{"gene":"SPRED3","run_date":"2026-04-28T20:42:08","timeline":{"discoveries":[{"year":2003,"finding":"SPRED3 suppresses growth factor-induced ERK/MAP kinase activation; the C-terminal SPR domain (rather than the KBD) is responsible for efficient ERK suppression, and the SPR domain is required for membrane localization. Unlike SPRED1/2, SPRED3 lacks a functional c-kit binding domain because the critical Arg residue is replaced by Gly.","method":"Overexpression of chimeric SPRED3/SPRED1 molecules in cells, ERK activation assays, domain-deletion analysis","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 2 — original cloning paper with domain-swap mutagenesis and functional ERK assays; foundational study with >100 citations","pmids":["12646235"],"is_preprint":false},{"year":2006,"finding":"SPRED3 (along with SPRED1 and SPRED2) is ubiquitinated in HEK293T cells upon EGF or pervanadate stimulation, indicating that ubiquitination-mediated degradation is a shared regulatory mechanism across SPRED family members.","method":"Co-immunoprecipitation and ubiquitination assays in HEK293T cells; proteasomal inhibitor (MG-132) treatment","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — SPRED3 ubiquitination shown by co-IP/western in overexpression system, single lab","pmids":["17094949"],"is_preprint":false},{"year":2014,"finding":"SPRED3 is palmitoylated (S-acylated) by the palmitoyl acyltransferase HIP14 (zDHHC17); HIP14 is the first enzyme identified to palmitoylate SPRED3.","method":"Yeast two-hybrid screen for HIP14 interactors; palmitoylation assays confirming HIP14-mediated S-acylation of SPRED3","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical palmitoylation assay confirming enzyme-substrate relationship, single lab","pmids":["24705354"],"is_preprint":false},{"year":2015,"finding":"Overexpression of SPRED3 in rat lens epithelial cells blocks TGFβ-induced epithelial-to-mesenchymal transition (EMT), establishing SPRED3 as a negative regulator of TGFβ-induced EMT in lens cells.","method":"Plasmid transfection of SPRED3 in rat lens epithelial explants followed by TGFβ treatment; morphological assessment and α-SMA immunolabeling","journal":"Experimental eye research","confidence":"Medium","confidence_rationale":"Tier 3 — overexpression with defined cellular phenotype readout, single lab","pmids":["25576668"],"is_preprint":false},{"year":2018,"finding":"Overexpression of SPRED3 in lens epithelial explants blocks FGF-induced fiber cell differentiation (cell elongation and ERK1/2-dependent signaling), demonstrating its role as a negative regulator of RTK-mediated MAPK signaling in lens differentiation.","method":"Transfection of SPRED3 in lens epithelial explants; FGF stimulation; assessment of cell elongation, ERK1/2 phosphorylation, and fiber-specific marker Prox1","journal":"Experimental eye research","confidence":"Medium","confidence_rationale":"Tier 3 — overexpression with defined molecular and morphological readouts, single lab","pmids":["29501879"],"is_preprint":false},{"year":2020,"finding":"Loss-of-function mutation in SPRED3 (c.120delG, p.E40fs) activates the Ras/Raf/MAPK pathway and confers resistance to EGFR tyrosine kinase inhibitor erlotinib in NSCLC cells; CRISPR/Cas9 knockout of SPRED3 phenocopies resistance and increased migration, while SPRED3 overexpression restores sensitivity.","method":"Whole-exome sequencing of resistant cells; CRISPR/Cas9 knockout; cDNA overexpression; western blotting of p-ERK1/2; MTS assay; Transwell migration assay","journal":"Translational cancer research","confidence":"High","confidence_rationale":"Tier 2 — reciprocal KO and overexpression with multiple orthogonal functional readouts in a single study","pmids":["35117614"],"is_preprint":false},{"year":2021,"finding":"miR-342-5p directly targets the 3'UTR of SPRED3 and suppresses its expression; Spred3 acts as a Raf1 regulator such that its loss (via miR-342-5p downregulation under hyperoxia) exacerbates neonatal bronchopulmonary dysplasia and pulmonary arterial hypertension in mice. Recombinant Spred3 treatment worsened the BPD phenotype.","method":"miR-342-5p mimic/overexpression in murine BPD models; 3'UTR luciferase reporter assay; recombinant Spred3 protein treatment; transgenic miR-342 mice; western blotting","journal":"British journal of pharmacology","confidence":"High","confidence_rationale":"Tier 2 — 3'UTR reporter assay plus reciprocal gain/loss-of-function in vivo with defined phenotype","pmids":["33434946"],"is_preprint":false},{"year":2022,"finding":"SPRED3 is S-acylated by zDHHC17 through a zDABM-independent mechanism; the cysteine-rich SPR domain of SPRED3 mediates interaction with zDHHC17 independently of the zDHHC17 ankyrin repeat domain (ANK17), revealing a novel mode of enzyme-substrate recognition.","method":"Mutational analysis of SPRED3; co-immunoprecipitation with zDHHC17 and ANK-deletion mutants; S-acylation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis combined with biochemical S-acylation assays and binding studies defining novel enzyme-substrate mechanism","pmids":["36442513"],"is_preprint":false},{"year":2023,"finding":"Affinity purification mass spectrometry revealed that RSK2 does not interact with SPRED3 (unlike SPRED2), establishing that SPRED family members have distinct binding partners and unique nodes of MAPK regulation.","method":"Affinity purification mass spectrometry (AP-MS) of SPRED1, SPRED2, and SPRED3 interactomes","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — AP-MS interactome distinguishing SPRED3 from SPRED2, single lab","pmids":["37149146"],"is_preprint":false},{"year":2024,"finding":"SPRED3 overexpression activates NF-κB transcriptional activity and enhances thyroid cancer cell proliferation and viability, while SPRED3 knockout reduces tumor growth in vivo, identifying NF-κB signaling as a pathway regulated by SPRED3 in thyroid cancer cells.","method":"Flag-SPRED3 overexpression and CRISPR knockout in thyroid cancer cells; luciferase NF-κB reporter assay; colony formation and CCK8 assays; in vivo mouse xenograft","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal gain/loss-of-function with reporter assay and in vivo confirmation, single lab","pmids":["39227612"],"is_preprint":false},{"year":2025,"finding":"SPRED3 knockout mice develop primary hypothyroidism (elevated TSH, reduced T4), mildly reduced thyroidal ERK signaling, and altered expression of autophagy regulators (reduced p62, increased ATG5, elevated LC3-II/I ratio, decreased pBeclin/Beclin), placing SPRED3 as a regulator of thyroidal homeostasis and autophagy. X-Gal staining localized Spred3 promoter activity to thyroid, adrenal gland, pituitary, cerebral cortex, and kidney.","method":"SPRED3 knockout mouse generation; hormonal profiling (TSH, T4); immunoblotting for ERK, p62, ATG5, LC3, Beclin; X-Gal staining","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 2 — constitutive KO mouse with multiple orthogonal molecular and hormonal readouts in a single study","pmids":["40806788"],"is_preprint":false}],"current_model":"SPRED3 is a brain-enriched negative regulator of the Ras/MAPK pathway that suppresses growth factor-induced ERK activation primarily through its C-terminal SPR domain (not its non-functional KBD); it is post-translationally regulated by S-acylation via zDHHC17 (through an SPR domain-mediated, ANK-independent mechanism) and by ubiquitination, and its loss activates Ras/Raf/MAPK signaling to drive EGFR-TKI resistance in NSCLC; in vivo, SPRED3 knockout mice develop primary hypothyroidism with altered thyroidal ERK signaling and dysregulated autophagy, and SPRED3 also modulates NF-κB signaling in thyroid cancer cells."},"narrative":{"teleology":[{"year":2003,"claim":"Establishing that SPRED3 suppresses growth factor-induced ERK activation resolved how this third family member functions and revealed that its SPR domain, rather than its non-functional KBD, drives both membrane targeting and ERK suppression — distinguishing it mechanistically from SPRED1/2.","evidence":"Domain-swap chimeras and deletion mutants with ERK activation assays in overexpression system","pmids":["12646235"],"confidence":"High","gaps":["Endogenous SPRED3 loss-of-function not tested","SPR domain binding partners mediating membrane localization unidentified","No in vivo validation"]},{"year":2006,"claim":"Demonstrating that SPRED3 is ubiquitinated upon growth factor stimulation established that proteasomal degradation regulates SPRED3 protein levels, paralleling the regulatory mechanism of SPRED1/2.","evidence":"Co-immunoprecipitation/ubiquitination assays in HEK293T cells with EGF stimulation and MG-132 treatment","pmids":["17094949"],"confidence":"Medium","gaps":["E3 ubiquitin ligase responsible not identified","Ubiquitination sites on SPRED3 not mapped","Functional consequence of ubiquitination on ERK suppression not tested"]},{"year":2014,"claim":"Identification of zDHHC17 (HIP14) as the palmitoyl acyltransferase that S-acylates SPRED3 provided the first enzyme-substrate link for SPRED3 lipid modification, suggesting a regulatory mechanism for its membrane association.","evidence":"Yeast two-hybrid screen for HIP14 interactors followed by palmitoylation assays","pmids":["24705354"],"confidence":"Medium","gaps":["Functional consequence of palmitoylation on SPRED3 ERK suppression not tested","Palmitoylation sites not mapped","Mechanism of enzyme-substrate recognition unclear"]},{"year":2015,"claim":"Showing that SPRED3 blocks TGFβ-induced EMT in lens epithelial cells extended its inhibitory role beyond classical RTK-ERK signaling to TGFβ-driven cellular reprogramming.","evidence":"SPRED3 overexpression in rat lens epithelial explants with TGFβ treatment; morphological and α-SMA marker assessment","pmids":["25576668"],"confidence":"Medium","gaps":["Whether SPRED3 directly inhibits TGFβ pathway components or acts via ERK cross-talk unclear","Endogenous SPRED3 expression in lens tissue not confirmed","Mechanism of EMT suppression not defined"]},{"year":2018,"claim":"Demonstration that SPRED3 blocks FGF-induced lens fiber differentiation by suppressing ERK1/2 phosphorylation confirmed its role as a general negative regulator of RTK-MAPK signaling in a developmental context.","evidence":"SPRED3 transfection in lens explants with FGF stimulation; p-ERK1/2 and Prox1 marker readouts","pmids":["29501879"],"confidence":"Medium","gaps":["Endogenous SPRED3 function in lens development not tested by knockout","Relative contribution of SPRED3 vs SPRED1/2 in lens tissue unknown"]},{"year":2020,"claim":"Discovery that loss-of-function SPRED3 mutations activate Ras/Raf/MAPK signaling and confer EGFR-TKI resistance in NSCLC established SPRED3 as a clinically relevant tumor suppressor, with reciprocal knockout and overexpression validating causality.","evidence":"Whole-exome sequencing of erlotinib-resistant cells; CRISPR knockout and cDNA rescue; p-ERK, MTS, and migration assays","pmids":["35117614"],"confidence":"High","gaps":["Frequency of SPRED3 loss in clinical EGFR-TKI resistance cohorts unknown","Direct interaction partner at the Raf level not defined","Whether SPRED3 loss cooperates with other resistance mechanisms untested"]},{"year":2021,"claim":"Identifying miR-342-5p as a direct negative regulator of SPRED3 via 3′UTR targeting linked SPRED3 downregulation to neonatal lung pathology, revealing a post-transcriptional regulatory axis controlling SPRED3 abundance in vivo.","evidence":"3′UTR luciferase reporter assay; miR-342-5p gain/loss in murine BPD models; recombinant Spred3 treatment","pmids":["33434946"],"confidence":"High","gaps":["Paradoxical worsening with recombinant Spred3 in BPD model not mechanistically explained","Whether Spred3 engages Raf1 directly or indirectly not resolved"]},{"year":2022,"claim":"Defining that zDHHC17 S-acylates SPRED3 through an ANK-independent, SPR domain-mediated mechanism revealed a non-canonical mode of enzyme-substrate recognition distinct from other zDHHC17 substrates.","evidence":"Mutational analysis of SPRED3 and zDHHC17 ANK-deletion mutants; co-immunoprecipitation and S-acylation assays","pmids":["36442513"],"confidence":"High","gaps":["Specific cysteine residues acylated not mapped","Whether S-acylation is required for SPRED3 membrane targeting and ERK suppression in cells not tested","Structural basis of SPR-zDHHC17 recognition unknown"]},{"year":2023,"claim":"AP-MS interactome comparison showing RSK2 binds SPRED2 but not SPRED3 established that SPRED paralogs occupy distinct signaling niches despite shared domain architecture.","evidence":"Affinity purification mass spectrometry of SPRED1/2/3 interactomes","pmids":["37149146"],"confidence":"Medium","gaps":["SPRED3-specific interactors not comprehensively characterized","Functional consequence of differential RSK2 binding on MAPK feedback not tested"]},{"year":2024,"claim":"Discovery that SPRED3 activates NF-κB transcriptional activity and promotes thyroid cancer cell proliferation uncovered a signaling role beyond MAPK inhibition, supported by reciprocal gain/loss-of-function and in vivo xenograft data.","evidence":"Flag-SPRED3 overexpression and CRISPR knockout in thyroid cancer cells; NF-κB luciferase reporter; mouse xenograft","pmids":["39227612"],"confidence":"Medium","gaps":["Mechanism by which SPRED3 activates NF-κB not defined","Reconciliation of tumor-suppressive MAPK role with tumor-promoting NF-κB role not addressed","Whether NF-κB activation is direct or indirect unknown"]},{"year":2025,"claim":"SPRED3 knockout mice developing primary hypothyroidism with reduced thyroidal ERK signaling and dysregulated autophagy established the first in vivo physiological function for SPRED3, linking it to thyroid hormone homeostasis.","evidence":"Constitutive SPRED3 KO mouse; serum TSH/T4 profiling; immunoblotting for ERK, p62, ATG5, LC3, Beclin; X-Gal promoter activity mapping","pmids":["40806788"],"confidence":"High","gaps":["Whether hypothyroidism results from direct thyroidal SPRED3 loss or secondary hypothalamic-pituitary effects not fully resolved","How a MAPK inhibitor's loss leads to mildly reduced (not increased) thyroidal ERK requires mechanistic explanation","Autophagy dysregulation could be secondary to hormonal changes"]},{"year":null,"claim":"The structural basis of SPRED3's SPR domain-mediated inhibition of Raf, the identity of SPRED3-specific protein interaction partners, and the mechanistic reconciliation of its MAPK-suppressive versus NF-κB-activating roles remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of SPRED3 or SPR domain in complex with Raf or zDHHC17","SPRED3-specific interactome incompletely defined","Context-dependent tumor-suppressive vs oncogenic functions not mechanistically explained"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,5,6]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,7]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,6,9]}],"complexes":[],"partners":["ZDHHC17","RAF1"],"other_free_text":[]},"mechanistic_narrative":"SPRED3 is a negative regulator of Ras/MAPK signaling that suppresses growth factor-induced ERK activation through its C-terminal cysteine-rich SPR domain, which also mediates membrane localization and a non-canonical interaction with the palmitoyl acyltransferase zDHHC17 for S-acylation [PMID:12646235, PMID:36442513]. Unlike SPRED1/2, SPRED3 lacks a functional c-Kit binding domain due to a critical Arg-to-Gly substitution, and it does not interact with RSK2, indicating paralog-specific regulatory wiring within the SPRED family [PMID:12646235, PMID:37149146]. Loss of SPRED3 activates Ras/Raf/MAPK signaling to drive EGFR-TKI resistance in NSCLC and causes primary hypothyroidism with dysregulated thyroidal ERK signaling and autophagy in knockout mice [PMID:35117614, PMID:40806788]. SPRED3 also modulates NF-κB signaling in thyroid cancer cells and blocks TGFβ-induced EMT and FGF-driven differentiation in lens epithelial cells [PMID:39227612, PMID:25576668, PMID:29501879]."},"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":11,"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 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40806788","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10284,"output_tokens":2766,"usd":0.036171},"stage2":{"model":"claude-opus-4-6","input_tokens":6116,"output_tokens":2853,"usd":0.152858},"total_usd":0.189029,"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\": 2003,\n      \"finding\": \"SPRED3 suppresses growth factor-induced ERK/MAP kinase activation; the C-terminal SPR domain (rather than the KBD) is responsible for efficient ERK suppression, and the SPR domain is required for membrane localization. Unlike SPRED1/2, SPRED3 lacks a functional c-kit binding domain because the critical Arg residue is replaced by Gly.\",\n      \"method\": \"Overexpression of chimeric SPRED3/SPRED1 molecules in cells, ERK activation assays, domain-deletion analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — original cloning paper with domain-swap mutagenesis and functional ERK assays; foundational study with >100 citations\",\n      \"pmids\": [\"12646235\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SPRED3 (along with SPRED1 and SPRED2) is ubiquitinated in HEK293T cells upon EGF or pervanadate stimulation, indicating that ubiquitination-mediated degradation is a shared regulatory mechanism across SPRED family members.\",\n      \"method\": \"Co-immunoprecipitation and ubiquitination assays in HEK293T cells; proteasomal inhibitor (MG-132) treatment\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — SPRED3 ubiquitination shown by co-IP/western in overexpression system, single lab\",\n      \"pmids\": [\"17094949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPRED3 is palmitoylated (S-acylated) by the palmitoyl acyltransferase HIP14 (zDHHC17); HIP14 is the first enzyme identified to palmitoylate SPRED3.\",\n      \"method\": \"Yeast two-hybrid screen for HIP14 interactors; palmitoylation assays confirming HIP14-mediated S-acylation of SPRED3\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical palmitoylation assay confirming enzyme-substrate relationship, single lab\",\n      \"pmids\": [\"24705354\"],\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), establishing SPRED3 as a negative regulator of TGFβ-induced EMT in lens cells.\",\n      \"method\": \"Plasmid transfection of SPRED3 in rat lens epithelial explants followed by TGFβ treatment; morphological assessment and α-SMA immunolabeling\",\n      \"journal\": \"Experimental eye research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — overexpression with defined cellular phenotype readout, single lab\",\n      \"pmids\": [\"25576668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Overexpression of SPRED3 in lens epithelial explants blocks FGF-induced fiber cell differentiation (cell elongation and ERK1/2-dependent signaling), demonstrating its role as a negative regulator of RTK-mediated MAPK signaling in lens differentiation.\",\n      \"method\": \"Transfection of SPRED3 in lens epithelial explants; FGF stimulation; assessment of cell elongation, ERK1/2 phosphorylation, and fiber-specific marker Prox1\",\n      \"journal\": \"Experimental eye research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — overexpression with defined molecular and morphological readouts, single lab\",\n      \"pmids\": [\"29501879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Loss-of-function mutation in SPRED3 (c.120delG, p.E40fs) activates the Ras/Raf/MAPK pathway and confers resistance to EGFR tyrosine kinase inhibitor erlotinib in NSCLC cells; CRISPR/Cas9 knockout of SPRED3 phenocopies resistance and increased migration, while SPRED3 overexpression restores sensitivity.\",\n      \"method\": \"Whole-exome sequencing of resistant cells; CRISPR/Cas9 knockout; cDNA overexpression; western blotting of p-ERK1/2; MTS assay; Transwell migration assay\",\n      \"journal\": \"Translational cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal KO and overexpression with multiple orthogonal functional readouts in a single study\",\n      \"pmids\": [\"35117614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"miR-342-5p directly targets the 3'UTR of SPRED3 and suppresses its expression; Spred3 acts as a Raf1 regulator such that its loss (via miR-342-5p downregulation under hyperoxia) exacerbates neonatal bronchopulmonary dysplasia and pulmonary arterial hypertension in mice. Recombinant Spred3 treatment worsened the BPD phenotype.\",\n      \"method\": \"miR-342-5p mimic/overexpression in murine BPD models; 3'UTR luciferase reporter assay; recombinant Spred3 protein treatment; transgenic miR-342 mice; western blotting\",\n      \"journal\": \"British journal of pharmacology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — 3'UTR reporter assay plus reciprocal gain/loss-of-function in vivo with defined phenotype\",\n      \"pmids\": [\"33434946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPRED3 is S-acylated by zDHHC17 through a zDABM-independent mechanism; the cysteine-rich SPR domain of SPRED3 mediates interaction with zDHHC17 independently of the zDHHC17 ankyrin repeat domain (ANK17), revealing a novel mode of enzyme-substrate recognition.\",\n      \"method\": \"Mutational analysis of SPRED3; co-immunoprecipitation with zDHHC17 and ANK-deletion mutants; S-acylation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis combined with biochemical S-acylation assays and binding studies defining novel enzyme-substrate mechanism\",\n      \"pmids\": [\"36442513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Affinity purification mass spectrometry revealed that RSK2 does not interact with SPRED3 (unlike SPRED2), establishing that SPRED family members have distinct binding partners and unique nodes of MAPK regulation.\",\n      \"method\": \"Affinity purification mass spectrometry (AP-MS) of SPRED1, SPRED2, and SPRED3 interactomes\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — AP-MS interactome distinguishing SPRED3 from SPRED2, single lab\",\n      \"pmids\": [\"37149146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPRED3 overexpression activates NF-κB transcriptional activity and enhances thyroid cancer cell proliferation and viability, while SPRED3 knockout reduces tumor growth in vivo, identifying NF-κB signaling as a pathway regulated by SPRED3 in thyroid cancer cells.\",\n      \"method\": \"Flag-SPRED3 overexpression and CRISPR knockout in thyroid cancer cells; luciferase NF-κB reporter assay; colony formation and CCK8 assays; in vivo mouse xenograft\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal gain/loss-of-function with reporter assay and in vivo confirmation, 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), mildly reduced thyroidal ERK signaling, and altered expression of autophagy regulators (reduced p62, increased ATG5, elevated LC3-II/I ratio, decreased pBeclin/Beclin), placing SPRED3 as a regulator of thyroidal homeostasis and autophagy. X-Gal staining localized Spred3 promoter activity to thyroid, adrenal gland, pituitary, cerebral cortex, and kidney.\",\n      \"method\": \"SPRED3 knockout mouse generation; hormonal profiling (TSH, T4); immunoblotting for ERK, p62, ATG5, LC3, Beclin; X-Gal staining\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — constitutive KO mouse with multiple orthogonal molecular and hormonal readouts in a single study\",\n      \"pmids\": [\"40806788\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPRED3 is a brain-enriched negative regulator of the Ras/MAPK pathway that suppresses growth factor-induced ERK activation primarily through its C-terminal SPR domain (not its non-functional KBD); it is post-translationally regulated by S-acylation via zDHHC17 (through an SPR domain-mediated, ANK-independent mechanism) and by ubiquitination, and its loss activates Ras/Raf/MAPK signaling to drive EGFR-TKI resistance in NSCLC; in vivo, SPRED3 knockout mice develop primary hypothyroidism with altered thyroidal ERK signaling and dysregulated autophagy, and SPRED3 also modulates NF-κB signaling in thyroid cancer cells.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SPRED3 is a negative regulator of Ras/MAPK signaling that suppresses growth factor-induced ERK activation through its C-terminal cysteine-rich SPR domain, which also mediates membrane localization and a non-canonical interaction with the palmitoyl acyltransferase zDHHC17 for S-acylation [PMID:12646235, PMID:36442513]. Unlike SPRED1/2, SPRED3 lacks a functional c-Kit binding domain due to a critical Arg-to-Gly substitution, and it does not interact with RSK2, indicating paralog-specific regulatory wiring within the SPRED family [PMID:12646235, PMID:37149146]. Loss of SPRED3 activates Ras/Raf/MAPK signaling to drive EGFR-TKI resistance in NSCLC and causes primary hypothyroidism with dysregulated thyroidal ERK signaling and autophagy in knockout mice [PMID:35117614, PMID:40806788]. SPRED3 also modulates NF-κB signaling in thyroid cancer cells and blocks TGFβ-induced EMT and FGF-driven differentiation in lens epithelial cells [PMID:39227612, PMID:25576668, PMID:29501879].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Establishing that SPRED3 suppresses growth factor-induced ERK activation resolved how this third family member functions and revealed that its SPR domain, rather than its non-functional KBD, drives both membrane targeting and ERK suppression — distinguishing it mechanistically from SPRED1/2.\",\n      \"evidence\": \"Domain-swap chimeras and deletion mutants with ERK activation assays in overexpression system\",\n      \"pmids\": [\"12646235\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous SPRED3 loss-of-function not tested\", \"SPR domain binding partners mediating membrane localization unidentified\", \"No in vivo validation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating that SPRED3 is ubiquitinated upon growth factor stimulation established that proteasomal degradation regulates SPRED3 protein levels, paralleling the regulatory mechanism of SPRED1/2.\",\n      \"evidence\": \"Co-immunoprecipitation/ubiquitination assays in HEK293T cells with EGF stimulation and MG-132 treatment\",\n      \"pmids\": [\"17094949\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ubiquitin ligase responsible not identified\", \"Ubiquitination sites on SPRED3 not mapped\", \"Functional consequence of ubiquitination on ERK suppression not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of zDHHC17 (HIP14) as the palmitoyl acyltransferase that S-acylates SPRED3 provided the first enzyme-substrate link for SPRED3 lipid modification, suggesting a regulatory mechanism for its membrane association.\",\n      \"evidence\": \"Yeast two-hybrid screen for HIP14 interactors followed by palmitoylation assays\",\n      \"pmids\": [\"24705354\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of palmitoylation on SPRED3 ERK suppression not tested\", \"Palmitoylation sites not mapped\", \"Mechanism of enzyme-substrate recognition unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showing that SPRED3 blocks TGFβ-induced EMT in lens epithelial cells extended its inhibitory role beyond classical RTK-ERK signaling to TGFβ-driven cellular reprogramming.\",\n      \"evidence\": \"SPRED3 overexpression in rat lens epithelial explants with TGFβ treatment; morphological and α-SMA marker assessment\",\n      \"pmids\": [\"25576668\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SPRED3 directly inhibits TGFβ pathway components or acts via ERK cross-talk unclear\", \"Endogenous SPRED3 expression in lens tissue not confirmed\", \"Mechanism of EMT suppression not defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstration that SPRED3 blocks FGF-induced lens fiber differentiation by suppressing ERK1/2 phosphorylation confirmed its role as a general negative regulator of RTK-MAPK signaling in a developmental context.\",\n      \"evidence\": \"SPRED3 transfection in lens explants with FGF stimulation; p-ERK1/2 and Prox1 marker readouts\",\n      \"pmids\": [\"29501879\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous SPRED3 function in lens development not tested by knockout\", \"Relative contribution of SPRED3 vs SPRED1/2 in lens tissue unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovery that loss-of-function SPRED3 mutations activate Ras/Raf/MAPK signaling and confer EGFR-TKI resistance in NSCLC established SPRED3 as a clinically relevant tumor suppressor, with reciprocal knockout and overexpression validating causality.\",\n      \"evidence\": \"Whole-exome sequencing of erlotinib-resistant cells; CRISPR knockout and cDNA rescue; p-ERK, MTS, and migration assays\",\n      \"pmids\": [\"35117614\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Frequency of SPRED3 loss in clinical EGFR-TKI resistance cohorts unknown\", \"Direct interaction partner at the Raf level not defined\", \"Whether SPRED3 loss cooperates with other resistance mechanisms untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identifying miR-342-5p as a direct negative regulator of SPRED3 via 3′UTR targeting linked SPRED3 downregulation to neonatal lung pathology, revealing a post-transcriptional regulatory axis controlling SPRED3 abundance in vivo.\",\n      \"evidence\": \"3′UTR luciferase reporter assay; miR-342-5p gain/loss in murine BPD models; recombinant Spred3 treatment\",\n      \"pmids\": [\"33434946\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Paradoxical worsening with recombinant Spred3 in BPD model not mechanistically explained\", \"Whether Spred3 engages Raf1 directly or indirectly not resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defining that zDHHC17 S-acylates SPRED3 through an ANK-independent, SPR domain-mediated mechanism revealed a non-canonical mode of enzyme-substrate recognition distinct from other zDHHC17 substrates.\",\n      \"evidence\": \"Mutational analysis of SPRED3 and zDHHC17 ANK-deletion mutants; co-immunoprecipitation and S-acylation assays\",\n      \"pmids\": [\"36442513\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific cysteine residues acylated not mapped\", \"Whether S-acylation is required for SPRED3 membrane targeting and ERK suppression in cells not tested\", \"Structural basis of SPR-zDHHC17 recognition unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"AP-MS interactome comparison showing RSK2 binds SPRED2 but not SPRED3 established that SPRED paralogs occupy distinct signaling niches despite shared domain architecture.\",\n      \"evidence\": \"Affinity purification mass spectrometry of SPRED1/2/3 interactomes\",\n      \"pmids\": [\"37149146\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SPRED3-specific interactors not comprehensively characterized\", \"Functional consequence of differential RSK2 binding on MAPK feedback not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that SPRED3 activates NF-κB transcriptional activity and promotes thyroid cancer cell proliferation uncovered a signaling role beyond MAPK inhibition, supported by reciprocal gain/loss-of-function and in vivo xenograft data.\",\n      \"evidence\": \"Flag-SPRED3 overexpression and CRISPR knockout in thyroid cancer cells; NF-κB luciferase reporter; mouse xenograft\",\n      \"pmids\": [\"39227612\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which SPRED3 activates NF-κB not defined\", \"Reconciliation of tumor-suppressive MAPK role with tumor-promoting NF-κB role not addressed\", \"Whether NF-κB activation is direct or indirect unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"SPRED3 knockout mice developing primary hypothyroidism with reduced thyroidal ERK signaling and dysregulated autophagy established the first in vivo physiological function for SPRED3, linking it to thyroid hormone homeostasis.\",\n      \"evidence\": \"Constitutive SPRED3 KO mouse; serum TSH/T4 profiling; immunoblotting for ERK, p62, ATG5, LC3, Beclin; X-Gal promoter activity mapping\",\n      \"pmids\": [\"40806788\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether hypothyroidism results from direct thyroidal SPRED3 loss or secondary hypothalamic-pituitary effects not fully resolved\", \"How a MAPK inhibitor's loss leads to mildly reduced (not increased) thyroidal ERK requires mechanistic explanation\", \"Autophagy dysregulation could be secondary to hormonal changes\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The structural basis of SPRED3's SPR domain-mediated inhibition of Raf, the identity of SPRED3-specific protein interaction partners, and the mechanistic reconciliation of its MAPK-suppressive versus NF-κB-activating roles remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of SPRED3 or SPR domain in complex with Raf or zDHHC17\", \"SPRED3-specific interactome incompletely defined\", \"Context-dependent tumor-suppressive vs oncogenic functions not mechanistically explained\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 5, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 6, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"zDHHC17\", \"RAF1\"],\n    \"other_free_text\": []\n  }\n}\n```"}