{"gene":"PPP6C","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2006,"finding":"Alpha4 regulatory subunit exerts opposing allosteric effects on PP6 and PP2A: alpha4·PP6 heterodimer has ~100-fold lower Vmax than alpha4·PP2A with MBP substrate, meaning alpha4 activates PP2A but inhibits PP6. Monomeric PP6 and PP2A show identical kinetics with pNPP and MBP, but PP6 is inhibited at 5-fold lower concentrations of toxins (okadaic acid, microcystin-LR, calyculin A, cantharidin) with MBP substrate, suggesting PP6 is a preferred in vivo target.","method":"In vitro phosphatase assays with recombinant monomeric and heterodimeric PP6 and PP2A; pNPP and 32P-MBP substrates; IC50 measurements with active-site inhibitors; transient expression of alpha4 in cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified recombinant proteins, multiple substrates, multiple inhibitors, and cell-based validation","pmids":["16895907"],"is_preprint":false},{"year":2003,"finding":"PP6 catalytic subunit (PP6C) is purified from bovine testes and shows relatively low phosphatase activity toward several substrates compared to PP2A(C) and PP4(C). PP6C does not bind GST-alpha4 in the same pulldown conditions where PP2A(C) binds alpha4.","method":"Parallel purification from bovine testes; microcystin-Sepharose affinity resin; GST-alpha4 pulldown; co-immunoprecipitation; in vitro phosphatase activity assays","journal":"Protein expression and purification","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro assays and pulldowns in single study, but the negative finding on alpha4 binding to PP6C contrasts with other data","pmids":["12963337"],"is_preprint":false},{"year":2003,"finding":"Fission yeast PP6 ortholog Ppe1 (scSit4/hPP6) and its binding partner Ekc1 (scSAP) negatively regulate kinetochore protein Mis12 localization and are required for faithful chromosome segregation; Gsk3 kinase counteracts Ppe1/PP6 at this step. Mis12 is phosphorylated at ≥2 sites as revealed by mass spectrometry, and Ppe1/Ekc1 co-fractionate with chromatin as non-histone chromatin-associated proteins.","method":"Suppressor screen of mis12 mutant in fission yeast; mass spectrometry of Mis12 phosphorylation; chromatin fractionation; genetic epistasis with gsk3; localization recovery assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (suppressor genetics, MS, fractionation, epistasis) in a single focused study on PP6 ortholog","pmids":["12773390"],"is_preprint":false},{"year":2007,"finding":"The N-terminal domain of human PP6 (first ~53 residues) restricts G1-to-S phase progression in human prostate cancer cells, suppresses cyclin D1 protein levels in G1, and reduces RB1 phosphorylation at Ser807/811, without significantly altering cyclin/cell-cycle gene transcripts. This function is distinct from the PP6 homolog Sit4 in yeast.","method":"Transient expression of GFP fusions of PP6 or PP2A N-termini in PC-3 cells; flow cytometry cell cycle analysis; cyclin D1 western blot; RB1 phosphorylation western blot; gene expression microarray","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal readouts (flow cytometry, protein levels, phosphorylation) in a single lab study","pmids":["17568194"],"is_preprint":false},{"year":2009,"finding":"Human PP6 regulatory subunits PP6R2 and PP6R3 (but not PP6R1) physically interact with yeast Sit4 and rescue growth defects, rapamycin hypersensitivity, cyclin G1 expression, and DNA synthesis of a yeast quadruple sap mutant in a Sit4-dependent manner, demonstrating functional conservation of the PP6 regulatory subunit–phosphatase interaction.","method":"Heterologous expression of human PP6R proteins in S. cerevisiae sap mutant; growth assays; rapamycin sensitivity; cyclin expression; DNA synthesis; genetic rescue","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic complementation with multiple phenotypic readouts in heterologous system, single lab","pmids":["19621075"],"is_preprint":false},{"year":2011,"finding":"PP6c-containing heterotrimeric complexes (produced in bacteria) exhibit phosphatase activity against γ-H2AX (phospho-H2AX Ser139) in vitro, establishing γ-H2AX as a substrate of PP6. Depletion of PP6c or its regulatory subunit PP6R2 leads to persistent γ-H2AX after DNA damage and defective homology-directed repair (HDR); PP6c is recruited to DSB sites as shown by ChIP.","method":"In vitro phosphatase assay with bacterially-produced PP6 heterotrimers; siRNA knockdown; γ-H2AX immunofluorescence; HDR reporter assay; chromatin immunoprecipitation","journal":"Cell cycle (Georgetown, Tex.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro phosphatase assay establishing substrate identity, corroborated by ChIP recruitment and cell-based HDR assay with multiple orthogonal methods","pmids":["21451261"],"is_preprint":false},{"year":2012,"finding":"PP6 regulatory subunit R1 (SAPSR1) acts as a bidentate anchor that bridges DNA-PK and PP6c: two distinct regions of R1 (residues 1–326 and 522–700) each bind DNA-PK, with residues 1–326 as the dominant domain; R1 is necessary to recruit PP6c to DNA-PK. Knockdown of R1 or PP6c prevents DNA-PK activation after ionizing radiation and radiosensitizes glioblastoma cells.","method":"R1 deletion mutant co-immunoprecipitation mapping; siRNA knockdown; clonogenic radiosensitivity assay; DNA-PK kinase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic deletion mapping with multiple constructs, reciprocal interaction evidence, functional knockdown readout, replicated domain assignment","pmids":["22298787"],"is_preprint":false},{"year":2013,"finding":"Yeast PP6 ortholog Sit4p dephosphorylates COPII coat subunits in vitro and in vivo; sit4Δ mutants accumulate hyperphosphorylated COPII coat subunits and show altered COPII distribution. Mammalian PP6 similarly regulates ER-to-Golgi trafficking, establishing PP6 as a regulator of COPII coat recycling.","method":"Phosphatase screen in yeast; in vitro dephosphorylation assay with Sit4p and COPII subunits; COPII localization in sit4Δ cells; mammalian PP6 knockdown ER-to-Golgi trafficking assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of Sit4p dephosphorylating COPII substrates, validated in vivo in yeast and with mammalian PP6 ortholog","pmids":["23864707"],"is_preprint":false},{"year":2013,"finding":"PP6 is stably associated with U1 snRNP and is present as part of the spliceosome throughout the splicing reaction, suggesting a role in pre-mRNA splicing regulation.","method":"Co-immunoprecipitation of PP6 with U1 snRNP; spliceosome pull-down; biochemical fractionation","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/pulldown, single lab, no functional mutant validation reported in abstract","pmids":["24064353"],"is_preprint":false},{"year":2014,"finding":"PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after ionizing radiation. PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis; phosphorylation of Thr3950 is DNA-PK-dependent. DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis in the apparent absence of Ku and DNA damage.","method":"In vitro kinase assay (PLK1 on DNA-PKcs); co-immunoprecipitation; phospho-specific antibodies; PP6 knockdown; mass spectrometry identification of phosphosites","journal":"Bioscience reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay combined with co-IP and cell-based phosphosite analysis, single lab","pmids":["24844881"],"is_preprint":false},{"year":2015,"finding":"PP6c depletion in mitotic HeLa cells causes phosphorylation changes in 408 phosphopeptides (272 proteins increased) and 298 phosphopeptides (220 proteins decreased). PP6c directly opposes casein kinase 2 (CK2)-dependent phosphorylation of condensin I subunit NCAP-G; PP6c depletion causes defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I.","method":"Baculovirus-mediated PP6c depletion in HeLa; quantitative mass spectrometry phosphoproteomics; biochemical phosphatase assay; chromosome condensation/segregation imaging","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1 / Strong — quantitative phosphoproteomics combined with biochemical assay and cellular phenotype with multiple orthogonal methods","pmids":["26462736"],"is_preprint":false},{"year":2015,"finding":"T cell lineage-specific ablation of PP6 in mice causes hyperactivation of multiple distal TCR signaling molecules including MAPKs, AKT, and NF-κB, enhanced thymic selection, preferential expansion of IL-17-producing Vγ6Vδ1+ T cells, and loss of naive T cell homeostasis. PP6 acts as a critical negative regulator of distal TCR signaling.","method":"Conditional PP6 knockout mice (T cell-specific); flow cytometry; western blot of downstream signaling molecules; T cell proliferation and differentiation assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean conditional KO with defined molecular (signaling pathway) and cellular (selection, homeostasis) phenotypes; multiple orthogonal readouts","pmids":["25609840"],"is_preprint":false},{"year":2016,"finding":"LCMT-1 is the major carboxyl methyltransferase for PP6 catalytic subunit (as well as PP2A and PP4) in mouse embryonic fibroblasts. PP6 is carboxyl-methylated on its C-terminal leucine. Unlike PP2A and PP4, loss of methylation (LCMT-1 KO) does not significantly affect PP6 holoenzyme assembly.","method":"Antibodies specific to unmethylated phosphatases; LCMT-1 knockout MEFs; blue native PAGE; co-immunoprecipitation; western blot","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods including KO MEFs and BN-PAGE, single lab; null effect on PP6 holoenzyme is negative but informative","pmids":["27507813"],"is_preprint":false},{"year":2016,"finding":"Homozygous deletion of the Ppp6c phosphatase domain in mice is embryonic lethal; mutant embryos degenerate by E7.5 with clear developmental defects at E8.5. Homozygous blastocysts exhibit growth failure of the inner cell mass in vitro, and Ppp6c-deficient MEFs show greatly reduced proliferation, establishing that PP6 phosphatase activity is indispensable for post-implantation embryogenesis.","method":"Conditional knockout mouse generation; embryo phenotyping; in vitro blastocyst culture; MEF proliferation assay","journal":"Mechanisms of development","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse model with defined lethal phenotype and cell-level validation across multiple assays","pmids":["26868000"],"is_preprint":false},{"year":2016,"finding":"TORC1 signaling suppresses the Tap42-regulated Sit4 (PP6) phosphatase complex; sit4Δ rescues histone acetylation under TORC1-repressive conditions. TORC1 inhibition activates PP6/Sit4, causing nuclear accumulation of sirtuin deacetylase Hst4 (reduced protein turnover), decreased histone H3/H4 acetylation. PP6 thus couples nutrient/TOR signaling to epigenetic regulation via sirtuin nuclear localization.","method":"Genetic epistasis (sit4Δ, hst3Δ, hst4Δ, tco89Δ); rapamycin treatment; histone acetylation western blot; Hst4-GFP nuclear localization imaging; protein stability assay","journal":"Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with multiple mutant combinations and multiple readouts; yeast ortholog study","pmids":["27343235"],"is_preprint":false},{"year":2017,"finding":"TRIM14 provides a mitochondrial docking platform for assembly of the WHIP–TRIM14–PPP6C signalosome required for RIG-I-mediated antiviral signaling. PPP6C dephosphorylates RIG-I within this complex, promoting its activation; WHIP bridges RIG-I with MAVS via polyubiquitin chains at RIG-I K164.","method":"Pooled RNAi screen; yeast two-hybrid; co-immunoprecipitation; RIG-I dephosphorylation assay; antiviral reporter assays; viral infection models","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (RNAi screen, Y2H, co-IP, dephosphorylation assay) identifying both complex composition and enzymatic activity","pmids":["29053956"],"is_preprint":false},{"year":2017,"finding":"In Drosophila, the PP6 regulatory subunit Fmt (fiery mountain) negatively regulates JNK signaling upstream of dTAK1; loss of Fmt or PpV (PP6 catalytic subunit) cooperates with oncogenic RasV12 to promote JNK-dependent tumor growth and invasion. Fmt and PpV act synergistically to suppress JNK-dependent tumorigenesis.","method":"Genetic screen in Drosophila; genetic epistasis (fmt, ppv, rasV12, dTAK1 alleles); tumor growth and invasion assays; reporter assays for JNK activity","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic genetic epistasis placing PP6 upstream of dTAK1 in JNK pathway, multiple allele combinations, functional tumor phenotypes","pmids":["28658615"],"is_preprint":false},{"year":2018,"finding":"PP6 directly interacts with and inactivates ASK3 kinase in an osmolality-dependent manner under hyperosmotic stress; PP6-ASK3 interaction promotes regulatory volume increase (RVI) and cell survival. A genome-wide siRNA screen identified PP6 as a direct ASK3 inactivator in the bidirectional osmotic stress response.","method":"Genome-wide siRNA screen; co-immunoprecipitation; kinase activity assay; cell volume measurement; cell viability assay; osmotic stress treatments","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide screen plus co-IP plus kinase activity assay plus functional readout (cell volume/survival), multiple orthogonal methods","pmids":["29539411"],"is_preprint":false},{"year":2018,"finding":"Plk1 inhibits the phosphatase PP6 toward Aurora A (Aurora kinase A), generating a Plk1–PP6 feedback loop that coordinates Plk1 and Aurora A activities during mitotic entry. PP6 interaction with Plk1 is phosphorylation-dependent and is terminated by Plk1 degradation during mitotic exit.","method":"Quantitative proteomics on HeLa cells with kinase inhibitors or Plk1 phospho-binding mutant; Aurora A activity assays; PP6 activity assays; co-immunoprecipitation","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative proteomics plus functional activity assays, multiple conditions establishing mechanistic feedback loop","pmids":["29764989"],"is_preprint":false},{"year":2020,"finding":"CK2 phosphorylates SAPS3 (a PP6 regulatory subunit) on multiple acidic motifs; CK2-phosphorylated SAPS3-PP6 complex shows significantly increased phosphatase activity toward pT288-Aurora A kinase substrate. Nine Ala substitutions in SAPS3 CK2 sites block this activation. CK2 inhibitors increase Aurora A phosphorylation in cells, consistent with reduced PP6 activity. SAPS3 knockdown/KO results in hyperactivated Aurora A and abnormal nuclei.","method":"In vitro CK2 kinase assay on SAPS3; PP6 phosphatase activity assay with pT288-AurA substrate; mutagenesis of CK2 sites in SAPS3; CK2 inhibitor cell treatment; CRISPR/siRNA depletion of SAPS3; nuclear morphology imaging","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay, in vitro phosphatase assay, mutagenesis, and cell-based validation; multiple orthogonal methods","pmids":["31904830"],"is_preprint":false},{"year":2020,"finding":"PPP6C is constitutively associated with cGAS in unstimulated cells. DNA virus infection causes rapid dissociation of PPP6C from cGAS, resulting in phosphorylation of human cGAS S435 (mouse cGAS S420) in the catalytic pocket. S420-phosphorylated mcGAS has higher affinity for GTP and higher enzymatic activity. PPP6C dephosphorylation of this site keeps cGAS inactive in the absence of infection to prevent autoimmune response.","method":"Co-immunoprecipitation; phospho-specific antibody; site-directed mutagenesis of cGAS S435/S420; in vitro cGAMP synthesis assay; PPP6C knockout cell lines; viral infection experiments","journal":"Protein & cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical substrate identification (co-IP, mutagenesis), in vitro enzymatic assay validating phospho-site function, KO cell validation","pmids":["32474700"],"is_preprint":false},{"year":2020,"finding":"PPP6C interacts with STING and negatively regulates the cGAS-STING pathway by dephosphorylating STING. Loss of PPP6C enhances STING phosphorylation, increases IRF3 activation (but not NF-κB activation) in response to dsDNA, restricts HSV-1 and VSV replication, and inhibits KSHV reactivation through increased type I interferon production.","method":"Co-immunoprecipitation of PPP6C with STING; PPP6C siRNA depletion; phospho-STING western blot; IRF3/NF-κB reporter assays; viral replication assays","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus phosphorylation analysis plus functional viral assays, single lab","pmids":["32753499"],"is_preprint":false},{"year":2021,"finding":"PPP6C is a major MEK phosphatase in cells with oncogenic ERK pathway activation; PPP6C is recruited to MEK through its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting ERK pathway signaling and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation.","method":"PPP6C knockdown/knockout; phospho-MEK western blot; co-immunoprecipitation with regulatory subunits; MEK inhibitor sensitivity assays; expression of melanoma-associated PPP6C mutants","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic depletion, co-IP, phospho-site analysis, mutant expression, and drug sensitivity readouts; multiple orthogonal methods establishing direct MEK dephosphorylation","pmids":["33789117"],"is_preprint":false},{"year":2021,"finding":"PP6 (Pp6) deficiency in Treg cells increases CpG methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhances Akt phosphorylation at Ser473/Thr308, leading to impaired FoxP3 expression and Treg cell instability. Conditional Pp6 KO in Treg cells causes spontaneous autoinflammation.","method":"Conditional PP6 KO mice (Treg-specific); bisulfite sequencing of FoxP3 locus; Dnmt1 dephosphorylation assay; Akt phosphorylation western blot; flow cytometry; autoimmune model experiments","journal":"Genes & diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional KO with mechanistic biochemical readouts (Dnmt1 phosphorylation, Akt phosphorylation), single lab","pmids":["35224167"],"is_preprint":false},{"year":2021,"finding":"Loss of PP6 C-terminal methylation does not affect PP6 holoenzyme assembly (interaction with ANKRD and SAPS regulatory subunits), in contrast to PP2A and PP4 where methylation is critical for regulatory subunit binding.","method":"Mass spectrometry-based proteomics; methylation-ablating mutations; genome editing (CRISPR); BN-PAGE; quantitative interaction proteomics","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (MS proteomics, CRISPR, BN-PAGE) establishing negative regulatory role of methylation for PP6 specifically","pmids":["34845248"],"is_preprint":false},{"year":2022,"finding":"PP6 is an identified component of TNF receptor complex I. PP6 loss protects cells from TNFα-mediated cell death in a phosphatase-activity-dependent manner. PP6 modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL, promoting RIPK1 activation and c-FLIPL degradation. Melanoma-associated PP6 inactivating mutations confer resistance to TNFα-mediated cell death.","method":"Co-immunoprecipitation identifying PP6 in complex I; PP6 KO/knockdown; cell death assays; RIPK1 and c-FLIPL ubiquitination assays; expression of melanoma PP6 mutants","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of complex component, KO functional assay, ubiquitination analysis; single lab","pmids":["36071040"],"is_preprint":false},{"year":2022,"finding":"PPP6C acts as a TAK1 phosphatase to inactivate its kinase activity. Deletion of PPP6C leads to hyperactivation of TAK1 and reduced RIPK1 kinase activity upon TNF stimulation, protecting cells from TNF-induced necroptosis. This was identified in a genome-wide CRISPR/Cas9 screen and validated mechanistically.","method":"Genome-wide CRISPR/Cas9 screen; PPP6C KO; TAK1 kinase activity assay; RIPK1 phosphorylation analysis; necroptosis/cell death assays; heterozygous Ppp6c mouse gut model","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR screen discovery, KO validation, kinase activity assay for TAK1 as substrate, in vivo mouse model corroboration","pmids":["35842423"],"is_preprint":false},{"year":2022,"finding":"AMPKγ subunit, when bound to AMP under energy starvation, sequesters PPP6C to block dephosphorylation of eukaryotic translation elongation factor 2 (eEF2), thereby inhibiting translation elongation and preserving energy. PPP6C is identified as an AMPKγ-regulated phosphatase of eEF2; this regulation is independent of AMPKα catalytic activity.","method":"AMPKγ and AMPKα knockout; co-immunoprecipitation of AMPKγ with PPP6C; eEF2 phosphorylation analysis; phosphoproteomics under energy starvation; cell survival assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO of both AMPK subunits, co-IP establishing direct interaction, phosphoproteomics, and functional translation assay; multiple orthogonal methods","pmids":["36384136"],"is_preprint":false},{"year":2022,"finding":"Rab40c (SOCS box protein/Cullin5 E3 ligase) binds the PP6 complex and ubiquitylates ANKRD28 (a PP6 regulatory subunit), leading to its lysosomal degradation and decreased PP6 activity. PP6 activity loss via this mechanism decreases FAK and MOB1 phosphorylation, affecting focal adhesion dynamics in migrating cells.","method":"Co-immunoprecipitation; ubiquitylation assay; Rab40c KO cells; focal adhesion quantification; phospho-FAK and phospho-MOB1 western blot; lysosome inhibitor experiments","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitylation assay, and KO phenotype analysis; single lab with multiple readouts","pmids":["35512830"],"is_preprint":false},{"year":2023,"finding":"PP6 (PPP6C) regulates Aurora A-TPX2-mediated phosphorylation of multiple N-terminal sites on NDC80 at checkpoint-silenced, microtubule-attached kinetochores during spindle formation. NDC80 phosphorylation by Aurora A-TPX2 is Aurora B-independent and persists until spindle disassembly in telophase; it is increased in PPP6C KO cells. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C KO cells. Synthetic lethality between PPP6C and NDC80 was identified by functional genomics.","method":"PPP6C knockout; phospho-specific antibodies; functional genomics screen; Aurora A-TPX2 in vitro kinase assay; NDC80-9A phospho-dead mutant rescue; spindle size measurement; nuclear structure imaging","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO combined with in vitro kinase assay, phospho-dead mutant rescue, functional genomics screen; multiple orthogonal methods, single focused study","pmids":["36897279"],"is_preprint":false},{"year":2024,"finding":"PPP6C and its regulatory subunits PPP6R1, PPP6R2, and PPP6R3 (with redundant roles) promote TAK1 inhibitor-induced, RIPK1-dependent PANoptosis. PPP6C promotes pro-death S166 auto-phosphorylation of RIPK1 and leads to reduction in pro-survival S321 phosphorylation of RIPK1. Loss of PPP6C significantly reduces PANoptosis.","method":"Cell death-based CRISPR screen; PPP6C and PPP6R1/2/3 KO/knockdown; RIPK1 phosphorylation (S166, S321) western blot; cell death assays; TAK1 inhibitor treatment","journal":"BMC biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR screen validated by KO, multiple regulatory subunit depletion, and specific phospho-site analysis of RIPK1; multiple orthogonal methods","pmids":["38807188"],"is_preprint":false},{"year":2025,"finding":"PPP6C (with regulatory subunit PPP6R3) dephosphorylates Sec16 at ER exit sites (ERES), counteracting FAM83A/CK1α-mediated phosphorylation of Sec16. Excessive dephosphorylation of Sec16 impairs secretion. A phosphorylation-coupled autoregulatory feedback loop involving FAM83A/CK1α and PP6 maintains balanced Sec16 phosphorylation state essential for proper ERES function and secretory activity.","method":"Phosphatase complex identification; phospho-Sec16 analysis; FAM83A/CK1α inhibitor/overexpression; ERES imaging; secretory assay; phospho-mutant rescue","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — preprint with defined complex (PPP6R3/PPP6C), phospho-Sec16 readout, and functional secretion assay; not yet peer-reviewed","pmids":["bio_10.1101_2025.06.18.660491"],"is_preprint":true},{"year":2025,"finding":"PP6 promotes intracellular Salmonella proliferation in macrophages by dephosphorylating Pfkfb1 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1); Pp6 deficiency elevates Pfkfb1 expression, which restricts bacterial growth by promoting NO production while suppressing Arg-1 expression and arginine metabolism. Yeast two-hybrid identified Pfkfb1 as a PP6 substrate.","method":"Fluorescence-dilution reporter in Salmonella-infected macrophages; conditional PP6 KO; yeast two-hybrid screening; Pfkfb1 KO; NO production assay; Arg-1 expression analysis","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid substrate identification, KO validation, and functional metabolic readouts; single lab","pmids":["41474810"],"is_preprint":false},{"year":2025,"finding":"SMURF2 E3 ubiquitin ligase interacts with PPP6C and promotes its ubiquitination and proteasomal/lysosomal degradation in keratinocytes, downstream of IL-17C signaling, leading to psoriasis-like changes. IL-17C upregulates SMURF2, which targets PPP6C for degradation.","method":"Co-immunoprecipitation (SMURF2–PPP6C); ubiquitination assay; SMURF2 knockdown/overexpression; PPP6C overexpression rescue; lentiviral/plasmid transfection; imiquimod mouse psoriasis model","journal":"Cell biology international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, and rescue experiment; single lab with multiple methods","pmids":["40244332"],"is_preprint":false}],"current_model":"PPP6C (PP6) is a heterotrimer serine/threonine phosphatase—composed of a catalytic subunit (PP6c/PPP6C), one of three SAPS/PPP6R regulatory subunits, and one of three ANKRD subunits—that dephosphorylates a defined set of substrates (including γ-H2AX, Aurora A, MEK, RIG-I, cGAS, STING, TAK1, RIPK1, DNA-PKcs, NCAP-G condensin I subunit, NDC80, eEF2, Dnmt1, COPII coat subunits, Sec16, Pfkfb1, and Mis12/kinetochore proteins) to regulate mitotic fidelity, DNA damage repair, innate immune signaling, cell cycle progression, T cell homeostasis, translation elongation, ER-to-Golgi trafficking, and cell death pathways; its activity is tuned by alpha4-mediated allosteric inhibition, CK2-dependent phosphorylation of SAPS3 that enhances Aurora A dephosphorylation, Plk1-dependent inhibition, TORC1 suppression, AMPKγ-dependent sequestration, and its own regulated ubiquitylation of ANKRD28 via Rab40c/CRL5 or SMURF2."},"narrative":{"mechanistic_narrative":"PPP6C is the catalytic subunit of the PP6 serine/threonine protein phosphatase, a heterotrimeric enzyme that assembles with SAPS/PPP6R and ANKRD regulatory subunits and dephosphorylates a defined substrate set to govern mitotic fidelity, the DNA damage response, innate immune signaling, and cell-death decisions [PMID:21451261, PMID:26462736, PMID:36897279]. In mitosis and the cell cycle, PP6 reverses kinase-driven phosphorylation events: it dephosphorylates the condensin I subunit NCAP-G to oppose CK2 and ensure proper chromosome condensation and segregation [PMID:26462736], controls Aurora A activity (a regulation tuned by a Plk1–PP6 feedback loop and by CK2 phosphorylation of SAPS3) [PMID:29764989, PMID:31904830], and restrains Aurora A–TPX2-mediated phosphorylation of the kinetochore protein NDC80 during spindle formation [PMID:36897279]. In the DNA damage response, PP6 dephosphorylates γ-H2AX to enable homology-directed repair and acts through the regulatory subunit R1 as a bidentate anchor that recruits the catalytic subunit to DNA-PK, supporting DNA-PK activation after ionizing radiation [PMID:21451261, PMID:22298787]. PP6 is a broad negative regulator of immune and stress signaling, dephosphorylating cGAS and STING to restrain the cGAS–STING–IRF3 axis, activating RIG-I within a mitochondrial WHIP–TRIM14–PPP6C signalosome, inactivating TAK1 and ASK3 kinases, and shaping TCR signaling and Treg stability via Dnmt1 and Akt [PMID:32474700, PMID:32753499, PMID:29053956, PMID:35842423, PMID:29539411, PMID:25609840, PMID:35224167]. It is a major MEK phosphatase whose recurrent melanoma-associated mutations cause MEK hyperphosphorylation and altered drug sensitivity [PMID:33789117], and it controls programmed cell death by modulating RIPK1 phosphorylation and ubiquitination within TNF receptor complex I and during PANoptosis [PMID:36071040, PMID:38807188]. PP6 additionally regulates ER-to-Golgi trafficking through dephosphorylation of COPII coat subunits and Sec16, and translation elongation through eEF2 [PMID:23864707, PMID:bio_10.1101_2025.06.18.660491, PMID:36384136]. PP6 phosphatase activity is essential for post-implantation mouse embryogenesis [PMID:26868000], and its activity is set by alpha4-mediated allosteric inhibition and by regulated degradation of its ANKRD28 subunit [PMID:16895907, PMID:35512830].","teleology":[{"year":2003,"claim":"Establishing PP6 as a distinct phosphatase required separating its catalytic and regulatory behavior from the related PP2A/PP4 enzymes and demonstrating conservation of the regulatory-subunit interaction.","evidence":"Purification of PP6C from bovine testes with activity assays and GST-alpha4 pulldowns, plus heterologous rescue of yeast sap mutants by human PP6R subunits","pmids":["12963337","19621075"],"confidence":"Medium","gaps":["Conflicting alpha4-binding result for PP6C left the regulatory relationship unresolved","Did not define mammalian substrates"]},{"year":2003,"claim":"Genetic work in fission yeast first linked the PP6 ortholog to chromosome segregation, placing the phosphatase opposite a kinase at the kinetochore.","evidence":"Suppressor screen of mis12, mass spectrometry of Mis12 phosphorylation, chromatin fractionation and gsk3 epistasis in S. pombe","pmids":["12773390"],"confidence":"High","gaps":["Direct dephosphorylation of Mis12 by PP6 not shown in vitro","Mammalian kinetochore relevance not established here"]},{"year":2006,"claim":"The question of how PP6 activity is tuned was addressed by showing alpha4 acts oppositely on PP6 versus PP2A, defining an allosteric brake specific to PP6.","evidence":"In vitro phosphatase assays with monomeric and alpha4-heterodimeric recombinant PP6/PP2A, inhibitor IC50 measurements, and cell expression","pmids":["16895907"],"confidence":"High","gaps":["Structural basis of differential alpha4 effect not resolved","Physiological substrates governed by alpha4 regulation not identified"]},{"year":2011,"claim":"PP6 gained its first mammalian molecular substrate when γ-H2AX dephosphorylation was reconstituted, linking the phosphatase to DNA double-strand break repair.","evidence":"In vitro phosphatase assay with bacterially produced PP6 heterotrimers, siRNA, γ-H2AX immunofluorescence, HDR reporter, and ChIP recruitment","pmids":["21451261"],"confidence":"High","gaps":["Which regulatory subunit directs γ-H2AX targeting in vivo not fully defined","Recruitment mechanism to DSBs incomplete"]},{"year":2012,"claim":"The mechanism of PP6 substrate targeting was clarified by showing the R1 regulatory subunit physically bridges the catalytic subunit to DNA-PK.","evidence":"Deletion-mapping co-IP of R1 with DNA-PK, siRNA knockdown, clonogenic radiosensitivity, and DNA-PK kinase assays","pmids":["22298787"],"confidence":"High","gaps":["Whether DNA-PKcs is a direct PP6 substrate here vs. an activator not fully separated","Structure of the R1-bridged complex unknown"]},{"year":2013,"claim":"PP6 was extended beyond the nucleus to membrane trafficking by showing it dephosphorylates COPII coat subunits to regulate ER-to-Golgi transport.","evidence":"Yeast phosphatase screen, in vitro dephosphorylation of COPII by Sit4p, COPII localization in sit4Δ, and mammalian PP6 trafficking assay","pmids":["23864707"],"confidence":"High","gaps":["Specific COPII phosphosites in mammals not mapped","Regulatory subunit directing COPII targeting not identified here"]},{"year":2014,"claim":"PP6 was implicated in mitotic DNA-PKcs regulation, integrating it with PLK1 signaling at Ser3205.","evidence":"In vitro PLK1 kinase assay on DNA-PKcs, co-IP, phospho-specific antibodies, PP6 knockdown, and MS phosphosite mapping","pmids":["24844881"],"confidence":"Medium","gaps":["Direct in vitro PP6 dephosphorylation of Ser3205 not reconstituted","Functional consequence of mitotic DNA-PKcs phosphorylation incomplete"]},{"year":2015,"claim":"System-wide phosphoproteomics defined the breadth of PP6 mitotic substrates and pinpointed condensin I (NCAP-G) as a CK2-opposed target controlling chromosome architecture.","evidence":"PP6c depletion with quantitative phosphoproteomics, biochemical phosphatase assay, and chromosome condensation/segregation imaging in HeLa","pmids":["26462736"],"confidence":"High","gaps":["Most of the hundreds of altered phosphosites not validated as direct substrates","Subunit-substrate assignment for NCAP-G not defined"]},{"year":2015,"claim":"Conditional knockout established PP6 as a physiological negative regulator of distal TCR signaling and T cell homeostasis.","evidence":"T cell-specific PP6 KO mice with flow cytometry, signaling western blots, and differentiation assays","pmids":["25609840"],"confidence":"High","gaps":["Direct PP6 substrates within the TCR cascade not identified","Which regulatory subunits operate in T cells unclear"]},{"year":2016,"claim":"Genetic and biochemical studies defined PP6 regulation by methylation and TOR signaling and demonstrated that its phosphatase activity is indispensable for development.","evidence":"LCMT-1 KO MEFs and BN-PAGE, yeast TORC1/Sit4 epistasis, and an embryonic-lethal Ppp6c phosphatase-domain deletion mouse","pmids":["27507813","27343235","26868000"],"confidence":"High","gaps":["Functional role of PP6 C-terminal methylation remains unexplained given no assembly effect","Mammalian TOR-PP6 coupling not directly demonstrated"]},{"year":2017,"claim":"PP6 was placed in innate immune and stress kinase pathways as an activator of RIG-I and a suppressor of TAK1/JNK signaling.","evidence":"RNAi screen, Y2H, co-IP and RIG-I dephosphorylation for the WHIP-TRIM14-PPP6C signalosome; Drosophila genetic epistasis placing PP6 upstream of dTAK1 in JNK signaling","pmids":["29053956","28658615"],"confidence":"High","gaps":["RIG-I phosphosites dephosphorylated by PP6 not fully mapped","Mammalian TAK1/JNK substrate validation deferred to later work"]},{"year":2018,"claim":"PP6 was shown to be embedded in mitotic kinase feedback loops, being inhibited by Plk1 to coordinate Aurora A activity, and to inactivate the stress kinase ASK3 under osmotic stress.","evidence":"Quantitative proteomics with kinase inhibitors and Plk1 phospho-binding mutants, Aurora A and PP6 activity assays; genome-wide siRNA screen with co-IP, kinase assay, and cell-volume readouts for ASK3","pmids":["29764989","29539411"],"confidence":"High","gaps":["Mechanism of Plk1-dependent PP6 inhibition at the molecular level incomplete","ASK3 dephosphorylation site not specified"]},{"year":2020,"claim":"Regulatory-subunit phosphorylation and substrate engagement were mechanistically detailed: CK2 phosphorylation of SAPS3 enhances Aurora A dephosphorylation, and PPP6C constitutively restrains cGAS-STING signaling.","evidence":"In vitro CK2 and PP6 activity assays with SAPS3 mutagenesis; co-IP, cGAS/STING phospho-site mutagenesis, in vitro cGAMP synthesis, and PPP6C KO with viral infection","pmids":["31904830","32474700","32753499"],"confidence":"High","gaps":["Coordination between cGAS and STING dephosphorylation by PP6 not integrated","Selectivity for IRF3 over NF-κB branch unexplained mechanistically"]},{"year":2021,"claim":"PP6 was established as a major MEK phosphatase relevant to oncogenic signaling and as a regulator of Treg stability, connecting recurrent melanoma mutations to MEK hyperactivation.","evidence":"PPP6C KO/knockdown with phospho-MEK analysis, regulatory-subunit co-IP, MEK-inhibitor sensitivity, melanoma mutant expression; Treg-specific KO with FoxP3 bisulfite sequencing and Dnmt1/Akt phospho-analysis","pmids":["33789117","35224167"],"confidence":"High","gaps":["Which regulatory subunit recruits PP6 to MEK not pinned down","Direct vs. indirect Dnmt1 dephosphorylation in Tregs needs reconstitution"]},{"year":2022,"claim":"PP6 was integrated into cell-death control by acting on TAK1 and RIPK1 within TNF receptor signaling, and additional layers of its own regulation (AMPKγ sequestration, Rab40c/CRL5-mediated ANKRD28 degradation) were defined.","evidence":"CRISPR screens with KO, TAK1 kinase and RIPK1 ubiquitination/phospho assays and gut mouse model; AMPK subunit KO with co-IP and eEF2 phosphoproteomics; Rab40c KO with ubiquitylation and focal-adhesion readouts","pmids":["35842423","36071040","36384136","35512830"],"confidence":"High","gaps":["Direct PP6 dephosphorylation sites on TAK1 and RIPK1 not all mapped","How energy state switches PP6 between substrates unresolved"]},{"year":2023,"claim":"PP6 was shown to restrain Aurora A-TPX2-driven NDC80 phosphorylation at attached kinetochores, with synthetic lethality to NDC80 defining its mitotic essentiality.","evidence":"PPP6C KO, in vitro Aurora A-TPX2 kinase assay, NDC80-9A phospho-dead rescue, functional genomics, and spindle/nuclear imaging","pmids":["36897279"],"confidence":"High","gaps":["Whether PP6 directly dephosphorylates NDC80 sites not shown in vitro","Spatial control of PP6 at kinetochores not defined"]},{"year":2024,"claim":"PP6 and its three regulatory subunits were shown to redundantly promote RIPK1-dependent PANoptosis through opposing phosphorylation of pro-death vs. pro-survival RIPK1 sites.","evidence":"Cell-death CRISPR screen, PPP6C and PPP6R1/2/3 KO/knockdown, RIPK1 S166/S321 phospho-analysis, and cell death assays","pmids":["38807188"],"confidence":"High","gaps":["Direct vs. indirect control of RIPK1 S166/S321 not separated","Substrate redundancy among the three PPP6R subunits not biochemically mapped"]},{"year":2025,"claim":"Recent work extended PP6 to secretory regulation (Sec16/ERES via a FAM83A/CK1α feedback loop), to host-pathogen metabolism (Pfkfb1 in Salmonella infection), and to a disease-relevant degradation axis (SMURF2/IL-17C in psoriasis).","evidence":"PPP6R3/PPP6C complex with phospho-Sec16 and secretion assays (preprint); Y2H, conditional KO and NO/Arg-1 readouts for Pfkfb1; co-IP, ubiquitination and rescue with psoriasis mouse model for SMURF2","pmids":["bio_10.1101_2025.06.18.660491","41474810","40244332"],"confidence":"Medium","gaps":["Sec16 work remains a preprint awaiting peer review","Direct Pfkfb1 dephosphorylation site not mapped","SMURF2-PPP6C degradation single-lab without reciprocal validation"]},{"year":null,"claim":"How a single catalytic subunit achieves substrate selectivity across mitosis, DNA repair, immunity, trafficking, and cell death—and how regulatory-subunit/holoenzyme composition and competing post-translational regulation dictate which substrate is engaged in a given context—remains the central open question.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model assigning specific SAPS/ANKRD combinations to specific substrates","Quantitative rules for substrate competition under different stimuli unknown","Most substrate dephosphorylation sites identified in cells lack in vitro reconstitution with defined holoenzymes"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,10,20,22,26,27,29]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,5,7,10]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[15,17,18,26]}],"localization":[{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[2,5]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[15]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[7,31]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,23]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[10,18,29]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[5,6]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[11,15,20,21]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[25,26,30]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[17,18,22]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[7,31]}],"complexes":["PP6 heterotrimer (PPP6C-SAPS/PPP6R-ANKRD)","WHIP-TRIM14-PPP6C signalosome","TNF receptor complex I","U1 snRNP/spliceosome"],"partners":["PPP6R1","PPP6R2","PPP6R3","ANKRD28","TRIM14","PLK1","STING1","SMURF2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O00743","full_name":"Serine/threonine-protein phosphatase 6 catalytic subunit","aliases":[],"length_aa":305,"mass_kda":35.1,"function":"Catalytic subunit of protein phosphatase 6 (PP6) (PubMed:17079228, PubMed:29053956, PubMed:32474700). PP6 is a component of a signaling pathway regulating cell cycle progression in response to IL2 receptor stimulation (PubMed:10227379). N-terminal domain restricts G1 to S phase progression in cancer cells, in part through control of cyclin D1 (PubMed:17568194). During mitosis, regulates spindle positioning (PubMed:27335426). Down-regulates MAP3K7 kinase activation of the IL1 signaling pathway by dephosphorylation of MAP3K7 (PubMed:17079228). Also participates in the innate immune defense against viruses by desphosphorylating RIGI, an essential step that triggers RIGI-mediated signaling activation (PubMed:29053956). Also regulates innate immunity by acting as a negative regulator of the cGAS-STING pathway: mediates dephosphorylation and inactivation of CGAS and STING1 (PubMed:32474700, PubMed:32753499). CGAS dephosphorylation at 'Ser-435' impairs its ability to bind GTP, thereby inactivating it (PubMed:32474700)","subcellular_location":"Mitochondrion; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O00743/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PPP6C","classification":"Not Classified","n_dependent_lines":661,"n_total_lines":1208,"dependency_fraction":0.5471854304635762},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ANKRD28","stoichiometry":10.0},{"gene":"ANKRD52","stoichiometry":10.0},{"gene":"MYL12B","stoichiometry":10.0},{"gene":"PPP6R1","stoichiometry":10.0},{"gene":"CCT2","stoichiometry":0.2},{"gene":"CLIP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPP6C","total_profiled":1310},"omim":[{"mim_id":"620862","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 52; ANKRD52","url":"https://www.omim.org/entry/620862"},{"mim_id":"612725","title":"PROTEIN PHOSPHATASE 6, CATALYTIC SUBUNIT; PPP6C","url":"https://www.omim.org/entry/612725"},{"mim_id":"611807","title":"TIP41-LIKE PROTEIN; TIPRL","url":"https://www.omim.org/entry/611807"},{"mim_id":"610879","title":"PROTEIN PHOSPHATASE 6, REGULATORY SUBUNIT 3; PPP6R3","url":"https://www.omim.org/entry/610879"},{"mim_id":"610877","title":"PROTEIN PHOSPHATASE 6, REGULATORY SUBUNIT 2; PPP6R2","url":"https://www.omim.org/entry/610877"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PPP6C"},"hgnc":{"alias_symbol":["PP6"],"prev_symbol":[]},"alphafold":{"accession":"O00743","domains":[{"cath_id":"3.60.21.10","chopping":"41-284","consensus_level":"medium","plddt":97.8315,"start":41,"end":284}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O00743","model_url":"https://alphafold.ebi.ac.uk/files/AF-O00743-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O00743-F1-predicted_aligned_error_v6.png","plddt_mean":95.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPP6C","jax_strain_url":"https://www.jax.org/strain/search?query=PPP6C"},"sequence":{"accession":"O00743","fasta_url":"https://rest.uniprot.org/uniprotkb/O00743.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O00743/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O00743"}},"corpus_meta":[{"pmid":"16895907","id":"PMC_16895907","title":"The alpha4 regulatory subunit exerts 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Monomeric PP6 and PP2A show identical kinetics with pNPP and MBP, but PP6 is inhibited at 5-fold lower concentrations of toxins (okadaic acid, microcystin-LR, calyculin A, cantharidin) with MBP substrate, suggesting PP6 is a preferred in vivo target.\",\n      \"method\": \"In vitro phosphatase assays with recombinant monomeric and heterodimeric PP6 and PP2A; pNPP and 32P-MBP substrates; IC50 measurements with active-site inhibitors; transient expression of alpha4 in cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified recombinant proteins, multiple substrates, multiple inhibitors, and cell-based validation\",\n      \"pmids\": [\"16895907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PP6 catalytic subunit (PP6C) is purified from bovine testes and shows relatively low phosphatase activity toward several substrates compared to PP2A(C) and PP4(C). PP6C does not bind GST-alpha4 in the same pulldown conditions where PP2A(C) binds alpha4.\",\n      \"method\": \"Parallel purification from bovine testes; microcystin-Sepharose affinity resin; GST-alpha4 pulldown; co-immunoprecipitation; in vitro phosphatase activity assays\",\n      \"journal\": \"Protein expression and purification\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro assays and pulldowns in single study, but the negative finding on alpha4 binding to PP6C contrasts with other data\",\n      \"pmids\": [\"12963337\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Fission yeast PP6 ortholog Ppe1 (scSit4/hPP6) and its binding partner Ekc1 (scSAP) negatively regulate kinetochore protein Mis12 localization and are required for faithful chromosome segregation; Gsk3 kinase counteracts Ppe1/PP6 at this step. Mis12 is phosphorylated at ≥2 sites as revealed by mass spectrometry, and Ppe1/Ekc1 co-fractionate with chromatin as non-histone chromatin-associated proteins.\",\n      \"method\": \"Suppressor screen of mis12 mutant in fission yeast; mass spectrometry of Mis12 phosphorylation; chromatin fractionation; genetic epistasis with gsk3; localization recovery assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (suppressor genetics, MS, fractionation, epistasis) in a single focused study on PP6 ortholog\",\n      \"pmids\": [\"12773390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The N-terminal domain of human PP6 (first ~53 residues) restricts G1-to-S phase progression in human prostate cancer cells, suppresses cyclin D1 protein levels in G1, and reduces RB1 phosphorylation at Ser807/811, without significantly altering cyclin/cell-cycle gene transcripts. This function is distinct from the PP6 homolog Sit4 in yeast.\",\n      \"method\": \"Transient expression of GFP fusions of PP6 or PP2A N-termini in PC-3 cells; flow cytometry cell cycle analysis; cyclin D1 western blot; RB1 phosphorylation western blot; gene expression microarray\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal readouts (flow cytometry, protein levels, phosphorylation) in a single lab study\",\n      \"pmids\": [\"17568194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Human PP6 regulatory subunits PP6R2 and PP6R3 (but not PP6R1) physically interact with yeast Sit4 and rescue growth defects, rapamycin hypersensitivity, cyclin G1 expression, and DNA synthesis of a yeast quadruple sap mutant in a Sit4-dependent manner, demonstrating functional conservation of the PP6 regulatory subunit–phosphatase interaction.\",\n      \"method\": \"Heterologous expression of human PP6R proteins in S. cerevisiae sap mutant; growth assays; rapamycin sensitivity; cyclin expression; DNA synthesis; genetic rescue\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic complementation with multiple phenotypic readouts in heterologous system, single lab\",\n      \"pmids\": [\"19621075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PP6c-containing heterotrimeric complexes (produced in bacteria) exhibit phosphatase activity against γ-H2AX (phospho-H2AX Ser139) in vitro, establishing γ-H2AX as a substrate of PP6. Depletion of PP6c or its regulatory subunit PP6R2 leads to persistent γ-H2AX after DNA damage and defective homology-directed repair (HDR); PP6c is recruited to DSB sites as shown by ChIP.\",\n      \"method\": \"In vitro phosphatase assay with bacterially-produced PP6 heterotrimers; siRNA knockdown; γ-H2AX immunofluorescence; HDR reporter assay; chromatin immunoprecipitation\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro phosphatase assay establishing substrate identity, corroborated by ChIP recruitment and cell-based HDR assay with multiple orthogonal methods\",\n      \"pmids\": [\"21451261\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PP6 regulatory subunit R1 (SAPSR1) acts as a bidentate anchor that bridges DNA-PK and PP6c: two distinct regions of R1 (residues 1–326 and 522–700) each bind DNA-PK, with residues 1–326 as the dominant domain; R1 is necessary to recruit PP6c to DNA-PK. Knockdown of R1 or PP6c prevents DNA-PK activation after ionizing radiation and radiosensitizes glioblastoma cells.\",\n      \"method\": \"R1 deletion mutant co-immunoprecipitation mapping; siRNA knockdown; clonogenic radiosensitivity assay; DNA-PK kinase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic deletion mapping with multiple constructs, reciprocal interaction evidence, functional knockdown readout, replicated domain assignment\",\n      \"pmids\": [\"22298787\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Yeast PP6 ortholog Sit4p dephosphorylates COPII coat subunits in vitro and in vivo; sit4Δ mutants accumulate hyperphosphorylated COPII coat subunits and show altered COPII distribution. Mammalian PP6 similarly regulates ER-to-Golgi trafficking, establishing PP6 as a regulator of COPII coat recycling.\",\n      \"method\": \"Phosphatase screen in yeast; in vitro dephosphorylation assay with Sit4p and COPII subunits; COPII localization in sit4Δ cells; mammalian PP6 knockdown ER-to-Golgi trafficking assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of Sit4p dephosphorylating COPII substrates, validated in vivo in yeast and with mammalian PP6 ortholog\",\n      \"pmids\": [\"23864707\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PP6 is stably associated with U1 snRNP and is present as part of the spliceosome throughout the splicing reaction, suggesting a role in pre-mRNA splicing regulation.\",\n      \"method\": \"Co-immunoprecipitation of PP6 with U1 snRNP; spliceosome pull-down; biochemical fractionation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/pulldown, single lab, no functional mutant validation reported in abstract\",\n      \"pmids\": [\"24064353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after ionizing radiation. PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis; phosphorylation of Thr3950 is DNA-PK-dependent. DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis in the apparent absence of Ku and DNA damage.\",\n      \"method\": \"In vitro kinase assay (PLK1 on DNA-PKcs); co-immunoprecipitation; phospho-specific antibodies; PP6 knockdown; mass spectrometry identification of phosphosites\",\n      \"journal\": \"Bioscience reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay combined with co-IP and cell-based phosphosite analysis, single lab\",\n      \"pmids\": [\"24844881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PP6c depletion in mitotic HeLa cells causes phosphorylation changes in 408 phosphopeptides (272 proteins increased) and 298 phosphopeptides (220 proteins decreased). PP6c directly opposes casein kinase 2 (CK2)-dependent phosphorylation of condensin I subunit NCAP-G; PP6c depletion causes defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I.\",\n      \"method\": \"Baculovirus-mediated PP6c depletion in HeLa; quantitative mass spectrometry phosphoproteomics; biochemical phosphatase assay; chromosome condensation/segregation imaging\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — quantitative phosphoproteomics combined with biochemical assay and cellular phenotype with multiple orthogonal methods\",\n      \"pmids\": [\"26462736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"T cell lineage-specific ablation of PP6 in mice causes hyperactivation of multiple distal TCR signaling molecules including MAPKs, AKT, and NF-κB, enhanced thymic selection, preferential expansion of IL-17-producing Vγ6Vδ1+ T cells, and loss of naive T cell homeostasis. PP6 acts as a critical negative regulator of distal TCR signaling.\",\n      \"method\": \"Conditional PP6 knockout mice (T cell-specific); flow cytometry; western blot of downstream signaling molecules; T cell proliferation and differentiation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean conditional KO with defined molecular (signaling pathway) and cellular (selection, homeostasis) phenotypes; multiple orthogonal readouts\",\n      \"pmids\": [\"25609840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LCMT-1 is the major carboxyl methyltransferase for PP6 catalytic subunit (as well as PP2A and PP4) in mouse embryonic fibroblasts. PP6 is carboxyl-methylated on its C-terminal leucine. Unlike PP2A and PP4, loss of methylation (LCMT-1 KO) does not significantly affect PP6 holoenzyme assembly.\",\n      \"method\": \"Antibodies specific to unmethylated phosphatases; LCMT-1 knockout MEFs; blue native PAGE; co-immunoprecipitation; western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods including KO MEFs and BN-PAGE, single lab; null effect on PP6 holoenzyme is negative but informative\",\n      \"pmids\": [\"27507813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Homozygous deletion of the Ppp6c phosphatase domain in mice is embryonic lethal; mutant embryos degenerate by E7.5 with clear developmental defects at E8.5. Homozygous blastocysts exhibit growth failure of the inner cell mass in vitro, and Ppp6c-deficient MEFs show greatly reduced proliferation, establishing that PP6 phosphatase activity is indispensable for post-implantation embryogenesis.\",\n      \"method\": \"Conditional knockout mouse generation; embryo phenotyping; in vitro blastocyst culture; MEF proliferation assay\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse model with defined lethal phenotype and cell-level validation across multiple assays\",\n      \"pmids\": [\"26868000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TORC1 signaling suppresses the Tap42-regulated Sit4 (PP6) phosphatase complex; sit4Δ rescues histone acetylation under TORC1-repressive conditions. TORC1 inhibition activates PP6/Sit4, causing nuclear accumulation of sirtuin deacetylase Hst4 (reduced protein turnover), decreased histone H3/H4 acetylation. PP6 thus couples nutrient/TOR signaling to epigenetic regulation via sirtuin nuclear localization.\",\n      \"method\": \"Genetic epistasis (sit4Δ, hst3Δ, hst4Δ, tco89Δ); rapamycin treatment; histone acetylation western blot; Hst4-GFP nuclear localization imaging; protein stability assay\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with multiple mutant combinations and multiple readouts; yeast ortholog study\",\n      \"pmids\": [\"27343235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRIM14 provides a mitochondrial docking platform for assembly of the WHIP–TRIM14–PPP6C signalosome required for RIG-I-mediated antiviral signaling. PPP6C dephosphorylates RIG-I within this complex, promoting its activation; WHIP bridges RIG-I with MAVS via polyubiquitin chains at RIG-I K164.\",\n      \"method\": \"Pooled RNAi screen; yeast two-hybrid; co-immunoprecipitation; RIG-I dephosphorylation assay; antiviral reporter assays; viral infection models\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (RNAi screen, Y2H, co-IP, dephosphorylation assay) identifying both complex composition and enzymatic activity\",\n      \"pmids\": [\"29053956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In Drosophila, the PP6 regulatory subunit Fmt (fiery mountain) negatively regulates JNK signaling upstream of dTAK1; loss of Fmt or PpV (PP6 catalytic subunit) cooperates with oncogenic RasV12 to promote JNK-dependent tumor growth and invasion. Fmt and PpV act synergistically to suppress JNK-dependent tumorigenesis.\",\n      \"method\": \"Genetic screen in Drosophila; genetic epistasis (fmt, ppv, rasV12, dTAK1 alleles); tumor growth and invasion assays; reporter assays for JNK activity\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic genetic epistasis placing PP6 upstream of dTAK1 in JNK pathway, multiple allele combinations, functional tumor phenotypes\",\n      \"pmids\": [\"28658615\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PP6 directly interacts with and inactivates ASK3 kinase in an osmolality-dependent manner under hyperosmotic stress; PP6-ASK3 interaction promotes regulatory volume increase (RVI) and cell survival. A genome-wide siRNA screen identified PP6 as a direct ASK3 inactivator in the bidirectional osmotic stress response.\",\n      \"method\": \"Genome-wide siRNA screen; co-immunoprecipitation; kinase activity assay; cell volume measurement; cell viability assay; osmotic stress treatments\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide screen plus co-IP plus kinase activity assay plus functional readout (cell volume/survival), multiple orthogonal methods\",\n      \"pmids\": [\"29539411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Plk1 inhibits the phosphatase PP6 toward Aurora A (Aurora kinase A), generating a Plk1–PP6 feedback loop that coordinates Plk1 and Aurora A activities during mitotic entry. PP6 interaction with Plk1 is phosphorylation-dependent and is terminated by Plk1 degradation during mitotic exit.\",\n      \"method\": \"Quantitative proteomics on HeLa cells with kinase inhibitors or Plk1 phospho-binding mutant; Aurora A activity assays; PP6 activity assays; co-immunoprecipitation\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative proteomics plus functional activity assays, multiple conditions establishing mechanistic feedback loop\",\n      \"pmids\": [\"29764989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CK2 phosphorylates SAPS3 (a PP6 regulatory subunit) on multiple acidic motifs; CK2-phosphorylated SAPS3-PP6 complex shows significantly increased phosphatase activity toward pT288-Aurora A kinase substrate. Nine Ala substitutions in SAPS3 CK2 sites block this activation. CK2 inhibitors increase Aurora A phosphorylation in cells, consistent with reduced PP6 activity. SAPS3 knockdown/KO results in hyperactivated Aurora A and abnormal nuclei.\",\n      \"method\": \"In vitro CK2 kinase assay on SAPS3; PP6 phosphatase activity assay with pT288-AurA substrate; mutagenesis of CK2 sites in SAPS3; CK2 inhibitor cell treatment; CRISPR/siRNA depletion of SAPS3; nuclear morphology imaging\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay, in vitro phosphatase assay, mutagenesis, and cell-based validation; multiple orthogonal methods\",\n      \"pmids\": [\"31904830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PPP6C is constitutively associated with cGAS in unstimulated cells. DNA virus infection causes rapid dissociation of PPP6C from cGAS, resulting in phosphorylation of human cGAS S435 (mouse cGAS S420) in the catalytic pocket. S420-phosphorylated mcGAS has higher affinity for GTP and higher enzymatic activity. PPP6C dephosphorylation of this site keeps cGAS inactive in the absence of infection to prevent autoimmune response.\",\n      \"method\": \"Co-immunoprecipitation; phospho-specific antibody; site-directed mutagenesis of cGAS S435/S420; in vitro cGAMP synthesis assay; PPP6C knockout cell lines; viral infection experiments\",\n      \"journal\": \"Protein & cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical substrate identification (co-IP, mutagenesis), in vitro enzymatic assay validating phospho-site function, KO cell validation\",\n      \"pmids\": [\"32474700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PPP6C interacts with STING and negatively regulates the cGAS-STING pathway by dephosphorylating STING. Loss of PPP6C enhances STING phosphorylation, increases IRF3 activation (but not NF-κB activation) in response to dsDNA, restricts HSV-1 and VSV replication, and inhibits KSHV reactivation through increased type I interferon production.\",\n      \"method\": \"Co-immunoprecipitation of PPP6C with STING; PPP6C siRNA depletion; phospho-STING western blot; IRF3/NF-κB reporter assays; viral replication assays\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus phosphorylation analysis plus functional viral assays, single lab\",\n      \"pmids\": [\"32753499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PPP6C is a major MEK phosphatase in cells with oncogenic ERK pathway activation; PPP6C is recruited to MEK through its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting ERK pathway signaling and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation.\",\n      \"method\": \"PPP6C knockdown/knockout; phospho-MEK western blot; co-immunoprecipitation with regulatory subunits; MEK inhibitor sensitivity assays; expression of melanoma-associated PPP6C mutants\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic depletion, co-IP, phospho-site analysis, mutant expression, and drug sensitivity readouts; multiple orthogonal methods establishing direct MEK dephosphorylation\",\n      \"pmids\": [\"33789117\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PP6 (Pp6) deficiency in Treg cells increases CpG methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhances Akt phosphorylation at Ser473/Thr308, leading to impaired FoxP3 expression and Treg cell instability. Conditional Pp6 KO in Treg cells causes spontaneous autoinflammation.\",\n      \"method\": \"Conditional PP6 KO mice (Treg-specific); bisulfite sequencing of FoxP3 locus; Dnmt1 dephosphorylation assay; Akt phosphorylation western blot; flow cytometry; autoimmune model experiments\",\n      \"journal\": \"Genes & diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional KO with mechanistic biochemical readouts (Dnmt1 phosphorylation, Akt phosphorylation), single lab\",\n      \"pmids\": [\"35224167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of PP6 C-terminal methylation does not affect PP6 holoenzyme assembly (interaction with ANKRD and SAPS regulatory subunits), in contrast to PP2A and PP4 where methylation is critical for regulatory subunit binding.\",\n      \"method\": \"Mass spectrometry-based proteomics; methylation-ablating mutations; genome editing (CRISPR); BN-PAGE; quantitative interaction proteomics\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (MS proteomics, CRISPR, BN-PAGE) establishing negative regulatory role of methylation for PP6 specifically\",\n      \"pmids\": [\"34845248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PP6 is an identified component of TNF receptor complex I. PP6 loss protects cells from TNFα-mediated cell death in a phosphatase-activity-dependent manner. PP6 modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIPL, promoting RIPK1 activation and c-FLIPL degradation. Melanoma-associated PP6 inactivating mutations confer resistance to TNFα-mediated cell death.\",\n      \"method\": \"Co-immunoprecipitation identifying PP6 in complex I; PP6 KO/knockdown; cell death assays; RIPK1 and c-FLIPL ubiquitination assays; expression of melanoma PP6 mutants\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of complex component, KO functional assay, ubiquitination analysis; single lab\",\n      \"pmids\": [\"36071040\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PPP6C acts as a TAK1 phosphatase to inactivate its kinase activity. Deletion of PPP6C leads to hyperactivation of TAK1 and reduced RIPK1 kinase activity upon TNF stimulation, protecting cells from TNF-induced necroptosis. This was identified in a genome-wide CRISPR/Cas9 screen and validated mechanistically.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen; PPP6C KO; TAK1 kinase activity assay; RIPK1 phosphorylation analysis; necroptosis/cell death assays; heterozygous Ppp6c mouse gut model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR screen discovery, KO validation, kinase activity assay for TAK1 as substrate, in vivo mouse model corroboration\",\n      \"pmids\": [\"35842423\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AMPKγ subunit, when bound to AMP under energy starvation, sequesters PPP6C to block dephosphorylation of eukaryotic translation elongation factor 2 (eEF2), thereby inhibiting translation elongation and preserving energy. PPP6C is identified as an AMPKγ-regulated phosphatase of eEF2; this regulation is independent of AMPKα catalytic activity.\",\n      \"method\": \"AMPKγ and AMPKα knockout; co-immunoprecipitation of AMPKγ with PPP6C; eEF2 phosphorylation analysis; phosphoproteomics under energy starvation; cell survival assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO of both AMPK subunits, co-IP establishing direct interaction, phosphoproteomics, and functional translation assay; multiple orthogonal methods\",\n      \"pmids\": [\"36384136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Rab40c (SOCS box protein/Cullin5 E3 ligase) binds the PP6 complex and ubiquitylates ANKRD28 (a PP6 regulatory subunit), leading to its lysosomal degradation and decreased PP6 activity. PP6 activity loss via this mechanism decreases FAK and MOB1 phosphorylation, affecting focal adhesion dynamics in migrating cells.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitylation assay; Rab40c KO cells; focal adhesion quantification; phospho-FAK and phospho-MOB1 western blot; lysosome inhibitor experiments\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitylation assay, and KO phenotype analysis; single lab with multiple readouts\",\n      \"pmids\": [\"35512830\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PP6 (PPP6C) regulates Aurora A-TPX2-mediated phosphorylation of multiple N-terminal sites on NDC80 at checkpoint-silenced, microtubule-attached kinetochores during spindle formation. NDC80 phosphorylation by Aurora A-TPX2 is Aurora B-independent and persists until spindle disassembly in telophase; it is increased in PPP6C KO cells. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C KO cells. Synthetic lethality between PPP6C and NDC80 was identified by functional genomics.\",\n      \"method\": \"PPP6C knockout; phospho-specific antibodies; functional genomics screen; Aurora A-TPX2 in vitro kinase assay; NDC80-9A phospho-dead mutant rescue; spindle size measurement; nuclear structure imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO combined with in vitro kinase assay, phospho-dead mutant rescue, functional genomics screen; multiple orthogonal methods, single focused study\",\n      \"pmids\": [\"36897279\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PPP6C and its regulatory subunits PPP6R1, PPP6R2, and PPP6R3 (with redundant roles) promote TAK1 inhibitor-induced, RIPK1-dependent PANoptosis. PPP6C promotes pro-death S166 auto-phosphorylation of RIPK1 and leads to reduction in pro-survival S321 phosphorylation of RIPK1. Loss of PPP6C significantly reduces PANoptosis.\",\n      \"method\": \"Cell death-based CRISPR screen; PPP6C and PPP6R1/2/3 KO/knockdown; RIPK1 phosphorylation (S166, S321) western blot; cell death assays; TAK1 inhibitor treatment\",\n      \"journal\": \"BMC biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR screen validated by KO, multiple regulatory subunit depletion, and specific phospho-site analysis of RIPK1; multiple orthogonal methods\",\n      \"pmids\": [\"38807188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PPP6C (with regulatory subunit PPP6R3) dephosphorylates Sec16 at ER exit sites (ERES), counteracting FAM83A/CK1α-mediated phosphorylation of Sec16. Excessive dephosphorylation of Sec16 impairs secretion. A phosphorylation-coupled autoregulatory feedback loop involving FAM83A/CK1α and PP6 maintains balanced Sec16 phosphorylation state essential for proper ERES function and secretory activity.\",\n      \"method\": \"Phosphatase complex identification; phospho-Sec16 analysis; FAM83A/CK1α inhibitor/overexpression; ERES imaging; secretory assay; phospho-mutant rescue\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — preprint with defined complex (PPP6R3/PPP6C), phospho-Sec16 readout, and functional secretion assay; not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.06.18.660491\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PP6 promotes intracellular Salmonella proliferation in macrophages by dephosphorylating Pfkfb1 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1); Pp6 deficiency elevates Pfkfb1 expression, which restricts bacterial growth by promoting NO production while suppressing Arg-1 expression and arginine metabolism. Yeast two-hybrid identified Pfkfb1 as a PP6 substrate.\",\n      \"method\": \"Fluorescence-dilution reporter in Salmonella-infected macrophages; conditional PP6 KO; yeast two-hybrid screening; Pfkfb1 KO; NO production assay; Arg-1 expression analysis\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid substrate identification, KO validation, and functional metabolic readouts; single lab\",\n      \"pmids\": [\"41474810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SMURF2 E3 ubiquitin ligase interacts with PPP6C and promotes its ubiquitination and proteasomal/lysosomal degradation in keratinocytes, downstream of IL-17C signaling, leading to psoriasis-like changes. IL-17C upregulates SMURF2, which targets PPP6C for degradation.\",\n      \"method\": \"Co-immunoprecipitation (SMURF2–PPP6C); ubiquitination assay; SMURF2 knockdown/overexpression; PPP6C overexpression rescue; lentiviral/plasmid transfection; imiquimod mouse psoriasis model\",\n      \"journal\": \"Cell biology international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, and rescue experiment; single lab with multiple methods\",\n      \"pmids\": [\"40244332\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPP6C (PP6) is a heterotrimer serine/threonine phosphatase—composed of a catalytic subunit (PP6c/PPP6C), one of three SAPS/PPP6R regulatory subunits, and one of three ANKRD subunits—that dephosphorylates a defined set of substrates (including γ-H2AX, Aurora A, MEK, RIG-I, cGAS, STING, TAK1, RIPK1, DNA-PKcs, NCAP-G condensin I subunit, NDC80, eEF2, Dnmt1, COPII coat subunits, Sec16, Pfkfb1, and Mis12/kinetochore proteins) to regulate mitotic fidelity, DNA damage repair, innate immune signaling, cell cycle progression, T cell homeostasis, translation elongation, ER-to-Golgi trafficking, and cell death pathways; its activity is tuned by alpha4-mediated allosteric inhibition, CK2-dependent phosphorylation of SAPS3 that enhances Aurora A dephosphorylation, Plk1-dependent inhibition, TORC1 suppression, AMPKγ-dependent sequestration, and its own regulated ubiquitylation of ANKRD28 via Rab40c/CRL5 or SMURF2.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP6C is the catalytic subunit of the PP6 serine/threonine protein phosphatase, a heterotrimeric enzyme that assembles with SAPS/PPP6R and ANKRD regulatory subunits and dephosphorylates a defined substrate set to govern mitotic fidelity, the DNA damage response, innate immune signaling, and cell-death decisions [#5, #10, #29]. In mitosis and the cell cycle, PP6 reverses kinase-driven phosphorylation events: it dephosphorylates the condensin I subunit NCAP-G to oppose CK2 and ensure proper chromosome condensation and segregation [#10], controls Aurora A activity (a regulation tuned by a Plk1\\u2013PP6 feedback loop and by CK2 phosphorylation of SAPS3) [#18, #19], and restrains Aurora A\\u2013TPX2-mediated phosphorylation of the kinetochore protein NDC80 during spindle formation [#29]. In the DNA damage response, PP6 dephosphorylates \\u03b3-H2AX to enable homology-directed repair and acts through the regulatory subunit R1 as a bidentate anchor that recruits the catalytic subunit to DNA-PK, supporting DNA-PK activation after ionizing radiation [#5, #6]. PP6 is a broad negative regulator of immune and stress signaling, dephosphorylating cGAS and STING to restrain the cGAS\\u2013STING\\u2013IRF3 axis, activating RIG-I within a mitochondrial WHIP\\u2013TRIM14\\u2013PPP6C signalosome, inactivating TAK1 and ASK3 kinases, and shaping TCR signaling and Treg stability via Dnmt1 and Akt [#20, #21, #15, #26, #17, #11, #23]. It is a major MEK phosphatase whose recurrent melanoma-associated mutations cause MEK hyperphosphorylation and altered drug sensitivity [#22], and it controls programmed cell death by modulating RIPK1 phosphorylation and ubiquitination within TNF receptor complex I and during PANoptosis [#25, #30]. PP6 additionally regulates ER-to-Golgi trafficking through dephosphorylation of COPII coat subunits and Sec16, and translation elongation through eEF2 [#7, #31, #27]. PP6 phosphatase activity is essential for post-implantation mouse embryogenesis [#13], and its activity is set by alpha4-mediated allosteric inhibition and by regulated degradation of its ANKRD28 subunit [#0, #28].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing PP6 as a distinct phosphatase required separating its catalytic and regulatory behavior from the related PP2A/PP4 enzymes and demonstrating conservation of the regulatory-subunit interaction.\",\n      \"evidence\": \"Purification of PP6C from bovine testes with activity assays and GST-alpha4 pulldowns, plus heterologous rescue of yeast sap mutants by human PP6R subunits\",\n      \"pmids\": [\"12963337\", \"19621075\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conflicting alpha4-binding result for PP6C left the regulatory relationship unresolved\", \"Did not define mammalian substrates\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Genetic work in fission yeast first linked the PP6 ortholog to chromosome segregation, placing the phosphatase opposite a kinase at the kinetochore.\",\n      \"evidence\": \"Suppressor screen of mis12, mass spectrometry of Mis12 phosphorylation, chromatin fractionation and gsk3 epistasis in S. pombe\",\n      \"pmids\": [\"12773390\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct dephosphorylation of Mis12 by PP6 not shown in vitro\", \"Mammalian kinetochore relevance not established here\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"The question of how PP6 activity is tuned was addressed by showing alpha4 acts oppositely on PP6 versus PP2A, defining an allosteric brake specific to PP6.\",\n      \"evidence\": \"In vitro phosphatase assays with monomeric and alpha4-heterodimeric recombinant PP6/PP2A, inhibitor IC50 measurements, and cell expression\",\n      \"pmids\": [\"16895907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of differential alpha4 effect not resolved\", \"Physiological substrates governed by alpha4 regulation not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"PP6 gained its first mammalian molecular substrate when \\u03b3-H2AX dephosphorylation was reconstituted, linking the phosphatase to DNA double-strand break repair.\",\n      \"evidence\": \"In vitro phosphatase assay with bacterially produced PP6 heterotrimers, siRNA, \\u03b3-H2AX immunofluorescence, HDR reporter, and ChIP recruitment\",\n      \"pmids\": [\"21451261\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which regulatory subunit directs \\u03b3-H2AX targeting in vivo not fully defined\", \"Recruitment mechanism to DSBs incomplete\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"The mechanism of PP6 substrate targeting was clarified by showing the R1 regulatory subunit physically bridges the catalytic subunit to DNA-PK.\",\n      \"evidence\": \"Deletion-mapping co-IP of R1 with DNA-PK, siRNA knockdown, clonogenic radiosensitivity, and DNA-PK kinase assays\",\n      \"pmids\": [\"22298787\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether DNA-PKcs is a direct PP6 substrate here vs. an activator not fully separated\", \"Structure of the R1-bridged complex unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"PP6 was extended beyond the nucleus to membrane trafficking by showing it dephosphorylates COPII coat subunits to regulate ER-to-Golgi transport.\",\n      \"evidence\": \"Yeast phosphatase screen, in vitro dephosphorylation of COPII by Sit4p, COPII localization in sit4\\u0394, and mammalian PP6 trafficking assay\",\n      \"pmids\": [\"23864707\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific COPII phosphosites in mammals not mapped\", \"Regulatory subunit directing COPII targeting not identified here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"PP6 was implicated in mitotic DNA-PKcs regulation, integrating it with PLK1 signaling at Ser3205.\",\n      \"evidence\": \"In vitro PLK1 kinase assay on DNA-PKcs, co-IP, phospho-specific antibodies, PP6 knockdown, and MS phosphosite mapping\",\n      \"pmids\": [\"24844881\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct in vitro PP6 dephosphorylation of Ser3205 not reconstituted\", \"Functional consequence of mitotic DNA-PKcs phosphorylation incomplete\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"System-wide phosphoproteomics defined the breadth of PP6 mitotic substrates and pinpointed condensin I (NCAP-G) as a CK2-opposed target controlling chromosome architecture.\",\n      \"evidence\": \"PP6c depletion with quantitative phosphoproteomics, biochemical phosphatase assay, and chromosome condensation/segregation imaging in HeLa\",\n      \"pmids\": [\"26462736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Most of the hundreds of altered phosphosites not validated as direct substrates\", \"Subunit-substrate assignment for NCAP-G not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Conditional knockout established PP6 as a physiological negative regulator of distal TCR signaling and T cell homeostasis.\",\n      \"evidence\": \"T cell-specific PP6 KO mice with flow cytometry, signaling western blots, and differentiation assays\",\n      \"pmids\": [\"25609840\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PP6 substrates within the TCR cascade not identified\", \"Which regulatory subunits operate in T cells unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Genetic and biochemical studies defined PP6 regulation by methylation and TOR signaling and demonstrated that its phosphatase activity is indispensable for development.\",\n      \"evidence\": \"LCMT-1 KO MEFs and BN-PAGE, yeast TORC1/Sit4 epistasis, and an embryonic-lethal Ppp6c phosphatase-domain deletion mouse\",\n      \"pmids\": [\"27507813\", \"27343235\", \"26868000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of PP6 C-terminal methylation remains unexplained given no assembly effect\", \"Mammalian TOR-PP6 coupling not directly demonstrated\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"PP6 was placed in innate immune and stress kinase pathways as an activator of RIG-I and a suppressor of TAK1/JNK signaling.\",\n      \"evidence\": \"RNAi screen, Y2H, co-IP and RIG-I dephosphorylation for the WHIP-TRIM14-PPP6C signalosome; Drosophila genetic epistasis placing PP6 upstream of dTAK1 in JNK signaling\",\n      \"pmids\": [\"29053956\", \"28658615\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RIG-I phosphosites dephosphorylated by PP6 not fully mapped\", \"Mammalian TAK1/JNK substrate validation deferred to later work\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"PP6 was shown to be embedded in mitotic kinase feedback loops, being inhibited by Plk1 to coordinate Aurora A activity, and to inactivate the stress kinase ASK3 under osmotic stress.\",\n      \"evidence\": \"Quantitative proteomics with kinase inhibitors and Plk1 phospho-binding mutants, Aurora A and PP6 activity assays; genome-wide siRNA screen with co-IP, kinase assay, and cell-volume readouts for ASK3\",\n      \"pmids\": [\"29764989\", \"29539411\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of Plk1-dependent PP6 inhibition at the molecular level incomplete\", \"ASK3 dephosphorylation site not specified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Regulatory-subunit phosphorylation and substrate engagement were mechanistically detailed: CK2 phosphorylation of SAPS3 enhances Aurora A dephosphorylation, and PPP6C constitutively restrains cGAS-STING signaling.\",\n      \"evidence\": \"In vitro CK2 and PP6 activity assays with SAPS3 mutagenesis; co-IP, cGAS/STING phospho-site mutagenesis, in vitro cGAMP synthesis, and PPP6C KO with viral infection\",\n      \"pmids\": [\"31904830\", \"32474700\", \"32753499\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination between cGAS and STING dephosphorylation by PP6 not integrated\", \"Selectivity for IRF3 over NF-\\u03baB branch unexplained mechanistically\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"PP6 was established as a major MEK phosphatase relevant to oncogenic signaling and as a regulator of Treg stability, connecting recurrent melanoma mutations to MEK hyperactivation.\",\n      \"evidence\": \"PPP6C KO/knockdown with phospho-MEK analysis, regulatory-subunit co-IP, MEK-inhibitor sensitivity, melanoma mutant expression; Treg-specific KO with FoxP3 bisulfite sequencing and Dnmt1/Akt phospho-analysis\",\n      \"pmids\": [\"33789117\", \"35224167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which regulatory subunit recruits PP6 to MEK not pinned down\", \"Direct vs. indirect Dnmt1 dephosphorylation in Tregs needs reconstitution\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"PP6 was integrated into cell-death control by acting on TAK1 and RIPK1 within TNF receptor signaling, and additional layers of its own regulation (AMPK\\u03b3 sequestration, Rab40c/CRL5-mediated ANKRD28 degradation) were defined.\",\n      \"evidence\": \"CRISPR screens with KO, TAK1 kinase and RIPK1 ubiquitination/phospho assays and gut mouse model; AMPK subunit KO with co-IP and eEF2 phosphoproteomics; Rab40c KO with ubiquitylation and focal-adhesion readouts\",\n      \"pmids\": [\"35842423\", \"36071040\", \"36384136\", \"35512830\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PP6 dephosphorylation sites on TAK1 and RIPK1 not all mapped\", \"How energy state switches PP6 between substrates unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"PP6 was shown to restrain Aurora A-TPX2-driven NDC80 phosphorylation at attached kinetochores, with synthetic lethality to NDC80 defining its mitotic essentiality.\",\n      \"evidence\": \"PPP6C KO, in vitro Aurora A-TPX2 kinase assay, NDC80-9A phospho-dead rescue, functional genomics, and spindle/nuclear imaging\",\n      \"pmids\": [\"36897279\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PP6 directly dephosphorylates NDC80 sites not shown in vitro\", \"Spatial control of PP6 at kinetochores not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"PP6 and its three regulatory subunits were shown to redundantly promote RIPK1-dependent PANoptosis through opposing phosphorylation of pro-death vs. pro-survival RIPK1 sites.\",\n      \"evidence\": \"Cell-death CRISPR screen, PPP6C and PPP6R1/2/3 KO/knockdown, RIPK1 S166/S321 phospho-analysis, and cell death assays\",\n      \"pmids\": [\"38807188\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs. indirect control of RIPK1 S166/S321 not separated\", \"Substrate redundancy among the three PPP6R subunits not biochemically mapped\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Recent work extended PP6 to secretory regulation (Sec16/ERES via a FAM83A/CK1\\u03b1 feedback loop), to host-pathogen metabolism (Pfkfb1 in Salmonella infection), and to a disease-relevant degradation axis (SMURF2/IL-17C in psoriasis).\",\n      \"evidence\": \"PPP6R3/PPP6C complex with phospho-Sec16 and secretion assays (preprint); Y2H, conditional KO and NO/Arg-1 readouts for Pfkfb1; co-IP, ubiquitination and rescue with psoriasis mouse model for SMURF2\",\n      \"pmids\": [\"bio_10.1101_2025.06.18.660491\", \"41474810\", \"40244332\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Sec16 work remains a preprint awaiting peer review\", \"Direct Pfkfb1 dephosphorylation site not mapped\", \"SMURF2-PPP6C degradation single-lab without reciprocal validation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single catalytic subunit achieves substrate selectivity across mitosis, DNA repair, immunity, trafficking, and cell death\\u2014and how regulatory-subunit/holoenzyme composition and competing post-translational regulation dictate which substrate is engaged in a given context\\u2014remains the central open question.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model assigning specific SAPS/ANKRD combinations to specific substrates\", \"Quantitative rules for substrate competition under different stimuli unknown\", \"Most substrate dephosphorylation sites identified in cells lack in vitro reconstitution with defined holoenzymes\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 10, 20, 22, 26, 27, 29]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 5, 7, 10]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [15, 17, 18, 26]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [2, 5]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [7, 31]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 23]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [10, 18, 29]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [11, 15, 20, 21]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [25, 26, 30]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [17, 18, 22]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [7, 31]}\n    ],\n    \"complexes\": [\"PP6 heterotrimer (PPP6C-SAPS/PPP6R-ANKRD)\", \"WHIP-TRIM14-PPP6C signalosome\", \"TNF receptor complex I\", \"U1 snRNP/spliceosome\"],\n    \"partners\": [\"PPP6R1\", \"PPP6R2\", \"PPP6R3\", \"ANKRD28\", \"TRIM14\", \"PLK1\", \"STING1\", \"SMURF2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}