{"gene":"SRC","run_date":"2026-06-10T07:46:41","timeline":{"discoveries":[{"year":1993,"finding":"The c-Src SH2 domain binds directly to the phosphorylated C-terminal tail (pTyr527) of c-Src in vitro, and this intramolecular interaction maintains c-Src in an inactive conformation. A competing phosphopeptide that binds the SH2 domain activates c-Src kinase activity in vitro, and this activation is blocked by excess purified SH2 domain.","method":"In vitro GST-SH2 domain pulldown with synthetic phosphopeptides, competition kinase assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis-equivalent peptide competition; multiple orthogonal biochemical assays in one study","pmids":["7683128"],"is_preprint":false},{"year":1992,"finding":"C-terminal Src kinase (Csk) suppresses c-Src kinase activity in vivo by phosphorylating Tyr527; overexpression of Csk reverses v-Crk-induced c-Src activation and cellular transformation, and this suppression is abolished when Tyr527 is mutated to Phe.","method":"Cotransfection/overexpression, kinase activity assay, morphological transformation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with point-mutant validation, replicated in multiple cell systems","pmids":["1383688"],"is_preprint":false},{"year":1996,"finding":"c-Src is required for osteoclast spreading downstream of CSF-1 receptor (c-Fms); c-Src is tyrosine-phosphorylated and its kinase activity increases ~3-fold upon CSF-1 stimulation, and src-null osteoclasts fail to spread and show altered downstream substrate phosphorylation (including alpha-actinin).","method":"Immunoprecipitation kinase assay, src-null mouse osteoclasts, confocal microscopy, Western blot","journal":"Molecular reproduction and development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with defined phenotype, multiple orthogonal methods, replicated across papers","pmids":["8981371"],"is_preprint":false},{"year":1996,"finding":"c-Cbl is tyrosine-phosphorylated in a c-Src-dependent manner in osteoclasts; c-Cbl and c-Src colocalize on vesicular structures; antisense knockdown of either c-src or c-cbl inhibits in vitro bone resorption, placing c-Cbl downstream of c-Src in a signaling pathway required for bone resorption.","method":"Antisense oligonucleotides, immunoprecipitation/phosphorylation assay, src-null osteoclasts, confocal colocalization, in vitro bone resorption assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal genetic and biochemical evidence, multiple orthogonal methods, published in high-impact journal","pmids":["8849724"],"is_preprint":false},{"year":1988,"finding":"pp60c-Src is concentrated at least 9-fold in nerve growth cone membrane fractions of developing rat brain and is an active tyrosine kinase in that compartment, with altered electrophoretic mobility characteristic of the neuronal form; it is present at lower levels in mature brain synaptosomal fractions.","method":"Subcellular fractionation, indirect immunofluorescence, in vitro kinase assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct biochemical fractionation combined with kinase assay and imaging; replicated across chick and rat systems","pmids":["2455889"],"is_preprint":false},{"year":1984,"finding":"Overexpression of wild-type c-Src in NIH 3T3 cells elevates tyrosine kinase activity and causes partial morphological transformation but does not induce foci or anchorage-independent growth, demonstrating that the mutations distinguishing v-Src from c-Src are required for full transformation.","method":"NIH 3T3 transfection/focus assay, soft-agar colony assay, in vitro kinase assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional gain-of-function with multiple readouts; foundational study replicated broadly","pmids":["6594680"],"is_preprint":false},{"year":1995,"finding":"c-Src directly phosphorylates the EGF receptor at Tyr845 within the c-Src–EGFR complex in an EGF-dependent manner, as demonstrated by in vitro kinase assay on cyanogen bromide fragments and phosphopeptide mapping co-migration with a synthetic pTyr845 standard.","method":"In vitro kinase assay, cyanogen bromide fragment phosphorylation, phosphopeptide mapping, in-cell EGF stimulation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro kinase reconstitution with peptide mapping, single lab, single paper","pmids":["7488034"],"is_preprint":false},{"year":2000,"finding":"Constitutively active c-Src phosphorylates connexin-43 at Tyr265, causing the phosphorylated C-terminus to bind the c-Src SH2 domain and displacing ZO-1; this reduces total and surface connexin-43 levels and diminishes gap junctional conductance. A Tyr265 mutant connexin-43 retains ZO-1 interaction despite active c-Src.","method":"Cotransfection, in vitro binding assay with recombinant proteins, cell-surface biotinylation, electrophysiology, site-directed mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution with recombinant proteins, mutagenesis at specific residue, multiple orthogonal readouts including electrophysiology","pmids":["11035005"],"is_preprint":false},{"year":2000,"finding":"Deletion/reduction of c-Src expression in mice enhances osteoblast differentiation and bone formation, increases alkaline phosphatase activity, nodule mineralization, and upregulates osteoblast differentiation markers (Osf2/Cbfa1, osteocalcin, collagen) in vitro and in vivo, demonstrating a negative regulatory role of c-Src in osteoblastogenesis.","method":"Src-null mice, bone histomorphometry, antisense oligonucleotide knockdown in primary osteoblasts, RT-PCR, in vitro differentiation assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout plus antisense knockdown with multiple orthogonal functional readouts","pmids":["11038178"],"is_preprint":false},{"year":2003,"finding":"c-Src localizes to mitochondria in osteoclasts and phosphorylates cytochrome c oxidase (Cox), increasing its enzymatic activity. Src deletion reduces Cox activity; re-expression of c-Src restores it. Increasing Src kinase activity reverses calcitonin-mediated inhibition of Cox and osteoclast function.","method":"Mitochondrial fractionation, kinase assay, c-Src knockout/rescue with adenoviral vectors, Cox activity assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — subcellular fractionation with functional enzyme activity assay, genetic rescue, multiple orthogonal readouts","pmids":["12615910"],"is_preprint":false},{"year":2000,"finding":"p130CAS (CAS) binds directly to both the SH2 and SH3 domains of c-Src and activates its tyrosine kinase activity; a single amino acid substitution in the CAS SH3-binding site disrupts both CAS–c-Src interaction and c-Src-dependent phosphorylation of cortactin and paxillin.","method":"Co-immunoprecipitation, overexpression in COS-1 cells, site-directed mutagenesis, tyrosine phosphorylation assay, soft-agar growth assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal binding with point-mutant validation, multiple substrates tested, functional transformation assay","pmids":["10913170"],"is_preprint":false},{"year":2003,"finding":"PLD2 (and to a lesser extent PLD1) are directly tyrosine-phosphorylated by c-Src via direct association; the interaction is mediated by the PLD2 pleckstrin homology domain and the c-Src catalytic domain. PLD catalytic activity (but not c-Src phosphorylation of PLD) in turn stimulates c-Src kinase activity and c-Src-mediated paxillin phosphorylation.","method":"Co-immunoprecipitation, in vitro kinase assay, domain-mapping mutagenesis, EGF stimulation in A431 cells","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct co-IP and in vitro phosphorylation, domain mapping; single lab","pmids":["12697812"],"is_preprint":false},{"year":2004,"finding":"PKCα activates c-Src via the scaffold protein AFAP-110: PKCα activation directs AFAP-110 to bind the c-Src SH3 domain, which activates c-Src kinase activity and promotes podosome formation containing cortactin, AFAP-110, actin, and c-Src. Cells lacking AFAP-110 cannot undergo PKCα-mediated c-Src activation or podosome formation, and rescue requires AFAP-110 capable of binding c-Src.","method":"Immunofluorescence, co-immunoprecipitation, kinase assay, AFAP-110-deficient cell line rescue, site-directed mutagenesis of AFAP-110","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic rescue with structure-function mutants, multiple orthogonal readouts","pmids":["15314167"],"is_preprint":false},{"year":2007,"finding":"c-Src regulates podosome formation, structure, life span, and actin flux in osteoclasts; Src−/− osteoclasts show fewer podosomes, decreased actin cloud, 4-fold increased podosome lifespan, and 40% reduced actin flux rate. Rescue with Src mutants shows both kinase activity AND either the SH2 or SH3 binding domain are required for normal podosome dynamics.","method":"Src-null osteoclasts, videomicroscopy, FRAP, adenoviral rescue with c-Src kinase/domain mutants, fluorescence microscopy","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with quantitative live imaging and FRAP, domain-mutant rescue panel","pmids":["17978100"],"is_preprint":false},{"year":2007,"finding":"c-Src forms an essential signaling complex with αvβ3 integrin and Syk in osteoclasts; upon integrin activation, c-Src phosphorylates Syk, and ITAM proteins Dap12 and FcRγ mediate the Syk–c-Src association and αvβ3-induced Syk phosphorylation required for cytoskeletal organization and bone resorption.","method":"Syk-null mice (in vitro and chimeric in vivo), co-immunoprecipitation, kinase assay, cytoskeletal staining, bone resorption assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout models combined with biochemical complex characterization and functional readouts","pmids":["17353363"],"is_preprint":false},{"year":2012,"finding":"VEGFR2 phosphorylated at Y951 binds the SH2 domain of TSAd, which in turn recruits and activates c-Src (increased pY418, decreased pY527); TSAd silencing blocks VEGF-induced c-Src activation and VE-cadherin junction rearrangement. TSAd, VEGFR2, and c-Src form a complex at endothelial junctions, and Tsad−/− mice show impaired VEGF-induced vascular permeability.","method":"TSAd knockout mice, siRNA silencing, co-immunoprecipitation, phosphorylation assays, Evans blue/dextran permeability assay, in vitro and in vivo experiments","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout in vivo, reciprocal co-IP, multiple orthogonal permeability readouts","pmids":["22689825"],"is_preprint":false},{"year":2012,"finding":"c-Src phosphorylates mitochondrial NADH dehydrogenase subunit NDUFV2 at Tyr193 (required for NADH dehydrogenase/complex I activity and cellular ATP) and SDHA at Tyr215 (no effect on enzyme activity but induces ROS via electron transfer perturbation); phosphorylation-defective mutants reduce cell viability.","method":"Mitochondrial kinase-dead c-Src with targeting sequence, phosphorylation-site mutagenesis, enzymatic activity assays, ROS measurement, cell viability assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vivo phosphorylation with site-specific mutants and enzyme activity readout; mitochondria-targeted kinase-dead construct","pmids":["22823520"],"is_preprint":false},{"year":2012,"finding":"c-Src phosphorylates FOXM1 on two tyrosine residues, stimulating FOXM1 nuclear localization and target gene expression (including G2/M regulators and c-Src itself), forming a positive feedback loop that drives mammary tumor cell proliferation.","method":"Genetically engineered mouse model with c-Src deletion, phosphorylation mapping, nuclear localization assay, target gene expression, patient-derived models","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mouse model deletion, direct phosphorylation site identification, multiple functional readouts including in vivo tumor progression","pmids":["36795481"],"is_preprint":false},{"year":1995,"finding":"c-Src associates with the prolactin receptor in rat hepatocytes and is activated upon prolactin stimulation, as shown by co-immunoprecipitation; prolactin treatment also induces c-src, c-fos, and c-jun gene expression.","method":"Co-immunoprecipitation kinase assay from hepatocyte lysates, Western blot","journal":"Molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single co-IP kinase assay, single lab, limited mechanistic follow-up","pmids":["8584023"],"is_preprint":false},{"year":2000,"finding":"c-Src stimulation by prolactin is independent of Jak2: a Box1-mutant PRLR that cannot activate Jak2 (and thus cannot phosphorylate the receptor) still activates c-Src equivalently to wild-type PRLR upon prolactin treatment.","method":"Expression of Box1-mutant PRLR and kinase-deleted Jak2 in chicken embryo fibroblasts, c-Src kinase activity assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic dissection with receptor and kinase mutants, single lab; epistasis result well controlled","pmids":["10600634"],"is_preprint":false},{"year":2003,"finding":"EphB1 recruits c-Src and p52Shc; activated EphB1 promotes c-Src tyrosine phosphorylation, and c-Src phosphorylates p52Shc, enabling p52Shc recruitment to EphB1 signaling complexes via its PTB domain. EphB1 tyrosines 600 and 778 are required for c-Src and p52Shc interaction. Dominant-negative c-Src reduces ERK1/2 activation and chemotaxis.","method":"Co-immunoprecipitation, site-directed mutagenesis of EphB1, dominant-negative c-Src expression, MEK inhibitor, ERK assay, migration assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal IP, receptor mutagenesis, dominant-negative, multiple functional readouts","pmids":["12925710"],"is_preprint":false},{"year":1997,"finding":"c-Src is required downstream of the PDGF and EGF receptors for mitogenesis; preferred c-Src substrates include cortactin, p190RhoGAP, and p130CAS (actin cytoskeleton/focal adhesion proteins), while EGF receptor substrates include SHC and PLCγ.","method":"C3H10T fibroblast model with wild-type and mutant c-Src, substrate phosphorylation comparison, temporal/spatial signaling analysis","journal":"Frontiers in bioscience","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — substrate identification by comparative phosphorylation, multiple cell-based experiments; review/methods paper","pmids":["9331427"],"is_preprint":false},{"year":1997,"finding":"c-Src activates both STAT1 and STAT3 in PDGF-stimulated NIH3T3 cells; STAT1 co-immunoprecipitates with c-Src, suggesting direct interaction; overexpression of dominant-negative c-Src reduces STAT1/3 tyrosine phosphorylation and DNA binding activity.","method":"Co-immunoprecipitation, overexpression of c-Src and dominant-negative Src, EMSA for DNA binding activity, tyrosine phosphorylation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single co-IP with dominant-negative, single lab; direct kinase assay for STATs not shown","pmids":["9344858"],"is_preprint":false},{"year":2004,"finding":"Pyk2 and c-Src synergistically activate Stat3 downstream of EGFR; EGF stimulation recruits c-Src, Pyk2, and Stat3 to EGFR; dominant-negative Pyk2 impairs c-Src-induced Stat3 activation; Pyk2 expression induces Stat3 phosphorylation at Tyr705 and Ser727.","method":"Co-immunoprecipitation, dominant-negative constructs, luciferase reporter assay, Western blot for phospho-Stat3","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP plus dominant-negative epistasis, single lab, multiple readouts","pmids":["14963038"],"is_preprint":false},{"year":2006,"finding":"Trans-interacting cadherin locally activates c-Src at cell-cell adhesion sites; c-Src then tyrosine-phosphorylates Vav2 (Rac-GEF) and activates Rap1 via C3G/Crk; both c-Src phosphorylation of Vav2 AND Rap1 activation (via PI3K) are jointly required for Rac activation.","method":"Inhibitor studies (PP2), dominant-negative constructs, co-immunoprecipitation, GTPase activity assays, cadherin trans-interaction model in fibroblasts and epithelial cells","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — pathway dissection with multiple inhibitors and dominant-negatives, single lab","pmids":["16170364"],"is_preprint":false},{"year":2005,"finding":"Aldosterone activates vascular c-Src through the mineralocorticoid receptor (eplerenone-sensitive); activated c-Src then mediates p38 MAPK phosphorylation and NADPH oxidase activation; c-Src-deficient (c-Src+/−) VSMCs fail to show aldosterone-induced cortactin or p38 MAPK phosphorylation.","method":"c-Src heterozygous mouse VSMCs, PP2 inhibitor, eplerenone, kinase assays, Western blot, [3H]proline incorporation","journal":"Hypertension","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic haploinsufficiency model combined with pharmacologic inhibition, multiple pathway readouts","pmids":["15699470"],"is_preprint":false},{"year":2008,"finding":"Endosomal NADPH oxidases (Nox1, Nox2) generate ROS that activate c-Src following hypoxia/reoxygenation; Rac1-dependent endocytosis recruits c-Src to endosomes where endosomal ROS activate it; activated c-Src then phosphorylates IκBα on tyrosine to activate NF-κB. Quenching endosomal ROS or Rac1 siRNA blocks c-Src activation.","method":"siRNA knockdown (Rac1), Nox-deficient primary fibroblasts, endosomal fractionation, intra-endosomal ROS quenching, phosphorylation assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockdown plus Nox-null cells, subcellular fractionation, multiple orthogonal methods","pmids":["18397177"],"is_preprint":false},{"year":2009,"finding":"c-Src associates with ErbB2 specifically through an interaction involving the ErbB2 kinase domain region surrounding Tyr877 (EGFR(YHAD) motif); this association does not require c-Src SH2 or SH3 domains or receptor phosphorylation, and confers enhanced transforming potential. EGFR mutants found in lung cancer that gain the Y877 equivalent motif also bind c-Src.","method":"Chimeric EGFR/ErbB2 receptors, co-immunoprecipitation, site-directed mutagenesis, in vitro and in vivo transformation assays, Stat3 activation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain-mapping with chimeric receptors and mutagenesis, reciprocal co-IP, functional transformation assay in vitro and in vivo","pmids":["19704002"],"is_preprint":false},{"year":2006,"finding":"c-Src overexpression enhances ErbB2/ErbB3 heterocomplex formation and their basal and heregulin-induced activation; kinase-inactive c-Src or PP2 treatment reduces heterocomplex formation and downstream signaling, indicating c-Src acts upstream to positively modulate ErbB2/ErbB3 association.","method":"Co-immunoprecipitation, wild-type vs. kinase-inactive c-Src overexpression, PP2 pharmacological inhibition, receptor activation Western blot, migration and anchorage-independent growth assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP plus kinase-dead mutant rescue, single lab, mechanistic placement upstream of receptor complex","pmids":["17173075"],"is_preprint":false},{"year":2017,"finding":"c-Src directly phosphorylates hexokinase 1 (HK1) at Tyr732 and HK2, dramatically increasing their catalytic activity (decreased Km, increased Vmax for HK1) by disrupting HK1 dimer formation. HK1-Y732F or HK2 phosphosite mutants abrogate c-Src-stimulated glycolysis, cell proliferation, tumorigenesis, and metastasis.","method":"In vitro kinase assay, Km/Vmax measurements, site-directed mutagenesis (Y732F knockin and knockin mice), xenograft and metastasis models, clinical sample correlation","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with kinetics, mutagenesis at specific residue, in vivo knockin mouse model","pmids":["28054552"],"is_preprint":false},{"year":2020,"finding":"c-Src phosphorylates PFKFB3 at Tyr194, activating this key glycolytic enzyme to boost fructose-2,6-bisphosphate production and PFK1 activity, replenishing PPP and serine pathways. PFKFB3-Y194F knockin mice show impaired glycolysis and reduced spontaneous colon cancer formation when crossed with APCmin/+ mice.","method":"In vitro kinase assay, site-directed mutagenesis (Y194F), PFKFB3 knockout cells, PFKFB3-Y194F knockin mice, metabolic flux assay, APCmin/+ cross, clinical sample correlation","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, knockin mouse model, multiple orthogonal metabolic and tumor readouts","pmids":["32209481"],"is_preprint":false},{"year":2021,"finding":"c-Src interacts with and phosphorylates G6PD at Tyr112, enhancing its catalytic activity (decreased Km, increased Kcat) for glucose-6-phosphate, thereby augmenting PPP flux for NADPH and ribose-5-phosphate production and promoting tumorigenesis.","method":"Co-immunoprecipitation, in vitro kinase assay, enzyme kinetics (Km/Kcat), site-directed mutagenesis (Y112), clinical colorectal cancer sample correlation","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with enzyme kinetics and site-specific mutagenesis, clinical validation","pmids":["33686238"],"is_preprint":false},{"year":2019,"finding":"c-Src phosphorylates E-cadherin at Tyr797, triggering RNF43-mediated ubiquitination of E-cadherin at Lys816 and subsequent proteasomal degradation, enabling nuclear β-catenin translocation and EMT in lung adenocarcinoma.","method":"Immunoprecipitation, ubiquitination assay, phospho-specific antibody, shRNA knockdown, xenograft model, site-directed mutagenesis","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — co-IP/ubiquitination with site-specific mutants, single lab","pmids":["31286874"],"is_preprint":false},{"year":2019,"finding":"Cdh1 (APC/C co-activator) suppresses c-Src kinase activity in an APC-independent manner; reciprocally, hyperactive c-Src phosphorylates Cdh1 at its N-terminus, disrupting Cdh1 interaction with the APC core complex and inhibiting APCCdh1 E3 ligase activity, forming a reciprocal feedback circuit.","method":"Co-immunoprecipitation, kinase assay, site-directed mutagenesis, ubiquitin E3 ligase assay, mouse mammary tumor model (PTEN loss), pharmacological c-Src inhibition","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, direct kinase phosphorylation of Cdh1, E3 ligase activity assay, in vivo mouse model","pmids":["31420536"],"is_preprint":false},{"year":2019,"finding":"RACK1 interacts with c-Src via RACK1 tyrosines 228 and 246 (binding the c-Src SH2 domain at Lys152); RACK1-Y228F/Y246F mutant fails to interact with c-Src and impairs osteoclast cytoskeletal integrity and bone resorption without affecting differentiation. c-Src K152R similarly impairs osteoclast resorption.","method":"Co-immunoprecipitation, site-directed mutagenesis of RACK1 and c-Src, osteoclast differentiation/resorption assays, cytoskeletal analysis","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — reciprocal mutagenesis of both binding partners with functional bone resorption readout, single lab","pmids":["31358728"],"is_preprint":false},{"year":2019,"finding":"c-Src autophosphorylation at Tyr416 causes global structural rearrangements: the kinase domain gains rigidity and stabilizes the ATP-binding site, while the regulatory SH2/SH3 domains become more flexible and detach from the kinase domain, resulting in a 4-fold increase in enzymatic activity.","method":"Hydrogen/deuterium exchange MS, biochemical kinase activity assay, molecular dynamics simulations","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — HDX-MS structural method plus kinase activity assay and simulations, multiple orthogonal approaches","pmids":["31331936"],"is_preprint":false},{"year":2013,"finding":"Molecular dynamics free-energy calculations demonstrate that phosphorylation of Tyr416 in the activation loop locks c-Src into a catalytically competent conformation by stabilizing the hydrophobic regulatory spine, HRD motif, and electrostatic switch; unphosphorylated A-loop shows high flexibility and the active conformation is only transiently visited.","method":"Molecular dynamics umbrella sampling free-energy simulations","journal":"Journal of molecular biology","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational only, no experimental validation in this paper","pmids":["24103328"],"is_preprint":false},{"year":2002,"finding":"Dominant-negative c-Src (K295M) prevents acid-induced activation of NHE3 in renal epithelial cells, placing c-Src upstream of NHE3 in the response to chronic acidosis; acid-induced ERK activation is independent of c-Src, demonstrating two parallel pathways both required for NHE3 activation.","method":"Dominant-negative c-Src transfection, NHE3 activity assay (pHi recovery), immune-complex kinase assay, MEK inhibitor (PD98059)","journal":"Kidney international","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — genetic epistasis with dominant-negative and pharmacologic tools, single lab","pmids":["12081562"],"is_preprint":false},{"year":2006,"finding":"c-Src controls functional co-localization of the proton pump and CLIC-5b chloride channel in osteoclast vesicles, which is required for vesicular acidification and bone resorption. CLIC-5b binds c-Src SH2 and SH3 domains. c-Src suppression reduces vesicular acidification (rescued by valinomycin), consistent with selective loss of chloride conductance.","method":"c-Src siRNA knockdown, CLIC-5b siRNA knockdown, vesicular acidification assay, valinomycin rescue, affinity pull-down with Src SH2/SH3 domains, bone resorption assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain pulldown plus siRNA knockdown, functional acidification rescue, multiple readouts","pmids":["16831863"],"is_preprint":false},{"year":2016,"finding":"Connexin43 recruits PTEN and Csk to its C-terminal region (residues 266–283) to inhibit c-Src; pull-down assays show this region is sufficient to recruit c-Src, PTEN, and Csk and inhibit oncogenic c-Src activity. Silencing Csk or PTEN reduces the antiproliferative effect of Cx43 in glioma cells.","method":"Pull-down assays with Cx43 peptide fragments, co-immunoprecipitation, confocal microscopy, siRNA silencing of Csk and PTEN, phosphorylation assays (pY527, pY416)","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — pull-down domain mapping combined with siRNA epistasis, single lab","pmids":["27391443"],"is_preprint":false},{"year":2009,"finding":"Apoptotic cell binding to MerTK on dendritic cells establishes a complex containing MerTK, c-Src, STAT3, and PI3K; this activates c-Src and STAT3 and mediates inhibition of DC maturation. Pharmacological inhibitors or siRNA against c-Src or STAT3 block apoptotic cell-induced DC inhibition.","method":"Co-immunoprecipitation, phosphorylation assay, siRNA knockdown, pharmacological inhibitors, MerTK-knockout DCs, DC maturation assay","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, genetic (MerTK KO) and pharmacologic epistasis, multiple readouts","pmids":["19667404"],"is_preprint":false},{"year":2002,"finding":"c-Src co-immunoprecipitates with c-Cbl and both localize to Golgi-enriched membrane fractions in CHO cells; activated (but not wild-type) c-Src increases the amount of c-Cbl co-immunoprecipitating with Src and the intensity of c-Cbl Golgi staining, with concomitant increased tyrosine phosphorylation of membrane-associated Cbl.","method":"Co-immunoprecipitation, subcellular fractionation (density centrifugation and free-flow electrophoresis), confocal immunofluorescence, activated c-Src transfection","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — co-IP and fractionation with activated Src mutant, single lab, localization without full functional consequence established","pmids":["11893076"],"is_preprint":false},{"year":2019,"finding":"c-Src phosphorylates HDAC3 at Tyr328 and Tyr331; phosphorylated HDAC3 shows higher deacetylase activity, is recruited to the plasma membrane upon EGF stimulation, and promotes breast cancer cell invasion. PP2 (c-Src inhibitor) blocks HDAC3 phosphorylation and reduces enzymatic activity.","method":"Site-directed mutagenesis (Y328/331A), phospho-specific antibody, in vitro kinase assay, TIRF microscopy, invasion assay, c-Src knockdown","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro phosphorylation with site-specific mutant and functional enzyme activity readout, single lab","pmids":["31430896"],"is_preprint":false},{"year":2021,"finding":"Inhibitor binding to c-Src induces a conformational change that promotes c-Src association with FAK in an active form; upon inhibitor dissociation, c-Src phosphorylates FAK and initiates FAK-Grb2-Erk signaling. A drug-resistant c-Src mutation that reduces inhibitor affinity paradoxically converts Src inhibitors into facilitators of FAK/Erk phosphorylation and cell proliferation.","method":"Co-immunoprecipitation with c-Src inhibitors, phosphorylation assay (FAK, Erk), drug-resistant c-Src mutant cells, cell proliferation assay","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — conformational mechanism inferred from inhibitor-dependent co-IP and phosphorylation, single lab","pmids":["33761359"],"is_preprint":false},{"year":2008,"finding":"c-Src is the specific kinase required for villin-mediated intestinal cell migration; reconstitution of SYF (Src/Yes/Fyn triple-knockout) cells individually with c-Src, c-Yes, or c-Fyn demonstrates an absolute requirement for c-Src specifically. SHP-2 and PTP-PEST are identified as negative regulators of c-Src activity in this context.","method":"SYF cells reconstituted with individual kinases, cell migration assay, villin phosphorylation assay, siRNA for phosphatases","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean genetic epistasis using triple-knockout cells reconstituted with individual kinases, specific functional readout","pmids":["18482983"],"is_preprint":false},{"year":2014,"finding":"c-Src drives intestinal stem cell (ISC) proliferation, regeneration, and tumorigenesis through upregulation of EGFR and activation of Ras/MAPK and Stat3 signaling, as shown by genetic gain- and loss-of-function in both Drosophila and mouse intestinal epithelium; c-Src plays a non-redundant role that cannot be substituted by Fyn or Yes.","method":"Genetic gain- and loss-of-function (Drosophila and conditional mouse knockout), ISC proliferation assay, EGFR/MAPK/Stat3 pathway activation readouts","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — orthogonal genetic experiments in two organisms, specificity demonstrated by Fyn/Yes non-rescue, mechanistic pathway placement","pmids":["24788409"],"is_preprint":false},{"year":2012,"finding":"c-Src stimulates IL-6 expression through STAT3; IL-6 in turn induces IGFBP5, which activates c-Src in immature (but not mature) osteoblasts, creating an amplifying loop that maintains osteoblasts in an immature state; IGFBP5 produced by osteoblasts also stimulates osteoclastogenesis.","method":"c-Src inhibition, siRNA knockdown, STAT3 reporter, cytokine measurements, in vitro and in vivo osteoblast/osteoclast assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — pathway dissection with multiple tools but largely pharmacologic, single lab","pmids":["22252554"],"is_preprint":false},{"year":2016,"finding":"The PDZ protein MPP2 interacts with c-Src via its PDZ domain in epithelial cells (identified by PDZ domain array screen and confirmed by co-immunoprecipitation); MPP2 negatively regulates c-Src kinase activity in cells and suppresses c-Src-dependent disorganization of the cortical actin cytoskeleton.","method":"PDZ domain array screen, co-immunoprecipitation, kinase activity assay, cytoskeletal imaging","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — co-IP plus kinase activity and phenotypic rescue, single lab","pmids":["19665017"],"is_preprint":false}],"current_model":"c-Src is a non-receptor tyrosine kinase regulated by an intramolecular inhibitory clamp in which the SH2 domain binds pTyr527 in the C-terminal tail (phosphorylated by Csk) and the SH3 domain engages the linker; release of this clamp, followed by autophosphorylation of Tyr416 in the activation loop (which locks the active conformation by rigidifying the kinase domain and detaching the regulatory domains), fully activates the enzyme. Active c-Src localizes to the plasma membrane, focal adhesions, endosomes, mitochondria, and Golgi, where it phosphorylates a broad range of substrates including EGFR (Y845), ErbB2 (Y877 region), connexin-43 (Y265), hexokinases HK1/HK2 (Y732), PFKFB3 (Y194), G6PD (Y112), FOXM1, HDAC3 (Y328/Y331), cytochrome c oxidase subunits, and mitochondrial OXPHOS complexes I/II, thereby regulating glycolysis, oxidative phosphorylation, actin dynamics (podosomes, invadopodia, cell migration), gap junction function, cell cycle progression (via FOXM1 and APCCdh1), and bone homeostasis through coordinated signaling with partners including c-Cbl, Syk/αvβ3/ITAM proteins, FAK, p130CAS, RACK1, TSAd/VEGFR2, MerTK/STAT3, and AFAP-110/PKCα."},"narrative":{"mechanistic_narrative":"c-Src is a non-receptor tyrosine kinase that transduces signals from membrane receptors and adhesion structures to control cell migration, proliferation, metabolism, and bone remodeling [PMID:8981371, PMID:9331427, PMID:28054552]. Its catalytic output is held in check by an intramolecular clamp: the SH2 domain binds the Csk-phosphorylated C-terminal tail at pTyr527, and a competing phosphopeptide that occupies the SH2 domain releases and activates the enzyme [PMID:7683128, PMID:1383688]. Activation is consolidated by autophosphorylation of Tyr416 in the activation loop, which rigidifies the kinase domain and detaches the regulatory SH2/SH3 modules to lock in the active conformation and raise activity ~4-fold [PMID:31331936]. The freed SH2/SH3 surfaces serve as docking sites that integrate diverse inputs and are themselves required for productive signaling: p130CAS engages both domains to stimulate kinase activity, PKCα drives AFAP-110 onto the SH3 domain to trigger podosome formation, VEGFR2-bound TSAd recruits Src through its SH2 domain, and RACK1 engages the SH2 domain at Lys152 [PMID:10913170, PMID:15314167, PMID:22689825, PMID:31358728]. Through these complexes c-Src governs the actin cytoskeleton — controlling podosome dynamics, cortactin/paxillin/p130CAS phosphorylation, and migration downstream of integrins, cadherins, and growth factor receptors [PMID:10913170, PMID:17978100, PMID:9331427, PMID:18482983]. In the osteoclast it is essential for cell spreading, podosome turnover, vesicular acidification, and bone resorption, signaling through c-Cbl, the αvβ3/Syk/ITAM module, RACK1, and CLIC-5b, while paradoxically restraining osteoblast differentiation [PMID:8981371, PMID:8849724, PMID:11038178, PMID:17978100, PMID:17353363, PMID:31358728, PMID:16831863]. c-Src also phosphorylates and activates an array of receptor and effector substrates — EGFR at Tyr845, the ErbB2 region around Tyr877, connexin-43 at Tyr265 (displacing ZO-1 and closing gap junctions), and FOXM1 in a proliferative feedback loop — and it reciprocally inhibits APC/C-Cdh1 by phosphorylating Cdh1 [PMID:7488034, PMID:11035005, PMID:36795481, PMID:19704002, PMID:31420536]. A prominent metabolic function emerges from its direct phosphorylation of glycolytic and pentose-phosphate enzymes (HK1/HK2 at Tyr732, PFKFB3 at Tyr194, G6PD at Tyr112) and mitochondrial complexes (cytochrome c oxidase, NDUFV2 at Tyr193, SDHA at Tyr215), coupling Src activity to glycolysis, oxidative phosphorylation, and tumor growth [PMID:12615910, PMID:22823520, PMID:28054552, PMID:32209481, PMID:33686238]. The mutations distinguishing v-Src from c-Src are required for full transformation, underscoring the tight physiological control of this oncogene [PMID:6594680].","teleology":[{"year":1984,"claim":"Established that wild-type c-Src is not by itself fully transforming, defining the difference between proto-oncogene and viral oncogene and motivating the search for c-Src's intrinsic regulation.","evidence":"NIH 3T3 transfection with focus and soft-agar assays comparing c-Src overexpression to v-Src","pmids":["6594680"],"confidence":"High","gaps":["Did not define the regulatory mechanism keeping c-Src inactive","Did not identify the specific activating lesions in v-Src"]},{"year":1988,"claim":"Showed that c-Src is enriched and catalytically active in a defined subcellular compartment (neuronal growth cone membranes), linking the kinase to membrane-localized developmental signaling.","evidence":"Subcellular fractionation, immunofluorescence, and in vitro kinase assay in developing rat/chick brain","pmids":["2455889"],"confidence":"High","gaps":["No substrates in the growth cone identified","Functional consequence for neuronal development not tested"]},{"year":1992,"claim":"Identified Csk-mediated phosphorylation of Tyr527 as the in vivo brake on c-Src, resolving how the kinase is held inactive in cells.","evidence":"Cotransfection/overexpression with Y527F point mutant, kinase and transformation assays","pmids":["1383688"],"confidence":"High","gaps":["Did not show the structural mechanism by which pTyr527 inhibits","Physiological signals controlling Csk activity not addressed"]},{"year":1993,"claim":"Defined the molecular basis of autoinhibition: the SH2 domain binds pTyr527 intramolecularly, and competition for the SH2 domain releases activity.","evidence":"In vitro GST-SH2 pulldowns with synthetic phosphopeptides and competition kinase assays","pmids":["7683128"],"confidence":"High","gaps":["SH3-linker contribution not resolved in this study","Did not address activation-loop autophosphorylation"]},{"year":1996,"claim":"Placed c-Src in osteoclast biology, showing it is activated downstream of CSF-1 and required for cell spreading, and identified c-Cbl as a downstream effector for bone resorption.","evidence":"src-null osteoclasts, IP kinase assays, antisense knockdown of src/cbl, confocal colocalization, in vitro resorption assay","pmids":["8981371","8849724"],"confidence":"High","gaps":["Direct phosphorylation of c-Cbl by c-Src not formally shown","Substrate(s) mediating spreading defect incompletely defined"]},{"year":1995,"claim":"Demonstrated that c-Src directly phosphorylates receptor tyrosine kinase substrates, identifying EGFR Tyr845 within the Src-EGFR complex.","evidence":"In vitro kinase assay on CNBr fragments and phosphopeptide mapping with EGF stimulation","pmids":["7488034"],"confidence":"Medium","gaps":["Single-lab in vitro result","Cellular consequence of Y845 phosphorylation not established here"]},{"year":2000,"claim":"Showed c-Src closes gap junctions by phosphorylating connexin-43 Tyr265, which then binds the Src SH2 domain and displaces ZO-1, and that scaffold partners (p130CAS) bidirectionally regulate Src activity.","evidence":"Reconstitution with recombinant proteins, electrophysiology, biotinylation, Y265 mutagenesis; reciprocal co-IP and point mutants for CAS","pmids":["11035005","10913170"],"confidence":"High","gaps":["Whether these mechanisms operate in the same physiological context not addressed","Connexin-43 turnover pathway not fully mapped"]},{"year":2000,"claim":"Revealed a negative role for c-Src in osteoblast differentiation, establishing that Src oppositely regulates the two bone cell lineages.","evidence":"src-null mice, bone histomorphometry, antisense knockdown, RT-PCR of differentiation markers","pmids":["11038178"],"confidence":"High","gaps":["Direct Src substrates restraining osteoblast genes not identified","Cell-autonomy versus osteoclast crosstalk not fully separated"]},{"year":2003,"claim":"Established a mitochondrial localization and function for c-Src, showing it phosphorylates and activates cytochrome c oxidase to support osteoclast bioenergetics.","evidence":"Mitochondrial fractionation, kinase assay, Src knockout/rescue, Cox activity assay","pmids":["12615910"],"confidence":"High","gaps":["Precise Cox phosphosite not defined in this study","How Src is imported/retained at mitochondria unclear"]},{"year":2007,"claim":"Defined how c-Src organizes the osteoclast cytoskeleton, showing both kinase activity and an intact SH2 or SH3 domain are needed for podosome dynamics, and that Src nucleates an αvβ3/Syk/ITAM complex for resorption.","evidence":"src-null osteoclasts with FRAP/videomicroscopy and domain-mutant rescue; Syk-null mice with co-IP and resorption assays","pmids":["17978100","17353363"],"confidence":"High","gaps":["Direct Src phosphosites on cytoskeletal components in osteoclasts not enumerated","Quantitative coupling of complex assembly to actin flux incomplete"]},{"year":2012,"claim":"Connected c-Src to receptor-proximal scaffolds and a proliferative transcription program: TSAd links VEGFR2 to Src for vascular permeability, and Src phosphorylates FOXM1 to drive a feed-forward proliferation loop.","evidence":"TSAd knockout mice with reciprocal co-IP and permeability assays; Src-deletion mouse model with FOXM1 phosphosite mapping and target gene readouts","pmids":["22689825","36795481"],"confidence":"High","gaps":["FOXM1 tyrosine sites only partially characterized","Generality of TSAd-Src axis beyond endothelium not addressed"]},{"year":2012,"claim":"Extended Src's mitochondrial reach to electron transport chain subunits, distinguishing activating (NDUFV2 Y193) from ROS-inducing (SDHA Y215) phosphorylation events tied to cell viability.","evidence":"Mitochondria-targeted kinase-dead Src construct, phosphosite mutants, enzyme activity and ROS assays","pmids":["22823520"],"confidence":"High","gaps":["Endogenous mitochondrial Src activity regulation unclear","Physiological versus pathological balance of complex I/II phosphorylation not resolved"]},{"year":2019,"claim":"Resolved the structural logic of activation, showing Tyr416 autophosphorylation rigidifies the kinase domain and frees the regulatory domains to lock the active state.","evidence":"Hydrogen/deuterium exchange MS with kinase assays and molecular dynamics","pmids":["31331936"],"confidence":"High","gaps":["Kinetics of the regulatory-domain release in cells not measured","Interplay with pTyr527 dephosphorylation not directly tested here"]},{"year":2019,"claim":"Showed c-Src feeds back on cell-cycle machinery and chromatin/EMT regulators, phosphorylating Cdh1 to inhibit APC/C-Cdh1, HDAC3 to enhance deacetylase activity and invasion, and E-cadherin to trigger its degradation and β-catenin nuclear translocation.","evidence":"Reciprocal co-IP and kinase/E3 ligase assays (Cdh1); in vitro kinase with phosphosite mutants and invasion assay (HDAC3); ubiquitination assay with phosphosite mutants (E-cadherin)","pmids":["31420536","31430896","31286874"],"confidence":"Medium","gaps":["HDAC3 and E-cadherin findings rest on single-lab data","In vivo relevance of the Src-Cdh1 circuit in normal cycling cells not established"]},{"year":2021,"claim":"Consolidated c-Src as a master kinase of cancer metabolism by direct phosphorylation of glycolytic and pentose-phosphate enzymes (HK1/HK2 Y732, PFKFB3 Y194, G6PD Y112) that boost flux and tumorigenesis, validated in knockin mouse models.","evidence":"In vitro kinase assays with enzyme kinetics, phosphosite knockin mice, metabolic flux and tumor models, clinical correlation","pmids":["28054552","32209481","33686238"],"confidence":"High","gaps":["Upstream signals directing Src to metabolic enzymes not fully defined","Subcellular site of these phosphorylation events not pinpointed"]},{"year":null,"claim":"How the multiple c-Src-regulating scaffolds and inhibitory partners (RACK1, MPP2, MerTK/STAT3, connexin-43/PTEN/Csk) are integrated to set Src activity in a given cell, and how its membrane/mitochondrial/nuclear substrate pools are spatially partitioned, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model linking compartment-specific Src pools to distinct substrate sets","Quantitative hierarchy among competing positive and negative regulators not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,7,16,29,30,31,42]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[6,17,29,33]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[10,12,33]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,4,15,42]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[9,16]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[41]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[26]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[12,13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,15,20,35]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[9,16,29,30,31]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[17,33]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,8,13,45]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[7,24]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[5,17,29,32]}],"complexes":["αvβ3 integrin–Syk–c-Src complex","VEGFR2–TSAd–c-Src complex","MerTK–c-Src–STAT3–PI3K complex"],"partners":["CSK","P130CAS","AFAP-110","RACK1","C-CBL","SYK","TSAD","FAK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P12931","full_name":"Proto-oncogene tyrosine-protein kinase Src","aliases":["Proto-oncogene c-Src","pp60c-src","p60-Src"],"length_aa":536,"mass_kda":59.8,"function":"Non-receptor protein tyrosine kinase which is activated following engagement of many different classes of cellular receptors including immune response receptors, integrins and other adhesion receptors, receptor protein tyrosine kinases, G protein-coupled receptors as well as cytokine receptors (PubMed:34234773). Participates in signaling pathways that control a diverse spectrum of biological activities including gene transcription, immune response, cell adhesion, cell cycle progression, apoptosis, migration, and transformation. Due to functional redundancy between members of the SRC kinase family, identification of the specific role of each SRC kinase is very difficult. SRC appears to be one of the primary kinases activated following engagement of receptors and plays a role in the activation of other protein tyrosine kinase (PTK) families. Receptor clustering or dimerization leads to recruitment of SRC to the receptor complexes where it phosphorylates the tyrosine residues within the receptor cytoplasmic domains. Plays an important role in the regulation of cytoskeletal organization through phosphorylation of specific substrates such as AFAP1. Phosphorylation of AFAP1 allows the SRC SH2 domain to bind AFAP1 and to localize to actin filaments. Cytoskeletal reorganization is also controlled through the phosphorylation of cortactin (CTTN) (Probable). When cells adhere via focal adhesions to the extracellular matrix, signals are transmitted by integrins into the cell resulting in tyrosine phosphorylation of a number of focal adhesion proteins, including PTK2/FAK1 and paxillin (PXN) (PubMed:21411625). In addition to phosphorylating focal adhesion proteins, SRC is also active at the sites of cell-cell contact adherens junctions and phosphorylates substrates such as beta-catenin (CTNNB1), delta-catenin (CTNND1), and plakoglobin (JUP). Another type of cell-cell junction, the gap junction, is also a target for SRC, which phosphorylates connexin-43 (GJA1). SRC is implicated in regulation of pre-mRNA-processing and phosphorylates RNA-binding proteins such as KHDRBS1 (Probable). Phosphorylates PKP3 at 'Tyr-195' in response to reactive oxygen species, which may cause the release of PKP3 from desmosome cell junctions into the cytoplasm (PubMed:25501895). Also plays a role in PDGF-mediated tyrosine phosphorylation of both STAT1 and STAT3, leading to increased DNA binding activity of these transcription factors (By similarity). Involved in the RAS pathway through phosphorylation of RASA1 and RASGRF1 (PubMed:11389730). Plays a role in EGF-mediated calcium-activated chloride channel activation (PubMed:18586953). Required for epidermal growth factor receptor (EGFR) internalization through phosphorylation of clathrin heavy chain (CLTC and CLTCL1) at 'Tyr-1477'. Involved in beta-arrestin (ARRB1 and ARRB2) desensitization through phosphorylation and activation of GRK2, leading to beta-arrestin phosphorylation and internalization. Has a critical role in the stimulation of the CDK20/MAPK3 mitogen-activated protein kinase cascade by epidermal growth factor (Probable). Might be involved not only in mediating the transduction of mitogenic signals at the level of the plasma membrane but also in controlling progression through the cell cycle via interaction with regulatory proteins in the nucleus (PubMed:7853507). Plays an important role in osteoclastic bone resorption in conjunction with PTK2B/PYK2. Both the formation of a SRC-PTK2B/PYK2 complex and SRC kinase activity are necessary for this function. Recruited to activated integrins by PTK2B/PYK2, thereby phosphorylating CBL, which in turn induces the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function (PubMed:14585963, PubMed:8755529). Upon activation of the G(q)-dependent KISS1/KISS1R signaling pathway, active SRC is recruited, together with the phosphatase DUSP18, to the KISS1R C-terminus (PubMed:38346942). This leads to DUSP18-mediated SRC dephosphorylation and inactivation, down-regulation of osteoclast differentiation and activity, and consequently suppression of bone resorption (By similarity). Promotes energy production in osteoclasts by activating mitochondrial cytochrome C oxidase (PubMed:12615910). Phosphorylates DDR2 on tyrosine residues, thereby promoting its subsequent autophosphorylation (PubMed:16186108). Phosphorylates RUNX3 and COX2 on tyrosine residues, TNK2 on 'Tyr-284' and CBL on 'Tyr-731' (PubMed:20100835, PubMed:21309750). Enhances RIGI-elicited antiviral signaling (PubMed:19419966). Phosphorylates PDPK1 at 'Tyr-9', 'Tyr-373' and 'Tyr-376' (PubMed:14585963). Phosphorylates BCAR1 at 'Tyr-128' (PubMed:22710723). Phosphorylates CBLC at multiple tyrosine residues, phosphorylation at 'Tyr-341' activates CBLC E3 activity (PubMed:20525694). Phosphorylates synaptic vesicle protein synaptophysin (SYP) (By similarity). Involved in anchorage-independent cell growth (PubMed:19307596). Required for podosome formation (By similarity). Mediates IL6 signaling by activating YAP1-NOTCH pathway to induce inflammation-induced epithelial regeneration (PubMed:25731159). Phosphorylates OTUB1, promoting deubiquitination of RPTOR (PubMed:35927303). Phosphorylates caspase CASP8 at 'Tyr-380' which negatively regulates CASP8 processing and activation, down-regulating CASP8 proapoptotic function (PubMed:16619028). Mediates laminin-induced activation of RAC1 signaling through phosphorylation of syntrophin (By similarity) Non-receptor protein tyrosine kinase which phosphorylates synaptophysin with high affinity Non-receptor protein tyrosine kinase which shows higher basal kinase activity than isoform 1, possibly due to weakened intramolecular interactions which enhance autophosphorylation of Tyr-419 and subsequent activation (By similarity). The SH3 domain shows reduced affinity with the linker sequence between the SH2 and kinase domains which may account for the increased basal activity (By similarity). Displays altered substrate specificity compared to isoform 1, showing weak affinity for synaptophysin and for peptide substrates containing class I or class II SH3 domain-binding motifs (By similarity). Plays a role in L1CAM-mediated neurite elongation, possibly by acting downstream of L1CAM to drive cytoskeletal rearrangements involved in neurite outgrowth (By similarity) Non-receptor protein tyrosine kinase which shows higher basal kinase activity than isoform 1, possibly due to weakened intramolecular interactions which enhance autophosphorylation of Tyr-419 and subsequent activation (By similarity). The SH3 domain shows reduced affinity with the linker sequence between the SH2 and kinase domains which may account for the increased basal activity (By similarity). Displays altered substrate specificity compared to isoform 1, showing weak affinity for synaptophysin and for peptide substrates containing class I or class II SH3 domain-binding motifs (By similarity). Plays a role in neurite elongation (By similarity)","subcellular_location":"Cell membrane; Mitochondrion inner membrane; Nucleus; Cytoplasm, cytoskeleton; Cytoplasm, perinuclear region; Cell junction, focal adhesion; Cell junction","url":"https://www.uniprot.org/uniprotkb/P12931/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SRC","classification":"Not Classified","n_dependent_lines":55,"n_total_lines":1208,"dependency_fraction":0.04552980132450331},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SRC","total_profiled":1310},"omim":[{"mim_id":"621391","title":"XK-RELATED PROTEIN 5; XKR5","url":"https://www.omim.org/entry/621391"},{"mim_id":"620955","title":"SORTING NEXIN 30; SNX30","url":"https://www.omim.org/entry/620955"},{"mim_id":"620678","title":"RAS AND RAB INTERACTOR-LIKE PROTEIN; RINL","url":"https://www.omim.org/entry/620678"},{"mim_id":"620664","title":"RHO GUANINE NUCLEOTIDE EXCHANGE FACTOR 37; ARHGEF37","url":"https://www.omim.org/entry/620664"},{"mim_id":"620484","title":"THROMBOCYTOPENIA 10; THC10","url":"https://www.omim.org/entry/620484"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Supported"},{"location":"Cell Junctions","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SRC"},"hgnc":{"alias_symbol":["ASV","c-src"],"prev_symbol":["SRC1"]},"alphafold":{"accession":"P12931","domains":[{"cath_id":"2.30.30.40","chopping":"87-147","consensus_level":"high","plddt":93.6366,"start":87,"end":147},{"cath_id":"3.30.505.10","chopping":"157-246","consensus_level":"high","plddt":93.6493,"start":157,"end":246},{"cath_id":"3.30.200.20","chopping":"261-343","consensus_level":"medium","plddt":93.5966,"start":261,"end":343},{"cath_id":"1.10.510.10","chopping":"348-524","consensus_level":"medium","plddt":92.0202,"start":348,"end":524}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P12931","model_url":"https://alphafold.ebi.ac.uk/files/AF-P12931-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P12931-F1-predicted_aligned_error_v6.png","plddt_mean":83.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SRC","jax_strain_url":"https://www.jax.org/strain/search?query=SRC"},"sequence":{"accession":"P12931","fasta_url":"https://rest.uniprot.org/uniprotkb/P12931.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P12931/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P12931"}},"corpus_meta":[{"pmid":"15380511","id":"PMC_15380511","title":"c-Src and cooperating partners in human cancer.","date":"2004","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/15380511","citation_count":506,"is_preprint":false},{"pmid":"8849724","id":"PMC_8849724","title":"c-Cbl is downstream of c-Src in a signalling pathway necessary for bone resorption.","date":"1996","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/8849724","citation_count":247,"is_preprint":false},{"pmid":"11038178","id":"PMC_11038178","title":"Decreased c-Src expression enhances osteoblast differentiation and bone formation.","date":"2000","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11038178","citation_count":236,"is_preprint":false},{"pmid":"17353363","id":"PMC_17353363","title":"Syk, c-Src, the alphavbeta3 integrin, and ITAM immunoreceptors, in concert, regulate osteoclastic bone resorption.","date":"2007","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/17353363","citation_count":234,"is_preprint":false},{"pmid":"2455889","id":"PMC_2455889","title":"c-src gene product in developing rat brain is enriched in nerve growth cone membranes.","date":"1988","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/2455889","citation_count":219,"is_preprint":false},{"pmid":"15699470","id":"PMC_15699470","title":"Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src.","date":"2005","source":"Hypertension (Dallas, Tex. : 1979)","url":"https://pubmed.ncbi.nlm.nih.gov/15699470","citation_count":211,"is_preprint":false},{"pmid":"16260764","id":"PMC_16260764","title":"High yield bacterial expression of active c-Abl and c-Src tyrosine kinases.","date":"2005","source":"Protein science : a publication of the Protein 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A competing phosphopeptide that binds the SH2 domain activates c-Src kinase activity in vitro, and this activation is blocked by excess purified SH2 domain.\",\n      \"method\": \"In vitro GST-SH2 domain pulldown with synthetic phosphopeptides, competition kinase assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis-equivalent peptide competition; multiple orthogonal biochemical assays in one study\",\n      \"pmids\": [\"7683128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"C-terminal Src kinase (Csk) suppresses c-Src kinase activity in vivo by phosphorylating Tyr527; overexpression of Csk reverses v-Crk-induced c-Src activation and cellular transformation, and this suppression is abolished when Tyr527 is mutated to Phe.\",\n      \"method\": \"Cotransfection/overexpression, kinase activity assay, morphological transformation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with point-mutant validation, replicated in multiple cell systems\",\n      \"pmids\": [\"1383688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"c-Src is required for osteoclast spreading downstream of CSF-1 receptor (c-Fms); c-Src is tyrosine-phosphorylated and its kinase activity increases ~3-fold upon CSF-1 stimulation, and src-null osteoclasts fail to spread and show altered downstream substrate phosphorylation (including alpha-actinin).\",\n      \"method\": \"Immunoprecipitation kinase assay, src-null mouse osteoclasts, confocal microscopy, Western blot\",\n      \"journal\": \"Molecular reproduction and development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with defined phenotype, multiple orthogonal methods, replicated across papers\",\n      \"pmids\": [\"8981371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"c-Cbl is tyrosine-phosphorylated in a c-Src-dependent manner in osteoclasts; c-Cbl and c-Src colocalize on vesicular structures; antisense knockdown of either c-src or c-cbl inhibits in vitro bone resorption, placing c-Cbl downstream of c-Src in a signaling pathway required for bone resorption.\",\n      \"method\": \"Antisense oligonucleotides, immunoprecipitation/phosphorylation assay, src-null osteoclasts, confocal colocalization, in vitro bone resorption assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal genetic and biochemical evidence, multiple orthogonal methods, published in high-impact journal\",\n      \"pmids\": [\"8849724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"pp60c-Src is concentrated at least 9-fold in nerve growth cone membrane fractions of developing rat brain and is an active tyrosine kinase in that compartment, with altered electrophoretic mobility characteristic of the neuronal form; it is present at lower levels in mature brain synaptosomal fractions.\",\n      \"method\": \"Subcellular fractionation, indirect immunofluorescence, in vitro kinase assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct biochemical fractionation combined with kinase assay and imaging; replicated across chick and rat systems\",\n      \"pmids\": [\"2455889\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1984,\n      \"finding\": \"Overexpression of wild-type c-Src in NIH 3T3 cells elevates tyrosine kinase activity and causes partial morphological transformation but does not induce foci or anchorage-independent growth, demonstrating that the mutations distinguishing v-Src from c-Src are required for full transformation.\",\n      \"method\": \"NIH 3T3 transfection/focus assay, soft-agar colony assay, in vitro kinase assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional gain-of-function with multiple readouts; foundational study replicated broadly\",\n      \"pmids\": [\"6594680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"c-Src directly phosphorylates the EGF receptor at Tyr845 within the c-Src–EGFR complex in an EGF-dependent manner, as demonstrated by in vitro kinase assay on cyanogen bromide fragments and phosphopeptide mapping co-migration with a synthetic pTyr845 standard.\",\n      \"method\": \"In vitro kinase assay, cyanogen bromide fragment phosphorylation, phosphopeptide mapping, in-cell EGF stimulation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro kinase reconstitution with peptide mapping, single lab, single paper\",\n      \"pmids\": [\"7488034\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Constitutively active c-Src phosphorylates connexin-43 at Tyr265, causing the phosphorylated C-terminus to bind the c-Src SH2 domain and displacing ZO-1; this reduces total and surface connexin-43 levels and diminishes gap junctional conductance. A Tyr265 mutant connexin-43 retains ZO-1 interaction despite active c-Src.\",\n      \"method\": \"Cotransfection, in vitro binding assay with recombinant proteins, cell-surface biotinylation, electrophysiology, site-directed mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution with recombinant proteins, mutagenesis at specific residue, multiple orthogonal readouts including electrophysiology\",\n      \"pmids\": [\"11035005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Deletion/reduction of c-Src expression in mice enhances osteoblast differentiation and bone formation, increases alkaline phosphatase activity, nodule mineralization, and upregulates osteoblast differentiation markers (Osf2/Cbfa1, osteocalcin, collagen) in vitro and in vivo, demonstrating a negative regulatory role of c-Src in osteoblastogenesis.\",\n      \"method\": \"Src-null mice, bone histomorphometry, antisense oligonucleotide knockdown in primary osteoblasts, RT-PCR, in vitro differentiation assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout plus antisense knockdown with multiple orthogonal functional readouts\",\n      \"pmids\": [\"11038178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"c-Src localizes to mitochondria in osteoclasts and phosphorylates cytochrome c oxidase (Cox), increasing its enzymatic activity. Src deletion reduces Cox activity; re-expression of c-Src restores it. Increasing Src kinase activity reverses calcitonin-mediated inhibition of Cox and osteoclast function.\",\n      \"method\": \"Mitochondrial fractionation, kinase assay, c-Src knockout/rescue with adenoviral vectors, Cox activity assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — subcellular fractionation with functional enzyme activity assay, genetic rescue, multiple orthogonal readouts\",\n      \"pmids\": [\"12615910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"p130CAS (CAS) binds directly to both the SH2 and SH3 domains of c-Src and activates its tyrosine kinase activity; a single amino acid substitution in the CAS SH3-binding site disrupts both CAS–c-Src interaction and c-Src-dependent phosphorylation of cortactin and paxillin.\",\n      \"method\": \"Co-immunoprecipitation, overexpression in COS-1 cells, site-directed mutagenesis, tyrosine phosphorylation assay, soft-agar growth assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal binding with point-mutant validation, multiple substrates tested, functional transformation assay\",\n      \"pmids\": [\"10913170\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PLD2 (and to a lesser extent PLD1) are directly tyrosine-phosphorylated by c-Src via direct association; the interaction is mediated by the PLD2 pleckstrin homology domain and the c-Src catalytic domain. PLD catalytic activity (but not c-Src phosphorylation of PLD) in turn stimulates c-Src kinase activity and c-Src-mediated paxillin phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, domain-mapping mutagenesis, EGF stimulation in A431 cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct co-IP and in vitro phosphorylation, domain mapping; single lab\",\n      \"pmids\": [\"12697812\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PKCα activates c-Src via the scaffold protein AFAP-110: PKCα activation directs AFAP-110 to bind the c-Src SH3 domain, which activates c-Src kinase activity and promotes podosome formation containing cortactin, AFAP-110, actin, and c-Src. Cells lacking AFAP-110 cannot undergo PKCα-mediated c-Src activation or podosome formation, and rescue requires AFAP-110 capable of binding c-Src.\",\n      \"method\": \"Immunofluorescence, co-immunoprecipitation, kinase assay, AFAP-110-deficient cell line rescue, site-directed mutagenesis of AFAP-110\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue with structure-function mutants, multiple orthogonal readouts\",\n      \"pmids\": [\"15314167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"c-Src regulates podosome formation, structure, life span, and actin flux in osteoclasts; Src−/− osteoclasts show fewer podosomes, decreased actin cloud, 4-fold increased podosome lifespan, and 40% reduced actin flux rate. Rescue with Src mutants shows both kinase activity AND either the SH2 or SH3 binding domain are required for normal podosome dynamics.\",\n      \"method\": \"Src-null osteoclasts, videomicroscopy, FRAP, adenoviral rescue with c-Src kinase/domain mutants, fluorescence microscopy\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with quantitative live imaging and FRAP, domain-mutant rescue panel\",\n      \"pmids\": [\"17978100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"c-Src forms an essential signaling complex with αvβ3 integrin and Syk in osteoclasts; upon integrin activation, c-Src phosphorylates Syk, and ITAM proteins Dap12 and FcRγ mediate the Syk–c-Src association and αvβ3-induced Syk phosphorylation required for cytoskeletal organization and bone resorption.\",\n      \"method\": \"Syk-null mice (in vitro and chimeric in vivo), co-immunoprecipitation, kinase assay, cytoskeletal staining, bone resorption assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout models combined with biochemical complex characterization and functional readouts\",\n      \"pmids\": [\"17353363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"VEGFR2 phosphorylated at Y951 binds the SH2 domain of TSAd, which in turn recruits and activates c-Src (increased pY418, decreased pY527); TSAd silencing blocks VEGF-induced c-Src activation and VE-cadherin junction rearrangement. TSAd, VEGFR2, and c-Src form a complex at endothelial junctions, and Tsad−/− mice show impaired VEGF-induced vascular permeability.\",\n      \"method\": \"TSAd knockout mice, siRNA silencing, co-immunoprecipitation, phosphorylation assays, Evans blue/dextran permeability assay, in vitro and in vivo experiments\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout in vivo, reciprocal co-IP, multiple orthogonal permeability readouts\",\n      \"pmids\": [\"22689825\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"c-Src phosphorylates mitochondrial NADH dehydrogenase subunit NDUFV2 at Tyr193 (required for NADH dehydrogenase/complex I activity and cellular ATP) and SDHA at Tyr215 (no effect on enzyme activity but induces ROS via electron transfer perturbation); phosphorylation-defective mutants reduce cell viability.\",\n      \"method\": \"Mitochondrial kinase-dead c-Src with targeting sequence, phosphorylation-site mutagenesis, enzymatic activity assays, ROS measurement, cell viability assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vivo phosphorylation with site-specific mutants and enzyme activity readout; mitochondria-targeted kinase-dead construct\",\n      \"pmids\": [\"22823520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"c-Src phosphorylates FOXM1 on two tyrosine residues, stimulating FOXM1 nuclear localization and target gene expression (including G2/M regulators and c-Src itself), forming a positive feedback loop that drives mammary tumor cell proliferation.\",\n      \"method\": \"Genetically engineered mouse model with c-Src deletion, phosphorylation mapping, nuclear localization assay, target gene expression, patient-derived models\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mouse model deletion, direct phosphorylation site identification, multiple functional readouts including in vivo tumor progression\",\n      \"pmids\": [\"36795481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"c-Src associates with the prolactin receptor in rat hepatocytes and is activated upon prolactin stimulation, as shown by co-immunoprecipitation; prolactin treatment also induces c-src, c-fos, and c-jun gene expression.\",\n      \"method\": \"Co-immunoprecipitation kinase assay from hepatocyte lysates, Western blot\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP kinase assay, single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"8584023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"c-Src stimulation by prolactin is independent of Jak2: a Box1-mutant PRLR that cannot activate Jak2 (and thus cannot phosphorylate the receptor) still activates c-Src equivalently to wild-type PRLR upon prolactin treatment.\",\n      \"method\": \"Expression of Box1-mutant PRLR and kinase-deleted Jak2 in chicken embryo fibroblasts, c-Src kinase activity assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic dissection with receptor and kinase mutants, single lab; epistasis result well controlled\",\n      \"pmids\": [\"10600634\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"EphB1 recruits c-Src and p52Shc; activated EphB1 promotes c-Src tyrosine phosphorylation, and c-Src phosphorylates p52Shc, enabling p52Shc recruitment to EphB1 signaling complexes via its PTB domain. EphB1 tyrosines 600 and 778 are required for c-Src and p52Shc interaction. Dominant-negative c-Src reduces ERK1/2 activation and chemotaxis.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis of EphB1, dominant-negative c-Src expression, MEK inhibitor, ERK assay, migration assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal IP, receptor mutagenesis, dominant-negative, multiple functional readouts\",\n      \"pmids\": [\"12925710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"c-Src is required downstream of the PDGF and EGF receptors for mitogenesis; preferred c-Src substrates include cortactin, p190RhoGAP, and p130CAS (actin cytoskeleton/focal adhesion proteins), while EGF receptor substrates include SHC and PLCγ.\",\n      \"method\": \"C3H10T fibroblast model with wild-type and mutant c-Src, substrate phosphorylation comparison, temporal/spatial signaling analysis\",\n      \"journal\": \"Frontiers in bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — substrate identification by comparative phosphorylation, multiple cell-based experiments; review/methods paper\",\n      \"pmids\": [\"9331427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"c-Src activates both STAT1 and STAT3 in PDGF-stimulated NIH3T3 cells; STAT1 co-immunoprecipitates with c-Src, suggesting direct interaction; overexpression of dominant-negative c-Src reduces STAT1/3 tyrosine phosphorylation and DNA binding activity.\",\n      \"method\": \"Co-immunoprecipitation, overexpression of c-Src and dominant-negative Src, EMSA for DNA binding activity, tyrosine phosphorylation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP with dominant-negative, single lab; direct kinase assay for STATs not shown\",\n      \"pmids\": [\"9344858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Pyk2 and c-Src synergistically activate Stat3 downstream of EGFR; EGF stimulation recruits c-Src, Pyk2, and Stat3 to EGFR; dominant-negative Pyk2 impairs c-Src-induced Stat3 activation; Pyk2 expression induces Stat3 phosphorylation at Tyr705 and Ser727.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative constructs, luciferase reporter assay, Western blot for phospho-Stat3\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP plus dominant-negative epistasis, single lab, multiple readouts\",\n      \"pmids\": [\"14963038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Trans-interacting cadherin locally activates c-Src at cell-cell adhesion sites; c-Src then tyrosine-phosphorylates Vav2 (Rac-GEF) and activates Rap1 via C3G/Crk; both c-Src phosphorylation of Vav2 AND Rap1 activation (via PI3K) are jointly required for Rac activation.\",\n      \"method\": \"Inhibitor studies (PP2), dominant-negative constructs, co-immunoprecipitation, GTPase activity assays, cadherin trans-interaction model in fibroblasts and epithelial cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — pathway dissection with multiple inhibitors and dominant-negatives, single lab\",\n      \"pmids\": [\"16170364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Aldosterone activates vascular c-Src through the mineralocorticoid receptor (eplerenone-sensitive); activated c-Src then mediates p38 MAPK phosphorylation and NADPH oxidase activation; c-Src-deficient (c-Src+/−) VSMCs fail to show aldosterone-induced cortactin or p38 MAPK phosphorylation.\",\n      \"method\": \"c-Src heterozygous mouse VSMCs, PP2 inhibitor, eplerenone, kinase assays, Western blot, [3H]proline incorporation\",\n      \"journal\": \"Hypertension\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic haploinsufficiency model combined with pharmacologic inhibition, multiple pathway readouts\",\n      \"pmids\": [\"15699470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Endosomal NADPH oxidases (Nox1, Nox2) generate ROS that activate c-Src following hypoxia/reoxygenation; Rac1-dependent endocytosis recruits c-Src to endosomes where endosomal ROS activate it; activated c-Src then phosphorylates IκBα on tyrosine to activate NF-κB. Quenching endosomal ROS or Rac1 siRNA blocks c-Src activation.\",\n      \"method\": \"siRNA knockdown (Rac1), Nox-deficient primary fibroblasts, endosomal fractionation, intra-endosomal ROS quenching, phosphorylation assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockdown plus Nox-null cells, subcellular fractionation, multiple orthogonal methods\",\n      \"pmids\": [\"18397177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"c-Src associates with ErbB2 specifically through an interaction involving the ErbB2 kinase domain region surrounding Tyr877 (EGFR(YHAD) motif); this association does not require c-Src SH2 or SH3 domains or receptor phosphorylation, and confers enhanced transforming potential. EGFR mutants found in lung cancer that gain the Y877 equivalent motif also bind c-Src.\",\n      \"method\": \"Chimeric EGFR/ErbB2 receptors, co-immunoprecipitation, site-directed mutagenesis, in vitro and in vivo transformation assays, Stat3 activation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-mapping with chimeric receptors and mutagenesis, reciprocal co-IP, functional transformation assay in vitro and in vivo\",\n      \"pmids\": [\"19704002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"c-Src overexpression enhances ErbB2/ErbB3 heterocomplex formation and their basal and heregulin-induced activation; kinase-inactive c-Src or PP2 treatment reduces heterocomplex formation and downstream signaling, indicating c-Src acts upstream to positively modulate ErbB2/ErbB3 association.\",\n      \"method\": \"Co-immunoprecipitation, wild-type vs. kinase-inactive c-Src overexpression, PP2 pharmacological inhibition, receptor activation Western blot, migration and anchorage-independent growth assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP plus kinase-dead mutant rescue, single lab, mechanistic placement upstream of receptor complex\",\n      \"pmids\": [\"17173075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"c-Src directly phosphorylates hexokinase 1 (HK1) at Tyr732 and HK2, dramatically increasing their catalytic activity (decreased Km, increased Vmax for HK1) by disrupting HK1 dimer formation. HK1-Y732F or HK2 phosphosite mutants abrogate c-Src-stimulated glycolysis, cell proliferation, tumorigenesis, and metastasis.\",\n      \"method\": \"In vitro kinase assay, Km/Vmax measurements, site-directed mutagenesis (Y732F knockin and knockin mice), xenograft and metastasis models, clinical sample correlation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with kinetics, mutagenesis at specific residue, in vivo knockin mouse model\",\n      \"pmids\": [\"28054552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"c-Src phosphorylates PFKFB3 at Tyr194, activating this key glycolytic enzyme to boost fructose-2,6-bisphosphate production and PFK1 activity, replenishing PPP and serine pathways. PFKFB3-Y194F knockin mice show impaired glycolysis and reduced spontaneous colon cancer formation when crossed with APCmin/+ mice.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (Y194F), PFKFB3 knockout cells, PFKFB3-Y194F knockin mice, metabolic flux assay, APCmin/+ cross, clinical sample correlation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, knockin mouse model, multiple orthogonal metabolic and tumor readouts\",\n      \"pmids\": [\"32209481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"c-Src interacts with and phosphorylates G6PD at Tyr112, enhancing its catalytic activity (decreased Km, increased Kcat) for glucose-6-phosphate, thereby augmenting PPP flux for NADPH and ribose-5-phosphate production and promoting tumorigenesis.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, enzyme kinetics (Km/Kcat), site-directed mutagenesis (Y112), clinical colorectal cancer sample correlation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with enzyme kinetics and site-specific mutagenesis, clinical validation\",\n      \"pmids\": [\"33686238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"c-Src phosphorylates E-cadherin at Tyr797, triggering RNF43-mediated ubiquitination of E-cadherin at Lys816 and subsequent proteasomal degradation, enabling nuclear β-catenin translocation and EMT in lung adenocarcinoma.\",\n      \"method\": \"Immunoprecipitation, ubiquitination assay, phospho-specific antibody, shRNA knockdown, xenograft model, site-directed mutagenesis\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — co-IP/ubiquitination with site-specific mutants, single lab\",\n      \"pmids\": [\"31286874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Cdh1 (APC/C co-activator) suppresses c-Src kinase activity in an APC-independent manner; reciprocally, hyperactive c-Src phosphorylates Cdh1 at its N-terminus, disrupting Cdh1 interaction with the APC core complex and inhibiting APCCdh1 E3 ligase activity, forming a reciprocal feedback circuit.\",\n      \"method\": \"Co-immunoprecipitation, kinase assay, site-directed mutagenesis, ubiquitin E3 ligase assay, mouse mammary tumor model (PTEN loss), pharmacological c-Src inhibition\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, direct kinase phosphorylation of Cdh1, E3 ligase activity assay, in vivo mouse model\",\n      \"pmids\": [\"31420536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RACK1 interacts with c-Src via RACK1 tyrosines 228 and 246 (binding the c-Src SH2 domain at Lys152); RACK1-Y228F/Y246F mutant fails to interact with c-Src and impairs osteoclast cytoskeletal integrity and bone resorption without affecting differentiation. c-Src K152R similarly impairs osteoclast resorption.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis of RACK1 and c-Src, osteoclast differentiation/resorption assays, cytoskeletal analysis\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — reciprocal mutagenesis of both binding partners with functional bone resorption readout, single lab\",\n      \"pmids\": [\"31358728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"c-Src autophosphorylation at Tyr416 causes global structural rearrangements: the kinase domain gains rigidity and stabilizes the ATP-binding site, while the regulatory SH2/SH3 domains become more flexible and detach from the kinase domain, resulting in a 4-fold increase in enzymatic activity.\",\n      \"method\": \"Hydrogen/deuterium exchange MS, biochemical kinase activity assay, molecular dynamics simulations\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — HDX-MS structural method plus kinase activity assay and simulations, multiple orthogonal approaches\",\n      \"pmids\": [\"31331936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Molecular dynamics free-energy calculations demonstrate that phosphorylation of Tyr416 in the activation loop locks c-Src into a catalytically competent conformation by stabilizing the hydrophobic regulatory spine, HRD motif, and electrostatic switch; unphosphorylated A-loop shows high flexibility and the active conformation is only transiently visited.\",\n      \"method\": \"Molecular dynamics umbrella sampling free-energy simulations\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational only, no experimental validation in this paper\",\n      \"pmids\": [\"24103328\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Dominant-negative c-Src (K295M) prevents acid-induced activation of NHE3 in renal epithelial cells, placing c-Src upstream of NHE3 in the response to chronic acidosis; acid-induced ERK activation is independent of c-Src, demonstrating two parallel pathways both required for NHE3 activation.\",\n      \"method\": \"Dominant-negative c-Src transfection, NHE3 activity assay (pHi recovery), immune-complex kinase assay, MEK inhibitor (PD98059)\",\n      \"journal\": \"Kidney international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — genetic epistasis with dominant-negative and pharmacologic tools, single lab\",\n      \"pmids\": [\"12081562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"c-Src controls functional co-localization of the proton pump and CLIC-5b chloride channel in osteoclast vesicles, which is required for vesicular acidification and bone resorption. CLIC-5b binds c-Src SH2 and SH3 domains. c-Src suppression reduces vesicular acidification (rescued by valinomycin), consistent with selective loss of chloride conductance.\",\n      \"method\": \"c-Src siRNA knockdown, CLIC-5b siRNA knockdown, vesicular acidification assay, valinomycin rescue, affinity pull-down with Src SH2/SH3 domains, bone resorption assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain pulldown plus siRNA knockdown, functional acidification rescue, multiple readouts\",\n      \"pmids\": [\"16831863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Connexin43 recruits PTEN and Csk to its C-terminal region (residues 266–283) to inhibit c-Src; pull-down assays show this region is sufficient to recruit c-Src, PTEN, and Csk and inhibit oncogenic c-Src activity. Silencing Csk or PTEN reduces the antiproliferative effect of Cx43 in glioma cells.\",\n      \"method\": \"Pull-down assays with Cx43 peptide fragments, co-immunoprecipitation, confocal microscopy, siRNA silencing of Csk and PTEN, phosphorylation assays (pY527, pY416)\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — pull-down domain mapping combined with siRNA epistasis, single lab\",\n      \"pmids\": [\"27391443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Apoptotic cell binding to MerTK on dendritic cells establishes a complex containing MerTK, c-Src, STAT3, and PI3K; this activates c-Src and STAT3 and mediates inhibition of DC maturation. Pharmacological inhibitors or siRNA against c-Src or STAT3 block apoptotic cell-induced DC inhibition.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation assay, siRNA knockdown, pharmacological inhibitors, MerTK-knockout DCs, DC maturation assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, genetic (MerTK KO) and pharmacologic epistasis, multiple readouts\",\n      \"pmids\": [\"19667404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"c-Src co-immunoprecipitates with c-Cbl and both localize to Golgi-enriched membrane fractions in CHO cells; activated (but not wild-type) c-Src increases the amount of c-Cbl co-immunoprecipitating with Src and the intensity of c-Cbl Golgi staining, with concomitant increased tyrosine phosphorylation of membrane-associated Cbl.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation (density centrifugation and free-flow electrophoresis), confocal immunofluorescence, activated c-Src transfection\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-IP and fractionation with activated Src mutant, single lab, localization without full functional consequence established\",\n      \"pmids\": [\"11893076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"c-Src phosphorylates HDAC3 at Tyr328 and Tyr331; phosphorylated HDAC3 shows higher deacetylase activity, is recruited to the plasma membrane upon EGF stimulation, and promotes breast cancer cell invasion. PP2 (c-Src inhibitor) blocks HDAC3 phosphorylation and reduces enzymatic activity.\",\n      \"method\": \"Site-directed mutagenesis (Y328/331A), phospho-specific antibody, in vitro kinase assay, TIRF microscopy, invasion assay, c-Src knockdown\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro phosphorylation with site-specific mutant and functional enzyme activity readout, single lab\",\n      \"pmids\": [\"31430896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Inhibitor binding to c-Src induces a conformational change that promotes c-Src association with FAK in an active form; upon inhibitor dissociation, c-Src phosphorylates FAK and initiates FAK-Grb2-Erk signaling. A drug-resistant c-Src mutation that reduces inhibitor affinity paradoxically converts Src inhibitors into facilitators of FAK/Erk phosphorylation and cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation with c-Src inhibitors, phosphorylation assay (FAK, Erk), drug-resistant c-Src mutant cells, cell proliferation assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — conformational mechanism inferred from inhibitor-dependent co-IP and phosphorylation, single lab\",\n      \"pmids\": [\"33761359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"c-Src is the specific kinase required for villin-mediated intestinal cell migration; reconstitution of SYF (Src/Yes/Fyn triple-knockout) cells individually with c-Src, c-Yes, or c-Fyn demonstrates an absolute requirement for c-Src specifically. SHP-2 and PTP-PEST are identified as negative regulators of c-Src activity in this context.\",\n      \"method\": \"SYF cells reconstituted with individual kinases, cell migration assay, villin phosphorylation assay, siRNA for phosphatases\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean genetic epistasis using triple-knockout cells reconstituted with individual kinases, specific functional readout\",\n      \"pmids\": [\"18482983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"c-Src drives intestinal stem cell (ISC) proliferation, regeneration, and tumorigenesis through upregulation of EGFR and activation of Ras/MAPK and Stat3 signaling, as shown by genetic gain- and loss-of-function in both Drosophila and mouse intestinal epithelium; c-Src plays a non-redundant role that cannot be substituted by Fyn or Yes.\",\n      \"method\": \"Genetic gain- and loss-of-function (Drosophila and conditional mouse knockout), ISC proliferation assay, EGFR/MAPK/Stat3 pathway activation readouts\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — orthogonal genetic experiments in two organisms, specificity demonstrated by Fyn/Yes non-rescue, mechanistic pathway placement\",\n      \"pmids\": [\"24788409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"c-Src stimulates IL-6 expression through STAT3; IL-6 in turn induces IGFBP5, which activates c-Src in immature (but not mature) osteoblasts, creating an amplifying loop that maintains osteoblasts in an immature state; IGFBP5 produced by osteoblasts also stimulates osteoclastogenesis.\",\n      \"method\": \"c-Src inhibition, siRNA knockdown, STAT3 reporter, cytokine measurements, in vitro and in vivo osteoblast/osteoclast assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — pathway dissection with multiple tools but largely pharmacologic, single lab\",\n      \"pmids\": [\"22252554\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The PDZ protein MPP2 interacts with c-Src via its PDZ domain in epithelial cells (identified by PDZ domain array screen and confirmed by co-immunoprecipitation); MPP2 negatively regulates c-Src kinase activity in cells and suppresses c-Src-dependent disorganization of the cortical actin cytoskeleton.\",\n      \"method\": \"PDZ domain array screen, co-immunoprecipitation, kinase activity assay, cytoskeletal imaging\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — co-IP plus kinase activity and phenotypic rescue, single lab\",\n      \"pmids\": [\"19665017\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"c-Src is a non-receptor tyrosine kinase regulated by an intramolecular inhibitory clamp in which the SH2 domain binds pTyr527 in the C-terminal tail (phosphorylated by Csk) and the SH3 domain engages the linker; release of this clamp, followed by autophosphorylation of Tyr416 in the activation loop (which locks the active conformation by rigidifying the kinase domain and detaching the regulatory domains), fully activates the enzyme. Active c-Src localizes to the plasma membrane, focal adhesions, endosomes, mitochondria, and Golgi, where it phosphorylates a broad range of substrates including EGFR (Y845), ErbB2 (Y877 region), connexin-43 (Y265), hexokinases HK1/HK2 (Y732), PFKFB3 (Y194), G6PD (Y112), FOXM1, HDAC3 (Y328/Y331), cytochrome c oxidase subunits, and mitochondrial OXPHOS complexes I/II, thereby regulating glycolysis, oxidative phosphorylation, actin dynamics (podosomes, invadopodia, cell migration), gap junction function, cell cycle progression (via FOXM1 and APCCdh1), and bone homeostasis through coordinated signaling with partners including c-Cbl, Syk/αvβ3/ITAM proteins, FAK, p130CAS, RACK1, TSAd/VEGFR2, MerTK/STAT3, and AFAP-110/PKCα.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"c-Src is a non-receptor tyrosine kinase that transduces signals from membrane receptors and adhesion structures to control cell migration, proliferation, metabolism, and bone remodeling [#2, #21, #29]. Its catalytic output is held in check by an intramolecular clamp: the SH2 domain binds the Csk-phosphorylated C-terminal tail at pTyr527, and a competing phosphopeptide that occupies the SH2 domain releases and activates the enzyme [#0, #1]. Activation is consolidated by autophosphorylation of Tyr416 in the activation loop, which rigidifies the kinase domain and detaches the regulatory SH2/SH3 modules to lock in the active conformation and raise activity ~4-fold [#35]. The freed SH2/SH3 surfaces serve as docking sites that integrate diverse inputs and are themselves required for productive signaling: p130CAS engages both domains to stimulate kinase activity, PKCα drives AFAP-110 onto the SH3 domain to trigger podosome formation, VEGFR2-bound TSAd recruits Src through its SH2 domain, and RACK1 engages the SH2 domain at Lys152 [#10, #12, #15, #34]. Through these complexes c-Src governs the actin cytoskeleton — controlling podosome dynamics, cortactin/paxillin/p130CAS phosphorylation, and migration downstream of integrins, cadherins, and growth factor receptors [#10, #13, #21, #44]. In the osteoclast it is essential for cell spreading, podosome turnover, vesicular acidification, and bone resorption, signaling through c-Cbl, the αvβ3/Syk/ITAM module, RACK1, and CLIC-5b, while paradoxically restraining osteoblast differentiation [#2, #3, #8, #13, #14, #34, #38]. c-Src also phosphorylates and activates an array of receptor and effector substrates — EGFR at Tyr845, the ErbB2 region around Tyr877, connexin-43 at Tyr265 (displacing ZO-1 and closing gap junctions), and FOXM1 in a proliferative feedback loop — and it reciprocally inhibits APC/C-Cdh1 by phosphorylating Cdh1 [#6, #7, #17, #27, #33]. A prominent metabolic function emerges from its direct phosphorylation of glycolytic and pentose-phosphate enzymes (HK1/HK2 at Tyr732, PFKFB3 at Tyr194, G6PD at Tyr112) and mitochondrial complexes (cytochrome c oxidase, NDUFV2 at Tyr193, SDHA at Tyr215), coupling Src activity to glycolysis, oxidative phosphorylation, and tumor growth [#9, #16, #29, #30, #31]. The mutations distinguishing v-Src from c-Src are required for full transformation, underscoring the tight physiological control of this oncogene [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 1984,\n      \"claim\": \"Established that wild-type c-Src is not by itself fully transforming, defining the difference between proto-oncogene and viral oncogene and motivating the search for c-Src's intrinsic regulation.\",\n      \"evidence\": \"NIH 3T3 transfection with focus and soft-agar assays comparing c-Src overexpression to v-Src\",\n      \"pmids\": [\"6594680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the regulatory mechanism keeping c-Src inactive\", \"Did not identify the specific activating lesions in v-Src\"]\n    },\n    {\n      \"year\": 1988,\n      \"claim\": \"Showed that c-Src is enriched and catalytically active in a defined subcellular compartment (neuronal growth cone membranes), linking the kinase to membrane-localized developmental signaling.\",\n      \"evidence\": \"Subcellular fractionation, immunofluorescence, and in vitro kinase assay in developing rat/chick brain\",\n      \"pmids\": [\"2455889\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No substrates in the growth cone identified\", \"Functional consequence for neuronal development not tested\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Identified Csk-mediated phosphorylation of Tyr527 as the in vivo brake on c-Src, resolving how the kinase is held inactive in cells.\",\n      \"evidence\": \"Cotransfection/overexpression with Y527F point mutant, kinase and transformation assays\",\n      \"pmids\": [\"1383688\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show the structural mechanism by which pTyr527 inhibits\", \"Physiological signals controlling Csk activity not addressed\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Defined the molecular basis of autoinhibition: the SH2 domain binds pTyr527 intramolecularly, and competition for the SH2 domain releases activity.\",\n      \"evidence\": \"In vitro GST-SH2 pulldowns with synthetic phosphopeptides and competition kinase assays\",\n      \"pmids\": [\"7683128\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SH3-linker contribution not resolved in this study\", \"Did not address activation-loop autophosphorylation\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Placed c-Src in osteoclast biology, showing it is activated downstream of CSF-1 and required for cell spreading, and identified c-Cbl as a downstream effector for bone resorption.\",\n      \"evidence\": \"src-null osteoclasts, IP kinase assays, antisense knockdown of src/cbl, confocal colocalization, in vitro resorption assay\",\n      \"pmids\": [\"8981371\", \"8849724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct phosphorylation of c-Cbl by c-Src not formally shown\", \"Substrate(s) mediating spreading defect incompletely defined\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Demonstrated that c-Src directly phosphorylates receptor tyrosine kinase substrates, identifying EGFR Tyr845 within the Src-EGFR complex.\",\n      \"evidence\": \"In vitro kinase assay on CNBr fragments and phosphopeptide mapping with EGF stimulation\",\n      \"pmids\": [\"7488034\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab in vitro result\", \"Cellular consequence of Y845 phosphorylation not established here\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed c-Src closes gap junctions by phosphorylating connexin-43 Tyr265, which then binds the Src SH2 domain and displaces ZO-1, and that scaffold partners (p130CAS) bidirectionally regulate Src activity.\",\n      \"evidence\": \"Reconstitution with recombinant proteins, electrophysiology, biotinylation, Y265 mutagenesis; reciprocal co-IP and point mutants for CAS\",\n      \"pmids\": [\"11035005\", \"10913170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether these mechanisms operate in the same physiological context not addressed\", \"Connexin-43 turnover pathway not fully mapped\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Revealed a negative role for c-Src in osteoblast differentiation, establishing that Src oppositely regulates the two bone cell lineages.\",\n      \"evidence\": \"src-null mice, bone histomorphometry, antisense knockdown, RT-PCR of differentiation markers\",\n      \"pmids\": [\"11038178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Src substrates restraining osteoblast genes not identified\", \"Cell-autonomy versus osteoclast crosstalk not fully separated\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Established a mitochondrial localization and function for c-Src, showing it phosphorylates and activates cytochrome c oxidase to support osteoclast bioenergetics.\",\n      \"evidence\": \"Mitochondrial fractionation, kinase assay, Src knockout/rescue, Cox activity assay\",\n      \"pmids\": [\"12615910\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise Cox phosphosite not defined in this study\", \"How Src is imported/retained at mitochondria unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined how c-Src organizes the osteoclast cytoskeleton, showing both kinase activity and an intact SH2 or SH3 domain are needed for podosome dynamics, and that Src nucleates an αvβ3/Syk/ITAM complex for resorption.\",\n      \"evidence\": \"src-null osteoclasts with FRAP/videomicroscopy and domain-mutant rescue; Syk-null mice with co-IP and resorption assays\",\n      \"pmids\": [\"17978100\", \"17353363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Src phosphosites on cytoskeletal components in osteoclasts not enumerated\", \"Quantitative coupling of complex assembly to actin flux incomplete\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Connected c-Src to receptor-proximal scaffolds and a proliferative transcription program: TSAd links VEGFR2 to Src for vascular permeability, and Src phosphorylates FOXM1 to drive a feed-forward proliferation loop.\",\n      \"evidence\": \"TSAd knockout mice with reciprocal co-IP and permeability assays; Src-deletion mouse model with FOXM1 phosphosite mapping and target gene readouts\",\n      \"pmids\": [\"22689825\", \"36795481\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"FOXM1 tyrosine sites only partially characterized\", \"Generality of TSAd-Src axis beyond endothelium not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Extended Src's mitochondrial reach to electron transport chain subunits, distinguishing activating (NDUFV2 Y193) from ROS-inducing (SDHA Y215) phosphorylation events tied to cell viability.\",\n      \"evidence\": \"Mitochondria-targeted kinase-dead Src construct, phosphosite mutants, enzyme activity and ROS assays\",\n      \"pmids\": [\"22823520\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous mitochondrial Src activity regulation unclear\", \"Physiological versus pathological balance of complex I/II phosphorylation not resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved the structural logic of activation, showing Tyr416 autophosphorylation rigidifies the kinase domain and frees the regulatory domains to lock the active state.\",\n      \"evidence\": \"Hydrogen/deuterium exchange MS with kinase assays and molecular dynamics\",\n      \"pmids\": [\"31331936\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetics of the regulatory-domain release in cells not measured\", \"Interplay with pTyr527 dephosphorylation not directly tested here\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed c-Src feeds back on cell-cycle machinery and chromatin/EMT regulators, phosphorylating Cdh1 to inhibit APC/C-Cdh1, HDAC3 to enhance deacetylase activity and invasion, and E-cadherin to trigger its degradation and β-catenin nuclear translocation.\",\n      \"evidence\": \"Reciprocal co-IP and kinase/E3 ligase assays (Cdh1); in vitro kinase with phosphosite mutants and invasion assay (HDAC3); ubiquitination assay with phosphosite mutants (E-cadherin)\",\n      \"pmids\": [\"31420536\", \"31430896\", \"31286874\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"HDAC3 and E-cadherin findings rest on single-lab data\", \"In vivo relevance of the Src-Cdh1 circuit in normal cycling cells not established\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Consolidated c-Src as a master kinase of cancer metabolism by direct phosphorylation of glycolytic and pentose-phosphate enzymes (HK1/HK2 Y732, PFKFB3 Y194, G6PD Y112) that boost flux and tumorigenesis, validated in knockin mouse models.\",\n      \"evidence\": \"In vitro kinase assays with enzyme kinetics, phosphosite knockin mice, metabolic flux and tumor models, clinical correlation\",\n      \"pmids\": [\"28054552\", \"32209481\", \"33686238\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals directing Src to metabolic enzymes not fully defined\", \"Subcellular site of these phosphorylation events not pinpointed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple c-Src-regulating scaffolds and inhibitory partners (RACK1, MPP2, MerTK/STAT3, connexin-43/PTEN/Csk) are integrated to set Src activity in a given cell, and how its membrane/mitochondrial/nuclear substrate pools are spatially partitioned, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model linking compartment-specific Src pools to distinct substrate sets\", \"Quantitative hierarchy among competing positive and negative regulators not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 7, 16, 29, 30, 31, 42]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [6, 17, 29, 33]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [10, 12, 33]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 4, 15, 42]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [9, 16]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [41]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [26]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [12, 13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 15, 20, 35]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [9, 16, 29, 30, 31]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [17, 33]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 8, 13, 45]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [7, 24]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [5, 17, 29, 32]}\n    ],\n    \"complexes\": [\n      \"αvβ3 integrin–Syk–c-Src complex\",\n      \"VEGFR2–TSAd–c-Src complex\",\n      \"MerTK–c-Src–STAT3–PI3K complex\"\n    ],\n    \"partners\": [\n      \"CSK\",\n      \"p130CAS\",\n      \"AFAP-110\",\n      \"RACK1\",\n      \"c-Cbl\",\n      \"SYK\",\n      \"TSAd\",\n      \"FAK\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}