Characterization of cullin-based E3 ubiquitin ligases in intact mammalian cells--evidence for cullin dimerization.

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.