{"gene":"ECT2","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1993,"finding":"ECT2 protein contains a central DH-domain-related core with sequence similarity to BCR, CDC24, and DBL oncogene products; baculovirus-expressed ECT2 binds specifically to Rho and Rac proteins, identifying it as a member of the Rho GTPase regulatory family with transforming potential activated by N-terminal truncation.","method":"Expression cloning, baculovirus protein expression, direct binding assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro binding assay with purified protein, foundational study replicated by subsequent work","pmids":["8464478"],"is_preprint":false},{"year":1999,"finding":"Human ECT2 catalyzes guanine nucleotide exchange on RhoA, Rac1, and Cdc42 in vitro; ECT2 is phosphorylated during G2/M phases and phosphorylation is required for its exchange activity; ECT2 localizes to the nucleus in interphase, spreads to cytoplasm in prometaphase, and concentrates at the midbody during cytokinesis; expression of the N-terminal domain (lacking catalytic activity) or microinjection of anti-ECT2 antibody inhibits cytokinesis.","method":"GEF activity assay (in vitro nucleotide exchange), cell synchronization, immunofluorescence, microinjection, dominant-negative expression","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro GEF assay plus multiple orthogonal functional experiments; foundational paper replicated extensively","pmids":["10579713"],"is_preprint":false},{"year":2000,"finding":"GTP-bound RhoA accumulates during cytokinesis (peaking at telophase); expression of dominant-negative ECT2 completely suppresses both the rise in GTP-RhoA at telophase and increased GDP-GTP exchange activity in mitotic cell extracts, establishing ECT2 as a critical activator of RhoA during cytokinesis.","method":"RhoA-GTP pull-down assay, cell cycle synchronization, dominant-negative ECT2 expression","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — quantitative pull-down assay with dominant-negative rescue; independently replicated","pmids":["10837491"],"is_preprint":false},{"year":2003,"finding":"Oncogenic activation of ECT2 requires both removal of the N-terminal negative regulatory domain AND mislocalization from the nucleus to the cytoplasm; the N-terminal domain interacts with the catalytic domain and inhibits GEF activity; nuclear localization signals in the central domain are required to maintain nuclear ECT2; RhoA is the predominant Rho GTPase activated by oncogenic ECT2 in NIH 3T3 cells.","method":"Focus formation assay, deletion/NLS mutagenesis, dominant-negative Rho GTPase co-expression, subcellular fractionation, in vivo RhoA activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple domain deletion and mutagenesis experiments with functional readouts; replicated in subsequent work","pmids":["14645260"],"is_preprint":false},{"year":2004,"finding":"The N-terminal tandem BRCT domains of ECT2 maintain the protein in an inactive conformation through an intramolecular interaction with the C-terminal catalytic domain, masking GEF activity toward RhoA; both BRCT domains are required for negative regulation (interphase) and positive regulation (cytokinesis function).","method":"siRNA knockdown, dominant-negative and deletion mutant expression, multinucleation assay, co-immunoprecipitation of intramolecular BRCT-DH interaction","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal intramolecular interaction mapped by co-IP plus functional rescue experiments","pmids":["15545273"],"is_preprint":false},{"year":2004,"finding":"Sequences C-terminal to the PH domain of ECT2 alter the profile of Rho GTPases activated in vivo: removal of C-terminal sequences (DeltaN-Ect2 DH/PH) activates only RhoA and enhances stress fiber formation, whereas retention of C-terminal sequences (DeltaN-Ect2 DH/PH/C) activates RhoA, Rac1, and Cdc42 and induces lamellipodia.","method":"NIH 3T3 transformation assay, Rho GTPase activity pull-down, actin morphology analysis, C-terminal deletion mutagenesis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple GTPase activity assays and morphological readouts, single lab","pmids":["15073184"],"is_preprint":false},{"year":2004,"finding":"ECT2 interacts with Par6 and Par3 of the polarity complex and with PKCζ; co-expression of Par6 and ECT2 efficiently activates Cdc42 in vivo; overexpression of ECT2 stimulates PKCζ activity; ECT2 localizes to sites of cell-cell contact and the nucleus in MDCK cells, and its localization is regulated by calcium.","method":"Co-immunoprecipitation, Cdc42-GTP pull-down assay, PKCζ kinase assay, immunofluorescence, calcium switch assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vivo GTPase activity and kinase assay, single lab","pmids":["15254234"],"is_preprint":false},{"year":2005,"finding":"ECT2 concentrates on the central spindle by binding to the centralspindlin component CYK-4/MgcRacGAP; this ECT2-CYK-4 interaction is cell cycle regulated via ECT2 phosphorylation; depletion of CYK-4 (but not MKLP1) prevents cortical accumulation of RhoA, F-actin, and myosin, placing CYK-4-ECT2 upstream of RhoA at the equatorial cortex.","method":"siRNA depletion, co-immunoprecipitation, immunofluorescence of RhoA/F-actin/myosin localization, phosphatase treatment","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP plus epistasis via RNAi with well-defined localization readouts; replicated by multiple groups","pmids":["16103226"],"is_preprint":false},{"year":2005,"finding":"Centralspindlin and ECT2 are both required for RhoA localization to the equatorial cortex before furrow initiation; centralspindlin localizes to central spindle and astral microtubule tips near the equatorial cortex and recruits ECT2; both Rho activity and microtubule organization are required for RhoA localization and furrowing.","method":"TCA fixation immunofluorescence, RNAi depletion, drug-mediated microtubule manipulation","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple RNAi depletion experiments with careful localization readouts; independently consistent with PMID 16103226","pmids":["16352658"],"is_preprint":false},{"year":2005,"finding":"ECT2 and MgcRacGAP regulate GTP-Cdc42 levels in metaphase; depletion of Ect2 by RNAi suppresses metaphase GTP-Cdc42 elevation, impairs microtubule attachment to kinetochores, and causes prometaphase delay and abnormal chromosome segregation.","method":"RNAi, GTP-Cdc42 pull-down assay, live cell microscopy, chromosome segregation analysis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi plus GTPase pull-down assay with defined phenotypic readouts, single lab","pmids":["15642749"],"is_preprint":false},{"year":2006,"finding":"CDK1 phosphorylates ECT2 at Thr-341 in G2/M phase (most likely via Cyclin B/Cdk1); phosphorylation at T341 induces a conformational change affecting the intramolecular interaction between N-terminal regulatory and C-terminal catalytic domains; phosphomimetic T341D weakly stimulates GEF catalytic activity via SRE reporter assay and increases self-association of ECT2.","method":"Cell synchronization, phospho-site mapping, site-directed mutagenesis, SRE luciferase reporter assay, co-immunoprecipitation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis with functional assays, single lab; kinase attribution based on cell cycle timing and inhibitor data","pmids":["16170345"],"is_preprint":false},{"year":2006,"finding":"CDK1 and Plk1 phosphorylate ECT2 in vitro; CDK1 phosphorylates ECT2 at T412, creating a phospho-epitope that recruits the Plk1 polo-box domain (PBD); phosphorylation of T412 is required for GTP-RhoA accumulation and cortical hyperactivity during cell division; ECT2 T412A (phospho-deficient) shows diminished RhoA activation.","method":"In vitro kinase assay, Plk1-PBD binding assay, phospho-mutant expression, RhoA-GTP pull-down, live cell imaging","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay plus PBD docking and RhoA activity assay with mutagenesis, single lab but multiple orthogonal methods","pmids":["16247472"],"is_preprint":false},{"year":2006,"finding":"ECT2 requires its BRCT domain for direct interaction with MKlp1-MgcRacGAP; central spindle localization also requires the MKlp2-Aurora B complex; a PH domain in ECT2 mediates cortical association; ECT2 displacement from the central spindle after cytokinesis onset (via N-terminal fragment overexpression) causes abscission failure, while RhoA and Citron kinase still localize to the cleavage furrow.","method":"RNAi depletion, GFP-fusion overexpression, immunofluorescence, abscission assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — domain deletion plus RNAi epistasis, single lab","pmids":["16803869"],"is_preprint":false},{"year":2006,"finding":"In C. elegans embryos, ECT-2 (a RhoGEF for RHO-1) is uniformly distributed at the cortex before polarization and is locally excluded from the posterior cortex by the centrosomal polarity cue; asymmetric ECT-2 generates an asymmetric RHO-1 distribution that drives cortical actomyosin flow to translocate PAR proteins and CDC-42 to the anterior cortex; polarized CDC-42 subsequently maintains the anterior cortical domain.","method":"Live imaging of GFP fusions, RNAi epistasis in C. elegans embryos, cortical flow analysis","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging with RNAi epistasis establishing pathway order; C. elegans ortholog with conserved mechanism","pmids":["16921365"],"is_preprint":false},{"year":2006,"finding":"ECT2 is identified as a direct E2F target gene: E2F1 and CUX1 bind ECT2 promoter upon S-phase entry and regulate its transcription; ECT2 expression is induced in S phase and peaks in G2/M.","method":"Chromatin immunoprecipitation, promoter-luciferase reporter assay, RNAi knockdown, E2F dominant-negative expression","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus reporter assay, single lab","pmids":["16862181"],"is_preprint":false},{"year":2006,"finding":"UBE3A ubiquitin E3 ligase physically interacts with ECT2 (and its Drosophila ortholog Pbl); Ect2 expression is regulated by Ube3a in mouse neurons, with dramatically altered Ect2 expression in the hippocampus and cerebellum of Ube3a null mice.","method":"2D gel/MALDI-TOF proteomics, co-immunoprecipitation, Ube3a knockout mouse analysis","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus in vivo knockout phenotype, single lab","pmids":["16905559"],"is_preprint":false},{"year":2007,"finding":"Plk1 promotes recruitment of ECT2 to the central spindle by phosphorylating HsCyk-4, creating a phospho-epitope recognized by the BRCT repeats of ECT2; inhibition of Plk1 (by BI 2536) abolishes the ECT2-HsCyk-4 interaction, prevents ECT2 central spindle localization, RhoA equatorial accumulation, and cleavage furrow formation; Plk1 acts after CDK1 inactivation and independently of Aurora B.","method":"Plk1 inhibitor (BI 2536), co-immunoprecipitation, immunofluorescence, cell cycle staging","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — pharmacological inhibition plus co-IP epistasis; replicated by Wolfe et al. 2009","pmids":["17488623"],"is_preprint":false},{"year":2008,"finding":"Plk1 phosphorylates the non-catalytic N terminus of HsCyk-4 at the central spindle, generating a phospho-epitope at Ser164 that is recognized by the BRCT repeats of ECT2, recruiting ECT2 to the central spindle to drive RhoA activation and furrowing; Prc1 and microtubules facilitate Plk1 phosphorylation of HsCyk-4; a phosphomimetic HsCyk-4 version promotes Ect2 recruitment.","method":"In vitro kinase assay (Plk1), phospho-peptide binding assay, mutagenesis, BRCT-phospho-epitope docking, immunofluorescence, Prc1 RNAi","journal":"PLoS biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus phospho-mutagenesis and functional rescue; replicates and extends Petronczki 2007","pmids":["19468300"],"is_preprint":false},{"year":2008,"finding":"Centralspindlin component Cyk-4 sequentially interacts with ECT2 (early cytokinesis) and then FIP3 (late telophase/abscission); the FIP3-binding region on Cyk-4 overlaps with the ECT2-binding domain; FIP3 and ECT2 form mutually exclusive complexes with Cyk-4; dissociation of ECT2 from the midbody is required for FIP3 and recycling endosome recruitment needed for abscission.","method":"Co-immunoprecipitation, domain mapping, immunofluorescence time course","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP plus domain competition analysis, single lab","pmids":["18511905"],"is_preprint":false},{"year":2008,"finding":"ECT2 in NSCLC is mislocalized to the cytoplasm where it binds the PKCι-Par6α complex; RNAi knockdown of PKCι or Par6α causes ECT2 to redistribute to the nucleus, indicating PKCι-Par6α regulates cytoplasmic ECT2 localization; cytoplasmic ECT2 activates Rac1 to drive transformed growth and invasion.","method":"RNAi knockdown, co-immunoprecipitation, subcellular fractionation, Rac1-GTP pull-down, colony formation/invasion assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus RNAi rescue epistasis, single lab","pmids":["19617897"],"is_preprint":false},{"year":2009,"finding":"PKCι directly phosphorylates ECT2 at Thr-328 in vitro; RNAi knockdown of PKCι or Par6 decreases phospho-Thr-328 ECT2 in NSCLC cells; phosphorylation-deficient T328A ECT2 fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation, whereas phosphomimetic T328D ECT2 retains all these activities.","method":"In vitro kinase assay (PKCι), site-directed mutagenesis, RNAi knockdown, Rac1-GTP pull-down, transformation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with phospho-site mutagenesis plus functional rescue, single lab but multiple orthogonal methods","pmids":["21189248"],"is_preprint":false},{"year":2011,"finding":"ECT2 membrane association during cytokinesis requires a pleckstrin homology domain and a polybasic cluster that bind phosphoinositide lipids; both GEF function and membrane targeting of ECT2 are essential for RhoA activation and cleavage furrow formation; membrane localization is spatially confined to the equator by centralspindlin and is temporally regulated by CDK1 activity.","method":"Live cell imaging, GFP-ECT2 constructs with PH domain and polybasic cluster mutations, phosphoinositide lipid binding assay, RhoA activity assay, CDK1 inhibitor treatment","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging with domain mutagenesis and lipid binding assay; replicated concept from Chalamalasetty 2006","pmids":["22172673"],"is_preprint":false},{"year":2011,"finding":"APC/C-Cdh1 ubiquitinates ECT2 after mitosis via K11-linked polyubiquitin chains, targeting it for proteasomal degradation; a bipartite NLS, a conventional D-box, and two TEK-like boxes in ECT2 are required for Cdh1-dependent degradation; proper nuclear localization of ECT2 is necessary for its APC-Cdh1-mediated degradation; degradation-resistant ECT2 mutants activate RhoA and transform NIH 3T3 cells.","method":"Co-immunoprecipitation, in vivo ubiquitination assay, site-directed mutagenesis, proteasome inhibitor treatment, NIH 3T3 transformation assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — ubiquitination assay plus mutagenesis of degradation signals with functional consequence, single lab but multiple orthogonal methods","pmids":["21886810"],"is_preprint":false},{"year":2011,"finding":"Nuclear GEFs Ect2 and Net1 activate RhoB after DNA damage (ionizing radiation); RNAi knockdown of Ect2 and Net1 inhibits IR-induced RhoB activity increase, reduces JNK phosphorylation and Bim induction, and protects cells from IR-induced cell death.","method":"RNAi, RhoB-GTP pull-down assay, Western blot for JNK phosphorylation and Bim, cell death assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi epistasis with GTPase activity assay and apoptosis markers, single lab","pmids":["21373644"],"is_preprint":false},{"year":2012,"finding":"Ect2 acts as a Cdk1 substrate that links mitotic entry to cortical rounding: in prophase, Ect2 is exported from the nucleus into the cytoplasm, where it activates RhoA to form a rigid rounded metaphase cortex; at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation for cytokinesis.","method":"Live cell imaging, RNAi, Cdk1 substrate mutagenesis, atomic force microscopy for cortical stiffness, immunofluorescence","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — live imaging with biophysical stiffness measurement and RNAi rescue; multiple orthogonal methods","pmids":["22898780"],"is_preprint":false},{"year":2012,"finding":"The PH domain of Ect2 interacts with anillin; this interaction may require Ect2 association with lipids since a PH domain mutation disrupting phospholipid binding weakens the Ect2-anillin interaction; the anillin-Ect2 complex stabilizes central spindle microtubule-cortical interactions at the division plane.","method":"Co-immunoprecipitation, PH domain mutagenesis, immunofluorescence","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus mutagenesis, single lab","pmids":["22514687"],"is_preprint":false},{"year":2013,"finding":"In ovarian cancer cells, nuclear ECT2 preferentially binds Rac1 (not RhoA), while cytoplasmic ECT2 binds RhoA; nuclear ECT2 GEF catalytic activity and nuclear localization sequences are both required for anchorage-independent growth; nuclear Rac1 activity is sufficient to rescue transformation caused by ECT2 knockdown.","method":"Subcellular fractionation, co-immunoprecipitation, NLS mutagenesis, DH domain mutagenesis, soft agar colony assay, constitutively active nuclear-targeted Rac1 rescue","journal":"Genes & cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical fractionation with co-IP plus mutagenesis rescue experiments, single lab","pmids":["24386507"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of the ECT2 triple-BRCT domain was solved; Ser164 on CYK-4 is the major Plk1 phosphorylation site that docks to the second ECT2 BRCT domain; systematic analysis of phospho-peptide interactions mapped the ECT2 BRCT-CYK-4 binding interface.","method":"X-ray crystallography, phospho-peptide binding assay, systematic mutagenesis of CYK-4 phosphorylation sites","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure plus biochemical binding mapping; single lab but structural validation","pmids":["25068414"],"is_preprint":false},{"year":2014,"finding":"Plk1 phosphorylation of MgcRacGAP at both S157 and S164 is required (neither alone is sufficient) for efficient Ect2 BRCT domain binding; central spindle assembly (requiring MKLP1 and the N-terminal domain of MgcRacGAP) is additionally required for Ect2 BRCT binding in early cytokinesis.","method":"Phospho-site mutagenesis, BRCT binding assay, siRNA depletion of MKLP1, co-immunoprecipitation","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis-based binding assay plus RNAi epistasis, single lab","pmids":["25486482"],"is_preprint":false},{"year":2014,"finding":"The BRCT domain of ECT2 directly binds poly(ADP-ribose) (PAR) both in vitro and in vivo; α-tubulin is PARylated during mitosis; PARylation of α-tubulin is recognized by ECT2 BRCT domain, recruiting ECT2 to the mitotic spindle.","method":"In vitro PAR binding assay, co-immunoprecipitation, immunofluorescence, mitosis analysis","journal":"Cell cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo PAR binding plus immunofluorescence co-localization, single lab","pmids":["25486481"],"is_preprint":false},{"year":2015,"finding":"CDK1 phosphorylates ECT2 at a non-S/T-P motif (a sequence matching P-X-S-X-[R/K]5 containing the NLS region) in vitro; this phosphorylation event is proposed to inhibitorily regulate ECT2 nuclear localization during mitosis.","method":"In vitro kinase assay with Cdk1, oriented peptide library screening, site-directed mutagenesis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro kinase assay, single lab, functional consequence inferred from NLS context","pmids":["25604483"],"is_preprint":false},{"year":2015,"finding":"Plasma membrane association of ECT2 during anaphase is required and sufficient for cytokinesis; local membrane targeting of ECT2 with optogenetics leads to unilateral furrowing; ECT2 mutations that prevent centralspindlin binding compromise midzone and equatorial membrane concentration but still sustain cytokinesis, indicating midzone recruitment is not essential.","method":"Chemical genetic membrane targeting, optogenetic local membrane targeting, ECT2 centralspindlin-binding mutants, immunofluorescence","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — optogenetic and chemical genetic approaches with spatial control; multiple orthogonal methods in single study","pmids":["27926870"],"is_preprint":false},{"year":2015,"finding":"In Drosophila and human cells, Pbl/ECT2 GEF activity negatively regulates Wg/Wnt target gene expression downstream of Armadillo/β-catenin stabilization; GEF activity is required for Wnt regulation whereas domains critical for cytokinesis are not.","method":"Drosophila genetic loss-of-function and gain-of-function, luciferase reporter assay for Wnt target genes in Drosophila and human cells, domain mutagenesis","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in Drosophila plus reporter assay in human cells, single lab","pmids":["24198276"],"is_preprint":false},{"year":2015,"finding":"In Drosophila epithelia, Pbl/Ect2 release from the nucleus at mitotic entry drives Rho-dependent Myosin-II activation and a switch from Arp2/3- to Diaphanous-mediated cortical actin nucleation that depends on Cdc42/aPKC/Par6, enabling assembly of an isotropic metaphase cortex.","method":"Drosophila genetics, RNAi, live imaging, actin polymerization pathway epistasis","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in model organism with live imaging; single lab","pmids":["25703349"],"is_preprint":false},{"year":2016,"finding":"E6AP E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of ECT2, acting as a negative regulator; loss of E6AP leads to elevated ECT2 and Rho GTPase activity and increased breast cancer invasiveness and metastasis.","method":"Co-immunoprecipitation, in vivo ubiquitination assay, proteasome inhibitor treatment, RhoA-GTP pull-down, invasion and metastasis assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ubiquitination assay plus functional consequence, single lab","pmids":["27231202"],"is_preprint":false},{"year":2017,"finding":"Nuclear ECT2 GEF activity is required for KRAS-driven lung tumorigenesis in vivo; ECT2 activates rRNA synthesis by binding the nucleolar transcription factor UBF1 on rDNA promoters; ECT2 recruits Rac1 and its effector nucleophosmin (NPM) to rDNA; PKCι-mediated ECT2 phosphorylation stimulates ECT2-dependent rDNA transcription.","method":"Mouse lung tumorigenesis model (Kras-Trp53), ChIP (ECT2/UBF1 on rDNA), Rac1-GTP pull-down, rRNA synthesis assay, PKCι phospho-site mutagenesis","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo tumorigenesis model plus ChIP and rRNA synthesis assay with phospho-mutagenesis; multiple orthogonal methods","pmids":["28110998"],"is_preprint":false},{"year":2019,"finding":"Aurora A kinase (AIR-1) in C. elegans acts upstream of ECT-2 to regulate cortical contractility and PAR-2 polarity axis singularity; AIR-1 depletion causes altered ECT-2 cortical localization and promiscuous PAR-2 domain formation; AIR-1 inhibition of ECT-2 is independent of microtubule nucleation.","method":"RNAi in C. elegans, live imaging, genetic epistasis","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging with RNAi epistasis in C. elegans, single lab","pmids":["31636075"],"is_preprint":false},{"year":2020,"finding":"PKCι directly phosphorylates UBF1 at Ser-412, generating a phosphopeptide-binding epitope that recruits the ECT2 BRCT domain to UBF1 on rDNA promoters; both a functional ECT2 BRCT domain and UBF1 Ser-412 phosphorylation are required for ECT2 rDNA recruitment, elevated rRNA synthesis, and transformed growth.","method":"In vitro kinase assay (PKCι on UBF1), MS-based phospho-site identification, BRCT domain mutagenesis, ChIP, rRNA synthesis assay, shRNA knockdown/reconstitution","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay with MS phospho-mapping plus BRCT mutagenesis and functional rescue; single lab but multiple rigorous methods","pmids":["32350115"],"is_preprint":false},{"year":2020,"finding":"FoxM1 binds to ECT2 through its N-terminal domain and inhibits ECT2 GEF activity, limiting RhoA GTPase and mDia1-mediated cortical actin nucleation; FoxM1 insufficiency leads to excess cortical actin, non-perpendicular mitotic spindles, chromosome missegregation, and tumorigenesis; low FOXM1 expression correlates with RhoA hyperactivity in human cancers.","method":"Co-immunoprecipitation, in vitro GEF inhibition assay, FoxM1 domain deletion analysis, cortical actin and spindle angle measurements, mouse tumorigenesis model","journal":"Nature cancer","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct co-IP plus in vitro GEF inhibition with domain mapping, functional mouse model, single lab but multiple orthogonal methods","pmids":["34841254"],"is_preprint":false},{"year":2021,"finding":"Each ECT2 BRCT domain (BRCT0, BRCT1, BRCT2) makes distinct contributions: BRCT0 contributes to and BRCT1 is essential for ECT2 activation in anaphase; BRCT2 integrates GEF inhibition and RACGAP1 binding to limit ECT2 activity to a narrow equatorial zone; BRCT2-dependent control of active RhoA zone dimension functions in addition to astral microtubule inhibitory signals.","method":"BRCT domain mutagenesis, live cell imaging, RhoA activity biosensor, RACGAP1 binding assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — systematic domain mutagenesis with live biosensor imaging; multiple orthogonal methods","pmids":["33657383"],"is_preprint":false},{"year":2021,"finding":"ECT2 physically associates with KU70-KU80 and BRCA1 via co-immunoprecipitation; ECT2 is recruited to DNA lesions in a PARP1-dependent manner; ECT2 deficiency impairs KU70 and BRCA1 recruitment to DNA damage sites, causing defective DSB repair and hypersensitivity to genotoxic agents; this DNA repair role is largely independent of ECT2 GEF catalytic activity.","method":"Co-immunoprecipitation, laser microirradiation/immunofluorescence, GEF catalytic mutant complementation, comet assay, genotoxin sensitivity assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus laser microirradiation foci and GEF-dead mutant rescue, single lab","pmids":["34343566"],"is_preprint":false},{"year":2021,"finding":"Nuclear ECT2 promotes ribosomal DNA transcription and ribosome biogenesis in colorectal cancer cells; both nuclear localization sequences and GEF catalytic activity of ECT2 are required for anchorage-independent growth and invasion independent of cytokinesis function.","method":"ECT2 knockdown/reconstitution with NLS and DH domain mutants, rDNA transcription assay, soft agar/invasion assays, mouse Kras/Apc colon cancer model","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis rescue with multiple functional readouts plus in vivo mouse model; multiple orthogonal methods","pmids":["34737214"],"is_preprint":false},{"year":2022,"finding":"DNA-PK phosphorylates the mTORC2 subunit Sin1 after DNA damage, enabling Sin1 interaction with ECT2; ECT2-Sin1 interaction and ECT2 GEF catalytic activity are required for DNA damage-induced AKT activation; depleting Sin1 or ECT2 or disrupting the protein interaction attenuates DNA damage-induced AKT activation and enhances cellular sensitivity to DNA-damaging agents.","method":"Co-immunoprecipitation (Sin1-ECT2), RNAi knockdown, ECT2 catalytic mutant, AKT phosphorylation assay, cell survival assay","journal":"Science signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus catalytic mutant and RNAi with defined signaling readout, single lab","pmids":["34982576"],"is_preprint":false},{"year":2022,"finding":"Centralspindlin (Cyk4/Mklp1) and ECT2 are required for exclusion of NuMA/dynein/dynactin from the equatorial cell membrane during anaphase; Ect2/Cyk4/Mklp1 and NuMA/dynein/dynactin occupy mutually exclusive membrane regions; equatorial Ect2-based complex enrichment coordinates spindle elongation with cleavage furrow formation.","method":"RNAi depletion, live cell imaging, immunofluorescence of membrane compartmentalization","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — RNAi epistasis with live imaging of membrane compartmentalization, single lab","pmids":["36197340"],"is_preprint":false},{"year":2022,"finding":"In C. elegans, Aurora A (AIR-1) breaks cortical symmetry by phosphorylating three putative sites in the PH domain of ECT-2, reducing ECT-2 cortical accumulation at the posterior cortex; myosin-dependent cortical flows amplify this local inhibition to generate regional ECT-2 asymmetry supporting both embryo polarization and cytokinesis.","method":"Live imaging, phospho-site mutagenesis of ECT-2 PH domain, AIR-1 depletion, myosin inhibition","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — phospho-site mutagenesis in live imaging context, single lab","pmids":["36533896"],"is_preprint":false},{"year":2025,"finding":"In confined migration, a cytoplasmic pool of anillin recruits ECT2 to the plasma membrane at cell poles; ECT2 GEF activity activates RhoA at the poles to drive myosin II-dependent bleb-based migration and invasion; confinement-induced nuclear envelope rupture releases additional anillin and ECT2 into the cytoplasm, amplifying the process; ROCK inhibition abolishes this ECT2-dependent confined migration.","method":"Microfluidic confinement assay, live imaging, RNAi/siRNA knockdown, GEF-dead ECT2 mutant, ROCK inhibitor (Y-27632), nuclear envelope rupture assay","journal":"Nature materials","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional assays in defined confinement context with GEF-dead mutant, single lab","pmids":["40571734"],"is_preprint":false}],"current_model":"ECT2 is a RhoGEF whose activity is tightly regulated through the cell cycle: in interphase it is sequestered in the nucleus in an autoinhibited conformation maintained by intramolecular BRCT–DH/PH interaction and APC/C-Cdh1-mediated degradation after mitosis; upon mitotic entry CDK1 exports ECT2 from the nucleus and phosphorylates it (at T341/T412), enabling Plk1 binding and further phosphorylation that unfolds the autoinhibition; at anaphase, Plk1 phosphorylates CYK-4/MgcRacGAP at S157 and S164, creating a BRCT-binding docking site that recruits ECT2 to centralspindlin at the central spindle, and a PH-domain/polybasic cluster targets ECT2 to the equatorial plasma membrane where it activates RhoA to drive contractile ring assembly and cytokinesis; in the nucleus of cancer cells, a PKCι–Thr328-phosphorylated ECT2 binds UBF1 on rDNA promoters (facilitated by PKCι-phospho-UBF1 Ser412 as a BRCT docking epitope) and recruits Rac1–NPM to stimulate rRNA synthesis and tumor growth; cytoplasmic ECT2 in cancer cells is stabilized by USP7 deubiquitination, activates Rac1 via the PKCι–Par6 complex, and promotes invasion, while nuclear ECT2 also participates in DNA double-strand break repair by recruiting KU70/KU80 and BRCA1 to lesions in a PARP1-dependent, GEF-independent manner."},"narrative":{"mechanistic_narrative":"ECT2 is a Rho-family guanine nucleotide exchange factor (RhoGEF) that serves as the principal activator of RhoA during cytokinesis and, when mislocalized, drives oncogenic Rho/Rac signaling [PMID:8464478, PMID:10579713, PMID:10837491]. Its catalytic DH/PH module exchanges nucleotide on RhoA, Rac1, and Cdc42 in vitro and is held autoinhibited in interphase by an intramolecular interaction between the N-terminal tandem BRCT domains and the C-terminal catalytic domain, with sequences C-terminal to the PH domain tuning which GTPase is activated [PMID:10579713, PMID:15545273, PMID:15073184]. Activation is gated through the cell cycle by a phosphorylation relay: CDK1 phosphorylates ECT2 at T341 and T412 during G2/M, the T412 phospho-epitope recruiting the Plk1 polo-box domain, and Plk1 in turn phosphorylates the centralspindlin subunit CYK-4/MgcRacGAP at S157/S164 to create a docking epitope read by the ECT2 BRCT repeats, recruiting ECT2 to the central spindle [PMID:16170345, PMID:16247472, PMID:17488623, PMID:19468300, PMID:25068414, PMID:25486482]. A PH domain and adjacent polybasic cluster bind equatorial-membrane phosphoinositides to spatially confine RhoA activation and contractile-ring assembly, while different BRCT domains partition between activation and zone restriction [PMID:22172673, PMID:33657383]. ECT2 levels are restrained by APC/C-Cdh1, E6AP, and other ubiquitin ligases [PMID:21886810, PMID:27231202]. Beyond cytokinesis, ECT2 establishes cell polarity through the centrosomal/Aurora-A axis acting on cortical RhoA and PAR proteins [PMID:16921365, PMID:31636075, PMID:36533896], and in cancer cells nuclear ECT2 phosphorylated by PKCι binds UBF1 on rDNA promoters to recruit Rac1-nucleophosmin and stimulate rRNA synthesis and KRAS-driven tumorigenesis [PMID:28110998, PMID:32350115, PMID:34737214], while cytoplasmic ECT2 acts through a PKCι-Par6 complex to activate Rac1 and promote invasion [PMID:19617897, PMID:21189248]. ECT2 additionally functions in DNA double-strand break repair, recruiting KU70/KU80 and BRCA1 to PARP1-dependent lesions independently of its GEF activity [PMID:34343566].","teleology":[{"year":1993,"claim":"Established ECT2 as a member of the Dbl/Rho-regulatory family, answering what protein class it belongs to and that truncation confers transforming potential.","evidence":"Expression cloning with baculovirus protein and direct Rho/Rac binding assay","pmids":["8464478"],"confidence":"High","gaps":["Did not establish exchange catalysis directly","No cellular function assigned"]},{"year":1999,"claim":"Defined ECT2 as a catalytically active GEF for RhoA/Rac1/Cdc42 and tied it functionally to cytokinesis, answering what cellular process it drives.","evidence":"In vitro nucleotide exchange assay, synchronization, immunofluorescence, microinjection and dominant-negative expression","pmids":["10579713"],"confidence":"High","gaps":["Which GTPase is physiologically relevant in vivo not resolved","Mechanism of phosphoregulation unknown"]},{"year":2000,"claim":"Showed ECT2 is required for the telophase rise in GTP-RhoA, establishing it as the critical RhoA activator in cytokinesis.","evidence":"RhoA-GTP pull-down with cell synchronization and dominant-negative ECT2","pmids":["10837491"],"confidence":"High","gaps":["Spatial targeting mechanism to furrow not addressed"]},{"year":2004,"claim":"Mapped autoinhibition to an intramolecular BRCT–catalytic domain interaction and showed C-terminal sequences select GTPase output, answering how activity is restrained and diversified.","evidence":"Co-IP of intramolecular interaction, deletion/NLS mutagenesis, GTPase pull-downs and multinucleation assays","pmids":["15545273","15073184","14645260"],"confidence":"High","gaps":["How phosphorylation relieves autoinhibition not yet defined","C-terminal partner mediating Rac/Cdc42 output unidentified"]},{"year":2005,"claim":"Identified centralspindlin/CYK-4 as the recruitment platform that positions ECT2 at the equatorial cortex, answering how RhoA activation is spatially confined.","evidence":"Reciprocal co-IP, RNAi epistasis, and immunofluorescence of RhoA/F-actin/myosin localization","pmids":["16103226","16352658","16803869","15642749"],"confidence":"High","gaps":["Molecular nature of the cell-cycle-regulated CYK-4 docking site not yet defined","Role in metaphase Cdc42 only Medium confidence"]},{"year":2006,"claim":"Defined the CDK1–Plk1 phosphorylation relay, showing CDK1 at T341/T412 creates a Plk1 docking epitope and links mitotic kinase activity to ECT2 activation and degradation.","evidence":"In vitro kinase assays, phospho-site mapping, Plk1-PBD binding, RhoA pull-down; APC/C E2F regulation by ChIP/reporter","pmids":["16170345","16247472","16862181"],"confidence":"High","gaps":["Quantitative contribution of each phospho-site to autoinhibition relief not resolved"]},{"year":2006,"claim":"Demonstrated a conserved polarity role in C. elegans, showing asymmetric ECT-2 patterns RHO-1 to drive cortical flows and PAR domain establishment.","evidence":"Live imaging of GFP fusions with RNAi epistasis in embryos","pmids":["16921365"],"confidence":"High","gaps":["Mechanism of centrosomal exclusion of ECT-2 not defined at this stage"]},{"year":2007,"claim":"Established that Plk1 phosphorylation of CYK-4 at S164 generates the BRCT docking epitope, mechanistically explaining ECT2 central spindle recruitment downstream of CDK1 inactivation.","evidence":"Plk1 inhibitor BI 2536, in vitro kinase assay, phospho-peptide binding, mutagenesis and immunofluorescence","pmids":["17488623","19468300"],"confidence":"High","gaps":["Whether S157 also contributes not resolved here"]},{"year":2008,"claim":"Revealed temporal handoff: CYK-4 binds ECT2 early then FIP3 late via overlapping sites, explaining how ECT2 dissociation enables abscission.","evidence":"Reciprocal co-IP, domain mapping, immunofluorescence time course","pmids":["18511905"],"confidence":"Medium","gaps":["Single lab","Trigger for the switch from ECT2 to FIP3 not defined"]},{"year":2008,"claim":"Linked cancer ECT2 to the PKCι–Par6 polarity complex, showing cytoplasmic mislocalized ECT2 activates Rac1 to drive transformation and invasion.","evidence":"RNAi, co-IP, fractionation, Rac1-GTP pull-down and invasion assays in NSCLC","pmids":["19617897","21189248"],"confidence":"Medium","gaps":["Single lab","How mislocalization is initiated in tumors not fully defined"]},{"year":2011,"claim":"Defined PH-domain/polybasic phosphoinositide binding as the equatorial membrane-targeting determinant and characterized ubiquitin-mediated turnover (APC/C-Cdh1, later E6AP).","evidence":"Live imaging with PH/polybasic mutants, lipid binding, RhoA assay; ubiquitination assays with degron mutagenesis","pmids":["22172673","21886810","34841254"],"confidence":"High","gaps":["How membrane and centralspindlin signals are integrated quantitatively unresolved"]},{"year":2012,"claim":"Showed CDK1 substrate ECT2 couples mitotic entry to cortical rounding and that nuclear export precedes its anaphase repositioning by RacGAP1, ordering its mitotic itinerary.","evidence":"Live imaging, Cdk1 substrate mutagenesis, atomic force microscopy and RNAi rescue","pmids":["22898780","22514687"],"confidence":"High","gaps":["Anillin–PH interaction only Medium confidence"]},{"year":2014,"claim":"Provided the structural basis of BRCT-phosphoepitope recognition and identified PAR/PARylation as an additional BRCT ligand, refining how ECT2 reads its docking sites.","evidence":"X-ray crystallography of triple-BRCT, phospho-peptide binding, in vitro/in vivo PAR binding and microscopy","pmids":["25068414","25486482","25486481"],"confidence":"Medium","gaps":["Functional importance of tubulin-PAR recruitment vs CYK-4 docking not weighted","PAR binding single lab"]},{"year":2015,"claim":"Used optogenetic/chemical membrane targeting to show equatorial membrane association is sufficient for furrowing and that midzone recruitment is dispensable, plus identified a GEF-dependent Wnt regulatory role.","evidence":"Optogenetic and chemical genetic membrane targeting; Drosophila/human Wnt reporter assays; Drosophila cortical actin epistasis","pmids":["27926870","24198276","25703349","25604483"],"confidence":"High","gaps":["Mechanism of GEF-dependent Wnt repression not defined","Relative weight of membrane vs midzone signals context-dependent"]},{"year":2017,"claim":"Established nuclear, GEF-dependent ECT2 as a driver of rRNA synthesis and KRAS-driven tumorigenesis via UBF1 binding and Rac1–NPM recruitment at rDNA.","evidence":"Kras mouse lung tumor model, ChIP, Rac1 pull-down, rRNA synthesis assays with PKCι phospho-mutagenesis","pmids":["28110998","34737214","24386507"],"confidence":"High","gaps":["How nuclear vs cytoplasmic GTPase selectivity is achieved mechanistically unresolved"]},{"year":2020,"claim":"Detailed the PKCι–UBF1 Ser412 docking mechanism for nuclear ECT2 and identified FoxM1 as a direct GEF inhibitor controlling cortical actin and spindle orientation.","evidence":"In vitro kinase/MS phospho-mapping with BRCT mutagenesis and rRNA assays; co-IP with in vitro GEF inhibition and mouse tumor model","pmids":["32350115","34841254"],"confidence":"High","gaps":["How FoxM1 inhibition is relieved during normal mitosis not defined"]},{"year":2021,"claim":"Dissected individual BRCT contributions to activation and zone restriction and uncovered a GEF-independent role for ECT2 in DSB repair recruiting KU70/80 and BRCA1.","evidence":"Systematic BRCT mutagenesis with RhoA biosensor imaging; co-IP, laser microirradiation, GEF-dead complementation and comet assays","pmids":["33657383","34343566"],"confidence":"Medium","gaps":["DSB repair role single lab","How ECT2 is recruited to PAR at lesions vs mitotic spindle distinguished only partially"]},{"year":2022,"claim":"Connected ECT2 to DNA-damage signaling (DNA-PK–Sin1–ECT2–AKT) and to membrane compartmentalization that coordinates spindle elongation with furrowing.","evidence":"Co-IP, RNAi, catalytic mutants and AKT/survival assays; RNAi with live imaging of NuMA/dynein membrane exclusion","pmids":["34982576","36197340","36533896"],"confidence":"Medium","gaps":["Single lab for each","How GEF activity feeds AKT activation mechanistically unclear"]},{"year":2025,"claim":"Extended ECT2 function to confined migration, where anillin recruits it to cell poles to activate RhoA-driven bleb migration amplified by nuclear envelope rupture.","evidence":"Microfluidic confinement, live imaging, GEF-dead mutant, ROCK inhibition and nuclear rupture assays","pmids":["40571734"],"confidence":"Medium","gaps":["Single lab","Generality across cell/tumor types untested"]},{"year":null,"claim":"How the multiple non-canonical nuclear roles (rDNA transcription, DSB repair, AKT signaling) are coordinated with the canonical cytokinetic function within a single cell remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model partitioning ECT2 pools across functions","No structural model of full-length activated ECT2 on membrane/centralspindlin","Disease-causal mutations not established in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,3,4]},{"term_id":"GO:0060089","term_label":"molecular transducer activity","supporting_discovery_ids":[1,2,11,21]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[29]},{"term_id":"GO:0008289","term_label":"lipid binding","supporting_discovery_ids":[21,25]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,3,22,26,41]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[1,19,24,45]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[21,31,45]},{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[35,37]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,2,7,24]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,11,19,42]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[35,37,41]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[40,42]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[19,34,35]}],"complexes":["centralspindlin (with CYK-4/MgcRacGAP)","PKCι-Par6 polarity complex"],"partners":["RACGAP1","PLK1","CDK1","PRKCI","PARD6A","UBF1","ANLN","RHOA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H8V3","full_name":"Protein ECT2","aliases":["Epithelial cell-transforming sequence 2 oncogene"],"length_aa":914,"mass_kda":103.5,"function":"Guanine nucleotide exchange factor (GEF) that catalyzes the exchange of GDP for GTP. Promotes guanine nucleotide exchange on the Rho family members of small GTPases, like RHOA, RHOC, RAC1 and CDC42. Required for signal transduction pathways involved in the regulation of cytokinesis. Component of the centralspindlin complex that serves as a microtubule-dependent and Rho-mediated signaling required for the myosin contractile ring formation during the cell cycle cytokinesis. Regulates the translocation of RHOA from the central spindle to the equatorial region. Plays a role in the control of mitotic spindle assembly; regulates the activation of CDC42 in metaphase for the process of spindle fibers attachment to kinetochores before chromosome congression. Involved in the regulation of epithelial cell polarity; participates in the formation of epithelial tight junctions in a polarity complex PARD3-PARD6-protein kinase PRKCQ-dependent manner. Plays a role in the regulation of neurite outgrowth. Inhibits phenobarbital (PB)-induced NR1I3 nuclear translocation. Stimulates the activity of RAC1 through its association with the oncogenic PARD6A-PRKCI complex in cancer cells, thereby acting to coordinately drive tumor cell proliferation and invasion. Also stimulates genotoxic stress-induced RHOB activity in breast cancer cells leading to their cell death","subcellular_location":"Nucleus; Cytoplasm; Cytoplasm, cytoskeleton, spindle; Cleavage furrow; Midbody; Cell junction; Cell junction, tight junction; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome","url":"https://www.uniprot.org/uniprotkb/Q9H8V3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/ECT2","classification":"Common Essential","n_dependent_lines":1175,"n_total_lines":1208,"dependency_fraction":0.972682119205298},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000114346","cell_line_id":"CID000575","localizations":[{"compartment":"cytoplasmic","grade":3},{"compartment":"nucleolus_gc","grade":2},{"compartment":"nucleoplasm","grade":2}],"interactors":[{"gene":"AHSA1","stoichiometry":0.2},{"gene":"PSME3","stoichiometry":0.2},{"gene":"RPAP2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID000575","total_profiled":1310},"omim":[{"mim_id":"617679","title":"KELCH-LIKE 20; KLHL20","url":"https://www.omim.org/entry/617679"},{"mim_id":"604980","title":"RAC GTPase-ACTIVATING PROTEIN 1; RACGAP1","url":"https://www.omim.org/entry/604980"},{"mim_id":"602098","title":"POLO-LIKE KINASE 1; PLK1","url":"https://www.omim.org/entry/602098"},{"mim_id":"600586","title":"EPITHELIAL CELL TRANSFORMING SEQUENCE 2 ONCOGENE; ECT2","url":"https://www.omim.org/entry/600586"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ECT2"},"hgnc":{"alias_symbol":["ARHGEF31"],"prev_symbol":[]},"alphafold":{"accession":"Q9H8V3","domains":[{"cath_id":"3.40.50.10190","chopping":"49-83_93-173","consensus_level":"medium","plddt":75.885,"start":49,"end":173},{"cath_id":"3.40.50.10190","chopping":"181-267","consensus_level":"medium","plddt":88.6098,"start":181,"end":267},{"cath_id":"3.40.50.10190","chopping":"276-352","consensus_level":"medium","plddt":86.8788,"start":276,"end":352},{"cath_id":"1.20.900.10","chopping":"453-645","consensus_level":"high","plddt":91.0539,"start":453,"end":645},{"cath_id":"2.30.29.30","chopping":"668-710_726-825","consensus_level":"high","plddt":81.1436,"start":668,"end":825}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H8V3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H8V3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H8V3-F1-predicted_aligned_error_v6.png","plddt_mean":70.44},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ECT2","jax_strain_url":"https://www.jax.org/strain/search?query=ECT2"},"sequence":{"accession":"Q9H8V3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H8V3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H8V3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H8V3"}},"corpus_meta":[{"pmid":"16103226","id":"PMC_16103226","title":"An 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\"discoveries\": [\n    {\n      \"year\": 1993,\n      \"finding\": \"ECT2 protein contains a central DH-domain-related core with sequence similarity to BCR, CDC24, and DBL oncogene products; baculovirus-expressed ECT2 binds specifically to Rho and Rac proteins, identifying it as a member of the Rho GTPase regulatory family with transforming potential activated by N-terminal truncation.\",\n      \"method\": \"Expression cloning, baculovirus protein expression, direct binding assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro binding assay with purified protein, foundational study replicated by subsequent work\",\n      \"pmids\": [\"8464478\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Human ECT2 catalyzes guanine nucleotide exchange on RhoA, Rac1, and Cdc42 in vitro; ECT2 is phosphorylated during G2/M phases and phosphorylation is required for its exchange activity; ECT2 localizes to the nucleus in interphase, spreads to cytoplasm in prometaphase, and concentrates at the midbody during cytokinesis; expression of the N-terminal domain (lacking catalytic activity) or microinjection of anti-ECT2 antibody inhibits cytokinesis.\",\n      \"method\": \"GEF activity assay (in vitro nucleotide exchange), cell synchronization, immunofluorescence, microinjection, dominant-negative expression\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro GEF assay plus multiple orthogonal functional experiments; foundational paper replicated extensively\",\n      \"pmids\": [\"10579713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"GTP-bound RhoA accumulates during cytokinesis (peaking at telophase); expression of dominant-negative ECT2 completely suppresses both the rise in GTP-RhoA at telophase and increased GDP-GTP exchange activity in mitotic cell extracts, establishing ECT2 as a critical activator of RhoA during cytokinesis.\",\n      \"method\": \"RhoA-GTP pull-down assay, cell cycle synchronization, dominant-negative ECT2 expression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — quantitative pull-down assay with dominant-negative rescue; independently replicated\",\n      \"pmids\": [\"10837491\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Oncogenic activation of ECT2 requires both removal of the N-terminal negative regulatory domain AND mislocalization from the nucleus to the cytoplasm; the N-terminal domain interacts with the catalytic domain and inhibits GEF activity; nuclear localization signals in the central domain are required to maintain nuclear ECT2; RhoA is the predominant Rho GTPase activated by oncogenic ECT2 in NIH 3T3 cells.\",\n      \"method\": \"Focus formation assay, deletion/NLS mutagenesis, dominant-negative Rho GTPase co-expression, subcellular fractionation, in vivo RhoA activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple domain deletion and mutagenesis experiments with functional readouts; replicated in subsequent work\",\n      \"pmids\": [\"14645260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The N-terminal tandem BRCT domains of ECT2 maintain the protein in an inactive conformation through an intramolecular interaction with the C-terminal catalytic domain, masking GEF activity toward RhoA; both BRCT domains are required for negative regulation (interphase) and positive regulation (cytokinesis function).\",\n      \"method\": \"siRNA knockdown, dominant-negative and deletion mutant expression, multinucleation assay, co-immunoprecipitation of intramolecular BRCT-DH interaction\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal intramolecular interaction mapped by co-IP plus functional rescue experiments\",\n      \"pmids\": [\"15545273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Sequences C-terminal to the PH domain of ECT2 alter the profile of Rho GTPases activated in vivo: removal of C-terminal sequences (DeltaN-Ect2 DH/PH) activates only RhoA and enhances stress fiber formation, whereas retention of C-terminal sequences (DeltaN-Ect2 DH/PH/C) activates RhoA, Rac1, and Cdc42 and induces lamellipodia.\",\n      \"method\": \"NIH 3T3 transformation assay, Rho GTPase activity pull-down, actin morphology analysis, C-terminal deletion mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple GTPase activity assays and morphological readouts, single lab\",\n      \"pmids\": [\"15073184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"ECT2 interacts with Par6 and Par3 of the polarity complex and with PKCζ; co-expression of Par6 and ECT2 efficiently activates Cdc42 in vivo; overexpression of ECT2 stimulates PKCζ activity; ECT2 localizes to sites of cell-cell contact and the nucleus in MDCK cells, and its localization is regulated by calcium.\",\n      \"method\": \"Co-immunoprecipitation, Cdc42-GTP pull-down assay, PKCζ kinase assay, immunofluorescence, calcium switch assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vivo GTPase activity and kinase assay, single lab\",\n      \"pmids\": [\"15254234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ECT2 concentrates on the central spindle by binding to the centralspindlin component CYK-4/MgcRacGAP; this ECT2-CYK-4 interaction is cell cycle regulated via ECT2 phosphorylation; depletion of CYK-4 (but not MKLP1) prevents cortical accumulation of RhoA, F-actin, and myosin, placing CYK-4-ECT2 upstream of RhoA at the equatorial cortex.\",\n      \"method\": \"siRNA depletion, co-immunoprecipitation, immunofluorescence of RhoA/F-actin/myosin localization, phosphatase treatment\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP plus epistasis via RNAi with well-defined localization readouts; replicated by multiple groups\",\n      \"pmids\": [\"16103226\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Centralspindlin and ECT2 are both required for RhoA localization to the equatorial cortex before furrow initiation; centralspindlin localizes to central spindle and astral microtubule tips near the equatorial cortex and recruits ECT2; both Rho activity and microtubule organization are required for RhoA localization and furrowing.\",\n      \"method\": \"TCA fixation immunofluorescence, RNAi depletion, drug-mediated microtubule manipulation\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple RNAi depletion experiments with careful localization readouts; independently consistent with PMID 16103226\",\n      \"pmids\": [\"16352658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ECT2 and MgcRacGAP regulate GTP-Cdc42 levels in metaphase; depletion of Ect2 by RNAi suppresses metaphase GTP-Cdc42 elevation, impairs microtubule attachment to kinetochores, and causes prometaphase delay and abnormal chromosome segregation.\",\n      \"method\": \"RNAi, GTP-Cdc42 pull-down assay, live cell microscopy, chromosome segregation analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi plus GTPase pull-down assay with defined phenotypic readouts, single lab\",\n      \"pmids\": [\"15642749\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CDK1 phosphorylates ECT2 at Thr-341 in G2/M phase (most likely via Cyclin B/Cdk1); phosphorylation at T341 induces a conformational change affecting the intramolecular interaction between N-terminal regulatory and C-terminal catalytic domains; phosphomimetic T341D weakly stimulates GEF catalytic activity via SRE reporter assay and increases self-association of ECT2.\",\n      \"method\": \"Cell synchronization, phospho-site mapping, site-directed mutagenesis, SRE luciferase reporter assay, co-immunoprecipitation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis with functional assays, single lab; kinase attribution based on cell cycle timing and inhibitor data\",\n      \"pmids\": [\"16170345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CDK1 and Plk1 phosphorylate ECT2 in vitro; CDK1 phosphorylates ECT2 at T412, creating a phospho-epitope that recruits the Plk1 polo-box domain (PBD); phosphorylation of T412 is required for GTP-RhoA accumulation and cortical hyperactivity during cell division; ECT2 T412A (phospho-deficient) shows diminished RhoA activation.\",\n      \"method\": \"In vitro kinase assay, Plk1-PBD binding assay, phospho-mutant expression, RhoA-GTP pull-down, live cell imaging\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay plus PBD docking and RhoA activity assay with mutagenesis, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16247472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ECT2 requires its BRCT domain for direct interaction with MKlp1-MgcRacGAP; central spindle localization also requires the MKlp2-Aurora B complex; a PH domain in ECT2 mediates cortical association; ECT2 displacement from the central spindle after cytokinesis onset (via N-terminal fragment overexpression) causes abscission failure, while RhoA and Citron kinase still localize to the cleavage furrow.\",\n      \"method\": \"RNAi depletion, GFP-fusion overexpression, immunofluorescence, abscission assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — domain deletion plus RNAi epistasis, single lab\",\n      \"pmids\": [\"16803869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In C. elegans embryos, ECT-2 (a RhoGEF for RHO-1) is uniformly distributed at the cortex before polarization and is locally excluded from the posterior cortex by the centrosomal polarity cue; asymmetric ECT-2 generates an asymmetric RHO-1 distribution that drives cortical actomyosin flow to translocate PAR proteins and CDC-42 to the anterior cortex; polarized CDC-42 subsequently maintains the anterior cortical domain.\",\n      \"method\": \"Live imaging of GFP fusions, RNAi epistasis in C. elegans embryos, cortical flow analysis\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging with RNAi epistasis establishing pathway order; C. elegans ortholog with conserved mechanism\",\n      \"pmids\": [\"16921365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"ECT2 is identified as a direct E2F target gene: E2F1 and CUX1 bind ECT2 promoter upon S-phase entry and regulate its transcription; ECT2 expression is induced in S phase and peaks in G2/M.\",\n      \"method\": \"Chromatin immunoprecipitation, promoter-luciferase reporter assay, RNAi knockdown, E2F dominant-negative expression\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus reporter assay, single lab\",\n      \"pmids\": [\"16862181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"UBE3A ubiquitin E3 ligase physically interacts with ECT2 (and its Drosophila ortholog Pbl); Ect2 expression is regulated by Ube3a in mouse neurons, with dramatically altered Ect2 expression in the hippocampus and cerebellum of Ube3a null mice.\",\n      \"method\": \"2D gel/MALDI-TOF proteomics, co-immunoprecipitation, Ube3a knockout mouse analysis\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus in vivo knockout phenotype, single lab\",\n      \"pmids\": [\"16905559\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Plk1 promotes recruitment of ECT2 to the central spindle by phosphorylating HsCyk-4, creating a phospho-epitope recognized by the BRCT repeats of ECT2; inhibition of Plk1 (by BI 2536) abolishes the ECT2-HsCyk-4 interaction, prevents ECT2 central spindle localization, RhoA equatorial accumulation, and cleavage furrow formation; Plk1 acts after CDK1 inactivation and independently of Aurora B.\",\n      \"method\": \"Plk1 inhibitor (BI 2536), co-immunoprecipitation, immunofluorescence, cell cycle staging\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — pharmacological inhibition plus co-IP epistasis; replicated by Wolfe et al. 2009\",\n      \"pmids\": [\"17488623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Plk1 phosphorylates the non-catalytic N terminus of HsCyk-4 at the central spindle, generating a phospho-epitope at Ser164 that is recognized by the BRCT repeats of ECT2, recruiting ECT2 to the central spindle to drive RhoA activation and furrowing; Prc1 and microtubules facilitate Plk1 phosphorylation of HsCyk-4; a phosphomimetic HsCyk-4 version promotes Ect2 recruitment.\",\n      \"method\": \"In vitro kinase assay (Plk1), phospho-peptide binding assay, mutagenesis, BRCT-phospho-epitope docking, immunofluorescence, Prc1 RNAi\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus phospho-mutagenesis and functional rescue; replicates and extends Petronczki 2007\",\n      \"pmids\": [\"19468300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Centralspindlin component Cyk-4 sequentially interacts with ECT2 (early cytokinesis) and then FIP3 (late telophase/abscission); the FIP3-binding region on Cyk-4 overlaps with the ECT2-binding domain; FIP3 and ECT2 form mutually exclusive complexes with Cyk-4; dissociation of ECT2 from the midbody is required for FIP3 and recycling endosome recruitment needed for abscission.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, immunofluorescence time course\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP plus domain competition analysis, single lab\",\n      \"pmids\": [\"18511905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ECT2 in NSCLC is mislocalized to the cytoplasm where it binds the PKCι-Par6α complex; RNAi knockdown of PKCι or Par6α causes ECT2 to redistribute to the nucleus, indicating PKCι-Par6α regulates cytoplasmic ECT2 localization; cytoplasmic ECT2 activates Rac1 to drive transformed growth and invasion.\",\n      \"method\": \"RNAi knockdown, co-immunoprecipitation, subcellular fractionation, Rac1-GTP pull-down, colony formation/invasion assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus RNAi rescue epistasis, single lab\",\n      \"pmids\": [\"19617897\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PKCι directly phosphorylates ECT2 at Thr-328 in vitro; RNAi knockdown of PKCι or Par6 decreases phospho-Thr-328 ECT2 in NSCLC cells; phosphorylation-deficient T328A ECT2 fails to bind the PKCι-Par6 complex, activate Rac1, or restore transformation, whereas phosphomimetic T328D ECT2 retains all these activities.\",\n      \"method\": \"In vitro kinase assay (PKCι), site-directed mutagenesis, RNAi knockdown, Rac1-GTP pull-down, transformation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with phospho-site mutagenesis plus functional rescue, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21189248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ECT2 membrane association during cytokinesis requires a pleckstrin homology domain and a polybasic cluster that bind phosphoinositide lipids; both GEF function and membrane targeting of ECT2 are essential for RhoA activation and cleavage furrow formation; membrane localization is spatially confined to the equator by centralspindlin and is temporally regulated by CDK1 activity.\",\n      \"method\": \"Live cell imaging, GFP-ECT2 constructs with PH domain and polybasic cluster mutations, phosphoinositide lipid binding assay, RhoA activity assay, CDK1 inhibitor treatment\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging with domain mutagenesis and lipid binding assay; replicated concept from Chalamalasetty 2006\",\n      \"pmids\": [\"22172673\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"APC/C-Cdh1 ubiquitinates ECT2 after mitosis via K11-linked polyubiquitin chains, targeting it for proteasomal degradation; a bipartite NLS, a conventional D-box, and two TEK-like boxes in ECT2 are required for Cdh1-dependent degradation; proper nuclear localization of ECT2 is necessary for its APC-Cdh1-mediated degradation; degradation-resistant ECT2 mutants activate RhoA and transform NIH 3T3 cells.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay, site-directed mutagenesis, proteasome inhibitor treatment, NIH 3T3 transformation assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ubiquitination assay plus mutagenesis of degradation signals with functional consequence, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"21886810\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Nuclear GEFs Ect2 and Net1 activate RhoB after DNA damage (ionizing radiation); RNAi knockdown of Ect2 and Net1 inhibits IR-induced RhoB activity increase, reduces JNK phosphorylation and Bim induction, and protects cells from IR-induced cell death.\",\n      \"method\": \"RNAi, RhoB-GTP pull-down assay, Western blot for JNK phosphorylation and Bim, cell death assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi epistasis with GTPase activity assay and apoptosis markers, single lab\",\n      \"pmids\": [\"21373644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Ect2 acts as a Cdk1 substrate that links mitotic entry to cortical rounding: in prophase, Ect2 is exported from the nucleus into the cytoplasm, where it activates RhoA to form a rigid rounded metaphase cortex; at anaphase, binding to RacGAP1 at the spindle midzone repositions Ect2 to induce local actomyosin ring formation for cytokinesis.\",\n      \"method\": \"Live cell imaging, RNAi, Cdk1 substrate mutagenesis, atomic force microscopy for cortical stiffness, immunofluorescence\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — live imaging with biophysical stiffness measurement and RNAi rescue; multiple orthogonal methods\",\n      \"pmids\": [\"22898780\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The PH domain of Ect2 interacts with anillin; this interaction may require Ect2 association with lipids since a PH domain mutation disrupting phospholipid binding weakens the Ect2-anillin interaction; the anillin-Ect2 complex stabilizes central spindle microtubule-cortical interactions at the division plane.\",\n      \"method\": \"Co-immunoprecipitation, PH domain mutagenesis, immunofluorescence\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus mutagenesis, single lab\",\n      \"pmids\": [\"22514687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In ovarian cancer cells, nuclear ECT2 preferentially binds Rac1 (not RhoA), while cytoplasmic ECT2 binds RhoA; nuclear ECT2 GEF catalytic activity and nuclear localization sequences are both required for anchorage-independent growth; nuclear Rac1 activity is sufficient to rescue transformation caused by ECT2 knockdown.\",\n      \"method\": \"Subcellular fractionation, co-immunoprecipitation, NLS mutagenesis, DH domain mutagenesis, soft agar colony assay, constitutively active nuclear-targeted Rac1 rescue\",\n      \"journal\": \"Genes & cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical fractionation with co-IP plus mutagenesis rescue experiments, single lab\",\n      \"pmids\": [\"24386507\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of the ECT2 triple-BRCT domain was solved; Ser164 on CYK-4 is the major Plk1 phosphorylation site that docks to the second ECT2 BRCT domain; systematic analysis of phospho-peptide interactions mapped the ECT2 BRCT-CYK-4 binding interface.\",\n      \"method\": \"X-ray crystallography, phospho-peptide binding assay, systematic mutagenesis of CYK-4 phosphorylation sites\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus biochemical binding mapping; single lab but structural validation\",\n      \"pmids\": [\"25068414\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Plk1 phosphorylation of MgcRacGAP at both S157 and S164 is required (neither alone is sufficient) for efficient Ect2 BRCT domain binding; central spindle assembly (requiring MKLP1 and the N-terminal domain of MgcRacGAP) is additionally required for Ect2 BRCT binding in early cytokinesis.\",\n      \"method\": \"Phospho-site mutagenesis, BRCT binding assay, siRNA depletion of MKLP1, co-immunoprecipitation\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis-based binding assay plus RNAi epistasis, single lab\",\n      \"pmids\": [\"25486482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The BRCT domain of ECT2 directly binds poly(ADP-ribose) (PAR) both in vitro and in vivo; α-tubulin is PARylated during mitosis; PARylation of α-tubulin is recognized by ECT2 BRCT domain, recruiting ECT2 to the mitotic spindle.\",\n      \"method\": \"In vitro PAR binding assay, co-immunoprecipitation, immunofluorescence, mitosis analysis\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo PAR binding plus immunofluorescence co-localization, single lab\",\n      \"pmids\": [\"25486481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CDK1 phosphorylates ECT2 at a non-S/T-P motif (a sequence matching P-X-S-X-[R/K]5 containing the NLS region) in vitro; this phosphorylation event is proposed to inhibitorily regulate ECT2 nuclear localization during mitosis.\",\n      \"method\": \"In vitro kinase assay with Cdk1, oriented peptide library screening, site-directed mutagenesis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro kinase assay, single lab, functional consequence inferred from NLS context\",\n      \"pmids\": [\"25604483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Plasma membrane association of ECT2 during anaphase is required and sufficient for cytokinesis; local membrane targeting of ECT2 with optogenetics leads to unilateral furrowing; ECT2 mutations that prevent centralspindlin binding compromise midzone and equatorial membrane concentration but still sustain cytokinesis, indicating midzone recruitment is not essential.\",\n      \"method\": \"Chemical genetic membrane targeting, optogenetic local membrane targeting, ECT2 centralspindlin-binding mutants, immunofluorescence\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — optogenetic and chemical genetic approaches with spatial control; multiple orthogonal methods in single study\",\n      \"pmids\": [\"27926870\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Drosophila and human cells, Pbl/ECT2 GEF activity negatively regulates Wg/Wnt target gene expression downstream of Armadillo/β-catenin stabilization; GEF activity is required for Wnt regulation whereas domains critical for cytokinesis are not.\",\n      \"method\": \"Drosophila genetic loss-of-function and gain-of-function, luciferase reporter assay for Wnt target genes in Drosophila and human cells, domain mutagenesis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in Drosophila plus reporter assay in human cells, single lab\",\n      \"pmids\": [\"24198276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In Drosophila epithelia, Pbl/Ect2 release from the nucleus at mitotic entry drives Rho-dependent Myosin-II activation and a switch from Arp2/3- to Diaphanous-mediated cortical actin nucleation that depends on Cdc42/aPKC/Par6, enabling assembly of an isotropic metaphase cortex.\",\n      \"method\": \"Drosophila genetics, RNAi, live imaging, actin polymerization pathway epistasis\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in model organism with live imaging; single lab\",\n      \"pmids\": [\"25703349\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"E6AP E3 ubiquitin ligase promotes ubiquitination and proteasomal degradation of ECT2, acting as a negative regulator; loss of E6AP leads to elevated ECT2 and Rho GTPase activity and increased breast cancer invasiveness and metastasis.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay, proteasome inhibitor treatment, RhoA-GTP pull-down, invasion and metastasis assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ubiquitination assay plus functional consequence, single lab\",\n      \"pmids\": [\"27231202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nuclear ECT2 GEF activity is required for KRAS-driven lung tumorigenesis in vivo; ECT2 activates rRNA synthesis by binding the nucleolar transcription factor UBF1 on rDNA promoters; ECT2 recruits Rac1 and its effector nucleophosmin (NPM) to rDNA; PKCι-mediated ECT2 phosphorylation stimulates ECT2-dependent rDNA transcription.\",\n      \"method\": \"Mouse lung tumorigenesis model (Kras-Trp53), ChIP (ECT2/UBF1 on rDNA), Rac1-GTP pull-down, rRNA synthesis assay, PKCι phospho-site mutagenesis\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo tumorigenesis model plus ChIP and rRNA synthesis assay with phospho-mutagenesis; multiple orthogonal methods\",\n      \"pmids\": [\"28110998\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Aurora A kinase (AIR-1) in C. elegans acts upstream of ECT-2 to regulate cortical contractility and PAR-2 polarity axis singularity; AIR-1 depletion causes altered ECT-2 cortical localization and promiscuous PAR-2 domain formation; AIR-1 inhibition of ECT-2 is independent of microtubule nucleation.\",\n      \"method\": \"RNAi in C. elegans, live imaging, genetic epistasis\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging with RNAi epistasis in C. elegans, single lab\",\n      \"pmids\": [\"31636075\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"PKCι directly phosphorylates UBF1 at Ser-412, generating a phosphopeptide-binding epitope that recruits the ECT2 BRCT domain to UBF1 on rDNA promoters; both a functional ECT2 BRCT domain and UBF1 Ser-412 phosphorylation are required for ECT2 rDNA recruitment, elevated rRNA synthesis, and transformed growth.\",\n      \"method\": \"In vitro kinase assay (PKCι on UBF1), MS-based phospho-site identification, BRCT domain mutagenesis, ChIP, rRNA synthesis assay, shRNA knockdown/reconstitution\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay with MS phospho-mapping plus BRCT mutagenesis and functional rescue; single lab but multiple rigorous methods\",\n      \"pmids\": [\"32350115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FoxM1 binds to ECT2 through its N-terminal domain and inhibits ECT2 GEF activity, limiting RhoA GTPase and mDia1-mediated cortical actin nucleation; FoxM1 insufficiency leads to excess cortical actin, non-perpendicular mitotic spindles, chromosome missegregation, and tumorigenesis; low FOXM1 expression correlates with RhoA hyperactivity in human cancers.\",\n      \"method\": \"Co-immunoprecipitation, in vitro GEF inhibition assay, FoxM1 domain deletion analysis, cortical actin and spindle angle measurements, mouse tumorigenesis model\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct co-IP plus in vitro GEF inhibition with domain mapping, functional mouse model, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"34841254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Each ECT2 BRCT domain (BRCT0, BRCT1, BRCT2) makes distinct contributions: BRCT0 contributes to and BRCT1 is essential for ECT2 activation in anaphase; BRCT2 integrates GEF inhibition and RACGAP1 binding to limit ECT2 activity to a narrow equatorial zone; BRCT2-dependent control of active RhoA zone dimension functions in addition to astral microtubule inhibitory signals.\",\n      \"method\": \"BRCT domain mutagenesis, live cell imaging, RhoA activity biosensor, RACGAP1 binding assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — systematic domain mutagenesis with live biosensor imaging; multiple orthogonal methods\",\n      \"pmids\": [\"33657383\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ECT2 physically associates with KU70-KU80 and BRCA1 via co-immunoprecipitation; ECT2 is recruited to DNA lesions in a PARP1-dependent manner; ECT2 deficiency impairs KU70 and BRCA1 recruitment to DNA damage sites, causing defective DSB repair and hypersensitivity to genotoxic agents; this DNA repair role is largely independent of ECT2 GEF catalytic activity.\",\n      \"method\": \"Co-immunoprecipitation, laser microirradiation/immunofluorescence, GEF catalytic mutant complementation, comet assay, genotoxin sensitivity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus laser microirradiation foci and GEF-dead mutant rescue, single lab\",\n      \"pmids\": [\"34343566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Nuclear ECT2 promotes ribosomal DNA transcription and ribosome biogenesis in colorectal cancer cells; both nuclear localization sequences and GEF catalytic activity of ECT2 are required for anchorage-independent growth and invasion independent of cytokinesis function.\",\n      \"method\": \"ECT2 knockdown/reconstitution with NLS and DH domain mutants, rDNA transcription assay, soft agar/invasion assays, mouse Kras/Apc colon cancer model\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis rescue with multiple functional readouts plus in vivo mouse model; multiple orthogonal methods\",\n      \"pmids\": [\"34737214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"DNA-PK phosphorylates the mTORC2 subunit Sin1 after DNA damage, enabling Sin1 interaction with ECT2; ECT2-Sin1 interaction and ECT2 GEF catalytic activity are required for DNA damage-induced AKT activation; depleting Sin1 or ECT2 or disrupting the protein interaction attenuates DNA damage-induced AKT activation and enhances cellular sensitivity to DNA-damaging agents.\",\n      \"method\": \"Co-immunoprecipitation (Sin1-ECT2), RNAi knockdown, ECT2 catalytic mutant, AKT phosphorylation assay, cell survival assay\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus catalytic mutant and RNAi with defined signaling readout, single lab\",\n      \"pmids\": [\"34982576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Centralspindlin (Cyk4/Mklp1) and ECT2 are required for exclusion of NuMA/dynein/dynactin from the equatorial cell membrane during anaphase; Ect2/Cyk4/Mklp1 and NuMA/dynein/dynactin occupy mutually exclusive membrane regions; equatorial Ect2-based complex enrichment coordinates spindle elongation with cleavage furrow formation.\",\n      \"method\": \"RNAi depletion, live cell imaging, immunofluorescence of membrane compartmentalization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RNAi epistasis with live imaging of membrane compartmentalization, single lab\",\n      \"pmids\": [\"36197340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In C. elegans, Aurora A (AIR-1) breaks cortical symmetry by phosphorylating three putative sites in the PH domain of ECT-2, reducing ECT-2 cortical accumulation at the posterior cortex; myosin-dependent cortical flows amplify this local inhibition to generate regional ECT-2 asymmetry supporting both embryo polarization and cytokinesis.\",\n      \"method\": \"Live imaging, phospho-site mutagenesis of ECT-2 PH domain, AIR-1 depletion, myosin inhibition\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — phospho-site mutagenesis in live imaging context, single lab\",\n      \"pmids\": [\"36533896\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In confined migration, a cytoplasmic pool of anillin recruits ECT2 to the plasma membrane at cell poles; ECT2 GEF activity activates RhoA at the poles to drive myosin II-dependent bleb-based migration and invasion; confinement-induced nuclear envelope rupture releases additional anillin and ECT2 into the cytoplasm, amplifying the process; ROCK inhibition abolishes this ECT2-dependent confined migration.\",\n      \"method\": \"Microfluidic confinement assay, live imaging, RNAi/siRNA knockdown, GEF-dead ECT2 mutant, ROCK inhibitor (Y-27632), nuclear envelope rupture assay\",\n      \"journal\": \"Nature materials\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional assays in defined confinement context with GEF-dead mutant, single lab\",\n      \"pmids\": [\"40571734\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ECT2 is a RhoGEF whose activity is tightly regulated through the cell cycle: in interphase it is sequestered in the nucleus in an autoinhibited conformation maintained by intramolecular BRCT–DH/PH interaction and APC/C-Cdh1-mediated degradation after mitosis; upon mitotic entry CDK1 exports ECT2 from the nucleus and phosphorylates it (at T341/T412), enabling Plk1 binding and further phosphorylation that unfolds the autoinhibition; at anaphase, Plk1 phosphorylates CYK-4/MgcRacGAP at S157 and S164, creating a BRCT-binding docking site that recruits ECT2 to centralspindlin at the central spindle, and a PH-domain/polybasic cluster targets ECT2 to the equatorial plasma membrane where it activates RhoA to drive contractile ring assembly and cytokinesis; in the nucleus of cancer cells, a PKCι–Thr328-phosphorylated ECT2 binds UBF1 on rDNA promoters (facilitated by PKCι-phospho-UBF1 Ser412 as a BRCT docking epitope) and recruits Rac1–NPM to stimulate rRNA synthesis and tumor growth; cytoplasmic ECT2 in cancer cells is stabilized by USP7 deubiquitination, activates Rac1 via the PKCι–Par6 complex, and promotes invasion, while nuclear ECT2 also participates in DNA double-strand break repair by recruiting KU70/KU80 and BRCA1 to lesions in a PARP1-dependent, GEF-independent manner.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ECT2 is a Rho-family guanine nucleotide exchange factor (RhoGEF) that serves as the principal activator of RhoA during cytokinesis and, when mislocalized, drives oncogenic Rho/Rac signaling [#0, #1, #2]. Its catalytic DH/PH module exchanges nucleotide on RhoA, Rac1, and Cdc42 in vitro and is held autoinhibited in interphase by an intramolecular interaction between the N-terminal tandem BRCT domains and the C-terminal catalytic domain, with sequences C-terminal to the PH domain tuning which GTPase is activated [#1, #4, #5]. Activation is gated through the cell cycle by a phosphorylation relay: CDK1 phosphorylates ECT2 at T341 and T412 during G2/M, the T412 phospho-epitope recruiting the Plk1 polo-box domain, and Plk1 in turn phosphorylates the centralspindlin subunit CYK-4/MgcRacGAP at S157/S164 to create a docking epitope read by the ECT2 BRCT repeats, recruiting ECT2 to the central spindle [#10, #11, #16, #17, #27, #28]. A PH domain and adjacent polybasic cluster bind equatorial-membrane phosphoinositides to spatially confine RhoA activation and contractile-ring assembly, while different BRCT domains partition between activation and zone restriction [#21, #39]. ECT2 levels are restrained by APC/C-Cdh1, E6AP, and other ubiquitin ligases [#22, #34]. Beyond cytokinesis, ECT2 establishes cell polarity through the centrosomal/Aurora-A axis acting on cortical RhoA and PAR proteins [#13, #36, #44], and in cancer cells nuclear ECT2 phosphorylated by PKCι binds UBF1 on rDNA promoters to recruit Rac1-nucleophosmin and stimulate rRNA synthesis and KRAS-driven tumorigenesis [#35, #37, #41], while cytoplasmic ECT2 acts through a PKCι-Par6 complex to activate Rac1 and promote invasion [#19, #20]. ECT2 additionally functions in DNA double-strand break repair, recruiting KU70/KU80 and BRCA1 to PARP1-dependent lesions independently of its GEF activity [#40].\",\n  \"teleology\": [\n    {\n      \"year\": 1993,\n      \"claim\": \"Established ECT2 as a member of the Dbl/Rho-regulatory family, answering what protein class it belongs to and that truncation confers transforming potential.\",\n      \"evidence\": \"Expression cloning with baculovirus protein and direct Rho/Rac binding assay\",\n      \"pmids\": [\"8464478\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish exchange catalysis directly\", \"No cellular function assigned\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined ECT2 as a catalytically active GEF for RhoA/Rac1/Cdc42 and tied it functionally to cytokinesis, answering what cellular process it drives.\",\n      \"evidence\": \"In vitro nucleotide exchange assay, synchronization, immunofluorescence, microinjection and dominant-negative expression\",\n      \"pmids\": [\"10579713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which GTPase is physiologically relevant in vivo not resolved\", \"Mechanism of phosphoregulation unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Showed ECT2 is required for the telophase rise in GTP-RhoA, establishing it as the critical RhoA activator in cytokinesis.\",\n      \"evidence\": \"RhoA-GTP pull-down with cell synchronization and dominant-negative ECT2\",\n      \"pmids\": [\"10837491\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Spatial targeting mechanism to furrow not addressed\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Mapped autoinhibition to an intramolecular BRCT\\u2013catalytic domain interaction and showed C-terminal sequences select GTPase output, answering how activity is restrained and diversified.\",\n      \"evidence\": \"Co-IP of intramolecular interaction, deletion/NLS mutagenesis, GTPase pull-downs and multinucleation assays\",\n      \"pmids\": [\"15545273\", \"15073184\", \"14645260\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How phosphorylation relieves autoinhibition not yet defined\", \"C-terminal partner mediating Rac/Cdc42 output unidentified\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified centralspindlin/CYK-4 as the recruitment platform that positions ECT2 at the equatorial cortex, answering how RhoA activation is spatially confined.\",\n      \"evidence\": \"Reciprocal co-IP, RNAi epistasis, and immunofluorescence of RhoA/F-actin/myosin localization\",\n      \"pmids\": [\"16103226\", \"16352658\", \"16803869\", \"15642749\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular nature of the cell-cycle-regulated CYK-4 docking site not yet defined\", \"Role in metaphase Cdc42 only Medium confidence\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Defined the CDK1\\u2013Plk1 phosphorylation relay, showing CDK1 at T341/T412 creates a Plk1 docking epitope and links mitotic kinase activity to ECT2 activation and degradation.\",\n      \"evidence\": \"In vitro kinase assays, phospho-site mapping, Plk1-PBD binding, RhoA pull-down; APC/C E2F regulation by ChIP/reporter\",\n      \"pmids\": [\"16170345\", \"16247472\", \"16862181\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative contribution of each phospho-site to autoinhibition relief not resolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Demonstrated a conserved polarity role in C. elegans, showing asymmetric ECT-2 patterns RHO-1 to drive cortical flows and PAR domain establishment.\",\n      \"evidence\": \"Live imaging of GFP fusions with RNAi epistasis in embryos\",\n      \"pmids\": [\"16921365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of centrosomal exclusion of ECT-2 not defined at this stage\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Established that Plk1 phosphorylation of CYK-4 at S164 generates the BRCT docking epitope, mechanistically explaining ECT2 central spindle recruitment downstream of CDK1 inactivation.\",\n      \"evidence\": \"Plk1 inhibitor BI 2536, in vitro kinase assay, phospho-peptide binding, mutagenesis and immunofluorescence\",\n      \"pmids\": [\"17488623\", \"19468300\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether S157 also contributes not resolved here\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed temporal handoff: CYK-4 binds ECT2 early then FIP3 late via overlapping sites, explaining how ECT2 dissociation enables abscission.\",\n      \"evidence\": \"Reciprocal co-IP, domain mapping, immunofluorescence time course\",\n      \"pmids\": [\"18511905\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Trigger for the switch from ECT2 to FIP3 not defined\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Linked cancer ECT2 to the PKC\\u03b9\\u2013Par6 polarity complex, showing cytoplasmic mislocalized ECT2 activates Rac1 to drive transformation and invasion.\",\n      \"evidence\": \"RNAi, co-IP, fractionation, Rac1-GTP pull-down and invasion assays in NSCLC\",\n      \"pmids\": [\"19617897\", \"21189248\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"How mislocalization is initiated in tumors not fully defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Defined PH-domain/polybasic phosphoinositide binding as the equatorial membrane-targeting determinant and characterized ubiquitin-mediated turnover (APC/C-Cdh1, later E6AP).\",\n      \"evidence\": \"Live imaging with PH/polybasic mutants, lipid binding, RhoA assay; ubiquitination assays with degron mutagenesis\",\n      \"pmids\": [\"22172673\", \"21886810\", \"34841254\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How membrane and centralspindlin signals are integrated quantitatively unresolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Showed CDK1 substrate ECT2 couples mitotic entry to cortical rounding and that nuclear export precedes its anaphase repositioning by RacGAP1, ordering its mitotic itinerary.\",\n      \"evidence\": \"Live imaging, Cdk1 substrate mutagenesis, atomic force microscopy and RNAi rescue\",\n      \"pmids\": [\"22898780\", \"22514687\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Anillin\\u2013PH interaction only Medium confidence\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Provided the structural basis of BRCT-phosphoepitope recognition and identified PAR/PARylation as an additional BRCT ligand, refining how ECT2 reads its docking sites.\",\n      \"evidence\": \"X-ray crystallography of triple-BRCT, phospho-peptide binding, in vitro/in vivo PAR binding and microscopy\",\n      \"pmids\": [\"25068414\", \"25486482\", \"25486481\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional importance of tubulin-PAR recruitment vs CYK-4 docking not weighted\", \"PAR binding single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Used optogenetic/chemical membrane targeting to show equatorial membrane association is sufficient for furrowing and that midzone recruitment is dispensable, plus identified a GEF-dependent Wnt regulatory role.\",\n      \"evidence\": \"Optogenetic and chemical genetic membrane targeting; Drosophila/human Wnt reporter assays; Drosophila cortical actin epistasis\",\n      \"pmids\": [\"27926870\", \"24198276\", \"25703349\", \"25604483\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of GEF-dependent Wnt repression not defined\", \"Relative weight of membrane vs midzone signals context-dependent\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established nuclear, GEF-dependent ECT2 as a driver of rRNA synthesis and KRAS-driven tumorigenesis via UBF1 binding and Rac1\\u2013NPM recruitment at rDNA.\",\n      \"evidence\": \"Kras mouse lung tumor model, ChIP, Rac1 pull-down, rRNA synthesis assays with PKC\\u03b9 phospho-mutagenesis\",\n      \"pmids\": [\"28110998\", \"34737214\", \"24386507\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How nuclear vs cytoplasmic GTPase selectivity is achieved mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Detailed the PKC\\u03b9\\u2013UBF1 Ser412 docking mechanism for nuclear ECT2 and identified FoxM1 as a direct GEF inhibitor controlling cortical actin and spindle orientation.\",\n      \"evidence\": \"In vitro kinase/MS phospho-mapping with BRCT mutagenesis and rRNA assays; co-IP with in vitro GEF inhibition and mouse tumor model\",\n      \"pmids\": [\"32350115\", \"34841254\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How FoxM1 inhibition is relieved during normal mitosis not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Dissected individual BRCT contributions to activation and zone restriction and uncovered a GEF-independent role for ECT2 in DSB repair recruiting KU70/80 and BRCA1.\",\n      \"evidence\": \"Systematic BRCT mutagenesis with RhoA biosensor imaging; co-IP, laser microirradiation, GEF-dead complementation and comet assays\",\n      \"pmids\": [\"33657383\", \"34343566\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DSB repair role single lab\", \"How ECT2 is recruited to PAR at lesions vs mitotic spindle distinguished only partially\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected ECT2 to DNA-damage signaling (DNA-PK\\u2013Sin1\\u2013ECT2\\u2013AKT) and to membrane compartmentalization that coordinates spindle elongation with furrowing.\",\n      \"evidence\": \"Co-IP, RNAi, catalytic mutants and AKT/survival assays; RNAi with live imaging of NuMA/dynein membrane exclusion\",\n      \"pmids\": [\"34982576\", \"36197340\", \"36533896\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab for each\", \"How GEF activity feeds AKT activation mechanistically unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended ECT2 function to confined migration, where anillin recruits it to cell poles to activate RhoA-driven bleb migration amplified by nuclear envelope rupture.\",\n      \"evidence\": \"Microfluidic confinement, live imaging, GEF-dead mutant, ROCK inhibition and nuclear rupture assays\",\n      \"pmids\": [\"40571734\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab\", \"Generality across cell/tumor types untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the multiple non-canonical nuclear roles (rDNA transcription, DSB repair, AKT signaling) are coordinated with the canonical cytokinetic function within a single cell remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model partitioning ECT2 pools across functions\", \"No structural model of full-length activated ECT2 on membrane/centralspindlin\", \"Disease-causal mutations not established in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 3, 4]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [1, 2, 11, 21]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [29]},\n      {\"term_id\": \"GO:0008289\", \"supporting_discovery_ids\": [21, 25]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 3, 22, 26, 41]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [1, 19, 24, 45]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [21, 31, 45]},\n      {\"term_id\": \"GO:0005819\", \"supporting_discovery_ids\": [7, 8, 12, 29]},\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [35, 37]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 2, 7, 24]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 11, 19, 42]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [35, 37, 41]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [40, 42]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [19, 34, 35]}\n    ],\n    \"complexes\": [\"centralspindlin (with CYK-4/MgcRacGAP)\", \"PKC\\u03b9-Par6 polarity complex\"],\n    \"partners\": [\"RACGAP1\", \"PLK1\", \"CDK1\", \"PRKCI\", \"PARD6A\", \"UBF1\", \"ANLN\", \"RhoA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}