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

Abstract

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 phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination 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 Nce1͞Ubc12 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 , independently 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.

Nedd8 on cullin: building an expressway to protein destruction

“…(Handeli and Weintraub, 1992;Chow et al, 1996;Lammer et al, 1998;Liakopoulos et al, 1998;Osaka et al, 1998;Gong and Yeh, 1999;Tateishi et al, 2001;Bohnsack and Haas, 2003;Walden et al, 2003). b (Liakopoulos et al, 1998;Osaka et al, 1998;Gong and Yeh, 1999;Jones and Candido, 2000;Podust et al, 2000;Kurz et al, 2002;Ohh et al, 2002;Dharmasiri et al, 2003). c (Wada et al, 1998;Linghu et al, 2002;Gan-Erdene et al, 2003).…”

AXR1-ECR1–Dependent Conjugation of RUB1 to the Arabidopsis Cullin AtCUL1 Is Required for Auxin Response

“…Unlike ubiquitination, RUB modification of the cullin does not affect its metabolic stability. Instead, genetic and biochemical evidence suggests that RUB modification regulates SCF function or subcellular localization (Lammer et al, 1998;Morimoto et al, 2000;Podust et al, 2000;Read et al, 2000).…”

“…Unlike ubiquitination, RUB modification of the cullin does not affect its metabolic stability. Instead, genetic and biochemical evidence suggests that RUB modification regulates SCF function or subcellular localization (Lammer et al, 1998;Morimoto et al, 2000;Podust et al, 2000;Read et al, 2000).Genetic studies in Arabidopsis indicate that a ubiquitinprotein ligase called SCF TIR1 is required for auxin response (Gray et al, 1999). This E3 is composed of the cullin AtCUL1, the SKP1-related protein ASK1 or ASK2, and an F-box protein called TIR1.…”

“…The precise function of RUB modification is unknown (Yeh et al, 2000). Several studies have reported that the NEDD8 pathway enhances E3 activity in vitro, but the basis for this enhancement is not clear (Morimoto et al, 2000;Podust et al, 2000;Read et al, 2000; Wu et al, 2000;Tanaka et al, 2001). NEDD8 does not appear to be required for the assembly or stability of the complex (Yeh et al, 2000).…”

The identification of candidate genes and SNP markers for classical bovine spongiform encephalopathy susceptibility

“…32 NEDD8 conjugation pathway has shown to be essential for proteolytic targeting by ubiquitination. 33 Impairment of this process may contribute to the amyloid plaques evident in classical BSE. NEDD8 has been shown to accumulate in both glial and neuronal inclusions in several neurodegenerative conditions such as Parkinson disease, Alcoholic liver disease and Astrocytoma.…”