{"gene":"MARCKSL1","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1992,"finding":"MacMARCKS (MARCKSL1) is a PKC substrate that binds calmodulin in a phosphorylation-regulated manner; phosphorylation by PKC disrupts calmodulin binding. It also binds actin and shares structural homology with MARCKS including an amino-terminal myristoylation sequence and a central effector/calmodulin-binding/PKC phosphorylation domain.","method":"Protein purification, cDNA cloning, calmodulin binding assays, PKC phosphorylation assays","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution with purified protein, replicated across two contemporaneous labs (PMID:1516135 and PMID:1618855)","pmids":["1516135","1618855"],"is_preprint":false},{"year":1992,"finding":"The F52/MARCKSL1 protein is myristoylated at its N-terminus; it is a PKC substrate with high-affinity calmodulin binding (Kd <3 nM) that is disrupted upon PKC phosphorylation. A 24-amino acid peptide from the effector domain recapitulated these properties (S0.5 for PKC = 173 nM, KH = 5.4).","method":"E. coli expression, co-expression with N-myristoyltransferase, PKC phosphorylation assay, calmodulin binding assay, peptide substrate kinetics","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical assays with defined kinetic parameters, replicated by independent lab","pmids":["1618855"],"is_preprint":false},{"year":1996,"finding":"Deletion of the MacMARCKS gene in mice prevents cranial neural tube closure, resulting in anencephaly, demonstrating an essential role for MARCKSL1 in PKC-dependent actin-based morphogenic movement of the anterior neural plate.","method":"Gene knockout (homologous recombination), developmental phenotypic analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent knockout mouse studies (PMID:8692805 and PMID:8700893) replicate neural tube defect phenotype","pmids":["8692805"],"is_preprint":false},{"year":1996,"finding":"F52/MARCKSL1-deficient mice develop neural tube defects (exencephaly and spina bifida) with partial penetrance (~60% homozygous), and ~10% of heterozygotes also show defects, establishing MARCKSL1 as haploinsufficient for neural tube closure.","method":"Gene targeting/knockout in mice, developmental phenotype analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — independent replication of KO phenotype by a second lab, consistent with PMID:8692805","pmids":["8700893"],"is_preprint":false},{"year":1995,"finding":"MacMARCKS concentrates around nascent phagosomes in macrophages during zymosan phagocytosis; expression of effector-domain deletion mutants reduced phagocytic capacity by ~90% without affecting receptor-mediated endocytosis of acetylated LDL, implicating the effector domain in phagocytosis.","method":"Immunofluorescence microscopy, stable transfection of effector-domain deletion mutants, phagocytosis assay, endocytosis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — dominant-negative mutant approach with functional readout; subsequent KO study (PMID:9837946) found normal phagocytosis, creating conflicting evidence","pmids":["7629059"],"is_preprint":false},{"year":1998,"finding":"MacMARCKS null macrophages phagocytose and spread normally, indicating that MacMARCKS is recruited to phagosomes but is not absolutely required for phagocytosis, contradicting findings from dominant-negative mutant studies.","method":"MacMARCKS null mouse macrophages (KO), phagocytosis assay, cell spreading assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic null (KO) provides cleaner evidence than dominant-negative; directly contradicts PMID:7629059","pmids":["9837946"],"is_preprint":false},{"year":1996,"finding":"MacMARCKS participates in integrin-dependent macrophage spreading and phorbol ester-stimulated spreading requiring multiple integrins; dominant-negative effector-domain mutant blocks macrophage binding to iC3b-opsonized targets (complement receptor 3/beta2 integrin), integrin-dependent paxillin tyrosine phosphorylation, and colocalizes with paxillin at leading-edge membrane ruffles.","method":"Dominant-negative mutant expression, cell spreading assay, rosette formation assay, immunofluorescence colocalization, paxillin phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — dominant-negative approach with multiple functional readouts in single lab","pmids":["8662782"],"is_preprint":false},{"year":1996,"finding":"MacMARCKS is phosphorylated in a Ca2+-dependent manner upon depolarization in PC12 cells and synaptosomes; it colocalizes with synaptophysin at neurite tips and associates with synaptic vesicles by subcellular fractionation, consistent with a role in integrating Ca2+/calmodulin and PKC signals in neurosecretion.","method":"Immunoprecipitation, immunofluorescence microscopy, subcellular fractionation, Percoll-purified synaptosomes, KCl depolarization, phorbol ester stimulation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation and localization with functional stimulus context; single lab","pmids":["8557647"],"is_preprint":false},{"year":1998,"finding":"MacMARCKS is targeted specifically to the basolateral membrane domain of polarized MDCK cells; the effector domain (24-amino acid basic region with PKC phosphorylation and calmodulin/actin-binding sites) combined with a myristoyl moiety is sufficient for basolateral targeting, and PKC activation displaces it from this location.","method":"Transfection into polarized MDCK cells, immunofluorescence microscopy, GFP-fusion domain targeting experiments, PKC activation","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain dissection with GFP fusion constructs in polarized cells, single lab","pmids":["9637918"],"is_preprint":false},{"year":1999,"finding":"Phosphorylated MacMARCKS is required for LFA-1 (beta2 integrin)-mediated cell-cell adhesion in U937 monocytic cells; phosphomimetic (phosphorylated) MacMARCKS enhances adhesion while unphosphorylatable mutant inhibits it, demonstrating that PKC-mediated phosphorylation state determines integrin activation.","method":"Transfection of wild-type and phosphorylation-site mutants, PMA stimulation, cell-cell adhesion assay, okadaic acid phosphatase inhibition","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — phospho-mutant approach with functional adhesion readout, single lab","pmids":["10497314"],"is_preprint":false},{"year":2001,"finding":"MacMARCKS interacts with dynamitin (a dynactin complex subunit) in living cells; interaction is concentrated at the cell periphery in resting macrophages and is lost upon PMA stimulation as both proteins redistribute to perinuclear regions, linking MacMARCKS to microtubule motor-dependent functions.","method":"FRET (CFP/YFP fusion proteins), in vitro pulldown, live-cell imaging in RAW macrophages and HEK293 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — FRET demonstrates in vivo interaction with spatial/temporal resolution; single lab, two orthogonal methods","pmids":["11278693"],"is_preprint":false},{"year":2002,"finding":"MacMARCKS interacts with the C-terminal cytoplasmic tail of the serotonin transporter (SERT) as identified by yeast two-hybrid and confirmed by co-transfection; MacMARCKS co-expression reduces maximal 5-HT uptake rate and attenuates PKC-induced SERT downregulation.","method":"Yeast two-hybrid screen, co-transfection in HEK293 cells, [3H]serotonin uptake assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid plus functional uptake assay; single lab","pmids":["12051706"],"is_preprint":false},{"year":2006,"finding":"MacMARCKS binds to the cytoplasmic C-terminal tail of metabotropic glutamate receptor 7 (mGluR7); binding is antagonized by Ca2+/calmodulin. Co-transfection of MacMARCKS with mGluR7 reduces G-protein-mediated tonic inhibition of voltage-sensitive Ca2+ channels (VSCCs), an effect dependent on MacMARCKS–mGluR7 interaction.","method":"Yeast two-hybrid, in vitro pulldown, co-immunoprecipitation, colocalization in transfected HEK293 and cerebellar granule cells, electrophysiology (VSCC inhibition)","journal":"Journal of neurochemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Y2H, pulldown, co-IP, colocalization, functional electrophysiology), single lab","pmids":["16987251"],"is_preprint":false},{"year":2012,"finding":"JNK directly phosphorylates MARCKSL1 on C-terminal residues S120, T148, and T183. Phosphorylation by JNK enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers/dynamics, and retard cell migration. Conversely, non-phosphorylatable MARCKSL1 enhances lamellipodia formation and cell migration while reducing filopodia. This mechanism operates in neurons and prostate cancer cells.","method":"In vitro kinase assay (JNK phosphorylation), site-directed mutagenesis (S120A/T148A/T183A and S120D/T148D/T183D), F-actin bundling/stabilization assay, live-cell imaging of filopodia/lamellipodia, migration assay (neurons and prostate cancer cells), siRNA knockdown","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus multiple functional readouts across two cell types in single rigorous study","pmids":["22751924"],"is_preprint":false},{"year":2014,"finding":"LOXL2 interacts with MARCKSL1 via its scavenger-receptor domain binding to the N-terminal domain of MARCKSL1; LOXL2 promotes cell proliferation by inhibiting MARCKSL1-induced apoptosis, and MARCKSL1 suppresses LOXL2-induced oncogenesis and reduces luciferase reporter activity in a dose-dependent manner.","method":"Co-immunoprecipitation, domain-mapping pulldown assays, luciferase reporter assay, flow cytometry (cell cycle/apoptosis), siRNA knockdown","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP with domain mapping plus functional assays; single lab","pmids":["24863880"],"is_preprint":false},{"year":2015,"finding":"MARCKSL1 suppresses angiogenesis by interacting with VEGFR-2 and inhibiting VEGF-induced phosphorylation of VEGFR-2 and downstream PI3K/Akt/PDK-1/mTOR signaling; it reduces VEGF-induced HUVEC proliferation and tubular structure formation in vitro, and decreases HIF-1α and VEGF expression.","method":"HUVEC proliferation assay, tube formation assay, Western blot for VEGFR-2 phosphorylation and downstream kinase phosphorylation, MARCKSL1 overexpression","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — functional assays with signaling readouts; interaction with VEGFR-2 inferred from phosphorylation data without direct binding assay; single lab","pmids":["26555156"],"is_preprint":false},{"year":2018,"finding":"MARCKSL1 overexpression in the amygdala increases dendritic spine formation in the central amygdala and elevates HPA axis activity and anxiety-like behaviors; knockdown of MARCKSL1 specifically in the amygdala normalizes both HPA axis activity and anxiety behaviors in transgenic mice.","method":"MARCKSL1 transgenic mice, site-specific knockdown in amygdala, behavioral assays (anxiety), spine morphology analysis, corticotropin-releasing hormone dependence assay","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — region-specific KD with behavioral and morphological readouts; single lab","pmids":["29580842"],"is_preprint":false},{"year":2019,"finding":"VPA-induced transcriptional downregulation of MARCKSL1 (an actin-stabilizing protein) underlies impairment of dendritic morphology and functional properties in developing but not mature human neurons; these effects are mediated through HDAC and GSK-3 pathway inhibition.","method":"Direct reprogramming of human neurons, VPA treatment, RNA sequencing, dendritic morphology quantification, functional electrophysiology, pathway inhibitor experiments","journal":"Cell stem cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function via VPA/pathway inhibition linked to MARCKSL1 downregulation with defined morphological and functional phenotype; single lab","pmids":["31155484"],"is_preprint":false},{"year":2020,"finding":"Marcksl1 modulates the mechanical properties of the endothelial cell cortex to regulate cell shape and vessel diameter during angiogenesis. Overexpression increases EC size, microvessel diameter, and induces ectopic blebbing suppressed by reduced blood flow; Marcksl1 promotes formation of linear actin bundles and decreases branched actin density at the cortex.","method":"In vivo overexpression/depletion in zebrafish, high-resolution live imaging, actin network analysis, microvessel diameter quantification, blood flow manipulation","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo gain- and loss-of-function with quantitative structural and functional readouts; multiple orthogonal approaches including imaging and flow manipulation","pmids":["33127887"],"is_preprint":false},{"year":2022,"finding":"MARCKSL1 promotes esophageal squamous cell carcinoma cell migration and invasion by interacting with F-actin and cortactin to regulate invadopodia formation and extracellular matrix degradation.","method":"siRNA knockdown and overexpression, immunofluorescence colocalization of F-actin and cortactin, gelatin degradation assay, Transwell/wound-healing assays, RNA sequencing","journal":"Cancer medicine","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — colocalization and functional assays; interaction inferred from colocalization rather than direct binding assay; single lab","pmids":["35894387"],"is_preprint":false},{"year":2023,"finding":"Endothelial JNK1 (but not JNK2) is activated by neutrophil adhesion and phosphorylates MARCKSL1 to promote formation of apical filopodia; these filopodia facilitate adhesion of secondary neutrophils and establish transmigration hotspots. Chemical JNK inhibition prevents neutrophil adhesion.","method":"Kinase translocation reporters, FRET-based Cdc42 biosensors, JNK1/JNK2 specific inhibition/knockdown, MARCKSL1 knockdown, live-cell imaging of filopodia, neutrophil adhesion assay","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — JNK1-specific activation linked to MARCKSL1-dependent filopodia with functional readout; single lab, multiple biosensor approaches","pmids":["37559902"],"is_preprint":false},{"year":2025,"finding":"MARCKSL1 promotes EV secretion from the plasma membrane (in part at the expense of late endosome-PM fusion); MARCKSL1 collaborates with PM-bridging cytoskeletal components (e.g., Radixin) and SNARE-associated proteins (e.g., STXBP3) in this process, as identified by CRISPR activation screen and genomic activation/ablation with microscopic and proteomic follow-up.","method":"Genome-wide CRISPR activation screen (CD63 surface levels), genomic activation/ablation, electron microscopy, proteomics, co-immunoprecipitation/interaction studies with Radixin and STXBP3","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — unbiased genetic screen plus orthogonal microscopic and proteomic validation; preprint, not yet peer-reviewed","pmids":["bio_10.1101_2025.07.24.665424"],"is_preprint":true},{"year":2026,"finding":"MARCKSL1 expression in dendritic cells is upregulated by TGF-β1 and is required for dendritic cell-mediated promotion of fibroblast differentiation into myofibroblasts during wound healing; MARCKSL1 shRNA knockdown in dendritic cells diminishes their ability to induce myofibroblast differentiation, and systemic MANS peptide inhibition attenuates scar formation in vivo.","method":"TGF-β1 stimulation of mouse dendritic cells, MARCKSL1 shRNA knockdown, co-culture with fibroblasts, myofibroblast differentiation assay, MANS peptide treatment in mouse wound model, single-cell RNA sequencing","journal":"Wound repair and regeneration","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with functional cellular readout and in vivo validation; single lab","pmids":["41782175"],"is_preprint":false}],"current_model":"MARCKSL1 (MacMARCKS/F52) is a myristoylated, peripherally membrane-associated PKC substrate whose basic effector domain binds F-actin and calmodulin in a phosphorylation-regulated manner; it integrates PKC and Ca2+/calmodulin signals to remodel cortical actin, controlling filopodium versus lamellipodium balance downstream of JNK-mediated phosphorylation (S120/T148/T183), is essential for cranial neural tube closure and dendritic morphogenesis, regulates endothelial cell cortical mechanics and vessel shape in response to hemodynamic forces, modulates integrin activation and neurotransmitter transporter trafficking, interacts with mGluR7 to control VSCC tonic inhibition, and promotes plasma membrane-derived extracellular vesicle secretion in collaboration with Radixin and STXBP3."},"narrative":{"mechanistic_narrative":"MARCKSL1 (MacMARCKS/F52) is a myristoylated, peripherally membrane-associated PKC substrate that integrates protein kinase C and Ca2+/calmodulin signals to remodel the cortical actin cytoskeleton [PMID:1516135, PMID:1618855]. Through a central basic effector domain it binds calmodulin with high affinity and binds and bundles F-actin, with PKC phosphorylation disrupting calmodulin binding; together with its N-terminal myristoylation this effector domain directs the protein to specific membrane domains and confers its actin-regulatory activity [PMID:1516135, PMID:1618855, PMID:9637918]. JNK directly phosphorylates MARCKSL1 at S120/T148/T183, switching it toward F-actin bundling and stabilization that increases filopodia and retards migration, whereas non-phosphorylatable protein favors lamellipodia and motility [PMID:22751924]. This actin-shaping function operates across contexts: it is genetically essential for cranial neural tube closure, where its loss causes neural tube defects and it behaves as haploinsufficient [PMID:8692805, PMID:8700893]; it governs dendritic spine and dendrite morphology in neurons with consequences for anxiety behavior and HPA axis activity [PMID:29580842, PMID:31155484]; and it controls endothelial cortical mechanics, vessel diameter, and JNK1-driven apical filopodia that establish neutrophil transmigration hotspots [PMID:33127887, PMID:37559902]. MARCKSL1 also modulates membrane signaling and trafficking, binding the C-terminal tails of the serotonin transporter and metabotropic glutamate receptor 7 to regulate transport activity and VSCC tonic inhibition in a calmodulin-antagonized manner [PMID:12051706, PMID:16987251], and influencing integrin-dependent adhesion in a phosphorylation-state-dependent fashion [PMID:10497314]. In cancer it promotes invadopodia formation and matrix degradation via F-actin and cortactin [PMID:35894387].","teleology":[{"year":1992,"claim":"Established the core biochemical identity of MARCKSL1 as a myristoylated PKC substrate whose effector domain reciprocally couples calmodulin binding and actin binding to phosphorylation state, defining the molecular switch underlying all later functions.","evidence":"Protein purification, cDNA cloning, in vitro PKC phosphorylation and calmodulin/actin binding assays with defined kinetics, replicated across two labs","pmids":["1516135","1618855"],"confidence":"High","gaps":["Did not establish which cellular processes the actin/calmodulin switch controls","No structural model of the membrane-bound effector domain"]},{"year":1996,"claim":"Demonstrated that MARCKSL1 is genetically essential in vivo, with knockout causing failure of cranial neural tube closure and dose-sensitive (haploinsufficient) penetrance, linking its actin function to morphogenetic movement.","evidence":"Independent gene knockout mouse studies with developmental phenotyping","pmids":["8692805","8700893"],"confidence":"High","gaps":["Did not resolve which cell movements at the neural plate require MARCKSL1","Phosphorylation-dependence of the developmental role untested at the time"]},{"year":1998,"claim":"Resolved a conflict over MARCKSL1's role in phagocytosis: dominant-negative effector-domain mutants implicated it, but genetic null macrophages phagocytose and spread normally, showing it is recruited but not absolutely required.","evidence":"Comparison of dominant-negative mutant phenotypes versus MacMARCKS null macrophage phagocytosis and spreading assays","pmids":["9837946"],"confidence":"High","gaps":["Possible redundancy with MARCKS not directly tested","Why dominant-negative and null phenotypes diverge mechanistically unresolved"]},{"year":1998,"claim":"Showed that the myristoyl moiety plus basic effector domain are sufficient for targeting MARCKSL1 to a specific (basolateral) membrane domain and that PKC activation displaces it, mechanistically connecting phosphorylation to membrane localization.","evidence":"GFP-fusion domain dissection in polarized MDCK cells with PKC activation","pmids":["9637918"],"confidence":"Medium","gaps":["Single lab","Targeting determinants in non-epithelial cells not addressed"]},{"year":2006,"claim":"Identified direct receptor-tail binding partners (SERT, mGluR7) showing MARCKSL1 modulates membrane transporter/receptor function, with mGluR7 binding antagonized by Ca2+/calmodulin and controlling VSCC tonic inhibition.","evidence":"Yeast two-hybrid, pulldown, co-IP, colocalization, transporter uptake and electrophysiology assays","pmids":["16987251","12051706"],"confidence":"High","gaps":["Physiological relevance of these interactions in vivo not established","Stoichiometry and regulation by PKC phosphorylation incompletely defined"]},{"year":2012,"claim":"Defined the JNK-MARCKSL1 axis: direct JNK phosphorylation at S120/T148/T183 switches MARCKSL1 toward F-actin bundling/stabilization that builds filopodia and slows migration, while the unphosphorylatable form drives lamellipodia and motility.","evidence":"In vitro JNK kinase assay, phosphosite mutagenesis, F-actin bundling assays, live imaging and migration assays in neurons and prostate cancer cells","pmids":["22751924"],"confidence":"High","gaps":["Upstream signals activating JNK toward MARCKSL1 not delineated here","Relationship between JNK sites and PKC/calmodulin switch unresolved"]},{"year":2020,"claim":"Extended the actin function to tissue mechanics, showing MARCKSL1 sets endothelial cortical stiffness by promoting linear over branched actin, thereby controlling cell size, vessel diameter, and flow-dependent blebbing.","evidence":"Zebrafish in vivo gain/loss-of-function, high-resolution imaging, actin network analysis, blood flow manipulation","pmids":["33127887"],"confidence":"High","gaps":["Molecular basis for linear vs branched actin preference not defined","Link to JNK phosphorylation in this context not tested"]},{"year":2023,"claim":"Connected upstream signaling to physiology, showing endothelial JNK1 (not JNK2) phosphorylates MARCKSL1 to form apical filopodia that establish neutrophil transmigration hotspots.","evidence":"Kinase translocation reporters, Cdc42 FRET biosensors, JNK1/2-specific perturbation, MARCKSL1 knockdown, neutrophil adhesion assays","pmids":["37559902"],"confidence":"Medium","gaps":["Single lab","Whether the same S120/T148/T183 sites mediate this remains inferential"]},{"year":2025,"claim":"Implicated MARCKSL1 in plasma-membrane-derived extracellular vesicle secretion in collaboration with Radixin and STXBP3, expanding its role from cortical actin to membrane budding/trafficking.","evidence":"Genome-wide CRISPR activation screen, genomic activation/ablation, electron microscopy, proteomics, interaction studies (preprint)","pmids":["bio_10.1101_2025.07.24.665424"],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Direct binding to Radixin/STXBP3 versus pathway co-dependence not fully separated"]},{"year":null,"claim":"How the JNK (S120/T148/T183), PKC, and Ca2+/calmodulin inputs are integrated on a single effector domain to select between filopodia, lamellipodia, cortical stiffening, and membrane trafficking outputs remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural/biophysical model reconciling the three phosphoregulatory inputs","Tissue-specific partner sets that bias output are incompletely mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,13,19]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[11,12,9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[8,18,21]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[13,18,19]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,3,18]}],"complexes":[],"partners":["CALM1","SLC6A4","GRM7","CTTN","RDX","STXBP3","LOXL2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P49006","full_name":"MARCKS-related protein","aliases":["MARCKS-like protein 1","Macrophage myristoylated alanine-rich C kinase substrate","Mac-MARCKS","MacMARCKS"],"length_aa":195,"mass_kda":19.5,"function":"Controls cell movement by regulating actin cytoskeleton homeostasis and filopodium and lamellipodium formation (PubMed:22751924). When unphosphorylated, induces cell migration (By similarity). When phosphorylated by MAPK8, induces actin bundles formation and stabilization, thereby reducing actin plasticity, hence restricting cell movement, including neuronal migration (By similarity). May be involved in coupling the protein kinase C and calmodulin signal transduction systems (By similarity)","subcellular_location":"Cytoplasm, cytoskeleton; Cell membrane","url":"https://www.uniprot.org/uniprotkb/P49006/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MARCKSL1","classification":"Not Classified","n_dependent_lines":33,"n_total_lines":1208,"dependency_fraction":0.027317880794701987},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CCAR1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MARCKSL1","total_profiled":1310},"omim":[{"mim_id":"602940","title":"MARCKS-LIKE PROTEIN 1; MARCKSL1","url":"https://www.omim.org/entry/602940"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Endoplasmic reticulum","reliability":"Additional"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Mitotic spindle","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":1097.7}],"url":"https://www.proteinatlas.org/search/MARCKSL1"},"hgnc":{"alias_symbol":["F52","MacMARCKS","MLP1"],"prev_symbol":["MLP"]},"alphafold":{"accession":"P49006","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P49006","model_url":"https://alphafold.ebi.ac.uk/files/AF-P49006-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P49006-F1-predicted_aligned_error_v6.png","plddt_mean":54.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MARCKSL1","jax_strain_url":"https://www.jax.org/strain/search?query=MARCKSL1"},"sequence":{"accession":"P49006","fasta_url":"https://rest.uniprot.org/uniprotkb/P49006.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P49006/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P49006"}},"corpus_meta":[{"pmid":"1516135","id":"PMC_1516135","title":"MacMARCKS, a novel member of the MARCKS family of protein kinase C substrates.","date":"1992","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/1516135","citation_count":117,"is_preprint":false},{"pmid":"8692805","id":"PMC_8692805","title":"Disruption of the MacMARCKS gene prevents cranial neural tube closure and results in anencephaly.","date":"1996","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8692805","citation_count":81,"is_preprint":false},{"pmid":"8700893","id":"PMC_8700893","title":"Neural tube defects and abnormal brain development in F52-deficient mice.","date":"1996","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8700893","citation_count":79,"is_preprint":false},{"pmid":"22751924","id":"PMC_22751924","title":"c-Jun N-terminal kinase phosphorylation of MARCKSL1 determines actin stability and migration in neurons and in cancer cells.","date":"2012","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/22751924","citation_count":76,"is_preprint":false},{"pmid":"1618855","id":"PMC_1618855","title":"Characteristics of the F52 protein, a MARCKS homologue.","date":"1992","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/1618855","citation_count":70,"is_preprint":false},{"pmid":"7629059","id":"PMC_7629059","title":"MacMARCKS mutation blocks macrophage phagocytosis of zymosan.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7629059","citation_count":55,"is_preprint":false},{"pmid":"35864549","id":"PMC_35864549","title":"MARCKSL1-2 reverses docetaxel-resistance of lung adenocarcinoma cells by recruiting SUZ12 to suppress HDAC1 and elevate miR-200b.","date":"2022","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/35864549","citation_count":48,"is_preprint":false},{"pmid":"8662782","id":"PMC_8662782","title":"Role of MacMARCKS in integrin-dependent macrophage spreading and tyrosine phosphorylation of paxillin.","date":"1996","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/8662782","citation_count":45,"is_preprint":false},{"pmid":"31155484","id":"PMC_31155484","title":"Direct Reprogramming of Human Neurons Identifies MARCKSL1 as a Pathogenic Mediator of Valproic Acid-Induced Teratogenicity.","date":"2019","source":"Cell stem cell","url":"https://pubmed.ncbi.nlm.nih.gov/31155484","citation_count":45,"is_preprint":false},{"pmid":"12051706","id":"PMC_12051706","title":"Interaction of the C-terminal region of the rat serotonin transporter with MacMARCKS modulates 5-HT uptake regulation by protein kinase C.","date":"2002","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12051706","citation_count":38,"is_preprint":false},{"pmid":"32194726","id":"PMC_32194726","title":"MARCKSL1 promotes the proliferation, migration and invasion of lung adenocarcinoma cells.","date":"2020","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/32194726","citation_count":33,"is_preprint":false},{"pmid":"24863880","id":"PMC_24863880","title":"Lysyl oxidase-like 2 (LOXL2) controls tumor-associated cell proliferation through the interaction with MARCKSL1.","date":"2014","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/24863880","citation_count":33,"is_preprint":false},{"pmid":"9837946","id":"PMC_9837946","title":"MacMARCKS is not essential for phagocytosis in macrophages.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9837946","citation_count":30,"is_preprint":false},{"pmid":"33127887","id":"PMC_33127887","title":"Marcksl1 modulates endothelial cell mechanoresponse to haemodynamic forces to control blood vessel shape and size.","date":"2020","source":"Nature 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samples","date":"2025-01-01","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.31.630863","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":19782,"output_tokens":5738,"usd":0.072708,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":14264,"output_tokens":3370,"usd":0.077785,"stage2_stop_reason":"end_turn"},"total_usd":0.150493,"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\": \"MacMARCKS (MARCKSL1) is a PKC substrate that binds calmodulin in a phosphorylation-regulated manner; phosphorylation by PKC disrupts calmodulin binding. It also binds actin and shares structural homology with MARCKS including an amino-terminal myristoylation sequence and a central effector/calmodulin-binding/PKC phosphorylation domain.\",\n      \"method\": \"Protein purification, cDNA cloning, calmodulin binding assays, PKC phosphorylation assays\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution with purified protein, replicated across two contemporaneous labs (PMID:1516135 and PMID:1618855)\",\n      \"pmids\": [\"1516135\", \"1618855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"The F52/MARCKSL1 protein is myristoylated at its N-terminus; it is a PKC substrate with high-affinity calmodulin binding (Kd <3 nM) that is disrupted upon PKC phosphorylation. A 24-amino acid peptide from the effector domain recapitulated these properties (S0.5 for PKC = 173 nM, KH = 5.4).\",\n      \"method\": \"E. coli expression, co-expression with N-myristoyltransferase, PKC phosphorylation assay, calmodulin binding assay, peptide substrate kinetics\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical assays with defined kinetic parameters, replicated by independent lab\",\n      \"pmids\": [\"1618855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Deletion of the MacMARCKS gene in mice prevents cranial neural tube closure, resulting in anencephaly, demonstrating an essential role for MARCKSL1 in PKC-dependent actin-based morphogenic movement of the anterior neural plate.\",\n      \"method\": \"Gene knockout (homologous recombination), developmental phenotypic analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent knockout mouse studies (PMID:8692805 and PMID:8700893) replicate neural tube defect phenotype\",\n      \"pmids\": [\"8692805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"F52/MARCKSL1-deficient mice develop neural tube defects (exencephaly and spina bifida) with partial penetrance (~60% homozygous), and ~10% of heterozygotes also show defects, establishing MARCKSL1 as haploinsufficient for neural tube closure.\",\n      \"method\": \"Gene targeting/knockout in mice, developmental phenotype analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — independent replication of KO phenotype by a second lab, consistent with PMID:8692805\",\n      \"pmids\": [\"8700893\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"MacMARCKS concentrates around nascent phagosomes in macrophages during zymosan phagocytosis; expression of effector-domain deletion mutants reduced phagocytic capacity by ~90% without affecting receptor-mediated endocytosis of acetylated LDL, implicating the effector domain in phagocytosis.\",\n      \"method\": \"Immunofluorescence microscopy, stable transfection of effector-domain deletion mutants, phagocytosis assay, endocytosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — dominant-negative mutant approach with functional readout; subsequent KO study (PMID:9837946) found normal phagocytosis, creating conflicting evidence\",\n      \"pmids\": [\"7629059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"MacMARCKS null macrophages phagocytose and spread normally, indicating that MacMARCKS is recruited to phagosomes but is not absolutely required for phagocytosis, contradicting findings from dominant-negative mutant studies.\",\n      \"method\": \"MacMARCKS null mouse macrophages (KO), phagocytosis assay, cell spreading assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic null (KO) provides cleaner evidence than dominant-negative; directly contradicts PMID:7629059\",\n      \"pmids\": [\"9837946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"MacMARCKS participates in integrin-dependent macrophage spreading and phorbol ester-stimulated spreading requiring multiple integrins; dominant-negative effector-domain mutant blocks macrophage binding to iC3b-opsonized targets (complement receptor 3/beta2 integrin), integrin-dependent paxillin tyrosine phosphorylation, and colocalizes with paxillin at leading-edge membrane ruffles.\",\n      \"method\": \"Dominant-negative mutant expression, cell spreading assay, rosette formation assay, immunofluorescence colocalization, paxillin phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — dominant-negative approach with multiple functional readouts in single lab\",\n      \"pmids\": [\"8662782\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"MacMARCKS is phosphorylated in a Ca2+-dependent manner upon depolarization in PC12 cells and synaptosomes; it colocalizes with synaptophysin at neurite tips and associates with synaptic vesicles by subcellular fractionation, consistent with a role in integrating Ca2+/calmodulin and PKC signals in neurosecretion.\",\n      \"method\": \"Immunoprecipitation, immunofluorescence microscopy, subcellular fractionation, Percoll-purified synaptosomes, KCl depolarization, phorbol ester stimulation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation and localization with functional stimulus context; single lab\",\n      \"pmids\": [\"8557647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"MacMARCKS is targeted specifically to the basolateral membrane domain of polarized MDCK cells; the effector domain (24-amino acid basic region with PKC phosphorylation and calmodulin/actin-binding sites) combined with a myristoyl moiety is sufficient for basolateral targeting, and PKC activation displaces it from this location.\",\n      \"method\": \"Transfection into polarized MDCK cells, immunofluorescence microscopy, GFP-fusion domain targeting experiments, PKC activation\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain dissection with GFP fusion constructs in polarized cells, single lab\",\n      \"pmids\": [\"9637918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Phosphorylated MacMARCKS is required for LFA-1 (beta2 integrin)-mediated cell-cell adhesion in U937 monocytic cells; phosphomimetic (phosphorylated) MacMARCKS enhances adhesion while unphosphorylatable mutant inhibits it, demonstrating that PKC-mediated phosphorylation state determines integrin activation.\",\n      \"method\": \"Transfection of wild-type and phosphorylation-site mutants, PMA stimulation, cell-cell adhesion assay, okadaic acid phosphatase inhibition\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — phospho-mutant approach with functional adhesion readout, single lab\",\n      \"pmids\": [\"10497314\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"MacMARCKS interacts with dynamitin (a dynactin complex subunit) in living cells; interaction is concentrated at the cell periphery in resting macrophages and is lost upon PMA stimulation as both proteins redistribute to perinuclear regions, linking MacMARCKS to microtubule motor-dependent functions.\",\n      \"method\": \"FRET (CFP/YFP fusion proteins), in vitro pulldown, live-cell imaging in RAW macrophages and HEK293 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — FRET demonstrates in vivo interaction with spatial/temporal resolution; single lab, two orthogonal methods\",\n      \"pmids\": [\"11278693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"MacMARCKS interacts with the C-terminal cytoplasmic tail of the serotonin transporter (SERT) as identified by yeast two-hybrid and confirmed by co-transfection; MacMARCKS co-expression reduces maximal 5-HT uptake rate and attenuates PKC-induced SERT downregulation.\",\n      \"method\": \"Yeast two-hybrid screen, co-transfection in HEK293 cells, [3H]serotonin uptake assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid plus functional uptake assay; single lab\",\n      \"pmids\": [\"12051706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MacMARCKS binds to the cytoplasmic C-terminal tail of metabotropic glutamate receptor 7 (mGluR7); binding is antagonized by Ca2+/calmodulin. Co-transfection of MacMARCKS with mGluR7 reduces G-protein-mediated tonic inhibition of voltage-sensitive Ca2+ channels (VSCCs), an effect dependent on MacMARCKS–mGluR7 interaction.\",\n      \"method\": \"Yeast two-hybrid, in vitro pulldown, co-immunoprecipitation, colocalization in transfected HEK293 and cerebellar granule cells, electrophysiology (VSCC inhibition)\",\n      \"journal\": \"Journal of neurochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Y2H, pulldown, co-IP, colocalization, functional electrophysiology), single lab\",\n      \"pmids\": [\"16987251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"JNK directly phosphorylates MARCKSL1 on C-terminal residues S120, T148, and T183. Phosphorylation by JNK enables MARCKSL1 to bundle and stabilize F-actin, increase filopodium numbers/dynamics, and retard cell migration. Conversely, non-phosphorylatable MARCKSL1 enhances lamellipodia formation and cell migration while reducing filopodia. This mechanism operates in neurons and prostate cancer cells.\",\n      \"method\": \"In vitro kinase assay (JNK phosphorylation), site-directed mutagenesis (S120A/T148A/T183A and S120D/T148D/T183D), F-actin bundling/stabilization assay, live-cell imaging of filopodia/lamellipodia, migration assay (neurons and prostate cancer cells), siRNA knockdown\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus multiple functional readouts across two cell types in single rigorous study\",\n      \"pmids\": [\"22751924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"LOXL2 interacts with MARCKSL1 via its scavenger-receptor domain binding to the N-terminal domain of MARCKSL1; LOXL2 promotes cell proliferation by inhibiting MARCKSL1-induced apoptosis, and MARCKSL1 suppresses LOXL2-induced oncogenesis and reduces luciferase reporter activity in a dose-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, domain-mapping pulldown assays, luciferase reporter assay, flow cytometry (cell cycle/apoptosis), siRNA knockdown\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP with domain mapping plus functional assays; single lab\",\n      \"pmids\": [\"24863880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"MARCKSL1 suppresses angiogenesis by interacting with VEGFR-2 and inhibiting VEGF-induced phosphorylation of VEGFR-2 and downstream PI3K/Akt/PDK-1/mTOR signaling; it reduces VEGF-induced HUVEC proliferation and tubular structure formation in vitro, and decreases HIF-1α and VEGF expression.\",\n      \"method\": \"HUVEC proliferation assay, tube formation assay, Western blot for VEGFR-2 phosphorylation and downstream kinase phosphorylation, MARCKSL1 overexpression\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — functional assays with signaling readouts; interaction with VEGFR-2 inferred from phosphorylation data without direct binding assay; single lab\",\n      \"pmids\": [\"26555156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MARCKSL1 overexpression in the amygdala increases dendritic spine formation in the central amygdala and elevates HPA axis activity and anxiety-like behaviors; knockdown of MARCKSL1 specifically in the amygdala normalizes both HPA axis activity and anxiety behaviors in transgenic mice.\",\n      \"method\": \"MARCKSL1 transgenic mice, site-specific knockdown in amygdala, behavioral assays (anxiety), spine morphology analysis, corticotropin-releasing hormone dependence assay\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — region-specific KD with behavioral and morphological readouts; single lab\",\n      \"pmids\": [\"29580842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"VPA-induced transcriptional downregulation of MARCKSL1 (an actin-stabilizing protein) underlies impairment of dendritic morphology and functional properties in developing but not mature human neurons; these effects are mediated through HDAC and GSK-3 pathway inhibition.\",\n      \"method\": \"Direct reprogramming of human neurons, VPA treatment, RNA sequencing, dendritic morphology quantification, functional electrophysiology, pathway inhibitor experiments\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function via VPA/pathway inhibition linked to MARCKSL1 downregulation with defined morphological and functional phenotype; single lab\",\n      \"pmids\": [\"31155484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Marcksl1 modulates the mechanical properties of the endothelial cell cortex to regulate cell shape and vessel diameter during angiogenesis. Overexpression increases EC size, microvessel diameter, and induces ectopic blebbing suppressed by reduced blood flow; Marcksl1 promotes formation of linear actin bundles and decreases branched actin density at the cortex.\",\n      \"method\": \"In vivo overexpression/depletion in zebrafish, high-resolution live imaging, actin network analysis, microvessel diameter quantification, blood flow manipulation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo gain- and loss-of-function with quantitative structural and functional readouts; multiple orthogonal approaches including imaging and flow manipulation\",\n      \"pmids\": [\"33127887\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"MARCKSL1 promotes esophageal squamous cell carcinoma cell migration and invasion by interacting with F-actin and cortactin to regulate invadopodia formation and extracellular matrix degradation.\",\n      \"method\": \"siRNA knockdown and overexpression, immunofluorescence colocalization of F-actin and cortactin, gelatin degradation assay, Transwell/wound-healing assays, RNA sequencing\",\n      \"journal\": \"Cancer medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — colocalization and functional assays; interaction inferred from colocalization rather than direct binding assay; single lab\",\n      \"pmids\": [\"35894387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Endothelial JNK1 (but not JNK2) is activated by neutrophil adhesion and phosphorylates MARCKSL1 to promote formation of apical filopodia; these filopodia facilitate adhesion of secondary neutrophils and establish transmigration hotspots. Chemical JNK inhibition prevents neutrophil adhesion.\",\n      \"method\": \"Kinase translocation reporters, FRET-based Cdc42 biosensors, JNK1/JNK2 specific inhibition/knockdown, MARCKSL1 knockdown, live-cell imaging of filopodia, neutrophil adhesion assay\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — JNK1-specific activation linked to MARCKSL1-dependent filopodia with functional readout; single lab, multiple biosensor approaches\",\n      \"pmids\": [\"37559902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MARCKSL1 promotes EV secretion from the plasma membrane (in part at the expense of late endosome-PM fusion); MARCKSL1 collaborates with PM-bridging cytoskeletal components (e.g., Radixin) and SNARE-associated proteins (e.g., STXBP3) in this process, as identified by CRISPR activation screen and genomic activation/ablation with microscopic and proteomic follow-up.\",\n      \"method\": \"Genome-wide CRISPR activation screen (CD63 surface levels), genomic activation/ablation, electron microscopy, proteomics, co-immunoprecipitation/interaction studies with Radixin and STXBP3\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — unbiased genetic screen plus orthogonal microscopic and proteomic validation; preprint, not yet peer-reviewed\",\n      \"pmids\": [\"bio_10.1101_2025.07.24.665424\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MARCKSL1 expression in dendritic cells is upregulated by TGF-β1 and is required for dendritic cell-mediated promotion of fibroblast differentiation into myofibroblasts during wound healing; MARCKSL1 shRNA knockdown in dendritic cells diminishes their ability to induce myofibroblast differentiation, and systemic MANS peptide inhibition attenuates scar formation in vivo.\",\n      \"method\": \"TGF-β1 stimulation of mouse dendritic cells, MARCKSL1 shRNA knockdown, co-culture with fibroblasts, myofibroblast differentiation assay, MANS peptide treatment in mouse wound model, single-cell RNA sequencing\",\n      \"journal\": \"Wound repair and regeneration\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with functional cellular readout and in vivo validation; single lab\",\n      \"pmids\": [\"41782175\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MARCKSL1 (MacMARCKS/F52) is a myristoylated, peripherally membrane-associated PKC substrate whose basic effector domain binds F-actin and calmodulin in a phosphorylation-regulated manner; it integrates PKC and Ca2+/calmodulin signals to remodel cortical actin, controlling filopodium versus lamellipodium balance downstream of JNK-mediated phosphorylation (S120/T148/T183), is essential for cranial neural tube closure and dendritic morphogenesis, regulates endothelial cell cortical mechanics and vessel shape in response to hemodynamic forces, modulates integrin activation and neurotransmitter transporter trafficking, interacts with mGluR7 to control VSCC tonic inhibition, and promotes plasma membrane-derived extracellular vesicle secretion in collaboration with Radixin and STXBP3.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MARCKSL1 (MacMARCKS/F52) is a myristoylated, peripherally membrane-associated PKC substrate that integrates protein kinase C and Ca2+/calmodulin signals to remodel the cortical actin cytoskeleton [#0, #1]. Through a central basic effector domain it binds calmodulin with high affinity and binds and bundles F-actin, with PKC phosphorylation disrupting calmodulin binding; together with its N-terminal myristoylation this effector domain directs the protein to specific membrane domains and confers its actin-regulatory activity [#0, #1, #8]. JNK directly phosphorylates MARCKSL1 at S120/T148/T183, switching it toward F-actin bundling and stabilization that increases filopodia and retards migration, whereas non-phosphorylatable protein favors lamellipodia and motility [#13]. This actin-shaping function operates across contexts: it is genetically essential for cranial neural tube closure, where its loss causes neural tube defects and it behaves as haploinsufficient [#2, #3]; it governs dendritic spine and dendrite morphology in neurons with consequences for anxiety behavior and HPA axis activity [#16, #17]; and it controls endothelial cortical mechanics, vessel diameter, and JNK1-driven apical filopodia that establish neutrophil transmigration hotspots [#18, #20]. MARCKSL1 also modulates membrane signaling and trafficking, binding the C-terminal tails of the serotonin transporter and metabotropic glutamate receptor 7 to regulate transport activity and VSCC tonic inhibition in a calmodulin-antagonized manner [#11, #12], and influencing integrin-dependent adhesion in a phosphorylation-state-dependent fashion [#9]. In cancer it promotes invadopodia formation and matrix degradation via F-actin and cortactin [#19].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established the core biochemical identity of MARCKSL1 as a myristoylated PKC substrate whose effector domain reciprocally couples calmodulin binding and actin binding to phosphorylation state, defining the molecular switch underlying all later functions.\",\n      \"evidence\": \"Protein purification, cDNA cloning, in vitro PKC phosphorylation and calmodulin/actin binding assays with defined kinetics, replicated across two labs\",\n      \"pmids\": [\"1516135\", \"1618855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish which cellular processes the actin/calmodulin switch controls\", \"No structural model of the membrane-bound effector domain\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstrated that MARCKSL1 is genetically essential in vivo, with knockout causing failure of cranial neural tube closure and dose-sensitive (haploinsufficient) penetrance, linking its actin function to morphogenetic movement.\",\n      \"evidence\": \"Independent gene knockout mouse studies with developmental phenotyping\",\n      \"pmids\": [\"8692805\", \"8700893\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which cell movements at the neural plate require MARCKSL1\", \"Phosphorylation-dependence of the developmental role untested at the time\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Resolved a conflict over MARCKSL1's role in phagocytosis: dominant-negative effector-domain mutants implicated it, but genetic null macrophages phagocytose and spread normally, showing it is recruited but not absolutely required.\",\n      \"evidence\": \"Comparison of dominant-negative mutant phenotypes versus MacMARCKS null macrophage phagocytosis and spreading assays\",\n      \"pmids\": [\"9837946\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Possible redundancy with MARCKS not directly tested\", \"Why dominant-negative and null phenotypes diverge mechanistically unresolved\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Showed that the myristoyl moiety plus basic effector domain are sufficient for targeting MARCKSL1 to a specific (basolateral) membrane domain and that PKC activation displaces it, mechanistically connecting phosphorylation to membrane localization.\",\n      \"evidence\": \"GFP-fusion domain dissection in polarized MDCK cells with PKC activation\",\n      \"pmids\": [\"9637918\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Targeting determinants in non-epithelial cells not addressed\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Identified direct receptor-tail binding partners (SERT, mGluR7) showing MARCKSL1 modulates membrane transporter/receptor function, with mGluR7 binding antagonized by Ca2+/calmodulin and controlling VSCC tonic inhibition.\",\n      \"evidence\": \"Yeast two-hybrid, pulldown, co-IP, colocalization, transporter uptake and electrophysiology assays\",\n      \"pmids\": [\"16987251\", \"12051706\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological relevance of these interactions in vivo not established\", \"Stoichiometry and regulation by PKC phosphorylation incompletely defined\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the JNK-MARCKSL1 axis: direct JNK phosphorylation at S120/T148/T183 switches MARCKSL1 toward F-actin bundling/stabilization that builds filopodia and slows migration, while the unphosphorylatable form drives lamellipodia and motility.\",\n      \"evidence\": \"In vitro JNK kinase assay, phosphosite mutagenesis, F-actin bundling assays, live imaging and migration assays in neurons and prostate cancer cells\",\n      \"pmids\": [\"22751924\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals activating JNK toward MARCKSL1 not delineated here\", \"Relationship between JNK sites and PKC/calmodulin switch unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Extended the actin function to tissue mechanics, showing MARCKSL1 sets endothelial cortical stiffness by promoting linear over branched actin, thereby controlling cell size, vessel diameter, and flow-dependent blebbing.\",\n      \"evidence\": \"Zebrafish in vivo gain/loss-of-function, high-resolution imaging, actin network analysis, blood flow manipulation\",\n      \"pmids\": [\"33127887\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for linear vs branched actin preference not defined\", \"Link to JNK phosphorylation in this context not tested\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected upstream signaling to physiology, showing endothelial JNK1 (not JNK2) phosphorylates MARCKSL1 to form apical filopodia that establish neutrophil transmigration hotspots.\",\n      \"evidence\": \"Kinase translocation reporters, Cdc42 FRET biosensors, JNK1/2-specific perturbation, MARCKSL1 knockdown, neutrophil adhesion assays\",\n      \"pmids\": [\"37559902\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Whether the same S120/T148/T183 sites mediate this remains inferential\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated MARCKSL1 in plasma-membrane-derived extracellular vesicle secretion in collaboration with Radixin and STXBP3, expanding its role from cortical actin to membrane budding/trafficking.\",\n      \"evidence\": \"Genome-wide CRISPR activation screen, genomic activation/ablation, electron microscopy, proteomics, interaction studies (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.07.24.665424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Direct binding to Radixin/STXBP3 versus pathway co-dependence not fully separated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the JNK (S120/T148/T183), PKC, and Ca2+/calmodulin inputs are integrated on a single effector domain to select between filopodia, lamellipodia, cortical stiffening, and membrane trafficking outputs remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural/biophysical model reconciling the three phosphoregulatory inputs\", \"Tissue-specific partner sets that bias output are incompletely mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 13, 19]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [11, 12, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [8, 18, 21]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [13, 18, 19]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 3, 18]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CALM1\", \"SLC6A4\", \"GRM7\", \"CTTN\", \"RDX\", \"STXBP3\", \"LOXL2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":6,"faith_pct":83.33333333333333}}