TTI1 untested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established TTI1 as a human disease gene, showing bi-allelic loss-of-function impairs TTT assembly and mTOR activity, partially rescuable by rapamycin.\",\n      \"evidence\": \"Patient-derived HEK293T, fibroblast and lymphoblastoid cell studies with western blot and rapamycin rescue\",\n      \"pmids\": [\"36724785\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; genotype-phenotype spectrum not fully defined\", \"Tissue-specific consequences of mTOR dysregulation not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated a therapeutically relevant role for TTI1 in tumor radioresistance through ATM-pathway-driven DNA damage repair.\",\n      \"evidence\": \"Knockdown/overexpression with colony formation, comet assay, flow cytometry, xenograft, organoid and PDX rectal cancer models\",\n      \"pmids\": [\"40514657\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the effect requires intact TTT or direct ATM stabilization not dissected\", \"Single cancer context\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Implicated TTI1 in a VEGFR2-mTORC1 axis controlling hypoxic erythroid differentiation of hematopoietic stem cells.\",\n      \"evidence\": \"Proteomics-guided siRNA knockdown, VEGFR2 neutralization and rapamycin in CD34+ erythroid differentiation assays\",\n      \"pmids\": [\"42152561\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Limited mechanistic detail on TTI1's direct role in the axis\", \"Correlative phenocopy rather than direct interaction mapping\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TTT achieves selectivity among its six PIKK clients, and how the opposing CK2-driven stabilizing and degradation-priming phosphorylation events are coordinated in vivo, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No residue-level map distinguishing stabilizing from degradative TTI1 phosphorylation\", \"Dynamics of client handoff to R2TP-HSP90 not captured\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 4]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [2, 5, 8]}\n    ],\n    \"complexes\": [\"TTT (TELO2-TTI1-TTI2) complex\", \"mTORC1\", \"mTORC2\", \"R2TP-TTT\"],\n    \"partners\": [\"TELO2\", \"TTI2\", \"MTOR\", \"ATM\", \"RUVBL1\", \"RUVBL2\", \"PIH1D1\", \"RPAP3\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}