Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization.
The SCF-ROC1 ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase complex targets the ubiquitination and subsequent degradation of protein substrates required for the regulation of cell cycle progression and signal transduction pathways. We have previously shown that ROC1-CUL1 is a core subassembly within the SCF-ROC1 complex, capable of supporting the polymerization of ubiquitin. This report describes that the CUL1 subunit of the bacterially expressed, unmodified ROC1-CUL1 complex is conjugated with Nedd8 at Lys-720 by HeLa cell extracts or by a purified Nedd8 conjugation system (consisting of APP-BP1/Uba3, Ubc12, and Nedd8). This covalent linkage of Nedd8 to CUL1 is both necessary and sufficient to markedly enhance the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization. A mutation of Lys-720 to arginine in CUL1 eliminates the Nedd8 modification, abolishes the activation of the ROC1-CUL1 ubiquitin ligase complex, and significantly reduces the ability of SCF(HOS/beta)(-TRCP)-ROC1 to support the ubiquitination of phosphorylated IkappaBalpha. Thus, although regulation of the SCF-ROC1 action has been previously shown to preside at the level of recognition of a phosphorylated substrate, we demonstrate that Nedd8 is a novel regulator of the efficiency of polyubiquitin chain synthesis and, hence, promotes rapid turnover of protein substrates.
Derald H. Ruttenberg Cancer Center, Mount Sinai School of Medicine, New York, New York 10029-6574, USA.
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D019943:Amino Acid Substitution; D064173:Anaphase-Promoting Complex-Cyclosome; D001704:Biopolymers / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044842:Cullin Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D009154:Mutation / Q000235:genetics; D000075687:NEDD8 Protein; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D025821:Polyubiquitin; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001704:Biopolymers; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D046911:Macromolecular Substances; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C000606204:NFKBIA protein, human; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D025821:Polyubiquitin; D000072000:NF-KappaB Inhibitor alpha; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases; D008239:Lysine