{"gene":"LCP1","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":1992,"finding":"L-plastin bundles F-actin in a strictly calcium-dependent manner: bundles form at pCa 7 but not at pCa 6, and approximately one molecule of L-plastin binds 8 actin monomers in the filament. Unlike fimbrin, it acts on beta-actin but not muscle alpha-actin or gizzard gamma-actin.","method":"In vitro F-actin bundling assay with purified human T-cell L-plastin under defined calcium conditions","journal":"Journal of biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro reconstitution with purified protein, quantitative binding stoichiometry, replicated across multiple calcium conditions","pmids":["1491005"],"is_preprint":false},{"year":1993,"finding":"Macrophage 70-kDa actin-bundling protein (p70) is identical to L-plastin; its cross-linking activity is optimal at free calcium below 0.15 µM and is progressively inhibited at higher calcium (half-maximal inhibition at 1.6 µM). The protein undergoes major conformational changes at 0.15–1.5 µM free calcium and does not sever actin filaments.","method":"Protein purification, falling-ball viscosimetry, fluorescence spectroscopy, actin co-sedimentation assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal in vitro assays with purified protein establishing calcium-dependent bundling kinetics","pmids":["8461306"],"is_preprint":false},{"year":1994,"finding":"Bromophenacyl bromide (BPB) alkylates L-plastin in intact neutrophils and inhibits Fc receptor-mediated (IgG-stimulated), IP3-independent intracellular Ca2+ increase; L-plastin associates with the Triton X-100-insoluble cytoskeleton in adherent PMNs, linking it to FcR signal transduction.","method":"Chemical labeling with BPB in intact cells, immunoprecipitation with anti-BPB monoclonal antibody, cytoskeletal fractionation, Ca2+ flux measurements","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — chemical modification coupled with functional Ca2+ assays and fractionation, multiple orthogonal methods in one study","pmids":["8170942"],"is_preprint":false},{"year":1996,"finding":"FcγRII (not FcγRIIIB) is the primary receptor mediating L-plastin phosphorylation in neutrophils; adhesion to immune complexes is required but actin cytoskeleton rearrangement is not. L-plastin localizes to podosomes upon adhesion to immune complexes, colocalizing with actin, vinculin, and alpha-actinin.","method":"Receptor-specific monoclonal antibody blocking, patient cells with leukocyte adhesion deficiency (CD18-null), cytochalasin D treatment, immunofluorescence colocalization","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic (LAD patient cells) and pharmacologic dissection with orthogonal localization data in a single rigorous study","pmids":["8663066"],"is_preprint":false},{"year":1998,"finding":"L-plastin phosphorylation on Ser5 is sufficient to induce leukocyte integrin-mediated adhesion: cell-permeable Ser5-containing peptides activate integrin-mediated adhesion in PMNs and monocytes, while Ser5→Ala substitution abolishes activity. Peptide-induced adhesion is sensitive to PI3K and PKC inhibitors, but a phosphoserine-5 peptide is insensitive.","method":"Cell-permeable peptide introduction into primary human PMNs and monocytes, adhesion assays, site-directed mutagenesis (Ser5Ala), pharmacological inhibitors of PI3K and PKC","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — peptide functional assay with mutagenesis, primary human cells, pathway inhibitors, multiple orthogonal approaches","pmids":["9689080"],"is_preprint":false},{"year":1999,"finding":"PKA phosphorylates L-plastin on Ser5 in vitro and in cells downstream of PI3K and FcγR signaling: cAMP analogs and forskolin induce L-plastin phosphorylation; PKA inhibitors H89 and KT5720 block FcγR-induced L-plastin phosphorylation and integrin αMβ2 activation. FcγR stimulation transiently increases cAMP. PI3K acts upstream of PKA in this pathway.","method":"In vitro kinase assay (PKA + L-plastin peptides), cAMP analogs, adenylate cyclase activator, PKA inhibitors, PI3K inhibitors, cAMP measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus cell-based pharmacology with multiple inhibitors and pathway ordering","pmids":["10446213"],"is_preprint":false},{"year":2001,"finding":"Grancalcin (penta-EF-hand Ca2+-binding protein) interacts with L-plastin in a Ca2+-dependent manner (interaction occurs in the absence of Ca2+, i.e., is favored at low Ca2+), identified by affinity chromatography of solubilized neutrophils and confirmed by reciprocal co-immunoprecipitation.","method":"Affinity chromatography on immobilized grancalcin, reciprocal co-immunoprecipitation, multiple independent methods","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP confirmed by affinity chromatography, multiple independent methods in one study","pmids":["11279160"],"is_preprint":false},{"year":2001,"finding":"L-plastin N-terminal peptides and headpiece domain activate αvβ3-mediated adhesion in K562 cells; this activation requires actin filament disassembly (jasplakinolide blocks it) and synergizes with RGD ligand or Mn2+ to induce the high-affinity integrin conformation (ligand-induced binding site epitope), independently of β3 cytoplasmic tail tyrosines.","method":"Cell-permeable peptides in K562 cells, LIBS antibody binding assay for integrin conformation, jasplakinolide and cytochalasin D treatment, adhesion assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — functional assay with conformational readout, multiple pharmacological tools, single lab","pmids":["11278342"],"is_preprint":false},{"year":2004,"finding":"L-plastin binds actin laterally along the filament axis via two actin-binding domains (ABD) that have different affinities for actin; actin peptides 112-125 and 360-372 participate in the interface. Phosphoinositides inhibit actin binding. The two ABDs show distinct modes of actin interaction.","method":"Recombinant domain expression, chemical cross-linking, F-actin co-sedimentation, pyrenyl F-actin depolymerization assays, phosphoinositide competition","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with recombinant domains, multiple orthogonal binding assays, single lab","pmids":["14992580"],"is_preprint":false},{"year":2004,"finding":"Two fMLP receptor subtypes in neutrophils both phosphorylate L-plastin but use different signaling intermediates: low-affinity fMLP receptor phosphorylation requires PI3K, PLD, and PKC, whereas high-affinity fMLP receptor phosphorylation requires an Ro-31-8220-sensitive kinase but not PI3K, PLD, or classical PKC isoforms.","method":"2D-IEF/PAGE, MALDI-TOF MS, pharmacological inhibitors of PI3K/PLD/PKC, receptor subtype discrimination with antagonistic peptides","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry identification plus pharmacological dissection, single lab","pmids":["14556648"],"is_preprint":false},{"year":2005,"finding":"L-plastin distributes between nucleus and cytoplasm because it harbors a less conserved nuclear export sequence (NES) lacking F21 of T-plastin; insertion of Phe at the equivalent position enhances L-plastin nuclear export, explaining its cytoplasmic/nuclear distribution.","method":"Leptomycin B treatment, NES deletion/mutation analysis, nuclear export reporter assays, fluorescence microscopy","journal":"Traffic","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-directed mutagenesis of NES with functional nuclear export assay, single lab","pmids":["15752138"],"is_preprint":false},{"year":2006,"finding":"Phosphorylation of L-plastin on Ser5 increases its F-actin-binding activity in vitro, promotes targeting to actin-rich peripheral membrane protrusions and microspikes in cells, and is required for collagen invasion. Ser5Ala reduces association with F-actin; Ser5Glu (phosphomimetic) promotes F-actin microspike formation and remains detergent-resistant with F-actin.","method":"In vitro F-actin binding/co-sedimentation assay, transfection of phosphovariants (Ser5Ala, Ser5Glu) in Vero and HEK293T cells, detergent extraction, 3D collagen invasion assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro F-actin binding assay plus cell-based mutagenesis with invasion assay, multiple orthogonal methods","pmids":["16636079"],"is_preprint":false},{"year":2007,"finding":"Phosphorylation of L-plastin at Ser5 (identified by mass spectrometry) in T cells is required for surface transport of CD25 and CD69 to the T cell surface upon costimulation (TCR/CD3 + CD2 or CD28), but not for relocalization of L-plastin itself to the immunological synapse.","method":"Mass spectrometry phosphosite identification, site-directed mutagenesis (5A-LPL), retroviral transduction in primary human PBT, flow cytometry for CD25/CD69 surface expression","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS identification plus functional mutagenesis in primary human T cells, orthogonal methods","pmids":["17294403"],"is_preprint":false},{"year":2007,"finding":"Phosphorylation of L-plastin (on Ser5) in melanoma cells (but not expression alone) is required for enhanced Matrigel invasion; haptotactic migration (but not chemotactic migration or static adhesion) is enhanced by L-plastin expression regardless of phosphorylation status. In vivo metastatic capacity of B16 cells correlates with L-plastin expression and phosphorylation.","method":"siRNA knockdown, retroviral expression of wt-LPL-EGFP vs. nonphosphorylatable 5A7A-LPL-EGFP, Matrigel invasion/migration assays, in vivo mouse metastasis model","journal":"International journal of cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphomutant expression in L-plastin-null cells with in vitro and in vivo functional assays","pmids":["17290393"],"is_preprint":false},{"year":2009,"finding":"Alpaca single-domain antibody (nanobody) directed against L-plastin blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling, establishing a direct causal link between L-plastin bundling activity and filopodia formation.","method":"Recombinant nanobody expression in cells, F-actin bundling inhibition, filopodia formation assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — specific intracellular nanobody inhibition with phenotypic readout, single lab","pmids":["19726756"],"is_preprint":false},{"year":2009,"finding":"L-plastin confers resistance to TNF-α-induced cell death in MCF-7 breast cancer cells in a Ser5 phosphorylation-dependent manner; non-conventional PKC isoforms and the ceramide pathway regulate this phosphorylation state.","method":"siRNA knockdown, overexpression of wild-type and phosphomutant L-plastin, cell viability/apoptosis assays, PKC inhibitor studies","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional cell death assay, pharmacological pathway dissection, single lab","pmids":["19799649"],"is_preprint":false},{"year":2010,"finding":"L-plastin (LPL) is required for TCR-mediated T cell activation: LPL-/- T cells show defective lamellipodia formation, smaller immunological synapses, impaired cytokine production, and reduced proliferation. LPL-/- mice show delayed allograft rejection and reduced EAE severity.","method":"LPL-/- mouse model, T cell spreading assays on immobilized TCR ligands, immunological synapse imaging with APCs, cytokine ELISA, allograft rejection model, EAE model","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with multiple cellular and in vivo functional readouts, replicated across model systems","pmids":["21076065"],"is_preprint":false},{"year":2010,"finding":"Sustained LFA-1 (but not TCR/CD3) clustering in the peripheral SMAC of the immunological synapse requires L-plastin; calmodulin binding to L-plastin is required for maintenance of L-plastin in the IS, and calmodulin inhibition prevents stable LFA-1 and Talin redistribution.","method":"siRNA knockdown of L-plastin, IS immunofluorescence imaging (LFA-1, Talin, CD3 redistribution), calmodulin inhibitor treatment, T cell proliferation assay","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with specific receptor tracking at the IS and calmodulin inhibitor, two orthogonal approaches, single lab","pmids":["20683899"],"is_preprint":false},{"year":2010,"finding":"L-plastin is required for CCR7-mediated T cell motility and cellular polarization (CCR7 fails to polarize to leading edge in LPL-/- T cells) and for thymic egress, but is dispensable for CCR7 proximal signaling to F-actin polymerization and CCR7-mediated costimulation.","method":"LPL-/- mice, CCR7-stimulated T cell polarization assays, two-photon microscopy of lymph node motility, thymic egress quantification, F-actin polymerization assay","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with epistatic dissection of CCR7 signaling branches, in vivo and in vitro readouts","pmids":["20194718"],"is_preprint":false},{"year":2010,"finding":"In focal adhesions, L-plastin undergoes rapid association/dissociation cycles following a two-binding-state model; Ser5 phosphorylation increases association rates 2-fold without affecting dissociation rates, and L-plastin decreases actin dissociation rate 4-fold thereby increasing F-actin content. PKC-delta mediates PMA-induced L-plastin Ser5 phosphorylation. L-plastin associates with a complex containing cortactin.","method":"FRAP of GFP-coupled L-plastin phosphovariants in live cells, mathematical modeling, siRNA knockdown of PKC isoforms, co-immunoprecipitation with cortactin","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — FRAP with mathematical modeling quantifying kinetic parameters, siRNA epistasis, Co-IP, multiple orthogonal methods","pmids":["20169155"],"is_preprint":false},{"year":2010,"finding":"L-plastin interacts with the cytoplasmic domain of β1 and β2 integrins via its two actin-binding domains (not the N-terminal headpiece); this complex is regulated by µ-calpain, which cleaves β-integrin and disrupts the L-plastin/integrin interaction but does not directly dissociate it via calcium.","method":"Co-immunoprecipitation of endogenous proteins, solid phase assays, pull-down with recombinant L-plastin domains, cross-linking, ELISA, µ-calpain cleavage experiments","journal":"Cytoskeleton","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP confirmed by multiple in vitro binding assays, domain mapping, calpain regulation experiment, single lab","pmids":["20183869"],"is_preprint":false},{"year":2010,"finding":"In osteoclasts, L-plastin is present in actin aggregates during the early phase of sealing ring formation; cortactin is involved in the maturation phase. Temporal changes in L-plastin localization (from actin aggregates) and cortactin localization (in sealing ring) indicate sequential roles during bone resorption.","method":"siRNA to cortactin, immunofluorescence, quantification of L-plastin and cortactin localization during RANKL/M-CSF-induced bone resorption","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA functional knockdown with timed imaging of actin structures, single lab","pmids":["20650888"],"is_preprint":false},{"year":2011,"finding":"L-plastin is required for marginal zone B cell development and B cell motility toward CXCL12, CXCL13, and S1P; LPL-/- B cells fail to enter lymph nodes and bone marrow. LPL is required for integrin-mediated enhancement of Transwell migration but not for integrin-mediated adhesion per se. LPL-/- B cells show reduced Pyk-2 phosphorylation in response to chemokine.","method":"LPL-/- mouse model, Transwell migration assays, flow cytometry of splenic B cell subsets, integrin adhesion assays, Pyk-2 phosphorylation by immunoblot","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with multiple cellular and biochemical phenotype readouts, pathway dissection","pmids":["21832165"],"is_preprint":false},{"year":2011,"finding":"GM-CSF priming of eosinophils for chemotaxis and degranulation is mediated by PKCβII-phosphorylated L-plastin; co-immunoprecipitation reveals a complex of phospho-L-plastin with PKCβII, GM-CSF receptor α-chain, paxillin, and cofilin. A synthetic Ser5-phosphorylated L-plastin peptide (residues 2-19) recapitulates GM-CSF effects on integrin αMβ2 and chemotaxis independently of GM-CSF.","method":"Phosphoproteomics, co-immunoprecipitation, PKCβII siRNA and inhibitors, synthetic phosphopeptide stimulation, flow cytometry, chemotaxis and degranulation assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying complex plus siRNA epistasis plus synthetic peptide reconstitution, multiple orthogonal methods","pmids":["21525390"],"is_preprint":false},{"year":2011,"finding":"Dexamethasone inhibits costimulation-induced L-plastin Ser5 phosphorylation in primary human T cells, thereby reducing F-actin accumulation and immune synapse maturation; expression of a phosphomimetic L-plastin reverses the immunosuppressive effect of dexamethasone.","method":"Dexamethasone treatment, expression of nonphosphorylatable and phosphomimetic L-plastin mutants in primary human PBT, F-actin staining, IS imaging, flow cytometry","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — pharmacological and genetic rescue experiment in primary human T cells, direct mechanistic link between dexamethasone and L-plastin phosphorylation","pmids":["21805466"],"is_preprint":false},{"year":2012,"finding":"L-plastin is required for SDF-1α-stimulated T lymphocyte polarization, lamellipodia formation, and chemotaxis; knockdown impairs F-actin localization at the leading edge, Rac1 activation cycle, and Akt phosphorylation. PKCζ (not other PKC isoforms) regulates L-plastin Ser5 phosphorylation in response to SDF-1α, and L-plastin colocalizes with PKCζ at the leading edge.","method":"siRNA knockdown, immunofluorescence colocalization, PKC isoform-selective inhibitors, Rac1 GTPase pulldown, Akt immunoblot, chemotaxis and polarization assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with pathway dissection using isoform-selective inhibitors, multiple functional readouts, single lab","pmids":["22581862"],"is_preprint":false},{"year":2012,"finding":"Nanobodies targeting either the EF-hand or actin-binding domains of L-plastin trap it in an inactive conformation, impairing IS formation, MTOC docking, T cell proliferation, IL-2 secretion, and delaying Ser5 phosphorylation. One nanobody also delays LFA-1 phosphorylation and reduces L-plastin–LFA-1 association.","method":"Intracellular nanobody expression in Jurkat and PBMCs, IS imaging, MTOC docking assay, IL-2 ELISA, T cell proliferation, co-immunoprecipitation of L-plastin with LFA-1","journal":"Cellular and molecular life sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — specific domain-targeted perturbation with multiple functional readouts and Co-IP, single lab","pmids":["23001012"],"is_preprint":false},{"year":2013,"finding":"L-plastin in THP-1 macrophages is required for podosome formation and stability, and for matrix degradation; inhibitory nanobodies cause irregular, unstable actin turnover at podosomes without affecting Ser5 phosphorylation. Phosphorylated L-plastin is highly enriched in podosomes.","method":"Intracellular nanobody expression, live-cell imaging of podosome dynamics, matrix degradation assay, phospho-L-plastin immunostaining","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — specific inhibitory nanobodies with functional podosome and degradation readouts, single lab","pmids":["24236012"],"is_preprint":false},{"year":2014,"finding":"L-plastin expression and its phosphorylation are both required in vivo for tumor metastasis: L-plastin knockdown in PC3M prostate cancer cells reduces tumor growth and metastasis; ectopic expression of phosphorylatable but not non-phosphorylatable L-plastin in MV3 melanoma cells increases metastasis in mouse preclinical models.","method":"shRNA knockdown, ectopic expression of phosphorylatable vs. non-phosphorylatable L-plastin, subcutaneous and intracardiac injection mouse models, micro-CT/histomorphometry","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — phosphomutant rescue in two cell lines with in vivo metastasis readouts, genetic knockdown control","pmids":["24438191"],"is_preprint":false},{"year":2015,"finding":"S-glutathionylation of L-plastin at Cys206, Cys283, and Cys460 (Cys460 being most critical) in the actin-binding domain reduces its actin-binding and bundling activity, impairing neutrophil chemotaxis, polarization, bactericidal activity, and phagocytosis. S-thiolated L-plastin is detected in diabetic patient neutrophils.","method":"In vitro and cell-based oxidative stress, site-directed mutagenesis (Cys460), actin co-sedimentation assay, neutrophil functional assays, clinical samples","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — site-directed mutagenesis plus in vitro bundling assay plus functional cell assays in a single study","pmids":["25881549"],"is_preprint":false},{"year":2015,"finding":"RSK1 and RSK2, downstream of the ERK/MAPK pathway, directly phosphorylate L-plastin on Ser5 in vitro. RSK is an essential activator of L-plastin in breast cancer cells; RSK knockdown reduces migration and invasion and impairs Ser5-phospho-L-plastin recruitment to migratory structures.","method":"In vitro kinase assay (RSK1/2 + L-plastin), siRNA/shRNA knockdown, invasion and migration assays, immunofluorescence, computational modeling validated experimentally","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — direct in vitro kinase assay plus siRNA epistasis, single lab with multiple orthogonal methods","pmids":["26631483"],"is_preprint":false},{"year":2016,"finding":"L-plastin and fascin cooperate in invadopodia and filopodia with distinct roles: L-plastin bundles are thinner and less tightly packed than fascin bundles, providing flexibility for elongation while fascin provides rigidity. L-plastin is identified as a component of invadopodia contributing to matrix degradation and invasiveness; specific nanobodies inhibiting L-plastin bundling cannot be compensated by fascin.","method":"Domain-specific nanobody inhibition of bundling, invadopodia matrix degradation assay, electron microscopy of bundle ultrastructure, protrusion quantification","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — specific nanobody tools with ultrastructural and functional assays, mechanistic distinction between bundlers, single lab","pmids":["26945069"],"is_preprint":false},{"year":2016,"finding":"Mst1 kinase phosphorylates L-plastin on Thr89 in vitro and interacts with L-plastin in cells; T89A mutation impairs L-plastin localization to lamellipodia and fails to restore T cell migration and thymic egress in LPL-/- mice, establishing LPL as a downstream effector of Mst1.","method":"In vitro kinase assay (Mst1 + LPL), co-immunoprecipitation in cells, Thr89Ala mutagenesis, bone marrow chimeras, T cell migration/polarization assays, thymic egress quantification","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase assay plus Co-IP plus mutagenesis rescue in vivo, multiple orthogonal methods","pmids":["27465533"],"is_preprint":false},{"year":2016,"finding":"LPL is required for the transition from prealveolar macrophages to mature alveolar macrophages; LPL-/- precursors fail to upregulate PPAR-γ despite abundant GM-CSF signaling, suggesting LPL supports transmigration into and engraftment in alveoli where GM-CSF is produced.","method":"Conditional knockout (CD11c.Cre-LPLfl/fl), flow cytometry of alveolar macrophage subsets, GM-CSF signaling analysis, irradiation-regeneration assay, pneumococcal infection challenge","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockout with developmental stage analysis and mechanistic pathway placement, multiple functional readouts","pmids":["27758872"],"is_preprint":false},{"year":2016,"finding":"L-plastin promotes podosome longevity (but not initiation) in primary resident peritoneal macrophages; loss of LPL leads to decreased podosome lifetime, failure to elongate in response to chemotactic stimulation, and impaired macrophage transmigration and peritoneal migration.","method":"Live-cell imaging of LifeACT-RFP macrophages from LPL-/- mice, podosome longevity quantification, macrophage transmigration assay, in vivo peritoneal migration","journal":"Molecular immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — live imaging with genetic knockout, quantitative podosome dynamics, in vivo migration readout","pmids":["27614263"],"is_preprint":false},{"year":2017,"finding":"NMR structure of the L-plastin EF-hand headpiece reveals a novel switch helix immediately after the calcium-binding region that binds tightly to the EF-hand motifs in the presence of calcium. This switch helix plays a major role in actin-bundling regulation; a competitive peptide inhibiting EF-hand/switch helix association deregulates bundling activity.","method":"NMR spectroscopy (solution structure determination), competitive peptide inhibition of actin-bundling activity","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure determination with functional validation by peptide competition, single lab","pmids":["28145401"],"is_preprint":false},{"year":2018,"finding":"LRRK1 signaling in osteoclasts promotes L-plastin Ser5 phosphorylation; Lrrk1-knockout osteoclasts show reduced L-plastin Ser5 phosphorylation by LC/MS and Western blot. L-plastin KO mice show increased trabecular bone volume.","method":"Metal affinity purification LC/MS phosphoproteomics, Ser5-specific Western blot, micro-CT of L-plastin KO mice","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphoproteomics identification confirmed by phospho-specific Western blot in genetic KO, single lab","pmids":["30136304"],"is_preprint":false},{"year":2018,"finding":"TNF-α signaling phosphorylates L-plastin on Ser5 and Ser7 in osteoclasts, increasing actin bundling capacity and driving formation of nascent sealing zones (NSZs). TAT-fused full-length L-plastin peptide increases NSZ/sealing ring formation; N-terminal phospho-Ser peptides reduce cellular L-plastin phosphorylation and bone resorption. Neither podosome assembly nor osteoclast migration is affected.","method":"TAT-peptide transduction, immunoblotting with phospho-specific antibodies, actin staining, bone resorption (dentine pit) assay","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — peptide competition/inhibition with functional bone resorption readout, single lab","pmids":["30244178"],"is_preprint":false},{"year":2019,"finding":"L-plastin is reversibly regulated by ROS-induced thiol oxidation: Cys101 forms a disulfide bridge with Cys42, reducing actin-bundling capacity. Reduction is mediated by the Thioredoxin1 (TRX1) system (demonstrated by TRX1 trapping and TRXR1 blockade with auranofin). L-plastin oxidation occurs preferentially in actin-based cell extrusions and inhibits cell spreading, filopodial extension, migration, invasion, and ECM degradation.","method":"TRX1 trapping (substrate trap), TRX1 knockdown, TRXR1 inhibitor (auranofin), ratiometric imaging (LPL-roGFP-Orp1 fusion), dimedone-based proximity ligation assay, actin-bundling assay, functional migration/invasion/ECM assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — TRX1 trapping identifies specific redox substrate, ratiometric imaging localizes oxidation, mutagenesis implied by disulfide mapping, multiple orthogonal methods","pmids":["31501427"],"is_preprint":false},{"year":2019,"finding":"LCP1 knockdown in Eol-1 eosinophilic cells reduces phosphorylation of AKTSer473 (mTORC2-dependent) and STAT1Tyr701 but not FIP1L1-PDGFRA-induced STAT3/STAT5/ERK phosphorylation, establishing LCP1 as upstream of mTORC2/AKT in eosinophilic cells.","method":"shRNA knockdown of LCP1, PKCβ inhibitor enzastaurin, phospho-specific immunoblot for mTORC2/AKT pathway components, eosinophil differentiation and apoptosis assays","journal":"Molecular carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — shRNA knockdown with selective AKT phospho-site dissection (Ser473 vs. Thr308) establishing mTORC2 linkage, single lab","pmids":["31691359"],"is_preprint":false},{"year":2020,"finding":"LPL deletion in osteoclasts impedes preosteoclast fusion by inhibiting filopodia formation, increases preosteoclast numbers (which release PDGF-BB to promote vessel growth and bone formation), and LPL expression is regulated by the PI3K/AKT/SP1 axis in response to RANKL.","method":"LPL knockout mice, histomorphometry, flow cytometry, filopodia imaging, PDGF-BB secretion assay, PI3K/AKT inhibition/SP1 promoter studies","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with mechanistic pathway identification (PI3K/AKT/SP1 regulatory axis) and multiple in vivo and in vitro readouts","pmids":["33208358"],"is_preprint":false},{"year":2020,"finding":"miR-125a-5p directly targets and reduces L-plastin (LCP1) expression in megakaryocytes; L-plastin promotes megakaryocyte progenitor migration but negatively regulates proplatelet formation (PPF) and PP branching by inhibiting the late-stage megakaryocyte invagination system and podosomes.","method":"miR overexpression/knockdown, LCP1 knockdown and overexpression in MKs, in vivo miR inhibition (platelet count), luciferase reporter assay (miR-125a-5p targeting LCP1 3'UTR), PPF quantification","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct target validation by luciferase assay, gain- and loss-of-function with quantitative PPF readout, in vivo platelet count, multiple orthogonal approaches","pmids":["32844999"],"is_preprint":false},{"year":2021,"finding":"LFA-1 cluster formation (avidity regulation) in T cells depends on L-plastin Ser5 phosphorylation mediated by an nPKC-MEK-p90RSK pathway and counter-regulated by PP2A. Ser5 phosphorylation is required for LFA-1 clustering in high-affinity conformation; cofilin dephosphorylation (not LPL phosphorylation) mediates LFA-1 recruitment to the T-cell/APC contact zone—a distinct, dichotomic spatial regulation.","method":"Phospho-specific imaging, nPKC/MEK/p90RSK and PP2A inhibitors, phosphomutant expression, super-resolution microscopy of LFA-1 nanoclusters","journal":"Cellular and molecular life sciences","confidence":"High","confidence_rationale":"Tier 2 / Moderate — pharmacological pathway dissection with phosphomutant validation and super-resolution imaging, mechanistic dissociation of clustering vs. recruitment, single lab","pmids":["33449151"],"is_preprint":false},{"year":2021,"finding":"PI3K/SGK3 pathway contributes to L-plastin Ser5 phosphorylation in breast cancer cells (in addition to ERK/MAPK-RSK), shown by computational modeling and experimental validation. Ser5 phosphorylation promotes L-plastin recruitment to invadopodia, MMP-9 activity, and ECM degradation.","method":"Computational network modeling, PI3K/SGK3 inhibition/overexpression, in vitro kinase validation, shRNA knockdown, invasion/migration assays, confocal imaging, gelatin degradation/zymography","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — computational model experimentally validated with kinase inhibition and functional assays, single lab","pmids":["33618712"],"is_preprint":false},{"year":2022,"finding":"LCP1 I232F missense mutation causes granulocytic dysplasia, reduced proliferation, G2/M cell cycle arrest, impaired cell motility/invasiveness, increased F-actin, and aberrant nuclear localization of the mutant protein (wild-type L-plastin is cytoplasmic). The mutation does not block differentiation or increase apoptosis.","method":"Lentiviral inducible expression of LCP1 I232F in 32D and HeLa cells, flow cytometry (cell cycle, F-actin), confocal imaging, subcellular fractionation, motility/invasion assays","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutant expression with subcellular fractionation confirming nuclear mislocalization and multiple functional readouts, single lab","pmids":["34991157"],"is_preprint":false},{"year":2022,"finding":"L-plastin (LPL) enhances NLRP3 inflammasome assembly by stabilizing ASC interactions with Pyk2 (a component of podosomes), enabling ASC oligomerization. LPL-/- alveolar macrophages exhibit reduced caspase-1 activity, IL-1β cleavage, and gasdermin-D processing upon NLRP3 activation; LPL-/- mice are resistant to bleomycin-induced lung fibrosis.","method":"LPL-/- mouse model, ASC oligomerization assay, co-immunoprecipitation (ASC-Pyk2-LPL), caspase-1 activity and IL-1β processing assays, bleomycin lung fibrosis model","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with Co-IP identifying ASC-Pyk2-LPL complex plus multiple NLRP3 functional readouts and in vivo disease model","pmids":["35294888"],"is_preprint":false},{"year":2022,"finding":"LCP1 splicing variant (c.740-1G>A) causes partial L-plastin deficiency resulting in lymphopenia, neutropenia, and thrombocytopenia. Functional analysis shows defective actin organization in T cells, reduced PBMC migration in response to CXCL12, impaired germinal center B-cell expansion, reduced T-cell cytokinesis, and aberrant platelet spreading on collagen.","method":"Human kindred genetic analysis, orthologous mouse knock-in, Jurkat cell mutation engineering, migration assays, flow cytometry, platelet spreading assay","journal":"The Journal of allergy and clinical immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — human disease variant validated in orthologous mouse model and cell line with multiple functional immune cell readouts","pmids":["38710235"],"is_preprint":false}],"current_model":"L-plastin (LCP1) is a leukocyte-specific, calcium-regulated actin-bundling protein whose activity is controlled primarily by phosphorylation of Ser5 in its N-terminal headpiece—a modification catalyzed by PKA (downstream of FcγR/PI3K/cAMP), PKCβII (in eosinophils), PKCδ (in cancer cells), RSK1/2 (downstream of ERK/MAPK), SGK3 (downstream of PI3K), and Mst1 (on Thr89)—and inhibited by dexamethasone; Ser5 phosphorylation increases F-actin association rate and bundling activity by switching the protein to a high-affinity state, driving lamellipodia and podosome formation, LFA-1 clustering in the immunological synapse (through an LPL–Pyk2–ASC pathway also governing NLRP3 inflammasome assembly), integrin inside-out activation, osteoclast sealing-ring formation, and T cell, B cell, macrophage, and NK cell migration; additionally, L-plastin is reversibly inactivated by ROS-induced Cys101–Cys42 disulfide formation reversed by Thioredoxin1, and by S-glutathionylation of Cys460 in the actin-binding domain, and its calmodulin-binding EF-hand headpiece harbors a calcium-dependent switch helix that regulates bundling; ectopic expression and phosphorylation of L-plastin in non-hematopoietic tumor cells drives invadopodia-mediated ECM degradation, invasion, and metastasis."},"narrative":{"mechanistic_narrative":"L-plastin (LCP1) is a leukocyte-enriched, calcium-regulated actin-bundling protein that cross-links F-actin into the higher-order structures driving immune cell migration, adhesion, and invasion [PMID:1491005, PMID:8461306]. It binds laterally along the filament through two actin-binding domains of differing affinity, contacts beta-actin selectively, and bundles only at low free calcium (optimal below ~0.15 µM, progressively inhibited above), reflecting a calcium-dependent conformational switch in its EF-hand headpiece [PMID:1491005, PMID:8461306, PMID:14992580]; an NMR structure localized this switch to a helix that binds the EF-hand motifs in the presence of calcium and governs bundling activity [PMID:28145401]. The dominant activating modification is phosphorylation of Ser5 in the N-terminal headpiece, which shifts the protein to a high-affinity state—increasing the F-actin association rate roughly two-fold and lowering actin dissociation—and is sufficient to drive integrin inside-out activation and adhesion in neutrophils and monocytes [PMID:9689080, PMID:20169155, PMID:16636079]. Ser5 is targeted by multiple kinases converging from distinct receptor inputs: PKA downstream of FcγR/PI3K/cAMP signaling [PMID:10446213], PKCβII in GM-CSF-primed eosinophils [PMID:21525390], RSK1/2 downstream of ERK/MAPK and SGK3 downstream of PI3K in cancer cells [PMID:26631483, PMID:33618712], and Mst1 on Thr89 [PMID:27465533], while dexamethasone suppresses Ser5 phosphorylation to blunt T cell synapse maturation [PMID:21805466]. Through this activity L-plastin builds and stabilizes lamellipodia, filopodia, and podosomes, sustains LFA-1 clustering and immunological synapse formation, and supports T, B, and macrophage migration and development, as defined by knockout, knockdown, and inhibitory-nanobody studies [PMID:21076065, PMID:20683899, PMID:21832165, PMID:27614263, PMID:19726756]; it further promotes alveolar macrophage maturation and scaffolds an ASC–Pyk2 complex required for NLRP3 inflammasome assembly [PMID:27758872, PMID:35294888]. L-plastin activity is reversibly inactivated by redox modification—an ROS-induced Cys101–Cys42 disulfide reversed by the Thioredoxin1 system, and S-glutathionylation at Cys460 in the actin-binding domain—both of which reduce bundling and impair cell motility and invasion [PMID:31501427, PMID:25881549]. Ectopic expression and Ser5 phosphorylation of L-plastin in non-hematopoietic tumor cells drive invadopodia-mediated ECM degradation, invasion, and metastasis [PMID:17290393, PMID:24438191, PMID:26945069]. Human LCP1 deficiency from a splice variant causes lymphopenia, neutropenia, and thrombocytopenia with defective actin organization and impaired immune cell migration, and an I232F missense mutation produces granulocytic dysplasia with aberrant nuclear mislocalization [PMID:38710235, PMID:34991157].","teleology":[{"year":1992,"claim":"Established the core biochemical activity: that L-plastin is a calcium-regulated actin cross-linker with defined isoform and stoichiometric specificity, distinguishing it from related bundlers.","evidence":"In vitro F-actin bundling and co-sedimentation assays with purified human T-cell L-plastin under defined calcium conditions","pmids":["1491005","8461306"],"confidence":"High","gaps":["Did not define how calcium is sensed structurally","Cellular triggers of bundling not addressed"]},{"year":1994,"claim":"Connected L-plastin biochemistry to receptor signaling by showing it associates with the cytoskeleton and participates in FcR-mediated calcium responses in intact neutrophils.","evidence":"Chemical labeling (BPB) in intact cells, immunoprecipitation, cytoskeletal fractionation, and Ca2+ flux measurements","pmids":["8170942"],"confidence":"High","gaps":["Mechanism linking L-plastin to Ca2+ flux not defined","No specific receptor identified"]},{"year":1996,"claim":"Identified FcγRII as the upstream receptor and podosomes as a localization target, separating adhesion signaling from cytoskeletal rearrangement requirements.","evidence":"Receptor-blocking antibodies, LAD (CD18-null) patient cells, cytochalasin D, and immunofluorescence colocalization","pmids":["8663066"],"confidence":"High","gaps":["Did not identify the kinase acting on L-plastin","Role of L-plastin within podosomes unresolved"]},{"year":1998,"claim":"Defined Ser5 phosphorylation as a sufficient molecular switch for integrin-mediated adhesion, shifting the model from passive bundler to actively regulated effector.","evidence":"Cell-permeable Ser5 and Ser5Ala peptides in primary human PMNs and monocytes with adhesion assays and PI3K/PKC inhibitors","pmids":["9689080"],"confidence":"High","gaps":["Did not identify the physiological Ser5 kinase","Mechanism of integrin activation by the headpiece unresolved"]},{"year":1999,"claim":"Identified PKA as a Ser5 kinase acting downstream of PI3K and FcγR-induced cAMP, providing the first complete signaling chain to L-plastin activation.","evidence":"In vitro PKA kinase assay on L-plastin peptides, cAMP analogs/forskolin, PKA and PI3K inhibitors, cAMP measurement","pmids":["10446213"],"confidence":"High","gaps":["Other receptors may use different kinases","Direct structural consequence of Ser5 phosphorylation not shown"]},{"year":2001,"claim":"Began mapping L-plastin's protein and integrin interactions, showing Ca2+-favored binding to grancalcin and headpiece-driven activation of αvβ3 conformational change.","evidence":"Affinity chromatography and reciprocal Co-IP (grancalcin); cell-permeable headpiece peptides with LIBS conformational antibodies in K562 cells","pmids":["11279160","11278342"],"confidence":"High","gaps":["Functional role of grancalcin binding not established","Whether full-length protein recapitulates peptide effects unclear"]},{"year":2004,"claim":"Defined the actin-binding architecture—two ABDs of distinct affinity binding laterally along the filament—and identified phosphoinositides as inhibitors of actin binding.","evidence":"Recombinant domain expression, cross-linking, F-actin co-sedimentation, pyrenyl depolymerization, phosphoinositide competition; fMLP receptor pharmacology with MS","pmids":["14992580","14556648"],"confidence":"High","gaps":["Atomic contact map of ABD-actin interface not resolved","Receptor-specific kinases for fMLP-induced phosphorylation not identified"]},{"year":2006,"claim":"Demonstrated directly that Ser5 phosphorylation increases F-actin binding and targets L-plastin to peripheral protrusions, linking the modification to invasive behavior.","evidence":"In vitro F-actin binding assay plus Ser5Ala/Ser5Glu phosphovariant transfection in Vero/HEK293T cells and 3D collagen invasion assay","pmids":["16636079"],"confidence":"High","gaps":["Did not address phosphorylation kinetics in living cells","In vivo relevance not tested"]},{"year":2007,"claim":"Extended the phospho-switch to functional immune and tumor outputs—T cell surface receptor transport and melanoma invasion/metastasis—showing expression and phosphorylation have separable roles.","evidence":"MS phosphosite mapping with 5A-LPL mutagenesis in primary T cells (CD25/CD69); siRNA and phosphomutant expression with Matrigel invasion and in vivo B16 metastasis","pmids":["17294403","17290393"],"confidence":"High","gaps":["Mechanism coupling L-plastin to surface receptor transport unresolved","Distinction between migration and invasion contributions incomplete"]},{"year":2009,"claim":"Causally tied L-plastin bundling activity to filopodia formation using domain-specific intracellular nanobodies, and linked Ser5 phosphorylation to TNF-α death resistance.","evidence":"Intracellular nanobody expression with filopodia and bundling readouts; phosphomutant overexpression with apoptosis assays and PKC inhibitors","pmids":["19726756","19799649"],"confidence":"Medium","gaps":["Single-lab nanobody tools","Mechanism of death resistance not defined beyond phosphorylation dependence"]},{"year":2010,"claim":"Multiple knockout and quantitative studies established L-plastin as essential for T cell synapse formation, LFA-1 clustering, chemokine-driven migration, integrin binding, and osteoclast structures, and quantified its kinetic effect on actin turnover.","evidence":"LPL-/- mice with synapse imaging, CCR7 polarization, two-photon node motility, EAE/allograft models; FRAP with modeling, PKCδ siRNA, cortactin Co-IP; integrin domain mapping with calpain; osteoclast imaging; NES analysis","pmids":["21076065","20683899","20194718","20169155","20183869","20650888","15752138"],"confidence":"High","gaps":["Integration of distinct kinase inputs across cell types not unified","Functional consequence of nucleocytoplasmic shuttling unclear"]},{"year":2011,"claim":"Defined L-plastin's roles in B cell development and eosinophil priming and identified cell-type-specific kinases (PKCβII) and signaling complexes (Pyk-2, GM-CSFRα, paxillin, cofilin).","evidence":"LPL-/- B cell migration and development assays with Pyk-2 phosphorylation; eosinophil phosphoproteomics, Co-IP, PKCβII siRNA, and synthetic phosphopeptide reconstitution","pmids":["21832165","21525390"],"confidence":"High","gaps":["How distinct kinases are selected per receptor unresolved","Stoichiometry of the eosinophil complex undefined"]},{"year":2012,"claim":"Showed dexamethasone acts through suppression of L-plastin Ser5 phosphorylation, and that L-plastin organizes leading-edge actin and Rac1/Akt signaling during chemotaxis via PKCζ.","evidence":"Dexamethasone with phosphomimetic rescue in primary T cells; siRNA knockdown with Rac1 pulldown, Akt immunoblot, PKC isoform inhibitors, and nanobody conformational trapping","pmids":["21805466","22581862","23001012"],"confidence":"High","gaps":["Direct kinase target of dexamethasone-regulated pathway not pinpointed","Order of Rac1/Akt relative to L-plastin not fully resolved"]},{"year":2013,"claim":"Established that L-plastin stabilizes podosomes and supports matrix degradation through its bundling activity independently of Ser5 phosphorylation status in macrophages.","evidence":"Intracellular nanobody expression with live podosome imaging, matrix degradation, and phospho-L-plastin immunostaining in THP-1 macrophages","pmids":["24236012"],"confidence":"Medium","gaps":["Single-lab nanobody approach","Relationship between bundling and phosphorylation contributions at podosomes not reconciled"]},{"year":2014,"claim":"Demonstrated in vivo that both L-plastin expression and Ser5 phosphorylation are required for tumor growth and metastasis across prostate and melanoma models.","evidence":"shRNA knockdown and phosphorylatable vs. non-phosphorylatable ectopic expression with subcutaneous and intracardiac mouse metastasis models","pmids":["24438191"],"confidence":"High","gaps":["Tumor-cell kinase driving Ser5 in vivo not identified here","Downstream invasion machinery not mapped"]},{"year":2015,"claim":"Identified redox modification—S-glutathionylation at Cys460 in the ABD—as an inactivating regulatory layer, and RSK1/2 as direct ERK/MAPK-driven Ser5 kinases in cancer.","evidence":"Cys460 mutagenesis with bundling and neutrophil functional assays and diabetic patient samples; in vitro RSK1/2 kinase assay with siRNA/shRNA and invasion assays","pmids":["25881549","26631483"],"confidence":"High","gaps":["Enzymes catalyzing/reversing glutathionylation not defined","Interplay between phosphorylation and oxidation not addressed"]},{"year":2016,"claim":"Resolved distinct biophysical roles versus fascin in invadopodia/filopodia, identified Mst1 as a Thr89 kinase, and placed L-plastin in alveolar macrophage maturation and podosome longevity.","evidence":"Nanobody inhibition with EM of bundle ultrastructure; in vitro Mst1 kinase assay, Co-IP, T89A rescue in LPL-/- mice; conditional macrophage knockout with PPAR-γ analysis; live podosome imaging in LPL-/- macrophages","pmids":["26945069","27465533","27758872","27614263"],"confidence":"High","gaps":["Functional role of Thr89 phosphorylation beyond localization unclear","How L-plastin couples to PPAR-γ upregulation unresolved"]},{"year":2017,"claim":"Provided a structural mechanism for calcium regulation by identifying a switch helix in the EF-hand headpiece that controls bundling activity.","evidence":"NMR solution structure of the EF-hand headpiece with competitive peptide inhibition of bundling","pmids":["28145401"],"confidence":"High","gaps":["Structure of full-length protein bound to actin not determined","How Ser5 phosphorylation intersects the switch helix unresolved"]},{"year":2018,"claim":"Extended kinase inputs to LRRK1 in osteoclasts and characterized osteoclast bone-resorption roles via Ser5/Ser7-dependent sealing-zone formation.","evidence":"LC/MS phosphoproteomics and phospho-specific Western in Lrrk1-KO osteoclasts with micro-CT; TAT-peptide transduction with bone resorption assays","pmids":["30136304","30244178"],"confidence":"Medium","gaps":["Whether LRRK1 phosphorylates L-plastin directly not shown","Single-lab peptide competition for bone resorption readout"]},{"year":2019,"claim":"Defined the Thioredoxin1-reversible Cys101–Cys42 disulfide as a localized redox switch and placed LCP1 upstream of mTORC2/AKT signaling in eosinophilic cells.","evidence":"TRX1 trapping/knockdown, auranofin, ratiometric LPL-roGFP-Orp1 imaging, proximity ligation, bundling and migration/invasion assays; shRNA knockdown with phospho-specific AKT/STAT immunoblots","pmids":["31501427","31691359"],"confidence":"High","gaps":["Trigger localizing oxidation to cell extrusions unresolved","Mechanism linking LCP1 to mTORC2 activity undefined"]},{"year":2020,"claim":"Defined opposing osteoclast and megakaryocyte roles and identified upstream transcriptional (PI3K/AKT/SP1) and post-transcriptional (miR-125a-5p) control of LCP1 expression.","evidence":"LPL-KO mice with histomorphometry, filopodia/fusion and PDGF-BB assays, SP1 promoter studies; miR overexpression/knockdown with luciferase reporter and proplatelet formation quantification","pmids":["33208358","32844999"],"confidence":"High","gaps":["How L-plastin negatively regulates the megakaryocyte invagination system unclear","Coupling of PI3K/AKT/SP1 to RANKL kinetics incomplete"]},{"year":2021,"claim":"Dissected spatial regulation of LFA-1 (Ser5-phosphorylation-dependent clustering via nPKC-MEK-p90RSK versus cofilin-mediated recruitment) and added the PI3K/SGK3 axis to cancer-cell Ser5 phosphorylation driving invadopodia and MMP-9-dependent degradation.","evidence":"Phospho-imaging, nPKC/MEK/p90RSK and PP2A inhibitors, phosphomutants, super-resolution microscopy; computational modeling with PI3K/SGK3 perturbation, kinase validation, and gelatin degradation/zymography","pmids":["33449151","33618712"],"confidence":"High","gaps":["Integration of redundant Ser5 kinases in a single cell unresolved","PP2A site specificity not defined"]},{"year":2022,"claim":"Established L-plastin as an NLRP3 inflammasome scaffold via ASC-Pyk2 stabilization and linked human LCP1 mutations to defined hematologic/immunologic disease, including nuclear mislocalization of an I232F mutant.","evidence":"LPL-/- mice with ASC oligomerization, ASC-Pyk2-LPL Co-IP, caspase-1/IL-1β/gasdermin-D assays and bleomycin fibrosis; lentiviral I232F expression with fractionation and functional assays","pmids":["35294888","34991157"],"confidence":"Medium","gaps":["Single-lab characterization of I232F mutant","How the inflammasome scaffolding role relates to actin bundling unresolved"]},{"year":2022,"claim":"Demonstrated that partial human L-plastin deficiency causes a multilineage cytopenia syndrome, defining a Mendelian disease link.","evidence":"Human kindred genetic analysis with orthologous mouse knock-in and Jurkat engineering, migration, flow cytometry, and platelet spreading assays","pmids":["38710235"],"confidence":"High","gaps":["Genotype-phenotype spectrum across additional patients not established","Tissue-specific molecular basis of each cytopenia incomplete"]},{"year":null,"claim":"How the many redundant and cell-type-specific Ser5 kinases, the Thr89 input, calcium switching, and redox modifications are integrated to set L-plastin bundling activity in a given physiological context remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model coupling phosphorylation, calcium, and redox states","No full-length actin-bound structure","Quantitative hierarchy of kinase inputs per cell type unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,8,11,14,19,35]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[20,45,23]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,7,17]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[2,3,11,19,27,34]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[10,44]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[10,44]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[11,17,25]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[16,17,22,23,45]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[13,28,31,44,46]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[5,9,25,30,42,43]}],"complexes":["NLRP3 inflammasome (ASC-Pyk2-LPL scaffold)","podosome","invadopodia","immunological synapse"],"partners":["ACTB","GCA","ITGB1","ITGB2","CTTN","PYCARD","PTK2B","PRKCB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P13796","full_name":"Plastin-2","aliases":["L-plastin","LC64P","Lymphocyte cytosolic protein 1","LCP-1"],"length_aa":627,"mass_kda":70.3,"function":"Actin-binding protein (PubMed:16636079, PubMed:17294403, PubMed:28493397). Plays a role in the activation of T-cells in response to costimulation through TCR/CD3 and CD2 or CD28 (PubMed:17294403). Modulates the cell surface expression of IL2RA/CD25 and CD69 (PubMed:17294403)","subcellular_location":"Cytoplasm, cytoskeleton; Cell junction; Cell projection; Cell projection, ruffle membrane","url":"https://www.uniprot.org/uniprotkb/P13796/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/LCP1","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/LCP1","total_profiled":1310},"omim":[{"mim_id":"614032","title":"TOX HIGH MOBILITY GROUP BOX FAMILY MEMBER 4; TOX4","url":"https://www.omim.org/entry/614032"},{"mim_id":"613004","title":"HUNTINGTIN; HTT","url":"https://www.omim.org/entry/613004"},{"mim_id":"610986","title":"LEUCINE-RICH REPEAT KINASE 1; LRRK1","url":"https://www.omim.org/entry/610986"},{"mim_id":"602734","title":"PLASTIN 1; PLS1","url":"https://www.omim.org/entry/602734"},{"mim_id":"601517","title":"ATAXIN 2; ATXN2","url":"https://www.omim.org/entry/601517"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Actin filaments","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":669.6},{"tissue":"epididymis","ntpm":380.4},{"tissue":"lymphoid tissue","ntpm":566.7}],"url":"https://www.proteinatlas.org/search/LCP1"},"hgnc":{"alias_symbol":["PLS2","CP64","L-PLASTIN","LC64P"],"prev_symbol":[]},"alphafold":{"accession":"P13796","domains":[{"cath_id":"1.10.238.10","chopping":"7-98","consensus_level":"high","plddt":78.5791,"start":7,"end":98},{"cath_id":"1.10.418.10","chopping":"119-236","consensus_level":"high","plddt":95.0499,"start":119,"end":236},{"cath_id":"1.10.418.10","chopping":"245-372","consensus_level":"high","plddt":94.3007,"start":245,"end":372},{"cath_id":"1.10.418.10","chopping":"394-508","consensus_level":"medium","plddt":95.4349,"start":394,"end":508},{"cath_id":"1.10.418.10","chopping":"517-624","consensus_level":"high","plddt":96.1956,"start":517,"end":624}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P13796","model_url":"https://alphafold.ebi.ac.uk/files/AF-P13796-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P13796-F1-predicted_aligned_error_v6.png","plddt_mean":89.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=LCP1","jax_strain_url":"https://www.jax.org/strain/search?query=LCP1"},"sequence":{"accession":"P13796","fasta_url":"https://rest.uniprot.org/uniprotkb/P13796.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P13796/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P13796"}},"corpus_meta":[{"pmid":"9689080","id":"PMC_9689080","title":"A role for the actin-bundling protein L-plastin in the regulation of leukocyte integrin function.","date":"1998","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9689080","citation_count":138,"is_preprint":false},{"pmid":"1491005","id":"PMC_1491005","title":"Human T cell L-plastin bundles actin filaments in a calcium-dependent manner.","date":"1992","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1491005","citation_count":131,"is_preprint":false},{"pmid":"16636079","id":"PMC_16636079","title":"Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells.","date":"2006","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16636079","citation_count":95,"is_preprint":false},{"pmid":"22194750","id":"PMC_22194750","title":"The actin-bundling protein L-plastin: a critical regulator of immune cell function.","date":"2011","source":"International journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/22194750","citation_count":90,"is_preprint":false},{"pmid":"17294403","id":"PMC_17294403","title":"Costimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25.","date":"2007","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/17294403","citation_count":89,"is_preprint":false},{"pmid":"33208358","id":"PMC_33208358","title":"Targeting actin-bundling protein L-plastin as an anabolic therapy for bone loss.","date":"2020","source":"Science advances","url":"https://pubmed.ncbi.nlm.nih.gov/33208358","citation_count":81,"is_preprint":false},{"pmid":"11735128","id":"PMC_11735128","title":"Differential expression of the L-plastin gene in human colorectal cancer progression and metastasis.","date":"2001","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/11735128","citation_count":79,"is_preprint":false},{"pmid":"36574182","id":"PMC_36574182","title":"Inhibition of the Glycolysis Prevents the Cerebral Infarction Progression Through Decreasing the Lactylation Levels of LCP1.","date":"2022","source":"Molecular biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/36574182","citation_count":73,"is_preprint":false},{"pmid":"16287074","id":"PMC_16287074","title":"The leukocyte protein L-plastin induces proliferation, invasion and loss of E-cadherin expression in colon cancer cells.","date":"2006","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/16287074","citation_count":72,"is_preprint":false},{"pmid":"30583289","id":"PMC_30583289","title":"Exosomal Release of L-Plastin by Breast Cancer Cells Facilitates Metastatic Bone Osteolysis.","date":"2018","source":"Translational 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Cancer Growth and Metastasis in a Helicobacter pylori cagA-ERK-SP1-Dependent Manner.","date":"2021","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/33771880","citation_count":8,"is_preprint":false},{"pmid":"31007850","id":"PMC_31007850","title":"Dual inhibition of ABCE1 and LCP1 by microRNA-96 results in an additive effect in breast cancer mouse model.","date":"2019","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/31007850","citation_count":8,"is_preprint":false},{"pmid":"31191656","id":"PMC_31191656","title":"Engineering of L-Plastin Peptide-Loaded Biodegradable Nanoparticles for Sustained Delivery and Suppression of Osteoclast Function In Vitro.","date":"2019","source":"International journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/31191656","citation_count":8,"is_preprint":false},{"pmid":"16625055","id":"PMC_16625055","title":"Analysis of a bacterial lipopolysaccharide-activated serine kinase that phosphorylates p65/L-plastin in macrophages.","date":"2006","source":"Microbiology and immunology","url":"https://pubmed.ncbi.nlm.nih.gov/16625055","citation_count":8,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":52297,"output_tokens":12075,"usd":0.169008,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":23367,"output_tokens":7779,"usd":0.155655,"stage2_stop_reason":"end_turn"},"total_usd":0.324663,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"L-plastin bundles F-actin in a strictly calcium-dependent manner: bundles form at pCa 7 but not at pCa 6, and approximately one molecule of L-plastin binds 8 actin monomers in the filament. Unlike fimbrin, it acts on beta-actin but not muscle alpha-actin or gizzard gamma-actin.\",\n      \"method\": \"In vitro F-actin bundling assay with purified human T-cell L-plastin under defined calcium conditions\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro reconstitution with purified protein, quantitative binding stoichiometry, replicated across multiple calcium conditions\",\n      \"pmids\": [\"1491005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Macrophage 70-kDa actin-bundling protein (p70) is identical to L-plastin; its cross-linking activity is optimal at free calcium below 0.15 µM and is progressively inhibited at higher calcium (half-maximal inhibition at 1.6 µM). The protein undergoes major conformational changes at 0.15–1.5 µM free calcium and does not sever actin filaments.\",\n      \"method\": \"Protein purification, falling-ball viscosimetry, fluorescence spectroscopy, actin co-sedimentation assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal in vitro assays with purified protein establishing calcium-dependent bundling kinetics\",\n      \"pmids\": [\"8461306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Bromophenacyl bromide (BPB) alkylates L-plastin in intact neutrophils and inhibits Fc receptor-mediated (IgG-stimulated), IP3-independent intracellular Ca2+ increase; L-plastin associates with the Triton X-100-insoluble cytoskeleton in adherent PMNs, linking it to FcR signal transduction.\",\n      \"method\": \"Chemical labeling with BPB in intact cells, immunoprecipitation with anti-BPB monoclonal antibody, cytoskeletal fractionation, Ca2+ flux measurements\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — chemical modification coupled with functional Ca2+ assays and fractionation, multiple orthogonal methods in one study\",\n      \"pmids\": [\"8170942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"FcγRII (not FcγRIIIB) is the primary receptor mediating L-plastin phosphorylation in neutrophils; adhesion to immune complexes is required but actin cytoskeleton rearrangement is not. L-plastin localizes to podosomes upon adhesion to immune complexes, colocalizing with actin, vinculin, and alpha-actinin.\",\n      \"method\": \"Receptor-specific monoclonal antibody blocking, patient cells with leukocyte adhesion deficiency (CD18-null), cytochalasin D treatment, immunofluorescence colocalization\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic (LAD patient cells) and pharmacologic dissection with orthogonal localization data in a single rigorous study\",\n      \"pmids\": [\"8663066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"L-plastin phosphorylation on Ser5 is sufficient to induce leukocyte integrin-mediated adhesion: cell-permeable Ser5-containing peptides activate integrin-mediated adhesion in PMNs and monocytes, while Ser5→Ala substitution abolishes activity. Peptide-induced adhesion is sensitive to PI3K and PKC inhibitors, but a phosphoserine-5 peptide is insensitive.\",\n      \"method\": \"Cell-permeable peptide introduction into primary human PMNs and monocytes, adhesion assays, site-directed mutagenesis (Ser5Ala), pharmacological inhibitors of PI3K and PKC\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — peptide functional assay with mutagenesis, primary human cells, pathway inhibitors, multiple orthogonal approaches\",\n      \"pmids\": [\"9689080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PKA phosphorylates L-plastin on Ser5 in vitro and in cells downstream of PI3K and FcγR signaling: cAMP analogs and forskolin induce L-plastin phosphorylation; PKA inhibitors H89 and KT5720 block FcγR-induced L-plastin phosphorylation and integrin αMβ2 activation. FcγR stimulation transiently increases cAMP. PI3K acts upstream of PKA in this pathway.\",\n      \"method\": \"In vitro kinase assay (PKA + L-plastin peptides), cAMP analogs, adenylate cyclase activator, PKA inhibitors, PI3K inhibitors, cAMP measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus cell-based pharmacology with multiple inhibitors and pathway ordering\",\n      \"pmids\": [\"10446213\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Grancalcin (penta-EF-hand Ca2+-binding protein) interacts with L-plastin in a Ca2+-dependent manner (interaction occurs in the absence of Ca2+, i.e., is favored at low Ca2+), identified by affinity chromatography of solubilized neutrophils and confirmed by reciprocal co-immunoprecipitation.\",\n      \"method\": \"Affinity chromatography on immobilized grancalcin, reciprocal co-immunoprecipitation, multiple independent methods\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP confirmed by affinity chromatography, multiple independent methods in one study\",\n      \"pmids\": [\"11279160\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"L-plastin N-terminal peptides and headpiece domain activate αvβ3-mediated adhesion in K562 cells; this activation requires actin filament disassembly (jasplakinolide blocks it) and synergizes with RGD ligand or Mn2+ to induce the high-affinity integrin conformation (ligand-induced binding site epitope), independently of β3 cytoplasmic tail tyrosines.\",\n      \"method\": \"Cell-permeable peptides in K562 cells, LIBS antibody binding assay for integrin conformation, jasplakinolide and cytochalasin D treatment, adhesion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assay with conformational readout, multiple pharmacological tools, single lab\",\n      \"pmids\": [\"11278342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"L-plastin binds actin laterally along the filament axis via two actin-binding domains (ABD) that have different affinities for actin; actin peptides 112-125 and 360-372 participate in the interface. Phosphoinositides inhibit actin binding. The two ABDs show distinct modes of actin interaction.\",\n      \"method\": \"Recombinant domain expression, chemical cross-linking, F-actin co-sedimentation, pyrenyl F-actin depolymerization assays, phosphoinositide competition\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with recombinant domains, multiple orthogonal binding assays, single lab\",\n      \"pmids\": [\"14992580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Two fMLP receptor subtypes in neutrophils both phosphorylate L-plastin but use different signaling intermediates: low-affinity fMLP receptor phosphorylation requires PI3K, PLD, and PKC, whereas high-affinity fMLP receptor phosphorylation requires an Ro-31-8220-sensitive kinase but not PI3K, PLD, or classical PKC isoforms.\",\n      \"method\": \"2D-IEF/PAGE, MALDI-TOF MS, pharmacological inhibitors of PI3K/PLD/PKC, receptor subtype discrimination with antagonistic peptides\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry identification plus pharmacological dissection, single lab\",\n      \"pmids\": [\"14556648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"L-plastin distributes between nucleus and cytoplasm because it harbors a less conserved nuclear export sequence (NES) lacking F21 of T-plastin; insertion of Phe at the equivalent position enhances L-plastin nuclear export, explaining its cytoplasmic/nuclear distribution.\",\n      \"method\": \"Leptomycin B treatment, NES deletion/mutation analysis, nuclear export reporter assays, fluorescence microscopy\",\n      \"journal\": \"Traffic\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-directed mutagenesis of NES with functional nuclear export assay, single lab\",\n      \"pmids\": [\"15752138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Phosphorylation of L-plastin on Ser5 increases its F-actin-binding activity in vitro, promotes targeting to actin-rich peripheral membrane protrusions and microspikes in cells, and is required for collagen invasion. Ser5Ala reduces association with F-actin; Ser5Glu (phosphomimetic) promotes F-actin microspike formation and remains detergent-resistant with F-actin.\",\n      \"method\": \"In vitro F-actin binding/co-sedimentation assay, transfection of phosphovariants (Ser5Ala, Ser5Glu) in Vero and HEK293T cells, detergent extraction, 3D collagen invasion assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro F-actin binding assay plus cell-based mutagenesis with invasion assay, multiple orthogonal methods\",\n      \"pmids\": [\"16636079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Phosphorylation of L-plastin at Ser5 (identified by mass spectrometry) in T cells is required for surface transport of CD25 and CD69 to the T cell surface upon costimulation (TCR/CD3 + CD2 or CD28), but not for relocalization of L-plastin itself to the immunological synapse.\",\n      \"method\": \"Mass spectrometry phosphosite identification, site-directed mutagenesis (5A-LPL), retroviral transduction in primary human PBT, flow cytometry for CD25/CD69 surface expression\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS identification plus functional mutagenesis in primary human T cells, orthogonal methods\",\n      \"pmids\": [\"17294403\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Phosphorylation of L-plastin (on Ser5) in melanoma cells (but not expression alone) is required for enhanced Matrigel invasion; haptotactic migration (but not chemotactic migration or static adhesion) is enhanced by L-plastin expression regardless of phosphorylation status. In vivo metastatic capacity of B16 cells correlates with L-plastin expression and phosphorylation.\",\n      \"method\": \"siRNA knockdown, retroviral expression of wt-LPL-EGFP vs. nonphosphorylatable 5A7A-LPL-EGFP, Matrigel invasion/migration assays, in vivo mouse metastasis model\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphomutant expression in L-plastin-null cells with in vitro and in vivo functional assays\",\n      \"pmids\": [\"17290393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Alpaca single-domain antibody (nanobody) directed against L-plastin blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling, establishing a direct causal link between L-plastin bundling activity and filopodia formation.\",\n      \"method\": \"Recombinant nanobody expression in cells, F-actin bundling inhibition, filopodia formation assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific intracellular nanobody inhibition with phenotypic readout, single lab\",\n      \"pmids\": [\"19726756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"L-plastin confers resistance to TNF-α-induced cell death in MCF-7 breast cancer cells in a Ser5 phosphorylation-dependent manner; non-conventional PKC isoforms and the ceramide pathway regulate this phosphorylation state.\",\n      \"method\": \"siRNA knockdown, overexpression of wild-type and phosphomutant L-plastin, cell viability/apoptosis assays, PKC inhibitor studies\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional cell death assay, pharmacological pathway dissection, single lab\",\n      \"pmids\": [\"19799649\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"L-plastin (LPL) is required for TCR-mediated T cell activation: LPL-/- T cells show defective lamellipodia formation, smaller immunological synapses, impaired cytokine production, and reduced proliferation. LPL-/- mice show delayed allograft rejection and reduced EAE severity.\",\n      \"method\": \"LPL-/- mouse model, T cell spreading assays on immobilized TCR ligands, immunological synapse imaging with APCs, cytokine ELISA, allograft rejection model, EAE model\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with multiple cellular and in vivo functional readouts, replicated across model systems\",\n      \"pmids\": [\"21076065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Sustained LFA-1 (but not TCR/CD3) clustering in the peripheral SMAC of the immunological synapse requires L-plastin; calmodulin binding to L-plastin is required for maintenance of L-plastin in the IS, and calmodulin inhibition prevents stable LFA-1 and Talin redistribution.\",\n      \"method\": \"siRNA knockdown of L-plastin, IS immunofluorescence imaging (LFA-1, Talin, CD3 redistribution), calmodulin inhibitor treatment, T cell proliferation assay\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with specific receptor tracking at the IS and calmodulin inhibitor, two orthogonal approaches, single lab\",\n      \"pmids\": [\"20683899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"L-plastin is required for CCR7-mediated T cell motility and cellular polarization (CCR7 fails to polarize to leading edge in LPL-/- T cells) and for thymic egress, but is dispensable for CCR7 proximal signaling to F-actin polymerization and CCR7-mediated costimulation.\",\n      \"method\": \"LPL-/- mice, CCR7-stimulated T cell polarization assays, two-photon microscopy of lymph node motility, thymic egress quantification, F-actin polymerization assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with epistatic dissection of CCR7 signaling branches, in vivo and in vitro readouts\",\n      \"pmids\": [\"20194718\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In focal adhesions, L-plastin undergoes rapid association/dissociation cycles following a two-binding-state model; Ser5 phosphorylation increases association rates 2-fold without affecting dissociation rates, and L-plastin decreases actin dissociation rate 4-fold thereby increasing F-actin content. PKC-delta mediates PMA-induced L-plastin Ser5 phosphorylation. L-plastin associates with a complex containing cortactin.\",\n      \"method\": \"FRAP of GFP-coupled L-plastin phosphovariants in live cells, mathematical modeling, siRNA knockdown of PKC isoforms, co-immunoprecipitation with cortactin\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — FRAP with mathematical modeling quantifying kinetic parameters, siRNA epistasis, Co-IP, multiple orthogonal methods\",\n      \"pmids\": [\"20169155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"L-plastin interacts with the cytoplasmic domain of β1 and β2 integrins via its two actin-binding domains (not the N-terminal headpiece); this complex is regulated by µ-calpain, which cleaves β-integrin and disrupts the L-plastin/integrin interaction but does not directly dissociate it via calcium.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, solid phase assays, pull-down with recombinant L-plastin domains, cross-linking, ELISA, µ-calpain cleavage experiments\",\n      \"journal\": \"Cytoskeleton\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP confirmed by multiple in vitro binding assays, domain mapping, calpain regulation experiment, single lab\",\n      \"pmids\": [\"20183869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"In osteoclasts, L-plastin is present in actin aggregates during the early phase of sealing ring formation; cortactin is involved in the maturation phase. Temporal changes in L-plastin localization (from actin aggregates) and cortactin localization (in sealing ring) indicate sequential roles during bone resorption.\",\n      \"method\": \"siRNA to cortactin, immunofluorescence, quantification of L-plastin and cortactin localization during RANKL/M-CSF-induced bone resorption\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA functional knockdown with timed imaging of actin structures, single lab\",\n      \"pmids\": [\"20650888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"L-plastin is required for marginal zone B cell development and B cell motility toward CXCL12, CXCL13, and S1P; LPL-/- B cells fail to enter lymph nodes and bone marrow. LPL is required for integrin-mediated enhancement of Transwell migration but not for integrin-mediated adhesion per se. LPL-/- B cells show reduced Pyk-2 phosphorylation in response to chemokine.\",\n      \"method\": \"LPL-/- mouse model, Transwell migration assays, flow cytometry of splenic B cell subsets, integrin adhesion assays, Pyk-2 phosphorylation by immunoblot\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with multiple cellular and biochemical phenotype readouts, pathway dissection\",\n      \"pmids\": [\"21832165\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GM-CSF priming of eosinophils for chemotaxis and degranulation is mediated by PKCβII-phosphorylated L-plastin; co-immunoprecipitation reveals a complex of phospho-L-plastin with PKCβII, GM-CSF receptor α-chain, paxillin, and cofilin. A synthetic Ser5-phosphorylated L-plastin peptide (residues 2-19) recapitulates GM-CSF effects on integrin αMβ2 and chemotaxis independently of GM-CSF.\",\n      \"method\": \"Phosphoproteomics, co-immunoprecipitation, PKCβII siRNA and inhibitors, synthetic phosphopeptide stimulation, flow cytometry, chemotaxis and degranulation assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying complex plus siRNA epistasis plus synthetic peptide reconstitution, multiple orthogonal methods\",\n      \"pmids\": [\"21525390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Dexamethasone inhibits costimulation-induced L-plastin Ser5 phosphorylation in primary human T cells, thereby reducing F-actin accumulation and immune synapse maturation; expression of a phosphomimetic L-plastin reverses the immunosuppressive effect of dexamethasone.\",\n      \"method\": \"Dexamethasone treatment, expression of nonphosphorylatable and phosphomimetic L-plastin mutants in primary human PBT, F-actin staining, IS imaging, flow cytometry\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and genetic rescue experiment in primary human T cells, direct mechanistic link between dexamethasone and L-plastin phosphorylation\",\n      \"pmids\": [\"21805466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"L-plastin is required for SDF-1α-stimulated T lymphocyte polarization, lamellipodia formation, and chemotaxis; knockdown impairs F-actin localization at the leading edge, Rac1 activation cycle, and Akt phosphorylation. PKCζ (not other PKC isoforms) regulates L-plastin Ser5 phosphorylation in response to SDF-1α, and L-plastin colocalizes with PKCζ at the leading edge.\",\n      \"method\": \"siRNA knockdown, immunofluorescence colocalization, PKC isoform-selective inhibitors, Rac1 GTPase pulldown, Akt immunoblot, chemotaxis and polarization assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with pathway dissection using isoform-selective inhibitors, multiple functional readouts, single lab\",\n      \"pmids\": [\"22581862\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Nanobodies targeting either the EF-hand or actin-binding domains of L-plastin trap it in an inactive conformation, impairing IS formation, MTOC docking, T cell proliferation, IL-2 secretion, and delaying Ser5 phosphorylation. One nanobody also delays LFA-1 phosphorylation and reduces L-plastin–LFA-1 association.\",\n      \"method\": \"Intracellular nanobody expression in Jurkat and PBMCs, IS imaging, MTOC docking assay, IL-2 ELISA, T cell proliferation, co-immunoprecipitation of L-plastin with LFA-1\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific domain-targeted perturbation with multiple functional readouts and Co-IP, single lab\",\n      \"pmids\": [\"23001012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"L-plastin in THP-1 macrophages is required for podosome formation and stability, and for matrix degradation; inhibitory nanobodies cause irregular, unstable actin turnover at podosomes without affecting Ser5 phosphorylation. Phosphorylated L-plastin is highly enriched in podosomes.\",\n      \"method\": \"Intracellular nanobody expression, live-cell imaging of podosome dynamics, matrix degradation assay, phospho-L-plastin immunostaining\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific inhibitory nanobodies with functional podosome and degradation readouts, single lab\",\n      \"pmids\": [\"24236012\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"L-plastin expression and its phosphorylation are both required in vivo for tumor metastasis: L-plastin knockdown in PC3M prostate cancer cells reduces tumor growth and metastasis; ectopic expression of phosphorylatable but not non-phosphorylatable L-plastin in MV3 melanoma cells increases metastasis in mouse preclinical models.\",\n      \"method\": \"shRNA knockdown, ectopic expression of phosphorylatable vs. non-phosphorylatable L-plastin, subcutaneous and intracardiac injection mouse models, micro-CT/histomorphometry\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — phosphomutant rescue in two cell lines with in vivo metastasis readouts, genetic knockdown control\",\n      \"pmids\": [\"24438191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"S-glutathionylation of L-plastin at Cys206, Cys283, and Cys460 (Cys460 being most critical) in the actin-binding domain reduces its actin-binding and bundling activity, impairing neutrophil chemotaxis, polarization, bactericidal activity, and phagocytosis. S-thiolated L-plastin is detected in diabetic patient neutrophils.\",\n      \"method\": \"In vitro and cell-based oxidative stress, site-directed mutagenesis (Cys460), actin co-sedimentation assay, neutrophil functional assays, clinical samples\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — site-directed mutagenesis plus in vitro bundling assay plus functional cell assays in a single study\",\n      \"pmids\": [\"25881549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"RSK1 and RSK2, downstream of the ERK/MAPK pathway, directly phosphorylate L-plastin on Ser5 in vitro. RSK is an essential activator of L-plastin in breast cancer cells; RSK knockdown reduces migration and invasion and impairs Ser5-phospho-L-plastin recruitment to migratory structures.\",\n      \"method\": \"In vitro kinase assay (RSK1/2 + L-plastin), siRNA/shRNA knockdown, invasion and migration assays, immunofluorescence, computational modeling validated experimentally\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct in vitro kinase assay plus siRNA epistasis, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26631483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"L-plastin and fascin cooperate in invadopodia and filopodia with distinct roles: L-plastin bundles are thinner and less tightly packed than fascin bundles, providing flexibility for elongation while fascin provides rigidity. L-plastin is identified as a component of invadopodia contributing to matrix degradation and invasiveness; specific nanobodies inhibiting L-plastin bundling cannot be compensated by fascin.\",\n      \"method\": \"Domain-specific nanobody inhibition of bundling, invadopodia matrix degradation assay, electron microscopy of bundle ultrastructure, protrusion quantification\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — specific nanobody tools with ultrastructural and functional assays, mechanistic distinction between bundlers, single lab\",\n      \"pmids\": [\"26945069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mst1 kinase phosphorylates L-plastin on Thr89 in vitro and interacts with L-plastin in cells; T89A mutation impairs L-plastin localization to lamellipodia and fails to restore T cell migration and thymic egress in LPL-/- mice, establishing LPL as a downstream effector of Mst1.\",\n      \"method\": \"In vitro kinase assay (Mst1 + LPL), co-immunoprecipitation in cells, Thr89Ala mutagenesis, bone marrow chimeras, T cell migration/polarization assays, thymic egress quantification\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase assay plus Co-IP plus mutagenesis rescue in vivo, multiple orthogonal methods\",\n      \"pmids\": [\"27465533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"LPL is required for the transition from prealveolar macrophages to mature alveolar macrophages; LPL-/- precursors fail to upregulate PPAR-γ despite abundant GM-CSF signaling, suggesting LPL supports transmigration into and engraftment in alveoli where GM-CSF is produced.\",\n      \"method\": \"Conditional knockout (CD11c.Cre-LPLfl/fl), flow cytometry of alveolar macrophage subsets, GM-CSF signaling analysis, irradiation-regeneration assay, pneumococcal infection challenge\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockout with developmental stage analysis and mechanistic pathway placement, multiple functional readouts\",\n      \"pmids\": [\"27758872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"L-plastin promotes podosome longevity (but not initiation) in primary resident peritoneal macrophages; loss of LPL leads to decreased podosome lifetime, failure to elongate in response to chemotactic stimulation, and impaired macrophage transmigration and peritoneal migration.\",\n      \"method\": \"Live-cell imaging of LifeACT-RFP macrophages from LPL-/- mice, podosome longevity quantification, macrophage transmigration assay, in vivo peritoneal migration\",\n      \"journal\": \"Molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with genetic knockout, quantitative podosome dynamics, in vivo migration readout\",\n      \"pmids\": [\"27614263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NMR structure of the L-plastin EF-hand headpiece reveals a novel switch helix immediately after the calcium-binding region that binds tightly to the EF-hand motifs in the presence of calcium. This switch helix plays a major role in actin-bundling regulation; a competitive peptide inhibiting EF-hand/switch helix association deregulates bundling activity.\",\n      \"method\": \"NMR spectroscopy (solution structure determination), competitive peptide inhibition of actin-bundling activity\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure determination with functional validation by peptide competition, single lab\",\n      \"pmids\": [\"28145401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"LRRK1 signaling in osteoclasts promotes L-plastin Ser5 phosphorylation; Lrrk1-knockout osteoclasts show reduced L-plastin Ser5 phosphorylation by LC/MS and Western blot. L-plastin KO mice show increased trabecular bone volume.\",\n      \"method\": \"Metal affinity purification LC/MS phosphoproteomics, Ser5-specific Western blot, micro-CT of L-plastin KO mice\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphoproteomics identification confirmed by phospho-specific Western blot in genetic KO, single lab\",\n      \"pmids\": [\"30136304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TNF-α signaling phosphorylates L-plastin on Ser5 and Ser7 in osteoclasts, increasing actin bundling capacity and driving formation of nascent sealing zones (NSZs). TAT-fused full-length L-plastin peptide increases NSZ/sealing ring formation; N-terminal phospho-Ser peptides reduce cellular L-plastin phosphorylation and bone resorption. Neither podosome assembly nor osteoclast migration is affected.\",\n      \"method\": \"TAT-peptide transduction, immunoblotting with phospho-specific antibodies, actin staining, bone resorption (dentine pit) assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — peptide competition/inhibition with functional bone resorption readout, single lab\",\n      \"pmids\": [\"30244178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"L-plastin is reversibly regulated by ROS-induced thiol oxidation: Cys101 forms a disulfide bridge with Cys42, reducing actin-bundling capacity. Reduction is mediated by the Thioredoxin1 (TRX1) system (demonstrated by TRX1 trapping and TRXR1 blockade with auranofin). L-plastin oxidation occurs preferentially in actin-based cell extrusions and inhibits cell spreading, filopodial extension, migration, invasion, and ECM degradation.\",\n      \"method\": \"TRX1 trapping (substrate trap), TRX1 knockdown, TRXR1 inhibitor (auranofin), ratiometric imaging (LPL-roGFP-Orp1 fusion), dimedone-based proximity ligation assay, actin-bundling assay, functional migration/invasion/ECM assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — TRX1 trapping identifies specific redox substrate, ratiometric imaging localizes oxidation, mutagenesis implied by disulfide mapping, multiple orthogonal methods\",\n      \"pmids\": [\"31501427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LCP1 knockdown in Eol-1 eosinophilic cells reduces phosphorylation of AKTSer473 (mTORC2-dependent) and STAT1Tyr701 but not FIP1L1-PDGFRA-induced STAT3/STAT5/ERK phosphorylation, establishing LCP1 as upstream of mTORC2/AKT in eosinophilic cells.\",\n      \"method\": \"shRNA knockdown of LCP1, PKCβ inhibitor enzastaurin, phospho-specific immunoblot for mTORC2/AKT pathway components, eosinophil differentiation and apoptosis assays\",\n      \"journal\": \"Molecular carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — shRNA knockdown with selective AKT phospho-site dissection (Ser473 vs. Thr308) establishing mTORC2 linkage, single lab\",\n      \"pmids\": [\"31691359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LPL deletion in osteoclasts impedes preosteoclast fusion by inhibiting filopodia formation, increases preosteoclast numbers (which release PDGF-BB to promote vessel growth and bone formation), and LPL expression is regulated by the PI3K/AKT/SP1 axis in response to RANKL.\",\n      \"method\": \"LPL knockout mice, histomorphometry, flow cytometry, filopodia imaging, PDGF-BB secretion assay, PI3K/AKT inhibition/SP1 promoter studies\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with mechanistic pathway identification (PI3K/AKT/SP1 regulatory axis) and multiple in vivo and in vitro readouts\",\n      \"pmids\": [\"33208358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-125a-5p directly targets and reduces L-plastin (LCP1) expression in megakaryocytes; L-plastin promotes megakaryocyte progenitor migration but negatively regulates proplatelet formation (PPF) and PP branching by inhibiting the late-stage megakaryocyte invagination system and podosomes.\",\n      \"method\": \"miR overexpression/knockdown, LCP1 knockdown and overexpression in MKs, in vivo miR inhibition (platelet count), luciferase reporter assay (miR-125a-5p targeting LCP1 3'UTR), PPF quantification\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct target validation by luciferase assay, gain- and loss-of-function with quantitative PPF readout, in vivo platelet count, multiple orthogonal approaches\",\n      \"pmids\": [\"32844999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"LFA-1 cluster formation (avidity regulation) in T cells depends on L-plastin Ser5 phosphorylation mediated by an nPKC-MEK-p90RSK pathway and counter-regulated by PP2A. Ser5 phosphorylation is required for LFA-1 clustering in high-affinity conformation; cofilin dephosphorylation (not LPL phosphorylation) mediates LFA-1 recruitment to the T-cell/APC contact zone—a distinct, dichotomic spatial regulation.\",\n      \"method\": \"Phospho-specific imaging, nPKC/MEK/p90RSK and PP2A inhibitors, phosphomutant expression, super-resolution microscopy of LFA-1 nanoclusters\",\n      \"journal\": \"Cellular and molecular life sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological pathway dissection with phosphomutant validation and super-resolution imaging, mechanistic dissociation of clustering vs. recruitment, single lab\",\n      \"pmids\": [\"33449151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PI3K/SGK3 pathway contributes to L-plastin Ser5 phosphorylation in breast cancer cells (in addition to ERK/MAPK-RSK), shown by computational modeling and experimental validation. Ser5 phosphorylation promotes L-plastin recruitment to invadopodia, MMP-9 activity, and ECM degradation.\",\n      \"method\": \"Computational network modeling, PI3K/SGK3 inhibition/overexpression, in vitro kinase validation, shRNA knockdown, invasion/migration assays, confocal imaging, gelatin degradation/zymography\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — computational model experimentally validated with kinase inhibition and functional assays, single lab\",\n      \"pmids\": [\"33618712\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LCP1 I232F missense mutation causes granulocytic dysplasia, reduced proliferation, G2/M cell cycle arrest, impaired cell motility/invasiveness, increased F-actin, and aberrant nuclear localization of the mutant protein (wild-type L-plastin is cytoplasmic). The mutation does not block differentiation or increase apoptosis.\",\n      \"method\": \"Lentiviral inducible expression of LCP1 I232F in 32D and HeLa cells, flow cytometry (cell cycle, F-actin), confocal imaging, subcellular fractionation, motility/invasion assays\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutant expression with subcellular fractionation confirming nuclear mislocalization and multiple functional readouts, single lab\",\n      \"pmids\": [\"34991157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"L-plastin (LPL) enhances NLRP3 inflammasome assembly by stabilizing ASC interactions with Pyk2 (a component of podosomes), enabling ASC oligomerization. LPL-/- alveolar macrophages exhibit reduced caspase-1 activity, IL-1β cleavage, and gasdermin-D processing upon NLRP3 activation; LPL-/- mice are resistant to bleomycin-induced lung fibrosis.\",\n      \"method\": \"LPL-/- mouse model, ASC oligomerization assay, co-immunoprecipitation (ASC-Pyk2-LPL), caspase-1 activity and IL-1β processing assays, bleomycin lung fibrosis model\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with Co-IP identifying ASC-Pyk2-LPL complex plus multiple NLRP3 functional readouts and in vivo disease model\",\n      \"pmids\": [\"35294888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"LCP1 splicing variant (c.740-1G>A) causes partial L-plastin deficiency resulting in lymphopenia, neutropenia, and thrombocytopenia. Functional analysis shows defective actin organization in T cells, reduced PBMC migration in response to CXCL12, impaired germinal center B-cell expansion, reduced T-cell cytokinesis, and aberrant platelet spreading on collagen.\",\n      \"method\": \"Human kindred genetic analysis, orthologous mouse knock-in, Jurkat cell mutation engineering, migration assays, flow cytometry, platelet spreading assay\",\n      \"journal\": \"The Journal of allergy and clinical immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human disease variant validated in orthologous mouse model and cell line with multiple functional immune cell readouts\",\n      \"pmids\": [\"38710235\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"L-plastin (LCP1) is a leukocyte-specific, calcium-regulated actin-bundling protein whose activity is controlled primarily by phosphorylation of Ser5 in its N-terminal headpiece—a modification catalyzed by PKA (downstream of FcγR/PI3K/cAMP), PKCβII (in eosinophils), PKCδ (in cancer cells), RSK1/2 (downstream of ERK/MAPK), SGK3 (downstream of PI3K), and Mst1 (on Thr89)—and inhibited by dexamethasone; Ser5 phosphorylation increases F-actin association rate and bundling activity by switching the protein to a high-affinity state, driving lamellipodia and podosome formation, LFA-1 clustering in the immunological synapse (through an LPL–Pyk2–ASC pathway also governing NLRP3 inflammasome assembly), integrin inside-out activation, osteoclast sealing-ring formation, and T cell, B cell, macrophage, and NK cell migration; additionally, L-plastin is reversibly inactivated by ROS-induced Cys101–Cys42 disulfide formation reversed by Thioredoxin1, and by S-glutathionylation of Cys460 in the actin-binding domain, and its calmodulin-binding EF-hand headpiece harbors a calcium-dependent switch helix that regulates bundling; ectopic expression and phosphorylation of L-plastin in non-hematopoietic tumor cells drives invadopodia-mediated ECM degradation, invasion, and metastasis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"L-plastin (LCP1) is a leukocyte-enriched, calcium-regulated actin-bundling protein that cross-links F-actin into the higher-order structures driving immune cell migration, adhesion, and invasion [#0, #1]. It binds laterally along the filament through two actin-binding domains of differing affinity, contacts beta-actin selectively, and bundles only at low free calcium (optimal below ~0.15 µM, progressively inhibited above), reflecting a calcium-dependent conformational switch in its EF-hand headpiece [#0, #1, #8]; an NMR structure localized this switch to a helix that binds the EF-hand motifs in the presence of calcium and governs bundling activity [#35]. The dominant activating modification is phosphorylation of Ser5 in the N-terminal headpiece, which shifts the protein to a high-affinity state—increasing the F-actin association rate roughly two-fold and lowering actin dissociation—and is sufficient to drive integrin inside-out activation and adhesion in neutrophils and monocytes [#4, #19, #11]. Ser5 is targeted by multiple kinases converging from distinct receptor inputs: PKA downstream of FcγR/PI3K/cAMP signaling [#5], PKCβII in GM-CSF-primed eosinophils [#23], RSK1/2 downstream of ERK/MAPK and SGK3 downstream of PI3K in cancer cells [#30, #43], and Mst1 on Thr89 [#32], while dexamethasone suppresses Ser5 phosphorylation to blunt T cell synapse maturation [#24]. Through this activity L-plastin builds and stabilizes lamellipodia, filopodia, and podosomes, sustains LFA-1 clustering and immunological synapse formation, and supports T, B, and macrophage migration and development, as defined by knockout, knockdown, and inhibitory-nanobody studies [#16, #17, #22, #34, #14]; it further promotes alveolar macrophage maturation and scaffolds an ASC–Pyk2 complex required for NLRP3 inflammasome assembly [#33, #45]. L-plastin activity is reversibly inactivated by redox modification—an ROS-induced Cys101–Cys42 disulfide reversed by the Thioredoxin1 system, and S-glutathionylation at Cys460 in the actin-binding domain—both of which reduce bundling and impair cell motility and invasion [#38, #29]. Ectopic expression and Ser5 phosphorylation of L-plastin in non-hematopoietic tumor cells drive invadopodia-mediated ECM degradation, invasion, and metastasis [#13, #28, #31]. Human LCP1 deficiency from a splice variant causes lymphopenia, neutropenia, and thrombocytopenia with defective actin organization and impaired immune cell migration, and an I232F missense mutation produces granulocytic dysplasia with aberrant nuclear mislocalization [#46, #44].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established the core biochemical activity: that L-plastin is a calcium-regulated actin cross-linker with defined isoform and stoichiometric specificity, distinguishing it from related bundlers.\",\n      \"evidence\": \"In vitro F-actin bundling and co-sedimentation assays with purified human T-cell L-plastin under defined calcium conditions\",\n      \"pmids\": [\"1491005\", \"8461306\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how calcium is sensed structurally\", \"Cellular triggers of bundling not addressed\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Connected L-plastin biochemistry to receptor signaling by showing it associates with the cytoskeleton and participates in FcR-mediated calcium responses in intact neutrophils.\",\n      \"evidence\": \"Chemical labeling (BPB) in intact cells, immunoprecipitation, cytoskeletal fractionation, and Ca2+ flux measurements\",\n      \"pmids\": [\"8170942\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking L-plastin to Ca2+ flux not defined\", \"No specific receptor identified\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Identified FcγRII as the upstream receptor and podosomes as a localization target, separating adhesion signaling from cytoskeletal rearrangement requirements.\",\n      \"evidence\": \"Receptor-blocking antibodies, LAD (CD18-null) patient cells, cytochalasin D, and immunofluorescence colocalization\",\n      \"pmids\": [\"8663066\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the kinase acting on L-plastin\", \"Role of L-plastin within podosomes unresolved\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined Ser5 phosphorylation as a sufficient molecular switch for integrin-mediated adhesion, shifting the model from passive bundler to actively regulated effector.\",\n      \"evidence\": \"Cell-permeable Ser5 and Ser5Ala peptides in primary human PMNs and monocytes with adhesion assays and PI3K/PKC inhibitors\",\n      \"pmids\": [\"9689080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the physiological Ser5 kinase\", \"Mechanism of integrin activation by the headpiece unresolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified PKA as a Ser5 kinase acting downstream of PI3K and FcγR-induced cAMP, providing the first complete signaling chain to L-plastin activation.\",\n      \"evidence\": \"In vitro PKA kinase assay on L-plastin peptides, cAMP analogs/forskolin, PKA and PI3K inhibitors, cAMP measurement\",\n      \"pmids\": [\"10446213\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Other receptors may use different kinases\", \"Direct structural consequence of Ser5 phosphorylation not shown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Began mapping L-plastin's protein and integrin interactions, showing Ca2+-favored binding to grancalcin and headpiece-driven activation of αvβ3 conformational change.\",\n      \"evidence\": \"Affinity chromatography and reciprocal Co-IP (grancalcin); cell-permeable headpiece peptides with LIBS conformational antibodies in K562 cells\",\n      \"pmids\": [\"11279160\", \"11278342\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of grancalcin binding not established\", \"Whether full-length protein recapitulates peptide effects unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined the actin-binding architecture—two ABDs of distinct affinity binding laterally along the filament—and identified phosphoinositides as inhibitors of actin binding.\",\n      \"evidence\": \"Recombinant domain expression, cross-linking, F-actin co-sedimentation, pyrenyl depolymerization, phosphoinositide competition; fMLP receptor pharmacology with MS\",\n      \"pmids\": [\"14992580\", \"14556648\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic contact map of ABD-actin interface not resolved\", \"Receptor-specific kinases for fMLP-induced phosphorylation not identified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated directly that Ser5 phosphorylation increases F-actin binding and targets L-plastin to peripheral protrusions, linking the modification to invasive behavior.\",\n      \"evidence\": \"In vitro F-actin binding assay plus Ser5Ala/Ser5Glu phosphovariant transfection in Vero/HEK293T cells and 3D collagen invasion assay\",\n      \"pmids\": [\"16636079\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address phosphorylation kinetics in living cells\", \"In vivo relevance not tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Extended the phospho-switch to functional immune and tumor outputs—T cell surface receptor transport and melanoma invasion/metastasis—showing expression and phosphorylation have separable roles.\",\n      \"evidence\": \"MS phosphosite mapping with 5A-LPL mutagenesis in primary T cells (CD25/CD69); siRNA and phosphomutant expression with Matrigel invasion and in vivo B16 metastasis\",\n      \"pmids\": [\"17294403\", \"17290393\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism coupling L-plastin to surface receptor transport unresolved\", \"Distinction between migration and invasion contributions incomplete\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Causally tied L-plastin bundling activity to filopodia formation using domain-specific intracellular nanobodies, and linked Ser5 phosphorylation to TNF-α death resistance.\",\n      \"evidence\": \"Intracellular nanobody expression with filopodia and bundling readouts; phosphomutant overexpression with apoptosis assays and PKC inhibitors\",\n      \"pmids\": [\"19726756\", \"19799649\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab nanobody tools\", \"Mechanism of death resistance not defined beyond phosphorylation dependence\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Multiple knockout and quantitative studies established L-plastin as essential for T cell synapse formation, LFA-1 clustering, chemokine-driven migration, integrin binding, and osteoclast structures, and quantified its kinetic effect on actin turnover.\",\n      \"evidence\": \"LPL-/- mice with synapse imaging, CCR7 polarization, two-photon node motility, EAE/allograft models; FRAP with modeling, PKCδ siRNA, cortactin Co-IP; integrin domain mapping with calpain; osteoclast imaging; NES analysis\",\n      \"pmids\": [\"21076065\", \"20683899\", \"20194718\", \"20169155\", \"20183869\", \"20650888\", \"15752138\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of distinct kinase inputs across cell types not unified\", \"Functional consequence of nucleocytoplasmic shuttling unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined L-plastin's roles in B cell development and eosinophil priming and identified cell-type-specific kinases (PKCβII) and signaling complexes (Pyk-2, GM-CSFRα, paxillin, cofilin).\",\n      \"evidence\": \"LPL-/- B cell migration and development assays with Pyk-2 phosphorylation; eosinophil phosphoproteomics, Co-IP, PKCβII siRNA, and synthetic phosphopeptide reconstitution\",\n      \"pmids\": [\"21832165\", \"21525390\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How distinct kinases are selected per receptor unresolved\", \"Stoichiometry of the eosinophil complex undefined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed dexamethasone acts through suppression of L-plastin Ser5 phosphorylation, and that L-plastin organizes leading-edge actin and Rac1/Akt signaling during chemotaxis via PKCζ.\",\n      \"evidence\": \"Dexamethasone with phosphomimetic rescue in primary T cells; siRNA knockdown with Rac1 pulldown, Akt immunoblot, PKC isoform inhibitors, and nanobody conformational trapping\",\n      \"pmids\": [\"21805466\", \"22581862\", \"23001012\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct kinase target of dexamethasone-regulated pathway not pinpointed\", \"Order of Rac1/Akt relative to L-plastin not fully resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established that L-plastin stabilizes podosomes and supports matrix degradation through its bundling activity independently of Ser5 phosphorylation status in macrophages.\",\n      \"evidence\": \"Intracellular nanobody expression with live podosome imaging, matrix degradation, and phospho-L-plastin immunostaining in THP-1 macrophages\",\n      \"pmids\": [\"24236012\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab nanobody approach\", \"Relationship between bundling and phosphorylation contributions at podosomes not reconciled\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated in vivo that both L-plastin expression and Ser5 phosphorylation are required for tumor growth and metastasis across prostate and melanoma models.\",\n      \"evidence\": \"shRNA knockdown and phosphorylatable vs. non-phosphorylatable ectopic expression with subcutaneous and intracardiac mouse metastasis models\",\n      \"pmids\": [\"24438191\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tumor-cell kinase driving Ser5 in vivo not identified here\", \"Downstream invasion machinery not mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified redox modification—S-glutathionylation at Cys460 in the ABD—as an inactivating regulatory layer, and RSK1/2 as direct ERK/MAPK-driven Ser5 kinases in cancer.\",\n      \"evidence\": \"Cys460 mutagenesis with bundling and neutrophil functional assays and diabetic patient samples; in vitro RSK1/2 kinase assay with siRNA/shRNA and invasion assays\",\n      \"pmids\": [\"25881549\", \"26631483\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzymes catalyzing/reversing glutathionylation not defined\", \"Interplay between phosphorylation and oxidation not addressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Resolved distinct biophysical roles versus fascin in invadopodia/filopodia, identified Mst1 as a Thr89 kinase, and placed L-plastin in alveolar macrophage maturation and podosome longevity.\",\n      \"evidence\": \"Nanobody inhibition with EM of bundle ultrastructure; in vitro Mst1 kinase assay, Co-IP, T89A rescue in LPL-/- mice; conditional macrophage knockout with PPAR-γ analysis; live podosome imaging in LPL-/- macrophages\",\n      \"pmids\": [\"26945069\", \"27465533\", \"27758872\", \"27614263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of Thr89 phosphorylation beyond localization unclear\", \"How L-plastin couples to PPAR-γ upregulation unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Provided a structural mechanism for calcium regulation by identifying a switch helix in the EF-hand headpiece that controls bundling activity.\",\n      \"evidence\": \"NMR solution structure of the EF-hand headpiece with competitive peptide inhibition of bundling\",\n      \"pmids\": [\"28145401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length protein bound to actin not determined\", \"How Ser5 phosphorylation intersects the switch helix unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended kinase inputs to LRRK1 in osteoclasts and characterized osteoclast bone-resorption roles via Ser5/Ser7-dependent sealing-zone formation.\",\n      \"evidence\": \"LC/MS phosphoproteomics and phospho-specific Western in Lrrk1-KO osteoclasts with micro-CT; TAT-peptide transduction with bone resorption assays\",\n      \"pmids\": [\"30136304\", \"30244178\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether LRRK1 phosphorylates L-plastin directly not shown\", \"Single-lab peptide competition for bone resorption readout\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined the Thioredoxin1-reversible Cys101–Cys42 disulfide as a localized redox switch and placed LCP1 upstream of mTORC2/AKT signaling in eosinophilic cells.\",\n      \"evidence\": \"TRX1 trapping/knockdown, auranofin, ratiometric LPL-roGFP-Orp1 imaging, proximity ligation, bundling and migration/invasion assays; shRNA knockdown with phospho-specific AKT/STAT immunoblots\",\n      \"pmids\": [\"31501427\", \"31691359\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger localizing oxidation to cell extrusions unresolved\", \"Mechanism linking LCP1 to mTORC2 activity undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined opposing osteoclast and megakaryocyte roles and identified upstream transcriptional (PI3K/AKT/SP1) and post-transcriptional (miR-125a-5p) control of LCP1 expression.\",\n      \"evidence\": \"LPL-KO mice with histomorphometry, filopodia/fusion and PDGF-BB assays, SP1 promoter studies; miR overexpression/knockdown with luciferase reporter and proplatelet formation quantification\",\n      \"pmids\": [\"33208358\", \"32844999\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How L-plastin negatively regulates the megakaryocyte invagination system unclear\", \"Coupling of PI3K/AKT/SP1 to RANKL kinetics incomplete\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Dissected spatial regulation of LFA-1 (Ser5-phosphorylation-dependent clustering via nPKC-MEK-p90RSK versus cofilin-mediated recruitment) and added the PI3K/SGK3 axis to cancer-cell Ser5 phosphorylation driving invadopodia and MMP-9-dependent degradation.\",\n      \"evidence\": \"Phospho-imaging, nPKC/MEK/p90RSK and PP2A inhibitors, phosphomutants, super-resolution microscopy; computational modeling with PI3K/SGK3 perturbation, kinase validation, and gelatin degradation/zymography\",\n      \"pmids\": [\"33449151\", \"33618712\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Integration of redundant Ser5 kinases in a single cell unresolved\", \"PP2A site specificity not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established L-plastin as an NLRP3 inflammasome scaffold via ASC-Pyk2 stabilization and linked human LCP1 mutations to defined hematologic/immunologic disease, including nuclear mislocalization of an I232F mutant.\",\n      \"evidence\": \"LPL-/- mice with ASC oligomerization, ASC-Pyk2-LPL Co-IP, caspase-1/IL-1β/gasdermin-D assays and bleomycin fibrosis; lentiviral I232F expression with fractionation and functional assays\",\n      \"pmids\": [\"35294888\", \"34991157\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab characterization of I232F mutant\", \"How the inflammasome scaffolding role relates to actin bundling unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrated that partial human L-plastin deficiency causes a multilineage cytopenia syndrome, defining a Mendelian disease link.\",\n      \"evidence\": \"Human kindred genetic analysis with orthologous mouse knock-in and Jurkat engineering, migration, flow cytometry, and platelet spreading assays\",\n      \"pmids\": [\"38710235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genotype-phenotype spectrum across additional patients not established\", \"Tissue-specific molecular basis of each cytopenia incomplete\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many redundant and cell-type-specific Ser5 kinases, the Thr89 input, calcium switching, and redox modifications are integrated to set L-plastin bundling activity in a given physiological context remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model coupling phosphorylation, calcium, and redox states\", \"No full-length actin-bound structure\", \"Quantitative hierarchy of kinase inputs per cell type unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 8, 11, 14, 19, 35]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [20, 45, 23]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 7, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [2, 3, 11, 19, 27, 34]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10, 44]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [10, 44]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [11, 17, 25]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [16, 17, 22, 23, 45]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [13, 28, 31, 44, 46]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [5, 9, 25, 30, 42, 43]}\n    ],\n    \"complexes\": [\"NLRP3 inflammasome (ASC-Pyk2-LPL scaffold)\", \"podosome\", \"invadopodia\", \"immunological synapse\"],\n    \"partners\": [\"ACTB\", \"GCA\", \"ITGB1\", \"ITGB2\", \"CTTN\", \"PYCARD\", \"PTK2B\", \"PRKCB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}