{"gene":"PPP4R2","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2000,"finding":"PPP4R2 (a 50 kDa protein) forms a regulatory subunit of protein phosphatase 4 (PPP4c), physically interacting with PPP4c as confirmed by co-sedimentation of PPP4c with bacterially expressed PPP4R2 and by formation of a 450 kDa complex with baculovirus-expressed His6-tagged PPP4R2. Native 450 kDa and 600 kDa PPP4 complexes are catalytically inactive but can be activated by basic proteins, suggesting PPP4R2 regulates PPP4c activity.","method":"Co-sedimentation assay, baculovirus/bacterial recombinant protein co-purification, native complex purification from mammalian skeletal muscle and testis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with recombinant proteins, biochemical co-purification, and enzymatic activity measurements in multiple systems","pmids":["10769191"],"is_preprint":false},{"year":2000,"finding":"PPP4R2 localizes to centrosomes by immunocytology, suggesting it targets PPP4c to centrosomal microtubule organizing centres.","method":"Immunocytological detection (immunofluorescence microscopy)","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — immunofluorescence localization in a single study; functional consequence inferred but not directly tested","pmids":["10769191"],"is_preprint":false},{"year":2012,"finding":"PPP4R2 loss of function impairs differentiation of the mouse motor-neuronal cell line NSC-34, and this effect can be rescued by SMN overexpression, establishing a functional cooperation between PPP4R2 and SMN in neuronal differentiation. PPP4R2 also protects NSC-34 cells from DNA damage-induced apoptosis and cooperates with SMN in this activity.","method":"RNAi-mediated loss-of-function, SMN overexpression rescue experiment, apoptosis assay in NSC-34 cells","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via KD/OE with defined cellular phenotype and rescue, single lab with two orthogonal functional readouts (differentiation and apoptosis)","pmids":["22559936"],"is_preprint":false},{"year":2012,"finding":"PPP4R2 displays dynamic intracellular localization in mouse and rat neuronal cell lines and in rat primary hippocampal neurons that strongly correlates with differentiation state.","method":"Immunofluorescence microscopy in neuronal cell lines and primary hippocampal neurons","journal":"European journal of cell biology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — localization observed by immunofluorescence, single lab, correlation with differentiation but no direct functional link established","pmids":["22559936"],"is_preprint":false},{"year":2017,"finding":"PPP4R2 deficiency impairs dephosphorylation of key DNA damage response proteins KAP1 (pKAP1), H2AX (γH2AX), p53 (pP53), and RPA2 (pRPA2) in hematopoietic and leukemic cells, establishing PPP4R2 as required for efficient DNA double-strand break repair via dephosphorylation of these substrates.","method":"RNAi knockdown of Ppp4r2 in murine hematopoietic stem/progenitor cells and leukemia cells; ectopic re-expression of PPP4R2 in a deficient human myeloid leukemic cell line; phospho-protein western blotting","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function and gain-of-function experiments with defined molecular readouts (phosphorylation of specific substrates), single lab","pmids":["29221109"],"is_preprint":false},{"year":2020,"finding":"PPP4R2 (Ppp4r2) physically interacts with Suppressor of fused (Sufu); Shh signaling promotes this interaction specifically in the nucleus. The PPP4 complex promotes dephosphorylation of Sufu (phosphorylated by PKA and GSK3β), leading to Sufu degradation and enhanced Gli1 transcriptional activity, thereby promoting Hedgehog signaling.","method":"Proteomic/co-immunoprecipitation identification of Sufu-Ppp4r2 interaction; subcellular fractionation; dephosphorylation assays; Gli1 reporter assays; proliferation assays in medulloblastoma cells","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP identification of binding partner, functional dephosphorylation assay, and transcriptional readout in a single lab with multiple orthogonal methods","pmids":["32826873"],"is_preprint":false},{"year":2021,"finding":"PPP4 with its regulatory subunit PPP4R2 is a critical component of the circadian clock; PPP4 binds BMAL1 and counteracts its phosphorylation, increasing CLOCK/BMAL1 DNA occupancy while decreasing transcriptional activity. Genetic depletion of PPP4 shortens circadian period; overexpression lengthens it. PPP4R2 was identified as a required component by systematic RNAi screen.","method":"Systematic RNAi screen in human cells; genetic depletion and overexpression in mammalian cells and Drosophila; CLOCK/BMAL1 transcription reporter assays; chromatin occupancy assay; period-length measurements","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic RNAi screen followed by genetic epistasis, binding assays, phosphorylation assays, and transcriptional readouts replicated in two organisms (mammals and Drosophila)","pmids":["34301769"],"is_preprint":false}],"current_model":"PPP4R2 is a regulatory subunit of protein phosphatase 4 (PPP4c) that forms large inactive complexes (450–600 kDa) activatable by basic proteins, targets PPP4c to centrosomes, dephosphorylates specific substrates including Sufu (promoting Hedgehog signaling via Gli1), KAP1/γH2AX/p53/RPA2 (enabling DNA double-strand break repair), and BMAL1 (modulating CLOCK/BMAL1 DNA occupancy to control circadian period length), and functionally cooperates with SMN to support neuronal differentiation and survival."},"narrative":{"mechanistic_narrative":"PPP4R2 is a regulatory subunit of protein phosphatase 4 (PPP4c) that directs the substrate specificity and subcellular targeting of the phosphatase across DNA repair, signaling, and circadian processes [PMID:10769191, PMID:29221109, PMID:34301769]. It binds PPP4c directly and assembles into large native PPP4 complexes (450–600 kDa) that are catalytically inactive until activated by basic proteins, identifying PPP4R2 as a key modulator of holoenzyme activity [PMID:10769191]. Through PPP4c, PPP4R2 promotes dephosphorylation of multiple substrates with distinct cellular consequences: it is required for dephosphorylation of the DNA damage response proteins KAP1, γH2AX, p53, and RPA2 and thereby for efficient double-strand break repair [PMID:29221109]; it drives nuclear dephosphorylation of Suppressor of fused (Sufu), promoting Sufu degradation and enhancing Gli1-dependent Hedgehog signaling [PMID:32826873]; and it counteracts BMAL1 phosphorylation to increase CLOCK/BMAL1 chromatin occupancy and set circadian period length, with depletion shortening and overexpression lengthening the period [PMID:34301769]. PPP4R2 also functionally cooperates with SMN to support motor-neuronal differentiation and to protect against DNA damage-induced apoptosis [PMID:22559936].","teleology":[{"year":2000,"claim":"Established that PPP4R2 is a bona fide regulatory subunit of PPP4c and that the resulting complexes are activity-regulated, defining PPP4R2 as a control point for phosphatase activity rather than a passive scaffold.","evidence":"Co-sedimentation and recombinant co-purification of PPP4c with PPP4R2, plus native complex purification and activity measurements from skeletal muscle and testis","pmids":["10769191"],"confidence":"High","gaps":["Mechanism by which basic proteins activate the inactive complex is not defined","No substrate identified in this study","Structural basis of the PPP4c–PPP4R2 interaction not resolved"]},{"year":2000,"claim":"Provided the first hint of where PPP4R2 acts in the cell by localizing it to centrosomes, implying it targets PPP4c to microtubule organizing centres.","evidence":"Immunofluorescence microscopy localization in mammalian cells","pmids":["10769191"],"confidence":"Medium","gaps":["Functional consequence of centrosomal localization not directly tested","No centrosomal substrate identified","Single-study localization without orthogonal validation"]},{"year":2012,"claim":"Connected PPP4R2 to a defined cellular program by showing it is required for motor-neuronal differentiation and survival, and that it acts in cooperation with SMN.","evidence":"RNAi loss-of-function with SMN-overexpression rescue and apoptosis assays in NSC-34 cells","pmids":["22559936"],"confidence":"Medium","gaps":["Molecular basis of PPP4R2–SMN cooperation not defined","No phosphatase substrate linked to the neuronal phenotype","In vivo relevance to neurodegeneration untested"]},{"year":2017,"claim":"Defined a direct biochemical role in genome maintenance by identifying the DNA damage response phospho-proteins whose dephosphorylation depends on PPP4R2.","evidence":"RNAi knockdown and re-expression in hematopoietic/leukemic cells with phospho-protein western blotting of KAP1, γH2AX, p53, and RPA2","pmids":["29221109"],"confidence":"Medium","gaps":["Direct vs indirect dephosphorylation of each substrate not distinguished","Recruitment of PPP4R2 to damage sites not characterized","Single-lab evidence"]},{"year":2020,"claim":"Extended PPP4R2 function into signal transduction by establishing a Sufu binding and dephosphorylation event that controls Hedgehog pathway output.","evidence":"Co-IP/proteomic identification of Sufu interaction, subcellular fractionation, dephosphorylation and Gli1 reporter assays in medulloblastoma cells","pmids":["32826873"],"confidence":"Medium","gaps":["Why Shh promotes the interaction specifically in the nucleus is unresolved","Direct catalytic action of PPP4c on Sufu vs scaffolding not separated","Single-lab evidence without reciprocal validation"]},{"year":2021,"claim":"Placed PPP4R2 in the core circadian clock by showing the PPP4–PPP4R2 complex tunes BMAL1 phosphorylation, CLOCK/BMAL1 chromatin occupancy, and period length.","evidence":"Systematic RNAi screen, genetic depletion/overexpression in human cells and Drosophila, BMAL1 binding and phosphorylation assays, chromatin occupancy and period-length measurements","pmids":["34301769"],"confidence":"High","gaps":["Specific BMAL1 phospho-sites controlled are not mapped","How elevated chromatin occupancy yields decreased transcriptional activity is mechanistically unexplained","Interplay with other clock kinases/phosphatases not resolved"]},{"year":null,"claim":"Whether PPP4R2's diverse substrate-targeting roles (DNA repair, Hedgehog, circadian) are governed by a shared recruitment logic or by context-specific cofactors remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of substrate selection by PPP4R2","Mechanism distinguishing nuclear vs centrosomal pools unknown","No unifying determinant of substrate specificity identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[4,5,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,5]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[4]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5]}],"complexes":["PPP4 complex"],"partners":["PPP4C","SUFU","BMAL1","SMN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9NY27","full_name":"Serine/threonine-protein phosphatase 4 regulatory subunit 2","aliases":[],"length_aa":417,"mass_kda":46.9,"function":"Regulatory subunit of serine/threonine-protein phosphatase 4 (PP4). May regulate the activity of PPP4C at centrosomal microtubule organizing centers. Its interaction with the SMN complex leads to enhance the temporal localization of snRNPs, suggesting a role of PPP4C in maturation of spliceosomal snRNPs. The PPP4C-PPP4R2-PPP4R3A PP4 complex specifically dephosphorylates H2AX phosphorylated on 'Ser-140' (gamma-H2AX) generated during DNA replication and required for DNA double strand break repair. Mediates RPA2 dephosphorylation by recruiting PPP4C to RPA2 in a DNA damage-dependent manner. RPA2 dephosphorylation is required for the efficient RPA2-mediated recruitment of RAD51 to chromatin following double strand breaks, an essential step for DNA repair","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9NY27/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/PPP4R2","classification":"Common Essential","n_dependent_lines":915,"n_total_lines":1208,"dependency_fraction":0.7574503311258278},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SF3B1","stoichiometry":4.0},{"gene":"SF3B4","stoichiometry":4.0},{"gene":"SF3B6","stoichiometry":4.0},{"gene":"DNAJC8","stoichiometry":0.2},{"gene":"SF3B2","stoichiometry":0.2},{"gene":"SF3B3","stoichiometry":0.2},{"gene":"SF3B5","stoichiometry":0.2},{"gene":"SNRPB2","stoichiometry":0.2},{"gene":"SNRPD2","stoichiometry":0.2},{"gene":"SRP9","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPP4R2","total_profiled":1310},"omim":[{"mim_id":"616790","title":"PROTEIN PHOSPHATASE 4, REGULATORY SUBUNIT 4; PPP4R4","url":"https://www.omim.org/entry/616790"},{"mim_id":"613822","title":"PROTEIN PHOSPHATASE 4, REGULATORY SUBUNIT 2; PPP4R2","url":"https://www.omim.org/entry/613822"},{"mim_id":"610351","title":"PROTEIN PHOSPHATASE 4, REGULATORY SUBUNIT 3, ALPHA; PPP4R3A","url":"https://www.omim.org/entry/610351"},{"mim_id":"609107","title":"F-BOX ONLY PROTEIN 40; FBXO40","url":"https://www.omim.org/entry/609107"},{"mim_id":"602035","title":"PROTEIN PHOSPHATASE 4, CATALYTIC SUBUNIT; PPP4C","url":"https://www.omim.org/entry/602035"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PPP4R2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q9NY27","domains":[{"cath_id":"-","chopping":"4-67_82-136","consensus_level":"high","plddt":93.1614,"start":4,"end":136}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NY27","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NY27-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NY27-F1-predicted_aligned_error_v6.png","plddt_mean":62.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPP4R2","jax_strain_url":"https://www.jax.org/strain/search?query=PPP4R2"},"sequence":{"accession":"Q9NY27","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NY27.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NY27/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NY27"}},"corpus_meta":[{"pmid":"10769191","id":"PMC_10769191","title":"A novel 50 kDa protein forms complexes with protein phosphatase 4 and is located at centrosomal microtubule organizing centres.","date":"2000","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/10769191","citation_count":50,"is_preprint":false},{"pmid":"12836054","id":"PMC_12836054","title":"Molecular characterisation of a 15 Mb constitutional de novo interstitial deletion of chromosome 3p in a boy with developmental delay and congenital anomalies.","date":"2003","source":"Journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/12836054","citation_count":23,"is_preprint":false},{"pmid":"22559936","id":"PMC_22559936","title":"PPP4R2 regulates neuronal cell differentiation and survival, functionally cooperating with SMN.","date":"2012","source":"European journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/22559936","citation_count":14,"is_preprint":false},{"pmid":"33515627","id":"PMC_33515627","title":"Next-generation sequencing analysis suggests varied multistep mutational pathogenesis for endocrine mucin-producing sweat gland carcinoma with comments on INSM1 and MUC2 suggesting a conjunctival origin.","date":"2021","source":"Journal of the American Academy of Dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/33515627","citation_count":14,"is_preprint":false},{"pmid":"32826873","id":"PMC_32826873","title":"Protein phosphatase 4 promotes Hedgehog signaling through dephosphorylation of Suppressor of fused.","date":"2020","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/32826873","citation_count":13,"is_preprint":false},{"pmid":"29221109","id":"PMC_29221109","title":"Protein phosphatase 4 regulatory subunit 2 (PPP4R2) is recurrently deleted in acute myeloid leukemia and required for efficient DNA double strand break repair.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29221109","citation_count":11,"is_preprint":false},{"pmid":"34301769","id":"PMC_34301769","title":"Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity.","date":"2021","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/34301769","citation_count":11,"is_preprint":false},{"pmid":"31142279","id":"PMC_31142279","title":"The association of germline variants with chronic lymphocytic leukemia outcome suggests the implication of novel genes and pathways in clinical evolution.","date":"2019","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31142279","citation_count":10,"is_preprint":false},{"pmid":"17556864","id":"PMC_17556864","title":"Familial pericentric inversion chromosome 3 and R448C mutation of CYP11B1 gene in Turkish kindred with 11beta-hydroxylase deficiency.","date":"2007","source":"Journal of endocrinological investigation","url":"https://pubmed.ncbi.nlm.nih.gov/17556864","citation_count":7,"is_preprint":false},{"pmid":"35865459","id":"PMC_35865459","title":"Genotypes of Papillary Thyroid Carcinoma With High Lateral Neck Metastasis in Chinese Population.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35865459","citation_count":6,"is_preprint":false},{"pmid":"39495984","id":"PMC_39495984","title":"Pancancer analysis of the interactions between CTNNB1 and infiltrating immune cell populations.","date":"2024","source":"Medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39495984","citation_count":3,"is_preprint":false},{"pmid":"36653584","id":"PMC_36653584","title":"Interactome of Paraoxonase PON2 Reveals New Pathways for Tumor Growth Regulation.","date":"2023","source":"Doklady. Biochemistry and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/36653584","citation_count":2,"is_preprint":false},{"pmid":"42026014","id":"PMC_42026014","title":"Whole Genome Sequence Analysis of Weight Loss in 16 972 Participants With COPD Reveals Novel Risk Loci in DRAIC and RFX3.","date":"2026","source":"Journal of cachexia, sarcopenia and muscle","url":"https://pubmed.ncbi.nlm.nih.gov/42026014","citation_count":0,"is_preprint":false},{"pmid":"41804841","id":"PMC_41804841","title":"Genetic architecture of plasma pTau217 and related biomarkers in Alzheimer's disease via genome-wide association studies.","date":"2026","source":"Alzheimer's & dementia : the journal of the Alzheimer's Association","url":"https://pubmed.ncbi.nlm.nih.gov/41804841","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9315,"output_tokens":1914,"usd":0.028327,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8785,"output_tokens":2571,"usd":0.0541,"stage2_stop_reason":"end_turn"},"total_usd":0.082427,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"PPP4R2 (a 50 kDa protein) forms a regulatory subunit of protein phosphatase 4 (PPP4c), physically interacting with PPP4c as confirmed by co-sedimentation of PPP4c with bacterially expressed PPP4R2 and by formation of a 450 kDa complex with baculovirus-expressed His6-tagged PPP4R2. Native 450 kDa and 600 kDa PPP4 complexes are catalytically inactive but can be activated by basic proteins, suggesting PPP4R2 regulates PPP4c activity.\",\n      \"method\": \"Co-sedimentation assay, baculovirus/bacterial recombinant protein co-purification, native complex purification from mammalian skeletal muscle and testis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with recombinant proteins, biochemical co-purification, and enzymatic activity measurements in multiple systems\",\n      \"pmids\": [\"10769191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PPP4R2 localizes to centrosomes by immunocytology, suggesting it targets PPP4c to centrosomal microtubule organizing centres.\",\n      \"method\": \"Immunocytological detection (immunofluorescence microscopy)\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — immunofluorescence localization in a single study; functional consequence inferred but not directly tested\",\n      \"pmids\": [\"10769191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PPP4R2 loss of function impairs differentiation of the mouse motor-neuronal cell line NSC-34, and this effect can be rescued by SMN overexpression, establishing a functional cooperation between PPP4R2 and SMN in neuronal differentiation. PPP4R2 also protects NSC-34 cells from DNA damage-induced apoptosis and cooperates with SMN in this activity.\",\n      \"method\": \"RNAi-mediated loss-of-function, SMN overexpression rescue experiment, apoptosis assay in NSC-34 cells\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via KD/OE with defined cellular phenotype and rescue, single lab with two orthogonal functional readouts (differentiation and apoptosis)\",\n      \"pmids\": [\"22559936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PPP4R2 displays dynamic intracellular localization in mouse and rat neuronal cell lines and in rat primary hippocampal neurons that strongly correlates with differentiation state.\",\n      \"method\": \"Immunofluorescence microscopy in neuronal cell lines and primary hippocampal neurons\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — localization observed by immunofluorescence, single lab, correlation with differentiation but no direct functional link established\",\n      \"pmids\": [\"22559936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PPP4R2 deficiency impairs dephosphorylation of key DNA damage response proteins KAP1 (pKAP1), H2AX (γH2AX), p53 (pP53), and RPA2 (pRPA2) in hematopoietic and leukemic cells, establishing PPP4R2 as required for efficient DNA double-strand break repair via dephosphorylation of these substrates.\",\n      \"method\": \"RNAi knockdown of Ppp4r2 in murine hematopoietic stem/progenitor cells and leukemia cells; ectopic re-expression of PPP4R2 in a deficient human myeloid leukemic cell line; phospho-protein western blotting\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function and gain-of-function experiments with defined molecular readouts (phosphorylation of specific substrates), single lab\",\n      \"pmids\": [\"29221109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PPP4R2 (Ppp4r2) physically interacts with Suppressor of fused (Sufu); Shh signaling promotes this interaction specifically in the nucleus. The PPP4 complex promotes dephosphorylation of Sufu (phosphorylated by PKA and GSK3β), leading to Sufu degradation and enhanced Gli1 transcriptional activity, thereby promoting Hedgehog signaling.\",\n      \"method\": \"Proteomic/co-immunoprecipitation identification of Sufu-Ppp4r2 interaction; subcellular fractionation; dephosphorylation assays; Gli1 reporter assays; proliferation assays in medulloblastoma cells\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP identification of binding partner, functional dephosphorylation assay, and transcriptional readout in a single lab with multiple orthogonal methods\",\n      \"pmids\": [\"32826873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PPP4 with its regulatory subunit PPP4R2 is a critical component of the circadian clock; PPP4 binds BMAL1 and counteracts its phosphorylation, increasing CLOCK/BMAL1 DNA occupancy while decreasing transcriptional activity. Genetic depletion of PPP4 shortens circadian period; overexpression lengthens it. PPP4R2 was identified as a required component by systematic RNAi screen.\",\n      \"method\": \"Systematic RNAi screen in human cells; genetic depletion and overexpression in mammalian cells and Drosophila; CLOCK/BMAL1 transcription reporter assays; chromatin occupancy assay; period-length measurements\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic RNAi screen followed by genetic epistasis, binding assays, phosphorylation assays, and transcriptional readouts replicated in two organisms (mammals and Drosophila)\",\n      \"pmids\": [\"34301769\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPP4R2 is a regulatory subunit of protein phosphatase 4 (PPP4c) that forms large inactive complexes (450–600 kDa) activatable by basic proteins, targets PPP4c to centrosomes, dephosphorylates specific substrates including Sufu (promoting Hedgehog signaling via Gli1), KAP1/γH2AX/p53/RPA2 (enabling DNA double-strand break repair), and BMAL1 (modulating CLOCK/BMAL1 DNA occupancy to control circadian period length), and functionally cooperates with SMN to support neuronal differentiation and survival.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP4R2 is a regulatory subunit of protein phosphatase 4 (PPP4c) that directs the substrate specificity and subcellular targeting of the phosphatase across DNA repair, signaling, and circadian processes [#0, #4, #6]. It binds PPP4c directly and assembles into large native PPP4 complexes (450\\u2013600 kDa) that are catalytically inactive until activated by basic proteins, identifying PPP4R2 as a key modulator of holoenzyme activity [#0]. Through PPP4c, PPP4R2 promotes dephosphorylation of multiple substrates with distinct cellular consequences: it is required for dephosphorylation of the DNA damage response proteins KAP1, \\u03b3H2AX, p53, and RPA2 and thereby for efficient double-strand break repair [#4]; it drives nuclear dephosphorylation of Suppressor of fused (Sufu), promoting Sufu degradation and enhancing Gli1-dependent Hedgehog signaling [#5]; and it counteracts BMAL1 phosphorylation to increase CLOCK/BMAL1 chromatin occupancy and set circadian period length, with depletion shortening and overexpression lengthening the period [#6]. PPP4R2 also functionally cooperates with SMN to support motor-neuronal differentiation and to protect against DNA damage-induced apoptosis [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established that PPP4R2 is a bona fide regulatory subunit of PPP4c and that the resulting complexes are activity-regulated, defining PPP4R2 as a control point for phosphatase activity rather than a passive scaffold.\",\n      \"evidence\": \"Co-sedimentation and recombinant co-purification of PPP4c with PPP4R2, plus native complex purification and activity measurements from skeletal muscle and testis\",\n      \"pmids\": [\"10769191\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which basic proteins activate the inactive complex is not defined\", \"No substrate identified in this study\", \"Structural basis of the PPP4c\\u2013PPP4R2 interaction not resolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Provided the first hint of where PPP4R2 acts in the cell by localizing it to centrosomes, implying it targets PPP4c to microtubule organizing centres.\",\n      \"evidence\": \"Immunofluorescence microscopy localization in mammalian cells\",\n      \"pmids\": [\"10769191\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of centrosomal localization not directly tested\", \"No centrosomal substrate identified\", \"Single-study localization without orthogonal validation\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected PPP4R2 to a defined cellular program by showing it is required for motor-neuronal differentiation and survival, and that it acts in cooperation with SMN.\",\n      \"evidence\": \"RNAi loss-of-function with SMN-overexpression rescue and apoptosis assays in NSC-34 cells\",\n      \"pmids\": [\"22559936\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of PPP4R2\\u2013SMN cooperation not defined\", \"No phosphatase substrate linked to the neuronal phenotype\", \"In vivo relevance to neurodegeneration untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined a direct biochemical role in genome maintenance by identifying the DNA damage response phospho-proteins whose dephosphorylation depends on PPP4R2.\",\n      \"evidence\": \"RNAi knockdown and re-expression in hematopoietic/leukemic cells with phospho-protein western blotting of KAP1, \\u03b3H2AX, p53, and RPA2\",\n      \"pmids\": [\"29221109\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect dephosphorylation of each substrate not distinguished\", \"Recruitment of PPP4R2 to damage sites not characterized\", \"Single-lab evidence\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended PPP4R2 function into signal transduction by establishing a Sufu binding and dephosphorylation event that controls Hedgehog pathway output.\",\n      \"evidence\": \"Co-IP/proteomic identification of Sufu interaction, subcellular fractionation, dephosphorylation and Gli1 reporter assays in medulloblastoma cells\",\n      \"pmids\": [\"32826873\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Why Shh promotes the interaction specifically in the nucleus is unresolved\", \"Direct catalytic action of PPP4c on Sufu vs scaffolding not separated\", \"Single-lab evidence without reciprocal validation\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed PPP4R2 in the core circadian clock by showing the PPP4\\u2013PPP4R2 complex tunes BMAL1 phosphorylation, CLOCK/BMAL1 chromatin occupancy, and period length.\",\n      \"evidence\": \"Systematic RNAi screen, genetic depletion/overexpression in human cells and Drosophila, BMAL1 binding and phosphorylation assays, chromatin occupancy and period-length measurements\",\n      \"pmids\": [\"34301769\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific BMAL1 phospho-sites controlled are not mapped\", \"How elevated chromatin occupancy yields decreased transcriptional activity is mechanistically unexplained\", \"Interplay with other clock kinases/phosphatases not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether PPP4R2's diverse substrate-targeting roles (DNA repair, Hedgehog, circadian) are governed by a shared recruitment logic or by context-specific cofactors remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of substrate selection by PPP4R2\", \"Mechanism distinguishing nuclear vs centrosomal pools unknown\", \"No unifying determinant of substrate specificity identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [4, 5, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5]}\n    ],\n    \"complexes\": [\"PPP4 complex\"],\n    \"partners\": [\"PPP4C\", \"SUFU\", \"BMAL1\", \"SMN\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}