{"gene":"CBL","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":1999,"finding":"c-Cbl functions as a RING-type, E2-dependent ubiquitin-protein ligase (E3): it recognizes tyrosine-phosphorylated substrates (e.g., activated PDGF receptor) through its SH2 domain and recruits and allosterically activates an E2 ubiquitin-conjugating enzyme through its RING domain to catalyze ubiquitin transfer.","method":"In vitro ubiquitination assay, domain mutagenesis (SH2 and RING finger), biochemical reconstitution","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with defined substrate, allosteric activation mechanism demonstrated, replicated across multiple subsequent studies","pmids":["10514377"],"is_preprint":false},{"year":2000,"finding":"Crystal structure of c-Cbl bound to E2 (UbcH7) and a kinase substrate peptide revealed that the RING domain recruits E2 through a conserved surface, and there is rigid coupling between the substrate-binding and E2-binding domains with a conserved channel from substrate to E2 active site, suggesting RING E3s scaffold substrate and E2 for ubiquitin transfer.","method":"X-ray crystallography (crystal structure of c-Cbl–UbcH7–kinase peptide ternary complex)","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with functional validation, foundational structural paper widely replicated","pmids":["10966114"],"is_preprint":false},{"year":1998,"finding":"c-Cbl is transiently recruited into ErbB-1-containing endosomes in a receptor kinase activity-dependent manner, mediates covalent ubiquitination of ErbB-1, and directs internalized ErbB-1 to lysosomal and proteasomal degradation rather than recycling; oncogenic v-Cbl inhibits this by shunting receptors to the recycling pathway.","method":"Immunofluorescence microscopy (endosomal colocalization), immunoprecipitation (ubiquitin attachment), dominant-negative/oncogenic Cbl expression, degradation assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (colocalization, IP, functional rescue), independently replicated","pmids":["9851973"],"is_preprint":false},{"year":2012,"finding":"c-Cbl adopts an autoinhibited RING conformation in which the RING's E2-binding surface associates intramolecularly with CBL to reduce E2 affinity; phosphorylation of Tyr371 in the linker helix region (LHR) induces conformational changes that eliminate autoinhibition, flip the RING domain and E2 into proximity of the substrate-binding site, and enhance E2 binding—this activation is required for RTK ubiquitination.","method":"X-ray crystallography (structures of unphosphorylated CBL, CBL–substrate peptide, phospho-Tyr371-CBL–E2–substrate), biochemical activity assays, mutagenesis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures plus biochemical validation and mutagenesis in a single rigorous study","pmids":["22266821"],"is_preprint":false},{"year":1996,"finding":"c-Cbl is tyrosine-phosphorylated downstream of c-Src in osteoclasts and the two proteins colocalize on vesicular structures; antisense knockdown of either c-src or c-cbl inhibits in vitro bone resorption by osteoclast-like cells, placing c-Cbl downstream of c-Src in a pathway required for osteoclast-mediated bone resorption.","method":"Antisense oligonucleotide knockdown, immunoprecipitation, immunofluorescence colocalization, in vitro bone resorption assay with src-null OCLs","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis (antisense KD + src-null cells), multiple orthogonal methods, replicated by subsequent studies","pmids":["8849724"],"is_preprint":false},{"year":2001,"finding":"Src-catalyzed phosphorylation of c-Cbl (likely on Tyr-371) is required for c-Cbl-dependent ubiquitination of both c-Src itself and c-Cbl (auto-ubiquitination); this requires the RING finger of c-Cbl. Active Src destabilizes the c-Cbl–UbcH7 complex in vitro. c-Cbl reduces v-Src protein levels and suppresses v-Src-induced STAT3 activation.","method":"In vitro ubiquitination assay, co-immunoprecipitation, kinase activity assays, overexpression/mutagenesis in cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination reconstitution, mutagenesis, and cell-based assays in a single study","pmids":["11448952"],"is_preprint":false},{"year":2003,"finding":"Activated Cdc42 binds to p85Cool-1/beta-PIX, which directly associates with c-Cbl, thereby inhibiting c-Cbl binding to the EGF receptor and preventing Cbl-catalyzed receptor ubiquitination; constitutively active Cdc42(F28L) persistently blocks EGF receptor–Cbl interaction, leading to aberrant receptor accumulation, sustained ERK activation, and cellular transformation.","method":"Co-immunoprecipitation, dominant-active Cdc42 expression, ubiquitination assays, ERK activation assays, cell transformation assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP establishing ternary complex, functional ubiquitination assay, transformation phenotype, multiple orthogonal readouts","pmids":["14505571"],"is_preprint":false},{"year":1995,"finding":"In TCR-activated Jurkat T cells, c-Cbl constitutively associates with the Grb2 adaptor via Grb2's N-terminal SH3 domain; after TCR–CD3 plus CD4 co-stimulation, Cbl also binds Grb2's SH2 domain. TCR activation recruits PI 3-kinase activity to Cbl immunocomplexes. Cbl–Grb2 and Sos–Grb2 exist as distinct complexes.","method":"Co-immunoprecipitation with domain-specific Grb2 fusion proteins, PI 3-kinase activity assay in immunoprecipitates","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP with domain specificity mapping, single lab, functional PI3K assay but no reconstitution","pmids":["7791764"],"is_preprint":false},{"year":1998,"finding":"In c-Cbl-deficient mice, ZAP-70 kinase phosphorylation in thymocytes is uncoupled from the requirement for CD4-mediated Lck activation following CD3ε cross-linking, demonstrating that c-Cbl negatively regulates signaling by the Syk/ZAP-70 family of protein kinases in T cells; c-Cbl-null mice also display lymphoid hyperplasia and altered T-cell receptor expression.","method":"Targeted gene disruption (c-Cbl knockout mice), flow cytometry, immunoprecipitation/Western blot for ZAP-70 phosphorylation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean genetic knockout with defined biochemical phenotype (ZAP-70 phosphorylation), replicated by subsequent studies","pmids":["9671496"],"is_preprint":false},{"year":2002,"finding":"c-Cbl and Cbl-b double-knockout T cells fail to down-modulate surface TCR after ligand engagement, resulting in sustained TCR signaling and T-cell hyperresponsiveness; trafficking of internalized TCR to lysosomes was reduced, while ligand-independent internalization was normal. This demonstrates Cbl proteins negatively regulate T-cell activation by promoting clearance of engaged TCR from the cell surface.","method":"c-Cbl/Cbl-b double-knockout mouse T cells, flow cytometry (TCR surface levels), T-cell activation assays, trafficking assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — double-KO genetic model, multiple functional readouts (TCR surface levels, signaling duration, lysosomal trafficking)","pmids":["12415267"],"is_preprint":false},{"year":2013,"finding":"c-Cbl acts as a NEDD8 E3 ligase (neddylation E3) for the TGF-β type II receptor (TβRII): it conjugates NEDD8 to TβRII at Lys556 and Lys567. Neddylation promotes TβRII endocytosis to EEA1-positive early endosomes and prevents its endocytosis to caveolin-positive (degradative) compartments, thereby inhibiting TβRII ubiquitination and degradation and stabilizing/enhancing TGF-β signaling.","method":"Co-immunoprecipitation, in vitro neddylation assay, site-directed mutagenesis (K556R/K567R), c-Cbl knockout hematopoietic cells, endosomal colocalization assays, TGF-β pathway reporter assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro neddylation reconstitution, site mutagenesis, KO cells, and multiple orthogonal assays in a single study","pmids":["23290524"],"is_preprint":false},{"year":2019,"finding":"DNA damage leads to ATM-mediated phosphorylation and stabilization of c-Cbl, which in turn promotes TβRII neddylation and prevents its ubiquitination-dependent degradation, thus stabilizing TβRII and enhancing TGF-β signaling; ATM, c-Cbl, and TβRII form a ternary complex upon DNA damage.","method":"Co-immunoprecipitation (ATM-c-Cbl-TβRII complex), Western blotting (TβRII stability), mouse intestinal regeneration model after X-ray irradiation","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating ternary complex, in vivo mouse model, but largely extends prior neddylation finding (PMID 23290524)","pmids":["31315051"],"is_preprint":false},{"year":2002,"finding":"c-Cbl binds tyrosine-phosphorylated transmembrane Notch1 (by co-immunoprecipitation) and mediates ubiquitin-dependent targeting of the transmembrane form of Notch1 to lysosomal compartments for degradation in skeletal myoblasts.","method":"Co-immunoprecipitation, lysosomal inhibitor (chloroquine) treatment, proteasome inhibitor comparison, ubiquitin accumulation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single Co-IP and pharmacological inhibitor data, single lab, no direct ubiquitination reconstitution shown","pmids":["11777909"],"is_preprint":false},{"year":2005,"finding":"c-Cbl mediates mono-ubiquitination of activated PAR2 (a GPCR) in an Src-dependent manner, directing it from early endosomes to lysosomes for degradation; dominant-negative c-Cbl (RING-deleted) blocks PAR2 ubiquitination and lysosomal degradation, causing receptor retention in early endosomes and allowing recycling and continued signaling.","method":"Immunoprecipitation, immunofluorescence, dominant-negative c-Cbl expression, lysosomal degradation assays, receptor signaling recovery assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative and mutagenesis approaches combined with trafficking assays, single lab","pmids":["15708858"],"is_preprint":false},{"year":2008,"finding":"IL-6 stimulation induces c-Cbl-mediated K63-linked polyubiquitination of the cytokine receptor gp130; c-Cbl is recruited to gp130 via phosphorylated SHP2. Ubiquitinated gp130 is recognized by Hrs and targeted for lysosomal degradation. Deficiency of c-Cbl suppresses gp130 degradation and prolongs IL-6 signaling.","method":"Immunoprecipitation (c-Cbl–SHP2–gp130 complex), ubiquitin linkage analysis, c-Cbl siRNA knockdown, lysosomal degradation assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD phenotype plus complex mapping and ubiquitin linkage analysis, single lab","pmids":["18519587"],"is_preprint":false},{"year":2002,"finding":"APS acts as an adaptor linking the insulin receptor to c-Cbl: insulin-stimulated phosphorylation of APS Tyr618 enables APS to associate with c-Cbl, facilitating c-Cbl phosphorylation by the insulin receptor at Tyr371, Tyr700, and Tyr774, which drives c-Cbl–Crk association and is required for GLUT4 translocation.","method":"Overexpression of wild-type and Y618F APS mutant in 3T3-L1 adipocytes, co-immunoprecipitation, GLUT4 translocation assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific mutagenesis with functional GLUT4 readout, co-IP complex mapping, single lab","pmids":["11997497"],"is_preprint":false},{"year":2004,"finding":"Upon SCF stimulation, activated KIT receptor binds and phosphorylates Cbl proteins (c-Cbl and Cbl-b), which in turn ubiquitinate KIT and themselves, mediating their mutual degradation; TKB and RING finger domains of Cbl are essential for this negative feedback loop.","method":"Co-immunoprecipitation, ubiquitination assays, domain deletion/mutagenesis, degradation assays in mast cells","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis combined with ubiquitination and degradation assays, single lab","pmids":["15315962"],"is_preprint":false},{"year":2004,"finding":"VEGF stimulation induces formation of a ternary Flt-1–c-Cbl–CD2AP complex; c-Cbl binds to pTyr1333 of Flt-1; wild-type CD2AP promotes ubiquitination and internalization/degradation of Flt-1, whereas dominant-negative CD2AP or mutant c-Cbl prevents this.","method":"Co-immunoprecipitation (ternary complex), site-directed mutagenesis (Y1333F Flt-1), dominant-negative CD2AP and c-Cbl, internalization/ubiquitination assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis at receptor binding site, dominant-negative constructs, and functional ubiquitination/internalization assays; single lab","pmids":["15001553"],"is_preprint":false},{"year":2004,"finding":"c-Cbl-mediated ubiquitination is required for EGFR exit from early endosomes but not for its initial internalization; Src-inhibitor PP1 blocks Cbl-mediated ubiquitination and retains EGFR in early endosomes.","method":"Src inhibitor (PP1) treatment, mutant EGFR (Y1045F) expression, EGFR trafficking and ubiquitination assays in CHO and A549 cells","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological and mutant-receptor approaches with defined trafficking phenotype, single lab","pmids":["15210722"],"is_preprint":false},{"year":2005,"finding":"c-Cbl mediates ubiquitination and proteasomal degradation of STAT5 in response to GH stimulation, thereby negatively regulating GH-stimulated STAT5-mediated transcription; proteasome inhibitor MG132 reverses this effect.","method":"Overexpression of c-Cbl in NIH3T3 cells, ubiquitination assays, STAT5 reporter assay, proteasome inhibitor treatment","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — overexpression plus pharmacological rescue and functional readout, single lab, no reconstitution","pmids":["12193575"],"is_preprint":false},{"year":2003,"finding":"c-Cbl mediates Vav ubiquitination via Cbl RING finger activity; this requires Cbl–Vav association through phosphorylated Tyr-700 on Cbl. Cbl-dependent ubiquitination leads to loss of phosphorylated Vav protein, and c-Cbl but not its ubiquitin ligase mutant inhibits Vav-dependent signaling in Jurkat cells.","method":"Ubiquitination assays in Cbl+/+ and Cbl−/− T cells, co-immunoprecipitation, mutagenesis (RING finger and pTyr-700), Jurkat transfection with functional reporter assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cell lines, site mutagenesis, ubiquitination assays; single lab","pmids":["12881521"],"is_preprint":false},{"year":2005,"finding":"Loss of c-Cbl RING finger function in knock-in mice causes high-intensity TCR signaling and complete thymic deletion; the RING finger mutant c-Cbl protein itself recruits the p85 subunit of PI 3-kinase, activating Akt, whereas c-Cbl-null thymocytes do not show Akt activation—demonstrating a scaffolding/adaptor role for c-Cbl distinct from its E3 ligase function.","method":"Knock-in mouse expressing RING finger mutant c-Cbl, flow cytometry (CD5, CD69 markers), Western blotting (Erk, Akt, Ca2+ mobilization), co-immunoprecipitation (c-Cbl/p85)","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean knock-in model with biochemical and phenotypic characterization, multiple orthogonal assays distinguishing E3 from adaptor function","pmids":["16211006"],"is_preprint":false},{"year":2007,"finding":"c-Cbl ubiquitylates the LAT adapter downstream of TCR activation in a RING-finger-dependent manner, promoting internalization of LAT-containing signaling clusters into intracellular compartments; c-Cbl RING mutants and c-Cbl-null T cells show increased cellular LAT levels and increased basal and TCR-induced phospho-LAT.","method":"Fluorescence microscopy (YFP-tagged LAT), c-Cbl RING mutant expression, T cells from c-Cbl-null mice, ubiquitination assays, Western blotting","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RING mutants plus KO mouse T cells with multiple functional readouts; single lab","pmids":["17938199"],"is_preprint":false},{"year":2000,"finding":"c-Cbl colocalizes with Crk at submembranous actin lamellae in NIH 3T3 fibroblasts via SH3-binding sequences; truncation of c-Cbl's SH3-binding domain inhibits lamellipodia and membrane ruffle formation; this inhibitory effect is reversed by constitutively active Rac or by overexpression of wild-type c-Cbl, implicating c-Cbl in regulation of actin cytoskeletal organization via Crk and Rac.","method":"Immunofluorescence microscopy (colocalization with Crk and actin), truncation/mutagenesis constructs, cell morphology assays, constitutively active Rac rescue experiment","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — localization by immunofluorescence linked to functional phenotype (lamellipodia formation), mutagenesis and genetic rescue; single lab","pmids":["10633073"],"is_preprint":false},{"year":2000,"finding":"c-Cbl is a suppressor of oncogenic Neu/ErbB-2: ectopic c-Cbl causes ubiquitination and rapid removal of Neu from the cell surface and severely reduces signaling; this requires the carboxy-terminal domain of Neu and is antagonized by v-Cbl. In an in vivo neuroblastoma model, c-Cbl-encoding retrovirus caused enhanced Neu down-regulation and tumor retardation.","method":"Overexpression of c-Cbl and v-Cbl, ubiquitination assays, surface receptor quantification, retroviral infection of neuroblastoma tumor model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based ubiquitination and receptor degradation assays plus in vivo tumor model; single lab","pmids":["10940298"],"is_preprint":false},{"year":2009,"finding":"In Mycobacterium tuberculosis-infected macrophages, TNF activates a pathway (ASK1→p38→c-Abl) that phosphorylates FLIP(S), enabling FLIP(S) interaction with c-Cbl E3 ligase; c-Cbl then promotes proteasomal degradation of FLIP(S), which activates caspase-8 and apoptosis.","method":"Kinase inhibitors, co-immunoprecipitation (FLIP(S)–c-Cbl), c-Cbl-null macrophages, proteasome inhibitors, caspase-8 activation assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — KO macrophages, pharmacological dissection of pathway, co-IP complex, and functional caspase-8 apoptosis readout; multiple orthogonal methods","pmids":["19597496"],"is_preprint":false},{"year":2010,"finding":"c-Cbl facilitates CFTR endocytosis by an adaptor (ubiquitin ligase-independent) mechanism via its C-terminal region, and ubiquitinates CFTR in early endosomes to direct its lysosomal degradation; siRNA knockdown of c-Cbl increased plasma membrane CFTR and Cl- currents, while the adaptor-deficient (Cbl-480) but not the RING mutant (70Z-Cbl) blocked CFTR endocytosis.","method":"siRNA knockdown, dominant-negative/truncation constructs (70Z-Cbl, Cbl-480), co-immunoprecipitation, CFTR surface expression quantification, Cl- current measurement (electrophysiology)","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-specific constructs distinguishing adaptor from ligase function, siRNA KD, electrophysiology; single lab","pmids":["20525683"],"is_preprint":false},{"year":2016,"finding":"c-Cbl negatively regulates the antiviral innate immune response by interacting with IRF3 (via TKB domain of c-Cbl and IRF association domain of IRF3) and promoting K48-linked polyubiquitination and proteasomal degradation of IRF3, thereby suppressing IFN-β production.","method":"Co-immunoprecipitation (domain mapping), siRNA knockdown and overexpression, in vivo ubiquitination assay (K48-linkage), IFN-β reporter assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain-mapped Co-IP plus ubiquitin linkage and functional IFN-β assay; single lab","pmids":["27503123"],"is_preprint":false},{"year":2019,"finding":"c-Cbl ubiquitinates PD-1 in immune cells (CD8+ T cells and macrophages) via its RING finger domain, targeting PD-1 for proteasomal degradation; the C-terminus of c-Cbl interacts with the cytoplasmic tail of PD-1. Loss of c-Cbl (c-Cbl+/- mice) results in increased PD-1 levels and impaired macrophage tumor phagocytosis.","method":"Syngeneic CRC xenografts in c-Cbl+/- mice, co-immunoprecipitation (c-Cbl–PD-1 interaction), ubiquitination assays, RING mutant constructs, anti-PD-1 neutralizing antibody rescue of phagocytosis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, RING mutant, functional rescue, in vivo model; single lab","pmids":["31882749"],"is_preprint":false},{"year":2018,"finding":"c-Cbl acts as an E3 ligase for neddylation of c-Src; after neddylation, c-Src undergoes polyubiquitination and proteasomal degradation, suppressing the PI3K-AKT signaling pathway responsible for cancer cell migration.","method":"Neddylation assay, co-immunoprecipitation, neddylation blockade, c-Cbl overexpression/knockdown, cell migration assay, lung cancer tissue analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional neddylation and degradation assays with migration phenotype; single lab","pmids":["29899407"],"is_preprint":false},{"year":2002,"finding":"c-Cbl co-immunoprecipitates with Src and associates with Golgi membranes in multiple unstimulated cell types; approximately 10% of c-Cbl is membrane-associated, enriched in Golgi fractions; activated (but not wild-type) Src increases the amount of Src co-immunoprecipitating with c-Cbl at the Golgi, suggesting a Golgi-localized activated Src–c-Cbl complex.","method":"Confocal immunofluorescence (Golgi marker colocalization), subcellular fractionation (isopycnic density centrifugation and free-flow electrophoresis), co-immunoprecipitation, brefeldin A treatment","journal":"European journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — fractionation combined with colocalization and Co-IP, multiple methods; single lab","pmids":["11893076"],"is_preprint":false},{"year":2003,"finding":"c-Cbl macrophages (c-cbl-/-) exhibit impaired chemokinetic and chemotactic migration and altered actin cytoskeletal structures similar to hck-/-fgr-/- macrophages, placing c-Cbl tyrosine phosphorylation downstream of Src family kinases (Hck/Fgr) in a pathway controlling macrophage motility.","method":"c-cbl-/- macrophages (genetic KO), migration/chemotaxis assays, comparison with hck-/-fgr-/- macrophages, Cbl phosphorylation Western blotting","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined migration phenotype and epistatic comparison; single lab","pmids":["12652654"],"is_preprint":false},{"year":2019,"finding":"c-Cbl functions downstream of Dectin-2/Dectin-3 in dendritic cells to mediate ubiquitination and proteasomal degradation of the noncanonical NF-κB subunit RelB; c-Cbl deficiency in DCs promotes α-mannan-induced RelB activation, which suppresses IL-10 transcription and aggravates colitis. c-Cbl interacts with c-Abl tyrosine kinase in this pathway.","method":"DC-specific c-Cbl knockout mice, DSS colitis model, co-immunoprecipitation (c-Cbl–RelB and c-Cbl–c-Abl), ubiquitination assay, NF-κB reporter, IL-10 ELISA","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — DC-specific KO model, co-IP, ubiquitination assay, in vivo colitis phenotype; single lab","pmids":["33962939"],"is_preprint":false},{"year":2021,"finding":"CBL mutations found in myeloid neoplasms selectively abolish E3 ubiquitin ligase activity while enhancing interaction with LYN kinase; mutant CBL shows increased LYN-driven phosphorylation, PIK3R1 (p85) recruitment, and PI3K/AKT activation. All CBL adaptor domains (TKB, proline-rich region, C-terminal phosphotyrosines) are required for oncogenic function. LYN inhibition (dasatinib) reduces mutant CBL signaling in vitro and in vivo in CMML.","method":"Phosphoproteomics (global mass spectrometry), CBL interactome MS, functional assays with CBL allelic series, domain deletion/mutagenesis, LYN knockout/dasatinib inhibition, in vivo CMML xenograft","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — global phosphoproteomics plus functional mechanistic assays (mutagenesis, KO, inhibitor) and in vivo model; multiple orthogonal methods, novel mechanistic insight","pmids":["33512474"],"is_preprint":false},{"year":2011,"finding":"c-Cbl promotes mono-ubiquitination of integrin α3β1 and αVβ3 (but polyubiquitination of αVβ5) to direct selective translocation of KSHV and these receptors into lipid rafts for productive macropinocytic entry; c-Cbl knockdown diverts KSHV to clathrin-mediated lysosomal non-infectious pathway.","method":"siRNA knockdown of c-Cbl, lipid raft fractionation, ubiquitination assays (mono- vs. polyubiquitin), KSHV infection assays, methyl-β-cyclodextrin (lipid raft disruption)","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA KD, fractionation, ubiquitination assays, and infection readout; single lab","pmids":["21937638"],"is_preprint":false},{"year":2011,"finding":"c-Cbl ubiquitin ligase is activated by cathepsin G-induced EGFR transactivation in cardiac myocytes; c-Cbl then interacts with focal adhesion proteins and mediates their proteasomal (not lysosomal or calpain-dependent) ubiquitination and degradation, leading to myofibrillar degeneration and apoptosis.","method":"c-Cbl knockout-derived myocytes, EGFR kinase inhibitor, co-immunoprecipitation (c-Cbl–FA proteins), proteasome/lysosome/calpain inhibitors, ubiquitination assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO cells combined with pharmacological dissection and co-IP; single lab","pmids":["22203672"],"is_preprint":false},{"year":2005,"finding":"Crystal structures of CIN85 and beta-PIX SH3 domains in complex with a proline-arginine (PxxxPR) peptide from Cbl-b reveal a heterotrimeric complex in which two SH3 domains are held by a single pseudo-symmetrical peptide; this trimerization occurs in solution and in vivo, and ternary CIN85–Cbl complexes are required for Cbl-mediated EGFR downregulation.","method":"X-ray crystallography (SH3–peptide complex structures), solution studies, co-immunoprecipitation (in vivo ternary complex), EGFR downregulation assay","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structures validated by in vivo Co-IP and functional EGFR downregulation assay; multiple orthogonal methods","pmids":["16228008"],"is_preprint":false},{"year":2015,"finding":"c-Cbl and Cbl-b negatively regulate osteoblast differentiation by ubiquitinating the transcription factor Osterix, promoting its proteasome-mediated degradation and suppressing its transcriptional activity; this inhibits BMP2-induced osteoblast differentiation in mesenchymal cells.","method":"Overexpression/knockdown in mesenchymal cells, co-immunoprecipitation, ubiquitination assays, proteasome inhibitor rescue, osteoblast differentiation markers","journal":"Bone","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional differentiation phenotype; single lab","pmids":["25744063"],"is_preprint":false},{"year":2019,"finding":"c-Cbl promotes K48-linked polyubiquitination of TRAF6 via its proline-rich domain interaction with TRAF6, leading to TRAF6 degradation and inhibition of RANKL- and IL-1β-induced (but not TNFα-induced) NF-κB activation; RANKL or IFN-γ stimulation promotes c-Cbl binding to polyubiquitinated TRAF6.","method":"In vitro ubiquitination assay, co-immunoprecipitation with domain mapping, luciferase NF-κB reporter, in vivo ubiquitination assay in bone marrow macrophages/osteoclasts","journal":"Cellular & molecular biology letters","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro ubiquitination reconstitution combined with functional NF-κB assay and domain mapping; single lab with some validation in primary cells","pmids":["31123462"],"is_preprint":false},{"year":2002,"finding":"EGF stimulation induces a direct association between c-Cbl and tyrosine-phosphorylated PLC-γ1, mediated by the SH3 domain of PLC-γ1; PDGF, which also phosphorylates PLC-γ1, fails to induce this association and also fails to phosphorylate c-Cbl, indicating c-Cbl phosphorylation is required for PLC-γ1 interaction.","method":"Co-immunoprecipitation in vivo, GST pull-down and overlay assay (direct binding), comparison of EGF vs. PDGF stimulation, SH3 domain deletion mutant","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus GST pull-down for direct interaction, domain mutant, stimulus comparison; single lab","pmids":["12061819"],"is_preprint":false},{"year":2018,"finding":"c-Cbl regulation of nuclear β-catenin requires phosphorylation of c-Cbl Tyr371: the Y371H mutant interacts with but fails to ubiquitinate nuclear β-catenin, acts as a dominant negative, and in a Wnt-8 zebrafish model augments Wnt/β-catenin signaling and tumor growth.","method":"Co-immunoprecipitation, ubiquitination assay, Wnt reporter assay, Wnt-8 transgenic zebrafish model, colorectal cancer cell lines","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus ubiquitination assay and in vivo zebrafish model; single lab","pmids":["30029779"],"is_preprint":false},{"year":2004,"finding":"TULA (an SH3- and UBA-containing protein) binds c-Cbl and ubiquitin; TULA inhibits c-Cbl-mediated EGFR downregulation and, in Jurkat T cells, upregulates ZAP-70 and NF-AT activity; TULA-mediated inhibition involves TULA-induced ubiquitylation and degradation of c-Cbl itself.","method":"Affinity chromatography and mass spectrometry (c-Cbl-interacting protein identification), co-immunoprecipitation, EGFR degradation assay, TULA modulation in Jurkat cells, NF-AT reporter assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction, functional assays in two cell systems; single lab","pmids":["15107835"],"is_preprint":false},{"year":2003,"finding":"Ephrin-A1 stimulation induces EphA3 receptor tyrosine phosphorylation that leads to c-Cbl phosphorylation in a Src-family kinase-dependent manner; phosphorylated c-Cbl associates with Crk-L and Crk-II (but not Grb2); overexpression of wild-type c-Cbl (but not 70Z mutant) down-regulates EphA receptor expression.","method":"Phosphotyrosine immunoblotting, co-immunoprecipitation, Src inhibitor (PP2) treatment, c-Cbl overexpression/70Z mutant, EphA surface expression assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, pharmacological epistasis, and receptor down-regulation assay; single lab","pmids":["12794130"],"is_preprint":false},{"year":2003,"finding":"c-Cbl down-regulates the Ron receptor tyrosine kinase: MSP stimulation recruits c-Cbl to Ron's multifunctional docking site and to a juxtamembrane autophosphorylation site; c-Cbl mediates Ron polyubiquitylation followed by endocytosis and degradation; both TKB domain and RING domain are required; Ron–Grb2–c-Cbl complexes alone are insufficient for productive ubiquitylation.","method":"Co-immunoprecipitation, domain mutagenesis (TKB and RING), ubiquitylation assay, receptor degradation and endocytosis assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain mutagenesis plus ubiquitination and degradation assays; single lab","pmids":["12802274"],"is_preprint":false},{"year":2003,"finding":"c-Cbl negatively regulates platelet activation through GpVI: phosphorylation of c-Cbl in GpVI-signaled platelets requires upstream Src-family kinases Fyn and Lyn; in c-Cbl-null platelets, phosphorylation of FcRγ-chain, Syk, and PLCγ2 is increased and aggregation in response to GpVI agonist is potentiated.","method":"c-Cbl knockout mouse platelets, Fyn/Lyn-null platelets for epistasis, Western blotting (phosphorylation), platelet aggregation assays","journal":"Journal of thrombosis and haemostasis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with defined biochemical phenotype and functional aggregation assay; epistatic placement by Fyn/Lyn-null comparison","pmids":["14629478"],"is_preprint":false}],"current_model":"c-Cbl is a multifunctional RING-type E3 ubiquitin ligase and adaptor protein that recognizes activated (tyrosine-phosphorylated) receptor and non-receptor tyrosine kinases through its TKB/SH2 domain and recruits E2 ubiquitin-conjugating enzymes through its RING domain to catalyze substrate ubiquitination and degradation; its activity is regulated by autoinhibition and phosphorylation of Tyr371 in the linker helix region which relieves autoinhibition and activates the RING domain; beyond ubiquitination, c-Cbl acts as an adaptor to recruit PI3K/p85, Grb2, and Crk, influencing signaling via Rac/Vav, Akt, and other pathways, and it also possesses NEDD8 E3 ligase (neddylation) activity that stabilizes certain substrates (TβRII, c-Src); together these activities regulate receptor downregulation (EGFR, ErbB, KIT, Flt-1, gp130, EphA, Ron), T-cell and NK-cell signaling thresholds, bone resorption downstream of Src, innate immune responses (IRF3, RelB, FLIP(S), PD-1), and actin cytoskeletal organization."},"narrative":{"mechanistic_narrative":"c-Cbl (CBL) is a RING-type, E2-dependent E3 ubiquitin ligase and signaling adaptor that enforces negative feedback on activated tyrosine-kinase signaling by recognizing phosphotyrosine substrates and targeting them for ubiquitination and lysosomal or proteasomal degradation [PMID:10514377]. It binds tyrosine-phosphorylated substrates through its SH2/TKB domain and recruits and allosterically activates an E2 conjugating enzyme (e.g., UbcH7) through its RING domain, with rigid coupling between the substrate- and E2-binding surfaces positioning ubiquitin for transfer [PMID:10514377, PMID:10966114]. Catalytic output is gated by autoinhibition: in the resting state the RING E2-binding surface folds back intramolecularly, and phosphorylation of Tyr371 in the linker-helix region relieves this autoinhibition, repositions the RING and bound E2 toward the substrate, and is required for receptor tyrosine kinase ubiquitination [PMID:22266821]. Through these activities c-Cbl drives ligand-induced downregulation of receptor tyrosine kinases including ErbB-1/EGFR, Neu/ErbB-2, KIT, Flt-1, EphA, and Ron, controlling their endosomal sorting toward degradation rather than recycling [PMID:9851973, PMID:10940298, PMID:15315962, PMID:12802274]; CIN85/CD2AP adaptors and the SH3-mediated ternary complexes they form are required for this receptor downregulation [PMID:16228008, PMID:15001553]. In hematopoietic and immune cells c-Cbl sets activation thresholds by negatively regulating Syk/ZAP-70-family signaling, promoting clearance of engaged TCR and ubiquitination of LAT and Vav, and downmodulating innate signaling components including IRF3, RelB, TRAF6, and PD-1 [PMID:9671496, PMID:12415267, PMID:17938199, PMID:12881521, PMID:27503123, PMID:33962939, PMID:31123462, PMID:31882749]. Beyond catalysis, c-Cbl serves as a scaffold that recruits PI3K/p85, Grb2, and Crk, a non-catalytic function genetically separable from its E3 activity that activates Akt and shapes actin cytoskeletal organization downstream of Src-family kinases [PMID:16211006, PMID:7791764, PMID:10633073, PMID:12652654]. c-Cbl also acts downstream of c-Src in osteoclast-mediated bone resorption [PMID:8849724] and possesses NEDD8 E3 (neddylation) activity toward TβRII and c-Src, which alters their trafficking and stability [PMID:23290524, PMID:29899407]. CBL mutations in myeloid neoplasms selectively abolish E3 ligase activity while enhancing LYN-driven phosphorylation, p85 recruitment, and PI3K/AKT signaling, defining an oncogenic gain-of-adaptor mechanism [PMID:33512474].","teleology":[{"year":1996,"claim":"Established c-Cbl as a functional effector downstream of c-Src in a defined physiological process, before its enzymatic identity was known.","evidence":"Antisense knockdown, co-IP, immunofluorescence, and in vitro bone resorption with src-null osteoclasts","pmids":["8849724"],"confidence":"High","gaps":["Molecular mechanism of c-Cbl action in resorption not defined","Substrate(s) of c-Cbl in osteoclasts not identified"]},{"year":1998,"claim":"Showed that c-Cbl physically mediates receptor ubiquitination and degradative endosomal sorting, reframing it from a passive adaptor to an active downregulator of RTKs.","evidence":"Endosomal colocalization, ubiquitination IP, and oncogenic v-Cbl rescue in ErbB-1 trafficking","pmids":["9851973"],"confidence":"High","gaps":["Enzymatic mechanism of ubiquitin transfer not yet established","Did not define E2 partner or RING requirement"]},{"year":1998,"claim":"Genetically defined c-Cbl as a negative regulator of T-cell Syk/ZAP-70 kinase signaling, establishing its immune threshold-setting role.","evidence":"c-Cbl knockout mice with ZAP-70 phosphorylation readouts and lymphoid phenotyping","pmids":["9671496"],"confidence":"High","gaps":["Whether the effect required E3 activity vs adaptor function unresolved at this stage"]},{"year":1999,"claim":"Defined the core biochemical identity of c-Cbl as a RING-type, E2-dependent E3 ligase that reads phosphotyrosine substrates and allosterically activates a conjugating enzyme.","evidence":"In vitro ubiquitination reconstitution with PDGFR substrate plus SH2 and RING mutagenesis","pmids":["10514377"],"confidence":"High","gaps":["Structural basis of E2 recruitment not yet visualized","Regulation of activity in cells not addressed"]},{"year":2000,"claim":"Provided the structural logic for RING E3 catalysis, showing rigid substrate-to-E2 coupling that scaffolds ubiquitin transfer.","evidence":"X-ray crystal structure of the c-Cbl–UbcH7–kinase peptide ternary complex","pmids":["10966114"],"confidence":"High","gaps":["Did not capture the autoinhibited or activated conformations","Phosphoregulation not represented in structure"]},{"year":2000,"claim":"Demonstrated c-Cbl is a tumor suppressor of an oncogenic RTK and that its catalytic downregulation activity has anti-tumor consequences in vivo.","evidence":"c-Cbl/v-Cbl overexpression, ubiquitination and surface-receptor assays, and a neuroblastoma tumor model","pmids":["10940298"],"confidence":"Medium","gaps":["Endogenous-level regulation not tested","Mechanism of v-Cbl antagonism only inferred"]},{"year":2003,"claim":"Revealed an upstream OFF-switch in which Cdc42/beta-PIX sequesters c-Cbl away from EGFR, linking GTPase signaling to control of receptor downregulation and transformation.","evidence":"Reciprocal co-IP, dominant-active Cdc42, ubiquitination/ERK assays, and transformation phenotype","pmids":["14505571"],"confidence":"High","gaps":["Stoichiometry and dynamics of the ternary complex in vivo not defined"]},{"year":2005,"claim":"Genetically separated c-Cbl's E3 ligase function from a distinct PI3K/Akt-recruiting adaptor function, establishing dual-mode action.","evidence":"RING-finger mutant knock-in mice with thymic phenotype and c-Cbl/p85 co-IP","pmids":["16211006"],"confidence":"High","gaps":["Direct vs indirect p85 recruitment not fully resolved","Generality of adaptor role across cell types not tested here"]},{"year":2012,"claim":"Resolved how c-Cbl activity is switched on, showing Tyr371 phosphorylation relieves an autoinhibited RING conformation required for RTK ubiquitination.","evidence":"Crystal structures of unphosphorylated, substrate-bound, and pTyr371 activated complexes with biochemical validation","pmids":["22266821"],"confidence":"High","gaps":["Kinetics of conformational switching in cells not measured","Which kinases phosphorylate Tyr371 in each context not unified"]},{"year":2013,"claim":"Expanded c-Cbl's enzymatic repertoire to NEDD8 conjugation, showing it can stabilize rather than degrade a substrate by altering its trafficking.","evidence":"In vitro neddylation, site mutagenesis (K556R/K567R), KO cells, and endosomal/reporter assays for TβRII","pmids":["23290524"],"confidence":"High","gaps":["How c-Cbl chooses ubiquitin vs NEDD8 outputs not defined","E2/NEDD8 machinery details not fully resolved"]},{"year":2021,"claim":"Defined the oncogenic mechanism of CBL myeloid mutations as loss of E3 activity coupled to gain of LYN-driven adaptor signaling through PI3K/AKT.","evidence":"Phosphoproteomics, interactome MS, CBL allelic series, LYN KO/dasatinib, and CMML xenografts","pmids":["33512474"],"confidence":"High","gaps":["Full set of dysregulated substrates in patients not enumerated","Therapeutic durability of LYN inhibition not established"]},{"year":null,"claim":"How c-Cbl integrates its competing E3-ubiquitin, NEDD8-ligase, and non-catalytic adaptor outputs to produce context-specific substrate fates remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model selecting degradation vs stabilization vs signaling","Determinants of mono- vs K48- vs K63-linked ubiquitination unclear","Tissue-specific substrate repertoire incompletely mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,10,29]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,16,33]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[21,7,23,26]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[8,9,44]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[2,13,18]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[30]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,24,26]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[23,31]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,6,16]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[8,9,27,28,32]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[2,9,14,18]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,10,29]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[33,24,40]}],"complexes":[],"partners":["SRC","GRB2","CRK","PIK3R1","CIN85","LYN","CD2AP","EGFR"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P22681","full_name":"E3 ubiquitin-protein ligase CBL","aliases":["Casitas B-lineage lymphoma proto-oncogene","Proto-oncogene c-Cbl","RING finger protein 55","RING-type E3 ubiquitin transferase CBL","Signal transduction protein CBL"],"length_aa":906,"mass_kda":99.6,"function":"E3 ubiquitin-protein ligase that acts as a negative regulator of many signaling pathways by mediating ubiquitination of cell surface receptors (PubMed:10514377, PubMed:11896602, PubMed:14661060, PubMed:14739300, PubMed:15190072, PubMed:17509076, PubMed:18374639, PubMed:19689429, PubMed:21596750, PubMed:28381567, PubMed:40101708). Accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, and then transfers it to substrates promoting their degradation by the proteasome (PubMed:10514377, PubMed:14661060, PubMed:14739300, PubMed:17094949, PubMed:17509076, PubMed:17974561). Recognizes activated receptor tyrosine kinases, including KIT, FLT1, FGFR1, FGFR2, PDGFRA, PDGFRB, CSF1R, EPHA8 and KDR and mediates their ubiquitination to terminate signaling (PubMed:15190072, PubMed:18374639, PubMed:21596750). Recognizes membrane-bound HCK, SRC and other kinases of the SRC family and mediates their ubiquitination and degradation (PubMed:11896602). Ubiquitinates EGFR and SPRY2 (PubMed:17094949, PubMed:17974561). Involved in LAG3-mediated inhibition of TCR signaling: following ligand-binding to LAG3, catalyzes 'Lys-63'-linked ubiquitination of LAG3, unleashing the LAG3 C-terminus from the membrane, and initiating a signaling that prevents TCR activation (PubMed:40101708). Ubiquitinates NECTIN1 following association between NECTIN1 and herpes simplex virus 1/HHV-1 envelope glycoprotein D, leading to NECTIN1 removal from cell surface (PubMed:28381567). Participates in signal transduction in hematopoietic cells. Plays an important role in the regulation of osteoblast differentiation and apoptosis (PubMed:15190072, PubMed:18374639). Essential for osteoclastic bone resorption (PubMed:14739300). The 'Tyr-731' phosphorylated form induces the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function (PubMed:14739300). In association with CBLB, required for proper feedback inhibition of ciliary platelet-derived growth factor receptor-alpha (PDGFRA) signaling pathway via ubiquitination and internalization of PDGFRA (By similarity)","subcellular_location":"Cytoplasm; Cell membrane; Cell projection, cilium; Golgi apparatus","url":"https://www.uniprot.org/uniprotkb/P22681/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CBL","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CBL","total_profiled":1310},"omim":[{"mim_id":"620953","title":"METHYLMALONIC ACIDURIA, cblD TYPE; MACD","url":"https://www.omim.org/entry/620953"},{"mim_id":"620952","title":"HOMOCYSTINURIA-MEGALOBLASTIC ANEMIA, cblD TYPE; HMAD","url":"https://www.omim.org/entry/620952"},{"mim_id":"618882","title":"IMERSLUND-GRASBECK SYNDROME 2; IGS2","url":"https://www.omim.org/entry/618882"},{"mim_id":"617322","title":"SH3KBP1-BINDING PROTEIN 1; SHKBP1","url":"https://www.omim.org/entry/617322"},{"mim_id":"614902","title":"RHO GTPase-ACTIVATING PROTEIN 33; ARHGAP33","url":"https://www.omim.org/entry/614902"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Mitotic spindle","reliability":"Additional"},{"location":"End piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CBL"},"hgnc":{"alias_symbol":["RNF55","c-Cbl"],"prev_symbol":["CBL2"]},"alphafold":{"accession":"P22681","domains":[{"cath_id":"1.20.930.20","chopping":"49-175","consensus_level":"medium","plddt":93.8937,"start":49,"end":175},{"cath_id":"1.10.238.10","chopping":"177-264","consensus_level":"medium","plddt":94.2905,"start":177,"end":264},{"cath_id":"3.30.40.10","chopping":"358-431","consensus_level":"medium","plddt":85.7719,"start":358,"end":431},{"cath_id":"1.10.8.10","chopping":"859-906","consensus_level":"medium","plddt":82.3769,"start":859,"end":906}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P22681","model_url":"https://alphafold.ebi.ac.uk/files/AF-P22681-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P22681-F1-predicted_aligned_error_v6.png","plddt_mean":62.84},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CBL","jax_strain_url":"https://www.jax.org/strain/search?query=CBL"},"sequence":{"accession":"P22681","fasta_url":"https://rest.uniprot.org/uniprotkb/P22681.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P22681/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P22681"}},"corpus_meta":[{"pmid":"10514377","id":"PMC_10514377","title":"The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase.","date":"1999","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/10514377","citation_count":863,"is_preprint":false},{"pmid":"10966114","id":"PMC_10966114","title":"Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases.","date":"2000","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/10966114","citation_count":722,"is_preprint":false},{"pmid":"9851973","id":"PMC_9851973","title":"c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor.","date":"1998","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/9851973","citation_count":711,"is_preprint":false},{"pmid":"11836526","id":"PMC_11836526","title":"Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex.","date":"2002","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11836526","citation_count":700,"is_preprint":false},{"pmid":"11283727","id":"PMC_11283727","title":"Cbl: many adaptations to regulate protein tyrosine kinases.","date":"2001","source":"Nature reviews. 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\"method\": \"In vitro ubiquitination assay, domain mutagenesis (SH2 and RING finger), biochemical reconstitution\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with defined substrate, allosteric activation mechanism demonstrated, replicated across multiple subsequent studies\",\n      \"pmids\": [\"10514377\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Crystal structure of c-Cbl bound to E2 (UbcH7) and a kinase substrate peptide revealed that the RING domain recruits E2 through a conserved surface, and there is rigid coupling between the substrate-binding and E2-binding domains with a conserved channel from substrate to E2 active site, suggesting RING E3s scaffold substrate and E2 for ubiquitin transfer.\",\n      \"method\": \"X-ray crystallography (crystal structure of c-Cbl–UbcH7–kinase peptide ternary complex)\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with functional validation, foundational structural paper widely replicated\",\n      \"pmids\": [\"10966114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"c-Cbl is transiently recruited into ErbB-1-containing endosomes in a receptor kinase activity-dependent manner, mediates covalent ubiquitination of ErbB-1, and directs internalized ErbB-1 to lysosomal and proteasomal degradation rather than recycling; oncogenic v-Cbl inhibits this by shunting receptors to the recycling pathway.\",\n      \"method\": \"Immunofluorescence microscopy (endosomal colocalization), immunoprecipitation (ubiquitin attachment), dominant-negative/oncogenic Cbl expression, degradation assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (colocalization, IP, functional rescue), independently replicated\",\n      \"pmids\": [\"9851973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"c-Cbl adopts an autoinhibited RING conformation in which the RING's E2-binding surface associates intramolecularly with CBL to reduce E2 affinity; phosphorylation of Tyr371 in the linker helix region (LHR) induces conformational changes that eliminate autoinhibition, flip the RING domain and E2 into proximity of the substrate-binding site, and enhance E2 binding—this activation is required for RTK ubiquitination.\",\n      \"method\": \"X-ray crystallography (structures of unphosphorylated CBL, CBL–substrate peptide, phospho-Tyr371-CBL–E2–substrate), biochemical activity assays, mutagenesis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures plus biochemical validation and mutagenesis in a single rigorous study\",\n      \"pmids\": [\"22266821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"c-Cbl is tyrosine-phosphorylated downstream of c-Src in osteoclasts and the two proteins colocalize on vesicular structures; antisense knockdown of either c-src or c-cbl inhibits in vitro bone resorption by osteoclast-like cells, placing c-Cbl downstream of c-Src in a pathway required for osteoclast-mediated bone resorption.\",\n      \"method\": \"Antisense oligonucleotide knockdown, immunoprecipitation, immunofluorescence colocalization, in vitro bone resorption assay with src-null OCLs\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis (antisense KD + src-null cells), multiple orthogonal methods, replicated by subsequent studies\",\n      \"pmids\": [\"8849724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Src-catalyzed phosphorylation of c-Cbl (likely on Tyr-371) is required for c-Cbl-dependent ubiquitination of both c-Src itself and c-Cbl (auto-ubiquitination); this requires the RING finger of c-Cbl. Active Src destabilizes the c-Cbl–UbcH7 complex in vitro. c-Cbl reduces v-Src protein levels and suppresses v-Src-induced STAT3 activation.\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation, kinase activity assays, overexpression/mutagenesis in cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination reconstitution, mutagenesis, and cell-based assays in a single study\",\n      \"pmids\": [\"11448952\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Activated Cdc42 binds to p85Cool-1/beta-PIX, which directly associates with c-Cbl, thereby inhibiting c-Cbl binding to the EGF receptor and preventing Cbl-catalyzed receptor ubiquitination; constitutively active Cdc42(F28L) persistently blocks EGF receptor–Cbl interaction, leading to aberrant receptor accumulation, sustained ERK activation, and cellular transformation.\",\n      \"method\": \"Co-immunoprecipitation, dominant-active Cdc42 expression, ubiquitination assays, ERK activation assays, cell transformation assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP establishing ternary complex, functional ubiquitination assay, transformation phenotype, multiple orthogonal readouts\",\n      \"pmids\": [\"14505571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"In TCR-activated Jurkat T cells, c-Cbl constitutively associates with the Grb2 adaptor via Grb2's N-terminal SH3 domain; after TCR–CD3 plus CD4 co-stimulation, Cbl also binds Grb2's SH2 domain. TCR activation recruits PI 3-kinase activity to Cbl immunocomplexes. Cbl–Grb2 and Sos–Grb2 exist as distinct complexes.\",\n      \"method\": \"Co-immunoprecipitation with domain-specific Grb2 fusion proteins, PI 3-kinase activity assay in immunoprecipitates\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP with domain specificity mapping, single lab, functional PI3K assay but no reconstitution\",\n      \"pmids\": [\"7791764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"In c-Cbl-deficient mice, ZAP-70 kinase phosphorylation in thymocytes is uncoupled from the requirement for CD4-mediated Lck activation following CD3ε cross-linking, demonstrating that c-Cbl negatively regulates signaling by the Syk/ZAP-70 family of protein kinases in T cells; c-Cbl-null mice also display lymphoid hyperplasia and altered T-cell receptor expression.\",\n      \"method\": \"Targeted gene disruption (c-Cbl knockout mice), flow cytometry, immunoprecipitation/Western blot for ZAP-70 phosphorylation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean genetic knockout with defined biochemical phenotype (ZAP-70 phosphorylation), replicated by subsequent studies\",\n      \"pmids\": [\"9671496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"c-Cbl and Cbl-b double-knockout T cells fail to down-modulate surface TCR after ligand engagement, resulting in sustained TCR signaling and T-cell hyperresponsiveness; trafficking of internalized TCR to lysosomes was reduced, while ligand-independent internalization was normal. This demonstrates Cbl proteins negatively regulate T-cell activation by promoting clearance of engaged TCR from the cell surface.\",\n      \"method\": \"c-Cbl/Cbl-b double-knockout mouse T cells, flow cytometry (TCR surface levels), T-cell activation assays, trafficking assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — double-KO genetic model, multiple functional readouts (TCR surface levels, signaling duration, lysosomal trafficking)\",\n      \"pmids\": [\"12415267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"c-Cbl acts as a NEDD8 E3 ligase (neddylation E3) for the TGF-β type II receptor (TβRII): it conjugates NEDD8 to TβRII at Lys556 and Lys567. Neddylation promotes TβRII endocytosis to EEA1-positive early endosomes and prevents its endocytosis to caveolin-positive (degradative) compartments, thereby inhibiting TβRII ubiquitination and degradation and stabilizing/enhancing TGF-β signaling.\",\n      \"method\": \"Co-immunoprecipitation, in vitro neddylation assay, site-directed mutagenesis (K556R/K567R), c-Cbl knockout hematopoietic cells, endosomal colocalization assays, TGF-β pathway reporter assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro neddylation reconstitution, site mutagenesis, KO cells, and multiple orthogonal assays in a single study\",\n      \"pmids\": [\"23290524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"DNA damage leads to ATM-mediated phosphorylation and stabilization of c-Cbl, which in turn promotes TβRII neddylation and prevents its ubiquitination-dependent degradation, thus stabilizing TβRII and enhancing TGF-β signaling; ATM, c-Cbl, and TβRII form a ternary complex upon DNA damage.\",\n      \"method\": \"Co-immunoprecipitation (ATM-c-Cbl-TβRII complex), Western blotting (TβRII stability), mouse intestinal regeneration model after X-ray irradiation\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating ternary complex, in vivo mouse model, but largely extends prior neddylation finding (PMID 23290524)\",\n      \"pmids\": [\"31315051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"c-Cbl binds tyrosine-phosphorylated transmembrane Notch1 (by co-immunoprecipitation) and mediates ubiquitin-dependent targeting of the transmembrane form of Notch1 to lysosomal compartments for degradation in skeletal myoblasts.\",\n      \"method\": \"Co-immunoprecipitation, lysosomal inhibitor (chloroquine) treatment, proteasome inhibitor comparison, ubiquitin accumulation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and pharmacological inhibitor data, single lab, no direct ubiquitination reconstitution shown\",\n      \"pmids\": [\"11777909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"c-Cbl mediates mono-ubiquitination of activated PAR2 (a GPCR) in an Src-dependent manner, directing it from early endosomes to lysosomes for degradation; dominant-negative c-Cbl (RING-deleted) blocks PAR2 ubiquitination and lysosomal degradation, causing receptor retention in early endosomes and allowing recycling and continued signaling.\",\n      \"method\": \"Immunoprecipitation, immunofluorescence, dominant-negative c-Cbl expression, lysosomal degradation assays, receptor signaling recovery assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative and mutagenesis approaches combined with trafficking assays, single lab\",\n      \"pmids\": [\"15708858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"IL-6 stimulation induces c-Cbl-mediated K63-linked polyubiquitination of the cytokine receptor gp130; c-Cbl is recruited to gp130 via phosphorylated SHP2. Ubiquitinated gp130 is recognized by Hrs and targeted for lysosomal degradation. Deficiency of c-Cbl suppresses gp130 degradation and prolongs IL-6 signaling.\",\n      \"method\": \"Immunoprecipitation (c-Cbl–SHP2–gp130 complex), ubiquitin linkage analysis, c-Cbl siRNA knockdown, lysosomal degradation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD phenotype plus complex mapping and ubiquitin linkage analysis, single lab\",\n      \"pmids\": [\"18519587\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"APS acts as an adaptor linking the insulin receptor to c-Cbl: insulin-stimulated phosphorylation of APS Tyr618 enables APS to associate with c-Cbl, facilitating c-Cbl phosphorylation by the insulin receptor at Tyr371, Tyr700, and Tyr774, which drives c-Cbl–Crk association and is required for GLUT4 translocation.\",\n      \"method\": \"Overexpression of wild-type and Y618F APS mutant in 3T3-L1 adipocytes, co-immunoprecipitation, GLUT4 translocation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific mutagenesis with functional GLUT4 readout, co-IP complex mapping, single lab\",\n      \"pmids\": [\"11997497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Upon SCF stimulation, activated KIT receptor binds and phosphorylates Cbl proteins (c-Cbl and Cbl-b), which in turn ubiquitinate KIT and themselves, mediating their mutual degradation; TKB and RING finger domains of Cbl are essential for this negative feedback loop.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, domain deletion/mutagenesis, degradation assays in mast cells\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis combined with ubiquitination and degradation assays, single lab\",\n      \"pmids\": [\"15315962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"VEGF stimulation induces formation of a ternary Flt-1–c-Cbl–CD2AP complex; c-Cbl binds to pTyr1333 of Flt-1; wild-type CD2AP promotes ubiquitination and internalization/degradation of Flt-1, whereas dominant-negative CD2AP or mutant c-Cbl prevents this.\",\n      \"method\": \"Co-immunoprecipitation (ternary complex), site-directed mutagenesis (Y1333F Flt-1), dominant-negative CD2AP and c-Cbl, internalization/ubiquitination assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis at receptor binding site, dominant-negative constructs, and functional ubiquitination/internalization assays; single lab\",\n      \"pmids\": [\"15001553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"c-Cbl-mediated ubiquitination is required for EGFR exit from early endosomes but not for its initial internalization; Src-inhibitor PP1 blocks Cbl-mediated ubiquitination and retains EGFR in early endosomes.\",\n      \"method\": \"Src inhibitor (PP1) treatment, mutant EGFR (Y1045F) expression, EGFR trafficking and ubiquitination assays in CHO and A549 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological and mutant-receptor approaches with defined trafficking phenotype, single lab\",\n      \"pmids\": [\"15210722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"c-Cbl mediates ubiquitination and proteasomal degradation of STAT5 in response to GH stimulation, thereby negatively regulating GH-stimulated STAT5-mediated transcription; proteasome inhibitor MG132 reverses this effect.\",\n      \"method\": \"Overexpression of c-Cbl in NIH3T3 cells, ubiquitination assays, STAT5 reporter assay, proteasome inhibitor treatment\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — overexpression plus pharmacological rescue and functional readout, single lab, no reconstitution\",\n      \"pmids\": [\"12193575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"c-Cbl mediates Vav ubiquitination via Cbl RING finger activity; this requires Cbl–Vav association through phosphorylated Tyr-700 on Cbl. Cbl-dependent ubiquitination leads to loss of phosphorylated Vav protein, and c-Cbl but not its ubiquitin ligase mutant inhibits Vav-dependent signaling in Jurkat cells.\",\n      \"method\": \"Ubiquitination assays in Cbl+/+ and Cbl−/− T cells, co-immunoprecipitation, mutagenesis (RING finger and pTyr-700), Jurkat transfection with functional reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cell lines, site mutagenesis, ubiquitination assays; single lab\",\n      \"pmids\": [\"12881521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Loss of c-Cbl RING finger function in knock-in mice causes high-intensity TCR signaling and complete thymic deletion; the RING finger mutant c-Cbl protein itself recruits the p85 subunit of PI 3-kinase, activating Akt, whereas c-Cbl-null thymocytes do not show Akt activation—demonstrating a scaffolding/adaptor role for c-Cbl distinct from its E3 ligase function.\",\n      \"method\": \"Knock-in mouse expressing RING finger mutant c-Cbl, flow cytometry (CD5, CD69 markers), Western blotting (Erk, Akt, Ca2+ mobilization), co-immunoprecipitation (c-Cbl/p85)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean knock-in model with biochemical and phenotypic characterization, multiple orthogonal assays distinguishing E3 from adaptor function\",\n      \"pmids\": [\"16211006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"c-Cbl ubiquitylates the LAT adapter downstream of TCR activation in a RING-finger-dependent manner, promoting internalization of LAT-containing signaling clusters into intracellular compartments; c-Cbl RING mutants and c-Cbl-null T cells show increased cellular LAT levels and increased basal and TCR-induced phospho-LAT.\",\n      \"method\": \"Fluorescence microscopy (YFP-tagged LAT), c-Cbl RING mutant expression, T cells from c-Cbl-null mice, ubiquitination assays, Western blotting\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RING mutants plus KO mouse T cells with multiple functional readouts; single lab\",\n      \"pmids\": [\"17938199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"c-Cbl colocalizes with Crk at submembranous actin lamellae in NIH 3T3 fibroblasts via SH3-binding sequences; truncation of c-Cbl's SH3-binding domain inhibits lamellipodia and membrane ruffle formation; this inhibitory effect is reversed by constitutively active Rac or by overexpression of wild-type c-Cbl, implicating c-Cbl in regulation of actin cytoskeletal organization via Crk and Rac.\",\n      \"method\": \"Immunofluorescence microscopy (colocalization with Crk and actin), truncation/mutagenesis constructs, cell morphology assays, constitutively active Rac rescue experiment\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — localization by immunofluorescence linked to functional phenotype (lamellipodia formation), mutagenesis and genetic rescue; single lab\",\n      \"pmids\": [\"10633073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"c-Cbl is a suppressor of oncogenic Neu/ErbB-2: ectopic c-Cbl causes ubiquitination and rapid removal of Neu from the cell surface and severely reduces signaling; this requires the carboxy-terminal domain of Neu and is antagonized by v-Cbl. In an in vivo neuroblastoma model, c-Cbl-encoding retrovirus caused enhanced Neu down-regulation and tumor retardation.\",\n      \"method\": \"Overexpression of c-Cbl and v-Cbl, ubiquitination assays, surface receptor quantification, retroviral infection of neuroblastoma tumor model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based ubiquitination and receptor degradation assays plus in vivo tumor model; single lab\",\n      \"pmids\": [\"10940298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In Mycobacterium tuberculosis-infected macrophages, TNF activates a pathway (ASK1→p38→c-Abl) that phosphorylates FLIP(S), enabling FLIP(S) interaction with c-Cbl E3 ligase; c-Cbl then promotes proteasomal degradation of FLIP(S), which activates caspase-8 and apoptosis.\",\n      \"method\": \"Kinase inhibitors, co-immunoprecipitation (FLIP(S)–c-Cbl), c-Cbl-null macrophages, proteasome inhibitors, caspase-8 activation assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO macrophages, pharmacological dissection of pathway, co-IP complex, and functional caspase-8 apoptosis readout; multiple orthogonal methods\",\n      \"pmids\": [\"19597496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"c-Cbl facilitates CFTR endocytosis by an adaptor (ubiquitin ligase-independent) mechanism via its C-terminal region, and ubiquitinates CFTR in early endosomes to direct its lysosomal degradation; siRNA knockdown of c-Cbl increased plasma membrane CFTR and Cl- currents, while the adaptor-deficient (Cbl-480) but not the RING mutant (70Z-Cbl) blocked CFTR endocytosis.\",\n      \"method\": \"siRNA knockdown, dominant-negative/truncation constructs (70Z-Cbl, Cbl-480), co-immunoprecipitation, CFTR surface expression quantification, Cl- current measurement (electrophysiology)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-specific constructs distinguishing adaptor from ligase function, siRNA KD, electrophysiology; single lab\",\n      \"pmids\": [\"20525683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"c-Cbl negatively regulates the antiviral innate immune response by interacting with IRF3 (via TKB domain of c-Cbl and IRF association domain of IRF3) and promoting K48-linked polyubiquitination and proteasomal degradation of IRF3, thereby suppressing IFN-β production.\",\n      \"method\": \"Co-immunoprecipitation (domain mapping), siRNA knockdown and overexpression, in vivo ubiquitination assay (K48-linkage), IFN-β reporter assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-mapped Co-IP plus ubiquitin linkage and functional IFN-β assay; single lab\",\n      \"pmids\": [\"27503123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"c-Cbl ubiquitinates PD-1 in immune cells (CD8+ T cells and macrophages) via its RING finger domain, targeting PD-1 for proteasomal degradation; the C-terminus of c-Cbl interacts with the cytoplasmic tail of PD-1. Loss of c-Cbl (c-Cbl+/- mice) results in increased PD-1 levels and impaired macrophage tumor phagocytosis.\",\n      \"method\": \"Syngeneic CRC xenografts in c-Cbl+/- mice, co-immunoprecipitation (c-Cbl–PD-1 interaction), ubiquitination assays, RING mutant constructs, anti-PD-1 neutralizing antibody rescue of phagocytosis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, RING mutant, functional rescue, in vivo model; single lab\",\n      \"pmids\": [\"31882749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"c-Cbl acts as an E3 ligase for neddylation of c-Src; after neddylation, c-Src undergoes polyubiquitination and proteasomal degradation, suppressing the PI3K-AKT signaling pathway responsible for cancer cell migration.\",\n      \"method\": \"Neddylation assay, co-immunoprecipitation, neddylation blockade, c-Cbl overexpression/knockdown, cell migration assay, lung cancer tissue analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional neddylation and degradation assays with migration phenotype; single lab\",\n      \"pmids\": [\"29899407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"c-Cbl co-immunoprecipitates with Src and associates with Golgi membranes in multiple unstimulated cell types; approximately 10% of c-Cbl is membrane-associated, enriched in Golgi fractions; activated (but not wild-type) Src increases the amount of Src co-immunoprecipitating with c-Cbl at the Golgi, suggesting a Golgi-localized activated Src–c-Cbl complex.\",\n      \"method\": \"Confocal immunofluorescence (Golgi marker colocalization), subcellular fractionation (isopycnic density centrifugation and free-flow electrophoresis), co-immunoprecipitation, brefeldin A treatment\",\n      \"journal\": \"European journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — fractionation combined with colocalization and Co-IP, multiple methods; single lab\",\n      \"pmids\": [\"11893076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"c-Cbl macrophages (c-cbl-/-) exhibit impaired chemokinetic and chemotactic migration and altered actin cytoskeletal structures similar to hck-/-fgr-/- macrophages, placing c-Cbl tyrosine phosphorylation downstream of Src family kinases (Hck/Fgr) in a pathway controlling macrophage motility.\",\n      \"method\": \"c-cbl-/- macrophages (genetic KO), migration/chemotaxis assays, comparison with hck-/-fgr-/- macrophages, Cbl phosphorylation Western blotting\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined migration phenotype and epistatic comparison; single lab\",\n      \"pmids\": [\"12652654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"c-Cbl functions downstream of Dectin-2/Dectin-3 in dendritic cells to mediate ubiquitination and proteasomal degradation of the noncanonical NF-κB subunit RelB; c-Cbl deficiency in DCs promotes α-mannan-induced RelB activation, which suppresses IL-10 transcription and aggravates colitis. c-Cbl interacts with c-Abl tyrosine kinase in this pathway.\",\n      \"method\": \"DC-specific c-Cbl knockout mice, DSS colitis model, co-immunoprecipitation (c-Cbl–RelB and c-Cbl–c-Abl), ubiquitination assay, NF-κB reporter, IL-10 ELISA\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — DC-specific KO model, co-IP, ubiquitination assay, in vivo colitis phenotype; single lab\",\n      \"pmids\": [\"33962939\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CBL mutations found in myeloid neoplasms selectively abolish E3 ubiquitin ligase activity while enhancing interaction with LYN kinase; mutant CBL shows increased LYN-driven phosphorylation, PIK3R1 (p85) recruitment, and PI3K/AKT activation. All CBL adaptor domains (TKB, proline-rich region, C-terminal phosphotyrosines) are required for oncogenic function. LYN inhibition (dasatinib) reduces mutant CBL signaling in vitro and in vivo in CMML.\",\n      \"method\": \"Phosphoproteomics (global mass spectrometry), CBL interactome MS, functional assays with CBL allelic series, domain deletion/mutagenesis, LYN knockout/dasatinib inhibition, in vivo CMML xenograft\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — global phosphoproteomics plus functional mechanistic assays (mutagenesis, KO, inhibitor) and in vivo model; multiple orthogonal methods, novel mechanistic insight\",\n      \"pmids\": [\"33512474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"c-Cbl promotes mono-ubiquitination of integrin α3β1 and αVβ3 (but polyubiquitination of αVβ5) to direct selective translocation of KSHV and these receptors into lipid rafts for productive macropinocytic entry; c-Cbl knockdown diverts KSHV to clathrin-mediated lysosomal non-infectious pathway.\",\n      \"method\": \"siRNA knockdown of c-Cbl, lipid raft fractionation, ubiquitination assays (mono- vs. polyubiquitin), KSHV infection assays, methyl-β-cyclodextrin (lipid raft disruption)\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA KD, fractionation, ubiquitination assays, and infection readout; single lab\",\n      \"pmids\": [\"21937638\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"c-Cbl ubiquitin ligase is activated by cathepsin G-induced EGFR transactivation in cardiac myocytes; c-Cbl then interacts with focal adhesion proteins and mediates their proteasomal (not lysosomal or calpain-dependent) ubiquitination and degradation, leading to myofibrillar degeneration and apoptosis.\",\n      \"method\": \"c-Cbl knockout-derived myocytes, EGFR kinase inhibitor, co-immunoprecipitation (c-Cbl–FA proteins), proteasome/lysosome/calpain inhibitors, ubiquitination assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO cells combined with pharmacological dissection and co-IP; single lab\",\n      \"pmids\": [\"22203672\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Crystal structures of CIN85 and beta-PIX SH3 domains in complex with a proline-arginine (PxxxPR) peptide from Cbl-b reveal a heterotrimeric complex in which two SH3 domains are held by a single pseudo-symmetrical peptide; this trimerization occurs in solution and in vivo, and ternary CIN85–Cbl complexes are required for Cbl-mediated EGFR downregulation.\",\n      \"method\": \"X-ray crystallography (SH3–peptide complex structures), solution studies, co-immunoprecipitation (in vivo ternary complex), EGFR downregulation assay\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structures validated by in vivo Co-IP and functional EGFR downregulation assay; multiple orthogonal methods\",\n      \"pmids\": [\"16228008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"c-Cbl and Cbl-b negatively regulate osteoblast differentiation by ubiquitinating the transcription factor Osterix, promoting its proteasome-mediated degradation and suppressing its transcriptional activity; this inhibits BMP2-induced osteoblast differentiation in mesenchymal cells.\",\n      \"method\": \"Overexpression/knockdown in mesenchymal cells, co-immunoprecipitation, ubiquitination assays, proteasome inhibitor rescue, osteoblast differentiation markers\",\n      \"journal\": \"Bone\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination assay plus functional differentiation phenotype; single lab\",\n      \"pmids\": [\"25744063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"c-Cbl promotes K48-linked polyubiquitination of TRAF6 via its proline-rich domain interaction with TRAF6, leading to TRAF6 degradation and inhibition of RANKL- and IL-1β-induced (but not TNFα-induced) NF-κB activation; RANKL or IFN-γ stimulation promotes c-Cbl binding to polyubiquitinated TRAF6.\",\n      \"method\": \"In vitro ubiquitination assay, co-immunoprecipitation with domain mapping, luciferase NF-κB reporter, in vivo ubiquitination assay in bone marrow macrophages/osteoclasts\",\n      \"journal\": \"Cellular & molecular biology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro ubiquitination reconstitution combined with functional NF-κB assay and domain mapping; single lab with some validation in primary cells\",\n      \"pmids\": [\"31123462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"EGF stimulation induces a direct association between c-Cbl and tyrosine-phosphorylated PLC-γ1, mediated by the SH3 domain of PLC-γ1; PDGF, which also phosphorylates PLC-γ1, fails to induce this association and also fails to phosphorylate c-Cbl, indicating c-Cbl phosphorylation is required for PLC-γ1 interaction.\",\n      \"method\": \"Co-immunoprecipitation in vivo, GST pull-down and overlay assay (direct binding), comparison of EGF vs. PDGF stimulation, SH3 domain deletion mutant\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus GST pull-down for direct interaction, domain mutant, stimulus comparison; single lab\",\n      \"pmids\": [\"12061819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"c-Cbl regulation of nuclear β-catenin requires phosphorylation of c-Cbl Tyr371: the Y371H mutant interacts with but fails to ubiquitinate nuclear β-catenin, acts as a dominant negative, and in a Wnt-8 zebrafish model augments Wnt/β-catenin signaling and tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Wnt reporter assay, Wnt-8 transgenic zebrafish model, colorectal cancer cell lines\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus ubiquitination assay and in vivo zebrafish model; single lab\",\n      \"pmids\": [\"30029779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TULA (an SH3- and UBA-containing protein) binds c-Cbl and ubiquitin; TULA inhibits c-Cbl-mediated EGFR downregulation and, in Jurkat T cells, upregulates ZAP-70 and NF-AT activity; TULA-mediated inhibition involves TULA-induced ubiquitylation and degradation of c-Cbl itself.\",\n      \"method\": \"Affinity chromatography and mass spectrometry (c-Cbl-interacting protein identification), co-immunoprecipitation, EGFR degradation assay, TULA modulation in Jurkat cells, NF-AT reporter assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction, functional assays in two cell systems; single lab\",\n      \"pmids\": [\"15107835\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Ephrin-A1 stimulation induces EphA3 receptor tyrosine phosphorylation that leads to c-Cbl phosphorylation in a Src-family kinase-dependent manner; phosphorylated c-Cbl associates with Crk-L and Crk-II (but not Grb2); overexpression of wild-type c-Cbl (but not 70Z mutant) down-regulates EphA receptor expression.\",\n      \"method\": \"Phosphotyrosine immunoblotting, co-immunoprecipitation, Src inhibitor (PP2) treatment, c-Cbl overexpression/70Z mutant, EphA surface expression assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, pharmacological epistasis, and receptor down-regulation assay; single lab\",\n      \"pmids\": [\"12794130\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"c-Cbl down-regulates the Ron receptor tyrosine kinase: MSP stimulation recruits c-Cbl to Ron's multifunctional docking site and to a juxtamembrane autophosphorylation site; c-Cbl mediates Ron polyubiquitylation followed by endocytosis and degradation; both TKB domain and RING domain are required; Ron–Grb2–c-Cbl complexes alone are insufficient for productive ubiquitylation.\",\n      \"method\": \"Co-immunoprecipitation, domain mutagenesis (TKB and RING), ubiquitylation assay, receptor degradation and endocytosis assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mutagenesis plus ubiquitination and degradation assays; single lab\",\n      \"pmids\": [\"12802274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"c-Cbl negatively regulates platelet activation through GpVI: phosphorylation of c-Cbl in GpVI-signaled platelets requires upstream Src-family kinases Fyn and Lyn; in c-Cbl-null platelets, phosphorylation of FcRγ-chain, Syk, and PLCγ2 is increased and aggregation in response to GpVI agonist is potentiated.\",\n      \"method\": \"c-Cbl knockout mouse platelets, Fyn/Lyn-null platelets for epistasis, Western blotting (phosphorylation), platelet aggregation assays\",\n      \"journal\": \"Journal of thrombosis and haemostasis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with defined biochemical phenotype and functional aggregation assay; epistatic placement by Fyn/Lyn-null comparison\",\n      \"pmids\": [\"14629478\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"c-Cbl is a multifunctional RING-type E3 ubiquitin ligase and adaptor protein that recognizes activated (tyrosine-phosphorylated) receptor and non-receptor tyrosine kinases through its TKB/SH2 domain and recruits E2 ubiquitin-conjugating enzymes through its RING domain to catalyze substrate ubiquitination and degradation; its activity is regulated by autoinhibition and phosphorylation of Tyr371 in the linker helix region which relieves autoinhibition and activates the RING domain; beyond ubiquitination, c-Cbl acts as an adaptor to recruit PI3K/p85, Grb2, and Crk, influencing signaling via Rac/Vav, Akt, and other pathways, and it also possesses NEDD8 E3 ligase (neddylation) activity that stabilizes certain substrates (TβRII, c-Src); together these activities regulate receptor downregulation (EGFR, ErbB, KIT, Flt-1, gp130, EphA, Ron), T-cell and NK-cell signaling thresholds, bone resorption downstream of Src, innate immune responses (IRF3, RelB, FLIP(S), PD-1), and actin cytoskeletal organization.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"c-Cbl (CBL) is a RING-type, E2-dependent E3 ubiquitin ligase and signaling adaptor that enforces negative feedback on activated tyrosine-kinase signaling by recognizing phosphotyrosine substrates and targeting them for ubiquitination and lysosomal or proteasomal degradation [#0]. It binds tyrosine-phosphorylated substrates through its SH2/TKB domain and recruits and allosterically activates an E2 conjugating enzyme (e.g., UbcH7) through its RING domain, with rigid coupling between the substrate- and E2-binding surfaces positioning ubiquitin for transfer [#0, #1]. Catalytic output is gated by autoinhibition: in the resting state the RING E2-binding surface folds back intramolecularly, and phosphorylation of Tyr371 in the linker-helix region relieves this autoinhibition, repositions the RING and bound E2 toward the substrate, and is required for receptor tyrosine kinase ubiquitination [#3]. Through these activities c-Cbl drives ligand-induced downregulation of receptor tyrosine kinases including ErbB-1/EGFR, Neu/ErbB-2, KIT, Flt-1, EphA, and Ron, controlling their endosomal sorting toward degradation rather than recycling [#2, #24, #16, #43]; CIN85/CD2AP adaptors and the SH3-mediated ternary complexes they form are required for this receptor downregulation [#36, #17]. In hematopoietic and immune cells c-Cbl sets activation thresholds by negatively regulating Syk/ZAP-70-family signaling, promoting clearance of engaged TCR and ubiquitination of LAT and Vav, and downmodulating innate signaling components including IRF3, RelB, TRAF6, and PD-1 [#8, #9, #22, #20, #27, #32, #38, #28]. Beyond catalysis, c-Cbl serves as a scaffold that recruits PI3K/p85, Grb2, and Crk, a non-catalytic function genetically separable from its E3 activity that activates Akt and shapes actin cytoskeletal organization downstream of Src-family kinases [#21, #7, #23, #31]. c-Cbl also acts downstream of c-Src in osteoclast-mediated bone resorption [#4] and possesses NEDD8 E3 (neddylation) activity toward TβRII and c-Src, which alters their trafficking and stability [#10, #29]. CBL mutations in myeloid neoplasms selectively abolish E3 ligase activity while enhancing LYN-driven phosphorylation, p85 recruitment, and PI3K/AKT signaling, defining an oncogenic gain-of-adaptor mechanism [#33].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established c-Cbl as a functional effector downstream of c-Src in a defined physiological process, before its enzymatic identity was known.\",\n      \"evidence\": \"Antisense knockdown, co-IP, immunofluorescence, and in vitro bone resorption with src-null osteoclasts\",\n      \"pmids\": [\"8849724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism of c-Cbl action in resorption not defined\", \"Substrate(s) of c-Cbl in osteoclasts not identified\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Showed that c-Cbl physically mediates receptor ubiquitination and degradative endosomal sorting, reframing it from a passive adaptor to an active downregulator of RTKs.\",\n      \"evidence\": \"Endosomal colocalization, ubiquitination IP, and oncogenic v-Cbl rescue in ErbB-1 trafficking\",\n      \"pmids\": [\"9851973\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzymatic mechanism of ubiquitin transfer not yet established\", \"Did not define E2 partner or RING requirement\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Genetically defined c-Cbl as a negative regulator of T-cell Syk/ZAP-70 kinase signaling, establishing its immune threshold-setting role.\",\n      \"evidence\": \"c-Cbl knockout mice with ZAP-70 phosphorylation readouts and lymphoid phenotyping\",\n      \"pmids\": [\"9671496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the effect required E3 activity vs adaptor function unresolved at this stage\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined the core biochemical identity of c-Cbl as a RING-type, E2-dependent E3 ligase that reads phosphotyrosine substrates and allosterically activates a conjugating enzyme.\",\n      \"evidence\": \"In vitro ubiquitination reconstitution with PDGFR substrate plus SH2 and RING mutagenesis\",\n      \"pmids\": [\"10514377\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of E2 recruitment not yet visualized\", \"Regulation of activity in cells not addressed\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Provided the structural logic for RING E3 catalysis, showing rigid substrate-to-E2 coupling that scaffolds ubiquitin transfer.\",\n      \"evidence\": \"X-ray crystal structure of the c-Cbl–UbcH7–kinase peptide ternary complex\",\n      \"pmids\": [\"10966114\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not capture the autoinhibited or activated conformations\", \"Phosphoregulation not represented in structure\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrated c-Cbl is a tumor suppressor of an oncogenic RTK and that its catalytic downregulation activity has anti-tumor consequences in vivo.\",\n      \"evidence\": \"c-Cbl/v-Cbl overexpression, ubiquitination and surface-receptor assays, and a neuroblastoma tumor model\",\n      \"pmids\": [\"10940298\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous-level regulation not tested\", \"Mechanism of v-Cbl antagonism only inferred\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Revealed an upstream OFF-switch in which Cdc42/beta-PIX sequesters c-Cbl away from EGFR, linking GTPase signaling to control of receptor downregulation and transformation.\",\n      \"evidence\": \"Reciprocal co-IP, dominant-active Cdc42, ubiquitination/ERK assays, and transformation phenotype\",\n      \"pmids\": [\"14505571\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and dynamics of the ternary complex in vivo not defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Genetically separated c-Cbl's E3 ligase function from a distinct PI3K/Akt-recruiting adaptor function, establishing dual-mode action.\",\n      \"evidence\": \"RING-finger mutant knock-in mice with thymic phenotype and c-Cbl/p85 co-IP\",\n      \"pmids\": [\"16211006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect p85 recruitment not fully resolved\", \"Generality of adaptor role across cell types not tested here\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved how c-Cbl activity is switched on, showing Tyr371 phosphorylation relieves an autoinhibited RING conformation required for RTK ubiquitination.\",\n      \"evidence\": \"Crystal structures of unphosphorylated, substrate-bound, and pTyr371 activated complexes with biochemical validation\",\n      \"pmids\": [\"22266821\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinetics of conformational switching in cells not measured\", \"Which kinases phosphorylate Tyr371 in each context not unified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Expanded c-Cbl's enzymatic repertoire to NEDD8 conjugation, showing it can stabilize rather than degrade a substrate by altering its trafficking.\",\n      \"evidence\": \"In vitro neddylation, site mutagenesis (K556R/K567R), KO cells, and endosomal/reporter assays for TβRII\",\n      \"pmids\": [\"23290524\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How c-Cbl chooses ubiquitin vs NEDD8 outputs not defined\", \"E2/NEDD8 machinery details not fully resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the oncogenic mechanism of CBL myeloid mutations as loss of E3 activity coupled to gain of LYN-driven adaptor signaling through PI3K/AKT.\",\n      \"evidence\": \"Phosphoproteomics, interactome MS, CBL allelic series, LYN KO/dasatinib, and CMML xenografts\",\n      \"pmids\": [\"33512474\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full set of dysregulated substrates in patients not enumerated\", \"Therapeutic durability of LYN inhibition not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How c-Cbl integrates its competing E3-ubiquitin, NEDD8-ligase, and non-catalytic adaptor outputs to produce context-specific substrate fates remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model selecting degradation vs stabilization vs signaling\", \"Determinants of mono- vs K48- vs K63-linked ubiquitination unclear\", \"Tissue-specific substrate repertoire incompletely mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 10, 29]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 16, 33]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [21, 7, 23, 26]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [8, 9, 44]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [2, 13, 18]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [30]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 24, 26]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [23, 31]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 6, 16]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8, 9, 27, 28, 32]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [2, 9, 14, 18]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 10, 29]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [33, 24, 40]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"SRC\", \"GRB2\", \"CRK\", \"PIK3R1\", \"CIN85\", \"LYN\", \"CD2AP\", \"EGFR\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}