{"gene":"ILKAP","run_date":"2026-04-28T18:06:54","timeline":{"discoveries":[{"year":2001,"finding":"ILKAP, a PP2C family serine/threonine phosphatase, was identified as a binding partner of integrin-linked kinase ILK1 via yeast two-hybrid screen, and their association is independent of the catalytic activity of either partner. Conditional ILKAP expression selectively inhibited ECM- and growth factor-stimulated ILK1 kinase activity; a catalytic dead mutant H154D failed to inhibit ILK1. ILKAP selectively inhibited GSK3β phosphorylation on Ser9 (downstream of ILK1) without affecting PKB/AKT Ser473 phosphorylation, and suppressed Tcf/Lef (TOPFlash) reporter transactivation, placing ILKAP in the ILK1–GSK3β–Wnt signaling axis.","method":"Yeast two-hybrid, co-precipitation, conditional expression in HEK293 cells, ILK immune complex kinase assay, catalytic mutant (H154D), Tcf/Lef luciferase reporter","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (two-hybrid, co-IP, kinase assay, reporter assay, mutagenesis) in a single foundational study, 120 citations","pmids":["11331582"],"is_preprint":false},{"year":2004,"finding":"Endogenous ILKAP selectively inhibits ILK-mediated GSK3β Ser9 phosphorylation without affecting PKB Ser473. siRNA silencing of ILKAP stimulated GSK3β-S9 phosphorylation and S-phase entry; ILKAP overexpression increased G1 fraction, reduced cyclin D1 levels, and suppressed anchorage-independent growth in LNCaP cells. Overexpression of ILK rescued ILKAP-mediated GSK3β inhibition; dominant-negative ILK did not, establishing ILKAP acts through ILK catalytic activity.","method":"siRNA knockdown, stable and transient overexpression, ILK immune complex kinase assay, flow cytometry, soft-agar anchorage-independent growth assay, Western blot","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — reciprocal gain/loss-of-function with multiple readouts replicating the foundational study, 69 citations","pmids":["14990992"],"is_preprint":false},{"year":2008,"finding":"ILKAP promotes CRM1-dependent nuclear export of ILK, restricting nuclear ILK accumulation. Nuclear ILK was associated with increased DNA synthesis in epidermal keratinocytes, and this proliferative effect was sensitive to ILKAP-mediated export, establishing ILKAP as a modulator of ILK subcellular localization with functional consequences for keratinocyte proliferation.","method":"Live-cell imaging, nuclear fractionation, CRM1 inhibition (leptomycin B), DNA synthesis assay (BrdU incorporation)","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization experiments with functional consequence, single lab","pmids":["18635968"],"is_preprint":false},{"year":2013,"finding":"ILKAP contains a nuclear localization signal (NLS) between residues 71–86 (critical residues Lys-78 and Arg-79) that mediates nuclear import via importin α1, α3, and α5. Nuclear ILKAP interacts with RSK2 and induces apoptosis by inhibiting RSK2 kinase activity and downregulating cyclin D1 expression.","method":"Immunofluorescence of endogenous and tagged ILKAP, co-immunoprecipitation with importin isoforms, NLS deletion mutant, RSK2 kinase assay, cyclin D1 Western blot, apoptosis assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 — multiple orthogonal methods (mutagenesis, co-IP, kinase assay, localization) in one study","pmids":["23329845"],"is_preprint":false},{"year":2012,"finding":"ILKAP binds phosphopeptides derived from known PP2C substrates including p38, ATM, Chk1, Chk2 and RSK2 in a phosphorylation-dependent and sequence-context-dependent manner, establishing these phosphoproteins as candidate ILKAP substrates and confirming ILKAP's substrate-binding selectivity.","method":"Solid-phase phosphopeptide affinity pull-down from cell lysates, competitive binding assays","journal":"Molecular bioSystems","confidence":"Medium","confidence_rationale":"Tier 3 — affinity pull-down establishing candidate substrate interactions, single lab","pmids":["22348942"],"is_preprint":false},{"year":2015,"finding":"ILKAP depletion sensitizes p53-wildtype glioblastoma cells to radiation, and radiation-induced phosphorylation of DNA-PK (DNAPK) is dependent on ILKAP, identifying DNAPK as a downstream mediator of ILKAP signaling in the DNA damage response.","method":"siRNA knockdown, γH2AX/53BP1 foci quantification, Western blot for DNAPK phosphorylation, clonogenic survival assay","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — functional KD with defined molecular readout, single lab","pmids":["26460618"],"is_preprint":false},{"year":2016,"finding":"In ovarian cancer cells, ILKAP dephosphorylates both p90RSK (RSK1/RSK2) and ILK/AKT. HGF pre-treatment upregulates ILKAP expression and reverts CDDP-induced RSK phosphorylation. ILKAP silencing protects cells from CDDP-induced death through simultaneous increased activity of RSK and ILK/AKT, requiring combined inhibition of p90RSK and ILK to rescue ILKAP-loss phenotype, establishing ILKAP as a regulatory hub controlling multiple pro-survival kinases.","method":"siRNA knockdown, pharmacological inhibitors, Western blot, cell viability and apoptosis assays","journal":"European journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 — combinatorial epistasis with kinase inhibitors and multiple siRNA knockdowns, single lab","pmids":["27065457"],"is_preprint":false},{"year":2017,"finding":"MAEL promotes lysosome-dependent degradation of ILKAP, leading to increased phosphorylation of ILKAP substrates p38, CHK1 and RSK2. ILKAP overexpression reversed the oncogenic effects of MAEL, placing MAEL upstream of ILKAP in a gastric cancer progression pathway.","method":"Co-expression/silencing experiments, Western blot, lysosome inhibitor treatment, adenovirus-mediated ILKAP overexpression, xenograft models","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis with rescue experiment and in vivo validation, single lab","pmids":["29371914"],"is_preprint":false},{"year":2018,"finding":"ILKAP physically interacts with HIF-1α (co-immunoprecipitation) and induces its dephosphorylation; both the HIF-1α–p53 interaction and hypoxia-induced apoptosis require ILKAP, identifying HIF-1α as a novel ILKAP substrate.","method":"Co-immunoprecipitation, HRE-luciferase reporter, trypan blue viability assay, shRNA knockdown, overexpression","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus functional gain/loss-of-function, single lab","pmids":["29742494"],"is_preprint":false},{"year":2024,"finding":"ILKAP interacts with β-catenin and dephosphorylates GSK3β and CK1, thereby reducing β-catenin ubiquitination and increasing β-catenin protein stability. ILKAP also mediates binding between TCF4 and β-catenin to enhance Wnt target gene expression (c-Myc, CyclinD1), promoting HCC metastasis in vitro and in a zebrafish xenograft model.","method":"Co-immunoprecipitation, Western blot, ubiquitination assay, Wnt reporter assay, zebrafish xenograft, siRNA knockdown","journal":"Advanced biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple biochemical assays with in vivo validation, single lab","pmids":["38379270"],"is_preprint":false},{"year":2025,"finding":"ILKAP knockdown suppresses PGAM1 expression in HCC cells; restoring PGAM1 in ILKAP-knockdown cells rescues proliferation and invasion, and ILKAP depletion reduces extracellular acidification rate, establishing an ILKAP–PGAM1 axis in glycolytic reprogramming and tumor progression.","method":"RNA sequencing, siRNA knockdown, PGAM1 rescue overexpression, Seahorse glycolysis assay, xenograft models","journal":"Frontiers of medicine","confidence":"Medium","confidence_rationale":"Tier 2 — epistasis by rescue experiment with metabolic readout, single lab, no citations yet","pmids":["41454076"],"is_preprint":false}],"current_model":"ILKAP is a PP2C-family serine/threonine phosphatase that localizes to both nucleus and cytoplasm (nuclear import via importin α–NLS interaction, nuclear export enhanced by CRM1); it binds ILK1 and selectively dephosphorylates GSK3β (suppressing Wnt/Tcf signaling and cyclin D1) without affecting AKT, dephosphorylates nuclear RSK2 (inducing apoptosis), dephosphorylates HIF-1α, GSK3β/CK1 (stabilizing β-catenin), and modulates PGAM1-driven glycolysis, while its own protein level is regulated by MAEL-dependent lysosomal degradation."},"narrative":{"teleology":[{"year":2001,"claim":"Established ILKAP as a PP2C-family phosphatase that physically associates with ILK1 and selectively dephosphorylates GSK3β Ser9 (but not AKT Ser473) to suppress Wnt/Tcf signaling, answering how ILK kinase activity is negatively regulated.","evidence":"Yeast two-hybrid, co-precipitation, catalytic mutant H154D, ILK immune complex kinase assay, Tcf/Lef luciferase reporter in HEK293 cells","pmids":["11331582"],"confidence":"High","gaps":["Direct phosphatase activity on GSK3β peptide not demonstrated in vitro","Endogenous interaction stoichiometry unknown","Mechanism of selectivity for GSK3β over AKT not resolved"]},{"year":2004,"claim":"Reciprocal gain- and loss-of-function confirmed endogenous ILKAP restrains ILK–GSK3β signaling to control cell cycle progression and anchorage-independent growth, establishing ILKAP as a tumor-suppressive phosphatase.","evidence":"siRNA knockdown and stable overexpression in LNCaP cells, flow cytometry, soft-agar colony assay, cyclin D1 Western blot","pmids":["14990992"],"confidence":"High","gaps":["Whether ILKAP directly dephosphorylates GSK3β or acts indirectly through ILK remains unresolved","Mechanism linking cyclin D1 loss to G1 arrest not dissected beyond GSK3β"]},{"year":2008,"claim":"Revealed that ILKAP promotes CRM1-dependent nuclear export of ILK, restricting nuclear ILK accumulation and ILK-driven DNA synthesis, thereby establishing ILKAP as a regulator of ILK subcellular distribution.","evidence":"Live-cell imaging, nuclear fractionation, leptomycin B treatment, BrdU incorporation in keratinocytes","pmids":["18635968"],"confidence":"Medium","gaps":["Whether ILKAP directly escorts ILK or modifies a separate export signal is unknown","Not tested in non-keratinocyte cell types"]},{"year":2012,"claim":"Phosphopeptide affinity studies identified p38, ATM, Chk1, Chk2, and RSK2 as candidate ILKAP substrates, expanding the substrate repertoire beyond GSK3β.","evidence":"Solid-phase phosphopeptide pull-down with competitive binding assays","pmids":["22348942"],"confidence":"Medium","gaps":["No in vitro dephosphorylation assays performed to confirm catalytic activity on these substrates","Binding ≠ dephosphorylation; functional validation needed for each candidate"]},{"year":2013,"claim":"Mapped an NLS (residues 71–86, critical K78/R79) mediating importin α–dependent nuclear import and showed nuclear ILKAP dephosphorylates RSK2 to induce apoptosis and reduce cyclin D1, answering how ILKAP reaches the nucleus and identifying its nuclear substrate.","evidence":"NLS deletion mutant, importin co-IP, RSK2 kinase assay, apoptosis assay, immunofluorescence in HeLa cells","pmids":["23329845"],"confidence":"High","gaps":["Direct dephosphorylation of RSK2 not shown with purified components","Relative contribution of nuclear vs. cytoplasmic ILKAP pools to apoptosis not quantified"]},{"year":2015,"claim":"ILKAP depletion sensitized glioblastoma cells to radiation and impaired DNA-PK phosphorylation, linking ILKAP to the DNA damage response for the first time.","evidence":"siRNA knockdown, γH2AX/53BP1 foci, DNA-PK phosphorylation Western blot, clonogenic survival in glioblastoma cells","pmids":["26460618"],"confidence":"Medium","gaps":["Whether ILKAP directly dephosphorylates DNA-PK or acts indirectly is unresolved","Only tested in p53-wildtype glioblastoma context"]},{"year":2016,"claim":"Showed ILKAP functions as a dual phosphatase hub controlling both RSK and ILK/AKT in ovarian cancer, with combined inhibition required to phenocopy ILKAP activity in cisplatin-induced death.","evidence":"siRNA knockdown, pharmacological inhibitors of RSK and ILK, apoptosis and viability assays in ovarian cancer cells","pmids":["27065457"],"confidence":"Medium","gaps":["Contradicts earlier finding that ILKAP does not affect AKT; cell-type dependence not clarified","ILKAP direct activity on AKT versus ILK not biochemically separated"]},{"year":2017,"claim":"Identified MAEL as an upstream negative regulator that targets ILKAP for lysosomal degradation, increasing phosphorylation of p38, CHK1, and RSK2—answering how ILKAP protein levels are controlled.","evidence":"Co-expression/silencing, lysosome inhibitor rescue, adenoviral ILKAP overexpression, xenograft in gastric cancer model","pmids":["29371914"],"confidence":"Medium","gaps":["Mechanism by which MAEL directs ILKAP to lysosomes (adapter, ubiquitin signal) is unknown","Not confirmed in non-gastric cancer contexts"]},{"year":2018,"claim":"Identified HIF-1α as a direct ILKAP-interacting substrate whose dephosphorylation is required for HIF-1α–p53 complex formation and hypoxia-induced apoptosis.","evidence":"Co-immunoprecipitation, HRE-luciferase reporter, shRNA knockdown, overexpression, viability assays","pmids":["29742494"],"confidence":"Medium","gaps":["Specific phosphosite(s) on HIF-1α targeted by ILKAP not mapped","In vitro phosphatase assay with purified ILKAP and HIF-1α not performed"]},{"year":2024,"claim":"Demonstrated ILKAP dephosphorylates GSK3β and CK1 to stabilize β-catenin by reducing its ubiquitination, and bridges TCF4–β-catenin interaction to promote Wnt target gene expression and HCC metastasis—revealing an unexpected pro-oncogenic role.","evidence":"Co-immunoprecipitation, ubiquitination assay, Wnt reporter, siRNA knockdown, zebrafish xenograft","pmids":["38379270"],"confidence":"Medium","gaps":["Contradicts earlier tumor-suppressive role via GSK3β dephosphorylation; context-dependent regulation not reconciled","TCF4–β-catenin bridging mechanism (scaffold vs. dephosphorylation) not dissected"]},{"year":2025,"claim":"Linked ILKAP to glycolytic reprogramming by showing ILKAP sustains PGAM1 expression and extracellular acidification in HCC, with PGAM1 rescue reversing ILKAP-knockdown proliferation defects.","evidence":"RNA sequencing, siRNA knockdown, PGAM1 rescue, Seahorse ECAR assay, xenograft in HCC cells","pmids":["41454076"],"confidence":"Medium","gaps":["Whether ILKAP regulates PGAM1 transcriptionally or post-translationally is unknown","No direct phosphatase–substrate relationship established between ILKAP and PGAM1"]},{"year":null,"claim":"A unifying model reconciling ILKAP's apparently contradictory tumor-suppressive (GSK3β dephosphorylation suppressing Wnt) and tumor-promoting (β-catenin stabilization, PGAM1-driven glycolysis) activities across tissue contexts is lacking.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural data for ILKAP exist to explain substrate selectivity","In vitro reconstitution of direct phosphatase activity on most claimed substrates has not been performed","Tissue- and context-specific regulation of ILKAP activity/expression is poorly defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,3,6,8,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,3]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1,2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,6,9]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,3]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,8]}],"complexes":[],"partners":["ILK","RSK2","GSK3B","HIF1A","CTNNB1","MAEL","PGAM1"],"other_free_text":[]},"mechanistic_narrative":"ILKAP is a PP2C-family serine/threonine phosphatase that functions as a negative regulator of multiple pro-survival and proliferative kinase pathways by selectively dephosphorylating GSK3β, RSK2, HIF-1α, and CK1. It was identified as a binding partner of integrin-linked kinase (ILK1) and selectively inhibits ILK1-mediated GSK3β Ser9 phosphorylation without affecting PKB/AKT Ser473, thereby suppressing Wnt/Tcf signaling and cyclin D1 expression, reducing S-phase entry and anchorage-independent growth [PMID:11331582, PMID:14990992]. ILKAP shuttles between nucleus and cytoplasm via an importin α–dependent NLS and CRM1-dependent export; nuclear ILKAP dephosphorylates RSK2 to induce apoptosis and promotes CRM1-dependent nuclear export of ILK to limit ILK-driven proliferation [PMID:23329845, PMID:18635968]. ILKAP protein stability is controlled by MAEL-dependent lysosomal degradation, and ILKAP additionally modulates β-catenin stability through dephosphorylation of GSK3β/CK1, regulates HIF-1α–p53–dependent apoptosis, and sustains PGAM1-driven glycolysis in hepatocellular carcinoma [PMID:29371914, PMID:38379270, PMID:29742494, PMID:41454076]."},"prefetch_data":{"uniprot":{"accession":"Q9H0C8","full_name":"Integrin-linked kinase-associated serine/threonine phosphatase 2C","aliases":[],"length_aa":392,"mass_kda":42.9,"function":"Protein phosphatase that may play a role in regulation of cell cycle progression via dephosphorylation of its substrates whose appropriate phosphorylation states might be crucial for cell proliferation. Selectively associates with integrin linked kinase (ILK), to modulate cell adhesion and growth factor signaling. Inhibits the ILK-GSK3B signaling axis and may play an important role in inhibiting oncogenic transformation","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9H0C8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ILKAP","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ILKAP","total_profiled":1310},"omim":[{"mim_id":"618909","title":"ILK-ASSOCIATED SERINE/THREONINE PHOSPHATASE; ILKAP","url":"https://www.omim.org/entry/618909"},{"mim_id":"603348","title":"HYPOXIA-INDUCIBLE FACTOR 1, ALPHA SUBUNIT; HIF1A","url":"https://www.omim.org/entry/603348"},{"mim_id":"602366","title":"INTEGRIN-LINKED KINASE; ILK","url":"https://www.omim.org/entry/602366"},{"mim_id":"300075","title":"RIBOSOMAL PROTEIN S6 KINASE A3; RPS6KA3","url":"https://www.omim.org/entry/300075"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ILKAP"},"hgnc":{"alias_symbol":["DKFZP434J2031","FLJ10181","PPM1O"],"prev_symbol":[]},"alphafold":{"accession":"Q9H0C8","domains":[{"cath_id":"3.60.40.10","chopping":"105-390","consensus_level":"medium","plddt":95.8969,"start":105,"end":390}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H0C8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H0C8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H0C8-F1-predicted_aligned_error_v6.png","plddt_mean":81.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ILKAP","jax_strain_url":"https://www.jax.org/strain/search?query=ILKAP"},"sequence":{"accession":"Q9H0C8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H0C8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H0C8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H0C8"}},"corpus_meta":[{"pmid":"11331582","id":"PMC_11331582","title":"Modulation of integrin signal transduction by ILKAP, a protein phosphatase 2C associating with the integrin-linked kinase, ILK1.","date":"2001","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11331582","citation_count":120,"is_preprint":false},{"pmid":"14990992","id":"PMC_14990992","title":"ILKAP regulates ILK signaling and inhibits anchorage-independent growth.","date":"2004","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/14990992","citation_count":69,"is_preprint":false},{"pmid":"18635968","id":"PMC_18635968","title":"Modulation of integrin-linked kinase nucleo-cytoplasmic shuttling by ILKAP and CRM1.","date":"2008","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/18635968","citation_count":22,"is_preprint":false},{"pmid":"29371914","id":"PMC_29371914","title":"MAEL contributes to gastric cancer progression by promoting ILKAP degradation.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29371914","citation_count":19,"is_preprint":false},{"pmid":"24743186","id":"PMC_24743186","title":"Involvement of ANXA5 and ILKAP in susceptibility to malignant melanoma.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24743186","citation_count":13,"is_preprint":false},{"pmid":"26460618","id":"PMC_26460618","title":"ILKAP, ILK and PINCH1 control cell survival of p53-wildtype glioblastoma cells after irradiation.","date":"2015","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/26460618","citation_count":13,"is_preprint":false},{"pmid":"23329845","id":"PMC_23329845","title":"Characterization of nuclear localization signal in the N terminus of integrin-linked kinase-associated phosphatase (ILKAP) and its essential role in the down-regulation of RSK2 protein signaling.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23329845","citation_count":10,"is_preprint":false},{"pmid":"27065457","id":"PMC_27065457","title":"The integrin-linked kinase-associated phosphatase (ILKAP) is a regulatory hub of ovarian cancer cell susceptibility to platinum drugs.","date":"2016","source":"European journal of cancer (Oxford, England : 1990)","url":"https://pubmed.ncbi.nlm.nih.gov/27065457","citation_count":9,"is_preprint":false},{"pmid":"22348942","id":"PMC_22348942","title":"Probing protein phosphatase substrate binding: affinity pull-down of ILKAP phosphatase 2C with phosphopeptides.","date":"2012","source":"Molecular bioSystems","url":"https://pubmed.ncbi.nlm.nih.gov/22348942","citation_count":6,"is_preprint":false},{"pmid":"29742494","id":"PMC_29742494","title":"ILKAP Binding to and Dephosphorylating HIF-1α is Essential for Apoptosis Induced by Severe Hypoxia.","date":"2018","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/29742494","citation_count":4,"is_preprint":false},{"pmid":"25872452","id":"PMC_25872452","title":"Endometrial ILKAP expression among patients with endometriosis and its association with clinical characteristics.","date":"2015","source":"International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics","url":"https://pubmed.ncbi.nlm.nih.gov/25872452","citation_count":2,"is_preprint":false},{"pmid":"38379270","id":"PMC_38379270","title":"ILKAP Promotes the Metastasis of Hepatocellular Carcinoma Cells by Inhibiting β-Catenin Degradation and Enhancing the WNT Signaling Pathway.","date":"2024","source":"Advanced biology","url":"https://pubmed.ncbi.nlm.nih.gov/38379270","citation_count":2,"is_preprint":false},{"pmid":"41454076","id":"PMC_41454076","title":"ILKAP drives hepatocellular carcinoma progression by modulating PGAM1-mediated glycolytic reprogramming.","date":"2025","source":"Frontiers of medicine","url":"https://pubmed.ncbi.nlm.nih.gov/41454076","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7894,"output_tokens":2926,"usd":0.033786},"stage2":{"model":"claude-opus-4-6","input_tokens":6364,"output_tokens":3184,"usd":0.16713},"total_usd":0.200916,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"ILKAP, a PP2C family serine/threonine phosphatase, was identified as a binding partner of integrin-linked kinase ILK1 via yeast two-hybrid screen, and their association is independent of the catalytic activity of either partner. Conditional ILKAP expression selectively inhibited ECM- and growth factor-stimulated ILK1 kinase activity; a catalytic dead mutant H154D failed to inhibit ILK1. ILKAP selectively inhibited GSK3β phosphorylation on Ser9 (downstream of ILK1) without affecting PKB/AKT Ser473 phosphorylation, and suppressed Tcf/Lef (TOPFlash) reporter transactivation, placing ILKAP in the ILK1–GSK3β–Wnt signaling axis.\",\n      \"method\": \"Yeast two-hybrid, co-precipitation, conditional expression in HEK293 cells, ILK immune complex kinase assay, catalytic mutant (H154D), Tcf/Lef luciferase reporter\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (two-hybrid, co-IP, kinase assay, reporter assay, mutagenesis) in a single foundational study, 120 citations\",\n      \"pmids\": [\"11331582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Endogenous ILKAP selectively inhibits ILK-mediated GSK3β Ser9 phosphorylation without affecting PKB Ser473. siRNA silencing of ILKAP stimulated GSK3β-S9 phosphorylation and S-phase entry; ILKAP overexpression increased G1 fraction, reduced cyclin D1 levels, and suppressed anchorage-independent growth in LNCaP cells. Overexpression of ILK rescued ILKAP-mediated GSK3β inhibition; dominant-negative ILK did not, establishing ILKAP acts through ILK catalytic activity.\",\n      \"method\": \"siRNA knockdown, stable and transient overexpression, ILK immune complex kinase assay, flow cytometry, soft-agar anchorage-independent growth assay, Western blot\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain/loss-of-function with multiple readouts replicating the foundational study, 69 citations\",\n      \"pmids\": [\"14990992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ILKAP promotes CRM1-dependent nuclear export of ILK, restricting nuclear ILK accumulation. Nuclear ILK was associated with increased DNA synthesis in epidermal keratinocytes, and this proliferative effect was sensitive to ILKAP-mediated export, establishing ILKAP as a modulator of ILK subcellular localization with functional consequences for keratinocyte proliferation.\",\n      \"method\": \"Live-cell imaging, nuclear fractionation, CRM1 inhibition (leptomycin B), DNA synthesis assay (BrdU incorporation)\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiments with functional consequence, single lab\",\n      \"pmids\": [\"18635968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ILKAP contains a nuclear localization signal (NLS) between residues 71–86 (critical residues Lys-78 and Arg-79) that mediates nuclear import via importin α1, α3, and α5. Nuclear ILKAP interacts with RSK2 and induces apoptosis by inhibiting RSK2 kinase activity and downregulating cyclin D1 expression.\",\n      \"method\": \"Immunofluorescence of endogenous and tagged ILKAP, co-immunoprecipitation with importin isoforms, NLS deletion mutant, RSK2 kinase assay, cyclin D1 Western blot, apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — multiple orthogonal methods (mutagenesis, co-IP, kinase assay, localization) in one study\",\n      \"pmids\": [\"23329845\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"ILKAP binds phosphopeptides derived from known PP2C substrates including p38, ATM, Chk1, Chk2 and RSK2 in a phosphorylation-dependent and sequence-context-dependent manner, establishing these phosphoproteins as candidate ILKAP substrates and confirming ILKAP's substrate-binding selectivity.\",\n      \"method\": \"Solid-phase phosphopeptide affinity pull-down from cell lysates, competitive binding assays\",\n      \"journal\": \"Molecular bioSystems\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — affinity pull-down establishing candidate substrate interactions, single lab\",\n      \"pmids\": [\"22348942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ILKAP depletion sensitizes p53-wildtype glioblastoma cells to radiation, and radiation-induced phosphorylation of DNA-PK (DNAPK) is dependent on ILKAP, identifying DNAPK as a downstream mediator of ILKAP signaling in the DNA damage response.\",\n      \"method\": \"siRNA knockdown, γH2AX/53BP1 foci quantification, Western blot for DNAPK phosphorylation, clonogenic survival assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional KD with defined molecular readout, single lab\",\n      \"pmids\": [\"26460618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In ovarian cancer cells, ILKAP dephosphorylates both p90RSK (RSK1/RSK2) and ILK/AKT. HGF pre-treatment upregulates ILKAP expression and reverts CDDP-induced RSK phosphorylation. ILKAP silencing protects cells from CDDP-induced death through simultaneous increased activity of RSK and ILK/AKT, requiring combined inhibition of p90RSK and ILK to rescue ILKAP-loss phenotype, establishing ILKAP as a regulatory hub controlling multiple pro-survival kinases.\",\n      \"method\": \"siRNA knockdown, pharmacological inhibitors, Western blot, cell viability and apoptosis assays\",\n      \"journal\": \"European journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — combinatorial epistasis with kinase inhibitors and multiple siRNA knockdowns, single lab\",\n      \"pmids\": [\"27065457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MAEL promotes lysosome-dependent degradation of ILKAP, leading to increased phosphorylation of ILKAP substrates p38, CHK1 and RSK2. ILKAP overexpression reversed the oncogenic effects of MAEL, placing MAEL upstream of ILKAP in a gastric cancer progression pathway.\",\n      \"method\": \"Co-expression/silencing experiments, Western blot, lysosome inhibitor treatment, adenovirus-mediated ILKAP overexpression, xenograft models\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis with rescue experiment and in vivo validation, single lab\",\n      \"pmids\": [\"29371914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ILKAP physically interacts with HIF-1α (co-immunoprecipitation) and induces its dephosphorylation; both the HIF-1α–p53 interaction and hypoxia-induced apoptosis require ILKAP, identifying HIF-1α as a novel ILKAP substrate.\",\n      \"method\": \"Co-immunoprecipitation, HRE-luciferase reporter, trypan blue viability assay, shRNA knockdown, overexpression\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus functional gain/loss-of-function, single lab\",\n      \"pmids\": [\"29742494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ILKAP interacts with β-catenin and dephosphorylates GSK3β and CK1, thereby reducing β-catenin ubiquitination and increasing β-catenin protein stability. ILKAP also mediates binding between TCF4 and β-catenin to enhance Wnt target gene expression (c-Myc, CyclinD1), promoting HCC metastasis in vitro and in a zebrafish xenograft model.\",\n      \"method\": \"Co-immunoprecipitation, Western blot, ubiquitination assay, Wnt reporter assay, zebrafish xenograft, siRNA knockdown\",\n      \"journal\": \"Advanced biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple biochemical assays with in vivo validation, single lab\",\n      \"pmids\": [\"38379270\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ILKAP knockdown suppresses PGAM1 expression in HCC cells; restoring PGAM1 in ILKAP-knockdown cells rescues proliferation and invasion, and ILKAP depletion reduces extracellular acidification rate, establishing an ILKAP–PGAM1 axis in glycolytic reprogramming and tumor progression.\",\n      \"method\": \"RNA sequencing, siRNA knockdown, PGAM1 rescue overexpression, Seahorse glycolysis assay, xenograft models\",\n      \"journal\": \"Frontiers of medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — epistasis by rescue experiment with metabolic readout, single lab, no citations yet\",\n      \"pmids\": [\"41454076\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ILKAP is a PP2C-family serine/threonine phosphatase that localizes to both nucleus and cytoplasm (nuclear import via importin α–NLS interaction, nuclear export enhanced by CRM1); it binds ILK1 and selectively dephosphorylates GSK3β (suppressing Wnt/Tcf signaling and cyclin D1) without affecting AKT, dephosphorylates nuclear RSK2 (inducing apoptosis), dephosphorylates HIF-1α, GSK3β/CK1 (stabilizing β-catenin), and modulates PGAM1-driven glycolysis, while its own protein level is regulated by MAEL-dependent lysosomal degradation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ILKAP is a PP2C-family serine/threonine phosphatase that functions as a negative regulator of multiple pro-survival and proliferative kinase pathways by selectively dephosphorylating GSK3β, RSK2, HIF-1α, and CK1. It was identified as a binding partner of integrin-linked kinase (ILK1) and selectively inhibits ILK1-mediated GSK3β Ser9 phosphorylation without affecting PKB/AKT Ser473, thereby suppressing Wnt/Tcf signaling and cyclin D1 expression, reducing S-phase entry and anchorage-independent growth [PMID:11331582, PMID:14990992]. ILKAP shuttles between nucleus and cytoplasm via an importin α–dependent NLS and CRM1-dependent export; nuclear ILKAP dephosphorylates RSK2 to induce apoptosis and promotes CRM1-dependent nuclear export of ILK to limit ILK-driven proliferation [PMID:23329845, PMID:18635968]. ILKAP protein stability is controlled by MAEL-dependent lysosomal degradation, and ILKAP additionally modulates β-catenin stability through dephosphorylation of GSK3β/CK1, regulates HIF-1α–p53–dependent apoptosis, and sustains PGAM1-driven glycolysis in hepatocellular carcinoma [PMID:29371914, PMID:38379270, PMID:29742494, PMID:41454076].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established ILKAP as a PP2C-family phosphatase that physically associates with ILK1 and selectively dephosphorylates GSK3β Ser9 (but not AKT Ser473) to suppress Wnt/Tcf signaling, answering how ILK kinase activity is negatively regulated.\",\n      \"evidence\": \"Yeast two-hybrid, co-precipitation, catalytic mutant H154D, ILK immune complex kinase assay, Tcf/Lef luciferase reporter in HEK293 cells\",\n      \"pmids\": [\"11331582\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct phosphatase activity on GSK3β peptide not demonstrated in vitro\",\n        \"Endogenous interaction stoichiometry unknown\",\n        \"Mechanism of selectivity for GSK3β over AKT not resolved\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Reciprocal gain- and loss-of-function confirmed endogenous ILKAP restrains ILK–GSK3β signaling to control cell cycle progression and anchorage-independent growth, establishing ILKAP as a tumor-suppressive phosphatase.\",\n      \"evidence\": \"siRNA knockdown and stable overexpression in LNCaP cells, flow cytometry, soft-agar colony assay, cyclin D1 Western blot\",\n      \"pmids\": [\"14990992\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether ILKAP directly dephosphorylates GSK3β or acts indirectly through ILK remains unresolved\",\n        \"Mechanism linking cyclin D1 loss to G1 arrest not dissected beyond GSK3β\"\n      ]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed that ILKAP promotes CRM1-dependent nuclear export of ILK, restricting nuclear ILK accumulation and ILK-driven DNA synthesis, thereby establishing ILKAP as a regulator of ILK subcellular distribution.\",\n      \"evidence\": \"Live-cell imaging, nuclear fractionation, leptomycin B treatment, BrdU incorporation in keratinocytes\",\n      \"pmids\": [\"18635968\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether ILKAP directly escorts ILK or modifies a separate export signal is unknown\",\n        \"Not tested in non-keratinocyte cell types\"\n      ]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Phosphopeptide affinity studies identified p38, ATM, Chk1, Chk2, and RSK2 as candidate ILKAP substrates, expanding the substrate repertoire beyond GSK3β.\",\n      \"evidence\": \"Solid-phase phosphopeptide pull-down with competitive binding assays\",\n      \"pmids\": [\"22348942\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No in vitro dephosphorylation assays performed to confirm catalytic activity on these substrates\",\n        \"Binding ≠ dephosphorylation; functional validation needed for each candidate\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapped an NLS (residues 71–86, critical K78/R79) mediating importin α–dependent nuclear import and showed nuclear ILKAP dephosphorylates RSK2 to induce apoptosis and reduce cyclin D1, answering how ILKAP reaches the nucleus and identifying its nuclear substrate.\",\n      \"evidence\": \"NLS deletion mutant, importin co-IP, RSK2 kinase assay, apoptosis assay, immunofluorescence in HeLa cells\",\n      \"pmids\": [\"23329845\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct dephosphorylation of RSK2 not shown with purified components\",\n        \"Relative contribution of nuclear vs. cytoplasmic ILKAP pools to apoptosis not quantified\"\n      ]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"ILKAP depletion sensitized glioblastoma cells to radiation and impaired DNA-PK phosphorylation, linking ILKAP to the DNA damage response for the first time.\",\n      \"evidence\": \"siRNA knockdown, γH2AX/53BP1 foci, DNA-PK phosphorylation Western blot, clonogenic survival in glioblastoma cells\",\n      \"pmids\": [\"26460618\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether ILKAP directly dephosphorylates DNA-PK or acts indirectly is unresolved\",\n        \"Only tested in p53-wildtype glioblastoma context\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed ILKAP functions as a dual phosphatase hub controlling both RSK and ILK/AKT in ovarian cancer, with combined inhibition required to phenocopy ILKAP activity in cisplatin-induced death.\",\n      \"evidence\": \"siRNA knockdown, pharmacological inhibitors of RSK and ILK, apoptosis and viability assays in ovarian cancer cells\",\n      \"pmids\": [\"27065457\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Contradicts earlier finding that ILKAP does not affect AKT; cell-type dependence not clarified\",\n        \"ILKAP direct activity on AKT versus ILK not biochemically separated\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified MAEL as an upstream negative regulator that targets ILKAP for lysosomal degradation, increasing phosphorylation of p38, CHK1, and RSK2—answering how ILKAP protein levels are controlled.\",\n      \"evidence\": \"Co-expression/silencing, lysosome inhibitor rescue, adenoviral ILKAP overexpression, xenograft in gastric cancer model\",\n      \"pmids\": [\"29371914\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism by which MAEL directs ILKAP to lysosomes (adapter, ubiquitin signal) is unknown\",\n        \"Not confirmed in non-gastric cancer contexts\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified HIF-1α as a direct ILKAP-interacting substrate whose dephosphorylation is required for HIF-1α–p53 complex formation and hypoxia-induced apoptosis.\",\n      \"evidence\": \"Co-immunoprecipitation, HRE-luciferase reporter, shRNA knockdown, overexpression, viability assays\",\n      \"pmids\": [\"29742494\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Specific phosphosite(s) on HIF-1α targeted by ILKAP not mapped\",\n        \"In vitro phosphatase assay with purified ILKAP and HIF-1α not performed\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstrated ILKAP dephosphorylates GSK3β and CK1 to stabilize β-catenin by reducing its ubiquitination, and bridges TCF4–β-catenin interaction to promote Wnt target gene expression and HCC metastasis—revealing an unexpected pro-oncogenic role.\",\n      \"evidence\": \"Co-immunoprecipitation, ubiquitination assay, Wnt reporter, siRNA knockdown, zebrafish xenograft\",\n      \"pmids\": [\"38379270\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Contradicts earlier tumor-suppressive role via GSK3β dephosphorylation; context-dependent regulation not reconciled\",\n        \"TCF4–β-catenin bridging mechanism (scaffold vs. dephosphorylation) not dissected\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked ILKAP to glycolytic reprogramming by showing ILKAP sustains PGAM1 expression and extracellular acidification in HCC, with PGAM1 rescue reversing ILKAP-knockdown proliferation defects.\",\n      \"evidence\": \"RNA sequencing, siRNA knockdown, PGAM1 rescue, Seahorse ECAR assay, xenograft in HCC cells\",\n      \"pmids\": [\"41454076\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether ILKAP regulates PGAM1 transcriptionally or post-translationally is unknown\",\n        \"No direct phosphatase–substrate relationship established between ILKAP and PGAM1\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A unifying model reconciling ILKAP's apparently contradictory tumor-suppressive (GSK3β dephosphorylation suppressing Wnt) and tumor-promoting (β-catenin stabilization, PGAM1-driven glycolysis) activities across tissue contexts is lacking.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No structural data for ILKAP exist to explain substrate selectivity\",\n        \"In vitro reconstitution of direct phosphatase activity on most claimed substrates has not been performed\",\n        \"Tissue- and context-specific regulation of ILKAP activity/expression is poorly defined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\n        \"term_id\": \"GO:0140096\",\n        \"supporting_discovery_ids\": [0, 1, 3, 6, 8, 9]\n      },\n      {\n        \"term_id\": \"GO:0098772\",\n        \"supporting_discovery_ids\": [0, 1, 2, 6]\n      }\n    ],\n    \"localization\": [\n      {\n        \"term_id\": \"GO:0005634\",\n        \"supporting_discovery_ids\": [2, 3]\n      },\n      {\n        \"term_id\": \"GO:0005829\",\n        \"supporting_discovery_ids\": [0, 1, 2]\n      }\n    ],\n    \"pathway\": [\n      {\n        \"term_id\": \"R-HSA-162582\",\n        \"supporting_discovery_ids\": [0, 1, 6, 9]\n      },\n      {\n        \"term_id\": \"R-HSA-1640170\",\n        \"supporting_discovery_ids\": [1, 3]\n      },\n      {\n        \"term_id\": \"R-HSA-5357801\",\n        \"supporting_discovery_ids\": [3, 8]\n      }\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"ILK\",\n      \"RSK2\",\n      \"GSK3B\",\n      \"HIF1A\",\n      \"CTNNB1\",\n      \"MAEL\",\n      \"PGAM1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}