{"gene":"PPP4R1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2014,"finding":"PP4R1 interacts with TRAF2 and TRAF6 in a RING finger domain-dependent manner, mediates dephosphorylation of TRAF2 Ser11, and inhibits TRAF6 polyubiquitination, thereby inhibiting NF-κB activation downstream of TRAF2, TRAF6, TNF, and EBV oncoprotein LMP1.","method":"Yeast two-hybrid screen, co-immunoprecipitation, RNA interference knockdown, NF-κB reporter assay, western blot for phospho-TRAF2 Ser11, ubiquitination assay","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction identified by Y2H and confirmed functionally by RNAi depletion and multiple signaling readouts in a single lab with orthogonal methods","pmids":["25134449"],"is_preprint":false},{"year":2017,"finding":"MCPyV small T antigen (tAg) forms a complex with PP4R1 and PP4c; this complex bridges tAg to the NEMO adaptor protein, enabling dephosphorylation-dependent deactivation of the NF-κB pathway. siRNA depletion of PP4R1 or tAg mutations that disrupt this interaction abolish tAg-mediated NF-κB inhibition and pro-inflammatory cytokine suppression.","method":"Co-immunoprecipitation, siRNA knockdown, NF-κB reporter assay, mutational analysis of tAg, cytokine production measurement","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, and RNAi with multiple functional readouts in a single study","pmids":["28445980"],"is_preprint":false},{"year":2017,"finding":"miR-338-3p directly targets the 3′UTR of PP4R1 mRNA; downregulation of miR-338-3p in TNF-α-treated hepatocytes increases PP4R1 and PP4 expression, impairing AKT/GSK3β signaling and glycogen synthesis, thereby contributing to hepatic insulin resistance.","method":"Luciferase reporter assay (direct target validation), western blot, miRNA mimic/inhibitor transfection, ChIP assay (HNF-4α transcriptional regulation of miR-338-3p), mouse models (db/db, HFD)","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase assay validates direct targeting, multiple in vivo and in vitro readouts, single lab","pmids":["28467989"],"is_preprint":false},{"year":2018,"finding":"PP4R1 mediates TNF-α-induced gluconeogenesis in hepatocytes; knockdown of PP4R1 attenuates the increase in glucose production caused by miR-338-3p inhibition, placing PP4R1 downstream of miR-338-3p in the regulation of FOXO1 phosphorylation and gluconeogenic gene expression (PGC-1α, PEPCK, G6Pase).","method":"Western blot, qPCR, pyruvate tolerance testing in mice, miR-338-3p inhibitor and PP4R1 siRNA in HEPA1-6 cells (genetic epistasis)","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis established by double knockdown, multiple metabolic readouts, single lab","pmids":["30132533"],"is_preprint":false},{"year":2024,"finding":"AMBRA1 competitively interacts with PP4R1 via its N-terminal F1 domain, disrupting the PP4R1/PP4c complex and antagonizing PP4R1/PP4c-mediated dephosphorylation of the IKK complex, thereby promoting sustained IKK phosphorylation and NF-κB-driven intestinal inflammation. In response to TNF-α, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 by reducing CUL4A-mediated K48-linked ubiquitination.","method":"Co-immunoprecipitation (reciprocal), domain-mapping experiments (N-term F1 deletion mutants), siRNA/knockout in colitis mouse model, phosphorylation site mutagenesis (AMBRA1 S1043), ubiquitination assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, domain mapping, mutagenesis, in vivo colitis model, multiple orthogonal methods in one study","pmids":["38424148"],"is_preprint":false},{"year":2020,"finding":"PP4R1 interacts with HMGA2 and promotes epithelial-mesenchymal transition in non-small-cell lung cancer via activation of the MAPK/ERK signaling pathway.","method":"Co-immunoprecipitation, wound-healing and Transwell invasion assays, western blot for EMT markers and ERK phosphorylation, PP4R1 overexpression/knockdown","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP identifying HMGA2 interaction, functional readouts present but limited mechanistic depth, single lab","pmids":["32077156"],"is_preprint":false},{"year":2024,"finding":"PP4R1 directly interacts with PKM2 and strengthens the interaction between ERK1/2 and PKM2, promoting ERK1/2-mediated PKM2 nuclear translocation and thereby enhancing glycolysis in gallbladder cancer cells.","method":"Co-immunoprecipitation, proximity ligation assay or pulldown (direct interaction), subcellular fractionation/nuclear translocation imaging, ERK inhibitor treatment, in vitro and in vivo tumor growth assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction shown by Co-IP, nuclear translocation measured, mechanistic link to glycolysis with in vivo validation, single lab","pmids":["38301910"],"is_preprint":false},{"year":2015,"finding":"Knockdown of PP4R1 in HepG2 hepatocellular carcinoma cells arrests cells at G2/M phase and inactivates p38 and JNK MAPK signaling cascades, indicating PP4R1 promotes cell proliferation through these kinase pathways. NOTE: The original article (PMID 26300649) was subsequently retracted (PMID 37396311).","method":"Lentiviral shRNA knockdown, flow cytometry (cell cycle), western blot for p38 and JNK activation, colony formation assay","journal":"OncoTargets and therapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single-method knockdown with signaling readouts; article was retracted, severely undermining reliability","pmids":["26300649","37396311"],"is_preprint":false},{"year":2023,"finding":"PP4R1 overexpression upregulates HSPA6 (an HSP70 family member) and activates endoplasmic reticulum stress, promoting proliferation, migration, and invasion of NSCLC cells; HSPA6 overexpression alone recapitulates the PP4R1 phenotype, placing HSPA6 downstream of PP4R1.","method":"RNA-seq after PP4R1 overexpression, HSPA6 overexpression/knockdown, ER stress marker western blot, in vitro proliferation/migration/invasion assays, in vivo mouse tumor and metastasis model","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transcriptomic profiling plus epistasis via HSPA6 rescue, in vivo validation, single lab with multiple orthogonal methods","pmids":["37739270"],"is_preprint":false}],"current_model":"PPP4R1 (PP4R1) is the regulatory subunit 1 of protein phosphatase 4 (PP4c) that directs the PP4 holoenzyme to specific substrates: it mediates dephosphorylation of TRAF2 Ser11 and antagonizes TRAF6 ubiquitin ligase activity to suppress NF-κB activation, forms a PP4R1/PP4c complex that dephosphorylates the IKK complex (counteracted by AMBRA1 competition for PP4R1 binding), can be co-opted by viral small T antigens via a NEMO-bridging mechanism to inhibit antiviral NF-κB responses, and in metabolic contexts is targeted by miR-338-3p to regulate hepatic insulin signaling, glycogen synthesis, and gluconeogenesis; additionally, PP4R1 interacts with HMGA2 to activate MAPK/ERK-driven EMT in lung cancer and promotes PKM2 nuclear translocation to enhance glycolysis in gallbladder cancer."},"narrative":{"mechanistic_narrative":"PPP4R1 (PP4R1) is the regulatory subunit of the protein phosphatase 4 (PP4c) holoenzyme that targets the phosphatase to substrates within the NF-κB signaling axis and acts as a negative regulator of inflammatory and immune responses [PMID:25134449, PMID:38424148]. It engages TRAF2 and TRAF6 in a RING finger domain-dependent manner, dephosphorylating TRAF2 at Ser11 and suppressing TRAF6 polyubiquitination to dampen NF-κB activation downstream of TNF and the EBV oncoprotein LMP1 [PMID:25134449]. As part of a PP4R1/PP4c complex, it dephosphorylates the IKK complex; this activity is antagonized by AMBRA1, which competitively binds PP4R1 through its N-terminal F1 domain to disrupt the complex and sustain IKK phosphorylation and NF-κB-driven intestinal inflammation [PMID:38424148]. This regulatory module is co-opted by Merkel cell polyomavirus small T antigen, which forms a complex with PP4R1/PP4c that bridges the viral protein to NEMO and deactivates NF-κB to suppress pro-inflammatory cytokine responses [PMID:28445980]. Beyond immune control, PP4R1 functions in hepatic metabolism as a target of miR-338-3p, where its derepression impairs AKT/GSK3β signaling and drives FOXO1-dependent gluconeogenesis and insulin resistance [PMID:28467989, PMID:30132533], and it promotes tumor progression by interacting with HMGA2 to activate MAPK/ERK-driven EMT in lung cancer [PMID:32077156], by binding PKM2 to enhance ERK1/2-mediated PKM2 nuclear translocation and glycolysis in gallbladder cancer [PMID:38301910], and by upregulating HSPA6 to activate ER stress in NSCLC [PMID:37739270].","teleology":[{"year":2014,"claim":"Established PP4R1 as a negative regulator of NF-κB by defining its direct substrates within TRAF signaling, answering how PP4 is targeted to this pathway.","evidence":"Yeast two-hybrid, reciprocal Co-IP, RNAi, NF-κB reporter, phospho-TRAF2 Ser11 and ubiquitination assays","pmids":["25134449"],"confidence":"High","gaps":["Whether PP4c catalytic activity is required for the observed TRAF2 dephosphorylation in cells was not isolated","Mechanism of TRAF6 ubiquitination suppression (direct vs indirect) not resolved"]},{"year":2017,"claim":"Showed that a viral oncoprotein hijacks the PP4R1/PP4c complex to silence antiviral NF-κB signaling, revealing the complex as a target of viral immune evasion.","evidence":"Reciprocal Co-IP, siRNA depletion, tAg mutagenesis, NF-κB reporter and cytokine measurements","pmids":["28445980"],"confidence":"High","gaps":["Identity of the NEMO-proximal phospho-substrate dephosphorylated in this context not pinned down","Structural basis of the tAg–PP4R1–NEMO bridge unresolved"]},{"year":2017,"claim":"Placed PP4R1 under post-transcriptional control of miR-338-3p in hepatocytes, linking its expression to insulin signaling and glycogen synthesis.","evidence":"Luciferase 3′UTR reporter, miRNA mimic/inhibitor, western blot, ChIP, db/db and HFD mouse models","pmids":["28467989"],"confidence":"Medium","gaps":["Whether PP4R1 acts on AKT/GSK3β components directly via PP4c not demonstrated","Single lab"]},{"year":2018,"claim":"Established PP4R1 as the downstream effector of miR-338-3p in driving hepatic gluconeogenesis through FOXO1, defining a metabolic epistatic relationship.","evidence":"Double knockdown epistasis, qPCR of gluconeogenic genes, pyruvate tolerance testing in mice","pmids":["30132533"],"confidence":"Medium","gaps":["Direct phosphatase action on FOXO1 not shown","Single lab"]},{"year":2020,"claim":"Extended PP4R1 function to cancer, linking it to HMGA2 and MAPK/ERK-driven EMT in lung cancer.","evidence":"Co-IP, migration/invasion assays, EMT marker and phospho-ERK western blot, overexpression/knockdown","pmids":["32077156"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal validation of the HMGA2 interaction","Whether the effect depends on PP4c phosphatase activity unknown"]},{"year":2023,"claim":"Identified HSPA6 induction and ER stress as a downstream mechanism for PP4R1-driven NSCLC progression.","evidence":"RNA-seq, HSPA6 rescue/epistasis, ER stress markers, in vivo tumor and metastasis models","pmids":["37739270"],"confidence":"Medium","gaps":["Mechanism connecting PP4R1 to HSPA6 transcription not defined","Single lab"]},{"year":2024,"claim":"Defined the AMBRA1–PP4R1 competition as a switch controlling IKK dephosphorylation, explaining how PP4R1's anti-inflammatory activity is reciprocally regulated.","evidence":"Reciprocal Co-IP, F1 domain mapping, AMBRA1 S1043 mutagenesis, ubiquitination assay, colitis mouse model","pmids":["38424148"],"confidence":"High","gaps":["Direct IKK phospho-site dephosphorylated by PP4R1/PP4c not mapped","Stoichiometry of AMBRA1 vs PP4c competition for PP4R1 unresolved"]},{"year":2024,"claim":"Showed PP4R1 promotes glycolysis in gallbladder cancer by strengthening ERK1/2–PKM2 interaction and driving PKM2 nuclear translocation.","evidence":"Co-IP, direct interaction pulldown, subcellular fractionation/imaging, ERK inhibitor, in vitro and in vivo tumor assays","pmids":["38301910"],"confidence":"Medium","gaps":["Role of PP4c phosphatase activity in PKM2 regulation not addressed","Single lab"]},{"year":null,"claim":"How a single PP4 regulatory subunit reconciles its phosphatase-targeting role in NF-κB suppression with its pro-tumorigenic, phosphatase-independent scaffolding of kinase complexes remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of PP4R1 substrate-targeting determinants","Whether catalytic PP4c activity is required across the diverse reported functions is unclear","Tissue-specific determinants of pro- vs anti-tumor roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[4,6]}],"localization":[],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,4]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[1,4]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[2,3,6]}],"complexes":["PP4R1/PP4c holoenzyme"],"partners":["PPP4C","TRAF2","TRAF6","AMBRA1","NEMO/IKBKG","HMGA2","PKM2","ERK1/2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8TF05","full_name":"Serine/threonine-protein phosphatase 4 regulatory subunit 1","aliases":[],"length_aa":950,"mass_kda":107.0,"function":"Regulatory subunit of serine/threonine-protein phosphatase 4. May play a role in regulation of cell division in renal glomeruli. The PPP4C-PPP4R1 PP4 complex may play a role in dephosphorylation and regulation of HDAC3. Plays a role in the inhibition of TNF-induced NF-kappa-B activation by regulating the dephosphorylation of TRAF2 (Microbial infection) Participates in merkel polyomavirus-mediated inhibition of NF-kappa-B by bridging viral small tumor antigen with NEMO","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q8TF05/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PPP4R1","classification":"Not Classified","n_dependent_lines":11,"n_total_lines":1208,"dependency_fraction":0.009105960264900662},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"PPP2CA","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PPP4R1","total_profiled":1310},"omim":[{"mim_id":"616790","title":"PROTEIN PHOSPHATASE 4, REGULATORY SUBUNIT 4; PPP4R4","url":"https://www.omim.org/entry/616790"},{"mim_id":"605166","title":"HISTONE DEACETYLASE 3; HDAC3","url":"https://www.omim.org/entry/605166"},{"mim_id":"604908","title":"PROTEIN PHOSPHATASE 4, REGULATORY SUBUNIT 1; PPP4R1","url":"https://www.omim.org/entry/604908"},{"mim_id":"602035","title":"PROTEIN PHOSPHATASE 4, CATALYTIC SUBUNIT; PPP4C","url":"https://www.omim.org/entry/602035"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Actin filaments","reliability":"Additional"},{"location":"Perinuclear theca","reliability":"Additional"},{"location":"Calyx","reliability":"Additional"},{"location":"Mid piece","reliability":"Additional"},{"location":"Principal piece","reliability":"Additional"},{"location":"End piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PPP4R1"},"hgnc":{"alias_symbol":["PP4R1"],"prev_symbol":[]},"alphafold":{"accession":"Q8TF05","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TF05","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TF05-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8TF05-F1-predicted_aligned_error_v6.png","plddt_mean":73.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PPP4R1","jax_strain_url":"https://www.jax.org/strain/search?query=PPP4R1"},"sequence":{"accession":"Q8TF05","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8TF05.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8TF05/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8TF05"}},"corpus_meta":[{"pmid":"28467989","id":"PMC_28467989","title":"Mir-338-3p Mediates Tnf-A-Induced Hepatic Insulin Resistance by Targeting PP4r1 to Regulate PP4 Expression.","date":"2017","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/28467989","citation_count":37,"is_preprint":false},{"pmid":"28445980","id":"PMC_28445980","title":"The PP4R1 sub-unit of protein phosphatase PP4 is essential for inhibition of NF-κB by merkel polyomavirus small tumour antigen.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/28445980","citation_count":34,"is_preprint":false},{"pmid":"25134449","id":"PMC_25134449","title":"The PP4R1 subunit of protein phosphatase PP4 targets TRAF2 and TRAF6 to mediate inhibition of NF-κB activation.","date":"2014","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/25134449","citation_count":22,"is_preprint":false},{"pmid":"32077156","id":"PMC_32077156","title":"PP4R1 interacts with HMGA2 to promote non-small-cell lung cancer migration and metastasis via activating MAPK/ERK-induced epithelial-mesenchymal transition.","date":"2020","source":"Molecular carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/32077156","citation_count":17,"is_preprint":false},{"pmid":"38301910","id":"PMC_38301910","title":"PP4R1 promotes glycolysis and gallbladder cancer progression through facilitating ERK1/2 mediated PKM2 nuclear translocation.","date":"2024","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/38301910","citation_count":13,"is_preprint":false},{"pmid":"30132533","id":"PMC_30132533","title":"miR‑338‑3p mediates gluconeogenesis via targeting of PP4R1 in hepatocytes.","date":"2018","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/30132533","citation_count":12,"is_preprint":false},{"pmid":"38424148","id":"PMC_38424148","title":"AMBRA1 promotes intestinal inflammation by antagonizing PP4R1/PP4c mediated IKK dephosphorylation in an autophagy-independent manner.","date":"2024","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/38424148","citation_count":11,"is_preprint":false},{"pmid":"26300649","id":"PMC_26300649","title":"PP4R1 accelerates cell growth and proliferation in HepG2 hepatocellular carcinoma.","date":"2015","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/26300649","citation_count":11,"is_preprint":false},{"pmid":"37739270","id":"PMC_37739270","title":"PP4R1 accelerates the malignant progression of NSCLC via up-regulating HSPA6 expression and HSPA6-mediated ER stress.","date":"2023","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/37739270","citation_count":11,"is_preprint":false},{"pmid":"23084354","id":"PMC_23084354","title":"The taming of the NF-κB: PP4R1 navigates while PP4c dephosphorylates.","date":"2012","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/23084354","citation_count":2,"is_preprint":false},{"pmid":"33750415","id":"PMC_33750415","title":"Diagnostic significance of serum PP4R1 and its predictive value for the development of chronic complications in patients with type 2 diabetes mellitus.","date":"2021","source":"Diabetology & metabolic syndrome","url":"https://pubmed.ncbi.nlm.nih.gov/33750415","citation_count":1,"is_preprint":false},{"pmid":"37396311","id":"PMC_37396311","title":"PP4R1 Accelerates Cell Growth and Proliferation in HepG2 Hepatocellular Carcinoma [Retraction].","date":"2023","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/37396311","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":7935,"output_tokens":2569,"usd":0.03117,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9786,"output_tokens":3185,"usd":0.064278,"stage2_stop_reason":"end_turn"},"total_usd":0.095448,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2014,\n      \"finding\": \"PP4R1 interacts with TRAF2 and TRAF6 in a RING finger domain-dependent manner, mediates dephosphorylation of TRAF2 Ser11, and inhibits TRAF6 polyubiquitination, thereby inhibiting NF-κB activation downstream of TRAF2, TRAF6, TNF, and EBV oncoprotein LMP1.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, RNA interference knockdown, NF-κB reporter assay, western blot for phospho-TRAF2 Ser11, ubiquitination assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction identified by Y2H and confirmed functionally by RNAi depletion and multiple signaling readouts in a single lab with orthogonal methods\",\n      \"pmids\": [\"25134449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MCPyV small T antigen (tAg) forms a complex with PP4R1 and PP4c; this complex bridges tAg to the NEMO adaptor protein, enabling dephosphorylation-dependent deactivation of the NF-κB pathway. siRNA depletion of PP4R1 or tAg mutations that disrupt this interaction abolish tAg-mediated NF-κB inhibition and pro-inflammatory cytokine suppression.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, NF-κB reporter assay, mutational analysis of tAg, cytokine production measurement\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, mutagenesis, and RNAi with multiple functional readouts in a single study\",\n      \"pmids\": [\"28445980\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"miR-338-3p directly targets the 3′UTR of PP4R1 mRNA; downregulation of miR-338-3p in TNF-α-treated hepatocytes increases PP4R1 and PP4 expression, impairing AKT/GSK3β signaling and glycogen synthesis, thereby contributing to hepatic insulin resistance.\",\n      \"method\": \"Luciferase reporter assay (direct target validation), western blot, miRNA mimic/inhibitor transfection, ChIP assay (HNF-4α transcriptional regulation of miR-338-3p), mouse models (db/db, HFD)\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase assay validates direct targeting, multiple in vivo and in vitro readouts, single lab\",\n      \"pmids\": [\"28467989\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PP4R1 mediates TNF-α-induced gluconeogenesis in hepatocytes; knockdown of PP4R1 attenuates the increase in glucose production caused by miR-338-3p inhibition, placing PP4R1 downstream of miR-338-3p in the regulation of FOXO1 phosphorylation and gluconeogenic gene expression (PGC-1α, PEPCK, G6Pase).\",\n      \"method\": \"Western blot, qPCR, pyruvate tolerance testing in mice, miR-338-3p inhibitor and PP4R1 siRNA in HEPA1-6 cells (genetic epistasis)\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis established by double knockdown, multiple metabolic readouts, single lab\",\n      \"pmids\": [\"30132533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AMBRA1 competitively interacts with PP4R1 via its N-terminal F1 domain, disrupting the PP4R1/PP4c complex and antagonizing PP4R1/PP4c-mediated dephosphorylation of the IKK complex, thereby promoting sustained IKK phosphorylation and NF-κB-driven intestinal inflammation. In response to TNF-α, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 by reducing CUL4A-mediated K48-linked ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation (reciprocal), domain-mapping experiments (N-term F1 deletion mutants), siRNA/knockout in colitis mouse model, phosphorylation site mutagenesis (AMBRA1 S1043), ubiquitination assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, domain mapping, mutagenesis, in vivo colitis model, multiple orthogonal methods in one study\",\n      \"pmids\": [\"38424148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PP4R1 interacts with HMGA2 and promotes epithelial-mesenchymal transition in non-small-cell lung cancer via activation of the MAPK/ERK signaling pathway.\",\n      \"method\": \"Co-immunoprecipitation, wound-healing and Transwell invasion assays, western blot for EMT markers and ERK phosphorylation, PP4R1 overexpression/knockdown\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP identifying HMGA2 interaction, functional readouts present but limited mechanistic depth, single lab\",\n      \"pmids\": [\"32077156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PP4R1 directly interacts with PKM2 and strengthens the interaction between ERK1/2 and PKM2, promoting ERK1/2-mediated PKM2 nuclear translocation and thereby enhancing glycolysis in gallbladder cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay or pulldown (direct interaction), subcellular fractionation/nuclear translocation imaging, ERK inhibitor treatment, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction shown by Co-IP, nuclear translocation measured, mechanistic link to glycolysis with in vivo validation, single lab\",\n      \"pmids\": [\"38301910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Knockdown of PP4R1 in HepG2 hepatocellular carcinoma cells arrests cells at G2/M phase and inactivates p38 and JNK MAPK signaling cascades, indicating PP4R1 promotes cell proliferation through these kinase pathways. NOTE: The original article (PMID 26300649) was subsequently retracted (PMID 37396311).\",\n      \"method\": \"Lentiviral shRNA knockdown, flow cytometry (cell cycle), western blot for p38 and JNK activation, colony formation assay\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single-method knockdown with signaling readouts; article was retracted, severely undermining reliability\",\n      \"pmids\": [\"26300649\", \"37396311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PP4R1 overexpression upregulates HSPA6 (an HSP70 family member) and activates endoplasmic reticulum stress, promoting proliferation, migration, and invasion of NSCLC cells; HSPA6 overexpression alone recapitulates the PP4R1 phenotype, placing HSPA6 downstream of PP4R1.\",\n      \"method\": \"RNA-seq after PP4R1 overexpression, HSPA6 overexpression/knockdown, ER stress marker western blot, in vitro proliferation/migration/invasion assays, in vivo mouse tumor and metastasis model\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptomic profiling plus epistasis via HSPA6 rescue, in vivo validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"37739270\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PPP4R1 (PP4R1) is the regulatory subunit 1 of protein phosphatase 4 (PP4c) that directs the PP4 holoenzyme to specific substrates: it mediates dephosphorylation of TRAF2 Ser11 and antagonizes TRAF6 ubiquitin ligase activity to suppress NF-κB activation, forms a PP4R1/PP4c complex that dephosphorylates the IKK complex (counteracted by AMBRA1 competition for PP4R1 binding), can be co-opted by viral small T antigens via a NEMO-bridging mechanism to inhibit antiviral NF-κB responses, and in metabolic contexts is targeted by miR-338-3p to regulate hepatic insulin signaling, glycogen synthesis, and gluconeogenesis; additionally, PP4R1 interacts with HMGA2 to activate MAPK/ERK-driven EMT in lung cancer and promotes PKM2 nuclear translocation to enhance glycolysis in gallbladder cancer.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PPP4R1 (PP4R1) is the regulatory subunit of the protein phosphatase 4 (PP4c) holoenzyme that targets the phosphatase to substrates within the NF-\\u03baB signaling axis and acts as a negative regulator of inflammatory and immune responses [#0, #4]. It engages TRAF2 and TRAF6 in a RING finger domain-dependent manner, dephosphorylating TRAF2 at Ser11 and suppressing TRAF6 polyubiquitination to dampen NF-\\u03baB activation downstream of TNF and the EBV oncoprotein LMP1 [#0]. As part of a PP4R1/PP4c complex, it dephosphorylates the IKK complex; this activity is antagonized by AMBRA1, which competitively binds PP4R1 through its N-terminal F1 domain to disrupt the complex and sustain IKK phosphorylation and NF-\\u03baB-driven intestinal inflammation [#4]. This regulatory module is co-opted by Merkel cell polyomavirus small T antigen, which forms a complex with PP4R1/PP4c that bridges the viral protein to NEMO and deactivates NF-\\u03baB to suppress pro-inflammatory cytokine responses [#1]. Beyond immune control, PP4R1 functions in hepatic metabolism as a target of miR-338-3p, where its derepression impairs AKT/GSK3\\u03b2 signaling and drives FOXO1-dependent gluconeogenesis and insulin resistance [#2, #3], and it promotes tumor progression by interacting with HMGA2 to activate MAPK/ERK-driven EMT in lung cancer [#5], by binding PKM2 to enhance ERK1/2-mediated PKM2 nuclear translocation and glycolysis in gallbladder cancer [#6], and by upregulating HSPA6 to activate ER stress in NSCLC [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Established PP4R1 as a negative regulator of NF-\\u03baB by defining its direct substrates within TRAF signaling, answering how PP4 is targeted to this pathway.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, RNAi, NF-\\u03baB reporter, phospho-TRAF2 Ser11 and ubiquitination assays\",\n      \"pmids\": [\"25134449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether PP4c catalytic activity is required for the observed TRAF2 dephosphorylation in cells was not isolated\",\n        \"Mechanism of TRAF6 ubiquitination suppression (direct vs indirect) not resolved\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed that a viral oncoprotein hijacks the PP4R1/PP4c complex to silence antiviral NF-\\u03baB signaling, revealing the complex as a target of viral immune evasion.\",\n      \"evidence\": \"Reciprocal Co-IP, siRNA depletion, tAg mutagenesis, NF-\\u03baB reporter and cytokine measurements\",\n      \"pmids\": [\"28445980\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Identity of the NEMO-proximal phospho-substrate dephosphorylated in this context not pinned down\",\n        \"Structural basis of the tAg\\u2013PP4R1\\u2013NEMO bridge unresolved\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Placed PP4R1 under post-transcriptional control of miR-338-3p in hepatocytes, linking its expression to insulin signaling and glycogen synthesis.\",\n      \"evidence\": \"Luciferase 3\\u2032UTR reporter, miRNA mimic/inhibitor, western blot, ChIP, db/db and HFD mouse models\",\n      \"pmids\": [\"28467989\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Whether PP4R1 acts on AKT/GSK3\\u03b2 components directly via PP4c not demonstrated\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established PP4R1 as the downstream effector of miR-338-3p in driving hepatic gluconeogenesis through FOXO1, defining a metabolic epistatic relationship.\",\n      \"evidence\": \"Double knockdown epistasis, qPCR of gluconeogenic genes, pyruvate tolerance testing in mice\",\n      \"pmids\": [\"30132533\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Direct phosphatase action on FOXO1 not shown\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended PP4R1 function to cancer, linking it to HMGA2 and MAPK/ERK-driven EMT in lung cancer.\",\n      \"evidence\": \"Co-IP, migration/invasion assays, EMT marker and phospho-ERK western blot, overexpression/knockdown\",\n      \"pmids\": [\"32077156\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Single Co-IP without reciprocal validation of the HMGA2 interaction\",\n        \"Whether the effect depends on PP4c phosphatase activity unknown\"\n      ]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified HSPA6 induction and ER stress as a downstream mechanism for PP4R1-driven NSCLC progression.\",\n      \"evidence\": \"RNA-seq, HSPA6 rescue/epistasis, ER stress markers, in vivo tumor and metastasis models\",\n      \"pmids\": [\"37739270\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Mechanism connecting PP4R1 to HSPA6 transcription not defined\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined the AMBRA1\\u2013PP4R1 competition as a switch controlling IKK dephosphorylation, explaining how PP4R1's anti-inflammatory activity is reciprocally regulated.\",\n      \"evidence\": \"Reciprocal Co-IP, F1 domain mapping, AMBRA1 S1043 mutagenesis, ubiquitination assay, colitis mouse model\",\n      \"pmids\": [\"38424148\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Direct IKK phospho-site dephosphorylated by PP4R1/PP4c not mapped\",\n        \"Stoichiometry of AMBRA1 vs PP4c competition for PP4R1 unresolved\"\n      ]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed PP4R1 promotes glycolysis in gallbladder cancer by strengthening ERK1/2\\u2013PKM2 interaction and driving PKM2 nuclear translocation.\",\n      \"evidence\": \"Co-IP, direct interaction pulldown, subcellular fractionation/imaging, ERK inhibitor, in vitro and in vivo tumor assays\",\n      \"pmids\": [\"38301910\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Role of PP4c phosphatase activity in PKM2 regulation not addressed\",\n        \"Single lab\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single PP4 regulatory subunit reconciles its phosphatase-targeting role in NF-\\u03baB suppression with its pro-tumorigenic, phosphatase-independent scaffolding of kinase complexes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"No structural model of PP4R1 substrate-targeting determinants\",\n        \"Whether catalytic PP4c activity is required across the diverse reported functions is unclear\",\n        \"Tissue-specific determinants of pro- vs anti-tumor roles undefined\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0019888\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [4, 6]}\n    ],\n    \"localization\": [],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 4]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [2, 3, 6]}\n    ],\n    \"complexes\": [\n      \"PP4R1/PP4c holoenzyme\"\n    ],\n    \"partners\": [\n      \"PPP4C\",\n      \"TRAF2\",\n      \"TRAF6\",\n      \"AMBRA1\",\n      \"NEMO/IKBKG\",\n      \"HMGA2\",\n      \"PKM2\",\n      \"ERK1/2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}