title: Nedd8 modification of cul-1 activates SCF(beta(TrCP))-dependent ubiquitination of IkappaBalpha.
abstract: Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.
authors.affiliation: LeukoSite, Inc., Cambridge, Massachusetts 02139, USA. mread@mpi.com
authors.forename: M A
authors.initials: MA
authors.lastname: Read
authors.forename: J E
authors.initials: JE
authors.lastname: Brownell
authors.forename: T B
authors.initials: TB
authors.lastname: Gladysheva
authors.forename: M
authors.initials: M
authors.lastname: Hottelet
authors.forename: L A
authors.initials: LA
authors.lastname: Parent
authors.forename: M B
authors.initials: MB
authors.lastname: Coggins
authors.forename: J W
authors.initials: JW
authors.lastname: Pierce
authors.forename: V N
authors.initials: VN
authors.lastname: Podust
authors.forename: R S
authors.initials: RS
authors.lastname: Luo
authors.forename: V
authors.initials: V
authors.lastname: Chau
authors.forename: V J
authors.initials: VJ
authors.lastname: Palombella
mesh_terms: D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins; D002460:Cell Line; D044842:Cullin Proteins; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020672:I-kappa B Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D000075687:NEDD8 Protein; D010453:Peptide Synthases / Q000378:metabolism*; D010766:Phosphorylation; D044843:SKP Cullin F-Box Protein Ligases; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D015534:Trans-Activators; D014162:Transfection; D014452:Ubiquitins / Q000378:metabolism*; D051176:beta Catenin; D044844:beta-Transducin Repeat-Containing Proteins
chemical_list: C471644:BTRC protein, human; C495270:CTNNB1 protein, human; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D015801:Helminth Proteins; C083487:I kappa B beta protein; D020672:I-kappa B Proteins; D009097:Multienzyme Complexes; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C112595:UBC12 protein, S cerevisiae; D014452:Ubiquitins; D051176:beta Catenin; D044844:beta-Transducin Repeat-Containing Proteins; D044843:SKP Cullin F-Box Protein Ligases; D019204:GTP-Binding Proteins; D010453:Peptide Synthases