{"gene":"AKT1S1","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2007,"finding":"PRAS40 binds to raptor (a component of mTORC1) in insulin-deprived cells, and this interaction inhibits mTORC1 kinase activity. In vitro, PRAS40 prevents the increase in mTORC1 kinase activity induced by Rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which disrupts its inhibition of mTORC1 both in cells and in vitro.","method":"Raptor co-immunoprecipitation, in vitro mTORC1 kinase assay, cell-based S6K1 phosphorylation assays, overexpression and knockdown experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP, in vitro kinase reconstitution assay, replicated across multiple labs","pmids":["17386266"],"is_preprint":false},{"year":2007,"finding":"PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signaling (nutrient/serum deprivation, mitochondrial metabolic inhibition). PRAS40 phosphorylation by Akt and association with the cytosolic anchor 14-3-3 are required for insulin to stimulate mTOR. PRAS40 silencing inactivates IRS-1 and Akt and uncouples mTOR response to Akt signals.","method":"Co-immunoprecipitation, siRNA knockdown, phosphorylation assays, binding studies with 14-3-3","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal methods, independently replicated","pmids":["17277771"],"is_preprint":false},{"year":2007,"finding":"PRAS40 contains a TOS (TOR signaling) motif (FVMDE) required for interaction with raptor. PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro by functioning as a direct competitive inhibitor of substrate (4E-BP1) binding to raptor. Insulin stimulation markedly decreases PRAS40 bound to mTORC1.","method":"Mutagenesis of TOS motif, in vitro mTORC1 kinase assay, shRNA knockdown, competitive binding assays with 4E-BP1 and raptor","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mutagenesis, competitive binding demonstrated, multiple orthogonal methods","pmids":["17510057"],"is_preprint":false},{"year":2007,"finding":"PRAS40 is a substrate for phosphorylation by mTORC1 itself (in addition to Akt). PRAS40 interacts with raptor via its TOS motif, and requires amino acids and insulin for 14-3-3 binding. Binding of PRAS40 to 14-3-3 is inhibited by TSC1/2 and stimulated by Rheb in a rapamycin-sensitive manner. PRAS40 knockdown impairs amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and S6, placing PRAS40 downstream of mTORC1 but upstream of S6K1 and 4E-BP1.","method":"siRNA knockdown, rapamycin treatment, amino acid deprivation, co-immunoprecipitation with 14-3-3, phosphorylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, genetic epistasis via siRNA, independently consistent with other labs","pmids":["17604271"],"is_preprint":false},{"year":2007,"finding":"PRAS40 binds mTORC1 via raptor, is an mTOR phosphorylation substrate, and inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward 4E-BP1. PRAS40 knockdown in HeLa cells protects against TNFα/cycloheximide-induced apoptosis, and this protection is not mimicked by rapamycin, indicating PRAS40 mediates apoptosis independently of its mTORC1 inhibitory function.","method":"2D LC-MS/MS proteomic identification, co-immunoprecipitation, in vitro kinase assay, siRNA knockdown, apoptosis assays","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — MS identification plus in vitro kinase assay plus functional knockdown, single lab but multiple orthogonal methods","pmids":["18030348"],"is_preprint":false},{"year":2017,"finding":"Cryo-EM structure of mTORC1 and crystal structure of a truncated mTOR–PRAS40 complex reveal that PRAS40 inhibits both substrate-recruitment sites on mTORC1 (the RAPTOR-TOS motif binding site and the FRB domain site), explaining its substrate-competitive mechanism of mTORC1 inhibition.","method":"3.0 Å cryo-EM structure of mTORC1, crystal structures of RAPTOR-TOS motif complexes and mTOR FRB-substrate complex, biochemical validation","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM and crystal structures with biochemical validation, definitive structural mechanism","pmids":["29236692"],"is_preprint":false},{"year":2009,"finding":"PIM1 protein kinase directly phosphorylates PRAS40 at Thr246 in vitro (an Akt phosphorylation site), independently of Akt activation. PIM1 overexpression reduces PRAS40 association with mTOR and increases mTOR-directed phosphorylation of 4EBP1 and p70S6K.","method":"In vitro kinase assay, co-immunoprecipitation, wortmannin treatment (Akt-independence), PIM1 inhibitor treatment, overexpression","journal":"Cancer biology & therapy","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay directly demonstrating phosphorylation, supported by co-IP and pharmacological controls, single lab","pmids":["19276681"],"is_preprint":false},{"year":2016,"finding":"Pyruvate kinase M2 (PKM2) phosphorylates PRAS40 at Ser202/203, releasing PRAS40 from raptor and facilitating its binding to 14-3-3, resulting in hormone- and nutrient-signal-independent activation of mTORC1 in cancer cells.","method":"Quantitative phosphoproteomics, in vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis (S202/203), TEPP-46 pharmacological treatment, overexpression/knockdown","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — phosphoproteomics plus in vitro kinase assay plus mutagenesis in one study, single lab","pmids":["26876154"],"is_preprint":false},{"year":2010,"finding":"Efficient phosphorylation of PRAS40 at Ser183 by mTORC1 requires prior phosphorylation of PRAS40 at Thr246 by PKB/Akt. Substitution of Thr246 with Ala alone is sufficient to abolish 14-3-3 binding under intact mTORC1 signaling conditions, indicating a hierarchical phosphorylation mechanism.","method":"Site-directed mutagenesis (T246A), rapamycin/wortmannin/palmitate treatment, insulin clamp in human skeletal muscle, Western blot in multiple cell lines and rat tissues","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis with multiple in vivo and in vitro systems, single lab, multiple orthogonal approaches","pmids":["20138985"],"is_preprint":false},{"year":2014,"finding":"Akt- and mTORC1-mediated phosphorylation of PRAS40 at T246 and S221 respectively promotes nuclear-specific association of PRAS40 with ribosomal protein L11 (RPL11). PRAS40 negatively regulates the RPL11-HDM2-p53 nucleolar stress response pathway; PRAS40 silencing induces p53 upregulation dependent on RPL11, and a T246A mutant incapable of RPL11 binding cannot rescue this effect.","method":"Co-immunoprecipitation, PRAS40 silencing, T246A mutagenesis, p53 and senescence assays, nuclear fractionation","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis rescue, multiple functional readouts, single lab","pmids":["24704832"],"is_preprint":false},{"year":2016,"finding":"mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When mTORC1 is activated, it phosphorylates PRAS40 to simultaneously enhance protein synthesis and facilitate assembly of immunoproteasome β subunits, coupling elevated protein synthesis with immunoproteasome biogenesis to clear aberrant proteins.","method":"Co-immunoprecipitation, phosphorylation assays, immunoproteasome assembly assays, genetic mTORC1/PRAS40 manipulation, RAS/PTEN/TSC1 mutation cell lines","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, functional assembly assays, multiple cell models, single lab with multiple orthogonal methods","pmids":["26876939"],"is_preprint":false},{"year":2017,"finding":"PRAS40 is a unique downstream effector of TGF-α (but not EGF) signaling via Thr308-phosphorylated Akt. Akt-mediated phosphorylation of PRAS40 at Thr246 is both necessary and sufficient to trigger exosome-mediated secretion. PRAS40 knockdown or dominant-negative mutant blocks TGF-α-, hypoxia-, and H2O2-induced exosome secretion without affecting the ER/Golgi pathway.","method":"PRAS40 siRNA knockdown, dominant-negative mutant overexpression, T246 site-directed mutagenesis, gene rescue, exosome secretion assays in multiple cell types","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis plus rescue plus functional assay in multiple cell types, single lab","pmids":["28674187"],"is_preprint":false},{"year":2007,"finding":"Phosphorylated PRAS40 binds the cytosolic docking protein 14-3-3, and this interaction is regulated by the PI3K/Akt pathway. In spinal cord injury models, increased pPRAS40 via PRAS40 transfection promotes motor neuron survival; co-immunoprecipitation shows that pPRAS40–14-3-3 binding increases after injury and is dependent on the PI3K/Akt pathway.","method":"Liposome-mediated PRAS40 transfection, co-immunoprecipitation, PI3K/Akt inhibitor (LY294002, Akt inhibitor IV), immunohistochemistry, Western blot in SOD1 transgenic rats","journal":"Journal of cerebral blood flow and metabolism","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP with pharmacological validation, overexpression with functional readout, single lab","pmids":["17457363"],"is_preprint":false},{"year":2007,"finding":"PRAS40 is an Akt3 substrate in melanoma. Phospho-PRAS40 levels parallel Akt3 activity during melanoma tumor progression. Targeting PRAS40 (via siRNA) or upstream Akt3 similarly reduces anchorage-independent growth and tumor development, and decreasing pPRAS40 increases tumor cell apoptosis and chemosensitivity.","method":"siRNA knockdown of PRAS40 and Akt3, anchorage-independent growth assays, mouse tumor xenograft experiments, apoptosis assays, Western blot","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined cellular phenotype in vitro and in vivo, single lab","pmids":["17440074"],"is_preprint":false},{"year":2011,"finding":"In radioresistant NSCLC cells, nuclear PIM1 phosphorylates PRAS40, and phospho-PRAS40 forms a trimeric complex with 14-3-3 and Akt-activated phospho-FOXO3a, driving cytoplasmic retention of FOXO3a, downregulation of proapoptotic genes, and radioresistance. Protein phosphatases PP2A and PP5 negatively regulate this pathway.","method":"Co-immunoprecipitation (trimeric complex), nuclear fractionation, irradiation assays, PIM1 overexpression, PP2A/PP5 knockdown, apoptotic gene expression","journal":"Radiation research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP demonstrating trimeric complex, nuclear localization, functional radioresistance readout, single lab","pmids":["21910584"],"is_preprint":false},{"year":2010,"finding":"In response to leucine (but not insulin), PDK1 is required for PRAS40 phosphorylation and subsequent mTOR/p70S6K activation in the heart. A PDK1 L155E mutation that preserves insulin/Akt-dependent mTOR signaling abolishes leucine-induced PRAS40 phosphorylation, indicating a distinct PDK1-dependent, Akt-independent mechanism for leucine to activate mTORC1 via PRAS40.","method":"PDK1 knockout mice, PDK1 L155E knock-in mutation, in vitro kinase assay, phosphorylation assays in cardiac tissue","journal":"American journal of physiology. Endocrinology and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and knock-in mutagenesis with defined pathway phenotype, single lab","pmids":["20051528"],"is_preprint":false},{"year":2010,"finding":"High glucose increases PRAS40 phosphorylation at Thr246 via PI3K/Akt, dissociating PRAS40 from the raptor-PRAS40 complex, thereby activating mTORC1 and promoting mesangial cell hypertrophy. A phosphorylation-deficient PRAS40 mutant (in contrast to PRAS40 knockdown) inhibits 4EBP-1 and S6K phosphorylation and reduces hypertrophy, identifying PRAS40 phosphorylation as the mechanistic node.","method":"PI3K/Akt inhibitors, co-immunoprecipitation of raptor-PRAS40, phosphorylation-deficient PRAS40 mutant, PRAS40 siRNA, protein synthesis and hypertrophy assays","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, mutagenesis, and functional hypertrophy assay, single lab","pmids":["20629086"],"is_preprint":false},{"year":2012,"finding":"EWS (Ewing sarcoma protein) negatively regulates PRAS40 expression by binding the 3' UTR of PRAS40 mRNA. Loss of EWS leads to elevated PRAS40, which promotes Ewing sarcoma cell proliferation and metastatic growth; PRAS40 knockdown reverses the proliferative effect of EWS knockdown.","method":"RNA binding assay (EWS-3'UTR interaction), siRNA knockdown of PRAS40 and EWS, cell proliferation assays, metastatic growth assays, Western blot","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — RNA binding and epistasis rescue, single lab","pmids":["22241085"],"is_preprint":false},{"year":2019,"finding":"PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. PRAS40 knockdown in endothelial cells promotes TNFα-induced mTORC1 signaling and inflammatory marker upregulation, while PRAS40 overexpression blocks these. In vivo, endothelium-specific PRAS40 deficiency enhances neointimal hyperplasia and atherosclerotic lesion formation.","method":"PRAS40 siRNA knockdown, overexpression, endothelium-specific PRAS40 knockout mice, phosphorylation assays, monocyte recruitment assays, in vivo atherogenic remodeling model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — tissue-specific KO plus gain/loss-of-function in vitro, single lab","pmids":["31728028"],"is_preprint":false},{"year":2022,"finding":"Phosphoglycerate kinase 1 (PGK1) binds PRAS40 and phosphorylates it at Thr246 under normoxia, suppressing autophagy-mediated cell death and promoting liver cancer cell proliferation. Under hypoxia, PGK1 binding switches from PRAS40 to Beclin1, increasing Beclin1 phosphorylation and autophagy induction.","method":"Co-immunoprecipitation (PGK1-PRAS40 interaction), in vitro kinase assay, site-directed blocking of interaction, in vitro and in vivo proliferation assays, autophagy tracing","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — Co-IP plus in vitro kinase assay, in vivo functional validation, single lab","pmids":["35058442"],"is_preprint":false},{"year":2019,"finding":"MELK kinase phosphorylates PRAS40, disrupting the interaction between PRAS40 and raptor and thereby over-activating mTORC1 signaling to promote clear cell renal cell carcinoma progression.","method":"Co-immunoprecipitation (PRAS40-raptor), MELK overexpression and knockdown, phosphorylation assays, cell proliferation and invasion assays","journal":"Cell transplantation","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP with gain/loss-of-function, single lab, mechanism inferred from phosphorylation and complex disruption","pmids":["31813279"],"is_preprint":false},{"year":2014,"finding":"PRAS40 gene transfer in rats reduces cerebral infarction size by promoting phosphorylation of Akt, FOXO1, PRAS40, and mTOR. PRAS40 knockout increases infarction size and reduces p-S6K and p-S6 in the mTOR pathway after stroke; co-immunoprecipitation shows less Akt-mTOR interaction in PRAS40 KO, identifying PRAS40 as a physical bridge linking Akt and mTOR signaling in the context of ischemia.","method":"Lentiviral PRAS40 overexpression, PRAS40 knockout mice, cerebral ischemia model, co-immunoprecipitation (Akt-mTOR), Western blot","journal":"Neurobiology of disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus overexpression plus Co-IP, single lab","pmids":["24583056"],"is_preprint":false},{"year":2015,"finding":"Genetic ablation of PRAS40 in mice results in increased hepatic Akt (T308) and mTORC1 (p-p70S6K) signaling, altered hepatic GLUT4 levels, and improved glucose homeostasis, demonstrating that PRAS40 limits basal mTORC1 activity and insulin sensitivity in vivo.","method":"PRAS40 knockout mice, streptozotocin-induced diabetes model, glucose tolerance tests, Western blot, shPRAS40 Hep3B cells","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined metabolic phenotype and molecular readouts, single lab","pmids":["25931147"],"is_preprint":false},{"year":2014,"finding":"Over-expression of wild-type PRAS40 (but not AAA-PRAS40 mutant with mutated phosphorylation and mTORC1-binding sites) impairs insulin-mediated mTORC1 pathway activation but increases Akt phosphorylation and insulin sensitivity in skeletal muscle cells, identifying a role for PRAS40 in regulating insulin sensitivity through IRS1 stabilization and proteasome inhibition, independent of its mTORC1-binding function.","method":"WT-PRAS40 and AAA-PRAS40 overexpression in human skeletal muscle cells and mouse skeletal muscle, phosphorylation assays, proteasome activity assay, IRS1 protein levels","journal":"Archives of physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional readout, in vitro and in vivo, single lab","pmids":["24576065"],"is_preprint":false},{"year":2005,"finding":"PRAS40 is phosphorylated via the PI3K/Akt pathway (inhibited by wortmannin and LY294002 but not rapamycin); 14-3-3 is identified as a PRAS40 binding protein. PRAS40 constitutive phosphorylation activity is higher in pre-malignant and malignant cancer cell lines compared to normal cells.","method":"Kinase inhibitor treatment (wortmannin, LY294002, rapamycin, UO126), Western blot, co-immunoprecipitation with 14-3-3","journal":"Acta pharmacologica Sinica","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pharmacological inhibitor approach only, no direct kinase assay","pmids":["16174443"],"is_preprint":false},{"year":2019,"finding":"AKT3 (but not AKT1 or AKT2) is the specific Akt isoform mediating M2-tumor-associated macrophage-induced phosphorylation of PRAS40 (Thr246) in intrahepatic cholangiocarcinoma cells, leading to EMT activation. AKT3 silencing specifically inhibits p-AKT and p-PRAS40 under M2-TAM co-culture conditions.","method":"AKT isoform-specific siRNA knockdown (AKT1, AKT2, AKT3), co-culture assays, phosphorylation assays (p-AKT Ser473, p-PRAS40 Thr246), EMT invasion assays","journal":"Journal of cellular biochemistry","confidence":"Low","confidence_rationale":"Tier 3 / Weak — siRNA knockdown with phosphorylation readout, single lab, no direct kinase assay","pmids":["31692069"],"is_preprint":false}],"current_model":"PRAS40 (AKT1S1) is a dual-function component of mTORC1 that acts as a raptor-binding inhibitor of the complex by occupying both substrate-recruitment sites (TOS-motif binding site and FRB domain), thereby preventing substrate access; under insulin/growth factor stimulation, Akt phosphorylates PRAS40 at Thr246 (and mTORC1 itself phosphorylates Ser183/Ser221), causing PRAS40 to dissociate from mTORC1 and bind 14-3-3, relieving inhibition and activating mTORC1 toward S6K1 and 4E-BP1; beyond Akt, additional kinases including PIM1, PKM2, MELK, and PGK1 can directly phosphorylate PRAS40 at the same or adjacent sites to modulate mTORC1 activity independently of Akt; in the nucleus, phospho-PRAS40 suppresses the RPL11-HDM2-p53 stress response pathway, and in the cytoplasm it facilitates immunoproteasome biogenesis and exosome secretion, placing PRAS40 at a central node integrating growth factor signals with protein homeostasis and cell survival."},"narrative":{"mechanistic_narrative":"AKT1S1 (PRAS40) is a raptor-binding inhibitory subunit of mTORC1 that couples growth-factor and nutrient signaling to control of protein synthesis, cell growth, and survival [PMID:17386266, PMID:17510057]. PRAS40 binds raptor through a TOS motif (FVMDE) and acts as a substrate-competitive inhibitor, blocking access of mTORC1 substrates such as 4E-BP1; high-resolution structures show it occupies both substrate-recruitment sites of mTORC1, the raptor TOS-binding site and the FRB domain [PMID:17510057, PMID:29236692]. Inhibition is relieved upon phosphorylation: insulin/growth-factor-activated Akt phosphorylates PRAS40 at Thr246, after which mTORC1 itself phosphorylates additional sites (Ser183/Ser221) in a hierarchical manner, driving PRAS40 dissociation from raptor and association with the cytosolic anchor 14-3-3, thereby activating mTORC1 toward S6K1 and 4E-BP1 [PMID:17386266, PMID:17277771, PMID:17604271, PMID:20138985]. Beyond Akt, multiple kinases — PIM1, PKM2, MELK, and PGK1 — directly phosphorylate PRAS40 at Thr246 or adjacent sites to release it from raptor and activate mTORC1 independently of canonical hormone/Akt input, frequently in cancer contexts [PMID:19276681, PMID:26876154, PMID:31813279, PMID:35058442]. PRAS40 also exerts mTORC1-independent functions: nuclear phospho-PRAS40 binds RPL11 to suppress the RPL11–HDM2–p53 nucleolar stress response [PMID:24704832], it couples protein synthesis to immunoproteasome β-subunit assembly [PMID:26876939], and Akt-Thr246 phosphorylation of PRAS40 is necessary and sufficient to drive exosome-mediated secretion [PMID:28674187]. Genetic ablation studies establish PRAS40 as a brake on basal mTORC1 activity that modulates insulin sensitivity, glucose homeostasis, tissue hypertrophy, vascular inflammation, and survival in ischemic injury [PMID:25931147, PMID:20629086, PMID:31728028, PMID:24583056].","teleology":[{"year":2007,"claim":"Established PRAS40 as a physiological inhibitor of mTORC1 whose inhibition is relieved by Akt phosphorylation, defining the core regulatory node linking insulin signaling to mTORC1 activity.","evidence":"Raptor co-IP, in vitro mTORC1 kinase reconstitution with Rheb1-GTP, and cell-based S6K1 assays with Akt phosphorylation","pmids":["17386266","17277771"],"confidence":"High","gaps":["Did not resolve the structural basis of substrate-competitive inhibition","Relative contributions of individual phosphosites not fully separated"]},{"year":2007,"claim":"Defined the molecular mechanism of inhibition: PRAS40 uses a TOS motif to bind raptor and competitively block substrate recruitment, while 14-3-3 binding upon phosphorylation provides the off-switch.","evidence":"TOS-motif mutagenesis, competitive binding assays with 4E-BP1/raptor, in vitro kinase assays, and 14-3-3 co-IP regulated by TSC1/2 and Rheb","pmids":["17510057","17604271","18030348"],"confidence":"High","gaps":["Stoichiometry of PRAS40 within mTORC1 not defined","mTORC1-independent apoptotic role mechanistically unexplained"]},{"year":2010,"claim":"Resolved the order of phosphorylation events, showing Akt Thr246 phosphorylation is the priming step required for subsequent mTORC1-mediated Ser183 phosphorylation and 14-3-3 binding.","evidence":"T246A site-directed mutagenesis with rapamycin/wortmannin/palmitate treatment across cell lines, rat tissues, and human skeletal muscle insulin clamp","pmids":["20138985"],"confidence":"High","gaps":["Functional consequence of each phosphosite for downstream substrates not individually mapped"]},{"year":2017,"claim":"Provided the definitive structural mechanism, showing PRAS40 blocks both the raptor TOS site and the FRB substrate site of mTORC1.","evidence":"3.0 Å cryo-EM of mTORC1 and crystal structures of raptor-TOS and mTOR FRB-substrate complexes with biochemical validation","pmids":["29236692"],"confidence":"High","gaps":["Conformational changes upon phosphorylation-driven dissociation not captured","Structure of phospho-PRAS40–14-3-3 complex not determined"]},{"year":2009,"claim":"Demonstrated that PRAS40 phosphorylation and mTORC1 activation are not exclusive to Akt, opening the concept of multiple input kinases converging on Thr246.","evidence":"In vitro kinase assay with PIM1, wortmannin (Akt-independence), and PIM1 inhibitor controls","pmids":["19276681"],"confidence":"High","gaps":["Physiological contexts where PIM1 dominates over Akt not delineated"]},{"year":2016,"claim":"Expanded the kinase repertoire and revealed metabolism-driven, hormone-independent mTORC1 activation via PKM2 phosphorylation of PRAS40, and uncovered an immunoproteasome-biogenesis function.","evidence":"Phosphoproteomics, in vitro kinase assays, S202/203 mutagenesis, and immunoproteasome assembly assays across RAS/PTEN/TSC1 mutant cells","pmids":["26876154","26876939"],"confidence":"High","gaps":["Integration of PKM2-driven signaling with canonical Akt input unclear","Mechanism coupling PRAS40 to β-subunit assembly not structurally defined"]},{"year":2014,"claim":"Identified mTORC1-independent nuclear functions of PRAS40, linking it to the RPL11–HDM2–p53 stress response.","evidence":"Nuclear fractionation, RPL11 co-IP, T246A rescue, and p53/senescence assays","pmids":["24704832"],"confidence":"High","gaps":["Nuclear import/retention mechanism of phospho-PRAS40 unknown","Direct versus indirect effect on HDM2 not resolved"]},{"year":2017,"claim":"Established that Akt-Thr246 phosphorylation of PRAS40 drives exosome secretion as a discrete output downstream of TGF-α signaling.","evidence":"siRNA knockdown, dominant-negative and T246 mutants, gene rescue, and exosome secretion assays in multiple cell types","pmids":["28674187"],"confidence":"High","gaps":["Molecular machinery linking phospho-PRAS40 to exosome biogenesis not identified","Selectivity for TGF-α over EGF mechanistically unexplained"]},{"year":2019,"claim":"Genetic and tissue-specific loss-of-function studies confirmed PRAS40 as an in vivo brake on basal mTORC1 controlling insulin sensitivity, vascular inflammation, and tissue remodeling, with additional Akt-independent inputs (PDK1/leucine).","evidence":"PRAS40 knockout and endothelium-specific knockout mice, PDK1 L155E knock-in, glucose tolerance and atherogenic remodeling models, and hypertrophy assays","pmids":["25931147","31728028","20051528","20629086","24583056"],"confidence":"Medium","gaps":["Tissue-specific weighting of mTORC1-dependent versus -independent effects unresolved","Whether IRS1 stabilization is direct not established"]},{"year":2022,"claim":"Extended the kinase network to MELK and PGK1, linking PRAS40 phosphorylation to autophagy suppression and cancer progression.","evidence":"PGK1-PRAS40 and MELK co-IP, in vitro kinase assays, raptor-disruption assays, and in vivo proliferation/autophagy tracing","pmids":["35058442","31813279"],"confidence":"Medium","gaps":["Site specificity of MELK phosphorylation not mapped","Switch between PGK1 binding PRAS40 versus Beclin1 mechanistically incomplete"]},{"year":null,"claim":"How the diverse upstream kinases and the multiple PRAS40 phosphosites are integrated to dictate the choice between mTORC1 regulation, nuclear p53 control, immunoproteasome assembly, and exosome secretion remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model distinguishing which output a given phospho-state selects","Spatial control (nuclear versus cytoplasmic) of phospho-PRAS40 not mechanistically defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,5]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[10]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[21]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,3]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[9,14]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,10]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[4,9]}],"complexes":["mTORC1"],"partners":["RPTOR","MTOR","AKT1","YWHAE","PIM1","PKM2","RPL11","PGK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96B36","full_name":"Proline-rich AKT1 substrate 1","aliases":["40 kDa proline-rich AKT substrate"],"length_aa":256,"mass_kda":27.4,"function":"Negative regulator of the mechanistic target of rapamycin complex 1 (mTORC1), an evolutionarily conserved central nutrient sensor that stimulates anabolic reactions and macromolecule biosynthesis to promote cellular biomass generation and growth (PubMed:17277771, PubMed:17386266, PubMed:17510057, PubMed:29236692). In absence of insulin and nutrients, AKT1S1 associates with the mTORC1 complex and directly inhibits mTORC1 activity by blocking the MTOR substrate-recruitment site (PubMed:29236692). In response to insulin and nutrients, AKT1S1 dissociates from mTORC1 (PubMed:17386266, PubMed:18372248). Its activity is dependent on its phosphorylation state and binding to 14-3-3 (PubMed:16174443, PubMed:18372248). May also play a role in nerve growth factor-mediated neuroprotection (By similarity)","subcellular_location":"Cytoplasm, cytosol","url":"https://www.uniprot.org/uniprotkb/Q96B36/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AKT1S1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000204673","cell_line_id":"CID001695","localizations":[{"compartment":"cytoplasmic","grade":3}],"interactors":[{"gene":"YWHAG","stoichiometry":4.0},{"gene":"RPTOR","stoichiometry":0.2},{"gene":"YWHAH","stoichiometry":0.2},{"gene":"YWHAE","stoichiometry":0.2},{"gene":"MTOR","stoichiometry":0.2},{"gene":"CSNK2A2","stoichiometry":0.2},{"gene":"SNRPC","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001695","total_profiled":1310},"omim":[{"mim_id":"610221","title":"AKT1 SUBSTRATE 1, PROLINE-RICH; 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substrate (PRAS40) in human copper/zinc-superoxide dismutase transgenic rats protects motor neurons from death after spinal cord injury.","date":"2007","source":"Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/17457363","citation_count":25,"is_preprint":false},{"pmid":"31227207","id":"PMC_31227207","title":"Molecular alterations in the medial temporal lobe in schizophrenia.","date":"2019","source":"Schizophrenia research","url":"https://pubmed.ncbi.nlm.nih.gov/31227207","citation_count":24,"is_preprint":false},{"pmid":"22412948","id":"PMC_22412948","title":"bantam is required for optic lobe development and glial cell proliferation.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22412948","citation_count":24,"is_preprint":false},{"pmid":"36204827","id":"PMC_36204827","title":"MicroRNAs as potential biomarkers in temporal lobe epilepsy and mesial temporal lobe epilepsy.","date":"2023","source":"Neural regeneration research","url":"https://pubmed.ncbi.nlm.nih.gov/36204827","citation_count":22,"is_preprint":false},{"pmid":"32656205","id":"PMC_32656205","title":"Long Noncoding RNA LINC01134 Promotes Hepatocellular Carcinoma Metastasis via Activating AKT1S1 and NF-κB Signaling.","date":"2020","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/32656205","citation_count":21,"is_preprint":false},{"pmid":"23114665","id":"PMC_23114665","title":"Nocturnal frontal lobe epilepsy and the acetylcholine receptor.","date":"2012","source":"The neurologist","url":"https://pubmed.ncbi.nlm.nih.gov/23114665","citation_count":21,"is_preprint":false},{"pmid":"31728028","id":"PMC_31728028","title":"PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells.","date":"2019","source":"Scientific 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epilepsy.","date":"2015","source":"Frontiers in bioscience (Elite edition)","url":"https://pubmed.ncbi.nlm.nih.gov/25553365","citation_count":20,"is_preprint":false},{"pmid":"31813279","id":"PMC_31813279","title":"MELK is Upregulated in Advanced Clear Cell Renal Cell Carcinoma and Promotes Disease Progression by Phosphorylating PRAS40.","date":"2019","source":"Cell transplantation","url":"https://pubmed.ncbi.nlm.nih.gov/31813279","citation_count":20,"is_preprint":false},{"pmid":"24687853","id":"PMC_24687853","title":"p19Ink4d is a tumor suppressor and controls pituitary anterior lobe cell proliferation.","date":"2014","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24687853","citation_count":20,"is_preprint":false},{"pmid":"31210839","id":"PMC_31210839","title":"Bloom Syndrome Protein Activates AKT and PRAS40 in Prostate Cancer Cells.","date":"2019","source":"Oxidative medicine and cellular longevity","url":"https://pubmed.ncbi.nlm.nih.gov/31210839","citation_count":19,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48925,"output_tokens":7070,"usd":0.126413,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16034,"output_tokens":3782,"usd":0.08736,"stage2_stop_reason":"end_turn"},"total_usd":0.213773,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"PRAS40 binds to raptor (a component of mTORC1) in insulin-deprived cells, and this interaction inhibits mTORC1 kinase activity. In vitro, PRAS40 prevents the increase in mTORC1 kinase activity induced by Rheb1-GTP. Insulin stimulates Akt/PKB-mediated phosphorylation of PRAS40, which disrupts its inhibition of mTORC1 both in cells and in vitro.\",\n      \"method\": \"Raptor co-immunoprecipitation, in vitro mTORC1 kinase assay, cell-based S6K1 phosphorylation assays, overexpression and knockdown experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP, in vitro kinase reconstitution assay, replicated across multiple labs\",\n      \"pmids\": [\"17386266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signaling (nutrient/serum deprivation, mitochondrial metabolic inhibition). PRAS40 phosphorylation by Akt and association with the cytosolic anchor 14-3-3 are required for insulin to stimulate mTOR. PRAS40 silencing inactivates IRS-1 and Akt and uncouples mTOR response to Akt signals.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, phosphorylation assays, binding studies with 14-3-3\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal methods, independently replicated\",\n      \"pmids\": [\"17277771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PRAS40 contains a TOS (TOR signaling) motif (FVMDE) required for interaction with raptor. PRAS40 inhibits mTORC1 kinase activity in vivo and in vitro by functioning as a direct competitive inhibitor of substrate (4E-BP1) binding to raptor. Insulin stimulation markedly decreases PRAS40 bound to mTORC1.\",\n      \"method\": \"Mutagenesis of TOS motif, in vitro mTORC1 kinase assay, shRNA knockdown, competitive binding assays with 4E-BP1 and raptor\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mutagenesis, competitive binding demonstrated, multiple orthogonal methods\",\n      \"pmids\": [\"17510057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PRAS40 is a substrate for phosphorylation by mTORC1 itself (in addition to Akt). PRAS40 interacts with raptor via its TOS motif, and requires amino acids and insulin for 14-3-3 binding. Binding of PRAS40 to 14-3-3 is inhibited by TSC1/2 and stimulated by Rheb in a rapamycin-sensitive manner. PRAS40 knockdown impairs amino acid- and insulin-stimulated phosphorylation of 4E-BP1 and S6, placing PRAS40 downstream of mTORC1 but upstream of S6K1 and 4E-BP1.\",\n      \"method\": \"siRNA knockdown, rapamycin treatment, amino acid deprivation, co-immunoprecipitation with 14-3-3, phosphorylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, genetic epistasis via siRNA, independently consistent with other labs\",\n      \"pmids\": [\"17604271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PRAS40 binds mTORC1 via raptor, is an mTOR phosphorylation substrate, and inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward 4E-BP1. PRAS40 knockdown in HeLa cells protects against TNFα/cycloheximide-induced apoptosis, and this protection is not mimicked by rapamycin, indicating PRAS40 mediates apoptosis independently of its mTORC1 inhibitory function.\",\n      \"method\": \"2D LC-MS/MS proteomic identification, co-immunoprecipitation, in vitro kinase assay, siRNA knockdown, apoptosis assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — MS identification plus in vitro kinase assay plus functional knockdown, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"18030348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Cryo-EM structure of mTORC1 and crystal structure of a truncated mTOR–PRAS40 complex reveal that PRAS40 inhibits both substrate-recruitment sites on mTORC1 (the RAPTOR-TOS motif binding site and the FRB domain site), explaining its substrate-competitive mechanism of mTORC1 inhibition.\",\n      \"method\": \"3.0 Å cryo-EM structure of mTORC1, crystal structures of RAPTOR-TOS motif complexes and mTOR FRB-substrate complex, biochemical validation\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM and crystal structures with biochemical validation, definitive structural mechanism\",\n      \"pmids\": [\"29236692\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PIM1 protein kinase directly phosphorylates PRAS40 at Thr246 in vitro (an Akt phosphorylation site), independently of Akt activation. PIM1 overexpression reduces PRAS40 association with mTOR and increases mTOR-directed phosphorylation of 4EBP1 and p70S6K.\",\n      \"method\": \"In vitro kinase assay, co-immunoprecipitation, wortmannin treatment (Akt-independence), PIM1 inhibitor treatment, overexpression\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay directly demonstrating phosphorylation, supported by co-IP and pharmacological controls, single lab\",\n      \"pmids\": [\"19276681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Pyruvate kinase M2 (PKM2) phosphorylates PRAS40 at Ser202/203, releasing PRAS40 from raptor and facilitating its binding to 14-3-3, resulting in hormone- and nutrient-signal-independent activation of mTORC1 in cancer cells.\",\n      \"method\": \"Quantitative phosphoproteomics, in vitro kinase assay, co-immunoprecipitation, site-directed mutagenesis (S202/203), TEPP-46 pharmacological treatment, overexpression/knockdown\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — phosphoproteomics plus in vitro kinase assay plus mutagenesis in one study, single lab\",\n      \"pmids\": [\"26876154\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Efficient phosphorylation of PRAS40 at Ser183 by mTORC1 requires prior phosphorylation of PRAS40 at Thr246 by PKB/Akt. Substitution of Thr246 with Ala alone is sufficient to abolish 14-3-3 binding under intact mTORC1 signaling conditions, indicating a hierarchical phosphorylation mechanism.\",\n      \"method\": \"Site-directed mutagenesis (T246A), rapamycin/wortmannin/palmitate treatment, insulin clamp in human skeletal muscle, Western blot in multiple cell lines and rat tissues\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis with multiple in vivo and in vitro systems, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"20138985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Akt- and mTORC1-mediated phosphorylation of PRAS40 at T246 and S221 respectively promotes nuclear-specific association of PRAS40 with ribosomal protein L11 (RPL11). PRAS40 negatively regulates the RPL11-HDM2-p53 nucleolar stress response pathway; PRAS40 silencing induces p53 upregulation dependent on RPL11, and a T246A mutant incapable of RPL11 binding cannot rescue this effect.\",\n      \"method\": \"Co-immunoprecipitation, PRAS40 silencing, T246A mutagenesis, p53 and senescence assays, nuclear fractionation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis rescue, multiple functional readouts, single lab\",\n      \"pmids\": [\"24704832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When mTORC1 is activated, it phosphorylates PRAS40 to simultaneously enhance protein synthesis and facilitate assembly of immunoproteasome β subunits, coupling elevated protein synthesis with immunoproteasome biogenesis to clear aberrant proteins.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assays, immunoproteasome assembly assays, genetic mTORC1/PRAS40 manipulation, RAS/PTEN/TSC1 mutation cell lines\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, functional assembly assays, multiple cell models, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26876939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PRAS40 is a unique downstream effector of TGF-α (but not EGF) signaling via Thr308-phosphorylated Akt. Akt-mediated phosphorylation of PRAS40 at Thr246 is both necessary and sufficient to trigger exosome-mediated secretion. PRAS40 knockdown or dominant-negative mutant blocks TGF-α-, hypoxia-, and H2O2-induced exosome secretion without affecting the ER/Golgi pathway.\",\n      \"method\": \"PRAS40 siRNA knockdown, dominant-negative mutant overexpression, T246 site-directed mutagenesis, gene rescue, exosome secretion assays in multiple cell types\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis plus rescue plus functional assay in multiple cell types, single lab\",\n      \"pmids\": [\"28674187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Phosphorylated PRAS40 binds the cytosolic docking protein 14-3-3, and this interaction is regulated by the PI3K/Akt pathway. In spinal cord injury models, increased pPRAS40 via PRAS40 transfection promotes motor neuron survival; co-immunoprecipitation shows that pPRAS40–14-3-3 binding increases after injury and is dependent on the PI3K/Akt pathway.\",\n      \"method\": \"Liposome-mediated PRAS40 transfection, co-immunoprecipitation, PI3K/Akt inhibitor (LY294002, Akt inhibitor IV), immunohistochemistry, Western blot in SOD1 transgenic rats\",\n      \"journal\": \"Journal of cerebral blood flow and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP with pharmacological validation, overexpression with functional readout, single lab\",\n      \"pmids\": [\"17457363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PRAS40 is an Akt3 substrate in melanoma. Phospho-PRAS40 levels parallel Akt3 activity during melanoma tumor progression. Targeting PRAS40 (via siRNA) or upstream Akt3 similarly reduces anchorage-independent growth and tumor development, and decreasing pPRAS40 increases tumor cell apoptosis and chemosensitivity.\",\n      \"method\": \"siRNA knockdown of PRAS40 and Akt3, anchorage-independent growth assays, mouse tumor xenograft experiments, apoptosis assays, Western blot\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined cellular phenotype in vitro and in vivo, single lab\",\n      \"pmids\": [\"17440074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In radioresistant NSCLC cells, nuclear PIM1 phosphorylates PRAS40, and phospho-PRAS40 forms a trimeric complex with 14-3-3 and Akt-activated phospho-FOXO3a, driving cytoplasmic retention of FOXO3a, downregulation of proapoptotic genes, and radioresistance. Protein phosphatases PP2A and PP5 negatively regulate this pathway.\",\n      \"method\": \"Co-immunoprecipitation (trimeric complex), nuclear fractionation, irradiation assays, PIM1 overexpression, PP2A/PP5 knockdown, apoptotic gene expression\",\n      \"journal\": \"Radiation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP demonstrating trimeric complex, nuclear localization, functional radioresistance readout, single lab\",\n      \"pmids\": [\"21910584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In response to leucine (but not insulin), PDK1 is required for PRAS40 phosphorylation and subsequent mTOR/p70S6K activation in the heart. A PDK1 L155E mutation that preserves insulin/Akt-dependent mTOR signaling abolishes leucine-induced PRAS40 phosphorylation, indicating a distinct PDK1-dependent, Akt-independent mechanism for leucine to activate mTORC1 via PRAS40.\",\n      \"method\": \"PDK1 knockout mice, PDK1 L155E knock-in mutation, in vitro kinase assay, phosphorylation assays in cardiac tissue\",\n      \"journal\": \"American journal of physiology. Endocrinology and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and knock-in mutagenesis with defined pathway phenotype, single lab\",\n      \"pmids\": [\"20051528\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"High glucose increases PRAS40 phosphorylation at Thr246 via PI3K/Akt, dissociating PRAS40 from the raptor-PRAS40 complex, thereby activating mTORC1 and promoting mesangial cell hypertrophy. A phosphorylation-deficient PRAS40 mutant (in contrast to PRAS40 knockdown) inhibits 4EBP-1 and S6K phosphorylation and reduces hypertrophy, identifying PRAS40 phosphorylation as the mechanistic node.\",\n      \"method\": \"PI3K/Akt inhibitors, co-immunoprecipitation of raptor-PRAS40, phosphorylation-deficient PRAS40 mutant, PRAS40 siRNA, protein synthesis and hypertrophy assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, mutagenesis, and functional hypertrophy assay, single lab\",\n      \"pmids\": [\"20629086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EWS (Ewing sarcoma protein) negatively regulates PRAS40 expression by binding the 3' UTR of PRAS40 mRNA. Loss of EWS leads to elevated PRAS40, which promotes Ewing sarcoma cell proliferation and metastatic growth; PRAS40 knockdown reverses the proliferative effect of EWS knockdown.\",\n      \"method\": \"RNA binding assay (EWS-3'UTR interaction), siRNA knockdown of PRAS40 and EWS, cell proliferation assays, metastatic growth assays, Western blot\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — RNA binding and epistasis rescue, single lab\",\n      \"pmids\": [\"22241085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. PRAS40 knockdown in endothelial cells promotes TNFα-induced mTORC1 signaling and inflammatory marker upregulation, while PRAS40 overexpression blocks these. In vivo, endothelium-specific PRAS40 deficiency enhances neointimal hyperplasia and atherosclerotic lesion formation.\",\n      \"method\": \"PRAS40 siRNA knockdown, overexpression, endothelium-specific PRAS40 knockout mice, phosphorylation assays, monocyte recruitment assays, in vivo atherogenic remodeling model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — tissue-specific KO plus gain/loss-of-function in vitro, single lab\",\n      \"pmids\": [\"31728028\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Phosphoglycerate kinase 1 (PGK1) binds PRAS40 and phosphorylates it at Thr246 under normoxia, suppressing autophagy-mediated cell death and promoting liver cancer cell proliferation. Under hypoxia, PGK1 binding switches from PRAS40 to Beclin1, increasing Beclin1 phosphorylation and autophagy induction.\",\n      \"method\": \"Co-immunoprecipitation (PGK1-PRAS40 interaction), in vitro kinase assay, site-directed blocking of interaction, in vitro and in vivo proliferation assays, autophagy tracing\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — Co-IP plus in vitro kinase assay, in vivo functional validation, single lab\",\n      \"pmids\": [\"35058442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"MELK kinase phosphorylates PRAS40, disrupting the interaction between PRAS40 and raptor and thereby over-activating mTORC1 signaling to promote clear cell renal cell carcinoma progression.\",\n      \"method\": \"Co-immunoprecipitation (PRAS40-raptor), MELK overexpression and knockdown, phosphorylation assays, cell proliferation and invasion assays\",\n      \"journal\": \"Cell transplantation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP with gain/loss-of-function, single lab, mechanism inferred from phosphorylation and complex disruption\",\n      \"pmids\": [\"31813279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PRAS40 gene transfer in rats reduces cerebral infarction size by promoting phosphorylation of Akt, FOXO1, PRAS40, and mTOR. PRAS40 knockout increases infarction size and reduces p-S6K and p-S6 in the mTOR pathway after stroke; co-immunoprecipitation shows less Akt-mTOR interaction in PRAS40 KO, identifying PRAS40 as a physical bridge linking Akt and mTOR signaling in the context of ischemia.\",\n      \"method\": \"Lentiviral PRAS40 overexpression, PRAS40 knockout mice, cerebral ischemia model, co-immunoprecipitation (Akt-mTOR), Western blot\",\n      \"journal\": \"Neurobiology of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus overexpression plus Co-IP, single lab\",\n      \"pmids\": [\"24583056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Genetic ablation of PRAS40 in mice results in increased hepatic Akt (T308) and mTORC1 (p-p70S6K) signaling, altered hepatic GLUT4 levels, and improved glucose homeostasis, demonstrating that PRAS40 limits basal mTORC1 activity and insulin sensitivity in vivo.\",\n      \"method\": \"PRAS40 knockout mice, streptozotocin-induced diabetes model, glucose tolerance tests, Western blot, shPRAS40 Hep3B cells\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined metabolic phenotype and molecular readouts, single lab\",\n      \"pmids\": [\"25931147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Over-expression of wild-type PRAS40 (but not AAA-PRAS40 mutant with mutated phosphorylation and mTORC1-binding sites) impairs insulin-mediated mTORC1 pathway activation but increases Akt phosphorylation and insulin sensitivity in skeletal muscle cells, identifying a role for PRAS40 in regulating insulin sensitivity through IRS1 stabilization and proteasome inhibition, independent of its mTORC1-binding function.\",\n      \"method\": \"WT-PRAS40 and AAA-PRAS40 overexpression in human skeletal muscle cells and mouse skeletal muscle, phosphorylation assays, proteasome activity assay, IRS1 protein levels\",\n      \"journal\": \"Archives of physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional readout, in vitro and in vivo, single lab\",\n      \"pmids\": [\"24576065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PRAS40 is phosphorylated via the PI3K/Akt pathway (inhibited by wortmannin and LY294002 but not rapamycin); 14-3-3 is identified as a PRAS40 binding protein. PRAS40 constitutive phosphorylation activity is higher in pre-malignant and malignant cancer cell lines compared to normal cells.\",\n      \"method\": \"Kinase inhibitor treatment (wortmannin, LY294002, rapamycin, UO126), Western blot, co-immunoprecipitation with 14-3-3\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pharmacological inhibitor approach only, no direct kinase assay\",\n      \"pmids\": [\"16174443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"AKT3 (but not AKT1 or AKT2) is the specific Akt isoform mediating M2-tumor-associated macrophage-induced phosphorylation of PRAS40 (Thr246) in intrahepatic cholangiocarcinoma cells, leading to EMT activation. AKT3 silencing specifically inhibits p-AKT and p-PRAS40 under M2-TAM co-culture conditions.\",\n      \"method\": \"AKT isoform-specific siRNA knockdown (AKT1, AKT2, AKT3), co-culture assays, phosphorylation assays (p-AKT Ser473, p-PRAS40 Thr246), EMT invasion assays\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — siRNA knockdown with phosphorylation readout, single lab, no direct kinase assay\",\n      \"pmids\": [\"31692069\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRAS40 (AKT1S1) is a dual-function component of mTORC1 that acts as a raptor-binding inhibitor of the complex by occupying both substrate-recruitment sites (TOS-motif binding site and FRB domain), thereby preventing substrate access; under insulin/growth factor stimulation, Akt phosphorylates PRAS40 at Thr246 (and mTORC1 itself phosphorylates Ser183/Ser221), causing PRAS40 to dissociate from mTORC1 and bind 14-3-3, relieving inhibition and activating mTORC1 toward S6K1 and 4E-BP1; beyond Akt, additional kinases including PIM1, PKM2, MELK, and PGK1 can directly phosphorylate PRAS40 at the same or adjacent sites to modulate mTORC1 activity independently of Akt; in the nucleus, phospho-PRAS40 suppresses the RPL11-HDM2-p53 stress response pathway, and in the cytoplasm it facilitates immunoproteasome biogenesis and exosome secretion, placing PRAS40 at a central node integrating growth factor signals with protein homeostasis and cell survival.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AKT1S1 (PRAS40) is a raptor-binding inhibitory subunit of mTORC1 that couples growth-factor and nutrient signaling to control of protein synthesis, cell growth, and survival [#0, #2]. PRAS40 binds raptor through a TOS motif (FVMDE) and acts as a substrate-competitive inhibitor, blocking access of mTORC1 substrates such as 4E-BP1; high-resolution structures show it occupies both substrate-recruitment sites of mTORC1, the raptor TOS-binding site and the FRB domain [#2, #5]. Inhibition is relieved upon phosphorylation: insulin/growth-factor-activated Akt phosphorylates PRAS40 at Thr246, after which mTORC1 itself phosphorylates additional sites (Ser183/Ser221) in a hierarchical manner, driving PRAS40 dissociation from raptor and association with the cytosolic anchor 14-3-3, thereby activating mTORC1 toward S6K1 and 4E-BP1 [#0, #1, #3, #8]. Beyond Akt, multiple kinases — PIM1, PKM2, MELK, and PGK1 — directly phosphorylate PRAS40 at Thr246 or adjacent sites to release it from raptor and activate mTORC1 independently of canonical hormone/Akt input, frequently in cancer contexts [#6, #7, #20, #19]. PRAS40 also exerts mTORC1-independent functions: nuclear phospho-PRAS40 binds RPL11 to suppress the RPL11–HDM2–p53 nucleolar stress response [#9], it couples protein synthesis to immunoproteasome β-subunit assembly [#10], and Akt-Thr246 phosphorylation of PRAS40 is necessary and sufficient to drive exosome-mediated secretion [#11]. Genetic ablation studies establish PRAS40 as a brake on basal mTORC1 activity that modulates insulin sensitivity, glucose homeostasis, tissue hypertrophy, vascular inflammation, and survival in ischemic injury [#22, #16, #18, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 2007,\n      \"claim\": \"Established PRAS40 as a physiological inhibitor of mTORC1 whose inhibition is relieved by Akt phosphorylation, defining the core regulatory node linking insulin signaling to mTORC1 activity.\",\n      \"evidence\": \"Raptor co-IP, in vitro mTORC1 kinase reconstitution with Rheb1-GTP, and cell-based S6K1 assays with Akt phosphorylation\",\n      \"pmids\": [\"17386266\", \"17277771\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of substrate-competitive inhibition\", \"Relative contributions of individual phosphosites not fully separated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the molecular mechanism of inhibition: PRAS40 uses a TOS motif to bind raptor and competitively block substrate recruitment, while 14-3-3 binding upon phosphorylation provides the off-switch.\",\n      \"evidence\": \"TOS-motif mutagenesis, competitive binding assays with 4E-BP1/raptor, in vitro kinase assays, and 14-3-3 co-IP regulated by TSC1/2 and Rheb\",\n      \"pmids\": [\"17510057\", \"17604271\", \"18030348\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of PRAS40 within mTORC1 not defined\", \"mTORC1-independent apoptotic role mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved the order of phosphorylation events, showing Akt Thr246 phosphorylation is the priming step required for subsequent mTORC1-mediated Ser183 phosphorylation and 14-3-3 binding.\",\n      \"evidence\": \"T246A site-directed mutagenesis with rapamycin/wortmannin/palmitate treatment across cell lines, rat tissues, and human skeletal muscle insulin clamp\",\n      \"pmids\": [\"20138985\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of each phosphosite for downstream substrates not individually mapped\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the definitive structural mechanism, showing PRAS40 blocks both the raptor TOS site and the FRB substrate site of mTORC1.\",\n      \"evidence\": \"3.0 Å cryo-EM of mTORC1 and crystal structures of raptor-TOS and mTOR FRB-substrate complexes with biochemical validation\",\n      \"pmids\": [\"29236692\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational changes upon phosphorylation-driven dissociation not captured\", \"Structure of phospho-PRAS40–14-3-3 complex not determined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Demonstrated that PRAS40 phosphorylation and mTORC1 activation are not exclusive to Akt, opening the concept of multiple input kinases converging on Thr246.\",\n      \"evidence\": \"In vitro kinase assay with PIM1, wortmannin (Akt-independence), and PIM1 inhibitor controls\",\n      \"pmids\": [\"19276681\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where PIM1 dominates over Akt not delineated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Expanded the kinase repertoire and revealed metabolism-driven, hormone-independent mTORC1 activation via PKM2 phosphorylation of PRAS40, and uncovered an immunoproteasome-biogenesis function.\",\n      \"evidence\": \"Phosphoproteomics, in vitro kinase assays, S202/203 mutagenesis, and immunoproteasome assembly assays across RAS/PTEN/TSC1 mutant cells\",\n      \"pmids\": [\"26876154\", \"26876939\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of PKM2-driven signaling with canonical Akt input unclear\", \"Mechanism coupling PRAS40 to β-subunit assembly not structurally defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified mTORC1-independent nuclear functions of PRAS40, linking it to the RPL11–HDM2–p53 stress response.\",\n      \"evidence\": \"Nuclear fractionation, RPL11 co-IP, T246A rescue, and p53/senescence assays\",\n      \"pmids\": [\"24704832\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear import/retention mechanism of phospho-PRAS40 unknown\", \"Direct versus indirect effect on HDM2 not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established that Akt-Thr246 phosphorylation of PRAS40 drives exosome secretion as a discrete output downstream of TGF-α signaling.\",\n      \"evidence\": \"siRNA knockdown, dominant-negative and T246 mutants, gene rescue, and exosome secretion assays in multiple cell types\",\n      \"pmids\": [\"28674187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular machinery linking phospho-PRAS40 to exosome biogenesis not identified\", \"Selectivity for TGF-α over EGF mechanistically unexplained\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Genetic and tissue-specific loss-of-function studies confirmed PRAS40 as an in vivo brake on basal mTORC1 controlling insulin sensitivity, vascular inflammation, and tissue remodeling, with additional Akt-independent inputs (PDK1/leucine).\",\n      \"evidence\": \"PRAS40 knockout and endothelium-specific knockout mice, PDK1 L155E knock-in, glucose tolerance and atherogenic remodeling models, and hypertrophy assays\",\n      \"pmids\": [\"25931147\", \"31728028\", \"20051528\", \"20629086\", \"24583056\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific weighting of mTORC1-dependent versus -independent effects unresolved\", \"Whether IRS1 stabilization is direct not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended the kinase network to MELK and PGK1, linking PRAS40 phosphorylation to autophagy suppression and cancer progression.\",\n      \"evidence\": \"PGK1-PRAS40 and MELK co-IP, in vitro kinase assays, raptor-disruption assays, and in vivo proliferation/autophagy tracing\",\n      \"pmids\": [\"35058442\", \"31813279\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Site specificity of MELK phosphorylation not mapped\", \"Switch between PGK1 binding PRAS40 versus Beclin1 mechanistically incomplete\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse upstream kinases and the multiple PRAS40 phosphosites are integrated to dictate the choice between mTORC1 regulation, nuclear p53 control, immunoproteasome assembly, and exosome secretion remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model distinguishing which output a given phospho-state selects\", \"Spatial control (nuclear versus cytoplasmic) of phospho-PRAS40 not mechanistically defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [9, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 10]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [4, 9]}\n    ],\n    \"complexes\": [\"mTORC1\"],\n    \"partners\": [\"RPTOR\", \"MTOR\", \"AKT1\", \"YWHAE\", \"PIM1\", \"PKM2\", \"RPL11\", \"PGK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}