{"gene":"PXN","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":1995,"finding":"Paxillin (PXN) and FAK both bind directly to the cytoplasmic domain of the beta1 integrin subunit in vitro; the N-terminal non-catalytic domain of FAK recognizes integrin sequences distinct from those bound by alpha-actinin, and paxillin binding is independent of FAK binding despite overlapping integrin sequences, establishing that integrins can simultaneously recruit both signaling molecules.","method":"In vitro binding assay using GST-fusion peptides mimicking beta1 integrin cytoplasmic domain, pull-down from chicken embryo cell lysates","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — direct in vitro reconstitution with domain mapping, foundational study replicated widely","pmids":["7657702"],"is_preprint":false},{"year":1996,"finding":"The LIM3 domain of PXN is the principal determinant of focal adhesion localization; vinculin binding maps to residues 143–164 (a single site), while FAK binding requires residues 143–164 plus a second site at 265–313; mutation E151Q abolishes vinculin binding without affecting FAK binding, demonstrating separable interaction interfaces on paxillin.","method":"Truncation and point-mutation mutagenesis, transfection of GFP-tagged constructs into CHO.K1 fibroblasts, co-immunoprecipitation, immunofluorescence localization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — systematic mutagenesis with multiple orthogonal readouts in intact cells, replicated across subsequent studies","pmids":["8922390"],"is_preprint":false},{"year":1999,"finding":"The paxillin LD4 motif serves as a selective docking site for a complex containing PAK, PIX (a Rac GEF), and Nck, bridged by a novel 95-kDa ARF-GAP protein p95PKL (paxillin-kinase linker) that binds directly to LD4 and to PIX; disruption of this interaction (via LD4 deletion mutant or microinjection of GST-LD4) inhibits lamellipodia formation and cell migration, placing paxillin upstream of Rac-mediated actin cytoskeletal remodeling.","method":"Yeast two-hybrid, co-immunoprecipitation, GST pull-down, microinjection of GST-LD4 into NIH3T3 cells, wound-healing assay, GFP-p95PKL localization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal binding assays plus functional rescue and loss-of-function in cells, replicated by subsequent studies","pmids":["10330411"],"is_preprint":false},{"year":2000,"finding":"Activation of EphA2 by ephrin-A1 recruits phosphatase SHP2 to EphA2 within one minute, leading to rapid dephosphorylation of both FAK and paxillin and dissociation of the FAK–EphA2 complex, demonstrating that Eph receptor signaling negatively regulates integrin–FAK–paxillin signaling.","method":"Co-immunoprecipitation, western blot with phospho-specific antibodies, time-course stimulation assay in intact cells","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — temporal co-IP and phospho-western with defined stimulus, published in high-impact journal","pmids":["10655584"],"is_preprint":false},{"year":2000,"finding":"GIT1 (a GRK-interacting ARF-GAP) directly binds paxillin via its C-terminal 125-residue domain; overexpression of GIT1 displaces paxillin from focal complexes and promotes cell motility; this activity is regulated by PIX, and GIT1 also couples to FAK via its Spa2 homology domain, placing the GIT1–PIX–PAK–paxillin complex as a coordinator of focal complex disassembly.","method":"Co-immunoprecipitation, overexpression in fibroblasts and epithelial cells, immunofluorescence, domain-deletion mapping","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — direct binding mapped with domain deletions plus functional motility readout, independently replicated","pmids":["10938112"],"is_preprint":false},{"year":2003,"finding":"JNK1 directly phosphorylates paxillin on serine 178 both in vitro and in intact cells; expression of the phosphorylation-deficient S178A mutant of paxillin causes cells to form stable focal adhesions and impairs rapid cell migration in wound-healing assays, demonstrating that JNK1-mediated phosphorylation of paxillin is required for maintaining labile adhesions during rapid cell migration.","method":"In vitro kinase assay, site-directed mutagenesis (S178A), transfection into NBT-II and other cell lines, single-cell migration assay, wound-healing assay, immunofluorescence","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis plus functional migration phenotype, published in Nature, replicated in multiple cell lines","pmids":["12853963"],"is_preprint":false},{"year":2011,"finding":"Proteomic analysis of isolated focal adhesions revealed that paxillin (PXN) is a core component of the myosin-II-responsive focal adhesion proteome; its abundance in focal adhesions is enhanced by myosin-II-mediated contractility, and it participates in Rho-mediated focal adhesion maturation pathways.","method":"Affinity isolation of focal adhesions, quantitative mass spectrometry comparing focal adhesions ± myosin II inhibition (blebbistatin), SILAC proteomics","journal":"Nature cell biology","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative proteomics with functional context, but paxillin not the primary focus of the study","pmids":["21423176"],"is_preprint":false},{"year":2014,"finding":"LSD1 (a histone demethylase) controls expression of paxillin (PXN) in androgen-independent prostate cancer cells; LSD1 depletion increases PXN mRNA and protein levels and leads to enhanced LPAR6 signaling, which promotes cell migration accompanied by phosphorylation of PXN; this identifies a LSD1–LPAR6–PXN axis controlling metastatic behavior.","method":"RNA-seq transcriptome analysis, ChIP-seq cistrome analysis, siRNA knockdown of LSD1, western blot for p-PXN, migration/invasion assays, in vivo mouse metastasis model","journal":"Oncogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — transcriptome and cistrome analysis with functional validation in cells and mice, but indirect regulation","pmids":["25285406"],"is_preprint":false},{"year":2015,"finding":"miR-212 directly targets the 3'UTR of PXN mRNA to suppress its expression; promoter hypermethylation silences miR-212 in gastric cancer, leading to PXN overexpression; restoration of miR-212 reduces PXN mRNA and protein and inhibits cell invasion and metastasis, while PXN re-expression rescues the miR-212-induced phenotype, establishing a methylation–miR-212–PXN axis in gastric cancer invasion.","method":"Luciferase 3'UTR reporter assay, RT-PCR, western blot, methylation analysis (5-aza treatment), transfection gain/loss-of-function, in vitro invasion assay, in vivo xenograft rescue experiment","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — direct 3'UTR reporter validation plus rescue experiments in vitro and in vivo","pmids":["26693054"],"is_preprint":false},{"year":2017,"finding":"Nobiletin inhibits tumor angiogenesis in ER+ breast cancer cells by suppressing Src/FAK/STAT3 signaling, with PXN acting as a downstream target; EMSA and ChIP assays demonstrated that nobiletin blocks STAT3 binding to a novel binding site in the PXN gene promoter, reducing PXN transcription.","method":"Western blotting, RT-PCR, EMSA, ChIP assay, HUVEC tube formation assay, migration/invasion assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 — EMSA and ChIP directly demonstrate STAT3 binding to PXN promoter with functional anti-angiogenic readout","pmids":["28468300"],"is_preprint":false},{"year":2017,"finding":"Paxillin (PXN) is a multifunctional focal adhesion scaffold phosphorylated on Tyr31, Tyr118, Ser188, and Ser190 upon integrin engagement; ERK-mediated phosphorylation of Ser106, Ser231, and Ser290 promotes paxillin disassembly from focal adhesions; the phosphatase PEST binds phosphorylated paxillin to drive disassembly; paxillin coordinates spatiotemporal Cdc42, Rac1, and RhoA GTPase activation by recruiting GEFs, GAPs, and GITs to focal adhesions.","method":"Synthesis/review of experimental literature including phospho-specific western blot, mutagenesis, co-immunoprecipitation studies compiled across multiple primary studies","journal":"Journal of hematology & oncology","confidence":"Medium","confidence_rationale":"Tier 3 — review consolidating multiple independently replicated experimental findings; individual underlying experiments are Tier 1–2","pmids":["28214467"],"is_preprint":false},{"year":2018,"finding":"PXN knockdown in cervical cancer cells suppresses Bcl-2 expression, induces apoptosis, and resensitizes cells to radiation, establishing PXN as a regulator of Bcl-2-dependent survival and radioresistance.","method":"siRNA knockdown of PXN, western blot for Bcl-2, colony formation assay, apoptosis assay, in vitro radiation sensitivity assay, in vivo xenograft","journal":"Future oncology","confidence":"Low","confidence_rationale":"Tier 3 — single lab, knockdown with phenotype but limited mechanistic depth between PXN and Bcl-2","pmids":["29318915"],"is_preprint":false},{"year":2018,"finding":"Cytoplasmic LIF (leukemia inhibitory factor, resulting from signal peptide mutations) promotes nasopharyngeal carcinoma invasion and vascular dissemination by activating YAP1, which in turn modulates FAK/paxillin (PXN) signaling; pharmaceutical inhibition with AZD0530 (Src/FAK inhibitor) reverses LIF-mediated dissemination and promotes cytoplasmic YAP1 accumulation while suppressing focal adhesion kinase activity.","method":"Immunohistochemistry of NPC biopsies, immunofluorescence, western blot, SNV sequencing of LIF signal peptide, pharmacological inhibition (AZD0530), in vivo invasion and dissemination models","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods in cells and in vivo, with pharmacological rescue confirming pathway placement","pmids":["30504771"],"is_preprint":false},{"year":2019,"finding":"ETV4 transcription factor directly upregulates PXN transcription in non-small cell lung cancer; luciferase reporter assays confirmed direct regulatory effect of ETV4 on the PXN promoter; PXN knockdown partially abolished ETV4-induced cell proliferation and migration, placing PXN as a functional downstream effector of ETV4-driven tumorigenesis.","method":"Microarray analysis, luciferase reporter assay, gain/loss-of-function (siRNA, overexpression), migration and proliferation assays, in vivo xenograft","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 — direct promoter reporter assay with functional rescue, but single lab","pmids":["31670855"],"is_preprint":false},{"year":2020,"finding":"Talin-1 overexpression promotes gastric cancer cell migration and invasion through the PTK2(FAK)–PXN–VCL–E-Cadherin–CAPN2–MAPK1 signaling axis; siRNA knockdown and overexpression of Talin-1 modulate PXN protein levels and downstream effectors.","method":"Western blot, immunohistochemistry, Transwell migration/invasion assay, overexpression and knockdown in MKN-45 cells","journal":"Journal of clinical laboratory analysis","confidence":"Low","confidence_rationale":"Tier 3 — single lab, western blot with limited mechanistic depth; pathway placement based on protein level changes","pmids":["32951272"],"is_preprint":false},{"year":2020,"finding":"miR-216b directly targets the 3'UTR of PXN, reducing its expression; miR-216b overexpression suppresses gastric cancer cell proliferation, migration and invasion; re-expression of PXN rescues these phenotypes; the miR-216b–PXN axis partially regulates PI3K/AKT signaling in gastric cancer cells.","method":"Luciferase 3'UTR reporter assay, western blot, gain/loss-of-function transfection, MTT, Transwell, flow cytometry, in vivo xenograft","journal":"Pathology, research and practice","confidence":"Medium","confidence_rationale":"Tier 2 — direct 3'UTR validation plus rescue experiments with pathway readout","pmids":["33422779"],"is_preprint":false},{"year":2021,"finding":"Matrix stiffness activates FAK and paxillin (PXN) phosphorylation at focal adhesions in endothelial cells, promoting transition of Rac1 from inactive to active state; this in turn activates YAP nuclear translocation, driving endothelial tip cell specification; phospho-PXN also loosens intercellular connections to facilitate tip cell emergence. This defines a p-PXN–Rac1–YAP mechanotransduction axis.","method":"Tunable hydrogel substrates, immunofluorescence, western blot for p-FAK and p-PXN, Rac1 activation assay, YAP localization imaging, EC spheroid sprouting assay, siRNA knockdown","journal":"Bioactive materials","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods linking substrate stiffness to PXN phosphorylation to downstream Rac1/YAP activation","pmids":["34466738"],"is_preprint":false},{"year":2021,"finding":"ITGB1 (integrin β1) drives hepatocellular carcinoma cell cycle progression through a signaling cascade involving PXN and YWHAZ (14-3-3ζ), with downstream AKT activation; ITGB1 knockdown by siRNA reduces PXN and YWHAZ protein expression, delays cell cycle progression, and impairs HCC aggressive behavior.","method":"Bioinformatics, siRNA knockdown, western blot, immunostaining, cell migration assay, drug resistance assay, colony formation, cell cycle analysis, xenograft","journal":"Frontiers in cell and developmental biology","confidence":"Low","confidence_rationale":"Tier 3 — single lab, primarily expression-level changes downstream of ITGB1; limited direct mechanistic evidence for PXN within the cascade","pmids":["34977001"],"is_preprint":false},{"year":2021,"finding":"miR-497 targets CDC42 and ITGB1 mRNAs; miR-497 overexpression decreases CDC42 and ITGB1 and inhibits phosphorylation of FAK, paxillin (PXN), and AKT; CDC42 restoration counteracts miR-497-mediated inhibition of focal adhesion and GC metastasis, placing PXN phosphorylation downstream of the miR-497/CDC42/ITGB1/FAK axis.","method":"RT-PCR, western blot for p-PXN, p-FAK, p-AKT, miR-497 knockout mice (NMU-induced GC model), gain/loss-of-function, in vivo metastasis assay","journal":"Molecular therapy. Nucleic acids","confidence":"Medium","confidence_rationale":"Tier 2 — includes in vivo KO mouse model, phospho-PXN readout, and restoration experiment confirming pathway","pmids":["34589278"],"is_preprint":false},{"year":2023,"finding":"ULK1 and ULK2 directly phosphorylate paxillin (PXN) at S32 and S119; this phosphorylation weakens homotypic PXN interactions and liquid-liquid phase separation of PXN, impairing focal adhesion assembly; ULK1/2 activity consequently reduces actin stress fiber assembly and focal adhesion formation, impeding cell contraction and migration independent of autophagy; ULK1/2 and FAK/Src have opposing effects on PXN and compete for phosphorylation of adjacent serine and tyrosine residues.","method":"In vitro kinase assay, site-directed mutagenesis (S32A/S119A), co-immunoprecipitation, FRAP, phase separation assay, focal adhesion immunofluorescence, traction force microscopy, breast cancer cell migration assay, ULK1/2 knockout cells","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus mutagenesis plus phase separation assay plus multiple functional readouts; rigorous mechanistic study","pmids":["37846507"],"is_preprint":false},{"year":2023,"finding":"ANGPTL4 binding to the NRP1 receptor activates ABL1 tyrosine kinase, which phosphorylates paxillin (PXN), promoting HNSCC tumor cell migration; siRNA silencing of NRP1 or ABL1, or treatment with the ABL1 inhibitor dasatinib, blocks PXN phosphorylation and cell migration, identifying NRP1/ABL1/PXN as a signaling axis downstream of ANGPTL4 in head and neck squamous cell carcinoma.","method":"Co-IP, siRNA knockdown of NRP1 and ABL1, pharmacological inhibition (dasatinib), western blot for p-PXN, migration assay, orthotopic HNSCC tumor model","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 — multiple genetic and pharmacological perturbations converging on p-PXN readout with in vivo validation","pmids":["37169211"],"is_preprint":false},{"year":2024,"finding":"CXCL5 activates phosphorylation of the Paxillin/AKT signaling cascade in lung cancer cells, leading to upregulation of PD-L1 expression through a positive feedback loop; combined anti-CXCL5 and anti-PD-L1 treatment inhibits tumor growth in vivo, with PXN phosphorylation serving as a mechanistic link between CXCL5 signaling and immune checkpoint upregulation.","method":"Western blot for p-PXN and p-AKT, gene silencing (siRNA), flow cytometry for PD-L1, confocal immunofluorescence, in vivo SCID/NOD mouse xenograft, ELISA","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods with in vivo validation; mechanistic link between p-PXN and PD-L1 established pharmacologically","pmids":["39034411"],"is_preprint":false},{"year":2025,"finding":"The disordered N-terminal domain of paxillin (PXN) undergoes significant compaction upon binding the FAK focal adhesion targeting domain (FAT), forming a flexible 48-kDa multi-modal complex with four major interconverting conformational states; each state involves unique sets of contacts from disordered PXN regions that are highly conserved; shifts in this multi-state equilibrium via ligand binding or phosphorylation may rewire cellular signaling networks.","method":"NMR spectroscopy, small-angle X-ray scattering (SAXS), ensemble modeling, structural validation of the PXN N-domain–FAT complex","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 — NMR/SAXS structural study with conformational ensemble analysis; novel mechanistic insight into PXN–FAK interaction","pmids":[],"is_preprint":true},{"year":2025,"finding":"Knockdown of PXN in premalignant Src-activated mammary epithelial cells prevents the transient increase in tensile forces at focal adhesions, which in turn abolishes the build-up of tensile forces at adherens junctions and suppresses cell proliferation; this demonstrates that PXN-dependent FA tension is required upstream of AJ strengthening and EGFR-ERK/MRTF-A-SRF activation during early malignant progression.","method":"siRNA knockdown of PXN, FRET-based tension sensors at focal adhesions and adherens junctions, immunofluorescence, proliferation assay, in vivo Drosophila E-cadherin knockdown model","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — tension sensors plus functional rescue with multiple perturbations; preprint not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2025,"finding":"STAT3 directly upregulates PXN transcription in GBM; PXN reciprocally activates STAT3 by regulating SRC transcription, forming a positive feedback loop; additionally, PXN stabilizes the YB-1 protein by inhibiting its ubiquitin-mediated proteasomal degradation, and this YB-1 stabilization contributes to GBM malignancy through multiple downstream pathways.","method":"qRT-PCR, western blot, immunohistochemistry, ChIP assay (STAT3 on PXN promoter), mRNA sequencing, co-immunoprecipitation (PXN–YB-1), ubiquitination assay, siRNA/overexpression functional assays","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP confirms STAT3 binding to PXN promoter; co-IP and ubiquitination assay support PXN–YB-1 mechanism; single lab","pmids":["41872167"],"is_preprint":false}],"current_model":"Paxillin (PXN) is a multidomain focal adhesion scaffold protein whose LIM3 domain targets it to focal adhesions, while its N-terminal LD motifs (particularly LD4) recruit FAK (via residues 143–164 and 265–313), vinculin (residues 143–164, E151 critical), and a PAK/PIX/p95PKL complex to coordinate integrin-mediated signaling; PXN is phosphorylated by multiple kinases—including FAK/Src (Tyr31, Tyr118), JNK1 (Ser178), ERK (Ser106/231/290), and ULK1/2 (Ser32/119)—with each phosphorylation event distinctly modulating focal adhesion assembly, disassembly, phase separation, and cell migration; dephosphorylation is triggered by SHP2 recruitment to activated Eph receptors, while GIT1 binding to PXN drives focal complex disassembly; PXN also acts downstream of CXCL5/AKT and ANGPTL4/NRP1/ABL1 to regulate PD-L1 expression and tumor cell invasion, and stabilizes YB-1 protein by inhibiting its ubiquitination, with the PXN N-domain forming a dynamic multi-modal fuzzy complex with the FAK FAT domain whose conformational equilibrium is modulated by phosphorylation and ligand binding."},"narrative":{"teleology":[{"year":1995,"claim":"Establishing that paxillin, like FAK, is a direct integrin cytoplasmic domain interactor resolved how adhesion receptors nucleate intracellular signaling scaffolds independently of each other.","evidence":"In vitro GST pull-down with beta1 integrin cytoplasmic peptides from chicken embryo cell lysates","pmids":["7657702"],"confidence":"High","gaps":["Binding affinities and stoichiometry not determined","No in vivo validation of simultaneous FAK–PXN co-recruitment"]},{"year":1996,"claim":"Systematic domain mapping revealed that PXN's LIM3 domain drives focal adhesion targeting while separable N-terminal interfaces independently bind vinculin and FAK, establishing PXN as a modular scaffold with distinct partner-binding surfaces.","evidence":"Truncation/point mutagenesis with GFP-tagged constructs in CHO.K1 cells, co-immunoprecipitation, and immunofluorescence","pmids":["8922390"],"confidence":"High","gaps":["Structural basis for LIM3-mediated targeting unresolved","Other LD motif functions not yet mapped"]},{"year":1999,"claim":"Discovery that the LD4 motif recruits a PAK/PIX/p95PKL complex to paxillin placed PXN upstream of Rac-mediated actin remodeling and explained how focal adhesions couple to lamellipodia and directional migration.","evidence":"Yeast two-hybrid, GST pull-down, microinjection of GST-LD4 into NIH3T3 cells, wound-healing assay","pmids":["10330411"],"confidence":"High","gaps":["Regulation of LD4 accessibility by phosphorylation not addressed","In vivo relevance in animal models not tested"]},{"year":2000,"claim":"Two contemporaneous studies defined opposing regulators of the PXN–FAK signaling module: EphA2-recruited SHP2 rapidly dephosphorylates FAK and PXN to dismantle integrin signaling, while GIT1 binding to PXN drives focal complex disassembly and promotes motility via PIX/PAK.","evidence":"Time-course co-IP with phospho-specific antibodies (Eph signaling); domain-deletion mapping and overexpression with motility readout (GIT1)","pmids":["10655584","10938112"],"confidence":"High","gaps":["Whether SHP2 dephosphorylates PXN directly or only via FAK not resolved","Quantitative relationship between GIT1 binding and adhesion lifetime unknown"]},{"year":2003,"claim":"Identification of JNK1 as a direct Ser178 kinase for paxillin, and the finding that S178A mutation stabilizes focal adhesions and blocks rapid migration, established that stress kinase signaling converges on PXN to regulate adhesion lability.","evidence":"In vitro kinase assay, S178A mutagenesis, wound-healing and single-cell migration assays in NBT-II cells","pmids":["12853963"],"confidence":"High","gaps":["Downstream reader of pS178 not identified","Interplay with ERK-mediated serine phosphorylation untested"]},{"year":2011,"claim":"Quantitative focal adhesion proteomics confirmed PXN as a core contractility-responsive component, situating it within the myosin-II/Rho-dependent adhesion maturation program.","evidence":"SILAC mass spectrometry of isolated focal adhesions ± blebbistatin","pmids":["21214176"],"confidence":"Medium","gaps":["PXN not primary focus of study; stoichiometric changes not deeply characterized","Post-translational modifications not captured"]},{"year":2017,"claim":"Consolidation of phosphorylation data clarified that ERK-mediated phosphorylation of PXN Ser106/231/290 promotes adhesion disassembly, complementing the earlier JNK1 and FAK/Src phosphorylation findings and establishing PXN as a multi-kinase integrator of adhesion dynamics.","evidence":"Review synthesizing phospho-specific western blot, mutagenesis, and co-immunoprecipitation studies across multiple primary publications","pmids":["28214467"],"confidence":"Medium","gaps":["Temporal order of multi-site phosphorylation during adhesion turnover unresolved","Combinatorial phospho-code not decoded"]},{"year":2021,"claim":"Demonstration that matrix stiffness drives FAK/PXN phosphorylation to activate Rac1 and nuclear YAP in endothelial cells extended PXN's role from cell migration to mechanotransduction-dependent cell fate specification (tip cell emergence).","evidence":"Tunable hydrogel substrates, Rac1 activation assay, YAP nuclear translocation imaging, EC spheroid sprouting assay","pmids":["34466738"],"confidence":"Medium","gaps":["Whether PXN phosphorylation is sufficient or merely necessary for YAP activation not tested","Identity of the Rac GEF recruited by p-PXN in this context unknown"]},{"year":2023,"claim":"Discovery that ULK1/2 directly phosphorylate PXN at S32/S119 to weaken homotypic interactions and suppress liquid-liquid phase separation of PXN revealed a novel autophagy-independent mechanism by which ULK kinases antagonize focal adhesion assembly and oppose FAK/Src signaling.","evidence":"In vitro kinase assay, S32A/S119A mutagenesis, phase separation assay, FRAP, traction force microscopy in ULK1/2 KO cells","pmids":["37846507"],"confidence":"High","gaps":["Phase separation of PXN in intact focal adhesions not directly visualized","Whether other kinases also regulate PXN LLPS unknown"]},{"year":2023,"claim":"Identification of an ANGPTL4/NRP1/ABL1 signaling axis converging on PXN phosphorylation to drive HNSCC migration expanded the upstream receptor inputs that funnel through PXN beyond classical integrins.","evidence":"siRNA knockdown of NRP1/ABL1, dasatinib inhibition, p-PXN western blot, orthotopic HNSCC tumor model","pmids":["37169211"],"confidence":"Medium","gaps":["Specific PXN tyrosine residues phosphorylated by ABL1 not mapped","Whether ABL1–PXN interaction is direct not confirmed"]},{"year":2024,"claim":"Linking CXCL5-induced PXN/AKT phosphorylation to PD-L1 upregulation in lung cancer revealed an unexpected role for PXN in immune checkpoint regulation, extending its function from adhesion signaling to tumor immune evasion.","evidence":"siRNA, p-PXN and p-AKT western blot, PD-L1 flow cytometry, in vivo combination anti-CXCL5/anti-PD-L1 therapy in SCID/NOD mice","pmids":["39034411"],"confidence":"Medium","gaps":["Molecular mechanism linking p-PXN to PD-L1 transcription/stabilization not defined","Generalizability beyond lung cancer not established"]},{"year":2025,"claim":"ChIP-validated STAT3–PXN transcriptional feedback loop and the discovery that PXN stabilizes YB-1 by blocking its ubiquitination revealed a non-canonical nuclear/signaling function for PXN beyond adhesion scaffolding.","evidence":"ChIP assay (STAT3 on PXN promoter), co-IP (PXN–YB-1), ubiquitination assay, mRNA-seq in GBM cells","pmids":["41872167"],"confidence":"Medium","gaps":["Whether PXN directly contacts the ubiquitin ligase targeting YB-1 is unknown","PXN subcellular compartment where YB-1 stabilization occurs not defined"]},{"year":null,"claim":"The combinatorial phospho-code on PXN — how simultaneous phosphorylation by FAK/Src, JNK1, ERK, and ULK1/2 is read by downstream effectors to switch between adhesion assembly, phase separation, and disassembly — remains unresolved, as does the structural basis of the multi-modal fuzzy PXN–FAK complex in the context of full-length proteins in cellulo.","evidence":"","pmids":[],"confidence":"High","gaps":["No systematic analysis of combinatorial phospho-states on PXN and their effector recruitment","Full-length PXN–FAK structural ensemble not resolved in cellular context","Phase separation of PXN at native expression levels in focal adhesions not demonstrated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,2,4,10]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[1,6,19]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,2,6,10,19]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[19,24]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,5,10,16,19,21]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[1,2,6,16,23]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[0,6,10]}],"complexes":["FAK-paxillin complex","PAK-PIX-GIT1-paxillin complex"],"partners":["PTK2","VCL","GIT1","PAK1","ARHGEF7","ABL1","YBX1","PTPN11"],"other_free_text":[]},"mechanistic_narrative":"Paxillin (PXN) is a multidomain scaffold protein that nucleates focal adhesion signaling complexes, coupling integrin-mediated adhesion to cytoskeletal remodeling, mechanotransduction, and cell migration. Its LIM3 domain targets it to focal adhesions, while N-terminal LD motifs recruit FAK (residues 143–164 and 265–313), vinculin (residue E151 critical), and a PAK/PIX/p95PKL complex (via LD4) that activates Rac-dependent lamellipodia formation [PMID:8922390, PMID:10330411]. PXN is phosphorylated by FAK/Src (Tyr31/Tyr118), JNK1 (Ser178), ERK (Ser106/231/290), and ULK1/2 (Ser32/S119), with each modification distinctly controlling focal adhesion assembly, phase separation, and turnover—ULK1/2 phosphorylation weakens homotypic PXN interactions and opposes FAK/Src-driven adhesion maturation, while JNK1-mediated Ser178 phosphorylation maintains labile adhesions required for rapid migration [PMID:37846507, PMID:12853963]. PXN also functions beyond canonical adhesion signaling: it stabilizes YB-1 protein by blocking its ubiquitin-mediated degradation, participates in a STAT3–PXN–SRC positive feedback loop, and links CXCL5/AKT signaling to PD-L1 immune checkpoint expression in tumor cells [PMID:41872167, PMID:39034411]."},"prefetch_data":{"uniprot":{"accession":"P49023","full_name":"Paxillin","aliases":[],"length_aa":591,"mass_kda":64.5,"function":"Cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the extracellular matrix (focal adhesion). 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translation elongation factors, inhibiting PXN translation, whereas the exon 4-containing transcript binds PXN mRNA 3'UTR and protects it from microRNA-24-AGO2-induced degradation, increasing PXN expression.\",\n      \"method\": \"Transcriptomic analysis, RNA pulldown, translation elongation factor co-immunoprecipitation, miRNA-AGO2 complex assays, knockdown/rescue experiments\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods, mechanistic detail at molecular level, replicated in functional and in vivo assays\",\n      \"pmids\": [\"28553938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ULK1/2 directly phosphorylate PXN at S32 and S119, weakening homotypic PXN interactions and liquid-liquid phase separation, impairing focal adhesion assembly, and thereby suppressing breast cancer cell migration and mechanotransduction; ULK1/2 and FAK/Src have opposing effects on PXN phosphorylation at adjacent serine and tyrosine residues.\",\n      \"method\": \"In vitro kinase assay, mutagenesis, FRAP/phase separation assays, focal adhesion imaging, traction force microscopy, KD/KO with defined migration phenotype\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct kinase-substrate relationship established in vitro, mutagenesis at specific sites, multiple orthogonal functional readouts\",\n      \"pmids\": [\"37846507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The disordered N-terminal domain of PXN undergoes significant compaction upon binding to the C-terminal focal adhesion targeting (FAT) domain of FAK, forming a 48-kDa multi-modal fuzzy complex with four major interconverting conformational states; each state has unique contact sets involving conserved disordered regions, providing a structural basis for how phosphorylation or ligand binding rewires PXN-dependent signaling.\",\n      \"method\": \"NMR spectroscopy, SAXS, ensemble analysis, structural modeling\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 structural — rigorous NMR/SAXS with functional interpretation, but preprint single study\",\n      \"pmids\": [\"bio_10.1101_2025.01.01.630265\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Matrix stiffness promotes FAK and PXN phosphorylation at focal adhesions in endothelial cells, which activates Rac1 (GDP→GTP transition) and subsequently nuclear YAP translocation, driving tip cell specification during angiogenesis; phospho-PXN also loosens intercellular junctions to facilitate tip cell formation.\",\n      \"method\": \"Tunable hydrogel substrates, western blot for phospho-FAK/PXN, Rac1 GTP-pulldown, YAP immunofluorescence, siRNA knockdown\",\n      \"journal\": \"Bioactive materials\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — phosphorylation and downstream Rac1/YAP pathway placement shown by multiple methods but single study\",\n      \"pmids\": [\"34466738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cytoplasmic LIF enhances cancer vascular dissemination and local invasion mechanistically through modulation of YAP1-FAK/PXN signaling in nasopharyngeal carcinoma; pharmaceutical inhibition with AZD0530 (Src/FAK inhibitor) reverses LIF-mediated effects by promoting cytoplasmic YAP1 accumulation and suppressing focal adhesion kinase activity.\",\n      \"method\": \"IHC correlation, pharmacological inhibition (AZD0530), immunofluorescence, functional invasion/dissemination assays, in vivo xenograft\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — pathway placement via pharmacological and KD approaches, multiple readouts, single lab\",\n      \"pmids\": [\"30504771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LSD1 demethylase regulates PXN expression in androgen-independent prostate cancer; upon LSD1 depletion, enhanced LPAR6 signaling promotes cell migration with concomitant phosphorylation of PXN.\",\n      \"method\": \"Transcriptome and cistrome (ChIP) analyses, siRNA knockdown, migration assays, western blot for phospho-PXN, in vivo mouse metastasis model\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — cistrome + transcriptome + functional assays establish LSD1→LPAR6→phospho-PXN pathway, single lab\",\n      \"pmids\": [\"25285406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"STAT3 binds to a novel binding site in the PXN gene promoter to regulate PXN transcription; PXN acts as a downstream target of the Src/FAK/STAT3 signaling axis in breast cancer angiogenesis.\",\n      \"method\": \"EMSA, ChIP assay, western blotting, RT-PCR, HUVEC angiogenesis assay\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — EMSA and ChIP directly demonstrate STAT3 binding to PXN promoter, single lab\",\n      \"pmids\": [\"28468300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ANGPTL4 binding to the NRP1 receptor leads to ABL1-dependent phosphorylation of PXN, driving head and neck squamous cell carcinoma cell migration; ABL1 inhibition (dasatinib) or NRP1/ABL1 siRNA silencing blocks PXN phosphorylation and tumor cell migration.\",\n      \"method\": \"siRNA silencing, pharmacological inhibition (dasatinib), western blot for phospho-PXN, migration assays, orthotopic tumor model\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — pathway placement (ANGPTL4/NRP1/ABL1→phospho-PXN) established by pharmacological and genetic approaches, single lab\",\n      \"pmids\": [\"37169211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CXCL5 activates phosphorylation of the PXN/AKT signaling cascade in lung cancer cells, leading to upregulation of PD-L1 expression and forming a positive feedback loop for immune escape.\",\n      \"method\": \"Gene silencing, western blot for phospho-PXN/AKT, flow cytometry, confocal immunofluorescence, in vivo SCID/NOD mouse model\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — phosphorylation cascade placement with functional immune-evasion readout, single lab\",\n      \"pmids\": [\"39034411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ITGB1 knockdown downregulates PXN expression in HCC cells, and the ITGB1/PXN/YWHAZ/AKT axis promotes HCC cell cycle progression and aggressive behavior.\",\n      \"method\": \"siRNA knockdown, western blot, immunostaining, cell cycle analysis, colony formation, xenograft\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — downstream pathway placement inferred from expression changes after ITGB1 siRNA, no direct interaction between ITGB1 and PXN demonstrated biochemically\",\n      \"pmids\": [\"34977001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"In GBM, STAT3 directly upregulates PXN transcription, and PXN reciprocally activates STAT3 by regulating SRC transcription, forming a positive feedback loop; additionally, PXN stabilizes YB-1 protein by inhibiting its ubiquitin-mediated degradation.\",\n      \"method\": \"qRT-PCR, western blotting, mRNA sequencing, functional knockdown assays, immunohistochemistry\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — transcriptional feedback loop and ubiquitination suppression identified by multiple methods, single lab, mechanistic claims need further validation\",\n      \"pmids\": [\"41872167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PXN (paxillin), a focal adhesion scaffold protein, is specifically expressed in pillar cells of the mouse organ of Corti during embryonic and early postnatal ages, suggesting a functional role in inner ear mechanosensory cell architecture.\",\n      \"method\": \"In situ hybridization, reporter expression analysis in mouse inner ear\",\n      \"journal\": \"Gene expression patterns\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — localization data without functional mechanistic follow-up\",\n      \"pmids\": [\"22446089\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PXN knockdown in premalignant Src-activated mammary epithelial cells prevents the transient increase in tensile forces at focal adhesions and at cell-cell adherens junctions, and abolishes the proliferative advantage, placing PXN as necessary for mechanotransduction coupling between focal adhesions and adherens junctions during early malignant transformation.\",\n      \"method\": \"siRNA knockdown, traction force microscopy, FRET-based tension biosensors, live-cell imaging, FAK inhibition\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct functional consequence of PXN KD on FA tension and AJ tension measured by FRET biosensors, preprint single study\",\n      \"pmids\": [\"bio_10.1101_2025.07.05.663275\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"Paxillin (PXN) is a focal adhesion scaffold protein whose disordered N-terminal domain forms a multi-modal fuzzy complex with the FAT domain of FAK to localize FAK to focal adhesions; it is phosphorylated at specific serine residues (S32, S119) by ULK1/2 (inhibiting FA assembly and cell migration) and at tyrosine residues by FAK/Src (promoting FA assembly and migration), and at additional sites downstream of LPAR6, ABL1, CXCL5, and matrix stiffness-activated FAK, coupling extracellular mechanical and chemical signals through Rac1, YAP, AKT, and STAT3 to control cell migration, invasion, and survival; its expression is post-transcriptionally regulated by the MBNL3/lncRNA-PXN-AS1 splicing axis and by multiple miRNAs targeting its 3'UTR.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries from KEPT papers only.\n\n**Classification:**\n\n**EXCLUDE (alt-locus/lncRNA products):**\n- Papers [1], [2] (corpus retrieved): About lncRNA-PXN-AS1 / PXN-AS1-IR3 — alt-locus products\n- Papers [7], [32] (corpus retrieved): About lncRNA PXN-AS1 in pancreatic cancer\n- Papers [14], [27] (corpus retrieved): About lncRNA PXN-AS1 in GBM/pituitary\n- Papers [16], [17] (corpus retrieved): About lncRNA PXN-AS1-L\n- Papers [29] (corpus retrieved): About lncRNA PXN-AS1\n- Paper [6] (corpus retrieved): XIST/miR-137/PXN — mainly about lncRNA XIST mechanism; PXN is downstream target only — BORDERLINE KEEP (PXN is target, miR-137 mechanism involves PXN 3'UTR)\n- Paper [22] (corpus retrieved): XIST/miR-132/PXN — same pattern\n- Paper [33] (corpus retrieved): Expression study only\n\n**EXCLUDE (symbol collision — non-mammalian peroxidasin):**\n- Paper [9] (corpus retrieved): Enterococcus faecium PXN-33 — probiotic bacterium, not the gene PXN\n- Paper [23] (corpus retrieved): C. elegans PXN-1 peroxidasin — symbol collision, not ortholog of human paxillin\n- Paper [31] (corpus retrieved): Crayfish peroxinectin PXN — symbol collision\n- Paper [37] (corpus retrieved): C. elegans PXN-2/peroxidasin — symbol collision\n\n**EXCLUDE (general genomics/proteomics resources, no PXN-specific mechanism):**\n- Gene2pubmed papers [1]-[14], [22]-[23], [25]-[27]: Human genome, phosphoproteomics atlases, interactome maps — no PXN-specific mechanism discovered\n\n**KEEP (canonical paxillin protein, mechanistic findings):**\n- Corpus [3]: Nobiletin/Src/FAK/STAT3/PXN signaling — KEEP\n- Corpus [4]: ETV4 transcriptionally upregulates PXN — KEEP\n- Corpus [5]: LSD1 regulates PXN expression/phosphorylation in PCa migration — KEEP\n- Corpus [8]: p-PXN-Rac1-YAP mechanotransduction — KEEP\n- Corpus [10]: ITGB1/PXN/YWHAZ/AKT cell cycle — KEEP\n- Corpus [11]: miR-212 targets PXN 3'UTR — KEEP\n- Corpus [12]: CXCL5/PXN/AKT/PD-L1 — KEEP\n- Corpus [13]: Cytoplasmic LIF/YAP1-FAK/PXN signaling — KEEP\n- Corpus [15]: ULK1/2 phosphorylates PXN at S32/S119, regulates FA assembly — KEEP\n- Corpus [18]: miR-497/CDC42/ITGB1/FAK/PXN/AKT — KEEP\n- Corpus [19]: PXN knockdown, Bcl-2 regulation, radioresistance — KEEP\n- Corpus [20]: Talin-1/PTK2-PXN-VCL signaling — KEEP\n- Corpus [21]: ITGB1/PTK2/PXN pathway in migration — KEEP\n- Corpus [24]: Specific expression of Pxn in organ of Corti — KEEP (localization)\n- Corpus [25]: Pxn expression in organ of Corti — same as [24], already noted\n- Corpus [26]: miR-216b targets PXN 3'UTR — KEEP\n- Corpus [28]: ANGPTL4/NRP1/ABL1/PXN phosphorylation — KEEP\n- Corpus [30]: Fendrr/miR-145-5p/PXN axis in VaD — KEEP (PXN is target)\n- Corpus [34]: STAT3-PXN feedback loop, YB-1 ubiquitination — KEEP\n- Corpus [35] (preprint): PXN N-domain/FAT complex structure, multi-modal states — KEEP\n- Corpus [36] (preprint): PXN knockdown impairs FA tensile forces — KEEP\n- Gene2pubmed [15]: FAK and paxillin bind beta integrin cytoplasmic domains — KEEP\n- Gene2pubmed [16]: Focal adhesion proteome, myosin-II-responsive — KEEP (PXN context)\n- Gene2pubmed [17]: EphA2/SHP2 causes FAK and paxillin dephosphorylation — KEEP\n- Gene2pubmed [18]: ZO-1/VE-cadherin — paxillin not central mechanism — BORDERLINE EXCLUDE\n- Gene2pubmed [19]: Phosphotyrosine profiling — PXN tyrosine phosphorylation detected — low specificity\n- Gene2pubmed [21]: JNK1 phosphorylates paxillin S178, regulates cell migration — KEEP\n- Gene2pubmed [24]: Paxillin LD4 binds PAK/PIX via p95PKL — KEEP\n- Gene2pubmed [28]: LIM3 is principal FA targeting motif; vinculin/FAK binding domains mapped — KEEP\n- Gene2pubmed [29]: GIT1 binds paxillin, promotes FC disassembly — KEEP\n- Gene2pubmed [30]: Review of paxillin phosphorylation and signaling — KEEP (mechanistic review grounded in experiments)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"Paxillin (PXN) and FAK both bind directly to the cytoplasmic domain of the beta1 integrin subunit in vitro; the N-terminal non-catalytic domain of FAK recognizes integrin sequences distinct from those bound by alpha-actinin, and paxillin binding is independent of FAK binding despite overlapping integrin sequences, establishing that integrins can simultaneously recruit both signaling molecules.\",\n      \"method\": \"In vitro binding assay using GST-fusion peptides mimicking beta1 integrin cytoplasmic domain, pull-down from chicken embryo cell lysates\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct in vitro reconstitution with domain mapping, foundational study replicated widely\",\n      \"pmids\": [\"7657702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"The LIM3 domain of PXN is the principal determinant of focal adhesion localization; vinculin binding maps to residues 143–164 (a single site), while FAK binding requires residues 143–164 plus a second site at 265–313; mutation E151Q abolishes vinculin binding without affecting FAK binding, demonstrating separable interaction interfaces on paxillin.\",\n      \"method\": \"Truncation and point-mutation mutagenesis, transfection of GFP-tagged constructs into CHO.K1 fibroblasts, co-immunoprecipitation, immunofluorescence localization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic mutagenesis with multiple orthogonal readouts in intact cells, replicated across subsequent studies\",\n      \"pmids\": [\"8922390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The paxillin LD4 motif serves as a selective docking site for a complex containing PAK, PIX (a Rac GEF), and Nck, bridged by a novel 95-kDa ARF-GAP protein p95PKL (paxillin-kinase linker) that binds directly to LD4 and to PIX; disruption of this interaction (via LD4 deletion mutant or microinjection of GST-LD4) inhibits lamellipodia formation and cell migration, placing paxillin upstream of Rac-mediated actin cytoskeletal remodeling.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, GST pull-down, microinjection of GST-LD4 into NIH3T3 cells, wound-healing assay, GFP-p95PKL localization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal binding assays plus functional rescue and loss-of-function in cells, replicated by subsequent studies\",\n      \"pmids\": [\"10330411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Activation of EphA2 by ephrin-A1 recruits phosphatase SHP2 to EphA2 within one minute, leading to rapid dephosphorylation of both FAK and paxillin and dissociation of the FAK–EphA2 complex, demonstrating that Eph receptor signaling negatively regulates integrin–FAK–paxillin signaling.\",\n      \"method\": \"Co-immunoprecipitation, western blot with phospho-specific antibodies, time-course stimulation assay in intact cells\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — temporal co-IP and phospho-western with defined stimulus, published in high-impact journal\",\n      \"pmids\": [\"10655584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"GIT1 (a GRK-interacting ARF-GAP) directly binds paxillin via its C-terminal 125-residue domain; overexpression of GIT1 displaces paxillin from focal complexes and promotes cell motility; this activity is regulated by PIX, and GIT1 also couples to FAK via its Spa2 homology domain, placing the GIT1–PIX–PAK–paxillin complex as a coordinator of focal complex disassembly.\",\n      \"method\": \"Co-immunoprecipitation, overexpression in fibroblasts and epithelial cells, immunofluorescence, domain-deletion mapping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding mapped with domain deletions plus functional motility readout, independently replicated\",\n      \"pmids\": [\"10938112\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"JNK1 directly phosphorylates paxillin on serine 178 both in vitro and in intact cells; expression of the phosphorylation-deficient S178A mutant of paxillin causes cells to form stable focal adhesions and impairs rapid cell migration in wound-healing assays, demonstrating that JNK1-mediated phosphorylation of paxillin is required for maintaining labile adhesions during rapid cell migration.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (S178A), transfection into NBT-II and other cell lines, single-cell migration assay, wound-healing assay, immunofluorescence\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis plus functional migration phenotype, published in Nature, replicated in multiple cell lines\",\n      \"pmids\": [\"12853963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Proteomic analysis of isolated focal adhesions revealed that paxillin (PXN) is a core component of the myosin-II-responsive focal adhesion proteome; its abundance in focal adhesions is enhanced by myosin-II-mediated contractility, and it participates in Rho-mediated focal adhesion maturation pathways.\",\n      \"method\": \"Affinity isolation of focal adhesions, quantitative mass spectrometry comparing focal adhesions ± myosin II inhibition (blebbistatin), SILAC proteomics\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative proteomics with functional context, but paxillin not the primary focus of the study\",\n      \"pmids\": [\"21423176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LSD1 (a histone demethylase) controls expression of paxillin (PXN) in androgen-independent prostate cancer cells; LSD1 depletion increases PXN mRNA and protein levels and leads to enhanced LPAR6 signaling, which promotes cell migration accompanied by phosphorylation of PXN; this identifies a LSD1–LPAR6–PXN axis controlling metastatic behavior.\",\n      \"method\": \"RNA-seq transcriptome analysis, ChIP-seq cistrome analysis, siRNA knockdown of LSD1, western blot for p-PXN, migration/invasion assays, in vivo mouse metastasis model\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — transcriptome and cistrome analysis with functional validation in cells and mice, but indirect regulation\",\n      \"pmids\": [\"25285406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"miR-212 directly targets the 3'UTR of PXN mRNA to suppress its expression; promoter hypermethylation silences miR-212 in gastric cancer, leading to PXN overexpression; restoration of miR-212 reduces PXN mRNA and protein and inhibits cell invasion and metastasis, while PXN re-expression rescues the miR-212-induced phenotype, establishing a methylation–miR-212–PXN axis in gastric cancer invasion.\",\n      \"method\": \"Luciferase 3'UTR reporter assay, RT-PCR, western blot, methylation analysis (5-aza treatment), transfection gain/loss-of-function, in vitro invasion assay, in vivo xenograft rescue experiment\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3'UTR reporter validation plus rescue experiments in vitro and in vivo\",\n      \"pmids\": [\"26693054\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nobiletin inhibits tumor angiogenesis in ER+ breast cancer cells by suppressing Src/FAK/STAT3 signaling, with PXN acting as a downstream target; EMSA and ChIP assays demonstrated that nobiletin blocks STAT3 binding to a novel binding site in the PXN gene promoter, reducing PXN transcription.\",\n      \"method\": \"Western blotting, RT-PCR, EMSA, ChIP assay, HUVEC tube formation assay, migration/invasion assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — EMSA and ChIP directly demonstrate STAT3 binding to PXN promoter with functional anti-angiogenic readout\",\n      \"pmids\": [\"28468300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Paxillin (PXN) is a multifunctional focal adhesion scaffold phosphorylated on Tyr31, Tyr118, Ser188, and Ser190 upon integrin engagement; ERK-mediated phosphorylation of Ser106, Ser231, and Ser290 promotes paxillin disassembly from focal adhesions; the phosphatase PEST binds phosphorylated paxillin to drive disassembly; paxillin coordinates spatiotemporal Cdc42, Rac1, and RhoA GTPase activation by recruiting GEFs, GAPs, and GITs to focal adhesions.\",\n      \"method\": \"Synthesis/review of experimental literature including phospho-specific western blot, mutagenesis, co-immunoprecipitation studies compiled across multiple primary studies\",\n      \"journal\": \"Journal of hematology & oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — review consolidating multiple independently replicated experimental findings; individual underlying experiments are Tier 1–2\",\n      \"pmids\": [\"28214467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PXN knockdown in cervical cancer cells suppresses Bcl-2 expression, induces apoptosis, and resensitizes cells to radiation, establishing PXN as a regulator of Bcl-2-dependent survival and radioresistance.\",\n      \"method\": \"siRNA knockdown of PXN, western blot for Bcl-2, colony formation assay, apoptosis assay, in vitro radiation sensitivity assay, in vivo xenograft\",\n      \"journal\": \"Future oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, knockdown with phenotype but limited mechanistic depth between PXN and Bcl-2\",\n      \"pmids\": [\"29318915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Cytoplasmic LIF (leukemia inhibitory factor, resulting from signal peptide mutations) promotes nasopharyngeal carcinoma invasion and vascular dissemination by activating YAP1, which in turn modulates FAK/paxillin (PXN) signaling; pharmaceutical inhibition with AZD0530 (Src/FAK inhibitor) reverses LIF-mediated dissemination and promotes cytoplasmic YAP1 accumulation while suppressing focal adhesion kinase activity.\",\n      \"method\": \"Immunohistochemistry of NPC biopsies, immunofluorescence, western blot, SNV sequencing of LIF signal peptide, pharmacological inhibition (AZD0530), in vivo invasion and dissemination models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods in cells and in vivo, with pharmacological rescue confirming pathway placement\",\n      \"pmids\": [\"30504771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ETV4 transcription factor directly upregulates PXN transcription in non-small cell lung cancer; luciferase reporter assays confirmed direct regulatory effect of ETV4 on the PXN promoter; PXN knockdown partially abolished ETV4-induced cell proliferation and migration, placing PXN as a functional downstream effector of ETV4-driven tumorigenesis.\",\n      \"method\": \"Microarray analysis, luciferase reporter assay, gain/loss-of-function (siRNA, overexpression), migration and proliferation assays, in vivo xenograft\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct promoter reporter assay with functional rescue, but single lab\",\n      \"pmids\": [\"31670855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Talin-1 overexpression promotes gastric cancer cell migration and invasion through the PTK2(FAK)–PXN–VCL–E-Cadherin–CAPN2–MAPK1 signaling axis; siRNA knockdown and overexpression of Talin-1 modulate PXN protein levels and downstream effectors.\",\n      \"method\": \"Western blot, immunohistochemistry, Transwell migration/invasion assay, overexpression and knockdown in MKN-45 cells\",\n      \"journal\": \"Journal of clinical laboratory analysis\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, western blot with limited mechanistic depth; pathway placement based on protein level changes\",\n      \"pmids\": [\"32951272\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-216b directly targets the 3'UTR of PXN, reducing its expression; miR-216b overexpression suppresses gastric cancer cell proliferation, migration and invasion; re-expression of PXN rescues these phenotypes; the miR-216b–PXN axis partially regulates PI3K/AKT signaling in gastric cancer cells.\",\n      \"method\": \"Luciferase 3'UTR reporter assay, western blot, gain/loss-of-function transfection, MTT, Transwell, flow cytometry, in vivo xenograft\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct 3'UTR validation plus rescue experiments with pathway readout\",\n      \"pmids\": [\"33422779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Matrix stiffness activates FAK and paxillin (PXN) phosphorylation at focal adhesions in endothelial cells, promoting transition of Rac1 from inactive to active state; this in turn activates YAP nuclear translocation, driving endothelial tip cell specification; phospho-PXN also loosens intercellular connections to facilitate tip cell emergence. This defines a p-PXN–Rac1–YAP mechanotransduction axis.\",\n      \"method\": \"Tunable hydrogel substrates, immunofluorescence, western blot for p-FAK and p-PXN, Rac1 activation assay, YAP localization imaging, EC spheroid sprouting assay, siRNA knockdown\",\n      \"journal\": \"Bioactive materials\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods linking substrate stiffness to PXN phosphorylation to downstream Rac1/YAP activation\",\n      \"pmids\": [\"34466738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ITGB1 (integrin β1) drives hepatocellular carcinoma cell cycle progression through a signaling cascade involving PXN and YWHAZ (14-3-3ζ), with downstream AKT activation; ITGB1 knockdown by siRNA reduces PXN and YWHAZ protein expression, delays cell cycle progression, and impairs HCC aggressive behavior.\",\n      \"method\": \"Bioinformatics, siRNA knockdown, western blot, immunostaining, cell migration assay, drug resistance assay, colony formation, cell cycle analysis, xenograft\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single lab, primarily expression-level changes downstream of ITGB1; limited direct mechanistic evidence for PXN within the cascade\",\n      \"pmids\": [\"34977001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-497 targets CDC42 and ITGB1 mRNAs; miR-497 overexpression decreases CDC42 and ITGB1 and inhibits phosphorylation of FAK, paxillin (PXN), and AKT; CDC42 restoration counteracts miR-497-mediated inhibition of focal adhesion and GC metastasis, placing PXN phosphorylation downstream of the miR-497/CDC42/ITGB1/FAK axis.\",\n      \"method\": \"RT-PCR, western blot for p-PXN, p-FAK, p-AKT, miR-497 knockout mice (NMU-induced GC model), gain/loss-of-function, in vivo metastasis assay\",\n      \"journal\": \"Molecular therapy. Nucleic acids\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — includes in vivo KO mouse model, phospho-PXN readout, and restoration experiment confirming pathway\",\n      \"pmids\": [\"34589278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ULK1 and ULK2 directly phosphorylate paxillin (PXN) at S32 and S119; this phosphorylation weakens homotypic PXN interactions and liquid-liquid phase separation of PXN, impairing focal adhesion assembly; ULK1/2 activity consequently reduces actin stress fiber assembly and focal adhesion formation, impeding cell contraction and migration independent of autophagy; ULK1/2 and FAK/Src have opposing effects on PXN and compete for phosphorylation of adjacent serine and tyrosine residues.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (S32A/S119A), co-immunoprecipitation, FRAP, phase separation assay, focal adhesion immunofluorescence, traction force microscopy, breast cancer cell migration assay, ULK1/2 knockout cells\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus mutagenesis plus phase separation assay plus multiple functional readouts; rigorous mechanistic study\",\n      \"pmids\": [\"37846507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ANGPTL4 binding to the NRP1 receptor activates ABL1 tyrosine kinase, which phosphorylates paxillin (PXN), promoting HNSCC tumor cell migration; siRNA silencing of NRP1 or ABL1, or treatment with the ABL1 inhibitor dasatinib, blocks PXN phosphorylation and cell migration, identifying NRP1/ABL1/PXN as a signaling axis downstream of ANGPTL4 in head and neck squamous cell carcinoma.\",\n      \"method\": \"Co-IP, siRNA knockdown of NRP1 and ABL1, pharmacological inhibition (dasatinib), western blot for p-PXN, migration assay, orthotopic HNSCC tumor model\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and pharmacological perturbations converging on p-PXN readout with in vivo validation\",\n      \"pmids\": [\"37169211\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CXCL5 activates phosphorylation of the Paxillin/AKT signaling cascade in lung cancer cells, leading to upregulation of PD-L1 expression through a positive feedback loop; combined anti-CXCL5 and anti-PD-L1 treatment inhibits tumor growth in vivo, with PXN phosphorylation serving as a mechanistic link between CXCL5 signaling and immune checkpoint upregulation.\",\n      \"method\": \"Western blot for p-PXN and p-AKT, gene silencing (siRNA), flow cytometry for PD-L1, confocal immunofluorescence, in vivo SCID/NOD mouse xenograft, ELISA\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods with in vivo validation; mechanistic link between p-PXN and PD-L1 established pharmacologically\",\n      \"pmids\": [\"39034411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"The disordered N-terminal domain of paxillin (PXN) undergoes significant compaction upon binding the FAK focal adhesion targeting domain (FAT), forming a flexible 48-kDa multi-modal complex with four major interconverting conformational states; each state involves unique sets of contacts from disordered PXN regions that are highly conserved; shifts in this multi-state equilibrium via ligand binding or phosphorylation may rewire cellular signaling networks.\",\n      \"method\": \"NMR spectroscopy, small-angle X-ray scattering (SAXS), ensemble modeling, structural validation of the PXN N-domain–FAT complex\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR/SAXS structural study with conformational ensemble analysis; novel mechanistic insight into PXN–FAK interaction\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Knockdown of PXN in premalignant Src-activated mammary epithelial cells prevents the transient increase in tensile forces at focal adhesions, which in turn abolishes the build-up of tensile forces at adherens junctions and suppresses cell proliferation; this demonstrates that PXN-dependent FA tension is required upstream of AJ strengthening and EGFR-ERK/MRTF-A-SRF activation during early malignant progression.\",\n      \"method\": \"siRNA knockdown of PXN, FRET-based tension sensors at focal adhesions and adherens junctions, immunofluorescence, proliferation assay, in vivo Drosophila E-cadherin knockdown model\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — tension sensors plus functional rescue with multiple perturbations; preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"STAT3 directly upregulates PXN transcription in GBM; PXN reciprocally activates STAT3 by regulating SRC transcription, forming a positive feedback loop; additionally, PXN stabilizes the YB-1 protein by inhibiting its ubiquitin-mediated proteasomal degradation, and this YB-1 stabilization contributes to GBM malignancy through multiple downstream pathways.\",\n      \"method\": \"qRT-PCR, western blot, immunohistochemistry, ChIP assay (STAT3 on PXN promoter), mRNA sequencing, co-immunoprecipitation (PXN–YB-1), ubiquitination assay, siRNA/overexpression functional assays\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP confirms STAT3 binding to PXN promoter; co-IP and ubiquitination assay support PXN–YB-1 mechanism; single lab\",\n      \"pmids\": [\"41872167\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Paxillin (PXN) is a multidomain focal adhesion scaffold protein whose LIM3 domain targets it to focal adhesions, while its N-terminal LD motifs (particularly LD4) recruit FAK (via residues 143–164 and 265–313), vinculin (residues 143–164, E151 critical), and a PAK/PIX/p95PKL complex to coordinate integrin-mediated signaling; PXN is phosphorylated by multiple kinases—including FAK/Src (Tyr31, Tyr118), JNK1 (Ser178), ERK (Ser106/231/290), and ULK1/2 (Ser32/119)—with each phosphorylation event distinctly modulating focal adhesion assembly, disassembly, phase separation, and cell migration; dephosphorylation is triggered by SHP2 recruitment to activated Eph receptors, while GIT1 binding to PXN drives focal complex disassembly; PXN also acts downstream of CXCL5/AKT and ANGPTL4/NRP1/ABL1 to regulate PD-L1 expression and tumor cell invasion, and stabilizes YB-1 protein by inhibiting its ubiquitination, with the PXN N-domain forming a dynamic multi-modal fuzzy complex with the FAK FAT domain whose conformational equilibrium is modulated by phosphorylation and ligand binding.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"Paxillin (PXN) is a focal adhesion scaffold protein that integrates mechanical and chemical signals to control cell migration, adhesion dynamics, and mechanotransduction. Its disordered N-terminal domain forms a multi-modal fuzzy complex with the FAT domain of FAK, and its phosphorylation state is tuned by opposing kinases: ULK1/2-mediated serine phosphorylation (S32, S119) weakens homotypic PXN interactions and liquid-liquid phase separation to suppress focal adhesion assembly and migration, whereas FAK/Src- and ABL1-dependent tyrosine phosphorylation promotes focal adhesion maturation, Rac1 activation, and downstream YAP nuclear translocation [PMID:37846507, PMID:34466738, PMID:37169211]. PXN expression is transcriptionally regulated by STAT3 binding to the PXN promoter and post-transcriptionally controlled by the MBNL3/lncRNA-PXN-AS1 splicing axis, which modulates miR-24-AGO2-mediated mRNA degradation versus translation elongation factor dissociation [PMID:28468300, PMID:28553938]. Phospho-PXN relays signals through AKT and STAT3 pathways to influence immune evasion via PD-L1 upregulation and angiogenesis, and PXN participates in a positive feedback loop with STAT3 and SRC in glioblastoma [PMID:39034411, PMID:41872167].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Establishing that PXN is expressed in mechanosensory support cells of the inner ear raised the question of whether its scaffold function extends beyond canonical focal adhesions to specialized mechanosensory tissues.\",\n      \"evidence\": \"In situ hybridization and reporter analysis in mouse organ of Corti\",\n      \"pmids\": [\"22446089\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No functional loss-of-function data in inner ear\", \"Expression pattern alone does not establish mechanistic role\", \"No link to hearing or mechanotransduction phenotype\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Placing PXN phosphorylation downstream of LPAR6 signaling revealed that PXN integrates GPCR-derived signals to promote migration in androgen-independent prostate cancer, expanding its role beyond integrin-only signaling.\",\n      \"evidence\": \"siRNA knockdown of LSD1, transcriptome/ChIP analyses, phospho-PXN western blot, migration assays, in vivo metastasis model\",\n      \"pmids\": [\"25285406\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link between LPAR6 and kinase(s) that phosphorylate PXN not identified\", \"Single-lab observation\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Discovery that MBNL3 controls PXN protein levels through alternative splicing of lncRNA-PXN-AS1 — toggling between translational repression and mRNA stabilization — established a post-transcriptional regulatory layer for PXN expression, while STAT3 was shown to directly bind the PXN promoter providing transcriptional control.\",\n      \"evidence\": \"RNA pulldown, translation elongation factor co-IP, miR-24-AGO2 assays, knockdown/rescue (MBNL3/PXN-AS1); EMSA and ChIP for STAT3–PXN promoter binding\",\n      \"pmids\": [\"28553938\", \"28468300\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MBNL3/PXN-AS1 axis operates in non-hepatocellular contexts is unknown\", \"Relative contribution of transcriptional vs. post-transcriptional regulation in vivo not quantified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrating that LIF signals through YAP1-FAK/PXN to drive vascular dissemination positioned PXN within a YAP1-centered mechanosignaling–invasion circuit and showed pharmacological tractability via Src/FAK inhibition.\",\n      \"evidence\": \"IHC, AZD0530 pharmacological inhibition, immunofluorescence, invasion assays, xenograft in nasopharyngeal carcinoma\",\n      \"pmids\": [\"30504771\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PXN phosphorylation is required or correlative in LIF-YAP1 axis not resolved by mutagenesis\", \"Single cancer type tested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showing that matrix stiffness drives FAK/PXN phosphorylation to activate Rac1-GTP and nuclear YAP in endothelial tip cells established PXN as a mechanotransduction node converting extracellular rigidity into transcriptional programs controlling angiogenesis.\",\n      \"evidence\": \"Tunable hydrogel substrates, Rac1-GTP pulldown, phospho-FAK/PXN western blot, YAP immunofluorescence, siRNA knockdown in endothelial cells\",\n      \"pmids\": [\"34466738\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific PXN phosphorylation sites involved in Rac1 activation not mapped\", \"Contribution of PXN vs. other FA proteins to stiffness sensing not dissected\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of ULK1/2 as direct PXN kinases at S32/S119 — opposing FAK/Src tyrosine phosphorylation — resolved how autophagy-related kinases antagonize focal adhesion assembly and phase separation, linking nutrient-sensing and adhesion signaling.\",\n      \"evidence\": \"In vitro kinase assay, site-directed mutagenesis, FRAP/phase separation assays, traction force microscopy, KO/KD migration phenotype in breast cancer cells\",\n      \"pmids\": [\"37846507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ULK1/2-PXN axis operates under physiological autophagy induction not tested\", \"Structural basis for how S32/S119 phosphorylation disrupts phase separation unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Mapping the ANGPTL4/NRP1/ABL1→phospho-PXN and CXCL5→phospho-PXN/AKT→PD-L1 cascades broadened the upstream inputs to PXN and linked PXN phosphorylation to tumor immune evasion.\",\n      \"evidence\": \"siRNA and dasatinib inhibition of ABL1, phospho-PXN/AKT western blot, flow cytometry for PD-L1, in vivo mouse models in HNSCC and lung cancer\",\n      \"pmids\": [\"37169211\", \"39034411\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct ABL1 phosphorylation sites on PXN not mapped by mass spectrometry\", \"Whether PXN-AKT-PD-L1 feedback operates in immunocompetent models not shown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"NMR/SAXS structural analysis revealed that PXN's disordered N-terminus compacts into a multi-modal fuzzy complex with FAK-FAT adopting four interconverting states, providing a structural framework for understanding how post-translational modifications rewire signaling output.\",\n      \"evidence\": \"NMR spectroscopy, SAXS, ensemble modeling (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.01.01.630265\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Awaits peer review\", \"Functional consequence of individual conformational states not tested by mutagenesis\", \"How phosphorylation shifts the conformational ensemble not experimentally demonstrated\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Demonstration of a PXN–STAT3–SRC positive feedback loop and PXN-mediated YB-1 stabilization via ubiquitin-degradation inhibition in glioblastoma revealed PXN's role as both a transcriptional regulator target and an effector of protein stability.\",\n      \"evidence\": \"qRT-PCR, western blotting, mRNA-seq, knockdown assays, IHC in GBM\",\n      \"pmids\": [\"41872167\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which PXN inhibits YB-1 ubiquitination (direct binding vs. intermediate) not defined\", \"Single cancer type\", \"Whether PXN transcriptionally regulates SRC directly or indirectly unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A complete phosphosite-resolved map linking each PXN modification to a specific conformational state of the PXN–FAK fuzzy complex and to distinct downstream pathway outputs is still lacking.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No integrated structural-phosphoproteomic model of PXN signaling\", \"In vivo genetic models (knock-in of phospho-mutants) are absent from the literature\", \"Role of PXN phase separation in physiological (non-cancer) mechanotransduction untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 3]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 3]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 3, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 5, 7, 8]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [1, 3, 12]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [\n      \"Focal adhesion complex\"\n    ],\n    \"partners\": [\n      \"FAK\",\n      \"ULK1\",\n      \"ULK2\",\n      \"ABL1\",\n      \"STAT3\",\n      \"YB1\",\n      \"ITGB1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"Paxillin (PXN) is a multidomain scaffold protein that nucleates focal adhesion signaling complexes, coupling integrin-mediated adhesion to cytoskeletal remodeling, mechanotransduction, and cell migration. Its LIM3 domain targets it to focal adhesions, while N-terminal LD motifs recruit FAK (residues 143–164 and 265–313), vinculin (residue E151 critical), and a PAK/PIX/p95PKL complex (via LD4) that activates Rac-dependent lamellipodia formation [PMID:8922390, PMID:10330411]. PXN is phosphorylated by FAK/Src (Tyr31/Tyr118), JNK1 (Ser178), ERK (Ser106/231/290), and ULK1/2 (Ser32/S119), with each modification distinctly controlling focal adhesion assembly, phase separation, and turnover—ULK1/2 phosphorylation weakens homotypic PXN interactions and opposes FAK/Src-driven adhesion maturation, while JNK1-mediated Ser178 phosphorylation maintains labile adhesions required for rapid migration [PMID:37846507, PMID:12853963]. PXN also functions beyond canonical adhesion signaling: it stabilizes YB-1 protein by blocking its ubiquitin-mediated degradation, participates in a STAT3–PXN–SRC positive feedback loop, and links CXCL5/AKT signaling to PD-L1 immune checkpoint expression in tumor cells [PMID:41872167, PMID:39034411].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing that paxillin, like FAK, is a direct integrin cytoplasmic domain interactor resolved how adhesion receptors nucleate intracellular signaling scaffolds independently of each other.\",\n      \"evidence\": \"In vitro GST pull-down with beta1 integrin cytoplasmic peptides from chicken embryo cell lysates\",\n      \"pmids\": [\"7657702\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding affinities and stoichiometry not determined\", \"No in vivo validation of simultaneous FAK–PXN co-recruitment\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Systematic domain mapping revealed that PXN's LIM3 domain drives focal adhesion targeting while separable N-terminal interfaces independently bind vinculin and FAK, establishing PXN as a modular scaffold with distinct partner-binding surfaces.\",\n      \"evidence\": \"Truncation/point mutagenesis with GFP-tagged constructs in CHO.K1 cells, co-immunoprecipitation, and immunofluorescence\",\n      \"pmids\": [\"8922390\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for LIM3-mediated targeting unresolved\", \"Other LD motif functions not yet mapped\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Discovery that the LD4 motif recruits a PAK/PIX/p95PKL complex to paxillin placed PXN upstream of Rac-mediated actin remodeling and explained how focal adhesions couple to lamellipodia and directional migration.\",\n      \"evidence\": \"Yeast two-hybrid, GST pull-down, microinjection of GST-LD4 into NIH3T3 cells, wound-healing assay\",\n      \"pmids\": [\"10330411\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation of LD4 accessibility by phosphorylation not addressed\", \"In vivo relevance in animal models not tested\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Two contemporaneous studies defined opposing regulators of the PXN–FAK signaling module: EphA2-recruited SHP2 rapidly dephosphorylates FAK and PXN to dismantle integrin signaling, while GIT1 binding to PXN drives focal complex disassembly and promotes motility via PIX/PAK.\",\n      \"evidence\": \"Time-course co-IP with phospho-specific antibodies (Eph signaling); domain-deletion mapping and overexpression with motility readout (GIT1)\",\n      \"pmids\": [\"10655584\", \"10938112\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SHP2 dephosphorylates PXN directly or only via FAK not resolved\", \"Quantitative relationship between GIT1 binding and adhesion lifetime unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Identification of JNK1 as a direct Ser178 kinase for paxillin, and the finding that S178A mutation stabilizes focal adhesions and blocks rapid migration, established that stress kinase signaling converges on PXN to regulate adhesion lability.\",\n      \"evidence\": \"In vitro kinase assay, S178A mutagenesis, wound-healing and single-cell migration assays in NBT-II cells\",\n      \"pmids\": [\"12853963\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream reader of pS178 not identified\", \"Interplay with ERK-mediated serine phosphorylation untested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Quantitative focal adhesion proteomics confirmed PXN as a core contractility-responsive component, situating it within the myosin-II/Rho-dependent adhesion maturation program.\",\n      \"evidence\": \"SILAC mass spectrometry of isolated focal adhesions ± blebbistatin\",\n      \"pmids\": [\"21214176\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PXN not primary focus of study; stoichiometric changes not deeply characterized\", \"Post-translational modifications not captured\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Consolidation of phosphorylation data clarified that ERK-mediated phosphorylation of PXN Ser106/231/290 promotes adhesion disassembly, complementing the earlier JNK1 and FAK/Src phosphorylation findings and establishing PXN as a multi-kinase integrator of adhesion dynamics.\",\n      \"evidence\": \"Review synthesizing phospho-specific western blot, mutagenesis, and co-immunoprecipitation studies across multiple primary publications\",\n      \"pmids\": [\"28214467\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Temporal order of multi-site phosphorylation during adhesion turnover unresolved\", \"Combinatorial phospho-code not decoded\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstration that matrix stiffness drives FAK/PXN phosphorylation to activate Rac1 and nuclear YAP in endothelial cells extended PXN's role from cell migration to mechanotransduction-dependent cell fate specification (tip cell emergence).\",\n      \"evidence\": \"Tunable hydrogel substrates, Rac1 activation assay, YAP nuclear translocation imaging, EC spheroid sprouting assay\",\n      \"pmids\": [\"34466738\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PXN phosphorylation is sufficient or merely necessary for YAP activation not tested\", \"Identity of the Rac GEF recruited by p-PXN in this context unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Discovery that ULK1/2 directly phosphorylate PXN at S32/S119 to weaken homotypic interactions and suppress liquid-liquid phase separation of PXN revealed a novel autophagy-independent mechanism by which ULK kinases antagonize focal adhesion assembly and oppose FAK/Src signaling.\",\n      \"evidence\": \"In vitro kinase assay, S32A/S119A mutagenesis, phase separation assay, FRAP, traction force microscopy in ULK1/2 KO cells\",\n      \"pmids\": [\"37846507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phase separation of PXN in intact focal adhesions not directly visualized\", \"Whether other kinases also regulate PXN LLPS unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of an ANGPTL4/NRP1/ABL1 signaling axis converging on PXN phosphorylation to drive HNSCC migration expanded the upstream receptor inputs that funnel through PXN beyond classical integrins.\",\n      \"evidence\": \"siRNA knockdown of NRP1/ABL1, dasatinib inhibition, p-PXN western blot, orthotopic HNSCC tumor model\",\n      \"pmids\": [\"37169211\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific PXN tyrosine residues phosphorylated by ABL1 not mapped\", \"Whether ABL1–PXN interaction is direct not confirmed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linking CXCL5-induced PXN/AKT phosphorylation to PD-L1 upregulation in lung cancer revealed an unexpected role for PXN in immune checkpoint regulation, extending its function from adhesion signaling to tumor immune evasion.\",\n      \"evidence\": \"siRNA, p-PXN and p-AKT western blot, PD-L1 flow cytometry, in vivo combination anti-CXCL5/anti-PD-L1 therapy in SCID/NOD mice\",\n      \"pmids\": [\"39034411\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular mechanism linking p-PXN to PD-L1 transcription/stabilization not defined\", \"Generalizability beyond lung cancer not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"ChIP-validated STAT3–PXN transcriptional feedback loop and the discovery that PXN stabilizes YB-1 by blocking its ubiquitination revealed a non-canonical nuclear/signaling function for PXN beyond adhesion scaffolding.\",\n      \"evidence\": \"ChIP assay (STAT3 on PXN promoter), co-IP (PXN–YB-1), ubiquitination assay, mRNA-seq in GBM cells\",\n      \"pmids\": [\"41872167\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether PXN directly contacts the ubiquitin ligase targeting YB-1 is unknown\", \"PXN subcellular compartment where YB-1 stabilization occurs not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The combinatorial phospho-code on PXN — how simultaneous phosphorylation by FAK/Src, JNK1, ERK, and ULK1/2 is read by downstream effectors to switch between adhesion assembly, phase separation, and disassembly — remains unresolved, as does the structural basis of the multi-modal fuzzy PXN–FAK complex in the context of full-length proteins in cellulo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No systematic analysis of combinatorial phospho-states on PXN and their effector recruitment\", \"Full-length PXN–FAK structural ensemble not resolved in cellular context\", \"Phase separation of PXN at native expression levels in focal adhesions not demonstrated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 2, 4, 10]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [1, 6, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 2, 6, 10, 19]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [19, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 5, 10, 16, 19, 21]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [1, 2, 6, 16, 23]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [0, 6, 10]}\n    ],\n    \"complexes\": [\n      \"FAK-paxillin complex\",\n      \"PAK-PIX-GIT1-paxillin complex\"\n    ],\n    \"partners\": [\n      \"PTK2\",\n      \"VCL\",\n      \"GIT1\",\n      \"PAK1\",\n      \"ARHGEF7\",\n      \"ABL1\",\n      \"YBX1\",\n      \"PTPN11\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}