{"gene":"CDC34","run_date":"2026-06-09T22:57:18","timeline":{"discoveries":[{"year":1988,"finding":"CDC34 encodes a ubiquitin-conjugating enzyme (E2); the bacterially expressed CDC34 protein catalyzes covalent attachment of ubiquitin to histones H2A and H2B in vitro, establishing its enzymatic activity and its role in G1-to-S cell cycle transition.","method":"In vitro ubiquitination assay with bacterially expressed protein; genetic complementation","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of enzymatic activity, foundational paper replicated extensively","pmids":["2842867"],"is_preprint":false},{"year":1991,"finding":"CDC34 is a bifunctional E2 enzyme competent in both E3-independent and E3-dependent ubiquitin conjugation reactions, and shows marked kinetic selectivity for processive multiubiquitination via Lys-48 of ubiquitin, in contrast to RAD6 which does not preferentially use K48.","method":"In vitro ubiquitin conjugation assays with purified recombinant proteins; kinetic analysis; K48R ubiquitin variants","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis of ubiquitin, replicated in multiple studies","pmids":["1848239"],"is_preprint":false},{"year":1992,"finding":"The CDC34 C-terminal tail (residues 171-244) is a portable determinant of cell cycle function; transplanting this tail onto RAD6 generates a chimeric E2 that performs CDC34 cell cycle functions, while the catalytic domain alone is insufficient for cell cycle activity.","method":"Chimeric E2 construction; in vivo complementation of cdc34 mutants; deletion analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mutagenesis plus functional complementation, independently replicated in companion paper (PMID:1639076)","pmids":["1639075","1639076"],"is_preprint":false},{"year":1993,"finding":"Bacterially expressed Cdc34 catalyzes its own ubiquitination (autoubiquitination) via intramolecular transfer, forming predominantly a single Lys48-linked multiubiquitin chain; the chain attaches within the C-terminal region (residues 215-295) on any of four lysines (K273, K277, K293, K294).","method":"In vitro autoubiquitination assay with purified recombinant CDC34; hydroxylamine cleavage; site-directed mutagenesis of lysine residues","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with systematic mutagenesis, single lab","pmids":["8383676"],"is_preprint":false},{"year":1993,"finding":"Human CDC34 cDNA functionally complements yeast CDC34 deletion, establishing that the human protein is a functional homolog of the yeast cell cycle ubiquitin-conjugating enzyme; the gene is located on chromosome 19p13.3.","method":"Yeast complementation; Southern blot; chromosomal mapping","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional complementation across species, single lab","pmids":["8248134"],"is_preprint":false},{"year":1994,"finding":"Cdc34 (Ubc3) is itself a substrate for ubiquitination and phosphorylation in vivo; immunochemical localization places the protein in the nucleus.","method":"In vivo ubiquitination assay; phosphorylation detection; immunofluorescence/immunochemical localization","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct detection of PTMs in vivo and localization, single lab","pmids":["8164658"],"is_preprint":false},{"year":1994,"finding":"A 39-residue region of the CDC34 C-terminal tail adjacent to the catalytic domain is necessary and sufficient for full cell cycle function and mediates CDC34 self-association (dimerization) in vitro, as shown by cross-linking.","method":"Deletion mutagenesis; biophysical characterization; chemical cross-linking; in vivo complementation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical methods in single lab","pmids":["7929378"],"is_preprint":false},{"year":1995,"finding":"Cdc34 contains a non-covalent ubiquitin binding site; overexpression of ubiquitin suppresses cdc34 temperature-sensitive mutations in an allele-specific manner, and chemical cross-linking confirms a specific noncovalent Ub-CDC34 interaction, indicating correct positioning of Ub on the E2 surface is required for function.","method":"Genetic suppression screen; chemical cross-linking; in vivo complementation with ubiquitin variants","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — genetic and biochemical evidence, single lab","pmids":["7721857"],"is_preprint":false},{"year":1995,"finding":"Cdc34 residues S73 and S97 (defining the Cdc34 class of E2 catalytic domains) are critical for function; S97 (near active-site C95) is essential, and intragenic suppression between S97D and S73K mutations, further rescued by deletion of residues 103-114 (the unique Cdc34 insertion), defines epistatic interactions within the catalytic domain.","method":"Site-directed mutagenesis; in vivo complementation; intragenic suppression analysis","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with functional readout, single lab","pmids":["7565715"],"is_preprint":false},{"year":1995,"finding":"A dominant-negative Cdc34 (C95S/L99S double mutant) blocks cell growth when overexpressed and inhibits in vitro ubiquitination of the Cdc34-specific substrate Cln2, establishing that Cys95 is the catalytic active-site residue.","method":"Site-directed mutagenesis; in vitro ubiquitination assay; overexpression phenotype analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — active-site mutagenesis with in vitro and in vivo functional validation","pmids":["7592826"],"is_preprint":false},{"year":1995,"finding":"Cbf2p/Ndc10p (a kinetochore protein) is an in vivo substrate of Cdc34; purified Cdc34p catalyzes monoubiquitin conjugation to Cbf2p in vitro, and cdc34-2 mutation alters Cbf2p modification pattern in vivo.","method":"In vitro ubiquitination assay; immunoprecipitation with anti-ubiquitin antibody; genetic suppression","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro reconstitution plus in vivo evidence, single lab","pmids":["7651401"],"is_preprint":false},{"year":1997,"finding":"SIC1 multiubiquitination is reconstituted in yeast extract and requires CDC34 (E2), CDC4, CDC53, SKP1, and CLN/CDC28 kinase; the N-terminal 160 residues of Sic1 are necessary and sufficient for CDC34-dependent ubiquitination.","method":"In vitro reconstitution in fractionated yeast extract; immunodepletion; deletion analysis of Sic1","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with multiple components, replicated in later studies","pmids":["9285816"],"is_preprint":false},{"year":1997,"finding":"In Xenopus egg extracts, Cdc34p present in a large complex is required for initiation of DNA replication and appears to regulate Cdk2-cyclin E function, possibly through degradation of the Xenopus CDK inhibitor Xic1.","method":"Xenopus egg extract reconstitution; immunodepletion of Cdc34","journal":"Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical reconstitution in Xenopus extract, single lab","pmids":["9287222"],"is_preprint":false},{"year":1998,"finding":"Cdc53 functions as a scaffold protein in SCF complexes, containing independent binding sites for Cdc34 and Skp1; Skp1 bridges Cdc53 to F-box proteins (Cdc4, Met30, Grr1) which provide substrate specificity, while the Cdc34-Cdc53-Skp1 E2/E3 core is required for all SCF functions.","method":"Co-immunoprecipitation; two-hybrid; in vivo genetic analysis; biochemical fractionation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple F-box specificity tested, replicated across labs","pmids":["9499404"],"is_preprint":false},{"year":1998,"finding":"SCF(Met30) and Cdc34 are required for degradation of the Cdk-inhibitory kinase Swe1; Met30 binds Swe1 in vivo (GST pulldown), and extracts from cdc34 or met30 mutants are defective in Swe1 polyubiquitination.","method":"GST pulldown; in vitro polyubiquitination assay with mutant extracts; genetic interactions","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — biochemical pulldown plus in vitro ubiquitination, single lab","pmids":["9716410"],"is_preprint":false},{"year":1998,"finding":"Human CUL-1 and CDC34 are partners of p45(SKP2) in vivo, forming a large multiprotein complex with p19(SKP1) and cyclin A, representing the human SCF-type E3 ubiquitin ligase complex.","method":"Co-immunoprecipitation from human cells; complex purification","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP from mammalian cells, single lab","pmids":["9430629"],"is_preprint":false},{"year":1998,"finding":"Wee1 kinase is degraded in a Cdc34-dependent fashion in Xenopus egg extracts; this proteolysis is required for timely entry into mitosis and is inhibited when DNA replication is blocked, coupling mitosis to completion of S phase.","method":"Xenopus egg extract assay; immunodepletion of Cdc34; DNA replication checkpoint experiments","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 / Moderate — biochemical reconstitution with immunodepletion, functional mitotic entry readout","pmids":["9836638"],"is_preprint":false},{"year":1999,"finding":"Hrt1 (RING-H2 protein) is a subunit of SCF that binds Cdc4, Cdc53, and Cdc34 (but not Skp1) individually, potently stimulates SCF E3 activity, and enables reconstitution of Cln2 ubiquitination; the Cdc53/Hrt1 subcomplex activates Cdc34 autoubiquitination by a mechanism not requiring a reactive thiol.","method":"Mass spectrometry identification; recombinant SCF reconstitution; binding assays; in vitro ubiquitination","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution with recombinant components, MS identification, replicated","pmids":["10385629"],"is_preprint":false},{"year":1999,"finding":"Human Cdc34 ubiquitinates repressors of cAMP-induced transcription hICERIIgamma and hATF5 in mammalian cells, targeting them for proteasomal degradation; this requires active ubiquitin-conjugating enzyme activity and abrogates repression of cAMP-induced transcription.","method":"Yeast two-hybrid; transfection with dominant-negative and antisense CDC34; ubiquitination assays in cells","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple cellular methods, single lab","pmids":["10373550"],"is_preprint":false},{"year":1999,"finding":"Phosphorylation-dependent ubiquitination of IκBα is catalyzed by Ubc3 (CDC34) together with SCF(β-TRCP); ubiquitin is transferred directly from the E2 to phospho-IκBα in an SCF(β-TRCP)-dependent reaction. Ubc4 is also capable but is 19-fold more efficient than Ubc3 in this reaction in THP.1 cells.","method":"In vitro reconstitution with recombinant components; co-immunoprecipitation from human cells","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — full biochemical reconstitution with phosphorylation-dependent ubiquitination","pmids":["10918611"],"is_preprint":false},{"year":1999,"finding":"A complex containing βTrCP, Skp1, and Cdc53/Cul1 recruits Cdc34 to catalyze polyubiquitination of phosphorylated IκBα peptide; Ubc5 only stimulates mono- and di-ubiquitination whereas Cdc34 is required for polyubiquitination.","method":"In vitro ubiquitination assay with phosphopeptide substrate; reconstituted multiprotein complex","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical reconstitution, single lab","pmids":["10437795"],"is_preprint":false},{"year":2000,"finding":"Met4 transcription factor is ubiquitinated by Cdc34/SCF(Met30) but remains stable (proteolysis-independent ubiquitination); ubiquitinated Met4 associates with target promoters but fails to form functional transcription complexes, revealing a non-proteolytic function of Cdc34/SCF ubiquitination.","method":"Genetic suppression (met4Δ suppresses met30Δ lethality); chromatin immunoprecipitation; ubiquitination assays; stability analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis plus biochemical assays, novel non-proteolytic mechanism confirmed by multiple approaches","pmids":["10975521"],"is_preprint":false},{"year":2000,"finding":"B-Myb physically interacts with CDC34 (E2) and p45Skp2 (E3), and ectopic expression of CDC34 or p45Skp2 accelerates B-Myb degradation; cyclin A promotes CDC34-SCF(p45Skp2)-dependent ubiquitination of B-Myb via its C-terminal domain.","method":"Co-immunoprecipitation; ubiquitination assay in cells; overexpression/deletion constructs","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and cellular ubiquitination, single lab","pmids":["10871850"],"is_preprint":false},{"year":2000,"finding":"During interphase, human CDC34 localizes to distinct nuclear and cytoplasmic speckles; nuclear localization requires C-terminal sequences; in anaphase, CDC34 co-localizes with β-tubulin at the mitotic spindle.","method":"Immunofluorescence; biochemical fractionation; deletion mutant analysis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — direct localization experiments with functional domain mapping, single lab","pmids":["10769200"],"is_preprint":false},{"year":2001,"finding":"HSV-1 ICP0 RING finger domain (exon 2) binds Cdc34 E2 and promotes its polyubiquitination; ICP0 functions as a unimolecular E3 ubiquitin ligase where the RING finger binds E2 while a separate C-terminal domain has ligase activity.","method":"In vitro substrate-independent ubiquitination; co-immunoprecipitation; RING finger domain analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding and ubiquitination assays, single lab","pmids":["11447293"],"is_preprint":false},{"year":2001,"finding":"Elevation of Cdc34 protein levels by microinjection into mammalian cells at prophase selectively blocks CENP-E association with kinetochores and inhibits chromosome congression, causing prometaphase arrest; this effect is not reversed by proteasome inhibitors.","method":"Microinjection of recombinant Cdc34 into mitotic mammalian cells; immunofluorescence; electron microscopy","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein injection with specific cellular phenotype, single lab","pmids":["11514588"],"is_preprint":false},{"year":2001,"finding":"CK2β (casein kinase 2 regulatory subunit) interacts with human CDC34 in vivo; CK2 phosphorylates human CDC34 at five sites within the C-terminal 36 amino acids in vitro; mutation of these CK2 sites shifts CDC34 localization from nucleus to cytoplasm.","method":"Yeast two-hybrid; co-immunoprecipitation; in vitro kinase assay; site-directed mutagenesis; immunofluorescence","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods, single lab","pmids":["11546811"],"is_preprint":false},{"year":2003,"finding":"Cdc34 self-associates in vivo (shown by co-immunoprecipitation); self-association is dependent on intact Cdc34-Ub thiolester and requires active-site Cys and residues S73, S97, and the catalytic domain insertion; self-association is a prerequisite for multi-Ub chain assembly.","method":"Co-immunoprecipitation; site-directed mutagenesis; in vitro thiolester formation assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP in vivo plus mutagenesis, single lab","pmids":["12861024"],"is_preprint":false},{"year":2003,"finding":"Release of ubiquitin-charged Cdc34~Ub from the RING domain of SCF(Cdc4) is essential for ubiquitination of the bound substrate Sic1; formation of the Cdc34~Ub thioester increases dissociation rate from RING, and an F72V Cdc34 mutant with increased RING affinity blocks substrate ubiquitination.","method":"Kinetic binding assays; F72V Cdc34 mutant; in vitro ubiquitination assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mechanistic dissection with mutagenesis and kinetic assays, published in high-profile journal","pmids":["13678584"],"is_preprint":false},{"year":2004,"finding":"Cdc34 mediates cell cycle-dependent degradation of c-Ski proto-oncoprotein; dominant-negative Cdc34 stabilizes Ski both in vitro and in vivo, enhancing Ski's ability to antagonize TGF-β signaling.","method":"In vitro ubiquitination assay; dominant-negative CDC34 overexpression; protein stability assay","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro and in vivo approaches, single lab","pmids":["15122324"],"is_preprint":false},{"year":2005,"finding":"Sic1 ubiquitination by SCF(Cdc4)/Cdc34 proceeds in two steps: rate-limiting attachment of the first ubiquitin, followed by rapid elongation of a K48-linked chain; a conserved acidic loop of Cdc34 has no effect on first ubiquitin attachment but is required for processivity and K48-linkage specificity of chain synthesis.","method":"In vitro reconstitution; mutagenesis of the Cdc34 acidic loop; kinetic analysis; linkage determination","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with mutagenesis and mechanistic dissection, widely cited","pmids":["16360039"],"is_preprint":false},{"year":2005,"finding":"Human Cdc34 activity in K48-linked polyubiquitin chain synthesis is dramatically enhanced by the SCF ROC1/CUL1-Nedd8 core ligase module; N-terminal GST-fusion or chemical-induced FKBP dimerization of Cdc34 activates it constitutively for K48-chain assembly, demonstrating that dimerization/proximity activates Cdc34 catalysis.","method":"In vitro ubiquitin chain synthesis assay; GST-fusion dimerization; chemical-induced dimerization with FKBP/AP20187","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical approaches, single lab","pmids":["16210246"],"is_preprint":false},{"year":2005,"finding":"Knockdown of human CDC34 by antisense oligonucleotides inhibits degradation of p27Kip1 and prevents cellular proliferation; reducing p27Kip1 with antisense reverses the anti-proliferative phenotype, establishing that CDC34 controls proliferation specifically through p27Kip1 levels.","method":"Antisense oligonucleotide knockdown; western blot; cell proliferation assay; epistasis by double knockdown","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis by double knockdown, single lab","pmids":["15652359"],"is_preprint":false},{"year":2007,"finding":"Human Cdc34 employs distinct sites for monoubiquitination vs. polyubiquitin chain assembly: the conserved charged stretch (143-153) and acidic loop residues D102/D103 are required for Ub-Ub ligation but not for mono-ubiquitination of IκBα; N85 stabilizes the oxyanion and coordinates, with S138, the attacking lysine for catalysis; S95 and E108/E112 are required for proper donor Ub positioning.","method":"Site-directed mutagenesis; in vitro ubiquitination assay; GST-fusion rescue experiments","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis with mechanistic in vitro readouts, single lab","pmids":["17698585"],"is_preprint":false},{"year":2007,"finding":"CK2 phosphorylates yeast Cdc34 at Ser207 and Ser216 (and human Cdc34 at S203, S222, S231) in the acidic tail domain; phosphorylation increases Cdc34 ubiquitination activity towards Sic1 with SCF(Cdc4) in vitro; phosphosite mutants with higher activity show accelerated cell cycle progression and Sic1 degradation in vivo.","method":"In vitro CK2 kinase assay; in vitro ubiquitination assay; site-directed mutagenesis; cell cycle synchronization and flow cytometry","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro kinase + ubiquitination assay + in vivo cell cycle readout, single lab","pmids":["17461777"],"is_preprint":false},{"year":2008,"finding":"ATF5 is a substrate of Cdc34-dependent ubiquitin-proteasome degradation; cisplatin prevents ATF5 ubiquitin-dependent degradation by promoting nuclear-to-cytoplasmic translocation of Cdc34 and reducing the ATF5-Cdc34 interaction.","method":"Co-immunoprecipitation; cellular fractionation; ubiquitination assay; cisplatin treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP and fractionation, single lab","pmids":["18458088"],"is_preprint":false},{"year":2008,"finding":"CK2 phosphorylates Cdc34 within its N-terminal catalytic domain at Ser130 and Ser167; this phosphorylation strongly stimulates Cdc34 ubiquitin charging in vitro; the S130A/S167A mutant has normal basal activity but cannot be activated by CK2 and fails to complement a cdc34-2 ts strain.","method":"Mass spectrometry; in vitro kinase assay; ubiquitin charging assay; in vivo complementation","journal":"Cell cycle","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — MS-confirmed phosphosites, in vitro charging assay, in vivo complementation","pmids":["18418079"],"is_preprint":false},{"year":2009,"finding":"The Cdc34 acidic C-terminal tail contributes to submicromolar Km for SCF(Cdc4) binding (electrostatic interaction) and also has an unexpected direct catalytic function in promoting ubiquitin transfer to substrate, independent of its role in E3 binding.","method":"Kinetic analysis; deletion mutants; Cdc34-Cul1 fusion constructs; in vitro ubiquitination assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical reconstitution with mechanistic dissection using fusions and deletions, single lab","pmids":["19875449"],"is_preprint":false},{"year":2010,"finding":"Polyubiquitination of IκBα by SCF(βTrCP2) involves a UbcH5/Cdc34 E2 handoff: UbcH5 rapidly transfers the first ubiquitin to IκBα K21/K22; Cdc34 then assembles the K48-linked polyubiquitin chain on the substrate-linked receptor ubiquitin, dependent on SCF and the substrate N-terminus.","method":"Biochemical reconstitution with recombinant components; ubiquitin fusion receptor constructs; kinetic analysis","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — full reconstitution with mechanistic dissection, single lab","pmids":["20347421"],"is_preprint":false},{"year":2010,"finding":"Human Cdc34 C-terminus contains two ubiquitin-binding sites (UBS1: residues 206-215; UBS2: 216-225) that interact non-covalently with ubiquitin near K48 and the C-terminal tail; aromatic residues (F206, Y207, Y210, Y211) are required for Ub binding and contribute to SCF-dependent ubiquitination; UBS1 is required for yeast complementation.","method":"NMR chemical shift perturbation; site-directed mutagenesis; in vitro IκBα ubiquitination assay; in vivo complementation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure of interaction plus mutagenesis and functional reconstitution, single lab","pmids":["20353940"],"is_preprint":false},{"year":2010,"finding":"Lysine selectivity by Cdc34 during polyubiquitination of Sic1 depends on amino acids proximal to acceptor lysines both on substrate and on ubiquitin, linked to key residues in the Cdc34 catalytic core; these core residues are independent of SCF and alter whether Cdc34 monoubiquitinates or polyubiquitinates Sic1.","method":"Site-directed mutagenesis; in vitro ubiquitination assay with Sic1 and ubiquitin variants; SCF-independent assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — systematic mutagenesis with mechanistic in vitro readout, single lab","pmids":["20194622"],"is_preprint":false},{"year":2011,"finding":"Ubiquitin I44A mutation profoundly inhibits its ability to serve as donor for Cdc34-mediated chain initiation or elongation; this is partially rescued by computationally designed compensatory mutations in Cdc34, indicating that Cdc34 interacts with donor ubiquitin to organize the active site for deprotonation of acceptor lysine.","method":"In vitro ubiquitination assay; mutagenesis of ubiquitin and Cdc34; hydroxylamine rescue; pH titration","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mechanistic mutagenesis with computational design and biochemical validation, single lab","pmids":["21474069"],"is_preprint":false},{"year":2011,"finding":"CC0651, a small molecule, selectively inhibits human Cdc34 by inserting into a cryptic allosteric pocket distant from the catalytic site, causing conformational changes without affecting E1/E3 interactions or thioester formation, but blocking ubiquitin discharge to acceptor lysines; inhibition causes p27Kip1 accumulation and cancer cell antiproliferation.","method":"Crystal structure determination; biochemical inhibition assays; cell-based p27 accumulation; mutagenesis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mechanistic biochemistry plus cellular validation","pmids":["21683433"],"is_preprint":false},{"year":2011,"finding":"The disordered CDC34 C-terminus intramolecularly interacts with catalytically bound ubiquitin in the CDC34~Ub complex, associating with a novel lysine-rich surface (K6, K11, K29, K33) on ubiquitin opposite the canonical hydrophobic patch; this interaction exists in two slowly exchanging conformations (compact vs. extended).","method":"NMR spectroscopy; CDC34-Ub disulfide mimetic; chemical shift perturbation mapping","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural data with mechanistic implications, single lab","pmids":["21296085"],"is_preprint":false},{"year":2011,"finding":"CK2 phosphorylation of conserved serine residues in the Cdc34 catalytic domain and the acidic β4α2 loop function as a molecular switch: phosphorylation creates electrostatic repulsion that promotes opening of the catalytic cleft, thereby enhancing ubiquitin charging.","method":"Molecular dynamics simulation (2.5 µs); biochemical assays; comparative sequence analysis","journal":"PLoS computational biology","confidence":"Medium","confidence_rationale":"Tier 1 (MD) + Tier 2 (biochemical) / Moderate — computational plus biochemical, single lab","pmids":["21637798"],"is_preprint":false},{"year":2011,"finding":"PKA and Sch9 (nutrient-sensing kinases) phosphorylate Cdc34 at S97 in the catalytic domain in vivo and in vitro; S97 phosphorylation is cell cycle regulated (elevated during active growth, decreased during arrest), providing a direct link between nutrient sensing and G1 cell division.","method":"In vitro kinase assay; mass spectrometry; cell cycle synchronization; genetic analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay and cell cycle regulation, single lab","pmids":["22087249"],"is_preprint":false},{"year":2013,"finding":"The acidic loop of human Cdc34 promotes ubiquitination by two mechanisms: (1) facilitating Cdc34-SCF interaction and (2) two glutamic acid residues on the distal side collaborate with a conserved histidine on the proximal side to suppress the pKa of an ionizing species critical for catalysis.","method":"In vitro ubiquitination assay; mutagenesis; binding assays; pKa analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple mechanistic modes identified biochemically, single lab","pmids":["24129577"],"is_preprint":false},{"year":2014,"finding":"The Cdc34-SCF interaction occurs in multiple conformations: several residues of the Cdc34 acidic tail contact a broad region of the SCF basic canyon via electrostatic interactions; similar patterns are seen with Cul2, implicating a common mechanism across cullin-RING ligases.","method":"Protein cross-linking; mass spectrometry; binding analysis","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cross-linking MS with multiple conformations identified, single lab","pmids":["25425648"],"is_preprint":false},{"year":2014,"finding":"Ube2r1 (Cdc34) requires its C-terminal extension (184-196) for efficient K48-linked polyubiquitination, while the acidic loop residues (Tyr-102/Tyr-104 in the related Ube2g1) control ubiquitin binding and K48-ubiquitylation; oxyester but not disulfide Ube2r1~Ub mimics are functional equivalents of the thioester.","method":"In vitro ubiquitylation assay; site-directed mutagenesis; NMR with oxyester E2~Ub mimics","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — NMR and biochemical assays, single lab","pmids":["25471371"],"is_preprint":false},{"year":2016,"finding":"Ube2R1/2 (Cdc34) has exquisite K48 specificity; computational docking and biochemical experiments identify a key electrostatic interaction between Arg54 on ubiquitin and Asp143 on Ube2R1/2 that guides K48 to the active site.","method":"Computational docking; site-directed mutagenesis; in vitro ubiquitination assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — computation confirmed by biochemistry, single lab","pmids":["27044868"],"is_preprint":false},{"year":2019,"finding":"Crystal structures of Cdc34 alone, in complex with E1 (Uba1), and as a Cdc34~Ub thioester mimetic reveal: (1) unique E1-E2 binding mode requiring conformational changes in both proteins for transthiolation; (2) contacts between the Cdc34 C-terminal extension and ubiquitin stabilize Cdc34~Ub in a closed conformation critical for ubiquitin discharge.","method":"X-ray crystallography; biochemical assays; cell-based functional assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple crystal structures with biochemical and cell-based validation in single study","pmids":["31341161"],"is_preprint":false},{"year":2020,"finding":"CDC34 competes with c-Cbl E3 ligase to bind EGFR at Y1045, thereby inhibiting EGFR polyubiquitination and degradation, and promoting NSCLC cell proliferation; knockdown of CDC34 inhibits EGFR-driven lung tumor growth in mouse models.","method":"siRNA library screen; co-immunoprecipitation; in vitro and in vivo tumor models; overexpression/knockdown","journal":"EBioMedicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP and in vivo mouse model, single lab","pmids":["32114396"],"is_preprint":false},{"year":2010,"finding":"CSN (COP9 signalosome) protects Cdc34/UBC3 from SCF(βTrCP)-mediated degradation; knockdown of CSN4 or CSN5 induces proteasomal degradation of Cdc34, and the acidic C-terminal extension of UBC3 is required and sufficient for SCF(βTrCP)-mediated ubiquitination.","method":"siRNA knockdown; co-immunoprecipitation; domain mapping with UBC3 C-terminal fusions","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical and genetic approaches, single lab","pmids":["20378537"],"is_preprint":false},{"year":2026,"finding":"A nanobody (VHH12R1) binding selectively to the Ube2R1 N-terminal extension transiently delays ubiquitin charging, promotes stable mono-ubiquitin conjugate accumulation, and markedly reduces self-directed polyubiquitination without impairing di-ubiquitin synthesis, revealing a regulatory role for the N-terminal extension in controlling processive self-elongation.","method":"Nanobody isolation and characterization; in vitro ubiquitin charging and chain synthesis assays; biochemical inhibition analysis","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical reconstitution with selective nanobody tool, single lab, preprint/recent publication","pmids":["41706475"],"is_preprint":false}],"current_model":"CDC34 (UBE2R1/Ubc3) is a ubiquitin-conjugating enzyme (E2) that forms a thioester with ubiquitin via its active-site Cys95, functions as the obligate E2 for SCF (Skp1-Cullin1-F-box) ubiquitin ligase complexes to catalyze K48-linked polyubiquitin chain assembly on cell cycle regulators (Sic1, Wee1, p27Kip1, cyclin-dependent kinase inhibitors) and other substrates (IκBα, Met4, ATF5, B-Myb, c-Ski), with chain specificity and processivity controlled by a conserved acidic loop and disordered acidic C-terminal tail that mediates SCF binding and positions donor ubiquitin for catalysis; CK2 phosphorylates both the catalytic domain and C-terminal tail to stimulate activity, while the COP9 signalosome protects CDC34 from SCF(βTrCP)-mediated autoubiquitination and degradation."},"narrative":{"mechanistic_narrative":"CDC34 (UBE2R1/Ubc3) is a ubiquitin-conjugating enzyme (E2) that drives the G1-to-S cell cycle transition by assembling K48-linked polyubiquitin chains on regulatory substrates marked for proteasomal degradation [PMID:2842867, PMID:1848239]. Catalysis proceeds through a thioester at the active-site Cys95, and a dominant-negative C95S mutant blocks both growth and substrate ubiquitination, establishing the catalytic center [PMID:7592826]. CDC34 shows intrinsic kinetic selectivity for processive K48-linked multiubiquitination [PMID:1848239], with chain processivity and linkage specificity governed by a conserved acidic loop and catalytic-core residues that position the donor ubiquitin and lower the pKa of the attacking acceptor lysine [PMID:16360039, PMID:17698585, PMID:21474069, PMID:24129577]. CDC34 functions as the obligate E2 for SCF (Skp1–Cullin/Cdc53–F-box) cullin-RING ligases: Cdc53 scaffolds independent binding sites for CDC34 and Skp1, the RING subunit Hrt1/ROC1 stimulates activity, and the disordered acidic C-terminal tail mediates high-affinity electrostatic binding to the SCF basic canyon while also directly promoting ubiquitin transfer [PMID:9499404, PMID:10385629, PMID:19875449, PMID:25425648]. Through SCF complexes it ubiquitinates cell cycle regulators including Sic1, Swe1/Wee1, and p27Kip1 to license replication and mitotic entry [PMID:9285816, PMID:9716410, PMID:9836638, PMID:15652359], and acts with SCF(βTrCP) on phospho-IκBα in a UbcH5/CDC34 handoff in which a priming E2 attaches the first ubiquitin and CDC34 elongates the K48 chain [PMID:10918611, PMID:20347421]. CDC34 also mediates degradation of additional substrates (ATF5, hICERIIγ, B-Myb, c-Ski) and, in the case of Met4, catalyzes a non-proteolytic ubiquitination that inactivates a transcription factor without destroying it [PMID:10373550, PMID:10975521, PMID:10871850, PMID:15122324]. Its activity is tuned by CK2 phosphorylation of both the catalytic domain and acidic tail, which stimulates ubiquitin charging and accelerates cell cycle progression [PMID:17461777, PMID:18418079]. CDC34 is itself a substrate for autoubiquitination and SCF(βTrCP)-mediated turnover, from which the COP9 signalosome protects it [PMID:8383676, PMID:20378537].","teleology":[{"year":1988,"claim":"Established the founding identity of CDC34 as an enzyme rather than merely a cell cycle gene, by showing the protein directly conjugates ubiquitin.","evidence":"In vitro ubiquitination of histones H2A/H2B with bacterially expressed protein plus genetic complementation in yeast","pmids":["2842867"],"confidence":"High","gaps":["Physiological substrates not yet identified","Mechanism of cell cycle coupling unknown"]},{"year":1991,"claim":"Defined CDC34's distinctive catalytic preference, showing it is a bifunctional E2 with marked kinetic selectivity for processive K48-linked chains, distinguishing it from RAD6.","evidence":"In vitro conjugation assays with K48R ubiquitin variants and kinetic analysis of purified recombinant proteins","pmids":["1848239"],"confidence":"High","gaps":["Structural basis of K48 selectivity unresolved","E3 partners not yet defined"]},{"year":1992,"claim":"Localized cell cycle function to a portable C-terminal tail rather than the catalytic core, by transplanting the tail onto RAD6 to confer CDC34 activity.","evidence":"Chimeric E2 construction and in vivo complementation of cdc34 mutants","pmids":["1639075","1639076"],"confidence":"High","gaps":["Molecular partner contacted by the tail unknown","Did not distinguish substrate targeting from E3 binding"]},{"year":1995,"claim":"Identified Cys95 as the catalytic active-site residue and demonstrated a dominant-negative blockade of an authentic substrate (Cln2).","evidence":"C95S/L99S mutagenesis with in vitro ubiquitination and overexpression growth-arrest phenotype","pmids":["7592826"],"confidence":"High","gaps":["E3 ligase context for Cln2 not yet defined"]},{"year":1995,"claim":"Mapped catalytic-domain residues (S73, S97, the Cdc34-class insertion) defining the enzyme family and their epistatic interactions.","evidence":"Site-directed mutagenesis with intragenic suppression and in vivo complementation","pmids":["7565715"],"confidence":"Medium","gaps":["Atomic-level role of these residues not structurally resolved at the time"]},{"year":1997,"claim":"Placed CDC34 within the SCF machinery by reconstituting Sic1 multiubiquitination requiring CDC34, CDC4, CDC53, SKP1, and Cln/Cdc28 kinase.","evidence":"In vitro reconstitution in fractionated yeast extract with immunodepletion and Sic1 deletion analysis","pmids":["9285816"],"confidence":"High","gaps":["Stoichiometry and architecture of the E2-E3 interaction undefined","RING subunit not yet identified"]},{"year":1998,"claim":"Defined SCF architecture, showing Cdc53 is a scaffold with separate CDC34 and Skp1 sites and that F-box proteins confer substrate specificity, with CDC34 as the shared E2.","evidence":"Co-IP, two-hybrid, and genetic analysis across multiple F-box proteins; parallel human SCF(SKP2) complex co-IP","pmids":["9499404","9430629"],"confidence":"High","gaps":["How CDC34 is positioned relative to substrate for catalysis unknown"]},{"year":1998,"claim":"Connected CDC34/SCF to cell cycle timing by showing CDC34-dependent degradation of Cdk-inhibitory kinases Swe1/Wee1 couples mitotic entry to S-phase completion.","evidence":"Yeast extract polyubiquitination assays and Xenopus egg extract immunodepletion with replication checkpoint experiments","pmids":["9716410","9836638"],"confidence":"High","gaps":["Checkpoint signal that gates CDC34 activity not identified"]},{"year":1999,"claim":"Identified the RING subunit Hrt1/ROC1 as an activator of CDC34 and reconstituted SCF ubiquitination with recombinant components.","evidence":"Mass spectrometry identification, recombinant SCF reconstitution, and binding/ubiquitination assays","pmids":["10385629"],"confidence":"High","gaps":["Conformational mechanism of RING activation of CDC34 not yet resolved"]},{"year":1999,"claim":"Extended CDC34 function beyond cell cycle to NF-κB signaling, showing it catalyzes SCF(βTrCP)-dependent polyubiquitination of phospho-IκBα.","evidence":"In vitro reconstitution with recombinant components and co-IP from human cells; phosphopeptide substrate assays","pmids":["10918611","10437795"],"confidence":"High","gaps":["Relative roles of CDC34 versus other E2s in vivo unresolved","Mechanism of chain initiation vs elongation not yet dissected"]},{"year":2000,"claim":"Revealed a non-proteolytic mode of CDC34/SCF ubiquitination, where ubiquitinated Met4 is stable but transcriptionally inactivated.","evidence":"Genetic epistasis, chromatin immunoprecipitation, ubiquitination and stability assays","pmids":["10975521"],"confidence":"High","gaps":["Reader machinery interpreting non-degradative chains not defined"]},{"year":2003,"claim":"Established that CDC34 self-association, dependent on the thioester and active-site residues, is a prerequisite for multiubiquitin chain assembly.","evidence":"In vivo co-IP, site-directed mutagenesis, and in vitro thiolester assays","pmids":["12861024"],"confidence":"Medium","gaps":["Whether dimerization occurs on the SCF in vivo not established"]},{"year":2003,"claim":"Defined the catalytic cycle dynamics, showing that release of charged CDC34~Ub from the SCF(Cdc4) RING is essential for substrate ubiquitination.","evidence":"Kinetic binding assays with F72V high-RING-affinity mutant and in vitro ubiquitination","pmids":["13678584"],"confidence":"High","gaps":["How charging-coupled affinity change is structurally encoded unresolved at the time"]},{"year":2005,"claim":"Dissected chain synthesis into rate-limiting initiation and rapid elongation and assigned processivity/K48 specificity to the conserved acidic loop.","evidence":"In vitro reconstitution with acidic-loop mutagenesis, kinetics, and linkage determination","pmids":["16360039"],"confidence":"High","gaps":["Atomic mechanism by which the loop enforces K48 not yet defined"]},{"year":2005,"claim":"Demonstrated proximity/dimerization-driven activation, with the ROC1/CUL1-Nedd8 module or forced dimerization constitutively activating K48 chain assembly.","evidence":"In vitro chain synthesis with GST-fusion and FKBP chemical-induced dimerization","pmids":["16210246"],"confidence":"Medium","gaps":["Physiological trigger for the activating proximity in vivo unclear"]},{"year":2005,"claim":"Linked CDC34 to mammalian proliferation control specifically through p27Kip1 degradation.","evidence":"Antisense knockdown with double-knockdown epistasis and proliferation assays in human cells","pmids":["15652359"],"confidence":"Medium","gaps":["Off-target contributions of antisense not fully excluded","F-box receptor for p27 not addressed here"]},{"year":2007,"claim":"Separated the active-site machinery for monoubiquitination from that for chain elongation and assigned individual catalytic residues.","evidence":"Systematic mutagenesis with in vitro IκBα and chain assembly assays","pmids":["17698585"],"confidence":"High","gaps":["Structural confirmation of proposed oxyanion/lysine coordination lacking at the time"]},{"year":2007,"claim":"Established CK2 phosphorylation of the acidic tail as a positive regulator linking phosphorylation to faster Sic1 degradation and cell cycle progression.","evidence":"In vitro kinase and ubiquitination assays plus in vivo cell cycle synchronization with phosphosite mutants","pmids":["17461777"],"confidence":"High","gaps":["How tail phosphorylation alters SCF binding or catalysis mechanistically unresolved"]},{"year":2008,"claim":"Identified CK2 phosphorylation within the catalytic domain (S130/S167) as required for stimulated ubiquitin charging and in vivo function.","evidence":"Mass spectrometry, in vitro charging assays, and complementation with S130A/S167A mutant","pmids":["18418079"],"confidence":"High","gaps":["Conformational consequence of catalytic-domain phosphorylation not directly visualized"]},{"year":2009,"claim":"Showed the acidic C-terminal tail has a dual role: submicromolar electrostatic binding to SCF and a direct catalytic contribution to ubiquitin transfer.","evidence":"Kinetics, deletion mutants, and CDC34-Cul1 fusion constructs in in vitro ubiquitination","pmids":["19875449"],"confidence":"High","gaps":["Structural basis of the catalytic tail function unresolved"]},{"year":2010,"claim":"Resolved E2 handoff in IκBα polyubiquitination: UbcH5 primes the substrate and CDC34 elongates the K48 chain on the receptor ubiquitin.","evidence":"Biochemical reconstitution with ubiquitin fusion receptor constructs and kinetics","pmids":["20347421"],"confidence":"High","gaps":["Whether handoff applies generally to other SCF substrates not established"]},{"year":2010,"claim":"Defined non-covalent ubiquitin-binding sites in the human CDC34 C-terminus and identified residues governing K48 lysine selectivity, mapping the structural basis of donor/acceptor positioning.","evidence":"NMR chemical shift perturbation, mutagenesis, in vitro ubiquitination, and yeast complementation; Sic1 lysine-selectivity assays","pmids":["20353940","20194622"],"confidence":"High","gaps":["How these surfaces operate within the assembled SCF in real time unresolved"]},{"year":2010,"claim":"Identified CDC34 as a regulated target of its own pathway, protected from SCF(βTrCP)-mediated degradation by the COP9 signalosome.","evidence":"siRNA knockdown of CSN4/CSN5, co-IP, and domain mapping of the UBC3 C-terminal extension","pmids":["20378537"],"confidence":"Medium","gaps":["Physiological conditions triggering CDC34 turnover unclear","Single-lab co-IP evidence"]},{"year":2011,"claim":"Provided structural and mechanistic detail on donor-ubiquitin engagement, showing the disordered C-terminus contacts a non-canonical lysine-rich ubiquitin surface and that Ile44 is critical for donor function.","evidence":"NMR with CDC34-Ub disulfide mimetics, ubiquitin/CDC34 mutagenesis, and computational compensatory design","pmids":["21296085","21474069"],"confidence":"High","gaps":["Functional weight of the two conformational states in catalysis not quantified"]},{"year":2011,"claim":"Validated CDC34 as a druggable target, identifying the allosteric inhibitor CC0651 that blocks ubiquitin discharge and causes p27Kip1 accumulation.","evidence":"Crystal structure of the inhibitor-bound enzyme, biochemical inhibition assays, and cell-based p27 accumulation","pmids":["21683433"],"confidence":"High","gaps":["In vivo efficacy and selectivity of the chemotype not established here"]},{"year":2011,"claim":"Proposed a molecular switch model in which CK2 phosphorylation opens the catalytic cleft via electrostatic repulsion to enhance charging.","evidence":"Microsecond molecular dynamics simulation with supporting biochemical assays","pmids":["21637798"],"confidence":"Medium","gaps":["Computational model not confirmed by direct structural observation","Single-lab study"]},{"year":2014,"claim":"Detailed the multi-conformational electrostatic CDC34 tail–SCF basic canyon interaction and showed it generalizes across cullin-RING ligases.","evidence":"Protein cross-linking mass spectrometry and binding analysis, including Cul2","pmids":["25425648"],"confidence":"Medium","gaps":["High-resolution structure of the encounter complex lacking"]},{"year":2019,"claim":"Delivered atomic-resolution mechanism by solving CDC34 structures alone, bound to E1, and as a CDC34~Ub thioester mimic, revealing the closed conformation required for discharge.","evidence":"X-ray crystallography with biochemical and cell-based functional validation","pmids":["31341161"],"confidence":"High","gaps":["Structure of the full CDC34~Ub–SCF–substrate complex during chain elongation not captured"]},{"year":2020,"claim":"Uncovered an SCF-independent, pro-oncogenic role where CDC34 competes with c-Cbl to shield EGFR from degradation, promoting NSCLC growth.","evidence":"siRNA screen, co-IP, and in vitro/in vivo tumor models with knockdown","pmids":["32114396"],"confidence":"Medium","gaps":["Whether CDC34 catalytic activity is required for EGFR stabilization unclear","Single-lab finding"]},{"year":2026,"claim":"Revealed a regulatory role for the N-terminal extension in controlling processive self-elongation using a selective nanobody tool.","evidence":"Nanobody isolation and in vitro charging/chain synthesis assays","pmids":["41706475"],"confidence":"Medium","gaps":["Physiological relevance of N-terminal control in cells not established","Single-lab biochemical study"]},{"year":null,"claim":"How CDC34's many regulatory inputs (CK2, PKA/Sch9 phosphorylation, COP9 protection, dimerization, conformational tail/Ub contacts) are integrated on an assembled SCF to determine substrate choice and proteolytic versus non-proteolytic outcomes in living cells remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of the complete CDC34~Ub–SCF–substrate elongation complex","Substrate-selection logic in vivo not fully mapped","Integration of multiple kinase inputs under physiological signaling untested"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,9,30,33,41]},{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,1,30,38]},{"term_id":"GO:0031386","term_label":"protein tag activity","supporting_discovery_ids":[0,1,21]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[7,39,43]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,23,26]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[23,26]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[23]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,11,16,32]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[11,13,17,19,38]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[19,20,38]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[18,21,22]}],"complexes":["SCF (Skp1-Cul1/Cdc53-F-box) ubiquitin ligase"],"partners":["CUL1","SKP1","RBX1","SKP2","BTRC","CSNK2B","EGFR","UBE2D1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P49427","full_name":"Ubiquitin-conjugating enzyme E2 R1","aliases":["(E3-independent) E2 ubiquitin-conjugating enzyme R1","E2 ubiquitin-conjugating enzyme R1","Ubiquitin-conjugating enzyme E2-32 kDa complementing","Ubiquitin-conjugating enzyme E2-CDC34","Ubiquitin-protein ligase R1"],"length_aa":236,"mass_kda":26.7,"function":"E2 ubiquitin-conjugating enzyme that accepts ubiquitin from an E1 ubiquitin-activating protein, and catalyzes its covalent attachment to other proteins by an E3 ubiquitin-protein ligase complex (PubMed:10329681, PubMed:17588522, PubMed:20061386, PubMed:38326650). In vitro catalyzes 'Lys-48'-linked polyubiquitination (PubMed:22496338). Cooperates with the E2 UBCH5C and the SCF(FBXW11) E3 ligase complex for the polyubiquitination of NFKBIA leading to its subsequent proteasomal degradation (PubMed:10329681, PubMed:10918611, PubMed:17698585). Performs ubiquitin chain elongation building ubiquitin chains from the UBE2D3-primed NFKBIA-linked ubiquitin. UBE2D3 acts as an initiator E2, priming the phosphorylated NFKBIA target at positions 'Lys-21' and/or 'Lys-22' with a monoubiquitin. Cooperates with the SCF(SKP2) E3 ligase complex to regulate cell proliferation through ubiquitination and degradation of MYBL2 and KIP1 (PubMed:10871850, PubMed:15652359, PubMed:19112177). Involved in ubiquitin conjugation and degradation of CREM isoform ICERIIgamma and ATF15 resulting in abrogation of ICERIIgamma- and ATF5-mediated repression of cAMP-induced transcription during both meiotic and mitotic cell cycles. Involved in the regulation of the cell cycle G2/M phase through its targeting of the WEE1 kinase for ubiquitination and degradation (PubMed:19126550). Also involved in the degradation of beta-catenin (PubMed:12037680). Is target of human herpes virus 1 protein ICP0, leading to ICP0-dependent dynamic interaction with proteasomes (PubMed:11805320, PubMed:12060736)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/P49427/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CDC34","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CDC34","total_profiled":1310},"omim":[{"mim_id":"613336","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 9; MARCHF9","url":"https://www.omim.org/entry/613336"},{"mim_id":"613335","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 8; MARCHF8","url":"https://www.omim.org/entry/613335"},{"mim_id":"613332","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 2; MARCHF2","url":"https://www.omim.org/entry/613332"},{"mim_id":"613331","title":"MEMBRANE-ASSOCIATED RING-CH FINGER PROTEIN 1; MARCHF1","url":"https://www.omim.org/entry/613331"},{"mim_id":"612598","title":"RING FINGER PROTEIN 11; RNF11","url":"https://www.omim.org/entry/612598"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nuclear speckles","reliability":"Supported"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CDC34"},"hgnc":{"alias_symbol":["E2-CDC34","UBE2R1","UBC3"],"prev_symbol":[]},"alphafold":{"accession":"P49427","domains":[{"cath_id":"3.10.110.10","chopping":"7-189","consensus_level":"high","plddt":94.9444,"start":7,"end":189}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P49427","model_url":"https://alphafold.ebi.ac.uk/files/AF-P49427-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P49427-F1-predicted_aligned_error_v6.png","plddt_mean":85.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CDC34","jax_strain_url":"https://www.jax.org/strain/search?query=CDC34"},"sequence":{"accession":"P49427","fasta_url":"https://rest.uniprot.org/uniprotkb/P49427.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P49427/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P49427"}},"corpus_meta":[{"pmid":"2842867","id":"PMC_2842867","title":"The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme.","date":"1988","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/2842867","citation_count":401,"is_preprint":false},{"pmid":"10385629","id":"PMC_10385629","title":"Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.","date":"1999","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/10385629","citation_count":352,"is_preprint":false},{"pmid":"10975521","id":"PMC_10975521","title":"Regulation of transcription by ubiquitination without proteolysis: Cdc34/SCF(Met30)-mediated inactivation of the transcription factor Met4.","date":"2000","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/10975521","citation_count":247,"is_preprint":false},{"pmid":"9499404","id":"PMC_9499404","title":"Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box proteincomplexes that regulate cell division and methionine biosynthesis in yeast.","date":"1998","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/9499404","citation_count":242,"is_preprint":false},{"pmid":"16360039","id":"PMC_16360039","title":"Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34.","date":"2005","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/16360039","citation_count":240,"is_preprint":false},{"pmid":"21683433","id":"PMC_21683433","title":"An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.","date":"2011","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/21683433","citation_count":197,"is_preprint":false},{"pmid":"9430629","id":"PMC_9430629","title":"Association of human CUL-1 and ubiquitin-conjugating enzyme CDC34 with the F-box protein p45(SKP2): evidence for evolutionary conservation in the subunit composition of the CDC34-SCF pathway.","date":"1998","source":"The EMBO 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microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/10564490","citation_count":128,"is_preprint":false},{"pmid":"9836638","id":"PMC_9836638","title":"Coupling of mitosis to the completion of S phase through Cdc34-mediated degradation of Wee1.","date":"1998","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/9836638","citation_count":119,"is_preprint":false},{"pmid":"20347421","id":"PMC_20347421","title":"Priming and extending: a UbcH5/Cdc34 E2 handoff mechanism for polyubiquitination on a SCF substrate.","date":"2010","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/20347421","citation_count":109,"is_preprint":false},{"pmid":"21474069","id":"PMC_21474069","title":"Essential role for ubiquitin-ubiquitin-conjugating enzyme interaction in ubiquitin discharge from Cdc34 to substrate.","date":"2011","source":"Molecular 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1988,\n      \"finding\": \"CDC34 encodes a ubiquitin-conjugating enzyme (E2); the bacterially expressed CDC34 protein catalyzes covalent attachment of ubiquitin to histones H2A and H2B in vitro, establishing its enzymatic activity and its role in G1-to-S cell cycle transition.\",\n      \"method\": \"In vitro ubiquitination assay with bacterially expressed protein; genetic complementation\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of enzymatic activity, foundational paper replicated extensively\",\n      \"pmids\": [\"2842867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"CDC34 is a bifunctional E2 enzyme competent in both E3-independent and E3-dependent ubiquitin conjugation reactions, and shows marked kinetic selectivity for processive multiubiquitination via Lys-48 of ubiquitin, in contrast to RAD6 which does not preferentially use K48.\",\n      \"method\": \"In vitro ubiquitin conjugation assays with purified recombinant proteins; kinetic analysis; K48R ubiquitin variants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis of ubiquitin, replicated in multiple studies\",\n      \"pmids\": [\"1848239\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"The CDC34 C-terminal tail (residues 171-244) is a portable determinant of cell cycle function; transplanting this tail onto RAD6 generates a chimeric E2 that performs CDC34 cell cycle functions, while the catalytic domain alone is insufficient for cell cycle activity.\",\n      \"method\": \"Chimeric E2 construction; in vivo complementation of cdc34 mutants; deletion analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mutagenesis plus functional complementation, independently replicated in companion paper (PMID:1639076)\",\n      \"pmids\": [\"1639075\", \"1639076\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Bacterially expressed Cdc34 catalyzes its own ubiquitination (autoubiquitination) via intramolecular transfer, forming predominantly a single Lys48-linked multiubiquitin chain; the chain attaches within the C-terminal region (residues 215-295) on any of four lysines (K273, K277, K293, K294).\",\n      \"method\": \"In vitro autoubiquitination assay with purified recombinant CDC34; hydroxylamine cleavage; site-directed mutagenesis of lysine residues\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with systematic mutagenesis, single lab\",\n      \"pmids\": [\"8383676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Human CDC34 cDNA functionally complements yeast CDC34 deletion, establishing that the human protein is a functional homolog of the yeast cell cycle ubiquitin-conjugating enzyme; the gene is located on chromosome 19p13.3.\",\n      \"method\": \"Yeast complementation; Southern blot; chromosomal mapping\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional complementation across species, single lab\",\n      \"pmids\": [\"8248134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Cdc34 (Ubc3) is itself a substrate for ubiquitination and phosphorylation in vivo; immunochemical localization places the protein in the nucleus.\",\n      \"method\": \"In vivo ubiquitination assay; phosphorylation detection; immunofluorescence/immunochemical localization\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct detection of PTMs in vivo and localization, single lab\",\n      \"pmids\": [\"8164658\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"A 39-residue region of the CDC34 C-terminal tail adjacent to the catalytic domain is necessary and sufficient for full cell cycle function and mediates CDC34 self-association (dimerization) in vitro, as shown by cross-linking.\",\n      \"method\": \"Deletion mutagenesis; biophysical characterization; chemical cross-linking; in vivo complementation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical methods in single lab\",\n      \"pmids\": [\"7929378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Cdc34 contains a non-covalent ubiquitin binding site; overexpression of ubiquitin suppresses cdc34 temperature-sensitive mutations in an allele-specific manner, and chemical cross-linking confirms a specific noncovalent Ub-CDC34 interaction, indicating correct positioning of Ub on the E2 surface is required for function.\",\n      \"method\": \"Genetic suppression screen; chemical cross-linking; in vivo complementation with ubiquitin variants\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — genetic and biochemical evidence, single lab\",\n      \"pmids\": [\"7721857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Cdc34 residues S73 and S97 (defining the Cdc34 class of E2 catalytic domains) are critical for function; S97 (near active-site C95) is essential, and intragenic suppression between S97D and S73K mutations, further rescued by deletion of residues 103-114 (the unique Cdc34 insertion), defines epistatic interactions within the catalytic domain.\",\n      \"method\": \"Site-directed mutagenesis; in vivo complementation; intragenic suppression analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with functional readout, single lab\",\n      \"pmids\": [\"7565715\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"A dominant-negative Cdc34 (C95S/L99S double mutant) blocks cell growth when overexpressed and inhibits in vitro ubiquitination of the Cdc34-specific substrate Cln2, establishing that Cys95 is the catalytic active-site residue.\",\n      \"method\": \"Site-directed mutagenesis; in vitro ubiquitination assay; overexpression phenotype analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — active-site mutagenesis with in vitro and in vivo functional validation\",\n      \"pmids\": [\"7592826\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Cbf2p/Ndc10p (a kinetochore protein) is an in vivo substrate of Cdc34; purified Cdc34p catalyzes monoubiquitin conjugation to Cbf2p in vitro, and cdc34-2 mutation alters Cbf2p modification pattern in vivo.\",\n      \"method\": \"In vitro ubiquitination assay; immunoprecipitation with anti-ubiquitin antibody; genetic suppression\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro reconstitution plus in vivo evidence, single lab\",\n      \"pmids\": [\"7651401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"SIC1 multiubiquitination is reconstituted in yeast extract and requires CDC34 (E2), CDC4, CDC53, SKP1, and CLN/CDC28 kinase; the N-terminal 160 residues of Sic1 are necessary and sufficient for CDC34-dependent ubiquitination.\",\n      \"method\": \"In vitro reconstitution in fractionated yeast extract; immunodepletion; deletion analysis of Sic1\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with multiple components, replicated in later studies\",\n      \"pmids\": [\"9285816\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"In Xenopus egg extracts, Cdc34p present in a large complex is required for initiation of DNA replication and appears to regulate Cdk2-cyclin E function, possibly through degradation of the Xenopus CDK inhibitor Xic1.\",\n      \"method\": \"Xenopus egg extract reconstitution; immunodepletion of Cdc34\",\n      \"journal\": \"Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical reconstitution in Xenopus extract, single lab\",\n      \"pmids\": [\"9287222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Cdc53 functions as a scaffold protein in SCF complexes, containing independent binding sites for Cdc34 and Skp1; Skp1 bridges Cdc53 to F-box proteins (Cdc4, Met30, Grr1) which provide substrate specificity, while the Cdc34-Cdc53-Skp1 E2/E3 core is required for all SCF functions.\",\n      \"method\": \"Co-immunoprecipitation; two-hybrid; in vivo genetic analysis; biochemical fractionation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple F-box specificity tested, replicated across labs\",\n      \"pmids\": [\"9499404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"SCF(Met30) and Cdc34 are required for degradation of the Cdk-inhibitory kinase Swe1; Met30 binds Swe1 in vivo (GST pulldown), and extracts from cdc34 or met30 mutants are defective in Swe1 polyubiquitination.\",\n      \"method\": \"GST pulldown; in vitro polyubiquitination assay with mutant extracts; genetic interactions\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical pulldown plus in vitro ubiquitination, single lab\",\n      \"pmids\": [\"9716410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Human CUL-1 and CDC34 are partners of p45(SKP2) in vivo, forming a large multiprotein complex with p19(SKP1) and cyclin A, representing the human SCF-type E3 ubiquitin ligase complex.\",\n      \"method\": \"Co-immunoprecipitation from human cells; complex purification\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP from mammalian cells, single lab\",\n      \"pmids\": [\"9430629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Wee1 kinase is degraded in a Cdc34-dependent fashion in Xenopus egg extracts; this proteolysis is required for timely entry into mitosis and is inhibited when DNA replication is blocked, coupling mitosis to completion of S phase.\",\n      \"method\": \"Xenopus egg extract assay; immunodepletion of Cdc34; DNA replication checkpoint experiments\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical reconstitution with immunodepletion, functional mitotic entry readout\",\n      \"pmids\": [\"9836638\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Hrt1 (RING-H2 protein) is a subunit of SCF that binds Cdc4, Cdc53, and Cdc34 (but not Skp1) individually, potently stimulates SCF E3 activity, and enables reconstitution of Cln2 ubiquitination; the Cdc53/Hrt1 subcomplex activates Cdc34 autoubiquitination by a mechanism not requiring a reactive thiol.\",\n      \"method\": \"Mass spectrometry identification; recombinant SCF reconstitution; binding assays; in vitro ubiquitination\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution with recombinant components, MS identification, replicated\",\n      \"pmids\": [\"10385629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Human Cdc34 ubiquitinates repressors of cAMP-induced transcription hICERIIgamma and hATF5 in mammalian cells, targeting them for proteasomal degradation; this requires active ubiquitin-conjugating enzyme activity and abrogates repression of cAMP-induced transcription.\",\n      \"method\": \"Yeast two-hybrid; transfection with dominant-negative and antisense CDC34; ubiquitination assays in cells\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple cellular methods, single lab\",\n      \"pmids\": [\"10373550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Phosphorylation-dependent ubiquitination of IκBα is catalyzed by Ubc3 (CDC34) together with SCF(β-TRCP); ubiquitin is transferred directly from the E2 to phospho-IκBα in an SCF(β-TRCP)-dependent reaction. Ubc4 is also capable but is 19-fold more efficient than Ubc3 in this reaction in THP.1 cells.\",\n      \"method\": \"In vitro reconstitution with recombinant components; co-immunoprecipitation from human cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — full biochemical reconstitution with phosphorylation-dependent ubiquitination\",\n      \"pmids\": [\"10918611\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"A complex containing βTrCP, Skp1, and Cdc53/Cul1 recruits Cdc34 to catalyze polyubiquitination of phosphorylated IκBα peptide; Ubc5 only stimulates mono- and di-ubiquitination whereas Cdc34 is required for polyubiquitination.\",\n      \"method\": \"In vitro ubiquitination assay with phosphopeptide substrate; reconstituted multiprotein complex\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical reconstitution, single lab\",\n      \"pmids\": [\"10437795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Met4 transcription factor is ubiquitinated by Cdc34/SCF(Met30) but remains stable (proteolysis-independent ubiquitination); ubiquitinated Met4 associates with target promoters but fails to form functional transcription complexes, revealing a non-proteolytic function of Cdc34/SCF ubiquitination.\",\n      \"method\": \"Genetic suppression (met4Δ suppresses met30Δ lethality); chromatin immunoprecipitation; ubiquitination assays; stability analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis plus biochemical assays, novel non-proteolytic mechanism confirmed by multiple approaches\",\n      \"pmids\": [\"10975521\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"B-Myb physically interacts with CDC34 (E2) and p45Skp2 (E3), and ectopic expression of CDC34 or p45Skp2 accelerates B-Myb degradation; cyclin A promotes CDC34-SCF(p45Skp2)-dependent ubiquitination of B-Myb via its C-terminal domain.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assay in cells; overexpression/deletion constructs\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and cellular ubiquitination, single lab\",\n      \"pmids\": [\"10871850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"During interphase, human CDC34 localizes to distinct nuclear and cytoplasmic speckles; nuclear localization requires C-terminal sequences; in anaphase, CDC34 co-localizes with β-tubulin at the mitotic spindle.\",\n      \"method\": \"Immunofluorescence; biochemical fractionation; deletion mutant analysis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — direct localization experiments with functional domain mapping, single lab\",\n      \"pmids\": [\"10769200\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HSV-1 ICP0 RING finger domain (exon 2) binds Cdc34 E2 and promotes its polyubiquitination; ICP0 functions as a unimolecular E3 ubiquitin ligase where the RING finger binds E2 while a separate C-terminal domain has ligase activity.\",\n      \"method\": \"In vitro substrate-independent ubiquitination; co-immunoprecipitation; RING finger domain analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding and ubiquitination assays, single lab\",\n      \"pmids\": [\"11447293\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Elevation of Cdc34 protein levels by microinjection into mammalian cells at prophase selectively blocks CENP-E association with kinetochores and inhibits chromosome congression, causing prometaphase arrest; this effect is not reversed by proteasome inhibitors.\",\n      \"method\": \"Microinjection of recombinant Cdc34 into mitotic mammalian cells; immunofluorescence; electron microscopy\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein injection with specific cellular phenotype, single lab\",\n      \"pmids\": [\"11514588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CK2β (casein kinase 2 regulatory subunit) interacts with human CDC34 in vivo; CK2 phosphorylates human CDC34 at five sites within the C-terminal 36 amino acids in vitro; mutation of these CK2 sites shifts CDC34 localization from nucleus to cytoplasm.\",\n      \"method\": \"Yeast two-hybrid; co-immunoprecipitation; in vitro kinase assay; site-directed mutagenesis; immunofluorescence\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods, single lab\",\n      \"pmids\": [\"11546811\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Cdc34 self-associates in vivo (shown by co-immunoprecipitation); self-association is dependent on intact Cdc34-Ub thiolester and requires active-site Cys and residues S73, S97, and the catalytic domain insertion; self-association is a prerequisite for multi-Ub chain assembly.\",\n      \"method\": \"Co-immunoprecipitation; site-directed mutagenesis; in vitro thiolester formation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP in vivo plus mutagenesis, single lab\",\n      \"pmids\": [\"12861024\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Release of ubiquitin-charged Cdc34~Ub from the RING domain of SCF(Cdc4) is essential for ubiquitination of the bound substrate Sic1; formation of the Cdc34~Ub thioester increases dissociation rate from RING, and an F72V Cdc34 mutant with increased RING affinity blocks substrate ubiquitination.\",\n      \"method\": \"Kinetic binding assays; F72V Cdc34 mutant; in vitro ubiquitination assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mechanistic dissection with mutagenesis and kinetic assays, published in high-profile journal\",\n      \"pmids\": [\"13678584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Cdc34 mediates cell cycle-dependent degradation of c-Ski proto-oncoprotein; dominant-negative Cdc34 stabilizes Ski both in vitro and in vivo, enhancing Ski's ability to antagonize TGF-β signaling.\",\n      \"method\": \"In vitro ubiquitination assay; dominant-negative CDC34 overexpression; protein stability assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro and in vivo approaches, single lab\",\n      \"pmids\": [\"15122324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Sic1 ubiquitination by SCF(Cdc4)/Cdc34 proceeds in two steps: rate-limiting attachment of the first ubiquitin, followed by rapid elongation of a K48-linked chain; a conserved acidic loop of Cdc34 has no effect on first ubiquitin attachment but is required for processivity and K48-linkage specificity of chain synthesis.\",\n      \"method\": \"In vitro reconstitution; mutagenesis of the Cdc34 acidic loop; kinetic analysis; linkage determination\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with mutagenesis and mechanistic dissection, widely cited\",\n      \"pmids\": [\"16360039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Human Cdc34 activity in K48-linked polyubiquitin chain synthesis is dramatically enhanced by the SCF ROC1/CUL1-Nedd8 core ligase module; N-terminal GST-fusion or chemical-induced FKBP dimerization of Cdc34 activates it constitutively for K48-chain assembly, demonstrating that dimerization/proximity activates Cdc34 catalysis.\",\n      \"method\": \"In vitro ubiquitin chain synthesis assay; GST-fusion dimerization; chemical-induced dimerization with FKBP/AP20187\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical approaches, single lab\",\n      \"pmids\": [\"16210246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Knockdown of human CDC34 by antisense oligonucleotides inhibits degradation of p27Kip1 and prevents cellular proliferation; reducing p27Kip1 with antisense reverses the anti-proliferative phenotype, establishing that CDC34 controls proliferation specifically through p27Kip1 levels.\",\n      \"method\": \"Antisense oligonucleotide knockdown; western blot; cell proliferation assay; epistasis by double knockdown\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis by double knockdown, single lab\",\n      \"pmids\": [\"15652359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Human Cdc34 employs distinct sites for monoubiquitination vs. polyubiquitin chain assembly: the conserved charged stretch (143-153) and acidic loop residues D102/D103 are required for Ub-Ub ligation but not for mono-ubiquitination of IκBα; N85 stabilizes the oxyanion and coordinates, with S138, the attacking lysine for catalysis; S95 and E108/E112 are required for proper donor Ub positioning.\",\n      \"method\": \"Site-directed mutagenesis; in vitro ubiquitination assay; GST-fusion rescue experiments\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis with mechanistic in vitro readouts, single lab\",\n      \"pmids\": [\"17698585\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"CK2 phosphorylates yeast Cdc34 at Ser207 and Ser216 (and human Cdc34 at S203, S222, S231) in the acidic tail domain; phosphorylation increases Cdc34 ubiquitination activity towards Sic1 with SCF(Cdc4) in vitro; phosphosite mutants with higher activity show accelerated cell cycle progression and Sic1 degradation in vivo.\",\n      \"method\": \"In vitro CK2 kinase assay; in vitro ubiquitination assay; site-directed mutagenesis; cell cycle synchronization and flow cytometry\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro kinase + ubiquitination assay + in vivo cell cycle readout, single lab\",\n      \"pmids\": [\"17461777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"ATF5 is a substrate of Cdc34-dependent ubiquitin-proteasome degradation; cisplatin prevents ATF5 ubiquitin-dependent degradation by promoting nuclear-to-cytoplasmic translocation of Cdc34 and reducing the ATF5-Cdc34 interaction.\",\n      \"method\": \"Co-immunoprecipitation; cellular fractionation; ubiquitination assay; cisplatin treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP and fractionation, single lab\",\n      \"pmids\": [\"18458088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"CK2 phosphorylates Cdc34 within its N-terminal catalytic domain at Ser130 and Ser167; this phosphorylation strongly stimulates Cdc34 ubiquitin charging in vitro; the S130A/S167A mutant has normal basal activity but cannot be activated by CK2 and fails to complement a cdc34-2 ts strain.\",\n      \"method\": \"Mass spectrometry; in vitro kinase assay; ubiquitin charging assay; in vivo complementation\",\n      \"journal\": \"Cell cycle\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — MS-confirmed phosphosites, in vitro charging assay, in vivo complementation\",\n      \"pmids\": [\"18418079\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The Cdc34 acidic C-terminal tail contributes to submicromolar Km for SCF(Cdc4) binding (electrostatic interaction) and also has an unexpected direct catalytic function in promoting ubiquitin transfer to substrate, independent of its role in E3 binding.\",\n      \"method\": \"Kinetic analysis; deletion mutants; Cdc34-Cul1 fusion constructs; in vitro ubiquitination assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical reconstitution with mechanistic dissection using fusions and deletions, single lab\",\n      \"pmids\": [\"19875449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Polyubiquitination of IκBα by SCF(βTrCP2) involves a UbcH5/Cdc34 E2 handoff: UbcH5 rapidly transfers the first ubiquitin to IκBα K21/K22; Cdc34 then assembles the K48-linked polyubiquitin chain on the substrate-linked receptor ubiquitin, dependent on SCF and the substrate N-terminus.\",\n      \"method\": \"Biochemical reconstitution with recombinant components; ubiquitin fusion receptor constructs; kinetic analysis\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — full reconstitution with mechanistic dissection, single lab\",\n      \"pmids\": [\"20347421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Human Cdc34 C-terminus contains two ubiquitin-binding sites (UBS1: residues 206-215; UBS2: 216-225) that interact non-covalently with ubiquitin near K48 and the C-terminal tail; aromatic residues (F206, Y207, Y210, Y211) are required for Ub binding and contribute to SCF-dependent ubiquitination; UBS1 is required for yeast complementation.\",\n      \"method\": \"NMR chemical shift perturbation; site-directed mutagenesis; in vitro IκBα ubiquitination assay; in vivo complementation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure of interaction plus mutagenesis and functional reconstitution, single lab\",\n      \"pmids\": [\"20353940\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Lysine selectivity by Cdc34 during polyubiquitination of Sic1 depends on amino acids proximal to acceptor lysines both on substrate and on ubiquitin, linked to key residues in the Cdc34 catalytic core; these core residues are independent of SCF and alter whether Cdc34 monoubiquitinates or polyubiquitinates Sic1.\",\n      \"method\": \"Site-directed mutagenesis; in vitro ubiquitination assay with Sic1 and ubiquitin variants; SCF-independent assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — systematic mutagenesis with mechanistic in vitro readout, single lab\",\n      \"pmids\": [\"20194622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Ubiquitin I44A mutation profoundly inhibits its ability to serve as donor for Cdc34-mediated chain initiation or elongation; this is partially rescued by computationally designed compensatory mutations in Cdc34, indicating that Cdc34 interacts with donor ubiquitin to organize the active site for deprotonation of acceptor lysine.\",\n      \"method\": \"In vitro ubiquitination assay; mutagenesis of ubiquitin and Cdc34; hydroxylamine rescue; pH titration\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mechanistic mutagenesis with computational design and biochemical validation, single lab\",\n      \"pmids\": [\"21474069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CC0651, a small molecule, selectively inhibits human Cdc34 by inserting into a cryptic allosteric pocket distant from the catalytic site, causing conformational changes without affecting E1/E3 interactions or thioester formation, but blocking ubiquitin discharge to acceptor lysines; inhibition causes p27Kip1 accumulation and cancer cell antiproliferation.\",\n      \"method\": \"Crystal structure determination; biochemical inhibition assays; cell-based p27 accumulation; mutagenesis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mechanistic biochemistry plus cellular validation\",\n      \"pmids\": [\"21683433\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The disordered CDC34 C-terminus intramolecularly interacts with catalytically bound ubiquitin in the CDC34~Ub complex, associating with a novel lysine-rich surface (K6, K11, K29, K33) on ubiquitin opposite the canonical hydrophobic patch; this interaction exists in two slowly exchanging conformations (compact vs. extended).\",\n      \"method\": \"NMR spectroscopy; CDC34-Ub disulfide mimetic; chemical shift perturbation mapping\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural data with mechanistic implications, single lab\",\n      \"pmids\": [\"21296085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"CK2 phosphorylation of conserved serine residues in the Cdc34 catalytic domain and the acidic β4α2 loop function as a molecular switch: phosphorylation creates electrostatic repulsion that promotes opening of the catalytic cleft, thereby enhancing ubiquitin charging.\",\n      \"method\": \"Molecular dynamics simulation (2.5 µs); biochemical assays; comparative sequence analysis\",\n      \"journal\": \"PLoS computational biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 (MD) + Tier 2 (biochemical) / Moderate — computational plus biochemical, single lab\",\n      \"pmids\": [\"21637798\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PKA and Sch9 (nutrient-sensing kinases) phosphorylate Cdc34 at S97 in the catalytic domain in vivo and in vitro; S97 phosphorylation is cell cycle regulated (elevated during active growth, decreased during arrest), providing a direct link between nutrient sensing and G1 cell division.\",\n      \"method\": \"In vitro kinase assay; mass spectrometry; cell cycle synchronization; genetic analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay and cell cycle regulation, single lab\",\n      \"pmids\": [\"22087249\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The acidic loop of human Cdc34 promotes ubiquitination by two mechanisms: (1) facilitating Cdc34-SCF interaction and (2) two glutamic acid residues on the distal side collaborate with a conserved histidine on the proximal side to suppress the pKa of an ionizing species critical for catalysis.\",\n      \"method\": \"In vitro ubiquitination assay; mutagenesis; binding assays; pKa analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple mechanistic modes identified biochemically, single lab\",\n      \"pmids\": [\"24129577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The Cdc34-SCF interaction occurs in multiple conformations: several residues of the Cdc34 acidic tail contact a broad region of the SCF basic canyon via electrostatic interactions; similar patterns are seen with Cul2, implicating a common mechanism across cullin-RING ligases.\",\n      \"method\": \"Protein cross-linking; mass spectrometry; binding analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cross-linking MS with multiple conformations identified, single lab\",\n      \"pmids\": [\"25425648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Ube2r1 (Cdc34) requires its C-terminal extension (184-196) for efficient K48-linked polyubiquitination, while the acidic loop residues (Tyr-102/Tyr-104 in the related Ube2g1) control ubiquitin binding and K48-ubiquitylation; oxyester but not disulfide Ube2r1~Ub mimics are functional equivalents of the thioester.\",\n      \"method\": \"In vitro ubiquitylation assay; site-directed mutagenesis; NMR with oxyester E2~Ub mimics\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — NMR and biochemical assays, single lab\",\n      \"pmids\": [\"25471371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Ube2R1/2 (Cdc34) has exquisite K48 specificity; computational docking and biochemical experiments identify a key electrostatic interaction between Arg54 on ubiquitin and Asp143 on Ube2R1/2 that guides K48 to the active site.\",\n      \"method\": \"Computational docking; site-directed mutagenesis; in vitro ubiquitination assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — computation confirmed by biochemistry, single lab\",\n      \"pmids\": [\"27044868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structures of Cdc34 alone, in complex with E1 (Uba1), and as a Cdc34~Ub thioester mimetic reveal: (1) unique E1-E2 binding mode requiring conformational changes in both proteins for transthiolation; (2) contacts between the Cdc34 C-terminal extension and ubiquitin stabilize Cdc34~Ub in a closed conformation critical for ubiquitin discharge.\",\n      \"method\": \"X-ray crystallography; biochemical assays; cell-based functional assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple crystal structures with biochemical and cell-based validation in single study\",\n      \"pmids\": [\"31341161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CDC34 competes with c-Cbl E3 ligase to bind EGFR at Y1045, thereby inhibiting EGFR polyubiquitination and degradation, and promoting NSCLC cell proliferation; knockdown of CDC34 inhibits EGFR-driven lung tumor growth in mouse models.\",\n      \"method\": \"siRNA library screen; co-immunoprecipitation; in vitro and in vivo tumor models; overexpression/knockdown\",\n      \"journal\": \"EBioMedicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP and in vivo mouse model, single lab\",\n      \"pmids\": [\"32114396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"CSN (COP9 signalosome) protects Cdc34/UBC3 from SCF(βTrCP)-mediated degradation; knockdown of CSN4 or CSN5 induces proteasomal degradation of Cdc34, and the acidic C-terminal extension of UBC3 is required and sufficient for SCF(βTrCP)-mediated ubiquitination.\",\n      \"method\": \"siRNA knockdown; co-immunoprecipitation; domain mapping with UBC3 C-terminal fusions\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical and genetic approaches, single lab\",\n      \"pmids\": [\"20378537\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"A nanobody (VHH12R1) binding selectively to the Ube2R1 N-terminal extension transiently delays ubiquitin charging, promotes stable mono-ubiquitin conjugate accumulation, and markedly reduces self-directed polyubiquitination without impairing di-ubiquitin synthesis, revealing a regulatory role for the N-terminal extension in controlling processive self-elongation.\",\n      \"method\": \"Nanobody isolation and characterization; in vitro ubiquitin charging and chain synthesis assays; biochemical inhibition analysis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical reconstitution with selective nanobody tool, single lab, preprint/recent publication\",\n      \"pmids\": [\"41706475\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CDC34 (UBE2R1/Ubc3) is a ubiquitin-conjugating enzyme (E2) that forms a thioester with ubiquitin via its active-site Cys95, functions as the obligate E2 for SCF (Skp1-Cullin1-F-box) ubiquitin ligase complexes to catalyze K48-linked polyubiquitin chain assembly on cell cycle regulators (Sic1, Wee1, p27Kip1, cyclin-dependent kinase inhibitors) and other substrates (IκBα, Met4, ATF5, B-Myb, c-Ski), with chain specificity and processivity controlled by a conserved acidic loop and disordered acidic C-terminal tail that mediates SCF binding and positions donor ubiquitin for catalysis; CK2 phosphorylates both the catalytic domain and C-terminal tail to stimulate activity, while the COP9 signalosome protects CDC34 from SCF(βTrCP)-mediated autoubiquitination and degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CDC34 (UBE2R1/Ubc3) is a ubiquitin-conjugating enzyme (E2) that drives the G1-to-S cell cycle transition by assembling K48-linked polyubiquitin chains on regulatory substrates marked for proteasomal degradation [#0, #1]. Catalysis proceeds through a thioester at the active-site Cys95, and a dominant-negative C95S mutant blocks both growth and substrate ubiquitination, establishing the catalytic center [#9]. CDC34 shows intrinsic kinetic selectivity for processive K48-linked multiubiquitination [#1], with chain processivity and linkage specificity governed by a conserved acidic loop and catalytic-core residues that position the donor ubiquitin and lower the pKa of the attacking acceptor lysine [#30, #33, #41, #46]. CDC34 functions as the obligate E2 for SCF (Skp1\\u2013Cullin/Cdc53\\u2013F-box) cullin-RING ligases: Cdc53 scaffolds independent binding sites for CDC34 and Skp1, the RING subunit Hrt1/ROC1 stimulates activity, and the disordered acidic C-terminal tail mediates high-affinity electrostatic binding to the SCF basic canyon while also directly promoting ubiquitin transfer [#13, #17, #37, #47]. Through SCF complexes it ubiquitinates cell cycle regulators including Sic1, Swe1/Wee1, and p27Kip1 to license replication and mitotic entry [#11, #14, #16, #32], and acts with SCF(\\u03b2TrCP) on phospho-I\\u03baB\\u03b1 in a UbcH5/CDC34 handoff in which a priming E2 attaches the first ubiquitin and CDC34 elongates the K48 chain [#19, #38]. CDC34 also mediates degradation of additional substrates (ATF5, hICERII\\u03b3, B-Myb, c-Ski) and, in the case of Met4, catalyzes a non-proteolytic ubiquitination that inactivates a transcription factor without destroying it [#18, #21, #22, #29]. Its activity is tuned by CK2 phosphorylation of both the catalytic domain and acidic tail, which stimulates ubiquitin charging and accelerates cell cycle progression [#34, #36]. CDC34 is itself a substrate for autoubiquitination and SCF(\\u03b2TrCP)-mediated turnover, from which the COP9 signalosome protects it [#3, #52].\",\n  \"teleology\": [\n    {\n      \"year\": 1988,\n      \"claim\": \"Established the founding identity of CDC34 as an enzyme rather than merely a cell cycle gene, by showing the protein directly conjugates ubiquitin.\",\n      \"evidence\": \"In vitro ubiquitination of histones H2A/H2B with bacterially expressed protein plus genetic complementation in yeast\",\n      \"pmids\": [\"2842867\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates not yet identified\", \"Mechanism of cell cycle coupling unknown\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Defined CDC34's distinctive catalytic preference, showing it is a bifunctional E2 with marked kinetic selectivity for processive K48-linked chains, distinguishing it from RAD6.\",\n      \"evidence\": \"In vitro conjugation assays with K48R ubiquitin variants and kinetic analysis of purified recombinant proteins\",\n      \"pmids\": [\"1848239\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of K48 selectivity unresolved\", \"E3 partners not yet defined\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Localized cell cycle function to a portable C-terminal tail rather than the catalytic core, by transplanting the tail onto RAD6 to confer CDC34 activity.\",\n      \"evidence\": \"Chimeric E2 construction and in vivo complementation of cdc34 mutants\",\n      \"pmids\": [\"1639075\", \"1639076\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular partner contacted by the tail unknown\", \"Did not distinguish substrate targeting from E3 binding\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Identified Cys95 as the catalytic active-site residue and demonstrated a dominant-negative blockade of an authentic substrate (Cln2).\",\n      \"evidence\": \"C95S/L99S mutagenesis with in vitro ubiquitination and overexpression growth-arrest phenotype\",\n      \"pmids\": [\"7592826\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase context for Cln2 not yet defined\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Mapped catalytic-domain residues (S73, S97, the Cdc34-class insertion) defining the enzyme family and their epistatic interactions.\",\n      \"evidence\": \"Site-directed mutagenesis with intragenic suppression and in vivo complementation\",\n      \"pmids\": [\"7565715\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Atomic-level role of these residues not structurally resolved at the time\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Placed CDC34 within the SCF machinery by reconstituting Sic1 multiubiquitination requiring CDC34, CDC4, CDC53, SKP1, and Cln/Cdc28 kinase.\",\n      \"evidence\": \"In vitro reconstitution in fractionated yeast extract with immunodepletion and Sic1 deletion analysis\",\n      \"pmids\": [\"9285816\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and architecture of the E2-E3 interaction undefined\", \"RING subunit not yet identified\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined SCF architecture, showing Cdc53 is a scaffold with separate CDC34 and Skp1 sites and that F-box proteins confer substrate specificity, with CDC34 as the shared E2.\",\n      \"evidence\": \"Co-IP, two-hybrid, and genetic analysis across multiple F-box proteins; parallel human SCF(SKP2) complex co-IP\",\n      \"pmids\": [\"9499404\", \"9430629\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CDC34 is positioned relative to substrate for catalysis unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Connected CDC34/SCF to cell cycle timing by showing CDC34-dependent degradation of Cdk-inhibitory kinases Swe1/Wee1 couples mitotic entry to S-phase completion.\",\n      \"evidence\": \"Yeast extract polyubiquitination assays and Xenopus egg extract immunodepletion with replication checkpoint experiments\",\n      \"pmids\": [\"9716410\", \"9836638\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Checkpoint signal that gates CDC34 activity not identified\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Identified the RING subunit Hrt1/ROC1 as an activator of CDC34 and reconstituted SCF ubiquitination with recombinant components.\",\n      \"evidence\": \"Mass spectrometry identification, recombinant SCF reconstitution, and binding/ubiquitination assays\",\n      \"pmids\": [\"10385629\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational mechanism of RING activation of CDC34 not yet resolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Extended CDC34 function beyond cell cycle to NF-\\u03baB signaling, showing it catalyzes SCF(\\u03b2TrCP)-dependent polyubiquitination of phospho-I\\u03baB\\u03b1.\",\n      \"evidence\": \"In vitro reconstitution with recombinant components and co-IP from human cells; phosphopeptide substrate assays\",\n      \"pmids\": [\"10918611\", \"10437795\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative roles of CDC34 versus other E2s in vivo unresolved\", \"Mechanism of chain initiation vs elongation not yet dissected\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Revealed a non-proteolytic mode of CDC34/SCF ubiquitination, where ubiquitinated Met4 is stable but transcriptionally inactivated.\",\n      \"evidence\": \"Genetic epistasis, chromatin immunoprecipitation, ubiquitination and stability assays\",\n      \"pmids\": [\"10975521\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Reader machinery interpreting non-degradative chains not defined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that CDC34 self-association, dependent on the thioester and active-site residues, is a prerequisite for multiubiquitin chain assembly.\",\n      \"evidence\": \"In vivo co-IP, site-directed mutagenesis, and in vitro thiolester assays\",\n      \"pmids\": [\"12861024\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether dimerization occurs on the SCF in vivo not established\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined the catalytic cycle dynamics, showing that release of charged CDC34~Ub from the SCF(Cdc4) RING is essential for substrate ubiquitination.\",\n      \"evidence\": \"Kinetic binding assays with F72V high-RING-affinity mutant and in vitro ubiquitination\",\n      \"pmids\": [\"13678584\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How charging-coupled affinity change is structurally encoded unresolved at the time\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Dissected chain synthesis into rate-limiting initiation and rapid elongation and assigned processivity/K48 specificity to the conserved acidic loop.\",\n      \"evidence\": \"In vitro reconstitution with acidic-loop mutagenesis, kinetics, and linkage determination\",\n      \"pmids\": [\"16360039\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic mechanism by which the loop enforces K48 not yet defined\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Demonstrated proximity/dimerization-driven activation, with the ROC1/CUL1-Nedd8 module or forced dimerization constitutively activating K48 chain assembly.\",\n      \"evidence\": \"In vitro chain synthesis with GST-fusion and FKBP chemical-induced dimerization\",\n      \"pmids\": [\"16210246\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological trigger for the activating proximity in vivo unclear\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Linked CDC34 to mammalian proliferation control specifically through p27Kip1 degradation.\",\n      \"evidence\": \"Antisense knockdown with double-knockdown epistasis and proliferation assays in human cells\",\n      \"pmids\": [\"15652359\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Off-target contributions of antisense not fully excluded\", \"F-box receptor for p27 not addressed here\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Separated the active-site machinery for monoubiquitination from that for chain elongation and assigned individual catalytic residues.\",\n      \"evidence\": \"Systematic mutagenesis with in vitro I\\u03baB\\u03b1 and chain assembly assays\",\n      \"pmids\": [\"17698585\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural confirmation of proposed oxyanion/lysine coordination lacking at the time\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Established CK2 phosphorylation of the acidic tail as a positive regulator linking phosphorylation to faster Sic1 degradation and cell cycle progression.\",\n      \"evidence\": \"In vitro kinase and ubiquitination assays plus in vivo cell cycle synchronization with phosphosite mutants\",\n      \"pmids\": [\"17461777\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How tail phosphorylation alters SCF binding or catalysis mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identified CK2 phosphorylation within the catalytic domain (S130/S167) as required for stimulated ubiquitin charging and in vivo function.\",\n      \"evidence\": \"Mass spectrometry, in vitro charging assays, and complementation with S130A/S167A mutant\",\n      \"pmids\": [\"18418079\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Conformational consequence of catalytic-domain phosphorylation not directly visualized\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed the acidic C-terminal tail has a dual role: submicromolar electrostatic binding to SCF and a direct catalytic contribution to ubiquitin transfer.\",\n      \"evidence\": \"Kinetics, deletion mutants, and CDC34-Cul1 fusion constructs in in vitro ubiquitination\",\n      \"pmids\": [\"19875449\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the catalytic tail function unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Resolved E2 handoff in I\\u03baB\\u03b1 polyubiquitination: UbcH5 primes the substrate and CDC34 elongates the K48 chain on the receptor ubiquitin.\",\n      \"evidence\": \"Biochemical reconstitution with ubiquitin fusion receptor constructs and kinetics\",\n      \"pmids\": [\"20347421\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether handoff applies generally to other SCF substrates not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined non-covalent ubiquitin-binding sites in the human CDC34 C-terminus and identified residues governing K48 lysine selectivity, mapping the structural basis of donor/acceptor positioning.\",\n      \"evidence\": \"NMR chemical shift perturbation, mutagenesis, in vitro ubiquitination, and yeast complementation; Sic1 lysine-selectivity assays\",\n      \"pmids\": [\"20353940\", \"20194622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How these surfaces operate within the assembled SCF in real time unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identified CDC34 as a regulated target of its own pathway, protected from SCF(\\u03b2TrCP)-mediated degradation by the COP9 signalosome.\",\n      \"evidence\": \"siRNA knockdown of CSN4/CSN5, co-IP, and domain mapping of the UBC3 C-terminal extension\",\n      \"pmids\": [\"20378537\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological conditions triggering CDC34 turnover unclear\", \"Single-lab co-IP evidence\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Provided structural and mechanistic detail on donor-ubiquitin engagement, showing the disordered C-terminus contacts a non-canonical lysine-rich ubiquitin surface and that Ile44 is critical for donor function.\",\n      \"evidence\": \"NMR with CDC34-Ub disulfide mimetics, ubiquitin/CDC34 mutagenesis, and computational compensatory design\",\n      \"pmids\": [\"21296085\", \"21474069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional weight of the two conformational states in catalysis not quantified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Validated CDC34 as a druggable target, identifying the allosteric inhibitor CC0651 that blocks ubiquitin discharge and causes p27Kip1 accumulation.\",\n      \"evidence\": \"Crystal structure of the inhibitor-bound enzyme, biochemical inhibition assays, and cell-based p27 accumulation\",\n      \"pmids\": [\"21683433\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy and selectivity of the chemotype not established here\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Proposed a molecular switch model in which CK2 phosphorylation opens the catalytic cleft via electrostatic repulsion to enhance charging.\",\n      \"evidence\": \"Microsecond molecular dynamics simulation with supporting biochemical assays\",\n      \"pmids\": [\"21637798\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Computational model not confirmed by direct structural observation\", \"Single-lab study\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Detailed the multi-conformational electrostatic CDC34 tail\\u2013SCF basic canyon interaction and showed it generalizes across cullin-RING ligases.\",\n      \"evidence\": \"Protein cross-linking mass spectrometry and binding analysis, including Cul2\",\n      \"pmids\": [\"25425648\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"High-resolution structure of the encounter complex lacking\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Delivered atomic-resolution mechanism by solving CDC34 structures alone, bound to E1, and as a CDC34~Ub thioester mimic, revealing the closed conformation required for discharge.\",\n      \"evidence\": \"X-ray crystallography with biochemical and cell-based functional validation\",\n      \"pmids\": [\"31341161\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full CDC34~Ub\\u2013SCF\\u2013substrate complex during chain elongation not captured\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Uncovered an SCF-independent, pro-oncogenic role where CDC34 competes with c-Cbl to shield EGFR from degradation, promoting NSCLC growth.\",\n      \"evidence\": \"siRNA screen, co-IP, and in vitro/in vivo tumor models with knockdown\",\n      \"pmids\": [\"32114396\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CDC34 catalytic activity is required for EGFR stabilization unclear\", \"Single-lab finding\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed a regulatory role for the N-terminal extension in controlling processive self-elongation using a selective nanobody tool.\",\n      \"evidence\": \"Nanobody isolation and in vitro charging/chain synthesis assays\",\n      \"pmids\": [\"41706475\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance of N-terminal control in cells not established\", \"Single-lab biochemical study\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How CDC34's many regulatory inputs (CK2, PKA/Sch9 phosphorylation, COP9 protection, dimerization, conformational tail/Ub contacts) are integrated on an assembled SCF to determine substrate choice and proteolytic versus non-proteolytic outcomes in living cells remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of the complete CDC34~Ub\\u2013SCF\\u2013substrate elongation complex\", \"Substrate-selection logic in vivo not fully mapped\", \"Integration of multiple kinase inputs under physiological signaling untested\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 9, 30, 33, 41]},\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 1, 30, 38]},\n      {\"term_id\": \"GO:0031386\", \"supporting_discovery_ids\": [0, 1, 21]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [7, 39, 43]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 23, 26]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [23, 26]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [23]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 11, 16, 32]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [11, 13, 17, 19, 38]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [19, 20, 38]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [18, 21, 22]}\n    ],\n    \"complexes\": [\"SCF (Skp1-Cul1/Cdc53-F-box) ubiquitin ligase\"],\n    \"partners\": [\"CUL1\", \"SKP1\", \"RBX1\", \"SKP2\", \"BTRC\", \"CSNK2B\", \"EGFR\", \"UBE2D1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}