{"gene":"PASK","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":2003,"finding":"PASK (SPAK) directly binds to and phosphorylates NKCC1 (Na-K-Cl cotransporter), activating it; a kinase-inactive dominant-negative PASK mutant reduces NKCC1 activity by 60-80% and blocks phosphorylation of two N-terminal regulatory threonines on NKCC1; co-immunoprecipitation confirmed constitutive PASK-NKCC1 association in HEK cells.","method":"Dominant-negative overexpression, 32Pi phosphorylation assay, co-immunoprecipitation, calyculin A rescue experiment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, dominant-negative functional rescue, phosphorylation assays with multiple orthogonal approaches in a single focused study","pmids":["12740379"],"is_preprint":false},{"year":2000,"finding":"Fray, the Drosophila ortholog of mammalian PASK, is required in peripheral glia for axonal ensheathment; rat PASK cDNA rescues fray null mutant nerve morphology defects, demonstrating functional conservation.","method":"Genetic null mutant analysis, transgenic rescue with rat PASK cDNA expressed in ensheathing glia","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic rescue with heterologous (rat) PASK demonstrates functional orthology; null mutant phenotype rigorously characterized","pmids":["11163267"],"is_preprint":false},{"year":2003,"finding":"Mouse PASKIN is strongly upregulated in postmeiotic germ cells during spermatogenesis; the Paskin knockout mouse is viable and fertile with no spermatogenesis defect. The Paskin gene shares its promoter with Ppp1r7 (Sds22, a regulatory subunit of protein phosphatase 1), and Sds22 co-localizes with PASKIN-expressing cell types in vivo.","method":"Targeted gene knockout (homologous recombination), lacZ reporter knock-in for expression mapping, promoter analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — constitutive knockout mouse with reporter, multiple tissues analyzed; negative fertility phenotype is a rigorous null result","pmids":["12972598"],"is_preprint":false},{"year":2010,"finding":"Crystal structure of the human PASK kinase domain reveals it adopts an active conformation and has catalytic activity in vitro and in vivo without activation loop phosphorylation; site-directed mutagenesis identified key structural features enabling this; combinatorial peptide library screening determined PASK prefers basic residues at P-3 and P-5 positions in substrates.","method":"X-ray crystallography, in vitro kinase assay, site-directed mutagenesis, combinatorial peptide library screening","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro kinase assays in a single rigorous study","pmids":["20943661"],"is_preprint":false},{"year":2007,"finding":"PASKIN localizes to nuclei of human testis germ cells and the midpiece of sperm tails; it also shows a speckle-like nuclear pattern in HeLa cells in addition to cytoplasmic localization. PASKIN interacts with eEF1A1 (eukaryotic translation elongation factor 1A1) via its PAS-A and kinase domains (mapped by mammalian two-hybrid and GST pulldown); PASKIN phosphorylates eEF1A1 primarily at Thr432 (confirmed by mass spectrometry and mutagenesis); wild-type but not kinase-inactive PASKIN increases in vitro translation of a reporter cRNA.","method":"Immunofluorescence/localization, yeast two-hybrid screening, mammalian two-hybrid, GST pulldown, in vitro kinase assay, mass spectrometry, site-directed mutagenesis, in vitro translation assay","journal":"Cellular physiology and biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal methods (pulldown, kinase assay, MS, mutagenesis, translation assay) in a single study; single lab","pmids":["17595531"],"is_preprint":false},{"year":2011,"finding":"Ribosomal protein S6 is identified as a novel PASKIN kinase substrate in addition to eEF1A1; phospholipids, particularly monophosphorylated phosphatidylinositols, bind PASKIN and stimulate autophosphorylation via the kinase domain (not the PAS domain); di- and tri-phosphorylated phosphatidylinositols inhibit both autophosphorylation and target phosphorylation, suggesting multiligand regulation of PASKIN activity.","method":"In vitro kinase assays, phospholipid binding assays, autophosphorylation assays","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assays with multiple substrates and lipid ligands; single lab, several orthogonal assays","pmids":["21418524"],"is_preprint":false},{"year":2011,"finding":"A gain-of-function mutation in PASK (p.G1117E), identified in a young-onset diabetes family, increases autophosphorylation ~25% and kinase activity ~2-fold toward exogenous substrates; mouse islets infected with adenovirus expressing p.G1117E PASK show a 4-fold increase in basal (low glucose) insulin release and attenuated glucose-stimulated insulin secretion. The p.L1051V mutation did not affect kinase activity.","method":"Affinity-purified kinase activity assay (autophosphorylation at Thr307, peptide substrate), adenoviral expression in mouse islets, insulin secretion assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — biochemical kinase assay with identified phosphosite, combined with functional insulin secretion in primary islets; single lab but multiple orthogonal methods","pmids":["22065581"],"is_preprint":false},{"year":2007,"finding":"PASKIN is not expressed in pancreatic islet beta-cells (no X-gal staining in beta-cells of Paskin-lacZ reporter mice at any glucose concentration); glucose-stimulated insulin production and blood glucose regulation are independent of PASKIN in mice.","method":"lacZ reporter knock-in mouse, X-gal staining, adenoviral lacZ control, insulin mRNA and release assays, glucose tolerance test","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Moderate — constitutive knockout reporter mouse with multiple negative results replicated by multiple methods; directly contradicts an earlier claim of PASK in beta-cell glucose sensing","pmids":["17472472"],"is_preprint":false},{"year":2016,"finding":"PASK phosphorylates Wdr5 (a member of H3K4 methyltransferase complexes) during myoblast differentiation, promoting conversion of H3K4me1 to H3K4me3 marks on the myogenin (Myog) promoter, enhancing MyoD accessibility and transcriptional activation of myogenin to initiate muscle differentiation; PASK also promotes differentiation of embryonic stem cells and adipogenic progenitor cells.","method":"Loss-of-function and gain-of-function experiments, chromatin immunoprecipitation (ChIP), in vitro kinase assay for Wdr5 phosphorylation, reporter assays, differentiation assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KO/KD, ChIP, kinase assay, differentiation assays across multiple progenitor cell types) in a single rigorous study","pmids":["27661449"],"is_preprint":false},{"year":2019,"finding":"mTORC1 phosphorylates PASK in muscle stem cells during differentiation; this mTORC1-dependent PASK phosphorylation is required for myogenin transcription, exit from self-renewal, and induction of the myogenesis program (early stage), acting via PASK-Wdr5 signaling, distinct from mTORC1-S6K signaling required for later myoblast fusion.","method":"Genetic epistasis (mTOR inhibitor rapamycin, S6K knockout), phosphorylation assays, muscle stem cell differentiation assays, myogenin reporter assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with pharmacological and genetic tools, multiple differentiation stage readouts, placing PASK downstream of mTORC1 in a defined pathway","pmids":["31072927"],"is_preprint":false},{"year":2019,"finding":"Nuclear PASK associates with the mammalian MLL2 H3K4 methyltransferase complex and enhances H3K4 di- and tri-methylation; PASK directly phosphorylates histone H3 at T3, T6, S10, and T11. Loss- or gain-of-function of PASK using CRISPR/Cas9 affects muscle satellite cell differentiation through regulation of these histone modifications.","method":"Co-immunoprecipitation, in vitro histone kinase assay, CRISPR/Cas9 loss/gain-of-function, muscle satellite cell differentiation assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and in vitro kinase assay with CRISPR functional validation; single lab","pmids":["31529049"],"is_preprint":false},{"year":2023,"finding":"Mitochondrial glutamine metabolism drives CBP/EP300-dependent acetylation of PASK, releasing it from cytoplasmic granules and enabling nuclear translocation; in the nucleus, PASK catalytically displaces WDR5 from the anaphase-promoting complex/cyclosome (APC/C), resulting in loss of post-mitotic Pax7 expression and exit from stem cell self-renewal to establish differentiation competence.","method":"Live-cell imaging, subcellular fractionation, genetic/pharmacological inhibition of glutamine metabolism, in vitro kinase competition assay, muscle regeneration assay in mice","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (imaging, fractionation, pharmacology, in vitro competition, in vivo mouse regeneration) in a single study; novel mechanism","pmids":["37052079"],"is_preprint":false},{"year":2024,"finding":"The PAS-A domain of PASK contains a monopartite nuclear localization sequence (NLS) that is inhibited by intramolecular association with a short linear motif (PAS Interacting Motif, PIM) located upstream of the kinase domain; this PAS-PIM interaction retains PASK in the cytosol in the absence of signaling; metabolic inputs disrupt this interaction to induce PASK nuclear import, with PIM recruitment and artificial ligand binding occurring at neighboring locations on PAS-A.","method":"Mutagenesis of NLS, intramolecular interaction mapping (biochemical assays), nuclear import assays, ligand binding assays","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical mapping of intramolecular interaction and functional nuclear import assays; single lab, multiple approaches","pmids":["38182104"],"is_preprint":false},{"year":2025,"finding":"PASK contains a previously unrecognized third PAS domain (PAS-C) formed through intramolecular interactions between an N-terminal PAS fold and a C-terminal PAC motif separated by an unstructured linker; PAS-C assembly is nutrient-responsive and drives quaternary structure reorganization that positions PAS-A near the kinase activation loop, stabilizing it for catalytic activation.","method":"Evolutionary sequence/domain mapping, deep learning structural modeling, residue-level cross-linking assays, biochemical kinase activity assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — computational structural models validated by cross-linking assays and kinase activity measurements; single lab, novel structural finding","pmids":["40106358"],"is_preprint":false},{"year":2008,"finding":"The kinase domain of PASK (SPAK) directly binds purified tubulin and microtubules in vitro; truncated PASK lacking the N-terminal non-catalytic domain promotes microtubule assembly at subcritical tubulin concentrations; FLAG-PASK expressed in COS-7 cells translocates to the cytoskeleton upon hypertonic NaCl stimulation and stabilizes microtubules against nocodazole-induced depolymerization.","method":"Tubulin binding assay, microtubule sedimentation (ultracentrifugation), in vitro microtubule assembly assay, FLAG-PASK overexpression with immunofluorescence and nocodazole treatment","journal":"Archives of biochemistry and biophysics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vitro and cell-based orthogonal assays; single lab","pmids":["18675246"],"is_preprint":false}],"current_model":"PASK (PASKIN) is an evolutionarily conserved PAS domain-containing serine/threonine kinase whose catalytic activity is regulated by nutrient-responsive structural assembly of its PAS domains (including a newly identified PAS-C) that stabilizes the activation loop without requiring activation loop phosphorylation; in the cytoplasm, an intramolecular PAS-A/PIM interaction masks a nuclear localization sequence to retain PASK in cytoplasmic granules, while metabolic signals (including CBP/EP300-dependent acetylation driven by glutamine metabolism) and mTORC1-mediated phosphorylation release and activate PASK, enabling nuclear translocation; nuclear PASK phosphorylates Wdr5 to remodel H3K4 methylation marks on differentiation gene promoters (e.g., myogenin), phosphorylates histone H3 directly, competes with WDR5-APC/C to disrupt post-mitotic self-renewal, and regulates ion transport by phosphorylating NKCC1 and eEF1A1 (at Thr432) to modulate protein translation."},"narrative":{"mechanistic_narrative":"PASK (PASKIN) is an evolutionarily conserved PAS domain–containing serine/threonine kinase that couples nutrient and metabolic signals to gene-regulatory and transport programs, most prominently the entry of progenitor cells into differentiation [PMID:27661449, PMID:37052079]. Its kinase domain adopts a catalytically active conformation and phosphorylates substrates without activation-loop phosphorylation, preferring basic residues at the P-3 and P-5 positions [PMID:20943661], with catalytic competence further stabilized by nutrient-responsive assembly of an intramolecular third PAS domain (PAS-C) that repositions PAS-A against the activation loop [PMID:40106358]. In the resting state, an intramolecular PAS-A/PIM interaction masks a nuclear localization sequence within PAS-A, retaining PASK in the cytosol [PMID:38182104]; metabolic inputs—including glutamine-metabolism-driven CBP/EP300-dependent acetylation and mTORC1-mediated phosphorylation—disrupt this restraint and license nuclear import [PMID:31072927, PMID:37052079]. Nuclear PASK drives differentiation by phosphorylating WDR5 to convert H3K4me1 to H3K4me3 at the myogenin promoter [PMID:27661449], associating with the MLL2 H3K4 methyltransferase complex and directly phosphorylating histone H3 [PMID:31529049], and catalytically displacing WDR5 from the APC/C to extinguish post-mitotic self-renewal (Pax7 loss) [PMID:37052079]. Independently of this nuclear role, PASK phosphorylates and activates the Na-K-Cl cotransporter NKCC1 [PMID:12740379] and phosphorylates eEF1A1 at Thr432 to stimulate translation [PMID:17595531]. Genetically, a gain-of-function PASK mutation (p.G1117E) identified in a young-onset diabetes family elevates kinase activity and dysregulates insulin secretion from mouse islets [PMID:22065581].","teleology":[{"year":2000,"claim":"Established that PASK has a conserved, essential physiological function by showing the mammalian gene can substitute for its Drosophila ortholog in vivo.","evidence":"Genetic null mutant analysis with transgenic rescue using rat PASK cDNA in fly ensheathing glia","pmids":["11163267"],"confidence":"High","gaps":["Does not define the molecular substrate or mechanism underlying glial ensheathment","Conservation of mechanism in mammals not directly tested"]},{"year":2003,"claim":"Identified the first direct PASK substrate and a transport function, showing PASK binds and phosphorylates NKCC1 to activate ion cotransport.","evidence":"Dominant-negative overexpression, 32Pi phosphorylation assay, and reciprocal co-immunoprecipitation in HEK cells","pmids":["12740379"],"confidence":"High","gaps":["Upstream signals controlling PASK-NKCC1 engagement not defined","Physiological context of NKCC1 regulation by PASK unclear"]},{"year":2003,"claim":"Defined PASK expression and tested a candidate reproductive role, finding germ-cell upregulation but no requirement for fertility, narrowing its essential functions.","evidence":"Targeted knockout mouse with lacZ reporter and promoter analysis","pmids":["12972598"],"confidence":"High","gaps":["Functional redundancy masking phenotype not excluded","Significance of shared promoter with Ppp1r7/Sds22 not mechanistically resolved"]},{"year":2007,"claim":"Resolved a controversy over a beta-cell glucose-sensing role by showing PASK is not expressed in islet beta-cells and is dispensable for glucose homeostasis in mice.","evidence":"lacZ reporter knock-in mouse, X-gal staining, insulin assays, and glucose tolerance testing","pmids":["17472472"],"confidence":"High","gaps":["Does not rule out PASK roles in other metabolic tissues","Species or context differences from gain-of-function human data unresolved"]},{"year":2007,"claim":"Linked PASK to translational control by identifying eEF1A1 as a direct substrate phosphorylated at Thr432 and showing PASK stimulates translation.","evidence":"Yeast/mammalian two-hybrid, GST pulldown, in vitro kinase assay, mass spectrometry, mutagenesis, and in vitro translation assay","pmids":["17595531"],"confidence":"High","gaps":["In vivo significance of Thr432 phosphorylation not established","Connection between translational and nuclear functions unclear"]},{"year":2010,"claim":"Explained how PASK is constitutively active, showing the kinase domain adopts an active fold without activation-loop phosphorylation and defining its substrate consensus.","evidence":"X-ray crystallography, in vitro kinase assays, mutagenesis, and combinatorial peptide library screening","pmids":["20943661"],"confidence":"High","gaps":["How full-length regulatory domains gate this intrinsic activity not addressed","Cellular activation triggers not defined by structure alone"]},{"year":2011,"claim":"Proposed multiligand regulation of PASK by showing phosphatidylinositol species and additional substrates (ribosomal protein S6) modulate kinase activity.","evidence":"In vitro kinase, autophosphorylation, and phospholipid binding assays","pmids":["21418524"],"confidence":"Medium","gaps":["Lipid regulation not demonstrated in cells","Physiological relevance of S6 phosphorylation unconfirmed"]},{"year":2011,"claim":"Connected PASK kinase activity to human disease by characterizing a young-onset diabetes gain-of-function mutation that alters insulin secretion.","evidence":"Affinity-purified kinase activity assays, adenoviral expression in mouse islets, and insulin secretion assays","pmids":["22065581"],"confidence":"High","gaps":["Tension with reporter data showing no PASK in beta-cells unresolved","Causal substrate mediating altered secretion not identified"]},{"year":2016,"claim":"Defined PASK's nuclear epigenetic function, showing it phosphorylates WDR5 to remodel H3K4 methylation and initiate differentiation gene transcription.","evidence":"Loss/gain-of-function, ChIP, in vitro WDR5 kinase assay, reporter and differentiation assays across multiple progenitor types","pmids":["27661449"],"confidence":"High","gaps":["Upstream signals triggering nuclear PASK activity not yet defined","Direct WDR5 phosphosite consequences on complex assembly incomplete"]},{"year":2019,"claim":"Placed PASK downstream of mTORC1, establishing an mTORC1–PASK–Wdr5 axis required for exit from self-renewal during early myogenesis.","evidence":"Genetic epistasis with rapamycin and S6K knockout, phosphorylation and myogenin reporter assays in muscle stem cells","pmids":["31072927"],"confidence":"High","gaps":["Direct mTORC1 phosphosite on PASK not mapped","How phosphorylation activates nuclear PASK function not detailed"]},{"year":2019,"claim":"Extended PASK's chromatin activity, showing direct histone H3 phosphorylation and association with the MLL2 H3K4 methyltransferase complex.","evidence":"Co-immunoprecipitation, in vitro histone kinase assay, and CRISPR/Cas9 functional studies in satellite cells","pmids":["31529049"],"confidence":"Medium","gaps":["In vivo histone phosphorylation sites not validated genetically","Reciprocal validation of MLL2 association limited to single lab"]},{"year":2023,"claim":"Identified the metabolic trigger and a non-transcriptional nuclear mechanism, showing glutamine-driven acetylation releases PASK to displace WDR5 from APC/C and terminate self-renewal.","evidence":"Live-cell imaging, fractionation, glutamine-metabolism perturbation, in vitro kinase competition, and mouse muscle regeneration assays","pmids":["37052079"],"confidence":"High","gaps":["Acetylation sites on PASK not mapped","Mechanism by which displacement alters APC/C substrate selection incomplete"]},{"year":2024,"claim":"Resolved how cytoplasmic retention is controlled, identifying a PAS-A NLS masked by an intramolecular PIM interaction that metabolic signals disrupt to permit nuclear import.","evidence":"NLS mutagenesis, intramolecular interaction mapping, nuclear import assays, and ligand binding assays","pmids":["38182104"],"confidence":"Medium","gaps":["Endogenous metabolic ligand disrupting PAS-PIM not identified","Quantitative kinetics of import switch not established"]},{"year":2025,"claim":"Revealed the structural basis of nutrient-responsive activation, defining a third PAS domain (PAS-C) whose assembly repositions PAS-A to stabilize the activation loop.","evidence":"Evolutionary domain mapping, deep-learning structural modeling, cross-linking assays, and biochemical kinase activity assays","pmids":["40106358"],"confidence":"Medium","gaps":["Experimental high-resolution structure of PAS-C lacking","Direct nutrient ligand sensed by PAS-C not identified"]},{"year":null,"claim":"The identity of the physiological metabolic ligands sensed by PASK's PAS domains and how these unify its transport, translational, and chromatin functions remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct endogenous PAS-domain ligand identified","Integration of cytoplasmic (NKCC1, eEF1A1) and nuclear (WDR5, histone, APC/C) functions not mechanistically reconciled"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,4,8,10,11]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3,4,8]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[5]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[14]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[8,10]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4,8,11,12]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4,11,12]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[14]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[8,9,11]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[8,10]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[9,11]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[0]}],"complexes":[],"partners":["NKCC1","EEF1A1","WDR5","MLL2","APC/C"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UEW8","full_name":"STE20/SPS1-related proline-alanine-rich protein kinase","aliases":["DCHT","Serine/threonine-protein kinase 39"],"length_aa":545,"mass_kda":59.5,"function":"Effector serine/threonine-protein kinase component of the WNK-SPAK/OSR1 kinase cascade, which is involved in various processes, such as ion transport, response to hypertonic stress and blood pressure (PubMed:16669787, PubMed:18270262, PubMed:21321328, PubMed:34289367). Specifically recognizes and binds proteins with a RFXV motif (PubMed:16669787, PubMed:21321328). Acts downstream of WNK kinases (WNK1, WNK2, WNK3 or WNK4): following activation by WNK kinases, catalyzes phosphorylation of ion cotransporters, such as SLC12A1/NKCC2, SLC12A2/NKCC1, SLC12A3/NCC, SLC12A5/KCC2 or SLC12A6/KCC3, regulating their activity (PubMed:21321328). Mediates regulatory volume increase in response to hyperosmotic stress by catalyzing phosphorylation of ion cotransporters SLC12A1/NKCC2, SLC12A2/NKCC1 and SLC12A6/KCC3 downstream of WNK1 and WNK3 kinases (PubMed:12740379, PubMed:16669787, PubMed:21321328). Phosphorylation of Na-K-Cl cotransporters SLC12A2/NKCC1 and SLC12A2/NKCC1 promote their activation and ion influx; simultaneously, phosphorylation of K-Cl cotransporters SLC12A5/KCC2 and SLC12A6/KCC3 inhibit their activity, blocking ion efflux (PubMed:16669787, PubMed:19665974, PubMed:21321328). Acts as a regulator of NaCl reabsorption in the distal nephron by mediating phosphorylation and activation of the thiazide-sensitive Na-Cl cotransporter SLC12A3/NCC in distal convoluted tubule cells of kidney downstream of WNK4 (PubMed:18270262). Mediates the inhibition of SLC4A4, SLC26A6 as well as CFTR activities (By similarity). Phosphorylates RELT (By similarity)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UEW8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PASK","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000115687","cell_line_id":"CID001231","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"big_aggregates","grade":2}],"interactors":[{"gene":"AKAP8","stoichiometry":0.2},{"gene":"POU2F1","stoichiometry":0.2},{"gene":"ERCC6L","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001231","total_profiled":1310},"omim":[{"mim_id":"616186","title":"H19/IGF2-IMPRINTING CONTROL REGION","url":"https://www.omim.org/entry/616186"},{"mim_id":"607505","title":"PAS DOMAIN-CONTAINING SERINE/THREONINE KINASE; PASK","url":"https://www.omim.org/entry/607505"},{"mim_id":"300032","title":"ATRX CHROMATIN REMODELER; ATRX","url":"https://www.omim.org/entry/300032"},{"mim_id":"103280","title":"H19, IMPRINTED MATERNALLY EXPRESSED NONCODING TRANSCRIPT; H19","url":"https://www.omim.org/entry/103280"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"lymphoid tissue","ntpm":21.3}],"url":"https://www.proteinatlas.org/search/PASK"},"hgnc":{"alias_symbol":["PASKIN","KIAA0135","STK37"],"prev_symbol":[]},"alphafold":{"accession":"Q9UEW8","domains":[{"cath_id":"3.30.200.20","chopping":"61-138","consensus_level":"medium","plddt":85.4486,"start":61,"end":138},{"cath_id":"1.10.510.10","chopping":"142-222_230-362","consensus_level":"high","plddt":87.1351,"start":142,"end":362},{"cath_id":"3.10.20.90","chopping":"453-545","consensus_level":"high","plddt":86.4892,"start":453,"end":545}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UEW8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UEW8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UEW8-F1-predicted_aligned_error_v6.png","plddt_mean":75.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PASK","jax_strain_url":"https://www.jax.org/strain/search?query=PASK"},"sequence":{"accession":"Q9UEW8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UEW8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UEW8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UEW8"}},"corpus_meta":[{"pmid":"12740379","id":"PMC_12740379","title":"PASK (proline-alanine-rich STE20-related kinase), a regulatory kinase of the Na-K-Cl cotransporter (NKCC1).","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12740379","citation_count":220,"is_preprint":false},{"pmid":"11163267","id":"PMC_11163267","title":"Fray, a Drosophila serine/threonine kinase homologous to mammalian PASK, is required for axonal ensheathment.","date":"2000","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/11163267","citation_count":109,"is_preprint":false},{"pmid":"35011564","id":"PMC_35011564","title":"Adipose-Specific PPARα Knockout Mice Have Increased Lipogenesis by PASK-SREBP1 Signaling and a Polarity Shift to Inflammatory Macrophages in White Adipose Tissue.","date":"2021","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/35011564","citation_count":60,"is_preprint":false},{"pmid":"12972598","id":"PMC_12972598","title":"Targeted disruption of the mouse PAS domain serine/threonine kinase PASKIN.","date":"2003","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12972598","citation_count":36,"is_preprint":false},{"pmid":"19365831","id":"PMC_19365831","title":"FARP2, HDLBP and PASK are downregulated in a patient with autism and 2q37.3 deletion syndrome.","date":"2009","source":"American journal of medical genetics. Part A","url":"https://pubmed.ncbi.nlm.nih.gov/19365831","citation_count":34,"is_preprint":false},{"pmid":"20943661","id":"PMC_20943661","title":"Structural bases of PAS domain-regulated kinase (PASK) activation in the absence of activation loop phosphorylation.","date":"2010","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20943661","citation_count":27,"is_preprint":false},{"pmid":"11688972","id":"PMC_11688972","title":"Mammalian PASKIN, a PAS-serine/threonine kinase related to bacterial oxygen sensors.","date":"2001","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/11688972","citation_count":26,"is_preprint":false},{"pmid":"31072927","id":"PMC_31072927","title":"Activation of PASK by mTORC1 is required for the onset of the terminal differentiation program.","date":"2019","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/31072927","citation_count":26,"is_preprint":false},{"pmid":"19189049","id":"PMC_19189049","title":"The PAS-domain kinase PASKIN: a new sensor in energy homeostasis.","date":"2009","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/19189049","citation_count":25,"is_preprint":false},{"pmid":"17595531","id":"PMC_17595531","title":"Male germ cell expression of the PAS domain kinase PASKIN and its novel target eukaryotic translation elongation factor eEF1A1.","date":"2007","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/17595531","citation_count":24,"is_preprint":false},{"pmid":"26371032","id":"PMC_26371032","title":"Per-Arnt-Sim Kinase (PASK): An Emerging Regulator of Mammalian Glucose and Lipid Metabolism.","date":"2015","source":"Nutrients","url":"https://pubmed.ncbi.nlm.nih.gov/26371032","citation_count":21,"is_preprint":false},{"pmid":"22065581","id":"PMC_22065581","title":"Human mutation within Per-Arnt-Sim (PAS) domain-containing protein kinase (PASK) causes basal insulin hypersecretion.","date":"2011","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/22065581","citation_count":19,"is_preprint":false},{"pmid":"17192472","id":"PMC_17192472","title":"Glucose-stimulated insulin production in mice deficient for the PAS kinase PASKIN.","date":"2007","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/17192472","citation_count":17,"is_preprint":false},{"pmid":"27661449","id":"PMC_27661449","title":"Pask integrates hormonal signaling with histone modification via Wdr5 phosphorylation to drive myogenesis.","date":"2016","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/27661449","citation_count":17,"is_preprint":false},{"pmid":"18509100","id":"PMC_18509100","title":"Ventilatory responses to acute and chronic hypoxia are altered in female but not male Paskin-deficient mice.","date":"2008","source":"American journal of physiology. Regulatory, integrative and comparative physiology","url":"https://pubmed.ncbi.nlm.nih.gov/18509100","citation_count":17,"is_preprint":false},{"pmid":"10990492","id":"PMC_10990492","title":"Isolation and expression of PASK, a serine/threonine kinase, during rat embryonic development, with special emphasis on the pancreas.","date":"2000","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/10990492","citation_count":14,"is_preprint":false},{"pmid":"34943132","id":"PMC_34943132","title":"Preventing Oxidative Stress in the Liver: An Opportunity for GLP-1 and/or PASK.","date":"2021","source":"Antioxidants (Basel, Switzerland)","url":"https://pubmed.ncbi.nlm.nih.gov/34943132","citation_count":12,"is_preprint":false},{"pmid":"21418524","id":"PMC_21418524","title":"Substrate preference and phosphatidylinositol monophosphate inhibition of the catalytic domain of the Per-Arnt-Sim domain kinase PASKIN.","date":"2011","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/21418524","citation_count":11,"is_preprint":false},{"pmid":"34444712","id":"PMC_34444712","title":"Storage and Utilization of Glycogen by Mouse Liver during Adaptation to Nutritional Changes Are GLP-1 and PASK Dependent.","date":"2021","source":"Nutrients","url":"https://pubmed.ncbi.nlm.nih.gov/34444712","citation_count":11,"is_preprint":false},{"pmid":"31529049","id":"PMC_31529049","title":"The metabolic sensor PASK is a histone 3 kinase that also regulates H3K4 methylation by associating with H3K4 MLL2 methyltransferase complex.","date":"2019","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/31529049","citation_count":11,"is_preprint":false},{"pmid":"37052079","id":"PMC_37052079","title":"PASK links cellular energy metabolism with a mitotic self-renewal network to establish differentiation competence.","date":"2023","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/37052079","citation_count":10,"is_preprint":false},{"pmid":"21327866","id":"PMC_21327866","title":"Per-arnt-sim (PAS) domain kinase (PASK) as a regulator of glucagon secretion.","date":"2011","source":"Diabetologia","url":"https://pubmed.ncbi.nlm.nih.gov/21327866","citation_count":10,"is_preprint":false},{"pmid":"30558306","id":"PMC_30558306","title":"Per-Arnt-Sim Kinase (PASK) Deficiency Increases Cellular Respiration on a Standard Diet and Decreases Liver Triglyceride Accumulation on a Western High-Fat High-Sugar Diet.","date":"2018","source":"Nutrients","url":"https://pubmed.ncbi.nlm.nih.gov/30558306","citation_count":7,"is_preprint":false},{"pmid":"18675246","id":"PMC_18675246","title":"PASK (proline-alanine-rich Ste20-related kinase) binds to tubulin and microtubules and is involved in microtubule stabilization.","date":"2008","source":"Archives of biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/18675246","citation_count":3,"is_preprint":false},{"pmid":"38182104","id":"PMC_38182104","title":"Signal-regulated Unmasking of Nuclear Localization Motif in the PAS Domain Regulates the Nuclear Translocation of PASK.","date":"2024","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/38182104","citation_count":3,"is_preprint":false},{"pmid":"37743322","id":"PMC_37743322","title":"Jasminoidin reduces ischemic stroke injury by regulating microglia polarization via PASK-EEF1A1 axis.","date":"2023","source":"Chemical biology & drug design","url":"https://pubmed.ncbi.nlm.nih.gov/37743322","citation_count":2,"is_preprint":false},{"pmid":"40106358","id":"PMC_40106358","title":"Structural assembly of the PAS domain drives the catalytic activation of metazoan PASK.","date":"2025","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/40106358","citation_count":2,"is_preprint":false},{"pmid":"38979257","id":"PMC_38979257","title":"Nutrient Signaling-Dependent Quaternary Structure Remodeling Drives the Catalytic Activation of metazoan PASK.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/38979257","citation_count":0,"is_preprint":false},{"pmid":"37732199","id":"PMC_37732199","title":"Signal-regulated unmasking of the nuclear localization motif in the PAS domain regulates the nuclear translocation of PASK.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37732199","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.02.12.637445","title":"Identification of polyphosphate-binding proteins in  <i>E. coli</i>  uncovers targets involved in translation control and ribosome biogenesis","date":"2025-02-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.12.637445","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16246,"output_tokens":4118,"usd":0.055254,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12004,"output_tokens":4367,"usd":0.084598,"stage2_stop_reason":"end_turn"},"total_usd":0.139852,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2003,\n      \"finding\": \"PASK (SPAK) directly binds to and phosphorylates NKCC1 (Na-K-Cl cotransporter), activating it; a kinase-inactive dominant-negative PASK mutant reduces NKCC1 activity by 60-80% and blocks phosphorylation of two N-terminal regulatory threonines on NKCC1; co-immunoprecipitation confirmed constitutive PASK-NKCC1 association in HEK cells.\",\n      \"method\": \"Dominant-negative overexpression, 32Pi phosphorylation assay, co-immunoprecipitation, calyculin A rescue experiment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, dominant-negative functional rescue, phosphorylation assays with multiple orthogonal approaches in a single focused study\",\n      \"pmids\": [\"12740379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Fray, the Drosophila ortholog of mammalian PASK, is required in peripheral glia for axonal ensheathment; rat PASK cDNA rescues fray null mutant nerve morphology defects, demonstrating functional conservation.\",\n      \"method\": \"Genetic null mutant analysis, transgenic rescue with rat PASK cDNA expressed in ensheathing glia\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic rescue with heterologous (rat) PASK demonstrates functional orthology; null mutant phenotype rigorously characterized\",\n      \"pmids\": [\"11163267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Mouse PASKIN is strongly upregulated in postmeiotic germ cells during spermatogenesis; the Paskin knockout mouse is viable and fertile with no spermatogenesis defect. The Paskin gene shares its promoter with Ppp1r7 (Sds22, a regulatory subunit of protein phosphatase 1), and Sds22 co-localizes with PASKIN-expressing cell types in vivo.\",\n      \"method\": \"Targeted gene knockout (homologous recombination), lacZ reporter knock-in for expression mapping, promoter analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — constitutive knockout mouse with reporter, multiple tissues analyzed; negative fertility phenotype is a rigorous null result\",\n      \"pmids\": [\"12972598\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Crystal structure of the human PASK kinase domain reveals it adopts an active conformation and has catalytic activity in vitro and in vivo without activation loop phosphorylation; site-directed mutagenesis identified key structural features enabling this; combinatorial peptide library screening determined PASK prefers basic residues at P-3 and P-5 positions in substrates.\",\n      \"method\": \"X-ray crystallography, in vitro kinase assay, site-directed mutagenesis, combinatorial peptide library screening\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and in vitro kinase assays in a single rigorous study\",\n      \"pmids\": [\"20943661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PASKIN localizes to nuclei of human testis germ cells and the midpiece of sperm tails; it also shows a speckle-like nuclear pattern in HeLa cells in addition to cytoplasmic localization. PASKIN interacts with eEF1A1 (eukaryotic translation elongation factor 1A1) via its PAS-A and kinase domains (mapped by mammalian two-hybrid and GST pulldown); PASKIN phosphorylates eEF1A1 primarily at Thr432 (confirmed by mass spectrometry and mutagenesis); wild-type but not kinase-inactive PASKIN increases in vitro translation of a reporter cRNA.\",\n      \"method\": \"Immunofluorescence/localization, yeast two-hybrid screening, mammalian two-hybrid, GST pulldown, in vitro kinase assay, mass spectrometry, site-directed mutagenesis, in vitro translation assay\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal methods (pulldown, kinase assay, MS, mutagenesis, translation assay) in a single study; single lab\",\n      \"pmids\": [\"17595531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Ribosomal protein S6 is identified as a novel PASKIN kinase substrate in addition to eEF1A1; phospholipids, particularly monophosphorylated phosphatidylinositols, bind PASKIN and stimulate autophosphorylation via the kinase domain (not the PAS domain); di- and tri-phosphorylated phosphatidylinositols inhibit both autophosphorylation and target phosphorylation, suggesting multiligand regulation of PASKIN activity.\",\n      \"method\": \"In vitro kinase assays, phospholipid binding assays, autophosphorylation assays\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assays with multiple substrates and lipid ligands; single lab, several orthogonal assays\",\n      \"pmids\": [\"21418524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"A gain-of-function mutation in PASK (p.G1117E), identified in a young-onset diabetes family, increases autophosphorylation ~25% and kinase activity ~2-fold toward exogenous substrates; mouse islets infected with adenovirus expressing p.G1117E PASK show a 4-fold increase in basal (low glucose) insulin release and attenuated glucose-stimulated insulin secretion. The p.L1051V mutation did not affect kinase activity.\",\n      \"method\": \"Affinity-purified kinase activity assay (autophosphorylation at Thr307, peptide substrate), adenoviral expression in mouse islets, insulin secretion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — biochemical kinase assay with identified phosphosite, combined with functional insulin secretion in primary islets; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"22065581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PASKIN is not expressed in pancreatic islet beta-cells (no X-gal staining in beta-cells of Paskin-lacZ reporter mice at any glucose concentration); glucose-stimulated insulin production and blood glucose regulation are independent of PASKIN in mice.\",\n      \"method\": \"lacZ reporter knock-in mouse, X-gal staining, adenoviral lacZ control, insulin mRNA and release assays, glucose tolerance test\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — constitutive knockout reporter mouse with multiple negative results replicated by multiple methods; directly contradicts an earlier claim of PASK in beta-cell glucose sensing\",\n      \"pmids\": [\"17472472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PASK phosphorylates Wdr5 (a member of H3K4 methyltransferase complexes) during myoblast differentiation, promoting conversion of H3K4me1 to H3K4me3 marks on the myogenin (Myog) promoter, enhancing MyoD accessibility and transcriptional activation of myogenin to initiate muscle differentiation; PASK also promotes differentiation of embryonic stem cells and adipogenic progenitor cells.\",\n      \"method\": \"Loss-of-function and gain-of-function experiments, chromatin immunoprecipitation (ChIP), in vitro kinase assay for Wdr5 phosphorylation, reporter assays, differentiation assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KO/KD, ChIP, kinase assay, differentiation assays across multiple progenitor cell types) in a single rigorous study\",\n      \"pmids\": [\"27661449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"mTORC1 phosphorylates PASK in muscle stem cells during differentiation; this mTORC1-dependent PASK phosphorylation is required for myogenin transcription, exit from self-renewal, and induction of the myogenesis program (early stage), acting via PASK-Wdr5 signaling, distinct from mTORC1-S6K signaling required for later myoblast fusion.\",\n      \"method\": \"Genetic epistasis (mTOR inhibitor rapamycin, S6K knockout), phosphorylation assays, muscle stem cell differentiation assays, myogenin reporter assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with pharmacological and genetic tools, multiple differentiation stage readouts, placing PASK downstream of mTORC1 in a defined pathway\",\n      \"pmids\": [\"31072927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Nuclear PASK associates with the mammalian MLL2 H3K4 methyltransferase complex and enhances H3K4 di- and tri-methylation; PASK directly phosphorylates histone H3 at T3, T6, S10, and T11. Loss- or gain-of-function of PASK using CRISPR/Cas9 affects muscle satellite cell differentiation through regulation of these histone modifications.\",\n      \"method\": \"Co-immunoprecipitation, in vitro histone kinase assay, CRISPR/Cas9 loss/gain-of-function, muscle satellite cell differentiation assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and in vitro kinase assay with CRISPR functional validation; single lab\",\n      \"pmids\": [\"31529049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Mitochondrial glutamine metabolism drives CBP/EP300-dependent acetylation of PASK, releasing it from cytoplasmic granules and enabling nuclear translocation; in the nucleus, PASK catalytically displaces WDR5 from the anaphase-promoting complex/cyclosome (APC/C), resulting in loss of post-mitotic Pax7 expression and exit from stem cell self-renewal to establish differentiation competence.\",\n      \"method\": \"Live-cell imaging, subcellular fractionation, genetic/pharmacological inhibition of glutamine metabolism, in vitro kinase competition assay, muscle regeneration assay in mice\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (imaging, fractionation, pharmacology, in vitro competition, in vivo mouse regeneration) in a single study; novel mechanism\",\n      \"pmids\": [\"37052079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The PAS-A domain of PASK contains a monopartite nuclear localization sequence (NLS) that is inhibited by intramolecular association with a short linear motif (PAS Interacting Motif, PIM) located upstream of the kinase domain; this PAS-PIM interaction retains PASK in the cytosol in the absence of signaling; metabolic inputs disrupt this interaction to induce PASK nuclear import, with PIM recruitment and artificial ligand binding occurring at neighboring locations on PAS-A.\",\n      \"method\": \"Mutagenesis of NLS, intramolecular interaction mapping (biochemical assays), nuclear import assays, ligand binding assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical mapping of intramolecular interaction and functional nuclear import assays; single lab, multiple approaches\",\n      \"pmids\": [\"38182104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PASK contains a previously unrecognized third PAS domain (PAS-C) formed through intramolecular interactions between an N-terminal PAS fold and a C-terminal PAC motif separated by an unstructured linker; PAS-C assembly is nutrient-responsive and drives quaternary structure reorganization that positions PAS-A near the kinase activation loop, stabilizing it for catalytic activation.\",\n      \"method\": \"Evolutionary sequence/domain mapping, deep learning structural modeling, residue-level cross-linking assays, biochemical kinase activity assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — computational structural models validated by cross-linking assays and kinase activity measurements; single lab, novel structural finding\",\n      \"pmids\": [\"40106358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The kinase domain of PASK (SPAK) directly binds purified tubulin and microtubules in vitro; truncated PASK lacking the N-terminal non-catalytic domain promotes microtubule assembly at subcritical tubulin concentrations; FLAG-PASK expressed in COS-7 cells translocates to the cytoskeleton upon hypertonic NaCl stimulation and stabilizes microtubules against nocodazole-induced depolymerization.\",\n      \"method\": \"Tubulin binding assay, microtubule sedimentation (ultracentrifugation), in vitro microtubule assembly assay, FLAG-PASK overexpression with immunofluorescence and nocodazole treatment\",\n      \"journal\": \"Archives of biochemistry and biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vitro and cell-based orthogonal assays; single lab\",\n      \"pmids\": [\"18675246\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PASK (PASKIN) is an evolutionarily conserved PAS domain-containing serine/threonine kinase whose catalytic activity is regulated by nutrient-responsive structural assembly of its PAS domains (including a newly identified PAS-C) that stabilizes the activation loop without requiring activation loop phosphorylation; in the cytoplasm, an intramolecular PAS-A/PIM interaction masks a nuclear localization sequence to retain PASK in cytoplasmic granules, while metabolic signals (including CBP/EP300-dependent acetylation driven by glutamine metabolism) and mTORC1-mediated phosphorylation release and activate PASK, enabling nuclear translocation; nuclear PASK phosphorylates Wdr5 to remodel H3K4 methylation marks on differentiation gene promoters (e.g., myogenin), phosphorylates histone H3 directly, competes with WDR5-APC/C to disrupt post-mitotic self-renewal, and regulates ion transport by phosphorylating NKCC1 and eEF1A1 (at Thr432) to modulate protein translation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PASK (PASKIN) is an evolutionarily conserved PAS domain–containing serine/threonine kinase that couples nutrient and metabolic signals to gene-regulatory and transport programs, most prominently the entry of progenitor cells into differentiation [#8, #11]. Its kinase domain adopts a catalytically active conformation and phosphorylates substrates without activation-loop phosphorylation, preferring basic residues at the P-3 and P-5 positions [#3], with catalytic competence further stabilized by nutrient-responsive assembly of an intramolecular third PAS domain (PAS-C) that repositions PAS-A against the activation loop [#13]. In the resting state, an intramolecular PAS-A/PIM interaction masks a nuclear localization sequence within PAS-A, retaining PASK in the cytosol [#12]; metabolic inputs—including glutamine-metabolism-driven CBP/EP300-dependent acetylation and mTORC1-mediated phosphorylation—disrupt this restraint and license nuclear import [#9, #11]. Nuclear PASK drives differentiation by phosphorylating WDR5 to convert H3K4me1 to H3K4me3 at the myogenin promoter [#8], associating with the MLL2 H3K4 methyltransferase complex and directly phosphorylating histone H3 [#10], and catalytically displacing WDR5 from the APC/C to extinguish post-mitotic self-renewal (Pax7 loss) [#11]. Independently of this nuclear role, PASK phosphorylates and activates the Na-K-Cl cotransporter NKCC1 [#0] and phosphorylates eEF1A1 at Thr432 to stimulate translation [#4]. Genetically, a gain-of-function PASK mutation (p.G1117E) identified in a young-onset diabetes family elevates kinase activity and dysregulates insulin secretion from mouse islets [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that PASK has a conserved, essential physiological function by showing the mammalian gene can substitute for its Drosophila ortholog in vivo.\",\n      \"evidence\": \"Genetic null mutant analysis with transgenic rescue using rat PASK cDNA in fly ensheathing glia\",\n      \"pmids\": [\"11163267\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not define the molecular substrate or mechanism underlying glial ensheathment\", \"Conservation of mechanism in mammals not directly tested\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identified the first direct PASK substrate and a transport function, showing PASK binds and phosphorylates NKCC1 to activate ion cotransport.\",\n      \"evidence\": \"Dominant-negative overexpression, 32Pi phosphorylation assay, and reciprocal co-immunoprecipitation in HEK cells\",\n      \"pmids\": [\"12740379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals controlling PASK-NKCC1 engagement not defined\", \"Physiological context of NKCC1 regulation by PASK unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined PASK expression and tested a candidate reproductive role, finding germ-cell upregulation but no requirement for fertility, narrowing its essential functions.\",\n      \"evidence\": \"Targeted knockout mouse with lacZ reporter and promoter analysis\",\n      \"pmids\": [\"12972598\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional redundancy masking phenotype not excluded\", \"Significance of shared promoter with Ppp1r7/Sds22 not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Resolved a controversy over a beta-cell glucose-sensing role by showing PASK is not expressed in islet beta-cells and is dispensable for glucose homeostasis in mice.\",\n      \"evidence\": \"lacZ reporter knock-in mouse, X-gal staining, insulin assays, and glucose tolerance testing\",\n      \"pmids\": [\"17472472\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not rule out PASK roles in other metabolic tissues\", \"Species or context differences from gain-of-function human data unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Linked PASK to translational control by identifying eEF1A1 as a direct substrate phosphorylated at Thr432 and showing PASK stimulates translation.\",\n      \"evidence\": \"Yeast/mammalian two-hybrid, GST pulldown, in vitro kinase assay, mass spectrometry, mutagenesis, and in vitro translation assay\",\n      \"pmids\": [\"17595531\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of Thr432 phosphorylation not established\", \"Connection between translational and nuclear functions unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Explained how PASK is constitutively active, showing the kinase domain adopts an active fold without activation-loop phosphorylation and defining its substrate consensus.\",\n      \"evidence\": \"X-ray crystallography, in vitro kinase assays, mutagenesis, and combinatorial peptide library screening\",\n      \"pmids\": [\"20943661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How full-length regulatory domains gate this intrinsic activity not addressed\", \"Cellular activation triggers not defined by structure alone\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Proposed multiligand regulation of PASK by showing phosphatidylinositol species and additional substrates (ribosomal protein S6) modulate kinase activity.\",\n      \"evidence\": \"In vitro kinase, autophosphorylation, and phospholipid binding assays\",\n      \"pmids\": [\"21418524\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Lipid regulation not demonstrated in cells\", \"Physiological relevance of S6 phosphorylation unconfirmed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected PASK kinase activity to human disease by characterizing a young-onset diabetes gain-of-function mutation that alters insulin secretion.\",\n      \"evidence\": \"Affinity-purified kinase activity assays, adenoviral expression in mouse islets, and insulin secretion assays\",\n      \"pmids\": [\"22065581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tension with reporter data showing no PASK in beta-cells unresolved\", \"Causal substrate mediating altered secretion not identified\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined PASK's nuclear epigenetic function, showing it phosphorylates WDR5 to remodel H3K4 methylation and initiate differentiation gene transcription.\",\n      \"evidence\": \"Loss/gain-of-function, ChIP, in vitro WDR5 kinase assay, reporter and differentiation assays across multiple progenitor types\",\n      \"pmids\": [\"27661449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals triggering nuclear PASK activity not yet defined\", \"Direct WDR5 phosphosite consequences on complex assembly incomplete\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Placed PASK downstream of mTORC1, establishing an mTORC1–PASK–Wdr5 axis required for exit from self-renewal during early myogenesis.\",\n      \"evidence\": \"Genetic epistasis with rapamycin and S6K knockout, phosphorylation and myogenin reporter assays in muscle stem cells\",\n      \"pmids\": [\"31072927\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mTORC1 phosphosite on PASK not mapped\", \"How phosphorylation activates nuclear PASK function not detailed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended PASK's chromatin activity, showing direct histone H3 phosphorylation and association with the MLL2 H3K4 methyltransferase complex.\",\n      \"evidence\": \"Co-immunoprecipitation, in vitro histone kinase assay, and CRISPR/Cas9 functional studies in satellite cells\",\n      \"pmids\": [\"31529049\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo histone phosphorylation sites not validated genetically\", \"Reciprocal validation of MLL2 association limited to single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified the metabolic trigger and a non-transcriptional nuclear mechanism, showing glutamine-driven acetylation releases PASK to displace WDR5 from APC/C and terminate self-renewal.\",\n      \"evidence\": \"Live-cell imaging, fractionation, glutamine-metabolism perturbation, in vitro kinase competition, and mouse muscle regeneration assays\",\n      \"pmids\": [\"37052079\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Acetylation sites on PASK not mapped\", \"Mechanism by which displacement alters APC/C substrate selection incomplete\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved how cytoplasmic retention is controlled, identifying a PAS-A NLS masked by an intramolecular PIM interaction that metabolic signals disrupt to permit nuclear import.\",\n      \"evidence\": \"NLS mutagenesis, intramolecular interaction mapping, nuclear import assays, and ligand binding assays\",\n      \"pmids\": [\"38182104\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous metabolic ligand disrupting PAS-PIM not identified\", \"Quantitative kinetics of import switch not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed the structural basis of nutrient-responsive activation, defining a third PAS domain (PAS-C) whose assembly repositions PAS-A to stabilize the activation loop.\",\n      \"evidence\": \"Evolutionary domain mapping, deep-learning structural modeling, cross-linking assays, and biochemical kinase activity assays\",\n      \"pmids\": [\"40106358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Experimental high-resolution structure of PAS-C lacking\", \"Direct nutrient ligand sensed by PAS-C not identified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The identity of the physiological metabolic ligands sensed by PASK's PAS domains and how these unify its transport, translational, and chromatin functions remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct endogenous PAS-domain ligand identified\", \"Integration of cytoplasmic (NKCC1, eEF1A1) and nuclear (WDR5, histone, APC/C) functions not mechanistically reconciled\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 4, 8, 10, 11]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3, 4, 8]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [8, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4, 8, 11, 12]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4, 11, 12]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [8, 9, 11]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [8, 10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [9, 11]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NKCC1\", \"EEF1A1\", \"WDR5\", \"MLL2\", \"APC/C\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}