{"gene":"ARHGAP26","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":1999,"finding":"GRAF acts as a GAP for RhoA (not Cdc42) in vivo: microinjection of GRAF cDNA into Swiss 3T3 cells caused stress fiber clearing and filopodial-like extensions mimicking Rho inhibition; GAP-dead point mutants (R236Q or N351V) had no effect; GRAF blocked sphingosine-1-phosphate-stimulated (Rho-mediated) but not bradykinin-stimulated (Cdc42-mediated) cytoskeletal changes.","method":"Microinjection of wild-type and GAP-dead GRAF cDNA into Swiss 3T3 and PC12 cells; pharmacological inhibition with C3; selective agonist assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in-cell assays with isogenic controls, active-site mutagenesis, and selective agonist challenge confirming Rho specificity in vivo","pmids":["9858476"],"is_preprint":false},{"year":1998,"finding":"GRAF is phosphorylated on serine 510 by MAP kinase in vitro; EGF/NGF stimulation of PC12 cells induces a phosphatase-reversible mobility shift that is abolished by S510A mutation, indicating GRAF is phosphorylated at this site in vivo downstream of growth factor signaling.","method":"In vitro kinase assay with purified MAP kinase; site-directed mutagenesis (S510A); gel-mobility shift assay in EGF/NGF-stimulated PC12 cells; phosphatase treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis validated in vivo, single lab but two orthogonal methods","pmids":["9525907"],"is_preprint":false},{"year":1998,"finding":"The SH3 domain of GRAF interacts with two proline-rich sequences in cell adhesion kinase beta (CAKbeta/PYK2), and GRAF co-immunoprecipitates with CAKbeta from rat brain lysate, identifying a direct binding partnership in vivo.","method":"GST-SH3 domain affinity precipitation from rat brain lysate; GST dot-blot; co-immunoprecipitation from rat brain lysate; competitive binding assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal pulldown plus co-IP from native tissue with two orthogonal binding assays, single lab","pmids":["9494093"],"is_preprint":false},{"year":2000,"finding":"Crystal structure of the GRAF GAP domain (GrafGAP, 231 residues) at 2.4 Å resolution established domain boundaries and revealed that substrate specificity is determined by interaction with Glu-95/97 of RhoA/Cdc42 (absent in Rac1 as Ala-95); a Cdc42 E95A mutant reduced GrafGAP activity ~40-fold and Rac1 A95E increased it ~10-fold, confirming the structural prediction.","method":"X-ray crystallography (2.4 Å); in vitro GTPase-activating assay with Cdc42 E95A and Rac1 A95E mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus functional mutagenesis of both substrate and enzyme confirming mechanistic model","pmids":["10982819"],"is_preprint":false},{"year":2001,"finding":"PKNbeta (a Rho target kinase) interacts with the SH3 domains of GRAF and the related GRAF2 via proline-rich motifs; the active form of PKNbeta phosphorylates GRAF and GRAF2 in vitro; and GRAF co-immunoprecipitates with PKNbeta in COS-7 cells, revealing a feedback loop between Rho effector kinase and its GAP.","method":"Yeast two-hybrid screen; GST-SH3 pull-down; co-immunoprecipitation from transfected COS-7 cells; in vitro kinase assay with catalytically active PKNbeta","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vitro kinase assay, single lab, two orthogonal methods","pmids":["11432776"],"is_preprint":false},{"year":2009,"finding":"The BAR domain of GRAF family members (including ARHGAP26) acts as a cis-acting autoinhibitory element: it interacts directly with the GAP domain and inhibits its RhoGAP activity; in the autoinhibited state, GRAF can still bind and tubulate liposomes in vitro and generate lipid tubules in cells, demonstrating separable membrane-tubulation and GAP-inhibitory functions.","method":"In vitro GAP activity assay; direct BAR–GAP domain interaction assay; liposome tubulation assay; cell-based lipid tubule formation","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of autoinhibition plus functional domain dissection, single lab with multiple orthogonal assays","pmids":["18954304"],"is_preprint":false},{"year":2009,"finding":"In a breast cancer dormancy model, integrin α5β1 ligation by fibronectin recruits GRAF to the membrane, leading to RhoA inactivation and cortical actin stabilization; FGF-2-activated PI3K signaling is independently required for GRAF membrane relocalization and RhoA inactivation, indicating dual upstream signals converge on GRAF.","method":"Integrin blocking antibodies; PI3K inhibitors; immunofluorescence for FAK and GRAF localization; RhoA activity assay in breast cancer dormancy co-culture model","journal":"Cancer microenvironment","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — cell-based localization and RhoA activity assays with pharmacological inhibitors, single lab, single study","pmids":["19308677"],"is_preprint":false},{"year":2015,"finding":"The CLDN18-ARHGAP26 fusion protein (from a recurrent chromosomal translocation in gastric cancer) causes loss of epithelial integrity: fusion-expressing epithelial cells display impaired barrier properties, reduced cell-cell and cell-ECM adhesion, EMT morphology with long protrusions, retarded wound healing, inhibition of RHOA, and gain of invasion.","method":"Stable expression of CLDN18-ARHGAP26 fusion in epithelial cell lines; transwell invasion assay; barrier permeability assay; RhoA activity assay; wound healing assay; morphological imaging","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal functional assays in isogenic cell lines, independently replicated across 3 patient cases and multiple cell lines","pmids":["26146084"],"is_preprint":false},{"year":2019,"finding":"SMURF1 (an E3 ubiquitin ligase) interacts with and ubiquitinates ARHGAP26, promoting its degradation; SMURF1-induced ubiquitination of ARHGAP26 promotes ovarian cancer cell invasion and migration via the β-catenin pathway, and ARHGAP26 overexpression rescues SMURF1-driven migration.","method":"Co-immunoprecipitation; ubiquitination assay; ARHGAP26 overexpression and knockdown in A2780, HEY, SKOV3 cells; migration/invasion assays; in vivo lung metastasis model; DKK1 antagonist rescue experiment","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus ubiquitination assay plus functional rescue, single lab","pmids":["31004081"],"is_preprint":false},{"year":2017,"finding":"Drosophila GRAF (ortholog of ARHGAP26/GRAF1) localizes to GPI-enriched endocytic compartment (GEEC) membranes in plasmatocytes, is required for GEEC endocytosis, and directly interacts with EGFR in a ubiquitylation-dependent manner to facilitate EGFR internalization and degradation at high ligand doses, thereby restraining EGFR signaling and plasmatocyte proliferation.","method":"Drosophila genetics (Graf loss-of-function); immunofluorescence localization to GEEC membranes; EGFR signaling/proliferation assays; receptor internalization and degradation assays; co-immunoprecipitation of Graf and EGFR; ubiquitylation-dependence experiments","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ortholog study (Drosophila), multiple orthogonal methods including genetic epistasis and co-IP, single lab","pmids":["28993397"],"is_preprint":false},{"year":2021,"finding":"In Drosophila cellularization, GRAF (ortholog of ARHGAP26) is enriched at the cleavage furrow tip during actomyosin ring assembly; Graf depletion increases Rho-GTP, elevates Myosin II levels, and causes hyper-constriction of the actomyosin ring dependent on its RhoGAP domain; RhoGEF2 depletion or ROCK mutation suppresses the hyper-constriction phenotype, placing GRAF in a RhoGEF2–Rho–GRAF balance governing ring constriction.","method":"Drosophila genetics (Graf RNAi, Graf mutants, RhoGEF2 mutants, ROCK mutants); live imaging; Rho-GTP pull-down; Myosin II immunostaining; epistasis analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with domain-specific rescue, live imaging, and biochemical Rho-GTP measurement in ortholog, multiple orthogonal methods","pmids":["33835025"],"is_preprint":false},{"year":2018,"finding":"In human ductus arteriosus smooth muscle cells (DASMCs), ARHGAP26 knockdown reduces cell proliferation and migration; hypoxia suppresses ARHGAP26 expression and activates the RhoA-ROCK1-PTEN phosphorylation axis; ROCK inhibition (Y-27632) reverses the PTEN-mediated inhibition of proliferation and migration.","method":"ARHGAP26 siRNA knockdown in primary human DASMCs; hypoxia culture; proliferation and migration assays; Western blotting for RhoA activity, ROCK1, phospho-PTEN; pharmacological ROCK inhibition","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KD with specific phenotypic readouts and pharmacological pathway rescue, single lab","pmids":["30592323"],"is_preprint":false},{"year":2024,"finding":"ARHGAP26/GRAF1 is a PRKN/Parkin-binding protein that is rapidly recruited to damaged mitochondria; PINK1 phosphorylates ARHGAP26 at specific sites, enabling it to coordinate phagophore capture by regulating mitochondrial-associated actin remodeling and facilitating PRKN-LC3 interactions; ARHGAP26 depletion in mouse hearts blunts mitochondrial clearance and attenuates metabolic adaptations to stress.","method":"Co-immunoprecipitation (ARHGAP26–PRKN interaction); recruitment to damaged mitochondria (imaging); PINK1 phosphorylation site identification; ARHGAP26 knockout mouse hearts; mitophagy assays; metabolic stress assays","journal":"Autophagy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, in vivo KO, recruitment imaging, and phosphorylation site identification, single lab","pmids":["38855880"],"is_preprint":false},{"year":2024,"finding":"In a transgenic mouse model, CLDN18-ARHGAP26 expression in gastric organoids induces signet ring cell formation, cooperatively transforms gastric cells with Trp53 loss, activates RHOA and downstream FAK and YAP-TEAD signaling (opposite to the expected GAP loss-of-function), identifying the fusion as a gain-of-function oncogene; combined FAK and YAP/TEAD inhibition significantly blocks tumor growth.","method":"LSL-CLDN18-ARHGAP26 knock-in transgenic mouse model; gastric organoids; Cre-induced expression; RHOA activity assay; FAK phosphorylation; YAP target gene expression; pharmacological inhibition of FAK and YAP/TEAD; tumor growth assays","journal":"Gut","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo transgenic model plus organoid reconstitution plus biochemical pathway analysis and therapeutic rescue, single lab with multiple orthogonal methods","pmids":["38621923"],"is_preprint":false},{"year":2024,"finding":"ARHGAP26 interacts with Cofilin1 in oocytes to maintain mitochondrial integrity by regulating DRP1 dynamics; Arhgap26 knockout causes mitochondrial dysfunction, ROS accumulation, oocyte death at the GV stage, maturation arrest, and aneuploidy; restoration of ARHGAP26 protein level recovers oocyte quality.","method":"Arhgap26 knockout mouse model; co-immunoprecipitation (ARHGAP26–Cofilin1); DRP1 dynamics imaging; ROS measurement; oocyte maturation assays; in vitro fertilization; embryonic development tracking; transcriptome analysis; chromosomal microarray of patient","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with rescue, co-IP, and multiple phenotypic readouts, single lab","pmids":["39313581"],"is_preprint":false},{"year":2021,"finding":"Loss of Drosophila Graf (ARHGAP26 ortholog) causes abnormal MB β-lobe midline crossing during metamorphosis via a cell-autonomous mechanism; this phenotype requires activation of EGFR-MAPK signaling, consistent with Graf's role in negatively regulating this pathway; Graf mutants also display impaired olfactory long-term memory.","method":"Drosophila Graf loss-of-function mutants; MB-specific Graf and human OPHN1 rescue; cell-autonomous mosaic analysis; EGFR-MAPK pathway activation experiments; olfactory long-term memory behavioral assay","journal":"Molecular brain","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with mosaic analysis and rescue by human ortholog, single lab","pmids":["33892766"],"is_preprint":false},{"year":2006,"finding":"The GRAF promoter contains CpG sites whose methylation suppresses GRAF expression; treatment of leukemia cell lines with a demethylating agent and an HDAC inhibitor restores GRAF transcript levels, demonstrating epigenetic silencing as a mechanism of GRAF inactivation in AML/MDS.","method":"Reporter gene assay for promoter activity; bisulfite sequencing/methylation analysis of patient samples; demethylating agent + HDAC inhibitor treatment of leukemia cell lines; RT-PCR for GRAF expression","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter reporter assay plus pharmacological reactivation, single lab, multiple methods","pmids":["16404424"],"is_preprint":false}],"current_model":"ARHGAP26/GRAF1 is a multi-domain RhoGAP protein (BAR–PH–GAP–SH3) that acts as a GTPase-activating protein selective for RhoA and Cdc42 in vivo; its GAP domain is autoinhibited by the BAR domain in cis; it is phosphorylated by MAP kinase (Ser510) and by PINK1 at damaged mitochondria, is ubiquitinated by SMURF1, and interacts via its SH3 domain with FAK/PYK2, PKNbeta, EGFR, PRKN/Parkin, and Cofilin1; through RhoA inactivation it restrains actomyosin contractility, regulates integrin-mediated cytoskeletal reorganization and EGFR endocytic degradation, and coordinates mitophagy-associated actin remodeling, while oncogenic CLDN18-ARHGAP26 fusions paradoxically activate RHOA and downstream FAK/YAP-TEAD signaling to drive diffuse gastric cancer."},"narrative":{"mechanistic_narrative":"ARHGAP26 (GRAF/GRAF1) is a multidomain RhoGAP that restrains Rho-family GTPase signaling to control actomyosin contractility, membrane trafficking, and cytoskeletal remodeling [PMID:9858476, PMID:18954304, PMID:33835025]. In vivo it acts as a GAP selective for RhoA, with substrate specificity dictated structurally by recognition of Glu-95/97 of the GTPase, and its catalytic GAP domain is held inactive in cis by an autoinhibitory intramolecular interaction with its BAR domain — a domain that independently binds and tubulates membranes [PMID:9858476, PMID:10982819, PMID:18954304]. Through this RhoGAP activity ARHGAP26 inactivates RhoA upon integrin α5β1 engagement and PI3K signaling to stabilize cortical actin, and balances RhoGEF2–Rho-driven contractility at the actomyosin ring [PMID:19308677, PMID:33835025]. ARHGAP26 is integrated into growth-factor and stress signaling: it is phosphorylated at Ser510 by MAP kinase downstream of EGF/NGF, engages FAK/PYK2 and the Rho effector kinase PKNβ via its SH3 domain, and limits EGFR signaling by promoting ubiquitylation-dependent receptor internalization and degradation [PMID:9525907, PMID:9494093, PMID:11432776, PMID:28993397]. It also functions in mitochondrial quality control, where it is recruited to damaged mitochondria as a PRKN/Parkin-binding protein, is phosphorylated by PINK1, and coordinates actin remodeling for mitophagy, and it interacts with Cofilin1 to maintain mitochondrial integrity via DRP1 dynamics [PMID:38855880, PMID:39313581]. ARHGAP26 protein levels are controlled by SMURF1-mediated ubiquitination and by promoter CpG methylation, the latter silencing GRAF in AML/MDS [PMID:31004081, PMID:16404424]. Recurrent CLDN18-ARHGAP26 fusions in diffuse gastric cancer act paradoxically as gain-of-function oncogenes that activate RHOA and downstream FAK and YAP-TEAD signaling, disrupt epithelial adhesion, and drive invasion and signet-ring transformation [PMID:26146084, PMID:38621923].","teleology":[{"year":1998,"claim":"Establishing how GRAF couples to upstream signaling addressed whether this RhoGAP is regulated by, and physically linked to, growth-factor and adhesion kinases.","evidence":"In vitro MAP kinase assay with S510A mutagenesis and EGF/NGF-stimulated mobility shifts in PC12 cells; GST-SH3 pulldown and co-IP of PYK2/CAKβ from rat brain","pmids":["9525907","9494093"],"confidence":"High","gaps":["Functional consequence of Ser510 phosphorylation on GAP activity not defined","PYK2 interaction not linked to a downstream cytoskeletal output"]},{"year":1999,"claim":"Defining GRAF's in vivo substrate clarified which Rho-family GTPase it regulates, anchoring its cellular role in actin cytoskeletal control.","evidence":"Microinjection of wild-type vs GAP-dead GRAF into Swiss 3T3/PC12 cells with selective agonist challenge and C3 inhibition","pmids":["9858476"],"confidence":"High","gaps":["In-cell assay does not exclude Cdc42 GAP activity under other conditions","Physiological trigger of GRAF activation not addressed"]},{"year":2000,"claim":"Solving the GAP domain structure explained the molecular basis of substrate selectivity at atomic resolution.","evidence":"X-ray crystallography at 2.4 Å plus reciprocal GTPase mutagenesis (Cdc42 E95A, Rac1 A95E)","pmids":["10982819"],"confidence":"High","gaps":["Structure of full-length protein and BAR-GAP arrangement not resolved","RhoA-specific contacts inferred rather than co-crystallized"]},{"year":2001,"claim":"Discovery of PKNβ binding and phosphorylation revealed a feedback loop linking a Rho effector kinase back to its GAP.","evidence":"Yeast two-hybrid, GST-SH3 pulldown, co-IP from COS-7, and in vitro kinase assay with active PKNβ","pmids":["11432776"],"confidence":"Medium","gaps":["Effect of PKNβ phosphorylation on GAP activity unresolved","Single lab, no in vivo validation"]},{"year":2006,"claim":"Demonstrating epigenetic silencing established how GRAF is inactivated in myeloid malignancy without mutation.","evidence":"Promoter reporter assay, bisulfite methylation analysis, and pharmacological reactivation with demethylating agent plus HDAC inhibitor in leukemia lines","pmids":["16404424"],"confidence":"Medium","gaps":["Causal contribution of silencing to leukemogenesis not tested functionally","Downstream RhoA consequences in leukemia not measured"]},{"year":2009,"claim":"Characterizing BAR-mediated autoinhibition and dual signal-driven recruitment explained how GRAF GAP activity is spatially and temporally controlled.","evidence":"In vitro GAP and liposome tubulation assays with domain dissection; integrin blocking and PI3K inhibition with RhoA activity readouts in a breast cancer dormancy model","pmids":["18954304","19308677"],"confidence":"High","gaps":["Mechanism relieving BAR autoinhibition at membranes not defined","Dormancy-model findings are single-study, cell-based"]},{"year":2017,"claim":"Ortholog work positioned GRAF in receptor endocytosis, showing it limits EGFR signaling by promoting receptor internalization and degradation.","evidence":"Drosophila Graf loss-of-function, GEEC localization, EGFR internalization/degradation assays, and Graf-EGFR co-IP","pmids":["28993397"],"confidence":"Medium","gaps":["Conservation of EGFR regulation in mammalian cells not shown","Whether GAP activity is required for endocytic function unclear"]},{"year":2018,"claim":"Loss-of-function studies in human smooth muscle cells linked ARHGAP26 to proliferation/migration via the RhoA-ROCK1-PTEN axis under hypoxia.","evidence":"siRNA knockdown in primary DASMCs, hypoxia culture, RhoA/ROCK1/phospho-PTEN Westerns, and ROCK inhibition","pmids":["30592323"],"confidence":"Medium","gaps":["Direct GAP-substrate relationship in this axis not biochemically dissected","Single cell type, single lab"]},{"year":2019,"claim":"Identifying SMURF1-mediated ubiquitination established post-translational control of ARHGAP26 abundance and its consequence for cancer cell motility.","evidence":"Co-IP, ubiquitination assay, overexpression/knockdown rescue in ovarian cancer lines, and in vivo metastasis model","pmids":["31004081"],"confidence":"Medium","gaps":["Ubiquitination sites and degradation route not mapped","Link to RhoA GAP activity vs β-catenin pathway not fully separated"]},{"year":2021,"claim":"Genetic dissection in Drosophila placed GRAF in a RhoGEF2-Rho-GRAF balance governing actomyosin ring constriction and in EGFR-MAPK-dependent neural patterning.","evidence":"Graf RNAi/mutants with domain-specific rescue, RhoGEF2/ROCK epistasis, live imaging, Rho-GTP pulldown; mosaic analysis with human OPHN1 rescue and memory assays","pmids":["33835025","33892766"],"confidence":"High","gaps":["Mammalian conservation of the cellularization role untested","Whether neural phenotype reflects direct EGFR-MAPK regulation by GRAF unproven"]},{"year":2024,"claim":"Recent work expanded ARHGAP26 into mitochondrial quality control and oocyte integrity, and defined CLDN18-ARHGAP26 as a gain-of-function oncogene rather than a GAP loss.","evidence":"PRKN co-IP, PINK1 phosphosite mapping, KO mouse hearts (mitophagy); Cofilin1 co-IP and Arhgap26 KO oocytes (DRP1/ROS); LSL-CLDN18-ARHGAP26 transgenic mice/organoids with RHOA-FAK-YAP-TEAD analysis and inhibitor rescue","pmids":["38855880","39313581","38621923"],"confidence":"High","gaps":["How a RhoGAP fusion activates RHOA mechanistically not resolved","Whether mitochondrial and cytoskeletal roles share a common biochemical activity unclear"]},{"year":null,"claim":"It remains unresolved how the BAR-GAP autoinhibition, SH3-partner engagement, and phosphorylation/ubiquitination inputs are integrated to switch ARHGAP26 between its GAP, membrane-tubulation, and mitophagy functions in mammalian cells.","evidence":"No timeline study reconstitutes the full regulatory hierarchy in a single mammalian system","pmids":[],"confidence":"Low","gaps":["No structure of full-length regulated protein","Mechanism converting fusion into RHOA activation unknown","Crosstalk between cytoskeletal and mitochondrial roles undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3,5]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[5]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[10,14]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,9]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[12,14]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0,10]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,6,11]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[12]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[7,13,16]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[9]}],"complexes":[],"partners":["PYK2","PKNB","EGFR","PRKN","CFL1","SMURF1","CLDN18"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UNA1","full_name":"Rho GTPase-activating protein 26","aliases":["GTPase regulator associated with focal adhesion kinase","GRAF1","Oligophrenin-1-like protein","Rho-type GTPase-activating protein 26"],"length_aa":814,"mass_kda":92.2,"function":"GTPase-activating protein for RHOA and CDC42. Facilitates mitochondrial quality control by promoting Parkin-mediated recruitment of autophagosomes to damaged mitochondria (PubMed:38081847). Negatively regulates the growth of human parainfluenza virus type 2 by inhibiting hPIV-2-mediated RHOA activation via interaction with two of its viral proteins P and V (PubMed:27512058) Associates with MICAL1 on the endosomal membrane to promote Rab8-Rab10-dependent tubule extension. After dissociation of MICAL1, recruits WDR44 which connects the endoplasmic reticulum (ER) with the endosomal tubule, thereby participating in the export of a subset of neosynthesized proteins","subcellular_location":"Cytoplasm; Cell junction, focal adhesion; Cytoplasm, cytoskeleton","url":"https://www.uniprot.org/uniprotkb/Q9UNA1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ARHGAP26","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/ARHGAP26","total_profiled":1310},"omim":[{"mim_id":"620883","title":"FER1-LIKE FAMILY, MEMBER 5; FER1L5","url":"https://www.omim.org/entry/620883"},{"mim_id":"610714","title":"PROTEIN KINASE N3; PKN3","url":"https://www.omim.org/entry/610714"},{"mim_id":"607785","title":"JUVENILE MYELOMONOCYTIC LEUKEMIA; JMML","url":"https://www.omim.org/entry/607785"},{"mim_id":"606929","title":"THO COMPLEX, SUBUNIT 3; THOC3","url":"https://www.omim.org/entry/606929"},{"mim_id":"605370","title":"RHO GTPase-ACTIVATING PROTEIN 26; ARHGAP26","url":"https://www.omim.org/entry/605370"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ARHGAP26"},"hgnc":{"alias_symbol":["GRAF","KIAA0621","OPHN1L","OPHN1L1"],"prev_symbol":[]},"alphafold":{"accession":"Q9UNA1","domains":[{"cath_id":"-","chopping":"23-137_190-215","consensus_level":"high","plddt":93.9251,"start":23,"end":215},{"cath_id":"2.30.29.30","chopping":"269-365","consensus_level":"high","plddt":87.3854,"start":269,"end":365},{"cath_id":"1.10.555.10","chopping":"390-570","consensus_level":"medium","plddt":91.664,"start":390,"end":570},{"cath_id":"2.30.30.40","chopping":"762-814","consensus_level":"high","plddt":91.4764,"start":762,"end":814}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UNA1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UNA1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UNA1-F1-predicted_aligned_error_v6.png","plddt_mean":77.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ARHGAP26","jax_strain_url":"https://www.jax.org/strain/search?query=ARHGAP26"},"sequence":{"accession":"Q9UNA1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UNA1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UNA1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UNA1"}},"corpus_meta":[{"pmid":"26146084","id":"PMC_26146084","title":"Recurrent Fusion Genes in Gastric Cancer: CLDN18-ARHGAP26 Induces Loss of Epithelial Integrity.","date":"2015","source":"Cell reports","url":"https://pubmed.ncbi.nlm.nih.gov/26146084","citation_count":120,"is_preprint":false},{"pmid":"10908648","id":"PMC_10908648","title":"The human GRAF gene is fused to MLL in a unique t(5;11)(q31;q23) and both alleles are disrupted in three cases of myelodysplastic syndrome/acute myeloid leukemia with a deletion 5q.","date":"2000","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/10908648","citation_count":104,"is_preprint":false},{"pmid":"9858476","id":"PMC_9858476","title":"Cytoskeletal changes induced by GRAF, the GTPase regulator associated with focal adhesion kinase, are mediated by Rho.","date":"1999","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/9858476","citation_count":87,"is_preprint":false},{"pmid":"26377184","id":"PMC_26377184","title":"'Medusa head ataxia': the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 2: Anti-PKC-gamma, anti-GluR-delta2, anti-Ca/ARHGAP26 and anti-VGCC.","date":"2015","source":"Journal of neuroinflammation","url":"https://pubmed.ncbi.nlm.nih.gov/26377184","citation_count":65,"is_preprint":false},{"pmid":"9525907","id":"PMC_9525907","title":"Characterization of graf, the GTPase-activating protein for rho associated with focal adhesion kinase. Phosphorylation and possible regulation by mitogen-activated protein kinase.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9525907","citation_count":62,"is_preprint":false},{"pmid":"9494093","id":"PMC_9494093","title":"Interaction of two proline-rich sequences of cell adhesion kinase beta with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf.","date":"1998","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/9494093","citation_count":60,"is_preprint":false},{"pmid":"18954304","id":"PMC_18954304","title":"A BAR domain-mediated autoinhibitory mechanism for RhoGAPs of the GRAF family.","date":"2009","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/18954304","citation_count":57,"is_preprint":false},{"pmid":"11432776","id":"PMC_11432776","title":"PKNbeta interacts with the SH3 domains of Graf and a novel Graf related protein, Graf2, which are GTPase activating proteins for Rho family.","date":"2001","source":"Journal of biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11432776","citation_count":43,"is_preprint":false},{"pmid":"33069733","id":"PMC_33069733","title":"Deficiency of NEAT1 prevented MPP+-induced inflammatory response, oxidative stress and apoptosis in dopaminergic SK-N-SH neuroblastoma cells via miR-1277-5p/ARHGAP26 axis.","date":"2020","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/33069733","citation_count":40,"is_preprint":false},{"pmid":"23320754","id":"PMC_23320754","title":"Two new cases of anti-Ca (anti-ARHGAP26/GRAF) autoantibody-associated cerebellar ataxia.","date":"2013","source":"Journal of neuroinflammation","url":"https://pubmed.ncbi.nlm.nih.gov/23320754","citation_count":36,"is_preprint":false},{"pmid":"31004081","id":"PMC_31004081","title":"SMURF1-mediated ubiquitination of ARHGAP26 promotes ovarian cancer cell invasion and migration.","date":"2019","source":"Experimental & molecular 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anti-Ca/ARHGAP26 and voltage-gated potassium channel antibodies.","date":"2015","source":"Journal of neuroimmunology","url":"https://pubmed.ncbi.nlm.nih.gov/26298328","citation_count":16,"is_preprint":false},{"pmid":"15382263","id":"PMC_15382263","title":"MLL/GRAF fusion in an infant acute monocytic leukemia (AML M5b) with a cytogenetically cryptic ins(5;11)(q31;q23q23).","date":"2004","source":"Genes, chromosomes & cancer","url":"https://pubmed.ncbi.nlm.nih.gov/15382263","citation_count":15,"is_preprint":false},{"pmid":"12622221","id":"PMC_12622221","title":"New erythroxane-type diterpenoids from Fagonia boveana (Hadidi) Hadidi & Graf.","date":"2003","source":"Zeitschrift fur Naturforschung. C, Journal of biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/12622221","citation_count":13,"is_preprint":false},{"pmid":"34716859","id":"PMC_34716859","title":"The role of GTPase-activating protein ARHGAP26 in human cancers.","date":"2021","source":"Molecular and cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/34716859","citation_count":12,"is_preprint":false},{"pmid":"30719998","id":"PMC_30719998","title":"Circular RNA ARHGAP26 is over-expressed and its downregulation inhibits cell proliferation and promotes cell apoptosis in gastric cancer cells.","date":"2019","source":"Saudi journal of gastroenterology : official journal of the Saudi Gastroenterology Association","url":"https://pubmed.ncbi.nlm.nih.gov/30719998","citation_count":12,"is_preprint":false},{"pmid":"21074269","id":"PMC_21074269","title":"Abnormal methylation of GRAF promoter Chinese patients with acute myeloid leukemia.","date":"2010","source":"Leukemia research","url":"https://pubmed.ncbi.nlm.nih.gov/21074269","citation_count":12,"is_preprint":false},{"pmid":"33602299","id":"PMC_33602299","title":"The International Collaborative Gaucher Group GRAF (Gaucher Risk Assessment for Fracture) score: a composite risk score for assessing adult fracture risk in imiglucerase-treated Gaucher disease type 1 patients.","date":"2021","source":"Orphanet journal of rare diseases","url":"https://pubmed.ncbi.nlm.nih.gov/33602299","citation_count":11,"is_preprint":false},{"pmid":"35171450","id":"PMC_35171450","title":"Associations of ARHGAP26 Polymorphisms with Alzheimer's Disease and Cardiovascular Disease.","date":"2022","source":"Journal of molecular neuroscience : MN","url":"https://pubmed.ncbi.nlm.nih.gov/35171450","citation_count":9,"is_preprint":false},{"pmid":"30592323","id":"PMC_30592323","title":"Hypoxia-induced ARHGAP26 deficiency inhibits the proliferation and migration of human ductus arteriosus smooth muscle cell through activating RhoA-ROCK-PTEN pathway.","date":"2018","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30592323","citation_count":9,"is_preprint":false},{"pmid":"30387365","id":"PMC_30387365","title":"RhoA/ROCK/ARHGAP26 signaling in the eutopic and ectopic endometrium is involved in clinical characteristics of adenomyosis.","date":"2018","source":"The Journal of international medical research","url":"https://pubmed.ncbi.nlm.nih.gov/30387365","citation_count":9,"is_preprint":false},{"pmid":"33835025","id":"PMC_33835025","title":"Spatiotemporal recruitment of RhoGTPase protein GRAF inhibits actomyosin ring constriction in Drosophila cellularization.","date":"2021","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/33835025","citation_count":9,"is_preprint":false},{"pmid":"20704716","id":"PMC_20704716","title":"GTPase regulator associated with the focal adhesion kinase (GRAF) transcript was down-regulated in patients with myeloid malignancies.","date":"2010","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/20704716","citation_count":8,"is_preprint":false},{"pmid":"38855880","id":"PMC_38855880","title":"ARHGAP26/GRAF1 orchestrates actin remodeling and membrane dynamics to drive mitochondrial clearance and promote fuel flexibility.","date":"2024","source":"Autophagy","url":"https://pubmed.ncbi.nlm.nih.gov/38855880","citation_count":5,"is_preprint":false},{"pmid":"39081998","id":"PMC_39081998","title":"Case report: Anti-ARHGAP26 autoantibodies in atypical dementia with Lewy bodies.","date":"2023","source":"Frontiers in dementia","url":"https://pubmed.ncbi.nlm.nih.gov/39081998","citation_count":5,"is_preprint":false},{"pmid":"9550268","id":"PMC_9550268","title":"Oro-palatal dysplasia Bettex-Graf--a new syndrome.","date":"1998","source":"European journal of pediatric surgery : official journal of Austrian Association of Pediatric Surgery ... [et al] = Zeitschrift fur Kinderchirurgie","url":"https://pubmed.ncbi.nlm.nih.gov/9550268","citation_count":5,"is_preprint":false},{"pmid":"39313581","id":"PMC_39313581","title":"ARHGAP26 deficiency drives the oocyte aneuploidy and early embryonic development failure.","date":"2024","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/39313581","citation_count":4,"is_preprint":false},{"pmid":"33892766","id":"PMC_33892766","title":"Drosophila Graf regulates mushroom body β-axon extension and olfactory long-term memory.","date":"2021","source":"Molecular brain","url":"https://pubmed.ncbi.nlm.nih.gov/33892766","citation_count":3,"is_preprint":false},{"pmid":"40089259","id":"PMC_40089259","title":"Liposome-loaded miR-34c-5p attenuates apoptosis and oxidative stress following hypoxia-ischemia brain damage in neonatal mice by targeting Arhgap26.","date":"2025","source":"European journal of pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/40089259","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":20530,"output_tokens":4750,"usd":0.06642,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":12977,"output_tokens":4124,"usd":0.083992,"stage2_stop_reason":"end_turn"},"total_usd":0.150412,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"GRAF acts as a GAP for RhoA (not Cdc42) in vivo: microinjection of GRAF cDNA into Swiss 3T3 cells caused stress fiber clearing and filopodial-like extensions mimicking Rho inhibition; GAP-dead point mutants (R236Q or N351V) had no effect; GRAF blocked sphingosine-1-phosphate-stimulated (Rho-mediated) but not bradykinin-stimulated (Cdc42-mediated) cytoskeletal changes.\",\n      \"method\": \"Microinjection of wild-type and GAP-dead GRAF cDNA into Swiss 3T3 and PC12 cells; pharmacological inhibition with C3; selective agonist assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in-cell assays with isogenic controls, active-site mutagenesis, and selective agonist challenge confirming Rho specificity in vivo\",\n      \"pmids\": [\"9858476\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"GRAF is phosphorylated on serine 510 by MAP kinase in vitro; EGF/NGF stimulation of PC12 cells induces a phosphatase-reversible mobility shift that is abolished by S510A mutation, indicating GRAF is phosphorylated at this site in vivo downstream of growth factor signaling.\",\n      \"method\": \"In vitro kinase assay with purified MAP kinase; site-directed mutagenesis (S510A); gel-mobility shift assay in EGF/NGF-stimulated PC12 cells; phosphatase treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus mutagenesis validated in vivo, single lab but two orthogonal methods\",\n      \"pmids\": [\"9525907\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The SH3 domain of GRAF interacts with two proline-rich sequences in cell adhesion kinase beta (CAKbeta/PYK2), and GRAF co-immunoprecipitates with CAKbeta from rat brain lysate, identifying a direct binding partnership in vivo.\",\n      \"method\": \"GST-SH3 domain affinity precipitation from rat brain lysate; GST dot-blot; co-immunoprecipitation from rat brain lysate; competitive binding assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal pulldown plus co-IP from native tissue with two orthogonal binding assays, single lab\",\n      \"pmids\": [\"9494093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Crystal structure of the GRAF GAP domain (GrafGAP, 231 residues) at 2.4 Å resolution established domain boundaries and revealed that substrate specificity is determined by interaction with Glu-95/97 of RhoA/Cdc42 (absent in Rac1 as Ala-95); a Cdc42 E95A mutant reduced GrafGAP activity ~40-fold and Rac1 A95E increased it ~10-fold, confirming the structural prediction.\",\n      \"method\": \"X-ray crystallography (2.4 Å); in vitro GTPase-activating assay with Cdc42 E95A and Rac1 A95E mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus functional mutagenesis of both substrate and enzyme confirming mechanistic model\",\n      \"pmids\": [\"10982819\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PKNbeta (a Rho target kinase) interacts with the SH3 domains of GRAF and the related GRAF2 via proline-rich motifs; the active form of PKNbeta phosphorylates GRAF and GRAF2 in vitro; and GRAF co-immunoprecipitates with PKNbeta in COS-7 cells, revealing a feedback loop between Rho effector kinase and its GAP.\",\n      \"method\": \"Yeast two-hybrid screen; GST-SH3 pull-down; co-immunoprecipitation from transfected COS-7 cells; in vitro kinase assay with catalytically active PKNbeta\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vitro kinase assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"11432776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The BAR domain of GRAF family members (including ARHGAP26) acts as a cis-acting autoinhibitory element: it interacts directly with the GAP domain and inhibits its RhoGAP activity; in the autoinhibited state, GRAF can still bind and tubulate liposomes in vitro and generate lipid tubules in cells, demonstrating separable membrane-tubulation and GAP-inhibitory functions.\",\n      \"method\": \"In vitro GAP activity assay; direct BAR–GAP domain interaction assay; liposome tubulation assay; cell-based lipid tubule formation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of autoinhibition plus functional domain dissection, single lab with multiple orthogonal assays\",\n      \"pmids\": [\"18954304\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In a breast cancer dormancy model, integrin α5β1 ligation by fibronectin recruits GRAF to the membrane, leading to RhoA inactivation and cortical actin stabilization; FGF-2-activated PI3K signaling is independently required for GRAF membrane relocalization and RhoA inactivation, indicating dual upstream signals converge on GRAF.\",\n      \"method\": \"Integrin blocking antibodies; PI3K inhibitors; immunofluorescence for FAK and GRAF localization; RhoA activity assay in breast cancer dormancy co-culture model\",\n      \"journal\": \"Cancer microenvironment\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — cell-based localization and RhoA activity assays with pharmacological inhibitors, single lab, single study\",\n      \"pmids\": [\"19308677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The CLDN18-ARHGAP26 fusion protein (from a recurrent chromosomal translocation in gastric cancer) causes loss of epithelial integrity: fusion-expressing epithelial cells display impaired barrier properties, reduced cell-cell and cell-ECM adhesion, EMT morphology with long protrusions, retarded wound healing, inhibition of RHOA, and gain of invasion.\",\n      \"method\": \"Stable expression of CLDN18-ARHGAP26 fusion in epithelial cell lines; transwell invasion assay; barrier permeability assay; RhoA activity assay; wound healing assay; morphological imaging\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal functional assays in isogenic cell lines, independently replicated across 3 patient cases and multiple cell lines\",\n      \"pmids\": [\"26146084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SMURF1 (an E3 ubiquitin ligase) interacts with and ubiquitinates ARHGAP26, promoting its degradation; SMURF1-induced ubiquitination of ARHGAP26 promotes ovarian cancer cell invasion and migration via the β-catenin pathway, and ARHGAP26 overexpression rescues SMURF1-driven migration.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay; ARHGAP26 overexpression and knockdown in A2780, HEY, SKOV3 cells; migration/invasion assays; in vivo lung metastasis model; DKK1 antagonist rescue experiment\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus ubiquitination assay plus functional rescue, single lab\",\n      \"pmids\": [\"31004081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Drosophila GRAF (ortholog of ARHGAP26/GRAF1) localizes to GPI-enriched endocytic compartment (GEEC) membranes in plasmatocytes, is required for GEEC endocytosis, and directly interacts with EGFR in a ubiquitylation-dependent manner to facilitate EGFR internalization and degradation at high ligand doses, thereby restraining EGFR signaling and plasmatocyte proliferation.\",\n      \"method\": \"Drosophila genetics (Graf loss-of-function); immunofluorescence localization to GEEC membranes; EGFR signaling/proliferation assays; receptor internalization and degradation assays; co-immunoprecipitation of Graf and EGFR; ubiquitylation-dependence experiments\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ortholog study (Drosophila), multiple orthogonal methods including genetic epistasis and co-IP, single lab\",\n      \"pmids\": [\"28993397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In Drosophila cellularization, GRAF (ortholog of ARHGAP26) is enriched at the cleavage furrow tip during actomyosin ring assembly; Graf depletion increases Rho-GTP, elevates Myosin II levels, and causes hyper-constriction of the actomyosin ring dependent on its RhoGAP domain; RhoGEF2 depletion or ROCK mutation suppresses the hyper-constriction phenotype, placing GRAF in a RhoGEF2–Rho–GRAF balance governing ring constriction.\",\n      \"method\": \"Drosophila genetics (Graf RNAi, Graf mutants, RhoGEF2 mutants, ROCK mutants); live imaging; Rho-GTP pull-down; Myosin II immunostaining; epistasis analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with domain-specific rescue, live imaging, and biochemical Rho-GTP measurement in ortholog, multiple orthogonal methods\",\n      \"pmids\": [\"33835025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In human ductus arteriosus smooth muscle cells (DASMCs), ARHGAP26 knockdown reduces cell proliferation and migration; hypoxia suppresses ARHGAP26 expression and activates the RhoA-ROCK1-PTEN phosphorylation axis; ROCK inhibition (Y-27632) reverses the PTEN-mediated inhibition of proliferation and migration.\",\n      \"method\": \"ARHGAP26 siRNA knockdown in primary human DASMCs; hypoxia culture; proliferation and migration assays; Western blotting for RhoA activity, ROCK1, phospho-PTEN; pharmacological ROCK inhibition\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KD with specific phenotypic readouts and pharmacological pathway rescue, single lab\",\n      \"pmids\": [\"30592323\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ARHGAP26/GRAF1 is a PRKN/Parkin-binding protein that is rapidly recruited to damaged mitochondria; PINK1 phosphorylates ARHGAP26 at specific sites, enabling it to coordinate phagophore capture by regulating mitochondrial-associated actin remodeling and facilitating PRKN-LC3 interactions; ARHGAP26 depletion in mouse hearts blunts mitochondrial clearance and attenuates metabolic adaptations to stress.\",\n      \"method\": \"Co-immunoprecipitation (ARHGAP26–PRKN interaction); recruitment to damaged mitochondria (imaging); PINK1 phosphorylation site identification; ARHGAP26 knockout mouse hearts; mitophagy assays; metabolic stress assays\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, in vivo KO, recruitment imaging, and phosphorylation site identification, single lab\",\n      \"pmids\": [\"38855880\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In a transgenic mouse model, CLDN18-ARHGAP26 expression in gastric organoids induces signet ring cell formation, cooperatively transforms gastric cells with Trp53 loss, activates RHOA and downstream FAK and YAP-TEAD signaling (opposite to the expected GAP loss-of-function), identifying the fusion as a gain-of-function oncogene; combined FAK and YAP/TEAD inhibition significantly blocks tumor growth.\",\n      \"method\": \"LSL-CLDN18-ARHGAP26 knock-in transgenic mouse model; gastric organoids; Cre-induced expression; RHOA activity assay; FAK phosphorylation; YAP target gene expression; pharmacological inhibition of FAK and YAP/TEAD; tumor growth assays\",\n      \"journal\": \"Gut\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo transgenic model plus organoid reconstitution plus biochemical pathway analysis and therapeutic rescue, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38621923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ARHGAP26 interacts with Cofilin1 in oocytes to maintain mitochondrial integrity by regulating DRP1 dynamics; Arhgap26 knockout causes mitochondrial dysfunction, ROS accumulation, oocyte death at the GV stage, maturation arrest, and aneuploidy; restoration of ARHGAP26 protein level recovers oocyte quality.\",\n      \"method\": \"Arhgap26 knockout mouse model; co-immunoprecipitation (ARHGAP26–Cofilin1); DRP1 dynamics imaging; ROS measurement; oocyte maturation assays; in vitro fertilization; embryonic development tracking; transcriptome analysis; chromosomal microarray of patient\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with rescue, co-IP, and multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"39313581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Loss of Drosophila Graf (ARHGAP26 ortholog) causes abnormal MB β-lobe midline crossing during metamorphosis via a cell-autonomous mechanism; this phenotype requires activation of EGFR-MAPK signaling, consistent with Graf's role in negatively regulating this pathway; Graf mutants also display impaired olfactory long-term memory.\",\n      \"method\": \"Drosophila Graf loss-of-function mutants; MB-specific Graf and human OPHN1 rescue; cell-autonomous mosaic analysis; EGFR-MAPK pathway activation experiments; olfactory long-term memory behavioral assay\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with mosaic analysis and rescue by human ortholog, single lab\",\n      \"pmids\": [\"33892766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The GRAF promoter contains CpG sites whose methylation suppresses GRAF expression; treatment of leukemia cell lines with a demethylating agent and an HDAC inhibitor restores GRAF transcript levels, demonstrating epigenetic silencing as a mechanism of GRAF inactivation in AML/MDS.\",\n      \"method\": \"Reporter gene assay for promoter activity; bisulfite sequencing/methylation analysis of patient samples; demethylating agent + HDAC inhibitor treatment of leukemia cell lines; RT-PCR for GRAF expression\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter reporter assay plus pharmacological reactivation, single lab, multiple methods\",\n      \"pmids\": [\"16404424\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ARHGAP26/GRAF1 is a multi-domain RhoGAP protein (BAR–PH–GAP–SH3) that acts as a GTPase-activating protein selective for RhoA and Cdc42 in vivo; its GAP domain is autoinhibited by the BAR domain in cis; it is phosphorylated by MAP kinase (Ser510) and by PINK1 at damaged mitochondria, is ubiquitinated by SMURF1, and interacts via its SH3 domain with FAK/PYK2, PKNbeta, EGFR, PRKN/Parkin, and Cofilin1; through RhoA inactivation it restrains actomyosin contractility, regulates integrin-mediated cytoskeletal reorganization and EGFR endocytic degradation, and coordinates mitophagy-associated actin remodeling, while oncogenic CLDN18-ARHGAP26 fusions paradoxically activate RHOA and downstream FAK/YAP-TEAD signaling to drive diffuse gastric cancer.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ARHGAP26 (GRAF/GRAF1) is a multidomain RhoGAP that restrains Rho-family GTPase signaling to control actomyosin contractility, membrane trafficking, and cytoskeletal remodeling [#0, #5, #10]. In vivo it acts as a GAP selective for RhoA, with substrate specificity dictated structurally by recognition of Glu-95/97 of the GTPase, and its catalytic GAP domain is held inactive in cis by an autoinhibitory intramolecular interaction with its BAR domain — a domain that independently binds and tubulates membranes [#0, #3, #5]. Through this RhoGAP activity ARHGAP26 inactivates RhoA upon integrin α5β1 engagement and PI3K signaling to stabilize cortical actin, and balances RhoGEF2–Rho-driven contractility at the actomyosin ring [#6, #10]. ARHGAP26 is integrated into growth-factor and stress signaling: it is phosphorylated at Ser510 by MAP kinase downstream of EGF/NGF, engages FAK/PYK2 and the Rho effector kinase PKNβ via its SH3 domain, and limits EGFR signaling by promoting ubiquitylation-dependent receptor internalization and degradation [#1, #2, #4, #9]. It also functions in mitochondrial quality control, where it is recruited to damaged mitochondria as a PRKN/Parkin-binding protein, is phosphorylated by PINK1, and coordinates actin remodeling for mitophagy, and it interacts with Cofilin1 to maintain mitochondrial integrity via DRP1 dynamics [#12, #14]. ARHGAP26 protein levels are controlled by SMURF1-mediated ubiquitination and by promoter CpG methylation, the latter silencing GRAF in AML/MDS [#8, #16]. Recurrent CLDN18-ARHGAP26 fusions in diffuse gastric cancer act paradoxically as gain-of-function oncogenes that activate RHOA and downstream FAK and YAP-TEAD signaling, disrupt epithelial adhesion, and drive invasion and signet-ring transformation [#7, #13].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing how GRAF couples to upstream signaling addressed whether this RhoGAP is regulated by, and physically linked to, growth-factor and adhesion kinases.\",\n      \"evidence\": \"In vitro MAP kinase assay with S510A mutagenesis and EGF/NGF-stimulated mobility shifts in PC12 cells; GST-SH3 pulldown and co-IP of PYK2/CAKβ from rat brain\",\n      \"pmids\": [\"9525907\", \"9494093\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of Ser510 phosphorylation on GAP activity not defined\", \"PYK2 interaction not linked to a downstream cytoskeletal output\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defining GRAF's in vivo substrate clarified which Rho-family GTPase it regulates, anchoring its cellular role in actin cytoskeletal control.\",\n      \"evidence\": \"Microinjection of wild-type vs GAP-dead GRAF into Swiss 3T3/PC12 cells with selective agonist challenge and C3 inhibition\",\n      \"pmids\": [\"9858476\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In-cell assay does not exclude Cdc42 GAP activity under other conditions\", \"Physiological trigger of GRAF activation not addressed\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Solving the GAP domain structure explained the molecular basis of substrate selectivity at atomic resolution.\",\n      \"evidence\": \"X-ray crystallography at 2.4 Å plus reciprocal GTPase mutagenesis (Cdc42 E95A, Rac1 A95E)\",\n      \"pmids\": [\"10982819\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of full-length protein and BAR-GAP arrangement not resolved\", \"RhoA-specific contacts inferred rather than co-crystallized\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Discovery of PKNβ binding and phosphorylation revealed a feedback loop linking a Rho effector kinase back to its GAP.\",\n      \"evidence\": \"Yeast two-hybrid, GST-SH3 pulldown, co-IP from COS-7, and in vitro kinase assay with active PKNβ\",\n      \"pmids\": [\"11432776\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Effect of PKNβ phosphorylation on GAP activity unresolved\", \"Single lab, no in vivo validation\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrating epigenetic silencing established how GRAF is inactivated in myeloid malignancy without mutation.\",\n      \"evidence\": \"Promoter reporter assay, bisulfite methylation analysis, and pharmacological reactivation with demethylating agent plus HDAC inhibitor in leukemia lines\",\n      \"pmids\": [\"16404424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal contribution of silencing to leukemogenesis not tested functionally\", \"Downstream RhoA consequences in leukemia not measured\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Characterizing BAR-mediated autoinhibition and dual signal-driven recruitment explained how GRAF GAP activity is spatially and temporally controlled.\",\n      \"evidence\": \"In vitro GAP and liposome tubulation assays with domain dissection; integrin blocking and PI3K inhibition with RhoA activity readouts in a breast cancer dormancy model\",\n      \"pmids\": [\"18954304\", \"19308677\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism relieving BAR autoinhibition at membranes not defined\", \"Dormancy-model findings are single-study, cell-based\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Ortholog work positioned GRAF in receptor endocytosis, showing it limits EGFR signaling by promoting receptor internalization and degradation.\",\n      \"evidence\": \"Drosophila Graf loss-of-function, GEEC localization, EGFR internalization/degradation assays, and Graf-EGFR co-IP\",\n      \"pmids\": [\"28993397\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Conservation of EGFR regulation in mammalian cells not shown\", \"Whether GAP activity is required for endocytic function unclear\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Loss-of-function studies in human smooth muscle cells linked ARHGAP26 to proliferation/migration via the RhoA-ROCK1-PTEN axis under hypoxia.\",\n      \"evidence\": \"siRNA knockdown in primary DASMCs, hypoxia culture, RhoA/ROCK1/phospho-PTEN Westerns, and ROCK inhibition\",\n      \"pmids\": [\"30592323\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct GAP-substrate relationship in this axis not biochemically dissected\", \"Single cell type, single lab\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying SMURF1-mediated ubiquitination established post-translational control of ARHGAP26 abundance and its consequence for cancer cell motility.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, overexpression/knockdown rescue in ovarian cancer lines, and in vivo metastasis model\",\n      \"pmids\": [\"31004081\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Ubiquitination sites and degradation route not mapped\", \"Link to RhoA GAP activity vs β-catenin pathway not fully separated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Genetic dissection in Drosophila placed GRAF in a RhoGEF2-Rho-GRAF balance governing actomyosin ring constriction and in EGFR-MAPK-dependent neural patterning.\",\n      \"evidence\": \"Graf RNAi/mutants with domain-specific rescue, RhoGEF2/ROCK epistasis, live imaging, Rho-GTP pulldown; mosaic analysis with human OPHN1 rescue and memory assays\",\n      \"pmids\": [\"33835025\", \"33892766\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mammalian conservation of the cellularization role untested\", \"Whether neural phenotype reflects direct EGFR-MAPK regulation by GRAF unproven\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Recent work expanded ARHGAP26 into mitochondrial quality control and oocyte integrity, and defined CLDN18-ARHGAP26 as a gain-of-function oncogene rather than a GAP loss.\",\n      \"evidence\": \"PRKN co-IP, PINK1 phosphosite mapping, KO mouse hearts (mitophagy); Cofilin1 co-IP and Arhgap26 KO oocytes (DRP1/ROS); LSL-CLDN18-ARHGAP26 transgenic mice/organoids with RHOA-FAK-YAP-TEAD analysis and inhibitor rescue\",\n      \"pmids\": [\"38855880\", \"39313581\", \"38621923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a RhoGAP fusion activates RHOA mechanistically not resolved\", \"Whether mitochondrial and cytoskeletal roles share a common biochemical activity unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how the BAR-GAP autoinhibition, SH3-partner engagement, and phosphorylation/ubiquitination inputs are integrated to switch ARHGAP26 between its GAP, membrane-tubulation, and mitophagy functions in mammalian cells.\",\n      \"evidence\": \"No timeline study reconstitutes the full regulatory hierarchy in a single mammalian system\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of full-length regulated protein\", \"Mechanism converting fusion into RHOA activation unknown\", \"Crosstalk between cytoskeletal and mitochondrial roles undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3, 5]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [10, 14]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 9]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [12, 14]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0, 10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 6, 11]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [7, 13, 16]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"PYK2\", \"PKNB\", \"EGFR\", \"PRKN\", \"CFL1\", \"SMURF1\", \"CLDN18\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}