Abstract
Cdc34 is an E2-conjugating enzyme required for catalyzing the polyubiquitination reaction mediated by the Skp1.CUL1.F-box (SCF) protein E3 ubiquitin (Ub) ligase. Here, we show that the activity of human Cdc34 in the Ub-Ub ligation reaction was enhanced dramatically by SCF's core Ub ligase module, composed of a heterodimeric complex formed by the ROC1 RING finger protein and the CUL1 C terminus that contains a Nedd8 moiety covalently conjugated at K720. Unexpectedly, we found that N-terminal fusion of a GST moiety to human Cdc34 generated dimeric GST-Cdc34 that was constitutively active in supporting the assembly of K48-linked polyUb chains independently of SCF. Furthermore, fusion of a FK506-binding protein (FKBP) to the N terminus of human Cdc34 yielded FKBP-Cdc34 that was induced to form a dimer upon treatment with the chemical inducer AP20187. The AP20187-induced dimeric form of FKBP-Cdc34 was substantially more active than the monomer in catalyzing Ub-Ub ligation. Thus, juxtaposition of human Cdc34 activates its catalytic capability, suggesting that the SCF-mediated polyubiquitination reaction may require the conversion of Cdc34 from an inactive monomer to a highly active dimeric form.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Anaphase-Promoting Complex-Cyclosome
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Carrier Proteins / metabolism
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Cell Cycle Proteins / metabolism
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Cullin Proteins / metabolism
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Dimerization
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Enzyme Activation
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Humans
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Polyubiquitin / biosynthesis
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Protein Structure, Quaternary*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Tacrolimus / analogs & derivatives
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Tacrolimus / metabolism
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Tacrolimus Binding Proteins / chemistry
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Tacrolimus Binding Proteins / genetics
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Tacrolimus Binding Proteins / metabolism
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Ubiquitin-Conjugating Enzymes / chemistry
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Ubiquitin-Conjugating Enzymes / genetics
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Ubiquitin-Conjugating Enzymes / metabolism
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Ubiquitin-Protein Ligase Complexes / chemistry*
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Ubiquitin-Protein Ligase Complexes / genetics
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Ubiquitin-Protein Ligase Complexes / metabolism*
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Ubiquitin-Protein Ligases / chemistry
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
Substances
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AP20187
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Carrier Proteins
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Cell Cycle Proteins
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Cullin 1
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Cullin Proteins
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RBX1 protein, human
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Recombinant Fusion Proteins
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Polyubiquitin
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CDC34 protein, human
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Ubiquitin-Conjugating Enzymes
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome
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Ubiquitin-Protein Ligases
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Tacrolimus Binding Proteins
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Tacrolimus