Modification of cullin-1 by ubiquitin-like protein Nedd8 enhances the activity of SCFskp2 toward p27kip1

The periodic expression of cell cycle proteins is important for the regulation of cell cycle progression. The amount of CDK inhibitor, p27(kip1), one such protein, seems to be regulated by the ubiquitin-proteasome system. The ubiquitin ligase (E3) toward p27(kip1) is thought to be SCFskp2. The activity of SCFskp2 was increased by the addition of Roc1 protein to the complex. Furthermore, the ubiquitination of p27(kip1) seemed to be dependent on the phosphorylation of T187 of p27(kip1) because the mutant T187A was not ubiquitinated at all in an in vitro ubiquitination system. Cullin-1, a component of SCF, is modified by ubiquitin-like protein Nedd8, The modification site of cullin-1 was shown to be K696 because the K696R mutant was not modified. When the effect of the Nedd8 modification on the SCFskp2 activity toward p27(kip1) was investigated, the activity was markedly decreased by using the Nedd8-unmodified mutant cullin-1 (K696R), indicating that the modification may play an important role on the SCFskp2 activity toward p27(kip1). (C) 2000 Academic Press.