{"gene":"NEK10","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2010,"finding":"NEK10 physically associates with Raf-1 and MEK1 in a Raf-1-dependent manner upon UV irradiation. This complex formation is necessary for NEK10-mediated MEK1 activation. NEK10 does not affect Raf-1 kinase activity but instead promotes the autophosphorylation-dependent activation of MEK1, leading to ERK1/2 activation and G2/M checkpoint maintenance after UV damage.","method":"Co-immunoprecipitation, kinase activity assays, siRNA knockdown with cell cycle analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and kinase assays in single lab with multiple orthogonal methods","pmids":["20956560"],"is_preprint":false},{"year":2010,"finding":"NEK10 is required for ERK1/2 activation in response to UV irradiation but not in response to mitogens such as EGF, indicating a UV-specific function upstream of the MEK-ERK pathway.","method":"siRNA knockdown, immunoblot for phospho-ERK1/2 after UV or EGF stimulation","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean knockdown with defined pathway readout, single lab","pmids":["20956560"],"is_preprint":false},{"year":2020,"finding":"NEK10 is a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport in primary human airway cultures; NEK10 loss does not affect ciliary number, radial structure, or beat frequency.","method":"Genetically modified primary human airway cultures (NEK10 loss-of-function), ciliary proteomics, mucociliary transport assay, electron microscopy","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — primary human airway cultures with genetic modification, multiple orthogonal methods (proteomics, functional assay, structural analysis), replicated in human patient context","pmids":["31959991"],"is_preprint":false},{"year":2020,"finding":"NEK10 directly phosphorylates p53 on tyrosine 327 (Y327), and a p53-Y327F mutant acts as a hypomorph with attenuated transcriptional response. NEK10 loss increases cellular proliferation by modulating p53-dependent transcriptional output and causes heightened sensitivity to DNA-damaging agents.","method":"In vitro kinase assay with purified proteins, phospho-specific site mutagenesis (Y327F), transcriptional reporter assays, siRNA/CRISPR knockdown with proliferation and drug sensitivity assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro phosphorylation with site-specific mutagenesis and functional validation, single lab but multiple orthogonal methods","pmids":["32561851"],"is_preprint":false},{"year":2020,"finding":"NEK10 interacts with mitochondria-associated proteins including SIRT3, ATAD3A, ATAD3B, and GLUD1 (confirmed by proximity ligation assay). NEK10 depletion causes mitochondrial fragmentation, altered mitochondrial ROS levels, decreased citrate synthase activity, inhibited mitochondrial respiration (particularly ATP-linked oxygen consumption and spare capacity), decreased mtDNA amplification ratio, and increased total mtDNA content.","method":"FLAG co-immunoprecipitation with LC-MS/MS proteomics, proximity ligation assay, confocal microscopy, stable shRNA knockdown, Seahorse respirometry, citrate synthase activity assay, mtDNA quantification","journal":"Proteome science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity ligation assay confirms key interaction, multiple functional readouts in single lab","pmids":["32368190"],"is_preprint":false},{"year":2020,"finding":"In C. elegans, NEKL-4 (NEK10 ortholog) localizes to the ciliary base but does not localize within cilia, suggesting an indirect role in ciliary processes. Loss-of-function of nekl-4 suppresses hyperglutamylation-induced ciliary degeneration in ccpp-1 mutants, placing NEKL-4 in a pathway modulating tubulin glutamylation effects.","method":"Forward genetic screen, fluorescence microscopy localization, epistasis analysis in C. elegans double mutants","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with localization in model organism, single study","pmids":["33064774"],"is_preprint":false},{"year":2024,"finding":"NEK10 associates with the Axin destruction complex and phosphorylates β-catenin at Tyr30 (within the regulatory region governing β-catenin turnover). In the absence of NEK10-mediated Tyr30 phosphorylation, GSK3-mediated phosphorylation of β-catenin—a prerequisite for its degradation—is impaired, leading to β-catenin stabilization. NEK10-deficient lung adenocarcinoma cells show reduced tumorsphere formation, soft-agar growth, and lung colonization in vivo.","method":"CRISPR knockout, co-immunoprecipitation with Axin complex, in vitro kinase assay, site-specific mutagenesis, immunoblot for β-catenin phosphorylation/stability, tumorsphere assay, soft-agar assay, tail-vein injection mouse model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct kinase assay with defined phosphorylation site, complex co-IP, in vitro and in vivo functional validation, multiple orthogonal methods in single rigorous study","pmids":["38683979"],"is_preprint":false},{"year":2024,"finding":"NEK10 knockout in HAP1 cells alters threonine phosphorylation status of mitochondrial/ER proteins including HSP60, NDUFB4, and TOM20, and affects steady-state levels of respiratory complex proteins and autophagy pathway components, supporting a role for NEK10 in mitochondrial homeostasis via phosphorylation-dependent regulation.","method":"HAP1 NEK10 knockout cells, LC-MS/MS proteomics and phosphoproteomics of mitochondrial fractions, mitochondrial respiration assay, morphology analysis","journal":"Proteome science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics with full KO and multiple functional readouts, single lab, replicates earlier findings","pmids":["39379991"],"is_preprint":false},{"year":2025,"finding":"NEK10 knockdown in tubular cells attenuates BSA-induced lipid droplet accumulation, G2/M arrest, and ERK1/2 phosphorylation, placing NEK10 upstream of ERK1/2 in an albumin-overload-driven metabolic and cell cycle disruption pathway; in vivo NEK10 knockdown (rAAV-shNEK10) reduces BSA-induced renal fibrosis in UUO mice.","method":"siRNA/rAAV shRNA knockdown in HK-2 cells and UUO mouse model, Western blotting for phospho-ERK1/2, flow cytometry cell cycle analysis, lipid droplet staining, fatty acid oxidation assay","journal":"American journal of nephrology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pathway placement based on knockdown + phosphorylation readout without direct mechanistic dissection","pmids":["40934128"],"is_preprint":false},{"year":2026,"finding":"ECM-derived mechanical forces reduce NEK10 expression via a cytoskeleton-dependent pathway, leading to suppression of the NEK10/p53/CDKN1A axis, activation of CDK2 signaling, and CDK4/6 inhibitor resistance in breast cancer cells.","method":"3D gel biomechanical models, scRNA-seq, transcriptomic analysis, Western blotting, NEK10-deficient cells/organoids, in vivo CDK2 inhibitor co-treatment","journal":"International journal of surgery (London, England)","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, pathway placement inferred from expression reduction and inhibitor rescue without direct biochemical dissection of NEK10 mechanism","pmids":["41427545"],"is_preprint":false}],"current_model":"NEK10 is a serine/threonine/tyrosine kinase that operates in multiple contexts: upon UV irradiation it forms a Raf-1-dependent complex with MEK1 to promote MEK1 autophosphorylation and ERK1/2 activation, thereby maintaining the G2/M checkpoint; it directly phosphorylates p53 on Tyr327 to modulate p53-dependent transcriptional responses to genotoxic stress; it phosphorylates β-catenin at Tyr30 within the Axin destruction complex, enabling subsequent GSK3-mediated β-catenin degradation; in ciliated airway cells it regulates the motile ciliary proteome to control ciliary length and mucociliary transport without affecting ciliary number, structure, or beat frequency; and it maintains mitochondrial homeostasis by modulating the threonine phosphorylation status of mitochondrial proteins including HSP60, NDUFB4, and TOM20."},"narrative":{"mechanistic_narrative":"NEK10 is a NIMA-related serine/threonine/tyrosine kinase that links DNA-damage signaling, motile cilia biology, Wnt/β-catenin turnover, and mitochondrial homeostasis [PMID:20956560, PMID:31959991, PMID:32561851, PMID:38683979]. In the DNA-damage response, NEK10 acts UV-specifically: upon UV irradiation it forms a Raf-1-dependent complex with MEK1 and drives MEK1 autophosphorylation to activate ERK1/2 and maintain the G2/M checkpoint, while leaving Raf-1 kinase activity and mitogen-driven ERK activation unaffected [PMID:20956560]. NEK10 also directly phosphorylates p53 on tyrosine 327 to tune p53-dependent transcriptional output, such that its loss increases proliferation and sensitizes cells to genotoxic agents [PMID:32561851]. In Wnt signaling, NEK10 associates with the Axin destruction complex and phosphorylates β-catenin at Tyr30, a modification required to license subsequent GSK3-mediated phosphorylation and β-catenin degradation; loss of this activity stabilizes β-catenin and promotes lung adenocarcinoma tumorigenicity in vitro and in vivo [PMID:38683979]. In ciliated airway cells, NEK10 is a ciliated-cell-specific kinase that shapes the motile ciliary proteome to control ciliary length and mucociliary transport without altering ciliary number, ultrastructure, or beat frequency [PMID:31959991]. NEK10 further interacts with mitochondria-associated proteins and modulates the threonine-phosphorylation status of mitochondrial proteins including HSP60, NDUFB4, and TOM20, with its depletion causing mitochondrial fragmentation, impaired respiration, and altered mtDNA content [PMID:32368190, PMID:39379991].","teleology":[{"year":2010,"claim":"Established the first signaling role for NEK10, placing it as a UV-specific activator upstream of the MEK-ERK cascade required for the G2/M checkpoint.","evidence":"Co-IP, kinase activity assays, and siRNA knockdown with cell cycle analysis showing NEK10 forms a Raf-1-dependent complex with MEK1 and promotes MEK1 autophosphorylation","pmids":["20956560"],"confidence":"Medium","gaps":["Direct NEK10 substrate(s) within the complex not defined (MEK1 activation is autophosphorylation-dependent rather than NEK10-catalyzed)","Structural basis of the Raf-1-dependent assembly unresolved","Why the requirement is UV-specific versus mitogen stimulation not mechanistically explained"]},{"year":2020,"claim":"Defined NEK10 as a ciliated-cell-specific kinase that controls ciliary length and mucociliary transport through the motile ciliary proteome rather than ciliary assembly.","evidence":"Genetically modified primary human airway cultures with ciliary proteomics, mucociliary transport assays, and electron microscopy","pmids":["31959991"],"confidence":"High","gaps":["Direct ciliary substrates of NEK10 not identified","Mechanism coupling proteome changes to length control unresolved"]},{"year":2020,"claim":"Identified p53 as a direct NEK10 substrate, connecting the kinase to DNA-damage transcriptional control and proliferation suppression.","evidence":"In vitro kinase assays with purified proteins, Y327F site mutagenesis, transcriptional reporters, and knockdown proliferation/drug-sensitivity assays","pmids":["32561851"],"confidence":"High","gaps":["How Y327 phosphorylation alters p53 conformation or cofactor binding not defined","Relationship between this axis and the earlier MEK-ERK function not integrated"]},{"year":2020,"claim":"Extended NEK10 function to mitochondria, showing physical association with mitochondrial proteins and a requirement for mitochondrial morphology and respiration.","evidence":"FLAG Co-IP/LC-MS/MS, proximity ligation assay confirming SIRT3/ATAD3A/ATAD3B/GLUD1 interactions, confocal microscopy, Seahorse respirometry, and mtDNA quantification in shRNA knockdown cells","pmids":["32368190"],"confidence":"Medium","gaps":["Direct mitochondrial phosphorylation substrates not yet established at this stage","Whether NEK10 localizes to mitochondria or acts indirectly unresolved"]},{"year":2020,"claim":"Ortholog analysis placed NEK10 (NEKL-4) at the ciliary base acting in a tubulin-glutamylation modulating pathway, supporting an indirect mode of ciliary regulation.","evidence":"Forward genetic screen, fluorescence localization, and epistasis with ccpp-1 mutants in C. elegans","pmids":["33064774"],"confidence":"Medium","gaps":["Molecular targets of NEKL-4 in the glutamylation pathway unknown","Conservation of the base-localized mechanism in human cells not demonstrated"]},{"year":2024,"claim":"Resolved a tumor-suppressive mechanism in which NEK10 primes β-catenin for degradation, linking the kinase to Wnt pathway control of lung cancer growth.","evidence":"CRISPR knockout, Axin-complex Co-IP, in vitro kinase assay, Tyr30 site mutagenesis, β-catenin stability immunoblots, and tumorsphere/soft-agar/tail-vein assays","pmids":["38683979"],"confidence":"High","gaps":["How Tyr30 phosphorylation gates subsequent GSK3 phosphorylation structurally unresolved","Regulation of NEK10 recruitment to the Axin complex not defined"]},{"year":2024,"claim":"Provided direct phosphoproteomic evidence that NEK10 sets the threonine-phosphorylation status of specific mitochondrial proteins, mechanistically anchoring its mitochondrial homeostasis role.","evidence":"HAP1 NEK10 knockout with mitochondrial-fraction phosphoproteomics identifying HSP60, NDUFB4, TOM20, plus respiration and morphology assays","pmids":["39379991"],"confidence":"Medium","gaps":["Direct versus indirect phosphorylation of the identified proteins not distinguished","Functional consequence of each phosphosite not individually tested"]},{"year":2025,"claim":"Placed NEK10 upstream of ERK1/2 in an albumin-overload pathway driving tubular metabolic and cell-cycle disruption and renal fibrosis.","evidence":"siRNA/rAAV-shNEK10 knockdown in HK-2 cells and a UUO mouse model with phospho-ERK1/2, cell cycle, lipid droplet, and fatty acid oxidation readouts","pmids":["40934128"],"confidence":"Low","gaps":["Pathway placement rests on knockdown plus phosphorylation readout without direct mechanistic dissection","Direct NEK10 substrate in this context not identified"]},{"year":2026,"claim":"Linked ECM mechanical force to NEK10 expression suppression and CDK4/6 inhibitor resistance via a NEK10/p53/CDKN1A axis in breast cancer.","evidence":"3D biomechanical gel models, scRNA-seq, transcriptomics, NEK10-deficient cells/organoids, and in vivo CDK2 inhibitor co-treatment","pmids":["41427545"],"confidence":"Low","gaps":["Axis inferred from expression changes and inhibitor rescue without biochemical dissection of NEK10 here","How cytoskeletal mechanotransduction regulates NEK10 expression unknown"]},{"year":null,"claim":"How NEK10's multiple substrate-specific activities (MEK1 complex, p53-Y327, β-catenin-Tyr30, mitochondrial threonine sites, ciliary proteome) are coordinated and contextually selected within a single kinase remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of NEK10 substrate recognition","No unified account of tissue- and stimulus-specific activation","Endogenous regulators of NEK10 activity not defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,6,7]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,3,6,7]}],"localization":[{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[2,5]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[4,7]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,3]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[0,3]}],"complexes":["Axin destruction complex"],"partners":["RAF1","MAP2K1","TP53","CTNNB1","SIRT3","ATAD3A","GLUD1","TOM20"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6ZWH5","full_name":"Serine/threonine-protein kinase Nek10","aliases":["Never in mitosis A-related kinase 10","NimA-related protein kinase 10"],"length_aa":1172,"mass_kda":133.3,"function":"Plays a role in the cellular response to UV irradiation. Mediates G2/M cell cycle arrest, MEK autoactivation and ERK1/2-signaling pathway activation in response to UV irradiation. In ciliated cells of airways, it is involved in the regulation of mucociliary transport (PubMed:31959991)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q6ZWH5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NEK10","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NEK10","total_profiled":1310},"omim":[{"mim_id":"620694","title":"TUBULIN TYROSINE LIGASE-LIKE 11; TTLL11","url":"https://www.omim.org/entry/620694"},{"mim_id":"618781","title":"CILIARY DYSKINESIA, PRIMARY, 44; CILD44","url":"https://www.omim.org/entry/618781"},{"mim_id":"618726","title":"NIMA-RELATED KINASE 10; NEK10","url":"https://www.omim.org/entry/618726"},{"mim_id":"244400","title":"CILIARY DYSKINESIA, PRIMARY, 1; CILD1","url":"https://www.omim.org/entry/244400"},{"mim_id":"114480","title":"BREAST CANCER","url":"https://www.omim.org/entry/114480"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"testis","ntpm":33.0},{"tissue":"tongue","ntpm":13.7}],"url":"https://www.proteinatlas.org/search/NEK10"},"hgnc":{"alias_symbol":["FLJ32685"],"prev_symbol":[]},"alphafold":{"accession":"Q6ZWH5","domains":[{"cath_id":"-","chopping":"253-349","consensus_level":"medium","plddt":90.5541,"start":253,"end":349},{"cath_id":"-","chopping":"413-507","consensus_level":"high","plddt":88.5189,"start":413,"end":507},{"cath_id":"3.30.200.20","chopping":"515-606","consensus_level":"high","plddt":78.6687,"start":515,"end":606},{"cath_id":"1.10.510.10","chopping":"611-775","consensus_level":"high","plddt":86.1765,"start":611,"end":775},{"cath_id":"1.10.287","chopping":"797-832_1035-1102_1132-1148","consensus_level":"medium","plddt":71.7234,"start":797,"end":1148}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZWH5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZWH5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZWH5-F1-predicted_aligned_error_v6.png","plddt_mean":69.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NEK10","jax_strain_url":"https://www.jax.org/strain/search?query=NEK10"},"sequence":{"accession":"Q6ZWH5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6ZWH5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6ZWH5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZWH5"}},"corpus_meta":[{"pmid":"20956560","id":"PMC_20956560","title":"Nek10 mediates G2/M cell cycle arrest and MEK autoactivation in response to UV irradiation.","date":"2010","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20956560","citation_count":57,"is_preprint":false},{"pmid":"31959991","id":"PMC_31959991","title":"A human ciliopathy reveals essential functions for NEK10 in airway mucociliary clearance.","date":"2020","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/31959991","citation_count":57,"is_preprint":false},{"pmid":"32368190","id":"PMC_32368190","title":"NEK10 interactome and depletion reveal new roles in mitochondria.","date":"2020","source":"Proteome science","url":"https://pubmed.ncbi.nlm.nih.gov/32368190","citation_count":26,"is_preprint":false},{"pmid":"32561851","id":"PMC_32561851","title":"NEK10 tyrosine phosphorylates p53 and controls its transcriptional activity.","date":"2020","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/32561851","citation_count":22,"is_preprint":false},{"pmid":"33064774","id":"PMC_33064774","title":"Mutation of NEKL-4/NEK10 and TTLL genes suppress neuronal ciliary degeneration caused by loss of CCPP-1 deglutamylase function.","date":"2020","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/33064774","citation_count":20,"is_preprint":false},{"pmid":"32414360","id":"PMC_32414360","title":"Homozygous truncating NEK10 mutation, associated with primary ciliary dyskinesia: a case report.","date":"2020","source":"BMC pulmonary medicine","url":"https://pubmed.ncbi.nlm.nih.gov/32414360","citation_count":16,"is_preprint":false},{"pmid":"38683979","id":"PMC_38683979","title":"β-catenin turnover is regulated by Nek10-mediated tyrosine phosphorylation in A549 lung adenocarcinoma cells.","date":"2024","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/38683979","citation_count":6,"is_preprint":false},{"pmid":"39379991","id":"PMC_39379991","title":"NEK10 kinase ablation affects mitochondrial morphology, function and protein phosphorylation status.","date":"2024","source":"Proteome science","url":"https://pubmed.ncbi.nlm.nih.gov/39379991","citation_count":2,"is_preprint":false},{"pmid":"41044802","id":"PMC_41044802","title":"In Silico Analysis of Human NEK10 Reveals Novel Domain Architecture and Protein-Protein Interactions.","date":"2025","source":"Proteins","url":"https://pubmed.ncbi.nlm.nih.gov/41044802","citation_count":0,"is_preprint":false},{"pmid":"41427545","id":"PMC_41427545","title":"Extracellular matrix-derived mechanical force induces CDK4/6 inhibitor resistance by inhibiting NEK10 dependent cell cycle regulation in breast cancer.","date":"2026","source":"International journal of surgery (London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/41427545","citation_count":0,"is_preprint":false},{"pmid":"40934128","id":"PMC_40934128","title":"NEK10 Drives Lipid Disturbances That Induce G2/M Phase Arrest in Renal Tubular Cells under Albumin Overload.","date":"2025","source":"American journal of nephrology","url":"https://pubmed.ncbi.nlm.nih.gov/40934128","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7760,"output_tokens":2700,"usd":0.03189,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10032,"output_tokens":3625,"usd":0.070392,"stage2_stop_reason":"end_turn"},"total_usd":0.102282,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2010,\n      \"finding\": \"NEK10 physically associates with Raf-1 and MEK1 in a Raf-1-dependent manner upon UV irradiation. This complex formation is necessary for NEK10-mediated MEK1 activation. NEK10 does not affect Raf-1 kinase activity but instead promotes the autophosphorylation-dependent activation of MEK1, leading to ERK1/2 activation and G2/M checkpoint maintenance after UV damage.\",\n      \"method\": \"Co-immunoprecipitation, kinase activity assays, siRNA knockdown with cell cycle analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and kinase assays in single lab with multiple orthogonal methods\",\n      \"pmids\": [\"20956560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"NEK10 is required for ERK1/2 activation in response to UV irradiation but not in response to mitogens such as EGF, indicating a UV-specific function upstream of the MEK-ERK pathway.\",\n      \"method\": \"siRNA knockdown, immunoblot for phospho-ERK1/2 after UV or EGF stimulation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knockdown with defined pathway readout, single lab\",\n      \"pmids\": [\"20956560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEK10 is a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport in primary human airway cultures; NEK10 loss does not affect ciliary number, radial structure, or beat frequency.\",\n      \"method\": \"Genetically modified primary human airway cultures (NEK10 loss-of-function), ciliary proteomics, mucociliary transport assay, electron microscopy\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — primary human airway cultures with genetic modification, multiple orthogonal methods (proteomics, functional assay, structural analysis), replicated in human patient context\",\n      \"pmids\": [\"31959991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEK10 directly phosphorylates p53 on tyrosine 327 (Y327), and a p53-Y327F mutant acts as a hypomorph with attenuated transcriptional response. NEK10 loss increases cellular proliferation by modulating p53-dependent transcriptional output and causes heightened sensitivity to DNA-damaging agents.\",\n      \"method\": \"In vitro kinase assay with purified proteins, phospho-specific site mutagenesis (Y327F), transcriptional reporter assays, siRNA/CRISPR knockdown with proliferation and drug sensitivity assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro phosphorylation with site-specific mutagenesis and functional validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"32561851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEK10 interacts with mitochondria-associated proteins including SIRT3, ATAD3A, ATAD3B, and GLUD1 (confirmed by proximity ligation assay). NEK10 depletion causes mitochondrial fragmentation, altered mitochondrial ROS levels, decreased citrate synthase activity, inhibited mitochondrial respiration (particularly ATP-linked oxygen consumption and spare capacity), decreased mtDNA amplification ratio, and increased total mtDNA content.\",\n      \"method\": \"FLAG co-immunoprecipitation with LC-MS/MS proteomics, proximity ligation assay, confocal microscopy, stable shRNA knockdown, Seahorse respirometry, citrate synthase activity assay, mtDNA quantification\",\n      \"journal\": \"Proteome science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity ligation assay confirms key interaction, multiple functional readouts in single lab\",\n      \"pmids\": [\"32368190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In C. elegans, NEKL-4 (NEK10 ortholog) localizes to the ciliary base but does not localize within cilia, suggesting an indirect role in ciliary processes. Loss-of-function of nekl-4 suppresses hyperglutamylation-induced ciliary degeneration in ccpp-1 mutants, placing NEKL-4 in a pathway modulating tubulin glutamylation effects.\",\n      \"method\": \"Forward genetic screen, fluorescence microscopy localization, epistasis analysis in C. elegans double mutants\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with localization in model organism, single study\",\n      \"pmids\": [\"33064774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NEK10 associates with the Axin destruction complex and phosphorylates β-catenin at Tyr30 (within the regulatory region governing β-catenin turnover). In the absence of NEK10-mediated Tyr30 phosphorylation, GSK3-mediated phosphorylation of β-catenin—a prerequisite for its degradation—is impaired, leading to β-catenin stabilization. NEK10-deficient lung adenocarcinoma cells show reduced tumorsphere formation, soft-agar growth, and lung colonization in vivo.\",\n      \"method\": \"CRISPR knockout, co-immunoprecipitation with Axin complex, in vitro kinase assay, site-specific mutagenesis, immunoblot for β-catenin phosphorylation/stability, tumorsphere assay, soft-agar assay, tail-vein injection mouse model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct kinase assay with defined phosphorylation site, complex co-IP, in vitro and in vivo functional validation, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"38683979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NEK10 knockout in HAP1 cells alters threonine phosphorylation status of mitochondrial/ER proteins including HSP60, NDUFB4, and TOM20, and affects steady-state levels of respiratory complex proteins and autophagy pathway components, supporting a role for NEK10 in mitochondrial homeostasis via phosphorylation-dependent regulation.\",\n      \"method\": \"HAP1 NEK10 knockout cells, LC-MS/MS proteomics and phosphoproteomics of mitochondrial fractions, mitochondrial respiration assay, morphology analysis\",\n      \"journal\": \"Proteome science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics with full KO and multiple functional readouts, single lab, replicates earlier findings\",\n      \"pmids\": [\"39379991\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NEK10 knockdown in tubular cells attenuates BSA-induced lipid droplet accumulation, G2/M arrest, and ERK1/2 phosphorylation, placing NEK10 upstream of ERK1/2 in an albumin-overload-driven metabolic and cell cycle disruption pathway; in vivo NEK10 knockdown (rAAV-shNEK10) reduces BSA-induced renal fibrosis in UUO mice.\",\n      \"method\": \"siRNA/rAAV shRNA knockdown in HK-2 cells and UUO mouse model, Western blotting for phospho-ERK1/2, flow cytometry cell cycle analysis, lipid droplet staining, fatty acid oxidation assay\",\n      \"journal\": \"American journal of nephrology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pathway placement based on knockdown + phosphorylation readout without direct mechanistic dissection\",\n      \"pmids\": [\"40934128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"ECM-derived mechanical forces reduce NEK10 expression via a cytoskeleton-dependent pathway, leading to suppression of the NEK10/p53/CDKN1A axis, activation of CDK2 signaling, and CDK4/6 inhibitor resistance in breast cancer cells.\",\n      \"method\": \"3D gel biomechanical models, scRNA-seq, transcriptomic analysis, Western blotting, NEK10-deficient cells/organoids, in vivo CDK2 inhibitor co-treatment\",\n      \"journal\": \"International journal of surgery (London, England)\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, pathway placement inferred from expression reduction and inhibitor rescue without direct biochemical dissection of NEK10 mechanism\",\n      \"pmids\": [\"41427545\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NEK10 is a serine/threonine/tyrosine kinase that operates in multiple contexts: upon UV irradiation it forms a Raf-1-dependent complex with MEK1 to promote MEK1 autophosphorylation and ERK1/2 activation, thereby maintaining the G2/M checkpoint; it directly phosphorylates p53 on Tyr327 to modulate p53-dependent transcriptional responses to genotoxic stress; it phosphorylates β-catenin at Tyr30 within the Axin destruction complex, enabling subsequent GSK3-mediated β-catenin degradation; in ciliated airway cells it regulates the motile ciliary proteome to control ciliary length and mucociliary transport without affecting ciliary number, structure, or beat frequency; and it maintains mitochondrial homeostasis by modulating the threonine phosphorylation status of mitochondrial proteins including HSP60, NDUFB4, and TOM20.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NEK10 is a NIMA-related serine/threonine/tyrosine kinase that links DNA-damage signaling, motile cilia biology, Wnt/β-catenin turnover, and mitochondrial homeostasis [#0, #2, #3, #6]. In the DNA-damage response, NEK10 acts UV-specifically: upon UV irradiation it forms a Raf-1-dependent complex with MEK1 and drives MEK1 autophosphorylation to activate ERK1/2 and maintain the G2/M checkpoint, while leaving Raf-1 kinase activity and mitogen-driven ERK activation unaffected [#0, #1]. NEK10 also directly phosphorylates p53 on tyrosine 327 to tune p53-dependent transcriptional output, such that its loss increases proliferation and sensitizes cells to genotoxic agents [#3]. In Wnt signaling, NEK10 associates with the Axin destruction complex and phosphorylates β-catenin at Tyr30, a modification required to license subsequent GSK3-mediated phosphorylation and β-catenin degradation; loss of this activity stabilizes β-catenin and promotes lung adenocarcinoma tumorigenicity in vitro and in vivo [#6]. In ciliated airway cells, NEK10 is a ciliated-cell-specific kinase that shapes the motile ciliary proteome to control ciliary length and mucociliary transport without altering ciliary number, ultrastructure, or beat frequency [#2]. NEK10 further interacts with mitochondria-associated proteins and modulates the threonine-phosphorylation status of mitochondrial proteins including HSP60, NDUFB4, and TOM20, with its depletion causing mitochondrial fragmentation, impaired respiration, and altered mtDNA content [#4, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2010,\n      \"claim\": \"Established the first signaling role for NEK10, placing it as a UV-specific activator upstream of the MEK-ERK cascade required for the G2/M checkpoint.\",\n      \"evidence\": \"Co-IP, kinase activity assays, and siRNA knockdown with cell cycle analysis showing NEK10 forms a Raf-1-dependent complex with MEK1 and promotes MEK1 autophosphorylation\",\n      \"pmids\": [\"20956560\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct NEK10 substrate(s) within the complex not defined (MEK1 activation is autophosphorylation-dependent rather than NEK10-catalyzed)\",\n        \"Structural basis of the Raf-1-dependent assembly unresolved\",\n        \"Why the requirement is UV-specific versus mitogen stimulation not mechanistically explained\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined NEK10 as a ciliated-cell-specific kinase that controls ciliary length and mucociliary transport through the motile ciliary proteome rather than ciliary assembly.\",\n      \"evidence\": \"Genetically modified primary human airway cultures with ciliary proteomics, mucociliary transport assays, and electron microscopy\",\n      \"pmids\": [\"31959991\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct ciliary substrates of NEK10 not identified\",\n        \"Mechanism coupling proteome changes to length control unresolved\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified p53 as a direct NEK10 substrate, connecting the kinase to DNA-damage transcriptional control and proliferation suppression.\",\n      \"evidence\": \"In vitro kinase assays with purified proteins, Y327F site mutagenesis, transcriptional reporters, and knockdown proliferation/drug-sensitivity assays\",\n      \"pmids\": [\"32561851\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How Y327 phosphorylation alters p53 conformation or cofactor binding not defined\",\n        \"Relationship between this axis and the earlier MEK-ERK function not integrated\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended NEK10 function to mitochondria, showing physical association with mitochondrial proteins and a requirement for mitochondrial morphology and respiration.\",\n      \"evidence\": \"FLAG Co-IP/LC-MS/MS, proximity ligation assay confirming SIRT3/ATAD3A/ATAD3B/GLUD1 interactions, confocal microscopy, Seahorse respirometry, and mtDNA quantification in shRNA knockdown cells\",\n      \"pmids\": [\"32368190\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct mitochondrial phosphorylation substrates not yet established at this stage\",\n        \"Whether NEK10 localizes to mitochondria or acts indirectly unresolved\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Ortholog analysis placed NEK10 (NEKL-4) at the ciliary base acting in a tubulin-glutamylation modulating pathway, supporting an indirect mode of ciliary regulation.\",\n      \"evidence\": \"Forward genetic screen, fluorescence localization, and epistasis with ccpp-1 mutants in C. elegans\",\n      \"pmids\": [\"33064774\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Molecular targets of NEKL-4 in the glutamylation pathway unknown\",\n        \"Conservation of the base-localized mechanism in human cells not demonstrated\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Resolved a tumor-suppressive mechanism in which NEK10 primes β-catenin for degradation, linking the kinase to Wnt pathway control of lung cancer growth.\",\n      \"evidence\": \"CRISPR knockout, Axin-complex Co-IP, in vitro kinase assay, Tyr30 site mutagenesis, β-catenin stability immunoblots, and tumorsphere/soft-agar/tail-vein assays\",\n      \"pmids\": [\"38683979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"How Tyr30 phosphorylation gates subsequent GSK3 phosphorylation structurally unresolved\",\n        \"Regulation of NEK10 recruitment to the Axin complex not defined\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided direct phosphoproteomic evidence that NEK10 sets the threonine-phosphorylation status of specific mitochondrial proteins, mechanistically anchoring its mitochondrial homeostasis role.\",\n      \"evidence\": \"HAP1 NEK10 knockout with mitochondrial-fraction phosphoproteomics identifying HSP60, NDUFB4, TOM20, plus respiration and morphology assays\",\n      \"pmids\": [\"39379991\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct versus indirect phosphorylation of the identified proteins not distinguished\",\n        \"Functional consequence of each phosphosite not individually tested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Placed NEK10 upstream of ERK1/2 in an albumin-overload pathway driving tubular metabolic and cell-cycle disruption and renal fibrosis.\",\n      \"evidence\": \"siRNA/rAAV-shNEK10 knockdown in HK-2 cells and a UUO mouse model with phospho-ERK1/2, cell cycle, lipid droplet, and fatty acid oxidation readouts\",\n      \"pmids\": [\"40934128\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Pathway placement rests on knockdown plus phosphorylation readout without direct mechanistic dissection\",\n        \"Direct NEK10 substrate in this context not identified\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Linked ECM mechanical force to NEK10 expression suppression and CDK4/6 inhibitor resistance via a NEK10/p53/CDKN1A axis in breast cancer.\",\n      \"evidence\": \"3D biomechanical gel models, scRNA-seq, transcriptomics, NEK10-deficient cells/organoids, and in vivo CDK2 inhibitor co-treatment\",\n      \"pmids\": [\"41427545\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Axis inferred from expression changes and inhibitor rescue without biochemical dissection of NEK10 here\",\n        \"How cytoskeletal mechanotransduction regulates NEK10 expression unknown\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NEK10's multiple substrate-specific activities (MEK1 complex, p53-Y327, β-catenin-Tyr30, mitochondrial threonine sites, ciliary proteome) are coordinated and contextually selected within a single kinase remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural model of NEK10 substrate recognition\",\n        \"No unified account of tissue- and stimulus-specific activation\",\n        \"Endogenous regulators of NEK10 activity not defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 6, 7]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 3, 6, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [4, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"complexes\": [\"Axin destruction complex\"],\n    \"partners\": [\"RAF1\", \"MAP2K1\", \"TP53\", \"CTNNB1\", \"SIRT3\", \"ATAD3A\", \"GLUD1\", \"TOM20\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}