{"gene":"HOOK3","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":2001,"finding":"HOOK3 is a cytosolic coiled-coil protein with a conserved NH2-terminal domain that mediates microtubule binding and a divergent COOH-terminal domain that mediates organelle binding. Human HOOK3 binds to Golgi membranes in vitro and is enriched at the cis-Golgi in vivo. Overexpression of HOOK3 caused fragmentation of the Golgi complex, indicating a role in defining Golgi architecture and localization.","method":"In vitro binding assay, subcellular fractionation, immunofluorescence, overexpression with phenotypic readout","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (in vitro binding, localization, functional overexpression phenotype) in a single foundational study","pmids":["11238449"],"is_preprint":false},{"year":1999,"finding":"Genetic analysis of Drosophila hook (ortholog of human HOOK proteins) demonstrated that all three domains (N-terminal, central coiled-coil, and C-terminal) are required for Hook function in endocytic trafficking; loss-of-function alleles block accumulation of both transmembrane and soluble ligands in multivesicular bodies. The importance of these domains is conserved between Drosophila and human Hook proteins.","method":"Genetic screen, null allele isolation, domain truncation analysis, endocytic trafficking assay","journal":"Genetics","confidence":"High","confidence_rationale":"Tier 1-2 — null allele genetic analysis with domain structure-function epistasis, foundational mechanistic paper","pmids":["9927460"],"is_preprint":false},{"year":2003,"finding":"Salmonella SpiC protein directly binds HOOK3 in macrophage cytosol; GST-SpiC pulled down HOOK3 from murine macrophages and anti-HOOK3 antibodies co-precipitated SpiC from infected cells. SpiC expression disrupted Golgi morphology and altered lysosome distribution, mimicking the phenotype of a hook3 dominant-negative mutant, indicating that SpiC targets HOOK3 function to inhibit phagosome-lysosome fusion.","method":"GST pulldown, co-immunoprecipitation, dominant-negative phenotypic comparison, Golgi morphology assay","journal":"Molecular microbiology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus functional phenotypic validation linking HOOK3 to lysosomal trafficking","pmids":["12950921"],"is_preprint":false},{"year":2004,"finding":"IIGP (a 47 kDa IFN-inducible GTPase) interacts with HOOK3 in a GTP-bound conformation-dependent manner. The physical complex was detected by co-immunoprecipitation in IFN-γ-stimulated macrophages. Both proteins co-localize in the cis-Golgi and perinuclear regions, suggesting HOOK3 links IFN-γ-inducible GTPase activity to cytoskeleton-based membrane trafficking.","method":"Yeast two-hybrid screening, co-immunoprecipitation, subcellular fractionation, immunofluorescence co-localization","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2/3 — yeast two-hybrid plus co-IP in primary cells, but interaction is minor subfraction and functional consequence not fully resolved","pmids":["15075236"],"is_preprint":false},{"year":2007,"finding":"HOOK3-RET is a novel oncogenic fusion protein arising from chromosomal rearrangement in papillary thyroid carcinoma, fusing exon 11 of HOOK3 to exon 12 of RET. The chimeric HOOK3-RET protein (~88 kDa) retains HOOK3 coiled-coil domains and the intact RET tyrosine kinase domain. Expression in NIH3T3 cells caused transformed foci formation and tumor growth in nude mice.","method":"5'RACE, Western blot with anti-RET antibody, NIH3T3 transformation assay, nude mouse xenograft","journal":"Endocrine-related cancer","confidence":"High","confidence_rationale":"Tier 2 — fusion protein identified and functionally validated by transformation and in vivo tumor assays","pmids":["17639057"],"is_preprint":false},{"year":2007,"finding":"HOOK3 interacts directly with the cytoplasmic domain of scavenger receptor A (SR-A). The interaction was identified by yeast two-hybrid and mass spectrometry, and confirmed by pulldown and co-immunoprecipitation of overexpressed and endogenous proteins. The C-terminal Val614-Ala717 region of HOOK3 is required for binding to negatively charged residues (Glu12, Asp13, Asp15) in the SR-A cytoplasmic domain. siRNA knockdown of HOOK3 promoted SR-A surface expression, ligand uptake, and protein stability, indicating HOOK3 participates in SR-A turnover.","method":"Yeast two-hybrid, mass spectrometry, pulldown, co-immunoprecipitation, domain truncation, siRNA knockdown with receptor trafficking assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including domain mapping and functional siRNA knockdown with defined trafficking phenotype","pmids":["17237231"],"is_preprint":false},{"year":2008,"finding":"HOOK3 is a component of the FHF complex (FTS/Hook/FHIP), a ~500 kDa multiprotein assembly containing FTS, Hook1, Hook2, Hook3, and FHIP. Hook proteins form homo- and heterodimers and interact with members of the HOPS complex. The FHF complex promotes endosomal clustering and EGF trafficking from early-to-late endosomes/lysosomes.","method":"Proteomic analysis (AP-MS), yeast two-hybrid, co-immunoprecipitation, siRNA depletion with EGF trafficking assay, lysosomal clustering assay","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — AP-MS complex identification confirmed by multiple biochemical methods with functional trafficking readout","pmids":["18799622"],"is_preprint":false},{"year":2010,"finding":"HOOK3 interacts with PCM1 (Pericentriolar Material 1) and is recruited to pericentriolar satellites through this interaction. Disruption of the HOOK3-PCM1 interaction in vivo impairs interkinetic nuclear migration in embryonic neural progenitors, leading to overproduction of neurons and premature depletion of the neural progenitor pool in the developing neocortex.","method":"Co-immunoprecipitation, in vivo disruption of protein-protein interaction, live imaging, cortical neurogenesis assay","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP plus in vivo loss-of-function with defined neurogenic phenotype","pmids":["20152126"],"is_preprint":false},{"year":2015,"finding":"HOOK3 is expressed in neurons and localizes to tau aggregates in Alzheimer's disease brain. Hook3 deficiency modeled in cultured cells slows endosomal transport and increases β-amyloid production, indicating that HOOK3 participates in endosomal transport relevant to amyloid precursor protein processing.","method":"Immunofluorescence in AD brain tissue, siRNA knockdown with endosomal transport assay, β-amyloid ELISA","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 — localization plus knockdown with defined transport and amyloid phenotype, single lab","pmids":["25799409"],"is_preprint":false},{"year":2016,"finding":"Hook3 activates dynein-dynactin motility by directly binding to the dynein light intermediate chain 1 (LIC1) through its conserved Hook domain. Crystal structure of the Hook domain was solved, and structure-based mutagenesis identified two conserved surface residues critical for LIC1 binding. Hook3 also contains a distinct region required for allosteric activation of processive dynein-dynactin motility.","method":"Crystal structure determination, structure-based mutagenesis, in vitro binding assay, single-molecule motility assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and in vitro reconstitution of motility","pmids":["27482052"],"is_preprint":false},{"year":2016,"finding":"Mammalian Hook1 and Hook3 activate cytoplasmic dynein by stabilizing the dynein-dynactin supramolecular complex. Optogenetic recruitment of Hook proteins to peroxisomes drove rapid perinuclear transport. Single-molecule TIRF assays showed Hook1 and Hook3 induce longer run lengths and higher velocities than the activator BICD2. The N-terminal domain (resembling a calponin-homology domain) is required for dynein-dynactin interaction.","method":"Optogenetics, single-molecule TIRF motility assay, biochemical co-sedimentation, domain mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro single-molecule reconstitution combined with optogenetic live-cell assay and biochemical domain analysis","pmids":["27365401"],"is_preprint":false},{"year":2019,"finding":"HOOK3 scaffolds both cytoplasmic dynein-1/dynactin and the kinesin-3 KIF1C into a tripartite complex. In vitro reconstitution with purified components showed full-length Hook3 activates dynein/dynactin motility and binds KIF1C tail, though without activating KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward microtubule minus ends, and KIF1C to transport dynein toward plus ends. In cells, KIF1C recruits Hook3 to the cell periphery.","method":"In vitro reconstitution with purified proteins, single-molecule motility assay, mass spectrometry interactome, live-cell imaging","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with purified components plus single-molecule assays and cellular validation","pmids":["31320392"],"is_preprint":false},{"year":2019,"finding":"HOOK3 binds the same region of KIF1C tail as the PTPN21 FERM domain and releases KIF1C autoinhibition by increasing KIF1C landing rate onto microtubules. Full-length human KIF1C is a processive plus-end motor, and its autoinhibitory interaction (motor domain with stalk) is relieved by Hook3 or PTPN21 FERM domain binding, enabling cargo-activated transport.","method":"In vitro single-molecule motility assay, pulldown, co-immunoprecipitation, domain mapping, neuronal transport assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro reconstitution demonstrating autoinhibition release mechanism, multiple orthogonal methods","pmids":["31217419"],"is_preprint":false},{"year":2021,"finding":"HOOK3 is phosphorylated by ERK1c early in mitosis, and subsequently by AuroraA. This dual phosphorylation causes HOOK3 to detach from microtubules and increases its interaction with GM130, leading to Golgi destabilization and fragmentation during mitosis. In cycling cells HOOK3 links microtubules to the Golgi via both interactions.","method":"Phosphorylation assay, mutagenesis, co-immunoprecipitation, live-cell imaging, Golgi morphology quantification","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — kinase substrate identification with mechanistic follow-up, single lab with multiple methods","pmids":["34189435"],"is_preprint":false},{"year":2022,"finding":"A novel in-frame HOOK3-FGFR1 fusion gene was identified in MDS/8p11 myeloproliferative syndrome, with breakpoints at HOOK3 exon 11 and FGFR1 exon 10. The chimeric protein retains the entire FGFR1 tyrosine kinase domain and is associated with NF-κB pathway activation, evidenced by increased phosphorylation of p65 (Ser536) and IKBα (Ser32).","method":"RNA-seq with STAR-Fusion, FISH, qRT-PCR, Sanger sequencing, Western blot, phosphorylation antibody array","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2/3 — fusion identified and signaling characterized biochemically, but functional oncogenic validation not as complete as HOOK3-RET study","pmids":["35081975"],"is_preprint":false},{"year":2024,"finding":"KIF1C facilitates retrograde lysosomal transport by interacting with dynein-activating adaptor HOOK3, which associates with the lysosome-anchored protein RUFY3. KIF1C motor activity is not required and actually inhibits this retrograde process; instead KIF1C acts as a scaffold via Hook3 to promote dynein-driven perinuclear lysosomal positioning critical for autophagic and endocytic cargo degradation.","method":"Co-immunoprecipitation, siRNA knockdown, live-cell imaging, lysosome positioning assay, autophagy flux assay","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods establishing non-motor scaffolding role, single lab","pmids":["39394274"],"is_preprint":false},{"year":2025,"finding":"Crystal structure of the Hook3(553-624)–KIF1C(714-809) complex was determined, revealing the structural basis for Hook3-KIF1C interaction. Structure-based mutagenesis showed this complex formation is necessary and sufficient for full-length protein interaction in HEK293T cells and for Hook3- and KIF1C-mediated anterograde transport in RPE1 cells. PTPN21 interaction with KIF1C is also part of this assembly.","method":"Crystal structure determination, structure-based mutagenesis, co-immunoprecipitation in HEK293T cells, live-cell transport assay in RPE1 cells","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — crystal structure combined with mutagenesis and functional cellular transport validation","pmids":["40312563"],"is_preprint":false}],"current_model":"HOOK3 is a cytosolic coiled-coil cargo adaptor that uses its conserved N-terminal Hook domain to bind dynein light intermediate chain 1 (LIC1) and allosterically activate processive dynein-dynactin motility toward microtubule minus ends, while its C-terminal domain anchors organelles (cis-Golgi, endosomes, lysosomes); it also scaffolds the opposite-polarity kinesin KIF1C through a structurally defined interaction at its C-terminal coiled-coil, enabling bidirectional cargo transport, and is regulated by ERK1c- and AuroraA-mediated phosphorylation that detaches it from microtubules to drive mitotic Golgi fragmentation."},"narrative":{"teleology":[{"year":1999,"claim":"Genetic analysis of Drosophila Hook established that all three protein domains—N-terminal, coiled-coil, and C-terminal—are functionally required for endocytic trafficking, defining the conserved tripartite architecture that underlies Hook protein function across species.","evidence":"Null allele isolation, domain truncation analysis, and endocytic trafficking assays in Drosophila","pmids":["9927460"],"confidence":"High","gaps":["Mammalian-specific domain functions not yet tested","Molecular targets of each domain unknown"]},{"year":2001,"claim":"Identification of human HOOK3 as a cis-Golgi-associated, microtubule-binding protein whose overexpression fragments the Golgi revealed a specific organelle-tethering role distinct from general endocytic trafficking, assigning the N-terminal domain to microtubule binding and the C-terminal domain to organelle attachment.","evidence":"In vitro binding assay, subcellular fractionation, immunofluorescence, and overexpression phenotype in mammalian cells","pmids":["11238449"],"confidence":"High","gaps":["Motor protein partners not yet identified","Mechanism of Golgi fragmentation upon overexpression unclear"]},{"year":2003,"claim":"The discovery that Salmonella effector SpiC directly binds HOOK3 to disrupt Golgi morphology and lysosome positioning demonstrated that HOOK3-dependent trafficking is exploited by intracellular pathogens to block phagosome-lysosome fusion.","evidence":"GST pulldown, reciprocal co-immunoprecipitation, and dominant-negative phenotypic comparison in macrophages","pmids":["12950921"],"confidence":"High","gaps":["Structural basis of SpiC–HOOK3 interaction unresolved","Whether SpiC directly inhibits motor recruitment unknown"]},{"year":2007,"claim":"Mapping the HOOK3 C-terminal domain interaction with scavenger receptor A (SR-A) cytoplasmic tail, and showing that HOOK3 depletion increases SR-A surface expression, established HOOK3 as an active participant in receptor turnover and endocytic cargo sorting.","evidence":"Yeast two-hybrid, mass spectrometry, domain truncation, co-IP, and siRNA knockdown with receptor trafficking assay","pmids":["17237231"],"confidence":"High","gaps":["Whether HOOK3 directs SR-A to lysosomes or recycling pathway not determined","Generality to other scavenger receptors untested"]},{"year":2008,"claim":"Proteomic identification of the FHF complex (FTS/Hook/FHIP) showed that HOOK3 operates as part of a ~500 kDa multiprotein assembly that promotes endosomal clustering and EGF trafficking from early to late endosomes, placing HOOK3 in a defined complex context.","evidence":"AP-MS, yeast two-hybrid, co-IP, siRNA depletion with EGF trafficking assay","pmids":["18799622"],"confidence":"High","gaps":["Stoichiometry of Hook homo- vs. heterodimers in the FHF complex unresolved","Whether FHF complex directly engages motors not tested"]},{"year":2010,"claim":"The interaction between HOOK3 and PCM1 at pericentriolar satellites, and its requirement for interkinetic nuclear migration in embryonic neural progenitors, revealed a non-Golgi role for HOOK3 in centrosome-based transport critical for neurogenesis.","evidence":"Co-IP, in vivo disruption of HOOK3-PCM1 interaction, live imaging, cortical neurogenesis assay in developing neocortex","pmids":["20152126"],"confidence":"High","gaps":["Whether HOOK3 at satellites acts through dynein not directly shown","Relevance to adult neurogenesis unknown"]},{"year":2016,"claim":"Crystal structure of the Hook domain and single-molecule reconstitution demonstrated that HOOK3 directly binds dynein LIC1 and allosterically activates processive dynein-dynactin motility, establishing HOOK3 as a bona fide dynein-activating adaptor with structurally defined motor-binding and activation regions.","evidence":"Crystal structure, structure-based mutagenesis, in vitro binding, single-molecule TIRF motility assay, optogenetic recruitment to peroxisomes","pmids":["27482052","27365401"],"confidence":"High","gaps":["Cryo-EM structure of the full HOOK3–dynein–dynactin supercomplex not available","In vivo contribution of individual LIC1-contact residues not tested"]},{"year":2019,"claim":"In vitro reconstitution revealed that HOOK3 simultaneously scaffolds dynein-dynactin and kinesin KIF1C into a single complex, enabling bidirectional transport and relieving KIF1C autoinhibition, answering how opposite-polarity motors are coordinated on the same adaptor.","evidence":"Purified protein reconstitution, single-molecule motility assay, mass spectrometry interactome, pulldown, neuronal transport assay","pmids":["31320392","31217419"],"confidence":"High","gaps":["Regulatory switch between dynein-dominant and kinesin-dominant states unknown","Whether the tripartite complex exists on specific cargo in vivo not visualized"]},{"year":2021,"claim":"Identification of ERK1c and Aurora A as sequential mitotic kinases that phosphorylate HOOK3, detach it from microtubules, and enhance its GM130 interaction explained how Golgi fragmentation is triggered during mitotic entry through regulated HOOK3 modification.","evidence":"Phosphorylation assays, mutagenesis, co-IP, live-cell imaging, Golgi morphology quantification","pmids":["34189435"],"confidence":"Medium","gaps":["Phosphorylation sites not mapped by mass spectrometry in vivo","Whether phosphorylation also affects dynein or KIF1C binding untested","Single-lab finding"]},{"year":2024,"claim":"Demonstration that KIF1C acts as a non-motor scaffold via HOOK3 and lysosome-anchored RUFY3 to promote dynein-driven perinuclear lysosomal positioning resolved how HOOK3 contributes to autophagic and endocytic degradation by positioning lysosomes near the cell center.","evidence":"Co-IP, siRNA knockdown, live-cell imaging, lysosome positioning assay, autophagy flux assay","pmids":["39394274"],"confidence":"Medium","gaps":["Direct HOOK3–RUFY3 binding not demonstrated with purified proteins","Contribution of the FHF complex to this pathway not addressed","Single-lab finding"]},{"year":2025,"claim":"Crystal structure of the Hook3(553–624)–KIF1C(714–809) complex provided the atomic-resolution basis for the adaptor–kinesin interaction, and structure-guided mutations confirmed this interface is necessary and sufficient for HOOK3–KIF1C-mediated transport in cells.","evidence":"Crystal structure, structure-based mutagenesis, co-IP in HEK293T, live-cell transport assay in RPE1 cells","pmids":["40312563"],"confidence":"High","gaps":["No structure of the full-length HOOK3 dimer with both dynein-dynactin and KIF1C simultaneously bound","Post-translational regulation of this interface not explored"]},{"year":null,"claim":"Key unresolved questions include how HOOK3-dependent transport is cargo-selectively regulated in vivo, whether distinct phosphorylation events differentially control dynein versus KIF1C engagement, and what structural features govern HOOK3 specificity relative to HOOK1 and HOOK2.","evidence":"","pmids":[],"confidence":"Low","gaps":["No cryo-EM structure of the HOOK3–dynein–dynactin–KIF1C supercomplex","Cargo-specific adaptor-receptor interactions beyond SR-A and RUFY3 are poorly defined","Paralog-specific functions among HOOK1/2/3 not systematically dissected"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[9,10,11,12,16]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0,1,9,10,13]}],"localization":[{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[0,2,3,13]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,2]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[1,6,8]},{"term_id":"GO:0005764","term_label":"lysosome","supporting_discovery_ids":[2,15]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,9,10]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[7]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[1,2,5,6,8,15]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[15]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[13]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,7,13]}],"complexes":["FHF complex (FTS/Hook/FHIP)","Dynein-dynactin-HOOK3 activating adaptor complex","HOOK3-KIF1C bidirectional transport complex"],"partners":["DYNC1LI1","KIF1C","PCM1","FTS","FHIP","GM130","RUFY3","MSR1"],"other_free_text":[]},"mechanistic_narrative":"HOOK3 is a coiled-coil cargo adaptor that coordinates bidirectional microtubule-based transport by simultaneously engaging the dynein-dynactin retrograde motor complex and the kinesin-3 KIF1C anterograde motor. Its conserved N-terminal Hook domain binds dynein light intermediate chain 1 (LIC1), allosterically activating processive dynein-dynactin motility toward microtubule minus ends, while its C-terminal coiled-coil domain anchors to organelle membranes—including the cis-Golgi, endosomes, and lysosomes—and scaffolds KIF1C through a structurally defined interface that also relieves KIF1C autoinhibition [PMID:27482052, PMID:31320392, PMID:31217419, PMID:40312563]. HOOK3 functions within the FHF complex (FTS/Hook/FHIP) to promote early-to-late endosome trafficking, interacts with PCM1 at pericentriolar satellites to support interkinetic nuclear migration in neural progenitors, and connects to lysosomes via RUFY3 to drive dynein-dependent perinuclear lysosomal positioning required for autophagic flux [PMID:18799622, PMID:20152126, PMID:39394274]. During mitosis, sequential phosphorylation by ERK1c and Aurora A detaches HOOK3 from microtubules and enhances its interaction with the Golgi matrix protein GM130, triggering Golgi fragmentation [PMID:34189435]."},"prefetch_data":{"uniprot":{"accession":"Q86VS8","full_name":"Protein Hook homolog 3","aliases":[],"length_aa":718,"mass_kda":83.1,"function":"Acts as an adapter protein linking the dynein motor complex to various cargos and converts dynein from a non-processive to a highly processive motor in the presence of dynactin. Facilitates the interaction between dynein and dynactin and activates dynein processivity (the ability to move along a microtubule for a long distance without falling off the track). Predominantly recruits 2 dyneins, which increases both the force and speed of the microtubule motor (PubMed:25035494, PubMed:33734450). Component of the FTS/Hook/FHIP complex (FHF complex). The FHF complex may function to promote vesicle trafficking and/or fusion via the homotypic vesicular protein sorting complex (the HOPS complex). May regulate clearance of endocytosed receptors such as MSR1. Participates in defining the architecture and localization of the Golgi complex. FHF complex promotes the distribution of AP-4 complex to the perinuclear area of the cell (PubMed:32073997) (Microbial infection) May serve as a target for the spiC protein from Salmonella typhimurium, which inactivates it, leading to a strong alteration in cellular trafficking","subcellular_location":"Cytoplasm, cytoskeleton; Golgi apparatus","url":"https://www.uniprot.org/uniprotkb/Q86VS8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HOOK3","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CLIP1","stoichiometry":0.2},{"gene":"DYNC1LI1","stoichiometry":0.2},{"gene":"TUBB4B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/HOOK3","total_profiled":1310},"omim":[{"mim_id":"620891","title":"COILED-COIL DOMAIN-CONTAINING PROTEIN 181; CCDC181","url":"https://www.omim.org/entry/620891"},{"mim_id":"620230","title":"FHF COMPLEX SUBUNIT HOOK-INTERACTING PROTEIN 2B; FHIP2B","url":"https://www.omim.org/entry/620230"},{"mim_id":"620229","title":"FHF COMPLEX SUBUNIT HOOK-INTERACTING PROTEIN 1B; FHIP1B","url":"https://www.omim.org/entry/620229"},{"mim_id":"617312","title":"FHF COMPLEX SUBUNIT HOOK-INTERACTING PROTEIN 2A; FHIP2A","url":"https://www.omim.org/entry/617312"},{"mim_id":"617002","title":"BICD FAMILY-LIKE CARGO ADAPTOR 1; BICDL1","url":"https://www.omim.org/entry/617002"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Centriolar satellite","reliability":"Supported"},{"location":"Golgi apparatus","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HOOK3"},"hgnc":{"alias_symbol":["HK3"],"prev_symbol":[]},"alphafold":{"accession":"Q86VS8","domains":[{"cath_id":"1.10.418.10","chopping":"3-162","consensus_level":"high","plddt":84.3988,"start":3,"end":162}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86VS8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q86VS8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q86VS8-F1-predicted_aligned_error_v6.png","plddt_mean":82.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HOOK3","jax_strain_url":"https://www.jax.org/strain/search?query=HOOK3"},"sequence":{"accession":"Q86VS8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q86VS8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q86VS8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q86VS8"}},"corpus_meta":[{"pmid":"11238449","id":"PMC_11238449","title":"The Golgi-associated hook3 protein is a member of a novel family of microtubule-binding proteins.","date":"2001","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11238449","citation_count":166,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"27482052","id":"PMC_27482052","title":"Assembly and activation of dynein-dynactin by the cargo adaptor protein Hook3.","date":"2016","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/27482052","citation_count":102,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"20152126","id":"PMC_20152126","title":"Hook3 interacts with PCM1 to regulate pericentriolar material assembly and the timing of neurogenesis.","date":"2010","source":"Neuron","url":"https://pubmed.ncbi.nlm.nih.gov/20152126","citation_count":98,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"9180162","id":"PMC_9180162","title":"Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), activates single-chain urokinase-type plasminogen activator.","date":"1997","source":"International journal of cancer","url":"https://pubmed.ncbi.nlm.nih.gov/9180162","citation_count":94,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31320392","id":"PMC_31320392","title":"Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C.","date":"2019","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/31320392","citation_count":65,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"22248077","id":"PMC_22248077","title":"ESR1, HK3 and BRSK1 gene variants are associated with both age at natural menopause and premature ovarian failure.","date":"2012","source":"Orphanet journal of rare diseases","url":"https://pubmed.ncbi.nlm.nih.gov/22248077","citation_count":62,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"31217419","id":"PMC_31217419","title":"PTPN21 and Hook3 relieve KIF1C autoinhibition and activate intracellular 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Overexpression of Hook3 causes Golgi fragmentation, and a large fraction maintains juxtanuclear localization after Brefeldin A treatment indicating a Golgi-independent localization mechanism.\",\n      \"method\": \"In vitro Golgi membrane binding assay, immunofluorescence, Brefeldin A treatment, overexpression with morphological readout\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (in vitro binding, in vivo localization, functional overexpression), foundational paper with 166 citations\",\n      \"pmids\": [\"11238449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The conserved Hook domain of HOOK3 interacts directly with dynein light intermediate chain 1 (LIC1). Crystal structure of the Hook domain was solved, and structure-based mutagenesis identified two conserved surface residues critical for LIC1 binding. A separate region of Hook3 is required for allosteric activation of processive dynein-dynactin motility.\",\n      \"method\": \"Crystal structure determination, structure-based mutagenesis, in vitro motility assays, co-immunoprecipitation, reconstitution of dynein-dynactin-Hook3 ternary complex\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and in vitro reconstitution in single rigorous study\",\n      \"pmids\": [\"27482052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HOOK3 is recruited to pericentriolar satellites through direct interaction with PCM1 (Pericentriolar Material 1). Disruption of the Hook3-PCM1 interaction impairs interkinetic nuclear migration in embryonic neural progenitors, leading to overproduction of neurons and premature depletion of the neural progenitor pool.\",\n      \"method\": \"Co-immunoprecipitation, in vivo disruption of interaction, interkinetic nuclear migration assay, cortical neurogenesis analysis in developing neocortex\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction demonstrated, in vivo loss-of-function with specific cellular phenotype, replicated across multiple experiments\",\n      \"pmids\": [\"20152126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HOOK3 acts as a scaffold that simultaneously binds dynein/dynactin (activating its motility) and the kinesin-3 KIF1C (via a short region in the KIF1C tail, without activating KIF1C). This allows dynein to transport KIF1C toward microtubule minus ends and KIF1C to transport dynein toward plus ends. KIF1C can recruit Hook3 to the cell periphery in cells.\",\n      \"method\": \"In vitro reconstitution with purified components, single-molecule motility assays, co-immunoprecipitation, live-cell imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified proteins and single-molecule imaging, plus cellular validation\",\n      \"pmids\": [\"31320392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Hook3 binds the same region of the KIF1C tail as PTPN21 FERM domain. Both Hook3 and PTPN21 binding increase the landing rate of KIF1C onto microtubules in vitro, relieving KIF1C autoinhibition (which involves interaction of the motor domain's microtubule-binding surface with its own stalk).\",\n      \"method\": \"In vitro single-molecule motility/landing rate assay with purified proteins, domain-mapping pulldowns, rescue of integrin trafficking in KIF1C-depleted cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified components demonstrating autoinhibition release, combined with cellular rescue experiments\",\n      \"pmids\": [\"31217419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Salmonella SpiC protein physically interacts with HOOK3 in macrophages. GST-SpiC pulls down Hook3 from macrophage lysates and anti-Hook3 antibodies co-precipitate SpiC from infected macrophage cytosol. SpiC expression disrupts Golgi morphology and alters lysosome distribution mimicking the Hook3 dominant-negative phenotype, suggesting SpiC inhibits phagosome-lysosome fusion by targeting Hook3 function.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, dominant-negative expression, immunofluorescence of Golgi and lysosome morphology\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus functional mimicry with dominant negative, multiple orthogonal approaches\",\n      \"pmids\": [\"12950921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The interferon-inducible 47 kDa GTPase IIGP interacts with Hook3 in a GTP-bound conformation-dependent manner. The interaction was identified by yeast two-hybrid and confirmed by co-immunoprecipitation from IFN-γ-stimulated macrophages. Both proteins co-localize and co-fractionate in Golgi-membrane-enriched fractions.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation from macrophages, subcellular fractionation, immunofluorescence co-localization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — yeast two-hybrid confirmed by co-IP, but interaction is minor/transient and functional consequence not fully established\",\n      \"pmids\": [\"15075236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Hook3 interacts with the cytoplasmic domain of scavenger receptor A (SR-A). The interaction was identified by yeast two-hybrid and mass spectrometry, confirmed by pulldown and co-immunoprecipitation. The positively charged C-terminal region (Val614-Ala717) of Hook3 binds negatively charged residues (Glu12, Asp13, Asp15) in the SR-A cytoplasmic domain. siRNA knockdown of Hook3 increases SR-A surface expression, ligand uptake, and protein stability.\",\n      \"method\": \"Yeast two-hybrid, mass spectrometry, pulldown assay, co-immunoprecipitation, siRNA knockdown, domain truncation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including domain mapping, reciprocal co-IP, and functional siRNA knockdown with specific readouts\",\n      \"pmids\": [\"17237231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HOOK3 participates in a chromosomal rearrangement (RET/PTC) in papillary thyroid carcinoma, generating a HOOK3-RET fusion protein. The fusion contains coiled-coil domains from HOOK3 and the intact tyrosine kinase domain of RET. Expression of HOOK3-RET in NIH3T3 cells causes foci formation and tumor formation in nude mice, confirming oncogenic activity.\",\n      \"method\": \"5'RACE identification of fusion, western blot, NIH3T3 transformation assay, nude mouse tumor formation\",\n      \"journal\": \"Endocrine-related cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional oncogenic assay with HOOK3-RET fusion, but mechanistic detail of HOOK3 contribution is limited\",\n      \"pmids\": [\"17639057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Hook3 deficiency in cultured cells slows endosomal transport and increases β-amyloid production, and Hook3 is expressed in neurons and localizes to tau aggregates in Alzheimer's disease brain.\",\n      \"method\": \"siRNA/shRNA knockdown, endosomal transport assay, β-amyloid ELISA, immunofluorescence co-localization in patient tissue\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — loss-of-function with defined cellular phenotypes, but single study without detailed pathway placement\",\n      \"pmids\": [\"25799409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ERK1c phosphorylates HOOK3 during mitosis, and a subsequent phosphorylation by AuroraA causes HOOK3 to detach from microtubules and increase interaction with GM130, destabilizing Golgi-microtubule connections and allowing Golgi fragmentation during mitosis.\",\n      \"method\": \"In vitro kinase assay (ERK1c and AuroraA phosphorylation of HOOK3), co-immunoprecipitation (HOOK3-GM130 interaction), microtubule co-sedimentation assay, phospho-mutant analysis, Golgi morphology imaging in cycling cells\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including in vitro kinase assays and pulldowns, but from a single lab\",\n      \"pmids\": [\"34189435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KIF1C facilitates dynein-driven retrograde lysosomal transport by interacting with the dynein-activating adaptor Hook3, which in turn associates with the lysosome-anchored protein RUFY3. KIF1C's motor activity is not required and actually inhibits this process; the non-motor scaffold role via Hook3 is critical.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, live-cell lysosome tracking, dominant-negative motor mutants, rescue experiments\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP and functional KD with specific lysosomal transport readout, single study\",\n      \"pmids\": [\"39394274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Crystal structure of the Hook3(553-624) – KIF1C(714-809) complex was solved, revealing the structural basis for Hook3-KIF1C interaction. Structure-based mutational analysis demonstrated that this complex formation is necessary and sufficient for interaction between full-length proteins and is required for Hook3- and KIF1C-mediated anterograde transport in RPE1 cells.\",\n      \"method\": \"Crystal structure determination, structure-based mutagenesis, co-immunoprecipitation in HEK293T cells, anterograde transport assay in RPE1 cells\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and cellular functional validation\",\n      \"pmids\": [\"40312563\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HOOK3 is a cytosolic coiled-coil cargo adaptor that links organelles (Golgi, endosomes, lysosomes) to the microtubule cytoskeleton via its conserved N-terminal microtubule-binding Hook domain; it directly binds dynein LIC1 to form and allosterically activate a processive dynein-dynactin motor complex, simultaneously scaffolds the opposite-polarity kinesin KIF1C (via a C-terminal region whose structure is now resolved) to regulate bidirectional cargo transport, interacts with PCM1 to traffic pericentriolar satellites required for interkinetic nuclear migration and neurogenesis, and is subject to sequential mitotic phosphorylation by ERK1c and AuroraA that releases it from microtubules to allow Golgi fragmentation during cell division.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"HOOK3 is a cytosolic coiled-coil protein with a conserved NH2-terminal domain that mediates microtubule binding and a divergent COOH-terminal domain that mediates organelle binding. Human HOOK3 binds to Golgi membranes in vitro and is enriched at the cis-Golgi in vivo. Overexpression of HOOK3 caused fragmentation of the Golgi complex, indicating a role in defining Golgi architecture and localization.\",\n      \"method\": \"In vitro binding assay, subcellular fractionation, immunofluorescence, overexpression with phenotypic readout\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (in vitro binding, localization, functional overexpression phenotype) in a single foundational study\",\n      \"pmids\": [\"11238449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Genetic analysis of Drosophila hook (ortholog of human HOOK proteins) demonstrated that all three domains (N-terminal, central coiled-coil, and C-terminal) are required for Hook function in endocytic trafficking; loss-of-function alleles block accumulation of both transmembrane and soluble ligands in multivesicular bodies. The importance of these domains is conserved between Drosophila and human Hook proteins.\",\n      \"method\": \"Genetic screen, null allele isolation, domain truncation analysis, endocytic trafficking assay\",\n      \"journal\": \"Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — null allele genetic analysis with domain structure-function epistasis, foundational mechanistic paper\",\n      \"pmids\": [\"9927460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Salmonella SpiC protein directly binds HOOK3 in macrophage cytosol; GST-SpiC pulled down HOOK3 from murine macrophages and anti-HOOK3 antibodies co-precipitated SpiC from infected cells. SpiC expression disrupted Golgi morphology and altered lysosome distribution, mimicking the phenotype of a hook3 dominant-negative mutant, indicating that SpiC targets HOOK3 function to inhibit phagosome-lysosome fusion.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, dominant-negative phenotypic comparison, Golgi morphology assay\",\n      \"journal\": \"Molecular microbiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus functional phenotypic validation linking HOOK3 to lysosomal trafficking\",\n      \"pmids\": [\"12950921\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"IIGP (a 47 kDa IFN-inducible GTPase) interacts with HOOK3 in a GTP-bound conformation-dependent manner. The physical complex was detected by co-immunoprecipitation in IFN-γ-stimulated macrophages. Both proteins co-localize in the cis-Golgi and perinuclear regions, suggesting HOOK3 links IFN-γ-inducible GTPase activity to cytoskeleton-based membrane trafficking.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, subcellular fractionation, immunofluorescence co-localization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — yeast two-hybrid plus co-IP in primary cells, but interaction is minor subfraction and functional consequence not fully resolved\",\n      \"pmids\": [\"15075236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HOOK3-RET is a novel oncogenic fusion protein arising from chromosomal rearrangement in papillary thyroid carcinoma, fusing exon 11 of HOOK3 to exon 12 of RET. The chimeric HOOK3-RET protein (~88 kDa) retains HOOK3 coiled-coil domains and the intact RET tyrosine kinase domain. Expression in NIH3T3 cells caused transformed foci formation and tumor growth in nude mice.\",\n      \"method\": \"5'RACE, Western blot with anti-RET antibody, NIH3T3 transformation assay, nude mouse xenograft\",\n      \"journal\": \"Endocrine-related cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — fusion protein identified and functionally validated by transformation and in vivo tumor assays\",\n      \"pmids\": [\"17639057\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"HOOK3 interacts directly with the cytoplasmic domain of scavenger receptor A (SR-A). The interaction was identified by yeast two-hybrid and mass spectrometry, and confirmed by pulldown and co-immunoprecipitation of overexpressed and endogenous proteins. The C-terminal Val614-Ala717 region of HOOK3 is required for binding to negatively charged residues (Glu12, Asp13, Asp15) in the SR-A cytoplasmic domain. siRNA knockdown of HOOK3 promoted SR-A surface expression, ligand uptake, and protein stability, indicating HOOK3 participates in SR-A turnover.\",\n      \"method\": \"Yeast two-hybrid, mass spectrometry, pulldown, co-immunoprecipitation, domain truncation, siRNA knockdown with receptor trafficking assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including domain mapping and functional siRNA knockdown with defined trafficking phenotype\",\n      \"pmids\": [\"17237231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HOOK3 is a component of the FHF complex (FTS/Hook/FHIP), a ~500 kDa multiprotein assembly containing FTS, Hook1, Hook2, Hook3, and FHIP. Hook proteins form homo- and heterodimers and interact with members of the HOPS complex. The FHF complex promotes endosomal clustering and EGF trafficking from early-to-late endosomes/lysosomes.\",\n      \"method\": \"Proteomic analysis (AP-MS), yeast two-hybrid, co-immunoprecipitation, siRNA depletion with EGF trafficking assay, lysosomal clustering assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — AP-MS complex identification confirmed by multiple biochemical methods with functional trafficking readout\",\n      \"pmids\": [\"18799622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"HOOK3 interacts with PCM1 (Pericentriolar Material 1) and is recruited to pericentriolar satellites through this interaction. Disruption of the HOOK3-PCM1 interaction in vivo impairs interkinetic nuclear migration in embryonic neural progenitors, leading to overproduction of neurons and premature depletion of the neural progenitor pool in the developing neocortex.\",\n      \"method\": \"Co-immunoprecipitation, in vivo disruption of protein-protein interaction, live imaging, cortical neurogenesis assay\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP plus in vivo loss-of-function with defined neurogenic phenotype\",\n      \"pmids\": [\"20152126\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HOOK3 is expressed in neurons and localizes to tau aggregates in Alzheimer's disease brain. Hook3 deficiency modeled in cultured cells slows endosomal transport and increases β-amyloid production, indicating that HOOK3 participates in endosomal transport relevant to amyloid precursor protein processing.\",\n      \"method\": \"Immunofluorescence in AD brain tissue, siRNA knockdown with endosomal transport assay, β-amyloid ELISA\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — localization plus knockdown with defined transport and amyloid phenotype, single lab\",\n      \"pmids\": [\"25799409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Hook3 activates dynein-dynactin motility by directly binding to the dynein light intermediate chain 1 (LIC1) through its conserved Hook domain. Crystal structure of the Hook domain was solved, and structure-based mutagenesis identified two conserved surface residues critical for LIC1 binding. Hook3 also contains a distinct region required for allosteric activation of processive dynein-dynactin motility.\",\n      \"method\": \"Crystal structure determination, structure-based mutagenesis, in vitro binding assay, single-molecule motility assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and in vitro reconstitution of motility\",\n      \"pmids\": [\"27482052\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Mammalian Hook1 and Hook3 activate cytoplasmic dynein by stabilizing the dynein-dynactin supramolecular complex. Optogenetic recruitment of Hook proteins to peroxisomes drove rapid perinuclear transport. Single-molecule TIRF assays showed Hook1 and Hook3 induce longer run lengths and higher velocities than the activator BICD2. The N-terminal domain (resembling a calponin-homology domain) is required for dynein-dynactin interaction.\",\n      \"method\": \"Optogenetics, single-molecule TIRF motility assay, biochemical co-sedimentation, domain mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro single-molecule reconstitution combined with optogenetic live-cell assay and biochemical domain analysis\",\n      \"pmids\": [\"27365401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HOOK3 scaffolds both cytoplasmic dynein-1/dynactin and the kinesin-3 KIF1C into a tripartite complex. In vitro reconstitution with purified components showed full-length Hook3 activates dynein/dynactin motility and binds KIF1C tail, though without activating KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward microtubule minus ends, and KIF1C to transport dynein toward plus ends. In cells, KIF1C recruits Hook3 to the cell periphery.\",\n      \"method\": \"In vitro reconstitution with purified proteins, single-molecule motility assay, mass spectrometry interactome, live-cell imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with purified components plus single-molecule assays and cellular validation\",\n      \"pmids\": [\"31320392\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HOOK3 binds the same region of KIF1C tail as the PTPN21 FERM domain and releases KIF1C autoinhibition by increasing KIF1C landing rate onto microtubules. Full-length human KIF1C is a processive plus-end motor, and its autoinhibitory interaction (motor domain with stalk) is relieved by Hook3 or PTPN21 FERM domain binding, enabling cargo-activated transport.\",\n      \"method\": \"In vitro single-molecule motility assay, pulldown, co-immunoprecipitation, domain mapping, neuronal transport assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro reconstitution demonstrating autoinhibition release mechanism, multiple orthogonal methods\",\n      \"pmids\": [\"31217419\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HOOK3 is phosphorylated by ERK1c early in mitosis, and subsequently by AuroraA. This dual phosphorylation causes HOOK3 to detach from microtubules and increases its interaction with GM130, leading to Golgi destabilization and fragmentation during mitosis. In cycling cells HOOK3 links microtubules to the Golgi via both interactions.\",\n      \"method\": \"Phosphorylation assay, mutagenesis, co-immunoprecipitation, live-cell imaging, Golgi morphology quantification\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — kinase substrate identification with mechanistic follow-up, single lab with multiple methods\",\n      \"pmids\": [\"34189435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"A novel in-frame HOOK3-FGFR1 fusion gene was identified in MDS/8p11 myeloproliferative syndrome, with breakpoints at HOOK3 exon 11 and FGFR1 exon 10. The chimeric protein retains the entire FGFR1 tyrosine kinase domain and is associated with NF-κB pathway activation, evidenced by increased phosphorylation of p65 (Ser536) and IKBα (Ser32).\",\n      \"method\": \"RNA-seq with STAR-Fusion, FISH, qRT-PCR, Sanger sequencing, Western blot, phosphorylation antibody array\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — fusion identified and signaling characterized biochemically, but functional oncogenic validation not as complete as HOOK3-RET study\",\n      \"pmids\": [\"35081975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"KIF1C facilitates retrograde lysosomal transport by interacting with dynein-activating adaptor HOOK3, which associates with the lysosome-anchored protein RUFY3. KIF1C motor activity is not required and actually inhibits this retrograde process; instead KIF1C acts as a scaffold via Hook3 to promote dynein-driven perinuclear lysosomal positioning critical for autophagic and endocytic cargo degradation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, live-cell imaging, lysosome positioning assay, autophagy flux assay\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods establishing non-motor scaffolding role, single lab\",\n      \"pmids\": [\"39394274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Crystal structure of the Hook3(553-624)–KIF1C(714-809) complex was determined, revealing the structural basis for Hook3-KIF1C interaction. Structure-based mutagenesis showed this complex formation is necessary and sufficient for full-length protein interaction in HEK293T cells and for Hook3- and KIF1C-mediated anterograde transport in RPE1 cells. PTPN21 interaction with KIF1C is also part of this assembly.\",\n      \"method\": \"Crystal structure determination, structure-based mutagenesis, co-immunoprecipitation in HEK293T cells, live-cell transport assay in RPE1 cells\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure combined with mutagenesis and functional cellular transport validation\",\n      \"pmids\": [\"40312563\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HOOK3 is a cytosolic coiled-coil cargo adaptor that uses its conserved N-terminal Hook domain to bind dynein light intermediate chain 1 (LIC1) and allosterically activate processive dynein-dynactin motility toward microtubule minus ends, while its C-terminal domain anchors organelles (cis-Golgi, endosomes, lysosomes); it also scaffolds the opposite-polarity kinesin KIF1C through a structurally defined interaction at its C-terminal coiled-coil, enabling bidirectional cargo transport, and is regulated by ERK1c- and AuroraA-mediated phosphorylation that detaches it from microtubules to drive mitotic Golgi fragmentation.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"HOOK3 is a coiled-coil cargo adaptor that couples intracellular organelles to microtubule-based motors, coordinating bidirectional transport and organelle positioning. Its conserved N-terminal Hook domain binds microtubules and directly engages dynein light intermediate chain 1 (LIC1) to form and allosterically activate a processive dynein–dynactin motor complex, while a structurally resolved C-terminal region simultaneously scaffolds the kinesin-3 KIF1C, enabling opposing-polarity motors to co-travel on a single adaptor and regulate transport of endosomes, lysosomes, and Golgi membranes [PMID:27482052, PMID:31320392, PMID:40312563]. HOOK3 also interacts with PCM1 to traffic pericentriolar satellites required for interkinetic nuclear migration during cortical neurogenesis [PMID:20152126], and with the lysosome-anchored protein RUFY3 to facilitate dynein-driven retrograde lysosomal transport [PMID:39394274]. During mitosis, sequential phosphorylation by ERK1c and Aurora A detaches HOOK3 from microtubules and enhances its interaction with GM130, destabilizing Golgi–microtubule connections to permit Golgi fragmentation [PMID:34189435].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Establishing the foundational architecture: the conserved N-terminal domain binds microtubules while the divergent C-terminus binds Golgi membranes, and overexpression causes Golgi fragmentation, defining HOOK3 as an organelle–cytoskeleton linker.\",\n      \"evidence\": \"In vitro Golgi membrane binding, immunofluorescence, Brefeldin A treatment, and overexpression in mammalian cells\",\n      \"pmids\": [\"11238449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No motor protein partner identified\", \"Mechanism of Golgi fragmentation upon overexpression not resolved\", \"Whether Hook3 acts as an adaptor versus a tether was unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrating that HOOK3 is a functional target during infection: Salmonella SpiC physically interacts with HOOK3 and disrupts Golgi/lysosome morphology, implicating HOOK3 in phagosome–lysosome fusion.\",\n      \"evidence\": \"Reciprocal GST pulldown and co-IP from infected macrophages, dominant-negative phenocopy\",\n      \"pmids\": [\"12950921\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct mechanism by which SpiC inhibits Hook3 function unknown\", \"Whether SpiC blocks Hook3–motor or Hook3–membrane interactions not determined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Expanding the cargo repertoire: HOOK3 binds the cytoplasmic tail of scavenger receptor A (SR-A) via its positively charged C-terminal region, and Hook3 depletion increases SR-A surface expression and ligand uptake, establishing a role in receptor trafficking and turnover.\",\n      \"evidence\": \"Yeast two-hybrid, mass spectrometry, domain-mapping pulldowns, co-IP, and siRNA knockdown with SR-A surface expression readout\",\n      \"pmids\": [\"17237231\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Hook3 promotes SR-A endocytosis, recycling, or degradation not resolved\", \"Motor dependence of SR-A trafficking via Hook3 not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Linking HOOK3 to a developmental process: HOOK3 interaction with PCM1 is required for pericentriolar satellite trafficking and interkinetic nuclear migration in neural progenitors; disruption causes premature neuronal differentiation.\",\n      \"evidence\": \"Co-IP, in vivo disruption of Hook3–PCM1 interaction in embryonic neocortex, interkinetic nuclear migration and neurogenesis assays\",\n      \"pmids\": [\"20152126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which motor(s) drive pericentriolar satellite movement via Hook3 not identified\", \"Whether the phenotype is specific to Hook3 or shared with other Hook paralogs not addressed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Solving how HOOK3 engages and activates dynein: crystal structure of the Hook domain reveals the LIC1-binding surface, and mutagenesis plus reconstitution show that a separate region allosterically activates processive dynein–dynactin motility.\",\n      \"evidence\": \"Crystal structure determination, structure-based mutagenesis, in vitro reconstitution of dynein–dynactin–Hook3 complex, single-molecule motility assays\",\n      \"pmids\": [\"27482052\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How allosteric activation is transmitted structurally not determined\", \"Relative contributions of Hook3 versus other activating adaptors in vivo unclear\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealing HOOK3 as a bidirectional transport scaffold: Hook3 simultaneously binds dynein/dynactin and kinesin-3 KIF1C, enabling opposing motors to co-travel, and both Hook3 and PTPN21 relieve KIF1C autoinhibition to increase microtubule landing rates.\",\n      \"evidence\": \"In vitro reconstitution with purified proteins, single-molecule motility assays, co-IP, live-cell imaging, integrin trafficking rescue\",\n      \"pmids\": [\"31320392\", \"31217419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the cell regulates which motor dominates on a Hook3 scaffold not determined\", \"Structural basis of Hook3–KIF1C interaction not yet resolved at this time\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Explaining mitotic Golgi fragmentation: sequential phosphorylation of HOOK3 by ERK1c and Aurora A during mitosis detaches it from microtubules and redirects it to GM130, destabilizing Golgi–microtubule connections.\",\n      \"evidence\": \"In vitro kinase assays, microtubule co-sedimentation with phospho-mutants, co-IP with GM130, Golgi morphology imaging\",\n      \"pmids\": [\"34189435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphorylation sites not mapped at residue resolution in the publication\", \"Single-lab finding; independent replication lacking\", \"Whether phosphorylation also affects dynein/KIF1C binding not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defining a lysosome-specific transport complex: KIF1C scaffolds (but does not motor) dynein-driven retrograde lysosomal transport via Hook3, which connects to lysosomes through RUFY3.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, live-cell lysosome tracking, dominant-negative motor mutants, rescue experiments\",\n      \"pmids\": [\"39394274\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How RUFY3–Hook3 interaction is regulated not addressed\", \"Single study; independent validation needed\", \"Whether Hook3 also mediates anterograde lysosomal transport via KIF1C motor activity unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Completing the structural picture of bidirectional transport: crystal structure of the Hook3(553–624)–KIF1C(714–809) complex reveals the molecular interface, and structure-guided mutations show this contact is necessary and sufficient for anterograde transport.\",\n      \"evidence\": \"Crystal structure determination, structure-based mutagenesis, co-IP in HEK293T cells, anterograde transport assay in RPE1 cells\",\n      \"pmids\": [\"40312563\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Hook3 simultaneously coordinates dynein and KIF1C binding at single-molecule resolution not visualized\", \"Full-length Hook3–KIF1C–dynein–dynactin complex structure not available\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Hook3-mediated bidirectional transport is spatiotemporally regulated in vivo — including the signaling inputs that switch between dynein-driven and kinesin-driven modes, the cargo specificity determinants at the C-terminus, and the in vivo redundancy with Hook1/Hook2 — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full reconstitution of a Hook3–dynein–dynactin–KIF1C quaternary complex on cargo membranes\", \"Paralog-specific versus shared functions not systematically tested in vivo\", \"Upstream signaling that phosphoregulates Hook3 outside mitosis not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 10]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 3, 11, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 6, 10]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 1, 10]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [5, 11]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 3, 7, 9, 11, 12]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 10]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"complexes\": [\n      \"dynein–dynactin–Hook3\",\n      \"Hook3–KIF1C\"\n    ],\n    \"partners\": [\n      \"DYNC1LI1\",\n      \"KIF1C\",\n      \"PCM1\",\n      \"RUFY3\",\n      \"GOLGA2\",\n      \"MSR1\",\n      \"SpiC\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"HOOK3 is a coiled-coil cargo adaptor that coordinates bidirectional microtubule-based transport by simultaneously engaging the dynein-dynactin retrograde motor complex and the kinesin-3 KIF1C anterograde motor. Its conserved N-terminal Hook domain binds dynein light intermediate chain 1 (LIC1), allosterically activating processive dynein-dynactin motility toward microtubule minus ends, while its C-terminal coiled-coil domain anchors to organelle membranes—including the cis-Golgi, endosomes, and lysosomes—and scaffolds KIF1C through a structurally defined interface that also relieves KIF1C autoinhibition [PMID:27482052, PMID:31320392, PMID:31217419, PMID:40312563]. HOOK3 functions within the FHF complex (FTS/Hook/FHIP) to promote early-to-late endosome trafficking, interacts with PCM1 at pericentriolar satellites to support interkinetic nuclear migration in neural progenitors, and connects to lysosomes via RUFY3 to drive dynein-dependent perinuclear lysosomal positioning required for autophagic flux [PMID:18799622, PMID:20152126, PMID:39394274]. During mitosis, sequential phosphorylation by ERK1c and Aurora A detaches HOOK3 from microtubules and enhances its interaction with the Golgi matrix protein GM130, triggering Golgi fragmentation [PMID:34189435].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Genetic analysis of Drosophila Hook established that all three protein domains—N-terminal, coiled-coil, and C-terminal—are functionally required for endocytic trafficking, defining the conserved tripartite architecture that underlies Hook protein function across species.\",\n      \"evidence\": \"Null allele isolation, domain truncation analysis, and endocytic trafficking assays in Drosophila\",\n      \"pmids\": [\"9927460\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian-specific domain functions not yet tested\", \"Molecular targets of each domain unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of human HOOK3 as a cis-Golgi-associated, microtubule-binding protein whose overexpression fragments the Golgi revealed a specific organelle-tethering role distinct from general endocytic trafficking, assigning the N-terminal domain to microtubule binding and the C-terminal domain to organelle attachment.\",\n      \"evidence\": \"In vitro binding assay, subcellular fractionation, immunofluorescence, and overexpression phenotype in mammalian cells\",\n      \"pmids\": [\"11238449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Motor protein partners not yet identified\", \"Mechanism of Golgi fragmentation upon overexpression unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"The discovery that Salmonella effector SpiC directly binds HOOK3 to disrupt Golgi morphology and lysosome positioning demonstrated that HOOK3-dependent trafficking is exploited by intracellular pathogens to block phagosome-lysosome fusion.\",\n      \"evidence\": \"GST pulldown, reciprocal co-immunoprecipitation, and dominant-negative phenotypic comparison in macrophages\",\n      \"pmids\": [\"12950921\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of SpiC–HOOK3 interaction unresolved\", \"Whether SpiC directly inhibits motor recruitment unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapping the HOOK3 C-terminal domain interaction with scavenger receptor A (SR-A) cytoplasmic tail, and showing that HOOK3 depletion increases SR-A surface expression, established HOOK3 as an active participant in receptor turnover and endocytic cargo sorting.\",\n      \"evidence\": \"Yeast two-hybrid, mass spectrometry, domain truncation, co-IP, and siRNA knockdown with receptor trafficking assay\",\n      \"pmids\": [\"17237231\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HOOK3 directs SR-A to lysosomes or recycling pathway not determined\", \"Generality to other scavenger receptors untested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Proteomic identification of the FHF complex (FTS/Hook/FHIP) showed that HOOK3 operates as part of a ~500 kDa multiprotein assembly that promotes endosomal clustering and EGF trafficking from early to late endosomes, placing HOOK3 in a defined complex context.\",\n      \"evidence\": \"AP-MS, yeast two-hybrid, co-IP, siRNA depletion with EGF trafficking assay\",\n      \"pmids\": [\"18799622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of Hook homo- vs. heterodimers in the FHF complex unresolved\", \"Whether FHF complex directly engages motors not tested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"The interaction between HOOK3 and PCM1 at pericentriolar satellites, and its requirement for interkinetic nuclear migration in embryonic neural progenitors, revealed a non-Golgi role for HOOK3 in centrosome-based transport critical for neurogenesis.\",\n      \"evidence\": \"Co-IP, in vivo disruption of HOOK3-PCM1 interaction, live imaging, cortical neurogenesis assay in developing neocortex\",\n      \"pmids\": [\"20152126\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HOOK3 at satellites acts through dynein not directly shown\", \"Relevance to adult neurogenesis unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Crystal structure of the Hook domain and single-molecule reconstitution demonstrated that HOOK3 directly binds dynein LIC1 and allosterically activates processive dynein-dynactin motility, establishing HOOK3 as a bona fide dynein-activating adaptor with structurally defined motor-binding and activation regions.\",\n      \"evidence\": \"Crystal structure, structure-based mutagenesis, in vitro binding, single-molecule TIRF motility assay, optogenetic recruitment to peroxisomes\",\n      \"pmids\": [\"27482052\", \"27365401\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cryo-EM structure of the full HOOK3–dynein–dynactin supercomplex not available\", \"In vivo contribution of individual LIC1-contact residues not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"In vitro reconstitution revealed that HOOK3 simultaneously scaffolds dynein-dynactin and kinesin KIF1C into a single complex, enabling bidirectional transport and relieving KIF1C autoinhibition, answering how opposite-polarity motors are coordinated on the same adaptor.\",\n      \"evidence\": \"Purified protein reconstitution, single-molecule motility assay, mass spectrometry interactome, pulldown, neuronal transport assay\",\n      \"pmids\": [\"31320392\", \"31217419\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulatory switch between dynein-dominant and kinesin-dominant states unknown\", \"Whether the tripartite complex exists on specific cargo in vivo not visualized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of ERK1c and Aurora A as sequential mitotic kinases that phosphorylate HOOK3, detach it from microtubules, and enhance its GM130 interaction explained how Golgi fragmentation is triggered during mitotic entry through regulated HOOK3 modification.\",\n      \"evidence\": \"Phosphorylation assays, mutagenesis, co-IP, live-cell imaging, Golgi morphology quantification\",\n      \"pmids\": [\"34189435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Phosphorylation sites not mapped by mass spectrometry in vivo\", \"Whether phosphorylation also affects dynein or KIF1C binding untested\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Demonstration that KIF1C acts as a non-motor scaffold via HOOK3 and lysosome-anchored RUFY3 to promote dynein-driven perinuclear lysosomal positioning resolved how HOOK3 contributes to autophagic and endocytic degradation by positioning lysosomes near the cell center.\",\n      \"evidence\": \"Co-IP, siRNA knockdown, live-cell imaging, lysosome positioning assay, autophagy flux assay\",\n      \"pmids\": [\"39394274\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct HOOK3–RUFY3 binding not demonstrated with purified proteins\", \"Contribution of the FHF complex to this pathway not addressed\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Crystal structure of the Hook3(553–624)–KIF1C(714–809) complex provided the atomic-resolution basis for the adaptor–kinesin interaction, and structure-guided mutations confirmed this interface is necessary and sufficient for HOOK3–KIF1C-mediated transport in cells.\",\n      \"evidence\": \"Crystal structure, structure-based mutagenesis, co-IP in HEK293T, live-cell transport assay in RPE1 cells\",\n      \"pmids\": [\"40312563\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structure of the full-length HOOK3 dimer with both dynein-dynactin and KIF1C simultaneously bound\", \"Post-translational regulation of this interface not explored\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include how HOOK3-dependent transport is cargo-selectively regulated in vivo, whether distinct phosphorylation events differentially control dynein versus KIF1C engagement, and what structural features govern HOOK3 specificity relative to HOOK1 and HOOK2.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No cryo-EM structure of the HOOK3–dynein–dynactin–KIF1C supercomplex\", \"Cargo-specific adaptor-receptor interactions beyond SR-A and RUFY3 are poorly defined\", \"Paralog-specific functions among HOOK1/2/3 not systematically dissected\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [9, 10, 11, 12, 16]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0, 1, 9, 10, 13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [0, 2, 3, 13]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [1, 6, 8]},\n      {\"term_id\": \"GO:0005764\", \"supporting_discovery_ids\": [2, 15]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 9, 10]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [1, 2, 5, 6, 8, 15]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [15]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 7, 13]}\n    ],\n    \"complexes\": [\n      \"FHF complex (FTS/Hook/FHIP)\",\n      \"Dynein-dynactin-HOOK3 activating adaptor complex\",\n      \"HOOK3-KIF1C bidirectional transport complex\"\n    ],\n    \"partners\": [\n      \"DYNC1LI1\",\n      \"KIF1C\",\n      \"PCM1\",\n      \"FTS\",\n      \"FHIP\",\n      \"GM130\",\n      \"RUFY3\",\n      \"MSR1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}