{"gene":"SPRED1","run_date":"2026-06-10T07:46:40","timeline":{"discoveries":[{"year":2007,"finding":"Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype (Legius syndrome). SPRED1 acts as a negative regulator of the RAS→RAF interaction and MAPK signaling pathway. Biallelic SPRED1 inactivation (germline + somatic mutation) was required to generate café-au-lait spots, establishing a tumor-suppressor two-hit mechanism.","method":"Germline mutation identification, somatic mutation analysis of melanocytes from café-au-lait spots","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct mutation identification with somatic second-hit confirmed in patient tissue, replicated across multiple families, foundational study","pmids":["17704776"],"is_preprint":false},{"year":2004,"finding":"SPRED1 negatively regulates IL-3-induced ERK/MAP kinase activation and cell proliferation in hematopoietic cells. Forced expression of wild-type SPRED1 reduced proliferation and ERK activation in response to both SCF and IL-3; a dominant-negative form (DeltaC-Spred) augmented both. SPRED1-deficient mast cells showed augmented ERK activation and proliferation in response to IL-3.","method":"Retroviral gene transfer, loss-of-function (Spred1 knockout cells), dominant-negative overexpression, proliferation assays, ERK phosphorylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal gain- and loss-of-function with biochemical readout, multiple cell types","pmids":["15465815"],"is_preprint":false},{"year":2005,"finding":"SPRED1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness by suppressing IL-5-dependent ERK activation and cell proliferation in eosinophils, without affecting T helper cell differentiation.","method":"Spred1-deficient mice, allergen challenge model, biochemical ERK activation assays, cell proliferation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout mouse model with defined cellular and biochemical phenotype, multiple orthogonal assays","pmids":["15630138"],"is_preprint":false},{"year":2005,"finding":"SPRED1 localizes in lipid raft/caveola membrane fractions and interacts with caveolin-1 (Cav-1). Co-localization with Cav-1 enhances ERK inhibition beyond either protein alone; forced Cav-1 expression in hematopoietic cells suppressed SCF/IL-3-induced ERK activation, and additional Spred-1 expression further suppressed it.","method":"Subcellular fractionation, co-immunoprecipitation, overexpression in hematopoietic cells, ERK activation assays","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus functional rescue, single lab, multiple methods","pmids":["16115197"],"is_preprint":false},{"year":2005,"finding":"Crystal structure of the Xenopus tropicalis Spred1 EVH1 domain solved to 1.15 Å resolution. The EVH1 domain adopts a pleckstrin-homology fold; a narrowed peptide-binding groove and structural flexibility at one end suggest Spred1 binds peptides less proline-rich than other EVH1 domains, likely via an induced-fit mechanism.","method":"X-ray crystallography","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — high-resolution crystal structure, single lab but unambiguous structural data","pmids":["15710406"],"is_preprint":false},{"year":2008,"finding":"SPRED1 is a binding partner of both MARKK/TAO1 and TESK1 kinases, linking microtubule and actin cytoskeleton regulation. SPRED1-MARKK binding does not affect MARKK activity or microtubule stability. SPRED1-TESK1 binding inhibits TESK1, which normally phosphorylates cofilin to stabilize F-actin stress fibers; thus SPRED1 promotes dynamic F-actin through TESK1 inhibition.","method":"Yeast two-hybrid, co-immunoprecipitation, kinase activity assays, CHO cell overexpression","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus functional assays, single lab","pmids":["18216281"],"is_preprint":false},{"year":2008,"finding":"Spred1 deficiency in mice causes defects in hippocampus-dependent learning, short- and long-term synaptic plasticity (including LTP/LTD imbalance in CA1), and increased ERK phosphorylation after LTP induction, demonstrating that SPRED1 limits Ras/ERK hyperactivation to maintain normal cognitive function and synaptic plasticity.","method":"Spred1 knockout mice, Morris water maze, T-maze, electrophysiology (LTP/LTD recordings), western blot for pERK","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout mouse, behavioral testing plus electrophysiology plus biochemistry, multiple orthogonal methods","pmids":["19118178"],"is_preprint":false},{"year":2010,"finding":"SPRED1 is highly enriched in CNS germinal zones and functions to dampen neural stem cell (NSC) self-renewal and progenitor proliferation cell-autonomously. SPRED1 localizes within distinct vesicles in cortical progenitor cells. Knockdown in vivo in embryonic mouse forebrain disrupts apical ventricular zone organization, impairs radial glia alignment, and causes periventricular heterotopia due to impaired late neuronal migration.","method":"shRNA knockdown in vivo and in vitro, overexpression, immunofluorescence/live imaging, subcellular fractionation/vesicle localization, cortical progenitor proliferation assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockdown plus overexpression with defined cellular phenotype, in vivo and in vitro, multiple orthogonal methods","pmids":["20047999"],"is_preprint":false},{"year":2010,"finding":"SPRED1 and SPRED2 directly interact with the novel kinase DYRK1A via their cysteine-rich domain (CRD), which binds the kinase domain of DYRK1A. SPRED1/2 inhibit DYRK1A-mediated phosphorylation of substrates Tau and STAT3 by competing for the substrate binding site on DYRK1A.","method":"Co-immunoprecipitation (endogenous and overexpressed), direct binding assays, kinase substrate phosphorylation assays, domain mapping","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous co-IP plus functional kinase assays, single lab","pmids":["20736167"],"is_preprint":false},{"year":2011,"finding":"SPRED1 is a substrate of the tyrosine phosphatase SHP2. SPRED proteins (but not Sprouty2) bind to the tyrosine phosphatase domain of SHP2. Overexpressed SHP2 dephosphorylates SPRED1. Two tyrosine residues on SPRED1, when phosphorylated, are required for Ras/ERK inhibition; Tyr-420 is a specific dephosphorylation target of SHP2, indicating that SHP2 attenuates the inhibitory activity of SPRED1.","method":"Co-immunoprecipitation, in vitro dephosphorylation assay, site-directed mutagenesis, ERK activation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzyme assay plus mutagenesis plus binding domain mapping, single lab with multiple orthogonal methods","pmids":["21531714"],"is_preprint":false},{"year":2011,"finding":"SPRED1 interacts with FGFRL1 via the C-terminal SPR domain of SPRED1 and the histidine-rich domain of FGFRL1. This interaction was verified by coprecipitation and co-distribution at the plasma membrane; SPRED1 increased the retention time of FGFRL1 at the plasma membrane.","method":"Yeast two-hybrid, coprecipitation, co-localization by immunofluorescence in COS1 and HEK293 cells","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid confirmed by co-precipitation and co-localization, single lab","pmids":["21616146"],"is_preprint":false},{"year":2011,"finding":"miR-126 positively regulates mast cell proliferation and FcεRI-mediated cytokine production by suppressing SPRED1 expression. Overexpression of miR-126 suppressed SPRED1 and enhanced ERK activity. Hematopoietic-specific Spred1 conditional knockout mice showed increased mast cell numbers and hyperactivation, establishing SPRED1 as the functional target of miR-126 in this context.","method":"miR-126 overexpression, conditional Spred1 knockout mice, SPRED1 western blot, ERK assays, cytokine production assays","journal":"Genes to cells","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout confirms functional target, complemented by overexpression and biochemical assays","pmids":["21668589"],"is_preprint":false},{"year":2015,"finding":"The SPRED1 EVH1 domain interacts with the N-terminal 16 aa and C-terminal 20 aa of the GTPase-activating protein-related domain (GRD) of neurofibromin (NF1), forming two crossing α-helix coils outside the GAP catalytic domain. These regions are dispensable for GAP activity and are absent from p120GAP. Pathogenic missense mutations in the EVH1 domain of SPRED1 (Legius syndrome) and in the GRD (NF1 patients) reduce binding affinity and disrupt ERK suppression, showing SPRED1 inhibits Ras-ERK by recruiting neurofibromin to Ras via EVH1-GRD interaction.","method":"Yeast two-hybrid, co-immunoprecipitation in HEK293 cells, ERK activation assays, mutational analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus reciprocal co-IP plus mutagenesis plus functional ERK assays, single lab with multiple orthogonal methods","pmids":["26635368"],"is_preprint":false},{"year":2016,"finding":"The SPRED1 EVH1 domain binds specifically to the noncatalytic GAPex subdomain of the neurofibromin GRD. This binding is compatible with simultaneous Ras binding and does not interfere with neurofibromin's GAP activity, establishing that SPRED1 functions as a membrane-targeting scaffold for neurofibromin rather than a direct modulator of its catalytic activity.","method":"Biochemical binding assays, mutational analysis, GAP activity assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical reconstitution of binding and GAP activity assays, independently consistent with PMID:26635368","pmids":["27313208"],"is_preprint":false},{"year":2016,"finding":"SPRED1 interaction with B-Raf and galectin-1 (Gal-1) facilitates SPRED1 translocation to the plasma membrane upon growth factor stimulation or pharmacological/Gal-1-mediated Raf dimer induction. At the plasma membrane, SPRED1 selectively perturbs membrane organization and ERK signaling of K-ras4B but not H-ras, and blocks positive effects of Gal-1 on H-ras nanoclusters. Two Legius syndrome mutations show diminished binding to both Gal-1 and B-Raf.","method":"Co-immunoprecipitation, super-resolution microscopy, FRET-based nanoclustering assays, mutational analysis, plasma membrane fractionation","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus nanoscale membrane assays plus mutagenesis, single lab","pmids":["27503857"],"is_preprint":false},{"year":2018,"finding":"SPRED1 functions as a tumor suppressor in mucosal melanoma, particularly in the context of KIT mutations. SPRED1 knockdown in zebrafish caused MAPK activation, increased cell proliferation, and conferred resistance to KIT tyrosine kinase inhibitors, establishing SPRED1 loss as a driver of KIT-mutant melanoma through MAPK pathway activation.","method":"Human tumor sequencing (43 mucosal melanomas), tissue-specific CRISPR in zebrafish, MAPK activation assays, cell proliferation assays, drug resistance assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo CRISPR modeling plus biochemical readout, human genomic data, multiple orthogonal methods","pmids":["30385465"],"is_preprint":false},{"year":2018,"finding":"SPRED1 negatively regulates hematopoietic stem cell (HSC) self-renewal and fitness under steady-state conditions, in part through Rho kinase (ROCK) activity. Under high-fat diet conditions, Spred1 deficiency leads to ERK hyperactivation and aberrant HSC self-renewal, causing functional HSC failure and myeloproliferative neoplasm-like disease.","method":"Spred1 conditional knockout mice, HSC functional assays, ERK activation assays, high-fat diet model","journal":"Cell stem cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout in multiple compartments plus biochemical pathway analysis, multiple phenotypic readouts","pmids":["29706577"],"is_preprint":false},{"year":2019,"finding":"A pathogenic Legius syndrome missense mutation in the EVH1 domain of SPRED1 (T102R) weakens the interaction with neurofibromin by approximately 3 orders of magnitude without perturbing the overall EVH1 protein fold. NMR spectroscopy was used to map the neurofibromin GRD binding site on the mutant Spred1 EVH1 domain.","method":"Binding affinity measurements, NMR spectroscopy, protein stability assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural mapping plus quantitative binding measurements, single lab with rigorous biophysical methods","pmids":["31401120"],"is_preprint":false},{"year":2020,"finding":"Crystal/cryo structure of the neurofibromin GAP-related domain complexed with the SPRED1 EVH1 domain and KRAS reveals how SPRED1 membrane-targeting of neurofibromin allows simultaneous interaction with activated KRAS. Oncogenic EGFR(L858R) signaling leads to phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. Analysis of the interface rationalizes Legius syndrome mutations and explains why SPRED1 binds neurofibromin but no other RasGAPs.","method":"Crystal structure (X-ray), biochemical binding assays, phosphorylation mapping, mutagenesis, cell-based ERK signaling assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure plus biochemical and functional validation, multiple orthogonal methods in single study","pmids":["32697994"],"is_preprint":false},{"year":2020,"finding":"Spred1 and Spred2 double-deficiency in lens results in elevated ERK1/2 phosphorylation, irregular lens epithelial and fiber cell activity, and impaired lens and eye development, demonstrating that Spred1 negatively regulates ERK1/2 activity to maintain normal lens morphogenesis.","method":"Spred1/2 double-knockout mice, ERK phosphorylation western blot, histological analysis of lens development","journal":"Experimental eye research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse with defined biochemical phenotype, single lab","pmids":["31923414"],"is_preprint":false},{"year":2021,"finding":"SPRED1 loss in human melanoma cell lines and primary zebrafish melanoma confers resistance to BRAFV600E inhibition by reactivating MAPK activity. Biallelic SPRED1 deletion was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy.","method":"SPRED1 inactivation in human cell lines, in vivo zebrafish melanoma model, MAPK pathway activation assays, drug resistance assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — combined human cell line plus in vivo zebrafish model plus patient sample, multiple orthogonal methods","pmids":["33306107"],"is_preprint":false},{"year":2021,"finding":"Spred1 deficiency in CML promotes transformation from chronic phase to accelerated/blast crisis CML. Spred1 knockout (in HSCs or endothelial cells) increased miR-126 in leukemic stem cells (LSKs), causing LSC expansion through MAPK/ERK hyperactivation, augmented Bcl-2 expression and stability, and enhanced Bcl-2-dependent oxidative phosphorylation.","method":"Conditional knockout mouse models, leukemia transformation assays, miR-126 quantification, pERK assays, Bcl-2 expression/stability assays","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple conditional KO models with mechanistic pathway dissection, single lab","pmids":["34564700"],"is_preprint":false},{"year":2021,"finding":"Spred1-deficient mice exhibit increased social dominance and impaired nesting behavior mediated by RAS-MAPK hyperactivation. Acute MEK inhibitor (PD325901) treatment in adulthood reversed the enhanced social dominance phenotype, demonstrating that Ras-MAPK dysregulation mediates social behavior phenotypes downstream of SPRED1 loss.","method":"Spred1 knockout mice, automated tube test, ultrasonic vocalization recording, nesting behavior, MEK inhibitor pharmacological rescue","journal":"Molecular autism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse behavioral phenotype with pharmacological pathway rescue, single lab","pmids":["34311771"],"is_preprint":false},{"year":2022,"finding":"Molecular dynamics simulation and Markov state model analysis of the KRAS-NF1-SPRED1 ternary complex suggest that SPRED1 loading allosterically enhances KRAS-NF1 binding at a site distal from the SPRED1-NF1 interface, but that this allosteric effect also hinders conformational transformation of the NF1 catalytic center required for RAS GTP hydrolysis. Allosteric pathways were identified by difference contact network analysis.","method":"Gaussian accelerated molecular dynamics simulations, Markov state model analysis, contact network analysis","journal":"Journal of molecular biology","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational modeling only, no experimental validation reported","pmids":["35872068"],"is_preprint":false},{"year":2015,"finding":"SPRED1 negatively regulates proliferation, apoptosis, and cytokine secretion of group 2 innate lymphoid cells (ILC2s) through the Ras-ERK pathway. Spred1 deficiency enhanced IL-33-induced ILC2 proliferation and cytokine production; MEK inhibitor suppressed these. Spred1 deficiency resulted in stabilization of GATA3, a key transcription factor for ILC2 function.","method":"Spred1 knockout mice, ILC2 proliferation and cytokine assays, MEK inhibitor treatment, GATA3 protein stability assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout mouse plus pharmacological rescue plus GATA3 mechanistic link, single lab","pmids":["26116510"],"is_preprint":false},{"year":2024,"finding":"Missense mutations in the C-terminal SPR (Sprouty-related) domain of SPRED1 (e.g., C368S, M369L, V408E, P415A, P415L, P422R) identified in Legius syndrome patients impair palmitoylation of SPRED1, causing loss of plasma membrane localization and formation of cytoplasmic granular aggregates. In knock-in mice, P415A and P415V mutations (but not a C-terminal deletion M417Afs*4) formed granular aggregates and led to Purkinje cell loss and cerebellar ataxia with age. Autophagy inducer spermidine rescued ataxia and Purkinje cell loss in Spred1P415A mice, suggesting aggregate toxicity as a potential neurodegenerative mechanism.","method":"Site-directed mutagenesis, knock-in mice, palmitoylation assay, live cell imaging/localization, histological analysis, behavioral (ataxia) testing, spermidine treatment rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro mutagenesis plus knock-in mouse models plus pharmacological rescue, multiple orthogonal methods","pmids":["39510187"],"is_preprint":false},{"year":2024,"finding":"EZH2 epigenetically suppresses SPRED1 transcriptional activity and expression; the EZH2 inhibitor Tazemetostat reverses this suppression. Overexpression of SPRED1 inhibits lung adenocarcinoma cell proliferation, migration, and invasion in vitro and retards tumor growth in vivo, while EZH2 elevation rescues these effects.","method":"Dual-luciferase reporter assay, EZH2 inhibitor treatment, overexpression gain-of-function, in vivo xenograft","journal":"Human cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — luciferase assay plus functional rescue plus in vivo, single lab","pmids":["36629984"],"is_preprint":false},{"year":2021,"finding":"Merlin (NF2 protein) can interact with both neurofibromin and SPRED1, forming a trimeric complex. Merlin directly binds the Ras-binding domain (RBD) and kinase domain (KiD) of Raf1, functioning as a 'selective Ras barrier' that blocks Ras binding to Raf1 without increasing neurofibromin's GAP activity.","method":"Co-immunoprecipitation, direct binding assays, RAS-GAP activity assays, Schwann cell models","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus GAP activity assays, single lab, multiple interactors mapped","pmids":["33331896"],"is_preprint":false},{"year":2026,"finding":"NF1 and SPRED1/2 cooperate through RAS-MAPK-independent functions. CRISPR-Cas9 ablation of NF1 or SPRED1/2 in 'RASless' MEFs showed that SPRED1/2 loss phenocopies NF1 loss and their cooperation modulates MAPK-AKT signaling. Additionally, NF1 or SPRED1/2 loss suppressed the RAS family GTPases RRAS and RRAS2 independently of RAS or AKT pathway activation. A RAS-independent, NF1-SPRED1/2-dependent gene signature was identified.","method":"CRISPR-Cas9 knockout in isogenic RASless MEFs, transcriptome microarray, signaling pathway assays, Schwann cell models from NF1 patients","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — isogenic CRISPR system plus transcriptomics plus patient-derived cells, single lab","pmids":["42201959"],"is_preprint":false}],"current_model":"SPRED1 is a negative regulator of the RAS-MAPK/ERK signaling pathway that functions primarily by recruiting neurofibromin (NF1 protein, a RasGAP) from the cytosol to membrane-anchored active RAS via a direct interaction between the SPRED1 EVH1 domain and the noncatalytic GAPex subdomain of neurofibromin's GAP-related domain; the C-terminal SPR domain mediates palmitoylation-dependent plasma membrane localization. SPRED1 can also inhibit signaling at the Ras/Raf interface (interacting with B-Raf and galectin-1 in a Ras isoform-selective manner), is a substrate of SHP2 (whose dephosphorylation of Tyr-420 attenuates SPRED1's inhibitory activity), interacts with DYRK1A (inhibiting its kinase activity via CRD-kinase domain contact) and TESK1 (inhibiting cofilin phosphorylation to promote dynamic F-actin), and colocalizes with caveolin-1 in lipid rafts for cooperative ERK suppression. Structural studies reveal that the EVH1 domain adopts a pleckstrin-homology fold that simultaneously allows neurofibromin binding and KRAS engagement, and oncogenic EGFR can disrupt this complex by phosphorylating SPRED1 at Ser105. Loss-of-function mutations in SPRED1 cause Legius syndrome through constitutive RAS-MAPK hyperactivation, and somatic SPRED1 loss acts as a tumor suppressor mechanism in melanoma and other cancers."},"narrative":{"mechanistic_narrative":"SPRED1 is a negative regulator of RAS-MAPK/ERK signaling that limits proliferation, self-renewal, and ERK-driven responses across hematopoietic, neural, immune, and ocular cell types [PMID:15465815, PMID:15630138, PMID:19118178, PMID:29706577]. Its central mechanism is a scaffolding one: the N-terminal EVH1 domain, which adopts a pleckstrin-homology fold [PMID:15710406], directly engages the noncatalytic GAPex region of the neurofibromin (NF1) GTPase-activating domain, thereby recruiting neurofibromin to membrane-anchored active KRAS without altering neurofibromin's intrinsic GAP catalysis [PMID:26635368, PMID:27313208, PMID:32697994]. Structural and biophysical analyses show this EVH1-GRD interface simultaneously accommodates KRAS, explains why SPRED1 binds neurofibromin but not other RasGAPs, and rationalizes Legius syndrome mutations that abolish binding and ERK suppression [PMID:31401120, PMID:32697994]. SPRED1 activity is controlled by post-translational and membrane-targeting inputs: the C-terminal SPR domain requires palmitoylation for plasma-membrane localization [PMID:39510187], oncogenic EGFR disrupts the neurofibromin complex by phosphorylating Ser105 [PMID:32697994], and SHP2 dephosphorylates Tyr-420 to attenuate SPRED1's inhibitory function [PMID:21531714]. Beyond RAS regulation, SPRED1 inhibits the kinases TESK1 and DYRK1A through its cysteine-rich domain, linking it to cytoskeletal dynamics and substrate phosphorylation [PMID:18216281, PMID:20736167]. Germline loss-of-function mutations in SPRED1 cause Legius syndrome through constitutive RAS-MAPK hyperactivation [PMID:17704776], and somatic SPRED1 loss reactivates MAPK signaling to drive melanoma progression and resistance to KIT and BRAF inhibitors [PMID:30385465, PMID:33306107].","teleology":[{"year":2004,"claim":"Established SPRED1 as a cell-intrinsic negative regulator of growth-factor-induced ERK activation, defining its core biological function.","evidence":"Reciprocal gain/loss-of-function with dominant-negative and knockout hematopoietic cells, ERK and proliferation assays","pmids":["15465815"],"confidence":"High","gaps":["Molecular target within the RAS-ERK cascade not yet identified","No structural basis for inhibition"]},{"year":2005,"claim":"Extended SPRED1's ERK-suppressing role to allergic airway inflammation and tied its activity to membrane microdomains via caveolin-1.","evidence":"Spred1-knockout mouse allergen model; subcellular fractionation and co-IP with Cav-1 plus ERK assays","pmids":["15630138","16115197"],"confidence":"High","gaps":["Direct molecular partner mediating ERK inhibition still unknown","Cav-1 interaction is single-lab, mechanism of cooperative suppression unresolved"]},{"year":2005,"claim":"Resolved the EVH1 domain fold, revealing a pleckstrin-homology architecture and a peptide-binding groove distinct from other EVH1 domains.","evidence":"1.15 Å X-ray crystal structure of Xenopus Spred1 EVH1","pmids":["15710406"],"confidence":"High","gaps":["Binding partner of the EVH1 groove not yet identified","Structure from Xenopus ortholog, not human"]},{"year":2007,"claim":"Identified SPRED1 loss-of-function as the cause of Legius syndrome, establishing it as a tumor suppressor acting through a two-hit mechanism on RAS-MAPK signaling.","evidence":"Germline and somatic mutation analysis in patient melanocytes","pmids":["17704776"],"confidence":"High","gaps":["Biochemical mechanism linking mutations to RAS hyperactivation not yet defined"]},{"year":2008,"claim":"Connected SPRED1 to cytoskeletal regulation by showing it inhibits TESK1 to promote dynamic F-actin, broadening its role beyond ERK.","evidence":"Yeast two-hybrid, co-IP, kinase assays in CHO cells","pmids":["18216281"],"confidence":"Medium","gaps":["Physiological relevance of cytoskeletal role in vivo unclear","Single-lab; MARKK/TAO1 binding has no functional consequence"]},{"year":2008,"claim":"Demonstrated that SPRED1 restrains Ras/ERK in the brain to maintain synaptic plasticity and learning, linking the gene to cognitive function.","evidence":"Spred1-knockout mice, behavioral testing, LTP/LTD electrophysiology, pERK western blot","pmids":["19118178"],"confidence":"High","gaps":["Cell-type and circuit specificity of ERK dysregulation not dissected"]},{"year":2010,"claim":"Showed SPRED1 dampens neural stem/progenitor proliferation and is required for cortical organization, and that it inhibits DYRK1A via its CRD.","evidence":"In vivo/in vitro shRNA knockdown, imaging, vesicle fractionation; co-IP and kinase substrate assays for DYRK1A","pmids":["20047999","20736167"],"confidence":"High","gaps":["Vesicular localization function not mechanistically explained","DYRK1A inhibition is Medium-confidence, single lab"]},{"year":2011,"claim":"Defined upstream regulation of SPRED1 by SHP2-mediated dephosphorylation and by miR-126, and identified an FGFRL1 membrane interaction, showing SPRED1 activity is tunable.","evidence":"In vitro dephosphorylation and mutagenesis (SHP2); conditional knockout and miRNA overexpression (miR-126); yeast two-hybrid and co-localization (FGFRL1)","pmids":["21531714","21668589","21616146"],"confidence":"High","gaps":["Functional consequence of FGFRL1 retention unclear","How phosphorylation at Tyr residues couples to neurofibromin recruitment not yet established"]},{"year":2016,"claim":"Defined the EVH1–neurofibromin GRD interface and established that SPRED1 acts as a membrane-targeting scaffold for neurofibromin rather than a direct GAP modulator.","evidence":"Yeast two-hybrid, reciprocal co-IP, mutagenesis, ERK assays, and biochemical reconstitution of binding and GAP activity","pmids":["26635368","27313208"],"confidence":"High","gaps":["Structure of the ternary SPRED1-neurofibromin-RAS complex not yet solved","Membrane delivery dynamics not directly visualized"]},{"year":2016,"claim":"Revealed isoform-selective regulation through B-Raf and galectin-1 binding, with SPRED1 perturbing KRAS but not HRAS membrane organization.","evidence":"Co-IP, super-resolution and FRET nanoclustering assays, mutagenesis","pmids":["27503857"],"confidence":"Medium","gaps":["Single-lab; mechanism of isoform selectivity at the membrane unresolved","Relationship to neurofibromin scaffolding pathway unclear"]},{"year":2019,"claim":"Quantified how a Legius mutation cripples neurofibromin binding without unfolding EVH1, pinpointing affinity loss as the disease mechanism.","evidence":"NMR mapping, binding affinity and protein stability measurements of the T102R EVH1 mutant","pmids":["31401120"],"confidence":"High","gaps":["Single mutation analyzed","Cellular and organismal consequences not measured in this study"]},{"year":2020,"claim":"Provided the structural basis for SPRED1's RasGAP specificity and revealed EGFR-driven Ser105 phosphorylation as a means to disrupt the complex.","evidence":"X-ray structure of NF1 GRD–SPRED1 EVH1–KRAS, phosphorylation mapping, mutagenesis, ERK assays","pmids":["32697994"],"confidence":"High","gaps":["Stoichiometry and kinetics of complex assembly at the membrane not defined","Generality of Ser105 phosphorylation across RTKs unknown"]},{"year":2020,"claim":"Showed SPRED1 (with SPRED2) is required for lens morphogenesis by restraining ERK1/2, extending its developmental role to the eye.","evidence":"Spred1/2 double-knockout mice, ERK western blot, histology","pmids":["31923414"],"confidence":"Medium","gaps":["Redundancy with SPRED2 not separated for SPRED1 alone","Single-lab"]},{"year":2018,"claim":"Established somatic SPRED1 loss as a melanoma tumor-suppressor event and as a driver of KIT-inhibitor resistance through MAPK reactivation.","evidence":"Human tumor sequencing, tissue-specific zebrafish CRISPR, MAPK and drug-resistance assays","pmids":["30385465"],"confidence":"High","gaps":["Mechanism of resistance downstream of MAPK reactivation not fully resolved"]},{"year":2018,"claim":"Defined SPRED1 as a restraint on hematopoietic stem cell self-renewal whose loss, under metabolic stress, produces myeloproliferative disease.","evidence":"Spred1 conditional knockout mice, HSC functional and ERK assays, high-fat diet model","pmids":["29706577"],"confidence":"High","gaps":["Contribution of ROCK versus ERK arms not quantitatively separated"]},{"year":2021,"claim":"Showed SPRED1 loss drives MAPK-targeted-therapy resistance in melanoma and promotes CML transformation via miR-126/ERK/Bcl-2 signaling, and that NF2/Merlin can join the neurofibromin-SPRED1 axis.","evidence":"Human cell lines, zebrafish models, patient sample (BRAFi resistance); conditional KO and pathway dissection (CML); co-IP and GAP assays (Merlin)","pmids":["33306107","34564700","33331896"],"confidence":"High","gaps":["Merlin trimeric complex is Medium-confidence, single lab","Whether miR-126 and Bcl-2 axis generalizes beyond CML unclear"]},{"year":2021,"claim":"Linked SPRED1 loss to social behavior and immune (ILC2) phenotypes mediated by RAS-MAPK, with pharmacological MEK inhibition reversing behavioral effects.","evidence":"Spred1-knockout mice, behavioral testing with MEK-inhibitor rescue; ILC2 assays with GATA3 stability analysis","pmids":["34311771","26116510"],"confidence":"Medium","gaps":["Single-lab phenotypes","Direct molecular link between SPRED1 and GATA3 stability not established"]},{"year":2024,"claim":"Showed that SPR-domain mutations impairing palmitoylation cause SPRED1 mislocalization and toxic cytoplasmic aggregates, defining a neurodegenerative mechanism distinct from simple RAS hyperactivation.","evidence":"Mutagenesis, knock-in mice, palmitoylation and localization assays, ataxia testing, spermidine rescue; EZH2 transcriptional suppression by luciferase and xenograft","pmids":["39510187","36629984"],"confidence":"High","gaps":["EZH2 regulation is Medium-confidence, single lab","Link between aggregate toxicity and Purkinje loss mechanism incompletely defined"]},{"year":2026,"claim":"Revealed RAS-MAPK-independent cooperation between NF1 and SPRED1/2, including suppression of RRAS/RRAS2 and a RAS-independent gene signature, broadening SPRED1's functional scope.","evidence":"CRISPR knockout in isogenic RASless MEFs, transcriptomics, signaling assays, NF1 patient Schwann cells","pmids":["42201959"],"confidence":"Medium","gaps":["Mechanism of RRAS/RRAS2 suppression undefined","Single-lab; RAS-independent signature not functionally validated"]},{"year":null,"claim":"How SPRED1 integrates its many regulatory inputs (palmitoylation, Tyr/Ser phosphorylation, miRNA, EZH2) to dynamically control neurofibromin delivery at the membrane, and the molecular basis of its RAS-independent functions, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model of membrane-targeting kinetics in vivo","RAS-independent NF1-SPRED1 mechanism uncharacterized","Interplay between scaffolding and kinase-inhibitory (DYRK1A/TESK1) functions unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,12,13,18]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[12,13,18]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,8]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[10,14,18,25]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12,25]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,12,18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,15,20]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[6,7,19]}],"complexes":["SPRED1–neurofibromin(NF1)–KRAS ternary complex"],"partners":["NF1","KRAS","SHP2","DYRK1A","TESK1","CAV1","BRAF","LGALS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7Z699","full_name":"Sprouty-related, EVH1 domain-containing protein 1","aliases":[],"length_aa":444,"mass_kda":50.5,"function":"Tyrosine kinase substrate that inhibits growth-factor-mediated activation of MAP kinase (By similarity). 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assignments","url":"https://pubmed.ncbi.nlm.nih.gov/28831766","citation_count":5,"is_preprint":false},{"pmid":"33624414","id":"PMC_33624414","title":"Impaired instrumental learning in Spred1-/- mice, a model for a rare RASopathy.","date":"2021","source":"Genes, brain, and behavior","url":"https://pubmed.ncbi.nlm.nih.gov/33624414","citation_count":4,"is_preprint":false},{"pmid":"38391903","id":"PMC_38391903","title":"Conditional Ablation of Spred1 and Spred2 in the Eye Lens Negatively Impacts Its Development and Growth.","date":"2024","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/38391903","citation_count":4,"is_preprint":false},{"pmid":"33073945","id":"PMC_33073945","title":"A Study on the Expression of SPRED1 and PBRM1 (Baf180) and their Clinical Significances in Patients with Gastric Cancer.","date":"2020","source":"Clinical laboratory","url":"https://pubmed.ncbi.nlm.nih.gov/33073945","citation_count":4,"is_preprint":false},{"pmid":"35359401","id":"PMC_35359401","title":"Methylation of SPRED1: A New Target in Acute Myeloid Leukemia.","date":"2022","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/35359401","citation_count":3,"is_preprint":false},{"pmid":"36745282","id":"PMC_36745282","title":"Circ_TEX2 Functions as a Tumor Suppressor in Hepatoma via miR-96-5p/SPRED1 Axis.","date":"2023","source":"Molecular biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/36745282","citation_count":3,"is_preprint":false},{"pmid":"40358892","id":"PMC_40358892","title":"miR-486-3p Suppresses Osteosarcoma Proliferation and Migration by Targeting the SPRED1-MAPK/ERK Pathway.","date":"2025","source":"Biochemical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/40358892","citation_count":3,"is_preprint":false},{"pmid":"27081556","id":"PMC_27081556","title":"Legius Syndrome: two novel mutations in the SPRED1 gene.","date":"2015","source":"Human genome variation","url":"https://pubmed.ncbi.nlm.nih.gov/27081556","citation_count":2,"is_preprint":false},{"pmid":"41094606","id":"PMC_41094606","title":"The role of SPRED1 mutation in melanoma.","date":"2025","source":"European journal of medical research","url":"https://pubmed.ncbi.nlm.nih.gov/41094606","citation_count":1,"is_preprint":false},{"pmid":"23998617","id":"PMC_23998617","title":"[Relationship between Spred1 and acute myeloid leukemia].","date":"2013","source":"Zhongguo shi yan xue ye xue za zhi","url":"https://pubmed.ncbi.nlm.nih.gov/23998617","citation_count":1,"is_preprint":false},{"pmid":"28150585","id":"PMC_28150585","title":"The first Slovak Legius syndrome patient carrying the SPRED1 gene mutation.","date":"2017","source":"General physiology and biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/28150585","citation_count":1,"is_preprint":false},{"pmid":"19966658","id":"PMC_19966658","title":"Expanding the phenotype of a neurofibromatosis type 1-like syndrome: a patient with a SPRED1 mutation and orbital manifestations.","date":"2009","source":"Ophthalmic plastic and reconstructive surgery","url":"https://pubmed.ncbi.nlm.nih.gov/19966658","citation_count":1,"is_preprint":false},{"pmid":"41208883","id":"PMC_41208883","title":"TTC36-Mediated Tumor Suppression via YBX3/SPRED1 Axis Paradoxically Reduces Sorafenib Sensitivity in Hepatocellular Carcinoma.","date":"2025","source":"International journal of biological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/41208883","citation_count":0,"is_preprint":false},{"pmid":"42201959","id":"PMC_42201959","title":"NF1 and SPRED1/2 cooperate through RAS-MAPK-independent functions.","date":"2026","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/42201959","citation_count":0,"is_preprint":false},{"pmid":"30531141","id":"PMC_30531141","title":"[Regulation of hematopoietic stem cell homeostasis by Spred1].","date":"2018","source":"[Rinsho ketsueki] The Japanese journal of clinical hematology","url":"https://pubmed.ncbi.nlm.nih.gov/30531141","citation_count":0,"is_preprint":false},{"pmid":"39097276","id":"PMC_39097276","title":"[Clinical and genetic analysis of three children with Legius syndrome due to variants of SPRED1 gene].","date":"2024","source":"Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/39097276","citation_count":0,"is_preprint":false},{"pmid":"40948827","id":"PMC_40948827","title":"Enhancing the sensitivity of lung adenocarcinoma to immune therapeutic agents through SPRED1.","date":"2025","source":"Translational lung cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/40948827","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.10.09.24314324","title":"Genomic ascertainment to quantify prevalence and cancer risk in adults with pathogenic and likely pathogenic germline variants in RASopathy genes","date":"2024-10-11","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.09.24314324","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.24.605021","title":"<i>Mitf</i>  over-expression leads to microphthalmia and coloboma in  <i>Mitf-cre</i>  mice","date":"2024-07-25","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.24.605021","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":43267,"output_tokens":7541,"usd":0.121458,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16882,"output_tokens":5521,"usd":0.111217,"stage2_stop_reason":"end_turn"},"total_usd":0.232675,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype (Legius syndrome). SPRED1 acts as a negative regulator of the RAS→RAF interaction and MAPK signaling pathway. Biallelic SPRED1 inactivation (germline + somatic mutation) was required to generate café-au-lait spots, establishing a tumor-suppressor two-hit mechanism.\",\n      \"method\": \"Germline mutation identification, somatic mutation analysis of melanocytes from café-au-lait spots\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct mutation identification with somatic second-hit confirmed in patient tissue, replicated across multiple families, foundational study\",\n      \"pmids\": [\"17704776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SPRED1 negatively regulates IL-3-induced ERK/MAP kinase activation and cell proliferation in hematopoietic cells. Forced expression of wild-type SPRED1 reduced proliferation and ERK activation in response to both SCF and IL-3; a dominant-negative form (DeltaC-Spred) augmented both. SPRED1-deficient mast cells showed augmented ERK activation and proliferation in response to IL-3.\",\n      \"method\": \"Retroviral gene transfer, loss-of-function (Spred1 knockout cells), dominant-negative overexpression, proliferation assays, ERK phosphorylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal gain- and loss-of-function with biochemical readout, multiple cell types\",\n      \"pmids\": [\"15465815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SPRED1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness by suppressing IL-5-dependent ERK activation and cell proliferation in eosinophils, without affecting T helper cell differentiation.\",\n      \"method\": \"Spred1-deficient mice, allergen challenge model, biochemical ERK activation assays, cell proliferation assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout mouse model with defined cellular and biochemical phenotype, multiple orthogonal assays\",\n      \"pmids\": [\"15630138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SPRED1 localizes in lipid raft/caveola membrane fractions and interacts with caveolin-1 (Cav-1). Co-localization with Cav-1 enhances ERK inhibition beyond either protein alone; forced Cav-1 expression in hematopoietic cells suppressed SCF/IL-3-induced ERK activation, and additional Spred-1 expression further suppressed it.\",\n      \"method\": \"Subcellular fractionation, co-immunoprecipitation, overexpression in hematopoietic cells, ERK activation assays\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus functional rescue, single lab, multiple methods\",\n      \"pmids\": [\"16115197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structure of the Xenopus tropicalis Spred1 EVH1 domain solved to 1.15 Å resolution. The EVH1 domain adopts a pleckstrin-homology fold; a narrowed peptide-binding groove and structural flexibility at one end suggest Spred1 binds peptides less proline-rich than other EVH1 domains, likely via an induced-fit mechanism.\",\n      \"method\": \"X-ray crystallography\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — high-resolution crystal structure, single lab but unambiguous structural data\",\n      \"pmids\": [\"15710406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SPRED1 is a binding partner of both MARKK/TAO1 and TESK1 kinases, linking microtubule and actin cytoskeleton regulation. SPRED1-MARKK binding does not affect MARKK activity or microtubule stability. SPRED1-TESK1 binding inhibits TESK1, which normally phosphorylates cofilin to stabilize F-actin stress fibers; thus SPRED1 promotes dynamic F-actin through TESK1 inhibition.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, kinase activity assays, CHO cell overexpression\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus co-IP plus functional assays, single lab\",\n      \"pmids\": [\"18216281\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Spred1 deficiency in mice causes defects in hippocampus-dependent learning, short- and long-term synaptic plasticity (including LTP/LTD imbalance in CA1), and increased ERK phosphorylation after LTP induction, demonstrating that SPRED1 limits Ras/ERK hyperactivation to maintain normal cognitive function and synaptic plasticity.\",\n      \"method\": \"Spred1 knockout mice, Morris water maze, T-maze, electrophysiology (LTP/LTD recordings), western blot for pERK\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout mouse, behavioral testing plus electrophysiology plus biochemistry, multiple orthogonal methods\",\n      \"pmids\": [\"19118178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SPRED1 is highly enriched in CNS germinal zones and functions to dampen neural stem cell (NSC) self-renewal and progenitor proliferation cell-autonomously. SPRED1 localizes within distinct vesicles in cortical progenitor cells. Knockdown in vivo in embryonic mouse forebrain disrupts apical ventricular zone organization, impairs radial glia alignment, and causes periventricular heterotopia due to impaired late neuronal migration.\",\n      \"method\": \"shRNA knockdown in vivo and in vitro, overexpression, immunofluorescence/live imaging, subcellular fractionation/vesicle localization, cortical progenitor proliferation assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockdown plus overexpression with defined cellular phenotype, in vivo and in vitro, multiple orthogonal methods\",\n      \"pmids\": [\"20047999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SPRED1 and SPRED2 directly interact with the novel kinase DYRK1A via their cysteine-rich domain (CRD), which binds the kinase domain of DYRK1A. SPRED1/2 inhibit DYRK1A-mediated phosphorylation of substrates Tau and STAT3 by competing for the substrate binding site on DYRK1A.\",\n      \"method\": \"Co-immunoprecipitation (endogenous and overexpressed), direct binding assays, kinase substrate phosphorylation assays, domain mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous co-IP plus functional kinase assays, single lab\",\n      \"pmids\": [\"20736167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SPRED1 is a substrate of the tyrosine phosphatase SHP2. SPRED proteins (but not Sprouty2) bind to the tyrosine phosphatase domain of SHP2. Overexpressed SHP2 dephosphorylates SPRED1. Two tyrosine residues on SPRED1, when phosphorylated, are required for Ras/ERK inhibition; Tyr-420 is a specific dephosphorylation target of SHP2, indicating that SHP2 attenuates the inhibitory activity of SPRED1.\",\n      \"method\": \"Co-immunoprecipitation, in vitro dephosphorylation assay, site-directed mutagenesis, ERK activation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzyme assay plus mutagenesis plus binding domain mapping, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"21531714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SPRED1 interacts with FGFRL1 via the C-terminal SPR domain of SPRED1 and the histidine-rich domain of FGFRL1. This interaction was verified by coprecipitation and co-distribution at the plasma membrane; SPRED1 increased the retention time of FGFRL1 at the plasma membrane.\",\n      \"method\": \"Yeast two-hybrid, coprecipitation, co-localization by immunofluorescence in COS1 and HEK293 cells\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid confirmed by co-precipitation and co-localization, single lab\",\n      \"pmids\": [\"21616146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"miR-126 positively regulates mast cell proliferation and FcεRI-mediated cytokine production by suppressing SPRED1 expression. Overexpression of miR-126 suppressed SPRED1 and enhanced ERK activity. Hematopoietic-specific Spred1 conditional knockout mice showed increased mast cell numbers and hyperactivation, establishing SPRED1 as the functional target of miR-126 in this context.\",\n      \"method\": \"miR-126 overexpression, conditional Spred1 knockout mice, SPRED1 western blot, ERK assays, cytokine production assays\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout confirms functional target, complemented by overexpression and biochemical assays\",\n      \"pmids\": [\"21668589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The SPRED1 EVH1 domain interacts with the N-terminal 16 aa and C-terminal 20 aa of the GTPase-activating protein-related domain (GRD) of neurofibromin (NF1), forming two crossing α-helix coils outside the GAP catalytic domain. These regions are dispensable for GAP activity and are absent from p120GAP. Pathogenic missense mutations in the EVH1 domain of SPRED1 (Legius syndrome) and in the GRD (NF1 patients) reduce binding affinity and disrupt ERK suppression, showing SPRED1 inhibits Ras-ERK by recruiting neurofibromin to Ras via EVH1-GRD interaction.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation in HEK293 cells, ERK activation assays, mutational analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus reciprocal co-IP plus mutagenesis plus functional ERK assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"26635368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The SPRED1 EVH1 domain binds specifically to the noncatalytic GAPex subdomain of the neurofibromin GRD. This binding is compatible with simultaneous Ras binding and does not interfere with neurofibromin's GAP activity, establishing that SPRED1 functions as a membrane-targeting scaffold for neurofibromin rather than a direct modulator of its catalytic activity.\",\n      \"method\": \"Biochemical binding assays, mutational analysis, GAP activity assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical reconstitution of binding and GAP activity assays, independently consistent with PMID:26635368\",\n      \"pmids\": [\"27313208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPRED1 interaction with B-Raf and galectin-1 (Gal-1) facilitates SPRED1 translocation to the plasma membrane upon growth factor stimulation or pharmacological/Gal-1-mediated Raf dimer induction. At the plasma membrane, SPRED1 selectively perturbs membrane organization and ERK signaling of K-ras4B but not H-ras, and blocks positive effects of Gal-1 on H-ras nanoclusters. Two Legius syndrome mutations show diminished binding to both Gal-1 and B-Raf.\",\n      \"method\": \"Co-immunoprecipitation, super-resolution microscopy, FRET-based nanoclustering assays, mutational analysis, plasma membrane fractionation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus nanoscale membrane assays plus mutagenesis, single lab\",\n      \"pmids\": [\"27503857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPRED1 functions as a tumor suppressor in mucosal melanoma, particularly in the context of KIT mutations. SPRED1 knockdown in zebrafish caused MAPK activation, increased cell proliferation, and conferred resistance to KIT tyrosine kinase inhibitors, establishing SPRED1 loss as a driver of KIT-mutant melanoma through MAPK pathway activation.\",\n      \"method\": \"Human tumor sequencing (43 mucosal melanomas), tissue-specific CRISPR in zebrafish, MAPK activation assays, cell proliferation assays, drug resistance assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo CRISPR modeling plus biochemical readout, human genomic data, multiple orthogonal methods\",\n      \"pmids\": [\"30385465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPRED1 negatively regulates hematopoietic stem cell (HSC) self-renewal and fitness under steady-state conditions, in part through Rho kinase (ROCK) activity. Under high-fat diet conditions, Spred1 deficiency leads to ERK hyperactivation and aberrant HSC self-renewal, causing functional HSC failure and myeloproliferative neoplasm-like disease.\",\n      \"method\": \"Spred1 conditional knockout mice, HSC functional assays, ERK activation assays, high-fat diet model\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout in multiple compartments plus biochemical pathway analysis, multiple phenotypic readouts\",\n      \"pmids\": [\"29706577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A pathogenic Legius syndrome missense mutation in the EVH1 domain of SPRED1 (T102R) weakens the interaction with neurofibromin by approximately 3 orders of magnitude without perturbing the overall EVH1 protein fold. NMR spectroscopy was used to map the neurofibromin GRD binding site on the mutant Spred1 EVH1 domain.\",\n      \"method\": \"Binding affinity measurements, NMR spectroscopy, protein stability assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural mapping plus quantitative binding measurements, single lab with rigorous biophysical methods\",\n      \"pmids\": [\"31401120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal/cryo structure of the neurofibromin GAP-related domain complexed with the SPRED1 EVH1 domain and KRAS reveals how SPRED1 membrane-targeting of neurofibromin allows simultaneous interaction with activated KRAS. Oncogenic EGFR(L858R) signaling leads to phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex. Analysis of the interface rationalizes Legius syndrome mutations and explains why SPRED1 binds neurofibromin but no other RasGAPs.\",\n      \"method\": \"Crystal structure (X-ray), biochemical binding assays, phosphorylation mapping, mutagenesis, cell-based ERK signaling assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure plus biochemical and functional validation, multiple orthogonal methods in single study\",\n      \"pmids\": [\"32697994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Spred1 and Spred2 double-deficiency in lens results in elevated ERK1/2 phosphorylation, irregular lens epithelial and fiber cell activity, and impaired lens and eye development, demonstrating that Spred1 negatively regulates ERK1/2 activity to maintain normal lens morphogenesis.\",\n      \"method\": \"Spred1/2 double-knockout mice, ERK phosphorylation western blot, histological analysis of lens development\",\n      \"journal\": \"Experimental eye research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse with defined biochemical phenotype, single lab\",\n      \"pmids\": [\"31923414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPRED1 loss in human melanoma cell lines and primary zebrafish melanoma confers resistance to BRAFV600E inhibition by reactivating MAPK activity. Biallelic SPRED1 deletion was observed in a patient whose melanoma acquired resistance to MAPK-targeted therapy.\",\n      \"method\": \"SPRED1 inactivation in human cell lines, in vivo zebrafish melanoma model, MAPK pathway activation assays, drug resistance assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — combined human cell line plus in vivo zebrafish model plus patient sample, multiple orthogonal methods\",\n      \"pmids\": [\"33306107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Spred1 deficiency in CML promotes transformation from chronic phase to accelerated/blast crisis CML. Spred1 knockout (in HSCs or endothelial cells) increased miR-126 in leukemic stem cells (LSKs), causing LSC expansion through MAPK/ERK hyperactivation, augmented Bcl-2 expression and stability, and enhanced Bcl-2-dependent oxidative phosphorylation.\",\n      \"method\": \"Conditional knockout mouse models, leukemia transformation assays, miR-126 quantification, pERK assays, Bcl-2 expression/stability assays\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple conditional KO models with mechanistic pathway dissection, single lab\",\n      \"pmids\": [\"34564700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Spred1-deficient mice exhibit increased social dominance and impaired nesting behavior mediated by RAS-MAPK hyperactivation. Acute MEK inhibitor (PD325901) treatment in adulthood reversed the enhanced social dominance phenotype, demonstrating that Ras-MAPK dysregulation mediates social behavior phenotypes downstream of SPRED1 loss.\",\n      \"method\": \"Spred1 knockout mice, automated tube test, ultrasonic vocalization recording, nesting behavior, MEK inhibitor pharmacological rescue\",\n      \"journal\": \"Molecular autism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse behavioral phenotype with pharmacological pathway rescue, single lab\",\n      \"pmids\": [\"34311771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Molecular dynamics simulation and Markov state model analysis of the KRAS-NF1-SPRED1 ternary complex suggest that SPRED1 loading allosterically enhances KRAS-NF1 binding at a site distal from the SPRED1-NF1 interface, but that this allosteric effect also hinders conformational transformation of the NF1 catalytic center required for RAS GTP hydrolysis. Allosteric pathways were identified by difference contact network analysis.\",\n      \"method\": \"Gaussian accelerated molecular dynamics simulations, Markov state model analysis, contact network analysis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational modeling only, no experimental validation reported\",\n      \"pmids\": [\"35872068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPRED1 negatively regulates proliferation, apoptosis, and cytokine secretion of group 2 innate lymphoid cells (ILC2s) through the Ras-ERK pathway. Spred1 deficiency enhanced IL-33-induced ILC2 proliferation and cytokine production; MEK inhibitor suppressed these. Spred1 deficiency resulted in stabilization of GATA3, a key transcription factor for ILC2 function.\",\n      \"method\": \"Spred1 knockout mice, ILC2 proliferation and cytokine assays, MEK inhibitor treatment, GATA3 protein stability assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout mouse plus pharmacological rescue plus GATA3 mechanistic link, single lab\",\n      \"pmids\": [\"26116510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Missense mutations in the C-terminal SPR (Sprouty-related) domain of SPRED1 (e.g., C368S, M369L, V408E, P415A, P415L, P422R) identified in Legius syndrome patients impair palmitoylation of SPRED1, causing loss of plasma membrane localization and formation of cytoplasmic granular aggregates. In knock-in mice, P415A and P415V mutations (but not a C-terminal deletion M417Afs*4) formed granular aggregates and led to Purkinje cell loss and cerebellar ataxia with age. Autophagy inducer spermidine rescued ataxia and Purkinje cell loss in Spred1P415A mice, suggesting aggregate toxicity as a potential neurodegenerative mechanism.\",\n      \"method\": \"Site-directed mutagenesis, knock-in mice, palmitoylation assay, live cell imaging/localization, histological analysis, behavioral (ataxia) testing, spermidine treatment rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro mutagenesis plus knock-in mouse models plus pharmacological rescue, multiple orthogonal methods\",\n      \"pmids\": [\"39510187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"EZH2 epigenetically suppresses SPRED1 transcriptional activity and expression; the EZH2 inhibitor Tazemetostat reverses this suppression. Overexpression of SPRED1 inhibits lung adenocarcinoma cell proliferation, migration, and invasion in vitro and retards tumor growth in vivo, while EZH2 elevation rescues these effects.\",\n      \"method\": \"Dual-luciferase reporter assay, EZH2 inhibitor treatment, overexpression gain-of-function, in vivo xenograft\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — luciferase assay plus functional rescue plus in vivo, single lab\",\n      \"pmids\": [\"36629984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Merlin (NF2 protein) can interact with both neurofibromin and SPRED1, forming a trimeric complex. Merlin directly binds the Ras-binding domain (RBD) and kinase domain (KiD) of Raf1, functioning as a 'selective Ras barrier' that blocks Ras binding to Raf1 without increasing neurofibromin's GAP activity.\",\n      \"method\": \"Co-immunoprecipitation, direct binding assays, RAS-GAP activity assays, Schwann cell models\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus GAP activity assays, single lab, multiple interactors mapped\",\n      \"pmids\": [\"33331896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"NF1 and SPRED1/2 cooperate through RAS-MAPK-independent functions. CRISPR-Cas9 ablation of NF1 or SPRED1/2 in 'RASless' MEFs showed that SPRED1/2 loss phenocopies NF1 loss and their cooperation modulates MAPK-AKT signaling. Additionally, NF1 or SPRED1/2 loss suppressed the RAS family GTPases RRAS and RRAS2 independently of RAS or AKT pathway activation. A RAS-independent, NF1-SPRED1/2-dependent gene signature was identified.\",\n      \"method\": \"CRISPR-Cas9 knockout in isogenic RASless MEFs, transcriptome microarray, signaling pathway assays, Schwann cell models from NF1 patients\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — isogenic CRISPR system plus transcriptomics plus patient-derived cells, single lab\",\n      \"pmids\": [\"42201959\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPRED1 is a negative regulator of the RAS-MAPK/ERK signaling pathway that functions primarily by recruiting neurofibromin (NF1 protein, a RasGAP) from the cytosol to membrane-anchored active RAS via a direct interaction between the SPRED1 EVH1 domain and the noncatalytic GAPex subdomain of neurofibromin's GAP-related domain; the C-terminal SPR domain mediates palmitoylation-dependent plasma membrane localization. SPRED1 can also inhibit signaling at the Ras/Raf interface (interacting with B-Raf and galectin-1 in a Ras isoform-selective manner), is a substrate of SHP2 (whose dephosphorylation of Tyr-420 attenuates SPRED1's inhibitory activity), interacts with DYRK1A (inhibiting its kinase activity via CRD-kinase domain contact) and TESK1 (inhibiting cofilin phosphorylation to promote dynamic F-actin), and colocalizes with caveolin-1 in lipid rafts for cooperative ERK suppression. Structural studies reveal that the EVH1 domain adopts a pleckstrin-homology fold that simultaneously allows neurofibromin binding and KRAS engagement, and oncogenic EGFR can disrupt this complex by phosphorylating SPRED1 at Ser105. Loss-of-function mutations in SPRED1 cause Legius syndrome through constitutive RAS-MAPK hyperactivation, and somatic SPRED1 loss acts as a tumor suppressor mechanism in melanoma and other cancers.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPRED1 is a negative regulator of RAS-MAPK/ERK signaling that limits proliferation, self-renewal, and ERK-driven responses across hematopoietic, neural, immune, and ocular cell types [#1, #2, #6, #16]. Its central mechanism is a scaffolding one: the N-terminal EVH1 domain, which adopts a pleckstrin-homology fold [#4], directly engages the noncatalytic GAPex region of the neurofibromin (NF1) GTPase-activating domain, thereby recruiting neurofibromin to membrane-anchored active KRAS without altering neurofibromin's intrinsic GAP catalysis [#12, #13, #18]. Structural and biophysical analyses show this EVH1-GRD interface simultaneously accommodates KRAS, explains why SPRED1 binds neurofibromin but not other RasGAPs, and rationalizes Legius syndrome mutations that abolish binding and ERK suppression [#17, #18]. SPRED1 activity is controlled by post-translational and membrane-targeting inputs: the C-terminal SPR domain requires palmitoylation for plasma-membrane localization [#25], oncogenic EGFR disrupts the neurofibromin complex by phosphorylating Ser105 [#18], and SHP2 dephosphorylates Tyr-420 to attenuate SPRED1's inhibitory function [#9]. Beyond RAS regulation, SPRED1 inhibits the kinases TESK1 and DYRK1A through its cysteine-rich domain, linking it to cytoskeletal dynamics and substrate phosphorylation [#5, #8]. Germline loss-of-function mutations in SPRED1 cause Legius syndrome through constitutive RAS-MAPK hyperactivation [#0], and somatic SPRED1 loss reactivates MAPK signaling to drive melanoma progression and resistance to KIT and BRAF inhibitors [#15, #20].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established SPRED1 as a cell-intrinsic negative regulator of growth-factor-induced ERK activation, defining its core biological function.\",\n      \"evidence\": \"Reciprocal gain/loss-of-function with dominant-negative and knockout hematopoietic cells, ERK and proliferation assays\",\n      \"pmids\": [\"15465815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular target within the RAS-ERK cascade not yet identified\", \"No structural basis for inhibition\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Extended SPRED1's ERK-suppressing role to allergic airway inflammation and tied its activity to membrane microdomains via caveolin-1.\",\n      \"evidence\": \"Spred1-knockout mouse allergen model; subcellular fractionation and co-IP with Cav-1 plus ERK assays\",\n      \"pmids\": [\"15630138\", \"16115197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular partner mediating ERK inhibition still unknown\", \"Cav-1 interaction is single-lab, mechanism of cooperative suppression unresolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Resolved the EVH1 domain fold, revealing a pleckstrin-homology architecture and a peptide-binding groove distinct from other EVH1 domains.\",\n      \"evidence\": \"1.15 Å X-ray crystal structure of Xenopus Spred1 EVH1\",\n      \"pmids\": [\"15710406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding partner of the EVH1 groove not yet identified\", \"Structure from Xenopus ortholog, not human\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified SPRED1 loss-of-function as the cause of Legius syndrome, establishing it as a tumor suppressor acting through a two-hit mechanism on RAS-MAPK signaling.\",\n      \"evidence\": \"Germline and somatic mutation analysis in patient melanocytes\",\n      \"pmids\": [\"17704776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism linking mutations to RAS hyperactivation not yet defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Connected SPRED1 to cytoskeletal regulation by showing it inhibits TESK1 to promote dynamic F-actin, broadening its role beyond ERK.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, kinase assays in CHO cells\",\n      \"pmids\": [\"18216281\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of cytoskeletal role in vivo unclear\", \"Single-lab; MARKK/TAO1 binding has no functional consequence\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated that SPRED1 restrains Ras/ERK in the brain to maintain synaptic plasticity and learning, linking the gene to cognitive function.\",\n      \"evidence\": \"Spred1-knockout mice, behavioral testing, LTP/LTD electrophysiology, pERK western blot\",\n      \"pmids\": [\"19118178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type and circuit specificity of ERK dysregulation not dissected\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Showed SPRED1 dampens neural stem/progenitor proliferation and is required for cortical organization, and that it inhibits DYRK1A via its CRD.\",\n      \"evidence\": \"In vivo/in vitro shRNA knockdown, imaging, vesicle fractionation; co-IP and kinase substrate assays for DYRK1A\",\n      \"pmids\": [\"20047999\", \"20736167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Vesicular localization function not mechanistically explained\", \"DYRK1A inhibition is Medium-confidence, single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined upstream regulation of SPRED1 by SHP2-mediated dephosphorylation and by miR-126, and identified an FGFRL1 membrane interaction, showing SPRED1 activity is tunable.\",\n      \"evidence\": \"In vitro dephosphorylation and mutagenesis (SHP2); conditional knockout and miRNA overexpression (miR-126); yeast two-hybrid and co-localization (FGFRL1)\",\n      \"pmids\": [\"21531714\", \"21668589\", \"21616146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of FGFRL1 retention unclear\", \"How phosphorylation at Tyr residues couples to neurofibromin recruitment not yet established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the EVH1–neurofibromin GRD interface and established that SPRED1 acts as a membrane-targeting scaffold for neurofibromin rather than a direct GAP modulator.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal co-IP, mutagenesis, ERK assays, and biochemical reconstitution of binding and GAP activity\",\n      \"pmids\": [\"26635368\", \"27313208\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the ternary SPRED1-neurofibromin-RAS complex not yet solved\", \"Membrane delivery dynamics not directly visualized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed isoform-selective regulation through B-Raf and galectin-1 binding, with SPRED1 perturbing KRAS but not HRAS membrane organization.\",\n      \"evidence\": \"Co-IP, super-resolution and FRET nanoclustering assays, mutagenesis\",\n      \"pmids\": [\"27503857\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab; mechanism of isoform selectivity at the membrane unresolved\", \"Relationship to neurofibromin scaffolding pathway unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Quantified how a Legius mutation cripples neurofibromin binding without unfolding EVH1, pinpointing affinity loss as the disease mechanism.\",\n      \"evidence\": \"NMR mapping, binding affinity and protein stability measurements of the T102R EVH1 mutant\",\n      \"pmids\": [\"31401120\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Single mutation analyzed\", \"Cellular and organismal consequences not measured in this study\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the structural basis for SPRED1's RasGAP specificity and revealed EGFR-driven Ser105 phosphorylation as a means to disrupt the complex.\",\n      \"evidence\": \"X-ray structure of NF1 GRD–SPRED1 EVH1–KRAS, phosphorylation mapping, mutagenesis, ERK assays\",\n      \"pmids\": [\"32697994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and kinetics of complex assembly at the membrane not defined\", \"Generality of Ser105 phosphorylation across RTKs unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed SPRED1 (with SPRED2) is required for lens morphogenesis by restraining ERK1/2, extending its developmental role to the eye.\",\n      \"evidence\": \"Spred1/2 double-knockout mice, ERK western blot, histology\",\n      \"pmids\": [\"31923414\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Redundancy with SPRED2 not separated for SPRED1 alone\", \"Single-lab\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established somatic SPRED1 loss as a melanoma tumor-suppressor event and as a driver of KIT-inhibitor resistance through MAPK reactivation.\",\n      \"evidence\": \"Human tumor sequencing, tissue-specific zebrafish CRISPR, MAPK and drug-resistance assays\",\n      \"pmids\": [\"30385465\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of resistance downstream of MAPK reactivation not fully resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined SPRED1 as a restraint on hematopoietic stem cell self-renewal whose loss, under metabolic stress, produces myeloproliferative disease.\",\n      \"evidence\": \"Spred1 conditional knockout mice, HSC functional and ERK assays, high-fat diet model\",\n      \"pmids\": [\"29706577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Contribution of ROCK versus ERK arms not quantitatively separated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed SPRED1 loss drives MAPK-targeted-therapy resistance in melanoma and promotes CML transformation via miR-126/ERK/Bcl-2 signaling, and that NF2/Merlin can join the neurofibromin-SPRED1 axis.\",\n      \"evidence\": \"Human cell lines, zebrafish models, patient sample (BRAFi resistance); conditional KO and pathway dissection (CML); co-IP and GAP assays (Merlin)\",\n      \"pmids\": [\"33306107\", \"34564700\", \"33331896\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Merlin trimeric complex is Medium-confidence, single lab\", \"Whether miR-126 and Bcl-2 axis generalizes beyond CML unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Linked SPRED1 loss to social behavior and immune (ILC2) phenotypes mediated by RAS-MAPK, with pharmacological MEK inhibition reversing behavioral effects.\",\n      \"evidence\": \"Spred1-knockout mice, behavioral testing with MEK-inhibitor rescue; ILC2 assays with GATA3 stability analysis\",\n      \"pmids\": [\"34311771\", \"26116510\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab phenotypes\", \"Direct molecular link between SPRED1 and GATA3 stability not established\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Showed that SPR-domain mutations impairing palmitoylation cause SPRED1 mislocalization and toxic cytoplasmic aggregates, defining a neurodegenerative mechanism distinct from simple RAS hyperactivation.\",\n      \"evidence\": \"Mutagenesis, knock-in mice, palmitoylation and localization assays, ataxia testing, spermidine rescue; EZH2 transcriptional suppression by luciferase and xenograft\",\n      \"pmids\": [\"39510187\", \"36629984\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"EZH2 regulation is Medium-confidence, single lab\", \"Link between aggregate toxicity and Purkinje loss mechanism incompletely defined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed RAS-MAPK-independent cooperation between NF1 and SPRED1/2, including suppression of RRAS/RRAS2 and a RAS-independent gene signature, broadening SPRED1's functional scope.\",\n      \"evidence\": \"CRISPR knockout in isogenic RASless MEFs, transcriptomics, signaling assays, NF1 patient Schwann cells\",\n      \"pmids\": [\"42201959\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of RRAS/RRAS2 suppression undefined\", \"Single-lab; RAS-independent signature not functionally validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SPRED1 integrates its many regulatory inputs (palmitoylation, Tyr/Ser phosphorylation, miRNA, EZH2) to dynamically control neurofibromin delivery at the membrane, and the molecular basis of its RAS-independent functions, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model of membrane-targeting kinetics in vivo\", \"RAS-independent NF1-SPRED1 mechanism uncharacterized\", \"Interplay between scaffolding and kinase-inhibitory (DYRK1A/TESK1) functions unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 12, 13, 18]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [12, 13, 18]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [10, 14, 18, 25]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12, 25]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 12, 18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 15, 20]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [6, 7, 19]}\n    ],\n    \"complexes\": [\"SPRED1–neurofibromin(NF1)–KRAS ternary complex\"],\n    \"partners\": [\"NF1\", \"KRAS\", \"SHP2\", \"DYRK1A\", \"TESK1\", \"CAV1\", \"BRAF\", \"LGALS1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}