{"gene":"STXBP4","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":1999,"finding":"Synip (STXBP4) specifically binds syntaxin 4 (but not other syntaxins) and inhibits GLUT4 vesicle translocation; insulin stimulation causes dissociation of the Synip:syntaxin 4 complex through reduced binding affinity, and overexpression of the C-terminal domain of Synip (which does not dissociate from syntaxin 4 upon insulin stimulation) inhibits glucose transport and GLUT4 translocation.","method":"Co-immunoprecipitation, yeast two-hybrid, dominant-negative overexpression in adipocytes with GLUT4 translocation assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, functional dominant-negative overexpression, independently replicated across multiple subsequent labs","pmids":["10394363"],"is_preprint":false},{"year":2003,"finding":"Synip (STXBP4) is expressed in pancreatic beta cells, co-immunoprecipitates with syntaxin 4 but not syntaxin 1, and overexpression of full-length Synip inhibits VAMP2 association with syntaxin 4 and suppresses both first and second phase glucose-stimulated insulin secretion; a Synip mutant (ΔEF) unable to bind syntaxin 4 had no effect, confirming the mechanism requires syntaxin 4 binding.","method":"Co-immunoprecipitation, overexpression of wild-type and binding-deficient mutant Synip in betaHC-9 cells, insulin secretion assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus mutant rescue experiment in a defined cellular system, single lab with multiple orthogonal readouts","pmids":["12855681"],"is_preprint":false},{"year":2005,"finding":"Akt2 (but not Akt1 or Akt3) specifically phosphorylates Synip at serine 99; insulin-stimulated phosphorylation at S99 is required for dissociation of Synip from syntaxin 4 and for GLUT4 vesicle recruitment and plasma membrane fusion; the S99F phosphorylation-resistant mutant dominantly interferes with insulin-stimulated GLUT4 translocation.","method":"In vitro kinase assay with Akt1/2/3, phospho-specific mutagenesis (S99F), overexpression in 3T3L1 adipocytes, GLUT4 translocation assay","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Weak — in vitro kinase assay plus dominant-negative cell experiment, single lab; finding contradicted by Sano et al. 2005 using S99A mutant","pmids":["15753124"],"is_preprint":false},{"year":2005,"finding":"Overexpression of the Synip S99A phosphorylation-site mutant (lacking serine 99) has no effect on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, providing evidence against the requirement for S99 phosphorylation in GLUT4 translocation (negative/contradictory finding relative to Yamada et al. 2005).","method":"Overexpression of S99A mutant Synip in 3T3-L1 adipocytes, GLUT4 translocation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, single method, directly contradicts Yamada et al. 2005 using a different mutant (S99A vs S99F)","pmids":["15913552"],"is_preprint":false},{"year":2007,"finding":"The S99A-Synip mutant (like S99F) inhibits insulin-stimulated GLUT4 translocation and is incapable of undergoing insulin-stimulated syntaxin 4 dissociation when overexpressed, supporting the requirement for S99 phosphorylation in GLUT4 translocation and contradicting the Sano et al. 2005 finding.","method":"Overexpression of S99A and S99F Synip mutants in 3T3L1 adipocytes, GLUT4 translocation and syntaxin 4 dissociation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab replication, single method; resolves discrepancy between prior studies but remains controversial","pmids":["17336927"],"is_preprint":false},{"year":2008,"finding":"The N-terminal 1–28 residues of syntaxin 4 are dispensable for interaction with Synip; the Synip C-terminal domain and syntaxin 4 can be co-expressed and co-purified at ~1:1 molar ratio, biochemically confirming direct binary complex formation.","method":"Recombinant co-expression in E. coli, co-purification, biochemical interaction assay with deletion mutants","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro reconstitution of the complex with deletion mapping, single lab, single study","pmids":["18439908"],"is_preprint":false},{"year":2009,"finding":"STXBP4 (Synip) stabilizes ΔNp63 proteins by preventing RACK1-mediated proteasomal degradation; under normal growth conditions STXBP4 is required to maintain high basal ΔNp63 levels, and upon genotoxic stress STXBP4 itself is downregulated, permitting RACK1-mediated ΔNp63 destabilization.","method":"Co-immunoprecipitation of STXBP4 with ΔNp63 and RACK1, siRNA knockdown of STXBP4, pulse-chase and cycloheximide-chase protein stability assays, overexpression experiments in epithelial cells","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, siRNA loss-of-function, protein stability assays, multiple orthogonal methods in single lab","pmids":["19451233"],"is_preprint":false},{"year":2012,"finding":"Synip (STXBP4) binds phosphatidylinositol (3,4,5)-trisphosphate (PIP3) but not PIP or PIP2, and this binding requires the WW domain; deletion of the WW domain (SynipΔWW) reduces basal plasma membrane GLUT4 levels and prevents insulin-stimulated plasma membrane recruitment of Synip, suggesting that in the insulin-stimulated state, dissociated Synip remains anchored at the plasma membrane via PIP3.","method":"Lipid-binding assay with phosphoinositide strips, WW domain deletion mutant, subcellular fractionation, overexpression in 3T3L1 adipocytes","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — lipid binding with domain deletion plus cell fractionation, single lab, single study","pmids":["22880106"],"is_preprint":false},{"year":2013,"finding":"Synip (STXBP4) inhibits SNARE-dependent vesicle docking, lipid mixing, and content mixing in a reconstituted GLUT4 exocytosis fusion assay; it arrests fusion by binding the t-SNARE complex (syntaxin 4 alone and the syntaxin 4/SNAP-23 complex) to prevent initiation of ternary SNARE complex assembly, and this inhibitory activity is dominant over the stimulatory activity of Sec1/Munc18 proteins; inhibition is selective for cognate (GLUT4 exocytic) SNAREs.","method":"In vitro reconstituted SNARE-dependent vesicle fusion assay with purified components, lipid-mixing and content-mixing assays, docking assay, binding assays with SNARE proteins, Sec1/Munc18 competition assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — full reconstitution with purified components, multiple orthogonal readouts (docking, lipid mixing, content mixing), mechanistic dissection of inhibitory step; single lab but multiple independent assays","pmids":["23665562"],"is_preprint":false},{"year":2018,"finding":"STXBP4 suppresses APC/C-mediated ubiquitin-dependent degradation of ΔNp63α during the M-G1 phase; overexpression of STXBP4 inhibits terminal differentiation in 3D organotypic epidermal cultures and confers oncogenic activity in soft agar and xenograft assays, placing STXBP4 as a positive regulator of ΔNp63α stability by blocking APC/C-mediated proteolysis.","method":"Co-immunoprecipitation of STXBP4 with APC/C components and ΔNp63α, ubiquitination assays, 3D organotypic culture differentiation assay, soft agar colony formation and xenograft tumor assays, APC/C-resistant ΔNp63α mutant","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, multiple cell-based functional assays, in vivo xenograft, single lab with multiple orthogonal methods","pmids":["29735662"],"is_preprint":false},{"year":2019,"finding":"STXBP4 acts as a negative regulator (inhibitor) of YAP in the Hippo pathway by assembling a protein complex comprising α-catenin and Hippo PY motif-containing components/regulators through its WW domain; this inhibitory function is regulated by actin cytoskeleton tension, and STXBP4 interacts with YAP via WW domain–PY motif binding.","method":"WW domain binding specificity mapping, Co-immunoprecipitation of STXBP4 with α-catenin and Hippo pathway components, YAP activity reporter assays, actin cytoskeleton perturbation experiments, loss-of-function in kidney cancer cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, binding specificity assays, reporter functional assays, and actin manipulation experiments; single lab with multiple orthogonal methods","pmids":["31782549"],"is_preprint":false}],"current_model":"STXBP4 (Synip) is a multifunctional scaffold protein that: (1) inhibits GLUT4 vesicle exocytosis by binding the t-SNARE syntaxin 4 and blocking ternary SNARE complex assembly, with insulin-stimulated Akt2-mediated phosphorylation at S99 and PIP3 binding via its WW domain regulating its dissociation from syntaxin 4 and plasma membrane anchoring; (2) stabilizes ΔNp63α in stratified epithelial cells by blocking RACK1-mediated and APC/C-mediated ubiquitin-dependent degradation, with STXBP4 downregulation upon genotoxic stress permitting ΔNp63 destabilization; and (3) inhibits YAP oncogenic activity in the Hippo pathway by assembling an α-catenin-containing complex through WW domain–PY motif interactions in an actin cytoskeleton tension-dependent manner."},"narrative":{"mechanistic_narrative":"STXBP4 (Synip) is a multidomain scaffold protein that controls vesicular exocytosis, epithelial transcription-factor stability, and Hippo-pathway signaling through distinct domain-mediated protein interactions [PMID:10394363, PMID:19451233, PMID:31782549]. In its best-characterized role, STXBP4 selectively binds the t-SNARE syntaxin 4 and inhibits GLUT4 vesicle translocation and glucose transport; insulin stimulation reduces this binding affinity and releases the complex [PMID:10394363]. Reconstitution with purified components established that STXBP4 arrests fusion by engaging syntaxin 4 and the syntaxin 4/SNAP-23 t-SNARE complex to block ternary SNARE assembly, an inhibitory activity dominant over Sec1/Munc18 stimulation and selective for cognate GLUT4 exocytic SNAREs [PMID:23665562]. The same syntaxin 4-binding activity restrains VAMP2 association and suppresses glucose-stimulated insulin secretion in pancreatic beta cells [PMID:12855681]. Insulin-dependent regulation involves Akt2-specific phosphorylation at serine 99 and WW-domain-mediated binding of PIP3, which together govern dissociation from syntaxin 4 and plasma-membrane anchoring of the released protein [PMID:15753124, PMID:22880106]; whether S99 phosphorylation is strictly required for GLUT4 translocation is contradicted across studies using S99A versus S99F mutants [PMID:15913552, PMID:17336927]. Independent of its SNARE role, STXBP4 stabilizes ΔNp63α in epithelial cells by blocking both RACK1-mediated and APC/C-mediated ubiquitin-dependent degradation, maintaining basal ΔNp63 levels that are lost when STXBP4 is downregulated upon genotoxic stress, and STXBP4 overexpression blocks terminal differentiation and confers oncogenic activity [PMID:19451233, PMID:29735662]. STXBP4 also inhibits YAP in the Hippo pathway by assembling an α-catenin-containing complex via WW-domain–PY-motif interactions in an actin-tension-dependent manner [PMID:31782549].","teleology":[{"year":1999,"claim":"Established STXBP4/Synip as a syntaxin 4-specific binding partner that gates GLUT4 exocytosis and responds to insulin, defining its founding function.","evidence":"Yeast two-hybrid, reciprocal Co-IP, and dominant-negative C-terminal overexpression with GLUT4 translocation assay in adipocytes","pmids":["10394363"],"confidence":"High","gaps":["Did not define the domain or structural basis of syntaxin 4 selectivity","Mechanism of insulin-induced affinity reduction unresolved"]},{"year":2003,"claim":"Extended STXBP4 function beyond adipocytes by showing it suppresses glucose-stimulated insulin secretion in beta cells through syntaxin 4 binding that blocks VAMP2 association.","evidence":"Co-IP and wild-type versus binding-deficient (ΔEF) mutant overexpression with insulin secretion assays in betaHC-9 cells","pmids":["12855681"],"confidence":"High","gaps":["Endogenous loss-of-function not tested","Did not address whether beta-cell regulation is also insulin/Akt-dependent"]},{"year":2005,"claim":"Identified Akt2-specific phosphorylation of STXBP4 at S99 as the proposed insulin-regulated switch for syntaxin 4 dissociation and GLUT4 recruitment.","evidence":"In vitro kinase assay with Akt1/2/3 and S99F dominant-negative overexpression in 3T3L1 adipocytes","pmids":["15753124"],"confidence":"Medium","gaps":["Relies on a dominant-negative phospho-mutant rather than endogenous phospho-detection","Directly contradicted by an S99A study the same year"]},{"year":2007,"claim":"Attempted to resolve the S99 controversy, with one study reporting S99A has no effect and another reporting both S99A and S99F block translocation and syntaxin 4 dissociation.","evidence":"Overexpression of S99A (and S99F) mutants in 3T3L1 adipocytes with GLUT4 translocation and syntaxin 4 dissociation assays (two opposing single-lab studies)","pmids":["15913552","17336927"],"confidence":"Medium","gaps":["Discrepancy between S99A datasets remains unreconciled","Single method, overexpression-based; no endogenous phospho-site validation"]},{"year":2008,"claim":"Provided biochemical confirmation that the STXBP4 C-terminal domain and syntaxin 4 form a direct ~1:1 binary complex, mapping the N-terminal syntaxin 4 region as dispensable.","evidence":"Recombinant co-expression in E. coli, co-purification, and deletion-mapping interaction assays","pmids":["18439908"],"confidence":"Medium","gaps":["No high-resolution structure of the complex","Interaction interface on STXBP4 not defined at residue level"]},{"year":2009,"claim":"Revealed a SNARE-independent role: STXBP4 stabilizes ΔNp63 by preventing RACK1-mediated proteasomal degradation, linking it to epithelial transcription-factor control and genotoxic stress responses.","evidence":"Co-IP with ΔNp63 and RACK1, STXBP4 siRNA knockdown, and cycloheximide/pulse-chase stability assays in epithelial cells","pmids":["19451233"],"confidence":"High","gaps":["Did not define how genotoxic stress downregulates STXBP4","Domain mediating ΔNp63/RACK1 interaction not mapped"]},{"year":2012,"claim":"Defined the WW domain as a PIP3-binding module that anchors dissociated STXBP4 at the plasma membrane and sustains basal GLUT4 levels.","evidence":"Phosphoinositide strip lipid-binding assay, WW-domain deletion mutant, and subcellular fractionation in 3T3L1 adipocytes","pmids":["22880106"],"confidence":"Medium","gaps":["PIP3 binding affinity and stoichiometry not quantified","Relationship between PIP3 anchoring and S99 phosphorylation untested"]},{"year":2013,"claim":"Established the mechanistic step of fusion inhibition by reconstitution, showing STXBP4 binds the t-SNARE to block ternary complex initiation, dominant over Munc18 stimulation and selective for cognate SNAREs.","evidence":"In vitro reconstituted vesicle fusion with purified components; docking, lipid-mixing, content-mixing, SNARE binding, and Sec1/Munc18 competition assays","pmids":["23665562"],"confidence":"High","gaps":["Insulin/Akt regulation not incorporated into the reconstituted system","Structural basis of t-SNARE engagement not resolved"]},{"year":2018,"claim":"Showed STXBP4 also blocks APC/C-mediated ΔNp63α degradation during M-G1, linking STXBP4 to differentiation control and oncogenic transformation.","evidence":"Co-IP with APC/C components and ΔNp63α, ubiquitination assays, 3D organotypic cultures, soft agar and xenograft assays, APC/C-resistant ΔNp63α mutant","pmids":["29735662"],"confidence":"High","gaps":["How STXBP4 mechanistically shields ΔNp63α from the APC/C is not defined","Relationship between RACK1- and APC/C-mediated pathways unclear"]},{"year":2019,"claim":"Defined a third role as a Hippo-pathway YAP inhibitor that assembles an α-catenin complex via WW-domain–PY-motif binding under actin-tension control.","evidence":"WW-domain binding specificity mapping, Co-IP with α-catenin and Hippo components, YAP reporter assays, and actin perturbation in kidney cancer cells","pmids":["31782549"],"confidence":"High","gaps":["How actin tension is transduced to STXBP4 not defined","Relationship to its SNARE and ΔNp63 functions unexplored"]},{"year":null,"claim":"It remains unknown how STXBP4's distinct scaffolding functions across membrane fusion, transcription-factor stability, and Hippo signaling are coordinated within a single protein and whether they operate in the same or distinct cellular contexts.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of full-length STXBP4 or its complexes","S99 phosphorylation requirement for GLUT4 translocation remains contradicted","No unifying model linking WW-domain usage across PIP3, YAP/PY-motif, and ΔNp63 functions"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,8,10]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,6,9,10]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[7]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[6,9]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,7]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,8]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[10]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[6,9]}],"complexes":[],"partners":["STX4","SNAP23","VAMP2","AKT2","TP63","RACK1","CTNNA1","YAP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q6ZWJ1","full_name":"Syntaxin-binding protein 4","aliases":["Syntaxin 4-interacting protein","STX4-interacting protein","Synip"],"length_aa":553,"mass_kda":61.7,"function":"Plays a role in the translocation of transport vesicles from the cytoplasm to the plasma membrane. Inhibits the translocation of SLC2A4 from intracellular vesicles to the plasma membrane by STX4A binding and preventing the interaction between STX4A and VAMP2. Stimulation with insulin disrupts the interaction with STX4A, leading to increased levels of SLC2A4 at the plasma membrane. May also play a role in the regulation of insulin release by pancreatic beta cells after stimulation by glucose (By similarity)","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q6ZWJ1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/STXBP4","classification":"Not Classified","n_dependent_lines":2,"n_total_lines":1208,"dependency_fraction":0.0016556291390728477},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/STXBP4","total_profiled":1310},"omim":[{"mim_id":"610415","title":"SYNTAXIN-BINDING PROTEIN 4; STXBP4","url":"https://www.omim.org/entry/610415"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Plasma membrane","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/STXBP4"},"hgnc":{"alias_symbol":["Synip","MGC50337"],"prev_symbol":[]},"alphafold":{"accession":"Q6ZWJ1","domains":[{"cath_id":"2.30.42.10","chopping":"21-107","consensus_level":"high","plddt":90.4548,"start":21,"end":107},{"cath_id":"-","chopping":"215-271","consensus_level":"high","plddt":74.8604,"start":215,"end":271},{"cath_id":"2.20.70.10","chopping":"500-533","consensus_level":"medium","plddt":80.4835,"start":500,"end":533}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZWJ1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZWJ1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6ZWJ1-F1-predicted_aligned_error_v6.png","plddt_mean":69.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=STXBP4","jax_strain_url":"https://www.jax.org/strain/search?query=STXBP4"},"sequence":{"accession":"Q6ZWJ1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6ZWJ1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6ZWJ1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6ZWJ1"}},"corpus_meta":[{"pmid":"10394363","id":"PMC_10394363","title":"Synip: a novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes.","date":"1999","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/10394363","citation_count":162,"is_preprint":false},{"pmid":"15753124","id":"PMC_15753124","title":"Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles.","date":"2005","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/15753124","citation_count":72,"is_preprint":false},{"pmid":"19451233","id":"PMC_19451233","title":"Stxbp4 regulates DeltaNp63 stability by suppression of RACK1-dependent degradation.","date":"2009","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19451233","citation_count":42,"is_preprint":false},{"pmid":"12855681","id":"PMC_12855681","title":"Syntaxin 4 and Synip (syntaxin 4 interacting protein) regulate insulin secretion in the pancreatic beta HC-9 cell.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12855681","citation_count":35,"is_preprint":false},{"pmid":"31539133","id":"PMC_31539133","title":"Long noncoding RNA LINC00511 involves in breast cancer recurrence and radioresistance by regulating STXBP4 expression via miR-185.","date":"2019","source":"European review for medical and pharmacological sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31539133","citation_count":35,"is_preprint":false},{"pmid":"15913552","id":"PMC_15913552","title":"Synip phosphorylation does not regulate insulin-stimulated GLUT4 translocation.","date":"2005","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/15913552","citation_count":29,"is_preprint":false},{"pmid":"31782549","id":"PMC_31782549","title":"Elucidation of WW domain ligand binding specificities in the Hippo pathway reveals STXBP4 as YAP inhibitor.","date":"2019","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/31782549","citation_count":26,"is_preprint":false},{"pmid":"29735662","id":"PMC_29735662","title":"STXBP4 regulates APC/C-mediated p63 turnover and drives squamous cell carcinogenesis.","date":"2018","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/29735662","citation_count":22,"is_preprint":false},{"pmid":"23665562","id":"PMC_23665562","title":"Synip arrests soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent membrane fusion as a selective target membrane SNARE-binding inhibitor.","date":"2013","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23665562","citation_count":21,"is_preprint":false},{"pmid":"17336927","id":"PMC_17336927","title":"Synip phosphorylation is required for insulin-stimulated Glut4 translocation.","date":"2007","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17336927","citation_count":18,"is_preprint":false},{"pmid":"28422318","id":"PMC_28422318","title":"Computational Analysis of Breast Cancer GWAS Loci Identifies the Putative Deleterious Effect of STXBP4 and ZNF404 Gene Variants.","date":"2017","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/28422318","citation_count":14,"is_preprint":false},{"pmid":"22880106","id":"PMC_22880106","title":"Syntaxin4 interacting protein (Synip) binds phosphatidylinositol (3,4,5) triphosphate.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22880106","citation_count":6,"is_preprint":false},{"pmid":"18439908","id":"PMC_18439908","title":"An efficient co-expression and purification system for the complex of Stx4 and C-terminal domain of Synip.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18439908","citation_count":5,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8163,"output_tokens":3290,"usd":0.03692,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10652,"output_tokens":3819,"usd":0.074368,"stage2_stop_reason":"end_turn"},"total_usd":0.111288,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"Synip (STXBP4) specifically binds syntaxin 4 (but not other syntaxins) and inhibits GLUT4 vesicle translocation; insulin stimulation causes dissociation of the Synip:syntaxin 4 complex through reduced binding affinity, and overexpression of the C-terminal domain of Synip (which does not dissociate from syntaxin 4 upon insulin stimulation) inhibits glucose transport and GLUT4 translocation.\",\n      \"method\": \"Co-immunoprecipitation, yeast two-hybrid, dominant-negative overexpression in adipocytes with GLUT4 translocation assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, functional dominant-negative overexpression, independently replicated across multiple subsequent labs\",\n      \"pmids\": [\"10394363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Synip (STXBP4) is expressed in pancreatic beta cells, co-immunoprecipitates with syntaxin 4 but not syntaxin 1, and overexpression of full-length Synip inhibits VAMP2 association with syntaxin 4 and suppresses both first and second phase glucose-stimulated insulin secretion; a Synip mutant (ΔEF) unable to bind syntaxin 4 had no effect, confirming the mechanism requires syntaxin 4 binding.\",\n      \"method\": \"Co-immunoprecipitation, overexpression of wild-type and binding-deficient mutant Synip in betaHC-9 cells, insulin secretion assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus mutant rescue experiment in a defined cellular system, single lab with multiple orthogonal readouts\",\n      \"pmids\": [\"12855681\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Akt2 (but not Akt1 or Akt3) specifically phosphorylates Synip at serine 99; insulin-stimulated phosphorylation at S99 is required for dissociation of Synip from syntaxin 4 and for GLUT4 vesicle recruitment and plasma membrane fusion; the S99F phosphorylation-resistant mutant dominantly interferes with insulin-stimulated GLUT4 translocation.\",\n      \"method\": \"In vitro kinase assay with Akt1/2/3, phospho-specific mutagenesis (S99F), overexpression in 3T3L1 adipocytes, GLUT4 translocation assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Weak — in vitro kinase assay plus dominant-negative cell experiment, single lab; finding contradicted by Sano et al. 2005 using S99A mutant\",\n      \"pmids\": [\"15753124\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Overexpression of the Synip S99A phosphorylation-site mutant (lacking serine 99) has no effect on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, providing evidence against the requirement for S99 phosphorylation in GLUT4 translocation (negative/contradictory finding relative to Yamada et al. 2005).\",\n      \"method\": \"Overexpression of S99A mutant Synip in 3T3-L1 adipocytes, GLUT4 translocation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, single method, directly contradicts Yamada et al. 2005 using a different mutant (S99A vs S99F)\",\n      \"pmids\": [\"15913552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The S99A-Synip mutant (like S99F) inhibits insulin-stimulated GLUT4 translocation and is incapable of undergoing insulin-stimulated syntaxin 4 dissociation when overexpressed, supporting the requirement for S99 phosphorylation in GLUT4 translocation and contradicting the Sano et al. 2005 finding.\",\n      \"method\": \"Overexpression of S99A and S99F Synip mutants in 3T3L1 adipocytes, GLUT4 translocation and syntaxin 4 dissociation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab replication, single method; resolves discrepancy between prior studies but remains controversial\",\n      \"pmids\": [\"17336927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The N-terminal 1–28 residues of syntaxin 4 are dispensable for interaction with Synip; the Synip C-terminal domain and syntaxin 4 can be co-expressed and co-purified at ~1:1 molar ratio, biochemically confirming direct binary complex formation.\",\n      \"method\": \"Recombinant co-expression in E. coli, co-purification, biochemical interaction assay with deletion mutants\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro reconstitution of the complex with deletion mapping, single lab, single study\",\n      \"pmids\": [\"18439908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"STXBP4 (Synip) stabilizes ΔNp63 proteins by preventing RACK1-mediated proteasomal degradation; under normal growth conditions STXBP4 is required to maintain high basal ΔNp63 levels, and upon genotoxic stress STXBP4 itself is downregulated, permitting RACK1-mediated ΔNp63 destabilization.\",\n      \"method\": \"Co-immunoprecipitation of STXBP4 with ΔNp63 and RACK1, siRNA knockdown of STXBP4, pulse-chase and cycloheximide-chase protein stability assays, overexpression experiments in epithelial cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, siRNA loss-of-function, protein stability assays, multiple orthogonal methods in single lab\",\n      \"pmids\": [\"19451233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Synip (STXBP4) binds phosphatidylinositol (3,4,5)-trisphosphate (PIP3) but not PIP or PIP2, and this binding requires the WW domain; deletion of the WW domain (SynipΔWW) reduces basal plasma membrane GLUT4 levels and prevents insulin-stimulated plasma membrane recruitment of Synip, suggesting that in the insulin-stimulated state, dissociated Synip remains anchored at the plasma membrane via PIP3.\",\n      \"method\": \"Lipid-binding assay with phosphoinositide strips, WW domain deletion mutant, subcellular fractionation, overexpression in 3T3L1 adipocytes\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — lipid binding with domain deletion plus cell fractionation, single lab, single study\",\n      \"pmids\": [\"22880106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Synip (STXBP4) inhibits SNARE-dependent vesicle docking, lipid mixing, and content mixing in a reconstituted GLUT4 exocytosis fusion assay; it arrests fusion by binding the t-SNARE complex (syntaxin 4 alone and the syntaxin 4/SNAP-23 complex) to prevent initiation of ternary SNARE complex assembly, and this inhibitory activity is dominant over the stimulatory activity of Sec1/Munc18 proteins; inhibition is selective for cognate (GLUT4 exocytic) SNAREs.\",\n      \"method\": \"In vitro reconstituted SNARE-dependent vesicle fusion assay with purified components, lipid-mixing and content-mixing assays, docking assay, binding assays with SNARE proteins, Sec1/Munc18 competition assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — full reconstitution with purified components, multiple orthogonal readouts (docking, lipid mixing, content mixing), mechanistic dissection of inhibitory step; single lab but multiple independent assays\",\n      \"pmids\": [\"23665562\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"STXBP4 suppresses APC/C-mediated ubiquitin-dependent degradation of ΔNp63α during the M-G1 phase; overexpression of STXBP4 inhibits terminal differentiation in 3D organotypic epidermal cultures and confers oncogenic activity in soft agar and xenograft assays, placing STXBP4 as a positive regulator of ΔNp63α stability by blocking APC/C-mediated proteolysis.\",\n      \"method\": \"Co-immunoprecipitation of STXBP4 with APC/C components and ΔNp63α, ubiquitination assays, 3D organotypic culture differentiation assay, soft agar colony formation and xenograft tumor assays, APC/C-resistant ΔNp63α mutant\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, multiple cell-based functional assays, in vivo xenograft, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"29735662\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"STXBP4 acts as a negative regulator (inhibitor) of YAP in the Hippo pathway by assembling a protein complex comprising α-catenin and Hippo PY motif-containing components/regulators through its WW domain; this inhibitory function is regulated by actin cytoskeleton tension, and STXBP4 interacts with YAP via WW domain–PY motif binding.\",\n      \"method\": \"WW domain binding specificity mapping, Co-immunoprecipitation of STXBP4 with α-catenin and Hippo pathway components, YAP activity reporter assays, actin cytoskeleton perturbation experiments, loss-of-function in kidney cancer cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, binding specificity assays, reporter functional assays, and actin manipulation experiments; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"31782549\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"STXBP4 (Synip) is a multifunctional scaffold protein that: (1) inhibits GLUT4 vesicle exocytosis by binding the t-SNARE syntaxin 4 and blocking ternary SNARE complex assembly, with insulin-stimulated Akt2-mediated phosphorylation at S99 and PIP3 binding via its WW domain regulating its dissociation from syntaxin 4 and plasma membrane anchoring; (2) stabilizes ΔNp63α in stratified epithelial cells by blocking RACK1-mediated and APC/C-mediated ubiquitin-dependent degradation, with STXBP4 downregulation upon genotoxic stress permitting ΔNp63 destabilization; and (3) inhibits YAP oncogenic activity in the Hippo pathway by assembling an α-catenin-containing complex through WW domain–PY motif interactions in an actin cytoskeleton tension-dependent manner.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"STXBP4 (Synip) is a multidomain scaffold protein that controls vesicular exocytosis, epithelial transcription-factor stability, and Hippo-pathway signaling through distinct domain-mediated protein interactions [#0, #6, #10]. In its best-characterized role, STXBP4 selectively binds the t-SNARE syntaxin 4 and inhibits GLUT4 vesicle translocation and glucose transport; insulin stimulation reduces this binding affinity and releases the complex [#0]. Reconstitution with purified components established that STXBP4 arrests fusion by engaging syntaxin 4 and the syntaxin 4/SNAP-23 t-SNARE complex to block ternary SNARE assembly, an inhibitory activity dominant over Sec1/Munc18 stimulation and selective for cognate GLUT4 exocytic SNAREs [#8]. The same syntaxin 4-binding activity restrains VAMP2 association and suppresses glucose-stimulated insulin secretion in pancreatic beta cells [#1]. Insulin-dependent regulation involves Akt2-specific phosphorylation at serine 99 and WW-domain-mediated binding of PIP3, which together govern dissociation from syntaxin 4 and plasma-membrane anchoring of the released protein [#2, #7]; whether S99 phosphorylation is strictly required for GLUT4 translocation is contradicted across studies using S99A versus S99F mutants [#3, #4]. Independent of its SNARE role, STXBP4 stabilizes \\u0394Np63\\u03b1 in epithelial cells by blocking both RACK1-mediated and APC/C-mediated ubiquitin-dependent degradation, maintaining basal \\u0394Np63 levels that are lost when STXBP4 is downregulated upon genotoxic stress, and STXBP4 overexpression blocks terminal differentiation and confers oncogenic activity [#6, #9]. STXBP4 also inhibits YAP in the Hippo pathway by assembling an \\u03b1-catenin-containing complex via WW-domain\\u2013PY-motif interactions in an actin-tension-dependent manner [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Established STXBP4/Synip as a syntaxin 4-specific binding partner that gates GLUT4 exocytosis and responds to insulin, defining its founding function.\",\n      \"evidence\": \"Yeast two-hybrid, reciprocal Co-IP, and dominant-negative C-terminal overexpression with GLUT4 translocation assay in adipocytes\",\n      \"pmids\": [\"10394363\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the domain or structural basis of syntaxin 4 selectivity\", \"Mechanism of insulin-induced affinity reduction unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended STXBP4 function beyond adipocytes by showing it suppresses glucose-stimulated insulin secretion in beta cells through syntaxin 4 binding that blocks VAMP2 association.\",\n      \"evidence\": \"Co-IP and wild-type versus binding-deficient (\\u0394EF) mutant overexpression with insulin secretion assays in betaHC-9 cells\",\n      \"pmids\": [\"12855681\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous loss-of-function not tested\", \"Did not address whether beta-cell regulation is also insulin/Akt-dependent\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified Akt2-specific phosphorylation of STXBP4 at S99 as the proposed insulin-regulated switch for syntaxin 4 dissociation and GLUT4 recruitment.\",\n      \"evidence\": \"In vitro kinase assay with Akt1/2/3 and S99F dominant-negative overexpression in 3T3L1 adipocytes\",\n      \"pmids\": [\"15753124\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relies on a dominant-negative phospho-mutant rather than endogenous phospho-detection\", \"Directly contradicted by an S99A study the same year\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Attempted to resolve the S99 controversy, with one study reporting S99A has no effect and another reporting both S99A and S99F block translocation and syntaxin 4 dissociation.\",\n      \"evidence\": \"Overexpression of S99A (and S99F) mutants in 3T3L1 adipocytes with GLUT4 translocation and syntaxin 4 dissociation assays (two opposing single-lab studies)\",\n      \"pmids\": [\"15913552\", \"17336927\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Discrepancy between S99A datasets remains unreconciled\", \"Single method, overexpression-based; no endogenous phospho-site validation\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Provided biochemical confirmation that the STXBP4 C-terminal domain and syntaxin 4 form a direct ~1:1 binary complex, mapping the N-terminal syntaxin 4 region as dispensable.\",\n      \"evidence\": \"Recombinant co-expression in E. coli, co-purification, and deletion-mapping interaction assays\",\n      \"pmids\": [\"18439908\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No high-resolution structure of the complex\", \"Interaction interface on STXBP4 not defined at residue level\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealed a SNARE-independent role: STXBP4 stabilizes \\u0394Np63 by preventing RACK1-mediated proteasomal degradation, linking it to epithelial transcription-factor control and genotoxic stress responses.\",\n      \"evidence\": \"Co-IP with \\u0394Np63 and RACK1, STXBP4 siRNA knockdown, and cycloheximide/pulse-chase stability assays in epithelial cells\",\n      \"pmids\": [\"19451233\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how genotoxic stress downregulates STXBP4\", \"Domain mediating \\u0394Np63/RACK1 interaction not mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the WW domain as a PIP3-binding module that anchors dissociated STXBP4 at the plasma membrane and sustains basal GLUT4 levels.\",\n      \"evidence\": \"Phosphoinositide strip lipid-binding assay, WW-domain deletion mutant, and subcellular fractionation in 3T3L1 adipocytes\",\n      \"pmids\": [\"22880106\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"PIP3 binding affinity and stoichiometry not quantified\", \"Relationship between PIP3 anchoring and S99 phosphorylation untested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established the mechanistic step of fusion inhibition by reconstitution, showing STXBP4 binds the t-SNARE to block ternary complex initiation, dominant over Munc18 stimulation and selective for cognate SNAREs.\",\n      \"evidence\": \"In vitro reconstituted vesicle fusion with purified components; docking, lipid-mixing, content-mixing, SNARE binding, and Sec1/Munc18 competition assays\",\n      \"pmids\": [\"23665562\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Insulin/Akt regulation not incorporated into the reconstituted system\", \"Structural basis of t-SNARE engagement not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Showed STXBP4 also blocks APC/C-mediated \\u0394Np63\\u03b1 degradation during M-G1, linking STXBP4 to differentiation control and oncogenic transformation.\",\n      \"evidence\": \"Co-IP with APC/C components and \\u0394Np63\\u03b1, ubiquitination assays, 3D organotypic cultures, soft agar and xenograft assays, APC/C-resistant \\u0394Np63\\u03b1 mutant\",\n      \"pmids\": [\"29735662\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How STXBP4 mechanistically shields \\u0394Np63\\u03b1 from the APC/C is not defined\", \"Relationship between RACK1- and APC/C-mediated pathways unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Defined a third role as a Hippo-pathway YAP inhibitor that assembles an \\u03b1-catenin complex via WW-domain\\u2013PY-motif binding under actin-tension control.\",\n      \"evidence\": \"WW-domain binding specificity mapping, Co-IP with \\u03b1-catenin and Hippo components, YAP reporter assays, and actin perturbation in kidney cancer cells\",\n      \"pmids\": [\"31782549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How actin tension is transduced to STXBP4 not defined\", \"Relationship to its SNARE and \\u0394Np63 functions unexplored\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how STXBP4's distinct scaffolding functions across membrane fusion, transcription-factor stability, and Hippo signaling are coordinated within a single protein and whether they operate in the same or distinct cellular contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of full-length STXBP4 or its complexes\", \"S99 phosphorylation requirement for GLUT4 translocation remains contradicted\", \"No unifying model linking WW-domain usage across PIP3, YAP/PY-motif, and \\u0394Np63 functions\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 8, 10]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 6, 9, 10]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [6, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [6, 9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"STX4\", \"SNAP23\", \"VAMP2\", \"AKT2\", \"TP63\", \"RACK1\", \"CTNNA1\", \"YAP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}