{"gene":"AKT1","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2005,"finding":"The rictor-mTOR complex (mTORC2) directly phosphorylates AKT1 on Ser473 in vitro and facilitates subsequent Thr308 phosphorylation by PDK1, establishing mTORC2 as the long-sought Ser473 kinase for AKT activation.","method":"In vitro kinase assay with purified rictor-mTOR complex, siRNA knockdown of rictor/mTOR in Drosophila and human cells, epistasis analysis","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro phosphorylation reconstituted, combined with genetic knockdown in two organisms, replicated across multiple labs subsequently","pmids":["15718470"],"is_preprint":false},{"year":2006,"finding":"Prolonged rapamycin treatment inhibits mTORC2 assembly, reducing Ser473 phosphorylation of AKT/PKB below levels needed to maintain AKT signaling; this mechanism underlies some proapoptotic and antitumor effects of rapamycin.","method":"mTORC2 assembly assays, Western blotting of phospho-AKT in multiple cell lines after prolonged rapamycin, rescue with rapamycin-resistant AKT mutant","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal biochemical assays, genetic rescue experiment, multiple cell types tested","pmids":["16603397"],"is_preprint":false},{"year":2006,"finding":"mSin1 is an essential component of mTORC2 required for mTORC2 assembly and its capacity to phosphorylate AKT/PKB on Ser473; alternative splicing generates at least three distinct mTORC2 complexes, only two of which are regulated by insulin.","method":"Co-immunoprecipitation, siRNA knockdown of mSin1, in vitro mTORC2 kinase assay, mass spectrometry identification of mSin1 isoforms","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro reconstitution of kinase activity, reciprocal co-IP, siRNA loss-of-function, multiple orthogonal methods","pmids":["16919458"],"is_preprint":false},{"year":1998,"finding":"PTEN negatively regulates AKT/PKB activity and phosphorylation by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in vitro and in vivo; PTEN-deficient cells show constitutively elevated AKT activity and resistance to apoptosis, which is reversed by re-expression of wild-type PTEN.","method":"In vitro lipid phosphatase assay, genetic rescue (PTEN re-expression in mutant cells), measurement of PI(3,4,5)P3 levels, PKB kinase assays in PTEN-deficient MEFs","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro phosphatase assay, genetic rescue, lipid measurements, replicated across labs","pmids":["9778245"],"is_preprint":false},{"year":1999,"finding":"PI3K-generated D3-phosphoinositides bind the AKT PH domain, inducing plasma membrane translocation and co-localization with PDK1, which then phosphorylates the AKT activation loop (Thr308); this multi-step activation mechanism is shared with other AGC kinases.","method":"Phosphoinositide binding assays, subcellular fractionation, kinase assays, domain mutagenesis","journal":"Annual review of biochemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — biochemical reconstitution, domain mutagenesis, widely replicated paradigm","pmids":["10872470"],"is_preprint":false},{"year":2004,"finding":"DNA-dependent protein kinase (DNA-PK) was purified as a major Ser473 kinase from HEK293 cells: it co-localizes and associates with AKT at the plasma membrane, phosphorylates AKT on Ser473 in vitro (~10-fold activation), and siRNA knockdown of DNA-PK inhibits insulin- and pervanadate-induced Ser473 phosphorylation; DNA-PK-deficient cells fail to phosphorylate Ser473 in response to insulin, restored by PRKDC complementation.","method":"Biochemical purification, in vitro kinase assay, siRNA knockdown, genetic complementation in DNA-PK-deficient cells, co-localization by microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — purification + reconstitution, siRNA, genetic complementation — multiple orthogonal methods in one study","pmids":["15262962"],"is_preprint":false},{"year":1998,"finding":"AKT/PKB directly phosphorylates CREB at Ser133 in serum-stimulated cells, promoting CBP recruitment and CREB-dependent target gene expression; this effect requires AKT kinase activity and is PI3K-dependent.","method":"Overexpression of AKT in serum-stimulated cells, phospho-Ser133 CREB detection, CBP co-immunoprecipitation, reporter gene assays, PI3K inhibitor (LY294002)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — cellular phosphorylation assay + functional reporter, single lab, two orthogonal methods","pmids":["9829964"],"is_preprint":false},{"year":1999,"finding":"AKT/PKB activation of NF-κB occurs at the level of IκB degradation and requires AKT kinase activity and its PH domain; AKT does not act alone but cooperates with other pathway signals to induce NF-κB-dependent cytokine promoters.","method":"Overexpression of kinase-dead and PH-domain mutant AKT in Jurkat cells, NF-κB reporter assays, IκB degradation assays","journal":"Current biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — kinase-dead and domain mutagenesis, reporter assays, single lab","pmids":["10359702"],"is_preprint":false},{"year":2002,"finding":"AKT/PKB phosphorylates p27Kip1 at Thr157 within its nuclear localization signal, impairing nuclear import of p27 and causing cytoplasmic retention; cells expressing the T157A mutant p27 are refractory to AKT-mediated nuclear exclusion and retain G1-arrest activity.","method":"In vitro kinase assay with recombinant AKT and p27, site-directed mutagenesis (T157A), subcellular fractionation/immunofluorescence, CDK2 activity assays, in vitro nuclear import assays","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay, mutagenesis, nuclear import reconstitution, independently replicated in companion paper (PMID:12244301)","pmids":["12244302","12244301"],"is_preprint":false},{"year":2001,"finding":"AKT/PKB directly phosphorylates p21Cip1/WAF1 at Thr145 and Ser146; Thr145 phosphorylation inhibits PCNA binding, while Ser146 phosphorylation stabilizes p21 protein, enhancing cell survival and CDK4/cyclin D1 complex assembly.","method":"In vitro kinase assay, site-directed mutagenesis, protein stability assays, co-immunoprecipitation for PCNA binding, kinase activity assays in glioblastoma cell lines","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro phosphorylation + mutagenesis of both sites + functional readouts in one rigorous study","pmids":["11756412"],"is_preprint":false},{"year":2004,"finding":"PKB/AKT physically interacts with Smad3 through a kinase-activity-independent mechanism; insulin induces complex formation while TGF-β inhibits it. AKT inhibits Smad3 by preventing its phosphorylation, Smad4 binding, and nuclear translocation, thereby suppressing TGF-β-induced apoptosis.","method":"Protein fragment complementation assay (PCA) in live cells, co-immunoprecipitation, siRNA knockdown of AKT, reporter assays for Smad3-dependent transcription, phosphorylation assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — PCA interaction assay, co-IP, siRNA rescue, functional reporter, multiple orthogonal methods","pmids":["15048128"],"is_preprint":false},{"year":2005,"finding":"AKT/PKB phosphorylates Girdin/APE at Ser1416; phosphorylated Girdin accumulates at the leading edge of migrating cells and is required for stress fiber/lamellipodia formation and cell migration. Cells expressing S1416A-Girdin show impaired migration and lamellipodia formation.","method":"Identification of Girdin as AKT substrate by mass spectrometry, in vitro kinase assay, site-directed mutagenesis (S1416A), immunofluorescence of phospho-Girdin localization, cell migration assays","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro phosphorylation, mutagenesis, live-cell localization, functional migration assay — multiple orthogonal methods","pmids":["16139227"],"is_preprint":false},{"year":2005,"finding":"Protein kinase CK2 phosphorylates AKT/PKB at Ser129 in vitro and in vivo; this phosphorylation of already-activated AKT further increases its catalytic activity, and CK2 inhibition (by two structurally distinct inhibitors or siRNA) reduces AKT activity and promotes apoptosis.","method":"In vitro kinase assay with CK2 and AKT, 32P incorporation, CK2 siRNA, two structurally distinct CK2 inhibitors, immunoprecipitate kinase assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro reconstitution, siRNA, two independent inhibitors confirming same result","pmids":["15818404"],"is_preprint":false},{"year":2010,"finding":"The non-receptor tyrosine kinase Ack1 directly phosphorylates AKT at the evolutionarily conserved Tyr176 in the kinase domain; Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes subsequent Thr308/Ser473 phosphorylation and AKT activation downstream of RTKs.","method":"Direct in vitro phosphorylation assay, site-directed mutagenesis (Y176F), subcellular fractionation, phospho-specific antibodies, mouse prostate cancer model with activated Ack1","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro reconstitution, mutagenesis, in vivo mouse model, phospho-specific antibody validation","pmids":["20333297"],"is_preprint":false},{"year":2000,"finding":"AKT/PKB optimal phosphorylation consensus motif was defined as Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) with Arg at −3, −5, and −7 being most critical for high Vmax/Km; this was validated against a cDNA library screen of HeLa cell substrates.","method":"Oriented peptide library screening, cDNA expression library screening (λGEX phage), kinetic analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution with peptide library + independent cDNA library validation, two orthogonal approaches","pmids":["10945990"],"is_preprint":false},{"year":2009,"finding":"AKT/PKB exists in an inactive closed conformation through an intramolecular interaction between its PH domain and kinase domain that prevents PDK1 phosphorylation of the activation loop; allosteric inhibitor AKT inhibitor VIII stabilizes this closed conformation and shows isoform selectivity based on differences at the PH-kinase domain interface.","method":"Molecular modeling, FRET/two-photon FLIM, classical biochemical kinase assays, domain mutagenesis","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — FRET/FLIM structural measurements, mutagenesis, biochemical assays — multiple orthogonal approaches in one study","pmids":["19166270"],"is_preprint":false},{"year":2009,"finding":"AKT/PKB directly phosphorylates Skp2, triggering SCF complex formation and E3 ligase activity; AKT-mediated phosphorylation also induces 14-3-3β-dependent cytoplasmic relocalization of Skp2, where cytosolic Skp2 promotes cell migration.","method":"Co-immunoprecipitation, direct in vitro kinase assay, phosphorylation-defective mutant (Skp2-S72A), ubiquitylation assays, subcellular fractionation, cell migration assays, analysis of human cancer specimens","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay, mutagenesis, 14-3-3β interaction, functional assays, human tissue validation","pmids":["19270694"],"is_preprint":false},{"year":1999,"finding":"In C. elegans, Akt/PKB homologs AKT-1 and AKT-2 transduce insulin receptor-like signals from AGE-1 PI3K downstream; loss-of-function of DAF-16 (Forkhead transcription factor) suppresses the akt-1/akt-2 requirement, establishing that AKT/PKB functions primarily to antagonize DAF-16 transcription factor activity.","method":"Genetic epistasis (daf-16 suppression of akt-1/akt-2 mutant phenotype), gain-of-function akt-1 screen, overexpression dosage experiments, expression pattern analysis in C. elegans","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis in C. elegans with suppressor analysis, highly replicated and foundational for the AKT-FOXO pathway","pmids":["9716402"],"is_preprint":false},{"year":2004,"finding":"ATM kinase is required for full AKT/PKB activation at Ser473 in response to insulin or gamma-radiation; this effect is mediated through the PI3K-like kinase domain of ATM and affects downstream AKT targets (Forkhead transcription factors) in ATM-deficient cells.","method":"Transient transfection, ATM inhibitors, ATM siRNA, cell lines from AT patients and ATM knockout mice, Ser473-specific phosphorylation analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA + pharmacological inhibitors + genetic model (AT patient cells and KO mice), single lab","pmids":["15546863"],"is_preprint":false},{"year":2001,"finding":"AKT1 directly associates with JIP1 (JNK-interacting protein 1/scaffold) in primary neurons; this interaction inhibits JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Excitotoxic stimuli disrupt the Akt1-JIP1 interaction, releasing JNK activation, and Akt1-deficient neurons show enhanced kainate susceptibility.","method":"Co-immunoprecipitation, direct binding assay, kinase assays, Akt1-deficient mouse neurons, overexpression of Akt1 mutants","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct protein interaction, kinase assay, KO mouse neurons, functional apoptosis readout — multiple orthogonal methods","pmids":["12194869"],"is_preprint":false},{"year":2007,"finding":"Akt2/PKBβ phosphorylates and inhibits PGC-1α, a global regulator of hepatic metabolism during fasting; phosphorylation prevents PGC-1α recruitment to cognate promoters, impairing gluconeogenesis and fatty acid oxidation programs.","method":"In vitro phosphorylation assay, chromatin immunoprecipitation, reporter assays, hepatocyte-specific Akt2 knockout, metabolic phenotyping","journal":"Nature","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro phosphorylation, ChIP, genetic KO with metabolic readout; primarily Akt2 isoform but mechanistically relevant to AKT family","pmids":["17554339"],"is_preprint":false},{"year":1999,"finding":"In Drosophila, Akt/PKB regulates cell and organ size in a cell-autonomous manner downstream of the insulin receptor/Chico/Dp110 (PI3K) pathway; ectopic Akt expression increases cell size without affecting apoptosis, cell fate, or proliferation rates.","method":"Drosophila genetic mosaic analysis, imaginal disc clonal analysis, gain- and loss-of-function transgenes, cell size measurement","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic epistasis in Drosophila, cell-autonomous clonal analysis, multiple transgenic lines","pmids":["10587646"],"is_preprint":false},{"year":1999,"finding":"AKT/PKB PH domain binds PI3K-generated 3'-phosphoinositides and localizes to membrane ruffles upon mitogen treatment; in epithelial cells, AKT PH domain localizes to sites of cell-cell and cell-matrix contact (distinct from focal contacts) in a PI3K-activity-dependent and phosphoinositide-binding-dependent manner, providing a constitutive survival signal.","method":"GFP-Akt and GFP-PH domain fusion live-cell imaging, immunofluorescence microscopy, PI3K inhibitor treatment, PH domain phosphoinositide-binding mutants","journal":"Current biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — live-cell localization with GFP fusions, pharmacological and mutagenesis controls, functional survival link established","pmids":["10226029"],"is_preprint":false},{"year":2003,"finding":"Akt/PKB phosphorylates tau at Thr212 and Ser214 independently in vitro; only one site per molecule is phosphorylated at a time (no AT100 double-phosphorylated tau produced by Akt alone). Prior Ser214 phosphorylation by Akt blocked subsequent PKA activity at that site, while GSK3β selectively blocked Ser214 phosphorylation.","method":"In vitro kinase assay with recombinant Akt and tau isoforms, phospho-site-specific antibodies, immunoprecipitation, mixed kinase assays","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution with rigorous site-specific antibody validation, single lab","pmids":["14636947"],"is_preprint":false},{"year":2001,"finding":"AKT/PKB promotes cancer cell invasion by increasing cell motility and MMP-9 production via NF-κB transcriptional activation; AKT/PKB localizes to the leading edge membrane of migrating cells in a PI3K- and PH-domain-dependent manner.","method":"Confocal live-cell imaging, invasion assays, MMP-9 production assays, NF-κB reporter assays, kinase-dead and PH-domain mutant AKT constructs, PI3K inhibitors","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — live-cell localization, domain mutagenesis, functional invasion and reporter assays, single lab","pmids":["11532975"],"is_preprint":false},{"year":2004,"finding":"AKT/PKB phosphorylates heme oxygenase-1 (HO-1) at Ser188 in vitro and in vivo; the phosphomimetic S188D mutant shows ~1.6-fold higher enzymatic activity and slightly altered binding to cytochrome P450 reductase and biliverdin reductase as measured by FRET.","method":"In vitro kinase assay with recombinant HO-1, site-directed mutagenesis (S188A, S188D), 32P metabolic labeling in HEK293T cells, FRET assays for protein-protein interactions, HO-1 enzymatic activity assay","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution + in vivo labeling + mutagenesis, but small effect size, single lab","pmids":["15581622"],"is_preprint":false},{"year":2006,"finding":"Akt1 phosphorylates FoxO3a to prevent its nuclear localization, thereby suppressing transactivation of the proapoptotic target gene Bim in osteoblasts; Akt1-deficient mice show low bone mass through increased osteoblast apoptosis via the Akt1/FoxO3a/Bim axis and decreased Runx2 transcriptional activity.","method":"Akt1 knockout mouse model, ex vivo osteoblast culture, apoptosis assays, FoxO3a nuclear localization by immunofluorescence, Bim expression analysis, Runx2 transcriptional activity assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO model with mechanistic follow-up, subcellular localization, transcriptional assays, single lab","pmids":["17957242"],"is_preprint":false},{"year":2006,"finding":"Akt1 has opposing roles to Akt2 in regulating cell migration and cytoskeletal organization: Akt1-deficient MEFs migrate more slowly and respond poorly to PDGF, while Akt2-deficient cells migrate faster. Akt2 inhibits Pak1 kinase activity in direct kinase assays; N-terminal PH domain and linker region distinguish the two isoforms' functions.","method":"Akt1/Akt2 knockout MEFs, cell migration assays, dorsal ruffling assays, domain-swap constructs between Akt1/Akt2, direct Pak1 kinase assays, Rac activity measurements","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cells, domain swaps, direct kinase assay for Pak1 inhibition by Akt2, single lab","pmids":["17012749"],"is_preprint":false},{"year":2003,"finding":"Akt/PKB directly binds the actin cytoskeleton via its N-terminal PH domain; PDGF stimulation increases the amount of Akt associated with the actin skeleton, and this association requires Ser473/Thr308 phosphorylation (abolished by S473A/T308A double mutant). Small GTPases Rac1 and Cdc42 facilitate actin binding.","method":"In situ cytoskeletal matrix preparations, co-immunoprecipitation, in vitro binding and overlay assays with recombinant proteins, subcellular fractionation, expression of Akt domain mutants","journal":"Cellular and molecular life sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — in vitro direct binding + co-IP + mutagenesis + fractionation, single lab","pmids":["14685694"],"is_preprint":false},{"year":2006,"finding":"Akt/PKB phosphorylates TopBP1 in vitro and in vivo; phosphorylation induces TopBP1 oligomerization through its 7th and 8th BRCT domains. Akt-dependent oligomerization is required for TopBP1 to interact with and repress E2F1 proapoptotic activity.","method":"In vitro kinase assay, site-directed mutagenesis, co-immunoprecipitation, TopBP1 oligomerization assays, E2F1 reporter assays, PI3K inhibitor treatment","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro phosphorylation, mutagenesis, oligomerization assay, functional E2F1 repression, single lab","pmids":["17006541"],"is_preprint":false},{"year":2000,"finding":"Caspases cleave AKT/PKB at three sites during apoptosis (TVAD108↓G, EEMD119↓F between PH and kinase domains, and SETD434↓T in the C-terminal regulatory domain), generating 40- and 44-kDa fragments; loss of the C-terminal domain reduces kinase activity and overexpression of the truncated fragment sensitizes cells to apoptosis.","method":"In vitro cleavage with purified caspases, caspase inhibitor rescue in cells, kinase activity assays of cleavage fragments, overexpression of truncated Akt constructs","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro reconstituted caspase cleavage, site identification, functional kinase assays, single lab","pmids":["10623893"],"is_preprint":false},{"year":2008,"finding":"Lipid raft nanodomains facilitate AKT/PKB recruitment and activation at the plasma membrane upon PI3K-generated PIP3 accumulation; specific inhibition of sphingolipid and cholesterol biosynthesis abolishes raft nanodomains and impairs AKT Ser473/Thr308 phosphorylation.","method":"Fluorescence correlation spectroscopy (FCS) in live cells, specific inhibition of sphingolipid and cholesterol biosynthesis, PIP3 measurement, phospho-AKT Western blotting","journal":"Nature chemical biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — novel FCS approach in live cells, pharmacological perturbation of rafts, single lab","pmids":["18641634"],"is_preprint":false},{"year":2008,"finding":"p53 stabilization in response to ionizing radiation requires AKT/PKB: DNA-PK is activated by ionizing radiation, phosphorylates and activates AKT/PKB, which in turn inactivates GSK-3; inactive GSK-3 fails to phosphorylate Mdm2 at sites required for p53 degradation, leading to p53 accumulation.","method":"Lymphoblasts from AT patients, ATR siRNA, AKT/PKB knockdown, DNA-PK knockdown, phospho-specific Western blotting of AKT, GSK-3, Mdm2, p53 accumulation assays","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genetic/siRNA perturbations, patient-derived cells, pathway epistasis, single lab","pmids":["18505846"],"is_preprint":false},{"year":2010,"finding":"Inhibition of AKT/PKB in late G2 cells restores DNA double-strand break processing (RPA, ATR, Rad51, CtIP recruitment to damage foci) and Chk1 checkpoint activation after irradiation; normally, AKT activity in late G2 suppresses DSB processing and prevents checkpoint activation.","method":"Chemical genetic Cdk1 inhibition to arrest cells in late G2, AKT inhibitor treatment, immunofluorescence for DSB repair factors (RPA, ATR, Rad51, CtIP, γ-H2AX), Chk1 phosphorylation Western blot","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chemical-genetic approach, multiple repair factor readouts, clean experimental design, single lab","pmids":["20679434"],"is_preprint":false},{"year":2011,"finding":"OX40 (CD134) assembles a signaling complex containing TRAF2, PI3K, and AKT/PKB upon ligation by OX40L; this complex forms in lipid microdomains independent of TCR engagement, but strong PI3K-AKT phosphorylation and functional activation only occur when antigen is also recognized, indicating OX40 augments TCR-induced AKT signaling quantitatively.","method":"Co-immunoprecipitation of OX40 complexes, detergent-resistant membrane fraction isolation, phospho-AKT Western blotting, TRAF2-deficient cells","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — reciprocal co-IP, lipid microdomain fractionation, genetic (TRAF2-KO) epistasis, single lab","pmids":["21289304"],"is_preprint":false},{"year":2007,"finding":"Pak1 (P21-activated kinase-1) directly phosphorylates AKT at Ser473 but not Thr308 in vitro; silencing or inactivating Pak1 reduces AKT Ser473 and Thr308 phosphorylation in cardiomyocytes, and Pak1 overexpression is cardioprotective in an AKT-dependent manner.","method":"In vitro kinase assay with purified Pak1 and AKT, Pak1 siRNA, dominant-negative Pak1, cardiomyocyte overexpression/knockdown, AKT inhibitor rescue","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase reconstitution, siRNA, dominant-negative, functional rescue — but potential conflict with mTORC2/DNA-PK as Ser473 kinase","pmids":["18054038"],"is_preprint":false},{"year":2019,"finding":"AKT/PKB promotes nuclear import of HDAC4 upon muscle denervation, enabling epigenetic changes and synaptic gene upregulation required for neuromuscular endplate remodeling; mTORC1 activation must be tightly balanced to allow dynamic autophagy regulation in denervated muscle.","method":"Muscle-specific transgenic mice with constitutive mTORC1 activation or AKT inhibition, HDAC4 nuclear localization assays, autophagy flux assays, NMJ morphology analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo mouse genetic models, subcellular localization assay, functional NMJ readout, single lab","pmids":["31320633"],"is_preprint":false},{"year":2007,"finding":"In mouse oocytes, Thr308-phosphorylated AKT localizes to pericentriolar materials while Ser473-phosphorylated AKT co-distributes with spindle microtubules; both are required for metaphase II spindle assembly. Ser473-phosphorylated AKT is specifically required for second polar body emission, while Thr308-phosphorylated AKT regulates microtubule organization during meiosis.","method":"Antibody injection into oocytes, immunofluorescence with phospho-specific antibodies, spindle morphology analysis, polar body emission assay","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — antibody injection (function-blocking), localization, distinct phenotypes for each phosphorylation site, single lab","pmids":["18177853"],"is_preprint":false},{"year":2011,"finding":"Inositol polyphosphate multikinase (IPMK) acts as a physiological PI3-kinase that generates PIP3 and activates AKT/PKB; IPMK deletion reduces growth factor-elicited AKT signaling uniquely through loss of its PI3K activity, and p110 PI3-kinases must act first to phosphorylate/activate IPMK in a sequential PIP3-generating cascade.","method":"IPMK knockout cells, in vitro PI3K activity assay, wortmannin inhibition, growth factor stimulation, AKT phosphorylation assays, cell proliferation assays","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro PI3K assay, genetic KO, pharmacological validation, single lab","pmids":["21220345"],"is_preprint":false},{"year":2011,"finding":"CK2-mediated phosphorylation of AKT at Ser129 is necessary for AKT-dependent upregulation of β-catenin transcriptional activity and nuclear localization; CK2α overexpression increased β-catenin activity and survivin expression, and these effects were abolished by expression of an AKT-S129A mutant.","method":"Overexpression of wild-type, constitutively active, and dominant-negative CK2 and AKT, AKT-S129A mutant, β-catenin transcriptional reporter, nuclear fractionation, survivin expression assay","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — mutagenesis, reporter assay, functional viability readout, extends PMID:15818404 mechanistically, single lab","pmids":["21506126"],"is_preprint":false},{"year":2000,"finding":"In platelets activated through integrin αIIbβ3, PtdIns(3,4)P2 that activates AKT/PKB is generated primarily via a novel pathway: PI3K generates PtdIns3P, which is then phosphorylated by PtdIns3P 4-kinase — not by 5'-phosphatase hydrolysis of PtdIns(3,4,5)P3, which is not detected in this context. Wortmannin and calpain inhibitor both block this pathway and AKT activation.","method":"Lipid analysis (PtdIns3P, PtdIns(3,4)P2, PtdIns(3,4,5)P3 measurement), wortmannin/calpeptin inhibition, PKB kinase assay in human platelets, integrin activation by anti-LIBS Fab","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lipid mass measurement, pharmacological dissection, in vitro kinase assay, single lab","pmids":["9417038"],"is_preprint":false}],"current_model":"AKT1/PKB is a pleckstrin-homology-domain-containing serine/threonine kinase activated by a sequential multi-step mechanism: PI3K-generated PIP3 recruits AKT to the plasma membrane via its PH domain (which also undergoes an intramolecular inhibitory interaction with the kinase domain in the resting state); PDK1 phosphorylates the activation-loop Thr308, while mTORC2 (requiring rictor and mSin1 for assembly) is the primary Ser473 hydrophobic-motif kinase — with DNA-PK and ATM also contributing in a stimulus-dependent manner, and Ack1 providing activating Tyr176 phosphorylation downstream of RTKs; additional regulatory phosphorylation by CK2 at Ser129 further enhances catalytic activity; active AKT phosphorylates a broad substrate network including FOXO transcription factors (cytoplasmic retention), GSK3β (inactivation), p27Kip1-Thr157 (nuclear exclusion), p21Cip1-Thr145/Ser146 (stabilization and PCNA uncoupling), CREB-Ser133 (transcriptional activation), Girdin/APE-Ser1416 (actin organization and cell migration), Skp2 (SCF E3 ligase activation and cytoplasmic relocalization), TopBP1 (oligomerization and E2F1 repression), Smad3 (kinase-independent sequestration blocking TGF-β signaling), and tau-Thr212/Ser214; AKT can also bind JIP1 to prevent JNK scaffold assembly, promote HDAC4 nuclear import in denervated muscle, and regulate p53 stability via GSK3β/Mdm2; its activity is negatively regulated by PTEN-mediated PIP3 dephosphorylation and by caspase cleavage during apoptosis."},"narrative":{"mechanistic_narrative":"AKT1/PKB is a plasma-membrane-recruited serine/threonine kinase that serves as the central effector of PI3K signaling, converting growth-factor and insulin inputs into pro-survival, growth, metabolic, and migratory outputs [PMID:10872470, PMID:9716402]. Its activation follows an ordered, multi-step mechanism: PI3K-generated 3'-phosphoinositides bind the AKT PH domain to drive plasma-membrane translocation and co-localization with PDK1, which phosphorylates the activation-loop Thr308 [PMID:10872470, PMID:10226029], while the rictor-mTOR complex (mTORC2), whose assembly requires mSin1, is the hydrophobic-motif Ser473 kinase that facilitates full activation [PMID:15718470, PMID:16919458]. In the resting state AKT is held in a closed, autoinhibited conformation via an intramolecular PH-domain–kinase-domain interaction that blocks activation-loop phosphorylation [PMID:19166270]; membrane recruitment within lipid raft nanodomains and additional inputs relieve this constraint [PMID:18641634]. Beyond mTORC2, Ser473 phosphorylation is contributed by DNA-PK and ATM in a stimulus-dependent manner [PMID:15262962, PMID:15546863], Ack1 supplies an activating Tyr176 phosphorylation downstream of RTKs [PMID:20333297], and CK2 phosphorylation at Ser129 further enhances catalytic output [PMID:15818404, PMID:21506126]. Active AKT recognizes the consensus motif RxRxxS/T [PMID:10945990] and phosphorylates a broad substrate network that executes its biological programs: it inactivates FOXO/DAF-16 transcription factors to suppress pro-apoptotic genes such as Bim [PMID:9716402, PMID:17957242], drives cytoplasmic retention or stabilization of the CDK inhibitors p27Kip1 and p21Cip1 [PMID:12244302, PMID:12244301, PMID:11756412], activates CREB- and NF-κB-dependent transcription [PMID:9829964, PMID:10359702], and phosphorylates cytoskeletal and migratory effectors including Girdin and Skp2 [PMID:16139227, PMID:19270694]. AKT also acts through kinase-independent protein binding, sequestering Smad3 to block TGF-β-induced apoptosis [PMID:15048128] and binding JIP1 to restrain JNK activation in neurons [PMID:12194869], and it controls cell and organ size cell-autonomously downstream of the insulin/PI3K axis [PMID:10587646]. AKT signaling is terminated by PTEN-mediated dephosphorylation of PIP3 and by caspase cleavage during apoptosis [PMID:9778245, PMID:10623893].","teleology":[{"year":1998,"claim":"Establishing how AKT activity is negatively gated, PTEN was shown to be the phosphatase that removes the PIP3 signal driving AKT, defining the off-switch of the pathway.","evidence":"In vitro lipid phosphatase assay, PIP3 measurement, and PTEN re-expression rescue in PTEN-deficient MEFs","pmids":["9778245"],"confidence":"High","gaps":["Does not address how PIP3 is generated","Does not resolve which AKT-activating kinases act downstream"]},{"year":1999,"claim":"Genetic epistasis in C. elegans and Drosophila placed AKT downstream of insulin/PI3K and defined its core physiological outputs — antagonizing FOXO/DAF-16 transcription and controlling cell-autonomous growth.","evidence":"daf-16 suppression of akt-1/akt-2 mutants in C. elegans; clonal mosaic cell-size analysis in Drosophila imaginal discs","pmids":["9716402","10587646"],"confidence":"High","gaps":["Does not define the biochemical activation steps","Substrate phosphorylation sites in mammals not addressed"]},{"year":1999,"claim":"Resolving the membrane-recruitment step, the AKT PH domain was shown to bind PI3K-generated 3'-phosphoinositides and localize AKT to the membrane where PDK1 phosphorylates Thr308, defining the multi-step AGC-kinase activation paradigm.","evidence":"Phosphoinositide binding assays, GFP-PH live-cell imaging, PI3K inhibitor and binding-mutant controls","pmids":["10872470","10226029"],"confidence":"High","gaps":["Identity of the Ser473 hydrophobic-motif kinase unresolved at this stage","Mechanism of resting-state autoinhibition not defined"]},{"year":2000,"claim":"Defining substrate selectivity, the optimal RxRxxS/T consensus motif was determined, enabling prediction and validation of AKT substrates.","evidence":"Oriented peptide library screening with cDNA expression-library validation and kinetic analysis","pmids":["10945990"],"confidence":"High","gaps":["Does not identify which physiological substrates are phosphorylated in vivo","Context-dependent substrate selection not addressed"]},{"year":2001,"claim":"Connecting AKT to cell-cycle control, AKT was shown to phosphorylate the CDK inhibitors p21Cip1 and p27Kip1 to alter their localization and stability, promoting proliferation and survival.","evidence":"In vitro kinase assays, T145/S146 and T157A mutagenesis, nuclear import reconstitution, and PCNA-binding assays","pmids":["11756412","12244302","12244301"],"confidence":"High","gaps":["In vivo contribution to tumor proliferation not quantified","Crosstalk with other p21/p27 kinases not resolved"]},{"year":2004,"claim":"Expanding AKT beyond kinase activity, AKT was shown to physically sequester Smad3 in a kinase-independent manner, defining a non-catalytic mechanism for suppressing TGF-β-induced apoptosis.","evidence":"Protein-fragment complementation, co-IP, AKT siRNA rescue, and Smad3 reporter assays","pmids":["15048128"],"confidence":"High","gaps":["Structural basis of the AKT-Smad3 interface unknown","Stoichiometry and regulation of the complex not defined"]},{"year":2004,"claim":"Addressing the long-standing Ser473 kinase question, DNA-PK and ATM were each implicated as stimulus-dependent contributors to AKT Ser473 phosphorylation.","evidence":"DNA-PK purification with in vitro phosphorylation and PRKDC complementation; ATM inhibitors/siRNA and AT-patient/KO cells","pmids":["15262962","15546863"],"confidence":"Medium","gaps":["Relative contribution versus mTORC2 across stimuli not quantified","Whether these act on membrane-recruited AKT or nuclear AKT unclear"]},{"year":2005,"claim":"Identifying the principal hydrophobic-motif kinase, mTORC2 (rictor-mTOR) was shown to directly phosphorylate AKT Ser473 in vitro and facilitate Thr308 phosphorylation, completing the canonical activation model.","evidence":"Reconstituted in vitro kinase assay with purified rictor-mTOR plus rictor/mTOR knockdown in Drosophila and human cells","pmids":["15718470"],"confidence":"High","gaps":["Does not reconcile multiplicity of reported Ser473 kinases","Spatial coordination of mTORC2 with membrane AKT not addressed"]},{"year":2005,"claim":"Adding a tuning input, CK2 was shown to phosphorylate already-active AKT at Ser129 to further enhance catalytic activity and downstream β-catenin signaling.","evidence":"In vitro CK2 kinase assay, two structurally distinct inhibitors plus siRNA; later S129A mutant analysis of β-catenin/survivin","pmids":["15818404","21506126"],"confidence":"High","gaps":["Physiological stimuli engaging CK2-AKT crosstalk not defined","Quantitative contribution to overall AKT output unclear"]},{"year":2006,"claim":"Defining the mTORC2 assembly requirement, mSin1 was identified as an essential subunit needed for mTORC2 to phosphorylate AKT Ser473, and prolonged rapamycin was shown to disrupt mTORC2 and reduce AKT signaling.","evidence":"Co-IP, mSin1 siRNA, in vitro mTORC2 kinase assay, mass spec isoform mapping; mTORC2 assembly assays with rapamycin-resistant AKT rescue","pmids":["16919458","16603397"],"confidence":"High","gaps":["Functional distinction among mSin1 isoform complexes not fully resolved","How rapamycin selectively disrupts assembly mechanistically unclear"]},{"year":2009,"claim":"Revealing the basis of resting-state inhibition, AKT was shown to adopt a closed PH-domain–kinase-domain conformation that blocks Thr308 phosphorylation and is exploited by isoform-selective allosteric inhibitors.","evidence":"Molecular modeling, FRET/two-photon FLIM, biochemical kinase assays, and domain mutagenesis","pmids":["19166270"],"confidence":"High","gaps":["High-resolution structure of the autoinhibited state not provided","How PIP3 binding kinetically relieves the closed state not detailed"]},{"year":2009,"claim":"Linking AKT to ubiquitin-ligase control and migration, AKT was shown to phosphorylate Skp2 (and earlier Girdin), driving SCF activity, 14-3-3-dependent cytoplasmic relocalization, and cell motility.","evidence":"In vitro kinase assays, phospho-defective mutants, ubiquitylation and migration assays, human tumor specimen analysis","pmids":["19270694","16139227"],"confidence":"High","gaps":["In vivo metastatic relevance of these substrates not established","Isoform specificity of cytoskeletal substrate phosphorylation not resolved"]},{"year":2010,"claim":"Adding tyrosine-kinase input, Ack1 was shown to phosphorylate AKT at Tyr176 to promote membrane localization and prime activation-loop/hydrophobic-motif phosphorylation downstream of RTKs.","evidence":"In vitro phosphorylation, Y176F mutagenesis, fractionation, and an Ack1-driven prostate cancer mouse model","pmids":["20333297"],"confidence":"High","gaps":["Generality across RTK contexts not established","Structural effect of Tyr176 phosphorylation on conformation unclear"]},{"year":null,"claim":"How the multiple reported Ser473 kinases (mTORC2, DNA-PK, ATM, Pak1) are deployed across distinct stimuli, subcellular compartments, and cell types — and how isoform-specific (AKT1 vs AKT2) substrate choice is achieved — remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model reconciling competing Ser473 kinases","Mechanistic basis of AKT1/AKT2 functional divergence incompletely defined","Compartment-specific substrate engagement not systematically mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,8,9,11,14,16]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[4,22,31]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[6,17,26]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,5,22,31]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[11,28]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[16]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4,17]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[8,9]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[10,26,30]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[6,17]}],"complexes":["mTORC2"],"partners":["MTOR","RICTOR","MAPKSIN1","PDPK1","PTEN","SMAD3","JIP1","ACK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P31749","full_name":"RAC-alpha serine/threonine-protein kinase","aliases":["Protein kinase B","PKB","Protein kinase B alpha","PKB alpha","Proto-oncogene c-Akt","RAC-PK-alpha"],"length_aa":480,"mass_kda":55.7,"function":"AKT1 is one of 3 closely related serine/threonine-protein kinases (AKT1, AKT2 and AKT3) called the AKT kinase, and which regulate many processes including metabolism, proliferation, cell survival, growth and angiogenesis (PubMed:11882383, PubMed:15526160, PubMed:15861136, PubMed:21432781, PubMed:21620960, PubMed:31204173). This is mediated through serine and/or threonine phosphorylation of a range of downstream substrates (PubMed:11882383, PubMed:15526160, PubMed:21432781, PubMed:21620960, PubMed:29343641, PubMed:31204173). Over 100 substrate candidates have been reported so far, but for most of them, no isoform specificity has been reported (PubMed:11882383, PubMed:15526160, PubMed:21432781, PubMed:21620960). AKT is responsible of the regulation of glucose uptake by mediating insulin-induced translocation of the SLC2A4/GLUT4 glucose transporter to the cell surface (By similarity). Phosphorylation of PTPN1 at 'Ser-50' negatively modulates its phosphatase activity preventing dephosphorylation of the insulin receptor and the attenuation of insulin signaling (By similarity). Phosphorylation of TBC1D4 triggers the binding of this effector to inhibitory 14-3-3 proteins, which is required for insulin-stimulated glucose transport (PubMed:11994271). AKT also regulates the storage of glucose in the form of glycogen by phosphorylating GSK3A at 'Ser-21' and GSK3B at 'Ser-9', resulting in inhibition of its kinase activity (By similarity). Phosphorylation of GSK3 isoforms by AKT is also thought to be one mechanism by which cell proliferation is driven (By similarity). AKT also regulates cell survival via the phosphorylation of MAP3K5 (apoptosis signal-related kinase) (PubMed:11154276). Phosphorylation of 'Ser-83' decreases MAP3K5 kinase activity stimulated by oxidative stress and thereby prevents apoptosis (PubMed:11154276). AKT mediates insulin-stimulated protein synthesis by phosphorylating TSC2 at 'Ser-939' and 'Thr-1462', thereby activating the mTORC1 signaling pathway, and leading to both phosphorylation of 4E-BP1 and in activation of RPS6KB1 (PubMed:12150915, PubMed:12172553). Also regulates the mTORC1 signaling pathway by catalyzing phosphorylation of CASTOR1 and DEPDC5 (PubMed:31548394, PubMed:33594058). AKT plays a role as key modulator of the AKT-mTOR signaling pathway controlling the tempo of the process of newborn neurons integration during adult neurogenesis, including correct neuron positioning, dendritic development and synapse formation (By similarity). Part of a positive feedback loop of mTORC2 signaling by mediating phosphorylation of MAPKAP1/SIN1, promoting mTORC2 activation (By similarity). AKT is involved in the phosphorylation of members of the FOXO factors (Forkhead family of transcription factors), leading to binding of 14-3-3 proteins and cytoplasmic localization (PubMed:10358075). In particular, FOXO1 is phosphorylated at 'Thr-24', 'Ser-256' and 'Ser-319' (PubMed:10358075). FOXO3 and FOXO4 are phosphorylated on equivalent sites (PubMed:10358075). AKT has an important role in the regulation of NF-kappa-B-dependent gene transcription and positively regulates the activity of CREB1 (cyclic AMP (cAMP)-response element binding protein) (PubMed:9829964). The phosphorylation of CREB1 induces the binding of accessory proteins that are necessary for the transcription of pro-survival genes such as BCL2 and MCL1 (PubMed:9829964). AKT phosphorylates 'Ser-454' on ATP citrate lyase (ACLY), thereby potentially regulating ACLY activity and fatty acid synthesis (By similarity). Activates the 3B isoform of cyclic nucleotide phosphodiesterase (PDE3B) via phosphorylation of 'Ser-273', resulting in reduced cyclic AMP levels and inhibition of lipolysis (By similarity). Phosphorylates PIKFYVE on 'Ser-318', which results in increased PI(3)P-5 activity (By similarity). The Rho GTPase-activating protein DLC1 is another substrate and its phosphorylation is implicated in the regulation cell proliferation and cell growth (By similarity). Signals downstream of phosphatidylinositol 3-kinase (PI(3)K) to mediate the effects of various growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin and insulin-like growth factor 1 (IGF1) (PubMed:12176338, PubMed:12964941). AKT mediates the antiapoptotic effects of IGF1 (By similarity). Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly (PubMed:19934221). May be involved in the regulation of the placental development (By similarity). Phosphorylates STK4/MST1 at 'Thr-120' and 'Thr-387' leading to inhibition of its: kinase activity, nuclear translocation, autophosphorylation and ability to phosphorylate FOXO3 (PubMed:17726016). Phosphorylates STK3/MST2 at 'Thr-117' and 'Thr-384' leading to inhibition of its: cleavage, kinase activity, autophosphorylation at Thr-180, binding to RASSF1 and nuclear translocation (PubMed:20086174). Phosphorylates SRPK2 and enhances its kinase activity towards SRSF2 and ACIN1 and promotes its nuclear translocation (PubMed:19592491). Phosphorylates RAF1 at 'Ser-259' and negatively regulates its activity (PubMed:10576742). Phosphorylation of BAD stimulates its pro-apoptotic activity (PubMed:10926925). Phosphorylates KAT6A at 'Thr-369' and this phosphorylation inhibits the interaction of KAT6A with PML and negatively regulates its acetylation activity towards p53/TP53 (PubMed:23431171). Phosphorylates palladin (PALLD), modulating cytoskeletal organization and cell motility (PubMed:20471940). Phosphorylates prohibitin (PHB), playing an important role in cell metabolism and proliferation (PubMed:18507042). Phosphorylates CDKN1A, for which phosphorylation at 'Thr-145' induces its release from CDK2 and cytoplasmic relocalization (PubMed:16982699). These recent findings indicate that the AKT1 isoform has a more specific role in cell motility and proliferation (PubMed:16139227). Phosphorylates CLK2 thereby controlling cell survival to ionizing radiation (PubMed:20682768). Phosphorylates PCK1 at 'Ser-90', reducing the binding affinity of PCK1 to oxaloacetate and changing PCK1 into an atypical protein kinase activity using GTP as donor (PubMed:32322062). Also acts as an activator of TMEM175 potassium channel activity in response to growth factors: forms the lysoK(GF) complex together with TMEM175 and acts by promoting TMEM175 channel activation, independently of its protein kinase activity (PubMed:32228865). Acts as a regulator of mitochondrial calcium uptake by mediating phosphorylation of MICU1 in the mitochondrial intermembrane space, impairing MICU1 maturation (PubMed:30504268). Acts as an inhibitor of tRNA methylation by mediating phosphorylation of the N-terminus of METTL1, thereby inhibiting METTL1 methyltransferase activity (PubMed:15861136). In response to LPAR1 receptor pathway activation, phosphorylates Rabin8/RAB3IP which alters its activity and phosphorylates WDR44 which induces WDR44 binding to Rab11, thereby switching Rab11 vesicular function from preciliary trafficking to endocytic recycling (PubMed:31204173)","subcellular_location":"Cytoplasm; Nucleus; Cell membrane; Mitochondrion intermembrane space","url":"https://www.uniprot.org/uniprotkb/P31749/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AKT1","classification":"Not Classified","n_dependent_lines":52,"n_total_lines":1208,"dependency_fraction":0.04304635761589404},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000142208","cell_line_id":"CID000618","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"membrane","grade":3},{"compartment":"nucleoplasm","grade":3},{"compartment":"vesicles","grade":2},{"compartment":"cell_contact","grade":1}],"interactors":[{"gene":"RPS6KB2;RPS6KB1","stoichiometry":4.0},{"gene":"VASP","stoichiometry":0.2},{"gene":"GPHN","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000618","total_profiled":1310},"omim":[{"mim_id":"621547","title":"ZDHHC PALMITOYLTRANSFERASE 4; ZDHHC4","url":"https://www.omim.org/entry/621547"},{"mim_id":"621477","title":"TORSIN FAMILY 4, MEMBER A; TOR4A","url":"https://www.omim.org/entry/621477"},{"mim_id":"621185","title":"HOUGE-JANSSENS SYNDROME 4; HJS4","url":"https://www.omim.org/entry/621185"},{"mim_id":"620997","title":"SEMAPHORIN 3G; SEMA3G","url":"https://www.omim.org/entry/620997"},{"mim_id":"620805","title":"ISOCHORISMATASE DOMAIN-CONTAINING PROTEIN 1; ISOC1","url":"https://www.omim.org/entry/620805"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Microtubules","reliability":"Approved"},{"location":"Principal piece","reliability":"Approved"},{"location":"End piece","reliability":"Approved"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"},{"location":"Perinuclear theca","reliability":"Additional"},{"location":"Calyx","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/AKT1"},"hgnc":{"alias_symbol":["RAC","PKB","PRKBA","AKT","RAC-alpha"],"prev_symbol":[]},"alphafold":{"accession":"P31749","domains":[{"cath_id":"2.30.29.30","chopping":"5-115","consensus_level":"high","plddt":82.3391,"start":5,"end":115},{"cath_id":"3.30.200.20","chopping":"146-229_430-450","consensus_level":"high","plddt":86.0435,"start":146,"end":450},{"cath_id":"1.10.510.10","chopping":"234-417","consensus_level":"high","plddt":91.9943,"start":234,"end":417}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P31749","model_url":"https://alphafold.ebi.ac.uk/files/AF-P31749-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P31749-F1-predicted_aligned_error_v6.png","plddt_mean":83.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=AKT1","jax_strain_url":"https://www.jax.org/strain/search?query=AKT1"},"sequence":{"accession":"P31749","fasta_url":"https://rest.uniprot.org/uniprotkb/P31749.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P31749/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P31749"}},"corpus_meta":[{"pmid":"15718470","id":"PMC_15718470","title":"Phosphorylation 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this mechanism underlies some proapoptotic and antitumor effects of rapamycin.\",\n      \"method\": \"mTORC2 assembly assays, Western blotting of phospho-AKT in multiple cell lines after prolonged rapamycin, rescue with rapamycin-resistant AKT mutant\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal biochemical assays, genetic rescue experiment, multiple cell types tested\",\n      \"pmids\": [\"16603397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"mSin1 is an essential component of mTORC2 required for mTORC2 assembly and its capacity to phosphorylate AKT/PKB on Ser473; alternative splicing generates at least three distinct mTORC2 complexes, only two of which are regulated by insulin.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown of mSin1, in vitro mTORC2 kinase assay, mass spectrometry identification of mSin1 isoforms\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro reconstitution of kinase activity, reciprocal co-IP, siRNA loss-of-function, multiple orthogonal methods\",\n      \"pmids\": [\"16919458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PTEN negatively regulates AKT/PKB activity and phosphorylation by dephosphorylating phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in vitro and in vivo; PTEN-deficient cells show constitutively elevated AKT activity and resistance to apoptosis, which is reversed by re-expression of wild-type PTEN.\",\n      \"method\": \"In vitro lipid phosphatase assay, genetic rescue (PTEN re-expression in mutant cells), measurement of PI(3,4,5)P3 levels, PKB kinase assays in PTEN-deficient MEFs\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro phosphatase assay, genetic rescue, lipid measurements, replicated across labs\",\n      \"pmids\": [\"9778245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PI3K-generated D3-phosphoinositides bind the AKT PH domain, inducing plasma membrane translocation and co-localization with PDK1, which then phosphorylates the AKT activation loop (Thr308); this multi-step activation mechanism is shared with other AGC kinases.\",\n      \"method\": \"Phosphoinositide binding assays, subcellular fractionation, kinase assays, domain mutagenesis\",\n      \"journal\": \"Annual review of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — biochemical reconstitution, domain mutagenesis, widely replicated paradigm\",\n      \"pmids\": [\"10872470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"DNA-dependent protein kinase (DNA-PK) was purified as a major Ser473 kinase from HEK293 cells: it co-localizes and associates with AKT at the plasma membrane, phosphorylates AKT on Ser473 in vitro (~10-fold activation), and siRNA knockdown of DNA-PK inhibits insulin- and pervanadate-induced Ser473 phosphorylation; DNA-PK-deficient cells fail to phosphorylate Ser473 in response to insulin, restored by PRKDC complementation.\",\n      \"method\": \"Biochemical purification, in vitro kinase assay, siRNA knockdown, genetic complementation in DNA-PK-deficient cells, co-localization by microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — purification + reconstitution, siRNA, genetic complementation — multiple orthogonal methods in one study\",\n      \"pmids\": [\"15262962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"AKT/PKB directly phosphorylates CREB at Ser133 in serum-stimulated cells, promoting CBP recruitment and CREB-dependent target gene expression; this effect requires AKT kinase activity and is PI3K-dependent.\",\n      \"method\": \"Overexpression of AKT in serum-stimulated cells, phospho-Ser133 CREB detection, CBP co-immunoprecipitation, reporter gene assays, PI3K inhibitor (LY294002)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — cellular phosphorylation assay + functional reporter, single lab, two orthogonal methods\",\n      \"pmids\": [\"9829964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"AKT/PKB activation of NF-κB occurs at the level of IκB degradation and requires AKT kinase activity and its PH domain; AKT does not act alone but cooperates with other pathway signals to induce NF-κB-dependent cytokine promoters.\",\n      \"method\": \"Overexpression of kinase-dead and PH-domain mutant AKT in Jurkat cells, NF-κB reporter assays, IκB degradation assays\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — kinase-dead and domain mutagenesis, reporter assays, single lab\",\n      \"pmids\": [\"10359702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"AKT/PKB phosphorylates p27Kip1 at Thr157 within its nuclear localization signal, impairing nuclear import of p27 and causing cytoplasmic retention; cells expressing the T157A mutant p27 are refractory to AKT-mediated nuclear exclusion and retain G1-arrest activity.\",\n      \"method\": \"In vitro kinase assay with recombinant AKT and p27, site-directed mutagenesis (T157A), subcellular fractionation/immunofluorescence, CDK2 activity assays, in vitro nuclear import assays\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay, mutagenesis, nuclear import reconstitution, independently replicated in companion paper (PMID:12244301)\",\n      \"pmids\": [\"12244302\", \"12244301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"AKT/PKB directly phosphorylates p21Cip1/WAF1 at Thr145 and Ser146; Thr145 phosphorylation inhibits PCNA binding, while Ser146 phosphorylation stabilizes p21 protein, enhancing cell survival and CDK4/cyclin D1 complex assembly.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, protein stability assays, co-immunoprecipitation for PCNA binding, kinase activity assays in glioblastoma cell lines\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro phosphorylation + mutagenesis of both sites + functional readouts in one rigorous study\",\n      \"pmids\": [\"11756412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PKB/AKT physically interacts with Smad3 through a kinase-activity-independent mechanism; insulin induces complex formation while TGF-β inhibits it. AKT inhibits Smad3 by preventing its phosphorylation, Smad4 binding, and nuclear translocation, thereby suppressing TGF-β-induced apoptosis.\",\n      \"method\": \"Protein fragment complementation assay (PCA) in live cells, co-immunoprecipitation, siRNA knockdown of AKT, reporter assays for Smad3-dependent transcription, phosphorylation assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — PCA interaction assay, co-IP, siRNA rescue, functional reporter, multiple orthogonal methods\",\n      \"pmids\": [\"15048128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AKT/PKB phosphorylates Girdin/APE at Ser1416; phosphorylated Girdin accumulates at the leading edge of migrating cells and is required for stress fiber/lamellipodia formation and cell migration. Cells expressing S1416A-Girdin show impaired migration and lamellipodia formation.\",\n      \"method\": \"Identification of Girdin as AKT substrate by mass spectrometry, in vitro kinase assay, site-directed mutagenesis (S1416A), immunofluorescence of phospho-Girdin localization, cell migration assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro phosphorylation, mutagenesis, live-cell localization, functional migration assay — multiple orthogonal methods\",\n      \"pmids\": [\"16139227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Protein kinase CK2 phosphorylates AKT/PKB at Ser129 in vitro and in vivo; this phosphorylation of already-activated AKT further increases its catalytic activity, and CK2 inhibition (by two structurally distinct inhibitors or siRNA) reduces AKT activity and promotes apoptosis.\",\n      \"method\": \"In vitro kinase assay with CK2 and AKT, 32P incorporation, CK2 siRNA, two structurally distinct CK2 inhibitors, immunoprecipitate kinase assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro reconstitution, siRNA, two independent inhibitors confirming same result\",\n      \"pmids\": [\"15818404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The non-receptor tyrosine kinase Ack1 directly phosphorylates AKT at the evolutionarily conserved Tyr176 in the kinase domain; Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes subsequent Thr308/Ser473 phosphorylation and AKT activation downstream of RTKs.\",\n      \"method\": \"Direct in vitro phosphorylation assay, site-directed mutagenesis (Y176F), subcellular fractionation, phospho-specific antibodies, mouse prostate cancer model with activated Ack1\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro reconstitution, mutagenesis, in vivo mouse model, phospho-specific antibody validation\",\n      \"pmids\": [\"20333297\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"AKT/PKB optimal phosphorylation consensus motif was defined as Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr) with Arg at −3, −5, and −7 being most critical for high Vmax/Km; this was validated against a cDNA library screen of HeLa cell substrates.\",\n      \"method\": \"Oriented peptide library screening, cDNA expression library screening (λGEX phage), kinetic analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution with peptide library + independent cDNA library validation, two orthogonal approaches\",\n      \"pmids\": [\"10945990\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"AKT/PKB exists in an inactive closed conformation through an intramolecular interaction between its PH domain and kinase domain that prevents PDK1 phosphorylation of the activation loop; allosteric inhibitor AKT inhibitor VIII stabilizes this closed conformation and shows isoform selectivity based on differences at the PH-kinase domain interface.\",\n      \"method\": \"Molecular modeling, FRET/two-photon FLIM, classical biochemical kinase assays, domain mutagenesis\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — FRET/FLIM structural measurements, mutagenesis, biochemical assays — multiple orthogonal approaches in one study\",\n      \"pmids\": [\"19166270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"AKT/PKB directly phosphorylates Skp2, triggering SCF complex formation and E3 ligase activity; AKT-mediated phosphorylation also induces 14-3-3β-dependent cytoplasmic relocalization of Skp2, where cytosolic Skp2 promotes cell migration.\",\n      \"method\": \"Co-immunoprecipitation, direct in vitro kinase assay, phosphorylation-defective mutant (Skp2-S72A), ubiquitylation assays, subcellular fractionation, cell migration assays, analysis of human cancer specimens\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay, mutagenesis, 14-3-3β interaction, functional assays, human tissue validation\",\n      \"pmids\": [\"19270694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"In C. elegans, Akt/PKB homologs AKT-1 and AKT-2 transduce insulin receptor-like signals from AGE-1 PI3K downstream; loss-of-function of DAF-16 (Forkhead transcription factor) suppresses the akt-1/akt-2 requirement, establishing that AKT/PKB functions primarily to antagonize DAF-16 transcription factor activity.\",\n      \"method\": \"Genetic epistasis (daf-16 suppression of akt-1/akt-2 mutant phenotype), gain-of-function akt-1 screen, overexpression dosage experiments, expression pattern analysis in C. elegans\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis in C. elegans with suppressor analysis, highly replicated and foundational for the AKT-FOXO pathway\",\n      \"pmids\": [\"9716402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ATM kinase is required for full AKT/PKB activation at Ser473 in response to insulin or gamma-radiation; this effect is mediated through the PI3K-like kinase domain of ATM and affects downstream AKT targets (Forkhead transcription factors) in ATM-deficient cells.\",\n      \"method\": \"Transient transfection, ATM inhibitors, ATM siRNA, cell lines from AT patients and ATM knockout mice, Ser473-specific phosphorylation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA + pharmacological inhibitors + genetic model (AT patient cells and KO mice), single lab\",\n      \"pmids\": [\"15546863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"AKT1 directly associates with JIP1 (JNK-interacting protein 1/scaffold) in primary neurons; this interaction inhibits JIP1-mediated potentiation of JNK activity by decreasing JIP1 binding to specific JNK pathway kinases. Excitotoxic stimuli disrupt the Akt1-JIP1 interaction, releasing JNK activation, and Akt1-deficient neurons show enhanced kainate susceptibility.\",\n      \"method\": \"Co-immunoprecipitation, direct binding assay, kinase assays, Akt1-deficient mouse neurons, overexpression of Akt1 mutants\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct protein interaction, kinase assay, KO mouse neurons, functional apoptosis readout — multiple orthogonal methods\",\n      \"pmids\": [\"12194869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Akt2/PKBβ phosphorylates and inhibits PGC-1α, a global regulator of hepatic metabolism during fasting; phosphorylation prevents PGC-1α recruitment to cognate promoters, impairing gluconeogenesis and fatty acid oxidation programs.\",\n      \"method\": \"In vitro phosphorylation assay, chromatin immunoprecipitation, reporter assays, hepatocyte-specific Akt2 knockout, metabolic phenotyping\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro phosphorylation, ChIP, genetic KO with metabolic readout; primarily Akt2 isoform but mechanistically relevant to AKT family\",\n      \"pmids\": [\"17554339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"In Drosophila, Akt/PKB regulates cell and organ size in a cell-autonomous manner downstream of the insulin receptor/Chico/Dp110 (PI3K) pathway; ectopic Akt expression increases cell size without affecting apoptosis, cell fate, or proliferation rates.\",\n      \"method\": \"Drosophila genetic mosaic analysis, imaginal disc clonal analysis, gain- and loss-of-function transgenes, cell size measurement\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic epistasis in Drosophila, cell-autonomous clonal analysis, multiple transgenic lines\",\n      \"pmids\": [\"10587646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"AKT/PKB PH domain binds PI3K-generated 3'-phosphoinositides and localizes to membrane ruffles upon mitogen treatment; in epithelial cells, AKT PH domain localizes to sites of cell-cell and cell-matrix contact (distinct from focal contacts) in a PI3K-activity-dependent and phosphoinositide-binding-dependent manner, providing a constitutive survival signal.\",\n      \"method\": \"GFP-Akt and GFP-PH domain fusion live-cell imaging, immunofluorescence microscopy, PI3K inhibitor treatment, PH domain phosphoinositide-binding mutants\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell localization with GFP fusions, pharmacological and mutagenesis controls, functional survival link established\",\n      \"pmids\": [\"10226029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Akt/PKB phosphorylates tau at Thr212 and Ser214 independently in vitro; only one site per molecule is phosphorylated at a time (no AT100 double-phosphorylated tau produced by Akt alone). Prior Ser214 phosphorylation by Akt blocked subsequent PKA activity at that site, while GSK3β selectively blocked Ser214 phosphorylation.\",\n      \"method\": \"In vitro kinase assay with recombinant Akt and tau isoforms, phospho-site-specific antibodies, immunoprecipitation, mixed kinase assays\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution with rigorous site-specific antibody validation, single lab\",\n      \"pmids\": [\"14636947\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"AKT/PKB promotes cancer cell invasion by increasing cell motility and MMP-9 production via NF-κB transcriptional activation; AKT/PKB localizes to the leading edge membrane of migrating cells in a PI3K- and PH-domain-dependent manner.\",\n      \"method\": \"Confocal live-cell imaging, invasion assays, MMP-9 production assays, NF-κB reporter assays, kinase-dead and PH-domain mutant AKT constructs, PI3K inhibitors\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — live-cell localization, domain mutagenesis, functional invasion and reporter assays, single lab\",\n      \"pmids\": [\"11532975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"AKT/PKB phosphorylates heme oxygenase-1 (HO-1) at Ser188 in vitro and in vivo; the phosphomimetic S188D mutant shows ~1.6-fold higher enzymatic activity and slightly altered binding to cytochrome P450 reductase and biliverdin reductase as measured by FRET.\",\n      \"method\": \"In vitro kinase assay with recombinant HO-1, site-directed mutagenesis (S188A, S188D), 32P metabolic labeling in HEK293T cells, FRET assays for protein-protein interactions, HO-1 enzymatic activity assay\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution + in vivo labeling + mutagenesis, but small effect size, single lab\",\n      \"pmids\": [\"15581622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Akt1 phosphorylates FoxO3a to prevent its nuclear localization, thereby suppressing transactivation of the proapoptotic target gene Bim in osteoblasts; Akt1-deficient mice show low bone mass through increased osteoblast apoptosis via the Akt1/FoxO3a/Bim axis and decreased Runx2 transcriptional activity.\",\n      \"method\": \"Akt1 knockout mouse model, ex vivo osteoblast culture, apoptosis assays, FoxO3a nuclear localization by immunofluorescence, Bim expression analysis, Runx2 transcriptional activity assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO model with mechanistic follow-up, subcellular localization, transcriptional assays, single lab\",\n      \"pmids\": [\"17957242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Akt1 has opposing roles to Akt2 in regulating cell migration and cytoskeletal organization: Akt1-deficient MEFs migrate more slowly and respond poorly to PDGF, while Akt2-deficient cells migrate faster. Akt2 inhibits Pak1 kinase activity in direct kinase assays; N-terminal PH domain and linker region distinguish the two isoforms' functions.\",\n      \"method\": \"Akt1/Akt2 knockout MEFs, cell migration assays, dorsal ruffling assays, domain-swap constructs between Akt1/Akt2, direct Pak1 kinase assays, Rac activity measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cells, domain swaps, direct kinase assay for Pak1 inhibition by Akt2, single lab\",\n      \"pmids\": [\"17012749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Akt/PKB directly binds the actin cytoskeleton via its N-terminal PH domain; PDGF stimulation increases the amount of Akt associated with the actin skeleton, and this association requires Ser473/Thr308 phosphorylation (abolished by S473A/T308A double mutant). Small GTPases Rac1 and Cdc42 facilitate actin binding.\",\n      \"method\": \"In situ cytoskeletal matrix preparations, co-immunoprecipitation, in vitro binding and overlay assays with recombinant proteins, subcellular fractionation, expression of Akt domain mutants\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — in vitro direct binding + co-IP + mutagenesis + fractionation, single lab\",\n      \"pmids\": [\"14685694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Akt/PKB phosphorylates TopBP1 in vitro and in vivo; phosphorylation induces TopBP1 oligomerization through its 7th and 8th BRCT domains. Akt-dependent oligomerization is required for TopBP1 to interact with and repress E2F1 proapoptotic activity.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, co-immunoprecipitation, TopBP1 oligomerization assays, E2F1 reporter assays, PI3K inhibitor treatment\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro phosphorylation, mutagenesis, oligomerization assay, functional E2F1 repression, single lab\",\n      \"pmids\": [\"17006541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Caspases cleave AKT/PKB at three sites during apoptosis (TVAD108↓G, EEMD119↓F between PH and kinase domains, and SETD434↓T in the C-terminal regulatory domain), generating 40- and 44-kDa fragments; loss of the C-terminal domain reduces kinase activity and overexpression of the truncated fragment sensitizes cells to apoptosis.\",\n      \"method\": \"In vitro cleavage with purified caspases, caspase inhibitor rescue in cells, kinase activity assays of cleavage fragments, overexpression of truncated Akt constructs\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro reconstituted caspase cleavage, site identification, functional kinase assays, single lab\",\n      \"pmids\": [\"10623893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Lipid raft nanodomains facilitate AKT/PKB recruitment and activation at the plasma membrane upon PI3K-generated PIP3 accumulation; specific inhibition of sphingolipid and cholesterol biosynthesis abolishes raft nanodomains and impairs AKT Ser473/Thr308 phosphorylation.\",\n      \"method\": \"Fluorescence correlation spectroscopy (FCS) in live cells, specific inhibition of sphingolipid and cholesterol biosynthesis, PIP3 measurement, phospho-AKT Western blotting\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — novel FCS approach in live cells, pharmacological perturbation of rafts, single lab\",\n      \"pmids\": [\"18641634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"p53 stabilization in response to ionizing radiation requires AKT/PKB: DNA-PK is activated by ionizing radiation, phosphorylates and activates AKT/PKB, which in turn inactivates GSK-3; inactive GSK-3 fails to phosphorylate Mdm2 at sites required for p53 degradation, leading to p53 accumulation.\",\n      \"method\": \"Lymphoblasts from AT patients, ATR siRNA, AKT/PKB knockdown, DNA-PK knockdown, phospho-specific Western blotting of AKT, GSK-3, Mdm2, p53 accumulation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genetic/siRNA perturbations, patient-derived cells, pathway epistasis, single lab\",\n      \"pmids\": [\"18505846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Inhibition of AKT/PKB in late G2 cells restores DNA double-strand break processing (RPA, ATR, Rad51, CtIP recruitment to damage foci) and Chk1 checkpoint activation after irradiation; normally, AKT activity in late G2 suppresses DSB processing and prevents checkpoint activation.\",\n      \"method\": \"Chemical genetic Cdk1 inhibition to arrest cells in late G2, AKT inhibitor treatment, immunofluorescence for DSB repair factors (RPA, ATR, Rad51, CtIP, γ-H2AX), Chk1 phosphorylation Western blot\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chemical-genetic approach, multiple repair factor readouts, clean experimental design, single lab\",\n      \"pmids\": [\"20679434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"OX40 (CD134) assembles a signaling complex containing TRAF2, PI3K, and AKT/PKB upon ligation by OX40L; this complex forms in lipid microdomains independent of TCR engagement, but strong PI3K-AKT phosphorylation and functional activation only occur when antigen is also recognized, indicating OX40 augments TCR-induced AKT signaling quantitatively.\",\n      \"method\": \"Co-immunoprecipitation of OX40 complexes, detergent-resistant membrane fraction isolation, phospho-AKT Western blotting, TRAF2-deficient cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — reciprocal co-IP, lipid microdomain fractionation, genetic (TRAF2-KO) epistasis, single lab\",\n      \"pmids\": [\"21289304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Pak1 (P21-activated kinase-1) directly phosphorylates AKT at Ser473 but not Thr308 in vitro; silencing or inactivating Pak1 reduces AKT Ser473 and Thr308 phosphorylation in cardiomyocytes, and Pak1 overexpression is cardioprotective in an AKT-dependent manner.\",\n      \"method\": \"In vitro kinase assay with purified Pak1 and AKT, Pak1 siRNA, dominant-negative Pak1, cardiomyocyte overexpression/knockdown, AKT inhibitor rescue\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase reconstitution, siRNA, dominant-negative, functional rescue — but potential conflict with mTORC2/DNA-PK as Ser473 kinase\",\n      \"pmids\": [\"18054038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"AKT/PKB promotes nuclear import of HDAC4 upon muscle denervation, enabling epigenetic changes and synaptic gene upregulation required for neuromuscular endplate remodeling; mTORC1 activation must be tightly balanced to allow dynamic autophagy regulation in denervated muscle.\",\n      \"method\": \"Muscle-specific transgenic mice with constitutive mTORC1 activation or AKT inhibition, HDAC4 nuclear localization assays, autophagy flux assays, NMJ morphology analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo mouse genetic models, subcellular localization assay, functional NMJ readout, single lab\",\n      \"pmids\": [\"31320633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In mouse oocytes, Thr308-phosphorylated AKT localizes to pericentriolar materials while Ser473-phosphorylated AKT co-distributes with spindle microtubules; both are required for metaphase II spindle assembly. Ser473-phosphorylated AKT is specifically required for second polar body emission, while Thr308-phosphorylated AKT regulates microtubule organization during meiosis.\",\n      \"method\": \"Antibody injection into oocytes, immunofluorescence with phospho-specific antibodies, spindle morphology analysis, polar body emission assay\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — antibody injection (function-blocking), localization, distinct phenotypes for each phosphorylation site, single lab\",\n      \"pmids\": [\"18177853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Inositol polyphosphate multikinase (IPMK) acts as a physiological PI3-kinase that generates PIP3 and activates AKT/PKB; IPMK deletion reduces growth factor-elicited AKT signaling uniquely through loss of its PI3K activity, and p110 PI3-kinases must act first to phosphorylate/activate IPMK in a sequential PIP3-generating cascade.\",\n      \"method\": \"IPMK knockout cells, in vitro PI3K activity assay, wortmannin inhibition, growth factor stimulation, AKT phosphorylation assays, cell proliferation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro PI3K assay, genetic KO, pharmacological validation, single lab\",\n      \"pmids\": [\"21220345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CK2-mediated phosphorylation of AKT at Ser129 is necessary for AKT-dependent upregulation of β-catenin transcriptional activity and nuclear localization; CK2α overexpression increased β-catenin activity and survivin expression, and these effects were abolished by expression of an AKT-S129A mutant.\",\n      \"method\": \"Overexpression of wild-type, constitutively active, and dominant-negative CK2 and AKT, AKT-S129A mutant, β-catenin transcriptional reporter, nuclear fractionation, survivin expression assay\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — mutagenesis, reporter assay, functional viability readout, extends PMID:15818404 mechanistically, single lab\",\n      \"pmids\": [\"21506126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"In platelets activated through integrin αIIbβ3, PtdIns(3,4)P2 that activates AKT/PKB is generated primarily via a novel pathway: PI3K generates PtdIns3P, which is then phosphorylated by PtdIns3P 4-kinase — not by 5'-phosphatase hydrolysis of PtdIns(3,4,5)P3, which is not detected in this context. Wortmannin and calpain inhibitor both block this pathway and AKT activation.\",\n      \"method\": \"Lipid analysis (PtdIns3P, PtdIns(3,4)P2, PtdIns(3,4,5)P3 measurement), wortmannin/calpeptin inhibition, PKB kinase assay in human platelets, integrin activation by anti-LIBS Fab\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lipid mass measurement, pharmacological dissection, in vitro kinase assay, single lab\",\n      \"pmids\": [\"9417038\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AKT1/PKB is a pleckstrin-homology-domain-containing serine/threonine kinase activated by a sequential multi-step mechanism: PI3K-generated PIP3 recruits AKT to the plasma membrane via its PH domain (which also undergoes an intramolecular inhibitory interaction with the kinase domain in the resting state); PDK1 phosphorylates the activation-loop Thr308, while mTORC2 (requiring rictor and mSin1 for assembly) is the primary Ser473 hydrophobic-motif kinase — with DNA-PK and ATM also contributing in a stimulus-dependent manner, and Ack1 providing activating Tyr176 phosphorylation downstream of RTKs; additional regulatory phosphorylation by CK2 at Ser129 further enhances catalytic activity; active AKT phosphorylates a broad substrate network including FOXO transcription factors (cytoplasmic retention), GSK3β (inactivation), p27Kip1-Thr157 (nuclear exclusion), p21Cip1-Thr145/Ser146 (stabilization and PCNA uncoupling), CREB-Ser133 (transcriptional activation), Girdin/APE-Ser1416 (actin organization and cell migration), Skp2 (SCF E3 ligase activation and cytoplasmic relocalization), TopBP1 (oligomerization and E2F1 repression), Smad3 (kinase-independent sequestration blocking TGF-β signaling), and tau-Thr212/Ser214; AKT can also bind JIP1 to prevent JNK scaffold assembly, promote HDAC4 nuclear import in denervated muscle, and regulate p53 stability via GSK3β/Mdm2; its activity is negatively regulated by PTEN-mediated PIP3 dephosphorylation and by caspase cleavage during apoptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AKT1/PKB is a plasma-membrane-recruited serine/threonine kinase that serves as the central effector of PI3K signaling, converting growth-factor and insulin inputs into pro-survival, growth, metabolic, and migratory outputs [#4, #17]. Its activation follows an ordered, multi-step mechanism: PI3K-generated 3'-phosphoinositides bind the AKT PH domain to drive plasma-membrane translocation and co-localization with PDK1, which phosphorylates the activation-loop Thr308 [#4, #22], while the rictor-mTOR complex (mTORC2), whose assembly requires mSin1, is the hydrophobic-motif Ser473 kinase that facilitates full activation [#0, #2]. In the resting state AKT is held in a closed, autoinhibited conformation via an intramolecular PH-domain–kinase-domain interaction that blocks activation-loop phosphorylation [#15]; membrane recruitment within lipid raft nanodomains and additional inputs relieve this constraint [#31]. Beyond mTORC2, Ser473 phosphorylation is contributed by DNA-PK and ATM in a stimulus-dependent manner [#5, #18], Ack1 supplies an activating Tyr176 phosphorylation downstream of RTKs [#13], and CK2 phosphorylation at Ser129 further enhances catalytic output [#12, #39]. Active AKT recognizes the consensus motif RxRxxS/T [#14] and phosphorylates a broad substrate network that executes its biological programs: it inactivates FOXO/DAF-16 transcription factors to suppress pro-apoptotic genes such as Bim [#17, #26], drives cytoplasmic retention or stabilization of the CDK inhibitors p27Kip1 and p21Cip1 [#8, #9], activates CREB- and NF-κB-dependent transcription [#6, #7], and phosphorylates cytoskeletal and migratory effectors including Girdin and Skp2 [#11, #16]. AKT also acts through kinase-independent protein binding, sequestering Smad3 to block TGF-β-induced apoptosis [#10] and binding JIP1 to restrain JNK activation in neurons [#19], and it controls cell and organ size cell-autonomously downstream of the insulin/PI3K axis [#21]. AKT signaling is terminated by PTEN-mediated dephosphorylation of PIP3 and by caspase cleavage during apoptosis [#3, #30].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing how AKT activity is negatively gated, PTEN was shown to be the phosphatase that removes the PIP3 signal driving AKT, defining the off-switch of the pathway.\",\n      \"evidence\": \"In vitro lipid phosphatase assay, PIP3 measurement, and PTEN re-expression rescue in PTEN-deficient MEFs\",\n      \"pmids\": [\"9778245\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address how PIP3 is generated\", \"Does not resolve which AKT-activating kinases act downstream\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Genetic epistasis in C. elegans and Drosophila placed AKT downstream of insulin/PI3K and defined its core physiological outputs — antagonizing FOXO/DAF-16 transcription and controlling cell-autonomous growth.\",\n      \"evidence\": \"daf-16 suppression of akt-1/akt-2 mutants in C. elegans; clonal mosaic cell-size analysis in Drosophila imaginal discs\",\n      \"pmids\": [\"9716402\", \"10587646\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define the biochemical activation steps\", \"Substrate phosphorylation sites in mammals not addressed\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Resolving the membrane-recruitment step, the AKT PH domain was shown to bind PI3K-generated 3'-phosphoinositides and localize AKT to the membrane where PDK1 phosphorylates Thr308, defining the multi-step AGC-kinase activation paradigm.\",\n      \"evidence\": \"Phosphoinositide binding assays, GFP-PH live-cell imaging, PI3K inhibitor and binding-mutant controls\",\n      \"pmids\": [\"10872470\", \"10226029\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the Ser473 hydrophobic-motif kinase unresolved at this stage\", \"Mechanism of resting-state autoinhibition not defined\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defining substrate selectivity, the optimal RxRxxS/T consensus motif was determined, enabling prediction and validation of AKT substrates.\",\n      \"evidence\": \"Oriented peptide library screening with cDNA expression-library validation and kinetic analysis\",\n      \"pmids\": [\"10945990\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify which physiological substrates are phosphorylated in vivo\", \"Context-dependent substrate selection not addressed\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Connecting AKT to cell-cycle control, AKT was shown to phosphorylate the CDK inhibitors p21Cip1 and p27Kip1 to alter their localization and stability, promoting proliferation and survival.\",\n      \"evidence\": \"In vitro kinase assays, T145/S146 and T157A mutagenesis, nuclear import reconstitution, and PCNA-binding assays\",\n      \"pmids\": [\"11756412\", \"12244302\", \"12244301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution to tumor proliferation not quantified\", \"Crosstalk with other p21/p27 kinases not resolved\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Expanding AKT beyond kinase activity, AKT was shown to physically sequester Smad3 in a kinase-independent manner, defining a non-catalytic mechanism for suppressing TGF-β-induced apoptosis.\",\n      \"evidence\": \"Protein-fragment complementation, co-IP, AKT siRNA rescue, and Smad3 reporter assays\",\n      \"pmids\": [\"15048128\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the AKT-Smad3 interface unknown\", \"Stoichiometry and regulation of the complex not defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Addressing the long-standing Ser473 kinase question, DNA-PK and ATM were each implicated as stimulus-dependent contributors to AKT Ser473 phosphorylation.\",\n      \"evidence\": \"DNA-PK purification with in vitro phosphorylation and PRKDC complementation; ATM inhibitors/siRNA and AT-patient/KO cells\",\n      \"pmids\": [\"15262962\", \"15546863\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution versus mTORC2 across stimuli not quantified\", \"Whether these act on membrane-recruited AKT or nuclear AKT unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identifying the principal hydrophobic-motif kinase, mTORC2 (rictor-mTOR) was shown to directly phosphorylate AKT Ser473 in vitro and facilitate Thr308 phosphorylation, completing the canonical activation model.\",\n      \"evidence\": \"Reconstituted in vitro kinase assay with purified rictor-mTOR plus rictor/mTOR knockdown in Drosophila and human cells\",\n      \"pmids\": [\"15718470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not reconcile multiplicity of reported Ser473 kinases\", \"Spatial coordination of mTORC2 with membrane AKT not addressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Adding a tuning input, CK2 was shown to phosphorylate already-active AKT at Ser129 to further enhance catalytic activity and downstream β-catenin signaling.\",\n      \"evidence\": \"In vitro CK2 kinase assay, two structurally distinct inhibitors plus siRNA; later S129A mutant analysis of β-catenin/survivin\",\n      \"pmids\": [\"15818404\", \"21506126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological stimuli engaging CK2-AKT crosstalk not defined\", \"Quantitative contribution to overall AKT output unclear\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defining the mTORC2 assembly requirement, mSin1 was identified as an essential subunit needed for mTORC2 to phosphorylate AKT Ser473, and prolonged rapamycin was shown to disrupt mTORC2 and reduce AKT signaling.\",\n      \"evidence\": \"Co-IP, mSin1 siRNA, in vitro mTORC2 kinase assay, mass spec isoform mapping; mTORC2 assembly assays with rapamycin-resistant AKT rescue\",\n      \"pmids\": [\"16919458\", \"16603397\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional distinction among mSin1 isoform complexes not fully resolved\", \"How rapamycin selectively disrupts assembly mechanistically unclear\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealing the basis of resting-state inhibition, AKT was shown to adopt a closed PH-domain–kinase-domain conformation that blocks Thr308 phosphorylation and is exploited by isoform-selective allosteric inhibitors.\",\n      \"evidence\": \"Molecular modeling, FRET/two-photon FLIM, biochemical kinase assays, and domain mutagenesis\",\n      \"pmids\": [\"19166270\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the autoinhibited state not provided\", \"How PIP3 binding kinetically relieves the closed state not detailed\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linking AKT to ubiquitin-ligase control and migration, AKT was shown to phosphorylate Skp2 (and earlier Girdin), driving SCF activity, 14-3-3-dependent cytoplasmic relocalization, and cell motility.\",\n      \"evidence\": \"In vitro kinase assays, phospho-defective mutants, ubiquitylation and migration assays, human tumor specimen analysis\",\n      \"pmids\": [\"19270694\", \"16139227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo metastatic relevance of these substrates not established\", \"Isoform specificity of cytoskeletal substrate phosphorylation not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Adding tyrosine-kinase input, Ack1 was shown to phosphorylate AKT at Tyr176 to promote membrane localization and prime activation-loop/hydrophobic-motif phosphorylation downstream of RTKs.\",\n      \"evidence\": \"In vitro phosphorylation, Y176F mutagenesis, fractionation, and an Ack1-driven prostate cancer mouse model\",\n      \"pmids\": [\"20333297\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality across RTK contexts not established\", \"Structural effect of Tyr176 phosphorylation on conformation unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple reported Ser473 kinases (mTORC2, DNA-PK, ATM, Pak1) are deployed across distinct stimuli, subcellular compartments, and cell types — and how isoform-specific (AKT1 vs AKT2) substrate choice is achieved — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model reconciling competing Ser473 kinases\", \"Mechanistic basis of AKT1/AKT2 functional divergence incompletely defined\", \"Compartment-specific substrate engagement not systematically mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 8, 9, 11, 14, 16]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [4, 22, 31]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [6, 17, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 5, 22, 31]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [11, 28]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4, 17]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [10, 26, 30]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [6, 17]}\n    ],\n    \"complexes\": [\"mTORC2\"],\n    \"partners\": [\"MTOR\", \"RICTOR\", \"MAPKSIN1\", \"PDPK1\", \"PTEN\", \"SMAD3\", \"JIP1\", \"ACK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}