{"gene":"PPP2R1A","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1999,"finding":"Crystal structure of PR65/Aα at 2.3 Å resolution revealed 15 tandemly repeated HEAT motifs forming an elongated double-layer alpha-helical scaffold with a novel left-hand superhelical conformation; the intrarepeat turns align to form a continuous ridge that constitutes the protein interaction interface for binding the catalytic and regulatory B subunits.","method":"X-ray crystallography (2.3 Å resolution crystal structure)","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with detailed structural validation, foundational structural paper widely replicated in field","pmids":["9989501"],"is_preprint":false},{"year":2000,"finding":"HEAT repeat 11 intra-repeat loop, specifically lysine 416, of PR65/A is critical for binding both the PP2A catalytic subunit and HSF2; HSF2 competes with the catalytic subunit for binding to PR65 at this residue, identifying a molecular basis for HSF2 as a PP2A regulatory protein.","method":"Point mutagenesis of PR65 HEAT repeat 11 loop; binding competition assays between HSF2 and catalytic subunit","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro mutagenesis with functional binding assays, single lab, single study","pmids":["10872807"],"is_preprint":false},{"year":2005,"finding":"Cancer-associated PPP2R1A (Aα) mutants exhibit defects in binding to other PP2A subunits and impair phosphatase activity; partial suppression of endogenous Aα activates the AKT pathway and permits tumor formation in immunodeficient mice, indicating that cancer-associated Aα mutations contribute to tumorigenesis through functional haploinsufficiency that disturbs PP2A holoenzyme composition.","method":"Introduction of cancer-associated Aα mutants into immortalized human cells; binding assays; phosphatase activity assays; tumor formation in immunodeficient mice","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding assays, phosphatase activity assays, and in vivo tumor model in single study with multiple orthogonal methods","pmids":["16166293"],"is_preprint":false},{"year":2009,"finding":"Transcription of PP2A-Aα (PPP2R1A) is regulated by multiple transcription factors (CREB, ETS-1, AP-2α, SP-1) binding to cis-elements in the proximal promoter; SP-1 negatively regulates while CREB, ETS-1, and AP-2α positively regulate promoter activity.","method":"Promoter dissection; gel mobility shift assay; in vitro mutagenesis; reporter gene assay; ChIP assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (EMSA, ChIP, reporter assay, mutagenesis) in single lab","pmids":["19750005"],"is_preprint":false},{"year":2013,"finding":"The PPP2R1A promoter contains a functional variant (-241 -/G) that influences NF-κB DNA-binding affinity; NF-κB regulates PPP2R1A transcription through this variant, and CpG island methylation in the promoter region also modulates PPP2R1A expression.","method":"Promoter reporter assay; NF-κB binding assay; methylation analysis; case-control study","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — reporter assay and binding assay in single lab; methylation analysis adds orthogonal support","pmids":["23555712"],"is_preprint":false},{"year":2014,"finding":"PP2A Aα scaffold subunit (PR65A) is phosphorylated in vivo at residues S303, T268, and S314 in cardiac tissue; phosphomimetic substitutions at these sites inhibit interaction of PR65A with the PP2A catalytic subunit and reduce PP2A holoenzyme signaling; failing hearts have less phosphorylated PR65A and increased PP2A activity.","method":"In vivo phosphorylation site identification; HEK cell transfection with phosphomimetic and non-phosphorylated PR65A mutants; co-immunoprecipitation; 2D-DIGE phospho-protein profiling; Western blot from Dahl rat failing hearts","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo site identification plus functional mutagenesis with multiple orthogonal readouts in single lab","pmids":["24465463"],"is_preprint":false},{"year":2016,"finding":"eEF-2 kinase restricts synthesis (translation) of PP2A-Aα (PPP2R1A); knockdown of eEF-2K increases PP2A-Aα protein synthesis, which promotes ubiquitin-proteasomal degradation of c-Myc and downregulates pyruvate kinase M2, reducing glycolysis (Warburg effect).","method":"eEF-2K knockdown in cancer cells; metabolic assays (glucose uptake, lactate, ATP); PP2A-A protein synthesis measurement; c-Myc and PKM2 Western blot; in vitro and in vivo tumor models","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal biochemical and in vivo methods, single lab; note: correction issued (PMID 29973686) on figure placement but conclusions unchanged","pmids":["27181208"],"is_preprint":false},{"year":2016,"finding":"Recurrent PPP2R1A cancer mutations (in HEAT repeats 5 and 7) act in a dominant-negative manner by gaining interaction with the PP2A inhibitor TIPRL1 while retaining binding to B56/B' family subunits, forming substrate-trapping complexes with impaired phosphatase activity; this leads to hyperphosphorylation of GSK3β, Akt, and mTOR/p70S6K substrates and promotes tumor growth; TIPRL1 silencing restores GSK3β phosphorylation and rescues the growth advantage.","method":"Co-immunoprecipitation; phosphatase activity assays; overexpression of Aα mutants in endometrial cancer cells; anchorage-independent growth and tumor formation assays; TIPRL1 silencing rescue experiments; phospho-substrate analysis","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, phosphatase assay, in vivo tumor model, siRNA rescue) across same study establishing gain-of-function mechanism","pmids":["27485451"],"is_preprint":false},{"year":2016,"finding":"PPP2R1A mutation W257G enhances cancer cell migration through activation of the SRC-JNK-c-Jun pathway; W257G loses the ability to bind most B56 regulatory subunits except B56δ; wild-type PPP2R1A overexpression increases binding to B56 subunits and total PP2A activity.","method":"Cell migration assays; pathway inhibitor studies; Co-immunoprecipitation of PPP2R1A with B56 subunits; PP2A activity assay; in vitro and in vivo proliferation assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (migration assay, Co-IP, phosphatase activity) in single lab","pmids":["27272709"],"is_preprint":false},{"year":2017,"finding":"Ppp2r1a (PP2A scaffolding subunit PR65α) is required for embryonic patterning, primitive streak formation, gastrulation, and mesoderm formation in mice; loss of Ppp2r1a impairs both WNT and Nodal signal transduction in the epiblast, causing patterning defects; identified by gene knockout, homologous recombination, and genetic rescue.","method":"Gene knockout by homologous recombination; genetic rescue; transcriptome analysis; marker gene analysis in mouse embryos","journal":"Biology open","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout + genetic rescue + pathway analysis establishing in vivo function, replicated by multiple genetic approaches","pmids":["28619992"],"is_preprint":false},{"year":2019,"finding":"The P179R missense mutation in PPP2R1A changes the stable conformation of the Aα subunit (shown by enhanced sampling molecular dynamics and a mutant crystal structure), significantly impairs binding to the PP2A catalytic subunit, disrupts holoenzyme formation, and reduces enzymatic activity; restoration of wild-type Aα in a P179R patient-derived cell line restores PP2A function and attenuates tumorigenesis and metastasis in vivo.","method":"Molecular dynamics simulations; X-ray crystallography of mutant PP2A Aα; biochemical binding assays; PP2A activity assay; in vivo tumor/metastasis models with Aα restoration; small molecule PP2A reactivation","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure + molecular dynamics + biochemical reconstitution + in vivo rescue in a single rigorous study","pmids":["31142515"],"is_preprint":false},{"year":2019,"finding":"The P179R heterozygous PPP2R1A mutation decreases PP2A holoenzyme assembly and intracellular targeting during mitosis and enhances centrosome clustering when centrosome number is increased (by cytokinesis failure or centrosome amplification), likely through PP2A Aα loss of function, providing a mechanism for increased cellular fitness after whole-genome doubling.","method":"CRISPR-Cas9 knock-in of P179R in human RPE-1 cells; centrosome clustering quantification; PP2A assembly assays; immunofluorescence for mitotic spindle analysis","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — isogenic CRISPR knock-in with multiple functional readouts, single lab","pmids":["31357169"],"is_preprint":false},{"year":2020,"finding":"BRG1 activates PPP2R1A (PR65A) transcription by interacting with ETS1 and being recruited to the PR65A promoter; this BRG1-ETS1-PP2A-Aα cascade regulates eNOS phosphorylation (Ser1177) and NO bioavailability in vascular endothelial cells; BRG1 or ETS1 depletion represses PR65A expression and rescues eNOS phosphorylation and NO biosynthesis under oxLDL treatment.","method":"BRG1 knockdown in endothelial cells and in Apoe-/- mice; NO biosynthesis measurement; eNOS phosphorylation Western blot; ChIP assay for BRG1 and ETS1 at PR65A promoter; Co-immunoprecipitation of BRG1 and ETS1","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, Co-IP, in vivo mouse model, and functional rescue in single lab with multiple orthogonal methods","pmids":["32903816"],"is_preprint":false},{"year":2020,"finding":"De novo PPP2R1A variants cause variable PP2A dysfunction: variants with combined impaired B55α binding and increased striatin binding are associated with more severe biochemical disruption; severity of B55α binding deficit correlates with phenotypic severity (profound ID, epilepsy, microcephaly), while variants without B55α binding deficit show macrocephaly and milder ID.","method":"Phosphatase activity assay; PP2A subunit interaction binding assays for B55α and striatin; clinical phenotyping of 30 individuals with 16 different PPP2R1A variants","journal":"Genetics in medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical characterization (phosphatase activity, subunit binding) with genotype-phenotype correlation across 30 patients in single study","pmids":["33106617"],"is_preprint":false},{"year":2023,"finding":"PPP2R1A associates with an alternative WAVE complex (WAVE Shell Complex, WSC) containing NHSL1 instead of the canonical Arp2/3-activating WAVE subunit, at the lamellipodial edge; this association is RAC1-activation-dependent; PPP2R1A is required for migration persistence and RAC1-dependent actin polymerization in cell extracts; PPP2R1A tumor mutations impair WSC binding; NHSL1 depletion abolishes the requirement for PPP2R1A in migration.","method":"Proteomics (differential Co-IP of ABI1 with/without RAC1 activation); Co-immunoprecipitation; live-cell imaging; random and directed migration assays; actin polymerization in cell extracts; NHSL1 depletion rescue experiments; analysis of tumor-associated PPP2R1A mutations on WSC binding","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — proteomics identification plus reciprocal Co-IP, live imaging, functional assays, and genetic epistasis (NHSL1 rescue) in single comprehensive study","pmids":["37322026"],"is_preprint":false},{"year":2024,"finding":"African swine fever virus p17 recruits the host scaffold protein PR65A (PPP2R1A), a PP2A subunit, along with interactions with STING and IRF3, to downregulate phospho-IRF3 levels, inhibiting IFN-β induction; p17 also targets STING for partial degradation via apoptosis induction, further inhibiting p-TBK1 and p-IRF3.","method":"Co-immunoprecipitation of p17 with PR65A, STING, and IRF3; IRF3 phosphorylation Western blot; IFN-β reporter assays; apoptosis assays","journal":"Frontiers in microbiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP binding and functional phosphorylation assays in single lab; viral system provides context for PPP2R1A mechanism in innate immune signaling","pmids":["38957619"],"is_preprint":false},{"year":2024,"finding":"PPP2R1A p.R183W gain-of-function mutation promotes dephosphorylation and inactivation of deoxycytidine kinase (dCK) via B56δ-containing PP2A complexes, causing resistance to the nucleoside analogue clofarabine; B56δ knockdown or PP2A inhibition rescued clofarabine sensitivity, confirming a gain-of-function mechanism for R183W through altered substrate targeting.","method":"Stable expression of R183W in uterine serous carcinoma cells; clofarabine resistance assays; apoptosis assays; γH2AX, ATM, Chk1/2 activation; dCK phosphorylation analysis; B56δ siRNA knockdown rescue; pharmacologic PP2A inhibition","journal":"Cellular oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (phospho-substrate analysis, siRNA rescue, pharmacologic rescue) establishing gain-of-function mechanism, single lab","pmids":["38888850"],"is_preprint":false},{"year":2024,"finding":"BRG1 occupies the transcriptional activation site of PPP2R1A and inhibits its expression, thereby activating the PI3K/AKT signaling pathway and upregulating c-Myc and BCL-2; BRG1 knockdown de-represses PPP2R1A, suppressing AKT signaling and leukemia progression.","method":"ChIP assay; PPP2R1A knockdown/overexpression in B-ALL cell lines; cell cycle and apoptosis assays; PI3K/AKT pathway Western blot; xenograft mouse model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus functional pathway analysis and in vivo model, single lab","pmids":["39187513"],"is_preprint":false},{"year":2025,"finding":"Sphingosine and constrained analogs (FTY720, SH-BC-893) directly bind PPP2R1A and trigger reversible unfolding of the scaffold subunit, resulting in PP2A activation; these compounds also bind structurally related karyopherins (importin-β1, transportin-1, importin-5, importin-7), simultaneously inhibiting nuclear import; ceramide does not bind PPP2R1A and does not activate PP2A through this mechanism.","method":"Direct binding assays of sphingosine/analogs to PPP2R1A; protein unfolding assays; PP2A activity assays; nuclear import assays; comparative binding of ceramide (negative result)","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding and activity assays with mechanistic follow-up; single lab; ceramide negative control strengthens specificity","pmids":["40588551"],"is_preprint":false},{"year":2025,"finding":"Ppp2r1a haploinsufficiency in forebrain excitatory neurons increases presynaptic release probability (via reduced 2-arachidonoyl glycerol/2-AG endocannabinoid), reduces inhibitory synapse numbers, and impairs spatial learning; the 2-AG reduction is caused by increased MAGL transcription driven by EZH2 destabilization; MAGL inhibitor JZL184 rescues synaptic and learning deficits.","method":"Conditional heterozygous Ppp2r1a knockout in forebrain neurons (NEX-het-cKO); electrophysiology (excitatory and inhibitory synaptic transmission); 2-AG level measurement; MAGL and EZH2 expression analysis; JZL184 pharmacologic rescue; spatial learning/memory behavioral assays","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with multiple orthogonal methods (electrophysiology, lipid measurement, molecular analysis, pharmacologic rescue) establishing mechanistic pathway in single rigorous study","pmids":["40839403"],"is_preprint":false},{"year":2025,"finding":"HAT1 succinylates PPP2R1A at lysine 541 (K541), blocking PP2A holoenzyme assembly and PPP2R1A interaction with PCK1; this prevents dephosphorylation of PCK1 at serine 90, inhibiting gluconeogenic enzyme activity and activating SREBP1-induced lipogenesis gene expression to promote liver tumor growth.","method":"Multi-omics (proteomics/metabolomics); HAT1 knockout mouse model; succinylation mapping; Co-immunoprecipitation of PPP2R1A with PCK1; PCK1 S90 phosphorylation analysis; SREBP1 nuclear translocation assay; in vivo tumor growth","journal":"Acta pharmaceutica Sinica. B","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multi-omics plus Co-IP and in vivo model; novel PTM with functional consequence, single lab","pmids":["41132830"],"is_preprint":false},{"year":2026,"finding":"Pseudolarolide B (PB) covalently binds Cys317 of PPP2R1A, enhancing structural stability of the A subunit and promoting recruitment of both catalytic (C) and regulatory (B) subunits, thereby facilitating PP2A holoenzyme assembly, augmenting phosphatase activity, and dephosphorylating key inflammatory signaling proteins.","method":"Activity-based protein profiling (ABPP) to identify PPP2R1A as direct target; covalent binding site identification at Cys317; holoenzyme assembly assays; PP2A phosphatase activity assay; molecular dynamics simulations; inflammatory signaling phospho-substrate analysis","journal":"Bioorganic chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ABPP covalent target identification with functional binding/activity assays and structural simulation; single lab","pmids":["41795341"],"is_preprint":false}],"current_model":"PPP2R1A (PR65A/PP2A-Aα) encodes the scaffold subunit of PP2A, comprising 15 HEAT motifs that form an elongated superhelical platform recruiting the catalytic C subunit (via HEAT repeat 11, Lys416) and variable B regulatory subunits to assemble functionally diverse heterotrimeric holoenzymes; cancer-associated mutations in HEAT repeats 5 and 7 impair catalytic subunit binding and holoenzyme activity while gaining aberrant interactions with the inhibitor TIPRL1 (dominant-negative) or causing haploinsufficiency, hyperactivating AKT, GSK3β, and mTOR substrates; PPP2R1A is phosphorylated in vivo at S303/T268/S314 to negatively regulate its interaction with the catalytic subunit; succinylation at K541 by HAT1 blocks holoenzyme assembly and PCK1 dephosphorylation to reprogram gluconeogenesis/lipogenesis; PPP2R1A also associates with the WAVE Shell Complex at lamellipodia to sustain RAC1-dependent actin polymerization and migration persistence, regulates endocannabinoid (2-AG) levels and inhibitory synaptogenesis in neurons through EZH2/MAGL, is essential for WNT/Nodal-dependent gastrulation in vivo, and its transcription is controlled by BRG1-ETS1, NF-κB, CREB, AP-2α, and SP-1."},"narrative":{"mechanistic_narrative":"PPP2R1A (PR65/Aα) encodes the scaffold subunit of protein phosphatase 2A (PP2A), built from 15 tandem HEAT motifs that fold into an elongated left-handed superhelix whose intra-repeat ridge serves as the docking platform for the catalytic (C) and variable regulatory (B) subunits, thereby templating the assembly of functionally diverse heterotrimeric holoenzymes [PMID:9989501]. Binding of the catalytic subunit depends on the HEAT repeat 11 loop, where Lys416 also mediates competitive recruitment of HSF2 [PMID:10872807], and holoenzyme assembly is negatively tuned in vivo by phosphorylation of the scaffold at S303/T268/S314, which weakens the scaffold–catalytic interaction [PMID:24465463]. Through these holoenzymes PPP2R1A controls major growth-signaling outputs, restraining AKT, GSK3β, and mTOR/p70S6K substrate phosphorylation [PMID:16166293, PMID:27485451]. Cancer-associated mutations corrupt scaffold function by two routes: P179R-type changes alter the scaffold conformation and impair catalytic subunit binding to cause loss of function and haploinsufficiency [PMID:16166293, PMID:31142515], whereas HEAT repeat 5/7 mutations act dominant-negatively by gaining interaction with the inhibitor TIPRL1 while retaining B56 binding, forming substrate-trapping complexes that drive tumor growth [PMID:27485451]; specific variants such as W257G and R183W reprogram B56-subunit selectivity to activate SRC-JNK-c-Jun migration signaling or to dephosphorylate dCK and confer nucleoside-analogue resistance [PMID:27272709, PMID:38888850]. Beyond signaling, PPP2R1A is required in vivo for WNT- and Nodal-dependent gastrulation and mesoderm formation [PMID:28619992], associates RAC1-dependently with the NHSL1-containing WAVE Shell Complex at the lamellipodial edge to sustain actin polymerization and migration persistence [PMID:37322026], and regulates inhibitory synaptogenesis and spatial learning in forebrain neurons via an EZH2–MAGL–2-AG endocannabinoid axis [PMID:40839403]. De novo PPP2R1A variants cause a neurodevelopmental disorder whose severity tracks with the degree of impaired B55α binding [PMID:33106617]. Scaffold activity is further modulated by post-translational modification (HAT1-mediated K541 succinylation blocks holoenzyme assembly and PCK1 dephosphorylation to reprogram hepatic gluconeogenesis/lipogenesis) [PMID:41132830] and by small molecules that bind the scaffold to either unfold it and activate PP2A or stabilize it to promote holoenzyme assembly [PMID:40588551, PMID:41795341].","teleology":[{"year":1999,"claim":"Established the structural logic of the PP2A scaffold, explaining how a single subunit can simultaneously present binding surfaces for catalytic and regulatory subunits.","evidence":"2.3 Å X-ray crystal structure of PR65/Aα revealing 15 HEAT repeats forming a superhelical platform","pmids":["9989501"],"confidence":"High","gaps":["Structure alone does not define which residues map to specific B-subunit families","No bound holoenzyme structure in this study"]},{"year":2000,"claim":"Mapped a discrete scaffold residue governing catalytic-subunit engagement and showed regulatory proteins can compete at that site.","evidence":"Point mutagenesis of HEAT repeat 11 (Lys416) with HSF2/catalytic-subunit binding competition assays","pmids":["10872807"],"confidence":"Medium","gaps":["Single in vitro study without structural validation of the HSF2 interface","Physiological consequence of HSF2 competition not established"]},{"year":2005,"claim":"Linked cancer-associated Aα mutations to tumorigenesis, identifying functional haploinsufficiency that perturbs holoenzyme composition and activates AKT.","evidence":"Cancer-mutant Aα expression in human cells with binding/phosphatase assays and tumor formation in immunodeficient mice","pmids":["16166293"],"confidence":"High","gaps":["Did not resolve which specific substrates drive transformation","Loss- vs gain-of-function distinction not fully separated"]},{"year":2009,"claim":"Defined the transcriptional control of PPP2R1A by identifying activating and repressing factors at the proximal promoter.","evidence":"Promoter dissection, EMSA, ChIP, and reporter assays for CREB, ETS-1, AP-2α, SP-1","pmids":["19750005"],"confidence":"Medium","gaps":["Physiological contexts driving each factor not defined","No in vivo confirmation"]},{"year":2013,"claim":"Extended transcriptional regulation to NF-κB and DNA methylation, connecting a promoter variant to expression control.","evidence":"Promoter reporter, NF-κB binding, and methylation analysis with case-control study","pmids":["23555712"],"confidence":"Medium","gaps":["Disease relevance of the variant not mechanistically established","Single-lab reporter system"]},{"year":2014,"claim":"Showed that scaffold phosphorylation is a physiological switch tuning holoenzyme assembly, relevant to cardiac PP2A activity.","evidence":"In vivo phosphosite identification (S303/T268/S314) plus phosphomimetic mutants and Co-IP in failing rat hearts","pmids":["24465463"],"confidence":"Medium","gaps":["Kinase(s) responsible for these sites not identified","Whether phosphorylation acts on all holoenzyme types unknown"]},{"year":2016,"claim":"Resolved the dominant-negative mechanism of recurrent HEAT 5/7 mutations: gain of TIPRL1 binding creates substrate-trapping complexes that hyperactivate growth signaling.","evidence":"Co-IP, phosphatase assays, anchorage-independent growth, tumor models, and TIPRL1 silencing rescue in endometrial cancer cells","pmids":["27485451"],"confidence":"High","gaps":["Structural basis of aberrant TIPRL1 recruitment not defined","Generality across all hotspot mutations not established"]},{"year":2016,"claim":"Connected scaffold abundance to metabolic and migratory phenotypes through mutation-specific B56 selectivity and translational control.","evidence":"W257G migration and B56-binding Co-IP assays (SRC-JNK-c-Jun); eEF-2K knockdown linking Aα synthesis to c-Myc/PKM2 and glycolysis","pmids":["27272709","27181208"],"confidence":"Medium","gaps":["B56δ-specific substrate set incompletely defined","eEF-2K study had a published figure correction"]},{"year":2017,"claim":"Demonstrated an essential developmental role: PPP2R1A is required for gastrulation by enabling WNT and Nodal signal transduction.","evidence":"Mouse Ppp2r1a knockout with genetic rescue and embryonic marker/transcriptome analysis","pmids":["28619992"],"confidence":"High","gaps":["Direct phosphatase substrates in WNT/Nodal signaling not identified","Which holoenzyme B-subunits mediate the effect unknown"]},{"year":2019,"claim":"Provided structural and in vivo proof that P179R is a conformation-altering loss-of-function mutation, with wild-type restoration reversing tumorigenicity.","evidence":"Mutant crystal structure, molecular dynamics, binding/activity assays, and in vivo rescue with PP2A reactivation","pmids":["31142515"],"confidence":"High","gaps":["Reconciliation with dominant-negative model for other hotspots","In-cell mitotic consequences addressed only in a separate study"]},{"year":2019,"claim":"Linked P179R loss of function to mitotic fitness via reduced holoenzyme targeting and enhanced centrosome clustering after whole-genome doubling.","evidence":"CRISPR knock-in of P179R in RPE-1 cells with centrosome clustering and PP2A assembly assays","pmids":["31357169"],"confidence":"Medium","gaps":["Mechanism connecting PP2A to centrosome clustering not detailed","Single isogenic cell system"]},{"year":2020,"claim":"Identified a chromatin-remodeling transcriptional cascade (BRG1-ETS1) controlling PPP2R1A and downstream endothelial eNOS/NO signaling.","evidence":"BRG1 knockdown, ChIP and Co-IP at the PR65A promoter, and NO/eNOS readouts in endothelial cells and Apoe-/- mice","pmids":["32903816"],"confidence":"Medium","gaps":["Direct phosphatase target controlling eNOS phosphorylation not defined","Apparent opposite BRG1 effect versus leukemia context unresolved"]},{"year":2020,"claim":"Established genotype-phenotype logic for de novo neurodevelopmental variants, tying severity to specific B-subunit binding deficits.","evidence":"Phosphatase and B55α/striatin binding assays with clinical phenotyping across 30 individuals","pmids":["33106617"],"confidence":"Medium","gaps":["Neuronal substrates underlying phenotypes not identified here","Mechanism of altered striatin recruitment not structurally resolved"]},{"year":2023,"claim":"Revealed a non-canonical cytoskeletal role: RAC1-dependent recruitment of PPP2R1A into the NHSL1-containing WAVE Shell Complex sustains lamellipodial actin dynamics and migration persistence.","evidence":"Differential proteomics, reciprocal Co-IP, live imaging, actin polymerization assays, and NHSL1-depletion epistasis","pmids":["37322026"],"confidence":"High","gaps":["Whether PP2A catalytic activity is required within the WSC unknown","Substrates dephosphorylated at the lamellipodium not identified"]},{"year":2024,"claim":"Showed PPP2R1A is exploited in innate-immune evasion, recruited by viral p17 to suppress IRF3 phosphorylation and IFN-β induction.","evidence":"Co-IP of p17 with PR65A/STING/IRF3 and IFN-β reporter/phosphorylation assays","pmids":["38957619"],"confidence":"Medium","gaps":["Direct dephosphorylation of IRF3 by PP2A not formally demonstrated","Single viral system"]},{"year":2024,"claim":"Defined a drug-resistance gain-of-function: R183W redirects B56δ-PP2A to inactivate dCK and blunt clofarabine cytotoxicity.","evidence":"R183W expression in carcinoma cells with dCK phosphorylation, B56δ knockdown, and pharmacologic PP2A inhibition rescues","pmids":["38888850"],"confidence":"Medium","gaps":["Structural basis of altered B56δ targeting not resolved","Generality to other nucleoside analogues unknown"]},{"year":2024,"claim":"Added a leukemia-context transcriptional axis where BRG1 represses PPP2R1A to license PI3K/AKT and c-Myc/BCL-2 signaling.","evidence":"ChIP, PPP2R1A knockdown/overexpression, pathway Western blots, and xenografts in B-ALL","pmids":["39187513"],"confidence":"Medium","gaps":["Opposing direction of BRG1 effect versus endothelial study not reconciled","Direct PP2A substrate in AKT pathway not pinpointed"]},{"year":2025,"claim":"Identified small-molecule modulation of the scaffold itself: sphingosine analogs bind and reversibly unfold PPP2R1A to activate PP2A while inhibiting karyopherins.","evidence":"Direct binding, unfolding and PP2A activity assays with ceramide negative control and nuclear import assays","pmids":["40588551"],"confidence":"Medium","gaps":["Binding site on the scaffold not mapped","Selectivity over karyopherin effects in cells unclear"]},{"year":2025,"claim":"Demonstrated that K541 succinylation by HAT1 is a metabolic switch blocking holoenzyme assembly and PCK1 dephosphorylation to reprogram hepatic metabolism.","evidence":"Multi-omics, HAT1 knockout mice, succinylation mapping, Co-IP with PCK1, and in vivo tumor growth","pmids":["41132830"],"confidence":"Medium","gaps":["Desuccinylase / dynamics of K541 modification not defined","Single-lab in vivo system"]},{"year":2025,"claim":"Established a neuronal mechanism whereby Ppp2r1a haploinsufficiency disrupts inhibitory synaptogenesis and learning through an EZH2-MAGL-2-AG endocannabinoid pathway.","evidence":"Forebrain conditional heterozygous knockout with electrophysiology, 2-AG measurement, EZH2/MAGL analysis, and JZL184 rescue","pmids":["40839403"],"confidence":"High","gaps":["Direct PP2A substrate upstream of EZH2 not identified","Relevance to specific human PPP2R1A variants not tested"]},{"year":2026,"claim":"Showed scaffold stabilization as a therapeutic strategy: covalent binding at Cys317 promotes holoenzyme assembly and PP2A-mediated dephosphorylation of inflammatory signaling.","evidence":"ABPP target identification, covalent site mapping, holoenzyme assembly and phosphatase assays, and molecular dynamics","pmids":["41795341"],"confidence":"Medium","gaps":["In vivo efficacy not established","Specificity of Cys317 engagement across the proteome not fully characterized"]},{"year":null,"claim":"How distinct hotspot mutations select between loss-of-function (haploinsufficiency) and dominant-negative gain-of-function outcomes, and which specific holoenzyme-substrate pairs mediate each tissue phenotype, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural framework reconciling P179R loss-of-function with HEAT 5/7 dominant-negative behavior","Direct physiological substrates for developmental, cytoskeletal, and immune roles largely unidentified","Mechanism linking specific B-subunit reassortment to each clinical phenotype incomplete"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,7,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,5,7,20]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[14]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[7,18]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[14]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,7,17]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[2,7,10,16]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[9]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[6,20]},{"term_id":"R-HSA-1640170","term_label":"Cell 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Upon interaction with GNA12 promotes dephosphorylation of microtubule associated protein TAU/MAPT (PubMed:15525651). Required for proper chromosome segregation and for centromeric localization of SGO1 in mitosis (PubMed:16580887). Together with RACK1 adapter, mediates dephosphorylation of AKT1 at 'Ser-473', preventing AKT1 activation and AKT-mTOR signaling pathway (By similarity). Dephosphorylation of AKT1 is essential for regulatory T-cells (Treg) homeostasis and stability (By similarity). Part of the striatin-interacting phosphatase and kinase (STRIPAK) complexes (PubMed:18782753, PubMed:33633399). STRIPAK complexes have critical roles in protein (de)phosphorylation and are regulators of multiple signaling pathways including Hippo, MAPK, nuclear receptor and cytoskeleton remodeling (PubMed:18782753, PubMed:33633399). 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the intrarepeat turns align to form a continuous ridge that constitutes the protein interaction interface for binding the catalytic and regulatory B subunits.\",\n      \"method\": \"X-ray crystallography (2.3 Å resolution crystal structure)\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with detailed structural validation, foundational structural paper widely replicated in field\",\n      \"pmids\": [\"9989501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"HEAT repeat 11 intra-repeat loop, specifically lysine 416, of PR65/A is critical for binding both the PP2A catalytic subunit and HSF2; HSF2 competes with the catalytic subunit for binding to PR65 at this residue, identifying a molecular basis for HSF2 as a PP2A regulatory protein.\",\n      \"method\": \"Point mutagenesis of PR65 HEAT repeat 11 loop; binding competition assays between HSF2 and catalytic subunit\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro mutagenesis with functional binding assays, single lab, single study\",\n      \"pmids\": [\"10872807\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Cancer-associated PPP2R1A (Aα) mutants exhibit defects in binding to other PP2A subunits and impair phosphatase activity; partial suppression of endogenous Aα activates the AKT pathway and permits tumor formation in immunodeficient mice, indicating that cancer-associated Aα mutations contribute to tumorigenesis through functional haploinsufficiency that disturbs PP2A holoenzyme composition.\",\n      \"method\": \"Introduction of cancer-associated Aα mutants into immortalized human cells; binding assays; phosphatase activity assays; tumor formation in immunodeficient mice\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding assays, phosphatase activity assays, and in vivo tumor model in single study with multiple orthogonal methods\",\n      \"pmids\": [\"16166293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Transcription of PP2A-Aα (PPP2R1A) is regulated by multiple transcription factors (CREB, ETS-1, AP-2α, SP-1) binding to cis-elements in the proximal promoter; SP-1 negatively regulates while CREB, ETS-1, and AP-2α positively regulate promoter activity.\",\n      \"method\": \"Promoter dissection; gel mobility shift assay; in vitro mutagenesis; reporter gene assay; ChIP assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (EMSA, ChIP, reporter assay, mutagenesis) in single lab\",\n      \"pmids\": [\"19750005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The PPP2R1A promoter contains a functional variant (-241 -/G) that influences NF-κB DNA-binding affinity; NF-κB regulates PPP2R1A transcription through this variant, and CpG island methylation in the promoter region also modulates PPP2R1A expression.\",\n      \"method\": \"Promoter reporter assay; NF-κB binding assay; methylation analysis; case-control study\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — reporter assay and binding assay in single lab; methylation analysis adds orthogonal support\",\n      \"pmids\": [\"23555712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PP2A Aα scaffold subunit (PR65A) is phosphorylated in vivo at residues S303, T268, and S314 in cardiac tissue; phosphomimetic substitutions at these sites inhibit interaction of PR65A with the PP2A catalytic subunit and reduce PP2A holoenzyme signaling; failing hearts have less phosphorylated PR65A and increased PP2A activity.\",\n      \"method\": \"In vivo phosphorylation site identification; HEK cell transfection with phosphomimetic and non-phosphorylated PR65A mutants; co-immunoprecipitation; 2D-DIGE phospho-protein profiling; Western blot from Dahl rat failing hearts\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo site identification plus functional mutagenesis with multiple orthogonal readouts in single lab\",\n      \"pmids\": [\"24465463\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"eEF-2 kinase restricts synthesis (translation) of PP2A-Aα (PPP2R1A); knockdown of eEF-2K increases PP2A-Aα protein synthesis, which promotes ubiquitin-proteasomal degradation of c-Myc and downregulates pyruvate kinase M2, reducing glycolysis (Warburg effect).\",\n      \"method\": \"eEF-2K knockdown in cancer cells; metabolic assays (glucose uptake, lactate, ATP); PP2A-A protein synthesis measurement; c-Myc and PKM2 Western blot; in vitro and in vivo tumor models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal biochemical and in vivo methods, single lab; note: correction issued (PMID 29973686) on figure placement but conclusions unchanged\",\n      \"pmids\": [\"27181208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Recurrent PPP2R1A cancer mutations (in HEAT repeats 5 and 7) act in a dominant-negative manner by gaining interaction with the PP2A inhibitor TIPRL1 while retaining binding to B56/B' family subunits, forming substrate-trapping complexes with impaired phosphatase activity; this leads to hyperphosphorylation of GSK3β, Akt, and mTOR/p70S6K substrates and promotes tumor growth; TIPRL1 silencing restores GSK3β phosphorylation and rescues the growth advantage.\",\n      \"method\": \"Co-immunoprecipitation; phosphatase activity assays; overexpression of Aα mutants in endometrial cancer cells; anchorage-independent growth and tumor formation assays; TIPRL1 silencing rescue experiments; phospho-substrate analysis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, phosphatase assay, in vivo tumor model, siRNA rescue) across same study establishing gain-of-function mechanism\",\n      \"pmids\": [\"27485451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PPP2R1A mutation W257G enhances cancer cell migration through activation of the SRC-JNK-c-Jun pathway; W257G loses the ability to bind most B56 regulatory subunits except B56δ; wild-type PPP2R1A overexpression increases binding to B56 subunits and total PP2A activity.\",\n      \"method\": \"Cell migration assays; pathway inhibitor studies; Co-immunoprecipitation of PPP2R1A with B56 subunits; PP2A activity assay; in vitro and in vivo proliferation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (migration assay, Co-IP, phosphatase activity) in single lab\",\n      \"pmids\": [\"27272709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Ppp2r1a (PP2A scaffolding subunit PR65α) is required for embryonic patterning, primitive streak formation, gastrulation, and mesoderm formation in mice; loss of Ppp2r1a impairs both WNT and Nodal signal transduction in the epiblast, causing patterning defects; identified by gene knockout, homologous recombination, and genetic rescue.\",\n      \"method\": \"Gene knockout by homologous recombination; genetic rescue; transcriptome analysis; marker gene analysis in mouse embryos\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout + genetic rescue + pathway analysis establishing in vivo function, replicated by multiple genetic approaches\",\n      \"pmids\": [\"28619992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The P179R missense mutation in PPP2R1A changes the stable conformation of the Aα subunit (shown by enhanced sampling molecular dynamics and a mutant crystal structure), significantly impairs binding to the PP2A catalytic subunit, disrupts holoenzyme formation, and reduces enzymatic activity; restoration of wild-type Aα in a P179R patient-derived cell line restores PP2A function and attenuates tumorigenesis and metastasis in vivo.\",\n      \"method\": \"Molecular dynamics simulations; X-ray crystallography of mutant PP2A Aα; biochemical binding assays; PP2A activity assay; in vivo tumor/metastasis models with Aα restoration; small molecule PP2A reactivation\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure + molecular dynamics + biochemical reconstitution + in vivo rescue in a single rigorous study\",\n      \"pmids\": [\"31142515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The P179R heterozygous PPP2R1A mutation decreases PP2A holoenzyme assembly and intracellular targeting during mitosis and enhances centrosome clustering when centrosome number is increased (by cytokinesis failure or centrosome amplification), likely through PP2A Aα loss of function, providing a mechanism for increased cellular fitness after whole-genome doubling.\",\n      \"method\": \"CRISPR-Cas9 knock-in of P179R in human RPE-1 cells; centrosome clustering quantification; PP2A assembly assays; immunofluorescence for mitotic spindle analysis\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isogenic CRISPR knock-in with multiple functional readouts, single lab\",\n      \"pmids\": [\"31357169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BRG1 activates PPP2R1A (PR65A) transcription by interacting with ETS1 and being recruited to the PR65A promoter; this BRG1-ETS1-PP2A-Aα cascade regulates eNOS phosphorylation (Ser1177) and NO bioavailability in vascular endothelial cells; BRG1 or ETS1 depletion represses PR65A expression and rescues eNOS phosphorylation and NO biosynthesis under oxLDL treatment.\",\n      \"method\": \"BRG1 knockdown in endothelial cells and in Apoe-/- mice; NO biosynthesis measurement; eNOS phosphorylation Western blot; ChIP assay for BRG1 and ETS1 at PR65A promoter; Co-immunoprecipitation of BRG1 and ETS1\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, Co-IP, in vivo mouse model, and functional rescue in single lab with multiple orthogonal methods\",\n      \"pmids\": [\"32903816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"De novo PPP2R1A variants cause variable PP2A dysfunction: variants with combined impaired B55α binding and increased striatin binding are associated with more severe biochemical disruption; severity of B55α binding deficit correlates with phenotypic severity (profound ID, epilepsy, microcephaly), while variants without B55α binding deficit show macrocephaly and milder ID.\",\n      \"method\": \"Phosphatase activity assay; PP2A subunit interaction binding assays for B55α and striatin; clinical phenotyping of 30 individuals with 16 different PPP2R1A variants\",\n      \"journal\": \"Genetics in medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical characterization (phosphatase activity, subunit binding) with genotype-phenotype correlation across 30 patients in single study\",\n      \"pmids\": [\"33106617\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PPP2R1A associates with an alternative WAVE complex (WAVE Shell Complex, WSC) containing NHSL1 instead of the canonical Arp2/3-activating WAVE subunit, at the lamellipodial edge; this association is RAC1-activation-dependent; PPP2R1A is required for migration persistence and RAC1-dependent actin polymerization in cell extracts; PPP2R1A tumor mutations impair WSC binding; NHSL1 depletion abolishes the requirement for PPP2R1A in migration.\",\n      \"method\": \"Proteomics (differential Co-IP of ABI1 with/without RAC1 activation); Co-immunoprecipitation; live-cell imaging; random and directed migration assays; actin polymerization in cell extracts; NHSL1 depletion rescue experiments; analysis of tumor-associated PPP2R1A mutations on WSC binding\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proteomics identification plus reciprocal Co-IP, live imaging, functional assays, and genetic epistasis (NHSL1 rescue) in single comprehensive study\",\n      \"pmids\": [\"37322026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"African swine fever virus p17 recruits the host scaffold protein PR65A (PPP2R1A), a PP2A subunit, along with interactions with STING and IRF3, to downregulate phospho-IRF3 levels, inhibiting IFN-β induction; p17 also targets STING for partial degradation via apoptosis induction, further inhibiting p-TBK1 and p-IRF3.\",\n      \"method\": \"Co-immunoprecipitation of p17 with PR65A, STING, and IRF3; IRF3 phosphorylation Western blot; IFN-β reporter assays; apoptosis assays\",\n      \"journal\": \"Frontiers in microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP binding and functional phosphorylation assays in single lab; viral system provides context for PPP2R1A mechanism in innate immune signaling\",\n      \"pmids\": [\"38957619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PPP2R1A p.R183W gain-of-function mutation promotes dephosphorylation and inactivation of deoxycytidine kinase (dCK) via B56δ-containing PP2A complexes, causing resistance to the nucleoside analogue clofarabine; B56δ knockdown or PP2A inhibition rescued clofarabine sensitivity, confirming a gain-of-function mechanism for R183W through altered substrate targeting.\",\n      \"method\": \"Stable expression of R183W in uterine serous carcinoma cells; clofarabine resistance assays; apoptosis assays; γH2AX, ATM, Chk1/2 activation; dCK phosphorylation analysis; B56δ siRNA knockdown rescue; pharmacologic PP2A inhibition\",\n      \"journal\": \"Cellular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (phospho-substrate analysis, siRNA rescue, pharmacologic rescue) establishing gain-of-function mechanism, single lab\",\n      \"pmids\": [\"38888850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"BRG1 occupies the transcriptional activation site of PPP2R1A and inhibits its expression, thereby activating the PI3K/AKT signaling pathway and upregulating c-Myc and BCL-2; BRG1 knockdown de-represses PPP2R1A, suppressing AKT signaling and leukemia progression.\",\n      \"method\": \"ChIP assay; PPP2R1A knockdown/overexpression in B-ALL cell lines; cell cycle and apoptosis assays; PI3K/AKT pathway Western blot; xenograft mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus functional pathway analysis and in vivo model, single lab\",\n      \"pmids\": [\"39187513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Sphingosine and constrained analogs (FTY720, SH-BC-893) directly bind PPP2R1A and trigger reversible unfolding of the scaffold subunit, resulting in PP2A activation; these compounds also bind structurally related karyopherins (importin-β1, transportin-1, importin-5, importin-7), simultaneously inhibiting nuclear import; ceramide does not bind PPP2R1A and does not activate PP2A through this mechanism.\",\n      \"method\": \"Direct binding assays of sphingosine/analogs to PPP2R1A; protein unfolding assays; PP2A activity assays; nuclear import assays; comparative binding of ceramide (negative result)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding and activity assays with mechanistic follow-up; single lab; ceramide negative control strengthens specificity\",\n      \"pmids\": [\"40588551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Ppp2r1a haploinsufficiency in forebrain excitatory neurons increases presynaptic release probability (via reduced 2-arachidonoyl glycerol/2-AG endocannabinoid), reduces inhibitory synapse numbers, and impairs spatial learning; the 2-AG reduction is caused by increased MAGL transcription driven by EZH2 destabilization; MAGL inhibitor JZL184 rescues synaptic and learning deficits.\",\n      \"method\": \"Conditional heterozygous Ppp2r1a knockout in forebrain neurons (NEX-het-cKO); electrophysiology (excitatory and inhibitory synaptic transmission); 2-AG level measurement; MAGL and EZH2 expression analysis; JZL184 pharmacologic rescue; spatial learning/memory behavioral assays\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with multiple orthogonal methods (electrophysiology, lipid measurement, molecular analysis, pharmacologic rescue) establishing mechanistic pathway in single rigorous study\",\n      \"pmids\": [\"40839403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"HAT1 succinylates PPP2R1A at lysine 541 (K541), blocking PP2A holoenzyme assembly and PPP2R1A interaction with PCK1; this prevents dephosphorylation of PCK1 at serine 90, inhibiting gluconeogenic enzyme activity and activating SREBP1-induced lipogenesis gene expression to promote liver tumor growth.\",\n      \"method\": \"Multi-omics (proteomics/metabolomics); HAT1 knockout mouse model; succinylation mapping; Co-immunoprecipitation of PPP2R1A with PCK1; PCK1 S90 phosphorylation analysis; SREBP1 nuclear translocation assay; in vivo tumor growth\",\n      \"journal\": \"Acta pharmaceutica Sinica. B\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multi-omics plus Co-IP and in vivo model; novel PTM with functional consequence, single lab\",\n      \"pmids\": [\"41132830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Pseudolarolide B (PB) covalently binds Cys317 of PPP2R1A, enhancing structural stability of the A subunit and promoting recruitment of both catalytic (C) and regulatory (B) subunits, thereby facilitating PP2A holoenzyme assembly, augmenting phosphatase activity, and dephosphorylating key inflammatory signaling proteins.\",\n      \"method\": \"Activity-based protein profiling (ABPP) to identify PPP2R1A as direct target; covalent binding site identification at Cys317; holoenzyme assembly assays; PP2A phosphatase activity assay; molecular dynamics simulations; inflammatory signaling phospho-substrate analysis\",\n      \"journal\": \"Bioorganic chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ABPP covalent target identification with functional binding/activity assays and structural simulation; single lab\",\n      \"pmids\": [\"41795341\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPP2R1A (PR65A/PP2A-Aα) encodes the scaffold subunit of PP2A, comprising 15 HEAT motifs that form an elongated superhelical platform recruiting the catalytic C subunit (via HEAT repeat 11, Lys416) and variable B regulatory subunits to assemble functionally diverse heterotrimeric holoenzymes; cancer-associated mutations in HEAT repeats 5 and 7 impair catalytic subunit binding and holoenzyme activity while gaining aberrant interactions with the inhibitor TIPRL1 (dominant-negative) or causing haploinsufficiency, hyperactivating AKT, GSK3β, and mTOR substrates; PPP2R1A is phosphorylated in vivo at S303/T268/S314 to negatively regulate its interaction with the catalytic subunit; succinylation at K541 by HAT1 blocks holoenzyme assembly and PCK1 dephosphorylation to reprogram gluconeogenesis/lipogenesis; PPP2R1A also associates with the WAVE Shell Complex at lamellipodia to sustain RAC1-dependent actin polymerization and migration persistence, regulates endocannabinoid (2-AG) levels and inhibitory synaptogenesis in neurons through EZH2/MAGL, is essential for WNT/Nodal-dependent gastrulation in vivo, and its transcription is controlled by BRG1-ETS1, NF-κB, CREB, AP-2α, and SP-1.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP2R1A (PR65/Aα) encodes the scaffold subunit of protein phosphatase 2A (PP2A), built from 15 tandem HEAT motifs that fold into an elongated left-handed superhelix whose intra-repeat ridge serves as the docking platform for the catalytic (C) and variable regulatory (B) subunits, thereby templating the assembly of functionally diverse heterotrimeric holoenzymes [#0]. Binding of the catalytic subunit depends on the HEAT repeat 11 loop, where Lys416 also mediates competitive recruitment of HSF2 [#1], and holoenzyme assembly is negatively tuned in vivo by phosphorylation of the scaffold at S303/T268/S314, which weakens the scaffold–catalytic interaction [#5]. Through these holoenzymes PPP2R1A controls major growth-signaling outputs, restraining AKT, GSK3β, and mTOR/p70S6K substrate phosphorylation [#2, #7]. Cancer-associated mutations corrupt scaffold function by two routes: P179R-type changes alter the scaffold conformation and impair catalytic subunit binding to cause loss of function and haploinsufficiency [#2, #10], whereas HEAT repeat 5/7 mutations act dominant-negatively by gaining interaction with the inhibitor TIPRL1 while retaining B56 binding, forming substrate-trapping complexes that drive tumor growth [#7]; specific variants such as W257G and R183W reprogram B56-subunit selectivity to activate SRC-JNK-c-Jun migration signaling or to dephosphorylate dCK and confer nucleoside-analogue resistance [#8, #16]. Beyond signaling, PPP2R1A is required in vivo for WNT- and Nodal-dependent gastrulation and mesoderm formation [#9], associates RAC1-dependently with the NHSL1-containing WAVE Shell Complex at the lamellipodial edge to sustain actin polymerization and migration persistence [#14], and regulates inhibitory synaptogenesis and spatial learning in forebrain neurons via an EZH2–MAGL–2-AG endocannabinoid axis [#19]. De novo PPP2R1A variants cause a neurodevelopmental disorder whose severity tracks with the degree of impaired B55α binding [#13]. Scaffold activity is further modulated by post-translational modification (HAT1-mediated K541 succinylation blocks holoenzyme assembly and PCK1 dephosphorylation to reprogram hepatic gluconeogenesis/lipogenesis) [#20] and by small molecules that bind the scaffold to either unfold it and activate PP2A or stabilize it to promote holoenzyme assembly [#18, #21].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established the structural logic of the PP2A scaffold, explaining how a single subunit can simultaneously present binding surfaces for catalytic and regulatory subunits.\",\n      \"evidence\": \"2.3 Å X-ray crystal structure of PR65/Aα revealing 15 HEAT repeats forming a superhelical platform\",\n      \"pmids\": [\"9989501\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure alone does not define which residues map to specific B-subunit families\", \"No bound holoenzyme structure in this study\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Mapped a discrete scaffold residue governing catalytic-subunit engagement and showed regulatory proteins can compete at that site.\",\n      \"evidence\": \"Point mutagenesis of HEAT repeat 11 (Lys416) with HSF2/catalytic-subunit binding competition assays\",\n      \"pmids\": [\"10872807\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single in vitro study without structural validation of the HSF2 interface\", \"Physiological consequence of HSF2 competition not established\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked cancer-associated Aα mutations to tumorigenesis, identifying functional haploinsufficiency that perturbs holoenzyme composition and activates AKT.\",\n      \"evidence\": \"Cancer-mutant Aα expression in human cells with binding/phosphatase assays and tumor formation in immunodeficient mice\",\n      \"pmids\": [\"16166293\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which specific substrates drive transformation\", \"Loss- vs gain-of-function distinction not fully separated\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the transcriptional control of PPP2R1A by identifying activating and repressing factors at the proximal promoter.\",\n      \"evidence\": \"Promoter dissection, EMSA, ChIP, and reporter assays for CREB, ETS-1, AP-2α, SP-1\",\n      \"pmids\": [\"19750005\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological contexts driving each factor not defined\", \"No in vivo confirmation\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended transcriptional regulation to NF-κB and DNA methylation, connecting a promoter variant to expression control.\",\n      \"evidence\": \"Promoter reporter, NF-κB binding, and methylation analysis with case-control study\",\n      \"pmids\": [\"23555712\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Disease relevance of the variant not mechanistically established\", \"Single-lab reporter system\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed that scaffold phosphorylation is a physiological switch tuning holoenzyme assembly, relevant to cardiac PP2A activity.\",\n      \"evidence\": \"In vivo phosphosite identification (S303/T268/S314) plus phosphomimetic mutants and Co-IP in failing rat hearts\",\n      \"pmids\": [\"24465463\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase(s) responsible for these sites not identified\", \"Whether phosphorylation acts on all holoenzyme types unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved the dominant-negative mechanism of recurrent HEAT 5/7 mutations: gain of TIPRL1 binding creates substrate-trapping complexes that hyperactivate growth signaling.\",\n      \"evidence\": \"Co-IP, phosphatase assays, anchorage-independent growth, tumor models, and TIPRL1 silencing rescue in endometrial cancer cells\",\n      \"pmids\": [\"27485451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of aberrant TIPRL1 recruitment not defined\", \"Generality across all hotspot mutations not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected scaffold abundance to metabolic and migratory phenotypes through mutation-specific B56 selectivity and translational control.\",\n      \"evidence\": \"W257G migration and B56-binding Co-IP assays (SRC-JNK-c-Jun); eEF-2K knockdown linking Aα synthesis to c-Myc/PKM2 and glycolysis\",\n      \"pmids\": [\"27272709\", \"27181208\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"B56δ-specific substrate set incompletely defined\", \"eEF-2K study had a published figure correction\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrated an essential developmental role: PPP2R1A is required for gastrulation by enabling WNT and Nodal signal transduction.\",\n      \"evidence\": \"Mouse Ppp2r1a knockout with genetic rescue and embryonic marker/transcriptome analysis\",\n      \"pmids\": [\"28619992\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phosphatase substrates in WNT/Nodal signaling not identified\", \"Which holoenzyme B-subunits mediate the effect unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided structural and in vivo proof that P179R is a conformation-altering loss-of-function mutation, with wild-type restoration reversing tumorigenicity.\",\n      \"evidence\": \"Mutant crystal structure, molecular dynamics, binding/activity assays, and in vivo rescue with PP2A reactivation\",\n      \"pmids\": [\"31142515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation with dominant-negative model for other hotspots\", \"In-cell mitotic consequences addressed only in a separate study\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Linked P179R loss of function to mitotic fitness via reduced holoenzyme targeting and enhanced centrosome clustering after whole-genome doubling.\",\n      \"evidence\": \"CRISPR knock-in of P179R in RPE-1 cells with centrosome clustering and PP2A assembly assays\",\n      \"pmids\": [\"31357169\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting PP2A to centrosome clustering not detailed\", \"Single isogenic cell system\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified a chromatin-remodeling transcriptional cascade (BRG1-ETS1) controlling PPP2R1A and downstream endothelial eNOS/NO signaling.\",\n      \"evidence\": \"BRG1 knockdown, ChIP and Co-IP at the PR65A promoter, and NO/eNOS readouts in endothelial cells and Apoe-/- mice\",\n      \"pmids\": [\"32903816\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphatase target controlling eNOS phosphorylation not defined\", \"Apparent opposite BRG1 effect versus leukemia context unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established genotype-phenotype logic for de novo neurodevelopmental variants, tying severity to specific B-subunit binding deficits.\",\n      \"evidence\": \"Phosphatase and B55α/striatin binding assays with clinical phenotyping across 30 individuals\",\n      \"pmids\": [\"33106617\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Neuronal substrates underlying phenotypes not identified here\", \"Mechanism of altered striatin recruitment not structurally resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Revealed a non-canonical cytoskeletal role: RAC1-dependent recruitment of PPP2R1A into the NHSL1-containing WAVE Shell Complex sustains lamellipodial actin dynamics and migration persistence.\",\n      \"evidence\": \"Differential proteomics, reciprocal Co-IP, live imaging, actin polymerization assays, and NHSL1-depletion epistasis\",\n      \"pmids\": [\"37322026\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PP2A catalytic activity is required within the WSC unknown\", \"Substrates dephosphorylated at the lamellipodium not identified\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed PPP2R1A is exploited in innate-immune evasion, recruited by viral p17 to suppress IRF3 phosphorylation and IFN-β induction.\",\n      \"evidence\": \"Co-IP of p17 with PR65A/STING/IRF3 and IFN-β reporter/phosphorylation assays\",\n      \"pmids\": [\"38957619\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct dephosphorylation of IRF3 by PP2A not formally demonstrated\", \"Single viral system\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a drug-resistance gain-of-function: R183W redirects B56δ-PP2A to inactivate dCK and blunt clofarabine cytotoxicity.\",\n      \"evidence\": \"R183W expression in carcinoma cells with dCK phosphorylation, B56δ knockdown, and pharmacologic PP2A inhibition rescues\",\n      \"pmids\": [\"38888850\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of altered B56δ targeting not resolved\", \"Generality to other nucleoside analogues unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Added a leukemia-context transcriptional axis where BRG1 represses PPP2R1A to license PI3K/AKT and c-Myc/BCL-2 signaling.\",\n      \"evidence\": \"ChIP, PPP2R1A knockdown/overexpression, pathway Western blots, and xenografts in B-ALL\",\n      \"pmids\": [\"39187513\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Opposing direction of BRG1 effect versus endothelial study not reconciled\", \"Direct PP2A substrate in AKT pathway not pinpointed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified small-molecule modulation of the scaffold itself: sphingosine analogs bind and reversibly unfold PPP2R1A to activate PP2A while inhibiting karyopherins.\",\n      \"evidence\": \"Direct binding, unfolding and PP2A activity assays with ceramide negative control and nuclear import assays\",\n      \"pmids\": [\"40588551\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Binding site on the scaffold not mapped\", \"Selectivity over karyopherin effects in cells unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that K541 succinylation by HAT1 is a metabolic switch blocking holoenzyme assembly and PCK1 dephosphorylation to reprogram hepatic metabolism.\",\n      \"evidence\": \"Multi-omics, HAT1 knockout mice, succinylation mapping, Co-IP with PCK1, and in vivo tumor growth\",\n      \"pmids\": [\"41132830\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Desuccinylase / dynamics of K541 modification not defined\", \"Single-lab in vivo system\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Established a neuronal mechanism whereby Ppp2r1a haploinsufficiency disrupts inhibitory synaptogenesis and learning through an EZH2-MAGL-2-AG endocannabinoid pathway.\",\n      \"evidence\": \"Forebrain conditional heterozygous knockout with electrophysiology, 2-AG measurement, EZH2/MAGL analysis, and JZL184 rescue\",\n      \"pmids\": [\"40839403\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PP2A substrate upstream of EZH2 not identified\", \"Relevance to specific human PPP2R1A variants not tested\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed scaffold stabilization as a therapeutic strategy: covalent binding at Cys317 promotes holoenzyme assembly and PP2A-mediated dephosphorylation of inflammatory signaling.\",\n      \"evidence\": \"ABPP target identification, covalent site mapping, holoenzyme assembly and phosphatase assays, and molecular dynamics\",\n      \"pmids\": [\"41795341\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo efficacy not established\", \"Specificity of Cys317 engagement across the proteome not fully characterized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How distinct hotspot mutations select between loss-of-function (haploinsufficiency) and dominant-negative gain-of-function outcomes, and which specific holoenzyme-substrate pairs mediate each tissue phenotype, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural framework reconciling P179R loss-of-function with HEAT 5/7 dominant-negative behavior\", \"Direct physiological substrates for developmental, cytoskeletal, and immune roles largely unidentified\", \"Mechanism linking specific B-subunit reassortment to each clinical phenotype incomplete\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 7, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 5, 7, 20]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [7, 18]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [14]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 7, 17]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [2, 7, 10, 16]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [6, 20]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"complexes\": [\n      \"PP2A holoenzyme (A-B-C heterotrimer)\",\n      \"WAVE Shell Complex (NHSL1-containing)\"\n    ],\n    \"partners\": [\n      \"PP2A catalytic subunit (C)\",\n      \"TIPRL1\",\n      \"B56/B' subunits\",\n      \"B55α\",\n      \"striatin\",\n      \"NHSL1\",\n      \"PCK1\",\n      \"HSF2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}