{"gene":"CBLB","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":2000,"finding":"Cbl-b loss uncouples T-cell proliferation, IL-2 production, and Vav1 phosphorylation from the requirement for CD28 co-stimulation, identifying Cbl-b as a key regulator of T-cell activation thresholds in mature lymphocytes.","method":"Gene-targeted knockout mice; hyperproliferation and cytokine production assays; Vav1 phosphorylation analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — two independent labs (PMID:10646608 and PMID:10646609) with complementary genetic and biochemical evidence, replicated","pmids":["10646608","10646609"],"is_preprint":false},{"year":2000,"finding":"Cbl-b selectively suppresses TCR-mediated Vav (guanine nucleotide exchange factor for Rac1/Rho/CDC42) activation to regulate CD28 dependence of T-cell activation; Cbl-b-null mutation fully restores T-cell-dependent antibody responses in CD28-deficient mice.","method":"Genetic epistasis (cbl-b-/- × CD28-/- double mutant mice); Vav activation assays; antibody response measurements","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — epistasis experiment across two genetic backgrounds with replicated finding","pmids":["10646609"],"is_preprint":false},{"year":2000,"finding":"Cbl-b interacts with and induces ubiquitin conjugation to the p85 regulatory subunit of PI3K; the RING finger domain is essential for p85 ubiquitination, and a distal proline-rich region of Cbl-b binds the SH3 domain of p85.","method":"Co-immunoprecipitation; in vitro ubiquitination assay; RING finger loss-of-function mutagenesis; deletion mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro ubiquitination assay with mutagenesis; replicated by PMID:11526404","pmids":["11087752"],"is_preprint":false},{"year":2001,"finding":"Cbl-b negatively regulates p85 (PI3K regulatory subunit) in a proteolysis-independent manner by controlling its recruitment to CD28 and TCR-zeta, requiring Cbl-b E3 ubiquitin ligase activity; enhanced activation of Cbl-b-/- T cells is suppressed by PI3K inhibition.","method":"Ubiquitination assay; PI3K inhibitor rescue; co-immunoprecipitation with CD28 and TCR-zeta","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 1 — in vitro ligase assay with functional rescue by PI3K inhibition; strong mechanistic follow-up","pmids":["11526404"],"is_preprint":false},{"year":2001,"finding":"Cbl-b is ubiquitinated and degraded upon EGFR activation; EGF-induced Cbl-b degradation requires intact RING finger and tyrosine kinase binding domains and binding to activated EGFR; Cbl-b mediates coordinated degradation of the EGFR signaling complex including Grb2 and Shc via lysosomal and proteasomal pathways.","method":"EGF stimulation; RING finger and TKB domain mutagenesis; lysosomal/proteasomal inhibitors; Western blot for complex components","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — mutagenesis with pharmacological pathway dissection, rigorous mechanistic follow-up","pmids":["11375397"],"is_preprint":false},{"year":2002,"finding":"CD28 co-stimulation selectively induces greater ubiquitination and degradation of Cbl-b than CD3 stimulation alone, establishing that CD28 regulates the T-cell activation threshold partly by promoting Cbl-b ubiquitination and degradation.","method":"Ubiquitination assay; stimulation of wild-type vs. CD28-deficient T cells; Western blot for Cbl-b levels","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — mechanistic biochemical experiment with genetic control (CD28-/- cells)","pmids":["12193687"],"is_preprint":false},{"year":2002,"finding":"CIN85 binds to the carboxyl terminus of Cbl-b; ligand-induced phosphorylation of Cbl-b increases CIN85 interaction; CIN85–Cbl-b binding is required for Cbl-b-mediated EGFR internalization (but dispensable for EGFR polyubiquitination), defining a CIN85/endophilin-dependent endocytic pathway.","method":"Co-immunoprecipitation; dominant-negative CIN85 interference; EGFR internalization assay; ubiquitination assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal pulldown plus functional internalization assay with mechanistic dissection","pmids":["12177062"],"is_preprint":false},{"year":2002,"finding":"Cbl-b positively regulates BCR-mediated Ca2+ signaling by interacting with PLC-γ2 and facilitating its association with Btk and BLNK; Cbl-b is required for Btk-dependent sustained intracellular Ca2+ increase; both N-terminal TKB domain and C-terminal half of Cbl-b are essential.","method":"Cbl-b-deficient DT40 B cells; overexpression in WEHI-231 cells; Ca2+ mobilization assay; co-immunoprecipitation of PLC-γ2/Btk/BLNK complex; domain deletion mutagenesis","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — loss-of-function and gain-of-function with biochemical complex analysis in two cell systems","pmids":["12093870"],"is_preprint":false},{"year":2004,"finding":"Cbl-b is upregulated in T cells after tolerizing signals and is essential for T-cell anergy induction; loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, attributable to Cbl-b-mediated regulation of PLCγ-1 phosphorylation.","method":"In vitro and in vivo T cell tolerance assays in Cbl-b-/- mice; calcium mobilization assay; PLCγ-1 phosphorylation analysis","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — KO with defined molecular readout (PLCγ-1 phosphorylation and Ca2+ flux), replicated in vivo","pmids":["15308098"],"is_preprint":false},{"year":2004,"finding":"Cbl-b-/- effector T cells are resistant to suppression by CD4+CD25+ regulatory T cells and by TGF-β in vitro, despite normal levels of TGF-β receptor II and normal Smad3 phosphorylation, indicating the resistance operates downstream of TGF-β receptor signaling.","method":"In vitro T cell suppression assays; TGF-β receptor expression analysis; Smad3 phosphorylation assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with defined phenotype but partial mechanistic placement (resistance downstream of Smad3)","pmids":["15240694"],"is_preprint":false},{"year":2007,"finding":"Cblb-/- CD8+ T cells are resistant to TGF-β suppression and do not require CD28 costimulation for activation in vitro; in vivo, Cblb-/- mice efficiently reject B7-negative tumors via massively infiltrating CD8+ T cells; adoptive transfer of purified Cblb-/- CD8+ T cells eradicates established tumors.","method":"Knockout mice; in vitro TGF-β suppression assay; tumor challenge with B7-negative cell lines; adoptive transfer of CD8+ T cells","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — multiple in vitro and in vivo orthogonal methods with defined molecular mechanism","pmids":["17364027"],"is_preprint":false},{"year":2007,"finding":"Cblb controls the association between TLR4 and the intracellular adaptor MyD88 and regulates TLR4 surface expression on neutrophils; E3 ligase activity (not adaptor function) of Cblb is required to suppress NF-κB-mediated inflammatory responses to LPS.","method":"Co-immunoprecipitation of TLR4-MyD88; NF-κB reporter assay with wild-type vs. RING-finger-mutant Cblb; flow cytometry of TLR4 surface levels on Cblb-/- neutrophils","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 — mechanistic mutagenesis (RING finger mutant) plus biochemical and functional assays","pmids":["17618294"],"is_preprint":false},{"year":2007,"finding":"Cbl-b interacts with Pseudomonas exotoxin T (ExoT) and promotes its polyubiquitination and proteasomal degradation in host cells; Cbl-b (but not c-Cbl) is specifically required in vivo to limit bacterial dissemination mediated by ExoT.","method":"Co-immunoprecipitation of Cbl-b with ExoT and Crk; proteasomal inhibitor experiments; in vivo mouse infection models with Cblb-/- mice","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 — biochemical pulldown plus in vivo genetic specificity (c-Cbl plays no role)","pmids":["17235393"],"is_preprint":false},{"year":2009,"finding":"Upon unloading stress, Cbl-b is induced in skeletal muscle and directly ubiquitinates IRS-1, targeting it for degradation; loss of IRS-1 activates FOXO3-dependent atrogin-1/MAFbx induction; Cbl-b-/- mice are resistant to unloading-induced atrophy.","method":"Co-immunoprecipitation of Cbl-b with IRS-1; ubiquitination assay; KO mice subjected to hindlimb unloading; peptide inhibitor (pY608-IRS-1 mimetic) blocks IRS-1 ubiquitination","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro ubiquitination plus KO plus peptide inhibitor, multiple orthogonal methods","pmids":["19546233"],"is_preprint":false},{"year":2009,"finding":"Cbl-b interacts with CARMA1 and promotes its monoubiquitination; this monoubiquitination disrupts CARMA1-Bcl10 complex formation (without affecting CARMA1 stability), thereby suppressing NF-κB activation and mediating NKT cell anergy; Cbl-b RING finger activity is critical.","method":"Co-immunoprecipitation; monoubiquitination assay; RING finger mutant analysis; CARMA1-/- NKT cell functional analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 — ubiquitination assay with mutagenesis plus complex disruption and genetic validation in KO cells","pmids":["19815501"],"is_preprint":false},{"year":2009,"finding":"Loss of Cbl-b increases osteoclast bone-resorbing activity and in vitro differentiation, and enhances RANKL-induced NF-κB, ERK, and p38 signaling; rescue by re-expressing Cbl-b (but not by c-Cbl) requires functional TKB and RING finger domains.","method":"Cbl-b-/- mice; in vitro bone resorption assay; RANKL signaling analysis; rescue by wild-type vs. domain-mutant Cbl-b re-expression","journal":"Journal of bone and mineral research","confidence":"High","confidence_rationale":"Tier 2 — KO phenotype with domain-mutant rescue establishing mechanistic requirements","pmids":["19257814"],"is_preprint":false},{"year":2011,"finding":"Selective genetic inactivation of Cbl-b E3 ligase activity (RING finger knock-in mutation) phenocopies total Cbl-b ablation in T-cell hyperactivation, spontaneous autoimmunity, impaired T-cell anergy, and spontaneous tumor rejection, demonstrating the catalytic E3 ligase activity is essential for all Cbl-b T-cell regulatory functions in vivo.","method":"RING finger knock-in mouse (E3 ligase-dead); comparison with total Cbl-b KO; autoimmunity scoring; tumor rejection assays; T-cell anergy induction assays","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 1 — knock-in mutagenesis in vivo phenocopying KO, definitive proof of catalytic requirement","pmids":["21248250"],"is_preprint":false},{"year":2012,"finding":"Cbl-b suppresses Pten inactivation (rather than directly inhibiting PI3K) in T cells by impeding Nedd4-mediated K63-linked polyubiquitination of Pten at K13; elevated Akt in Cbl-b-/- T cells is due to heightened Pten inactivation; this function is independent of Cbl-b's own E3 ligase activity.","method":"Pten ubiquitination analysis; Nedd4 co-immunoprecipitation; double KO (Cbl-b-/- × Nedd4-/-); Akt activity measurement; ubiquitin linkage-specific assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis (double KO rescue) plus biochemical ubiquitination mechanism, multiple orthogonal approaches","pmids":["22763434"],"is_preprint":false},{"year":2014,"finding":"Cbl-b ubiquitylates TAM receptor tyrosine kinases (Tyro3, Axl, Mertk) in NK cells, limiting NK cell anti-metastatic activity; genetic deletion or E3 ligase inactivation of Cbl-b licenses NK cells to reject metastatic tumors; a TAM kinase inhibitor phenocopies Cbl-b loss.","method":"Ubiquitylation assay identifying TAM receptors as Cbl-b substrates; E3-ligase-dead knock-in mice; in vivo tumor metastasis models; TAM kinase inhibitor treatment","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 — substrate identification by ubiquitylation assay with genetic and pharmacological validation in vivo","pmids":["24553136"],"is_preprint":false},{"year":2014,"finding":"Cbl-b ubiquitylates IGF-IR in gastric cancer cells leading to its degradation, thereby inhibiting the Akt/ERK-miR-200c-ZEB2 axis and suppressing IGF-I-induced epithelial-mesenchymal transition.","method":"Cbl-b shRNA knockdown; co-immunoprecipitation; ubiquitination assay; Akt/ERK inhibitors; miR-200c and ZEB2 expression analysis","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 3 — single lab with co-IP and functional assays but limited orthogonal confirmation","pmids":["24885194"],"is_preprint":false},{"year":2015,"finding":"Upon NGF stimulation of neuroblastoma cells, TrkA recruits Cbl-b, which becomes phosphorylated and ubiquitylated; Cbl-b promotes TrkA ubiquitylation and degradation; Cbl-b depletion increases ERK phosphorylation and neurite outgrowth.","method":"Quantitative interactome/proteome/phosphoproteome mass spectrometry; Cbl-b depletion; neurite outgrowth measurement","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1-2 — temporal proteomics with KD rescue, multiple orthogonal datasets","pmids":["25921289"],"is_preprint":false},{"year":2015,"finding":"Cbl-b binds Foxp3 upon TCR stimulation and, together with Stub1, targets Foxp3 for ubiquitination and proteasomal degradation, thereby regulating thymic Treg development; proteasome inhibition rescues defective tTreg development in CD28-/- mice.","method":"Co-immunoprecipitation of Cbl-b with Foxp3; ubiquitination assay; Cbl-b-/- × CD28-/- genetic rescue; proteasome inhibitor rescue of tTreg development","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — biochemical ubiquitination assay plus genetic epistasis plus pharmacological rescue","pmids":["25560411"],"is_preprint":false},{"year":2015,"finding":"NFATc1 induces Nur77 expression during osteoclast differentiation; Nur77 transcriptionally upregulates Cbl-b; Cbl-b then triggers NFATc1 protein degradation via ubiquitination, creating a self-limiting NFATc1→Nur77→Cbl-b→NFATc1 feedback loop that prevents excessive osteoclastogenesis.","method":"Genetic KO (Nur77-/-); ChIP/reporter assay linking Nur77 to Cbl-b promoter; ubiquitination assay showing Cbl-b ubiquitinates NFATc1; bone mass phenotyping","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (transcription assay, ubiquitination, KO phenotype) establishing a complete pathway","pmids":["26173181"],"is_preprint":false},{"year":2016,"finding":"CBLB associates with SYK and ubiquitinates SYK, dectin-1, and dectin-2 following fungal recognition via C-type lectin receptors; CBLB deficiency results in increased inflammasome activation, enhanced ROS production, and enhanced fungal killing.","method":"Co-immunoprecipitation of CBLB with SYK/dectin-1/dectin-2; ubiquitination assay; Cblb-/- macrophage/DC functional assays; lethal C. albicans infection model","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 — substrate identification by ubiquitination assay replicated in vivo with KO mice; confirmed by companion paper PMID:27428899","pmids":["27428901","27428899"],"is_preprint":false},{"year":2016,"finding":"CBLB directs polyubiquitination of dectin-1 and dectin-2 and their downstream kinase SYK, inhibiting dectin-mediated innate immune responses; dectin-1 and dectin-2 deficiency in Cblb-/- mice abrogates the protection against systemic C. albicans infection.","method":"Ubiquitination assay; genetic epistasis (Cblb-/- × dectin-1-/- or dectin-2-/- double KO); in vivo lethal C. albicans infection model","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 — epistasis experiment with KO rescue plus substrate ubiquitination assay, replicated by PMID:27428901","pmids":["27428899"],"is_preprint":false},{"year":2016,"finding":"Cbl-b mediates ubiquitination of activated Dectin-2 and Dectin-3 via adapter FcR-γ and kinase Syk in a Syk-dependent manner; ubiquitinated CLRs are sorted into lysosomes via the ESCRT system for degradation.","method":"Co-immunoprecipitation of Cbl-b with FcR-γ/Syk/CLRs; ubiquitination assay; ESCRT subunit knockdown; CLR degradation analysis","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 — mechanistic ubiquitination and trafficking pathway established with multiple components","pmids":["27432944"],"is_preprint":false},{"year":2016,"finding":"TCR stimulation dynamics of CBL and CBLB signaling complexes were characterized by affinity purification coupled to quantitative mass spectrometry; CD5 transmembrane receptor was identified as a key scaffold for CBLB-mediated ubiquitylation following TCR engagement.","method":"Affinity purification coupled to quantitative MS over 600 s TCR stimulation time course; biochemical validation of CD5 as a scaffold","journal":"Molecular systems biology","confidence":"High","confidence_rationale":"Tier 1-2 — unbiased quantitative interactome with biochemical validation, strong methodology","pmids":["27474268"],"is_preprint":false},{"year":2019,"finding":"TAM receptors (Tyro3, Axl, Mer) phosphorylate Cbl-b at tyrosine residues Y133 and Y363 to activate it; Gas6-mediated TAM receptor ligation suppresses NK-cell functions (IFN-γ production, degranulation) via Cbl-b; Cbl-b promotes degradation of LAT1 (transmembrane adaptor) in response to Gas6.","method":"TAM receptor ligation with Gas6; phosphorylation site mutagenesis (Y133, Y363); Cbl-b KO NK cells; LAT1 degradation assay","journal":"European journal of immunology","confidence":"High","confidence_rationale":"Tier 2 — phosphorylation site mutagenesis with KO genetic validation and defined substrate (LAT1)","pmids":["31531847"],"is_preprint":false},{"year":2020,"finding":"Cbl-b is enriched in spinal cord dorsal horn neurons; it interacts with GluN2B via its N-terminal TKB domain and ubiquitinates GluN2B, reducing synaptic GluN2B-containing NMDARs; peripheral inflammation induces dephosphorylation of Cbl-b at Y363, impairing its binding to and ubiquitylation of GluN2B and enabling GluN2B reappearance at synapses.","method":"Co-immunoprecipitation; ubiquitination assay; in vivo Cbl-b knockdown; phosphomimetic Cbl-b mutant expression; electrophysiology of synaptic currents","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical substrate identification with mutagenesis plus in vivo electrophysiology","pmids":["32606037"],"is_preprint":false},{"year":2023,"finding":"The co-crystal structure of Cbl-b with inhibitor C7683 (analogue of Nx-1607) reveals the compound binds both the TKBD and LHR domains but not the RING domain, locking Cbl-b in an inactive conformation by acting as an intramolecular glue; confirmed by biophysical assays.","method":"X-ray crystallography (co-crystal structure); biophysical binding assays (SPR, thermal shift); cellular ubiquitination assay","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with biophysical and cellular validation","pmids":["38104184"],"is_preprint":false},{"year":2023,"finding":"TCR stimulation induces a molecular complex between Cbl-b and the pH-sensitive unconventional phosphatase STS1 via a proline motif in Cbl-b interacting with the STS1 SH3 domain; STS1 dephosphorylates Cbl-b-interacting phosphoproteins; deficiency of STS1 or Cbl-b reduces T-cell sensitivity to acidic environments and promotes anti-tumor T-cell activity.","method":"Co-immunoprecipitation; STS1 and Cbl-b KO mice; proline-motif mutagenesis; in vitro and in vivo pH sensitivity assays; tumor growth experiments","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — biochemical complex characterization with mutagenesis and dual KO genetic validation in vivo","pmids":["38091950"],"is_preprint":false},{"year":2003,"finding":"Cbl-b undergoes tyrosine phosphorylation and plasma membrane translocation in response to insulin in adipocytes; it constitutively associates with CAP and interacts with Crk upon insulin stimulation; Cbl proteins form homo- and heterodimers via a conserved leucine zipper domain required for APS interaction and tyrosine phosphorylation.","method":"Phosphorylation assays in 3T3-L1 adipocytes; co-immunoprecipitation with CAP and Crk; leucine zipper dimerization mutagenesis; membrane fractionation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 — co-IP and mutagenesis in cell line model, single lab","pmids":["12842890"],"is_preprint":false},{"year":2006,"finding":"Cbl-b is upregulated in osteoblastic cells after denervation and ubiquitinates IRS-1, promoting its degradation, thereby suppressing IGF-I signaling (PI3K, Akt-1) in osteoblasts and causing reduced bone formation; Cbl-b-/- mice maintain bone mass after denervation.","method":"Cbl-b-/- denervation model; IRS-1 ubiquitination assay in primary osteoblasts; Western blot for IGF-I signaling; bone histomorphometry","journal":"Journal of bone and mineral research","confidence":"High","confidence_rationale":"Tier 2 — KO with defined molecular mechanism (IRS-1 ubiquitination) confirmed in primary cells","pmids":["16734387"],"is_preprint":false},{"year":2023,"finding":"Cbl-b and Cbl-b interact with EGFR through distinct modes: Cbl-b preferentially binds pY1045 of EGFR directly, while c-Cbl relies mainly on Grb2-dependent indirect binding; each operates independently to jointly control EGFR ubiquitination, endocytic trafficking, and chemotaxis signaling.","method":"Inducible expression of E3-ligase-dead Cbl-b and c-Cbl mutants; EGFR ubiquitination and degradation assays; chemotaxis migration assay; cells with differential Cbl-b endogenous levels","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — domain-specific mutagenesis with functional readouts across multiple cell lines","pmids":["37903221"],"is_preprint":false},{"year":1999,"finding":"Cbl-b is phosphorylated and recruited to EGFR upon EGF stimulation and binds Grb2; overexpression of Cbl-b in EGFR-dependent cells decreases AKT activation amplitude/duration and shortens MAP kinase/JNK activation, inhibiting EGF-induced cell growth and promoting apoptosis.","method":"Overexpression in 32D/EGFR cells; growth assays; AKT and MAPK activation time course; apoptosis assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 — gain-of-function overexpression with kinase activation readouts, single lab","pmids":["10086340"],"is_preprint":false},{"year":1998,"finding":"CBLB is phosphorylated on tyrosine following FLT3 ligand or IL-7 stimulation in pro-B cells; tyrosine-phosphorylated SHC and p85 of PI3K associate with CBLB upon FL or IL-7 treatment; CBLB constitutively binds GRB2 predominantly through GRB2's N-terminal SH3 domain.","method":"Tyrosine phosphorylation assay; co-immunoprecipitation of SHC, p85, GRB2 with CBLB","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP and phosphorylation analysis in hematopoietic cell lines, single lab","pmids":["9614102"],"is_preprint":false},{"year":2018,"finding":"CBLB forms a complex with CD81 and calpain-5 (CAPN5) in human liver cells; CBLB is required for a post-binding, pre-replication step in HCV entry; CBLB knockout reduces susceptibility to all tested HCV genotypes but not to other enveloped viruses.","method":"Quantitative proteomics of CD81 interactome; CBLB/CAPN5 knockout; HCV infection assay across genotypes","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 — proteomics interactome with KO validation and specificity controls, single lab","pmids":["30024968"],"is_preprint":false},{"year":2017,"finding":"DR5-Cbl-b/c-Cbl-TRAF2 form a complex in TRAIL-resistant gastric cancer cells; Cbl-b and c-Cbl serve as adaptors linking DR5 to TRAF2; TRAIL-induced caspase-8 translocation into this complex leads to TRAF2-mediated K48-linked polyubiquitination and proteasomal degradation of caspase-8, inhibiting apoptosis.","method":"Co-immunoprecipitation; ubiquitination assay (K48-linkage-specific); TRAF2 and Cbl-b knockdown; proteasome inhibitor (bortezomib) rescue","journal":"Molecular oncology","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical complex identification with linkage-specific ubiquitination and genetic validation, single lab","pmids":["28972304"],"is_preprint":false},{"year":2022,"finding":"Cbl-b and c-Cbl ubiquitinate STAT5a and STAT5b in dendritic cells to promote their degradation and cell apoptosis; Cbl-b mediates K27-linked ubiquitination of STAT5a at K164, while c-Cbl induces K29-linked ubiquitination of STAT5a K696 and K27-linked ubiquitination of STAT5b K140 and K694.","method":"DC-specific double KO mice (Cbl-b-/-×c-Cblflox/flox CD11c-Cre); STAT5 ubiquitination assays with linkage-specific ubiquitin mutants; site-directed mutagenesis of STAT5 lysine residues","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 — ubiquitination assay with linkage and site mutagenesis, but single lab","pmids":["35354799"],"is_preprint":false},{"year":2021,"finding":"Cbl-b is upregulated in exhausted (PD1+Tim3+) CD8+ tumor-infiltrating lymphocytes; CRISPR-Cas9-mediated deletion of Cbl-b in CD8+ T cells and CAR T cells restores effector function of exhausted cells, reduces PD1+Tim3+ populations, and increases IFN-γ/TNF-α production and tumor cell killing.","method":"RNA-seq of TILs; syngeneic MC38 tumor model; CRISPR-Cas9 Cbl-b KO in CAR T cells; tumor growth and cytokine assays","journal":"Journal for immunotherapy of cancer","confidence":"High","confidence_rationale":"Tier 2 — CRISPR KO with functional readouts in both endogenous and CAR T cells, multiple tumor models","pmids":["33462140"],"is_preprint":false}],"current_model":"CBLB encodes a RING-type E3 ubiquitin ligase that acts as a master negative regulator of immune cell activation by ubiquitinating and/or modulating a diverse set of substrates—including PI3K p85, IRS-1, PLCγ-1, Vav1, TAM receptors, SYK, dectin-1/2, CARMA1, NFATc1, GluN2B, Foxp3, and EGFR/TrkA—downstream of antigen, growth-factor, and pattern-recognition receptors; its catalytic RING finger activity and adaptor functions (via TKB, leucine zipper, and proline-rich domains) set T-cell, NK-cell, and myeloid activation thresholds, enforce peripheral T-cell tolerance and anergy, regulate bone remodeling and muscle mass, control innate antifungal immunity, and suppress tumor immune evasion, with the protein itself subject to regulation by CD28-induced auto-ubiquitination, TAM receptor-mediated phosphorylation (Y133, Y363), and complex formation with the pH-sensitive phosphatase STS1."},"narrative":{"teleology":[{"year":1999,"claim":"Initial biochemical work established that Cbl-b is phosphorylated upon growth factor (EGF, FLT3L, IL-7) stimulation and associates with key signaling adaptors (Grb2, SHC, PI3K p85), positioning it as a signaling hub downstream of receptor tyrosine kinases before its E3 ligase function was appreciated.","evidence":"Co-immunoprecipitation and phosphorylation assays in hematopoietic and EGFR-expressing cell lines","pmids":["9614102","10086340"],"confidence":"Medium","gaps":["E3 ligase activity not yet tested","overexpression system without loss-of-function validation","physiological relevance in primary cells unknown"]},{"year":2000,"claim":"Two independent knockout studies revealed that Cbl-b sets the CD28 co-stimulation requirement for T-cell activation by suppressing Vav1 phosphorylation, establishing Cbl-b as the critical gatekeeper of peripheral T-cell activation thresholds.","evidence":"Gene-targeted Cbl-b KO mice; genetic epistasis with CD28-/- mice; Vav activation and T-cell proliferation/cytokine assays","pmids":["10646608","10646609"],"confidence":"High","gaps":["Direct enzymatic mechanism on Vav not defined","whether Cbl-b ubiquitinates Vav directly or acts indirectly remained unknown"]},{"year":2001,"claim":"Identification of PI3K p85 as a direct Cbl-b ubiquitination substrate revealed a RING-finger-dependent, proteolysis-independent mechanism whereby Cbl-b controls PI3K recruitment to CD28 and TCRζ, explaining how Cbl-b restrains PI3K/Akt signaling in T cells.","evidence":"In vitro ubiquitination assay with RING finger mutagenesis; PI3K inhibitor rescue of Cbl-b-/- T-cell hyperactivation; co-IP with CD28/TCRζ","pmids":["11087752","11526404"],"confidence":"High","gaps":["Non-degradative ubiquitin signal on p85 not fully characterized (chain type unknown)","whether this is the sole PI3K-regulatory mechanism of Cbl-b was unclear"]},{"year":2001,"claim":"Demonstration that Cbl-b coordinates EGFR signaling complex degradation through its TKB and RING domains, and that it is itself degraded upon EGFR activation, established Cbl-b as a self-limiting negative regulator of receptor tyrosine kinase signaling beyond the immune system.","evidence":"EGF stimulation with RING/TKB mutagenesis; lysosomal and proteasomal inhibitors; Western blot for EGFR/Grb2/Shc complex degradation","pmids":["11375397"],"confidence":"High","gaps":["Relative contributions of Cbl-b vs. c-Cbl to EGFR degradation not resolved","in vivo relevance in epithelial tissues not tested"]},{"year":2002,"claim":"CD28 co-stimulation was shown to selectively induce Cbl-b ubiquitination and degradation, establishing a feedforward mechanism: CD28 removes Cbl-b to lower the activation threshold, while CIN85 binding to Cbl-b was found necessary for receptor internalization but dispensable for polyubiquitination.","evidence":"Ubiquitination assay comparing CD3 vs. CD28 stimulation in WT and CD28-/- T cells; CIN85 dominant-negative and EGFR internalization assay","pmids":["12193687","12177062"],"confidence":"High","gaps":["Identity of the E3 ligase ubiquitinating Cbl-b itself not determined","CIN85-Cbl-b function in T cells (vs. EGFR context) not tested"]},{"year":2002,"claim":"Discovery that Cbl-b positively regulates BCR-mediated calcium signaling by scaffolding PLC-γ2/Btk/BLNK complexes revealed a context-dependent positive signaling role, contrasting with its inhibitory function in T cells.","evidence":"Cbl-b-deficient DT40 B cells; gain-of-function in WEHI-231; Ca2+ mobilization; co-IP of PLCγ2/Btk/BLNK; domain deletion mutagenesis","pmids":["12093870"],"confidence":"High","gaps":["Whether this positive role depends on E3 ligase activity or adaptor function was not resolved","relevance to primary mammalian B cells not confirmed"]},{"year":2004,"claim":"Cbl-b was established as essential for T-cell anergy induction and identified as a mediator of regulatory T-cell suppression: tolerizing signals upregulate Cbl-b to suppress PLCγ-1 phosphorylation and calcium mobilization, while Cbl-b-/- T cells escape Treg- and TGF-β-mediated suppression.","evidence":"In vitro/in vivo tolerance assays in Cbl-b KO mice; PLCγ-1 phosphorylation and Ca2+ flux; Treg suppression and TGF-β assays with Smad3 analysis","pmids":["15308098","15240694"],"confidence":"High","gaps":["Direct substrate linking Cbl-b to TGF-β resistance not identified","mechanism downstream of Smad3 not resolved"]},{"year":2006,"claim":"Cbl-b was shown to ubiquitinate IRS-1 for degradation in osteoblasts after denervation and later in skeletal muscle upon unloading, establishing a conserved Cbl-b→IRS-1 degradation axis that suppresses IGF-I/PI3K/Akt signaling to cause bone loss and muscle atrophy.","evidence":"Cbl-b KO denervation/unloading models; IRS-1 ubiquitination assay in primary cells; bone histomorphometry; peptide inhibitor blocking IRS-1 ubiquitination","pmids":["16734387","19546233"],"confidence":"High","gaps":["Whether Cbl-b directly ubiquitinates IRS-1 or requires cofactors not fully resolved","therapeutic utility of peptide inhibitor in vivo not established"]},{"year":2007,"claim":"The biological consequence of Cbl-b loss for tumor immunity was demonstrated: Cbl-b-/- CD8+ T cells reject B7-negative tumors and resist TGF-β, while Cbl-b's RING finger E3 ligase activity was shown to suppress TLR4-MyD88 association and NF-κB signaling in innate immune cells.","evidence":"Tumor challenge and adoptive transfer in KO mice; TLR4-MyD88 co-IP; RING finger mutant NF-κB reporter assay; neutrophil TLR4 surface expression","pmids":["17364027","17618294"],"confidence":"High","gaps":["Direct TLR4/MyD88 ubiquitination substrate identity not confirmed","whether TGF-β resistance involves a direct Cbl-b substrate was unknown"]},{"year":2009,"claim":"CARMA1 and NFATc1 were identified as Cbl-b substrates: Cbl-b monoubiquitinates CARMA1 to disrupt CARMA1-Bcl10 association and suppress NF-κB (mediating NKT cell anergy), while Cbl-b ubiquitinates NFATc1 for degradation as part of an NFATc1→Nur77→Cbl-b feedback loop limiting osteoclastogenesis.","evidence":"Co-IP; monoubiquitination and polyubiquitination assays; RING finger mutagenesis; Nur77 ChIP; bone phenotyping in KO mice","pmids":["19815501","25257814","26173181"],"confidence":"High","gaps":["Mono- vs. polyubiquitination fate determinants unclear","structural basis of CARMA1-Bcl10 disruption by monoubiquitin not defined"]},{"year":2011,"claim":"A RING finger knock-in mouse definitively proved that all major T-cell regulatory functions of Cbl-b—including tolerance, anergy, autoimmunity prevention, and tumor rejection—require its catalytic E3 ubiquitin ligase activity rather than adaptor functions alone.","evidence":"E3-ligase-dead knock-in mouse phenocopying full KO in autoimmunity, anergy, and tumor rejection assays","pmids":["21248250"],"confidence":"High","gaps":["Whether non-catalytic functions contribute in non-T-cell lineages remained untested"]},{"year":2012,"claim":"An E3-ligase-independent function of Cbl-b was uncovered: Cbl-b suppresses Nedd4-mediated K63-linked polyubiquitination and inactivation of PTEN, providing a distinct, non-catalytic mechanism by which Cbl-b restrains PI3K/Akt signaling in T cells.","evidence":"Pten ubiquitination analysis; Cbl-b/Nedd4 double KO epistasis; K63-linkage-specific assays; Akt activity measurement","pmids":["22763434"],"confidence":"High","gaps":["How Cbl-b physically interferes with Nedd4-Pten interaction not structurally defined","whether this applies beyond T cells unknown"]},{"year":2014,"claim":"TAM receptor tyrosine kinases were identified as Cbl-b ubiquitination substrates in NK cells, establishing that Cbl-b limits NK-cell anti-metastatic activity; a TAM kinase inhibitor phenocopied Cbl-b loss, opening a pharmacological strategy.","evidence":"Ubiquitylation assay; E3-ligase-dead knock-in mice; in vivo metastasis models; TAM kinase inhibitor treatment","pmids":["24553136"],"confidence":"High","gaps":["Which specific ubiquitin chain types on TAM receptors not defined","downstream effectors of TAM stabilization in NK cells incompletely mapped"]},{"year":2015,"claim":"Cbl-b was shown to ubiquitinate Foxp3 (with Stub1) for proteasomal degradation to regulate thymic Treg development, and to promote TrkA ubiquitination and degradation upon NGF stimulation, expanding the substrate repertoire to transcription factor and neurotrophin receptor signaling.","evidence":"Co-IP and ubiquitination assay for Foxp3; genetic epistasis (Cbl-b-/- × CD28-/-); temporal proteomics for TrkA; neurite outgrowth upon Cbl-b depletion","pmids":["25560411","25921289"],"confidence":"High","gaps":["Relative contribution of Cbl-b vs. Stub1 to Foxp3 degradation not quantified","physiological role in neuronal TrkA signaling in vivo not established"]},{"year":2016,"claim":"CBLB was identified as a key negative regulator of innate antifungal immunity by ubiquitinating SYK, dectin-1, dectin-2, and dectin-3 downstream of C-type lectin receptors; Cbl-b targets ubiquitinated CLRs for ESCRT-mediated lysosomal degradation, and loss of Cbl-b protects mice from lethal systemic candidiasis.","evidence":"Ubiquitination assays; Cbl-b KO and double KO (Cbl-b × dectin) epistasis in C. albicans infection models; ESCRT knockdown; CLR trafficking analysis","pmids":["27428901","27428899","27432944"],"confidence":"High","gaps":["Whether pharmacological Cbl-b inhibition recapitulates antifungal protection not tested","role in other fungal pathogens beyond C. albicans not established"]},{"year":2016,"claim":"Quantitative temporal interactomics of TCR-stimulated T cells identified CD5 as a key scaffold for CBLB-mediated ubiquitylation following TCR engagement, providing an unbiased map of the dynamic CBLB signaling complex.","evidence":"AP-MS time course over 600 s of TCR stimulation with biochemical validation","pmids":["27474268"],"confidence":"High","gaps":["Functional consequence of CD5-scaffolded Cbl-b activity not fully dissected","direct vs. indirect interactions in the complex not resolved for all partners"]},{"year":2019,"claim":"TAM receptor-mediated phosphorylation of Cbl-b at Y133 and Y363 was shown to activate Cbl-b in NK cells, with Gas6/TAM ligation triggering Cbl-b-dependent degradation of the adaptor LAT1 to suppress NK-cell effector functions.","evidence":"Gas6 ligation; Y133/Y363 phosphosite mutagenesis; Cbl-b KO NK cells; LAT1 degradation assay","pmids":["31531847"],"confidence":"High","gaps":["Whether Y133/Y363 phosphorylation equally regulates Cbl-b in T cells and myeloid cells not tested","kinase(s) besides TAM receptors that phosphorylate these sites not mapped"]},{"year":2020,"claim":"Cbl-b was found to ubiquitinate NMDA receptor subunit GluN2B in spinal dorsal horn neurons via its TKB domain, with inflammation-induced Y363 dephosphorylation impairing Cbl-b–GluN2B binding and enabling synaptic GluN2B accumulation, linking Cbl-b to nociceptive sensitization.","evidence":"Co-IP and ubiquitination assay; phosphomimetic Y363 mutagenesis; in vivo knockdown; electrophysiology of synaptic NMDAR currents","pmids":["32606037"],"confidence":"High","gaps":["Identity of the phosphatase dephosphorylating Y363 in neurons not determined","behavioral pain outcomes of Cbl-b manipulation not fully characterized"]},{"year":2021,"claim":"CRISPR-mediated Cbl-b deletion was shown to rescue exhausted CD8+ tumor-infiltrating lymphocytes and CAR T cells, reducing PD1+Tim3+ populations and restoring effector function, establishing Cbl-b as a druggable checkpoint for reversing T-cell exhaustion.","evidence":"CRISPR-Cas9 Cbl-b KO in CD8+ T cells and CAR T cells; syngeneic MC38 tumor model; cytokine and killing assays","pmids":["33462140"],"confidence":"High","gaps":["Direct substrate(s) linking Cbl-b to the exhaustion transcriptional program not identified","long-term safety of Cbl-b deletion in therapeutic T cells not assessed"]},{"year":2023,"claim":"A co-crystal structure of Cbl-b with a small-molecule inhibitor revealed that the compound acts as an intramolecular glue bridging the TKBD and LHR domains (not the RING domain), locking Cbl-b in an autoinhibited conformation—providing the first structural basis for pharmacological Cbl-b inhibition.","evidence":"X-ray crystallography; SPR and thermal shift biophysical assays; cellular ubiquitination assay","pmids":["38104184"],"confidence":"High","gaps":["In vivo efficacy and selectivity of TKBD-LHR glue inhibitors not yet demonstrated","whether this mechanism generalizes to other Cbl family members unknown"]},{"year":2023,"claim":"Cbl-b was found to form a TCR-induced complex with the pH-sensitive phosphatase STS1 via a proline motif, with STS1 dephosphorylating Cbl-b-associated phosphoproteins; this complex renders T cells sensitive to acidic tumor microenvironments, and loss of either partner enhances anti-tumor immunity.","evidence":"Co-IP with proline-motif mutagenesis; STS1 and Cbl-b KO mice; pH sensitivity assays; tumor growth experiments","pmids":["38091950"],"confidence":"High","gaps":["Which specific phosphoproteins STS1 dephosphorylates in the Cbl-b complex not mapped","structural basis of pH sensing by STS1-Cbl-b not resolved"]},{"year":null,"claim":"Key remaining questions include: the structural basis of Cbl-b substrate selectivity across its >15 known substrates; whether non-catalytic functions (e.g., PTEN protection) operate broadly beyond T cells; the identity of the E3 ligase(s) that ubiquitinate Cbl-b itself for degradation upon CD28 signaling; and whether pharmacological Cbl-b inhibitors achieve selective immune activation without autoimmune toxicity in vivo.","evidence":"","pmids":[],"confidence":"Low","gaps":["No comprehensive structural model for multi-substrate recognition","E3 ligase targeting Cbl-b for degradation unidentified","in vivo therapeutic window of Cbl-b inhibitors not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,3,4,13,14,16,18,21,22,23,25,28,33,38]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[2,3,16,18,23]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[7,37]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,8,9,17]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,3,14,30]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,31,33]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,1,8,9,10,11,16,18,23,24,27,30,39]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,4,6,17,19,20,34]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,3,13,14,18,21,23,28,38]}],"complexes":["Cbl-b–STS1 complex","Cbl-b–CIN85–endophilin complex","DR5–Cbl-b/c-Cbl–TRAF2 complex"],"partners":["PIK3R1","VAV1","SYK","CLEC7A","CIN85","STS1","NEDD4","GRB2"],"other_free_text":[]},"mechanistic_narrative":"CBLB encodes a RING-type E3 ubiquitin ligase that functions as a central negative regulator of immune cell activation thresholds across T cells, NK cells, and myeloid lineages, while also controlling receptor tyrosine kinase signaling, bone remodeling, and skeletal muscle homeostasis. Its catalytic RING finger domain ubiquitinates a broad substrate repertoire—including PI3K p85, Vav1, PLCγ-1, CARMA1, NFATc1, TAM receptors, SYK, dectin-1/2, IRS-1, GluN2B, Foxp3, EGFR, TrkA, and STAT5—to enforce peripheral T-cell tolerance and anergy, suppress NF-κB and calcium signaling, limit innate antifungal responses, and attenuate growth factor receptor signaling through ubiquitin-dependent degradation or non-degradative signaling modulation [PMID:10646608, PMID:11526404, PMID:15308098, PMID:21248250, PMID:27428901, PMID:19546233, PMID:24553136, PMID:32606037]. Cbl-b activity is itself regulated by CD28-induced auto-ubiquitination and degradation, TAM receptor-mediated phosphorylation at Y133 and Y363, complex formation with the pH-sensitive phosphatase STS1 that tunes T-cell function in acidic tumor microenvironments, and intramolecular conformational control exploited by small-molecule inhibitors that lock the TKBD–LHR interface [PMID:12193687, PMID:31531847, PMID:38091950, PMID:38104184]. Loss of Cbl-b or its E3 ligase activity unleashes CD8+ T-cell and NK-cell anti-tumor immunity, enabling rejection of established tumors and metastases, and reverses T-cell exhaustion in tumor-infiltrating and CAR T-cell settings [PMID:17364027, PMID:24553136, PMID:33462140]."},"prefetch_data":{"uniprot":{"accession":"Q13191","full_name":"E3 ubiquitin-protein ligase CBL-B","aliases":["Casitas B-lineage lymphoma proto-oncogene b","RING finger protein 56","RING-type E3 ubiquitin transferase CBL-B","SH3-binding protein CBL-B","Signal transduction protein CBL-B"],"length_aa":982,"mass_kda":109.5,"function":"E3 ubiquitin-protein ligase which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, and transfers it to substrates, generally promoting their degradation by the proteasome (PubMed:20525694, PubMed:40101708). Negatively regulates TCR (T-cell receptor), BCR (B-cell receptor) and FCER1 (high affinity immunoglobulin epsilon receptor) signal transduction pathways (PubMed:40101708). In naive T-cells, inhibits VAV1 activation upon TCR engagement and imposes a requirement for CD28 costimulation for proliferation and IL-2 production (By similarity). Also acts by promoting PIK3R1/p85 ubiquitination, which impairs its recruitment to the TCR and subsequent activation (PubMed:11087752, PubMed:11526404). In activated T-cells, inhibits PLCG1 activation and calcium mobilization upon restimulation and promotes anergy (By similarity). 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:39030301, PubMed:40101708). In B-cells, acts by ubiquitinating SYK and promoting its proteasomal degradation (By similarity). Slightly promotes SRC ubiquitination (PubMed:14661060). May be involved in EGFR ubiquitination and internalization (PubMed:10086340). May be functionally coupled with the E2 ubiquitin-protein ligase UB2D3. In association with CBL, 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","url":"https://www.uniprot.org/uniprotkb/Q13191/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CBLB","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CBLB","total_profiled":1310},"omim":[{"mim_id":"620953","title":"METHYLMALONIC ACIDURIA, cblD TYPE; MACD","url":"https://www.omim.org/entry/620953"},{"mim_id":"620430","title":"AUTOIMMUNE DISEASE, MULTISYSTEM, INFANTILE-ONSET, 3; ADMIO3","url":"https://www.omim.org/entry/620430"},{"mim_id":"615952","title":"AUTOIMMUNE DISEASE, MULTISYSTEM, INFANTILE-ONSET, 1; ADMIO1","url":"https://www.omim.org/entry/615952"},{"mim_id":"609201","title":"UBIQUITIN-ASSOCIATED AND SH3 DOMAIN-CONTAINING PROTEIN B; UBASH3B","url":"https://www.omim.org/entry/609201"},{"mim_id":"608453","title":"CAS-BR-M MURINE ECOTROPIC RETROVIRAL TRANSFORMING SEQUENCE C; CBLC","url":"https://www.omim.org/entry/608453"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CBLB"},"hgnc":{"alias_symbol":["RNF56","Cbl-b"],"prev_symbol":[]},"alphafold":{"accession":"Q13191","domains":[{"cath_id":"1.20.930.20","chopping":"36-168","consensus_level":"medium","plddt":90.7944,"start":36,"end":168},{"cath_id":"1.10.238.10","chopping":"169-257","consensus_level":"medium","plddt":94.2811,"start":169,"end":257},{"cath_id":"3.30.40.10","chopping":"352-425","consensus_level":"high","plddt":88.2638,"start":352,"end":425},{"cath_id":"1.10.8.10","chopping":"932-971","consensus_level":"high","plddt":89.7442,"start":932,"end":971}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13191","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13191-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13191-F1-predicted_aligned_error_v6.png","plddt_mean":61.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CBLB","jax_strain_url":"https://www.jax.org/strain/search?query=CBLB"},"sequence":{"accession":"Q13191","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13191.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13191/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13191"}},"corpus_meta":[{"pmid":"10646608","id":"PMC_10646608","title":"Negative 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\"confidence_rationale\": \"Tier 2 — two independent labs (PMID:10646608 and PMID:10646609) with complementary genetic and biochemical evidence, replicated\",\n      \"pmids\": [\"10646608\", \"10646609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Cbl-b selectively suppresses TCR-mediated Vav (guanine nucleotide exchange factor for Rac1/Rho/CDC42) activation to regulate CD28 dependence of T-cell activation; Cbl-b-null mutation fully restores T-cell-dependent antibody responses in CD28-deficient mice.\",\n      \"method\": \"Genetic epistasis (cbl-b-/- × CD28-/- double mutant mice); Vav activation assays; antibody response measurements\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis experiment across two genetic backgrounds with replicated finding\",\n      \"pmids\": [\"10646609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Cbl-b interacts with and induces ubiquitin conjugation to the p85 regulatory subunit of PI3K; the RING finger domain is essential for p85 ubiquitination, and a distal proline-rich region of Cbl-b binds the SH3 domain of p85.\",\n      \"method\": \"Co-immunoprecipitation; in vitro ubiquitination assay; RING finger loss-of-function mutagenesis; deletion mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro ubiquitination assay with mutagenesis; replicated by PMID:11526404\",\n      \"pmids\": [\"11087752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cbl-b negatively regulates p85 (PI3K regulatory subunit) in a proteolysis-independent manner by controlling its recruitment to CD28 and TCR-zeta, requiring Cbl-b E3 ubiquitin ligase activity; enhanced activation of Cbl-b-/- T cells is suppressed by PI3K inhibition.\",\n      \"method\": \"Ubiquitination assay; PI3K inhibitor rescue; co-immunoprecipitation with CD28 and TCR-zeta\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro ligase assay with functional rescue by PI3K inhibition; strong mechanistic follow-up\",\n      \"pmids\": [\"11526404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Cbl-b is ubiquitinated and degraded upon EGFR activation; EGF-induced Cbl-b degradation requires intact RING finger and tyrosine kinase binding domains and binding to activated EGFR; Cbl-b mediates coordinated degradation of the EGFR signaling complex including Grb2 and Shc via lysosomal and proteasomal pathways.\",\n      \"method\": \"EGF stimulation; RING finger and TKB domain mutagenesis; lysosomal/proteasomal inhibitors; Western blot for complex components\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — mutagenesis with pharmacological pathway dissection, rigorous mechanistic follow-up\",\n      \"pmids\": [\"11375397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CD28 co-stimulation selectively induces greater ubiquitination and degradation of Cbl-b than CD3 stimulation alone, establishing that CD28 regulates the T-cell activation threshold partly by promoting Cbl-b ubiquitination and degradation.\",\n      \"method\": \"Ubiquitination assay; stimulation of wild-type vs. CD28-deficient T cells; Western blot for Cbl-b levels\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic biochemical experiment with genetic control (CD28-/- cells)\",\n      \"pmids\": [\"12193687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CIN85 binds to the carboxyl terminus of Cbl-b; ligand-induced phosphorylation of Cbl-b increases CIN85 interaction; CIN85–Cbl-b binding is required for Cbl-b-mediated EGFR internalization (but dispensable for EGFR polyubiquitination), defining a CIN85/endophilin-dependent endocytic pathway.\",\n      \"method\": \"Co-immunoprecipitation; dominant-negative CIN85 interference; EGFR internalization assay; ubiquitination assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal pulldown plus functional internalization assay with mechanistic dissection\",\n      \"pmids\": [\"12177062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Cbl-b positively regulates BCR-mediated Ca2+ signaling by interacting with PLC-γ2 and facilitating its association with Btk and BLNK; Cbl-b is required for Btk-dependent sustained intracellular Ca2+ increase; both N-terminal TKB domain and C-terminal half of Cbl-b are essential.\",\n      \"method\": \"Cbl-b-deficient DT40 B cells; overexpression in WEHI-231 cells; Ca2+ mobilization assay; co-immunoprecipitation of PLC-γ2/Btk/BLNK complex; domain deletion mutagenesis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — loss-of-function and gain-of-function with biochemical complex analysis in two cell systems\",\n      \"pmids\": [\"12093870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cbl-b is upregulated in T cells after tolerizing signals and is essential for T-cell anergy induction; loss of Cbl-b rescues reduced calcium mobilization of anergic T cells, attributable to Cbl-b-mediated regulation of PLCγ-1 phosphorylation.\",\n      \"method\": \"In vitro and in vivo T cell tolerance assays in Cbl-b-/- mice; calcium mobilization assay; PLCγ-1 phosphorylation analysis\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with defined molecular readout (PLCγ-1 phosphorylation and Ca2+ flux), replicated in vivo\",\n      \"pmids\": [\"15308098\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cbl-b-/- effector T cells are resistant to suppression by CD4+CD25+ regulatory T cells and by TGF-β in vitro, despite normal levels of TGF-β receptor II and normal Smad3 phosphorylation, indicating the resistance operates downstream of TGF-β receptor signaling.\",\n      \"method\": \"In vitro T cell suppression assays; TGF-β receptor expression analysis; Smad3 phosphorylation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined phenotype but partial mechanistic placement (resistance downstream of Smad3)\",\n      \"pmids\": [\"15240694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Cblb-/- CD8+ T cells are resistant to TGF-β suppression and do not require CD28 costimulation for activation in vitro; in vivo, Cblb-/- mice efficiently reject B7-negative tumors via massively infiltrating CD8+ T cells; adoptive transfer of purified Cblb-/- CD8+ T cells eradicates established tumors.\",\n      \"method\": \"Knockout mice; in vitro TGF-β suppression assay; tumor challenge with B7-negative cell lines; adoptive transfer of CD8+ T cells\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple in vitro and in vivo orthogonal methods with defined molecular mechanism\",\n      \"pmids\": [\"17364027\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Cblb controls the association between TLR4 and the intracellular adaptor MyD88 and regulates TLR4 surface expression on neutrophils; E3 ligase activity (not adaptor function) of Cblb is required to suppress NF-κB-mediated inflammatory responses to LPS.\",\n      \"method\": \"Co-immunoprecipitation of TLR4-MyD88; NF-κB reporter assay with wild-type vs. RING-finger-mutant Cblb; flow cytometry of TLR4 surface levels on Cblb-/- neutrophils\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic mutagenesis (RING finger mutant) plus biochemical and functional assays\",\n      \"pmids\": [\"17618294\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Cbl-b interacts with Pseudomonas exotoxin T (ExoT) and promotes its polyubiquitination and proteasomal degradation in host cells; Cbl-b (but not c-Cbl) is specifically required in vivo to limit bacterial dissemination mediated by ExoT.\",\n      \"method\": \"Co-immunoprecipitation of Cbl-b with ExoT and Crk; proteasomal inhibitor experiments; in vivo mouse infection models with Cblb-/- mice\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical pulldown plus in vivo genetic specificity (c-Cbl plays no role)\",\n      \"pmids\": [\"17235393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Upon unloading stress, Cbl-b is induced in skeletal muscle and directly ubiquitinates IRS-1, targeting it for degradation; loss of IRS-1 activates FOXO3-dependent atrogin-1/MAFbx induction; Cbl-b-/- mice are resistant to unloading-induced atrophy.\",\n      \"method\": \"Co-immunoprecipitation of Cbl-b with IRS-1; ubiquitination assay; KO mice subjected to hindlimb unloading; peptide inhibitor (pY608-IRS-1 mimetic) blocks IRS-1 ubiquitination\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro ubiquitination plus KO plus peptide inhibitor, multiple orthogonal methods\",\n      \"pmids\": [\"19546233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cbl-b interacts with CARMA1 and promotes its monoubiquitination; this monoubiquitination disrupts CARMA1-Bcl10 complex formation (without affecting CARMA1 stability), thereby suppressing NF-κB activation and mediating NKT cell anergy; Cbl-b RING finger activity is critical.\",\n      \"method\": \"Co-immunoprecipitation; monoubiquitination assay; RING finger mutant analysis; CARMA1-/- NKT cell functional analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — ubiquitination assay with mutagenesis plus complex disruption and genetic validation in KO cells\",\n      \"pmids\": [\"19815501\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Loss of Cbl-b increases osteoclast bone-resorbing activity and in vitro differentiation, and enhances RANKL-induced NF-κB, ERK, and p38 signaling; rescue by re-expressing Cbl-b (but not by c-Cbl) requires functional TKB and RING finger domains.\",\n      \"method\": \"Cbl-b-/- mice; in vitro bone resorption assay; RANKL signaling analysis; rescue by wild-type vs. domain-mutant Cbl-b re-expression\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO phenotype with domain-mutant rescue establishing mechanistic requirements\",\n      \"pmids\": [\"19257814\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Selective genetic inactivation of Cbl-b E3 ligase activity (RING finger knock-in mutation) phenocopies total Cbl-b ablation in T-cell hyperactivation, spontaneous autoimmunity, impaired T-cell anergy, and spontaneous tumor rejection, demonstrating the catalytic E3 ligase activity is essential for all Cbl-b T-cell regulatory functions in vivo.\",\n      \"method\": \"RING finger knock-in mouse (E3 ligase-dead); comparison with total Cbl-b KO; autoimmunity scoring; tumor rejection assays; T-cell anergy induction assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — knock-in mutagenesis in vivo phenocopying KO, definitive proof of catalytic requirement\",\n      \"pmids\": [\"21248250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cbl-b suppresses Pten inactivation (rather than directly inhibiting PI3K) in T cells by impeding Nedd4-mediated K63-linked polyubiquitination of Pten at K13; elevated Akt in Cbl-b-/- T cells is due to heightened Pten inactivation; this function is independent of Cbl-b's own E3 ligase activity.\",\n      \"method\": \"Pten ubiquitination analysis; Nedd4 co-immunoprecipitation; double KO (Cbl-b-/- × Nedd4-/-); Akt activity measurement; ubiquitin linkage-specific assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis (double KO rescue) plus biochemical ubiquitination mechanism, multiple orthogonal approaches\",\n      \"pmids\": [\"22763434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cbl-b ubiquitylates TAM receptor tyrosine kinases (Tyro3, Axl, Mertk) in NK cells, limiting NK cell anti-metastatic activity; genetic deletion or E3 ligase inactivation of Cbl-b licenses NK cells to reject metastatic tumors; a TAM kinase inhibitor phenocopies Cbl-b loss.\",\n      \"method\": \"Ubiquitylation assay identifying TAM receptors as Cbl-b substrates; E3-ligase-dead knock-in mice; in vivo tumor metastasis models; TAM kinase inhibitor treatment\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — substrate identification by ubiquitylation assay with genetic and pharmacological validation in vivo\",\n      \"pmids\": [\"24553136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cbl-b ubiquitylates IGF-IR in gastric cancer cells leading to its degradation, thereby inhibiting the Akt/ERK-miR-200c-ZEB2 axis and suppressing IGF-I-induced epithelial-mesenchymal transition.\",\n      \"method\": \"Cbl-b shRNA knockdown; co-immunoprecipitation; ubiquitination assay; Akt/ERK inhibitors; miR-200c and ZEB2 expression analysis\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab with co-IP and functional assays but limited orthogonal confirmation\",\n      \"pmids\": [\"24885194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Upon NGF stimulation of neuroblastoma cells, TrkA recruits Cbl-b, which becomes phosphorylated and ubiquitylated; Cbl-b promotes TrkA ubiquitylation and degradation; Cbl-b depletion increases ERK phosphorylation and neurite outgrowth.\",\n      \"method\": \"Quantitative interactome/proteome/phosphoproteome mass spectrometry; Cbl-b depletion; neurite outgrowth measurement\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — temporal proteomics with KD rescue, multiple orthogonal datasets\",\n      \"pmids\": [\"25921289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Cbl-b binds Foxp3 upon TCR stimulation and, together with Stub1, targets Foxp3 for ubiquitination and proteasomal degradation, thereby regulating thymic Treg development; proteasome inhibition rescues defective tTreg development in CD28-/- mice.\",\n      \"method\": \"Co-immunoprecipitation of Cbl-b with Foxp3; ubiquitination assay; Cbl-b-/- × CD28-/- genetic rescue; proteasome inhibitor rescue of tTreg development\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical ubiquitination assay plus genetic epistasis plus pharmacological rescue\",\n      \"pmids\": [\"25560411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"NFATc1 induces Nur77 expression during osteoclast differentiation; Nur77 transcriptionally upregulates Cbl-b; Cbl-b then triggers NFATc1 protein degradation via ubiquitination, creating a self-limiting NFATc1→Nur77→Cbl-b→NFATc1 feedback loop that prevents excessive osteoclastogenesis.\",\n      \"method\": \"Genetic KO (Nur77-/-); ChIP/reporter assay linking Nur77 to Cbl-b promoter; ubiquitination assay showing Cbl-b ubiquitinates NFATc1; bone mass phenotyping\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (transcription assay, ubiquitination, KO phenotype) establishing a complete pathway\",\n      \"pmids\": [\"26173181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CBLB associates with SYK and ubiquitinates SYK, dectin-1, and dectin-2 following fungal recognition via C-type lectin receptors; CBLB deficiency results in increased inflammasome activation, enhanced ROS production, and enhanced fungal killing.\",\n      \"method\": \"Co-immunoprecipitation of CBLB with SYK/dectin-1/dectin-2; ubiquitination assay; Cblb-/- macrophage/DC functional assays; lethal C. albicans infection model\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — substrate identification by ubiquitination assay replicated in vivo with KO mice; confirmed by companion paper PMID:27428899\",\n      \"pmids\": [\"27428901\", \"27428899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CBLB directs polyubiquitination of dectin-1 and dectin-2 and their downstream kinase SYK, inhibiting dectin-mediated innate immune responses; dectin-1 and dectin-2 deficiency in Cblb-/- mice abrogates the protection against systemic C. albicans infection.\",\n      \"method\": \"Ubiquitination assay; genetic epistasis (Cblb-/- × dectin-1-/- or dectin-2-/- double KO); in vivo lethal C. albicans infection model\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis experiment with KO rescue plus substrate ubiquitination assay, replicated by PMID:27428901\",\n      \"pmids\": [\"27428899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cbl-b mediates ubiquitination of activated Dectin-2 and Dectin-3 via adapter FcR-γ and kinase Syk in a Syk-dependent manner; ubiquitinated CLRs are sorted into lysosomes via the ESCRT system for degradation.\",\n      \"method\": \"Co-immunoprecipitation of Cbl-b with FcR-γ/Syk/CLRs; ubiquitination assay; ESCRT subunit knockdown; CLR degradation analysis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic ubiquitination and trafficking pathway established with multiple components\",\n      \"pmids\": [\"27432944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TCR stimulation dynamics of CBL and CBLB signaling complexes were characterized by affinity purification coupled to quantitative mass spectrometry; CD5 transmembrane receptor was identified as a key scaffold for CBLB-mediated ubiquitylation following TCR engagement.\",\n      \"method\": \"Affinity purification coupled to quantitative MS over 600 s TCR stimulation time course; biochemical validation of CD5 as a scaffold\",\n      \"journal\": \"Molecular systems biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — unbiased quantitative interactome with biochemical validation, strong methodology\",\n      \"pmids\": [\"27474268\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TAM receptors (Tyro3, Axl, Mer) phosphorylate Cbl-b at tyrosine residues Y133 and Y363 to activate it; Gas6-mediated TAM receptor ligation suppresses NK-cell functions (IFN-γ production, degranulation) via Cbl-b; Cbl-b promotes degradation of LAT1 (transmembrane adaptor) in response to Gas6.\",\n      \"method\": \"TAM receptor ligation with Gas6; phosphorylation site mutagenesis (Y133, Y363); Cbl-b KO NK cells; LAT1 degradation assay\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — phosphorylation site mutagenesis with KO genetic validation and defined substrate (LAT1)\",\n      \"pmids\": [\"31531847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cbl-b is enriched in spinal cord dorsal horn neurons; it interacts with GluN2B via its N-terminal TKB domain and ubiquitinates GluN2B, reducing synaptic GluN2B-containing NMDARs; peripheral inflammation induces dephosphorylation of Cbl-b at Y363, impairing its binding to and ubiquitylation of GluN2B and enabling GluN2B reappearance at synapses.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; in vivo Cbl-b knockdown; phosphomimetic Cbl-b mutant expression; electrophysiology of synaptic currents\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical substrate identification with mutagenesis plus in vivo electrophysiology\",\n      \"pmids\": [\"32606037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The co-crystal structure of Cbl-b with inhibitor C7683 (analogue of Nx-1607) reveals the compound binds both the TKBD and LHR domains but not the RING domain, locking Cbl-b in an inactive conformation by acting as an intramolecular glue; confirmed by biophysical assays.\",\n      \"method\": \"X-ray crystallography (co-crystal structure); biophysical binding assays (SPR, thermal shift); cellular ubiquitination assay\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with biophysical and cellular validation\",\n      \"pmids\": [\"38104184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TCR stimulation induces a molecular complex between Cbl-b and the pH-sensitive unconventional phosphatase STS1 via a proline motif in Cbl-b interacting with the STS1 SH3 domain; STS1 dephosphorylates Cbl-b-interacting phosphoproteins; deficiency of STS1 or Cbl-b reduces T-cell sensitivity to acidic environments and promotes anti-tumor T-cell activity.\",\n      \"method\": \"Co-immunoprecipitation; STS1 and Cbl-b KO mice; proline-motif mutagenesis; in vitro and in vivo pH sensitivity assays; tumor growth experiments\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical complex characterization with mutagenesis and dual KO genetic validation in vivo\",\n      \"pmids\": [\"38091950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cbl-b undergoes tyrosine phosphorylation and plasma membrane translocation in response to insulin in adipocytes; it constitutively associates with CAP and interacts with Crk upon insulin stimulation; Cbl proteins form homo- and heterodimers via a conserved leucine zipper domain required for APS interaction and tyrosine phosphorylation.\",\n      \"method\": \"Phosphorylation assays in 3T3-L1 adipocytes; co-immunoprecipitation with CAP and Crk; leucine zipper dimerization mutagenesis; membrane fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP and mutagenesis in cell line model, single lab\",\n      \"pmids\": [\"12842890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cbl-b is upregulated in osteoblastic cells after denervation and ubiquitinates IRS-1, promoting its degradation, thereby suppressing IGF-I signaling (PI3K, Akt-1) in osteoblasts and causing reduced bone formation; Cbl-b-/- mice maintain bone mass after denervation.\",\n      \"method\": \"Cbl-b-/- denervation model; IRS-1 ubiquitination assay in primary osteoblasts; Western blot for IGF-I signaling; bone histomorphometry\",\n      \"journal\": \"Journal of bone and mineral research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO with defined molecular mechanism (IRS-1 ubiquitination) confirmed in primary cells\",\n      \"pmids\": [\"16734387\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cbl-b and Cbl-b interact with EGFR through distinct modes: Cbl-b preferentially binds pY1045 of EGFR directly, while c-Cbl relies mainly on Grb2-dependent indirect binding; each operates independently to jointly control EGFR ubiquitination, endocytic trafficking, and chemotaxis signaling.\",\n      \"method\": \"Inducible expression of E3-ligase-dead Cbl-b and c-Cbl mutants; EGFR ubiquitination and degradation assays; chemotaxis migration assay; cells with differential Cbl-b endogenous levels\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific mutagenesis with functional readouts across multiple cell lines\",\n      \"pmids\": [\"37903221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Cbl-b is phosphorylated and recruited to EGFR upon EGF stimulation and binds Grb2; overexpression of Cbl-b in EGFR-dependent cells decreases AKT activation amplitude/duration and shortens MAP kinase/JNK activation, inhibiting EGF-induced cell growth and promoting apoptosis.\",\n      \"method\": \"Overexpression in 32D/EGFR cells; growth assays; AKT and MAPK activation time course; apoptosis assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function overexpression with kinase activation readouts, single lab\",\n      \"pmids\": [\"10086340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"CBLB is phosphorylated on tyrosine following FLT3 ligand or IL-7 stimulation in pro-B cells; tyrosine-phosphorylated SHC and p85 of PI3K associate with CBLB upon FL or IL-7 treatment; CBLB constitutively binds GRB2 predominantly through GRB2's N-terminal SH3 domain.\",\n      \"method\": \"Tyrosine phosphorylation assay; co-immunoprecipitation of SHC, p85, GRB2 with CBLB\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP and phosphorylation analysis in hematopoietic cell lines, single lab\",\n      \"pmids\": [\"9614102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"CBLB forms a complex with CD81 and calpain-5 (CAPN5) in human liver cells; CBLB is required for a post-binding, pre-replication step in HCV entry; CBLB knockout reduces susceptibility to all tested HCV genotypes but not to other enveloped viruses.\",\n      \"method\": \"Quantitative proteomics of CD81 interactome; CBLB/CAPN5 knockout; HCV infection assay across genotypes\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proteomics interactome with KO validation and specificity controls, single lab\",\n      \"pmids\": [\"30024968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"DR5-Cbl-b/c-Cbl-TRAF2 form a complex in TRAIL-resistant gastric cancer cells; Cbl-b and c-Cbl serve as adaptors linking DR5 to TRAF2; TRAIL-induced caspase-8 translocation into this complex leads to TRAF2-mediated K48-linked polyubiquitination and proteasomal degradation of caspase-8, inhibiting apoptosis.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay (K48-linkage-specific); TRAF2 and Cbl-b knockdown; proteasome inhibitor (bortezomib) rescue\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical complex identification with linkage-specific ubiquitination and genetic validation, single lab\",\n      \"pmids\": [\"28972304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cbl-b and c-Cbl ubiquitinate STAT5a and STAT5b in dendritic cells to promote their degradation and cell apoptosis; Cbl-b mediates K27-linked ubiquitination of STAT5a at K164, while c-Cbl induces K29-linked ubiquitination of STAT5a K696 and K27-linked ubiquitination of STAT5b K140 and K694.\",\n      \"method\": \"DC-specific double KO mice (Cbl-b-/-×c-Cblflox/flox CD11c-Cre); STAT5 ubiquitination assays with linkage-specific ubiquitin mutants; site-directed mutagenesis of STAT5 lysine residues\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assay with linkage and site mutagenesis, but single lab\",\n      \"pmids\": [\"35354799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Cbl-b is upregulated in exhausted (PD1+Tim3+) CD8+ tumor-infiltrating lymphocytes; CRISPR-Cas9-mediated deletion of Cbl-b in CD8+ T cells and CAR T cells restores effector function of exhausted cells, reduces PD1+Tim3+ populations, and increases IFN-γ/TNF-α production and tumor cell killing.\",\n      \"method\": \"RNA-seq of TILs; syngeneic MC38 tumor model; CRISPR-Cas9 Cbl-b KO in CAR T cells; tumor growth and cytokine assays\",\n      \"journal\": \"Journal for immunotherapy of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — CRISPR KO with functional readouts in both endogenous and CAR T cells, multiple tumor models\",\n      \"pmids\": [\"33462140\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CBLB encodes a RING-type E3 ubiquitin ligase that acts as a master negative regulator of immune cell activation by ubiquitinating and/or modulating a diverse set of substrates—including PI3K p85, IRS-1, PLCγ-1, Vav1, TAM receptors, SYK, dectin-1/2, CARMA1, NFATc1, GluN2B, Foxp3, and EGFR/TrkA—downstream of antigen, growth-factor, and pattern-recognition receptors; its catalytic RING finger activity and adaptor functions (via TKB, leucine zipper, and proline-rich domains) set T-cell, NK-cell, and myeloid activation thresholds, enforce peripheral T-cell tolerance and anergy, regulate bone remodeling and muscle mass, control innate antifungal immunity, and suppress tumor immune evasion, with the protein itself subject to regulation by CD28-induced auto-ubiquitination, TAM receptor-mediated phosphorylation (Y133, Y363), and complex formation with the pH-sensitive phosphatase STS1.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CBLB encodes a RING-type E3 ubiquitin ligase that functions as a central negative regulator of immune cell activation thresholds across T cells, NK cells, and myeloid lineages, while also controlling receptor tyrosine kinase signaling, bone remodeling, and skeletal muscle homeostasis. Its catalytic RING finger domain ubiquitinates a broad substrate repertoire—including PI3K p85, Vav1, PLCγ-1, CARMA1, NFATc1, TAM receptors, SYK, dectin-1/2, IRS-1, GluN2B, Foxp3, EGFR, TrkA, and STAT5—to enforce peripheral T-cell tolerance and anergy, suppress NF-κB and calcium signaling, limit innate antifungal responses, and attenuate growth factor receptor signaling through ubiquitin-dependent degradation or non-degradative signaling modulation [PMID:10646608, PMID:11526404, PMID:15308098, PMID:21248250, PMID:27428901, PMID:19546233, PMID:24553136, PMID:32606037]. Cbl-b activity is itself regulated by CD28-induced auto-ubiquitination and degradation, TAM receptor-mediated phosphorylation at Y133 and Y363, complex formation with the pH-sensitive phosphatase STS1 that tunes T-cell function in acidic tumor microenvironments, and intramolecular conformational control exploited by small-molecule inhibitors that lock the TKBD–LHR interface [PMID:12193687, PMID:31531847, PMID:38091950, PMID:38104184]. Loss of Cbl-b or its E3 ligase activity unleashes CD8+ T-cell and NK-cell anti-tumor immunity, enabling rejection of established tumors and metastases, and reverses T-cell exhaustion in tumor-infiltrating and CAR T-cell settings [PMID:17364027, PMID:24553136, PMID:33462140].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Initial biochemical work established that Cbl-b is phosphorylated upon growth factor (EGF, FLT3L, IL-7) stimulation and associates with key signaling adaptors (Grb2, SHC, PI3K p85), positioning it as a signaling hub downstream of receptor tyrosine kinases before its E3 ligase function was appreciated.\",\n      \"evidence\": \"Co-immunoprecipitation and phosphorylation assays in hematopoietic and EGFR-expressing cell lines\",\n      \"pmids\": [\"9614102\", \"10086340\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase activity not yet tested\", \"overexpression system without loss-of-function validation\", \"physiological relevance in primary cells unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Two independent knockout studies revealed that Cbl-b sets the CD28 co-stimulation requirement for T-cell activation by suppressing Vav1 phosphorylation, establishing Cbl-b as the critical gatekeeper of peripheral T-cell activation thresholds.\",\n      \"evidence\": \"Gene-targeted Cbl-b KO mice; genetic epistasis with CD28-/- mice; Vav activation and T-cell proliferation/cytokine assays\",\n      \"pmids\": [\"10646608\", \"10646609\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic mechanism on Vav not defined\", \"whether Cbl-b ubiquitinates Vav directly or acts indirectly remained unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of PI3K p85 as a direct Cbl-b ubiquitination substrate revealed a RING-finger-dependent, proteolysis-independent mechanism whereby Cbl-b controls PI3K recruitment to CD28 and TCRζ, explaining how Cbl-b restrains PI3K/Akt signaling in T cells.\",\n      \"evidence\": \"In vitro ubiquitination assay with RING finger mutagenesis; PI3K inhibitor rescue of Cbl-b-/- T-cell hyperactivation; co-IP with CD28/TCRζ\",\n      \"pmids\": [\"11087752\", \"11526404\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Non-degradative ubiquitin signal on p85 not fully characterized (chain type unknown)\", \"whether this is the sole PI3K-regulatory mechanism of Cbl-b was unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstration that Cbl-b coordinates EGFR signaling complex degradation through its TKB and RING domains, and that it is itself degraded upon EGFR activation, established Cbl-b as a self-limiting negative regulator of receptor tyrosine kinase signaling beyond the immune system.\",\n      \"evidence\": \"EGF stimulation with RING/TKB mutagenesis; lysosomal and proteasomal inhibitors; Western blot for EGFR/Grb2/Shc complex degradation\",\n      \"pmids\": [\"11375397\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of Cbl-b vs. c-Cbl to EGFR degradation not resolved\", \"in vivo relevance in epithelial tissues not tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"CD28 co-stimulation was shown to selectively induce Cbl-b ubiquitination and degradation, establishing a feedforward mechanism: CD28 removes Cbl-b to lower the activation threshold, while CIN85 binding to Cbl-b was found necessary for receptor internalization but dispensable for polyubiquitination.\",\n      \"evidence\": \"Ubiquitination assay comparing CD3 vs. CD28 stimulation in WT and CD28-/- T cells; CIN85 dominant-negative and EGFR internalization assay\",\n      \"pmids\": [\"12193687\", \"12177062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the E3 ligase ubiquitinating Cbl-b itself not determined\", \"CIN85-Cbl-b function in T cells (vs. EGFR context) not tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Discovery that Cbl-b positively regulates BCR-mediated calcium signaling by scaffolding PLC-γ2/Btk/BLNK complexes revealed a context-dependent positive signaling role, contrasting with its inhibitory function in T cells.\",\n      \"evidence\": \"Cbl-b-deficient DT40 B cells; gain-of-function in WEHI-231; Ca2+ mobilization; co-IP of PLCγ2/Btk/BLNK; domain deletion mutagenesis\",\n      \"pmids\": [\"12093870\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this positive role depends on E3 ligase activity or adaptor function was not resolved\", \"relevance to primary mammalian B cells not confirmed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Cbl-b was established as essential for T-cell anergy induction and identified as a mediator of regulatory T-cell suppression: tolerizing signals upregulate Cbl-b to suppress PLCγ-1 phosphorylation and calcium mobilization, while Cbl-b-/- T cells escape Treg- and TGF-β-mediated suppression.\",\n      \"evidence\": \"In vitro/in vivo tolerance assays in Cbl-b KO mice; PLCγ-1 phosphorylation and Ca2+ flux; Treg suppression and TGF-β assays with Smad3 analysis\",\n      \"pmids\": [\"15308098\", \"15240694\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrate linking Cbl-b to TGF-β resistance not identified\", \"mechanism downstream of Smad3 not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Cbl-b was shown to ubiquitinate IRS-1 for degradation in osteoblasts after denervation and later in skeletal muscle upon unloading, establishing a conserved Cbl-b→IRS-1 degradation axis that suppresses IGF-I/PI3K/Akt signaling to cause bone loss and muscle atrophy.\",\n      \"evidence\": \"Cbl-b KO denervation/unloading models; IRS-1 ubiquitination assay in primary cells; bone histomorphometry; peptide inhibitor blocking IRS-1 ubiquitination\",\n      \"pmids\": [\"16734387\", \"19546233\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Cbl-b directly ubiquitinates IRS-1 or requires cofactors not fully resolved\", \"therapeutic utility of peptide inhibitor in vivo not established\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"The biological consequence of Cbl-b loss for tumor immunity was demonstrated: Cbl-b-/- CD8+ T cells reject B7-negative tumors and resist TGF-β, while Cbl-b's RING finger E3 ligase activity was shown to suppress TLR4-MyD88 association and NF-κB signaling in innate immune cells.\",\n      \"evidence\": \"Tumor challenge and adoptive transfer in KO mice; TLR4-MyD88 co-IP; RING finger mutant NF-κB reporter assay; neutrophil TLR4 surface expression\",\n      \"pmids\": [\"17364027\", \"17618294\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct TLR4/MyD88 ubiquitination substrate identity not confirmed\", \"whether TGF-β resistance involves a direct Cbl-b substrate was unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"CARMA1 and NFATc1 were identified as Cbl-b substrates: Cbl-b monoubiquitinates CARMA1 to disrupt CARMA1-Bcl10 association and suppress NF-κB (mediating NKT cell anergy), while Cbl-b ubiquitinates NFATc1 for degradation as part of an NFATc1→Nur77→Cbl-b feedback loop limiting osteoclastogenesis.\",\n      \"evidence\": \"Co-IP; monoubiquitination and polyubiquitination assays; RING finger mutagenesis; Nur77 ChIP; bone phenotyping in KO mice\",\n      \"pmids\": [\"19815501\", \"25257814\", \"26173181\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mono- vs. polyubiquitination fate determinants unclear\", \"structural basis of CARMA1-Bcl10 disruption by monoubiquitin not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"A RING finger knock-in mouse definitively proved that all major T-cell regulatory functions of Cbl-b—including tolerance, anergy, autoimmunity prevention, and tumor rejection—require its catalytic E3 ubiquitin ligase activity rather than adaptor functions alone.\",\n      \"evidence\": \"E3-ligase-dead knock-in mouse phenocopying full KO in autoimmunity, anergy, and tumor rejection assays\",\n      \"pmids\": [\"21248250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether non-catalytic functions contribute in non-T-cell lineages remained untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"An E3-ligase-independent function of Cbl-b was uncovered: Cbl-b suppresses Nedd4-mediated K63-linked polyubiquitination and inactivation of PTEN, providing a distinct, non-catalytic mechanism by which Cbl-b restrains PI3K/Akt signaling in T cells.\",\n      \"evidence\": \"Pten ubiquitination analysis; Cbl-b/Nedd4 double KO epistasis; K63-linkage-specific assays; Akt activity measurement\",\n      \"pmids\": [\"22763434\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Cbl-b physically interferes with Nedd4-Pten interaction not structurally defined\", \"whether this applies beyond T cells unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"TAM receptor tyrosine kinases were identified as Cbl-b ubiquitination substrates in NK cells, establishing that Cbl-b limits NK-cell anti-metastatic activity; a TAM kinase inhibitor phenocopied Cbl-b loss, opening a pharmacological strategy.\",\n      \"evidence\": \"Ubiquitylation assay; E3-ligase-dead knock-in mice; in vivo metastasis models; TAM kinase inhibitor treatment\",\n      \"pmids\": [\"24553136\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific ubiquitin chain types on TAM receptors not defined\", \"downstream effectors of TAM stabilization in NK cells incompletely mapped\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Cbl-b was shown to ubiquitinate Foxp3 (with Stub1) for proteasomal degradation to regulate thymic Treg development, and to promote TrkA ubiquitination and degradation upon NGF stimulation, expanding the substrate repertoire to transcription factor and neurotrophin receptor signaling.\",\n      \"evidence\": \"Co-IP and ubiquitination assay for Foxp3; genetic epistasis (Cbl-b-/- × CD28-/-); temporal proteomics for TrkA; neurite outgrowth upon Cbl-b depletion\",\n      \"pmids\": [\"25560411\", \"25921289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of Cbl-b vs. Stub1 to Foxp3 degradation not quantified\", \"physiological role in neuronal TrkA signaling in vivo not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"CBLB was identified as a key negative regulator of innate antifungal immunity by ubiquitinating SYK, dectin-1, dectin-2, and dectin-3 downstream of C-type lectin receptors; Cbl-b targets ubiquitinated CLRs for ESCRT-mediated lysosomal degradation, and loss of Cbl-b protects mice from lethal systemic candidiasis.\",\n      \"evidence\": \"Ubiquitination assays; Cbl-b KO and double KO (Cbl-b × dectin) epistasis in C. albicans infection models; ESCRT knockdown; CLR trafficking analysis\",\n      \"pmids\": [\"27428901\", \"27428899\", \"27432944\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether pharmacological Cbl-b inhibition recapitulates antifungal protection not tested\", \"role in other fungal pathogens beyond C. albicans not established\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Quantitative temporal interactomics of TCR-stimulated T cells identified CD5 as a key scaffold for CBLB-mediated ubiquitylation following TCR engagement, providing an unbiased map of the dynamic CBLB signaling complex.\",\n      \"evidence\": \"AP-MS time course over 600 s of TCR stimulation with biochemical validation\",\n      \"pmids\": [\"27474268\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of CD5-scaffolded Cbl-b activity not fully dissected\", \"direct vs. indirect interactions in the complex not resolved for all partners\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"TAM receptor-mediated phosphorylation of Cbl-b at Y133 and Y363 was shown to activate Cbl-b in NK cells, with Gas6/TAM ligation triggering Cbl-b-dependent degradation of the adaptor LAT1 to suppress NK-cell effector functions.\",\n      \"evidence\": \"Gas6 ligation; Y133/Y363 phosphosite mutagenesis; Cbl-b KO NK cells; LAT1 degradation assay\",\n      \"pmids\": [\"31531847\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Y133/Y363 phosphorylation equally regulates Cbl-b in T cells and myeloid cells not tested\", \"kinase(s) besides TAM receptors that phosphorylate these sites not mapped\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Cbl-b was found to ubiquitinate NMDA receptor subunit GluN2B in spinal dorsal horn neurons via its TKB domain, with inflammation-induced Y363 dephosphorylation impairing Cbl-b–GluN2B binding and enabling synaptic GluN2B accumulation, linking Cbl-b to nociceptive sensitization.\",\n      \"evidence\": \"Co-IP and ubiquitination assay; phosphomimetic Y363 mutagenesis; in vivo knockdown; electrophysiology of synaptic NMDAR currents\",\n      \"pmids\": [\"32606037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the phosphatase dephosphorylating Y363 in neurons not determined\", \"behavioral pain outcomes of Cbl-b manipulation not fully characterized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"CRISPR-mediated Cbl-b deletion was shown to rescue exhausted CD8+ tumor-infiltrating lymphocytes and CAR T cells, reducing PD1+Tim3+ populations and restoring effector function, establishing Cbl-b as a druggable checkpoint for reversing T-cell exhaustion.\",\n      \"evidence\": \"CRISPR-Cas9 Cbl-b KO in CD8+ T cells and CAR T cells; syngeneic MC38 tumor model; cytokine and killing assays\",\n      \"pmids\": [\"33462140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrate(s) linking Cbl-b to the exhaustion transcriptional program not identified\", \"long-term safety of Cbl-b deletion in therapeutic T cells not assessed\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"A co-crystal structure of Cbl-b with a small-molecule inhibitor revealed that the compound acts as an intramolecular glue bridging the TKBD and LHR domains (not the RING domain), locking Cbl-b in an autoinhibited conformation—providing the first structural basis for pharmacological Cbl-b inhibition.\",\n      \"evidence\": \"X-ray crystallography; SPR and thermal shift biophysical assays; cellular ubiquitination assay\",\n      \"pmids\": [\"38104184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy and selectivity of TKBD-LHR glue inhibitors not yet demonstrated\", \"whether this mechanism generalizes to other Cbl family members unknown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Cbl-b was found to form a TCR-induced complex with the pH-sensitive phosphatase STS1 via a proline motif, with STS1 dephosphorylating Cbl-b-associated phosphoproteins; this complex renders T cells sensitive to acidic tumor microenvironments, and loss of either partner enhances anti-tumor immunity.\",\n      \"evidence\": \"Co-IP with proline-motif mutagenesis; STS1 and Cbl-b KO mice; pH sensitivity assays; tumor growth experiments\",\n      \"pmids\": [\"38091950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which specific phosphoproteins STS1 dephosphorylates in the Cbl-b complex not mapped\", \"structural basis of pH sensing by STS1-Cbl-b not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key remaining questions include: the structural basis of Cbl-b substrate selectivity across its >15 known substrates; whether non-catalytic functions (e.g., PTEN protection) operate broadly beyond T cells; the identity of the E3 ligase(s) that ubiquitinate Cbl-b itself for degradation upon CD28 signaling; and whether pharmacological Cbl-b inhibitors achieve selective immune activation without autoimmune toxicity in vivo.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No comprehensive structural model for multi-substrate recognition\", \"E3 ligase targeting Cbl-b for degradation unidentified\", \"in vivo therapeutic window of Cbl-b inhibitors not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 3, 4, 13, 14, 16, 18, 21, 22, 23, 25, 28, 33, 38]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [2, 3, 16, 18, 23]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [7, 37]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 8, 9, 17]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 3, 14, 30]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 31, 33]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0392499\", \"supporting_discovery_ids\": [2, 3, 13, 14, 18, 21, 23, 28, 38]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 1, 8, 9, 10, 11, 16, 18, 23, 24, 27, 30, 39]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 4, 6, 17, 19, 20, 34]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 3, 13, 14, 18, 21, 23, 28, 38]}\n    ],\n    \"complexes\": [\n      \"Cbl-b–STS1 complex\",\n      \"Cbl-b–CIN85–endophilin complex\",\n      \"DR5–Cbl-b/c-Cbl–TRAF2 complex\"\n    ],\n    \"partners\": [\n      \"PIK3R1\",\n      \"VAV1\",\n      \"SYK\",\n      \"CLEC7A\",\n      \"CIN85\",\n      \"STS1\",\n      \"NEDD4\",\n      \"GRB2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}