{"gene":"BNIP2","run_date":"2026-06-09T22:02:45","timeline":{"discoveries":[{"year":1999,"finding":"BNIP-2 is a substrate of FGF receptor-1 (FGFR1/Flg) tyrosine kinase; FGFR1 phosphorylates BNIP-2 on tyrosine residues both in co-transfected 293T cells and in vitro using recombinant proteins. Tyrosine phosphorylation of BNIP-2 prevents its binding to Cdc42GAP and Cdc42, and abolishes its GAP-like activity toward Cdc42.","method":"Co-transfection/capture experiments with kinase-dead mutant in 293T cells; in vitro phosphorylation of bacterially expressed BNIP-2 by active FGFR1; co-immunoprecipitation; GST pulldown; GTPase activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro phosphorylation assay plus mutagenesis, multiple orthogonal methods (co-IP, pulldown, GTPase assay) in single rigorous study","pmids":["10551883"],"is_preprint":false},{"year":2000,"finding":"The BCH domain of BNIP-2 binds Cdc42 and stimulates its GTPase activity via a novel arginine-patch motif (Arg-235, Arg-238). Site-directed mutagenesis of R235K or R238K severely impairs GAP activity without affecting Cdc42 binding. The Cdc42 binding involves residues 288EYV290 and the Switch I and Insert regions of Cdc42.","method":"GST pulldown with recombinant BCH domain; site-directed mutagenesis; in vitro GTPase activity assay; deletion studies","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis, multiple orthogonal approaches in one rigorous study","pmids":["10799524"],"is_preprint":false},{"year":2000,"finding":"BNIP-2 and Cdc42GAP form homo- and heterocomplexes via their conserved BCH domains. The major BCH-BCH interaction site within BNIP-2 is the region 217RRKMP221, distinct from the arginine-patch required for GAP activity (235RRLRK239) or the Cdc42 binding sequence (288EYV290).","method":"GST recombinant protein pulldown; co-immunoprecipitation; yeast two-hybrid assay; deletion mutagenesis; molecular modeling","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal methods (GST pulldown, co-IP, yeast two-hybrid, mutagenesis) in one study","pmids":["10954711"],"is_preprint":false},{"year":2003,"finding":"BPGAP1, a novel RhoGAP containing a BCH domain, interacts with BCH domain-containing proteins including BNIP-2 via homophilic and heterophilic BCH-BCH interactions, as shown by pulldown and co-immunoprecipitation. BNIP-2's BCH domain mediates formation of these complexes.","method":"GST pulldown; co-immunoprecipitation; fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and pulldown, single lab, two orthogonal methods","pmids":["12944407"],"is_preprint":false},{"year":2005,"finding":"BNIP-2 induces cell elongation and membrane protrusions via its BCH domain by binding Cdc42 through a unique motif 285VPMEYVGI292, distinct from canonical CRIB motifs. Dominant-negative Cdc42 completely blocked BNIP-2-induced cell elongation. Subcellular localization of BNIP-2 is to the cytoplasm and concentrated at the leading edge of cellular extensions.","method":"Transient expression; dominant-negative GTPase co-expression; deletional mutagenesis; binding studies; fluorescence microscopy","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis identifying specific binding motif combined with functional assays and multiple orthogonal methods in one study","pmids":["15652341"],"is_preprint":false},{"year":2006,"finding":"BNIP-Salpha (a BNIP-2 family member) activates RhoA by competing with p50RhoGAP/Cdc42GAP for RhoA binding via overlapping motifs (residues 133-147 and 148-177) in its BCH domain, leading to cell rounding and apoptosis. Only dominant-negative RhoA prevented this effect; BNIP-2's BCH domain interaction with p50RhoGAP involves overlapping regions with its RhoA-binding site.","method":"Mutagenesis; co-immunoprecipitation; dominant-negative/constitutively active GTPase expression; cell morphology assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis and co-IP combined with functional readout, single lab","pmids":["16331259"],"is_preprint":false},{"year":2008,"finding":"BNIP-2 interacts with Cdo (a promyogenic cell surface receptor), JLP (a p38 scaffold), and Cdc42GAP, forming a multi-scaffold complex. Cdo-BNIP-2 interaction stimulates Cdc42 activity, which promotes p38alpha/beta MAPK activity and myoblast differentiation. BNIP-2 and JLP are brought together through mutual interaction with Cdo.","method":"Co-immunoprecipitation; gain- and loss-of-function experiments in myoblasts; p38 activity assays; Cdc42 activation assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP combined with gain- and loss-of-function experiments in physiologically relevant cells, multiple orthogonal methods","pmids":["18678706"],"is_preprint":false},{"year":2008,"finding":"BNIPXL (BNIP2 Extra Long) BCH domain inhibits RhoA activity by binding specific conformers of RhoA (fast-cycling F30L and dominant-negative T19N, but not constitutively active G14V or Q63L) and interacts with the DH-PH catalytic domains of Lbc RhoGEF, suppressing Lbc-induced oncogenic transformation. Knockdown of BNIPXL increases active RhoA levels.","method":"Co-immunoprecipitation; RhoA pulldown activity assay; knockdown; overexpression; transformation assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus gain/loss-of-function with functional readout, single lab, two orthogonal methods","pmids":["18445682"],"is_preprint":false},{"year":2007,"finding":"BNIP-2 and BNIP-XL are cleaved by caspases during apoptosis. Caspase cleavage sites on BNIP-2 are located on its N-terminal EF-hand motif. Caspase-mediated cleavage releases the BCH domain or smaller fragments implicated in pro-apoptotic activities.","method":"In vitro caspase cleavage assay; identification of cleavage sites by mutagenesis/biochemical analysis; cell-based apoptosis assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro cleavage assay with site mapping, single lab","pmids":["17961507"],"is_preprint":false},{"year":2010,"finding":"BNIP-2 is a substrate of granzyme B during natural killer cell-mediated killing. Granzyme B cleaves recombinant BNIP-2 in vitro at a defined site (bioinformatically identified), and endogenous BNIP-2 is cleaved during NK cell-mediated tumor cell killing in a caspase-independent manner. Full-length BNIP-2 and the truncated granzyme B-cleaved form are both pro-apoptotic and lead to subsequent caspase-dependent cleavage of BNIP-2 at a distinct site. Inhibition of BNIP-2 expression did not affect susceptibility to NK cell killing.","method":"In vitro granzyme B cleavage assay; NK cell killing assay; siRNA knockdown; site-directed mutagenesis; immunoprecipitation","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of cleavage with site identification plus cell-based validation, multiple orthogonal methods","pmids":["20704564"],"is_preprint":false},{"year":2014,"finding":"BNIP-2 interacts with kinesin-1 (KIF5B) via its BCH domain, binding both the motor and tail domains of KIF5B. BNIP-2 undergoes microtubule-dependent anterograde transport on endosomes in C2C12 cells; disruption by dominant-negative KIF5B or KIF5B knockdown causes aberrant aggregation of BNIP-2. KIF5B-mediated anterograde transport of BNIP-2 is required for its pro-myogenic effects on p38MAPK activity and myogenic differentiation.","method":"Co-immunoprecipitation; far-Western blot; organelle marker co-localization; live cell microscopy; dominant-negative expression; siRNA knockdown; p38 activity assay; differentiation assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding confirmed by far-Western, multiple orthogonal methods including live imaging, gain/loss-of-function with functional readout","pmids":["25378581"],"is_preprint":false},{"year":2014,"finding":"BNIP-2 binds phosphatidylserine via its CRAL-TRIO domain and localizes to Golgi apparatus, early and recycling endosomes, and mitochondria aligned with microtubules. BNIP-2 interacts with kinesin light chains (KLC) through a conserved WED motif in its N-terminal region and is transported by kinesin-1. Vesicular localization requires phosphatidylserine binding; BNIP-2 mutants that do not bind phosphatidylserine fail to induce morphological changes.","method":"Lipid-binding assay; co-immunoprecipitation with KLC; live cell imaging; speed measurement; mutagenesis; subcellular fractionation/organelle markers","journal":"Genes to cells","confidence":"High","confidence_rationale":"Tier 1 / Strong — lipid binding reconstitution, KLC interaction, live imaging of transport, mutagenesis linking phosphatidylserine binding to function, multiple orthogonal methods","pmids":["25472445"],"is_preprint":false},{"year":2014,"finding":"Mouse granzyme B efficiently cleaves BNIP-2 at the IEAD28 tetrapeptide motif in vitro. Extended substrate context beyond P4-P1 positions (particularly P1' and P3' positions) differentially influences cleavage efficiency by human vs. mouse granzyme B. Mutagenesis of P1' (I29>T) yields a 4-fold increase in mouse granzyme B cleavage efficiency.","method":"In vitro degradomics/kinetic cleavage assay; mutagenesis of cleavage site residues","journal":"BMC biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis identifying specific cleavage determinants, single lab","pmids":["25208769"],"is_preprint":false},{"year":2020,"finding":"BNIP-2 scaffolds GEF-H1 and RhoA on microtubules, coupling microtubule disassembly to RhoA activation. BNIP-2 binds both RhoA and GEF-H1, and traffics with kinesin-1 on microtubules. Upon nocodazole-induced microtubule disassembly, BNIP-2–GEF-H1 interaction increases. Depletion of BNIP-2 in MDA-MB-231 cells reduces RhoA activity, uncouples RhoA-GEF-H1 interaction, reduces cell rounding, and promotes cell migration.","method":"Co-immunoprecipitation; knockdown (siRNA); RhoA activation assay; live cell imaging; nocodazole treatment; migration assay","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, multiple knockdown experiments with defined molecular and phenotypic readouts, pharmacological perturbation, multiple orthogonal methods","pmids":["32789168"],"is_preprint":false},{"year":2022,"finding":"BNIP-2 promotes cardiomyoblast differentiation by scaffolding LATS1 to phosphorylate and inactivate YAP (causing its cytosolic retention), in a process requiring BNIP-2 activation of cellular contractility via RhoA/Myosin II. Turbo-ID proximity labeling, super-resolution microscopy, and biochemical pulldown data together revealed BNIP-2 as a scaffold integrating RhoA/Myosin II and LATS1/YAP signaling.","method":"Turbo-ID proximity labeling; super-resolution microscopy; co-immunoprecipitation/pulldown; YAP phosphorylation assay; knockdown; overexpression; cardiac gene expression (cTnT, Myl2)","journal":"Advanced science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (proximity labeling, super-resolution, biochemical, functional), single lab but rigorous mechanistic dissection","pmids":["35975420"],"is_preprint":false}],"current_model":"BNIP-2 is a BCH domain-containing scaffold protein that: (1) is phosphorylated by FGFR1 on tyrosine residues, which abolishes its GAP-like activity toward Cdc42 and its binding to Cdc42GAP; (2) acts as a GAP for Cdc42 via an arginine-patch motif in its BCH domain; (3) binds phosphatidylserine through its CRAL-TRIO domain to localize to vesicles (Golgi, endosomes, mitochondria) and is transported anterogradely along microtubules by kinesin-1 (KIF5B) via a WED–KLC interaction; (4) scaffolds Cdo, JLP, and Cdc42GAP to stimulate Cdc42 and p38alpha/beta MAPK activity during myoblast differentiation, with KIF5B-mediated transport being required for this promyogenic signaling; (5) scaffolds GEF-H1 and RhoA on microtubules, coupling microtubule disassembly to RhoA activation and thereby restraining breast cancer cell migration; (6) scaffolds LATS1 to phosphorylate and inactivate YAP, integrating RhoA/Myosin II and Hippo mechanotransduction to drive cardiomyoblast differentiation; and (7) is cleaved by caspases (at its N-terminal EF-hand region) and by granzyme B (at IEAD28), generating pro-apoptotic BCH-containing fragments."},"narrative":{"mechanistic_narrative":"BNIP2 is a BCH domain-containing scaffold protein that organizes Rho-family GTPase signaling on intracellular membranes and microtubules to control cell shape, differentiation, and apoptosis [PMID:10799524, PMID:18678706]. Through an arginine-patch motif (Arg-235/Arg-238) in its BCH domain it acts as a GAP-like activator of Cdc42, while a distinct motif (288EYV290 / 285VPMEYVGI292) mediates Cdc42 binding and drives cell elongation and membrane protrusions [PMID:10799524, PMID:15652341]; FGFR1 phosphorylates BNIP2 on tyrosine residues, abolishing both its Cdc42GAP binding and its GAP-like activity, providing a kinase-controlled switch over this output [PMID:10551883]. The BCH domain also engages partner proteins through homophilic and heterophilic BCH-BCH interactions, including with Cdc42GAP [PMID:10954711]. BNIP2 binds phosphatidylserine through its CRAL-TRIO domain to localize to the Golgi, endosomes, and mitochondria, and is carried anterogradely along microtubules by kinesin-1, binding both KIF5B and the kinesin light chains via an N-terminal WED motif [PMID:25378581, PMID:25472445]. This transport platform is used in distinct contexts: BNIP2 assembles a Cdo–JLP–Cdc42GAP complex that stimulates Cdc42 and p38 MAPK to drive myoblast differentiation, an output requiring KIF5B-mediated transport [PMID:18678706, PMID:25378581]; it scaffolds GEF-H1 and RhoA on microtubules so that microtubule disassembly activates RhoA, thereby promoting cell rounding and restraining breast cancer cell migration [PMID:32789168]; and it scaffolds LATS1 to phosphorylate and inactivate YAP, coupling RhoA/Myosin II contractility to Hippo signaling during cardiomyoblast differentiation [PMID:35975420]. Independently, BNIP2 is a pro-apoptotic substrate cleaved by caspases at its N-terminal EF-hand region and by granzyme B at the IEAD28 motif, generating BCH-containing fragments [PMID:17961507, PMID:20704564, PMID:25208769].","teleology":[{"year":1999,"claim":"Established that BNIP2 function is under tyrosine-kinase control, linking it to FGFR1 signaling and revealing a regulatory switch over its GTPase-directed activity.","evidence":"In vitro phosphorylation of recombinant BNIP2 by active FGFR1 plus co-IP, pulldown, and GTPase assays in 293T cells","pmids":["10551883"],"confidence":"High","gaps":["Specific phosphorylated tyrosine residues not all mapped","Physiological cellular context of FGFR1-BNIP2 regulation not defined"]},{"year":2000,"claim":"Defined the molecular basis of BNIP2's GAP-like activity and its self/partner assembly, showing the BCH domain uses separable motifs for Cdc42 stimulation, Cdc42 binding, and BCH-BCH complex formation.","evidence":"GST pulldown, site-directed mutagenesis, in vitro GTPase assays, yeast two-hybrid and co-IP with recombinant BCH domain","pmids":["10799524","10954711"],"confidence":"High","gaps":["Functional consequence of BNIP2/Cdc42GAP heterocomplex in cells not established","No structural model of the BCH-GTPase interface"]},{"year":2003,"claim":"Extended the BCH-BCH interaction network by showing BNIP2 forms complexes with another BCH-domain RhoGAP, BPGAP1.","evidence":"GST pulldown, co-IP, and fluorescence microscopy","pmids":["12944407"],"confidence":"Medium","gaps":["Functional output of the BNIP2-BPGAP1 complex unresolved","Single-lab, two orthogonal methods only"]},{"year":2005,"claim":"Connected BNIP2 to cell morphology, demonstrating that Cdc42 binding via a non-canonical motif drives cell elongation and protrusion formation at the leading edge.","evidence":"Transient and dominant-negative Cdc42 expression, deletion mutagenesis, binding studies, fluorescence microscopy","pmids":["15652341"],"confidence":"High","gaps":["Endogenous role in cell migration not tested here","Upstream signals controlling the morphological response unknown"]},{"year":2007,"claim":"Identified BNIP2 as a caspase substrate, showing apoptotic cleavage at its N-terminal EF-hand region liberates pro-apoptotic BCH-containing fragments.","evidence":"In vitro caspase cleavage with site mapping and cell-based apoptosis assays","pmids":["17961507"],"confidence":"Medium","gaps":["Effector mechanism of the released fragments not defined","Physiological apoptotic trigger not identified"]},{"year":2008,"claim":"Revealed BNIP2 as a multi-scaffold for promyogenic signaling, bridging the Cdo receptor, JLP, and Cdc42GAP to activate Cdc42 and p38 MAPK during myoblast differentiation.","evidence":"Reciprocal co-IP with gain/loss-of-function in myoblasts, Cdc42 and p38 activity assays","pmids":["18678706"],"confidence":"High","gaps":["Spatial regulation of the complex not yet resolved","How tyrosine phosphorylation intersects with this complex untested"]},{"year":2008,"claim":"Characterized RhoA regulation by BNIP2 family members, showing BCH-domain proteins can bind specific RhoA conformers and RhoGEF catalytic domains to suppress oncogenic transformation.","evidence":"Co-IP, RhoA pulldown activity assays, knockdown, overexpression, and transformation assays (BNIPXL/BNIP-Salpha)","pmids":["18445682","16331259"],"confidence":"Medium","gaps":["Findings concern BNIP2 family members rather than BNIP2 itself","Conformer selectivity mechanism not structurally defined"]},{"year":2010,"claim":"Established BNIP2 as a granzyme B substrate during NK-cell killing, broadening its pro-apoptotic role to caspase-independent cleavage with subsequent caspase-dependent processing.","evidence":"In vitro granzyme B cleavage, NK killing assay, siRNA knockdown, mutagenesis, immunoprecipitation","pmids":["20704564"],"confidence":"High","gaps":["BNIP2 knockdown did not alter NK killing susceptibility, leaving physiological significance open","Downstream apoptotic targets of the fragments unknown"]},{"year":2014,"claim":"Defined the membrane-targeting and transport machinery of BNIP2, showing phosphatidylserine binding via CRAL-TRIO and kinesin-1-driven anterograde transport that is required for its promyogenic p38 signaling.","evidence":"Lipid-binding assays, co-IP with KIF5B/KLC, far-Western, live imaging, mutagenesis, organelle markers, p38 and differentiation assays","pmids":["25378581","25472445"],"confidence":"High","gaps":["Cargo composition delivered by BNIP2-bearing vesicles not fully cataloged","Regulation of the WED-KLC interaction unknown"]},{"year":2014,"claim":"Refined the granzyme B cleavage determinants, mapping the IEAD28 site and showing extended substrate context governs human vs. mouse cleavage efficiency.","evidence":"In vitro kinetic cleavage assays with cleavage-site mutagenesis","pmids":["25208769"],"confidence":"High","gaps":["In vivo relevance of species-specific cleavage efficiency not addressed","Single-lab biochemical study"]},{"year":2020,"claim":"Showed BNIP2 transduces microtubule dynamics into RhoA activity by scaffolding GEF-H1 and RhoA, coupling microtubule disassembly to RhoA activation and restraint of cancer cell migration.","evidence":"Reciprocal co-IP, siRNA knockdown, RhoA activation assay, nocodazole treatment, live imaging, migration assays in MDA-MB-231 cells","pmids":["32789168"],"confidence":"High","gaps":["How the same scaffold partitions between Cdc42 and RhoA outputs unresolved","In vivo tumor relevance not tested"]},{"year":2022,"claim":"Connected BNIP2 to Hippo mechanotransduction, demonstrating it scaffolds LATS1 to inactivate YAP downstream of RhoA/Myosin II contractility to drive cardiomyoblast differentiation.","evidence":"Turbo-ID proximity labeling, super-resolution microscopy, pulldown, YAP phosphorylation assays, knockdown/overexpression, cardiac gene markers","pmids":["35975420"],"confidence":"High","gaps":["Mechanism selecting LATS1 versus other scaffolding partners unclear","In vivo cardiac developmental requirement not established"]},{"year":null,"claim":"How BNIP2 integrates and switches between its distinct outputs — Cdc42-driven morphology, RhoA/GEF-H1 microtubule sensing, LATS1/YAP Hippo signaling, and apoptotic fragment generation — within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the full-length scaffold with its multiple partners","Signals that route BNIP2 toward Cdc42 versus RhoA versus apoptotic cleavage unknown","In vivo physiological loss-of-function phenotype not characterized in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[6,13,14]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,0,13]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[11]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[10,11,13]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[11]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[10,11]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[11]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[10,13]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6,13,14]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[6,14]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[8,9]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[10,11]}],"complexes":[],"partners":["CDC42","ARHGAP1","RHOA","ARHGEF2","KIF5B","CDON","SPAG9","LATS1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q12982","full_name":"BCL2/adenovirus E1B 19 kDa protein-interacting protein 2","aliases":[],"length_aa":314,"mass_kda":36.0,"function":"Implicated in the suppression of cell death. Interacts with the BCL-2 and adenovirus E1B 19 kDa proteins","subcellular_location":"Cytoplasm; Cytoplasm, perinuclear region","url":"https://www.uniprot.org/uniprotkb/Q12982/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/BNIP2","classification":"Not Classified","n_dependent_lines":65,"n_total_lines":383,"dependency_fraction":0.16971279373368145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/BNIP2","total_profiled":1310},"omim":[{"mim_id":"611275","title":"BCL2/ADENOVIRUS E1B 19-KD PROTEIN-INTERACTING PROTEIN 2-LIKE; BNIPL","url":"https://www.omim.org/entry/611275"},{"mim_id":"610691","title":"PRUNE HOMOLOG 2 WITH BCH DOMAIN; PRUNE2","url":"https://www.omim.org/entry/610691"},{"mim_id":"609405","title":"RHO GTPase-ACTIVATING PROTEIN 8; ARHGAP8","url":"https://www.omim.org/entry/609405"},{"mim_id":"608179","title":"CAYTAXIN; ATCAY","url":"https://www.omim.org/entry/608179"},{"mim_id":"603292","title":"BCL2/ADENOVIRUS E1B 19-KD PROTEIN-INTERACTING PROTEIN 2; BNIP2","url":"https://www.omim.org/entry/603292"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"},{"location":"Nucleoli","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":122.0}],"url":"https://www.proteinatlas.org/search/BNIP2"},"hgnc":{"alias_symbol":["Nip2","BNIP-2"],"prev_symbol":[]},"alphafold":{"accession":"Q12982","domains":[{"cath_id":"3.40.525.10","chopping":"119-307","consensus_level":"medium","plddt":88.4671,"start":119,"end":307}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12982","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q12982-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q12982-F1-predicted_aligned_error_v6.png","plddt_mean":72.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=BNIP2","jax_strain_url":"https://www.jax.org/strain/search?query=BNIP2"},"sequence":{"accession":"Q12982","fasta_url":"https://rest.uniprot.org/uniprotkb/Q12982.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q12982/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12982"}},"corpus_meta":[{"pmid":"18678706","id":"PMC_18678706","title":"A Cdo-Bnip-2-Cdc42 signaling pathway regulates p38alpha/beta MAPK activity and myogenic differentiation.","date":"2008","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/18678706","citation_count":91,"is_preprint":false},{"pmid":"21242194","id":"PMC_21242194","title":"miR-20a targets BNIP2 and contributes chemotherapeutic resistance in colorectal adenocarcinoma SW480 and SW620 cell lines.","date":"2011","source":"Acta biochimica et biophysica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/21242194","citation_count":89,"is_preprint":false},{"pmid":"17535851","id":"PMC_17535851","title":"Characterization of NIP2/centrobin, a novel substrate of Nek2, and its potential role in microtubule stabilization.","date":"2007","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/17535851","citation_count":74,"is_preprint":false},{"pmid":"28323408","id":"PMC_28323408","title":"Monodispersed Carbon-Coated Cubic NiP2 Nanoparticles Anchored on Carbon Nanotubes as Ultra-Long-Life Anodes for Reversible Lithium Storage.","date":"2017","source":"ACS nano","url":"https://pubmed.ncbi.nlm.nih.gov/28323408","citation_count":58,"is_preprint":false},{"pmid":"16899818","id":"PMC_16899818","title":"Brain-specific BNIP-2-homology protein Caytaxin relocalises glutaminase to neurite terminals and reduces glutamate levels.","date":"2006","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/16899818","citation_count":55,"is_preprint":false},{"pmid":"33309839","id":"PMC_33309839","title":"Astrocyte-derived exosomes carry microRNA-17-5p to protect neonatal rats from hypoxic-ischemic brain damage via inhibiting BNIP-2 expression.","date":"2020","source":"Neurotoxicology","url":"https://pubmed.ncbi.nlm.nih.gov/33309839","citation_count":54,"is_preprint":false},{"pmid":"12944407","id":"PMC_12944407","title":"Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like, proline-rich, and GTPase-activating protein domains of a novel Rho GTPase-activating protein, BPGAP1.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12944407","citation_count":44,"is_preprint":false},{"pmid":"10551883","id":"PMC_10551883","title":"Tyrosine phosphorylation of the Bcl-2-associated protein BNIP-2 by fibroblast growth factor receptor-1 prevents its binding to Cdc42GAP and Cdc42.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10551883","citation_count":42,"is_preprint":false},{"pmid":"10954711","id":"PMC_10954711","title":"The BNIP-2 and Cdc42GAP homology domain of BNIP-2 mediates its homophilic association and heterophilic interaction with Cdc42GAP.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10954711","citation_count":39,"is_preprint":false},{"pmid":"11741952","id":"PMC_11741952","title":"The BNIP-2 and Cdc42GAP homology/Sec14p-like domain of BNIP-Salpha is a novel apoptosis-inducing sequence.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11741952","citation_count":38,"is_preprint":false},{"pmid":"18445682","id":"PMC_18445682","title":"BNIP2 extra long inhibits RhoA and cellular transformation by Lbc RhoGEF via its BCH domain.","date":"2008","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/18445682","citation_count":38,"is_preprint":false},{"pmid":"16331259","id":"PMC_16331259","title":"BNIP-Salpha induces cell rounding and apoptosis by displacing p50RhoGAP and facilitating RhoA activation via its unique motifs in the BNIP-2 and Cdc42GAP homology domain.","date":"2006","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/16331259","citation_count":36,"is_preprint":false},{"pmid":"11069120","id":"PMC_11069120","title":"Oestrogen prevention of neural cell death correlates with decreased expression of mRNA for the pro-apoptotic protein 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/20511645","citation_count":24,"is_preprint":false},{"pmid":"12901880","id":"PMC_12901880","title":"BNIPL-2, a novel homologue of BNIP-2, interacts with Bcl-2 and Cdc42GAP in apoptosis.","date":"2003","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12901880","citation_count":23,"is_preprint":false},{"pmid":"22710163","id":"PMC_22710163","title":"Functional plasticity of the BNIP-2 and Cdc42GAP Homology (BCH) domain in cell signaling and cell dynamics.","date":"2012","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/22710163","citation_count":22,"is_preprint":false},{"pmid":"19117032","id":"PMC_19117032","title":"Nip2/centrobin may be a substrate of Nek2 that is required for proper spindle assembly during mitosis in early mouse embryos.","date":"2009","source":"Molecular reproduction and 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BNIP-2 to regulate p38 mitogen-activated protein kinase activation and myoblast differentiation.","date":"2014","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/25378581","citation_count":16,"is_preprint":false},{"pmid":"20569513","id":"PMC_20569513","title":"Nek2 and its substrate, centrobin/Nip2, are required for proper meiotic spindle formation of the mouse oocytes.","date":"2010","source":"Zygote (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/20569513","citation_count":15,"is_preprint":false},{"pmid":"10715586","id":"PMC_10715586","title":"Expression of the estrogen-regulated gene Nip2 during rat brain maturation.","date":"2000","source":"International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/10715586","citation_count":12,"is_preprint":false},{"pmid":"20704564","id":"PMC_20704564","title":"Identification of the BCL2/adenovirus E1B-19K protein-interacting protein 2 (BNIP-2) as a granzyme B target during human natural killer cell-mediated killing.","date":"2010","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/20704564","citation_count":12,"is_preprint":false},{"pmid":"35975420","id":"PMC_35975420","title":"BNIP-2 Activation of Cellular Contractility Inactivates YAP for H9c2 Cardiomyoblast Differentiation.","date":"2022","source":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/35975420","citation_count":11,"is_preprint":false},{"pmid":"19951366","id":"PMC_19951366","title":"Pro-apoptotic activity of mBNIP-21 depends on its BNIP-2 and Cdc42GAP homology (BCH) domain and is enhanced by coxsackievirus B3 infection.","date":"2009","source":"Cellular microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/19951366","citation_count":10,"is_preprint":false},{"pmid":"35469395","id":"PMC_35469395","title":"Superhydrophilic/Superaerophobic Hierarchical NiP2@MoO2/Co(Ni)MoO4 Core-Shell Array Electrocatalysts for Efficient Hydrogen Production at Large Current Densities.","date":"2022","source":"ACS applied materials & interfaces","url":"https://pubmed.ncbi.nlm.nih.gov/35469395","citation_count":10,"is_preprint":false},{"pmid":"19533028","id":"PMC_19533028","title":"Centrobin/Nip2 expression in vivo suggests its involvement in cell proliferation.","date":"2009","source":"Molecules and cells","url":"https://pubmed.ncbi.nlm.nih.gov/19533028","citation_count":6,"is_preprint":false},{"pmid":"25208769","id":"PMC_25208769","title":"Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B.","date":"2014","source":"BMC biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25208769","citation_count":4,"is_preprint":false},{"pmid":"25472445","id":"PMC_25472445","title":"BNIP-2 binds phosphatidylserine, localizes to vesicles, and is transported by kinesin-1.","date":"2014","source":"Genes to cells : devoted to molecular & cellular mechanisms","url":"https://pubmed.ncbi.nlm.nih.gov/25472445","citation_count":3,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.10.15.618441","title":"Revisiting the evolution and function of NIP2 parologs in the  <i>Rhynchosporium spp.</i>  complex","date":"2024-10-18","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.15.618441","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":19572,"output_tokens":4426,"usd":0.062553,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12207,"output_tokens":4334,"usd":0.084693,"stage2_stop_reason":"end_turn"},"total_usd":0.147246,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"BNIP-2 is a substrate of FGF receptor-1 (FGFR1/Flg) tyrosine kinase; FGFR1 phosphorylates BNIP-2 on tyrosine residues both in co-transfected 293T cells and in vitro using recombinant proteins. Tyrosine phosphorylation of BNIP-2 prevents its binding to Cdc42GAP and Cdc42, and abolishes its GAP-like activity toward Cdc42.\",\n      \"method\": \"Co-transfection/capture experiments with kinase-dead mutant in 293T cells; in vitro phosphorylation of bacterially expressed BNIP-2 by active FGFR1; co-immunoprecipitation; GST pulldown; GTPase activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro phosphorylation assay plus mutagenesis, multiple orthogonal methods (co-IP, pulldown, GTPase assay) in single rigorous study\",\n      \"pmids\": [\"10551883\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"The BCH domain of BNIP-2 binds Cdc42 and stimulates its GTPase activity via a novel arginine-patch motif (Arg-235, Arg-238). Site-directed mutagenesis of R235K or R238K severely impairs GAP activity without affecting Cdc42 binding. The Cdc42 binding involves residues 288EYV290 and the Switch I and Insert regions of Cdc42.\",\n      \"method\": \"GST pulldown with recombinant BCH domain; site-directed mutagenesis; in vitro GTPase activity assay; deletion studies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis, multiple orthogonal approaches in one rigorous study\",\n      \"pmids\": [\"10799524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"BNIP-2 and Cdc42GAP form homo- and heterocomplexes via their conserved BCH domains. The major BCH-BCH interaction site within BNIP-2 is the region 217RRKMP221, distinct from the arginine-patch required for GAP activity (235RRLRK239) or the Cdc42 binding sequence (288EYV290).\",\n      \"method\": \"GST recombinant protein pulldown; co-immunoprecipitation; yeast two-hybrid assay; deletion mutagenesis; molecular modeling\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal methods (GST pulldown, co-IP, yeast two-hybrid, mutagenesis) in one study\",\n      \"pmids\": [\"10954711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"BPGAP1, a novel RhoGAP containing a BCH domain, interacts with BCH domain-containing proteins including BNIP-2 via homophilic and heterophilic BCH-BCH interactions, as shown by pulldown and co-immunoprecipitation. BNIP-2's BCH domain mediates formation of these complexes.\",\n      \"method\": \"GST pulldown; co-immunoprecipitation; fluorescence microscopy\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and pulldown, single lab, two orthogonal methods\",\n      \"pmids\": [\"12944407\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"BNIP-2 induces cell elongation and membrane protrusions via its BCH domain by binding Cdc42 through a unique motif 285VPMEYVGI292, distinct from canonical CRIB motifs. Dominant-negative Cdc42 completely blocked BNIP-2-induced cell elongation. Subcellular localization of BNIP-2 is to the cytoplasm and concentrated at the leading edge of cellular extensions.\",\n      \"method\": \"Transient expression; dominant-negative GTPase co-expression; deletional mutagenesis; binding studies; fluorescence microscopy\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis identifying specific binding motif combined with functional assays and multiple orthogonal methods in one study\",\n      \"pmids\": [\"15652341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"BNIP-Salpha (a BNIP-2 family member) activates RhoA by competing with p50RhoGAP/Cdc42GAP for RhoA binding via overlapping motifs (residues 133-147 and 148-177) in its BCH domain, leading to cell rounding and apoptosis. Only dominant-negative RhoA prevented this effect; BNIP-2's BCH domain interaction with p50RhoGAP involves overlapping regions with its RhoA-binding site.\",\n      \"method\": \"Mutagenesis; co-immunoprecipitation; dominant-negative/constitutively active GTPase expression; cell morphology assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis and co-IP combined with functional readout, single lab\",\n      \"pmids\": [\"16331259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BNIP-2 interacts with Cdo (a promyogenic cell surface receptor), JLP (a p38 scaffold), and Cdc42GAP, forming a multi-scaffold complex. Cdo-BNIP-2 interaction stimulates Cdc42 activity, which promotes p38alpha/beta MAPK activity and myoblast differentiation. BNIP-2 and JLP are brought together through mutual interaction with Cdo.\",\n      \"method\": \"Co-immunoprecipitation; gain- and loss-of-function experiments in myoblasts; p38 activity assays; Cdc42 activation assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP combined with gain- and loss-of-function experiments in physiologically relevant cells, multiple orthogonal methods\",\n      \"pmids\": [\"18678706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"BNIPXL (BNIP2 Extra Long) BCH domain inhibits RhoA activity by binding specific conformers of RhoA (fast-cycling F30L and dominant-negative T19N, but not constitutively active G14V or Q63L) and interacts with the DH-PH catalytic domains of Lbc RhoGEF, suppressing Lbc-induced oncogenic transformation. Knockdown of BNIPXL increases active RhoA levels.\",\n      \"method\": \"Co-immunoprecipitation; RhoA pulldown activity assay; knockdown; overexpression; transformation assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus gain/loss-of-function with functional readout, single lab, two orthogonal methods\",\n      \"pmids\": [\"18445682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"BNIP-2 and BNIP-XL are cleaved by caspases during apoptosis. Caspase cleavage sites on BNIP-2 are located on its N-terminal EF-hand motif. Caspase-mediated cleavage releases the BCH domain or smaller fragments implicated in pro-apoptotic activities.\",\n      \"method\": \"In vitro caspase cleavage assay; identification of cleavage sites by mutagenesis/biochemical analysis; cell-based apoptosis assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro cleavage assay with site mapping, single lab\",\n      \"pmids\": [\"17961507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"BNIP-2 is a substrate of granzyme B during natural killer cell-mediated killing. Granzyme B cleaves recombinant BNIP-2 in vitro at a defined site (bioinformatically identified), and endogenous BNIP-2 is cleaved during NK cell-mediated tumor cell killing in a caspase-independent manner. Full-length BNIP-2 and the truncated granzyme B-cleaved form are both pro-apoptotic and lead to subsequent caspase-dependent cleavage of BNIP-2 at a distinct site. Inhibition of BNIP-2 expression did not affect susceptibility to NK cell killing.\",\n      \"method\": \"In vitro granzyme B cleavage assay; NK cell killing assay; siRNA knockdown; site-directed mutagenesis; immunoprecipitation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of cleavage with site identification plus cell-based validation, multiple orthogonal methods\",\n      \"pmids\": [\"20704564\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"BNIP-2 interacts with kinesin-1 (KIF5B) via its BCH domain, binding both the motor and tail domains of KIF5B. BNIP-2 undergoes microtubule-dependent anterograde transport on endosomes in C2C12 cells; disruption by dominant-negative KIF5B or KIF5B knockdown causes aberrant aggregation of BNIP-2. KIF5B-mediated anterograde transport of BNIP-2 is required for its pro-myogenic effects on p38MAPK activity and myogenic differentiation.\",\n      \"method\": \"Co-immunoprecipitation; far-Western blot; organelle marker co-localization; live cell microscopy; dominant-negative expression; siRNA knockdown; p38 activity assay; differentiation assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding confirmed by far-Western, multiple orthogonal methods including live imaging, gain/loss-of-function with functional readout\",\n      \"pmids\": [\"25378581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"BNIP-2 binds phosphatidylserine via its CRAL-TRIO domain and localizes to Golgi apparatus, early and recycling endosomes, and mitochondria aligned with microtubules. BNIP-2 interacts with kinesin light chains (KLC) through a conserved WED motif in its N-terminal region and is transported by kinesin-1. Vesicular localization requires phosphatidylserine binding; BNIP-2 mutants that do not bind phosphatidylserine fail to induce morphological changes.\",\n      \"method\": \"Lipid-binding assay; co-immunoprecipitation with KLC; live cell imaging; speed measurement; mutagenesis; subcellular fractionation/organelle markers\",\n      \"journal\": \"Genes to cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — lipid binding reconstitution, KLC interaction, live imaging of transport, mutagenesis linking phosphatidylserine binding to function, multiple orthogonal methods\",\n      \"pmids\": [\"25472445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mouse granzyme B efficiently cleaves BNIP-2 at the IEAD28 tetrapeptide motif in vitro. Extended substrate context beyond P4-P1 positions (particularly P1' and P3' positions) differentially influences cleavage efficiency by human vs. mouse granzyme B. Mutagenesis of P1' (I29>T) yields a 4-fold increase in mouse granzyme B cleavage efficiency.\",\n      \"method\": \"In vitro degradomics/kinetic cleavage assay; mutagenesis of cleavage site residues\",\n      \"journal\": \"BMC biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis identifying specific cleavage determinants, single lab\",\n      \"pmids\": [\"25208769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"BNIP-2 scaffolds GEF-H1 and RhoA on microtubules, coupling microtubule disassembly to RhoA activation. BNIP-2 binds both RhoA and GEF-H1, and traffics with kinesin-1 on microtubules. Upon nocodazole-induced microtubule disassembly, BNIP-2–GEF-H1 interaction increases. Depletion of BNIP-2 in MDA-MB-231 cells reduces RhoA activity, uncouples RhoA-GEF-H1 interaction, reduces cell rounding, and promotes cell migration.\",\n      \"method\": \"Co-immunoprecipitation; knockdown (siRNA); RhoA activation assay; live cell imaging; nocodazole treatment; migration assay\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, multiple knockdown experiments with defined molecular and phenotypic readouts, pharmacological perturbation, multiple orthogonal methods\",\n      \"pmids\": [\"32789168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"BNIP-2 promotes cardiomyoblast differentiation by scaffolding LATS1 to phosphorylate and inactivate YAP (causing its cytosolic retention), in a process requiring BNIP-2 activation of cellular contractility via RhoA/Myosin II. Turbo-ID proximity labeling, super-resolution microscopy, and biochemical pulldown data together revealed BNIP-2 as a scaffold integrating RhoA/Myosin II and LATS1/YAP signaling.\",\n      \"method\": \"Turbo-ID proximity labeling; super-resolution microscopy; co-immunoprecipitation/pulldown; YAP phosphorylation assay; knockdown; overexpression; cardiac gene expression (cTnT, Myl2)\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (proximity labeling, super-resolution, biochemical, functional), single lab but rigorous mechanistic dissection\",\n      \"pmids\": [\"35975420\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"BNIP-2 is a BCH domain-containing scaffold protein that: (1) is phosphorylated by FGFR1 on tyrosine residues, which abolishes its GAP-like activity toward Cdc42 and its binding to Cdc42GAP; (2) acts as a GAP for Cdc42 via an arginine-patch motif in its BCH domain; (3) binds phosphatidylserine through its CRAL-TRIO domain to localize to vesicles (Golgi, endosomes, mitochondria) and is transported anterogradely along microtubules by kinesin-1 (KIF5B) via a WED–KLC interaction; (4) scaffolds Cdo, JLP, and Cdc42GAP to stimulate Cdc42 and p38alpha/beta MAPK activity during myoblast differentiation, with KIF5B-mediated transport being required for this promyogenic signaling; (5) scaffolds GEF-H1 and RhoA on microtubules, coupling microtubule disassembly to RhoA activation and thereby restraining breast cancer cell migration; (6) scaffolds LATS1 to phosphorylate and inactivate YAP, integrating RhoA/Myosin II and Hippo mechanotransduction to drive cardiomyoblast differentiation; and (7) is cleaved by caspases (at its N-terminal EF-hand region) and by granzyme B (at IEAD28), generating pro-apoptotic BCH-containing fragments.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"BNIP2 is a BCH domain-containing scaffold protein that organizes Rho-family GTPase signaling on intracellular membranes and microtubules to control cell shape, differentiation, and apoptosis [#1, #6]. Through an arginine-patch motif (Arg-235/Arg-238) in its BCH domain it acts as a GAP-like activator of Cdc42, while a distinct motif (288EYV290 / 285VPMEYVGI292) mediates Cdc42 binding and drives cell elongation and membrane protrusions [#1, #4]; FGFR1 phosphorylates BNIP2 on tyrosine residues, abolishing both its Cdc42GAP binding and its GAP-like activity, providing a kinase-controlled switch over this output [#0]. The BCH domain also engages partner proteins through homophilic and heterophilic BCH-BCH interactions, including with Cdc42GAP [#2]. BNIP2 binds phosphatidylserine through its CRAL-TRIO domain to localize to the Golgi, endosomes, and mitochondria, and is carried anterogradely along microtubules by kinesin-1, binding both KIF5B and the kinesin light chains via an N-terminal WED motif [#10, #11]. This transport platform is used in distinct contexts: BNIP2 assembles a Cdo–JLP–Cdc42GAP complex that stimulates Cdc42 and p38 MAPK to drive myoblast differentiation, an output requiring KIF5B-mediated transport [#6, #10]; it scaffolds GEF-H1 and RhoA on microtubules so that microtubule disassembly activates RhoA, thereby promoting cell rounding and restraining breast cancer cell migration [#13]; and it scaffolds LATS1 to phosphorylate and inactivate YAP, coupling RhoA/Myosin II contractility to Hippo signaling during cardiomyoblast differentiation [#14]. Independently, BNIP2 is a pro-apoptotic substrate cleaved by caspases at its N-terminal EF-hand region and by granzyme B at the IEAD28 motif, generating BCH-containing fragments [#8, #9, #12].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established that BNIP2 function is under tyrosine-kinase control, linking it to FGFR1 signaling and revealing a regulatory switch over its GTPase-directed activity.\",\n      \"evidence\": \"In vitro phosphorylation of recombinant BNIP2 by active FGFR1 plus co-IP, pulldown, and GTPase assays in 293T cells\",\n      \"pmids\": [\"10551883\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific phosphorylated tyrosine residues not all mapped\", \"Physiological cellular context of FGFR1-BNIP2 regulation not defined\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined the molecular basis of BNIP2's GAP-like activity and its self/partner assembly, showing the BCH domain uses separable motifs for Cdc42 stimulation, Cdc42 binding, and BCH-BCH complex formation.\",\n      \"evidence\": \"GST pulldown, site-directed mutagenesis, in vitro GTPase assays, yeast two-hybrid and co-IP with recombinant BCH domain\",\n      \"pmids\": [\"10799524\", \"10954711\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of BNIP2/Cdc42GAP heterocomplex in cells not established\", \"No structural model of the BCH-GTPase interface\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended the BCH-BCH interaction network by showing BNIP2 forms complexes with another BCH-domain RhoGAP, BPGAP1.\",\n      \"evidence\": \"GST pulldown, co-IP, and fluorescence microscopy\",\n      \"pmids\": [\"12944407\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional output of the BNIP2-BPGAP1 complex unresolved\", \"Single-lab, two orthogonal methods only\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Connected BNIP2 to cell morphology, demonstrating that Cdc42 binding via a non-canonical motif drives cell elongation and protrusion formation at the leading edge.\",\n      \"evidence\": \"Transient and dominant-negative Cdc42 expression, deletion mutagenesis, binding studies, fluorescence microscopy\",\n      \"pmids\": [\"15652341\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous role in cell migration not tested here\", \"Upstream signals controlling the morphological response unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified BNIP2 as a caspase substrate, showing apoptotic cleavage at its N-terminal EF-hand region liberates pro-apoptotic BCH-containing fragments.\",\n      \"evidence\": \"In vitro caspase cleavage with site mapping and cell-based apoptosis assays\",\n      \"pmids\": [\"17961507\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effector mechanism of the released fragments not defined\", \"Physiological apoptotic trigger not identified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed BNIP2 as a multi-scaffold for promyogenic signaling, bridging the Cdo receptor, JLP, and Cdc42GAP to activate Cdc42 and p38 MAPK during myoblast differentiation.\",\n      \"evidence\": \"Reciprocal co-IP with gain/loss-of-function in myoblasts, Cdc42 and p38 activity assays\",\n      \"pmids\": [\"18678706\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial regulation of the complex not yet resolved\", \"How tyrosine phosphorylation intersects with this complex untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Characterized RhoA regulation by BNIP2 family members, showing BCH-domain proteins can bind specific RhoA conformers and RhoGEF catalytic domains to suppress oncogenic transformation.\",\n      \"evidence\": \"Co-IP, RhoA pulldown activity assays, knockdown, overexpression, and transformation assays (BNIPXL/BNIP-Salpha)\",\n      \"pmids\": [\"18445682\", \"16331259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Findings concern BNIP2 family members rather than BNIP2 itself\", \"Conformer selectivity mechanism not structurally defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Established BNIP2 as a granzyme B substrate during NK-cell killing, broadening its pro-apoptotic role to caspase-independent cleavage with subsequent caspase-dependent processing.\",\n      \"evidence\": \"In vitro granzyme B cleavage, NK killing assay, siRNA knockdown, mutagenesis, immunoprecipitation\",\n      \"pmids\": [\"20704564\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"BNIP2 knockdown did not alter NK killing susceptibility, leaving physiological significance open\", \"Downstream apoptotic targets of the fragments unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the membrane-targeting and transport machinery of BNIP2, showing phosphatidylserine binding via CRAL-TRIO and kinesin-1-driven anterograde transport that is required for its promyogenic p38 signaling.\",\n      \"evidence\": \"Lipid-binding assays, co-IP with KIF5B/KLC, far-Western, live imaging, mutagenesis, organelle markers, p38 and differentiation assays\",\n      \"pmids\": [\"25378581\", \"25472445\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cargo composition delivered by BNIP2-bearing vesicles not fully cataloged\", \"Regulation of the WED-KLC interaction unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Refined the granzyme B cleavage determinants, mapping the IEAD28 site and showing extended substrate context governs human vs. mouse cleavage efficiency.\",\n      \"evidence\": \"In vitro kinetic cleavage assays with cleavage-site mutagenesis\",\n      \"pmids\": [\"25208769\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of species-specific cleavage efficiency not addressed\", \"Single-lab biochemical study\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showed BNIP2 transduces microtubule dynamics into RhoA activity by scaffolding GEF-H1 and RhoA, coupling microtubule disassembly to RhoA activation and restraint of cancer cell migration.\",\n      \"evidence\": \"Reciprocal co-IP, siRNA knockdown, RhoA activation assay, nocodazole treatment, live imaging, migration assays in MDA-MB-231 cells\",\n      \"pmids\": [\"32789168\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the same scaffold partitions between Cdc42 and RhoA outputs unresolved\", \"In vivo tumor relevance not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected BNIP2 to Hippo mechanotransduction, demonstrating it scaffolds LATS1 to inactivate YAP downstream of RhoA/Myosin II contractility to drive cardiomyoblast differentiation.\",\n      \"evidence\": \"Turbo-ID proximity labeling, super-resolution microscopy, pulldown, YAP phosphorylation assays, knockdown/overexpression, cardiac gene markers\",\n      \"pmids\": [\"35975420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism selecting LATS1 versus other scaffolding partners unclear\", \"In vivo cardiac developmental requirement not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How BNIP2 integrates and switches between its distinct outputs — Cdc42-driven morphology, RhoA/GEF-H1 microtubule sensing, LATS1/YAP Hippo signaling, and apoptotic fragment generation — within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the full-length scaffold with its multiple partners\", \"Signals that route BNIP2 toward Cdc42 versus RhoA versus apoptotic cleavage unknown\", \"In vivo physiological loss-of-function phenotype not characterized in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [6, 13, 14]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 0, 13]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [10, 11, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [10, 11]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [10, 13]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6, 13, 14]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [6, 14]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [8, 9]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [10, 11]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CDC42\", \"ARHGAP1\", \"RHOA\", \"ARHGEF2\", \"KIF5B\", \"CDON\", \"SPAG9\", \"LATS1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}