{"gene":"PRAG1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2006,"finding":"Pragmin (PRAG1) was identified as a novel effector of Rnd2 GTPase; it binds specifically to GTP-loaded Rnd2 (but not other Rho family GTPases) in a GTP-dependent manner, and this interaction stimulates RhoA activity and induces cell contraction through the RhoA/Rho-kinase pathway. Knockdown of Pragmin by siRNA enhances neurite elongation in PC12 cells, placing Pragmin downstream of Rnd2 and upstream of RhoA.","method":"Yeast two-hybrid screen; in vivo and in vitro binding assays; RhoA activity assays; siRNA knockdown in PC12 cells; morphological readout in HeLa cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays, activity assays, and siRNA loss-of-function with specific phenotypic readout; multiple orthogonal methods in one study","pmids":["16481321"],"is_preprint":false},{"year":2011,"finding":"Pragmin/SgK223 is tyrosine-phosphorylated at its EPIYA motif by Src family kinases (SFKs), and phosphorylated Pragmin binds the SH2 domain of C-terminal Src kinase (Csk), sequestering Csk in the cytoplasm away from the membrane and preventing inactivation of membrane-associated SFKs. This establishes a positive feedback loop of SFK activation, because SFKs phosphorylate Pragmin, which then sequesters Csk.","method":"Co-immunoprecipitation; subcellular fractionation; tyrosine phosphorylation assays; functional reporter assays; comparison with H. pylori CagA EPIYA effector","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, fractionation, phosphorylation assays) in one study with mechanistically coherent model","pmids":["21873224"],"is_preprint":false},{"year":2015,"finding":"SgK223/Pragmin overexpression in human pancreatic ductal epithelial cells promotes cell migration and invasion through enhanced JAK1 activation and subsequent Stat3 Tyr705 phosphorylation and transcriptional activity; SgK223 and Stat3 physically associate in vivo, and pharmacological inhibition of JAK or Stat3 blocks SgK223-driven motility and invasion.","method":"Retroviral overexpression; siRNA knockdown; immunoprecipitation; Western blot; luciferase reporter assay; selective kinase inhibitors; transwell migration/invasion assays","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, reporter assay, pharmacological inhibition) with defined phenotypic readout; single lab but comprehensive","pmids":["26215634"],"is_preprint":false},{"year":2016,"finding":"Pragmin directly binds Csk via its tyrosine-phosphorylated EPIYA motif; this complex formation potentiates Csk kinase activity, and Csk in turn phosphorylates Pragmin on Y238, Y343, and Y391 (the EPIYA motif), creating a feed-forward Csk activation loop. Pragmin and Csk co-localize to focal adhesions, and their interaction induces elongated cell morphology and elevated cell scattering in a mutually dependent manner.","method":"Co-immunoprecipitation; in vitro kinase assay; confocal co-localization; cell morphology assays; mutational analysis of phosphorylation sites","journal":"Cancer science","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase assay plus Co-IP and localization; single lab with multiple orthogonal methods","pmids":["27116701"],"is_preprint":false},{"year":2016,"finding":"SgK223 and SgK269/PEAK1 form both homotypic and heterotypic complexes dependent on their CH (α-helical) and pseudokinase (PK) domains; SgK269 bridges SgK223 to Grb2, but SgK269 cannot activate Stat3 or efficiently enhance migration in SgK223 knockout cells, demonstrating that SgK223 is functionally required downstream of SgK269 for these outputs.","method":"Mass spectrometry-based proteomics; Co-immunoprecipitation; pulldown; size-exclusion chromatography; CRISPR/Cas9 knockout; cell migration assays; Stat3 activation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (MS, Co-IP, pulldown, SEC, CRISPR KO) across a single comprehensive study","pmids":["27531744"],"is_preprint":false},{"year":2017,"finding":"Crystal structure of the SgK223 pseudokinase domain with flanking N- and C-terminal helices reveals that these helices engage in a novel fold (later termed SHED) to mediate high-affinity homodimerization, and additional regulatory interfaces on the pseudokinase domain support assembly of large open-ended oligomers. Both homo- and heterotypic association with SgK269 is mediated by the CH and PK regions.","method":"X-ray crystallography; structure-function mutagenesis; biochemical oligomerization assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with functional validation of dimerization interfaces via mutagenesis","pmids":["29079850"],"is_preprint":false},{"year":2018,"finding":"Crystal structure of the Pragmin C-terminus (residues 906–1368) confirms a classical protein-kinase fold devoid of catalytic activity despite a conserved catalytic lysine (K997). Flanking N- and C-terminal extensions form an original dimerization domain; the A1329E mutation in the C-terminal extension destabilizes dimerization and reduces Csk activation. Pragmin uses the tyrosine kinase Csk to induce protein tyrosine phosphorylation in human cells, identified by proteomics.","method":"X-ray crystallography; site-directed mutagenesis (A1329E); mass spectrometry proteomics; in-cell tyrosine phosphorylation assays; Csk kinase activity assays","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis plus proteomics; single lab but multiple orthogonal methods","pmids":["29503074"],"is_preprint":false},{"year":2014,"finding":"NACK (SgK223/PRAG1) associates with the Notch transcriptional activation complex on DNA, acts as a Notch transcriptional coactivator, and is required for Notch-mediated tumorigenesis. Homozygous NACK loss is embryonic lethal in mice. NACK is also a Notch target gene, establishing a feed-forward loop.","method":"Co-immunoprecipitation; chromatin immunoprecipitation (ChIP); luciferase reporter assays; siRNA knockdown; mouse genetic knockout; xenograft tumorigenesis assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP (DNA occupancy), Co-IP (complex membership), reporter assays, and in vivo genetic evidence in a single comprehensive study","pmids":["25038227"],"is_preprint":false},{"year":2017,"finding":"p300/CBP acetylates Mastermind-like 1 (Maml1) at K188 and K189, and this acetylation is required to recruit NACK (SgK223) to the Notch1 ternary complex, which then leads to RNA polymerase II recruitment and transcriptional initiation. NACK is recruited by Maml1 and Maml3 but not Maml2.","method":"Co-immunoprecipitation; ChIP; luciferase reporter assays; site-directed mutagenesis of acetylation sites; p300/CBP inhibitor treatment","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, ChIP, mutagenesis, and pharmacological validation; single lab with multiple orthogonal methods","pmids":["28625977"],"is_preprint":false},{"year":2021,"finding":"NACK (SgK223) is required for recruitment of RNA polymerase II to Notch-dependent promoters, while the Integrator complex (INT) is required for RNAPII phosphorylation at serine 5 (RNAPII-S5P) to initiate transcription; both NACK and INT act coordinately as components of the Notch transcriptional supercomplex.","method":"Size exclusion chromatography; CSL-DNA affinity FPLC; LC-MS/MS; ChIP; siRNA knockdown; HEK293T transfection reconstitution assay; xenograft assays","journal":"Cell communication and signaling : CCS","confidence":"High","confidence_rationale":"Tier 2 / Moderate — biochemical purification of complex by SEC/FPLC, MS identification, ChIP showing promoter occupancy; multiple orthogonal methods","pmids":["34551776"],"is_preprint":false},{"year":2023,"finding":"NACK (SgK223/PRAG1) binds and hydrolyzes ATP, and only ATP-bound NACK can bind to the Notch ternary complex (NTC); a small-molecule inhibitor (Z271-0326) targeting this ATP-dependent function blocks Notch-mediated transcription.","method":"ATPase activity assay; nucleotide-binding assay; co-immunoprecipitation with NTC; small-molecule inhibitor screen; PDX mouse models","journal":"Molecular therapy oncolytics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — ATPase and binding assays establish nucleotide dependence; single lab, mechanistic follow-up limited by abstract detail","pmids":["36938545"],"is_preprint":false},{"year":2022,"finding":"PEAK2 (PRAG1/SgK223) localizes to focal adhesions of colorectal cancer cells, induces cellular protein tyrosine phosphorylation (despite catalytic inactivity), and activates ABL tyrosine kinase; ABL-dependent formation of actin-rich filopodia drives cell invasion. The main phosphorylation site Tyr413 (phosphorylated by SRC) is required for all these transforming activities.","method":"Phosphoproteomic analysis; siRNA knockdown; overexpression; confocal localization; actin cytoskeleton imaging; ABL kinase assays; site-directed mutagenesis (Y413); nude mouse xenograft","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics, mutagenesis, localization, and in vivo xenograft; single lab","pmids":["35740644"],"is_preprint":false},{"year":2022,"finding":"SGK223 (PRAG1) is phosphorylated at Y411 by c-Src and in response to EGFR; tyrosine-phosphorylated SGK223 at Y411 interacts with CSK, upregulating c-Src activity and promoting cell migration. hAMSC secretome suppresses this pathway to inhibit Panc1 cancer cell invasion.","method":"Co-culture system; Western blot for phospho-SGK223 Y411, phospho-Src Y416/Y530; co-immunoprecipitation; invasion assay","journal":"Medical oncology (Northwood, London, England)","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP and phospho-blot, single lab, limited mechanistic follow-up","pmids":["35059869"],"is_preprint":false},{"year":2025,"finding":"PRAG1 forms dynamic phase-separated condensates in cells mediated by its αN and αJ helices; these condensates are required for mediating cell contraction, as condensate-formation-deficient mutants lose this function. Spherical PRAG1 condensates form under diverse stress conditions and in dopaminergic neurons from a Parkinson's disease patient.","method":"Live-cell imaging of condensates; mutagenesis of αN and αJ helices; stress induction assays; cell contraction phenotypic readout; iPSC-derived dopaminergic neurons","journal":"Biomolecules","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging plus mutagenesis with functional (contraction) readout; single lab, single study","pmids":["40149915"],"is_preprint":false},{"year":2025,"finding":"PRAG1 binds to F-box protein FBXO11 and reverses FBXO11-mediated inhibition of SNAIL1 protein expression (i.e., PRAG1 prevents FBXO11-dependent degradation of SNAIL1), thereby promoting EMT and cell proliferation in biliary epithelial cells.","method":"Co-immunoprecipitation (PRAG1–FBXO11 interaction); overexpression/knockdown with Western blot for SNAIL1, EMT markers; cell proliferation assays","journal":"Biochimica et biophysica acta. Molecular basis of disease","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP plus expression readouts; single lab, limited mechanistic depth","pmids":["40743806"],"is_preprint":false}],"current_model":"PRAG1 (Pragmin/SgK223/PEAK2/NACK) is a catalytically inactive pseudokinase that functions as a scaffolding protein with multiple mechanistic roles: it binds GTP-loaded Rnd2 to activate RhoA signaling; it undergoes SFK-mediated EPIYA tyrosine phosphorylation (at Y391/Y411) to recruit and sequester Csk, sustaining a positive feedback loop of SFK/Csk activation at focal adhesions; it dimerizes via a split helical dimerization (SHED) domain to assemble signaling complexes that activate the ABL tyrosine kinase and promote actin-based invasion; it forms heterotypic complexes with SgK269/PEAK1 to regulate Stat3 and Grb2-dependent signaling; it acts as an ATPase-dependent transcriptional coactivator of Notch by associating with the Notch ternary complex (recruited via p300-acetylated Maml1) and recruiting RNA polymerase II; it cooperates with the Integrator complex to drive RNAPII-S5P-dependent Notch target gene transcription; it forms phase-separated condensates via αN/αJ helices to mediate stress-induced cell contraction; and it binds FBXO11 to stabilize SNAIL1 and promote EMT."},"narrative":{"mechanistic_narrative":"PRAG1 (Pragmin/SgK223/PEAK2/NACK) is a catalytically inactive pseudokinase that operates as a multifunctional scaffold coordinating Rho-family GTPase signaling, Src-family kinase (SFK) regulation, and Notch-dependent transcription [PMID:16481321, PMID:21873224, PMID:25038227]. It was first defined as a GTP-dependent effector of Rnd2 that stimulates RhoA/Rho-kinase signaling and cell contraction [PMID:16481321]. PRAG1 is tyrosine-phosphorylated on its EPIYA motif (Y391/Y411/Y413) by SFKs and, in response to EGFR, then binds and sequesters the SH2 domain of C-terminal Src kinase (Csk); this both potentiates Csk activity in a feed-forward loop and sustains membrane-associated SFK activation, driving cell migration and invasion at focal adhesions [PMID:21873224, PMID:27116701, PMID:35740644, PMID:35059869]. Despite lacking catalytic activity, PRAG1 induces cellular protein tyrosine phosphorylation by recruiting active kinases such as Csk and ABL, the latter promoting actin-rich filopodia and invasion [PMID:29503074, PMID:35740644]. Structurally, flanking N- and C-terminal helices around the pseudokinase domain form a split helical dimerization (SHED) fold mediating high-affinity homodimerization and higher-order oligomerization, and also support heterotypic association with the related pseudokinase SgK269/PEAK1, which bridges PRAG1 to Grb2 and is required for JAK1/Stat3 activation [PMID:26215634, PMID:27531744, PMID:29079850, PMID:29503074]. In a distinct nuclear role, PRAG1/NACK associates with the Notch ternary complex on DNA as a transcriptional coactivator, recruited via p300/CBP-acetylated Maml1 and required for RNA polymerase II recruitment and, together with the Integrator complex, RNAPII-S5P-dependent transcription of Notch targets; this function depends on ATP binding and hydrolysis, and PRAG1 is itself a Notch target gene forming a feed-forward loop essential for Notch-driven tumorigenesis and embryonic viability [PMID:25038227, PMID:28625977, PMID:34551776, PMID:36938545]. PRAG1 additionally forms stress-induced phase-separated condensates via its αN/αJ helices that mediate cell contraction [PMID:40149915], and stabilizes SNAIL1 by binding FBXO11 to promote EMT [PMID:40743806].","teleology":[{"year":2006,"claim":"Established the founding function of PRAG1 by placing it in a GTPase signaling axis, answering what upstream and downstream partners control its biological output.","evidence":"Yeast two-hybrid, GTP-dependent binding and RhoA activity assays, and siRNA knockdown with morphological readout in PC12/HeLa cells","pmids":["16481321"],"confidence":"High","gaps":["Did not define the molecular mechanism linking Rnd2 binding to RhoA activation","No structural basis for the Rnd2 interaction"]},{"year":2011,"claim":"Defined how PRAG1 regulates SFK signaling, showing that EPIYA phosphorylation converts it into a Csk-sequestering scaffold that sustains SFK activity.","evidence":"Co-IP, subcellular fractionation, and tyrosine phosphorylation assays compared against H. pylori CagA","pmids":["21873224"],"confidence":"High","gaps":["Did not resolve which SFK residues PRAG1 protects","Stoichiometry of cytoplasmic Csk sequestration unquantified"]},{"year":2014,"claim":"Revealed an unexpected nuclear function as a Notch transcriptional coactivator and demonstrated its requirement for tumorigenesis and development.","evidence":"ChIP, Co-IP, reporter assays, siRNA, mouse knockout, and xenograft tumorigenesis","pmids":["25038227"],"confidence":"High","gaps":["Mechanism of recruitment to the Notch complex not yet defined","Unclear how a focal-adhesion scaffold reaches chromatin"]},{"year":2015,"claim":"Connected PRAG1 overexpression to JAK1/Stat3-driven invasion, identifying a cytokine-signaling output downstream of the scaffold.","evidence":"Retroviral overexpression, siRNA, Co-IP, luciferase reporter, kinase inhibitors, and transwell assays in pancreatic cells","pmids":["26215634"],"confidence":"High","gaps":["Direct vs indirect nature of the SgK223-Stat3 association unresolved","How JAK1 is activated by a pseudokinase scaffold unknown"]},{"year":2016,"claim":"Showed PRAG1 functions within heterotypic PEAK-family complexes, establishing it as the functionally required node downstream of SgK269/PEAK1 for Stat3 and migration outputs.","evidence":"MS proteomics, Co-IP, pulldown, SEC, and CRISPR knockout with migration/Stat3 assays","pmids":["27531744"],"confidence":"High","gaps":["Functional difference between homo- and heterotypic complexes not fully dissected"]},{"year":2016,"claim":"Clarified the Csk interaction as a reciprocal feed-forward loop in which Csk phosphorylates PRAG1 and PRAG1 potentiates Csk activity at focal adhesions.","evidence":"Co-IP, in vitro kinase assay, confocal co-localization, and phosphosite mutagenesis","pmids":["27116701"],"confidence":"High","gaps":["Net cellular consequence of simultaneous Csk activation and sequestration not reconciled"]},{"year":2017,"claim":"Provided the structural basis for PRAG1 oligomerization, defining the SHED fold that drives homodimerization and PEAK1 heterotypic assembly.","evidence":"X-ray crystallography of the pseudokinase domain with flanking helices plus mutagenesis and oligomerization assays","pmids":["29079850"],"confidence":"High","gaps":["Link between specific oligomeric states and signaling outputs not established"]},{"year":2017,"claim":"Defined the recruitment mechanism to the Notch complex, showing p300/CBP acetylation of Maml1 is required to bring NACK in for RNAPII recruitment.","evidence":"Co-IP, ChIP, reporter assays, acetylation-site mutagenesis, and p300/CBP inhibition","pmids":["28625977"],"confidence":"High","gaps":["Why Maml2 fails to recruit NACK not explained at the structural level"]},{"year":2018,"claim":"Confirmed catalytic inactivity by structure while showing PRAG1 still drives tyrosine phosphorylation through Csk, and identified a dimerization residue (A1329) controlling Csk activation.","evidence":"X-ray crystallography of the C-terminus, A1329E mutagenesis, proteomics, and Csk activity assays","pmids":["29503074"],"confidence":"High","gaps":["Full substrate repertoire of the Csk-PRAG1 axis incompletely mapped"]},{"year":2021,"claim":"Resolved the division of labor in Notch transcription, with NACK recruiting RNAPII and the Integrator complex driving its S5 phosphorylation.","evidence":"SEC, CSL-DNA affinity FPLC, LC-MS/MS, ChIP, siRNA, and HEK293T reconstitution","pmids":["34551776"],"confidence":"High","gaps":["Direct physical contacts between NACK and RNAPII not mapped","Order of supercomplex assembly unresolved"]},{"year":2023,"claim":"Identified an ATP-dependent function gating PRAG1's coactivator role, with ATP binding required for Notch complex association and druggable by a small molecule.","evidence":"ATPase and nucleotide-binding assays, Co-IP with the NTC, inhibitor screen, and PDX models","pmids":["36938545"],"confidence":"Medium","gaps":["Structural basis for ATP-dependent NTC binding not determined","Mechanism limited by abstract-level detail"]},{"year":2022,"claim":"Extended the SFK/Csk and ABL invasion mechanism to colorectal and pancreatic contexts, pinpointing Y413/Y411 as the SRC/EGFR-driven phosphosite required for transformation.","evidence":"Phosphoproteomics, mutagenesis (Y413), localization, ABL kinase assays, xenografts, co-culture, and phospho-blots","pmids":["35740644","35059869"],"confidence":"Medium","gaps":["Y411 vs Y413 numbering/usage across studies not unified","Y411 single Co-IP without reciprocal validation in the secretome study"]},{"year":2025,"claim":"Demonstrated that PRAG1 forms stress-induced phase-separated condensates via αN/αJ helices that are functionally required for cell contraction, linking it to neuronal stress states.","evidence":"Live-cell condensate imaging, αN/αJ mutagenesis, stress induction, contraction readout, and iPSC-derived dopaminergic neurons","pmids":["40149915"],"confidence":"Medium","gaps":["Relationship of condensates to the SHED-mediated oligomers unclear","Disease relevance in Parkinson's not causally established"]},{"year":2025,"claim":"Identified a new role in EMT through FBXO11 binding that stabilizes SNAIL1, connecting PRAG1 to ubiquitin-pathway regulation.","evidence":"Co-IP, overexpression/knockdown with EMT-marker Western blots, and proliferation assays in biliary epithelial cells","pmids":["40743806"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal validation","Whether PRAG1 directly competes with FBXO11 substrate binding unknown"]},{"year":null,"claim":"How PRAG1 partitions between its cytoplasmic scaffolding/SFK roles, nuclear Notch coactivator role, and condensate-forming state, and what signals govern this switching, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking ATP-dependence, oligomerization, and condensate formation","Spatial regulation between focal adhesions, chromatin, and condensates undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,6,11]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,4,5]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[7,8,9]},{"term_id":"GO:0140657","term_label":"ATP-dependent activity","supporting_discovery_ids":[10]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[7,8,9]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[11]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[7,8,9]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[7]}],"complexes":["Notch transcriptional activation complex","PEAK family (SgK223/SgK269) complex"],"partners":["RND2","CSK","STAT3","SGK269/PEAK1","GRB2","MAML1","ABL","FBXO11"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q86YV5","full_name":"Inactive tyrosine-protein kinase PRAG1","aliases":["PEAK1-related kinase-activating pseudokinase 1","Pragmin","Sugen kinase 223","SgK223"],"length_aa":1406,"mass_kda":149.6,"function":"Catalytically inactive protein kinase that acts as a scaffold protein. Functions as an effector of the small GTPase RND2, which stimulates RhoA activity and inhibits NGF-induced neurite outgrowth (By similarity). Promotes Src family kinase (SFK) signaling by regulating the subcellular localization of CSK, a negative regulator of these kinases, leading to the regulation of cell morphology and motility by a CSK-dependent mechanism (By similarity). Acts as a critical coactivator of Notch signaling (By similarity)","subcellular_location":"Cytoplasm; Cell junction, focal adhesion; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q86YV5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PRAG1","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/PRAG1","total_profiled":1310},"omim":[{"mim_id":"618526","title":"PEAK FAMILY MEMBER 3; PEAK3","url":"https://www.omim.org/entry/618526"},{"mim_id":"617344","title":"PEAK1-RELATED KINASE-ACTIVATING PSEUDOKINASE 1; PRAG1","url":"https://www.omim.org/entry/617344"},{"mim_id":"601555","title":"RHO FAMILY GTPase 2; RND2","url":"https://www.omim.org/entry/601555"},{"mim_id":"190198","title":"NOTCH RECEPTOR 1; NOTCH1","url":"https://www.omim.org/entry/190198"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoli","reliability":"Uncertain"},{"location":"Focal adhesion sites","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":41.0},{"tissue":"thyroid gland","ntpm":22.0}],"url":"https://www.proteinatlas.org/search/PRAG1"},"hgnc":{"alias_symbol":["DKFZp761P0423","SgK223","PRAGMIN","PEAK2","NACK"],"prev_symbol":[]},"alphafold":{"accession":"Q86YV5","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86YV5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86YV5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86YV5-F1-predicted_aligned_error_v6.png","plddt_mean":50.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PRAG1","jax_strain_url":"https://www.jax.org/strain/search?query=PRAG1"},"sequence":{"accession":"Q86YV5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86YV5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86YV5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86YV5"}},"corpus_meta":[{"pmid":"16481321","id":"PMC_16481321","title":"Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity.","date":"2006","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16481321","citation_count":70,"is_preprint":false},{"pmid":"21873224","id":"PMC_21873224","title":"Mammalian Pragmin regulates Src family kinases via the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif that is exploited by bacterial effectors.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21873224","citation_count":53,"is_preprint":false},{"pmid":"26215634","id":"PMC_26215634","title":"The pseudokinase SgK223 promotes invasion of pancreatic ductal epithelial cells through JAK1/Stat3 signaling.","date":"2015","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/26215634","citation_count":46,"is_preprint":false},{"pmid":"29079850","id":"PMC_29079850","title":"Structure of SgK223 pseudokinase reveals novel mechanisms of homotypic and heterotypic association.","date":"2017","source":"Nature 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research","url":"https://pubmed.ncbi.nlm.nih.gov/28625977","citation_count":21,"is_preprint":false},{"pmid":"25748359","id":"PMC_25748359","title":"NACK kinesin is required for metaphase chromosome alignment and cytokinesis in the moss Physcomitrella patens.","date":"2015","source":"Cell structure and function","url":"https://pubmed.ncbi.nlm.nih.gov/25748359","citation_count":20,"is_preprint":false},{"pmid":"26782286","id":"PMC_26782286","title":"Silencing NACK by siRNA inhibits tumorigenesis in non-small cell lung cancer via targeting Notch1 signaling pathway.","date":"2016","source":"Oncology reports","url":"https://pubmed.ncbi.nlm.nih.gov/26782286","citation_count":17,"is_preprint":false},{"pmid":"35059869","id":"PMC_35059869","title":"The inhibition of Panc1 cancer cells invasion by hAMSCs secretome through suppression of tyrosine phosphorylation of SGK223 (at Y411 site), c-Src (at Y416, Y530 sites), AKT activity, and JAK1/Stat3 signaling.","date":"2022","source":"Medical oncology (Northwood, London, England)","url":"https://pubmed.ncbi.nlm.nih.gov/35059869","citation_count":14,"is_preprint":false},{"pmid":"29105536","id":"PMC_29105536","title":"The pseudokinases SgK269 and SgK223: A novel oncogenic alliance in human cancer.","date":"2017","source":"Cell adhesion & migration","url":"https://pubmed.ncbi.nlm.nih.gov/29105536","citation_count":12,"is_preprint":false},{"pmid":"34551776","id":"PMC_34551776","title":"NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis.","date":"2021","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/34551776","citation_count":10,"is_preprint":false},{"pmid":"30906642","id":"PMC_30906642","title":"SHEDding light on the role of Pragmin pseudo-kinases in cancer.","date":"2019","source":"American journal of cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/30906642","citation_count":9,"is_preprint":false},{"pmid":"35740644","id":"PMC_35740644","title":"Oncogenic Signalling of PEAK2 Pseudokinase in Colon Cancer.","date":"2022","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/35740644","citation_count":6,"is_preprint":false},{"pmid":"40743806","id":"PMC_40743806","title":"PRAG1 promotes cholangiocyte epithelial-mesenchymal transition and liver fibrosis in biliary atresia.","date":"2025","source":"Biochimica et biophysica acta. Molecular basis of disease","url":"https://pubmed.ncbi.nlm.nih.gov/40743806","citation_count":4,"is_preprint":false},{"pmid":"36938545","id":"PMC_36938545","title":"A novel chemical attack on Notch-mediated transcription by targeting the NACK ATPase.","date":"2023","source":"Molecular therapy oncolytics","url":"https://pubmed.ncbi.nlm.nih.gov/36938545","citation_count":2,"is_preprint":false},{"pmid":"40149915","id":"PMC_40149915","title":"PRAG1 Condensation Drives Cell Contraction Under Stress.","date":"2025","source":"Biomolecules","url":"https://pubmed.ncbi.nlm.nih.gov/40149915","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11736,"output_tokens":4232,"usd":0.049344,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12084,"output_tokens":4557,"usd":0.087172,"stage2_stop_reason":"end_turn"},"total_usd":0.136516,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"Pragmin (PRAG1) was identified as a novel effector of Rnd2 GTPase; it binds specifically to GTP-loaded Rnd2 (but not other Rho family GTPases) in a GTP-dependent manner, and this interaction stimulates RhoA activity and induces cell contraction through the RhoA/Rho-kinase pathway. Knockdown of Pragmin by siRNA enhances neurite elongation in PC12 cells, placing Pragmin downstream of Rnd2 and upstream of RhoA.\",\n      \"method\": \"Yeast two-hybrid screen; in vivo and in vitro binding assays; RhoA activity assays; siRNA knockdown in PC12 cells; morphological readout in HeLa cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays, activity assays, and siRNA loss-of-function with specific phenotypic readout; multiple orthogonal methods in one study\",\n      \"pmids\": [\"16481321\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Pragmin/SgK223 is tyrosine-phosphorylated at its EPIYA motif by Src family kinases (SFKs), and phosphorylated Pragmin binds the SH2 domain of C-terminal Src kinase (Csk), sequestering Csk in the cytoplasm away from the membrane and preventing inactivation of membrane-associated SFKs. This establishes a positive feedback loop of SFK activation, because SFKs phosphorylate Pragmin, which then sequesters Csk.\",\n      \"method\": \"Co-immunoprecipitation; subcellular fractionation; tyrosine phosphorylation assays; functional reporter assays; comparison with H. pylori CagA EPIYA effector\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, fractionation, phosphorylation assays) in one study with mechanistically coherent model\",\n      \"pmids\": [\"21873224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SgK223/Pragmin overexpression in human pancreatic ductal epithelial cells promotes cell migration and invasion through enhanced JAK1 activation and subsequent Stat3 Tyr705 phosphorylation and transcriptional activity; SgK223 and Stat3 physically associate in vivo, and pharmacological inhibition of JAK or Stat3 blocks SgK223-driven motility and invasion.\",\n      \"method\": \"Retroviral overexpression; siRNA knockdown; immunoprecipitation; Western blot; luciferase reporter assay; selective kinase inhibitors; transwell migration/invasion assays\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, reporter assay, pharmacological inhibition) with defined phenotypic readout; single lab but comprehensive\",\n      \"pmids\": [\"26215634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Pragmin directly binds Csk via its tyrosine-phosphorylated EPIYA motif; this complex formation potentiates Csk kinase activity, and Csk in turn phosphorylates Pragmin on Y238, Y343, and Y391 (the EPIYA motif), creating a feed-forward Csk activation loop. Pragmin and Csk co-localize to focal adhesions, and their interaction induces elongated cell morphology and elevated cell scattering in a mutually dependent manner.\",\n      \"method\": \"Co-immunoprecipitation; in vitro kinase assay; confocal co-localization; cell morphology assays; mutational analysis of phosphorylation sites\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase assay plus Co-IP and localization; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"27116701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SgK223 and SgK269/PEAK1 form both homotypic and heterotypic complexes dependent on their CH (α-helical) and pseudokinase (PK) domains; SgK269 bridges SgK223 to Grb2, but SgK269 cannot activate Stat3 or efficiently enhance migration in SgK223 knockout cells, demonstrating that SgK223 is functionally required downstream of SgK269 for these outputs.\",\n      \"method\": \"Mass spectrometry-based proteomics; Co-immunoprecipitation; pulldown; size-exclusion chromatography; CRISPR/Cas9 knockout; cell migration assays; Stat3 activation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (MS, Co-IP, pulldown, SEC, CRISPR KO) across a single comprehensive study\",\n      \"pmids\": [\"27531744\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure of the SgK223 pseudokinase domain with flanking N- and C-terminal helices reveals that these helices engage in a novel fold (later termed SHED) to mediate high-affinity homodimerization, and additional regulatory interfaces on the pseudokinase domain support assembly of large open-ended oligomers. Both homo- and heterotypic association with SgK269 is mediated by the CH and PK regions.\",\n      \"method\": \"X-ray crystallography; structure-function mutagenesis; biochemical oligomerization assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with functional validation of dimerization interfaces via mutagenesis\",\n      \"pmids\": [\"29079850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structure of the Pragmin C-terminus (residues 906–1368) confirms a classical protein-kinase fold devoid of catalytic activity despite a conserved catalytic lysine (K997). Flanking N- and C-terminal extensions form an original dimerization domain; the A1329E mutation in the C-terminal extension destabilizes dimerization and reduces Csk activation. Pragmin uses the tyrosine kinase Csk to induce protein tyrosine phosphorylation in human cells, identified by proteomics.\",\n      \"method\": \"X-ray crystallography; site-directed mutagenesis (A1329E); mass spectrometry proteomics; in-cell tyrosine phosphorylation assays; Csk kinase activity assays\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis plus proteomics; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"29503074\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NACK (SgK223/PRAG1) associates with the Notch transcriptional activation complex on DNA, acts as a Notch transcriptional coactivator, and is required for Notch-mediated tumorigenesis. Homozygous NACK loss is embryonic lethal in mice. NACK is also a Notch target gene, establishing a feed-forward loop.\",\n      \"method\": \"Co-immunoprecipitation; chromatin immunoprecipitation (ChIP); luciferase reporter assays; siRNA knockdown; mouse genetic knockout; xenograft tumorigenesis assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP (DNA occupancy), Co-IP (complex membership), reporter assays, and in vivo genetic evidence in a single comprehensive study\",\n      \"pmids\": [\"25038227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"p300/CBP acetylates Mastermind-like 1 (Maml1) at K188 and K189, and this acetylation is required to recruit NACK (SgK223) to the Notch1 ternary complex, which then leads to RNA polymerase II recruitment and transcriptional initiation. NACK is recruited by Maml1 and Maml3 but not Maml2.\",\n      \"method\": \"Co-immunoprecipitation; ChIP; luciferase reporter assays; site-directed mutagenesis of acetylation sites; p300/CBP inhibitor treatment\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ChIP, mutagenesis, and pharmacological validation; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28625977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NACK (SgK223) is required for recruitment of RNA polymerase II to Notch-dependent promoters, while the Integrator complex (INT) is required for RNAPII phosphorylation at serine 5 (RNAPII-S5P) to initiate transcription; both NACK and INT act coordinately as components of the Notch transcriptional supercomplex.\",\n      \"method\": \"Size exclusion chromatography; CSL-DNA affinity FPLC; LC-MS/MS; ChIP; siRNA knockdown; HEK293T transfection reconstitution assay; xenograft assays\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical purification of complex by SEC/FPLC, MS identification, ChIP showing promoter occupancy; multiple orthogonal methods\",\n      \"pmids\": [\"34551776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NACK (SgK223/PRAG1) binds and hydrolyzes ATP, and only ATP-bound NACK can bind to the Notch ternary complex (NTC); a small-molecule inhibitor (Z271-0326) targeting this ATP-dependent function blocks Notch-mediated transcription.\",\n      \"method\": \"ATPase activity assay; nucleotide-binding assay; co-immunoprecipitation with NTC; small-molecule inhibitor screen; PDX mouse models\",\n      \"journal\": \"Molecular therapy oncolytics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — ATPase and binding assays establish nucleotide dependence; single lab, mechanistic follow-up limited by abstract detail\",\n      \"pmids\": [\"36938545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PEAK2 (PRAG1/SgK223) localizes to focal adhesions of colorectal cancer cells, induces cellular protein tyrosine phosphorylation (despite catalytic inactivity), and activates ABL tyrosine kinase; ABL-dependent formation of actin-rich filopodia drives cell invasion. The main phosphorylation site Tyr413 (phosphorylated by SRC) is required for all these transforming activities.\",\n      \"method\": \"Phosphoproteomic analysis; siRNA knockdown; overexpression; confocal localization; actin cytoskeleton imaging; ABL kinase assays; site-directed mutagenesis (Y413); nude mouse xenograft\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics, mutagenesis, localization, and in vivo xenograft; single lab\",\n      \"pmids\": [\"35740644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SGK223 (PRAG1) is phosphorylated at Y411 by c-Src and in response to EGFR; tyrosine-phosphorylated SGK223 at Y411 interacts with CSK, upregulating c-Src activity and promoting cell migration. hAMSC secretome suppresses this pathway to inhibit Panc1 cancer cell invasion.\",\n      \"method\": \"Co-culture system; Western blot for phospho-SGK223 Y411, phospho-Src Y416/Y530; co-immunoprecipitation; invasion assay\",\n      \"journal\": \"Medical oncology (Northwood, London, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and phospho-blot, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"35059869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PRAG1 forms dynamic phase-separated condensates in cells mediated by its αN and αJ helices; these condensates are required for mediating cell contraction, as condensate-formation-deficient mutants lose this function. Spherical PRAG1 condensates form under diverse stress conditions and in dopaminergic neurons from a Parkinson's disease patient.\",\n      \"method\": \"Live-cell imaging of condensates; mutagenesis of αN and αJ helices; stress induction assays; cell contraction phenotypic readout; iPSC-derived dopaminergic neurons\",\n      \"journal\": \"Biomolecules\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging plus mutagenesis with functional (contraction) readout; single lab, single study\",\n      \"pmids\": [\"40149915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PRAG1 binds to F-box protein FBXO11 and reverses FBXO11-mediated inhibition of SNAIL1 protein expression (i.e., PRAG1 prevents FBXO11-dependent degradation of SNAIL1), thereby promoting EMT and cell proliferation in biliary epithelial cells.\",\n      \"method\": \"Co-immunoprecipitation (PRAG1–FBXO11 interaction); overexpression/knockdown with Western blot for SNAIL1, EMT markers; cell proliferation assays\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular basis of disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus expression readouts; single lab, limited mechanistic depth\",\n      \"pmids\": [\"40743806\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PRAG1 (Pragmin/SgK223/PEAK2/NACK) is a catalytically inactive pseudokinase that functions as a scaffolding protein with multiple mechanistic roles: it binds GTP-loaded Rnd2 to activate RhoA signaling; it undergoes SFK-mediated EPIYA tyrosine phosphorylation (at Y391/Y411) to recruit and sequester Csk, sustaining a positive feedback loop of SFK/Csk activation at focal adhesions; it dimerizes via a split helical dimerization (SHED) domain to assemble signaling complexes that activate the ABL tyrosine kinase and promote actin-based invasion; it forms heterotypic complexes with SgK269/PEAK1 to regulate Stat3 and Grb2-dependent signaling; it acts as an ATPase-dependent transcriptional coactivator of Notch by associating with the Notch ternary complex (recruited via p300-acetylated Maml1) and recruiting RNA polymerase II; it cooperates with the Integrator complex to drive RNAPII-S5P-dependent Notch target gene transcription; it forms phase-separated condensates via αN/αJ helices to mediate stress-induced cell contraction; and it binds FBXO11 to stabilize SNAIL1 and promote EMT.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PRAG1 (Pragmin/SgK223/PEAK2/NACK) is a catalytically inactive pseudokinase that operates as a multifunctional scaffold coordinating Rho-family GTPase signaling, Src-family kinase (SFK) regulation, and Notch-dependent transcription [#0, #1, #7]. It was first defined as a GTP-dependent effector of Rnd2 that stimulates RhoA/Rho-kinase signaling and cell contraction [#0]. PRAG1 is tyrosine-phosphorylated on its EPIYA motif (Y391/Y411/Y413) by SFKs and, in response to EGFR, then binds and sequesters the SH2 domain of C-terminal Src kinase (Csk); this both potentiates Csk activity in a feed-forward loop and sustains membrane-associated SFK activation, driving cell migration and invasion at focal adhesions [#1, #3, #11, #12]. Despite lacking catalytic activity, PRAG1 induces cellular protein tyrosine phosphorylation by recruiting active kinases such as Csk and ABL, the latter promoting actin-rich filopodia and invasion [#6, #11]. Structurally, flanking N- and C-terminal helices around the pseudokinase domain form a split helical dimerization (SHED) fold mediating high-affinity homodimerization and higher-order oligomerization, and also support heterotypic association with the related pseudokinase SgK269/PEAK1, which bridges PRAG1 to Grb2 and is required for JAK1/Stat3 activation [#2, #4, #5, #6]. In a distinct nuclear role, PRAG1/NACK associates with the Notch ternary complex on DNA as a transcriptional coactivator, recruited via p300/CBP-acetylated Maml1 and required for RNA polymerase II recruitment and, together with the Integrator complex, RNAPII-S5P-dependent transcription of Notch targets; this function depends on ATP binding and hydrolysis, and PRAG1 is itself a Notch target gene forming a feed-forward loop essential for Notch-driven tumorigenesis and embryonic viability [#7, #8, #9, #10]. PRAG1 additionally forms stress-induced phase-separated condensates via its \\u03b1N/\\u03b1J helices that mediate cell contraction [#13], and stabilizes SNAIL1 by binding FBXO11 to promote EMT [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2006,\n      \"claim\": \"Established the founding function of PRAG1 by placing it in a GTPase signaling axis, answering what upstream and downstream partners control its biological output.\",\n      \"evidence\": \"Yeast two-hybrid, GTP-dependent binding and RhoA activity assays, and siRNA knockdown with morphological readout in PC12/HeLa cells\",\n      \"pmids\": [\"16481321\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the molecular mechanism linking Rnd2 binding to RhoA activation\", \"No structural basis for the Rnd2 interaction\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined how PRAG1 regulates SFK signaling, showing that EPIYA phosphorylation converts it into a Csk-sequestering scaffold that sustains SFK activity.\",\n      \"evidence\": \"Co-IP, subcellular fractionation, and tyrosine phosphorylation assays compared against H. pylori CagA\",\n      \"pmids\": [\"21873224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which SFK residues PRAG1 protects\", \"Stoichiometry of cytoplasmic Csk sequestration unquantified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed an unexpected nuclear function as a Notch transcriptional coactivator and demonstrated its requirement for tumorigenesis and development.\",\n      \"evidence\": \"ChIP, Co-IP, reporter assays, siRNA, mouse knockout, and xenograft tumorigenesis\",\n      \"pmids\": [\"25038227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of recruitment to the Notch complex not yet defined\", \"Unclear how a focal-adhesion scaffold reaches chromatin\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Connected PRAG1 overexpression to JAK1/Stat3-driven invasion, identifying a cytokine-signaling output downstream of the scaffold.\",\n      \"evidence\": \"Retroviral overexpression, siRNA, Co-IP, luciferase reporter, kinase inhibitors, and transwell assays in pancreatic cells\",\n      \"pmids\": [\"26215634\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect nature of the SgK223-Stat3 association unresolved\", \"How JAK1 is activated by a pseudokinase scaffold unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed PRAG1 functions within heterotypic PEAK-family complexes, establishing it as the functionally required node downstream of SgK269/PEAK1 for Stat3 and migration outputs.\",\n      \"evidence\": \"MS proteomics, Co-IP, pulldown, SEC, and CRISPR knockout with migration/Stat3 assays\",\n      \"pmids\": [\"27531744\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional difference between homo- and heterotypic complexes not fully dissected\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Clarified the Csk interaction as a reciprocal feed-forward loop in which Csk phosphorylates PRAG1 and PRAG1 potentiates Csk activity at focal adhesions.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, confocal co-localization, and phosphosite mutagenesis\",\n      \"pmids\": [\"27116701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Net cellular consequence of simultaneous Csk activation and sequestration not reconciled\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided the structural basis for PRAG1 oligomerization, defining the SHED fold that drives homodimerization and PEAK1 heterotypic assembly.\",\n      \"evidence\": \"X-ray crystallography of the pseudokinase domain with flanking helices plus mutagenesis and oligomerization assays\",\n      \"pmids\": [\"29079850\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Link between specific oligomeric states and signaling outputs not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined the recruitment mechanism to the Notch complex, showing p300/CBP acetylation of Maml1 is required to bring NACK in for RNAPII recruitment.\",\n      \"evidence\": \"Co-IP, ChIP, reporter assays, acetylation-site mutagenesis, and p300/CBP inhibition\",\n      \"pmids\": [\"28625977\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why Maml2 fails to recruit NACK not explained at the structural level\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Confirmed catalytic inactivity by structure while showing PRAG1 still drives tyrosine phosphorylation through Csk, and identified a dimerization residue (A1329) controlling Csk activation.\",\n      \"evidence\": \"X-ray crystallography of the C-terminus, A1329E mutagenesis, proteomics, and Csk activity assays\",\n      \"pmids\": [\"29503074\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full substrate repertoire of the Csk-PRAG1 axis incompletely mapped\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Resolved the division of labor in Notch transcription, with NACK recruiting RNAPII and the Integrator complex driving its S5 phosphorylation.\",\n      \"evidence\": \"SEC, CSL-DNA affinity FPLC, LC-MS/MS, ChIP, siRNA, and HEK293T reconstitution\",\n      \"pmids\": [\"34551776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct physical contacts between NACK and RNAPII not mapped\", \"Order of supercomplex assembly unresolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified an ATP-dependent function gating PRAG1's coactivator role, with ATP binding required for Notch complex association and druggable by a small molecule.\",\n      \"evidence\": \"ATPase and nucleotide-binding assays, Co-IP with the NTC, inhibitor screen, and PDX models\",\n      \"pmids\": [\"36938545\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for ATP-dependent NTC binding not determined\", \"Mechanism limited by abstract-level detail\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended the SFK/Csk and ABL invasion mechanism to colorectal and pancreatic contexts, pinpointing Y413/Y411 as the SRC/EGFR-driven phosphosite required for transformation.\",\n      \"evidence\": \"Phosphoproteomics, mutagenesis (Y413), localization, ABL kinase assays, xenografts, co-culture, and phospho-blots\",\n      \"pmids\": [\"35740644\", \"35059869\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Y411 vs Y413 numbering/usage across studies not unified\", \"Y411 single Co-IP without reciprocal validation in the secretome study\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated that PRAG1 forms stress-induced phase-separated condensates via \\u03b1N/\\u03b1J helices that are functionally required for cell contraction, linking it to neuronal stress states.\",\n      \"evidence\": \"Live-cell condensate imaging, \\u03b1N/\\u03b1J mutagenesis, stress induction, contraction readout, and iPSC-derived dopaminergic neurons\",\n      \"pmids\": [\"40149915\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship of condensates to the SHED-mediated oligomers unclear\", \"Disease relevance in Parkinson's not causally established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified a new role in EMT through FBXO11 binding that stabilizes SNAIL1, connecting PRAG1 to ubiquitin-pathway regulation.\",\n      \"evidence\": \"Co-IP, overexpression/knockdown with EMT-marker Western blots, and proliferation assays in biliary epithelial cells\",\n      \"pmids\": [\"40743806\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal validation\", \"Whether PRAG1 directly competes with FBXO11 substrate binding unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PRAG1 partitions between its cytoplasmic scaffolding/SFK roles, nuclear Notch coactivator role, and condensate-forming state, and what signals govern this switching, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking ATP-dependence, oligomerization, and condensate formation\", \"Spatial regulation between focal adhesions, chromatin, and condensates undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 6, 11]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 4, 5]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [7, 8, 9]},\n      {\"term_id\": \"GO:0140657\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [7, 8, 9]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [7, 8, 9]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"complexes\": [\"Notch transcriptional activation complex\", \"PEAK family (SgK223/SgK269) complex\"],\n    \"partners\": [\"Rnd2\", \"CSK\", \"STAT3\", \"SgK269/PEAK1\", \"GRB2\", \"MAML1\", \"ABL\", \"FBXO11\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}