title: The SCF(HOS/beta-TRCP)-ROC1 E3 ubiquitin ligase utilizes two 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 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.
authors.affiliation: Derald H. Ruttenberg Cancer Center, The Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
authors.forename: K
authors.initials: K
authors.lastname: Wu
authors.forename: S Y
authors.initials: SY
authors.lastname: Fuchs
authors.forename: A
authors.initials: A
authors.lastname: Chen
authors.forename: P
authors.initials: P
authors.lastname: Tan
authors.forename: C
authors.initials: C
authors.lastname: Gomez
authors.forename: Z
authors.initials: Z
authors.lastname: Ronai
authors.forename: Z Q
authors.initials: ZQ
authors.lastname: Pan
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D044842:Cullin Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D066298:In Vitro Techniques; D008025:Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D051379:Mice; D008954:Models, Biological; D000072000:NF-KappaB Inhibitor alpha; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases; D013379:Substrate Specificity; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D044844:beta-Transducin Repeat-Containing Proteins
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D004268:DNA-Binding Proteins; C471645:FBXW11 protein, human; C486774:Fbxw1b protein, mouse; D020672:I-kappa B Proteins; D046911:Macromolecular Substances; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D011994:Recombinant Proteins; D014452:Ubiquitins; D044844:beta-Transducin Repeat-Containing Proteins; D000072000:NF-KappaB Inhibitor alpha; C577447:CDC34 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D010453:Peptide Synthases