{"gene":"SPOP","run_date":"2026-06-10T07:46:40","timeline":{"discoveries":[{"year":2017,"finding":"SPOP functions as a substrate adaptor for the CUL3-RING E3 ubiquitin ligase to ubiquitinate and target PD-L1 for proteasome-mediated degradation. Cyclin D-CDK4 phosphorylates SPOP, promoting SPOP's own degradation by the APC/C activator FZR1, thereby stabilizing PD-L1. Loss-of-function SPOP mutations compromise ubiquitination-mediated PD-L1 degradation.","method":"Co-immunoprecipitation, in vivo ubiquitination assays, pharmacological CDK4/6 inhibition, mouse tumor models, phosphorylation mapping","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo ubiquitination, multiple orthogonal methods, replicated in mouse models and primary human specimens","pmids":["29160310"],"is_preprint":false},{"year":2017,"finding":"CUL3-SPOP E3 ligase complex ubiquitinates BET proteins (BRD2, BRD3, BRD4) for proteasomal degradation. Prostate cancer-associated SPOP mutants fail to interact with BET proteins, leading to their accumulation and resistance to BET inhibitors.","method":"Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, prostate cancer organoids, Western blot of patient specimens","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus ubiquitination assays, independently replicated in two concurrent papers (PMIDs 28805820 and 28805822)","pmids":["28805820","28805822"],"is_preprint":false},{"year":2017,"finding":"Wild-type SPOP recognizes a degron motif common among BET proteins and promotes their proteasomal degradation; prostate cancer SPOP mutants show impaired binding to BET proteins. BRD4 stabilization activates RAC1 and AKT-mTORC1 signaling as a consequence.","method":"Degron binding assay, ubiquitination assay, transcriptome and BRD4 cistrome analyses, prostate cancer cell lines and patient specimens","journal":"Nature medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — degron mapping, ubiquitination assays, transcriptome analysis, replicated across two concurrent labs","pmids":["28805822"],"is_preprint":false},{"year":2014,"finding":"SPOP recognizes a Ser/Thr-rich degron in the hinge domain of full-length androgen receptor (AR) and induces its ubiquitination and proteasomal degradation, inhibiting AR-mediated transcription and prostate cancer cell growth. AR splice variants lacking the hinge domain escape SPOP-mediated degradation. Prostate cancer SPOP mutants cannot bind or promote AR destruction. Androgens antagonize SPOP-mediated AR degradation, whereas antiandrogens promote it.","method":"Co-immunoprecipitation, ubiquitination assays, degron mapping, siRNA knockdown, cell growth assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, degron mapping, ubiquitination, functional cell assays), single rigorous study","pmids":["24508459"],"is_preprint":false},{"year":2013,"finding":"SPOP directly interacts with SRC-3 (NCOA3/AIB1) and promotes its CUL3-dependent ubiquitination and proteolysis, suppressing androgen receptor transcriptional activity. Prostate cancer-associated SPOP mutants cannot interact with SRC-3 or promote its ubiquitination and degradation.","method":"Co-immunoprecipitation, ubiquitination assays, proteasome inhibition, prostate cancer cell lines","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP and ubiquitination assays with mutant characterization, single rigorous study with multiple orthogonal methods","pmids":["23559371"],"is_preprint":false},{"year":2015,"finding":"SPOP recognizes ERG via the CUL3 ubiquitin ligase system and promotes ERG ubiquitination and proteasomal degradation. The SPOP/ERG interaction is modulated by CKI-mediated phosphorylation. Truncated ERG (ΔERG) encoded by the fusion gene evades SPOP-mediated destruction. Prostate cancer SPOP mutants are deficient in promoting ERG ubiquitination. DNA damage drugs (topoisomerase inhibitors) trigger CKI activation to restore SPOP/ΔERG interaction.","method":"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, drug treatment experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination, phosphorylation, drug treatment), single rigorous study","pmids":["26344095"],"is_preprint":false},{"year":2016,"finding":"SPOP self-associates into higher-order oligomers through BTB-mediated dimerization forming linear oligomers via BACK domain dimerization. Higher-order SPOP oligomerization is required for localization to liquid nuclear speckles; self-association-deficient SPOP mutants show diffuse nuclear distribution. Higher-order oligomerization stimulates CRL3(SPOP) ubiquitination efficiency for substrate Gli3, indicating nuclear speckles are hotspots of ubiquitination.","method":"Analytical ultracentrifugation, biophysical sizing, live-cell imaging, in vitro ubiquitination assays, domain mapping with mutants","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — biophysical reconstitution, in vitro ubiquitination assay, live imaging, multiple orthogonal methods in one study","pmids":["27220849"],"is_preprint":false},{"year":2023,"finding":"Cryo-EM/X-ray crystal structure of SPOP in its oligomeric form reveals new interfaces important for SPOP self-assembly. Many endometrial cancer mutations localize to newly identified oligomeric interfaces, providing the structural basis for their gain-of-function or altered-assembly cancer-causing properties.","method":"X-ray crystallography (oligomeric structure determination), mutagenesis, biochemical characterization of cancer mutants","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure of oligomeric SPOP with mutational validation, single rigorous study","pmids":["36693379"],"is_preprint":false},{"year":2021,"finding":"ATM kinase phosphorylates SPOP in response to DNA damage, causing a conformational change (revealed by X-ray crystal structures) that stabilizes SPOP's interaction with 53BP1. SPOP then induces polyubiquitination of 53BP1, leading to 53BP1 extraction from chromatin by a p97/VCP segregase complex, promoting homologous recombination over NHEJ during S phase. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects.","method":"X-ray crystallography, in vivo and in vitro ubiquitination assays, Co-immunoprecipitation, phosphorylation mapping, cell cycle analysis","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 1 / Strong — X-ray crystal structure plus multiple functional biochemical assays (Co-IP, ubiquitination, phosphorylation mapping), single rigorous study with orthogonal methods","pmids":["34144977"],"is_preprint":false},{"year":2015,"finding":"SPOP mutations are associated with genomic instability in prostate cancer. SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation, impairing homology-directed repair (HDR) of DNA double-strand breaks. SPOP mutation sensitizes cells to PARP inhibitors.","method":"Epistasis/transcriptional profiling, DNA repair functional assays, in vivo mouse models, PARP inhibitor sensitivity assay","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple assays in mouse models and cell lines, single lab study with functional DNA repair readouts","pmids":["26374986"],"is_preprint":false},{"year":2021,"finding":"SPOP promotes K27-linked non-degradative polyubiquitination of Geminin at K100 and K127, preventing DNA replication over-firing by blocking Cdt1 association with the MCM complex. Cancer-associated SPOP mutations impair Geminin K27-linked polyubiquitination and induce replication origin over-firing and re-replication.","method":"Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, ubiquitin chain linkage analysis, DNA replication assays, cancer mutant analysis","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reconstitution of non-degradative ubiquitination plus mechanistic dissection of Cdt1-MCM interaction, multiple orthogonal methods","pmids":["34599168"],"is_preprint":false},{"year":2016,"finding":"SPOP is critically involved in SETD2 protein stability control; the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro. Modulation of SPOP expression confers differential H3K36me3 levels on SETD2 target genes and induces H3K36me3-coupled alternative splicing events.","method":"Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, ChIP-Seq, RNA splicing analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro ubiquitination reconstitution plus ChIP-Seq and splicing analysis, multiple orthogonal methods","pmids":["27614073"],"is_preprint":false},{"year":2017,"finding":"SPOP recognizes INF2 via a Ser/Thr-rich motif in its C-terminal region and triggers atypical (non-degradative) polyubiquitination. This modification reduces INF2 localization in the ER and mitochondrially associated DRP1 puncta, impairing INF2-mediated mitochondrial fission. Prostate cancer SPOP mutants increase INF2 ER localization and promote mitochondrial fission via dominant-negative effect.","method":"Co-immunoprecipitation, ubiquitination assays, immunofluorescence, mitochondrial fission assays, dominant-negative mutant analysis","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination, localization imaging, functional fission assays), single rigorous study","pmids":["28448495"],"is_preprint":false},{"year":2018,"finding":"SPOP physically interacts with Nanog via a conserved degron motif and promotes Nanog poly-ubiquitination and subsequent proteasomal degradation. Cancer-derived mutations in SPOP or at the Nanog degron (S68Y) disrupt this destruction. Pin1 oncoprotein acts as an upstream regulator that impairs Nanog recognition by SPOP, stabilizing Nanog.","method":"Co-immunoprecipitation, ubiquitination assays, degron mutagenesis, cancer mutant analysis, Pin1 interaction studies","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, degron mutagenesis, ubiquitination assays, multiple orthogonal methods; independently replicated in a concurrent paper (PMID 30595535)","pmids":["30595538","30595535"],"is_preprint":false},{"year":2018,"finding":"AMPK-BRAF signaling controls SPOP-mediated NANOG degradation through phosphorylation of NANOG at Ser68, which blocks the SPOP-NANOG interaction. Cancer-associated mutations of SPOP or the NANOG S68Y mutation abrogates SPOP-mediated degradation.","method":"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, kinase assays","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — kinase phosphorylation mapping plus Co-IP and ubiquitination, single lab study","pmids":["30595535"],"is_preprint":false},{"year":2021,"finding":"SPOP promotes ubiquitination and degradation of PDK1 in a CK1/GSK3β-mediated phosphorylation-dependent manner, by directly binding a degron in PDK1. This suppresses AKT kinase activity. Prostate cancer SPOP mutations impair PDK1 degradation and activate AKT.","method":"CRISPR-based E3 ligase screen, Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, in vitro kinase assays, mass spectrometry, xenograft models","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — CRISPR screen plus reconstituted in vitro ubiquitination plus kinase assays and mass spectrometry, multiple orthogonal methods","pmids":["34353330"],"is_preprint":false},{"year":2017,"finding":"SPOP physically interacts with c-MYC protein and promotes c-MYC ubiquitination and degradation. PC-associated SPOP mutants or SPOP knockdown increase c-MYC protein levels. Prostate-specific biallelic Spop knockout mice develop prostatic intraepithelial neoplasia with elevated c-MYC.","method":"Co-immunoprecipitation, ubiquitination assays, prostate-specific knockout mouse model, gene expression analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ubiquitination assays, and in vivo knockout model with phenotypic readout, multiple orthogonal methods","pmids":["28414305"],"is_preprint":false},{"year":2015,"finding":"SPOP recognizes multiple Ser/Thr-rich degrons in the AF2 domain of estrogen receptor-α (ERα) and triggers ERα ubiquitin-proteasome pathway degradation as part of the SPOP-CUL3-RBX1 E3 complex. SPOP participates in estrogen-induced ERα degradation. Endometrial cancer SPOP mutants are defective in promoting ERα ubiquitination and degradation.","method":"Co-immunoprecipitation, in vivo and in vitro ubiquitination assays, degron mapping, siRNA knockdown, endometrial cancer mutant analysis","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination, degron mapping, cancer mutant analysis), single rigorous study","pmids":["25766326"],"is_preprint":false},{"year":2022,"finding":"Cytoplasmic SPOP binds p62/SQSTM1 and induces non-degradative ubiquitination of p62 at residue K420 within the UBA domain. This modification decreases p62 puncta formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, suppressing p62-dependent autophagy. SPOP also relieves p62-mediated Keap1 sequestration, reducing Nrf2-mediated antioxidant transcription. Prostate cancer SPOP mutants fail to ubiquitinate p62 and promote autophagy and Nrf2 activation in a dominant-negative manner.","method":"Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, site-specific mutagenesis, autophagy assays, liquid-liquid phase separation experiments","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — non-degradative ubiquitination mapped to specific residue (K420), liquid-liquid phase separation assays, multiple orthogonal functional readouts","pmids":["34987184"],"is_preprint":false},{"year":2021,"finding":"SPOP binds and promotes polyubiquitination and proteasomal degradation of the histone methyltransferase GLP, and by extension its partner G9a. SPOP mutation induces GLP/G9a stabilization, aberrant global DNA hypermethylation in prostate cancer cells, and silencing of tumor suppressor genes (FOXO3, GATA5, NDRG1).","method":"Co-immunoprecipitation, ubiquitination assays, genome-wide DNA methylome analysis, gene expression analysis, cancer mutant validation","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, ubiquitination assays, genome-wide methylome analysis, multiple orthogonal methods","pmids":["34588438"],"is_preprint":false},{"year":2018,"finding":"TRIM28 interacts with TRIM24 (a known SPOP substrate) to prevent its ubiquitination and degradation by SPOP, acting as a competitive upstream regulator. TRIM28 facilitates TRIM24 chromatin occupancy and augments AR signaling.","method":"Co-immunoprecipitation, ubiquitination assays, chromatin immunoprecipitation, xenograft studies","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays plus ChIP, single lab study","pmids":["30479348"],"is_preprint":false},{"year":2019,"finding":"SPOP recognizes and triggers ubiquitin-dependent proteasomal degradation of Caprin1 (a stress granule nucleating protein). Prostate cancer SPOP mutants fail to degrade Caprin1, leading to elevated Caprin1 and enhanced stress granule assembly, conferring resistance to docetaxel and other stress inducers.","method":"Yeast two-hybrid screen, Co-immunoprecipitation, ubiquitination assays, stress granule assays, xenograft models, patient specimen analysis","journal":"Molecular cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus Co-IP and ubiquitination assays, single lab study","pmids":["31771591"],"is_preprint":false},{"year":2016,"finding":"In mouse models, Spop directly targets the Gli3 repressor for ubiquitination and degradation, positively regulating Indian Hedgehog (Ihh) signaling and skeletal development. Loss of Spop results in upregulation of full-length and repressor forms of Gli3, down-regulation of Ihh target genes (Ptch1, Pthlh), and skeletal defects rescued by reducing Gli3 dosage.","method":"Spop null mouse model, conditional mouse model, genetic epistasis (reducing Gli3 dosage), in vivo ubiquitination assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic epistasis with Gli3 dosage rescue, ubiquitination assays, multiple mouse models","pmids":["27930311"],"is_preprint":false},{"year":2014,"finding":"SPOP is recruited to DNA double-strand break sites, forms nuclear foci co-localizing with γ-H2AX foci, and interacts with ATM kinase in response to DNA damage. SPOP recruitment is predominantly dependent on ATM kinase activity. SPOP knockdown results in impaired DNA damage response and hypersensitivity to ionizing irradiation.","method":"Immunofluorescence (foci formation), Co-immunoprecipitation, siRNA knockdown, ionizing radiation sensitivity assays","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and localization imaging plus functional KD phenotype, single lab study","pmids":["24451148"],"is_preprint":false},{"year":2022,"finding":"SPOP negatively regulates PD-L1 expression at the transcriptional level by binding to IRF1 (a transcription factor for PD-L1) and triggering its ubiquitin-proteasomal degradation. Endometrial cancer SPOP mutants lose capacity to degrade IRF1, instead stabilizing it and upregulating PD-L1 expression.","method":"Co-immunoprecipitation, ubiquitination assays, luciferase reporter assays, xenograft tumor models","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, reporter assay, and in vivo model, single lab study","pmids":["36481790"],"is_preprint":false},{"year":2021,"finding":"SPOP promotes ATF2 ubiquitination and degradation via recognition of multiple Ser/Thr-rich degrons in ATF2 by the SPOP-CUL3-RBX1 complex. Prostate cancer SPOP mutants are defective in promoting ATF2 degradation.","method":"Yeast two-hybrid screen, Co-immunoprecipitation, ubiquitination assays, degron mapping, Transwell migration and invasion assays","journal":"Journal of experimental & clinical cancer research : CR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus Co-IP and ubiquitination assays, single lab study","pmids":["29996942"],"is_preprint":false},{"year":2016,"finding":"SPOP interacts with Cdc20 and promotes its poly-ubiquitination and subsequent proteasomal degradation in a degron-dependent manner via CUL3. Prostate cancer-derived SPOP mutants fail to interact with Cdc20 to promote its degradation.","method":"Co-immunoprecipitation, ubiquitination assays, pharmacological CUL inhibition (MLN4924), half-life assays","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays plus pharmacological validation, single lab study","pmids":["27780719"],"is_preprint":false},{"year":2018,"finding":"SPOP directly interacts with CYCLIN E1 and regulates its stability through poly-ubiquitination by the SPOP/CUL3/RBX1 complex. CDK2 competes with SPOP for CYCLIN E1 interaction, suggesting SPOP preferentially targets CDK2-free CYCLIN E1. Loss of CYCLIN E1 regulation by SPOP contributes to prostate cancer cell proliferation.","method":"Co-immunoprecipitation, ubiquitination assays, competition binding assays, prostate cancer cell lines","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ubiquitination and competition assays, single lab study","pmids":["30237511"],"is_preprint":false},{"year":2014,"finding":"SPOP recognizes a Ser/Thr-rich degron in the transactivation domain of DDIT3/CHOP and triggers its degradation via the ubiquitin-proteasome pathway through the SPOP-CUL3-RBX1 complex. Prostate cancer SPOP mutants are defective in promoting DDIT3 degradation.","method":"Co-immunoprecipitation, ubiquitination assays, degron mapping, prostate cancer mutant analysis","journal":"Human mutation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, degron mapping, and ubiquitination assays, single lab study","pmids":["24990631"],"is_preprint":false},{"year":2021,"finding":"SPOP is phosphorylated at Ser119 by ATM kinase upon DNA damage, enhancing SPOP binding to HIPK2. SPOP induces non-degradative ubiquitination of HIPK2, which increases HIPK2 phosphorylation of HP1γ and promotes HP1γ dissociation from H3K9me3 to initiate DNA damage repair. Prostate cancer SPOP mutations abrogate this SPOP-HIPK2 axis.","method":"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, DNA damage repair assays, cancer mutant analysis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple Co-IP and phosphorylation assays with functional DNA repair readout, single lab study","pmids":["34133717"],"is_preprint":false},{"year":2019,"finding":"ILF3 is a substrate of SPOP; EGF-MEK-ERK pathway-mediated phosphorylation of ILF3 hinders SPOP-mediated poly-ubiquitination and degradation of ILF3. ILF3 regulates the SGOC (Serine-Glycine-One-Carbon) metabolic pathway by regulating SGOC gene mRNA stability.","method":"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, patient-derived xenografts","journal":"Cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, phosphorylation mapping in a single lab study","pmids":["31772275"],"is_preprint":false},{"year":2022,"finding":"CUL3-SPOP E3 ligase promotes ASCT2/SLC1A5 (glutamine transporter) ubiquitylation and degradation, controlling glutamine uptake. MLN4924 (neddylation inhibitor) inactivates CRL3-SPOP, causing ASCT2 accumulation. SPOP itself undergoes auto-ubiquitylation upon glutamine deprivation.","method":"Co-immunoprecipitation, ubiquitination assays, pharmacological neddylation inhibition, siRNA knockdown, breast cancer specimens","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays plus auto-ubiquitination finding, single lab study","pmids":["35641493"],"is_preprint":false},{"year":2021,"finding":"SPOP binds and promotes non-degradative K27- and K29-linked polyubiquitination of 17βHSD4 at a functional SBC motif (315RATST319). This stabilizes 17βHSD4. SGK3-mediated phosphorylation of S318 in the SBC blocks SPOP binding and instead leads to SKP2-dependent K48-linked degradative ubiquitination of 17βHSD4. Prostate cancer SPOP mutations impair SPOP-17βHSD4 interaction, causing 17βHSD4 destruction and increased testosterone production.","method":"Co-immunoprecipitation, in vivo and in vitro ubiquitination assays (linkage-specific), kinase assays, xenograft mouse models, patient specimens","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — non-degradative ubiquitination characterized with linkage specificity, competing E3 ligase system defined, in vivo models, multiple orthogonal methods","pmids":["33762355"],"is_preprint":false},{"year":2016,"finding":"SPOP promotes DAXX ubiquitination and degradation as part of a CUL3-SPOP-DAXX axis in vascular endothelial cells. DAXX degradation by SPOP is required for VEGFR2 mRNA expression. Knockdown of SPOP or CUL3 upregulates DAXX protein and downregulates VEGFR2 levels. Simultaneous knockdown of SPOP and DAXX reverses VEGFR2 downregulation.","method":"siRNA knockdown, Western blotting, epistasis by double-knockdown, endothelial cell functional assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis by double knockdown plus functional assays, single lab study","pmids":["28216678"],"is_preprint":false},{"year":2021,"finding":"SPOP targets and destabilizes STING1 protein. Prostate cancer-associated SPOP mutations result in upregulated non-canonical STING-NF-κB signaling. PARP inhibitor treatment shifts this signaling from immunosuppressive non-canonical STING-NF-κB to antitumor canonical cGAS-STING-IFNβ signaling in SPOP-mutant prostate cancer.","method":"Proteomics analysis, genetically modified cell line models, in vitro and in vivo PARP inhibitor treatment, transcriptomic analysis","journal":"Clinical cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomics plus functional cell line models, single lab study","pmids":["37581614"],"is_preprint":false},{"year":2020,"finding":"SPOP negatively regulates TLR-induced inflammation by disrupting MyD88 self-association. SPOP is recruited to MyD88 following TLR4 activation and translocates from the nucleus to the cytoplasm upon TLR4 activation. SPOP depletion promotes MyD88 aggregation and recruitment of downstream signaling kinases IRAK4, IRAK1, and IRAK2.","method":"Co-immunoprecipitation, SPOP overexpression and knockdown, immunofluorescence (subcellular localization), NF-κB reporter assays","journal":"Cellular & molecular immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus localization imaging and NF-κB reporter assays, single lab study","pmids":["32235916"],"is_preprint":false},{"year":2020,"finding":"SPOP promotes ubiquitination and proteasomal degradation of MyD88 via recognizing the intermediate domain of MyD88. Knockdown or genetic ablation of SPOP leads to aberrant elevation of MyD88 protein. SPOP negatively regulates NF-κB pathway activity and IL-1β production upon LPS challenge. Spop-deficient mice are more susceptible to Salmonella typhimurium infection.","method":"Co-immunoprecipitation, ubiquitination assays, Spop-knockout mice, infection model","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, and in vivo knockout mouse infection model, single lab study","pmids":["32365080"],"is_preprint":false},{"year":2021,"finding":"SPOP promotes ubiquitination and degradation of LATS1 (a Hippo pathway tumor suppressor) via a degron-dependent interaction with CUL3. SPOP overexpression promotes kidney cancer cell proliferation and invasion partly through LATS1 degradation.","method":"Co-immunoprecipitation, ubiquitination assays, protein half-life assays, xenograft models","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays plus in vivo model, single lab study","pmids":["32460168"],"is_preprint":false},{"year":2021,"finding":"SPOP promotes CDCA5 poly-ubiquitination and proteasomal degradation in a degron-dependent manner. Prostate cancer SPOP mutations impair this effect. CDCA5 depletion leads to G2M arrest, sister chromatid cohesion defects, and apoptosis via the AKT pathway.","method":"Co-immunoprecipitation, ubiquitination assays, cell cycle analysis, functional knockdown assays","journal":"Neoplasia (New York, N.Y.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assays, and functional cell cycle readout, single lab study","pmids":["34509929"],"is_preprint":false},{"year":2017,"finding":"SPOP mutation activates both PI3K/mTOR and androgen receptor signaling in prostate cancer in vivo, effectively uncoupling the normal negative feedback between these two pathways, as demonstrated in a conditional mutant SPOP mouse model with Pten loss.","method":"Conditional mouse model (prostate-specific mutant SPOP expression), prostate organoids, human prostate cancer sample analysis, transcriptional profiling","journal":"Cancer cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo mouse genetic model plus organoids and human samples, single lab study","pmids":["28292441"],"is_preprint":false},{"year":2020,"finding":"AURORA A (AURKA) kinase directly phosphorylates SPOP at three sites, causing SPOP ubiquitylation and degradation. Conversely, SPOP degrades AURKA via a feedback loop. SPOP degradation by AURKA stabilizes AR, ARv7, and c-Myc, promoting oncogenic phenotypes. Phospho-resistant SPOP fully abrogates tumorigenesis in vivo.","method":"In vitro kinase assay (direct phosphorylation), ubiquitination assays, Co-immunoprecipitation, xenograft studies","journal":"Cancers","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus ubiquitination and in vivo xenograft, single lab study","pmids":["33158056"],"is_preprint":false},{"year":2020,"finding":"LIMK2 kinase directly phosphorylates SPOP at three sites, causing SPOP degradation. SPOP promotes LIMK2 ubiquitylation, creating a feedback loop. Phospho-resistant SPOP completely suppresses tumorigenesis in vivo.","method":"In vitro kinase assay, ubiquitination assays, Co-immunoprecipitation, xenograft studies","journal":"British journal of cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus ubiquitination and xenograft, single lab study; conceptually replicated in concurrent AURKA paper (PMID 33158056)","pmids":["33311589"],"is_preprint":false},{"year":2021,"finding":"ERG upregulates wild-type SPOP to dampen AR signaling and sustain ERG activity through SPOP-mediated degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG function and enable oncogenic AR signaling. This antagonism makes SPOP-mutant and ERG tumors synthetic sick.","method":"Co-immunoprecipitation, ubiquitination assays, transcriptomic analysis, genetic epistasis in prostate cancer models","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, and transcriptomic analyses in a single study, moderate evidence for ZMYND11 substrate","pmids":["33531470"],"is_preprint":false},{"year":2021,"finding":"G3BP1 interacts with SPOP and functions as a competitive inhibitor of CUL3-SPOP E3 ligase activity, providing a mechanism to inactivate SPOP tumor suppressor function without SPOP mutation in prostate cancer. AR directly upregulates G3BP1 transcription, creating a feed-forward amplification loop.","method":"Co-immunoprecipitation, transcriptomic analysis, functional knockdown assays, AR ChIP analysis","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and transcriptomic analyses plus AR ChIP, single lab study","pmids":["34795264"],"is_preprint":false},{"year":2019,"finding":"SPOP controls fetal hemoglobin (HbF) expression in adult erythroid cells as a CUL3-RING ligase adaptor. SPOP depletion or dominant-negative SPOP overexpression significantly raises fetal globin mRNA and protein levels, independently of BCL11A and LRF HbF repressors.","method":"CRISPR-Cas9 library screen, siRNA knockdown, dominant-negative overexpression, transcriptome and proteome analyses","journal":"Blood advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen plus functional depletion and dominant-negative overexpression, single lab study","pmids":["31126914"],"is_preprint":false},{"year":2021,"finding":"Intrinsically disordered substrates dictate SPOP subnuclear localization. Pdx1, which has only moderate valency (two SPOP-binding motifs), does not phase separate with SPOP and instead prompts SPOP relocalization from nuclear speckles to the diffuse nucleoplasm. SPOP-mediated ubiquitination of Pdx1 occurs in the nucleoplasm, and both SB motifs are required for efficient Pdx1 turnover.","method":"NMR spectroscopy (SB motif identification), live-cell imaging (SPOP relocalization), ubiquitination assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — NMR plus live imaging and ubiquitination assays, single lab study","pmids":["33894201"],"is_preprint":false},{"year":2018,"finding":"SPOP promotes transcriptional expression of DNA repair and replication factors including BRCA2, ATR, CHK1, and RAD51. SPOP knockdown leads to spontaneous replication stress, impaired RAD51 foci formation, impaired CHK1 activation, and compromised recovery from replication fork stalling. SPOP interactome analysis shows wild-type SPOP (but not mutant SPOP) associates with transcription, mRNA splicing and export complexes.","method":"Proteomic interactome analysis, siRNA knockdown, RAD51/CHK1 foci formation, DNA fiber assays, gene expression analysis","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proteomic interactome plus DNA repair functional assays and gene expression, single lab study","pmids":["30124983"],"is_preprint":false},{"year":2021,"finding":"SPOP promotes SIRT2 binding and degradation by the 26S proteasome. SPOP loss or mutations impair SIRT2 degradation in non-small cell lung cancer cells.","method":"Co-immunoprecipitation, proteasome inhibition assays, Western blot in cancer cell lines","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP plus proteasome inhibition in cell lines, single lab study","pmids":["28073696"],"is_preprint":false},{"year":2002,"finding":"SPOP (Spop) physically interacts with macroH2A1.2 histone variant via its MATH domain, which binds the putative leucine zipper domain of macroH2A1.2. This was identified by yeast two-hybrid and confirmed by GST pull-down.","method":"Yeast two-hybrid screen, GST pull-down, domain mapping","journal":"Biochimica et biophysica acta","confidence":"Low","confidence_rationale":"Tier 3 / Weak — yeast two-hybrid and GST pull-down only, no functional validation, single study","pmids":["12183056"],"is_preprint":false},{"year":2023,"finding":"SPOP promotes ubiquitination and degradation of EWS-FLI1 fusion oncoprotein in Ewing sarcoma. Casein kinase 1-mediated phosphorylation of the VTSSS degron in the FLI1 domain enhances SPOP-mediated EWS-FLI1 degradation. OTUD7A deubiquitinase opposes SPOP by deubiquitinating and stabilizing EWS-FLI1.","method":"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, Ewing sarcoma cell line knockdown, xenograft mouse models","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, phosphorylation mapping plus in vivo xenograft, single lab study","pmids":["34060252"],"is_preprint":false},{"year":2019,"finding":"SPOP recognizes and promotes ubiquitination and degradation of ZBTB3 transcription factor via two Ser/Thr-rich degrons by the SPOP-CUL3-RBX1 E3 complex. Endometrial cancer SPOP mutants are defective in regulating ZBTB3 stability. SPOP inactivation promotes endometrial cell proliferation via ZBTB3-dependent SHH upregulation.","method":"Co-immunoprecipitation, ubiquitination assays, degron mapping, luciferase reporter assays, cancer mutant analysis","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, degron mapping, and reporter assays, single lab study","pmids":["31911863"],"is_preprint":false},{"year":2021,"finding":"SPOP promotes ubiquitination and degradation of HMGCS1 (HMG-CoA synthase 1), and CSN6 antagonizes SPOP to stabilize HMGCS1. HMGCS1 stabilization activates YAP1 to promote hepatocellular carcinoma growth.","method":"Co-immunoprecipitation, ubiquitination assays, xenograft and orthotopic liver cancer models","journal":"Advanced science (Weinheim, Baden-Wurttemberg, Germany)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays plus in vivo models, single lab study","pmids":["38308184"],"is_preprint":false},{"year":2020,"finding":"SPOP promotes SPOP-mediated ubiquitination and degradation of PTEN in kidney cancer when SPOP is mislocalized to the cytoplasm. Small molecule inhibitors of the SPOP-substrate protein interaction increase PTEN levels, decrease PTEN ubiquitination, and reduce phosphorylated AKT and ERK in ccRCC cell lines.","method":"Structure-based small molecule design, in vitro binding assays, Co-immunoprecipitation, ubiquitination assays, cell viability assays","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — structure-based inhibitor plus biochemical validation in cell lines, single lab study","pmids":["32297747"],"is_preprint":false},{"year":2020,"finding":"O-GlcNAcylation of SPOP at Ser96 by OGT increases nuclear positioning of SPOP in hepatoma cells, alleviating SPOP-mediated ubiquitination of the Nogo-B/RTN4B oncoprotein and thereby promoting HCC progression. In normal liver cells, cytoplasmic SPOP ubiquitinates Nogo-B via N-terminal SBC motifs. Abolition of O-GlcNAcylation by S96A mutation increased cytoplasmic SPOP localization.","method":"Co-immunoprecipitation, ubiquitination assays, subcellular fractionation, site-specific mutagenesis, in vitro and in vivo cancer models","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — PTM identification and subcellular localization with functional consequence, ubiquitination assays, single lab study","pmids":["36604567"],"is_preprint":false},{"year":2020,"finding":"SPOP was identified as a novel substrate of STAT3 in bladder cancer; SPOP deficiency increases STAT3 protein stability, leading to elevated CCL2 secretion and macrophage M2 polarization. VEZF1 directly activates SPOP transcription, and its overexpression suppresses STAT3/CCL2/IL-6 axis effects.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, luciferase reporter, ChIP assay, co-culture system","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assays plus ChIP and reporter assays, single lab study","pmids":["39479456"],"is_preprint":false}],"current_model":"SPOP functions as the substrate-binding adaptor subunit of the CUL3-RBX1 RING E3 ubiquitin ligase complex, recognizing substrates through Ser/Thr-rich degron motifs via its MATH domain, and mediating both degradative (K48-linked) and non-degradative (K27/K29/K63-linked) polyubiquitination of a diverse array of substrates—including AR, BET proteins (BRD2/3/4), ERG, SRC-3, PD-L1, Nanog, c-MYC, Gli2/3, 53BP1, GLP, Geminin, SETD2, CYCLIN E1, and many others—to regulate androgen/nuclear receptor signaling, DNA damage response and replication fidelity, Hedgehog signaling, immune checkpoints, cellular metabolism, and mitochondrial dynamics; SPOP self-assembles into higher-order oligomers through BTB and BACK domain interactions to localize to liquid nuclear speckles where ubiquitination efficiency is enhanced, and is itself regulated by upstream kinases (ATM, CDK4, AURKA, LIMK2) and post-translational modifications (phosphorylation, O-GlcNAcylation), while cancer-associated missense mutations clustered in the MATH domain impair substrate binding and broadly dysregulate these proteostatic functions."},"narrative":{"mechanistic_narrative":"SPOP is the substrate-recognition adaptor of a CUL3-RBX1 RING E3 ubiquitin ligase that selects substrates bearing Ser/Thr-rich degron motifs through its MATH domain, thereby controlling the abundance and activity of a broad array of nuclear regulators [PMID:24508459, PMID:23559371, PMID:25766326]. Through its BTB and BACK domains SPOP self-assembles into higher-order oligomers that concentrate the ligase in liquid nuclear speckles, where ubiquitination of substrates such as Gli3 is most efficient; substrate valency itself can redistribute SPOP between speckles and the diffuse nucleoplasm [PMID:27220849, PMID:33894201], and crystallographic analysis of the oligomeric form maps cancer mutations to self-assembly interfaces [PMID:36693379]. A dominant theme is the proteasomal destruction of transcriptional and signaling regulators—androgen receptor and its coactivator SRC-3, the ETS factor ERG, BET bromodomain proteins BRD2/3/4, c-MYC, ERα, the pluripotency factor Nanog, and the histone methyltransferases SETD2 and GLP—linking SPOP to nuclear receptor signaling, chromatin state, and proliferative control [PMID:28805820, PMID:28805822, PMID:24508459, PMID:23559371, PMID:26344095, PMID:27614073, PMID:30595538, PMID:30595535, PMID:28414305, PMID:34588438]. Beyond canonical K48-linked degradation, SPOP also deposits non-degradative K27/K29-linked chains: it ubiquitinates Geminin to restrain replication origin re-firing, modifies INF2 to suppress mitochondrial fission, marks p62/SQSTM1 to inhibit autophagy and Nrf2 signaling, and stabilizes 17βHSD4 to limit androgen synthesis [PMID:34599168, PMID:28448495, PMID:34987184, PMID:33762355]. In the DNA damage response, ATM-mediated phosphorylation of SPOP triggers a conformational switch that drives 53BP1 ubiquitination and chromatin extraction to favor homologous recombination, and SPOP loss produces a BRCA-like repair defect that sensitizes cells to PARP inhibition [PMID:34144977, PMID:26374986, PMID:34133717]. SPOP activity is itself governed by upstream kinases and modifications—CDK4, AURKA, and LIMK2 phosphorylation drive SPOP turnover, while O-GlcNAcylation tunes its nuclear/cytoplasmic distribution—and cancer-associated MATH-domain mutations recurrent in prostate and endometrial tumors abolish substrate binding, broadly dysregulating these proteostatic programs [PMID:29160310, PMID:33158056, PMID:33311589, PMID:36604567].","teleology":[{"year":2002,"claim":"Before its E3 role was known, the question was what SPOP binds; the MATH domain was shown to engage a histone variant, hinting at a nuclear/chromatin-associated function.","evidence":"Yeast two-hybrid and GST pull-down mapping MATH-domain binding to macroH2A1.2","pmids":["12183056"],"confidence":"Low","gaps":["Interaction not confirmed in cells and no functional consequence established","No link to ubiquitination demonstrated"]},{"year":2013,"claim":"Establishing SPOP as a CUL3 substrate adaptor in cancer, SRC-3 was identified as a degradation target whose loss explains SPOP's suppression of androgen receptor activity.","evidence":"Co-IP and CUL3-dependent ubiquitination assays with prostate cancer mutant characterization","pmids":["23559371"],"confidence":"High","gaps":["Degron not precisely mapped here","Did not address direct AR turnover"]},{"year":2014,"claim":"The defining recognition principle—a Ser/Thr-rich degron read by SPOP—was demonstrated by direct degradation of full-length AR and DDIT3/CHOP, with splice variants and cancer mutants escaping turnover.","evidence":"Degron mapping, Co-IP, ubiquitination assays, and growth assays in prostate cancer cells","pmids":["24508459","24990631"],"confidence":"High","gaps":["Did not resolve which ubiquitin linkage types are used","In vivo physiological relevance not yet tested"]},{"year":2015,"claim":"SPOP was tied to genome integrity and to phospho-regulated substrate selection, showing ERG recognition is gated by CKI phosphorylation and that SPOP mutation produces a BRCA1-like HDR defect.","evidence":"Phosphorylation mapping and ubiquitination assays (ERG); transcriptional profiling, DNA repair assays, and PARP inhibitor sensitivity in mouse models","pmids":["26344095","26374986"],"confidence":"Medium","gaps":["The direct repair substrate underlying the HDR defect was not defined in 2015","Mechanism connecting adaptor loss to repair gene expression unclear"]},{"year":2016,"claim":"The biophysical basis of SPOP function was established: BTB/BACK-driven higher-order oligomerization localizes the ligase to nuclear speckles and boosts ubiquitination efficiency, reframing speckles as ubiquitination hotspots.","evidence":"Analytical ultracentrifugation, live-cell imaging, in vitro ubiquitination of Gli3, and domain-mapping mutants","pmids":["27220849"],"confidence":"High","gaps":["Did not address how individual substrates partition relative to speckles","Quantitative contribution of oligomerization to specific substrate turnover not measured"]},{"year":2016,"claim":"In vivo developmental and oncogenic roles were anchored by knockout genetics: Spop regulates Gli3 and Hedgehog-dependent skeletal development, and SETD2 turnover links SPOP to chromatin methylation and splicing.","evidence":"Spop-null/conditional mice with Gli3 dosage rescue; ChIP-Seq and splicing analysis for SETD2","pmids":["27930311","27614073"],"confidence":"High","gaps":["Tissue-specificity of substrate selection not fully resolved","Whether speckle localization governs these substrates untested"]},{"year":2017,"claim":"SPOP's role in oncogenic transcription and immune evasion was consolidated through BET protein, c-MYC, and PD-L1 degradation, with CDK4-driven SPOP turnover linking the cell cycle to checkpoint ligand stability.","evidence":"Co-IP, in vivo ubiquitination, organoids, knockout mice, and CDK4/6 inhibitor and phosphorylation mapping","pmids":["28805820","28805822","28414305","29160310"],"confidence":"High","gaps":["Relative substrate hierarchy in tumors not established","Did not quantify combined effect of simultaneous substrate stabilization"]},{"year":2017,"claim":"Non-degradative ubiquitination emerged as a distinct SPOP output, shown by INF2 modification that suppresses mitochondrial fission, extending SPOP beyond proteasomal targeting.","evidence":"Co-IP, ubiquitination assays, immunofluorescence, and mitochondrial fission assays with dominant-negative mutants","pmids":["28448495"],"confidence":"High","gaps":["Chain linkage type not fully defined in this study","Downstream effectors of altered fission not mapped"]},{"year":2018,"claim":"SPOP was shown to control pluripotency and replication-stress responses, degrading Nanog under kinase control and promoting expression of DNA repair/replication factors via its interactome.","evidence":"Degron mutagenesis and ubiquitination (Nanog); proteomic interactome with DNA fiber and foci assays","pmids":["30595538","30595535","30124983"],"confidence":"Medium","gaps":["Mechanism by which SPOP promotes repair-gene transcription not resolved","Direct versus indirect effects on replication factors unclear"]},{"year":2021,"claim":"ATM-triggered conformational activation of SPOP was structurally defined, explaining how DNA damage redirects SPOP to ubiquitinate 53BP1 (for p97-mediated chromatin extraction) and HIPK2 to favor homologous recombination.","evidence":"X-ray crystallography, phosphorylation mapping, ubiquitination assays, and cell-cycle/repair readouts","pmids":["34144977","34133717"],"confidence":"High","gaps":["In vivo contribution to tumor repair phenotypes not fully quantified","Interplay between the 53BP1 and HIPK2 arms not resolved"]},{"year":2021,"claim":"The breadth of linkage-specific and non-degradative signaling was extended: K27-linked Geminin ubiquitination restrains re-replication, K27/K29 chains stabilize 17βHSD4 to limit androgen synthesis, and PDK1 and GLP degradation couple SPOP to AKT and DNA methylation.","evidence":"Linkage-specific ubiquitination, kinase assays, replication assays, methylome analysis, and CRISPR E3 screen with xenografts","pmids":["34599168","33762355","34353330","34588438"],"confidence":"High","gaps":["Determinants selecting degradative versus non-degradative chains not generalized","Substrate-specific deubiquitinase counterparts mostly unidentified"]},{"year":2022,"claim":"SPOP was shown to regulate autophagy and antioxidant signaling through non-degradative ubiquitination of p62/SQSTM1, controlling its phase separation and Keap1-Nrf2 sequestration.","evidence":"Site-specific ubiquitination (K420), autophagy assays, and liquid-liquid phase separation experiments","pmids":["34987184"],"confidence":"High","gaps":["Cytoplasmic versus nuclear SPOP pool governing p62 not fully delineated","Physiological autophagy thresholds in vivo not established"]},{"year":2023,"claim":"Oligomeric structure determination provided the structural rationale for non-MATH cancer mutations, linking endometrial cancer mutations to altered self-assembly interfaces.","evidence":"X-ray crystallography of oligomeric SPOP with mutational and biochemical validation","pmids":["36693379"],"confidence":"High","gaps":["Functional consequences of altered assembly on specific substrates not exhaustively tested","Whether these mutants are gain- or loss-of-function context-dependent"]},{"year":null,"claim":"It remains unresolved what determines, for any given substrate, whether SPOP deposits degradative versus non-degradative ubiquitin chains and how subnuclear partitioning, oligomerization state, and the spectrum of MATH-domain versus assembly-interface mutations integrate to specify substrate fate.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking chain-type choice to substrate or localization","Quantitative substrate hierarchy across tissues unknown","Functional distinction between prostate (MATH) and endometrial (interface) mutations incompletely mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,3,4,10,12,18,32]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[0,4,17,22]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,4,17]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,16,22]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[6,45]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6,23,53]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[18,35,53]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[8,23]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,1,3,4,17]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[8,9,29]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[10]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,5,15,22]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,24,35,36]},{"term_id":"R-HSA-9612973","term_label":"Autophagy","supporting_discovery_ids":[18]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[11,19]}],"complexes":["CUL3-RBX1 RING E3 ubiquitin ligase (CRL3-SPOP)"],"partners":["CUL3","RBX1","AR","BRD4","ERG","ATM","53BP1","P62/SQSTM1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O43791","full_name":"Speckle-type POZ protein","aliases":["HIB homolog 1","Roadkill homolog 1"],"length_aa":374,"mass_kda":42.1,"function":"Component of a cullin-RING-based BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex that mediates the ubiquitination of target proteins, leading most often to their proteasomal degradation. In complex with CUL3, involved in ubiquitination and proteasomal degradation of BRMS1, DAXX, PDX1/IPF1, GLI2 and GLI3. In complex with CUL3, involved in ubiquitination of MACROH2A1 and BMI1; this does not lead to their proteasomal degradation. Inhibits transcriptional activation of PDX1/IPF1 targets, such as insulin, by promoting PDX1/IPF1 degradation. The cullin-RING-based BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex containing homodimeric SPOP has higher ubiquitin ligase activity than the complex that contains the heterodimer formed by SPOP and SPOPL. Involved in the regulation of bromodomain and extra-terminal motif (BET) proteins BRD2, BRD3, BRD4 stability (PubMed:32109420). Plays an essential role for proper translation, but not for their degradation, of critical DNA replication licensing factors CDT1 and CDC6, thereby participating in DNA synthesis and cell proliferation (PubMed:36791496). Regulates interferon regulatory factor 1/IRF1 proteasomal turnover by targeting S/T-rich degrons in IRF1 (PubMed:37622993). Facilitates the lysosome-dependent degradation of enterovirus EV71 protease 2A by inducing its 'Lys-48'-linked polyubiquitination, which ultimately restricts EV71 replication (PubMed:37796126). Acts as an antiviral factor also against hepatitis B virus/HBV by promoting ubiquitination and subsequent degradation of HNF1A (PubMed:38018242). In turn, inhibits HBV transcription and replication by preventing HNF1A stimulating activity of HBV preS1 promoter and enhancer II (PubMed:38018242). Involved in ubiquitination of BRDT and promotes its degradation, thereby regulates histone removal in early condensing spermatids prior to histone-to-protamine exchange (By similarity)","subcellular_location":"Nucleus; Nucleus speckle; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/O43791/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SPOP","classification":"Not Classified","n_dependent_lines":22,"n_total_lines":1208,"dependency_fraction":0.018211920529801324},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SPOP","total_profiled":1310},"omim":[{"mim_id":"620057","title":"PHD FINGER PROTEIN 7; PHF7","url":"https://www.omim.org/entry/620057"},{"mim_id":"618829","title":"NABAIS SA-DE VRIES SYNDROME, TYPE 2; NSDVS2","url":"https://www.omim.org/entry/618829"},{"mim_id":"618828","title":"NABAIS SA-DE VRIES SYNDROME, TYPE 1; NSDVS1","url":"https://www.omim.org/entry/618828"},{"mim_id":"608089","title":"ENDOMETRIAL CANCER","url":"https://www.omim.org/entry/608089"},{"mim_id":"605402","title":"CD274 MOLECULE; CD274","url":"https://www.omim.org/entry/605402"}],"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/SPOP"},"hgnc":{"alias_symbol":["TEF2","BTBD32"],"prev_symbol":[]},"alphafold":{"accession":"O43791","domains":[{"cath_id":"2.60.210.10","chopping":"25-167","consensus_level":"high","plddt":95.5452,"start":25,"end":167}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O43791","model_url":"https://alphafold.ebi.ac.uk/files/AF-O43791-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O43791-F1-predicted_aligned_error_v6.png","plddt_mean":90.12},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SPOP","jax_strain_url":"https://www.jax.org/strain/search?query=SPOP"},"sequence":{"accession":"O43791","fasta_url":"https://rest.uniprot.org/uniprotkb/O43791.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O43791/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O43791"}},"corpus_meta":[{"pmid":"29160310","id":"PMC_29160310","title":"Cyclin D-CDK4 kinase destabilizes PD-L1 via cullin 3-SPOP to control cancer immune surveillance.","date":"2017","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/29160310","citation_count":857,"is_preprint":false},{"pmid":"28805820","id":"PMC_28805820","title":"Prostate cancer-associated SPOP mutations confer resistance to BET inhibitors through stabilization of BRD4.","date":"2017","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28805820","citation_count":285,"is_preprint":false},{"pmid":"28805822","id":"PMC_28805822","title":"Intrinsic BET inhibitor resistance in SPOP-mutated prostate cancer is mediated by BET protein stabilization and AKT-mTORC1 activation.","date":"2017","source":"Nature medicine","url":"https://pubmed.ncbi.nlm.nih.gov/28805822","citation_count":253,"is_preprint":false},{"pmid":"24508459","id":"PMC_24508459","title":"Destruction of full-length androgen receptor by wild-type SPOP, but not prostate-cancer-associated mutants.","date":"2014","source":"Cell 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Abiraterone Sensitivity.","date":"2018","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/30068710","citation_count":120,"is_preprint":false},{"pmid":"34168048","id":"PMC_34168048","title":"ATR Inhibition Induces CDK1-SPOP Signaling and Enhances Anti-PD-L1 Cytotoxicity in Prostate Cancer.","date":"2021","source":"Clinical cancer research : an official journal of the American Association for Cancer Research","url":"https://pubmed.ncbi.nlm.nih.gov/34168048","citation_count":117,"is_preprint":false},{"pmid":"31771591","id":"PMC_31771591","title":"Prostate Cancer-associated SPOP mutations enhance cancer cell survival and docetaxel resistance by upregulating Caprin1-dependent stress granule assembly.","date":"2019","source":"Molecular cancer","url":"https://pubmed.ncbi.nlm.nih.gov/31771591","citation_count":107,"is_preprint":false},{"pmid":"28414305","id":"PMC_28414305","title":"SPOP regulates prostate epithelial cell proliferation and promotes ubiquitination and turnover of c-MYC oncoprotein.","date":"2017","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/28414305","citation_count":103,"is_preprint":false},{"pmid":"30479348","id":"PMC_30479348","title":"TRIM28 protects TRIM24 from SPOP-mediated degradation and promotes prostate cancer progression.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/30479348","citation_count":100,"is_preprint":false},{"pmid":"31772275","id":"PMC_31772275","title":"ILF3 is a substrate of SPOP for regulating serine biosynthesis in colorectal cancer.","date":"2019","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/31772275","citation_count":92,"is_preprint":false},{"pmid":"31901237","id":"PMC_31901237","title":"The emerging role of SPOP protein in tumorigenesis and cancer therapy.","date":"2020","source":"Molecular 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target PD-L1 for proteasome-mediated degradation. Cyclin D-CDK4 phosphorylates SPOP, promoting SPOP's own degradation by the APC/C activator FZR1, thereby stabilizing PD-L1. Loss-of-function SPOP mutations compromise ubiquitination-mediated PD-L1 degradation.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assays, pharmacological CDK4/6 inhibition, mouse tumor models, phosphorylation mapping\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo ubiquitination, multiple orthogonal methods, replicated in mouse models and primary human specimens\",\n      \"pmids\": [\"29160310\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CUL3-SPOP E3 ligase complex ubiquitinates BET proteins (BRD2, BRD3, BRD4) for proteasomal degradation. Prostate cancer-associated SPOP mutants fail to interact with BET proteins, leading to their accumulation and resistance to BET inhibitors.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, prostate cancer organoids, Western blot of patient specimens\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus ubiquitination assays, independently replicated in two concurrent papers (PMIDs 28805820 and 28805822)\",\n      \"pmids\": [\"28805820\", \"28805822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Wild-type SPOP recognizes a degron motif common among BET proteins and promotes their proteasomal degradation; prostate cancer SPOP mutants show impaired binding to BET proteins. BRD4 stabilization activates RAC1 and AKT-mTORC1 signaling as a consequence.\",\n      \"method\": \"Degron binding assay, ubiquitination assay, transcriptome and BRD4 cistrome analyses, prostate cancer cell lines and patient specimens\",\n      \"journal\": \"Nature medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — degron mapping, ubiquitination assays, transcriptome analysis, replicated across two concurrent labs\",\n      \"pmids\": [\"28805822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPOP recognizes a Ser/Thr-rich degron in the hinge domain of full-length androgen receptor (AR) and induces its ubiquitination and proteasomal degradation, inhibiting AR-mediated transcription and prostate cancer cell growth. AR splice variants lacking the hinge domain escape SPOP-mediated degradation. Prostate cancer SPOP mutants cannot bind or promote AR destruction. Androgens antagonize SPOP-mediated AR degradation, whereas antiandrogens promote it.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, degron mapping, siRNA knockdown, cell growth assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, degron mapping, ubiquitination, functional cell assays), single rigorous study\",\n      \"pmids\": [\"24508459\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"SPOP directly interacts with SRC-3 (NCOA3/AIB1) and promotes its CUL3-dependent ubiquitination and proteolysis, suppressing androgen receptor transcriptional activity. Prostate cancer-associated SPOP mutants cannot interact with SRC-3 or promote its ubiquitination and degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, proteasome inhibition, prostate cancer cell lines\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP and ubiquitination assays with mutant characterization, single rigorous study with multiple orthogonal methods\",\n      \"pmids\": [\"23559371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPOP recognizes ERG via the CUL3 ubiquitin ligase system and promotes ERG ubiquitination and proteasomal degradation. The SPOP/ERG interaction is modulated by CKI-mediated phosphorylation. Truncated ERG (ΔERG) encoded by the fusion gene evades SPOP-mediated destruction. Prostate cancer SPOP mutants are deficient in promoting ERG ubiquitination. DNA damage drugs (topoisomerase inhibitors) trigger CKI activation to restore SPOP/ΔERG interaction.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, drug treatment experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination, phosphorylation, drug treatment), single rigorous study\",\n      \"pmids\": [\"26344095\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPOP self-associates into higher-order oligomers through BTB-mediated dimerization forming linear oligomers via BACK domain dimerization. Higher-order SPOP oligomerization is required for localization to liquid nuclear speckles; self-association-deficient SPOP mutants show diffuse nuclear distribution. Higher-order oligomerization stimulates CRL3(SPOP) ubiquitination efficiency for substrate Gli3, indicating nuclear speckles are hotspots of ubiquitination.\",\n      \"method\": \"Analytical ultracentrifugation, biophysical sizing, live-cell imaging, in vitro ubiquitination assays, domain mapping with mutants\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — biophysical reconstitution, in vitro ubiquitination assay, live imaging, multiple orthogonal methods in one study\",\n      \"pmids\": [\"27220849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Cryo-EM/X-ray crystal structure of SPOP in its oligomeric form reveals new interfaces important for SPOP self-assembly. Many endometrial cancer mutations localize to newly identified oligomeric interfaces, providing the structural basis for their gain-of-function or altered-assembly cancer-causing properties.\",\n      \"method\": \"X-ray crystallography (oligomeric structure determination), mutagenesis, biochemical characterization of cancer mutants\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure of oligomeric SPOP with mutational validation, single rigorous study\",\n      \"pmids\": [\"36693379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ATM kinase phosphorylates SPOP in response to DNA damage, causing a conformational change (revealed by X-ray crystal structures) that stabilizes SPOP's interaction with 53BP1. SPOP then induces polyubiquitination of 53BP1, leading to 53BP1 extraction from chromatin by a p97/VCP segregase complex, promoting homologous recombination over NHEJ during S phase. Cancer-derived SPOP mutations block SPOP interaction with 53BP1, inducing HR defects.\",\n      \"method\": \"X-ray crystallography, in vivo and in vitro ubiquitination assays, Co-immunoprecipitation, phosphorylation mapping, cell cycle analysis\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — X-ray crystal structure plus multiple functional biochemical assays (Co-IP, ubiquitination, phosphorylation mapping), single rigorous study with orthogonal methods\",\n      \"pmids\": [\"34144977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPOP mutations are associated with genomic instability in prostate cancer. SPOP mutation results in a transcriptional response consistent with BRCA1 inactivation, impairing homology-directed repair (HDR) of DNA double-strand breaks. SPOP mutation sensitizes cells to PARP inhibitors.\",\n      \"method\": \"Epistasis/transcriptional profiling, DNA repair functional assays, in vivo mouse models, PARP inhibitor sensitivity assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple assays in mouse models and cell lines, single lab study with functional DNA repair readouts\",\n      \"pmids\": [\"26374986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP promotes K27-linked non-degradative polyubiquitination of Geminin at K100 and K127, preventing DNA replication over-firing by blocking Cdt1 association with the MCM complex. Cancer-associated SPOP mutations impair Geminin K27-linked polyubiquitination and induce replication origin over-firing and re-replication.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, ubiquitin chain linkage analysis, DNA replication assays, cancer mutant analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reconstitution of non-degradative ubiquitination plus mechanistic dissection of Cdt1-MCM interaction, multiple orthogonal methods\",\n      \"pmids\": [\"34599168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPOP is critically involved in SETD2 protein stability control; the SPOP/CUL3 complex is responsible for SETD2 polyubiquitination both in vivo and in vitro. Modulation of SPOP expression confers differential H3K36me3 levels on SETD2 target genes and induces H3K36me3-coupled alternative splicing events.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, ChIP-Seq, RNA splicing analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro ubiquitination reconstitution plus ChIP-Seq and splicing analysis, multiple orthogonal methods\",\n      \"pmids\": [\"27614073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPOP recognizes INF2 via a Ser/Thr-rich motif in its C-terminal region and triggers atypical (non-degradative) polyubiquitination. This modification reduces INF2 localization in the ER and mitochondrially associated DRP1 puncta, impairing INF2-mediated mitochondrial fission. Prostate cancer SPOP mutants increase INF2 ER localization and promote mitochondrial fission via dominant-negative effect.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, immunofluorescence, mitochondrial fission assays, dominant-negative mutant analysis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination, localization imaging, functional fission assays), single rigorous study\",\n      \"pmids\": [\"28448495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPOP physically interacts with Nanog via a conserved degron motif and promotes Nanog poly-ubiquitination and subsequent proteasomal degradation. Cancer-derived mutations in SPOP or at the Nanog degron (S68Y) disrupt this destruction. Pin1 oncoprotein acts as an upstream regulator that impairs Nanog recognition by SPOP, stabilizing Nanog.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, degron mutagenesis, cancer mutant analysis, Pin1 interaction studies\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, degron mutagenesis, ubiquitination assays, multiple orthogonal methods; independently replicated in a concurrent paper (PMID 30595535)\",\n      \"pmids\": [\"30595538\", \"30595535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"AMPK-BRAF signaling controls SPOP-mediated NANOG degradation through phosphorylation of NANOG at Ser68, which blocks the SPOP-NANOG interaction. Cancer-associated mutations of SPOP or the NANOG S68Y mutation abrogates SPOP-mediated degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, kinase assays\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — kinase phosphorylation mapping plus Co-IP and ubiquitination, single lab study\",\n      \"pmids\": [\"30595535\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP promotes ubiquitination and degradation of PDK1 in a CK1/GSK3β-mediated phosphorylation-dependent manner, by directly binding a degron in PDK1. This suppresses AKT kinase activity. Prostate cancer SPOP mutations impair PDK1 degradation and activate AKT.\",\n      \"method\": \"CRISPR-based E3 ligase screen, Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, in vitro kinase assays, mass spectrometry, xenograft models\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — CRISPR screen plus reconstituted in vitro ubiquitination plus kinase assays and mass spectrometry, multiple orthogonal methods\",\n      \"pmids\": [\"34353330\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPOP physically interacts with c-MYC protein and promotes c-MYC ubiquitination and degradation. PC-associated SPOP mutants or SPOP knockdown increase c-MYC protein levels. Prostate-specific biallelic Spop knockout mice develop prostatic intraepithelial neoplasia with elevated c-MYC.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, prostate-specific knockout mouse model, gene expression analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ubiquitination assays, and in vivo knockout model with phenotypic readout, multiple orthogonal methods\",\n      \"pmids\": [\"28414305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"SPOP recognizes multiple Ser/Thr-rich degrons in the AF2 domain of estrogen receptor-α (ERα) and triggers ERα ubiquitin-proteasome pathway degradation as part of the SPOP-CUL3-RBX1 E3 complex. SPOP participates in estrogen-induced ERα degradation. Endometrial cancer SPOP mutants are defective in promoting ERα ubiquitination and degradation.\",\n      \"method\": \"Co-immunoprecipitation, in vivo and in vitro ubiquitination assays, degron mapping, siRNA knockdown, endometrial cancer mutant analysis\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination, degron mapping, cancer mutant analysis), single rigorous study\",\n      \"pmids\": [\"25766326\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cytoplasmic SPOP binds p62/SQSTM1 and induces non-degradative ubiquitination of p62 at residue K420 within the UBA domain. This modification decreases p62 puncta formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, suppressing p62-dependent autophagy. SPOP also relieves p62-mediated Keap1 sequestration, reducing Nrf2-mediated antioxidant transcription. Prostate cancer SPOP mutants fail to ubiquitinate p62 and promote autophagy and Nrf2 activation in a dominant-negative manner.\",\n      \"method\": \"Co-immunoprecipitation, in vitro and in vivo ubiquitination assays, site-specific mutagenesis, autophagy assays, liquid-liquid phase separation experiments\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — non-degradative ubiquitination mapped to specific residue (K420), liquid-liquid phase separation assays, multiple orthogonal functional readouts\",\n      \"pmids\": [\"34987184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP binds and promotes polyubiquitination and proteasomal degradation of the histone methyltransferase GLP, and by extension its partner G9a. SPOP mutation induces GLP/G9a stabilization, aberrant global DNA hypermethylation in prostate cancer cells, and silencing of tumor suppressor genes (FOXO3, GATA5, NDRG1).\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, genome-wide DNA methylome analysis, gene expression analysis, cancer mutant validation\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, ubiquitination assays, genome-wide methylome analysis, multiple orthogonal methods\",\n      \"pmids\": [\"34588438\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRIM28 interacts with TRIM24 (a known SPOP substrate) to prevent its ubiquitination and degradation by SPOP, acting as a competitive upstream regulator. TRIM28 facilitates TRIM24 chromatin occupancy and augments AR signaling.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, chromatin immunoprecipitation, xenograft studies\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays plus ChIP, single lab study\",\n      \"pmids\": [\"30479348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPOP recognizes and triggers ubiquitin-dependent proteasomal degradation of Caprin1 (a stress granule nucleating protein). Prostate cancer SPOP mutants fail to degrade Caprin1, leading to elevated Caprin1 and enhanced stress granule assembly, conferring resistance to docetaxel and other stress inducers.\",\n      \"method\": \"Yeast two-hybrid screen, Co-immunoprecipitation, ubiquitination assays, stress granule assays, xenograft models, patient specimen analysis\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus Co-IP and ubiquitination assays, single lab study\",\n      \"pmids\": [\"31771591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"In mouse models, Spop directly targets the Gli3 repressor for ubiquitination and degradation, positively regulating Indian Hedgehog (Ihh) signaling and skeletal development. Loss of Spop results in upregulation of full-length and repressor forms of Gli3, down-regulation of Ihh target genes (Ptch1, Pthlh), and skeletal defects rescued by reducing Gli3 dosage.\",\n      \"method\": \"Spop null mouse model, conditional mouse model, genetic epistasis (reducing Gli3 dosage), in vivo ubiquitination assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic epistasis with Gli3 dosage rescue, ubiquitination assays, multiple mouse models\",\n      \"pmids\": [\"27930311\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPOP is recruited to DNA double-strand break sites, forms nuclear foci co-localizing with γ-H2AX foci, and interacts with ATM kinase in response to DNA damage. SPOP recruitment is predominantly dependent on ATM kinase activity. SPOP knockdown results in impaired DNA damage response and hypersensitivity to ionizing irradiation.\",\n      \"method\": \"Immunofluorescence (foci formation), Co-immunoprecipitation, siRNA knockdown, ionizing radiation sensitivity assays\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and localization imaging plus functional KD phenotype, single lab study\",\n      \"pmids\": [\"24451148\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SPOP negatively regulates PD-L1 expression at the transcriptional level by binding to IRF1 (a transcription factor for PD-L1) and triggering its ubiquitin-proteasomal degradation. Endometrial cancer SPOP mutants lose capacity to degrade IRF1, instead stabilizing it and upregulating PD-L1 expression.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, luciferase reporter assays, xenograft tumor models\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, reporter assay, and in vivo model, single lab study\",\n      \"pmids\": [\"36481790\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP promotes ATF2 ubiquitination and degradation via recognition of multiple Ser/Thr-rich degrons in ATF2 by the SPOP-CUL3-RBX1 complex. Prostate cancer SPOP mutants are defective in promoting ATF2 degradation.\",\n      \"method\": \"Yeast two-hybrid screen, Co-immunoprecipitation, ubiquitination assays, degron mapping, Transwell migration and invasion assays\",\n      \"journal\": \"Journal of experimental & clinical cancer research : CR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus Co-IP and ubiquitination assays, single lab study\",\n      \"pmids\": [\"29996942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPOP interacts with Cdc20 and promotes its poly-ubiquitination and subsequent proteasomal degradation in a degron-dependent manner via CUL3. Prostate cancer-derived SPOP mutants fail to interact with Cdc20 to promote its degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, pharmacological CUL inhibition (MLN4924), half-life assays\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays plus pharmacological validation, single lab study\",\n      \"pmids\": [\"27780719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPOP directly interacts with CYCLIN E1 and regulates its stability through poly-ubiquitination by the SPOP/CUL3/RBX1 complex. CDK2 competes with SPOP for CYCLIN E1 interaction, suggesting SPOP preferentially targets CDK2-free CYCLIN E1. Loss of CYCLIN E1 regulation by SPOP contributes to prostate cancer cell proliferation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, competition binding assays, prostate cancer cell lines\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ubiquitination and competition assays, single lab study\",\n      \"pmids\": [\"30237511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SPOP recognizes a Ser/Thr-rich degron in the transactivation domain of DDIT3/CHOP and triggers its degradation via the ubiquitin-proteasome pathway through the SPOP-CUL3-RBX1 complex. Prostate cancer SPOP mutants are defective in promoting DDIT3 degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, degron mapping, prostate cancer mutant analysis\",\n      \"journal\": \"Human mutation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, degron mapping, and ubiquitination assays, single lab study\",\n      \"pmids\": [\"24990631\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP is phosphorylated at Ser119 by ATM kinase upon DNA damage, enhancing SPOP binding to HIPK2. SPOP induces non-degradative ubiquitination of HIPK2, which increases HIPK2 phosphorylation of HP1γ and promotes HP1γ dissociation from H3K9me3 to initiate DNA damage repair. Prostate cancer SPOP mutations abrogate this SPOP-HIPK2 axis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, DNA damage repair assays, cancer mutant analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple Co-IP and phosphorylation assays with functional DNA repair readout, single lab study\",\n      \"pmids\": [\"34133717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"ILF3 is a substrate of SPOP; EGF-MEK-ERK pathway-mediated phosphorylation of ILF3 hinders SPOP-mediated poly-ubiquitination and degradation of ILF3. ILF3 regulates the SGOC (Serine-Glycine-One-Carbon) metabolic pathway by regulating SGOC gene mRNA stability.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, patient-derived xenografts\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, phosphorylation mapping in a single lab study\",\n      \"pmids\": [\"31772275\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CUL3-SPOP E3 ligase promotes ASCT2/SLC1A5 (glutamine transporter) ubiquitylation and degradation, controlling glutamine uptake. MLN4924 (neddylation inhibitor) inactivates CRL3-SPOP, causing ASCT2 accumulation. SPOP itself undergoes auto-ubiquitylation upon glutamine deprivation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, pharmacological neddylation inhibition, siRNA knockdown, breast cancer specimens\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays plus auto-ubiquitination finding, single lab study\",\n      \"pmids\": [\"35641493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP binds and promotes non-degradative K27- and K29-linked polyubiquitination of 17βHSD4 at a functional SBC motif (315RATST319). This stabilizes 17βHSD4. SGK3-mediated phosphorylation of S318 in the SBC blocks SPOP binding and instead leads to SKP2-dependent K48-linked degradative ubiquitination of 17βHSD4. Prostate cancer SPOP mutations impair SPOP-17βHSD4 interaction, causing 17βHSD4 destruction and increased testosterone production.\",\n      \"method\": \"Co-immunoprecipitation, in vivo and in vitro ubiquitination assays (linkage-specific), kinase assays, xenograft mouse models, patient specimens\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — non-degradative ubiquitination characterized with linkage specificity, competing E3 ligase system defined, in vivo models, multiple orthogonal methods\",\n      \"pmids\": [\"33762355\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"SPOP promotes DAXX ubiquitination and degradation as part of a CUL3-SPOP-DAXX axis in vascular endothelial cells. DAXX degradation by SPOP is required for VEGFR2 mRNA expression. Knockdown of SPOP or CUL3 upregulates DAXX protein and downregulates VEGFR2 levels. Simultaneous knockdown of SPOP and DAXX reverses VEGFR2 downregulation.\",\n      \"method\": \"siRNA knockdown, Western blotting, epistasis by double-knockdown, endothelial cell functional assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis by double knockdown plus functional assays, single lab study\",\n      \"pmids\": [\"28216678\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP targets and destabilizes STING1 protein. Prostate cancer-associated SPOP mutations result in upregulated non-canonical STING-NF-κB signaling. PARP inhibitor treatment shifts this signaling from immunosuppressive non-canonical STING-NF-κB to antitumor canonical cGAS-STING-IFNβ signaling in SPOP-mutant prostate cancer.\",\n      \"method\": \"Proteomics analysis, genetically modified cell line models, in vitro and in vivo PARP inhibitor treatment, transcriptomic analysis\",\n      \"journal\": \"Clinical cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomics plus functional cell line models, single lab study\",\n      \"pmids\": [\"37581614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPOP negatively regulates TLR-induced inflammation by disrupting MyD88 self-association. SPOP is recruited to MyD88 following TLR4 activation and translocates from the nucleus to the cytoplasm upon TLR4 activation. SPOP depletion promotes MyD88 aggregation and recruitment of downstream signaling kinases IRAK4, IRAK1, and IRAK2.\",\n      \"method\": \"Co-immunoprecipitation, SPOP overexpression and knockdown, immunofluorescence (subcellular localization), NF-κB reporter assays\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus localization imaging and NF-κB reporter assays, single lab study\",\n      \"pmids\": [\"32235916\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPOP promotes ubiquitination and proteasomal degradation of MyD88 via recognizing the intermediate domain of MyD88. Knockdown or genetic ablation of SPOP leads to aberrant elevation of MyD88 protein. SPOP negatively regulates NF-κB pathway activity and IL-1β production upon LPS challenge. Spop-deficient mice are more susceptible to Salmonella typhimurium infection.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, Spop-knockout mice, infection model\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, and in vivo knockout mouse infection model, single lab study\",\n      \"pmids\": [\"32365080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP promotes ubiquitination and degradation of LATS1 (a Hippo pathway tumor suppressor) via a degron-dependent interaction with CUL3. SPOP overexpression promotes kidney cancer cell proliferation and invasion partly through LATS1 degradation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, protein half-life assays, xenograft models\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays plus in vivo model, single lab study\",\n      \"pmids\": [\"32460168\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP promotes CDCA5 poly-ubiquitination and proteasomal degradation in a degron-dependent manner. Prostate cancer SPOP mutations impair this effect. CDCA5 depletion leads to G2M arrest, sister chromatid cohesion defects, and apoptosis via the AKT pathway.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, cell cycle analysis, functional knockdown assays\",\n      \"journal\": \"Neoplasia (New York, N.Y.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assays, and functional cell cycle readout, single lab study\",\n      \"pmids\": [\"34509929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SPOP mutation activates both PI3K/mTOR and androgen receptor signaling in prostate cancer in vivo, effectively uncoupling the normal negative feedback between these two pathways, as demonstrated in a conditional mutant SPOP mouse model with Pten loss.\",\n      \"method\": \"Conditional mouse model (prostate-specific mutant SPOP expression), prostate organoids, human prostate cancer sample analysis, transcriptional profiling\",\n      \"journal\": \"Cancer cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo mouse genetic model plus organoids and human samples, single lab study\",\n      \"pmids\": [\"28292441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AURORA A (AURKA) kinase directly phosphorylates SPOP at three sites, causing SPOP ubiquitylation and degradation. Conversely, SPOP degrades AURKA via a feedback loop. SPOP degradation by AURKA stabilizes AR, ARv7, and c-Myc, promoting oncogenic phenotypes. Phospho-resistant SPOP fully abrogates tumorigenesis in vivo.\",\n      \"method\": \"In vitro kinase assay (direct phosphorylation), ubiquitination assays, Co-immunoprecipitation, xenograft studies\",\n      \"journal\": \"Cancers\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus ubiquitination and in vivo xenograft, single lab study\",\n      \"pmids\": [\"33158056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LIMK2 kinase directly phosphorylates SPOP at three sites, causing SPOP degradation. SPOP promotes LIMK2 ubiquitylation, creating a feedback loop. Phospho-resistant SPOP completely suppresses tumorigenesis in vivo.\",\n      \"method\": \"In vitro kinase assay, ubiquitination assays, Co-immunoprecipitation, xenograft studies\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus ubiquitination and xenograft, single lab study; conceptually replicated in concurrent AURKA paper (PMID 33158056)\",\n      \"pmids\": [\"33311589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ERG upregulates wild-type SPOP to dampen AR signaling and sustain ERG activity through SPOP-mediated degradation of the bromodomain histone reader ZMYND11. Conversely, SPOP-mutant tumors stabilize ZMYND11 to repress ERG function and enable oncogenic AR signaling. This antagonism makes SPOP-mutant and ERG tumors synthetic sick.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, transcriptomic analysis, genetic epistasis in prostate cancer models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, and transcriptomic analyses in a single study, moderate evidence for ZMYND11 substrate\",\n      \"pmids\": [\"33531470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"G3BP1 interacts with SPOP and functions as a competitive inhibitor of CUL3-SPOP E3 ligase activity, providing a mechanism to inactivate SPOP tumor suppressor function without SPOP mutation in prostate cancer. AR directly upregulates G3BP1 transcription, creating a feed-forward amplification loop.\",\n      \"method\": \"Co-immunoprecipitation, transcriptomic analysis, functional knockdown assays, AR ChIP analysis\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and transcriptomic analyses plus AR ChIP, single lab study\",\n      \"pmids\": [\"34795264\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPOP controls fetal hemoglobin (HbF) expression in adult erythroid cells as a CUL3-RING ligase adaptor. SPOP depletion or dominant-negative SPOP overexpression significantly raises fetal globin mRNA and protein levels, independently of BCL11A and LRF HbF repressors.\",\n      \"method\": \"CRISPR-Cas9 library screen, siRNA knockdown, dominant-negative overexpression, transcriptome and proteome analyses\",\n      \"journal\": \"Blood advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen plus functional depletion and dominant-negative overexpression, single lab study\",\n      \"pmids\": [\"31126914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Intrinsically disordered substrates dictate SPOP subnuclear localization. Pdx1, which has only moderate valency (two SPOP-binding motifs), does not phase separate with SPOP and instead prompts SPOP relocalization from nuclear speckles to the diffuse nucleoplasm. SPOP-mediated ubiquitination of Pdx1 occurs in the nucleoplasm, and both SB motifs are required for efficient Pdx1 turnover.\",\n      \"method\": \"NMR spectroscopy (SB motif identification), live-cell imaging (SPOP relocalization), ubiquitination assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — NMR plus live imaging and ubiquitination assays, single lab study\",\n      \"pmids\": [\"33894201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SPOP promotes transcriptional expression of DNA repair and replication factors including BRCA2, ATR, CHK1, and RAD51. SPOP knockdown leads to spontaneous replication stress, impaired RAD51 foci formation, impaired CHK1 activation, and compromised recovery from replication fork stalling. SPOP interactome analysis shows wild-type SPOP (but not mutant SPOP) associates with transcription, mRNA splicing and export complexes.\",\n      \"method\": \"Proteomic interactome analysis, siRNA knockdown, RAD51/CHK1 foci formation, DNA fiber assays, gene expression analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proteomic interactome plus DNA repair functional assays and gene expression, single lab study\",\n      \"pmids\": [\"30124983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP promotes SIRT2 binding and degradation by the 26S proteasome. SPOP loss or mutations impair SIRT2 degradation in non-small cell lung cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, proteasome inhibition assays, Western blot in cancer cell lines\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP plus proteasome inhibition in cell lines, single lab study\",\n      \"pmids\": [\"28073696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SPOP (Spop) physically interacts with macroH2A1.2 histone variant via its MATH domain, which binds the putative leucine zipper domain of macroH2A1.2. This was identified by yeast two-hybrid and confirmed by GST pull-down.\",\n      \"method\": \"Yeast two-hybrid screen, GST pull-down, domain mapping\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — yeast two-hybrid and GST pull-down only, no functional validation, single study\",\n      \"pmids\": [\"12183056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SPOP promotes ubiquitination and degradation of EWS-FLI1 fusion oncoprotein in Ewing sarcoma. Casein kinase 1-mediated phosphorylation of the VTSSS degron in the FLI1 domain enhances SPOP-mediated EWS-FLI1 degradation. OTUD7A deubiquitinase opposes SPOP by deubiquitinating and stabilizing EWS-FLI1.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, phosphorylation mapping, Ewing sarcoma cell line knockdown, xenograft mouse models\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, phosphorylation mapping plus in vivo xenograft, single lab study\",\n      \"pmids\": [\"34060252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SPOP recognizes and promotes ubiquitination and degradation of ZBTB3 transcription factor via two Ser/Thr-rich degrons by the SPOP-CUL3-RBX1 E3 complex. Endometrial cancer SPOP mutants are defective in regulating ZBTB3 stability. SPOP inactivation promotes endometrial cell proliferation via ZBTB3-dependent SHH upregulation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, degron mapping, luciferase reporter assays, cancer mutant analysis\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, degron mapping, and reporter assays, single lab study\",\n      \"pmids\": [\"31911863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"SPOP promotes ubiquitination and degradation of HMGCS1 (HMG-CoA synthase 1), and CSN6 antagonizes SPOP to stabilize HMGCS1. HMGCS1 stabilization activates YAP1 to promote hepatocellular carcinoma growth.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, xenograft and orthotopic liver cancer models\",\n      \"journal\": \"Advanced science (Weinheim, Baden-Wurttemberg, Germany)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays plus in vivo models, single lab study\",\n      \"pmids\": [\"38308184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPOP promotes SPOP-mediated ubiquitination and degradation of PTEN in kidney cancer when SPOP is mislocalized to the cytoplasm. Small molecule inhibitors of the SPOP-substrate protein interaction increase PTEN levels, decrease PTEN ubiquitination, and reduce phosphorylated AKT and ERK in ccRCC cell lines.\",\n      \"method\": \"Structure-based small molecule design, in vitro binding assays, Co-immunoprecipitation, ubiquitination assays, cell viability assays\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — structure-based inhibitor plus biochemical validation in cell lines, single lab study\",\n      \"pmids\": [\"32297747\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"O-GlcNAcylation of SPOP at Ser96 by OGT increases nuclear positioning of SPOP in hepatoma cells, alleviating SPOP-mediated ubiquitination of the Nogo-B/RTN4B oncoprotein and thereby promoting HCC progression. In normal liver cells, cytoplasmic SPOP ubiquitinates Nogo-B via N-terminal SBC motifs. Abolition of O-GlcNAcylation by S96A mutation increased cytoplasmic SPOP localization.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, subcellular fractionation, site-specific mutagenesis, in vitro and in vivo cancer models\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — PTM identification and subcellular localization with functional consequence, ubiquitination assays, single lab study\",\n      \"pmids\": [\"36604567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"SPOP was identified as a novel substrate of STAT3 in bladder cancer; SPOP deficiency increases STAT3 protein stability, leading to elevated CCL2 secretion and macrophage M2 polarization. VEZF1 directly activates SPOP transcription, and its overexpression suppresses STAT3/CCL2/IL-6 axis effects.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, luciferase reporter, ChIP assay, co-culture system\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assays plus ChIP and reporter assays, single lab study\",\n      \"pmids\": [\"39479456\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SPOP functions as the substrate-binding adaptor subunit of the CUL3-RBX1 RING E3 ubiquitin ligase complex, recognizing substrates through Ser/Thr-rich degron motifs via its MATH domain, and mediating both degradative (K48-linked) and non-degradative (K27/K29/K63-linked) polyubiquitination of a diverse array of substrates—including AR, BET proteins (BRD2/3/4), ERG, SRC-3, PD-L1, Nanog, c-MYC, Gli2/3, 53BP1, GLP, Geminin, SETD2, CYCLIN E1, and many others—to regulate androgen/nuclear receptor signaling, DNA damage response and replication fidelity, Hedgehog signaling, immune checkpoints, cellular metabolism, and mitochondrial dynamics; SPOP self-assembles into higher-order oligomers through BTB and BACK domain interactions to localize to liquid nuclear speckles where ubiquitination efficiency is enhanced, and is itself regulated by upstream kinases (ATM, CDK4, AURKA, LIMK2) and post-translational modifications (phosphorylation, O-GlcNAcylation), while cancer-associated missense mutations clustered in the MATH domain impair substrate binding and broadly dysregulate these proteostatic functions.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SPOP is the substrate-recognition adaptor of a CUL3-RBX1 RING E3 ubiquitin ligase that selects substrates bearing Ser/Thr-rich degron motifs through its MATH domain, thereby controlling the abundance and activity of a broad array of nuclear regulators [#3, #4, #17]. Through its BTB and BACK domains SPOP self-assembles into higher-order oligomers that concentrate the ligase in liquid nuclear speckles, where ubiquitination of substrates such as Gli3 is most efficient; substrate valency itself can redistribute SPOP between speckles and the diffuse nucleoplasm [#6, #45], and crystallographic analysis of the oligomeric form maps cancer mutations to self-assembly interfaces [#7]. A dominant theme is the proteasomal destruction of transcriptional and signaling regulators—androgen receptor and its coactivator SRC-3, the ETS factor ERG, BET bromodomain proteins BRD2/3/4, c-MYC, ERα, the pluripotency factor Nanog, and the histone methyltransferases SETD2 and GLP—linking SPOP to nuclear receptor signaling, chromatin state, and proliferative control [#1, #2, #3, #4, #5, #11, #13, #16, #19]. Beyond canonical K48-linked degradation, SPOP also deposits non-degradative K27/K29-linked chains: it ubiquitinates Geminin to restrain replication origin re-firing, modifies INF2 to suppress mitochondrial fission, marks p62/SQSTM1 to inhibit autophagy and Nrf2 signaling, and stabilizes 17βHSD4 to limit androgen synthesis [#10, #12, #18, #32]. In the DNA damage response, ATM-mediated phosphorylation of SPOP triggers a conformational switch that drives 53BP1 ubiquitination and chromatin extraction to favor homologous recombination, and SPOP loss produces a BRCA-like repair defect that sensitizes cells to PARP inhibition [#8, #9, #29]. SPOP activity is itself governed by upstream kinases and modifications—CDK4, AURKA, and LIMK2 phosphorylation drive SPOP turnover, while O-GlcNAcylation tunes its nuclear/cytoplasmic distribution—and cancer-associated MATH-domain mutations recurrent in prostate and endometrial tumors abolish substrate binding, broadly dysregulating these proteostatic programs [#0, #40, #41, #53].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Before its E3 role was known, the question was what SPOP binds; the MATH domain was shown to engage a histone variant, hinting at a nuclear/chromatin-associated function.\",\n      \"evidence\": \"Yeast two-hybrid and GST pull-down mapping MATH-domain binding to macroH2A1.2\",\n      \"pmids\": [\"12183056\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Interaction not confirmed in cells and no functional consequence established\", \"No link to ubiquitination demonstrated\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Establishing SPOP as a CUL3 substrate adaptor in cancer, SRC-3 was identified as a degradation target whose loss explains SPOP's suppression of androgen receptor activity.\",\n      \"evidence\": \"Co-IP and CUL3-dependent ubiquitination assays with prostate cancer mutant characterization\",\n      \"pmids\": [\"23559371\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degron not precisely mapped here\", \"Did not address direct AR turnover\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The defining recognition principle—a Ser/Thr-rich degron read by SPOP—was demonstrated by direct degradation of full-length AR and DDIT3/CHOP, with splice variants and cancer mutants escaping turnover.\",\n      \"evidence\": \"Degron mapping, Co-IP, ubiquitination assays, and growth assays in prostate cancer cells\",\n      \"pmids\": [\"24508459\", \"24990631\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve which ubiquitin linkage types are used\", \"In vivo physiological relevance not yet tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"SPOP was tied to genome integrity and to phospho-regulated substrate selection, showing ERG recognition is gated by CKI phosphorylation and that SPOP mutation produces a BRCA1-like HDR defect.\",\n      \"evidence\": \"Phosphorylation mapping and ubiquitination assays (ERG); transcriptional profiling, DNA repair assays, and PARP inhibitor sensitivity in mouse models\",\n      \"pmids\": [\"26344095\", \"26374986\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The direct repair substrate underlying the HDR defect was not defined in 2015\", \"Mechanism connecting adaptor loss to repair gene expression unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"The biophysical basis of SPOP function was established: BTB/BACK-driven higher-order oligomerization localizes the ligase to nuclear speckles and boosts ubiquitination efficiency, reframing speckles as ubiquitination hotspots.\",\n      \"evidence\": \"Analytical ultracentrifugation, live-cell imaging, in vitro ubiquitination of Gli3, and domain-mapping mutants\",\n      \"pmids\": [\"27220849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address how individual substrates partition relative to speckles\", \"Quantitative contribution of oligomerization to specific substrate turnover not measured\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"In vivo developmental and oncogenic roles were anchored by knockout genetics: Spop regulates Gli3 and Hedgehog-dependent skeletal development, and SETD2 turnover links SPOP to chromatin methylation and splicing.\",\n      \"evidence\": \"Spop-null/conditional mice with Gli3 dosage rescue; ChIP-Seq and splicing analysis for SETD2\",\n      \"pmids\": [\"27930311\", \"27614073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specificity of substrate selection not fully resolved\", \"Whether speckle localization governs these substrates untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"SPOP's role in oncogenic transcription and immune evasion was consolidated through BET protein, c-MYC, and PD-L1 degradation, with CDK4-driven SPOP turnover linking the cell cycle to checkpoint ligand stability.\",\n      \"evidence\": \"Co-IP, in vivo ubiquitination, organoids, knockout mice, and CDK4/6 inhibitor and phosphorylation mapping\",\n      \"pmids\": [\"28805820\", \"28805822\", \"28414305\", \"29160310\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative substrate hierarchy in tumors not established\", \"Did not quantify combined effect of simultaneous substrate stabilization\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Non-degradative ubiquitination emerged as a distinct SPOP output, shown by INF2 modification that suppresses mitochondrial fission, extending SPOP beyond proteasomal targeting.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, immunofluorescence, and mitochondrial fission assays with dominant-negative mutants\",\n      \"pmids\": [\"28448495\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chain linkage type not fully defined in this study\", \"Downstream effectors of altered fission not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"SPOP was shown to control pluripotency and replication-stress responses, degrading Nanog under kinase control and promoting expression of DNA repair/replication factors via its interactome.\",\n      \"evidence\": \"Degron mutagenesis and ubiquitination (Nanog); proteomic interactome with DNA fiber and foci assays\",\n      \"pmids\": [\"30595538\", \"30595535\", \"30124983\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which SPOP promotes repair-gene transcription not resolved\", \"Direct versus indirect effects on replication factors unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"ATM-triggered conformational activation of SPOP was structurally defined, explaining how DNA damage redirects SPOP to ubiquitinate 53BP1 (for p97-mediated chromatin extraction) and HIPK2 to favor homologous recombination.\",\n      \"evidence\": \"X-ray crystallography, phosphorylation mapping, ubiquitination assays, and cell-cycle/repair readouts\",\n      \"pmids\": [\"34144977\", \"34133717\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution to tumor repair phenotypes not fully quantified\", \"Interplay between the 53BP1 and HIPK2 arms not resolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"The breadth of linkage-specific and non-degradative signaling was extended: K27-linked Geminin ubiquitination restrains re-replication, K27/K29 chains stabilize 17βHSD4 to limit androgen synthesis, and PDK1 and GLP degradation couple SPOP to AKT and DNA methylation.\",\n      \"evidence\": \"Linkage-specific ubiquitination, kinase assays, replication assays, methylome analysis, and CRISPR E3 screen with xenografts\",\n      \"pmids\": [\"34599168\", \"33762355\", \"34353330\", \"34588438\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants selecting degradative versus non-degradative chains not generalized\", \"Substrate-specific deubiquitinase counterparts mostly unidentified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"SPOP was shown to regulate autophagy and antioxidant signaling through non-degradative ubiquitination of p62/SQSTM1, controlling its phase separation and Keap1-Nrf2 sequestration.\",\n      \"evidence\": \"Site-specific ubiquitination (K420), autophagy assays, and liquid-liquid phase separation experiments\",\n      \"pmids\": [\"34987184\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cytoplasmic versus nuclear SPOP pool governing p62 not fully delineated\", \"Physiological autophagy thresholds in vivo not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Oligomeric structure determination provided the structural rationale for non-MATH cancer mutations, linking endometrial cancer mutations to altered self-assembly interfaces.\",\n      \"evidence\": \"X-ray crystallography of oligomeric SPOP with mutational and biochemical validation\",\n      \"pmids\": [\"36693379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequences of altered assembly on specific substrates not exhaustively tested\", \"Whether these mutants are gain- or loss-of-function context-dependent\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved what determines, for any given substrate, whether SPOP deposits degradative versus non-degradative ubiquitin chains and how subnuclear partitioning, oligomerization state, and the spectrum of MATH-domain versus assembly-interface mutations integrate to specify substrate fate.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking chain-type choice to substrate or localization\", \"Quantitative substrate hierarchy across tissues unknown\", \"Functional distinction between prostate (MATH) and endometrial (interface) mutations incompletely mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 3, 4, 10, 12, 18, 32]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [0, 4, 17, 22]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 4, 17]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 16, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [6, 45]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6, 23, 53]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [18, 35, 53]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [8, 23]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 1, 3, 4, 17]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [8, 9, 29]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [10]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 5, 15, 22]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 24, 35, 36]},\n      {\"term_id\": \"R-HSA-9612973\", \"supporting_discovery_ids\": [18]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [11, 19]}\n    ],\n    \"complexes\": [\"CUL3-RBX1 RING E3 ubiquitin ligase (CRL3-SPOP)\"],\n    \"partners\": [\"CUL3\", \"RBX1\", \"AR\", \"BRD4\", \"ERG\", \"ATM\", \"53BP1\", \"p62/SQSTM1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}