{"gene":"CASKIN1","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2011,"finding":"A short linear EEIWVLRK peptide motif from Caskin1 is necessary and sufficient for binding the CASK calmodulin kinase domain. This motif competes with a related peptide from Mint1 (EPIWVMRQ) for the same conserved binding site on CASK, explaining how Caskin1 and Mint1 form mutually exclusive competing complexes with CASK.","method":"Peptide binding assays, site-directed mutagenesis of CASK binding site, in vitro binding experiments","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with defined peptide motif and mutagenesis of the binding site, two orthogonal methods, single lab","pmids":["21763699"],"is_preprint":false},{"year":2011,"finding":"The SAM domain of Caskin1 forms homopolymers, as identified by a native gel screen using negGFP-SAM fusions and confirmed by electron microscopy.","method":"Native gel electrophoresis with negGFP-SAM fusions, electron microscopy","journal":"Protein science : a publication of the Protein Society","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (native gel + EM), but a screening study with limited mechanistic depth for Caskin1 specifically","pmids":["21805519"],"is_preprint":false},{"year":2012,"finding":"EphB1 receptor tyrosine kinase recruits Caskin1 through the adaptor protein Nck: Nck's SH2 domain binds to a phosphotyrosine on EphB1, while Nck's SH3 domains interact with the proline-rich domain of Caskin1. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain at tyrosines Y296 and Y336, which causes significant structural changes in the SH3 domain as measured by CD spectroscopy.","method":"Co-immunoprecipitation, mass spectrometry (phosphosite identification), CD spectroscopy","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, MS-identified phosphosites, and CD spectroscopy for structural consequence; single lab, multiple orthogonal methods","pmids":["23181695"],"is_preprint":false},{"year":2014,"finding":"Caskin1 localizes to a subset of CASK-positive synapses in the bovine retina, distinct from CASK which is present in virtually all retinal synapses; Caskin1 is enriched at only a subset of ribbon and conventional synapses, suggesting specialized rather than universal synaptic functions.","method":"Immunohistochemistry, fractionation/subcellular localization in bovine retina","journal":"Molecular and cellular neurosciences","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct immunolocalization in tissue, replicated with multiple antibodies; no functional consequence directly demonstrated","pmids":["25123431"],"is_preprint":false},{"year":2017,"finding":"The atypical SH3 domain of human Caskin1 selectively binds lysophosphatidic acid (LPA) in vitro, with nanomolar affinity to LPA-containing membranous surfaces. The binding involves beta-strand residues distinct from the canonical proline-rich ligand-binding groove. No proline-rich protein interacting partner for this SH3 domain was identified.","method":"In vitro lipid-binding assays, NMR/structural analysis of binding interface","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro binding assay with defined binding site and mutagenesis-informed analysis; single lab, later confirmed by NMR structure","pmids":["28104445"],"is_preprint":false},{"year":2018,"finding":"Caskin1 localizes primarily at synapses throughout the brain and spinal cord. Caskin1-knockout mice exhibit enhanced nociception, anxiety-like behavior, increased fear conditioning, and impaired spatial memory, establishing that Caskin1 contributes to nociception, memory, and stress response in the CNS.","method":"Caskin1-KO mouse generation, comprehensive behavioral test battery, immunohistochemistry, biochemical fractionation","journal":"Molecular brain","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO with defined behavioral phenotypes and synaptic localization confirmed by immunohistochemistry; single lab, multiple behavioral assays","pmids":["30359304"],"is_preprint":false},{"year":2019,"finding":"Caskin1 is enriched in dendritic spine heads postsynaptically and increases mushroom-shaped dendritic spine formation when overexpressed in hippocampal neurons. Caskin1 co-immunoprecipitates and co-localizes with the postsynaptic scaffold protein Shank2. Loss of Caskin proteins (double KO of Caskin1 and Caskin2) reduces synaptic profiles and dendritic spine area, impairs LTP, alters AMPA receptor phosphorylation, and causes deficits in novelty recognition and spatial memory.","method":"Double knockout mice, LTP recordings in hippocampal slices, immunoprecipitation, immunocytochemistry, ultrastructural analysis, overexpression in cultured neurons","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (KO, electrophysiology, Co-IP, ultrastructure, live imaging), replicated across multiple assays in single study","pmids":["31727973"],"is_preprint":false},{"year":2021,"finding":"Solution NMR structure of the human Caskin1 SH3 domain reveals that the canonical proline-rich peptide binding groove is absent due to missing key aromatic residues, supporting the conclusion that this SH3 domain does not bind proline-rich protein motifs. The LPA binding site is structurally distinct from the altered protein-binding groove.","method":"Solution NMR structure determination","journal":"Cells","confidence":"High","confidence_rationale":"Tier 1 / Moderate — atomic-resolution NMR structure with functional validation; confirms prior biochemical findings; single lab","pmids":["33467043"],"is_preprint":false},{"year":2024,"finding":"SASH1 was identified as a novel binding partner of Caskin1, interacting via SAM-SAM domain interaction at the end-helix (EH)/mid-loop (ML) interface. This heterogeneous SAM-SAM interaction disrupts Caskin1 tandem SAM homopolymers, as validated by sedimentation, TEM, co-IP, GST pulldown, and immunofluorescence. Key interface residues were identified by mutagenesis of AlphaFold2-predicted complex models.","method":"Yeast two-hybrid screening, size-exclusion chromatography, isothermal titration calorimetry, GST pulldown, co-immunoprecipitation, transmission electron microscopy, sedimentation assay, mutagenesis, immunofluorescence","journal":"The FEBS journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal biochemical and structural methods (ITC, SEC, TEM, Co-IP, mutagenesis) in single study establishing the interaction and its functional consequence on polymer disassembly","pmids":["39688081"],"is_preprint":false},{"year":2025,"finding":"Conditional knockout analysis in mice showed that CASKIN1 (unlike CASKIN2) is not critical for synaptic transmission, synaptic strength, or AZ protein arrangement at glutamatergic synapses. Combined CASKIN1/2 deletion recapitulates CASKIN2-cKO phenotypes, indicating CASKIN1 plays a redundant or secondary role at hippocampal glutamatergic synapses compared to CASKIN2.","method":"Conditional knockout mice, electrophysiology, synaptic transmission assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean conditional KO with defined electrophysiological readouts; CASKIN1-specific result is a negative finding within a study focused on CASKIN2","pmids":["41223222"],"is_preprint":false}],"current_model":"CASKIN1 is a multidomain neuronal scaffold protein that binds CASK via a conserved EEIWVLRK peptide motif competing with Mint1, localizes primarily to a subset of presynaptic and postsynaptic sites, forms SAM-domain-mediated homopolymers that can be disrupted by SASH1 through heterogeneous SAM-SAM interaction, contains an atypical SH3 domain (NMR-validated) that binds lysophosphatidic acid rather than proline-rich protein motifs, is recruited to the EphB1 receptor complex via Nck leading to phosphorylation of its SH3 domain at Y296/Y336 with attendant structural changes, and contributes postsynaptically to dendritic spine morphology, AMPA receptor localization, and LTP—with its loss (particularly combined with Caskin2) causing deficits in spatial memory and novelty recognition, while CASKIN2 rather than CASKIN1 appears to be the primary mediator of transsynaptic NMDAR-related functions at glutamatergic synapses."},"narrative":{"mechanistic_narrative":"CASKIN1 is a multidomain neuronal scaffold protein that organizes synaptic protein complexes and contributes to dendritic spine architecture and synaptic plasticity [PMID:31727973]. It engages the core synaptic scaffold CASK through a short EEIWVLRK peptide motif that binds the CASK CaMK domain and competes with a homologous Mint1 motif for the same site, establishing mutually exclusive CASK complexes [PMID:21763699]. CASKIN1 self-assembles through SAM-domain-mediated homopolymerization [PMID:21805519], an oligomeric state that SASH1 disrupts via a heterogeneous SAM-SAM interaction at the end-helix/mid-loop interface [PMID:39688081]. Its atypical SH3 domain lacks the aromatic residues that form the canonical proline-rich binding groove and instead selectively binds lysophosphatidic acid, as established by NMR structure and lipid-binding assays [PMID:28104445, PMID:33467043]. CASKIN1 is recruited to the EphB1 receptor tyrosine kinase via the adaptor Nck, which couples a phosphotyrosine on EphB1 to the CASKIN1 proline-rich region and triggers phosphorylation of its SH3 domain at Y296/Y336 with attendant conformational change [PMID:23181695]. Postsynaptically, CASKIN1 is enriched in dendritic spine heads, associates with Shank2, and promotes mushroom spine formation, with combined loss of Caskin1 and Caskin2 impairing LTP, altering AMPA receptor phosphorylation, and causing spatial memory and novelty-recognition deficits [PMID:31727973]; at hippocampal glutamatergic synapses CASKIN1 plays a secondary, redundant role relative to CASKIN2 in synaptic transmission [PMID:41223222].","teleology":[{"year":2011,"claim":"Defined how CASKIN1 docks onto the central synaptic scaffold CASK and why this is exclusive with Mint1, explaining competitive assembly of CASK complexes.","evidence":"Peptide binding assays and site-directed mutagenesis of the CASK CaMK-domain binding site in vitro","pmids":["21763699"],"confidence":"High","gaps":["Affinity and stoichiometry in a full-length cellular context not established","Functional consequence of CASK-Mint1-Caskin1 competition at synapses not demonstrated"]},{"year":2011,"claim":"Established that the CASKIN1 SAM domain self-associates into higher-order homopolymers, defining a structural basis for scaffold clustering.","evidence":"Native gel screen with negGFP-SAM fusions and electron microscopy","pmids":["21805519"],"confidence":"Medium","gaps":["Polymer geometry and physiological regulation not resolved","Screening study with limited Caskin1-specific depth"]},{"year":2012,"claim":"Showed CASKIN1 is an EphB1 receptor signaling effector, recruited through Nck and post-translationally modified in a way that remodels its SH3 domain.","evidence":"Reciprocal co-immunoprecipitation, mass-spectrometry phosphosite mapping, and CD spectroscopy","pmids":["23181695"],"confidence":"Medium","gaps":["Downstream signaling output of Y296/Y336 phosphorylation not defined","Single lab; in vivo relevance of EphB1-Nck-Caskin1 axis untested"]},{"year":2014,"claim":"Demonstrated CASKIN1 marks only a subset of CASK-positive synapses, implying specialized rather than universal synaptic functions.","evidence":"Immunohistochemistry and subcellular fractionation in bovine retina","pmids":["25123431"],"confidence":"Medium","gaps":["No functional consequence demonstrated","Molecular determinant of selective synaptic targeting unknown"]},{"year":2017,"claim":"Reassigned the CASKIN1 SH3 domain function from a protein-interaction module to a lipid sensor by showing selective high-affinity binding to lysophosphatidic acid.","evidence":"In vitro lipid-binding assays and structural analysis of the binding interface","pmids":["28104445"],"confidence":"Medium","gaps":["Cellular role of LPA binding not established","No proline-rich protein ligand identified"]},{"year":2018,"claim":"Established CASKIN1 as a CNS-wide synaptic protein with defined behavioral functions in nociception, anxiety, fear, and spatial memory.","evidence":"Caskin1-knockout mice, behavioral test battery, immunohistochemistry, fractionation","pmids":["30359304"],"confidence":"Medium","gaps":["Circuit and synaptic mechanism underlying behaviors not resolved","Single lab"]},{"year":2019,"claim":"Localized CASKIN1 to the postsynaptic spine head and linked it mechanistically to spine morphology, AMPA receptor regulation, and LTP via association with Shank2.","evidence":"Caskin1/2 double-knockout mice, LTP recordings, co-IP, immunocytochemistry, ultrastructure, and overexpression in cultured neurons","pmids":["31727973"],"confidence":"High","gaps":["Caskin1-specific versus Caskin2-shared contribution not separated","Mechanism linking Shank2 binding to AMPA receptor phosphorylation unresolved"]},{"year":2021,"claim":"Provided the atomic-resolution basis for why the CASKIN1 SH3 domain cannot bind proline-rich motifs and confirmed a distinct LPA binding site.","evidence":"Solution NMR structure determination of the human Caskin1 SH3 domain","pmids":["33467043"],"confidence":"High","gaps":["In vivo LPA engagement not demonstrated","Functional impact of Y296/Y336 phosphorylation on the structure not directly resolved here"]},{"year":2024,"claim":"Identified SASH1 as a regulator that disassembles CASKIN1 SAM homopolymers through a heterogeneous SAM-SAM interaction, defining a mechanism for controlling scaffold oligomerization.","evidence":"Yeast two-hybrid, SEC, ITC, GST pulldown, co-IP, TEM, sedimentation, mutagenesis guided by AlphaFold2 models, and immunofluorescence","pmids":["39688081"],"confidence":"High","gaps":["Physiological context and neuronal consequence of SASH1-driven depolymerization not established","Regulatory triggers for the interaction unknown"]},{"year":2025,"claim":"Resolved the relative contribution of CASKIN1 at glutamatergic synapses, showing it is dispensable for synaptic transmission and redundant with the dominant CASKIN2.","evidence":"Conditional knockout mice with electrophysiology and synaptic transmission assays","pmids":["41223222"],"confidence":"Medium","gaps":["CASKIN1-specific role at non-glutamatergic or peripheral synapses untested","Negative finding embedded in a CASKIN2-focused study"]},{"year":null,"claim":"How CASKIN1's distinct modules — CASK binding, SAM polymerization, LPA-sensing SH3, and EphB1/Nck phosphorylation — are integrated into a single regulated signaling event at the synapse remains unknown.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking lipid binding, phosphorylation, and polymer state","Cellular trigger and consequence of SASH1-mediated depolymerization in neurons undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,6]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[4,7]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,5,6]}],"pathway":[{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[5,6]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2]}],"complexes":[],"partners":["CASK","SASH1","SHANK2","NCK","EPHB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8WXD9","full_name":"Caskin-1","aliases":["CASK-interacting protein 1"],"length_aa":1431,"mass_kda":149.8,"function":"May link the scaffolding protein CASK to downstream intracellular effectors","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q8WXD9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CASKIN1","classification":"Not Classified","n_dependent_lines":8,"n_total_lines":1208,"dependency_fraction":0.006622516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CASKIN1","total_profiled":1310},"omim":[{"mim_id":"612185","title":"CASK-INTERACTING PROTEIN 2; CASKIN2","url":"https://www.omim.org/entry/612185"},{"mim_id":"612184","title":"CASK-INTERACTING PROTEIN 1; CASKIN1","url":"https://www.omim.org/entry/612184"},{"mim_id":"300172","title":"CALCIUM/CALMODULIN-DEPENDENT SERINE PROTEIN KINASE; CASK","url":"https://www.omim.org/entry/300172"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Nuclear bodies","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Tissue enriched","tissue_distribution":"Detected in some","driving_tissues":[{"tissue":"brain","ntpm":57.3}],"url":"https://www.proteinatlas.org/search/CASKIN1"},"hgnc":{"alias_symbol":["KIAA1306","ANKS5A"],"prev_symbol":[]},"alphafold":{"accession":"Q8WXD9","domains":[{"cath_id":"1.10.150.50","chopping":"470-613","consensus_level":"medium","plddt":85.0847,"start":470,"end":613}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WXD9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WXD9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8WXD9-F1-predicted_aligned_error_v6.png","plddt_mean":53.41},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CASKIN1","jax_strain_url":"https://www.jax.org/strain/search?query=CASKIN1"},"sequence":{"accession":"Q8WXD9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8WXD9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8WXD9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8WXD9"}},"corpus_meta":[{"pmid":"21805519","id":"PMC_21805519","title":"A human sterile alpha motif domain polymerizome.","date":"2011","source":"Protein science : a publication of the Protein Society","url":"https://pubmed.ncbi.nlm.nih.gov/21805519","citation_count":89,"is_preprint":false},{"pmid":"21763699","id":"PMC_21763699","title":"The molecular basis of the Caskin1 and Mint1 interaction with CASK.","date":"2011","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/21763699","citation_count":35,"is_preprint":false},{"pmid":"30359304","id":"PMC_30359304","title":"Distribution of Caskin1 protein and phenotypic characterization of its knockout mice using a comprehensive behavioral test battery.","date":"2018","source":"Molecular brain","url":"https://pubmed.ncbi.nlm.nih.gov/30359304","citation_count":31,"is_preprint":false},{"pmid":"31727973","id":"PMC_31727973","title":"Dendritic spine morphology and memory formation depend on postsynaptic Caskin proteins.","date":"2019","source":"Scientific 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CCS","url":"https://pubmed.ncbi.nlm.nih.gov/27549312","citation_count":16,"is_preprint":false},{"pmid":"28298907","id":"PMC_28298907","title":"miR-21a-5p Contributes to Porcine Hemagglutinating Encephalomyelitis Virus Proliferation via Targeting CASK-Interactive Protein1 In vivo and vitro.","date":"2017","source":"Frontiers in microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/28298907","citation_count":13,"is_preprint":false},{"pmid":"25123431","id":"PMC_25123431","title":"Differential synaptic distribution of the scaffold proteins Cask and Caskin1 in the bovine retina.","date":"2014","source":"Molecular and cellular neurosciences","url":"https://pubmed.ncbi.nlm.nih.gov/25123431","citation_count":12,"is_preprint":false},{"pmid":"17185137","id":"PMC_17185137","title":"Gross genomic rearrangement involving the TSC2-PKD1 contiguous deletion syndrome: characterization of the deletion event by quantitative polymerase chain reaction deletion assay.","date":"2007","source":"American 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Methods.","date":"2025","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/40243907","citation_count":3,"is_preprint":false},{"pmid":"35510126","id":"PMC_35510126","title":"Identification of Common Hub Genes in Human Dermal Fibroblasts Stimulated by Mechanical Stretch at Both the Early and Late Stages.","date":"2022","source":"Frontiers in surgery","url":"https://pubmed.ncbi.nlm.nih.gov/35510126","citation_count":3,"is_preprint":false},{"pmid":"41279801","id":"PMC_41279801","title":"Beyond the Genotype: A Multi-Omic Analysis of APOEe4's Role in Alzheimer's Disease.","date":"2025","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/41279801","citation_count":2,"is_preprint":false},{"pmid":"39688081","id":"PMC_39688081","title":"SASH1 is a novel binding partner to disassemble Caskin1 tandem SAM homopolymer through heterogeneous SAM-SAM interaction.","date":"2024","source":"The FEBS 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Caskin1 is necessary and sufficient for binding the CASK calmodulin kinase domain. This motif competes with a related peptide from Mint1 (EPIWVMRQ) for the same conserved binding site on CASK, explaining how Caskin1 and Mint1 form mutually exclusive competing complexes with CASK.\",\n      \"method\": \"Peptide binding assays, site-directed mutagenesis of CASK binding site, in vitro binding experiments\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with defined peptide motif and mutagenesis of the binding site, two orthogonal methods, single lab\",\n      \"pmids\": [\"21763699\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The SAM domain of Caskin1 forms homopolymers, as identified by a native gel screen using negGFP-SAM fusions and confirmed by electron microscopy.\",\n      \"method\": \"Native gel electrophoresis with negGFP-SAM fusions, electron microscopy\",\n      \"journal\": \"Protein science : a publication of the Protein Society\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (native gel + EM), but a screening study with limited mechanistic depth for Caskin1 specifically\",\n      \"pmids\": [\"21805519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"EphB1 receptor tyrosine kinase recruits Caskin1 through the adaptor protein Nck: Nck's SH2 domain binds to a phosphotyrosine on EphB1, while Nck's SH3 domains interact with the proline-rich domain of Caskin1. Complex formation results in tyrosine phosphorylation of the Caskin1 SH3 domain at tyrosines Y296 and Y336, which causes significant structural changes in the SH3 domain as measured by CD spectroscopy.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry (phosphosite identification), CD spectroscopy\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, MS-identified phosphosites, and CD spectroscopy for structural consequence; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"23181695\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Caskin1 localizes to a subset of CASK-positive synapses in the bovine retina, distinct from CASK which is present in virtually all retinal synapses; Caskin1 is enriched at only a subset of ribbon and conventional synapses, suggesting specialized rather than universal synaptic functions.\",\n      \"method\": \"Immunohistochemistry, fractionation/subcellular localization in bovine retina\",\n      \"journal\": \"Molecular and cellular neurosciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct immunolocalization in tissue, replicated with multiple antibodies; no functional consequence directly demonstrated\",\n      \"pmids\": [\"25123431\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The atypical SH3 domain of human Caskin1 selectively binds lysophosphatidic acid (LPA) in vitro, with nanomolar affinity to LPA-containing membranous surfaces. The binding involves beta-strand residues distinct from the canonical proline-rich ligand-binding groove. No proline-rich protein interacting partner for this SH3 domain was identified.\",\n      \"method\": \"In vitro lipid-binding assays, NMR/structural analysis of binding interface\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assay with defined binding site and mutagenesis-informed analysis; single lab, later confirmed by NMR structure\",\n      \"pmids\": [\"28104445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Caskin1 localizes primarily at synapses throughout the brain and spinal cord. Caskin1-knockout mice exhibit enhanced nociception, anxiety-like behavior, increased fear conditioning, and impaired spatial memory, establishing that Caskin1 contributes to nociception, memory, and stress response in the CNS.\",\n      \"method\": \"Caskin1-KO mouse generation, comprehensive behavioral test battery, immunohistochemistry, biochemical fractionation\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined behavioral phenotypes and synaptic localization confirmed by immunohistochemistry; single lab, multiple behavioral assays\",\n      \"pmids\": [\"30359304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Caskin1 is enriched in dendritic spine heads postsynaptically and increases mushroom-shaped dendritic spine formation when overexpressed in hippocampal neurons. Caskin1 co-immunoprecipitates and co-localizes with the postsynaptic scaffold protein Shank2. Loss of Caskin proteins (double KO of Caskin1 and Caskin2) reduces synaptic profiles and dendritic spine area, impairs LTP, alters AMPA receptor phosphorylation, and causes deficits in novelty recognition and spatial memory.\",\n      \"method\": \"Double knockout mice, LTP recordings in hippocampal slices, immunoprecipitation, immunocytochemistry, ultrastructural analysis, overexpression in cultured neurons\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (KO, electrophysiology, Co-IP, ultrastructure, live imaging), replicated across multiple assays in single study\",\n      \"pmids\": [\"31727973\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Solution NMR structure of the human Caskin1 SH3 domain reveals that the canonical proline-rich peptide binding groove is absent due to missing key aromatic residues, supporting the conclusion that this SH3 domain does not bind proline-rich protein motifs. The LPA binding site is structurally distinct from the altered protein-binding groove.\",\n      \"method\": \"Solution NMR structure determination\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — atomic-resolution NMR structure with functional validation; confirms prior biochemical findings; single lab\",\n      \"pmids\": [\"33467043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SASH1 was identified as a novel binding partner of Caskin1, interacting via SAM-SAM domain interaction at the end-helix (EH)/mid-loop (ML) interface. This heterogeneous SAM-SAM interaction disrupts Caskin1 tandem SAM homopolymers, as validated by sedimentation, TEM, co-IP, GST pulldown, and immunofluorescence. Key interface residues were identified by mutagenesis of AlphaFold2-predicted complex models.\",\n      \"method\": \"Yeast two-hybrid screening, size-exclusion chromatography, isothermal titration calorimetry, GST pulldown, co-immunoprecipitation, transmission electron microscopy, sedimentation assay, mutagenesis, immunofluorescence\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal biochemical and structural methods (ITC, SEC, TEM, Co-IP, mutagenesis) in single study establishing the interaction and its functional consequence on polymer disassembly\",\n      \"pmids\": [\"39688081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Conditional knockout analysis in mice showed that CASKIN1 (unlike CASKIN2) is not critical for synaptic transmission, synaptic strength, or AZ protein arrangement at glutamatergic synapses. Combined CASKIN1/2 deletion recapitulates CASKIN2-cKO phenotypes, indicating CASKIN1 plays a redundant or secondary role at hippocampal glutamatergic synapses compared to CASKIN2.\",\n      \"method\": \"Conditional knockout mice, electrophysiology, synaptic transmission assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional KO with defined electrophysiological readouts; CASKIN1-specific result is a negative finding within a study focused on CASKIN2\",\n      \"pmids\": [\"41223222\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CASKIN1 is a multidomain neuronal scaffold protein that binds CASK via a conserved EEIWVLRK peptide motif competing with Mint1, localizes primarily to a subset of presynaptic and postsynaptic sites, forms SAM-domain-mediated homopolymers that can be disrupted by SASH1 through heterogeneous SAM-SAM interaction, contains an atypical SH3 domain (NMR-validated) that binds lysophosphatidic acid rather than proline-rich protein motifs, is recruited to the EphB1 receptor complex via Nck leading to phosphorylation of its SH3 domain at Y296/Y336 with attendant structural changes, and contributes postsynaptically to dendritic spine morphology, AMPA receptor localization, and LTP—with its loss (particularly combined with Caskin2) causing deficits in spatial memory and novelty recognition, while CASKIN2 rather than CASKIN1 appears to be the primary mediator of transsynaptic NMDAR-related functions at glutamatergic synapses.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CASKIN1 is a multidomain neuronal scaffold protein that organizes synaptic protein complexes and contributes to dendritic spine architecture and synaptic plasticity [#6]. It engages the core synaptic scaffold CASK through a short EEIWVLRK peptide motif that binds the CASK CaMK domain and competes with a homologous Mint1 motif for the same site, establishing mutually exclusive CASK complexes [#0]. CASKIN1 self-assembles through SAM-domain-mediated homopolymerization [#1], an oligomeric state that SASH1 disrupts via a heterogeneous SAM-SAM interaction at the end-helix/mid-loop interface [#8]. Its atypical SH3 domain lacks the aromatic residues that form the canonical proline-rich binding groove and instead selectively binds lysophosphatidic acid, as established by NMR structure and lipid-binding assays [#4, #7]. CASKIN1 is recruited to the EphB1 receptor tyrosine kinase via the adaptor Nck, which couples a phosphotyrosine on EphB1 to the CASKIN1 proline-rich region and triggers phosphorylation of its SH3 domain at Y296/Y336 with attendant conformational change [#2]. Postsynaptically, CASKIN1 is enriched in dendritic spine heads, associates with Shank2, and promotes mushroom spine formation, with combined loss of Caskin1 and Caskin2 impairing LTP, altering AMPA receptor phosphorylation, and causing spatial memory and novelty-recognition deficits [#6]; at hippocampal glutamatergic synapses CASKIN1 plays a secondary, redundant role relative to CASKIN2 in synaptic transmission [#9].\",\n  \"teleology\": [\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined how CASKIN1 docks onto the central synaptic scaffold CASK and why this is exclusive with Mint1, explaining competitive assembly of CASK complexes.\",\n      \"evidence\": \"Peptide binding assays and site-directed mutagenesis of the CASK CaMK-domain binding site in vitro\",\n      \"pmids\": [\"21763699\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Affinity and stoichiometry in a full-length cellular context not established\", \"Functional consequence of CASK-Mint1-Caskin1 competition at synapses not demonstrated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established that the CASKIN1 SAM domain self-associates into higher-order homopolymers, defining a structural basis for scaffold clustering.\",\n      \"evidence\": \"Native gel screen with negGFP-SAM fusions and electron microscopy\",\n      \"pmids\": [\"21805519\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Polymer geometry and physiological regulation not resolved\", \"Screening study with limited Caskin1-specific depth\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed CASKIN1 is an EphB1 receptor signaling effector, recruited through Nck and post-translationally modified in a way that remodels its SH3 domain.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, mass-spectrometry phosphosite mapping, and CD spectroscopy\",\n      \"pmids\": [\"23181695\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Downstream signaling output of Y296/Y336 phosphorylation not defined\", \"Single lab; in vivo relevance of EphB1-Nck-Caskin1 axis untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated CASKIN1 marks only a subset of CASK-positive synapses, implying specialized rather than universal synaptic functions.\",\n      \"evidence\": \"Immunohistochemistry and subcellular fractionation in bovine retina\",\n      \"pmids\": [\"25123431\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional consequence demonstrated\", \"Molecular determinant of selective synaptic targeting unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Reassigned the CASKIN1 SH3 domain function from a protein-interaction module to a lipid sensor by showing selective high-affinity binding to lysophosphatidic acid.\",\n      \"evidence\": \"In vitro lipid-binding assays and structural analysis of the binding interface\",\n      \"pmids\": [\"28104445\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cellular role of LPA binding not established\", \"No proline-rich protein ligand identified\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established CASKIN1 as a CNS-wide synaptic protein with defined behavioral functions in nociception, anxiety, fear, and spatial memory.\",\n      \"evidence\": \"Caskin1-knockout mice, behavioral test battery, immunohistochemistry, fractionation\",\n      \"pmids\": [\"30359304\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Circuit and synaptic mechanism underlying behaviors not resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Localized CASKIN1 to the postsynaptic spine head and linked it mechanistically to spine morphology, AMPA receptor regulation, and LTP via association with Shank2.\",\n      \"evidence\": \"Caskin1/2 double-knockout mice, LTP recordings, co-IP, immunocytochemistry, ultrastructure, and overexpression in cultured neurons\",\n      \"pmids\": [\"31727973\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Caskin1-specific versus Caskin2-shared contribution not separated\", \"Mechanism linking Shank2 binding to AMPA receptor phosphorylation unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided the atomic-resolution basis for why the CASKIN1 SH3 domain cannot bind proline-rich motifs and confirmed a distinct LPA binding site.\",\n      \"evidence\": \"Solution NMR structure determination of the human Caskin1 SH3 domain\",\n      \"pmids\": [\"33467043\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo LPA engagement not demonstrated\", \"Functional impact of Y296/Y336 phosphorylation on the structure not directly resolved here\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified SASH1 as a regulator that disassembles CASKIN1 SAM homopolymers through a heterogeneous SAM-SAM interaction, defining a mechanism for controlling scaffold oligomerization.\",\n      \"evidence\": \"Yeast two-hybrid, SEC, ITC, GST pulldown, co-IP, TEM, sedimentation, mutagenesis guided by AlphaFold2 models, and immunofluorescence\",\n      \"pmids\": [\"39688081\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological context and neuronal consequence of SASH1-driven depolymerization not established\", \"Regulatory triggers for the interaction unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolved the relative contribution of CASKIN1 at glutamatergic synapses, showing it is dispensable for synaptic transmission and redundant with the dominant CASKIN2.\",\n      \"evidence\": \"Conditional knockout mice with electrophysiology and synaptic transmission assays\",\n      \"pmids\": [\"41223222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CASKIN1-specific role at non-glutamatergic or peripheral synapses untested\", \"Negative finding embedded in a CASKIN2-focused study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CASKIN1's distinct modules — CASK binding, SAM polymerization, LPA-sensing SH3, and EphB1/Nck phosphorylation — are integrated into a single regulated signaling event at the synapse remains unknown.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking lipid binding, phosphorylation, and polymer state\", \"Cellular trigger and consequence of SASH1-mediated depolymerization in neurons undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [4, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CASK\", \"SASH1\", \"Shank2\", \"Nck\", \"EphB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}