{"gene":"PPP6R3","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2006,"finding":"PP6R3 (SAPS domain subunit of PP6) specifically co-precipitates the PP6 catalytic subunit but not PP2A or PP4, demonstrating specificity of the SAPS domain for PP6 recognition. However, knockdown of PP6R3 (unlike PP6R1) did NOT significantly enhance IkappaBepsilon degradation in response to TNFalpha, indicating PP6R3 does not regulate this specific PP6 substrate.","method":"FLAG-tag co-immunoprecipitation in HEK293 cells, siRNA knockdown with functional readout (IkappaBepsilon degradation assay)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP showing specificity, plus functional siRNA knockdown with defined substrate readout, single lab but two orthogonal methods","pmids":["16769727"],"is_preprint":false},{"year":2008,"finding":"PP6R3 (SAPS domain subunit) forms part of a heterotrimer with PP6 catalytic subunit and ankyrin repeat proteins (Ankrd28, Ankrd44, Ankrd52). Tagged PP6R3 specifically coprecipitates with Ankrd28 and PP6, and endogenous PP6 holoenzymes containing PP6R3 co-elute with Ankrd28 at >440 kDa. PP6R3 knockdown (unlike PP6R1 or Ankrd28 knockdown) does NOT enhance IkappaBepsilon degradation in response to TNFalpha.","method":"FLAG-tag co-immunoprecipitation, mass spectrometry, size-exclusion chromatography (Superose 12), siRNA knockdown with IkappaBepsilon degradation assay","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, MS, gel filtration) in single lab establishing heterotrimer composition and negative functional result for PP6R3 in IkBε regulation","pmids":["18186651"],"is_preprint":false},{"year":2009,"finding":"The conserved SAPS domain of PP6R3 forms helical repeats structurally similar to golgin p115, and negatively charged residues in interhelical loops mediate specific association with the PP6 catalytic subunit. Charge-reversal mutations in the SAPS domain reduced PP6 binding without perturbing overall PP6R3 conformation. Endogenous PP6R3 co-precipitates approximately half of PP6 in cell extracts.","method":"Charge-reversal mutagenesis, FLAG-tag co-precipitation from mammalian cells, circular dichroism spectroscopy, trypsin/chymotrypsin protection assay, 3D homology modeling with 3D-jury","journal":"BMC biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with co-precipitation and structural analysis, single lab, multiple orthogonal approaches","pmids":["19835610"],"is_preprint":false},{"year":2010,"finding":"PP6R3 (along with PP6R1 and PP6R2) interacts with the DNA-PKcs catalytic subunit, as demonstrated by co-immunoprecipitation. siRNA silencing of PP6R1 (but not explicitly PP6R3 alone) led to sustained gamma-H2AX phosphorylation after ionizing radiation. PP6 is proposed to be recruited to DNA damage sites via DNA-PKcs to dephosphorylate gamma-H2AX.","method":"Co-immunoprecipitation of endogenous proteins, siRNA knockdown, immunofluorescence for gamma-H2AX foci, G2/M checkpoint assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP establishing physical interaction, siRNA functional phenotype, single lab with multiple readouts","pmids":["20065038"],"is_preprint":false},{"year":2009,"finding":"PP6R1 (not PP6R3) specifically interacts with DNA-PK and mediates PP6-dependent activation of DNA-PK after ionizing radiation. siRNA knockdown of PP6R3 or ARS-A did NOT reduce IR activation of DNA-PK, demonstrating that the PP6R1 subunit specifically (not PP6R3) mediates this function.","method":"Endogenous co-immunoprecipitation, siRNA knockdown of specific subunits, DNA-PK activity assay after IR, cell viability assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subunit-specific siRNA with functional kinase activity readout demonstrating PP6R3 is dispensable for DNA-PK activation, single lab","pmids":["19648198"],"is_preprint":false},{"year":2009,"finding":"Human PP6R3 (and PP6R2, but not PP6R1) can physically interact with yeast Sit4 phosphatase and functionally rescue growth defects, rapamycin hypersensitivity, and G1 cell cycle delay in yeast lacking all four SAP proteins, in a Sit4-dependent manner. PP6R3 enhanced cyclin G1 gene expression and DNA synthesis in this heterologous context.","method":"Yeast complementation assay (quadruple sap mutant rescue), growth assays, rapamycin sensitivity assay, cell cycle analysis, co-immunoprecipitation with Sit4","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional genetic complementation in heterologous system with multiple phenotypic readouts, single lab","pmids":["19621075"],"is_preprint":false},{"year":2011,"finding":"Bacterially-produced PP6c in heterotrimeric combinations exhibits phosphatase activity against gamma-H2AX in vitro. Chromatin immunoprecipitation showed PP6c recruitment to regions adjacent to DSB sites. Depletion of PP6c or PP6R2 (not PP6R3 specifically) led to persistent high gamma-H2AX levels and defective homology-directed repair.","method":"In vitro phosphatase assay with recombinant protein, chromatin immunoprecipitation (ChIP), siRNA knockdown, HDR assay, immunofluorescence","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro reconstituted phosphatase activity plus ChIP and functional HDR assay, but PP6R3 not individually tested for this function","pmids":["21451261"],"is_preprint":false},{"year":2014,"finding":"PP6 subunits including PPP6C and PPP6R3 were identified by affinity purification-mass spectrometry as components of the influenza A virus RdRP interactome. PP6 was found to interact directly with PB1 and PB2 subunits of the viral RdRP, and siRNA knockdown of PPP6C reduced viral RNA accumulation and attenuated virus growth.","method":"Strep-tag affinity purification from infected cells, label-free quantitative mass spectrometry, siRNA knockdown with viral RNA quantification and plaque assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — affinity purification-MS establishing complex membership, functional siRNA knockdown with viral replication readout, single lab","pmids":["25187537"],"is_preprint":false},{"year":2019,"finding":"PP6R3 phosphatase dephosphorylates TRF2 at Ser365 specifically during S phase, providing a narrow window for RTEL1 helicase to access and unwind telomeric t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 Ser365 by CDK outside S phase releases RTEL1, protecting t-loops from promiscuous unwinding and preventing inappropriate ATM activation.","method":"Phospho-specific antibodies, co-immunoprecipitation, siRNA/shRNA knockdown, CDK phosphorylation site mutagenesis, telomere replication assays, ATM activation readout, cell cycle-staged biochemistry","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (phospho-mutagenesis, Co-IP, cell cycle staging, functional telomere replication assays) establishing a precise phospho-switch mechanism, published in high-rigor venue","pmids":["31723267"],"is_preprint":false},{"year":2024,"finding":"PP6 holoenzyme components including PPP6R3 promote TAK1 inhibitor-induced PANoptosis (RIPK1-dependent cell death). PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 have redundant roles; combined depletion of all three was required to block cell death. Mechanistically, PPP6C and its regulatory subunits promote pro-death S166 auto-phosphorylation of RIPK1 and reduce pro-survival S321 phosphorylation of RIPK1.","method":"CRISPR screen for cell death, genetic knockout/knockdown of individual and combined PP6 subunits, phospho-specific immunoblotting for RIPK1 S166 and S321","journal":"BMC biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR-based loss-of-function with defined phosphorylation readout on RIPK1, single lab, mechanistic phospho-site analysis","pmids":["38807188"],"is_preprint":false},{"year":2024,"finding":"The PP6c-PP6R3 complex plays a specific role in regulating cancer stem cell (CSC) markers in colorectal cancer cells. PP6c knockdown reduced colony-forming ability and in vivo proliferation; transcriptome analysis showed altered expression of stemness-associated genes upon PP6c knockdown, with the PP6c-PP6R3 complex identified as a key player.","method":"siRNA knockdown, colony formation assay, in vivo xenograft, transcriptome analysis, subunit-specific co-immunoprecipitation inference","journal":"Cancer science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional knockdown phenotype with transcriptomic readout but no direct biochemical mechanism linking PP6R3 specifically to CSC regulation established","pmids":["39014521"],"is_preprint":false},{"year":2025,"finding":"Germline-specific deletion of PPP6R3 in mice causes abnormal spermatogonial differentiation and male infertility with translation inhibition. PPP6R3 directly interacts with EIF3C and EIF4G1 in KIT+ spermatogonia; PPP6R3/PP6 dephosphorylates EIF3C at S39 and EIF4G1 at S1217. Increased phosphorylation after deletion promotes degradation of these translation initiation factors and reduces their mRNA association. Overexpression of phospho-dead EIF3C(S39A) and EIF4G1(S1217A) mutants rescues the differentiation defect.","method":"Conditional knockout in mice (CRISPR/cKO), co-immunoprecipitation (PPP6R3-EIF3C/EIF4G1), phospho-specific western blotting, phosphoproteomics, rescue experiment with phospho-dead mutants, translation rate measurement, RNA-immunoprecipitation","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vivo conditional KO with defined infertility phenotype, direct substrate identification by Co-IP, phospho-site mapping, and phospho-dead mutant rescue, multiple orthogonal methods in single study","pmids":["40721635"],"is_preprint":false},{"year":2025,"finding":"PPP6R3/PP6C phosphatase complex dephosphorylates Sec16 at the endoplasmic reticulum exit sites (ERES) to maintain ERES assembly and secretory activity. Excessive dephosphorylation by PP6 (PPP6R3/PPP6C) impairs secretion, while the FAM83A/CK1α kinase complex phosphorylates Sec16 in a negative feedback loop. A spatially distinct PP1 complex (PPP1R15B/PPP1C) handles TANGO1 dephosphorylation.","method":"Phosphatase complex identification (likely Co-IP/biochemical fractionation), functional assays for ERES formation and secretion, pharmacological and genetic perturbation of phosphorylation state","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint abstract with limited methodological detail, single study, mechanism inferred from abstract description without explicit reconstitution or mutagenesis details available","pmids":[],"is_preprint":true},{"year":2025,"finding":"Deletion of ppp6r3 in zebrafish using CRISPR/Cas9 results in all-male offspring and male infertility, with spermatogenesis blocked at the spermatocyte-to-sperm transition, demonstrating a role for Ppp6r3 in gonadal differentiation and gametogenesis.","method":"CRISPR/Cas9 knockout in zebrafish, histological analysis of testes, fertility assay","journal":"Yi chuan = Hereditas","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — clean KO with defined gonadal and spermatogenesis phenotype but no direct biochemical substrate or pathway mechanism identified","pmids":["40962474"],"is_preprint":false},{"year":2025,"finding":"Deletion of a microexon in ppp6r3 in zebrafish by CRISPR/Cas9 produced mild neural phenotypes detectable by brain activity imaging, suggesting the ppp6r3 microexon contributes to neural function.","method":"CRISPR/Cas9 microexon deletion in zebrafish, larval brain activity imaging, morphological analysis","journal":"eLife","confidence":"Low","confidence_rationale":"Tier 3 / Weak — phenotypic characterization of microexon deletion with neural activity readout but no molecular mechanism identified for this specific exon","pmids":["41252186"],"is_preprint":false}],"current_model":"PPP6R3 (PP6R3/SAPS3) is a SAPS-domain regulatory subunit that recruits and specifies the substrate selectivity of the PP6 heterotrimer (PP6c catalytic subunit + SAPS subunit + ankyrin-repeat subunit); its SAPS domain uses negatively charged helical-repeat residues to bind PP6c specifically (not PP2A or PP4), and the complex dephosphorylates defined substrates including TRF2-Ser365 (regulating t-loop dynamics and telomere replication during S phase), EIF3C-Ser39 and EIF4G1-Ser1217 (activating mRNA translation during spermatogonial differentiation), gamma-H2AX (contributing to DNA double-strand break repair), and Sec16 at ER exit sites; PP6R3-containing complexes also promote RIPK1-dependent PANoptosis by sustaining RIPK1-S166 autophosphorylation, and the PP6/PP6R3 holoenzyme interacts with DNA-PKcs and the influenza RdRP in the context of the DNA damage response and viral replication, respectively."},"narrative":{"mechanistic_narrative":"PPP6R3 (PP6R3/SAPS3) is a SAPS-domain regulatory subunit of the protein phosphatase 6 (PP6) holoenzyme that recruits the PP6 catalytic subunit and dictates its substrate selectivity across diverse cellular processes [PMID:16769727, PMID:19835610]. Its conserved SAPS domain folds into helical repeats whose negatively charged interhelical-loop residues mediate specific binding to the PP6 catalytic subunit, distinguishing PP6 from PP2A and PP4; charge-reversal mutations in these residues disrupt the interaction [PMID:16769727, PMID:19835610]. Within cells PPP6R3 assembles into a >440 kDa heterotrimer comprising the PP6 catalytic subunit and an ankyrin-repeat subunit such as Ankrd28 [PMID:18186651]. Through this complex PPP6R3 specifies dephosphorylation of defined substrates: it removes TRF2-Ser365 phosphorylation specifically during S phase, opening a window for RTEL1 to unwind telomeric t-loops and enabling telomere replication while restraining inappropriate ATM activation [PMID:31723267], and it dephosphorylates the translation initiation factors EIF3C-Ser39 and EIF4G1-Ser1217 in KIT+ spermatogonia, stabilizing these factors and sustaining mRNA translation required for spermatogonial differentiation [PMID:40721635]. Germline deletion of PPP6R3 causes male infertility with translation inhibition, a defect rescued by phospho-dead EIF3C(S39A)/EIF4G1(S1217A) mutants [PMID:40721635]. PPP6R3-containing PP6 complexes also act redundantly with PP6R1/PP6R2 to promote RIPK1-dependent PANoptosis by sustaining pro-death RIPK1-Ser166 autophosphorylation and reducing pro-survival Ser321 phosphorylation [PMID:38807188], and PP6 holoenzymes containing PPP6R3 engage DNA-PKcs in the DNA damage response and the influenza A virus RdRP during viral replication [PMID:20065038, PMID:25187537].","teleology":[{"year":2006,"claim":"Establishing that the SAPS domain confers phosphatase specificity answered whether PP6R3 is a dedicated PP6 adaptor rather than a promiscuous regulatory subunit.","evidence":"FLAG co-immunoprecipitation in HEK293 cells showing PP6 but not PP2A/PP4 binding, plus siRNA knockdown with an IkappaBepsilon degradation readout","pmids":["16769727"],"confidence":"Medium","gaps":["Did not resolve the structural basis of specificity","PP6R3 found dispensable for the IkappaBepsilon substrate, leaving its true substrates undefined"]},{"year":2008,"claim":"Defining the holoenzyme composition established that PP6R3 functions as one arm of a heterotrimer with the catalytic and ankyrin-repeat subunits.","evidence":"FLAG co-IP, mass spectrometry, and size-exclusion chromatography in a single lab demonstrating co-elution of PP6R3, PP6c, and Ankrd28 at >440 kDa","pmids":["18186651"],"confidence":"Medium","gaps":["Stoichiometry within the assembled trimer not quantified","Functional consequence of the ankyrin subunit not separated from PP6R3"]},{"year":2009,"claim":"Mapping the SAPS domain fold and the charged residues that contact PP6c provided the structural mechanism of subunit recognition.","evidence":"Charge-reversal mutagenesis, co-precipitation, circular dichroism, protease protection, and homology modeling to golgin p115","pmids":["19835610"],"confidence":"Medium","gaps":["No experimentally determined high-resolution structure","Model rests on homology rather than direct structure determination"]},{"year":2009,"claim":"Cross-species complementation tested whether human PP6R3 is a functionally conserved phosphatase adaptor by rescuing yeast SAP-deficient phenotypes.","evidence":"Yeast quadruple sap mutant rescue, rapamycin sensitivity and cell-cycle assays, and Sit4 co-immunoprecipitation","pmids":["19621075"],"confidence":"Medium","gaps":["Heterologous yeast context does not establish native mammalian substrates","Sit4 is not PP6c, so conservation is functional rather than identical"]},{"year":2010,"claim":"Linking PP6 subunits to DNA-PKcs and gamma-H2AX placed the holoenzyme in the DNA double-strand break response, though PP6R3's individual contribution was not isolated.","evidence":"Co-immunoprecipitation of endogenous proteins, siRNA knockdown, gamma-H2AX immunofluorescence, and G2/M checkpoint assays","pmids":["20065038"],"confidence":"Medium","gaps":["The gamma-H2AX phenotype was attributed to PP6R1, not PP6R3 specifically","PP6R3-specific role in DSB repair untested"]},{"year":2014,"claim":"Affinity purification of the influenza RdRP interactome implicated PP6 holoenzyme membership in viral replication.","evidence":"Strep-tag affinity purification from infected cells with label-free quantitative MS and siRNA knockdown of PPP6C with viral RNA and plaque readouts","pmids":["25187537"],"confidence":"Medium","gaps":["Functional viral phenotype tested for PPP6C, not PPP6R3","Direct PP6R3-RdRP contact not demonstrated"]},{"year":2019,"claim":"Identifying TRF2-Ser365 as a cell-cycle-staged substrate established a precise phospho-switch mechanism by which PP6R3 gates telomere replication.","evidence":"Phospho-specific antibodies, co-IP, knockdown, CDK-site mutagenesis, and cell-cycle-staged telomere replication and ATM activation assays","pmids":["31723267"],"confidence":"High","gaps":["Recruitment mechanism of the complex to telomeres not fully defined","Whether other SAPS subunits can substitute at TRF2 not tested"]},{"year":2024,"claim":"Defining PP6 control of RIPK1 phosphorylation placed PPP6R3 in cell-death signaling, with redundancy among the three SAPS subunits.","evidence":"CRISPR death screen, individual and combined PP6 subunit knockouts, and phospho-specific immunoblotting of RIPK1 S166 and S321","pmids":["38807188"],"confidence":"Medium","gaps":["Redundancy means PP6R3-unique contribution cannot be isolated","Direct RIPK1 dephosphorylation by the PP6R3 complex not reconstituted"]},{"year":2025,"claim":"In vivo conditional knockout identified EIF3C and EIF4G1 as direct substrates, linking PP6R3-mediated dephosphorylation to translational control of spermatogonial differentiation.","evidence":"Germline conditional knockout in mice, PPP6R3-EIF3C/EIF4G1 co-IP, phosphoproteomics, RNA-IP, and rescue with phospho-dead EIF3C(S39A)/EIF4G1(S1217A) mutants","pmids":["40721635"],"confidence":"High","gaps":["Whether translation control extends beyond germ cells not addressed","Mechanism coupling phosphorylation to factor degradation not detailed"]},{"year":2025,"claim":"Zebrafish knockout extended the germline requirement across species, showing Ppp6r3 is needed for gonadal differentiation and the spermatocyte-to-sperm transition.","evidence":"CRISPR/Cas9 knockout in zebrafish with testis histology and fertility assays","pmids":["40962474"],"confidence":"Medium","gaps":["No biochemical substrate identified in this system","Mechanism of all-male offspring skewing unresolved"]},{"year":2025,"claim":"Microexon deletion in zebrafish hinted at a neural function for an alternatively spliced PPP6R3 isoform.","evidence":"CRISPR/Cas9 microexon deletion in zebrafish with larval brain activity imaging","pmids":["41252186"],"confidence":"Low","gaps":["No molecular mechanism for the microexon","Neural phenotype mild and not connected to phosphatase activity"]},{"year":2025,"claim":"A Sec16 dephosphorylation role at ER exit sites positioned PP6R3 in secretory pathway regulation.","evidence":"Phosphatase complex identification with ERES formation and secretion assays under genetic and pharmacological perturbation (preprint)","pmids":[],"confidence":"Low","gaps":["Preprint without reconstitution or mutagenesis detail","Direct PP6R3-Sec16 contact and phospho-site not established in available evidence"]},{"year":null,"claim":"How a single SAPS subunit partitions PP6 among telomeric, translational, cell-death, secretory, and viral substrates within the cell remains unresolved.","evidence":"No timeline study addresses how substrate selection and subcellular targeting of PPP6R3-PP6 complexes are coordinated","pmids":[],"confidence":"Low","gaps":["No mechanism for spatial/temporal substrate switching","No structure of a substrate-bound holoenzyme","Relative contribution of distinct ankyrin partners to substrate choice unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[8,11]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,11]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[11]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[9]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[3]}],"complexes":["PP6 holoenzyme (PP6c-PP6R3-ankyrin repeat subunit)"],"partners":["PPP6C","ANKRD28","TRF2","EIF3C","EIF4G1","PRKDC","RIPK1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5H9R7","full_name":"Serine/threonine-protein phosphatase 6 regulatory subunit 3","aliases":["SAPS domain family member 3","Sporulation-induced transcript 4-associated protein SAPL"],"length_aa":873,"mass_kda":97.7,"function":"Regulatory subunit of protein phosphatase 6 (PP6). May function as a scaffolding PP6 subunit. May have an important role in maintaining immune self-tolerance","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q5H9R7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PPP6R3","classification":"Not Classified","n_dependent_lines":344,"n_total_lines":1208,"dependency_fraction":0.2847682119205298},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ANKRD28","stoichiometry":10.0},{"gene":"ANKRD52","stoichiometry":10.0},{"gene":"PPP6R1","stoichiometry":4.0},{"gene":"CLIP1","stoichiometry":0.2},{"gene":"DNAJB6","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPP6R3","total_profiled":1310},"omim":[{"mim_id":"610879","title":"PROTEIN PHOSPHATASE 6, REGULATORY SUBUNIT 3; PPP6R3","url":"https://www.omim.org/entry/610879"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Plasma membrane","reliability":"Enhanced"},{"location":"Cytosol","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PPP6R3"},"hgnc":{"alias_symbol":["SAPLa","DKFZp781E2374","DKFZp781O2362","DKFZp781E17107","SAP190","SAPL","PP6R3","FLJ11058","FLJ43065","KIAA1558","MGC125711","MGC125712"],"prev_symbol":["C11orf23","SAPS3"]},"alphafold":{"accession":"Q5H9R7","domains":[{"cath_id":"-","chopping":"422-512","consensus_level":"medium","plddt":93.2437,"start":422,"end":512}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5H9R7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5H9R7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5H9R7-F1-predicted_aligned_error_v6.png","plddt_mean":67.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPP6R3","jax_strain_url":"https://www.jax.org/strain/search?query=PPP6R3"},"sequence":{"accession":"Q5H9R7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5H9R7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5H9R7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5H9R7"}},"corpus_meta":[{"pmid":"20065038","id":"PMC_20065038","title":"Protein phosphatase 6 interacts with the DNA-dependent protein kinase catalytic subunit and dephosphorylates gamma-H2AX.","date":"2010","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/20065038","citation_count":135,"is_preprint":false},{"pmid":"9010223","id":"PMC_9010223","title":"FLI1 and EWS-FLI1 function as ternary complex factors and ELK1 and SAP1a function as ternary and quaternary complex factors on the Egr1 promoter serum response elements.","date":"1997","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/9010223","citation_count":98,"is_preprint":false},{"pmid":"18186651","id":"PMC_18186651","title":"Protein phosphatase 6 regulatory subunits composed of ankyrin repeat domains.","date":"2008","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/18186651","citation_count":96,"is_preprint":false},{"pmid":"16769727","id":"PMC_16769727","title":"Protein phosphatase 6 subunit with conserved Sit4-associated protein domain targets 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However, knockdown of PP6R3 (unlike PP6R1) did NOT significantly enhance IkappaBepsilon degradation in response to TNFalpha, indicating PP6R3 does not regulate this specific PP6 substrate.\",\n      \"method\": \"FLAG-tag co-immunoprecipitation in HEK293 cells, siRNA knockdown with functional readout (IkappaBepsilon degradation assay)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP showing specificity, plus functional siRNA knockdown with defined substrate readout, single lab but two orthogonal methods\",\n      \"pmids\": [\"16769727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PP6R3 (SAPS domain subunit) forms part of a heterotrimer with PP6 catalytic subunit and ankyrin repeat proteins (Ankrd28, Ankrd44, Ankrd52). Tagged PP6R3 specifically coprecipitates with Ankrd28 and PP6, and endogenous PP6 holoenzymes containing PP6R3 co-elute with Ankrd28 at >440 kDa. PP6R3 knockdown (unlike PP6R1 or Ankrd28 knockdown) does NOT enhance IkappaBepsilon degradation in response to TNFalpha.\",\n      \"method\": \"FLAG-tag co-immunoprecipitation, mass spectrometry, size-exclusion chromatography (Superose 12), siRNA knockdown with IkappaBepsilon degradation assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, MS, gel filtration) in single lab establishing heterotrimer composition and negative functional result for PP6R3 in IkBε regulation\",\n      \"pmids\": [\"18186651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The conserved SAPS domain of PP6R3 forms helical repeats structurally similar to golgin p115, and negatively charged residues in interhelical loops mediate specific association with the PP6 catalytic subunit. Charge-reversal mutations in the SAPS domain reduced PP6 binding without perturbing overall PP6R3 conformation. Endogenous PP6R3 co-precipitates approximately half of PP6 in cell extracts.\",\n      \"method\": \"Charge-reversal mutagenesis, FLAG-tag co-precipitation from mammalian cells, circular dichroism spectroscopy, trypsin/chymotrypsin protection assay, 3D homology modeling with 3D-jury\",\n      \"journal\": \"BMC biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with co-precipitation and structural analysis, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"19835610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PP6R3 (along with PP6R1 and PP6R2) interacts with the DNA-PKcs catalytic subunit, as demonstrated by co-immunoprecipitation. siRNA silencing of PP6R1 (but not explicitly PP6R3 alone) led to sustained gamma-H2AX phosphorylation after ionizing radiation. PP6 is proposed to be recruited to DNA damage sites via DNA-PKcs to dephosphorylate gamma-H2AX.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, siRNA knockdown, immunofluorescence for gamma-H2AX foci, G2/M checkpoint assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP establishing physical interaction, siRNA functional phenotype, single lab with multiple readouts\",\n      \"pmids\": [\"20065038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PP6R1 (not PP6R3) specifically interacts with DNA-PK and mediates PP6-dependent activation of DNA-PK after ionizing radiation. siRNA knockdown of PP6R3 or ARS-A did NOT reduce IR activation of DNA-PK, demonstrating that the PP6R1 subunit specifically (not PP6R3) mediates this function.\",\n      \"method\": \"Endogenous co-immunoprecipitation, siRNA knockdown of specific subunits, DNA-PK activity assay after IR, cell viability assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subunit-specific siRNA with functional kinase activity readout demonstrating PP6R3 is dispensable for DNA-PK activation, single lab\",\n      \"pmids\": [\"19648198\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Human PP6R3 (and PP6R2, but not PP6R1) can physically interact with yeast Sit4 phosphatase and functionally rescue growth defects, rapamycin hypersensitivity, and G1 cell cycle delay in yeast lacking all four SAP proteins, in a Sit4-dependent manner. PP6R3 enhanced cyclin G1 gene expression and DNA synthesis in this heterologous context.\",\n      \"method\": \"Yeast complementation assay (quadruple sap mutant rescue), growth assays, rapamycin sensitivity assay, cell cycle analysis, co-immunoprecipitation with Sit4\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional genetic complementation in heterologous system with multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"19621075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Bacterially-produced PP6c in heterotrimeric combinations exhibits phosphatase activity against gamma-H2AX in vitro. Chromatin immunoprecipitation showed PP6c recruitment to regions adjacent to DSB sites. Depletion of PP6c or PP6R2 (not PP6R3 specifically) led to persistent high gamma-H2AX levels and defective homology-directed repair.\",\n      \"method\": \"In vitro phosphatase assay with recombinant protein, chromatin immunoprecipitation (ChIP), siRNA knockdown, HDR assay, immunofluorescence\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro reconstituted phosphatase activity plus ChIP and functional HDR assay, but PP6R3 not individually tested for this function\",\n      \"pmids\": [\"21451261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PP6 subunits including PPP6C and PPP6R3 were identified by affinity purification-mass spectrometry as components of the influenza A virus RdRP interactome. PP6 was found to interact directly with PB1 and PB2 subunits of the viral RdRP, and siRNA knockdown of PPP6C reduced viral RNA accumulation and attenuated virus growth.\",\n      \"method\": \"Strep-tag affinity purification from infected cells, label-free quantitative mass spectrometry, siRNA knockdown with viral RNA quantification and plaque assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — affinity purification-MS establishing complex membership, functional siRNA knockdown with viral replication readout, single lab\",\n      \"pmids\": [\"25187537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PP6R3 phosphatase dephosphorylates TRF2 at Ser365 specifically during S phase, providing a narrow window for RTEL1 helicase to access and unwind telomeric t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 Ser365 by CDK outside S phase releases RTEL1, protecting t-loops from promiscuous unwinding and preventing inappropriate ATM activation.\",\n      \"method\": \"Phospho-specific antibodies, co-immunoprecipitation, siRNA/shRNA knockdown, CDK phosphorylation site mutagenesis, telomere replication assays, ATM activation readout, cell cycle-staged biochemistry\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (phospho-mutagenesis, Co-IP, cell cycle staging, functional telomere replication assays) establishing a precise phospho-switch mechanism, published in high-rigor venue\",\n      \"pmids\": [\"31723267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PP6 holoenzyme components including PPP6R3 promote TAK1 inhibitor-induced PANoptosis (RIPK1-dependent cell death). PP6 regulatory subunits PPP6R1, PPP6R2, and PPP6R3 have redundant roles; combined depletion of all three was required to block cell death. Mechanistically, PPP6C and its regulatory subunits promote pro-death S166 auto-phosphorylation of RIPK1 and reduce pro-survival S321 phosphorylation of RIPK1.\",\n      \"method\": \"CRISPR screen for cell death, genetic knockout/knockdown of individual and combined PP6 subunits, phospho-specific immunoblotting for RIPK1 S166 and S321\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR-based loss-of-function with defined phosphorylation readout on RIPK1, single lab, mechanistic phospho-site analysis\",\n      \"pmids\": [\"38807188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The PP6c-PP6R3 complex plays a specific role in regulating cancer stem cell (CSC) markers in colorectal cancer cells. PP6c knockdown reduced colony-forming ability and in vivo proliferation; transcriptome analysis showed altered expression of stemness-associated genes upon PP6c knockdown, with the PP6c-PP6R3 complex identified as a key player.\",\n      \"method\": \"siRNA knockdown, colony formation assay, in vivo xenograft, transcriptome analysis, subunit-specific co-immunoprecipitation inference\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional knockdown phenotype with transcriptomic readout but no direct biochemical mechanism linking PP6R3 specifically to CSC regulation established\",\n      \"pmids\": [\"39014521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Germline-specific deletion of PPP6R3 in mice causes abnormal spermatogonial differentiation and male infertility with translation inhibition. PPP6R3 directly interacts with EIF3C and EIF4G1 in KIT+ spermatogonia; PPP6R3/PP6 dephosphorylates EIF3C at S39 and EIF4G1 at S1217. Increased phosphorylation after deletion promotes degradation of these translation initiation factors and reduces their mRNA association. Overexpression of phospho-dead EIF3C(S39A) and EIF4G1(S1217A) mutants rescues the differentiation defect.\",\n      \"method\": \"Conditional knockout in mice (CRISPR/cKO), co-immunoprecipitation (PPP6R3-EIF3C/EIF4G1), phospho-specific western blotting, phosphoproteomics, rescue experiment with phospho-dead mutants, translation rate measurement, RNA-immunoprecipitation\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vivo conditional KO with defined infertility phenotype, direct substrate identification by Co-IP, phospho-site mapping, and phospho-dead mutant rescue, multiple orthogonal methods in single study\",\n      \"pmids\": [\"40721635\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PPP6R3/PP6C phosphatase complex dephosphorylates Sec16 at the endoplasmic reticulum exit sites (ERES) to maintain ERES assembly and secretory activity. Excessive dephosphorylation by PP6 (PPP6R3/PPP6C) impairs secretion, while the FAM83A/CK1α kinase complex phosphorylates Sec16 in a negative feedback loop. A spatially distinct PP1 complex (PPP1R15B/PPP1C) handles TANGO1 dephosphorylation.\",\n      \"method\": \"Phosphatase complex identification (likely Co-IP/biochemical fractionation), functional assays for ERES formation and secretion, pharmacological and genetic perturbation of phosphorylation state\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint abstract with limited methodological detail, single study, mechanism inferred from abstract description without explicit reconstitution or mutagenesis details available\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Deletion of ppp6r3 in zebrafish using CRISPR/Cas9 results in all-male offspring and male infertility, with spermatogenesis blocked at the spermatocyte-to-sperm transition, demonstrating a role for Ppp6r3 in gonadal differentiation and gametogenesis.\",\n      \"method\": \"CRISPR/Cas9 knockout in zebrafish, histological analysis of testes, fertility assay\",\n      \"journal\": \"Yi chuan = Hereditas\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — clean KO with defined gonadal and spermatogenesis phenotype but no direct biochemical substrate or pathway mechanism identified\",\n      \"pmids\": [\"40962474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Deletion of a microexon in ppp6r3 in zebrafish by CRISPR/Cas9 produced mild neural phenotypes detectable by brain activity imaging, suggesting the ppp6r3 microexon contributes to neural function.\",\n      \"method\": \"CRISPR/Cas9 microexon deletion in zebrafish, larval brain activity imaging, morphological analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — phenotypic characterization of microexon deletion with neural activity readout but no molecular mechanism identified for this specific exon\",\n      \"pmids\": [\"41252186\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPP6R3 (PP6R3/SAPS3) is a SAPS-domain regulatory subunit that recruits and specifies the substrate selectivity of the PP6 heterotrimer (PP6c catalytic subunit + SAPS subunit + ankyrin-repeat subunit); its SAPS domain uses negatively charged helical-repeat residues to bind PP6c specifically (not PP2A or PP4), and the complex dephosphorylates defined substrates including TRF2-Ser365 (regulating t-loop dynamics and telomere replication during S phase), EIF3C-Ser39 and EIF4G1-Ser1217 (activating mRNA translation during spermatogonial differentiation), gamma-H2AX (contributing to DNA double-strand break repair), and Sec16 at ER exit sites; PP6R3-containing complexes also promote RIPK1-dependent PANoptosis by sustaining RIPK1-S166 autophosphorylation, and the PP6/PP6R3 holoenzyme interacts with DNA-PKcs and the influenza RdRP in the context of the DNA damage response and viral replication, respectively.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP6R3 (PP6R3/SAPS3) is a SAPS-domain regulatory subunit of the protein phosphatase 6 (PP6) holoenzyme that recruits the PP6 catalytic subunit and dictates its substrate selectivity across diverse cellular processes [#0, #2]. Its conserved SAPS domain folds into helical repeats whose negatively charged interhelical-loop residues mediate specific binding to the PP6 catalytic subunit, distinguishing PP6 from PP2A and PP4; charge-reversal mutations in these residues disrupt the interaction [#0, #2]. Within cells PPP6R3 assembles into a >440 kDa heterotrimer comprising the PP6 catalytic subunit and an ankyrin-repeat subunit such as Ankrd28 [#1]. Through this complex PPP6R3 specifies dephosphorylation of defined substrates: it removes TRF2-Ser365 phosphorylation specifically during S phase, opening a window for RTEL1 to unwind telomeric t-loops and enabling telomere replication while restraining inappropriate ATM activation [#8], and it dephosphorylates the translation initiation factors EIF3C-Ser39 and EIF4G1-Ser1217 in KIT+ spermatogonia, stabilizing these factors and sustaining mRNA translation required for spermatogonial differentiation [#11]. Germline deletion of PPP6R3 causes male infertility with translation inhibition, a defect rescued by phospho-dead EIF3C(S39A)/EIF4G1(S1217A) mutants [#11]. PPP6R3-containing PP6 complexes also act redundantly with PP6R1/PP6R2 to promote RIPK1-dependent PANoptosis by sustaining pro-death RIPK1-Ser166 autophosphorylation and reducing pro-survival Ser321 phosphorylation [#9], and PP6 holoenzymes containing PPP6R3 engage DNA-PKcs in the DNA damage response and the influenza A virus RdRP during viral replication [#3, #7].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Establishing that the SAPS domain confers phosphatase specificity answered whether PP6R3 is a dedicated PP6 adaptor rather than a promiscuous regulatory subunit.\",\n      \"evidence\": \"FLAG co-immunoprecipitation in HEK293 cells showing PP6 but not PP2A/PP4 binding, plus siRNA knockdown with an IkappaBepsilon degradation readout\",\n      \"pmids\": [\"16769727\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve the structural basis of specificity\", \"PP6R3 found dispensable for the IkappaBepsilon substrate, leaving its true substrates undefined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defining the holoenzyme composition established that PP6R3 functions as one arm of a heterotrimer with the catalytic and ankyrin-repeat subunits.\",\n      \"evidence\": \"FLAG co-IP, mass spectrometry, and size-exclusion chromatography in a single lab demonstrating co-elution of PP6R3, PP6c, and Ankrd28 at >440 kDa\",\n      \"pmids\": [\"18186651\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry within the assembled trimer not quantified\", \"Functional consequence of the ankyrin subunit not separated from PP6R3\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Mapping the SAPS domain fold and the charged residues that contact PP6c provided the structural mechanism of subunit recognition.\",\n      \"evidence\": \"Charge-reversal mutagenesis, co-precipitation, circular dichroism, protease protection, and homology modeling to golgin p115\",\n      \"pmids\": [\"19835610\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No experimentally determined high-resolution structure\", \"Model rests on homology rather than direct structure determination\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Cross-species complementation tested whether human PP6R3 is a functionally conserved phosphatase adaptor by rescuing yeast SAP-deficient phenotypes.\",\n      \"evidence\": \"Yeast quadruple sap mutant rescue, rapamycin sensitivity and cell-cycle assays, and Sit4 co-immunoprecipitation\",\n      \"pmids\": [\"19621075\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Heterologous yeast context does not establish native mammalian substrates\", \"Sit4 is not PP6c, so conservation is functional rather than identical\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linking PP6 subunits to DNA-PKcs and gamma-H2AX placed the holoenzyme in the DNA double-strand break response, though PP6R3's individual contribution was not isolated.\",\n      \"evidence\": \"Co-immunoprecipitation of endogenous proteins, siRNA knockdown, gamma-H2AX immunofluorescence, and G2/M checkpoint assays\",\n      \"pmids\": [\"20065038\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The gamma-H2AX phenotype was attributed to PP6R1, not PP6R3 specifically\", \"PP6R3-specific role in DSB repair untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Affinity purification of the influenza RdRP interactome implicated PP6 holoenzyme membership in viral replication.\",\n      \"evidence\": \"Strep-tag affinity purification from infected cells with label-free quantitative MS and siRNA knockdown of PPP6C with viral RNA and plaque readouts\",\n      \"pmids\": [\"25187537\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional viral phenotype tested for PPP6C, not PPP6R3\", \"Direct PP6R3-RdRP contact not demonstrated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying TRF2-Ser365 as a cell-cycle-staged substrate established a precise phospho-switch mechanism by which PP6R3 gates telomere replication.\",\n      \"evidence\": \"Phospho-specific antibodies, co-IP, knockdown, CDK-site mutagenesis, and cell-cycle-staged telomere replication and ATM activation assays\",\n      \"pmids\": [\"31723267\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recruitment mechanism of the complex to telomeres not fully defined\", \"Whether other SAPS subunits can substitute at TRF2 not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defining PP6 control of RIPK1 phosphorylation placed PPP6R3 in cell-death signaling, with redundancy among the three SAPS subunits.\",\n      \"evidence\": \"CRISPR death screen, individual and combined PP6 subunit knockouts, and phospho-specific immunoblotting of RIPK1 S166 and S321\",\n      \"pmids\": [\"38807188\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Redundancy means PP6R3-unique contribution cannot be isolated\", \"Direct RIPK1 dephosphorylation by the PP6R3 complex not reconstituted\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"In vivo conditional knockout identified EIF3C and EIF4G1 as direct substrates, linking PP6R3-mediated dephosphorylation to translational control of spermatogonial differentiation.\",\n      \"evidence\": \"Germline conditional knockout in mice, PPP6R3-EIF3C/EIF4G1 co-IP, phosphoproteomics, RNA-IP, and rescue with phospho-dead EIF3C(S39A)/EIF4G1(S1217A) mutants\",\n      \"pmids\": [\"40721635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether translation control extends beyond germ cells not addressed\", \"Mechanism coupling phosphorylation to factor degradation not detailed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Zebrafish knockout extended the germline requirement across species, showing Ppp6r3 is needed for gonadal differentiation and the spermatocyte-to-sperm transition.\",\n      \"evidence\": \"CRISPR/Cas9 knockout in zebrafish with testis histology and fertility assays\",\n      \"pmids\": [\"40962474\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No biochemical substrate identified in this system\", \"Mechanism of all-male offspring skewing unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Microexon deletion in zebrafish hinted at a neural function for an alternatively spliced PPP6R3 isoform.\",\n      \"evidence\": \"CRISPR/Cas9 microexon deletion in zebrafish with larval brain activity imaging\",\n      \"pmids\": [\"41252186\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No molecular mechanism for the microexon\", \"Neural phenotype mild and not connected to phosphatase activity\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"A Sec16 dephosphorylation role at ER exit sites positioned PP6R3 in secretory pathway regulation.\",\n      \"evidence\": \"Phosphatase complex identification with ERES formation and secretion assays under genetic and pharmacological perturbation (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint without reconstitution or mutagenesis detail\", \"Direct PP6R3-Sec16 contact and phospho-site not established in available evidence\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single SAPS subunit partitions PP6 among telomeric, translational, cell-death, secretory, and viral substrates within the cell remains unresolved.\",\n      \"evidence\": \"No timeline study addresses how substrate selection and subcellular targeting of PPP6R3-PP6 complexes are coordinated\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No mechanism for spatial/temporal substrate switching\", \"No structure of a substrate-bound holoenzyme\", \"Relative contribution of distinct ankyrin partners to substrate choice unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [8, 11]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [9]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\"PP6 holoenzyme (PP6c-PP6R3-ankyrin repeat subunit)\"],\n    \"partners\": [\"PPP6C\", \"ANKRD28\", \"TRF2\", \"EIF3C\", \"EIF4G1\", \"PRKDC\", \"RIPK1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}