The SCF(HOS/beta-TRCP)-ROC1 E3 ubiquitin ligase utilizes two distinct domains within CUL1 for substrate targeting and ubiquitin ligation.
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.
Mesh Terms:
Animals, Base Sequence, Carrier Proteins, Cell Cycle Proteins, Cell Line, Cullin Proteins, DNA-Binding Proteins, Hela Cells, Humans, I-kappa B Proteins, Ligases, Macromolecular Substances, Mice, Models, Biological, Peptide Synthases, Plasmids, Protein Structure, Tertiary, Recombinant Proteins, SKP Cullin F-Box Protein Ligases, Substrate Specificity, Transfection, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases, Ubiquitins, beta-Transducin Repeat-Containing Proteins
Animals, Base Sequence, Carrier Proteins, Cell Cycle Proteins, Cell Line, Cullin Proteins, DNA-Binding Proteins, Hela Cells, Humans, I-kappa B Proteins, Ligases, Macromolecular Substances, Mice, Models, Biological, Peptide Synthases, Plasmids, Protein Structure, Tertiary, Recombinant Proteins, SKP Cullin F-Box Protein Ligases, Substrate Specificity, Transfection, Ubiquitin-Protein Ligase Complexes, Ubiquitin-Protein Ligases, Ubiquitins, beta-Transducin Repeat-Containing Proteins
Mol. Cell. Biol.
Date: Feb. 01, 2000
PubMed ID: 10648623
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