{"gene":"SPRED2","run_date":"2026-06-10T07:46:40","timeline":{"discoveries":[{"year":2009,"finding":"SPRED2 interacts directly with the late endosomal protein NBR1 via its EVH1 domain, and this interaction is required for SPRED2-mediated attenuation of FGF signaling by redirecting activated FGF receptors to the lysosomal degradation pathway.","method":"Co-immunoprecipitation, colocalization, loss-of-function (NBR1 depletion), receptor trafficking assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, colocalization, functional rescue, single lab with multiple orthogonal methods","pmids":["19822672"],"is_preprint":false},{"year":2010,"finding":"SPRED2 directly interacts with the kinase domain of DYRK1A via the cysteine-rich domain (CRD) of SPRED2, and this interaction inhibits DYRK1A-mediated phosphorylation of its substrates Tau and STAT3 by competing for the substrate-binding site on DYRK1A.","method":"Co-immunoprecipitation (endogenous), domain-mapping pulldowns, in vitro kinase assays with substrate competition","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — endogenous Co-IP, domain mapping, in vitro kinase assay, single lab with multiple orthogonal methods","pmids":["20736167"],"is_preprint":false},{"year":2012,"finding":"Tyrosines Y303, Y343, and Y353 within the SPR domain of SPRED2 are critical for EGF-induced binding to the p85 subunit of PI3K; this p85 interaction enhances SPRED2-mediated inhibition of Ras/ERK by increasing Ras binding to SPRED2 and decreasing SPRED2 ubiquitination. SPRED2 also constitutively associates with EGFR via its SPR domain and dissociates upon EGF stimulation; mutation of these tyrosines enhances EGFR binding.","method":"Site-directed mutagenesis, Co-immunoprecipitation, cell proliferation assays, neurite outgrowth assays, ubiquitination assays","journal":"The international journal of biochemistry & cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis combined with Co-IP and functional readouts, single lab, multiple orthogonal methods","pmids":["22305891"],"is_preprint":false},{"year":2006,"finding":"SPRED2 is ubiquitinated in an EGF/pervanadate-stimulated manner; tyrosines Y228 and/or Y231 in the Kit-binding domain are required for SPRED2 ubiquitination. The E3 ubiquitin ligases Cbl and Cbl-b mediate SPRED2 ubiquitination, requiring the Cbl SH2 domain; this ubiquitination reduces SPRED2 steady-state levels via proteasomal degradation.","method":"Ubiquitination assays, site-directed mutagenesis, RNAi depletion of Cbl/Cbl-b, co-immunoprecipitation, proteasome inhibitor (MG-132) treatment","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis, RNAi, Co-IP, pharmacological inhibition; single lab, multiple orthogonal methods","pmids":["17094949"],"is_preprint":false},{"year":2023,"finding":"SPRED2 specifically interacts with the N-terminal kinase domain of RSK2 (but not SPRED1 or SPRED3) via SPRED2 residues 123–201, with F145 being critical for the interaction as determined by X-ray crystallography. This SPRED2–RSK2 complex formation is regulated by MAPK signaling, and SPRED2 knockdown increases RSK substrate phosphorylation (YB1, CREB) and alters phospho-RSK subcellular localization.","method":"Affinity purification mass spectrometry, X-ray crystallography, site-directed mutagenesis (F145A), Co-immunoprecipitation, knockdown with substrate phosphorylation and localization readouts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — X-ray crystal structure combined with mutagenesis, AP-MS, and functional phosphorylation assays in a single study","pmids":["37149146"],"is_preprint":false},{"year":2004,"finding":"Spred-2 functions as a negative regulator of the MAP kinase pathway in AGM hematopoiesis; overexpression of Spred-2 reduced CD45+ hematopoietic cell production in AGM culture, and Spred-2-null mice showed elevated CD45+ cell production and enhanced hematopoietic colony formation from VE-cadherin+ cells.","method":"Gain-of-function (overexpression) in AGM explant culture, Spred-2 knockout mouse analysis, colony-forming assays","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO and overexpression with defined cellular phenotype, replicated across multiple experimental systems","pmids":["14981116"],"is_preprint":false},{"year":2005,"finding":"Loss of Spred-2 in mice causes dwarfism similar to achondroplasia; Spred-2-deficient chondrocytes show earlier and augmented ERK phosphorylation in response to FGF stimulation, indicating that Spred-2 normally restrains the FGFR3/MAPK pathway to control bone growth via chondrocyte differentiation.","method":"Gene-trap knockout mouse, ERK phosphorylation assays in primary chondrocytes, skeletal measurements, histology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO, signaling measurements in primary cells, multiple phenotypic readouts, single lab","pmids":["15946934"],"is_preprint":false},{"year":2011,"finding":"SPRED2 deficiency leads to upregulated ERK/MAPK signaling in the hypothalamus, increasing ERK phosphorylation and Ets-dependent CRH promoter activity. Overexpressed SPRED2 suppresses CRH production in hypothalamic cells, linking SPRED2 to negative regulation of the hypothalamic–pituitary–adrenal (HPA) axis.","method":"SPRED2 KO mouse phenotyping, CRH promoter reporter assays in hypothalamic cells, Western blotting of ERK phosphorylation, hormone measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO combined with reporter assays and overexpression in hypothalamic cells, multiple readouts, single lab","pmids":["21199868"],"is_preprint":false},{"year":2016,"finding":"SPRED2 promotes autophagosome maturation and autophagy-dependent cell death in cancer cells via direct interaction with LC3 through LIR motifs in its SPR domain; mutations of LIR motifs or deletion of the SPR domain impair autophagosome maturation and cell death. SPRED2 also interacts and colocalizes with p62/SQSTM1 through its SPR domain.","method":"Co-immunoprecipitation, colocalization (GFP-LC3 puncta), LIR motif mutagenesis, SPR domain deletion, gene silencing (ATG5, LC3, p62), lysosomal inhibitor treatment","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis of defined motifs, multiple Co-IPs and functional readouts, single lab","pmids":["27028858"],"is_preprint":false},{"year":2019,"finding":"SPRED2 deficiency causes impaired autophagy, cardiomyocyte hypertrophy, cardiac fibrosis, and life-threatening arrhythmias via ERK hyperactivation; SPRED2 physically interacts with p62/SQSTM1, NBR1, and Cathepsin D in wild-type hearts, indicating a role in autophagolysosome formation. MEK inhibition with selumetinib restores autophagic flux in vivo.","method":"SPRED2 KO mouse cardiac phenotyping, Co-immunoprecipitation, LC3-II/LC3-I ratio, Atg protein expression, MEK inhibitor treatment in vivo","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO, Co-IP of endogenous proteins, in vivo rescue with MEK inhibitor, multiple orthogonal methods","pmids":["30771306"],"is_preprint":false},{"year":2017,"finding":"SPRED2 deficiency in mice causes OCD-like excessive grooming mediated by upregulated TrkB/ERK-MAPK signaling in the amygdala, with increased activity of TrkB, Ras, and ERK. Electrophysiology reveals altered thalamo-amygdala synaptic transmission. MEK inhibition with selumetinib reduces OCD-like behavior in SPRED2 KO mice.","method":"SPRED2 KO mouse behavioral analysis, electrophysiology, Western blotting of pathway components, MEK inhibitor treatment in vivo, fluoxetine treatment","journal":"Molecular psychiatry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic KO, electrophysiology, in vivo pharmacological rescue, multiple orthogonal methods, single lab","pmids":["28070119"],"is_preprint":false},{"year":2021,"finding":"SPRED2 loss-of-function variants (p.Arg63*, p.Leu100Pro, p.Leu381Hisfs*95) cause a recessive Noonan syndrome-like RASopathy; all variants impair protein stability and fail to negatively modulate EGF-induced RAF1, MEK, and ERK phosphorylation. Primary fibroblasts with these variants show increased and prolonged MAPK cascade activation in response to EGF. Morpholino knockdown of spred2a/b in zebrafish causes convergence/extension defects rescued by wild-type SPRED2 but not by the frameshift variant.","method":"Variant overexpression in cells, EGF stimulation/time-course signaling assays (RAF1/MEK/ERK phosphorylation), primary fibroblast experiments, zebrafish morpholino knockdown with rescue experiments","journal":"American journal of human genetics","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple patient variants tested by in vitro signaling assays and in vivo zebrafish rescue, multiple orthogonal methods","pmids":["34626534"],"is_preprint":false},{"year":2010,"finding":"Spred2 inhibits TGF-β1-induced ERK1/2 activation (but not Smad2 activation), blocking TGF-β1-induced uPA expression, EMT (E-cadherin disruption, actin reorganization, vimentin upregulation), and cell migration in transformed keratinocytes. Knockdown of Spred2 enhances TGF-β1-induced ERK activation.","method":"Stable overexpression and knockdown, luciferase reporter assay, Western blotting (ERK/Smad phosphorylation), cell migration assays, EMT marker analysis","journal":"International journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression and knockdown with multiple functional readouts including signaling specificity (ERK vs Smad), single lab","pmids":["19908229"],"is_preprint":false},{"year":2010,"finding":"Adenovirus-mediated Spred2 overexpression in CML cells inhibits constitutive and SCF-stimulated sphingosine kinase-1 (SPHK1) expression, Mcl-1 expression, and the Ras-ERK cascade, promoting apoptosis. Imatinib induces endogenous Spred2 expression in CML cells, and Spred2 silencing partially protects K562 cells from imatinib-induced apoptosis.","method":"Adenoviral overexpression, stable RNAi knockdown, imatinib treatment, apoptosis assays, Western blotting","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with defined signaling readouts (SPHK1, Mcl-1, ERK), single lab","pmids":["20153728"],"is_preprint":false},{"year":2014,"finding":"Spred-2 negatively regulates LPS-induced acute lung inflammation by inhibiting the ERK-MAPK pathway; Spred-2 KO mice show augmented ERK activation, increased cytokines/chemokines (TNF-α, CXCL2, CCL2), and enhanced leukocyte infiltration. MEK inhibitor U0126 reduces the augmented inflammation in Spred-2 KO mice.","method":"Spred-2 KO mouse model, siRNA knockdown and overexpression in macrophage/lung epithelial cell lines, MEK inhibitor treatment, cytokine ELISA, ERK phosphorylation assays","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO, siRNA, overexpression, and pharmacological rescue in vitro and in vivo, single lab","pmids":["25275324"],"is_preprint":false},{"year":2011,"finding":"Adenoviral Spred2 overexpression in hepatocellular carcinoma (HCC) cells reduces ERK activation, inhibits proliferation and migration, activates caspase-3-mediated apoptosis, and reduces Mcl-1 expression; Spred2 knockdown markedly enhances tumor growth in vivo.","method":"Adenoviral overexpression, RNAi knockdown, tumor xenograft in vivo, caspase-3 activation assay, Western blotting","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with in vivo xenograft and defined signaling readouts, single lab","pmids":["21703232"],"is_preprint":false},{"year":2022,"finding":"SPRED2 knockdown in ERα-positive breast cancer cells increases ERK1/2 activation, enhances ERα transcriptional activity, increases proliferation, and causes tamoxifen resistance. Combined treatment with ERK1/2 inhibitor ulixertinib and 4-OHT overcomes this resistance.","method":"siRNA knockdown, cell proliferation assays, ERK phosphorylation assays, ERα transcriptional reporter, pharmacological inhibitor combination treatment","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with defined signaling mechanism and pharmacological rescue, single lab, single set of methods","pmids":["35205702"],"is_preprint":false},{"year":2025,"finding":"Neurofibromin (NF1 gene product) forms a protein complex with SPRED2 and facilitates its translocation to the plasma membrane; NF or SPRED2 downregulation in breast cancer cells enhances RAF/ERK activation, cell proliferation, migration and invasion, while overexpression has opposite effects. Membrane localization of SPRED2 is absent in NF1-negative breast cancers.","method":"Co-immunoprecipitation, overexpression and knockdown, cell functional assays, immunohistochemistry of clinical samples, database analysis","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional assays in multiple cell lines, clinical IHC validation, single lab","pmids":["41155378"],"is_preprint":false},{"year":2024,"finding":"SPRED2 overexpression in HCC cells increases autophagosomes/autophagic vacuoles, decreases p62, and increases LC3-II via reduction of ERK activation and downstream mTORC1-mediated signaling; SPRED2 deficiency shows the opposite pattern. SPRED2-deficient mice show impaired hepatic autophagy with lipid droplet accumulation during starvation.","method":"Overexpression and KO in HCC cell lines, LC3-II/p62/TOM20 Western blotting, electron microscopy of autophagic vacuoles, SPRED2 KO mouse starvation model, mTORC1 signaling assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function in vitro plus in vivo KO mouse model, multiple markers, single lab","pmids":["38892460"],"is_preprint":false},{"year":2026,"finding":"SPRED2 forms a protein complex with p53 and cooperatively upregulates miR-506 gene transcription by binding to its promoter region; increased miR-506-3p then downregulates KLF4 mRNA, suppressing HCC cell stemness. SPRED2-KO HCC cells show elevated KLF4, Nanog, and c-Myc expression in an ERK-dependent manner.","method":"Co-immunoprecipitation, ChIP-qPCR, overexpression/knockdown, Western blotting, RT-qPCR, 3D sphere formation assays","journal":"Cancer biology & medicine","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — ChIP-qPCR and Co-IP establish physical complex with functional promoter evidence; single lab","pmids":["41560328"],"is_preprint":false},{"year":2020,"finding":"SPRED2 knockout in HCC cells promotes EMT (elongated morphology, cadherin switching) and cancer stem cell features (sphere/colony formation, elevated CD44/CD90/stemness markers, cisplatin resistance) via ERK1/2 pathway activation; endogenous SPRED2 expression is lower in CD44+CD90+ stem-like populations and in 3D culture conditions.","method":"CRISPR/gene-KO, overexpression, knockdown, EMT marker analysis, sphere/colony formation, flow cytometry for stem cell markers, drug resistance assays","journal":"International journal of molecular sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO plus functional readouts in multiple contexts, single lab","pmids":["36902429"],"is_preprint":false},{"year":2019,"finding":"Spred2 in macrophages negatively regulates high-fat diet-induced ERK activation; Spred2 KO bone marrow-derived macrophages produce higher TNFα and MCP-1 upon palmitate stimulation and show enhanced ERK activation, which is reversed by MEK inhibitor U0126, linking Spred2 to adipose tissue inflammation and metabolic dysregulation.","method":"Spred2 KO mouse model, bone marrow-derived macrophage stimulation assays, MEK inhibitor treatment, ELISA, ERK phosphorylation assays","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with primary cell functional assays and pharmacological rescue, single lab","pmids":["30723473"],"is_preprint":false},{"year":2024,"finding":"SPRED2 interacts with RSK kinases through a DDVF-like short linear motif (SLiM) at the same interface used by pathogen-derived proteins, as confirmed by co-immunoprecipitation experiments; this interaction is consistent with the previously crystallographically documented SPRED2–RSK2 interface.","method":"AlphaFold docking prediction, co-immunoprecipitation","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP, computational docking, preprint, corroborating but not extending prior crystallography","pmids":["bio_10.1101_2024.08.08.607128"],"is_preprint":true}],"current_model":"SPRED2 is a membrane-associated negative regulator of the RAS/RAF/ERK-MAPK pathway that operates through multiple molecular mechanisms: it binds NBR1 via its EVH1 domain to redirect activated growth factor receptors to lysosomal degradation; its SPR domain tyrosines (Y303/Y343/Y353) mediate binding to p85 (PI3K) and EGFR to enhance Ras inhibition; its CRD binds and inhibits DYRK1A kinase activity; it specifically interacts with RSK2 (but not RSK1/RSK3) via residues 123–201 (critical F145) to limit RSK substrate phosphorylation; it is degraded by Cbl/Cbl-b-mediated ubiquitination triggered by tyrosine phosphorylation at Y228/Y231; it forms a complex with p53 to drive miR-506-3p-mediated KLF4 suppression in cancer cells; it promotes autophagosome maturation by interacting with LC3 via LIR motifs in its SPR domain and with p62/SQSTM1 and NBR1; and neurofibromin facilitates its membrane translocation to enable Ras inhibition, with deficiency causing RASopathy (recessive Noonan syndrome), dwarfism, OCD-like behavior, cardiac arrhythmias, and exacerbated inflammatory responses across multiple tissues."},"narrative":{"mechanistic_narrative":"SPRED2 is a membrane-associated negative regulator of the RAS/RAF/ERK-MAPK pathway that restrains growth-factor signaling across many tissues and developmental contexts [PMID:15946934, PMID:34626534]. It attenuates receptor signaling by routing activated receptors for degradation: its EVH1 domain binds the late endosomal adaptor NBR1 to redirect activated FGF receptors to the lysosomal pathway [PMID:19822672], while tyrosines Y303/Y343/Y353 in its SPR domain mediate EGF-induced binding to the p85 subunit of PI3K, increasing Ras association with SPRED2 and reinforcing Ras/ERK inhibition [PMID:22305891]. SPRED2 protein levels are set by Cbl/Cbl-b-mediated ubiquitination triggered by phosphorylation at Y228/Y231, coupling its abundance to receptor activation [PMID:17094949]. Beyond Ras, SPRED2 directly engages additional kinases—inhibiting DYRK1A substrate phosphorylation through its cysteine-rich domain [PMID:20736167] and binding the N-terminal kinase domain of RSK2 via residues 123–201 (critical F145) to limit RSK substrate phosphorylation [PMID:37149146]. Through LIR motifs in its SPR domain SPRED2 binds LC3 and associates with p62/SQSTM1 and NBR1 to promote autophagosome maturation and autophagic flux [PMID:27028858, PMID:30771306]. Loss of SPRED2 produces ERK hyperactivation with phenotypes including dwarfism via unrestrained FGFR3 signaling in chondrocytes [PMID:15946934], OCD-like grooming through amygdalar TrkB/ERK signaling [PMID:28070119], cardiomyocyte hypertrophy and arrhythmia with impaired autophagy [PMID:30771306], and exaggerated inflammatory responses [PMID:25275324]; in cancer, SPRED2 acts as a tumor suppressor that restrains proliferation, EMT, and stemness [PMID:21703232, PMID:36902429]. Biallelic loss-of-function variants in SPRED2 cause a recessive Noonan syndrome-like RASopathy, with patient variants destabilizing the protein and failing to dampen EGF-induced RAF1/MEK/ERK activation [PMID:34626534].","teleology":[{"year":2004,"claim":"Established SPRED2 as a functional negative regulator of the MAPK pathway with a defined cellular consequence, moving it from sequence to physiology.","evidence":"Spred-2 overexpression and knockout in AGM hematopoiesis explant cultures and mice","pmids":["14981116"],"confidence":"High","gaps":["Molecular mechanism of MAPK inhibition not resolved","No defined direct binding partners at this stage"]},{"year":2005,"claim":"Linked SPRED2 to a specific developmental signaling axis by showing it restrains FGFR3/MAPK in chondrocytes to control bone growth.","evidence":"Gene-trap knockout mouse with ERK phosphorylation assays in primary chondrocytes and skeletal phenotyping","pmids":["15946934"],"confidence":"High","gaps":["Direct molecular interaction with FGFR machinery not demonstrated","Does not address receptor trafficking mechanism"]},{"year":2006,"claim":"Defined how SPRED2 abundance is controlled, showing tyrosine-phosphorylation-dependent Cbl/Cbl-b ubiquitination drives its proteasomal degradation.","evidence":"Ubiquitination assays, Y228/Y231 mutagenesis, Cbl/Cbl-b RNAi, MG-132 treatment","pmids":["17094949"],"confidence":"High","gaps":["Kinase responsible for Y228/Y231 phosphorylation not identified","Functional consequence of degradation on downstream signaling not quantified here"]},{"year":2009,"claim":"Provided a degradation-based mechanism for signal attenuation, showing the EVH1 domain binds NBR1 to route activated FGF receptors to lysosomes.","evidence":"Reciprocal Co-IP, colocalization, NBR1 depletion, receptor trafficking assays","pmids":["19822672"],"confidence":"High","gaps":["Generality to receptors beyond FGFR not established","Structural basis of EVH1–NBR1 binding unknown"]},{"year":2010,"claim":"Expanded SPRED2's targets beyond Ras by showing its CRD directly inhibits DYRK1A kinase activity via substrate competition.","evidence":"Endogenous Co-IP, domain-mapping pulldowns, in vitro kinase assays with substrate competition","pmids":["20736167"],"confidence":"High","gaps":["Cellular contexts where DYRK1A inhibition is physiologically relevant not defined","Relationship to ERK regulation unclear"]},{"year":2010,"claim":"Demonstrated SPRED2 selectively blocks the ERK branch of TGF-β1 signaling, controlling EMT and migration without affecting Smad activation.","evidence":"Stable overexpression/knockdown, reporter assays, ERK vs Smad phosphorylation, migration and EMT marker analysis in keratinocytes","pmids":["19908229"],"confidence":"Medium","gaps":["Mechanism of ERK-branch selectivity within TGF-β signaling not resolved","Limited to a single transformed cell context"]},{"year":2012,"claim":"Identified phosphotyrosine-dependent recruitment of p85/PI3K and EGFR to the SPR domain as a mechanism that tunes Ras inhibition and SPRED2 stability.","evidence":"Y303/Y343/Y353 mutagenesis, Co-IP, proliferation and neurite outgrowth assays, ubiquitination assays","pmids":["22305891"],"confidence":"High","gaps":["Kinase phosphorylating the SPR tyrosines not identified","Quantitative contribution of p85 binding versus EGFR dissociation not separated"]},{"year":2016,"claim":"Connected SPRED2 to autophagy directly, showing SPR-domain LIR motifs bind LC3 and p62 to drive autophagosome maturation.","evidence":"Co-IP, GFP-LC3 puncta colocalization, LIR motif and SPR-deletion mutagenesis, ATG5/LC3/p62 silencing","pmids":["27028858"],"confidence":"High","gaps":["Whether autophagy role is independent of ERK inhibition not fully separated","In vivo relevance not yet shown in this study"]},{"year":2019,"claim":"Extended the autophagy role to cardiac and metabolic physiology, showing SPRED2 complexes with p62/NBR1/Cathepsin D and that ERK-driven autophagy failure underlies cardiac pathology.","evidence":"SPRED2 KO cardiac phenotyping and macrophage assays, endogenous Co-IP, LC3 ratios, in vivo selumetinib and U0126 rescue","pmids":["30771306","30723473"],"confidence":"High","gaps":["Direct versus ERK-mediated contributions to autophagy in vivo not fully dissected","Tissue-specific partner stoichiometry unknown"]},{"year":2021,"claim":"Established SPRED2 as a Mendelian disease gene, showing biallelic loss-of-function variants cause recessive Noonan-like RASopathy via destabilization and failed MAPK suppression.","evidence":"Patient variant overexpression with EGF time-course signaling, primary fibroblasts, zebrafish morpholino knockdown with rescue","pmids":["34626534"],"confidence":"High","gaps":["Genotype–phenotype correlations across the variant spectrum incomplete","Tissue-specific disease mechanisms not resolved"]},{"year":2023,"claim":"Provided structural definition of a SPRED2 kinase interaction, showing residues 123–201 (F145) bind specifically to the RSK2 N-terminal kinase domain to limit RSK substrate phosphorylation.","evidence":"AP-MS, X-ray crystallography, F145A mutagenesis, Co-IP, knockdown with YB1/CREB phosphorylation and localization readouts","pmids":["37149146"],"confidence":"High","gaps":["Selectivity for RSK2 over RSK1/RSK3 mechanism beyond the interface not fully explained","Physiological output of RSK regulation in vivo undefined"]},{"year":2025,"claim":"Defined how SPRED2 reaches its site of action, showing neurofibromin forms a complex with SPRED2 and drives its plasma membrane translocation for RAF/ERK inhibition.","evidence":"Co-IP, overexpression/knockdown functional assays, IHC of NF1-negative breast cancers","pmids":["41155378"],"confidence":"Medium","gaps":["Structural basis of the NF1–SPRED2 membrane-targeting complex not defined","Single-lab Co-IP without orthogonal interaction validation"]},{"year":2026,"claim":"Identified a transcriptional axis for SPRED2 tumor suppression, showing a SPRED2–p53 complex activates miR-506 to suppress KLF4 and limit HCC stemness.","evidence":"Co-IP, ChIP-qPCR, overexpression/knockdown, 3D sphere assays, RT-qPCR/Western blotting","pmids":["41560328"],"confidence":"Medium","gaps":["How a cytoplasmic MAPK regulator engages nuclear p53 not mechanistically explained","Generality beyond HCC not tested"]},{"year":null,"claim":"How SPRED2's multiple direct activities (NBR1/lysosomal targeting, LC3-autophagy, DYRK1A/RSK2 inhibition, p53/transcriptional roles) are integrated and prioritized within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of how SPRED2 partitions between membrane Ras inhibition, autophagy scaffolding, and nuclear functions","Upstream kinases setting SPRED2 phospho-state across contexts not fully mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,4,6,11]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[1,4]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,8,9]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[8]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[17]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[0]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8,9]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,6,11,17]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[8,9,18]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[14,21]}],"complexes":[],"partners":["NBR1","DYRK1A","RSK2","CBL","PIK3R1","MAP1LC3","SQSTM1","NF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7Z698","full_name":"Sprouty-related, EVH1 domain-containing protein 2","aliases":[],"length_aa":418,"mass_kda":47.6,"function":"Negatively regulates Ras signaling pathways and downstream activation of MAP kinases (PubMed:15683364, PubMed:34626534). Recruits and translocates NF1 to the cell membrane, thereby enabling NF1-dependent hydrolysis of active GTP-bound Ras to inactive GDP-bound Ras (PubMed:34626534). 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; Cytoplasmic vesicle, secretory vesicle membrane; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q7Z698/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPRED2","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SPRED2","total_profiled":1310},"omim":[{"mim_id":"619745","title":"NOONAN SYNDROME 14; NS14","url":"https://www.omim.org/entry/619745"},{"mim_id":"609293","title":"SPROUTY-RELATED EVH1 DOMAIN-CONTAINING PROTEIN 3; SPRED3","url":"https://www.omim.org/entry/609293"},{"mim_id":"609292","title":"SPROUTY-RELATED EVH1 DOMAIN-CONTAINING PROTEIN 2; SPRED2","url":"https://www.omim.org/entry/609292"},{"mim_id":"609291","title":"SPROUTY-RELATED EVH1 DOMAIN-CONTAINING PROTEIN 1; SPRED1","url":"https://www.omim.org/entry/609291"},{"mim_id":"163950","title":"NOONAN SYNDROME 1; NS1","url":"https://www.omim.org/entry/163950"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Nucleoplasm","reliability":"Uncertain"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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SPRED2 also constitutively associates with EGFR via its SPR domain and dissociates upon EGF stimulation; mutation of these tyrosines enhances EGFR binding.\",\n      \"method\": \"Site-directed mutagenesis, Co-immunoprecipitation, cell proliferation assays, neurite outgrowth assays, ubiquitination assays\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis combined with Co-IP and functional readouts, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"22305891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SPRED2 is ubiquitinated in an EGF/pervanadate-stimulated manner; tyrosines Y228 and/or Y231 in the Kit-binding domain are required for SPRED2 ubiquitination. The E3 ubiquitin ligases Cbl and Cbl-b mediate SPRED2 ubiquitination, requiring the Cbl SH2 domain; this ubiquitination reduces SPRED2 steady-state levels via proteasomal degradation.\",\n      \"method\": \"Ubiquitination assays, site-directed mutagenesis, RNAi depletion of Cbl/Cbl-b, co-immunoprecipitation, proteasome inhibitor (MG-132) treatment\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis, RNAi, Co-IP, pharmacological inhibition; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"17094949\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SPRED2 specifically interacts with the N-terminal kinase domain of RSK2 (but not SPRED1 or SPRED3) via SPRED2 residues 123–201, with F145 being critical for the interaction as determined by X-ray crystallography. This SPRED2–RSK2 complex formation is regulated by MAPK signaling, and SPRED2 knockdown increases RSK substrate phosphorylation (YB1, CREB) and alters phospho-RSK subcellular localization.\",\n      \"method\": \"Affinity purification mass spectrometry, X-ray crystallography, site-directed mutagenesis (F145A), Co-immunoprecipitation, knockdown with substrate phosphorylation and localization readouts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — X-ray crystal structure combined with mutagenesis, AP-MS, and functional phosphorylation assays in a single study\",\n      \"pmids\": [\"37149146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Spred-2 functions as a negative regulator of the MAP kinase pathway in AGM hematopoiesis; overexpression of Spred-2 reduced CD45+ hematopoietic cell production in AGM culture, and Spred-2-null mice showed elevated CD45+ cell production and enhanced hematopoietic colony formation from VE-cadherin+ cells.\",\n      \"method\": \"Gain-of-function (overexpression) in AGM explant culture, Spred-2 knockout mouse analysis, colony-forming assays\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO and overexpression with defined cellular phenotype, replicated across multiple experimental systems\",\n      \"pmids\": [\"14981116\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Loss of Spred-2 in mice causes dwarfism similar to achondroplasia; Spred-2-deficient chondrocytes show earlier and augmented ERK phosphorylation in response to FGF stimulation, indicating that Spred-2 normally restrains the FGFR3/MAPK pathway to control bone growth via chondrocyte differentiation.\",\n      \"method\": \"Gene-trap knockout mouse, ERK phosphorylation assays in primary chondrocytes, skeletal measurements, histology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO, signaling measurements in primary cells, multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"15946934\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SPRED2 deficiency leads to upregulated ERK/MAPK signaling in the hypothalamus, increasing ERK phosphorylation and Ets-dependent CRH promoter activity. Overexpressed SPRED2 suppresses CRH production in hypothalamic cells, linking SPRED2 to negative regulation of the hypothalamic–pituitary–adrenal (HPA) axis.\",\n      \"method\": \"SPRED2 KO mouse phenotyping, CRH promoter reporter assays in hypothalamic cells, Western blotting of ERK phosphorylation, hormone measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO combined with reporter assays and overexpression in hypothalamic cells, multiple readouts, single lab\",\n      \"pmids\": [\"21199868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPRED2 promotes autophagosome maturation and autophagy-dependent cell death in cancer cells via direct interaction with LC3 through LIR motifs in its SPR domain; mutations of LIR motifs or deletion of the SPR domain impair autophagosome maturation and cell death. SPRED2 also interacts and colocalizes with p62/SQSTM1 through its SPR domain.\",\n      \"method\": \"Co-immunoprecipitation, colocalization (GFP-LC3 puncta), LIR motif mutagenesis, SPR domain deletion, gene silencing (ATG5, LC3, p62), lysosomal inhibitor treatment\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis of defined motifs, multiple Co-IPs and functional readouts, single lab\",\n      \"pmids\": [\"27028858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPRED2 deficiency causes impaired autophagy, cardiomyocyte hypertrophy, cardiac fibrosis, and life-threatening arrhythmias via ERK hyperactivation; SPRED2 physically interacts with p62/SQSTM1, NBR1, and Cathepsin D in wild-type hearts, indicating a role in autophagolysosome formation. MEK inhibition with selumetinib restores autophagic flux in vivo.\",\n      \"method\": \"SPRED2 KO mouse cardiac phenotyping, Co-immunoprecipitation, LC3-II/LC3-I ratio, Atg protein expression, MEK inhibitor treatment in vivo\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO, Co-IP of endogenous proteins, in vivo rescue with MEK inhibitor, multiple orthogonal methods\",\n      \"pmids\": [\"30771306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPRED2 deficiency in mice causes OCD-like excessive grooming mediated by upregulated TrkB/ERK-MAPK signaling in the amygdala, with increased activity of TrkB, Ras, and ERK. Electrophysiology reveals altered thalamo-amygdala synaptic transmission. MEK inhibition with selumetinib reduces OCD-like behavior in SPRED2 KO mice.\",\n      \"method\": \"SPRED2 KO mouse behavioral analysis, electrophysiology, Western blotting of pathway components, MEK inhibitor treatment in vivo, fluoxetine treatment\",\n      \"journal\": \"Molecular psychiatry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO, electrophysiology, in vivo pharmacological rescue, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"28070119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPRED2 loss-of-function variants (p.Arg63*, p.Leu100Pro, p.Leu381Hisfs*95) cause a recessive Noonan syndrome-like RASopathy; all variants impair protein stability and fail to negatively modulate EGF-induced RAF1, MEK, and ERK phosphorylation. Primary fibroblasts with these variants show increased and prolonged MAPK cascade activation in response to EGF. Morpholino knockdown of spred2a/b in zebrafish causes convergence/extension defects rescued by wild-type SPRED2 but not by the frameshift variant.\",\n      \"method\": \"Variant overexpression in cells, EGF stimulation/time-course signaling assays (RAF1/MEK/ERK phosphorylation), primary fibroblast experiments, zebrafish morpholino knockdown with rescue experiments\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple patient variants tested by in vitro signaling assays and in vivo zebrafish rescue, multiple orthogonal methods\",\n      \"pmids\": [\"34626534\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Spred2 inhibits TGF-β1-induced ERK1/2 activation (but not Smad2 activation), blocking TGF-β1-induced uPA expression, EMT (E-cadherin disruption, actin reorganization, vimentin upregulation), and cell migration in transformed keratinocytes. Knockdown of Spred2 enhances TGF-β1-induced ERK activation.\",\n      \"method\": \"Stable overexpression and knockdown, luciferase reporter assay, Western blotting (ERK/Smad phosphorylation), cell migration assays, EMT marker analysis\",\n      \"journal\": \"International journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression and knockdown with multiple functional readouts including signaling specificity (ERK vs Smad), single lab\",\n      \"pmids\": [\"19908229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Adenovirus-mediated Spred2 overexpression in CML cells inhibits constitutive and SCF-stimulated sphingosine kinase-1 (SPHK1) expression, Mcl-1 expression, and the Ras-ERK cascade, promoting apoptosis. Imatinib induces endogenous Spred2 expression in CML cells, and Spred2 silencing partially protects K562 cells from imatinib-induced apoptosis.\",\n      \"method\": \"Adenoviral overexpression, stable RNAi knockdown, imatinib treatment, apoptosis assays, Western blotting\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with defined signaling readouts (SPHK1, Mcl-1, ERK), single lab\",\n      \"pmids\": [\"20153728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Spred-2 negatively regulates LPS-induced acute lung inflammation by inhibiting the ERK-MAPK pathway; Spred-2 KO mice show augmented ERK activation, increased cytokines/chemokines (TNF-α, CXCL2, CCL2), and enhanced leukocyte infiltration. MEK inhibitor U0126 reduces the augmented inflammation in Spred-2 KO mice.\",\n      \"method\": \"Spred-2 KO mouse model, siRNA knockdown and overexpression in macrophage/lung epithelial cell lines, MEK inhibitor treatment, cytokine ELISA, ERK phosphorylation assays\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO, siRNA, overexpression, and pharmacological rescue in vitro and in vivo, single lab\",\n      \"pmids\": [\"25275324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Adenoviral Spred2 overexpression in hepatocellular carcinoma (HCC) cells reduces ERK activation, inhibits proliferation and migration, activates caspase-3-mediated apoptosis, and reduces Mcl-1 expression; Spred2 knockdown markedly enhances tumor growth in vivo.\",\n      \"method\": \"Adenoviral overexpression, RNAi knockdown, tumor xenograft in vivo, caspase-3 activation assay, Western blotting\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with in vivo xenograft and defined signaling readouts, single lab\",\n      \"pmids\": [\"21703232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPRED2 knockdown in ERα-positive breast cancer cells increases ERK1/2 activation, enhances ERα transcriptional activity, increases proliferation, and causes tamoxifen resistance. Combined treatment with ERK1/2 inhibitor ulixertinib and 4-OHT overcomes this resistance.\",\n      \"method\": \"siRNA knockdown, cell proliferation assays, ERK phosphorylation assays, ERα transcriptional reporter, pharmacological inhibitor combination treatment\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with defined signaling mechanism and pharmacological rescue, single lab, single set of methods\",\n      \"pmids\": [\"35205702\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Neurofibromin (NF1 gene product) forms a protein complex with SPRED2 and facilitates its translocation to the plasma membrane; NF or SPRED2 downregulation in breast cancer cells enhances RAF/ERK activation, cell proliferation, migration and invasion, while overexpression has opposite effects. Membrane localization of SPRED2 is absent in NF1-negative breast cancers.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and knockdown, cell functional assays, immunohistochemistry of clinical samples, database analysis\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional assays in multiple cell lines, clinical IHC validation, single lab\",\n      \"pmids\": [\"41155378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPRED2 overexpression in HCC cells increases autophagosomes/autophagic vacuoles, decreases p62, and increases LC3-II via reduction of ERK activation and downstream mTORC1-mediated signaling; SPRED2 deficiency shows the opposite pattern. SPRED2-deficient mice show impaired hepatic autophagy with lipid droplet accumulation during starvation.\",\n      \"method\": \"Overexpression and KO in HCC cell lines, LC3-II/p62/TOM20 Western blotting, electron microscopy of autophagic vacuoles, SPRED2 KO mouse starvation model, mTORC1 signaling assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function in vitro plus in vivo KO mouse model, multiple markers, single lab\",\n      \"pmids\": [\"38892460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"SPRED2 forms a protein complex with p53 and cooperatively upregulates miR-506 gene transcription by binding to its promoter region; increased miR-506-3p then downregulates KLF4 mRNA, suppressing HCC cell stemness. SPRED2-KO HCC cells show elevated KLF4, Nanog, and c-Myc expression in an ERK-dependent manner.\",\n      \"method\": \"Co-immunoprecipitation, ChIP-qPCR, overexpression/knockdown, Western blotting, RT-qPCR, 3D sphere formation assays\",\n      \"journal\": \"Cancer biology & medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — ChIP-qPCR and Co-IP establish physical complex with functional promoter evidence; single lab\",\n      \"pmids\": [\"41560328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPRED2 knockout in HCC cells promotes EMT (elongated morphology, cadherin switching) and cancer stem cell features (sphere/colony formation, elevated CD44/CD90/stemness markers, cisplatin resistance) via ERK1/2 pathway activation; endogenous SPRED2 expression is lower in CD44+CD90+ stem-like populations and in 3D culture conditions.\",\n      \"method\": \"CRISPR/gene-KO, overexpression, knockdown, EMT marker analysis, sphere/colony formation, flow cytometry for stem cell markers, drug resistance assays\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO plus functional readouts in multiple contexts, single lab\",\n      \"pmids\": [\"36902429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Spred2 in macrophages negatively regulates high-fat diet-induced ERK activation; Spred2 KO bone marrow-derived macrophages produce higher TNFα and MCP-1 upon palmitate stimulation and show enhanced ERK activation, which is reversed by MEK inhibitor U0126, linking Spred2 to adipose tissue inflammation and metabolic dysregulation.\",\n      \"method\": \"Spred2 KO mouse model, bone marrow-derived macrophage stimulation assays, MEK inhibitor treatment, ELISA, ERK phosphorylation assays\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with primary cell functional assays and pharmacological rescue, single lab\",\n      \"pmids\": [\"30723473\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SPRED2 interacts with RSK kinases through a DDVF-like short linear motif (SLiM) at the same interface used by pathogen-derived proteins, as confirmed by co-immunoprecipitation experiments; this interaction is consistent with the previously crystallographically documented SPRED2–RSK2 interface.\",\n      \"method\": \"AlphaFold docking prediction, co-immunoprecipitation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP, computational docking, preprint, corroborating but not extending prior crystallography\",\n      \"pmids\": [\"bio_10.1101_2024.08.08.607128\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SPRED2 is a membrane-associated negative regulator of the RAS/RAF/ERK-MAPK pathway that operates through multiple molecular mechanisms: it binds NBR1 via its EVH1 domain to redirect activated growth factor receptors to lysosomal degradation; its SPR domain tyrosines (Y303/Y343/Y353) mediate binding to p85 (PI3K) and EGFR to enhance Ras inhibition; its CRD binds and inhibits DYRK1A kinase activity; it specifically interacts with RSK2 (but not RSK1/RSK3) via residues 123–201 (critical F145) to limit RSK substrate phosphorylation; it is degraded by Cbl/Cbl-b-mediated ubiquitination triggered by tyrosine phosphorylation at Y228/Y231; it forms a complex with p53 to drive miR-506-3p-mediated KLF4 suppression in cancer cells; it promotes autophagosome maturation by interacting with LC3 via LIR motifs in its SPR domain and with p62/SQSTM1 and NBR1; and neurofibromin facilitates its membrane translocation to enable Ras inhibition, with deficiency causing RASopathy (recessive Noonan syndrome), dwarfism, OCD-like behavior, cardiac arrhythmias, and exacerbated inflammatory responses across multiple tissues.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPRED2 is a membrane-associated negative regulator of the RAS/RAF/ERK-MAPK pathway that restrains growth-factor signaling across many tissues and developmental contexts [#6, #11]. It attenuates receptor signaling by routing activated receptors for degradation: its EVH1 domain binds the late endosomal adaptor NBR1 to redirect activated FGF receptors to the lysosomal pathway [#0], while tyrosines Y303/Y343/Y353 in its SPR domain mediate EGF-induced binding to the p85 subunit of PI3K, increasing Ras association with SPRED2 and reinforcing Ras/ERK inhibition [#2]. SPRED2 protein levels are set by Cbl/Cbl-b-mediated ubiquitination triggered by phosphorylation at Y228/Y231, coupling its abundance to receptor activation [#3]. Beyond Ras, SPRED2 directly engages additional kinases—inhibiting DYRK1A substrate phosphorylation through its cysteine-rich domain [#1] and binding the N-terminal kinase domain of RSK2 via residues 123–201 (critical F145) to limit RSK substrate phosphorylation [#4]. Through LIR motifs in its SPR domain SPRED2 binds LC3 and associates with p62/SQSTM1 and NBR1 to promote autophagosome maturation and autophagic flux [#8, #9]. Loss of SPRED2 produces ERK hyperactivation with phenotypes including dwarfism via unrestrained FGFR3 signaling in chondrocytes [#6], OCD-like grooming through amygdalar TrkB/ERK signaling [#10], cardiomyocyte hypertrophy and arrhythmia with impaired autophagy [#9], and exaggerated inflammatory responses [#14]; in cancer, SPRED2 acts as a tumor suppressor that restrains proliferation, EMT, and stemness [#15, #20]. Biallelic loss-of-function variants in SPRED2 cause a recessive Noonan syndrome-like RASopathy, with patient variants destabilizing the protein and failing to dampen EGF-induced RAF1/MEK/ERK activation [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established SPRED2 as a functional negative regulator of the MAPK pathway with a defined cellular consequence, moving it from sequence to physiology.\",\n      \"evidence\": \"Spred-2 overexpression and knockout in AGM hematopoiesis explant cultures and mice\",\n      \"pmids\": [\"14981116\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of MAPK inhibition not resolved\", \"No defined direct binding partners at this stage\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked SPRED2 to a specific developmental signaling axis by showing it restrains FGFR3/MAPK in chondrocytes to control bone growth.\",\n      \"evidence\": \"Gene-trap knockout mouse with ERK phosphorylation assays in primary chondrocytes and skeletal phenotyping\",\n      \"pmids\": [\"15946934\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular interaction with FGFR machinery not demonstrated\", \"Does not address receptor trafficking mechanism\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined how SPRED2 abundance is controlled, showing tyrosine-phosphorylation-dependent Cbl/Cbl-b ubiquitination drives its proteasomal degradation.\",\n      \"evidence\": \"Ubiquitination assays, Y228/Y231 mutagenesis, Cbl/Cbl-b RNAi, MG-132 treatment\",\n      \"pmids\": [\"17094949\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for Y228/Y231 phosphorylation not identified\", \"Functional consequence of degradation on downstream signaling not quantified here\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Provided a degradation-based mechanism for signal attenuation, showing the EVH1 domain binds NBR1 to route activated FGF receptors to lysosomes.\",\n      \"evidence\": \"Reciprocal Co-IP, colocalization, NBR1 depletion, receptor trafficking assays\",\n      \"pmids\": [\"19822672\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality to receptors beyond FGFR not established\", \"Structural basis of EVH1–NBR1 binding unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Expanded SPRED2's targets beyond Ras by showing its CRD directly inhibits DYRK1A kinase activity via substrate competition.\",\n      \"evidence\": \"Endogenous Co-IP, domain-mapping pulldowns, in vitro kinase assays with substrate competition\",\n      \"pmids\": [\"20736167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular contexts where DYRK1A inhibition is physiologically relevant not defined\", \"Relationship to ERK regulation unclear\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Demonstrated SPRED2 selectively blocks the ERK branch of TGF-β1 signaling, controlling EMT and migration without affecting Smad activation.\",\n      \"evidence\": \"Stable overexpression/knockdown, reporter assays, ERK vs Smad phosphorylation, migration and EMT marker analysis in keratinocytes\",\n      \"pmids\": [\"19908229\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of ERK-branch selectivity within TGF-β signaling not resolved\", \"Limited to a single transformed cell context\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified phosphotyrosine-dependent recruitment of p85/PI3K and EGFR to the SPR domain as a mechanism that tunes Ras inhibition and SPRED2 stability.\",\n      \"evidence\": \"Y303/Y343/Y353 mutagenesis, Co-IP, proliferation and neurite outgrowth assays, ubiquitination assays\",\n      \"pmids\": [\"22305891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase phosphorylating the SPR tyrosines not identified\", \"Quantitative contribution of p85 binding versus EGFR dissociation not separated\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected SPRED2 to autophagy directly, showing SPR-domain LIR motifs bind LC3 and p62 to drive autophagosome maturation.\",\n      \"evidence\": \"Co-IP, GFP-LC3 puncta colocalization, LIR motif and SPR-deletion mutagenesis, ATG5/LC3/p62 silencing\",\n      \"pmids\": [\"27028858\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether autophagy role is independent of ERK inhibition not fully separated\", \"In vivo relevance not yet shown in this study\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Extended the autophagy role to cardiac and metabolic physiology, showing SPRED2 complexes with p62/NBR1/Cathepsin D and that ERK-driven autophagy failure underlies cardiac pathology.\",\n      \"evidence\": \"SPRED2 KO cardiac phenotyping and macrophage assays, endogenous Co-IP, LC3 ratios, in vivo selumetinib and U0126 rescue\",\n      \"pmids\": [\"30771306\", \"30723473\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct versus ERK-mediated contributions to autophagy in vivo not fully dissected\", \"Tissue-specific partner stoichiometry unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established SPRED2 as a Mendelian disease gene, showing biallelic loss-of-function variants cause recessive Noonan-like RASopathy via destabilization and failed MAPK suppression.\",\n      \"evidence\": \"Patient variant overexpression with EGF time-course signaling, primary fibroblasts, zebrafish morpholino knockdown with rescue\",\n      \"pmids\": [\"34626534\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genotype–phenotype correlations across the variant spectrum incomplete\", \"Tissue-specific disease mechanisms not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Provided structural definition of a SPRED2 kinase interaction, showing residues 123–201 (F145) bind specifically to the RSK2 N-terminal kinase domain to limit RSK substrate phosphorylation.\",\n      \"evidence\": \"AP-MS, X-ray crystallography, F145A mutagenesis, Co-IP, knockdown with YB1/CREB phosphorylation and localization readouts\",\n      \"pmids\": [\"37149146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity for RSK2 over RSK1/RSK3 mechanism beyond the interface not fully explained\", \"Physiological output of RSK regulation in vivo undefined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined how SPRED2 reaches its site of action, showing neurofibromin forms a complex with SPRED2 and drives its plasma membrane translocation for RAF/ERK inhibition.\",\n      \"evidence\": \"Co-IP, overexpression/knockdown functional assays, IHC of NF1-negative breast cancers\",\n      \"pmids\": [\"41155378\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of the NF1–SPRED2 membrane-targeting complex not defined\", \"Single-lab Co-IP without orthogonal interaction validation\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Identified a transcriptional axis for SPRED2 tumor suppression, showing a SPRED2–p53 complex activates miR-506 to suppress KLF4 and limit HCC stemness.\",\n      \"evidence\": \"Co-IP, ChIP-qPCR, overexpression/knockdown, 3D sphere assays, RT-qPCR/Western blotting\",\n      \"pmids\": [\"41560328\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How a cytoplasmic MAPK regulator engages nuclear p53 not mechanistically explained\", \"Generality beyond HCC not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SPRED2's multiple direct activities (NBR1/lysosomal targeting, LC3-autophagy, DYRK1A/RSK2 inhibition, p53/transcriptional roles) are integrated and prioritized within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of how SPRED2 partitions between membrane Ras inhibition, autophagy scaffolding, and nuclear functions\", \"Upstream kinases setting SPRED2 phospho-state across contexts not fully mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 4, 6, 11]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 8, 9]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 6, 11, 17]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [8, 9, 18]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [14, 21]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"NBR1\", \"DYRK1A\", \"RSK2\", \"CBL\", \"PIK3R1\", \"MAP1LC3\", \"SQSTM1\", \"NF1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}