{"gene":"PJA2","run_date":"2026-04-28T19:45:44","timeline":{"discoveries":[{"year":2011,"finding":"Praja2 (PJA2) forms a stable complex with PKA regulatory (R) subunits and is phosphorylated by PKA. Rising cAMP levels promote praja2-mediated ubiquitylation and subsequent proteasomal degradation of R subunits, leading to sustained catalytic subunit activity and downstream substrate phosphorylation. This mechanism is required for efficient nuclear cAMP signalling and PKA-mediated long-term memory.","method":"Co-immunoprecipitation, ubiquitylation assays, proteasome inhibitor experiments, in vivo mouse memory assays, loss-of-function knockdown with defined phosphorylation readouts","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, functional KD phenotype, in vivo validation, multiple orthogonal methods in a high-citation study","pmids":["21423175"],"is_preprint":false},{"year":2013,"finding":"Praja2 ubiquitylates and targets MOB1 (Mob), a core component of NDR/LATS kinases and positive regulator of the Hippo cascade, for proteasomal degradation. This attenuates Hippo signalling and sustains glioblastoma growth in vivo.","method":"Co-immunoprecipitation, in vitro ubiquitylation assay, proteasome inhibitor experiments, in vivo xenograft tumor model, knockdown with defined Hippo pathway readouts","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus in vitro ubiquitylation plus in vivo model, high citation count indicating broad acceptance","pmids":["23652010"],"is_preprint":false},{"year":2014,"finding":"PJA2 forms a complex with the AMPK-related kinase SIK2 and the CDK5 activator p35. Following glucose stimulation, SIK2 phosphorylates p35 at Ser91, which triggers p35 ubiquitylation by PJA2 and promotes insulin secretion. β-cell-specific SIK2 knockout leads to p35 accumulation and impaired secretion.","method":"Affinity purification-mass spectrometry (AP-MS), Co-immunoprecipitation, in vitro ubiquitylation assay, β-cell-specific knockout mouse, glucose-stimulated insulin secretion assay","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1–2 — AP-MS complex identification, in vitro ubiquitylation, conditional KO mouse with defined physiological phenotype","pmids":["24561619"],"is_preprint":false},{"year":2016,"finding":"Praja2 acts as the E3 ubiquitin ligase that polyubiquitylates KSR1, a scaffold of the Ras/MAPK pathway, leading to its proteasomal degradation. This attenuates ERK1/2 signalling downstream of receptor stimulation, controls cancer cell growth, and is required for maintenance of mouse embryonic stem cell pluripotency.","method":"Co-immunoprecipitation, in vitro ubiquitylation assay, proteasome inhibitor rescue, overexpression/knockdown with ERK phosphorylation and colony formation readouts, mouse ESC differentiation assays","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, in vitro ubiquitylation, KD/OE phenotypes) with defined pathway readouts","pmids":["27195677"],"is_preprint":false},{"year":2017,"finding":"PJA2 ubiquitylates the HIV-1 Tat protein with atypical, non-degradative polyubiquitin chain linkages, specifically regulating the transcription elongation step of HIV transcription. Ubiquitin acceptor sites on Tat and the ubiquitin linkage type are both variable. Proper chain assembly by PJA2 requires Tat to first bind P-TEFb.","method":"RNAi knockdown screen, in vivo ubiquitylation assay, mutational mapping of ubiquitin acceptor lysines, HIV transcription elongation reporter assay, viral replication assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — functional RNAi screen plus mutagenesis plus mechanistic assay, single lab","pmids":["28345603"],"is_preprint":false},{"year":2017,"finding":"Praja2 directly binds MFHAS1 (confirmed by pulldown and co-immunoprecipitation) and promotes its non-degradative ubiquitylation. This ubiquitylation positively regulates TLR2-mediated JNK/p38 pathway activation and promotes M1 macrophage polarization.","method":"In vitro pulldown, co-immunoprecipitation, in situ immunostaining colocalization, ubiquitylation assay, macrophage polarization assays with pathway readouts","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP plus ubiquitylation assay plus functional polarization readout, single lab","pmids":["28471450"],"is_preprint":false},{"year":2018,"finding":"Pja2 binds TCF/LEF1 transcription factors and promotes their ubiquitylation-dependent degradation, thereby downregulating Wnt/β-catenin signalling activity and influencing embryonic stem cell differentiation.","method":"Co-immunoprecipitation, ubiquitylation assay, Wnt reporter assay, gain- and loss-of-function in stem cells","journal":"International journal of stem cells","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus ubiquitylation assay plus reporter assay, single lab with moderate follow-up","pmids":["30021253"],"is_preprint":false},{"year":2021,"finding":"Praja2 assembles a novel centrosomal complex with TBC1D31, PKA, and OFD1. Upon GPCR-cAMP stimulation, PKA phosphorylates OFD1 at Ser735, triggering OFD1 ubiquitylation and proteasomal degradation by praja2. This pathway is essential for primary ciliogenesis; a non-phosphorylatable OFD1 mutant dramatically affects cilium morphology. Disruption of this network impairs ciliogenesis in Medaka fish.","method":"Co-immunoprecipitation, proximity ligation assay, in vitro ubiquitylation assay, site-directed mutagenesis (S735A OFD1), GPCR-cAMP stimulation, ciliogenesis assays, in vivo Medaka zebrafish model","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 — reconstitution-style complex identification, mutagenesis, in vitro ubiquitylation, in vivo vertebrate validation","pmids":["33934390"],"is_preprint":false},{"year":2021,"finding":"PJA2 reduces KSR1 protein levels through ubiquitylation and degradation, which inhibits MEK-ERK signalling in gastric cancer cells. Overexpression of praja2 suppresses cancer cell proliferation, migration, and invasion, and inhibits tumor growth in vivo via the KSR1-MEK-ERK axis.","method":"Co-immunoprecipitation, ubiquitylation assay, proteasome inhibitor rescue (MG132), in vitro proliferation/migration/invasion assays, in vivo xenograft model","journal":"Aging","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP plus ubiquitylation plus in vivo model, single lab, consistent with prior KSR1 work","pmids":["33461174"],"is_preprint":false},{"year":2021,"finding":"PJA2 ubiquitylates KSR1, promoting its degradation. In the KDM5A-PJA2-KSR1 axis in gastric cancer macrophages, KDM5A suppresses PJA2 expression by removing H3K4me3 from the PJA2 promoter, thereby stabilizing KSR1 and promoting macrophage M2 polarization. ELK4 drives this axis through transcriptional activation of KDM5A.","method":"Dual luciferase reporter assay, ChIP assay, Co-immunoprecipitation, cycloheximide chase for KSR1 stability, gain- and loss-of-function assays, xenograft model","journal":"Journal of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2–3 — ChIP, Co-IP, CHX chase, functional assays in single lab; positions PJA2 in an epigenetically regulated axis","pmids":["34372882"],"is_preprint":false},{"year":2021,"finding":"FTO demethylates m6A modification on PJA2 mRNA, reducing its decay and restoring PJA2 expression. Increased PJA2 suppresses Wnt signalling and restrains proliferation, invasion, and metastasis of pancreatic cancer cells.","method":"m6A methylation assay, mRNA stability assay, Wnt signalling reporter, gain- and loss-of-function with proliferation/invasion readouts","journal":"Molecular therapy. Nucleic acids","confidence":"Medium","confidence_rationale":"Tier 2–3 — m6A assay plus mRNA stability plus functional readouts, single lab","pmids":["34484859"],"is_preprint":false},{"year":2024,"finding":"Praja2 forms a complex with the AP2 adapter complex and ubiquitylates it, contributing to EGFR endocytosis and clearance. Downregulation of praja2 by oncomiRs impairs EGFR endocytosis, amplifies downstream mitogenic signalling, and promotes kidney cancer cell growth. Genetic ablation of praja2 in mice upregulates EGFR and VEGFR and induces epithelial and vascular alterations in kidney tissue.","method":"Co-immunoprecipitation, ubiquitylation assay, endocytosis assay, oncomiR overexpression, praja2 restoration experiments, praja2 knockout mouse model with RTK readout","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, ubiquitylation assay, KO mouse with defined molecular phenotype, multiple orthogonal approaches","pmids":["38379085"],"is_preprint":false},{"year":2024,"finding":"PJA2 interacts with TYK2 and JAK1 within the type I IFN signalling cascade, promotes their non-degradative ubiquitination, and limits the activating phosphorylation of TYK2, thereby restraining downstream STAT signalling and acting as a negative regulator of IFN signalling.","method":"TurboID proximity labeling coupled with affinity purification-mass spectrometry, functional RNAi screen, co-immunoprecipitation, ubiquitination assays, phosphorylation assays for TYK2/STAT","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — proximity proteomics plus AP-MS plus functional screen plus mechanistic Co-IP and ubiquitination assays","pmids":["38802340"],"is_preprint":false},{"year":2025,"finding":"Praja2 forms a multimeric complex with the RNA helicase DDX6 and promotes non-proteolytic polyubiquitylation of DDX6 in response to cAMP/GPCR signalling, inducing P-body assembly and translational repression of target RNAs. Genetic inactivation of praja2 reshapes DDX6/mRNA complexes and polysomes, promoting cellular senescence and GBM growth arrest. An ubiquitylation-defective DDX6 mutant suppresses P-body assembly and sustains GBM growth.","method":"Co-immunoprecipitation, in vitro ubiquitylation assay, site-directed mutagenesis (ubiquitylation-defective DDX6), polysome profiling, P-body imaging, cAMP stimulation, loss-of-function with senescence readout","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1–2 — Co-IP, in vitro ubiquitylation, mutagenesis, polysome profiling, multiple orthogonal mechanistic methods","pmids":["40148504"],"is_preprint":false},{"year":2025,"finding":"PJA2 recognizes HDAC2 via its RING-B-box domain (RBD), binds the N-terminal of HDAC2, and facilitates its ubiquitylation at Lys90, promoting HDAC2 degradation. This de-represses the IFIT family of interferon-induced genes, suppressing colorectal cancer progression. HDAC2 reciprocally regulates PJA2 expression, forming a positive feedback loop.","method":"Co-immunoprecipitation, proximity ligation assay, ubiquitylation assay, ChIP, RNA-seq, AOM/DSS mouse CRC model, colony formation assay, domain-deletion mutagenesis","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 1–2 — Co-IP, PLA, site-specific ubiquitylation (K90), ChIP, RNA-seq, in vivo CRC model, multiple orthogonal methods","pmids":["39928532"],"is_preprint":false},{"year":2025,"finding":"PJA2 (RING E3 ligase) directly interacts with CHRM3 (muscarinic acetylcholine receptor M3), ubiquitinates it, and promotes its degradation, thereby suppressing downstream TGFβ-pSMAD3 signalling and gastric tumor cell progression. The catalytically dead ΔRING PJA2 mutant cannot suppress CHRM3-driven organoid growth.","method":"Mass spectrometry, co-immunoprecipitation, ubiquitination assay, degradation assay, patient-derived organoids, ΔRING mutant rescue, xenograft metastasis model","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 — MS identification, Co-IP, ubiquitination/degradation assays, mutagenesis, PDO model; single lab, recent publication","pmids":["40858831"],"is_preprint":false}],"current_model":"PJA2 is a RING-domain E3 ubiquitin ligase that directly ubiquitylates a broad set of substrates (including PKA regulatory subunits, MOB1, KSR1, OFD1, TCF/LEF1, MFHAS1, HDAC2, CHRM3, AP2 adapter complex, DDX6, TYK2/JAK1, and HIV-1 Tat) to control their stability or activity in a context-dependent manner (degradative or non-degradative), thereby regulating cAMP/PKA signalling duration, Hippo and Wnt pathway activity, MAPK/ERK signalling, primary ciliogenesis, receptor endocytosis, type I interferon signalling, and mRNA translation in P-bodies, and is itself regulated by PKA-mediated phosphorylation and m6A-dependent mRNA stability."},"narrative":{"teleology":[{"year":2011,"claim":"Establishing PJA2 as a cAMP-responsive E3 ligase that degrades PKA regulatory subunits resolved how cells terminate PKA holoenzyme reassembly and sustain catalytic activity during prolonged cAMP signalling, linking ubiquitin-dependent proteolysis to long-term memory formation.","evidence":"Co-IP, ubiquitylation assays, proteasome inhibitor rescue, PKA phosphorylation mapping, and mouse long-term memory assays","pmids":["21423175"],"confidence":"High","gaps":["Whether PKA phosphorylation of PJA2 is required for all subsequent substrate interactions or is context-specific","Structural basis for PJA2–R subunit recognition"]},{"year":2013,"claim":"Identifying MOB1 as a PJA2 substrate established the ligase as a negative regulator of Hippo signalling and showed that PJA2-mediated proteolysis sustains glioblastoma growth, broadening its substrate repertoire beyond the PKA axis.","evidence":"Reciprocal Co-IP, in vitro ubiquitylation, proteasome inhibitor rescue, and in vivo xenograft tumour model","pmids":["23652010"],"confidence":"High","gaps":["Whether PJA2 targets additional Hippo pathway components","Upstream signal that triggers MOB1 ubiquitylation"]},{"year":2014,"claim":"Demonstration that PJA2 operates within a SIK2–p35 complex to ubiquitylate phosphorylated p35 upon glucose stimulation revealed a phosphorylation-primed degron mechanism coupling nutrient sensing to insulin secretion.","evidence":"AP-MS complex identification, in vitro ubiquitylation, β-cell-specific SIK2 knockout mouse with insulin secretion phenotype","pmids":["24561619"],"confidence":"High","gaps":["Direct in vivo confirmation using β-cell-specific PJA2 knockout","Whether PJA2 ubiquitylates additional CDK5-related substrates in β-cells"]},{"year":2016,"claim":"Showing that PJA2 ubiquitylates the Ras/MAPK scaffold KSR1 for degradation established a direct mechanism for attenuating ERK signalling intensity and linked this axis to embryonic stem cell pluripotency maintenance.","evidence":"Co-IP, in vitro ubiquitylation, proteasome inhibitor rescue, ERK phosphorylation readouts, ESC differentiation assays","pmids":["27195677"],"confidence":"High","gaps":["Whether KSR2 is similarly targeted","Structural determinants of KSR1 recognition by PJA2"]},{"year":2017,"claim":"Discovery that PJA2 attaches atypical, non-degradative polyubiquitin chains to HIV-1 Tat—requiring prior Tat–P-TEFb binding—revealed a non-proteolytic ubiquitylation mode of PJA2 and a role in viral transcription elongation.","evidence":"RNAi screen, ubiquitylation assay with linkage analysis, ubiquitin acceptor site mutagenesis, HIV transcription elongation reporter","pmids":["28345603"],"confidence":"Medium","gaps":["Specific ubiquitin chain linkage types remain incompletely defined","Independent replication in primary CD4+ T cells","Whether PJA2 affects latency reversal"]},{"year":2017,"claim":"Identification of MFHAS1 as a non-degradative substrate showed PJA2 can positively regulate innate immune signalling (TLR2–JNK/p38) and macrophage M1 polarization through ubiquitylation that does not lead to target destruction.","evidence":"In vitro pulldown, Co-IP, ubiquitylation assay, macrophage polarization readouts","pmids":["28471450"],"confidence":"Medium","gaps":["Ubiquitin chain type on MFHAS1 not determined","In vivo confirmation of PJA2-dependent macrophage polarization"]},{"year":2018,"claim":"Demonstration that PJA2 ubiquitylates TCF/LEF1 for degradation extended its reach to Wnt/β-catenin signalling and embryonic stem cell fate decisions.","evidence":"Co-IP, ubiquitylation assay, Wnt reporter assay, gain/loss-of-function in stem cells","pmids":["30021253"],"confidence":"Medium","gaps":["Which TCF/LEF family member is preferentially targeted","Independent replication outside the original lab"]},{"year":2021,"claim":"Reconstitution of a centrosomal PJA2–TBC1D31–PKA–OFD1 complex, and showing that PKA phosphorylation of OFD1 triggers its PJA2-mediated degradation to enable ciliogenesis, established a GPCR–cAMP–ubiquitin axis at the centrosome governing primary cilium formation.","evidence":"Co-IP, PLA, in vitro ubiquitylation, S735A OFD1 mutagenesis, ciliogenesis assays, in vivo Medaka fish model","pmids":["33934390"],"confidence":"High","gaps":["Whether PJA2 targets other centrosomal ciliogenesis regulators","Human ciliopathy genetic evidence linking PJA2"]},{"year":2021,"claim":"Convergent studies on KSR1 in gastric cancer confirmed PJA2's tumour-suppressive role through ERK attenuation and uncovered upstream epigenetic regulation of PJA2 expression by KDM5A-mediated H3K4me3 removal and FTO-dependent m6A demethylation of PJA2 mRNA, placing PJA2 within transcriptional and epitranscriptomic regulatory circuits.","evidence":"ChIP, dual-luciferase reporter, CHX chase, m6A methylation and mRNA stability assays, xenograft models","pmids":["33461174","34372882","34484859"],"confidence":"Medium","gaps":["Functional interplay between epigenetic and epitranscriptomic PJA2 regulation in the same cell type","In vivo confirmation in genetic mouse models of gastric/pancreatic cancer"]},{"year":2024,"claim":"Identification of the AP2 adapter complex as a PJA2 substrate linked the ligase to receptor endocytosis: PJA2 loss impairs EGFR clearance, amplifies mitogenic signalling, and phenocopies vascular and epithelial alterations in knockout mice, establishing PJA2 as a gatekeeper of RTK surface levels.","evidence":"Co-IP, ubiquitylation assay, endocytosis assay, PJA2 knockout mouse with EGFR/VEGFR readouts","pmids":["38379085"],"confidence":"High","gaps":["Whether PJA2 regulates endocytosis of RTKs other than EGFR","Specific ubiquitin chain linkage on AP2 subunits"]},{"year":2024,"claim":"Proximity proteomics and functional screening identified TYK2 and JAK1 as PJA2 substrates undergoing non-degradative ubiquitylation that limits TYK2 phosphorylation, establishing PJA2 as a negative regulator of type I interferon/STAT signalling.","evidence":"TurboID proximity labeling–AP-MS, RNAi screen, Co-IP, ubiquitination and phosphorylation assays","pmids":["38802340"],"confidence":"High","gaps":["Ubiquitin chain topology on TYK2/JAK1","In vivo consequence of PJA2 loss for antiviral immunity"]},{"year":2025,"claim":"Discovery that PJA2 non-proteolytically ubiquitylates the P-body helicase DDX6 in response to cAMP/GPCR signalling, driving P-body assembly and translational repression, revealed a new role in post-transcriptional gene regulation; an ubiquitylation-defective DDX6 mutant sustained glioblastoma growth, linking this axis to cellular senescence.","evidence":"Co-IP, in vitro ubiquitylation, DDX6 ubiquitylation-site mutagenesis, polysome profiling, P-body imaging, senescence assays","pmids":["40148504"],"confidence":"High","gaps":["Identity of translationally repressed mRNAs critical for senescence","Whether PJA2 targets other P-body components"]},{"year":2025,"claim":"Mapping PJA2's RING-B-box domain as the HDAC2-binding interface and Lys90 of HDAC2 as the ubiquitylation site, with consequent de-repression of IFIT innate immune genes, connected PJA2 to chromatin-level regulation of interferon-stimulated genes and colorectal cancer suppression.","evidence":"Co-IP, PLA, domain-deletion mutagenesis, site-specific ubiquitylation (K90), ChIP, RNA-seq, AOM/DSS mouse CRC model","pmids":["39928532"],"confidence":"High","gaps":["Whether other HDACs are PJA2 substrates","Crystal structure of PJA2 RBD–HDAC2 interface"]},{"year":2025,"claim":"Identification of CHRM3 as a PJA2 substrate whose ubiquitylation-dependent degradation suppresses TGFβ–pSMAD3 signalling extended PJA2's reach to GPCR turnover and gastric cancer progression.","evidence":"Mass spectrometry, Co-IP, ubiquitination/degradation assays, ΔRING mutant rescue, patient-derived organoids, xenograft metastasis model","pmids":["40858831"],"confidence":"Medium","gaps":["Whether PJA2 degrades other muscarinic receptors","Independent replication outside the original study","Ubiquitylation site(s) on CHRM3 not mapped"]},{"year":null,"claim":"Despite extensive substrate cataloguing, no crystal or cryo-EM structure of PJA2 exists; the basis for its remarkably broad substrate selectivity, the determinants specifying degradative versus non-degradative ubiquitin chain assembly, and the in vivo physiological consequences of whole-organism PJA2 loss in mammals remain incompletely defined.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of PJA2 or any PJA2–substrate complex","Rules governing chain-type selectivity (K48 vs. K63 vs. atypical) unknown","Comprehensive phenotyping of PJA2 knockout mice beyond kidney reported only for AP2/EGFR axis"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,3,4,5,6,7,8,11,12,13,14,15]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,2,3,7,11,12,13,14,15]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[7]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,14]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,2,3,4,5,6,7,11,12,13,14,15]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,3,6,8,9,10,12,15]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,12,14]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[11]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[7]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,8,9,14,15]}],"complexes":["PJA2–TBC1D31–PKA–OFD1 centrosomal complex","PJA2–SIK2–p35 complex"],"partners":["PRKAR1A","MOB1A","KSR1","OFD1","DDX6","TYK2","AP2A1","HDAC2"],"other_free_text":[]},"mechanistic_narrative":"PJA2 is a RING-domain E3 ubiquitin ligase that controls the amplitude and duration of multiple signalling pathways by ubiquitylating a diverse set of substrates for either proteasomal degradation or non-degradative regulatory modification. PJA2 attenuates cAMP/PKA signalling by degrading PKA regulatory subunits upon cAMP stimulation [PMID:21423175], suppresses Hippo signalling through MOB1 degradation [PMID:23652010], limits MAPK/ERK output by targeting the scaffold KSR1 [PMID:27195677], restrains Wnt/β-catenin signalling via TCF/LEF1 degradation [PMID:30021253], negatively regulates type I interferon signalling through non-degradative ubiquitylation of TYK2/JAK1 [PMID:38802340], promotes primary ciliogenesis by degrading OFD1 at the centrosome [PMID:33934390], facilitates EGFR endocytosis by ubiquitylating the AP2 adapter complex [PMID:38379085], and drives P-body assembly and translational repression through non-proteolytic ubiquitylation of the RNA helicase DDX6 [PMID:40148504]. PJA2 itself is regulated at transcriptional and post-transcriptional levels, including PKA-mediated phosphorylation that activates its ligase function [PMID:21423175], epigenetic silencing via KDM5A-mediated H3K4me3 removal at its promoter [PMID:34372882], and FTO-dependent m6A demethylation that stabilizes its mRNA [PMID:34484859]. Through these activities PJA2 functions as a versatile signalling rheostat in processes spanning long-term memory, ciliogenesis, receptor trafficking, innate immunity, macrophage polarization, and tumour suppression [PMID:21423175, PMID:33934390, PMID:38802340, PMID:38379085]."},"prefetch_data":{"uniprot":{"accession":"O43164","full_name":"E3 ubiquitin-protein ligase Praja-2","aliases":["RING finger protein 131","RING-type E3 ubiquitin transferase Praja-2"],"length_aa":708,"mass_kda":78.2,"function":"Has E2-dependent E3 ubiquitin-protein ligase activity (PubMed:12036302, PubMed:21423175). Responsible for ubiquitination of cAMP-dependent protein kinase type I and type II-alpha/beta regulatory subunits and for targeting them for proteasomal degradation. Essential for PKA-mediated long-term memory processes (PubMed:21423175). Through the ubiquitination of MFHAS1, positively regulates the TLR2 signaling pathway that leads to the activation of the downstream p38 and JNK MAP kinases and promotes the polarization of macrophages toward the pro-inflammatory M1 phenotype (PubMed:28471450). Plays a role in ciliogenesis by ubiquitinating OFD1 (PubMed:33934390)","subcellular_location":"Cytoplasm; Cell membrane; Endoplasmic reticulum membrane; Golgi apparatus membrane; Synapse; Postsynaptic density; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome","url":"https://www.uniprot.org/uniprotkb/O43164/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PJA2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PJA2","total_profiled":1310},"omim":[{"mim_id":"621444","title":"TBC1 DOMAIN FAMILY, MEMBER 31; TBC1D31","url":"https://www.omim.org/entry/621444"},{"mim_id":"619341","title":"PRAJA RING FINGER UBIQUITIN LIGASE 2; PJA2","url":"https://www.omim.org/entry/619341"},{"mim_id":"601639","title":"PROTEIN KINASE, cAMP-DEPENDENT, CATALYTIC, ALPHA; PRKACA","url":"https://www.omim.org/entry/601639"},{"mim_id":"601132","title":"KINASE SUPPRESSOR OF RAS 1; KSR1","url":"https://www.omim.org/entry/601132"},{"mim_id":"300420","title":"PRAJA RING FINGER UBIQUITIN LIGASE 1; PJA1","url":"https://www.omim.org/entry/300420"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Microtubules","reliability":"Supported"},{"location":"Basal body","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"},{"location":"Primary cilium","reliability":"Additional"},{"location":"Primary cilium transition zone","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PJA2"},"hgnc":{"alias_symbol":["KIAA0438","Neurodap1","PRAJA2"],"prev_symbol":["RNF131"]},"alphafold":{"accession":"O43164","domains":[{"cath_id":"3.30.40.10","chopping":"606-669","consensus_level":"high","plddt":82.1,"start":606,"end":669},{"cath_id":"1.20.5","chopping":"571-604","consensus_level":"medium","plddt":83.4226,"start":571,"end":604}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43164","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43164-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43164-F1-predicted_aligned_error_v6.png","plddt_mean":47.97},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PJA2","jax_strain_url":"https://www.jax.org/strain/search?query=PJA2"},"sequence":{"accession":"O43164","fasta_url":"https://rest.uniprot.org/uniprotkb/O43164.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43164/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43164"}},"corpus_meta":[{"pmid":"23652010","id":"PMC_23652010","title":"Proteolysis of MOB1 by the ubiquitin ligase praja2 attenuates Hippo signalling and supports glioblastoma growth.","date":"2013","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/23652010","citation_count":102,"is_preprint":false},{"pmid":"21423175","id":"PMC_21423175","title":"Control of PKA stability and signalling by the RING ligase praja2.","date":"2011","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/21423175","citation_count":78,"is_preprint":false},{"pmid":"24561619","id":"PMC_24561619","title":"Role of the SIK2-p35-PJA2 complex in pancreatic β-cell functional compensation.","date":"2014","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/24561619","citation_count":69,"is_preprint":false},{"pmid":"28471450","id":"PMC_28471450","title":"Ubiquitylation of MFHAS1 by the ubiquitin ligase praja2 promotes M1 macrophage polarization by activating JNK and p38 pathways.","date":"2017","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/28471450","citation_count":63,"is_preprint":false},{"pmid":"34372882","id":"PMC_34372882","title":"ELK4 promotes the development of gastric cancer by inducing M2 polarization of macrophages through regulation of the KDM5A-PJA2-KSR1 axis.","date":"2021","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34372882","citation_count":41,"is_preprint":false},{"pmid":"33934390","id":"PMC_33934390","title":"The TBC1D31/praja2 complex controls primary ciliogenesis through PKA-directed OFD1 ubiquitylation.","date":"2021","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/33934390","citation_count":33,"is_preprint":false},{"pmid":"28345603","id":"PMC_28345603","title":"PJA2 ubiquitinates the HIV-1 Tat protein with atypical chain linkages to activate viral transcription.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28345603","citation_count":27,"is_preprint":false},{"pmid":"34484859","id":"PMC_34484859","title":"m6A demethylase FTO suppresses pancreatic cancer tumorigenesis by demethylating PJA2 and inhibiting Wnt signaling.","date":"2021","source":"Molecular therapy. Nucleic acids","url":"https://pubmed.ncbi.nlm.nih.gov/34484859","citation_count":26,"is_preprint":false},{"pmid":"27195677","id":"PMC_27195677","title":"praja2 regulates KSR1 stability and mitogenic signaling.","date":"2016","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/27195677","citation_count":25,"is_preprint":false},{"pmid":"22948757","id":"PMC_22948757","title":"Expression of the ring ligase PRAJA2 in thyroid cancer.","date":"2012","source":"The Journal of clinical endocrinology and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/22948757","citation_count":18,"is_preprint":false},{"pmid":"30021253","id":"PMC_30021253","title":"Pja2 Inhibits Wnt/β-catenin Signaling by Reducing the Level of TCF/LEF1.","date":"2018","source":"International journal of stem cells","url":"https://pubmed.ncbi.nlm.nih.gov/30021253","citation_count":16,"is_preprint":false},{"pmid":"33461174","id":"PMC_33461174","title":"Praja2 suppresses the growth of gastric cancer by ubiquitylation of KSR1 and inhibiting MEK-ERK signal pathways.","date":"2021","source":"Aging","url":"https://pubmed.ncbi.nlm.nih.gov/33461174","citation_count":12,"is_preprint":false},{"pmid":"30651076","id":"PMC_30651076","title":"CD1d- and PJA2-related immune microenvironment differs between invasive breast carcinomas with and without a micropapillary feature.","date":"2019","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30651076","citation_count":11,"is_preprint":false},{"pmid":"38802340","id":"PMC_38802340","title":"Proximal protein landscapes of the type I interferon signaling cascade reveal negative regulation by PJA2.","date":"2024","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38802340","citation_count":8,"is_preprint":false},{"pmid":"38379085","id":"PMC_38379085","title":"Downregulation of praja2 restrains endocytosis and boosts tyrosine kinase receptors in kidney cancer.","date":"2024","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/38379085","citation_count":7,"is_preprint":false},{"pmid":"39928532","id":"PMC_39928532","title":"PJA2 Suppresses Colorectal Cancer Progression by Controlling HDAC2 Degradation and Stability.","date":"2025","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/39928532","citation_count":6,"is_preprint":false},{"pmid":"39614918","id":"PMC_39614918","title":"ZC3H13 promotes autophagy in bladder cancer through m6A methylation modification of PJA2 and ubiquitination of KSR1.","date":"2024","source":"Human cell","url":"https://pubmed.ncbi.nlm.nih.gov/39614918","citation_count":4,"is_preprint":false},{"pmid":"40148504","id":"PMC_40148504","title":"Praja2 controls P-body assembly and translation in glioblastoma by non-proteolytic ubiquitylation of DDX6.","date":"2025","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/40148504","citation_count":2,"is_preprint":false},{"pmid":"40858831","id":"PMC_40858831","title":"Aberrant PJA2-CHRM3 signaling creates a therapeutic vulnerability in gastric tumor.","date":"2025","source":"British journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/40858831","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.06.10.598176","title":"E3 ligase Praja1 mediates ubiquitination and degradation of microtubule-associated protein Tau","date":"2024-06-10","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.10.598176","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.10.19.683343","title":"Transcriptomic profiling of the middle temporal gyrus reveals differential glial/neuronal dysregulation across Alzheimer’s disease and aging","date":"2025-10-20","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.19.683343","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11895,"output_tokens":4327,"usd":0.050295},"stage2":{"model":"claude-opus-4-6","input_tokens":7781,"output_tokens":4146,"usd":0.213833},"total_usd":0.264128,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"Praja2 (PJA2) forms a stable complex with PKA regulatory (R) subunits and is phosphorylated by PKA. Rising cAMP levels promote praja2-mediated ubiquitylation and subsequent proteasomal degradation of R subunits, leading to sustained catalytic subunit activity and downstream substrate phosphorylation. This mechanism is required for efficient nuclear cAMP signalling and PKA-mediated long-term memory.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays, proteasome inhibitor experiments, in vivo mouse memory assays, loss-of-function knockdown with defined phosphorylation readouts\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, functional KD phenotype, in vivo validation, multiple orthogonal methods in a high-citation study\",\n      \"pmids\": [\"21423175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Praja2 ubiquitylates and targets MOB1 (Mob), a core component of NDR/LATS kinases and positive regulator of the Hippo cascade, for proteasomal degradation. This attenuates Hippo signalling and sustains glioblastoma growth in vivo.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitylation assay, proteasome inhibitor experiments, in vivo xenograft tumor model, knockdown with defined Hippo pathway readouts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus in vitro ubiquitylation plus in vivo model, high citation count indicating broad acceptance\",\n      \"pmids\": [\"23652010\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PJA2 forms a complex with the AMPK-related kinase SIK2 and the CDK5 activator p35. Following glucose stimulation, SIK2 phosphorylates p35 at Ser91, which triggers p35 ubiquitylation by PJA2 and promotes insulin secretion. β-cell-specific SIK2 knockout leads to p35 accumulation and impaired secretion.\",\n      \"method\": \"Affinity purification-mass spectrometry (AP-MS), Co-immunoprecipitation, in vitro ubiquitylation assay, β-cell-specific knockout mouse, glucose-stimulated insulin secretion assay\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — AP-MS complex identification, in vitro ubiquitylation, conditional KO mouse with defined physiological phenotype\",\n      \"pmids\": [\"24561619\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Praja2 acts as the E3 ubiquitin ligase that polyubiquitylates KSR1, a scaffold of the Ras/MAPK pathway, leading to its proteasomal degradation. This attenuates ERK1/2 signalling downstream of receptor stimulation, controls cancer cell growth, and is required for maintenance of mouse embryonic stem cell pluripotency.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitylation assay, proteasome inhibitor rescue, overexpression/knockdown with ERK phosphorylation and colony formation readouts, mouse ESC differentiation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, in vitro ubiquitylation, KD/OE phenotypes) with defined pathway readouts\",\n      \"pmids\": [\"27195677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PJA2 ubiquitylates the HIV-1 Tat protein with atypical, non-degradative polyubiquitin chain linkages, specifically regulating the transcription elongation step of HIV transcription. Ubiquitin acceptor sites on Tat and the ubiquitin linkage type are both variable. Proper chain assembly by PJA2 requires Tat to first bind P-TEFb.\",\n      \"method\": \"RNAi knockdown screen, in vivo ubiquitylation assay, mutational mapping of ubiquitin acceptor lysines, HIV transcription elongation reporter assay, viral replication assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional RNAi screen plus mutagenesis plus mechanistic assay, single lab\",\n      \"pmids\": [\"28345603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Praja2 directly binds MFHAS1 (confirmed by pulldown and co-immunoprecipitation) and promotes its non-degradative ubiquitylation. This ubiquitylation positively regulates TLR2-mediated JNK/p38 pathway activation and promotes M1 macrophage polarization.\",\n      \"method\": \"In vitro pulldown, co-immunoprecipitation, in situ immunostaining colocalization, ubiquitylation assay, macrophage polarization assays with pathway readouts\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP plus ubiquitylation assay plus functional polarization readout, single lab\",\n      \"pmids\": [\"28471450\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Pja2 binds TCF/LEF1 transcription factors and promotes their ubiquitylation-dependent degradation, thereby downregulating Wnt/β-catenin signalling activity and influencing embryonic stem cell differentiation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, Wnt reporter assay, gain- and loss-of-function in stem cells\",\n      \"journal\": \"International journal of stem cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus ubiquitylation assay plus reporter assay, single lab with moderate follow-up\",\n      \"pmids\": [\"30021253\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Praja2 assembles a novel centrosomal complex with TBC1D31, PKA, and OFD1. Upon GPCR-cAMP stimulation, PKA phosphorylates OFD1 at Ser735, triggering OFD1 ubiquitylation and proteasomal degradation by praja2. This pathway is essential for primary ciliogenesis; a non-phosphorylatable OFD1 mutant dramatically affects cilium morphology. Disruption of this network impairs ciliogenesis in Medaka fish.\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay, in vitro ubiquitylation assay, site-directed mutagenesis (S735A OFD1), GPCR-cAMP stimulation, ciliogenesis assays, in vivo Medaka zebrafish model\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — reconstitution-style complex identification, mutagenesis, in vitro ubiquitylation, in vivo vertebrate validation\",\n      \"pmids\": [\"33934390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PJA2 reduces KSR1 protein levels through ubiquitylation and degradation, which inhibits MEK-ERK signalling in gastric cancer cells. Overexpression of praja2 suppresses cancer cell proliferation, migration, and invasion, and inhibits tumor growth in vivo via the KSR1-MEK-ERK axis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, proteasome inhibitor rescue (MG132), in vitro proliferation/migration/invasion assays, in vivo xenograft model\",\n      \"journal\": \"Aging\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP plus ubiquitylation plus in vivo model, single lab, consistent with prior KSR1 work\",\n      \"pmids\": [\"33461174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PJA2 ubiquitylates KSR1, promoting its degradation. In the KDM5A-PJA2-KSR1 axis in gastric cancer macrophages, KDM5A suppresses PJA2 expression by removing H3K4me3 from the PJA2 promoter, thereby stabilizing KSR1 and promoting macrophage M2 polarization. ELK4 drives this axis through transcriptional activation of KDM5A.\",\n      \"method\": \"Dual luciferase reporter assay, ChIP assay, Co-immunoprecipitation, cycloheximide chase for KSR1 stability, gain- and loss-of-function assays, xenograft model\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — ChIP, Co-IP, CHX chase, functional assays in single lab; positions PJA2 in an epigenetically regulated axis\",\n      \"pmids\": [\"34372882\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"FTO demethylates m6A modification on PJA2 mRNA, reducing its decay and restoring PJA2 expression. Increased PJA2 suppresses Wnt signalling and restrains proliferation, invasion, and metastasis of pancreatic cancer cells.\",\n      \"method\": \"m6A methylation assay, mRNA stability assay, Wnt signalling reporter, gain- and loss-of-function with proliferation/invasion readouts\",\n      \"journal\": \"Molecular therapy. Nucleic acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — m6A assay plus mRNA stability plus functional readouts, single lab\",\n      \"pmids\": [\"34484859\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Praja2 forms a complex with the AP2 adapter complex and ubiquitylates it, contributing to EGFR endocytosis and clearance. Downregulation of praja2 by oncomiRs impairs EGFR endocytosis, amplifies downstream mitogenic signalling, and promotes kidney cancer cell growth. Genetic ablation of praja2 in mice upregulates EGFR and VEGFR and induces epithelial and vascular alterations in kidney tissue.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay, endocytosis assay, oncomiR overexpression, praja2 restoration experiments, praja2 knockout mouse model with RTK readout\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitylation assay, KO mouse with defined molecular phenotype, multiple orthogonal approaches\",\n      \"pmids\": [\"38379085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PJA2 interacts with TYK2 and JAK1 within the type I IFN signalling cascade, promotes their non-degradative ubiquitination, and limits the activating phosphorylation of TYK2, thereby restraining downstream STAT signalling and acting as a negative regulator of IFN signalling.\",\n      \"method\": \"TurboID proximity labeling coupled with affinity purification-mass spectrometry, functional RNAi screen, co-immunoprecipitation, ubiquitination assays, phosphorylation assays for TYK2/STAT\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — proximity proteomics plus AP-MS plus functional screen plus mechanistic Co-IP and ubiquitination assays\",\n      \"pmids\": [\"38802340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Praja2 forms a multimeric complex with the RNA helicase DDX6 and promotes non-proteolytic polyubiquitylation of DDX6 in response to cAMP/GPCR signalling, inducing P-body assembly and translational repression of target RNAs. Genetic inactivation of praja2 reshapes DDX6/mRNA complexes and polysomes, promoting cellular senescence and GBM growth arrest. An ubiquitylation-defective DDX6 mutant suppresses P-body assembly and sustains GBM growth.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitylation assay, site-directed mutagenesis (ubiquitylation-defective DDX6), polysome profiling, P-body imaging, cAMP stimulation, loss-of-function with senescence readout\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — Co-IP, in vitro ubiquitylation, mutagenesis, polysome profiling, multiple orthogonal mechanistic methods\",\n      \"pmids\": [\"40148504\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PJA2 recognizes HDAC2 via its RING-B-box domain (RBD), binds the N-terminal of HDAC2, and facilitates its ubiquitylation at Lys90, promoting HDAC2 degradation. This de-represses the IFIT family of interferon-induced genes, suppressing colorectal cancer progression. HDAC2 reciprocally regulates PJA2 expression, forming a positive feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay, ubiquitylation assay, ChIP, RNA-seq, AOM/DSS mouse CRC model, colony formation assay, domain-deletion mutagenesis\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — Co-IP, PLA, site-specific ubiquitylation (K90), ChIP, RNA-seq, in vivo CRC model, multiple orthogonal methods\",\n      \"pmids\": [\"39928532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PJA2 (RING E3 ligase) directly interacts with CHRM3 (muscarinic acetylcholine receptor M3), ubiquitinates it, and promotes its degradation, thereby suppressing downstream TGFβ-pSMAD3 signalling and gastric tumor cell progression. The catalytically dead ΔRING PJA2 mutant cannot suppress CHRM3-driven organoid growth.\",\n      \"method\": \"Mass spectrometry, co-immunoprecipitation, ubiquitination assay, degradation assay, patient-derived organoids, ΔRING mutant rescue, xenograft metastasis model\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS identification, Co-IP, ubiquitination/degradation assays, mutagenesis, PDO model; single lab, recent publication\",\n      \"pmids\": [\"40858831\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PJA2 is a RING-domain E3 ubiquitin ligase that directly ubiquitylates a broad set of substrates (including PKA regulatory subunits, MOB1, KSR1, OFD1, TCF/LEF1, MFHAS1, HDAC2, CHRM3, AP2 adapter complex, DDX6, TYK2/JAK1, and HIV-1 Tat) to control their stability or activity in a context-dependent manner (degradative or non-degradative), thereby regulating cAMP/PKA signalling duration, Hippo and Wnt pathway activity, MAPK/ERK signalling, primary ciliogenesis, receptor endocytosis, type I interferon signalling, and mRNA translation in P-bodies, and is itself regulated by PKA-mediated phosphorylation and m6A-dependent mRNA stability.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"PJA2 is a RING-domain E3 ubiquitin ligase that controls the amplitude and duration of multiple signalling pathways by ubiquitylating a diverse set of substrates for either proteasomal degradation or non-degradative regulatory modification. PJA2 attenuates cAMP/PKA signalling by degrading PKA regulatory subunits upon cAMP stimulation [PMID:21423175], suppresses Hippo signalling through MOB1 degradation [PMID:23652010], limits MAPK/ERK output by targeting the scaffold KSR1 [PMID:27195677], restrains Wnt/β-catenin signalling via TCF/LEF1 degradation [PMID:30021253], negatively regulates type I interferon signalling through non-degradative ubiquitylation of TYK2/JAK1 [PMID:38802340], promotes primary ciliogenesis by degrading OFD1 at the centrosome [PMID:33934390], facilitates EGFR endocytosis by ubiquitylating the AP2 adapter complex [PMID:38379085], and drives P-body assembly and translational repression through non-proteolytic ubiquitylation of the RNA helicase DDX6 [PMID:40148504]. PJA2 itself is regulated at transcriptional and post-transcriptional levels, including PKA-mediated phosphorylation that activates its ligase function [PMID:21423175], epigenetic silencing via KDM5A-mediated H3K4me3 removal at its promoter [PMID:34372882], and FTO-dependent m6A demethylation that stabilizes its mRNA [PMID:34484859]. Through these activities PJA2 functions as a versatile signalling rheostat in processes spanning long-term memory, ciliogenesis, receptor trafficking, innate immunity, macrophage polarization, and tumour suppression [PMID:21423175, PMID:33934390, PMID:38802340, PMID:38379085].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Establishing PJA2 as a cAMP-responsive E3 ligase that degrades PKA regulatory subunits resolved how cells terminate PKA holoenzyme reassembly and sustain catalytic activity during prolonged cAMP signalling, linking ubiquitin-dependent proteolysis to long-term memory formation.\",\n      \"evidence\": \"Co-IP, ubiquitylation assays, proteasome inhibitor rescue, PKA phosphorylation mapping, and mouse long-term memory assays\",\n      \"pmids\": [\"21423175\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PKA phosphorylation of PJA2 is required for all subsequent substrate interactions or is context-specific\", \"Structural basis for PJA2–R subunit recognition\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identifying MOB1 as a PJA2 substrate established the ligase as a negative regulator of Hippo signalling and showed that PJA2-mediated proteolysis sustains glioblastoma growth, broadening its substrate repertoire beyond the PKA axis.\",\n      \"evidence\": \"Reciprocal Co-IP, in vitro ubiquitylation, proteasome inhibitor rescue, and in vivo xenograft tumour model\",\n      \"pmids\": [\"23652010\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PJA2 targets additional Hippo pathway components\", \"Upstream signal that triggers MOB1 ubiquitylation\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstration that PJA2 operates within a SIK2–p35 complex to ubiquitylate phosphorylated p35 upon glucose stimulation revealed a phosphorylation-primed degron mechanism coupling nutrient sensing to insulin secretion.\",\n      \"evidence\": \"AP-MS complex identification, in vitro ubiquitylation, β-cell-specific SIK2 knockout mouse with insulin secretion phenotype\",\n      \"pmids\": [\"24561619\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct in vivo confirmation using β-cell-specific PJA2 knockout\", \"Whether PJA2 ubiquitylates additional CDK5-related substrates in β-cells\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showing that PJA2 ubiquitylates the Ras/MAPK scaffold KSR1 for degradation established a direct mechanism for attenuating ERK signalling intensity and linked this axis to embryonic stem cell pluripotency maintenance.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitylation, proteasome inhibitor rescue, ERK phosphorylation readouts, ESC differentiation assays\",\n      \"pmids\": [\"27195677\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether KSR2 is similarly targeted\", \"Structural determinants of KSR1 recognition by PJA2\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Discovery that PJA2 attaches atypical, non-degradative polyubiquitin chains to HIV-1 Tat—requiring prior Tat–P-TEFb binding—revealed a non-proteolytic ubiquitylation mode of PJA2 and a role in viral transcription elongation.\",\n      \"evidence\": \"RNAi screen, ubiquitylation assay with linkage analysis, ubiquitin acceptor site mutagenesis, HIV transcription elongation reporter\",\n      \"pmids\": [\"28345603\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific ubiquitin chain linkage types remain incompletely defined\", \"Independent replication in primary CD4+ T cells\", \"Whether PJA2 affects latency reversal\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of MFHAS1 as a non-degradative substrate showed PJA2 can positively regulate innate immune signalling (TLR2–JNK/p38) and macrophage M1 polarization through ubiquitylation that does not lead to target destruction.\",\n      \"evidence\": \"In vitro pulldown, Co-IP, ubiquitylation assay, macrophage polarization readouts\",\n      \"pmids\": [\"28471450\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitin chain type on MFHAS1 not determined\", \"In vivo confirmation of PJA2-dependent macrophage polarization\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstration that PJA2 ubiquitylates TCF/LEF1 for degradation extended its reach to Wnt/β-catenin signalling and embryonic stem cell fate decisions.\",\n      \"evidence\": \"Co-IP, ubiquitylation assay, Wnt reporter assay, gain/loss-of-function in stem cells\",\n      \"pmids\": [\"30021253\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Which TCF/LEF family member is preferentially targeted\", \"Independent replication outside the original lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Reconstitution of a centrosomal PJA2–TBC1D31–PKA–OFD1 complex, and showing that PKA phosphorylation of OFD1 triggers its PJA2-mediated degradation to enable ciliogenesis, established a GPCR–cAMP–ubiquitin axis at the centrosome governing primary cilium formation.\",\n      \"evidence\": \"Co-IP, PLA, in vitro ubiquitylation, S735A OFD1 mutagenesis, ciliogenesis assays, in vivo Medaka fish model\",\n      \"pmids\": [\"33934390\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PJA2 targets other centrosomal ciliogenesis regulators\", \"Human ciliopathy genetic evidence linking PJA2\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Convergent studies on KSR1 in gastric cancer confirmed PJA2's tumour-suppressive role through ERK attenuation and uncovered upstream epigenetic regulation of PJA2 expression by KDM5A-mediated H3K4me3 removal and FTO-dependent m6A demethylation of PJA2 mRNA, placing PJA2 within transcriptional and epitranscriptomic regulatory circuits.\",\n      \"evidence\": \"ChIP, dual-luciferase reporter, CHX chase, m6A methylation and mRNA stability assays, xenograft models\",\n      \"pmids\": [\"33461174\", \"34372882\", \"34484859\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional interplay between epigenetic and epitranscriptomic PJA2 regulation in the same cell type\", \"In vivo confirmation in genetic mouse models of gastric/pancreatic cancer\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of the AP2 adapter complex as a PJA2 substrate linked the ligase to receptor endocytosis: PJA2 loss impairs EGFR clearance, amplifies mitogenic signalling, and phenocopies vascular and epithelial alterations in knockout mice, establishing PJA2 as a gatekeeper of RTK surface levels.\",\n      \"evidence\": \"Co-IP, ubiquitylation assay, endocytosis assay, PJA2 knockout mouse with EGFR/VEGFR readouts\",\n      \"pmids\": [\"38379085\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PJA2 regulates endocytosis of RTKs other than EGFR\", \"Specific ubiquitin chain linkage on AP2 subunits\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Proximity proteomics and functional screening identified TYK2 and JAK1 as PJA2 substrates undergoing non-degradative ubiquitylation that limits TYK2 phosphorylation, establishing PJA2 as a negative regulator of type I interferon/STAT signalling.\",\n      \"evidence\": \"TurboID proximity labeling–AP-MS, RNAi screen, Co-IP, ubiquitination and phosphorylation assays\",\n      \"pmids\": [\"38802340\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain topology on TYK2/JAK1\", \"In vivo consequence of PJA2 loss for antiviral immunity\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Discovery that PJA2 non-proteolytically ubiquitylates the P-body helicase DDX6 in response to cAMP/GPCR signalling, driving P-body assembly and translational repression, revealed a new role in post-transcriptional gene regulation; an ubiquitylation-defective DDX6 mutant sustained glioblastoma growth, linking this axis to cellular senescence.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitylation, DDX6 ubiquitylation-site mutagenesis, polysome profiling, P-body imaging, senescence assays\",\n      \"pmids\": [\"40148504\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of translationally repressed mRNAs critical for senescence\", \"Whether PJA2 targets other P-body components\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Mapping PJA2's RING-B-box domain as the HDAC2-binding interface and Lys90 of HDAC2 as the ubiquitylation site, with consequent de-repression of IFIT innate immune genes, connected PJA2 to chromatin-level regulation of interferon-stimulated genes and colorectal cancer suppression.\",\n      \"evidence\": \"Co-IP, PLA, domain-deletion mutagenesis, site-specific ubiquitylation (K90), ChIP, RNA-seq, AOM/DSS mouse CRC model\",\n      \"pmids\": [\"39928532\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other HDACs are PJA2 substrates\", \"Crystal structure of PJA2 RBD–HDAC2 interface\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of CHRM3 as a PJA2 substrate whose ubiquitylation-dependent degradation suppresses TGFβ–pSMAD3 signalling extended PJA2's reach to GPCR turnover and gastric cancer progression.\",\n      \"evidence\": \"Mass spectrometry, Co-IP, ubiquitination/degradation assays, ΔRING mutant rescue, patient-derived organoids, xenograft metastasis model\",\n      \"pmids\": [\"40858831\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PJA2 degrades other muscarinic receptors\", \"Independent replication outside the original study\", \"Ubiquitylation site(s) on CHRM3 not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Despite extensive substrate cataloguing, no crystal or cryo-EM structure of PJA2 exists; the basis for its remarkably broad substrate selectivity, the determinants specifying degradative versus non-degradative ubiquitin chain assembly, and the in vivo physiological consequences of whole-organism PJA2 loss in mammals remain incompletely defined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of PJA2 or any PJA2–substrate complex\", \"Rules governing chain-type selectivity (K48 vs. K63 vs. atypical) unknown\", \"Comprehensive phenotyping of PJA2 knockout mice beyond kidney reported only for AP2/EGFR axis\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6, 7, 8, 11, 12, 13, 14, 15]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 2, 3, 7, 11, 12, 13, 14, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4, 5, 6, 7, 11, 12, 13, 14, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3, 6, 8, 9, 10, 12, 15]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 12, 14]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 8, 9, 14, 15]}\n    ],\n    \"complexes\": [\n      \"PJA2–TBC1D31–PKA–OFD1 centrosomal complex\",\n      \"PJA2–SIK2–p35 complex\"\n    ],\n    \"partners\": [\n      \"PRKAR1A\",\n      \"MOB1A\",\n      \"KSR1\",\n      \"OFD1\",\n      \"DDX6\",\n      \"TYK2\",\n      \"AP2A1\",\n      \"HDAC2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}