Title: Distinct Domains within CUL1 for Substrate Targeting and Ubiquitin Ligation
Abstract: We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IkBa. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCF HOS/b-TRCP -ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/b-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 IkB 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. Regulation of protein stability by ubiquitin (Ub)-dependent proteolysis plays major roles in the control of multiple aspects of cell function, such as transcriptional control, cell cycle progression, and signal transduction (17). Ubiquitination involves three distinct enzymatic events that ultimately lead to the co
Publication Year: 2000
Publication Date: 2000-01-01
Language: en
Type: article
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