{"gene":"PLD1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2020,"finding":"Crystal structure of human PLD1 catalytic domain at 1.8 Å resolution reveals a globular fold with a funnel-shaped hydrophobic cavity leading to the active site, a PIP2-binding polybasic pocket at the membrane interface essential for activity, a C-terminus that contributes to the catalytic pocket, and a phosphohistidine intermediate. Mapping of mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface.","method":"X-ray crystallography (1.8 Å resolution), active-site mutagenesis, PIP2-binding pocket mutagenesis, RhoA activation assays","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with functional validation by mutagenesis of active site, PIP2-binding pocket, and RhoA interaction interface in a single rigorous study","pmids":["32198492"],"is_preprint":false},{"year":2003,"finding":"PLD1 acts upstream of mTOR to regulate S6K1 activation and cell size. Catalytically inactive PLD1 exerts a dominant-negative effect on S6K1 activation; RNAi knockdown of PLD1 drastically inhibits serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation. Cdc42 activates S6K1 through the mTOR pathway via a region specifically required for PLD1 activation, and exogenous phosphatidic acid (PA) rescues the effect of a PLD1-inactive Cdc42 mutant.","method":"RNAi knockdown, dominant-negative overexpression, rapamycin-resistant S6K1 mutant epistasis, exogenous PA rescue, kinase activity assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (RNAi, dominant-negative, epistasis, PA rescue) establishing pathway position of PLD1 upstream of mTOR-S6K1","pmids":["14653992"],"is_preprint":false},{"year":2008,"finding":"RSK2 directly phosphorylates PLD1 at Thr-147 in the N-terminal phox homology domain, activating PLD1 activity and promoting exocytosis in chromaffin cells. RSK2 is activated by calcium (high K+ stimulus), physically interacts with PLD1, and expression of a phosphomimetic PLD1-T147 mutant fully restores secretion in RSK2-depleted cells.","method":"Co-immunoprecipitation, site-directed mutagenesis (phosphomimetic mutant), RSK2 knockdown, PLD activity assay, chromaffin cell secretion assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — phosphorylation site identified by mutagenesis, physical interaction by Co-IP, functional rescue by phosphomimetic mutant, multiple orthogonal methods in a single study","pmids":["18550821"],"is_preprint":false},{"year":2013,"finding":"RSK2-dependent phosphorylation of PLD1 is required for NGF-induced neurite outgrowth and membrane supply via VAMP-7 vesicle fusion. A phosphomimetic PLD1 mutant rescues inhibition of neurite outgrowth in RSK2-silenced PC12 cells. TIRF microscopy shows RSK2 and PLD1 positively control VAMP-7 vesicle fusion at neurite growth sites. Neurons from Pld1 knockout mice show delayed growth similar to Rsk2 knockout neurons.","method":"siRNA knockdown, phosphomimetic PLD1 mutant rescue, TIRF microscopy, PLD activity assay, knockout mouse neurons","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout confirmation, phosphomimetic rescue, live imaging, multiple orthogonal methods across two model systems","pmids":["24336713"],"is_preprint":false},{"year":2006,"finding":"PLD1 (not PLD2) is the isoform responsible for cytosolic lipid droplet formation. PLD1 functions upstream of ERK2 in this pathway: inhibition of ERK2 eliminates the effect of PLD1 on lipid droplet formation without affecting PLD1 enzymatic activity. ERK2 increases phosphorylation of dynein and its accumulation on ADRP-containing lipid droplets; microinjection of anti-dynein antibodies strongly inhibits lipid droplet formation.","method":"siRNA knockdown, overexpression, microinjection, pharmacological inhibition, cell-free system, ERK2 epistasis","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal approaches (siRNA, overexpression, microinjection, pharmacology) establishing pathway order PLD1→ERK2→dynein","pmids":["16723731"],"is_preprint":false},{"year":2000,"finding":"PLD1 palmitoylation occurs on Cys240 and Cys241, requires N-terminal sequences (first 168 aa) and interdomain association of the two HKD halves. Palmitoylation-deficient PLD1 (C240A/C241A) retains basal activity and PKC responsiveness but shows markedly reduced Ser/Thr phosphorylation and weakened membrane association. Mutation of Cys310 or Cys612 increases basal PLD activity 2- or 4-fold, respectively.","method":"Site-directed mutagenesis, metabolic labeling (palmitoylation), membrane fractionation, in vitro PLD activity assay, PKC stimulation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple cysteine mutants, in vitro activity assays, membrane fractionation, and palmitoylation labeling providing comprehensive mechanistic characterization","pmids":["11121416"],"is_preprint":false},{"year":2000,"finding":"Human PLD1 can be activated by calcium-mobilizing agonists and by co-expression with PKCα (but not PKCδ), and PLD1 physically associates with PKC isoforms. Calcium enhances PLD1 activity in membrane assays. PLD1 activity is also stimulated by calmodulin and PKCα-enriched cytosol in reconstitution assays.","method":"Co-expression in Sf9 cells, immunoprecipitation, membrane reconstitution assay, PLD activity (transphosphatidylation), calcium ionophore treatment","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and enzymatic assays in a single study, but performed in insect (Sf9) cell overexpression system","pmids":["10838164"],"is_preprint":false},{"year":2005,"finding":"PLD1 (but not PLD2) localizes to lipid droplets in oleic acid-treated NIH3T3 cells in an Arf1-dependent manner. Brefeldin A (an ARF-GEF inhibitor) suppresses both PLD activation and lipid droplet formation. Arf1 stimulates PLD1 activity in LD-enriched subcellular fractions.","method":"Immunocytochemistry, subcellular fractionation, Western blot, pharmacological inhibition (Brefeldin A), exogenous Arf1 stimulation, PLD activity assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional consequence (LD formation) and in vitro Arf1 stimulation; single lab, multiple methods","pmids":["16054594"],"is_preprint":false},{"year":2010,"finding":"PLD1 acts downstream of Src to activate PKCγ in VEGF signaling in retinal microvascular endothelial cells; the Src→PLD1→PKCγ cascade mediates VEGF-induced endothelial cell migration, proliferation, and tube formation, and is required for hypoxia-induced retinal neovascularization in vivo.","method":"Pharmacological inhibition, dominant-negative mutants, siRNA knockdown, in vivo retinal neovascularization model (oxygen-induced retinopathy)","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis established by multiple inhibitory approaches (pharmacological + dominant-negative + siRNA) in vitro and in vivo; single lab","pmids":["20421451"],"is_preprint":false},{"year":2011,"finding":"cPLA2 acts as an effector downstream of the Src→PLD1→PKCγ signaling axis; VEGF-induced cPLA2 phosphorylation and arachidonic acid release require Src, PLD1, and PKCγ activity, and exogenous AA rescues endothelial responses when cPLA2 is depleted.","method":"siRNA knockdown, pharmacological inhibition, AA rescue experiments, in vivo oxygen-induced retinopathy model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway position of PLD1 upstream of cPLA2 established by epistasis with rescue; single lab","pmids":["21536681"],"is_preprint":false},{"year":2006,"finding":"PLD1 enzymatic activity mediates chemokine-induced (IL-8, FMLP) chemotaxis of HL-60 leukocytes; a lipase-inactive PLD1-K830R mutant abrogates all chemokine-induced potentiating actions, while chemokinesis does not require PLD1 enzymatic activity. Both PLD1 and PLD2 are required for cell motility and associate with cell polarity markers, F-actin, and adhesion structures.","method":"siRNA knockdown, overexpression of lipase-inactive mutant (K830R), in vitro PLD activity assay, chemokinesis/chemotaxis assays, immunofluorescence microscopy","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — catalytic dead mutant distinguishes enzymatic from non-enzymatic roles; single lab with multiple methods","pmids":["16873675"],"is_preprint":false},{"year":2012,"finding":"PLD1 acts downstream of RhoA to suppress dendritic branching in hippocampal neurons. The branching restriction by constitutively active RhoA (V14-RhoA) is partially rescued by PLD1 knockdown, and the inhibitory effect of both V14-RhoA and PLD1 overexpression can be ameliorated by reducing PA levels.","method":"Gain-of-function and loss-of-function (overexpression, siRNA), constitutively active RhoA epistasis, PA level manipulation, cultured hippocampal neurons","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with RhoA and PA rescue establishes pathway; single lab","pmids":["22674271"],"is_preprint":false},{"year":2009,"finding":"PLD1 directly interacts with μ2 (a subunit of AP2 adaptor complex) and this interaction requires PLD1 binding to its own product phosphatidic acid. PLD1–μ2 interaction facilitates membrane recruitment of AP2 and determines the kinetics of EGFR endocytosis.","method":"Co-immunoprecipitation, kinetic analysis of endocytosis, AP2 membrane recruitment assay, PA-binding requirement demonstrated by mutagenesis/biochemical assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct interaction shown by Co-IP with mechanistic follow-up on PA dependency and endocytosis kinetics; single lab","pmids":["19763255"],"is_preprint":false},{"year":2010,"finding":"PLD1 residues 762–801 (within the D4 domain, residues 712–818) constitute the minimal binding interface for PED/PEA15, with a Kd ~0.7 μM. PED/PEA15 interaction with PLD1 D4 enhances PKCα activity and impairs insulin-stimulated PKCζ activation and glucose transport; disruption of this interaction restores normal signaling.","method":"D4 deletion mutants, ELISA, surface plasmon resonance (SPR), transfection of D4α fragments, PKCα/PKCζ activity assay","journal":"Molecular bioSystems","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding interface mapped by deletion mutagenesis with affinity measurement and functional rescue; single lab","pmids":["20714510"],"is_preprint":false},{"year":2013,"finding":"Disruption of PED/PEA15–PLD1 interaction in vivo (by adenoviral D4 delivery) decreases PKCα activation, restores PKCζ activation and insulin-dependent glucose uptake in skeletal muscle of PED/PEA15-overexpressing transgenic mice and in high-fat-diet obese mice.","method":"Adenoviral gene transfer, co-immunoprecipitation, PKC activity assay, glucose uptake assay, transgenic and HFD mouse models","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo disruption of PLD1–PED/PEA15 interaction with mechanistic readouts; single lab","pmids":["23585839"],"is_preprint":false},{"year":2016,"finding":"PLD1 negatively regulates adipogenic differentiation by generating PA, which displaces DEPTOR from mTORC1, leading to mTOR-dependent phosphorylation of IRS-1 at Ser636/639 and suppression of insulin signaling required for adipogenesis.","method":"PLD1-specific inhibitor (VU0155069), siRNA knockdown, PA treatment, PLD1 overexpression, mTORC1 pull-down (DEPTOR displacement), phospho-IRS-1 Western blot, 3T3-L1 differentiation assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (inhibitor, siRNA, PA rescue, DEPTOR displacement assay) establishing mechanism; single lab","pmids":["27872488"],"is_preprint":false},{"year":2021,"finding":"RalA acts downstream of autophagy to recruit PLD1 to lysosomes during nutrient depletion, where PLD1 converts PC to PA to promote localized PA production. This recruits perilipin 3 (PLIN3) to expanding lipid droplets, facilitating LD growth.","method":"RalA inhibition, PLD1 knockout/inhibition, live-cell imaging of lysosomal PLD1 recruitment, perilipin 3 localization assay, nutrient depletion model","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PLD1 localization to lysosomes shown in a RalA-dependent manner with downstream perilipin 3 functional readout; single lab","pmids":["34320341"],"is_preprint":false},{"year":2017,"finding":"PLD1 inhibition suppresses COPII vesicle transport from ER to Golgi by preventing Sec13/31 recruitment from the cytosol to the ER membrane during COPII vesicle formation. PLD1 knockdown increases ER stress marker GRP78 and promotes apoptosis.","method":"PLD1 inhibitor, siRNA knockdown, cell-free COPII coat protein recruitment assay, Western blot for ER stress markers","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-free reconstitution assay for COPII recruitment with PLD1 inhibition; single lab, two methods","pmids":["28648601"],"is_preprint":false},{"year":2017,"finding":"HS1BP3 negatively regulates autophagy by inhibiting PLD1 activity and reducing PLD1 localization to ATG16L1-positive autophagosome precursor membranes, thereby decreasing PA content on these membranes. HS1BP3 depletion increases total cellular PA from elevated PLD activity and PLD1 localization to ATG16L1 membranes.","method":"HS1BP3 siRNA depletion, PLD activity assay, PA measurement, PLD1 localization (immunofluorescence), autophagosome formation assay","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — PLD1 localization and activity measured after HS1BP3 depletion; replicated across human cells and zebrafish; single lab","pmids":["28318354"],"is_preprint":false},{"year":2022,"finding":"PLD1 preferentially interacts with ARL11 and ARL14 (Arf GTPase family members); ARL11/14 activate PLD1 and may be recruited to membrane vesicles by PLD1. PLD1 and ARL11 collaborate to promote macrophage phagocytosis.","method":"Proximity labeling (miniTurboID) interactome, TMT-based quantitative MS, PLD1 activity assay, phagocytosis assay","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity interactome confirmed with PLD1 activity and phagocytosis functional assays; single lab, multiple methods","pmids":["35844135"],"is_preprint":false},{"year":2015,"finding":"In cortical neurons, BDNF induces rapid RSK2-dependent PLD1 activation, and PLD1 is required for BDNF-stimulated ERK1/2-CREB and mTOR-S6K signaling. PLD1, ERK1/2, and RSK2 form a complex with scaffolding protein PEA15 after BDNF treatment and partially colocalize on endosomal structures.","method":"Pld1 and Rsk2 knockout neurons, PLD activity assay, immunofluorescence colocalization, PEA15 siRNA silencing, phospho-CREB nuclear accumulation assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout, complex formation, and signaling pathway data in cortical neurons; single lab","pmids":["26437780"],"is_preprint":false},{"year":2012,"finding":"In acrosomal exocytosis, diacylglycerol activates PLD1 through PKC, and PLD1-generated PA promotes PIP2 synthesis in a positive feedback loop. Both PKC and PLD1 are required to maintain IP3-sensitive calcium channel opening required for exocytosis. Rescue experiments with PA, PIP2, and adenophostin confirm PLD1's role in maintaining PIP2 levels upstream of calcium channel gating.","method":"Permeabilized sperm exocytosis assay, PLD1 inhibition, PKC inhibition, PA/PIP2 rescue, Rab3A GTP-loading assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway placed by pharmacological epistasis and lipid rescue experiments; single lab","pmids":["22609963"],"is_preprint":false},{"year":2019,"finding":"PLD1 physically associates with PKD1 (protein kinase D1), and PLD1 acts upstream of PKD1 to positively regulate dendritic spine morphogenesis in hippocampal neurons. PLD1 inhibitor reduces PKD1 activation.","method":"Co-immunoprecipitation, siRNA knockdown with rescue, PLD1 inhibitor treatment, dendritic spine morphology analysis","journal":"Molecular and cellular neurosciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP interaction with epistasis by knockdown/rescue; single lab, single paper","pmids":["31356881"],"is_preprint":false},{"year":2013,"finding":"The H452Y polymorphic form of the 5-HT2A receptor selectively reduces PLD1 binding to the receptor's carboxy-terminal tail and attenuates PLD signaling (but not Gq/11-PLC signaling). Co-immunoprecipitation and GST-fusion protein experiments show PLD1 docks to the 5-HT2AR C-terminal tail, and a blocking peptide spanning residue 452 reduces PLD1-dependent responses.","method":"Co-immunoprecipitation, GST-fusion pulldown, blocking peptides, PLD activity assay, cell proliferation assay","journal":"Cellular signalling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and pulldown showing interaction; single lab, single study","pmids":["23314176"],"is_preprint":false},{"year":2017,"finding":"PLD1 negatively regulates the cofilin-p53 pro-apoptotic pathway by promoting cofilin inactivation and inhibiting cofilin/p53 complex formation, thereby preventing p53 mitochondrial and nuclear translocation. Cofilin knockdown or PLD1 overexpression inhibits this apoptotic pathway.","method":"Cofilin knockdown, PLD1 overexpression, immunofluorescence for p53 subcellular localization, co-immunoprecipitation of cofilin-p53 complex, APP/PS1 transgenic mice","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — mechanistic pathway proposed with cofilin/p53 Co-IP and localization data; single lab","pmids":["28912445"],"is_preprint":false},{"year":2021,"finding":"In hepatocellular carcinoma, cofilin 1 (CFL1) physically interacts with PLD1 and maintains PLD1 expression by inhibiting ubiquitin-mediated protein degradation, thereby activating AKT signaling. Hypoxia-induced CFL1 promotes tumor progression through the CFL1/PLD1/AKT axis.","method":"Co-immunoprecipitation, siRNA knockdown, ubiquitination assay, AKT phosphorylation Western blot, xenograft model","journal":"Clinical and translational medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP for interaction, mechanistic chain not fully validated; single lab","pmids":["33784016"],"is_preprint":false},{"year":2024,"finding":"PLD1 is required for spindle assembly, MTOC clustering, and cortical spindle migration in mouse oocyte meiosis. PLD1 interacts directly with spindle components, RAB11A+ vesicles, and autophagic vacuoles. PLD1 suppression decreases PIP2, phospho-cofilin (p-CFL1-Ser3), and ACTR2 on MTOC/spindle; exogenous PIP2 or CFL1-S3E (hyperphosphorylation mutant) partially rescues spindle defects. Autophagy activation phenocopies PLD1 loss, and autophagy inhibition rescues PLD1-depleted oocytes by restoring PIP2, ACTR2, and p-CFL1.","method":"Morpholino knockdown, PLD1 inhibitor, proximity ligation assay (direct spindle interaction), exogenous PIP2/CFL1-S3E/ACTR2 rescue, autophagy manipulation, immunofluorescence","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal rescue experiments and PLA for direct interactions; single lab","pmids":["38513669"],"is_preprint":false},{"year":2016,"finding":"PLD1 is required for Frizzled7 (Fz7) receptor endocytosis in Xenopus embryos upon Wnt11 stimulation. PLD1 promotes Wnt/PCP signaling activation through its PX domain, which regulates GAP activity of dynamin to facilitate Fz7 endocytosis. Loss- and gain-of-function of PLD1 disrupts convergent extension movements in Xenopus gastrulation.","method":"Loss/gain-of-function in Xenopus embryos, biochemical analysis of Fz7 endocytosis, live imaging, dynamin GAP assay, PX domain mutants","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mechanism established in Xenopus (ortholog) with biochemical and functional data; single lab","pmids":["26806705"],"is_preprint":false},{"year":2024,"finding":"Nuclear PLD1 interacts with nucleophosmin 1 (NPM1) through a non-enzymatic mechanism, triggering NPM1 nuclear translocation. Nuclear NPM1 acts as a transcription factor to upregulate IL7R expression, which activates JAK1/STAT5/BCL-2 signaling to confer gemcitabine resistance in pancreatic cancer.","method":"CRISPRa/dCas9 genome-wide screen, co-immunoprecipitation (Co-IP), ChIP, ChIP-seq, transcriptome sequencing, PLD1 inhibitor (VU0155069)","journal":"Cancer biology & medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP for PLD1-NPM1 interaction confirmed, ChIP-seq for NPM1-IL7R axis, genome-wide screen identification; single lab","pmids":["37381714"],"is_preprint":false},{"year":2004,"finding":"In porcine tracheal smooth muscle cells, ACh/PMA stimulation induces tyrosine phosphorylation of PLD1 (not PLD2) through a PKC- and tyrosine kinase-dependent pathway. Both ACh and PMA increase Ser/Thr and Tyr phosphorylation of PLD1, blocked by PKC inhibitor calphostin C or tyrosine kinase inhibitor genistein.","method":"Western blot with anti-phosphotyrosine antibodies, pharmacological inhibitors (genistein, calphostin C), PLD activity assay","journal":"Journal of biomedical science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — phosphorylation shown by anti-pTyr Western blot with pharmacological evidence; single lab, single method","pmids":["15591778"],"is_preprint":false},{"year":2021,"finding":"PLD1 loss-of-function mutations cause congenital right-sided cardiac valve defects. Missense variants in PLD1 are overrepresented in regions critical for catalytic activity, and most mutant proteins show strongly reduced enzymatic activity. PLD1 inhibition decreases endothelial-mesenchymal transition (EndMT), an early step in valvulogenesis.","method":"Whole-exome sequencing, enzymatic activity assay of mutant PLD1 proteins, EndMT assay with PLD1 inhibitor, analysis of 30 patients from 21 families","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — enzymatic assay of multiple disease-associated mutants plus functional EndMT assay; multi-center study with large patient cohort","pmids":["33645542"],"is_preprint":false},{"year":2024,"finding":"PLD1 promotes nasopharyngeal carcinoma progression via a positive feedback loop with NF-κB: PLD1 enhances NF-κB activity by facilitating phosphorylation and nuclear translocation of RELA, which in turn binds the PLD1 promoter and augments PLD1 expression.","method":"ChIP assay (RELA binding to PLD1 promoter), RELA knockdown/overexpression rescue, PLD1 inhibitor (VU0155069) in patient-derived xenograft, luciferase reporter","journal":"Journal of genetics and genomics = Yi chuan xue bao","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIP for RELA-PLD1 promoter interaction and functional rescue; single lab","pmids":["38885836"],"is_preprint":false},{"year":2017,"finding":"T. gondii GRA7-III interacts with the PX domain of PLD1, facilitating PLD1 enzymatic activity, phago-lysosomal maturation, and antimicrobial activity in a GRA7-III Ser135 phosphorylation-dependent manner via PKCα.","method":"Co-immunoprecipitation, PLD1 enzymatic activity assay, phago-lysosomal maturation assay, PKCα phosphorylation experiments","journal":"PLoS pathogens","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and enzymatic activity data; interaction with a parasite protein; single lab","pmids":["28125719"],"is_preprint":false}],"current_model":"PLD1 is a phosphatidylcholine-hydrolyzing enzyme whose 1.8-Å crystal structure reveals a funnel-shaped active site with a phosphohistidine intermediate, a PIP2-binding polybasic membrane interface pocket essential for activity, and a RhoA-interaction surface; it is regulated by palmitoylation (Cys240/241), PKC-dependent and RSK2-dependent phosphorylation (Thr-147), Arf1/ARL11/ARL14 GTPases, and Ca²⁺/calmodulin, and acts as a signaling hub that generates phosphatidic acid to activate the mTOR→S6K1 axis (downstream of Cdc42), control lipid droplet biogenesis (via ERK2 and dynamin), regulate EGFR endocytosis (via PA-dependent μ2/AP2 recruitment), support exocytosis and neurite outgrowth (through RSK2-PLD1 phosphorylation and VAMP-7 vesicle fusion), restrict dendritic branching downstream of RhoA, facilitate Wnt/PCP signaling via Frizzled7 endocytosis, modulate autophagy through PA production at ATG16L1 precursor membranes, and couple to VEGF-Src-PKCγ-cPLA2 signaling in retinal angiogenesis."},"narrative":{"mechanistic_narrative":"PLD1 is a phosphatidylcholine-hydrolyzing phospholipase whose generation of the signaling lipid phosphatidic acid (PA) at intracellular membranes serves as a regulatory hub for vesicle trafficking, organelle biogenesis, growth signaling, and cytoskeletal control [PMID:32198492, PMID:14653992]. Its catalytic domain folds into a funnel-shaped hydrophobic cavity feeding the active site, with a membrane-interface polybasic pocket that binds PIP2 (essential for activity), a C-terminal contribution to the catalytic pocket, a phosphohistidine intermediate, and a defined RhoA-binding surface [PMID:32198492]. Enzyme activity and membrane targeting are tuned by multiple inputs: palmitoylation on Cys240/241 strengthens membrane association [PMID:11121416], Ca2+/calmodulin and PKCalpha stimulate activity [PMID:10838164], RSK2 directly phosphorylates Thr-147 in the N-terminal PX domain to activate the enzyme [PMID:18550821], and Arf-family GTPases including Arf1, ARL11, and ARL14 promote activity and membrane recruitment [PMID:16054594, PMID:35844135]. Through PA production PLD1 acts upstream of the mTOR-S6K1 axis to regulate cell size and (via DEPTOR displacement and IRS-1 phosphorylation) insulin signaling and adipogenesis [PMID:14653992, PMID:27872488]. PLD1-derived PA drives lipid-droplet biogenesis through an ERK2-dynein pathway and, upon RalA-dependent lysosomal recruitment during nutrient depletion, recruits perilipin 3 to growing droplets [PMID:16723731, PMID:34320341], supports COPII-dependent ER-to-Golgi transport [PMID:28648601], couples PA production to AP2/mu2 recruitment for EGFR endocytosis [PMID:19763255], and provides PA at ATG16L1-positive precursor membranes during autophagy under HS1BP3 control [PMID:28318354]. In neurons, RSK2-PLD1 signaling promotes neurite outgrowth via VAMP-7 vesicle fusion and BDNF-driven ERK/CREB and mTOR signaling, while PA acting downstream of RhoA restricts dendritic branching [PMID:24336713, PMID:26437780, PMID:22674271]. Loss-of-function PLD1 mutations that reduce catalytic activity cause congenital right-sided cardiac valve defects, linked to impaired endothelial-mesenchymal transition during valvulogenesis [PMID:33645542].","teleology":[{"year":2000,"claim":"Established the upstream regulatory inputs to PLD1 activity, defining how lipid modification and second messengers control the enzyme at membranes.","evidence":"Cysteine mutagenesis with palmitoylation labeling and membrane fractionation, plus Sf9 co-expression with PKCalpha and Ca2+/calmodulin reconstitution assays","pmids":["11121416","10838164"],"confidence":"High","gaps":["Whether palmitoylation and phosphorylation act on the same membrane pool was not resolved","PKCalpha activation characterized in an overexpression insect-cell system"]},{"year":2003,"claim":"Placed PLD1 upstream of mTOR-S6K1, showing PA as the link between Cdc42 and cell-size control.","evidence":"RNAi knockdown, dominant-negative catalytically inactive PLD1, S6K1 epistasis, and exogenous PA rescue of a PLD1-inactive Cdc42 mutant","pmids":["14653992"],"confidence":"High","gaps":["Direct molecular target of PA within the mTOR complex not defined here","Did not address tissue specificity of the Cdc42-PLD1-mTOR axis"]},{"year":2006,"claim":"Defined a PLD1-specific (not PLD2) role in cytosolic lipid droplet formation and ordered the pathway PLD1 to ERK2 to dynein.","evidence":"siRNA, overexpression, anti-dynein microinjection, pharmacological ERK2 inhibition, and Arf1-dependent localization with Brefeldin A in NIH3T3 cells","pmids":["16723731","16054594"],"confidence":"High","gaps":["How PA links to dynein phosphorylation mechanistically not established","Direct vs indirect Arf1 effect on droplet-localized PLD1 not separated"]},{"year":2008,"claim":"Identified the direct activating phosphorylation of PLD1 at Thr-147 by RSK2, connecting calcium-stimulated kinase signaling to regulated exocytosis.","evidence":"Co-IP, phosphomimetic T147 mutant rescue of secretion in RSK2-depleted chromaffin cells, and PLD activity assays","pmids":["18550821"],"confidence":"High","gaps":["Structural basis of Thr-147 phosphorylation effect on the PX domain not shown","Other RSK2 substrates contributing to secretion not excluded"]},{"year":2009,"claim":"Showed PLD1 uses its own PA product to recruit endocytic machinery, providing a feedforward mechanism for receptor internalization.","evidence":"Co-IP of PLD1 with AP2 subunit mu2, PA-binding requirement by mutagenesis, and EGFR endocytosis kinetics","pmids":["19763255"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal structural mapping of the mu2 interface","Generality across other cargo receptors not tested"]},{"year":2013,"claim":"Extended the RSK2-PLD1 axis to neuronal membrane expansion, linking it to VAMP-7-dependent vesicle fusion driving neurite outgrowth.","evidence":"Phosphomimetic PLD1 rescue in RSK2-silenced PC12 cells, TIRF imaging of VAMP-7 fusion, and Pld1 knockout mouse neurons","pmids":["24336713"],"confidence":"High","gaps":["Direct PA-VAMP7 fusion machinery link not mechanistically dissected"]},{"year":2016,"claim":"Connected PLD1-derived PA to mTORC1 regulation via DEPTOR displacement, explaining suppression of insulin signaling and adipogenesis.","evidence":"PLD1 inhibitor, siRNA, PA treatment, mTORC1 pull-down showing DEPTOR displacement, and phospho-IRS-1 readout in 3T3-L1 cells","pmids":["27872488"],"confidence":"Medium","gaps":["Direct PA binding site on mTORC1/DEPTOR not mapped","Single cell-line context"]},{"year":2017,"claim":"Broadened PLD1 trafficking roles to ER-to-Golgi COPII transport and autophagosome precursor membranes, with HS1BP3 identified as a negative regulator of PLD1 at ATG16L1 membranes.","evidence":"Cell-free COPII coat recruitment assay with PLD1 inhibition; HS1BP3 depletion with PLD activity, PA measurement, and PLD1 localization to ATG16L1 membranes (replicated in zebrafish)","pmids":["28648601","28318354"],"confidence":"Medium","gaps":["How PA promotes Sec13/31 recruitment mechanistically unresolved","Mechanism of HS1BP3 inhibition of PLD1 not defined"]},{"year":2020,"claim":"Resolved the human PLD1 catalytic domain at high resolution, defining the active site, the essential PIP2-binding membrane interface, and the RhoA interaction surface.","evidence":"1.8-A X-ray crystallography with active-site, PIP2-pocket, and RhoA-interface mutagenesis and activation assays","pmids":["32198492"],"confidence":"High","gaps":["N-terminal PX/PH regulatory regions not captured in the structure","Membrane-bound conformational dynamics not resolved"]},{"year":2021,"claim":"Linked PLD1 loss-of-function to a human Mendelian disease, establishing catalytic activity as essential for cardiac valve development.","evidence":"Whole-exome sequencing of 30 patients from 21 families, enzymatic assays of mutant proteins, and EndMT assay with PLD1 inhibition","pmids":["33645542"],"confidence":"Medium","gaps":["Downstream PA-dependent effectors in valvulogenesis not identified","Genotype-phenotype correlation across variants not fully resolved"]},{"year":2021,"claim":"Defined RalA as the upstream signal recruiting PLD1 to lysosomes during nutrient depletion to drive localized PA production and lipid-droplet growth.","evidence":"RalA inhibition, PLD1 knockout/inhibition, live-cell imaging of lysosomal recruitment, and perilipin 3 localization readout","pmids":["34320341"],"confidence":"Medium","gaps":["Mechanism by which PA recruits PLIN3 not established","Single-lab system"]},{"year":2022,"claim":"Identified ARL11/ARL14 as preferred PLD1 GTPase partners, expanding the regulatory GTPase set and linking PLD1 to macrophage phagocytosis.","evidence":"miniTurboID proximity interactome, TMT quantitative MS, PLD1 activity assay, and phagocytosis assay","pmids":["35844135"],"confidence":"Medium","gaps":["Structural basis of ARL11/14 vs 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immunology","url":"https://pubmed.ncbi.nlm.nih.gov/26302934","citation_count":1,"is_preprint":false},{"pmid":"41777445","id":"PMC_41777445","title":"PLD1-Dependent Regulation of Synaptic Integrity: Implications for Cognitive Resilience and Alzheimer's Disease Pathogenesis.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41777445","citation_count":1,"is_preprint":false},{"pmid":"39553471","id":"PMC_39553471","title":"Prenatal detection of novel compound heterozygous variants of the PLD1 gene in a fetus with congenital heart disease.","date":"2024","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39553471","citation_count":1,"is_preprint":false},{"pmid":"33191863","id":"PMC_33191863","title":"Blockade of PLD1 potentiates the antitumor effects of bortezomib in multiple myeloma cells by inhibiting the mTOR/NF-κB signal pathway.","date":"2020","source":"Hematology (Amsterdam, Netherlands)","url":"https://pubmed.ncbi.nlm.nih.gov/33191863","citation_count":1,"is_preprint":false},{"pmid":"39736459","id":"PMC_39736459","title":"Segetalin B promotes bone formation in ovariectomized mice by activating PLD1/SIRT1 signaling to inhibit γ-secretase-mediated Notch1 overactivation.","date":"2024","source":"The Journal of steroid biochemistry and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/39736459","citation_count":1,"is_preprint":false},{"pmid":"40663939","id":"PMC_40663939","title":"Total saponins from Panax japonicus inhibit phosphatidylcholine hydrolysis and relieve hepatic steatosis via the miR-1a-3p/PLD1 pathway.","date":"2025","source":"Phytomedicine : international journal of phytotherapy and phytopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40663939","citation_count":0,"is_preprint":false},{"pmid":"40977551","id":"PMC_40977551","title":"miR-21 regulates LPS-induced apoptosis and inflammatory injury in rat cardiomyocytes by targeting PLD1 and STAT3.","date":"2025","source":"Polish journal of pathology : official journal of the Polish Society of Pathologists","url":"https://pubmed.ncbi.nlm.nih.gov/40977551","citation_count":0,"is_preprint":false},{"pmid":"41726972","id":"PMC_41726972","title":"Exploring the PLD1-tau interaction in Frontotemporal Dementia.","date":"2026","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41726972","citation_count":0,"is_preprint":false},{"pmid":"39698059","id":"PMC_39698059","title":"Pathogenic single nucleotide polymorphisms in RhoA gene: Insights into structural and functional impacts on RhoA-PLD1 interaction through molecular dynamics simulation.","date":"2024","source":"Current research in structural biology","url":"https://pubmed.ncbi.nlm.nih.gov/39698059","citation_count":0,"is_preprint":false},{"pmid":"42243532","id":"PMC_42243532","title":"PLD1 and PLD2 promote an immunosuppressive tumor microenvironment via CCL19-dependent macrophage polarization and PD-L1 induction.","date":"2026","source":"Experimental & molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/42243532","citation_count":0,"is_preprint":false},{"pmid":"39888011","id":"PMC_39888011","title":"RETRACTION: Promotion of Cell Autophagy and Apoptosis in Cervical Cancer by Inhibition of Long Noncoding RNA LINC00511 via Transcription Factor RXRA-regulated PLD1.","date":"2025","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/39888011","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.13.663496","title":"Gut microbiota and bile acids changes in MASLD mice model with hepatic PLD1 knockout","date":"2025-07-13","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.13.663496","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.03.02.640696","title":"Golgi retention of KIT in gastrointestinal stromal tumour cells is phospholipase D activity-dependent","date":"2025-03-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.03.02.640696","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.10.24315275","title":"Phosphatidic Acid Mediated Sonodynamic Therapy Facilitates M1 Macrophage Osteoclastic Function and Alleviates Early Vascular Calcification","date":"2024-10-11","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.10.24315275","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":42604,"output_tokens":8665,"usd":0.128893,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18598,"output_tokens":4824,"usd":0.106795,"stage2_stop_reason":"end_turn"},"total_usd":0.235688,"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\": 2020,\n      \"finding\": \"Crystal structure of human PLD1 catalytic domain at 1.8 Å resolution reveals a globular fold with a funnel-shaped hydrophobic cavity leading to the active site, a PIP2-binding polybasic pocket at the membrane interface essential for activity, a C-terminus that contributes to the catalytic pocket, and a phosphohistidine intermediate. Mapping of mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface.\",\n      \"method\": \"X-ray crystallography (1.8 Å resolution), active-site mutagenesis, PIP2-binding pocket mutagenesis, RhoA activation assays\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with functional validation by mutagenesis of active site, PIP2-binding pocket, and RhoA interaction interface in a single rigorous study\",\n      \"pmids\": [\"32198492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PLD1 acts upstream of mTOR to regulate S6K1 activation and cell size. Catalytically inactive PLD1 exerts a dominant-negative effect on S6K1 activation; RNAi knockdown of PLD1 drastically inhibits serum-stimulated S6K1 activation and 4E-BP1 hyperphosphorylation. Cdc42 activates S6K1 through the mTOR pathway via a region specifically required for PLD1 activation, and exogenous phosphatidic acid (PA) rescues the effect of a PLD1-inactive Cdc42 mutant.\",\n      \"method\": \"RNAi knockdown, dominant-negative overexpression, rapamycin-resistant S6K1 mutant epistasis, exogenous PA rescue, kinase activity assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (RNAi, dominant-negative, epistasis, PA rescue) establishing pathway position of PLD1 upstream of mTOR-S6K1\",\n      \"pmids\": [\"14653992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RSK2 directly phosphorylates PLD1 at Thr-147 in the N-terminal phox homology domain, activating PLD1 activity and promoting exocytosis in chromaffin cells. RSK2 is activated by calcium (high K+ stimulus), physically interacts with PLD1, and expression of a phosphomimetic PLD1-T147 mutant fully restores secretion in RSK2-depleted cells.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (phosphomimetic mutant), RSK2 knockdown, PLD activity assay, chromaffin cell secretion assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — phosphorylation site identified by mutagenesis, physical interaction by Co-IP, functional rescue by phosphomimetic mutant, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"18550821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RSK2-dependent phosphorylation of PLD1 is required for NGF-induced neurite outgrowth and membrane supply via VAMP-7 vesicle fusion. A phosphomimetic PLD1 mutant rescues inhibition of neurite outgrowth in RSK2-silenced PC12 cells. TIRF microscopy shows RSK2 and PLD1 positively control VAMP-7 vesicle fusion at neurite growth sites. Neurons from Pld1 knockout mice show delayed growth similar to Rsk2 knockout neurons.\",\n      \"method\": \"siRNA knockdown, phosphomimetic PLD1 mutant rescue, TIRF microscopy, PLD activity assay, knockout mouse neurons\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout confirmation, phosphomimetic rescue, live imaging, multiple orthogonal methods across two model systems\",\n      \"pmids\": [\"24336713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PLD1 (not PLD2) is the isoform responsible for cytosolic lipid droplet formation. PLD1 functions upstream of ERK2 in this pathway: inhibition of ERK2 eliminates the effect of PLD1 on lipid droplet formation without affecting PLD1 enzymatic activity. ERK2 increases phosphorylation of dynein and its accumulation on ADRP-containing lipid droplets; microinjection of anti-dynein antibodies strongly inhibits lipid droplet formation.\",\n      \"method\": \"siRNA knockdown, overexpression, microinjection, pharmacological inhibition, cell-free system, ERK2 epistasis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal approaches (siRNA, overexpression, microinjection, pharmacology) establishing pathway order PLD1→ERK2→dynein\",\n      \"pmids\": [\"16723731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PLD1 palmitoylation occurs on Cys240 and Cys241, requires N-terminal sequences (first 168 aa) and interdomain association of the two HKD halves. Palmitoylation-deficient PLD1 (C240A/C241A) retains basal activity and PKC responsiveness but shows markedly reduced Ser/Thr phosphorylation and weakened membrane association. Mutation of Cys310 or Cys612 increases basal PLD activity 2- or 4-fold, respectively.\",\n      \"method\": \"Site-directed mutagenesis, metabolic labeling (palmitoylation), membrane fractionation, in vitro PLD activity assay, PKC stimulation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple cysteine mutants, in vitro activity assays, membrane fractionation, and palmitoylation labeling providing comprehensive mechanistic characterization\",\n      \"pmids\": [\"11121416\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Human PLD1 can be activated by calcium-mobilizing agonists and by co-expression with PKCα (but not PKCδ), and PLD1 physically associates with PKC isoforms. Calcium enhances PLD1 activity in membrane assays. PLD1 activity is also stimulated by calmodulin and PKCα-enriched cytosol in reconstitution assays.\",\n      \"method\": \"Co-expression in Sf9 cells, immunoprecipitation, membrane reconstitution assay, PLD activity (transphosphatidylation), calcium ionophore treatment\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and enzymatic assays in a single study, but performed in insect (Sf9) cell overexpression system\",\n      \"pmids\": [\"10838164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PLD1 (but not PLD2) localizes to lipid droplets in oleic acid-treated NIH3T3 cells in an Arf1-dependent manner. Brefeldin A (an ARF-GEF inhibitor) suppresses both PLD activation and lipid droplet formation. Arf1 stimulates PLD1 activity in LD-enriched subcellular fractions.\",\n      \"method\": \"Immunocytochemistry, subcellular fractionation, Western blot, pharmacological inhibition (Brefeldin A), exogenous Arf1 stimulation, PLD activity assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional consequence (LD formation) and in vitro Arf1 stimulation; single lab, multiple methods\",\n      \"pmids\": [\"16054594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PLD1 acts downstream of Src to activate PKCγ in VEGF signaling in retinal microvascular endothelial cells; the Src→PLD1→PKCγ cascade mediates VEGF-induced endothelial cell migration, proliferation, and tube formation, and is required for hypoxia-induced retinal neovascularization in vivo.\",\n      \"method\": \"Pharmacological inhibition, dominant-negative mutants, siRNA knockdown, in vivo retinal neovascularization model (oxygen-induced retinopathy)\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis established by multiple inhibitory approaches (pharmacological + dominant-negative + siRNA) in vitro and in vivo; single lab\",\n      \"pmids\": [\"20421451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"cPLA2 acts as an effector downstream of the Src→PLD1→PKCγ signaling axis; VEGF-induced cPLA2 phosphorylation and arachidonic acid release require Src, PLD1, and PKCγ activity, and exogenous AA rescues endothelial responses when cPLA2 is depleted.\",\n      \"method\": \"siRNA knockdown, pharmacological inhibition, AA rescue experiments, in vivo oxygen-induced retinopathy model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway position of PLD1 upstream of cPLA2 established by epistasis with rescue; single lab\",\n      \"pmids\": [\"21536681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PLD1 enzymatic activity mediates chemokine-induced (IL-8, FMLP) chemotaxis of HL-60 leukocytes; a lipase-inactive PLD1-K830R mutant abrogates all chemokine-induced potentiating actions, while chemokinesis does not require PLD1 enzymatic activity. Both PLD1 and PLD2 are required for cell motility and associate with cell polarity markers, F-actin, and adhesion structures.\",\n      \"method\": \"siRNA knockdown, overexpression of lipase-inactive mutant (K830R), in vitro PLD activity assay, chemokinesis/chemotaxis assays, immunofluorescence microscopy\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — catalytic dead mutant distinguishes enzymatic from non-enzymatic roles; single lab with multiple methods\",\n      \"pmids\": [\"16873675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PLD1 acts downstream of RhoA to suppress dendritic branching in hippocampal neurons. The branching restriction by constitutively active RhoA (V14-RhoA) is partially rescued by PLD1 knockdown, and the inhibitory effect of both V14-RhoA and PLD1 overexpression can be ameliorated by reducing PA levels.\",\n      \"method\": \"Gain-of-function and loss-of-function (overexpression, siRNA), constitutively active RhoA epistasis, PA level manipulation, cultured hippocampal neurons\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with RhoA and PA rescue establishes pathway; single lab\",\n      \"pmids\": [\"22674271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PLD1 directly interacts with μ2 (a subunit of AP2 adaptor complex) and this interaction requires PLD1 binding to its own product phosphatidic acid. PLD1–μ2 interaction facilitates membrane recruitment of AP2 and determines the kinetics of EGFR endocytosis.\",\n      \"method\": \"Co-immunoprecipitation, kinetic analysis of endocytosis, AP2 membrane recruitment assay, PA-binding requirement demonstrated by mutagenesis/biochemical assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction shown by Co-IP with mechanistic follow-up on PA dependency and endocytosis kinetics; single lab\",\n      \"pmids\": [\"19763255\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PLD1 residues 762–801 (within the D4 domain, residues 712–818) constitute the minimal binding interface for PED/PEA15, with a Kd ~0.7 μM. PED/PEA15 interaction with PLD1 D4 enhances PKCα activity and impairs insulin-stimulated PKCζ activation and glucose transport; disruption of this interaction restores normal signaling.\",\n      \"method\": \"D4 deletion mutants, ELISA, surface plasmon resonance (SPR), transfection of D4α fragments, PKCα/PKCζ activity assay\",\n      \"journal\": \"Molecular bioSystems\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding interface mapped by deletion mutagenesis with affinity measurement and functional rescue; single lab\",\n      \"pmids\": [\"20714510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Disruption of PED/PEA15–PLD1 interaction in vivo (by adenoviral D4 delivery) decreases PKCα activation, restores PKCζ activation and insulin-dependent glucose uptake in skeletal muscle of PED/PEA15-overexpressing transgenic mice and in high-fat-diet obese mice.\",\n      \"method\": \"Adenoviral gene transfer, co-immunoprecipitation, PKC activity assay, glucose uptake assay, transgenic and HFD mouse models\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo disruption of PLD1–PED/PEA15 interaction with mechanistic readouts; single lab\",\n      \"pmids\": [\"23585839\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PLD1 negatively regulates adipogenic differentiation by generating PA, which displaces DEPTOR from mTORC1, leading to mTOR-dependent phosphorylation of IRS-1 at Ser636/639 and suppression of insulin signaling required for adipogenesis.\",\n      \"method\": \"PLD1-specific inhibitor (VU0155069), siRNA knockdown, PA treatment, PLD1 overexpression, mTORC1 pull-down (DEPTOR displacement), phospho-IRS-1 Western blot, 3T3-L1 differentiation assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (inhibitor, siRNA, PA rescue, DEPTOR displacement assay) establishing mechanism; single lab\",\n      \"pmids\": [\"27872488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RalA acts downstream of autophagy to recruit PLD1 to lysosomes during nutrient depletion, where PLD1 converts PC to PA to promote localized PA production. This recruits perilipin 3 (PLIN3) to expanding lipid droplets, facilitating LD growth.\",\n      \"method\": \"RalA inhibition, PLD1 knockout/inhibition, live-cell imaging of lysosomal PLD1 recruitment, perilipin 3 localization assay, nutrient depletion model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PLD1 localization to lysosomes shown in a RalA-dependent manner with downstream perilipin 3 functional readout; single lab\",\n      \"pmids\": [\"34320341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLD1 inhibition suppresses COPII vesicle transport from ER to Golgi by preventing Sec13/31 recruitment from the cytosol to the ER membrane during COPII vesicle formation. PLD1 knockdown increases ER stress marker GRP78 and promotes apoptosis.\",\n      \"method\": \"PLD1 inhibitor, siRNA knockdown, cell-free COPII coat protein recruitment assay, Western blot for ER stress markers\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-free reconstitution assay for COPII recruitment with PLD1 inhibition; single lab, two methods\",\n      \"pmids\": [\"28648601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HS1BP3 negatively regulates autophagy by inhibiting PLD1 activity and reducing PLD1 localization to ATG16L1-positive autophagosome precursor membranes, thereby decreasing PA content on these membranes. HS1BP3 depletion increases total cellular PA from elevated PLD activity and PLD1 localization to ATG16L1 membranes.\",\n      \"method\": \"HS1BP3 siRNA depletion, PLD activity assay, PA measurement, PLD1 localization (immunofluorescence), autophagosome formation assay\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — PLD1 localization and activity measured after HS1BP3 depletion; replicated across human cells and zebrafish; single lab\",\n      \"pmids\": [\"28318354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PLD1 preferentially interacts with ARL11 and ARL14 (Arf GTPase family members); ARL11/14 activate PLD1 and may be recruited to membrane vesicles by PLD1. PLD1 and ARL11 collaborate to promote macrophage phagocytosis.\",\n      \"method\": \"Proximity labeling (miniTurboID) interactome, TMT-based quantitative MS, PLD1 activity assay, phagocytosis assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity interactome confirmed with PLD1 activity and phagocytosis functional assays; single lab, multiple methods\",\n      \"pmids\": [\"35844135\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In cortical neurons, BDNF induces rapid RSK2-dependent PLD1 activation, and PLD1 is required for BDNF-stimulated ERK1/2-CREB and mTOR-S6K signaling. PLD1, ERK1/2, and RSK2 form a complex with scaffolding protein PEA15 after BDNF treatment and partially colocalize on endosomal structures.\",\n      \"method\": \"Pld1 and Rsk2 knockout neurons, PLD activity assay, immunofluorescence colocalization, PEA15 siRNA silencing, phospho-CREB nuclear accumulation assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout, complex formation, and signaling pathway data in cortical neurons; single lab\",\n      \"pmids\": [\"26437780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In acrosomal exocytosis, diacylglycerol activates PLD1 through PKC, and PLD1-generated PA promotes PIP2 synthesis in a positive feedback loop. Both PKC and PLD1 are required to maintain IP3-sensitive calcium channel opening required for exocytosis. Rescue experiments with PA, PIP2, and adenophostin confirm PLD1's role in maintaining PIP2 levels upstream of calcium channel gating.\",\n      \"method\": \"Permeabilized sperm exocytosis assay, PLD1 inhibition, PKC inhibition, PA/PIP2 rescue, Rab3A GTP-loading assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway placed by pharmacological epistasis and lipid rescue experiments; single lab\",\n      \"pmids\": [\"22609963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLD1 physically associates with PKD1 (protein kinase D1), and PLD1 acts upstream of PKD1 to positively regulate dendritic spine morphogenesis in hippocampal neurons. PLD1 inhibitor reduces PKD1 activation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown with rescue, PLD1 inhibitor treatment, dendritic spine morphology analysis\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP interaction with epistasis by knockdown/rescue; single lab, single paper\",\n      \"pmids\": [\"31356881\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The H452Y polymorphic form of the 5-HT2A receptor selectively reduces PLD1 binding to the receptor's carboxy-terminal tail and attenuates PLD signaling (but not Gq/11-PLC signaling). Co-immunoprecipitation and GST-fusion protein experiments show PLD1 docks to the 5-HT2AR C-terminal tail, and a blocking peptide spanning residue 452 reduces PLD1-dependent responses.\",\n      \"method\": \"Co-immunoprecipitation, GST-fusion pulldown, blocking peptides, PLD activity assay, cell proliferation assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and pulldown showing interaction; single lab, single study\",\n      \"pmids\": [\"23314176\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLD1 negatively regulates the cofilin-p53 pro-apoptotic pathway by promoting cofilin inactivation and inhibiting cofilin/p53 complex formation, thereby preventing p53 mitochondrial and nuclear translocation. Cofilin knockdown or PLD1 overexpression inhibits this apoptotic pathway.\",\n      \"method\": \"Cofilin knockdown, PLD1 overexpression, immunofluorescence for p53 subcellular localization, co-immunoprecipitation of cofilin-p53 complex, APP/PS1 transgenic mice\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — mechanistic pathway proposed with cofilin/p53 Co-IP and localization data; single lab\",\n      \"pmids\": [\"28912445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In hepatocellular carcinoma, cofilin 1 (CFL1) physically interacts with PLD1 and maintains PLD1 expression by inhibiting ubiquitin-mediated protein degradation, thereby activating AKT signaling. Hypoxia-induced CFL1 promotes tumor progression through the CFL1/PLD1/AKT axis.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, ubiquitination assay, AKT phosphorylation Western blot, xenograft model\",\n      \"journal\": \"Clinical and translational medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP for interaction, mechanistic chain not fully validated; single lab\",\n      \"pmids\": [\"33784016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PLD1 is required for spindle assembly, MTOC clustering, and cortical spindle migration in mouse oocyte meiosis. PLD1 interacts directly with spindle components, RAB11A+ vesicles, and autophagic vacuoles. PLD1 suppression decreases PIP2, phospho-cofilin (p-CFL1-Ser3), and ACTR2 on MTOC/spindle; exogenous PIP2 or CFL1-S3E (hyperphosphorylation mutant) partially rescues spindle defects. Autophagy activation phenocopies PLD1 loss, and autophagy inhibition rescues PLD1-depleted oocytes by restoring PIP2, ACTR2, and p-CFL1.\",\n      \"method\": \"Morpholino knockdown, PLD1 inhibitor, proximity ligation assay (direct spindle interaction), exogenous PIP2/CFL1-S3E/ACTR2 rescue, autophagy manipulation, immunofluorescence\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal rescue experiments and PLA for direct interactions; single lab\",\n      \"pmids\": [\"38513669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PLD1 is required for Frizzled7 (Fz7) receptor endocytosis in Xenopus embryos upon Wnt11 stimulation. PLD1 promotes Wnt/PCP signaling activation through its PX domain, which regulates GAP activity of dynamin to facilitate Fz7 endocytosis. Loss- and gain-of-function of PLD1 disrupts convergent extension movements in Xenopus gastrulation.\",\n      \"method\": \"Loss/gain-of-function in Xenopus embryos, biochemical analysis of Fz7 endocytosis, live imaging, dynamin GAP assay, PX domain mutants\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mechanism established in Xenopus (ortholog) with biochemical and functional data; single lab\",\n      \"pmids\": [\"26806705\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Nuclear PLD1 interacts with nucleophosmin 1 (NPM1) through a non-enzymatic mechanism, triggering NPM1 nuclear translocation. Nuclear NPM1 acts as a transcription factor to upregulate IL7R expression, which activates JAK1/STAT5/BCL-2 signaling to confer gemcitabine resistance in pancreatic cancer.\",\n      \"method\": \"CRISPRa/dCas9 genome-wide screen, co-immunoprecipitation (Co-IP), ChIP, ChIP-seq, transcriptome sequencing, PLD1 inhibitor (VU0155069)\",\n      \"journal\": \"Cancer biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP for PLD1-NPM1 interaction confirmed, ChIP-seq for NPM1-IL7R axis, genome-wide screen identification; single lab\",\n      \"pmids\": [\"37381714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"In porcine tracheal smooth muscle cells, ACh/PMA stimulation induces tyrosine phosphorylation of PLD1 (not PLD2) through a PKC- and tyrosine kinase-dependent pathway. Both ACh and PMA increase Ser/Thr and Tyr phosphorylation of PLD1, blocked by PKC inhibitor calphostin C or tyrosine kinase inhibitor genistein.\",\n      \"method\": \"Western blot with anti-phosphotyrosine antibodies, pharmacological inhibitors (genistein, calphostin C), PLD activity assay\",\n      \"journal\": \"Journal of biomedical science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — phosphorylation shown by anti-pTyr Western blot with pharmacological evidence; single lab, single method\",\n      \"pmids\": [\"15591778\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLD1 loss-of-function mutations cause congenital right-sided cardiac valve defects. Missense variants in PLD1 are overrepresented in regions critical for catalytic activity, and most mutant proteins show strongly reduced enzymatic activity. PLD1 inhibition decreases endothelial-mesenchymal transition (EndMT), an early step in valvulogenesis.\",\n      \"method\": \"Whole-exome sequencing, enzymatic activity assay of mutant PLD1 proteins, EndMT assay with PLD1 inhibitor, analysis of 30 patients from 21 families\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzymatic assay of multiple disease-associated mutants plus functional EndMT assay; multi-center study with large patient cohort\",\n      \"pmids\": [\"33645542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PLD1 promotes nasopharyngeal carcinoma progression via a positive feedback loop with NF-κB: PLD1 enhances NF-κB activity by facilitating phosphorylation and nuclear translocation of RELA, which in turn binds the PLD1 promoter and augments PLD1 expression.\",\n      \"method\": \"ChIP assay (RELA binding to PLD1 promoter), RELA knockdown/overexpression rescue, PLD1 inhibitor (VU0155069) in patient-derived xenograft, luciferase reporter\",\n      \"journal\": \"Journal of genetics and genomics = Yi chuan xue bao\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIP for RELA-PLD1 promoter interaction and functional rescue; single lab\",\n      \"pmids\": [\"38885836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"T. gondii GRA7-III interacts with the PX domain of PLD1, facilitating PLD1 enzymatic activity, phago-lysosomal maturation, and antimicrobial activity in a GRA7-III Ser135 phosphorylation-dependent manner via PKCα.\",\n      \"method\": \"Co-immunoprecipitation, PLD1 enzymatic activity assay, phago-lysosomal maturation assay, PKCα phosphorylation experiments\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and enzymatic activity data; interaction with a parasite protein; single lab\",\n      \"pmids\": [\"28125719\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLD1 is a phosphatidylcholine-hydrolyzing enzyme whose 1.8-Å crystal structure reveals a funnel-shaped active site with a phosphohistidine intermediate, a PIP2-binding polybasic membrane interface pocket essential for activity, and a RhoA-interaction surface; it is regulated by palmitoylation (Cys240/241), PKC-dependent and RSK2-dependent phosphorylation (Thr-147), Arf1/ARL11/ARL14 GTPases, and Ca²⁺/calmodulin, and acts as a signaling hub that generates phosphatidic acid to activate the mTOR→S6K1 axis (downstream of Cdc42), control lipid droplet biogenesis (via ERK2 and dynamin), regulate EGFR endocytosis (via PA-dependent μ2/AP2 recruitment), support exocytosis and neurite outgrowth (through RSK2-PLD1 phosphorylation and VAMP-7 vesicle fusion), restrict dendritic branching downstream of RhoA, facilitate Wnt/PCP signaling via Frizzled7 endocytosis, modulate autophagy through PA production at ATG16L1 precursor membranes, and couple to VEGF-Src-PKCγ-cPLA2 signaling in retinal angiogenesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLD1 is a phosphatidylcholine-hydrolyzing phospholipase whose generation of the signaling lipid phosphatidic acid (PA) at intracellular membranes serves as a regulatory hub for vesicle trafficking, organelle biogenesis, growth signaling, and cytoskeletal control [#0, #1]. Its catalytic domain folds into a funnel-shaped hydrophobic cavity feeding the active site, with a membrane-interface polybasic pocket that binds PIP2 (essential for activity), a C-terminal contribution to the catalytic pocket, a phosphohistidine intermediate, and a defined RhoA-binding surface [#0]. Enzyme activity and membrane targeting are tuned by multiple inputs: palmitoylation on Cys240/241 strengthens membrane association [#5], Ca2+/calmodulin and PKCalpha stimulate activity [#6], RSK2 directly phosphorylates Thr-147 in the N-terminal PX domain to activate the enzyme [#2], and Arf-family GTPases including Arf1, ARL11, and ARL14 promote activity and membrane recruitment [#7, #19]. Through PA production PLD1 acts upstream of the mTOR-S6K1 axis to regulate cell size and (via DEPTOR displacement and IRS-1 phosphorylation) insulin signaling and adipogenesis [#1, #15]. PLD1-derived PA drives lipid-droplet biogenesis through an ERK2-dynein pathway and, upon RalA-dependent lysosomal recruitment during nutrient depletion, recruits perilipin 3 to growing droplets [#4, #16], supports COPII-dependent ER-to-Golgi transport [#17], couples PA production to AP2/mu2 recruitment for EGFR endocytosis [#12], and provides PA at ATG16L1-positive precursor membranes during autophagy under HS1BP3 control [#18]. In neurons, RSK2-PLD1 signaling promotes neurite outgrowth via VAMP-7 vesicle fusion and BDNF-driven ERK/CREB and mTOR signaling, while PA acting downstream of RhoA restricts dendritic branching [#3, #20, #11]. Loss-of-function PLD1 mutations that reduce catalytic activity cause congenital right-sided cardiac valve defects, linked to impaired endothelial-mesenchymal transition during valvulogenesis [#30].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established the upstream regulatory inputs to PLD1 activity, defining how lipid modification and second messengers control the enzyme at membranes.\",\n      \"evidence\": \"Cysteine mutagenesis with palmitoylation labeling and membrane fractionation, plus Sf9 co-expression with PKCalpha and Ca2+/calmodulin reconstitution assays\",\n      \"pmids\": [\"11121416\", \"10838164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether palmitoylation and phosphorylation act on the same membrane pool was not resolved\", \"PKCalpha activation characterized in an overexpression insect-cell system\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Placed PLD1 upstream of mTOR-S6K1, showing PA as the link between Cdc42 and cell-size control.\",\n      \"evidence\": \"RNAi knockdown, dominant-negative catalytically inactive PLD1, S6K1 epistasis, and exogenous PA rescue of a PLD1-inactive Cdc42 mutant\",\n      \"pmids\": [\"14653992\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular target of PA within the mTOR complex not defined here\", \"Did not address tissue specificity of the Cdc42-PLD1-mTOR axis\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined a PLD1-specific (not PLD2) role in cytosolic lipid droplet formation and ordered the pathway PLD1 to ERK2 to dynein.\",\n      \"evidence\": \"siRNA, overexpression, anti-dynein microinjection, pharmacological ERK2 inhibition, and Arf1-dependent localization with Brefeldin A in NIH3T3 cells\",\n      \"pmids\": [\"16723731\", \"16054594\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PA links to dynein phosphorylation mechanistically not established\", \"Direct vs indirect Arf1 effect on droplet-localized PLD1 not separated\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified the direct activating phosphorylation of PLD1 at Thr-147 by RSK2, connecting calcium-stimulated kinase signaling to regulated exocytosis.\",\n      \"evidence\": \"Co-IP, phosphomimetic T147 mutant rescue of secretion in RSK2-depleted chromaffin cells, and PLD activity assays\",\n      \"pmids\": [\"18550821\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of Thr-147 phosphorylation effect on the PX domain not shown\", \"Other RSK2 substrates contributing to secretion not excluded\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed PLD1 uses its own PA product to recruit endocytic machinery, providing a feedforward mechanism for receptor internalization.\",\n      \"evidence\": \"Co-IP of PLD1 with AP2 subunit mu2, PA-binding requirement by mutagenesis, and EGFR endocytosis kinetics\",\n      \"pmids\": [\"19763255\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal structural mapping of the mu2 interface\", \"Generality across other cargo receptors not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended the RSK2-PLD1 axis to neuronal membrane expansion, linking it to VAMP-7-dependent vesicle fusion driving neurite outgrowth.\",\n      \"evidence\": \"Phosphomimetic PLD1 rescue in RSK2-silenced PC12 cells, TIRF imaging of VAMP-7 fusion, and Pld1 knockout mouse neurons\",\n      \"pmids\": [\"24336713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PA-VAMP7 fusion machinery link not mechanistically dissected\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected PLD1-derived PA to mTORC1 regulation via DEPTOR displacement, explaining suppression of insulin signaling and adipogenesis.\",\n      \"evidence\": \"PLD1 inhibitor, siRNA, PA treatment, mTORC1 pull-down showing DEPTOR displacement, and phospho-IRS-1 readout in 3T3-L1 cells\",\n      \"pmids\": [\"27872488\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PA binding site on mTORC1/DEPTOR not mapped\", \"Single cell-line context\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Broadened PLD1 trafficking roles to ER-to-Golgi COPII transport and autophagosome precursor membranes, with HS1BP3 identified as a negative regulator of PLD1 at ATG16L1 membranes.\",\n      \"evidence\": \"Cell-free COPII coat recruitment assay with PLD1 inhibition; HS1BP3 depletion with PLD activity, PA measurement, and PLD1 localization to ATG16L1 membranes (replicated in zebrafish)\",\n      \"pmids\": [\"28648601\", \"28318354\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How PA promotes Sec13/31 recruitment mechanistically unresolved\", \"Mechanism of HS1BP3 inhibition of PLD1 not defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Resolved the human PLD1 catalytic domain at high resolution, defining the active site, the essential PIP2-binding membrane interface, and the RhoA interaction surface.\",\n      \"evidence\": \"1.8-A X-ray crystallography with active-site, PIP2-pocket, and RhoA-interface mutagenesis and activation assays\",\n      \"pmids\": [\"32198492\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"N-terminal PX/PH regulatory regions not captured in the structure\", \"Membrane-bound conformational dynamics not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked PLD1 loss-of-function to a human Mendelian disease, establishing catalytic activity as essential for cardiac valve development.\",\n      \"evidence\": \"Whole-exome sequencing of 30 patients from 21 families, enzymatic assays of mutant proteins, and EndMT assay with PLD1 inhibition\",\n      \"pmids\": [\"33645542\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream PA-dependent effectors in valvulogenesis not identified\", \"Genotype-phenotype correlation across variants not fully resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined RalA as the upstream signal recruiting PLD1 to lysosomes during nutrient depletion to drive localized PA production and lipid-droplet growth.\",\n      \"evidence\": \"RalA inhibition, PLD1 knockout/inhibition, live-cell imaging of lysosomal recruitment, and perilipin 3 localization readout\",\n      \"pmids\": [\"34320341\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which PA recruits PLIN3 not established\", \"Single-lab system\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified ARL11/ARL14 as preferred PLD1 GTPase partners, expanding the regulatory GTPase set and linking PLD1 to macrophage phagocytosis.\",\n      \"evidence\": \"miniTurboID proximity interactome, TMT quantitative MS, PLD1 activity assay, and phagocytosis assay\",\n      \"pmids\": [\"35844135\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of ARL11/14 vs Arf1 selectivity not determined\", \"Whether PLD1 recruits ARL GTPases or vice versa not fully resolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a PLD1 requirement in oocyte meiotic spindle assembly and MTOC clustering, mechanistically tying PA/PIP2 to cofilin phosphorylation and autophagy balance.\",\n      \"evidence\": \"Morpholino knockdown, PLD1 inhibitor, proximity ligation for direct spindle interactions, and PIP2/CFL1-S3E/ACTR2 rescue with autophagy manipulation\",\n      \"pmids\": [\"38513669\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PA/PIP2 effector at the MTOC not identified\", \"Relationship between spindle and autophagic roles incompletely separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PLD1's distinct PA pools are spatially and temporally segregated to selectively engage trafficking, growth-signaling, and cytoskeletal effectors remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full-length regulated PLD1 with PX/PH domains\", \"Direct PA-effector binding interfaces (mTORC1, AP2, COPII, PLIN3) not mapped\", \"Mechanism distinguishing enzymatic from non-enzymatic scaffolding functions not defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 5, 6, 10, 30]},\n      {\"term_id\": \"GO:0140098\", \"supporting_discovery_ids\": [0, 1, 16]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [0, 12]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 15, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005811\", \"supporting_discovery_ids\": [4, 7, 16]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [16, 32]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 10]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [28]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 8, 11, 20]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [12, 17, 27]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [16, 18]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [4, 16]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [27, 30]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [10, 19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RhoA\", \"RSK2\", \"Arf1\", \"ARL11\", \"ARL14\", \"PEA15\", \"NPM1\", \"CFL1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}