{"gene":"DBNL","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":1999,"finding":"SH3P7/HIP-55 is a substrate for Src and Syk family kinases (tyrosine phosphorylation at YXXP motifs), contains an SCAD region homologous to actin-binding proteins that mediates actin binding, and colocalizes with actin filaments of the cytoskeleton, implicating it as an adaptor linking antigen receptor signaling to the cytoskeleton.","method":"In vitro kinase assay, colocalization immunofluorescence, sequence analysis, SDS-PAGE migration analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct kinase substrate assay and colocalization; single lab but multiple methods","pmids":["9891087"],"is_preprint":false},{"year":1999,"finding":"HIP-55 binds HPK1 (hematopoietic progenitor kinase 1) both in vitro and in vivo via its SH3 domain interacting with HPK1 proline-rich domains. Co-expression of HIP-55 increases HPK1 kinase activity and JNK1 kinase activity. A dominant-negative HPK1 mutant blocked HIP-55-mediated JNK1 activation, placing HIP-55 upstream of HPK1 in the JNK signaling cascade.","method":"In vitro binding assay, co-immunoprecipitation, kinase activity assay, dominant-negative epistasis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro binding + co-IP + kinase assay + epistasis with dominant-negative mutant, replicated across multiple papers","pmids":["10567356"],"is_preprint":false},{"year":2000,"finding":"Mouse Abp1 (mAbp1/SH3P7/DBNL) binds actin filaments using two independent actin-binding domains with a 1:5 saturation stoichiometry. In cells, mAbp1 colocalizes with cortical F-actin and the Arp2/3 complex at lamellipodia; dominant-positive Rac1 expression mimics growth factor-induced mAbp1 recruitment, and this recruitment depends on de novo actin polymerization.","method":"In vitro F-actin binding assay (stoichiometry), live-cell fluorescence imaging, dominant-positive Rac1 expression, actin polymerization inhibition","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstituted binding assay with stoichiometry plus multiple cellular methods; independently consistent with other reports","pmids":["10637315"],"is_preprint":false},{"year":2001,"finding":"HIP-55 is a direct substrate of caspase-3 during apoptosis; it is cleaved at EHID(361), and the D361A mutant is resistant to caspase cleavage. Caspase cleavage dissociates the actin-binding domain from the SH3 domain of HIP-55.","method":"In vitro caspase cleavage assay, site-directed mutagenesis (D361A), Western blot during apoptosis","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro caspase assay with specific site mutagenesis confirming cleavage site; single lab but direct biochemical evidence","pmids":["11689006"],"is_preprint":false},{"year":2001,"finding":"SH3P7 localizes primarily to dendrites of neurons in rat brain and to peripheral protrusive structures of astrocytes, as determined by immunohistochemistry with isoform-specific antibodies, with immunoreactivity absent from presynaptic terminals.","method":"Immunohistochemistry, immunofluorescence double-labeling, RT-PCR for isoforms","journal":"The European journal of neuroscience","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by immunostaining; single lab, multiple brain regions examined","pmids":["11595038"],"is_preprint":false},{"year":2003,"finding":"HIP-55 is recruited to glycolipid-enriched microdomains (lipid rafts) upon TCR stimulation. ZAP-70 interacts with HIP-55 in a TCR-stimulation-dependent manner and phosphorylates HIP-55 at Tyr-334 and Tyr-344 in vitro and in vivo; the Y334F/Y344F mutant is not phosphorylated in stimulated T cells. RNAi-mediated depletion of HIP-55 in Jurkat cells decreased TCR-stimulated (but not UV-stimulated) JNK activation and HPK1 activation.","method":"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis (Y334F/Y344F), RNA interference, sucrose density gradient fractionation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis + RNAi epistasis + co-IP; multiple orthogonal methods in one study","pmids":["14557276"],"is_preprint":false},{"year":2004,"finding":"HIP-55 localizes to the T cell–APC immunological synapse in an antigen-dependent manner, requiring both its SH3 and actin-binding domains for recruitment. HIP-55 binds and activates HPK1 at the synapse, forming a complex that negatively regulates NFAT activation downstream of TCR. HIP-55 also promotes basal and ligand-dependent TCR down-modulation/endocytosis, requiring both ADF-H and SH3 domains.","method":"GFP fusion live imaging, RNA interference, overexpression, co-immunoprecipitation, NFAT-luciferase reporter assay, flow cytometry for TCR surface expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (live imaging, RNAi, Co-IP, reporter assay, flow cytometry) in one study; domain-deletion experiments define requirements","pmids":["14729663"],"is_preprint":false},{"year":2004,"finding":"The cytoplasmic tail of ASFV CD2v protein binds to the SH3 domain of SH3P7/HIP-55 via proline-rich PPPKPC repeats, as shown by yeast two-hybrid and direct binding studies. CD2v and SH3P7 co-localize in Golgi-derived membranes surrounding perinuclear virus factories in ASFV-infected cells.","method":"Yeast two-hybrid, direct binding assay, deletion mutagenesis, co-localization immunofluorescence in infected cells","journal":"The Journal of general virology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — yeast two-hybrid + direct binding + colocalization; single lab, viral interactor context","pmids":["14718626"],"is_preprint":false},{"year":2005,"finding":"HIP-55 knockout mice show defective T-cell proliferation, decreased cytokine production, reduced activation marker upregulation, and partially defective TCR signaling (LAT/PLCγ1 phosphorylation, HPK1/JNK activation) upon TCR stimulation. HIP-55 interacts with and is phosphorylated by ZAP-70, and is required for JNK and HPK1 activation in TCR signaling.","method":"Gene knockout mouse model, T-cell proliferation assay, cytokine ELISA, flow cytometry, Western blot signaling assays, co-immunoprecipitation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with defined cellular phenotypes, multiple signaling readouts, replicated findings from prior RNAi study","pmids":["16055701"],"is_preprint":false},{"year":2006,"finding":"SH3P7/mAbp1-deficient mice show reduced receptor-mediated endocytosis in embryonic fibroblasts and reduced synaptic vesicle endocytosis in hippocampal neurons; recycling of synaptic vesicles is severely delayed ~4-fold. These results place mAbp1 function downstream of vesicle fission in clathrin-mediated endocytosis (CME).","method":"Targeted gene disruption (KO mouse), transferrin uptake assay (CME), live imaging of synaptic vesicle recycling in hippocampal boutons, FM dye loading/unloading","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean KO mouse with multiple cell-type-specific functional assays and quantitative imaging; replicated finding","pmids":["16601697"],"is_preprint":false},{"year":2007,"finding":"The adapter protein 3BP2 directly interacts with the SH3 domain of HIP-55 via a proline-rich domain of 3BP2. 3BP2 co-localizes with HIP-55 in T cell lipid rafts and at the T cell/APC synapse.","method":"Direct binding assay, co-localization immunofluorescence, deletion mapping","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct binding assay plus colocalization; single lab","pmids":["17306257"],"is_preprint":false},{"year":2009,"finding":"Upon platelet activation by thrombin, HIP-55 becomes increasingly associated with both Syk kinase and integrin β3. HIP-55-deficient platelets show reduced fibrinogen binding upon thrombin stimulation, demonstrating a role for HIP-55 in integrin activation and platelet function.","method":"Proteomics/mass spectrometry, NeutrAvidin affinity chromatography, co-immunoprecipitation, fibrinogen binding assay on HIP-55-deficient platelets","journal":"Proteomics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — functional KO platelet assay + co-IP; single lab, combined proteomic and genetic approach","pmids":["19725075"],"is_preprint":false},{"year":2012,"finding":"mAbp1/DBNL localizes to podosomes in Src-transformed fibroblasts and is required for formation of podosome rosettes but not individual podosome dots (which require cortactin). Src phosphorylates mAbp1 at Tyr337 and Tyr347, and these phosphorylation events are required for podosome rosette formation and ECM degradation. Depletion of mAbp1 increased invasive cell migration. Interaction with WIP was not required for podosome rosette formation.","method":"siRNA knockdown, overexpression of phospho-mutants (Y337F/Y347F), immunofluorescence, gelatin degradation assay, Boyden chamber invasion assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function + phospho-mutant rescue + functional ECM degradation assay; multiple orthogonal methods","pmids":["22303001"],"is_preprint":false},{"year":2014,"finding":"HIP-55 forms a complex with 14-3-3 proteins via phospho-Ser269/Thr291 sites, identified by affinity chromatography. S269A/T291A mutant HIP-55 (HIP-55AA) abolishes 14-3-3 binding. Tumors expressing HIP-55AA show significantly reduced growth in xenograft models compared to wild-type HIP-55, supporting a pro-oncogenic role mediated by the HIP-55/14-3-3 interaction node that antagonizes HPK1 tumor suppressor function.","method":"Affinity chromatography (14-3-3 pull-down), site-directed mutagenesis (S269A/T291A), xenograft tumor model, colony formation and invasion assays","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — affinity chromatography + mutagenesis + in vivo xenograft; multiple orthogonal methods in one study","pmids":["24912570"],"is_preprint":false},{"year":2014,"finding":"HIP-55 negatively regulates β-adrenergic receptor-activated ERK1/2 signaling and cardiac fibroblast proliferation; both HIP-55 overexpression and knockdown studies confirmed this regulatory role.","method":"Proteomics, HIP-55 overexpression and knockdown in cardiac fibroblasts, ERK1/2 phosphorylation assay, proliferation assay","journal":"Molecular bioSystems","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — gain- and loss-of-function in primary cells with defined signaling readout; single lab","pmids":["24802081"],"is_preprint":false},{"year":2014,"finding":"14-3-3 interacts with HIP-55 through S269/T291 sites (confirmed by BiFC and co-IP); the 14-3-3/HIP-55 complex increases HIP-55 protein stability, as the S269A/T291A mutant (defective in 14-3-3 binding) shows reduced stability after puromycin block of new protein synthesis.","method":"Bimolecular fluorescence complementation (BiFC), co-immunoprecipitation, pulse-chase with puromycin, site-directed mutagenesis","journal":"Journal of Peking University. Health sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — BiFC + co-IP + stability assay with mutant; single lab, multiple methods","pmids":["26679646"],"is_preprint":false},{"year":2014,"finding":"HIP-55 negatively regulates myocardial contractility; knockdown increased and overexpression decreased contractile traction forces in single cardiomyocytes. HIP-55 co-localizes with F-actin in cardiomyocytes, suggesting the mechanism involves HIP-55–F-actin interaction.","method":"Adenoviral overexpression and knockdown, traction force microscopy, immunofluorescence co-localization","journal":"Journal of biomechanics","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — bidirectional manipulation (KD + OE) with quantitative traction force readout; single lab","pmids":["24880669"],"is_preprint":false},{"year":2015,"finding":"mAbp1/HIP-55 binds clathrin, actin, and dynamin simultaneously, acting as an adaptor that links dynamin to actin at clathrin-coated pits (CCPs). Live-cell imaging shows mAbp1 is recruited specifically at a late stage to long-lived CCPs. mAbp1 knockdown reduced dynamin recruitment at the plasma membrane and impaired CCP scission. Actin disruption eliminated both mAbp1 and dynamin recruitment, indicating mAbp1 acts downstream of F-actin assembly to recruit dynamin.","method":"Live-cell imaging, siRNA knockdown, co-immunoprecipitation/binding assays with clathrin/actin/dynamin, actin disruption experiments","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging + KD + binding assays for three partners + actin disruption epistasis; multiple orthogonal methods","pmids":["25690657"],"is_preprint":false},{"year":2016,"finding":"mAbp1/HIP-55 interacts with FHL2 through its N-terminal ADF homology (ADFH) domain, identified by yeast two-hybrid. Depletion of mAbp1 or ectopic expression of its ADFH domain increased Rho GTPase signaling and breast cancer cell invasion; ADFH-domain-induced invasion required FHL2 expression, placing FHL2 downstream of mAbp1 in the Rho/invasion axis.","method":"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, domain overexpression, Rho GTPase activity assay, Boyden chamber invasion assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — yeast two-hybrid + co-IP + epistasis with FHL2 KD; single lab, multiple methods","pmids":["27129278"],"is_preprint":false},{"year":2018,"finding":"Dbnl (DBNL) is required for neuronal migration, multipolar morphology, and polarity in the developing cerebral cortex. Dbnl knockdown decreased plasma membrane-localized N-cadherin in cortical neurons. Neuronal migration defects were rescued by N-cadherin/αN-catenin overexpression or by a phospho-mimetic Dbnl (Y337E/Y347E) but not by phospho-resistant Dbnl (Y337F/Y347F), placing Tyr337/347 phosphorylation upstream of N-cadherin regulation.","method":"In utero electroporation knockdown, time-lapse imaging, rescue with N-cadherin/αN-catenin and phospho-mutants, surface N-cadherin quantification","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KD + time-lapse imaging + phospho-mutant rescue + N-cadherin surface quantification; multiple orthogonal methods","pmids":["30504273"],"is_preprint":false},{"year":2021,"finding":"Dbnl interacts with N-cadherin in a β-catenin-dependent manner during anterograde Golgi trafficking and promotes pro-N-cadherin propeptide excision and maturation. Dbnl knockdown causes pro-N-cadherin accumulation and limits adherens junction formation, disrupting apico-basal polarity in the neural tube.","method":"Co-immunoprecipitation, Dbnl knockdown, Western blot for pro-N-cadherin vs mature N-cadherin, immunofluorescence for AJ markers","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP + KD with N-cadherin processing readout; single lab, two methods","pmids":["33939796"],"is_preprint":false},{"year":2021,"finding":"HIP-55 interacts with Smad7 and competes with Smad7/Axin complex formation, thereby inhibiting Axin-mediated Smad7 degradation. HIP-55 couples Smad7 to TβRI (but not TβRII) to drive TβRI degradation; loss of HIP-55 causes TGF-β signaling overactivation and abnormal ECM gene accumulation.","method":"Co-immunoprecipitation, overexpression and knockdown, Western blot for signaling pathway components and ECM genes","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP + gain/loss-of-function + receptor degradation assay; single lab","pmids":["34331017"],"is_preprint":false},{"year":2023,"finding":"HIP-55 is a new AKT substrate phosphorylated at S269 and T291. AKT-phosphorylated HIP-55 directs AKT signaling to negatively regulate the MAP4K1 pathway, protecting against MI-induced ferroptosis. S269A/T291A-mutated HIP-55 fails to inhibit the MAP4K1/GPX4 ferroptosis pathway and fails to protect against MI injury in vivo.","method":"In vitro kinase assay (AKT phosphorylation), site-directed mutagenesis (S269A/T291A), cardiac-specific overexpression mouse model, co-immunoprecipitation, GPX4 assay, MI model","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay + phospho-mutant + in vivo cardiac-specific OE mouse; multiple orthogonal methods","pmids":["36639542"],"is_preprint":false},{"year":2023,"finding":"HIP-55 interacts with clathrin and F-actin to promote clathrin-mediated endocytosis of nanoparticles (quantum dots). A HIP-55 ΔADF mutant defective in F-actin binding fails to promote nanoparticle endocytosis. HIP-55 knockout inhibits QD endocytosis in vivo.","method":"Co-immunoprecipitation (HIP-55–clathrin interaction), ΔADF domain mutant overexpression, pharmacological clathrin inhibition, HIP-55 KO mouse, live imaging","journal":"Nano letters","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — co-IP + domain mutant + KO mouse; single lab, multiple approaches","pmids":["38084909"],"is_preprint":false},{"year":2024,"finding":"AKT phosphorylates HIP-55 at S269 and T291, which is required for normal liquid-liquid phase separation of HIP-55. Failure of AKT-mediated phosphorylation leads to abnormal HIP-55 aggregation. HIP-55 phase separation inhibits β-adrenergic receptor-mediated P38/MAPK signaling. Phospho-deficient HIP-55 (S269A/T291A) undergoes massive aberrant phase separation, loses protective activity against heart failure, and phospho-resistant knock-in mice show aggravated HF.","method":"FRAP assay, DIC microscopy, pull-down, immunofluorescence, phospho-mutant knock-in mice, cardiac-specific KO and OE mouse models, P38/MAPK signaling assays","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — FRAP + DIC (phase separation), mutagenesis, multiple in vivo mouse models with functional cardiac readouts; multiple orthogonal methods","pmids":["38328928"],"is_preprint":false},{"year":2024,"finding":"HIP-55 facilitates AP-1 complex activation in macrophages by promoting ERK1/2 and JNK phosphorylation in response to Ang II; blocking AP-1 attenuates HIP-55-mediated macrophage M1 polarization. HIP-55 expression is upregulated in M1 macrophages, and its genetic deletion inhibits Ang II-induced M1 polarization.","method":"HIP-55 overexpression and genetic deletion in macrophages, AP-1 reporter/inhibitor assay, ERK1/2 and JNK phosphorylation Western blot, M1 polarization marker assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — bidirectional manipulation + AP-1 inhibitor epistasis + signaling readouts; single lab","pmids":["38417633"],"is_preprint":false},{"year":2025,"finding":"RSK1 phosphorylates HIP-55 at S269/T291 to promote 14-3-3/HIP-55 complex formation, which suppresses the ASK1 apoptotic pathway. S269A/T291A HIP-55 (RSK1-phosphorylation-deficient) fails to form the 14-3-3/HIP-55 complex and fails to protect cardiomyocytes against MI-induced apoptosis in vivo and in vitro.","method":"In vitro kinase assay (RSK1), site-directed mutagenesis (S269A/T291A), co-immunoprecipitation, in vivo MI model with phospho-resistant knock-in, apoptosis assays","journal":"Journal of molecular and cellular cardiology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay + mutagenesis + co-IP + in vivo knock-in mouse; multiple orthogonal methods","pmids":["40675322"],"is_preprint":false}],"current_model":"DBNL/HIP-55/mAbp1 is a multidomain adaptor protein with an N-terminal ADF-H (actin-binding) domain and a C-terminal SH3 domain that binds F-actin (1:5 stoichiometry) and scaffolds diverse signaling complexes: it is phosphorylated by Src/Syk/ZAP-70 at Tyr334/344 (and Tyr337/347) to regulate actin dynamics, podosome rosette formation, and neuronal migration via N-cadherin; it activates HPK1 and the JNK cascade (and negatively regulates NFAT) downstream of antigen receptors; it links dynamin and clathrin to F-actin to drive CCP scission in clathrin-mediated endocytosis and synaptic vesicle recycling; it undergoes AKT- and RSK1-mediated phosphorylation at S269/T291, controlling its liquid-liquid phase separation, 14-3-3 complex formation (which stabilizes HIP-55 and suppresses ASK1-mediated apoptosis), and MAP4K1/GPX4 ferroptosis pathway regulation, thereby protecting against myocardial infarction and heart failure; it is cleaved by caspase-3 at D361 during apoptosis; and it promotes AP-1-mediated macrophage M1 polarization and modulates TGF-β signaling by coupling Smad7 to TβRI degradation."},"narrative":{"mechanistic_narrative":"DBNL (HIP-55/mAbp1/SH3P7) is a multidomain actin-associated adaptor protein that couples cytoskeletal dynamics to receptor signaling, membrane trafficking, and cell survival across immune, neuronal, and cardiac contexts [PMID:9891087, PMID:10637315]. Through an N-terminal actin-binding (ADF-H/SCAD) region and a C-terminal SH3 domain, it binds F-actin with a defined 1:5 stoichiometry and is recruited to cortical actin and the Arp2/3 complex at lamellipodia downstream of Rac1-driven actin polymerization [PMID:10637315]. The SH3 domain scaffolds proline-rich partners: DBNL binds and activates HPK1 to drive the JNK cascade downstream of antigen receptors, where it is recruited to lipid rafts and the immunological synapse, phosphorylated by ZAP-70 at Tyr334/344, and is required for TCR-stimulated JNK/HPK1 activation while negatively regulating NFAT [PMID:10567356, PMID:14557276, PMID:14729663, PMID:16055701]. In membrane trafficking, DBNL acts downstream of F-actin assembly to link clathrin, actin, and dynamin at clathrin-coated pits, driving late-stage dynamin recruitment and coated-pit scission in receptor-mediated endocytosis and synaptic vesicle recycling [PMID:16601697, PMID:25690657, PMID:38084909]. Tyrosine phosphorylation by Src at Tyr337/347 controls its role in podosome rosette formation and ECM degradation, and the same residues, in phospho-mimetic form, govern N-cadherin surface delivery, propeptide maturation, and neuronal migration in the developing cortex [PMID:22303001, PMID:30504273, PMID:33939796]. A distinct AKT/RSK1-dependent phosphorylation node at Ser269/Thr291 directs 14-3-3 complex formation, protein stabilization, and liquid-liquid phase separation, suppressing ASK1-mediated apoptosis and MAP4K1/GPX4-driven ferroptosis to protect the heart against myocardial infarction and heart failure [PMID:24912570, PMID:26679646, PMID:36639542, PMID:38328928, PMID:40675322]. DBNL is also cleaved by caspase-3 at Asp361 during apoptosis, severing the actin-binding domain from the SH3 domain [PMID:11689006].","teleology":[{"year":1999,"claim":"Established DBNL as an actin-associated adaptor that physically couples antigen-receptor kinase signaling to the cytoskeleton, defining its core molecular identity.","evidence":"In vitro kinase assays with Src/Syk family kinases, actin colocalization, and sequence analysis of the actin-binding SCAD region","pmids":["9891087"],"confidence":"Medium","gaps":["Functional consequence of tyrosine phosphorylation not defined","Specific signaling pathway downstream not yet identified"]},{"year":1999,"claim":"Placed DBNL upstream of HPK1 in the JNK cascade, showing its SH3 domain activates a defined kinase module rather than acting as a passive scaffold.","evidence":"In vitro binding, co-IP, kinase activity assays, and dominant-negative HPK1 epistasis","pmids":["10567356"],"confidence":"High","gaps":["Receptor input driving the cascade not yet established","Mechanism by which binding stimulates HPK1 activity unresolved"]},{"year":2000,"claim":"Quantified DBNL's F-actin binding (1:5 stoichiometry) and linked its recruitment to Rac1-driven actin polymerization at lamellipodia, defining how it senses cytoskeletal remodeling.","evidence":"In vitro F-actin binding stoichiometry, live-cell imaging, dominant-positive Rac1, and actin polymerization inhibition","pmids":["10637315"],"confidence":"High","gaps":["Relationship between two actin-binding domains and signaling unclear","Direct Arp2/3 interaction not biochemically reconstituted"]},{"year":2001,"claim":"Identified DBNL as a caspase-3 substrate cleaved at Asp361, revealing a regulated mechanism that uncouples its actin-binding and adaptor modules during apoptosis.","evidence":"In vitro caspase cleavage, D361A site-directed mutagenesis, and Western blot during apoptosis","pmids":["11689006"],"confidence":"High","gaps":["Functional consequence of cleavage fragments not characterized","Whether cleavage promotes or limits apoptosis unknown"]},{"year":2003,"claim":"Defined the receptor-proximal mechanism in T cells: ZAP-70 phosphorylates DBNL at Tyr334/344 upon TCR engagement, making it required for TCR-specific JNK/HPK1 activation.","evidence":"Co-IP, in vitro kinase assay, Y334F/Y344F mutagenesis, RNAi, and sucrose gradient raft fractionation","pmids":["14557276"],"confidence":"High","gaps":["Structural basis of raft recruitment unresolved","Distinct roles of Tyr334 vs Tyr344 not separated"]},{"year":2004,"claim":"Localized DBNL function to the immunological synapse and showed dual-domain requirement for synapse recruitment, HPK1 activation, NFAT suppression, and TCR endocytosis.","evidence":"GFP live imaging, RNAi, co-IP, NFAT-luciferase reporter, and flow cytometry for TCR surface levels","pmids":["14729663"],"confidence":"High","gaps":["Mechanism linking TCR endocytosis to NFAT regulation not dissected"]},{"year":2004,"claim":"Showed the DBNL SH3 domain is targeted by a viral proline-rich ligand (ASFV CD2v), implicating it in pathogen exploitation of the host actin/trafficking machinery.","evidence":"Yeast two-hybrid, direct binding, deletion mapping, and colocalization in infected cells","pmids":["14718626"],"confidence":"Medium","gaps":["Functional consequence for viral replication not established","Single-lab interactor not independently confirmed"]},{"year":2005,"claim":"Confirmed the in vivo requirement for DBNL in T-cell activation using knockout mice, validating RNAi-based placement in TCR-driven HPK1/JNK signaling.","evidence":"KO mouse, proliferation assays, cytokine ELISA, flow cytometry, signaling Western blots, and co-IP","pmids":["16055701"],"confidence":"High","gaps":["Whether actin-binding or scaffolding drives the phenotype not separated"]},{"year":2006,"claim":"Established DBNL as required for clathrin-mediated and synaptic vesicle endocytosis, placing its function downstream of vesicle fission.","evidence":"KO mouse, transferrin uptake, live imaging of synaptic vesicle recycling, and FM dye assays","pmids":["16601697"],"confidence":"High","gaps":["Molecular partners at the fission step not yet identified in this study"]},{"year":2015,"claim":"Defined the molecular mechanism of DBNL in endocytosis: it simultaneously binds clathrin, actin, and dynamin, recruiting dynamin to coated pits downstream of F-actin to drive scission.","evidence":"Live-cell imaging, siRNA, binding assays for three partners, and actin disruption epistasis","pmids":["25690657"],"confidence":"High","gaps":["Stoichiometry of the clathrin/actin/dynamin assembly unresolved"]},{"year":2012,"claim":"Linked Src phosphorylation at Tyr337/347 to DBNL-driven podosome rosette formation and ECM degradation, distinguishing it from cortactin-dependent podosome dots.","evidence":"siRNA, Y337F/Y347F phospho-mutant rescue, gelatin degradation, and invasion assays","pmids":["22303001"],"confidence":"High","gaps":["Effectors downstream of phospho-DBNL at rosettes not identified"]},{"year":2018,"claim":"Showed Tyr337/347 phosphorylation governs DBNL control of surface N-cadherin and neuronal migration in vivo, extending its actin-adaptor role to cortical development.","evidence":"In utero electroporation knockdown, time-lapse imaging, and phospho-mutant/N-cadherin rescue","pmids":["30504273"],"confidence":"High","gaps":["How phospho-DBNL controls N-cadherin trafficking mechanistically not fully resolved"]},{"year":2021,"claim":"Defined a trafficking mechanism whereby DBNL binds N-cadherin β-catenin-dependently in the Golgi to promote pro-N-cadherin maturation and adherens junction formation.","evidence":"Co-IP, knockdown, pro- versus mature N-cadherin Western blot, and AJ marker immunofluorescence","pmids":["33939796"],"confidence":"Medium","gaps":["Identity of the protease excising the propeptide not established"]},{"year":2014,"claim":"Discovered the Ser269/Thr291 14-3-3 binding node and linked it to DBNL protein stability and a pro-oncogenic role antagonizing HPK1.","evidence":"Affinity chromatography, S269A/T291A mutagenesis, BiFC, puromycin pulse-chase, and xenograft tumor models","pmids":["24912570","26679646"],"confidence":"High","gaps":["Kinase generating the phospho-sites not identified at this stage"]},{"year":2014,"claim":"Identified DBNL as a negative regulator of β-adrenergic ERK1/2 signaling and contractility in cardiac cells, opening its cardiovascular role.","evidence":"Proteomics, gain/loss-of-function in cardiac fibroblasts and cardiomyocytes, ERK1/2 phosphorylation, proliferation, and traction force microscopy","pmids":["24802081","24880669"],"confidence":"Medium","gaps":["Molecular link between DBNL and the β-AR/ERK pathway not defined","Single-lab findings"]},{"year":2016,"claim":"Linked the DBNL ADF-H domain to FHL2-dependent suppression of Rho-driven invasion, defining a domain-specific tumor-suppressive axis distinct from its scaffolding functions.","evidence":"Yeast two-hybrid, co-IP, siRNA, ADFH domain overexpression, Rho activity, and invasion assays","pmids":["27129278"],"confidence":"Medium","gaps":["Reconciliation of pro-oncogenic (14-3-3) versus anti-invasive (FHL2) roles unresolved"]},{"year":2021,"claim":"Showed DBNL couples Smad7 to TβRI degradation by competing with the Smad7/Axin complex, identifying a mechanism restraining TGF-β signaling and ECM accumulation.","evidence":"Co-IP, gain/loss-of-function, and Western blot for pathway components and ECM genes","pmids":["34331017"],"confidence":"Medium","gaps":["Single-lab finding; receptor-degradation route (lysosomal vs proteasomal) not defined"]},{"year":2023,"claim":"Identified AKT as a kinase for the S269/T291 node, channeling AKT signaling against MAP4K1/GPX4 ferroptosis to protect against myocardial infarction.","evidence":"In vitro AKT kinase assay, S269A/T291A mutagenesis, cardiac-specific overexpression mouse, co-IP, GPX4 assay, and MI model","pmids":["36639542"],"confidence":"High","gaps":["Direct biochemical link between phospho-DBNL and MAP4K1 inhibition not detailed"]},{"year":2024,"claim":"Established that AKT phosphorylation of S269/T291 governs DBNL liquid-liquid phase separation, with aberrant aggregation suppressing protective β-AR/P38 signaling in failing hearts.","evidence":"FRAP, DIC microscopy, pull-down, phospho-mutant knock-in mice, cardiac KO/OE models, and P38/MAPK assays","pmids":["38328928"],"confidence":"High","gaps":["Composition of phase-separated condensates not defined","Relationship between condensates and 14-3-3 binding unresolved"]},{"year":2024,"claim":"Linked DBNL to macrophage M1 polarization via AP-1 activation through ERK1/2 and JNK, extending its adaptor role to inflammatory signaling.","evidence":"Overexpression/genetic deletion in macrophages, AP-1 reporter/inhibitor epistasis, MAPK Western blots, and M1 marker assays","pmids":["38417633"],"confidence":"Medium","gaps":["Direct molecular partners upstream of MAPK in macrophages not identified"]},{"year":2025,"claim":"Identified RSK1 as a second kinase phosphorylating S269/T291 to drive 14-3-3 complex formation and ASK1-apoptosis suppression, cementing the cardioprotective phospho-node.","evidence":"In vitro RSK1 kinase assay, S269A/T291A mutagenesis, co-IP, phospho-resistant knock-in MI model, and apoptosis assays","pmids":["40675322"],"confidence":"High","gaps":["Coordination between AKT and RSK1 inputs at the same sites not resolved"]},{"year":null,"claim":"How DBNL's competing actin-cytoskeletal, endocytic, and S269/T291 phase-separation/14-3-3 functions are coordinated within a single cell, and how phospho-state integrates these roles, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified structural model integrating ADF-H, SH3, and disordered phospho-region","Tissue-specific partitioning of functions not mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,2,16,17]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,5,17,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,6,21,22]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,2,16]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[5,17,19]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[13,24]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[7,20]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[9,17,23]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,6,8,25]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,14,21,22]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[3,22,26]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[19,20]}],"complexes":["HIP-55/14-3-3 complex","HIP-55/HPK1 complex","clathrin–actin–dynamin coat assembly"],"partners":["HPK1","ZAP-70","14-3-3","DYNAMIN","CLATHRIN","N-CADHERIN","FHL2","SMAD7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UJU6","full_name":"Drebrin-like protein","aliases":["Cervical SH3P7","Cervical mucin-associated protein","Drebrin-F","HPK1-interacting protein of 55 kDa","HIP-55","SH3 domain-containing protein 7"],"length_aa":430,"mass_kda":48.2,"function":"Adapter protein that binds F-actin and DNM1, and thereby plays a role in receptor-mediated endocytosis. Plays a role in the reorganization of the actin cytoskeleton, formation of cell projections, such as neurites, in neuron morphogenesis and synapse formation via its interaction with WASL and COBL. Does not bind G-actin and promote actin polymerization by itself. Required for the formation of organized podosome rosettes (By similarity). May act as a common effector of antigen receptor-signaling pathways in leukocytes. Acts as a key component of the immunological synapse that regulates T-cell activation by bridging TCRs and the actin cytoskeleton to gene activation and endocytic processes","subcellular_location":"Cytoplasm, cytoskeleton; Cell projection, lamellipodium; Cell projection, ruffle; Cytoplasm, cell cortex; Cytoplasm, cytosol; Synapse; Perikaryon; Cell projection, neuron projection; Cell membrane; Cytoplasmic vesicle, clathrin-coated vesicle membrane; Golgi apparatus membrane; Cell projection, podosome; Early endosome; Cell projection, dendrite; Postsynaptic density","url":"https://www.uniprot.org/uniprotkb/Q9UJU6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/DBNL","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000136279","cell_line_id":"CID000522","localizations":[{"compartment":"vesicles","grade":3},{"compartment":"cell_contact","grade":2},{"compartment":"membrane","grade":2}],"interactors":[{"gene":"CALD1","stoichiometry":0.2},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"ARPC5","stoichiometry":0.2},{"gene":"ARPC2","stoichiometry":0.2},{"gene":"ZNF536","stoichiometry":0.2},{"gene":"CAPZA2","stoichiometry":0.2},{"gene":"ARPC5L","stoichiometry":0.2},{"gene":"ARPC1A","stoichiometry":0.2},{"gene":"HIRA","stoichiometry":0.2},{"gene":"UBN2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000522","total_profiled":1310},"omim":[{"mim_id":"610106","title":"DREBRIN-LIKE; DBNL","url":"https://www.omim.org/entry/610106"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Vesicles","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/DBNL"},"hgnc":{"alias_symbol":["SH3P7","HIP-55","ABP1"],"prev_symbol":[]},"alphafold":{"accession":"Q9UJU6","domains":[{"cath_id":"3.40.20.10","chopping":"9-137","consensus_level":"high","plddt":95.9932,"start":9,"end":137},{"cath_id":"2.30.30.40","chopping":"376-430","consensus_level":"high","plddt":93.8935,"start":376,"end":430}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJU6","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJU6-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJU6-F1-predicted_aligned_error_v6.png","plddt_mean":74.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=DBNL","jax_strain_url":"https://www.jax.org/strain/search?query=DBNL"},"sequence":{"accession":"Q9UJU6","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UJU6.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UJU6/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJU6"}},"corpus_meta":[{"pmid":"10637315","id":"PMC_10637315","title":"Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation.","date":"2000","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/10637315","citation_count":130,"is_preprint":false},{"pmid":"9891087","id":"PMC_9891087","title":"SH3P7 is a cytoskeleton adapter protein and is coupled to signal transduction from lymphocyte antigen receptors.","date":"1999","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9891087","citation_count":77,"is_preprint":false},{"pmid":"14729663","id":"PMC_14729663","title":"Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/14729663","citation_count":74,"is_preprint":false},{"pmid":"10567356","id":"PMC_10567356","title":"A novel src homology 3 domain-containing adaptor protein, HIP-55, that interacts with hematopoietic progenitor kinase 1.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10567356","citation_count":56,"is_preprint":false},{"pmid":"16055701","id":"PMC_16055701","title":"HIP-55 is important for T-cell proliferation, cytokine production, and immune responses.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16055701","citation_count":49,"is_preprint":false},{"pmid":"14718626","id":"PMC_14718626","title":"The CD2v protein of African swine fever virus interacts with the actin-binding adaptor protein SH3P7.","date":"2004","source":"The Journal of general virology","url":"https://pubmed.ncbi.nlm.nih.gov/14718626","citation_count":48,"is_preprint":false},{"pmid":"14557276","id":"PMC_14557276","title":"The SH3 domain-containing adaptor HIP-55 mediates c-Jun N-terminal kinase activation in T cell receptor 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Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/30504273","citation_count":22,"is_preprint":false},{"pmid":"38328928","id":"PMC_38328928","title":"Phosphorylation-Regulated Dynamic Phase Separation of HIP-55 Protects Against Heart Failure.","date":"2024","source":"Circulation","url":"https://pubmed.ncbi.nlm.nih.gov/38328928","citation_count":19,"is_preprint":false},{"pmid":"38084909","id":"PMC_38084909","title":"Nanoparticles Internalization through HIP-55-Dependent Clathrin Endocytosis Pathway.","date":"2023","source":"Nano letters","url":"https://pubmed.ncbi.nlm.nih.gov/38084909","citation_count":18,"is_preprint":false},{"pmid":"24802081","id":"PMC_24802081","title":"HIP-55/DBNL-dependent regulation of adrenergic receptor mediates the ERK1/2 proliferative pathway.","date":"2014","source":"Molecular bioSystems","url":"https://pubmed.ncbi.nlm.nih.gov/24802081","citation_count":17,"is_preprint":false},{"pmid":"17306257","id":"PMC_17306257","title":"Abl-SH3 binding protein 2, 3BP2, interacts with CIN85 and HIP-55.","date":"2007","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/17306257","citation_count":17,"is_preprint":false},{"pmid":"24912570","id":"PMC_24912570","title":"Pro-oncogenic function of HIP-55/Drebrin-like (DBNL) through Ser269/Thr291-phospho-sensor motifs.","date":"2014","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/24912570","citation_count":15,"is_preprint":false},{"pmid":"11595038","id":"PMC_11595038","title":"Molecular cloning and dendritic localization of rat SH3P7.","date":"2001","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/11595038","citation_count":12,"is_preprint":false},{"pmid":"27129278","id":"PMC_27129278","title":"Mammalian Actin-binding Protein-1/Hip-55 Interacts with FHL2 and Negatively Regulates Cell Invasion.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/27129278","citation_count":12,"is_preprint":false},{"pmid":"33939796","id":"PMC_33939796","title":"Dbnl and β-catenin promote pro-N-cadherin processing to maintain apico-basal polarity.","date":"2021","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/33939796","citation_count":9,"is_preprint":false},{"pmid":"24668193","id":"PMC_24668193","title":"Regulation of cell survival by the HIP-55 signaling network.","date":"2014","source":"Molecular bioSystems","url":"https://pubmed.ncbi.nlm.nih.gov/24668193","citation_count":8,"is_preprint":false},{"pmid":"25690657","id":"PMC_25690657","title":"Mammalian actin-binding protein 1/HIP-55 is essential for the scission of clathrin-coated pits by regulating dynamin-actin interaction.","date":"2015","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/25690657","citation_count":7,"is_preprint":false},{"pmid":"38417633","id":"PMC_38417633","title":"Adaptor protein HIP-55 promotes macrophage M1 polarization through promoting AP-1 complex activation.","date":"2024","source":"Cellular signalling","url":"https://pubmed.ncbi.nlm.nih.gov/38417633","citation_count":4,"is_preprint":false},{"pmid":"34331017","id":"PMC_34331017","title":"The multifunctional adaptor protein HIP-55 couples Smad7 to accelerate TGF-β type I receptor degradation.","date":"2021","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/34331017","citation_count":2,"is_preprint":false},{"pmid":"32134471","id":"PMC_32134471","title":"Multi-omic analysis reveals HIP-55-dependent regulation of cytokines release.","date":"2020","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/32134471","citation_count":2,"is_preprint":false},{"pmid":"24651461","id":"PMC_24651461","title":"Phosphosite mapping of HIP-55 protein in mammalian cells.","date":"2014","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/24651461","citation_count":2,"is_preprint":false},{"pmid":"31377908","id":"PMC_31377908","title":"Identification of a new type of haematopoietic progenitor kinase-interacting protein (HIP-55) in Aedes aegypti mosquito haemocytes and its involvement in immunity-like functions in mosquito: a molecular study.","date":"2019","source":"Parasitology research","url":"https://pubmed.ncbi.nlm.nih.gov/31377908","citation_count":2,"is_preprint":false},{"pmid":"24880669","id":"PMC_24880669","title":"HIP-55 negatively regulates myocardial contractility at the single-cell level.","date":"2014","source":"Journal of biomechanics","url":"https://pubmed.ncbi.nlm.nih.gov/24880669","citation_count":2,"is_preprint":false},{"pmid":"40675322","id":"PMC_40675322","title":"14-3-3/HIP-55 complex attenuates cardiomyocyte apoptosis.","date":"2025","source":"Journal of molecular and cellular cardiology","url":"https://pubmed.ncbi.nlm.nih.gov/40675322","citation_count":1,"is_preprint":false},{"pmid":"26679646","id":"PMC_26679646","title":"[14-3-3/HIP-55 complex increases the stability of HIP-55].","date":"2015","source":"Beijing da xue xue bao. Yi xue ban = Journal of Peking University. Health sciences","url":"https://pubmed.ncbi.nlm.nih.gov/26679646","citation_count":0,"is_preprint":false},{"pmid":"41553874","id":"PMC_41553874","title":"Molecular Mechanisms of DBNL in Heart Failure: From Macrophage Immunometabolism to Therapeutic Implications.","date":"2026","source":"Journal of visualized experiments : JoVE","url":"https://pubmed.ncbi.nlm.nih.gov/41553874","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17362,"output_tokens":7299,"usd":0.080785,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16346,"output_tokens":6048,"usd":0.116465,"stage2_stop_reason":"end_turn"},"total_usd":0.19725,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"SH3P7/HIP-55 is a substrate for Src and Syk family kinases (tyrosine phosphorylation at YXXP motifs), contains an SCAD region homologous to actin-binding proteins that mediates actin binding, and colocalizes with actin filaments of the cytoskeleton, implicating it as an adaptor linking antigen receptor signaling to the cytoskeleton.\",\n      \"method\": \"In vitro kinase assay, colocalization immunofluorescence, sequence analysis, SDS-PAGE migration analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct kinase substrate assay and colocalization; single lab but multiple methods\",\n      \"pmids\": [\"9891087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"HIP-55 binds HPK1 (hematopoietic progenitor kinase 1) both in vitro and in vivo via its SH3 domain interacting with HPK1 proline-rich domains. Co-expression of HIP-55 increases HPK1 kinase activity and JNK1 kinase activity. A dominant-negative HPK1 mutant blocked HIP-55-mediated JNK1 activation, placing HIP-55 upstream of HPK1 in the JNK signaling cascade.\",\n      \"method\": \"In vitro binding assay, co-immunoprecipitation, kinase activity assay, dominant-negative epistasis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro binding + co-IP + kinase assay + epistasis with dominant-negative mutant, replicated across multiple papers\",\n      \"pmids\": [\"10567356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Mouse Abp1 (mAbp1/SH3P7/DBNL) binds actin filaments using two independent actin-binding domains with a 1:5 saturation stoichiometry. In cells, mAbp1 colocalizes with cortical F-actin and the Arp2/3 complex at lamellipodia; dominant-positive Rac1 expression mimics growth factor-induced mAbp1 recruitment, and this recruitment depends on de novo actin polymerization.\",\n      \"method\": \"In vitro F-actin binding assay (stoichiometry), live-cell fluorescence imaging, dominant-positive Rac1 expression, actin polymerization inhibition\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstituted binding assay with stoichiometry plus multiple cellular methods; independently consistent with other reports\",\n      \"pmids\": [\"10637315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HIP-55 is a direct substrate of caspase-3 during apoptosis; it is cleaved at EHID(361), and the D361A mutant is resistant to caspase cleavage. Caspase cleavage dissociates the actin-binding domain from the SH3 domain of HIP-55.\",\n      \"method\": \"In vitro caspase cleavage assay, site-directed mutagenesis (D361A), Western blot during apoptosis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro caspase assay with specific site mutagenesis confirming cleavage site; single lab but direct biochemical evidence\",\n      \"pmids\": [\"11689006\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SH3P7 localizes primarily to dendrites of neurons in rat brain and to peripheral protrusive structures of astrocytes, as determined by immunohistochemistry with isoform-specific antibodies, with immunoreactivity absent from presynaptic terminals.\",\n      \"method\": \"Immunohistochemistry, immunofluorescence double-labeling, RT-PCR for isoforms\",\n      \"journal\": \"The European journal of neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by immunostaining; single lab, multiple brain regions examined\",\n      \"pmids\": [\"11595038\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"HIP-55 is recruited to glycolipid-enriched microdomains (lipid rafts) upon TCR stimulation. ZAP-70 interacts with HIP-55 in a TCR-stimulation-dependent manner and phosphorylates HIP-55 at Tyr-334 and Tyr-344 in vitro and in vivo; the Y334F/Y344F mutant is not phosphorylated in stimulated T cells. RNAi-mediated depletion of HIP-55 in Jurkat cells decreased TCR-stimulated (but not UV-stimulated) JNK activation and HPK1 activation.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, site-directed mutagenesis (Y334F/Y344F), RNA interference, sucrose density gradient fractionation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay with mutagenesis + RNAi epistasis + co-IP; multiple orthogonal methods in one study\",\n      \"pmids\": [\"14557276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"HIP-55 localizes to the T cell–APC immunological synapse in an antigen-dependent manner, requiring both its SH3 and actin-binding domains for recruitment. HIP-55 binds and activates HPK1 at the synapse, forming a complex that negatively regulates NFAT activation downstream of TCR. HIP-55 also promotes basal and ligand-dependent TCR down-modulation/endocytosis, requiring both ADF-H and SH3 domains.\",\n      \"method\": \"GFP fusion live imaging, RNA interference, overexpression, co-immunoprecipitation, NFAT-luciferase reporter assay, flow cytometry for TCR surface expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (live imaging, RNAi, Co-IP, reporter assay, flow cytometry) in one study; domain-deletion experiments define requirements\",\n      \"pmids\": [\"14729663\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The cytoplasmic tail of ASFV CD2v protein binds to the SH3 domain of SH3P7/HIP-55 via proline-rich PPPKPC repeats, as shown by yeast two-hybrid and direct binding studies. CD2v and SH3P7 co-localize in Golgi-derived membranes surrounding perinuclear virus factories in ASFV-infected cells.\",\n      \"method\": \"Yeast two-hybrid, direct binding assay, deletion mutagenesis, co-localization immunofluorescence in infected cells\",\n      \"journal\": \"The Journal of general virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — yeast two-hybrid + direct binding + colocalization; single lab, viral interactor context\",\n      \"pmids\": [\"14718626\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"HIP-55 knockout mice show defective T-cell proliferation, decreased cytokine production, reduced activation marker upregulation, and partially defective TCR signaling (LAT/PLCγ1 phosphorylation, HPK1/JNK activation) upon TCR stimulation. HIP-55 interacts with and is phosphorylated by ZAP-70, and is required for JNK and HPK1 activation in TCR signaling.\",\n      \"method\": \"Gene knockout mouse model, T-cell proliferation assay, cytokine ELISA, flow cytometry, Western blot signaling assays, co-immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with defined cellular phenotypes, multiple signaling readouts, replicated findings from prior RNAi study\",\n      \"pmids\": [\"16055701\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"SH3P7/mAbp1-deficient mice show reduced receptor-mediated endocytosis in embryonic fibroblasts and reduced synaptic vesicle endocytosis in hippocampal neurons; recycling of synaptic vesicles is severely delayed ~4-fold. These results place mAbp1 function downstream of vesicle fission in clathrin-mediated endocytosis (CME).\",\n      \"method\": \"Targeted gene disruption (KO mouse), transferrin uptake assay (CME), live imaging of synaptic vesicle recycling in hippocampal boutons, FM dye loading/unloading\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean KO mouse with multiple cell-type-specific functional assays and quantitative imaging; replicated finding\",\n      \"pmids\": [\"16601697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The adapter protein 3BP2 directly interacts with the SH3 domain of HIP-55 via a proline-rich domain of 3BP2. 3BP2 co-localizes with HIP-55 in T cell lipid rafts and at the T cell/APC synapse.\",\n      \"method\": \"Direct binding assay, co-localization immunofluorescence, deletion mapping\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct binding assay plus colocalization; single lab\",\n      \"pmids\": [\"17306257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Upon platelet activation by thrombin, HIP-55 becomes increasingly associated with both Syk kinase and integrin β3. HIP-55-deficient platelets show reduced fibrinogen binding upon thrombin stimulation, demonstrating a role for HIP-55 in integrin activation and platelet function.\",\n      \"method\": \"Proteomics/mass spectrometry, NeutrAvidin affinity chromatography, co-immunoprecipitation, fibrinogen binding assay on HIP-55-deficient platelets\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — functional KO platelet assay + co-IP; single lab, combined proteomic and genetic approach\",\n      \"pmids\": [\"19725075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"mAbp1/DBNL localizes to podosomes in Src-transformed fibroblasts and is required for formation of podosome rosettes but not individual podosome dots (which require cortactin). Src phosphorylates mAbp1 at Tyr337 and Tyr347, and these phosphorylation events are required for podosome rosette formation and ECM degradation. Depletion of mAbp1 increased invasive cell migration. Interaction with WIP was not required for podosome rosette formation.\",\n      \"method\": \"siRNA knockdown, overexpression of phospho-mutants (Y337F/Y347F), immunofluorescence, gelatin degradation assay, Boyden chamber invasion assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function + phospho-mutant rescue + functional ECM degradation assay; multiple orthogonal methods\",\n      \"pmids\": [\"22303001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HIP-55 forms a complex with 14-3-3 proteins via phospho-Ser269/Thr291 sites, identified by affinity chromatography. S269A/T291A mutant HIP-55 (HIP-55AA) abolishes 14-3-3 binding. Tumors expressing HIP-55AA show significantly reduced growth in xenograft models compared to wild-type HIP-55, supporting a pro-oncogenic role mediated by the HIP-55/14-3-3 interaction node that antagonizes HPK1 tumor suppressor function.\",\n      \"method\": \"Affinity chromatography (14-3-3 pull-down), site-directed mutagenesis (S269A/T291A), xenograft tumor model, colony formation and invasion assays\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — affinity chromatography + mutagenesis + in vivo xenograft; multiple orthogonal methods in one study\",\n      \"pmids\": [\"24912570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HIP-55 negatively regulates β-adrenergic receptor-activated ERK1/2 signaling and cardiac fibroblast proliferation; both HIP-55 overexpression and knockdown studies confirmed this regulatory role.\",\n      \"method\": \"Proteomics, HIP-55 overexpression and knockdown in cardiac fibroblasts, ERK1/2 phosphorylation assay, proliferation assay\",\n      \"journal\": \"Molecular bioSystems\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — gain- and loss-of-function in primary cells with defined signaling readout; single lab\",\n      \"pmids\": [\"24802081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"14-3-3 interacts with HIP-55 through S269/T291 sites (confirmed by BiFC and co-IP); the 14-3-3/HIP-55 complex increases HIP-55 protein stability, as the S269A/T291A mutant (defective in 14-3-3 binding) shows reduced stability after puromycin block of new protein synthesis.\",\n      \"method\": \"Bimolecular fluorescence complementation (BiFC), co-immunoprecipitation, pulse-chase with puromycin, site-directed mutagenesis\",\n      \"journal\": \"Journal of Peking University. Health sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — BiFC + co-IP + stability assay with mutant; single lab, multiple methods\",\n      \"pmids\": [\"26679646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HIP-55 negatively regulates myocardial contractility; knockdown increased and overexpression decreased contractile traction forces in single cardiomyocytes. HIP-55 co-localizes with F-actin in cardiomyocytes, suggesting the mechanism involves HIP-55–F-actin interaction.\",\n      \"method\": \"Adenoviral overexpression and knockdown, traction force microscopy, immunofluorescence co-localization\",\n      \"journal\": \"Journal of biomechanics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — bidirectional manipulation (KD + OE) with quantitative traction force readout; single lab\",\n      \"pmids\": [\"24880669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"mAbp1/HIP-55 binds clathrin, actin, and dynamin simultaneously, acting as an adaptor that links dynamin to actin at clathrin-coated pits (CCPs). Live-cell imaging shows mAbp1 is recruited specifically at a late stage to long-lived CCPs. mAbp1 knockdown reduced dynamin recruitment at the plasma membrane and impaired CCP scission. Actin disruption eliminated both mAbp1 and dynamin recruitment, indicating mAbp1 acts downstream of F-actin assembly to recruit dynamin.\",\n      \"method\": \"Live-cell imaging, siRNA knockdown, co-immunoprecipitation/binding assays with clathrin/actin/dynamin, actin disruption experiments\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging + KD + binding assays for three partners + actin disruption epistasis; multiple orthogonal methods\",\n      \"pmids\": [\"25690657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"mAbp1/HIP-55 interacts with FHL2 through its N-terminal ADF homology (ADFH) domain, identified by yeast two-hybrid. Depletion of mAbp1 or ectopic expression of its ADFH domain increased Rho GTPase signaling and breast cancer cell invasion; ADFH-domain-induced invasion required FHL2 expression, placing FHL2 downstream of mAbp1 in the Rho/invasion axis.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, siRNA knockdown, domain overexpression, Rho GTPase activity assay, Boyden chamber invasion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — yeast two-hybrid + co-IP + epistasis with FHL2 KD; single lab, multiple methods\",\n      \"pmids\": [\"27129278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Dbnl (DBNL) is required for neuronal migration, multipolar morphology, and polarity in the developing cerebral cortex. Dbnl knockdown decreased plasma membrane-localized N-cadherin in cortical neurons. Neuronal migration defects were rescued by N-cadherin/αN-catenin overexpression or by a phospho-mimetic Dbnl (Y337E/Y347E) but not by phospho-resistant Dbnl (Y337F/Y347F), placing Tyr337/347 phosphorylation upstream of N-cadherin regulation.\",\n      \"method\": \"In utero electroporation knockdown, time-lapse imaging, rescue with N-cadherin/αN-catenin and phospho-mutants, surface N-cadherin quantification\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KD + time-lapse imaging + phospho-mutant rescue + N-cadherin surface quantification; multiple orthogonal methods\",\n      \"pmids\": [\"30504273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Dbnl interacts with N-cadherin in a β-catenin-dependent manner during anterograde Golgi trafficking and promotes pro-N-cadherin propeptide excision and maturation. Dbnl knockdown causes pro-N-cadherin accumulation and limits adherens junction formation, disrupting apico-basal polarity in the neural tube.\",\n      \"method\": \"Co-immunoprecipitation, Dbnl knockdown, Western blot for pro-N-cadherin vs mature N-cadherin, immunofluorescence for AJ markers\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP + KD with N-cadherin processing readout; single lab, two methods\",\n      \"pmids\": [\"33939796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HIP-55 interacts with Smad7 and competes with Smad7/Axin complex formation, thereby inhibiting Axin-mediated Smad7 degradation. HIP-55 couples Smad7 to TβRI (but not TβRII) to drive TβRI degradation; loss of HIP-55 causes TGF-β signaling overactivation and abnormal ECM gene accumulation.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and knockdown, Western blot for signaling pathway components and ECM genes\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP + gain/loss-of-function + receptor degradation assay; single lab\",\n      \"pmids\": [\"34331017\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HIP-55 is a new AKT substrate phosphorylated at S269 and T291. AKT-phosphorylated HIP-55 directs AKT signaling to negatively regulate the MAP4K1 pathway, protecting against MI-induced ferroptosis. S269A/T291A-mutated HIP-55 fails to inhibit the MAP4K1/GPX4 ferroptosis pathway and fails to protect against MI injury in vivo.\",\n      \"method\": \"In vitro kinase assay (AKT phosphorylation), site-directed mutagenesis (S269A/T291A), cardiac-specific overexpression mouse model, co-immunoprecipitation, GPX4 assay, MI model\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay + phospho-mutant + in vivo cardiac-specific OE mouse; multiple orthogonal methods\",\n      \"pmids\": [\"36639542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HIP-55 interacts with clathrin and F-actin to promote clathrin-mediated endocytosis of nanoparticles (quantum dots). A HIP-55 ΔADF mutant defective in F-actin binding fails to promote nanoparticle endocytosis. HIP-55 knockout inhibits QD endocytosis in vivo.\",\n      \"method\": \"Co-immunoprecipitation (HIP-55–clathrin interaction), ΔADF domain mutant overexpression, pharmacological clathrin inhibition, HIP-55 KO mouse, live imaging\",\n      \"journal\": \"Nano letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — co-IP + domain mutant + KO mouse; single lab, multiple approaches\",\n      \"pmids\": [\"38084909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AKT phosphorylates HIP-55 at S269 and T291, which is required for normal liquid-liquid phase separation of HIP-55. Failure of AKT-mediated phosphorylation leads to abnormal HIP-55 aggregation. HIP-55 phase separation inhibits β-adrenergic receptor-mediated P38/MAPK signaling. Phospho-deficient HIP-55 (S269A/T291A) undergoes massive aberrant phase separation, loses protective activity against heart failure, and phospho-resistant knock-in mice show aggravated HF.\",\n      \"method\": \"FRAP assay, DIC microscopy, pull-down, immunofluorescence, phospho-mutant knock-in mice, cardiac-specific KO and OE mouse models, P38/MAPK signaling assays\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — FRAP + DIC (phase separation), mutagenesis, multiple in vivo mouse models with functional cardiac readouts; multiple orthogonal methods\",\n      \"pmids\": [\"38328928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HIP-55 facilitates AP-1 complex activation in macrophages by promoting ERK1/2 and JNK phosphorylation in response to Ang II; blocking AP-1 attenuates HIP-55-mediated macrophage M1 polarization. HIP-55 expression is upregulated in M1 macrophages, and its genetic deletion inhibits Ang II-induced M1 polarization.\",\n      \"method\": \"HIP-55 overexpression and genetic deletion in macrophages, AP-1 reporter/inhibitor assay, ERK1/2 and JNK phosphorylation Western blot, M1 polarization marker assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — bidirectional manipulation + AP-1 inhibitor epistasis + signaling readouts; single lab\",\n      \"pmids\": [\"38417633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RSK1 phosphorylates HIP-55 at S269/T291 to promote 14-3-3/HIP-55 complex formation, which suppresses the ASK1 apoptotic pathway. S269A/T291A HIP-55 (RSK1-phosphorylation-deficient) fails to form the 14-3-3/HIP-55 complex and fails to protect cardiomyocytes against MI-induced apoptosis in vivo and in vitro.\",\n      \"method\": \"In vitro kinase assay (RSK1), site-directed mutagenesis (S269A/T291A), co-immunoprecipitation, in vivo MI model with phospho-resistant knock-in, apoptosis assays\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay + mutagenesis + co-IP + in vivo knock-in mouse; multiple orthogonal methods\",\n      \"pmids\": [\"40675322\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"DBNL/HIP-55/mAbp1 is a multidomain adaptor protein with an N-terminal ADF-H (actin-binding) domain and a C-terminal SH3 domain that binds F-actin (1:5 stoichiometry) and scaffolds diverse signaling complexes: it is phosphorylated by Src/Syk/ZAP-70 at Tyr334/344 (and Tyr337/347) to regulate actin dynamics, podosome rosette formation, and neuronal migration via N-cadherin; it activates HPK1 and the JNK cascade (and negatively regulates NFAT) downstream of antigen receptors; it links dynamin and clathrin to F-actin to drive CCP scission in clathrin-mediated endocytosis and synaptic vesicle recycling; it undergoes AKT- and RSK1-mediated phosphorylation at S269/T291, controlling its liquid-liquid phase separation, 14-3-3 complex formation (which stabilizes HIP-55 and suppresses ASK1-mediated apoptosis), and MAP4K1/GPX4 ferroptosis pathway regulation, thereby protecting against myocardial infarction and heart failure; it is cleaved by caspase-3 at D361 during apoptosis; and it promotes AP-1-mediated macrophage M1 polarization and modulates TGF-β signaling by coupling Smad7 to TβRI degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"DBNL (HIP-55/mAbp1/SH3P7) is a multidomain actin-associated adaptor protein that couples cytoskeletal dynamics to receptor signaling, membrane trafficking, and cell survival across immune, neuronal, and cardiac contexts [#0, #2]. Through an N-terminal actin-binding (ADF-H/SCAD) region and a C-terminal SH3 domain, it binds F-actin with a defined 1:5 stoichiometry and is recruited to cortical actin and the Arp2/3 complex at lamellipodia downstream of Rac1-driven actin polymerization [#2]. The SH3 domain scaffolds proline-rich partners: DBNL binds and activates HPK1 to drive the JNK cascade downstream of antigen receptors, where it is recruited to lipid rafts and the immunological synapse, phosphorylated by ZAP-70 at Tyr334/344, and is required for TCR-stimulated JNK/HPK1 activation while negatively regulating NFAT [#1, #5, #6, #8]. In membrane trafficking, DBNL acts downstream of F-actin assembly to link clathrin, actin, and dynamin at clathrin-coated pits, driving late-stage dynamin recruitment and coated-pit scission in receptor-mediated endocytosis and synaptic vesicle recycling [#9, #17, #23]. Tyrosine phosphorylation by Src at Tyr337/347 controls its role in podosome rosette formation and ECM degradation, and the same residues, in phospho-mimetic form, govern N-cadherin surface delivery, propeptide maturation, and neuronal migration in the developing cortex [#12, #19, #20]. A distinct AKT/RSK1-dependent phosphorylation node at Ser269/Thr291 directs 14-3-3 complex formation, protein stabilization, and liquid-liquid phase separation, suppressing ASK1-mediated apoptosis and MAP4K1/GPX4-driven ferroptosis to protect the heart against myocardial infarction and heart failure [#13, #15, #22, #24, #26]. DBNL is also cleaved by caspase-3 at Asp361 during apoptosis, severing the actin-binding domain from the SH3 domain [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established DBNL as an actin-associated adaptor that physically couples antigen-receptor kinase signaling to the cytoskeleton, defining its core molecular identity.\",\n      \"evidence\": \"In vitro kinase assays with Src/Syk family kinases, actin colocalization, and sequence analysis of the actin-binding SCAD region\",\n      \"pmids\": [\"9891087\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of tyrosine phosphorylation not defined\", \"Specific signaling pathway downstream not yet identified\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Placed DBNL upstream of HPK1 in the JNK cascade, showing its SH3 domain activates a defined kinase module rather than acting as a passive scaffold.\",\n      \"evidence\": \"In vitro binding, co-IP, kinase activity assays, and dominant-negative HPK1 epistasis\",\n      \"pmids\": [\"10567356\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Receptor input driving the cascade not yet established\", \"Mechanism by which binding stimulates HPK1 activity unresolved\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Quantified DBNL's F-actin binding (1:5 stoichiometry) and linked its recruitment to Rac1-driven actin polymerization at lamellipodia, defining how it senses cytoskeletal remodeling.\",\n      \"evidence\": \"In vitro F-actin binding stoichiometry, live-cell imaging, dominant-positive Rac1, and actin polymerization inhibition\",\n      \"pmids\": [\"10637315\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relationship between two actin-binding domains and signaling unclear\", \"Direct Arp2/3 interaction not biochemically reconstituted\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified DBNL as a caspase-3 substrate cleaved at Asp361, revealing a regulated mechanism that uncouples its actin-binding and adaptor modules during apoptosis.\",\n      \"evidence\": \"In vitro caspase cleavage, D361A site-directed mutagenesis, and Western blot during apoptosis\",\n      \"pmids\": [\"11689006\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of cleavage fragments not characterized\", \"Whether cleavage promotes or limits apoptosis unknown\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the receptor-proximal mechanism in T cells: ZAP-70 phosphorylates DBNL at Tyr334/344 upon TCR engagement, making it required for TCR-specific JNK/HPK1 activation.\",\n      \"evidence\": \"Co-IP, in vitro kinase assay, Y334F/Y344F mutagenesis, RNAi, and sucrose gradient raft fractionation\",\n      \"pmids\": [\"14557276\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of raft recruitment unresolved\", \"Distinct roles of Tyr334 vs Tyr344 not separated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Localized DBNL function to the immunological synapse and showed dual-domain requirement for synapse recruitment, HPK1 activation, NFAT suppression, and TCR endocytosis.\",\n      \"evidence\": \"GFP live imaging, RNAi, co-IP, NFAT-luciferase reporter, and flow cytometry for TCR surface levels\",\n      \"pmids\": [\"14729663\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking TCR endocytosis to NFAT regulation not dissected\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Showed the DBNL SH3 domain is targeted by a viral proline-rich ligand (ASFV CD2v), implicating it in pathogen exploitation of the host actin/trafficking machinery.\",\n      \"evidence\": \"Yeast two-hybrid, direct binding, deletion mapping, and colocalization in infected cells\",\n      \"pmids\": [\"14718626\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence for viral replication not established\", \"Single-lab interactor not independently confirmed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Confirmed the in vivo requirement for DBNL in T-cell activation using knockout mice, validating RNAi-based placement in TCR-driven HPK1/JNK signaling.\",\n      \"evidence\": \"KO mouse, proliferation assays, cytokine ELISA, flow cytometry, signaling Western blots, and co-IP\",\n      \"pmids\": [\"16055701\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether actin-binding or scaffolding drives the phenotype not separated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Established DBNL as required for clathrin-mediated and synaptic vesicle endocytosis, placing its function downstream of vesicle fission.\",\n      \"evidence\": \"KO mouse, transferrin uptake, live imaging of synaptic vesicle recycling, and FM dye assays\",\n      \"pmids\": [\"16601697\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partners at the fission step not yet identified in this study\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined the molecular mechanism of DBNL in endocytosis: it simultaneously binds clathrin, actin, and dynamin, recruiting dynamin to coated pits downstream of F-actin to drive scission.\",\n      \"evidence\": \"Live-cell imaging, siRNA, binding assays for three partners, and actin disruption epistasis\",\n      \"pmids\": [\"25690657\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the clathrin/actin/dynamin assembly unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked Src phosphorylation at Tyr337/347 to DBNL-driven podosome rosette formation and ECM degradation, distinguishing it from cortactin-dependent podosome dots.\",\n      \"evidence\": \"siRNA, Y337F/Y347F phospho-mutant rescue, gelatin degradation, and invasion assays\",\n      \"pmids\": [\"22303001\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Effectors downstream of phospho-DBNL at rosettes not identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed Tyr337/347 phosphorylation governs DBNL control of surface N-cadherin and neuronal migration in vivo, extending its actin-adaptor role to cortical development.\",\n      \"evidence\": \"In utero electroporation knockdown, time-lapse imaging, and phospho-mutant/N-cadherin rescue\",\n      \"pmids\": [\"30504273\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How phospho-DBNL controls N-cadherin trafficking mechanistically not fully resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined a trafficking mechanism whereby DBNL binds N-cadherin β-catenin-dependently in the Golgi to promote pro-N-cadherin maturation and adherens junction formation.\",\n      \"evidence\": \"Co-IP, knockdown, pro- versus mature N-cadherin Western blot, and AJ marker immunofluorescence\",\n      \"pmids\": [\"33939796\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the protease excising the propeptide not established\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovered the Ser269/Thr291 14-3-3 binding node and linked it to DBNL protein stability and a pro-oncogenic role antagonizing HPK1.\",\n      \"evidence\": \"Affinity chromatography, S269A/T291A mutagenesis, BiFC, puromycin pulse-chase, and xenograft tumor models\",\n      \"pmids\": [\"24912570\", \"26679646\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase generating the phospho-sites not identified at this stage\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified DBNL as a negative regulator of β-adrenergic ERK1/2 signaling and contractility in cardiac cells, opening its cardiovascular role.\",\n      \"evidence\": \"Proteomics, gain/loss-of-function in cardiac fibroblasts and cardiomyocytes, ERK1/2 phosphorylation, proliferation, and traction force microscopy\",\n      \"pmids\": [\"24802081\", \"24880669\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular link between DBNL and the β-AR/ERK pathway not defined\", \"Single-lab findings\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Linked the DBNL ADF-H domain to FHL2-dependent suppression of Rho-driven invasion, defining a domain-specific tumor-suppressive axis distinct from its scaffolding functions.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, siRNA, ADFH domain overexpression, Rho activity, and invasion assays\",\n      \"pmids\": [\"27129278\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation of pro-oncogenic (14-3-3) versus anti-invasive (FHL2) roles unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Showed DBNL couples Smad7 to TβRI degradation by competing with the Smad7/Axin complex, identifying a mechanism restraining TGF-β signaling and ECM accumulation.\",\n      \"evidence\": \"Co-IP, gain/loss-of-function, and Western blot for pathway components and ECM genes\",\n      \"pmids\": [\"34331017\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding; receptor-degradation route (lysosomal vs proteasomal) not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identified AKT as a kinase for the S269/T291 node, channeling AKT signaling against MAP4K1/GPX4 ferroptosis to protect against myocardial infarction.\",\n      \"evidence\": \"In vitro AKT kinase assay, S269A/T291A mutagenesis, cardiac-specific overexpression mouse, co-IP, GPX4 assay, and MI model\",\n      \"pmids\": [\"36639542\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical link between phospho-DBNL and MAP4K1 inhibition not detailed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established that AKT phosphorylation of S269/T291 governs DBNL liquid-liquid phase separation, with aberrant aggregation suppressing protective β-AR/P38 signaling in failing hearts.\",\n      \"evidence\": \"FRAP, DIC microscopy, pull-down, phospho-mutant knock-in mice, cardiac KO/OE models, and P38/MAPK assays\",\n      \"pmids\": [\"38328928\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Composition of phase-separated condensates not defined\", \"Relationship between condensates and 14-3-3 binding unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked DBNL to macrophage M1 polarization via AP-1 activation through ERK1/2 and JNK, extending its adaptor role to inflammatory signaling.\",\n      \"evidence\": \"Overexpression/genetic deletion in macrophages, AP-1 reporter/inhibitor epistasis, MAPK Western blots, and M1 marker assays\",\n      \"pmids\": [\"38417633\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular partners upstream of MAPK in macrophages not identified\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified RSK1 as a second kinase phosphorylating S269/T291 to drive 14-3-3 complex formation and ASK1-apoptosis suppression, cementing the cardioprotective phospho-node.\",\n      \"evidence\": \"In vitro RSK1 kinase assay, S269A/T291A mutagenesis, co-IP, phospho-resistant knock-in MI model, and apoptosis assays\",\n      \"pmids\": [\"40675322\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination between AKT and RSK1 inputs at the same sites not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How DBNL's competing actin-cytoskeletal, endocytic, and S269/T291 phase-separation/14-3-3 functions are coordinated within a single cell, and how phospho-state integrates these roles, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified structural model integrating ADF-H, SH3, and disordered phospho-region\", \"Tissue-specific partitioning of functions not mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 2, 16, 17]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 5, 17, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 6, 21, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 2, 16]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [5, 17, 19]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [13, 24]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [7, 20]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [9, 17, 23]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 6, 8, 25]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 14, 21, 22]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [3, 22, 26]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [19, 20]}\n    ],\n    \"complexes\": [\n      \"HIP-55/14-3-3 complex\",\n      \"HIP-55/HPK1 complex\",\n      \"clathrin–actin–dynamin coat assembly\"\n    ],\n    \"partners\": [\n      \"HPK1\",\n      \"ZAP-70\",\n      \"14-3-3\",\n      \"dynamin\",\n      \"clathrin\",\n      \"N-cadherin\",\n      \"FHL2\",\n      \"Smad7\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}