Abstract
We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IkappaBalpha. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCF(HOS/beta-TRCP)-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/beta-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IkappaB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.
Publication types
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Research Support, Non-U.S. Gov't
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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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Animals
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Base Sequence
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Carrier Proteins / chemistry
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Line
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Cullin Proteins*
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DNA-Binding Proteins / metabolism
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HeLa Cells
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Humans
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I-kappa B Proteins*
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In Vitro Techniques
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Ligases / chemistry
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Ligases / genetics
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Ligases / metabolism*
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Macromolecular Substances
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Mice
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Models, Biological
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NF-KappaB Inhibitor alpha
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Peptide Synthases / chemistry
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Peptide Synthases / genetics
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Peptide Synthases / metabolism*
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Plasmids / genetics
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Protein Structure, Tertiary
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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SKP Cullin F-Box Protein Ligases
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Substrate Specificity
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Transfection
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Ubiquitin-Conjugating Enzymes
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Ubiquitin-Protein Ligase Complexes*
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Ubiquitin-Protein Ligases
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Ubiquitins / metabolism*
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beta-Transducin Repeat-Containing Proteins*
Substances
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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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DNA-Binding Proteins
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FBXW11 protein, human
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Fbxw1b protein, mouse
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I-kappa B Proteins
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Macromolecular Substances
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NFKBIA protein, human
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Nfkbia protein, mouse
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Recombinant Proteins
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Ubiquitins
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beta-Transducin Repeat-Containing Proteins
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NF-KappaB Inhibitor alpha
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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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SKP Cullin F-Box Protein Ligases
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Ubiquitin-Protein Ligases
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Ligases
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Peptide Synthases