{"gene":"PPP2R5E","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2003,"finding":"PP2A:B56epsilon is required for Wnt/beta-catenin signaling in Xenopus embryogenesis, acting upstream of Dishevelled and downstream of the Wnt ligand; loss-of-function disrupts dorsal development, Wnt target gene (engrailed) expression, midbrain-hindbrain boundary formation, and neural tube closure, demonstrating a positive role for PP2A:B56epsilon in the Wnt pathway.","method":"Loss-of-function analysis in Xenopus embryos (morpholino/dominant-negative injection), epistasis experiments placing B56epsilon upstream of Dishevelled and downstream of Wnt ligand","journal":"Development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple loss-of-function readouts and pathway placement, replicated across multiple developmental contexts in a focused mechanistic study","pmids":["14522869"],"is_preprint":false},{"year":2006,"finding":"B56epsilon is required for eye induction via the IGF/PI3K/Akt signaling pathway and for eye field separation via the Hedgehog pathway in Xenopus; loss-of-function of B56epsilon inhibits both processes, and inhibition of PI3K/Akt phenocopies loss of B56epsilon for eye induction.","method":"Loss-of-function in Xenopus embryos, epistasis with PI3K/Akt inhibitors and Hedgehog pathway components","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function with specific phenotypic readouts combined with epistasis placing B56epsilon in two distinct signaling pathways, single lab but multiple orthogonal approaches","pmids":["17074314"],"is_preprint":false},{"year":2009,"finding":"B56epsilon is alternatively translated to produce a full-length form and a shorter 48-kDa isoform lacking the N-terminal 76 amino acids via a cap-dependent mechanism. The N-terminus contains a nuclear localization signal (NLS) and the C-terminus contains a nuclear export signal (NES); the shorter isoform lacking the NLS is restricted to the cytoplasm, while the full-length form can localize to the nucleus in a cell type-specific manner. The shorter cytoplasmic isoform is specifically required for Wnt signaling and midbrain-hindbrain boundary formation.","method":"Alternative translation mapping, NLS/NES mutagenesis, subcellular fractionation/localization, Xenopus loss-of-function rescue experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — multiple orthogonal methods (translation mapping, mutagenesis, localization, functional rescue in vivo) in a single focused study","pmids":["19129191"],"is_preprint":false},{"year":2016,"finding":"PPP2R5E (B56epsilon) physically interacts with microtubule crosslinking factor 1 (MTCL1) and stabilizes MTCL1 protein abundance; PPP2R5E depletion reduces MTCL1 levels, exogenous PPP2R5E expression increases endogenous MTCL1, and phosphatase inhibition by okadaic acid reduces MTCL1 (restored by proteasome inhibitor MG132), indicating PP2A phosphatase activity protects MTCL1 from proteasomal degradation. Cells depleted of PPP2R5E and MTCL1 exhibit defects in microtubule organization.","method":"Mass spectrometry interactome screen, co-immunoprecipitation, siRNA knockdown, pharmacological inhibition (okadaic acid, MG132), immunofluorescence of microtubule organization","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — MS-identified interaction confirmed by co-IP, mechanistic follow-up with phosphatase inhibition and proteasome inhibitor rescue, single lab","pmids":["27521566"],"is_preprint":false},{"year":2014,"finding":"PPP2R5E is a direct target of miR-23a; miR-23a downregulates PPP2R5E expression, and overexpression of PPP2R5E reverses miR-23a-induced suppression of apoptosis and promotion of cell growth in gastric cancer cells.","method":"Overexpression/knockdown of PPP2R5E and miR-23a in gastric cancer cell lines, in vivo xenograft assay, functional rescue experiments","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — functional rescue confirms PPP2R5E as downstream mediator of miR-23a, single lab with in vitro and in vivo evidence","pmids":["24997345"],"is_preprint":false},{"year":2023,"finding":"miR-19b is a direct negative regulator of PPP2R5E in colorectal cancer cells, confirmed by luciferase assay; miR-19b overexpression decreases PP2A activity, and PPP2R5E downregulation mediates miR-19b-induced oncogenic effects including increased cell viability, colonosphere formation, cell migration, and 5-FU resistance.","method":"Luciferase reporter assay, PP2A activity assay, siRNA knockdown, cell viability/migration/sphere formation assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct target validation by luciferase assay combined with PP2A activity measurement and functional knockdown phenotypes, single lab","pmids":["37175484"],"is_preprint":false},{"year":2024,"finding":"A de novo heterozygous variant Glu191Lys in PPP2R5E, within a conserved LFDSEDPRER motif, results in decreased interaction with PP2A A and C subunits, disrupting holoenzyme formation, as demonstrated by biochemical assays in a patient with learning disorders, motor coordination problems, hypotonia, and myopathy.","method":"Whole exome sequencing, biochemical co-immunoprecipitation/interaction assays for PP2A holoenzyme assembly","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — biochemical interaction assay directly demonstrates impaired holoenzyme assembly, single case study/single lab","pmids":["39284558"],"is_preprint":false},{"year":2026,"finding":"PPP2R5E (a PP2A subunit) is a critical mediator of temozolomide sensitization in glioblastoma; miR-19b attenuation upregulates PPP2R5E, which induces genotoxic stress via elevated nuclear ROS, promoting senescence and ferroptosis. Pharmacological PP2A activation with FTY720 phenocopies miR-19b suppression, and PPP2R5E knockdown reverses temozolomide sensitization in vitro, in vivo, and ex vivo.","method":"MicroRNA screens, phosphoprotein analysis, clonogenic and spheroid assays, γH2AX foci formation, cell cycle/senescence/ferroptosis assays, orthotopic xenograft model, syngeneic mouse model, siRNA knockdown","journal":"British journal of cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including in vitro, in vivo (two animal models), and ex vivo validation; pharmacological phenocopy and genetic rescue confirm mechanism","pmids":["42203893"],"is_preprint":false},{"year":2025,"finding":"PPP2R5E is identified as a key target of EXOSC2 (RNA exosome component 2) in stem cells and cancer cells; EXOSC2 regulation of PPP2R5E is preserved across stem cells and cancer, suggesting PPP2R5E abundance is post-transcriptionally regulated by the RNA exosome.","method":"Genetic/molecular analysis in embryonic stem cells and cancer cells (EXOSC2 perturbation with monitoring of PPP2R5E)","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint with limited methodological detail in abstract, single lab, mechanism of regulation not fully described","pmids":[],"is_preprint":true}],"current_model":"PPP2R5E (B56epsilon) is a regulatory subunit of PP2A that positively regulates Wnt/beta-catenin signaling (upstream of Dishevelled), IGF/PI3K/Akt signaling, and Hedgehog signaling during development; it is alternatively translated to produce a cytoplasmic isoform (lacking an NLS) required for Wnt signaling and a nuclear-competent full-length form, with the C-terminus containing a nuclear export signal; it stabilizes MTCL1 protein via PP2A phosphatase activity to support microtubule organization; its interaction with the PP2A A and C subunits (holoenzyme assembly) is disrupted by pathogenic variants such as Glu191Lys; and it acts as a tumor suppressor whose downregulation by miRNAs (miR-23a, miR-19b) promotes cancer cell growth, while its upregulation sensitizes glioblastoma cells to temozolomide through nuclear ROS-induced DNA damage, senescence, and ferroptosis."},"narrative":{"mechanistic_narrative":"PPP2R5E (B56epsilon) is a regulatory subunit of protein phosphatase 2A (PP2A) that confers substrate specificity on the holoenzyme to control developmental signaling, cytoskeletal organization, and tumor cell fate [PMID:14522869, PMID:39284558]. During vertebrate embryogenesis it acts as a positive regulator of multiple pathways: it is required for Wnt/beta-catenin signaling upstream of Dishevelled and downstream of the Wnt ligand, and it promotes eye induction through the IGF/PI3K/Akt pathway and eye field separation through the Hedgehog pathway [PMID:14522869, PMID:17074314]. The subunit is alternatively translated into a full-length, nuclear-competent form bearing an N-terminal nuclear localization signal and a C-terminal nuclear export signal, and a shorter cytoplasmic isoform lacking the NLS; the cytoplasmic isoform is specifically required for Wnt signaling and midbrain-hindbrain boundary formation [PMID:19129191]. Through PP2A phosphatase activity, PPP2R5E physically binds and stabilizes the microtubule crosslinking factor MTCL1 against proteasomal degradation, supporting proper microtubule organization [PMID:27521566]. Holoenzyme assembly depends on PPP2R5E contacting the PP2A A and C subunits; the de novo Glu191Lys variant in a conserved motif weakens these contacts and is associated with a neurodevelopmental and myopathic phenotype [PMID:39284558]. PPP2R5E functions as a tumor suppressor: it is a direct target of miR-23a and miR-19b, whose elevation suppresses PPP2R5E and drives proliferation, survival, and chemoresistance [PMID:24997345, PMID:37175484], whereas restoring PPP2R5E in glioblastoma induces nuclear ROS-driven genotoxic stress, senescence, and ferroptosis to sensitize cells to temozolomide [PMID:42203893].","teleology":[{"year":2003,"claim":"Established that a specific PP2A regulatory subunit positively gates Wnt signaling, defining where PP2A:B56epsilon acts within the pathway rather than treating PP2A as a generic phosphatase.","evidence":"Loss-of-function and epistasis in Xenopus embryos placing B56epsilon upstream of Dishevelled and downstream of Wnt ligand","pmids":["14522869"],"confidence":"High","gaps":["Direct phosphatase substrate within the Wnt pathway not identified","Mechanism connecting PP2A activity to Dishevelled regulation unresolved"]},{"year":2006,"claim":"Extended B56epsilon's developmental role beyond Wnt to IGF/PI3K/Akt and Hedgehog signaling, showing it is a multi-pathway node in eye development.","evidence":"Loss-of-function in Xenopus with epistasis using PI3K/Akt inhibitors and Hedgehog pathway components","pmids":["17074314"],"confidence":"High","gaps":["Molecular substrates in PI3K/Akt and Hedgehog pathways not defined","Whether the same isoform mediates all three pathways unknown at this stage"]},{"year":2009,"claim":"Resolved that distinct subcellular pools of B56epsilon carry distinct functions, showing alternative translation produces a cytoplasm-restricted isoform required specifically for Wnt signaling.","evidence":"Alternative translation mapping, NLS/NES mutagenesis, subcellular localization, and rescue in Xenopus","pmids":["19129191"],"confidence":"High","gaps":["Regulation of isoform choice in mammalian tissues not characterized","Nuclear function of the full-length form not assigned a substrate"]},{"year":2014,"claim":"Connected PPP2R5E to oncogenic miRNA regulation, establishing it as a growth-suppressive effector silenced by miR-23a in gastric cancer.","evidence":"Overexpression/knockdown with functional rescue in gastric cancer cells and xenografts","pmids":["24997345"],"confidence":"Medium","gaps":["Phosphatase substrates mediating the anti-growth effect not identified","Direct binding of miR-23a to the PPP2R5E transcript not shown here"]},{"year":2016,"claim":"Identified a non-signaling, cytoskeletal role: PPP2R5E-directed PP2A activity protects MTCL1 from proteasomal turnover to maintain microtubule organization.","evidence":"Mass spectrometry interactome, co-IP, siRNA, okadaic acid/MG132 rescue, and microtubule immunofluorescence","pmids":["27521566"],"confidence":"Medium","gaps":["The phosphosite on MTCL1 dephosphorylated by PP2A not mapped","Single-lab interaction without reciprocal in vivo validation"]},{"year":2023,"claim":"Generalized miRNA control of PPP2R5E to a second cancer type and linked it to PP2A enzymatic output, with miR-19b directly repressing PPP2R5E to drive chemoresistance.","evidence":"Luciferase reporter, PP2A activity assay, and knockdown phenotypes in colorectal cancer cells","pmids":["37175484"],"confidence":"Medium","gaps":["Downstream PP2A substrates driving 5-FU resistance not defined","Single lab, in vitro only"]},{"year":2024,"claim":"Provided a direct biochemical mechanism for human disease by showing a de novo variant disrupts PPP2R5E incorporation into the PP2A holoenzyme.","evidence":"Whole exome sequencing and biochemical interaction assays for A/C subunit binding in a patient","pmids":["39284558"],"confidence":"Medium","gaps":["Single case; genotype-phenotype causality not established in a cohort","Downstream signaling consequence of holoenzyme loss not measured"]},{"year":2026,"claim":"Demonstrated that restoring PPP2R5E actively reprograms tumor cell fate, inducing nuclear ROS, senescence, and ferroptosis to sensitize glioblastoma to temozolomide.","evidence":"miRNA screens, γH2AX foci, senescence/ferroptosis assays, FTY720 phenocopy, knockdown rescue, and orthotopic/syngeneic mouse models","pmids":["42203893"],"confidence":"High","gaps":["Phosphatase substrate linking PPP2R5E to nuclear ROS generation unknown","Whether the nuclear full-length isoform specifically drives this effect untested"]},{"year":2025,"claim":"Raised the possibility that PPP2R5E abundance is set post-transcriptionally by the RNA exosome via EXOSC2 across stem and cancer cells.","evidence":"EXOSC2 perturbation with PPP2R5E monitoring in embryonic stem and cancer cells (preprint)","pmids":[],"confidence":"Low","gaps":["Preprint with limited methodological detail; mechanism of regulation not described","Direct exosome targeting of the PPP2R5E transcript not demonstrated"]},{"year":null,"claim":"The specific phosphoprotein substrates dephosphorylated by PPP2R5E-containing PP2A across its Wnt, PI3K/Akt, Hedgehog, cytoskeletal, and tumor-suppressive roles remain unidentified.","evidence":"","pmids":[],"confidence":"Low","gaps":["No direct PP2A:B56epsilon substrate mapped in any pathway","Structural basis of substrate selection not resolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,6]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7]}],"complexes":["PP2A holoenzyme"],"partners":["MTCL1","PPP2CA","PPP2R1A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q16537","full_name":"Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit epsilon isoform","aliases":["PP2A B subunit isoform B'-epsilon","PP2A B subunit isoform B56-epsilon","PP2A B subunit isoform PR61-epsilon","PP2A B subunit isoform R5-epsilon"],"length_aa":467,"mass_kda":54.7,"function":"The B regulatory subunit might modulate substrate selectivity and catalytic activity, and might also direct the localization of the catalytic enzyme to a particular subcellular compartment","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q16537/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PPP2R5E","classification":"Not Classified","n_dependent_lines":13,"n_total_lines":1208,"dependency_fraction":0.01076158940397351},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"DDX56","stoichiometry":0.2},{"gene":"PPP2CA","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPP2R5E","total_profiled":1310},"omim":[{"mim_id":"601647","title":"PROTEIN PHOSPHATASE 2, REGULATORY SUBUNIT B (B56), EPSILON; PPP2R5E","url":"https://www.omim.org/entry/601647"},{"mim_id":"601646","title":"PROTEIN PHOSPHATASE 2, REGULATORY SUBUNIT B (B56), DELTA; PPP2R5D","url":"https://www.omim.org/entry/601646"},{"mim_id":"601645","title":"PROTEIN PHOSPHATASE 2, REGULATORY SUBUNIT B (B56), GAMMA; PPP2R5C","url":"https://www.omim.org/entry/601645"},{"mim_id":"601644","title":"PROTEIN PHOSPHATASE 2, REGULATORY SUBUNIT B (B56), BETA; PPP2R5B","url":"https://www.omim.org/entry/601644"},{"mim_id":"601643","title":"PROTEIN PHOSPHATASE 2, REGULATORY SUBUNIT B (B56), ALPHA; PPP2R5A","url":"https://www.omim.org/entry/601643"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Mid piece","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PPP2R5E"},"hgnc":{"alias_symbol":["B56E","B56epsilon"],"prev_symbol":[]},"alphafold":{"accession":"Q16537","domains":[{"cath_id":"1.25.40","chopping":"46-127_145-209","consensus_level":"medium","plddt":94.765,"start":46,"end":209}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16537","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q16537-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q16537-F1-predicted_aligned_error_v6.png","plddt_mean":88.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPP2R5E","jax_strain_url":"https://www.jax.org/strain/search?query=PPP2R5E"},"sequence":{"accession":"Q16537","fasta_url":"https://rest.uniprot.org/uniprotkb/Q16537.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q16537/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q16537"}},"corpus_meta":[{"pmid":"14522869","id":"PMC_14522869","title":"PP2A:B56epsilon is required for Wnt/beta-catenin signaling during embryonic development.","date":"2003","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/14522869","citation_count":78,"is_preprint":false},{"pmid":"8812429","id":"PMC_8812429","title":"Assignment of human protein phosphatase 2A regulatory subunit genes b56alpha, b56beta, b56gamma, b56delta, and b56epsilon (PPP2R5A-PPP2R5E), highly expressed in muscle and brain, to chromosome regions 1q41, 11q12, 3p21, 6p21.1, and 7p11.2 --> p12.","date":"1996","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/8812429","citation_count":58,"is_preprint":false},{"pmid":"17074314","id":"PMC_17074314","title":"PP2A:B56epsilon is required for eye induction and eye field separation.","date":"2006","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/17074314","citation_count":49,"is_preprint":false},{"pmid":"24997345","id":"PMC_24997345","title":"Downregulation of PPP2R5E expression by miR-23a suppresses apoptosis to facilitate the growth of gastric cancer cells.","date":"2014","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/24997345","citation_count":30,"is_preprint":false},{"pmid":"19129191","id":"PMC_19129191","title":"The 48-kDa alternative translation isoform of PP2A:B56epsilon is required for Wnt signaling during midbrain-hindbrain boundary formation.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19129191","citation_count":27,"is_preprint":false},{"pmid":"30352868","id":"PMC_30352868","title":"Mapping of breakpoints in balanced chromosomal translocations by shallow whole-genome sequencing points to EFNA5, BAHD1 and PPP2R5E as novel candidates for genes causing human Mendelian disorders.","date":"2018","source":"Journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30352868","citation_count":14,"is_preprint":false},{"pmid":"27521566","id":"PMC_27521566","title":"A regulatory subunit of protein phosphatase 2A, PPP2R5E, regulates the abundance of microtubule crosslinking factor 1.","date":"2016","source":"The FEBS journal","url":"https://pubmed.ncbi.nlm.nih.gov/27521566","citation_count":10,"is_preprint":false},{"pmid":"37175484","id":"PMC_37175484","title":"Deregulation of the miR-19b/PPP2R5E Signaling Axis Shows High Functional Impact in Colorectal Cancer Cells.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37175484","citation_count":5,"is_preprint":false},{"pmid":"39284558","id":"PMC_39284558","title":"PPP2R5E: New gene potentially involved in specific learning disorders and myopathy.","date":"2024","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/39284558","citation_count":4,"is_preprint":false},{"pmid":"38425404","id":"PMC_38425404","title":"Comprehensive analysis of the protein phosphatase 2A regulatory subunit B56ε in pan-cancer and its role and mechanism in hepatocellular carcinoma.","date":"2024","source":"World journal of gastrointestinal oncology","url":"https://pubmed.ncbi.nlm.nih.gov/38425404","citation_count":2,"is_preprint":false},{"pmid":"40921709","id":"PMC_40921709","title":"The Function of Hsa_circ_0001862 Regulating miR-23a-IRF1/PPP2R5E Axis to Oral Squamous Cell Carcinoma Development.","date":"2025","source":"Journal of oral pathology & medicine : official publication of the International Association of Oral Pathologists and the American Academy of Oral Pathology","url":"https://pubmed.ncbi.nlm.nih.gov/40921709","citation_count":0,"is_preprint":false},{"pmid":"42203893","id":"PMC_42203893","title":"Targeting the miR-19b/PPP2R5E axis enhances temozolomide response in glioblastoma via ROS-induced DNA damage.","date":"2026","source":"British journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/42203893","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.04.22.649861","title":"Physiological α-synuclein S129 phosphorylation mediates postsynaptic and nuclear interactions in the human brain","date":"2025-04-26","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.22.649861","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.04.04.647279","title":"Aging-dependent dysregulation of EXOSC2 is maintained in cancer as a dependency","date":"2025-04-07","source":"bioRxiv","url":"https://doi.org/10.1101/2025.04.04.647279","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8800,"output_tokens":2593,"usd":0.032648,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9820,"output_tokens":3300,"usd":0.0658,"stage2_stop_reason":"end_turn"},"total_usd":0.098448,"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\": 2003,\n      \"finding\": \"PP2A:B56epsilon is required for Wnt/beta-catenin signaling in Xenopus embryogenesis, acting upstream of Dishevelled and downstream of the Wnt ligand; loss-of-function disrupts dorsal development, Wnt target gene (engrailed) expression, midbrain-hindbrain boundary formation, and neural tube closure, demonstrating a positive role for PP2A:B56epsilon in the Wnt pathway.\",\n      \"method\": \"Loss-of-function analysis in Xenopus embryos (morpholino/dominant-negative injection), epistasis experiments placing B56epsilon upstream of Dishevelled and downstream of Wnt ligand\",\n      \"journal\": \"Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple loss-of-function readouts and pathway placement, replicated across multiple developmental contexts in a focused mechanistic study\",\n      \"pmids\": [\"14522869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"B56epsilon is required for eye induction via the IGF/PI3K/Akt signaling pathway and for eye field separation via the Hedgehog pathway in Xenopus; loss-of-function of B56epsilon inhibits both processes, and inhibition of PI3K/Akt phenocopies loss of B56epsilon for eye induction.\",\n      \"method\": \"Loss-of-function in Xenopus embryos, epistasis with PI3K/Akt inhibitors and Hedgehog pathway components\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with specific phenotypic readouts combined with epistasis placing B56epsilon in two distinct signaling pathways, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"17074314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"B56epsilon is alternatively translated to produce a full-length form and a shorter 48-kDa isoform lacking the N-terminal 76 amino acids via a cap-dependent mechanism. The N-terminus contains a nuclear localization signal (NLS) and the C-terminus contains a nuclear export signal (NES); the shorter isoform lacking the NLS is restricted to the cytoplasm, while the full-length form can localize to the nucleus in a cell type-specific manner. The shorter cytoplasmic isoform is specifically required for Wnt signaling and midbrain-hindbrain boundary formation.\",\n      \"method\": \"Alternative translation mapping, NLS/NES mutagenesis, subcellular fractionation/localization, Xenopus loss-of-function rescue experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — multiple orthogonal methods (translation mapping, mutagenesis, localization, functional rescue in vivo) in a single focused study\",\n      \"pmids\": [\"19129191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PPP2R5E (B56epsilon) physically interacts with microtubule crosslinking factor 1 (MTCL1) and stabilizes MTCL1 protein abundance; PPP2R5E depletion reduces MTCL1 levels, exogenous PPP2R5E expression increases endogenous MTCL1, and phosphatase inhibition by okadaic acid reduces MTCL1 (restored by proteasome inhibitor MG132), indicating PP2A phosphatase activity protects MTCL1 from proteasomal degradation. Cells depleted of PPP2R5E and MTCL1 exhibit defects in microtubule organization.\",\n      \"method\": \"Mass spectrometry interactome screen, co-immunoprecipitation, siRNA knockdown, pharmacological inhibition (okadaic acid, MG132), immunofluorescence of microtubule organization\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — MS-identified interaction confirmed by co-IP, mechanistic follow-up with phosphatase inhibition and proteasome inhibitor rescue, single lab\",\n      \"pmids\": [\"27521566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PPP2R5E is a direct target of miR-23a; miR-23a downregulates PPP2R5E expression, and overexpression of PPP2R5E reverses miR-23a-induced suppression of apoptosis and promotion of cell growth in gastric cancer cells.\",\n      \"method\": \"Overexpression/knockdown of PPP2R5E and miR-23a in gastric cancer cell lines, in vivo xenograft assay, functional rescue experiments\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — functional rescue confirms PPP2R5E as downstream mediator of miR-23a, single lab with in vitro and in vivo evidence\",\n      \"pmids\": [\"24997345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"miR-19b is a direct negative regulator of PPP2R5E in colorectal cancer cells, confirmed by luciferase assay; miR-19b overexpression decreases PP2A activity, and PPP2R5E downregulation mediates miR-19b-induced oncogenic effects including increased cell viability, colonosphere formation, cell migration, and 5-FU resistance.\",\n      \"method\": \"Luciferase reporter assay, PP2A activity assay, siRNA knockdown, cell viability/migration/sphere formation assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct target validation by luciferase assay combined with PP2A activity measurement and functional knockdown phenotypes, single lab\",\n      \"pmids\": [\"37175484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A de novo heterozygous variant Glu191Lys in PPP2R5E, within a conserved LFDSEDPRER motif, results in decreased interaction with PP2A A and C subunits, disrupting holoenzyme formation, as demonstrated by biochemical assays in a patient with learning disorders, motor coordination problems, hypotonia, and myopathy.\",\n      \"method\": \"Whole exome sequencing, biochemical co-immunoprecipitation/interaction assays for PP2A holoenzyme assembly\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — biochemical interaction assay directly demonstrates impaired holoenzyme assembly, single case study/single lab\",\n      \"pmids\": [\"39284558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"PPP2R5E (a PP2A subunit) is a critical mediator of temozolomide sensitization in glioblastoma; miR-19b attenuation upregulates PPP2R5E, which induces genotoxic stress via elevated nuclear ROS, promoting senescence and ferroptosis. Pharmacological PP2A activation with FTY720 phenocopies miR-19b suppression, and PPP2R5E knockdown reverses temozolomide sensitization in vitro, in vivo, and ex vivo.\",\n      \"method\": \"MicroRNA screens, phosphoprotein analysis, clonogenic and spheroid assays, γH2AX foci formation, cell cycle/senescence/ferroptosis assays, orthotopic xenograft model, syngeneic mouse model, siRNA knockdown\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including in vitro, in vivo (two animal models), and ex vivo validation; pharmacological phenocopy and genetic rescue confirm mechanism\",\n      \"pmids\": [\"42203893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PPP2R5E is identified as a key target of EXOSC2 (RNA exosome component 2) in stem cells and cancer cells; EXOSC2 regulation of PPP2R5E is preserved across stem cells and cancer, suggesting PPP2R5E abundance is post-transcriptionally regulated by the RNA exosome.\",\n      \"method\": \"Genetic/molecular analysis in embryonic stem cells and cancer cells (EXOSC2 perturbation with monitoring of PPP2R5E)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint with limited methodological detail in abstract, single lab, mechanism of regulation not fully described\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PPP2R5E (B56epsilon) is a regulatory subunit of PP2A that positively regulates Wnt/beta-catenin signaling (upstream of Dishevelled), IGF/PI3K/Akt signaling, and Hedgehog signaling during development; it is alternatively translated to produce a cytoplasmic isoform (lacking an NLS) required for Wnt signaling and a nuclear-competent full-length form, with the C-terminus containing a nuclear export signal; it stabilizes MTCL1 protein via PP2A phosphatase activity to support microtubule organization; its interaction with the PP2A A and C subunits (holoenzyme assembly) is disrupted by pathogenic variants such as Glu191Lys; and it acts as a tumor suppressor whose downregulation by miRNAs (miR-23a, miR-19b) promotes cancer cell growth, while its upregulation sensitizes glioblastoma cells to temozolomide through nuclear ROS-induced DNA damage, senescence, and ferroptosis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP2R5E (B56epsilon) is a regulatory subunit of protein phosphatase 2A (PP2A) that confers substrate specificity on the holoenzyme to control developmental signaling, cytoskeletal organization, and tumor cell fate [#0, #6]. During vertebrate embryogenesis it acts as a positive regulator of multiple pathways: it is required for Wnt/beta-catenin signaling upstream of Dishevelled and downstream of the Wnt ligand, and it promotes eye induction through the IGF/PI3K/Akt pathway and eye field separation through the Hedgehog pathway [#0, #1]. The subunit is alternatively translated into a full-length, nuclear-competent form bearing an N-terminal nuclear localization signal and a C-terminal nuclear export signal, and a shorter cytoplasmic isoform lacking the NLS; the cytoplasmic isoform is specifically required for Wnt signaling and midbrain-hindbrain boundary formation [#2]. Through PP2A phosphatase activity, PPP2R5E physically binds and stabilizes the microtubule crosslinking factor MTCL1 against proteasomal degradation, supporting proper microtubule organization [#3]. Holoenzyme assembly depends on PPP2R5E contacting the PP2A A and C subunits; the de novo Glu191Lys variant in a conserved motif weakens these contacts and is associated with a neurodevelopmental and myopathic phenotype [#6]. PPP2R5E functions as a tumor suppressor: it is a direct target of miR-23a and miR-19b, whose elevation suppresses PPP2R5E and drives proliferation, survival, and chemoresistance [#4, #5], whereas restoring PPP2R5E in glioblastoma induces nuclear ROS-driven genotoxic stress, senescence, and ferroptosis to sensitize cells to temozolomide [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that a specific PP2A regulatory subunit positively gates Wnt signaling, defining where PP2A:B56epsilon acts within the pathway rather than treating PP2A as a generic phosphatase.\",\n      \"evidence\": \"Loss-of-function and epistasis in Xenopus embryos placing B56epsilon upstream of Dishevelled and downstream of Wnt ligand\",\n      \"pmids\": [\"14522869\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct phosphatase substrate within the Wnt pathway not identified\",\n        \"Mechanism connecting PP2A activity to Dishevelled regulation unresolved\"\n      ]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended B56epsilon's developmental role beyond Wnt to IGF/PI3K/Akt and Hedgehog signaling, showing it is a multi-pathway node in eye development.\",\n      \"evidence\": \"Loss-of-function in Xenopus with epistasis using PI3K/Akt inhibitors and Hedgehog pathway components\",\n      \"pmids\": [\"17074314\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Molecular substrates in PI3K/Akt and Hedgehog pathways not defined\",\n        \"Whether the same isoform mediates all three pathways unknown at this stage\"\n      ]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolved that distinct subcellular pools of B56epsilon carry distinct functions, showing alternative translation produces a cytoplasm-restricted isoform required specifically for Wnt signaling.\",\n      \"evidence\": \"Alternative translation mapping, NLS/NES mutagenesis, subcellular localization, and rescue in Xenopus\",\n      \"pmids\": [\"19129191\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Regulation of isoform choice in mammalian tissues not characterized\",\n        \"Nuclear function of the full-length form not assigned a substrate\"\n      ]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected PPP2R5E to oncogenic miRNA regulation, establishing it as a growth-suppressive effector silenced by miR-23a in gastric cancer.\",\n      \"evidence\": \"Overexpression/knockdown with functional rescue in gastric cancer cells and xenografts\",\n      \"pmids\": [\"24997345\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Phosphatase substrates mediating the anti-growth effect not identified\",\n        \"Direct binding of miR-23a to the PPP2R5E transcript not shown here\"\n      ]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified a non-signaling, cytoskeletal role: PPP2R5E-directed PP2A activity protects MTCL1 from proteasomal turnover to maintain microtubule organization.\",\n      \"evidence\": \"Mass spectrometry interactome, co-IP, siRNA, okadaic acid/MG132 rescue, and microtubule immunofluorescence\",\n      \"pmids\": [\"27521566\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The phosphosite on MTCL1 dephosphorylated by PP2A not mapped\",\n        \"Single-lab interaction without reciprocal in vivo validation\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Generalized miRNA control of PPP2R5E to a second cancer type and linked it to PP2A enzymatic output, with miR-19b directly repressing PPP2R5E to drive chemoresistance.\",\n      \"evidence\": \"Luciferase reporter, PP2A activity assay, and knockdown phenotypes in colorectal cancer cells\",\n      \"pmids\": [\"37175484\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Downstream PP2A substrates driving 5-FU resistance not defined\",\n        \"Single lab, in vitro only\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided a direct biochemical mechanism for human disease by showing a de novo variant disrupts PPP2R5E incorporation into the PP2A holoenzyme.\",\n      \"evidence\": \"Whole exome sequencing and biochemical interaction assays for A/C subunit binding in a patient\",\n      \"pmids\": [\"39284558\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single case; genotype-phenotype causality not established in a cohort\",\n        \"Downstream signaling consequence of holoenzyme loss not measured\"\n      ]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstrated that restoring PPP2R5E actively reprograms tumor cell fate, inducing nuclear ROS, senescence, and ferroptosis to sensitize glioblastoma to temozolomide.\",\n      \"evidence\": \"miRNA screens, γH2AX foci, senescence/ferroptosis assays, FTY720 phenocopy, knockdown rescue, and orthotopic/syngeneic mouse models\",\n      \"pmids\": [\"42203893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Phosphatase substrate linking PPP2R5E to nuclear ROS generation unknown\",\n        \"Whether the nuclear full-length isoform specifically drives this effect untested\"\n      ]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Raised the possibility that PPP2R5E abundance is set post-transcriptionally by the RNA exosome via EXOSC2 across stem and cancer cells.\",\n      \"evidence\": \"EXOSC2 perturbation with PPP2R5E monitoring in embryonic stem and cancer cells (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"Preprint with limited methodological detail; mechanism of regulation not described\",\n        \"Direct exosome targeting of the PPP2R5E transcript not demonstrated\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The specific phosphoprotein substrates dephosphorylated by PPP2R5E-containing PP2A across its Wnt, PI3K/Akt, Hedgehog, cytoskeletal, and tumor-suppressive roles remain unidentified.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\n        \"No direct PP2A:B56epsilon substrate mapped in any pathway\",\n        \"Structural basis of substrate selection not resolved\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"PP2A holoenzyme\"],\n    \"partners\": [\"MTCL1\", \"PPP2CA\", \"PPP2R1A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}