{"gene":"MLST8","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2003,"finding":"GbetaL (mLST8) binds directly to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. GbetaL binding strongly stimulates mTOR kinase activity toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Nutrients and rapamycin regulate the mTOR-raptor association only in complexes that also contain GbetaL.","method":"Co-immunoprecipitation, in vitro kinase assay, biochemical fractionation","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct binding, kinase activity assay, and nutrient-regulation established in a foundational study with multiple orthogonal methods","pmids":["12718876"],"is_preprint":false},{"year":2006,"finding":"Genetic ablation of mLST8 in mice phenocopies rictor (mTORC2) loss, not raptor (mTORC1) loss. mLST8 is required to maintain the rictor-mTOR interaction but not the raptor-mTOR interaction. Both mLST8 and rictor are required for hydrophobic motif phosphorylation of Akt/PKB and PKCalpha, but not S6K1. Insulin signaling to FOXO3 but not to TSC2 or GSK3beta requires mLST8 and rictor.","method":"Conditional knockout mice, immunoprecipitation, immunoblot for pathway phosphorylation, genetic epistasis","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic knockout with multiple defined phosphorylation readouts, replicated by genetic epistasis with raptor/rictor knockouts","pmids":["17141160"],"is_preprint":false},{"year":2003,"finding":"Yeast Lst8p associates with both Tor1p and Tor2p and is a peripheral membrane protein localizing to endosomal or Golgi membranes, cofractioning with Tor1p. lst8 mutants display hypersensitivity to rapamycin and derepressed Gln3p activity, establishing Lst8p as a TOR pathway component. Different lst8 alleles selectively affect either Rtg1/3p or Gln3p transcription factor outputs, revealing two genetically separable TOR-Lst8 downstream branches.","method":"Co-immunoprecipitation, subcellular fractionation, genetic epistasis (rapamycin sensitivity, transcription factor reporter assays), temperature-sensitive mutant analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, fractionation, and multiple genetic readouts in a single focused study of yeast Lst8","pmids":["12719473"],"is_preprint":false},{"year":2012,"finding":"In Drosophila, LST8 functions exclusively in TORC2 and is not required for TORC1 activity. In lst8 knockout mutants, expression of TOR, RAPTOR, and upstream activator Rheb was sufficient to provide TORC1 activity and stimulate cell and organ growth. TORC2 regulates cell growth cell-autonomously but not via its canonical target AKT.","method":"Genetic knockout in Drosophila, epistasis with Rheb overexpression, cell-autonomous growth assays, phosphorylation readouts","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic knockout with epistasis and multiple growth phenotype readouts, ortholog study in Drosophila","pmids":["22493059"],"is_preprint":false},{"year":2019,"finding":"mLST8 loss blocks mTOR association with mTORC2 cofactors RICTOR and SIN1, abrogating mTORC2 activity, but has little to no impact on mTORC1 assembly or activity. A direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor SIN1 was identified. A single pair of mutations on mLST8 with a corresponding mutation on mTOR selectively disrupts mTORC2 assembly and activity without affecting mTORC1.","method":"Co-immunoprecipitation, site-directed mutagenesis, in vivo xenograft tumor model, phosphorylation assays (AKT S473)","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — reciprocal Co-IP, mutagenesis, and in vivo functional validation with multiple orthogonal methods in a single study","pmids":["31085701"],"is_preprint":false},{"year":2015,"finding":"mLST8 knockdown significantly suppresses mTORC1 and mTORC2 complex formation and inhibits tumor growth and invasiveness. mLST8 knockdown reduced mTORC2-mediated AKT phosphorylation in both cancer and normal cells, but potently inhibited mTORC1-mediated 4E-BP1 phosphorylation specifically in cancer cells, indicating distinct roles depending on expression level.","method":"siRNA knockdown, co-immunoprecipitation, phosphorylation assays, anchorage-independent growth assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with co-IP and multiple phosphorylation readouts, single lab","pmids":["25906254"],"is_preprint":false},{"year":2008,"finding":"GbetaL (mLST8) interacts with IKKalpha and IKKbeta both in vitro and in vivo. The C-terminal WD domains of GbetaL are required for interaction with both the kinase domain and leucine zipper domain of IKKbeta. GbetaL overexpression inhibits TNFα-induced NF-κB signaling, while GbetaL knockdown enhances NF-κB activity. GbetaL constitutively interacts with IKKbeta, and this interaction is enhanced by TNFα treatment.","method":"Yeast two-hybrid screening, co-immunoprecipitation (in vitro and in vivo), siRNA knockdown, NF-κB reporter assays, domain mapping with deletion mutants","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, domain mapping, and functional reporter assays, single lab","pmids":["18755269"],"is_preprint":false},{"year":2010,"finding":"GbetaL (mLST8) recruits protein phosphatases PP4, PP2A, and PP6 to IKKβ via immunoprecipitation. By mediating association of these phosphatases (which do not directly bind IKKβ) with the IKK complex, GbetaL acts as a scaffold to negatively regulate IKK activation and NF-κB signaling. siRNA knockdown of GbetaL diminishes the inhibitory effect of overexpressed phosphatases on NF-κB signaling.","method":"Co-immunoprecipitation, proteomic analysis, siRNA knockdown, NF-κB reporter assays","journal":"Molecules and cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with proteomic identification of binding partners plus functional siRNA rescue, single lab","pmids":["21110129"],"is_preprint":false},{"year":2013,"finding":"mLST8 associates with CAD, a multifunctional protein catalyzing the initial three steps in de novo pyrimidine synthesis. mLST8 bridges CAD and mTOR, with mLST8 recognizing CAD and mTOR in distinct ways. CAD enzymatic activity decreases upon amino acid and serum depletion (conditions that suppress mTOR activity), suggesting mLST8 links mTOR pathway status to pyrimidine biosynthesis regulation.","method":"FLAG immunoprecipitation from transfected HEK293 cells, co-immunoprecipitation of endogenous proteins, domain mapping with deletion mutants, CAD enzyme activity assay","journal":"Journal of biomedical science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of both exogenous and endogenous proteins plus enzymatic activity assay, single lab","pmids":["23594158"],"is_preprint":false},{"year":2021,"finding":"CDK1 phosphorylates MLST8 at a consensus (T/S)PXX(S/T/D/E) motif, which is required for FBXW7-mediated recognition and ubiquitin-proteasome degradation of MLST8. FBXW7 (E3 ubiquitin ligase) directly interacts with MLST8 and promotes its ubiquitination and degradation.","method":"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, phosphorylation analysis with CDK1, domain/motif mapping","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, in vitro ubiquitination, and phosphorylation assays from a single lab","pmids":["34741373"],"is_preprint":false},{"year":2022,"finding":"YB-1 promotes translation of CCT4 mRNA by binding its 5'UTR; CCT4 (a subunit of the CCT chaperone complex) in turn promotes mLST8 protein folding, thereby activating both mTORC1 and mTORC2 signaling. This establishes a YB-1/CCT4/mLST8/mTOR axis in glioblastoma.","method":"RNA immunoprecipitation, polysome profiling, co-immunoprecipitation, knockdown/overexpression experiments, xenograft mouse model","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (RIP, polysome profiling, Co-IP, in vivo) establishing CCT4-mLST8 folding interaction, single lab","pmids":["35239512"],"is_preprint":false},{"year":2022,"finding":"ALKBH5-mediated m6A demethylation of MLST8 mRNA stabilizes the transcript; when ALKBH5 is downregulated (by bioactive peptide), m6A levels on MLST8 mRNA increase, leading to mRNA decay and reduced MLST8 protein, which inhibits AML cell proliferation.","method":"MeRIP-seq, RNA-seq, MeRIP-qPCR, RNA stability assays, dual-luciferase reporter assays, Western blot","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal RNA modification detection methods in a single study, single lab","pmids":["35579750"],"is_preprint":false},{"year":2023,"finding":"Keap1 binds to mLST8 via a conserved ETGE motif. The CRL3-Keap1 ubiquitin ligase complex promotes non-degradative ubiquitination of mLST8, reducing mTORC2 complex integrity and mTORC2-AKT activation. Oxidative stress and ROS burst prevent this effect. Cancer-derived Keap1 or mLST8 mutations disrupt the Keap1-mLST8 interaction, allowing mLST8 to evade Keap1-mediated ubiquitination and enhancing mTORC2-AKT activation.","method":"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (ETGE motif), mTORC2 assembly assays, AKT phosphorylation readout","journal":"Redox biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and mutagenesis in single lab study with multiple readouts","pmids":["37688978"],"is_preprint":false},{"year":2024,"finding":"SUMOylation of GβL (mLST8) at lysine residues K86, K215, K245, K261, and K305 by SUMO1, 2, and 3 isoforms promotes mTOR-Raptor and mTOR-Rictor complex formation and nutrient-induced mTOR signaling. Reconstitution with wild-type GbetaL but not SUMOylation-defective KR mutant GβL restores mTOR signaling in GβL-depleted cells.","method":"Mutagenesis, mass spectrometry, co-immunoprecipitation (mTOR complex assembly), rescue experiments with SUMOylation-defective mutant","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with mass spectrometry identification of SUMOylation sites and functional rescue, single lab","pmids":["38395307"],"is_preprint":false},{"year":2025,"finding":"SLAP interacts with mLST8 and facilitates its non-degradative ubiquitination at lysines K86 and K215 via the E3 ubiquitin ligase UBE3C, thereby reducing mTORC2 integrity and mTORC2-AKT signaling. SLAP inhibition of colorectal cancer cell growth and invasion is dependent on mTORC2 signaling inhibition.","method":"Co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis, loss-of-function cell assays, in vivo xenograft model","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, site mapping, and in vivo validation, single lab","pmids":["41398047"],"is_preprint":false},{"year":2023,"finding":"mLST8 knockout reduces mTORC1-dependent phosphorylation of ULK1, promoting autophagy activation, which in turn inhibits coronavirus double-membrane vesicle formation and replication. mTORC1 (not mTORC2) is essential for coronavirus replication, establishing mLST8 as a host factor required for mTORC1-ULK1-autophagy regulation during CoV infection.","method":"CRISPR knockout, pharmacological inhibitors, transmission electron microscopy, autophagy flux assays, phosphorylation assays (ULK1), viral replication assays","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with multiple orthogonal assays establishing mechanistic pathway, single lab","pmids":["37377422"],"is_preprint":false},{"year":2012,"finding":"LST8 expression level determines basal mTORC1 and mTORC2 activities, as LST8 is the rate-limiting shared component present at the lowest level in both complexes. mTORC1 (Raptor-mTOR) and mTORC2 (Rictor-mTOR) complexes compete for association with LST8; displacement of Raptor from LST8 leads to reciprocal enhancement of mTORC2 activity.","method":"siRNA knockdown, overexpression of Raptor deletion mutant (Raptor-ΔCT), co-immunoprecipitation, phosphorylation assays (S6K1, Akt)","journal":"Obesity research & clinical practice","confidence":"Low","confidence_rationale":"Tier 3 / Weak — co-IP and phosphorylation assays with indirect competition model, single lab, single study","pmids":["24331524"],"is_preprint":false},{"year":2023,"finding":"In fission yeast, the mLST8 ortholog Wat1 undergoes hyper-phosphorylation at S116 in response to osmotic stress. Wat1 interacts with the C-terminal/FATC domain-containing region of Tor1. Phosphorylation of Wat1 at S116 is required for its physical interaction with Gad8 (AGC kinase, AKT ortholog), and this phosphorylation is required for vacuolar integrity and sexual differentiation.","method":"Co-immunoprecipitation, molecular modelling, phosphomutant analysis, growth/phenotypic assays in fission yeast","journal":"European journal of cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and phosphomutant analysis in yeast ortholog, single lab, partial mechanistic follow-up","pmids":["29699848"],"is_preprint":false},{"year":2025,"finding":"MLST8 overexpression in retinal pigment epithelium (RPE) cells drives upregulation of mTORC1 and mTORC2 complexes, which disrupts autophagy by suppressing autophagosome formation genes and impairing LC3 processing, leading to autophagosome accumulation and defective autolysosome formation. Torin1 (mTOR inhibitor) or CRYBA1 overexpression rescues these autophagy defects.","method":"RPE-specific MLST8 knock-in mouse model, Western blot (LC3 processing), autophagosome quantification, pharmacological rescue with Torin1","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knock-in animal model with pharmacological rescue and multiple autophagy readouts, single lab","pmids":["40205682"],"is_preprint":false},{"year":2023,"finding":"In a maternal hyperglycemia (GDM) model, high glucose promotes TRAF2-dependent ubiquitination of GβL (mLST8), which increases the GβL/Raptor association (favoring mTORC1) while decreasing GβL/Rictor and GβL/Sin1 association (reducing mTORC2), impairing pulmonary vasculogenesis. TRAF2 knockdown inhibited high-glucose-induced GβL ubiquitination and GβL/Raptor association and restored tube formation.","method":"Immunoprecipitation of ubiquitinated GβL, co-IP of GβL with Raptor/Rictor/Sin1, TRAF2 knockdown, tube formation assay","journal":"Diabetology & metabolic syndrome","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and knockdown with functional readout, single lab, single study","pmids":["36927703"],"is_preprint":false},{"year":2024,"finding":"Cryo-EM structural reconstitution of mTORC1 (comprising mTOR, Raptor, and mLST8) on lysosomal membranes shows that Raptor and mTOR directly interact with the membrane at anchor points separated by up to 230 Å. Full membrane engagement is required for maximal mTORC1 kinase activation via alignment of catalytic residues in the mTOR active site, with mLST8 as part of the complex.","method":"Cryo-EM structure determination, biochemical reconstitution on membranes with physiological concentrations of Rheb, Rags, and Ragulator","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure with biochemical reconstitution, preprint not yet peer-reviewed","pmids":["bio_10.1101_2024.11.15.623810"],"is_preprint":true}],"current_model":"mLST8 (GbetaL) is a WD-repeat scaffold protein that constitutively associates with the mTOR kinase domain in both mTORC1 and mTORC2 complexes: it stimulates mTOR kinase activity, is required to maintain the rictor-mTOR (mTORC2) interaction and downstream phosphorylation of Akt/PKCalpha/FOXO, and its SUMOylation, non-degradative ubiquitination (by CRL3-Keap1, UBE3C/SLAP, or TRAF2), and phosphorylation-dependent degradation (by CDK1/FBXW7) dynamically regulate mTOR complex assembly and activity; outside the mTOR pathway, mLST8 acts as a scaffold to recruit protein phosphatases PP4/PP2A/PP6 to IKKβ, negatively regulating TNFα-induced NF-κB signaling."},"narrative":{"mechanistic_narrative":"mLST8 (GβL) is a WD-repeat scaffold protein that binds directly to the kinase domain of mTOR and serves as a shared, rate-limiting structural component of both mTOR complexes, coupling nutrient status to mTOR kinase output [PMID:12718876, PMID:24331524]. In mTORC1 it stabilizes the raptor-mTOR association and stimulates mTOR kinase activity toward S6K1 and 4E-BP1 in a nutrient- and rapamycin-regulated manner [PMID:12718876]. Its more essential role is in mTORC2: genetic ablation of mLST8 phenocopies rictor loss, and mLST8 is required to maintain mTOR association with the mTORC2 cofactors RICTOR and SIN1 — through a direct interaction with the SIN1 N-terminal domain — to drive hydrophobic-motif phosphorylation of AKT, PKCα, and FOXO3 [PMID:17141160, PMID:31085701]. This division of labor is conserved, with the ortholog functioning selectively in TORC2 in Drosophila and in the TOR pathway in yeast [PMID:12719473, PMID:22493059]. mLST8 abundance and complex partitioning are tightly controlled: CCT4-mediated folding supports its accumulation [PMID:35239512], while post-translational modifications dynamically tune mTOR complex assembly — SUMOylation promotes both mTOR-Raptor and mTOR-Rictor formation [PMID:38395307], and non-degradative ubiquitination by CRL3-Keap1 (via an ETGE motif), by UBE3C/SLAP, or by TRAF2 selectively reduces mTORC2 integrity and AKT activation [PMID:37688978, PMID:41398047, PMID:36927703], whereas CDK1 phosphorylation triggers FBXW7-mediated proteasomal degradation [PMID:34741373]. Through control of mTORC1, mLST8 governs ULK1 phosphorylation and autophagy, a function exploited as a host factor during coronavirus replication and implicated in retinal pigment epithelium pathology [PMID:37377422, PMID:40205682]. Independently of mTOR, mLST8 binds IKKα/IKKβ and acts as a scaffold recruiting PP4, PP2A, and PP6 phosphatases to IKKβ, negatively regulating TNFα-induced NF-κB signaling [PMID:18755269, PMID:21110129].","teleology":[{"year":2003,"claim":"Established that mLST8 is a direct mTOR-binding subunit that positively controls kinase activity and gates nutrient regulation, defining it as a core mTOR pathway component rather than a passive associate.","evidence":"Co-immunoprecipitation, in vitro kinase assay, and biochemical fractionation in mammalian cells; reciprocal co-IP and genetic epistasis in yeast Lst8","pmids":["12718876","12719473"],"confidence":"High","gaps":["Did not resolve whether mLST8 functions identically in mTORC2","Membrane localization observed in yeast not structurally explained","No structure of the mLST8-mTOR interface"]},{"year":2006,"claim":"Resolved the long-standing question of which complex mLST8 is essential for, showing in vivo that it is dispensable for mTORC1/S6K1 signaling but required for mTORC2 integrity and AKT/PKCα/FOXO3 phosphorylation.","evidence":"Conditional knockout mice with genetic epistasis against raptor and rictor knockouts and defined phosphorylation readouts","pmids":["17141160"],"confidence":"High","gaps":["Molecular basis for selective mTORC2 requirement unknown","Did not identify the direct mTORC2 cofactor contact"]},{"year":2012,"claim":"Tested conservation of complex specificity, confirming the mTORC2-selective role of the ortholog in an intact animal and showing TORC2 controls growth independently of canonical AKT.","evidence":"Drosophila genetic knockout with Rheb-overexpression epistasis and cell-autonomous growth assays","pmids":["22493059"],"confidence":"High","gaps":["AKT-independent TORC2 effectors not defined","Relevance to mammalian growth control not addressed"]},{"year":2019,"claim":"Pinpointed the structural mechanism of mTORC2-selectivity by identifying a direct mLST8-SIN1 N-terminal interaction and engineering a mutation pair that disrupts mTORC2 without affecting mTORC1.","evidence":"Co-IP, site-directed mutagenesis, and in vivo xenograft validation with AKT-S473 readout","pmids":["31085701"],"confidence":"High","gaps":["No atomic structure of the mLST8-SIN1 interface","Whether the interaction is regulated dynamically unknown"]},{"year":2012,"claim":"Proposed that mLST8 abundance is rate-limiting and that mTORC1 and mTORC2 compete for it, providing a quantitative model for reciprocal complex regulation.","evidence":"siRNA knockdown, Raptor-ΔCT overexpression, co-IP and S6K1/Akt phosphorylation","pmids":["24331524"],"confidence":"Low","gaps":["Competition model is indirect from a single study","Stoichiometry not measured directly","Not confirmed in vivo"]},{"year":2008,"claim":"Revealed an mTOR-independent function by showing mLST8 binds IKKβ and negatively regulates TNFα-induced NF-κB signaling.","evidence":"Yeast two-hybrid, reciprocal co-IP, domain mapping, siRNA, and NF-κB reporter assays","pmids":["18755269"],"confidence":"Medium","gaps":["Mechanism of inhibition not defined in this study","Relationship to mTOR complex pool unclear"]},{"year":2010,"claim":"Defined the mechanism of NF-κB inhibition, showing mLST8 scaffolds PP4/PP2A/PP6 phosphatases onto IKKβ to suppress its activation.","evidence":"Co-IP with proteomic partner identification, siRNA rescue, and NF-κB reporter assays","pmids":["21110129"],"confidence":"Medium","gaps":["Single lab; not independently replicated","Physiological context of phosphatase recruitment unclear"]},{"year":2013,"claim":"Linked mTOR pathway status to metabolism by showing mLST8 bridges mTOR and the pyrimidine-synthesis enzyme CAD.","evidence":"FLAG and endogenous co-IP, domain mapping, and CAD enzyme activity assays under nutrient depletion","pmids":["23594158"],"confidence":"Medium","gaps":["Direct phosphorylation of CAD by mTOR via mLST8 not demonstrated here","Single lab"]},{"year":2021,"claim":"Identified degradative control of mLST8, showing CDK1 phosphorylation creates a phosphodegron recognized by FBXW7 for proteasomal degradation.","evidence":"Co-IP, in vitro ubiquitination, proteasome inhibition, and motif mapping","pmids":["34741373"],"confidence":"Medium","gaps":["Cell-cycle context of CDK1-driven turnover not fully defined","Effect on mTORC1 vs mTORC2 partitioning not separated"]},{"year":2022,"claim":"Established upstream control of mLST8 levels via folding and mRNA stability, showing CCT4 chaperones mLST8 folding and ALKBH5/m6A regulates MLST8 transcript stability.","evidence":"RIP, polysome profiling, co-IP and xenografts (CCT4 axis); MeRIP-seq, RNA stability and luciferase assays (ALKBH5 axis)","pmids":["35239512","35579750"],"confidence":"Medium","gaps":["Generality beyond glioblastoma/AML untested","Quantitative contribution to mTOR signaling output not measured"]},{"year":2024,"claim":"Built a layered post-translational regulatory model in which SUMOylation promotes mTOR complex assembly while distinct non-degradative ubiquitination events selectively dismantle mTORC2.","evidence":"Mass-spec site mapping, mutagenesis and rescue (SUMO); co-IP, ubiquitination and ETGE-motif mutagenesis for CRL3-Keap1, UBE3C/SLAP, and TRAF2","pmids":["38395307","37688978","41398047","36927703"],"confidence":"Medium","gaps":["Interplay/hierarchy among these modifications not resolved","TRAF2 axis is Low-confidence single study","Structural consequence of ubiquitination on mTORC2 not visualized"]},{"year":2023,"claim":"Connected mLST8 to autophagy and disease physiology, showing mTORC1-dependent ULK1 phosphorylation controls autophagy with consequences for coronavirus replication and RPE pathology.","evidence":"CRISPR KO, autophagy flux, EM and viral replication assays; RPE-specific knock-in mouse with Torin1 rescue","pmids":["37377422","40205682"],"confidence":"Medium","gaps":["Direct vs indirect contribution of mLST8 to ULK1 control not isolated","Tissue specificity of autophagy phenotypes unexplained"]},{"year":2024,"claim":"Began to place mLST8 in the activated mTORC1 architecture, showing membrane engagement aligns the mTOR active site with mLST8 as part of the assembled complex.","evidence":"Cryo-EM and membrane reconstitution with Rheb, Rags, and Ragulator (preprint)","pmids":["bio_10.1101_2024.11.15.623810"],"confidence":"Medium","gaps":["Specific catalytic contribution of mLST8 not isolated structurally","No comparable mTORC2 membrane structure","Preprint, not peer-reviewed"]},{"year":null,"claim":"How the competing post-translational modifications (SUMOylation, multiple ubiquitination routes, phosphodegron turnover) are integrated to dynamically allocate the limited mLST8 pool between mTORC1 and mTORC2 in vivo remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified quantitative model of mLST8 partitioning","No atomic structure of the mLST8-SIN1/mTORC2 interface","Crosstalk between mTOR-dependent and NF-κB scaffolding functions unexplored"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,4]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,4,16]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[20]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[2]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,4]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[15,18]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[9,13,12]}],"complexes":["mTORC1","mTORC2","IKK complex"],"partners":["MTOR","RPTOR","RICTOR","MAPKAP1","IKBKB","KEAP1","FBXW7","CAD"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BVC4","full_name":"Target of rapamycin complex subunit LST8","aliases":["G protein beta subunit-like","Gable","Protein GbetaL","Mammalian lethal with SEC13 protein 8","mLST8"],"length_aa":326,"mass_kda":35.9,"function":"Subunit of both mTORC1 and mTORC2, which regulates cell growth and survival in response to nutrient and hormonal signals (PubMed:12718876, PubMed:15268862, PubMed:15467718, PubMed:24403073, PubMed:28489822). mTORC1 is activated in response to growth factors or amino acids (PubMed:12718876, PubMed:15268862, PubMed:15467718, PubMed:24403073). In response to nutrients, mTORC1 is recruited to the lysosome membrane and promotes protein, lipid and nucleotide synthesis by phosphorylating several substrates, such as ribosomal protein S6 kinase (RPS6KB1 and RPS6KB2) and EIF4EBP1 (4E-BP1) (PubMed:12718876, PubMed:15268862, PubMed:15467718, PubMed:24403073). In the same time, it inhibits catabolic pathways by phosphorylating the autophagy initiation components ULK1 and ATG13, as well as transcription factor TFEB, a master regulators of lysosomal biogenesis and autophagy (PubMed:24403073). The mTORC1 complex is inhibited in response to starvation and amino acid depletion (PubMed:24403073). Within mTORC1, MLST8 interacts directly with MTOR and enhances its kinase activity (PubMed:12718876). In nutrient-poor conditions, stabilizes the MTOR-RPTOR interaction and favors RPTOR-mediated inhibition of MTOR activity (PubMed:12718876). As part of the mTORC2 complex, transduces signals from growth factors to pathways involved in proliferation, cytoskeletal organization, lipogenesis and anabolic output (PubMed:15467718, PubMed:35926713). mTORC2 is also activated by growth factors, but seems to be nutrient-insensitive (PubMed:15467718, PubMed:35926713). In response to growth factors, mTORC2 phosphorylates and activates AGC protein kinase family members, including AKT (AKT1, AKT2 and AKT3), PKC (PRKCA, PRKCB and PRKCE) and SGK1 (PubMed:15467718, PubMed:35926713). mTORC2 functions upstream of Rho GTPases to regulate the actin cytoskeleton, probably by activating one or more Rho-type guanine nucleotide exchange factors (PubMed:15467718). mTORC2 promotes the serum-induced formation of stress-fibers or F-actin (PubMed:15467718). mTORC2 plays a critical role in AKT1 activation by mediating phosphorylation of different sites depending on the context, such as 'Thr-450', 'Ser-473', 'Ser-477' or 'Thr-479', facilitating the phosphorylation of the activation loop of AKT1 on 'Thr-308' by PDPK1/PDK1 which is a prerequisite for full activation (PubMed:15467718). mTORC2 regulates the phosphorylation of SGK1 at 'Ser-422' (PubMed:15467718). mTORC2 also modulates the phosphorylation of PRKCA on 'Ser-657' (PubMed:15467718). Within mTORC2, MLST8 acts as a bridge between MAPKAP1/SIN1 and MTOR (PubMed:31085701)","subcellular_location":"Lysosome membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9BVC4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MLST8","classification":"Common Essential","n_dependent_lines":873,"n_total_lines":1208,"dependency_fraction":0.722682119205298},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"COPB2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MLST8","total_profiled":1310},"omim":[{"mim_id":"616899","title":"TBC1 DOMAIN-CONTAINING KINASE; TBCK","url":"https://www.omim.org/entry/616899"},{"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"},{"mim_id":"612190","title":"MTOR-ASSOCIATED PROTEIN LST8; MLST8","url":"https://www.omim.org/entry/612190"},{"mim_id":"611140","title":"TELOMERE MAINTENANCE 2; TELO2","url":"https://www.omim.org/entry/611140"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Cell Junctions","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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GbetaL binding strongly stimulates mTOR kinase activity toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Nutrients and rapamycin regulate the mTOR-raptor association only in complexes that also contain GbetaL.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, biochemical fractionation\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct binding, kinase activity assay, and nutrient-regulation established in a foundational study with multiple orthogonal methods\",\n      \"pmids\": [\"12718876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Genetic ablation of mLST8 in mice phenocopies rictor (mTORC2) loss, not raptor (mTORC1) loss. mLST8 is required to maintain the rictor-mTOR interaction but not the raptor-mTOR interaction. Both mLST8 and rictor are required for hydrophobic motif phosphorylation of Akt/PKB and PKCalpha, but not S6K1. Insulin signaling to FOXO3 but not to TSC2 or GSK3beta requires mLST8 and rictor.\",\n      \"method\": \"Conditional knockout mice, immunoprecipitation, immunoblot for pathway phosphorylation, genetic epistasis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic knockout with multiple defined phosphorylation readouts, replicated by genetic epistasis with raptor/rictor knockouts\",\n      \"pmids\": [\"17141160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Yeast Lst8p associates with both Tor1p and Tor2p and is a peripheral membrane protein localizing to endosomal or Golgi membranes, cofractioning with Tor1p. lst8 mutants display hypersensitivity to rapamycin and derepressed Gln3p activity, establishing Lst8p as a TOR pathway component. Different lst8 alleles selectively affect either Rtg1/3p or Gln3p transcription factor outputs, revealing two genetically separable TOR-Lst8 downstream branches.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, genetic epistasis (rapamycin sensitivity, transcription factor reporter assays), temperature-sensitive mutant analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, fractionation, and multiple genetic readouts in a single focused study of yeast Lst8\",\n      \"pmids\": [\"12719473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In Drosophila, LST8 functions exclusively in TORC2 and is not required for TORC1 activity. In lst8 knockout mutants, expression of TOR, RAPTOR, and upstream activator Rheb was sufficient to provide TORC1 activity and stimulate cell and organ growth. TORC2 regulates cell growth cell-autonomously but not via its canonical target AKT.\",\n      \"method\": \"Genetic knockout in Drosophila, epistasis with Rheb overexpression, cell-autonomous growth assays, phosphorylation readouts\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout with epistasis and multiple growth phenotype readouts, ortholog study in Drosophila\",\n      \"pmids\": [\"22493059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"mLST8 loss blocks mTOR association with mTORC2 cofactors RICTOR and SIN1, abrogating mTORC2 activity, but has little to no impact on mTORC1 assembly or activity. A direct interaction between mLST8 and the NH2-terminal domain of the mTORC2 cofactor SIN1 was identified. A single pair of mutations on mLST8 with a corresponding mutation on mTOR selectively disrupts mTORC2 assembly and activity without affecting mTORC1.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis, in vivo xenograft tumor model, phosphorylation assays (AKT S473)\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — reciprocal Co-IP, mutagenesis, and in vivo functional validation with multiple orthogonal methods in a single study\",\n      \"pmids\": [\"31085701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"mLST8 knockdown significantly suppresses mTORC1 and mTORC2 complex formation and inhibits tumor growth and invasiveness. mLST8 knockdown reduced mTORC2-mediated AKT phosphorylation in both cancer and normal cells, but potently inhibited mTORC1-mediated 4E-BP1 phosphorylation specifically in cancer cells, indicating distinct roles depending on expression level.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, phosphorylation assays, anchorage-independent growth assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with co-IP and multiple phosphorylation readouts, single lab\",\n      \"pmids\": [\"25906254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"GbetaL (mLST8) interacts with IKKalpha and IKKbeta both in vitro and in vivo. The C-terminal WD domains of GbetaL are required for interaction with both the kinase domain and leucine zipper domain of IKKbeta. GbetaL overexpression inhibits TNFα-induced NF-κB signaling, while GbetaL knockdown enhances NF-κB activity. GbetaL constitutively interacts with IKKbeta, and this interaction is enhanced by TNFα treatment.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation (in vitro and in vivo), siRNA knockdown, NF-κB reporter assays, domain mapping with deletion mutants\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, domain mapping, and functional reporter assays, single lab\",\n      \"pmids\": [\"18755269\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"GbetaL (mLST8) recruits protein phosphatases PP4, PP2A, and PP6 to IKKβ via immunoprecipitation. By mediating association of these phosphatases (which do not directly bind IKKβ) with the IKK complex, GbetaL acts as a scaffold to negatively regulate IKK activation and NF-κB signaling. siRNA knockdown of GbetaL diminishes the inhibitory effect of overexpressed phosphatases on NF-κB signaling.\",\n      \"method\": \"Co-immunoprecipitation, proteomic analysis, siRNA knockdown, NF-κB reporter assays\",\n      \"journal\": \"Molecules and cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with proteomic identification of binding partners plus functional siRNA rescue, single lab\",\n      \"pmids\": [\"21110129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"mLST8 associates with CAD, a multifunctional protein catalyzing the initial three steps in de novo pyrimidine synthesis. mLST8 bridges CAD and mTOR, with mLST8 recognizing CAD and mTOR in distinct ways. CAD enzymatic activity decreases upon amino acid and serum depletion (conditions that suppress mTOR activity), suggesting mLST8 links mTOR pathway status to pyrimidine biosynthesis regulation.\",\n      \"method\": \"FLAG immunoprecipitation from transfected HEK293 cells, co-immunoprecipitation of endogenous proteins, domain mapping with deletion mutants, CAD enzyme activity assay\",\n      \"journal\": \"Journal of biomedical science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of both exogenous and endogenous proteins plus enzymatic activity assay, single lab\",\n      \"pmids\": [\"23594158\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CDK1 phosphorylates MLST8 at a consensus (T/S)PXX(S/T/D/E) motif, which is required for FBXW7-mediated recognition and ubiquitin-proteasome degradation of MLST8. FBXW7 (E3 ubiquitin ligase) directly interacts with MLST8 and promotes its ubiquitination and degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasome inhibitor treatment, phosphorylation analysis with CDK1, domain/motif mapping\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, in vitro ubiquitination, and phosphorylation assays from a single lab\",\n      \"pmids\": [\"34741373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"YB-1 promotes translation of CCT4 mRNA by binding its 5'UTR; CCT4 (a subunit of the CCT chaperone complex) in turn promotes mLST8 protein folding, thereby activating both mTORC1 and mTORC2 signaling. This establishes a YB-1/CCT4/mLST8/mTOR axis in glioblastoma.\",\n      \"method\": \"RNA immunoprecipitation, polysome profiling, co-immunoprecipitation, knockdown/overexpression experiments, xenograft mouse model\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (RIP, polysome profiling, Co-IP, in vivo) establishing CCT4-mLST8 folding interaction, single lab\",\n      \"pmids\": [\"35239512\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ALKBH5-mediated m6A demethylation of MLST8 mRNA stabilizes the transcript; when ALKBH5 is downregulated (by bioactive peptide), m6A levels on MLST8 mRNA increase, leading to mRNA decay and reduced MLST8 protein, which inhibits AML cell proliferation.\",\n      \"method\": \"MeRIP-seq, RNA-seq, MeRIP-qPCR, RNA stability assays, dual-luciferase reporter assays, Western blot\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal RNA modification detection methods in a single study, single lab\",\n      \"pmids\": [\"35579750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Keap1 binds to mLST8 via a conserved ETGE motif. The CRL3-Keap1 ubiquitin ligase complex promotes non-degradative ubiquitination of mLST8, reducing mTORC2 complex integrity and mTORC2-AKT activation. Oxidative stress and ROS burst prevent this effect. Cancer-derived Keap1 or mLST8 mutations disrupt the Keap1-mLST8 interaction, allowing mLST8 to evade Keap1-mediated ubiquitination and enhancing mTORC2-AKT activation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (ETGE motif), mTORC2 assembly assays, AKT phosphorylation readout\",\n      \"journal\": \"Redox biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and mutagenesis in single lab study with multiple readouts\",\n      \"pmids\": [\"37688978\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SUMOylation of GβL (mLST8) at lysine residues K86, K215, K245, K261, and K305 by SUMO1, 2, and 3 isoforms promotes mTOR-Raptor and mTOR-Rictor complex formation and nutrient-induced mTOR signaling. Reconstitution with wild-type GbetaL but not SUMOylation-defective KR mutant GβL restores mTOR signaling in GβL-depleted cells.\",\n      \"method\": \"Mutagenesis, mass spectrometry, co-immunoprecipitation (mTOR complex assembly), rescue experiments with SUMOylation-defective mutant\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with mass spectrometry identification of SUMOylation sites and functional rescue, single lab\",\n      \"pmids\": [\"38395307\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SLAP interacts with mLST8 and facilitates its non-degradative ubiquitination at lysines K86 and K215 via the E3 ubiquitin ligase UBE3C, thereby reducing mTORC2 integrity and mTORC2-AKT signaling. SLAP inhibition of colorectal cancer cell growth and invasion is dependent on mTORC2 signaling inhibition.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, site-directed mutagenesis, loss-of-function cell assays, in vivo xenograft model\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, site mapping, and in vivo validation, single lab\",\n      \"pmids\": [\"41398047\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"mLST8 knockout reduces mTORC1-dependent phosphorylation of ULK1, promoting autophagy activation, which in turn inhibits coronavirus double-membrane vesicle formation and replication. mTORC1 (not mTORC2) is essential for coronavirus replication, establishing mLST8 as a host factor required for mTORC1-ULK1-autophagy regulation during CoV infection.\",\n      \"method\": \"CRISPR knockout, pharmacological inhibitors, transmission electron microscopy, autophagy flux assays, phosphorylation assays (ULK1), viral replication assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with multiple orthogonal assays establishing mechanistic pathway, single lab\",\n      \"pmids\": [\"37377422\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"LST8 expression level determines basal mTORC1 and mTORC2 activities, as LST8 is the rate-limiting shared component present at the lowest level in both complexes. mTORC1 (Raptor-mTOR) and mTORC2 (Rictor-mTOR) complexes compete for association with LST8; displacement of Raptor from LST8 leads to reciprocal enhancement of mTORC2 activity.\",\n      \"method\": \"siRNA knockdown, overexpression of Raptor deletion mutant (Raptor-ΔCT), co-immunoprecipitation, phosphorylation assays (S6K1, Akt)\",\n      \"journal\": \"Obesity research & clinical practice\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-IP and phosphorylation assays with indirect competition model, single lab, single study\",\n      \"pmids\": [\"24331524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In fission yeast, the mLST8 ortholog Wat1 undergoes hyper-phosphorylation at S116 in response to osmotic stress. Wat1 interacts with the C-terminal/FATC domain-containing region of Tor1. Phosphorylation of Wat1 at S116 is required for its physical interaction with Gad8 (AGC kinase, AKT ortholog), and this phosphorylation is required for vacuolar integrity and sexual differentiation.\",\n      \"method\": \"Co-immunoprecipitation, molecular modelling, phosphomutant analysis, growth/phenotypic assays in fission yeast\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and phosphomutant analysis in yeast ortholog, single lab, partial mechanistic follow-up\",\n      \"pmids\": [\"29699848\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MLST8 overexpression in retinal pigment epithelium (RPE) cells drives upregulation of mTORC1 and mTORC2 complexes, which disrupts autophagy by suppressing autophagosome formation genes and impairing LC3 processing, leading to autophagosome accumulation and defective autolysosome formation. Torin1 (mTOR inhibitor) or CRYBA1 overexpression rescues these autophagy defects.\",\n      \"method\": \"RPE-specific MLST8 knock-in mouse model, Western blot (LC3 processing), autophagosome quantification, pharmacological rescue with Torin1\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knock-in animal model with pharmacological rescue and multiple autophagy readouts, single lab\",\n      \"pmids\": [\"40205682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In a maternal hyperglycemia (GDM) model, high glucose promotes TRAF2-dependent ubiquitination of GβL (mLST8), which increases the GβL/Raptor association (favoring mTORC1) while decreasing GβL/Rictor and GβL/Sin1 association (reducing mTORC2), impairing pulmonary vasculogenesis. TRAF2 knockdown inhibited high-glucose-induced GβL ubiquitination and GβL/Raptor association and restored tube formation.\",\n      \"method\": \"Immunoprecipitation of ubiquitinated GβL, co-IP of GβL with Raptor/Rictor/Sin1, TRAF2 knockdown, tube formation assay\",\n      \"journal\": \"Diabetology & metabolic syndrome\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and knockdown with functional readout, single lab, single study\",\n      \"pmids\": [\"36927703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Cryo-EM structural reconstitution of mTORC1 (comprising mTOR, Raptor, and mLST8) on lysosomal membranes shows that Raptor and mTOR directly interact with the membrane at anchor points separated by up to 230 Å. Full membrane engagement is required for maximal mTORC1 kinase activation via alignment of catalytic residues in the mTOR active site, with mLST8 as part of the complex.\",\n      \"method\": \"Cryo-EM structure determination, biochemical reconstitution on membranes with physiological concentrations of Rheb, Rags, and Ragulator\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure with biochemical reconstitution, preprint not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2024.11.15.623810\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"mLST8 (GbetaL) is a WD-repeat scaffold protein that constitutively associates with the mTOR kinase domain in both mTORC1 and mTORC2 complexes: it stimulates mTOR kinase activity, is required to maintain the rictor-mTOR (mTORC2) interaction and downstream phosphorylation of Akt/PKCalpha/FOXO, and its SUMOylation, non-degradative ubiquitination (by CRL3-Keap1, UBE3C/SLAP, or TRAF2), and phosphorylation-dependent degradation (by CDK1/FBXW7) dynamically regulate mTOR complex assembly and activity; outside the mTOR pathway, mLST8 acts as a scaffold to recruit protein phosphatases PP4/PP2A/PP6 to IKKβ, negatively regulating TNFα-induced NF-κB signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"mLST8 (GβL) is a WD-repeat scaffold protein that binds directly to the kinase domain of mTOR and serves as a shared, rate-limiting structural component of both mTOR complexes, coupling nutrient status to mTOR kinase output [#0, #16]. In mTORC1 it stabilizes the raptor-mTOR association and stimulates mTOR kinase activity toward S6K1 and 4E-BP1 in a nutrient- and rapamycin-regulated manner [#0]. Its more essential role is in mTORC2: genetic ablation of mLST8 phenocopies rictor loss, and mLST8 is required to maintain mTOR association with the mTORC2 cofactors RICTOR and SIN1 — through a direct interaction with the SIN1 N-terminal domain — to drive hydrophobic-motif phosphorylation of AKT, PKCα, and FOXO3 [#1, #4]. This division of labor is conserved, with the ortholog functioning selectively in TORC2 in Drosophila and in the TOR pathway in yeast [#2, #3]. mLST8 abundance and complex partitioning are tightly controlled: CCT4-mediated folding supports its accumulation [#10], while post-translational modifications dynamically tune mTOR complex assembly — SUMOylation promotes both mTOR-Raptor and mTOR-Rictor formation [#13], and non-degradative ubiquitination by CRL3-Keap1 (via an ETGE motif), by UBE3C/SLAP, or by TRAF2 selectively reduces mTORC2 integrity and AKT activation [#12, #14, #19], whereas CDK1 phosphorylation triggers FBXW7-mediated proteasomal degradation [#9]. Through control of mTORC1, mLST8 governs ULK1 phosphorylation and autophagy, a function exploited as a host factor during coronavirus replication and implicated in retinal pigment epithelium pathology [#15, #18]. Independently of mTOR, mLST8 binds IKKα/IKKβ and acts as a scaffold recruiting PP4, PP2A, and PP6 phosphatases to IKKβ, negatively regulating TNFα-induced NF-κB signaling [#6, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that mLST8 is a direct mTOR-binding subunit that positively controls kinase activity and gates nutrient regulation, defining it as a core mTOR pathway component rather than a passive associate.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro kinase assay, and biochemical fractionation in mammalian cells; reciprocal co-IP and genetic epistasis in yeast Lst8\",\n      \"pmids\": [\"12718876\", \"12719473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve whether mLST8 functions identically in mTORC2\", \"Membrane localization observed in yeast not structurally explained\", \"No structure of the mLST8-mTOR interface\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved the long-standing question of which complex mLST8 is essential for, showing in vivo that it is dispensable for mTORC1/S6K1 signaling but required for mTORC2 integrity and AKT/PKCα/FOXO3 phosphorylation.\",\n      \"evidence\": \"Conditional knockout mice with genetic epistasis against raptor and rictor knockouts and defined phosphorylation readouts\",\n      \"pmids\": [\"17141160\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for selective mTORC2 requirement unknown\", \"Did not identify the direct mTORC2 cofactor contact\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Tested conservation of complex specificity, confirming the mTORC2-selective role of the ortholog in an intact animal and showing TORC2 controls growth independently of canonical AKT.\",\n      \"evidence\": \"Drosophila genetic knockout with Rheb-overexpression epistasis and cell-autonomous growth assays\",\n      \"pmids\": [\"22493059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"AKT-independent TORC2 effectors not defined\", \"Relevance to mammalian growth control not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Pinpointed the structural mechanism of mTORC2-selectivity by identifying a direct mLST8-SIN1 N-terminal interaction and engineering a mutation pair that disrupts mTORC2 without affecting mTORC1.\",\n      \"evidence\": \"Co-IP, site-directed mutagenesis, and in vivo xenograft validation with AKT-S473 readout\",\n      \"pmids\": [\"31085701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No atomic structure of the mLST8-SIN1 interface\", \"Whether the interaction is regulated dynamically unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Proposed that mLST8 abundance is rate-limiting and that mTORC1 and mTORC2 compete for it, providing a quantitative model for reciprocal complex regulation.\",\n      \"evidence\": \"siRNA knockdown, Raptor-ΔCT overexpression, co-IP and S6K1/Akt phosphorylation\",\n      \"pmids\": [\"24331524\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Competition model is indirect from a single study\", \"Stoichiometry not measured directly\", \"Not confirmed in vivo\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed an mTOR-independent function by showing mLST8 binds IKKβ and negatively regulates TNFα-induced NF-κB signaling.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, domain mapping, siRNA, and NF-κB reporter assays\",\n      \"pmids\": [\"18755269\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of inhibition not defined in this study\", \"Relationship to mTOR complex pool unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the mechanism of NF-κB inhibition, showing mLST8 scaffolds PP4/PP2A/PP6 phosphatases onto IKKβ to suppress its activation.\",\n      \"evidence\": \"Co-IP with proteomic partner identification, siRNA rescue, and NF-κB reporter assays\",\n      \"pmids\": [\"21110129\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; not independently replicated\", \"Physiological context of phosphatase recruitment unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Linked mTOR pathway status to metabolism by showing mLST8 bridges mTOR and the pyrimidine-synthesis enzyme CAD.\",\n      \"evidence\": \"FLAG and endogenous co-IP, domain mapping, and CAD enzyme activity assays under nutrient depletion\",\n      \"pmids\": [\"23594158\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphorylation of CAD by mTOR via mLST8 not demonstrated here\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified degradative control of mLST8, showing CDK1 phosphorylation creates a phosphodegron recognized by FBXW7 for proteasomal degradation.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination, proteasome inhibition, and motif mapping\",\n      \"pmids\": [\"34741373\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-cycle context of CDK1-driven turnover not fully defined\", \"Effect on mTORC1 vs mTORC2 partitioning not separated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established upstream control of mLST8 levels via folding and mRNA stability, showing CCT4 chaperones mLST8 folding and ALKBH5/m6A regulates MLST8 transcript stability.\",\n      \"evidence\": \"RIP, polysome profiling, co-IP and xenografts (CCT4 axis); MeRIP-seq, RNA stability and luciferase assays (ALKBH5 axis)\",\n      \"pmids\": [\"35239512\", \"35579750\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality beyond glioblastoma/AML untested\", \"Quantitative contribution to mTOR signaling output not measured\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Built a layered post-translational regulatory model in which SUMOylation promotes mTOR complex assembly while distinct non-degradative ubiquitination events selectively dismantle mTORC2.\",\n      \"evidence\": \"Mass-spec site mapping, mutagenesis and rescue (SUMO); co-IP, ubiquitination and ETGE-motif mutagenesis for CRL3-Keap1, UBE3C/SLAP, and TRAF2\",\n      \"pmids\": [\"38395307\", \"37688978\", \"41398047\", \"36927703\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Interplay/hierarchy among these modifications not resolved\", \"TRAF2 axis is Low-confidence single study\", \"Structural consequence of ubiquitination on mTORC2 not visualized\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected mLST8 to autophagy and disease physiology, showing mTORC1-dependent ULK1 phosphorylation controls autophagy with consequences for coronavirus replication and RPE pathology.\",\n      \"evidence\": \"CRISPR KO, autophagy flux, EM and viral replication assays; RPE-specific knock-in mouse with Torin1 rescue\",\n      \"pmids\": [\"37377422\", \"40205682\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect contribution of mLST8 to ULK1 control not isolated\", \"Tissue specificity of autophagy phenotypes unexplained\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Began to place mLST8 in the activated mTORC1 architecture, showing membrane engagement aligns the mTOR active site with mLST8 as part of the assembled complex.\",\n      \"evidence\": \"Cryo-EM and membrane reconstitution with Rheb, Rags, and Ragulator (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.11.15.623810\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific catalytic contribution of mLST8 not isolated structurally\", \"No comparable mTORC2 membrane structure\", \"Preprint, not peer-reviewed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the competing post-translational modifications (SUMOylation, multiple ubiquitination routes, phosphodegron turnover) are integrated to dynamically allocate the limited mLST8 pool between mTORC1 and mTORC2 in vivo remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified quantitative model of mLST8 partitioning\", \"No atomic structure of the mLST8-SIN1/mTORC2 interface\", \"Crosstalk between mTOR-dependent and NF-κB scaffolding functions unexplored\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 4, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 4]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [15, 18]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [9, 13, 12]}\n    ],\n    \"complexes\": [\"mTORC1\", \"mTORC2\", \"IKK complex\"],\n    \"partners\": [\"MTOR\", \"RPTOR\", \"RICTOR\", \"MAPKAP1\", \"IKBKB\", \"KEAP1\", \"FBXW7\", \"CAD\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}