{"gene":"SPRED3","run_date":"2026-06-10T07:46:40","timeline":{"discoveries":[{"year":2003,"finding":"SPRED3 suppresses growth factor-induced MAP kinase (ERK) activation; its SPR domain (C-terminal cysteine-rich Sprouty-related domain), rather than the KBD, is responsible for efficient ERK suppression, as determined by chimeric molecule analysis between Spred-3 and Spred-1. SPRED3 lacks a functional c-kit binding domain due to substitution of the critical Arg residue with Gly.","method":"Chimeric protein analysis, overexpression in cells, ERK activation assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — chimeric molecule dissection with functional ERK assays in a single lab, two complementary approaches","pmids":["12646235"],"is_preprint":false},{"year":2003,"finding":"SPRED3 is expressed exclusively in the brain and localizes to the membrane via its SPR domain.","method":"RT-PCR expression analysis; subcellular localization inferred from SPR domain function in chimeric constructs","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, localization inferred from domain function without direct live-imaging or fractionation","pmids":["12646235"],"is_preprint":false},{"year":2006,"finding":"SPRED3 is ubiquitinated in HEK293T cells stimulated with EGF or pervanadate, indicating it undergoes post-translational ubiquitination in response to receptor tyrosine kinase signaling.","method":"Ubiquitination assay in HEK293T cells with EGF/pervanadate stimulation; Western blotting","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single method, SPRED3 ubiquitination noted but not characterized further (study focused on SPRED2)","pmids":["17094949"],"is_preprint":false},{"year":2014,"finding":"SPRED3 is palmitoylated by the palmitoyl acyltransferase HIP14 (zDHHC17), making HIP14 the first enzyme known to palmitoylate SPRED3.","method":"Yeast two-hybrid interactome screen; palmitoylation assay confirming HIP14 as the acyltransferase for SPRED3","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical palmitoylation assay confirming enzymatic modification, single lab","pmids":["24705354"],"is_preprint":false},{"year":2021,"finding":"miR-342-5p directly targets the 3'UTR of Spred3, inhibiting Spred3 expression. Spred3 functions as a Raf1 regulator; recombinant Spred3 treatment exacerbated the BPD phenotype and pulmonary arterial hypertension in a murine hyperoxia model, consistent with Spred3 acting downstream of miR-342-5p to modulate Raf1/MAPK signaling in type II alveolar epithelial cells.","method":"3'UTR reporter assay (miR-342-5p targeting Spred3); transgenic mouse models; recombinant Spred3 treatment; murine hyperoxia models","journal":"British journal of pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — 3'UTR reporter assay plus in vivo gain/loss-of-function, single lab, multiple orthogonal approaches","pmids":["33434946"],"is_preprint":false},{"year":2022,"finding":"SPRED3 is S-acylated by zDHHC17 via a zDABM-independent mechanism. The interaction of SPRED3 with zDHHC17 is mediated through the SPR (cysteine-rich Sprouty-related) domain of SPRED3 rather than via the canonical zDHHC ANK binding motif (zDABM), and the interaction is independent of the ankyrin repeat domain (ANK17) of zDHHC17.","method":"Mutational analysis of SPRED3 SPR domain; co-immunoprecipitation with zDHHC17 constructs; S-acylation assays; comparison with zDABM-deleted mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutational dissection of enzyme-substrate interaction combined with biochemical S-acylation assays and multiple zDHHC17 domain mutants in a single rigorous study","pmids":["36442513"],"is_preprint":false},{"year":2020,"finding":"Loss-of-function mutation in SPRED3 (c.120delG, p.E40fs) leads to activation of the Ras/Raf/MAPK pathway and confers acquired resistance to EGFR-TKI erlotinib in NSCLC cells. CRISPR/Cas9 knockout of SPRED3 in HCC827 cells increased erlotinib resistance and cell migration; overexpression of SPRED3 in resistant cells reduced resistance and migration. ERK1/2 inhibition in SPRED3-knockout cells reduced erlotinib resistance and migration.","method":"Whole-exome sequencing; CRISPR/Cas9 knockout; cDNA overexpression; Western blotting for p-ERK; MTS assay (IC50); Transwell migration assay; ERK inhibition epistasis","journal":"Translational cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal KO and overexpression with epistasis (ERK inhibitor rescue), single lab","pmids":["35117614"],"is_preprint":false},{"year":2018,"finding":"Overexpression of SPRED3 in lens epithelial cells suppresses FGF-induced ERK1/2 phosphorylation and blocks FGF-induced lens fiber cell differentiation (cell elongation), placing SPRED3 as a negative regulator of RTK-mediated MAPK signaling in the context of lens differentiation.","method":"Transfection of lens epithelial explants; FGF stimulation; ERK1/2 phosphorylation assay; morphological cell elongation scoring","journal":"Experimental eye research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, overexpression study in explants with limited mechanistic depth for SPRED3 specifically","pmids":["29501879"],"is_preprint":false},{"year":2015,"finding":"Overexpression of SPRED3 in rat lens epithelial cells blocks TGFβ-induced epithelial-to-mesenchymal transition (EMT), as measured by reduced fibrotic cell elongation and α-SMA expression, indicating SPRED3 negatively regulates TGFβ-induced EMT in lens cells.","method":"Plasmid transfection of lens epithelial explants; TGFβ treatment; morphological scoring; α-SMA immunolabeling","journal":"Experimental eye research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, overexpression only, limited mechanistic dissection of SPRED3's pathway","pmids":["25576668"],"is_preprint":false},{"year":2024,"finding":"SPRED3 overexpression enhances thyroid cancer (THCA) cell viability and colony formation, while SPRED3 depletion reduces cell growth and tumor growth in vivo. Mechanistically, SPRED3 activates NF-κB signaling as demonstrated by luciferase reporter assays, placing SPRED3 as a positive regulator of NF-κB in thyroid cancer cells.","method":"Flag-SPRED3 overexpression; SPRED3 knockout; CCK8 and colony formation assays; in vivo mouse tumor model; NF-κB luciferase reporter assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal gain/loss-of-function with orthogonal pathway readout (luciferase reporter), single lab","pmids":["39227612"],"is_preprint":false},{"year":2025,"finding":"SPRED3 knockout mice develop primary hypothyroidism (elevated TSH, reduced T4), with mildly reduced thyroidal ERK signaling and altered autophagy regulator expression (reduced p62, increased ATG5, elevated LC3-II/I ratio, decreased pBeclin/Beclin), indicating SPRED3 regulates thyroidal homeostasis and autophagy processes in the thyroid gland.","method":"SPRED3 knockout mouse generation; hormonal profiling (TSH, T4); X-Gal staining for promoter activity; immunoblotting for ERK, autophagy markers","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO mouse model with hormonal and molecular phenotyping using multiple readouts, single lab","pmids":["40806788"],"is_preprint":false}],"current_model":"SPRED3 is a brain-enriched (and thyroid-expressed) member of the SPRED family that negatively regulates Ras/RAF/MAPK (ERK) signaling primarily through its C-terminal SPR domain, which also mediates membrane localization and S-acylation by zDHHC17 via a novel zDABM-independent mechanism; loss of SPRED3 activates the MAPK pathway to drive EGFR-TKI resistance, whereas in thyroid cells SPRED3 unexpectedly activates NF-κB to promote proliferation, and SPRED3 knockout mice develop primary hypothyroidism with altered thyroidal ERK signaling and dysregulated autophagy."},"narrative":{"mechanistic_narrative":"SPRED3 is a SPRED-family negative regulator of receptor tyrosine kinase-driven Ras/RAF/MAPK (ERK) signaling whose suppressive activity is encoded by its C-terminal cysteine-rich Sprouty-related (SPR) domain rather than the kinase-binding domain, and which lacks a functional c-kit binding domain [PMID:12646235]. SPRED3 dampens ERK activation downstream of growth factor receptors in several contexts: it suppresses FGF-induced ERK phosphorylation and lens fiber differentiation [PMID:29501879], and its loss activates the Ras/RAF/MAPK pathway to confer EGFR-TKI (erlotinib) resistance and increased migration in NSCLC cells, a phenotype reversed by ERK1/2 inhibition [PMID:35117614]. The same SPR domain directs membrane localization [PMID:12646235] and mediates S-acylation by the palmitoyl acyltransferase zDHHC17 (HIP14) through a zDABM-independent interaction that does not require the enzyme's ankyrin-repeat domain [PMID:24705354, PMID:36442513]. Beyond canonical MAPK suppression, SPRED3 has context-specific roles in the thyroid, where it acts as a positive regulator of NF-κB to promote thyroid cancer cell growth [PMID:39227612], and where its knockout in mice produces primary hypothyroidism accompanied by altered thyroidal ERK signaling and dysregulated autophagy markers [PMID:40806788].","teleology":[{"year":2003,"claim":"Established that SPRED3 is a brain-expressed MAPK suppressor and localized the suppressive and membrane-targeting activity to the SPR domain rather than the kinase-binding domain, distinguishing it functionally within the SPRED family.","evidence":"Chimeric Spred-3/Spred-1 protein analysis with overexpression and ERK activation assays; RT-PCR expression profiling","pmids":["12646235"],"confidence":"Medium","gaps":["Membrane localization inferred from domain function, not from direct imaging or fractionation","No identification of the direct molecular target within the Ras/RAF/MAPK cascade","Mechanism of ERK suppression by the SPR domain not resolved"]},{"year":2006,"claim":"Showed SPRED3 is post-translationally ubiquitinated upon RTK stimulation, hinting at regulated turnover, but the modification was not characterized.","evidence":"Ubiquitination assay in HEK293T cells with EGF/pervanadate stimulation and Western blotting","pmids":["17094949"],"confidence":"Low","gaps":["E3 ligase not identified","Functional consequence of ubiquitination unknown","Observed incidentally in a SPRED2-focused study"]},{"year":2014,"claim":"Identified the first enzyme modifying SPRED3, establishing that it is a palmitoylation substrate of zDHHC17 (HIP14).","evidence":"Yeast two-hybrid interactome screen plus palmitoylation assay","pmids":["24705354"],"confidence":"Medium","gaps":["Palmitoylation sites on SPRED3 not mapped","Functional impact on localization or MAPK suppression not tested"]},{"year":2015,"claim":"Extended SPRED3 negative regulation beyond ERK to TGFβ-induced EMT in lens epithelial cells.","evidence":"Plasmid overexpression in rat lens epithelial explants with TGFβ treatment, morphological scoring, and α-SMA immunolabeling","pmids":["25576668"],"confidence":"Low","gaps":["Overexpression-only without loss-of-function","Direct molecular link between SPRED3 and the TGFβ pathway not defined","Single explant system"]},{"year":2018,"claim":"Confirmed SPRED3 suppresses FGF/RTK-driven ERK signaling and the downstream differentiation program in lens cells.","evidence":"Transfection of lens epithelial explants, FGF stimulation, ERK1/2 phosphorylation assay, morphological elongation scoring","pmids":["29501879"],"confidence":"Low","gaps":["Overexpression-based; endogenous role not tested","Limited mechanistic depth specific to SPRED3"]},{"year":2020,"claim":"Provided reciprocal genetic evidence that endogenous SPRED3 restrains the Ras/RAF/MAPK pathway, with loss driving EGFR-TKI resistance and migration in lung cancer.","evidence":"Whole-exome sequencing, CRISPR/Cas9 knockout and overexpression in HCC827 cells, p-ERK Western blotting, IC50/migration assays, and ERK-inhibitor epistasis","pmids":["35117614"],"confidence":"Medium","gaps":["Direct biochemical target in the cascade not identified","Single cell-line context","Whether the c.120delG variant occurs in patient tumors not addressed"]},{"year":2021,"claim":"Placed SPRED3 in a miRNA-regulated Raf1/MAPK axis in lung disease, with miR-342-5p directly repressing Spred3 and recombinant Spred3 worsening hyperoxia-induced pathology in vivo.","evidence":"3'UTR luciferase reporter assay, transgenic mouse models, and recombinant protein treatment in murine hyperoxia models","pmids":["33434946"],"confidence":"Medium","gaps":["Mechanism by which extracellular recombinant Spred3 acts not defined","Direction of Spred3's effect on Raf1 in this model not fully reconciled with its suppressor role"]},{"year":2022,"claim":"Defined the molecular basis of SPRED3 S-acylation, showing the SPR domain mediates a zDABM-independent interaction with zDHHC17 that bypasses the enzyme's ankyrin-repeat domain.","evidence":"Mutational dissection of the SPR domain, co-immunoprecipitation with zDHHC17 domain mutants, and S-acylation assays","pmids":["36442513"],"confidence":"High","gaps":["Functional consequence of S-acylation for MAPK suppression not established","In vivo relevance of the modification untested"]},{"year":2024,"claim":"Revealed a context-dependent, non-canonical role in which SPRED3 positively regulates NF-κB to promote thyroid cancer growth, contrasting its MAPK-suppressing role elsewhere.","evidence":"Reciprocal Flag-SPRED3 overexpression and knockout, CCK8/colony formation assays, in vivo tumor model, and NF-κB luciferase reporter","pmids":["39227612"],"confidence":"Medium","gaps":["Mechanism linking SPRED3 to NF-κB activation unknown","How a MAPK suppressor activates NF-κB not reconciled"]},{"year":2025,"claim":"Demonstrated a physiological requirement for SPRED3 in thyroid homeostasis, with knockout mice developing primary hypothyroidism alongside altered thyroidal ERK and autophagy markers.","evidence":"SPRED3 knockout mouse hormonal profiling (TSH, T4), X-Gal promoter staining, and immunoblotting for ERK and autophagy regulators","pmids":["40806788"],"confidence":"Medium","gaps":["Causal link between altered ERK/autophagy and hypothyroidism not established","Cell-type responsible for the phenotype within the thyroid not resolved"]},{"year":null,"claim":"How SPRED3 mechanistically engages the Ras/RAF/MAPK cascade, and how it switches between MAPK suppression and NF-κB activation in a tissue-specific manner, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No direct MAPK-pathway binding partner or substrate identified","Functional role of S-acylation and palmitoylation in SPRED3 activity unknown","Molecular basis for the thyroid-specific NF-κB-activating role undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,6,7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6]}],"complexes":[],"partners":["ZDHHC17"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q2MJR0","full_name":"Sprouty-related, EVH1 domain-containing protein 3","aliases":[],"length_aa":410,"mass_kda":42.7,"function":"Tyrosine kinase substrate that inhibits growth-factor-mediated activation of MAP kinase (By similarity). Inhibits fibroblast growth factor (FGF)-induced retinal lens fiber differentiation, probably by inhibiting FGF-mediated phosphorylation of ERK1/2 (By similarity). Inhibits TGFB-induced epithelial-to-mesenchymal transition in lens epithelial cells (By similarity)","subcellular_location":"Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q2MJR0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPRED3","classification":"Not Classified","n_dependent_lines":10,"n_total_lines":1208,"dependency_fraction":0.008278145695364239},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SPRED3","total_profiled":1310},"omim":[{"mim_id":"609293","title":"SPROUTY-RELATED EVH1 DOMAIN-CONTAINING PROTEIN 3; SPRED3","url":"https://www.omim.org/entry/609293"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Plasma membrane","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":3.5}],"url":"https://www.proteinatlas.org/search/SPRED3"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q2MJR0","domains":[{"cath_id":"2.30.29.30","chopping":"4-37_45-114","consensus_level":"high","plddt":88.8908,"start":4,"end":114},{"cath_id":"-","chopping":"294-406","consensus_level":"medium","plddt":81.6883,"start":294,"end":406}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q2MJR0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q2MJR0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q2MJR0-F1-predicted_aligned_error_v6.png","plddt_mean":65.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPRED3","jax_strain_url":"https://www.jax.org/strain/search?query=SPRED3"},"sequence":{"accession":"Q2MJR0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q2MJR0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q2MJR0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q2MJR0"}},"corpus_meta":[{"pmid":"12646235","id":"PMC_12646235","title":"Molecular cloning of mammalian Spred-3 which suppresses tyrosine kinase-mediated Erk activation.","date":"2003","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12646235","citation_count":103,"is_preprint":false},{"pmid":"24705354","id":"PMC_24705354","title":"The palmitoyl acyltransferase HIP14 shares a high proportion of interactors with huntingtin: implications for a role in the pathogenesis of Huntington's disease.","date":"2014","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/24705354","citation_count":58,"is_preprint":false},{"pmid":"16465403","id":"PMC_16465403","title":"FGF signaling inhibitor, SPRY4, is evolutionarily conserved target of WNT signaling pathway in progenitor cells.","date":"2006","source":"International journal of molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/16465403","citation_count":54,"is_preprint":false},{"pmid":"25576668","id":"PMC_25576668","title":"Negative regulation of TGFβ-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists.","date":"2015","source":"Experimental eye research","url":"https://pubmed.ncbi.nlm.nih.gov/25576668","citation_count":30,"is_preprint":false},{"pmid":"34620846","id":"PMC_34620846","title":"Genomic alterations associated with mutational signatures, DNA damage repair and chromatin remodeling pathways in cervical carcinoma.","date":"2021","source":"NPJ genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/34620846","citation_count":16,"is_preprint":false},{"pmid":"33434946","id":"PMC_33434946","title":"Hyperoxia-induced miR-342-5p down-regulation exacerbates neonatal bronchopulmonary dysplasia via the Raf1 regulator Spred3.","date":"2021","source":"British journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/33434946","citation_count":14,"is_preprint":false},{"pmid":"36442513","id":"PMC_36442513","title":"S-acylation of Sprouty and SPRED proteins by the S-acyltransferase zDHHC17 involves a novel mode of enzyme-substrate interaction.","date":"2022","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/36442513","citation_count":12,"is_preprint":false},{"pmid":"29501879","id":"PMC_29501879","title":"Negative regulation of lens fiber cell differentiation by RTK antagonists Spry and Spred.","date":"2018","source":"Experimental eye research","url":"https://pubmed.ncbi.nlm.nih.gov/29501879","citation_count":11,"is_preprint":false},{"pmid":"38041506","id":"PMC_38041506","title":"Comparison of first-tier whole-exome sequencing with a multi-step traditional approach for diagnosing paediatric outpatients: An Italian prospective study.","date":"2023","source":"Molecular genetics & genomic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/38041506","citation_count":11,"is_preprint":false},{"pmid":"35117614","id":"PMC_35117614","title":"Spred-3 mutation and Ras/Raf/MAPK activation confer acquired resistance to EGFR tyrosine kinase inhibitor in an EGFR mutated NSCLC cell line.","date":"2020","source":"Translational cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/35117614","citation_count":10,"is_preprint":false},{"pmid":"37149146","id":"PMC_37149146","title":"The ribosomal S6 kinase 2 (RSK2)-SPRED2 complex regulates the phosphorylation of RSK substrates and MAPK signaling.","date":"2023","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37149146","citation_count":10,"is_preprint":false},{"pmid":"17094949","id":"PMC_17094949","title":"Spred-2 steady-state levels are regulated by phosphorylation and Cbl-mediated ubiquitination.","date":"2006","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17094949","citation_count":10,"is_preprint":false},{"pmid":"39227612","id":"PMC_39227612","title":"SPRED3 regulates the NF-κB signaling pathway in thyroid cancer and promotes the proliferation.","date":"2024","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/39227612","citation_count":6,"is_preprint":false},{"pmid":"39627016","id":"PMC_39627016","title":"Intrauterine fetal growth restriction in sheep leads to sexually dimorphic programming of Preadipocytes' differentiation potential.","date":"2024","source":"Physiological reports","url":"https://pubmed.ncbi.nlm.nih.gov/39627016","citation_count":2,"is_preprint":false},{"pmid":"40806788","id":"PMC_40806788","title":"Loss of SPRED3 Causes Primary Hypothyroidism and Alters Thyroidal Expression of Autophagy Regulators LC3, p62, and ATG5 in Mice.","date":"2025","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40806788","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11003,"output_tokens":2811,"usd":0.037587,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10042,"output_tokens":3282,"usd":0.06613,"stage2_stop_reason":"end_turn"},"total_usd":0.103717,"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\": 2003,\n      \"finding\": \"SPRED3 suppresses growth factor-induced MAP kinase (ERK) activation; its SPR domain (C-terminal cysteine-rich Sprouty-related domain), rather than the KBD, is responsible for efficient ERK suppression, as determined by chimeric molecule analysis between Spred-3 and Spred-1. SPRED3 lacks a functional c-kit binding domain due to substitution of the critical Arg residue with Gly.\",\n      \"method\": \"Chimeric protein analysis, overexpression in cells, ERK activation assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chimeric molecule dissection with functional ERK assays in a single lab, two complementary approaches\",\n      \"pmids\": [\"12646235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"SPRED3 is expressed exclusively in the brain and localizes to the membrane via its SPR domain.\",\n      \"method\": \"RT-PCR expression analysis; subcellular localization inferred from SPR domain function in chimeric constructs\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, localization inferred from domain function without direct live-imaging or fractionation\",\n      \"pmids\": [\"12646235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SPRED3 is ubiquitinated in HEK293T cells stimulated with EGF or pervanadate, indicating it undergoes post-translational ubiquitination in response to receptor tyrosine kinase signaling.\",\n      \"method\": \"Ubiquitination assay in HEK293T cells with EGF/pervanadate stimulation; Western blotting\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single method, SPRED3 ubiquitination noted but not characterized further (study focused on SPRED2)\",\n      \"pmids\": [\"17094949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPRED3 is palmitoylated by the palmitoyl acyltransferase HIP14 (zDHHC17), making HIP14 the first enzyme known to palmitoylate SPRED3.\",\n      \"method\": \"Yeast two-hybrid interactome screen; palmitoylation assay confirming HIP14 as the acyltransferase for SPRED3\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical palmitoylation assay confirming enzymatic modification, single lab\",\n      \"pmids\": [\"24705354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-342-5p directly targets the 3'UTR of Spred3, inhibiting Spred3 expression. Spred3 functions as a Raf1 regulator; recombinant Spred3 treatment exacerbated the BPD phenotype and pulmonary arterial hypertension in a murine hyperoxia model, consistent with Spred3 acting downstream of miR-342-5p to modulate Raf1/MAPK signaling in type II alveolar epithelial cells.\",\n      \"method\": \"3'UTR reporter assay (miR-342-5p targeting Spred3); transgenic mouse models; recombinant Spred3 treatment; murine hyperoxia models\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — 3'UTR reporter assay plus in vivo gain/loss-of-function, single lab, multiple orthogonal approaches\",\n      \"pmids\": [\"33434946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPRED3 is S-acylated by zDHHC17 via a zDABM-independent mechanism. The interaction of SPRED3 with zDHHC17 is mediated through the SPR (cysteine-rich Sprouty-related) domain of SPRED3 rather than via the canonical zDHHC ANK binding motif (zDABM), and the interaction is independent of the ankyrin repeat domain (ANK17) of zDHHC17.\",\n      \"method\": \"Mutational analysis of SPRED3 SPR domain; co-immunoprecipitation with zDHHC17 constructs; S-acylation assays; comparison with zDABM-deleted mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutational dissection of enzyme-substrate interaction combined with biochemical S-acylation assays and multiple zDHHC17 domain mutants in a single rigorous study\",\n      \"pmids\": [\"36442513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss-of-function mutation in SPRED3 (c.120delG, p.E40fs) leads to activation of the Ras/Raf/MAPK pathway and confers acquired resistance to EGFR-TKI erlotinib in NSCLC cells. CRISPR/Cas9 knockout of SPRED3 in HCC827 cells increased erlotinib resistance and cell migration; overexpression of SPRED3 in resistant cells reduced resistance and migration. ERK1/2 inhibition in SPRED3-knockout cells reduced erlotinib resistance and migration.\",\n      \"method\": \"Whole-exome sequencing; CRISPR/Cas9 knockout; cDNA overexpression; Western blotting for p-ERK; MTS assay (IC50); Transwell migration assay; ERK inhibition epistasis\",\n      \"journal\": \"Translational cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal KO and overexpression with epistasis (ERK inhibitor rescue), single lab\",\n      \"pmids\": [\"35117614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Overexpression of SPRED3 in lens epithelial cells suppresses FGF-induced ERK1/2 phosphorylation and blocks FGF-induced lens fiber cell differentiation (cell elongation), placing SPRED3 as a negative regulator of RTK-mediated MAPK signaling in the context of lens differentiation.\",\n      \"method\": \"Transfection of lens epithelial explants; FGF stimulation; ERK1/2 phosphorylation assay; morphological cell elongation scoring\",\n      \"journal\": \"Experimental eye research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, overexpression study in explants with limited mechanistic depth for SPRED3 specifically\",\n      \"pmids\": [\"29501879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Overexpression of SPRED3 in rat lens epithelial cells blocks TGFβ-induced epithelial-to-mesenchymal transition (EMT), as measured by reduced fibrotic cell elongation and α-SMA expression, indicating SPRED3 negatively regulates TGFβ-induced EMT in lens cells.\",\n      \"method\": \"Plasmid transfection of lens epithelial explants; TGFβ treatment; morphological scoring; α-SMA immunolabeling\",\n      \"journal\": \"Experimental eye research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, overexpression only, limited mechanistic dissection of SPRED3's pathway\",\n      \"pmids\": [\"25576668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPRED3 overexpression enhances thyroid cancer (THCA) cell viability and colony formation, while SPRED3 depletion reduces cell growth and tumor growth in vivo. Mechanistically, SPRED3 activates NF-κB signaling as demonstrated by luciferase reporter assays, placing SPRED3 as a positive regulator of NF-κB in thyroid cancer cells.\",\n      \"method\": \"Flag-SPRED3 overexpression; SPRED3 knockout; CCK8 and colony formation assays; in vivo mouse tumor model; NF-κB luciferase reporter assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal gain/loss-of-function with orthogonal pathway readout (luciferase reporter), single lab\",\n      \"pmids\": [\"39227612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SPRED3 knockout mice develop primary hypothyroidism (elevated TSH, reduced T4), with mildly reduced thyroidal ERK signaling and altered autophagy regulator expression (reduced p62, increased ATG5, elevated LC3-II/I ratio, decreased pBeclin/Beclin), indicating SPRED3 regulates thyroidal homeostasis and autophagy processes in the thyroid gland.\",\n      \"method\": \"SPRED3 knockout mouse generation; hormonal profiling (TSH, T4); X-Gal staining for promoter activity; immunoblotting for ERK, autophagy markers\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO mouse model with hormonal and molecular phenotyping using multiple readouts, single lab\",\n      \"pmids\": [\"40806788\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPRED3 is a brain-enriched (and thyroid-expressed) member of the SPRED family that negatively regulates Ras/RAF/MAPK (ERK) signaling primarily through its C-terminal SPR domain, which also mediates membrane localization and S-acylation by zDHHC17 via a novel zDABM-independent mechanism; loss of SPRED3 activates the MAPK pathway to drive EGFR-TKI resistance, whereas in thyroid cells SPRED3 unexpectedly activates NF-κB to promote proliferation, and SPRED3 knockout mice develop primary hypothyroidism with altered thyroidal ERK signaling and dysregulated autophagy.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPRED3 is a SPRED-family negative regulator of receptor tyrosine kinase-driven Ras/RAF/MAPK (ERK) signaling whose suppressive activity is encoded by its C-terminal cysteine-rich Sprouty-related (SPR) domain rather than the kinase-binding domain, and which lacks a functional c-kit binding domain [#0]. SPRED3 dampens ERK activation downstream of growth factor receptors in several contexts: it suppresses FGF-induced ERK phosphorylation and lens fiber differentiation [#7], and its loss activates the Ras/RAF/MAPK pathway to confer EGFR-TKI (erlotinib) resistance and increased migration in NSCLC cells, a phenotype reversed by ERK1/2 inhibition [#6]. The same SPR domain directs membrane localization [#1] and mediates S-acylation by the palmitoyl acyltransferase zDHHC17 (HIP14) through a zDABM-independent interaction that does not require the enzyme's ankyrin-repeat domain [#3, #5]. Beyond canonical MAPK suppression, SPRED3 has context-specific roles in the thyroid, where it acts as a positive regulator of NF-\\u03baB to promote thyroid cancer cell growth [#9], and where its knockout in mice produces primary hypothyroidism accompanied by altered thyroidal ERK signaling and dysregulated autophagy markers [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that SPRED3 is a brain-expressed MAPK suppressor and localized the suppressive and membrane-targeting activity to the SPR domain rather than the kinase-binding domain, distinguishing it functionally within the SPRED family.\",\n      \"evidence\": \"Chimeric Spred-3/Spred-1 protein analysis with overexpression and ERK activation assays; RT-PCR expression profiling\",\n      \"pmids\": [\"12646235\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Membrane localization inferred from domain function, not from direct imaging or fractionation\", \"No identification of the direct molecular target within the Ras/RAF/MAPK cascade\", \"Mechanism of ERK suppression by the SPR domain not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed SPRED3 is post-translationally ubiquitinated upon RTK stimulation, hinting at regulated turnover, but the modification was not characterized.\",\n      \"evidence\": \"Ubiquitination assay in HEK293T cells with EGF/pervanadate stimulation and Western blotting\",\n      \"pmids\": [\"17094949\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"E3 ligase not identified\", \"Functional consequence of ubiquitination unknown\", \"Observed incidentally in a SPRED2-focused study\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified the first enzyme modifying SPRED3, establishing that it is a palmitoylation substrate of zDHHC17 (HIP14).\",\n      \"evidence\": \"Yeast two-hybrid interactome screen plus palmitoylation assay\",\n      \"pmids\": [\"24705354\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Palmitoylation sites on SPRED3 not mapped\", \"Functional impact on localization or MAPK suppression not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Extended SPRED3 negative regulation beyond ERK to TGF\\u03b2-induced EMT in lens epithelial cells.\",\n      \"evidence\": \"Plasmid overexpression in rat lens epithelial explants with TGF\\u03b2 treatment, morphological scoring, and \\u03b1-SMA immunolabeling\",\n      \"pmids\": [\"25576668\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Overexpression-only without loss-of-function\", \"Direct molecular link between SPRED3 and the TGF\\u03b2 pathway not defined\", \"Single explant system\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Confirmed SPRED3 suppresses FGF/RTK-driven ERK signaling and the downstream differentiation program in lens cells.\",\n      \"evidence\": \"Transfection of lens epithelial explants, FGF stimulation, ERK1/2 phosphorylation assay, morphological elongation scoring\",\n      \"pmids\": [\"29501879\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Overexpression-based; endogenous role not tested\", \"Limited mechanistic depth specific to SPRED3\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided reciprocal genetic evidence that endogenous SPRED3 restrains the Ras/RAF/MAPK pathway, with loss driving EGFR-TKI resistance and migration in lung cancer.\",\n      \"evidence\": \"Whole-exome sequencing, CRISPR/Cas9 knockout and overexpression in HCC827 cells, p-ERK Western blotting, IC50/migration assays, and ERK-inhibitor epistasis\",\n      \"pmids\": [\"35117614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical target in the cascade not identified\", \"Single cell-line context\", \"Whether the c.120delG variant occurs in patient tumors not addressed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed SPRED3 in a miRNA-regulated Raf1/MAPK axis in lung disease, with miR-342-5p directly repressing Spred3 and recombinant Spred3 worsening hyperoxia-induced pathology in vivo.\",\n      \"evidence\": \"3'UTR luciferase reporter assay, transgenic mouse models, and recombinant protein treatment in murine hyperoxia models\",\n      \"pmids\": [\"33434946\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which extracellular recombinant Spred3 acts not defined\", \"Direction of Spred3's effect on Raf1 in this model not fully reconciled with its suppressor role\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined the molecular basis of SPRED3 S-acylation, showing the SPR domain mediates a zDABM-independent interaction with zDHHC17 that bypasses the enzyme's ankyrin-repeat domain.\",\n      \"evidence\": \"Mutational dissection of the SPR domain, co-immunoprecipitation with zDHHC17 domain mutants, and S-acylation assays\",\n      \"pmids\": [\"36442513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of S-acylation for MAPK suppression not established\", \"In vivo relevance of the modification untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Revealed a context-dependent, non-canonical role in which SPRED3 positively regulates NF-\\u03baB to promote thyroid cancer growth, contrasting its MAPK-suppressing role elsewhere.\",\n      \"evidence\": \"Reciprocal Flag-SPRED3 overexpression and knockout, CCK8/colony formation assays, in vivo tumor model, and NF-\\u03baB luciferase reporter\",\n      \"pmids\": [\"39227612\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking SPRED3 to NF-\\u03baB activation unknown\", \"How a MAPK suppressor activates NF-\\u03baB not reconciled\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated a physiological requirement for SPRED3 in thyroid homeostasis, with knockout mice developing primary hypothyroidism alongside altered thyroidal ERK and autophagy markers.\",\n      \"evidence\": \"SPRED3 knockout mouse hormonal profiling (TSH, T4), X-Gal promoter staining, and immunoblotting for ERK and autophagy regulators\",\n      \"pmids\": [\"40806788\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal link between altered ERK/autophagy and hypothyroidism not established\", \"Cell-type responsible for the phenotype within the thyroid not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SPRED3 mechanistically engages the Ras/RAF/MAPK cascade, and how it switches between MAPK suppression and NF-\\u03baB activation in a tissue-specific manner, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No direct MAPK-pathway binding partner or substrate identified\", \"Functional role of S-acylation and palmitoylation in SPRED3 activity unknown\", \"Molecular basis for the thyroid-specific NF-\\u03baB-activating role undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 6, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"ZDHHC17\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":3,"faith_total":4,"faith_pct":75.0}}