{"gene":"RBCK1","run_date":"2026-04-28T19:45:45","timeline":{"discoveries":[{"year":2008,"finding":"RBCK1 functions as an E3 ubiquitin ligase that catalyzes K48-linked ubiquitination and proteasomal degradation of IRF3, acting as a negative feedback regulator of virus-triggered type I interferon induction.","method":"Overexpression and knockdown experiments, ubiquitination assays, plaque assays, viral infection-induced RBCK1 induction demonstrated","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (OE, KD, ubiquitination assay, plaque assay), replicated across conditions, 129 citations","pmids":["18711448"],"is_preprint":false},{"year":2007,"finding":"RBCK1 physically interacts with TAB2 and TAB3 adaptor proteins and facilitates their proteasome-dependent degradation, thereby negatively regulating TNF- and IL-1-triggered NF-κB activation.","method":"Co-immunoprecipitation, overexpression, RNAi knockdown, proteasome inhibitor (MG-132) rescue, NF-κB reporter assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, proteasome inhibitor rescue, OE and KD with defined signaling phenotype; 66 citations","pmids":["17449468"],"is_preprint":false},{"year":2008,"finding":"RBCK1 possesses intrinsic E3 ubiquitin ligase activity (self-ubiquitination in vitro) that is inhibited by interaction with its splice variant RBCK2 (which lacks the RING-IBR domain) and by phosphorylation by PKCβ.","method":"In vitro ubiquitination assay, coexpression studies, PKCβ phosphorylation assay, proteasomal degradation rescue","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay with mutagenesis-equivalent inhibition studies; multiple orthogonal mechanisms demonstrated","pmids":["18303026"],"is_preprint":false},{"year":1998,"finding":"RBCK1 has transcriptional activation activity requiring both its RING-finger and B-Box motifs; PKA enhances this activity ~8-fold while MEK1 and MEKK1 repress it.","method":"GAL4 chimeric transcription regulator system, deletion/mutant constructs, kinase co-expression","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — defined domain requirements by mutagenesis and functional reporter assay; single lab","pmids":["9642138"],"is_preprint":false},{"year":1998,"finding":"RBCK2, a splice variant of RBCK1 lacking the RING-IBR domain, forms a hetero-oligomeric complex with RBCK1 and inhibits its transcriptional activity; RBCK1 homo- and hetero-dimerizes but RBCK2 does not self-interact.","method":"In vitro interaction assays, GAL4 transcription reporter system, analysis of alternative splicing","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro interaction and reporter assay; single lab, moderate evidence","pmids":["9755849"],"is_preprint":false},{"year":2005,"finding":"RBCK1 shuttles between cytoplasm and nucleus via an N-terminal nuclear export signal (Leu-142/145) and a C-terminal RING-IBR nuclear localization signal; in the nucleus it localizes to nuclear bodies and interacts with CBP (enhancing transcription) and PML (repressing CBP-enhanced activity).","method":"Leptomycin B treatment, mutational analysis of NES/NLS, nuclear body co-localization, co-immunoprecipitation with CBP and PML, transcriptional reporter assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — direct localization with functional consequence, mutagenesis of shuttling signals, Co-IP of interactors with reporter assays; multiple orthogonal methods","pmids":["15833741"],"is_preprint":false},{"year":2005,"finding":"RBCK2 (RING-lacking splice variant) tethers RBCK1 in the cytoplasm by forming a hetero-oligomeric complex, relocating even NES-disrupted RBCK1 from nucleus to cytoplasm.","method":"Subcellular localization assays, co-expression of NES-disrupted RBCK1 with RBCK2, fluorescence microscopy","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — direct localization experiment with functional consequence, single lab","pmids":["16083853"],"is_preprint":false},{"year":2006,"finding":"RBCK1 physically associates with PKCβI in cardiac myocytes; this interaction increases upon phenylephrine stimulation, and RBCK1-mediated hypertrophy requires PKCβ activity, establishing RBCK1 as a key regulator of PKCβI function in cardiac hypertrophy.","method":"Co-immunoprecipitation in neonatal myocytes, adenovirus-based overexpression, PKCβ-selective antagonists, RNAi knockdown with cell size measurement","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, pharmacological inhibition epistasis, KD with defined phenotype; multiple orthogonal approaches","pmids":["17121852"],"is_preprint":false},{"year":2010,"finding":"RBCK1 promotes ERα-positive breast cancer cell cycle progression by driving transcription of ERα and cyclin B1; it is recruited to the ERα promoter (by ChIP), and its depletion causes G2-M arrest.","method":"Chromatin immunoprecipitation (ChIP), siRNA knockdown, flow cytometry cell cycle analysis, RT-PCR for target genes","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — ChIP demonstrating direct promoter recruitment, KD with defined cell-cycle phenotype, multiple orthogonal methods","pmids":["20103625"],"is_preprint":false},{"year":2010,"finding":"RBCK1 interacts with Eya1 (Eyes absent 1 protein) and enhances Eya protein function as co-activators for Six transcription factors; knockdown of the Rbck1 ortholog in zebrafish causes a BOR syndrome-like phenotype with ear and branchial arch defects.","method":"GST pulldown, co-immunoprecipitation, zebrafish morpholino knockdown, in situ hybridization","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — GST pulldown + Co-IP + in vivo morpholino phenotype; multiple orthogonal methods","pmids":["20956555"],"is_preprint":false},{"year":2012,"finding":"RBCK1 interacts with human PXR (pregnane X receptor) via all RBCK1 domains, ubiquitinates PXR, and promotes its proteasomal degradation, thereby reducing PXR target gene induction by rifampicin in primary human hepatocytes.","method":"Yeast two-hybrid screening, co-immunoprecipitation, domain mapping, ubiquitination assay, proteasome inhibitor (MG-132) rescue, siRNA knockdown in primary hepatocytes","journal":"Drug metabolism and disposition","confidence":"High","confidence_rationale":"Tier 1–2 — yeast 2-hybrid + Co-IP + in vitro ubiquitination + proteasome rescue + primary human cell validation","pmids":["23160820"],"is_preprint":false},{"year":2013,"finding":"RBCK1 interacts with FKBPL within HSP90 chaperone complexes together with ERα, regulates FKBPL stability via ubiquitination, and together with FKBPL associates with ERα at the pS2 gene promoter to regulate its transcription.","method":"Co-immunoprecipitation, ubiquitination assay, stable overexpression clones, siRNA knockdown, ChIP at pS2 promoter","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — Co-IP of multi-protein complex, ubiquitination assay, ChIP, multiple cell line validation","pmids":["23912458"],"is_preprint":false},{"year":2016,"finding":"RBCK1 interacts with HOXA1 and TRAF2; RBCK1 and TRAF2 act epistatically downstream of HOXA1 in NF-κB activation, and the HOXA1 11-His repeat and homeodomain are required for RBCK1 binding and NF-κB stimulation.","method":"Proteome-wide interaction screening, Co-IP, epistasis analysis by overexpression/knockdown, NF-κB luciferase reporter assays, domain deletion mapping","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, epistasis, domain mapping, reporter assays; multiple orthogonal methods","pmids":["27382069"],"is_preprint":false},{"year":2019,"finding":"RBCK1 directly interacts with and facilitates poly-ubiquitination and proteasomal degradation of p53 in renal cell carcinoma cells; the proliferative effect of RBCK1 is rescued by p53 knockdown.","method":"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, rescue experiment with p53 knockdown, in vivo xenograft","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, ubiquitination assay, genetic rescue experiment; multiple orthogonal methods","pmids":["30874541"],"is_preprint":false},{"year":2020,"finding":"MTSS1 interacts with RBCK1 to facilitate RBCK1-mediated K48-linked ubiquitination and degradation of NF-κB p65, suppressing NF-κB signaling; ACTBL2 competes with RBCK1 for MTSS1 binding, leading to p65 stabilization.","method":"Co-immunoprecipitation, ubiquitination assay (K48-specific), Mtss1 knockout mouse models, competition binding assays, organoid and xenograft models","journal":"Nature cancer","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, K48-specific ubiquitination assay, KO mouse model, competition assay; multiple orthogonal methods","pmids":["35122005"],"is_preprint":false},{"year":2022,"finding":"RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC) and generates linear ubiquitin chains; RBCK1 deficiency causes glycogen hyperphosphorylation and accumulation of polyglucosan bodies with overlong glucan branches in muscle and brain, recapitulating malin-deficient Lafora disease pathology.","method":"RBCK1-deficient mouse model, glycogen structural analysis (branch length, phosphorylation), neuroinflammation assays, behavioral tests, glycogen synthase downregulation rescue","journal":"Brain : a journal of neurology","confidence":"High","confidence_rationale":"Tier 2 — mouse KO model with multiple biochemical and structural analyses, genetic rescue experiment; strong mechanistic characterization","pmids":["35084461"],"is_preprint":false},{"year":2022,"finding":"RBCK1 interacts with PTEN and promotes its K48-linked ubiquitination and proteasomal degradation in ovarian cancer cells.","method":"Co-immunoprecipitation, K48-linked ubiquitination assay, siRNA knockdown, cell proliferation and apoptosis assays","journal":"Human cell","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and K48 ubiquitination assay; single lab, moderate evidence","pmids":["35174471"],"is_preprint":false},{"year":2022,"finding":"RBCK1 interacts with YAP protein and promotes K48-linked poly-ubiquitination of YAP at lysines K76, K204, and K321, leading to YAP degradation and suppression of Hippo/YAP signaling in triple-negative breast cancer.","method":"Co-immunoprecipitation, ubiquitin-based IP assay with K48-specific linkage detection, protein stability assays, in vitro and in vivo overexpression/depletion, RNA sequencing, luciferase reporter","journal":"Cell communication and signaling","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP + site-specific ubiquitination mapping + stability assay + RNA-seq; single lab but multiple methods","pmids":["36280829"],"is_preprint":false},{"year":2022,"finding":"RBCK1 interacts with RNF31 (HOIP), a partner subunit of LUBAC, and stabilizes RNF31 by repressing its ubiquitination and proteasomal degradation in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown, in vivo xenograft","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP and ubiquitination assay demonstrating stabilization; single lab","pmids":["35869046"],"is_preprint":false},{"year":2022,"finding":"RBCK1 promotes PPARγ ubiquitination and degradation, thereby disrupting the PPARγ/PGC1α complex and enhancing WNT/β-catenin/GLUT1-mediated aerobic glycolysis in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown, WNT/β-catenin reporter, GLUT1 expression analysis","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, ubiquitination assay, pathway reporter; single lab","pmids":["35411229"],"is_preprint":false},{"year":2024,"finding":"RBCK1 interacts with and polyubiquitylates mitofusin 2 (MFN2) to promote its proteasomal degradation under ferroptotic stress, leading to decreased mitochondrial ROS and lipid peroxidation and conferring ferroptosis resistance in pancreatic cancer cells.","method":"Co-immunoprecipitation, ubiquitination assay, RBCK1 genetic depletion, ferroptosis induction with IKE, mitochondrial ROS measurement, xenograft model","journal":"Free radical biology & medicine","confidence":"High","confidence_rationale":"Tier 2 — Co-IP, ubiquitination assay, defined molecular mechanism with functional readout (ROS, lipid peroxidation), in vivo validation","pmids":["38763208"],"is_preprint":false},{"year":2023,"finding":"RBCK1 promotes degradation of PICK1 via ubiquitination under hypoxia; HIF-1α transcriptionally regulates RBCK1 to mediate this effect, and RBCK1 knockdown inhibits nasopharyngeal carcinoma cell proliferation that is rescued by co-knockdown of PICK1.","method":"Co-immunoprecipitation, immunofluorescence co-localization, ubiquitination/protein stability assays, siRNA knockdown with genetic rescue, xenograft model","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP, ubiquitination assay, genetic rescue; single lab","pmids":["36934971"],"is_preprint":false},{"year":2023,"finding":"RBCK1 promotes degradation of ANKRD35 via ubiquitination; ANKRD35 destabilizes MITD1 by binding SUMO2, and the resulting RBCK1-ANKRD35-MITD1-ANXA1 axis regulates AKT and ERK phosphorylation and sunitinib resistance in renal cell carcinoma.","method":"Bioinformatic analysis, in vitro/in vivo RBCK1 knockdown, Co-immunoprecipitation, pathway phosphorylation analysis (AKT, ERK)","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 — Co-IP and pathway analysis; mechanistic chain partially supported; single lab","pmids":["36732658"],"is_preprint":false},{"year":2022,"finding":"RBCK1 regulates HIF1α signaling in ER-positive breast cancer; RBCK1 depletion affects HIF1α pathway activity, identifying RBCK1 as a regulator of HIF1α in this cancer context.","method":"siRNA knockdown, western blotting for HIF1α pathway components, cell proliferation assays","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 — KD with pathway readout but no direct biochemical interaction or substrate ubiquitination demonstrated; single lab, limited mechanistic detail","pmids":["36473847"],"is_preprint":false}],"current_model":"RBCK1 (HOIL-1L) is an RBR-type E3 ubiquitin ligase that, as a component of the LUBAC complex, generates linear ubiquitin chains and also directly ubiquitinates multiple substrates (including IRF3, TAB2/3, p53, PTEN, YAP, PPARγ, PXR, MFN2, and PICK1) for proteasomal degradation; its activity is regulated by interaction with its RING-lacking splice variant RBCK2 (which sequesters it in the cytoplasm and inhibits its E3 activity) and by PKCβ phosphorylation, while in the nucleus RBCK1 acts as a transcriptional activator requiring both RING-finger and B-Box motifs, regulated by CBP and PML interactions, collectively positioning RBCK1 as a multi-functional ubiquitin ligase controlling NF-κB signaling, antiviral responses, glycogen metabolism, and cell cycle progression."},"narrative":{"teleology":[{"year":1998,"claim":"Establishing that RBCK1 possesses intrinsic transcriptional activation capacity dependent on its RING-finger and B-Box motifs, and that an alternative splice variant RBCK2 acts as a dominant-negative inhibitor through hetero-oligomerization, defined RBCK1 as a regulated transcription factor before its E3 ligase activity was recognized.","evidence":"GAL4 chimeric reporter system with domain deletions, in vitro interaction assays of RBCK1/RBCK2 homo- and hetero-oligomers","pmids":["9642138","9755849"],"confidence":"Medium","gaps":["Transcriptional targets not identified","Physiological relevance of RBCK2 inhibition not validated in vivo","Endogenous stoichiometry of RBCK1:RBCK2 unknown"]},{"year":2005,"claim":"Mapping a nuclear export signal (Leu-142/145) and a RING-IBR-dependent nuclear localization signal resolved how RBCK1 shuttles between cytoplasm and nucleus, and identifying CBP and PML as nuclear interaction partners explained how its transcriptional activity is enhanced and repressed, respectively.","evidence":"Leptomycin B treatment, NES/NLS mutagenesis, nuclear body co-localization, Co-IP with CBP/PML, transcriptional reporters; RBCK2 co-expression relocalization assays","pmids":["15833741","16083853"],"confidence":"High","gaps":["Chromatin targets of RBCK1 nuclear activity not identified at this stage","Structural basis of RBCK2-mediated cytoplasmic tethering unknown"]},{"year":2006,"claim":"Discovery that RBCK1 physically associates with PKCβI in cardiac myocytes and is required for phenylephrine-induced hypertrophy connected RBCK1 to signal-dependent cardiac remodeling and identified PKCβ as both a binding partner and a functional regulator.","evidence":"Co-IP in neonatal myocytes, PKCβ-selective antagonists, adenoviral overexpression, RNAi with cell size measurement","pmids":["17121852"],"confidence":"High","gaps":["Whether RBCK1 ubiquitinates PKCβI or acts as a scaffold not resolved","In vivo cardiac phenotype not established"]},{"year":2007,"claim":"Demonstrating that RBCK1 targets TAB2 and TAB3 adaptors for proteasome-dependent degradation established RBCK1 as a negative feedback regulator of TNF/IL-1-triggered NF-κB signaling, the first clear link between RBCK1 E3 activity and a major inflammatory pathway.","evidence":"Reciprocal Co-IP, proteasome inhibitor rescue, RNAi knockdown, NF-κB reporter assays","pmids":["17449468"],"confidence":"High","gaps":["Ubiquitin chain linkage type on TAB2/3 not specified","In vivo role in inflammation not tested"]},{"year":2008,"claim":"Biochemical reconstitution of RBCK1 E3 ligase activity in vitro, together with identification of IRF3 as a K48-ubiquitination substrate degraded during viral infection, established RBCK1 as a bona fide ubiquitin ligase with dual roles in NF-κB and antiviral innate immunity, while revealing regulation by PKCβ phosphorylation and RBCK2 inhibition.","evidence":"In vitro self-ubiquitination assay, PKCβ phosphorylation, RBCK2 co-expression inhibition, IRF3 ubiquitination/degradation upon viral infection, plaque assays","pmids":["18303026","18711448"],"confidence":"High","gaps":["PKCβ phosphorylation sites on RBCK1 not mapped","Relative contribution of K48-linked versus linear ubiquitin ligase activities not delineated"]},{"year":2010,"claim":"ChIP-based demonstration that RBCK1 is recruited to the ERα promoter to drive ERα and cyclin B1 transcription, with depletion causing G2-M arrest, established a direct nuclear transcriptional role in breast cancer cell proliferation; concurrently, interaction with Eya1 and a zebrafish loss-of-function phenotype mimicking BOR syndrome expanded RBCK1 function to developmental transcription.","evidence":"ChIP at ERα promoter, siRNA/flow cytometry in breast cancer cells; GST pulldown/Co-IP with Eya1, zebrafish morpholino with BOR-like phenotype","pmids":["20103625","20956555"],"confidence":"High","gaps":["Whether transcriptional roles are ubiquitin-dependent or -independent unclear","Mammalian developmental phenotype not established"]},{"year":2012,"claim":"Identification of PXR as an RBCK1 ubiquitination substrate in primary human hepatocytes extended the substrate repertoire to nuclear receptors controlling drug metabolism.","evidence":"Yeast two-hybrid, Co-IP, domain mapping, ubiquitination assay, MG-132 rescue, siRNA in primary hepatocytes","pmids":["23160820"],"confidence":"High","gaps":["Ubiquitin chain linkage type on PXR not determined","In vivo pharmacokinetic consequences not tested"]},{"year":2019,"claim":"Showing that RBCK1 directly ubiquitinates p53 for proteasomal degradation, with genetic rescue by p53 knockdown, established RBCK1 as an alternative p53 E3 ligase promoting renal cell carcinoma proliferation.","evidence":"Co-IP, ubiquitination assay, siRNA with p53 co-knockdown rescue, xenograft","pmids":["30874541"],"confidence":"High","gaps":["Ubiquitin chain type on p53 not specified","Relationship to MDM2-mediated p53 degradation not addressed"]},{"year":2020,"claim":"Discovering that MTSS1 recruits RBCK1 to catalyze K48-linked ubiquitination of NF-κB p65, and that ACTBL2 competes for MTSS1 binding to stabilize p65, revealed a cofactor-dependent mechanism by which RBCK1 directly degrades a key NF-κB transcription factor.","evidence":"Reciprocal Co-IP, K48-specific ubiquitination assay, Mtss1 KO mice, competition binding, organoid and xenograft models","pmids":["35122005"],"confidence":"High","gaps":["Structural basis of MTSS1-mediated RBCK1 recruitment unknown","Whether this mechanism operates in non-cancer contexts not tested"]},{"year":2022,"claim":"Multiple studies in 2022 greatly expanded the RBCK1 substrate repertoire—identifying PTEN, YAP, PPARγ, and PICK1 as K48-ubiquitination targets—while RBCK1 deficiency in mice was shown to cause glycogen hyperphosphorylation and polyglucosan body formation mimicking Lafora disease, revealing an unexpected LUBAC-dependent role in glycogen quality control.","evidence":"RBCK1-KO mouse glycogen analysis, neuroinflammation/behavioral tests, genetic rescue (glycogen synthase); Co-IP and K48-ubiquitination assays for PTEN, YAP, PPARγ, PICK1 in cancer cell models; RNF31 stabilization by RBCK1 in HCC","pmids":["35084461","35174471","36280829","35411229","35869046","36934971"],"confidence":"High","gaps":["Molecular mechanism linking LUBAC linear ubiquitination to glycogen metabolism not resolved","Whether substrate selectivity between K48 and linear activities is context-dependent remains unclear","In vivo validation of most cancer-context substrates lacking"]},{"year":2024,"claim":"Identification of MFN2 as an RBCK1 ubiquitination substrate whose degradation reduces mitochondrial ROS and lipid peroxidation under ferroptotic stress connected RBCK1 to ferroptosis resistance in pancreatic cancer.","evidence":"Co-IP, ubiquitination assay, RBCK1 depletion, IKE-induced ferroptosis, mitochondrial ROS measurement, xenograft","pmids":["38763208"],"confidence":"High","gaps":["Ubiquitin chain linkage type on MFN2 not specified","Whether RBCK1-MFN2 axis operates outside pancreatic cancer unknown"]},{"year":null,"claim":"The molecular logic determining whether RBCK1 acts via linear (LUBAC-dependent) versus K48-linked (autonomous) ubiquitination on specific substrates remains unresolved, as does the structural basis for its remarkably broad substrate recognition.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of RBCK1 in complex with any substrate","Switch mechanism between LUBAC-linear and autonomous-K48 activity unknown","Comprehensive in vivo substrate mapping not performed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,2,10,13,14,16,17,19,20,21,22]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,2,15]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[3,5,8,9]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,5,8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5,6]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[1,7,12,14,17]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,10,13,16,20]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[8]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[3,8,9]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[20]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[15,19]}],"complexes":["LUBAC"],"partners":["RNF31","PKCΒ","MTSS1","CBP","PML","EYA1","FKBPL","TAB2"],"other_free_text":[]},"mechanistic_narrative":"RBCK1 (HOIL-1L) is an RBR-type E3 ubiquitin ligase that functions both as a catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC) and as an autonomous K48-linked ubiquitin ligase targeting diverse substrates for proteasomal degradation. As a LUBAC component, RBCK1 generates linear ubiquitin chains essential for NF-κB signaling and glycogen metabolism, and its deficiency causes polyglucosan body accumulation recapitulating Lafora disease pathology [PMID:35084461]. Acting independently, RBCK1 catalyzes K48-linked ubiquitination and degradation of substrates including IRF3, TAB2/TAB3, p53, PTEN, YAP, PPARγ, PXR, MFN2, and PICK1, thereby regulating antiviral innate immunity, NF-κB activation, cell cycle progression, Hippo signaling, metabolic reprogramming, and ferroptosis resistance [PMID:18711448, PMID:17449468, PMID:30874541, PMID:36280829, PMID:38763208]. RBCK1 shuttles between cytoplasm and nucleus via defined NES and NLS sequences; in the nucleus it acts as a transcriptional co-activator at ER-responsive and other promoters, with its activity modulated by CBP, PML, PKCβ phosphorylation, and sequestration by its RING-lacking splice variant RBCK2 [PMID:15833741, PMID:18303026, PMID:20103625]."},"prefetch_data":{"uniprot":{"accession":"Q9BYM8","full_name":"RanBP-type and C3HC4-type zinc finger-containing protein 1","aliases":["HBV-associated factor 4","Heme-oxidized IRP2 ubiquitin ligase 1","HOIL-1","Hepatitis B virus X-associated protein 4","RING finger protein 54","RING-type E3 ubiquitin transferase HOIL-1","Ubiquitin-conjugating enzyme 7-interacting protein 3"],"length_aa":510,"mass_kda":57.6,"function":"E3 ubiquitin-protein ligase, which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, such as UBE2L3/UBCM4, and then transfers it to substrates (PubMed:12629548, PubMed:17449468, PubMed:18711448). Functions as an E3 ligase for oxidized IREB2 and both heme and oxygen are necessary for IREB2 ubiquitination (PubMed:12629548). Promotes ubiquitination of TAB2 and IRF3 and their degradation by the proteasome (PubMed:17449468, PubMed:18711448). Component of the LUBAC complex which conjugates linear ('Met-1'-linked) polyubiquitin chains to substrates and plays a key role in NF-kappa-B activation and regulation of inflammation (PubMed:17006537, PubMed:19136968, PubMed:21455173, PubMed:21455180, PubMed:21455181). LUBAC conjugates linear polyubiquitin to IKBKG and RIPK1 and is involved in activation of the canonical NF-kappa-B and the JNK signaling pathways (PubMed:17006537, PubMed:19136968, PubMed:21455173, PubMed:21455180, PubMed:21455181). Linear ubiquitination mediated by the LUBAC complex interferes with TNF-induced cell death and thereby prevents inflammation (PubMed:17006537, PubMed:21455173, PubMed:21455180, PubMed:21455181). LUBAC is recruited to the TNF-R1 signaling complex (TNF-RSC) following polyubiquitination of TNF-RSC components by BIRC2 and/or BIRC3 and to conjugate linear polyubiquitin to IKBKG and possibly other components contributing to the stability of the complex (PubMed:17006537, PubMed:19136968, PubMed:21455173, PubMed:21455180, PubMed:21455181). The LUBAC complex is also involved in innate immunity by conjugating linear polyubiquitin chains at the surface of bacteria invading the cytosol to form the ubiquitin coat surrounding bacteria (PubMed:28481331). LUBAC is not able to initiate formation of the bacterial ubiquitin coat, and can only promote formation of linear polyubiquitins on pre-existing ubiquitin (PubMed:28481331). The bacterial ubiquitin coat acts as an 'eat-me' signal for xenophagy and promotes NF-kappa-B activation (PubMed:28481331). Together with OTULIN, the LUBAC complex regulates the canonical Wnt signaling during angiogenesis (PubMed:23708998). Binds polyubiquitin of different linkage types (PubMed:20005846, PubMed:21455181)","subcellular_location":"","url":"https://www.uniprot.org/uniprotkb/Q9BYM8/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/RBCK1","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/RBCK1","total_profiled":1310},"omim":[{"mim_id":"621505","title":"ANKYRIN REPEAT DOMAIN-CONTAINING PROTEIN 35; ANKRD35","url":"https://www.omim.org/entry/621505"},{"mim_id":"621486","title":"MICROTUBULE-INTERACTING AND TRAFFICKING DOMAIN-CONTAINING PROTEIN 1; MITD1","url":"https://www.omim.org/entry/621486"},{"mim_id":"620632","title":"IMMUNODEFICIENCY 115 WITH AUTOINFLAMMATION; IMD115","url":"https://www.omim.org/entry/620632"},{"mim_id":"617076","title":"FK506-BINDING PROTEIN-LIKE; FKBPL","url":"https://www.omim.org/entry/617076"},{"mim_id":"615895","title":"POLYGLUCOSAN BODY MYOPATHY 1 WITH OR WITHOUT IMMUNODEFICIENCY; PGBM1","url":"https://www.omim.org/entry/615895"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/RBCK1"},"hgnc":{"alias_symbol":["RBCK2","XAP4","RNF54","ZRANB4","UBCE7IP3","HOIL1"],"prev_symbol":["C20orf18"]},"alphafold":{"accession":"Q9BYM8","domains":[{"cath_id":"-","chopping":"1-43","consensus_level":"medium","plddt":89.1098,"start":1,"end":43},{"cath_id":"3.10.20.90","chopping":"54-131","consensus_level":"high","plddt":88.3024,"start":54,"end":131},{"cath_id":"3.30.40.10","chopping":"279-364","consensus_level":"high","plddt":94.8028,"start":279,"end":364},{"cath_id":"-","chopping":"442-510","consensus_level":"medium","plddt":88.5671,"start":442,"end":510}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BYM8","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BYM8-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BYM8-F1-predicted_aligned_error_v6.png","plddt_mean":84.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=RBCK1","jax_strain_url":"https://www.jax.org/strain/search?query=RBCK1"},"sequence":{"accession":"Q9BYM8","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BYM8.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BYM8/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BYM8"}},"corpus_meta":[{"pmid":"18711448","id":"PMC_18711448","title":"Negative 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Pengze.","date":"2025","source":"Fish & shellfish immunology","url":"https://pubmed.ncbi.nlm.nih.gov/40588086","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":21749,"output_tokens":5310,"usd":0.072448},"stage2":{"model":"claude-opus-4-6","input_tokens":9002,"output_tokens":3467,"usd":0.197527},"total_usd":0.269975,"stage1_batch_id":"msgbatch_01SUR8NDzVrNmyszWyJeCct7","stage2_batch_id":"msgbatch_01G7Yfutko3cDWzejrwH5AMx","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2008,\n      \"finding\": \"RBCK1 functions as an E3 ubiquitin ligase that catalyzes K48-linked ubiquitination and proteasomal degradation of IRF3, acting as a negative feedback regulator of virus-triggered type I interferon induction.\",\n      \"method\": \"Overexpression and knockdown experiments, ubiquitination assays, plaque assays, viral infection-induced RBCK1 induction demonstrated\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (OE, KD, ubiquitination assay, plaque assay), replicated across conditions, 129 citations\",\n      \"pmids\": [\"18711448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"RBCK1 physically interacts with TAB2 and TAB3 adaptor proteins and facilitates their proteasome-dependent degradation, thereby negatively regulating TNF- and IL-1-triggered NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation, overexpression, RNAi knockdown, proteasome inhibitor (MG-132) rescue, NF-κB reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, proteasome inhibitor rescue, OE and KD with defined signaling phenotype; 66 citations\",\n      \"pmids\": [\"17449468\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"RBCK1 possesses intrinsic E3 ubiquitin ligase activity (self-ubiquitination in vitro) that is inhibited by interaction with its splice variant RBCK2 (which lacks the RING-IBR domain) and by phosphorylation by PKCβ.\",\n      \"method\": \"In vitro ubiquitination assay, coexpression studies, PKCβ phosphorylation assay, proteasomal degradation rescue\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with mutagenesis-equivalent inhibition studies; multiple orthogonal mechanisms demonstrated\",\n      \"pmids\": [\"18303026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"RBCK1 has transcriptional activation activity requiring both its RING-finger and B-Box motifs; PKA enhances this activity ~8-fold while MEK1 and MEKK1 repress it.\",\n      \"method\": \"GAL4 chimeric transcription regulator system, deletion/mutant constructs, kinase co-expression\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined domain requirements by mutagenesis and functional reporter assay; single lab\",\n      \"pmids\": [\"9642138\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"RBCK2, a splice variant of RBCK1 lacking the RING-IBR domain, forms a hetero-oligomeric complex with RBCK1 and inhibits its transcriptional activity; RBCK1 homo- and hetero-dimerizes but RBCK2 does not self-interact.\",\n      \"method\": \"In vitro interaction assays, GAL4 transcription reporter system, analysis of alternative splicing\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro interaction and reporter assay; single lab, moderate evidence\",\n      \"pmids\": [\"9755849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RBCK1 shuttles between cytoplasm and nucleus via an N-terminal nuclear export signal (Leu-142/145) and a C-terminal RING-IBR nuclear localization signal; in the nucleus it localizes to nuclear bodies and interacts with CBP (enhancing transcription) and PML (repressing CBP-enhanced activity).\",\n      \"method\": \"Leptomycin B treatment, mutational analysis of NES/NLS, nuclear body co-localization, co-immunoprecipitation with CBP and PML, transcriptional reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization with functional consequence, mutagenesis of shuttling signals, Co-IP of interactors with reporter assays; multiple orthogonal methods\",\n      \"pmids\": [\"15833741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"RBCK2 (RING-lacking splice variant) tethers RBCK1 in the cytoplasm by forming a hetero-oligomeric complex, relocating even NES-disrupted RBCK1 from nucleus to cytoplasm.\",\n      \"method\": \"Subcellular localization assays, co-expression of NES-disrupted RBCK1 with RBCK2, fluorescence microscopy\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct localization experiment with functional consequence, single lab\",\n      \"pmids\": [\"16083853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"RBCK1 physically associates with PKCβI in cardiac myocytes; this interaction increases upon phenylephrine stimulation, and RBCK1-mediated hypertrophy requires PKCβ activity, establishing RBCK1 as a key regulator of PKCβI function in cardiac hypertrophy.\",\n      \"method\": \"Co-immunoprecipitation in neonatal myocytes, adenovirus-based overexpression, PKCβ-selective antagonists, RNAi knockdown with cell size measurement\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, pharmacological inhibition epistasis, KD with defined phenotype; multiple orthogonal approaches\",\n      \"pmids\": [\"17121852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RBCK1 promotes ERα-positive breast cancer cell cycle progression by driving transcription of ERα and cyclin B1; it is recruited to the ERα promoter (by ChIP), and its depletion causes G2-M arrest.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), siRNA knockdown, flow cytometry cell cycle analysis, RT-PCR for target genes\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP demonstrating direct promoter recruitment, KD with defined cell-cycle phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"20103625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"RBCK1 interacts with Eya1 (Eyes absent 1 protein) and enhances Eya protein function as co-activators for Six transcription factors; knockdown of the Rbck1 ortholog in zebrafish causes a BOR syndrome-like phenotype with ear and branchial arch defects.\",\n      \"method\": \"GST pulldown, co-immunoprecipitation, zebrafish morpholino knockdown, in situ hybridization\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — GST pulldown + Co-IP + in vivo morpholino phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"20956555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"RBCK1 interacts with human PXR (pregnane X receptor) via all RBCK1 domains, ubiquitinates PXR, and promotes its proteasomal degradation, thereby reducing PXR target gene induction by rifampicin in primary human hepatocytes.\",\n      \"method\": \"Yeast two-hybrid screening, co-immunoprecipitation, domain mapping, ubiquitination assay, proteasome inhibitor (MG-132) rescue, siRNA knockdown in primary hepatocytes\",\n      \"journal\": \"Drug metabolism and disposition\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — yeast 2-hybrid + Co-IP + in vitro ubiquitination + proteasome rescue + primary human cell validation\",\n      \"pmids\": [\"23160820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"RBCK1 interacts with FKBPL within HSP90 chaperone complexes together with ERα, regulates FKBPL stability via ubiquitination, and together with FKBPL associates with ERα at the pS2 gene promoter to regulate its transcription.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, stable overexpression clones, siRNA knockdown, ChIP at pS2 promoter\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP of multi-protein complex, ubiquitination assay, ChIP, multiple cell line validation\",\n      \"pmids\": [\"23912458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"RBCK1 interacts with HOXA1 and TRAF2; RBCK1 and TRAF2 act epistatically downstream of HOXA1 in NF-κB activation, and the HOXA1 11-His repeat and homeodomain are required for RBCK1 binding and NF-κB stimulation.\",\n      \"method\": \"Proteome-wide interaction screening, Co-IP, epistasis analysis by overexpression/knockdown, NF-κB luciferase reporter assays, domain deletion mapping\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, epistasis, domain mapping, reporter assays; multiple orthogonal methods\",\n      \"pmids\": [\"27382069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"RBCK1 directly interacts with and facilitates poly-ubiquitination and proteasomal degradation of p53 in renal cell carcinoma cells; the proliferative effect of RBCK1 is rescued by p53 knockdown.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, siRNA knockdown, rescue experiment with p53 knockdown, in vivo xenograft\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination assay, genetic rescue experiment; multiple orthogonal methods\",\n      \"pmids\": [\"30874541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MTSS1 interacts with RBCK1 to facilitate RBCK1-mediated K48-linked ubiquitination and degradation of NF-κB p65, suppressing NF-κB signaling; ACTBL2 competes with RBCK1 for MTSS1 binding, leading to p65 stabilization.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay (K48-specific), Mtss1 knockout mouse models, competition binding assays, organoid and xenograft models\",\n      \"journal\": \"Nature cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, K48-specific ubiquitination assay, KO mouse model, competition assay; multiple orthogonal methods\",\n      \"pmids\": [\"35122005\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 is a component of the linear ubiquitin chain assembly complex (LUBAC) and generates linear ubiquitin chains; RBCK1 deficiency causes glycogen hyperphosphorylation and accumulation of polyglucosan bodies with overlong glucan branches in muscle and brain, recapitulating malin-deficient Lafora disease pathology.\",\n      \"method\": \"RBCK1-deficient mouse model, glycogen structural analysis (branch length, phosphorylation), neuroinflammation assays, behavioral tests, glycogen synthase downregulation rescue\",\n      \"journal\": \"Brain : a journal of neurology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — mouse KO model with multiple biochemical and structural analyses, genetic rescue experiment; strong mechanistic characterization\",\n      \"pmids\": [\"35084461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 interacts with PTEN and promotes its K48-linked ubiquitination and proteasomal degradation in ovarian cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, K48-linked ubiquitination assay, siRNA knockdown, cell proliferation and apoptosis assays\",\n      \"journal\": \"Human cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and K48 ubiquitination assay; single lab, moderate evidence\",\n      \"pmids\": [\"35174471\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 interacts with YAP protein and promotes K48-linked poly-ubiquitination of YAP at lysines K76, K204, and K321, leading to YAP degradation and suppression of Hippo/YAP signaling in triple-negative breast cancer.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitin-based IP assay with K48-specific linkage detection, protein stability assays, in vitro and in vivo overexpression/depletion, RNA sequencing, luciferase reporter\",\n      \"journal\": \"Cell communication and signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP + site-specific ubiquitination mapping + stability assay + RNA-seq; single lab but multiple methods\",\n      \"pmids\": [\"36280829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 interacts with RNF31 (HOIP), a partner subunit of LUBAC, and stabilizes RNF31 by repressing its ubiquitination and proteasomal degradation in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown, in vivo xenograft\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP and ubiquitination assay demonstrating stabilization; single lab\",\n      \"pmids\": [\"35869046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 promotes PPARγ ubiquitination and degradation, thereby disrupting the PPARγ/PGC1α complex and enhancing WNT/β-catenin/GLUT1-mediated aerobic glycolysis in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, overexpression/knockdown, WNT/β-catenin reporter, GLUT1 expression analysis\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination assay, pathway reporter; single lab\",\n      \"pmids\": [\"35411229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"RBCK1 interacts with and polyubiquitylates mitofusin 2 (MFN2) to promote its proteasomal degradation under ferroptotic stress, leading to decreased mitochondrial ROS and lipid peroxidation and conferring ferroptosis resistance in pancreatic cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, RBCK1 genetic depletion, ferroptosis induction with IKE, mitochondrial ROS measurement, xenograft model\",\n      \"journal\": \"Free radical biology & medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination assay, defined molecular mechanism with functional readout (ROS, lipid peroxidation), in vivo validation\",\n      \"pmids\": [\"38763208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RBCK1 promotes degradation of PICK1 via ubiquitination under hypoxia; HIF-1α transcriptionally regulates RBCK1 to mediate this effect, and RBCK1 knockdown inhibits nasopharyngeal carcinoma cell proliferation that is rescued by co-knockdown of PICK1.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, ubiquitination/protein stability assays, siRNA knockdown with genetic rescue, xenograft model\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP, ubiquitination assay, genetic rescue; single lab\",\n      \"pmids\": [\"36934971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"RBCK1 promotes degradation of ANKRD35 via ubiquitination; ANKRD35 destabilizes MITD1 by binding SUMO2, and the resulting RBCK1-ANKRD35-MITD1-ANXA1 axis regulates AKT and ERK phosphorylation and sunitinib resistance in renal cell carcinoma.\",\n      \"method\": \"Bioinformatic analysis, in vitro/in vivo RBCK1 knockdown, Co-immunoprecipitation, pathway phosphorylation analysis (AKT, ERK)\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 — Co-IP and pathway analysis; mechanistic chain partially supported; single lab\",\n      \"pmids\": [\"36732658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"RBCK1 regulates HIF1α signaling in ER-positive breast cancer; RBCK1 depletion affects HIF1α pathway activity, identifying RBCK1 as a regulator of HIF1α in this cancer context.\",\n      \"method\": \"siRNA knockdown, western blotting for HIF1α pathway components, cell proliferation assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — KD with pathway readout but no direct biochemical interaction or substrate ubiquitination demonstrated; single lab, limited mechanistic detail\",\n      \"pmids\": [\"36473847\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"RBCK1 (HOIL-1L) is an RBR-type E3 ubiquitin ligase that, as a component of the LUBAC complex, generates linear ubiquitin chains and also directly ubiquitinates multiple substrates (including IRF3, TAB2/3, p53, PTEN, YAP, PPARγ, PXR, MFN2, and PICK1) for proteasomal degradation; its activity is regulated by interaction with its RING-lacking splice variant RBCK2 (which sequesters it in the cytoplasm and inhibits its E3 activity) and by PKCβ phosphorylation, while in the nucleus RBCK1 acts as a transcriptional activator requiring both RING-finger and B-Box motifs, regulated by CBP and PML interactions, collectively positioning RBCK1 as a multi-functional ubiquitin ligase controlling NF-κB signaling, antiviral responses, glycogen metabolism, and cell cycle progression.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"RBCK1 (HOIL-1L) is an RBR-type E3 ubiquitin ligase that functions both as a catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC) and as an autonomous K48-linked ubiquitin ligase targeting diverse substrates for proteasomal degradation. As a LUBAC component, RBCK1 generates linear ubiquitin chains essential for NF-κB signaling and glycogen metabolism, and its deficiency causes polyglucosan body accumulation recapitulating Lafora disease pathology [PMID:35084461]. Acting independently, RBCK1 catalyzes K48-linked ubiquitination and degradation of substrates including IRF3, TAB2/TAB3, p53, PTEN, YAP, PPARγ, PXR, MFN2, and PICK1, thereby regulating antiviral innate immunity, NF-κB activation, cell cycle progression, Hippo signaling, metabolic reprogramming, and ferroptosis resistance [PMID:18711448, PMID:17449468, PMID:30874541, PMID:36280829, PMID:38763208]. RBCK1 shuttles between cytoplasm and nucleus via defined NES and NLS sequences; in the nucleus it acts as a transcriptional co-activator at ER-responsive and other promoters, with its activity modulated by CBP, PML, PKCβ phosphorylation, and sequestration by its RING-lacking splice variant RBCK2 [PMID:15833741, PMID:18303026, PMID:20103625].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing that RBCK1 possesses intrinsic transcriptional activation capacity dependent on its RING-finger and B-Box motifs, and that an alternative splice variant RBCK2 acts as a dominant-negative inhibitor through hetero-oligomerization, defined RBCK1 as a regulated transcription factor before its E3 ligase activity was recognized.\",\n      \"evidence\": \"GAL4 chimeric reporter system with domain deletions, in vitro interaction assays of RBCK1/RBCK2 homo- and hetero-oligomers\",\n      \"pmids\": [\"9642138\", \"9755849\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional targets not identified\", \"Physiological relevance of RBCK2 inhibition not validated in vivo\", \"Endogenous stoichiometry of RBCK1:RBCK2 unknown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Mapping a nuclear export signal (Leu-142/145) and a RING-IBR-dependent nuclear localization signal resolved how RBCK1 shuttles between cytoplasm and nucleus, and identifying CBP and PML as nuclear interaction partners explained how its transcriptional activity is enhanced and repressed, respectively.\",\n      \"evidence\": \"Leptomycin B treatment, NES/NLS mutagenesis, nuclear body co-localization, Co-IP with CBP/PML, transcriptional reporters; RBCK2 co-expression relocalization assays\",\n      \"pmids\": [\"15833741\", \"16083853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chromatin targets of RBCK1 nuclear activity not identified at this stage\", \"Structural basis of RBCK2-mediated cytoplasmic tethering unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Discovery that RBCK1 physically associates with PKCβI in cardiac myocytes and is required for phenylephrine-induced hypertrophy connected RBCK1 to signal-dependent cardiac remodeling and identified PKCβ as both a binding partner and a functional regulator.\",\n      \"evidence\": \"Co-IP in neonatal myocytes, PKCβ-selective antagonists, adenoviral overexpression, RNAi with cell size measurement\",\n      \"pmids\": [\"17121852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether RBCK1 ubiquitinates PKCβI or acts as a scaffold not resolved\", \"In vivo cardiac phenotype not established\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating that RBCK1 targets TAB2 and TAB3 adaptors for proteasome-dependent degradation established RBCK1 as a negative feedback regulator of TNF/IL-1-triggered NF-κB signaling, the first clear link between RBCK1 E3 activity and a major inflammatory pathway.\",\n      \"evidence\": \"Reciprocal Co-IP, proteasome inhibitor rescue, RNAi knockdown, NF-κB reporter assays\",\n      \"pmids\": [\"17449468\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain linkage type on TAB2/3 not specified\", \"In vivo role in inflammation not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Biochemical reconstitution of RBCK1 E3 ligase activity in vitro, together with identification of IRF3 as a K48-ubiquitination substrate degraded during viral infection, established RBCK1 as a bona fide ubiquitin ligase with dual roles in NF-κB and antiviral innate immunity, while revealing regulation by PKCβ phosphorylation and RBCK2 inhibition.\",\n      \"evidence\": \"In vitro self-ubiquitination assay, PKCβ phosphorylation, RBCK2 co-expression inhibition, IRF3 ubiquitination/degradation upon viral infection, plaque assays\",\n      \"pmids\": [\"18303026\", \"18711448\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"PKCβ phosphorylation sites on RBCK1 not mapped\", \"Relative contribution of K48-linked versus linear ubiquitin ligase activities not delineated\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"ChIP-based demonstration that RBCK1 is recruited to the ERα promoter to drive ERα and cyclin B1 transcription, with depletion causing G2-M arrest, established a direct nuclear transcriptional role in breast cancer cell proliferation; concurrently, interaction with Eya1 and a zebrafish loss-of-function phenotype mimicking BOR syndrome expanded RBCK1 function to developmental transcription.\",\n      \"evidence\": \"ChIP at ERα promoter, siRNA/flow cytometry in breast cancer cells; GST pulldown/Co-IP with Eya1, zebrafish morpholino with BOR-like phenotype\",\n      \"pmids\": [\"20103625\", \"20956555\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether transcriptional roles are ubiquitin-dependent or -independent unclear\", \"Mammalian developmental phenotype not established\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identification of PXR as an RBCK1 ubiquitination substrate in primary human hepatocytes extended the substrate repertoire to nuclear receptors controlling drug metabolism.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, domain mapping, ubiquitination assay, MG-132 rescue, siRNA in primary hepatocytes\",\n      \"pmids\": [\"23160820\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain linkage type on PXR not determined\", \"In vivo pharmacokinetic consequences not tested\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showing that RBCK1 directly ubiquitinates p53 for proteasomal degradation, with genetic rescue by p53 knockdown, established RBCK1 as an alternative p53 E3 ligase promoting renal cell carcinoma proliferation.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, siRNA with p53 co-knockdown rescue, xenograft\",\n      \"pmids\": [\"30874541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain type on p53 not specified\", \"Relationship to MDM2-mediated p53 degradation not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Discovering that MTSS1 recruits RBCK1 to catalyze K48-linked ubiquitination of NF-κB p65, and that ACTBL2 competes for MTSS1 binding to stabilize p65, revealed a cofactor-dependent mechanism by which RBCK1 directly degrades a key NF-κB transcription factor.\",\n      \"evidence\": \"Reciprocal Co-IP, K48-specific ubiquitination assay, Mtss1 KO mice, competition binding, organoid and xenograft models\",\n      \"pmids\": [\"35122005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of MTSS1-mediated RBCK1 recruitment unknown\", \"Whether this mechanism operates in non-cancer contexts not tested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Multiple studies in 2022 greatly expanded the RBCK1 substrate repertoire—identifying PTEN, YAP, PPARγ, and PICK1 as K48-ubiquitination targets—while RBCK1 deficiency in mice was shown to cause glycogen hyperphosphorylation and polyglucosan body formation mimicking Lafora disease, revealing an unexpected LUBAC-dependent role in glycogen quality control.\",\n      \"evidence\": \"RBCK1-KO mouse glycogen analysis, neuroinflammation/behavioral tests, genetic rescue (glycogen synthase); Co-IP and K48-ubiquitination assays for PTEN, YAP, PPARγ, PICK1 in cancer cell models; RNF31 stabilization by RBCK1 in HCC\",\n      \"pmids\": [\"35084461\", \"35174471\", \"36280829\", \"35411229\", \"35869046\", \"36934971\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism linking LUBAC linear ubiquitination to glycogen metabolism not resolved\", \"Whether substrate selectivity between K48 and linear activities is context-dependent remains unclear\", \"In vivo validation of most cancer-context substrates lacking\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identification of MFN2 as an RBCK1 ubiquitination substrate whose degradation reduces mitochondrial ROS and lipid peroxidation under ferroptotic stress connected RBCK1 to ferroptosis resistance in pancreatic cancer.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, RBCK1 depletion, IKE-induced ferroptosis, mitochondrial ROS measurement, xenograft\",\n      \"pmids\": [\"38763208\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ubiquitin chain linkage type on MFN2 not specified\", \"Whether RBCK1-MFN2 axis operates outside pancreatic cancer unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular logic determining whether RBCK1 acts via linear (LUBAC-dependent) versus K48-linked (autonomous) ubiquitination on specific substrates remains unresolved, as does the structural basis for its remarkably broad substrate recognition.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of RBCK1 in complex with any substrate\", \"Switch mechanism between LUBAC-linear and autonomous-K48 activity unknown\", \"Comprehensive in vivo substrate mapping not performed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 2, 10, 13, 14, 16, 17, 19, 20, 21, 22]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 2, 15]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [3, 5, 8, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 5, 8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0168256\", \"supporting_discovery_ids\": [0, 1, 12, 14, 15]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [1, 7, 12, 14, 17]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 10, 13, 16, 20]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [3, 8, 9]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [20]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [15, 19]}\n    ],\n    \"complexes\": [\n      \"LUBAC\"\n    ],\n    \"partners\": [\n      \"RNF31\",\n      \"PKCβ\",\n      \"MTSS1\",\n      \"CBP\",\n      \"PML\",\n      \"EYA1\",\n      \"FKBPL\",\n      \"TAB2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}