{"gene":"CBLL1","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2002,"finding":"Hakai (CBLL1) is an E3 ubiquitin ligase that interacts with E-cadherin in a tyrosine phosphorylation-dependent manner, induces ubiquitination of the E-cadherin complex, and promotes its endocytosis, disrupting cell-cell contacts and enhancing cell motility. Hakai contains SH2, RING, zinc-finger and proline-rich domains.","method":"Modified yeast 2-hybrid, co-immunoprecipitation, ubiquitination assays, endocytosis assays, overexpression in epithelial cells","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — foundational paper with multiple orthogonal methods (Y2H, Co-IP, in vitro ubiquitination, endocytosis assay), widely replicated across subsequent studies","pmids":["11836526"],"is_preprint":false},{"year":2009,"finding":"Hakai interacts with PTB-associated splicing factor (PSF), an RNA-binding protein, and enhances PSF's ability to bind cancer-related mRNAs; knockdown of PSF suppresses Hakai-induced cell proliferation, placing Hakai upstream of PSF-mediated post-transcriptional regulation.","method":"Co-immunoprecipitation, cDNA arrays, siRNA knockdown, BrdU proliferation assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP interaction identified, functional RNA-binding consequence shown by arrays, epistasis confirmed by shRNA rescue, single lab","pmids":["19535458"],"is_preprint":false},{"year":2009,"finding":"Drosophila Hakai forms a complex with E-cadherin (Shotgun) in a manner distinct from the mammalian interaction; maternal Hakai mutants show stochastic loss of E-cadherin expression and reduction of aPKC, epithelial integrity defects, and defects in cell specification and migration, demonstrating essential in vivo roles beyond E-cadherin ubiquitination alone.","method":"Drosophila genetics (null mutants, maternal mutants), co-immunoprecipitation in S2 cells, immunostaining","journal":"Genes to cells : devoted to molecular & cellular mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic null and maternal mutant analysis with cellular phenotyping, Co-IP in cultured cells, single lab","pmids":["19682089"],"is_preprint":false},{"year":2010,"finding":"Hakai acts as a corepressor of estrogen receptor alpha (ERα) in breast cancer cells by binding directly to the DNA-binding domain of ERα and competing with coactivators SRC-1 and GRIP-1; this activity does not require Hakai's ubiquitin-ligase activity and results in inhibited ERα transcriptional activity, cell proliferation, and migration.","method":"Co-immunoprecipitation, reporter gene assays, domain mapping, overexpression/knockdown, proliferation assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with domain mapping, reporter assay for transcriptional activity, catalytically inactive mutant used to dissect mechanism, single lab","pmids":["20608937"],"is_preprint":false},{"year":2011,"finding":"Slit2/Robo1 signaling recruits Hakai to ubiquitinate E-cadherin, leading to its lysosomal degradation and EMT; knockdown of Hakai rescues E-cadherin levels and reverses EMT induced by Slit2/Robo1.","method":"siRNA knockdown, recombinant protein treatment, immunoblot, xenograft model","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — epistasis established by Hakai knockdown rescuing Slit2/Robo1-induced phenotype, in vivo xenograft validation, single lab","pmids":["21283129"],"is_preprint":false},{"year":2012,"finding":"Ubiquitination of the E-cadherin juxtamembrane domain (JMD) at lysines K5 and K83 by Hakai inhibits p120-catenin binding and targets E-cadherin for degradation; mutation of these lysines stabilizes JMD and allows p120-catenin binding, establishing competitive regulation between ubiquitination and p120-catenin association.","method":"Mitochondrial targeting of JMD, site-directed mutagenesis, co-immunoprecipitation, proteasome inhibitor treatment, immunofluorescence","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — mutagenesis of specific ubiquitination sites with functional consequences, orthogonal localization and interaction assays, single lab","pmids":["22693575"],"is_preprint":false},{"year":2012,"finding":"miR-203 post-transcriptionally represses Hakai expression by binding two sites in the Hakai mRNA 3'-UTR; inhibition of miR-203 elevates Hakai levels and increases cell proliferation in a Hakai-dependent manner.","method":"miRNA precursor/inhibitor overexpression, luciferase reporter assays, siRNA knockdown epistasis, BrdU incorporation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assay confirms direct 3'-UTR targeting, epistasis experiment (Hakai siRNA suppresses anti-miR-203 proliferative effect), single lab","pmids":["23285092"],"is_preprint":false},{"year":2014,"finding":"The Hakai phosphotyrosine-binding (HYB) domain forms an atypical zinc-coordinated tight homodimer essential for recognizing phosphotyrosine motifs of substrates including E-cadherin, cortactin, and DOK1; a C-terminal truncation mutant (HYB-ΔC) is monomeric but dimerizes upon phosphotyrosine substrate binding, demonstrating that the dimeric architecture is required for phosphotyrosine binding.","method":"NMR structure determination, isothermal titration calorimetry, analytical ultracentrifugation, size-exclusion chromatography, dynamic light scattering, circular dichroism","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with multiple orthogonal biophysical validation methods (ITC, AUC, SEC, DLS, CD) in one rigorous study","pmids":["25074933"],"is_preprint":false},{"year":2017,"finding":"Hakai stabilizes δ-catenin independently of its E3 ligase activity by stabilizing Src kinase; Src in turn inhibits binding between GSK-3β and δ-catenin, reducing proteasomal degradation of δ-catenin. Hakai and Src act synergistically to increase δ-catenin stability.","method":"Co-immunoprecipitation, overexpression/knockdown, immunoblot, pharmacological inhibition of Src","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP interactions, catalytically inactive mutant used, Src inhibitor epistasis, single lab","pmids":["28069439"],"is_preprint":false},{"year":2018,"finding":"Hakai interacts with Ajuba LIM domain protein via its HYB domain and induces Ajuba neddylation (not proteasomal ubiquitination), destabilizing Ajuba; this is blocked by neddylation inhibitor MLN4924 but not proteasome inhibitor MG132.","method":"Co-immunoprecipitation, confocal microscopy, siRNA knockdown, pharmacological inhibition (MLN4924, MG132), immunoblot","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP with domain mapping, neddylation versus proteasome dissection using specific inhibitors, single lab","pmids":["30041665"],"is_preprint":false},{"year":2020,"finding":"Hakai is a client protein of the HSP90 chaperone complex; pharmacological inhibition of Hsp90 with geldanamycin causes lysosome-dependent degradation of Hakai, accompanied by increased E-cadherin and Annexin A2 expression and suppressed cell motility.","method":"Co-immunoprecipitation, geldanamycin treatment, lysosome/proteasome inhibitor dissection, immunoblot, cell motility assays","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — Co-IP identifies Hsp90-Hakai interaction, lysosomal pathway confirmed by inhibitor panel, functional motility consequence shown, single lab","pmids":["31952268"],"is_preprint":false},{"year":2021,"finding":"Drosophila Hakai colocalizes and physically interacts with other m6A writer complex components (Vir, Fl(2)d, Flacc); Hakai mutants display reduced m6A levels in mRNA, aberrant Sxl alternative splicing, and wing/behavior defects. Disruption of Hakai, Vir, or Fl(2)d causes degradation of the other complex components, indicating mutual stabilization. m6A modification is deposited on Sxl mRNA in a sex-specific, writer-dependent manner.","method":"Drosophila genetics (Hakai mutants), co-immunoprecipitation, MeRIP-seq, RT-PCR for alternative splicing, behavioral assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — Drosophila genetic loss-of-function with transcriptome-wide m6A mapping (MeRIP-seq), Co-IP of complex, multiple phenotypic readouts, published in high-impact journal","pmids":["33846330"],"is_preprint":false},{"year":2022,"finding":"Hakai interacts with Fatty Acid Synthase (FASN) via interactome analysis and induces FASN ubiquitination and lysosomal degradation, thereby regulating FASN-mediated lipid accumulation; inverse expression of FASN and Hakai was observed in an inflammatory AOM/DSS mouse model.","method":"Hakai interactome/proteomic analysis, co-immunoprecipitation, ubiquitination assay, lysosome inhibitor treatment, immunohistochemistry in mouse colitis model","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — interactome identification followed by Co-IP and functional ubiquitination assay, in vivo model validation, single lab","pmids":["36266428"],"is_preprint":false},{"year":2024,"finding":"Hakai physically interacts with Runx2 transcription factor and rescues it from Smurf2-mediated proteasomal degradation by inducing proteasome-dependent degradation of Smurf2 itself; catalytically inactive Hakai-C109A mutant has minimal effect, indicating dependence on E3 ligase activity. This promotes osteoblast differentiation.","method":"Affinity pulldown-based proteomics, co-immunoprecipitation, overexpression of wild-type vs. C109A mutant Hakai, shRNA knockdown, osteoblast differentiation assays in vitro and in vivo (ovariectomized rat model)","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics-based identification confirmed by Co-IP, catalytic mutant dissects mechanism, in vivo model, single lab","pmids":["39034451"],"is_preprint":false},{"year":2025,"finding":"CBLL1/Hakai is identified as a substrate of UBE3C ubiquitin ligase; the UBE3C-Cbll1 axis drives N6-methyladenosine (m6A) mRNA methylation in neural progenitors, and hyperactivation of m6A writers in UBE3C-deficient forebrains impairs cell cycle exit during cortical neurogenesis.","method":"Proteomic profiling of UBE3C-deficient mouse forebrains and human brain organoids, genetic complementation, METTL3 inhibitor (STM2457) rescue in vivo","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic identification of Cbll1 as UBE3C substrate, in vivo genetic and pharmacological rescue, preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"Hakai interacts with LRP4 (a negative regulator of Wnt/β-catenin signaling), promotes LRP4 ubiquitination and degradation, and thereby induces hyperactivation of Wnt/β-catenin signaling; pharmacological inhibition of Hakai's HYB domain with Hakin-1 restores LRP4 levels and attenuates Wnt/β-catenin activity in colorectal cancer tumourspheres.","method":"Co-immunoprecipitation, ubiquitination assay, TOPFlash Wnt reporter assay, inducible shRNA knockdown, immunofluorescence, Western blot, RT-qPCR, Hakin-1 inhibitor treatment","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP confirms interaction, ubiquitination assay and reporter assay show functional consequence, Hakin-1 pharmacological validation, preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2010,"finding":"CBLL1 knockdown using specific siRNAs strongly inhibits endocytosis of Listeria monocytogenes, confirming a role for CBLL1 in bacterial invasion; however, the same CBLL1 knockdown does not impair WNV, dengue virus, or yellow fever virus infection, indicating CBLL1 is not required for flavivirus entry.","method":"siRNA knockdown, viral infection assays, bacterial internalization assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple siRNAs tested, both bacterial and viral infection assays performed, functional negative result for flavivirus robustly established, single lab","pmids":["21191016"],"is_preprint":false}],"current_model":"CBLL1/Hakai is an E3 ubiquitin ligase with an atypical dimeric phosphotyrosine-binding (HYB) domain that recognizes tyrosine-phosphorylated substrates including E-cadherin, promoting their ubiquitination and lysosomal or proteasomal degradation to drive epithelial-mesenchymal transition; it also functions as an essential structural component of the m6A mRNA writer complex (interacting with METTL3/METTL14 complex partners), acts as a corepressor of ERα, stabilizes δ-catenin via Src, ubiquitinates FASN and LRP4, and is itself regulated by HSP90 (for stability), miR-203 (post-transcriptional repression), and UBE3C (ubiquitination)."},"narrative":{"mechanistic_narrative":"CBLL1/Hakai is a RING-type E3 ubiquitin ligase that controls epithelial cell adhesion and motility by recognizing tyrosine-phosphorylated substrates and targeting them for degradation [PMID:11836526]. It binds the E-cadherin complex in a phosphotyrosine-dependent manner, ubiquitinates the E-cadherin juxtamembrane domain at lysines K5 and K83 to block p120-catenin association, and drives E-cadherin endocytosis and lysosomal degradation, thereby disrupting cell-cell contacts and promoting epithelial-mesenchymal transition downstream of Slit2/Robo1 signaling [PMID:11836526, PMID:21283129, PMID:22693575]. Substrate recognition is mediated by an atypical dimeric phosphotyrosine-binding (HYB) domain whose zinc-coordinated homodimerization is required to engage phosphotyrosine motifs of substrates such as E-cadherin, cortactin, and DOK1 [PMID:25074933]. Beyond cadherin turnover, Hakai is an essential, mutually stabilized structural component of the m6A mRNA methyltransferase writer complex, where it associates with other writer subunits and is required for normal m6A deposition and downstream sex-specific alternative splicing [PMID:33846330]. Hakai also acts independently of its ligase activity as an ERα corepressor that competes with coactivators SRC-1 and GRIP-1 [PMID:20608937] and stabilizes δ-catenin through Src [PMID:28069439], while extending its catalytic activity to additional substrates including FASN (lysosomal degradation, lipid regulation) [PMID:36266428] and the Runx2 stabilizer Smurf2 (promoting osteoblast differentiation) [PMID:39034451]. Its own abundance is set post-transcriptionally by miR-203 [PMID:23285092] and by HSP90 chaperone-dependent stabilization [PMID:31952268].","teleology":[{"year":2002,"claim":"Established Hakai as an E3 ligase that couples tyrosine phosphorylation of E-cadherin to its ubiquitination and endocytosis, providing a mechanism for dynamic regulation of cell-cell adhesion.","evidence":"Modified yeast two-hybrid, Co-IP, in vitro ubiquitination, and endocytosis assays in epithelial cells","pmids":["11836526"],"confidence":"High","gaps":["Did not resolve the structural basis of phosphotyrosine recognition","Degradation route (lysosomal vs proteasomal) not fully dissected"]},{"year":2009,"claim":"Extended Hakai's reach beyond adhesion by linking it to post-transcriptional RNA regulation via the splicing factor PSF, hinting at a non-cadherin proliferative function.","evidence":"Co-IP, cDNA arrays, siRNA knockdown and BrdU proliferation epistasis","pmids":["19535458"],"confidence":"Medium","gaps":["Direct vs indirect effect on RNA binding not separated","Single-lab finding"]},{"year":2009,"claim":"Drosophila genetics demonstrated essential in vivo roles for Hakai in epithelial integrity and cell specification that exceed simple E-cadherin ubiquitination.","evidence":"Null and maternal mutant analysis, S2-cell Co-IP, immunostaining","pmids":["19682089"],"confidence":"Medium","gaps":["Molecular basis of the non-cadherin phenotypes unresolved","Relationship to later-defined m6A role not yet appreciated"]},{"year":2010,"claim":"Revealed a ligase-independent transcriptional role for Hakai as an ERα corepressor, showing it acts through protein-protein competition as well as catalysis.","evidence":"Co-IP with domain mapping, reporter assays, catalytically inactive mutant","pmids":["20608937"],"confidence":"Medium","gaps":["Genomic occupancy of Hakai at ERα targets not mapped","Single-lab finding"]},{"year":2010,"claim":"Distinguished Hakai's requirement for bacterial endocytosis from flavivirus entry, defining the specificity of its membrane-trafficking contribution.","evidence":"Multiple siRNAs with bacterial internalization and viral infection assays","pmids":["21191016"],"confidence":"Medium","gaps":["Substrate or trafficking step mediating Listeria entry not identified"]},{"year":2011,"claim":"Placed Hakai downstream of Slit2/Robo1 signaling as the effector routing E-cadherin to lysosomal degradation during EMT.","evidence":"siRNA knockdown rescue, recombinant ligand treatment, xenograft model","pmids":["21283129"],"confidence":"Medium","gaps":["How Robo1 signaling recruits/activates Hakai mechanistically unknown"]},{"year":2012,"claim":"Mapped the specific E-cadherin lysines ubiquitinated by Hakai and showed ubiquitination directly competes with p120-catenin binding.","evidence":"Mitochondrial JMD targeting, site-directed mutagenesis, Co-IP, proteasome inhibition","pmids":["22693575"],"confidence":"Medium","gaps":["Polyubiquitin chain topology not defined"]},{"year":2012,"claim":"Identified miR-203 as a direct post-transcriptional repressor of Hakai, defining an upstream control on its proliferative output.","evidence":"Luciferase 3'-UTR reporter, miRNA mimic/inhibitor, siRNA epistasis, BrdU assays","pmids":["23285092"],"confidence":"Medium","gaps":["Physiological contexts where miR-203 regulates Hakai not established"]},{"year":2014,"claim":"Solved the structural basis of substrate recognition, showing the HYB domain must homodimerize via zinc coordination to bind phosphotyrosine motifs.","evidence":"NMR structure with ITC, AUC, SEC, DLS, and CD validation","pmids":["25074933"],"confidence":"High","gaps":["Full-length ligase architecture and catalytic coupling not resolved"]},{"year":2017,"claim":"Showed Hakai can stabilize rather than degrade a partner (δ-catenin) by acting through Src, expanding its repertoire beyond destructive ubiquitination.","evidence":"Co-IP, catalytically inactive mutant, Src pharmacological inhibition","pmids":["28069439"],"confidence":"Medium","gaps":["Whether Src is a direct Hakai target unclear","Single-lab finding"]},{"year":2018,"claim":"Demonstrated Hakai can route a substrate to neddylation rather than proteasomal ubiquitination, diversifying its modification outputs.","evidence":"Co-IP, domain mapping, MLN4924 vs MG132 inhibitor dissection","pmids":["30041665"],"confidence":"Medium","gaps":["Mechanism by which Hakai promotes neddylation not defined"]},{"year":2021,"claim":"Established Hakai as an essential, mutually stabilizing subunit of the m6A writer complex, defining a non-ligase structural role in mRNA methylation and splicing control.","evidence":"Drosophila mutants, Co-IP of writer components, MeRIP-seq, splicing RT-PCR, behavioral assays","pmids":["33846330"],"confidence":"High","gaps":["Precise structural contribution of Hakai within the writer complex unresolved","Generality to mammalian writer complex not directly shown here"]},{"year":2020,"claim":"Identified HSP90 as a chaperone that maintains Hakai stability, linking proteostasis to E-cadherin and motility outcomes.","evidence":"Co-IP, geldanamycin treatment, lysosome/proteasome inhibitor dissection, motility assays","pmids":["31952268"],"confidence":"Medium","gaps":["Co-chaperones and recognition determinants not identified"]},{"year":2022,"claim":"Expanded the catalytic substrate set to FASN, connecting Hakai to lysosomal control of lipid metabolism in an inflammatory model.","evidence":"Interactome analysis, Co-IP, ubiquitination assay, lysosome inhibition, IHC in AOM/DSS mice","pmids":["36266428"],"confidence":"Medium","gaps":["Ubiquitin linkage and physiological lipid impact not fully defined"]},{"year":2024,"claim":"Showed Hakai stabilizes Runx2 by degrading its antagonist Smurf2 in a catalysis-dependent manner, demonstrating indirect substrate stabilization driving osteoblast differentiation.","evidence":"Affinity proteomics, Co-IP, WT vs C109A mutant, shRNA, in vitro/in vivo differentiation (ovariectomized rat)","pmids":["39034451"],"confidence":"Medium","gaps":["Direct Smurf2 ubiquitination by Hakai not shown biochemically"]},{"year":2025,"claim":"Positioned Hakai within a UBE3C-controlled axis governing m6A levels during cortical neurogenesis, linking its turnover to brain development.","evidence":"Proteomics of UBE3C-deficient mouse forebrain and human organoids, genetic complementation, METTL3-inhibitor rescue (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","Direct UBE3C-mediated ubiquitination of Hakai not biochemically reconstituted"]},{"year":2025,"claim":"Implicated Hakai-mediated LRP4 degradation in Wnt/β-catenin hyperactivation and validated HYB-domain inhibition as a pharmacological strategy in colorectal cancer.","evidence":"Co-IP, ubiquitination assay, TOPFlash reporter, inducible shRNA, Hakin-1 HYB inhibitor (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint not yet peer-reviewed","LRP4 ubiquitin linkage and selectivity not defined"]},{"year":null,"claim":"How Hakai's distinct activities — catalytic phosphotyrosine-dependent ubiquitination versus its structural role in the m6A writer and its ligase-independent transcriptional/stabilizing functions — are partitioned and coordinated within the cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of full-length Hakai engaged with the writer complex","Determinants selecting degradative vs stabilizing vs neddylation outcomes unknown","Mammalian counterparts of Drosophila m6A findings not directly demonstrated in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,5,12,13]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,5,9,12]},{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[11]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[3]}],"localization":[],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,5,12]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[11]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,13]}],"complexes":["m6A mRNA writer complex"],"partners":["CDH1","SRC","FASN","LRP4","RUNX2","AJUBA","UBE3C"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q75N03","full_name":"E3 ubiquitin-protein ligase Hakai","aliases":["Casitas B-lineage lymphoma-transforming sequence-like protein 1","c-Cbl-like protein 1","RING finger protein 188","RING-type E3 ubiquitin transferase Hakai"],"length_aa":491,"mass_kda":54.5,"function":"E3 ubiquitin-protein ligase that mediates ubiquitination of several tyrosine-phosphorylated Src substrates, including CDH1, CTTN and DOK1 (By similarity). Targets CDH1 for endocytosis and degradation (By similarity). Associated component of the WMM complex, a complex that mediates N6-methyladenosine (m6A) methylation of RNAs, a modification that plays a role in the efficiency of mRNA splicing and RNA processing (PubMed:29507755). Its function in the WMM complex is unknown (PubMed:29507755)","subcellular_location":"Nucleus speckle; Nucleus, nucleoplasm; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q75N03/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CBLL1","classification":"Not Classified","n_dependent_lines":442,"n_total_lines":1208,"dependency_fraction":0.3658940397350993},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CBLL1","total_profiled":1310},"omim":[{"mim_id":"616453","title":"ZINC FINGER CCCH DOMAIN-CONTAINING PROTEIN 13; ZC3H13","url":"https://www.omim.org/entry/616453"},{"mim_id":"616447","title":"VIR-LIKE M6A METHYLTRANSFERASE-ASSOCIATED PROTEIN; VIRMA","url":"https://www.omim.org/entry/616447"},{"mim_id":"612588","title":"BCL2-ASSOCIATED TRANSCRIPTION FACTOR 1; BCLAF1","url":"https://www.omim.org/entry/612588"},{"mim_id":"606872","title":"CAS-BR-M MURINE ECTOPIC RETROVIRAL TRANSFORMING SEQUENCE LIKE-1; CBLL1","url":"https://www.omim.org/entry/606872"},{"mim_id":"606077","title":"RNA-BINDING MOTIF PROTEIN 15; RBM15","url":"https://www.omim.org/entry/606077"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear speckles","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CBLL1"},"hgnc":{"alias_symbol":["HAKAI","FLJ23109","RNF188"],"prev_symbol":[]},"alphafold":{"accession":"Q75N03","domains":[{"cath_id":"3.30.40.10","chopping":"118-189","consensus_level":"medium","plddt":92.1642,"start":118,"end":189}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q75N03","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q75N03-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q75N03-F1-predicted_aligned_error_v6.png","plddt_mean":56.59},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CBLL1","jax_strain_url":"https://www.jax.org/strain/search?query=CBLL1"},"sequence":{"accession":"Q75N03","fasta_url":"https://rest.uniprot.org/uniprotkb/Q75N03.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q75N03/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q75N03"}},"corpus_meta":[{"pmid":"11836526","id":"PMC_11836526","title":"Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex.","date":"2002","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11836526","citation_count":700,"is_preprint":false},{"pmid":"21283129","id":"PMC_21283129","title":"Slit-Robo signaling induces malignant transformation through Hakai-mediated E-cadherin degradation during colorectal epithelial cell carcinogenesis.","date":"2011","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/21283129","citation_count":125,"is_preprint":false},{"pmid":"21191016","id":"PMC_21191016","title":"Appraising the roles of CBLL1 and the ubiquitin/proteasome system for flavivirus entry and replication.","date":"2010","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/21191016","citation_count":64,"is_preprint":false},{"pmid":"19535458","id":"PMC_19535458","title":"Novel roles of hakai in cell proliferation and oncogenesis.","date":"2009","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/19535458","citation_count":63,"is_preprint":false},{"pmid":"33846330","id":"PMC_33846330","title":"Role of Hakai in m6A modification pathway in Drosophila.","date":"2021","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/33846330","citation_count":51,"is_preprint":false},{"pmid":"32190002","id":"PMC_32190002","title":"Circ_0072083 interference enhances growth-inhibiting effects of cisplatin in non-small-cell lung cancer cells via miR-545-3p/CBLL1 axis.","date":"2020","source":"Cancer cell international","url":"https://pubmed.ncbi.nlm.nih.gov/32190002","citation_count":49,"is_preprint":false},{"pmid":"30041665","id":"PMC_30041665","title":"Ajuba inhibits hepatocellular carcinoma cell growth via targeting of β-catenin and YAP signaling and is regulated by E3 ligase Hakai through neddylation.","date":"2018","source":"Journal of experimental & clinical cancer research : 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antagonizes plant m6A modification by promoting the autophagic degradation of the m6A writer HAKAI.","date":"2023","source":"aBIOTECH","url":"https://pubmed.ncbi.nlm.nih.gov/37581026","citation_count":31,"is_preprint":false},{"pmid":"19682089","id":"PMC_19682089","title":"Essential requirement for RING finger E3 ubiquitin ligase Hakai in early embryonic development of Drosophila.","date":"2009","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/19682089","citation_count":29,"is_preprint":false},{"pmid":"31124298","id":"PMC_31124298","title":"CBLL1 is highly expressed in non-small cell lung cancer and promotes cell proliferation and invasion.","date":"2019","source":"Thoracic cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31124298","citation_count":27,"is_preprint":false},{"pmid":"29472634","id":"PMC_29472634","title":"Hakai overexpression effectively induces tumour progression and metastasis in 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Hakai contains SH2, RING, zinc-finger and proline-rich domains.\",\n      \"method\": \"Modified yeast 2-hybrid, co-immunoprecipitation, ubiquitination assays, endocytosis assays, overexpression in epithelial cells\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — foundational paper with multiple orthogonal methods (Y2H, Co-IP, in vitro ubiquitination, endocytosis assay), widely replicated across subsequent studies\",\n      \"pmids\": [\"11836526\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Hakai interacts with PTB-associated splicing factor (PSF), an RNA-binding protein, and enhances PSF's ability to bind cancer-related mRNAs; knockdown of PSF suppresses Hakai-induced cell proliferation, placing Hakai upstream of PSF-mediated post-transcriptional regulation.\",\n      \"method\": \"Co-immunoprecipitation, cDNA arrays, siRNA knockdown, BrdU proliferation assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP interaction identified, functional RNA-binding consequence shown by arrays, epistasis confirmed by shRNA rescue, single lab\",\n      \"pmids\": [\"19535458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Drosophila Hakai forms a complex with E-cadherin (Shotgun) in a manner distinct from the mammalian interaction; maternal Hakai mutants show stochastic loss of E-cadherin expression and reduction of aPKC, epithelial integrity defects, and defects in cell specification and migration, demonstrating essential in vivo roles beyond E-cadherin ubiquitination alone.\",\n      \"method\": \"Drosophila genetics (null mutants, maternal mutants), co-immunoprecipitation in S2 cells, immunostaining\",\n      \"journal\": \"Genes to cells : devoted to molecular & cellular mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic null and maternal mutant analysis with cellular phenotyping, Co-IP in cultured cells, single lab\",\n      \"pmids\": [\"19682089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Hakai acts as a corepressor of estrogen receptor alpha (ERα) in breast cancer cells by binding directly to the DNA-binding domain of ERα and competing with coactivators SRC-1 and GRIP-1; this activity does not require Hakai's ubiquitin-ligase activity and results in inhibited ERα transcriptional activity, cell proliferation, and migration.\",\n      \"method\": \"Co-immunoprecipitation, reporter gene assays, domain mapping, overexpression/knockdown, proliferation assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with domain mapping, reporter assay for transcriptional activity, catalytically inactive mutant used to dissect mechanism, single lab\",\n      \"pmids\": [\"20608937\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Slit2/Robo1 signaling recruits Hakai to ubiquitinate E-cadherin, leading to its lysosomal degradation and EMT; knockdown of Hakai rescues E-cadherin levels and reverses EMT induced by Slit2/Robo1.\",\n      \"method\": \"siRNA knockdown, recombinant protein treatment, immunoblot, xenograft model\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — epistasis established by Hakai knockdown rescuing Slit2/Robo1-induced phenotype, in vivo xenograft validation, single lab\",\n      \"pmids\": [\"21283129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ubiquitination of the E-cadherin juxtamembrane domain (JMD) at lysines K5 and K83 by Hakai inhibits p120-catenin binding and targets E-cadherin for degradation; mutation of these lysines stabilizes JMD and allows p120-catenin binding, establishing competitive regulation between ubiquitination and p120-catenin association.\",\n      \"method\": \"Mitochondrial targeting of JMD, site-directed mutagenesis, co-immunoprecipitation, proteasome inhibitor treatment, immunofluorescence\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — mutagenesis of specific ubiquitination sites with functional consequences, orthogonal localization and interaction assays, single lab\",\n      \"pmids\": [\"22693575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"miR-203 post-transcriptionally represses Hakai expression by binding two sites in the Hakai mRNA 3'-UTR; inhibition of miR-203 elevates Hakai levels and increases cell proliferation in a Hakai-dependent manner.\",\n      \"method\": \"miRNA precursor/inhibitor overexpression, luciferase reporter assays, siRNA knockdown epistasis, BrdU incorporation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assay confirms direct 3'-UTR targeting, epistasis experiment (Hakai siRNA suppresses anti-miR-203 proliferative effect), single lab\",\n      \"pmids\": [\"23285092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The Hakai phosphotyrosine-binding (HYB) domain forms an atypical zinc-coordinated tight homodimer essential for recognizing phosphotyrosine motifs of substrates including E-cadherin, cortactin, and DOK1; a C-terminal truncation mutant (HYB-ΔC) is monomeric but dimerizes upon phosphotyrosine substrate binding, demonstrating that the dimeric architecture is required for phosphotyrosine binding.\",\n      \"method\": \"NMR structure determination, isothermal titration calorimetry, analytical ultracentrifugation, size-exclusion chromatography, dynamic light scattering, circular dichroism\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with multiple orthogonal biophysical validation methods (ITC, AUC, SEC, DLS, CD) in one rigorous study\",\n      \"pmids\": [\"25074933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Hakai stabilizes δ-catenin independently of its E3 ligase activity by stabilizing Src kinase; Src in turn inhibits binding between GSK-3β and δ-catenin, reducing proteasomal degradation of δ-catenin. Hakai and Src act synergistically to increase δ-catenin stability.\",\n      \"method\": \"Co-immunoprecipitation, overexpression/knockdown, immunoblot, pharmacological inhibition of Src\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP interactions, catalytically inactive mutant used, Src inhibitor epistasis, single lab\",\n      \"pmids\": [\"28069439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Hakai interacts with Ajuba LIM domain protein via its HYB domain and induces Ajuba neddylation (not proteasomal ubiquitination), destabilizing Ajuba; this is blocked by neddylation inhibitor MLN4924 but not proteasome inhibitor MG132.\",\n      \"method\": \"Co-immunoprecipitation, confocal microscopy, siRNA knockdown, pharmacological inhibition (MLN4924, MG132), immunoblot\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP with domain mapping, neddylation versus proteasome dissection using specific inhibitors, single lab\",\n      \"pmids\": [\"30041665\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Hakai is a client protein of the HSP90 chaperone complex; pharmacological inhibition of Hsp90 with geldanamycin causes lysosome-dependent degradation of Hakai, accompanied by increased E-cadherin and Annexin A2 expression and suppressed cell motility.\",\n      \"method\": \"Co-immunoprecipitation, geldanamycin treatment, lysosome/proteasome inhibitor dissection, immunoblot, cell motility assays\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — Co-IP identifies Hsp90-Hakai interaction, lysosomal pathway confirmed by inhibitor panel, functional motility consequence shown, single lab\",\n      \"pmids\": [\"31952268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Drosophila Hakai colocalizes and physically interacts with other m6A writer complex components (Vir, Fl(2)d, Flacc); Hakai mutants display reduced m6A levels in mRNA, aberrant Sxl alternative splicing, and wing/behavior defects. Disruption of Hakai, Vir, or Fl(2)d causes degradation of the other complex components, indicating mutual stabilization. m6A modification is deposited on Sxl mRNA in a sex-specific, writer-dependent manner.\",\n      \"method\": \"Drosophila genetics (Hakai mutants), co-immunoprecipitation, MeRIP-seq, RT-PCR for alternative splicing, behavioral assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — Drosophila genetic loss-of-function with transcriptome-wide m6A mapping (MeRIP-seq), Co-IP of complex, multiple phenotypic readouts, published in high-impact journal\",\n      \"pmids\": [\"33846330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Hakai interacts with Fatty Acid Synthase (FASN) via interactome analysis and induces FASN ubiquitination and lysosomal degradation, thereby regulating FASN-mediated lipid accumulation; inverse expression of FASN and Hakai was observed in an inflammatory AOM/DSS mouse model.\",\n      \"method\": \"Hakai interactome/proteomic analysis, co-immunoprecipitation, ubiquitination assay, lysosome inhibitor treatment, immunohistochemistry in mouse colitis model\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — interactome identification followed by Co-IP and functional ubiquitination assay, in vivo model validation, single lab\",\n      \"pmids\": [\"36266428\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Hakai physically interacts with Runx2 transcription factor and rescues it from Smurf2-mediated proteasomal degradation by inducing proteasome-dependent degradation of Smurf2 itself; catalytically inactive Hakai-C109A mutant has minimal effect, indicating dependence on E3 ligase activity. This promotes osteoblast differentiation.\",\n      \"method\": \"Affinity pulldown-based proteomics, co-immunoprecipitation, overexpression of wild-type vs. C109A mutant Hakai, shRNA knockdown, osteoblast differentiation assays in vitro and in vivo (ovariectomized rat model)\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics-based identification confirmed by Co-IP, catalytic mutant dissects mechanism, in vivo model, single lab\",\n      \"pmids\": [\"39034451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CBLL1/Hakai is identified as a substrate of UBE3C ubiquitin ligase; the UBE3C-Cbll1 axis drives N6-methyladenosine (m6A) mRNA methylation in neural progenitors, and hyperactivation of m6A writers in UBE3C-deficient forebrains impairs cell cycle exit during cortical neurogenesis.\",\n      \"method\": \"Proteomic profiling of UBE3C-deficient mouse forebrains and human brain organoids, genetic complementation, METTL3 inhibitor (STM2457) rescue in vivo\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic identification of Cbll1 as UBE3C substrate, in vivo genetic and pharmacological rescue, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Hakai interacts with LRP4 (a negative regulator of Wnt/β-catenin signaling), promotes LRP4 ubiquitination and degradation, and thereby induces hyperactivation of Wnt/β-catenin signaling; pharmacological inhibition of Hakai's HYB domain with Hakin-1 restores LRP4 levels and attenuates Wnt/β-catenin activity in colorectal cancer tumourspheres.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, TOPFlash Wnt reporter assay, inducible shRNA knockdown, immunofluorescence, Western blot, RT-qPCR, Hakin-1 inhibitor treatment\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP confirms interaction, ubiquitination assay and reporter assay show functional consequence, Hakin-1 pharmacological validation, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CBLL1 knockdown using specific siRNAs strongly inhibits endocytosis of Listeria monocytogenes, confirming a role for CBLL1 in bacterial invasion; however, the same CBLL1 knockdown does not impair WNV, dengue virus, or yellow fever virus infection, indicating CBLL1 is not required for flavivirus entry.\",\n      \"method\": \"siRNA knockdown, viral infection assays, bacterial internalization assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple siRNAs tested, both bacterial and viral infection assays performed, functional negative result for flavivirus robustly established, single lab\",\n      \"pmids\": [\"21191016\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CBLL1/Hakai is an E3 ubiquitin ligase with an atypical dimeric phosphotyrosine-binding (HYB) domain that recognizes tyrosine-phosphorylated substrates including E-cadherin, promoting their ubiquitination and lysosomal or proteasomal degradation to drive epithelial-mesenchymal transition; it also functions as an essential structural component of the m6A mRNA writer complex (interacting with METTL3/METTL14 complex partners), acts as a corepressor of ERα, stabilizes δ-catenin via Src, ubiquitinates FASN and LRP4, and is itself regulated by HSP90 (for stability), miR-203 (post-transcriptional repression), and UBE3C (ubiquitination).\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CBLL1/Hakai is a RING-type E3 ubiquitin ligase that controls epithelial cell adhesion and motility by recognizing tyrosine-phosphorylated substrates and targeting them for degradation [#0]. It binds the E-cadherin complex in a phosphotyrosine-dependent manner, ubiquitinates the E-cadherin juxtamembrane domain at lysines K5 and K83 to block p120-catenin association, and drives E-cadherin endocytosis and lysosomal degradation, thereby disrupting cell-cell contacts and promoting epithelial-mesenchymal transition downstream of Slit2/Robo1 signaling [#0, #4, #5]. Substrate recognition is mediated by an atypical dimeric phosphotyrosine-binding (HYB) domain whose zinc-coordinated homodimerization is required to engage phosphotyrosine motifs of substrates such as E-cadherin, cortactin, and DOK1 [#7]. Beyond cadherin turnover, Hakai is an essential, mutually stabilized structural component of the m6A mRNA methyltransferase writer complex, where it associates with other writer subunits and is required for normal m6A deposition and downstream sex-specific alternative splicing [#11]. Hakai also acts independently of its ligase activity as an ERα corepressor that competes with coactivators SRC-1 and GRIP-1 [#3] and stabilizes δ-catenin through Src [#8], while extending its catalytic activity to additional substrates including FASN (lysosomal degradation, lipid regulation) [#12] and the Runx2 stabilizer Smurf2 (promoting osteoblast differentiation) [#13]. Its own abundance is set post-transcriptionally by miR-203 [#6] and by HSP90 chaperone-dependent stabilization [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established Hakai as an E3 ligase that couples tyrosine phosphorylation of E-cadherin to its ubiquitination and endocytosis, providing a mechanism for dynamic regulation of cell-cell adhesion.\",\n      \"evidence\": \"Modified yeast two-hybrid, Co-IP, in vitro ubiquitination, and endocytosis assays in epithelial cells\",\n      \"pmids\": [\"11836526\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of phosphotyrosine recognition\", \"Degradation route (lysosomal vs proteasomal) not fully dissected\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Extended Hakai's reach beyond adhesion by linking it to post-transcriptional RNA regulation via the splicing factor PSF, hinting at a non-cadherin proliferative function.\",\n      \"evidence\": \"Co-IP, cDNA arrays, siRNA knockdown and BrdU proliferation epistasis\",\n      \"pmids\": [\"19535458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect effect on RNA binding not separated\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Drosophila genetics demonstrated essential in vivo roles for Hakai in epithelial integrity and cell specification that exceed simple E-cadherin ubiquitination.\",\n      \"evidence\": \"Null and maternal mutant analysis, S2-cell Co-IP, immunostaining\",\n      \"pmids\": [\"19682089\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of the non-cadherin phenotypes unresolved\", \"Relationship to later-defined m6A role not yet appreciated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Revealed a ligase-independent transcriptional role for Hakai as an ERα corepressor, showing it acts through protein-protein competition as well as catalysis.\",\n      \"evidence\": \"Co-IP with domain mapping, reporter assays, catalytically inactive mutant\",\n      \"pmids\": [\"20608937\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genomic occupancy of Hakai at ERα targets not mapped\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Distinguished Hakai's requirement for bacterial endocytosis from flavivirus entry, defining the specificity of its membrane-trafficking contribution.\",\n      \"evidence\": \"Multiple siRNAs with bacterial internalization and viral infection assays\",\n      \"pmids\": [\"21191016\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Substrate or trafficking step mediating Listeria entry not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Placed Hakai downstream of Slit2/Robo1 signaling as the effector routing E-cadherin to lysosomal degradation during EMT.\",\n      \"evidence\": \"siRNA knockdown rescue, recombinant ligand treatment, xenograft model\",\n      \"pmids\": [\"21283129\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How Robo1 signaling recruits/activates Hakai mechanistically unknown\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Mapped the specific E-cadherin lysines ubiquitinated by Hakai and showed ubiquitination directly competes with p120-catenin binding.\",\n      \"evidence\": \"Mitochondrial JMD targeting, site-directed mutagenesis, Co-IP, proteasome inhibition\",\n      \"pmids\": [\"22693575\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Polyubiquitin chain topology not defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified miR-203 as a direct post-transcriptional repressor of Hakai, defining an upstream control on its proliferative output.\",\n      \"evidence\": \"Luciferase 3'-UTR reporter, miRNA mimic/inhibitor, siRNA epistasis, BrdU assays\",\n      \"pmids\": [\"23285092\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological contexts where miR-203 regulates Hakai not established\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Solved the structural basis of substrate recognition, showing the HYB domain must homodimerize via zinc coordination to bind phosphotyrosine motifs.\",\n      \"evidence\": \"NMR structure with ITC, AUC, SEC, DLS, and CD validation\",\n      \"pmids\": [\"25074933\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length ligase architecture and catalytic coupling not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed Hakai can stabilize rather than degrade a partner (δ-catenin) by acting through Src, expanding its repertoire beyond destructive ubiquitination.\",\n      \"evidence\": \"Co-IP, catalytically inactive mutant, Src pharmacological inhibition\",\n      \"pmids\": [\"28069439\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Src is a direct Hakai target unclear\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated Hakai can route a substrate to neddylation rather than proteasomal ubiquitination, diversifying its modification outputs.\",\n      \"evidence\": \"Co-IP, domain mapping, MLN4924 vs MG132 inhibitor dissection\",\n      \"pmids\": [\"30041665\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which Hakai promotes neddylation not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established Hakai as an essential, mutually stabilizing subunit of the m6A writer complex, defining a non-ligase structural role in mRNA methylation and splicing control.\",\n      \"evidence\": \"Drosophila mutants, Co-IP of writer components, MeRIP-seq, splicing RT-PCR, behavioral assays\",\n      \"pmids\": [\"33846330\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise structural contribution of Hakai within the writer complex unresolved\", \"Generality to mammalian writer complex not directly shown here\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified HSP90 as a chaperone that maintains Hakai stability, linking proteostasis to E-cadherin and motility outcomes.\",\n      \"evidence\": \"Co-IP, geldanamycin treatment, lysosome/proteasome inhibitor dissection, motility assays\",\n      \"pmids\": [\"31952268\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Co-chaperones and recognition determinants not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Expanded the catalytic substrate set to FASN, connecting Hakai to lysosomal control of lipid metabolism in an inflammatory model.\",\n      \"evidence\": \"Interactome analysis, Co-IP, ubiquitination assay, lysosome inhibition, IHC in AOM/DSS mice\",\n      \"pmids\": [\"36266428\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin linkage and physiological lipid impact not fully defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed Hakai stabilizes Runx2 by degrading its antagonist Smurf2 in a catalysis-dependent manner, demonstrating indirect substrate stabilization driving osteoblast differentiation.\",\n      \"evidence\": \"Affinity proteomics, Co-IP, WT vs C109A mutant, shRNA, in vitro/in vivo differentiation (ovariectomized rat)\",\n      \"pmids\": [\"39034451\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct Smurf2 ubiquitination by Hakai not shown biochemically\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Positioned Hakai within a UBE3C-controlled axis governing m6A levels during cortical neurogenesis, linking its turnover to brain development.\",\n      \"evidence\": \"Proteomics of UBE3C-deficient mouse forebrain and human organoids, genetic complementation, METTL3-inhibitor rescue (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"Direct UBE3C-mediated ubiquitination of Hakai not biochemically reconstituted\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated Hakai-mediated LRP4 degradation in Wnt/β-catenin hyperactivation and validated HYB-domain inhibition as a pharmacological strategy in colorectal cancer.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, TOPFlash reporter, inducible shRNA, Hakin-1 HYB inhibitor (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not yet peer-reviewed\", \"LRP4 ubiquitin linkage and selectivity not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Hakai's distinct activities — catalytic phosphotyrosine-dependent ubiquitination versus its structural role in the m6A writer and its ligase-independent transcriptional/stabilizing functions — are partitioned and coordinated within the cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full-length Hakai engaged with the writer complex\", \"Determinants selecting degradative vs stabilizing vs neddylation outcomes unknown\", \"Mammalian counterparts of Drosophila m6A findings not directly demonstrated in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 5, 12, 13]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 5, 9, 12]},\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 5, 12]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 13]}\n    ],\n    \"complexes\": [\"m6A mRNA writer complex\"],\n    \"partners\": [\"CDH1\", \"Src\", \"FASN\", \"LRP4\", \"RUNX2\", \"AJUBA\", \"UBE3C\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}