{"gene":"PABIR1","run_date":"2026-06-10T05:19:53","timeline":{"discoveries":[{"year":2016,"finding":"FAM122A directly interacts with PP2A-Aα and B55α subunits (but not B56α) and inhibits the phosphatase activity of the PP2A-Aα/B55α/Cα holoenzyme; additionally, FAM122A promotes poly-ubiquitination and proteasomal degradation of the catalytic subunit PP2A-Cα.","method":"Co-immunoprecipitation, phosphatase activity assay, ubiquitination assay, overexpression and knockdown in cell lines","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and in-cell phosphatase activity assay, single lab, two orthogonal methods","pmids":["27588481"],"is_preprint":false},{"year":2018,"finding":"FAM122A is SUMOylated at lysine 89, a modification that can be removed by the SUMO protease SENP1; SUMOylation of FAM122A reduces PP2A-Cα protein levels and PP2A phosphatase activity, thereby suppressing cell proliferation.","method":"Site-directed mutagenesis (K89 mutation), Co-IP/SUMO conjugation assay, SENP1 de-conjugation assay, phosphatase activity assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis identifying specific SUMO site plus functional read-out, single lab, two orthogonal methods","pmids":["29678583"],"is_preprint":false},{"year":2020,"finding":"CHK1 directly phosphorylates FAM122A; loss of FAM122A (CRISPR KO) activates PP2A-B55α, which dephosphorylates WEE1 and rescues it from ubiquitin-mediated degradation, elevating WEE1 protein and thereby reducing replication stress and conferring resistance to CHK1 inhibitors.","method":"CRISPR knockout, in vitro kinase assay (CHK1 phosphorylation of FAM122A), immunoblot for WEE1 ubiquitination and protein level, PP2A activity assay, genetic rescue experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (kinase assay, KO, ubiquitination, phosphatase activity) in a single rigorous study","pmids":["33108758"],"is_preprint":false},{"year":2020,"finding":"FAM122A directly interacts with the C-terminal zinc finger domain of GATA1, reducing GATA1 chromatin occupancy on target gene promoters and suppressing GATA1 transcriptional activity, thereby inhibiting erythroid differentiation.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), overexpression in primary human hematopoietic progenitor cells and erythroleukemia cells","journal":"Stem cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ChIP demonstrating reduced chromatin occupancy, single lab, two orthogonal methods","pmids":["32763160"],"is_preprint":false},{"year":2020,"finding":"FAM122A interacts with TOP2α (but not TOP2β); FAM122A deletion increases TOP2α protein level and enhances TOP2 poison-induced DNA damage in cancer cells without affecting ROS levels or short-term DNA repair, suggesting FAM122A maintains DNA stability by modulating TOP2α.","method":"Co-immunoprecipitation, CRISPR KO, γH2AX immunofluorescence/foci, comet assay, ROS measurement","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP showing interaction specificity plus multiple damage readouts, single lab","pmids":["32866497"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM structures of PP2A:B55 bound to FAM122A (and separately to phosphorylated ARPP19) reveal that FAM122A, an intrinsically disordered protein, binds PP2A:B55 through multiple distinct sites on the B55 subunit in a manner different from ARPP19; NMR and biochemical data show how substrates and inhibitors are recruited to PP2A:B55.","method":"Single-particle cryo-EM, NMR spectroscopy, biochemical binding assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution cryo-EM structures with complementary NMR and biochemical validation in a single rigorous study","pmids":["38123684"],"is_preprint":false},{"year":2023,"finding":"FAM122A contains a conserved short linear motif (SLiM) [RK]-V-x-x-[VI]-R that forms a short α-helix required for binding to the top groove of B55α; a second adjacent helix occludes the catalytic subunit, making FAM122A a substrate-competitive inhibitor of B55α/PP2A that prevents CDK substrate dephosphorylation; FAM122A deficiency abrogates G1/S and intra-S phase checkpoints and attenuates CHK1/CHK2 activation under replication stress.","method":"Mutagenesis of SLiM, AlphaFold2 structural prediction, in vitro competition/binding assays, dephosphorylation assays in cell lysates, CRISPR KO in HEK293, flow cytometry cell-cycle analysis","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis, structural modeling, and biochemical competition assays; preprint later peer-reviewed (PMID 38982062)","pmids":["36945596"],"is_preprint":true},{"year":2024,"finding":"FAM122A uses helical motifs (a substrate-docking SHeM [RK]-V-x-x-[VI]-R helix and an adjacent catalytic-subunit-occluding helix) to act as a substrate-competitive inhibitor of B55α/PP2A; FAM122A deficiency impairs G1/S and intra-S phase checkpoint responses and attenuates CHK1 and CHK2 activation after replication stress.","method":"Mutagenesis of helical motifs, in vitro competition assay, CDK-substrate dephosphorylation assay in cell lysates, CRISPR KO, flow cytometry, immunoblot for checkpoint kinases","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis identifying functional helices, biochemical reconstitution of competitive inhibition, and clean KO phenotypes; single lab but multiple orthogonal methods","pmids":["38982062"],"is_preprint":false},{"year":2024,"finding":"FAM122A-dependent inhibition of PP2A-B55 is the initial mitotic-entry event: FAM122A permits phosphorylation of early mitotic substrates by cyclin A/Cdk, enabling full cyclin B/Cdk1 and Greatwall kinase activation; subsequently, Greatwall-phosphorylated Arpp19/ENSA competes with FAM122A and displaces it from PP2A-B55, taking over phosphatase inhibition for the remainder of mitosis. Loss of the FAM122A orthologue in C. elegans prevents germline stem cells from entering mitosis.","method":"Xenopus egg extract biochemistry (add-back/depletion), C. elegans RNAi (germline mitotic entry assay), immunoblot for Cdk substrate phosphorylation, Greatwall/ENSA phosphorylation assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — Xenopus egg extract reconstitution plus C. elegans genetic loss-of-function, multiple orthogonal readouts, independent from structural studies","pmids":["38378890"],"is_preprint":false},{"year":2023,"finding":"FAM122A is required for mesendodermal specification and cardiac differentiation from mouse embryonic stem cells; Fam122a conditional cardiac KO causes embryonic lethality with cardiovascular defects; the differentiation defect is mechanistically linked to dysregulation of histone modifications and Wnt and Hippo signalling pathways through modulation of PP2A activity.","method":"Conditional KO mice, mouse ESC differentiation assays, PP2A activity assay, histone modification immunoblot, Wnt/Hippo pathway reporter assays","journal":"Stem cells (Dayton, Ohio)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean genetic KO with defined developmental phenotypes and pathway readouts, single lab","pmids":["36715298"],"is_preprint":false},{"year":2026,"finding":"FAM122A inhibits PP2A-B55 through a bipartite binding mechanism: the N-terminal helices and a novel C-terminal region (residues 150–170) are both required for efficient binding; conserved Ser158 within this C-terminal region is important for PP2A-B55 inhibition in human cells and for stimulation of mitotic entry in Xenopus egg extracts; Ser158 phosphorylation occupancy increases at cell cycle stages requiring PP2A-B55 inhibition.","method":"Systematic alanine scanning of residues 150–170, phospho-site mutagenesis (Ser158), Co-IP in human cells, Xenopus egg extract mitotic-entry assay, phosphoproteomics/cell-cycle staging","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional read-out in two orthogonal systems (human cells + Xenopus extracts), preprint, single lab","pmids":["41928937"],"is_preprint":true}],"current_model":"PABIR1/FAM122A is an intrinsically disordered, endogenous inhibitor of the PP2A:B55α holoenzyme that uses N-terminal helical motifs (docking via a conserved [RK]-V-x-x-[VI]-R SLiM) and a C-terminal bipartite binding region (including phospho-Ser158) to competitively block substrate access to both the B55α regulatory subunit and the catalytic subunit; it triggers mitotic entry by inhibiting PP2A-B55 ahead of ARPP19/ENSA, is directly phosphorylated by CHK1 to modulate WEE1 stability and replication-stress responses, can be SUMOylated at K89 (reversed by SENP1) to further reduce PP2A-Cα levels, interacts with GATA1 to suppress erythroid differentiation, and is required for mesendodermal/cardiac differentiation through PP2A-dependent regulation of histone modifications and Wnt/Hippo signalling."},"narrative":{"mechanistic_narrative":"PABIR1 (FAM122A) is an intrinsically disordered, endogenous substrate-competitive inhibitor of the PP2A:B55α holoenzyme that couples phosphatase regulation to cell-cycle progression, the DNA replication-stress response, and developmental signalling [PMID:27588481, PMID:38123684, PMID:38378890]. It binds the PP2A-Aα and B55α subunits (but not B56α) and inhibits holoenzyme activity, and additionally promotes poly-ubiquitination and proteasomal degradation of the catalytic PP2A-Cα subunit [PMID:27588481]. Structurally, FAM122A engages B55α through a conserved N-terminal SLiM ([RK]-V-x-x-[VI]-R) that folds into a short helix docking in the B55α top groove, while an adjacent helix occludes the catalytic subunit to block CDK-substrate dephosphorylation; efficient inhibition also requires a C-terminal region (residues 150–170) containing a cell-cycle-regulated phospho-Ser158, defining a bipartite binding mode [PMID:38123684, PMID:38982062, PMID:41928937]. Functionally, FAM122A-dependent inhibition of PP2A-B55 is the initiating event of mitotic entry, permitting cyclin A/Cdk substrate phosphorylation and full cyclin B/Cdk1 and Greatwall activation before Greatwall-phosphorylated ARPP19/ENSA displaces it to sustain phosphatase inhibition through mitosis [PMID:38378890]. CHK1 directly phosphorylates FAM122A, and its loss activates PP2A-B55α to dephosphorylate and stabilize WEE1, abrogating G1/S and intra-S checkpoints and conferring resistance to CHK1 inhibitors [PMID:33108758, PMID:38982062]. Beyond cell-cycle control, FAM122A is SUMOylated at K89 (reversed by SENP1) to further reduce PP2A-Cα [PMID:29678583], interacts with the GATA1 zinc-finger to suppress erythroid differentiation [PMID:32763160], and is required for mesendodermal/cardiac differentiation via PP2A-dependent regulation of histone modifications and Wnt/Hippo signalling [PMID:36715298].","teleology":[{"year":2016,"claim":"Established FAM122A as a direct binding partner and inhibitor of a specific PP2A holoenzyme, defining its core molecular activity.","evidence":"Reciprocal Co-IP, in-cell phosphatase and ubiquitination assays with overexpression/knockdown in cell lines","pmids":["27588481"],"confidence":"Medium","gaps":["Did not resolve binding interface or distinguish inhibition from catalytic-subunit degradation as the primary mechanism","B-subunit specificity tested only against B56α"]},{"year":2018,"claim":"Showed FAM122A activity is itself post-translationally tuned, linking SUMOylation to PP2A-Cα abundance and proliferation control.","evidence":"K89 site-directed mutagenesis, SUMO conjugation/SENP1 de-conjugation assays and phosphatase activity readout in cells","pmids":["29678583"],"confidence":"Medium","gaps":["SUMO E3 ligase and physiological trigger for K89 SUMOylation not identified","Mechanistic link between FAM122A SUMOylation and reduced PP2A-Cα not resolved"]},{"year":2020,"claim":"Placed FAM122A in the replication-stress checkpoint by showing CHK1 phosphorylates it and its loss stabilizes WEE1 via PP2A-B55α, explaining CHK1-inhibitor resistance.","evidence":"In vitro CHK1 kinase assay, CRISPR KO, WEE1 ubiquitination/immunoblot, PP2A activity assay and genetic rescue","pmids":["33108758"],"confidence":"High","gaps":["CHK1 phosphosite(s) on FAM122A and their functional consequence not mapped here","Whether WEE1 is a direct PP2A-B55α substrate not fully resolved"]},{"year":2020,"claim":"Extended FAM122A function beyond PP2A by identifying transcriptional (GATA1) and genome-stability (TOP2α) partners.","evidence":"Co-IP and ChIP in hematopoietic progenitors/erythroleukemia cells (GATA1); Co-IP, CRISPR KO, γH2AX and comet assays (TOP2α)","pmids":["32763160","32866497"],"confidence":"Medium","gaps":["Whether GATA1 and TOP2α effects are PP2A-dependent or independent not established","Direct vs indirect nature of TOP2α protein-level regulation unclear"]},{"year":2023,"claim":"Resolved the structural basis of inhibition, revealing FAM122A as a disordered protein engaging B55α at multiple sites distinct from ARPP19.","evidence":"Single-particle cryo-EM of PP2A:B55–FAM122A complexes, NMR and biochemical binding assays","pmids":["38123684"],"confidence":"High","gaps":["Functional dissection of individual contact sites limited within structural study","C-terminal phospho-dependent contacts not yet defined"]},{"year":2024,"claim":"Defined the inhibitory mechanism at residue resolution: a docking SLiM helix plus a catalytic-subunit-occluding helix make FAM122A substrate-competitive and checkpoint-essential.","evidence":"Helical-motif mutagenesis, in vitro competition and CDK-substrate dephosphorylation assays, CRISPR KO, flow cytometry, checkpoint-kinase immunoblot","pmids":["36945596","38982062"],"confidence":"High","gaps":["How FAM122A inhibition is relieved at mitotic exit not addressed","Contribution of C-terminal region not yet incorporated"]},{"year":2024,"claim":"Established FAM122A as the initiating PP2A-B55 inhibitor at mitotic entry, ordering its action ahead of the ARPP19/ENSA hand-off and showing conservation in vivo.","evidence":"Xenopus egg extract add-back/depletion biochemistry and C. elegans germline RNAi with Cdk-substrate and Greatwall/ENSA phosphorylation readouts","pmids":["38378890"],"confidence":"High","gaps":["Trigger that switches inhibition from FAM122A to ARPP19/ENSA not fully defined","Regulation of FAM122A levels/activity across the cell cycle not resolved here"]},{"year":2023,"claim":"Connected FAM122A to embryonic development, showing it is required for mesendoderm/cardiac differentiation through PP2A-dependent chromatin and Wnt/Hippo control.","evidence":"Conditional KO mice, mouse ESC differentiation, PP2A activity assays, histone-modification immunoblot and Wnt/Hippo reporters","pmids":["36715298"],"confidence":"Medium","gaps":["Direct PP2A substrates mediating histone and Wnt/Hippo changes not identified","Cell-type specificity of the requirement not resolved"]},{"year":2026,"claim":"Revised the binding model to bipartite, adding a C-terminal region and a cell-cycle-regulated phospho-Ser158 as determinants of efficient PP2A-B55 inhibition.","evidence":"Alanine scanning of residues 150–170, Ser158 phospho-mutagenesis, Co-IP in human cells, Xenopus mitotic-entry assay and phosphoproteomic staging (preprint)","pmids":["41928937"],"confidence":"Medium","gaps":["Preprint; kinase phosphorylating Ser158 not identified","Structural basis of the C-terminal contact not yet resolved"]},{"year":null,"claim":"How FAM122A integrates its PP2A-inhibitory role with its PP2A-independent partners (GATA1, TOP2α) and how its activity is switched on and off across the cell cycle remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Upstream signals regulating FAM122A abundance/modification not unified","Whether transcriptional and genome-stability functions are PP2A-dependent unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5,7,8]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[7]}],"localization":[],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[8,7]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[2,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[9]}],"complexes":[],"partners":["PPP2R1A","PPP2R2A","PPP2CA","CHEK1","GATA1","TOP2A","SENP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96E09","full_name":"PPP2R1A-PPP2R2A-interacting phosphatase regulator 1","aliases":["PABIR family member 1"],"length_aa":287,"mass_kda":30.5,"function":"Acts as an inhibitor of serine/threonine-protein phosphatase 2A (PP2A) activity (PubMed:27588481, PubMed:33108758, PubMed:38123684). Inhibits PP2A activity by blocking the substrate binding site on PPP2R2A and the active site of PPP2CA (PubMed:38123684). Potentiates ubiquitin-mediated proteasomal degradation of serine/threonine-protein phosphatase 2A catalytic subunit alpha (PPP2CA) (PubMed:27588481). Inhibits PP2A-mediated dephosphorylation of WEE1, promoting ubiquitin-mediated proteolysis of WEE1, thereby releasing G2/M checkpoint (PubMed:33108758)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q96E09/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PABIR1","classification":"Not Classified","n_dependent_lines":310,"n_total_lines":1208,"dependency_fraction":0.25662251655629137},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PABIR1","total_profiled":1310},"omim":[],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear bodies","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PABIR1"},"hgnc":{"alias_symbol":["MGC17347"],"prev_symbol":["C9orf42","FAM122A"]},"alphafold":{"accession":"Q96E09","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96E09","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96E09-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96E09-F1-predicted_aligned_error_v6.png","plddt_mean":59.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PABIR1","jax_strain_url":"https://www.jax.org/strain/search?query=PABIR1"},"sequence":{"accession":"Q96E09","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96E09.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96E09/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96E09"}},"corpus_meta":[{"pmid":"33108758","id":"PMC_33108758","title":"CHK1 Inhibitor Blocks Phosphorylation of FAM122A and Promotes Replication Stress.","date":"2020","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/33108758","citation_count":50,"is_preprint":false},{"pmid":"27588481","id":"PMC_27588481","title":"FAM122A, a new endogenous inhibitor of protein phosphatase 2A.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/27588481","citation_count":34,"is_preprint":false},{"pmid":"38123684","id":"PMC_38123684","title":"Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.","date":"2023","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/38123684","citation_count":32,"is_preprint":false},{"pmid":"31711919","id":"PMC_31711919","title":"FAM122A supports the growth of hepatocellular carcinoma cells and its deletion enhances Doxorubicin-induced cytotoxicity.","date":"2019","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/31711919","citation_count":13,"is_preprint":false},{"pmid":"38982062","id":"PMC_38982062","title":"FAM122A ensures cell cycle interphase progression and checkpoint control by inhibiting B55α/PP2A through helical motifs.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38982062","citation_count":8,"is_preprint":false},{"pmid":"32763160","id":"PMC_32763160","title":"FAM122A Inhibits Erythroid Differentiation through GATA1.","date":"2020","source":"Stem cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/32763160","citation_count":6,"is_preprint":false},{"pmid":"38378890","id":"PMC_38378890","title":"Increases in cyclin A/Cdk activity and in PP2A-B55 inhibition by FAM122A are key mitosis-inducing events.","date":"2024","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/38378890","citation_count":5,"is_preprint":false},{"pmid":"29678583","id":"PMC_29678583","title":"Identifying the SUMO1 modification of FAM122A leading to the degradation of PP2A-Cα by ubiquitin-proteasome system.","date":"2018","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/29678583","citation_count":4,"is_preprint":false},{"pmid":"32866497","id":"PMC_32866497","title":"FAM122A maintains DNA stability possibly through the regulation of topoisomerase IIα expression.","date":"2020","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/32866497","citation_count":3,"is_preprint":false},{"pmid":"36945596","id":"PMC_36945596","title":"FAM122A ensures cell cycle interphase progression and checkpoint control as a SLiM-dependent substrate-competitive inhibitor to the B55⍺/PP2A phosphatase.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/36945596","citation_count":3,"is_preprint":false},{"pmid":"36715298","id":"PMC_36715298","title":"FAM122A Is Required for Mesendodermal and Cardiac Differentiation of Embryonic Stem Cells.","date":"2023","source":"Stem cells (Dayton, Ohio)","url":"https://pubmed.ncbi.nlm.nih.gov/36715298","citation_count":2,"is_preprint":false},{"pmid":"39009214","id":"PMC_39009214","title":"FAM122A functions as a tumor suppressor in oral squamous cell carcinoma.","date":"2024","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/39009214","citation_count":2,"is_preprint":false},{"pmid":"37693408","id":"PMC_37693408","title":"Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.","date":"2023","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/37693408","citation_count":0,"is_preprint":false},{"pmid":"41928937","id":"PMC_41928937","title":"FAM122A inhibition of PP2A-B55 through a bipartite binding mechanism.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41928937","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8363,"output_tokens":3087,"usd":0.035697,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10540,"output_tokens":3643,"usd":0.071887,"stage2_stop_reason":"end_turn"},"total_usd":0.107584,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2016,\n      \"finding\": \"FAM122A directly interacts with PP2A-Aα and B55α subunits (but not B56α) and inhibits the phosphatase activity of the PP2A-Aα/B55α/Cα holoenzyme; additionally, FAM122A promotes poly-ubiquitination and proteasomal degradation of the catalytic subunit PP2A-Cα.\",\n      \"method\": \"Co-immunoprecipitation, phosphatase activity assay, ubiquitination assay, overexpression and knockdown in cell lines\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and in-cell phosphatase activity assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"27588481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"FAM122A is SUMOylated at lysine 89, a modification that can be removed by the SUMO protease SENP1; SUMOylation of FAM122A reduces PP2A-Cα protein levels and PP2A phosphatase activity, thereby suppressing cell proliferation.\",\n      \"method\": \"Site-directed mutagenesis (K89 mutation), Co-IP/SUMO conjugation assay, SENP1 de-conjugation assay, phosphatase activity assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis identifying specific SUMO site plus functional read-out, single lab, two orthogonal methods\",\n      \"pmids\": [\"29678583\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CHK1 directly phosphorylates FAM122A; loss of FAM122A (CRISPR KO) activates PP2A-B55α, which dephosphorylates WEE1 and rescues it from ubiquitin-mediated degradation, elevating WEE1 protein and thereby reducing replication stress and conferring resistance to CHK1 inhibitors.\",\n      \"method\": \"CRISPR knockout, in vitro kinase assay (CHK1 phosphorylation of FAM122A), immunoblot for WEE1 ubiquitination and protein level, PP2A activity assay, genetic rescue experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (kinase assay, KO, ubiquitination, phosphatase activity) in a single rigorous study\",\n      \"pmids\": [\"33108758\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FAM122A directly interacts with the C-terminal zinc finger domain of GATA1, reducing GATA1 chromatin occupancy on target gene promoters and suppressing GATA1 transcriptional activity, thereby inhibiting erythroid differentiation.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), overexpression in primary human hematopoietic progenitor cells and erythroleukemia cells\",\n      \"journal\": \"Stem cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ChIP demonstrating reduced chromatin occupancy, single lab, two orthogonal methods\",\n      \"pmids\": [\"32763160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FAM122A interacts with TOP2α (but not TOP2β); FAM122A deletion increases TOP2α protein level and enhances TOP2 poison-induced DNA damage in cancer cells without affecting ROS levels or short-term DNA repair, suggesting FAM122A maintains DNA stability by modulating TOP2α.\",\n      \"method\": \"Co-immunoprecipitation, CRISPR KO, γH2AX immunofluorescence/foci, comet assay, ROS measurement\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP showing interaction specificity plus multiple damage readouts, single lab\",\n      \"pmids\": [\"32866497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM structures of PP2A:B55 bound to FAM122A (and separately to phosphorylated ARPP19) reveal that FAM122A, an intrinsically disordered protein, binds PP2A:B55 through multiple distinct sites on the B55 subunit in a manner different from ARPP19; NMR and biochemical data show how substrates and inhibitors are recruited to PP2A:B55.\",\n      \"method\": \"Single-particle cryo-EM, NMR spectroscopy, biochemical binding assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution cryo-EM structures with complementary NMR and biochemical validation in a single rigorous study\",\n      \"pmids\": [\"38123684\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FAM122A contains a conserved short linear motif (SLiM) [RK]-V-x-x-[VI]-R that forms a short α-helix required for binding to the top groove of B55α; a second adjacent helix occludes the catalytic subunit, making FAM122A a substrate-competitive inhibitor of B55α/PP2A that prevents CDK substrate dephosphorylation; FAM122A deficiency abrogates G1/S and intra-S phase checkpoints and attenuates CHK1/CHK2 activation under replication stress.\",\n      \"method\": \"Mutagenesis of SLiM, AlphaFold2 structural prediction, in vitro competition/binding assays, dephosphorylation assays in cell lysates, CRISPR KO in HEK293, flow cytometry cell-cycle analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis, structural modeling, and biochemical competition assays; preprint later peer-reviewed (PMID 38982062)\",\n      \"pmids\": [\"36945596\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FAM122A uses helical motifs (a substrate-docking SHeM [RK]-V-x-x-[VI]-R helix and an adjacent catalytic-subunit-occluding helix) to act as a substrate-competitive inhibitor of B55α/PP2A; FAM122A deficiency impairs G1/S and intra-S phase checkpoint responses and attenuates CHK1 and CHK2 activation after replication stress.\",\n      \"method\": \"Mutagenesis of helical motifs, in vitro competition assay, CDK-substrate dephosphorylation assay in cell lysates, CRISPR KO, flow cytometry, immunoblot for checkpoint kinases\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis identifying functional helices, biochemical reconstitution of competitive inhibition, and clean KO phenotypes; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"38982062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"FAM122A-dependent inhibition of PP2A-B55 is the initial mitotic-entry event: FAM122A permits phosphorylation of early mitotic substrates by cyclin A/Cdk, enabling full cyclin B/Cdk1 and Greatwall kinase activation; subsequently, Greatwall-phosphorylated Arpp19/ENSA competes with FAM122A and displaces it from PP2A-B55, taking over phosphatase inhibition for the remainder of mitosis. Loss of the FAM122A orthologue in C. elegans prevents germline stem cells from entering mitosis.\",\n      \"method\": \"Xenopus egg extract biochemistry (add-back/depletion), C. elegans RNAi (germline mitotic entry assay), immunoblot for Cdk substrate phosphorylation, Greatwall/ENSA phosphorylation assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — Xenopus egg extract reconstitution plus C. elegans genetic loss-of-function, multiple orthogonal readouts, independent from structural studies\",\n      \"pmids\": [\"38378890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"FAM122A is required for mesendodermal specification and cardiac differentiation from mouse embryonic stem cells; Fam122a conditional cardiac KO causes embryonic lethality with cardiovascular defects; the differentiation defect is mechanistically linked to dysregulation of histone modifications and Wnt and Hippo signalling pathways through modulation of PP2A activity.\",\n      \"method\": \"Conditional KO mice, mouse ESC differentiation assays, PP2A activity assay, histone modification immunoblot, Wnt/Hippo pathway reporter assays\",\n      \"journal\": \"Stem cells (Dayton, Ohio)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic KO with defined developmental phenotypes and pathway readouts, single lab\",\n      \"pmids\": [\"36715298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"FAM122A inhibits PP2A-B55 through a bipartite binding mechanism: the N-terminal helices and a novel C-terminal region (residues 150–170) are both required for efficient binding; conserved Ser158 within this C-terminal region is important for PP2A-B55 inhibition in human cells and for stimulation of mitotic entry in Xenopus egg extracts; Ser158 phosphorylation occupancy increases at cell cycle stages requiring PP2A-B55 inhibition.\",\n      \"method\": \"Systematic alanine scanning of residues 150–170, phospho-site mutagenesis (Ser158), Co-IP in human cells, Xenopus egg extract mitotic-entry assay, phosphoproteomics/cell-cycle staging\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional read-out in two orthogonal systems (human cells + Xenopus extracts), preprint, single lab\",\n      \"pmids\": [\"41928937\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PABIR1/FAM122A is an intrinsically disordered, endogenous inhibitor of the PP2A:B55α holoenzyme that uses N-terminal helical motifs (docking via a conserved [RK]-V-x-x-[VI]-R SLiM) and a C-terminal bipartite binding region (including phospho-Ser158) to competitively block substrate access to both the B55α regulatory subunit and the catalytic subunit; it triggers mitotic entry by inhibiting PP2A-B55 ahead of ARPP19/ENSA, is directly phosphorylated by CHK1 to modulate WEE1 stability and replication-stress responses, can be SUMOylated at K89 (reversed by SENP1) to further reduce PP2A-Cα levels, interacts with GATA1 to suppress erythroid differentiation, and is required for mesendodermal/cardiac differentiation through PP2A-dependent regulation of histone modifications and Wnt/Hippo signalling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PABIR1 (FAM122A) is an intrinsically disordered, endogenous substrate-competitive inhibitor of the PP2A:B55\\u03b1 holoenzyme that couples phosphatase regulation to cell-cycle progression, the DNA replication-stress response, and developmental signalling [#0, #5, #8]. It binds the PP2A-A\\u03b1 and B55\\u03b1 subunits (but not B56\\u03b1) and inhibits holoenzyme activity, and additionally promotes poly-ubiquitination and proteasomal degradation of the catalytic PP2A-C\\u03b1 subunit [#0]. Structurally, FAM122A engages B55\\u03b1 through a conserved N-terminal SLiM ([RK]-V-x-x-[VI]-R) that folds into a short helix docking in the B55\\u03b1 top groove, while an adjacent helix occludes the catalytic subunit to block CDK-substrate dephosphorylation; efficient inhibition also requires a C-terminal region (residues 150\\u2013170) containing a cell-cycle-regulated phospho-Ser158, defining a bipartite binding mode [#5, #7, #10]. Functionally, FAM122A-dependent inhibition of PP2A-B55 is the initiating event of mitotic entry, permitting cyclin A/Cdk substrate phosphorylation and full cyclin B/Cdk1 and Greatwall activation before Greatwall-phosphorylated ARPP19/ENSA displaces it to sustain phosphatase inhibition through mitosis [#8]. CHK1 directly phosphorylates FAM122A, and its loss activates PP2A-B55\\u03b1 to dephosphorylate and stabilize WEE1, abrogating G1/S and intra-S checkpoints and conferring resistance to CHK1 inhibitors [#2, #7]. Beyond cell-cycle control, FAM122A is SUMOylated at K89 (reversed by SENP1) to further reduce PP2A-C\\u03b1 [#1], interacts with the GATA1 zinc-finger to suppress erythroid differentiation [#3], and is required for mesendodermal/cardiac differentiation via PP2A-dependent regulation of histone modifications and Wnt/Hippo signalling [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2016,\n      \"claim\": \"Established FAM122A as a direct binding partner and inhibitor of a specific PP2A holoenzyme, defining its core molecular activity.\",\n      \"evidence\": \"Reciprocal Co-IP, in-cell phosphatase and ubiquitination assays with overexpression/knockdown in cell lines\",\n      \"pmids\": [\"27588481\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not resolve binding interface or distinguish inhibition from catalytic-subunit degradation as the primary mechanism\", \"B-subunit specificity tested only against B56\\u03b1\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed FAM122A activity is itself post-translationally tuned, linking SUMOylation to PP2A-C\\u03b1 abundance and proliferation control.\",\n      \"evidence\": \"K89 site-directed mutagenesis, SUMO conjugation/SENP1 de-conjugation assays and phosphatase activity readout in cells\",\n      \"pmids\": [\"29678583\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"SUMO E3 ligase and physiological trigger for K89 SUMOylation not identified\", \"Mechanistic link between FAM122A SUMOylation and reduced PP2A-C\\u03b1 not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed FAM122A in the replication-stress checkpoint by showing CHK1 phosphorylates it and its loss stabilizes WEE1 via PP2A-B55\\u03b1, explaining CHK1-inhibitor resistance.\",\n      \"evidence\": \"In vitro CHK1 kinase assay, CRISPR KO, WEE1 ubiquitination/immunoblot, PP2A activity assay and genetic rescue\",\n      \"pmids\": [\"33108758\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"CHK1 phosphosite(s) on FAM122A and their functional consequence not mapped here\", \"Whether WEE1 is a direct PP2A-B55\\u03b1 substrate not fully resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended FAM122A function beyond PP2A by identifying transcriptional (GATA1) and genome-stability (TOP2\\u03b1) partners.\",\n      \"evidence\": \"Co-IP and ChIP in hematopoietic progenitors/erythroleukemia cells (GATA1); Co-IP, CRISPR KO, \\u03b3H2AX and comet assays (TOP2\\u03b1)\",\n      \"pmids\": [\"32763160\", \"32866497\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether GATA1 and TOP2\\u03b1 effects are PP2A-dependent or independent not established\", \"Direct vs indirect nature of TOP2\\u03b1 protein-level regulation unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Resolved the structural basis of inhibition, revealing FAM122A as a disordered protein engaging B55\\u03b1 at multiple sites distinct from ARPP19.\",\n      \"evidence\": \"Single-particle cryo-EM of PP2A:B55\\u2013FAM122A complexes, NMR and biochemical binding assays\",\n      \"pmids\": [\"38123684\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Functional dissection of individual contact sites limited within structural study\", \"C-terminal phospho-dependent contacts not yet defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the inhibitory mechanism at residue resolution: a docking SLiM helix plus a catalytic-subunit-occluding helix make FAM122A substrate-competitive and checkpoint-essential.\",\n      \"evidence\": \"Helical-motif mutagenesis, in vitro competition and CDK-substrate dephosphorylation assays, CRISPR KO, flow cytometry, checkpoint-kinase immunoblot\",\n      \"pmids\": [\"36945596\", \"38982062\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"How FAM122A inhibition is relieved at mitotic exit not addressed\", \"Contribution of C-terminal region not yet incorporated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established FAM122A as the initiating PP2A-B55 inhibitor at mitotic entry, ordering its action ahead of the ARPP19/ENSA hand-off and showing conservation in vivo.\",\n      \"evidence\": \"Xenopus egg extract add-back/depletion biochemistry and C. elegans germline RNAi with Cdk-substrate and Greatwall/ENSA phosphorylation readouts\",\n      \"pmids\": [\"38378890\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Trigger that switches inhibition from FAM122A to ARPP19/ENSA not fully defined\", \"Regulation of FAM122A levels/activity across the cell cycle not resolved here\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected FAM122A to embryonic development, showing it is required for mesendoderm/cardiac differentiation through PP2A-dependent chromatin and Wnt/Hippo control.\",\n      \"evidence\": \"Conditional KO mice, mouse ESC differentiation, PP2A activity assays, histone-modification immunoblot and Wnt/Hippo reporters\",\n      \"pmids\": [\"36715298\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Direct PP2A substrates mediating histone and Wnt/Hippo changes not identified\", \"Cell-type specificity of the requirement not resolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revised the binding model to bipartite, adding a C-terminal region and a cell-cycle-regulated phospho-Ser158 as determinants of efficient PP2A-B55 inhibition.\",\n      \"evidence\": \"Alanine scanning of residues 150\\u2013170, Ser158 phospho-mutagenesis, Co-IP in human cells, Xenopus mitotic-entry assay and phosphoproteomic staging (preprint)\",\n      \"pmids\": [\"41928937\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Preprint; kinase phosphorylating Ser158 not identified\", \"Structural basis of the C-terminal contact not yet resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How FAM122A integrates its PP2A-inhibitory role with its PP2A-independent partners (GATA1, TOP2\\u03b1) and how its activity is switched on and off across the cell cycle remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Upstream signals regulating FAM122A abundance/modification not unified\", \"Whether transcriptional and genome-stability functions are PP2A-dependent unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5, 7, 8]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [8, 7]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PPP2R1A\", \"PPP2R2A\", \"PPP2CA\", \"CHEK1\", \"GATA1\", \"TOP2A\", \"SENP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}