{"gene":"RAP2C","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2005,"finding":"Rap2C is a new member of the Rap2 subfamily of small GTPases that binds GTP in a Mg2+-dependent manner, with slower GDP release and lower relative GTP affinity compared to Rap2B. When expressed in eukaryotic cells, Rap2C localizes to the plasma membrane via its C-terminal CAAX motif.","method":"In vitro nucleotide binding assays with recombinant purified protein, eukaryotic cell expression with subcellular localization","journal":"Biochimie","confidence":"High","confidence_rationale":"Tier 1 — in vitro biochemical characterization with purified recombinant protein, multiple orthogonal methods","pmids":["16213650"],"is_preprint":false},{"year":2007,"finding":"Overexpression of RAP2C in HEK293T cells activates transcriptional activity of the serum response element (SRE), suggesting involvement in SRE-mediated gene transcription. RAP2C protein localizes to the cytoplasm when overexpressed in COS-7 cells.","method":"Reporter gene (luciferase) assays, RT-PCR, overexpression in COS-7 and HEK293T cells","journal":"Molecular biology reports","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, reporter assay with overexpression, no endogenous protein validation","pmids":["17447155"],"is_preprint":false},{"year":2013,"finding":"JAM-A associates directly with ZO-2 and indirectly with afadin; this complex, together with PDZ-GEF1, activates the small GTPase Rap2c to regulate epithelial barrier function by controlling apical actomyosin contraction via RhoA activity and nonmuscle myosin phosphorylation.","method":"Co-immunoprecipitation, siRNA knockdown with paracellular permeability assays, JAM-A-deficient mice, RhoA activity assays, myosin phosphorylation western blot","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, epistasis via siRNA, multiple orthogonal readouts, in vivo mouse model, replicated across cell and animal systems","pmids":["23885123"],"is_preprint":false},{"year":2018,"finding":"Rap2c overexpression promotes migration and invasion of osteosarcoma cells by increasing MMP2 activity and phosphorylation of Akt, while decreasing TIMP2; Rap2c knockdown has the opposite effect with no impact on proliferation or apoptosis.","method":"Overexpression and siRNA knockdown, Transwell migration/invasion assay, MMP2 activity assay, western blot for pAkt and TIMP2","journal":"Oncology letters","confidence":"Medium","confidence_rationale":"Tier 3 — single lab, loss- and gain-of-function with defined phenotypic readouts, no direct pathway reconstitution","pmids":["29552178"],"is_preprint":false},{"year":2018,"finding":"Rap2c is the only Rap2 isoform expressed in A20 B cells and is required for BCR-induced reorientation of the microtubule-organizing center (MTOC) and F-actin remodeling at the antigen contact site.","method":"siRNA knockdown of Rap2c, live imaging of MTOC polarization and F-actin remodeling in B cells stimulated on antigen-coated surfaces","journal":"Small GTPases","confidence":"Medium","confidence_rationale":"Tier 2 — clean KD with specific cellular phenotype (MTOC reorientation), direct imaging readout","pmids":["29457987"],"is_preprint":false},{"year":2019,"finding":"miR-188-5p targets the 3'UTR of Rap2c mRNA (confirmed by dual-luciferase assay), and Rap2c acts as a molecular switch for MAPK signaling to promote proliferation and migration while decreasing apoptosis in breast cancer cells.","method":"Dual-luciferase reporter assay, siRNA knockdown, overexpression, CCK-8, flow cytometry, Transwell assay","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 3 — luciferase validation of miRNA-target interaction, functional knockdown/overexpression with MAPK pathway readout, single lab","pmids":["31541458"],"is_preprint":false},{"year":2020,"finding":"miR-195 binds to Rap2C mRNA (confirmed by luciferase reporter assay) and inhibits Rap2C expression, thereby suppressing MAPK signaling and promoting apoptosis while inhibiting proliferation in small cell lung cancer cells.","method":"Luciferase reporter assay, qRT-PCR, western blot, overexpression and knockdown, CCK-8, flow cytometry, subcutaneous mouse model","journal":"Technology in cancer research & treatment","confidence":"Medium","confidence_rationale":"Tier 3 — luciferase validation, functional knockdown/overexpression with MAPK pathway readout, single lab","pmids":["33302819"],"is_preprint":false},{"year":2020,"finding":"circRNA_100290 acts as a sponge for miR-136-5p, relieving miR-136-5p-mediated repression of RAP2C, thereby promoting proliferation, migration, and invasion in laryngeal squamous cell carcinoma cells.","method":"Dual-luciferase reporter assay, siRNA knockdown, overexpression, CCK-8, Transwell assay, in vivo xenograft","journal":"Biomedicine & pharmacotherapy","confidence":"Medium","confidence_rationale":"Tier 3 — luciferase validation of both interactions, functional rescue experiments, single lab","pmids":["32014687"],"is_preprint":false},{"year":2021,"finding":"In fibroblasts, GPCR agonism activates cAMP effectors EPAC1/2, which activate RAP2C, which in turn activates the serine/threonine kinase MAP4K7, leading to LATS1/2-mediated phosphorylation and nuclear exclusion (inactivation) of YAP/TAZ, reducing fibroblast proliferation, contraction, and extracellular matrix production.","method":"siRNA knockdown of EPAC1/2, RAP2C, and MAP4K7 in human lung fibroblasts; YAP/TAZ nuclear localization imaging; fibroblast proliferation, contraction, and ECM assays","journal":"Journal of cellular physiology","confidence":"High","confidence_rationale":"Tier 2 — epistatic ordering of pathway components with sequential knockdowns and multiple orthogonal functional readouts in primary human cells","pmids":["34046891"],"is_preprint":false},{"year":2021,"finding":"RAP2C promotes apoptosis, inflammatory cytokine release, and extracellular matrix degradation in nucleus pulposus cells via activation of ERK signaling; miR-200c-3p targets RAP2C's 3'UTR (confirmed by luciferase assay) to suppress this pathway.","method":"Luciferase reporter assay, miRNA mimic/inhibitor transfection, RAP2C knockdown, western blot for ERK signaling, cell apoptosis assay","journal":"Molecular medicine reports","confidence":"Medium","confidence_rationale":"Tier 3 — luciferase validation, loss-of-function with defined pathway readout, single lab","pmids":["34676879"],"is_preprint":false},{"year":2022,"finding":"The lncRNA LENOX promotes association of RAP2C GTPase with mitochondrial fission regulator DRP1, increasing DRP1 S637 phosphorylation, promoting mitochondrial fusion, and enhancing oxidative phosphorylation in melanoma cells, facilitating resistance to MAPK inhibitors.","method":"Co-immunoprecipitation, siRNA knockdown of LENOX and RAP2C, phosphorylation western blot (DRP1 S637), mitochondrial morphology imaging, metabolic assays, in vivo melanoma models","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP of RAP2C with DRP1, multiple orthogonal functional readouts (metabolic, morphological, phosphorylation), in vivo validation","pmids":["36214632"],"is_preprint":false},{"year":2024,"finding":"RAP2C interacts with MAP4K4 (confirmed by co-immunoprecipitation and immunofluorescence), and this interaction is upregulated by hypoxia/reoxygenation; RAP2C overexpression activates the MAPK pathway (JNK, P38, ERK phosphorylation) and promotes cardiomyocyte apoptosis via MAP4K4, while ischemic postconditioning reduces RAP2C–MAP4K4 interaction and cardiomyocyte apoptosis.","method":"Co-immunoprecipitation, immunofluorescence co-localization, RAP2C knockdown and overexpression, MAP4K4 knockdown, western blot for phospho-JNK/P38/ERK, apoptosis assay, rat I/R model","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP confirmation of RAP2C–MAP4K4 interaction with epistatic functional validation, preprint","pmids":["bio_10.1101_2024.12.04.626922"],"is_preprint":true}],"current_model":"RAP2C is a plasma membrane-localized small GTPase of the Rap2 subfamily that is activated by upstream signals including JAM-A/ZO-2/afadin/PDZ-GEF1 complexes and EPAC1/2 (downstream of Gαs-coupled GPCRs); once active, it regulates epithelial barrier function via actomyosin contraction (RhoA/myosin phosphorylation), controls YAP/TAZ nuclear localization in fibroblasts via MAP4K7→LATS1/2, promotes mitochondrial fusion and oxidative phosphorylation by associating with DRP1 (facilitated by lncRNA LENOX), drives MAPK signaling through MAP4K4 to regulate apoptosis in cardiomyocytes, and supports B cell MTOC polarization and actin remodeling upon BCR engagement."},"narrative":{"teleology":[{"year":2005,"claim":"Establishing RAP2C as a bona fide GTPase defined a new Rap2 subfamily member and showed it differs from RAP2B in nucleotide handling and reaches the plasma membrane via its CAAX motif.","evidence":"In vitro nucleotide binding assays with purified recombinant protein and subcellular localization in eukaryotic cells","pmids":["16213650"],"confidence":"High","gaps":["No GTPase-activating protein (GAP) or guanine nucleotide exchange factor (GEF) identified at this stage","Functional role in intact cells unknown","No comparison of isoform-specific signaling outputs"]},{"year":2007,"claim":"Demonstrating that RAP2C overexpression activates serum response element (SRE) transcription provided the first link between this GTPase and gene regulation, though the pathway was undefined.","evidence":"Luciferase reporter assays in HEK293T cells with RAP2C overexpression","pmids":["17447155"],"confidence":"Medium","gaps":["Overexpression-only system without endogenous protein validation","Downstream effectors mediating SRE activation not identified","No loss-of-function data"]},{"year":2013,"claim":"Placing RAP2C downstream of a JAM-A/ZO-2/afadin/PDZ-GEF1 junctional complex that regulates RhoA-dependent actomyosin contraction provided the first complete signaling axis and physiological context for RAP2C in epithelial barrier maintenance.","evidence":"Reciprocal co-immunoprecipitation, siRNA epistasis, paracellular permeability assays, JAM-A-deficient mice, RhoA activity and myosin phosphorylation readouts","pmids":["23885123"],"confidence":"High","gaps":["Direct GEF activity of PDZ-GEF1 on RAP2C not reconstituted in vitro","How RAP2C activates RhoA mechanistically is unclear","Relative contributions of Rap2 isoforms in vivo not resolved"]},{"year":2018,"claim":"Identifying RAP2C as the sole Rap2 isoform in A20 B cells required for BCR-induced MTOC reorientation and actin remodeling extended its cytoskeletal control function to immune cell polarity.","evidence":"siRNA knockdown of Rap2c in B cells with live imaging of MTOC polarization and F-actin dynamics on antigen-coated surfaces","pmids":["29457987"],"confidence":"Medium","gaps":["Effector kinase or scaffold linking RAP2C to MTOC reorientation machinery not identified","Single cell line (A20); generalizability to primary B cells untested","Upstream GEF activating RAP2C upon BCR engagement unknown"]},{"year":2019,"claim":"Multiple studies converged on RAP2C as a positive regulator of MAPK (ERK/JNK/P38) signaling in cancer and disease models, with its expression controlled by miRNAs (miR-188-5p, miR-195, miR-136-5p, miR-200c-3p), establishing RAP2C as a MAPK-activating node subject to extensive post-transcriptional regulation.","evidence":"Dual-luciferase 3′UTR reporter assays confirming miRNA–RAP2C interactions; knockdown and overexpression with MAPK phosphorylation readouts in breast cancer, SCLC, LSCC, and nucleus pulposus cells","pmids":["31541458","33302819","32014687","34676879"],"confidence":"Medium","gaps":["Direct biochemical mechanism by which RAP2C activates MAPK kinases not resolved","Isoform specificity (RAP2A/B vs C) in MAPK activation not tested","miRNA studies are individually single-lab without independent replication"]},{"year":2021,"claim":"Demonstrating that EPAC1/2→RAP2C→MAP4K7→LATS1/2 signaling excludes YAP/TAZ from the nucleus in fibroblasts connected RAP2C to the Hippo pathway and explained how Gαs-coupled GPCRs suppress fibrotic responses.","evidence":"Sequential siRNA knockdowns of EPAC1/2, RAP2C, and MAP4K7 in primary human lung fibroblasts with YAP/TAZ localization imaging, proliferation, contraction, and ECM assays","pmids":["34046891"],"confidence":"High","gaps":["Whether RAP2C directly binds and activates MAP4K7 or acts through an intermediate is unknown","Structural basis of RAP2C–MAP4K7 interaction not determined","In vivo relevance in fibrotic disease models not shown"]},{"year":2022,"claim":"Discovering that RAP2C associates with DRP1 and promotes DRP1 S637 phosphorylation to drive mitochondrial fusion and oxidative phosphorylation revealed a non-canonical mitochondrial function for this GTPase, with therapeutic relevance to MAPK-inhibitor resistance in melanoma.","evidence":"Reciprocal co-immunoprecipitation of RAP2C–DRP1, siRNA knockdown of LENOX and RAP2C, DRP1 phosphorylation western blot, mitochondrial morphology imaging, metabolic assays, in vivo melanoma models","pmids":["36214632"],"confidence":"High","gaps":["Whether RAP2C directly phosphorylates DRP1 or recruits a kinase is unresolved","Role of GTP-bound vs GDP-bound state in DRP1 interaction not tested","Generalizability beyond melanoma not established"]},{"year":null,"claim":"Key unresolved questions include the structural basis for RAP2C effector selectivity (MAP4K4/MAP4K7, DRP1, RhoA regulators), whether its GTPase cycle is differentially controlled by specific GAPs in different tissues, and how its mitochondrial versus plasma membrane pools are partitioned.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal or cryo-EM structure of RAP2C in complex with any effector","GAP specificity for RAP2C not characterized","Conditional knockout mouse models are lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[0]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[2,8,10]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,5,6,8,9,11]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[2]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[9,11]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[10]}],"complexes":[],"partners":["JAM-A","PDZ-GEF1","MAP4K7","DRP1","MAP4K4","EPAC1","EPAC2"],"other_free_text":[]},"mechanistic_narrative":"RAP2C is a plasma membrane-associated small GTPase of the Rap2 subfamily that integrates upstream signals from junctional complexes, GPCRs, and B cell receptors to regulate cytoskeletal dynamics, Hippo–YAP signaling, MAPK cascades, and mitochondrial morphology. At epithelial tight junctions, RAP2C is activated by a JAM-A/ZO-2/afadin/PDZ-GEF1 complex to control apical actomyosin contraction and barrier permeability through RhoA and nonmuscle myosin phosphorylation [PMID:23885123]. In fibroblasts, Gαs-coupled GPCR signaling activates RAP2C via EPAC1/2, which engages MAP4K7 to phosphorylate LATS1/2 and exclude YAP/TAZ from the nucleus, thereby suppressing proliferation and extracellular matrix production [PMID:34046891]. RAP2C also associates with the mitochondrial fission regulator DRP1, promoting DRP1 S637 phosphorylation and mitochondrial fusion to enhance oxidative phosphorylation [PMID:36214632], and activates MAPK signaling (ERK, JNK, P38) through MAP4K family kinases in multiple cell types [PMID:31541458, PMID:34676879]."},"prefetch_data":{"uniprot":{"accession":"Q9Y3L5","full_name":"Ras-related protein Rap-2c","aliases":[],"length_aa":183,"mass_kda":20.7,"function":"Small GTP-binding protein which cycles between a GDP-bound inactive and a GTP-bound active form. May play a role in cytoskeletal rearrangements and regulate cell spreading through activation of the effector TNIK. May play a role in SRE-mediated gene transcription","subcellular_location":"Cytoplasm; Recycling endosome membrane","url":"https://www.uniprot.org/uniprotkb/Q9Y3L5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAP2C","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RAP2C","total_profiled":1310},"omim":[{"mim_id":"301016","title":"RAS-RELATED PROTEIN 2C; RAP2C","url":"https://www.omim.org/entry/301016"},{"mim_id":"179541","title":"RAS-RELATED PROTEIN 2B; RAP2B","url":"https://www.omim.org/entry/179541"},{"mim_id":"179540","title":"RAS-RELATED PROTEIN 2A; RAP2A","url":"https://www.omim.org/entry/179540"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"endometrium 1","ntpm":86.4}],"url":"https://www.proteinatlas.org/search/RAP2C"},"hgnc":{"alias_symbol":["DKFZp313B211"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y3L5","domains":[{"cath_id":"3.40.50.300","chopping":"1-166","consensus_level":"high","plddt":95.7387,"start":1,"end":166}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3L5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3L5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y3L5-F1-predicted_aligned_error_v6.png","plddt_mean":91.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RAP2C","jax_strain_url":"https://www.jax.org/strain/search?query=RAP2C"},"sequence":{"accession":"Q9Y3L5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y3L5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y3L5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y3L5"}},"corpus_meta":[{"pmid":"23885123","id":"PMC_23885123","title":"JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function.","date":"2013","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/23885123","citation_count":102,"is_preprint":false},{"pmid":"16213650","id":"PMC_16213650","title":"Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins.","date":"2005","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/16213650","citation_count":43,"is_preprint":false},{"pmid":"32014687","id":"PMC_32014687","title":"Overexpression of circRNA_100290 promotes the progression of laryngeal squamous cell carcinoma through the miR-136-5p/RAP2C axis.","date":"2020","source":"Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie","url":"https://pubmed.ncbi.nlm.nih.gov/32014687","citation_count":38,"is_preprint":false},{"pmid":"31541458","id":"PMC_31541458","title":"MicroRNA-188-5p promotes apoptosis and inhibits cell proliferation of breast cancer cells via the MAPK signaling pathway by targeting Rap2c.","date":"2019","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/31541458","citation_count":38,"is_preprint":false},{"pmid":"34132374","id":"PMC_34132374","title":"Serum long non‑coding RNA NNT‑AS1 protected by exosome is a potential biomarker and functions as an oncogene via the miR‑496/RAP2C axis in colorectal cancer.","date":"2021","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/34132374","citation_count":33,"is_preprint":false},{"pmid":"36214632","id":"PMC_36214632","title":"The LncRNA LENOX Interacts with RAP2C to Regulate Metabolism and Promote Resistance to MAPK Inhibition in Melanoma.","date":"2022","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/36214632","citation_count":31,"is_preprint":false},{"pmid":"29552178","id":"PMC_29552178","title":"Ras-related protein Rap2c promotes the migration and invasion of human osteosarcoma cells.","date":"2018","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/29552178","citation_count":22,"is_preprint":false},{"pmid":"17447155","id":"PMC_17447155","title":"Cloning and characterization of the human gene RAP2C, a novel member of Ras family, which activates transcriptional activities of SRE.","date":"2007","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/17447155","citation_count":20,"is_preprint":false},{"pmid":"34046891","id":"PMC_34046891","title":"GPCR-mediated YAP/TAZ inactivation in fibroblasts via EPAC1/2, RAP2C, and MAP4K7.","date":"2021","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/34046891","citation_count":18,"is_preprint":false},{"pmid":"33302819","id":"PMC_33302819","title":"microRNA-195 Promotes Small Cell Lung Cancer Cell Apoptosis via Inhibiting Rap2C Protein-Dependent MAPK Signal Transduction.","date":"2020","source":"Technology in cancer research & treatment","url":"https://pubmed.ncbi.nlm.nih.gov/33302819","citation_count":9,"is_preprint":false},{"pmid":"34676879","id":"PMC_34676879","title":"MicroRNA‑200c‑3p suppresses intervertebral disc degeneration by targeting RAP2C/ERK signaling.","date":"2021","source":"Molecular medicine reports","url":"https://pubmed.ncbi.nlm.nih.gov/34676879","citation_count":8,"is_preprint":false},{"pmid":"28714326","id":"PMC_28714326","title":"Spinal cord ischemia-reperfusion causes damage of neurocyte by inhibiting RAP2C.","date":"2017","source":"Neurological research","url":"https://pubmed.ncbi.nlm.nih.gov/28714326","citation_count":8,"is_preprint":false},{"pmid":"29457987","id":"PMC_29457987","title":"The Rap2c GTPase facilitates B cell receptor-induced reorientation of the microtubule-organizing center.","date":"2018","source":"Small GTPases","url":"https://pubmed.ncbi.nlm.nih.gov/29457987","citation_count":4,"is_preprint":false},{"pmid":"33907572","id":"PMC_33907572","title":"Erratum: Ras-related protein Rap2c promotes the migration and invasion of human osteosarcoma cells.","date":"2021","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/33907572","citation_count":1,"is_preprint":false},{"pmid":"41280817","id":"PMC_41280817","title":"Stability of G/C DNA Tetraplexes at Physiological pH Formed at the Promoter-UTR Intersection of the RAP2C Gene: Structural Multitude of a G/C Rich Genomic Segment.","date":"2025","source":"ACS omega","url":"https://pubmed.ncbi.nlm.nih.gov/41280817","citation_count":0,"is_preprint":false},{"pmid":"41280826","id":"PMC_41280826","title":"Multiformity of a G/C Rich Segment at the Promoter-UTR Intersection of RAP2C Gene: Structural Validation by Circular Dichroism.","date":"2025","source":"ACS omega","url":"https://pubmed.ncbi.nlm.nih.gov/41280826","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.12.04.626922","title":"The Cardioprotective Effect of Ischemic Postconditioning is Mediated by Inhibiting RAP2C-MAP4K4 Pathway","date":"2024-12-05","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.04.626922","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9684,"output_tokens":2972,"usd":0.036816},"stage2":{"model":"claude-opus-4-6","input_tokens":6327,"output_tokens":2439,"usd":0.138915},"total_usd":0.175731,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"Rap2C is a new member of the Rap2 subfamily of small GTPases that binds GTP in a Mg2+-dependent manner, with slower GDP release and lower relative GTP affinity compared to Rap2B. When expressed in eukaryotic cells, Rap2C localizes to the plasma membrane via its C-terminal CAAX motif.\",\n      \"method\": \"In vitro nucleotide binding assays with recombinant purified protein, eukaryotic cell expression with subcellular localization\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical characterization with purified recombinant protein, multiple orthogonal methods\",\n      \"pmids\": [\"16213650\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Overexpression of RAP2C in HEK293T cells activates transcriptional activity of the serum response element (SRE), suggesting involvement in SRE-mediated gene transcription. RAP2C protein localizes to the cytoplasm when overexpressed in COS-7 cells.\",\n      \"method\": \"Reporter gene (luciferase) assays, RT-PCR, overexpression in COS-7 and HEK293T cells\",\n      \"journal\": \"Molecular biology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, reporter assay with overexpression, no endogenous protein validation\",\n      \"pmids\": [\"17447155\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"JAM-A associates directly with ZO-2 and indirectly with afadin; this complex, together with PDZ-GEF1, activates the small GTPase Rap2c to regulate epithelial barrier function by controlling apical actomyosin contraction via RhoA activity and nonmuscle myosin phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown with paracellular permeability assays, JAM-A-deficient mice, RhoA activity assays, myosin phosphorylation western blot\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, epistasis via siRNA, multiple orthogonal readouts, in vivo mouse model, replicated across cell and animal systems\",\n      \"pmids\": [\"23885123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Rap2c overexpression promotes migration and invasion of osteosarcoma cells by increasing MMP2 activity and phosphorylation of Akt, while decreasing TIMP2; Rap2c knockdown has the opposite effect with no impact on proliferation or apoptosis.\",\n      \"method\": \"Overexpression and siRNA knockdown, Transwell migration/invasion assay, MMP2 activity assay, western blot for pAkt and TIMP2\",\n      \"journal\": \"Oncology letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab, loss- and gain-of-function with defined phenotypic readouts, no direct pathway reconstitution\",\n      \"pmids\": [\"29552178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Rap2c is the only Rap2 isoform expressed in A20 B cells and is required for BCR-induced reorientation of the microtubule-organizing center (MTOC) and F-actin remodeling at the antigen contact site.\",\n      \"method\": \"siRNA knockdown of Rap2c, live imaging of MTOC polarization and F-actin remodeling in B cells stimulated on antigen-coated surfaces\",\n      \"journal\": \"Small GTPases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KD with specific cellular phenotype (MTOC reorientation), direct imaging readout\",\n      \"pmids\": [\"29457987\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-188-5p targets the 3'UTR of Rap2c mRNA (confirmed by dual-luciferase assay), and Rap2c acts as a molecular switch for MAPK signaling to promote proliferation and migration while decreasing apoptosis in breast cancer cells.\",\n      \"method\": \"Dual-luciferase reporter assay, siRNA knockdown, overexpression, CCK-8, flow cytometry, Transwell assay\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — luciferase validation of miRNA-target interaction, functional knockdown/overexpression with MAPK pathway readout, single lab\",\n      \"pmids\": [\"31541458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"miR-195 binds to Rap2C mRNA (confirmed by luciferase reporter assay) and inhibits Rap2C expression, thereby suppressing MAPK signaling and promoting apoptosis while inhibiting proliferation in small cell lung cancer cells.\",\n      \"method\": \"Luciferase reporter assay, qRT-PCR, western blot, overexpression and knockdown, CCK-8, flow cytometry, subcutaneous mouse model\",\n      \"journal\": \"Technology in cancer research & treatment\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — luciferase validation, functional knockdown/overexpression with MAPK pathway readout, single lab\",\n      \"pmids\": [\"33302819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"circRNA_100290 acts as a sponge for miR-136-5p, relieving miR-136-5p-mediated repression of RAP2C, thereby promoting proliferation, migration, and invasion in laryngeal squamous cell carcinoma cells.\",\n      \"method\": \"Dual-luciferase reporter assay, siRNA knockdown, overexpression, CCK-8, Transwell assay, in vivo xenograft\",\n      \"journal\": \"Biomedicine & pharmacotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — luciferase validation of both interactions, functional rescue experiments, single lab\",\n      \"pmids\": [\"32014687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In fibroblasts, GPCR agonism activates cAMP effectors EPAC1/2, which activate RAP2C, which in turn activates the serine/threonine kinase MAP4K7, leading to LATS1/2-mediated phosphorylation and nuclear exclusion (inactivation) of YAP/TAZ, reducing fibroblast proliferation, contraction, and extracellular matrix production.\",\n      \"method\": \"siRNA knockdown of EPAC1/2, RAP2C, and MAP4K7 in human lung fibroblasts; YAP/TAZ nuclear localization imaging; fibroblast proliferation, contraction, and ECM assays\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistatic ordering of pathway components with sequential knockdowns and multiple orthogonal functional readouts in primary human cells\",\n      \"pmids\": [\"34046891\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RAP2C promotes apoptosis, inflammatory cytokine release, and extracellular matrix degradation in nucleus pulposus cells via activation of ERK signaling; miR-200c-3p targets RAP2C's 3'UTR (confirmed by luciferase assay) to suppress this pathway.\",\n      \"method\": \"Luciferase reporter assay, miRNA mimic/inhibitor transfection, RAP2C knockdown, western blot for ERK signaling, cell apoptosis assay\",\n      \"journal\": \"Molecular medicine reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — luciferase validation, loss-of-function with defined pathway readout, single lab\",\n      \"pmids\": [\"34676879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The lncRNA LENOX promotes association of RAP2C GTPase with mitochondrial fission regulator DRP1, increasing DRP1 S637 phosphorylation, promoting mitochondrial fusion, and enhancing oxidative phosphorylation in melanoma cells, facilitating resistance to MAPK inhibitors.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown of LENOX and RAP2C, phosphorylation western blot (DRP1 S637), mitochondrial morphology imaging, metabolic assays, in vivo melanoma models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP of RAP2C with DRP1, multiple orthogonal functional readouts (metabolic, morphological, phosphorylation), in vivo validation\",\n      \"pmids\": [\"36214632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RAP2C interacts with MAP4K4 (confirmed by co-immunoprecipitation and immunofluorescence), and this interaction is upregulated by hypoxia/reoxygenation; RAP2C overexpression activates the MAPK pathway (JNK, P38, ERK phosphorylation) and promotes cardiomyocyte apoptosis via MAP4K4, while ischemic postconditioning reduces RAP2C–MAP4K4 interaction and cardiomyocyte apoptosis.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, RAP2C knockdown and overexpression, MAP4K4 knockdown, western blot for phospho-JNK/P38/ERK, apoptosis assay, rat I/R model\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP confirmation of RAP2C–MAP4K4 interaction with epistatic functional validation, preprint\",\n      \"pmids\": [\"bio_10.1101_2024.12.04.626922\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"RAP2C is a plasma membrane-localized small GTPase of the Rap2 subfamily that is activated by upstream signals including JAM-A/ZO-2/afadin/PDZ-GEF1 complexes and EPAC1/2 (downstream of Gαs-coupled GPCRs); once active, it regulates epithelial barrier function via actomyosin contraction (RhoA/myosin phosphorylation), controls YAP/TAZ nuclear localization in fibroblasts via MAP4K7→LATS1/2, promotes mitochondrial fusion and oxidative phosphorylation by associating with DRP1 (facilitated by lncRNA LENOX), drives MAPK signaling through MAP4K4 to regulate apoptosis in cardiomyocytes, and supports B cell MTOC polarization and actin remodeling upon BCR engagement.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RAP2C is a plasma membrane-associated small GTPase of the Rap2 subfamily that integrates upstream signals from junctional complexes, GPCRs, and B cell receptors to regulate cytoskeletal dynamics, Hippo–YAP signaling, MAPK cascades, and mitochondrial morphology. At epithelial tight junctions, RAP2C is activated by a JAM-A/ZO-2/afadin/PDZ-GEF1 complex to control apical actomyosin contraction and barrier permeability through RhoA and nonmuscle myosin phosphorylation [PMID:23885123]. In fibroblasts, Gαs-coupled GPCR signaling activates RAP2C via EPAC1/2, which engages MAP4K7 to phosphorylate LATS1/2 and exclude YAP/TAZ from the nucleus, thereby suppressing proliferation and extracellular matrix production [PMID:34046891]. RAP2C also associates with the mitochondrial fission regulator DRP1, promoting DRP1 S637 phosphorylation and mitochondrial fusion to enhance oxidative phosphorylation [PMID:36214632], and activates MAPK signaling (ERK, JNK, P38) through MAP4K family kinases in multiple cell types [PMID:31541458, PMID:34676879].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing RAP2C as a bona fide GTPase defined a new Rap2 subfamily member and showed it differs from RAP2B in nucleotide handling and reaches the plasma membrane via its CAAX motif.\",\n      \"evidence\": \"In vitro nucleotide binding assays with purified recombinant protein and subcellular localization in eukaryotic cells\",\n      \"pmids\": [\"16213650\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No GTPase-activating protein (GAP) or guanine nucleotide exchange factor (GEF) identified at this stage\", \"Functional role in intact cells unknown\", \"No comparison of isoform-specific signaling outputs\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating that RAP2C overexpression activates serum response element (SRE) transcription provided the first link between this GTPase and gene regulation, though the pathway was undefined.\",\n      \"evidence\": \"Luciferase reporter assays in HEK293T cells with RAP2C overexpression\",\n      \"pmids\": [\"17447155\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Overexpression-only system without endogenous protein validation\", \"Downstream effectors mediating SRE activation not identified\", \"No loss-of-function data\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placing RAP2C downstream of a JAM-A/ZO-2/afadin/PDZ-GEF1 junctional complex that regulates RhoA-dependent actomyosin contraction provided the first complete signaling axis and physiological context for RAP2C in epithelial barrier maintenance.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, siRNA epistasis, paracellular permeability assays, JAM-A-deficient mice, RhoA activity and myosin phosphorylation readouts\",\n      \"pmids\": [\"23885123\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct GEF activity of PDZ-GEF1 on RAP2C not reconstituted in vitro\", \"How RAP2C activates RhoA mechanistically is unclear\", \"Relative contributions of Rap2 isoforms in vivo not resolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identifying RAP2C as the sole Rap2 isoform in A20 B cells required for BCR-induced MTOC reorientation and actin remodeling extended its cytoskeletal control function to immune cell polarity.\",\n      \"evidence\": \"siRNA knockdown of Rap2c in B cells with live imaging of MTOC polarization and F-actin dynamics on antigen-coated surfaces\",\n      \"pmids\": [\"29457987\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effector kinase or scaffold linking RAP2C to MTOC reorientation machinery not identified\", \"Single cell line (A20); generalizability to primary B cells untested\", \"Upstream GEF activating RAP2C upon BCR engagement unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Multiple studies converged on RAP2C as a positive regulator of MAPK (ERK/JNK/P38) signaling in cancer and disease models, with its expression controlled by miRNAs (miR-188-5p, miR-195, miR-136-5p, miR-200c-3p), establishing RAP2C as a MAPK-activating node subject to extensive post-transcriptional regulation.\",\n      \"evidence\": \"Dual-luciferase 3′UTR reporter assays confirming miRNA–RAP2C interactions; knockdown and overexpression with MAPK phosphorylation readouts in breast cancer, SCLC, LSCC, and nucleus pulposus cells\",\n      \"pmids\": [\"31541458\", \"33302819\", \"32014687\", \"34676879\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical mechanism by which RAP2C activates MAPK kinases not resolved\", \"Isoform specificity (RAP2A/B vs C) in MAPK activation not tested\", \"miRNA studies are individually single-lab without independent replication\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that EPAC1/2→RAP2C→MAP4K7→LATS1/2 signaling excludes YAP/TAZ from the nucleus in fibroblasts connected RAP2C to the Hippo pathway and explained how Gαs-coupled GPCRs suppress fibrotic responses.\",\n      \"evidence\": \"Sequential siRNA knockdowns of EPAC1/2, RAP2C, and MAP4K7 in primary human lung fibroblasts with YAP/TAZ localization imaging, proliferation, contraction, and ECM assays\",\n      \"pmids\": [\"34046891\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RAP2C directly binds and activates MAP4K7 or acts through an intermediate is unknown\", \"Structural basis of RAP2C–MAP4K7 interaction not determined\", \"In vivo relevance in fibrotic disease models not shown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Discovering that RAP2C associates with DRP1 and promotes DRP1 S637 phosphorylation to drive mitochondrial fusion and oxidative phosphorylation revealed a non-canonical mitochondrial function for this GTPase, with therapeutic relevance to MAPK-inhibitor resistance in melanoma.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation of RAP2C–DRP1, siRNA knockdown of LENOX and RAP2C, DRP1 phosphorylation western blot, mitochondrial morphology imaging, metabolic assays, in vivo melanoma models\",\n      \"pmids\": [\"36214632\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RAP2C directly phosphorylates DRP1 or recruits a kinase is unresolved\", \"Role of GTP-bound vs GDP-bound state in DRP1 interaction not tested\", \"Generalizability beyond melanoma not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the structural basis for RAP2C effector selectivity (MAP4K4/MAP4K7, DRP1, RhoA regulators), whether its GTPase cycle is differentially controlled by specific GAPs in different tissues, and how its mitochondrial versus plasma membrane pools are partitioned.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal or cryo-EM structure of RAP2C in complex with any effector\", \"GAP specificity for RAP2C not characterized\", \"Conditional knockout mouse models are lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [2, 8, 10]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 5, 6, 8, 9, 11]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [9, 11]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"JAM-A\",\n      \"PDZ-GEF1\",\n      \"MAP4K7\",\n      \"DRP1\",\n      \"MAP4K4\",\n      \"EPAC1\",\n      \"EPAC2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}