{"gene":"GIT2","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2000,"finding":"GIT2 (including both long and short splice variants) possesses ARF GTPase-activating protein (ARF-GAP) activity toward ARF1 in vitro, interacts with G protein-coupled receptor kinase 2 (GRK2), and interacts with PIX-PAK complexes. The longest GIT2 variant inhibits beta2-adrenergic receptor sequestration when overexpressed, whereas the shortest splice variant is inactive in this assay.","method":"In vitro GAP assay, co-immunoprecipitation, cellular overexpression with receptor sequestration readout","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro ARF-GAP activity demonstrated with multiple variants, co-IP binding partners identified, functional receptor sequestration assay in cells; multiple orthogonal methods in one study","pmids":["10896954"],"is_preprint":false},{"year":2001,"finding":"GIT2/PKL (paxillin kinase linker) interacts with paxillin via the paxillin LD4 motif; this interaction is required for PKL localization to focal contacts and for normal Rac-dependent cell spreading and directional motility. Loss of the paxillin-PKL interaction leads to prolonged Rac activation, multiple broad lamellipodia, and impaired directional motility without affecting FAK activity.","method":"Overexpression of deletion mutants (paxillinΔLD4, PKLδPBS2) in CHO.K1 fibroblasts; immunofluorescence localization; cell spreading/motility assays; Rac activity measurements","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal deletion mutant approach with multiple orthogonal readouts (localization, Rac activity, motility), consistent with parallel studies","pmids":["11448998"],"is_preprint":false},{"year":2001,"finding":"GIT2-short (KIAA0148), a short isoform of GIT2, co-localizes with paxillin at perinuclear areas and acts as an ARF-GAP for ARF1 in vivo. Overexpression of wild-type GIT2-short (but not its GAP-inactive mutant) redistributes Golgi protein β-COP, reduces focal adhesions and actin stress fibers, and alters perinuclear paxillin localization, demonstrating that the ARF-GAP catalytic activity is required for these effects.","method":"Overexpression and GAP-inactive mutant rescue in cells; immunofluorescence co-localization; Golgi distribution assay; focal adhesion and actin stress fiber quantification","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — catalytic mutant used to assign phenotype to GAP activity, multiple orthogonal cellular readouts in one study","pmids":["11251077"],"is_preprint":false},{"year":2001,"finding":"PAK1, PIX (β-PIX), and p95PKL (GIT2) form a stable trimolecular complex in T cells. This complex is activated downstream of the T-cell receptor via ZAP-70/Syk kinases and a LAT/Slp-76-independent pathway; PIX GEF activity within the complex is required for Rho GTPase activation upstream of PAK1.","method":"Co-immunoprecipitation demonstrating trimolecular complex; dominant-negative PIX overexpression; TCR stimulation assays with kinase inhibitors and adaptor-deficient Jurkat cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — stable trimolecular complex demonstrated by Co-IP, functional dissection using dominant-negative and adaptor-null cell lines with multiple readouts","pmids":["11157752"],"is_preprint":false},{"year":2003,"finding":"CrkII/CrkL adapter proteins associate with a Paxillin/GIT2/β-PIX complex; this association promotes Rac-dependent relocalization of paxillin to focal contacts and lamellipodia formation. Paxillin mutants unable to associate with Crk or GIT2 block Crk-dependent cell spreading.","method":"Co-immunoprecipitation; stable cell line overexpression; dominant-negative Crk mutants; Rac inhibition; immunofluorescence localization","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP establishing complex, multiple mutant controls, Rac-dependence tested orthogonally","pmids":["12857867"],"is_preprint":false},{"year":2006,"finding":"Endogenous GIT2 represses lamellipodial extension via Rac1-dependent signaling and represses focal adhesion turnover via Cdc42-dependent signaling. GIT2 knockdown is sufficient to induce migration of non-transformed MCF10A epithelial cells. The SH2-SH3 adaptor Crk is identified as an essential downstream target of GIT2 inhibition, whereas β-PIX is dispensable for GIT2-mediated effects.","method":"siRNA knockdown of endogenous GIT2; lamellipodia and FA turnover assays; epistasis with Rac1, Cdc42, Crk, and β-PIX","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis by knockdown of endogenous protein, multiple pathway components tested with defined phenotypic readouts, negative result for β-PIX also mechanistically informative","pmids":["16628223"],"is_preprint":false},{"year":2006,"finding":"GIT2 is required in neutrophils for directional chemotaxis and for suppression of superoxide production in response to G protein-coupled receptor stimulation. GIT2 suppresses ARF1 activity and functions downstream of Gβγ subunits in the direction-sensing machinery. Loss of GIT2 in vivo leads to an immunodeficient state.","method":"GIT2-knockout mouse neutrophils; directional chemotaxis assays; superoxide production measurements; epistasis placing GIT2 downstream of Gβγ and upstream of ARF1","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with multiple orthogonal functional readouts, in vivo confirmation","pmids":["16715100"],"is_preprint":false},{"year":2010,"finding":"GIT2 is required for efficient thymocyte positive selection. Git2-/- double-positive thymocytes show elevated Rac GTPase activation, increased actin polymerization, and enhanced chemokine-directed migration in vitro. Two-photon microscopy revealed that scanning activity of Git2-/- thymocytes was compromised in the thymic cortex, indicating GIT2 negatively regulates Rac-mediated chemotactic motility in thymocytes.","method":"Git2-knockout mice; Rac activation assay; actin polymerization assay; in vitro chemotaxis; two-photon laser-scanning microscopy in intact thymus","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout mouse with multiple orthogonal in vitro and intravital imaging readouts","pmids":["20431621"],"is_preprint":false},{"year":2010,"finding":"Zebrafish Git2a is required for cell movements during gastrulation; its depletion arrests directed cell migration toward the vegetal pole and reduces cell contractility. Git2a regulates phosphorylation of myosin light chain (MLC), thereby controlling myosin II-mediated cell contractility. The phenotype is rescued by chicken GIT2, confirming functional conservation.","method":"Antisense morpholino knockdown; time-lapse microscopy; myosin light chain phosphorylation assay; pharmacological inhibition with Blebbistatin; rescue with chicken GIT2","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockdown with molecular rescue, direct phosphorylation readout, pharmacological epistasis with myosin II inhibitor","pmids":["21034731"],"is_preprint":false},{"year":2013,"finding":"PKL/GIT2 regulates activity of the Rac1 GEF Vav2 through a phosphorylation-dependent interaction. PKL is required for Vav2 activation downstream of integrin engagement and EGF stimulation. Vav2 in turn regulates redistribution of PKL and β-PIX to focal adhesions after EGF stimulation, forming a feedforward signaling loop. Vav2 knockdown reduces directional persistence and polarization of migrating cells.","method":"Co-immunoprecipitation; PKL and Vav2 knockdown; Rac1 activation assays; immunofluorescence localization of PKL and β-PIX; cell migration directionality assays","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, knockdown of both partners, multiple orthogonal functional readouts in one study","pmids":["23615439"],"is_preprint":false},{"year":2013,"finding":"Loss of Git2 promotes epithelial-mesenchymal transition (EMT) through a pathway involving enhanced maturation of miR-146a, which suppresses Cnot6L (a deadenylase), leading to stabilization of Zeb1 mRNA and increased Zeb1 expression.","method":"Git2 knockdown/knockout; miR-146a maturation assay; Cnot6L manipulation; Zeb1 mRNA stability assay; EMT marker analysis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pathway dissection with multiple components tested, single lab, abstract does not fully detail all controls","pmids":["23591815"],"is_preprint":false},{"year":2014,"finding":"GIT2 negatively regulates TLR-induced NF-κB and MAPK signaling by recruiting the deubiquitinating enzyme Cylindromatosis (CYLD) to inhibit K63-linked ubiquitination of TRAF6, thereby terminating downstream inflammatory signaling. Git2-deficient mice and macrophages show dramatically increased pro-inflammatory cytokine production in response to TLR stimulation.","method":"Git2-knockout mice and macrophages; TLR stimulation assays; NF-κB and MAPK activation measurements; Co-immunoprecipitation of GIT2-CYLD complex; TRAF6 ubiquitination assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP demonstrating GIT2-CYLD complex, ubiquitination assay, in vivo knockout confirmation, multiple orthogonal methods","pmids":["24879442"],"is_preprint":false},{"year":2015,"finding":"GIT2 localizes to the nucleus, is phosphorylated by ATM kinase following DNA damage, and forms complexes with multiple DNA damage response (DDR) factors. GIT2 targeting to DNA double-strand breaks depends on H2AX, ATM, and MRE11 but is independent of MDC1 and RNF8. GIT2 promotes DNA repair by stabilizing BRCA1 in repair complexes, upregulating HMGN1 and RFC1, and regulating PARP activity. GIT2-knockout mice show increased susceptibility to irradiation-induced DNA damage.","method":"Nuclear fractionation; Co-immunoprecipitation with DDR factors; ATM kinase phosphorylation assay; DDR factor dependency analysis (H2AX/ATM/MRE11/MDC1/RNF8); GIT2-KO mice irradiation; PARP activity assay; immunofluorescence foci analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, kinase assay, KO mice), epistasis with multiple DDR components, functional rescue experiments","pmids":["25605334"],"is_preprint":false},{"year":2016,"finding":"GIT2 restricts focal adhesion recruitment of DOCK5 and inhibits DOCK5 interaction with Crk, thereby suppressing DOCK5-dependent activation of the Crk-p130Cas signaling cascade, Rac1-mediated lamellipodial protrusion, and FA turnover. GIT2 is recruited to focal adhesions in response to Rho-ROCK signaling and actomyosin contractility.","method":"GIT2 knockdown/overexpression; Co-immunoprecipitation of GIT2-DOCK5-Crk interactions; ROCK and MLC inhibition; Rac1 activation assay; invasion assays in epithelial cells","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP demonstrating GIT2-DOCK5-Crk interactions, pharmacological pathway dissection, multiple cell line validation","pmids":["27669437"],"is_preprint":false},{"year":2016,"finding":"RUSC2 interacts with the Spa Homology Domain (SHD) of GIT2 in lung cancer cells, stabilizes GIT2 by decreasing its degradation and increasing its phosphorylation, and promotes Golgi reorientation and directional migration. EGF stimulation transiently increases RUSC2-GIT2 interaction, while prolonged EGF stimulation decreases it via Rab35 activation. Rab35 silencing reduces GIT2 stability and phosphorylation.","method":"Co-immunoprecipitation (RUSC2-GIT2 interaction); RUSC2 and Rab35 silencing; GIT2 stability assays; Golgi reorientation assay; directional migration assay","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with SHD domain specificity, functional knockdown experiments, single lab","pmids":["27238570"],"is_preprint":false},{"year":2016,"finding":"GIT2 physically interacts with the insulin receptor and insulin receptor substrate 2 (IRS-2) in pancreatic tissue; this interaction is diminished in diabetic db/db mice. Genomic deletion of GIT2 disrupts pancreatic beta cell mass and reduces insulin secretion, leading to elevated plasma glucose and insulin resistance.","method":"Co-immunoprecipitation of GIT2 with insulin receptor and IRS-2; GIT2-KO mouse metabolic phenotyping; pancreatic islet transcriptomics","journal":"Frontiers in endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating direct interaction, KO mouse metabolic phenotype, single lab","pmids":["26834700"],"is_preprint":false},{"year":2018,"finding":"GIT2/PKL mediates endothelial progenitor cell (EPC) migration downstream of CXCR2 via the Src-PKL/Vav2-Rac1 signaling pathway. Phosphorylation and co-localization of PKL and Vav2 are required for Rac1 activation and development of lamellipodia/filopodia driving EPC migration.","method":"Transwell migration assays; shRNA knockdown; signaling inhibitors; immunofluorescence co-localization of PKL and Vav2; Rac1 phosphorylation assays","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockdown and inhibitor-based pathway dissection, co-localization, single lab","pmids":["29229683"],"is_preprint":false},{"year":2024,"finding":"GIT2 associates with centrosomes and γ-tubulin complex proteins in mast cells. Depletion of GIT2 enhances centrosomal microtubule nucleation. Phosphorylation of GIT2 by conventional protein kinase C (PKC) promotes its centrosomal localization and microtubule nucleation during FcεRI-induced activation. GIT2 (unlike GIT1) acts as a negative regulator of microtubule nucleation in mast cells and also participates in antigen-induced degranulation and chemotaxis.","method":"shRNA depletion; immunofluorescence and Co-IP with γ-tubulin complex proteins; time-lapse microtubule nucleation assay; PKC inhibitor treatment; site-directed mutagenesis of phosphorylation sites; phenotypic rescue","journal":"Frontiers in immunology","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — mutagenesis of phosphorylation sites, reconstitution-level kinase assay, Co-IP with γTuRC components, functional rescue; single lab but multiple orthogonal methods","pmids":["38370406"],"is_preprint":false},{"year":2025,"finding":"GIT2 directly binds NF-κB components p65 (canonical) and p52 (non-canonical) to inhibit their activation, and positively regulates TRAF3 expression to further suppress both canonical and non-canonical NF-κB signaling. GIT2 thereby promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and alleviates DNA damage-induced cellular senescence.","method":"Co-immunoprecipitation (GIT2 with p65 and p52); Western blotting of NF-κB pathway components; GIT2 overexpression/knockdown in BMSCs; comet assay; in vivo ovariectomy mouse model; micro-CT bone analysis","journal":"Tissue & cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating direct binding to p65/p52, multiple functional assays in vitro and in vivo, single lab","pmids":["39954559"],"is_preprint":false},{"year":2025,"finding":"THRAP3 recruits the splicing factor SLU7 to facilitate skipping of GIT2 Exon14, generating a GIT2 splice variant that promotes ferroptosis resistance in AML cells by inhibiting iron accumulation and promoting GSH synthesis. Inhibition of GIT2 Exon14 skipping reverses THRAP3-induced ferroptosis resistance in vitro and in vivo.","method":"THRAP3 knockdown/overexpression; Co-immunoprecipitation of THRAP3-SLU7; RT-PCR/splicing assays for Exon14; ferroptosis assays (RSL3/erastin); iron and GSH measurement; orthotopic mouse models","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of THRAP3-SLU7 complex, splicing assay for GIT2 exon14, functional rescue, in vivo tumor model; single lab","pmids":["41326370"],"is_preprint":false},{"year":2025,"finding":"In glioblastoma cells, GIT2 associates with γ-tubulin ring complex (γTuRC) proteins and localizes to centrosomes. Depletion of GIT2 enhances centrosomal microtubule nucleation. The N-terminal ArfGAP domain of GIT2 is responsible for centrosomal localization and regulation of microtubule nucleation. PKC phosphorylates GIT2 at serine 46 (S46) on the ArfGAP domain, and phosphomimetic S46 promotes microtubule nucleation.","method":"shRNA depletion; immunofluorescence; time-lapse microtubule nucleation; immunoprecipitation with γTuRC components; site-directed mutagenesis (S46); kinase assay with PKC inhibitors","journal":"Cancer cell international","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — site-directed mutagenesis identifying specific phosphorylation site (S46), kinase assay, Co-IP with γTuRC, multiple orthogonal methods, single lab","pmids":["40176062"],"is_preprint":false}],"current_model":"GIT2 (also known as PKL/KIAA0148) is a multifunctional ARF GTPase-activating protein (ARF-GAP) that scaffolds signaling complexes at focal adhesions, centrosomes, and the nucleus: it inactivates ARF1 to control Golgi organization and focal adhesion dynamics; associates with paxillin (via LD4), β-PIX, PAK1, Crk, DOCK5, Vav2, and GRK2 to spatiotemporally regulate Rac1/Cdc42 activity and thereby control cell spreading, polarity, and directional migration; is phosphorylated by ATM at DNA double-strand breaks to promote DDR complex assembly and repair; is phosphorylated by PKC at S46 of its ArfGAP domain to regulate centrosomal microtubule nucleation; recruits CYLD to deubiquitinate TRAF6 and terminate TLR-induced NF-κB signaling; and directly binds p65/p52 NF-κB subunits to suppress inflammatory gene activation, collectively establishing GIT2 as a signaling hub linking cytoskeletal remodeling, immune signaling, and genome integrity."},"narrative":{"mechanistic_narrative":"GIT2 is a multifunctional ARF GTPase-activating protein that operates as a signaling scaffold coupling cytoskeletal remodeling to cell migration, immune signaling, and genome maintenance [PMID:10896954, PMID:16628223]. Through its ARF-GAP domain it inactivates ARF1 to control Golgi organization and focal adhesion/actin architecture, an activity that is catalytically required for these effects [PMID:11251077]. GIT2 nucleates a paxillin–β-PIX–PAK1 complex—binding paxillin via the LD4 motif to localize at focal contacts—and integrates this module with Crk/CrkL adaptors, the Rac1 GEF Vav2, and DOCK5 to spatiotemporally restrain Rac1 and Cdc42 activity; loss of GIT2 produces sustained Rac activation, excess lamellipodia, faster focal adhesion turnover, and loss of directional motility [PMID:11448998, PMID:11157752, PMID:12857867, PMID:16628223, PMID:23615439, PMID:27669437]. Consistent with a brake on Rac-driven motility, GIT2 acts downstream of Gβγ and CXCR2 to enforce directional chemotaxis and suppress superoxide in neutrophils, governs thymocyte selection and migration, and controls myosin II-mediated contractility during gastrulation [PMID:16715100, PMID:20431621, PMID:21034731, PMID:29229683]. Beyond the cytoskeleton, GIT2 is a negative regulator of inflammatory signaling: it recruits the deubiquitinase CYLD to remove K63-linked ubiquitin from TRAF6 to terminate TLR-induced NF-κB/MAPK signaling, and directly binds the NF-κB subunits p65 and p52 while elevating TRAF3 to suppress canonical and non-canonical NF-κB output [PMID:24879442, PMID:39954559]. A nuclear pool of GIT2 is phosphorylated by ATM and recruited to DNA double-strand breaks in an H2AX/MRE11-dependent manner, where it stabilizes BRCA1 and promotes repair [PMID:25605334]. PKC-mediated phosphorylation at S46 in the ArfGAP domain drives centrosomal localization where GIT2 negatively regulates γ-tubulin-dependent microtubule nucleation [PMID:38370406, PMID:40176062].","teleology":[{"year":2000,"claim":"Established GIT2's core biochemical identity by showing it is an ARF1-directed GAP that also binds GRK2 and PIX-PAK complexes and regulates receptor sequestration, defining it as both an enzyme and a scaffold.","evidence":"In vitro GAP assay, co-immunoprecipitation, and receptor sequestration readout across splice variants","pmids":["10896954"],"confidence":"High","gaps":["Cellular substrate specificity beyond ARF1 not defined","Functional distinction between long/short variants only assayed via receptor sequestration"]},{"year":2001,"claim":"Defined how GIT2 reaches focal adhesions and what it does there—paxillin LD4 binding targets it to focal contacts to restrain Rac and enforce directional motility, while its GAP activity reorganizes Golgi and adhesions.","evidence":"Reciprocal deletion mutants in fibroblasts, GAP-inactive mutant rescue, immunofluorescence and Rac/motility assays","pmids":["11448998","11251077"],"confidence":"High","gaps":["Direct in vivo ARF1 GTP-loading not measured","Link between Golgi reorganization and motility phenotype not mechanistically connected"]},{"year":2001,"claim":"Showed the PAK1–β-PIX–GIT2 module is a stable trimolecular complex activated downstream of receptor signaling, placing PIX GEF activity within the complex upstream of Rho/PAK.","evidence":"Co-IP and dominant-negative PIX in TCR-stimulated Jurkat cells with kinase inhibitors and adaptor-null lines","pmids":["11157752"],"confidence":"High","gaps":["Stoichiometry of the complex not determined","Direct GIT2 contribution to complex assembly versus PIX/PAK not isolated"]},{"year":2003,"claim":"Integrated Crk/CrkL adaptors into the paxillin/GIT2/β-PIX complex, linking the scaffold to Rac-dependent paxillin relocalization and lamellipodia formation.","evidence":"Reciprocal Co-IP, dominant-negative Crk mutants, Rac inhibition, immunofluorescence","pmids":["12857867"],"confidence":"High","gaps":["Direct versus indirect Crk-GIT2 contact not resolved","Quantitative ordering of complex assembly steps unknown"]},{"year":2006,"claim":"Demonstrated endogenous GIT2 is a brake on motility, repressing lamellipodia via Rac1 and focal adhesion turnover via Cdc42 with Crk as the essential effector and β-PIX dispensable, refining the pathway logic.","evidence":"siRNA knockdown in MCF10A epithelial cells with epistasis across Rac1, Cdc42, Crk, β-PIX","pmids":["16628223"],"confidence":"High","gaps":["Mechanism by which GIT2 selectively suppresses Crk not defined","Why β-PIX is dispensable here but required in other contexts unresolved"]},{"year":2006,"claim":"Extended GIT2's motility-suppressing role into innate immunity in vivo, showing it acts downstream of Gβγ and upstream of ARF1 to direct neutrophil chemotaxis and limit superoxide, with knockout causing immunodeficiency.","evidence":"GIT2-knockout mouse neutrophils, chemotaxis and superoxide assays, epistasis","pmids":["16715100"],"confidence":"High","gaps":["Molecular link between Gβγ and GIT2 recruitment not identified","Mechanism of superoxide suppression not detailed"]},{"year":2010,"claim":"Confirmed conserved GIT2 control of cell movement in distinct in vivo systems—negatively regulating Rac-driven thymocyte motility and controlling myosin II contractility via MLC phosphorylation in gastrulation.","evidence":"Git2-knockout mice with intravital two-photon imaging; zebrafish morpholino with chicken GIT2 rescue and MLC phosphorylation/blebbistatin assays","pmids":["20431621","21034731"],"confidence":"High","gaps":["How GIT2 regulates MLC phosphorylation mechanistically unknown","Connection between Rac suppression and contractility control not unified"]},{"year":2013,"claim":"Identified a phosphorylation-dependent GIT2-Vav2 feedforward loop downstream of integrin and EGF that controls Rac1 activation and directional persistence, and linked GIT2 loss to EMT via a miR-146a/Cnot6L/Zeb1 axis.","evidence":"Co-IP, knockdown of both partners, Rac1 assays, migration directionality; separate miR-146a maturation and Zeb1 mRNA stability assays","pmids":["23615439","23591815"],"confidence":"High","gaps":["Kinase phosphorylating GIT2 to drive Vav2 interaction not identified","EMT/miR-146a axis is Medium-confidence and pathway not independently confirmed"]},{"year":2014,"claim":"Revealed a non-cytoskeletal role: GIT2 terminates TLR-induced NF-κB/MAPK signaling by recruiting CYLD to deubiquitinate TRAF6, establishing it as a negative regulator of inflammation.","evidence":"Git2-knockout mice/macrophages, TLR stimulation, GIT2-CYLD Co-IP, TRAF6 ubiquitination assay","pmids":["24879442"],"confidence":"High","gaps":["Whether ARF-GAP activity is required for CYLD recruitment unknown","Direct GIT2-TRAF6 contact not established"]},{"year":2015,"claim":"Placed GIT2 in genome maintenance, showing a nuclear pool is phosphorylated by ATM and recruited to DSBs via H2AX/ATM/MRE11 to stabilize BRCA1 and promote repair.","evidence":"Nuclear fractionation, Co-IP with DDR factors, ATM kinase assay, dependency analysis, GIT2-KO mouse irradiation","pmids":["25605334"],"confidence":"High","gaps":["ATM phosphorylation sites not mapped","How a cytoskeletal scaffold mechanistically stabilizes BRCA1 unresolved"]},{"year":2016,"claim":"Refined the migration machinery and GIT2 regulation: GIT2 restrains DOCK5-Crk-p130Cas signaling and FA turnover under Rho-ROCK contractility, while RUSC2 (and Rab35) and the insulin receptor/IRS-2 control GIT2 stability and tissue-specific functions.","evidence":"Co-IP of GIT2-DOCK5-Crk, ROCK/MLC inhibition, invasion assays; RUSC2/Rab35 silencing and stability assays; GIT2-IR/IRS-2 Co-IP and KO mouse metabolic phenotyping","pmids":["27669437","27238570","26834700"],"confidence":"High","gaps":["RUSC2 and insulin receptor findings are Medium-confidence and single-lab","Mechanism of GIT2 stabilization/degradation control incomplete"]},{"year":2024,"claim":"Established a centrosomal function distinct from GIT1: PKC phosphorylation of GIT2 at S46 in the ArfGAP domain drives centrosomal localization where GIT2 negatively regulates γ-tubulin-dependent microtubule nucleation.","evidence":"shRNA depletion, Co-IP with γTuRC components, microtubule nucleation assays, S46 mutagenesis and PKC inhibitors in mast cells and glioblastoma","pmids":["38370406","40176062"],"confidence":"High","gaps":["Whether ARF-GAP catalytic activity is required for nucleation control unknown","Mechanism of negative regulation of γTuRC not defined"]},{"year":2025,"claim":"Extended NF-κB suppression to direct subunit binding and exposed a disease-relevant splicing axis: GIT2 binds p65 and p52 and elevates TRAF3 to suppress NF-κB and senescence, while THRAP3/SLU7-driven Exon14 skipping yields a variant conferring ferroptosis resistance in AML.","evidence":"GIT2-p65/p52 Co-IP, BMSC functional assays and ovariectomy model; THRAP3-SLU7 Co-IP, Exon14 splicing assays, ferroptosis and orthotopic tumor models","pmids":["39954559","41326370"],"confidence":"Medium","gaps":["Both are Medium-confidence single-lab studies","Functional contribution of specific GIT2 splice variants to NF-κB and ferroptosis not reconciled"]},{"year":null,"claim":"How GIT2's distinct functional pools—focal adhesion scaffold, nuclear DDR factor, NF-κB suppressor, and centrosomal regulator—are coordinated, and whether its ARF-GAP catalytic activity is required for each, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking cytoskeletal, nuclear, immune, and centrosomal roles","Catalytic requirement for non-adhesion functions untested","No structural model of GIT2 in any complex"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,5]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[1,3,4,11]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,6]}],"localization":[{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[12]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[17,20]},{"term_id":"GO:0005794","term_label":"Golgi apparatus","supporting_discovery_ids":[2,14]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,3,9]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,7,11,18]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[12]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[8]}],"complexes":["paxillin–β-PIX–PAK1 (GIT2/PKL) complex","γ-tubulin ring complex (γTuRC) association"],"partners":["PXN","ARHGEF7","PAK1","CRK","VAV2","DOCK5","CYLD","GRK2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14161","full_name":"ARF GTPase-activating protein GIT2","aliases":["Cool-interacting tyrosine-phosphorylated protein 2","CAT-2","CAT2","G protein-coupled receptor kinase-interactor 2","GRK-interacting protein 2"],"length_aa":759,"mass_kda":84.5,"function":"GTPase-activating protein for ADP ribosylation factor family members, including ARF1","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q14161/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/GIT2","classification":"Not Classified","n_dependent_lines":10,"n_total_lines":1208,"dependency_fraction":0.008278145695364239},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ARHGEF7","stoichiometry":10.0},{"gene":"PAK1","stoichiometry":10.0},{"gene":"PAK2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/GIT2","total_profiled":1310},"omim":[{"mim_id":"608564","title":"GIT ArfGAP 2; GIT2","url":"https://www.omim.org/entry/608564"},{"mim_id":"608434","title":"GIT ArfGAP 1; GIT1","url":"https://www.omim.org/entry/608434"},{"mim_id":"300724","title":"CONNECTOR ENHANCER OF KINASE SUPPRESSOR OF RAS 2; CNKSR2","url":"https://www.omim.org/entry/300724"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Microtubules","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GIT2"},"hgnc":{"alias_symbol":["KIAA0148","PKL"],"prev_symbol":[]},"alphafold":{"accession":"Q14161","domains":[{"cath_id":"1.10.220.150","chopping":"8-113","consensus_level":"high","plddt":93.754,"start":8,"end":113},{"cath_id":"1.25.40.20","chopping":"128-226","consensus_level":"high","plddt":97.1352,"start":128,"end":226},{"cath_id":"-","chopping":"262-359","consensus_level":"high","plddt":91.0169,"start":262,"end":359},{"cath_id":"1.20.120.330","chopping":"641-759","consensus_level":"high","plddt":87.5258,"start":641,"end":759}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14161","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14161-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14161-F1-predicted_aligned_error_v6.png","plddt_mean":75.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GIT2","jax_strain_url":"https://www.jax.org/strain/search?query=GIT2"},"sequence":{"accession":"Q14161","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14161.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14161/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14161"}},"corpus_meta":[{"pmid":"11448998","id":"PMC_11448998","title":"The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).","date":"2001","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11448998","citation_count":144,"is_preprint":false},{"pmid":"10896954","id":"PMC_10896954","title":"The GIT family of ADP-ribosylation factor GTPase-activating proteins. 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possesses ARF GTPase-activating protein (ARF-GAP) activity toward ARF1 in vitro, interacts with G protein-coupled receptor kinase 2 (GRK2), and interacts with PIX-PAK complexes. The longest GIT2 variant inhibits beta2-adrenergic receptor sequestration when overexpressed, whereas the shortest splice variant is inactive in this assay.\",\n      \"method\": \"In vitro GAP assay, co-immunoprecipitation, cellular overexpression with receptor sequestration readout\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro ARF-GAP activity demonstrated with multiple variants, co-IP binding partners identified, functional receptor sequestration assay in cells; multiple orthogonal methods in one study\",\n      \"pmids\": [\"10896954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"GIT2/PKL (paxillin kinase linker) interacts with paxillin via the paxillin LD4 motif; this interaction is required for PKL localization to focal contacts and for normal Rac-dependent cell spreading and directional motility. Loss of the paxillin-PKL interaction leads to prolonged Rac activation, multiple broad lamellipodia, and impaired directional motility without affecting FAK activity.\",\n      \"method\": \"Overexpression of deletion mutants (paxillinΔLD4, PKLδPBS2) in CHO.K1 fibroblasts; immunofluorescence localization; cell spreading/motility assays; Rac activity measurements\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal deletion mutant approach with multiple orthogonal readouts (localization, Rac activity, motility), consistent with parallel studies\",\n      \"pmids\": [\"11448998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"GIT2-short (KIAA0148), a short isoform of GIT2, co-localizes with paxillin at perinuclear areas and acts as an ARF-GAP for ARF1 in vivo. Overexpression of wild-type GIT2-short (but not its GAP-inactive mutant) redistributes Golgi protein β-COP, reduces focal adhesions and actin stress fibers, and alters perinuclear paxillin localization, demonstrating that the ARF-GAP catalytic activity is required for these effects.\",\n      \"method\": \"Overexpression and GAP-inactive mutant rescue in cells; immunofluorescence co-localization; Golgi distribution assay; focal adhesion and actin stress fiber quantification\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — catalytic mutant used to assign phenotype to GAP activity, multiple orthogonal cellular readouts in one study\",\n      \"pmids\": [\"11251077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PAK1, PIX (β-PIX), and p95PKL (GIT2) form a stable trimolecular complex in T cells. This complex is activated downstream of the T-cell receptor via ZAP-70/Syk kinases and a LAT/Slp-76-independent pathway; PIX GEF activity within the complex is required for Rho GTPase activation upstream of PAK1.\",\n      \"method\": \"Co-immunoprecipitation demonstrating trimolecular complex; dominant-negative PIX overexpression; TCR stimulation assays with kinase inhibitors and adaptor-deficient Jurkat cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — stable trimolecular complex demonstrated by Co-IP, functional dissection using dominant-negative and adaptor-null cell lines with multiple readouts\",\n      \"pmids\": [\"11157752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"CrkII/CrkL adapter proteins associate with a Paxillin/GIT2/β-PIX complex; this association promotes Rac-dependent relocalization of paxillin to focal contacts and lamellipodia formation. Paxillin mutants unable to associate with Crk or GIT2 block Crk-dependent cell spreading.\",\n      \"method\": \"Co-immunoprecipitation; stable cell line overexpression; dominant-negative Crk mutants; Rac inhibition; immunofluorescence localization\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP establishing complex, multiple mutant controls, Rac-dependence tested orthogonally\",\n      \"pmids\": [\"12857867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Endogenous GIT2 represses lamellipodial extension via Rac1-dependent signaling and represses focal adhesion turnover via Cdc42-dependent signaling. GIT2 knockdown is sufficient to induce migration of non-transformed MCF10A epithelial cells. The SH2-SH3 adaptor Crk is identified as an essential downstream target of GIT2 inhibition, whereas β-PIX is dispensable for GIT2-mediated effects.\",\n      \"method\": \"siRNA knockdown of endogenous GIT2; lamellipodia and FA turnover assays; epistasis with Rac1, Cdc42, Crk, and β-PIX\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis by knockdown of endogenous protein, multiple pathway components tested with defined phenotypic readouts, negative result for β-PIX also mechanistically informative\",\n      \"pmids\": [\"16628223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"GIT2 is required in neutrophils for directional chemotaxis and for suppression of superoxide production in response to G protein-coupled receptor stimulation. GIT2 suppresses ARF1 activity and functions downstream of Gβγ subunits in the direction-sensing machinery. Loss of GIT2 in vivo leads to an immunodeficient state.\",\n      \"method\": \"GIT2-knockout mouse neutrophils; directional chemotaxis assays; superoxide production measurements; epistasis placing GIT2 downstream of Gβγ and upstream of ARF1\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with multiple orthogonal functional readouts, in vivo confirmation\",\n      \"pmids\": [\"16715100\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"GIT2 is required for efficient thymocyte positive selection. Git2-/- double-positive thymocytes show elevated Rac GTPase activation, increased actin polymerization, and enhanced chemokine-directed migration in vitro. Two-photon microscopy revealed that scanning activity of Git2-/- thymocytes was compromised in the thymic cortex, indicating GIT2 negatively regulates Rac-mediated chemotactic motility in thymocytes.\",\n      \"method\": \"Git2-knockout mice; Rac activation assay; actin polymerization assay; in vitro chemotaxis; two-photon laser-scanning microscopy in intact thymus\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout mouse with multiple orthogonal in vitro and intravital imaging readouts\",\n      \"pmids\": [\"20431621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Zebrafish Git2a is required for cell movements during gastrulation; its depletion arrests directed cell migration toward the vegetal pole and reduces cell contractility. Git2a regulates phosphorylation of myosin light chain (MLC), thereby controlling myosin II-mediated cell contractility. The phenotype is rescued by chicken GIT2, confirming functional conservation.\",\n      \"method\": \"Antisense morpholino knockdown; time-lapse microscopy; myosin light chain phosphorylation assay; pharmacological inhibition with Blebbistatin; rescue with chicken GIT2\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockdown with molecular rescue, direct phosphorylation readout, pharmacological epistasis with myosin II inhibitor\",\n      \"pmids\": [\"21034731\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PKL/GIT2 regulates activity of the Rac1 GEF Vav2 through a phosphorylation-dependent interaction. PKL is required for Vav2 activation downstream of integrin engagement and EGF stimulation. Vav2 in turn regulates redistribution of PKL and β-PIX to focal adhesions after EGF stimulation, forming a feedforward signaling loop. Vav2 knockdown reduces directional persistence and polarization of migrating cells.\",\n      \"method\": \"Co-immunoprecipitation; PKL and Vav2 knockdown; Rac1 activation assays; immunofluorescence localization of PKL and β-PIX; cell migration directionality assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, knockdown of both partners, multiple orthogonal functional readouts in one study\",\n      \"pmids\": [\"23615439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Loss of Git2 promotes epithelial-mesenchymal transition (EMT) through a pathway involving enhanced maturation of miR-146a, which suppresses Cnot6L (a deadenylase), leading to stabilization of Zeb1 mRNA and increased Zeb1 expression.\",\n      \"method\": \"Git2 knockdown/knockout; miR-146a maturation assay; Cnot6L manipulation; Zeb1 mRNA stability assay; EMT marker analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pathway dissection with multiple components tested, single lab, abstract does not fully detail all controls\",\n      \"pmids\": [\"23591815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GIT2 negatively regulates TLR-induced NF-κB and MAPK signaling by recruiting the deubiquitinating enzyme Cylindromatosis (CYLD) to inhibit K63-linked ubiquitination of TRAF6, thereby terminating downstream inflammatory signaling. Git2-deficient mice and macrophages show dramatically increased pro-inflammatory cytokine production in response to TLR stimulation.\",\n      \"method\": \"Git2-knockout mice and macrophages; TLR stimulation assays; NF-κB and MAPK activation measurements; Co-immunoprecipitation of GIT2-CYLD complex; TRAF6 ubiquitination assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP demonstrating GIT2-CYLD complex, ubiquitination assay, in vivo knockout confirmation, multiple orthogonal methods\",\n      \"pmids\": [\"24879442\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"GIT2 localizes to the nucleus, is phosphorylated by ATM kinase following DNA damage, and forms complexes with multiple DNA damage response (DDR) factors. GIT2 targeting to DNA double-strand breaks depends on H2AX, ATM, and MRE11 but is independent of MDC1 and RNF8. GIT2 promotes DNA repair by stabilizing BRCA1 in repair complexes, upregulating HMGN1 and RFC1, and regulating PARP activity. GIT2-knockout mice show increased susceptibility to irradiation-induced DNA damage.\",\n      \"method\": \"Nuclear fractionation; Co-immunoprecipitation with DDR factors; ATM kinase phosphorylation assay; DDR factor dependency analysis (H2AX/ATM/MRE11/MDC1/RNF8); GIT2-KO mice irradiation; PARP activity assay; immunofluorescence foci analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, kinase assay, KO mice), epistasis with multiple DDR components, functional rescue experiments\",\n      \"pmids\": [\"25605334\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GIT2 restricts focal adhesion recruitment of DOCK5 and inhibits DOCK5 interaction with Crk, thereby suppressing DOCK5-dependent activation of the Crk-p130Cas signaling cascade, Rac1-mediated lamellipodial protrusion, and FA turnover. GIT2 is recruited to focal adhesions in response to Rho-ROCK signaling and actomyosin contractility.\",\n      \"method\": \"GIT2 knockdown/overexpression; Co-immunoprecipitation of GIT2-DOCK5-Crk interactions; ROCK and MLC inhibition; Rac1 activation assay; invasion assays in epithelial cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP demonstrating GIT2-DOCK5-Crk interactions, pharmacological pathway dissection, multiple cell line validation\",\n      \"pmids\": [\"27669437\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RUSC2 interacts with the Spa Homology Domain (SHD) of GIT2 in lung cancer cells, stabilizes GIT2 by decreasing its degradation and increasing its phosphorylation, and promotes Golgi reorientation and directional migration. EGF stimulation transiently increases RUSC2-GIT2 interaction, while prolonged EGF stimulation decreases it via Rab35 activation. Rab35 silencing reduces GIT2 stability and phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation (RUSC2-GIT2 interaction); RUSC2 and Rab35 silencing; GIT2 stability assays; Golgi reorientation assay; directional migration assay\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with SHD domain specificity, functional knockdown experiments, single lab\",\n      \"pmids\": [\"27238570\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GIT2 physically interacts with the insulin receptor and insulin receptor substrate 2 (IRS-2) in pancreatic tissue; this interaction is diminished in diabetic db/db mice. Genomic deletion of GIT2 disrupts pancreatic beta cell mass and reduces insulin secretion, leading to elevated plasma glucose and insulin resistance.\",\n      \"method\": \"Co-immunoprecipitation of GIT2 with insulin receptor and IRS-2; GIT2-KO mouse metabolic phenotyping; pancreatic islet transcriptomics\",\n      \"journal\": \"Frontiers in endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating direct interaction, KO mouse metabolic phenotype, single lab\",\n      \"pmids\": [\"26834700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GIT2/PKL mediates endothelial progenitor cell (EPC) migration downstream of CXCR2 via the Src-PKL/Vav2-Rac1 signaling pathway. Phosphorylation and co-localization of PKL and Vav2 are required for Rac1 activation and development of lamellipodia/filopodia driving EPC migration.\",\n      \"method\": \"Transwell migration assays; shRNA knockdown; signaling inhibitors; immunofluorescence co-localization of PKL and Vav2; Rac1 phosphorylation assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockdown and inhibitor-based pathway dissection, co-localization, single lab\",\n      \"pmids\": [\"29229683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"GIT2 associates with centrosomes and γ-tubulin complex proteins in mast cells. Depletion of GIT2 enhances centrosomal microtubule nucleation. Phosphorylation of GIT2 by conventional protein kinase C (PKC) promotes its centrosomal localization and microtubule nucleation during FcεRI-induced activation. GIT2 (unlike GIT1) acts as a negative regulator of microtubule nucleation in mast cells and also participates in antigen-induced degranulation and chemotaxis.\",\n      \"method\": \"shRNA depletion; immunofluorescence and Co-IP with γ-tubulin complex proteins; time-lapse microtubule nucleation assay; PKC inhibitor treatment; site-directed mutagenesis of phosphorylation sites; phenotypic rescue\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — mutagenesis of phosphorylation sites, reconstitution-level kinase assay, Co-IP with γTuRC components, functional rescue; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"38370406\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GIT2 directly binds NF-κB components p65 (canonical) and p52 (non-canonical) to inhibit their activation, and positively regulates TRAF3 expression to further suppress both canonical and non-canonical NF-κB signaling. GIT2 thereby promotes osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and alleviates DNA damage-induced cellular senescence.\",\n      \"method\": \"Co-immunoprecipitation (GIT2 with p65 and p52); Western blotting of NF-κB pathway components; GIT2 overexpression/knockdown in BMSCs; comet assay; in vivo ovariectomy mouse model; micro-CT bone analysis\",\n      \"journal\": \"Tissue & cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating direct binding to p65/p52, multiple functional assays in vitro and in vivo, single lab\",\n      \"pmids\": [\"39954559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"THRAP3 recruits the splicing factor SLU7 to facilitate skipping of GIT2 Exon14, generating a GIT2 splice variant that promotes ferroptosis resistance in AML cells by inhibiting iron accumulation and promoting GSH synthesis. Inhibition of GIT2 Exon14 skipping reverses THRAP3-induced ferroptosis resistance in vitro and in vivo.\",\n      \"method\": \"THRAP3 knockdown/overexpression; Co-immunoprecipitation of THRAP3-SLU7; RT-PCR/splicing assays for Exon14; ferroptosis assays (RSL3/erastin); iron and GSH measurement; orthotopic mouse models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of THRAP3-SLU7 complex, splicing assay for GIT2 exon14, functional rescue, in vivo tumor model; single lab\",\n      \"pmids\": [\"41326370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In glioblastoma cells, GIT2 associates with γ-tubulin ring complex (γTuRC) proteins and localizes to centrosomes. Depletion of GIT2 enhances centrosomal microtubule nucleation. The N-terminal ArfGAP domain of GIT2 is responsible for centrosomal localization and regulation of microtubule nucleation. PKC phosphorylates GIT2 at serine 46 (S46) on the ArfGAP domain, and phosphomimetic S46 promotes microtubule nucleation.\",\n      \"method\": \"shRNA depletion; immunofluorescence; time-lapse microtubule nucleation; immunoprecipitation with γTuRC components; site-directed mutagenesis (S46); kinase assay with PKC inhibitors\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — site-directed mutagenesis identifying specific phosphorylation site (S46), kinase assay, Co-IP with γTuRC, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"40176062\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GIT2 (also known as PKL/KIAA0148) is a multifunctional ARF GTPase-activating protein (ARF-GAP) that scaffolds signaling complexes at focal adhesions, centrosomes, and the nucleus: it inactivates ARF1 to control Golgi organization and focal adhesion dynamics; associates with paxillin (via LD4), β-PIX, PAK1, Crk, DOCK5, Vav2, and GRK2 to spatiotemporally regulate Rac1/Cdc42 activity and thereby control cell spreading, polarity, and directional migration; is phosphorylated by ATM at DNA double-strand breaks to promote DDR complex assembly and repair; is phosphorylated by PKC at S46 of its ArfGAP domain to regulate centrosomal microtubule nucleation; recruits CYLD to deubiquitinate TRAF6 and terminate TLR-induced NF-κB signaling; and directly binds p65/p52 NF-κB subunits to suppress inflammatory gene activation, collectively establishing GIT2 as a signaling hub linking cytoskeletal remodeling, immune signaling, and genome integrity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GIT2 is a multifunctional ARF GTPase-activating protein that operates as a signaling scaffold coupling cytoskeletal remodeling to cell migration, immune signaling, and genome maintenance [#0, #5]. Through its ARF-GAP domain it inactivates ARF1 to control Golgi organization and focal adhesion/actin architecture, an activity that is catalytically required for these effects [#2]. GIT2 nucleates a paxillin–\\u03b2-PIX–PAK1 complex—binding paxillin via the LD4 motif to localize at focal contacts—and integrates this module with Crk/CrkL adaptors, the Rac1 GEF Vav2, and DOCK5 to spatiotemporally restrain Rac1 and Cdc42 activity; loss of GIT2 produces sustained Rac activation, excess lamellipodia, faster focal adhesion turnover, and loss of directional motility [#1, #3, #4, #5, #9, #13]. Consistent with a brake on Rac-driven motility, GIT2 acts downstream of G\\u03b2\\u03b3 and CXCR2 to enforce directional chemotaxis and suppress superoxide in neutrophils, governs thymocyte selection and migration, and controls myosin II-mediated contractility during gastrulation [#6, #7, #8, #16]. Beyond the cytoskeleton, GIT2 is a negative regulator of inflammatory signaling: it recruits the deubiquitinase CYLD to remove K63-linked ubiquitin from TRAF6 to terminate TLR-induced NF-\\u03baB/MAPK signaling, and directly binds the NF-\\u03baB subunits p65 and p52 while elevating TRAF3 to suppress canonical and non-canonical NF-\\u03baB output [#11, #18]. A nuclear pool of GIT2 is phosphorylated by ATM and recruited to DNA double-strand breaks in an H2AX/MRE11-dependent manner, where it stabilizes BRCA1 and promotes repair [#12]. PKC-mediated phosphorylation at S46 in the ArfGAP domain drives centrosomal localization where GIT2 negatively regulates \\u03b3-tubulin-dependent microtubule nucleation [#17, #20].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established GIT2's core biochemical identity by showing it is an ARF1-directed GAP that also binds GRK2 and PIX-PAK complexes and regulates receptor sequestration, defining it as both an enzyme and a scaffold.\",\n      \"evidence\": \"In vitro GAP assay, co-immunoprecipitation, and receptor sequestration readout across splice variants\",\n      \"pmids\": [\"10896954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular substrate specificity beyond ARF1 not defined\", \"Functional distinction between long/short variants only assayed via receptor sequestration\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Defined how GIT2 reaches focal adhesions and what it does there—paxillin LD4 binding targets it to focal contacts to restrain Rac and enforce directional motility, while its GAP activity reorganizes Golgi and adhesions.\",\n      \"evidence\": \"Reciprocal deletion mutants in fibroblasts, GAP-inactive mutant rescue, immunofluorescence and Rac/motility assays\",\n      \"pmids\": [\"11448998\", \"11251077\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct in vivo ARF1 GTP-loading not measured\", \"Link between Golgi reorganization and motility phenotype not mechanistically connected\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showed the PAK1\\u2013\\u03b2-PIX\\u2013GIT2 module is a stable trimolecular complex activated downstream of receptor signaling, placing PIX GEF activity within the complex upstream of Rho/PAK.\",\n      \"evidence\": \"Co-IP and dominant-negative PIX in TCR-stimulated Jurkat cells with kinase inhibitors and adaptor-null lines\",\n      \"pmids\": [\"11157752\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the complex not determined\", \"Direct GIT2 contribution to complex assembly versus PIX/PAK not isolated\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Integrated Crk/CrkL adaptors into the paxillin/GIT2/\\u03b2-PIX complex, linking the scaffold to Rac-dependent paxillin relocalization and lamellipodia formation.\",\n      \"evidence\": \"Reciprocal Co-IP, dominant-negative Crk mutants, Rac inhibition, immunofluorescence\",\n      \"pmids\": [\"12857867\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct versus indirect Crk-GIT2 contact not resolved\", \"Quantitative ordering of complex assembly steps unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated endogenous GIT2 is a brake on motility, repressing lamellipodia via Rac1 and focal adhesion turnover via Cdc42 with Crk as the essential effector and \\u03b2-PIX dispensable, refining the pathway logic.\",\n      \"evidence\": \"siRNA knockdown in MCF10A epithelial cells with epistasis across Rac1, Cdc42, Crk, \\u03b2-PIX\",\n      \"pmids\": [\"16628223\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which GIT2 selectively suppresses Crk not defined\", \"Why \\u03b2-PIX is dispensable here but required in other contexts unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Extended GIT2's motility-suppressing role into innate immunity in vivo, showing it acts downstream of G\\u03b2\\u03b3 and upstream of ARF1 to direct neutrophil chemotaxis and limit superoxide, with knockout causing immunodeficiency.\",\n      \"evidence\": \"GIT2-knockout mouse neutrophils, chemotaxis and superoxide assays, epistasis\",\n      \"pmids\": [\"16715100\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link between G\\u03b2\\u03b3 and GIT2 recruitment not identified\", \"Mechanism of superoxide suppression not detailed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Confirmed conserved GIT2 control of cell movement in distinct in vivo systems—negatively regulating Rac-driven thymocyte motility and controlling myosin II contractility via MLC phosphorylation in gastrulation.\",\n      \"evidence\": \"Git2-knockout mice with intravital two-photon imaging; zebrafish morpholino with chicken GIT2 rescue and MLC phosphorylation/blebbistatin assays\",\n      \"pmids\": [\"20431621\", \"21034731\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How GIT2 regulates MLC phosphorylation mechanistically unknown\", \"Connection between Rac suppression and contractility control not unified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified a phosphorylation-dependent GIT2-Vav2 feedforward loop downstream of integrin and EGF that controls Rac1 activation and directional persistence, and linked GIT2 loss to EMT via a miR-146a/Cnot6L/Zeb1 axis.\",\n      \"evidence\": \"Co-IP, knockdown of both partners, Rac1 assays, migration directionality; separate miR-146a maturation and Zeb1 mRNA stability assays\",\n      \"pmids\": [\"23615439\", \"23591815\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Kinase phosphorylating GIT2 to drive Vav2 interaction not identified\", \"EMT/miR-146a axis is Medium-confidence and pathway not independently confirmed\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Revealed a non-cytoskeletal role: GIT2 terminates TLR-induced NF-\\u03baB/MAPK signaling by recruiting CYLD to deubiquitinate TRAF6, establishing it as a negative regulator of inflammation.\",\n      \"evidence\": \"Git2-knockout mice/macrophages, TLR stimulation, GIT2-CYLD Co-IP, TRAF6 ubiquitination assay\",\n      \"pmids\": [\"24879442\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ARF-GAP activity is required for CYLD recruitment unknown\", \"Direct GIT2-TRAF6 contact not established\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed GIT2 in genome maintenance, showing a nuclear pool is phosphorylated by ATM and recruited to DSBs via H2AX/ATM/MRE11 to stabilize BRCA1 and promote repair.\",\n      \"evidence\": \"Nuclear fractionation, Co-IP with DDR factors, ATM kinase assay, dependency analysis, GIT2-KO mouse irradiation\",\n      \"pmids\": [\"25605334\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ATM phosphorylation sites not mapped\", \"How a cytoskeletal scaffold mechanistically stabilizes BRCA1 unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Refined the migration machinery and GIT2 regulation: GIT2 restrains DOCK5-Crk-p130Cas signaling and FA turnover under Rho-ROCK contractility, while RUSC2 (and Rab35) and the insulin receptor/IRS-2 control GIT2 stability and tissue-specific functions.\",\n      \"evidence\": \"Co-IP of GIT2-DOCK5-Crk, ROCK/MLC inhibition, invasion assays; RUSC2/Rab35 silencing and stability assays; GIT2-IR/IRS-2 Co-IP and KO mouse metabolic phenotyping\",\n      \"pmids\": [\"27669437\", \"27238570\", \"26834700\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RUSC2 and insulin receptor findings are Medium-confidence and single-lab\", \"Mechanism of GIT2 stabilization/degradation control incomplete\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a centrosomal function distinct from GIT1: PKC phosphorylation of GIT2 at S46 in the ArfGAP domain drives centrosomal localization where GIT2 negatively regulates \\u03b3-tubulin-dependent microtubule nucleation.\",\n      \"evidence\": \"shRNA depletion, Co-IP with \\u03b3TuRC components, microtubule nucleation assays, S46 mutagenesis and PKC inhibitors in mast cells and glioblastoma\",\n      \"pmids\": [\"38370406\", \"40176062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ARF-GAP catalytic activity is required for nucleation control unknown\", \"Mechanism of negative regulation of \\u03b3TuRC not defined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended NF-\\u03baB suppression to direct subunit binding and exposed a disease-relevant splicing axis: GIT2 binds p65 and p52 and elevates TRAF3 to suppress NF-\\u03baB and senescence, while THRAP3/SLU7-driven Exon14 skipping yields a variant conferring ferroptosis resistance in AML.\",\n      \"evidence\": \"GIT2-p65/p52 Co-IP, BMSC functional assays and ovariectomy model; THRAP3-SLU7 Co-IP, Exon14 splicing assays, ferroptosis and orthotopic tumor models\",\n      \"pmids\": [\"39954559\", \"41326370\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Both are Medium-confidence single-lab studies\", \"Functional contribution of specific GIT2 splice variants to NF-\\u03baB and ferroptosis not reconciled\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GIT2's distinct functional pools—focal adhesion scaffold, nuclear DDR factor, NF-\\u03baB suppressor, and centrosomal regulator—are coordinated, and whether its ARF-GAP catalytic activity is required for each, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking cytoskeletal, nuclear, immune, and centrosomal roles\", \"Catalytic requirement for non-adhesion functions untested\", \"No structural model of GIT2 in any complex\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 5]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [1, 3, 4, 11]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [17, 20]},\n      {\"term_id\": \"GO:0005794\", \"supporting_discovery_ids\": [2, 14]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 3, 9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 7, 11, 18]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\n      \"paxillin\\u2013\\u03b2-PIX\\u2013PAK1 (GIT2/PKL) complex\",\n      \"\\u03b3-tubulin ring complex (\\u03b3TuRC) association\"\n    ],\n    \"partners\": [\n      \"PXN\",\n      \"ARHGEF7\",\n      \"PAK1\",\n      \"CRK\",\n      \"VAV2\",\n      \"DOCK5\",\n      \"CYLD\",\n      \"GRK2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}