An intact NEDD8 pathway is required for Cullin-dependent ubiquitylation in mammalian cells.

Skp1-Cdc53/Cul1-F-box (SCF) complexes constitute a class of E3 ubiquitin ligases. Recently, a multiprotein complex containing pVHL, elongin C and Cul2 (VEC) was shown to structurally and functionally resemble SCF complexes. Cdc53 and the Cullins can become covalently linked to the ubiquitin-like molecule Rub1/NEDD8. Inhibition of neddylation inhibits SCF function in vitro and in yeast and plants. Here we show that ongoing neddylation is likewise required for VEC function in vitro and for the degradation of SCF and VEC targets in mammalian cells. Thus, neddylation regulates the activity of two specific subclasses of mammalian ubiquitin ligases.