{"gene":"RNF6","run_date":"2026-06-10T06:43:37","timeline":{"discoveries":[{"year":2005,"finding":"RNF6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. RNAi-mediated knockdown of Rnf6 alters axon outgrowth, and this phenotype can be rescued by modulating LIMK1 expression, placing RNF6 upstream of LIMK1 in the control of axonal growth cone actin dynamics.","method":"Co-IP/pulldown, in vitro ubiquitination assay, RNAi knockdown with phenotypic rescue, primary hippocampal neuron cultures","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding, polyubiquitination assay, genetic epistasis via rescue experiments, multiple orthogonal methods in one study","pmids":["16204183"],"is_preprint":false},{"year":2009,"finding":"RNF6 is an E3 ubiquitin ligase that associates with the androgen receptor (AR), induces AR ubiquitination, and promotes AR transcriptional activity. Knockdown of RNF6 or mutation of RNF6-induced ubiquitination acceptor sites on AR selectively alters expression of a subset of AR target genes and diminishes recruitment of AR and its coactivators to androgen-responsive elements.","method":"Proteomic screen (Co-IP), ubiquitination assays, site-directed mutagenesis of AR ubiquitination acceptor sites, ChIP, RNAi knockdown with transcriptional readout","journal":"Cancer cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods including mutagenesis of substrate sites, ChIP, ubiquitination assay, and functional transcriptional readout in a single rigorous study","pmids":["19345326"],"is_preprint":false},{"year":2002,"finding":"RNF6 localizes to punctate nuclear bodies together with PML and Daxx proteins in mouse Sertoli cells, and its re-expression in a Sertoli tumor cell line (where both Inha and Rnf6 are reduced) restores Inha expression and reverts cells to normal growth control, identifying RNF6 as a transcriptional regulator of the Inha promoter through binding of a GGGGC repeat motif.","method":"Expression screening, transient and stable transfection with reporter assays, co-localization by immunofluorescence, cell growth assays","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct DNA-binding and promoter reporter assays, co-localization, functional rescue; single lab with multiple complementary methods","pmids":["11971979"],"is_preprint":false},{"year":2018,"finding":"RNF6 binds and ubiquitylates TLE3, a transcriptional repressor of the β-catenin/TCF4 complex, leading to TLE3 proteasomal degradation. RNF6-mediated TLE3 loss releases β-catenin to TCF4/LEF, activating Wnt/β-catenin signaling and promoting colorectal cancer cell growth and metastasis.","method":"Co-IP, ubiquitination assay, rescue experiments with TLE3 re-expression, in vivo tumor/metastasis models","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ubiquitination assay, substrate rescue experiments, and in vivo validation across multiple methods","pmids":["29374067"],"is_preprint":false},{"year":2018,"finding":"RNF6 directly binds and ubiquitylates FoxA1, a transcriptional repressor of EMT, leading to its degradation; the oncogenic effects of RNF6 in hepatocellular carcinoma are partially dependent on FoxA1 degradation.","method":"Co-IP, ubiquitination assay, RNF6 knockdown with phenotypic rescue by FoxA1, in vivo metastasis models","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays with rescue experiments, single lab","pmids":["30496760"],"is_preprint":false},{"year":2018,"finding":"RNF6 regulates STAT3 signaling in gastric cancer by inducing polyubiquitination and degradation of the phosphatase SHP-1, thereby relieving SHP-1-mediated inhibition of STAT3 phosphorylation and promoting expression of STAT3 target genes MCL1 and XIAP.","method":"Immunoprecipitation/ubiquitination assay, luciferase reporter assay, Western blot, RNF6 knockdown/overexpression","journal":"OncoTargets and therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — IP-based ubiquitination assay and luciferase reporter, multiple readouts, single lab","pmids":["30323630"],"is_preprint":false},{"year":2019,"finding":"RNF6 interacts with the glucocorticoid receptor (GR) and induces K63-linked polyubiquitination of GR, which stabilizes GR protein (rather than degrading it) and promotes GR transcriptional activity, leading to increased expression of pro-survival genes Bcl-xL and Mcl-1 and resistance to dexamethasone in multiple myeloma cells.","method":"Co-IP, ubiquitination assay specifying K63 linkage, luciferase reporter assay, RNF6 overexpression/knockdown","journal":"Acta pharmacologica Sinica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, linkage-specific ubiquitination assay, luciferase reporter; single lab, multiple methods","pmids":["31645658"],"is_preprint":false},{"year":2022,"finding":"RNF6 undergoes auto-ubiquitination (K48-linked polyubiquitination) in a manner dependent on its RING domain; deletion of the RING domain (ΔRING) or mutation of core cysteine residues (C632S/C635S) abolishes this auto-ubiquitination. USP7 was identified as a deubiquitinase of RNF6 by tandem mass spectrometry; USP7 interacts with RNF6 and removes K48-linked polyubiquitin chains, preventing RNF6 degradation. Anti-cancer drugs Nilotinib and Panobinostat induce RNF6 K48-linked auto-ubiquitination and degradation in an in vitro ubiquitination system without other E3 ligases.","method":"RING domain deletion mutagenesis, cysteine-to-serine point mutagenesis, tandem mass spectrometry identification of USP7, Co-IP, in vitro ubiquitination reconstitution assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of auto-ubiquitination, active-site mutagenesis, MS-identified deubiquitinase, multiple orthogonal methods in one study","pmids":["35926709"],"is_preprint":false},{"year":2022,"finding":"RNF6 interacts with p27 (Kip1) via its KIL domain in a phosphorylation-independent manner, enhances ubiquitination and subsequent proteasomal degradation of p27 in the early G0/G1 phase of the cell cycle, and thereby promotes CDK2/Cyclin E complex activity, Rb phosphorylation, and cell cycle progression in prostate cancer cells.","method":"Co-IP, ubiquitination assay, shRNA knockdown with cell cycle (G1 arrest) readout, domain mapping","journal":"Pharmaceutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, ubiquitination assay, functional cell-cycle readout; single lab","pmids":["35456636"],"is_preprint":false},{"year":2021,"finding":"RNF6 interacts with MST1 (a kinase regulating YAP), promotes MST1 ubiquitination and degradation, and thereby upregulates YAP signaling to promote breast cancer cell metastasis.","method":"Co-IP, ubiquitination assay, RNF6 knockdown/overexpression with YAP and metastasis readouts","journal":"Experimental and therapeutic medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — Co-IP and ubiquitination, single lab, single paper with limited mechanistic depth","pmids":["34970341"],"is_preprint":false},{"year":2021,"finding":"RNF6 promotes colorectal cancer invasion and migration by increasing GSK3β phosphorylation (inhibiting GSK3β activity), which in turn activates the Wnt/β-catenin pathway.","method":"RNF6 overexpression/silencing with Western blot, GSK3β inhibitor rescue, in vivo rat CRC model","journal":"Pathology, research and practice","confidence":"Low","confidence_rationale":"Tier 3 / Weak — indirect mechanism (no direct ubiquitination of GSK3β demonstrated), single lab, no Co-IP for direct interaction shown in abstract","pmids":["34352441"],"is_preprint":false},{"year":2022,"finding":"RNF6 mediates ubiquitination and degradation of the RNA-binding protein QKI in macrophages; this RNF6-mediated QKI degradation activates PI3K-p110β-dependent autophagy and enhances phagocytic clearance of MRSA.","method":"Co-IP, iTRAQ mass spectrometry, immunoprecipitation-ubiquitination assay, QKI knockout mouse model, MRSA infection model","journal":"Cell & bioscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified interaction, Co-IP, ubiquitination assay, in vivo model; single lab but multiple methods","pmids":["36088389"],"is_preprint":false},{"year":2023,"finding":"In gastric cancer cells, RNF6 localizes to the nucleus and directly binds to the promoter regions of CCNA1 and CREBBP, acting as a transcription factor to regulate their expression and promote tumor cell cycle progression and apoptosis resistance.","method":"ChIP-seq, ChIP-PCR, RNF6 overexpression/silencing with CCNA1/CREBBP rescue, luciferase assay","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-seq with functional validation and rescue experiments; single lab","pmids":["37904524"],"is_preprint":false},{"year":2025,"finding":"RNF6 physically binds cyclin D2 (CCND2) and mediates its K48-linked polyubiquitination and proteasomal degradation in lung adenocarcinoma cells. RING-domain-deleted RNF6 (ΔRING) fails to ubiquitinate CCND2. Thr280 on CCND2 is critical for its stability; T280 mutation makes CCND2 more stable and less susceptible to RNF6-mediated ubiquitination. RNF6 arrests LUAD cells in G1 by inhibiting CCND2/phospho-Rb signaling.","method":"Affinity purification/tandem MS, Co-IP, ubiquitination assay, RING domain deletion mutagenesis, Thr280 point mutation, cell cycle analysis, in vivo xenograft","journal":"Molecular cancer research : MCR","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — MS-based substrate identification, in vitro ubiquitination with domain and substrate site mutagenesis, multiple orthogonal methods in one study","pmids":["39918413"],"is_preprint":false},{"year":2025,"finding":"RNF6 interacts with p27 and directly ubiquitinates it via E3 ligase activity, and also indirectly accelerates p27 degradation by acting as a transcription factor to activate SKP2 promoter activity and by interacting with SKP2 to stabilize its protein levels in an Akt-dependent manner, thereby driving glioblastoma cell proliferation.","method":"Co-IP, ubiquitination assay, SKP2 promoter reporter assay, Western blot, RNF6 knockdown/overexpression with cell growth readout","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, promoter reporter assays; single lab, multiple methods","pmids":["39938447"],"is_preprint":false},{"year":2025,"finding":"CXCL13 enhances RNF6 expression in endothelial cells, and RNF6 facilitates ubiquitination of Sqstm1 at the K314 residue, thereby promoting autophagy and damaging blood spinal cord barrier integrity in experimental allergic encephalomyelitis.","method":"Co-IP, ubiquitination assay (K314 site-specific), Western blot, in vivo EAE model with barrier permeability readout","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — site-specific ubiquitination, Co-IP, in vivo model; single lab","pmids":["40231770"],"is_preprint":false},{"year":2026,"finding":"RNF6 directly binds NME4 and facilitates its K48-linked polyubiquitination and proteasomal degradation; NME4 degradation activates the JNK/c-JUN pathway in ovarian cancer cells, promoting proliferation, invasion, and EMT.","method":"Co-IP, CHX chase assay, ubiquitination assay (K48-specific), RNF6/NME4 knockdown rescue experiments, in vivo xenograft","journal":"Pathology, research and practice","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, linkage-specific ubiquitination, substrate rescue, in vivo model; single lab","pmids":["41616518"],"is_preprint":false},{"year":2026,"finding":"RNF6 promotes ubiquitination and proteasomal degradation of OTULIN (a linear deubiquitinase) in bronchial epithelial cells; RNF6 knockdown stabilizes OTULIN and suppresses TGF-β1-induced partial EMT-like phenotypic changes, while OTULIN knockdown reverses the protective effect of RNF6 silencing.","method":"Co-IP/mass spectrometry (label-free proteomics), Co-IP validation, immunofluorescence co-localization, cycloheximide/MG132 stability assay, RNF6 and OTULIN knockdown rescue experiments","journal":"Biochemical pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-identified substrate, Co-IP, stability assays, functional rescue; single lab","pmids":["42055142"],"is_preprint":false},{"year":2026,"finding":"RNF6 interacts with the Zika virus NS5 protein and acts as a negative regulator of type I interferon and MAPK signaling pathways, functioning as a proviral host factor for ZIKV replication in brain microvascular endothelial cells; RNF6 knockout reduces ZIKV infection while overexpression enhances it.","method":"Genome-wide CRISPR/Cas9 screen, RNF6 knockout/knockdown/overexpression with viral replication readout, Co-IP for NS5 interaction, molecular docking","journal":"Viruses","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen hit validated by Co-IP and gain/loss-of-function; single lab, docking is computational","pmids":["41902231"],"is_preprint":false},{"year":2016,"finding":"PBX1 directly binds a PBX1 recognition element (PRE) between -144 and -99 upstream of the RNF6 transcription start site and, in collaboration with PREP1 (but not MEIS1), activates RNF6 transcription; mutation of the PRE completely abolishes RNF6 transcription. Doxorubicin suppresses RNF6 expression by down-regulating PBX1.","method":"Luciferase reporter assay with truncated RNF6 promoter constructs, PRE mutation, ChIP, knockdown experiments","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter deletion and mutagenesis reporter assays, ChIP; single lab, multiple complementary methods","pmids":["26971355"],"is_preprint":false}],"current_model":"RNF6 is a RING-H2 domain E3 ubiquitin ligase that targets multiple substrates—including LIMK1, AR, TLE3, FoxA1, SHP-1, GR (via atypical K63 ubiquitination that stabilizes rather than degrades), p27/Kip1, MST1, CCND2, NME4, QKI, OTULIN, and Sqstm1—for K48-linked polyubiquitination and proteasomal degradation (or, in the case of GR, K63-linked stabilizing ubiquitination), and also functions in the nucleus as a direct transcriptional regulator; it undergoes RING-domain-dependent auto-ubiquitination that is counteracted by the deubiquitinase USP7, and its substrates collectively place it at the intersection of axon growth cone actin dynamics, androgen/glucocorticoid receptor signaling, Wnt/β-catenin, SHP-1/STAT3, Hippo/YAP, JNK/c-JUN, and cell-cycle G1/S control."},"narrative":{"mechanistic_narrative":"RNF6 is a RING-domain E3 ubiquitin ligase that controls the abundance of diverse regulatory proteins to govern cytoskeletal dynamics, nuclear receptor signaling, and proliferative control, and additionally acts as a sequence-specific nuclear transcriptional regulator [PMID:16204183, PMID:19345326, PMID:11971979]. Its founding activity was defined in neurons, where it binds and polyubiquitinates LIMK1 to drive its proteasomal degradation and thereby tune growth-cone actin dynamics and axon outgrowth [PMID:16204183]. Across cancer contexts RNF6 acts predominantly as an oncogenic ligase by destroying negative regulators of growth and survival pathways: it ubiquitinates and degrades the Wnt/β-catenin repressor TLE3 [PMID:29374067], the EMT repressor FoxA1 [PMID:30496760], the STAT3-inhibitory phosphatase SHP-1 [PMID:30323630], the Hippo kinase MST1 to elevate YAP signaling [PMID:34970341], and the CDK inhibitor p27/Kip1 to promote CDK2/Cyclin E activity and G1/S progression [PMID:35456636, PMID:39938447]. It directs K48-linked polyubiquitination and degradation of cyclin D2 via substrate residue Thr280 to enforce G1 arrest in lung adenocarcinoma [PMID:39918413] and degrades NME4 to activate JNK/c-JUN signaling [PMID:41616518]. Beyond degradative ubiquitination, RNF6 can stabilize substrates: it attaches K63-linked chains to the glucocorticoid receptor, increasing GR protein levels and pro-survival transcription [PMID:31645658]. RNF6 also ubiquitinates substrates that reprogram autophagy and barrier integrity, including QKI in macrophages and Sqstm1 at K314 in endothelial cells [PMID:36088389, PMID:40231770]. In the nucleus it binds defined promoter elements—a GGGGC repeat in the Inha promoter and the CCNA1/CREBBP promoters—to directly regulate transcription [PMID:11971979, PMID:37904524]. RNF6 itself is regulated by RING-dependent K48-linked auto-ubiquitination that is reversed by the deubiquitinase USP7, and its transcription is activated by PBX1/PREP1 [PMID:35926709, PMID:26971355].","teleology":[{"year":2002,"claim":"Established RNF6 as a nuclear factor capable of direct promoter binding and transcriptional control, an activity distinct from its later-defined ligase function.","evidence":"Expression screening, reporter assays, and immunofluorescence co-localization with PML/Daxx in Sertoli cells","pmids":["11971979"],"confidence":"Medium","gaps":["Did not connect transcriptional activity to ubiquitin ligase function","Sequence-specificity of the GGGGC-motif binding not structurally defined"]},{"year":2005,"claim":"Defined RNF6's first molecular substrate, showing it is an E3 ligase that degrades LIMK1 to control axonal actin dynamics.","evidence":"Co-IP, in vitro ubiquitination, RNAi with LIMK1 rescue in primary hippocampal neurons","pmids":["16204183"],"confidence":"High","gaps":["Ubiquitin chain linkage type not specified","Did not address RNF6 substrate selectivity beyond LIMK1"]},{"year":2009,"claim":"Linked RNF6 to nuclear receptor signaling, showing it ubiquitinates AR and selectively reprograms AR target-gene transcription rather than simply degrading the receptor.","evidence":"Proteomic Co-IP, AR ubiquitination-site mutagenesis, ChIP, and RNAi transcriptional readout","pmids":["19345326"],"confidence":"High","gaps":["Mechanism by which AR ubiquitination alters coactivator recruitment not fully resolved","Linkage type and fate of ubiquitinated AR not detailed"]},{"year":2016,"claim":"Identified upstream transcriptional control of RNF6, defining how its expression is set rather than how it acts.","evidence":"RNF6 promoter truncation/PRE-mutation luciferase reporters and ChIP for PBX1/PREP1","pmids":["26971355"],"confidence":"Medium","gaps":["Tissue contexts of PBX1-driven RNF6 expression not broadly mapped","Does not address post-translational regulation of RNF6"]},{"year":2018,"claim":"Established RNF6 as an oncogenic ligase across multiple cancers by degrading distinct tumor-suppressive substrates feeding Wnt, EMT, and STAT3 pathways.","evidence":"Co-IP, ubiquitination assays, and substrate-rescue/in vivo models for TLE3, FoxA1, and SHP-1","pmids":["29374067","30496760","30323630"],"confidence":"High","gaps":["Whether one ligase engages all substrates simultaneously or context-specifically is unclear","Ubiquitination linkage detail varies between substrates"]},{"year":2019,"claim":"Revealed a non-degradative mode, showing RNF6 stabilizes GR via K63-linked ubiquitination to drive pro-survival transcription and drug resistance.","evidence":"Co-IP, K63-linkage-specific ubiquitination assay, luciferase reporter in multiple myeloma cells","pmids":["31645658"],"confidence":"Medium","gaps":["Structural basis for K48-versus-K63 substrate-dependent chain choice unknown","Single-lab finding"]},{"year":2021,"claim":"Extended RNF6 substrate range to Hippo signaling via MST1 degradation, though with limited mechanistic depth.","evidence":"Co-IP and ubiquitination assay with YAP and metastasis readouts in breast cancer cells","pmids":["34970341"],"confidence":"Low","gaps":["Single lab, limited mechanistic depth; chain linkage and direct ubiquitination sites not defined","Indirect Wnt/GSK3β effects reported separately remain non-direct"]},{"year":2022,"claim":"Resolved how RNF6 is itself controlled, demonstrating RING-dependent K48 auto-ubiquitination opposed by the deubiquitinase USP7.","evidence":"RING deletion and C632S/C635S active-site mutagenesis, MS identification of USP7, in vitro ubiquitination reconstitution","pmids":["35926709"],"confidence":"High","gaps":["Physiological signals controlling the RNF6/USP7 balance not mapped","Whether USP7 also edits RNF6 substrate chains untested"]},{"year":2022,"claim":"Connected RNF6 to direct cell-cycle control via p27/Kip1 degradation through a defined KIL domain interaction.","evidence":"Co-IP with domain mapping, ubiquitination assay, shRNA with G1-arrest readout in prostate cancer cells","pmids":["35456636"],"confidence":"Medium","gaps":["Generality of KIL-domain-mediated substrate recognition unknown","Single lab"]},{"year":2023,"claim":"Reaffirmed RNF6's direct transcription-factor activity by mapping genome-wide promoter binding to CCNA1 and CREBBP.","evidence":"ChIP-seq/ChIP-PCR with rescue and luciferase validation in gastric cancer cells","pmids":["37904524"],"confidence":"Medium","gaps":["DNA-binding mode of a RING ligase acting as a transcription factor not structurally explained","Relationship between ligase and transcriptional roles unresolved"]},{"year":2025,"claim":"Defined substrate-residue-level control of cyclin D2 and broadened RNF6's degradative targets to NME4 and to SKP2-coupled p27 regulation.","evidence":"MS substrate ID, RING-deletion and substrate-site (CCND2 Thr280) mutagenesis, K48 linkage assays, SKP2 promoter reporter, in vivo xenografts","pmids":["39918413","39938447","41616518"],"confidence":"High","gaps":["Context determining whether RNF6 arrests (CCND2) or drives (p27/SKP2) the cycle is unclear","Dual direct-and-transcriptional control of p27 not fully integrated"]},{"year":2025,"claim":"Implicated RNF6 in autophagy and barrier biology via site-specific Sqstm1 ubiquitination downstream of CXCL13.","evidence":"K314 site-specific ubiquitination assay, Co-IP, in vivo EAE barrier-permeability model","pmids":["40231770"],"confidence":"Medium","gaps":["Whether Sqstm1 K314 ubiquitination is degradative or signaling-type not stated","Single disease model"]},{"year":2026,"claim":"Extended RNF6 substrates to deubiquitinase regulation (OTULIN) and host-pathogen biology (ZIKV NS5), revealing innate-immune and EMT-modulatory roles.","evidence":"MS-based substrate ID with stability assays for OTULIN; CRISPR screen and Co-IP for ZIKV NS5 interaction","pmids":["42055142","41902231"],"confidence":"Medium","gaps":["Whether RNF6 ubiquitinates NS5 or acts non-catalytically as proviral factor unresolved","OTULIN chain linkage and broader linear-ubiquitin consequences untested"]},{"year":null,"claim":"How RNF6 selects among its many substrates and chooses degradative (K48) versus stabilizing (K63) chain linkages, and how its E3-ligase and direct transcription-factor activities are coordinated within the same protein, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of substrate recognition or chain-type determination","No unified account linking nuclear DNA-binding and cytoplasmic ligase functions","Most substrate findings are single-lab"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,3,7,13,16]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,3,13,16]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[7,13]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,12]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[2,12]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[2,12]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,7,13]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[8,13,14]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,5,6,9]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,12]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[11,15]}],"complexes":[],"partners":["LIMK1","AR","TLE3","FOXA1","SHP-1","GR","MST1","USP7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9Y252","full_name":"E3 ubiquitin-protein ligase RNF6","aliases":[],"length_aa":685,"mass_kda":78.1,"function":"E3 ubiquitin-protein ligase mediating 'Lys-48'-linked polyubiquitination of LIMK1 and its subsequent targeting to the proteasome for degradation (By similarity). Negatively regulates axonal outgrowth through regulation of the LIMK1 turnover (By similarity). Mediates 'Lys-6' and 'Lys-27'-linked polyubiquitination of AR/androgen receptor thereby modulating its transcriptional activity (PubMed:19345326). May also bind DNA and function as a transcriptional regulator (By similarity). Mediates polyubiquitination of QKI in macrophages, leading to its degradation (By similarity)","subcellular_location":"Nucleus; Cytoplasm; Cell projection, axon; Nucleus, PML body","url":"https://www.uniprot.org/uniprotkb/Q9Y252/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RNF6","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RNF6","total_profiled":1310},"omim":[{"mim_id":"604242","title":"RING FINGER PROTEIN 6; RNF6","url":"https://www.omim.org/entry/604242"},{"mim_id":"133239","title":"ESOPHAGEAL CANCER","url":"https://www.omim.org/entry/133239"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear membrane","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RNF6"},"hgnc":{"alias_symbol":["DKFZp686P0776"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y252","domains":[{"cath_id":"-","chopping":"34-76","consensus_level":"medium","plddt":87.1142,"start":34,"end":76},{"cath_id":"-","chopping":"118-157_397-418_449-467","consensus_level":"medium","plddt":60.5033,"start":118,"end":467},{"cath_id":"3.30.40.10","chopping":"607-664","consensus_level":"high","plddt":86.0324,"start":607,"end":664}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y252","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y252-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y252-F1-predicted_aligned_error_v6.png","plddt_mean":49.47},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RNF6","jax_strain_url":"https://www.jax.org/strain/search?query=RNF6"},"sequence":{"accession":"Q9Y252","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y252.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y252/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y252"}},"corpus_meta":[{"pmid":"19345326","id":"PMC_19345326","title":"Regulation of androgen receptor transcriptional activity and specificity by RNF6-induced ubiquitination.","date":"2009","source":"Cancer cell","url":"https://pubmed.ncbi.nlm.nih.gov/19345326","citation_count":191,"is_preprint":false},{"pmid":"16204183","id":"PMC_16204183","title":"The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones.","date":"2005","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/16204183","citation_count":90,"is_preprint":false},{"pmid":"29374067","id":"PMC_29374067","title":"RNF6 Promotes Colorectal Cancer by Activating the Wnt/β-Catenin Pathway via Ubiquitination of TLE3.","date":"2018","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/29374067","citation_count":79,"is_preprint":false},{"pmid":"26971355","id":"PMC_26971355","title":"The Ring Finger Protein RNF6 Induces Leukemia Cell Proliferation as a Direct Target of Pre-B-cell Leukemia Homeobox 1.","date":"2016","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/26971355","citation_count":53,"is_preprint":false},{"pmid":"31063232","id":"PMC_31063232","title":"MicroRNA-26a-5p inhibits breast cancer cell growth by suppressing RNF6 expression.","date":"2019","source":"The Kaohsiung journal of medical sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31063232","citation_count":39,"is_preprint":false},{"pmid":"30496760","id":"PMC_30496760","title":"RNF6 facilitates metastasis and radioresistance in hepatocellular carcinoma through ubiquitination of FoxA1.","date":"2018","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/30496760","citation_count":29,"is_preprint":false},{"pmid":"12154016","id":"PMC_12154016","title":"Identification of somatic mutations of the RNF6 gene in human esophageal squamous cell carcinoma.","date":"2002","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/12154016","citation_count":29,"is_preprint":false},{"pmid":"30323630","id":"PMC_30323630","title":"Knockdown of RNF6 inhibits gastric cancer cell growth by suppressing STAT3 signaling.","date":"2018","source":"OncoTargets and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/30323630","citation_count":28,"is_preprint":false},{"pmid":"31645658","id":"PMC_31645658","title":"RNF6 promotes myeloma cell proliferation and survival by inducing glucocorticoid receptor polyubiquitination.","date":"2019","source":"Acta pharmacologica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/31645658","citation_count":25,"is_preprint":false},{"pmid":"10331950","id":"PMC_10331950","title":"Cloning and characterization of RNF6, a novel RING finger gene mapping to 13q12.","date":"1999","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/10331950","citation_count":23,"is_preprint":false},{"pmid":"11971979","id":"PMC_11971979","title":"Gene control in germinal differentiation: RNF6, a transcription regulatory protein in the mouse sertoli cell.","date":"2002","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11971979","citation_count":16,"is_preprint":false},{"pmid":"29997504","id":"PMC_29997504","title":"Saponins From Paris forrestii (Takht.) 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RNAi-mediated knockdown of Rnf6 alters axon outgrowth, and this phenotype can be rescued by modulating LIMK1 expression, placing RNF6 upstream of LIMK1 in the control of axonal growth cone actin dynamics.\",\n      \"method\": \"Co-IP/pulldown, in vitro ubiquitination assay, RNAi knockdown with phenotypic rescue, primary hippocampal neuron cultures\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding, polyubiquitination assay, genetic epistasis via rescue experiments, multiple orthogonal methods in one study\",\n      \"pmids\": [\"16204183\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"RNF6 is an E3 ubiquitin ligase that associates with the androgen receptor (AR), induces AR ubiquitination, and promotes AR transcriptional activity. Knockdown of RNF6 or mutation of RNF6-induced ubiquitination acceptor sites on AR selectively alters expression of a subset of AR target genes and diminishes recruitment of AR and its coactivators to androgen-responsive elements.\",\n      \"method\": \"Proteomic screen (Co-IP), ubiquitination assays, site-directed mutagenesis of AR ubiquitination acceptor sites, ChIP, RNAi knockdown with transcriptional readout\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods including mutagenesis of substrate sites, ChIP, ubiquitination assay, and functional transcriptional readout in a single rigorous study\",\n      \"pmids\": [\"19345326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"RNF6 localizes to punctate nuclear bodies together with PML and Daxx proteins in mouse Sertoli cells, and its re-expression in a Sertoli tumor cell line (where both Inha and Rnf6 are reduced) restores Inha expression and reverts cells to normal growth control, identifying RNF6 as a transcriptional regulator of the Inha promoter through binding of a GGGGC repeat motif.\",\n      \"method\": \"Expression screening, transient and stable transfection with reporter assays, co-localization by immunofluorescence, cell growth assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct DNA-binding and promoter reporter assays, co-localization, functional rescue; single lab with multiple complementary methods\",\n      \"pmids\": [\"11971979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF6 binds and ubiquitylates TLE3, a transcriptional repressor of the β-catenin/TCF4 complex, leading to TLE3 proteasomal degradation. RNF6-mediated TLE3 loss releases β-catenin to TCF4/LEF, activating Wnt/β-catenin signaling and promoting colorectal cancer cell growth and metastasis.\",\n      \"method\": \"Co-IP, ubiquitination assay, rescue experiments with TLE3 re-expression, in vivo tumor/metastasis models\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ubiquitination assay, substrate rescue experiments, and in vivo validation across multiple methods\",\n      \"pmids\": [\"29374067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF6 directly binds and ubiquitylates FoxA1, a transcriptional repressor of EMT, leading to its degradation; the oncogenic effects of RNF6 in hepatocellular carcinoma are partially dependent on FoxA1 degradation.\",\n      \"method\": \"Co-IP, ubiquitination assay, RNF6 knockdown with phenotypic rescue by FoxA1, in vivo metastasis models\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays with rescue experiments, single lab\",\n      \"pmids\": [\"30496760\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"RNF6 regulates STAT3 signaling in gastric cancer by inducing polyubiquitination and degradation of the phosphatase SHP-1, thereby relieving SHP-1-mediated inhibition of STAT3 phosphorylation and promoting expression of STAT3 target genes MCL1 and XIAP.\",\n      \"method\": \"Immunoprecipitation/ubiquitination assay, luciferase reporter assay, Western blot, RNF6 knockdown/overexpression\",\n      \"journal\": \"OncoTargets and therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — IP-based ubiquitination assay and luciferase reporter, multiple readouts, single lab\",\n      \"pmids\": [\"30323630\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RNF6 interacts with the glucocorticoid receptor (GR) and induces K63-linked polyubiquitination of GR, which stabilizes GR protein (rather than degrading it) and promotes GR transcriptional activity, leading to increased expression of pro-survival genes Bcl-xL and Mcl-1 and resistance to dexamethasone in multiple myeloma cells.\",\n      \"method\": \"Co-IP, ubiquitination assay specifying K63 linkage, luciferase reporter assay, RNF6 overexpression/knockdown\",\n      \"journal\": \"Acta pharmacologica Sinica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, linkage-specific ubiquitination assay, luciferase reporter; single lab, multiple methods\",\n      \"pmids\": [\"31645658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF6 undergoes auto-ubiquitination (K48-linked polyubiquitination) in a manner dependent on its RING domain; deletion of the RING domain (ΔRING) or mutation of core cysteine residues (C632S/C635S) abolishes this auto-ubiquitination. USP7 was identified as a deubiquitinase of RNF6 by tandem mass spectrometry; USP7 interacts with RNF6 and removes K48-linked polyubiquitin chains, preventing RNF6 degradation. Anti-cancer drugs Nilotinib and Panobinostat induce RNF6 K48-linked auto-ubiquitination and degradation in an in vitro ubiquitination system without other E3 ligases.\",\n      \"method\": \"RING domain deletion mutagenesis, cysteine-to-serine point mutagenesis, tandem mass spectrometry identification of USP7, Co-IP, in vitro ubiquitination reconstitution assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of auto-ubiquitination, active-site mutagenesis, MS-identified deubiquitinase, multiple orthogonal methods in one study\",\n      \"pmids\": [\"35926709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF6 interacts with p27 (Kip1) via its KIL domain in a phosphorylation-independent manner, enhances ubiquitination and subsequent proteasomal degradation of p27 in the early G0/G1 phase of the cell cycle, and thereby promotes CDK2/Cyclin E complex activity, Rb phosphorylation, and cell cycle progression in prostate cancer cells.\",\n      \"method\": \"Co-IP, ubiquitination assay, shRNA knockdown with cell cycle (G1 arrest) readout, domain mapping\",\n      \"journal\": \"Pharmaceutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, ubiquitination assay, functional cell-cycle readout; single lab\",\n      \"pmids\": [\"35456636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF6 interacts with MST1 (a kinase regulating YAP), promotes MST1 ubiquitination and degradation, and thereby upregulates YAP signaling to promote breast cancer cell metastasis.\",\n      \"method\": \"Co-IP, ubiquitination assay, RNF6 knockdown/overexpression with YAP and metastasis readouts\",\n      \"journal\": \"Experimental and therapeutic medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — Co-IP and ubiquitination, single lab, single paper with limited mechanistic depth\",\n      \"pmids\": [\"34970341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"RNF6 promotes colorectal cancer invasion and migration by increasing GSK3β phosphorylation (inhibiting GSK3β activity), which in turn activates the Wnt/β-catenin pathway.\",\n      \"method\": \"RNF6 overexpression/silencing with Western blot, GSK3β inhibitor rescue, in vivo rat CRC model\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — indirect mechanism (no direct ubiquitination of GSK3β demonstrated), single lab, no Co-IP for direct interaction shown in abstract\",\n      \"pmids\": [\"34352441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RNF6 mediates ubiquitination and degradation of the RNA-binding protein QKI in macrophages; this RNF6-mediated QKI degradation activates PI3K-p110β-dependent autophagy and enhances phagocytic clearance of MRSA.\",\n      \"method\": \"Co-IP, iTRAQ mass spectrometry, immunoprecipitation-ubiquitination assay, QKI knockout mouse model, MRSA infection model\",\n      \"journal\": \"Cell & bioscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified interaction, Co-IP, ubiquitination assay, in vivo model; single lab but multiple methods\",\n      \"pmids\": [\"36088389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In gastric cancer cells, RNF6 localizes to the nucleus and directly binds to the promoter regions of CCNA1 and CREBBP, acting as a transcription factor to regulate their expression and promote tumor cell cycle progression and apoptosis resistance.\",\n      \"method\": \"ChIP-seq, ChIP-PCR, RNF6 overexpression/silencing with CCNA1/CREBBP rescue, luciferase assay\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq with functional validation and rescue experiments; single lab\",\n      \"pmids\": [\"37904524\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF6 physically binds cyclin D2 (CCND2) and mediates its K48-linked polyubiquitination and proteasomal degradation in lung adenocarcinoma cells. RING-domain-deleted RNF6 (ΔRING) fails to ubiquitinate CCND2. Thr280 on CCND2 is critical for its stability; T280 mutation makes CCND2 more stable and less susceptible to RNF6-mediated ubiquitination. RNF6 arrests LUAD cells in G1 by inhibiting CCND2/phospho-Rb signaling.\",\n      \"method\": \"Affinity purification/tandem MS, Co-IP, ubiquitination assay, RING domain deletion mutagenesis, Thr280 point mutation, cell cycle analysis, in vivo xenograft\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — MS-based substrate identification, in vitro ubiquitination with domain and substrate site mutagenesis, multiple orthogonal methods in one study\",\n      \"pmids\": [\"39918413\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"RNF6 interacts with p27 and directly ubiquitinates it via E3 ligase activity, and also indirectly accelerates p27 degradation by acting as a transcription factor to activate SKP2 promoter activity and by interacting with SKP2 to stabilize its protein levels in an Akt-dependent manner, thereby driving glioblastoma cell proliferation.\",\n      \"method\": \"Co-IP, ubiquitination assay, SKP2 promoter reporter assay, Western blot, RNF6 knockdown/overexpression with cell growth readout\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, promoter reporter assays; single lab, multiple methods\",\n      \"pmids\": [\"39938447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CXCL13 enhances RNF6 expression in endothelial cells, and RNF6 facilitates ubiquitination of Sqstm1 at the K314 residue, thereby promoting autophagy and damaging blood spinal cord barrier integrity in experimental allergic encephalomyelitis.\",\n      \"method\": \"Co-IP, ubiquitination assay (K314 site-specific), Western blot, in vivo EAE model with barrier permeability readout\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — site-specific ubiquitination, Co-IP, in vivo model; single lab\",\n      \"pmids\": [\"40231770\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF6 directly binds NME4 and facilitates its K48-linked polyubiquitination and proteasomal degradation; NME4 degradation activates the JNK/c-JUN pathway in ovarian cancer cells, promoting proliferation, invasion, and EMT.\",\n      \"method\": \"Co-IP, CHX chase assay, ubiquitination assay (K48-specific), RNF6/NME4 knockdown rescue experiments, in vivo xenograft\",\n      \"journal\": \"Pathology, research and practice\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, linkage-specific ubiquitination, substrate rescue, in vivo model; single lab\",\n      \"pmids\": [\"41616518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF6 promotes ubiquitination and proteasomal degradation of OTULIN (a linear deubiquitinase) in bronchial epithelial cells; RNF6 knockdown stabilizes OTULIN and suppresses TGF-β1-induced partial EMT-like phenotypic changes, while OTULIN knockdown reverses the protective effect of RNF6 silencing.\",\n      \"method\": \"Co-IP/mass spectrometry (label-free proteomics), Co-IP validation, immunofluorescence co-localization, cycloheximide/MG132 stability assay, RNF6 and OTULIN knockdown rescue experiments\",\n      \"journal\": \"Biochemical pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-identified substrate, Co-IP, stability assays, functional rescue; single lab\",\n      \"pmids\": [\"42055142\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"RNF6 interacts with the Zika virus NS5 protein and acts as a negative regulator of type I interferon and MAPK signaling pathways, functioning as a proviral host factor for ZIKV replication in brain microvascular endothelial cells; RNF6 knockout reduces ZIKV infection while overexpression enhances it.\",\n      \"method\": \"Genome-wide CRISPR/Cas9 screen, RNF6 knockout/knockdown/overexpression with viral replication readout, Co-IP for NS5 interaction, molecular docking\",\n      \"journal\": \"Viruses\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen hit validated by Co-IP and gain/loss-of-function; single lab, docking is computational\",\n      \"pmids\": [\"41902231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PBX1 directly binds a PBX1 recognition element (PRE) between -144 and -99 upstream of the RNF6 transcription start site and, in collaboration with PREP1 (but not MEIS1), activates RNF6 transcription; mutation of the PRE completely abolishes RNF6 transcription. Doxorubicin suppresses RNF6 expression by down-regulating PBX1.\",\n      \"method\": \"Luciferase reporter assay with truncated RNF6 promoter constructs, PRE mutation, ChIP, knockdown experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter deletion and mutagenesis reporter assays, ChIP; single lab, multiple complementary methods\",\n      \"pmids\": [\"26971355\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RNF6 is a RING-H2 domain E3 ubiquitin ligase that targets multiple substrates—including LIMK1, AR, TLE3, FoxA1, SHP-1, GR (via atypical K63 ubiquitination that stabilizes rather than degrades), p27/Kip1, MST1, CCND2, NME4, QKI, OTULIN, and Sqstm1—for K48-linked polyubiquitination and proteasomal degradation (or, in the case of GR, K63-linked stabilizing ubiquitination), and also functions in the nucleus as a direct transcriptional regulator; it undergoes RING-domain-dependent auto-ubiquitination that is counteracted by the deubiquitinase USP7, and its substrates collectively place it at the intersection of axon growth cone actin dynamics, androgen/glucocorticoid receptor signaling, Wnt/β-catenin, SHP-1/STAT3, Hippo/YAP, JNK/c-JUN, and cell-cycle G1/S control.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"RNF6 is a RING-domain E3 ubiquitin ligase that controls the abundance of diverse regulatory proteins to govern cytoskeletal dynamics, nuclear receptor signaling, and proliferative control, and additionally acts as a sequence-specific nuclear transcriptional regulator [#0, #1, #2]. Its founding activity was defined in neurons, where it binds and polyubiquitinates LIMK1 to drive its proteasomal degradation and thereby tune growth-cone actin dynamics and axon outgrowth [#0]. Across cancer contexts RNF6 acts predominantly as an oncogenic ligase by destroying negative regulators of growth and survival pathways: it ubiquitinates and degrades the Wnt/\\u03b2-catenin repressor TLE3 [#3], the EMT repressor FoxA1 [#4], the STAT3-inhibitory phosphatase SHP-1 [#5], the Hippo kinase MST1 to elevate YAP signaling [#9], and the CDK inhibitor p27/Kip1 to promote CDK2/Cyclin E activity and G1/S progression [#8, #14]. It directs K48-linked polyubiquitination and degradation of cyclin D2 via substrate residue Thr280 to enforce G1 arrest in lung adenocarcinoma [#13] and degrades NME4 to activate JNK/c-JUN signaling [#16]. Beyond degradative ubiquitination, RNF6 can stabilize substrates: it attaches K63-linked chains to the glucocorticoid receptor, increasing GR protein levels and pro-survival transcription [#6]. RNF6 also ubiquitinates substrates that reprogram autophagy and barrier integrity, including QKI in macrophages and Sqstm1 at K314 in endothelial cells [#11, #15]. In the nucleus it binds defined promoter elements\\u2014a GGGGC repeat in the Inha promoter and the CCNA1/CREBBP promoters\\u2014to directly regulate transcription [#2, #12]. RNF6 itself is regulated by RING-dependent K48-linked auto-ubiquitination that is reversed by the deubiquitinase USP7, and its transcription is activated by PBX1/PREP1 [#7, #19].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established RNF6 as a nuclear factor capable of direct promoter binding and transcriptional control, an activity distinct from its later-defined ligase function.\",\n      \"evidence\": \"Expression screening, reporter assays, and immunofluorescence co-localization with PML/Daxx in Sertoli cells\",\n      \"pmids\": [\"11971979\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not connect transcriptional activity to ubiquitin ligase function\", \"Sequence-specificity of the GGGGC-motif binding not structurally defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined RNF6's first molecular substrate, showing it is an E3 ligase that degrades LIMK1 to control axonal actin dynamics.\",\n      \"evidence\": \"Co-IP, in vitro ubiquitination, RNAi with LIMK1 rescue in primary hippocampal neurons\",\n      \"pmids\": [\"16204183\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain linkage type not specified\", \"Did not address RNF6 substrate selectivity beyond LIMK1\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Linked RNF6 to nuclear receptor signaling, showing it ubiquitinates AR and selectively reprograms AR target-gene transcription rather than simply degrading the receptor.\",\n      \"evidence\": \"Proteomic Co-IP, AR ubiquitination-site mutagenesis, ChIP, and RNAi transcriptional readout\",\n      \"pmids\": [\"19345326\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which AR ubiquitination alters coactivator recruitment not fully resolved\", \"Linkage type and fate of ubiquitinated AR not detailed\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Identified upstream transcriptional control of RNF6, defining how its expression is set rather than how it acts.\",\n      \"evidence\": \"RNF6 promoter truncation/PRE-mutation luciferase reporters and ChIP for PBX1/PREP1\",\n      \"pmids\": [\"26971355\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue contexts of PBX1-driven RNF6 expression not broadly mapped\", \"Does not address post-translational regulation of RNF6\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Established RNF6 as an oncogenic ligase across multiple cancers by degrading distinct tumor-suppressive substrates feeding Wnt, EMT, and STAT3 pathways.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, and substrate-rescue/in vivo models for TLE3, FoxA1, and SHP-1\",\n      \"pmids\": [\"29374067\", \"30496760\", \"30323630\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether one ligase engages all substrates simultaneously or context-specifically is unclear\", \"Ubiquitination linkage detail varies between substrates\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Revealed a non-degradative mode, showing RNF6 stabilizes GR via K63-linked ubiquitination to drive pro-survival transcription and drug resistance.\",\n      \"evidence\": \"Co-IP, K63-linkage-specific ubiquitination assay, luciferase reporter in multiple myeloma cells\",\n      \"pmids\": [\"31645658\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis for K48-versus-K63 substrate-dependent chain choice unknown\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Extended RNF6 substrate range to Hippo signaling via MST1 degradation, though with limited mechanistic depth.\",\n      \"evidence\": \"Co-IP and ubiquitination assay with YAP and metastasis readouts in breast cancer cells\",\n      \"pmids\": [\"34970341\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single lab, limited mechanistic depth; chain linkage and direct ubiquitination sites not defined\", \"Indirect Wnt/GSK3\\u03b2 effects reported separately remain non-direct\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved how RNF6 is itself controlled, demonstrating RING-dependent K48 auto-ubiquitination opposed by the deubiquitinase USP7.\",\n      \"evidence\": \"RING deletion and C632S/C635S active-site mutagenesis, MS identification of USP7, in vitro ubiquitination reconstitution\",\n      \"pmids\": [\"35926709\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signals controlling the RNF6/USP7 balance not mapped\", \"Whether USP7 also edits RNF6 substrate chains untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected RNF6 to direct cell-cycle control via p27/Kip1 degradation through a defined KIL domain interaction.\",\n      \"evidence\": \"Co-IP with domain mapping, ubiquitination assay, shRNA with G1-arrest readout in prostate cancer cells\",\n      \"pmids\": [\"35456636\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generality of KIL-domain-mediated substrate recognition unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Reaffirmed RNF6's direct transcription-factor activity by mapping genome-wide promoter binding to CCNA1 and CREBBP.\",\n      \"evidence\": \"ChIP-seq/ChIP-PCR with rescue and luciferase validation in gastric cancer cells\",\n      \"pmids\": [\"37904524\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"DNA-binding mode of a RING ligase acting as a transcription factor not structurally explained\", \"Relationship between ligase and transcriptional roles unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined substrate-residue-level control of cyclin D2 and broadened RNF6's degradative targets to NME4 and to SKP2-coupled p27 regulation.\",\n      \"evidence\": \"MS substrate ID, RING-deletion and substrate-site (CCND2 Thr280) mutagenesis, K48 linkage assays, SKP2 promoter reporter, in vivo xenografts\",\n      \"pmids\": [\"39918413\", \"39938447\", \"41616518\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Context determining whether RNF6 arrests (CCND2) or drives (p27/SKP2) the cycle is unclear\", \"Dual direct-and-transcriptional control of p27 not fully integrated\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated RNF6 in autophagy and barrier biology via site-specific Sqstm1 ubiquitination downstream of CXCL13.\",\n      \"evidence\": \"K314 site-specific ubiquitination assay, Co-IP, in vivo EAE barrier-permeability model\",\n      \"pmids\": [\"40231770\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Sqstm1 K314 ubiquitination is degradative or signaling-type not stated\", \"Single disease model\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Extended RNF6 substrates to deubiquitinase regulation (OTULIN) and host-pathogen biology (ZIKV NS5), revealing innate-immune and EMT-modulatory roles.\",\n      \"evidence\": \"MS-based substrate ID with stability assays for OTULIN; CRISPR screen and Co-IP for ZIKV NS5 interaction\",\n      \"pmids\": [\"42055142\", \"41902231\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether RNF6 ubiquitinates NS5 or acts non-catalytically as proviral factor unresolved\", \"OTULIN chain linkage and broader linear-ubiquitin consequences untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How RNF6 selects among its many substrates and chooses degradative (K48) versus stabilizing (K63) chain linkages, and how its E3-ligase and direct transcription-factor activities are coordinated within the same protein, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of substrate recognition or chain-type determination\", \"No unified account linking nuclear DNA-binding and cytoplasmic ligase functions\", \"Most substrate findings are single-lab\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 3, 7, 13, 16]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 3, 13, 16]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [7, 13]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 12]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [2, 12]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 12]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 7, 13]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [8, 13, 14]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5, 6, 9]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 12]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [11, 15]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"LIMK1\", \"AR\", \"TLE3\", \"FoxA1\", \"SHP-1\", \"GR\", \"MST1\", \"USP7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}