{"gene":"PPP4R3A","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2006,"finding":"SMK-1 (C. elegans ortholog of PPP4R3A/SMEK1) acts downstream of DAF-16 nuclear translocation to modulate DAF-16 transcriptional specificity, being required for DAF-16-dependent innate immune, UV, and oxidative stress functions but not thermal stress functions, placing SMK-1 as a pathway component that controls which subset of DAF-16 target genes are activated.","method":"Genetic epistasis (localization analysis of DAF-16::GFP in smk-1 mutants), transcriptional analysis of DAF-16 target genes, physiological assays in C. elegans loss-of-function mutants","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with multiple orthogonal phenotypic readouts (longevity, immunity, stress resistance, transcriptional targets), replicated in follow-up studies","pmids":["16530049"],"is_preprint":false},{"year":2005,"finding":"PP4R3 (human PPP4R3A) forms a stable trimeric complex with PP4C (catalytic subunit) and PP4R2 regulatory subunit (termed PP4cs); the yeast ortholog Psy2 and Drosophila ortholog Flfl/PP4R3 are required for DNA damage repair, and human PP4R3 functionally complements the yeast psy2 deletion. PP4R3 may target the complex to the DNA damage repair machinery via an interaction with Rad53 (CHK2 ortholog).","method":"Tandem affinity purification (TAP) tagging combined with mass spectrometry, yeast complementation, cisplatin sensitivity assays in yeast and Drosophila, co-immunoprecipitation","journal":"Molecular & cellular proteomics : MCP","confidence":"High","confidence_rationale":"Tier 2 / Strong — TAP-MS complex identification, cross-species complementation, and multiple orthogonal functional assays across three organisms","pmids":["16085932"],"is_preprint":false},{"year":2008,"finding":"PP4R3 (PPP4R3A) forms part of a PP4c·PP4R2·PP4R3 heterotrimeric complex that exhibits phosphatase activity toward γH2AX; this complex has higher phosphatase activity than the PP4c·PP4R4 complex but lower than free PP4c, demonstrating that regulatory subunits modulate catalytic activity. Mutations analogous to those disrupting PP2Ac–PP2A-A interaction also disrupt PP4c–PP4R4 association.","method":"Affinity purification/mass spectrometry, in vitro phosphatase activity assay toward fluorogenic substrate and γH2AX, mutagenesis of PP4c interaction interface","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro phosphatase activity assay with defined substrates plus mutagenesis, single lab with multiple orthogonal methods","pmids":["18715871"],"is_preprint":false},{"year":2009,"finding":"PP4R3/Falafel (Drosophila ortholog of PPP4R3A) is required for asymmetric cortical localization of Miranda and associated cell fate determinants during neuroblast asymmetric division. Flfl is nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown; nuclear Flfl excludes Prospero/Miranda from the nucleus during interphase, while cytoplasmic Flfl mediates cortical Mira localization during mitosis. Flfl directly interacts with Miranda, and genetic analysis places flfl in parallel to or downstream of lgl.","method":"Clonal screen, loss-of-function analysis, live imaging of Flfl subcellular localization, nuclear-excluded and membrane-targeted Flfl constructs, direct protein interaction (co-immunoprecipitation/pulldown with Miranda), genetic epistasis with lgl","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal approaches (localization mutants, direct binding, genetic epistasis, knockdown of PP4C and PP4R2 producing same phenotype), single lab","pmids":["19204120"],"is_preprint":false},{"year":2007,"finding":"SMK-1 (PPP4R3A ortholog in C. elegans) recruits the PP4 catalytic subunit PPH-4.1 to replicating chromatin, where the SMK-1–PPH-4.1 complex silences the CHK-1 DNA damage checkpoint response during early embryogenesis, enabling on-schedule P lineage cell divisions.","method":"Genetic analysis (smk-1 mutant phenotype recapitulating rad-2 mutation), chromatin fractionation showing SMK-1-dependent PPH-4.1 recruitment to replicating chromatin, C. elegans loss-of-function","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic epistasis combined with biochemical chromatin fractionation establishing localization-function link, single lab with two orthogonal methods","pmids":["17908915"],"is_preprint":false},{"year":2007,"finding":"In Dictyostelium, SMEK (PPP4R3A ortholog) functions as the PP4R3 homolog and positively regulates a subset of PP4C functions including developmental progression, chemotaxis, and stress/cell movement gene expression. SMEK does not control absolute PP4C activity levels but regulates PP4C by controlling its nuclear localization. Genetic epistasis places mek1 upstream of pppC-smkA.","method":"Genetic epistasis (mek1, smkA, pppC mutant combinations), gene expression profiling, biochemical PP4C activity assays in smkA mutants, localization analysis of PP4C","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with gene expression profiling and PP4C activity assay, single lab, model organism study","pmids":["17353263"],"is_preprint":false},{"year":2020,"finding":"SMK-1/SMEK functions as part of a specific PP4 complex (PP4^SMK-1) that is required for transcriptional initiation at DAF-16-activated genes by facilitating RNA polymerase II recruitment to their promoters. Phosphoproteomics identified SPT-5/SUPT5H as a relevant substrate of PP4^SMK-1; SPT-5 knockdown phenocopies loss of PP4^SMK-1, indicating that PP4^SMK-1-mediated dephosphorylation of SPT-5 regulates transcription initiation at rate-limiting DAF-16 target genes.","method":"Phosphoproteomics (mass spectrometry-based substrate identification), RNA polymerase II ChIP or equivalent (promoter recruitment assay), RNAi knockdown epistasis in C. elegans, transcriptional reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphoproteomics substrate identification combined with epistasis knockdown and RNAPII recruitment assay, multiple orthogonal methods in single study","pmids":["31919361"],"is_preprint":false},{"year":2020,"finding":"SMK-1, as a regulatory subunit of the PP4 phosphatase complex, is required for DAF-16-mediated innate immunity specifically during adulthood in C. elegans; SMK-1 is epistatic to DAF-16 for adult pathogen resistance.","method":"Genetic epistasis (smk-1 and daf-16 mutants and double mutants), pathogen infection survival assays, DAF-16 transcriptional target analysis in adult C. elegans","journal":"G3 (Bethesda, Md.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple phenotypic readouts (survival, transcription), single lab","pmids":["32161087"],"is_preprint":false},{"year":2020,"finding":"PP4 complex members PPH-4.1/4.2 and SMK-1 regulate DAF-16 transcriptional activity in an age-dependent manner in adult C. elegans and contribute with PPFR-2 to innate immunity.","method":"Reverse genetics (RNAi knockdown of all C. elegans PP2A/4/6 subunit orthologs), DAF-16 transcriptional reporter assays, pathogen survival assays in postreproductive adults","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic reverse genetics screen with functional readouts, single lab, confirms prior findings in aging context","pmids":["33315870"],"is_preprint":false},{"year":2015,"finding":"Loss of Falafel/PP4R3 (Drosophila ortholog of PPP4R3A) promotes JNK pathway activation and JNK-dependent cell death in developing eye and wing, while ectopic Flfl expression suppresses TNF-triggered JNK-dependent cell death, identifying Flfl as a negative regulator of the JNK signaling pathway.","method":"Gain-of-function and loss-of-function analysis in Drosophila, epistasis with JNK pathway components, cell death assays (TUNEL/activated caspase) in developing tissues","journal":"BioMed research international","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — loss-of-function and gain-of-function genetic analysis with JNK pathway epistasis, single lab, single organism","pmids":["26583122"],"is_preprint":false},{"year":2020,"finding":"The EVH1 domain of Drosophila PP4R3/Falafel (ortholog of PPP4R3A) recognizes atypical FxxP and MxPP short linear motifs rather than canonical proline-rich sequences; this specificity is conferred by a conserved leucine that replaces the invariant phenylalanine of canonical EVH1 domains. The conserved Smk-1 domain of Falafel also participates in target-binding.","method":"Identification of novel EVH1 binding partners (pulldown/interaction assays), mutagenesis of the conserved leucine residue, domain-function mapping of the Smk-1 domain","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding partner identification combined with mutagenesis of critical residue establishing mechanism, single lab","pmids":["33352067"],"is_preprint":false},{"year":2022,"finding":"PPP4R3A loss-of-function in mouse cortex and hippocampus leads to downregulated mTORC1 signaling, reduced synthesis of synaptic proteins, and impaired synaptic function resulting in depression- and anxiety-like behaviors; overexpression of PPP4R3A in these regions activates mTORC1 and rescues synaptic protein synthesis, an effect blocked by rapamycin, placing PPP4R3A upstream of mTORC1.","method":"Conditional knockout of Ppp4r3a in mouse cortex/hippocampus, viral overexpression, rapamycin pharmacological inhibition, Western blot for mTORC1 pathway components and synaptic proteins, behavioral assays","journal":"Disease models & mechanisms","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO and OE with rescue and pharmacological blockade establishing pathway position, single lab with multiple orthogonal approaches","pmids":["35314861"],"is_preprint":false},{"year":2024,"finding":"Wild-type PPP4R3A exerts tumor-suppressive effects via the Akt-mTOR-P70 S6K/4E-BP1 axis in thyroid cancer cells; the missense variant PPP4R3A Asp409Asn loses tumor-suppressive function and instead promotes cell proliferation, migration, and invasion by activating Akt/mTOR signaling.","method":"Overexpression of wild-type vs. Asp409Asn mutant PPP4R3A in thyroid cancer cell lines, Western blot for Akt-mTOR-P70 S6K/4E-BP1 pathway, cell invasion/proliferation/migration assays","journal":"Biomedicines","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — gain-of-function with mutant comparison and pathway readout, single lab, no in vitro reconstitution","pmids":["38275415"],"is_preprint":false},{"year":2024,"finding":"In C. elegans, cathepsin B (CPR-6) promotes Aβ proteotoxicity by elevating SWSN-3 expression and reducing SMK-1 protein levels; knockdown of cpr-6 alleviates Aβ toxicity by increasing SMK-1 levels, placing SMK-1 downstream of CPR-6 in the regulation of proteotoxicity.","method":"RNAi knockdown of cpr-6, activity-based protease probes, SMK-1 protein level measurement, genetic epistasis in C. elegans Aβ toxicity model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — activity-based probe and genetic epistasis with protein level readout, single lab, single study","pmids":["39362844"],"is_preprint":false},{"year":2016,"finding":"In C. elegans, SMK-1 is a direct target of mir-231; loss of smk-1 causes susceptibility to graphene oxide toxicity while intestinal overexpression confers resistance; SMK-1 acts upstream of DAF-16/FOXO in the insulin signaling pathway to regulate toxicity responses.","method":"C. elegans smk-1 deletion mutant, tissue-specific overexpression, epistasis with daf-16, mir-231 target validation","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, genetic epistasis only, no direct biochemical mechanism for the SMK-1/DAF-16 relationship","pmids":["27558892"],"is_preprint":false}],"current_model":"PPP4R3A/SMEK1 is a regulatory subunit of the Protein Phosphatase 4 (PP4) holoenzyme that forms a trimeric complex with PP4C and PP4R2; its EVH1 domain recruits the complex to substrates bearing FxxP/MxPP motifs, and its Smk-1 domain also participates in target binding. The PP4^PPP4R3A complex dephosphorylates substrates including γH2AX and SPT-5/SUPT5H, thereby regulating DNA damage checkpoint silencing (via chromatin recruitment of PPH-4.1/PP4C), transcriptional initiation at FOXO/DAF-16 target genes (by controlling RNA polymerase II recruitment through SPT-5 dephosphorylation), asymmetric cell fate determinant localization in neuroblasts (via Miranda dephosphorylation), and in mammals it positively regulates mTORC1 signaling to support synaptic protein synthesis, with loss of function leading to depression-like phenotypes and gain-of-function mutations disrupting tumor suppression through the Akt-mTOR axis."},"narrative":{"mechanistic_narrative":"PPP4R3A (SMEK1/SMK-1) is a substrate-targeting regulatory subunit of the Protein Phosphatase 4 (PP4) holoenzyme, in which it assembles into a stable heterotrimeric complex with the PP4 catalytic subunit (PP4C) and the regulatory subunit PP4R2; this complex dephosphorylates γH2AX, and the regulatory subunits tune the catalytic activity of PP4C [PMID:16085932, PMID:18715871]. PPP4R3A directs the phosphatase to its targets through its EVH1 domain, which atypically recognizes FxxP and MxPP short linear motifs owing to a conserved leucine in place of the invariant EVH1 phenylalanine, with the Smk-1 domain also contributing to target binding [PMID:33352067]. A recurring theme across species is that PPP4R3A governs PP4C subcellular targeting and substrate selection rather than bulk catalytic output — it recruits PP4C to replicating chromatin to silence the DNA damage checkpoint [PMID:17908915], and it controls PP4C nuclear localization to enable a developmental and stress-response transcriptional program [PMID:17353263]. Through PP4-mediated dephosphorylation of the transcription elongation factor SPT-5/SUPT5H, PPP4R3A promotes RNA polymerase II recruitment and transcriptional initiation at FOXO/DAF-16 target genes, thereby specifying which stress-response and innate-immunity genes are activated [PMID:16530049, PMID:31919361]. In neuroblast asymmetric division it directly binds Miranda and, in a cell-cycle-dependent manner dictated by its own nuclear/cytoplasmic shuttling, controls cortical localization of cell-fate determinants [PMID:19204120]. In mammals, PPP4R3A positively regulates mTORC1 signaling to support synaptic protein synthesis, with loss of function producing depression- and anxiety-like phenotypes [PMID:35314861], and wild-type protein acts as a tumor suppressor through the Akt-mTOR-P70 S6K/4E-BP1 axis, a function lost by the Asp409Asn variant [PMID:38275415].","teleology":[{"year":2005,"claim":"Establishing PPP4R3A as a defined PP4 holoenzyme subunit answered whether it acts as a phosphatase-targeting protein and connected it to DNA damage repair across species.","evidence":"TAP-MS complex purification, yeast complementation, and cisplatin-sensitivity assays in yeast, Drosophila, and human cells","pmids":["16085932"],"confidence":"High","gaps":["Did not define how PP4R3 selects substrates","Rad53/CHK2 interaction not mapped to a direct binding interface"]},{"year":2006,"claim":"Genetic placement of SMK-1 downstream of DAF-16 nuclear entry showed it acts as a transcriptional-specificity determinant rather than a regulator of FOXO localization, defining its role in selecting target-gene subsets.","evidence":"Genetic epistasis with DAF-16::GFP localization and stress/immunity phenotypes in C. elegans loss-of-function mutants","pmids":["16530049"],"confidence":"High","gaps":["Molecular mechanism of transcriptional specificity not resolved at this stage","No biochemical substrate identified"]},{"year":2007,"claim":"Chromatin fractionation showed PPP4R3A controls PP4C recruitment to a site of action, establishing the subunit as a spatial targeting factor for the catalytic subunit.","evidence":"Genetic analysis plus SMK-1-dependent PPH-4.1 chromatin fractionation in C. elegans embryos; parallel Dictyostelium work showed SMEK controls PP4C nuclear localization without altering bulk activity","pmids":["17908915","17353263"],"confidence":"Medium","gaps":["Direct chromatin-binding partner of SMK-1 not identified","Checkpoint substrate dephosphorylated on chromatin not defined"]},{"year":2008,"claim":"Reconstitution of the PP4c·PP4R2·PP4R3 trimer with in vitro phosphatase assays answered whether regulatory subunits modulate catalysis, demonstrating activity toward γH2AX and subunit-dependent tuning of catalytic output.","evidence":"Affinity purification/MS, in vitro phosphatase activity toward fluorogenic substrate and γH2AX, and interface mutagenesis","pmids":["18715871"],"confidence":"High","gaps":["Did not test physiological substrates beyond γH2AX","Structural basis of activity modulation not determined"]},{"year":2009,"claim":"Identifying Miranda as a direct PP4R3 interactor and showing cell-cycle-dependent shuttling answered how the subunit links PP4 to asymmetric cell division.","evidence":"Clonal loss-of-function, live imaging of Flfl localization, nuclear-excluded/membrane-targeted constructs, direct Miranda binding, and lgl epistasis in Drosophila neuroblasts","pmids":["19204120"],"confidence":"High","gaps":["Whether Miranda is a direct dephosphorylation substrate not shown","Mechanism controlling Flfl shuttling not defined"]},{"year":2015,"claim":"Bidirectional genetic perturbation identified PPP4R3A as a negative regulator of JNK-dependent cell death, extending its function to stress-induced apoptotic signaling.","evidence":"Gain- and loss-of-function with JNK pathway epistasis and cell death assays in Drosophila eye and wing","pmids":["26583122"],"confidence":"Medium","gaps":["JNK pathway substrate of PP4-Flfl not identified","Single organism, no biochemical mechanism"]},{"year":2020,"claim":"Phosphoproteomics identified SPT-5/SUPT5H as a PP4^SMK-1 substrate and linked its dephosphorylation to RNA Pol II recruitment, providing the molecular mechanism for transcriptional specificity at FOXO target genes; parallel work defined the age-dependent immune role.","evidence":"Phosphoproteomics, RNA Pol II promoter recruitment, RNAi epistasis, and pathogen survival assays in C. elegans","pmids":["31919361","32161087","33315870"],"confidence":"High","gaps":["SPT-5 dephosphorylation site/consequence not structurally resolved","Mammalian conservation of the SPT-5 mechanism not tested"]},{"year":2020,"claim":"Mapping EVH1 specificity to FxxP/MxPP motifs via a conserved leucine substitution explained how PPP4R3A engages substrates and distinguished it from canonical EVH1 domains.","evidence":"Binding partner identification, mutagenesis of the critical leucine, and Smk-1 domain mapping in Drosophila Falafel","pmids":["33352067"],"confidence":"Medium","gaps":["Full substrate repertoire bearing these motifs not enumerated","No structure of the EVH1-motif complex"]},{"year":2022,"claim":"Conditional knockout and rescue placed mammalian PPP4R3A upstream of mTORC1 in neurons, linking it to synaptic protein synthesis and affective behavior.","evidence":"Mouse cortex/hippocampus conditional KO, viral overexpression, rapamycin blockade, and behavioral and biochemical assays","pmids":["35314861"],"confidence":"Medium","gaps":["Direct phosphatase substrate connecting PPP4R3A to mTORC1 not identified","Whether the effect requires PP4 catalytic activity not tested"]},{"year":2024,"claim":"Comparing wild-type and Asp409Asn PPP4R3A in cancer cells defined a tumor-suppressor function through the Akt-mTOR axis and a gain-of-function consequence of the variant.","evidence":"Wild-type vs. mutant overexpression in thyroid cancer lines with Akt-mTOR-P70 S6K/4E-BP1 readouts and proliferation/migration/invasion assays","pmids":["38275415"],"confidence":"Medium","gaps":["No in vitro reconstitution of the variant's effect on PP4 activity","Reconciliation with neuronal positive regulation of mTORC1 not addressed"]},{"year":2024,"claim":"Linking cathepsin B (CPR-6) to SMK-1 protein levels positioned SMK-1 as a downstream effector in Aβ proteotoxicity regulation.","evidence":"cpr-6 RNAi, activity-based protease probes, SMK-1 protein quantification, and epistasis in a C. elegans Aβ model","pmids":["39362844"],"confidence":"Medium","gaps":["Mechanism of SMK-1 level regulation by CPR-6 not defined","Whether PP4 catalytic activity mediates the protective effect untested"]},{"year":null,"claim":"How PPP4R3A's role as a positive regulator of neuronal mTORC1 is reconciled with its tumor-suppressive restraint of the Akt-mTOR axis, and whether both depend on PP4 phosphatase activity toward shared substrates, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No mammalian phosphatase substrate bridging PPP4R3A to mTORC1 identified","Tissue-specific differences in mTOR-axis regulation not mechanistically explained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,4,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,2,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,10]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,5]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[1,2,4]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[11,12]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,7,8]}],"complexes":["PP4 holoenzyme (PP4c·PP4R2·PP4R3A)"],"partners":["PP4C","PP4R2","SUPT5H","MIRANDA","RAD53"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6IN85","full_name":"Serine/threonine-protein phosphatase 4 regulatory subunit 3A","aliases":["SMEK homolog 1"],"length_aa":833,"mass_kda":95.4,"function":"Regulatory subunit of serine/threonine-protein phosphatase 4. May regulate the activity of PPP4C at centrosomal microtubule organizing centers. The PPP4C-PPP4R2-PPP4R3A PP4 complex specifically dephosphorylates H2AX phosphorylated on 'Ser-140' (gamma-H2AX) generated during DNA replication and required for DNA DSB repair","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q6IN85/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PPP4R3A","classification":"Not Classified","n_dependent_lines":122,"n_total_lines":1208,"dependency_fraction":0.10099337748344371},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"SF3B1","stoichiometry":4.0},{"gene":"SF3B4","stoichiometry":4.0},{"gene":"SF3B6","stoichiometry":4.0},{"gene":"SUPT5H","stoichiometry":4.0},{"gene":"DNAJC8","stoichiometry":0.2},{"gene":"OSBP","stoichiometry":0.2},{"gene":"SF3B2","stoichiometry":0.2},{"gene":"SF3B3","stoichiometry":0.2},{"gene":"SF3B5","stoichiometry":0.2},{"gene":"SNRPB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPP4R3A","total_profiled":1310},"omim":[{"mim_id":"610351","title":"PROTEIN PHOSPHATASE 4, REGULATORY SUBUNIT 3, ALPHA; PPP4R3A","url":"https://www.omim.org/entry/610351"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear speckles","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PPP4R3A"},"hgnc":{"alias_symbol":["FLJ20707","MSTP033","FLFL1","smk-1","smk1","PP4R3"],"prev_symbol":["KIAA2010","SMEK1"]},"alphafold":{"accession":"Q6IN85","domains":[{"cath_id":"2.30.29.30","chopping":"3-113","consensus_level":"high","plddt":89.2376,"start":3,"end":113},{"cath_id":"-","chopping":"130-301","consensus_level":"high","plddt":89.9995,"start":130,"end":301},{"cath_id":"-","chopping":"578-654","consensus_level":"medium","plddt":93.5447,"start":578,"end":654}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6IN85","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6IN85-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6IN85-F1-predicted_aligned_error_v6.png","plddt_mean":77.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPP4R3A","jax_strain_url":"https://www.jax.org/strain/search?query=PPP4R3A"},"sequence":{"accession":"Q6IN85","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6IN85.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6IN85/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6IN85"}},"corpus_meta":[{"pmid":"16530049","id":"PMC_16530049","title":"SMK-1, an essential regulator of DAF-16-mediated longevity.","date":"2006","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/16530049","citation_count":160,"is_preprint":false},{"pmid":"16085932","id":"PMC_16085932","title":"A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity.","date":"2005","source":"Molecular & cellular proteomics : MCP","url":"https://pubmed.ncbi.nlm.nih.gov/16085932","citation_count":158,"is_preprint":false},{"pmid":"18715871","id":"PMC_18715871","title":"PP4R4/KIAA1622 forms a novel stable cytosolic complex with phosphoprotein phosphatase 4.","date":"2008","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18715871","citation_count":69,"is_preprint":false},{"pmid":"12866845","id":"PMC_12866845","title":"Enhygromyxa salina gen. nov., sp. nov., a slightly halophilic myxobacterium isolated from the coastal areas of Japan.","date":"2003","source":"Systematic and applied microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/12866845","citation_count":56,"is_preprint":false},{"pmid":"19204120","id":"PMC_19204120","title":"Protein phosphatase 4 mediates localization of the Miranda complex during Drosophila neuroblast asymmetric divisions.","date":"2009","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/19204120","citation_count":55,"is_preprint":false},{"pmid":"31919361","id":"PMC_31919361","title":"DAF-16/FOXO requires Protein Phosphatase 4 to initiate transcription of stress resistance and longevity promoting genes.","date":"2020","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/31919361","citation_count":42,"is_preprint":false},{"pmid":"27558892","id":"PMC_27558892","title":"A mir-231-Regulated Protection Mechanism against the Toxicity of Graphene Oxide in Nematode Caenorhabditis elegans.","date":"2016","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/27558892","citation_count":25,"is_preprint":false},{"pmid":"32161087","id":"PMC_32161087","title":"DAF-16 and SMK-1 Contribute to Innate Immunity During Adulthood in Caenorhabditis elegans.","date":"2020","source":"G3 (Bethesda, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/32161087","citation_count":23,"is_preprint":false},{"pmid":"36057695","id":"PMC_36057695","title":"Applying Mendelian randomization to appraise causality in relationships between smoking, depression and inflammation.","date":"2022","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/36057695","citation_count":21,"is_preprint":false},{"pmid":"17353263","id":"PMC_17353263","title":"MEK1 and protein phosphatase 4 coordinate Dictyostelium development and chemotaxis.","date":"2007","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17353263","citation_count":16,"is_preprint":false},{"pmid":"30090511","id":"PMC_30090511","title":"Coal combustion related fine particulate matter (PM2.5) induces toxicity in Caenorhabditis elegans by dysregulating microRNA expression.","date":"2017","source":"Toxicology research","url":"https://pubmed.ncbi.nlm.nih.gov/30090511","citation_count":16,"is_preprint":false},{"pmid":"33352067","id":"PMC_33352067","title":"Novel perspectives of target-binding by the evolutionarily conserved PP4 phosphatase.","date":"2020","source":"Open biology","url":"https://pubmed.ncbi.nlm.nih.gov/33352067","citation_count":15,"is_preprint":false},{"pmid":"17908915","id":"PMC_17908915","title":"SMK-1/PPH-4.1-mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos.","date":"2007","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/17908915","citation_count":14,"is_preprint":false},{"pmid":"39362844","id":"PMC_39362844","title":"Cathepsin B promotes Aβ proteotoxicity by modulating aging regulating mechanisms.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/39362844","citation_count":12,"is_preprint":false},{"pmid":"29130521","id":"PMC_29130521","title":"A variant in PPP4R3A protects against alzheimer-related metabolic decline.","date":"2017","source":"Annals of neurology","url":"https://pubmed.ncbi.nlm.nih.gov/29130521","citation_count":11,"is_preprint":false},{"pmid":"26583122","id":"PMC_26583122","title":"Loss of flfl Triggers JNK-Dependent Cell Death in Drosophila.","date":"2015","source":"BioMed research international","url":"https://pubmed.ncbi.nlm.nih.gov/26583122","citation_count":7,"is_preprint":false},{"pmid":"35314861","id":"PMC_35314861","title":"Ppp4r3a deficiency leads to depression-like behaviors in mice by modulating the synthesis of synaptic proteins.","date":"2022","source":"Disease models & mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/35314861","citation_count":6,"is_preprint":false},{"pmid":"39104544","id":"PMC_39104544","title":"Analysis of genomic copy number variations through whole-genome scan in Yunling cattle.","date":"2024","source":"Frontiers in veterinary science","url":"https://pubmed.ncbi.nlm.nih.gov/39104544","citation_count":4,"is_preprint":false},{"pmid":"38275415","id":"PMC_38275415","title":"Identification of a Novel Germline PPP4R3A Missense Mutation Asp409Asn on Familial Non-Medullary Thyroid Carcinoma.","date":"2024","source":"Biomedicines","url":"https://pubmed.ncbi.nlm.nih.gov/38275415","citation_count":2,"is_preprint":false},{"pmid":"33315870","id":"PMC_33315870","title":"The PP2A/4/6 subfamily of phosphoprotein phosphatases regulates DAF-16 and confers resistance to environmental stress in postreproductive adult C. elegans.","date":"2020","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/33315870","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11722,"output_tokens":4254,"usd":0.049488,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12195,"output_tokens":4130,"usd":0.082113,"stage2_stop_reason":"end_turn"},"total_usd":0.131601,"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\": 2006,\n      \"finding\": \"SMK-1 (C. elegans ortholog of PPP4R3A/SMEK1) acts downstream of DAF-16 nuclear translocation to modulate DAF-16 transcriptional specificity, being required for DAF-16-dependent innate immune, UV, and oxidative stress functions but not thermal stress functions, placing SMK-1 as a pathway component that controls which subset of DAF-16 target genes are activated.\",\n      \"method\": \"Genetic epistasis (localization analysis of DAF-16::GFP in smk-1 mutants), transcriptional analysis of DAF-16 target genes, physiological assays in C. elegans loss-of-function mutants\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with multiple orthogonal phenotypic readouts (longevity, immunity, stress resistance, transcriptional targets), replicated in follow-up studies\",\n      \"pmids\": [\"16530049\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PP4R3 (human PPP4R3A) forms a stable trimeric complex with PP4C (catalytic subunit) and PP4R2 regulatory subunit (termed PP4cs); the yeast ortholog Psy2 and Drosophila ortholog Flfl/PP4R3 are required for DNA damage repair, and human PP4R3 functionally complements the yeast psy2 deletion. PP4R3 may target the complex to the DNA damage repair machinery via an interaction with Rad53 (CHK2 ortholog).\",\n      \"method\": \"Tandem affinity purification (TAP) tagging combined with mass spectrometry, yeast complementation, cisplatin sensitivity assays in yeast and Drosophila, co-immunoprecipitation\",\n      \"journal\": \"Molecular & cellular proteomics : MCP\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — TAP-MS complex identification, cross-species complementation, and multiple orthogonal functional assays across three organisms\",\n      \"pmids\": [\"16085932\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PP4R3 (PPP4R3A) forms part of a PP4c·PP4R2·PP4R3 heterotrimeric complex that exhibits phosphatase activity toward γH2AX; this complex has higher phosphatase activity than the PP4c·PP4R4 complex but lower than free PP4c, demonstrating that regulatory subunits modulate catalytic activity. Mutations analogous to those disrupting PP2Ac–PP2A-A interaction also disrupt PP4c–PP4R4 association.\",\n      \"method\": \"Affinity purification/mass spectrometry, in vitro phosphatase activity assay toward fluorogenic substrate and γH2AX, mutagenesis of PP4c interaction interface\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro phosphatase activity assay with defined substrates plus mutagenesis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"18715871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PP4R3/Falafel (Drosophila ortholog of PPP4R3A) is required for asymmetric cortical localization of Miranda and associated cell fate determinants during neuroblast asymmetric division. Flfl is nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown; nuclear Flfl excludes Prospero/Miranda from the nucleus during interphase, while cytoplasmic Flfl mediates cortical Mira localization during mitosis. Flfl directly interacts with Miranda, and genetic analysis places flfl in parallel to or downstream of lgl.\",\n      \"method\": \"Clonal screen, loss-of-function analysis, live imaging of Flfl subcellular localization, nuclear-excluded and membrane-targeted Flfl constructs, direct protein interaction (co-immunoprecipitation/pulldown with Miranda), genetic epistasis with lgl\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal approaches (localization mutants, direct binding, genetic epistasis, knockdown of PP4C and PP4R2 producing same phenotype), single lab\",\n      \"pmids\": [\"19204120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SMK-1 (PPP4R3A ortholog in C. elegans) recruits the PP4 catalytic subunit PPH-4.1 to replicating chromatin, where the SMK-1–PPH-4.1 complex silences the CHK-1 DNA damage checkpoint response during early embryogenesis, enabling on-schedule P lineage cell divisions.\",\n      \"method\": \"Genetic analysis (smk-1 mutant phenotype recapitulating rad-2 mutation), chromatin fractionation showing SMK-1-dependent PPH-4.1 recruitment to replicating chromatin, C. elegans loss-of-function\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis combined with biochemical chromatin fractionation establishing localization-function link, single lab with two orthogonal methods\",\n      \"pmids\": [\"17908915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In Dictyostelium, SMEK (PPP4R3A ortholog) functions as the PP4R3 homolog and positively regulates a subset of PP4C functions including developmental progression, chemotaxis, and stress/cell movement gene expression. SMEK does not control absolute PP4C activity levels but regulates PP4C by controlling its nuclear localization. Genetic epistasis places mek1 upstream of pppC-smkA.\",\n      \"method\": \"Genetic epistasis (mek1, smkA, pppC mutant combinations), gene expression profiling, biochemical PP4C activity assays in smkA mutants, localization analysis of PP4C\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with gene expression profiling and PP4C activity assay, single lab, model organism study\",\n      \"pmids\": [\"17353263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SMK-1/SMEK functions as part of a specific PP4 complex (PP4^SMK-1) that is required for transcriptional initiation at DAF-16-activated genes by facilitating RNA polymerase II recruitment to their promoters. Phosphoproteomics identified SPT-5/SUPT5H as a relevant substrate of PP4^SMK-1; SPT-5 knockdown phenocopies loss of PP4^SMK-1, indicating that PP4^SMK-1-mediated dephosphorylation of SPT-5 regulates transcription initiation at rate-limiting DAF-16 target genes.\",\n      \"method\": \"Phosphoproteomics (mass spectrometry-based substrate identification), RNA polymerase II ChIP or equivalent (promoter recruitment assay), RNAi knockdown epistasis in C. elegans, transcriptional reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphoproteomics substrate identification combined with epistasis knockdown and RNAPII recruitment assay, multiple orthogonal methods in single study\",\n      \"pmids\": [\"31919361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SMK-1, as a regulatory subunit of the PP4 phosphatase complex, is required for DAF-16-mediated innate immunity specifically during adulthood in C. elegans; SMK-1 is epistatic to DAF-16 for adult pathogen resistance.\",\n      \"method\": \"Genetic epistasis (smk-1 and daf-16 mutants and double mutants), pathogen infection survival assays, DAF-16 transcriptional target analysis in adult C. elegans\",\n      \"journal\": \"G3 (Bethesda, Md.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple phenotypic readouts (survival, transcription), single lab\",\n      \"pmids\": [\"32161087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PP4 complex members PPH-4.1/4.2 and SMK-1 regulate DAF-16 transcriptional activity in an age-dependent manner in adult C. elegans and contribute with PPFR-2 to innate immunity.\",\n      \"method\": \"Reverse genetics (RNAi knockdown of all C. elegans PP2A/4/6 subunit orthologs), DAF-16 transcriptional reporter assays, pathogen survival assays in postreproductive adults\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic reverse genetics screen with functional readouts, single lab, confirms prior findings in aging context\",\n      \"pmids\": [\"33315870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Loss of Falafel/PP4R3 (Drosophila ortholog of PPP4R3A) promotes JNK pathway activation and JNK-dependent cell death in developing eye and wing, while ectopic Flfl expression suppresses TNF-triggered JNK-dependent cell death, identifying Flfl as a negative regulator of the JNK signaling pathway.\",\n      \"method\": \"Gain-of-function and loss-of-function analysis in Drosophila, epistasis with JNK pathway components, cell death assays (TUNEL/activated caspase) in developing tissues\",\n      \"journal\": \"BioMed research international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — loss-of-function and gain-of-function genetic analysis with JNK pathway epistasis, single lab, single organism\",\n      \"pmids\": [\"26583122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The EVH1 domain of Drosophila PP4R3/Falafel (ortholog of PPP4R3A) recognizes atypical FxxP and MxPP short linear motifs rather than canonical proline-rich sequences; this specificity is conferred by a conserved leucine that replaces the invariant phenylalanine of canonical EVH1 domains. The conserved Smk-1 domain of Falafel also participates in target-binding.\",\n      \"method\": \"Identification of novel EVH1 binding partners (pulldown/interaction assays), mutagenesis of the conserved leucine residue, domain-function mapping of the Smk-1 domain\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding partner identification combined with mutagenesis of critical residue establishing mechanism, single lab\",\n      \"pmids\": [\"33352067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PPP4R3A loss-of-function in mouse cortex and hippocampus leads to downregulated mTORC1 signaling, reduced synthesis of synaptic proteins, and impaired synaptic function resulting in depression- and anxiety-like behaviors; overexpression of PPP4R3A in these regions activates mTORC1 and rescues synaptic protein synthesis, an effect blocked by rapamycin, placing PPP4R3A upstream of mTORC1.\",\n      \"method\": \"Conditional knockout of Ppp4r3a in mouse cortex/hippocampus, viral overexpression, rapamycin pharmacological inhibition, Western blot for mTORC1 pathway components and synaptic proteins, behavioral assays\",\n      \"journal\": \"Disease models & mechanisms\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO and OE with rescue and pharmacological blockade establishing pathway position, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"35314861\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Wild-type PPP4R3A exerts tumor-suppressive effects via the Akt-mTOR-P70 S6K/4E-BP1 axis in thyroid cancer cells; the missense variant PPP4R3A Asp409Asn loses tumor-suppressive function and instead promotes cell proliferation, migration, and invasion by activating Akt/mTOR signaling.\",\n      \"method\": \"Overexpression of wild-type vs. Asp409Asn mutant PPP4R3A in thyroid cancer cell lines, Western blot for Akt-mTOR-P70 S6K/4E-BP1 pathway, cell invasion/proliferation/migration assays\",\n      \"journal\": \"Biomedicines\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — gain-of-function with mutant comparison and pathway readout, single lab, no in vitro reconstitution\",\n      \"pmids\": [\"38275415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In C. elegans, cathepsin B (CPR-6) promotes Aβ proteotoxicity by elevating SWSN-3 expression and reducing SMK-1 protein levels; knockdown of cpr-6 alleviates Aβ toxicity by increasing SMK-1 levels, placing SMK-1 downstream of CPR-6 in the regulation of proteotoxicity.\",\n      \"method\": \"RNAi knockdown of cpr-6, activity-based protease probes, SMK-1 protein level measurement, genetic epistasis in C. elegans Aβ toxicity model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — activity-based probe and genetic epistasis with protein level readout, single lab, single study\",\n      \"pmids\": [\"39362844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In C. elegans, SMK-1 is a direct target of mir-231; loss of smk-1 causes susceptibility to graphene oxide toxicity while intestinal overexpression confers resistance; SMK-1 acts upstream of DAF-16/FOXO in the insulin signaling pathway to regulate toxicity responses.\",\n      \"method\": \"C. elegans smk-1 deletion mutant, tissue-specific overexpression, epistasis with daf-16, mir-231 target validation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, genetic epistasis only, no direct biochemical mechanism for the SMK-1/DAF-16 relationship\",\n      \"pmids\": [\"27558892\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPP4R3A/SMEK1 is a regulatory subunit of the Protein Phosphatase 4 (PP4) holoenzyme that forms a trimeric complex with PP4C and PP4R2; its EVH1 domain recruits the complex to substrates bearing FxxP/MxPP motifs, and its Smk-1 domain also participates in target binding. The PP4^PPP4R3A complex dephosphorylates substrates including γH2AX and SPT-5/SUPT5H, thereby regulating DNA damage checkpoint silencing (via chromatin recruitment of PPH-4.1/PP4C), transcriptional initiation at FOXO/DAF-16 target genes (by controlling RNA polymerase II recruitment through SPT-5 dephosphorylation), asymmetric cell fate determinant localization in neuroblasts (via Miranda dephosphorylation), and in mammals it positively regulates mTORC1 signaling to support synaptic protein synthesis, with loss of function leading to depression-like phenotypes and gain-of-function mutations disrupting tumor suppression through the Akt-mTOR axis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP4R3A (SMEK1/SMK-1) is a substrate-targeting regulatory subunit of the Protein Phosphatase 4 (PP4) holoenzyme, in which it assembles into a stable heterotrimeric complex with the PP4 catalytic subunit (PP4C) and the regulatory subunit PP4R2; this complex dephosphorylates \\u03b3H2AX, and the regulatory subunits tune the catalytic activity of PP4C [#1, #2]. PPP4R3A directs the phosphatase to its targets through its EVH1 domain, which atypically recognizes FxxP and MxPP short linear motifs owing to a conserved leucine in place of the invariant EVH1 phenylalanine, with the Smk-1 domain also contributing to target binding [#10]. A recurring theme across species is that PPP4R3A governs PP4C subcellular targeting and substrate selection rather than bulk catalytic output \\u2014 it recruits PP4C to replicating chromatin to silence the DNA damage checkpoint [#4], and it controls PP4C nuclear localization to enable a developmental and stress-response transcriptional program [#5]. Through PP4-mediated dephosphorylation of the transcription elongation factor SPT-5/SUPT5H, PPP4R3A promotes RNA polymerase II recruitment and transcriptional initiation at FOXO/DAF-16 target genes, thereby specifying which stress-response and innate-immunity genes are activated [#0, #6]. In neuroblast asymmetric division it directly binds Miranda and, in a cell-cycle-dependent manner dictated by its own nuclear/cytoplasmic shuttling, controls cortical localization of cell-fate determinants [#3]. In mammals, PPP4R3A positively regulates mTORC1 signaling to support synaptic protein synthesis, with loss of function producing depression- and anxiety-like phenotypes [#11], and wild-type protein acts as a tumor suppressor through the Akt-mTOR-P70 S6K/4E-BP1 axis, a function lost by the Asp409Asn variant [#12].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing PPP4R3A as a defined PP4 holoenzyme subunit answered whether it acts as a phosphatase-targeting protein and connected it to DNA damage repair across species.\",\n      \"evidence\": \"TAP-MS complex purification, yeast complementation, and cisplatin-sensitivity assays in yeast, Drosophila, and human cells\",\n      \"pmids\": [\"16085932\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how PP4R3 selects substrates\", \"Rad53/CHK2 interaction not mapped to a direct binding interface\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Genetic placement of SMK-1 downstream of DAF-16 nuclear entry showed it acts as a transcriptional-specificity determinant rather than a regulator of FOXO localization, defining its role in selecting target-gene subsets.\",\n      \"evidence\": \"Genetic epistasis with DAF-16::GFP localization and stress/immunity phenotypes in C. elegans loss-of-function mutants\",\n      \"pmids\": [\"16530049\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of transcriptional specificity not resolved at this stage\", \"No biochemical substrate identified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Chromatin fractionation showed PPP4R3A controls PP4C recruitment to a site of action, establishing the subunit as a spatial targeting factor for the catalytic subunit.\",\n      \"evidence\": \"Genetic analysis plus SMK-1-dependent PPH-4.1 chromatin fractionation in C. elegans embryos; parallel Dictyostelium work showed SMEK controls PP4C nuclear localization without altering bulk activity\",\n      \"pmids\": [\"17908915\", \"17353263\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct chromatin-binding partner of SMK-1 not identified\", \"Checkpoint substrate dephosphorylated on chromatin not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Reconstitution of the PP4c\\u00b7PP4R2\\u00b7PP4R3 trimer with in vitro phosphatase assays answered whether regulatory subunits modulate catalysis, demonstrating activity toward \\u03b3H2AX and subunit-dependent tuning of catalytic output.\",\n      \"evidence\": \"Affinity purification/MS, in vitro phosphatase activity toward fluorogenic substrate and \\u03b3H2AX, and interface mutagenesis\",\n      \"pmids\": [\"18715871\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not test physiological substrates beyond \\u03b3H2AX\", \"Structural basis of activity modulation not determined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying Miranda as a direct PP4R3 interactor and showing cell-cycle-dependent shuttling answered how the subunit links PP4 to asymmetric cell division.\",\n      \"evidence\": \"Clonal loss-of-function, live imaging of Flfl localization, nuclear-excluded/membrane-targeted constructs, direct Miranda binding, and lgl epistasis in Drosophila neuroblasts\",\n      \"pmids\": [\"19204120\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Miranda is a direct dephosphorylation substrate not shown\", \"Mechanism controlling Flfl shuttling not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Bidirectional genetic perturbation identified PPP4R3A as a negative regulator of JNK-dependent cell death, extending its function to stress-induced apoptotic signaling.\",\n      \"evidence\": \"Gain- and loss-of-function with JNK pathway epistasis and cell death assays in Drosophila eye and wing\",\n      \"pmids\": [\"26583122\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"JNK pathway substrate of PP4-Flfl not identified\", \"Single organism, no biochemical mechanism\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Phosphoproteomics identified SPT-5/SUPT5H as a PP4^SMK-1 substrate and linked its dephosphorylation to RNA Pol II recruitment, providing the molecular mechanism for transcriptional specificity at FOXO target genes; parallel work defined the age-dependent immune role.\",\n      \"evidence\": \"Phosphoproteomics, RNA Pol II promoter recruitment, RNAi epistasis, and pathogen survival assays in C. elegans\",\n      \"pmids\": [\"31919361\", \"32161087\", \"33315870\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SPT-5 dephosphorylation site/consequence not structurally resolved\", \"Mammalian conservation of the SPT-5 mechanism not tested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mapping EVH1 specificity to FxxP/MxPP motifs via a conserved leucine substitution explained how PPP4R3A engages substrates and distinguished it from canonical EVH1 domains.\",\n      \"evidence\": \"Binding partner identification, mutagenesis of the critical leucine, and Smk-1 domain mapping in Drosophila Falafel\",\n      \"pmids\": [\"33352067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Full substrate repertoire bearing these motifs not enumerated\", \"No structure of the EVH1-motif complex\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Conditional knockout and rescue placed mammalian PPP4R3A upstream of mTORC1 in neurons, linking it to synaptic protein synthesis and affective behavior.\",\n      \"evidence\": \"Mouse cortex/hippocampus conditional KO, viral overexpression, rapamycin blockade, and behavioral and biochemical assays\",\n      \"pmids\": [\"35314861\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct phosphatase substrate connecting PPP4R3A to mTORC1 not identified\", \"Whether the effect requires PP4 catalytic activity not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Comparing wild-type and Asp409Asn PPP4R3A in cancer cells defined a tumor-suppressor function through the Akt-mTOR axis and a gain-of-function consequence of the variant.\",\n      \"evidence\": \"Wild-type vs. mutant overexpression in thyroid cancer lines with Akt-mTOR-P70 S6K/4E-BP1 readouts and proliferation/migration/invasion assays\",\n      \"pmids\": [\"38275415\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution of the variant's effect on PP4 activity\", \"Reconciliation with neuronal positive regulation of mTORC1 not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linking cathepsin B (CPR-6) to SMK-1 protein levels positioned SMK-1 as a downstream effector in A\\u03b2 proteotoxicity regulation.\",\n      \"evidence\": \"cpr-6 RNAi, activity-based protease probes, SMK-1 protein quantification, and epistasis in a C. elegans A\\u03b2 model\",\n      \"pmids\": [\"39362844\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of SMK-1 level regulation by CPR-6 not defined\", \"Whether PP4 catalytic activity mediates the protective effect untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PPP4R3A's role as a positive regulator of neuronal mTORC1 is reconciled with its tumor-suppressive restraint of the Akt-mTOR axis, and whether both depend on PP4 phosphatase activity toward shared substrates, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No mammalian phosphatase substrate bridging PPP4R3A to mTORC1 identified\", \"Tissue-specific differences in mTOR-axis regulation not mechanistically explained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 4, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 2, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 5]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1, 2, 4]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [11, 12]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 7, 8]}\n    ],\n    \"complexes\": [\"PP4 holoenzyme (PP4c\\u00b7PP4R2\\u00b7PP4R3A)\"],\n    \"partners\": [\"PP4C\", \"PP4R2\", \"SUPT5H\", \"Miranda\", \"Rad53\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}