title: Characterization of cullin-based E3 ubiquitin ligases in intact mammalian cells--evidence for cullin dimerization.
abstract: Cullin-based E3 ligases are a large family of ubiquitin ligases with diverse cellular functions. They are composed of one of six mammalian cullin homologues, the Ring finger containing protein Roc1/Rbx1 and cullin homologue-specific adapter and substrate recognition subunits. To be active, cullin-based ligases require the covalent modification of a conserved lysine residue in the cullin protein with the ubiquitin-like protein Nedd8. To characterize this family of E3 ligases in intact cells, we generated a cell line with tetracycline-inducible expression of a dominant-negative mutant of the Nedd8-conjugating enzyme Ubc12, a reported inhibitor of cullin neddylation. Using this cell line, we demonstrate that the substrate recognition subunit Skp2 and the adaptor protein Skp1 are subject to Ubc12-dependent autoubiquitination and degradation. In contrast, cullin protein stability is not regulated by neddylation in mammalian cells. We also provide evidence that Cul1 and Cul3, as well as their associated substrate recognition subunits Skp2 and Keap1, respectively, homooligomerize in intact cells, suggesting that cullin-based ligases are dimeric. Cul3, but not Cul1 homooligomerization is dependent on substrate recognition subunit dimer formation. As shown for other E3 ubiquitin ligases, dimerization may play a role in regulating the activity of cullin-based E3 ligases.
authors.affiliation: Wolfson Digestive Diseases Centre, University of Nottingham, Queen's Medical Centre, Nottingham, NG7 2UH, UK.
authors.forename: Eng-Hui
authors.initials: EH
authors.lastname: Chew
authors.forename: Thurka
authors.initials: T
authors.lastname: Poobalasingam
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Hawkey
authors.forename: Thilo
authors.initials: T
authors.lastname: Hagen
mesh_terms: D000818:Animals; D002352:Carrier Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D044842:Cullin Proteins / Q000378:metabolism*; D050760:Cyclin-Dependent Kinase Inhibitor p27; D019281:Dimerization; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D000072019:Kelch-Like ECH-Associated Protein 1; D051379:Mice; D000075687:NEDD8 Protein; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D044843:SKP Cullin F-Box Protein Ligases / Q000378:metabolism; D013752:Tetracycline / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
chemical_list: C486317:CAND1 protein, human; C436291:CDKN1B protein, human; C503645:CUL3 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C100318:Cullin 1; D044842:Cullin Proteins; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D000075687:NEDD8 Protein; C082860:NEDD8 protein, human; C479529:RBX1 protein, human; D044786:S-Phase Kinase-Associated Proteins; D014157:Transcription Factors; D014452:Ubiquitins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases; D013752:Tetracycline