{"gene":"ANKRD54","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2008,"finding":"ANKRD54/Liar was identified as a Lyn-interacting protein via yeast two-hybrid screen and forms a multiprotein complex with Lyn and HS1 in erythroid cells. Three ankyrin repeats of Liar define a novel SH3-binding region mediating interaction with Lyn and HS1. Liar contains functional nuclear localization and nuclear export sequences, shuttles rapidly between nucleus and cytoplasm, and ectopic expression of Liar inhibited differentiation of normal and immortalized erythroid progenitors while affecting Epo-activated signaling molecules Erk2, STAT5, Akt, and Lyn.","method":"Yeast two-hybrid screen, co-immunoprecipitation, subcellular fractionation/nuclear export assay, ectopic overexpression with erythroid differentiation readout, western blotting for signaling molecules","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, functional overexpression with defined cellular phenotype, localization studies; single lab with multiple orthogonal methods","pmids":["19064729"],"is_preprint":false},{"year":2012,"finding":"ANKRD54/Liar mediates nuclear export of Bruton's tyrosine kinase (Btk) and another Tec family kinase Txk/Rlk in a CRM1-dependent manner. The interaction is exclusively dependent on Btk's SH3 domain; Liar preferentially binds non-phosphorylated (activation loop) Btk and shows reduced binding to a phosphomimetic SH3 mutant. The interaction site was mapped to the C terminus of the Btk SH3 domain, and a synthetic biotinylated peptide (ARDKNGQEGYIPSNYVTEAEDS) was sufficient for interaction. Three other nuclear proteins (Abl, ERβ, T-bet) were unaffected by Liar, demonstrating specificity.","method":"Affinity purification of Flag-tagged Btk combined with tandem mass spectrometry (AP-MS), co-immunoprecipitation, nuclear export assays, SH3 domain mutagenesis, synthetic peptide pulldown, CRM1 inhibitor (leptomycin B) treatment","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — AP-MS identification, reciprocal Co-IP, domain mutagenesis, peptide-level mapping, mechanistic CRM1-dependence assay, multiple orthogonal methods in single focused study","pmids":["22527282"],"is_preprint":false},{"year":2017,"finding":"ANKRD54 preferentially selects the BTK SH3 domain from a phage-display library of all 296 human SH3 domains, confirming that BTK-SH3 is the prime interactor of ANKRD54 among the entire human SH3 domain repertoire.","method":"Phage-display screening of a library containing all 296 human SH3 domains, quantitative western blotting validation","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic phage-display library screen with quantitative western blot validation; single lab, two orthogonal methods confirming SH3 specificity","pmids":["28369144"],"is_preprint":false},{"year":2017,"finding":"PKCδ phosphorylates ANKRD54 at Ser18 (within an N-terminal cluster Ser14/17/18/19) to promote its nuclear export and cytoplasmic localization. PMA-induced PKC activation increased ANKRD54 phosphorylation and cytoplasmic accumulation; alanine substitution of Ser14/17/18/19 reduced both PMA-induced cytoplasmic localization and phosphorylation. PKCδ co-expression also enhanced co-immunoprecipitation and co-localization of ANKRD54 with Lyn in the cytoplasm.","method":"Immunofluorescence imaging of eGFP-tagged ANKRD54 in HEK293T cells, subcellular fractionation/immunoblotting, Phos-tag gel retardation assay, site-directed alanine mutagenesis, multiple-reaction-monitoring (MRM) proteomic phosphopeptide analysis, co-immunoprecipitation","journal":"World journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct phosphorylation site identification by MRM proteomics, mutagenesis, subcellular fractionation with functional localization consequence; single lab with multiple orthogonal methods","pmids":["28924458"],"is_preprint":false}],"current_model":"ANKRD54 (Liar) is an ankyrin repeat-containing nucleocytoplasmic shuttling protein that uses its ankyrin repeats as an SH3-binding module to interact preferentially with the SH3 domain of Bruton's tyrosine kinase (BTK) and the Src-family kinase Lyn, mediating CRM1-dependent nuclear export of non-phosphorylated BTK and Txk/Rlk; its own shuttling is regulated by PKCδ-mediated phosphorylation at Ser18, and its expression modulates erythropoietin-induced erythroid differentiation signaling downstream of Lyn."},"narrative":{"mechanistic_narrative":"ANKRD54 (Liar) is an ankyrin repeat-containing nucleocytoplasmic shuttling protein that functions as a specificity adaptor controlling the subcellular distribution of SH3-domain-bearing tyrosine kinases [PMID:19064729, PMID:22527282]. Its three ankyrin repeats constitute a non-canonical SH3-binding module, and unbiased phage-display screening of the entire human SH3 repertoire establishes the BTK SH3 domain as its prime interactor [PMID:28369144]. Through this interaction ANKRD54 drives CRM1-dependent nuclear export of the Tec-family kinases BTK and Txk/Rlk, binding preferentially to non-phosphorylated kinase and discriminating against an activation-state-mimicking SH3 mutant, conferring selectivity over other nuclear proteins [PMID:22527282]. ANKRD54 was originally isolated as a Lyn-interacting protein forming a complex with Lyn and HS1 in erythroid cells, where its ectopic expression perturbs erythropoietin-activated signaling (Erk2, STAT5, Akt, Lyn) and inhibits erythroid differentiation [PMID:19064729]. Its own nuclear export and cytoplasmic accumulation are regulated by PKCδ-mediated phosphorylation at Ser18 within an N-terminal serine cluster, which also enhances its cytoplasmic association with Lyn [PMID:28924458].","teleology":[{"year":2008,"claim":"Establishing that ANKRD54/Liar physically links a Src-family kinase to a nucleocytoplasmic shuttling machinery answered whether ankyrin repeats could serve as a kinase-binding and trafficking module relevant to erythroid signaling.","evidence":"Yeast two-hybrid screen, reciprocal co-IP, nuclear export/fractionation assays, and ectopic overexpression with erythroid differentiation and Epo-signaling readouts","pmids":["19064729"],"confidence":"Medium","gaps":["Did not define which kinase SH3 domain is the preferred ANKRD54 ligand","Mechanistic basis for differentiation inhibition versus shuttling not separated","Direct nuclear export carrier not identified"]},{"year":2012,"claim":"Identifying BTK and Txk/Rlk as CRM1-dependent export cargoes and mapping the interaction to the kinase SH3 domain converted ANKRD54 from a binding partner into a defined export adaptor with activation-state selectivity.","evidence":"AP-MS of Flag-BTK, reciprocal co-IP, SH3 domain mutagenesis, synthetic peptide pulldown, and leptomycin B CRM1-inhibition assays","pmids":["22527282"],"confidence":"High","gaps":["Physiological consequence of BTK/Txk nuclear export not established","Structure of the ankyrin-repeat:SH3 interface not solved","Whether export is constitutive or signal-regulated in B cells unclear"]},{"year":2017,"claim":"Screening all 296 human SH3 domains answered whether BTK-SH3 selectivity was genuine or one of many partners, confirming BTK-SH3 as the prime interactor across the human SH3 repertoire.","evidence":"Phage-display screening of a complete human SH3-domain library with quantitative western blot validation","pmids":["28369144"],"confidence":"Medium","gaps":["In vitro selection may not reflect cellular partner availability","Affinity ranking of secondary SH3 partners not quantified","Functional outcome of preferential BTK binding not addressed"]},{"year":2017,"claim":"Mapping PKCδ phosphorylation at Ser18 answered how ANKRD54's own localization is controlled, linking a signaling kinase to its cytoplasmic redistribution and enhanced Lyn association.","evidence":"MRM phosphopeptide proteomics, Phos-tag gels, alanine mutagenesis, PMA stimulation, fractionation/immunofluorescence, and co-IP in HEK293T cells","pmids":["28924458"],"confidence":"Medium","gaps":["Whether PKCδ phosphorylation directly regulates kinase-cargo export not tested","Done in HEK293T rather than physiological lymphoid/erythroid context","Kinase responsible in vivo not confirmed beyond co-expression"]},{"year":null,"claim":"The physiological and disease relevance of ANKRD54-mediated kinase export in B-cell or erythroid biology remains undefined.","evidence":"","pmids":[],"confidence":"Low","gaps":["No loss-of-function/knockout phenotype reported","No structural model of the ankyrin:SH3 interaction","Consequence of sequestering non-phosphorylated BTK in the nucleus versus cytoplasm unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,2]},{"term_id":"GO:0140104","term_label":"molecular carrier activity","supporting_discovery_ids":[1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,3]}],"pathway":[{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[1]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0]}],"complexes":[],"partners":["BTK","LYN","TXK","HCLS1","PRKCD"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6NXT1","full_name":"Ankyrin repeat domain-containing protein 54","aliases":["Lyn-interacting ankyrin repeat protein"],"length_aa":300,"mass_kda":32.5,"function":"Plays an important role in regulating intracellular signaling events associated with erythroid terminal differentiation","subcellular_location":"Nucleus; Cytoplasm; Midbody","url":"https://www.uniprot.org/uniprotkb/Q6NXT1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ANKRD54","classification":"Not Classified","n_dependent_lines":29,"n_total_lines":1208,"dependency_fraction":0.024006622516556293},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000100124","cell_line_id":"CID001926","localizations":[{"compartment":"nucleoplasm","grade":3},{"compartment":"nuclear_punctae","grade":2}],"interactors":[{"gene":"HNRNPUL2-BSCL2;HNRNPUL2","stoichiometry":4.0},{"gene":"SNX33","stoichiometry":0.2},{"gene":"FAM192A;NIP30","stoichiometry":0.2},{"gene":"KIAA1279","stoichiometry":0.2},{"gene":"DOCK1","stoichiometry":0.2},{"gene":"KPNA2","stoichiometry":0.2},{"gene":"PSME3","stoichiometry":0.2},{"gene":"TULP3","stoichiometry":0.2},{"gene":"CSNK2A2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001926","total_profiled":1310},"omim":[{"mim_id":"613383","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 54; ANKRD54","url":"https://www.omim.org/entry/613383"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"},{"location":"Midbody","reliability":"Approved"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ANKRD54"},"hgnc":{"alias_symbol":["LIAR"],"prev_symbol":[]},"alphafold":{"accession":"Q6NXT1","domains":[{"cath_id":"1.25.40.20","chopping":"106-205","consensus_level":"medium","plddt":91.3023,"start":106,"end":205},{"cath_id":"-","chopping":"212-300","consensus_level":"medium","plddt":76.806,"start":212,"end":300}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6NXT1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6NXT1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6NXT1-F1-predicted_aligned_error_v6.png","plddt_mean":68.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ANKRD54","jax_strain_url":"https://www.jax.org/strain/search?query=ANKRD54"},"sequence":{"accession":"Q6NXT1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6NXT1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6NXT1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6NXT1"}},"corpus_meta":[{"pmid":"20447076","id":"PMC_20447076","title":"Eosinophils in health and disease: the LIAR hypothesis.","date":"2010","source":"Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology","url":"https://pubmed.ncbi.nlm.nih.gov/20447076","citation_count":272,"is_preprint":false},{"pmid":"20639339","id":"PMC_20639339","title":"In-depth profiling of the LiaR response of Bacillus subtilis.","date":"2010","source":"Journal of bacteriology","url":"https://pubmed.ncbi.nlm.nih.gov/20639339","citation_count":100,"is_preprint":false},{"pmid":"25362197","id":"PMC_25362197","title":"A liaR deletion restores susceptibility to daptomycin and antimicrobial peptides in multidrug-resistant Enterococcus faecalis.","date":"2014","source":"The Journal of infectious diseases","url":"https://pubmed.ncbi.nlm.nih.gov/25362197","citation_count":79,"is_preprint":false},{"pmid":"23851083","id":"PMC_23851083","title":"Nisin resistance of Listeria monocytogenes is increased by exposure to salt stress and is mediated via LiaR.","date":"2013","source":"Applied and environmental microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/23851083","citation_count":69,"is_preprint":false},{"pmid":"23704792","id":"PMC_23704792","title":"The two-component response regulator LiaR regulates cell wall stress responses, pili expression and virulence in group B Streptococcus.","date":"2013","source":"Microbiology (Reading, England)","url":"https://pubmed.ncbi.nlm.nih.gov/23704792","citation_count":51,"is_preprint":false},{"pmid":"21128282","id":"PMC_21128282","title":"LRIG1 and the liar paradox in prostate cancer: a study of the expression and clinical significance of LRIG1 in prostate cancer.","date":"2011","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/21128282","citation_count":39,"is_preprint":false},{"pmid":"27208105","id":"PMC_27208105","title":"Exogenous Fatty Acids Protect Enterococcus faecalis from Daptomycin-Induced Membrane Stress Independently of the Response Regulator LiaR.","date":"2016","source":"Applied and environmental microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/27208105","citation_count":39,"is_preprint":false},{"pmid":"25897118","id":"PMC_25897118","title":"A variable DNA recognition site organization establishes the LiaR-mediated cell envelope stress response of enterococci to daptomycin.","date":"2015","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/25897118","citation_count":37,"is_preprint":false},{"pmid":"26369959","id":"PMC_26369959","title":"Deletion of liaR Reverses Daptomycin Resistance in Enterococcus faecium Independent of the Genetic Background.","date":"2015","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/26369959","citation_count":36,"is_preprint":false},{"pmid":"30582877","id":"PMC_30582877","title":"LiaR-independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics.","date":"2019","source":"Molecular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/30582877","citation_count":31,"is_preprint":false},{"pmid":"27670715","id":"PMC_27670715","title":"An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State.","date":"2016","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/27670715","citation_count":24,"is_preprint":false},{"pmid":"22527282","id":"PMC_22527282","title":"Regulation of nucleocytoplasmic shuttling of Bruton's tyrosine kinase (Btk) through a novel SH3-dependent interaction with ankyrin repeat domain 54 (ANKRD54).","date":"2012","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/22527282","citation_count":20,"is_preprint":false},{"pmid":"19064729","id":"PMC_19064729","title":"Liar, a novel Lyn-binding nuclear/cytoplasmic shuttling protein that influences erythropoietin-induced differentiation.","date":"2008","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/19064729","citation_count":16,"is_preprint":false},{"pmid":"32043713","id":"PMC_32043713","title":"Streptococcus mutans SpxA2 relays the signal of cell envelope stress from LiaR to effectors that maintain cell wall and membrane homeostasis.","date":"2020","source":"Molecular oral microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/32043713","citation_count":14,"is_preprint":false},{"pmid":"19722183","id":"PMC_19722183","title":"Gene networks and liar paradoxes.","date":"2009","source":"BioEssays : news and reviews in molecular, cellular and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/19722183","citation_count":14,"is_preprint":false},{"pmid":"30403130","id":"PMC_30403130","title":"Two Mutations Commonly Associated with Daptomycin Resistance in Enterococcus faecium LiaST120A and LiaRW73C Appear To Function Epistatically in LiaFSR Signaling.","date":"2018","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30403130","citation_count":11,"is_preprint":false},{"pmid":"32349670","id":"PMC_32349670","title":"Low phosphatase activity of LiaS and strong LiaR-DNA affinity explain the unusual LiaS to LiaR in vivo stoichiometry.","date":"2020","source":"BMC microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/32349670","citation_count":6,"is_preprint":false},{"pmid":"34168597","id":"PMC_34168597","title":"Catching a Liar Through Facial Expression of Fear.","date":"2021","source":"Frontiers in psychology","url":"https://pubmed.ncbi.nlm.nih.gov/34168597","citation_count":6,"is_preprint":false},{"pmid":"25308550","id":"PMC_25308550","title":"A Streptococcus uberis transposon mutant screen reveals a negative role for LiaR homologue in biofilm formation.","date":"2014","source":"Journal of applied microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/25308550","citation_count":5,"is_preprint":false},{"pmid":"28369144","id":"PMC_28369144","title":"ANKRD54 preferentially selects Bruton's Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library.","date":"2017","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/28369144","citation_count":3,"is_preprint":false},{"pmid":"40659638","id":"PMC_40659638","title":"Genome-wide antibiotic-CRISPRi profiling identifies LiaR activation as a strategy to resensitize fluoroquinolone-resistant Streptococcus pneumoniae.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/40659638","citation_count":2,"is_preprint":false},{"pmid":"28924458","id":"PMC_28924458","title":"Control of nuclear-cytoplasmic shuttling of Ankrd54 by PKCδ.","date":"2017","source":"World journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28924458","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.19.660511","title":"LiaS-dependent activation of the MadR regulon enables cross-talk between  <i>Enterococcus faecalis</i>  cell envelope defense systems","date":"2025-06-20","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.19.660511","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.30.621020","title":"Genome-wide antibiotic-CRISPRi profiling identifies LiaR activation as a strategy to resensitize fluoroquinolone-resistant  <i>Streptococcus pneumoniae</i>","date":"2024-10-30","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.30.621020","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14415,"output_tokens":1790,"usd":0.035048,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8163,"output_tokens":2157,"usd":0.04737,"stage2_stop_reason":"end_turn"},"total_usd":0.082418,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"Looking at the corpus, I need to identify papers about ANKRD54 (also known as LIAR/Liar in the context of the human/mammalian protein). \n\nPapers [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [14], [15], [16], [17], [19], [21], [23], [24] describe bacterial LiaR proteins (Bacillus subtilis, Enterococcus, Listeria, Streptococcus) — these are symbol collisions with a completely different protein family (two-component response regulators). The mammalian ANKRD54/Liar is an ankyrin repeat domain protein involved in tyrosine kinase shuttling. These bacterial LiaR papers are NOT orthologs — they describe fundamentally different proteins. I will skip them.\n\nPaper [1] is about eosinophil biology (LIAR hypothesis as an acronym) — not the ANKRD54 protein. Skip.\nPaper [6] is about LRIG1 — different gene. Skip.\nPaper [15] is about gene network logic paradoxes — not the protein. Skip.\nPaper [18] is about facial expression/deception detection — not the protein. Skip.\n\nPapers [12], [13], [20], [22] are clearly about the mammalian ANKRD54/Liar protein. Extract from these.\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"ANKRD54/Liar was identified as a Lyn-interacting protein via yeast two-hybrid screen and forms a multiprotein complex with Lyn and HS1 in erythroid cells. Three ankyrin repeats of Liar define a novel SH3-binding region mediating interaction with Lyn and HS1. Liar contains functional nuclear localization and nuclear export sequences, shuttles rapidly between nucleus and cytoplasm, and ectopic expression of Liar inhibited differentiation of normal and immortalized erythroid progenitors while affecting Epo-activated signaling molecules Erk2, STAT5, Akt, and Lyn.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, subcellular fractionation/nuclear export assay, ectopic overexpression with erythroid differentiation readout, western blotting for signaling molecules\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, functional overexpression with defined cellular phenotype, localization studies; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"19064729\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ANKRD54/Liar mediates nuclear export of Bruton's tyrosine kinase (Btk) and another Tec family kinase Txk/Rlk in a CRM1-dependent manner. The interaction is exclusively dependent on Btk's SH3 domain; Liar preferentially binds non-phosphorylated (activation loop) Btk and shows reduced binding to a phosphomimetic SH3 mutant. The interaction site was mapped to the C terminus of the Btk SH3 domain, and a synthetic biotinylated peptide (ARDKNGQEGYIPSNYVTEAEDS) was sufficient for interaction. Three other nuclear proteins (Abl, ERβ, T-bet) were unaffected by Liar, demonstrating specificity.\",\n      \"method\": \"Affinity purification of Flag-tagged Btk combined with tandem mass spectrometry (AP-MS), co-immunoprecipitation, nuclear export assays, SH3 domain mutagenesis, synthetic peptide pulldown, CRM1 inhibitor (leptomycin B) treatment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — AP-MS identification, reciprocal Co-IP, domain mutagenesis, peptide-level mapping, mechanistic CRM1-dependence assay, multiple orthogonal methods in single focused study\",\n      \"pmids\": [\"22527282\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ANKRD54 preferentially selects the BTK SH3 domain from a phage-display library of all 296 human SH3 domains, confirming that BTK-SH3 is the prime interactor of ANKRD54 among the entire human SH3 domain repertoire.\",\n      \"method\": \"Phage-display screening of a library containing all 296 human SH3 domains, quantitative western blotting validation\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic phage-display library screen with quantitative western blot validation; single lab, two orthogonal methods confirming SH3 specificity\",\n      \"pmids\": [\"28369144\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PKCδ phosphorylates ANKRD54 at Ser18 (within an N-terminal cluster Ser14/17/18/19) to promote its nuclear export and cytoplasmic localization. PMA-induced PKC activation increased ANKRD54 phosphorylation and cytoplasmic accumulation; alanine substitution of Ser14/17/18/19 reduced both PMA-induced cytoplasmic localization and phosphorylation. PKCδ co-expression also enhanced co-immunoprecipitation and co-localization of ANKRD54 with Lyn in the cytoplasm.\",\n      \"method\": \"Immunofluorescence imaging of eGFP-tagged ANKRD54 in HEK293T cells, subcellular fractionation/immunoblotting, Phos-tag gel retardation assay, site-directed alanine mutagenesis, multiple-reaction-monitoring (MRM) proteomic phosphopeptide analysis, co-immunoprecipitation\",\n      \"journal\": \"World journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct phosphorylation site identification by MRM proteomics, mutagenesis, subcellular fractionation with functional localization consequence; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28924458\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANKRD54 (Liar) is an ankyrin repeat-containing nucleocytoplasmic shuttling protein that uses its ankyrin repeats as an SH3-binding module to interact preferentially with the SH3 domain of Bruton's tyrosine kinase (BTK) and the Src-family kinase Lyn, mediating CRM1-dependent nuclear export of non-phosphorylated BTK and Txk/Rlk; its own shuttling is regulated by PKCδ-mediated phosphorylation at Ser18, and its expression modulates erythropoietin-induced erythroid differentiation signaling downstream of Lyn.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ANKRD54 (Liar) is an ankyrin repeat-containing nucleocytoplasmic shuttling protein that functions as a specificity adaptor controlling the subcellular distribution of SH3-domain-bearing tyrosine kinases [#0, #1]. Its three ankyrin repeats constitute a non-canonical SH3-binding module, and unbiased phage-display screening of the entire human SH3 repertoire establishes the BTK SH3 domain as its prime interactor [#2]. Through this interaction ANKRD54 drives CRM1-dependent nuclear export of the Tec-family kinases BTK and Txk/Rlk, binding preferentially to non-phosphorylated kinase and discriminating against an activation-state-mimicking SH3 mutant, conferring selectivity over other nuclear proteins [#1]. ANKRD54 was originally isolated as a Lyn-interacting protein forming a complex with Lyn and HS1 in erythroid cells, where its ectopic expression perturbs erythropoietin-activated signaling (Erk2, STAT5, Akt, Lyn) and inhibits erythroid differentiation [#0]. Its own nuclear export and cytoplasmic accumulation are regulated by PKC\\u03b4-mediated phosphorylation at Ser18 within an N-terminal serine cluster, which also enhances its cytoplasmic association with Lyn [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 2008,\n      \"claim\": \"Establishing that ANKRD54/Liar physically links a Src-family kinase to a nucleocytoplasmic shuttling machinery answered whether ankyrin repeats could serve as a kinase-binding and trafficking module relevant to erythroid signaling.\",\n      \"evidence\": \"Yeast two-hybrid screen, reciprocal co-IP, nuclear export/fractionation assays, and ectopic overexpression with erythroid differentiation and Epo-signaling readouts\",\n      \"pmids\": [\"19064729\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define which kinase SH3 domain is the preferred ANKRD54 ligand\", \"Mechanistic basis for differentiation inhibition versus shuttling not separated\", \"Direct nuclear export carrier not identified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying BTK and Txk/Rlk as CRM1-dependent export cargoes and mapping the interaction to the kinase SH3 domain converted ANKRD54 from a binding partner into a defined export adaptor with activation-state selectivity.\",\n      \"evidence\": \"AP-MS of Flag-BTK, reciprocal co-IP, SH3 domain mutagenesis, synthetic peptide pulldown, and leptomycin B CRM1-inhibition assays\",\n      \"pmids\": [\"22527282\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological consequence of BTK/Txk nuclear export not established\", \"Structure of the ankyrin-repeat:SH3 interface not solved\", \"Whether export is constitutive or signal-regulated in B cells unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Screening all 296 human SH3 domains answered whether BTK-SH3 selectivity was genuine or one of many partners, confirming BTK-SH3 as the prime interactor across the human SH3 repertoire.\",\n      \"evidence\": \"Phage-display screening of a complete human SH3-domain library with quantitative western blot validation\",\n      \"pmids\": [\"28369144\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro selection may not reflect cellular partner availability\", \"Affinity ranking of secondary SH3 partners not quantified\", \"Functional outcome of preferential BTK binding not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Mapping PKC\\u03b4 phosphorylation at Ser18 answered how ANKRD54's own localization is controlled, linking a signaling kinase to its cytoplasmic redistribution and enhanced Lyn association.\",\n      \"evidence\": \"MRM phosphopeptide proteomics, Phos-tag gels, alanine mutagenesis, PMA stimulation, fractionation/immunofluorescence, and co-IP in HEK293T cells\",\n      \"pmids\": [\"28924458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PKC\\u03b4 phosphorylation directly regulates kinase-cargo export not tested\", \"Done in HEK293T rather than physiological lymphoid/erythroid context\", \"Kinase responsible in vivo not confirmed beyond co-expression\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The physiological and disease relevance of ANKRD54-mediated kinase export in B-cell or erythroid biology remains undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No loss-of-function/knockout phenotype reported\", \"No structural model of the ankyrin:SH3 interaction\", \"Consequence of sequestering non-phosphorylated BTK in the nucleus versus cytoplasm unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0140104\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"BTK\", \"LYN\", \"TXK\", \"HCLS1\", \"PRKCD\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}