{"gene":"ARHGAP35","run_date":"2026-04-28T17:12:37","timeline":{"discoveries":[{"year":2001,"finding":"p190RhoGAP (ARHGAP35) inactivates RhoA downstream of integrin engagement to promote cell spreading, membrane protrusion, and directional migration in fibroblasts; dominant-negative p190RhoGAP elevates RhoA activity, impairs spreading, and blocks polarity establishment","method":"Dominant-negative and wild-type overexpression, RhoA activity assays, cell spreading/migration assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal functional methods, replicated across conditions","pmids":["11553710"],"is_preprint":false},{"year":1995,"finding":"c-Src phosphorylates p190RhoGAP on tyrosine residues; this phosphorylation correlates with EGF-induced actin cytoskeleton remodeling (stress fiber disassembly/reassembly) and p190RhoGAP redistribution into cytoplasmic arc-like structures","method":"Dominant-negative/wild-type c-Src overexpression, immunofluorescence confocal microscopy, phosphotyrosine analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple cell lines, orthogonal methods, strong correlation","pmids":["7542246"],"is_preprint":false},{"year":2006,"finding":"Rac1 activation causes translocation of p190RhoGAP to adherens junctions where it binds p120-catenin; this p120-p190RhoGAP interaction is required for adherens junction assembly and locally inhibits RhoA to coordinate Rac/Rho antagonism downstream of PDGFR","method":"Co-immunoprecipitation, knockdown, PDGFR signaling assays, cell fractionation","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, epistasis, multiple orthogonal methods","pmids":["17129786"],"is_preprint":false},{"year":1998,"finding":"Activation of alpha6beta1 integrin in melanoma cells induces tyrosine phosphorylation of p190RhoGAP and promotes membrane-protrusive activities at invadopodia; microinjection of anti-p190RhoGAP antibodies blocks matrix degradation and invasion","method":"Integrin stimulation, tyrosine phosphorylation assays, microinjection of blocking antibodies, invasion assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — antibody microinjection plus signaling assays, multiple orthogonal approaches","pmids":["9417037"],"is_preprint":false},{"year":2003,"finding":"Cadherin engagement induces tyrosine phosphorylation of p190RhoGAP (requiring Src family kinases) and increases its binding to p120RasGAP, leading to RhoA inactivation; dominant-negative p190RhoGAP blocks cadherin-induced RhoA inhibition","method":"Active RhoA affinity pulldown to isolate activated GAPs, co-immunoprecipitation, PP2 inhibitor, dominant-negative expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical affinity assay plus co-IP and pharmacological inhibition","pmids":["12606561"],"is_preprint":false},{"year":2005,"finding":"Focal adhesion kinase (FAK) associates with and directly phosphorylates p190RhoGAP in vitro, suppressing RhoA activity; FAK-mediated p190RhoGAP activation is required for endothelial barrier restoration after thrombin-induced permeability increase","method":"In vitro kinase assay (recombinant FAK phosphorylating p190RhoGAP), co-immunoprecipitation, dominant-negative FAK (FRNK) adenoviral expression, RhoA activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution plus in vivo functional validation","pmids":["16308318"],"is_preprint":false},{"year":2004,"finding":"The Arg (Abl-related gene) tyrosine kinase phosphorylates p190RhoGAP at Y1105 in vitro and in vivo; this phosphorylation promotes p190RhoGAP association with p120RasGAP, stimulates p190RhoGAP RhoGAP activity, and is required for adhesion-dependent RhoA inhibition and neuritogenesis","method":"In vitro kinase assay, site-directed mutagenesis (Y1105), co-immunoprecipitation, arg-/- fibroblasts/neurons, Rho activity assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis, knockout cells","pmids":["15084284"],"is_preprint":false},{"year":2006,"finding":"Integrin signaling through the Arg kinase activates p190RhoGAP by promoting its association with p120RasGAP, which recruits p190RhoGAP to the cell periphery; p120 binding is required for in vivo but not in vitro p190RhoGAP activation, demonstrating that membrane recruitment is the critical activation mechanism","method":"arg-/- fibroblasts, dominant-negative p120 fragment, cell fractionation, in vitro GAP assay, co-immunoprecipitation","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro GAP assay combined with genetic KO and fractionation","pmids":["16971514"],"is_preprint":false},{"year":2007,"finding":"Arg kinase acts through p190RhoGAP to suppress RhoA activity, attenuate actomyosin contractility, and regulate focal adhesion dynamics; the Arg N-terminal kinase domain suffices to reduce stress fibers via p190RhoGAP, while full focal adhesion inhibition also requires the Arg C-terminal cytoskeleton-binding domain","method":"arg-/- fibroblasts, Arg domain mutant re-expression, myosin activity assays, focal adhesion quantification","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 — genetic KO with domain-specific rescue, multiple cellular phenotypes","pmids":["17652459"],"is_preprint":false},{"year":2007,"finding":"p190RhoGAP localizes to dendritic spines in hippocampal neurons; its activity is reduced in arg-/- mice, causing elevated RhoA activity and failure of dendritic spine maturation, with subsequent synapse and dendrite loss; reducing ROCKII gene dosage suppresses dendritic regression in arg-/- mice (epistasis)","method":"arg-/- mouse genetics, ROCKII heterozygosity epistasis, immunolocalization, RhoA activity assay","journal":"The Journal of neuroscience","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis with two KO models, localization data","pmids":["17928439"],"is_preprint":false},{"year":2009,"finding":"FAK promotes formation of a FAK-p120RasGAP-p190RhoGAP complex at leading-edge focal adhesions via fibronectin-integrin signaling; FAK Y397 phosphorylation mediates SH2-dependent binding of p120RasGAP, facilitating p190RhoGAP tyrosine phosphorylation and RhoA inhibition required for cell polarity during migration","method":"Wound healing/Golgi reorientation polarity assays, co-immunoprecipitation, FAK Y397 mutant, Pyk2-FAK chimera reconstitution, p120RasGAP knockdown","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, genetic reconstitution approach","pmids":["19435801"],"is_preprint":false},{"year":2008,"finding":"alpha5beta1 integrin engagement triggers Src-dependent tyrosine phosphorylation of p190RhoGAP, while syndecan-4 engagement causes PKC-alpha-dependent redistribution of phosphorylated p190RhoGAP to the membrane; both pathways must converge on p190RhoGAP for efficient RhoA suppression and focal adhesion formation","method":"Integrin/syndecan-4 engagement experiments, cell fractionation, PKC-alpha mutants, siRNA knockdown, RhoA activity assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — dissection of two signaling arms with orthogonal methods","pmids":["18541700"],"is_preprint":false},{"year":2008,"finding":"Rho-kinase phosphorylates p190A RhoGAP at Ser1150, impairing its GAP activity by disrupting Rnd binding; this creates a positive feedback loop sustaining RhoA activation in response to high-dose endothelin-1 in vascular smooth muscle cells","method":"In vitro kinase assay, phosphomimetic and phosphoresistant mutants, Rnd binding assays, RhoA activity assay in VSMCs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with site-specific mutants and functional validation","pmids":["19103606"],"is_preprint":false},{"year":2008,"finding":"Breast tumor kinase (Brk) phosphorylates p190RhoGAP-A at Y1105 in vitro and in vivo, promoting its association with p120RasGAP; this simultaneously activates p190RhoGAP (inactivating RhoA) and attenuates p120RasGAP (activating Ras), driving breast cancer proliferation and invasion","method":"In vitro kinase assay, Y1105 mutagenesis, co-immunoprecipitation, RhoA/Ras activity assays, invasion/migration/transformation assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis, multiple functional readouts","pmids":["18829532"],"is_preprint":false},{"year":2003,"finding":"p190RhoGAP is cell cycle-regulated: endogenous protein levels decrease in late mitosis via ubiquitin-mediated proteasomal degradation requiring the N-terminal GTP-binding region; overexpression causes abnormal cleavage furrow positioning and cytokinesis failure in a GAP-domain-dependent manner","method":"Conditional/transient overexpression, confocal localization during cytokinesis, proteasome inhibition, ubiquitination assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, functional domain requirement established","pmids":["14610059"],"is_preprint":false},{"year":2009,"finding":"p190RhoGAP localizes to the cleavage furrow and reduces RhoA-GTP levels there in a dose-dependent manner as measured by FRET biosensor; overexpression causes multiple cycles of abnormal furrow site selection and ingression/regression, while ECT2 (RhoGEF) and p190RhoGAP colocalize at the furrow and physically interact to antagonistically regulate RhoA-GTP","method":"FRET-based RhoA biosensor, time-lapse microscopy, co-immunoprecipitation of ECT2 and p190RhoGAP","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1-2 — FRET biosensor plus co-IP, real-time imaging","pmids":["19254711","18642445"],"is_preprint":false},{"year":2010,"finding":"Mitotic reduction of p190RhoGAP levels is required for successful cytokinesis; a degradation-resistant p190RhoGAP mutant causes cytokinesis failure dependent on GAP activity; the N-terminal GBDS1 region (four residues) is necessary and sufficient for mitotic ubiquitination and degradation","method":"RNAi reconstitution with degradation-resistant mutant, domain mapping, cytokinesis assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — clean RNAi rescue experiment with defined mutants","pmids":["20534586"],"is_preprint":false},{"year":2014,"finding":"p190RhoGAP-A forms a complex with the cytokinetic organizer anillin; p190RhoGAP-A depletion causes excess RhoA-GTP at the furrow and cytokinesis failure; mutants unable to bind anillin or lacking GAP activity fail to rescue; excess contractility is the proximate cause (rescued by myosin II inhibitor blebbistatin)","method":"Depletion/rescue with anillin-binding and GAP-dead mutants, RhoA-GTP measurement, blebbistatin rescue","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — structure-function dissection with clean rescue experiments","pmids":["25359885"],"is_preprint":false},{"year":2009,"finding":"GSK-3beta phosphorylates p190A RhoGAP in a priming-dependent manner at C-terminal tail residues, inhibiting p190A GAP activity in vitro and in vivo; p190A-deficient fibroblasts show directional migration defects, and this requires GSK-3beta-mediated phosphorylation","method":"In vitro GAP activity assay with GSK-3beta phosphorylation, p190A-deficient fibroblasts, phospho-site mutagenesis, directional migration assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis plus cellular phenotype","pmids":["18502760"],"is_preprint":false},{"year":2009,"finding":"Acidic phospholipids inhibit p190A RhoGAP activity toward Rho and promote its RacGAP activity; PKC phosphorylation at Ser1221 and Thr1226 within a polybasic region prevents phospholipid binding, thereby reversing this substrate preference switch","method":"In vitro GAP/RacGAP activity assays, liposome binding assays, PKC phosphorylation, site-directed mutagenesis","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution with mutagenesis and lipid binding assays","pmids":["19673492"],"is_preprint":false},{"year":2013,"finding":"p190RhoGAP has cellular RacGAP activity that requires an intact polybasic region adjacent to the GAP domain; this same region inhibits RhoGAP activity in cells, enabling p190RhoGAP to alternately suppress Rac or Rho depending on polybasic region status","method":"Cellular Rac/Rho activity assays with polybasic region mutants","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 — single lab, cellular assay with defined mutants","pmids":["23499677"],"is_preprint":false},{"year":2007,"finding":"Actin cytoskeleton rearrangements during cell spreading suppress RhoA by promoting accumulation of p190RhoGAP in lipid rafts; the cytoskeletal protein filamin controls this p190RhoGAP redistribution, as cells lacking filamin (or expressing calpain-resistant filamin) fail to accumulate p190RhoGAP in rafts","method":"Lipid raft fractionation, siRNA knockdown of p190RhoGAP, filamin knockout/mutant cells, RhoA activity assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — fractionation plus genetic KO/mutant plus functional assay","pmids":["17227794"],"is_preprint":false},{"year":2011,"finding":"Caveolin-1 deficiency leads to eNOS activation and peroxynitrite generation, which selectively nitrates p190RhoGAP-A at Tyr1105, impairing its GAP activity and activating RhoA, causing adherens junction disassembly and endothelial hyperpermeability; thrombin similarly induces nitration of p120-catenin-associated p190RhoGAP-A","method":"Cav-1-/- endothelial cells, nitrotyrosine immunoprecipitation, eNOS inhibition, in vitro GAP activity assay with nitrated protein","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1-2 — KO cells, specific PTM identification, functional GAP assay","pmids":["21624953"],"is_preprint":false},{"year":2009,"finding":"SHP2 phosphatase maintains p190A RhoGAP basally phosphorylated and active (via c-Abl); angiotensin II AT1R activation induces SHP2-mediated dephosphorylation of p190A, inactivating it and enabling RhoA activation; phosphomimetic p190A mutants block ANG II-induced RhoA activation","method":"siRNA knockdown of p190A and SHP2, phospho-mutant overexpression (phosphoresistant, phosphomimetic), RhoA-kinase activity assays in VSMCs","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 — siRNA plus gain-of-function mutants, mechanistic dissection","pmids":["19692654"],"is_preprint":false},{"year":2010,"finding":"ERK activity is required for fibronectin-stimulated RhoA-GTP loading; ERK phosphorylates the C-terminus of p190A RhoGAP to reduce its peripheral localization and GAP activity, thereby promoting Rho-dependent focal adhesion maturation","method":"MEK/ERK inhibitors, phospho-site mapping of p190A C-terminus, localization assays, focal adhesion quantification, RhoA activity assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — pharmacological inhibition plus site-specific phospho-mutants with functional readout","pmids":["20439493"],"is_preprint":false},{"year":2006,"finding":"Cell surface transglutaminase (tTG) activates RhoA by clustering integrins, which inhibits Src kinase activity and decreases Src-mediated activation of p190RhoGAP; pharmacological Src inhibition reproduces the RhoA activation, placing tTG upstream of Src-p190RhoGAP","method":"tTG overexpression, integrin cross-linking, Src inhibitor (PP2), p190RhoGAP activation measurement, RhoA activity assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — pathway epistasis with pharmacological tools, single lab","pmids":["16452636"],"is_preprint":false},{"year":2016,"finding":"p190A RhoGAP contains a protrusion localization sequence (PLS) that is necessary and sufficient for targeting to leading-edge actin protrusions; cortactin binds the PLS and is required for p190A recruitment to protrusions; the PLS also negatively regulates GAP activity; cancer-associated PLS mutations disrupt both localization and function","method":"Truncation mutagenesis, co-immunoprecipitation with cortactin, subcellular localization assays, RhoA activity assays, cancer mutation analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — domain mapping with multiple mutants plus co-IP and functional assays","pmids":["27646271"],"is_preprint":false},{"year":2022,"finding":"The PLS of p190A RhoGAP acts as an autoinhibitory domain that masks the GAP domain; the PLS interacts with a region adjacent to the GAP domain in cis (intramolecular interaction); cancer-associated PLS mutations (S866F, Δ865-870) abolish this autoinhibitory interaction, constitutively activating GAP function","method":"Two-hybrid screen, co-immunoprecipitation, cancer mutation analysis, cellular RhoA activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — two-hybrid plus co-IP plus functional mutant analysis","pmids":["36516886"],"is_preprint":false},{"year":2013,"finding":"p190RhoGAP interacts with a 23-amino-acid stretch (residues 820-843, CRAD) in the C-terminal domain of p120-catenin; this interaction is required for membrane targeting of p190RhoGAP and activation of its GAP activity at cell-cell junctions, reciprocally suppressing RhoA and activating Rac1 to protect endothelial barrier function","method":"p120-catenin truncation mutants, p190RhoGAP membrane fractionation, RhoA/Rac1 activity assays, barrier permeability assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — minimal domain mapping combined with functional barrier assays","pmids":["23653363"],"is_preprint":false},{"year":2018,"finding":"Crystal structure of the N-terminal GTPase domain of p190RhoGAP-A co-purified with GTP reveals an unusual GTP-Mg2+ binding pocket with six inserts that prevent catalytic activity and interactions with canonical GTPase regulators; mutational analysis shows GTP/Mg2+ binding stabilizes the domain; this establishes N-GTPase as a third pseudoGTPase domain in p190RhoGAP","method":"X-ray crystallography (2.8 Å), biochemical GTP hydrolysis assay, mutagenesis, nucleotide exchange assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus biochemical validation and mutagenesis","pmids":["30174148"],"is_preprint":false},{"year":2019,"finding":"The p120RasGAP N-terminal SH2 domain binds the phosphorylated Y1105-containing peptide of p190RhoGAP (EEENI[pY]SVPHDST) via the conserved FLVR arginine (R207); crystal structure at 1.6 Å reveals peptide binding stabilizes the βE-βF loop and specific arginine conformations; dissociation constant ~0.3 μM","method":"X-ray crystallography (1.75 and 1.6 Å co-crystal), isothermal titration calorimetry, site-directed mutagenesis, native gel shift","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 — crystal structure with ITC and mutagenesis validation","pmids":["31891593"],"is_preprint":false},{"year":2009,"finding":"NMR structure of the first FF domain of p190A RhoGAP (RhoGAPFF1) reveals a non-canonical α1-α2-α3-α4 topology; Y308 is buried in the hydrophobic core and inaccessible to kinases in the folded state; phosphorylation by PDGFR-alpha requires prior domain unfolding and causes irreversible destabilization, providing a mechanism for inhibition of TFII-I interaction","method":"NMR spectroscopy, thermal unfolding assays, in vitro phosphorylation at different temperatures","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 — NMR structure with biochemical validation of phosphorylation mechanism","pmids":["19393245"],"is_preprint":false},{"year":2021,"finding":"HNRNPL facilitates back-splicing of ARHGAP35 exons 2-3 to produce circARHGAP35, which is translated via an m6A-modified start codon into a truncated protein containing four FF domains but lacking the RhoGAP domain; this truncated protein promotes cancer progression by interacting with TFII-I in the nucleus","method":"circRNA identification, m6A sequencing, ORF translation assay, co-immunoprecipitation with TFII-I, HNRNPL knockdown","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 — novel mechanism with co-IP and translation assays, single lab","pmids":["34258149"],"is_preprint":false},{"year":2016,"finding":"p190A RhoGAP is required for primary cilium formation in renal nephrons; a GAP-domain mutation (L1396Q) reduces GAP activity toward RhoA and Rac1, causes glomerulocystic kidneys in mice; p190A localizes to the base of cilia; ROCK inhibition or F-actin polymerization blockade rescues ciliogenesis defects","method":"ENU mutagenesis, in vitro GAP activity assay with L1396Q mutant, immunolocalization, ROCK/F-actin inhibitor rescue, kidney phenotype analysis","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro GAP assay with mutant plus genetic model and rescue experiments","pmids":["26859289"],"is_preprint":false},{"year":2020,"finding":"Polycystin-1 (PC1) regulates ARHGAP35 localization at centrosomes; PKD1-null cystic cells show decreased centrosomal ARHGAP35 associated with increased centrosomal active RhoA and ROCK signaling; ROCK inhibition reduces cyst expansion in vitro and in Pkd1 mouse models","method":"Centrosome-targeted proximity ligation assay, dual immunofluorescence, cilia length as phenotypic readout, ROCK inhibitor in 3D cyst assay and mouse model","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 — proximity ligation assay plus functional in vivo rescue, single lab","pmids":["32663194"],"is_preprint":false},{"year":2019,"finding":"The E3 ubiquitin ligase TRIM65 directly ubiquitinates and promotes proteasomal degradation of ARHGAP35; TRIM65 overexpression elevates RhoA GTPase activity and enhances CRC metastasis; forced ARHGAP35 expression rescues TRIM65-induced migration phenotypes","method":"Co-immunoprecipitation, ubiquitination assay, rescue experiments, in vivo metastasis mouse model","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — ubiquitination assay plus co-IP plus in vivo rescue","pmids":["31332286"],"is_preprint":false},{"year":2020,"finding":"p190A RhoGAP promotes mesenchymal-to-epithelial transition, induces CDH1/E-cadherin expression, and cooperates with E-cadherin to activate LATS kinases and phosphorylate YAP, suppressing tumor growth; p190A is obligatory for E-cadherin to activate LATS kinases; cancer-associated p190A mutants lack these activities","method":"Xenograft mouse model, LATS kinase assay, YAP phosphorylation assay, cancer mutant functional analysis, contact inhibition assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal functional assays plus in vivo model","pmids":["32641858"],"is_preprint":false},{"year":2023,"finding":"p190A RhoGAP activates the Hippo pathway via a complex requiring both p120 RasGAP and the tight junction protein ZO-2; ZO-2 interaction with p190A is dependent on RasGAP; RasGAP and ZO-2 are both necessary for p190A-mediated LATS kinase activation, MET, contact inhibition, and tumor suppression","method":"Co-immunoprecipitation, LATS kinase assay, tumorigenesis assays, siRNA knockdown of ZO-2 and RasGAP","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 — multiple interactors identified by co-IP with functional epistasis","pmids":["37995182"],"is_preprint":false},{"year":2016,"finding":"p190A interacts with all 13 eIF3 subunits and other translational preinitiation factors via its first FF motif (FF1) and the winged helix/PCI domain of eIF3A; the interaction is phosphorylation-dependent (S296 in FF1), serum-stimulated, and the p190A/eIF3 complex is distinct from eIF3-S6K1 or eIF3-mTOR complexes","method":"Tandem mass spectrometry of endogenous p190A complex, co-immunoprecipitation, site-directed mutagenesis (S296A, Y308), phosphatase treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — MS interactome plus orthogonal co-IP plus mutagenesis, functional implications not fully tested","pmids":["28007963"],"is_preprint":false},{"year":2019,"finding":"p190RhoGAP acts as a critical regulator of motor axon guidance in mice via two mechanisms: a GAP-independent mode that transiently suppresses attraction to Netrin-1 while axons exit the spinal cord, and a GAP-dependent mode that enables RhoA inhibition for targeting specific muscles; identified by mouse mutagenesis screen","method":"Mouse mutagenesis screen, GAP-dead mutant analysis, motor axon guidance phenotyping","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 — genetic screen plus GAP-dead mutant functional dissection in vivo","pmids":["30902550"],"is_preprint":false},{"year":2024,"finding":"The CPLANE protein Fuzzy interacts with ARHGAP35 (p190A RhoGAP) and recruits it to the basal body of primary cilia; loss of Fuzzy reduces ARHGAP35 at the basal body, increases RhoA activity and actin polymerization there, and impairs ciliogenesis; genetic interaction between Fuzzy and Arhgap35 alleles confirmed","method":"Co-immunoprecipitation of Fuzzy and ARHGAP35, immunolocalization at basal body, Fuzzy-/- mouse genetics, genetic interaction assay","journal":"Development","confidence":"High","confidence_rationale":"Tier 2 — co-IP plus KO genetics plus localization with functional consequence","pmids":["38546045"],"is_preprint":false},{"year":2018,"finding":"In IPF fibroblasts, Rnd3 expression and p190RhoGAP activity are both suppressed; restoration of Rnd3 levels increases p190RhoGAP activity and decreases RhoA activity, reducing the fibrotic phenotype; the IPF drugs nintedanib and pirfenidone decrease fibrosis through upregulation of Rnd3 and p190RhoGAP activity","method":"Rnd3 restoration in IPF fibroblasts, p190RhoGAP GAP activity assay, RhoA activity assay, pharmacological treatment","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 — GAP activity assay plus genetic restoration, single lab","pmids":["29995590"],"is_preprint":false},{"year":2009,"finding":"p190RhoGAP and ECT2 (RhoGEF) physically associate (co-immunoprecipitation) and colocalize at the cleavage furrow; ECT2 rescues p190-induced multinucleation in a dose-dependent manner; their opposing activities on RhoA-GTP together determine cytokinesis outcome","method":"Co-immunoprecipitation, colocalization, Rho pull-down assay, multinucleation rescue assay","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP plus functional rescue, single lab","pmids":["18642445"],"is_preprint":false},{"year":2014,"finding":"HPV16 E7 protein binds p190RhoGAP via conserved region 3 (CR3) of E7 and the middle domain of p190RhoGAP; E7 proteins from multiple HPV types bind p190RhoGAP; this interaction dysregulates p190RhoGAP GAP activity, alters the actin cytoskeleton, and negatively regulates cell spreading on fibronectin","method":"Mass spectrometry identification, co-immunoprecipitation, CR3 mutagenesis, domain mapping, cell spreading assay","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 — MS discovery plus co-IP domain mapping plus functional readout","pmids":["24403595"],"is_preprint":false},{"year":2018,"finding":"p190RhoGAP depletion causes multipolar spindle formation and prolonged mitotic arrest; p190RhoGAP-depleted multipolar cells localize Aurora A to all poles but activate it only at centriolar poles; Eg5 inhibitor rescue indicates excess Eg5-generated forces underlie the multipolar spindle; defining a role for p190RhoGAP in spindle pole integrity independent of RhoA","method":"siRNA depletion, Aurora A activation assay, Eg5 inhibitor rescue, spindle pole analysis by microscopy","journal":"Chromosoma","confidence":"Medium","confidence_rationale":"Tier 2 — depletion with pharmacological rescue and localization, single lab","pmids":["29656322"],"is_preprint":false},{"year":2023,"finding":"p120RasGAP SH2 domains interact with three doubly phosphorylated partners with distinct binding modes: p190RhoGAP binding engages both SH2 domains with high affinity (~μM), EphB4 binds with ~100-fold lower affinity via single SH2 domain; SAXS reveals distinct conformational states; none of these interactions affect RasGAP catalytic activity","method":"Isothermal titration calorimetry, small-angle X-ray scattering (SAXS), in vitro GAP activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — ITC plus SAXS plus in vitro activity assay","pmids":["37507023"],"is_preprint":false}],"current_model":"ARHGAP35 (p190RhoGAP-A) is a multidomain RhoGAP that serves as the primary negative regulator of RhoA (and, context-dependently, Rac1) downstream of integrin, cadherin, and growth factor receptor signaling; it is activated by tyrosine phosphorylation at Y1105 by Src-family kinases (c-Src, Arg/Abl2, FAK, Brk, Blk), which promotes its association with p120RasGAP via SH2 domain–pTyr interactions and recruitment to the cell periphery/lipid rafts/adherens junctions where it suppresses local RhoA activity; its activity is further regulated by Rho-kinase phosphorylation at Ser1150 (autoinhibitory feedback), GSK-3β phosphorylation of its C-terminal tail, PKC-mediated phosphorylation of a polybasic region that switches substrate preference between Rho and Rac, eNOS-derived nitration at Y1105 (inactivating), TRIM65-mediated ubiquitin-proteasomal degradation, and an intramolecular autoinhibitory interaction between its PLS domain and the GAP domain; during cytokinesis it localizes to the cleavage furrow where it antagonizes ECT2 and forms a complex with anillin to tune RhoA-GTP levels, and its mitotic degradation via an N-terminal degron is required for successful cell division; in neurons it acts downstream of Arg kinase to maintain dendritic spine maturation and synapse stability; it also suppresses tumor growth by activating the Hippo–LATS–YAP pathway in cooperation with p120RasGAP and ZO-2, and its N-terminal domain is a pseudoGTPase that constitutively binds GTP without hydrolysis."},"narrative":{"teleology":[{"year":1995,"claim":"Establishing c-Src as the upstream kinase linking growth factor signaling to p190RhoGAP activation resolved how receptor tyrosine kinase signals reach Rho-family GTPases through a cytoplasmic GAP.","evidence":"Dominant-negative/wild-type c-Src overexpression with immunofluorescence and phosphotyrosine analysis in EGF-stimulated fibroblasts","pmids":["7542246"],"confidence":"High","gaps":["Specific tyrosine site(s) phosphorylated by c-Src not yet mapped","Whether Src phosphorylation directly stimulates GAP catalytic activity or acts via recruitment not distinguished"]},{"year":1998,"claim":"Demonstrating that integrin α6β1 activates p190RhoGAP and that blocking p190RhoGAP inhibits matrix invasion established it as a critical effector linking integrin engagement to Rho suppression during cell motility.","evidence":"Integrin stimulation, tyrosine phosphorylation assays, anti-p190RhoGAP antibody microinjection, and invasion assays in melanoma cells","pmids":["9417037"],"confidence":"High","gaps":["Identity of the kinase mediating integrin-induced p190RhoGAP phosphorylation unresolved","In vivo relevance to metastasis not tested"]},{"year":2001,"claim":"Using dominant-negative p190RhoGAP to show that it is required for integrin-dependent RhoA inactivation, cell spreading, and polarity establishment placed it as the central RhoA-inactivating mechanism during adhesion.","evidence":"Dominant-negative and wild-type overexpression with RhoA activity assays, cell spreading, and migration assays in fibroblasts","pmids":["11553710"],"confidence":"High","gaps":["Genetic loss-of-function not yet performed","Contribution of p190B paralog not assessed"]},{"year":2003,"claim":"Two discoveries—cadherin-induced Src-dependent phosphorylation promoting p120RasGAP binding, and cell-cycle-regulated proteasomal degradation during mitosis—revealed that p190RhoGAP operates in both adhesion signaling and cytokinesis under distinct regulatory modes.","evidence":"RhoA affinity pulldown, co-IP, PP2 inhibition for cadherin axis; conditional overexpression, ubiquitination assays, proteasome inhibition for mitotic degradation","pmids":["12606561","14610059"],"confidence":"High","gaps":["E3 ligase mediating mitotic degradation not identified","Whether cadherin and integrin pathways converge on the same phospho-site unclear"]},{"year":2004,"claim":"Identification of Y1105 as the specific Arg kinase phosphorylation site that triggers p120RasGAP binding and GAP activation provided the first defined molecular switch controlling p190RhoGAP activity.","evidence":"In vitro kinase assay, Y1105 site-directed mutagenesis, co-IP, RhoA activity assay in arg−/− fibroblasts and neurons","pmids":["15084284"],"confidence":"High","gaps":["Whether other kinases also target Y1105 not yet shown","Structural basis for how pY1105 engages p120RasGAP SH2 domain unknown"]},{"year":2006,"claim":"Three studies collectively established that p120RasGAP binding serves primarily as a membrane recruitment mechanism rather than an allosteric activator, and that p190RhoGAP translocation to adherens junctions via p120-catenin mediates Rac/Rho antagonism.","evidence":"arg−/− fibroblasts with fractionation and in vitro GAP assay; p120-catenin co-IP and PDGFR signaling in epithelial cells; tTG/Src epistasis experiments","pmids":["16971514","17129786","16452636"],"confidence":"High","gaps":["Whether p120-catenin and p120RasGAP compete for p190RhoGAP binding not resolved","How p190RhoGAP distinguishes RhoA from Rac1 at junctions unclear"]},{"year":2007,"claim":"Work in arg−/− neurons showed that p190RhoGAP localizes to dendritic spines and is essential for spine maturation and synapse stability via RhoA–ROCKII suppression, extending its function to postmitotic neuronal physiology.","evidence":"arg−/− mouse genetics with ROCKII heterozygosity epistasis, immunolocalization in hippocampal neurons, RhoA activity assay","pmids":["17928439","17652459","17227794"],"confidence":"High","gaps":["Spine-specific activating kinase not confirmed in vivo","Whether p190RhoGAP loss contributes to neurodegenerative disease not tested"]},{"year":2008,"claim":"Multiple inhibitory phosphorylation events were discovered—Rho-kinase at S1150 (feedback loop), Brk at Y1105 (dual Ras/Rho modulation), and syndecan-4/PKCα-mediated membrane redistribution—revealing that p190RhoGAP is a multi-input signaling integrator.","evidence":"In vitro kinase assays with phosphomutants, Rnd binding assays in VSMCs, PKCα mutants with membrane fractionation, and Brk in vitro kinase assay in breast cancer cells","pmids":["19103606","18829532","18541700"],"confidence":"High","gaps":["Relative contributions of activating vs. inhibiting kinases in a single cell type not quantified","No structural model of how S1150 phosphorylation disrupts Rnd binding"]},{"year":2009,"claim":"A cluster of discoveries established additional regulatory layers: GSK-3β C-terminal phosphorylation inhibits GAP activity, PKC phosphorylation at S1221/T1226 switches substrate preference from Rac to Rho via polybasic region–lipid interactions, SHP2 controls basal phosphorylation state, and p190RhoGAP antagonizes ECT2 at the cleavage furrow.","evidence":"In vitro GAP/RacGAP assays with liposomes and PKC; GSK-3β phosphomutants with migration assays; SHP2 siRNA with RhoA assays in VSMCs; FRET RhoA biosensor at the furrow with ECT2 co-IP","pmids":["18502760","19673492","19692654","19254711","18642445","19393245","19803801"],"confidence":"High","gaps":["How PKC-mediated substrate switching operates in vivo not demonstrated","Whether SHP2 directly dephosphorylates Y1105 not confirmed"]},{"year":2010,"claim":"Mapping the minimal N-terminal degron (four residues in GBDS1) sufficient for mitotic ubiquitination, and showing ERK phosphorylation reduces peripheral localization, completed the picture of how p190RhoGAP is temporally and spatially silenced during mitosis and adhesion maturation.","evidence":"RNAi reconstitution with degradation-resistant mutants and domain mapping; MEK/ERK inhibitors with phospho-site mutants and focal adhesion quantification","pmids":["20534586","20439493"],"confidence":"High","gaps":["Identity of the E3 ligase recognizing the N-terminal degron still unknown","How ERK phosphorylation reduces membrane association mechanistically unclear"]},{"year":2013,"claim":"Mapping the minimal p120-catenin CRAD region (residues 820–843) required for p190RhoGAP membrane targeting at junctions, and demonstrating cellular RacGAP activity requiring the polybasic region, established p190RhoGAP as a dual-specificity GAP whose substrate selectivity depends on membrane context.","evidence":"p120-catenin truncation mutants with membrane fractionation and barrier assays; cellular Rac/Rho activity assays with polybasic region mutants","pmids":["23653363","23499677"],"confidence":"High","gaps":["Structural basis for polybasic region-mediated substrate switching not determined","Whether RacGAP activity is physiologically relevant in vivo untested"]},{"year":2014,"claim":"Discovery that p190RhoGAP forms a complex with anillin at the cleavage furrow and that both anillin binding and GAP activity are required for cytokinesis defined the molecular scaffold through which p190RhoGAP tunes contractility during cell division.","evidence":"Depletion/rescue with anillin-binding and GAP-dead mutants, RhoA-GTP measurement, blebbistatin rescue","pmids":["25359885"],"confidence":"High","gaps":["How anillin recruits p190RhoGAP to the furrow structurally unknown","Whether anillin interaction is direct or via a bridging factor not fully resolved"]},{"year":2016,"claim":"Identification of the PLS as a protrusion-targeting sequence that binds cortactin and also functions as an intramolecular autoinhibitory domain, together with the finding that a GAP-domain mutation causes ciliogenesis failure and glomerulocystic kidneys, revealed new regulatory and physiological dimensions of p190RhoGAP.","evidence":"Truncation mutagenesis with co-IP of cortactin and localization/RhoA assays; ENU mutagenesis screen with L1396Q in vitro GAP assay and ROCK inhibitor rescue in mouse kidneys","pmids":["27646271","26859289"],"confidence":"High","gaps":["Structural basis for PLS autoinhibition not determined at atomic resolution","Whether ciliogenesis role is GAP-dependent or scaffolding-dependent not fully dissected"]},{"year":2018,"claim":"Crystal structure of the N-terminal pseudoGTPase domain bound to GTP revealed six structural inserts that prevent catalysis and regulator binding, establishing a third pseudoGTPase in p190RhoGAP and suggesting a structural/scaffolding rather than catalytic role for this domain.","evidence":"X-ray crystallography at 2.8 Å with GTP hydrolysis assay, mutagenesis, and nucleotide exchange assays","pmids":["30174148"],"confidence":"High","gaps":["Cellular function of constitutive GTP binding unknown","Whether pseudoGTPase domain mediates protein–protein interactions not tested"]},{"year":2019,"claim":"Co-crystal structure of the p120RasGAP N-SH2 domain with pY1105 peptide at 1.6 Å defined the atomic basis for the central activating interaction, while in vivo studies showed p190RhoGAP controls motor axon guidance via both GAP-dependent and GAP-independent mechanisms.","evidence":"X-ray crystallography with ITC (Kd ~0.3 µM) and mutagenesis; mouse mutagenesis screen with GAP-dead mutant axon guidance phenotyping","pmids":["31891593","30902550","31332286"],"confidence":"High","gaps":["Full-length p190RhoGAP–p120RasGAP complex structure unavailable","Molecular basis of GAP-independent axon guidance function unknown"]},{"year":2020,"claim":"Demonstrating that p190RhoGAP cooperates with E-cadherin to activate LATS kinases and phosphorylate YAP revealed a direct mechanism for tumor suppression beyond Rho inactivation, linking p190RhoGAP to Hippo pathway regulation.","evidence":"Xenograft mouse model, LATS kinase assay, YAP phosphorylation, cancer mutant analysis, contact inhibition assays","pmids":["32641858"],"confidence":"High","gaps":["Whether p190RhoGAP activates LATS directly or via an intermediary not resolved","Mechanism by which p190RhoGAP induces E-cadherin expression unknown"]},{"year":2022,"claim":"Demonstrating that the PLS autoinhibits the GAP domain via an intramolecular interaction, and that cancer-associated PLS mutations (S866F, Δ865–870) abolish autoinhibition, provided a structural explanation for how somatic mutations constitutively activate p190RhoGAP in tumors.","evidence":"Two-hybrid screen, co-IP, cancer mutation analysis, cellular RhoA activity assays","pmids":["36516886"],"confidence":"High","gaps":["Atomic-resolution structure of PLS–GAP interaction not available","Whether constitutive GAP activation is oncogenic or tumor-suppressive in different contexts not clarified"]},{"year":2023,"claim":"Identification of ZO-2 as a required partner in a tripartite p190RhoGAP–p120RasGAP–ZO-2 complex for Hippo pathway activation established the tight junction as a signaling platform for p190RhoGAP-mediated tumor suppression and contact inhibition.","evidence":"Co-IP, LATS kinase assay, tumorigenesis assays, siRNA knockdown of ZO-2 and RasGAP","pmids":["37995182"],"confidence":"High","gaps":["How ZO-2 connects p190RhoGAP to LATS mechanistically not determined","Whether other tight junction proteins participate not tested"]},{"year":2024,"claim":"Discovery that the CPLANE protein Fuzzy recruits ARHGAP35 to the basal body for ciliogenesis, confirmed by genetic interaction, identified the upstream targeting mechanism for p190RhoGAP's ciliary function.","evidence":"Co-IP of Fuzzy and ARHGAP35, immunolocalization at basal body, Fuzzy−/− mouse genetics, genetic interaction assay","pmids":["38546045"],"confidence":"High","gaps":["Whether Fuzzy directly binds ARHGAP35 or via a bridging protein not resolved","Structural basis of basal body targeting unknown"]},{"year":null,"claim":"A full-length structural model of p190RhoGAP, the mechanism by which it activates LATS kinases in the Hippo pathway, and the identity of the E3 ubiquitin ligase responsible for mitotic degradation via the N-terminal degron remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No full-length structure or cryo-EM model exists","E3 ligase for mitotic N-terminal degron unidentified","How PLS autoinhibition is relieved by upstream signals is structurally undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,6,7,12,15,17,19,20,33]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,15,17,19,33]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[26,27]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[2,7,11,21,26,28]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[15,17,26]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[34,40]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[33,40]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,2,4,5,6,7,11,36,37]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[14,15,16,17,44]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[9,39]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[2,4,28]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[33,40]}],"complexes":["p190RhoGAP–p120RasGAP","p190RhoGAP–anillin (cytokinesis)","p190RhoGAP–p120RasGAP–ZO-2 (Hippo activation)"],"partners":["RASA1","CTNND1","ABL2","ANLN","ECT2","TJP2","CTTN","FUZ"],"other_free_text":[]},"mechanistic_narrative":"ARHGAP35 (p190RhoGAP-A) is the principal GTPase-activating protein for RhoA, integrating signals from integrins, cadherins, and growth factor receptors to locally suppress RhoA-GTP and coordinate cytoskeletal remodeling, cell adhesion, migration, and cytokinesis. Its activation requires Src-family or Arg kinase-mediated phosphorylation at Y1105, which promotes high-affinity binding to the p120RasGAP SH2 domains and recruitment to the plasma membrane, adherens junctions, or lipid rafts where it exerts its GAP activity [PMID:15084284, PMID:31891593, PMID:16971514]; an intramolecular autoinhibitory interaction between the protrusion-localization sequence (PLS) and the GAP domain, along with inhibitory phosphorylation by Rho-kinase (S1150), GSK-3β, and ERK, provides multilayered negative regulation [PMID:36516886, PMID:19103606, PMID:18502760, PMID:20439493]. A polybasic region adjacent to the GAP domain, regulated by PKC phosphorylation and acidic phospholipids, switches substrate preference between RhoA and Rac1 [PMID:19673492, PMID:23499677]. During mitosis, ARHGAP35 localizes to the cleavage furrow where it antagonizes the RhoGEF ECT2 and complexes with anillin to fine-tune contractility, and its timely proteasomal degradation via an N-terminal degron is essential for successful cytokinesis; in neurons it maintains dendritic spine stability downstream of Arg kinase, it is required for primary ciliogenesis at the basal body, and it suppresses tumorigenesis by activating the Hippo–LATS–YAP pathway in cooperation with p120RasGAP and ZO-2 [PMID:25359885, PMID:20534586, PMID:17928439, PMID:26859289, PMID:32641858, PMID:37995182]."},"prefetch_data":{"uniprot":{"accession":"Q9NRY4","full_name":"Rho GTPase-activating protein 35","aliases":["Glucocorticoid receptor DNA-binding factor 1","Glucocorticoid receptor repression factor 1","GRF-1","Rho GAP p190A","p190-A"],"length_aa":1499,"mass_kda":170.5,"function":"Rho GTPase-activating protein (GAP) (PubMed:19673492, PubMed:28894085). Binds several acidic phospholipids which inhibits the Rho GAP activity to promote the Rac GAP activity (PubMed:19673492). This binding is inhibited by phosphorylation by PRKCA (PubMed:19673492). Involved in cell differentiation as well as cell adhesion and migration, plays an important role in retinal tissue morphogenesis, neural tube fusion, midline fusion of the cerebral hemispheres and mammary gland branching morphogenesis (By similarity). Transduces signals from p21-ras to the nucleus, acting via the ras GTPase-activating protein (GAP) (By similarity). Transduces SRC-dependent signals from cell-surface adhesion molecules, such as laminin, to promote neurite outgrowth. Regulates axon outgrowth, guidance and fasciculation (By similarity). Modulates Rho GTPase-dependent F-actin polymerization, organization and assembly, is involved in polarized cell migration and in the positive regulation of ciliogenesis and cilia elongation (By similarity). During mammary gland development, is required in both the epithelial and stromal compartments for ductal outgrowth (By similarity). Represses transcription of the glucocorticoid receptor by binding to the cis-acting regulatory sequence 5'-GAGAAAAGAAACTGGAGAAACTC-3'; this function is however unclear and would need additional experimental evidences (PubMed:1894621)","subcellular_location":"Cytoplasm, cytoskeleton, cilium basal body; Cytoplasm; Nucleus; Cell membrane","url":"https://www.uniprot.org/uniprotkb/Q9NRY4/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ARHGAP35","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000160007","cell_line_id":"CID000558","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"membrane","grade":2},{"compartment":"nucleoplasm","grade":1}],"interactors":[{"gene":"RASA1","stoichiometry":0.2},{"gene":"PA2G4","stoichiometry":0.2},{"gene":"KIAA1211L","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000558","total_profiled":1310},"omim":[{"mim_id":"605277","title":"RHO GTPase-ACTIVATING PROTEIN 35; ARHGAP35","url":"https://www.omim.org/entry/605277"},{"mim_id":"602680","title":"RHO GTPase-ACTIVATING PROTEIN 5; ARHGAP5","url":"https://www.omim.org/entry/602680"},{"mim_id":"601702","title":"RHO-ASSOCIATED COILED-COIL-CONTAINING PROTEIN KINASE 1; ROCK1","url":"https://www.omim.org/entry/601702"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nuclear bodies","reliability":"Approved"},{"location":"Microtubules","reliability":"Additional"},{"location":"Aggresome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ARHGAP35"},"hgnc":{"alias_symbol":["GRF-1","p190ARhoGAP","P190A","KIAA1722","p190RhoGAP"],"prev_symbol":["GRLF1"]},"alphafold":{"accession":"Q9NRY4","domains":[{"cath_id":"3.40.50.300","chopping":"14-258","consensus_level":"high","plddt":91.4193,"start":14,"end":258},{"cath_id":"-","chopping":"282-328","consensus_level":"medium","plddt":93.6353,"start":282,"end":328},{"cath_id":"-","chopping":"338-426","consensus_level":"high","plddt":90.7542,"start":338,"end":426},{"cath_id":"-","chopping":"444-553","consensus_level":"medium","plddt":84.7015,"start":444,"end":553},{"cath_id":"3.40.50.300","chopping":"597-685_692-763","consensus_level":"high","plddt":77.3319,"start":597,"end":763},{"cath_id":"3.40.50.300","chopping":"780-959","consensus_level":"high","plddt":81.4874,"start":780,"end":959},{"cath_id":"1.10.555.10","chopping":"1252-1438","consensus_level":"high","plddt":91.9069,"start":1252,"end":1438}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NRY4","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NRY4-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9NRY4-F1-predicted_aligned_error_v6.png","plddt_mean":73.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ARHGAP35","jax_strain_url":"https://www.jax.org/strain/search?query=ARHGAP35"},"sequence":{"accession":"Q9NRY4","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9NRY4.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9NRY4/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9NRY4"}},"corpus_meta":[{"pmid":"11553710","id":"PMC_11553710","title":"RhoA 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activation causes translocation of p190RhoGAP to adherens junctions where it binds p120-catenin; this p120-p190RhoGAP interaction is required for adherens junction assembly and locally inhibits RhoA to coordinate Rac/Rho antagonism downstream of PDGFR\",\n      \"method\": \"Co-immunoprecipitation, knockdown, PDGFR signaling assays, cell fractionation\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, epistasis, multiple orthogonal methods\",\n      \"pmids\": [\"17129786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Activation of alpha6beta1 integrin in melanoma cells induces tyrosine phosphorylation of p190RhoGAP and promotes membrane-protrusive activities at invadopodia; microinjection of anti-p190RhoGAP antibodies blocks matrix degradation and invasion\",\n      \"method\": \"Integrin stimulation, tyrosine phosphorylation assays, microinjection of blocking antibodies, invasion assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — antibody microinjection plus signaling assays, multiple orthogonal approaches\",\n      \"pmids\": [\"9417037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cadherin engagement induces tyrosine phosphorylation of p190RhoGAP (requiring Src family kinases) and increases its binding to p120RasGAP, leading to RhoA inactivation; dominant-negative p190RhoGAP blocks cadherin-induced RhoA inhibition\",\n      \"method\": \"Active RhoA affinity pulldown to isolate activated GAPs, co-immunoprecipitation, PP2 inhibitor, dominant-negative expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical affinity assay plus co-IP and pharmacological inhibition\",\n      \"pmids\": [\"12606561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Focal adhesion kinase (FAK) associates with and directly phosphorylates p190RhoGAP in vitro, suppressing RhoA activity; FAK-mediated p190RhoGAP activation is required for endothelial barrier restoration after thrombin-induced permeability increase\",\n      \"method\": \"In vitro kinase assay (recombinant FAK phosphorylating p190RhoGAP), co-immunoprecipitation, dominant-negative FAK (FRNK) adenoviral expression, RhoA activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution plus in vivo functional validation\",\n      \"pmids\": [\"16308318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The Arg (Abl-related gene) tyrosine kinase phosphorylates p190RhoGAP at Y1105 in vitro and in vivo; this phosphorylation promotes p190RhoGAP association with p120RasGAP, stimulates p190RhoGAP RhoGAP activity, and is required for adhesion-dependent RhoA inhibition and neuritogenesis\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis (Y1105), co-immunoprecipitation, arg-/- fibroblasts/neurons, Rho activity assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis, knockout cells\",\n      \"pmids\": [\"15084284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Integrin signaling through the Arg kinase activates p190RhoGAP by promoting its association with p120RasGAP, which recruits p190RhoGAP to the cell periphery; p120 binding is required for in vivo but not in vitro p190RhoGAP activation, demonstrating that membrane recruitment is the critical activation mechanism\",\n      \"method\": \"arg-/- fibroblasts, dominant-negative p120 fragment, cell fractionation, in vitro GAP assay, co-immunoprecipitation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro GAP assay combined with genetic KO and fractionation\",\n      \"pmids\": [\"16971514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Arg kinase acts through p190RhoGAP to suppress RhoA activity, attenuate actomyosin contractility, and regulate focal adhesion dynamics; the Arg N-terminal kinase domain suffices to reduce stress fibers via p190RhoGAP, while full focal adhesion inhibition also requires the Arg C-terminal cytoskeleton-binding domain\",\n      \"method\": \"arg-/- fibroblasts, Arg domain mutant re-expression, myosin activity assays, focal adhesion quantification\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic KO with domain-specific rescue, multiple cellular phenotypes\",\n      \"pmids\": [\"17652459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"p190RhoGAP localizes to dendritic spines in hippocampal neurons; its activity is reduced in arg-/- mice, causing elevated RhoA activity and failure of dendritic spine maturation, with subsequent synapse and dendrite loss; reducing ROCKII gene dosage suppresses dendritic regression in arg-/- mice (epistasis)\",\n      \"method\": \"arg-/- mouse genetics, ROCKII heterozygosity epistasis, immunolocalization, RhoA activity assay\",\n      \"journal\": \"The Journal of neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with two KO models, localization data\",\n      \"pmids\": [\"17928439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"FAK promotes formation of a FAK-p120RasGAP-p190RhoGAP complex at leading-edge focal adhesions via fibronectin-integrin signaling; FAK Y397 phosphorylation mediates SH2-dependent binding of p120RasGAP, facilitating p190RhoGAP tyrosine phosphorylation and RhoA inhibition required for cell polarity during migration\",\n      \"method\": \"Wound healing/Golgi reorientation polarity assays, co-immunoprecipitation, FAK Y397 mutant, Pyk2-FAK chimera reconstitution, p120RasGAP knockdown\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, genetic reconstitution approach\",\n      \"pmids\": [\"19435801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"alpha5beta1 integrin engagement triggers Src-dependent tyrosine phosphorylation of p190RhoGAP, while syndecan-4 engagement causes PKC-alpha-dependent redistribution of phosphorylated p190RhoGAP to the membrane; both pathways must converge on p190RhoGAP for efficient RhoA suppression and focal adhesion formation\",\n      \"method\": \"Integrin/syndecan-4 engagement experiments, cell fractionation, PKC-alpha mutants, siRNA knockdown, RhoA activity assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — dissection of two signaling arms with orthogonal methods\",\n      \"pmids\": [\"18541700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Rho-kinase phosphorylates p190A RhoGAP at Ser1150, impairing its GAP activity by disrupting Rnd binding; this creates a positive feedback loop sustaining RhoA activation in response to high-dose endothelin-1 in vascular smooth muscle cells\",\n      \"method\": \"In vitro kinase assay, phosphomimetic and phosphoresistant mutants, Rnd binding assays, RhoA activity assay in VSMCs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with site-specific mutants and functional validation\",\n      \"pmids\": [\"19103606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Breast tumor kinase (Brk) phosphorylates p190RhoGAP-A at Y1105 in vitro and in vivo, promoting its association with p120RasGAP; this simultaneously activates p190RhoGAP (inactivating RhoA) and attenuates p120RasGAP (activating Ras), driving breast cancer proliferation and invasion\",\n      \"method\": \"In vitro kinase assay, Y1105 mutagenesis, co-immunoprecipitation, RhoA/Ras activity assays, invasion/migration/transformation assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis, multiple functional readouts\",\n      \"pmids\": [\"18829532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"p190RhoGAP is cell cycle-regulated: endogenous protein levels decrease in late mitosis via ubiquitin-mediated proteasomal degradation requiring the N-terminal GTP-binding region; overexpression causes abnormal cleavage furrow positioning and cytokinesis failure in a GAP-domain-dependent manner\",\n      \"method\": \"Conditional/transient overexpression, confocal localization during cytokinesis, proteasome inhibition, ubiquitination assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, functional domain requirement established\",\n      \"pmids\": [\"14610059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"p190RhoGAP localizes to the cleavage furrow and reduces RhoA-GTP levels there in a dose-dependent manner as measured by FRET biosensor; overexpression causes multiple cycles of abnormal furrow site selection and ingression/regression, while ECT2 (RhoGEF) and p190RhoGAP colocalize at the furrow and physically interact to antagonistically regulate RhoA-GTP\",\n      \"method\": \"FRET-based RhoA biosensor, time-lapse microscopy, co-immunoprecipitation of ECT2 and p190RhoGAP\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — FRET biosensor plus co-IP, real-time imaging\",\n      \"pmids\": [\"19254711\", \"18642445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mitotic reduction of p190RhoGAP levels is required for successful cytokinesis; a degradation-resistant p190RhoGAP mutant causes cytokinesis failure dependent on GAP activity; the N-terminal GBDS1 region (four residues) is necessary and sufficient for mitotic ubiquitination and degradation\",\n      \"method\": \"RNAi reconstitution with degradation-resistant mutant, domain mapping, cytokinesis assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean RNAi rescue experiment with defined mutants\",\n      \"pmids\": [\"20534586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"p190RhoGAP-A forms a complex with the cytokinetic organizer anillin; p190RhoGAP-A depletion causes excess RhoA-GTP at the furrow and cytokinesis failure; mutants unable to bind anillin or lacking GAP activity fail to rescue; excess contractility is the proximate cause (rescued by myosin II inhibitor blebbistatin)\",\n      \"method\": \"Depletion/rescue with anillin-binding and GAP-dead mutants, RhoA-GTP measurement, blebbistatin rescue\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — structure-function dissection with clean rescue experiments\",\n      \"pmids\": [\"25359885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"GSK-3beta phosphorylates p190A RhoGAP in a priming-dependent manner at C-terminal tail residues, inhibiting p190A GAP activity in vitro and in vivo; p190A-deficient fibroblasts show directional migration defects, and this requires GSK-3beta-mediated phosphorylation\",\n      \"method\": \"In vitro GAP activity assay with GSK-3beta phosphorylation, p190A-deficient fibroblasts, phospho-site mutagenesis, directional migration assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis plus cellular phenotype\",\n      \"pmids\": [\"18502760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Acidic phospholipids inhibit p190A RhoGAP activity toward Rho and promote its RacGAP activity; PKC phosphorylation at Ser1221 and Thr1226 within a polybasic region prevents phospholipid binding, thereby reversing this substrate preference switch\",\n      \"method\": \"In vitro GAP/RacGAP activity assays, liposome binding assays, PKC phosphorylation, site-directed mutagenesis\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution with mutagenesis and lipid binding assays\",\n      \"pmids\": [\"19673492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"p190RhoGAP has cellular RacGAP activity that requires an intact polybasic region adjacent to the GAP domain; this same region inhibits RhoGAP activity in cells, enabling p190RhoGAP to alternately suppress Rac or Rho depending on polybasic region status\",\n      \"method\": \"Cellular Rac/Rho activity assays with polybasic region mutants\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — single lab, cellular assay with defined mutants\",\n      \"pmids\": [\"23499677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Actin cytoskeleton rearrangements during cell spreading suppress RhoA by promoting accumulation of p190RhoGAP in lipid rafts; the cytoskeletal protein filamin controls this p190RhoGAP redistribution, as cells lacking filamin (or expressing calpain-resistant filamin) fail to accumulate p190RhoGAP in rafts\",\n      \"method\": \"Lipid raft fractionation, siRNA knockdown of p190RhoGAP, filamin knockout/mutant cells, RhoA activity assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — fractionation plus genetic KO/mutant plus functional assay\",\n      \"pmids\": [\"17227794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Caveolin-1 deficiency leads to eNOS activation and peroxynitrite generation, which selectively nitrates p190RhoGAP-A at Tyr1105, impairing its GAP activity and activating RhoA, causing adherens junction disassembly and endothelial hyperpermeability; thrombin similarly induces nitration of p120-catenin-associated p190RhoGAP-A\",\n      \"method\": \"Cav-1-/- endothelial cells, nitrotyrosine immunoprecipitation, eNOS inhibition, in vitro GAP activity assay with nitrated protein\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — KO cells, specific PTM identification, functional GAP assay\",\n      \"pmids\": [\"21624953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SHP2 phosphatase maintains p190A RhoGAP basally phosphorylated and active (via c-Abl); angiotensin II AT1R activation induces SHP2-mediated dephosphorylation of p190A, inactivating it and enabling RhoA activation; phosphomimetic p190A mutants block ANG II-induced RhoA activation\",\n      \"method\": \"siRNA knockdown of p190A and SHP2, phospho-mutant overexpression (phosphoresistant, phosphomimetic), RhoA-kinase activity assays in VSMCs\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — siRNA plus gain-of-function mutants, mechanistic dissection\",\n      \"pmids\": [\"19692654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ERK activity is required for fibronectin-stimulated RhoA-GTP loading; ERK phosphorylates the C-terminus of p190A RhoGAP to reduce its peripheral localization and GAP activity, thereby promoting Rho-dependent focal adhesion maturation\",\n      \"method\": \"MEK/ERK inhibitors, phospho-site mapping of p190A C-terminus, localization assays, focal adhesion quantification, RhoA activity assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — pharmacological inhibition plus site-specific phospho-mutants with functional readout\",\n      \"pmids\": [\"20439493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cell surface transglutaminase (tTG) activates RhoA by clustering integrins, which inhibits Src kinase activity and decreases Src-mediated activation of p190RhoGAP; pharmacological Src inhibition reproduces the RhoA activation, placing tTG upstream of Src-p190RhoGAP\",\n      \"method\": \"tTG overexpression, integrin cross-linking, Src inhibitor (PP2), p190RhoGAP activation measurement, RhoA activity assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — pathway epistasis with pharmacological tools, single lab\",\n      \"pmids\": [\"16452636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"p190A RhoGAP contains a protrusion localization sequence (PLS) that is necessary and sufficient for targeting to leading-edge actin protrusions; cortactin binds the PLS and is required for p190A recruitment to protrusions; the PLS also negatively regulates GAP activity; cancer-associated PLS mutations disrupt both localization and function\",\n      \"method\": \"Truncation mutagenesis, co-immunoprecipitation with cortactin, subcellular localization assays, RhoA activity assays, cancer mutation analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain mapping with multiple mutants plus co-IP and functional assays\",\n      \"pmids\": [\"27646271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The PLS of p190A RhoGAP acts as an autoinhibitory domain that masks the GAP domain; the PLS interacts with a region adjacent to the GAP domain in cis (intramolecular interaction); cancer-associated PLS mutations (S866F, Δ865-870) abolish this autoinhibitory interaction, constitutively activating GAP function\",\n      \"method\": \"Two-hybrid screen, co-immunoprecipitation, cancer mutation analysis, cellular RhoA activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — two-hybrid plus co-IP plus functional mutant analysis\",\n      \"pmids\": [\"36516886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"p190RhoGAP interacts with a 23-amino-acid stretch (residues 820-843, CRAD) in the C-terminal domain of p120-catenin; this interaction is required for membrane targeting of p190RhoGAP and activation of its GAP activity at cell-cell junctions, reciprocally suppressing RhoA and activating Rac1 to protect endothelial barrier function\",\n      \"method\": \"p120-catenin truncation mutants, p190RhoGAP membrane fractionation, RhoA/Rac1 activity assays, barrier permeability assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — minimal domain mapping combined with functional barrier assays\",\n      \"pmids\": [\"23653363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Crystal structure of the N-terminal GTPase domain of p190RhoGAP-A co-purified with GTP reveals an unusual GTP-Mg2+ binding pocket with six inserts that prevent catalytic activity and interactions with canonical GTPase regulators; mutational analysis shows GTP/Mg2+ binding stabilizes the domain; this establishes N-GTPase as a third pseudoGTPase domain in p190RhoGAP\",\n      \"method\": \"X-ray crystallography (2.8 Å), biochemical GTP hydrolysis assay, mutagenesis, nucleotide exchange assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus biochemical validation and mutagenesis\",\n      \"pmids\": [\"30174148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The p120RasGAP N-terminal SH2 domain binds the phosphorylated Y1105-containing peptide of p190RhoGAP (EEENI[pY]SVPHDST) via the conserved FLVR arginine (R207); crystal structure at 1.6 Å reveals peptide binding stabilizes the βE-βF loop and specific arginine conformations; dissociation constant ~0.3 μM\",\n      \"method\": \"X-ray crystallography (1.75 and 1.6 Å co-crystal), isothermal titration calorimetry, site-directed mutagenesis, native gel shift\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure with ITC and mutagenesis validation\",\n      \"pmids\": [\"31891593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NMR structure of the first FF domain of p190A RhoGAP (RhoGAPFF1) reveals a non-canonical α1-α2-α3-α4 topology; Y308 is buried in the hydrophobic core and inaccessible to kinases in the folded state; phosphorylation by PDGFR-alpha requires prior domain unfolding and causes irreversible destabilization, providing a mechanism for inhibition of TFII-I interaction\",\n      \"method\": \"NMR spectroscopy, thermal unfolding assays, in vitro phosphorylation at different temperatures\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — NMR structure with biochemical validation of phosphorylation mechanism\",\n      \"pmids\": [\"19393245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HNRNPL facilitates back-splicing of ARHGAP35 exons 2-3 to produce circARHGAP35, which is translated via an m6A-modified start codon into a truncated protein containing four FF domains but lacking the RhoGAP domain; this truncated protein promotes cancer progression by interacting with TFII-I in the nucleus\",\n      \"method\": \"circRNA identification, m6A sequencing, ORF translation assay, co-immunoprecipitation with TFII-I, HNRNPL knockdown\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — novel mechanism with co-IP and translation assays, single lab\",\n      \"pmids\": [\"34258149\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"p190A RhoGAP is required for primary cilium formation in renal nephrons; a GAP-domain mutation (L1396Q) reduces GAP activity toward RhoA and Rac1, causes glomerulocystic kidneys in mice; p190A localizes to the base of cilia; ROCK inhibition or F-actin polymerization blockade rescues ciliogenesis defects\",\n      \"method\": \"ENU mutagenesis, in vitro GAP activity assay with L1396Q mutant, immunolocalization, ROCK/F-actin inhibitor rescue, kidney phenotype analysis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro GAP assay with mutant plus genetic model and rescue experiments\",\n      \"pmids\": [\"26859289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Polycystin-1 (PC1) regulates ARHGAP35 localization at centrosomes; PKD1-null cystic cells show decreased centrosomal ARHGAP35 associated with increased centrosomal active RhoA and ROCK signaling; ROCK inhibition reduces cyst expansion in vitro and in Pkd1 mouse models\",\n      \"method\": \"Centrosome-targeted proximity ligation assay, dual immunofluorescence, cilia length as phenotypic readout, ROCK inhibitor in 3D cyst assay and mouse model\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — proximity ligation assay plus functional in vivo rescue, single lab\",\n      \"pmids\": [\"32663194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The E3 ubiquitin ligase TRIM65 directly ubiquitinates and promotes proteasomal degradation of ARHGAP35; TRIM65 overexpression elevates RhoA GTPase activity and enhances CRC metastasis; forced ARHGAP35 expression rescues TRIM65-induced migration phenotypes\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, rescue experiments, in vivo metastasis mouse model\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ubiquitination assay plus co-IP plus in vivo rescue\",\n      \"pmids\": [\"31332286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"p190A RhoGAP promotes mesenchymal-to-epithelial transition, induces CDH1/E-cadherin expression, and cooperates with E-cadherin to activate LATS kinases and phosphorylate YAP, suppressing tumor growth; p190A is obligatory for E-cadherin to activate LATS kinases; cancer-associated p190A mutants lack these activities\",\n      \"method\": \"Xenograft mouse model, LATS kinase assay, YAP phosphorylation assay, cancer mutant functional analysis, contact inhibition assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal functional assays plus in vivo model\",\n      \"pmids\": [\"32641858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"p190A RhoGAP activates the Hippo pathway via a complex requiring both p120 RasGAP and the tight junction protein ZO-2; ZO-2 interaction with p190A is dependent on RasGAP; RasGAP and ZO-2 are both necessary for p190A-mediated LATS kinase activation, MET, contact inhibition, and tumor suppression\",\n      \"method\": \"Co-immunoprecipitation, LATS kinase assay, tumorigenesis assays, siRNA knockdown of ZO-2 and RasGAP\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple interactors identified by co-IP with functional epistasis\",\n      \"pmids\": [\"37995182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"p190A interacts with all 13 eIF3 subunits and other translational preinitiation factors via its first FF motif (FF1) and the winged helix/PCI domain of eIF3A; the interaction is phosphorylation-dependent (S296 in FF1), serum-stimulated, and the p190A/eIF3 complex is distinct from eIF3-S6K1 or eIF3-mTOR complexes\",\n      \"method\": \"Tandem mass spectrometry of endogenous p190A complex, co-immunoprecipitation, site-directed mutagenesis (S296A, Y308), phosphatase treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS interactome plus orthogonal co-IP plus mutagenesis, functional implications not fully tested\",\n      \"pmids\": [\"28007963\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"p190RhoGAP acts as a critical regulator of motor axon guidance in mice via two mechanisms: a GAP-independent mode that transiently suppresses attraction to Netrin-1 while axons exit the spinal cord, and a GAP-dependent mode that enables RhoA inhibition for targeting specific muscles; identified by mouse mutagenesis screen\",\n      \"method\": \"Mouse mutagenesis screen, GAP-dead mutant analysis, motor axon guidance phenotyping\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic screen plus GAP-dead mutant functional dissection in vivo\",\n      \"pmids\": [\"30902550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The CPLANE protein Fuzzy interacts with ARHGAP35 (p190A RhoGAP) and recruits it to the basal body of primary cilia; loss of Fuzzy reduces ARHGAP35 at the basal body, increases RhoA activity and actin polymerization there, and impairs ciliogenesis; genetic interaction between Fuzzy and Arhgap35 alleles confirmed\",\n      \"method\": \"Co-immunoprecipitation of Fuzzy and ARHGAP35, immunolocalization at basal body, Fuzzy-/- mouse genetics, genetic interaction assay\",\n      \"journal\": \"Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus KO genetics plus localization with functional consequence\",\n      \"pmids\": [\"38546045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In IPF fibroblasts, Rnd3 expression and p190RhoGAP activity are both suppressed; restoration of Rnd3 levels increases p190RhoGAP activity and decreases RhoA activity, reducing the fibrotic phenotype; the IPF drugs nintedanib and pirfenidone decrease fibrosis through upregulation of Rnd3 and p190RhoGAP activity\",\n      \"method\": \"Rnd3 restoration in IPF fibroblasts, p190RhoGAP GAP activity assay, RhoA activity assay, pharmacological treatment\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — GAP activity assay plus genetic restoration, single lab\",\n      \"pmids\": [\"29995590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"p190RhoGAP and ECT2 (RhoGEF) physically associate (co-immunoprecipitation) and colocalize at the cleavage furrow; ECT2 rescues p190-induced multinucleation in a dose-dependent manner; their opposing activities on RhoA-GTP together determine cytokinesis outcome\",\n      \"method\": \"Co-immunoprecipitation, colocalization, Rho pull-down assay, multinucleation rescue assay\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP plus functional rescue, single lab\",\n      \"pmids\": [\"18642445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HPV16 E7 protein binds p190RhoGAP via conserved region 3 (CR3) of E7 and the middle domain of p190RhoGAP; E7 proteins from multiple HPV types bind p190RhoGAP; this interaction dysregulates p190RhoGAP GAP activity, alters the actin cytoskeleton, and negatively regulates cell spreading on fibronectin\",\n      \"method\": \"Mass spectrometry identification, co-immunoprecipitation, CR3 mutagenesis, domain mapping, cell spreading assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — MS discovery plus co-IP domain mapping plus functional readout\",\n      \"pmids\": [\"24403595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"p190RhoGAP depletion causes multipolar spindle formation and prolonged mitotic arrest; p190RhoGAP-depleted multipolar cells localize Aurora A to all poles but activate it only at centriolar poles; Eg5 inhibitor rescue indicates excess Eg5-generated forces underlie the multipolar spindle; defining a role for p190RhoGAP in spindle pole integrity independent of RhoA\",\n      \"method\": \"siRNA depletion, Aurora A activation assay, Eg5 inhibitor rescue, spindle pole analysis by microscopy\",\n      \"journal\": \"Chromosoma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — depletion with pharmacological rescue and localization, single lab\",\n      \"pmids\": [\"29656322\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"p120RasGAP SH2 domains interact with three doubly phosphorylated partners with distinct binding modes: p190RhoGAP binding engages both SH2 domains with high affinity (~μM), EphB4 binds with ~100-fold lower affinity via single SH2 domain; SAXS reveals distinct conformational states; none of these interactions affect RasGAP catalytic activity\",\n      \"method\": \"Isothermal titration calorimetry, small-angle X-ray scattering (SAXS), in vitro GAP activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — ITC plus SAXS plus in vitro activity assay\",\n      \"pmids\": [\"37507023\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ARHGAP35 (p190RhoGAP-A) is a multidomain RhoGAP that serves as the primary negative regulator of RhoA (and, context-dependently, Rac1) downstream of integrin, cadherin, and growth factor receptor signaling; it is activated by tyrosine phosphorylation at Y1105 by Src-family kinases (c-Src, Arg/Abl2, FAK, Brk, Blk), which promotes its association with p120RasGAP via SH2 domain–pTyr interactions and recruitment to the cell periphery/lipid rafts/adherens junctions where it suppresses local RhoA activity; its activity is further regulated by Rho-kinase phosphorylation at Ser1150 (autoinhibitory feedback), GSK-3β phosphorylation of its C-terminal tail, PKC-mediated phosphorylation of a polybasic region that switches substrate preference between Rho and Rac, eNOS-derived nitration at Y1105 (inactivating), TRIM65-mediated ubiquitin-proteasomal degradation, and an intramolecular autoinhibitory interaction between its PLS domain and the GAP domain; during cytokinesis it localizes to the cleavage furrow where it antagonizes ECT2 and forms a complex with anillin to tune RhoA-GTP levels, and its mitotic degradation via an N-terminal degron is required for successful cell division; in neurons it acts downstream of Arg kinase to maintain dendritic spine maturation and synapse stability; it also suppresses tumor growth by activating the Hippo–LATS–YAP pathway in cooperation with p120RasGAP and ZO-2, and its N-terminal domain is a pseudoGTPase that constitutively binds GTP without hydrolysis.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ARHGAP35 (p190RhoGAP-A) is the principal GTPase-activating protein for RhoA, integrating signals from integrins, cadherins, and growth factor receptors to locally suppress RhoA-GTP and coordinate cytoskeletal remodeling, cell adhesion, migration, and cytokinesis. Its activation requires Src-family or Arg kinase-mediated phosphorylation at Y1105, which promotes high-affinity binding to the p120RasGAP SH2 domains and recruitment to the plasma membrane, adherens junctions, or lipid rafts where it exerts its GAP activity [PMID:15084284, PMID:31891593, PMID:16971514]; an intramolecular autoinhibitory interaction between the protrusion-localization sequence (PLS) and the GAP domain, along with inhibitory phosphorylation by Rho-kinase (S1150), GSK-3β, and ERK, provides multilayered negative regulation [PMID:36516886, PMID:19103606, PMID:18502760, PMID:20439493]. A polybasic region adjacent to the GAP domain, regulated by PKC phosphorylation and acidic phospholipids, switches substrate preference between RhoA and Rac1 [PMID:19673492, PMID:23499677]. During mitosis, ARHGAP35 localizes to the cleavage furrow where it antagonizes the RhoGEF ECT2 and complexes with anillin to fine-tune contractility, and its timely proteasomal degradation via an N-terminal degron is essential for successful cytokinesis; in neurons it maintains dendritic spine stability downstream of Arg kinase, it is required for primary ciliogenesis at the basal body, and it suppresses tumorigenesis by activating the Hippo–LATS–YAP pathway in cooperation with p120RasGAP and ZO-2 [PMID:25359885, PMID:20534586, PMID:17928439, PMID:26859289, PMID:32641858, PMID:37995182].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Establishing c-Src as the upstream kinase linking growth factor signaling to p190RhoGAP activation resolved how receptor tyrosine kinase signals reach Rho-family GTPases through a cytoplasmic GAP.\",\n      \"evidence\": \"Dominant-negative/wild-type c-Src overexpression with immunofluorescence and phosphotyrosine analysis in EGF-stimulated fibroblasts\",\n      \"pmids\": [\"7542246\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific tyrosine site(s) phosphorylated by c-Src not yet mapped\", \"Whether Src phosphorylation directly stimulates GAP catalytic activity or acts via recruitment not distinguished\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Demonstrating that integrin α6β1 activates p190RhoGAP and that blocking p190RhoGAP inhibits matrix invasion established it as a critical effector linking integrin engagement to Rho suppression during cell motility.\",\n      \"evidence\": \"Integrin stimulation, tyrosine phosphorylation assays, anti-p190RhoGAP antibody microinjection, and invasion assays in melanoma cells\",\n      \"pmids\": [\"9417037\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the kinase mediating integrin-induced p190RhoGAP phosphorylation unresolved\", \"In vivo relevance to metastasis not tested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Using dominant-negative p190RhoGAP to show that it is required for integrin-dependent RhoA inactivation, cell spreading, and polarity establishment placed it as the central RhoA-inactivating mechanism during adhesion.\",\n      \"evidence\": \"Dominant-negative and wild-type overexpression with RhoA activity assays, cell spreading, and migration assays in fibroblasts\",\n      \"pmids\": [\"11553710\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genetic loss-of-function not yet performed\", \"Contribution of p190B paralog not assessed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Two discoveries—cadherin-induced Src-dependent phosphorylation promoting p120RasGAP binding, and cell-cycle-regulated proteasomal degradation during mitosis—revealed that p190RhoGAP operates in both adhesion signaling and cytokinesis under distinct regulatory modes.\",\n      \"evidence\": \"RhoA affinity pulldown, co-IP, PP2 inhibition for cadherin axis; conditional overexpression, ubiquitination assays, proteasome inhibition for mitotic degradation\",\n      \"pmids\": [\"12606561\", \"14610059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase mediating mitotic degradation not identified\", \"Whether cadherin and integrin pathways converge on the same phospho-site unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identification of Y1105 as the specific Arg kinase phosphorylation site that triggers p120RasGAP binding and GAP activation provided the first defined molecular switch controlling p190RhoGAP activity.\",\n      \"evidence\": \"In vitro kinase assay, Y1105 site-directed mutagenesis, co-IP, RhoA activity assay in arg−/− fibroblasts and neurons\",\n      \"pmids\": [\"15084284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether other kinases also target Y1105 not yet shown\", \"Structural basis for how pY1105 engages p120RasGAP SH2 domain unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Three studies collectively established that p120RasGAP binding serves primarily as a membrane recruitment mechanism rather than an allosteric activator, and that p190RhoGAP translocation to adherens junctions via p120-catenin mediates Rac/Rho antagonism.\",\n      \"evidence\": \"arg−/− fibroblasts with fractionation and in vitro GAP assay; p120-catenin co-IP and PDGFR signaling in epithelial cells; tTG/Src epistasis experiments\",\n      \"pmids\": [\"16971514\", \"17129786\", \"16452636\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p120-catenin and p120RasGAP compete for p190RhoGAP binding not resolved\", \"How p190RhoGAP distinguishes RhoA from Rac1 at junctions unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Work in arg−/− neurons showed that p190RhoGAP localizes to dendritic spines and is essential for spine maturation and synapse stability via RhoA–ROCKII suppression, extending its function to postmitotic neuronal physiology.\",\n      \"evidence\": \"arg−/− mouse genetics with ROCKII heterozygosity epistasis, immunolocalization in hippocampal neurons, RhoA activity assay\",\n      \"pmids\": [\"17928439\", \"17652459\", \"17227794\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spine-specific activating kinase not confirmed in vivo\", \"Whether p190RhoGAP loss contributes to neurodegenerative disease not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Multiple inhibitory phosphorylation events were discovered—Rho-kinase at S1150 (feedback loop), Brk at Y1105 (dual Ras/Rho modulation), and syndecan-4/PKCα-mediated membrane redistribution—revealing that p190RhoGAP is a multi-input signaling integrator.\",\n      \"evidence\": \"In vitro kinase assays with phosphomutants, Rnd binding assays in VSMCs, PKCα mutants with membrane fractionation, and Brk in vitro kinase assay in breast cancer cells\",\n      \"pmids\": [\"19103606\", \"18829532\", \"18541700\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contributions of activating vs. inhibiting kinases in a single cell type not quantified\", \"No structural model of how S1150 phosphorylation disrupts Rnd binding\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"A cluster of discoveries established additional regulatory layers: GSK-3β C-terminal phosphorylation inhibits GAP activity, PKC phosphorylation at S1221/T1226 switches substrate preference from Rac to Rho via polybasic region–lipid interactions, SHP2 controls basal phosphorylation state, and p190RhoGAP antagonizes ECT2 at the cleavage furrow.\",\n      \"evidence\": \"In vitro GAP/RacGAP assays with liposomes and PKC; GSK-3β phosphomutants with migration assays; SHP2 siRNA with RhoA assays in VSMCs; FRET RhoA biosensor at the furrow with ECT2 co-IP\",\n      \"pmids\": [\"18502760\", \"19673492\", \"19692654\", \"19254711\", \"18642445\", \"19393245\", \"19803801\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PKC-mediated substrate switching operates in vivo not demonstrated\", \"Whether SHP2 directly dephosphorylates Y1105 not confirmed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Mapping the minimal N-terminal degron (four residues in GBDS1) sufficient for mitotic ubiquitination, and showing ERK phosphorylation reduces peripheral localization, completed the picture of how p190RhoGAP is temporally and spatially silenced during mitosis and adhesion maturation.\",\n      \"evidence\": \"RNAi reconstitution with degradation-resistant mutants and domain mapping; MEK/ERK inhibitors with phospho-site mutants and focal adhesion quantification\",\n      \"pmids\": [\"20534586\", \"20439493\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the E3 ligase recognizing the N-terminal degron still unknown\", \"How ERK phosphorylation reduces membrane association mechanistically unclear\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapping the minimal p120-catenin CRAD region (residues 820–843) required for p190RhoGAP membrane targeting at junctions, and demonstrating cellular RacGAP activity requiring the polybasic region, established p190RhoGAP as a dual-specificity GAP whose substrate selectivity depends on membrane context.\",\n      \"evidence\": \"p120-catenin truncation mutants with membrane fractionation and barrier assays; cellular Rac/Rho activity assays with polybasic region mutants\",\n      \"pmids\": [\"23653363\", \"23499677\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for polybasic region-mediated substrate switching not determined\", \"Whether RacGAP activity is physiologically relevant in vivo untested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that p190RhoGAP forms a complex with anillin at the cleavage furrow and that both anillin binding and GAP activity are required for cytokinesis defined the molecular scaffold through which p190RhoGAP tunes contractility during cell division.\",\n      \"evidence\": \"Depletion/rescue with anillin-binding and GAP-dead mutants, RhoA-GTP measurement, blebbistatin rescue\",\n      \"pmids\": [\"25359885\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How anillin recruits p190RhoGAP to the furrow structurally unknown\", \"Whether anillin interaction is direct or via a bridging factor not fully resolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identification of the PLS as a protrusion-targeting sequence that binds cortactin and also functions as an intramolecular autoinhibitory domain, together with the finding that a GAP-domain mutation causes ciliogenesis failure and glomerulocystic kidneys, revealed new regulatory and physiological dimensions of p190RhoGAP.\",\n      \"evidence\": \"Truncation mutagenesis with co-IP of cortactin and localization/RhoA assays; ENU mutagenesis screen with L1396Q in vitro GAP assay and ROCK inhibitor rescue in mouse kidneys\",\n      \"pmids\": [\"27646271\", \"26859289\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis for PLS autoinhibition not determined at atomic resolution\", \"Whether ciliogenesis role is GAP-dependent or scaffolding-dependent not fully dissected\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Crystal structure of the N-terminal pseudoGTPase domain bound to GTP revealed six structural inserts that prevent catalysis and regulator binding, establishing a third pseudoGTPase in p190RhoGAP and suggesting a structural/scaffolding rather than catalytic role for this domain.\",\n      \"evidence\": \"X-ray crystallography at 2.8 Å with GTP hydrolysis assay, mutagenesis, and nucleotide exchange assays\",\n      \"pmids\": [\"30174148\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular function of constitutive GTP binding unknown\", \"Whether pseudoGTPase domain mediates protein–protein interactions not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Co-crystal structure of the p120RasGAP N-SH2 domain with pY1105 peptide at 1.6 Å defined the atomic basis for the central activating interaction, while in vivo studies showed p190RhoGAP controls motor axon guidance via both GAP-dependent and GAP-independent mechanisms.\",\n      \"evidence\": \"X-ray crystallography with ITC (Kd ~0.3 µM) and mutagenesis; mouse mutagenesis screen with GAP-dead mutant axon guidance phenotyping\",\n      \"pmids\": [\"31891593\", \"30902550\", \"31332286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length p190RhoGAP–p120RasGAP complex structure unavailable\", \"Molecular basis of GAP-independent axon guidance function unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrating that p190RhoGAP cooperates with E-cadherin to activate LATS kinases and phosphorylate YAP revealed a direct mechanism for tumor suppression beyond Rho inactivation, linking p190RhoGAP to Hippo pathway regulation.\",\n      \"evidence\": \"Xenograft mouse model, LATS kinase assay, YAP phosphorylation, cancer mutant analysis, contact inhibition assays\",\n      \"pmids\": [\"32641858\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p190RhoGAP activates LATS directly or via an intermediary not resolved\", \"Mechanism by which p190RhoGAP induces E-cadherin expression unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Demonstrating that the PLS autoinhibits the GAP domain via an intramolecular interaction, and that cancer-associated PLS mutations (S866F, Δ865–870) abolish autoinhibition, provided a structural explanation for how somatic mutations constitutively activate p190RhoGAP in tumors.\",\n      \"evidence\": \"Two-hybrid screen, co-IP, cancer mutation analysis, cellular RhoA activity assays\",\n      \"pmids\": [\"36516886\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of PLS–GAP interaction not available\", \"Whether constitutive GAP activation is oncogenic or tumor-suppressive in different contexts not clarified\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identification of ZO-2 as a required partner in a tripartite p190RhoGAP–p120RasGAP–ZO-2 complex for Hippo pathway activation established the tight junction as a signaling platform for p190RhoGAP-mediated tumor suppression and contact inhibition.\",\n      \"evidence\": \"Co-IP, LATS kinase assay, tumorigenesis assays, siRNA knockdown of ZO-2 and RasGAP\",\n      \"pmids\": [\"37995182\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ZO-2 connects p190RhoGAP to LATS mechanistically not determined\", \"Whether other tight junction proteins participate not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Discovery that the CPLANE protein Fuzzy recruits ARHGAP35 to the basal body for ciliogenesis, confirmed by genetic interaction, identified the upstream targeting mechanism for p190RhoGAP's ciliary function.\",\n      \"evidence\": \"Co-IP of Fuzzy and ARHGAP35, immunolocalization at basal body, Fuzzy−/− mouse genetics, genetic interaction assay\",\n      \"pmids\": [\"38546045\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Fuzzy directly binds ARHGAP35 or via a bridging protein not resolved\", \"Structural basis of basal body targeting unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A full-length structural model of p190RhoGAP, the mechanism by which it activates LATS kinases in the Hippo pathway, and the identity of the E3 ubiquitin ligase responsible for mitotic degradation via the N-terminal degron remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No full-length structure or cryo-EM model exists\", \"E3 ligase for mitotic N-terminal degron unidentified\", \"How PLS autoinhibition is relieved by upstream signals is structurally undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 6, 7, 12, 15, 17, 19, 20, 33]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 15, 17, 19, 33]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [26, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [2, 7, 11, 21, 26, 28]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [15, 17, 26]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [34, 40]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [33, 40]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 2, 4, 5, 6, 7, 11, 36, 37]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [14, 15, 16, 17, 44]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [9, 39]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [2, 4, 28]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [33, 40]}\n    ],\n    \"complexes\": [\n      \"p190RhoGAP–p120RasGAP\",\n      \"p190RhoGAP–anillin (cytokinesis)\",\n      \"p190RhoGAP–p120RasGAP–ZO-2 (Hippo activation)\"\n    ],\n    \"partners\": [\n      \"RASA1\",\n      \"CTNND1\",\n      \"ABL2\",\n      \"ANLN\",\n      \"ECT2\",\n      \"TJP2\",\n      \"CTTN\",\n      \"FUZ\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}