{"gene":"ERRFI1","run_date":"2026-04-28T17:46:03","timeline":{"discoveries":[{"year":2000,"finding":"RALT (ERRFI1/Gene 33) was identified as an ErbB-2-interacting protein via yeast two-hybrid screen; the interaction requires active ErbB-2 catalytic function (but not autophosphorylation), is mediated by residues 282–395 of RALT, and results in inhibition of ErbB-2-driven cell proliferation, transformation, and sustained ERK1/2 activation. RALT was also found to associate with GRB2 in living cells.","method":"Yeast two-hybrid, GST pulldown, Co-IP, cell proliferation and transformation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, in vitro pulldown, multiple orthogonal functional assays in single study","pmids":["11003669"],"is_preprint":false},{"year":2001,"finding":"Mig-6 (ERRFI1) was identified in a yeast two-hybrid screen with the EGFR kinase domain; upon EGF stimulation Mig-6 binds to EGFR via an acidic region (aa 985–995) in a kinase-activity-dependent manner, overexpression reduces EGF-induced ERK2 activation and enhances EGFR internalization, and Mig-6 inhibits EGFR-overexpression-induced transformation.","method":"Yeast two-hybrid, Co-IP, ERK2 activation assay, receptor internalization assay, transformation assay","journal":"Biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods, replicated across labs","pmids":["11843178"],"is_preprint":false},{"year":2000,"finding":"Gene 33/Mig-6 interacts in vivo and in a GTP-dependent manner in vitro with Cdc42Hs (a Rho-family GTPase), and transient expression of Gene 33 selectively activates SAPK/JNK kinases.","method":"Co-IP in vivo, in vitro GTP-dependent pulldown, SAPK/JNK activation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro reconstitution (GTP-dependent binding) plus cellular functional assay","pmids":["10749885"],"is_preprint":false},{"year":2003,"finding":"RALT/ERRFI1 binds ligand-activated EGFR, ErbB-4, and ErbB-2:ErbB-3 dimers, functioning as a pan-ErbB inhibitor; a mutant (RALT deltaEBR) unable to bind ErbB RTKs loses the ability to suppress ERK and AKT activation at saturating ligand concentrations, demonstrating that suppressive activity is primarily receptor-proximal. RALT deltaEBR retains partial suppression of ErbB-2:ErbB-3 mitogenic signals at a receptor-distal level.","method":"Co-IP, cell proliferation assays, ERK and AKT phosphorylation assays, mutant analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP plus multiple functional assays with structure-function mutant","pmids":["12833145"],"is_preprint":false},{"year":2002,"finding":"RALT/ERRFI1 protein is ubiquitinated and degraded by the proteasome; its expression is driven transcriptionally through the Ras-Raf-ERK pathway (pharmacological ERK inhibition blocks RALT induction; activated Raf:ER chimera is sufficient to induce RALT), providing integrated transcriptional and post-translational control.","method":"Ubiquitin Co-IP, proteasome inhibitor treatment, pharmacological kinase inhibitors, inducible Raf:ER system","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (ubiquitination detection, genetic epistasis via pharmacological tools, inducible system) in single study","pmids":["12226756"],"is_preprint":false},{"year":2004,"finding":"Gene 33/ERRFI1 inhibits EGFR autophosphorylation and downstream activation of Ras, ERK, JNK, Akt/PKB, and Rb; the Ack-homology (EBR) domain is necessary and sufficient for this inhibition. Dexamethasone-induced Gene 33 expression mediates glucocorticoid suppression of EGF signaling, as demonstrated by RNA interference reversal.","method":"In vitro EGFR autophosphorylation assay, domain deletion mutants, RNAi knockdown, Western blot","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro enzymatic assay with domain mutants plus RNAi rescue","pmids":["15556944"],"is_preprint":false},{"year":2007,"finding":"The evolutionarily conserved ErbB-binding region (EBR) of RALT/MIG6 is necessary and sufficient to suppress EGFR kinase catalytic activity in vitro and in intact cells; the mechanism involves binding of the EBR to the 953RYLVIQ958 sequence in the αI helix of the EGFR kinase domain, which participates in allosteric control of EGFR activity.","method":"In vitro kinase assay, domain mutant overexpression, EBR peptide binding, site-directed mutagenesis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase inhibition assay with defined domain mutants and mechanistic mapping to allosteric site","pmids":["17599051"],"is_preprint":false},{"year":2010,"finding":"RALT/MIG6 mediates EGFR endocytosis and lysosomal degradation via a domain distinct from the EGFR kinase-suppressive domain; RALT drives EGFR endocytosis by binding to AP-2 and Intersectins, rescues endocytic deficits of EGFR mutants (Dc214 and Y1045F), integrating kinase suppression with receptor removal for durable repression of EGFR signaling.","method":"Co-IP (RALT with AP-2 and Intersectins), endocytosis assays with EGFR mutants, lysosomal degradation assays, domain mutant analysis","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP of endocytic proteins, multiple EGFR mutants, domain dissection; replicated concept across labs","pmids":["20421427"],"is_preprint":false},{"year":2010,"finding":"Mig-6 drives EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation by binding to the SNARE protein STX8 (required for late endosome trafficking), physically linking EGFR to STX8 during ligand-stimulated receptor trafficking.","method":"Co-IP (Mig-6 with STX8 and EGFR), EGFR trafficking assay, late endosome/lysosome localization","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — Co-IP identifying novel binding partner STX8 with functional trafficking consequence, combined with in vivo GBM data","pmids":["20351267"],"is_preprint":false},{"year":2002,"finding":"Full-length Mig-6 (but not CRIB domain-deleted Mig-6 or uncleavable Mig-6-S38A mutant) activates NF-κB transcription; Mig-6 undergoes limited proteolytic cleavage, and the processed N-terminal CRIB-containing fragment binds IκBα through its NF-κB binding region, competing with NF-κB for IκBα and releasing NF-κB.","method":"Luciferase reporter assay, Co-IP of CRIB fragment with IκBα, mutagenesis (CRIB deletion, S38A mutant)","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and reporter assay with domain mutants, single lab","pmids":["12384522"],"is_preprint":false},{"year":2005,"finding":"Targeted expression of RALT/MIG6 in mouse skin (K14-RALT transgene) generates a dose-dependent Waved-like phenotype (wavy coat, curly whiskers, open eyes at birth) due to suppression of EGFR signaling; ex vivo keratinocytes show reduced ErbB-ligand-induced responses, and RNAi knockdown of RALT enhances ErbB mitogenic signaling.","method":"Transgenic mouse (K14-RALT), ex vivo keratinocyte stimulation assays, RNAi knockdown","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 — in vivo gain-of-function with defined phenotype plus RNAi loss-of-function, consistent mechanistic interpretation","pmids":["16007071"],"is_preprint":false},{"year":2005,"finding":"Targeted disruption of Mig-6 (ERRFI1) in mice leads to early onset degenerative joint disease with articular cartilage degradation and osteophyte formation; absence of Rag2 does not rescue the phenotype, excluding the acquired immune system. Mig-6 knockout chondrocytes show enhanced EGFR signaling and excessive proliferation.","method":"Germline knockout mouse, histology, Rag2 double-knockout epistasis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined joint phenotype, epistasis experiment, replicated in multiple follow-up studies","pmids":["16087873"],"is_preprint":false},{"year":2006,"finding":"Gene 33/ERRFI1 is induced in cardiomyocytes by hypoxia; adenoviral overexpression promotes cardiomyocyte death by reducing Akt and ERK signaling, and RNAi knockdown of endogenous Gene 33 reduces hypoxia-induced cardiomyocyte death, demonstrating that Gene 33 is a pro-death component in ischemic injury.","method":"Adenoviral overexpression, RNAi knockdown, Akt/ERK phosphorylation assay, cardiomyocyte death assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — gain-of-function and RNAi loss-of-function with defined death phenotype and signaling readout","pmids":["16782890"],"is_preprint":false},{"year":2009,"finding":"Mig-6 (ERRFI1) is a downstream target of progesterone receptor (PR) and SRC-1 in the uterus; absence of Mig-6 prevents progesterone from inhibiting estrogen-induced uterine responses, and ovariectomized Mig-6-deficient mice treated with estrogen develop endometrial adenocarcinoma, placing Mig-6 in a PR–SRC-1–Mig-6 pathway that suppresses endometrial cancer.","method":"Germline knockout mouse, ovariectomy/hormone treatment, uterine weight assay, gene expression analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined hormone-response phenotype, genetic pathway placement","pmids":["19439667"],"is_preprint":false},{"year":2012,"finding":"Chk1 kinase phosphorylates Mig-6 at Ser251 in vivo and in vitro; EGF stimulation promotes this phosphorylation via PI3K–Chk1 axis; the Ser251Ala substitution increases Mig-6 inhibitory activity against EGFR; Chk1 depletion inhibits EGF-dependent EGFR activation and cell growth, which is rescued by co-depletion of Mig-6, placing Chk1 as a positive regulator of EGFR signaling through Mig-6 inactivation.","method":"In vitro kinase assay, mass spectrometry phosphosite mapping, site-directed mutagenesis, RNAi epistasis, EGFR activation assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay + MS phosphosite mapping + mutagenesis + genetic epistasis","pmids":["22505024"],"is_preprint":false},{"year":2017,"finding":"Mig-6 inhibits EGF-induced cell migration via its CRIB domain binding to Cdc42; four specific CRIB residues (I11, R12, M26, R30) mediate Cdc42 binding, which is necessary and sufficient to inhibit filopodia formation and migration. The CRIB domain alone is sufficient to inhibit migration; Mig-6 binding to EGFR is dispensable for this anti-migratory function.","method":"Co-IP, site-directed mutagenesis of CRIB domain, cell migration assay, filopodia formation assay, domain rescue experiments","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 2 — domain mutagenesis with Co-IP and defined cell migration phenotype; mechanistic dissection from EGFR binding","pmids":["27341132"],"is_preprint":false},{"year":2017,"finding":"MIG-6 negatively regulates STAT3 phosphorylation in uterine epithelial cells through protein–protein interaction; Mig-6 epithelial-specific knockout mice develop endometrial hyperplasia with aberrant STAT3 phosphorylation, and immunoprecipitation confirmed MIG-6–STAT3 interaction.","method":"Conditional epithelial-specific KO mouse, immunoprecipitation, STAT3 phosphorylation assay","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — Co-IP identifying direct interaction plus clean conditional KO with defined phosphorylation phenotype","pmids":["28925396"],"is_preprint":false},{"year":2017,"finding":"Gene 33/Mig-6 localizes to the nucleus and chromatin fractions; nuclear localization is regulated by its 14-3-3-binding domain; chromatin association is partially dependent on its ErbB-binding domain. Nuclear Gene 33 promotes ATM-dependent DNA damage response (DDR), associates with histone H2AX, and promotes ATM–H2AX interaction without triggering DNA damage. Gene 33 may regulate c-Abl targeting to chromatin.","method":"Subcellular fractionation, Co-IP (Gene 33 with H2AX and ATM), domain mutagenesis, ectopic expression with ATM inhibition","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2/3 — fractionation, Co-IP, and domain mutants, but single lab; ATM dependence shown pharmacologically","pmids":["28842482"],"is_preprint":false},{"year":2018,"finding":"ERRFI1 has a dual role in AKT regulation: in EGFR-high cells, ERRFI1 inhibits AKT signaling through EGFR inhibition; in EGFR-low cells, ERRFI1 positively modulates AKT signaling by interfering with the interaction between the inactivating phosphatase PHLPP and AKT, thereby promoting AKT activity.","method":"Co-IP (ERRFI1 with PHLPP and AKT), knockdown/overexpression, AKT phosphorylation assay, proliferation assay","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP identifying PHLPP–AKT disruption plus functional assays, single lab","pmids":["29335246"],"is_preprint":false},{"year":2010,"finding":"Co-immunoprecipitation identified ERK2 as an MIG-6 interacting protein; Mig-6 ablation (alone or combined with Pten ablation) increases ERK2 phosphorylation, and Mig-6 loss promotes endometrial tumorigenesis partly through upregulation of apoptotic inhibitor Birc1.","method":"Co-IP, conditional double KO mouse, Western blot for phospho-ERK2","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP identifying ERK2 interaction, supported by KO phenotype; single lab","pmids":["20418913"],"is_preprint":false},{"year":2021,"finding":"MIG-6 recruits HAUSP (USP7) deubiquitinase to stabilize HIF1α protein, leading to upregulation of GLUT1 and other HIF1α-regulated glycolytic genes in triple-negative breast cancer, thereby driving metabolic reprogramming toward glycolysis.","method":"Co-IP (MIG-6 with HAUSP), HIF1α protein stability assay, GLUT1 expression assay, metabolic array, mouse tumor xenograft","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP identifying HAUSP interaction with functional consequence on HIF1α stability; single lab","pmids":["33655623"],"is_preprint":false},{"year":2021,"finding":"ERRFI1 induces apoptosis in hepatocellular carcinoma cells by binding PDCD2; PDCD2 knockdown decreases ERRFI1-induced apoptosis, establishing PDCD2 as a functional effector of ERRFI1-mediated apoptosis.","method":"Co-IP (ERRFI1 with PDCD2), RNAi knockdown, apoptosis assay","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP plus RNAi rescue; single lab, single study","pmids":["34608122"],"is_preprint":false},{"year":2022,"finding":"Loss of MIG-6 in the endometrium causes ERBB2 overexpression and endometrial progesterone resistance; genetic ablation of Erbb2 in Mig-6 knockout mice rescues all Mig-6d/d phenotypes (non-receptive endometrium, infertility, hyperplasia), placing ERBB2 downstream of MIG-6 loss.","method":"Double conditional KO mouse (Mig-6d/dErbb2d/d), transcriptomics, fertility/implantation assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 — clean genetic epistasis via double KO with full phenotypic rescue and transcriptomics","pmids":["35232969"],"is_preprint":false},{"year":2024,"finding":"ERRFI1 interacts with GRB2 and maintains GRB2 stability by hindering its proteasomal degradation; hepatocyte-specific ERRFI1 knockout reduces apoptosis and ferroptosis in hepatic ischemia–reperfusion injury, and GRB2 overexpression reverses the protective effects of ERRFI1 silencing.","method":"Hepatocyte-specific KO mouse, Co-IP (ERRFI1–GRB2), proteasomal degradation assay, apoptosis and ferroptosis assays","journal":"Molecular medicine (Cambridge, Mass.)","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP with functional rescue by GRB2 overexpression, clean conditional KO; single lab","pmids":["38862918"],"is_preprint":false},{"year":2020,"finding":"ANRIL lncRNA binds EZH2 and maintains H3K27me3 at the ERRFI1 promoter, epigenetically repressing ERRFI1 expression in cholangiocarcinoma; ANRIL knockdown increases ERRFI1 and inhibits tumor growth.","method":"RNA-seq after ANRIL knockdown, ChIP for H3K27me3 at ERRFI1 promoter, Co-IP (ANRIL-EZH2), in vitro and in vivo tumor assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP plus RIP/Co-IP identifying epigenetic writer; functional tumor assays; single lab","pmids":["32378752"],"is_preprint":false},{"year":2018,"finding":"In MET-TKI-resistant cancer cells, epigenetically induced miR-205 downregulates ERRFI1, which in turn causes EGFR activation and sustains resistance; anti-miR-205 reverts crizotinib resistance in vivo, and this mechanism operates in the absence of EGFR genetic alterations.","method":"miRNA profiling, RNA-seq, anti-miR-205 transduction, in vivo xenograft, patient sample analysis","journal":"EMBO molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 — miRNA-target axis validated with in vivo rescue experiment and patient sample; single lab","pmids":["30021798"],"is_preprint":false},{"year":2014,"finding":"Cartilage-specific deletion of Mig-6 in chondrocytes (Col2a1-Cre) causes excessive articular chondrocyte proliferation and an OA-like phenotype in knees (but not all joints affected in full KO), demonstrating that Mig-6 in chondrocytes regulates EGF receptor signaling and chondrocyte proliferation in joint homeostasis.","method":"Cartilage-specific conditional KO mouse, histology, EGFR phosphorylation immunostaining, proliferation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — clean cell-type-specific KO with defined cellular phenotype and EGFR signaling readout","pmids":["24550287"],"is_preprint":false},{"year":2018,"finding":"MIG-6 interacts with AKT and inhibits AKT phosphorylation in uterine epithelial cells; in vitro studies revealed a direct MIG-6–AKT protein interaction, and Mig-6 epithelial-specific KO mice develop endometrial hyperplasia with increased phospho-AKT.","method":"Co-IP (MIG-6 with AKT), conditional epithelial KO mouse, AKT phosphorylation assay","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP identifying interaction plus KO phenotype; single lab","pmids":["29843645"],"is_preprint":false}],"current_model":"ERRFI1/MIG-6/RALT is a multifunctional adapter protein that acts as a feedback inhibitor of ErbB receptor tyrosine kinases: its evolutionarily conserved EBR domain directly suppresses EGFR kinase activity by binding the αI-helix allosteric site, while a separate domain drives EGFR endocytosis and lysosomal degradation by recruiting AP-2, Intersectin, and the SNARE protein STX8; ERRFI1 also inhibits cell migration by binding Cdc42 via its CRIB domain, regulates STAT3 and AKT phosphorylation through direct protein interactions, interacts with GRB2 and PDCD2 to modulate apoptosis, recruits HAUSP to stabilize HIF1α in certain cancer contexts, promotes ATM-dependent DNA damage responses from the nucleus, and is itself phosphorylated by Chk1 on Ser251 to attenuate its inhibitory activity—collectively placing ERRFI1 as a central node integrating receptor signaling, cytoskeletal dynamics, DNA damage response, and hormone-mediated transcriptional control."},"narrative":{"teleology":[{"year":2000,"claim":"Establishing ERRFI1 as a direct ErbB-2/EGFR-interacting inhibitor and Cdc42 effector resolved the dual question of how an immediate-early gene product feeds back on receptor signaling and connects to Rho-family GTPase pathways.","evidence":"Yeast two-hybrid, Co-IP, GST pulldown identifying ErbB-2 and Cdc42 as binding partners, with proliferation/transformation assays and SAPK/JNK activation","pmids":["11003669","10749885"],"confidence":"High","gaps":["Structural basis of ERRFI1–ErbB-2 interaction unknown","Whether Cdc42 binding and ErbB binding are functionally coupled was not tested","Endogenous stoichiometry not determined"]},{"year":2001,"claim":"Demonstrating that Mig-6 binds EGFR in a kinase-activity-dependent manner and enhances EGFR internalization established it as a pan-ErbB feedback inhibitor that operates both at the kinase level and at receptor trafficking.","evidence":"Yeast two-hybrid with EGFR kinase domain, Co-IP, receptor internalization and transformation assays","pmids":["11843178"],"confidence":"High","gaps":["Mechanism of enhanced internalization not dissected","ErbB-3/ErbB-4 binding not yet tested"]},{"year":2002,"claim":"Showing that ERRFI1 is transcriptionally induced by the Ras–Raf–ERK pathway and degraded by the proteasome established the complete negative-feedback loop architecture: ERK induces ERRFI1, which then suppresses ErbB–ERK signaling and is itself cleared by ubiquitination.","evidence":"Pharmacological ERK inhibition, inducible Raf:ER chimera, ubiquitin Co-IP, proteasome inhibitor treatment","pmids":["12226756"],"confidence":"High","gaps":["E3 ubiquitin ligase responsible for ERRFI1 degradation not identified","Half-life quantification not performed"]},{"year":2002,"claim":"Discovery that a proteolytically processed CRIB-containing fragment of Mig-6 activates NF-κB by competing with NF-κB for IκBα binding revealed a non-ErbB signaling function downstream of limited proteolysis.","evidence":"Luciferase reporter, Co-IP of CRIB fragment with IκBα, CRIB deletion and S38A mutants","pmids":["12384522"],"confidence":"Medium","gaps":["Protease responsible for Mig-6 cleavage not identified","Physiological relevance of NF-κB activation not confirmed in vivo","Not independently replicated"]},{"year":2003,"claim":"Extending ERRFI1 inhibition to ErbB-4 and ErbB-2:ErbB-3 heterodimers, and showing that an EBR-deletion mutant loses receptor-proximal but retains partial receptor-distal suppression, dissected the architecture of ERRFI1's inhibitory mechanism into two functional layers.","evidence":"Co-IP with multiple ErbB family members, ERK/AKT phosphorylation assays, RALT-ΔEBR mutant","pmids":["12833145"],"confidence":"High","gaps":["Identity of the receptor-distal suppressive mechanism unknown","Quantitative contribution of each layer not measured"]},{"year":2004,"claim":"Demonstrating that the EBR domain is necessary and sufficient to block EGFR autophosphorylation in vitro and that dexamethasone-induced ERRFI1 mediates glucocorticoid suppression of EGF signaling linked hormonal regulation to ERRFI1's kinase-inhibitory function.","evidence":"In vitro EGFR autophosphorylation assay, domain deletions, RNAi reversal of dexamethasone effect","pmids":["15556944"],"confidence":"High","gaps":["Glucocorticoid receptor binding site in ERRFI1 promoter not mapped","Whether glucocorticoid–ERRFI1 axis operates in all tissues not tested"]},{"year":2005,"claim":"In vivo gain-of-function (K14-RALT transgene producing Waved-like phenotype) and loss-of-function (Mig-6 knockout causing degenerative joint disease) in mice validated ERRFI1 as a physiologically essential EGFR inhibitor in skin and cartilage homeostasis.","evidence":"Transgenic mouse with dose-dependent phenotype; germline KO mouse with joint degeneration; Rag2 double-KO epistasis excluding immune involvement","pmids":["16007071","16087873"],"confidence":"High","gaps":["Molecular events downstream of EGFR hyperactivation in chondrocytes not detailed","Whether joint phenotype is solely EGFR-dependent not formally tested"]},{"year":2007,"claim":"Mapping the EBR–EGFR interaction to the αI-helix (953RYLVIQ958) allosteric site provided a structural mechanism: ERRFI1 locks EGFR in an inactive conformation by blocking the asymmetric dimer interface.","evidence":"In vitro kinase assay, EBR peptide binding, site-directed mutagenesis of αI-helix residues","pmids":["17599051"],"confidence":"High","gaps":["No crystal structure of the ERRFI1-EBR:EGFR complex at this time","Whether this mechanism applies equally to all ErbB family members not tested"]},{"year":2009,"claim":"Placing ERRFI1 downstream of progesterone receptor–SRC-1 signaling and showing that Mig-6-deficient mice develop estrogen-driven endometrial adenocarcinoma revealed ERRFI1 as a hormone-regulated tumor suppressor in the uterus.","evidence":"Germline KO mouse with ovariectomy/hormone treatment, endometrial adenocarcinoma development","pmids":["19439667"],"confidence":"High","gaps":["Direct transcriptional regulation of ERRFI1 by PR not shown at the promoter level","Whether human endometrial cancer recapitulates this mechanism not established"]},{"year":2010,"claim":"Identification of AP-2, Intersectin, and STX8 as ERRFI1 binding partners that mediate EGFR endocytosis and late-endosome/lysosomal sorting established a bipartite mechanism: one domain suppresses kinase activity, a separate domain ensures receptor destruction.","evidence":"Co-IP with AP-2, Intersectins, and STX8; EGFR trafficking and degradation assays; rescue of endocytosis-defective EGFR mutants","pmids":["20421427","20351267"],"confidence":"High","gaps":["Precise ERRFI1 residues mediating AP-2 and STX8 binding not mapped","Whether ERRFI1-driven endocytosis operates independently of Cbl-mediated ubiquitination not resolved"]},{"year":2012,"claim":"Discovery that Chk1 phosphorylates ERRFI1 at Ser251 to attenuate its EGFR-inhibitory activity uncovered a mechanism by which DNA damage checkpoint signaling cross-talks with receptor tyrosine kinase output, converting ERRFI1 from an active inhibitor to an inactive form.","evidence":"In vitro kinase assay, MS phosphosite mapping, S251A mutagenesis, RNAi epistasis (Chk1 + Mig-6 co-depletion rescue)","pmids":["22505024"],"confidence":"High","gaps":["Phosphatase that reverses Ser251 phosphorylation not identified","Whether other kinases also regulate ERRFI1 function not tested"]},{"year":2014,"claim":"Cartilage-specific deletion of Mig-6 confirmed that ERRFI1 acts cell-autonomously in chondrocytes to restrain EGFR-driven proliferation and protect against osteoarthritis-like degeneration.","evidence":"Col2a1-Cre conditional KO mouse, EGFR phosphorylation immunostaining, chondrocyte proliferation assays","pmids":["24550287"],"confidence":"High","gaps":["Specific EGFR ligand driving joint pathology not identified","Therapeutic rescue by ERRFI1 restoration not attempted"]},{"year":2017,"claim":"Dissecting ERRFI1's CRIB domain showed that Cdc42 binding (via I11, R12, M26, R30) is necessary and sufficient to inhibit filopodia formation and cell migration, independently of EGFR binding, formally separating the cytoskeletal and receptor-inhibitory functions of the protein.","evidence":"CRIB point mutagenesis, Co-IP with Cdc42, migration and filopodia assays, domain rescue experiments","pmids":["27341132"],"confidence":"High","gaps":["Whether CRIB–Cdc42 interaction also affects polarity or invasion not tested","In vivo relevance of CRIB-mediated migration inhibition not established"]},{"year":2017,"claim":"Demonstrating direct MIG-6–STAT3 interaction and showing that epithelial-specific Mig-6 loss causes endometrial hyperplasia with aberrant STAT3 phosphorylation extended ERRFI1's inhibitory reach beyond receptor kinases to a transcription factor, providing a new mechanism for its tumor-suppressive activity in the uterus.","evidence":"Conditional epithelial KO mouse, immunoprecipitation, STAT3 phosphorylation assay","pmids":["28925396"],"confidence":"High","gaps":["Binding interface between MIG-6 and STAT3 not mapped","Whether STAT3 inhibition is direct or requires an intermediate not resolved"]},{"year":2017,"claim":"Finding that ERRFI1 localizes to chromatin and promotes ATM–H2AX interaction without inducing DNA damage revealed a nuclear scaffolding function that places ERRFI1 in DNA damage response signaling independent of its cytoplasmic ErbB-inhibitory role.","evidence":"Subcellular fractionation, Co-IP with H2AX and ATM, domain mutagenesis, ATM pharmacological inhibition","pmids":["28842482"],"confidence":"Medium","gaps":["Not independently replicated","Whether nuclear ERRFI1 function is physiologically relevant in DNA repair outcomes not tested","ATM dependence shown only pharmacologically"]},{"year":2018,"claim":"Revealing that ERRFI1 has a context-dependent dual role in AKT regulation — inhibiting AKT via EGFR in EGFR-high cells but promoting AKT by disrupting the PHLPP–AKT interaction in EGFR-low cells — explained contradictory reports and established ERRFI1 as a context-dependent signaling switch.","evidence":"Co-IP of ERRFI1 with PHLPP and AKT, knockdown/overexpression in EGFR-high versus EGFR-low cell lines","pmids":["29335246"],"confidence":"Medium","gaps":["Structural basis of PHLPP displacement not determined","Threshold of EGFR expression for mode-switching not defined","Not replicated by independent laboratory"]},{"year":2021,"claim":"Discovery that MIG-6 recruits the HAUSP deubiquitinase to stabilize HIF1α and drive glycolytic reprogramming in triple-negative breast cancer revealed a pro-tumorigenic function that contrasts with ERRFI1's canonical tumor-suppressive role.","evidence":"Co-IP of MIG-6 with HAUSP, HIF1α stability assay, metabolic profiling, mouse xenograft","pmids":["33655623"],"confidence":"Medium","gaps":["Whether HAUSP recruitment is ErbB-dependent not tested","Generalizability beyond triple-negative breast cancer unknown","Not independently replicated"]},{"year":2022,"claim":"Genetic epistasis showing that Erbb2 ablation fully rescues all phenotypes of Mig-6-deficient endometrium (infertility, hyperplasia, progesterone resistance) placed ERBB2 overexpression as the principal effector downstream of ERRFI1 loss in the uterus.","evidence":"Double conditional KO mouse (Mig-6d/d Erbb2d/d), fertility and implantation assays, transcriptomics","pmids":["35232969"],"confidence":"High","gaps":["Whether ERRFI1 directly degrades ERBB2 protein or merely restrains its signaling not distinguished","Whether ERBB2 is the sole relevant ErbB target in all endometrial contexts unclear"]},{"year":2024,"claim":"Identification of GRB2 stabilization by ERRFI1 (hindering proteasomal degradation) and its role in promoting hepatocyte apoptosis and ferroptosis during ischemia–reperfusion injury added a new cell-death axis to ERRFI1's functional repertoire.","evidence":"Hepatocyte-specific KO mouse, Co-IP of ERRFI1–GRB2, proteasomal degradation assay, GRB2 overexpression rescue","pmids":["38862918"],"confidence":"Medium","gaps":["Whether GRB2 stabilization depends on the EBR domain not tested","Ferroptosis mechanism downstream of GRB2 not elucidated","Not independently replicated"]},{"year":null,"claim":"Key unresolved questions include the identity of the E3 ligase that ubiquitinates ERRFI1, the structural basis of the full-length ERRFI1–EGFR complex, how nuclear versus cytoplasmic ERRFI1 pools are partitioned and regulated, and whether the pro-tumorigenic HAUSP/HIF1α-stabilizing function and the anti-tumorigenic ErbB-inhibitory function coexist or are mutually exclusive in specific cancer contexts.","evidence":"Unresolved in current literature","pmids":[],"confidence":"Low","gaps":["E3 ligase for ERRFI1 ubiquitination not identified","Full-length ERRFI1 crystal structure unavailable","Nuclear–cytoplasmic partitioning regulation not systematically studied"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,3,5,6,7,14,16,27]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[7,8,20,23]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[2,15]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,1,7,15]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[17]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[7,8]},{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[1,7]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,1,3,5,6,14,16,18,27]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11,13,22,26]},{"term_id":"R-HSA-9609507","term_label":"Protein localization","supporting_discovery_ids":[7,8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[12,21,23]}],"complexes":[],"partners":["EGFR","ERBB2","CDC42","GRB2","STX8","STAT3","AKT1","PHLPP1"],"other_free_text":[]},"mechanistic_narrative":"ERRFI1 (also known as MIG-6/RALT/Gene 33) is a stress-inducible, multifunctional adaptor protein that serves as a central feedback inhibitor of ErbB receptor tyrosine kinase signaling while also integrating cytoskeletal, DNA damage, and metabolic pathways. Its conserved ErbB-binding region (EBR) directly suppresses EGFR kinase activity by engaging the αI-helix allosteric site, while a separate domain drives EGFR endocytosis and lysosomal degradation through interactions with AP-2, Intersectins, and the SNARE protein STX8, thereby achieving durable signal extinction [PMID:17599051, PMID:20421427, PMID:20351267]. Independent of EGFR binding, ERRFI1 inhibits Cdc42-dependent cell migration via its CRIB domain, negatively regulates STAT3 and AKT phosphorylation through direct protein–protein interactions, and promotes ATM-dependent DNA damage responses from the nucleus [PMID:27341132, PMID:28925396, PMID:29843645, PMID:28842482]. ERRFI1 itself is transcriptionally induced by the Ras–Raf–ERK pathway and by progesterone receptor signaling, is subject to proteasomal degradation, and is phosphorylated by Chk1 at Ser251 to attenuate its inhibitory function, establishing multilayered feedback control; loss of ERRFI1 in mice causes degenerative joint disease, endometrial hyperplasia and cancer, and enhanced ErbB-driven proliferation [PMID:12226756, PMID:22505024, PMID:16087873, PMID:19439667]."},"prefetch_data":{"uniprot":{"accession":"Q9UJM3","full_name":"ERBB receptor feedback inhibitor 1","aliases":["Mitogen-inducible gene 6 protein","MIG-6"],"length_aa":462,"mass_kda":50.6,"function":"Negative regulator of EGFR signaling in skin morphogenesis. Acts as a negative regulator for several EGFR family members, including ERBB2, ERBB3 and ERBB4. Inhibits EGFR catalytic activity by interfering with its dimerization. Inhibits autophosphorylation of EGFR, ERBB2 and ERBB4. Important for normal keratinocyte proliferation and differentiation. Plays a role in modulating the response to steroid hormones in the uterus. Required for normal response to progesterone in the uterus and for fertility. Mediates epithelial estrogen responses in the uterus by regulating ESR1 levels and activation. Important for regulation of endometrium cell proliferation. Important for normal prenatal and perinatal lung development (By similarity)","subcellular_location":"Cytoplasm; Cell membrane; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9UJM3/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ERRFI1","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ERRFI1","total_profiled":1310},"omim":[{"mim_id":"608069","title":"ERBB RECEPTOR FEEDBACK INHIBITOR 1; ERRFI1","url":"https://www.omim.org/entry/608069"},{"mim_id":"131550","title":"EPIDERMAL GROWTH FACTOR RECEPTOR; EGFR","url":"https://www.omim.org/entry/131550"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Cytosol","reliability":"Enhanced"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"liver","ntpm":695.4},{"tissue":"pancreas","ntpm":640.2}],"url":"https://www.proteinatlas.org/search/ERRFI1"},"hgnc":{"alias_symbol":["MIG-6","GENE-33","RALT"],"prev_symbol":[]},"alphafold":{"accession":"Q9UJM3","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJM3","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJM3-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9UJM3-F1-predicted_aligned_error_v6.png","plddt_mean":56.56},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ERRFI1","jax_strain_url":"https://www.jax.org/strain/search?query=ERRFI1"},"sequence":{"accession":"Q9UJM3","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9UJM3.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9UJM3/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9UJM3"}},"corpus_meta":[{"pmid":"11843178","id":"PMC_11843178","title":"Mig-6 is a negative regulator of the epidermal growth factor receptor signal.","date":"2001","source":"Biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11843178","citation_count":130,"is_preprint":false},{"pmid":"11003669","id":"PMC_11003669","title":"Inhibition of ErbB-2 mitogenic and transforming activity by RALT, a mitogen-induced signal transducer which binds to the ErbB-2 kinase domain.","date":"2000","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11003669","citation_count":121,"is_preprint":false},{"pmid":"10749885","id":"PMC_10749885","title":"Gene 33/Mig-6, a transcriptionally inducible adapter protein that binds GTP-Cdc42 and activates SAPK/JNK. 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development","url":"https://pubmed.ncbi.nlm.nih.gov/27358163","citation_count":7,"is_preprint":false},{"pmid":"34845855","id":"PMC_34845855","title":"Mig-6 could inhibit cell proliferation and induce apoptosis in esophageal squamous cell carcinoma.","date":"2021","source":"Thoracic cancer","url":"https://pubmed.ncbi.nlm.nih.gov/34845855","citation_count":7,"is_preprint":false},{"pmid":"9292001","id":"PMC_9292001","title":"Characterization of the proteinase specified by varicella-zoster virus gene 33.","date":"1997","source":"The Journal of general virology","url":"https://pubmed.ncbi.nlm.nih.gov/9292001","citation_count":7,"is_preprint":false},{"pmid":"18056042","id":"PMC_18056042","title":"Expression of MIG-6, WNT-9A, and WNT-7B during osteoarthritis.","date":"2007","source":"Annals of the New York Academy of Sciences","url":"https://pubmed.ncbi.nlm.nih.gov/18056042","citation_count":7,"is_preprint":false},{"pmid":"1472065","id":"PMC_1472065","title":"Vanadate and insulin stimulate gene 33 expression.","date":"1992","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/1472065","citation_count":7,"is_preprint":false},{"pmid":"33869036","id":"PMC_33869036","title":"Capilliposide C from Lysimachia capillipes Restores Radiosensitivity in Ionizing Radiation-Resistant Lung Cancer Cells Through Regulation of ERRFI1/EGFR/STAT3 Signaling Pathway.","date":"2021","source":"Frontiers in oncology","url":"https://pubmed.ncbi.nlm.nih.gov/33869036","citation_count":6,"is_preprint":false},{"pmid":"25342879","id":"PMC_25342879","title":"Decreased Mitogen Inducible Gene 6 (MIG-6) Associated with Symptom Severity in Children with Autism.","date":"2014","source":"Biomarker insights","url":"https://pubmed.ncbi.nlm.nih.gov/25342879","citation_count":6,"is_preprint":false},{"pmid":"9184943","id":"PMC_9184943","title":"Secondary structure of T4 gene 33 protein. Fourier transform infrared and circular dichroic spectroscopic studies.","date":"1997","source":"International journal of biological macromolecules","url":"https://pubmed.ncbi.nlm.nih.gov/9184943","citation_count":5,"is_preprint":false},{"pmid":"6684608","id":"PMC_6684608","title":"[Transmission of amber mutants in bacteriophage T4. II. Thermal sensitivity of the multiplication of gene 26 amber mutants in Escherichia coli B cells and the absence of such sensitivity in the case of gene 33].","date":"1983","source":"Genetika","url":"https://pubmed.ncbi.nlm.nih.gov/6684608","citation_count":5,"is_preprint":false},{"pmid":"36498921","id":"PMC_36498921","title":"MIG-6 Is Critical for Progesterone Responsiveness in Human Complex Atypical Hyperplasia and Early-Stage Endometrial Cancer.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36498921","citation_count":4,"is_preprint":false},{"pmid":"23324575","id":"PMC_23324575","title":"Novel ERBB receptor feedback inhibitor 1 (ERRFI1) + 808 T/G polymorphism confers protective effect on diabetic nephropathy in a Korean population.","date":"2013","source":"Disease markers","url":"https://pubmed.ncbi.nlm.nih.gov/23324575","citation_count":4,"is_preprint":false},{"pmid":"30773264","id":"PMC_30773264","title":"A calcium-dependent phospholipase A2 (cPLA2) expression is regulated by MIG-6 during endometrial tumorigenesis.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/30773264","citation_count":4,"is_preprint":false},{"pmid":"30854112","id":"PMC_30854112","title":"AAV-Mig-6 Increase the Efficacy of TAE in VX2 Rabbit Model, Is Associated With JNK Mediated Autophagy.","date":"2019","source":"Journal of Cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30854112","citation_count":4,"is_preprint":false},{"pmid":"35340414","id":"PMC_35340414","title":"KLF4 Affects Acute Renal Allograft Injury via Binding to MicroRNA-155-5p Promoter to Regulate ERRFI1.","date":"2022","source":"Disease markers","url":"https://pubmed.ncbi.nlm.nih.gov/35340414","citation_count":3,"is_preprint":false},{"pmid":"39563082","id":"PMC_39563082","title":"Construction of ceRNA Network and Disease Diagnosis Model for Keloid Based on Tumor Suppressor ERRFI1.","date":"2024","source":"Experimental dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/39563082","citation_count":2,"is_preprint":false},{"pmid":"39096122","id":"PMC_39096122","title":"Potentially functional variants of ERRFI1 in hypoxia-related genes predict survival of non-small cell lung cancer patients.","date":"2024","source":"Cancer medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39096122","citation_count":2,"is_preprint":false},{"pmid":"10547870","id":"PMC_10547870","title":"Insulin and phorbol ester regulation of gene 33 expression in CHO cells.","date":"1999","source":"Puerto Rico health sciences journal","url":"https://pubmed.ncbi.nlm.nih.gov/10547870","citation_count":2,"is_preprint":false},{"pmid":"37854204","id":"PMC_37854204","title":"Precision oncology strategy in cetuximab-resistant ERRFI1-mutant colon cancer: case report of disease progression on afatinib.","date":"2023","source":"American journal of translational research","url":"https://pubmed.ncbi.nlm.nih.gov/37854204","citation_count":1,"is_preprint":false},{"pmid":"40905903","id":"PMC_40905903","title":"MIG-6 Plays a Critical Role as a PGR Mediator in Maintaining Epithelial and Stromal Cells for Uterine Receptivity.","date":"2025","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/40905903","citation_count":1,"is_preprint":false},{"pmid":"37070291","id":"PMC_37070291","title":"The increased expression of cytokeratin 13 leads to an increase in radiosensitivity of nasopharyngeal carcinoma HNE-3 cells by upregulating ERRFI1.","date":"2023","source":"IUBMB life","url":"https://pubmed.ncbi.nlm.nih.gov/37070291","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":49227,"output_tokens":6881,"usd":0.125448},"stage2":{"model":"claude-opus-4-6","input_tokens":10618,"output_tokens":4865,"usd":0.262072},"total_usd":0.38752,"stage1_batch_id":"msgbatch_0118RScg3WGaGkaZRtWZbtce","stage2_batch_id":"msgbatch_01Cpfzvtbj42aqp5oYZ5Te2s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"RALT (ERRFI1/Gene 33) was identified as an ErbB-2-interacting protein via yeast two-hybrid screen; the interaction requires active ErbB-2 catalytic function (but not autophosphorylation), is mediated by residues 282–395 of RALT, and results in inhibition of ErbB-2-driven cell proliferation, transformation, and sustained ERK1/2 activation. RALT was also found to associate with GRB2 in living cells.\",\n      \"method\": \"Yeast two-hybrid, GST pulldown, Co-IP, cell proliferation and transformation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, in vitro pulldown, multiple orthogonal functional assays in single study\",\n      \"pmids\": [\"11003669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Mig-6 (ERRFI1) was identified in a yeast two-hybrid screen with the EGFR kinase domain; upon EGF stimulation Mig-6 binds to EGFR via an acidic region (aa 985–995) in a kinase-activity-dependent manner, overexpression reduces EGF-induced ERK2 activation and enhances EGFR internalization, and Mig-6 inhibits EGFR-overexpression-induced transformation.\",\n      \"method\": \"Yeast two-hybrid, Co-IP, ERK2 activation assay, receptor internalization assay, transformation assay\",\n      \"journal\": \"Biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, replicated across labs\",\n      \"pmids\": [\"11843178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Gene 33/Mig-6 interacts in vivo and in a GTP-dependent manner in vitro with Cdc42Hs (a Rho-family GTPase), and transient expression of Gene 33 selectively activates SAPK/JNK kinases.\",\n      \"method\": \"Co-IP in vivo, in vitro GTP-dependent pulldown, SAPK/JNK activation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro reconstitution (GTP-dependent binding) plus cellular functional assay\",\n      \"pmids\": [\"10749885\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"RALT/ERRFI1 binds ligand-activated EGFR, ErbB-4, and ErbB-2:ErbB-3 dimers, functioning as a pan-ErbB inhibitor; a mutant (RALT deltaEBR) unable to bind ErbB RTKs loses the ability to suppress ERK and AKT activation at saturating ligand concentrations, demonstrating that suppressive activity is primarily receptor-proximal. RALT deltaEBR retains partial suppression of ErbB-2:ErbB-3 mitogenic signals at a receptor-distal level.\",\n      \"method\": \"Co-IP, cell proliferation assays, ERK and AKT phosphorylation assays, mutant analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP plus multiple functional assays with structure-function mutant\",\n      \"pmids\": [\"12833145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"RALT/ERRFI1 protein is ubiquitinated and degraded by the proteasome; its expression is driven transcriptionally through the Ras-Raf-ERK pathway (pharmacological ERK inhibition blocks RALT induction; activated Raf:ER chimera is sufficient to induce RALT), providing integrated transcriptional and post-translational control.\",\n      \"method\": \"Ubiquitin Co-IP, proteasome inhibitor treatment, pharmacological kinase inhibitors, inducible Raf:ER system\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (ubiquitination detection, genetic epistasis via pharmacological tools, inducible system) in single study\",\n      \"pmids\": [\"12226756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Gene 33/ERRFI1 inhibits EGFR autophosphorylation and downstream activation of Ras, ERK, JNK, Akt/PKB, and Rb; the Ack-homology (EBR) domain is necessary and sufficient for this inhibition. Dexamethasone-induced Gene 33 expression mediates glucocorticoid suppression of EGF signaling, as demonstrated by RNA interference reversal.\",\n      \"method\": \"In vitro EGFR autophosphorylation assay, domain deletion mutants, RNAi knockdown, Western blot\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro enzymatic assay with domain mutants plus RNAi rescue\",\n      \"pmids\": [\"15556944\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"The evolutionarily conserved ErbB-binding region (EBR) of RALT/MIG6 is necessary and sufficient to suppress EGFR kinase catalytic activity in vitro and in intact cells; the mechanism involves binding of the EBR to the 953RYLVIQ958 sequence in the αI helix of the EGFR kinase domain, which participates in allosteric control of EGFR activity.\",\n      \"method\": \"In vitro kinase assay, domain mutant overexpression, EBR peptide binding, site-directed mutagenesis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase inhibition assay with defined domain mutants and mechanistic mapping to allosteric site\",\n      \"pmids\": [\"17599051\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RALT/MIG6 mediates EGFR endocytosis and lysosomal degradation via a domain distinct from the EGFR kinase-suppressive domain; RALT drives EGFR endocytosis by binding to AP-2 and Intersectins, rescues endocytic deficits of EGFR mutants (Dc214 and Y1045F), integrating kinase suppression with receptor removal for durable repression of EGFR signaling.\",\n      \"method\": \"Co-IP (RALT with AP-2 and Intersectins), endocytosis assays with EGFR mutants, lysosomal degradation assays, domain mutant analysis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP of endocytic proteins, multiple EGFR mutants, domain dissection; replicated concept across labs\",\n      \"pmids\": [\"20421427\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mig-6 drives EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation by binding to the SNARE protein STX8 (required for late endosome trafficking), physically linking EGFR to STX8 during ligand-stimulated receptor trafficking.\",\n      \"method\": \"Co-IP (Mig-6 with STX8 and EGFR), EGFR trafficking assay, late endosome/lysosome localization\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifying novel binding partner STX8 with functional trafficking consequence, combined with in vivo GBM data\",\n      \"pmids\": [\"20351267\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Full-length Mig-6 (but not CRIB domain-deleted Mig-6 or uncleavable Mig-6-S38A mutant) activates NF-κB transcription; Mig-6 undergoes limited proteolytic cleavage, and the processed N-terminal CRIB-containing fragment binds IκBα through its NF-κB binding region, competing with NF-κB for IκBα and releasing NF-κB.\",\n      \"method\": \"Luciferase reporter assay, Co-IP of CRIB fragment with IκBα, mutagenesis (CRIB deletion, S38A mutant)\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and reporter assay with domain mutants, single lab\",\n      \"pmids\": [\"12384522\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Targeted expression of RALT/MIG6 in mouse skin (K14-RALT transgene) generates a dose-dependent Waved-like phenotype (wavy coat, curly whiskers, open eyes at birth) due to suppression of EGFR signaling; ex vivo keratinocytes show reduced ErbB-ligand-induced responses, and RNAi knockdown of RALT enhances ErbB mitogenic signaling.\",\n      \"method\": \"Transgenic mouse (K14-RALT), ex vivo keratinocyte stimulation assays, RNAi knockdown\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo gain-of-function with defined phenotype plus RNAi loss-of-function, consistent mechanistic interpretation\",\n      \"pmids\": [\"16007071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Targeted disruption of Mig-6 (ERRFI1) in mice leads to early onset degenerative joint disease with articular cartilage degradation and osteophyte formation; absence of Rag2 does not rescue the phenotype, excluding the acquired immune system. Mig-6 knockout chondrocytes show enhanced EGFR signaling and excessive proliferation.\",\n      \"method\": \"Germline knockout mouse, histology, Rag2 double-knockout epistasis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined joint phenotype, epistasis experiment, replicated in multiple follow-up studies\",\n      \"pmids\": [\"16087873\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Gene 33/ERRFI1 is induced in cardiomyocytes by hypoxia; adenoviral overexpression promotes cardiomyocyte death by reducing Akt and ERK signaling, and RNAi knockdown of endogenous Gene 33 reduces hypoxia-induced cardiomyocyte death, demonstrating that Gene 33 is a pro-death component in ischemic injury.\",\n      \"method\": \"Adenoviral overexpression, RNAi knockdown, Akt/ERK phosphorylation assay, cardiomyocyte death assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function and RNAi loss-of-function with defined death phenotype and signaling readout\",\n      \"pmids\": [\"16782890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Mig-6 (ERRFI1) is a downstream target of progesterone receptor (PR) and SRC-1 in the uterus; absence of Mig-6 prevents progesterone from inhibiting estrogen-induced uterine responses, and ovariectomized Mig-6-deficient mice treated with estrogen develop endometrial adenocarcinoma, placing Mig-6 in a PR–SRC-1–Mig-6 pathway that suppresses endometrial cancer.\",\n      \"method\": \"Germline knockout mouse, ovariectomy/hormone treatment, uterine weight assay, gene expression analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined hormone-response phenotype, genetic pathway placement\",\n      \"pmids\": [\"19439667\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Chk1 kinase phosphorylates Mig-6 at Ser251 in vivo and in vitro; EGF stimulation promotes this phosphorylation via PI3K–Chk1 axis; the Ser251Ala substitution increases Mig-6 inhibitory activity against EGFR; Chk1 depletion inhibits EGF-dependent EGFR activation and cell growth, which is rescued by co-depletion of Mig-6, placing Chk1 as a positive regulator of EGFR signaling through Mig-6 inactivation.\",\n      \"method\": \"In vitro kinase assay, mass spectrometry phosphosite mapping, site-directed mutagenesis, RNAi epistasis, EGFR activation assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay + MS phosphosite mapping + mutagenesis + genetic epistasis\",\n      \"pmids\": [\"22505024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Mig-6 inhibits EGF-induced cell migration via its CRIB domain binding to Cdc42; four specific CRIB residues (I11, R12, M26, R30) mediate Cdc42 binding, which is necessary and sufficient to inhibit filopodia formation and migration. The CRIB domain alone is sufficient to inhibit migration; Mig-6 binding to EGFR is dispensable for this anti-migratory function.\",\n      \"method\": \"Co-IP, site-directed mutagenesis of CRIB domain, cell migration assay, filopodia formation assay, domain rescue experiments\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain mutagenesis with Co-IP and defined cell migration phenotype; mechanistic dissection from EGFR binding\",\n      \"pmids\": [\"27341132\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MIG-6 negatively regulates STAT3 phosphorylation in uterine epithelial cells through protein–protein interaction; Mig-6 epithelial-specific knockout mice develop endometrial hyperplasia with aberrant STAT3 phosphorylation, and immunoprecipitation confirmed MIG-6–STAT3 interaction.\",\n      \"method\": \"Conditional epithelial-specific KO mouse, immunoprecipitation, STAT3 phosphorylation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifying direct interaction plus clean conditional KO with defined phosphorylation phenotype\",\n      \"pmids\": [\"28925396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Gene 33/Mig-6 localizes to the nucleus and chromatin fractions; nuclear localization is regulated by its 14-3-3-binding domain; chromatin association is partially dependent on its ErbB-binding domain. Nuclear Gene 33 promotes ATM-dependent DNA damage response (DDR), associates with histone H2AX, and promotes ATM–H2AX interaction without triggering DNA damage. Gene 33 may regulate c-Abl targeting to chromatin.\",\n      \"method\": \"Subcellular fractionation, Co-IP (Gene 33 with H2AX and ATM), domain mutagenesis, ectopic expression with ATM inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — fractionation, Co-IP, and domain mutants, but single lab; ATM dependence shown pharmacologically\",\n      \"pmids\": [\"28842482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"ERRFI1 has a dual role in AKT regulation: in EGFR-high cells, ERRFI1 inhibits AKT signaling through EGFR inhibition; in EGFR-low cells, ERRFI1 positively modulates AKT signaling by interfering with the interaction between the inactivating phosphatase PHLPP and AKT, thereby promoting AKT activity.\",\n      \"method\": \"Co-IP (ERRFI1 with PHLPP and AKT), knockdown/overexpression, AKT phosphorylation assay, proliferation assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifying PHLPP–AKT disruption plus functional assays, single lab\",\n      \"pmids\": [\"29335246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Co-immunoprecipitation identified ERK2 as an MIG-6 interacting protein; Mig-6 ablation (alone or combined with Pten ablation) increases ERK2 phosphorylation, and Mig-6 loss promotes endometrial tumorigenesis partly through upregulation of apoptotic inhibitor Birc1.\",\n      \"method\": \"Co-IP, conditional double KO mouse, Western blot for phospho-ERK2\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP identifying ERK2 interaction, supported by KO phenotype; single lab\",\n      \"pmids\": [\"20418913\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"MIG-6 recruits HAUSP (USP7) deubiquitinase to stabilize HIF1α protein, leading to upregulation of GLUT1 and other HIF1α-regulated glycolytic genes in triple-negative breast cancer, thereby driving metabolic reprogramming toward glycolysis.\",\n      \"method\": \"Co-IP (MIG-6 with HAUSP), HIF1α protein stability assay, GLUT1 expression assay, metabolic array, mouse tumor xenograft\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP identifying HAUSP interaction with functional consequence on HIF1α stability; single lab\",\n      \"pmids\": [\"33655623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ERRFI1 induces apoptosis in hepatocellular carcinoma cells by binding PDCD2; PDCD2 knockdown decreases ERRFI1-induced apoptosis, establishing PDCD2 as a functional effector of ERRFI1-mediated apoptosis.\",\n      \"method\": \"Co-IP (ERRFI1 with PDCD2), RNAi knockdown, apoptosis assay\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP plus RNAi rescue; single lab, single study\",\n      \"pmids\": [\"34608122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Loss of MIG-6 in the endometrium causes ERBB2 overexpression and endometrial progesterone resistance; genetic ablation of Erbb2 in Mig-6 knockout mice rescues all Mig-6d/d phenotypes (non-receptive endometrium, infertility, hyperplasia), placing ERBB2 downstream of MIG-6 loss.\",\n      \"method\": \"Double conditional KO mouse (Mig-6d/dErbb2d/d), transcriptomics, fertility/implantation assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic epistasis via double KO with full phenotypic rescue and transcriptomics\",\n      \"pmids\": [\"35232969\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ERRFI1 interacts with GRB2 and maintains GRB2 stability by hindering its proteasomal degradation; hepatocyte-specific ERRFI1 knockout reduces apoptosis and ferroptosis in hepatic ischemia–reperfusion injury, and GRB2 overexpression reverses the protective effects of ERRFI1 silencing.\",\n      \"method\": \"Hepatocyte-specific KO mouse, Co-IP (ERRFI1–GRB2), proteasomal degradation assay, apoptosis and ferroptosis assays\",\n      \"journal\": \"Molecular medicine (Cambridge, Mass.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP with functional rescue by GRB2 overexpression, clean conditional KO; single lab\",\n      \"pmids\": [\"38862918\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ANRIL lncRNA binds EZH2 and maintains H3K27me3 at the ERRFI1 promoter, epigenetically repressing ERRFI1 expression in cholangiocarcinoma; ANRIL knockdown increases ERRFI1 and inhibits tumor growth.\",\n      \"method\": \"RNA-seq after ANRIL knockdown, ChIP for H3K27me3 at ERRFI1 promoter, Co-IP (ANRIL-EZH2), in vitro and in vivo tumor assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus RIP/Co-IP identifying epigenetic writer; functional tumor assays; single lab\",\n      \"pmids\": [\"32378752\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"In MET-TKI-resistant cancer cells, epigenetically induced miR-205 downregulates ERRFI1, which in turn causes EGFR activation and sustains resistance; anti-miR-205 reverts crizotinib resistance in vivo, and this mechanism operates in the absence of EGFR genetic alterations.\",\n      \"method\": \"miRNA profiling, RNA-seq, anti-miR-205 transduction, in vivo xenograft, patient sample analysis\",\n      \"journal\": \"EMBO molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — miRNA-target axis validated with in vivo rescue experiment and patient sample; single lab\",\n      \"pmids\": [\"30021798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Cartilage-specific deletion of Mig-6 in chondrocytes (Col2a1-Cre) causes excessive articular chondrocyte proliferation and an OA-like phenotype in knees (but not all joints affected in full KO), demonstrating that Mig-6 in chondrocytes regulates EGF receptor signaling and chondrocyte proliferation in joint homeostasis.\",\n      \"method\": \"Cartilage-specific conditional KO mouse, histology, EGFR phosphorylation immunostaining, proliferation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean cell-type-specific KO with defined cellular phenotype and EGFR signaling readout\",\n      \"pmids\": [\"24550287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MIG-6 interacts with AKT and inhibits AKT phosphorylation in uterine epithelial cells; in vitro studies revealed a direct MIG-6–AKT protein interaction, and Mig-6 epithelial-specific KO mice develop endometrial hyperplasia with increased phospho-AKT.\",\n      \"method\": \"Co-IP (MIG-6 with AKT), conditional epithelial KO mouse, AKT phosphorylation assay\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP identifying interaction plus KO phenotype; single lab\",\n      \"pmids\": [\"29843645\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ERRFI1/MIG-6/RALT is a multifunctional adapter protein that acts as a feedback inhibitor of ErbB receptor tyrosine kinases: its evolutionarily conserved EBR domain directly suppresses EGFR kinase activity by binding the αI-helix allosteric site, while a separate domain drives EGFR endocytosis and lysosomal degradation by recruiting AP-2, Intersectin, and the SNARE protein STX8; ERRFI1 also inhibits cell migration by binding Cdc42 via its CRIB domain, regulates STAT3 and AKT phosphorylation through direct protein interactions, interacts with GRB2 and PDCD2 to modulate apoptosis, recruits HAUSP to stabilize HIF1α in certain cancer contexts, promotes ATM-dependent DNA damage responses from the nucleus, and is itself phosphorylated by Chk1 on Ser251 to attenuate its inhibitory activity—collectively placing ERRFI1 as a central node integrating receptor signaling, cytoskeletal dynamics, DNA damage response, and hormone-mediated transcriptional control.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"ERRFI1 (also known as MIG-6/RALT/Gene 33) is a stress-inducible, multifunctional adaptor protein that serves as a central feedback inhibitor of ErbB receptor tyrosine kinase signaling while also integrating cytoskeletal, DNA damage, and metabolic pathways. Its conserved ErbB-binding region (EBR) directly suppresses EGFR kinase activity by engaging the αI-helix allosteric site, while a separate domain drives EGFR endocytosis and lysosomal degradation through interactions with AP-2, Intersectins, and the SNARE protein STX8, thereby achieving durable signal extinction [PMID:17599051, PMID:20421427, PMID:20351267]. Independent of EGFR binding, ERRFI1 inhibits Cdc42-dependent cell migration via its CRIB domain, negatively regulates STAT3 and AKT phosphorylation through direct protein–protein interactions, and promotes ATM-dependent DNA damage responses from the nucleus [PMID:27341132, PMID:28925396, PMID:29843645, PMID:28842482]. ERRFI1 itself is transcriptionally induced by the Ras–Raf–ERK pathway and by progesterone receptor signaling, is subject to proteasomal degradation, and is phosphorylated by Chk1 at Ser251 to attenuate its inhibitory function, establishing multilayered feedback control; loss of ERRFI1 in mice causes degenerative joint disease, endometrial hyperplasia and cancer, and enhanced ErbB-driven proliferation [PMID:12226756, PMID:22505024, PMID:16087873, PMID:19439667].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing ERRFI1 as a direct ErbB-2/EGFR-interacting inhibitor and Cdc42 effector resolved the dual question of how an immediate-early gene product feeds back on receptor signaling and connects to Rho-family GTPase pathways.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, GST pulldown identifying ErbB-2 and Cdc42 as binding partners, with proliferation/transformation assays and SAPK/JNK activation\",\n      \"pmids\": [\"11003669\", \"10749885\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of ERRFI1–ErbB-2 interaction unknown\", \"Whether Cdc42 binding and ErbB binding are functionally coupled was not tested\", \"Endogenous stoichiometry not determined\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that Mig-6 binds EGFR in a kinase-activity-dependent manner and enhances EGFR internalization established it as a pan-ErbB feedback inhibitor that operates both at the kinase level and at receptor trafficking.\",\n      \"evidence\": \"Yeast two-hybrid with EGFR kinase domain, Co-IP, receptor internalization and transformation assays\",\n      \"pmids\": [\"11843178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of enhanced internalization not dissected\", \"ErbB-3/ErbB-4 binding not yet tested\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showing that ERRFI1 is transcriptionally induced by the Ras–Raf–ERK pathway and degraded by the proteasome established the complete negative-feedback loop architecture: ERK induces ERRFI1, which then suppresses ErbB–ERK signaling and is itself cleared by ubiquitination.\",\n      \"evidence\": \"Pharmacological ERK inhibition, inducible Raf:ER chimera, ubiquitin Co-IP, proteasome inhibitor treatment\",\n      \"pmids\": [\"12226756\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ubiquitin ligase responsible for ERRFI1 degradation not identified\", \"Half-life quantification not performed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Discovery that a proteolytically processed CRIB-containing fragment of Mig-6 activates NF-κB by competing with NF-κB for IκBα binding revealed a non-ErbB signaling function downstream of limited proteolysis.\",\n      \"evidence\": \"Luciferase reporter, Co-IP of CRIB fragment with IκBα, CRIB deletion and S38A mutants\",\n      \"pmids\": [\"12384522\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Protease responsible for Mig-6 cleavage not identified\", \"Physiological relevance of NF-κB activation not confirmed in vivo\", \"Not independently replicated\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extending ERRFI1 inhibition to ErbB-4 and ErbB-2:ErbB-3 heterodimers, and showing that an EBR-deletion mutant loses receptor-proximal but retains partial receptor-distal suppression, dissected the architecture of ERRFI1's inhibitory mechanism into two functional layers.\",\n      \"evidence\": \"Co-IP with multiple ErbB family members, ERK/AKT phosphorylation assays, RALT-ΔEBR mutant\",\n      \"pmids\": [\"12833145\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the receptor-distal suppressive mechanism unknown\", \"Quantitative contribution of each layer not measured\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrating that the EBR domain is necessary and sufficient to block EGFR autophosphorylation in vitro and that dexamethasone-induced ERRFI1 mediates glucocorticoid suppression of EGF signaling linked hormonal regulation to ERRFI1's kinase-inhibitory function.\",\n      \"evidence\": \"In vitro EGFR autophosphorylation assay, domain deletions, RNAi reversal of dexamethasone effect\",\n      \"pmids\": [\"15556944\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Glucocorticoid receptor binding site in ERRFI1 promoter not mapped\", \"Whether glucocorticoid–ERRFI1 axis operates in all tissues not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"In vivo gain-of-function (K14-RALT transgene producing Waved-like phenotype) and loss-of-function (Mig-6 knockout causing degenerative joint disease) in mice validated ERRFI1 as a physiologically essential EGFR inhibitor in skin and cartilage homeostasis.\",\n      \"evidence\": \"Transgenic mouse with dose-dependent phenotype; germline KO mouse with joint degeneration; Rag2 double-KO epistasis excluding immune involvement\",\n      \"pmids\": [\"16007071\", \"16087873\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular events downstream of EGFR hyperactivation in chondrocytes not detailed\", \"Whether joint phenotype is solely EGFR-dependent not formally tested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapping the EBR–EGFR interaction to the αI-helix (953RYLVIQ958) allosteric site provided a structural mechanism: ERRFI1 locks EGFR in an inactive conformation by blocking the asymmetric dimer interface.\",\n      \"evidence\": \"In vitro kinase assay, EBR peptide binding, site-directed mutagenesis of αI-helix residues\",\n      \"pmids\": [\"17599051\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No crystal structure of the ERRFI1-EBR:EGFR complex at this time\", \"Whether this mechanism applies equally to all ErbB family members not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Placing ERRFI1 downstream of progesterone receptor–SRC-1 signaling and showing that Mig-6-deficient mice develop estrogen-driven endometrial adenocarcinoma revealed ERRFI1 as a hormone-regulated tumor suppressor in the uterus.\",\n      \"evidence\": \"Germline KO mouse with ovariectomy/hormone treatment, endometrial adenocarcinoma development\",\n      \"pmids\": [\"19439667\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcriptional regulation of ERRFI1 by PR not shown at the promoter level\", \"Whether human endometrial cancer recapitulates this mechanism not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of AP-2, Intersectin, and STX8 as ERRFI1 binding partners that mediate EGFR endocytosis and late-endosome/lysosomal sorting established a bipartite mechanism: one domain suppresses kinase activity, a separate domain ensures receptor destruction.\",\n      \"evidence\": \"Co-IP with AP-2, Intersectins, and STX8; EGFR trafficking and degradation assays; rescue of endocytosis-defective EGFR mutants\",\n      \"pmids\": [\"20421427\", \"20351267\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise ERRFI1 residues mediating AP-2 and STX8 binding not mapped\", \"Whether ERRFI1-driven endocytosis operates independently of Cbl-mediated ubiquitination not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Discovery that Chk1 phosphorylates ERRFI1 at Ser251 to attenuate its EGFR-inhibitory activity uncovered a mechanism by which DNA damage checkpoint signaling cross-talks with receptor tyrosine kinase output, converting ERRFI1 from an active inhibitor to an inactive form.\",\n      \"evidence\": \"In vitro kinase assay, MS phosphosite mapping, S251A mutagenesis, RNAi epistasis (Chk1 + Mig-6 co-depletion rescue)\",\n      \"pmids\": [\"22505024\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphatase that reverses Ser251 phosphorylation not identified\", \"Whether other kinases also regulate ERRFI1 function not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Cartilage-specific deletion of Mig-6 confirmed that ERRFI1 acts cell-autonomously in chondrocytes to restrain EGFR-driven proliferation and protect against osteoarthritis-like degeneration.\",\n      \"evidence\": \"Col2a1-Cre conditional KO mouse, EGFR phosphorylation immunostaining, chondrocyte proliferation assays\",\n      \"pmids\": [\"24550287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific EGFR ligand driving joint pathology not identified\", \"Therapeutic rescue by ERRFI1 restoration not attempted\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Dissecting ERRFI1's CRIB domain showed that Cdc42 binding (via I11, R12, M26, R30) is necessary and sufficient to inhibit filopodia formation and cell migration, independently of EGFR binding, formally separating the cytoskeletal and receptor-inhibitory functions of the protein.\",\n      \"evidence\": \"CRIB point mutagenesis, Co-IP with Cdc42, migration and filopodia assays, domain rescue experiments\",\n      \"pmids\": [\"27341132\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CRIB–Cdc42 interaction also affects polarity or invasion not tested\", \"In vivo relevance of CRIB-mediated migration inhibition not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstrating direct MIG-6–STAT3 interaction and showing that epithelial-specific Mig-6 loss causes endometrial hyperplasia with aberrant STAT3 phosphorylation extended ERRFI1's inhibitory reach beyond receptor kinases to a transcription factor, providing a new mechanism for its tumor-suppressive activity in the uterus.\",\n      \"evidence\": \"Conditional epithelial KO mouse, immunoprecipitation, STAT3 phosphorylation assay\",\n      \"pmids\": [\"28925396\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding interface between MIG-6 and STAT3 not mapped\", \"Whether STAT3 inhibition is direct or requires an intermediate not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Finding that ERRFI1 localizes to chromatin and promotes ATM–H2AX interaction without inducing DNA damage revealed a nuclear scaffolding function that places ERRFI1 in DNA damage response signaling independent of its cytoplasmic ErbB-inhibitory role.\",\n      \"evidence\": \"Subcellular fractionation, Co-IP with H2AX and ATM, domain mutagenesis, ATM pharmacological inhibition\",\n      \"pmids\": [\"28842482\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not independently replicated\", \"Whether nuclear ERRFI1 function is physiologically relevant in DNA repair outcomes not tested\", \"ATM dependence shown only pharmacologically\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Revealing that ERRFI1 has a context-dependent dual role in AKT regulation — inhibiting AKT via EGFR in EGFR-high cells but promoting AKT by disrupting the PHLPP–AKT interaction in EGFR-low cells — explained contradictory reports and established ERRFI1 as a context-dependent signaling switch.\",\n      \"evidence\": \"Co-IP of ERRFI1 with PHLPP and AKT, knockdown/overexpression in EGFR-high versus EGFR-low cell lines\",\n      \"pmids\": [\"29335246\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of PHLPP displacement not determined\", \"Threshold of EGFR expression for mode-switching not defined\", \"Not replicated by independent laboratory\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Discovery that MIG-6 recruits the HAUSP deubiquitinase to stabilize HIF1α and drive glycolytic reprogramming in triple-negative breast cancer revealed a pro-tumorigenic function that contrasts with ERRFI1's canonical tumor-suppressive role.\",\n      \"evidence\": \"Co-IP of MIG-6 with HAUSP, HIF1α stability assay, metabolic profiling, mouse xenograft\",\n      \"pmids\": [\"33655623\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether HAUSP recruitment is ErbB-dependent not tested\", \"Generalizability beyond triple-negative breast cancer unknown\", \"Not independently replicated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Genetic epistasis showing that Erbb2 ablation fully rescues all phenotypes of Mig-6-deficient endometrium (infertility, hyperplasia, progesterone resistance) placed ERBB2 overexpression as the principal effector downstream of ERRFI1 loss in the uterus.\",\n      \"evidence\": \"Double conditional KO mouse (Mig-6d/d Erbb2d/d), fertility and implantation assays, transcriptomics\",\n      \"pmids\": [\"35232969\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ERRFI1 directly degrades ERBB2 protein or merely restrains its signaling not distinguished\", \"Whether ERBB2 is the sole relevant ErbB target in all endometrial contexts unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of GRB2 stabilization by ERRFI1 (hindering proteasomal degradation) and its role in promoting hepatocyte apoptosis and ferroptosis during ischemia–reperfusion injury added a new cell-death axis to ERRFI1's functional repertoire.\",\n      \"evidence\": \"Hepatocyte-specific KO mouse, Co-IP of ERRFI1–GRB2, proteasomal degradation assay, GRB2 overexpression rescue\",\n      \"pmids\": [\"38862918\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether GRB2 stabilization depends on the EBR domain not tested\", \"Ferroptosis mechanism downstream of GRB2 not elucidated\", \"Not independently replicated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of the E3 ligase that ubiquitinates ERRFI1, the structural basis of the full-length ERRFI1–EGFR complex, how nuclear versus cytoplasmic ERRFI1 pools are partitioned and regulated, and whether the pro-tumorigenic HAUSP/HIF1α-stabilizing function and the anti-tumorigenic ErbB-inhibitory function coexist or are mutually exclusive in specific cancer contexts.\",\n      \"evidence\": \"Unresolved in current literature\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"E3 ligase for ERRFI1 ubiquitination not identified\", \"Full-length ERRFI1 crystal structure unavailable\", \"Nuclear–cytoplasmic partitioning regulation not systematically studied\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 3, 5, 6, 7, 14, 16, 27]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [7, 8, 20, 23]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [2, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 1, 7, 15]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [17]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [1, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 1, 3, 5, 6, 14, 16, 18, 27]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11, 13, 22, 26]},\n      {\"term_id\": \"R-HSA-9609507\", \"supporting_discovery_ids\": [7, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [12, 21, 23]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"EGFR\", \"ERBB2\", \"CDC42\", \"GRB2\", \"STX8\", \"STAT3\", \"AKT1\", \"PHLPP1\"],\n    \"other_free_text\": []\n  }\n}\n```"}