{"gene":"PRKAG1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2010,"finding":"Targeted disruption of the mouse AMPKγ1 gene (Prkag1) causes regenerative hemolytic anemia by increasing sequestration of abnormal erythrocytes, leading to splenomegaly and iron accumulation. AMPKγ1-deficient erythrocytes show greater membrane rigidity, reduced deformability under shear stress, and elevated phosphorylation of adducin (a protein promoting spectrin-actin binding), demonstrating that AMPKγ1 is required for maintenance of erythrocyte membrane elasticity.","method":"Genetic knockout (Prkag1-/- mice), osmotic hemolysis assay, ektacytometry (deformability under shear stress), phosphorylation analysis of cytoskeletal proteins, histology/iron staining","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with well-defined cellular and molecular phenotype, multiple orthogonal methods (osmotic hemolysis, deformability, phospho-protein analysis), single rigorous study","pmids":["20881209"],"is_preprint":false},{"year":2020,"finding":"PRKDC (DNA-PKcs) phosphorylates PRKAG1/AMPKγ1 at Ser192 and Thr284 under basal conditions. Alanine substitution of these sites (S192A/T284A double mutant) reduces AMPK complex activation and inhibits lysosomal localization of the AMPK complex and its starvation-induced association with STK11/LKB1, without affecting nucleotide-sensing capacity. Thus, PRKDC-mediated phosphorylation of PRKAG1 primes AMPK for lysosomal activation by STK11.","method":"Co-immunoprecipitation (PRKDC–AMPK complex), in vitro kinase assay, site-directed mutagenesis (S192A/T284A), immunofluorescence/subcellular fractionation for lysosomal localization, siRNA knockdown, autophagic flux assays","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro phosphorylation assay, site-directed mutagenesis, reciprocal Co-IP, and functional localization readout, all in a single rigorous study","pmids":["31983282"],"is_preprint":false},{"year":2023,"finding":"During the fasting-refeeding cycle, AMPKγ1 (Prkag1) and AMPKγ2 (Prkag2) show inverse oscillatory expression in adipose tissue of young killifish; aging blunts this regulation and reduces Prkag1 expression. Transgenic overexpression of AMPKγ1 in aged fish counteracts the fasting-like transcriptional quiescence program in adipose tissue, improves metabolic health, and extends longevity, establishing a direct role for AMPKγ1 complex activity in metabolic homeostasis and healthy aging.","method":"Transcriptomic analysis (RNA-seq), transgenic overexpression of Prkag1 in killifish, longitudinal survival and metabolic phenotyping","journal":"Nature aging","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic rescue with defined phenotypic readouts (longevity, metabolic health) and transcriptomic support, but single-organism model and no in vitro biochemical reconstitution","pmids":["37957359"],"is_preprint":false},{"year":2020,"finding":"Knockdown of AMPKγ1 (PRKAG1) in cardiac myocytes abolishes adiponectin-induced enhancement of mitochondrial oxygen consumption rate, ATP production, and spare respiratory capacity, placing AMPKγ1 as a required component of the AMPK complex mediating adiponectin's bioenergetic effects through a succinate dehydrogenase (SDH)- and Sirt3-dependent mechanism.","method":"siRNA knockdown of AMPKγ1, Seahorse mitochondrial respiration assay, SDH assembly/activity assays, overexpression of Sdhaf1 and Sirt3","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with specific metabolic readout (OCR/ATP), multiple pathway components tested, but single lab and no direct biochemical reconstitution of the AMPK complex","pmids":["33271223"],"is_preprint":false},{"year":2025,"finding":"PRKAG1 interacts with nitazoxanide (NTZ); knockdown of PRKAG1 enhances cellular autophagy and inhibits Japanese encephalitis virus (JEV) replication in a manner synergistic with NTZ, while PRKAG1 overexpression reduces autophagy and promotes JEV replication. Phosphorylation proteomics of PRKAG1-silenced cells revealed increased phosphorylation of TP53 and GSK3B, indicating PRKAG1 modulates these signaling pathways to regulate autophagy and antiviral responses.","method":"Western blotting, solvent-induced protein precipitation (SIP), RNA interference (siRNA knockdown), overexpression, JEV replication assay, phosphorylation quantitative proteomics","journal":"Veterinary microbiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — KD and OE with specific viral and autophagy readouts, plus phospho-proteomics; binding shown by SIP but not reconstituted in vitro; single lab","pmids":["41033165"],"is_preprint":false},{"year":2019,"finding":"Transient knockdown of Prkag1 in rat insulinoma INS-1E cells elevated glucose-stimulated insulin secretion (GSIS), possibly through mTOR signaling, suggesting PRKAG1 negatively regulates insulin secretion in pancreatic beta cells.","method":"siRNA knockdown in INS-1E cells, GSIS assay","journal":"Environment international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single KD experiment with one functional readout (GSIS), no pathway mechanistic follow-up, single lab","pmids":["31195220"],"is_preprint":false},{"year":2015,"finding":"In a genome-wide RNAi screen in Drosophila cells, SNF4Aγ (the Drosophila ortholog of PRKAG1) was identified as a regulator of nuclear actin localization. This role was validated in mammalian cells by showing that knockdown of PRKAG1 affects nuclear actin levels.","method":"Genome-wide RNAi screen in Drosophila cells, validation by RNAi knockdown in mammalian cells, fluorescence microscopy for nuclear actin","journal":"Journal of cell science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — RNAi screen hit validated by single KD experiment in mammalian cells; mechanistic basis of nuclear actin effect not established","pmids":["26021350"],"is_preprint":false},{"year":2024,"finding":"Joint affinity chromatography and solvent-induced protein precipitation / thermal proteome profiling identified PRKAG1 as a candidate binding target of nitazoxanide in mammalian (VERO) cells; fluorescent probes localized the binding around the nuclear membrane.","method":"Affinity chromatography with tizoxanide-biotin, mass spectrometry, drug affinity response target stability (DARTS), solvent-induced protein precipitation (SIP), thermal proteome profiling (TPP), fluorescent probe localization","journal":"Current drug targets","confidence":"Low","confidence_rationale":"Tier 3 / Weak — chemical proteomics binding evidence without mutagenesis or functional reconstitution; single study","pmids":["39171461"],"is_preprint":false}],"current_model":"PRKAG1 encodes the γ1 regulatory subunit of the AMPK heterotrimer, which senses cellular energy status; it is required for erythrocyte membrane elasticity (loss causes hemolytic anemia with elevated adducin phosphorylation), is phosphorylated by PRKDC/DNA-PKcs at Ser192/Thr284 to prime lysosomal AMPK activation by STK11, mediates adiponectin-driven mitochondrial bioenergetics in cardiomyocytes via an SDH/Sirt3-dependent pathway, and regulates a fasting-refeeding transcriptional program in adipose tissue whose age-related decline contributes to metabolic dysfunction."},"narrative":{"mechanistic_narrative":"PRKAG1 encodes the γ1 regulatory subunit of the AMP-activated protein kinase (AMPK) heterotrimer, integrating cellular energy status into diverse physiological programs spanning erythrocyte mechanics, lysosomal AMPK activation, and metabolic homeostasis [PMID:20881209, PMID:31983282, PMID:37957359]. Its activity is gated upstream by PRKDC/DNA-PKcs, which phosphorylates PRKAG1 at Ser192 and Thr284 to prime the AMPK complex for lysosomal localization and starvation-induced association with STK11/LKB1, independently of the subunit's nucleotide-sensing function [PMID:31983282]. Functionally, PRKAG1 is required to maintain erythrocyte membrane elasticity: its loss increases adducin phosphorylation, rigidifies the membrane, and produces regenerative hemolytic anemia [PMID:20881209]. In metabolic tissues it drives a fasting–refeeding transcriptional program in adipose tissue whose age-related decline impairs metabolic health, such that restoring PRKAG1 extends longevity [PMID:37957359], and in cardiomyocytes it is a required component for adiponectin-induced mitochondrial respiration through an SDH- and Sirt3-dependent pathway [PMID:33271223].","teleology":[{"year":2010,"claim":"Established that the AMPK γ1 subunit has a non-redundant cellular role beyond classical energy sensing by demonstrating its requirement for erythrocyte membrane integrity.","evidence":"Prkag1-/- knockout mice with osmotic hemolysis, ektacytometry, and phospho-protein analysis of cytoskeletal adducin","pmids":["20881209"],"confidence":"High","gaps":["Whether AMPK directly phosphorylates/dephosphorylates adducin or acts through intermediary kinases is not resolved","Does not address PRKAG1's role in nucleated cells or other tissues"]},{"year":2020,"claim":"Defined an upstream regulatory input on PRKAG1, showing that DNA-PKcs phosphorylation primes the AMPK complex for lysosomal activation rather than acting through nucleotide sensing.","evidence":"In vitro kinase assay, S192A/T284A site-directed mutagenesis, reciprocal Co-IP, and lysosomal localization/autophagic flux readouts","pmids":["31983282"],"confidence":"High","gaps":["Structural basis for how Ser192/Thr284 phosphorylation promotes STK11 association is unknown","Physiological contexts in which PRKDC controls AMPK activation in vivo are not established"]},{"year":2020,"claim":"Placed PRKAG1 as a required node linking adiponectin signaling to mitochondrial bioenergetics in cardiac cells.","evidence":"siRNA knockdown of AMPKγ1 in cardiac myocytes, Seahorse respirometry, SDH activity assays, Sdhaf1/Sirt3 overexpression","pmids":["33271223"],"confidence":"Medium","gaps":["Direct biochemical reconstitution of the AMPK complex with SDH/Sirt3 not performed","Single lab, single cell-type readout"]},{"year":2023,"claim":"Demonstrated a causal role for PRKAG1 in the fasting–refeeding transcriptional program and in healthy aging via gain-of-function rescue.","evidence":"RNA-seq, transgenic Prkag1 overexpression in killifish, longitudinal survival and metabolic phenotyping","pmids":["37957359"],"confidence":"Medium","gaps":["No in vitro biochemical reconstitution of the underlying transcriptional mechanism","Single-organism (killifish) model; mammalian validation absent"]},{"year":2025,"claim":"Linked PRKAG1 levels to autophagy and antiviral responses, implicating downstream TP53 and GSK3B phosphorylation.","evidence":"siRNA knockdown and overexpression with JEV replication and autophagy assays, plus phospho-proteomics in mammalian cells","pmids":["41033165"],"confidence":"Medium","gaps":["Nitazoxanide binding shown by solvent-induced precipitation but not reconstituted in vitro","Whether TP53/GSK3B phosphorylation changes are direct AMPK-dependent effects is unresolved"]},{"year":2019,"claim":"Suggested a tissue-specific negative role for PRKAG1 in pancreatic beta-cell insulin secretion.","evidence":"Transient siRNA knockdown in INS-1E insulinoma cells with glucose-stimulated insulin secretion assay","pmids":["31195220"],"confidence":"Low","gaps":["Single knockdown with one functional readout; no mechanistic follow-up on the proposed mTOR link","Not validated in primary beta cells or in vivo"]},{"year":2015,"claim":"Identified an unexpected role for the PRKAG1 ortholog in regulating nuclear actin localization.","evidence":"Genome-wide RNAi screen in Drosophila with validation by knockdown and fluorescence microscopy in mammalian cells","pmids":["26021350"],"confidence":"Low","gaps":["Mechanistic basis of the nuclear actin effect not established","Validated only by single knockdown in mammalian cells"]},{"year":null,"claim":"How the diverse PRKAG1-dependent phenotypes (membrane mechanics, lysosomal activation, mitochondrial respiration, aging, autophagy) are unified at the level of AMPK complex assembly and substrate selection remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of how PRKAG1 phosphorylation or expression level reroutes AMPK output across tissues","Direct substrates downstream of PRKAG1 in each context largely undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,3]}],"localization":[{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[1,4]}],"complexes":["AMPK heterotrimer"],"partners":["PRKDC","STK11"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P54619","full_name":"5'-AMP-activated protein kinase subunit gamma-1","aliases":[],"length_aa":331,"mass_kda":37.6,"function":"AMP/ATP-binding subunit of AMP-activated protein kinase (AMPK), an energy sensor protein kinase that plays a key role in regulating cellular energy metabolism (PubMed:21680840, PubMed:24563466). 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:21680840, PubMed:24563466). AMPK acts via direct phosphorylation of metabolic enzymes, and by longer-term effects via phosphorylation of transcription regulators (PubMed:21680840, PubMed:24563466). Also acts as a regulator of cellular polarity by remodeling the actin cytoskeleton; probably by indirectly activating myosin (PubMed:21680840, PubMed:24563466). Gamma non-catalytic subunit mediates binding to AMP, ADP and ATP, leading to activate or inhibit AMPK: AMP-binding results in allosteric activation of alpha catalytic subunit (PRKAA1 or PRKAA2) both by inducing phosphorylation and preventing dephosphorylation of catalytic subunits (PubMed:21680840, PubMed:24563466). ADP also stimulates phosphorylation, without stimulating already phosphorylated catalytic subunit (PubMed:21680840, PubMed:24563466). ATP promotes dephosphorylation of catalytic subunit, rendering the AMPK enzyme inactive (PubMed:21680840, PubMed:24563466)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/P54619/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PRKAG1","classification":"Not Classified","n_dependent_lines":59,"n_total_lines":1208,"dependency_fraction":0.048841059602649006},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PRKAA1","stoichiometry":10.0}],"url":"https://opencell.sf.czbiohub.org/search/PRKAG1","total_profiled":1310},"omim":[{"mim_id":"615002","title":"CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE KINASE 2, BETA; CAMKK2","url":"https://www.omim.org/entry/615002"},{"mim_id":"610594","title":"FOLLICULIN-INTERACTING PROTEIN 1; FNIP1","url":"https://www.omim.org/entry/610594"},{"mim_id":"604976","title":"PROTEIN KINASE, AMP-ACTIVATED, NONCATALYTIC, GAMMA-3; PRKAG3","url":"https://www.omim.org/entry/604976"},{"mim_id":"602743","title":"PROTEIN KINASE, AMP-ACTIVATED, NONCATALYTIC, GAMMA-2; PRKAG2","url":"https://www.omim.org/entry/602743"},{"mim_id":"602742","title":"PROTEIN KINASE, AMP-ACTIVATED, NONCATALYTIC, GAMMA-1; PRKAG1","url":"https://www.omim.org/entry/602742"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nuclear bodies","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Basal body","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PRKAG1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P54619","domains":[{"cath_id":"3.10.580.10","chopping":"37-179","consensus_level":"medium","plddt":89.2052,"start":37,"end":179},{"cath_id":"3.10.580.10","chopping":"186-324","consensus_level":"medium","plddt":91.2493,"start":186,"end":324}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P54619","model_url":"https://alphafold.ebi.ac.uk/files/AF-P54619-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P54619-F1-predicted_aligned_error_v6.png","plddt_mean":86.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PRKAG1","jax_strain_url":"https://www.jax.org/strain/search?query=PRKAG1"},"sequence":{"accession":"P54619","fasta_url":"https://rest.uniprot.org/uniprotkb/P54619.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P54619/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P54619"}},"corpus_meta":[{"pmid":"17547415","id":"PMC_17547415","title":"New 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medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39137672","citation_count":0,"is_preprint":false},{"pmid":"41666498","id":"PMC_41666498","title":"Genome-wide association study identified genomic regions and putative candidate genes affecting different backfat layers in Landrace pigs.","date":"2026","source":"Animal : an international journal of animal bioscience","url":"https://pubmed.ncbi.nlm.nih.gov/41666498","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":26235,"output_tokens":2384,"usd":0.057232,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9501,"output_tokens":2576,"usd":0.055953,"stage2_stop_reason":"end_turn"},"total_usd":0.113185,"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\": 2010,\n      \"finding\": \"Targeted disruption of the mouse AMPKγ1 gene (Prkag1) causes regenerative hemolytic anemia by increasing sequestration of abnormal erythrocytes, leading to splenomegaly and iron accumulation. AMPKγ1-deficient erythrocytes show greater membrane rigidity, reduced deformability under shear stress, and elevated phosphorylation of adducin (a protein promoting spectrin-actin binding), demonstrating that AMPKγ1 is required for maintenance of erythrocyte membrane elasticity.\",\n      \"method\": \"Genetic knockout (Prkag1-/- mice), osmotic hemolysis assay, ektacytometry (deformability under shear stress), phosphorylation analysis of cytoskeletal proteins, histology/iron staining\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with well-defined cellular and molecular phenotype, multiple orthogonal methods (osmotic hemolysis, deformability, phospho-protein analysis), single rigorous study\",\n      \"pmids\": [\"20881209\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PRKDC (DNA-PKcs) phosphorylates PRKAG1/AMPKγ1 at Ser192 and Thr284 under basal conditions. Alanine substitution of these sites (S192A/T284A double mutant) reduces AMPK complex activation and inhibits lysosomal localization of the AMPK complex and its starvation-induced association with STK11/LKB1, without affecting nucleotide-sensing capacity. Thus, PRKDC-mediated phosphorylation of PRKAG1 primes AMPK for lysosomal activation by STK11.\",\n      \"method\": \"Co-immunoprecipitation (PRKDC–AMPK complex), in vitro kinase assay, site-directed mutagenesis (S192A/T284A), immunofluorescence/subcellular fractionation for lysosomal localization, siRNA knockdown, autophagic flux assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro phosphorylation assay, site-directed mutagenesis, reciprocal Co-IP, and functional localization readout, all in a single rigorous study\",\n      \"pmids\": [\"31983282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"During the fasting-refeeding cycle, AMPKγ1 (Prkag1) and AMPKγ2 (Prkag2) show inverse oscillatory expression in adipose tissue of young killifish; aging blunts this regulation and reduces Prkag1 expression. Transgenic overexpression of AMPKγ1 in aged fish counteracts the fasting-like transcriptional quiescence program in adipose tissue, improves metabolic health, and extends longevity, establishing a direct role for AMPKγ1 complex activity in metabolic homeostasis and healthy aging.\",\n      \"method\": \"Transcriptomic analysis (RNA-seq), transgenic overexpression of Prkag1 in killifish, longitudinal survival and metabolic phenotyping\",\n      \"journal\": \"Nature aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic rescue with defined phenotypic readouts (longevity, metabolic health) and transcriptomic support, but single-organism model and no in vitro biochemical reconstitution\",\n      \"pmids\": [\"37957359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Knockdown of AMPKγ1 (PRKAG1) in cardiac myocytes abolishes adiponectin-induced enhancement of mitochondrial oxygen consumption rate, ATP production, and spare respiratory capacity, placing AMPKγ1 as a required component of the AMPK complex mediating adiponectin's bioenergetic effects through a succinate dehydrogenase (SDH)- and Sirt3-dependent mechanism.\",\n      \"method\": \"siRNA knockdown of AMPKγ1, Seahorse mitochondrial respiration assay, SDH assembly/activity assays, overexpression of Sdhaf1 and Sirt3\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with specific metabolic readout (OCR/ATP), multiple pathway components tested, but single lab and no direct biochemical reconstitution of the AMPK complex\",\n      \"pmids\": [\"33271223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PRKAG1 interacts with nitazoxanide (NTZ); knockdown of PRKAG1 enhances cellular autophagy and inhibits Japanese encephalitis virus (JEV) replication in a manner synergistic with NTZ, while PRKAG1 overexpression reduces autophagy and promotes JEV replication. Phosphorylation proteomics of PRKAG1-silenced cells revealed increased phosphorylation of TP53 and GSK3B, indicating PRKAG1 modulates these signaling pathways to regulate autophagy and antiviral responses.\",\n      \"method\": \"Western blotting, solvent-induced protein precipitation (SIP), RNA interference (siRNA knockdown), overexpression, JEV replication assay, phosphorylation quantitative proteomics\",\n      \"journal\": \"Veterinary microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — KD and OE with specific viral and autophagy readouts, plus phospho-proteomics; binding shown by SIP but not reconstituted in vitro; single lab\",\n      \"pmids\": [\"41033165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Transient knockdown of Prkag1 in rat insulinoma INS-1E cells elevated glucose-stimulated insulin secretion (GSIS), possibly through mTOR signaling, suggesting PRKAG1 negatively regulates insulin secretion in pancreatic beta cells.\",\n      \"method\": \"siRNA knockdown in INS-1E cells, GSIS assay\",\n      \"journal\": \"Environment international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single KD experiment with one functional readout (GSIS), no pathway mechanistic follow-up, single lab\",\n      \"pmids\": [\"31195220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In a genome-wide RNAi screen in Drosophila cells, SNF4Aγ (the Drosophila ortholog of PRKAG1) was identified as a regulator of nuclear actin localization. This role was validated in mammalian cells by showing that knockdown of PRKAG1 affects nuclear actin levels.\",\n      \"method\": \"Genome-wide RNAi screen in Drosophila cells, validation by RNAi knockdown in mammalian cells, fluorescence microscopy for nuclear actin\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — RNAi screen hit validated by single KD experiment in mammalian cells; mechanistic basis of nuclear actin effect not established\",\n      \"pmids\": [\"26021350\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Joint affinity chromatography and solvent-induced protein precipitation / thermal proteome profiling identified PRKAG1 as a candidate binding target of nitazoxanide in mammalian (VERO) cells; fluorescent probes localized the binding around the nuclear membrane.\",\n      \"method\": \"Affinity chromatography with tizoxanide-biotin, mass spectrometry, drug affinity response target stability (DARTS), solvent-induced protein precipitation (SIP), thermal proteome profiling (TPP), fluorescent probe localization\",\n      \"journal\": \"Current drug targets\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — chemical proteomics binding evidence without mutagenesis or functional reconstitution; single study\",\n      \"pmids\": [\"39171461\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRKAG1 encodes the γ1 regulatory subunit of the AMPK heterotrimer, which senses cellular energy status; it is required for erythrocyte membrane elasticity (loss causes hemolytic anemia with elevated adducin phosphorylation), is phosphorylated by PRKDC/DNA-PKcs at Ser192/Thr284 to prime lysosomal AMPK activation by STK11, mediates adiponectin-driven mitochondrial bioenergetics in cardiomyocytes via an SDH/Sirt3-dependent pathway, and regulates a fasting-refeeding transcriptional program in adipose tissue whose age-related decline contributes to metabolic dysfunction.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PRKAG1 encodes the γ1 regulatory subunit of the AMP-activated protein kinase (AMPK) heterotrimer, integrating cellular energy status into diverse physiological programs spanning erythrocyte mechanics, lysosomal AMPK activation, and metabolic homeostasis [#0, #1, #2]. Its activity is gated upstream by PRKDC/DNA-PKcs, which phosphorylates PRKAG1 at Ser192 and Thr284 to prime the AMPK complex for lysosomal localization and starvation-induced association with STK11/LKB1, independently of the subunit's nucleotide-sensing function [#1]. Functionally, PRKAG1 is required to maintain erythrocyte membrane elasticity: its loss increases adducin phosphorylation, rigidifies the membrane, and produces regenerative hemolytic anemia [#0]. In metabolic tissues it drives a fasting–refeeding transcriptional program in adipose tissue whose age-related decline impairs metabolic health, such that restoring PRKAG1 extends longevity [#2], and in cardiomyocytes it is a required component for adiponectin-induced mitochondrial respiration through an SDH- and Sirt3-dependent pathway [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established that the AMPK γ1 subunit has a non-redundant cellular role beyond classical energy sensing by demonstrating its requirement for erythrocyte membrane integrity.\",\n      \"evidence\": \"Prkag1-/- knockout mice with osmotic hemolysis, ektacytometry, and phospho-protein analysis of cytoskeletal adducin\",\n      \"pmids\": [\"20881209\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether AMPK directly phosphorylates/dephosphorylates adducin or acts through intermediary kinases is not resolved\",\n        \"Does not address PRKAG1's role in nucleated cells or other tissues\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined an upstream regulatory input on PRKAG1, showing that DNA-PKcs phosphorylation primes the AMPK complex for lysosomal activation rather than acting through nucleotide sensing.\",\n      \"evidence\": \"In vitro kinase assay, S192A/T284A site-directed mutagenesis, reciprocal Co-IP, and lysosomal localization/autophagic flux readouts\",\n      \"pmids\": [\"31983282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for how Ser192/Thr284 phosphorylation promotes STK11 association is unknown\",\n        \"Physiological contexts in which PRKDC controls AMPK activation in vivo are not established\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed PRKAG1 as a required node linking adiponectin signaling to mitochondrial bioenergetics in cardiac cells.\",\n      \"evidence\": \"siRNA knockdown of AMPKγ1 in cardiac myocytes, Seahorse respirometry, SDH activity assays, Sdhaf1/Sirt3 overexpression\",\n      \"pmids\": [\"33271223\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct biochemical reconstitution of the AMPK complex with SDH/Sirt3 not performed\",\n        \"Single lab, single cell-type readout\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated a causal role for PRKAG1 in the fasting–refeeding transcriptional program and in healthy aging via gain-of-function rescue.\",\n      \"evidence\": \"RNA-seq, transgenic Prkag1 overexpression in killifish, longitudinal survival and metabolic phenotyping\",\n      \"pmids\": [\"37957359\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No in vitro biochemical reconstitution of the underlying transcriptional mechanism\",\n        \"Single-organism (killifish) model; mammalian validation absent\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked PRKAG1 levels to autophagy and antiviral responses, implicating downstream TP53 and GSK3B phosphorylation.\",\n      \"evidence\": \"siRNA knockdown and overexpression with JEV replication and autophagy assays, plus phospho-proteomics in mammalian cells\",\n      \"pmids\": [\"41033165\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Nitazoxanide binding shown by solvent-induced precipitation but not reconstituted in vitro\",\n        \"Whether TP53/GSK3B phosphorylation changes are direct AMPK-dependent effects is unresolved\"\n      ]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Suggested a tissue-specific negative role for PRKAG1 in pancreatic beta-cell insulin secretion.\",\n      \"evidence\": \"Transient siRNA knockdown in INS-1E insulinoma cells with glucose-stimulated insulin secretion assay\",\n      \"pmids\": [\"31195220\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Single knockdown with one functional readout; no mechanistic follow-up on the proposed mTOR link\",\n        \"Not validated in primary beta cells or in vivo\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified an unexpected role for the PRKAG1 ortholog in regulating nuclear actin localization.\",\n      \"evidence\": \"Genome-wide RNAi screen in Drosophila with validation by knockdown and fluorescence microscopy in mammalian cells\",\n      \"pmids\": [\"26021350\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Mechanistic basis of the nuclear actin effect not established\",\n        \"Validated only by single knockdown in mammalian cells\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the diverse PRKAG1-dependent phenotypes (membrane mechanics, lysosomal activation, mitochondrial respiration, aging, autophagy) are unified at the level of AMPK complex assembly and substrate selection remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural model of how PRKAG1 phosphorylation or expression level reroutes AMPK output across tissues\",\n        \"Direct substrates downstream of PRKAG1 in each context largely undefined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"complexes\": [\"AMPK heterotrimer\"],\n    \"partners\": [\"PRKDC\", \"STK11\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}