{"gene":"NUAK1","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":2013,"finding":"LKB1 directly activates NUAK1 kinase, and the LKB1-NUAK1 pathway controls axon branching in cortical neurons by promoting mitochondria immobilization at nascent presynaptic sites; conditional deletion of LKB1 or NUAK1 knockdown drastically reduces axon branching in vivo, while overexpression increases branching, and manipulation of Syntaphilin (a mitochondrial anchor) phenocopies NUAK1 effects.","method":"Conditional knockout, in utero knockdown/overexpression, live imaging, Syntaphilin genetic manipulation in mouse cortical neurons","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal genetic methods in vivo, replicated with mechanistic rescue experiment","pmids":["23791179"],"is_preprint":false},{"year":2016,"finding":"NUAK1 phosphorylates tau at Ser356, stabilizing tau protein levels; inhibition of NUAK1 in Drosophila suppresses neurodegeneration in tau-expressing flies, and NUAK1 haploinsufficiency rescues tauopathy phenotypes in a mouse model.","method":"Cell-based and Drosophila genetic screens, in vitro phosphorylation assay, tauopathy mouse model","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay identifying Ser356 phosphorylation, validated in Drosophila and mouse models","pmids":["27720485"],"is_preprint":false},{"year":2003,"finding":"ARK5/NUAK1 suppresses cell death (induced by nutrient starvation, TRAIL, and TNF-α) by inhibiting caspase 8 activation, delaying Bid cleavage and FLIP degradation.","method":"Overexpression in HepG2 cells, Western blot for caspase 8 activation, Bid cleavage, FLIP degradation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — clean overexpression with defined molecular readouts, single lab","pmids":["13679856"],"is_preprint":false},{"year":2004,"finding":"ARK5/NUAK1 directly phosphorylates caspase-6 at Ser257 in vitro, preventing procaspase-6 activation and thereby suppressing Fas-mediated cell death in colorectal cancer cells; mutation of Ser257 to Ala abolishes this protection.","method":"In vitro kinase assay with wild-type and S257A mutant caspase-6, antisense RNA knockdown, Western blot","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with mutagenesis validating specific phosphorylation site","pmids":["15273717"],"is_preprint":false},{"year":2004,"finding":"ARK5/NUAK1 is activated downstream of Akt and promotes tumor invasion by inducing activation of MMP-2, MMP-9, and MT1-MMP via rapamycin-sensitive signaling; ARK5 overexpression in PANC-1 cells leads to invasion and metastasis in nude mice.","method":"Matrigel invasion assay, nude mouse xenograft, Western blot for MMP activation, pharmacological inhibition","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — overexpression with in vivo metastasis readout, single lab","pmids":["15060171"],"is_preprint":false},{"year":2006,"finding":"NDR2 kinase phosphorylates ARK5/NUAK1 at Thr211 on the T-loop during IGF-1 signaling, providing full kinase activation; Akt phosphorylates ARK5 at Ser600 in the regulatory domain, and combined phosphorylation at T211 (by NDR2) and S600 (by Akt) is required for full ARK5 activity.","method":"In vitro kinase assay, mutant constructs, co-immunoprecipitation, IGF-1 stimulation in colorectal cancer cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of phosphorylation with mutagenesis identifying specific residues","pmids":["16488889"],"is_preprint":false},{"year":2005,"finding":"ARK5/NUAK1 is transcriptionally regulated by the Large-MAF family transcription factors (c-MAF and MAFB) through MAF-recognition element (MARE) sequences in the ARK5 promoter; ARK5 mediates IGF-1-induced cell invasion in multiple myeloma.","method":"Chromatin immunoprecipitation, promoter reporter assay with MARE deletion/mutation, gene expression profiling","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 — ChIP and promoter mutagenesis with functional validation","pmids":["16044163"],"is_preprint":false},{"year":2009,"finding":"NUAK1 phosphorylates LATS1 at Ser464, promoting its degradation and thereby controlling cellular ploidy and senescence; NUAK1 overexpression triggers premature senescence and aneuploidies, while a dominant-negative LATS1 phenocopies NUAK1 effects.","method":"Loss-of-function screen, shRNA depletion, overexpression, in vitro kinase assay for LATS1 phosphorylation","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay identifying phosphorylation site, multiple genetic approaches in human fibroblasts","pmids":["19927127"],"is_preprint":false},{"year":2011,"finding":"NUAK1 directly interacts with and phosphorylates p53 in vitro and in vivo in a LKB1-dependent manner; phosphorylation of NUAK1 at Thr211 by LKB1 is required for NUAK1 kinase activity; the LKB1/NUAK1/p53 axis induces G1/S cell cycle arrest via p21/WAF1 and NUAK1 associates with p53 in the nucleus at the p21 promoter.","method":"Co-immunoprecipitation, in vitro kinase assay, chromatin immunoprecipitation, cell cycle analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro kinase assay, ChIP, co-IP, multiple orthogonal methods","pmids":["21317932"],"is_preprint":false},{"year":2012,"finding":"Muscle-specific knockout of NUAK1 prevents high-fat diet-induced glucose intolerance; NUAK1 suppresses insulin signaling by promoting phosphorylation of IRS1 at Ser1097; loss of NUAK1 enhances phosphorylation of IRS1 Tyr608, AKT Thr308, and TBC1D4 Thr649, improving glucose uptake.","method":"Muscle-specific knockout mice, glucose tolerance test, quantitative phosphoproteomics of soleus muscle","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — tissue-specific KO with phosphoproteomics identifying IRS1 Ser1097 as downstream effector","pmids":["22418434"],"is_preprint":false},{"year":2012,"finding":"NUAK1 and NUAK2 together (but not individually) are required for apical constriction and apico-basal elongation during dorsolateral hinge point formation in neural plate; double mutants show defective apical concentration of phosphorylated myosin light chain 2, F-actin, and cortactin, causing neural tube defects.","method":"Double knockout mouse model, immunofluorescence for cytoskeletal markers","journal":"Developmental dynamics","confidence":"Medium","confidence_rationale":"Tier 2 — double KO with specific cytoskeletal phenotype readout, single study","pmids":["22689267"],"is_preprint":false},{"year":2014,"finding":"NUAK1 expression is controlled through the cell cycle by CDK phosphorylation at Ser445, which triggers PLK1 binding; PLK1 then phosphorylates NUAK1 at Ser476 and Ser480, promoting binding to βTrCP and SCFβTrCP-mediated ubiquitylation and degradation; NUAK1 inhibits the PP1β-MYPT1 phosphatase complex and thereby promotes PLK1 T-loop phosphorylation at Thr210, establishing a reciprocal regulatory loop.","method":"Biochemical fractionation, co-immunoprecipitation, in vitro phosphorylation, ubiquitylation assay, NUAK1 inhibitors (WZ4003, HTH-01-015), site-directed mutagenesis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1-2 — multiple biochemical assays with mutagenesis, phosphorylation reconstitution, cell-cycle staging","pmids":["24785407"],"is_preprint":false},{"year":2013,"finding":"NUAK1 is a direct target of miR-211, which is regulated by the MITF transcription factor; miR-211 suppresses NUAK1 expression and promotes melanoma cell adhesion, thereby inhibiting the invasive program.","method":"miRNA overexpression/inhibition, luciferase reporter assay for 3'UTR targeting, adhesion assays","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 3 — luciferase assay with functional adhesion readout, single lab","pmids":["23934065"],"is_preprint":false},{"year":2018,"finding":"NUAK1 is activated by oxidative stress and facilitates nuclear import of the antioxidant master regulator NRF2 by coordinating PP1β inhibition with AKT activation to suppress GSK3β-dependent inhibition of NRF2 nuclear import; deletion of NUAK1 suppresses colorectal tumor formation and causes regression of established tumors.","method":"NUAK1 conditional knockout, autochthonous colorectal tumor models, Western blot for PP1β, AKT, GSK3β, NRF2 nuclear fractionation","journal":"Cancer discovery","confidence":"High","confidence_rationale":"Tier 2 — genetic deletion in autochthonous tumor model with defined molecular pathway, multiple orthogonal approaches","pmids":["29500295"],"is_preprint":false},{"year":2020,"finding":"NUAK1 is a predominantly nuclear kinase that phosphorylates PNUTS (a nuclear regulatory subunit of PP1) to locally inhibit PP1 activity; NUAK1-PNUTS interaction is required for chromatin association of PNUTS, spliceosome activity, and nascent RNA synthesis; in MYC-overexpressing cells, NUAK1 inhibition causes global RNAPII accumulation at the pause site and first exon-intron boundary without increasing mRNA synthesis.","method":"Nuclear/cytoplasmic fractionation, co-immunoprecipitation of PP1 interactor network, in vitro kinase assay of PNUTS, NUAK1 inhibition, RNAPII ChIP, nascent RNA synthesis assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 — MS interactome, in vitro phosphorylation, RNAPII ChIP, spliceosome activity assays, multiple methods","pmids":["32006464"],"is_preprint":false},{"year":2022,"finding":"TGF-β induces rapid NUAK1 upregulation in fibroblasts; NUAK1 in turn promotes YAP/TAZ and TGF-β/SMAD profibrotic signaling; activated YAP/TAZ further induces NUAK1 expression, creating a positive feedback loop; fibroblast-specific loss or pharmacological inhibition of NUAK1 markedly attenuates kidney, lung, and liver fibrosis in mouse models.","method":"Fibroblast-specific NUAK1 knockout, mouse models of organ fibrosis, Western blot for SMAD/YAP signaling, pharmacological inhibition","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific KO in multiple organ fibrosis models with defined signaling pathway, multiple orthogonal approaches","pmids":["35320001"],"is_preprint":false},{"year":2019,"finding":"NUAK1 nuclear import is mediated by a bipartite nuclear localization signal at its N-terminal domain and requires importin-β members KPNB1, IPO7, and IPO9; oxidative stress induces NUAK1 cytoplasmic accumulation by affecting its nuclear transport.","method":"Cell fractionation, mass spectrometry identification of importin interactors, importin-β knockdown, Importazole treatment, NLS mutant constructs","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — MS-identified interactors validated by KD and chemical inhibition, single lab","pmids":["31090959"],"is_preprint":false},{"year":2017,"finding":"In tumor cells lacking LKB1, NUAK1 kinase activity is maintained by an alternative calcium-dependent pathway involving PKCα activation; calcium/PKCα-dependent NUAK1 activation supports AMPK-TORC1 metabolic checkpoint engagement, protecting tumor cells from MYC-driven cell death; MYC transcriptionally regulates PKCα and ITPR to promote this pathway.","method":"LKB1-null cell models, PKCα knockdown/inhibition, calcium manipulation, MYC transcriptomics, TORC1 signaling assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — genetic and pharmacological manipulation identifying alternative activation pathway, single lab","pmids":["29106388"],"is_preprint":false},{"year":2020,"finding":"Cytosolic NUAK1 increases ATP production by enhancing mitochondrial respiration; NUAK1 inhibition leads to mitochondrial morphology changes ('donut-like' structures); nuclear NUAK1 is involved in the metabolic switch to glycolysis; NUAK1 subcellular localization differentially affects metabolic outcomes in cancer cells.","method":"Subcellular fractionation, mitochondrial respiration assays, ATP measurements, live-cell imaging of mitochondrial morphology, NUAK1 inhibition","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 — fractionation linked to functional metabolic readouts, single lab","pmids":["32754444"],"is_preprint":false},{"year":2024,"finding":"NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration in cortical neurons; upregulation of mitochondrial function rescues axon branching defects in NUAK1-null neurons in vitro and in vivo; NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN, demonstrating a dual function controlling both mitochondrial distribution and metabolic activity.","method":"NUAK1 conditional knockout, mitochondrial function assays, ATP biosensors, BRAWNIN knockdown/overexpression, in vivo axon branching quantification","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic and metabolic approaches in vitro and in vivo, identifying BRAWNIN as downstream effector","pmids":["38514619"],"is_preprint":false},{"year":2020,"finding":"LKB1 phosphorylates and stabilizes NUAK1 in ovarian cancer spheroids; NUAK1 loss reduces spheroid integrity and fibronectin (FN1) expression (745-fold decrease); fibronectin is required for compact spheroid formation and metastasis; NUAK1 loss extends survival in a xenograft model of intraperitoneal metastasis.","method":"NUAK1 knockout, transcriptome analysis, fibronectin rescue experiments, xenograft metastasis model","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 — KO with transcriptome, molecular rescue, and in vivo model, single lab","pmids":["32429240"],"is_preprint":false},{"year":2022,"finding":"ARK5/NUAK1 directly interacts with and phosphorylates hnRNP A1 on serine residues within the F-peptide region; the M9 motif of hnRNP A1 is required for ARK5 interaction and phosphorylation; ARK5-mediated phosphorylation controls hnRNP A1 cytoplasmic relocalization during hypertonic stress; ARK5 silencing increases Bcl-xL expression and delays caspase activation.","method":"Co-immunoprecipitation, in vitro kinase assay, RNAi-based kinome screen, flow cytometry for caspase activation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay identifying hnRNP A1 as substrate, with mutagenesis of M9 motif and functional cellular readouts","pmids":["35963429"],"is_preprint":false},{"year":2023,"finding":"NUAK1 governs accurate centrosome duplication via MYPT1/PP1β and GSK3β-dependent regulation of PLK4; loss of NUAK1 triggers genomic instability in both pancreatic cancer cells and primary fibroblasts.","method":"NUAK1 inhibition/depletion, centrosome counting, genomic instability assays, pathway analysis in PDAC cells","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 — functional KO/inhibition with defined pathway (MYPT1/PP1β/GSK3β/PLK4) and centrosome phenotype, single lab","pmids":["36975767"],"is_preprint":false},{"year":2023,"finding":"NUAK1 promotes metastasis in esophageal squamous cell carcinoma by activating the JNK/c-Jun pathway, which upregulates Slug transcription; silencing Slug completely abolishes NUAK1-overexpression-driven migration and invasion.","method":"Co-immunoprecipitation, immunofluorescence, dual-luciferase reporter gene assay for Slug promoter, in vivo pulmonary metastasis model","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2-3 — reporter assay and co-IP identifying JNK/c-Jun/Slug pathway, single lab","pmids":["37919754"],"is_preprint":false},{"year":2024,"finding":"NUAK1 directly phosphorylates NADK (nicotinamide adenine dinucleotide kinase) at Ser64, reducing ROS accumulation and thereby conferring osimertinib resistance in NSCLC; genetic or pharmacological blockade of NUAK1 restores osimertinib sensitivity.","method":"In vitro kinase assay identifying NADK S64 phosphorylation, NUAK1 knockdown/overexpression, ROS measurements, in vitro and in vivo drug sensitivity assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1-2 — direct phosphorylation identified by in vitro kinase assay with specific substrate residue, validated in vitro and in vivo","pmids":["39159134"],"is_preprint":false},{"year":2023,"finding":"NUAK1 interacts with mTORC2 components and regulates mTORC2-dependent Akt activation by controlling lysosome positioning and mTOR association with lysosomes; NUAK1 also directly phosphorylates Akt at Ser473; NUAK1 is associated with early endosomes and selectively promotes Akt/FOXO1/3a signaling but not Akt/TSC2 signaling.","method":"Co-immunoprecipitation of mTORC2 components, in vitro kinase assay for Akt S473, lysosome positioning assay, subcellular fractionation, Akt substrate phosphorylation analysis","journal":"Cell & bioscience","confidence":"Medium","confidence_rationale":"Tier 1-2 — in vitro kinase assay and co-IP with mTORC2, supported by organelle localization data, single lab","pmids":["38135881"],"is_preprint":false},{"year":2024,"finding":"NUAK1 upregulates GLI1 expression by activating STAT5-mediated transcription and stabilizing GLI1 protein; depletion or inhibition of NUAK1 impairs cancer cell expansion and tumor formation via the NUAK1/STAT5/GLI1 axis in gastric cancer.","method":"Patient-derived xenograft, transgenic mouse model, siRNA knockdown, pharmacological inhibition, Western blot and reporter assays for STAT5/GLI1","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 — multiple in vivo models with defined pathway, single lab","pmids":["38996065"],"is_preprint":false},{"year":2020,"finding":"In yeast, Tda1 (the ortholog of human NUAK1) directly phosphorylates histone H3 at Thr11 upon low glucose stress; yeast AMPK (Snf1) phosphorylates Tda1 to govern its activity, while CK2 regulates Tda1 nuclear localization; AMPK and CK2 signaling converge on histone kinase Tda1 to link glucose stress to chromatin regulation.","method":"In vitro kinase assay, H3pT11 ChIP, mutant analysis, epistasis in budding yeast","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of H3 phosphorylation by Tda1, with epistasis and localization experiments; yeast ortholog","pmids":["33372657"],"is_preprint":false},{"year":2025,"finding":"USP9X deubiquitinates and thereby activates ARK5/NUAK1; USP9X-activated NUAK1 promotes TGF-β1-induced myofibroblast transformation via the Rho kinase pathway; myofibroblast-specific USP9X knockout or pharmacological inhibition reduces NUAK1 activity and attenuates cardiac fibrosis.","method":"Co-immunoprecipitation, USP9X knockout (myofibroblast-specific), in vivo myocardial infarction model, Western blot for NUAK1 ubiquitination/activation and Rho kinase pathway","journal":"JACC. Basic to translational science","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP and cell-type-specific KO with defined pathway, single lab","pmids":["40310323"],"is_preprint":false},{"year":2024,"finding":"ARK5/NUAK1 regulates mitochondrial morphological dynamics, promoting fission over fusion in multiple myeloma cells; ARK5 knockout cells show enhanced mitochondrial fusion, reduced phosphorylation of DRP1 at Ser616, higher MFN1/MFN2 expression, lower ATP, higher lactate, and higher ROS levels.","method":"Biallelic ARK5 knockout in myeloma cell lines, mitochondrial morphology imaging, Western blot for DRP1/MFN1/MFN2, ATP and lactate measurements","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 — clean biallelic KO with multiple orthogonal mitochondrial readouts, single lab","pmids":["38282096"],"is_preprint":false},{"year":2021,"finding":"NUAK1 facilitates nuclear import of NRF2 by inhibiting PP1β, which leads to AKT activation and suppression of GSK3β-dependent phosphorylation of NRF2, thus reducing GSK3β-mediated inhibition of NRF2 nuclear import; silencing NUAK1 upregulates PP1β and downregulates phospho-GSK3βSer9, suppressing NRF2 nuclear accumulation and GPX4 expression.","method":"siRNA knockdown, Western blot for PP1β, p-GSK3βSer9, NRF2 nuclear fractionation, NUAK1 agonist rescue experiment","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2-3 — defined pharmacological and genetic dissection of PP1β/GSK3β/NRF2 pathway, single lab","pmids":["33819186"],"is_preprint":false},{"year":2018,"finding":"Haploinsufficiency of NUAK1 in mice impairs cortical axon branching and leads to behavioral deficits including defective spatial memory consolidation, impaired social novelty, and abnormal sensorimotor gating, confirming NUAK1 as a haploinsufficient autism spectrum disorder candidate gene.","method":"NUAK1 heterozygous knockout mice, cortical connectivity analysis, behavioral test battery","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — haploinsufficiency mouse model with multiple behavioral and anatomical readouts, replicated across tests","pmids":["30327473"],"is_preprint":false},{"year":2024,"finding":"NUAK1 promotes PD-L1 transcription in hepatocellular carcinoma by phosphorylating GSK3β at Ser9, which activates β-catenin nuclear accumulation and PD-L1 gene expression; knockdown of β-catenin reverses NUAK1-mediated PD-L1 upregulation.","method":"Gain and loss of function experiments, Western blot and immunofluorescence for GSK3β/β-catenin/PD-L1, β-catenin knockdown rescue, IHC of patient samples","journal":"Molecular medicine (Cambridge, Mass.)","confidence":"Medium","confidence_rationale":"Tier 2-3 — genetic and rescue experiments defining GSK3β/β-catenin/PD-L1 pathway, single lab","pmids":["39901136"],"is_preprint":false},{"year":2025,"finding":"NUAK1 is present in human erythrocytes and undergoes increased phosphorylation in P. falciparum-infected cells; NUAK1 inhibition reduces MYPT1 phosphorylation in erythroid cells; genetic overexpression of NUAK1 in erythroid cells partially rescues both signaling and parasite invasion phenotypes caused by NUAK1 inhibitors.","method":"Chemoproteomic profiling confirming inhibitor selectivity, phosphoproteomic analysis, NUAK1 overexpression in erythroid cells, P. falciparum invasion assays","journal":"bioRxiv (PMID available: 41278793)","confidence":"Medium","confidence_rationale":"Tier 2 — chemoproteomic validation of inhibitor selectivity, genetic rescue of MYPT1 signaling, novel host context","pmids":["41278793"],"is_preprint":true},{"year":2025,"finding":"NUAK1 phosphorylates the splicing co-factor SON, regulating a conserved set of alternative splicing events in genes crucial for neurodevelopment in cortical neurons; SON is required for proper cortical neuron development consistent with ZTTK syndrome phenotypes.","method":"NUAK1 conditional knockout in cortical neurons, RNA splicing analysis, identification of SON as phosphorylation substrate, SON knockdown","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO with splicing transcriptomics and identification of SON as substrate, preprint only","pmids":[],"is_preprint":true},{"year":2024,"finding":"NUAK1 kinase activity correlates with MYPT1 phosphorylation at Ser445 and Ser910 in vascular smooth muscle cells; NUAK1 knockdown decreases myosin light chain phosphorylation, potentially by increasing MYPT1 phosphatase activity, reducing contractility and altering F-actin cytoskeletal organization.","method":"Phosphoproteomic analysis, NUAK1 knockdown, pMLC and MYPT1 phosphorylation Western blot, impedance-based contraction assay","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 — phosphoproteomic correlation and KD, preprint, single lab","pmids":[],"is_preprint":true},{"year":2024,"finding":"First crystal structures of a NUAK1 chimeric protein were determined, providing structural insights into inhibitor binding modes and identifying a unique glutamate residue (Glu139) that can be exploited for selective NUAK1 targeting.","method":"X-ray crystallography of NUAK1 chimeric protein with bound inhibitors","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 1 — crystal structure with functional validation of binding mode, single study","pmids":["39588908"],"is_preprint":false},{"year":2022,"finding":"ARK5/NUAK1 prevents degradation of TGF-β type I receptor (TβRI) and Smad4 proteins by inhibiting Smurf2 expression in hepatic stellate cells, thereby maintaining TGF-β signaling; in hepatocytes, ARK5 induces EMT and promotes inflammatory cytokine secretion, synergistically driving liver fibrosis.","method":"ARK5 knockdown in HSCs and hepatocytes, Western blot for TβRI/Smad4/Smurf2, mouse CCl4-fibrosis model with HTH-01-015 inhibitor","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2-3 — mechanistic pathway identified by KD with defined molecular readouts and in vivo pharmacology, single lab","pmids":["36361872"],"is_preprint":false},{"year":2024,"finding":"NUAK1 promotes pyroptosis and inflammation in MASH by activating Caspase 6; NUAK1/Caspase 6 axis inhibition accelerates the interaction between TAK1 and RIPK1, leading to RIPK1 degradation and suppression of hepatic inflammation.","method":"NUAK1 inhibition in vivo and in vitro MASH models, Western blot for Caspase 6, TAK1, RIPK1, caspase 6 disruption experiments","journal":"Hepatology communications","confidence":"Medium","confidence_rationale":"Tier 2-3 — in vivo and in vitro NUAK1 inhibition with defined Caspase 6/TAK1/RIPK1 pathway dissection, single lab","pmids":["38967580"],"is_preprint":false}],"current_model":"NUAK1 is an LKB1-activated AMPK-related serine/threonine kinase that phosphorylates a broad spectrum of substrates—including MYPT1/PP1β (to regulate myosin activity and NRF2 nuclear import), tau (Ser356), caspase-6 (Ser257), LATS1 (Ser464), p53, PNUTS (to support spliceosome activity), Akt (Ser473), NADK (Ser64), and hnRNP A1—and operates in multiple cellular contexts including axon branching via mitochondrial immobilization and metabolic support, cellular senescence, the antioxidant stress response, fibrogenesis through YAP/TGF-β signaling, cell cycle regulation via PLK1/SCFβTrCP, and nuclear RNA splicing through a MYC-dependent PP1/PNUTS axis, with its own activity regulated by Akt phosphorylation (Ser600), NDR2 phosphorylation (Thr211), calcium/PKCα signaling (in LKB1-null contexts), CDK/PLK1/SCFβTrCP-mediated degradation, USP9X-mediated stabilization, and oxidative stress-driven cytoplasmic relocalization."},"narrative":{"teleology":[{"year":2003,"claim":"NUAK1 was first linked to cell survival signaling when overexpression was shown to suppress nutrient-starvation- and death-receptor-induced apoptosis by inhibiting caspase-8 activation, establishing it as a pro-survival kinase.","evidence":"Overexpression in HepG2 cells with caspase-8, Bid, and FLIP readouts","pmids":["13679856"],"confidence":"Medium","gaps":["Mechanism of caspase-8 inhibition not defined at the substrate level","Single cell line, no genetic loss-of-function"]},{"year":2004,"claim":"The first direct substrate of NUAK1 was identified—caspase-6 at Ser257—providing a molecular mechanism for its anti-apoptotic function and demonstrating it is a bona fide kinase with defined substrates.","evidence":"In vitro kinase assay with S257A mutagenesis abolishing protection from Fas-mediated death in colorectal cancer cells","pmids":["15273717"],"confidence":"High","gaps":["Whether caspase-6 phosphorylation is relevant in non-cancer contexts","Structural basis of kinase-substrate recognition unknown"]},{"year":2006,"claim":"The activation mechanism of NUAK1 was elucidated: NDR2 phosphorylates Thr211 on the T-loop and Akt phosphorylates Ser600 in the regulatory domain, with both events required for full kinase activity, placing NUAK1 downstream of growth factor signaling.","evidence":"In vitro kinase assay with mutant constructs and co-IP upon IGF-1 stimulation in colorectal cancer cells","pmids":["16488889"],"confidence":"High","gaps":["Whether LKB1 and NDR2 phosphorylate the same site independently or cooperatively","Context-dependence of NDR2 vs. LKB1 activation not resolved"]},{"year":2009,"claim":"NUAK1 was connected to cell cycle and ploidy control through its phosphorylation of LATS1 at Ser464, triggering LATS1 degradation and linking NUAK1 to Hippo pathway regulation and cellular senescence.","evidence":"Loss-of-function screen, in vitro kinase assay, and dominant-negative LATS1 phenocopy in human fibroblasts","pmids":["19927127"],"confidence":"High","gaps":["Whether NUAK1-LATS1 interaction operates in Hippo-dependent tissue growth contexts","Ubiquitin ligase mediating LATS1 degradation downstream of Ser464 phosphorylation not identified"]},{"year":2011,"claim":"NUAK1 was shown to phosphorylate p53 in an LKB1-dependent manner and to occupy the p21 promoter with p53, establishing a tumor-suppressive LKB1/NUAK1/p53/p21 axis that induces G1/S arrest.","evidence":"Co-IP, in vitro kinase assay, ChIP at the p21 promoter, cell cycle analysis","pmids":["21317932"],"confidence":"High","gaps":["Specific p53 phosphorylation site(s) not mapped","How this tumor-suppressive function is reconciled with NUAK1's pro-survival roles"]},{"year":2013,"claim":"NUAK1 was placed in a neurodevelopmental pathway: the LKB1–NUAK1 axis controls cortical axon branching by immobilizing mitochondria at nascent presynaptic sites via Syntaphilin, revealing a cell-biological function beyond cancer.","evidence":"Conditional LKB1 knockout, in utero NUAK1 knockdown/overexpression, Syntaphilin rescue in mouse cortical neurons","pmids":["23791179"],"confidence":"High","gaps":["Whether NUAK1 directly phosphorylates Syntaphilin or acts indirectly","Mechanism linking mitochondrial immobilization to branch stabilization unknown"]},{"year":2014,"claim":"A cell-cycle-linked regulatory circuit was defined: CDK phosphorylation primes NUAK1 for PLK1-mediated phosphorylation and SCFβTrCP-dependent degradation, while NUAK1 reciprocally promotes PLK1 activation by inhibiting PP1β-MYPT1, establishing the MYPT1/PP1β complex as a central NUAK1 effector.","evidence":"Biochemical reconstitution of CDK→PLK1→βTrCP ubiquitylation cascade, site-directed mutagenesis, NUAK1 inhibitors WZ4003/HTH-01-015","pmids":["24785407"],"confidence":"High","gaps":["Whether PP1β-MYPT1 inhibition by NUAK1 involves direct MYPT1 phosphorylation or an indirect mechanism","Functional consequence of NUAK1 degradation timing on mitotic fidelity"]},{"year":2016,"claim":"NUAK1 was identified as a kinase that phosphorylates tau at Ser356, stabilizing pathological tau; genetic reduction of NUAK1 suppressed neurodegeneration in both Drosophila and mouse tauopathy models, establishing NUAK1 as a therapeutic target for tauopathies.","evidence":"In vitro kinase assay, Drosophila genetic screen, NUAK1 haploinsufficiency rescue in P301S tau mouse model","pmids":["27720485"],"confidence":"High","gaps":["Whether NUAK1 phosphorylates tau directly in neurons or through an intermediate","Therapeutic window of NUAK1 inhibition given its essential neurodevelopmental roles"]},{"year":2017,"claim":"An alternative, LKB1-independent activation mechanism was uncovered: in LKB1-null tumors, calcium/PKCα signaling maintains NUAK1 activity to engage the AMPK-TORC1 metabolic checkpoint, explaining how NUAK1 remains functional in LKB1-deficient cancers.","evidence":"PKCα knockdown/inhibition and calcium manipulation in LKB1-null cell models with TORC1 readouts","pmids":["29106388"],"confidence":"Medium","gaps":["Whether PKCα directly phosphorylates NUAK1 and at which site","Generalizability beyond MYC-driven LKB1-null contexts"]},{"year":2018,"claim":"NUAK1 was connected to the antioxidant stress response: it facilitates NRF2 nuclear import by inhibiting PP1β, thereby activating AKT and suppressing GSK3β-dependent NRF2 phosphorylation; conditional NUAK1 deletion caused regression of established colorectal tumors, positioning NUAK1 as a druggable vulnerability in ROS-dependent cancers.","evidence":"Conditional KO in autochthonous colorectal tumor models with NRF2/PP1β/AKT/GSK3β pathway dissection","pmids":["29500295"],"confidence":"High","gaps":["Whether NUAK1 directly phosphorylates PP1β or MYPT1 in this context","NRF2-independent mechanisms of tumor regression upon NUAK1 loss"]},{"year":2018,"claim":"NUAK1 haploinsufficiency in mice was shown to impair cortical axon branching and produce behavioral deficits including defective spatial memory, impaired social novelty, and abnormal sensorimotor gating, validating NUAK1 as a haploinsufficient autism spectrum disorder candidate gene.","evidence":"Heterozygous NUAK1 knockout mice with cortical connectivity analysis and behavioral test battery","pmids":["30327473"],"confidence":"High","gaps":["No human family studies confirming NUAK1 haploinsufficiency as causative for ASD","Molecular mechanism linking reduced kinase dosage to circuit-level defects unclear"]},{"year":2020,"claim":"The nuclear function of NUAK1 was defined: it phosphorylates the PP1 regulatory subunit PNUTS to locally inhibit PP1, supporting spliceosome activity and nascent RNA synthesis; in MYC-overexpressing cells, NUAK1 inhibition causes global RNAPII pausing, revealing a transcription-coupled splicing role.","evidence":"Nuclear interactome by mass spectrometry, in vitro PNUTS phosphorylation, RNAPII ChIP, spliceosome and nascent RNA assays","pmids":["32006464"],"confidence":"High","gaps":["Specific PNUTS phosphorylation sites not mapped","Whether NUAK1-PNUTS axis is MYC-selective or generalizable"]},{"year":2020,"claim":"Subcellular compartmentalization of NUAK1 was shown to differentially control metabolism: cytosolic NUAK1 enhances mitochondrial respiration and ATP production, whereas nuclear NUAK1 promotes glycolysis, establishing NUAK1 as a localization-dependent metabolic switch.","evidence":"Subcellular fractionation with mitochondrial respiration assays and ATP measurements in cancer cells","pmids":["32754444"],"confidence":"Medium","gaps":["Cytoplasmic substrates mediating mitochondrial effects not identified","Single study; compartment-specific constructs not used"]},{"year":2022,"claim":"NUAK1 was shown to drive organ fibrosis through a TGF-β–YAP/TAZ positive feedback loop: TGF-β induces NUAK1, which promotes YAP/TAZ activity, and YAP/TAZ in turn upregulates NUAK1; fibroblast-specific NUAK1 loss attenuated kidney, lung, and liver fibrosis.","evidence":"Fibroblast-specific NUAK1 KO in multiple organ fibrosis mouse models with SMAD/YAP signaling analysis","pmids":["35320001"],"confidence":"High","gaps":["Direct NUAK1 substrate in YAP/TAZ activation pathway not identified","Whether PP1β-MYPT1 mediates the fibrogenic loop"]},{"year":2022,"claim":"hnRNP A1 was identified as a direct NUAK1 substrate: NUAK1 phosphorylates serine residues within the F-peptide region of hnRNP A1, controlling its cytoplasmic relocalization during hypertonic stress, expanding NUAK1's role into RNA-binding protein regulation.","evidence":"In vitro kinase assay, M9-motif mutagenesis, RNAi kinome screen, co-IP","pmids":["35963429"],"confidence":"High","gaps":["Specific phosphorylation sites on hnRNP A1 not individually mapped","Functional consequences for mRNA splicing or stability not determined"]},{"year":2023,"claim":"NUAK1 was found to govern centrosome duplication fidelity through the MYPT1/PP1β/GSK3β/PLK4 axis, with NUAK1 loss triggering centrosome amplification and genomic instability in both cancer cells and primary fibroblasts.","evidence":"NUAK1 depletion/inhibition with centrosome counting and genomic instability assays in PDAC cells and fibroblasts","pmids":["36975767"],"confidence":"Medium","gaps":["Direct phosphorylation of PLK4 pathway components by NUAK1 not shown","Whether centrosome defects contribute to the senescence phenotype previously observed"]},{"year":2023,"claim":"NUAK1 was shown to interact with mTORC2 components and directly phosphorylate Akt at Ser473 while controlling lysosome positioning to regulate mTOR–lysosome association, selectively activating Akt/FOXO signaling over Akt/TSC2 signaling.","evidence":"Co-IP of mTORC2 components, in vitro kinase assay for Akt S473, lysosome positioning and subcellular fractionation","pmids":["38135881"],"confidence":"Medium","gaps":["Mechanism of substrate-selective Akt signaling not explained","Single lab; independent validation needed","Whether NUAK1 phosphorylation of Akt is physiologically relevant given mTORC2 dominance"]},{"year":2024,"claim":"The metabolic function of NUAK1 in neurons was deepened: NUAK1 deficiency impairs mitochondrial metabolism and axonal ATP levels, and upregulation of mitochondrial function via the microprotein BRAWNIN rescues axon branching defects, demonstrating NUAK1 controls both mitochondrial distribution and metabolic output.","evidence":"Conditional NUAK1 KO, ATP biosensors, BRAWNIN knockdown/overexpression, in vivo axon branching","pmids":["38514619"],"confidence":"High","gaps":["Whether NUAK1 directly regulates BRAWNIN expression or activity","How mitochondrial metabolic and anchoring functions are coordinated"]},{"year":2024,"claim":"NADK was identified as a direct NUAK1 substrate (Ser64), linking NUAK1 to NADPH metabolism and ROS control; this phosphorylation confers osimertinib resistance in NSCLC, extending NUAK1's roles to drug resistance via redox balance.","evidence":"In vitro kinase assay identifying NADK S64, NUAK1 knockdown/overexpression, ROS measurements, in vivo drug sensitivity","pmids":["39159134"],"confidence":"High","gaps":["Whether NADK Ser64 phosphorylation alters enzymatic kinetics directly","Generalizability to other EGFR-TKI resistance contexts"]},{"year":2024,"claim":"The first crystal structures of NUAK1 were determined, revealing inhibitor binding modes and a unique Glu139 residue exploitable for selective inhibitor design, providing a structural foundation for drug development.","evidence":"X-ray crystallography of NUAK1 chimeric protein with bound inhibitors","pmids":["39588908"],"confidence":"Medium","gaps":["Structure is of a chimeric construct, not full-length wild-type NUAK1","No structure of NUAK1 in complex with a substrate or regulatory subunit"]},{"year":2025,"claim":"USP9X was identified as a deubiquitinase that stabilizes and activates NUAK1, with the USP9X–NUAK1 axis promoting TGF-β1-induced myofibroblast transformation via Rho kinase signaling and driving cardiac fibrosis.","evidence":"Co-IP, myofibroblast-specific USP9X KO, in vivo myocardial infarction model","pmids":["40310323"],"confidence":"Medium","gaps":["Direct ubiquitination sites on NUAK1 targeted by USP9X not mapped","Relationship between USP9X stabilization and SCFβTrCP-mediated degradation not clarified"]},{"year":null,"claim":"Key unresolved questions include: the full catalog of direct NUAK1 phosphorylation sites on MYPT1 and PNUTS; how nuclear versus cytoplasmic NUAK1 pools are coordinated to produce context-dependent metabolic and transcriptional outcomes; and whether NUAK1 haploinsufficiency is causative for human neurodevelopmental disorders.","evidence":"","pmids":[],"confidence":"Low","gaps":["No human genetic validation of NUAK1 as a Mendelian disease gene","No full-length wild-type NUAK1 structure","Mechanism by which NUAK1 selectively engages different substrates in different compartments unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,3,5,7,8,11,14,21,24,25]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,11,13,30]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,14,16,18]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[16,18]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[0,19,29]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[25]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,5,13,15,25,26,32]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,3,38]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[7,11,22]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[14,34]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,10,31]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[13,30]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[9,18,19,24,29]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,15,37]}],"complexes":[],"partners":["MYPT1","PP1B","PNUTS","LKB1","PLK1","USP9X","LATS1","TP53"],"other_free_text":[]},"mechanistic_narrative":"NUAK1 is an AMPK-related serine/threonine kinase that functions as a central signaling node integrating metabolic stress, cytoskeletal regulation, and gene expression control across diverse cellular contexts. Activated by LKB1 phosphorylation at Thr211 and cooperatively by Akt at Ser600 and NDR2 at the T-loop, NUAK1 phosphorylates the PP1β-MYPT1 phosphatase complex to regulate myosin phosphorylation, centrosome duplication, and NRF2 nuclear import via the GSK3β axis, and directly phosphorylates substrates including caspase-6 (Ser257), LATS1 (Ser464), p53, tau (Ser356), PNUTS, Akt (Ser473), NADK (Ser64), and hnRNP A1 [PMID:16488889, PMID:15273717, PMID:19927127, PMID:21317932, PMID:27720485, PMID:32006464, PMID:38135881, PMID:39159134, PMID:35963429]. In neurons, the LKB1–NUAK1 pathway controls axon branching by immobilizing mitochondria at nascent presynaptic sites and by sustaining mitochondrial metabolic activity through the microprotein BRAWNIN; NUAK1 haploinsufficiency impairs cortical connectivity and causes behavioral deficits consistent with autism spectrum disorder [PMID:23791179, PMID:38514619, PMID:30327473]. In the nucleus, NUAK1 phosphorylates PNUTS to locally inhibit PP1 and support spliceosome function and nascent RNA synthesis, while in fibroblasts it drives a positive-feedback loop with YAP/TAZ and TGF-β/SMAD signaling that promotes organ fibrosis [PMID:32006464, PMID:35320001, PMID:40310323]."},"prefetch_data":{"uniprot":{"accession":"O60285","full_name":"NUAK family SNF1-like kinase 1","aliases":["AMPK-related protein kinase 5","ARK5","Omphalocele kinase 1"],"length_aa":661,"mass_kda":74.3,"function":"Serine/threonine-protein kinase involved in various processes such as cell adhesion, regulation of cell ploidy and senescence, cell proliferation and tumor progression. Phosphorylates ATM, CASP6, LATS1, PPP1R12A and p53/TP53. Acts as a regulator of cellular senescence and cellular ploidy by mediating phosphorylation of 'Ser-464' of LATS1, thereby controlling its stability. Controls cell adhesion by regulating activity of the myosin protein phosphatase 1 (PP1) complex. Acts by mediating phosphorylation of PPP1R12A subunit of myosin PP1: phosphorylated PPP1R12A then interacts with 14-3-3, leading to reduced dephosphorylation of myosin MLC2 by myosin PP1. May be involved in DNA damage response: phosphorylates p53/TP53 at 'Ser-15' and 'Ser-392' and is recruited to the CDKN1A/WAF1 promoter to participate in transcription activation by p53/TP53. May also act as a tumor malignancy-associated factor by promoting tumor invasion and metastasis under regulation and phosphorylation by AKT1. Suppresses Fas-induced apoptosis by mediating phosphorylation of CASP6, thereby suppressing the activation of the caspase and the subsequent cleavage of CFLAR. Regulates UV radiation-induced DNA damage response mediated by CDKN1A. In association with STK11, phosphorylates CDKN1A in response to UV radiation and contributes to its degradation which is necessary for optimal DNA repair (PubMed:25329316)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O60285/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NUAK1","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NUAK1","total_profiled":1310},"omim":[{"mim_id":"608130","title":"NUAK FAMILY, SNF1-LIKE KINASE, 1; NUAK1","url":"https://www.omim.org/entry/608130"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoli fibrillar center","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Microtubules","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NUAK1"},"hgnc":{"alias_symbol":["ARK5","KIAA0537"],"prev_symbol":[]},"alphafold":{"accession":"O60285","domains":[{"cath_id":"3.30.200.20","chopping":"44-133","consensus_level":"high","plddt":90.9592,"start":44,"end":133},{"cath_id":"1.10.510.10","chopping":"139-315","consensus_level":"high","plddt":93.4659,"start":139,"end":315}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O60285","model_url":"https://alphafold.ebi.ac.uk/files/AF-O60285-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O60285-F1-predicted_aligned_error_v6.png","plddt_mean":61.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NUAK1","jax_strain_url":"https://www.jax.org/strain/search?query=NUAK1"},"sequence":{"accession":"O60285","fasta_url":"https://rest.uniprot.org/uniprotkb/O60285.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O60285/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O60285"}},"corpus_meta":[{"pmid":"23791179","id":"PMC_23791179","title":"Terminal 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and TGF-β/SMAD signaling.","date":"2022","source":"Science translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35320001","citation_count":85,"is_preprint":false},{"pmid":"29500295","id":"PMC_29500295","title":"Colorectal Tumors Require NUAK1 for Protection from Oxidative Stress.","date":"2018","source":"Cancer discovery","url":"https://pubmed.ncbi.nlm.nih.gov/29500295","citation_count":79,"is_preprint":false},{"pmid":"13679856","id":"PMC_13679856","title":"ARK5 suppresses the cell death induced by nutrient starvation and death receptors via inhibition of caspase 8 activation, but not by chemotherapeutic agents or UV irradiation.","date":"2003","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/13679856","citation_count":77,"is_preprint":false},{"pmid":"15273717","id":"PMC_15273717","title":"Regulation of caspase-6 and FLIP by the AMPK family member ARK5.","date":"2004","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/15273717","citation_count":76,"is_preprint":false},{"pmid":"29575772","id":"PMC_29575772","title":"LncRNA SNHG1 functions as a ceRNA to antagonize the effect of miR-145a-5p on the down-regulation of NUAK1 in nasopharyngeal carcinoma cell.","date":"2018","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/29575772","citation_count":67,"is_preprint":false},{"pmid":"21317932","id":"PMC_21317932","title":"A new role of NUAK1: directly phosphorylating p53 and regulating cell proliferation.","date":"2011","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/21317932","citation_count":65,"is_preprint":false},{"pmid":"16044163","id":"PMC_16044163","title":"ARK5 is transcriptionally regulated by the Large-MAF family and mediates IGF-1-induced cell invasion in multiple myeloma: ARK5 as a new molecular determinant of malignant multiple myeloma.","date":"2005","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/16044163","citation_count":60,"is_preprint":false},{"pmid":"14982852","id":"PMC_14982852","title":"ARK5 expression in colorectal cancer and its implications for tumor progression.","date":"2004","source":"The American journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/14982852","citation_count":59,"is_preprint":false},{"pmid":"23063350","id":"PMC_23063350","title":"ARK5 promotes glioma cell invasion, and its elevated expression is correlated with poor clinical outcome.","date":"2012","source":"European journal of cancer (Oxford, England : 1990)","url":"https://pubmed.ncbi.nlm.nih.gov/23063350","citation_count":58,"is_preprint":false},{"pmid":"26882562","id":"PMC_26882562","title":"microRNA-203 suppresses invasion and epithelial-mesenchymal transition induction via targeting NUAK1 in head and neck 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kinase 5 (ARK5).","date":"2014","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24417566","citation_count":48,"is_preprint":false},{"pmid":"32006464","id":"PMC_32006464","title":"Localized Inhibition of Protein Phosphatase 1 by NUAK1 Promotes Spliceosome Activity and Reveals a MYC-Sensitive Feedback Control of Transcription.","date":"2020","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/32006464","citation_count":45,"is_preprint":false},{"pmid":"26151663","id":"PMC_26151663","title":"Activation of ARK5/miR-1181/HOXA10 axis promotes epithelial-mesenchymal transition in ovarian cancer.","date":"2015","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/26151663","citation_count":45,"is_preprint":false},{"pmid":"27126361","id":"PMC_27126361","title":"ARK5 promotes doxorubicin resistance in hepatocellular carcinoma via epithelial-mesenchymal transition.","date":"2016","source":"Cancer 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J Cell Physiol. 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Basic to translational science","url":"https://pubmed.ncbi.nlm.nih.gov/40310323","citation_count":2,"is_preprint":false},{"pmid":"32844054","id":"PMC_32844054","title":"Silencing of the ARK5 gene reverses the drug resistance of multidrug-resistant SGC7901/DDP gastric cancer cells.","date":"2020","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/32844054","citation_count":2,"is_preprint":false},{"pmid":"41264843","id":"PMC_41264843","title":"NUAK1 Promotes Diabetic Kidney Disease by Accelerating Renal Tubular Senescence via the ROS/P53 Axis.","date":"2025","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/41264843","citation_count":1,"is_preprint":false},{"pmid":"39967649","id":"PMC_39967649","title":"Development of the Pyrido[2,3-d]pyrimidin-7(8H)-one Scaffold toward Potent and Selective NUAK1 Inhibitors.","date":"2025","source":"ACS medicinal chemistry 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GPX4.","date":"2026","source":"Cellular oncology (Dordrecht, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/41848996","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.30.685469","title":"<i>Plasmodium falciparum</i>  exploits NUAK1 to establish infection in human erythrocytes","date":"2025-10-30","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.30.685469","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.10.10.681550","title":"The AMPK-related kinase NUAK1 regulates neuronal morphogenesis through the RNA splicing co-factor SON","date":"2025-10-10","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.10.681550","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.19.629332","title":"NUAK1 regulates contraction in vascular smooth muscle cells derived from abdominal aortic aneurysm 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controls axon branching in cortical neurons by promoting mitochondria immobilization at nascent presynaptic sites; conditional deletion of LKB1 or NUAK1 knockdown drastically reduces axon branching in vivo, while overexpression increases branching, and manipulation of Syntaphilin (a mitochondrial anchor) phenocopies NUAK1 effects.\",\n      \"method\": \"Conditional knockout, in utero knockdown/overexpression, live imaging, Syntaphilin genetic manipulation in mouse cortical neurons\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal genetic methods in vivo, replicated with mechanistic rescue experiment\",\n      \"pmids\": [\"23791179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NUAK1 phosphorylates tau at Ser356, stabilizing tau protein levels; inhibition of NUAK1 in Drosophila suppresses neurodegeneration in tau-expressing flies, and NUAK1 haploinsufficiency rescues tauopathy phenotypes in a mouse model.\",\n      \"method\": \"Cell-based and Drosophila genetic screens, in vitro phosphorylation assay, tauopathy mouse model\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay identifying Ser356 phosphorylation, validated in Drosophila and mouse models\",\n      \"pmids\": [\"27720485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ARK5/NUAK1 suppresses cell death (induced by nutrient starvation, TRAIL, and TNF-α) by inhibiting caspase 8 activation, delaying Bid cleavage and FLIP degradation.\",\n      \"method\": \"Overexpression in HepG2 cells, Western blot for caspase 8 activation, Bid cleavage, FLIP degradation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean overexpression with defined molecular readouts, single lab\",\n      \"pmids\": [\"13679856\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ARK5/NUAK1 directly phosphorylates caspase-6 at Ser257 in vitro, preventing procaspase-6 activation and thereby suppressing Fas-mediated cell death in colorectal cancer cells; mutation of Ser257 to Ala abolishes this protection.\",\n      \"method\": \"In vitro kinase assay with wild-type and S257A mutant caspase-6, antisense RNA knockdown, Western blot\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with mutagenesis validating specific phosphorylation site\",\n      \"pmids\": [\"15273717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ARK5/NUAK1 is activated downstream of Akt and promotes tumor invasion by inducing activation of MMP-2, MMP-9, and MT1-MMP via rapamycin-sensitive signaling; ARK5 overexpression in PANC-1 cells leads to invasion and metastasis in nude mice.\",\n      \"method\": \"Matrigel invasion assay, nude mouse xenograft, Western blot for MMP activation, pharmacological inhibition\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — overexpression with in vivo metastasis readout, single lab\",\n      \"pmids\": [\"15060171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"NDR2 kinase phosphorylates ARK5/NUAK1 at Thr211 on the T-loop during IGF-1 signaling, providing full kinase activation; Akt phosphorylates ARK5 at Ser600 in the regulatory domain, and combined phosphorylation at T211 (by NDR2) and S600 (by Akt) is required for full ARK5 activity.\",\n      \"method\": \"In vitro kinase assay, mutant constructs, co-immunoprecipitation, IGF-1 stimulation in colorectal cancer cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of phosphorylation with mutagenesis identifying specific residues\",\n      \"pmids\": [\"16488889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ARK5/NUAK1 is transcriptionally regulated by the Large-MAF family transcription factors (c-MAF and MAFB) through MAF-recognition element (MARE) sequences in the ARK5 promoter; ARK5 mediates IGF-1-induced cell invasion in multiple myeloma.\",\n      \"method\": \"Chromatin immunoprecipitation, promoter reporter assay with MARE deletion/mutation, gene expression profiling\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — ChIP and promoter mutagenesis with functional validation\",\n      \"pmids\": [\"16044163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NUAK1 phosphorylates LATS1 at Ser464, promoting its degradation and thereby controlling cellular ploidy and senescence; NUAK1 overexpression triggers premature senescence and aneuploidies, while a dominant-negative LATS1 phenocopies NUAK1 effects.\",\n      \"method\": \"Loss-of-function screen, shRNA depletion, overexpression, in vitro kinase assay for LATS1 phosphorylation\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay identifying phosphorylation site, multiple genetic approaches in human fibroblasts\",\n      \"pmids\": [\"19927127\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"NUAK1 directly interacts with and phosphorylates p53 in vitro and in vivo in a LKB1-dependent manner; phosphorylation of NUAK1 at Thr211 by LKB1 is required for NUAK1 kinase activity; the LKB1/NUAK1/p53 axis induces G1/S cell cycle arrest via p21/WAF1 and NUAK1 associates with p53 in the nucleus at the p21 promoter.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, chromatin immunoprecipitation, cell cycle analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay, ChIP, co-IP, multiple orthogonal methods\",\n      \"pmids\": [\"21317932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Muscle-specific knockout of NUAK1 prevents high-fat diet-induced glucose intolerance; NUAK1 suppresses insulin signaling by promoting phosphorylation of IRS1 at Ser1097; loss of NUAK1 enhances phosphorylation of IRS1 Tyr608, AKT Thr308, and TBC1D4 Thr649, improving glucose uptake.\",\n      \"method\": \"Muscle-specific knockout mice, glucose tolerance test, quantitative phosphoproteomics of soleus muscle\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — tissue-specific KO with phosphoproteomics identifying IRS1 Ser1097 as downstream effector\",\n      \"pmids\": [\"22418434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NUAK1 and NUAK2 together (but not individually) are required for apical constriction and apico-basal elongation during dorsolateral hinge point formation in neural plate; double mutants show defective apical concentration of phosphorylated myosin light chain 2, F-actin, and cortactin, causing neural tube defects.\",\n      \"method\": \"Double knockout mouse model, immunofluorescence for cytoskeletal markers\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — double KO with specific cytoskeletal phenotype readout, single study\",\n      \"pmids\": [\"22689267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NUAK1 expression is controlled through the cell cycle by CDK phosphorylation at Ser445, which triggers PLK1 binding; PLK1 then phosphorylates NUAK1 at Ser476 and Ser480, promoting binding to βTrCP and SCFβTrCP-mediated ubiquitylation and degradation; NUAK1 inhibits the PP1β-MYPT1 phosphatase complex and thereby promotes PLK1 T-loop phosphorylation at Thr210, establishing a reciprocal regulatory loop.\",\n      \"method\": \"Biochemical fractionation, co-immunoprecipitation, in vitro phosphorylation, ubiquitylation assay, NUAK1 inhibitors (WZ4003, HTH-01-015), site-directed mutagenesis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple biochemical assays with mutagenesis, phosphorylation reconstitution, cell-cycle staging\",\n      \"pmids\": [\"24785407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"NUAK1 is a direct target of miR-211, which is regulated by the MITF transcription factor; miR-211 suppresses NUAK1 expression and promotes melanoma cell adhesion, thereby inhibiting the invasive program.\",\n      \"method\": \"miRNA overexpression/inhibition, luciferase reporter assay for 3'UTR targeting, adhesion assays\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — luciferase assay with functional adhesion readout, single lab\",\n      \"pmids\": [\"23934065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"NUAK1 is activated by oxidative stress and facilitates nuclear import of the antioxidant master regulator NRF2 by coordinating PP1β inhibition with AKT activation to suppress GSK3β-dependent inhibition of NRF2 nuclear import; deletion of NUAK1 suppresses colorectal tumor formation and causes regression of established tumors.\",\n      \"method\": \"NUAK1 conditional knockout, autochthonous colorectal tumor models, Western blot for PP1β, AKT, GSK3β, NRF2 nuclear fractionation\",\n      \"journal\": \"Cancer discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic deletion in autochthonous tumor model with defined molecular pathway, multiple orthogonal approaches\",\n      \"pmids\": [\"29500295\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NUAK1 is a predominantly nuclear kinase that phosphorylates PNUTS (a nuclear regulatory subunit of PP1) to locally inhibit PP1 activity; NUAK1-PNUTS interaction is required for chromatin association of PNUTS, spliceosome activity, and nascent RNA synthesis; in MYC-overexpressing cells, NUAK1 inhibition causes global RNAPII accumulation at the pause site and first exon-intron boundary without increasing mRNA synthesis.\",\n      \"method\": \"Nuclear/cytoplasmic fractionation, co-immunoprecipitation of PP1 interactor network, in vitro kinase assay of PNUTS, NUAK1 inhibition, RNAPII ChIP, nascent RNA synthesis assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — MS interactome, in vitro phosphorylation, RNAPII ChIP, spliceosome activity assays, multiple methods\",\n      \"pmids\": [\"32006464\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TGF-β induces rapid NUAK1 upregulation in fibroblasts; NUAK1 in turn promotes YAP/TAZ and TGF-β/SMAD profibrotic signaling; activated YAP/TAZ further induces NUAK1 expression, creating a positive feedback loop; fibroblast-specific loss or pharmacological inhibition of NUAK1 markedly attenuates kidney, lung, and liver fibrosis in mouse models.\",\n      \"method\": \"Fibroblast-specific NUAK1 knockout, mouse models of organ fibrosis, Western blot for SMAD/YAP signaling, pharmacological inhibition\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific KO in multiple organ fibrosis models with defined signaling pathway, multiple orthogonal approaches\",\n      \"pmids\": [\"35320001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NUAK1 nuclear import is mediated by a bipartite nuclear localization signal at its N-terminal domain and requires importin-β members KPNB1, IPO7, and IPO9; oxidative stress induces NUAK1 cytoplasmic accumulation by affecting its nuclear transport.\",\n      \"method\": \"Cell fractionation, mass spectrometry identification of importin interactors, importin-β knockdown, Importazole treatment, NLS mutant constructs\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS-identified interactors validated by KD and chemical inhibition, single lab\",\n      \"pmids\": [\"31090959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In tumor cells lacking LKB1, NUAK1 kinase activity is maintained by an alternative calcium-dependent pathway involving PKCα activation; calcium/PKCα-dependent NUAK1 activation supports AMPK-TORC1 metabolic checkpoint engagement, protecting tumor cells from MYC-driven cell death; MYC transcriptionally regulates PKCα and ITPR to promote this pathway.\",\n      \"method\": \"LKB1-null cell models, PKCα knockdown/inhibition, calcium manipulation, MYC transcriptomics, TORC1 signaling assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic and pharmacological manipulation identifying alternative activation pathway, single lab\",\n      \"pmids\": [\"29106388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cytosolic NUAK1 increases ATP production by enhancing mitochondrial respiration; NUAK1 inhibition leads to mitochondrial morphology changes ('donut-like' structures); nuclear NUAK1 is involved in the metabolic switch to glycolysis; NUAK1 subcellular localization differentially affects metabolic outcomes in cancer cells.\",\n      \"method\": \"Subcellular fractionation, mitochondrial respiration assays, ATP measurements, live-cell imaging of mitochondrial morphology, NUAK1 inhibition\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — fractionation linked to functional metabolic readouts, single lab\",\n      \"pmids\": [\"32754444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration in cortical neurons; upregulation of mitochondrial function rescues axon branching defects in NUAK1-null neurons in vitro and in vivo; NUAK1 regulates axon branching through the mitochondria-targeted microprotein BRAWNIN, demonstrating a dual function controlling both mitochondrial distribution and metabolic activity.\",\n      \"method\": \"NUAK1 conditional knockout, mitochondrial function assays, ATP biosensors, BRAWNIN knockdown/overexpression, in vivo axon branching quantification\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and metabolic approaches in vitro and in vivo, identifying BRAWNIN as downstream effector\",\n      \"pmids\": [\"38514619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LKB1 phosphorylates and stabilizes NUAK1 in ovarian cancer spheroids; NUAK1 loss reduces spheroid integrity and fibronectin (FN1) expression (745-fold decrease); fibronectin is required for compact spheroid formation and metastasis; NUAK1 loss extends survival in a xenograft model of intraperitoneal metastasis.\",\n      \"method\": \"NUAK1 knockout, transcriptome analysis, fibronectin rescue experiments, xenograft metastasis model\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO with transcriptome, molecular rescue, and in vivo model, single lab\",\n      \"pmids\": [\"32429240\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ARK5/NUAK1 directly interacts with and phosphorylates hnRNP A1 on serine residues within the F-peptide region; the M9 motif of hnRNP A1 is required for ARK5 interaction and phosphorylation; ARK5-mediated phosphorylation controls hnRNP A1 cytoplasmic relocalization during hypertonic stress; ARK5 silencing increases Bcl-xL expression and delays caspase activation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, RNAi-based kinome screen, flow cytometry for caspase activation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay identifying hnRNP A1 as substrate, with mutagenesis of M9 motif and functional cellular readouts\",\n      \"pmids\": [\"35963429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NUAK1 governs accurate centrosome duplication via MYPT1/PP1β and GSK3β-dependent regulation of PLK4; loss of NUAK1 triggers genomic instability in both pancreatic cancer cells and primary fibroblasts.\",\n      \"method\": \"NUAK1 inhibition/depletion, centrosome counting, genomic instability assays, pathway analysis in PDAC cells\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional KO/inhibition with defined pathway (MYPT1/PP1β/GSK3β/PLK4) and centrosome phenotype, single lab\",\n      \"pmids\": [\"36975767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NUAK1 promotes metastasis in esophageal squamous cell carcinoma by activating the JNK/c-Jun pathway, which upregulates Slug transcription; silencing Slug completely abolishes NUAK1-overexpression-driven migration and invasion.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, dual-luciferase reporter gene assay for Slug promoter, in vivo pulmonary metastasis model\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reporter assay and co-IP identifying JNK/c-Jun/Slug pathway, single lab\",\n      \"pmids\": [\"37919754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NUAK1 directly phosphorylates NADK (nicotinamide adenine dinucleotide kinase) at Ser64, reducing ROS accumulation and thereby conferring osimertinib resistance in NSCLC; genetic or pharmacological blockade of NUAK1 restores osimertinib sensitivity.\",\n      \"method\": \"In vitro kinase assay identifying NADK S64 phosphorylation, NUAK1 knockdown/overexpression, ROS measurements, in vitro and in vivo drug sensitivity assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct phosphorylation identified by in vitro kinase assay with specific substrate residue, validated in vitro and in vivo\",\n      \"pmids\": [\"39159134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NUAK1 interacts with mTORC2 components and regulates mTORC2-dependent Akt activation by controlling lysosome positioning and mTOR association with lysosomes; NUAK1 also directly phosphorylates Akt at Ser473; NUAK1 is associated with early endosomes and selectively promotes Akt/FOXO1/3a signaling but not Akt/TSC2 signaling.\",\n      \"method\": \"Co-immunoprecipitation of mTORC2 components, in vitro kinase assay for Akt S473, lysosome positioning assay, subcellular fractionation, Akt substrate phosphorylation analysis\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro kinase assay and co-IP with mTORC2, supported by organelle localization data, single lab\",\n      \"pmids\": [\"38135881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NUAK1 upregulates GLI1 expression by activating STAT5-mediated transcription and stabilizing GLI1 protein; depletion or inhibition of NUAK1 impairs cancer cell expansion and tumor formation via the NUAK1/STAT5/GLI1 axis in gastric cancer.\",\n      \"method\": \"Patient-derived xenograft, transgenic mouse model, siRNA knockdown, pharmacological inhibition, Western blot and reporter assays for STAT5/GLI1\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple in vivo models with defined pathway, single lab\",\n      \"pmids\": [\"38996065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In yeast, Tda1 (the ortholog of human NUAK1) directly phosphorylates histone H3 at Thr11 upon low glucose stress; yeast AMPK (Snf1) phosphorylates Tda1 to govern its activity, while CK2 regulates Tda1 nuclear localization; AMPK and CK2 signaling converge on histone kinase Tda1 to link glucose stress to chromatin regulation.\",\n      \"method\": \"In vitro kinase assay, H3pT11 ChIP, mutant analysis, epistasis in budding yeast\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of H3 phosphorylation by Tda1, with epistasis and localization experiments; yeast ortholog\",\n      \"pmids\": [\"33372657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"USP9X deubiquitinates and thereby activates ARK5/NUAK1; USP9X-activated NUAK1 promotes TGF-β1-induced myofibroblast transformation via the Rho kinase pathway; myofibroblast-specific USP9X knockout or pharmacological inhibition reduces NUAK1 activity and attenuates cardiac fibrosis.\",\n      \"method\": \"Co-immunoprecipitation, USP9X knockout (myofibroblast-specific), in vivo myocardial infarction model, Western blot for NUAK1 ubiquitination/activation and Rho kinase pathway\",\n      \"journal\": \"JACC. Basic to translational science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP and cell-type-specific KO with defined pathway, single lab\",\n      \"pmids\": [\"40310323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ARK5/NUAK1 regulates mitochondrial morphological dynamics, promoting fission over fusion in multiple myeloma cells; ARK5 knockout cells show enhanced mitochondrial fusion, reduced phosphorylation of DRP1 at Ser616, higher MFN1/MFN2 expression, lower ATP, higher lactate, and higher ROS levels.\",\n      \"method\": \"Biallelic ARK5 knockout in myeloma cell lines, mitochondrial morphology imaging, Western blot for DRP1/MFN1/MFN2, ATP and lactate measurements\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean biallelic KO with multiple orthogonal mitochondrial readouts, single lab\",\n      \"pmids\": [\"38282096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NUAK1 facilitates nuclear import of NRF2 by inhibiting PP1β, which leads to AKT activation and suppression of GSK3β-dependent phosphorylation of NRF2, thus reducing GSK3β-mediated inhibition of NRF2 nuclear import; silencing NUAK1 upregulates PP1β and downregulates phospho-GSK3βSer9, suppressing NRF2 nuclear accumulation and GPX4 expression.\",\n      \"method\": \"siRNA knockdown, Western blot for PP1β, p-GSK3βSer9, NRF2 nuclear fractionation, NUAK1 agonist rescue experiment\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — defined pharmacological and genetic dissection of PP1β/GSK3β/NRF2 pathway, single lab\",\n      \"pmids\": [\"33819186\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Haploinsufficiency of NUAK1 in mice impairs cortical axon branching and leads to behavioral deficits including defective spatial memory consolidation, impaired social novelty, and abnormal sensorimotor gating, confirming NUAK1 as a haploinsufficient autism spectrum disorder candidate gene.\",\n      \"method\": \"NUAK1 heterozygous knockout mice, cortical connectivity analysis, behavioral test battery\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — haploinsufficiency mouse model with multiple behavioral and anatomical readouts, replicated across tests\",\n      \"pmids\": [\"30327473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NUAK1 promotes PD-L1 transcription in hepatocellular carcinoma by phosphorylating GSK3β at Ser9, which activates β-catenin nuclear accumulation and PD-L1 gene expression; knockdown of β-catenin reverses NUAK1-mediated PD-L1 upregulation.\",\n      \"method\": \"Gain and loss of function experiments, Western blot and immunofluorescence for GSK3β/β-catenin/PD-L1, β-catenin knockdown rescue, IHC of patient samples\",\n      \"journal\": \"Molecular medicine (Cambridge, Mass.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — genetic and rescue experiments defining GSK3β/β-catenin/PD-L1 pathway, single lab\",\n      \"pmids\": [\"39901136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NUAK1 is present in human erythrocytes and undergoes increased phosphorylation in P. falciparum-infected cells; NUAK1 inhibition reduces MYPT1 phosphorylation in erythroid cells; genetic overexpression of NUAK1 in erythroid cells partially rescues both signaling and parasite invasion phenotypes caused by NUAK1 inhibitors.\",\n      \"method\": \"Chemoproteomic profiling confirming inhibitor selectivity, phosphoproteomic analysis, NUAK1 overexpression in erythroid cells, P. falciparum invasion assays\",\n      \"journal\": \"bioRxiv (PMID available: 41278793)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — chemoproteomic validation of inhibitor selectivity, genetic rescue of MYPT1 signaling, novel host context\",\n      \"pmids\": [\"41278793\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NUAK1 phosphorylates the splicing co-factor SON, regulating a conserved set of alternative splicing events in genes crucial for neurodevelopment in cortical neurons; SON is required for proper cortical neuron development consistent with ZTTK syndrome phenotypes.\",\n      \"method\": \"NUAK1 conditional knockout in cortical neurons, RNA splicing analysis, identification of SON as phosphorylation substrate, SON knockdown\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with splicing transcriptomics and identification of SON as substrate, preprint only\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NUAK1 kinase activity correlates with MYPT1 phosphorylation at Ser445 and Ser910 in vascular smooth muscle cells; NUAK1 knockdown decreases myosin light chain phosphorylation, potentially by increasing MYPT1 phosphatase activity, reducing contractility and altering F-actin cytoskeletal organization.\",\n      \"method\": \"Phosphoproteomic analysis, NUAK1 knockdown, pMLC and MYPT1 phosphorylation Western blot, impedance-based contraction assay\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — phosphoproteomic correlation and KD, preprint, single lab\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"First crystal structures of a NUAK1 chimeric protein were determined, providing structural insights into inhibitor binding modes and identifying a unique glutamate residue (Glu139) that can be exploited for selective NUAK1 targeting.\",\n      \"method\": \"X-ray crystallography of NUAK1 chimeric protein with bound inhibitors\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with functional validation of binding mode, single study\",\n      \"pmids\": [\"39588908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"ARK5/NUAK1 prevents degradation of TGF-β type I receptor (TβRI) and Smad4 proteins by inhibiting Smurf2 expression in hepatic stellate cells, thereby maintaining TGF-β signaling; in hepatocytes, ARK5 induces EMT and promotes inflammatory cytokine secretion, synergistically driving liver fibrosis.\",\n      \"method\": \"ARK5 knockdown in HSCs and hepatocytes, Western blot for TβRI/Smad4/Smurf2, mouse CCl4-fibrosis model with HTH-01-015 inhibitor\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — mechanistic pathway identified by KD with defined molecular readouts and in vivo pharmacology, single lab\",\n      \"pmids\": [\"36361872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NUAK1 promotes pyroptosis and inflammation in MASH by activating Caspase 6; NUAK1/Caspase 6 axis inhibition accelerates the interaction between TAK1 and RIPK1, leading to RIPK1 degradation and suppression of hepatic inflammation.\",\n      \"method\": \"NUAK1 inhibition in vivo and in vitro MASH models, Western blot for Caspase 6, TAK1, RIPK1, caspase 6 disruption experiments\",\n      \"journal\": \"Hepatology communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — in vivo and in vitro NUAK1 inhibition with defined Caspase 6/TAK1/RIPK1 pathway dissection, single lab\",\n      \"pmids\": [\"38967580\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NUAK1 is an LKB1-activated AMPK-related serine/threonine kinase that phosphorylates a broad spectrum of substrates—including MYPT1/PP1β (to regulate myosin activity and NRF2 nuclear import), tau (Ser356), caspase-6 (Ser257), LATS1 (Ser464), p53, PNUTS (to support spliceosome activity), Akt (Ser473), NADK (Ser64), and hnRNP A1—and operates in multiple cellular contexts including axon branching via mitochondrial immobilization and metabolic support, cellular senescence, the antioxidant stress response, fibrogenesis through YAP/TGF-β signaling, cell cycle regulation via PLK1/SCFβTrCP, and nuclear RNA splicing through a MYC-dependent PP1/PNUTS axis, with its own activity regulated by Akt phosphorylation (Ser600), NDR2 phosphorylation (Thr211), calcium/PKCα signaling (in LKB1-null contexts), CDK/PLK1/SCFβTrCP-mediated degradation, USP9X-mediated stabilization, and oxidative stress-driven cytoplasmic relocalization.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NUAK1 is an AMPK-related serine/threonine kinase that functions as a central signaling node integrating metabolic stress, cytoskeletal regulation, and gene expression control across diverse cellular contexts. Activated by LKB1 phosphorylation at Thr211 and cooperatively by Akt at Ser600 and NDR2 at the T-loop, NUAK1 phosphorylates the PP1β-MYPT1 phosphatase complex to regulate myosin phosphorylation, centrosome duplication, and NRF2 nuclear import via the GSK3β axis, and directly phosphorylates substrates including caspase-6 (Ser257), LATS1 (Ser464), p53, tau (Ser356), PNUTS, Akt (Ser473), NADK (Ser64), and hnRNP A1 [PMID:16488889, PMID:15273717, PMID:19927127, PMID:21317932, PMID:27720485, PMID:32006464, PMID:38135881, PMID:39159134, PMID:35963429]. In neurons, the LKB1–NUAK1 pathway controls axon branching by immobilizing mitochondria at nascent presynaptic sites and by sustaining mitochondrial metabolic activity through the microprotein BRAWNIN; NUAK1 haploinsufficiency impairs cortical connectivity and causes behavioral deficits consistent with autism spectrum disorder [PMID:23791179, PMID:38514619, PMID:30327473]. In the nucleus, NUAK1 phosphorylates PNUTS to locally inhibit PP1 and support spliceosome function and nascent RNA synthesis, while in fibroblasts it drives a positive-feedback loop with YAP/TAZ and TGF-β/SMAD signaling that promotes organ fibrosis [PMID:32006464, PMID:35320001, PMID:40310323].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"NUAK1 was first linked to cell survival signaling when overexpression was shown to suppress nutrient-starvation- and death-receptor-induced apoptosis by inhibiting caspase-8 activation, establishing it as a pro-survival kinase.\",\n      \"evidence\": \"Overexpression in HepG2 cells with caspase-8, Bid, and FLIP readouts\",\n      \"pmids\": [\"13679856\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of caspase-8 inhibition not defined at the substrate level\", \"Single cell line, no genetic loss-of-function\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The first direct substrate of NUAK1 was identified—caspase-6 at Ser257—providing a molecular mechanism for its anti-apoptotic function and demonstrating it is a bona fide kinase with defined substrates.\",\n      \"evidence\": \"In vitro kinase assay with S257A mutagenesis abolishing protection from Fas-mediated death in colorectal cancer cells\",\n      \"pmids\": [\"15273717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether caspase-6 phosphorylation is relevant in non-cancer contexts\", \"Structural basis of kinase-substrate recognition unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The activation mechanism of NUAK1 was elucidated: NDR2 phosphorylates Thr211 on the T-loop and Akt phosphorylates Ser600 in the regulatory domain, with both events required for full kinase activity, placing NUAK1 downstream of growth factor signaling.\",\n      \"evidence\": \"In vitro kinase assay with mutant constructs and co-IP upon IGF-1 stimulation in colorectal cancer cells\",\n      \"pmids\": [\"16488889\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether LKB1 and NDR2 phosphorylate the same site independently or cooperatively\", \"Context-dependence of NDR2 vs. LKB1 activation not resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"NUAK1 was connected to cell cycle and ploidy control through its phosphorylation of LATS1 at Ser464, triggering LATS1 degradation and linking NUAK1 to Hippo pathway regulation and cellular senescence.\",\n      \"evidence\": \"Loss-of-function screen, in vitro kinase assay, and dominant-negative LATS1 phenocopy in human fibroblasts\",\n      \"pmids\": [\"19927127\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NUAK1-LATS1 interaction operates in Hippo-dependent tissue growth contexts\", \"Ubiquitin ligase mediating LATS1 degradation downstream of Ser464 phosphorylation not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"NUAK1 was shown to phosphorylate p53 in an LKB1-dependent manner and to occupy the p21 promoter with p53, establishing a tumor-suppressive LKB1/NUAK1/p53/p21 axis that induces G1/S arrest.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, ChIP at the p21 promoter, cell cycle analysis\",\n      \"pmids\": [\"21317932\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific p53 phosphorylation site(s) not mapped\", \"How this tumor-suppressive function is reconciled with NUAK1's pro-survival roles\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"NUAK1 was placed in a neurodevelopmental pathway: the LKB1–NUAK1 axis controls cortical axon branching by immobilizing mitochondria at nascent presynaptic sites via Syntaphilin, revealing a cell-biological function beyond cancer.\",\n      \"evidence\": \"Conditional LKB1 knockout, in utero NUAK1 knockdown/overexpression, Syntaphilin rescue in mouse cortical neurons\",\n      \"pmids\": [\"23791179\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NUAK1 directly phosphorylates Syntaphilin or acts indirectly\", \"Mechanism linking mitochondrial immobilization to branch stabilization unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"A cell-cycle-linked regulatory circuit was defined: CDK phosphorylation primes NUAK1 for PLK1-mediated phosphorylation and SCFβTrCP-dependent degradation, while NUAK1 reciprocally promotes PLK1 activation by inhibiting PP1β-MYPT1, establishing the MYPT1/PP1β complex as a central NUAK1 effector.\",\n      \"evidence\": \"Biochemical reconstitution of CDK→PLK1→βTrCP ubiquitylation cascade, site-directed mutagenesis, NUAK1 inhibitors WZ4003/HTH-01-015\",\n      \"pmids\": [\"24785407\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PP1β-MYPT1 inhibition by NUAK1 involves direct MYPT1 phosphorylation or an indirect mechanism\", \"Functional consequence of NUAK1 degradation timing on mitotic fidelity\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"NUAK1 was identified as a kinase that phosphorylates tau at Ser356, stabilizing pathological tau; genetic reduction of NUAK1 suppressed neurodegeneration in both Drosophila and mouse tauopathy models, establishing NUAK1 as a therapeutic target for tauopathies.\",\n      \"evidence\": \"In vitro kinase assay, Drosophila genetic screen, NUAK1 haploinsufficiency rescue in P301S tau mouse model\",\n      \"pmids\": [\"27720485\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NUAK1 phosphorylates tau directly in neurons or through an intermediate\", \"Therapeutic window of NUAK1 inhibition given its essential neurodevelopmental roles\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"An alternative, LKB1-independent activation mechanism was uncovered: in LKB1-null tumors, calcium/PKCα signaling maintains NUAK1 activity to engage the AMPK-TORC1 metabolic checkpoint, explaining how NUAK1 remains functional in LKB1-deficient cancers.\",\n      \"evidence\": \"PKCα knockdown/inhibition and calcium manipulation in LKB1-null cell models with TORC1 readouts\",\n      \"pmids\": [\"29106388\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PKCα directly phosphorylates NUAK1 and at which site\", \"Generalizability beyond MYC-driven LKB1-null contexts\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"NUAK1 was connected to the antioxidant stress response: it facilitates NRF2 nuclear import by inhibiting PP1β, thereby activating AKT and suppressing GSK3β-dependent NRF2 phosphorylation; conditional NUAK1 deletion caused regression of established colorectal tumors, positioning NUAK1 as a druggable vulnerability in ROS-dependent cancers.\",\n      \"evidence\": \"Conditional KO in autochthonous colorectal tumor models with NRF2/PP1β/AKT/GSK3β pathway dissection\",\n      \"pmids\": [\"29500295\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NUAK1 directly phosphorylates PP1β or MYPT1 in this context\", \"NRF2-independent mechanisms of tumor regression upon NUAK1 loss\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"NUAK1 haploinsufficiency in mice was shown to impair cortical axon branching and produce behavioral deficits including defective spatial memory, impaired social novelty, and abnormal sensorimotor gating, validating NUAK1 as a haploinsufficient autism spectrum disorder candidate gene.\",\n      \"evidence\": \"Heterozygous NUAK1 knockout mice with cortical connectivity analysis and behavioral test battery\",\n      \"pmids\": [\"30327473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No human family studies confirming NUAK1 haploinsufficiency as causative for ASD\", \"Molecular mechanism linking reduced kinase dosage to circuit-level defects unclear\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"The nuclear function of NUAK1 was defined: it phosphorylates the PP1 regulatory subunit PNUTS to locally inhibit PP1, supporting spliceosome activity and nascent RNA synthesis; in MYC-overexpressing cells, NUAK1 inhibition causes global RNAPII pausing, revealing a transcription-coupled splicing role.\",\n      \"evidence\": \"Nuclear interactome by mass spectrometry, in vitro PNUTS phosphorylation, RNAPII ChIP, spliceosome and nascent RNA assays\",\n      \"pmids\": [\"32006464\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific PNUTS phosphorylation sites not mapped\", \"Whether NUAK1-PNUTS axis is MYC-selective or generalizable\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Subcellular compartmentalization of NUAK1 was shown to differentially control metabolism: cytosolic NUAK1 enhances mitochondrial respiration and ATP production, whereas nuclear NUAK1 promotes glycolysis, establishing NUAK1 as a localization-dependent metabolic switch.\",\n      \"evidence\": \"Subcellular fractionation with mitochondrial respiration assays and ATP measurements in cancer cells\",\n      \"pmids\": [\"32754444\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cytoplasmic substrates mediating mitochondrial effects not identified\", \"Single study; compartment-specific constructs not used\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"NUAK1 was shown to drive organ fibrosis through a TGF-β–YAP/TAZ positive feedback loop: TGF-β induces NUAK1, which promotes YAP/TAZ activity, and YAP/TAZ in turn upregulates NUAK1; fibroblast-specific NUAK1 loss attenuated kidney, lung, and liver fibrosis.\",\n      \"evidence\": \"Fibroblast-specific NUAK1 KO in multiple organ fibrosis mouse models with SMAD/YAP signaling analysis\",\n      \"pmids\": [\"35320001\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct NUAK1 substrate in YAP/TAZ activation pathway not identified\", \"Whether PP1β-MYPT1 mediates the fibrogenic loop\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"hnRNP A1 was identified as a direct NUAK1 substrate: NUAK1 phosphorylates serine residues within the F-peptide region of hnRNP A1, controlling its cytoplasmic relocalization during hypertonic stress, expanding NUAK1's role into RNA-binding protein regulation.\",\n      \"evidence\": \"In vitro kinase assay, M9-motif mutagenesis, RNAi kinome screen, co-IP\",\n      \"pmids\": [\"35963429\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphorylation sites on hnRNP A1 not individually mapped\", \"Functional consequences for mRNA splicing or stability not determined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"NUAK1 was found to govern centrosome duplication fidelity through the MYPT1/PP1β/GSK3β/PLK4 axis, with NUAK1 loss triggering centrosome amplification and genomic instability in both cancer cells and primary fibroblasts.\",\n      \"evidence\": \"NUAK1 depletion/inhibition with centrosome counting and genomic instability assays in PDAC cells and fibroblasts\",\n      \"pmids\": [\"36975767\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphorylation of PLK4 pathway components by NUAK1 not shown\", \"Whether centrosome defects contribute to the senescence phenotype previously observed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"NUAK1 was shown to interact with mTORC2 components and directly phosphorylate Akt at Ser473 while controlling lysosome positioning to regulate mTOR–lysosome association, selectively activating Akt/FOXO signaling over Akt/TSC2 signaling.\",\n      \"evidence\": \"Co-IP of mTORC2 components, in vitro kinase assay for Akt S473, lysosome positioning and subcellular fractionation\",\n      \"pmids\": [\"38135881\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of substrate-selective Akt signaling not explained\", \"Single lab; independent validation needed\", \"Whether NUAK1 phosphorylation of Akt is physiologically relevant given mTORC2 dominance\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The metabolic function of NUAK1 in neurons was deepened: NUAK1 deficiency impairs mitochondrial metabolism and axonal ATP levels, and upregulation of mitochondrial function via the microprotein BRAWNIN rescues axon branching defects, demonstrating NUAK1 controls both mitochondrial distribution and metabolic output.\",\n      \"evidence\": \"Conditional NUAK1 KO, ATP biosensors, BRAWNIN knockdown/overexpression, in vivo axon branching\",\n      \"pmids\": [\"38514619\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NUAK1 directly regulates BRAWNIN expression or activity\", \"How mitochondrial metabolic and anchoring functions are coordinated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"NADK was identified as a direct NUAK1 substrate (Ser64), linking NUAK1 to NADPH metabolism and ROS control; this phosphorylation confers osimertinib resistance in NSCLC, extending NUAK1's roles to drug resistance via redox balance.\",\n      \"evidence\": \"In vitro kinase assay identifying NADK S64, NUAK1 knockdown/overexpression, ROS measurements, in vivo drug sensitivity\",\n      \"pmids\": [\"39159134\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NADK Ser64 phosphorylation alters enzymatic kinetics directly\", \"Generalizability to other EGFR-TKI resistance contexts\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"The first crystal structures of NUAK1 were determined, revealing inhibitor binding modes and a unique Glu139 residue exploitable for selective inhibitor design, providing a structural foundation for drug development.\",\n      \"evidence\": \"X-ray crystallography of NUAK1 chimeric protein with bound inhibitors\",\n      \"pmids\": [\"39588908\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structure is of a chimeric construct, not full-length wild-type NUAK1\", \"No structure of NUAK1 in complex with a substrate or regulatory subunit\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"USP9X was identified as a deubiquitinase that stabilizes and activates NUAK1, with the USP9X–NUAK1 axis promoting TGF-β1-induced myofibroblast transformation via Rho kinase signaling and driving cardiac fibrosis.\",\n      \"evidence\": \"Co-IP, myofibroblast-specific USP9X KO, in vivo myocardial infarction model\",\n      \"pmids\": [\"40310323\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ubiquitination sites on NUAK1 targeted by USP9X not mapped\", \"Relationship between USP9X stabilization and SCFβTrCP-mediated degradation not clarified\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include: the full catalog of direct NUAK1 phosphorylation sites on MYPT1 and PNUTS; how nuclear versus cytoplasmic NUAK1 pools are coordinated to produce context-dependent metabolic and transcriptional outcomes; and whether NUAK1 haploinsufficiency is causative for human neurodevelopmental disorders.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No human genetic validation of NUAK1 as a Mendelian disease gene\", \"No full-length wild-type NUAK1 structure\", \"Mechanism by which NUAK1 selectively engages different substrates in different compartments unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 3, 5, 7, 8, 11, 14, 21, 24, 25]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 11, 13, 30]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 14, 16, 18]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [16, 18]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [0, 19, 29]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 5, 13, 15, 25, 26, 32]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 3, 38]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7, 11, 22]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [14, 34]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 10, 31]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [13, 30]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [9, 18, 19, 24, 29]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 15, 37]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MYPT1\",\n      \"PP1B\",\n      \"PNUTS\",\n      \"LKB1\",\n      \"PLK1\",\n      \"USP9X\",\n      \"LATS1\",\n      \"TP53\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}