{"gene":"PLD2","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1998,"finding":"PLD2 associates with the EGF receptor in a ligand-independent manner and undergoes tyrosine phosphorylation at Tyr-11 upon EGF receptor activation; mutation of Tyr-11 to phenylalanine enhanced basal PLD2 activity approximately 2-fold but did not alter EGF-mediated increase.","method":"Co-immunoprecipitation, site-directed mutagenesis, in vivo PLD activity assay in transiently transfected HEK293 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus mutagenesis identifying specific phosphorylation site, single lab, two orthogonal methods","pmids":["9837959"],"is_preprint":false},{"year":2000,"finding":"PLD2 interacts with Type Iα PIPkinase (PI4P 5-kinase α) and recruits it to its intracellular location; PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase, establishing that PtdIns(4,5)P2 generated locally by the PIPkinase is required for PLD2 activity.","method":"Co-immunoprecipitation, co-transfection, in vivo PLD activity assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional rescue experiments, single lab, two orthogonal methods","pmids":["11032811"],"is_preprint":false},{"year":2000,"finding":"PLD1 and PLD2 are both activated by calcium-mobilizing agonists and by co-expression with PKCα (but not PKCδ) in Sf9 cells; PLD1 and PLD2 physically associate with PKC isoforms by immunoprecipitation; PLD2 (but not PLD1) activity is also enhanced by co-expression with PKCδ in the presence of calcium ionophore.","method":"Sf9 cell expression system, in vivo PtdEth assay, immunoprecipitation, membrane reconstitution assay","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (in vivo assay + Co-IP + reconstitution), single lab","pmids":["10838164"],"is_preprint":false},{"year":2003,"finding":"S100B-RAGE interaction triggers activation of PLD2, leading to ROS production and augmentation of Ang II-induced JAK2 tyrosine phosphorylation and VSMC proliferation; PLD2 is positioned between RAGE signaling and ROS/JAK2 activation in this pathway.","method":"Pharmacological inhibition of PLD, siRNA/dominant-negative constructs, cell proliferation and signaling assays in VSMCs","journal":"Diabetes","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pathway placement via pharmacological inhibition and dominant-negative, single lab, multiple readouts","pmids":["12941779"],"is_preprint":false},{"year":2004,"finding":"Alpha-synuclein inhibits PLD2 in vitro; PLD2 inhibition requires a lipid-stabilized alpha-helical structure in exon 4 (residues 56-102) and C-terminal residues 130-140 (exon 6) of alpha-synuclein; phosphorylation at Ser129, Tyr125, or Tyr136 of alpha-synuclein abolishes PLD2 inhibition; A53T mutant is a more potent PLD2 inhibitor than WT.","method":"In vitro PLD2 activity assay with recombinant proteins, deletion and point mutants of alpha-synuclein, phosphomimetic mutations","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with systematic mutagenesis defining structural determinants, single lab, multiple orthogonal methods","pmids":["15033366"],"is_preprint":false},{"year":2004,"finding":"VEGFR-2 and PLD2 are co-localized in caveolae-enriched fractions of endothelial cells; VEGF stimulates PLD activity via VEGFR-2/PKC-δ; PLD-generated phosphatidic acid mediates VEGF-induced MEK/ERK phosphorylation and cellular proliferation; intact caveolae are required for this signaling cascade.","method":"Membrane fractionation, 1-butanol inhibition, exogenous PA rescue, pharmacological inhibitor panel, cholesterol depletion with MβCD","journal":"American journal of physiology. Heart and circulatory physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation localization plus functional pathway dissection, single lab, multiple orthogonal approaches","pmids":["14704231"],"is_preprint":false},{"year":2005,"finding":"PLD2 exists in a ternary complex with protein tyrosine phosphatase PTP1B and the adaptor protein Grb2; PTP1B treatment of PLD2 immunoprecipitates paradoxically increases both lipase activity and tyrosine phosphorylation; Grb2 addition to cell extracts elevates PLD2 tyrosine phosphorylation >10-fold.","method":"Co-immunoprecipitation, immunoblotting, in vitro phosphatase treatment, lipase activity assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus functional lipase assay, single lab, two orthogonal methods","pmids":["15896299"],"is_preprint":false},{"year":2006,"finding":"PLD2 activity is essential for chemotaxis of HL-60 cells toward FMLP and IL-8 (via CXCR-1), while ENA-78 (CXCR-2) selectively activates endogenous PLD2; a lipase-inactive PLD1-K830R mutant negates chemotactic responses; both PLD isoforms associate with cell polarity markers and F-actin polymerization in response to IL-8.","method":"siRNA knockdown, overexpression of WT and lipase-inactive mutants, in vitro PLD activity assay, cell migration assay (chemokinesis/chemotaxis), immunofluorescence microscopy","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function (siRNA) and gain-of-function plus lipase-dead mutant with defined migration phenotype, replicated across multiple chemokines","pmids":["16873675"],"is_preprint":false},{"year":2006,"finding":"PLD2 forms a functional complex with mTOR/raptor via a TOS-like motif (Phe-Glu-Val-Gln-Val, residues 265-269) in PLD2; siRNA-mediated knockdown of PLD2 (but not PLD1) profoundly reduces mitogen-induced phosphorylation of S6K1 and 4EBP1; PLD2-dependent mTOR activation requires both raptor binding and lipase activity, as raptor-binding-deficient or lipase-inactive PLD2 cannot restore mTOR activation.","method":"siRNA knockdown, co-immunoprecipitation, mutagenesis of TOS motif, in vivo S6K1/4EBP1 phosphorylation assay, rescue experiments","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, siRNA knockdown, domain mutagenesis, and rescue experiments all in one study with multiple orthogonal methods","pmids":["16837165"],"is_preprint":false},{"year":2006,"finding":"PLD2 contains two SH2-binding sites at Tyr-169 and Tyr-179 that mediate interaction with the SH2 domain of Grb2; Tyr-169 modulates enzymatic activity while Tyr-179 regulates total tyrosine phosphorylation; Grb2 binds PLD2 independently of lipase activity; PLD2-Y179F (but not WT) causes increased Ras activity, ERK phosphorylation and DNA synthesis, mediated through Sos recruitment.","method":"Deletion and point mutagenesis, GST pulldown, co-immunoprecipitation, in vitro PLD activity assay, ERK/Ras activation assays, DNA synthesis assay","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — systematic mutagenesis plus GST pulldown plus functional assays, single lab, multiple orthogonal methods","pmids":["16407827"],"is_preprint":false},{"year":2007,"finding":"Grb2 is essential for PLD2 activity in vivo; shRNA silencing of Grb2 reduces PLD2 activity and is rescued only by SH2-competent Grb2; Grb2 and PLD2 re-localize to perinuclear Golgi-like structures after EGF stimulation in a manner dependent on PLD2 residues Y169/Y179 and the Grb2 SH2 domain.","method":"shRNA-mediated Grb2 silencing, rescue with SH2-deficient mutant, co-immunoprecipitation, immunofluorescence microscopy, in vitro PLD activity assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function with rescue plus subcellular localization plus activity assay, single lab, three orthogonal methods","pmids":["17276458"],"is_preprint":false},{"year":2007,"finding":"PLD2-Y179F mutation (unavailability of Y179 for phosphorylation) leads to increased basal Akt phosphorylation at T308 and S473, enhanced DNA synthesis, ERK phosphorylation, and G0/G1 transition markers in a PI3K-dependent manner; active Akt in turn phosphorylates PLD2 at Thr-175; lipase-inactive double mutant Y179F-K758R abolishes the DNA synthesis effect, indicating PLD2 enzymatic activity is required.","method":"Site-directed mutagenesis, transfection in COS7 cells, Akt/ERK phosphorylation Western blot, PI3K inhibitor (LY294002), DNA synthesis assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus pharmacological inhibition plus functional readout, single lab, multiple methods","pmids":["18006275"],"is_preprint":false},{"year":2008,"finding":"Cyclin-dependent kinase 5 (Cdk5) phosphorylates PLD2 at Ser-134 in vitro and in cells; this phosphorylation is critical for EGF-dependent PLD2 activation and insulin secretion; PLD2-S134A mutant fails to show EGF-dependent phosphorylation, activation, or insulin secretion in pancreatic beta-cell lines.","method":"In vitro kinase assay, Cdk5 inhibitor (roscovitine), dominant-negative Cdk5, site-directed mutagenesis (S134A), co-immunoprecipitation, insulin secretion assay","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional cellular readout, single lab, multiple orthogonal methods","pmids":["18625302"],"is_preprint":false},{"year":2009,"finding":"Phosphorylated PLD2 (aided by Grb2) mediates lipase activity, whereas dephosphorylated PLD2 (dependent on phosphatase CD45) mediates induction of cell proliferation; Y179F and Y511F mutations both enhance DNA synthesis through a CD45-dependent mechanism; purified PLD2 is activated by Grb2 and deactivated by CD45 in vitro.","method":"In vitro activity assay with purified proteins, siRNA knockdown of CD45, phosphorylation-deficient mutants (Y179F, Y511F), proliferation markers (PCNA, p27KIP1, phospho-histone H3)","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro assay with purified proteins plus siRNA knockdown plus mutagenesis, single lab","pmids":["19715678"],"is_preprint":false},{"year":2011,"finding":"PLD2 directly binds the small GTPase Rac2 and functions as a guanine nucleotide exchange factor (GEF), switching Rac2 from GDP-bound to GTP-bound state; GEF activity is demonstrable in vitro with recombinant proteins without lipid substrates; a catalytically inactive lipase mutant (PLD2-K758R) retains GEF activity; PLD2 PH domain residues 263-266 (CRIB region) and PX domain mediate Rac2 binding; Rac2 Switch-1 residue N17 is required for PLD2 binding; PLD2-initiated Rac2 activation enhances cell adhesion, chemotaxis, and phagocytosis.","method":"In vitro GEF assay with recombinant proteins, GDP dissociation and GTP association kinetics, co-immunoprecipitation, mutagenesis, cell functional assays (adhesion, chemotaxis, phagocytosis), siRNA knockdown","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins plus mutagenesis plus cellular functional assays, multiple orthogonal methods, single lab but highly rigorous","pmids":["22106281"],"is_preprint":false},{"year":2011,"finding":"PLD2 contains two CRIB motifs (CRIB-1 and CRIB-2) in and around the PH domain that mediate specific binding to Rac2; binding is saturable with apparent Kd ~3 nM; PLD2 binds more efficiently to Rac2-GTP than Rac2-GDP; increasing Rac2-GTP concentrations inhibit PLD2 lipase activity, creating a negative feedback termination signal.","method":"Co-immunoprecipitation, FRET with CFP-Rac2/YFP-PLD2 in living cells, in vitro binding with affinity-purified recombinant proteins, deletion mutants (ΔCRIB-1/2), lipase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with Kd measurement plus FRET in living cells plus mutagenesis, multiple orthogonal methods","pmids":["21378159"],"is_preprint":false},{"year":2011,"finding":"PLD2 co-localizes with Grb2 at actin-rich membrane protrusions; PLD2-Y169 interacts with the SH2 domain of Grb2 (confirmed by immunoprecipitation); Grb2 binding enhances PLD2 activity; Rac2 is a third required component for full actin polymerization and membrane ruffle formation in vivo; PLD2 Y169F or Grb2 R86K mutations negate this effect.","method":"YFP/CFP fluorescent chimeras, co-immunoprecipitation, Western blot, in vitro PLD activity assay, cell ruffling assay with M-CSF stimulation","journal":"Cellular signalling","confidence":"High","confidence_rationale":"Tier 2 / Moderate — live-cell imaging, reciprocal Co-IP, mutagenesis, and functional assay, single lab, multiple orthogonal methods","pmids":["21419846"],"is_preprint":false},{"year":2011,"finding":"PLD2 interacts with and activates the oncogenic tyrosine kinase Fes/Fps; PLD2 overexpression elevates Fes activity in a manner dependent on phosphatidic acid and PIP2; Co-immunoprecipitation demonstrates PLD2-Fes physical interaction requiring Fes SH2 domain (R483K mutant negates interaction); this PLD2/Fes axis shortens time required for myeloid leukemic cell differentiation.","method":"Co-immunoprecipitation, Fes kinase activity assay, overexpression and siRNA knockdown, cell differentiation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mutant plus kinase activity assay plus functional differentiation assay, single lab","pmids":["22094461"],"is_preprint":false},{"year":2011,"finding":"JAK3 phosphorylates PLD2 at Tyr-415, enabling PLD2 activation; JAK3 knockdown abrogates PLD2 lipase activity and EGF-stimulated cancer cell invasion; JAK3 activation of PLD2 for invasion operates independently of the canonical STAT pathway.","method":"siRNA knockdown of JAK3, site-directed mutagenesis (Y415), in vitro PLD activity assay, Matrigel invasion assay","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis identifying phosphorylation site plus siRNA knockdown plus functional invasion assay, single lab","pmids":["21414324"],"is_preprint":false},{"year":2012,"finding":"The PX domain of PLD2 is sufficient for GEF activity toward Rac2; the GEF catalytic site is formed by hydrophobic residues Phe-107, Phe-129, Leu-166, and Leu-173 plus Arg-172 in the PX domain; mutations at these residues abolish GEF activity without affecting Rac2 binding; the PH domain (Ile-306 to Ala-310) provides an auxiliary docking site for Rac2 during catalysis; PX/PH mutants abolish chemotaxis and phagocytosis.","method":"GST fusion protein GEF assay, purified recombinant protein assay, site-directed mutagenesis, cell functional assays (chemotaxis, phagocytosis)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with multiple point mutants identifying catalytic residues plus cellular functional validation, rigorous domain mapping","pmids":["23035122"],"is_preprint":false},{"year":2012,"finding":"Serum-deprived MDA-MB-231 breast cancer cells upregulate an EGFR/JAK3/PLD2-PA system; both EGFR and JAK3 directly regulate PLD2 activity to mediate cell invasion; combined inhibition of JAK3 and PLD2 is especially effective in serum-deprived cells.","method":"Flavonoid kinase inhibitor (apigenin), RNA silencing, in vitro PLD activity assay, Matrigel invasion assay","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — pharmacological inhibition plus siRNA with functional invasion readout, single lab","pmids":["23238254"],"is_preprint":false},{"year":2014,"finding":"ARF6 and its effector PLD2 regulate syntenin exosome biogenesis by controlling budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs); ARF6 also controls EGFR degradation through degradative MVBs but does not affect HIV-1 budding, excluding general ESCRT effects.","method":"Co-immunoprecipitation, siRNA knockdown, electron microscopy, nanoparticle tracking, functional exosome assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus EM plus functional readout with controls, replicated across multiple approaches","pmids":["24637612"],"is_preprint":false},{"year":2014,"finding":"PLD2 inhibitor FIPI acts at the catalytic site (Ser-757 in HKD2) with mixed-kinetics; PLD2-specific inhibitor NFOT acts at two sites: the catalytic site (Ser-757/Ser-648) and an allosteric PIP2-binding site (Arg-210/Arg-212) in a hydrophobic pocket (Phe-244/Leu-245/Leu-246) in the PH domain; NFOT prevents cancer cell invasion, and this is lost in cells overexpressing PLD2-F244A/L245A/L246A or PLD2-R210A/R212A or PLD2-S757/S648 mutants.","method":"Enzyme kinetics, site-directed mutagenesis, mixed-inhibition kinetics analysis, cancer cell invasion assay","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Moderate — enzyme kinetics with mutagenesis identifying catalytic and allosteric sites, plus functional validation, single lab, multiple orthogonal methods","pmids":["25532944"],"is_preprint":false},{"year":2016,"finding":"Elevated membrane tension acts through PLD2 and mTORC2 to limit actin nucleation and cell protrusion; in the absence of PLD2, neutrophils exhibit larger leading edges, higher membrane tension, and defective chemotaxis; this biochemical feedback loop (via PLD2 and mTORC2) is distinct from direct mechanical inhibition of actin assembly.","method":"PLD2 knockout (genetic), membrane tension measurement, actin dynamics assay, chemotaxis assay, mathematical modeling","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined mechanosensory phenotype plus physical measurements plus pathway placement via mTORC2, multiple orthogonal methods","pmids":["27280401"],"is_preprint":false},{"year":2016,"finding":"RalA activates PLD2 in endothelial cells; PLD2-generated phosphatidic acid (PA) facilitates caveolae-mediated endocytosis and trafficking; PA co-localizes with caveolin-1 upon albumin stimulation; dominant-negative PLD2 blocks PA accumulation at caveolae and inhibits caveolae fusion.","method":"siRNA knockdown of RalA and PLD2, co-immunoprecipitation, PA biosensor (GFP-PASS), TIRF microscopy of Cav-1-RFP, fluorescent BSA uptake assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — live-cell biosensor imaging plus siRNA plus dominant-negative plus TIRF microscopy, multiple orthogonal methods, clear mechanism","pmids":["27510034"],"is_preprint":false},{"year":2017,"finding":"PLD2-generated PA specifically and directly binds to the C-terminus of KIF5B (kinesin-1 heavy chain), identified by liposome pulldown screen; PA binding is required for vesicular association of KIF5B, surface localization of MT1-MMP, invadopodia formation, and cancer cell invasion; PLD2 knockout inhibits lung metastases in MMTV-Neu transgenic mice.","method":"Liposome pulldown screen, in vitro PA-KIF5B binding assay, PLD2 knockout mouse model, surface biotinylation, invasion assay, lung metastasis quantification","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro direct binding assay plus KO mouse model plus multiple cellular functional assays, multiple orthogonal methods","pmids":["29033361"],"is_preprint":false},{"year":2017,"finding":"PLD2 mediates phosphorylation of occludin and induces its proteasomal degradation via a c-Src kinase-dependent pathway; intestinal-epithelial-cell-specific Pld2 knockout mice are protected from DSS-induced colitis; DSS induces PLD2 expression which downregulates occludin in colon epithelial cells.","method":"Intestinal-specific Pld2 knockout mice, DSS colitis model, Western blot for occludin phosphorylation and degradation, proteasome inhibitor experiments, c-Src inhibitor","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific KO mouse model plus mechanistic pathway identification (c-Src/occludin/proteasome), multiple orthogonal methods","pmids":["28484281"],"is_preprint":false},{"year":2018,"finding":"PLD2 (but not PLD1)-null macrophages cannot fully phagocytose aggregated oxidized LDL; PLD2 couples Agg-oxLDL phagocytosis with WASP, Grb2, and actin; CD36 and PLD2 exhibit mutual dependence: without PLD2, CD36 cannot engage in Agg-oxLDL removal; without CD36, PLD2 cannot form protein complexes with WASP or actin.","method":"Bone marrow-derived macrophages from PLD-null mice, PLD2-selective inhibitor, co-immunoprecipitation, phagocytosis assay, CD36 blocking antibody","journal":"Journal of leukocyte biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO plus pharmacological inhibition plus Co-IP defining molecular complex, multiple orthogonal methods","pmids":["29656494"],"is_preprint":false},{"year":2019,"finding":"PLD2-generated PA specifically binds IQGAP1 scaffold protein; PA-IQGAP1 binding is required for plasma membrane recruitment of IQGAP1; similar to PLD2 inhibition, IQGAP1 knockdown blocks membrane ruffle formation and VSMC migration, which is rescued by WT IQGAP1 but not PA-binding-deficient IQGAP1 mutant; PLD2 deficiency reduces neointimal formation in carotid artery ligation model.","method":"In vitro PA-IQGAP1 binding assay, PLD2 knockout mice, carotid artery ligation model, siRNA knockdown, membrane fractionation, cell migration assay","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro direct binding assay plus KO mouse model plus rescue with PA-binding-deficient mutant, multiple orthogonal methods","pmids":["30811216"],"is_preprint":false},{"year":2019,"finding":"An acute decrease in plasma membrane tension activates PLD2 by causing nanodomain disintegration; PLD2 activation generates PA leading to PI(4,5)P2-enriched dorsal membrane ruffling and macropinocytosis; this pathway is prominent in myotubes and represents a PM tension homeostasis mechanism.","method":"Plasma membrane tension manipulation, PLD2 inhibitor, PA biosensor, F-actin/PI(4,5)P2 imaging, macropinocytosis assay, siRNA knockdown","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition plus biosensor imaging plus siRNA, single lab, multiple orthogonal methods","pmids":["31391241"],"is_preprint":false},{"year":2020,"finding":"PLD1 selectively couples to TLR4/MyD88 to regulate M1 macrophage polarization, while PLD2 selectively couples to IL-4 receptor/JAK3 to regulate M2 macrophage polarization; LPS enhances TLR4/MyD88 interaction with PLD1; IL-4 induces IL-4R/JAK3 association with PLD2; PLD2 ablation intensifies M1-predominant disease severity.","method":"Co-immunoprecipitation showing isoform-specific receptor associations, PLD1/PLD2 knockout mice, macrophage polarization assays, in vivo sepsis and injury models","journal":"Journal of cellular physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP showing isoform-specific complexes plus genetic KO with defined M1/M2 polarization phenotypes, multiple orthogonal methods","pmids":["33368247"],"is_preprint":false},{"year":2021,"finding":"PLD2 deletion in adipose tissue or pharmacological PLD2 inhibition augments adaptive thermogenesis via p62-mediated improvement in mitochondrial quality and quantity in adipocytes; adipocyte-specific Pld2 knockout mice are resistant to high-fat diet-induced obesity, glucose intolerance, and insulin resistance.","method":"Adipocyte-specific Pld2 knockout mice, high-fat diet model, PLD2-specific inhibitor, mitochondrial biogenesis assays, p62 pathway analysis","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — tissue-specific KO plus pharmacological inhibition plus mechanistic pathway (p62/mitochondria), multiple orthogonal methods","pmids":["34940790"],"is_preprint":false},{"year":2022,"finding":"PLD2-generated PA increases STAT3 phosphorylation; activated STAT3 mediates PA-induced degradation of endothelial tight junction proteins (claudin-5, occludin, ZO-1) in LPS-induced ALI; PLD2 knockout reduces PA production, STAT3 phosphorylation, and TJ protein degradation.","method":"PLD2 knockout mice, LPS-induced ALI model, HUVEC in vitro model, exogenous PA treatment, STAT3 inhibitor, Western blot, ELISA for PA","journal":"International immunopharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO plus exogenous PA rescue plus pharmacological pathway inhibition, single lab, multiple orthogonal methods","pmids":["36700766"],"is_preprint":false},{"year":2024,"finding":"HIF-1α activates PLD2 transcription through hypoxia response elements; PLD2 overexpression increases chromatin accessibility around stemness genes at AP-1 bound sites (detected by ATAC-seq), leading to upregulation of SOX2, SOX9, and NOTCH1 and promoting cancer stem cell formation and chemoresistance in ovarian cancer.","method":"ATAC-seq, ChIP/hypoxia response element reporter, CRISPR/siRNA, xenograft models, iPSC reprogramming assay, in vitro and in vivo pharmacological inhibition","journal":"Journal of experimental & clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ATAC-seq plus functional KO plus in vivo models, single lab, mechanistic pathway from HIF-1α to chromatin remodeling","pmids":["38403587"],"is_preprint":false},{"year":2025,"finding":"Oleate (OA) enhances PLD2 S-acylation at Cys-223 and Cys-224, disrupting its lipid raft localization and increasing its colocalization with PIP2-enriched microdomains; PLD2 acts as a GEF for Cdc42 (in addition to Rac2); OA-dependent S-acylation and lipid raft dynamics regulate PLD2's GEF activity toward Cdc42; mutation of S-acylation sites or disruption of lipid rafts abolishes PLD2-mediated Cdc42 activation and filopodia-like protrusion formation.","method":"Confocal microscopy, lipid raft isolation, S-acylation assay, Cdc42 GEF activity assay, site-directed mutagenesis of Cys223/Cys224, methyl-β-cyclodextrin lipid raft disruption, filopodia quantification","journal":"Journal of lipid research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical S-acylation assay plus functional GEF assay plus mutagenesis plus live-cell imaging, single lab, multiple orthogonal methods","pmids":["41223946"],"is_preprint":false},{"year":2025,"finding":"PLD2 is downstream of the PLCγ2-PKD2 pathway activated by constitutively active KIT mutant in GIST cells; PKD2 activates PLD2 (but not PI4KIIIβ) for Golgi/TGN retention of KIT mutant; PLD2 activity is required for association of γ-adaptin with GGA1 at the Golgi/TGN; knockdown of PLD2 releases KIT mutant from Golgi for lysosomal degradation.","method":"PLD inhibitor (CAY10594), siRNA knockdown of PLD2, immunofluorescence microscopy, co-immunoprecipitation of GGA1/γ-adaptin, Western blot for KIT localization and degradation","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown plus pharmacological inhibition plus Co-IP plus functional localization assay, single lab, preprint, multiple orthogonal methods","pmids":["bio_10.1101_2025.03.02.640696"],"is_preprint":true},{"year":2012,"finding":"PLD2's C-terminal domain (residues 578-933) interacts with the N-terminal domain of CKIIβ subunit in HCT116 cells; PLD2 overexpression relocates CKIIβ to the plasma membrane and promotes its ubiquitin-dependent proteasomal degradation; the C-terminal domain of PLD2 is sufficient for CKIIβ degradation and lipase activity is not required.","method":"Co-immunoprecipitation with domain deletion mutants, subcellular fractionation/immunofluorescence, proteasome inhibitor, ubiquitination assay, siRNA knockdown","journal":"BMB reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping plus proteasome inhibitor plus lipase-dead controls, single lab, multiple methods","pmids":["21944249"],"is_preprint":false}],"current_model":"PLD2 is a phosphatidylcholine-hydrolyzing enzyme that generates the second messenger phosphatidic acid (PA) at the plasma membrane and intracellular membranes; it is regulated by PIP2 (via direct binding to its PH domain and local PIPkinase recruitment), Grb2 (SH2 interaction at Tyr-169/Tyr-179 enhances activity and directs Golgi localization), tyrosine phosphorylation by EGFR (Tyr-11), JAK3 (Tyr-415), and serine phosphorylation by Cdk5 (Ser-134); it forms functional complexes with mTOR/raptor (via its TOS motif) to transduce mitogenic signals through S6K1/4EBP1, with ARF6 to control ILV budding and exosome biogenesis, and with RalA to drive caveolae-mediated endocytosis; PLD2-generated PA directly binds KIF5B (kinesin-1) and IQGAP1 to promote MT1-MMP surface trafficking/invasion and VSMC membrane ruffle formation/neointima, respectively; independently of its lipase activity, PLD2 functions as a guanine nucleotide exchange factor (GEF) for Rac2 (and Cdc42) through hydrophobic residues in its PX domain, promoting actin polymerization, membrane ruffling, chemotaxis, and phagocytosis; membrane tension mechanosensing acts through PLD2 and mTORC2 to limit actin nucleation during neutrophil migration; PLD2 isoform-specifically couples to IL-4R/JAK3 to drive M2 macrophage polarization and regulates adipocyte thermogenesis via p62-dependent mitochondrial quality control."},"narrative":{"mechanistic_narrative":"PLD2 is a phosphatidylcholine-hydrolyzing enzyme that generates the second messenger phosphatidic acid (PA) at the plasma membrane to drive cytoskeletal remodeling, directed cell migration, membrane trafficking, and mitogenic signaling [PMID:16873675, PMID:22106281, PMID:29033361]. Its lipase activity depends on local PtdIns(4,5)P2: PLD2 recruits Type Iα PIPkinase to its membrane location, and the resulting PIP2 is required for catalysis through a defined allosteric PIP2-binding pocket in the PH domain [PMID:11032811, PMID:25532944]. Lipase output is gated by a tyrosine-phosphorylation circuit in which the adaptor Grb2 binds PLD2 at Tyr-169/Tyr-179 to potentiate activity and to direct Grb2/PLD2 co-relocalization to perinuclear Golgi-like structures, while site-specific phosphorylation by Cdk5 (Ser-134) and JAK3 (Tyr-415) further controls activation [PMID:16407827, PMID:17276458, PMID:18625302, PMID:21414324]. PLD2-generated PA acts as a direct effector ligand: it binds the kinesin-1 heavy chain KIF5B to promote MT1-MMP surface delivery, invadopodia formation, and metastasis, and binds the scaffold IQGAP1 to drive membrane ruffling, vascular smooth muscle migration, and neointimal formation [PMID:29033361, PMID:30811216]. Independently of its lipase function, PLD2 is a guanine nucleotide exchange factor for Rac2 (and Cdc42), using a catalytic site built from hydrophobic PX-domain residues with the PH domain providing CRIB-mediated auxiliary docking, thereby driving actin polymerization, chemotaxis, and phagocytosis [PMID:22106281, PMID:21378159, PMID:23035122, PMID:41223946]. PLD2 also serves as a membrane-tension mechanosensor that, together with mTORC2, limits actin nucleation during neutrophil migration, and couples to ARF6 and RalA to control intraluminal-vesicle/exosome biogenesis and caveolae-mediated endocytosis [PMID:27280401, PMID:24637612, PMID:27510034]. Through a TOS-like motif PLD2 forms a raptor-containing mTOR complex required for mitogen-induced S6K1/4EBP1 phosphorylation, linking it to growth control [PMID:16837165]. In vivo, PLD2 governs epithelial and endothelial barrier integrity via degradation of tight-junction proteins, M2 macrophage polarization through IL-4R/JAK3, and adipocyte thermogenesis via p62-dependent mitochondrial quality control [PMID:28484281, PMID:33368247, PMID:34940790].","teleology":[{"year":1998,"claim":"Established that PLD2 is physically and functionally coupled to receptor tyrosine kinase signaling, defining a phosphoregulatory input to its activity.","evidence":"Co-IP and Tyr-11 mutagenesis with in vivo PLD activity assay in HEK293 cells","pmids":["9837959"],"confidence":"High","gaps":["Did not establish the downstream consequence of EGFR coupling for a cellular process","Kinase directly phosphorylating Tyr-11 not resolved here"]},{"year":2000,"claim":"Showed PLD2 catalysis requires locally generated PtdIns(4,5)P2, defining a lipid cofactor dependence linked to a recruited PIPkinase.","evidence":"Co-IP, co-transfection and in vivo PLD activity assay with Type Iα PIPkinase","pmids":["11032811"],"confidence":"High","gaps":["Structural basis of PIP2 recognition not defined at this stage","Whether PIPkinase recruitment is constitutive or stimulus-dependent unresolved"]},{"year":2003,"claim":"Placed PLD2 within RAGE-driven oxidative signaling in vascular smooth muscle, broadening its role beyond growth-factor inputs.","evidence":"Pharmacological PLD inhibition and dominant-negative constructs in VSMCs","pmids":["12941779"],"confidence":"Medium","gaps":["Pharmacological inhibition does not isolate PLD2 from PLD1","Direct molecular link from RAGE to PLD2 not mapped"]},{"year":2004,"claim":"Identified alpha-synuclein as a structural inhibitor of PLD2 and mapped the inhibitory determinants, providing a regulatory and disease-relevant antagonist.","evidence":"In vitro PLD2 activity assay with recombinant alpha-synuclein deletion/point/phosphomimetic mutants","pmids":["15033366"],"confidence":"High","gaps":["In vivo significance of alpha-synuclein inhibition not demonstrated","Whether endogenous alpha-synuclein controls PLD2 in neurons untested here"]},{"year":2006,"claim":"Defined the Grb2 interaction surface (Tyr-169/Tyr-179) and resolved how distinct tyrosines partition PLD2 between catalytic and mitogenic (Ras/Sos) outputs.","evidence":"Mutagenesis, GST pulldown, Co-IP and Ras/ERK/DNA-synthesis assays","pmids":["16407827","15896299"],"confidence":"High","gaps":["Kinase generating the relevant phosphotyrosines not fully defined","Stoichiometry within the PTP1B/Grb2/PLD2 ternary complex unresolved"]},{"year":2006,"claim":"Demonstrated PLD2 is a raptor-binding component required for mTOR-dependent S6K1/4EBP1 signaling, linking it to translational/growth control via both raptor binding and lipase activity.","evidence":"siRNA knockdown, TOS-motif mutagenesis, Co-IP and rescue with phosphorylation readouts","pmids":["16837165"],"confidence":"High","gaps":["Whether PA is the proximate mTOR-activating signal not directly shown","Isoform specificity established only against PLD1"]},{"year":2006,"claim":"Established PLD2 lipase activity as essential for chemokine-directed chemotaxis, tying PA generation to migratory cell behavior.","evidence":"siRNA, lipase-dead mutants, PLD activity and migration assays in HL-60 cells","pmids":["16873675"],"confidence":"High","gaps":["Direct PA effectors driving migration not identified at this stage","Relative contributions of PLD1 vs PLD2 across chemokines incompletely separated"]},{"year":2007,"claim":"Showed Grb2 is required for cellular PLD2 activity and for EGF-driven Golgi relocalization, integrating adaptor binding with subcellular targeting.","evidence":"shRNA silencing with SH2-mutant rescue, Co-IP and immunofluorescence","pmids":["17276458","18006275"],"confidence":"High","gaps":["Functional consequence of Golgi-localized PLD2 not defined","Feedback from Akt (Thr-175) on PLD2 output not mechanistically resolved"]},{"year":2008,"claim":"Identified Cdk5-mediated Ser-134 phosphorylation as a required activating event for EGF-dependent PLD2 activation and insulin secretion.","evidence":"In vitro kinase assay, S134A mutagenesis and insulin secretion assays in beta-cells","pmids":["18625302"],"confidence":"High","gaps":["How Ser-134 phosphorylation alters catalysis mechanistically unresolved","Interplay with tyrosine-phosphorylation circuit not tested"]},{"year":2009,"claim":"Resolved a phosphorylation switch in which Grb2-bound phospho-PLD2 favors lipase activity while CD45-dependent dephosphorylation drives proliferation.","evidence":"In vitro assays with purified proteins, CD45 siRNA and phospho-deficient mutants","pmids":["19715678"],"confidence":"Medium","gaps":["Mechanism by which dephosphorylated PLD2 drives proliferation not defined","Physiological CD45 relevance outside the assay system unclear"]},{"year":2011,"claim":"Established the lipase-independent GEF function of PLD2 toward Rac2, redefining PLD2 as a bifunctional enzyme driving actin-dependent processes.","evidence":"In vitro GEF assays with recombinant proteins, FRET, Kd measurement, mutagenesis and functional assays","pmids":["22106281","21378159","21419846"],"confidence":"High","gaps":["How lipase and GEF activities are coordinated in time/space partially open","Negative-feedback inhibition by Rac2-GTP characterized only in vitro"]},{"year":2011,"claim":"Extended PLD2 partner repertoire to oncogenic kinase Fes/Fps and JAK3, linking PLD2 to leukemic differentiation and cancer cell invasion via Tyr-415.","evidence":"Co-IP with domain mutants, kinase/PLD activity assays, siRNA and invasion assays","pmids":["22094461","21414324"],"confidence":"Medium","gaps":["Direct PA effectors in invasion not identified at this stage","STAT-independence of JAK3-PLD2 axis shown but mechanism incompletely defined"]},{"year":2012,"claim":"Mapped the PX-domain catalytic residues sufficient for GEF activity and a separable PH-domain docking site, mechanistically dissociating GEF from lipase function.","evidence":"GST-fusion GEF assays with point mutants and chemotaxis/phagocytosis assays","pmids":["23035122"],"confidence":"High","gaps":["Structural model of the GEF active site not solved","How PX-GEF and HKD-lipase domains influence each other unresolved"]},{"year":2012,"claim":"Showed PLD2 promotes proteasomal degradation of CKIIβ via its C-terminal domain independently of lipase activity, indicating a scaffolding/adaptor role.","evidence":"Co-IP domain mapping, ubiquitination and proteasome-inhibitor assays in HCT116","pmids":["21944249"],"confidence":"Medium","gaps":["E3 ligase mediating CKIIβ degradation not identified","Physiological significance of CKIIβ regulation untested in vivo"]},{"year":2014,"claim":"Connected PLD2 to ARF6- and RalA-controlled membrane trafficking, defining roles in exosome biogenesis and caveolae-mediated endocytosis.","evidence":"Co-IP, siRNA, EM, PA biosensor imaging and TIRF/uptake assays","pmids":["24637612","27510034"],"confidence":"High","gaps":["Direct PA effectors at MVB and caveolae steps not fully enumerated","How ARF6 versus RalA inputs are coordinated unresolved"]},{"year":2014,"claim":"Defined catalytic (Ser-757/Ser-648) and allosteric PIP2-binding (Arg-210/Arg-212) sites through inhibitor pharmacology, providing the structural logic of PLD2 regulation and druggability.","evidence":"Enzyme kinetics with FIPI/NFOT, site-directed mutagenesis and invasion assays","pmids":["25532944"],"confidence":"High","gaps":["Full atomic structure of PLD2 not determined","Allosteric coupling between PIP2 pocket and catalysis not structurally visualized"]},{"year":2016,"claim":"Established PLD2 as a membrane-tension mechanosensor acting through mTORC2 to limit actin nucleation, embedding it in a migratory feedback loop.","evidence":"Genetic KO neutrophils, membrane tension measurement, actin/chemotaxis assays and modeling","pmids":["27280401"],"confidence":"High","gaps":["How membrane tension is transduced into PLD2 activation not molecularly defined","Relationship to its GEF versus lipase activity in this context unclear"]},{"year":2017,"claim":"Identified PA-binding effectors KIF5B and the c-Src/occludin axis, mechanistically linking PLD2-generated PA to MT1-MMP trafficking, metastasis, and barrier control.","evidence":"Liposome pulldown, in vitro PA-binding, KO mouse models, biotinylation and colitis/DSS assays","pmids":["29033361","28484281"],"confidence":"High","gaps":["Selectivity of PA recognition among KIF5B and other effectors not fully mapped","Whether occludin phosphorylation is direct or PA-effector-mediated unresolved"]},{"year":2018,"claim":"Defined an isoform-specific PLD2 requirement coupling CD36-dependent oxLDL phagocytosis to WASP/Grb2/actin assembly in macrophages.","evidence":"PLD-null BMDMs, selective inhibitor, Co-IP and phagocytosis assays","pmids":["29656494"],"confidence":"High","gaps":["Whether PLD2 lipase or GEF activity drives this complex not separated","Direct PLD2-CD36 contact versus indirect coupling unresolved"]},{"year":2019,"claim":"Extended the PA-effector model to IQGAP1 in vascular remodeling and established a tension-driven PLD2 pathway for ruffling and macropinocytosis.","evidence":"In vitro PA-IQGAP1 binding, KO mice, carotid ligation, PA biosensor and macropinocytosis assays","pmids":["30811216","31391241"],"confidence":"High","gaps":["How tension-induced nanodomain disintegration activates PLD2 mechanistically open","PA-effector hierarchy at the ruffle not fully defined"]},{"year":2020,"claim":"Established isoform-segregated receptor coupling whereby PLD2 selectively partners IL-4R/JAK3 to drive M2 macrophage polarization.","evidence":"Reciprocal Co-IP, PLD1/PLD2 KO mice and in vivo inflammation models","pmids":["33368247"],"confidence":"High","gaps":["Downstream PA targets in M2 programming not identified","Whether PLD2 catalytic or GEF activity drives polarization untested"]},{"year":2021,"claim":"Defined a metabolic role for PLD2 in adipocyte thermogenesis through p62-dependent mitochondrial quality control, with whole-body metabolic consequences.","evidence":"Adipocyte-specific KO mice, HFD model, selective inhibitor and mitochondrial/p62 analyses","pmids":["34940790"],"confidence":"High","gaps":["Molecular link from PLD2 to p62 not defined","Whether PA directly modulates mitochondrial quality control unresolved"]},{"year":2024,"claim":"Placed PLD2 in a HIF-1α-driven transcriptional-epigenetic axis promoting cancer stemness and chemoresistance.","evidence":"ATAC-seq, HRE reporter, CRISPR/siRNA, xenografts and reprogramming assays in ovarian cancer","pmids":["38403587"],"confidence":"Medium","gaps":["How PLD2 activity alters AP-1-bound chromatin accessibility mechanistically unclear","Whether lipase product PA is required for the chromatin effect untested"]},{"year":2025,"claim":"Expanded PLD2 GEF function to Cdc42 and showed S-acylation at Cys-223/Cys-224 controls lipid-raft partitioning and GEF output, adding a lipid-modification layer of regulation.","evidence":"S-acylation and Cdc42 GEF assays, Cys mutagenesis, raft isolation and filopodia imaging","pmids":["41223946"],"confidence":"Medium","gaps":["Enzyme mediating PLD2 S-acylation not identified","How raft partitioning mechanistically toggles GEF specificity unresolved"]},{"year":2025,"claim":"Positioned PLD2 downstream of PLCγ2-PKD2 in oncogenic KIT signaling to retain mutant KIT at the Golgi/TGN via GGA1/γ-adaptin trafficking.","evidence":"PLD inhibitor, siRNA, Co-IP of GGA1/γ-adaptin and KIT localization assays in GIST cells (preprint)","pmids":["bio_10.1101_2025.03.02.640696"],"confidence":"Medium","gaps":["Preprint not peer-reviewed","Whether PA directly recruits the GGA1/γ-adaptin machinery not shown"]},{"year":null,"claim":"How PLD2's two enzymatic activities (PA-generating lipase and Rac2/Cdc42 GEF), its scaffolding functions, and its multiple post-translational and lipid modifications are coordinated in space and time to select among migration, trafficking, metabolic, and transcriptional outputs remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated structural model coupling lipase, GEF, and scaffolding domains","Determinants of context-specific output selection unknown","PA effector hierarchy across cell types incompletely mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[1,7,8,22,25]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[14,15,19,34]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[1,22,25,28]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[27,36]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[14,15,19]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,24,29,34]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[10,35]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[7,16,28]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,8,9,18,23]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[21,24,25,35]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[7,27,30]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[25,26,33]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[31]}],"complexes":["mTOR/raptor complex","PLD2/Grb2/PTP1B ternary complex"],"partners":["GRB2","RAC2","KIF5B","IQGAP1","ARF6","RALA","JAK3","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O14939","full_name":"Phospholipase D2","aliases":["Choline phosphatase 2","PLD1C","Phosphatidylcholine-hydrolyzing phospholipase D2"],"length_aa":933,"mass_kda":106.0,"function":"Function as phospholipase selective for phosphatidylcholine (PubMed:9582313). May have a role in signal-induced cytoskeletal regulation and/or endocytosis (By similarity)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/O14939/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PLD2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000129219","cell_line_id":"CID000170","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"membrane","grade":1},{"compartment":"vesicles","grade":1}],"interactors":[{"gene":"FKBP5","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000170","total_profiled":1310},"omim":[{"mim_id":"621519","title":"SCAFFOLDING CK1-ANCHORING PROTEIN B; SACK1B","url":"https://www.omim.org/entry/621519"},{"mim_id":"612334","title":"N-ACYL PHOSPHATIDYLETHANOLAMINE-HYDROLYZING PHOSPHOLIPASE D; NAPEPLD","url":"https://www.omim.org/entry/612334"},{"mim_id":"603122","title":"DEDICATOR OF CYTOKINESIS 2; DOCK2","url":"https://www.omim.org/entry/603122"},{"mim_id":"602384","title":"PHOSPHOLIPASE D2; PLD2","url":"https://www.omim.org/entry/602384"},{"mim_id":"602382","title":"PHOSPHOLIPASE D1, PHOSPHATIDYLCHOLINE-SPECIFIC; PLD1","url":"https://www.omim.org/entry/602382"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"esophagus","ntpm":62.1}],"url":"https://www.proteinatlas.org/search/PLD2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"O14939","domains":[{"cath_id":"3.30.1520.10","chopping":"38-76_86-117_162-198","consensus_level":"medium","plddt":87.7857,"start":38,"end":198},{"cath_id":"2.30.29.30","chopping":"203-218_231-320","consensus_level":"high","plddt":90.1025,"start":203,"end":320},{"cath_id":"3.30.870.10","chopping":"331-477_501-565","consensus_level":"medium","plddt":96.7864,"start":331,"end":565}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O14939","model_url":"https://alphafold.ebi.ac.uk/files/AF-O14939-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O14939-F1-predicted_aligned_error_v6.png","plddt_mean":88.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PLD2","jax_strain_url":"https://www.jax.org/strain/search?query=PLD2"},"sequence":{"accession":"O14939","fasta_url":"https://rest.uniprot.org/uniprotkb/O14939.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O14939/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O14939"}},"corpus_meta":[{"pmid":"24637612","id":"PMC_24637612","title":"Syntenin-ALIX exosome biogenesis and budding into multivesicular bodies are controlled by ARF6 and PLD2.","date":"2014","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/24637612","citation_count":456,"is_preprint":false},{"pmid":"27280401","id":"PMC_27280401","title":"Membrane Tension Acts Through PLD2 and mTORC2 to Limit Actin Network Assembly During Neutrophil Migration.","date":"2016","source":"PLoS biology","url":"https://pubmed.ncbi.nlm.nih.gov/27280401","citation_count":151,"is_preprint":false},{"pmid":"11032811","id":"PMC_11032811","title":"Interaction of the type Ialpha PIPkinase with phospholipase D: a role for the local generation of phosphatidylinositol 4, 5-bisphosphate in the regulation of PLD2 activity.","date":"2000","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11032811","citation_count":107,"is_preprint":false},{"pmid":"9837959","id":"PMC_9837959","title":"PLD2 complexes with the EGF receptor and undergoes tyrosine phosphorylation at a single site upon agonist stimulation.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9837959","citation_count":101,"is_preprint":false},{"pmid":"9761774","id":"PMC_9761774","title":"Characterization of human PLD2 and the analysis of PLD isoform splice variants.","date":"1998","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/9761774","citation_count":101,"is_preprint":false},{"pmid":"15033366","id":"PMC_15033366","title":"Structural determinants of PLD2 inhibition by alpha-synuclein.","date":"2004","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15033366","citation_count":83,"is_preprint":false},{"pmid":"16873675","id":"PMC_16873675","title":"Phagocyte cell migration is mediated by phospholipases PLD1 and PLD2.","date":"2006","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/16873675","citation_count":81,"is_preprint":false},{"pmid":"12941779","id":"PMC_12941779","title":"S100B-RAGE-mediated augmentation of angiotensin II-induced activation of JAK2 in vascular smooth muscle cells is dependent on PLD2.","date":"2003","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/12941779","citation_count":80,"is_preprint":false},{"pmid":"10838164","id":"PMC_10838164","title":"Regulation of human PLD1 and PLD2 by calcium and protein kinase C.","date":"2000","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/10838164","citation_count":60,"is_preprint":false},{"pmid":"14704231","id":"PMC_14704231","title":"Localization of VEGFR-2 and PLD2 in endothelial caveolae is involved in VEGF-induced phosphorylation of MEK and ERK.","date":"2004","source":"American journal of physiology. 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[et al.]","url":"https://pubmed.ncbi.nlm.nih.gov/38630134","citation_count":12,"is_preprint":false},{"pmid":"30811216","id":"PMC_30811216","title":"Phosphatidic acid generated by PLD2 promotes the plasma membrane recruitment of IQGAP1 and neointima formation.","date":"2019","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/30811216","citation_count":12,"is_preprint":false},{"pmid":"22750546","id":"PMC_22750546","title":"Biochemical and cellular implications of a dual lipase-GEF function of phospholipase D2 (PLD2).","date":"2012","source":"Journal of leukocyte biology","url":"https://pubmed.ncbi.nlm.nih.gov/22750546","citation_count":11,"is_preprint":false},{"pmid":"32205175","id":"PMC_32205175","title":"HIF1α/PLD2 axis linked to glycolysis induces T-cell immunity in oral lichen planus.","date":"2020","source":"Biochimica et biophysica acta. 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GTPases","url":"https://pubmed.ncbi.nlm.nih.gov/22858691","citation_count":6,"is_preprint":false},{"pmid":"38706911","id":"PMC_38706911","title":"Decreased Expression of PLD2 Promotes EMT in Colorectal Cancer Invasion and Metastasis.","date":"2024","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/38706911","citation_count":5,"is_preprint":false},{"pmid":"38563085","id":"PMC_38563085","title":"β-Hydroxy-β-methylbutyrate (HMB) leads to phospholipase D2 (PLD2) activation and alters circadian rhythms in myotubes.","date":"2024","source":"Food & function","url":"https://pubmed.ncbi.nlm.nih.gov/38563085","citation_count":5,"is_preprint":false},{"pmid":"22426721","id":"PMC_22426721","title":"Cloning of PLD2 from baculovirus for studies in inflammatory responses.","date":"2012","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/22426721","citation_count":5,"is_preprint":false},{"pmid":"38703555","id":"PMC_38703555","title":"PLD2 deficiency alleviates endothelial glycocalyx degradation in LPS-induced ARDS/ALI.","date":"2024","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/38703555","citation_count":4,"is_preprint":false},{"pmid":"39461986","id":"PMC_39461986","title":"The molecular mechanism of PLD2-mediated regulation of apoptosis and cell edema in pancreatic cells via the Nrf2/NF-κB pathway.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39461986","citation_count":4,"is_preprint":false},{"pmid":"23649737","id":"PMC_23649737","title":"Analysis of non-synonymous single-nucleotide polymorphisms and population variability of PLD2 gene associated with hypertension.","date":"2013","source":"International journal of bioinformatics research and applications","url":"https://pubmed.ncbi.nlm.nih.gov/23649737","citation_count":4,"is_preprint":false},{"pmid":"37455419","id":"PMC_37455419","title":"Importance of PLD2 in an IL-23 driven psoriasiform dermatitis model and potential link to human psoriasis.","date":"2023","source":"The Journal of dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/37455419","citation_count":3,"is_preprint":false},{"pmid":"28533957","id":"PMC_28533957","title":"PLD2 regulates microtubule stability and spindle migration in mouse oocytes during meiotic division.","date":"2017","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/28533957","citation_count":3,"is_preprint":false},{"pmid":"21944249","id":"PMC_21944249","title":"The C-terminal domain of PLD2 participates in degradation of protein kinase CKII β subunit in human colorectal carcinoma cells.","date":"2011","source":"BMB reports","url":"https://pubmed.ncbi.nlm.nih.gov/21944249","citation_count":3,"is_preprint":false},{"pmid":"41449865","id":"PMC_41449865","title":"Astragaloside IV Alleviates Colorectal Cancer Metastases by Regulating RALY/PLD2 Axis and Inhibiting Tumoral Exosome Biogenesis.","date":"2025","source":"Phytotherapy research : PTR","url":"https://pubmed.ncbi.nlm.nih.gov/41449865","citation_count":2,"is_preprint":false},{"pmid":"41177242","id":"PMC_41177242","title":"Jieyu Wuwei Sinisan formula alleviates depression-like behaviors in CUMS mice by regulating synaptic plasticity through PLD2 pathway.","date":"2025","source":"Journal of ethnopharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/41177242","citation_count":1,"is_preprint":false},{"pmid":"32524116","id":"PMC_32524116","title":"[Effect of mi-138 targeting PLD2 gene on proliferation and migration of oral cancer cells].","date":"2020","source":"Shanghai kou qiang yi xue = Shanghai journal of stomatology","url":"https://pubmed.ncbi.nlm.nih.gov/32524116","citation_count":1,"is_preprint":false},{"pmid":"41308722","id":"PMC_41308722","title":"Optimizing functional recovery after acute ischemic stroke through intensity and frequency of rehabilitation: The critical role of HIF-1α/PLD2/mTOR signaling mechanisms.","date":"2025","source":"Experimental neurology","url":"https://pubmed.ncbi.nlm.nih.gov/41308722","citation_count":1,"is_preprint":false},{"pmid":"41223946","id":"PMC_41223946","title":"Oleate activates PLD2 lipase and GEF activity by modulating membrane microdomain dynamics via S-acylation.","date":"2025","source":"Journal of lipid research","url":"https://pubmed.ncbi.nlm.nih.gov/41223946","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":"39692536","id":"PMC_39692536","title":"Differential Control of T-Cell Subsets by Recombinant Human PLD2 in a Mouse Model of Allergic Asthma.","date":"2024","source":"Immunological investigations","url":"https://pubmed.ncbi.nlm.nih.gov/39692536","citation_count":0,"is_preprint":false},{"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}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":39498,"output_tokens":10234,"usd":0.136002,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":20625,"output_tokens":7307,"usd":0.1429,"stage2_stop_reason":"end_turn"},"total_usd":0.278902,"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\": 1998,\n      \"finding\": \"PLD2 associates with the EGF receptor in a ligand-independent manner and undergoes tyrosine phosphorylation at Tyr-11 upon EGF receptor activation; mutation of Tyr-11 to phenylalanine enhanced basal PLD2 activity approximately 2-fold but did not alter EGF-mediated increase.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis, in vivo PLD activity assay in transiently transfected HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus mutagenesis identifying specific phosphorylation site, single lab, two orthogonal methods\",\n      \"pmids\": [\"9837959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PLD2 interacts with Type Iα PIPkinase (PI4P 5-kinase α) and recruits it to its intracellular location; PLD2 activity in vivo can be regulated solely by the expression of this lipid kinase, establishing that PtdIns(4,5)P2 generated locally by the PIPkinase is required for PLD2 activity.\",\n      \"method\": \"Co-immunoprecipitation, co-transfection, in vivo PLD activity assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional rescue experiments, single lab, two orthogonal methods\",\n      \"pmids\": [\"11032811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PLD1 and PLD2 are both activated by calcium-mobilizing agonists and by co-expression with PKCα (but not PKCδ) in Sf9 cells; PLD1 and PLD2 physically associate with PKC isoforms by immunoprecipitation; PLD2 (but not PLD1) activity is also enhanced by co-expression with PKCδ in the presence of calcium ionophore.\",\n      \"method\": \"Sf9 cell expression system, in vivo PtdEth assay, immunoprecipitation, membrane reconstitution assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (in vivo assay + Co-IP + reconstitution), single lab\",\n      \"pmids\": [\"10838164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"S100B-RAGE interaction triggers activation of PLD2, leading to ROS production and augmentation of Ang II-induced JAK2 tyrosine phosphorylation and VSMC proliferation; PLD2 is positioned between RAGE signaling and ROS/JAK2 activation in this pathway.\",\n      \"method\": \"Pharmacological inhibition of PLD, siRNA/dominant-negative constructs, cell proliferation and signaling assays in VSMCs\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pathway placement via pharmacological inhibition and dominant-negative, single lab, multiple readouts\",\n      \"pmids\": [\"12941779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Alpha-synuclein inhibits PLD2 in vitro; PLD2 inhibition requires a lipid-stabilized alpha-helical structure in exon 4 (residues 56-102) and C-terminal residues 130-140 (exon 6) of alpha-synuclein; phosphorylation at Ser129, Tyr125, or Tyr136 of alpha-synuclein abolishes PLD2 inhibition; A53T mutant is a more potent PLD2 inhibitor than WT.\",\n      \"method\": \"In vitro PLD2 activity assay with recombinant proteins, deletion and point mutants of alpha-synuclein, phosphomimetic mutations\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with systematic mutagenesis defining structural determinants, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"15033366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"VEGFR-2 and PLD2 are co-localized in caveolae-enriched fractions of endothelial cells; VEGF stimulates PLD activity via VEGFR-2/PKC-δ; PLD-generated phosphatidic acid mediates VEGF-induced MEK/ERK phosphorylation and cellular proliferation; intact caveolae are required for this signaling cascade.\",\n      \"method\": \"Membrane fractionation, 1-butanol inhibition, exogenous PA rescue, pharmacological inhibitor panel, cholesterol depletion with MβCD\",\n      \"journal\": \"American journal of physiology. Heart and circulatory physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation localization plus functional pathway dissection, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"14704231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PLD2 exists in a ternary complex with protein tyrosine phosphatase PTP1B and the adaptor protein Grb2; PTP1B treatment of PLD2 immunoprecipitates paradoxically increases both lipase activity and tyrosine phosphorylation; Grb2 addition to cell extracts elevates PLD2 tyrosine phosphorylation >10-fold.\",\n      \"method\": \"Co-immunoprecipitation, immunoblotting, in vitro phosphatase treatment, lipase activity assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus functional lipase assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"15896299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PLD2 activity is essential for chemotaxis of HL-60 cells toward FMLP and IL-8 (via CXCR-1), while ENA-78 (CXCR-2) selectively activates endogenous PLD2; a lipase-inactive PLD1-K830R mutant negates chemotactic responses; both PLD isoforms associate with cell polarity markers and F-actin polymerization in response to IL-8.\",\n      \"method\": \"siRNA knockdown, overexpression of WT and lipase-inactive mutants, in vitro PLD activity assay, cell migration assay (chemokinesis/chemotaxis), immunofluorescence microscopy\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function (siRNA) and gain-of-function plus lipase-dead mutant with defined migration phenotype, replicated across multiple chemokines\",\n      \"pmids\": [\"16873675\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PLD2 forms a functional complex with mTOR/raptor via a TOS-like motif (Phe-Glu-Val-Gln-Val, residues 265-269) in PLD2; siRNA-mediated knockdown of PLD2 (but not PLD1) profoundly reduces mitogen-induced phosphorylation of S6K1 and 4EBP1; PLD2-dependent mTOR activation requires both raptor binding and lipase activity, as raptor-binding-deficient or lipase-inactive PLD2 cannot restore mTOR activation.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, mutagenesis of TOS motif, in vivo S6K1/4EBP1 phosphorylation assay, rescue experiments\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, siRNA knockdown, domain mutagenesis, and rescue experiments all in one study with multiple orthogonal methods\",\n      \"pmids\": [\"16837165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PLD2 contains two SH2-binding sites at Tyr-169 and Tyr-179 that mediate interaction with the SH2 domain of Grb2; Tyr-169 modulates enzymatic activity while Tyr-179 regulates total tyrosine phosphorylation; Grb2 binds PLD2 independently of lipase activity; PLD2-Y179F (but not WT) causes increased Ras activity, ERK phosphorylation and DNA synthesis, mediated through Sos recruitment.\",\n      \"method\": \"Deletion and point mutagenesis, GST pulldown, co-immunoprecipitation, in vitro PLD activity assay, ERK/Ras activation assays, DNA synthesis assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — systematic mutagenesis plus GST pulldown plus functional assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"16407827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Grb2 is essential for PLD2 activity in vivo; shRNA silencing of Grb2 reduces PLD2 activity and is rescued only by SH2-competent Grb2; Grb2 and PLD2 re-localize to perinuclear Golgi-like structures after EGF stimulation in a manner dependent on PLD2 residues Y169/Y179 and the Grb2 SH2 domain.\",\n      \"method\": \"shRNA-mediated Grb2 silencing, rescue with SH2-deficient mutant, co-immunoprecipitation, immunofluorescence microscopy, in vitro PLD activity assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with rescue plus subcellular localization plus activity assay, single lab, three orthogonal methods\",\n      \"pmids\": [\"17276458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PLD2-Y179F mutation (unavailability of Y179 for phosphorylation) leads to increased basal Akt phosphorylation at T308 and S473, enhanced DNA synthesis, ERK phosphorylation, and G0/G1 transition markers in a PI3K-dependent manner; active Akt in turn phosphorylates PLD2 at Thr-175; lipase-inactive double mutant Y179F-K758R abolishes the DNA synthesis effect, indicating PLD2 enzymatic activity is required.\",\n      \"method\": \"Site-directed mutagenesis, transfection in COS7 cells, Akt/ERK phosphorylation Western blot, PI3K inhibitor (LY294002), DNA synthesis assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus pharmacological inhibition plus functional readout, single lab, multiple methods\",\n      \"pmids\": [\"18006275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Cyclin-dependent kinase 5 (Cdk5) phosphorylates PLD2 at Ser-134 in vitro and in cells; this phosphorylation is critical for EGF-dependent PLD2 activation and insulin secretion; PLD2-S134A mutant fails to show EGF-dependent phosphorylation, activation, or insulin secretion in pancreatic beta-cell lines.\",\n      \"method\": \"In vitro kinase assay, Cdk5 inhibitor (roscovitine), dominant-negative Cdk5, site-directed mutagenesis (S134A), co-immunoprecipitation, insulin secretion assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis plus functional cellular readout, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"18625302\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Phosphorylated PLD2 (aided by Grb2) mediates lipase activity, whereas dephosphorylated PLD2 (dependent on phosphatase CD45) mediates induction of cell proliferation; Y179F and Y511F mutations both enhance DNA synthesis through a CD45-dependent mechanism; purified PLD2 is activated by Grb2 and deactivated by CD45 in vitro.\",\n      \"method\": \"In vitro activity assay with purified proteins, siRNA knockdown of CD45, phosphorylation-deficient mutants (Y179F, Y511F), proliferation markers (PCNA, p27KIP1, phospho-histone H3)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro assay with purified proteins plus siRNA knockdown plus mutagenesis, single lab\",\n      \"pmids\": [\"19715678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PLD2 directly binds the small GTPase Rac2 and functions as a guanine nucleotide exchange factor (GEF), switching Rac2 from GDP-bound to GTP-bound state; GEF activity is demonstrable in vitro with recombinant proteins without lipid substrates; a catalytically inactive lipase mutant (PLD2-K758R) retains GEF activity; PLD2 PH domain residues 263-266 (CRIB region) and PX domain mediate Rac2 binding; Rac2 Switch-1 residue N17 is required for PLD2 binding; PLD2-initiated Rac2 activation enhances cell adhesion, chemotaxis, and phagocytosis.\",\n      \"method\": \"In vitro GEF assay with recombinant proteins, GDP dissociation and GTP association kinetics, co-immunoprecipitation, mutagenesis, cell functional assays (adhesion, chemotaxis, phagocytosis), siRNA knockdown\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins plus mutagenesis plus cellular functional assays, multiple orthogonal methods, single lab but highly rigorous\",\n      \"pmids\": [\"22106281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PLD2 contains two CRIB motifs (CRIB-1 and CRIB-2) in and around the PH domain that mediate specific binding to Rac2; binding is saturable with apparent Kd ~3 nM; PLD2 binds more efficiently to Rac2-GTP than Rac2-GDP; increasing Rac2-GTP concentrations inhibit PLD2 lipase activity, creating a negative feedback termination signal.\",\n      \"method\": \"Co-immunoprecipitation, FRET with CFP-Rac2/YFP-PLD2 in living cells, in vitro binding with affinity-purified recombinant proteins, deletion mutants (ΔCRIB-1/2), lipase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with Kd measurement plus FRET in living cells plus mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"21378159\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PLD2 co-localizes with Grb2 at actin-rich membrane protrusions; PLD2-Y169 interacts with the SH2 domain of Grb2 (confirmed by immunoprecipitation); Grb2 binding enhances PLD2 activity; Rac2 is a third required component for full actin polymerization and membrane ruffle formation in vivo; PLD2 Y169F or Grb2 R86K mutations negate this effect.\",\n      \"method\": \"YFP/CFP fluorescent chimeras, co-immunoprecipitation, Western blot, in vitro PLD activity assay, cell ruffling assay with M-CSF stimulation\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live-cell imaging, reciprocal Co-IP, mutagenesis, and functional assay, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21419846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PLD2 interacts with and activates the oncogenic tyrosine kinase Fes/Fps; PLD2 overexpression elevates Fes activity in a manner dependent on phosphatidic acid and PIP2; Co-immunoprecipitation demonstrates PLD2-Fes physical interaction requiring Fes SH2 domain (R483K mutant negates interaction); this PLD2/Fes axis shortens time required for myeloid leukemic cell differentiation.\",\n      \"method\": \"Co-immunoprecipitation, Fes kinase activity assay, overexpression and siRNA knockdown, cell differentiation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mutant plus kinase activity assay plus functional differentiation assay, single lab\",\n      \"pmids\": [\"22094461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"JAK3 phosphorylates PLD2 at Tyr-415, enabling PLD2 activation; JAK3 knockdown abrogates PLD2 lipase activity and EGF-stimulated cancer cell invasion; JAK3 activation of PLD2 for invasion operates independently of the canonical STAT pathway.\",\n      \"method\": \"siRNA knockdown of JAK3, site-directed mutagenesis (Y415), in vitro PLD activity assay, Matrigel invasion assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis identifying phosphorylation site plus siRNA knockdown plus functional invasion assay, single lab\",\n      \"pmids\": [\"21414324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The PX domain of PLD2 is sufficient for GEF activity toward Rac2; the GEF catalytic site is formed by hydrophobic residues Phe-107, Phe-129, Leu-166, and Leu-173 plus Arg-172 in the PX domain; mutations at these residues abolish GEF activity without affecting Rac2 binding; the PH domain (Ile-306 to Ala-310) provides an auxiliary docking site for Rac2 during catalysis; PX/PH mutants abolish chemotaxis and phagocytosis.\",\n      \"method\": \"GST fusion protein GEF assay, purified recombinant protein assay, site-directed mutagenesis, cell functional assays (chemotaxis, phagocytosis)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with multiple point mutants identifying catalytic residues plus cellular functional validation, rigorous domain mapping\",\n      \"pmids\": [\"23035122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Serum-deprived MDA-MB-231 breast cancer cells upregulate an EGFR/JAK3/PLD2-PA system; both EGFR and JAK3 directly regulate PLD2 activity to mediate cell invasion; combined inhibition of JAK3 and PLD2 is especially effective in serum-deprived cells.\",\n      \"method\": \"Flavonoid kinase inhibitor (apigenin), RNA silencing, in vitro PLD activity assay, Matrigel invasion assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — pharmacological inhibition plus siRNA with functional invasion readout, single lab\",\n      \"pmids\": [\"23238254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ARF6 and its effector PLD2 regulate syntenin exosome biogenesis by controlling budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs); ARF6 also controls EGFR degradation through degradative MVBs but does not affect HIV-1 budding, excluding general ESCRT effects.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, electron microscopy, nanoparticle tracking, functional exosome assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus EM plus functional readout with controls, replicated across multiple approaches\",\n      \"pmids\": [\"24637612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PLD2 inhibitor FIPI acts at the catalytic site (Ser-757 in HKD2) with mixed-kinetics; PLD2-specific inhibitor NFOT acts at two sites: the catalytic site (Ser-757/Ser-648) and an allosteric PIP2-binding site (Arg-210/Arg-212) in a hydrophobic pocket (Phe-244/Leu-245/Leu-246) in the PH domain; NFOT prevents cancer cell invasion, and this is lost in cells overexpressing PLD2-F244A/L245A/L246A or PLD2-R210A/R212A or PLD2-S757/S648 mutants.\",\n      \"method\": \"Enzyme kinetics, site-directed mutagenesis, mixed-inhibition kinetics analysis, cancer cell invasion assay\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — enzyme kinetics with mutagenesis identifying catalytic and allosteric sites, plus functional validation, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"25532944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Elevated membrane tension acts through PLD2 and mTORC2 to limit actin nucleation and cell protrusion; in the absence of PLD2, neutrophils exhibit larger leading edges, higher membrane tension, and defective chemotaxis; this biochemical feedback loop (via PLD2 and mTORC2) is distinct from direct mechanical inhibition of actin assembly.\",\n      \"method\": \"PLD2 knockout (genetic), membrane tension measurement, actin dynamics assay, chemotaxis assay, mathematical modeling\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined mechanosensory phenotype plus physical measurements plus pathway placement via mTORC2, multiple orthogonal methods\",\n      \"pmids\": [\"27280401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RalA activates PLD2 in endothelial cells; PLD2-generated phosphatidic acid (PA) facilitates caveolae-mediated endocytosis and trafficking; PA co-localizes with caveolin-1 upon albumin stimulation; dominant-negative PLD2 blocks PA accumulation at caveolae and inhibits caveolae fusion.\",\n      \"method\": \"siRNA knockdown of RalA and PLD2, co-immunoprecipitation, PA biosensor (GFP-PASS), TIRF microscopy of Cav-1-RFP, fluorescent BSA uptake assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live-cell biosensor imaging plus siRNA plus dominant-negative plus TIRF microscopy, multiple orthogonal methods, clear mechanism\",\n      \"pmids\": [\"27510034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLD2-generated PA specifically and directly binds to the C-terminus of KIF5B (kinesin-1 heavy chain), identified by liposome pulldown screen; PA binding is required for vesicular association of KIF5B, surface localization of MT1-MMP, invadopodia formation, and cancer cell invasion; PLD2 knockout inhibits lung metastases in MMTV-Neu transgenic mice.\",\n      \"method\": \"Liposome pulldown screen, in vitro PA-KIF5B binding assay, PLD2 knockout mouse model, surface biotinylation, invasion assay, lung metastasis quantification\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro direct binding assay plus KO mouse model plus multiple cellular functional assays, multiple orthogonal methods\",\n      \"pmids\": [\"29033361\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PLD2 mediates phosphorylation of occludin and induces its proteasomal degradation via a c-Src kinase-dependent pathway; intestinal-epithelial-cell-specific Pld2 knockout mice are protected from DSS-induced colitis; DSS induces PLD2 expression which downregulates occludin in colon epithelial cells.\",\n      \"method\": \"Intestinal-specific Pld2 knockout mice, DSS colitis model, Western blot for occludin phosphorylation and degradation, proteasome inhibitor experiments, c-Src inhibitor\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific KO mouse model plus mechanistic pathway identification (c-Src/occludin/proteasome), multiple orthogonal methods\",\n      \"pmids\": [\"28484281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PLD2 (but not PLD1)-null macrophages cannot fully phagocytose aggregated oxidized LDL; PLD2 couples Agg-oxLDL phagocytosis with WASP, Grb2, and actin; CD36 and PLD2 exhibit mutual dependence: without PLD2, CD36 cannot engage in Agg-oxLDL removal; without CD36, PLD2 cannot form protein complexes with WASP or actin.\",\n      \"method\": \"Bone marrow-derived macrophages from PLD-null mice, PLD2-selective inhibitor, co-immunoprecipitation, phagocytosis assay, CD36 blocking antibody\",\n      \"journal\": \"Journal of leukocyte biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO plus pharmacological inhibition plus Co-IP defining molecular complex, multiple orthogonal methods\",\n      \"pmids\": [\"29656494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLD2-generated PA specifically binds IQGAP1 scaffold protein; PA-IQGAP1 binding is required for plasma membrane recruitment of IQGAP1; similar to PLD2 inhibition, IQGAP1 knockdown blocks membrane ruffle formation and VSMC migration, which is rescued by WT IQGAP1 but not PA-binding-deficient IQGAP1 mutant; PLD2 deficiency reduces neointimal formation in carotid artery ligation model.\",\n      \"method\": \"In vitro PA-IQGAP1 binding assay, PLD2 knockout mice, carotid artery ligation model, siRNA knockdown, membrane fractionation, cell migration assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro direct binding assay plus KO mouse model plus rescue with PA-binding-deficient mutant, multiple orthogonal methods\",\n      \"pmids\": [\"30811216\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"An acute decrease in plasma membrane tension activates PLD2 by causing nanodomain disintegration; PLD2 activation generates PA leading to PI(4,5)P2-enriched dorsal membrane ruffling and macropinocytosis; this pathway is prominent in myotubes and represents a PM tension homeostasis mechanism.\",\n      \"method\": \"Plasma membrane tension manipulation, PLD2 inhibitor, PA biosensor, F-actin/PI(4,5)P2 imaging, macropinocytosis assay, siRNA knockdown\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition plus biosensor imaging plus siRNA, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"31391241\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PLD1 selectively couples to TLR4/MyD88 to regulate M1 macrophage polarization, while PLD2 selectively couples to IL-4 receptor/JAK3 to regulate M2 macrophage polarization; LPS enhances TLR4/MyD88 interaction with PLD1; IL-4 induces IL-4R/JAK3 association with PLD2; PLD2 ablation intensifies M1-predominant disease severity.\",\n      \"method\": \"Co-immunoprecipitation showing isoform-specific receptor associations, PLD1/PLD2 knockout mice, macrophage polarization assays, in vivo sepsis and injury models\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP showing isoform-specific complexes plus genetic KO with defined M1/M2 polarization phenotypes, multiple orthogonal methods\",\n      \"pmids\": [\"33368247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PLD2 deletion in adipose tissue or pharmacological PLD2 inhibition augments adaptive thermogenesis via p62-mediated improvement in mitochondrial quality and quantity in adipocytes; adipocyte-specific Pld2 knockout mice are resistant to high-fat diet-induced obesity, glucose intolerance, and insulin resistance.\",\n      \"method\": \"Adipocyte-specific Pld2 knockout mice, high-fat diet model, PLD2-specific inhibitor, mitochondrial biogenesis assays, p62 pathway analysis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — tissue-specific KO plus pharmacological inhibition plus mechanistic pathway (p62/mitochondria), multiple orthogonal methods\",\n      \"pmids\": [\"34940790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PLD2-generated PA increases STAT3 phosphorylation; activated STAT3 mediates PA-induced degradation of endothelial tight junction proteins (claudin-5, occludin, ZO-1) in LPS-induced ALI; PLD2 knockout reduces PA production, STAT3 phosphorylation, and TJ protein degradation.\",\n      \"method\": \"PLD2 knockout mice, LPS-induced ALI model, HUVEC in vitro model, exogenous PA treatment, STAT3 inhibitor, Western blot, ELISA for PA\",\n      \"journal\": \"International immunopharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO plus exogenous PA rescue plus pharmacological pathway inhibition, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"36700766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HIF-1α activates PLD2 transcription through hypoxia response elements; PLD2 overexpression increases chromatin accessibility around stemness genes at AP-1 bound sites (detected by ATAC-seq), leading to upregulation of SOX2, SOX9, and NOTCH1 and promoting cancer stem cell formation and chemoresistance in ovarian cancer.\",\n      \"method\": \"ATAC-seq, ChIP/hypoxia response element reporter, CRISPR/siRNA, xenograft models, iPSC reprogramming assay, in vitro and in vivo pharmacological inhibition\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ATAC-seq plus functional KO plus in vivo models, single lab, mechanistic pathway from HIF-1α to chromatin remodeling\",\n      \"pmids\": [\"38403587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Oleate (OA) enhances PLD2 S-acylation at Cys-223 and Cys-224, disrupting its lipid raft localization and increasing its colocalization with PIP2-enriched microdomains; PLD2 acts as a GEF for Cdc42 (in addition to Rac2); OA-dependent S-acylation and lipid raft dynamics regulate PLD2's GEF activity toward Cdc42; mutation of S-acylation sites or disruption of lipid rafts abolishes PLD2-mediated Cdc42 activation and filopodia-like protrusion formation.\",\n      \"method\": \"Confocal microscopy, lipid raft isolation, S-acylation assay, Cdc42 GEF activity assay, site-directed mutagenesis of Cys223/Cys224, methyl-β-cyclodextrin lipid raft disruption, filopodia quantification\",\n      \"journal\": \"Journal of lipid research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical S-acylation assay plus functional GEF assay plus mutagenesis plus live-cell imaging, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"41223946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PLD2 is downstream of the PLCγ2-PKD2 pathway activated by constitutively active KIT mutant in GIST cells; PKD2 activates PLD2 (but not PI4KIIIβ) for Golgi/TGN retention of KIT mutant; PLD2 activity is required for association of γ-adaptin with GGA1 at the Golgi/TGN; knockdown of PLD2 releases KIT mutant from Golgi for lysosomal degradation.\",\n      \"method\": \"PLD inhibitor (CAY10594), siRNA knockdown of PLD2, immunofluorescence microscopy, co-immunoprecipitation of GGA1/γ-adaptin, Western blot for KIT localization and degradation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown plus pharmacological inhibition plus Co-IP plus functional localization assay, single lab, preprint, multiple orthogonal methods\",\n      \"pmids\": [\"bio_10.1101_2025.03.02.640696\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"PLD2's C-terminal domain (residues 578-933) interacts with the N-terminal domain of CKIIβ subunit in HCT116 cells; PLD2 overexpression relocates CKIIβ to the plasma membrane and promotes its ubiquitin-dependent proteasomal degradation; the C-terminal domain of PLD2 is sufficient for CKIIβ degradation and lipase activity is not required.\",\n      \"method\": \"Co-immunoprecipitation with domain deletion mutants, subcellular fractionation/immunofluorescence, proteasome inhibitor, ubiquitination assay, siRNA knockdown\",\n      \"journal\": \"BMB reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping plus proteasome inhibitor plus lipase-dead controls, single lab, multiple methods\",\n      \"pmids\": [\"21944249\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PLD2 is a phosphatidylcholine-hydrolyzing enzyme that generates the second messenger phosphatidic acid (PA) at the plasma membrane and intracellular membranes; it is regulated by PIP2 (via direct binding to its PH domain and local PIPkinase recruitment), Grb2 (SH2 interaction at Tyr-169/Tyr-179 enhances activity and directs Golgi localization), tyrosine phosphorylation by EGFR (Tyr-11), JAK3 (Tyr-415), and serine phosphorylation by Cdk5 (Ser-134); it forms functional complexes with mTOR/raptor (via its TOS motif) to transduce mitogenic signals through S6K1/4EBP1, with ARF6 to control ILV budding and exosome biogenesis, and with RalA to drive caveolae-mediated endocytosis; PLD2-generated PA directly binds KIF5B (kinesin-1) and IQGAP1 to promote MT1-MMP surface trafficking/invasion and VSMC membrane ruffle formation/neointima, respectively; independently of its lipase activity, PLD2 functions as a guanine nucleotide exchange factor (GEF) for Rac2 (and Cdc42) through hydrophobic residues in its PX domain, promoting actin polymerization, membrane ruffling, chemotaxis, and phagocytosis; membrane tension mechanosensing acts through PLD2 and mTORC2 to limit actin nucleation during neutrophil migration; PLD2 isoform-specifically couples to IL-4R/JAK3 to drive M2 macrophage polarization and regulates adipocyte thermogenesis via p62-dependent mitochondrial quality control.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PLD2 is a phosphatidylcholine-hydrolyzing enzyme that generates the second messenger phosphatidic acid (PA) at the plasma membrane to drive cytoskeletal remodeling, directed cell migration, membrane trafficking, and mitogenic signaling [#7, #14, #25]. Its lipase activity depends on local PtdIns(4,5)P2: PLD2 recruits Type Iα PIPkinase to its membrane location, and the resulting PIP2 is required for catalysis through a defined allosteric PIP2-binding pocket in the PH domain [#1, #22]. Lipase output is gated by a tyrosine-phosphorylation circuit in which the adaptor Grb2 binds PLD2 at Tyr-169/Tyr-179 to potentiate activity and to direct Grb2/PLD2 co-relocalization to perinuclear Golgi-like structures, while site-specific phosphorylation by Cdk5 (Ser-134) and JAK3 (Tyr-415) further controls activation [#9, #10, #12, #18]. PLD2-generated PA acts as a direct effector ligand: it binds the kinesin-1 heavy chain KIF5B to promote MT1-MMP surface delivery, invadopodia formation, and metastasis, and binds the scaffold IQGAP1 to drive membrane ruffling, vascular smooth muscle migration, and neointimal formation [#25, #28]. Independently of its lipase function, PLD2 is a guanine nucleotide exchange factor for Rac2 (and Cdc42), using a catalytic site built from hydrophobic PX-domain residues with the PH domain providing CRIB-mediated auxiliary docking, thereby driving actin polymerization, chemotaxis, and phagocytosis [#14, #15, #19, #34]. PLD2 also serves as a membrane-tension mechanosensor that, together with mTORC2, limits actin nucleation during neutrophil migration, and couples to ARF6 and RalA to control intraluminal-vesicle/exosome biogenesis and caveolae-mediated endocytosis [#23, #21, #24]. Through a TOS-like motif PLD2 forms a raptor-containing mTOR complex required for mitogen-induced S6K1/4EBP1 phosphorylation, linking it to growth control [#8]. In vivo, PLD2 governs epithelial and endothelial barrier integrity via degradation of tight-junction proteins, M2 macrophage polarization through IL-4R/JAK3, and adipocyte thermogenesis via p62-dependent mitochondrial quality control [#26, #30, #31].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that PLD2 is physically and functionally coupled to receptor tyrosine kinase signaling, defining a phosphoregulatory input to its activity.\",\n      \"evidence\": \"Co-IP and Tyr-11 mutagenesis with in vivo PLD activity assay in HEK293 cells\",\n      \"pmids\": [\"9837959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish the downstream consequence of EGFR coupling for a cellular process\", \"Kinase directly phosphorylating Tyr-11 not resolved here\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed PLD2 catalysis requires locally generated PtdIns(4,5)P2, defining a lipid cofactor dependence linked to a recruited PIPkinase.\",\n      \"evidence\": \"Co-IP, co-transfection and in vivo PLD activity assay with Type Iα PIPkinase\",\n      \"pmids\": [\"11032811\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PIP2 recognition not defined at this stage\", \"Whether PIPkinase recruitment is constitutive or stimulus-dependent unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Placed PLD2 within RAGE-driven oxidative signaling in vascular smooth muscle, broadening its role beyond growth-factor inputs.\",\n      \"evidence\": \"Pharmacological PLD inhibition and dominant-negative constructs in VSMCs\",\n      \"pmids\": [\"12941779\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Pharmacological inhibition does not isolate PLD2 from PLD1\", \"Direct molecular link from RAGE to PLD2 not mapped\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified alpha-synuclein as a structural inhibitor of PLD2 and mapped the inhibitory determinants, providing a regulatory and disease-relevant antagonist.\",\n      \"evidence\": \"In vitro PLD2 activity assay with recombinant alpha-synuclein deletion/point/phosphomimetic mutants\",\n      \"pmids\": [\"15033366\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of alpha-synuclein inhibition not demonstrated\", \"Whether endogenous alpha-synuclein controls PLD2 in neurons untested here\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the Grb2 interaction surface (Tyr-169/Tyr-179) and resolved how distinct tyrosines partition PLD2 between catalytic and mitogenic (Ras/Sos) outputs.\",\n      \"evidence\": \"Mutagenesis, GST pulldown, Co-IP and Ras/ERK/DNA-synthesis assays\",\n      \"pmids\": [\"16407827\", \"15896299\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase generating the relevant phosphotyrosines not fully defined\", \"Stoichiometry within the PTP1B/Grb2/PLD2 ternary complex unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated PLD2 is a raptor-binding component required for mTOR-dependent S6K1/4EBP1 signaling, linking it to translational/growth control via both raptor binding and lipase activity.\",\n      \"evidence\": \"siRNA knockdown, TOS-motif mutagenesis, Co-IP and rescue with phosphorylation readouts\",\n      \"pmids\": [\"16837165\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PA is the proximate mTOR-activating signal not directly shown\", \"Isoform specificity established only against PLD1\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Established PLD2 lipase activity as essential for chemokine-directed chemotaxis, tying PA generation to migratory cell behavior.\",\n      \"evidence\": \"siRNA, lipase-dead mutants, PLD activity and migration assays in HL-60 cells\",\n      \"pmids\": [\"16873675\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PA effectors driving migration not identified at this stage\", \"Relative contributions of PLD1 vs PLD2 across chemokines incompletely separated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed Grb2 is required for cellular PLD2 activity and for EGF-driven Golgi relocalization, integrating adaptor binding with subcellular targeting.\",\n      \"evidence\": \"shRNA silencing with SH2-mutant rescue, Co-IP and immunofluorescence\",\n      \"pmids\": [\"17276458\", \"18006275\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Golgi-localized PLD2 not defined\", \"Feedback from Akt (Thr-175) on PLD2 output not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified Cdk5-mediated Ser-134 phosphorylation as a required activating event for EGF-dependent PLD2 activation and insulin secretion.\",\n      \"evidence\": \"In vitro kinase assay, S134A mutagenesis and insulin secretion assays in beta-cells\",\n      \"pmids\": [\"18625302\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Ser-134 phosphorylation alters catalysis mechanistically unresolved\", \"Interplay with tyrosine-phosphorylation circuit not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Resolved a phosphorylation switch in which Grb2-bound phospho-PLD2 favors lipase activity while CD45-dependent dephosphorylation drives proliferation.\",\n      \"evidence\": \"In vitro assays with purified proteins, CD45 siRNA and phospho-deficient mutants\",\n      \"pmids\": [\"19715678\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which dephosphorylated PLD2 drives proliferation not defined\", \"Physiological CD45 relevance outside the assay system unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established the lipase-independent GEF function of PLD2 toward Rac2, redefining PLD2 as a bifunctional enzyme driving actin-dependent processes.\",\n      \"evidence\": \"In vitro GEF assays with recombinant proteins, FRET, Kd measurement, mutagenesis and functional assays\",\n      \"pmids\": [\"22106281\", \"21378159\", \"21419846\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How lipase and GEF activities are coordinated in time/space partially open\", \"Negative-feedback inhibition by Rac2-GTP characterized only in vitro\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended PLD2 partner repertoire to oncogenic kinase Fes/Fps and JAK3, linking PLD2 to leukemic differentiation and cancer cell invasion via Tyr-415.\",\n      \"evidence\": \"Co-IP with domain mutants, kinase/PLD activity assays, siRNA and invasion assays\",\n      \"pmids\": [\"22094461\", \"21414324\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PA effectors in invasion not identified at this stage\", \"STAT-independence of JAK3-PLD2 axis shown but mechanism incompletely defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Mapped the PX-domain catalytic residues sufficient for GEF activity and a separable PH-domain docking site, mechanistically dissociating GEF from lipase function.\",\n      \"evidence\": \"GST-fusion GEF assays with point mutants and chemotaxis/phagocytosis assays\",\n      \"pmids\": [\"23035122\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural model of the GEF active site not solved\", \"How PX-GEF and HKD-lipase domains influence each other unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed PLD2 promotes proteasomal degradation of CKIIβ via its C-terminal domain independently of lipase activity, indicating a scaffolding/adaptor role.\",\n      \"evidence\": \"Co-IP domain mapping, ubiquitination and proteasome-inhibitor assays in HCT116\",\n      \"pmids\": [\"21944249\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase mediating CKIIβ degradation not identified\", \"Physiological significance of CKIIβ regulation untested in vivo\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected PLD2 to ARF6- and RalA-controlled membrane trafficking, defining roles in exosome biogenesis and caveolae-mediated endocytosis.\",\n      \"evidence\": \"Co-IP, siRNA, EM, PA biosensor imaging and TIRF/uptake assays\",\n      \"pmids\": [\"24637612\", \"27510034\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PA effectors at MVB and caveolae steps not fully enumerated\", \"How ARF6 versus RalA inputs are coordinated unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined catalytic (Ser-757/Ser-648) and allosteric PIP2-binding (Arg-210/Arg-212) sites through inhibitor pharmacology, providing the structural logic of PLD2 regulation and druggability.\",\n      \"evidence\": \"Enzyme kinetics with FIPI/NFOT, site-directed mutagenesis and invasion assays\",\n      \"pmids\": [\"25532944\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full atomic structure of PLD2 not determined\", \"Allosteric coupling between PIP2 pocket and catalysis not structurally visualized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Established PLD2 as a membrane-tension mechanosensor acting through mTORC2 to limit actin nucleation, embedding it in a migratory feedback loop.\",\n      \"evidence\": \"Genetic KO neutrophils, membrane tension measurement, actin/chemotaxis assays and modeling\",\n      \"pmids\": [\"27280401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How membrane tension is transduced into PLD2 activation not molecularly defined\", \"Relationship to its GEF versus lipase activity in this context unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified PA-binding effectors KIF5B and the c-Src/occludin axis, mechanistically linking PLD2-generated PA to MT1-MMP trafficking, metastasis, and barrier control.\",\n      \"evidence\": \"Liposome pulldown, in vitro PA-binding, KO mouse models, biotinylation and colitis/DSS assays\",\n      \"pmids\": [\"29033361\", \"28484281\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity of PA recognition among KIF5B and other effectors not fully mapped\", \"Whether occludin phosphorylation is direct or PA-effector-mediated unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined an isoform-specific PLD2 requirement coupling CD36-dependent oxLDL phagocytosis to WASP/Grb2/actin assembly in macrophages.\",\n      \"evidence\": \"PLD-null BMDMs, selective inhibitor, Co-IP and phagocytosis assays\",\n      \"pmids\": [\"29656494\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PLD2 lipase or GEF activity drives this complex not separated\", \"Direct PLD2-CD36 contact versus indirect coupling unresolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended the PA-effector model to IQGAP1 in vascular remodeling and established a tension-driven PLD2 pathway for ruffling and macropinocytosis.\",\n      \"evidence\": \"In vitro PA-IQGAP1 binding, KO mice, carotid ligation, PA biosensor and macropinocytosis assays\",\n      \"pmids\": [\"30811216\", \"31391241\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How tension-induced nanodomain disintegration activates PLD2 mechanistically open\", \"PA-effector hierarchy at the ruffle not fully defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established isoform-segregated receptor coupling whereby PLD2 selectively partners IL-4R/JAK3 to drive M2 macrophage polarization.\",\n      \"evidence\": \"Reciprocal Co-IP, PLD1/PLD2 KO mice and in vivo inflammation models\",\n      \"pmids\": [\"33368247\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream PA targets in M2 programming not identified\", \"Whether PLD2 catalytic or GEF activity drives polarization untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a metabolic role for PLD2 in adipocyte thermogenesis through p62-dependent mitochondrial quality control, with whole-body metabolic consequences.\",\n      \"evidence\": \"Adipocyte-specific KO mice, HFD model, selective inhibitor and mitochondrial/p62 analyses\",\n      \"pmids\": [\"34940790\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link from PLD2 to p62 not defined\", \"Whether PA directly modulates mitochondrial quality control unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed PLD2 in a HIF-1α-driven transcriptional-epigenetic axis promoting cancer stemness and chemoresistance.\",\n      \"evidence\": \"ATAC-seq, HRE reporter, CRISPR/siRNA, xenografts and reprogramming assays in ovarian cancer\",\n      \"pmids\": [\"38403587\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How PLD2 activity alters AP-1-bound chromatin accessibility mechanistically unclear\", \"Whether lipase product PA is required for the chromatin effect untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Expanded PLD2 GEF function to Cdc42 and showed S-acylation at Cys-223/Cys-224 controls lipid-raft partitioning and GEF output, adding a lipid-modification layer of regulation.\",\n      \"evidence\": \"S-acylation and Cdc42 GEF assays, Cys mutagenesis, raft isolation and filopodia imaging\",\n      \"pmids\": [\"41223946\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Enzyme mediating PLD2 S-acylation not identified\", \"How raft partitioning mechanistically toggles GEF specificity unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Positioned PLD2 downstream of PLCγ2-PKD2 in oncogenic KIT signaling to retain mutant KIT at the Golgi/TGN via GGA1/γ-adaptin trafficking.\",\n      \"evidence\": \"PLD inhibitor, siRNA, Co-IP of GGA1/γ-adaptin and KIT localization assays in GIST cells (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.03.02.640696\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not peer-reviewed\", \"Whether PA directly recruits the GGA1/γ-adaptin machinery not shown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PLD2's two enzymatic activities (PA-generating lipase and Rac2/Cdc42 GEF), its scaffolding functions, and its multiple post-translational and lipid modifications are coordinated in space and time to select among migration, trafficking, metabolic, and transcriptional outputs remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated structural model coupling lipase, GEF, and scaffolding domains\", \"Determinants of context-specific output selection unknown\", \"PA effector hierarchy across cell types incompletely mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [1, 7, 8, 22, 25]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [14, 15, 19, 34]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [1, 22, 25, 28]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [27, 36]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [14, 15, 19]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 24, 29, 34]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [10, 35]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [7, 16, 28]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 8, 9, 18, 23]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [21, 24, 25, 35]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [7, 27, 30]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [25, 26, 33]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [31]}\n    ],\n    \"complexes\": [\n      \"mTOR/raptor complex\",\n      \"PLD2/Grb2/PTP1B ternary complex\"\n    ],\n    \"partners\": [\n      \"GRB2\",\n      \"RAC2\",\n      \"KIF5B\",\n      \"IQGAP1\",\n      \"ARF6\",\n      \"RALA\",\n      \"JAK3\",\n      \"EGFR\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}