{"gene":"RAP1GAP","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2004,"finding":"Crystal structure of the catalytic domain of Rap1GAP at 2.9 Å resolution revealed that Rap1GAP uses a catalytic asparagine (not the arginine used by other GAPs) to stimulate GTP hydrolysis by Rap1. Mutational analysis, fluorescence titration, and stopped-flow kinetic assays confirmed the catalytic asparagine mechanism.","method":"X-ray crystallography, active-site mutagenesis, fluorescence titration, stopped-flow kinetics","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and two orthogonal kinetic assays in a single rigorous study","pmids":["15141215"],"is_preprint":false},{"year":1992,"finding":"The catalytic domain of Rap1GAP was mapped to amino acids 75–416; the phosphorylation sites by cAMP-dependent kinase (Ser490, Ser499) and p34cdc2 (Ser484) are localized to the C-terminal region outside the catalytic domain.","method":"Deletion mutagenesis of cDNA constructs, purification of truncation mutants, in vitro GAP activity assays, phosphopeptide mapping, point mutagenesis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with purified proteins, multiple deletion mutants, and orthogonal phosphopeptide mapping in one study","pmids":["1406653"],"is_preprint":false},{"year":1992,"finding":"Rap1GAP is phosphorylated in vitro by cAMP-dependent kinase (~3 mol phosphate/mol) and p34cdc2 kinase (~2 mol phosphate/mol) at sites in the C-terminal 100-residue segment; dibutyryl-cAMP treatment of SK-MEL-3 cells promotes Rap1GAP phosphorylation in vivo at sites identical to the in vitro cAMP-dependent kinase sites.","method":"In vitro kinase assays with purified kinases, 32Pi labeling, comparative phosphopeptide mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified proteins replicated by in vivo labeling and phosphopeptide mapping in one study, consistent with PMID:1406653","pmids":["1587853"],"is_preprint":false},{"year":1995,"finding":"Tuberin (TSC2 product) immunoprecipitates contain Rap1GAP-like activity that specifically stimulates intrinsic GTPase of Rap1a but not Rap2, Ha-Ras, Rac, or Rho, establishing tuberin as a Rap1-specific GAP distinct from Rap1GAP.","method":"Immunoprecipitation of native tuberin followed by GTPase activity assay; bacterial and Sf9 cell-expressed C-terminal fragment assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assay on immunoprecipitated endogenous protein plus recombinant fragment, single lab; relevant because it defines Rap1GAP's functional specificity by comparison","pmids":["7608212"],"is_preprint":false},{"year":1999,"finding":"A novel isoform of Rap1GAP, rap1GAPII, binds specifically to Gαi-family α-subunits; stimulation of the Gi-coupled m2-muscarinic receptor translocates rap1GAPII from cytosol to membrane, reduces GTP-bound Rap1, and thereby activates ERK/MAPK.","method":"Co-immunoprecipitation, subcellular fractionation, Rap1-GTP pull-down, ERK activation assays, receptor stimulation experiments","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, fractionation, and functional Rap1-GTP/ERK readouts together in one study; published in Nature with multiple orthogonal methods","pmids":["10476970"],"is_preprint":false},{"year":1999,"finding":"Gαz binds Rap1GAP via the N-terminal 74 amino acids of Rap1GAP (a region distinct from the catalytic domain); this interaction blocks RGS-stimulated GTP hydrolysis of Gαz, attenuates Gαz-mediated adenylyl cyclase inhibition, and allows formation of a stable Gαz–Rap1GAP–Rap1 complex.","method":"Yeast two-hybrid, co-precipitation with purified recombinant proteins, adenylyl cyclase assay, N-terminal deletion analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — yeast two-hybrid plus biochemical reconstitution with purified proteins plus functional adenylyl cyclase assay and domain mapping in one study","pmids":["10593970"],"is_preprint":false},{"year":2005,"finding":"Gαo/i directly interacts with Rap1GAPII and targets it for ubiquitination and proteasomal degradation; this reduces Rap1GAP levels, activates Rap1, and drives CB1 cannabinoid receptor-induced neurite outgrowth in Neuro-2A cells. Proteasomal inhibitor lactacystin blocks Gαo/i-induced Rap1 activation and neurite outgrowth.","method":"Co-immunoprecipitation, ubiquitination assay, proteasomal inhibitor treatment (lactacystin), dominant-negative Rap1, siRNA knockdown, pertussis toxin treatment","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal approaches (Co-IP, ubiquitination assay, pharmacological inhibition, genetic dominant-negative, siRNA) in one study","pmids":["15657046"],"is_preprint":false},{"year":2003,"finding":"GSK3β phosphorylates Rap1GAP (at Ser525 identified by mutagenesis) and promotes its proteasome-mediated degradation; GSK3β inhibitors prevent phosphorylation and degradation of endogenous Rap1GAP. TSH/cAMP signaling stabilizes Rap1GAP, while TSH withdrawal leads to GSK3β-dependent Rap1GAP turnover.","method":"In vitro kinase assay (GSK3β on immunoprecipitated Rap1GAP), pharmacological GSK3β inhibitors, co-expression of GSK3β + Rap1GAP with proteasome inhibitors, point mutagenesis (Ser525)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay plus mutagenesis plus pharmacological rescue, multiple orthogonal methods in one study","pmids":["14660640"],"is_preprint":false},{"year":2009,"finding":"PKA phosphorylates Rap1GAP at Ser-441 and Ser-499 in striatal neurons in response to D1 dopamine receptor activation; this phosphorylation inhibits Rap1GAP GAP activity (increasing Rap1-GTP levels) and is associated with decreased dendritic spine head size.","method":"Mass spectrometry identification of PKA substrate, in vitro phosphorylation, Rap1-GTP pull-down in striatal neurons, D1 receptor activation, phospho-specific analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS-identified substrate, in vitro phosphorylation, and functional Rap1-GTP readout in neuronal system with receptor stimulation","pmids":["19218462"],"is_preprint":false},{"year":1994,"finding":"Rap1GAP is hyperphosphorylated during mitosis; p34cdc2 kinase co-immunoprecipitated from mitotic (but not interphase) HeLa cell lysates phosphorylates wild-type Rap1GAP but not a Ser484 mutant, indicating p34cdc2 phosphorylates Rap1GAP at Ser484 during mitosis. This phosphorylation does not affect GAP catalytic activity toward Rap1.","method":"Cell cycle synchronization, co-immunoprecipitation of cdc2/cyclin B1, in vitro kinase assay with wild-type and Ser484 mutant Rap1GAP, SDS-PAGE mobility shift","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP of endogenous cdc2 plus mutagenesis, single lab, consistent with earlier in vitro data (PMID:1587853)","pmids":["8048970"],"is_preprint":false},{"year":2014,"finding":"PLK1 phosphorylates Ser525 within the 524DSGHVS529 degron of Rap1GAP, promoting its interaction with the β-TrCP ubiquitin ligase complex and subsequent proteasomal degradation during mitosis; PLK1 binds Rap1GAP via recognition of an SSP motif within Rap1GAP.","method":"Co-immunoprecipitation, in vitro kinase assay, ubiquitination assay, proteasome inhibitor treatment, mutagenesis of Ser525 and SSP motif","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro kinase assay plus mutagenesis plus Co-IP and ubiquitination assay, multiple orthogonal methods","pmids":["25329897"],"is_preprint":false},{"year":2010,"finding":"Rap1GAP physically interacts with the RET receptor tyrosine kinase via Tyr981 in RET's intracellular domain; endogenous Rap1GAP co-immunoprecipitates with RET in neural tissues; GDNF treatment enhances RET–Rap1GAP interaction; overexpression of Rap1GAP attenuates GDNF-induced ERK activation and neurite outgrowth, while Rap1GAP knockdown enhances them.","method":"Yeast two-hybrid, co-immunoprecipitation (endogenous proteins in neural tissue), RNAi knockdown, mutagenesis of RET Tyr981, neurite outgrowth assay, ERK phosphorylation assay","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid confirmed by endogenous Co-IP in tissue plus mutagenesis plus functional readouts, multiple orthogonal methods","pmids":["20877310"],"is_preprint":false},{"year":2010,"finding":"Silencing Rap1GAP in human colon carcinoma cells enhances Rap activity, impairs cell–cell adhesion (aberrant distribution of E-cadherin, β-catenin, p120-catenin), and enhances spreading/adhesion on collagen; silencing Rap expression rescues these defects; Src activity is increased in Rap1GAP-depleted cells and Src inhibition restores E-cadherin at cell–cell contacts.","method":"siRNA knockdown, Rap1-GTP pull-down assay, immunofluorescence of adherens junction proteins, Src kinase inhibitor treatment","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (Rap1GAP KD then Rap KD rescue), multiple cell biology readouts, single lab","pmids":["20439492"],"is_preprint":false},{"year":2007,"finding":"Oncogenic Ras downregulates Rap1GAP expression via the Raf/MEK/ERK cascade in rat thyroid cells; restoring Rap1GAP inhibits Rap1 and Rac1 activity and blocks cell migration, invasion, DNA synthesis, and anchorage-independent growth.","method":"Ras transformation, MEK inhibitor treatment, Rap1-GTP/Rac1-GTP pull-down, siRNA knockdown, Rap1GAP re-expression, migration/invasion assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — epistasis via MEK inhibitor plus functional readouts, single lab","pmids":["17646383"],"is_preprint":false},{"year":2013,"finding":"Depletion of Rap1GAP in colon cancer cells enhances migration via increased endogenous Rap activity, suppresses ROCK-mediated contractility, and switches migratory mode to Rac1-dependent mesenchymal motility; Rac1 inhibition restores membrane blebbing and ROCK activity in Rap1GAP-depleted cells.","method":"siRNA knockdown, Rap1-GTP pull-down, ROCK inhibitor, Rac1 inhibitor, live-cell migration tracking, morphological analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis (Rap1GAP KD, then Rap KD, then Rac1 inhibition) with multiple cellular readouts, single lab","pmids":["23864657"],"is_preprint":false},{"year":2014,"finding":"Podocyte-specific inactivation of both Rap1a and Rap1b (the targets of Rap1GAP) causes massive glomerulosclerosis; increased Rap1GAP expression in injured podocytes reduces active β1 integrin, leading to podocyte detachment; preventing RAP1GAP elevation maintains β1 integrin-mediated adhesion and prevents detachment.","method":"Insertional mutagenesis screen, podocyte-specific conditional knockout mice, β1 integrin activation assays, kidney biopsy immunostaining","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional knockout with defined glomerular phenotype plus mechanistic β1 integrin readout, replicated in human biopsies","pmids":["24642466"],"is_preprint":false},{"year":2021,"finding":"Degradation of Rap1GAP in HPV16/18-positive cervical cancer cells is mediated by the E6AP ubiquitin ligase via the proteasome pathway; co-immunoprecipitation showed E6AP binds Rap1GAP in HPV-positive cells; knockdown of E6AP shifts degradation from proteasomal to autophagy-mediated.","method":"Co-immunoprecipitation, MG132 proteasome inhibitor treatment, siRNA knockdown of E6AP, rapamycin-induced autophagy, Western blotting","journal":"Infectious agents and cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus pharmacological and genetic manipulation, single lab","pmids":["34952616"],"is_preprint":false},{"year":2025,"finding":"Gαo-GTP (not GDP) binds and activates Rap1GAP1 via a GoLoco/GPR motif; specific residues in the GoLoco/GPR motif confer differential recognition of Gαo guanine-nucleotide-binding status; GNAO1 encephalopathy mutations in Gαo prevent it from attaining the conformation needed for Rap1GAP1 effector binding.","method":"Proximity-based proteomics (BioID) screen, co-immunoprecipitation, in vitro binding assays with GDP- and GTP-locked Gαo mutants, Rap1GAP1 activity assays, point mutagenesis of GoLoco/GPR motif","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — proteomics screen confirmed by Co-IP plus in vitro binding with nucleotide-state mutants plus GAP activity assay plus mutagenesis, multiple orthogonal methods","pmids":["40615045"],"is_preprint":false},{"year":2022,"finding":"In the subventricular zone niche, ADAM10 cleavage of JAMC increases Rap1GAP activity, which promotes neural stem cell transit from the apical to basal compartment and subsequent lineage progression.","method":"ADAM10 loss-of-function in vivo, NSC positioning assay, molecular pathway analysis linking JAMC processing to Rap1GAP activity","journal":"Neural regeneration research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, indirect pathway placement inferred from ADAM10 KO phenotype with Rap1GAP as downstream effector, limited direct mechanistic data on Rap1GAP itself in the abstract","pmids":["35535899"],"is_preprint":false},{"year":2011,"finding":"EZH2 represses Rap1GAP by facilitating H3K27me3 at the Rap1GAP locus and promoting Rap1GAP promoter hypermethylation; loss of miR-101 leads to EZH2 upregulation and concomitant Rap1GAP downregulation in head and neck cancer.","method":"ChIP for H3K27me3, bisulfite methylation analysis, miR-101 overexpression/inhibition, Western blotting","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and methylation analyses linking EZH2 to Rap1GAP promoter, single lab, two orthogonal epigenetic methods","pmids":["21532618"],"is_preprint":false},{"year":2021,"finding":"Rap1GAP overexpression in cardiomyocytes inhibits the AMPK/AKT/mTOR signaling pathway, exacerbates Ang II-induced cardiomyocyte hypertrophy, increases ROS generation, and inhibits autophagy; conversely, Rap1GAP knockdown activates AMPK/AKT/mTOR and reduces hypertrophy. Co-immunoprecipitation showed exogenous Rap1GAP interacts with AMPK.","method":"siRNA knockdown, adenoviral overexpression, AMPK/AKT/mTOR pathway Western blotting, ROS assay, autophagy markers, co-immunoprecipitation","journal":"Oxidative medicine and cellular longevity","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP plus gain/loss-of-function with multiple downstream readouts, single lab","pmids":["33936386"],"is_preprint":false}],"current_model":"Rap1GAP is a Rap1-specific GTPase-activating protein that uses a catalytic asparagine (rather than the arginine employed by Ras GAPs) to stimulate GTP hydrolysis by Rap1; its catalytic domain spans residues 75–416, while its C-terminal region harbors phosphorylation sites for PKA (Ser490/499, inhibiting GAP activity), p34cdc2/PLK1 (Ser484/525, the latter promoting β-TrCP-dependent proteasomal degradation in mitosis), and GSK3β (Ser525, promoting proteasomal turnover); an N-terminal region (first 74 aa) mediates binding to Gαz and Gαo (via a GoLoco/GPR motif), with Gαo-GTP activating Rap1GAP1 and Gαi/o targeting the rap1GAPII isoform for membrane recruitment or proteasomal degradation to modulate Rap1-GTP levels and downstream ERK/MAPK and integrin signaling; Rap1GAP also physically interacts with the RET receptor kinase and with AMPK, placing it at the nexus of neurotrophin, adhesion, and metabolic signaling."},"narrative":{"mechanistic_narrative":"RAP1GAP is a Rap1-specific GTPase-activating protein that terminates Rap1 signaling and thereby controls cell adhesion, migration, and receptor-driven ERK/MAPK output [PMID:10476970, PMID:20439492]. Its catalytic domain (residues 75–416) stimulates GTP hydrolysis on Rap1 through a non-canonical catalytic asparagine, distinguishing it from the arginine-finger mechanism of Ras GAPs [PMID:15141215, PMID:1406653]. Activity and abundance are tightly regulated by phosphorylation in a C-terminal regulatory region outside the catalytic domain: PKA phosphorylation (downstream of cAMP and D1 dopamine receptor signaling) inhibits GAP activity to raise Rap1-GTP [PMID:1587853, PMID:19218462], while GSK3β (Ser525) and mitotic PLK1 (Ser525, within a β-TrCP degron) and p34cdc2 (Ser484) phosphorylation route the protein toward proteasomal degradation [PMID:14660640, PMID:8048970, PMID:25329897]. An N-terminal region (first 74 aa) binds heterotrimeric Gα subunits, coupling Rap1GAP to receptor signaling: Gαz binding blocks RGS-stimulated GTP hydrolysis and forms a stable Gαz–Rap1GAP–Rap1 complex [PMID:10593970], Gαo-GTP binds and activates Rap1GAP1 through a GoLoco/GPR motif [PMID:40615045], and Gi/o coupling translocates or degrades the rap1GAPII isoform to modulate Rap1 levels and drive ERK activation and neurite outgrowth [PMID:10476970, PMID:15657046]. Through suppression of Rap1, Rap1GAP maintains E-cadherin-based cell–cell adhesion and β1 integrin-mediated attachment, and its loss enhances Src activity, Rac1-dependent migration, and invasion [PMID:20439492, PMID:23864657, PMID:24642466]; consistent with a tumor-suppressive role, it is downregulated by oncogenic Ras/MEK/ERK signaling and by EZH2/miR-101-mediated epigenetic silencing in cancer [PMID:17646383, PMID:21532618]. Rap1GAP also physically interacts with the RET receptor tyrosine kinase to restrain GDNF-induced signaling [PMID:20877310].","teleology":[{"year":1992,"claim":"Establishing the modular architecture of Rap1GAP separated catalysis from regulation, defining where activity is generated versus where it is controlled.","evidence":"Deletion mutagenesis, in vitro GAP assays, and phosphopeptide mapping localizing the catalytic domain to aa 75–416 and PKA/cdc2 sites to the C-terminus","pmids":["1406653","1587853"],"confidence":"High","gaps":["Functional consequence of each phosphorylation on activity not resolved here","No structural basis for catalysis yet"]},{"year":1994,"claim":"Mitotic phosphorylation at Ser484 by p34cdc2 raised the possibility of cell-cycle regulation of Rap1GAP, even though catalytic activity was unchanged.","evidence":"Cell-cycle synchronization with co-IP of cdc2/cyclin B1 and in vitro kinase assay on wild-type vs Ser484 mutant","pmids":["8048970"],"confidence":"Medium","gaps":["No catalytic or abundance effect identified for Ser484 phosphorylation","Downstream functional role left open until later degron work"]},{"year":1999,"claim":"Discovery of Gα subunit binding via the N-terminus and of the rap1GAPII isoform connected Rap1GAP to GPCR signaling and to ERK/MAPK activation.","evidence":"Yeast two-hybrid, co-precipitation, subcellular fractionation, Rap1-GTP pull-down and adenylyl cyclase/ERK readouts for Gαz and Gαi binding","pmids":["10593970","10476970"],"confidence":"High","gaps":["Nucleotide-state dependence of Gα binding not yet defined","How translocation alters local Rap1 pools mechanistically unresolved"]},{"year":2003,"claim":"GSK3β phosphorylation at Ser525 linking Rap1GAP stability to hormonal cAMP signaling showed that abundance, not only activity, is a regulated variable.","evidence":"In vitro kinase assay, GSK3β inhibitors, proteasome inhibitors, Ser525 mutagenesis in TSH-responsive cells","pmids":["14660640"],"confidence":"High","gaps":["Identity of the ubiquitin ligase not defined here","Physiological scope beyond thyroid cells unclear"]},{"year":2004,"claim":"The crystal structure resolved the catalytic mechanism, revealing an asparagine thumb rather than the arginine finger used by Ras GAPs.","evidence":"X-ray crystallography of the catalytic domain with active-site mutagenesis, fluorescence titration, and stopped-flow kinetics","pmids":["15141215"],"confidence":"High","gaps":["No co-structure with Rap1 in the published work","Structure of full-length regulatory regions not determined"]},{"year":2005,"claim":"Gαo/i-driven ubiquitination and degradation of rap1GAPII provided a mechanism for receptor-induced Rap1 activation and neurite outgrowth.","evidence":"Co-IP, ubiquitination assay, lactacystin, dominant-negative Rap1, siRNA, and pertussis toxin in Neuro-2A cells","pmids":["15657046"],"confidence":"High","gaps":["Specific ligase mediating Gαo/i-directed degradation not identified","Relationship to GSK3β-driven turnover unresolved"]},{"year":2009,"claim":"PKA phosphorylation at Ser441/Ser499 inhibiting GAP activity placed Rap1GAP in dopamine-receptor-controlled regulation of dendritic spine morphology.","evidence":"Mass spectrometry, in vitro phosphorylation, and Rap1-GTP pull-down in striatal neurons after D1 receptor activation","pmids":["19218462"],"confidence":"High","gaps":["Direct causal link between Rap1-GTP and spine size not fully dissected","Interplay with degradative phosphosites unaddressed"]},{"year":2010,"claim":"RET binding and adhesion-junction control defined Rap1GAP as a brake on receptor tyrosine kinase signaling and a maintainer of epithelial integrity.","evidence":"Yeast two-hybrid and endogenous Co-IP with RET (Tyr981), plus siRNA, immunofluorescence of junction proteins, and Src inhibition in colon carcinoma cells","pmids":["20877310","20439492"],"confidence":"Medium","gaps":["Whether RET binding directly alters GAP activity not established","Mechanism connecting Rap to Src activation incompletely defined"]},{"year":2013,"claim":"Rap1GAP loss was shown to switch migratory mode by suppressing ROCK contractility and promoting Rac1-dependent mesenchymal motility.","evidence":"siRNA, Rap1-GTP pull-down, ROCK and Rac1 inhibitors, and live-cell migration tracking","pmids":["23864657"],"confidence":"Medium","gaps":["Direct effectors linking Rap to ROCK/Rac1 not identified","In vivo relevance of mode-switching not tested"]},{"year":2014,"claim":"Mitotic PLK1 phosphorylation within a β-TrCP degron and in vivo podocyte studies established degron-driven turnover and the physiological consequence of Rap1GAP excess on integrin-mediated adhesion.","evidence":"In vitro kinase, ubiquitination, and Co-IP for PLK1/β-TrCP; podocyte-specific conditional knockouts and β1 integrin assays with human biopsy validation","pmids":["25329897","24642466"],"confidence":"High","gaps":["Coordination of PLK1, GSK3β, and cdc2 phosphorylation across the cycle unresolved","How Rap1GAP elevation lowers active β1 integrin mechanistically incomplete"]},{"year":2021,"claim":"Identification of E6AP-mediated degradation and an AMPK interaction extended Rap1GAP regulation into viral oncogenesis and cardiomyocyte metabolic/autophagy control.","evidence":"Co-IP, MG132, E6AP siRNA in HPV+ cervical cancer; gain/loss-of-function with AMPK/AKT/mTOR Western blots, ROS, autophagy markers, and AMPK Co-IP in cardiomyocytes","pmids":["34952616","33936386"],"confidence":"Medium","gaps":["Direct vs indirect nature of AMPK interaction not resolved","Whether E6AP recognizes a defined Rap1GAP degron unknown"]},{"year":2025,"claim":"Resolving that Gαo-GTP, not GDP, binds and activates Rap1GAP1 via a GoLoco/GPR motif tied the regulator to GNAO1 encephalopathy through conformation-dependent effector recognition.","evidence":"BioID proteomics, Co-IP, in vitro binding with nucleotide-locked Gαo mutants, GAP activity assays, and GoLoco/GPR motif mutagenesis","pmids":["40615045"],"confidence":"High","gaps":["Downstream Rap1-dependent consequences in neurons not mapped","Relationship to the earlier Gαz/Gαi binding modes not reconciled"]},{"year":null,"claim":"How the many regulatory inputs—Gα nucleotide-state binding, multiple inhibitory and degradative phosphorylations, and distinct E3 ligases—are integrated to set Rap1-GTP levels in a given cell type remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unified model of competing phosphorylation and degradation pathways","Cell-type-specific isoform usage (Rap1GAP1 vs rap1GAPII) not systematically mapped","Structural basis of Gα–Rap1GAP–Rap1 ternary complex undetermined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,4,12]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[4,6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,5,6,8,17]},{"term_id":"R-HSA-1474244","term_label":"Extracellular matrix organization","supporting_discovery_ids":[12,15]}],"complexes":[],"partners":["RAP1A","GNAZ","GNAO1","RET","PLK1","BTRC","GSK3B","PRKAA1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P47736","full_name":"Rap1 GTPase-activating protein 1","aliases":[],"length_aa":663,"mass_kda":73.4,"function":"GTPase activator for the nuclear Ras-related regulatory protein RAP-1A (KREV-1), converting it to the putatively inactive GDP-bound state","subcellular_location":"Golgi apparatus membrane","url":"https://www.uniprot.org/uniprotkb/P47736/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RAP1GAP","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RAP1GAP","total_profiled":1310},"omim":[{"mim_id":"618714","title":"RAP1 GTPase-ACTIVATING PROTEIN 2; RAP1GAP2","url":"https://www.omim.org/entry/618714"},{"mim_id":"617504","title":"SIPA1-LIKE PROTEIN 1; SIPA1L1","url":"https://www.omim.org/entry/617504"},{"mim_id":"602180","title":"SIGNAL-INDUCED PROLIFERATION-ASSOCIATED GENE 1; SIPA1","url":"https://www.omim.org/entry/602180"},{"mim_id":"600278","title":"RAP1 GTPase-ACTIVATING PROTEIN; RAP1GAP","url":"https://www.omim.org/entry/600278"},{"mim_id":"191092","title":"TSC COMPLEX SUBUNIT 2; TSC2","url":"https://www.omim.org/entry/191092"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Cytosol","reliability":"Approved"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"brain","ntpm":242.8},{"tissue":"kidney","ntpm":179.1},{"tissue":"thyroid gland","ntpm":150.3}],"url":"https://www.proteinatlas.org/search/RAP1GAP"},"hgnc":{"alias_symbol":["KIAA0474","RAP1GAP1","RAP1GAPII"],"prev_symbol":["RAP1GA1"]},"alphafold":{"accession":"P47736","domains":[{"cath_id":"3.30.1120.160","chopping":"87-185","consensus_level":"high","plddt":94.5274,"start":87,"end":185},{"cath_id":"3.40.50.11210","chopping":"190-378","consensus_level":"high","plddt":94.6958,"start":190,"end":378}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P47736","model_url":"https://alphafold.ebi.ac.uk/files/AF-P47736-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P47736-F1-predicted_aligned_error_v6.png","plddt_mean":69.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RAP1GAP","jax_strain_url":"https://www.jax.org/strain/search?query=RAP1GAP"},"sequence":{"accession":"P47736","fasta_url":"https://rest.uniprot.org/uniprotkb/P47736.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P47736/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P47736"}},"corpus_meta":[{"pmid":"7608212","id":"PMC_7608212","title":"Identification of tuberin, the tuberous sclerosis-2 product. Tuberin possesses specific Rap1GAP activity.","date":"1995","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/7608212","citation_count":324,"is_preprint":false},{"pmid":"10476970","id":"PMC_10476970","title":"Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G alpha(i).","date":"1999","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/10476970","citation_count":198,"is_preprint":false},{"pmid":"15141215","id":"PMC_15141215","title":"The GTPase-activating protein Rap1GAP uses a catalytic asparagine.","date":"2004","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/15141215","citation_count":125,"is_preprint":false},{"pmid":"15657046","id":"PMC_15657046","title":"Cannabinoid receptor-induced neurite outgrowth is mediated by Rap1 activation through G(alpha)o/i-triggered proteasomal degradation of Rap1GAPII.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15657046","citation_count":107,"is_preprint":false},{"pmid":"21532618","id":"PMC_21532618","title":"The tumor suppressor gene rap1GAP is silenced by miR-101-mediated EZH2 overexpression in invasive squamous cell carcinoma.","date":"2011","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/21532618","citation_count":92,"is_preprint":false},{"pmid":"16424023","id":"PMC_16424023","title":"Identification of a putative tumor suppressor gene Rap1GAP in pancreatic cancer.","date":"2006","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/16424023","citation_count":82,"is_preprint":false},{"pmid":"20124489","id":"PMC_20124489","title":"Downregulation of Rap1GAP through epigenetic silencing and loss of heterozygosity promotes invasion and progression of thyroid tumors.","date":"2010","source":"Cancer 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Mutational analysis, fluorescence titration, and stopped-flow kinetic assays confirmed the catalytic asparagine mechanism.\",\n      \"method\": \"X-ray crystallography, active-site mutagenesis, fluorescence titration, stopped-flow kinetics\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and two orthogonal kinetic assays in a single rigorous study\",\n      \"pmids\": [\"15141215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"The catalytic domain of Rap1GAP was mapped to amino acids 75–416; the phosphorylation sites by cAMP-dependent kinase (Ser490, Ser499) and p34cdc2 (Ser484) are localized to the C-terminal region outside the catalytic domain.\",\n      \"method\": \"Deletion mutagenesis of cDNA constructs, purification of truncation mutants, in vitro GAP activity assays, phosphopeptide mapping, point mutagenesis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with purified proteins, multiple deletion mutants, and orthogonal phosphopeptide mapping in one study\",\n      \"pmids\": [\"1406653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Rap1GAP is phosphorylated in vitro by cAMP-dependent kinase (~3 mol phosphate/mol) and p34cdc2 kinase (~2 mol phosphate/mol) at sites in the C-terminal 100-residue segment; dibutyryl-cAMP treatment of SK-MEL-3 cells promotes Rap1GAP phosphorylation in vivo at sites identical to the in vitro cAMP-dependent kinase sites.\",\n      \"method\": \"In vitro kinase assays with purified kinases, 32Pi labeling, comparative phosphopeptide mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified proteins replicated by in vivo labeling and phosphopeptide mapping in one study, consistent with PMID:1406653\",\n      \"pmids\": [\"1587853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Tuberin (TSC2 product) immunoprecipitates contain Rap1GAP-like activity that specifically stimulates intrinsic GTPase of Rap1a but not Rap2, Ha-Ras, Rac, or Rho, establishing tuberin as a Rap1-specific GAP distinct from Rap1GAP.\",\n      \"method\": \"Immunoprecipitation of native tuberin followed by GTPase activity assay; bacterial and Sf9 cell-expressed C-terminal fragment assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assay on immunoprecipitated endogenous protein plus recombinant fragment, single lab; relevant because it defines Rap1GAP's functional specificity by comparison\",\n      \"pmids\": [\"7608212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"A novel isoform of Rap1GAP, rap1GAPII, binds specifically to Gαi-family α-subunits; stimulation of the Gi-coupled m2-muscarinic receptor translocates rap1GAPII from cytosol to membrane, reduces GTP-bound Rap1, and thereby activates ERK/MAPK.\",\n      \"method\": \"Co-immunoprecipitation, subcellular fractionation, Rap1-GTP pull-down, ERK activation assays, receptor stimulation experiments\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, fractionation, and functional Rap1-GTP/ERK readouts together in one study; published in Nature with multiple orthogonal methods\",\n      \"pmids\": [\"10476970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Gαz binds Rap1GAP via the N-terminal 74 amino acids of Rap1GAP (a region distinct from the catalytic domain); this interaction blocks RGS-stimulated GTP hydrolysis of Gαz, attenuates Gαz-mediated adenylyl cyclase inhibition, and allows formation of a stable Gαz–Rap1GAP–Rap1 complex.\",\n      \"method\": \"Yeast two-hybrid, co-precipitation with purified recombinant proteins, adenylyl cyclase assay, N-terminal deletion analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — yeast two-hybrid plus biochemical reconstitution with purified proteins plus functional adenylyl cyclase assay and domain mapping in one study\",\n      \"pmids\": [\"10593970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Gαo/i directly interacts with Rap1GAPII and targets it for ubiquitination and proteasomal degradation; this reduces Rap1GAP levels, activates Rap1, and drives CB1 cannabinoid receptor-induced neurite outgrowth in Neuro-2A cells. Proteasomal inhibitor lactacystin blocks Gαo/i-induced Rap1 activation and neurite outgrowth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, proteasomal inhibitor treatment (lactacystin), dominant-negative Rap1, siRNA knockdown, pertussis toxin treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal approaches (Co-IP, ubiquitination assay, pharmacological inhibition, genetic dominant-negative, siRNA) in one study\",\n      \"pmids\": [\"15657046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"GSK3β phosphorylates Rap1GAP (at Ser525 identified by mutagenesis) and promotes its proteasome-mediated degradation; GSK3β inhibitors prevent phosphorylation and degradation of endogenous Rap1GAP. TSH/cAMP signaling stabilizes Rap1GAP, while TSH withdrawal leads to GSK3β-dependent Rap1GAP turnover.\",\n      \"method\": \"In vitro kinase assay (GSK3β on immunoprecipitated Rap1GAP), pharmacological GSK3β inhibitors, co-expression of GSK3β + Rap1GAP with proteasome inhibitors, point mutagenesis (Ser525)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay plus mutagenesis plus pharmacological rescue, multiple orthogonal methods in one study\",\n      \"pmids\": [\"14660640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PKA phosphorylates Rap1GAP at Ser-441 and Ser-499 in striatal neurons in response to D1 dopamine receptor activation; this phosphorylation inhibits Rap1GAP GAP activity (increasing Rap1-GTP levels) and is associated with decreased dendritic spine head size.\",\n      \"method\": \"Mass spectrometry identification of PKA substrate, in vitro phosphorylation, Rap1-GTP pull-down in striatal neurons, D1 receptor activation, phospho-specific analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS-identified substrate, in vitro phosphorylation, and functional Rap1-GTP readout in neuronal system with receptor stimulation\",\n      \"pmids\": [\"19218462\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Rap1GAP is hyperphosphorylated during mitosis; p34cdc2 kinase co-immunoprecipitated from mitotic (but not interphase) HeLa cell lysates phosphorylates wild-type Rap1GAP but not a Ser484 mutant, indicating p34cdc2 phosphorylates Rap1GAP at Ser484 during mitosis. This phosphorylation does not affect GAP catalytic activity toward Rap1.\",\n      \"method\": \"Cell cycle synchronization, co-immunoprecipitation of cdc2/cyclin B1, in vitro kinase assay with wild-type and Ser484 mutant Rap1GAP, SDS-PAGE mobility shift\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP of endogenous cdc2 plus mutagenesis, single lab, consistent with earlier in vitro data (PMID:1587853)\",\n      \"pmids\": [\"8048970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PLK1 phosphorylates Ser525 within the 524DSGHVS529 degron of Rap1GAP, promoting its interaction with the β-TrCP ubiquitin ligase complex and subsequent proteasomal degradation during mitosis; PLK1 binds Rap1GAP via recognition of an SSP motif within Rap1GAP.\",\n      \"method\": \"Co-immunoprecipitation, in vitro kinase assay, ubiquitination assay, proteasome inhibitor treatment, mutagenesis of Ser525 and SSP motif\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro kinase assay plus mutagenesis plus Co-IP and ubiquitination assay, multiple orthogonal methods\",\n      \"pmids\": [\"25329897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Rap1GAP physically interacts with the RET receptor tyrosine kinase via Tyr981 in RET's intracellular domain; endogenous Rap1GAP co-immunoprecipitates with RET in neural tissues; GDNF treatment enhances RET–Rap1GAP interaction; overexpression of Rap1GAP attenuates GDNF-induced ERK activation and neurite outgrowth, while Rap1GAP knockdown enhances them.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation (endogenous proteins in neural tissue), RNAi knockdown, mutagenesis of RET Tyr981, neurite outgrowth assay, ERK phosphorylation assay\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid confirmed by endogenous Co-IP in tissue plus mutagenesis plus functional readouts, multiple orthogonal methods\",\n      \"pmids\": [\"20877310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Silencing Rap1GAP in human colon carcinoma cells enhances Rap activity, impairs cell–cell adhesion (aberrant distribution of E-cadherin, β-catenin, p120-catenin), and enhances spreading/adhesion on collagen; silencing Rap expression rescues these defects; Src activity is increased in Rap1GAP-depleted cells and Src inhibition restores E-cadherin at cell–cell contacts.\",\n      \"method\": \"siRNA knockdown, Rap1-GTP pull-down assay, immunofluorescence of adherens junction proteins, Src kinase inhibitor treatment\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (Rap1GAP KD then Rap KD rescue), multiple cell biology readouts, single lab\",\n      \"pmids\": [\"20439492\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Oncogenic Ras downregulates Rap1GAP expression via the Raf/MEK/ERK cascade in rat thyroid cells; restoring Rap1GAP inhibits Rap1 and Rac1 activity and blocks cell migration, invasion, DNA synthesis, and anchorage-independent growth.\",\n      \"method\": \"Ras transformation, MEK inhibitor treatment, Rap1-GTP/Rac1-GTP pull-down, siRNA knockdown, Rap1GAP re-expression, migration/invasion assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — epistasis via MEK inhibitor plus functional readouts, single lab\",\n      \"pmids\": [\"17646383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Depletion of Rap1GAP in colon cancer cells enhances migration via increased endogenous Rap activity, suppresses ROCK-mediated contractility, and switches migratory mode to Rac1-dependent mesenchymal motility; Rac1 inhibition restores membrane blebbing and ROCK activity in Rap1GAP-depleted cells.\",\n      \"method\": \"siRNA knockdown, Rap1-GTP pull-down, ROCK inhibitor, Rac1 inhibitor, live-cell migration tracking, morphological analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis (Rap1GAP KD, then Rap KD, then Rac1 inhibition) with multiple cellular readouts, single lab\",\n      \"pmids\": [\"23864657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Podocyte-specific inactivation of both Rap1a and Rap1b (the targets of Rap1GAP) causes massive glomerulosclerosis; increased Rap1GAP expression in injured podocytes reduces active β1 integrin, leading to podocyte detachment; preventing RAP1GAP elevation maintains β1 integrin-mediated adhesion and prevents detachment.\",\n      \"method\": \"Insertional mutagenesis screen, podocyte-specific conditional knockout mice, β1 integrin activation assays, kidney biopsy immunostaining\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional knockout with defined glomerular phenotype plus mechanistic β1 integrin readout, replicated in human biopsies\",\n      \"pmids\": [\"24642466\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Degradation of Rap1GAP in HPV16/18-positive cervical cancer cells is mediated by the E6AP ubiquitin ligase via the proteasome pathway; co-immunoprecipitation showed E6AP binds Rap1GAP in HPV-positive cells; knockdown of E6AP shifts degradation from proteasomal to autophagy-mediated.\",\n      \"method\": \"Co-immunoprecipitation, MG132 proteasome inhibitor treatment, siRNA knockdown of E6AP, rapamycin-induced autophagy, Western blotting\",\n      \"journal\": \"Infectious agents and cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus pharmacological and genetic manipulation, single lab\",\n      \"pmids\": [\"34952616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Gαo-GTP (not GDP) binds and activates Rap1GAP1 via a GoLoco/GPR motif; specific residues in the GoLoco/GPR motif confer differential recognition of Gαo guanine-nucleotide-binding status; GNAO1 encephalopathy mutations in Gαo prevent it from attaining the conformation needed for Rap1GAP1 effector binding.\",\n      \"method\": \"Proximity-based proteomics (BioID) screen, co-immunoprecipitation, in vitro binding assays with GDP- and GTP-locked Gαo mutants, Rap1GAP1 activity assays, point mutagenesis of GoLoco/GPR motif\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — proteomics screen confirmed by Co-IP plus in vitro binding with nucleotide-state mutants plus GAP activity assay plus mutagenesis, multiple orthogonal methods\",\n      \"pmids\": [\"40615045\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In the subventricular zone niche, ADAM10 cleavage of JAMC increases Rap1GAP activity, which promotes neural stem cell transit from the apical to basal compartment and subsequent lineage progression.\",\n      \"method\": \"ADAM10 loss-of-function in vivo, NSC positioning assay, molecular pathway analysis linking JAMC processing to Rap1GAP activity\",\n      \"journal\": \"Neural regeneration research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, indirect pathway placement inferred from ADAM10 KO phenotype with Rap1GAP as downstream effector, limited direct mechanistic data on Rap1GAP itself in the abstract\",\n      \"pmids\": [\"35535899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"EZH2 represses Rap1GAP by facilitating H3K27me3 at the Rap1GAP locus and promoting Rap1GAP promoter hypermethylation; loss of miR-101 leads to EZH2 upregulation and concomitant Rap1GAP downregulation in head and neck cancer.\",\n      \"method\": \"ChIP for H3K27me3, bisulfite methylation analysis, miR-101 overexpression/inhibition, Western blotting\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and methylation analyses linking EZH2 to Rap1GAP promoter, single lab, two orthogonal epigenetic methods\",\n      \"pmids\": [\"21532618\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rap1GAP overexpression in cardiomyocytes inhibits the AMPK/AKT/mTOR signaling pathway, exacerbates Ang II-induced cardiomyocyte hypertrophy, increases ROS generation, and inhibits autophagy; conversely, Rap1GAP knockdown activates AMPK/AKT/mTOR and reduces hypertrophy. Co-immunoprecipitation showed exogenous Rap1GAP interacts with AMPK.\",\n      \"method\": \"siRNA knockdown, adenoviral overexpression, AMPK/AKT/mTOR pathway Western blotting, ROS assay, autophagy markers, co-immunoprecipitation\",\n      \"journal\": \"Oxidative medicine and cellular longevity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP plus gain/loss-of-function with multiple downstream readouts, single lab\",\n      \"pmids\": [\"33936386\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"Rap1GAP is a Rap1-specific GTPase-activating protein that uses a catalytic asparagine (rather than the arginine employed by Ras GAPs) to stimulate GTP hydrolysis by Rap1; its catalytic domain spans residues 75–416, while its C-terminal region harbors phosphorylation sites for PKA (Ser490/499, inhibiting GAP activity), p34cdc2/PLK1 (Ser484/525, the latter promoting β-TrCP-dependent proteasomal degradation in mitosis), and GSK3β (Ser525, promoting proteasomal turnover); an N-terminal region (first 74 aa) mediates binding to Gαz and Gαo (via a GoLoco/GPR motif), with Gαo-GTP activating Rap1GAP1 and Gαi/o targeting the rap1GAPII isoform for membrane recruitment or proteasomal degradation to modulate Rap1-GTP levels and downstream ERK/MAPK and integrin signaling; Rap1GAP also physically interacts with the RET receptor kinase and with AMPK, placing it at the nexus of neurotrophin, adhesion, and metabolic signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RAP1GAP is a Rap1-specific GTPase-activating protein that terminates Rap1 signaling and thereby controls cell adhesion, migration, and receptor-driven ERK/MAPK output [#4, #12]. Its catalytic domain (residues 75\\u2013416) stimulates GTP hydrolysis on Rap1 through a non-canonical catalytic asparagine, distinguishing it from the arginine-finger mechanism of Ras GAPs [#0, #1]. Activity and abundance are tightly regulated by phosphorylation in a C-terminal regulatory region outside the catalytic domain: PKA phosphorylation (downstream of cAMP and D1 dopamine receptor signaling) inhibits GAP activity to raise Rap1-GTP [#2, #8], while GSK3\\u03b2 (Ser525) and mitotic PLK1 (Ser525, within a \\u03b2-TrCP degron) and p34cdc2 (Ser484) phosphorylation route the protein toward proteasomal degradation [#7, #9, #10]. An N-terminal region (first 74 aa) binds heterotrimeric G\\u03b1 subunits, coupling Rap1GAP to receptor signaling: G\\u03b1z binding blocks RGS-stimulated GTP hydrolysis and forms a stable G\\u03b1z\\u2013Rap1GAP\\u2013Rap1 complex [#5], G\\u03b1o-GTP binds and activates Rap1GAP1 through a GoLoco/GPR motif [#17], and Gi/o coupling translocates or degrades the rap1GAPII isoform to modulate Rap1 levels and drive ERK activation and neurite outgrowth [#4, #6]. Through suppression of Rap1, Rap1GAP maintains E-cadherin-based cell\\u2013cell adhesion and \\u03b21 integrin-mediated attachment, and its loss enhances Src activity, Rac1-dependent migration, and invasion [#12, #14, #15]; consistent with a tumor-suppressive role, it is downregulated by oncogenic Ras/MEK/ERK signaling and by EZH2/miR-101-mediated epigenetic silencing in cancer [#13, #19]. Rap1GAP also physically interacts with the RET receptor tyrosine kinase to restrain GDNF-induced signaling [#11].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Establishing the modular architecture of Rap1GAP separated catalysis from regulation, defining where activity is generated versus where it is controlled.\",\n      \"evidence\": \"Deletion mutagenesis, in vitro GAP assays, and phosphopeptide mapping localizing the catalytic domain to aa 75\\u2013416 and PKA/cdc2 sites to the C-terminus\",\n      \"pmids\": [\"1406653\", \"1587853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of each phosphorylation on activity not resolved here\", \"No structural basis for catalysis yet\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Mitotic phosphorylation at Ser484 by p34cdc2 raised the possibility of cell-cycle regulation of Rap1GAP, even though catalytic activity was unchanged.\",\n      \"evidence\": \"Cell-cycle synchronization with co-IP of cdc2/cyclin B1 and in vitro kinase assay on wild-type vs Ser484 mutant\",\n      \"pmids\": [\"8048970\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No catalytic or abundance effect identified for Ser484 phosphorylation\", \"Downstream functional role left open until later degron work\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Discovery of G\\u03b1 subunit binding via the N-terminus and of the rap1GAPII isoform connected Rap1GAP to GPCR signaling and to ERK/MAPK activation.\",\n      \"evidence\": \"Yeast two-hybrid, co-precipitation, subcellular fractionation, Rap1-GTP pull-down and adenylyl cyclase/ERK readouts for G\\u03b1z and G\\u03b1i binding\",\n      \"pmids\": [\"10593970\", \"10476970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nucleotide-state dependence of G\\u03b1 binding not yet defined\", \"How translocation alters local Rap1 pools mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"GSK3\\u03b2 phosphorylation at Ser525 linking Rap1GAP stability to hormonal cAMP signaling showed that abundance, not only activity, is a regulated variable.\",\n      \"evidence\": \"In vitro kinase assay, GSK3\\u03b2 inhibitors, proteasome inhibitors, Ser525 mutagenesis in TSH-responsive cells\",\n      \"pmids\": [\"14660640\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the ubiquitin ligase not defined here\", \"Physiological scope beyond thyroid cells unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"The crystal structure resolved the catalytic mechanism, revealing an asparagine thumb rather than the arginine finger used by Ras GAPs.\",\n      \"evidence\": \"X-ray crystallography of the catalytic domain with active-site mutagenesis, fluorescence titration, and stopped-flow kinetics\",\n      \"pmids\": [\"15141215\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No co-structure with Rap1 in the published work\", \"Structure of full-length regulatory regions not determined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"G\\u03b1o/i-driven ubiquitination and degradation of rap1GAPII provided a mechanism for receptor-induced Rap1 activation and neurite outgrowth.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, lactacystin, dominant-negative Rap1, siRNA, and pertussis toxin in Neuro-2A cells\",\n      \"pmids\": [\"15657046\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific ligase mediating G\\u03b1o/i-directed degradation not identified\", \"Relationship to GSK3\\u03b2-driven turnover unresolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"PKA phosphorylation at Ser441/Ser499 inhibiting GAP activity placed Rap1GAP in dopamine-receptor-controlled regulation of dendritic spine morphology.\",\n      \"evidence\": \"Mass spectrometry, in vitro phosphorylation, and Rap1-GTP pull-down in striatal neurons after D1 receptor activation\",\n      \"pmids\": [\"19218462\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct causal link between Rap1-GTP and spine size not fully dissected\", \"Interplay with degradative phosphosites unaddressed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"RET binding and adhesion-junction control defined Rap1GAP as a brake on receptor tyrosine kinase signaling and a maintainer of epithelial integrity.\",\n      \"evidence\": \"Yeast two-hybrid and endogenous Co-IP with RET (Tyr981), plus siRNA, immunofluorescence of junction proteins, and Src inhibition in colon carcinoma cells\",\n      \"pmids\": [\"20877310\", \"20439492\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RET binding directly alters GAP activity not established\", \"Mechanism connecting Rap to Src activation incompletely defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Rap1GAP loss was shown to switch migratory mode by suppressing ROCK contractility and promoting Rac1-dependent mesenchymal motility.\",\n      \"evidence\": \"siRNA, Rap1-GTP pull-down, ROCK and Rac1 inhibitors, and live-cell migration tracking\",\n      \"pmids\": [\"23864657\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct effectors linking Rap to ROCK/Rac1 not identified\", \"In vivo relevance of mode-switching not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mitotic PLK1 phosphorylation within a \\u03b2-TrCP degron and in vivo podocyte studies established degron-driven turnover and the physiological consequence of Rap1GAP excess on integrin-mediated adhesion.\",\n      \"evidence\": \"In vitro kinase, ubiquitination, and Co-IP for PLK1/\\u03b2-TrCP; podocyte-specific conditional knockouts and \\u03b21 integrin assays with human biopsy validation\",\n      \"pmids\": [\"25329897\", \"24642466\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Coordination of PLK1, GSK3\\u03b2, and cdc2 phosphorylation across the cycle unresolved\", \"How Rap1GAP elevation lowers active \\u03b21 integrin mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identification of E6AP-mediated degradation and an AMPK interaction extended Rap1GAP regulation into viral oncogenesis and cardiomyocyte metabolic/autophagy control.\",\n      \"evidence\": \"Co-IP, MG132, E6AP siRNA in HPV+ cervical cancer; gain/loss-of-function with AMPK/AKT/mTOR Western blots, ROS, autophagy markers, and AMPK Co-IP in cardiomyocytes\",\n      \"pmids\": [\"34952616\", \"33936386\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect nature of AMPK interaction not resolved\", \"Whether E6AP recognizes a defined Rap1GAP degron unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Resolving that G\\u03b1o-GTP, not GDP, binds and activates Rap1GAP1 via a GoLoco/GPR motif tied the regulator to GNAO1 encephalopathy through conformation-dependent effector recognition.\",\n      \"evidence\": \"BioID proteomics, Co-IP, in vitro binding with nucleotide-locked G\\u03b1o mutants, GAP activity assays, and GoLoco/GPR motif mutagenesis\",\n      \"pmids\": [\"40615045\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream Rap1-dependent consequences in neurons not mapped\", \"Relationship to the earlier G\\u03b1z/G\\u03b1i binding modes not reconciled\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the many regulatory inputs\\u2014G\\u03b1 nucleotide-state binding, multiple inhibitory and degradative phosphorylations, and distinct E3 ligases\\u2014are integrated to set Rap1-GTP levels in a given cell type remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unified model of competing phosphorylation and degradation pathways\", \"Cell-type-specific isoform usage (Rap1GAP1 vs rap1GAPII) not systematically mapped\", \"Structural basis of G\\u03b1\\u2013Rap1GAP\\u2013Rap1 ternary complex undetermined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 4, 12]},\n      {\"term_id\": \"GO:0005096\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [4, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 5, 6, 8, 17]},\n      {\"term_id\": \"R-HSA-1474244\", \"supporting_discovery_ids\": [12, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"RAP1A\", \"GNAZ\", \"GNAO1\", \"RET\", \"PLK1\", \"BTRC\", \"GSK3B\", \"PRKAA1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}