{"gene":"RAB40B","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2013,"finding":"Rab40b GTPase is required for sorting of MMP2 and MMP9 into VAMP4-containing secretory vesicles and for transport of these vesicles to invadopodia, thereby regulating invadopodia-dependent extracellular matrix degradation and breast cancer cell invasion in vitro.","method":"Rab siRNA library screen, siRNA knockdown, co-localization, in vitro invasion assay, ECM degradation assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (siRNA screen, co-localization, functional invasion/ECM assays), replicated across subsequent studies","pmids":["23902685"],"is_preprint":false},{"year":2016,"finding":"Rab40b interacts with Tks5 (SH3PXD2A), a Src kinase substrate, as an effector protein; Tks5 functions as a tether mediating Rab40b-dependent targeting of MMP2/MMP9-containing transport vesicles to extending invadopodia. Both Rab40b and Tks5 levels are post-transcriptionally regulated by the tumor suppressor miR-204.","method":"Co-immunoprecipitation, vesicle targeting assays, siRNA knockdown, miR-204 overexpression/inhibition, ECM degradation assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP identifying effector interaction, combined with functional vesicle-targeting and miRNA regulatory experiments in the same study","pmids":["27789576"],"is_preprint":false},{"year":2021,"finding":"Rab40b interacts with Cullin5 via its C-terminal SOCS box domain to form an E3 ubiquitin ligase complex. Loss of Rab40b-Cullin5 binding decreases cell motility and invasion, increases stress fibers and stable focal adhesions, and decreases invadopodia formation and actin dynamics at the leading edge. EPLIN (epithelial protein lost in neoplasm) is identified as a direct binding partner of Rab40b and a substrate for Rab40b-Cullin5-dependent ubiquitylation and degradation.","method":"Co-immunoprecipitation, SOCS-domain mutant constructs, siRNA/CRISPR knockdown/knockout, actin dynamics imaging, focal adhesion assays, ubiquitylation assay, mass spectrometry substrate identification","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — domain mutagenesis, Co-IP, in-cell ubiquitylation assay, multiple orthogonal functional readouts in a single rigorous study","pmids":["33999101"],"is_preprint":false},{"year":2022,"finding":"The Rab40b/Cullin5 E3 ubiquitin ligase complex ubiquitylates Rap2, a Ras-like small GTPase. Ubiquitylation by Rab40b/Cullin5 regulates Rap2 activation and its recycling from the endolysosomal compartment to the lamellipodia of migrating cells, thereby controlling actin dynamics at the leading edge and breast cancer cell migration and invasion.","method":"Co-immunoprecipitation, in-cell ubiquitylation assay, dominant-negative and constitutively-active Rab40b constructs, live-cell imaging, Rap2 activation assay (GST-RalGDS pulldown), loss-of-function cell migration and invasion assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including ubiquitylation assay, GTPase activation pulldown, and live imaging, all in a single focused study","pmids":["35293963"],"is_preprint":false},{"year":2021,"finding":"Rab40b contains a C-terminal SOCS box that mediates binding to Cullin5, forming a CRL5-type E3 ubiquitin ligase complex; this complex can be used to identify substrates through unbiased proteomic approaches. The SOCS box is required for Rab40b's role in 3D breast cancer cell migration.","method":"SOCS-domain mutant constructs, Co-immunoprecipitation with Cullin5, proteomic substrate identification, 3D migration assay","journal":"Methods in molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — methodological description paper with supporting Co-IP and functional data, single lab, building on prior published work","pmids":["34453716"],"is_preprint":false},{"year":2023,"finding":"A nonsense mutation (Y83X) in RAB40B that deletes the C-terminal SOCS box causes axonal peripheral neuropathy (Charcot-Marie-Tooth disease type 2) in humans. In zebrafish, expression of mutant hRAB40B-Y83X caused defective swimming and reduced synaptic markers at neuromuscular junctions. In Drosophila, pan-glial (but not neuronal) expression of the mutant caused progressive locomotion decline. Genetic downregulation of Cul5 exacerbated the locomotion defect, consistent with the mutation causing loss of SOCS box–Cul5 interaction.","method":"Human genetic analysis, zebrafish transgenic expression, Drosophila ectopic expression (GAL4/UAS), neuromuscular junction immunostaining, behavioral locomotion assays, Cul5 genetic interaction","journal":"Experimental neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo model organism validation with genetic epistasis and multiple readouts, single lab","pmids":["38196136"],"is_preprint":false},{"year":2025,"finding":"DCLK1 forms a complex with KIF16B and RAB40B to traffic MMP9-containing cargo along invadopodia to the distal end, enabling ECM degradation in head and neck squamous cell carcinoma. DCLK1 knockdown disrupted this trafficking and reduced invadopodia numbers, in vitro invasion, and ECM degradation.","method":"Proximity ligation assay, co-immunoprecipitation, gelatin invadopodia assay, TMT-based proteomics, super-resolution confocal microscopy, siRNA knockdown, Boyden chamber invasion assay","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PLA and Co-IP confirm RAB40B-DCLK1-KIF16B complex, combined with functional knockdown readouts, single lab","pmids":["39994636"],"is_preprint":false},{"year":2025,"finding":"ARV oncolytic virus protein p17 transcriptionally downregulates Rab40b, Tks5, and Nck1, disrupting TKs5-Rab40b and TKs5-Nck1 complexes and inhibiting invadopodia formation. p17 also inhibits the Rab40b-PI3K-Akt signaling axis. Co-transfection with Rab40b or Tks5 rescued invadopodia formation and matrix degradation suppressed by p17.","method":"qRT-PCR, Western blot, co-immunoprecipitation, fluorescence imaging, gelatin degradation assay, rescue co-transfection experiments","journal":"Frontiers in cellular and infection microbiology","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple methods including rescue experiments confirming Rab40b role, but mechanistic detail on the Rab40b-PI3K-Akt axis is limited to expression-level evidence","pmids":["40575485"],"is_preprint":false}],"current_model":"RAB40B is an atypical Rab GTPase that contains a C-terminal SOCS box domain enabling it to bind Cullin5 and form a CRL5-type E3 ubiquitin ligase complex; this complex ubiquitylates substrates including EPLIN and Rap2 to regulate actin cytoskeleton dynamics, focal adhesion turnover, and endosomal recycling of Rap2 to lamellipodia, while RAB40B also mediates vesicular sorting of MMP2/MMP9 into VAMP4-positive secretory vesicles and, through interaction with the effector Tks5, targets these vesicles to invadopodia for ECM degradation, collectively driving breast cancer cell migration and invasion."},"narrative":{"mechanistic_narrative":"RAB40B is an atypical Rab GTPase that couples vesicular trafficking to substrate ubiquitylation, functioning as the substrate-recognition module of a CRL5-type E3 ubiquitin ligase to drive actin remodeling, invadopodia function, and breast cancer cell invasion [PMID:23902685, PMID:33999101]. Its C-terminal SOCS box mediates binding to Cullin5, and loss of this interaction increases stress fibers and stable focal adhesions while suppressing invadopodia formation, actin dynamics at the leading edge, and 3D migration [PMID:33999101, PMID:34453716]. Through this complex RAB40B ubiquitylates the actin regulator EPLIN, targeting it for degradation, and ubiquitylates the Ras-like GTPase Rap2 to control its activation and recycling from the endolysosomal compartment to lamellipodia [PMID:33999101, PMID:35293963]. In parallel, RAB40B sorts MMP2 and MMP9 into VAMP4-positive secretory vesicles and, via its effector Tks5, tethers and targets these vesicles to extending invadopodia for extracellular matrix degradation; RAB40B and Tks5 are jointly regulated by the tumor suppressor miR-204 [PMID:23902685, PMID:27789576]. A nonsense mutation (Y83X) that deletes the SOCS box causes axonal Charcot-Marie-Tooth disease type 2, and model-organism epistasis with Cul5 ties the neuropathy to loss of SOCS box–Cullin5 function [PMID:38196136].","teleology":[{"year":2013,"claim":"Established RAB40B's cellular role by showing it is required to sort matrix metalloproteinases into secretory vesicles and deliver them to invadopodia, defining a trafficking step needed for tumor cell invasion.","evidence":"Rab siRNA library screen with knockdown, co-localization, and in vitro ECM degradation/invasion assays in breast cancer cells","pmids":["23902685"],"confidence":"High","gaps":["Did not identify the effector tethering vesicles to invadopodia","GTPase biochemistry and nucleotide cycling not characterized"]},{"year":2016,"claim":"Identified Tks5 as the RAB40B effector that physically tethers MMP-containing vesicles to invadopodia, and placed both proteins under miR-204 control, linking the trafficking machinery to a tumor-suppressive regulatory input.","evidence":"Reciprocal co-immunoprecipitation, vesicle-targeting assays, siRNA knockdown, and miR-204 gain/loss of function with ECM degradation readout","pmids":["27789576"],"confidence":"High","gaps":["Did not address whether RAB40B has enzymatic/ligase activity beyond trafficking","Direct vs indirect nature of the Tks5 interaction surface unresolved"]},{"year":2021,"claim":"Reframed RAB40B as a CRL5 E3 ligase component by showing its SOCS box binds Cullin5 and that the complex ubiquitylates and degrades EPLIN, mechanistically connecting RAB40B to actin, focal adhesion, and invadopodia dynamics.","evidence":"SOCS-domain mutant constructs, Co-IP with Cullin5, in-cell ubiquitylation assay, mass spectrometry substrate ID, and actin/focal adhesion imaging plus a methods paper formalizing substrate discovery and 3D migration","pmids":["33999101","34453716"],"confidence":"High","gaps":["Full substrate repertoire beyond EPLIN not defined","How GTPase state regulates ligase assembly unresolved"]},{"year":2022,"claim":"Extended the ligase model to a second substrate by showing RAB40B/Cullin5 ubiquitylates Rap2 to control its activation and endolysosome-to-lamellipodia recycling, linking ubiquitylation directly to leading-edge actin and migration.","evidence":"Co-IP, in-cell ubiquitylation assay, dominant-negative/constitutively-active constructs, Rap2 activation pulldown, and live-cell imaging with migration/invasion assays","pmids":["35293963"],"confidence":"High","gaps":["Whether EPLIN and Rap2 ubiquitylation are coordinated or independent unknown","Lysine sites and ubiquitin chain topology not defined"]},{"year":2023,"claim":"Connected RAB40B to human disease by showing a SOCS box-truncating Y83X mutation causes axonal CMT2, with model-organism epistasis attributing the defect to lost Cullin5 interaction—extending its biology beyond cancer to neuromuscular function.","evidence":"Human genetic analysis, zebrafish and Drosophila transgenic expression with NMJ immunostaining, locomotion assays, and Cul5 genetic interaction","pmids":["38196136"],"confidence":"Medium","gaps":["Disease-relevant substrate(s) in neurons/glia not identified","Glial vs neuronal site-of-action mechanism unresolved"]},{"year":2025,"claim":"Broadened the trafficking machinery by placing RAB40B in a complex with DCLK1 and the kinesin KIF16B for motor-driven MMP9 cargo transport along invadopodia, and showed viral suppression of RAB40B disrupts invadopodia, indicating the pathway is a target for invasion control.","evidence":"Proximity ligation assay, Co-IP, super-resolution microscopy, TMT proteomics, and gelatin/Boyden invasion assays (DCLK1/KIF16B study); qRT-PCR, Western, Co-IP, and rescue co-transfection (ARV p17 study)","pmids":["39994636","40575485"],"confidence":"Medium","gaps":["Whether DCLK1/KIF16B coupling requires RAB40B ligase activity unknown","Mechanistic basis of the proposed RAB40B-PI3K-Akt axis limited to expression-level evidence"]},{"year":null,"claim":"How RAB40B integrates its GTPase nucleotide cycle with CRL5 ligase assembly and substrate selection, and the full substrate repertoire across cancer and neuronal contexts, remain open.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the RAB40B-Cullin5 complex or substrate engagement","GAP/GEF regulators and nucleotide state effects on ligase function uncharacterized","Substrates relevant to the neuropathy phenotype unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[0,3]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[2,3]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[2,4]}],"localization":[{"term_id":"GO:0031410","term_label":"cytoplasmic vesicle","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3]}],"pathway":[{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[0,1,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[0,6]}],"complexes":["CRL5 (Rab40b-Cullin5) E3 ubiquitin ligase","DCLK1-KIF16B-RAB40B trafficking complex"],"partners":["CUL5","SH3PXD2A","EPLIN","RAP2","DCLK1","KIF16B","VAMP4"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q12829","full_name":"Ras-related protein Rab-40B","aliases":["SOCS box-containing protein RAR","Protein Rar"],"length_aa":278,"mass_kda":31.0,"function":"RAB40B small GTPase acts as substrate-recognition components of the ECS(RAB40B) E3 ubiquitin ligase complex which mediates the ubiquitination of target proteins (PubMed:33999101, PubMed:35293963). The Rab40 subfamily belongs to the Rab family that are key regulators of intracellular membrane trafficking, from the formation of transport vesicles to their fusion with membranes. Rabs cycle between an inactive GDP-bound form and an active GTP-bound form that is able to recruit to membranes different sets of downstream effectors directly responsible for vesicle formation, movement, tethering and fusion (PubMed:27789576). As part of the ECS(RAB40B) complex, GTP-bound RAB40B promotes LIMA1/EPLIN ubiquitination and degradation, thereby regulating leading-edge actin dynamics during cell migration (PubMed:33999101). As part of the ECS(RAB40B) complex, GTP-bound RAB40B also ubiquitinates RAP2A GTPase which promotes its localization to lamellipodia and activation to drive cell migration. The ECS(RAB40B) complex does not mediate canonical ubiquitin-dependent degradation of RAP2 (PubMed:35293963). RAB40B also binds TKS5/SH3PXD2A effector independently from ECS complex to promote invadopodia-mediated extracellular matrix degradation (PubMed:27789576)","subcellular_location":"Cell membrane; Cytoplasm, cytosol; Cell projection, lamellipodium membrane; Cell projection, ruffle","url":"https://www.uniprot.org/uniprotkb/Q12829/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAB40B","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RAB40B","total_profiled":1310},"omim":[{"mim_id":"619551","title":"RAB40C, MEMBER RAS ONCOGENE FAMILY; RAB40C","url":"https://www.omim.org/entry/619551"},{"mim_id":"619550","title":"RAB40B, MEMBER RAS ONCOGENE FAMILY; RAB40B","url":"https://www.omim.org/entry/619550"},{"mim_id":"619455","title":"SH3 AND PX DOMAINS-CONTAINING PROTEIN 2A; SH3PXD2A","url":"https://www.omim.org/entry/619455"},{"mim_id":"610942","title":"MICRO RNA 204; MIR204","url":"https://www.omim.org/entry/610942"},{"mim_id":"607151","title":"MOYAMOYA DISEASE 2; MYMY2","url":"https://www.omim.org/entry/607151"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Vesicles","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":106.4}],"url":"https://www.proteinatlas.org/search/RAB40B"},"hgnc":{"alias_symbol":["SEC4L","RAR"],"prev_symbol":[]},"alphafold":{"accession":"Q12829","domains":[{"cath_id":"3.40.50.300","chopping":"12-177","consensus_level":"high","plddt":86.8359,"start":12,"end":177},{"cath_id":"-","chopping":"198-227","consensus_level":"medium","plddt":84.831,"start":198,"end":227}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12829","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q12829-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q12829-F1-predicted_aligned_error_v6.png","plddt_mean":75.69},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RAB40B","jax_strain_url":"https://www.jax.org/strain/search?query=RAB40B"},"sequence":{"accession":"Q12829","fasta_url":"https://rest.uniprot.org/uniprotkb/Q12829.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q12829/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q12829"}},"corpus_meta":[{"pmid":"25699257","id":"PMC_25699257","title":"The regulation of MMP targeting to invadopodia during cancer metastasis.","date":"2015","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/25699257","citation_count":235,"is_preprint":false},{"pmid":"23902685","id":"PMC_23902685","title":"Rab40b regulates trafficking of MMP2 and MMP9 during invadopodia formation and invasion of breast cancer cells.","date":"2013","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/23902685","citation_count":124,"is_preprint":false},{"pmid":"21282569","id":"PMC_21282569","title":"Role of miR-204 in the regulation of apoptosis, endoplasmic reticulum stress response, and inflammation in human trabecular meshwork cells.","date":"2011","source":"Investigative ophthalmology & visual science","url":"https://pubmed.ncbi.nlm.nih.gov/21282569","citation_count":107,"is_preprint":false},{"pmid":"19709424","id":"PMC_19709424","title":"Depletion of T-cell intracellular antigen proteins promotes cell proliferation.","date":"2009","source":"Genome biology","url":"https://pubmed.ncbi.nlm.nih.gov/19709424","citation_count":57,"is_preprint":false},{"pmid":"27789576","id":"PMC_27789576","title":"The role and regulation of Rab40b-Tks5 complex during invadopodia formation and cancer cell invasion.","date":"2016","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/27789576","citation_count":53,"is_preprint":false},{"pmid":"20615631","id":"PMC_20615631","title":"Proteomics of the radioresistant phenotype in head-and-neck cancer: Gp96 as a novel prediction marker and sensitizing target for radiotherapy.","date":"2010","source":"International journal of radiation oncology, biology, physics","url":"https://pubmed.ncbi.nlm.nih.gov/20615631","citation_count":48,"is_preprint":false},{"pmid":"29976164","id":"PMC_29976164","title":"Bulk tumour cell migration in lung carcinomas might be more common than epithelial-mesenchymal transition and be differently regulated.","date":"2018","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/29976164","citation_count":35,"is_preprint":false},{"pmid":"15160388","id":"PMC_15160388","title":"Vesicle transport in oligodendrocytes: probable role of Rab40c protein.","date":"2004","source":"Journal of neuroscience research","url":"https://pubmed.ncbi.nlm.nih.gov/15160388","citation_count":33,"is_preprint":false},{"pmid":"35293963","id":"PMC_35293963","title":"Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migration.","date":"2022","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/35293963","citation_count":23,"is_preprint":false},{"pmid":"27893846","id":"PMC_27893846","title":"Cerebellum Transcriptome of Mice Bred for High Voluntary Activity Offers Insights into Locomotor Control and Reward-Dependent Behaviors.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27893846","citation_count":22,"is_preprint":false},{"pmid":"33999101","id":"PMC_33999101","title":"Rab40-Cullin5 complex regulates EPLIN and actin cytoskeleton dynamics during cell migration.","date":"2021","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/33999101","citation_count":21,"is_preprint":false},{"pmid":"34453716","id":"PMC_34453716","title":"Methods to Study the Unique SOCS Box Domain of the Rab40 Small GTPase Subfamily.","date":"2021","source":"Methods in molecular biology (Clifton, N.J.)","url":"https://pubmed.ncbi.nlm.nih.gov/34453716","citation_count":8,"is_preprint":false},{"pmid":"36467401","id":"PMC_36467401","title":"The complex, dynamic SpliceOme of the small GTPase transcripts altered by technique, sex, genetics, tissue specificity, and RNA base editing.","date":"2022","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/36467401","citation_count":7,"is_preprint":false},{"pmid":"33116869","id":"PMC_33116869","title":"Overexpression of Rab40b Promotes Hepatocellular Carcinoma Cell Proliferation and Metastasis via PI3K/AKT Signaling Pathway.","date":"2020","source":"Cancer management and research","url":"https://pubmed.ncbi.nlm.nih.gov/33116869","citation_count":6,"is_preprint":false},{"pmid":"39994636","id":"PMC_39994636","title":"DCLK1-mediated regulation of invadopodia dynamics and matrix metalloproteinase trafficking drives invasive progression in head and neck squamous cell carcinoma.","date":"2025","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/39994636","citation_count":5,"is_preprint":false},{"pmid":"39066315","id":"PMC_39066315","title":"The Oncolytic Avian Reovirus p17 Protein Inhibits Invadopodia Formation in Murine Melanoma Cancer Cells by Suppressing the FAK/Src Pathway and the Formation of theTKs5/NCK1 Complex.","date":"2024","source":"Viruses","url":"https://pubmed.ncbi.nlm.nih.gov/39066315","citation_count":4,"is_preprint":false},{"pmid":"38196136","id":"PMC_38196136","title":"Peripheral Neuropathy and Decreased Locomotion of a RAB40B Mutation in Human and Model Animals.","date":"2023","source":"Experimental neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/38196136","citation_count":2,"is_preprint":false},{"pmid":"39376611","id":"PMC_39376611","title":"Analysis of chromatin accessibility in peripheral blood mononuclear cells from patients with early-stage breast cancer.","date":"2024","source":"Frontiers in pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/39376611","citation_count":2,"is_preprint":false},{"pmid":"36915458","id":"PMC_36915458","title":"Network and pathway-based analysis of candidate genes associated with esophageal adenocarcinoma.","date":"2023","source":"Journal of gastrointestinal oncology","url":"https://pubmed.ncbi.nlm.nih.gov/36915458","citation_count":2,"is_preprint":false},{"pmid":"40575485","id":"PMC_40575485","title":"The oncolytic avian reovirus p17 protein suppresses invadopodia formation via disruption of TKs5 complexes and oncogenic signaling pathways.","date":"2025","source":"Frontiers in cellular and infection microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/40575485","citation_count":1,"is_preprint":false},{"pmid":"35333280","id":"PMC_35333280","title":"Non-canonical ubiquitylation makes its mark on Rap2 and cell motility.","date":"2022","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/35333280","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12947,"output_tokens":2390,"usd":0.037345,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9445,"output_tokens":2907,"usd":0.05995,"stage2_stop_reason":"end_turn"},"total_usd":0.097295,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"Rab40b GTPase is required for sorting of MMP2 and MMP9 into VAMP4-containing secretory vesicles and for transport of these vesicles to invadopodia, thereby regulating invadopodia-dependent extracellular matrix degradation and breast cancer cell invasion in vitro.\",\n      \"method\": \"Rab siRNA library screen, siRNA knockdown, co-localization, in vitro invasion assay, ECM degradation assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (siRNA screen, co-localization, functional invasion/ECM assays), replicated across subsequent studies\",\n      \"pmids\": [\"23902685\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Rab40b interacts with Tks5 (SH3PXD2A), a Src kinase substrate, as an effector protein; Tks5 functions as a tether mediating Rab40b-dependent targeting of MMP2/MMP9-containing transport vesicles to extending invadopodia. Both Rab40b and Tks5 levels are post-transcriptionally regulated by the tumor suppressor miR-204.\",\n      \"method\": \"Co-immunoprecipitation, vesicle targeting assays, siRNA knockdown, miR-204 overexpression/inhibition, ECM degradation assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP identifying effector interaction, combined with functional vesicle-targeting and miRNA regulatory experiments in the same study\",\n      \"pmids\": [\"27789576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rab40b interacts with Cullin5 via its C-terminal SOCS box domain to form an E3 ubiquitin ligase complex. Loss of Rab40b-Cullin5 binding decreases cell motility and invasion, increases stress fibers and stable focal adhesions, and decreases invadopodia formation and actin dynamics at the leading edge. EPLIN (epithelial protein lost in neoplasm) is identified as a direct binding partner of Rab40b and a substrate for Rab40b-Cullin5-dependent ubiquitylation and degradation.\",\n      \"method\": \"Co-immunoprecipitation, SOCS-domain mutant constructs, siRNA/CRISPR knockdown/knockout, actin dynamics imaging, focal adhesion assays, ubiquitylation assay, mass spectrometry substrate identification\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — domain mutagenesis, Co-IP, in-cell ubiquitylation assay, multiple orthogonal functional readouts in a single rigorous study\",\n      \"pmids\": [\"33999101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The Rab40b/Cullin5 E3 ubiquitin ligase complex ubiquitylates Rap2, a Ras-like small GTPase. Ubiquitylation by Rab40b/Cullin5 regulates Rap2 activation and its recycling from the endolysosomal compartment to the lamellipodia of migrating cells, thereby controlling actin dynamics at the leading edge and breast cancer cell migration and invasion.\",\n      \"method\": \"Co-immunoprecipitation, in-cell ubiquitylation assay, dominant-negative and constitutively-active Rab40b constructs, live-cell imaging, Rap2 activation assay (GST-RalGDS pulldown), loss-of-function cell migration and invasion assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including ubiquitylation assay, GTPase activation pulldown, and live imaging, all in a single focused study\",\n      \"pmids\": [\"35293963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rab40b contains a C-terminal SOCS box that mediates binding to Cullin5, forming a CRL5-type E3 ubiquitin ligase complex; this complex can be used to identify substrates through unbiased proteomic approaches. The SOCS box is required for Rab40b's role in 3D breast cancer cell migration.\",\n      \"method\": \"SOCS-domain mutant constructs, Co-immunoprecipitation with Cullin5, proteomic substrate identification, 3D migration assay\",\n      \"journal\": \"Methods in molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — methodological description paper with supporting Co-IP and functional data, single lab, building on prior published work\",\n      \"pmids\": [\"34453716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"A nonsense mutation (Y83X) in RAB40B that deletes the C-terminal SOCS box causes axonal peripheral neuropathy (Charcot-Marie-Tooth disease type 2) in humans. In zebrafish, expression of mutant hRAB40B-Y83X caused defective swimming and reduced synaptic markers at neuromuscular junctions. In Drosophila, pan-glial (but not neuronal) expression of the mutant caused progressive locomotion decline. Genetic downregulation of Cul5 exacerbated the locomotion defect, consistent with the mutation causing loss of SOCS box–Cul5 interaction.\",\n      \"method\": \"Human genetic analysis, zebrafish transgenic expression, Drosophila ectopic expression (GAL4/UAS), neuromuscular junction immunostaining, behavioral locomotion assays, Cul5 genetic interaction\",\n      \"journal\": \"Experimental neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo model organism validation with genetic epistasis and multiple readouts, single lab\",\n      \"pmids\": [\"38196136\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"DCLK1 forms a complex with KIF16B and RAB40B to traffic MMP9-containing cargo along invadopodia to the distal end, enabling ECM degradation in head and neck squamous cell carcinoma. DCLK1 knockdown disrupted this trafficking and reduced invadopodia numbers, in vitro invasion, and ECM degradation.\",\n      \"method\": \"Proximity ligation assay, co-immunoprecipitation, gelatin invadopodia assay, TMT-based proteomics, super-resolution confocal microscopy, siRNA knockdown, Boyden chamber invasion assay\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PLA and Co-IP confirm RAB40B-DCLK1-KIF16B complex, combined with functional knockdown readouts, single lab\",\n      \"pmids\": [\"39994636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"ARV oncolytic virus protein p17 transcriptionally downregulates Rab40b, Tks5, and Nck1, disrupting TKs5-Rab40b and TKs5-Nck1 complexes and inhibiting invadopodia formation. p17 also inhibits the Rab40b-PI3K-Akt signaling axis. Co-transfection with Rab40b or Tks5 rescued invadopodia formation and matrix degradation suppressed by p17.\",\n      \"method\": \"qRT-PCR, Western blot, co-immunoprecipitation, fluorescence imaging, gelatin degradation assay, rescue co-transfection experiments\",\n      \"journal\": \"Frontiers in cellular and infection microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple methods including rescue experiments confirming Rab40b role, but mechanistic detail on the Rab40b-PI3K-Akt axis is limited to expression-level evidence\",\n      \"pmids\": [\"40575485\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RAB40B is an atypical Rab GTPase that contains a C-terminal SOCS box domain enabling it to bind Cullin5 and form a CRL5-type E3 ubiquitin ligase complex; this complex ubiquitylates substrates including EPLIN and Rap2 to regulate actin cytoskeleton dynamics, focal adhesion turnover, and endosomal recycling of Rap2 to lamellipodia, while RAB40B also mediates vesicular sorting of MMP2/MMP9 into VAMP4-positive secretory vesicles and, through interaction with the effector Tks5, targets these vesicles to invadopodia for ECM degradation, collectively driving breast cancer cell migration and invasion.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAB40B is an atypical Rab GTPase that couples vesicular trafficking to substrate ubiquitylation, functioning as the substrate-recognition module of a CRL5-type E3 ubiquitin ligase to drive actin remodeling, invadopodia function, and breast cancer cell invasion [#0, #2]. Its C-terminal SOCS box mediates binding to Cullin5, and loss of this interaction increases stress fibers and stable focal adhesions while suppressing invadopodia formation, actin dynamics at the leading edge, and 3D migration [#2, #4]. Through this complex RAB40B ubiquitylates the actin regulator EPLIN, targeting it for degradation, and ubiquitylates the Ras-like GTPase Rap2 to control its activation and recycling from the endolysosomal compartment to lamellipodia [#2, #3]. In parallel, RAB40B sorts MMP2 and MMP9 into VAMP4-positive secretory vesicles and, via its effector Tks5, tethers and targets these vesicles to extending invadopodia for extracellular matrix degradation; RAB40B and Tks5 are jointly regulated by the tumor suppressor miR-204 [#0, #1]. A nonsense mutation (Y83X) that deletes the SOCS box causes axonal Charcot-Marie-Tooth disease type 2, and model-organism epistasis with Cul5 ties the neuropathy to loss of SOCS box–Cullin5 function [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established RAB40B's cellular role by showing it is required to sort matrix metalloproteinases into secretory vesicles and deliver them to invadopodia, defining a trafficking step needed for tumor cell invasion.\",\n      \"evidence\": \"Rab siRNA library screen with knockdown, co-localization, and in vitro ECM degradation/invasion assays in breast cancer cells\",\n      \"pmids\": [\"23902685\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the effector tethering vesicles to invadopodia\", \"GTPase biochemistry and nucleotide cycling not characterized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified Tks5 as the RAB40B effector that physically tethers MMP-containing vesicles to invadopodia, and placed both proteins under miR-204 control, linking the trafficking machinery to a tumor-suppressive regulatory input.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, vesicle-targeting assays, siRNA knockdown, and miR-204 gain/loss of function with ECM degradation readout\",\n      \"pmids\": [\"27789576\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address whether RAB40B has enzymatic/ligase activity beyond trafficking\", \"Direct vs indirect nature of the Tks5 interaction surface unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Reframed RAB40B as a CRL5 E3 ligase component by showing its SOCS box binds Cullin5 and that the complex ubiquitylates and degrades EPLIN, mechanistically connecting RAB40B to actin, focal adhesion, and invadopodia dynamics.\",\n      \"evidence\": \"SOCS-domain mutant constructs, Co-IP with Cullin5, in-cell ubiquitylation assay, mass spectrometry substrate ID, and actin/focal adhesion imaging plus a methods paper formalizing substrate discovery and 3D migration\",\n      \"pmids\": [\"33999101\", \"34453716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full substrate repertoire beyond EPLIN not defined\", \"How GTPase state regulates ligase assembly unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended the ligase model to a second substrate by showing RAB40B/Cullin5 ubiquitylates Rap2 to control its activation and endolysosome-to-lamellipodia recycling, linking ubiquitylation directly to leading-edge actin and migration.\",\n      \"evidence\": \"Co-IP, in-cell ubiquitylation assay, dominant-negative/constitutively-active constructs, Rap2 activation pulldown, and live-cell imaging with migration/invasion assays\",\n      \"pmids\": [\"35293963\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EPLIN and Rap2 ubiquitylation are coordinated or independent unknown\", \"Lysine sites and ubiquitin chain topology not defined\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Connected RAB40B to human disease by showing a SOCS box-truncating Y83X mutation causes axonal CMT2, with model-organism epistasis attributing the defect to lost Cullin5 interaction—extending its biology beyond cancer to neuromuscular function.\",\n      \"evidence\": \"Human genetic analysis, zebrafish and Drosophila transgenic expression with NMJ immunostaining, locomotion assays, and Cul5 genetic interaction\",\n      \"pmids\": [\"38196136\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Disease-relevant substrate(s) in neurons/glia not identified\", \"Glial vs neuronal site-of-action mechanism unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Broadened the trafficking machinery by placing RAB40B in a complex with DCLK1 and the kinesin KIF16B for motor-driven MMP9 cargo transport along invadopodia, and showed viral suppression of RAB40B disrupts invadopodia, indicating the pathway is a target for invasion control.\",\n      \"evidence\": \"Proximity ligation assay, Co-IP, super-resolution microscopy, TMT proteomics, and gelatin/Boyden invasion assays (DCLK1/KIF16B study); qRT-PCR, Western, Co-IP, and rescue co-transfection (ARV p17 study)\",\n      \"pmids\": [\"39994636\", \"40575485\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether DCLK1/KIF16B coupling requires RAB40B ligase activity unknown\", \"Mechanistic basis of the proposed RAB40B-PI3K-Akt axis limited to expression-level evidence\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RAB40B integrates its GTPase nucleotide cycle with CRL5 ligase assembly and substrate selection, and the full substrate repertoire across cancer and neuronal contexts, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the RAB40B-Cullin5 complex or substrate engagement\", \"GAP/GEF regulators and nucleotide state effects on ligase function uncharacterized\", \"Substrates relevant to the neuropathy phenotype unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [2, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0031410\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"complexes\": [\"CRL5 (Rab40b-Cullin5) E3 ubiquitin ligase\", \"DCLK1-KIF16B-RAB40B trafficking complex\"],\n    \"partners\": [\"CUL5\", \"SH3PXD2A\", \"EPLIN\", \"RAP2\", \"DCLK1\", \"KIF16B\", \"VAMP4\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}