{"gene":"SH3KBP1","run_date":"2026-06-10T07:46:31","timeline":{"discoveries":[{"year":2002,"finding":"CIN85 constitutively associates with endophilins via SH3 domain interactions, and is rapidly recruited by Cbl to form a trimeric complex with activated EGF receptors upon EGF stimulation, controlling receptor internalization independently of Cbl ubiquitin ligase activity. Inhibition of CIN85-Cbl or CIN85-endophilin interactions blocks EGFR internalization and delays receptor degradation.","method":"Co-immunoprecipitation, dominant-negative inhibition, EGF receptor internalization/degradation assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, functional rescue experiments, replicated independently in same issue by two labs (PMIDs 11894095 and 11894096)","pmids":["11894095"],"is_preprint":false},{"year":2002,"finding":"The endophilin-CIN85-Cbl complex controls HGF receptor (c-Met) internalization; Cbl ubiquitinates activated c-Met and recruits the endophilin-CIN85 complex to regulate receptor internalization. Inhibition of complex formation blocks HGF receptor internalization and enhances HGF-induced signaling.","method":"Co-immunoprecipitation, dominant-negative inhibition, HGF receptor internalization assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus functional inhibition experiments, independent replication in same issue","pmids":["11894096"],"is_preprint":false},{"year":2000,"finding":"CIN85 binds to c-Cbl via its second SH3 domain; this association is basally present and enhanced after EGF stimulation correlating with Cbl tyrosine phosphorylation. CIN85 self-associates through its coiled-coil region to form tetramers, and both SH3 domains and coiled-coil region contribute to subcellular localization.","method":"Co-immunoprecipitation, yeast two-hybrid, domain deletion mutagenesis, EGF stimulation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mutagenesis, single lab, multiple methods","pmids":["10679202"],"is_preprint":false},{"year":2000,"finding":"CIN85 associates with BLNK (B cell linker protein) via its SH3 domains, with both proteins co-localizing in the cytoplasm, implicating CIN85 in BCR-mediated signaling. CIN85 also forms complexes with Crk-I, Crk-II, p130Cas, p85-PI3K, Grb2, and Sos1.","method":"Yeast two-hybrid screen, co-immunoprecipitation, immunostaining","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and immunolocalization, single lab, two orthogonal methods","pmids":["11071869"],"is_preprint":false},{"year":2002,"finding":"CIN85 and its homologue CMS are monoubiquitinated by Cbl/Cbl-b after EGF stimulation. Monoubiquitination requires direct CIN85-Cbl interaction, intact RING finger domain of Cbl, and a ubiquitin acceptor site in the CIN85 C-terminus. Cbl-b and monoubiquitinated CIN85 are degraded together with polyubiquitinated EGFRs in the lysosome.","method":"Co-immunoprecipitation, ubiquitination assay, RING finger domain mutagenesis, lysosomal degradation assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination assay with mutagenesis, single lab but multiple orthogonal methods","pmids":["12218189"],"is_preprint":false},{"year":2003,"finding":"CIN85 SH3 domains specifically recognize a novel polyproline-arginine motif (PXXXPR) present in Cbl/Cbl-b. Individual SH3 domains bind with micromolar affinity, while extended two- or three-SH3-domain constructs bind with higher stoichiometry and affinity. This enables full-length CIN85 to simultaneously bind multiple Cbl molecules, promoting their clustering, which is important for EGFR degradation.","method":"Peptide binding assays, isothermal titration calorimetry, mutagenesis, co-immunoprecipitation, receptor degradation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding assays with mutagenesis, ITC, and functional EGFR degradation readout, single lab with multiple orthogonal methods","pmids":["12874286"],"is_preprint":false},{"year":2002,"finding":"Cbl-b, but not Cbl-3, utilizes the same CIN85/endophilin pathway as Cbl to down-regulate multiple RTKs (EGFR, PDGFR). CIN85 binds to the C-terminus of Cbl-b; inhibiting CIN85-Cbl-b interaction impairs EGFR internalization but not receptor polyubiquitination.","method":"Co-immunoprecipitation, dominant-negative inhibition, EGFR internalization and ubiquitination assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional internalization and ubiquitination assays, epistasis between CIN85-Cbl-b interaction and receptor internalization vs ubiquitination","pmids":["12177062"],"is_preprint":false},{"year":2004,"finding":"CIN85 SH3 domains bind to PxxxPR motifs in multiple endocytic effectors including SHIP-1, synaptojanin 2B1, ASAP1, ARAP3, Hip1R, STAP1, and p115RhoGEF, functioning as a molecular scaffold that clusters these proteins into high-molecular-weight complexes. ASAP1 overexpression increases EGFR recycling, an effect dependent on its intact PxxxPR motif.","method":"Co-immunoprecipitation, pulldown, overexpression/mutant studies, EGFR recycling assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple binding partners identified by Co-IP/pulldown, single lab with functional receptor recycling readout","pmids":["15090612"],"is_preprint":false},{"year":2003,"finding":"CIN85 and CMS/CD2AP SH3 domains bind to the CD2 cytoplasmic tail proline-rich segment, and CIN85/CMS directly bind to the actin-capping protein CAPZ via their C-terminal half, providing a molecular link between CD2 and the actin cytoskeleton. Overexpression of CMS/CIN85 fragments in T cells enhanced IL-2 production and reduced TCR downmodulation.","method":"Peptide affinity chromatography, BIAcore surface plasmon resonance, co-immunoprecipitation, functional T cell assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — SPR quantitative binding data, Co-IP, functional T cell readout, single lab","pmids":["12690097"],"is_preprint":false},{"year":2004,"finding":"Alix/AIP1 antagonizes the Cbl-CIN85 complex by weakening the CIN85-Cbl interaction and reducing Cbl-mediated ubiquitination of EGFR and CIN85, thereby diminishing EGFR internalization. siRNA knockdown of Alix promoted EGFR internalization, confirming the inhibitory role.","method":"Co-immunoprecipitation, siRNA knockdown, ubiquitination assay, EGFR internalization assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with functional internalization readout, Co-IP, single lab","pmids":["15456872"],"is_preprint":false},{"year":2003,"finding":"CIN85 SH3 domains bind to an atypical recognition consensus PX(P/A)XXR, confirmed by mutagenesis and in vitro binding. This motif maps CIN85-binding sites in c-Cbl, Cbl-b, BLNK, AIP1/Alix, and CD2. Novel interactors synaptojanin 1, PAK2, ZO-2, and TAFII70 were identified by GST-pulldown from brain lysates.","method":"Target-assisted iterative peptide library screening, mutagenesis, in vitro binding assays, GST pulldown, Far Western blotting","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding with mutagenesis and multiple orthogonal methods (Far Western, pulldown), single lab","pmids":["12829691"],"is_preprint":false},{"year":2003,"finding":"Disabled-2 (Dab2) associates with all three SH3 domains of CIN85 via a PKPAPR motif and recruits CIN85 to the clathrin coat. This association is modulated by growth factor stimulation, whereby Dab2 and clathrin dissociate from CIN85 after growth factor treatment, enabling Cbl to bind.","method":"Co-immunoprecipitation, peptide binding, stimulation-dependent association studies","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP with dynamic stimulation-dependent analysis, single lab","pmids":["14596919"],"is_preprint":false},{"year":2004,"finding":"Yeast Sla1 is a functional ortholog of CIN85, with similar domain structure and direct binding to endophilin-like Rvs167. Rsp5 (yeast ubiquitin ligase) directly binds and monoubiquitinates Rvs167 at Lys481 in its SH3 domain, mediated through Rsp5 WW domains and PXY motifs in Rvs167.","method":"Genetic analysis, in vitro binding, ubiquitination assay, mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination with mutagenesis in yeast ortholog system, single lab","pmids":["14761940"],"is_preprint":false},{"year":2008,"finding":"All three SH3 domains of CIN85 bind to ubiquitin at the canonical proline-rich ligand-binding surface, with ubiquitin mimicking a PPII helix. Ubiquitin and proline-rich ligands compete for the same binding surface. Disruption of ubiquitin binding results in constitutive CIN85 ubiquitination and elevated basal EGFR ubiquitination in the absence of EGF.","method":"NMR structural modeling, ITC, mutagenesis, EGFR ubiquitination assay","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure with mutagenesis and functional ubiquitination readout, single lab with multiple orthogonal methods","pmids":["18680311"],"is_preprint":false},{"year":2010,"finding":"CIN85 binds to the coiled-coil domain of nephrin and podocin (not via SH3 domains), mediates nephrin internalization after FGF-4 stimulation, and promotes nephrin ubiquitination. CD2AP competes with CIN85 for binding to nephrin/podocin. In CD2AP-deficient podocytes, CIN85 accumulation correlates with increased nephrin ubiquitination.","method":"Co-immunoprecipitation, binding assays with domain mutants, immunofluorescence, internalization assay, CD2AP knockout mouse analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mutant binding assays, KO mouse data, functional internalization assay, replicated in multiple experimental systems","pmids":["20457601"],"is_preprint":false},{"year":2010,"finding":"CIN85 directly interacts with dynamin 2 (Dyn2) in a manner induced by EGFR stimulation; this interaction occurs late in the endocytic process at late endosomes. Disruption of CIN85-Dyn2 interaction causes accumulation of internalized EGFR in aberrantly elongated late endosomal tubules and sustained downstream signaling.","method":"Co-immunoprecipitation, dominant-negative studies, fluorescence microscopy of EGFR trafficking","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional dominant-negative perturbation and morphological phenotype, single lab","pmids":["20711168"],"is_preprint":false},{"year":2005,"finding":"Sprouty2 associates with CIN85 via SH3 domains A and C binding proline-arginine motifs in Sprouty2, and acts at the Cbl-CIN85 interface to inhibit EGFR endocytosis and degradation. Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation.","method":"Co-immunoprecipitation, EGFR endocytosis/degradation assay, PC12 differentiation assay, domain mutagenesis","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mutagenesis, functional EGFR endocytosis and differentiation readouts, single lab","pmids":["15962011"],"is_preprint":false},{"year":2011,"finding":"CIN85 constitutively associates with SLP65 (BLNK) in a stimulation-independent manner in B cells, which is required for SLP65 phosphorylation and inducible plasma membrane translocation. In the absence of a stable SLP65/CIN85 complex, BCR-induced Ca2+ and NF-κB responses are abrogated.","method":"Mass spectrometry interactome, co-immunoprecipitation, live cell imaging, loss-of-function studies, Ca2+ flux assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative MS interactome plus functional loss-of-function with defined BCR signaling readouts, multiple orthogonal methods","pmids":["21822214"],"is_preprint":false},{"year":2011,"finding":"CIN85 links the BCR to IKK-β activation to drive canonical NF-κB signaling in B cells. B cell-specific CIN85 knockout mice show impaired T cell-independent type II antibody responses, diminished IKK-β activation, and defective BCR cross-linking responses, rescued by constitutively active IKK-β.","method":"B cell-specific conditional knockout mouse (Mb1-cre), in vivo antibody responses, IKK-β activation assay, constitutively active IKK-β rescue","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with in vivo functional readouts and genetic rescue, single lab","pmids":["21708930"],"is_preprint":false},{"year":2016,"finding":"CIN85 oligomerizes SLP-65 in B cells through its trimeric coiled-coil domain and multiple SH3 domains, forming large signaling condensates/oligomeric complexes. This pre-formed oligomeric complex in resting B cells is required for efficient BCR signaling initiation.","method":"Biochemical oligomerization assays, co-immunoprecipitation, structure-function analysis with domain mutants, B cell functional assays","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal biochemical and functional methods, identifies mechanistic basis of CIN85 oligomerization in B cell signaling","pmids":["27353366"],"is_preprint":false},{"year":2010,"finding":"CIN85 deficiency in mice (CIN85Δex2) leads to insufficient endophilin-D2 dopamine receptor (D2DR) complex formation in the striatum and decreased D2DR endocytosis in striatal neurons in response to dopamine, resulting in elevated striatal D2DR levels and dopamine, and hyperactive behavior. CIN85 localizes to the post-synaptic compartment of striatal neurons and co-clusters with D2DRs.","method":"Conditional knockout mouse, receptor endocytosis assay in striatal neurons, co-immunoprecipitation, immunofluorescence localization, behavioral analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO mouse with direct endocytosis assay, protein co-localization, and behavioral phenotype all linked to mechanism","pmids":["20551902"],"is_preprint":false},{"year":2008,"finding":"Drosophila Cindr (CD2AP/CIN85 ortholog) links E-cadherin and Roughest (IgCAM adhesion receptor) at cell junctions with actin cytoskeletal components including capping protein alpha and beta. Reduction of cindr activity causes defects in E-cadherin and Roughest localization, local cell movement, tissue patterning, and cell death in the developing retina.","method":"Drosophila genetics (loss-of-function), immunofluorescence, protein interaction studies, tissue phenotype analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function in Drosophila ortholog with defined molecular and cellular phenotypes","pmids":["18362180"],"is_preprint":false},{"year":2007,"finding":"CIN85 colocalizes with AMAP1 at invadopodia in breast cancer cells; binding of AMAP1 to CIN85 is required for invasive activity including matrix degradation. CIN85 siRNA silencing inhibits invasion. Cbl monoubiquitinates AMAP1, and this monoubiquitination is important for AMAP1's role in invasion.","method":"siRNA knockdown, co-immunoprecipitation, invasion assay, immunofluorescence","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA functional studies with defined invasion phenotype, Co-IP, single lab","pmids":["17255943"],"is_preprint":false},{"year":2005,"finding":"CIN85 associates with the TNFR1 signaling complex via Src family kinases (not directly with TNFR1 cytoplasmic domain); Src binds directly to TNFR1. Ectopic CIN85 expression increases susceptibility to TNF-α-induced apoptosis 10-fold. The three SH3 domains and proline-rich regions of CIN85 are essential for this pro-apoptotic effect.","method":"Co-immunoprecipitation, overexpression, apoptosis assay, domain deletion mutagenesis","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mutagenesis and functional apoptosis readout, single lab","pmids":["15707590"],"is_preprint":false},{"year":2005,"finding":"CIN85 overexpression in mast cells drives IgE receptor (FcεRI) internalization into early endosomes and lysosomes, reduces FcεRI expression levels, and dramatically impairs mast cell degranulation after antigen stimulation.","method":"Overexpression, confocal microscopy, receptor internalization/degradation assay, degranulation assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — overexpression with functional receptor trafficking and degranulation readouts, single lab","pmids":["16177060"],"is_preprint":false},{"year":2009,"finding":"CIN85 associates with endosomal membranes through its positively charged C-terminus binding to phosphatidic acid (PA), with the coiled-coil domain essential for this protein-lipid interaction. Deletion of the coiled-coil domain abolishes membrane association, reduces c-Cbl interaction, and blocks EGFR downregulation. A significant pool of CIN85 localizes to EEA1-positive endosomal compartments.","method":"Lipid binding assay, domain deletion mutagenesis, subcellular fractionation, immunofluorescence, EGFR degradation assay","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — lipid binding assay with domain mutagenesis and functional EGFR degradation readout, single lab","pmids":["19417776"],"is_preprint":false},{"year":2011,"finding":"SHIP-1 constitutively associates with all three SH3 domains of CIN85 in B cells via a 79-amino acid region near the SHIP-1 C-terminus, identified by systematic pulldown proteomics.","method":"Pulldown proteomics, co-immunoprecipitation, domain mapping","journal":"Molecular & cellular proteomics","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP/pulldown with domain mapping, single lab, multiple orthogonal methods","pmids":["21725061"],"is_preprint":false},{"year":2012,"finding":"Src kinase mediates tyrosine phosphorylation of CIN85 following EGFR activation; phospho-CIN85 interacts with Rab5-positive early endosomes and mediates sequestration of ubiquitinated EGFR into multivesicular bodies (MVBs). CIN85 knockdown decreases EGF-induced EGFR ubiquitination and reduces EGFR sorting to MVBs.","method":"siRNA knockdown, Src inhibition, phospho-specific analysis, co-immunoprecipitation, subcellular fractionation, immunofluorescence","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA with specific phospho-CIN85 mechanistic studies, single lab","pmids":["22833562"],"is_preprint":false},{"year":2015,"finding":"CIN85 interacts with TβRI (TGFβ receptor type I) SH3 domains in a TRAF6-dependent manner upon TGFβ stimulation. CIN85 knockdown causes accumulation of TβRI in intracellular compartments and diminished Smad2 phosphorylation. CIN85 overexpression increases TβRI at the cell surface by promoting receptor recycling via a Rab11-dependent pathway, enhancing TGFβ-stimulated Smad2 phosphorylation, transcriptional responses, and cell migration.","method":"siRNA knockdown, overexpression, dominant-negative Rab11, Smad2 phosphorylation assay, transcriptional reporter, cell migration assay, Co-IP","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA/OE with multiple functional readouts and dominant-negative epistasis, single lab","pmids":["26169354"],"is_preprint":false},{"year":2011,"finding":"CIN85 regulates Syk protein levels in mast cells by promoting c-Cbl-mediated Syk ubiquitination and proteasomal degradation. CIN85 overexpression limits Cbl binding to Sts1 (a negative regulator of Cbl), while CIN85 knockdown favors Cbl/Sts1 complex formation, reducing Cbl-directed Syk ubiquitination.","method":"Overexpression, siRNA knockdown, co-immunoprecipitation, proteasome inhibition, ubiquitination assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA/OE with proteasome inhibitor rescue and mechanistic Co-IP, single lab","pmids":["17675467"],"is_preprint":false},{"year":2011,"finding":"In human B cells, CIN85 is constitutively associated with c-Cbl and Cbl-b, and increases c-Cbl phosphorylation. CIN85 overexpression inhibits BCR-induced calcium flux and phosphorylation of Syk and PLCγ2, correlating with increased Syk ubiquitination and degradation. CIN85 knockdown enhances BCR-induced survival, growth, and differentiation-associated gene expression.","method":"Overexpression, siRNA knockdown, co-immunoprecipitation, calcium flux assay, phosphorylation analysis, ubiquitination assay","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — paired OE/KD studies with multiple signaling readouts, single lab","pmids":["22262777"],"is_preprint":false},{"year":2007,"finding":"CMS/CIN85 can crosslink filamentous actin (F-actin) into bundles, a function dependent on the proline-rich region and coiled-coil domain. CMS/CIN85 are found in podosomes. CMS and CIN85 can form heterotypic complexes via their coiled-coil domains. Removal of these actin-bundling domains reduces cell migration.","method":"F-actin binding/bundling assays, domain deletion mutagenesis, cell migration assay, immunofluorescence","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro actin bundling assay with domain mutagenesis and functional migration readout, single lab","pmids":["17606992"],"is_preprint":false},{"year":2011,"finding":"CIN85 co-localizes with actin at focal adhesions and with microtubules, and CIN85 interacts with focal adhesion kinases FAK and PYK-2. CIN85 promotes cell adhesion as assessed by ECIS.","method":"Immunofluorescence co-localization, co-immunoprecipitation, ECIS (electrical cell-substrate impedance sensing) adhesion assay","journal":"Journal of cell science","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and localization without direct mechanistic follow-up for CIN85-FAK interaction, single lab","pmids":["12771190"],"is_preprint":false},{"year":2006,"finding":"CIN85 forms a complex with S-SCAM and dendrin at synapses; the first SH3 domain and the C-terminal region of CIN85 bind the proline-rich and N-terminal regions of dendrin, respectively. CIN85 localizes to synaptic compartments as shown by immunocytochemistry and subcellular fractionation.","method":"Yeast two-hybrid, co-immunoprecipitation, immunocytochemistry, subcellular fractionation, in vitro binding assay","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — multiple orthogonal binding assays with synaptic localization, single lab","pmids":["16751601"],"is_preprint":false},{"year":2004,"finding":"CIN85 interacts with the p85α regulatory subunit of PI3K through multiple domains: CIN85 SH3 domains are required and sufficient for binding full-length p85α, but the SH3 domain of p85α is required to activate this interaction by relieving an intramolecular autoinhibitory interaction between CIN85 SH3 domains and its proline-rich region.","method":"Co-immunoprecipitation, domain deletion/mutagenesis, in vitro binding assays","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding with domain mapping and intramolecular interaction analysis, single lab","pmids":["15476827"],"is_preprint":false},{"year":2009,"finding":"Intersectin 1 (ITSN1) forms a constitutive complex with CIN85 mediated by the SH3A domain of ITSN1 and the third or fourth proline-rich blocks of CIN85, independent of EGF stimulation.","method":"Co-immunoprecipitation, domain mapping, immunofluorescence co-localization","journal":"Cellular signalling","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP with domain mapping, single lab","pmids":["19166927"],"is_preprint":false},{"year":2008,"finding":"CIN85 is distributed in multiple membrane trafficking compartments in human cells; the major endogenous pool associates with COPI-coated vesicles of the Golgi complex involved in retrograde ER-Golgi transport. This localization depends on Golgi complex integrity and intact microtubules.","method":"Immunofluorescence with compartment markers, subcellular fractionation, antibody characterization, Golgi disruption experiments","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — detailed localization study with functional perturbation of organelle integrity, single lab","pmids":["18266907"],"is_preprint":false},{"year":2010,"finding":"CIN85 binds the catalytic subunit of protein phosphatase 2A (PP2Ac) via the P3 block (PAIPPKKPRP) in CIN85's proline-rich region; this interaction suppresses PP2Ac phosphatase activity. Disruption of PP2Ac-CIN85 interaction decreases platelet spreading and fibrin clot retraction and reduces phosphorylation of Src and GSK3β.","method":"Yeast two-hybrid, truncation/alanine mutagenesis, in vitro phosphatase activity assay, cell adhesion assay, clot retraction assay, cell-permeable peptide","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro phosphatase activity assay with mutagenesis, functional platelet assay, single lab","pmids":["27334924"],"is_preprint":false},{"year":2005,"finding":"CIN85 SH3 domains interact with MEKK4 via three PxxxPR motifs; disruption of this interaction demonstrates that CIN85 binding to MEKK4 enhances MKK6 and p38 MAP kinase activation following oxidative stress and growth factor stimulation. CIN85 also regulates MEKK4 activation by GADD45 proteins and promotes MEKK4 multi-ubiquitination.","method":"Co-immunoprecipitation, dominant-negative inhibition, kinase activation assays, ubiquitination assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with functional kinase assays and specific PxxxPR motif disruption, single lab","pmids":["16256071"],"is_preprint":false},{"year":2010,"finding":"CD2AP regulates SUMOylation of CIN85 in podocytes; full-length CIN85 is SUMOylated by SUMO-1, -2, and -3, and SUMOylation is enhanced in the presence of CD2AP. Mutation of lysine 598 to arginine abolishes CIN85 SUMOylation and leads to increased CIN85 binding to nephrin.","method":"SUMOylation assay, site-directed mutagenesis (K598R), co-immunoprecipitation, podocyte cell culture","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro/cell-based SUMO modification assay with site mutagenesis and functional binding consequences, single lab","pmids":["22037207"],"is_preprint":false},{"year":2016,"finding":"CIN85 (RukL isoform) deficiency in mice preserves nephrin surface expression on the slit diaphragm and reduces proteinuria under diabetic conditions; CIN85/RukL overexpression in zebrafish causes severe edema and disruption of the filtration barrier. CIN85/RukL mediates nephrin endocytosis via ubiquitination in podocytes.","method":"CIN85 knockout mouse under diabetic conditions, zebrafish overexpression, nephrin surface expression assay, proteinuria measurement","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function in mice and gain-of-function in zebrafish with functional filtration barrier readouts, replicated across species","pmids":["27531950"],"is_preprint":false},{"year":2014,"finding":"Basic amino acids K645, K646, R648, and R650 in the CIN85 coiled-coil domain are required for both phosphatidic acid binding and c-Cbl interaction but not endophilin interaction. CIN85 also interacts with ESCRT components, and mutations in the coiled-coil domain dissociate CIN85 from endosomes and prevent ESCRT assembly on endosomal membranes, inhibiting EGFR sorting.","method":"Site-directed mutagenesis, lipid binding assay, co-immunoprecipitation, EGFR sorting/degradation assay, immunofluorescence","journal":"BMC biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro lipid binding with mutagenesis and functional EGFR sorting readout, single lab","pmids":["25005938"],"is_preprint":false},{"year":2011,"finding":"CIN85 depletion by siRNA in neutrophils prevents FcγRIIa ubiquitination and degradation by c-Cbl following receptor cross-linking, increases IgG-mediated phagocytosis, and disrupts proper FcγRIIa endosomal sorting. CIN85 is a substrate of classical PKCs, which positively regulate FcγRIIa ubiquitination/degradation.","method":"siRNA knockdown, co-immunoprecipitation, ubiquitination assay, phagocytosis assay, PKC inhibition, confocal microscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA functional studies with multiple mechanistic readouts, single lab","pmids":["21372129"],"is_preprint":false},{"year":2019,"finding":"CIN85 inhibits T cell activation by being recruited to the TCR signaling complex upon stimulation; CIN85-deficient T cells show enhanced TCR signaling (Zap70, SLP76, Erk phosphorylation) and increased IL-2 production. The inhibitory function requires CIN85 SH3 and PR regions, which associate with the phosphatase Sts-2 after TCR stimulation.","method":"Conditional knockout T cells, T cell activation assays, signaling phosphorylation analysis, co-immunoprecipitation, domain mutant studies","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with defined signaling phenotypes, mechanistic Co-IP for Sts-2, domain mutant epistasis, single lab","pmids":["30723173"],"is_preprint":false},{"year":2018,"finding":"Germline deletion of CIN85 in humans with X-linked antibody deficiency causes B cell-intrinsic defects in BCR effector pathways, most notably NF-κB activation and CD86 upregulation, without affecting immune cell compartment development.","method":"Human genetic analysis, functional B cell assays (NF-κB activation, CD86 upregulation), immune cell phenotyping","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — human germline loss-of-function with defined B cell signaling phenotypes, natural experiment","pmids":["29636373"],"is_preprint":false},{"year":2019,"finding":"CIN85 binds directly to PHD2 (the main HIF prolyl hydroxylase) via CIN85 N-terminal SH3 domains interacting with the proline-arginine-rich N-terminus of PHD2, but not PHD1 or PHD3. This interaction inhibits PHD2 hydroxylase activity and HIF degradation. CRISPR/Cas9 disruption of the CIN85-PHD2 interaction in cells affects growth, migration, and tumor growth in mice.","method":"Co-immunoprecipitation, PHD2 hydroxylase activity assay, CRISPR/Cas9 knock-in, tumor xenograft","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — enzymatic activity assay with binding data and CRISPR functional validation in vivo, single lab with multiple orthogonal methods","pmids":["31142511"],"is_preprint":false},{"year":2022,"finding":"CIN85 interacts directly with the N-terminal region of anillin and with SEPT9 to facilitate SEPT9-containing septin filament localization to the intercellular bridge (ICB) plasma membrane during cytokinesis. CIN85 is required for robust and timely cytokinesis and ICB elongation and maturation.","method":"Co-immunoprecipitation, direct binding assay, siRNA knockdown, live-cell imaging of cytokinesis, immunofluorescence","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding assay with siRNA functional cytokinesis phenotype and live-cell imaging, single lab with multiple orthogonal methods","pmids":["36044846"],"is_preprint":false},{"year":2023,"finding":"An intramolecular autoinhibitory interaction occurs between the CIN85 SH3C domain and an adjacent proline-rich motif (PRM) in the linker region. Phosphorylation of a serine adjacent to this PRM regulates the intramolecular SH3:PRM interaction, modulating CIN85 valency toward SLP65. Disruption of this interaction impairs SLP65/CIN85 condensate formation, CIN85 membrane recruitment, and Ca2+ mobilization in B cells.","method":"NMR spectroscopy of multidomain constructs, phosphomimetic mutagenesis, B cell functional assays (Ca2+ flux, membrane recruitment)","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural evidence with mutagenesis and B cell functional validation, single lab with multiple orthogonal methods","pmids":["38111344"],"is_preprint":false},{"year":2025,"finding":"SH3KBP1 N-terminus binds directly to dynamin-2 and C-terminus associates with the ER through calnexin; these interactions control myonuclei dynamics and ER integrity respectively during myofiber formation. SH3KBP1 is required for myoblast fusion, myonuclear positioning, myotube elongation, triad formation, and efficient excitation-contraction coupling.","method":"siRNA screen, co-immunoprecipitation (dynamin-2, calnexin), domain binding assays, live-cell imaging, functional excitation-contraction coupling assay, Dnm2R465W/+ mouse model","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain binding and functional myogenesis readouts, single lab","pmids":["40065183"],"is_preprint":false},{"year":2025,"finding":"SH3KBP1 recruits PI3K to bleb necks via liquid-liquid phase separation through interactions with septin, forming a Septin-SH3KBP1-PI3K axis that establishes differential phosphoinositide (PI3P/PIP2) distribution at bleb vs non-bleb membrane and confers anoikis resistance.","method":"Phase separation assay, co-immunoprecipitation, immunofluorescence with phosphoinositide biosensors, anoikis assay","journal":"American journal of physiology. Cell physiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, single set of assays, phase separation claim based on indirect evidence","pmids":["40445717"],"is_preprint":false},{"year":2023,"finding":"ASAP2 directly binds CIN85, disrupting the CIN85-c-MET interaction and preventing CIN85-induced c-MET internalization and lysosomal degradation, thereby sustaining HGF/c-MET signaling. CIN85 knockdown rescues the inhibitory effect of ASAP2 knockdown on c-MET signaling.","method":"Co-immunoprecipitation, cycloheximide chase, siRNA knockdown (ASAP2, CIN85), c-MET signaling assays","journal":"Experimental hematology & oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus genetic epistasis (CIN85 KD rescues ASAP2 KD phenotype), single lab","pmids":["37061723"],"is_preprint":false},{"year":2011,"finding":"SH3KBP1-binding protein 1 (SHKBP1) constitutively binds to CIN85 SH3 domains via its two PXXXPR motifs, competing with c-Cbl for CIN85 binding. This competition prevents CIN85 translocation to EGFR-containing vesicles, reduces EGFR degradation, and enhances EGF-induced signaling.","method":"Co-immunoprecipitation, dominant-negative inhibition, EGFR degradation assay, reporter gene assay","journal":"Cell biochemistry and function","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — competitive binding Co-IP with functional EGFR degradation readout, single lab","pmids":["21830225"],"is_preprint":false},{"year":2025,"finding":"CIN85 and CD2AP are recruited to tubular recycling endosomes (TREs) via interactions with MICAL-L1 through their SH3 domains. Depletion of either CIN85 or CD2AP impairs receptor recycling (CD98 cargo), demonstrating their roles in TRE function.","method":"siRNA knockdown, co-immunoprecipitation, live-cell fluorescence imaging of TRE dynamics, recycling assays","journal":"Traffic (Copenhagen, Denmark)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA functional recycling assay with Co-IP, single lab","pmids":["40740057"],"is_preprint":false},{"year":2011,"finding":"Cbl/Cbl-b double knockout MEFs retain largely normal EGFR internalization, and inducible siRNA knockdown of CIN85 in WT or Cbl/Cbl-b DKO MEFs has no impact on EGFR internalization. This demonstrates that endogenous CIN85, Cbl, and Cbl-b are largely dispensable for initial EGFR internalization, while Cbl is required for EGFR ubiquitination and degradation.","method":"Cbl/Cbl-b double-knockout MEFs, inducible siRNA knockdown of CIN85, 125I-EGF and fluorescent EGF internalization assay","journal":"The international journal of biochemistry & cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic double KO plus siRNA knockdown with quantitative internalization assays, rigorous experimental controls","pmids":["25449262"],"is_preprint":false},{"year":2024,"finding":"SH3KBP1 enhances RIG-I signal transduction by promoting K63-linked polyubiquitination through interaction with E3 ubiquitin ligase TRIM25. PRRSV NSP2 induces autophagic degradation of SH3KBP1 via its third polyproline-arginine motif (453PVPAPR458), counteracting innate immune signaling. Sh3kbp1 knockout mice show increased susceptibility to VSV with reduced IFN-β.","method":"Co-immunoprecipitation, mass spectrometry, ubiquitination assay, autophagy assay, knockout mouse, recombinant virus with motif deletion","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with in vivo KO mouse and recombinant virus, multiple orthogonal methods, single lab","pmids":["39466846"],"is_preprint":false},{"year":2010,"finding":"HEV ORF3 protein (pORF3) interacts with CIN85 and thereby competes with growth factor receptor-Cbl-CIN85 complex formation, resulting in reduced CIN85 ubiquitination and delayed trafficking of the growth factor receptor complex to late endosomes/lysosomes.","method":"Co-immunoprecipitation, receptor trafficking assay, ubiquitination assay, pORF3 mutant analysis","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional receptor trafficking assay with competitive binding mechanism, single lab","pmids":["20130058"],"is_preprint":false},{"year":2011,"finding":"SOX10 transcription factor directly regulates expression of an alternative CIN85 isoform in Schwann cells via a highly conserved SOX10 binding site within an alternative promoter at the Sh3kbp1 locus. Mutation of the SOX10 binding site ablates promoter activity, and ectopic SOX10 promotes endogenous Sh3kbp1 expression.","method":"ChIP, promoter reporter assay, site-directed mutagenesis, ectopic SOX10 expression","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assays with mutagenesis confirming direct SOX10-dependent transcriptional regulation, single lab","pmids":["22037207"],"is_preprint":false},{"year":2023,"finding":"The EHBP1L1-CIN85/CD2AP axis controls ciliary length via actin network remodeling; CIN85 and CD2AP localize to the ciliary sheath in an EHBP1L1-dependent manner, and their depletion causes elongated cilia with actin nucleation and branching defects around the ciliary base.","method":"siRNA knockdown, immunofluorescence microscopy, co-immunoprecipitation, rescue with EHBP1L1 mutants","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA knockdown with morphological and actin phenotypes, domain mutant rescue, single lab","pmids":["36754282"],"is_preprint":false}],"current_model":"SH3KBP1/CIN85 is a multidomain adaptor protein (three SH3 domains, proline-rich region, coiled-coil domain) that acts as a central scaffold in receptor tyrosine kinase endocytosis and downregulation: upon growth factor stimulation, its SH3 domains bind a PXXXPR motif in Cbl/Cbl-b, enabling CIN85 to bridge activated RTKs (EGFR, c-Met, ErbB2, D2DR, FcεRI, FcγRIIa, TGFβR, nephrin) with endophilins, dynamin-2, and endosomal membrane (via phosphatidic acid binding at its coiled-coil domain), facilitating clathrin-mediated internalization and lysosomal degradation; Cbl monoubiquitinates CIN85 and Src phosphorylates it to regulate endosomal sorting into MVBs; in B cells, CIN85 constitutively pre-assembles with SLP65 into phase-separated oligomeric condensates (regulated by an intramolecular SH3C:PRM autoinhibitory interaction and serine phosphorylation) that prime BCR signaling by linking the BCR to IKK-β-dependent NF-κB activation; in T cells, CIN85 is recruited to the TCR complex and inhibits activation via Sts-2; beyond vesicular trafficking, CIN85 interacts with PHD2 to inhibit HIF-α hydroxylation, promotes p38 MAPK activation via MEKK4, suppresses PP2Ac phosphatase activity, crosslinks F-actin, localizes to COPI-Golgi membranes, intercellular bridges, and cilia, and is required for cytokinesis, muscle fiber formation, and innate immune RIG-I signaling via TRIM25."},"narrative":{"mechanistic_narrative":"SH3KBP1 (CIN85) is a multidomain adaptor that organizes receptor downregulation and assembly of multiprotein signaling complexes through its three SH3 domains, proline-rich region, and self-associating coiled-coil [PMID:11894095, PMID:10679202, PMID:12874286]. Its SH3 domains recognize an atypical PX(P/A)XXR/PXXXPR proline-arginine motif, allowing it to bind Cbl/Cbl-b and a wide array of endocytic effectors and to cluster them into high-molecular-weight complexes; full-length CIN85 can simultaneously engage multiple Cbl molecules to promote receptor degradation [PMID:12874286, PMID:12829691, PMID:15090612]. Upon growth factor stimulation it is recruited by Cbl to activated RTKs, where together with constitutively bound endophilins it controls internalization and lysosomal degradation of EGFR and c-Met [PMID:11894095, PMID:11894096]. CIN85 couples to the endocytic and sorting machinery through stimulation-induced binding to dynamin-2 at late endosomes and Src-dependent phosphorylation that drives ubiquitinated EGFR into multivesicular bodies, while its coiled-coil/basic C-terminus binds phosphatidic acid on endosomal membranes to enable ESCRT assembly and cargo sorting [PMID:20711168, PMID:22833562, PMID:19417776, PMID:25005938]. It is itself a substrate of regulatory modification, being monoubiquitinated by Cbl/Cbl-b and competing with ubiquitin for its own SH3 ligand-binding surface, with disruption of ubiquitin binding causing constitutive CIN85 and EGFR ubiquitination [PMID:12218189, PMID:18680311]. CIN85 broadly tunes receptor trafficking across many systems, mediating endocytosis of nephrin in podocytes, D2 dopamine receptors in striatal neurons, FcεRI in mast cells, FcγRIIa in neutrophils, and recycling of TβRI, and its activity is gated by competitive binding partners (Sprouty2, SHKBP1, ASAP2, Alix) that displace it from Cbl or its receptor targets [PMID:20457601, PMID:20551902, PMID:16177060, PMID:21372129, PMID:26169354, PMID:15962011, PMID:21830225, PMID:37061723, PMID:15456872]. In B cells CIN85 constitutively pre-assembles with SLP65/BLNK into oligomeric, condensate-forming complexes via its coiled-coil and SH3 domains, an interaction gated by an intramolecular SH3C:proline-rich autoinhibition under serine-phosphorylation control, priming BCR signaling and linking the receptor to IKK-β–dependent NF-κB activation [PMID:21822214, PMID:27353366, PMID:38111344, PMID:21708930]. Germline loss of CIN85 in humans causes an X-linked antibody deficiency with B cell-intrinsic defects in BCR-driven NF-κB activation [PMID:29636373]. Beyond trafficking and lymphocyte signaling, CIN85 inhibits HIF prolyl hydroxylase PHD2 to stabilize HIF-α [PMID:31142511], activates p38 MAPK through MEKK4 [PMID:16256071], suppresses PP2Ac phosphatase activity [PMID:27334924], crosslinks F-actin [PMID:17606992], and acts in cytokinesis, myofiber formation, ciliary length control, and RIG-I innate immune signaling through interactions with septins/anillin, dynamin-2/calnexin, EHBP1L1, and TRIM25 respectively [PMID:36044846, PMID:40065183, PMID:36754282, PMID:39466846].","teleology":[{"year":2000,"claim":"Established the core architecture and first partners: how CIN85 engages Cbl and self-associates, defining it as a clustering adaptor rather than a simple linker.","evidence":"Yeast two-hybrid, Co-IP, and domain-deletion mutagenesis in EGF-stimulated cells, identifying Cbl binding via the second SH3 domain and coiled-coil-mediated tetramerization, plus BLNK/Crk/PI3K associations","pmids":["10679202","11071869"],"confidence":"Medium","gaps":["Functional consequence of self-association not yet defined","No structural basis for SH3-ligand recognition"]},{"year":2002,"claim":"Defined CIN85's central role in RTK downregulation by showing it forms an endophilin-Cbl-receptor complex that drives internalization, and that it is itself monoubiquitinated and co-degraded with the receptor.","evidence":"Reciprocal Co-IP, dominant-negative inhibition, EGFR/c-Met internalization and degradation assays (two independent labs), plus in vitro ubiquitination with Cbl RING and acceptor-site mutagenesis","pmids":["11894095","11894096","12218189","12177062"],"confidence":"High","gaps":["Whether internalization requirement is cell-type dependent","Endosomal sorting steps downstream of internalization not resolved"]},{"year":2003,"claim":"Defined the molecular grammar of CIN85 recognition — an atypical PX(P/A)XXR proline-arginine motif — explaining how it binds many partners and clusters multiple Cbl molecules to promote receptor degradation.","evidence":"Peptide library screening, ITC, mutagenesis, Far Western and GST-pulldown mapping the motif across Cbl, BLNK, Alix, CD2 and identifying additional interactors; CD2/CAPZ link to actin","pmids":["12874286","12829691","12690097"],"confidence":"High","gaps":["Stoichiometry of multivalent complexes in cells not measured","Selectivity among the three SH3 domains incompletely defined"]},{"year":2004,"claim":"Showed CIN85 functions as a scaffold clustering endocytic effectors and revealed antagonistic regulators and an intramolecular autoinhibition relieved by partners.","evidence":"Co-IP/pulldown identifying SHIP-1, synaptojanin, ASAP1, ARAP3, Hip1R partners; siRNA and Co-IP for Alix antagonism; in vitro binding showing p85α SH3 relieves CIN85 SH3:PR autoinhibition; yeast Sla1/Rvs167 ortholog ubiquitination","pmids":["15090612","15456872","15476827","14761940"],"confidence":"Medium","gaps":["Physiological balance between scaffolding and antagonist binding unquantified","Autoinhibition regulation in cells not yet shown"]},{"year":2005,"claim":"Extended CIN85 regulation to receptor trafficking control by competitive inhibitors and to MAPK and death-receptor signaling beyond pure endocytosis.","evidence":"Co-IP, domain mutagenesis and functional EGFR/FcεRI/TNFR1 assays for Sprouty2 inhibition, MEKK4-p38 activation, FcεRI internalization, and Src-mediated TNFR1 association","pmids":["15962011","16256071","16177060","15707590"],"confidence":"Medium","gaps":["Whether p38 and apoptosis roles are direct or trafficking-dependent unclear","Most based on overexpression rather than endogenous loss-of-function"]},{"year":2008,"claim":"Provided the structural basis for ubiquitin/proline-rich ligand competition and mapped the endogenous CIN85 pool to Golgi COPI vesicles, broadening its trafficking remit.","evidence":"NMR modeling, ITC, mutagenesis showing ubiquitin binds the SH3 PPII surface and competes with ligands; immunofluorescence/fractionation localizing CIN85 to COPI-Golgi membranes","pmids":["18680311","18266907"],"confidence":"High","gaps":["Functional role of ubiquitin-binding in vivo not fully resolved","Golgi function of CIN85 not mechanistically connected to RTK role"]},{"year":2010,"claim":"Defined the membrane-targeting and late-endosomal steps and broadened receptor scope, showing CIN85 binds phosphatidic acid and dynamin-2 to route receptors through endosomal sorting, and controls nephrin and D2 dopamine receptor endocytosis in vivo.","evidence":"Lipid-binding and domain-deletion assays; Co-IP and dominant-negative dynamin-2 studies; CD2AP-knockout podocyte and CIN85-knockout mouse analyses with endocytosis and behavioral readouts","pmids":["19417776","20711168","20457601","20551902"],"confidence":"High","gaps":["Direct PA-binding residues not yet pinpointed","Tissue-specific receptor selectivity mechanism unclear"]},{"year":2011,"claim":"Established CIN85 as a constitutive organizer of the B-cell SLP65/BLNK complex required for BCR-driven Ca2+ and NF-κB responses via IKK-β, and as a regulator of immunoreceptor degradation across mast cells, neutrophils and B cells.","evidence":"MS interactome, live imaging, Ca2+ flux, and B cell-specific conditional knockout with IKK-β rescue; siRNA/overexpression Co-IP studies of Syk and FcγRIIa ubiquitination","pmids":["21822214","21708930","17675467","22262777","21372129","21725061","21830225"],"confidence":"High","gaps":["Direct link between SLP65 scaffolding and NF-κB output mechanistically incomplete","How CIN85 switches between negative (Syk degradation) and positive (NF-κB) roles unclear"]},{"year":2016,"claim":"Revealed the biophysical basis of B-cell priming — CIN85 oligomerizes SLP65 into pre-formed condensates via its trimeric coiled-coil and multiple SH3 domains — and linked CIN85 to PP2Ac suppression and nephrin endocytosis in vivo.","evidence":"Biochemical oligomerization and structure-function assays in B cells; phosphatase activity assay and platelet functional readouts for PP2Ac; CIN85-knockout mouse and zebrafish overexpression for nephrin filtration barrier","pmids":["27353366","27334924","27531950"],"confidence":"High","gaps":["Quantitative properties of condensates in resting cells unresolved","Phase behavior in vivo not directly demonstrated"]},{"year":2019,"claim":"Showed CIN85 acts as a negative regulator of TCR signaling via Sts-2 recruitment and inhibits PHD2 to stabilize HIF-α, expanding its roles into T-cell tolerance and hypoxia/tumor biology.","evidence":"Conditional knockout T cells with signaling and IL-2 readouts and Sts-2 Co-IP; binding, PHD2 hydroxylase activity assay, and CRISPR knock-in with tumor xenograft","pmids":["30723173","31142511"],"confidence":"High","gaps":["How CIN85 partitions between activating and inhibitory immunoreceptor roles unresolved","Structural basis of PHD2 inhibition not defined"]},{"year":2022,"claim":"Established cytoskeletal and developmental roles, placing CIN85 at the intercellular bridge in cytokinesis, in myofiber formation, ciliary length control, and RIG-I innate immune signaling.","evidence":"Direct binding and live-cell imaging for anillin/SEPT9 in cytokinesis; Co-IP, domain binding and Dnm2 mouse for dynamin-2/calnexin in myogenesis; siRNA/EHBP1L1 rescue for cilia; Co-IP, KO mouse and recombinant virus for TRIM25/RIG-I","pmids":["36044846","40065183","36754282","39466846"],"confidence":"Medium","gaps":["Mechanistic connection between adaptor scaffolding and septin filament assembly incomplete","Whether these roles share a common biochemical activity unclear"]},{"year":2023,"claim":"Defined phosphorylation-gated intramolecular autoinhibition controlling CIN85 valency toward SLP65 and identified further trafficking modulators that displace CIN85 from receptors.","evidence":"NMR of multidomain constructs with phosphomimetic mutagenesis and B cell Ca2+/recruitment assays; Co-IP and epistasis for ASAP2-c-MET","pmids":["38111344","37061723"],"confidence":"High","gaps":["Kinase responsible for the regulatory serine phosphorylation not identified","How autoinhibition is relieved in vivo unresolved"]},{"year":2025,"claim":"Tested necessity of CIN85/Cbl for EGFR internalization and extended its trafficking roles to tubular recycling endosomes and bleb-associated phosphoinositide patterning.","evidence":"Cbl/Cbl-b double-knockout MEFs with inducible CIN85 siRNA and quantitative EGF internalization assays; siRNA and Co-IP for MICAL-L1/TRE recycling; phase-separation and biosensor assays for septin-PI3K bleb axis","pmids":["25449262","40740057","40445717"],"confidence":"High","gaps":["Reconciliation of dispensability for internalization with earlier dominant-negative data unaddressed","Bleb phase-separation role rests on indirect evidence (Low confidence)"]},{"year":null,"claim":"How CIN85 selects among its dozens of partners to switch between promoting versus inhibiting a given receptor, and how its condensate/oligomerization behavior is controlled across the many cell types in which it acts, remain unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model for partner-selection logic","Cell-type-specific regulatory inputs (kinases, modifications) largely uncharacterized","In vivo significance of phase separation beyond B cells unestablished"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,5,7,17,19]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[31,8]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[25,41]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[45,37,38]}],"localization":[{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[15,25,27,41,52]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,17]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[36]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[20,47,49]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[31,32]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[57]},{"term_id":"GO:0005783","term_label":"endoplasmic reticulum","supporting_discovery_ids":[48]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1,15,27,41,52]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[38,45,37]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[17,18,43,44,54]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[46]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[4,13,29]}],"complexes":["SLP65/CIN85 oligomeric condensate","endophilin-CIN85-Cbl complex"],"partners":["CBL","CBLB","SLP65/BLNK","ENDOPHILIN","DNM2","PHD2/EGLN1","TRIM25","SEPT9"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96B97","full_name":"SH3 domain-containing kinase-binding protein 1","aliases":["CD2-binding protein 3","CD2BP3","Cbl-interacting protein of 85 kDa","Human Src family kinase-binding protein 1","HSB-1"],"length_aa":665,"mass_kda":73.1,"function":"Adapter protein involved in regulating diverse signal transduction pathways. Involved in the regulation of endocytosis and lysosomal degradation of ligand-induced receptor tyrosine kinases, including EGFR and MET/hepatocyte growth factor receptor, through an association with CBL and endophilins. The association with CBL, and thus the receptor internalization, may be inhibited by an interaction with PDCD6IP and/or SPRY2. Involved in regulation of ligand-dependent endocytosis of the IgE receptor. Attenuates phosphatidylinositol 3-kinase activity by interaction with its regulatory subunit (By similarity). May be involved in regulation of cell adhesion; promotes the interaction between TTK2B and PDCD6IP. May be involved in the regulation of cellular stress response via the MAPK pathways through its interaction with MAP3K4. Is involved in modulation of tumor necrosis factor mediated apoptosis. Plays a role in the regulation of cell morphology and cytoskeletal organization. Required in the control of cell shape and migration. Has an essential role in the stimulation of B cell activation (PubMed:29636373)","subcellular_location":"Cytoplasm; Cytoplasm, cytoskeleton; Cytoplasmic vesicle membrane; Synapse, synaptosome; Cell junction, focal adhesion","url":"https://www.uniprot.org/uniprotkb/Q96B97/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SH3KBP1","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":[{"gene":"CAPZB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/SH3KBP1","total_profiled":1310},"omim":[{"mim_id":"621453","title":"MULTIVESICULAR BODY SUBUNIT 12A; MVB12A","url":"https://www.omim.org/entry/621453"},{"mim_id":"619583","title":"EH DOMAIN-BINDING PROTEIN 1-LIKE 1; EHBP1L1","url":"https://www.omim.org/entry/619583"},{"mim_id":"617322","title":"SH3KBP1-BINDING PROTEIN 1; SHKBP1","url":"https://www.omim.org/entry/617322"},{"mim_id":"610591","title":"RHO GTPase-ACTIVATING PROTEIN 27; ARHGAP27","url":"https://www.omim.org/entry/610591"},{"mim_id":"604241","title":"CD2-ASSOCIATED PROTEIN; CD2AP","url":"https://www.omim.org/entry/604241"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SH3KBP1"},"hgnc":{"alias_symbol":["CIN85"],"prev_symbol":[]},"alphafold":{"accession":"Q96B97","domains":[{"cath_id":"2.30.30.40","chopping":"3-57","consensus_level":"high","plddt":92.1855,"start":3,"end":57},{"cath_id":"2.30.30.40","chopping":"103-155","consensus_level":"high","plddt":92.3677,"start":103,"end":155},{"cath_id":"2.30.30.40","chopping":"272-334","consensus_level":"high","plddt":89.7308,"start":272,"end":334}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96B97","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96B97-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96B97-F1-predicted_aligned_error_v6.png","plddt_mean":62.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SH3KBP1","jax_strain_url":"https://www.jax.org/strain/search?query=SH3KBP1"},"sequence":{"accession":"Q96B97","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96B97.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96B97/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96B97"}},"corpus_meta":[{"pmid":"11894095","id":"PMC_11894095","title":"Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors.","date":"2002","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/11894095","citation_count":471,"is_preprint":false},{"pmid":"11894096","id":"PMC_11894096","title":"The endophilin-CIN85-Cbl complex mediates ligand-dependent downregulation of c-Met.","date":"2002","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/11894096","citation_count":371,"is_preprint":false},{"pmid":"12354621","id":"PMC_12354621","title":"CIN85/CMS family of adaptor molecules.","date":"2002","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/12354621","citation_count":163,"is_preprint":false},{"pmid":"10679202","id":"PMC_10679202","title":"Cloning and characterization of a novel adaptor protein, CIN85, that interacts with c-Cbl.","date":"2000","source":"Biochemical and biophysical research 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Cell physiology","url":"https://pubmed.ncbi.nlm.nih.gov/40445717","citation_count":3,"is_preprint":false},{"pmid":"39955954","id":"PMC_39955954","title":"Feature gene selection and functional validation of SH3KBP1 in infantile hemangioma using machine learning.","date":"2025","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/39955954","citation_count":3,"is_preprint":false},{"pmid":"40740057","id":"PMC_40740057","title":"CIN85 and CD2AP Are Novel Constituents of Dynamic Tubular Recycling Endosomes That Regulate Recycling Upon Recruitment by MICAL-L1.","date":"2025","source":"Traffic (Copenhagen, Denmark)","url":"https://pubmed.ncbi.nlm.nih.gov/40740057","citation_count":3,"is_preprint":false},{"pmid":"21400643","id":"PMC_21400643","title":"Lentiviral vector-mediated siRNA knockdown and concurrent rescue of Murine CIN85.","date":"2011","source":"Journal of biochemical and molecular toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/21400643","citation_count":1,"is_preprint":false},{"pmid":"25816594","id":"PMC_25816594","title":"Multiple molecular forms of adaptor protein Ruk/CIN85 specifically associate with different subcellular compartments in human breast adenocarcinoma MCF-7 cells.","date":"2014","source":"Ukrainian biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/25816594","citation_count":1,"is_preprint":false},{"pmid":"33672244","id":"PMC_33672244","title":"Exploring Molecular Contacts of MUC1 at CIN85 Binding Interface to Address Future Drug Design Efforts.","date":"2021","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33672244","citation_count":1,"is_preprint":false},{"pmid":"41998852","id":"PMC_41998852","title":"Adaptor protein CIN85 potentiates the motility of osteosarcoma cells via the Akt/mTOR and MMP2-COL3A1 axis.","date":"2026","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/41998852","citation_count":0,"is_preprint":false},{"pmid":"40990523","id":"PMC_40990523","title":"Herpes simplex virus diverts CIN85 endosomal cargo for exocytosis to evade antiviral responses: a novel role for the viral immediate-early protein ICP0.","date":"2025","source":"mBio","url":"https://pubmed.ncbi.nlm.nih.gov/40990523","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":48718,"output_tokens":14054,"usd":0.178482,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":26061,"output_tokens":5966,"usd":0.139728,"stage2_stop_reason":"end_turn"},"total_usd":0.31821,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"CIN85 constitutively associates with endophilins via SH3 domain interactions, and is rapidly recruited by Cbl to form a trimeric complex with activated EGF receptors upon EGF stimulation, controlling receptor internalization independently of Cbl ubiquitin ligase activity. Inhibition of CIN85-Cbl or CIN85-endophilin interactions blocks EGFR internalization and delays receptor degradation.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative inhibition, EGF receptor internalization/degradation assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, functional rescue experiments, replicated independently in same issue by two labs (PMIDs 11894095 and 11894096)\",\n      \"pmids\": [\"11894095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The endophilin-CIN85-Cbl complex controls HGF receptor (c-Met) internalization; Cbl ubiquitinates activated c-Met and recruits the endophilin-CIN85 complex to regulate receptor internalization. Inhibition of complex formation blocks HGF receptor internalization and enhances HGF-induced signaling.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative inhibition, HGF receptor internalization assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus functional inhibition experiments, independent replication in same issue\",\n      \"pmids\": [\"11894096\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CIN85 binds to c-Cbl via its second SH3 domain; this association is basally present and enhanced after EGF stimulation correlating with Cbl tyrosine phosphorylation. CIN85 self-associates through its coiled-coil region to form tetramers, and both SH3 domains and coiled-coil region contribute to subcellular localization.\",\n      \"method\": \"Co-immunoprecipitation, yeast two-hybrid, domain deletion mutagenesis, EGF stimulation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mutagenesis, single lab, multiple methods\",\n      \"pmids\": [\"10679202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CIN85 associates with BLNK (B cell linker protein) via its SH3 domains, with both proteins co-localizing in the cytoplasm, implicating CIN85 in BCR-mediated signaling. CIN85 also forms complexes with Crk-I, Crk-II, p130Cas, p85-PI3K, Grb2, and Sos1.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, immunostaining\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and immunolocalization, single lab, two orthogonal methods\",\n      \"pmids\": [\"11071869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"CIN85 and its homologue CMS are monoubiquitinated by Cbl/Cbl-b after EGF stimulation. Monoubiquitination requires direct CIN85-Cbl interaction, intact RING finger domain of Cbl, and a ubiquitin acceptor site in the CIN85 C-terminus. Cbl-b and monoubiquitinated CIN85 are degraded together with polyubiquitinated EGFRs in the lysosome.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RING finger domain mutagenesis, lysosomal degradation assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination assay with mutagenesis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12218189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CIN85 SH3 domains specifically recognize a novel polyproline-arginine motif (PXXXPR) present in Cbl/Cbl-b. Individual SH3 domains bind with micromolar affinity, while extended two- or three-SH3-domain constructs bind with higher stoichiometry and affinity. This enables full-length CIN85 to simultaneously bind multiple Cbl molecules, promoting their clustering, which is important for EGFR degradation.\",\n      \"method\": \"Peptide binding assays, isothermal titration calorimetry, mutagenesis, co-immunoprecipitation, receptor degradation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assays with mutagenesis, ITC, and functional EGFR degradation readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"12874286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Cbl-b, but not Cbl-3, utilizes the same CIN85/endophilin pathway as Cbl to down-regulate multiple RTKs (EGFR, PDGFR). CIN85 binds to the C-terminus of Cbl-b; inhibiting CIN85-Cbl-b interaction impairs EGFR internalization but not receptor polyubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative inhibition, EGFR internalization and ubiquitination assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional internalization and ubiquitination assays, epistasis between CIN85-Cbl-b interaction and receptor internalization vs ubiquitination\",\n      \"pmids\": [\"12177062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CIN85 SH3 domains bind to PxxxPR motifs in multiple endocytic effectors including SHIP-1, synaptojanin 2B1, ASAP1, ARAP3, Hip1R, STAP1, and p115RhoGEF, functioning as a molecular scaffold that clusters these proteins into high-molecular-weight complexes. ASAP1 overexpression increases EGFR recycling, an effect dependent on its intact PxxxPR motif.\",\n      \"method\": \"Co-immunoprecipitation, pulldown, overexpression/mutant studies, EGFR recycling assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple binding partners identified by Co-IP/pulldown, single lab with functional receptor recycling readout\",\n      \"pmids\": [\"15090612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CIN85 and CMS/CD2AP SH3 domains bind to the CD2 cytoplasmic tail proline-rich segment, and CIN85/CMS directly bind to the actin-capping protein CAPZ via their C-terminal half, providing a molecular link between CD2 and the actin cytoskeleton. Overexpression of CMS/CIN85 fragments in T cells enhanced IL-2 production and reduced TCR downmodulation.\",\n      \"method\": \"Peptide affinity chromatography, BIAcore surface plasmon resonance, co-immunoprecipitation, functional T cell assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — SPR quantitative binding data, Co-IP, functional T cell readout, single lab\",\n      \"pmids\": [\"12690097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Alix/AIP1 antagonizes the Cbl-CIN85 complex by weakening the CIN85-Cbl interaction and reducing Cbl-mediated ubiquitination of EGFR and CIN85, thereby diminishing EGFR internalization. siRNA knockdown of Alix promoted EGFR internalization, confirming the inhibitory role.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, ubiquitination assay, EGFR internalization assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with functional internalization readout, Co-IP, single lab\",\n      \"pmids\": [\"15456872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CIN85 SH3 domains bind to an atypical recognition consensus PX(P/A)XXR, confirmed by mutagenesis and in vitro binding. This motif maps CIN85-binding sites in c-Cbl, Cbl-b, BLNK, AIP1/Alix, and CD2. Novel interactors synaptojanin 1, PAK2, ZO-2, and TAFII70 were identified by GST-pulldown from brain lysates.\",\n      \"method\": \"Target-assisted iterative peptide library screening, mutagenesis, in vitro binding assays, GST pulldown, Far Western blotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding with mutagenesis and multiple orthogonal methods (Far Western, pulldown), single lab\",\n      \"pmids\": [\"12829691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Disabled-2 (Dab2) associates with all three SH3 domains of CIN85 via a PKPAPR motif and recruits CIN85 to the clathrin coat. This association is modulated by growth factor stimulation, whereby Dab2 and clathrin dissociate from CIN85 after growth factor treatment, enabling Cbl to bind.\",\n      \"method\": \"Co-immunoprecipitation, peptide binding, stimulation-dependent association studies\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP with dynamic stimulation-dependent analysis, single lab\",\n      \"pmids\": [\"14596919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Yeast Sla1 is a functional ortholog of CIN85, with similar domain structure and direct binding to endophilin-like Rvs167. Rsp5 (yeast ubiquitin ligase) directly binds and monoubiquitinates Rvs167 at Lys481 in its SH3 domain, mediated through Rsp5 WW domains and PXY motifs in Rvs167.\",\n      \"method\": \"Genetic analysis, in vitro binding, ubiquitination assay, mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination with mutagenesis in yeast ortholog system, single lab\",\n      \"pmids\": [\"14761940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"All three SH3 domains of CIN85 bind to ubiquitin at the canonical proline-rich ligand-binding surface, with ubiquitin mimicking a PPII helix. Ubiquitin and proline-rich ligands compete for the same binding surface. Disruption of ubiquitin binding results in constitutive CIN85 ubiquitination and elevated basal EGFR ubiquitination in the absence of EGF.\",\n      \"method\": \"NMR structural modeling, ITC, mutagenesis, EGFR ubiquitination assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure with mutagenesis and functional ubiquitination readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"18680311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CIN85 binds to the coiled-coil domain of nephrin and podocin (not via SH3 domains), mediates nephrin internalization after FGF-4 stimulation, and promotes nephrin ubiquitination. CD2AP competes with CIN85 for binding to nephrin/podocin. In CD2AP-deficient podocytes, CIN85 accumulation correlates with increased nephrin ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, binding assays with domain mutants, immunofluorescence, internalization assay, CD2AP knockout mouse analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mutant binding assays, KO mouse data, functional internalization assay, replicated in multiple experimental systems\",\n      \"pmids\": [\"20457601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CIN85 directly interacts with dynamin 2 (Dyn2) in a manner induced by EGFR stimulation; this interaction occurs late in the endocytic process at late endosomes. Disruption of CIN85-Dyn2 interaction causes accumulation of internalized EGFR in aberrantly elongated late endosomal tubules and sustained downstream signaling.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative studies, fluorescence microscopy of EGFR trafficking\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional dominant-negative perturbation and morphological phenotype, single lab\",\n      \"pmids\": [\"20711168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Sprouty2 associates with CIN85 via SH3 domains A and C binding proline-arginine motifs in Sprouty2, and acts at the Cbl-CIN85 interface to inhibit EGFR endocytosis and degradation. Sprouty4, which lacks CIN85-binding sites, does not inhibit EGFR downregulation.\",\n      \"method\": \"Co-immunoprecipitation, EGFR endocytosis/degradation assay, PC12 differentiation assay, domain mutagenesis\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mutagenesis, functional EGFR endocytosis and differentiation readouts, single lab\",\n      \"pmids\": [\"15962011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CIN85 constitutively associates with SLP65 (BLNK) in a stimulation-independent manner in B cells, which is required for SLP65 phosphorylation and inducible plasma membrane translocation. In the absence of a stable SLP65/CIN85 complex, BCR-induced Ca2+ and NF-κB responses are abrogated.\",\n      \"method\": \"Mass spectrometry interactome, co-immunoprecipitation, live cell imaging, loss-of-function studies, Ca2+ flux assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative MS interactome plus functional loss-of-function with defined BCR signaling readouts, multiple orthogonal methods\",\n      \"pmids\": [\"21822214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CIN85 links the BCR to IKK-β activation to drive canonical NF-κB signaling in B cells. B cell-specific CIN85 knockout mice show impaired T cell-independent type II antibody responses, diminished IKK-β activation, and defective BCR cross-linking responses, rescued by constitutively active IKK-β.\",\n      \"method\": \"B cell-specific conditional knockout mouse (Mb1-cre), in vivo antibody responses, IKK-β activation assay, constitutively active IKK-β rescue\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with in vivo functional readouts and genetic rescue, single lab\",\n      \"pmids\": [\"21708930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CIN85 oligomerizes SLP-65 in B cells through its trimeric coiled-coil domain and multiple SH3 domains, forming large signaling condensates/oligomeric complexes. This pre-formed oligomeric complex in resting B cells is required for efficient BCR signaling initiation.\",\n      \"method\": \"Biochemical oligomerization assays, co-immunoprecipitation, structure-function analysis with domain mutants, B cell functional assays\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal biochemical and functional methods, identifies mechanistic basis of CIN85 oligomerization in B cell signaling\",\n      \"pmids\": [\"27353366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CIN85 deficiency in mice (CIN85Δex2) leads to insufficient endophilin-D2 dopamine receptor (D2DR) complex formation in the striatum and decreased D2DR endocytosis in striatal neurons in response to dopamine, resulting in elevated striatal D2DR levels and dopamine, and hyperactive behavior. CIN85 localizes to the post-synaptic compartment of striatal neurons and co-clusters with D2DRs.\",\n      \"method\": \"Conditional knockout mouse, receptor endocytosis assay in striatal neurons, co-immunoprecipitation, immunofluorescence localization, behavioral analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO mouse with direct endocytosis assay, protein co-localization, and behavioral phenotype all linked to mechanism\",\n      \"pmids\": [\"20551902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Drosophila Cindr (CD2AP/CIN85 ortholog) links E-cadherin and Roughest (IgCAM adhesion receptor) at cell junctions with actin cytoskeletal components including capping protein alpha and beta. Reduction of cindr activity causes defects in E-cadherin and Roughest localization, local cell movement, tissue patterning, and cell death in the developing retina.\",\n      \"method\": \"Drosophila genetics (loss-of-function), immunofluorescence, protein interaction studies, tissue phenotype analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function in Drosophila ortholog with defined molecular and cellular phenotypes\",\n      \"pmids\": [\"18362180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CIN85 colocalizes with AMAP1 at invadopodia in breast cancer cells; binding of AMAP1 to CIN85 is required for invasive activity including matrix degradation. CIN85 siRNA silencing inhibits invasion. Cbl monoubiquitinates AMAP1, and this monoubiquitination is important for AMAP1's role in invasion.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, invasion assay, immunofluorescence\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA functional studies with defined invasion phenotype, Co-IP, single lab\",\n      \"pmids\": [\"17255943\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CIN85 associates with the TNFR1 signaling complex via Src family kinases (not directly with TNFR1 cytoplasmic domain); Src binds directly to TNFR1. Ectopic CIN85 expression increases susceptibility to TNF-α-induced apoptosis 10-fold. The three SH3 domains and proline-rich regions of CIN85 are essential for this pro-apoptotic effect.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, apoptosis assay, domain deletion mutagenesis\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mutagenesis and functional apoptosis readout, single lab\",\n      \"pmids\": [\"15707590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CIN85 overexpression in mast cells drives IgE receptor (FcεRI) internalization into early endosomes and lysosomes, reduces FcεRI expression levels, and dramatically impairs mast cell degranulation after antigen stimulation.\",\n      \"method\": \"Overexpression, confocal microscopy, receptor internalization/degradation assay, degranulation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — overexpression with functional receptor trafficking and degranulation readouts, single lab\",\n      \"pmids\": [\"16177060\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"CIN85 associates with endosomal membranes through its positively charged C-terminus binding to phosphatidic acid (PA), with the coiled-coil domain essential for this protein-lipid interaction. Deletion of the coiled-coil domain abolishes membrane association, reduces c-Cbl interaction, and blocks EGFR downregulation. A significant pool of CIN85 localizes to EEA1-positive endosomal compartments.\",\n      \"method\": \"Lipid binding assay, domain deletion mutagenesis, subcellular fractionation, immunofluorescence, EGFR degradation assay\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — lipid binding assay with domain mutagenesis and functional EGFR degradation readout, single lab\",\n      \"pmids\": [\"19417776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SHIP-1 constitutively associates with all three SH3 domains of CIN85 in B cells via a 79-amino acid region near the SHIP-1 C-terminus, identified by systematic pulldown proteomics.\",\n      \"method\": \"Pulldown proteomics, co-immunoprecipitation, domain mapping\",\n      \"journal\": \"Molecular & cellular proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP/pulldown with domain mapping, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21725061\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Src kinase mediates tyrosine phosphorylation of CIN85 following EGFR activation; phospho-CIN85 interacts with Rab5-positive early endosomes and mediates sequestration of ubiquitinated EGFR into multivesicular bodies (MVBs). CIN85 knockdown decreases EGF-induced EGFR ubiquitination and reduces EGFR sorting to MVBs.\",\n      \"method\": \"siRNA knockdown, Src inhibition, phospho-specific analysis, co-immunoprecipitation, subcellular fractionation, immunofluorescence\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA with specific phospho-CIN85 mechanistic studies, single lab\",\n      \"pmids\": [\"22833562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CIN85 interacts with TβRI (TGFβ receptor type I) SH3 domains in a TRAF6-dependent manner upon TGFβ stimulation. CIN85 knockdown causes accumulation of TβRI in intracellular compartments and diminished Smad2 phosphorylation. CIN85 overexpression increases TβRI at the cell surface by promoting receptor recycling via a Rab11-dependent pathway, enhancing TGFβ-stimulated Smad2 phosphorylation, transcriptional responses, and cell migration.\",\n      \"method\": \"siRNA knockdown, overexpression, dominant-negative Rab11, Smad2 phosphorylation assay, transcriptional reporter, cell migration assay, Co-IP\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA/OE with multiple functional readouts and dominant-negative epistasis, single lab\",\n      \"pmids\": [\"26169354\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CIN85 regulates Syk protein levels in mast cells by promoting c-Cbl-mediated Syk ubiquitination and proteasomal degradation. CIN85 overexpression limits Cbl binding to Sts1 (a negative regulator of Cbl), while CIN85 knockdown favors Cbl/Sts1 complex formation, reducing Cbl-directed Syk ubiquitination.\",\n      \"method\": \"Overexpression, siRNA knockdown, co-immunoprecipitation, proteasome inhibition, ubiquitination assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA/OE with proteasome inhibitor rescue and mechanistic Co-IP, single lab\",\n      \"pmids\": [\"17675467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"In human B cells, CIN85 is constitutively associated with c-Cbl and Cbl-b, and increases c-Cbl phosphorylation. CIN85 overexpression inhibits BCR-induced calcium flux and phosphorylation of Syk and PLCγ2, correlating with increased Syk ubiquitination and degradation. CIN85 knockdown enhances BCR-induced survival, growth, and differentiation-associated gene expression.\",\n      \"method\": \"Overexpression, siRNA knockdown, co-immunoprecipitation, calcium flux assay, phosphorylation analysis, ubiquitination assay\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — paired OE/KD studies with multiple signaling readouts, single lab\",\n      \"pmids\": [\"22262777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CMS/CIN85 can crosslink filamentous actin (F-actin) into bundles, a function dependent on the proline-rich region and coiled-coil domain. CMS/CIN85 are found in podosomes. CMS and CIN85 can form heterotypic complexes via their coiled-coil domains. Removal of these actin-bundling domains reduces cell migration.\",\n      \"method\": \"F-actin binding/bundling assays, domain deletion mutagenesis, cell migration assay, immunofluorescence\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro actin bundling assay with domain mutagenesis and functional migration readout, single lab\",\n      \"pmids\": [\"17606992\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CIN85 co-localizes with actin at focal adhesions and with microtubules, and CIN85 interacts with focal adhesion kinases FAK and PYK-2. CIN85 promotes cell adhesion as assessed by ECIS.\",\n      \"method\": \"Immunofluorescence co-localization, co-immunoprecipitation, ECIS (electrical cell-substrate impedance sensing) adhesion assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and localization without direct mechanistic follow-up for CIN85-FAK interaction, single lab\",\n      \"pmids\": [\"12771190\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CIN85 forms a complex with S-SCAM and dendrin at synapses; the first SH3 domain and the C-terminal region of CIN85 bind the proline-rich and N-terminal regions of dendrin, respectively. CIN85 localizes to synaptic compartments as shown by immunocytochemistry and subcellular fractionation.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, immunocytochemistry, subcellular fractionation, in vitro binding assay\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — multiple orthogonal binding assays with synaptic localization, single lab\",\n      \"pmids\": [\"16751601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"CIN85 interacts with the p85α regulatory subunit of PI3K through multiple domains: CIN85 SH3 domains are required and sufficient for binding full-length p85α, but the SH3 domain of p85α is required to activate this interaction by relieving an intramolecular autoinhibitory interaction between CIN85 SH3 domains and its proline-rich region.\",\n      \"method\": \"Co-immunoprecipitation, domain deletion/mutagenesis, in vitro binding assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding with domain mapping and intramolecular interaction analysis, single lab\",\n      \"pmids\": [\"15476827\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Intersectin 1 (ITSN1) forms a constitutive complex with CIN85 mediated by the SH3A domain of ITSN1 and the third or fourth proline-rich blocks of CIN85, independent of EGF stimulation.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, immunofluorescence co-localization\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP with domain mapping, single lab\",\n      \"pmids\": [\"19166927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CIN85 is distributed in multiple membrane trafficking compartments in human cells; the major endogenous pool associates with COPI-coated vesicles of the Golgi complex involved in retrograde ER-Golgi transport. This localization depends on Golgi complex integrity and intact microtubules.\",\n      \"method\": \"Immunofluorescence with compartment markers, subcellular fractionation, antibody characterization, Golgi disruption experiments\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — detailed localization study with functional perturbation of organelle integrity, single lab\",\n      \"pmids\": [\"18266907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CIN85 binds the catalytic subunit of protein phosphatase 2A (PP2Ac) via the P3 block (PAIPPKKPRP) in CIN85's proline-rich region; this interaction suppresses PP2Ac phosphatase activity. Disruption of PP2Ac-CIN85 interaction decreases platelet spreading and fibrin clot retraction and reduces phosphorylation of Src and GSK3β.\",\n      \"method\": \"Yeast two-hybrid, truncation/alanine mutagenesis, in vitro phosphatase activity assay, cell adhesion assay, clot retraction assay, cell-permeable peptide\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro phosphatase activity assay with mutagenesis, functional platelet assay, single lab\",\n      \"pmids\": [\"27334924\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"CIN85 SH3 domains interact with MEKK4 via three PxxxPR motifs; disruption of this interaction demonstrates that CIN85 binding to MEKK4 enhances MKK6 and p38 MAP kinase activation following oxidative stress and growth factor stimulation. CIN85 also regulates MEKK4 activation by GADD45 proteins and promotes MEKK4 multi-ubiquitination.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative inhibition, kinase activation assays, ubiquitination assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with functional kinase assays and specific PxxxPR motif disruption, single lab\",\n      \"pmids\": [\"16256071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CD2AP regulates SUMOylation of CIN85 in podocytes; full-length CIN85 is SUMOylated by SUMO-1, -2, and -3, and SUMOylation is enhanced in the presence of CD2AP. Mutation of lysine 598 to arginine abolishes CIN85 SUMOylation and leads to increased CIN85 binding to nephrin.\",\n      \"method\": \"SUMOylation assay, site-directed mutagenesis (K598R), co-immunoprecipitation, podocyte cell culture\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro/cell-based SUMO modification assay with site mutagenesis and functional binding consequences, single lab\",\n      \"pmids\": [\"22037207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CIN85 (RukL isoform) deficiency in mice preserves nephrin surface expression on the slit diaphragm and reduces proteinuria under diabetic conditions; CIN85/RukL overexpression in zebrafish causes severe edema and disruption of the filtration barrier. CIN85/RukL mediates nephrin endocytosis via ubiquitination in podocytes.\",\n      \"method\": \"CIN85 knockout mouse under diabetic conditions, zebrafish overexpression, nephrin surface expression assay, proteinuria measurement\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function in mice and gain-of-function in zebrafish with functional filtration barrier readouts, replicated across species\",\n      \"pmids\": [\"27531950\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Basic amino acids K645, K646, R648, and R650 in the CIN85 coiled-coil domain are required for both phosphatidic acid binding and c-Cbl interaction but not endophilin interaction. CIN85 also interacts with ESCRT components, and mutations in the coiled-coil domain dissociate CIN85 from endosomes and prevent ESCRT assembly on endosomal membranes, inhibiting EGFR sorting.\",\n      \"method\": \"Site-directed mutagenesis, lipid binding assay, co-immunoprecipitation, EGFR sorting/degradation assay, immunofluorescence\",\n      \"journal\": \"BMC biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro lipid binding with mutagenesis and functional EGFR sorting readout, single lab\",\n      \"pmids\": [\"25005938\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CIN85 depletion by siRNA in neutrophils prevents FcγRIIa ubiquitination and degradation by c-Cbl following receptor cross-linking, increases IgG-mediated phagocytosis, and disrupts proper FcγRIIa endosomal sorting. CIN85 is a substrate of classical PKCs, which positively regulate FcγRIIa ubiquitination/degradation.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, ubiquitination assay, phagocytosis assay, PKC inhibition, confocal microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA functional studies with multiple mechanistic readouts, single lab\",\n      \"pmids\": [\"21372129\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CIN85 inhibits T cell activation by being recruited to the TCR signaling complex upon stimulation; CIN85-deficient T cells show enhanced TCR signaling (Zap70, SLP76, Erk phosphorylation) and increased IL-2 production. The inhibitory function requires CIN85 SH3 and PR regions, which associate with the phosphatase Sts-2 after TCR stimulation.\",\n      \"method\": \"Conditional knockout T cells, T cell activation assays, signaling phosphorylation analysis, co-immunoprecipitation, domain mutant studies\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with defined signaling phenotypes, mechanistic Co-IP for Sts-2, domain mutant epistasis, single lab\",\n      \"pmids\": [\"30723173\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Germline deletion of CIN85 in humans with X-linked antibody deficiency causes B cell-intrinsic defects in BCR effector pathways, most notably NF-κB activation and CD86 upregulation, without affecting immune cell compartment development.\",\n      \"method\": \"Human genetic analysis, functional B cell assays (NF-κB activation, CD86 upregulation), immune cell phenotyping\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — human germline loss-of-function with defined B cell signaling phenotypes, natural experiment\",\n      \"pmids\": [\"29636373\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CIN85 binds directly to PHD2 (the main HIF prolyl hydroxylase) via CIN85 N-terminal SH3 domains interacting with the proline-arginine-rich N-terminus of PHD2, but not PHD1 or PHD3. This interaction inhibits PHD2 hydroxylase activity and HIF degradation. CRISPR/Cas9 disruption of the CIN85-PHD2 interaction in cells affects growth, migration, and tumor growth in mice.\",\n      \"method\": \"Co-immunoprecipitation, PHD2 hydroxylase activity assay, CRISPR/Cas9 knock-in, tumor xenograft\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — enzymatic activity assay with binding data and CRISPR functional validation in vivo, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31142511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CIN85 interacts directly with the N-terminal region of anillin and with SEPT9 to facilitate SEPT9-containing septin filament localization to the intercellular bridge (ICB) plasma membrane during cytokinesis. CIN85 is required for robust and timely cytokinesis and ICB elongation and maturation.\",\n      \"method\": \"Co-immunoprecipitation, direct binding assay, siRNA knockdown, live-cell imaging of cytokinesis, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding assay with siRNA functional cytokinesis phenotype and live-cell imaging, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"36044846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"An intramolecular autoinhibitory interaction occurs between the CIN85 SH3C domain and an adjacent proline-rich motif (PRM) in the linker region. Phosphorylation of a serine adjacent to this PRM regulates the intramolecular SH3:PRM interaction, modulating CIN85 valency toward SLP65. Disruption of this interaction impairs SLP65/CIN85 condensate formation, CIN85 membrane recruitment, and Ca2+ mobilization in B cells.\",\n      \"method\": \"NMR spectroscopy of multidomain constructs, phosphomimetic mutagenesis, B cell functional assays (Ca2+ flux, membrane recruitment)\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural evidence with mutagenesis and B cell functional validation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38111344\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SH3KBP1 N-terminus binds directly to dynamin-2 and C-terminus associates with the ER through calnexin; these interactions control myonuclei dynamics and ER integrity respectively during myofiber formation. SH3KBP1 is required for myoblast fusion, myonuclear positioning, myotube elongation, triad formation, and efficient excitation-contraction coupling.\",\n      \"method\": \"siRNA screen, co-immunoprecipitation (dynamin-2, calnexin), domain binding assays, live-cell imaging, functional excitation-contraction coupling assay, Dnm2R465W/+ mouse model\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain binding and functional myogenesis readouts, single lab\",\n      \"pmids\": [\"40065183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SH3KBP1 recruits PI3K to bleb necks via liquid-liquid phase separation through interactions with septin, forming a Septin-SH3KBP1-PI3K axis that establishes differential phosphoinositide (PI3P/PIP2) distribution at bleb vs non-bleb membrane and confers anoikis resistance.\",\n      \"method\": \"Phase separation assay, co-immunoprecipitation, immunofluorescence with phosphoinositide biosensors, anoikis assay\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, single set of assays, phase separation claim based on indirect evidence\",\n      \"pmids\": [\"40445717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"ASAP2 directly binds CIN85, disrupting the CIN85-c-MET interaction and preventing CIN85-induced c-MET internalization and lysosomal degradation, thereby sustaining HGF/c-MET signaling. CIN85 knockdown rescues the inhibitory effect of ASAP2 knockdown on c-MET signaling.\",\n      \"method\": \"Co-immunoprecipitation, cycloheximide chase, siRNA knockdown (ASAP2, CIN85), c-MET signaling assays\",\n      \"journal\": \"Experimental hematology & oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus genetic epistasis (CIN85 KD rescues ASAP2 KD phenotype), single lab\",\n      \"pmids\": [\"37061723\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SH3KBP1-binding protein 1 (SHKBP1) constitutively binds to CIN85 SH3 domains via its two PXXXPR motifs, competing with c-Cbl for CIN85 binding. This competition prevents CIN85 translocation to EGFR-containing vesicles, reduces EGFR degradation, and enhances EGF-induced signaling.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative inhibition, EGFR degradation assay, reporter gene assay\",\n      \"journal\": \"Cell biochemistry and function\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — competitive binding Co-IP with functional EGFR degradation readout, single lab\",\n      \"pmids\": [\"21830225\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CIN85 and CD2AP are recruited to tubular recycling endosomes (TREs) via interactions with MICAL-L1 through their SH3 domains. Depletion of either CIN85 or CD2AP impairs receptor recycling (CD98 cargo), demonstrating their roles in TRE function.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, live-cell fluorescence imaging of TRE dynamics, recycling assays\",\n      \"journal\": \"Traffic (Copenhagen, Denmark)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA functional recycling assay with Co-IP, single lab\",\n      \"pmids\": [\"40740057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Cbl/Cbl-b double knockout MEFs retain largely normal EGFR internalization, and inducible siRNA knockdown of CIN85 in WT or Cbl/Cbl-b DKO MEFs has no impact on EGFR internalization. This demonstrates that endogenous CIN85, Cbl, and Cbl-b are largely dispensable for initial EGFR internalization, while Cbl is required for EGFR ubiquitination and degradation.\",\n      \"method\": \"Cbl/Cbl-b double-knockout MEFs, inducible siRNA knockdown of CIN85, 125I-EGF and fluorescent EGF internalization assay\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic double KO plus siRNA knockdown with quantitative internalization assays, rigorous experimental controls\",\n      \"pmids\": [\"25449262\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SH3KBP1 enhances RIG-I signal transduction by promoting K63-linked polyubiquitination through interaction with E3 ubiquitin ligase TRIM25. PRRSV NSP2 induces autophagic degradation of SH3KBP1 via its third polyproline-arginine motif (453PVPAPR458), counteracting innate immune signaling. Sh3kbp1 knockout mice show increased susceptibility to VSV with reduced IFN-β.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, ubiquitination assay, autophagy assay, knockout mouse, recombinant virus with motif deletion\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with in vivo KO mouse and recombinant virus, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"39466846\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HEV ORF3 protein (pORF3) interacts with CIN85 and thereby competes with growth factor receptor-Cbl-CIN85 complex formation, resulting in reduced CIN85 ubiquitination and delayed trafficking of the growth factor receptor complex to late endosomes/lysosomes.\",\n      \"method\": \"Co-immunoprecipitation, receptor trafficking assay, ubiquitination assay, pORF3 mutant analysis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional receptor trafficking assay with competitive binding mechanism, single lab\",\n      \"pmids\": [\"20130058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"SOX10 transcription factor directly regulates expression of an alternative CIN85 isoform in Schwann cells via a highly conserved SOX10 binding site within an alternative promoter at the Sh3kbp1 locus. Mutation of the SOX10 binding site ablates promoter activity, and ectopic SOX10 promotes endogenous Sh3kbp1 expression.\",\n      \"method\": \"ChIP, promoter reporter assay, site-directed mutagenesis, ectopic SOX10 expression\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assays with mutagenesis confirming direct SOX10-dependent transcriptional regulation, single lab\",\n      \"pmids\": [\"22037207\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The EHBP1L1-CIN85/CD2AP axis controls ciliary length via actin network remodeling; CIN85 and CD2AP localize to the ciliary sheath in an EHBP1L1-dependent manner, and their depletion causes elongated cilia with actin nucleation and branching defects around the ciliary base.\",\n      \"method\": \"siRNA knockdown, immunofluorescence microscopy, co-immunoprecipitation, rescue with EHBP1L1 mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA knockdown with morphological and actin phenotypes, domain mutant rescue, single lab\",\n      \"pmids\": [\"36754282\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SH3KBP1/CIN85 is a multidomain adaptor protein (three SH3 domains, proline-rich region, coiled-coil domain) that acts as a central scaffold in receptor tyrosine kinase endocytosis and downregulation: upon growth factor stimulation, its SH3 domains bind a PXXXPR motif in Cbl/Cbl-b, enabling CIN85 to bridge activated RTKs (EGFR, c-Met, ErbB2, D2DR, FcεRI, FcγRIIa, TGFβR, nephrin) with endophilins, dynamin-2, and endosomal membrane (via phosphatidic acid binding at its coiled-coil domain), facilitating clathrin-mediated internalization and lysosomal degradation; Cbl monoubiquitinates CIN85 and Src phosphorylates it to regulate endosomal sorting into MVBs; in B cells, CIN85 constitutively pre-assembles with SLP65 into phase-separated oligomeric condensates (regulated by an intramolecular SH3C:PRM autoinhibitory interaction and serine phosphorylation) that prime BCR signaling by linking the BCR to IKK-β-dependent NF-κB activation; in T cells, CIN85 is recruited to the TCR complex and inhibits activation via Sts-2; beyond vesicular trafficking, CIN85 interacts with PHD2 to inhibit HIF-α hydroxylation, promotes p38 MAPK activation via MEKK4, suppresses PP2Ac phosphatase activity, crosslinks F-actin, localizes to COPI-Golgi membranes, intercellular bridges, and cilia, and is required for cytokinesis, muscle fiber formation, and innate immune RIG-I signaling via TRIM25.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SH3KBP1 (CIN85) is a multidomain adaptor that organizes receptor downregulation and assembly of multiprotein signaling complexes through its three SH3 domains, proline-rich region, and self-associating coiled-coil [#0, #2, #5]. Its SH3 domains recognize an atypical PX(P/A)XXR/PXXXPR proline-arginine motif, allowing it to bind Cbl/Cbl-b and a wide array of endocytic effectors and to cluster them into high-molecular-weight complexes; full-length CIN85 can simultaneously engage multiple Cbl molecules to promote receptor degradation [#5, #10, #7]. Upon growth factor stimulation it is recruited by Cbl to activated RTKs, where together with constitutively bound endophilins it controls internalization and lysosomal degradation of EGFR and c-Met [#0, #1]. CIN85 couples to the endocytic and sorting machinery through stimulation-induced binding to dynamin-2 at late endosomes and Src-dependent phosphorylation that drives ubiquitinated EGFR into multivesicular bodies, while its coiled-coil/basic C-terminus binds phosphatidic acid on endosomal membranes to enable ESCRT assembly and cargo sorting [#15, #27, #25, #41]. It is itself a substrate of regulatory modification, being monoubiquitinated by Cbl/Cbl-b and competing with ubiquitin for its own SH3 ligand-binding surface, with disruption of ubiquitin binding causing constitutive CIN85 and EGFR ubiquitination [#4, #13]. CIN85 broadly tunes receptor trafficking across many systems, mediating endocytosis of nephrin in podocytes, D2 dopamine receptors in striatal neurons, FcεRI in mast cells, FcγRIIa in neutrophils, and recycling of TβRI, and its activity is gated by competitive binding partners (Sprouty2, SHKBP1, ASAP2, Alix) that displace it from Cbl or its receptor targets [#14, #20, #24, #42, #28, #16, #51, #50, #9]. In B cells CIN85 constitutively pre-assembles with SLP65/BLNK into oligomeric, condensate-forming complexes via its coiled-coil and SH3 domains, an interaction gated by an intramolecular SH3C:proline-rich autoinhibition under serine-phosphorylation control, priming BCR signaling and linking the receptor to IKK-β–dependent NF-κB activation [#17, #19, #47, #18]. Germline loss of CIN85 in humans causes an X-linked antibody deficiency with B cell-intrinsic defects in BCR-driven NF-κB activation [#44]. Beyond trafficking and lymphocyte signaling, CIN85 inhibits HIF prolyl hydroxylase PHD2 to stabilize HIF-α [#45], activates p38 MAPK through MEKK4 [#38], suppresses PP2Ac phosphatase activity [#37], crosslinks F-actin [#31], and acts in cytokinesis, myofiber formation, ciliary length control, and RIG-I innate immune signaling through interactions with septins/anillin, dynamin-2/calnexin, EHBP1L1, and TRIM25 respectively [#46, #48, #57, #54].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established the core architecture and first partners: how CIN85 engages Cbl and self-associates, defining it as a clustering adaptor rather than a simple linker.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, and domain-deletion mutagenesis in EGF-stimulated cells, identifying Cbl binding via the second SH3 domain and coiled-coil-mediated tetramerization, plus BLNK/Crk/PI3K associations\",\n      \"pmids\": [\"10679202\", \"11071869\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of self-association not yet defined\", \"No structural basis for SH3-ligand recognition\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined CIN85's central role in RTK downregulation by showing it forms an endophilin-Cbl-receptor complex that drives internalization, and that it is itself monoubiquitinated and co-degraded with the receptor.\",\n      \"evidence\": \"Reciprocal Co-IP, dominant-negative inhibition, EGFR/c-Met internalization and degradation assays (two independent labs), plus in vitro ubiquitination with Cbl RING and acceptor-site mutagenesis\",\n      \"pmids\": [\"11894095\", \"11894096\", \"12218189\", \"12177062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether internalization requirement is cell-type dependent\", \"Endosomal sorting steps downstream of internalization not resolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the molecular grammar of CIN85 recognition — an atypical PX(P/A)XXR proline-arginine motif — explaining how it binds many partners and clusters multiple Cbl molecules to promote receptor degradation.\",\n      \"evidence\": \"Peptide library screening, ITC, mutagenesis, Far Western and GST-pulldown mapping the motif across Cbl, BLNK, Alix, CD2 and identifying additional interactors; CD2/CAPZ link to actin\",\n      \"pmids\": [\"12874286\", \"12829691\", \"12690097\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of multivalent complexes in cells not measured\", \"Selectivity among the three SH3 domains incompletely defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed CIN85 functions as a scaffold clustering endocytic effectors and revealed antagonistic regulators and an intramolecular autoinhibition relieved by partners.\",\n      \"evidence\": \"Co-IP/pulldown identifying SHIP-1, synaptojanin, ASAP1, ARAP3, Hip1R partners; siRNA and Co-IP for Alix antagonism; in vitro binding showing p85α SH3 relieves CIN85 SH3:PR autoinhibition; yeast Sla1/Rvs167 ortholog ubiquitination\",\n      \"pmids\": [\"15090612\", \"15456872\", \"15476827\", \"14761940\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological balance between scaffolding and antagonist binding unquantified\", \"Autoinhibition regulation in cells not yet shown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Extended CIN85 regulation to receptor trafficking control by competitive inhibitors and to MAPK and death-receptor signaling beyond pure endocytosis.\",\n      \"evidence\": \"Co-IP, domain mutagenesis and functional EGFR/FcεRI/TNFR1 assays for Sprouty2 inhibition, MEKK4-p38 activation, FcεRI internalization, and Src-mediated TNFR1 association\",\n      \"pmids\": [\"15962011\", \"16256071\", \"16177060\", \"15707590\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether p38 and apoptosis roles are direct or trafficking-dependent unclear\", \"Most based on overexpression rather than endogenous loss-of-function\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Provided the structural basis for ubiquitin/proline-rich ligand competition and mapped the endogenous CIN85 pool to Golgi COPI vesicles, broadening its trafficking remit.\",\n      \"evidence\": \"NMR modeling, ITC, mutagenesis showing ubiquitin binds the SH3 PPII surface and competes with ligands; immunofluorescence/fractionation localizing CIN85 to COPI-Golgi membranes\",\n      \"pmids\": [\"18680311\", \"18266907\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of ubiquitin-binding in vivo not fully resolved\", \"Golgi function of CIN85 not mechanistically connected to RTK role\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined the membrane-targeting and late-endosomal steps and broadened receptor scope, showing CIN85 binds phosphatidic acid and dynamin-2 to route receptors through endosomal sorting, and controls nephrin and D2 dopamine receptor endocytosis in vivo.\",\n      \"evidence\": \"Lipid-binding and domain-deletion assays; Co-IP and dominant-negative dynamin-2 studies; CD2AP-knockout podocyte and CIN85-knockout mouse analyses with endocytosis and behavioral readouts\",\n      \"pmids\": [\"19417776\", \"20711168\", \"20457601\", \"20551902\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct PA-binding residues not yet pinpointed\", \"Tissue-specific receptor selectivity mechanism unclear\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established CIN85 as a constitutive organizer of the B-cell SLP65/BLNK complex required for BCR-driven Ca2+ and NF-κB responses via IKK-β, and as a regulator of immunoreceptor degradation across mast cells, neutrophils and B cells.\",\n      \"evidence\": \"MS interactome, live imaging, Ca2+ flux, and B cell-specific conditional knockout with IKK-β rescue; siRNA/overexpression Co-IP studies of Syk and FcγRIIa ubiquitination\",\n      \"pmids\": [\"21822214\", \"21708930\", \"17675467\", \"22262777\", \"21372129\", \"21725061\", \"21830225\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct link between SLP65 scaffolding and NF-κB output mechanistically incomplete\", \"How CIN85 switches between negative (Syk degradation) and positive (NF-κB) roles unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed the biophysical basis of B-cell priming — CIN85 oligomerizes SLP65 into pre-formed condensates via its trimeric coiled-coil and multiple SH3 domains — and linked CIN85 to PP2Ac suppression and nephrin endocytosis in vivo.\",\n      \"evidence\": \"Biochemical oligomerization and structure-function assays in B cells; phosphatase activity assay and platelet functional readouts for PP2Ac; CIN85-knockout mouse and zebrafish overexpression for nephrin filtration barrier\",\n      \"pmids\": [\"27353366\", \"27334924\", \"27531950\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative properties of condensates in resting cells unresolved\", \"Phase behavior in vivo not directly demonstrated\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed CIN85 acts as a negative regulator of TCR signaling via Sts-2 recruitment and inhibits PHD2 to stabilize HIF-α, expanding its roles into T-cell tolerance and hypoxia/tumor biology.\",\n      \"evidence\": \"Conditional knockout T cells with signaling and IL-2 readouts and Sts-2 Co-IP; binding, PHD2 hydroxylase activity assay, and CRISPR knock-in with tumor xenograft\",\n      \"pmids\": [\"30723173\", \"31142511\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CIN85 partitions between activating and inhibitory immunoreceptor roles unresolved\", \"Structural basis of PHD2 inhibition not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Established cytoskeletal and developmental roles, placing CIN85 at the intercellular bridge in cytokinesis, in myofiber formation, ciliary length control, and RIG-I innate immune signaling.\",\n      \"evidence\": \"Direct binding and live-cell imaging for anillin/SEPT9 in cytokinesis; Co-IP, domain binding and Dnm2 mouse for dynamin-2/calnexin in myogenesis; siRNA/EHBP1L1 rescue for cilia; Co-IP, KO mouse and recombinant virus for TRIM25/RIG-I\",\n      \"pmids\": [\"36044846\", \"40065183\", \"36754282\", \"39466846\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanistic connection between adaptor scaffolding and septin filament assembly incomplete\", \"Whether these roles share a common biochemical activity unclear\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Defined phosphorylation-gated intramolecular autoinhibition controlling CIN85 valency toward SLP65 and identified further trafficking modulators that displace CIN85 from receptors.\",\n      \"evidence\": \"NMR of multidomain constructs with phosphomimetic mutagenesis and B cell Ca2+/recruitment assays; Co-IP and epistasis for ASAP2-c-MET\",\n      \"pmids\": [\"38111344\", \"37061723\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase responsible for the regulatory serine phosphorylation not identified\", \"How autoinhibition is relieved in vivo unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Tested necessity of CIN85/Cbl for EGFR internalization and extended its trafficking roles to tubular recycling endosomes and bleb-associated phosphoinositide patterning.\",\n      \"evidence\": \"Cbl/Cbl-b double-knockout MEFs with inducible CIN85 siRNA and quantitative EGF internalization assays; siRNA and Co-IP for MICAL-L1/TRE recycling; phase-separation and biosensor assays for septin-PI3K bleb axis\",\n      \"pmids\": [\"25449262\", \"40740057\", \"40445717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reconciliation of dispensability for internalization with earlier dominant-negative data unaddressed\", \"Bleb phase-separation role rests on indirect evidence (Low confidence)\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CIN85 selects among its dozens of partners to switch between promoting versus inhibiting a given receptor, and how its condensate/oligomerization behavior is controlled across the many cell types in which it acts, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model for partner-selection logic\", \"Cell-type-specific regulatory inputs (kinases, modifications) largely uncharacterized\", \"In vivo significance of phase separation beyond B cells unestablished\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 5, 7, 17, 19]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [31, 8]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [25, 41]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [45, 37, 38]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [15, 25, 27, 41, 52]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 17]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [36]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [20, 47, 49]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [31, 32]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [57]},\n      {\"term_id\": \"GO:0005783\", \"supporting_discovery_ids\": [48]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0005215\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 15, 27, 41, 52]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [38, 45, 37]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [17, 18, 43, 44, 54]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [46]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [4, 13, 29]}\n    ],\n    \"complexes\": [\n      \"SLP65/CIN85 oligomeric condensate\",\n      \"endophilin-CIN85-Cbl complex\"\n    ],\n    \"partners\": [\n      \"CBL\",\n      \"CBLB\",\n      \"SLP65/BLNK\",\n      \"endophilin\",\n      \"DNM2\",\n      \"PHD2/EGLN1\",\n      \"TRIM25\",\n      \"SEPT9\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}