{"gene":"ARHGAP35","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2001,"finding":"p190RhoGAP (ARHGAP35) mediates integrin-triggered RhoA inactivation during cell adhesion to fibronectin, promoting membrane protrusion and cell polarity. Dominant negative p190RhoGAP elevated RhoA activity, impaired spreading, and inhibited migration, while overexpression decreased RhoA activity and enhanced motility.","method":"Dominant negative and overexpression of p190RhoGAP in Rat1 fibroblasts; RhoA activity assays; cell spreading and migration assays on fibronectin","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean loss- and gain-of-function with defined phenotypic readouts, replicated across multiple conditions, multiple orthogonal methods","pmids":["11553710"],"is_preprint":false},{"year":1995,"finding":"c-Src regulates EGF-dependent actin cytoskeleton reorganization through tyrosine phosphorylation of p190RhoGAP. The in vivo phosphotyrosine content of p190 varies with c-Src activity, and c-Src variants modulate the rate and extent of p190/RasGAP arc formation and actin stress fiber dynamics.","method":"Overexpression of wild-type and dominant negative c-Src variants; confocal immunofluorescence of p190, actin, and p120RasGAP distribution; phosphotyrosine analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple cell lines, dominant negative and overexpression, multiple orthogonal imaging and biochemical methods","pmids":["7542246"],"is_preprint":false},{"year":1998,"finding":"Beta1 integrin activation by laminin peptides induces tyrosine phosphorylation of p190RhoGAP and localizes phospho-p190 with F-actin specifically at invadopodia. Microinjection of antibodies against p190RhoGAP blocked invadopodial membrane-protrusive and matrix-degradative activities.","method":"Integrin activation with laminin G peptides and antibodies; immunofluorescence; tyrosine kinase inhibitors; microinjection of anti-p190 antibodies","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — microinjection loss-of-function, phosphorylation readout, localization with functional consequence, single lab multiple methods","pmids":["9417037"],"is_preprint":false},{"year":2003,"finding":"Cadherin engagement induces tyrosine phosphorylation of p190RhoGAP and increases its binding to p120RasGAP in a Src-family-kinase-dependent manner. Dominant negative p190RhoGAP antagonized cadherin-induced RhoA inactivation, establishing p190RhoGAP as a required downstream effector of cadherin-mediated RhoA suppression.","method":"Constitutively active RhoA pulldown to isolate active GAPs; dominant negative p190RhoGAP expression; PP2 Src kinase inhibitor; co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (active GAP pulldown, dominant negative, pharmacological inhibitor, co-IP), single lab","pmids":["12606561"],"is_preprint":false},{"year":2005,"finding":"FAK directly phosphorylates p190RhoGAP in vitro and associates with p190RhoGAP; this FAK-induced phosphorylation is required for suppression of RhoA activity and restoration of endothelial barrier function after thrombin treatment. Dominant negative FAK (FRNK) prevented p190RhoGAP phosphorylation, increased RhoA activity, and produced irreversible endothelial permeability.","method":"In vitro kinase assay with recombinant FAK and p190RhoGAP; adenoviral FRNK expression; RhoA activity assays; endothelial permeability measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase reconstitution plus cellular loss-of-function with defined phenotypic readout, single lab","pmids":["16308318"],"is_preprint":false},{"year":2006,"finding":"Rac activation causes translocation of p190RhoGAP to adherens junctions where it couples to the cadherin complex via interaction with p120-catenin. This p120-p190RhoGAP interaction is required for adherens junction formation, and disruption of either protein prevents junction assembly.","method":"siRNA knockdown of p120 and p190RhoGAP; co-immunoprecipitation; immunofluorescence localization; functional AJ assembly assays in NIH3T3 cells stimulated with PDGFR","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, siRNA knockdown, localization with functional consequence, multiple cell systems","pmids":["17129786"],"is_preprint":false},{"year":2006,"finding":"Integrin signaling through the Arg tyrosine kinase activates p190RhoGAP by promoting its association with p120RasGAP and recruiting the p190/p120 complex to the cell periphery. p120 binding does not activate p190RhoGAP activity in vitro; instead, membrane recruitment is required for in vivo activation.","method":"arg-/- fibroblasts; dominant-negative p120 fragment; in vitro GAP activity assays; immunofluorescence localization; co-immunoprecipitation","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro assay combined with genetic null cells, dominant negative, and localization studies; dissects mechanism of activation","pmids":["16971514"],"is_preprint":false},{"year":2004,"finding":"Arg tyrosine kinase phosphorylates p190RhoGAP at Y1105 in vitro and in vivo. This phosphorylation is adhesion-dependent, promotes p190RhoGAP binding to p120RasGAP, stimulates p190RhoGAP GAP activity to inhibit Rho, and induces neuritogenesis in neuroblastoma cells.","method":"In vitro kinase assay; arg-/- mouse brain extracts and fibroblasts; site-directed mutagenesis (Y1105); co-immunoprecipitation; neuritogenesis assay","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis, genetic null cells, multiple functional readouts, single lab","pmids":["15084284"],"is_preprint":false},{"year":2007,"finding":"Arg kinase acts through p190RhoGAP to inhibit actomyosin contractility (stress fiber formation) via RhoA suppression upon adhesion to fibronectin. The Arg N-terminal kinase domain is sufficient to act through p190RhoGAP; Arg also requires its C-terminal cytoskeleton-binding half to fully regulate focal adhesion dynamics.","method":"arg-/- fibroblasts; Arg re-expression; traction force microscopy; myosin light chain phosphorylation assays; immunofluorescence of stress fibers and focal adhesions","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic null with domain dissection, multiple phenotypic readouts, single lab","pmids":["17652459"],"is_preprint":false},{"year":2007,"finding":"The Arg tyrosine kinase and p190RhoGAP pathway is essential for dendritic spine maturation and synapse/dendrite stability during late postnatal hippocampal development. p190RhoGAP localizes to dendritic spines; p190RhoGAP activity is reduced in arg-/- hippocampus, leading to elevated RhoA. Reducing ROCKII gene dosage suppresses dendritic regression in arg-/- mice.","method":"arg-/- mice; genetic epistasis (p190rhogap heterozygous/arg-/- double mutants; ROCKII haploinsufficiency in arg-/- background); immunofluorescence localization; RhoA activity assays; behavioral testing","journal":"The Journal of neuroscience : the official journal of the Society for Neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in multiple mouse mutant combinations with defined cellular and behavioral phenotypes","pmids":["17928439"],"is_preprint":false},{"year":2009,"finding":"FAK forms a complex with p120RasGAP and p190RhoGAP-A (p190A) at leading-edge focal adhesions. Fibronectin-integrin-mediated FAK activation promotes SH2-mediated p120RasGAP binding to FAK, which facilitates FAK-mediated p190A tyrosine phosphorylation. This complex is required for cell polarity during migration; knockdown of p120RasGAP or mutation of FAK Y397 prevents p190A association, tyrosine phosphorylation, and cell polarity.","method":"Co-immunoprecipitation; siRNA knockdown; FAK Y397F mutation; wound healing and Golgi reorientation polarity assays; fibronectin-integrin stimulation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP, mutagenesis, siRNA, multiple functional readouts, single lab","pmids":["19435801"],"is_preprint":false},{"year":2003,"finding":"p190RhoGAP overexpression induces a multinucleated phenotype dependent on the GAP domain, and endogenous p190RhoGAP localizes to the cleavage furrow of dividing cells. Endogenous p190 protein levels are transiently decreased in late mitosis via ubiquitin-mediated degradation requiring the N-terminal GTP-binding region.","method":"Conditional/transient overexpression; GAP domain mutants; confocal immunofluorescence of cleavage furrow localization; ubiquitin-proteasome inhibitors; cell cycle synchronization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain-dependent phenotype, localization, and degradation mechanism with multiple orthogonal methods, single lab","pmids":["14610059"],"is_preprint":false},{"year":2006,"finding":"Cell surface transglutaminase activates RhoA by suppressing the Src-p190RhoGAP regulatory pathway through integrin clustering. tTG-induced integrin aggregation inhibits Src kinase activity, decreasing activation of the Src substrate p190RhoGAP, leading to elevated RhoA-GTP levels.","method":"tTG overexpression; Src kinase activity assays; p190RhoGAP phosphorylation measurements; RhoA-GTP pulldown; pharmacological Src inhibition","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods but p190RhoGAP studied as part of a broader pathway, single lab","pmids":["16452636"],"is_preprint":false},{"year":2008,"finding":"Alpha5beta1 integrin engagement stimulates tyrosine phosphorylation of p190RhoGAP (Src-dependent), while parallel syndecan-4 engagement redistributes tyrosine-phosphorylated p190RhoGAP between membrane and cytosolic fractions via PKCalpha activation. Both pathways must be activated for efficient RhoA suppression and focal adhesion formation; p190RhoGAP is the convergence point of these two adhesion receptor signals.","method":"Function-blocking antibodies against alpha5beta1 integrin and syndecan-4; siRNA knockdown; fractionation; phosphotyrosine blotting; RhoA-GTP pulldown; focal adhesion scoring","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple receptor perturbations, fractionation, siRNA, RhoA activity assays; mechanistically dissects two pathways converging on p190","pmids":["18541700"],"is_preprint":false},{"year":2007,"finding":"p190RhoGAP accumulates in lipid rafts during early cell spreading, where it exerts Rho inhibitory activity. Filamin controls this accumulation: cells lacking filamin fail to accumulate p190RhoGAP in lipid rafts and maintain high Rho activity even when spread. Calpain-resistant filamin also prevents spreading-induced raft accumulation of p190RhoGAP.","method":"siRNA knockdown of p190RhoGAP; filamin-null cell lines; calpain-resistant filamin expression; lipid raft fractionation; RhoA activity assays; cell rounding/spreading assays","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic null and siRNA, lipid raft fractionation, multiple orthogonal methods linking localization to function, single lab","pmids":["17227794"],"is_preprint":false},{"year":2008,"finding":"Rho-kinase (ROCK) phosphorylates p190A RhoGAP at Ser1150 and attenuates its GAP activity. This phosphorylation impairs Rnd binding to p190A (Rnd binding normally enhances p190A activation). A phosphomimetic S1150 mutation of p190A weakened Rnd binding and GAP activity. ET-1-induced sustained RhoA activation in vascular smooth muscle cells involves this ROCK-p190A feedback loop.","method":"In vitro phosphorylation assays; co-immunoprecipitation of Rnd and p190A; phosphomimetic/phosphoresistant mutations; Rho-kinase inhibitor Y-27632; COS7 and vascular smooth muscle cell experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with mutagenesis, binding assays, cellular validation, single lab","pmids":["19103606"],"is_preprint":false},{"year":2008,"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 and contributing to cell polarity during directional migration. p190A-deficient fibroblasts exhibit a defect in directional migration that reflects a requirement for GSK-3beta-mediated phosphorylation.","method":"In vitro kinase assay; phosphorylation mapping; p190A-deficient fibroblasts; reconstitution with phosphomimetic/phosphoresistant mutants; wound healing assays; RhoA activity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with reconstitution, site mapping, genetic null cells, multiple phenotypic readouts, single lab","pmids":["18502760"],"is_preprint":false},{"year":2008,"finding":"Breast tumor kinase (Brk) phosphorylates p190RhoGAP-A at Y1105 in vitro and in vivo, promoting p190 association with p120RasGAP. This stimulates p190 GAP activity (RhoA inactivation) and attenuates p120RasGAP activity (Ras activation). Disruption of the p190/p120 complex abolishes Brk-mediated regulation of RhoA and Ras and Brk-driven proliferation, migration, invasion, and tumorigenicity.","method":"In vitro kinase assay; site-directed mutagenesis (Y1105); co-immunoprecipitation; RhoA and Ras activity assays; proliferation, migration, invasion, and xenograft tumor assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase reconstitution, mutagenesis, co-IP, multiple functional assays, single lab","pmids":["18829532"],"is_preprint":false},{"year":2009,"finding":"PTP-PEST directly dephosphorylates and thereby regulates p190RhoGAP activity. PTP-PEST null fibroblasts show enhanced p190RhoGAP activity (decreased RhoA) and exaggerated leading-edge protrusions; PTP-PEST acts to couple protrusion and retraction by reciprocally modulating VAV2 (Rac1 GEF) and p190RhoGAP (RhoA GAP).","method":"PTP-PEST null fibroblasts; RhoA and Rac1 activity assays; direct substrate phosphatase assays; co-immunoprecipitation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic null cells with defined phenotypes, direct substrate assay, single lab","pmids":["16513648"],"is_preprint":false},{"year":2009,"finding":"Angiotensin II activates RhoA in vascular smooth muscle cells by causing SHP2-dependent dephosphorylation and inactivation of p190A RhoGAP. SHP2 maintains basal p190A phosphorylation via c-Abl; AT1R activation induces SHP2-mediated p190A dephosphorylation and RhoA activation. Phosphomimetic p190A mutant inhibits, and phosphoresistant mutant increases, basal RhoA-Rho kinase activity.","method":"siRNA knockdown of p190A and SHP2; phosphomimetic/phosphoresistant p190A mutants; RhoA/Rho-kinase activity assays; co-immunoprecipitation","journal":"American journal of physiology. Cell physiology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — siRNA, mutagenesis, activity assays, multiple orthogonal approaches, single lab","pmids":["19692654"],"is_preprint":false},{"year":2009,"finding":"Protein kinase C phosphorylates p190A RhoGAP at Ser1221 and Thr1226 within a polybasic region, preventing binding of acidic phospholipids to this region. Phospholipid binding to this region inhibits RhoGAP activity and promotes RacGAP activity. PKC-mediated phosphorylation thus indirectly alters substrate specificity by blocking phospholipid binding.","method":"In vitro PKC phosphorylation; liposome binding assays; site-directed mutagenesis; in vitro GAP activity assays with RhoA and Rac substrates; COS-7 cell morphology assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, lipid binding, and substrate specificity assays, single lab","pmids":["19673492"],"is_preprint":false},{"year":2006,"finding":"Tiam1/Rac1 signaling downregulates Rho activity through p190-RhoGAP. p190-RhoGAP is required for this pathway; Src-kinase-dependent tyrosine phosphorylation of p190-RhoGAP and its recruitment to the membrane through p120-RasGAP SH2 domains are both necessary for Tiam1-mediated Rho downregulation.","method":"Dominant negative/active constructs of Tiam1, Rac isoforms, and p190-RhoGAP; phosphorylation mutants; dominant-negative p120 fragment; RhoA activity assays in COS-7 cells","journal":"Biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple dominant negative constructs and activity assays, pathway placement, single lab","pmids":["16542153"],"is_preprint":false},{"year":2009,"finding":"p190RhoGAP negatively regulates RhoA activity at the cleavage furrow, modulating cytokinetic furrow site selection and ring contraction. FRET analysis showed that wild-type p190 overexpression reduces RhoA-GTP at the furrow in a dose-dependent manner, causing abnormal furrow positioning and failed cytokinesis, while dominant-negative p190 causes hyper-activated Rho.","method":"FRET-based RhoA activity biosensor; time-lapse microscopy; overexpression of wild-type and dominant-negative p190RhoGAP; cleavage furrow localization by confocal imaging","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — FRET biosensor for direct RhoA activity measurement at furrow, combined with live imaging, multiple constructs","pmids":["19254711"],"is_preprint":false},{"year":2008,"finding":"p190RhoGAP and Ect2 RhoGEF physically associate and colocalize at the cleavage furrow with opposing roles on RhoA activity and cytokinesis outcome; Ect2 can dose-dependently reduce p190-induced multinucleation. Their functional and physical interactions regulate RhoA activity levels at the furrow.","method":"Co-immunoprecipitation; immunofluorescence colocalization at cleavage furrow; multinucleation assays; RhoA pulldown activity assays","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP and functional epistasis in same cellular system, single lab","pmids":["18642445"],"is_preprint":false},{"year":2010,"finding":"Mitotic down-regulation of p190RhoGAP via ubiquitin-proteasome-mediated degradation is required for successful cytokinesis. The N-terminal GBDS1 region (and four specific N-terminal residues) is necessary and sufficient for p190A mitotic ubiquitination and degradation; a degradation-resistant p190A mutant causes cytokinesis failure in an RNAi reconstitution approach.","method":"RNAi reconstitution with degradation-resistant mutant; N-terminal deletion mapping; ubiquitin-proteasome inhibitors; cell cycle synchronization; multinucleation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reconstitution approach with defined mutants, domain mapping, clear causal link between degradation and phenotype, single lab","pmids":["20534586"],"is_preprint":false},{"year":2014,"finding":"p190RhoGAP-A forms a complex with anillin at the cytokinetic furrow. p190RhoGAP-A depletion causes accumulation of high RhoA-GTP in the furrow and failure to progress to abscission. Mutants of p190RhoGAP-A unable to bind anillin or lacking GAP activity fail to rescue cytokinesis defects; low-dose blebbistatin (myosin II inhibitor) rescues the cytokinesis failure of p190RhoGAP-A-depleted cells.","method":"Co-immunoprecipitation; siRNA knockdown; anillin-binding mutant and GAP-dead mutants; RhoA-GTP quantification at furrow; blebbistatin rescue experiment","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, siRNA depletion with specific mutant rescue, mechanistic epistasis via blebbistatin, multiple orthogonal approaches","pmids":["25359885"],"is_preprint":false},{"year":2010,"finding":"ERK promotes Rho-dependent focal adhesion formation by suppressing p190A RhoGAP. ERK activity is required for RhoA-GTP loading and focal adhesion maturation during cell spreading on fibronectin. ERK phosphorylates the C-terminus of p190A, affecting its peripheral localization and activity.","method":"MEK/ERK inhibitors; ERK phosphorylation site mapping; p190A localization by immunofluorescence; RhoA-GTP pulldown; focal adhesion maturation assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phosphorylation site identification, localization and activity readouts, pharmacological inhibition, single lab","pmids":["20439493"],"is_preprint":false},{"year":2011,"finding":"Caveolin-1-deficiency leads to eNOS activation, peroxynitrite generation, and selective nitration of p190RhoGAP-A at Tyr1105, impairing its GAP activity and causing RhoA activation and adherens junction disassembly. Thrombin also induces nitration of p120-catenin-associated p190RhoGAP-A.","method":"Cav-1-/- endothelial cells; nitration assays (tyrosine nitration); eNOS inhibition; S-nitrosylation/nitration profiling; RhoA activity assays; endothelial permeability measurements; adherens junction imaging","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic null cells, specific nitration site identified, functional rescue by eNOS/RhoA inhibition, multiple orthogonal methods","pmids":["21624953"],"is_preprint":false},{"year":2009,"finding":"p190RhoGAP has RacGAP activity in cells, not only RhoGAP activity. The cellular RacGAP activity requires an intact polybasic region adjacent to the GAP domain (which inhibits RhoGAP activity), revealing that this polybasic region inversely regulates substrate preference between Rac and Rho.","method":"Mutagenesis of polybasic region; in vitro GAP activity assays with RhoA and Rac substrates; cellular Rac/RhoA activity readouts","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — confirmed in cells and in vitro with mutagenesis, single lab; builds on prior in vitro work","pmids":["23499677"],"is_preprint":false},{"year":2013,"finding":"p120-catenin interacts with p190RhoGAP via a 23-amino acid stretch (CRAD, amino acids 820–843) in its C-terminal domain. This interaction is required for membrane translocation of p190RhoGAP, suppression of RhoA and upregulation of Rac1/PAK1/cortactin signaling. Expression of p120-catenin lacking CRAD prevents p190RhoGAP recruitment to the cell periphery and impairs endothelial barrier recovery.","method":"Truncation mutants of p120-catenin; co-immunoprecipitation; immunofluorescence localization; RhoA and Rac1 activity assays; endothelial permeability measurements","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain mapping with truncation mutants, co-IP, localization with functional consequences, multiple readouts, single lab","pmids":["23653363"],"is_preprint":false},{"year":2009,"finding":"p190A RhoGAP is phosphorylated by PDGF receptor alpha on Y308 within the FF1 domain, but phosphorylation requires prior domain unfolding since Y308 is buried in the hydrophobic core. Phosphorylation irreversibly destabilizes the FF1 structure, likely accounting for its inhibitory effect on TFII-I interaction.","method":"NMR structure determination of FF1 domain; in vitro phosphorylation at different temperatures; thermal denaturation studies","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure determination with in vitro phosphorylation and mechanistic characterization of domain stability, single lab","pmids":["19393245"],"is_preprint":false},{"year":2018,"finding":"The N-terminal domain of p190RhoGAP proteins is a pseudoGTPase (N-GTPase) that constitutively binds GTP but does not hydrolyze it. Crystal structure at 2.8 Å shows an unusual GTP-Mg2+ binding pocket with six inserts perturbing catalytic activity. GTP/Mg2+ binding stabilizes the domain, suggesting it functions as a protein-protein interaction platform.","method":"X-ray crystal structure (2.8 Å); biochemical GTP hydrolysis assays; nucleotide exchange under Mg2+ chelation; mutational analysis of GTP/Mg2+ binding","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus biochemical and mutagenesis validation, rigorous characterization of pseudoenzyme properties","pmids":["30174148"],"is_preprint":false},{"year":2019,"finding":"The p120RasGAP N-terminal SH2 domain binds the p190RhoGAP phosphopeptide centered on pTyr-1105 with a dissociation constant of 0.3 μM via a canonical SH2-pTyr interaction requiring the conserved FLVR motif arginine R207. Crystal structure at 1.6 Å shows that peptide binding stabilizes specific conformations of the βE-βF loop and key arginine residues.","method":"X-ray co-crystal structure (1.6 Å); site-directed mutagenesis (R207); native gel shifts; isothermal titration calorimetry","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, ITC binding measurement, mutagenesis confirmation, rigorous biophysical characterization","pmids":["31891593"],"is_preprint":false},{"year":2016,"finding":"p190A localizes to membrane protrusions via a novel domain termed the PLS (protrusion localization sequence). The PLS is required for targeting to the leading edge and also negatively regulates p190A GAP activity. Cortactin binds the PLS and is required for p190A targeting to protrusions. Cancer-associated mutations in PLS disrupt localization, GAP activity regulation, and tumor cell migration.","method":"Truncation mutants; subcellular localization (immunofluorescence); GAP activity assays; cortactin co-immunoprecipitation; cancer-mutation panel analysis; cell migration assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — domain mapping with multiple mutants including cancer variants, co-IP, localization tied to functional consequences, single lab","pmids":["27646271"],"is_preprint":false},{"year":2016,"finding":"A point mutation (L1396Q) in the GAP domain of p190A RhoGAP (Arhgap35) decreases GAP activity for RhoA and Rac1 and impairs primary cilia formation in renal nephrons. p190A localizes to the base of cilia; its GAP activity is required for axoneme elongation. Pharmacological inhibition of ROCK or F-actin polymerization rescues ciliogenesis defects, placing p190A upstream of Rho-ROCK-actin in cilia formation.","method":"ENU mutagenesis; in vitro GAP activity assay with L1396Q mutant; mouse glomerulocystic kidney model; immunofluorescence localization to cilia base; ROCK inhibitor and actin polymerization inhibitor rescue experiments","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro GAP activity mutagenesis, genetic mouse model, localization, pharmacological epistasis, single lab","pmids":["26859289"],"is_preprint":false},{"year":2019,"finding":"p190RhoGAP acts in a GAP-independent mode to transiently suppress attraction to Netrin-1 while motor axons exit the spinal cord, and also uses GAP-dependent RhoA inhibition to guide a subset of axons to specific muscles. Identified by a mouse mutagenesis screen; multifunctional activity emerges from modular design.","method":"Mouse mutagenesis screen; in vivo motor axon guidance assays; genetic analysis distinguishing GAP-dependent and GAP-independent activities","journal":"Neuron","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic screen plus in vivo functional dissection of GAP-dependent vs. GAP-independent modes, single lab","pmids":["30902550"],"is_preprint":false},{"year":2020,"finding":"p190A (ARHGAP35) is an upstream regulator of the Hippo-YAP signaling pathway. p190A knockout promotes YAP nuclear localization and transcriptional activity; wild-type but not cancer-associated p190A mutants suppress YAP. p190A promotes MET and CDH1 (E-cadherin) expression; E-cadherin amplifies p190A-mediated LATS activation and is required for contact inhibition of proliferation.","method":"CRISPR/Cas9 knockout; lentiviral expression of cancer mutants; YAP localization/activity assays; LATS kinase activity; xenograft mouse model; CDH1 expression analysis","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with specific rescue by WT but not cancer mutants, in vivo xenograft validation, LATS kinase assay, YAP localization","pmids":["32457342"],"is_preprint":false},{"year":2020,"finding":"Polycystin-1 (PC1) regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling. PKD1-null cells show decreased centrosomal ARHGAP35, increased total and centrosomal active RhoA, and ROCK signaling. ARHGAP35 knockdown reduces primary ciliation in normal renal tubular cells. ROCK inhibitor hydroxyfasudil reduces cyst expansion in PKD1 3D cyst assays and an inducible Pkd1 mouse model.","method":"Centrosome-targeted proximity ligation assay; dual immunofluorescence; siRNA knockdown; cilia length phenotypic assay; 3D cyst assay; inducible Pkd1 mouse model","journal":"JCI insight","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity ligation and localization assays, siRNA, in vivo model, single lab","pmids":["32663194"],"is_preprint":false},{"year":2020,"finding":"p190A RhoGAP promotes CDH1 (E-cadherin) expression and cooperates with E-cadherin to activate LATS kinases and suppress tumor cell growth. p190A and E-cadherin are mutually required for LATS activation and contact inhibition of proliferation (CIP); p190A cancer mutations lose this function.","method":"Xenograft mouse model; LATS kinase assays; YAP phosphorylation; CDH1 induction assays; reconstitution with cancer mutant panel; co-expression/knockdown experiments","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple cancer mutants, in vivo xenograft validation, LATS kinase activity assays, E-cadherin epistasis, single lab","pmids":["32641858"],"is_preprint":false},{"year":2019,"finding":"TRIM65 E3 ubiquitin ligase mediates ubiquitination and proteasomal degradation of ARHGAP35 (p190A), leading to elevated Rho GTPase activity, increased migration-related structures (focal adhesions, filopodia), and enhanced CRC metastasis.","method":"Co-immunoprecipitation; ubiquitination assays; TRIM65 overexpression and knockdown; in vivo mouse metastasis model (liver, lung); RhoA activity assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP, ubiquitination assay, in vivo metastasis model, RhoA activity readout, single lab","pmids":["31332286"],"is_preprint":false},{"year":2023,"finding":"p120 RasGAP and ZO-2 are both necessary for p190A to activate LATS kinases, elicit MET, promote contact inhibition of proliferation, and suppress tumorigenesis. The interaction of p190A with ZO-2 is dependent on RasGAP; RasGAP and ZO-2 are required for transcriptional modulation by p190A.","method":"Co-immunoprecipitation; siRNA knockdown of RasGAP and ZO-2; LATS kinase activity assays; contact inhibition assays; xenograft tumor assays; transcriptional reporter assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP, siRNA, kinase activity assays, in vivo validation, multiple functional readouts; independent confirmation also as preprint","pmids":["37995182"],"is_preprint":false},{"year":2016,"finding":"Interaction of p190A RhoGAP with eIF3A (and other translation preinitiation factors) involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A; interaction is enhanced by serum and reduced by phosphatase treatment. Disrupted by S296A mutation in the first FF domain but not by Y308 mutation.","method":"Tandem mass spectrometry of endogenous p190A interactors; co-immunoprecipitation; site-directed mutagenesis (S296A, Y308); phosphatase treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS interactome with co-IP validation and mutagenesis; functional consequence not demonstrated beyond complex formation, single lab","pmids":["28007963"],"is_preprint":false},{"year":2014,"finding":"HPV16 E7 protein binds p190RhoGAP via conserved region 3 (CR3) of E7 and the middle domain of p190RhoGAP. This interaction dysregulates p190RhoGAP and alters the actin cytoskeleton, and negatively regulates cell spreading on fibronectin.","method":"Mass spectrometry identification; co-immunoprecipitation; domain-mapping with E7 CR3 mutants and p190 deletion constructs; cell spreading assays on fibronectin; actin cytoskeleton immunofluorescence","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified, co-IP with domain mapping, functional cell spreading assay, single lab","pmids":["24403595"],"is_preprint":false},{"year":2018,"finding":"A Rnd3/p190RhoGAP pathway operates in idiopathic pulmonary fibrosis (IPF) fibroblasts. Rnd3 expression and p190RhoGAP activity are suppressed in IPF; restoration of Rnd3 increases p190 activity and decreases RhoA activity and fibrotic phenotype. IPF drugs nintedanib and pirfenidone decrease RhoA activity through up-regulation of Rnd3 and p190 activity.","method":"Rnd3 overexpression in IPF fibroblasts; p190RhoGAP activity assays; RhoA activity assays; nintedanib/pirfenidone treatment; collagen gel contraction (fibrotic phenotype) assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain of function rescue, direct p190 activity measurement, pharmacological epistasis, single lab","pmids":["29995590"],"is_preprint":false},{"year":2022,"finding":"The PLS of p190A acts as an autoinhibitory domain that masks the GAP domain through intramolecular interaction. Co-immunoprecipitation demonstrates that the PLS interacts with a region near the GAP domain; p190A lacking PLS (p190AΔPLS) has stronger RhoA-inhibiting activity. This intramolecular autoinhibition is disrupted by cancer-associated mutations S866F and Δ865-870 in the PLS.","method":"Yeast two-hybrid screen; co-immunoprecipitation of PLS with p190A GAP-proximal region; RhoA inactivation assays comparing WT vs. ΔPLS; cancer-associated PLS mutants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — two-hybrid and co-IP for intramolecular interaction, functional GAP activity comparison, cancer mutation panel, single lab","pmids":["36516886"],"is_preprint":false},{"year":2024,"finding":"Fuzzy (CPLANE protein) recruits ARHGAP35 (p190A RhoGAP) to the basal body/base of primary cilia to restrict actin polymerization. Loss of Fuzzy in mice reduces cilia length with increased RhoA activity and excessive basal body actin; genetic interaction between Fuzzy and Arhgap35 alleles confirms their functional cooperation at the basal body.","method":"Co-immunoprecipitation of Fuzzy with ARHGAP35; immunofluorescence localization at basal body; Fuzzy knockout mice; Fuzzy/Arhgap35 compound mutant genetic epistasis; actin and RhoA activity measurements","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — co-IP, localization, genetic KO, and compound mutant epistasis with quantitative phenotypic readouts, single lab","pmids":["38546045"],"is_preprint":false},{"year":2023,"finding":"In cardiac myocytes, M2 muscarinic receptor (M2R) stimulation by carbachol induces eNOS activation in caveolae, producing NO/peroxynitrite that nitrates p190A at Tyr1105. This nitration promotes p190A binding to RGS3L and shifts p190A substrate preference from RhoA to Rac1, resulting in net RhoA activation. Complex of eNOS, p190A, RGS3L, and caveolin-3 detected by co-immunoprecipitation.","method":"Carbachol stimulation; eNOS inhibitors (L-NIO, L-NAME); nitration assays; co-immunoprecipitation (eNOS/p190A/RGS3L/caveolin-3); GAP activity assays for RhoA vs. Rac1; caveolae disruption by methyl-β-cyclodextrin","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP, enzymatic activity assay, pharmacological inhibition of eNOS, caveolae disruption; single lab, single paper","pmids":["37887276"],"is_preprint":false},{"year":2018,"finding":"p190RhoGAP is required to prevent mitotic spindle fragmentation and to activate Aurora A kinase at acentriolar poles. Depletion of p190RhoGAP causes prolonged mitotic arrest with multipolar spindles. Low-dose Eg5 inhibitor rescues the multipolar phenotype. Aurora A localizes to all poles in p190-depleted cells but is only activated at centriolar poles.","method":"siRNA knockdown of p190RhoGAP; live-cell imaging of spindle dynamics; Aurora A activation (immunofluorescence with activation-specific antibody); Eg5 inhibitor rescue experiment","journal":"Chromosoma","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA depletion with defined spindle phenotype, pharmacological epistasis, Aurora A activity readout, single lab","pmids":["29656322"],"is_preprint":false}],"current_model":"ARHGAP35/p190RhoGAP is a multi-domain RhoGAP (with three pseudoGTPase domains, four FF domains, and a GAP domain) that serves as a central hub for RhoA inactivation downstream of multiple upstream signals—including integrins (via Arg/Abl2 kinase, FAK, Src), cadherins, receptor tyrosine kinases (EGFR, PDGFR, Brk), and GPCRs—by becoming tyrosine-phosphorylated (primarily at Y1105 by Src/Arg/FAK/Brk), which promotes its binding to p120RasGAP and membrane/junction recruitment to suppress RhoA-GTP; its activity is further tuned by phospholipid binding and PKC-dependent serine/threonine phosphorylation (altering RhoA vs Rac1 substrate preference), ROCK-mediated Ser1150 phosphorylation (feedback inhibition), GSK-3β-mediated C-terminal phosphorylation (polarity), eNOS-dependent nitration at Y1105 (GAP inactivation), autoinhibition via intramolecular PLS-GAP domain interaction, ubiquitin-proteasome-mediated mitotic degradation (required for cytokinesis completion), and localization to lipid rafts (filamin-dependent) or protrusions (cortactin-PLS-dependent); downstream it suppresses actomyosin contractility, promotes cell spreading, migration and polarity, adherens junction assembly, dendritic spine maturation, ciliogenesis (at the basal body via Fuzzy), and cytokinetic furrow progression (in complex with anillin), while also activating the Hippo-LATS-YAP pathway (requiring p120RasGAP and ZO-2) to suppress tumor cell proliferation."},"narrative":{"mechanistic_narrative":"ARHGAP35 (p190RhoGAP-A) is a multidomain GTPase-activating protein that functions as a central convergence node for inactivating RhoA downstream of adhesion and growth-factor signaling, thereby controlling actomyosin contractility, cell spreading, migration, and polarity [PMID:11553710, PMID:18541700]. Its activation is driven by adhesion-dependent tyrosine phosphorylation—catalyzed by c-Src, the Arg/Abl2 kinase, FAK, and the tumor kinase Brk—primarily at Tyr1105, a modification that creates a phosphopeptide bound with high affinity by the p120RasGAP SH2 domain via the FLVR-motif arginine R207 [PMID:7542246, PMID:15084284, PMID:16308318, PMID:18829532, PMID:31891593]. This p190–p120RasGAP association does not directly stimulate catalysis but instead drives recruitment of the complex to the membrane, leading-edge focal adhesions, lipid rafts, and cadherin junctions, where RhoA suppression occurs [PMID:16971514, PMID:19435801, PMID:17227794, PMID:17129786]. At adherens junctions p190 couples to the cadherin complex through p120-catenin, which binds a defined C-terminal CRAD segment and is required for membrane translocation, RhoA suppression, and junction assembly [PMID:17129786, PMID:23653363]. ARHGAP35 integrates signals from integrins, syndecan-4/PKCα, RTKs, and Rac/Tiam1, acting as the shared point at which these inputs converge to suppress RhoA [PMID:18541700, PMID:16542153]. Its output is tuned by an extensive regulatory layer: ROCK-mediated Ser1150 phosphorylation provides feedback inhibition by impairing Rnd binding [PMID:19103606], GSK-3β and ERK phosphorylate the C-terminus to control polarity and localization [PMID:18502760, PMID:20439493], PKC phosphorylation within a polybasic region blocks acidic-phospholipid binding and shifts substrate preference between RhoA and Rac1 [PMID:19673492, PMID:23499677], and eNOS-dependent nitration at Tyr1105 inactivates the GAP and can redirect substrate specificity toward Rac1 [PMID:21624953, PMID:37887276]. The protein is also autoinhibited by an intramolecular interaction between its protrusion-localization sequence (PLS) and the GAP domain, an interaction disrupted by cancer-associated mutations [PMID:36516886, PMID:27646271]; its N-terminal domain is a pseudoGTPase that constitutively binds GTP without hydrolysis and serves as a protein-interaction platform [PMID:30174148]. ARHGAP35 governs cytokinesis by suppressing RhoA at the cleavage furrow in complex with anillin, and its ubiquitin-proteasome-mediated mitotic degradation is required for furrow progression and abscission [PMID:19254711, PMID:25359885, PMID:20534586]. Beyond contractility, it promotes ciliogenesis at the basal body—where it is recruited by Fuzzy to restrict actin polymerization—and acts as an upstream activator of the Hippo–LATS–YAP pathway, cooperating with p120RasGAP, ZO-2, and E-cadherin to drive contact inhibition of proliferation and suppress tumorigenesis [PMID:38546045, PMID:26859289, PMID:32457342, PMID:32641858, PMID:37995182].","teleology":[{"year":1995,"claim":"Established that p190RhoGAP is a tyrosine-phosphorylation substrate linking Src-family kinase activity to actin cytoskeleton remodeling, framing it as a regulated effector rather than a constitutive enzyme.","evidence":"c-Src wild-type/dominant-negative overexpression with imaging of p190/p120RasGAP distribution and phosphotyrosine analysis","pmids":["7542246"],"confidence":"High","gaps":["Did not identify the specific phosphosite or define how phosphorylation alters GAP catalysis","Did not establish the RhoA target quantitatively"]},{"year":2001,"claim":"Defined the core physiological role: integrin-triggered RhoA inactivation by p190RhoGAP promotes spreading, polarity, and migration, placing it in adhesion signaling.","evidence":"Dominant-negative and overexpression in Rat1 fibroblasts with RhoA activity, spreading, and migration assays on fibronectin","pmids":["11553710"],"confidence":"High","gaps":["Upstream kinase coupling integrins to p190 not identified","Membrane recruitment mechanism not addressed"]},{"year":2004,"claim":"Identified Tyr1105 as the key adhesion-dependent phosphosite, phosphorylated by Arg kinase, that promotes p120RasGAP binding and stimulates GAP activity — resolving the molecular switch.","evidence":"In vitro kinase assay, arg-/- cells, Y1105 mutagenesis, co-IP, and neuritogenesis assay","pmids":["15084284"],"confidence":"High","gaps":["How p120RasGAP binding converts to RhoA suppression not mechanistically resolved at this stage"]},{"year":2006,"claim":"Showed that p120RasGAP binding does not activate the GAP enzymatically in vitro but instead drives membrane recruitment required for in vivo activity, redefining 'activation' as relocalization.","evidence":"arg-/- fibroblasts, dominant-negative p120 fragment, in vitro GAP assays, and localization studies","pmids":["16971514"],"confidence":"High","gaps":["Membrane anchoring partners at the periphery not fully defined","Did not address junctional vs. raft recruitment"]},{"year":2006,"claim":"Connected p190RhoGAP to adherens junction assembly through a p120-catenin interaction, extending its role from focal adhesions to cell-cell contacts.","evidence":"siRNA of p120 and p190, reciprocal co-IP, and AJ assembly assays in PDGFR-stimulated cells","pmids":["17129786"],"confidence":"High","gaps":["The catenin-binding region on p190 not yet mapped (later defined as CRAD)"]},{"year":2008,"claim":"Demonstrated that p190RhoGAP is the convergence point of multiple adhesion receptors (integrin + syndecan-4/PKCα) and is subject to ROCK feedback and GSK-3β/ERK control, establishing a multi-input regulatory architecture.","evidence":"Receptor-blocking antibodies, siRNA, fractionation, in vitro kinase assays with phosphosite mapping and phosphomutants","pmids":["18541700","19103606","18502760"],"confidence":"High","gaps":["Quantitative integration of opposing phosphorylation inputs not modeled","Crosstalk between distinct phosphosites unresolved"]},{"year":2009,"claim":"Defined a polybasic/phospholipid-based mechanism by which PKC phosphorylation toggles substrate preference between RhoA and Rac1, revealing p190 as a dual GAP.","evidence":"In vitro PKC phosphorylation, liposome binding, mutagenesis, and dual-substrate GAP assays","pmids":["19673492","23499677"],"confidence":"High","gaps":["Physiological triggers selecting Rho vs Rac mode in vivo not fully mapped"]},{"year":2010,"claim":"Established that mitotic ubiquitin-proteasome degradation of p190 via its N-terminal region is required for cytokinesis, linking its turnover to cell division.","evidence":"RNAi reconstitution with degradation-resistant mutant, N-terminal deletion mapping, and proteasome inhibitors","pmids":["20534586","14610059"],"confidence":"High","gaps":["The E3 ligase mediating mitotic degradation not identified here"]},{"year":2014,"claim":"Resolved the cytokinetic mechanism: p190 forms a complex with anillin at the furrow and locally suppresses RhoA-GTP to permit abscission.","evidence":"Co-IP, siRNA with anillin-binding and GAP-dead rescue mutants, and blebbistatin rescue","pmids":["25359885","19254711"],"confidence":"High","gaps":["How anillin binding spatially restricts GAP activity not structurally defined"]},{"year":2016,"claim":"Identified the PLS as a dual-function domain that targets p190 to protrusions via cortactin and regulates GAP activity, with cancer mutations disrupting both.","evidence":"Truncation mutants, cortactin co-IP, localization, GAP assays, and cancer-mutation panel","pmids":["27646271"],"confidence":"High","gaps":["Mechanism of GAP regulation by PLS not resolved (later shown autoinhibitory)"]},{"year":2019,"claim":"Structurally characterized the regulatory interfaces: the N-terminal domain is a GTP-binding pseudoGTPase and the p120RasGAP SH2 binds pTyr1105 with sub-micromolar affinity via R207.","evidence":"X-ray crystallography of N-GTPase and SH2–phosphopeptide complex, ITC, and mutagenesis","pmids":["30174148","31891593"],"confidence":"High","gaps":["Full-length architecture and how domains coordinate not solved","Interaction partners of the pseudoGTPase platform unidentified"]},{"year":2019,"claim":"Identified TRIM65 as an E3 ligase degrading p190A, linking its loss to elevated Rho activity and metastasis in colorectal cancer.","evidence":"Co-IP, ubiquitination assays, TRIM65 perturbation, and in vivo metastasis model","pmids":["31332286"],"confidence":"High","gaps":["Whether TRIM65 mediates the mitotic degradation pathway not addressed"]},{"year":2020,"claim":"Revealed a tumor-suppressive role through Hippo signaling: p190A activates LATS and suppresses YAP, requiring E-cadherin, p120RasGAP, and ZO-2, with cancer mutants losing this function.","evidence":"CRISPR KO, cancer-mutant reconstitution, LATS kinase and YAP assays, CDH1 analysis, and xenografts","pmids":["32457342","32641858","37995182"],"confidence":"High","gaps":["How GAP activity mechanistically couples to LATS activation not fully defined","Direct molecular link between junctional p190 and the Hippo core kinases unresolved"]},{"year":2024,"claim":"Established a ciliogenesis role: p190A is recruited to the basal body by Fuzzy to restrict actin polymerization and RhoA, controlling cilium formation.","evidence":"Co-IP, basal-body localization, Fuzzy KO mice, and Fuzzy/Arhgap35 compound mutant epistasis","pmids":["38546045","26859289","32663194"],"confidence":"High","gaps":["Centrosomal/ciliary recruitment partners beyond Fuzzy and PC1 not fully mapped"]},{"year":null,"claim":"How the many competing phosphorylation, nitration, lipid-binding, autoinhibition, and degradation inputs are quantitatively integrated to set RhoA-vs-Rac1 output in a given cellular context remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No full-length structure showing how regulatory domains coordinate","No unified model reconciling GAP-dependent and GAP-independent functions","Spatiotemporal logic of substrate-preference switching in vivo undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,6,20,25,34]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3,22,36]},{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[31]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[20]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[13,21]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,14,29]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[8,33]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[34,37,45]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[34,45]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[13]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,13,36]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[22,24,25,47]},{"term_id":"R-HSA-1500931","term_label":"Cell-Cell communication","supporting_discovery_ids":[5,29]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[9,35,34,45]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[17,36,38,39]}],"complexes":["p190RhoGAP–p120RasGAP complex","p190A–anillin furrow complex","eNOS–p190A–RGS3L–caveolin-3 complex"],"partners":["RASA1","CTNND1","PTK2","ABL2","SRC","ANLN","CTTN","FUZ"],"other_free_text":[]}},"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 inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity.","date":"2001","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/11553710","citation_count":388,"is_preprint":false},{"pmid":"17129786","id":"PMC_17129786","title":"p120-catenin and p190RhoGAP regulate cell-cell adhesion by coordinating antagonism between Rac and Rho.","date":"2006","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/17129786","citation_count":338,"is_preprint":false},{"pmid":"7542246","id":"PMC_7542246","title":"c-Src regulates the simultaneous rearrangement of actin cytoskeleton, p190RhoGAP, and p120RasGAP following epidermal growth factor stimulation.","date":"1995","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/7542246","citation_count":224,"is_preprint":false},{"pmid":"9417037","id":"PMC_9417037","title":"Activation of beta1 integrin signaling stimulates tyrosine phosphorylation of p190RhoGAP and membrane-protrusive activities at invadopodia.","date":"1998","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9417037","citation_count":170,"is_preprint":false},{"pmid":"12606561","id":"PMC_12606561","title":"Cadherin engagement inhibits RhoA via p190RhoGAP.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12606561","citation_count":143,"is_preprint":false},{"pmid":"16308318","id":"PMC_16308318","title":"Suppression of RhoA activity by focal adhesion kinase-induced activation of p190RhoGAP: role in regulation of endothelial permeability.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16308318","citation_count":131,"is_preprint":false},{"pmid":"19435801","id":"PMC_19435801","title":"A FAK-p120RasGAP-p190RhoGAP complex regulates polarity in migrating cells.","date":"2009","source":"Journal of cell 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biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16971514","citation_count":103,"is_preprint":false},{"pmid":"16452636","id":"PMC_16452636","title":"Cell surface transglutaminase promotes RhoA activation via integrin clustering and suppression of the Src-p190RhoGAP signaling pathway.","date":"2006","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16452636","citation_count":102,"is_preprint":false},{"pmid":"18541700","id":"PMC_18541700","title":"p190RhoGAP is the convergence point of adhesion signals from alpha 5 beta 1 integrin and syndecan-4.","date":"2008","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/18541700","citation_count":91,"is_preprint":false},{"pmid":"17227794","id":"PMC_17227794","title":"Filamin links cell shape and cytoskeletal structure to Rho regulation by controlling accumulation of p190RhoGAP in lipid rafts.","date":"2007","source":"Journal of cell 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assignments","url":"https://pubmed.ncbi.nlm.nih.gov/39402262","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49495,"output_tokens":12935,"usd":0.171255,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24445,"output_tokens":5110,"usd":0.124988,"stage2_stop_reason":"end_turn"},"total_usd":0.296243,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"p190RhoGAP (ARHGAP35) mediates integrin-triggered RhoA inactivation during cell adhesion to fibronectin, promoting membrane protrusion and cell polarity. Dominant negative p190RhoGAP elevated RhoA activity, impaired spreading, and inhibited migration, while overexpression decreased RhoA activity and enhanced motility.\",\n      \"method\": \"Dominant negative and overexpression of p190RhoGAP in Rat1 fibroblasts; RhoA activity assays; cell spreading and migration assays on fibronectin\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean loss- and gain-of-function with defined phenotypic readouts, replicated across multiple conditions, multiple orthogonal methods\",\n      \"pmids\": [\"11553710\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"c-Src regulates EGF-dependent actin cytoskeleton reorganization through tyrosine phosphorylation of p190RhoGAP. The in vivo phosphotyrosine content of p190 varies with c-Src activity, and c-Src variants modulate the rate and extent of p190/RasGAP arc formation and actin stress fiber dynamics.\",\n      \"method\": \"Overexpression of wild-type and dominant negative c-Src variants; confocal immunofluorescence of p190, actin, and p120RasGAP distribution; phosphotyrosine analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple cell lines, dominant negative and overexpression, multiple orthogonal imaging and biochemical methods\",\n      \"pmids\": [\"7542246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Beta1 integrin activation by laminin peptides induces tyrosine phosphorylation of p190RhoGAP and localizes phospho-p190 with F-actin specifically at invadopodia. Microinjection of antibodies against p190RhoGAP blocked invadopodial membrane-protrusive and matrix-degradative activities.\",\n      \"method\": \"Integrin activation with laminin G peptides and antibodies; immunofluorescence; tyrosine kinase inhibitors; microinjection of anti-p190 antibodies\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — microinjection loss-of-function, phosphorylation readout, localization with functional consequence, single lab multiple methods\",\n      \"pmids\": [\"9417037\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cadherin engagement induces tyrosine phosphorylation of p190RhoGAP and increases its binding to p120RasGAP in a Src-family-kinase-dependent manner. Dominant negative p190RhoGAP antagonized cadherin-induced RhoA inactivation, establishing p190RhoGAP as a required downstream effector of cadherin-mediated RhoA suppression.\",\n      \"method\": \"Constitutively active RhoA pulldown to isolate active GAPs; dominant negative p190RhoGAP expression; PP2 Src kinase inhibitor; co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (active GAP pulldown, dominant negative, pharmacological inhibitor, co-IP), single lab\",\n      \"pmids\": [\"12606561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"FAK directly phosphorylates p190RhoGAP in vitro and associates with p190RhoGAP; this FAK-induced phosphorylation is required for suppression of RhoA activity and restoration of endothelial barrier function after thrombin treatment. Dominant negative FAK (FRNK) prevented p190RhoGAP phosphorylation, increased RhoA activity, and produced irreversible endothelial permeability.\",\n      \"method\": \"In vitro kinase assay with recombinant FAK and p190RhoGAP; adenoviral FRNK expression; RhoA activity assays; endothelial permeability measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase reconstitution plus cellular loss-of-function with defined phenotypic readout, single lab\",\n      \"pmids\": [\"16308318\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Rac activation causes translocation of p190RhoGAP to adherens junctions where it couples to the cadherin complex via interaction with p120-catenin. This p120-p190RhoGAP interaction is required for adherens junction formation, and disruption of either protein prevents junction assembly.\",\n      \"method\": \"siRNA knockdown of p120 and p190RhoGAP; co-immunoprecipitation; immunofluorescence localization; functional AJ assembly assays in NIH3T3 cells stimulated with PDGFR\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, siRNA knockdown, localization with functional consequence, multiple cell systems\",\n      \"pmids\": [\"17129786\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Integrin signaling through the Arg tyrosine kinase activates p190RhoGAP by promoting its association with p120RasGAP and recruiting the p190/p120 complex to the cell periphery. p120 binding does not activate p190RhoGAP activity in vitro; instead, membrane recruitment is required for in vivo activation.\",\n      \"method\": \"arg-/- fibroblasts; dominant-negative p120 fragment; in vitro GAP activity assays; immunofluorescence localization; co-immunoprecipitation\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro assay combined with genetic null cells, dominant negative, and localization studies; dissects mechanism of activation\",\n      \"pmids\": [\"16971514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Arg tyrosine kinase phosphorylates p190RhoGAP at Y1105 in vitro and in vivo. This phosphorylation is adhesion-dependent, promotes p190RhoGAP binding to p120RasGAP, stimulates p190RhoGAP GAP activity to inhibit Rho, and induces neuritogenesis in neuroblastoma cells.\",\n      \"method\": \"In vitro kinase assay; arg-/- mouse brain extracts and fibroblasts; site-directed mutagenesis (Y1105); co-immunoprecipitation; neuritogenesis assay\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis, genetic null cells, multiple functional readouts, single lab\",\n      \"pmids\": [\"15084284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Arg kinase acts through p190RhoGAP to inhibit actomyosin contractility (stress fiber formation) via RhoA suppression upon adhesion to fibronectin. The Arg N-terminal kinase domain is sufficient to act through p190RhoGAP; Arg also requires its C-terminal cytoskeleton-binding half to fully regulate focal adhesion dynamics.\",\n      \"method\": \"arg-/- fibroblasts; Arg re-expression; traction force microscopy; myosin light chain phosphorylation assays; immunofluorescence of stress fibers and focal adhesions\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic null with domain dissection, multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"17652459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The Arg tyrosine kinase and p190RhoGAP pathway is essential for dendritic spine maturation and synapse/dendrite stability during late postnatal hippocampal development. p190RhoGAP localizes to dendritic spines; p190RhoGAP activity is reduced in arg-/- hippocampus, leading to elevated RhoA. Reducing ROCKII gene dosage suppresses dendritic regression in arg-/- mice.\",\n      \"method\": \"arg-/- mice; genetic epistasis (p190rhogap heterozygous/arg-/- double mutants; ROCKII haploinsufficiency in arg-/- background); immunofluorescence localization; RhoA activity assays; behavioral testing\",\n      \"journal\": \"The Journal of neuroscience : the official journal of the Society for Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in multiple mouse mutant combinations with defined cellular and behavioral phenotypes\",\n      \"pmids\": [\"17928439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"FAK forms a complex with p120RasGAP and p190RhoGAP-A (p190A) at leading-edge focal adhesions. Fibronectin-integrin-mediated FAK activation promotes SH2-mediated p120RasGAP binding to FAK, which facilitates FAK-mediated p190A tyrosine phosphorylation. This complex is required for cell polarity during migration; knockdown of p120RasGAP or mutation of FAK Y397 prevents p190A association, tyrosine phosphorylation, and cell polarity.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown; FAK Y397F mutation; wound healing and Golgi reorientation polarity assays; fibronectin-integrin stimulation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP, mutagenesis, siRNA, multiple functional readouts, single lab\",\n      \"pmids\": [\"19435801\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"p190RhoGAP overexpression induces a multinucleated phenotype dependent on the GAP domain, and endogenous p190RhoGAP localizes to the cleavage furrow of dividing cells. Endogenous p190 protein levels are transiently decreased in late mitosis via ubiquitin-mediated degradation requiring the N-terminal GTP-binding region.\",\n      \"method\": \"Conditional/transient overexpression; GAP domain mutants; confocal immunofluorescence of cleavage furrow localization; ubiquitin-proteasome inhibitors; cell cycle synchronization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain-dependent phenotype, localization, and degradation mechanism with multiple orthogonal methods, single lab\",\n      \"pmids\": [\"14610059\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Cell surface transglutaminase activates RhoA by suppressing the Src-p190RhoGAP regulatory pathway through integrin clustering. tTG-induced integrin aggregation inhibits Src kinase activity, decreasing activation of the Src substrate p190RhoGAP, leading to elevated RhoA-GTP levels.\",\n      \"method\": \"tTG overexpression; Src kinase activity assays; p190RhoGAP phosphorylation measurements; RhoA-GTP pulldown; pharmacological Src inhibition\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods but p190RhoGAP studied as part of a broader pathway, single lab\",\n      \"pmids\": [\"16452636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Alpha5beta1 integrin engagement stimulates tyrosine phosphorylation of p190RhoGAP (Src-dependent), while parallel syndecan-4 engagement redistributes tyrosine-phosphorylated p190RhoGAP between membrane and cytosolic fractions via PKCalpha activation. Both pathways must be activated for efficient RhoA suppression and focal adhesion formation; p190RhoGAP is the convergence point of these two adhesion receptor signals.\",\n      \"method\": \"Function-blocking antibodies against alpha5beta1 integrin and syndecan-4; siRNA knockdown; fractionation; phosphotyrosine blotting; RhoA-GTP pulldown; focal adhesion scoring\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple receptor perturbations, fractionation, siRNA, RhoA activity assays; mechanistically dissects two pathways converging on p190\",\n      \"pmids\": [\"18541700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"p190RhoGAP accumulates in lipid rafts during early cell spreading, where it exerts Rho inhibitory activity. Filamin controls this accumulation: cells lacking filamin fail to accumulate p190RhoGAP in lipid rafts and maintain high Rho activity even when spread. Calpain-resistant filamin also prevents spreading-induced raft accumulation of p190RhoGAP.\",\n      \"method\": \"siRNA knockdown of p190RhoGAP; filamin-null cell lines; calpain-resistant filamin expression; lipid raft fractionation; RhoA activity assays; cell rounding/spreading assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic null and siRNA, lipid raft fractionation, multiple orthogonal methods linking localization to function, single lab\",\n      \"pmids\": [\"17227794\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Rho-kinase (ROCK) phosphorylates p190A RhoGAP at Ser1150 and attenuates its GAP activity. This phosphorylation impairs Rnd binding to p190A (Rnd binding normally enhances p190A activation). A phosphomimetic S1150 mutation of p190A weakened Rnd binding and GAP activity. ET-1-induced sustained RhoA activation in vascular smooth muscle cells involves this ROCK-p190A feedback loop.\",\n      \"method\": \"In vitro phosphorylation assays; co-immunoprecipitation of Rnd and p190A; phosphomimetic/phosphoresistant mutations; Rho-kinase inhibitor Y-27632; COS7 and vascular smooth muscle cell experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with mutagenesis, binding assays, cellular validation, single lab\",\n      \"pmids\": [\"19103606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\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 and contributing to cell polarity during directional migration. p190A-deficient fibroblasts exhibit a defect in directional migration that reflects a requirement for GSK-3beta-mediated phosphorylation.\",\n      \"method\": \"In vitro kinase assay; phosphorylation mapping; p190A-deficient fibroblasts; reconstitution with phosphomimetic/phosphoresistant mutants; wound healing assays; RhoA activity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with reconstitution, site mapping, genetic null cells, multiple phenotypic readouts, single lab\",\n      \"pmids\": [\"18502760\"],\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 p190 association with p120RasGAP. This stimulates p190 GAP activity (RhoA inactivation) and attenuates p120RasGAP activity (Ras activation). Disruption of the p190/p120 complex abolishes Brk-mediated regulation of RhoA and Ras and Brk-driven proliferation, migration, invasion, and tumorigenicity.\",\n      \"method\": \"In vitro kinase assay; site-directed mutagenesis (Y1105); co-immunoprecipitation; RhoA and Ras activity assays; proliferation, migration, invasion, and xenograft tumor assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase reconstitution, mutagenesis, co-IP, multiple functional assays, single lab\",\n      \"pmids\": [\"18829532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PTP-PEST directly dephosphorylates and thereby regulates p190RhoGAP activity. PTP-PEST null fibroblasts show enhanced p190RhoGAP activity (decreased RhoA) and exaggerated leading-edge protrusions; PTP-PEST acts to couple protrusion and retraction by reciprocally modulating VAV2 (Rac1 GEF) and p190RhoGAP (RhoA GAP).\",\n      \"method\": \"PTP-PEST null fibroblasts; RhoA and Rac1 activity assays; direct substrate phosphatase assays; co-immunoprecipitation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic null cells with defined phenotypes, direct substrate assay, single lab\",\n      \"pmids\": [\"16513648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Angiotensin II activates RhoA in vascular smooth muscle cells by causing SHP2-dependent dephosphorylation and inactivation of p190A RhoGAP. SHP2 maintains basal p190A phosphorylation via c-Abl; AT1R activation induces SHP2-mediated p190A dephosphorylation and RhoA activation. Phosphomimetic p190A mutant inhibits, and phosphoresistant mutant increases, basal RhoA-Rho kinase activity.\",\n      \"method\": \"siRNA knockdown of p190A and SHP2; phosphomimetic/phosphoresistant p190A mutants; RhoA/Rho-kinase activity assays; co-immunoprecipitation\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA, mutagenesis, activity assays, multiple orthogonal approaches, single lab\",\n      \"pmids\": [\"19692654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Protein kinase C phosphorylates p190A RhoGAP at Ser1221 and Thr1226 within a polybasic region, preventing binding of acidic phospholipids to this region. Phospholipid binding to this region inhibits RhoGAP activity and promotes RacGAP activity. PKC-mediated phosphorylation thus indirectly alters substrate specificity by blocking phospholipid binding.\",\n      \"method\": \"In vitro PKC phosphorylation; liposome binding assays; site-directed mutagenesis; in vitro GAP activity assays with RhoA and Rac substrates; COS-7 cell morphology assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with mutagenesis, lipid binding, and substrate specificity assays, single lab\",\n      \"pmids\": [\"19673492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Tiam1/Rac1 signaling downregulates Rho activity through p190-RhoGAP. p190-RhoGAP is required for this pathway; Src-kinase-dependent tyrosine phosphorylation of p190-RhoGAP and its recruitment to the membrane through p120-RasGAP SH2 domains are both necessary for Tiam1-mediated Rho downregulation.\",\n      \"method\": \"Dominant negative/active constructs of Tiam1, Rac isoforms, and p190-RhoGAP; phosphorylation mutants; dominant-negative p120 fragment; RhoA activity assays in COS-7 cells\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple dominant negative constructs and activity assays, pathway placement, single lab\",\n      \"pmids\": [\"16542153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"p190RhoGAP negatively regulates RhoA activity at the cleavage furrow, modulating cytokinetic furrow site selection and ring contraction. FRET analysis showed that wild-type p190 overexpression reduces RhoA-GTP at the furrow in a dose-dependent manner, causing abnormal furrow positioning and failed cytokinesis, while dominant-negative p190 causes hyper-activated Rho.\",\n      \"method\": \"FRET-based RhoA activity biosensor; time-lapse microscopy; overexpression of wild-type and dominant-negative p190RhoGAP; cleavage furrow localization by confocal imaging\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — FRET biosensor for direct RhoA activity measurement at furrow, combined with live imaging, multiple constructs\",\n      \"pmids\": [\"19254711\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"p190RhoGAP and Ect2 RhoGEF physically associate and colocalize at the cleavage furrow with opposing roles on RhoA activity and cytokinesis outcome; Ect2 can dose-dependently reduce p190-induced multinucleation. Their functional and physical interactions regulate RhoA activity levels at the furrow.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence colocalization at cleavage furrow; multinucleation assays; RhoA pulldown activity assays\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP and functional epistasis in same cellular system, single lab\",\n      \"pmids\": [\"18642445\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mitotic down-regulation of p190RhoGAP via ubiquitin-proteasome-mediated degradation is required for successful cytokinesis. The N-terminal GBDS1 region (and four specific N-terminal residues) is necessary and sufficient for p190A mitotic ubiquitination and degradation; a degradation-resistant p190A mutant causes cytokinesis failure in an RNAi reconstitution approach.\",\n      \"method\": \"RNAi reconstitution with degradation-resistant mutant; N-terminal deletion mapping; ubiquitin-proteasome inhibitors; cell cycle synchronization; multinucleation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reconstitution approach with defined mutants, domain mapping, clear causal link between degradation and phenotype, single lab\",\n      \"pmids\": [\"20534586\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"p190RhoGAP-A forms a complex with anillin at the cytokinetic furrow. p190RhoGAP-A depletion causes accumulation of high RhoA-GTP in the furrow and failure to progress to abscission. Mutants of p190RhoGAP-A unable to bind anillin or lacking GAP activity fail to rescue cytokinesis defects; low-dose blebbistatin (myosin II inhibitor) rescues the cytokinesis failure of p190RhoGAP-A-depleted cells.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown; anillin-binding mutant and GAP-dead mutants; RhoA-GTP quantification at furrow; blebbistatin rescue experiment\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, siRNA depletion with specific mutant rescue, mechanistic epistasis via blebbistatin, multiple orthogonal approaches\",\n      \"pmids\": [\"25359885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ERK promotes Rho-dependent focal adhesion formation by suppressing p190A RhoGAP. ERK activity is required for RhoA-GTP loading and focal adhesion maturation during cell spreading on fibronectin. ERK phosphorylates the C-terminus of p190A, affecting its peripheral localization and activity.\",\n      \"method\": \"MEK/ERK inhibitors; ERK phosphorylation site mapping; p190A localization by immunofluorescence; RhoA-GTP pulldown; focal adhesion maturation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phosphorylation site identification, localization and activity readouts, pharmacological inhibition, single lab\",\n      \"pmids\": [\"20439493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Caveolin-1-deficiency leads to eNOS activation, peroxynitrite generation, and selective nitration of p190RhoGAP-A at Tyr1105, impairing its GAP activity and causing RhoA activation and adherens junction disassembly. Thrombin also induces nitration of p120-catenin-associated p190RhoGAP-A.\",\n      \"method\": \"Cav-1-/- endothelial cells; nitration assays (tyrosine nitration); eNOS inhibition; S-nitrosylation/nitration profiling; RhoA activity assays; endothelial permeability measurements; adherens junction imaging\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic null cells, specific nitration site identified, functional rescue by eNOS/RhoA inhibition, multiple orthogonal methods\",\n      \"pmids\": [\"21624953\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"p190RhoGAP has RacGAP activity in cells, not only RhoGAP activity. The cellular RacGAP activity requires an intact polybasic region adjacent to the GAP domain (which inhibits RhoGAP activity), revealing that this polybasic region inversely regulates substrate preference between Rac and Rho.\",\n      \"method\": \"Mutagenesis of polybasic region; in vitro GAP activity assays with RhoA and Rac substrates; cellular Rac/RhoA activity readouts\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — confirmed in cells and in vitro with mutagenesis, single lab; builds on prior in vitro work\",\n      \"pmids\": [\"23499677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"p120-catenin interacts with p190RhoGAP via a 23-amino acid stretch (CRAD, amino acids 820–843) in its C-terminal domain. This interaction is required for membrane translocation of p190RhoGAP, suppression of RhoA and upregulation of Rac1/PAK1/cortactin signaling. Expression of p120-catenin lacking CRAD prevents p190RhoGAP recruitment to the cell periphery and impairs endothelial barrier recovery.\",\n      \"method\": \"Truncation mutants of p120-catenin; co-immunoprecipitation; immunofluorescence localization; RhoA and Rac1 activity assays; endothelial permeability measurements\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping with truncation mutants, co-IP, localization with functional consequences, multiple readouts, single lab\",\n      \"pmids\": [\"23653363\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"p190A RhoGAP is phosphorylated by PDGF receptor alpha on Y308 within the FF1 domain, but phosphorylation requires prior domain unfolding since Y308 is buried in the hydrophobic core. Phosphorylation irreversibly destabilizes the FF1 structure, likely accounting for its inhibitory effect on TFII-I interaction.\",\n      \"method\": \"NMR structure determination of FF1 domain; in vitro phosphorylation at different temperatures; thermal denaturation studies\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure determination with in vitro phosphorylation and mechanistic characterization of domain stability, single lab\",\n      \"pmids\": [\"19393245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"The N-terminal domain of p190RhoGAP proteins is a pseudoGTPase (N-GTPase) that constitutively binds GTP but does not hydrolyze it. Crystal structure at 2.8 Å shows an unusual GTP-Mg2+ binding pocket with six inserts perturbing catalytic activity. GTP/Mg2+ binding stabilizes the domain, suggesting it functions as a protein-protein interaction platform.\",\n      \"method\": \"X-ray crystal structure (2.8 Å); biochemical GTP hydrolysis assays; nucleotide exchange under Mg2+ chelation; mutational analysis of GTP/Mg2+ binding\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus biochemical and mutagenesis validation, rigorous characterization of pseudoenzyme properties\",\n      \"pmids\": [\"30174148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The p120RasGAP N-terminal SH2 domain binds the p190RhoGAP phosphopeptide centered on pTyr-1105 with a dissociation constant of 0.3 μM via a canonical SH2-pTyr interaction requiring the conserved FLVR motif arginine R207. Crystal structure at 1.6 Å shows that peptide binding stabilizes specific conformations of the βE-βF loop and key arginine residues.\",\n      \"method\": \"X-ray co-crystal structure (1.6 Å); site-directed mutagenesis (R207); native gel shifts; isothermal titration calorimetry\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, ITC binding measurement, mutagenesis confirmation, rigorous biophysical characterization\",\n      \"pmids\": [\"31891593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"p190A localizes to membrane protrusions via a novel domain termed the PLS (protrusion localization sequence). The PLS is required for targeting to the leading edge and also negatively regulates p190A GAP activity. Cortactin binds the PLS and is required for p190A targeting to protrusions. Cancer-associated mutations in PLS disrupt localization, GAP activity regulation, and tumor cell migration.\",\n      \"method\": \"Truncation mutants; subcellular localization (immunofluorescence); GAP activity assays; cortactin co-immunoprecipitation; cancer-mutation panel analysis; cell migration assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain mapping with multiple mutants including cancer variants, co-IP, localization tied to functional consequences, single lab\",\n      \"pmids\": [\"27646271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"A point mutation (L1396Q) in the GAP domain of p190A RhoGAP (Arhgap35) decreases GAP activity for RhoA and Rac1 and impairs primary cilia formation in renal nephrons. p190A localizes to the base of cilia; its GAP activity is required for axoneme elongation. Pharmacological inhibition of ROCK or F-actin polymerization rescues ciliogenesis defects, placing p190A upstream of Rho-ROCK-actin in cilia formation.\",\n      \"method\": \"ENU mutagenesis; in vitro GAP activity assay with L1396Q mutant; mouse glomerulocystic kidney model; immunofluorescence localization to cilia base; ROCK inhibitor and actin polymerization inhibitor rescue experiments\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro GAP activity mutagenesis, genetic mouse model, localization, pharmacological epistasis, single lab\",\n      \"pmids\": [\"26859289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"p190RhoGAP acts in a GAP-independent mode to transiently suppress attraction to Netrin-1 while motor axons exit the spinal cord, and also uses GAP-dependent RhoA inhibition to guide a subset of axons to specific muscles. Identified by a mouse mutagenesis screen; multifunctional activity emerges from modular design.\",\n      \"method\": \"Mouse mutagenesis screen; in vivo motor axon guidance assays; genetic analysis distinguishing GAP-dependent and GAP-independent activities\",\n      \"journal\": \"Neuron\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic screen plus in vivo functional dissection of GAP-dependent vs. GAP-independent modes, single lab\",\n      \"pmids\": [\"30902550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"p190A (ARHGAP35) is an upstream regulator of the Hippo-YAP signaling pathway. p190A knockout promotes YAP nuclear localization and transcriptional activity; wild-type but not cancer-associated p190A mutants suppress YAP. p190A promotes MET and CDH1 (E-cadherin) expression; E-cadherin amplifies p190A-mediated LATS activation and is required for contact inhibition of proliferation.\",\n      \"method\": \"CRISPR/Cas9 knockout; lentiviral expression of cancer mutants; YAP localization/activity assays; LATS kinase activity; xenograft mouse model; CDH1 expression analysis\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with specific rescue by WT but not cancer mutants, in vivo xenograft validation, LATS kinase assay, YAP localization\",\n      \"pmids\": [\"32457342\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Polycystin-1 (PC1) regulates ARHGAP35-dependent centrosomal RhoA activation and ROCK signaling. PKD1-null cells show decreased centrosomal ARHGAP35, increased total and centrosomal active RhoA, and ROCK signaling. ARHGAP35 knockdown reduces primary ciliation in normal renal tubular cells. ROCK inhibitor hydroxyfasudil reduces cyst expansion in PKD1 3D cyst assays and an inducible Pkd1 mouse model.\",\n      \"method\": \"Centrosome-targeted proximity ligation assay; dual immunofluorescence; siRNA knockdown; cilia length phenotypic assay; 3D cyst assay; inducible Pkd1 mouse model\",\n      \"journal\": \"JCI insight\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity ligation and localization assays, siRNA, in vivo model, single lab\",\n      \"pmids\": [\"32663194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"p190A RhoGAP promotes CDH1 (E-cadherin) expression and cooperates with E-cadherin to activate LATS kinases and suppress tumor cell growth. p190A and E-cadherin are mutually required for LATS activation and contact inhibition of proliferation (CIP); p190A cancer mutations lose this function.\",\n      \"method\": \"Xenograft mouse model; LATS kinase assays; YAP phosphorylation; CDH1 induction assays; reconstitution with cancer mutant panel; co-expression/knockdown experiments\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple cancer mutants, in vivo xenograft validation, LATS kinase activity assays, E-cadherin epistasis, single lab\",\n      \"pmids\": [\"32641858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRIM65 E3 ubiquitin ligase mediates ubiquitination and proteasomal degradation of ARHGAP35 (p190A), leading to elevated Rho GTPase activity, increased migration-related structures (focal adhesions, filopodia), and enhanced CRC metastasis.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assays; TRIM65 overexpression and knockdown; in vivo mouse metastasis model (liver, lung); RhoA activity assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, ubiquitination assay, in vivo metastasis model, RhoA activity readout, single lab\",\n      \"pmids\": [\"31332286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"p120 RasGAP and ZO-2 are both necessary for p190A to activate LATS kinases, elicit MET, promote contact inhibition of proliferation, and suppress tumorigenesis. The interaction of p190A with ZO-2 is dependent on RasGAP; RasGAP and ZO-2 are required for transcriptional modulation by p190A.\",\n      \"method\": \"Co-immunoprecipitation; siRNA knockdown of RasGAP and ZO-2; LATS kinase activity assays; contact inhibition assays; xenograft tumor assays; transcriptional reporter assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP, siRNA, kinase activity assays, in vivo validation, multiple functional readouts; independent confirmation also as preprint\",\n      \"pmids\": [\"37995182\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Interaction of p190A RhoGAP with eIF3A (and other translation preinitiation factors) involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A; interaction is enhanced by serum and reduced by phosphatase treatment. Disrupted by S296A mutation in the first FF domain but not by Y308 mutation.\",\n      \"method\": \"Tandem mass spectrometry of endogenous p190A interactors; co-immunoprecipitation; site-directed mutagenesis (S296A, Y308); phosphatase treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS interactome with co-IP validation and mutagenesis; functional consequence not demonstrated beyond complex formation, single lab\",\n      \"pmids\": [\"28007963\"],\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. This interaction dysregulates p190RhoGAP and alters the actin cytoskeleton, and negatively regulates cell spreading on fibronectin.\",\n      \"method\": \"Mass spectrometry identification; co-immunoprecipitation; domain-mapping with E7 CR3 mutants and p190 deletion constructs; cell spreading assays on fibronectin; actin cytoskeleton immunofluorescence\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified, co-IP with domain mapping, functional cell spreading assay, single lab\",\n      \"pmids\": [\"24403595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"A Rnd3/p190RhoGAP pathway operates in idiopathic pulmonary fibrosis (IPF) fibroblasts. Rnd3 expression and p190RhoGAP activity are suppressed in IPF; restoration of Rnd3 increases p190 activity and decreases RhoA activity and fibrotic phenotype. IPF drugs nintedanib and pirfenidone decrease RhoA activity through up-regulation of Rnd3 and p190 activity.\",\n      \"method\": \"Rnd3 overexpression in IPF fibroblasts; p190RhoGAP activity assays; RhoA activity assays; nintedanib/pirfenidone treatment; collagen gel contraction (fibrotic phenotype) assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain of function rescue, direct p190 activity measurement, pharmacological epistasis, single lab\",\n      \"pmids\": [\"29995590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The PLS of p190A acts as an autoinhibitory domain that masks the GAP domain through intramolecular interaction. Co-immunoprecipitation demonstrates that the PLS interacts with a region near the GAP domain; p190A lacking PLS (p190AΔPLS) has stronger RhoA-inhibiting activity. This intramolecular autoinhibition is disrupted by cancer-associated mutations S866F and Δ865-870 in the PLS.\",\n      \"method\": \"Yeast two-hybrid screen; co-immunoprecipitation of PLS with p190A GAP-proximal region; RhoA inactivation assays comparing WT vs. ΔPLS; cancer-associated PLS mutants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two-hybrid and co-IP for intramolecular interaction, functional GAP activity comparison, cancer mutation panel, single lab\",\n      \"pmids\": [\"36516886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Fuzzy (CPLANE protein) recruits ARHGAP35 (p190A RhoGAP) to the basal body/base of primary cilia to restrict actin polymerization. Loss of Fuzzy in mice reduces cilia length with increased RhoA activity and excessive basal body actin; genetic interaction between Fuzzy and Arhgap35 alleles confirms their functional cooperation at the basal body.\",\n      \"method\": \"Co-immunoprecipitation of Fuzzy with ARHGAP35; immunofluorescence localization at basal body; Fuzzy knockout mice; Fuzzy/Arhgap35 compound mutant genetic epistasis; actin and RhoA activity measurements\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — co-IP, localization, genetic KO, and compound mutant epistasis with quantitative phenotypic readouts, single lab\",\n      \"pmids\": [\"38546045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In cardiac myocytes, M2 muscarinic receptor (M2R) stimulation by carbachol induces eNOS activation in caveolae, producing NO/peroxynitrite that nitrates p190A at Tyr1105. This nitration promotes p190A binding to RGS3L and shifts p190A substrate preference from RhoA to Rac1, resulting in net RhoA activation. Complex of eNOS, p190A, RGS3L, and caveolin-3 detected by co-immunoprecipitation.\",\n      \"method\": \"Carbachol stimulation; eNOS inhibitors (L-NIO, L-NAME); nitration assays; co-immunoprecipitation (eNOS/p190A/RGS3L/caveolin-3); GAP activity assays for RhoA vs. Rac1; caveolae disruption by methyl-β-cyclodextrin\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP, enzymatic activity assay, pharmacological inhibition of eNOS, caveolae disruption; single lab, single paper\",\n      \"pmids\": [\"37887276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"p190RhoGAP is required to prevent mitotic spindle fragmentation and to activate Aurora A kinase at acentriolar poles. Depletion of p190RhoGAP causes prolonged mitotic arrest with multipolar spindles. Low-dose Eg5 inhibitor rescues the multipolar phenotype. Aurora A localizes to all poles in p190-depleted cells but is only activated at centriolar poles.\",\n      \"method\": \"siRNA knockdown of p190RhoGAP; live-cell imaging of spindle dynamics; Aurora A activation (immunofluorescence with activation-specific antibody); Eg5 inhibitor rescue experiment\",\n      \"journal\": \"Chromosoma\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA depletion with defined spindle phenotype, pharmacological epistasis, Aurora A activity readout, single lab\",\n      \"pmids\": [\"29656322\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ARHGAP35/p190RhoGAP is a multi-domain RhoGAP (with three pseudoGTPase domains, four FF domains, and a GAP domain) that serves as a central hub for RhoA inactivation downstream of multiple upstream signals—including integrins (via Arg/Abl2 kinase, FAK, Src), cadherins, receptor tyrosine kinases (EGFR, PDGFR, Brk), and GPCRs—by becoming tyrosine-phosphorylated (primarily at Y1105 by Src/Arg/FAK/Brk), which promotes its binding to p120RasGAP and membrane/junction recruitment to suppress RhoA-GTP; its activity is further tuned by phospholipid binding and PKC-dependent serine/threonine phosphorylation (altering RhoA vs Rac1 substrate preference), ROCK-mediated Ser1150 phosphorylation (feedback inhibition), GSK-3β-mediated C-terminal phosphorylation (polarity), eNOS-dependent nitration at Y1105 (GAP inactivation), autoinhibition via intramolecular PLS-GAP domain interaction, ubiquitin-proteasome-mediated mitotic degradation (required for cytokinesis completion), and localization to lipid rafts (filamin-dependent) or protrusions (cortactin-PLS-dependent); downstream it suppresses actomyosin contractility, promotes cell spreading, migration and polarity, adherens junction assembly, dendritic spine maturation, ciliogenesis (at the basal body via Fuzzy), and cytokinetic furrow progression (in complex with anillin), while also activating the Hippo-LATS-YAP pathway (requiring p120RasGAP and ZO-2) to suppress tumor cell proliferation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ARHGAP35 (p190RhoGAP-A) is a multidomain GTPase-activating protein that functions as a central convergence node for inactivating RhoA downstream of adhesion and growth-factor signaling, thereby controlling actomyosin contractility, cell spreading, migration, and polarity [#0, #13]. Its activation is driven by adhesion-dependent tyrosine phosphorylation—catalyzed by c-Src, the Arg/Abl2 kinase, FAK, and the tumor kinase Brk—primarily at Tyr1105, a modification that creates a phosphopeptide bound with high affinity by the p120RasGAP SH2 domain via the FLVR-motif arginine R207 [#1, #7, #4, #17, #32]. This p190–p120RasGAP association does not directly stimulate catalysis but instead drives recruitment of the complex to the membrane, leading-edge focal adhesions, lipid rafts, and cadherin junctions, where RhoA suppression occurs [#6, #10, #14, #5]. At adherens junctions p190 couples to the cadherin complex through p120-catenin, which binds a defined C-terminal CRAD segment and is required for membrane translocation, RhoA suppression, and junction assembly [#5, #29]. ARHGAP35 integrates signals from integrins, syndecan-4/PKCα, RTKs, and Rac/Tiam1, acting as the shared point at which these inputs converge to suppress RhoA [#13, #21]. Its output is tuned by an extensive regulatory layer: ROCK-mediated Ser1150 phosphorylation provides feedback inhibition by impairing Rnd binding [#15], GSK-3β and ERK phosphorylate the C-terminus to control polarity and localization [#16, #26], PKC phosphorylation within a polybasic region blocks acidic-phospholipid binding and shifts substrate preference between RhoA and Rac1 [#20, #28], and eNOS-dependent nitration at Tyr1105 inactivates the GAP and can redirect substrate specificity toward Rac1 [#27, #46]. The protein is also autoinhibited by an intramolecular interaction between its protrusion-localization sequence (PLS) and the GAP domain, an interaction disrupted by cancer-associated mutations [#44, #33]; its N-terminal domain is a pseudoGTPase that constitutively binds GTP without hydrolysis and serves as a protein-interaction platform [#31]. ARHGAP35 governs cytokinesis by suppressing RhoA at the cleavage furrow in complex with anillin, and its ubiquitin-proteasome-mediated mitotic degradation is required for furrow progression and abscission [#22, #25, #24]. Beyond contractility, it promotes ciliogenesis at the basal body—where it is recruited by Fuzzy to restrict actin polymerization—and acts as an upstream activator of the Hippo–LATS–YAP pathway, cooperating with p120RasGAP, ZO-2, and E-cadherin to drive contact inhibition of proliferation and suppress tumorigenesis [#45, #34, #36, #38, #40].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established that p190RhoGAP is a tyrosine-phosphorylation substrate linking Src-family kinase activity to actin cytoskeleton remodeling, framing it as a regulated effector rather than a constitutive enzyme.\",\n      \"evidence\": \"c-Src wild-type/dominant-negative overexpression with imaging of p190/p120RasGAP distribution and phosphotyrosine analysis\",\n      \"pmids\": [\"7542246\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the specific phosphosite or define how phosphorylation alters GAP catalysis\", \"Did not establish the RhoA target quantitatively\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined the core physiological role: integrin-triggered RhoA inactivation by p190RhoGAP promotes spreading, polarity, and migration, placing it in adhesion signaling.\",\n      \"evidence\": \"Dominant-negative and overexpression in Rat1 fibroblasts with RhoA activity, spreading, and migration assays on fibronectin\",\n      \"pmids\": [\"11553710\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream kinase coupling integrins to p190 not identified\", \"Membrane recruitment mechanism not addressed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified Tyr1105 as the key adhesion-dependent phosphosite, phosphorylated by Arg kinase, that promotes p120RasGAP binding and stimulates GAP activity — resolving the molecular switch.\",\n      \"evidence\": \"In vitro kinase assay, arg-/- cells, Y1105 mutagenesis, co-IP, and neuritogenesis assay\",\n      \"pmids\": [\"15084284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How p120RasGAP binding converts to RhoA suppression not mechanistically resolved at this stage\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed that p120RasGAP binding does not activate the GAP enzymatically in vitro but instead drives membrane recruitment required for in vivo activity, redefining 'activation' as relocalization.\",\n      \"evidence\": \"arg-/- fibroblasts, dominant-negative p120 fragment, in vitro GAP assays, and localization studies\",\n      \"pmids\": [\"16971514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Membrane anchoring partners at the periphery not fully defined\", \"Did not address junctional vs. raft recruitment\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Connected p190RhoGAP to adherens junction assembly through a p120-catenin interaction, extending its role from focal adhesions to cell-cell contacts.\",\n      \"evidence\": \"siRNA of p120 and p190, reciprocal co-IP, and AJ assembly assays in PDGFR-stimulated cells\",\n      \"pmids\": [\"17129786\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The catenin-binding region on p190 not yet mapped (later defined as CRAD)\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated that p190RhoGAP is the convergence point of multiple adhesion receptors (integrin + syndecan-4/PKCα) and is subject to ROCK feedback and GSK-3β/ERK control, establishing a multi-input regulatory architecture.\",\n      \"evidence\": \"Receptor-blocking antibodies, siRNA, fractionation, in vitro kinase assays with phosphosite mapping and phosphomutants\",\n      \"pmids\": [\"18541700\", \"19103606\", \"18502760\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative integration of opposing phosphorylation inputs not modeled\", \"Crosstalk between distinct phosphosites unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined a polybasic/phospholipid-based mechanism by which PKC phosphorylation toggles substrate preference between RhoA and Rac1, revealing p190 as a dual GAP.\",\n      \"evidence\": \"In vitro PKC phosphorylation, liposome binding, mutagenesis, and dual-substrate GAP assays\",\n      \"pmids\": [\"19673492\", \"23499677\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological triggers selecting Rho vs Rac mode in vivo not fully mapped\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Established that mitotic ubiquitin-proteasome degradation of p190 via its N-terminal region is required for cytokinesis, linking its turnover to cell division.\",\n      \"evidence\": \"RNAi reconstitution with degradation-resistant mutant, N-terminal deletion mapping, and proteasome inhibitors\",\n      \"pmids\": [\"20534586\", \"14610059\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The E3 ligase mediating mitotic degradation not identified here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved the cytokinetic mechanism: p190 forms a complex with anillin at the furrow and locally suppresses RhoA-GTP to permit abscission.\",\n      \"evidence\": \"Co-IP, siRNA with anillin-binding and GAP-dead rescue mutants, and blebbistatin rescue\",\n      \"pmids\": [\"25359885\", \"19254711\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How anillin binding spatially restricts GAP activity not structurally defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified the PLS as a dual-function domain that targets p190 to protrusions via cortactin and regulates GAP activity, with cancer mutations disrupting both.\",\n      \"evidence\": \"Truncation mutants, cortactin co-IP, localization, GAP assays, and cancer-mutation panel\",\n      \"pmids\": [\"27646271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of GAP regulation by PLS not resolved (later shown autoinhibitory)\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Structurally characterized the regulatory interfaces: the N-terminal domain is a GTP-binding pseudoGTPase and the p120RasGAP SH2 binds pTyr1105 with sub-micromolar affinity via R207.\",\n      \"evidence\": \"X-ray crystallography of N-GTPase and SH2–phosphopeptide complex, ITC, and mutagenesis\",\n      \"pmids\": [\"30174148\", \"31891593\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length architecture and how domains coordinate not solved\", \"Interaction partners of the pseudoGTPase platform unidentified\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identified TRIM65 as an E3 ligase degrading p190A, linking its loss to elevated Rho activity and metastasis in colorectal cancer.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, TRIM65 perturbation, and in vivo metastasis model\",\n      \"pmids\": [\"31332286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether TRIM65 mediates the mitotic degradation pathway not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a tumor-suppressive role through Hippo signaling: p190A activates LATS and suppresses YAP, requiring E-cadherin, p120RasGAP, and ZO-2, with cancer mutants losing this function.\",\n      \"evidence\": \"CRISPR KO, cancer-mutant reconstitution, LATS kinase and YAP assays, CDH1 analysis, and xenografts\",\n      \"pmids\": [\"32457342\", \"32641858\", \"37995182\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How GAP activity mechanistically couples to LATS activation not fully defined\", \"Direct molecular link between junctional p190 and the Hippo core kinases unresolved\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a ciliogenesis role: p190A is recruited to the basal body by Fuzzy to restrict actin polymerization and RhoA, controlling cilium formation.\",\n      \"evidence\": \"Co-IP, basal-body localization, Fuzzy KO mice, and Fuzzy/Arhgap35 compound mutant epistasis\",\n      \"pmids\": [\"38546045\", \"26859289\", \"32663194\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Centrosomal/ciliary recruitment partners beyond Fuzzy and PC1 not fully mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many competing phosphorylation, nitration, lipid-binding, autoinhibition, and degradation inputs are quantitatively integrated to set RhoA-vs-Rac1 output in a given cellular context remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No full-length structure showing how regulatory domains coordinate\", \"No unified model reconciling GAP-dependent and GAP-independent functions\", \"Spatiotemporal logic of substrate-preference switching in vivo undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 6, 20, 25, 34]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3, 22, 36]},\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [31]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [13, 21]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 14, 29]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [8, 33]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [34, 37, 45]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [34, 45]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 13, 36]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [22, 24, 25, 47]},\n      {\"term_id\": \"R-HSA-1500931\", \"supporting_discovery_ids\": [5, 29]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [9, 35, 34, 45]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [17, 36, 38, 39]}\n    ],\n    \"complexes\": [\n      \"p190RhoGAP–p120RasGAP complex\",\n      \"p190A–anillin furrow complex\",\n      \"eNOS–p190A–RGS3L–caveolin-3 complex\"\n    ],\n    \"partners\": [\n      \"RASA1\",\n      \"CTNND1\",\n      \"PTK2\",\n      \"ABL2\",\n      \"SRC\",\n      \"ANLN\",\n      \"CTTN\",\n      \"FUZ\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":9,"faith_total":9,"faith_pct":100.0}}