A Nedd8 conjugation pathway is essential for proteolytic targeting of p27~Kip1 by ubiquitination

摘要

Temporal control of p27~Kip1 (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphor- ylation of p27 at Thr-187, which marks the protein for ubiquitina- tion by SCF~skp2 and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF~skp2 also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27~Kip1, inde- pendently of p27 phosphorylation. and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.