title: The CUL1 C-terminal sequence and ROC1 are required for efficient nuclear accumulation, NEDD8 modification, and ubiquitin ligase activity of CUL1.
abstract: Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IkappaBalpha ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity-ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.
authors.affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7295, USA.
authors.forename: M
authors.initials: M
authors.lastname: Furukawa
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: McCarville
authors.forename: T
authors.initials: T
authors.lastname: Ohta
authors.forename: Y
authors.initials: Y
authors.lastname: Xiong
mesh_terms: D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D044842:Cullin Proteins; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006801:Humans; D020672:I-kappa B Proteins; D007150:Immunohistochemistry; D008025:Ligases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000075687:NEDD8 Protein; D000072000:NF-KappaB Inhibitor alpha; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000502:physiology*
chemical_list: D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C000606204:NFKBIA protein, human; D014452:Ubiquitins; D000072000:NF-KappaB Inhibitor alpha; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases