{"gene":"PRKAA2","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2011,"finding":"AMPK directly phosphorylates ULK1 at Ser317 and Ser777 to activate autophagy under glucose starvation. Under nutrient sufficiency, mTOR phosphorylates ULK1 at Ser757, disrupting the AMPK-ULK1 interaction and preventing ULK1 activation.","method":"In vitro phosphorylation assay, mutagenesis, co-immunoprecipitation, genetic knockout cells","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — direct phosphorylation demonstrated in vitro and in cells with mutagenesis, replicated across multiple experimental systems","pmids":["21258367"],"is_preprint":false},{"year":2008,"finding":"AMPK directly phosphorylates raptor (the mTOR binding partner) on two conserved serine residues, inducing 14-3-3 binding to raptor; this phosphorylation is required for mTORC1 inhibition and cell-cycle arrest induced by energy stress.","method":"Proteomic/bioinformatic substrate identification, in vitro kinase assay, Co-IP, mutagenesis, genetic epistasis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay plus mutagenesis plus genetic epistasis in a single rigorous study","pmids":["18439900"],"is_preprint":false},{"year":2007,"finding":"AMPK directly phosphorylates PGC-1alpha at Thr177 and Ser538 both in vitro and in cells; these phosphorylations are required for AMPK-dependent induction of the PGC-1alpha promoter and downstream gene expression (GLUT4, mitochondrial genes) in skeletal muscle.","method":"In vitro kinase assay, site-directed mutagenesis, PGC-1alpha-knockout primary muscle cells, reporter assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, mutagenesis, and genetic validation in knockout cells","pmids":["17609368"],"is_preprint":false},{"year":2015,"finding":"The cancer-germline ubiquitin ligase MAGE-A3/6-TRIM28 ubiquitinates and degrades AMPKα1 (PRKAA1), leading to inhibition of autophagy and activation of mTOR signaling; this represents a mechanism by which cancer cells suppress AMPK activity.","method":"Substrate screen, Co-IP, ubiquitination assay, genetic gain/loss-of-function, cell viability assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, biochemical ubiquitination assay, multiple orthogonal functional readouts in a single rigorous study","pmids":["25679763"],"is_preprint":false},{"year":2017,"finding":"AMPK negatively regulates β1-integrin activity in fibroblasts by suppressing expression of the integrin-binding proteins tensin1 and tensin3; loss of AMPK upregulates tensins, which bind β1-integrins to promote fibrillar adhesion formation, cell spreading, traction stress, and fibronectin fibrillogenesis.","method":"Loss-of-function (siRNA/genetic deletion), integrin activity assay, traction force microscopy, tensin rescue/silencing experiments","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KO, KD, rescue) with defined mechanistic pathway in a single study","pmids":["28289092"],"is_preprint":false},{"year":2014,"finding":"AMPK directly phosphorylates OGT (O-GlcNAc transferase), and while this phosphorylation does not alter OGT enzymatic activity, it inhibits OGT-chromatin association, reducing histone H2B O-GlcNAcylation and gene transcription. Conversely, OGT O-GlcNAcylates AMPK and positively regulates AMPK activity, creating a feedback loop.","method":"In vitro kinase assay, Co-IP, chromatin immunoprecipitation, mutagenesis, gene transcription reporter assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay plus ChIP plus functional transcription readout, single lab with multiple orthogonal methods","pmids":["24692660"],"is_preprint":false},{"year":2018,"finding":"STIM2 (a calcium sensor) physically interacts with both AMPK and CaMKK2; increased intracellular calcium promotes AMPK colocalization with STIM2, and STIM2 deficiency attenuates calcium-induced but not energy-stress-induced AMPK activation, indicating STIM2 is a regulator of the CaMKK2-AMPK calcium-signaling axis.","method":"Co-immunoprecipitation, fluorescence microscopy colocalization, genetic knockdown, selective AMPK activation assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and genetic KD with two mechanistic readouts, single lab","pmids":["30335546"],"is_preprint":false},{"year":2018,"finding":"Mitochondria-derived ROS activate AMPK indirectly (via effects on mitochondrial ATP production and changes in ATP/ADP ratio) rather than by direct oxidation of redox-sensitive cysteine residues (Cys299/Cys304) on the AMPK α subunit; mutation of these cysteines to alanine did not alter the AMPK response to H2O2.","method":"Mutagenesis (Cys→Ala), exogenous H2O2 treatment, mitochondria-targeted ROS generator (MitoParaquat), ATP/ADP ratio measurement, redox-sensitive fluorescent proteins","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis with multiple orthogonal ROS-generation tools and parallel energetic measurements; negative result for direct ROS sensing is well-controlled","pmids":["30232152"],"is_preprint":false},{"year":2017,"finding":"Deletion of PRKAA (AMPKα, including the α2 isoform) causes defective autophagy, leading to accumulation of DNM1L (dynamin-1-like) and aberrant mitochondrial fragmentation in vascular endothelial cells; the autophagy receptor SQSTM1/p62 binds DNM1L and directs it to autophagosomes for degradation, linking PRKAA activity to mitochondrial fission control.","method":"Genetic knockout (Prkaa1/Prkaa2 KO mice), autophagy inhibition (chloroquine, ATG7 siRNA), autophagy activation (ATG7 overexpression, rapamycin), Co-IP of SQSTM1-DNM1L, isolated aorta contractility assay","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KO model plus multiple pharmacological/genetic rescue experiments with defined molecular mechanism","pmids":["28085543"],"is_preprint":false},{"year":2024,"finding":"AMPK directly inhibits NIX-dependent (programmed) mitophagy by phosphorylating ULK1 at Ser556 and a newly identified site Ser694, triggering 14-3-3-mediated sequestration of ULK1. Conversely, AMPK enhances depolarization-induced (damage-induced) mitophagy by increasing Parkin phosphorylation, independently of ULK1.","method":"In vitro kinase assay, mutagenesis, mito-QC mouse model (in vivo), cell-based mitophagy assays, phosphoproteomic identification of Ser694","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro phosphorylation, mutagenesis, and in vivo validation in mito-QC mouse model with multiple orthogonal readouts","pmids":["39532100"],"is_preprint":false},{"year":2022,"finding":"Blocking FBP (fructose-1,6-bisphosphate) binding to aldolase with the small molecule aldometanib prevents aldolase from associating with v-ATPase on lysosomes, thereby selectively activating the lysosomal pool of AMPK and mimicking cellular glucose starvation; this demonstrates that aldolase acts as a glucose sensor upstream of lysosomal AMPK.","method":"Chemical screen, biochemical binding assays, lysosomal AMPK activity measurement, metabolic phenotyping in rodents, C. elegans and mouse lifespan assays","journal":"Nature metabolism","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — chemical probe plus biochemical mechanism plus in vivo validation across multiple model organisms","pmids":["36217034"],"is_preprint":false},{"year":2024,"finding":"LCA (lithocholic acid) binds TULP3, which allosterically activates sirtuins; activated sirtuins deacetylate the V1E1 subunit of v-ATPase at residues K52, K99, and K191, which inhibits v-ATPase and activates AMPK through the lysosomal glucose-sensing pathway.","method":"Proteomics/Co-IP identifying TULP3 as sirtuin-interacting LCA receptor, in vitro deacetylation assays, mutagenesis (3KR deacetylation mimic), muscle-specific viral expression in aged mice, AMPK activity assays, lifespan assays in C. elegans and flies","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — biochemical reconstitution, mutagenesis, in vivo rescue, and cross-species validation in a single study","pmids":["39695235"],"is_preprint":false},{"year":2020,"finding":"Cordycepin (3'-deoxyadenosine) is converted intracellularly into cordycepin monophosphate, which mimics all three effects of AMP on AMPK (activation, protection from dephosphorylation, allosteric activation); AMPK activation by cordycepin is blocked by a γ-subunit mutation that prevents AMP binding, confirming the AMP-mimicry mechanism.","method":"Nucleotide quantification in intact cells, cell-free AMPK assays, genetic AMPK knockout, γ-subunit mutagenesis","journal":"Cell chemical biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro reconstitution, mutagenesis, and genetic KO validation with multiple orthogonal readouts","pmids":["31991096"],"is_preprint":false},{"year":2017,"finding":"Deconvolution of AMPK adenine nucleotide binding established that CBS3 (not CBS1) is the high-affinity exchangeable AMP/ADP/ATP-binding site in the γ-subunit; AMP binding at CBS4 increases AMP affinity at CBS3 by ~100-fold and reverses CBS3's AMP/ATP preference. NADPH (in addition to NADH) directly and competitively binds AMPK at the CBS3 site.","method":"Quantitative competition binding assays, hydrogen-deuterium exchange mass spectrometry, wild-type and mutant AMPK protein complexes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — HDX-MS structural method combined with quantitative binding assays and mutagenesis in a single comprehensive study","pmids":["28615457"],"is_preprint":false},{"year":2017,"finding":"AMPK deficiency in myeloid cells increases PKM2-dependent aerobic glycolysis, leading to enhanced HMGB1 release from macrophages/monocytes and promoting sepsis; pharmacological AMPK activation (A-769662) protects against endotoxic shock, while PKM2 inhibition rescues the pro-inflammatory phenotype of AMPKα-deficient mice.","method":"Myeloid-specific AMPKα knockout mice, pharmacological activation/inhibition, HMGB1 measurement, polymicrobial sepsis model, glycolysis/lactate assays","journal":"Brain, behavior, and immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific KO plus pharmacological rescue plus defined mechanistic pathway in vivo","pmids":["29109024"],"is_preprint":false},{"year":2003,"finding":"AMPKα2-deficient (Prkaa2−/−) mice display elevated blood glucose, reduced plasma insulin in the fed state, in vivo insulin resistance, and reduced muscle glycogen synthesis; the insulin resistance was not intrinsic to skeletal muscle (isolated muscle glucose transport was normal), suggesting AMPKα2 regulates systemic glucose homeostasis partly through modulation of the autonomic nervous system (increased catecholamine excretion).","method":"Genetic knockout mouse (AMPKα2−/−), hyperinsulinemic-euglycaemic clamp, isolated muscle glucose transport assay, dominant-negative AMPK transgenic muscle mice, catecholamine measurement","journal":"Biochemical Society transactions","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KO with clamp studies plus mechanistic dissection using dominant-negative transgenic and isolated tissue experiments","pmids":["12546688"],"is_preprint":false},{"year":2021,"finding":"AMPKα2 nuclear localization employs a nuclear localization signal (NLS) present on the AMPKα2 kinase domain, while nuclear export involves RanGTPase-CRM1-mediated recognition of a nuclear export sequence (NES) on the α subunit; nucleo-cytoplasmic shuttling is regulated by starvation, exercise, heat shock, oxidants, cell density, and circadian rhythm.","method":"Subcellular fractionation, nuclear export sequence deletion/mutation analysis, CRM1 inhibition, fluorescence localization studies (compiled from multiple studies reviewed)","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — review synthesizing multiple localization studies; individual NLS/NES experiments documented but full methodology not detailed in abstract","pmids":["34681581"],"is_preprint":false},{"year":2020,"finding":"AMPK-glycogen binding via the β2 subunit CBM (disrupted by W98A knock-in mutation) is required to stabilize AMPK protein and kinase activity in skeletal muscle; β2-W98A KI mice show reduced total AMPK protein and kinase activity in muscle, increased adiposity, impaired whole-body glucose handling, and reduced maximal exercise capacity.","method":"Whole-body knock-in mouse (β2-W98A), systematic metabolic phenotyping, AMPK kinase activity assays, tissue AMPK protein quantification","journal":"Molecular metabolism","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knock-in genetic model with comprehensive molecular and physiological phenotyping","pmids":["32610071"],"is_preprint":false},{"year":2024,"finding":"Crizotinib inhibits PRKAA/AMPK phosphorylation at Ser485/491, impairing autophagosome-lysosome fusion and preventing MET protein degradation; metformin restores PRKAA (Ser485/491) phosphorylation, re-activates autophagy flux, and rescues crizotinib-induced cardiomyocyte injury.","method":"In vitro cardiomyocyte models, in vivo mouse cardiotoxicity model, autophagy flux assay, PRKAA phosphorylation analysis, metformin rescue experiment, MET silencing","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo plus in vitro models with pharmacological and genetic intervention, single lab","pmids":["37733896"],"is_preprint":false},{"year":2016,"finding":"PRKAA/AMPK is activated by HBV-induced oxidative stress and restricts HBV production by promoting autolysosome-dependent degradation through stimulation of cellular ATP levels, leading to depletion of autophagic vacuoles that HBV depends on for replication.","method":"HBV infection cell model, PRKAA activation/inhibition, autophagy flux assay, ATP measurement, autophagic vacuole quantification","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanistic dissection with pharmacological and genetic tools in a defined cellular model, single lab","pmids":["27305174"],"is_preprint":false},{"year":2016,"finding":"AMPK promotes osteogenesis and inhibits adipogenesis through downregulation of the transcriptional repressor Gfi1, which dissociates from the osteopontin (OPN) promoter upon AMPK activation, resulting in OPN upregulation; overexpression or dominant-negative Gfi1 modulates osteogenesis and adipogenesis accordingly.","method":"Lentiviral AMPKα overexpression, Gfi1 overexpression/dominant-negative constructs, luciferase reporter (OPN promoter), ChIP (Gfi1 binding), ectopic bone formation assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assay plus ChIP plus in vivo ectopic bone formation, single lab with multiple orthogonal methods","pmids":["27283242"],"is_preprint":false},{"year":2016,"finding":"AMPK activates LXRα expression in macrophages, which then transcriptionally upregulates ABCA1 via binding to the LXR-responsive element in the ABCA1 promoter, resulting in increased cholesterol efflux; LXRβ silencing did not affect this pathway, establishing LXRα specificity.","method":"Pharmacological and genetic AMPK activation/knockdown, luciferase reporter assay, chromatin immunoprecipitation (ChIP), LXRα/β siRNA silencing, cholesterol efflux assay","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus reporter assay plus genetic KD with functional cholesterol efflux readout, single lab","pmids":["27343431"],"is_preprint":false}],"current_model":"PRKAA2 (AMPKα2) is the catalytic subunit of the heterotrimeric AMPK complex, an energy sensor activated by elevated AMP/ADP:ATP ratios (sensed via γ-subunit CBS sites, particularly CBS3) and calcium signaling (via CaMKK2/STIM2); once activated, it directly phosphorylates a broad set of substrates—including ULK1 (Ser317/777) to initiate autophagy, raptor to suppress mTORC1, PGC-1α (Thr177/Ser538) to drive mitochondrial biogenesis, OGT to modulate chromatin O-GlcNAcylation, NIX-pathway effectors (ULK1 Ser556/694) to restrain programmed mitophagy while enhancing damage-induced mitophagy via Parkin, and tensins to regulate integrin activity—while the AMPKα2 protein itself is subject to degradation by the MAGE-A3/6-TRIM28 ubiquitin ligase in cancer cells and requires glycogen binding (via the β-subunit) for stability."},"narrative":{"mechanistic_narrative":"PRKAA2 (AMPKα2) is the catalytic subunit of the AMPK energy-sensing kinase, which couples cellular energy status to a broad transcriptional and post-translational program controlling autophagy, mitochondrial homeostasis, and systemic glucose metabolism [PMID:21258367, PMID:12546688]. Sensing of low energy occurs through adenine-nucleotide binding to the γ-subunit, where CBS3 is the high-affinity exchangeable AMP/ADP/ATP site whose nucleotide preference is allosterically tuned by AMP occupancy at CBS4 and which also binds NADPH competitively [PMID:28615457]; this AMP-sensing mechanism is exploited pharmacologically by cordycepin monophosphate, which mimics all three effects of AMP and is abolished by a γ-subunit AMP-binding mutation [PMID:31991096]. A parallel lysosomal glucose-sensing branch activates AMPK when low fructose-1,6-bisphosphate releases aldolase from v-ATPase, a circuit that can be engaged chemically (aldometanib) or via sirtuin-dependent deacetylation of the v-ATPase V1E1 subunit [PMID:36217034, PMID:39695235], while a calcium branch operates through STIM2-coordinated CaMKK2 signaling [PMID:30335546]. Once active, AMPKα2 directly phosphorylates a defined substrate set: ULK1 (Ser317/Ser777) to initiate starvation autophagy in opposition to mTOR [PMID:21258367], raptor to suppress mTORC1 and enforce energy-stress cell-cycle arrest [PMID:18439900], PGC-1α (Thr177/Ser538) to drive mitochondrial and GLUT4 gene expression in muscle [PMID:17609368], and ULK1 (Ser556/Ser694) to restrain NIX-dependent programmed mitophagy while promoting Parkin-dependent damage-induced mitophagy [PMID:39532100]. Through its control of autophagic flux, AMPKα2 also governs SQSTM1/p62-mediated clearance of the fission factor DNM1L to maintain mitochondrial morphology [PMID:28085543]. Beyond canonical metabolic targets, AMPK phosphorylates OGT to displace it from chromatin and reduce histone H2B O-GlcNAcylation in a reciprocal regulatory loop [PMID:24692660], and suppresses tensin expression to limit β1-integrin activity and fibrillar adhesion [PMID:28289092]. Physiologically, Prkaa2 loss produces systemic insulin resistance and hyperglycemia involving autonomic signaling rather than a muscle-intrinsic transport defect [PMID:12546688], and AMPK stability and activity depend on β-subunit glycogen binding [PMID:32610071]. The catalytic α-subunit carries its own NLS and CRM1-dependent NES enabling regulated nucleo-cytoplasmic shuttling [PMID:34681581].","teleology":[{"year":2003,"claim":"Established the physiological role of AMPKα2 in systemic glucose homeostasis and showed its insulin-resistance phenotype is not muscle-intrinsic, redirecting attention to whole-body neuroendocrine control.","evidence":"AMPKα2 knockout mice with hyperinsulinemic-euglycaemic clamp, isolated muscle transport assays, and catecholamine measurement","pmids":["12546688"],"confidence":"High","gaps":["Does not identify the AMPKα2 substrates mediating autonomic effects","Tissue source of systemic insulin resistance not pinpointed"]},{"year":2007,"claim":"Defined a direct transcriptional output of AMPK by showing it phosphorylates PGC-1α to drive mitochondrial biogenesis gene programs.","evidence":"In vitro kinase assay, mutagenesis, and PGC-1α-knockout primary muscle cells with promoter reporter","pmids":["17609368"],"confidence":"High","gaps":["Relative contribution of α2 vs α1 not isolated","Downstream coactivator partners on the promoter not mapped"]},{"year":2008,"claim":"Connected AMPK to mTORC1 inhibition by identifying raptor as a direct substrate whose phosphorylation recruits 14-3-3 and enforces energy-stress cell-cycle arrest.","evidence":"Proteomic substrate identification, in vitro kinase assay, Co-IP, mutagenesis, and genetic epistasis","pmids":["18439900"],"confidence":"High","gaps":["Quantitative balance between raptor and other mTORC1 inputs unresolved","α-isoform specificity not addressed"]},{"year":2011,"claim":"Resolved how AMPK initiates autophagy, showing direct ULK1 phosphorylation and reciprocal mTOR-mediated disruption of the AMPK-ULK1 interaction under nutrient sufficiency.","evidence":"In vitro phosphorylation, mutagenesis, Co-IP, and genetic knockout cells","pmids":["21258367"],"confidence":"High","gaps":["Does not separate α1 from α2 catalytic contribution","In vivo relevance of specific sites in tissues not tested here"]},{"year":2014,"claim":"Extended AMPK function to chromatin regulation by showing it phosphorylates OGT to block chromatin association and reduce histone O-GlcNAcylation, with reciprocal OGT regulation of AMPK.","evidence":"In vitro kinase assay, Co-IP, ChIP, mutagenesis, and transcription reporter","pmids":["24692660"],"confidence":"High","gaps":["Genome-wide scope of affected loci not defined","Physiological contexts where the feedback loop dominates unclear"]},{"year":2015,"claim":"Revealed how cancer cells suppress AMPK by identifying MAGE-A3/6-TRIM28 as a ubiquitin ligase that degrades the catalytic α-subunit, inhibiting autophagy and activating mTOR.","evidence":"Substrate screen, Co-IP, ubiquitination assay, and gain/loss-of-function with viability readouts (demonstrated for α1)","pmids":["25679763"],"confidence":"High","gaps":["Direct demonstration on the α2 isoform not shown","Degron residues on AMPKα not mapped"]},{"year":2016,"claim":"Linked AMPK to lineage and lipid-handling transcriptional programs, including osteogenesis/adipogenesis via Gfi1-OPN, macrophage cholesterol efflux via LXRα-ABCA1, and antiviral restriction of HBV through autolysosomal degradation.","evidence":"Reporter assays, ChIP, siRNA silencing, ectopic bone formation, and HBV infection autophagy-flux models","pmids":["27283242","27343431","27305174"],"confidence":"Medium","gaps":["AMPKα2-specific requirement not isolated from α1","Direct kinase substrates in each pathway not identified","Single-lab observations per pathway"]},{"year":2017,"claim":"Mapped the nucleotide-sensing core, establishing CBS3 as the high-affinity exchangeable site with CBS4-driven allostery, and dissected AMPK's roles in mitochondrial fission control, integrin/adhesion regulation, and macrophage inflammation.","evidence":"HDX-MS and competition binding (CBS3); Prkaa1/2 KO mice with SQSTM1-DNM1L Co-IP; tensin loss/rescue with traction microscopy; myeloid-specific KO sepsis model with PKM2 rescue","pmids":["28615457","28085543","28289092","29109024"],"confidence":"High","gaps":["CBS3 nucleotide work uses reconstituted complexes, not in-cell occupancy","Direct kinase substrates for tensin and PKM2 effects not defined"]},{"year":2018,"claim":"Distinguished AMPK's energy and calcium activation inputs and excluded direct ROS oxidation, showing STIM2 regulates the calcium/CaMKK2 axis while mitochondrial ROS act indirectly via ATP/ADP changes.","evidence":"Reciprocal Co-IP and colocalization with STIM2; Cys→Ala mutagenesis with mitochondria-targeted ROS generators and ATP/ADP measurement","pmids":["30335546","30232152"],"confidence":"High","gaps":["STIM2 finding is single-lab, Medium confidence","Molecular mechanism of STIM2-AMPK colocalization not resolved"]},{"year":2020,"claim":"Defined determinants of AMPK pharmacology and stability, validating AMP-mimicry by cordycepin monophosphate via the γ-subunit and establishing β-subunit glycogen binding as required for AMPK protein stability and activity in muscle.","evidence":"Nucleotide quantification, cell-free assays, γ-subunit mutagenesis (cordycepin); β2-W98A knock-in mice with metabolic phenotyping","pmids":["31991096","32610071"],"confidence":"High","gaps":["Glycogen-binding stabilization mechanism at the molecular level not detailed","α2-specific stability dependence not isolated"]},{"year":2021,"claim":"Synthesized the regulated nucleo-cytoplasmic shuttling of AMPKα2, attributing nuclear import to an NLS on the kinase domain and export to RanGTPase-CRM1 recognition of an α-subunit NES.","evidence":"Review compiling fractionation, NES deletion/mutation, and CRM1-inhibition localization studies","pmids":["34681581"],"confidence":"Medium","gaps":["Review-level synthesis, not primary experiments","Stimulus-specific shuttling kinetics not quantified"]},{"year":2022,"claim":"Established a lysosomal glucose-sensing branch upstream of AMPK in which low FBP releases aldolase from v-ATPase to activate the lysosomal AMPK pool, druggable by aldometanib.","evidence":"Chemical screen, biochemical binding assays, lysosomal AMPK activity, and lifespan/metabolic phenotyping across rodents and C. elegans","pmids":["36217034"],"confidence":"High","gaps":["Molecular bridge from v-ATPase to AMPK activation not fully defined","α2-specific contribution to lysosomal pool not isolated"]},{"year":2024,"claim":"Refined AMPK's mitophagy control by showing it phosphorylates ULK1 (Ser556/Ser694) to restrain NIX-dependent programmed mitophagy while promoting Parkin-dependent damage mitophagy, and uncovered an upstream LCA-TULP3-sirtuin-v-ATPase route to AMPK activation.","evidence":"In vitro kinase assays, mutagenesis, mito-QC and aged-mouse in vivo models, phosphoproteomics; LCA-TULP3 Co-IP with deacetylation assays and cross-species lifespan","pmids":["39532100","39695235"],"confidence":"High","gaps":["How AMPK switches between restraining and promoting mitophagy contextually not fully resolved","Direct AMPK substrate in the Parkin arm not specified"]},{"year":2024,"claim":"Illustrated drug-induced AMPK dysregulation, showing crizotinib lowers AMPK Ser485/491 phosphorylation to impair autophagosome-lysosome fusion and cardiomyocyte clearance of MET, reversible by metformin.","evidence":"In vitro and in vivo cardiotoxicity models with autophagy-flux assays, phosphorylation analysis, and MET silencing","pmids":["37733896"],"confidence":"Medium","gaps":["Single-lab Medium-confidence study","Whether Ser485/491 change is direct or indirect not resolved"]},{"year":null,"claim":"It remains unresolved which AMPK functions are specifically executed by the α2 (PRKAA2) catalytic isoform versus α1, since most substrate and phenotype data are reported for AMPKα generically.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No isoform-resolved substrate map","α2-specific structural and localization determinants incompletely defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,5,9]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,2,9]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[12,13]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[13,10]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[16,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[16]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[0,8,9,19]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,10,15,21]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[7,13,12]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,6]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,5,20,21]}],"complexes":["AMPK heterotrimer"],"partners":["ULK1","RPTOR","PPARGC1A","OGT","STIM2","CAMKK2","PRKN","TRIM28"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P54646","full_name":"5'-AMP-activated protein kinase catalytic subunit alpha-2","aliases":["Acetyl-CoA carboxylase kinase","ACACA kinase","Hydroxymethylglutaryl-CoA reductase kinase","HMGCR kinase"],"length_aa":552,"mass_kda":62.3,"function":"Catalytic subunit of AMP-activated protein kinase (AMPK), an energy sensor protein kinase that plays a key role in regulating cellular energy metabolism (PubMed:17307971, PubMed:17712357). In response to reduction of intracellular ATP levels, AMPK activates energy-producing pathways and inhibits energy-consuming processes: inhibits protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation (PubMed:17307971, PubMed:17712357). AMPK acts via direct phosphorylation of metabolic enzymes, and by longer-term effects via phosphorylation of transcription regulators (PubMed:17307971, PubMed:17712357). Regulates lipid synthesis by phosphorylating and inactivating lipid metabolic enzymes such as ACACA, ACACB, GYS1, HMGCR and LIPE; regulates fatty acid and cholesterol synthesis by phosphorylating acetyl-CoA carboxylase (ACACA and ACACB) and hormone-sensitive lipase (LIPE) enzymes, respectively (PubMed:7959015). Promotes lipolysis of lipid droplets by mediating phosphorylation of isoform 1 of CHKA (CHKalpha2) (PubMed:34077757). Regulates insulin-signaling and glycolysis by phosphorylating IRS1, PFKFB2 and PFKFB3 (By similarity). Involved in insulin receptor/INSR internalization (PubMed:25687571). AMPK stimulates glucose uptake in muscle by increasing the translocation of the glucose transporter SLC2A4/GLUT4 to the plasma membrane, possibly by mediating phosphorylation of TBC1D4/AS160 (By similarity). Regulates transcription and chromatin structure by phosphorylating transcription regulators involved in energy metabolism such as CRTC2/TORC2, FOXO3, histone H2B, HDAC5, MEF2C, MLXIPL/ChREBP, EP300, HNF4A, p53/TP53, SREBF1, SREBF2 and PPARGC1A (PubMed:11518699, PubMed:11554766, PubMed:15866171, PubMed:17711846, PubMed:18184930). Acts as a key regulator of glucose homeostasis in liver by phosphorylating CRTC2/TORC2, leading to CRTC2/TORC2 sequestration in the cytoplasm (By similarity). In response to stress, phosphorylates 'Ser-36' of histone H2B (H2BS36ph), leading to promote transcription (By similarity). Acts as a key regulator of cell growth and proliferation by phosphorylating FNIP1, TSC2, RPTOR, WDR24 and ATG1/ULK1: in response to nutrient limitation, negatively regulates the mTORC1 complex by phosphorylating RPTOR component of the mTORC1 complex and by phosphorylating and activating TSC2 (PubMed:14651849, PubMed:20160076, PubMed:21205641). Also phosphorylates and inhibits GATOR2 subunit WDR24 in response to nutrient limitation, leading to suppress glucose-mediated mTORC1 activation (PubMed:36732624). In response to energetic stress, phosphorylates FNIP1, inactivating the non-canonical mTORC1 signaling, thereby promoting nuclear translocation of TFEB and TFE3, and inducing transcription of lysosomal or autophagy genes (PubMed:37079666). In response to nutrient limitation, promotes autophagy by phosphorylating and activating ATG1/ULK1 (PubMed:21205641). In that process, it also activates WDR45/WIPI4 (PubMed:28561066). Phosphorylates CASP6, thereby preventing its autoprocessing and subsequent activation (PubMed:32029622). AMPK also acts as a regulator of circadian rhythm by mediating phosphorylation of CRY1, leading to destabilize it (By similarity). May regulate the Wnt signaling pathway by phosphorylating CTNNB1, leading to stabilize it (By similarity). Also acts as a regulator of cellular polarity by remodeling the actin cytoskeleton; probably by indirectly activating myosin (PubMed:17486097). Also phosphorylates CFTR, EEF2K, KLC1, NOS3 and SLC12A1 (PubMed:12519745, PubMed:20074060). Plays an important role in the differential regulation of pro-autophagy (composed of PIK3C3, BECN1, PIK3R4 and UVRAG or ATG14) and non-autophagy (composed of PIK3C3, BECN1 and PIK3R4) complexes, in response to glucose starvation (By similarity). Can inhibit the non-autophagy complex by phosphorylating PIK3C3 and can activate the pro-autophagy complex by phosphorylating BECN1 (By similarity). Upon glucose starvation, promotes ARF6 activation in a kinase-independent manner leading to cell migration (PubMed:36017701). Upon glucose deprivation mediates the phosphorylation of ACSS2 at 'Ser-659', which exposes the nuclear localization signal of ACSS2, required for its interaction with KPNA1 and nuclear translocation (PubMed:28552616). 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Neurodegenerative Diseases: Implications and Therapeutic Perspectives.","date":"2016","source":"Current drug targets","url":"https://pubmed.ncbi.nlm.nih.gov/26073858","citation_count":45,"is_preprint":false},{"pmid":"32944623","id":"PMC_32944623","title":"Energy stress inhibits ferroptosis via AMPK.","date":"2020","source":"Molecular & cellular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/32944623","citation_count":44,"is_preprint":false},{"pmid":"27812981","id":"PMC_27812981","title":"AMPK in Cardiovascular Diseases.","date":"2016","source":"Experientia supplementum (2012)","url":"https://pubmed.ncbi.nlm.nih.gov/27812981","citation_count":42,"is_preprint":false},{"pmid":"37733896","id":"PMC_37733896","title":"Inhibition of PRKAA/AMPK (Ser485/491) phosphorylation by crizotinib induces cardiotoxicity via perturbing autophagosome-lysosome fusion.","date":"2024","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/37733896","citation_count":41,"is_preprint":false},{"pmid":"30935723","id":"PMC_30935723","title":"AMPK in microvascular complications of diabetes and the beneficial effects of AMPK activators from plants.","date":"2018","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/30935723","citation_count":41,"is_preprint":false},{"pmid":"27343431","id":"PMC_27343431","title":"AMPK activates LXRα and ABCA1 expression in human macrophages.","date":"2016","source":"The international journal of biochemistry & cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27343431","citation_count":41,"is_preprint":false},{"pmid":"39532100","id":"PMC_39532100","title":"Opposing roles for AMPK in regulating distinct mitophagy pathways.","date":"2024","source":"Molecular 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of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and their pharmacological activities.","date":"2018","source":"Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association","url":"https://pubmed.ncbi.nlm.nih.gov/30290216","citation_count":35,"is_preprint":false},{"pmid":"32610071","id":"PMC_32610071","title":"Genetic loss of AMPK-glycogen binding destabilises AMPK and disrupts metabolism.","date":"2020","source":"Molecular metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/32610071","citation_count":35,"is_preprint":false},{"pmid":"34681581","id":"PMC_34681581","title":"AMPK Localization: A Key to Differential Energy Regulation.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/34681581","citation_count":34,"is_preprint":false},{"pmid":"30304773","id":"PMC_30304773","title":"AMP-Activated Protein (AMPK) in Pathophysiology of Pregnancy Complications.","date":"2018","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/30304773","citation_count":34,"is_preprint":false},{"pmid":"19273172","id":"PMC_19273172","title":"Cytokine Regulation of AMPK signalling.","date":"2009","source":"Frontiers in bioscience (Landmark edition)","url":"https://pubmed.ncbi.nlm.nih.gov/19273172","citation_count":34,"is_preprint":false},{"pmid":"17950019","id":"PMC_17950019","title":"Single nucleotide polymorphisms in genes encoding LKB1 (STK11), TORC2 (CRTC2) and AMPK alpha2-subunit (PRKAA2) and risk of type 2 diabetes.","date":"2007","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/17950019","citation_count":34,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":40550,"output_tokens":5607,"usd":0.102877,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14284,"output_tokens":5346,"usd":0.102535,"stage2_stop_reason":"end_turn"},"total_usd":0.205412,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"AMPK directly phosphorylates ULK1 at Ser317 and Ser777 to activate autophagy under glucose starvation. Under nutrient sufficiency, mTOR phosphorylates ULK1 at Ser757, disrupting the AMPK-ULK1 interaction and preventing ULK1 activation.\",\n      \"method\": \"In vitro phosphorylation assay, mutagenesis, co-immunoprecipitation, genetic knockout cells\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — direct phosphorylation demonstrated in vitro and in cells with mutagenesis, replicated across multiple experimental systems\",\n      \"pmids\": [\"21258367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"AMPK directly phosphorylates raptor (the mTOR binding partner) on two conserved serine residues, inducing 14-3-3 binding to raptor; this phosphorylation is required for mTORC1 inhibition and cell-cycle arrest induced by energy stress.\",\n      \"method\": \"Proteomic/bioinformatic substrate identification, in vitro kinase assay, Co-IP, mutagenesis, genetic epistasis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay plus mutagenesis plus genetic epistasis in a single rigorous study\",\n      \"pmids\": [\"18439900\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"AMPK directly phosphorylates PGC-1alpha at Thr177 and Ser538 both in vitro and in cells; these phosphorylations are required for AMPK-dependent induction of the PGC-1alpha promoter and downstream gene expression (GLUT4, mitochondrial genes) in skeletal muscle.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, PGC-1alpha-knockout primary muscle cells, reporter assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, mutagenesis, and genetic validation in knockout cells\",\n      \"pmids\": [\"17609368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The cancer-germline ubiquitin ligase MAGE-A3/6-TRIM28 ubiquitinates and degrades AMPKα1 (PRKAA1), leading to inhibition of autophagy and activation of mTOR signaling; this represents a mechanism by which cancer cells suppress AMPK activity.\",\n      \"method\": \"Substrate screen, Co-IP, ubiquitination assay, genetic gain/loss-of-function, cell viability assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, biochemical ubiquitination assay, multiple orthogonal functional readouts in a single rigorous study\",\n      \"pmids\": [\"25679763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AMPK negatively regulates β1-integrin activity in fibroblasts by suppressing expression of the integrin-binding proteins tensin1 and tensin3; loss of AMPK upregulates tensins, which bind β1-integrins to promote fibrillar adhesion formation, cell spreading, traction stress, and fibronectin fibrillogenesis.\",\n      \"method\": \"Loss-of-function (siRNA/genetic deletion), integrin activity assay, traction force microscopy, tensin rescue/silencing experiments\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KO, KD, rescue) with defined mechanistic pathway in a single study\",\n      \"pmids\": [\"28289092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"AMPK directly phosphorylates OGT (O-GlcNAc transferase), and while this phosphorylation does not alter OGT enzymatic activity, it inhibits OGT-chromatin association, reducing histone H2B O-GlcNAcylation and gene transcription. Conversely, OGT O-GlcNAcylates AMPK and positively regulates AMPK activity, creating a feedback loop.\",\n      \"method\": \"In vitro kinase assay, Co-IP, chromatin immunoprecipitation, mutagenesis, gene transcription reporter assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay plus ChIP plus functional transcription readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"24692660\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"STIM2 (a calcium sensor) physically interacts with both AMPK and CaMKK2; increased intracellular calcium promotes AMPK colocalization with STIM2, and STIM2 deficiency attenuates calcium-induced but not energy-stress-induced AMPK activation, indicating STIM2 is a regulator of the CaMKK2-AMPK calcium-signaling axis.\",\n      \"method\": \"Co-immunoprecipitation, fluorescence microscopy colocalization, genetic knockdown, selective AMPK activation assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and genetic KD with two mechanistic readouts, single lab\",\n      \"pmids\": [\"30335546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Mitochondria-derived ROS activate AMPK indirectly (via effects on mitochondrial ATP production and changes in ATP/ADP ratio) rather than by direct oxidation of redox-sensitive cysteine residues (Cys299/Cys304) on the AMPK α subunit; mutation of these cysteines to alanine did not alter the AMPK response to H2O2.\",\n      \"method\": \"Mutagenesis (Cys→Ala), exogenous H2O2 treatment, mitochondria-targeted ROS generator (MitoParaquat), ATP/ADP ratio measurement, redox-sensitive fluorescent proteins\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis with multiple orthogonal ROS-generation tools and parallel energetic measurements; negative result for direct ROS sensing is well-controlled\",\n      \"pmids\": [\"30232152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Deletion of PRKAA (AMPKα, including the α2 isoform) causes defective autophagy, leading to accumulation of DNM1L (dynamin-1-like) and aberrant mitochondrial fragmentation in vascular endothelial cells; the autophagy receptor SQSTM1/p62 binds DNM1L and directs it to autophagosomes for degradation, linking PRKAA activity to mitochondrial fission control.\",\n      \"method\": \"Genetic knockout (Prkaa1/Prkaa2 KO mice), autophagy inhibition (chloroquine, ATG7 siRNA), autophagy activation (ATG7 overexpression, rapamycin), Co-IP of SQSTM1-DNM1L, isolated aorta contractility assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KO model plus multiple pharmacological/genetic rescue experiments with defined molecular mechanism\",\n      \"pmids\": [\"28085543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AMPK directly inhibits NIX-dependent (programmed) mitophagy by phosphorylating ULK1 at Ser556 and a newly identified site Ser694, triggering 14-3-3-mediated sequestration of ULK1. Conversely, AMPK enhances depolarization-induced (damage-induced) mitophagy by increasing Parkin phosphorylation, independently of ULK1.\",\n      \"method\": \"In vitro kinase assay, mutagenesis, mito-QC mouse model (in vivo), cell-based mitophagy assays, phosphoproteomic identification of Ser694\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro phosphorylation, mutagenesis, and in vivo validation in mito-QC mouse model with multiple orthogonal readouts\",\n      \"pmids\": [\"39532100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Blocking FBP (fructose-1,6-bisphosphate) binding to aldolase with the small molecule aldometanib prevents aldolase from associating with v-ATPase on lysosomes, thereby selectively activating the lysosomal pool of AMPK and mimicking cellular glucose starvation; this demonstrates that aldolase acts as a glucose sensor upstream of lysosomal AMPK.\",\n      \"method\": \"Chemical screen, biochemical binding assays, lysosomal AMPK activity measurement, metabolic phenotyping in rodents, C. elegans and mouse lifespan assays\",\n      \"journal\": \"Nature metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — chemical probe plus biochemical mechanism plus in vivo validation across multiple model organisms\",\n      \"pmids\": [\"36217034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"LCA (lithocholic acid) binds TULP3, which allosterically activates sirtuins; activated sirtuins deacetylate the V1E1 subunit of v-ATPase at residues K52, K99, and K191, which inhibits v-ATPase and activates AMPK through the lysosomal glucose-sensing pathway.\",\n      \"method\": \"Proteomics/Co-IP identifying TULP3 as sirtuin-interacting LCA receptor, in vitro deacetylation assays, mutagenesis (3KR deacetylation mimic), muscle-specific viral expression in aged mice, AMPK activity assays, lifespan assays in C. elegans and flies\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — biochemical reconstitution, mutagenesis, in vivo rescue, and cross-species validation in a single study\",\n      \"pmids\": [\"39695235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cordycepin (3'-deoxyadenosine) is converted intracellularly into cordycepin monophosphate, which mimics all three effects of AMP on AMPK (activation, protection from dephosphorylation, allosteric activation); AMPK activation by cordycepin is blocked by a γ-subunit mutation that prevents AMP binding, confirming the AMP-mimicry mechanism.\",\n      \"method\": \"Nucleotide quantification in intact cells, cell-free AMPK assays, genetic AMPK knockout, γ-subunit mutagenesis\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro reconstitution, mutagenesis, and genetic KO validation with multiple orthogonal readouts\",\n      \"pmids\": [\"31991096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Deconvolution of AMPK adenine nucleotide binding established that CBS3 (not CBS1) is the high-affinity exchangeable AMP/ADP/ATP-binding site in the γ-subunit; AMP binding at CBS4 increases AMP affinity at CBS3 by ~100-fold and reverses CBS3's AMP/ATP preference. NADPH (in addition to NADH) directly and competitively binds AMPK at the CBS3 site.\",\n      \"method\": \"Quantitative competition binding assays, hydrogen-deuterium exchange mass spectrometry, wild-type and mutant AMPK protein complexes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — HDX-MS structural method combined with quantitative binding assays and mutagenesis in a single comprehensive study\",\n      \"pmids\": [\"28615457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AMPK deficiency in myeloid cells increases PKM2-dependent aerobic glycolysis, leading to enhanced HMGB1 release from macrophages/monocytes and promoting sepsis; pharmacological AMPK activation (A-769662) protects against endotoxic shock, while PKM2 inhibition rescues the pro-inflammatory phenotype of AMPKα-deficient mice.\",\n      \"method\": \"Myeloid-specific AMPKα knockout mice, pharmacological activation/inhibition, HMGB1 measurement, polymicrobial sepsis model, glycolysis/lactate assays\",\n      \"journal\": \"Brain, behavior, and immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific KO plus pharmacological rescue plus defined mechanistic pathway in vivo\",\n      \"pmids\": [\"29109024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"AMPKα2-deficient (Prkaa2−/−) mice display elevated blood glucose, reduced plasma insulin in the fed state, in vivo insulin resistance, and reduced muscle glycogen synthesis; the insulin resistance was not intrinsic to skeletal muscle (isolated muscle glucose transport was normal), suggesting AMPKα2 regulates systemic glucose homeostasis partly through modulation of the autonomic nervous system (increased catecholamine excretion).\",\n      \"method\": \"Genetic knockout mouse (AMPKα2−/−), hyperinsulinemic-euglycaemic clamp, isolated muscle glucose transport assay, dominant-negative AMPK transgenic muscle mice, catecholamine measurement\",\n      \"journal\": \"Biochemical Society transactions\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KO with clamp studies plus mechanistic dissection using dominant-negative transgenic and isolated tissue experiments\",\n      \"pmids\": [\"12546688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"AMPKα2 nuclear localization employs a nuclear localization signal (NLS) present on the AMPKα2 kinase domain, while nuclear export involves RanGTPase-CRM1-mediated recognition of a nuclear export sequence (NES) on the α subunit; nucleo-cytoplasmic shuttling is regulated by starvation, exercise, heat shock, oxidants, cell density, and circadian rhythm.\",\n      \"method\": \"Subcellular fractionation, nuclear export sequence deletion/mutation analysis, CRM1 inhibition, fluorescence localization studies (compiled from multiple studies reviewed)\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — review synthesizing multiple localization studies; individual NLS/NES experiments documented but full methodology not detailed in abstract\",\n      \"pmids\": [\"34681581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AMPK-glycogen binding via the β2 subunit CBM (disrupted by W98A knock-in mutation) is required to stabilize AMPK protein and kinase activity in skeletal muscle; β2-W98A KI mice show reduced total AMPK protein and kinase activity in muscle, increased adiposity, impaired whole-body glucose handling, and reduced maximal exercise capacity.\",\n      \"method\": \"Whole-body knock-in mouse (β2-W98A), systematic metabolic phenotyping, AMPK kinase activity assays, tissue AMPK protein quantification\",\n      \"journal\": \"Molecular metabolism\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knock-in genetic model with comprehensive molecular and physiological phenotyping\",\n      \"pmids\": [\"32610071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Crizotinib inhibits PRKAA/AMPK phosphorylation at Ser485/491, impairing autophagosome-lysosome fusion and preventing MET protein degradation; metformin restores PRKAA (Ser485/491) phosphorylation, re-activates autophagy flux, and rescues crizotinib-induced cardiomyocyte injury.\",\n      \"method\": \"In vitro cardiomyocyte models, in vivo mouse cardiotoxicity model, autophagy flux assay, PRKAA phosphorylation analysis, metformin rescue experiment, MET silencing\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo plus in vitro models with pharmacological and genetic intervention, single lab\",\n      \"pmids\": [\"37733896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PRKAA/AMPK is activated by HBV-induced oxidative stress and restricts HBV production by promoting autolysosome-dependent degradation through stimulation of cellular ATP levels, leading to depletion of autophagic vacuoles that HBV depends on for replication.\",\n      \"method\": \"HBV infection cell model, PRKAA activation/inhibition, autophagy flux assay, ATP measurement, autophagic vacuole quantification\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanistic dissection with pharmacological and genetic tools in a defined cellular model, single lab\",\n      \"pmids\": [\"27305174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"AMPK promotes osteogenesis and inhibits adipogenesis through downregulation of the transcriptional repressor Gfi1, which dissociates from the osteopontin (OPN) promoter upon AMPK activation, resulting in OPN upregulation; overexpression or dominant-negative Gfi1 modulates osteogenesis and adipogenesis accordingly.\",\n      \"method\": \"Lentiviral AMPKα overexpression, Gfi1 overexpression/dominant-negative constructs, luciferase reporter (OPN promoter), ChIP (Gfi1 binding), ectopic bone formation assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assay plus ChIP plus in vivo ectopic bone formation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27283242\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"AMPK activates LXRα expression in macrophages, which then transcriptionally upregulates ABCA1 via binding to the LXR-responsive element in the ABCA1 promoter, resulting in increased cholesterol efflux; LXRβ silencing did not affect this pathway, establishing LXRα specificity.\",\n      \"method\": \"Pharmacological and genetic AMPK activation/knockdown, luciferase reporter assay, chromatin immunoprecipitation (ChIP), LXRα/β siRNA silencing, cholesterol efflux assay\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus reporter assay plus genetic KD with functional cholesterol efflux readout, single lab\",\n      \"pmids\": [\"27343431\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRKAA2 (AMPKα2) is the catalytic subunit of the heterotrimeric AMPK complex, an energy sensor activated by elevated AMP/ADP:ATP ratios (sensed via γ-subunit CBS sites, particularly CBS3) and calcium signaling (via CaMKK2/STIM2); once activated, it directly phosphorylates a broad set of substrates—including ULK1 (Ser317/777) to initiate autophagy, raptor to suppress mTORC1, PGC-1α (Thr177/Ser538) to drive mitochondrial biogenesis, OGT to modulate chromatin O-GlcNAcylation, NIX-pathway effectors (ULK1 Ser556/694) to restrain programmed mitophagy while enhancing damage-induced mitophagy via Parkin, and tensins to regulate integrin activity—while the AMPKα2 protein itself is subject to degradation by the MAGE-A3/6-TRIM28 ubiquitin ligase in cancer cells and requires glycogen binding (via the β-subunit) for stability.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PRKAA2 (AMPK\\u03b12) is the catalytic subunit of the AMPK energy-sensing kinase, which couples cellular energy status to a broad transcriptional and post-translational program controlling autophagy, mitochondrial homeostasis, and systemic glucose metabolism [#0, #15]. Sensing of low energy occurs through adenine-nucleotide binding to the \\u03b3-subunit, where CBS3 is the high-affinity exchangeable AMP/ADP/ATP site whose nucleotide preference is allosterically tuned by AMP occupancy at CBS4 and which also binds NADPH competitively [#13]; this AMP-sensing mechanism is exploited pharmacologically by cordycepin monophosphate, which mimics all three effects of AMP and is abolished by a \\u03b3-subunit AMP-binding mutation [#12]. A parallel lysosomal glucose-sensing branch activates AMPK when low fructose-1,6-bisphosphate releases aldolase from v-ATPase, a circuit that can be engaged chemically (aldometanib) or via sirtuin-dependent deacetylation of the v-ATPase V1E1 subunit [#10, #11], while a calcium branch operates through STIM2-coordinated CaMKK2 signaling [#6]. Once active, AMPK\\u03b12 directly phosphorylates a defined substrate set: ULK1 (Ser317/Ser777) to initiate starvation autophagy in opposition to mTOR [#0], raptor to suppress mTORC1 and enforce energy-stress cell-cycle arrest [#1], PGC-1\\u03b1 (Thr177/Ser538) to drive mitochondrial and GLUT4 gene expression in muscle [#2], and ULK1 (Ser556/Ser694) to restrain NIX-dependent programmed mitophagy while promoting Parkin-dependent damage-induced mitophagy [#9]. Through its control of autophagic flux, AMPK\\u03b12 also governs SQSTM1/p62-mediated clearance of the fission factor DNM1L to maintain mitochondrial morphology [#8]. Beyond canonical metabolic targets, AMPK phosphorylates OGT to displace it from chromatin and reduce histone H2B O-GlcNAcylation in a reciprocal regulatory loop [#5], and suppresses tensin expression to limit \\u03b21-integrin activity and fibrillar adhesion [#4]. Physiologically, Prkaa2 loss produces systemic insulin resistance and hyperglycemia involving autonomic signaling rather than a muscle-intrinsic transport defect [#15], and AMPK stability and activity depend on \\u03b2-subunit glycogen binding [#17]. The catalytic \\u03b1-subunit carries its own NLS and CRM1-dependent NES enabling regulated nucleo-cytoplasmic shuttling [#16].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established the physiological role of AMPK\\u03b12 in systemic glucose homeostasis and showed its insulin-resistance phenotype is not muscle-intrinsic, redirecting attention to whole-body neuroendocrine control.\",\n      \"evidence\": \"AMPK\\u03b12 knockout mice with hyperinsulinemic-euglycaemic clamp, isolated muscle transport assays, and catecholamine measurement\",\n      \"pmids\": [\"12546688\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not identify the AMPK\\u03b12 substrates mediating autonomic effects\", \"Tissue source of systemic insulin resistance not pinpointed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined a direct transcriptional output of AMPK by showing it phosphorylates PGC-1\\u03b1 to drive mitochondrial biogenesis gene programs.\",\n      \"evidence\": \"In vitro kinase assay, mutagenesis, and PGC-1\\u03b1-knockout primary muscle cells with promoter reporter\",\n      \"pmids\": [\"17609368\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of \\u03b12 vs \\u03b11 not isolated\", \"Downstream coactivator partners on the promoter not mapped\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Connected AMPK to mTORC1 inhibition by identifying raptor as a direct substrate whose phosphorylation recruits 14-3-3 and enforces energy-stress cell-cycle arrest.\",\n      \"evidence\": \"Proteomic substrate identification, in vitro kinase assay, Co-IP, mutagenesis, and genetic epistasis\",\n      \"pmids\": [\"18439900\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative balance between raptor and other mTORC1 inputs unresolved\", \"\\u03b1-isoform specificity not addressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved how AMPK initiates autophagy, showing direct ULK1 phosphorylation and reciprocal mTOR-mediated disruption of the AMPK-ULK1 interaction under nutrient sufficiency.\",\n      \"evidence\": \"In vitro phosphorylation, mutagenesis, Co-IP, and genetic knockout cells\",\n      \"pmids\": [\"21258367\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not separate \\u03b11 from \\u03b12 catalytic contribution\", \"In vivo relevance of specific sites in tissues not tested here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Extended AMPK function to chromatin regulation by showing it phosphorylates OGT to block chromatin association and reduce histone O-GlcNAcylation, with reciprocal OGT regulation of AMPK.\",\n      \"evidence\": \"In vitro kinase assay, Co-IP, ChIP, mutagenesis, and transcription reporter\",\n      \"pmids\": [\"24692660\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide scope of affected loci not defined\", \"Physiological contexts where the feedback loop dominates unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed how cancer cells suppress AMPK by identifying MAGE-A3/6-TRIM28 as a ubiquitin ligase that degrades the catalytic \\u03b1-subunit, inhibiting autophagy and activating mTOR.\",\n      \"evidence\": \"Substrate screen, Co-IP, ubiquitination assay, and gain/loss-of-function with viability readouts (demonstrated for \\u03b11)\",\n      \"pmids\": [\"25679763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration on the \\u03b12 isoform not shown\", \"Degron residues on AMPK\\u03b1 not mapped\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked AMPK to lineage and lipid-handling transcriptional programs, including osteogenesis/adipogenesis via Gfi1-OPN, macrophage cholesterol efflux via LXR\\u03b1-ABCA1, and antiviral restriction of HBV through autolysosomal degradation.\",\n      \"evidence\": \"Reporter assays, ChIP, siRNA silencing, ectopic bone formation, and HBV infection autophagy-flux models\",\n      \"pmids\": [\"27283242\", \"27343431\", \"27305174\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"AMPK\\u03b12-specific requirement not isolated from \\u03b11\", \"Direct kinase substrates in each pathway not identified\", \"Single-lab observations per pathway\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mapped the nucleotide-sensing core, establishing CBS3 as the high-affinity exchangeable site with CBS4-driven allostery, and dissected AMPK's roles in mitochondrial fission control, integrin/adhesion regulation, and macrophage inflammation.\",\n      \"evidence\": \"HDX-MS and competition binding (CBS3); Prkaa1/2 KO mice with SQSTM1-DNM1L Co-IP; tensin loss/rescue with traction microscopy; myeloid-specific KO sepsis model with PKM2 rescue\",\n      \"pmids\": [\"28615457\", \"28085543\", \"28289092\", \"29109024\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CBS3 nucleotide work uses reconstituted complexes, not in-cell occupancy\", \"Direct kinase substrates for tensin and PKM2 effects not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Distinguished AMPK's energy and calcium activation inputs and excluded direct ROS oxidation, showing STIM2 regulates the calcium/CaMKK2 axis while mitochondrial ROS act indirectly via ATP/ADP changes.\",\n      \"evidence\": \"Reciprocal Co-IP and colocalization with STIM2; Cys\\u2192Ala mutagenesis with mitochondria-targeted ROS generators and ATP/ADP measurement\",\n      \"pmids\": [\"30335546\", \"30232152\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"STIM2 finding is single-lab, Medium confidence\", \"Molecular mechanism of STIM2-AMPK colocalization not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined determinants of AMPK pharmacology and stability, validating AMP-mimicry by cordycepin monophosphate via the \\u03b3-subunit and establishing \\u03b2-subunit glycogen binding as required for AMPK protein stability and activity in muscle.\",\n      \"evidence\": \"Nucleotide quantification, cell-free assays, \\u03b3-subunit mutagenesis (cordycepin); \\u03b22-W98A knock-in mice with metabolic phenotyping\",\n      \"pmids\": [\"31991096\", \"32610071\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Glycogen-binding stabilization mechanism at the molecular level not detailed\", \"\\u03b12-specific stability dependence not isolated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Synthesized the regulated nucleo-cytoplasmic shuttling of AMPK\\u03b12, attributing nuclear import to an NLS on the kinase domain and export to RanGTPase-CRM1 recognition of an \\u03b1-subunit NES.\",\n      \"evidence\": \"Review compiling fractionation, NES deletion/mutation, and CRM1-inhibition localization studies\",\n      \"pmids\": [\"34681581\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Review-level synthesis, not primary experiments\", \"Stimulus-specific shuttling kinetics not quantified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established a lysosomal glucose-sensing branch upstream of AMPK in which low FBP releases aldolase from v-ATPase to activate the lysosomal AMPK pool, druggable by aldometanib.\",\n      \"evidence\": \"Chemical screen, biochemical binding assays, lysosomal AMPK activity, and lifespan/metabolic phenotyping across rodents and C. elegans\",\n      \"pmids\": [\"36217034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular bridge from v-ATPase to AMPK activation not fully defined\", \"\\u03b12-specific contribution to lysosomal pool not isolated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Refined AMPK's mitophagy control by showing it phosphorylates ULK1 (Ser556/Ser694) to restrain NIX-dependent programmed mitophagy while promoting Parkin-dependent damage mitophagy, and uncovered an upstream LCA-TULP3-sirtuin-v-ATPase route to AMPK activation.\",\n      \"evidence\": \"In vitro kinase assays, mutagenesis, mito-QC and aged-mouse in vivo models, phosphoproteomics; LCA-TULP3 Co-IP with deacetylation assays and cross-species lifespan\",\n      \"pmids\": [\"39532100\", \"39695235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How AMPK switches between restraining and promoting mitophagy contextually not fully resolved\", \"Direct AMPK substrate in the Parkin arm not specified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Illustrated drug-induced AMPK dysregulation, showing crizotinib lowers AMPK Ser485/491 phosphorylation to impair autophagosome-lysosome fusion and cardiomyocyte clearance of MET, reversible by metformin.\",\n      \"evidence\": \"In vitro and in vivo cardiotoxicity models with autophagy-flux assays, phosphorylation analysis, and MET silencing\",\n      \"pmids\": [\"37733896\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab Medium-confidence study\", \"Whether Ser485/491 change is direct or indirect not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved which AMPK functions are specifically executed by the \\u03b12 (PRKAA2) catalytic isoform versus \\u03b11, since most substrate and phenotype data are reported for AMPK\\u03b1 generically.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No isoform-resolved substrate map\", \"\\u03b12-specific structural and localization determinants incompletely defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 5, 9]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 2, 9]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [12, 13]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [13, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [16, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [16]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [0, 8, 9, 19]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 10, 15, 21]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [7, 13, 12]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 5, 20, 21]}\n    ],\n    \"complexes\": [\"AMPK heterotrimer\"],\n    \"partners\": [\"ULK1\", \"RPTOR\", \"PPARGC1A\", \"OGT\", \"STIM2\", \"CAMKK2\", \"PRKN\", \"TRIM28\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}