{"gene":"RHOU","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2001,"finding":"Wrch-1 (RHOU) was identified as a Wnt-1-responsive Cdc42 homolog that activates PAK-1 and JNK-1, induces filopodium formation and stress fiber dissolution, stimulates cell cycle re-entry, and morphologically phenocopies Wnt-1 in transformation of mouse mammary epithelial cells.","method":"Functional assays in cell lines: PAK-1/JNK-1 activation assays, morphological analysis, cell cycle analysis, transformation assays","journal":"Genes & development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal functional assays in a single foundational study; single lab","pmids":["11459829"],"is_preprint":false},{"year":2004,"finding":"Wrch-1 (RHOU) possesses an extremely rapid intrinsic guanine nucleotide exchange activity (unlike Cdc42) and essentially no GTPase activity, rendering it constitutively GTP-bound. The unique N-terminal extension negatively regulates PAK interaction and transformation, and associates with the Grb2 SH3 domain adaptor protein, which overcomes N-terminal inhibition to promote effector interaction.","method":"In vitro biochemical nucleotide exchange and GTPase assays; co-immunoprecipitation with Grb2; N-terminal truncation mutant analysis; transformation assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical reconstitution of exchange/GTPase activity combined with mutagenesis and binding assays; replicated in companion paper (PMID:15350535)","pmids":["15556869"],"is_preprint":false},{"year":2004,"finding":"Wrch1 (RHOU) has no detectable GTPase activity in vitro and very high intrinsic nucleotide exchange rate. It interacts with PAK1 and NCKβ; the NCKβ interaction is mediated via PxxP motifs in the N-terminal extension binding to the second and third SH3 domains of NCKβ.","method":"In vitro GTPase activity assay; pull-down and co-immunoprecipitation identifying PAK1 and NCKβ as binding partners; mapping of interaction to PxxP motifs","journal":"Experimental cell research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical assay for GTPase activity plus binding partner identification with domain mapping; corroborates PMID:15556869","pmids":["15350535"],"is_preprint":false},{"year":2005,"finding":"Wrch-1 (RHOU) is modified by palmitoylation rather than prenylation, with membrane localization dependent on the second cysteine of the C-terminal CCFV motif. Mutation of this cysteine (C→S) abrogated membrane localization and anchorage-independent transformation. Inhibitors of palmitoylation caused mislocalization, while prenylation inhibitors had no effect.","method":"Metabolic labeling with palmitate and isoprenoids; pharmacological inhibition of palmitoylation/prenylation; site-directed mutagenesis of CCFV motif; subcellular fractionation; transformation assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — biochemical labeling plus mutagenesis plus functional transformation assay; multiple orthogonal methods in one study","pmids":["16046391"],"is_preprint":false},{"year":2007,"finding":"RhoU/Wrch-1 localizes to focal adhesions via its C-terminal extension and effector binding loop (N-terminal extension and palmitoylation site dispensable for FA targeting). Activated RhoU reduces focal adhesion number and redistributes them; RhoU silencing increases focal adhesion number. RhoU also localizes to podosomes in osteoclasts and Src-expressing cells. RhoU transiently associated with adhesion structures promotes adhesion turnover and increases cell migration.","method":"Fluorescence microscopy; RNAi knockdown; expression of deletion/point mutants; cell migration assays","journal":"Biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization by imaging with functional domain mapping and siRNA knockdown phenotype; single lab","pmids":["17620058"],"is_preprint":false},{"year":2007,"finding":"Wrch-1 (RHOU) depletion by siRNA increases focal adhesion formation, inhibits myosin light chain phosphorylation, and inhibits cell migration. Wrch-1 depletion also inhibits Akt and JNK activation. These results place Wrch-1 upstream of myosin light chain phosphorylation and Akt/JNK pathways controlling focal adhesion dynamics and migration.","method":"siRNA knockdown; myosin light chain phosphorylation assay; focal adhesion quantification; wound healing migration assay; pharmacological inhibitors of Akt and JNK","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean siRNA knockdown with multiple defined readouts; single lab","pmids":["17504809"],"is_preprint":false},{"year":2007,"finding":"Wrch1 (RHOU) binds to the nonreceptor tyrosine kinase Pyk2 in a GTP-dependent manner requiring both the N-terminal proline-rich extension and intact effector loop. Pyk2 is required for Wrch1-induced filopodium formation. Src activity is required for formation of the Wrch1-Pyk2 complex and for Wrch1-induced filopodia.","method":"Co-immunoprecipitation; GTP-loading experiments with constitutively active/dominant negative mutants; siRNA knockdown of Pyk2; Src inhibitor treatment; morphological analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction mapping with GTP-dependence and functional rescue; single lab","pmids":["18086875"],"is_preprint":false},{"year":2008,"finding":"Wrch1/RhoU binds integrin β3 cytoplasmic domain and interferes with adhesion-induced Pyk2 and paxillin phosphorylation. Wrch1 expression increases osteoclast precursor aggregation, reduces adhesion onto vitronectin (but not fibronectin), and inhibits M-CSF-induced prefusion osteoclast migration. High Wrch1 activity inhibits podosome belt formation in mature osteoclasts.","method":"Co-immunoprecipitation (Wrch1-integrin β3); phosphorylation assays (Pyk2, paxillin); adhesion assays; migration assays; RNAi knockdown; osteoclast differentiation assays","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay plus downstream phosphorylation readouts and functional cell-based assays; single lab","pmids":["19135548"],"is_preprint":false},{"year":2008,"finding":"Activated Wrch-1 (RHOU) binds the cell polarity protein Par6 in a GTP-dependent manner. Activated Wrch-1 negatively regulates tight junction assembly kinetics and disrupts epithelial cystogenesis in 3D culture. A Wrch-1 effector domain mutant that inhibits Par6 binding abrogates tight junction disruption, actin reorganization, and morphogenesis defects, placing Par6 binding as necessary for these effects.","method":"Co-immunoprecipitation (GTP-dependent Par6 binding); tight junction assembly assays; 3D cystogenesis assay; effector domain mutant analysis; shRNA knockdown","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — GTP-dependent binding assay plus effector mutant causally linking Par6 interaction to tight junction and morphogenesis phenotypes, with multiple orthogonal readouts","pmids":["19064640"],"is_preprint":false},{"year":2009,"finding":"RhoU transcription is induced by Wnt-1 at the transcriptional level via the non-canonical Wnt/planar cell polarity pathway through JNK activation (independent of β-catenin). RhoU is also transcriptionally induced by gp130 cytokines via STAT3, with two functional STAT3-binding sites identified on the mouse RhoU promoter.","method":"Reporter assays; promoter deletion/mutation analysis; ChIP or EMSA for STAT3 binding sites; pathway inhibitor experiments; β-catenin loss-of-function","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter mapping with functional STAT3 binding sites plus pathway dissection using genetic tools; single lab","pmids":["19397496"],"is_preprint":false},{"year":2010,"finding":"Wrch-1 (RHOU) is phosphorylated by Src at C-terminal residue Y254. This phosphorylation causes rapid relocalization from plasma membrane to endosomes upon serum stimulation. Y254 phosphorylation decreases active (GTP-bound) Wrch-1, reduces PAK recruitment and activation, and is required for proper cystogenesis in 3D culture. Phospho-deficient Y254F remains plasma membrane-localized and GTP-bound, sustaining PAK activation.","method":"Site-directed mutagenesis (Y254F, Y254E); Src genetic/pharmacological inhibition; subcellular fractionation and imaging; GTP-loading assay; PAK co-immunoprecipitation; 3D cystogenesis assay; anchorage-independent growth assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis identifying specific phosphorylation site plus biochemical GTP/PAK assays plus multiple functional readouts; single lab but multiple orthogonal methods","pmids":["20547754"],"is_preprint":false},{"year":2010,"finding":"RhoU activates pathways cooperating with PAK1 and Rac1 in epithelial adhesion, cell spreading, and directional cell migration in cranial neural crest (CNC) cells. Loss or gain of RhoU function in Xenopus impairs CNC cell migration and subsequent craniofacial cartilage differentiation.","method":"Gain- and loss-of-function experiments in Xenopus embryos; in vitro cell migration, spreading, and adhesion assays; epistasis with PAK1 and Rac1","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo loss/gain-of-function with in vitro mechanistic follow-up; single lab","pmids":["21156169"],"is_preprint":false},{"year":2011,"finding":"Rhou (RHOU) maintains the epithelial architecture and F-actin cortical organization of foregut endoderm in vivo. Rhou-deficient embryos show flattened foregut, loss of microvilli, reduced sub-apical F-actin, impaired endoderm differentiation, and reduced c-Jun/AP-1 target gene expression consistent with impaired JNK activity.","method":"Rhou knockdown ES cell-derived embryos; embryoid body differentiation; phalloidin staining (F-actin); gene expression analysis","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo knockdown with specific structural and molecular readouts; single lab","pmids":["21903671"],"is_preprint":false},{"year":2011,"finding":"GRB2 couples RhoU to EGFR signaling: after EGF stimulation, RhoU co-localizes with EGFR on endosomes and physically associates with activated EGFR via Grb2 through N-terminal proline-rich motifs. GRB2 knockdown or mutation of proline-rich sequences abolishes the EGFR-RhoU interaction and abrogates EGF-stimulated RhoU GTP loading. RhoU in this complex mediates AP-1 transcriptional activity and cell migration in pancreatic cancer cells.","method":"Co-immunoprecipitation; GRB2 RNAi; proline-rich motif mutagenesis; GTP-loading assay; AP-1 reporter assay; cell migration assay","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP with mutagenesis and GTP-loading assay; single lab with multiple functional readouts","pmids":["21508312"],"is_preprint":false},{"year":2011,"finding":"ARHGAP30 was identified as a Wrch-1 (RHOU)-interacting protein in a binding-partner screen. CdGAP also binds Wrch-1. Ectopic expression of ARHGAP30 results in membrane blebbing and dissolution of stress fibers and focal adhesions downstream of Wrch-1.","method":"Binding-partner screen; co-immunoprecipitation/pull-down; overexpression morphological analysis","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single co-IP/pulldown screen with morphological readout; single lab, limited mechanistic follow-up","pmids":["21565175"],"is_preprint":false},{"year":2012,"finding":"NOTCH1 signaling upregulates RhoU expression in T-ALL cells, and Notch1 or RhoU depletion inhibits T-ALL cell adhesion, migration, and chemotaxis, placing RhoU downstream of NOTCH1 in regulating T-ALL cell migration.","method":"γ-secretase inhibitor treatment; Notch1 RNAi; constitutively active Notch1 expression; RhoU RNAi; adhesion, migration, and chemotaxis assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (Notch1→RhoU) with multiple functional readouts; single lab","pmids":["22349824"],"is_preprint":false},{"year":2013,"finding":"The N-terminal extension of Wrch1/RhoU contains a central PxxP motif with an essential arginine that mediates high-avidity interactions with full-length Grb2 and Nck1 (but not Crk, c-Src, or p120) in cells, and in vitro. Individual SH3 domains of these adaptors bind with low affinity, but the multivalent full-length proteins achieve tight binding.","method":"Sedimentation assays; isothermal titration calorimetry (ITC); co-immunoprecipitation; peptide competition analysis","journal":"Biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — ITC quantitative binding measurements plus sedimentation assays and cell-based co-IP; single lab but rigorous biophysical methods","pmids":["23183748"],"is_preprint":false},{"year":2014,"finding":"RhoU regulates cell junctions between cardiomyocytes through the Arhgef7b/PAK kinase pathway to guide atrioventricular canal development and cardiac looping in zebrafish. Loss of RhoU recapitulates cardiac defects seen with ROCK inhibition, and PAK kinase overexpression rescues the RhoU loss-of-function cardiac defect.","method":"Zebrafish loss-of-function (morpholino); chemical genetic screen; PAK overexpression rescue; epistasis with Arhgef7b/PAK pathway","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic epistasis with rescue experiment in zebrafish; single lab","pmids":["24607366"],"is_preprint":false},{"year":2015,"finding":"PAK4 protects RhoU from ubiquitination and proteasomal degradation in a kinase-independent manner. RhoU is targeted for ubiquitination by the Rab40A-Cullin 5 E3 ubiquitin ligase complex. PAK4 depletion leads to concomitant loss of RhoU protein; overexpression of RhoU rescues the PAK4 depletion adhesion turnover phenotype. RhoU and PAK4 together drive adhesion turnover and cell migration.","method":"Ubiquitination assays; co-immunoprecipitation (Rab40A-Cullin 5 with RhoU; PAK4 with RhoU); PAK4 depletion/kinase-dead mutants; RhoU rescue overexpression; adhesion dynamics assays","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ubiquitination assay identifying E3 complex, co-IP identifying protector mechanism, rescue experiment; multiple orthogonal methods","pmids":["26598620"],"is_preprint":false},{"year":2019,"finding":"RhoU loss-of-function in mouse gut epithelium or DLD-1 cells causes hyperplasia through reduced apoptosis and increased proliferation, associated with increased RhoA activity and elevated phosphorylated Myosin Light Chain-2, linking RhoU activity to actomyosin-dependent apoptosis control.","method":"Conditional Rhou knockout mice; RNAi in DLD-1 cells; RhoA activity assay; pMLC-2 western blot; TUNEL apoptosis assay; BrdU proliferation assay","journal":"Biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo conditional knockout with mechanistic molecular readouts (RhoA activity, pMLC-2); single lab","pmids":["30834544"],"is_preprint":false},{"year":2020,"finding":"RhoU interacts with intersectin-1 and intersectin-2 (ITSN1, ITSN2) via the second PxxP motif in its N-terminus binding to ITSN SH3 domains. Silencing of RhoU or ITSN2 (but not ITSN1) increases transferrin accumulation in early endosomes due to a defect in fast vesicle recycling. RhoU and ITSN2 co-localize on Rab4-positive fast recycling endosomes.","method":"Co-immunoprecipitation; PxxP motif mutagenesis; fluorescent transferrin uptake/recycling assay; siRNA knockdown; co-localization imaging with Rab4 marker","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — binding partner mapping with mutagenesis plus functional trafficking assay and co-localization; single lab","pmids":["32737221"],"is_preprint":false},{"year":2024,"finding":"RhoU forms homo-oligomers (homodimers) in cells, mediated by the C-terminal extension; C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension acts as a dominant negative, reducing RhoU-induced PAK activation and causing morphological changes consistent with RhoU inhibition. Self-association is required for full RhoU activity.","method":"Co-immunoprecipitation of tagged RhoU variants; C-terminal extension deletion/expression; palmitoylation-deficient mutants; PAK activation assay; cell morphology analysis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with mutagenesis plus functional PAK activation readout; single lab","pmids":["38180080"],"is_preprint":false},{"year":2024,"finding":"SUMOylated annexin A6 (AnxA6) binds RhoU; when AnxA6 is deSUMOylated (by SENP1 or K579R mutation), the AnxA6-RhoU interaction is lost, leading to increased RHOU-mediated p-AKT1(Ser473) and facilitation of EMT and cell migration in hepatocellular carcinoma.","method":"LC-MS/MS identification of SUMOylation sites; site-directed mutagenesis (K579R); co-immunoprecipitation (AnxA6–RhoU); western blot for p-AKT1; EMT and migration assays","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP with mutagenesis and downstream signaling readout; single lab","pmids":["38566133"],"is_preprint":false}],"current_model":"RHOU/Wrch-1 is an atypical Rho GTPase with constitutively high intrinsic GDP/GTP exchange activity and negligible GTPase activity, rendering it constitutively GTP-bound; it associates with the plasma membrane and endosomes via C-terminal palmitoylation (not prenylation), is transcriptionally induced by non-canonical Wnt/JNK and STAT3 pathways, is negatively regulated by Src-mediated phosphorylation at Y254 causing endosomal relocalization and GTP unloading, and is stabilized against ubiquitin-mediated (Rab40A-Cullin5) degradation by PAK4; its N-terminal PxxP motifs recruit SH3 adaptor proteins (Grb2, Nck) to couple growth factor receptor (EGFR) signaling to RhoU GTP loading, while its GTP-dependent effectors include PAK1/PAK4, Pyk2, and Par6, through which it regulates focal adhesion turnover, tight junction assembly, filopodium formation, cell migration, endosomal recycling (via ITSN2 on Rab4-positive endosomes), and actomyosin-dependent apoptosis; RhoU also forms C-terminal-dependent homo-oligomers required for full PAK activation."},"narrative":{"mechanistic_narrative":"RHOU (Wrch-1) is an atypical Rho-family GTPase that couples non-canonical Wnt and cytokine signaling to actin-cytoskeleton remodeling, cell adhesion, and migration [PMID:11459829, PMID:19397496]. Biochemically it is distinguished from Cdc42 by an extremely rapid intrinsic guanine-nucleotide exchange rate and negligible GTPase activity, leaving it constitutively GTP-bound, while its N-terminal extension autoinhibits effector engagement until relieved by SH3-adaptor binding [PMID:15556869, PMID:15350535]. Membrane targeting is achieved through C-terminal palmitoylation rather than prenylation, and the conserved CCFV cysteine is required for membrane localization and transformation [PMID:16046391]. Its N-terminal proline-rich (PxxP) motifs recruit the SH3 adaptors Grb2 and Nck through high-avidity multivalent binding, linking activated EGFR on endosomes to RhoU GTP loading and downstream AP-1 activity and migration [PMID:21508312, PMID:23183748]. In its GTP-bound state RhoU engages effectors including PAK1/PAK4, Pyk2 and the polarity protein Par6 to control filopodium formation, focal adhesion turnover, tight-junction assembly and epithelial morphogenesis [PMID:18086875, PMID:19064640, PMID:20547754]. RhoU activity is regulated post-translationally: Src phosphorylation at Y254 relocalizes it from the plasma membrane to endosomes, unloads GTP and dampens PAK activation [PMID:20547754], whereas PAK4 stabilizes RhoU against Rab40A-Cullin5-mediated ubiquitination and degradation [PMID:26598620]. RhoU additionally forms C-terminal palmitoylation-dependent homo-oligomers needed for full PAK activation [PMID:38180080], and regulates fast endosomal recycling through intersectin-2 on Rab4-positive endosomes [PMID:32737221]. In vivo, RhoU maintains epithelial architecture of foregut endoderm, guides neural crest and cardiac development, and restrains intestinal hyperplasia by enabling actomyosin-dependent apoptosis [PMID:21156169, PMID:21903671, PMID:24607366, PMID:30834544].","teleology":[{"year":2001,"claim":"Established RHOU as a Wnt-responsive Cdc42-like GTPase, defining its place as a signaling node linking Wnt to cytoskeletal and proliferative outputs.","evidence":"PAK-1/JNK-1 activation, morphology, cell cycle, and transformation assays in mouse mammary epithelial cells","pmids":["11459829"],"confidence":"Medium","gaps":["Did not resolve the unusual biochemistry distinguishing RhoU from Cdc42","Direct effector partners not yet mapped"]},{"year":2004,"claim":"Resolved why RhoU behaves as a constitutively active GTPase—fast intrinsic exchange with no GTPase activity—and showed the N-terminal extension autoinhibits effector binding until relieved by SH3 adaptors.","evidence":"In vitro nucleotide exchange/GTPase assays, N-terminal truncation, and co-IP with Grb2, PAK1, and NCKβ (two companion studies)","pmids":["15556869","15350535"],"confidence":"High","gaps":["Physiological trigger for relieving N-terminal autoinhibition in vivo not defined","Did not address membrane targeting"]},{"year":2005,"claim":"Identified palmitoylation, not prenylation, as the membrane-anchoring modification, an atypical feature for a Rho GTPase that governs localization and transforming activity.","evidence":"Metabolic labeling, palmitoylation/prenylation inhibitors, CCFV mutagenesis, fractionation, transformation assays","pmids":["16046391"],"confidence":"High","gaps":["Palmitoyltransferase responsible not identified","Dynamics of palmitoylation/depalmitoylation cycling unknown"]},{"year":2007,"claim":"Linked RhoU to focal adhesion turnover and migration, and identified Pyk2 as a GTP-dependent, Src-requiring effector for filopodium formation.","evidence":"Imaging, RNAi, domain mutants, migration assays, MLC phosphorylation, and Pyk2 co-IP across three studies","pmids":["17620058","17504809","18086875"],"confidence":"Medium","gaps":["Quantitative hierarchy of PAK vs Pyk2 vs MLC outputs not established","Direct vs indirect control of MLC phosphorylation unresolved"]},{"year":2008,"claim":"Demonstrated GTP-dependent Par6 binding as the effector route by which RhoU controls tight-junction assembly and epithelial morphogenesis, and showed integrin β3 binding modulates osteoclast adhesion.","evidence":"GTP-dependent co-IP, effector-domain mutants, 3D cystogenesis, and osteoclast adhesion/migration assays","pmids":["19064640","19135548"],"confidence":"High","gaps":["How Par6 binding integrates with the Par polarity complex downstream of RhoU not detailed","Structural basis of effector selection unknown"]},{"year":2009,"claim":"Defined the upstream transcriptional control of RhoU by non-canonical Wnt/JNK and gp130/STAT3 pathways, explaining how the constitutively active GTPase is regulated at the expression level.","evidence":"Reporter assays, promoter mapping of STAT3 sites, pathway inhibitors, and β-catenin loss-of-function","pmids":["19397496"],"confidence":"Medium","gaps":["Human promoter regulation not directly tested","Quantitative contribution of each pathway in physiological contexts unknown"]},{"year":2010,"claim":"Identified Src phosphorylation of Y254 as a negative regulatory switch that relocalizes RhoU to endosomes and unloads GTP, providing post-translational control of an otherwise constitutively active enzyme.","evidence":"Y254F/Y254E mutagenesis, Src inhibition, fractionation/imaging, GTP-loading and PAK co-IP, 3D cystogenesis","pmids":["20547754"],"confidence":"High","gaps":["How endosomal relocalization mechanistically unloads GTP not defined","Phosphatase reversing Y254 not identified"]},{"year":2011,"claim":"Mapped the receptor-coupling mechanism: Grb2/Nck bind RhoU PxxP motifs with high avidity to link activated EGFR on endosomes to RhoU GTP loading, AP-1 activity, and migration, and extended RhoU's role into neural crest and endoderm development.","evidence":"Co-IP, GRB2 RNAi, proline-rich mutagenesis, GTP-loading and AP-1 assays; Xenopus and ES-cell embryo loss/gain of function; ITC binding measurements","pmids":["21508312","23183748","21156169","21903671"],"confidence":"High","gaps":["Whether GTP loading is driven by adaptor-mediated localization or a GEF remains unresolved","Selectivity for Grb2/Nck over other SH3 adaptors mechanistically explained only by avidity"]},{"year":2011,"claim":"Identified candidate GAP-family interactors (ARHGAP30, CdGAP) potentially shaping RhoU-driven cytoskeletal output.","evidence":"Binding-partner screen, co-IP/pull-down, overexpression morphology","pmids":["21565175"],"confidence":"Low","gaps":["Single co-IP/pulldown screen without reciprocal validation or GAP-activity demonstration on RhoU","Functional consequence on RhoU nucleotide state untested"]},{"year":2012,"claim":"Placed RhoU downstream of NOTCH1 in controlling leukemic cell adhesion and migration, broadening its upstream regulatory inputs.","evidence":"γ-secretase inhibition, Notch1 and RhoU RNAi, active Notch1 expression, adhesion/migration/chemotaxis assays in T-ALL","pmids":["22349824"],"confidence":"Medium","gaps":["Whether NOTCH1 acts transcriptionally on RhoU not directly shown","Effectors mediating the T-ALL phenotype not defined"]},{"year":2014,"claim":"Demonstrated an in vivo developmental requirement for RhoU acting through Arhgef7b/PAK to control cardiomyocyte junctions and cardiac looping.","evidence":"Zebrafish morpholino, chemical genetic screen, PAK overexpression rescue, epistasis","pmids":["24607366"],"confidence":"Medium","gaps":["Direct biochemical link between RhoU and Arhgef7b not established","Cell-type-specific effectors not resolved"]},{"year":2015,"claim":"Revealed proteostatic control of RhoU: PAK4 stabilizes RhoU kinase-independently against Rab40A-Cullin5-mediated ubiquitination, coupling RhoU abundance to adhesion turnover.","evidence":"Ubiquitination assays, Rab40A-Cullin5 and PAK4 co-IPs, PAK4 depletion/kinase-dead, RhoU rescue, adhesion dynamics","pmids":["26598620"],"confidence":"High","gaps":["Structural basis of PAK4 protection vs Rab40A recognition unknown","Signals controlling Rab40A-Cullin5 activity toward RhoU undefined"]},{"year":2019,"claim":"Connected RhoU activity to actomyosin-dependent apoptosis in vivo, showing RhoU loss causes intestinal hyperplasia via elevated RhoA and pMLC-2 and reduced apoptosis.","evidence":"Conditional Rhou knockout mice and DLD-1 RNAi with RhoA activity, pMLC-2, TUNEL, and BrdU readouts","pmids":["30834544"],"confidence":"Medium","gaps":["Mechanism by which RhoU restrains RhoA not defined","Direct apoptotic effector pathway unidentified"]},{"year":2020,"claim":"Assigned RhoU a role in endosomal fast recycling through PxxP-mediated binding to intersectin-2 on Rab4-positive endosomes.","evidence":"Co-IP, PxxP mutagenesis, transferrin recycling assay, siRNA, Rab4 co-localization","pmids":["32737221"],"confidence":"Medium","gaps":["Whether GTP state regulates ITSN2 binding not tested","Cargo selectivity of RhoU-dependent recycling unknown"]},{"year":2024,"claim":"Identified C-terminal palmitoylation-dependent homo-oligomerization as a requirement for full RhoU activity, and showed deSUMOylated annexin A6 releases RhoU to enhance AKT signaling and EMT.","evidence":"Co-IP of tagged variants, C-terminal/palmitoylation mutants, PAK activation assay; LC-MS/MS, AnxA6 K579R mutant, p-AKT1 and EMT/migration assays","pmids":["38180080","38566133"],"confidence":"Medium","gaps":["Stoichiometry and structural architecture of the oligomer unresolved","How AnxA6 sequestration intersects with palmitoylation/oligomerization not addressed"]},{"year":null,"claim":"It remains unresolved how the constitutively GTP-bound RhoU is spatially and temporally switched between its many effectors and locations to produce distinct adhesion, junction, recycling, and apoptotic outputs.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of the autoinhibited vs adaptor-bound state","No definitive GAP or GEF demonstrated to set RhoU nucleotide state in cells","Integration of palmitoylation, Y254 phosphorylation, oligomerization, and degradation into a single regulatory logic untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003924","term_label":"GTPase activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[0,13]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[10,18]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[4,5]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[3,10]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[10,13,20]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,9,13]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[11,12,17]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[20]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[19]}],"complexes":[],"partners":["GRB2","NCK1","PAK1","PAK4","PYK2","PARD6","ITSN2","RAB40A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q7L0Q8","full_name":"Rho-related GTP-binding protein RhoU","aliases":["CDC42-like GTPase 1","GTP-binding protein-like 1","Rho GTPase-like protein ARHU","Ryu GTPase","Wnt-1 responsive Cdc42 homolog 1","WRCH-1"],"length_aa":258,"mass_kda":28.2,"function":"Binds to and activates protein kinase PAK1 (PubMed:11459829). Plays a role in the regulation of cell morphology, cytoskeletal organization and focal adhesion assembly during cell migration (PubMed:11459829, PubMed:17620058, PubMed:18086875, PubMed:21834987). Also stimulates quiescent cells to reenter the cell cycle (PubMed:11459829). Has no detectable GTPase activity but its high intrinsic guanine nucleotide exchange activity suggests it is constitutively GTP-bound (PubMed:16472646)","subcellular_location":"Cell membrane; Golgi apparatus membrane; Cell junction, focal adhesion; Cell projection, podosome","url":"https://www.uniprot.org/uniprotkb/Q7L0Q8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RHOU","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RHOU","total_profiled":1310},"omim":[{"mim_id":"614264","title":"RHO GTPase-ACTIVATING PROTEIN 30; ARHGAP30","url":"https://www.omim.org/entry/614264"},{"mim_id":"613058","title":"BASAL CELL CARCINOMA, SUSCEPTIBILITY TO, 2; BCC2","url":"https://www.omim.org/entry/613058"},{"mim_id":"606366","title":"RAS HOMOLOG GENE FAMILY, MEMBER U; RHOU","url":"https://www.omim.org/entry/606366"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Uncertain","locations":[{"location":"Microtubules","reliability":"Uncertain"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"brain","ntpm":67.0}],"url":"https://www.proteinatlas.org/search/RHOU"},"hgnc":{"alias_symbol":["WRCH-1","DJ646B12.2","FLJ10616","WRCH1","CDC42L1","hG28K","fJ646B12.2"],"prev_symbol":["ARHU"]},"alphafold":{"accession":"Q7L0Q8","domains":[{"cath_id":"3.40.50.300","chopping":"48-229","consensus_level":"high","plddt":94.5408,"start":48,"end":229}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7L0Q8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q7L0Q8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q7L0Q8-F1-predicted_aligned_error_v6.png","plddt_mean":79.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RHOU","jax_strain_url":"https://www.jax.org/strain/search?query=RHOU"},"sequence":{"accession":"Q7L0Q8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q7L0Q8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q7L0Q8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q7L0Q8"}},"corpus_meta":[{"pmid":"11459829","id":"PMC_11459829","title":"Wrch-1, a novel member of the Rho gene family that is regulated by Wnt-1.","date":"2001","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/11459829","citation_count":182,"is_preprint":false},{"pmid":"16046391","id":"PMC_16046391","title":"Transforming activity of the Rho family GTPase, Wrch-1, a Wnt-regulated Cdc42 homolog, is dependent on a novel carboxyl-terminal palmitoylation motif.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16046391","citation_count":69,"is_preprint":false},{"pmid":"15556869","id":"PMC_15556869","title":"Atypical mechanism of regulation of the Wrch-1 Rho family small GTPase.","date":"2004","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/15556869","citation_count":67,"is_preprint":false},{"pmid":"26598620","id":"PMC_26598620","title":"PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion.","date":"2015","source":"The Journal of cell 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Wrch-1 disrupts epithelial cell tight junctions and epithelial morphogenesis.","date":"2008","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19064640","citation_count":43,"is_preprint":false},{"pmid":"17504809","id":"PMC_17504809","title":"The atypical Rho family GTPase Wrch-1 regulates focal adhesion formation and cell migration.","date":"2007","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/17504809","citation_count":43,"is_preprint":false},{"pmid":"21903671","id":"PMC_21903671","title":"Rhou maintains the epithelial architecture and facilitates differentiation of the foregut endoderm.","date":"2011","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/21903671","citation_count":42,"is_preprint":false},{"pmid":"22349824","id":"PMC_22349824","title":"Analysis of Rho GTPase expression in T-ALL identifies RhoU as a target for Notch involved in T-ALL cell migration.","date":"2012","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/22349824","citation_count":36,"is_preprint":false},{"pmid":"36221055","id":"PMC_36221055","title":"LncRNA SNHG16 promotes development of oesophageal squamous cell carcinoma by interacting with EIF4A3 and modulating RhoU mRNA stability.","date":"2022","source":"Cellular & molecular biology letters","url":"https://pubmed.ncbi.nlm.nih.gov/36221055","citation_count":34,"is_preprint":false},{"pmid":"21508312","id":"PMC_21508312","title":"GRB2 couples RhoU to epidermal growth factor receptor signaling and cell migration.","date":"2011","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/21508312","citation_count":32,"is_preprint":false},{"pmid":"20547754","id":"PMC_20547754","title":"Regulation of the Rho family small GTPase Wrch-1/RhoU by C-terminal tyrosine phosphorylation requires Src.","date":"2010","source":"Molecular and cellular 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chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23183748","citation_count":13,"is_preprint":false},{"pmid":"38566133","id":"PMC_38566133","title":"SUMOylation of annexin A6 retards cell migration and tumor growth by suppressing RHOU/AKT1-involved EMT in hepatocellular carcinoma.","date":"2024","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/38566133","citation_count":12,"is_preprint":false},{"pmid":"30834544","id":"PMC_30834544","title":"The atypical RhoU/Wrch1 Rho GTPase controls cell proliferation and apoptosis in the gut epithelium.","date":"2019","source":"Biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/30834544","citation_count":12,"is_preprint":false},{"pmid":"22545228","id":"PMC_22545228","title":"Atypical RhoV and RhoU GTPases control development of the neural crest.","date":"2011","source":"Small 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Cdc42 homolog that activates PAK-1 and JNK-1, induces filopodium formation and stress fiber dissolution, stimulates cell cycle re-entry, and morphologically phenocopies Wnt-1 in transformation of mouse mammary epithelial cells.\",\n      \"method\": \"Functional assays in cell lines: PAK-1/JNK-1 activation assays, morphological analysis, cell cycle analysis, transformation assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal functional assays in a single foundational study; single lab\",\n      \"pmids\": [\"11459829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Wrch-1 (RHOU) possesses an extremely rapid intrinsic guanine nucleotide exchange activity (unlike Cdc42) and essentially no GTPase activity, rendering it constitutively GTP-bound. The unique N-terminal extension negatively regulates PAK interaction and transformation, and associates with the Grb2 SH3 domain adaptor protein, which overcomes N-terminal inhibition to promote effector interaction.\",\n      \"method\": \"In vitro biochemical nucleotide exchange and GTPase assays; co-immunoprecipitation with Grb2; N-terminal truncation mutant analysis; transformation assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical reconstitution of exchange/GTPase activity combined with mutagenesis and binding assays; replicated in companion paper (PMID:15350535)\",\n      \"pmids\": [\"15556869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Wrch1 (RHOU) has no detectable GTPase activity in vitro and very high intrinsic nucleotide exchange rate. It interacts with PAK1 and NCKβ; the NCKβ interaction is mediated via PxxP motifs in the N-terminal extension binding to the second and third SH3 domains of NCKβ.\",\n      \"method\": \"In vitro GTPase activity assay; pull-down and co-immunoprecipitation identifying PAK1 and NCKβ as binding partners; mapping of interaction to PxxP motifs\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical assay for GTPase activity plus binding partner identification with domain mapping; corroborates PMID:15556869\",\n      \"pmids\": [\"15350535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Wrch-1 (RHOU) is modified by palmitoylation rather than prenylation, with membrane localization dependent on the second cysteine of the C-terminal CCFV motif. Mutation of this cysteine (C→S) abrogated membrane localization and anchorage-independent transformation. Inhibitors of palmitoylation caused mislocalization, while prenylation inhibitors had no effect.\",\n      \"method\": \"Metabolic labeling with palmitate and isoprenoids; pharmacological inhibition of palmitoylation/prenylation; site-directed mutagenesis of CCFV motif; subcellular fractionation; transformation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — biochemical labeling plus mutagenesis plus functional transformation assay; multiple orthogonal methods in one study\",\n      \"pmids\": [\"16046391\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RhoU/Wrch-1 localizes to focal adhesions via its C-terminal extension and effector binding loop (N-terminal extension and palmitoylation site dispensable for FA targeting). Activated RhoU reduces focal adhesion number and redistributes them; RhoU silencing increases focal adhesion number. RhoU also localizes to podosomes in osteoclasts and Src-expressing cells. RhoU transiently associated with adhesion structures promotes adhesion turnover and increases cell migration.\",\n      \"method\": \"Fluorescence microscopy; RNAi knockdown; expression of deletion/point mutants; cell migration assays\",\n      \"journal\": \"Biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization by imaging with functional domain mapping and siRNA knockdown phenotype; single lab\",\n      \"pmids\": [\"17620058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Wrch-1 (RHOU) depletion by siRNA increases focal adhesion formation, inhibits myosin light chain phosphorylation, and inhibits cell migration. Wrch-1 depletion also inhibits Akt and JNK activation. These results place Wrch-1 upstream of myosin light chain phosphorylation and Akt/JNK pathways controlling focal adhesion dynamics and migration.\",\n      \"method\": \"siRNA knockdown; myosin light chain phosphorylation assay; focal adhesion quantification; wound healing migration assay; pharmacological inhibitors of Akt and JNK\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean siRNA knockdown with multiple defined readouts; single lab\",\n      \"pmids\": [\"17504809\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Wrch1 (RHOU) binds to the nonreceptor tyrosine kinase Pyk2 in a GTP-dependent manner requiring both the N-terminal proline-rich extension and intact effector loop. Pyk2 is required for Wrch1-induced filopodium formation. Src activity is required for formation of the Wrch1-Pyk2 complex and for Wrch1-induced filopodia.\",\n      \"method\": \"Co-immunoprecipitation; GTP-loading experiments with constitutively active/dominant negative mutants; siRNA knockdown of Pyk2; Src inhibitor treatment; morphological analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction mapping with GTP-dependence and functional rescue; single lab\",\n      \"pmids\": [\"18086875\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Wrch1/RhoU binds integrin β3 cytoplasmic domain and interferes with adhesion-induced Pyk2 and paxillin phosphorylation. Wrch1 expression increases osteoclast precursor aggregation, reduces adhesion onto vitronectin (but not fibronectin), and inhibits M-CSF-induced prefusion osteoclast migration. High Wrch1 activity inhibits podosome belt formation in mature osteoclasts.\",\n      \"method\": \"Co-immunoprecipitation (Wrch1-integrin β3); phosphorylation assays (Pyk2, paxillin); adhesion assays; migration assays; RNAi knockdown; osteoclast differentiation assays\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay plus downstream phosphorylation readouts and functional cell-based assays; single lab\",\n      \"pmids\": [\"19135548\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Activated Wrch-1 (RHOU) binds the cell polarity protein Par6 in a GTP-dependent manner. Activated Wrch-1 negatively regulates tight junction assembly kinetics and disrupts epithelial cystogenesis in 3D culture. A Wrch-1 effector domain mutant that inhibits Par6 binding abrogates tight junction disruption, actin reorganization, and morphogenesis defects, placing Par6 binding as necessary for these effects.\",\n      \"method\": \"Co-immunoprecipitation (GTP-dependent Par6 binding); tight junction assembly assays; 3D cystogenesis assay; effector domain mutant analysis; shRNA knockdown\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — GTP-dependent binding assay plus effector mutant causally linking Par6 interaction to tight junction and morphogenesis phenotypes, with multiple orthogonal readouts\",\n      \"pmids\": [\"19064640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RhoU transcription is induced by Wnt-1 at the transcriptional level via the non-canonical Wnt/planar cell polarity pathway through JNK activation (independent of β-catenin). RhoU is also transcriptionally induced by gp130 cytokines via STAT3, with two functional STAT3-binding sites identified on the mouse RhoU promoter.\",\n      \"method\": \"Reporter assays; promoter deletion/mutation analysis; ChIP or EMSA for STAT3 binding sites; pathway inhibitor experiments; β-catenin loss-of-function\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter mapping with functional STAT3 binding sites plus pathway dissection using genetic tools; single lab\",\n      \"pmids\": [\"19397496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Wrch-1 (RHOU) is phosphorylated by Src at C-terminal residue Y254. This phosphorylation causes rapid relocalization from plasma membrane to endosomes upon serum stimulation. Y254 phosphorylation decreases active (GTP-bound) Wrch-1, reduces PAK recruitment and activation, and is required for proper cystogenesis in 3D culture. Phospho-deficient Y254F remains plasma membrane-localized and GTP-bound, sustaining PAK activation.\",\n      \"method\": \"Site-directed mutagenesis (Y254F, Y254E); Src genetic/pharmacological inhibition; subcellular fractionation and imaging; GTP-loading assay; PAK co-immunoprecipitation; 3D cystogenesis assay; anchorage-independent growth assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis identifying specific phosphorylation site plus biochemical GTP/PAK assays plus multiple functional readouts; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"20547754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RhoU activates pathways cooperating with PAK1 and Rac1 in epithelial adhesion, cell spreading, and directional cell migration in cranial neural crest (CNC) cells. Loss or gain of RhoU function in Xenopus impairs CNC cell migration and subsequent craniofacial cartilage differentiation.\",\n      \"method\": \"Gain- and loss-of-function experiments in Xenopus embryos; in vitro cell migration, spreading, and adhesion assays; epistasis with PAK1 and Rac1\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo loss/gain-of-function with in vitro mechanistic follow-up; single lab\",\n      \"pmids\": [\"21156169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Rhou (RHOU) maintains the epithelial architecture and F-actin cortical organization of foregut endoderm in vivo. Rhou-deficient embryos show flattened foregut, loss of microvilli, reduced sub-apical F-actin, impaired endoderm differentiation, and reduced c-Jun/AP-1 target gene expression consistent with impaired JNK activity.\",\n      \"method\": \"Rhou knockdown ES cell-derived embryos; embryoid body differentiation; phalloidin staining (F-actin); gene expression analysis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo knockdown with specific structural and molecular readouts; single lab\",\n      \"pmids\": [\"21903671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"GRB2 couples RhoU to EGFR signaling: after EGF stimulation, RhoU co-localizes with EGFR on endosomes and physically associates with activated EGFR via Grb2 through N-terminal proline-rich motifs. GRB2 knockdown or mutation of proline-rich sequences abolishes the EGFR-RhoU interaction and abrogates EGF-stimulated RhoU GTP loading. RhoU in this complex mediates AP-1 transcriptional activity and cell migration in pancreatic cancer cells.\",\n      \"method\": \"Co-immunoprecipitation; GRB2 RNAi; proline-rich motif mutagenesis; GTP-loading assay; AP-1 reporter assay; cell migration assay\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP with mutagenesis and GTP-loading assay; single lab with multiple functional readouts\",\n      \"pmids\": [\"21508312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ARHGAP30 was identified as a Wrch-1 (RHOU)-interacting protein in a binding-partner screen. CdGAP also binds Wrch-1. Ectopic expression of ARHGAP30 results in membrane blebbing and dissolution of stress fibers and focal adhesions downstream of Wrch-1.\",\n      \"method\": \"Binding-partner screen; co-immunoprecipitation/pull-down; overexpression morphological analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single co-IP/pulldown screen with morphological readout; single lab, limited mechanistic follow-up\",\n      \"pmids\": [\"21565175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"NOTCH1 signaling upregulates RhoU expression in T-ALL cells, and Notch1 or RhoU depletion inhibits T-ALL cell adhesion, migration, and chemotaxis, placing RhoU downstream of NOTCH1 in regulating T-ALL cell migration.\",\n      \"method\": \"γ-secretase inhibitor treatment; Notch1 RNAi; constitutively active Notch1 expression; RhoU RNAi; adhesion, migration, and chemotaxis assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (Notch1→RhoU) with multiple functional readouts; single lab\",\n      \"pmids\": [\"22349824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The N-terminal extension of Wrch1/RhoU contains a central PxxP motif with an essential arginine that mediates high-avidity interactions with full-length Grb2 and Nck1 (but not Crk, c-Src, or p120) in cells, and in vitro. Individual SH3 domains of these adaptors bind with low affinity, but the multivalent full-length proteins achieve tight binding.\",\n      \"method\": \"Sedimentation assays; isothermal titration calorimetry (ITC); co-immunoprecipitation; peptide competition analysis\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — ITC quantitative binding measurements plus sedimentation assays and cell-based co-IP; single lab but rigorous biophysical methods\",\n      \"pmids\": [\"23183748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"RhoU regulates cell junctions between cardiomyocytes through the Arhgef7b/PAK kinase pathway to guide atrioventricular canal development and cardiac looping in zebrafish. Loss of RhoU recapitulates cardiac defects seen with ROCK inhibition, and PAK kinase overexpression rescues the RhoU loss-of-function cardiac defect.\",\n      \"method\": \"Zebrafish loss-of-function (morpholino); chemical genetic screen; PAK overexpression rescue; epistasis with Arhgef7b/PAK pathway\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic epistasis with rescue experiment in zebrafish; single lab\",\n      \"pmids\": [\"24607366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PAK4 protects RhoU from ubiquitination and proteasomal degradation in a kinase-independent manner. RhoU is targeted for ubiquitination by the Rab40A-Cullin 5 E3 ubiquitin ligase complex. PAK4 depletion leads to concomitant loss of RhoU protein; overexpression of RhoU rescues the PAK4 depletion adhesion turnover phenotype. RhoU and PAK4 together drive adhesion turnover and cell migration.\",\n      \"method\": \"Ubiquitination assays; co-immunoprecipitation (Rab40A-Cullin 5 with RhoU; PAK4 with RhoU); PAK4 depletion/kinase-dead mutants; RhoU rescue overexpression; adhesion dynamics assays\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ubiquitination assay identifying E3 complex, co-IP identifying protector mechanism, rescue experiment; multiple orthogonal methods\",\n      \"pmids\": [\"26598620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RhoU loss-of-function in mouse gut epithelium or DLD-1 cells causes hyperplasia through reduced apoptosis and increased proliferation, associated with increased RhoA activity and elevated phosphorylated Myosin Light Chain-2, linking RhoU activity to actomyosin-dependent apoptosis control.\",\n      \"method\": \"Conditional Rhou knockout mice; RNAi in DLD-1 cells; RhoA activity assay; pMLC-2 western blot; TUNEL apoptosis assay; BrdU proliferation assay\",\n      \"journal\": \"Biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo conditional knockout with mechanistic molecular readouts (RhoA activity, pMLC-2); single lab\",\n      \"pmids\": [\"30834544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"RhoU interacts with intersectin-1 and intersectin-2 (ITSN1, ITSN2) via the second PxxP motif in its N-terminus binding to ITSN SH3 domains. Silencing of RhoU or ITSN2 (but not ITSN1) increases transferrin accumulation in early endosomes due to a defect in fast vesicle recycling. RhoU and ITSN2 co-localize on Rab4-positive fast recycling endosomes.\",\n      \"method\": \"Co-immunoprecipitation; PxxP motif mutagenesis; fluorescent transferrin uptake/recycling assay; siRNA knockdown; co-localization imaging with Rab4 marker\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — binding partner mapping with mutagenesis plus functional trafficking assay and co-localization; single lab\",\n      \"pmids\": [\"32737221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RhoU forms homo-oligomers (homodimers) in cells, mediated by the C-terminal extension; C-terminal palmitoylation is required for self-association. Expression of the isolated C-terminal extension acts as a dominant negative, reducing RhoU-induced PAK activation and causing morphological changes consistent with RhoU inhibition. Self-association is required for full RhoU activity.\",\n      \"method\": \"Co-immunoprecipitation of tagged RhoU variants; C-terminal extension deletion/expression; palmitoylation-deficient mutants; PAK activation assay; cell morphology analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with mutagenesis plus functional PAK activation readout; single lab\",\n      \"pmids\": [\"38180080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SUMOylated annexin A6 (AnxA6) binds RhoU; when AnxA6 is deSUMOylated (by SENP1 or K579R mutation), the AnxA6-RhoU interaction is lost, leading to increased RHOU-mediated p-AKT1(Ser473) and facilitation of EMT and cell migration in hepatocellular carcinoma.\",\n      \"method\": \"LC-MS/MS identification of SUMOylation sites; site-directed mutagenesis (K579R); co-immunoprecipitation (AnxA6–RhoU); western blot for p-AKT1; EMT and migration assays\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP with mutagenesis and downstream signaling readout; single lab\",\n      \"pmids\": [\"38566133\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RHOU/Wrch-1 is an atypical Rho GTPase with constitutively high intrinsic GDP/GTP exchange activity and negligible GTPase activity, rendering it constitutively GTP-bound; it associates with the plasma membrane and endosomes via C-terminal palmitoylation (not prenylation), is transcriptionally induced by non-canonical Wnt/JNK and STAT3 pathways, is negatively regulated by Src-mediated phosphorylation at Y254 causing endosomal relocalization and GTP unloading, and is stabilized against ubiquitin-mediated (Rab40A-Cullin5) degradation by PAK4; its N-terminal PxxP motifs recruit SH3 adaptor proteins (Grb2, Nck) to couple growth factor receptor (EGFR) signaling to RhoU GTP loading, while its GTP-dependent effectors include PAK1/PAK4, Pyk2, and Par6, through which it regulates focal adhesion turnover, tight junction assembly, filopodium formation, cell migration, endosomal recycling (via ITSN2 on Rab4-positive endosomes), and actomyosin-dependent apoptosis; RhoU also forms C-terminal-dependent homo-oligomers required for full PAK activation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RHOU (Wrch-1) is an atypical Rho-family GTPase that couples non-canonical Wnt and cytokine signaling to actin-cytoskeleton remodeling, cell adhesion, and migration [#0, #9]. Biochemically it is distinguished from Cdc42 by an extremely rapid intrinsic guanine-nucleotide exchange rate and negligible GTPase activity, leaving it constitutively GTP-bound, while its N-terminal extension autoinhibits effector engagement until relieved by SH3-adaptor binding [#1, #2]. Membrane targeting is achieved through C-terminal palmitoylation rather than prenylation, and the conserved CCFV cysteine is required for membrane localization and transformation [#3]. Its N-terminal proline-rich (PxxP) motifs recruit the SH3 adaptors Grb2 and Nck through high-avidity multivalent binding, linking activated EGFR on endosomes to RhoU GTP loading and downstream AP-1 activity and migration [#13, #16]. In its GTP-bound state RhoU engages effectors including PAK1/PAK4, Pyk2 and the polarity protein Par6 to control filopodium formation, focal adhesion turnover, tight-junction assembly and epithelial morphogenesis [#6, #8, #10]. RhoU activity is regulated post-translationally: Src phosphorylation at Y254 relocalizes it from the plasma membrane to endosomes, unloads GTP and dampens PAK activation [#10], whereas PAK4 stabilizes RhoU against Rab40A-Cullin5-mediated ubiquitination and degradation [#18]. RhoU additionally forms C-terminal palmitoylation-dependent homo-oligomers needed for full PAK activation [#21], and regulates fast endosomal recycling through intersectin-2 on Rab4-positive endosomes [#20]. In vivo, RhoU maintains epithelial architecture of foregut endoderm, guides neural crest and cardiac development, and restrains intestinal hyperplasia by enabling actomyosin-dependent apoptosis [#11, #12, #17, #19].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established RHOU as a Wnt-responsive Cdc42-like GTPase, defining its place as a signaling node linking Wnt to cytoskeletal and proliferative outputs.\",\n      \"evidence\": \"PAK-1/JNK-1 activation, morphology, cell cycle, and transformation assays in mouse mammary epithelial cells\",\n      \"pmids\": [\"11459829\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not resolve the unusual biochemistry distinguishing RhoU from Cdc42\", \"Direct effector partners not yet mapped\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Resolved why RhoU behaves as a constitutively active GTPase—fast intrinsic exchange with no GTPase activity—and showed the N-terminal extension autoinhibits effector binding until relieved by SH3 adaptors.\",\n      \"evidence\": \"In vitro nucleotide exchange/GTPase assays, N-terminal truncation, and co-IP with Grb2, PAK1, and NCKβ (two companion studies)\",\n      \"pmids\": [\"15556869\", \"15350535\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological trigger for relieving N-terminal autoinhibition in vivo not defined\", \"Did not address membrane targeting\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified palmitoylation, not prenylation, as the membrane-anchoring modification, an atypical feature for a Rho GTPase that governs localization and transforming activity.\",\n      \"evidence\": \"Metabolic labeling, palmitoylation/prenylation inhibitors, CCFV mutagenesis, fractionation, transformation assays\",\n      \"pmids\": [\"16046391\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Palmitoyltransferase responsible not identified\", \"Dynamics of palmitoylation/depalmitoylation cycling unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Linked RhoU to focal adhesion turnover and migration, and identified Pyk2 as a GTP-dependent, Src-requiring effector for filopodium formation.\",\n      \"evidence\": \"Imaging, RNAi, domain mutants, migration assays, MLC phosphorylation, and Pyk2 co-IP across three studies\",\n      \"pmids\": [\"17620058\", \"17504809\", \"18086875\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative hierarchy of PAK vs Pyk2 vs MLC outputs not established\", \"Direct vs indirect control of MLC phosphorylation unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated GTP-dependent Par6 binding as the effector route by which RhoU controls tight-junction assembly and epithelial morphogenesis, and showed integrin β3 binding modulates osteoclast adhesion.\",\n      \"evidence\": \"GTP-dependent co-IP, effector-domain mutants, 3D cystogenesis, and osteoclast adhesion/migration assays\",\n      \"pmids\": [\"19064640\", \"19135548\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Par6 binding integrates with the Par polarity complex downstream of RhoU not detailed\", \"Structural basis of effector selection unknown\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defined the upstream transcriptional control of RhoU by non-canonical Wnt/JNK and gp130/STAT3 pathways, explaining how the constitutively active GTPase is regulated at the expression level.\",\n      \"evidence\": \"Reporter assays, promoter mapping of STAT3 sites, pathway inhibitors, and β-catenin loss-of-function\",\n      \"pmids\": [\"19397496\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Human promoter regulation not directly tested\", \"Quantitative contribution of each pathway in physiological contexts unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified Src phosphorylation of Y254 as a negative regulatory switch that relocalizes RhoU to endosomes and unloads GTP, providing post-translational control of an otherwise constitutively active enzyme.\",\n      \"evidence\": \"Y254F/Y254E mutagenesis, Src inhibition, fractionation/imaging, GTP-loading and PAK co-IP, 3D cystogenesis\",\n      \"pmids\": [\"20547754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How endosomal relocalization mechanistically unloads GTP not defined\", \"Phosphatase reversing Y254 not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Mapped the receptor-coupling mechanism: Grb2/Nck bind RhoU PxxP motifs with high avidity to link activated EGFR on endosomes to RhoU GTP loading, AP-1 activity, and migration, and extended RhoU's role into neural crest and endoderm development.\",\n      \"evidence\": \"Co-IP, GRB2 RNAi, proline-rich mutagenesis, GTP-loading and AP-1 assays; Xenopus and ES-cell embryo loss/gain of function; ITC binding measurements\",\n      \"pmids\": [\"21508312\", \"23183748\", \"21156169\", \"21903671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether GTP loading is driven by adaptor-mediated localization or a GEF remains unresolved\", \"Selectivity for Grb2/Nck over other SH3 adaptors mechanistically explained only by avidity\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified candidate GAP-family interactors (ARHGAP30, CdGAP) potentially shaping RhoU-driven cytoskeletal output.\",\n      \"evidence\": \"Binding-partner screen, co-IP/pull-down, overexpression morphology\",\n      \"pmids\": [\"21565175\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single co-IP/pulldown screen without reciprocal validation or GAP-activity demonstration on RhoU\", \"Functional consequence on RhoU nucleotide state untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Placed RhoU downstream of NOTCH1 in controlling leukemic cell adhesion and migration, broadening its upstream regulatory inputs.\",\n      \"evidence\": \"γ-secretase inhibition, Notch1 and RhoU RNAi, active Notch1 expression, adhesion/migration/chemotaxis assays in T-ALL\",\n      \"pmids\": [\"22349824\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether NOTCH1 acts transcriptionally on RhoU not directly shown\", \"Effectors mediating the T-ALL phenotype not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrated an in vivo developmental requirement for RhoU acting through Arhgef7b/PAK to control cardiomyocyte junctions and cardiac looping.\",\n      \"evidence\": \"Zebrafish morpholino, chemical genetic screen, PAK overexpression rescue, epistasis\",\n      \"pmids\": [\"24607366\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct biochemical link between RhoU and Arhgef7b not established\", \"Cell-type-specific effectors not resolved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Revealed proteostatic control of RhoU: PAK4 stabilizes RhoU kinase-independently against Rab40A-Cullin5-mediated ubiquitination, coupling RhoU abundance to adhesion turnover.\",\n      \"evidence\": \"Ubiquitination assays, Rab40A-Cullin5 and PAK4 co-IPs, PAK4 depletion/kinase-dead, RhoU rescue, adhesion dynamics\",\n      \"pmids\": [\"26598620\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of PAK4 protection vs Rab40A recognition unknown\", \"Signals controlling Rab40A-Cullin5 activity toward RhoU undefined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected RhoU activity to actomyosin-dependent apoptosis in vivo, showing RhoU loss causes intestinal hyperplasia via elevated RhoA and pMLC-2 and reduced apoptosis.\",\n      \"evidence\": \"Conditional Rhou knockout mice and DLD-1 RNAi with RhoA activity, pMLC-2, TUNEL, and BrdU readouts\",\n      \"pmids\": [\"30834544\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which RhoU restrains RhoA not defined\", \"Direct apoptotic effector pathway unidentified\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Assigned RhoU a role in endosomal fast recycling through PxxP-mediated binding to intersectin-2 on Rab4-positive endosomes.\",\n      \"evidence\": \"Co-IP, PxxP mutagenesis, transferrin recycling assay, siRNA, Rab4 co-localization\",\n      \"pmids\": [\"32737221\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether GTP state regulates ITSN2 binding not tested\", \"Cargo selectivity of RhoU-dependent recycling unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identified C-terminal palmitoylation-dependent homo-oligomerization as a requirement for full RhoU activity, and showed deSUMOylated annexin A6 releases RhoU to enhance AKT signaling and EMT.\",\n      \"evidence\": \"Co-IP of tagged variants, C-terminal/palmitoylation mutants, PAK activation assay; LC-MS/MS, AnxA6 K579R mutant, p-AKT1 and EMT/migration assays\",\n      \"pmids\": [\"38180080\", \"38566133\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and structural architecture of the oligomer unresolved\", \"How AnxA6 sequestration intersects with palmitoylation/oligomerization not addressed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how the constitutively GTP-bound RhoU is spatially and temporally switched between its many effectors and locations to produce distinct adhesion, junction, recycling, and apoptotic outputs.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of the autoinhibited vs adaptor-bound state\", \"No definitive GAP or GEF demonstrated to set RhoU nucleotide state in cells\", \"Integration of palmitoylation, Y254 phosphorylation, oligomerization, and degradation into a single regulatory logic untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003924\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0, 13]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [10, 18]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [3, 10]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [10, 13, 20]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 9, 13]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [11, 12, 17]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [19]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"GRB2\", \"NCK1\", \"PAK1\", \"PAK4\", \"PYK2\", \"PARD6\", \"ITSN2\", \"RAB40A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}