{"gene":"SPRED1","run_date":"2026-04-28T20:42:08","timeline":{"discoveries":[{"year":2007,"finding":"Germline loss-of-function mutations in SPRED1 cause a neurofibromatosis 1-like phenotype (Legius syndrome) by impairing negative regulation of RAS→RAF interaction and MAPK signaling; melanocytes from café-au-lait spots showed biallelic SPRED1 inactivation (germline + somatic mutation), indicating complete loss is required for the phenotype.","method":"Germline mutation identification + somatic mutation analysis in melanocytes (LOH/second-hit analysis)","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — foundational discovery paper with mutational and LOH evidence, widely replicated","pmids":["17704776"],"is_preprint":false},{"year":2004,"finding":"SPRED1 negatively regulates IL-3- and SCF-induced ERK/MAP kinase activation and cell proliferation in hematopoietic cells; a dominant-negative SPRED1 (ΔC-Spred) augmented IL-3-induced ERK activation, and Spred-1-deficient bone marrow mast cells showed augmented ERK activation in response to IL-3.","method":"Retroviral gene transfer of wild-type and dominant-negative SPRED1 into hematopoietic cell lines; Spred1-deficient bone marrow mast cells; ERK activation assays; colony formation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (dominant-negative, KO cells, proliferation/ERK assays)","pmids":["15465815"],"is_preprint":false},{"year":2005,"finding":"Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness by suppressing IL-5-dependent cell proliferation and ERK activation in eosinophils.","method":"Spred-1-deficient mice; allergen challenge model; biochemical ERK activation assays; cell proliferation assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — KO mouse model with defined cellular phenotype and biochemical readout, orthogonal methods","pmids":["15630138"],"is_preprint":false},{"year":2005,"finding":"Spred-1 localizes to lipid raft/caveola membrane fractions and physically interacts with caveolin-1 (Cav-1), collaborating with Cav-1 to inhibit SCF- and IL-3-induced ERK activation and cell proliferation.","method":"Subcellular fractionation (lipid raft isolation); co-immunoprecipitation; forced expression of Cav-1 and Spred-1 in hematopoietic cells; proliferation and ERK activation assays","journal":"Genes to cells","confidence":"Medium","confidence_rationale":"Tier 2/3 — co-IP and fractionation with functional readout, single lab","pmids":["16115197"],"is_preprint":false},{"year":2005,"finding":"Crystal structure of the Xenopus tropicalis Spred1 EVH1 domain (1.15 Å resolution) reveals a pleckstrin-homology fold with a narrowed peptide-binding groove, suggesting a distinct peptide-binding mechanism compared to other EVH1 domains, likely binding less proline-rich peptides.","method":"X-ray crystallography at 1.15 Å resolution","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure","pmids":["15710406"],"is_preprint":false},{"year":2008,"finding":"SPRED1 physically interacts with MARKK/TAO1 kinase (no effect on MARKK activity) and with TESK1 kinase; Spred1 binding to TESK1 inhibits TESK1, thereby preventing TESK1-mediated phosphorylation of cofilin and making F-actin stress fibers dynamic. This three-way Spred1/MARKK/TESK1 interaction links regulation of both microtubule and F-actin cytoskeleton.","method":"Yeast two-hybrid; co-immunoprecipitation; kinase activity assays; overexpression in CHO cells with cytoskeletal readouts","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — multiple binding/activity assays, single lab","pmids":["18216281"],"is_preprint":false},{"year":2008,"finding":"Spred1 deficiency in mice causes defects in hippocampus-dependent learning and memory, short- and long-term synaptic plasticity (including LTP/LTD imbalance in CA1), and increased ERK phosphorylation after LTP induction, demonstrating that Spred1 suppresses the Ras/ERK pathway in neurons to regulate synaptic plasticity.","method":"Spred1-/- mice; Morris water maze; T-maze; electrophysiological recordings (LTP/LTD); biochemical ERK phosphorylation assays","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — KO mouse with multiple behavioral, electrophysiological, and biochemical endpoints","pmids":["19118178"],"is_preprint":false},{"year":2010,"finding":"Spred1 is highly enriched in CNS germinal zones; knockdown increases neural stem cell self-renewal and progenitor proliferation cell-autonomously, while overexpression causes premature differentiation. In vivo knockdown disrupts apical ventricular zone integrity, impairs late neuronal migration, and causes periventricular heterotopia. Spred1 localizes within distinct vesicles in cortical progenitors.","method":"In utero electroporation knockdown/overexpression; EdU proliferation assays; live imaging; subcellular localization (vesicle fractionation/immunofluorescence); Spred1-/- mice","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (KD, OE, localization, in vivo), defined cellular phenotypes","pmids":["20047999"],"is_preprint":false},{"year":2010,"finding":"SPRED1 and SPRED2 directly interact with DYRK1A kinase via their CRD domain binding to the kinase domain of DYRK1A, and this interaction competitively inhibits DYRK1A-mediated phosphorylation of its substrates Tau and STAT3.","method":"Yeast two-hybrid; endogenous co-immunoprecipitation; in vitro kinase activity assays; domain mapping (CRD required)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal binding and in vitro kinase assay, single lab","pmids":["20736167"],"is_preprint":false},{"year":2011,"finding":"SPRED1 is a substrate of the tyrosine phosphatase SHP2: SPRED1 binds to the catalytic (phosphatase) domain of SHP2 (unlike Sprouty2 which binds the C-terminal tail), SHP2 dephosphorylates SPRED1, and two tyrosine residues on SPRED1 (including Tyr-420) are required when phosphorylated to inhibit Ras/ERK activation. SHP2-mediated dephosphorylation of Tyr-420 attenuates SPRED1's inhibitory action.","method":"Co-immunoprecipitation; in vitro dephosphorylation assays; site-directed mutagenesis of SPRED1 tyrosines; ERK activation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro enzymatic assay + mutagenesis + binding domain mapping","pmids":["21531714"],"is_preprint":false},{"year":2011,"finding":"FGFRL1 interacts with SPRED1 via the SPR (Sprouty) domain of SPRED1 binding to the C-terminal histidine-rich domain of FGFRL1; this interaction was verified by co-precipitation and co-distribution at the plasma membrane. Spred1 increases the retention time of FGFRL1 at the plasma membrane.","method":"Yeast two-hybrid; co-precipitation; co-localization at cell membrane in COS1 and HEK293 cells; truncation experiments","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 3 — yeast 2-hybrid + co-precipitation + localization, single lab","pmids":["21616146"],"is_preprint":false},{"year":2011,"finding":"miR-126 positively regulates mast cell proliferation and cytokine production by suppressing Spred1 expression; Spred1 overexpression or deficiency inversely regulated ERK activity and FcεRI-mediated cytokine production. Hematopoietic cell-specific Spred1 conditional KO mice showed increased mast cell numbers and heightened activation.","method":"miR-126 overexpression; Spred1 conditional knockout mice; ERK activity assays; cytokine production assays","journal":"Genes to cells","confidence":"High","confidence_rationale":"Tier 2 — conditional KO mouse model with defined cellular phenotype + mechanistic biochemistry","pmids":["21668589"],"is_preprint":false},{"year":2015,"finding":"The SPRED1 EVH1 domain interacts with the N-terminal 16 and C-terminal 20 amino acids of the GAP-related domain (GRD) of neurofibromin (two crossing α-helix coils outside the GAP catalytic site, not present in p120GAP). Pathogenic missense mutations in the EVH1 domain of SPRED1 (Legius syndrome) and in these GRD regions (NF1) reduce binding affinity. EVH1 domain mutations that disrupt GRD binding also abolish SPRED1's ERK suppression activity.","method":"Yeast two-hybrid; co-immunoprecipitation in HEK293 cells; domain mapping with truncations; mutagenesis; ERK suppression assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Y2H, Co-IP, mutagenesis, functional assay), clear mechanistic link","pmids":["26635368"],"is_preprint":false},{"year":2016,"finding":"The EVH1 domain of SPRED1 binds to the noncatalytic GAPex subdomain of neurofibromin's GRD; this binding is compatible with simultaneous Ras binding and does not interfere with neurofibromin's GAP activity, supporting a targeting/recruitment function rather than GAP modulation.","method":"Biochemical binding assays; domain mapping; GAP activity assays in the presence and absence of Spred1","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro reconstitution with functional (GAP) activity assay, clear mechanistic finding","pmids":["27313208"],"is_preprint":false},{"year":2016,"finding":"SPRED1 is translocated to the plasma membrane upon growth factor stimulation or pharmacological/galectin-1-mediated induction of B/C-Raf dimers; this translocation involves SPRED1 interaction with B-Raf and, via its N terminus, with galectin-1 (Gal-1). On the plasma membrane, SPRED1 perturbs membrane organization and ERK signaling specifically of active K-ras4B but not H-ras. Legius syndrome mutations show diminished binding to both Gal-1 and B-Raf.","method":"Live-cell imaging; FRET/FLIM nanoscale membrane analysis; co-immunoprecipitation; pharmacological treatments; mutagenesis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (imaging, co-IP, mutagenesis), single lab","pmids":["27503857"],"is_preprint":false},{"year":2018,"finding":"SPRED1 functions as a tumor suppressor in 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.","method":"CRISPR-based tissue-specific zebrafish melanoma model; human mucosal melanoma sequencing; MAPK activation assays; drug resistance assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — in vivo CRISPR model + mechanistic biochemistry (MAPK activation), replicated in human tumors","pmids":["30385465"],"is_preprint":false},{"year":2018,"finding":"Spred1 safeguards hematopoietic stem cell (HSC) homeostasis partly through Rho kinase (ROCK) activity under steady-state conditions. Under high-fat diet, Spred1 deficiency causes ERK hyperactivation and aberrant HSC self-renewal, leading to functional HSC failure, anemia, and myeloproliferative disease; compensatory upregulation of Spred2 prevents leukemogenesis in steady-state Spred1 KO.","method":"Spred1 knockout mice; HSC transplantation assays; ERK phosphorylation assays; ROCK inhibitor treatment; gut microbiota manipulation","journal":"Cell stem cell","confidence":"High","confidence_rationale":"Tier 2 — in vivo KO model with multiple defined phenotypic readouts and mechanistic pathway analysis","pmids":["29706577"],"is_preprint":false},{"year":2019,"finding":"A pathogenic Legius syndrome missense mutation (T102R) in the EVH1 domain of Spred1 weakens interaction with neurofibromin by ~1000-fold without perturbing the protein fold; NMR spectroscopy identified the binding site of NF1-GAP on the mutant Spred1(EVH1) variant.","method":"NMR spectroscopy; binding affinity measurements; mutagenesis; protein folding analysis","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — NMR structural/binding analysis with mutagenesis and quantitative affinity data","pmids":["31401120"],"is_preprint":false},{"year":2020,"finding":"Crystal/cryo structure of the neurofibromin GRD complexed with SPRED1 EVH1 domain and KRAS reveals how SPRED1 membrane-targets neurofibromin to allow simultaneous interaction with activated KRAS. Oncogenic EGFR(L858R) signaling leads to phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex.","method":"X-ray crystallography (structure of neurofibromin GRD-SPRED1 EVH1-KRAS ternary complex); biochemical binding assays; mutagenesis; cell-based phosphorylation assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biochemical and mutational validation, mechanistic insight into phospho-regulation","pmids":["32697994"],"is_preprint":false},{"year":2021,"finding":"SPRED1 deletion confers resistance to BRAFV600E inhibition in melanoma by reactivating MAPK activity; this was demonstrated in human melanoma cell lines and primary zebrafish melanoma models in vivo.","method":"SPRED1 KO in human melanoma cell lines; zebrafish melanoma in vivo model; MAPK activity assays; BRAF inhibitor treatment","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — combined human cell and in vivo zebrafish model with mechanistic MAPK readout","pmids":["33306107"],"is_preprint":false},{"year":2021,"finding":"Merlin interacts with both neurofibromin and Spred1 in a complex via merlin-binding sites on both proteins; as a third component, merlin blocks Ras binding to Raf1 (acting as a 'selective Ras barrier') without increasing neurofibromin's GAP activity. Merlin-deficient Schwann cells require Ras-ERK pathway for proliferation.","method":"Co-immunoprecipitation; binding domain analysis; Ras-GAP activity assays; cell proliferation assays in merlin-deficient Schwann cells","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2/3 — co-IP with functional assays, single lab","pmids":["33331896"],"is_preprint":false},{"year":2021,"finding":"Spred1 deficiency in CML promotes disease transformation from chronic phase to accelerated/blast crisis CML by expanding leukemic stem cells (LSCs) through hyperactivation of MAPK/ERK, increased miR-126 in LSKs, augmented Bcl-2 expression and stability, and enhanced Bcl-2-dependent oxidative phosphorylation supporting LSC survival.","method":"Spred1 KO in CML mouse models (global, HSC-specific, endothelial cell-specific); ERK phosphorylation; miR-126 quantification; Bcl-2 assays; oxidative phosphorylation measurements","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 — multiple KO models with defined pathway analysis, single lab","pmids":["34564700"],"is_preprint":false},{"year":2022,"finding":"Molecular dynamics simulations and Markov state model analysis revealed that SPRED1 loading allosterically enhances KRAS-NF1 (neurofibromin) binding but hinders conformational transformation of the NF1 catalytic center required for RAS GTP hydrolysis; allosteric signal transduction pathways upon SPRED1 binding were mapped through 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 — computational only, no experimental validation","pmids":["35872068"],"is_preprint":false},{"year":2024,"finding":"Missense mutations in the SPR (Sprouty-related) C-terminal domain of SPRED1 (e.g., C368S, M369L, V408E, P415A, P415L, P422R) cause loss of palmitoylation, loss of plasma membrane localization, and formation of cytoplasmic granular aggregates. Knock-in mice with P415A and P415V (but not M417Afs*4 which lacks the C-terminal region) developed cerebellar ataxia and Purkinje cell loss, which was rescued by the autophagy inducer spermidine.","method":"Mutagenesis of palmitoylation sites; subcellular localization (live imaging/immunofluorescence); knock-in mouse generation; behavioral/cerebellar phenotype analysis; spermidine treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1/2 — mutagenesis + in vivo KI mice + pharmacological rescue, multiple orthogonal methods","pmids":["39510187"],"is_preprint":false},{"year":2015,"finding":"Spred1 negatively regulates the expansion and function of group 2 innate lymphoid cells (ILC2s) through suppression of the Ras-ERK pathway; Spred1 deficiency leads to faster ILC2 proliferation, higher cytokine (IL-5, IL-13) production in response to IL-33, and stabilization of GATA3. A MEK inhibitor suppressed ILC2 proliferation and cytokine production.","method":"Spred1-/- mice; in vivo papain challenge; in vitro ILC2 stimulation; MEK inhibitor treatment; GATA3 protein stability assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with defined pathway/cellular phenotype, pharmacological validation","pmids":["26116510"],"is_preprint":false},{"year":2020,"finding":"Combined loss of Spred1 and Spred2 in mouse lens leads to increased ERK1/2 phosphorylation, irregular lens epithelial and fiber cell activity, and impaired lens and eye development, demonstrating that these proteins negatively regulate ERK1/2 to control lens cell proliferation and differentiation.","method":"Spred1/Spred2 double-KO mice; ERK phosphorylation assays; histological analysis of lens development","journal":"Experimental eye research","confidence":"Medium","confidence_rationale":"Tier 2 — genetic KO model with defined cellular phenotype and biochemical ERK readout","pmids":["31923414"],"is_preprint":false},{"year":2009,"finding":"TGF-β upregulates SPRED1 expression in CD8+ tumor-infiltrating lymphocytes, and elevated SPRED1 correlates with reduced TCR-mediated ITK and ERK kinase activation, contributing to T cell hyporesponsiveness in the tumor microenvironment.","method":"Gene expression analysis; TGF-β inhibitor treatment; ITK and ERK activation assays in TILs vs. splenic CD8+ T cells","journal":"Cancer immunology, immunotherapy","confidence":"Low","confidence_rationale":"Tier 3 — correlative expression + pharmacological inhibitor, no direct KD/KO of SPRED1","pmids":["19319531"],"is_preprint":false}],"current_model":"SPRED1 is a negative regulator of RAS-MAPK/ERK signaling that acts primarily by recruiting neurofibromin (a RasGAP) from the cytosol to membrane-anchored RAS via its N-terminal EVH1 domain binding to the GAPex subdomain of neurofibromin's GRD, while its C-terminal SPR domain mediates palmitoylation-dependent plasma membrane localization; additional regulatory inputs include SHP2-mediated dephosphorylation of SPRED1 Tyr-420 (attenuating its inhibitory function), oncogenic EGFR-driven phosphorylation of Ser-105 (disrupting the SPRED1-neurofibromin complex), interaction with Caveolin-1 to enhance ERK suppression, and interaction with TESK1 to regulate actin cytoskeletal dynamics, with loss-of-function mutations causing Legius syndrome and acting as a tumor suppressor across multiple cancer contexts."},"narrative":{"teleology":[{"year":2004,"claim":"Establishing that SPRED1 is a functional negative regulator of ERK/MAPK signaling in hematopoietic cells answered whether this Sprouty-related protein has pathway-suppressive activity in a physiological context beyond overexpression systems.","evidence":"Dominant-negative SPRED1, wild-type retroviral expression, and Spred1-deficient bone marrow mast cells with ERK and proliferation assays","pmids":["15465815"],"confidence":"High","gaps":["Mechanism of ERK suppression not defined—direct target unknown","Relevance beyond hematopoietic lineage not tested"]},{"year":2005,"claim":"Knockout mouse studies extended SPRED1's inhibitory role to in vivo immune (eosinophilic airway inflammation) and structural (lipid raft/caveola localization with Caveolin-1) contexts, establishing tissue-breadth and membrane compartmentalization of its function.","evidence":"Spred1-/- mice in allergen challenge; subcellular fractionation and Cav-1 co-IP in hematopoietic cells","pmids":["15630138","16115197"],"confidence":"High","gaps":["Caveolin-1 interaction shown in single lab without reciprocal validation","Whether raft localization is essential for SPRED1 function not tested genetically"]},{"year":2005,"claim":"Determination of the EVH1 domain crystal structure revealed a PH-fold with an atypically narrow peptide groove, predicting that SPRED1 recognizes non-canonical (less proline-rich) ligands distinct from classical EVH1 targets.","evidence":"X-ray crystallography of Xenopus tropicalis Spred1 EVH1 at 1.15 Å resolution","pmids":["15710406"],"confidence":"High","gaps":["Cognate binding peptide/partner not identified from the structure alone","Mammalian EVH1 structure not yet determined"]},{"year":2007,"claim":"Identification of germline SPRED1 loss-of-function mutations as the cause of Legius syndrome, with biallelic inactivation in melanocyte café-au-lait spots, established SPRED1 as a bona fide RASopathy gene and implicated two-hit tumor-suppressor-like behavior.","evidence":"Patient mutation screening plus somatic LOH analysis in melanocytes","pmids":["17704776"],"confidence":"High","gaps":["Molecular mechanism linking SPRED1 loss to melanocyte phenotype not resolved","Genotype–phenotype correlations across mutation types not defined"]},{"year":2008,"claim":"Discovery of SPRED1 interaction with TESK1 and MARKK/TAO1 revealed a non-MAPK function—inhibition of TESK1-mediated cofilin phosphorylation to regulate actin dynamics—broadening SPRED1's role beyond RAS-ERK to cytoskeletal regulation.","evidence":"Yeast two-hybrid, co-IP, kinase assays, and cytoskeletal readouts in CHO cells","pmids":["18216281"],"confidence":"Medium","gaps":["Physiological relevance of SPRED1–TESK1 axis not confirmed in vivo","Whether this interaction is independent of MAPK suppression is unclear"]},{"year":2008,"claim":"Spred1-/- mice exhibited hippocampal learning deficits and LTP/LTD imbalance with increased ERK phosphorylation, demonstrating that SPRED1 constrains RAS-ERK in neurons to regulate synaptic plasticity—paralleling cognitive features of RASopathies.","evidence":"Behavioral testing (Morris water maze, T-maze), electrophysiology, and ERK assays in Spred1-/- mice","pmids":["19118178"],"confidence":"High","gaps":["Cell-type specificity within the hippocampus not dissected","Whether neurofibromin recruitment underlies the neuronal phenotype not tested"]},{"year":2010,"claim":"Spred1 was shown to govern neural stem cell self-renewal and cortical progenitor migration; knockdown caused periventricular heterotopia, linking SPRED1 to neurodevelopmental architecture and Legius syndrome brain phenotypes.","evidence":"In utero electroporation knockdown/overexpression, EdU assays, live imaging, and Spred1-/- mice","pmids":["20047999"],"confidence":"High","gaps":["Downstream ERK targets mediating migration defects not identified","Vesicular localization in progenitors not functionally characterized"]},{"year":2011,"claim":"Identification of SHP2-mediated dephosphorylation of SPRED1 Tyr-420 as a mechanism that attenuates SPRED1's inhibitory activity established a direct phosphatase-based regulatory switch controlling SPRED1 function, explaining how SHP2 (a positive RAS-ERK regulator) counteracts SPRED1.","evidence":"Co-IP, in vitro dephosphorylation, site-directed mutagenesis, and ERK activation assays","pmids":["21531714"],"confidence":"High","gaps":["Kinase that phosphorylates Tyr-420 not identified","In vivo significance of Tyr-420 phosphorylation not confirmed in animal models"]},{"year":2015,"claim":"Mapping of the SPRED1 EVH1–neurofibromin GRD interaction to the noncatalytic GAPex subdomain, with Legius and NF1 disease mutations disrupting binding, unified the molecular basis of Legius syndrome and NF1 into a shared SPRED1–NF1 complex disruption model.","evidence":"Yeast two-hybrid, co-IP, domain truncations, disease mutation mutagenesis, and ERK suppression assays","pmids":["26635368"],"confidence":"High","gaps":["Whether SPRED1 binding modifies NF1 conformation was not resolved","Quantitative affinity of disease mutants not fully determined"]},{"year":2016,"claim":"Biochemical demonstration that the SPRED1–NF1 complex binds RAS simultaneously without altering GAP catalytic activity confirmed SPRED1 as a membrane-targeting adaptor rather than an allosteric GAP modulator, clarifying its mechanistic role.","evidence":"Reconstituted binding and GAP activity assays with purified components","pmids":["27313208"],"confidence":"High","gaps":["Whether membrane lipid context affects GAP efficiency of the complex not tested","Role of the SPR domain in the ternary complex not addressed"]},{"year":2018,"claim":"SPRED1 was established as a melanoma tumor suppressor: loss conferred MAPK reactivation and resistance to KIT inhibitors, linking Legius syndrome biology to cancer vulnerability.","evidence":"CRISPR zebrafish melanoma model and human mucosal melanoma sequencing with MAPK assays","pmids":["30385465"],"confidence":"High","gaps":["Whether SPRED1 loss cooperates with specific oncogenic drivers beyond KIT not fully explored","Mechanism by which SPRED1 loss causes drug resistance (neurofibromin-dependent or not) not dissected"]},{"year":2018,"claim":"Spred1 was found essential for hematopoietic stem cell homeostasis partly through ROCK activity; under metabolic stress (high-fat diet), its loss caused ERK hyperactivation, HSC failure, and myeloproliferative disease, revealing a context-dependent tumor-suppressor function in hematopoiesis.","evidence":"Spred1 KO mice, HSC transplantation, ROCK inhibitor treatment, and dietary stress models","pmids":["29706577"],"confidence":"High","gaps":["How SPRED1 connects to ROCK signaling mechanistically is not defined","Compensatory role of SPRED2 complicates interpretation of single-KO steady-state phenotypes"]},{"year":2020,"claim":"A crystal structure of the ternary NF1-GRD–SPRED1-EVH1–KRAS complex revealed the structural basis for membrane targeting, and discovery that oncogenic EGFR phosphorylates SPRED1 Ser-105 to disrupt the complex provided a mechanism for EGFR-driven MAPK reactivation.","evidence":"X-ray crystallography, binding assays, mutagenesis, and phosphorylation analysis in EGFR-mutant cells","pmids":["32697994"],"confidence":"High","gaps":["Kinase(s) other than EGFR that target Ser-105 not identified","In vivo consequences of Ser-105 phosphorylation not tested in animal models"]},{"year":2021,"claim":"SPRED1 deletion was shown to confer resistance to BRAFV600E inhibitors in melanoma by reactivating MAPK, extending its tumor-suppressor role to the most common targeted therapy in melanoma and nominating SPRED1 status as a resistance biomarker.","evidence":"SPRED1 KO human melanoma cell lines and zebrafish in vivo melanoma models treated with BRAF inhibitors","pmids":["33306107"],"confidence":"High","gaps":["Whether SPRED1 re-expression can re-sensitize resistant tumors not tested","Interaction with RAF dimerization-based resistance mechanisms not explored"]},{"year":2024,"claim":"SPR domain missense mutations were shown to abolish palmitoylation and membrane localization, causing cytoplasmic aggregation and Purkinje cell degeneration (cerebellar ataxia) in knock-in mice—rescued by the autophagy inducer spermidine—revealing a gain-of-toxic-function mechanism for certain SPRED1 mutations distinct from simple loss-of-function.","evidence":"Palmitoylation assays, live imaging, knock-in mouse models, behavioral/cerebellar phenotyping, and spermidine rescue","pmids":["39510187"],"confidence":"High","gaps":["Whether aggregate toxicity operates through autophagy impairment or proteasomal overload not distinguished","Relevance of this mechanism to human Legius syndrome patients with SPR domain mutations not established"]},{"year":null,"claim":"Key open questions include: the structural basis of full-length SPRED1 autoinhibition and activation, the identity of the kinase phosphorylating Tyr-420, the in vivo relevance of SPRED1 interactions with DYRK1A and TESK1, and whether SPRED1 status predicts clinical response to MAPK-pathway inhibitors in cancer.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length SPRED1 structure or autoinhibition model exists","Kinase for Tyr-420 phosphorylation unknown","Clinical utility of SPRED1 as a drug-resistance biomarker not validated in patients"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[12,13,18]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,5,8,9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,10,14,23]},{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[7]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[23]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,2,9,12,13,18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,15,19,21]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[2,11,24]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[7,25]}],"complexes":["SPRED1-neurofibromin-KRAS ternary complex","SPRED1-merlin-neurofibromin complex"],"partners":["NF1","KRAS","SHP2","CAV1","TESK1","NF2","DYRK1A","FGFRL1"],"other_free_text":[]},"mechanistic_narrative":"SPRED1 is a membrane-associated negative regulator of the RAS-MAPK/ERK signaling pathway that controls cell proliferation, differentiation, and synaptic plasticity across diverse tissues including hematopoietic, neural, and immune compartments. SPRED1 suppresses RAS signaling primarily by recruiting neurofibromin (NF1) to the plasma membrane via its N-terminal EVH1 domain binding the noncatalytic GAPex subdomain of neurofibromin's GRD, forming a ternary complex with KRAS that enables RAS-GAP activity without directly modulating it [PMID:26635368, PMID:27313208, PMID:32697994]; its C-terminal SPR domain mediates palmitoylation-dependent membrane anchoring, and mutations disrupting palmitoylation cause cytoplasmic aggregation and cerebellar neurodegeneration in mice [PMID:39510187]. Regulatory inputs include SHP2-mediated dephosphorylation of SPRED1 Tyr-420, which attenuates its inhibitory function [PMID:21531714], and oncogenic EGFR-driven phosphorylation of Ser-105, which disrupts the SPRED1–neurofibromin complex [PMID:32697994]. Germline loss-of-function mutations in SPRED1 cause Legius syndrome (a neurofibromatosis 1-like disorder with café-au-lait macules requiring biallelic inactivation in melanocytes), and SPRED1 loss acts as a tumor suppressor mechanism whose disruption confers resistance to BRAF and KIT inhibitors in melanoma [PMID:17704776, PMID:30385465, PMID:33306107]."},"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). Negatively regulates hematopoiesis of bone marrow (By similarity). Inhibits fibroblast growth factor (FGF)-induced retinal lens fiber differentiation, probably by inhibiting FGF-mediated phosphorylation of ERK1/2 (By similarity). Attenuates actin stress fiber formation via inhibition of TESK1-mediated phosphorylation of cofilin (PubMed:18216281). 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hematology","url":"https://pubmed.ncbi.nlm.nih.gov/30531141","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":"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":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":41883,"output_tokens":6488,"usd":0.111485},"stage2":{"model":"claude-opus-4-6","input_tokens":10138,"output_tokens":3919,"usd":0.222997},"total_usd":0.334482,"stage1_batch_id":"msgbatch_011rkcQdVV1RaFq9XhcG6df6","stage2_batch_id":"msgbatch_01RzgrJafdF4EWHhj5mNi6dh","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) by impairing negative regulation of RAS→RAF interaction and MAPK signaling; melanocytes from café-au-lait spots showed biallelic SPRED1 inactivation (germline + somatic mutation), indicating complete loss is required for the phenotype.\",\n      \"method\": \"Germline mutation identification + somatic mutation analysis in melanocytes (LOH/second-hit analysis)\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — foundational discovery paper with mutational and LOH evidence, widely replicated\",\n      \"pmids\": [\"17704776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SPRED1 negatively regulates IL-3- and SCF-induced ERK/MAP kinase activation and cell proliferation in hematopoietic cells; a dominant-negative SPRED1 (ΔC-Spred) augmented IL-3-induced ERK activation, and Spred-1-deficient bone marrow mast cells showed augmented ERK activation in response to IL-3.\",\n      \"method\": \"Retroviral gene transfer of wild-type and dominant-negative SPRED1 into hematopoietic cell lines; Spred1-deficient bone marrow mast cells; ERK activation assays; colony formation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (dominant-negative, KO cells, proliferation/ERK assays)\",\n      \"pmids\": [\"15465815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Spred-1 negatively regulates allergen-induced airway eosinophilia and hyperresponsiveness by suppressing IL-5-dependent cell proliferation and ERK activation in eosinophils.\",\n      \"method\": \"Spred-1-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 — KO mouse model with defined cellular phenotype and biochemical readout, orthogonal methods\",\n      \"pmids\": [\"15630138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Spred-1 localizes to lipid raft/caveola membrane fractions and physically interacts with caveolin-1 (Cav-1), collaborating with Cav-1 to inhibit SCF- and IL-3-induced ERK activation and cell proliferation.\",\n      \"method\": \"Subcellular fractionation (lipid raft isolation); co-immunoprecipitation; forced expression of Cav-1 and Spred-1 in hematopoietic cells; proliferation and ERK activation assays\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — co-IP and fractionation with functional readout, single lab\",\n      \"pmids\": [\"16115197\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structure of the Xenopus tropicalis Spred1 EVH1 domain (1.15 Å resolution) reveals a pleckstrin-homology fold with a narrowed peptide-binding groove, suggesting a distinct peptide-binding mechanism compared to other EVH1 domains, likely binding less proline-rich peptides.\",\n      \"method\": \"X-ray crystallography at 1.15 Å resolution\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure\",\n      \"pmids\": [\"15710406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"SPRED1 physically interacts with MARKK/TAO1 kinase (no effect on MARKK activity) and with TESK1 kinase; Spred1 binding to TESK1 inhibits TESK1, thereby preventing TESK1-mediated phosphorylation of cofilin and making F-actin stress fibers dynamic. This three-way Spred1/MARKK/TESK1 interaction links regulation of both microtubule and F-actin cytoskeleton.\",\n      \"method\": \"Yeast two-hybrid; co-immunoprecipitation; kinase activity assays; overexpression in CHO cells with cytoskeletal readouts\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple binding/activity 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 and memory, short- and long-term synaptic plasticity (including LTP/LTD imbalance in CA1), and increased ERK phosphorylation after LTP induction, demonstrating that Spred1 suppresses the Ras/ERK pathway in neurons to regulate synaptic plasticity.\",\n      \"method\": \"Spred1-/- mice; Morris water maze; T-maze; electrophysiological recordings (LTP/LTD); biochemical ERK phosphorylation assays\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with multiple behavioral, electrophysiological, and biochemical endpoints\",\n      \"pmids\": [\"19118178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Spred1 is highly enriched in CNS germinal zones; knockdown increases neural stem cell self-renewal and progenitor proliferation cell-autonomously, while overexpression causes premature differentiation. In vivo knockdown disrupts apical ventricular zone integrity, impairs late neuronal migration, and causes periventricular heterotopia. Spred1 localizes within distinct vesicles in cortical progenitors.\",\n      \"method\": \"In utero electroporation knockdown/overexpression; EdU proliferation assays; live imaging; subcellular localization (vesicle fractionation/immunofluorescence); Spred1-/- mice\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (KD, OE, localization, in vivo), defined cellular phenotypes\",\n      \"pmids\": [\"20047999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"SPRED1 and SPRED2 directly interact with DYRK1A kinase via their CRD domain binding to the kinase domain of DYRK1A, and this interaction competitively inhibits DYRK1A-mediated phosphorylation of its substrates Tau and STAT3.\",\n      \"method\": \"Yeast two-hybrid; endogenous co-immunoprecipitation; in vitro kinase activity assays; domain mapping (CRD required)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal binding and in vitro kinase assay, 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: SPRED1 binds to the catalytic (phosphatase) domain of SHP2 (unlike Sprouty2 which binds the C-terminal tail), SHP2 dephosphorylates SPRED1, and two tyrosine residues on SPRED1 (including Tyr-420) are required when phosphorylated to inhibit Ras/ERK activation. SHP2-mediated dephosphorylation of Tyr-420 attenuates SPRED1's inhibitory action.\",\n      \"method\": \"Co-immunoprecipitation; in vitro dephosphorylation assays; site-directed mutagenesis of SPRED1 tyrosines; ERK activation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro enzymatic assay + mutagenesis + binding domain mapping\",\n      \"pmids\": [\"21531714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FGFRL1 interacts with SPRED1 via the SPR (Sprouty) domain of SPRED1 binding to the C-terminal histidine-rich domain of FGFRL1; this interaction was verified by co-precipitation and co-distribution at the plasma membrane. Spred1 increases the retention time of FGFRL1 at the plasma membrane.\",\n      \"method\": \"Yeast two-hybrid; co-precipitation; co-localization at cell membrane in COS1 and HEK293 cells; truncation experiments\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — yeast 2-hybrid + co-precipitation + 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 cytokine production by suppressing Spred1 expression; Spred1 overexpression or deficiency inversely regulated ERK activity and FcεRI-mediated cytokine production. Hematopoietic cell-specific Spred1 conditional KO mice showed increased mast cell numbers and heightened activation.\",\n      \"method\": \"miR-126 overexpression; Spred1 conditional knockout mice; ERK activity assays; cytokine production assays\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — conditional KO mouse model with defined cellular phenotype + mechanistic biochemistry\",\n      \"pmids\": [\"21668589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The SPRED1 EVH1 domain interacts with the N-terminal 16 and C-terminal 20 amino acids of the GAP-related domain (GRD) of neurofibromin (two crossing α-helix coils outside the GAP catalytic site, not present in p120GAP). Pathogenic missense mutations in the EVH1 domain of SPRED1 (Legius syndrome) and in these GRD regions (NF1) reduce binding affinity. EVH1 domain mutations that disrupt GRD binding also abolish SPRED1's ERK suppression activity.\",\n      \"method\": \"Yeast two-hybrid; co-immunoprecipitation in HEK293 cells; domain mapping with truncations; mutagenesis; ERK suppression assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Y2H, Co-IP, mutagenesis, functional assay), clear mechanistic link\",\n      \"pmids\": [\"26635368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The EVH1 domain of SPRED1 binds to the noncatalytic GAPex subdomain of neurofibromin's GRD; this binding is compatible with simultaneous Ras binding and does not interfere with neurofibromin's GAP activity, supporting a targeting/recruitment function rather than GAP modulation.\",\n      \"method\": \"Biochemical binding assays; domain mapping; GAP activity assays in the presence and absence of Spred1\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro reconstitution with functional (GAP) activity assay, clear mechanistic finding\",\n      \"pmids\": [\"27313208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPRED1 is translocated to the plasma membrane upon growth factor stimulation or pharmacological/galectin-1-mediated induction of B/C-Raf dimers; this translocation involves SPRED1 interaction with B-Raf and, via its N terminus, with galectin-1 (Gal-1). On the plasma membrane, SPRED1 perturbs membrane organization and ERK signaling specifically of active K-ras4B but not H-ras. Legius syndrome mutations show diminished binding to both Gal-1 and B-Raf.\",\n      \"method\": \"Live-cell imaging; FRET/FLIM nanoscale membrane analysis; co-immunoprecipitation; pharmacological treatments; mutagenesis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (imaging, co-IP, mutagenesis), single lab\",\n      \"pmids\": [\"27503857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPRED1 functions as a tumor suppressor in 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.\",\n      \"method\": \"CRISPR-based tissue-specific zebrafish melanoma model; human mucosal melanoma sequencing; MAPK activation assays; drug resistance assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo CRISPR model + mechanistic biochemistry (MAPK activation), replicated in human tumors\",\n      \"pmids\": [\"30385465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Spred1 safeguards hematopoietic stem cell (HSC) homeostasis partly through Rho kinase (ROCK) activity under steady-state conditions. Under high-fat diet, Spred1 deficiency causes ERK hyperactivation and aberrant HSC self-renewal, leading to functional HSC failure, anemia, and myeloproliferative disease; compensatory upregulation of Spred2 prevents leukemogenesis in steady-state Spred1 KO.\",\n      \"method\": \"Spred1 knockout mice; HSC transplantation assays; ERK phosphorylation assays; ROCK inhibitor treatment; gut microbiota manipulation\",\n      \"journal\": \"Cell stem cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KO model with multiple defined phenotypic readouts and mechanistic pathway analysis\",\n      \"pmids\": [\"29706577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A pathogenic Legius syndrome missense mutation (T102R) in the EVH1 domain of Spred1 weakens interaction with neurofibromin by ~1000-fold without perturbing the protein fold; NMR spectroscopy identified the binding site of NF1-GAP on the mutant Spred1(EVH1) variant.\",\n      \"method\": \"NMR spectroscopy; binding affinity measurements; mutagenesis; protein folding analysis\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structural/binding analysis with mutagenesis and quantitative affinity data\",\n      \"pmids\": [\"31401120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal/cryo structure of the neurofibromin GRD complexed with SPRED1 EVH1 domain and KRAS reveals how SPRED1 membrane-targets neurofibromin to allow simultaneous interaction with activated KRAS. Oncogenic EGFR(L858R) signaling leads to phosphorylation of SPRED1 on serine 105, disrupting the SPRED1-neurofibromin complex.\",\n      \"method\": \"X-ray crystallography (structure of neurofibromin GRD-SPRED1 EVH1-KRAS ternary complex); biochemical binding assays; mutagenesis; cell-based phosphorylation assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biochemical and mutational validation, mechanistic insight into phospho-regulation\",\n      \"pmids\": [\"32697994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPRED1 deletion confers resistance to BRAFV600E inhibition in melanoma by reactivating MAPK activity; this was demonstrated in human melanoma cell lines and primary zebrafish melanoma models in vivo.\",\n      \"method\": \"SPRED1 KO in human melanoma cell lines; zebrafish melanoma in vivo model; MAPK activity assays; BRAF inhibitor treatment\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — combined human cell and in vivo zebrafish model with mechanistic MAPK readout\",\n      \"pmids\": [\"33306107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Merlin interacts with both neurofibromin and Spred1 in a complex via merlin-binding sites on both proteins; as a third component, merlin blocks Ras binding to Raf1 (acting as a 'selective Ras barrier') without increasing neurofibromin's GAP activity. Merlin-deficient Schwann cells require Ras-ERK pathway for proliferation.\",\n      \"method\": \"Co-immunoprecipitation; binding domain analysis; Ras-GAP activity assays; cell proliferation assays in merlin-deficient Schwann cells\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — co-IP with functional assays, single lab\",\n      \"pmids\": [\"33331896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Spred1 deficiency in CML promotes disease transformation from chronic phase to accelerated/blast crisis CML by expanding leukemic stem cells (LSCs) through hyperactivation of MAPK/ERK, increased miR-126 in LSKs, augmented Bcl-2 expression and stability, and enhanced Bcl-2-dependent oxidative phosphorylation supporting LSC survival.\",\n      \"method\": \"Spred1 KO in CML mouse models (global, HSC-specific, endothelial cell-specific); ERK phosphorylation; miR-126 quantification; Bcl-2 assays; oxidative phosphorylation measurements\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple KO models with defined pathway analysis, single lab\",\n      \"pmids\": [\"34564700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Molecular dynamics simulations and Markov state model analysis revealed that SPRED1 loading allosterically enhances KRAS-NF1 (neurofibromin) binding but hinders conformational transformation of the NF1 catalytic center required for RAS GTP hydrolysis; allosteric signal transduction pathways upon SPRED1 binding were mapped through 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 — computational only, no experimental validation\",\n      \"pmids\": [\"35872068\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Missense mutations in the SPR (Sprouty-related) C-terminal domain of SPRED1 (e.g., C368S, M369L, V408E, P415A, P415L, P422R) cause loss of palmitoylation, loss of plasma membrane localization, and formation of cytoplasmic granular aggregates. Knock-in mice with P415A and P415V (but not M417Afs*4 which lacks the C-terminal region) developed cerebellar ataxia and Purkinje cell loss, which was rescued by the autophagy inducer spermidine.\",\n      \"method\": \"Mutagenesis of palmitoylation sites; subcellular localization (live imaging/immunofluorescence); knock-in mouse generation; behavioral/cerebellar phenotype analysis; spermidine treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — mutagenesis + in vivo KI mice + pharmacological rescue, multiple orthogonal methods\",\n      \"pmids\": [\"39510187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Spred1 negatively regulates the expansion and function of group 2 innate lymphoid cells (ILC2s) through suppression of the Ras-ERK pathway; Spred1 deficiency leads to faster ILC2 proliferation, higher cytokine (IL-5, IL-13) production in response to IL-33, and stabilization of GATA3. A MEK inhibitor suppressed ILC2 proliferation and cytokine production.\",\n      \"method\": \"Spred1-/- mice; in vivo papain challenge; in vitro ILC2 stimulation; MEK inhibitor treatment; GATA3 protein stability assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined pathway/cellular phenotype, pharmacological validation\",\n      \"pmids\": [\"26116510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Combined loss of Spred1 and Spred2 in mouse lens leads to increased ERK1/2 phosphorylation, irregular lens epithelial and fiber cell activity, and impaired lens and eye development, demonstrating that these proteins negatively regulate ERK1/2 to control lens cell proliferation and differentiation.\",\n      \"method\": \"Spred1/Spred2 double-KO mice; ERK phosphorylation assays; histological analysis of lens development\",\n      \"journal\": \"Experimental eye research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO model with defined cellular phenotype and biochemical ERK readout\",\n      \"pmids\": [\"31923414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"TGF-β upregulates SPRED1 expression in CD8+ tumor-infiltrating lymphocytes, and elevated SPRED1 correlates with reduced TCR-mediated ITK and ERK kinase activation, contributing to T cell hyporesponsiveness in the tumor microenvironment.\",\n      \"method\": \"Gene expression analysis; TGF-β inhibitor treatment; ITK and ERK activation assays in TILs vs. splenic CD8+ T cells\",\n      \"journal\": \"Cancer immunology, immunotherapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — correlative expression + pharmacological inhibitor, no direct KD/KO of SPRED1\",\n      \"pmids\": [\"19319531\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPRED1 is a negative regulator of RAS-MAPK/ERK signaling that acts primarily by recruiting neurofibromin (a RasGAP) from the cytosol to membrane-anchored RAS via its N-terminal EVH1 domain binding to the GAPex subdomain of neurofibromin's GRD, while its C-terminal SPR domain mediates palmitoylation-dependent plasma membrane localization; additional regulatory inputs include SHP2-mediated dephosphorylation of SPRED1 Tyr-420 (attenuating its inhibitory function), oncogenic EGFR-driven phosphorylation of Ser-105 (disrupting the SPRED1-neurofibromin complex), interaction with Caveolin-1 to enhance ERK suppression, and interaction with TESK1 to regulate actin cytoskeletal dynamics, with loss-of-function mutations causing Legius syndrome and acting as a tumor suppressor across multiple cancer contexts.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SPRED1 is a membrane-associated negative regulator of the RAS-MAPK/ERK signaling pathway that controls cell proliferation, differentiation, and synaptic plasticity across diverse tissues including hematopoietic, neural, and immune compartments. SPRED1 suppresses RAS signaling primarily by recruiting neurofibromin (NF1) to the plasma membrane via its N-terminal EVH1 domain binding the noncatalytic GAPex subdomain of neurofibromin's GRD, forming a ternary complex with KRAS that enables RAS-GAP activity without directly modulating it [PMID:26635368, PMID:27313208, PMID:32697994]; its C-terminal SPR domain mediates palmitoylation-dependent membrane anchoring, and mutations disrupting palmitoylation cause cytoplasmic aggregation and cerebellar neurodegeneration in mice [PMID:39510187]. Regulatory inputs include SHP2-mediated dephosphorylation of SPRED1 Tyr-420, which attenuates its inhibitory function [PMID:21531714], and oncogenic EGFR-driven phosphorylation of Ser-105, which disrupts the SPRED1–neurofibromin complex [PMID:32697994]. Germline loss-of-function mutations in SPRED1 cause Legius syndrome (a neurofibromatosis 1-like disorder with café-au-lait macules requiring biallelic inactivation in melanocytes), and SPRED1 loss acts as a tumor suppressor mechanism whose disruption confers resistance to BRAF and KIT inhibitors in melanoma [PMID:17704776, PMID:30385465, PMID:33306107].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Establishing that SPRED1 is a functional negative regulator of ERK/MAPK signaling in hematopoietic cells answered whether this Sprouty-related protein has pathway-suppressive activity in a physiological context beyond overexpression systems.\",\n      \"evidence\": \"Dominant-negative SPRED1, wild-type retroviral expression, and Spred1-deficient bone marrow mast cells with ERK and proliferation assays\",\n      \"pmids\": [\"15465815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of ERK suppression not defined—direct target unknown\", \"Relevance beyond hematopoietic lineage not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Knockout mouse studies extended SPRED1's inhibitory role to in vivo immune (eosinophilic airway inflammation) and structural (lipid raft/caveola localization with Caveolin-1) contexts, establishing tissue-breadth and membrane compartmentalization of its function.\",\n      \"evidence\": \"Spred1-/- mice in allergen challenge; subcellular fractionation and Cav-1 co-IP in hematopoietic cells\",\n      \"pmids\": [\"15630138\", \"16115197\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Caveolin-1 interaction shown in single lab without reciprocal validation\", \"Whether raft localization is essential for SPRED1 function not tested genetically\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Determination of the EVH1 domain crystal structure revealed a PH-fold with an atypically narrow peptide groove, predicting that SPRED1 recognizes non-canonical (less proline-rich) ligands distinct from classical EVH1 targets.\",\n      \"evidence\": \"X-ray crystallography of Xenopus tropicalis Spred1 EVH1 at 1.15 Å resolution\",\n      \"pmids\": [\"15710406\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cognate binding peptide/partner not identified from the structure alone\", \"Mammalian EVH1 structure not yet determined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of germline SPRED1 loss-of-function mutations as the cause of Legius syndrome, with biallelic inactivation in melanocyte café-au-lait spots, established SPRED1 as a bona fide RASopathy gene and implicated two-hit tumor-suppressor-like behavior.\",\n      \"evidence\": \"Patient mutation screening plus somatic LOH analysis in melanocytes\",\n      \"pmids\": [\"17704776\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism linking SPRED1 loss to melanocyte phenotype not resolved\", \"Genotype–phenotype correlations across mutation types not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Discovery of SPRED1 interaction with TESK1 and MARKK/TAO1 revealed a non-MAPK function—inhibition of TESK1-mediated cofilin phosphorylation to regulate actin dynamics—broadening SPRED1's role beyond RAS-ERK to cytoskeletal regulation.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, kinase assays, and cytoskeletal readouts in CHO cells\",\n      \"pmids\": [\"18216281\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of SPRED1–TESK1 axis not confirmed in vivo\", \"Whether this interaction is independent of MAPK suppression is unclear\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Spred1-/- mice exhibited hippocampal learning deficits and LTP/LTD imbalance with increased ERK phosphorylation, demonstrating that SPRED1 constrains RAS-ERK in neurons to regulate synaptic plasticity—paralleling cognitive features of RASopathies.\",\n      \"evidence\": \"Behavioral testing (Morris water maze, T-maze), electrophysiology, and ERK assays in Spred1-/- mice\",\n      \"pmids\": [\"19118178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell-type specificity within the hippocampus not dissected\", \"Whether neurofibromin recruitment underlies the neuronal phenotype not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Spred1 was shown to govern neural stem cell self-renewal and cortical progenitor migration; knockdown caused periventricular heterotopia, linking SPRED1 to neurodevelopmental architecture and Legius syndrome brain phenotypes.\",\n      \"evidence\": \"In utero electroporation knockdown/overexpression, EdU assays, live imaging, and Spred1-/- mice\",\n      \"pmids\": [\"20047999\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream ERK targets mediating migration defects not identified\", \"Vesicular localization in progenitors not functionally characterized\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identification of SHP2-mediated dephosphorylation of SPRED1 Tyr-420 as a mechanism that attenuates SPRED1's inhibitory activity established a direct phosphatase-based regulatory switch controlling SPRED1 function, explaining how SHP2 (a positive RAS-ERK regulator) counteracts SPRED1.\",\n      \"evidence\": \"Co-IP, in vitro dephosphorylation, site-directed mutagenesis, and ERK activation assays\",\n      \"pmids\": [\"21531714\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase that phosphorylates Tyr-420 not identified\", \"In vivo significance of Tyr-420 phosphorylation not confirmed in animal models\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapping of the SPRED1 EVH1–neurofibromin GRD interaction to the noncatalytic GAPex subdomain, with Legius and NF1 disease mutations disrupting binding, unified the molecular basis of Legius syndrome and NF1 into a shared SPRED1–NF1 complex disruption model.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, domain truncations, disease mutation mutagenesis, and ERK suppression assays\",\n      \"pmids\": [\"26635368\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SPRED1 binding modifies NF1 conformation was not resolved\", \"Quantitative affinity of disease mutants not fully determined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Biochemical demonstration that the SPRED1–NF1 complex binds RAS simultaneously without altering GAP catalytic activity confirmed SPRED1 as a membrane-targeting adaptor rather than an allosteric GAP modulator, clarifying its mechanistic role.\",\n      \"evidence\": \"Reconstituted binding and GAP activity assays with purified components\",\n      \"pmids\": [\"27313208\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether membrane lipid context affects GAP efficiency of the complex not tested\", \"Role of the SPR domain in the ternary complex not addressed\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"SPRED1 was established as a melanoma tumor suppressor: loss conferred MAPK reactivation and resistance to KIT inhibitors, linking Legius syndrome biology to cancer vulnerability.\",\n      \"evidence\": \"CRISPR zebrafish melanoma model and human mucosal melanoma sequencing with MAPK assays\",\n      \"pmids\": [\"30385465\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SPRED1 loss cooperates with specific oncogenic drivers beyond KIT not fully explored\", \"Mechanism by which SPRED1 loss causes drug resistance (neurofibromin-dependent or not) not dissected\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Spred1 was found essential for hematopoietic stem cell homeostasis partly through ROCK activity; under metabolic stress (high-fat diet), its loss caused ERK hyperactivation, HSC failure, and myeloproliferative disease, revealing a context-dependent tumor-suppressor function in hematopoiesis.\",\n      \"evidence\": \"Spred1 KO mice, HSC transplantation, ROCK inhibitor treatment, and dietary stress models\",\n      \"pmids\": [\"29706577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How SPRED1 connects to ROCK signaling mechanistically is not defined\", \"Compensatory role of SPRED2 complicates interpretation of single-KO steady-state phenotypes\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A crystal structure of the ternary NF1-GRD–SPRED1-EVH1–KRAS complex revealed the structural basis for membrane targeting, and discovery that oncogenic EGFR phosphorylates SPRED1 Ser-105 to disrupt the complex provided a mechanism for EGFR-driven MAPK reactivation.\",\n      \"evidence\": \"X-ray crystallography, binding assays, mutagenesis, and phosphorylation analysis in EGFR-mutant cells\",\n      \"pmids\": [\"32697994\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase(s) other than EGFR that target Ser-105 not identified\", \"In vivo consequences of Ser-105 phosphorylation not tested in animal models\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"SPRED1 deletion was shown to confer resistance to BRAFV600E inhibitors in melanoma by reactivating MAPK, extending its tumor-suppressor role to the most common targeted therapy in melanoma and nominating SPRED1 status as a resistance biomarker.\",\n      \"evidence\": \"SPRED1 KO human melanoma cell lines and zebrafish in vivo melanoma models treated with BRAF inhibitors\",\n      \"pmids\": [\"33306107\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SPRED1 re-expression can re-sensitize resistant tumors not tested\", \"Interaction with RAF dimerization-based resistance mechanisms not explored\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"SPR domain missense mutations were shown to abolish palmitoylation and membrane localization, causing cytoplasmic aggregation and Purkinje cell degeneration (cerebellar ataxia) in knock-in mice—rescued by the autophagy inducer spermidine—revealing a gain-of-toxic-function mechanism for certain SPRED1 mutations distinct from simple loss-of-function.\",\n      \"evidence\": \"Palmitoylation assays, live imaging, knock-in mouse models, behavioral/cerebellar phenotyping, and spermidine rescue\",\n      \"pmids\": [\"39510187\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether aggregate toxicity operates through autophagy impairment or proteasomal overload not distinguished\", \"Relevance of this mechanism to human Legius syndrome patients with SPR domain mutations not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: the structural basis of full-length SPRED1 autoinhibition and activation, the identity of the kinase phosphorylating Tyr-420, the in vivo relevance of SPRED1 interactions with DYRK1A and TESK1, and whether SPRED1 status predicts clinical response to MAPK-pathway inhibitors in cancer.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length SPRED1 structure or autoinhibition model exists\", \"Kinase for Tyr-420 phosphorylation unknown\", \"Clinical utility of SPRED1 as a drug-resistance biomarker not validated in patients\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [12, 13, 18]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 5, 8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 10, 14, 23]},\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [23]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 2, 9, 12, 13, 18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 15, 19, 21]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [2, 11, 24]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [7, 25]}\n    ],\n    \"complexes\": [\n      \"SPRED1-neurofibromin-KRAS ternary complex\",\n      \"SPRED1-merlin-neurofibromin complex\"\n    ],\n    \"partners\": [\n      \"NF1\",\n      \"KRAS\",\n      \"SHP2\",\n      \"CAV1\",\n      \"TESK1\",\n      \"NF2\",\n      \"DYRK1A\",\n      \"FGFRL1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}