{"gene":"NKD1","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2001,"finding":"Human NKD1 protein contains an EF-hand motif in its NH2 domain and shows 43.8% amino acid identity with NKD2; it was cloned and characterized as a Dishevelled-binding protein functioning as a negative regulator of the WNT-beta-catenin-TCF signaling pathway, based on homology to mouse Nkd.","method":"Molecular cloning, sequence analysis, domain prediction","journal":"International journal of oncology","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational/sequence analysis only, no direct functional assay on human NKD1 in this paper","pmids":["11604995"],"is_preprint":false},{"year":2004,"finding":"The EF-hand motif of Nkd1 is required for its inhibitory function in the Wnt/beta-catenin signaling pathway; targeted deletion of the EF-hand in mice resulted in increased nuclear beta-catenin in elongating spermatids and reduced sperm count, demonstrating that the EF-hand is necessary for Nkd1-mediated inhibition of Wnt/beta-catenin signaling in spermatogenesis.","method":"Targeted knock-in mutagenesis (EF-hand deletion), mouse genetic model, nuclear beta-catenin immunostaining, sperm count analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo loss-of-function with defined domain-specific mutation and mechanistic phenotypic readout","pmids":["15546883"],"is_preprint":false},{"year":2007,"finding":"Mouse Nkd1 and Nkd2 proteins bind Dvl (Dishevelled) proteins and inhibit Wnt signaling; targeted replacement of nkd exons encoding Dvl-binding sequences with IRES-lacZ/neomycin cassettes generated viable double-knockout mice with subtle cranial bone morphology alterations, showing that nkd1 and nkd2 are dispensable for murine embryonic development but play a role in Wnt/beta-catenin antagonism via Dvl binding.","method":"Gene targeting (Dvl-binding domain replacement), double-knockout mouse generation, morphological analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function with domain-specific targeting, replicated across both paralogs in vivo","pmids":["17438140"],"is_preprint":false},{"year":2009,"finding":"Mutations in human NKD1 found in colorectal cancer reduce its ability to inhibit Wnt signaling, stabilize beta-catenin, promote cell proliferation, and reduce NKD1's ability to bind and destabilize Dvl proteins, establishing NKD1 as a functional Wnt pathway antagonist acting through Dvl binding and destabilization.","method":"Mutation identification in colorectal tumors, functional assays (Wnt signaling reporter, beta-catenin stabilization, cell proliferation), co-immunoprecipitation for Dvl binding","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays (signaling reporters, protein stability, proliferation, Co-IP) in a single study","pmids":["19956716"],"is_preprint":false},{"year":2010,"finding":"Zebrafish Nkd1 promotes Dvl protein degradation upon overexpression; knockdown of Nkd1 specifically in dorsal forerunner cells leads to beta-catenin nuclear localization, transcriptional activation, defects in DFC migration, Kupffer's vesicle formation, ciliogenesis, and left-right patterning, establishing Nkd1 as a beta-catenin antagonist required for left-right axis establishment.","method":"Nkd1 overexpression (Dvl degradation assay), morpholino knockdown targeted to DFCs, beta-catenin nuclear localization assay, left-right patterning analysis, ciliogenesis analysis","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal in vivo methods (overexpression, cell-type-specific knockdown, molecular and morphological readouts) in single study","pmids":["20858476"],"is_preprint":false},{"year":2013,"finding":"Nkd1 functions as a passive antagonist of Wnt/beta-catenin signaling in zebrafish, meaning its antagonistic activity is enhanced only when canonical Wnt signaling levels have been destabilized (e.g., in Wnt/PCP mutants silberblick/wnt11 and trilobite/vangl2), rather than actively suppressing normal Wnt levels.","method":"Genetic epistasis using zebrafish Wnt/PCP mutant lines (slb/wnt11, tri/vangl2), Wnt8a overexpression, phenotypic rescue by Nkd1","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in multiple mutant backgrounds, single lab","pmids":["24009776"],"is_preprint":false},{"year":2015,"finding":"Nkd1 activity is specifically dependent on Wnt ligand activation of the receptor; Nkd1 is recruited to the Wnt signalosome with Dvl2 upon Wnt ligand stimulation, then moves into the cytoplasm to interact with beta-catenin and inhibit its nuclear accumulation.","method":"Wnt-responsive zebrafish blastula cell assays, signalosome recruitment assay, beta-catenin nuclear localization assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based functional assays with defined pathway placement, single lab","pmids":["25904337"],"is_preprint":false},{"year":2015,"finding":"NKD1 is an immediate early target gene induced by FGF receptor signaling; NKD1 suppresses canonical WNT signaling during the transition from endoderm to hepatic progenitor cells, and loss of NKD1 impairs hepatic progenitor cell formation from human iPSCs in a manner rescued by pharmacological WNT antagonism.","method":"FGFR inhibitor treatment, NKD1 knockdown in human iPSC differentiation, pharmacological WNT antagonist rescue","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined rescue, pathway placement via pharmacological epistasis, human stem cell model","pmids":["26637527"],"is_preprint":false},{"year":2016,"finding":"NKD1 interacts with Rac1 in the cytoplasm and promotes its degradation via the ubiquitin-proteasome pathway; NKD1 overexpression in HCC cells reduces Rac1 expression and activity, affecting cytoskeletal organization and E-cadherin expression; conversely, Rac1 overexpression enhances NKD1 transcription by negatively regulating EZH2, forming a feedback loop.","method":"Co-immunoprecipitation, overexpression/knockdown, ubiquitin-proteasome inhibitor assay, in vitro and in vivo invasion assays, E-cadherin expression analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal functional relationship with Co-IP and proteasome inhibitor assay, single lab, multiple readouts","pmids":["27231134"],"is_preprint":false},{"year":2016,"finding":"Rnf25/AO7 E3 ubiquitin ligase physically interacts with both Nkd1 and Axin in an E3 ligase-independent manner, disrupting the Nkd1-Axin inhibitory complex and thereby positively regulating Wnt signaling; this interaction distinguishes Nkd1 from Nkd2 in their feedback regulation of Wnt signaling.","method":"Co-immunoprecipitation, Wnt target gene expression assay, morpholino knockdown in zebrafish","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP with in vivo zebrafish knockdown, single lab, two methods","pmids":["27007149"],"is_preprint":false},{"year":2022,"finding":"NKD1 binds APC protein and promotes its ubiquitination degradation by restraining expression of the deubiquitinating enzyme USP15 and blocking the USP15-APC interaction, thereby enhancing beta-catenin nuclear accumulation and promoting colon cancer cell proliferation and migration.","method":"Co-immunoprecipitation, ubiquitination assay, USP15 interaction analysis, luciferase reporter assay, loss-of-function/overexpression experiments","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical assays (Co-IP, ubiquitination assay, reporter) in single lab establishing mechanistic interaction","pmids":["36445120"],"is_preprint":false},{"year":2023,"finding":"NKD1 protein binds to the YWHAE gene promoter region and activates its transcription, thereby promoting glucose uptake in colon cancer cells; NKD1 and YWHAE proteins also co-localize in colon cancer cells.","method":"ChIP assay, dual-luciferase reporter gene assay, NKD1 overexpression/knockout, immunofluorescence, glucose uptake assay","journal":"Nan fang yi ke da xue xue bao","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase assay establish direct transcriptional activation, single lab","pmids":["37202194"],"is_preprint":false},{"year":2023,"finding":"NKD1 interacts with PCM1 and promotes PCM1 degradation through the ubiquitin-proteasome pathway; this NKD1/PCM1 interaction mediates NKD1-regulated cell proliferation and cell cycle progression in colorectal cancer cells.","method":"Quantitative proteomics, co-immunoprecipitation, immunofluorescence, siRNA knockdown, cell cycle analysis","journal":"Molecular biology reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus proteomic identification plus functional rescue, single lab","pmids":["37338734"],"is_preprint":false},{"year":2024,"finding":"YTHDF3, an m6A reader, suppresses NKD1 transcription and translation in an m6A-dependent manner; reduced NKD1 expression activates the WNT/beta-catenin signaling pathway, promoting HCC cell migration and invasion, establishing NKD1 as a downstream target of YTHDF3-mediated m6A modification.","method":"RNA-seq, meRIP-seq, Lace-seq, Western blot, in vitro and in vivo functional assays","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multi-omics identification with functional validation, single lab","pmids":["39127439"],"is_preprint":false},{"year":2024,"finding":"Nkd1 functions downstream of Axin2 in Wnt signaling feedback; genetic epistasis in axin2/nkd1 double-mutant zebrafish demonstrated that the double mutant phenotype (including Wnt target gene expression profile by qRT-PCR/RNA-seq and protein expression by mass spectrometry) resembles nkd1 single mutant, placing Nkd1 downstream of Axin2 in the pathway.","method":"CRISPR/Cas9 double-mutant zebrafish generation, qRT-PCR, RNA-seq, mass spectrometry protein expression, Wnt sensitivity assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple orthogonal molecular readouts (transcriptomics, proteomics, functional) in a rigorous double-mutant study","pmids":["38656801"],"is_preprint":false},{"year":2025,"finding":"NKD1 binds MYC protein through its EF-hand domain, inhibits autophagic degradation of MYC by suppressing the LC3B-MYC interaction, and facilitates MYC nuclear entry; PPARdelta acts as a transcription factor for NKD1; this PPARdelta/NKD1/MYC axis promotes colon cancer cell proliferation, migration, and angiogenesis.","method":"Differential protein expression profiling (SW620 vs SW620-nkd1-/-), Co-IP, autophagy pathway analysis, EF-hand domain mutant analysis, ChIP for PPARdelta on NKD1 promoter, NKD1 knockout cell line","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-specific interaction (EF-hand mutant), proteomic profiling, and functional assays, single lab","pmids":["40675969"],"is_preprint":false},{"year":2026,"finding":"Wnt3a specifically induces NKD1 expression and triggers NKD1 membrane detachment; NKD1 then directly interacts with MSX1 (identified as a key transcription factor via SCENIC analysis), facilitating MSX1 nuclear translocation to promote odontogenic gene activation; MSX1 occupancy at odontogenic gene promoters was validated by CUT&Tag, and Wnt3a-activated NKD1-MSX1 signaling enhances reparative dentin formation in vivo.","method":"Single-cell transcriptomics, SCENIC gene regulatory network analysis, CUT&Tag, co-localization assay, in vivo murine pulp exposure model, overexpression experiments","journal":"International journal of oral science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (scRNA-seq, CUT&Tag, in vivo) establishing mechanistic axis, single lab","pmids":["41526338"],"is_preprint":false}],"current_model":"NKD1 is a Wnt-inducible negative feedback regulator that is recruited to the Wnt signalosome upon ligand stimulation, where it binds and destabilizes Dvl proteins to inhibit beta-catenin nuclear accumulation; its EF-hand domain is required for canonical Wnt inhibition and also mediates direct binding to MYC to suppress its autophagic degradation; NKD1 additionally interacts with Rac1 and PCM1 to promote their proteasomal degradation, binds APC to facilitate its ubiquitination, and can translocate to the nucleus to act as a transcriptional activator of YWHAE; NKD1 expression itself is regulated by FGFR signaling, m6A modification via YTHDF3, EZH2-mediated epigenetic repression, and acts downstream of Axin2 in the Wnt feedback hierarchy."},"narrative":{"mechanistic_narrative":"NKD1 is a Wnt-inducible negative feedback regulator of the canonical Wnt/beta-catenin pathway, encoding an EF-hand-containing protein that antagonizes signaling by binding Dishevelled (Dvl) and limiting beta-catenin nuclear accumulation [PMID:15546883, PMID:19956716]. Upon Wnt ligand stimulation NKD1 is recruited with Dvl2 to the Wnt signalosome and subsequently relocates to the cytoplasm to engage beta-catenin and block its nuclear entry, with activity that is strictly dependent on receptor-level ligand activation [PMID:25904337]; it binds and destabilizes Dvl proteins, and cancer-derived mutations that weaken Dvl binding stabilize beta-catenin and drive proliferation [PMID:19956716, PMID:20858476]. The EF-hand motif is required for this inhibitory function in vivo, as its deletion in mice elevates nuclear beta-catenin and disrupts spermatogenesis [PMID:15546883]. NKD1 operates within a layered feedback hierarchy, acting downstream of Axin2 [PMID:38656801], and its abundance is set by FGFR signaling, which induces NKD1 as an immediate-early gene to restrain Wnt during hepatic progenitor specification [PMID:26637527], and by YTHDF3-mediated m6A modification that suppresses its expression [PMID:39127439]. Beyond Wnt antagonism, NKD1 has context-dependent pro-tumorigenic activities in colorectal and hepatocellular cancer: it interacts with and promotes ubiquitin-proteasomal degradation of Rac1 and PCM1 [PMID:27231134, PMID:37338734], binds APC to enhance its ubiquitination by restraining USP15 [PMID:36445120], stabilizes MYC through its EF-hand by blocking LC3B-mediated autophagic degradation [PMID:40675969], and can act as a nuclear transcriptional activator of YWHAE to promote glucose uptake [PMID:37202194]. NKD1 also partners with the transcription factors MSX1 and PPARdelta in differentiation and proliferation programs [PMID:40675969, PMID:41526338].","teleology":[{"year":2001,"claim":"Established human NKD1 as a Dishevelled-binding, EF-hand-containing candidate negative regulator of Wnt/beta-catenin/TCF signaling by homology to mouse Nkd.","evidence":"Molecular cloning, sequence and domain analysis","pmids":["11604995"],"confidence":"Low","gaps":["Computational/sequence inference only, no direct functional assay on human NKD1","Dvl binding not biochemically demonstrated for the human protein here"]},{"year":2004,"claim":"Defined the EF-hand motif as functionally necessary for Wnt antagonism in vivo, showing domain-specific loss elevates nuclear beta-catenin and impairs spermatogenesis.","evidence":"Targeted EF-hand deletion knock-in mouse, nuclear beta-catenin immunostaining, sperm count","pmids":["15546883"],"confidence":"High","gaps":["Does not resolve the molecular ligand or partner engaged by the EF-hand","Mechanism linking EF-hand to Dvl destabilization not addressed"]},{"year":2007,"claim":"Genetic loss-of-function showed Nkd1/Nkd2 bind Dvl and antagonize Wnt but are individually dispensable for murine development, defining them as modulatory rather than essential.","evidence":"Dvl-binding-domain replacement, double-knockout mouse, morphological analysis","pmids":["17438140"],"confidence":"High","gaps":["Subtle phenotype leaves the in vivo physiological context underdefined","Paralog redundancy not fully dissected"]},{"year":2009,"claim":"Connected NKD1 dysfunction to colorectal cancer by demonstrating tumor-derived mutations reduce Dvl binding/destabilization, stabilize beta-catenin and drive proliferation.","evidence":"Tumor mutation analysis, Wnt reporter, beta-catenin stability, proliferation, Co-IP","pmids":["19956716"],"confidence":"High","gaps":["Does not establish whether mutations act dominantly or recessively in tumors","Mechanism of Dvl destabilization not resolved"]},{"year":2010,"claim":"Showed Nkd1 promotes Dvl degradation and is required in dorsal forerunner cells for ciliogenesis and left-right axis establishment, extending its antagonism to developmental patterning.","evidence":"Zebrafish overexpression Dvl-degradation assay, DFC-targeted morpholino knockdown, patterning analysis","pmids":["20858476"],"confidence":"High","gaps":["Biochemical mechanism of Dvl degradation unresolved","Link between ciliogenesis defect and beta-catenin not fully separated"]},{"year":2013,"claim":"Refined NKD1 as a passive antagonist whose activity manifests only when Wnt signaling is destabilized, clarifying it is a buffering rather than constitutive suppressor.","evidence":"Genetic epistasis in zebrafish Wnt/PCP mutants, Wnt8a overexpression, Nkd1 rescue","pmids":["24009776"],"confidence":"Medium","gaps":["Molecular basis of conditional/passive behavior not defined","Single-lab epistasis"]},{"year":2015,"claim":"Placed NKD1 dynamically in the pathway: ligand-triggered recruitment to the signalosome with Dvl2 followed by cytoplasmic relocation to block beta-catenin nuclear accumulation.","evidence":"Wnt-responsive zebrafish blastula cell assays, signalosome recruitment and beta-catenin localization assays","pmids":["25904337"],"confidence":"Medium","gaps":["Recruitment mechanism to signalosome not defined","Direct beta-catenin binding not biochemically isolated"]},{"year":2015,"claim":"Identified NKD1 as an FGFR-induced immediate-early gene that restrains Wnt during endoderm-to-hepatic-progenitor transition, linking it to lineage specification.","evidence":"FGFR inhibition, NKD1 knockdown in human iPSC differentiation, pharmacological Wnt-antagonist rescue","pmids":["26637527"],"confidence":"High","gaps":["Direct FGFR-to-NKD1 transcriptional mechanism not mapped","Whether Dvl binding mediates this hepatic role untested"]},{"year":2016,"claim":"Revealed a non-Wnt activity: NKD1 binds Rac1 and drives its proteasomal degradation affecting cytoskeleton and E-cadherin, within a Rac1-EZH2-NKD1 feedback loop.","evidence":"Co-IP, overexpression/knockdown, proteasome inhibitor assay, invasion and E-cadherin assays in HCC","pmids":["27231134"],"confidence":"Medium","gaps":["No identified E3 ligase mediating Rac1 degradation","Single-lab; reciprocal loop mechanism incomplete"]},{"year":2016,"claim":"Distinguished Nkd1 from Nkd2 mechanistically by showing Rnf25/AO7 disrupts the Nkd1-Axin inhibitory complex to positively regulate Wnt.","evidence":"Co-IP, Wnt target gene expression, zebrafish morpholino knockdown","pmids":["27007149"],"confidence":"Medium","gaps":["E3-ligase-independent disruption mechanism unclear","Stoichiometry of Nkd1-Axin complex undefined"]},{"year":2022,"claim":"Showed a pro-oncogenic Wnt-activating role in colon cancer: NKD1 promotes APC ubiquitination by restraining USP15, enhancing beta-catenin nuclear accumulation.","evidence":"Co-IP, ubiquitination assay, USP15 interaction, luciferase reporter, loss/gain-of-function","pmids":["36445120"],"confidence":"Medium","gaps":["Reconciliation with NKD1's Wnt-antagonist role not addressed","Direct E3 ligase for APC not identified"]},{"year":2023,"claim":"Demonstrated a nuclear transcriptional function: NKD1 binds the YWHAE promoter to activate transcription and promote glucose uptake in colon cancer.","evidence":"ChIP, dual-luciferase reporter, overexpression/knockout, immunofluorescence, glucose uptake assay","pmids":["37202194"],"confidence":"Medium","gaps":["No DNA-binding domain defined for NKD1","Mechanism of nuclear translocation unresolved"]},{"year":2023,"claim":"Identified PCM1 as an NKD1 interactor degraded via the ubiquitin-proteasome pathway, linking NKD1 to cell-cycle progression in colorectal cancer.","evidence":"Quantitative proteomics, Co-IP, immunofluorescence, siRNA, cell cycle analysis","pmids":["37338734"],"confidence":"Medium","gaps":["E3 ligase mediating PCM1 degradation not identified","Single-lab; reciprocal validation limited"]},{"year":2024,"claim":"Placed Nkd1 downstream of Axin2 in the Wnt feedback hierarchy via rigorous double-mutant epistasis.","evidence":"CRISPR axin2/nkd1 double-mutant zebrafish, qRT-PCR, RNA-seq, mass spectrometry, Wnt sensitivity assays","pmids":["38656801"],"confidence":"High","gaps":["Direct molecular link between Axin2 and Nkd1 not defined","Does not address non-Wnt NKD1 functions"]},{"year":2024,"claim":"Established m6A control of NKD1: YTHDF3 suppresses NKD1 transcription and translation, derepressing Wnt/beta-catenin to promote HCC migration and invasion.","evidence":"RNA-seq, meRIP-seq, Lace-seq, Western blot, in vitro/in vivo functional assays","pmids":["39127439"],"confidence":"Medium","gaps":["Specific m6A sites on NKD1 transcript not finely mapped","Single-lab"]},{"year":2025,"claim":"Defined an EF-hand-dependent MYC-stabilizing axis: NKD1 binds MYC, blocks LC3B-mediated autophagic degradation, and promotes its nuclear entry, downstream of PPARdelta.","evidence":"Differential proteomics (SW620 vs nkd1-/-), Co-IP, autophagy analysis, EF-hand mutant, PPARdelta ChIP on NKD1 promoter","pmids":["40675969"],"confidence":"Medium","gaps":["How EF-hand binding blocks LC3B-MYC interaction structurally unresolved","Reconciliation with NKD1 tumor-suppressive activity not addressed"]},{"year":2026,"claim":"Showed Wnt3a induces NKD1 and triggers its membrane detachment, enabling direct MSX1 interaction and nuclear translocation to drive odontogenic gene activation and reparative dentin formation.","evidence":"scRNA-seq, SCENIC, CUT&Tag, co-localization, in vivo murine pulp exposure model, overexpression","pmids":["41526338"],"confidence":"Medium","gaps":["Mechanism of NKD1 membrane detachment undefined","Direct vs indirect NKD1-MSX1 binding not biochemically isolated"]},{"year":null,"claim":"How NKD1 switches between Wnt-antagonist and Wnt/proliferation-promoting roles, and how its cytoplasmic-degradation and nuclear-transcriptional activities are coordinated, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of EF-hand engagement with distinct partners (MYC, beta-catenin, Dvl)","No defined E3 ligase for NKD1-promoted degradation of Rac1/PCM1/APC","Context determinants of antagonist vs oncogenic behavior undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,6,9]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[11,16]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[12,8]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,8]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[11,16]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,16]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,6,14]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[3,10,15]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,7,16]}],"complexes":["Wnt signalosome"],"partners":["DVL2","RAC1","PCM1","APC","MYC","MSX1","AXIN","YWHAE"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q969G9","full_name":"Protein naked cuticle homolog 1","aliases":[],"length_aa":470,"mass_kda":52.3,"function":"Cell autonomous antagonist of the canonical Wnt signaling pathway. May activate a second Wnt signaling pathway that controls planar cell polarity","subcellular_location":"Cell membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q969G9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NKD1","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NKD1","total_profiled":1310},"omim":[{"mim_id":"607852","title":"NKD INHIBITOR OF WNT SIGNALING PATHWAY 2; NKD2","url":"https://www.omim.org/entry/607852"},{"mim_id":"607851","title":"NKD INHIBITOR OF WNT SIGNALING PATHWAY 1; NKD1","url":"https://www.omim.org/entry/607851"},{"mim_id":"190198","title":"NOTCH RECEPTOR 1; NOTCH1","url":"https://www.omim.org/entry/190198"},{"mim_id":"164820","title":"WINGLESS-TYPE MMTV INTEGRATION SITE FAMILY, MEMBER 1; WNT1","url":"https://www.omim.org/entry/164820"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoli fibrillar center","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"blood vessel","ntpm":14.7}],"url":"https://www.proteinatlas.org/search/NKD1"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q969G9","domains":[{"cath_id":"1.20.5","chopping":"137-169","consensus_level":"medium","plddt":83.963,"start":137,"end":169}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969G9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q969G9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q969G9-F1-predicted_aligned_error_v6.png","plddt_mean":54.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NKD1","jax_strain_url":"https://www.jax.org/strain/search?query=NKD1"},"sequence":{"accession":"Q969G9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q969G9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q969G9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q969G9"}},"corpus_meta":[{"pmid":"11604995","id":"PMC_11604995","title":"Molecular cloning, gene structure, and expression analyses of NKD1 and NKD2.","date":"2001","source":"International journal of oncology","url":"https://pubmed.ncbi.nlm.nih.gov/11604995","citation_count":115,"is_preprint":false},{"pmid":"28356225","id":"PMC_28356225","title":"miR-532 promoted gastric cancer migration and invasion by targeting NKD1.","date":"2017","source":"Life sciences","url":"https://pubmed.ncbi.nlm.nih.gov/28356225","citation_count":54,"is_preprint":false},{"pmid":"31435644","id":"PMC_31435644","title":"Exosome-mediated transfer of miR-1290 promotes cell proliferation and invasion in gastric cancer via NKD1.","date":"2019","source":"Acta biochimica et biophysica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/31435644","citation_count":42,"is_preprint":false},{"pmid":"19956716","id":"PMC_19956716","title":"Mutations in the human naked cuticle homolog NKD1 found in colorectal cancer alter Wnt/Dvl/beta-catenin signaling.","date":"2009","source":"PloS 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NKD1/Rac1 feedback loop regulates the invasion and migration ability of hepatocarcinoma cells.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27231134","citation_count":20,"is_preprint":false},{"pmid":"36445120","id":"PMC_36445120","title":"Let-7b-5p inhibits colon cancer progression by prohibiting APC ubiquitination degradation and the Wnt pathway by targeting NKD1.","date":"2022","source":"Cancer science","url":"https://pubmed.ncbi.nlm.nih.gov/36445120","citation_count":16,"is_preprint":false},{"pmid":"25706354","id":"PMC_25706354","title":"Expression pattern and clinicopathologic significance of NKD1 in human primary hepatocellular carcinoma.","date":"2015","source":"APMIS : acta pathologica, microbiologica, et immunologica Scandinavica","url":"https://pubmed.ncbi.nlm.nih.gov/25706354","citation_count":12,"is_preprint":false},{"pmid":"16763811","id":"PMC_16763811","title":"Expression and regulation of Nkd-1, an intracellular component of Wnt signalling pathway in the chick embryo.","date":"2006","source":"Anatomy and embryology","url":"https://pubmed.ncbi.nlm.nih.gov/16763811","citation_count":9,"is_preprint":false},{"pmid":"39127439","id":"PMC_39127439","title":"YTHDF3-mediated m6A modification of NKD1 regulates hepatocellular carcinoma invasion and metastasis by activating the WNT/β-catenin signaling axis.","date":"2024","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/39127439","citation_count":8,"is_preprint":false},{"pmid":"27007149","id":"PMC_27007149","title":"Rnf25/AO7 positively regulates wnt signaling via disrupting Nkd1-Axin inhibitory complex independent of its ubiquitin ligase activity.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27007149","citation_count":8,"is_preprint":false},{"pmid":"29115501","id":"PMC_29115501","title":"Downregulation of NKD1 in human osteosarcoma and its clinical significance.","date":"2017","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/29115501","citation_count":7,"is_preprint":false},{"pmid":"38656801","id":"PMC_38656801","title":"Nkd1 functions downstream of Axin2 to attenuate Wnt signaling.","date":"2024","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/38656801","citation_count":6,"is_preprint":false},{"pmid":"36969985","id":"PMC_36969985","title":"Clinical Significance of NKD Inhibitor of WNT Signaling Pathway 1 (NKD1) in Glioblastoma.","date":"2023","source":"Genetics research","url":"https://pubmed.ncbi.nlm.nih.gov/36969985","citation_count":3,"is_preprint":false},{"pmid":"40675969","id":"PMC_40675969","title":"NKD1 enhances colon cancer progression by inhibiting the autophagic degradation of MYC.","date":"2025","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/40675969","citation_count":1,"is_preprint":false},{"pmid":"37202194","id":"PMC_37202194","title":"[NKD1 promotes glucose uptake in colon cancer cells by activating YWHAE transcription].","date":"2023","source":"Nan fang yi ke da xue xue bao = Journal of Southern Medical University","url":"https://pubmed.ncbi.nlm.nih.gov/37202194","citation_count":1,"is_preprint":false},{"pmid":"37338734","id":"PMC_37338734","title":"NKD1 targeting PCM1 regulates the therapeutic effects of homoharringtonine on colorectal cancer.","date":"2023","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/37338734","citation_count":1,"is_preprint":false},{"pmid":"31472047","id":"PMC_31472047","title":"miR-532 promotes colorectal cancer invasion and metastasis by targeting NKD1.","date":"2019","source":"Cellular and molecular biology (Noisy-le-Grand, France)","url":"https://pubmed.ncbi.nlm.nih.gov/31472047","citation_count":1,"is_preprint":false},{"pmid":"41526338","id":"PMC_41526338","title":"Wnt3a promotes in situ dentin formation through NKD1-MSX1 axis-mediated odontogenic differentiation of dental pulp stem cells.","date":"2026","source":"International journal of oral science","url":"https://pubmed.ncbi.nlm.nih.gov/41526338","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":15412,"output_tokens":4284,"usd":0.055248,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12199,"output_tokens":5141,"usd":0.09476,"stage2_stop_reason":"end_turn"},"total_usd":0.150008,"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\": 2001,\n      \"finding\": \"Human NKD1 protein contains an EF-hand motif in its NH2 domain and shows 43.8% amino acid identity with NKD2; it was cloned and characterized as a Dishevelled-binding protein functioning as a negative regulator of the WNT-beta-catenin-TCF signaling pathway, based on homology to mouse Nkd.\",\n      \"method\": \"Molecular cloning, sequence analysis, domain prediction\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational/sequence analysis only, no direct functional assay on human NKD1 in this paper\",\n      \"pmids\": [\"11604995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The EF-hand motif of Nkd1 is required for its inhibitory function in the Wnt/beta-catenin signaling pathway; targeted deletion of the EF-hand in mice resulted in increased nuclear beta-catenin in elongating spermatids and reduced sperm count, demonstrating that the EF-hand is necessary for Nkd1-mediated inhibition of Wnt/beta-catenin signaling in spermatogenesis.\",\n      \"method\": \"Targeted knock-in mutagenesis (EF-hand deletion), mouse genetic model, nuclear beta-catenin immunostaining, sperm count analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo loss-of-function with defined domain-specific mutation and mechanistic phenotypic readout\",\n      \"pmids\": [\"15546883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Mouse Nkd1 and Nkd2 proteins bind Dvl (Dishevelled) proteins and inhibit Wnt signaling; targeted replacement of nkd exons encoding Dvl-binding sequences with IRES-lacZ/neomycin cassettes generated viable double-knockout mice with subtle cranial bone morphology alterations, showing that nkd1 and nkd2 are dispensable for murine embryonic development but play a role in Wnt/beta-catenin antagonism via Dvl binding.\",\n      \"method\": \"Gene targeting (Dvl-binding domain replacement), double-knockout mouse generation, morphological analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function with domain-specific targeting, replicated across both paralogs in vivo\",\n      \"pmids\": [\"17438140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mutations in human NKD1 found in colorectal cancer reduce its ability to inhibit Wnt signaling, stabilize beta-catenin, promote cell proliferation, and reduce NKD1's ability to bind and destabilize Dvl proteins, establishing NKD1 as a functional Wnt pathway antagonist acting through Dvl binding and destabilization.\",\n      \"method\": \"Mutation identification in colorectal tumors, functional assays (Wnt signaling reporter, beta-catenin stabilization, cell proliferation), co-immunoprecipitation for Dvl binding\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays (signaling reporters, protein stability, proliferation, Co-IP) in a single study\",\n      \"pmids\": [\"19956716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Zebrafish Nkd1 promotes Dvl protein degradation upon overexpression; knockdown of Nkd1 specifically in dorsal forerunner cells leads to beta-catenin nuclear localization, transcriptional activation, defects in DFC migration, Kupffer's vesicle formation, ciliogenesis, and left-right patterning, establishing Nkd1 as a beta-catenin antagonist required for left-right axis establishment.\",\n      \"method\": \"Nkd1 overexpression (Dvl degradation assay), morpholino knockdown targeted to DFCs, beta-catenin nuclear localization assay, left-right patterning analysis, ciliogenesis analysis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal in vivo methods (overexpression, cell-type-specific knockdown, molecular and morphological readouts) in single study\",\n      \"pmids\": [\"20858476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Nkd1 functions as a passive antagonist of Wnt/beta-catenin signaling in zebrafish, meaning its antagonistic activity is enhanced only when canonical Wnt signaling levels have been destabilized (e.g., in Wnt/PCP mutants silberblick/wnt11 and trilobite/vangl2), rather than actively suppressing normal Wnt levels.\",\n      \"method\": \"Genetic epistasis using zebrafish Wnt/PCP mutant lines (slb/wnt11, tri/vangl2), Wnt8a overexpression, phenotypic rescue by Nkd1\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in multiple mutant backgrounds, single lab\",\n      \"pmids\": [\"24009776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Nkd1 activity is specifically dependent on Wnt ligand activation of the receptor; Nkd1 is recruited to the Wnt signalosome with Dvl2 upon Wnt ligand stimulation, then moves into the cytoplasm to interact with beta-catenin and inhibit its nuclear accumulation.\",\n      \"method\": \"Wnt-responsive zebrafish blastula cell assays, signalosome recruitment assay, beta-catenin nuclear localization assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based functional assays with defined pathway placement, single lab\",\n      \"pmids\": [\"25904337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NKD1 is an immediate early target gene induced by FGF receptor signaling; NKD1 suppresses canonical WNT signaling during the transition from endoderm to hepatic progenitor cells, and loss of NKD1 impairs hepatic progenitor cell formation from human iPSCs in a manner rescued by pharmacological WNT antagonism.\",\n      \"method\": \"FGFR inhibitor treatment, NKD1 knockdown in human iPSC differentiation, pharmacological WNT antagonist rescue\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined rescue, pathway placement via pharmacological epistasis, human stem cell model\",\n      \"pmids\": [\"26637527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NKD1 interacts with Rac1 in the cytoplasm and promotes its degradation via the ubiquitin-proteasome pathway; NKD1 overexpression in HCC cells reduces Rac1 expression and activity, affecting cytoskeletal organization and E-cadherin expression; conversely, Rac1 overexpression enhances NKD1 transcription by negatively regulating EZH2, forming a feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown, ubiquitin-proteasome inhibitor assay, in vitro and in vivo invasion assays, E-cadherin expression analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal functional relationship with Co-IP and proteasome inhibitor assay, single lab, multiple readouts\",\n      \"pmids\": [\"27231134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rnf25/AO7 E3 ubiquitin ligase physically interacts with both Nkd1 and Axin in an E3 ligase-independent manner, disrupting the Nkd1-Axin inhibitory complex and thereby positively regulating Wnt signaling; this interaction distinguishes Nkd1 from Nkd2 in their feedback regulation of Wnt signaling.\",\n      \"method\": \"Co-immunoprecipitation, Wnt target gene expression assay, morpholino knockdown in zebrafish\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP with in vivo zebrafish knockdown, single lab, two methods\",\n      \"pmids\": [\"27007149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NKD1 binds APC protein and promotes its ubiquitination degradation by restraining expression of the deubiquitinating enzyme USP15 and blocking the USP15-APC interaction, thereby enhancing beta-catenin nuclear accumulation and promoting colon cancer cell proliferation and migration.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, USP15 interaction analysis, luciferase reporter assay, loss-of-function/overexpression experiments\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical assays (Co-IP, ubiquitination assay, reporter) in single lab establishing mechanistic interaction\",\n      \"pmids\": [\"36445120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NKD1 protein binds to the YWHAE gene promoter region and activates its transcription, thereby promoting glucose uptake in colon cancer cells; NKD1 and YWHAE proteins also co-localize in colon cancer cells.\",\n      \"method\": \"ChIP assay, dual-luciferase reporter gene assay, NKD1 overexpression/knockout, immunofluorescence, glucose uptake assay\",\n      \"journal\": \"Nan fang yi ke da xue xue bao\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase assay establish direct transcriptional activation, single lab\",\n      \"pmids\": [\"37202194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NKD1 interacts with PCM1 and promotes PCM1 degradation through the ubiquitin-proteasome pathway; this NKD1/PCM1 interaction mediates NKD1-regulated cell proliferation and cell cycle progression in colorectal cancer cells.\",\n      \"method\": \"Quantitative proteomics, co-immunoprecipitation, immunofluorescence, siRNA knockdown, cell cycle analysis\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus proteomic identification plus functional rescue, single lab\",\n      \"pmids\": [\"37338734\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"YTHDF3, an m6A reader, suppresses NKD1 transcription and translation in an m6A-dependent manner; reduced NKD1 expression activates the WNT/beta-catenin signaling pathway, promoting HCC cell migration and invasion, establishing NKD1 as a downstream target of YTHDF3-mediated m6A modification.\",\n      \"method\": \"RNA-seq, meRIP-seq, Lace-seq, Western blot, in vitro and in vivo functional assays\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multi-omics identification with functional validation, single lab\",\n      \"pmids\": [\"39127439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Nkd1 functions downstream of Axin2 in Wnt signaling feedback; genetic epistasis in axin2/nkd1 double-mutant zebrafish demonstrated that the double mutant phenotype (including Wnt target gene expression profile by qRT-PCR/RNA-seq and protein expression by mass spectrometry) resembles nkd1 single mutant, placing Nkd1 downstream of Axin2 in the pathway.\",\n      \"method\": \"CRISPR/Cas9 double-mutant zebrafish generation, qRT-PCR, RNA-seq, mass spectrometry protein expression, Wnt sensitivity assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple orthogonal molecular readouts (transcriptomics, proteomics, functional) in a rigorous double-mutant study\",\n      \"pmids\": [\"38656801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"NKD1 binds MYC protein through its EF-hand domain, inhibits autophagic degradation of MYC by suppressing the LC3B-MYC interaction, and facilitates MYC nuclear entry; PPARdelta acts as a transcription factor for NKD1; this PPARdelta/NKD1/MYC axis promotes colon cancer cell proliferation, migration, and angiogenesis.\",\n      \"method\": \"Differential protein expression profiling (SW620 vs SW620-nkd1-/-), Co-IP, autophagy pathway analysis, EF-hand domain mutant analysis, ChIP for PPARdelta on NKD1 promoter, NKD1 knockout cell line\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific interaction (EF-hand mutant), proteomic profiling, and functional assays, single lab\",\n      \"pmids\": [\"40675969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Wnt3a specifically induces NKD1 expression and triggers NKD1 membrane detachment; NKD1 then directly interacts with MSX1 (identified as a key transcription factor via SCENIC analysis), facilitating MSX1 nuclear translocation to promote odontogenic gene activation; MSX1 occupancy at odontogenic gene promoters was validated by CUT&Tag, and Wnt3a-activated NKD1-MSX1 signaling enhances reparative dentin formation in vivo.\",\n      \"method\": \"Single-cell transcriptomics, SCENIC gene regulatory network analysis, CUT&Tag, co-localization assay, in vivo murine pulp exposure model, overexpression experiments\",\n      \"journal\": \"International journal of oral science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (scRNA-seq, CUT&Tag, in vivo) establishing mechanistic axis, single lab\",\n      \"pmids\": [\"41526338\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NKD1 is a Wnt-inducible negative feedback regulator that is recruited to the Wnt signalosome upon ligand stimulation, where it binds and destabilizes Dvl proteins to inhibit beta-catenin nuclear accumulation; its EF-hand domain is required for canonical Wnt inhibition and also mediates direct binding to MYC to suppress its autophagic degradation; NKD1 additionally interacts with Rac1 and PCM1 to promote their proteasomal degradation, binds APC to facilitate its ubiquitination, and can translocate to the nucleus to act as a transcriptional activator of YWHAE; NKD1 expression itself is regulated by FGFR signaling, m6A modification via YTHDF3, EZH2-mediated epigenetic repression, and acts downstream of Axin2 in the Wnt feedback hierarchy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NKD1 is a Wnt-inducible negative feedback regulator of the canonical Wnt/beta-catenin pathway, encoding an EF-hand-containing protein that antagonizes signaling by binding Dishevelled (Dvl) and limiting beta-catenin nuclear accumulation [#1, #3]. Upon Wnt ligand stimulation NKD1 is recruited with Dvl2 to the Wnt signalosome and subsequently relocates to the cytoplasm to engage beta-catenin and block its nuclear entry, with activity that is strictly dependent on receptor-level ligand activation [#6]; it binds and destabilizes Dvl proteins, and cancer-derived mutations that weaken Dvl binding stabilize beta-catenin and drive proliferation [#3, #4]. The EF-hand motif is required for this inhibitory function in vivo, as its deletion in mice elevates nuclear beta-catenin and disrupts spermatogenesis [#1]. NKD1 operates within a layered feedback hierarchy, acting downstream of Axin2 [#14], and its abundance is set by FGFR signaling, which induces NKD1 as an immediate-early gene to restrain Wnt during hepatic progenitor specification [#7], and by YTHDF3-mediated m6A modification that suppresses its expression [#13]. Beyond Wnt antagonism, NKD1 has context-dependent pro-tumorigenic activities in colorectal and hepatocellular cancer: it interacts with and promotes ubiquitin-proteasomal degradation of Rac1 and PCM1 [#8, #12], binds APC to enhance its ubiquitination by restraining USP15 [#10], stabilizes MYC through its EF-hand by blocking LC3B-mediated autophagic degradation [#15], and can act as a nuclear transcriptional activator of YWHAE to promote glucose uptake [#11]. NKD1 also partners with the transcription factors MSX1 and PPARdelta in differentiation and proliferation programs [#15, #16].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established human NKD1 as a Dishevelled-binding, EF-hand-containing candidate negative regulator of Wnt/beta-catenin/TCF signaling by homology to mouse Nkd.\",\n      \"evidence\": \"Molecular cloning, sequence and domain analysis\",\n      \"pmids\": [\"11604995\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Computational/sequence inference only, no direct functional assay on human NKD1\", \"Dvl binding not biochemically demonstrated for the human protein here\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the EF-hand motif as functionally necessary for Wnt antagonism in vivo, showing domain-specific loss elevates nuclear beta-catenin and impairs spermatogenesis.\",\n      \"evidence\": \"Targeted EF-hand deletion knock-in mouse, nuclear beta-catenin immunostaining, sperm count\",\n      \"pmids\": [\"15546883\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not resolve the molecular ligand or partner engaged by the EF-hand\", \"Mechanism linking EF-hand to Dvl destabilization not addressed\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Genetic loss-of-function showed Nkd1/Nkd2 bind Dvl and antagonize Wnt but are individually dispensable for murine development, defining them as modulatory rather than essential.\",\n      \"evidence\": \"Dvl-binding-domain replacement, double-knockout mouse, morphological analysis\",\n      \"pmids\": [\"17438140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Subtle phenotype leaves the in vivo physiological context underdefined\", \"Paralog redundancy not fully dissected\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connected NKD1 dysfunction to colorectal cancer by demonstrating tumor-derived mutations reduce Dvl binding/destabilization, stabilize beta-catenin and drive proliferation.\",\n      \"evidence\": \"Tumor mutation analysis, Wnt reporter, beta-catenin stability, proliferation, Co-IP\",\n      \"pmids\": [\"19956716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not establish whether mutations act dominantly or recessively in tumors\", \"Mechanism of Dvl destabilization not resolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed Nkd1 promotes Dvl degradation and is required in dorsal forerunner cells for ciliogenesis and left-right axis establishment, extending its antagonism to developmental patterning.\",\n      \"evidence\": \"Zebrafish overexpression Dvl-degradation assay, DFC-targeted morpholino knockdown, patterning analysis\",\n      \"pmids\": [\"20858476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism of Dvl degradation unresolved\", \"Link between ciliogenesis defect and beta-catenin not fully separated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Refined NKD1 as a passive antagonist whose activity manifests only when Wnt signaling is destabilized, clarifying it is a buffering rather than constitutive suppressor.\",\n      \"evidence\": \"Genetic epistasis in zebrafish Wnt/PCP mutants, Wnt8a overexpression, Nkd1 rescue\",\n      \"pmids\": [\"24009776\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of conditional/passive behavior not defined\", \"Single-lab epistasis\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed NKD1 dynamically in the pathway: ligand-triggered recruitment to the signalosome with Dvl2 followed by cytoplasmic relocation to block beta-catenin nuclear accumulation.\",\n      \"evidence\": \"Wnt-responsive zebrafish blastula cell assays, signalosome recruitment and beta-catenin localization assays\",\n      \"pmids\": [\"25904337\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Recruitment mechanism to signalosome not defined\", \"Direct beta-catenin binding not biochemically isolated\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified NKD1 as an FGFR-induced immediate-early gene that restrains Wnt during endoderm-to-hepatic-progenitor transition, linking it to lineage specification.\",\n      \"evidence\": \"FGFR inhibition, NKD1 knockdown in human iPSC differentiation, pharmacological Wnt-antagonist rescue\",\n      \"pmids\": [\"26637527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct FGFR-to-NKD1 transcriptional mechanism not mapped\", \"Whether Dvl binding mediates this hepatic role untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed a non-Wnt activity: NKD1 binds Rac1 and drives its proteasomal degradation affecting cytoskeleton and E-cadherin, within a Rac1-EZH2-NKD1 feedback loop.\",\n      \"evidence\": \"Co-IP, overexpression/knockdown, proteasome inhibitor assay, invasion and E-cadherin assays in HCC\",\n      \"pmids\": [\"27231134\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No identified E3 ligase mediating Rac1 degradation\", \"Single-lab; reciprocal loop mechanism incomplete\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Distinguished Nkd1 from Nkd2 mechanistically by showing Rnf25/AO7 disrupts the Nkd1-Axin inhibitory complex to positively regulate Wnt.\",\n      \"evidence\": \"Co-IP, Wnt target gene expression, zebrafish morpholino knockdown\",\n      \"pmids\": [\"27007149\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3-ligase-independent disruption mechanism unclear\", \"Stoichiometry of Nkd1-Axin complex undefined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed a pro-oncogenic Wnt-activating role in colon cancer: NKD1 promotes APC ubiquitination by restraining USP15, enhancing beta-catenin nuclear accumulation.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, USP15 interaction, luciferase reporter, loss/gain-of-function\",\n      \"pmids\": [\"36445120\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with NKD1's Wnt-antagonist role not addressed\", \"Direct E3 ligase for APC not identified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Demonstrated a nuclear transcriptional function: NKD1 binds the YWHAE promoter to activate transcription and promote glucose uptake in colon cancer.\",\n      \"evidence\": \"ChIP, dual-luciferase reporter, overexpression/knockout, immunofluorescence, glucose uptake assay\",\n      \"pmids\": [\"37202194\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No DNA-binding domain defined for NKD1\", \"Mechanism of nuclear translocation unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified PCM1 as an NKD1 interactor degraded via the ubiquitin-proteasome pathway, linking NKD1 to cell-cycle progression in colorectal cancer.\",\n      \"evidence\": \"Quantitative proteomics, Co-IP, immunofluorescence, siRNA, cell cycle analysis\",\n      \"pmids\": [\"37338734\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase mediating PCM1 degradation not identified\", \"Single-lab; reciprocal validation limited\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed Nkd1 downstream of Axin2 in the Wnt feedback hierarchy via rigorous double-mutant epistasis.\",\n      \"evidence\": \"CRISPR axin2/nkd1 double-mutant zebrafish, qRT-PCR, RNA-seq, mass spectrometry, Wnt sensitivity assays\",\n      \"pmids\": [\"38656801\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular link between Axin2 and Nkd1 not defined\", \"Does not address non-Wnt NKD1 functions\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established m6A control of NKD1: YTHDF3 suppresses NKD1 transcription and translation, derepressing Wnt/beta-catenin to promote HCC migration and invasion.\",\n      \"evidence\": \"RNA-seq, meRIP-seq, Lace-seq, Western blot, in vitro/in vivo functional assays\",\n      \"pmids\": [\"39127439\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific m6A sites on NKD1 transcript not finely mapped\", \"Single-lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined an EF-hand-dependent MYC-stabilizing axis: NKD1 binds MYC, blocks LC3B-mediated autophagic degradation, and promotes its nuclear entry, downstream of PPARdelta.\",\n      \"evidence\": \"Differential proteomics (SW620 vs nkd1-/-), Co-IP, autophagy analysis, EF-hand mutant, PPARdelta ChIP on NKD1 promoter\",\n      \"pmids\": [\"40675969\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How EF-hand binding blocks LC3B-MYC interaction structurally unresolved\", \"Reconciliation with NKD1 tumor-suppressive activity not addressed\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed Wnt3a induces NKD1 and triggers its membrane detachment, enabling direct MSX1 interaction and nuclear translocation to drive odontogenic gene activation and reparative dentin formation.\",\n      \"evidence\": \"scRNA-seq, SCENIC, CUT&Tag, co-localization, in vivo murine pulp exposure model, overexpression\",\n      \"pmids\": [\"41526338\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of NKD1 membrane detachment undefined\", \"Direct vs indirect NKD1-MSX1 binding not biochemically isolated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How NKD1 switches between Wnt-antagonist and Wnt/proliferation-promoting roles, and how its cytoplasmic-degradation and nuclear-transcriptional activities are coordinated, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of EF-hand engagement with distinct partners (MYC, beta-catenin, Dvl)\", \"No defined E3 ligase for NKD1-promoted degradation of Rac1/PCM1/APC\", \"Context determinants of antagonist vs oncogenic behavior undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 6, 9]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [11, 16]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [12, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [11, 16]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 16]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 6, 14]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [3, 10, 15]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 7, 16]}\n    ],\n    \"complexes\": [\"Wnt signalosome\"],\n    \"partners\": [\"DVL2\", \"RAC1\", \"PCM1\", \"APC\", \"MYC\", \"MSX1\", \"AXIN\", \"YWHAE\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}