RESUMO
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 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), 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.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Inibidores Enzimáticos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Supressoras de Tumor , Complexos Ubiquitina-Proteína Ligase , Ubiquitinas/metabolismo , Substituição de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Sítios de Ligação , Domínio Catalítico , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Cisteína , Escherichia coli , Células HeLa , Humanos , Cinética , Ligases/metabolismo , Mutagênese Sítio-Dirigida , Proteína NEDD8 , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Serina , Ubiquitina-Proteína LigasesRESUMO
Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.
Assuntos
Proteínas de Ciclo Celular , Proteínas Culina , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Helminto/metabolismo , Proteínas I-kappa B , Peptídeo Sintases/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Helminto/genética , Humanos , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Proteína NEDD8 , Fosforilação , Proteínas Ligases SKP Culina F-Box , Alinhamento de Sequência , Transfecção , beta Catenina , Proteínas Contendo Repetições de beta-TransducinaRESUMO
A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-proteasome pathway was investigated. Cloning of the colon-26 tumor produced a cell line, termed R-1, which induced cytokine (noninterleukin-1beta, interleukin-6 and tumor necrosis factor-alpha)-independent cachexia. Implantation of R-1 cells in mice elicited significant (20-30%) weight loss and decreased blood glucose by 70%, and adipose tissue levels declined by 95% and muscle weights decreased by 20-25%. Food intake was unaffected. The decrease in muscle weight reflected a decline in insoluble, but not soluble, muscle protein that was associated with a significant increase in net protein degradation. The rate of ubiquitin conjugation of proteins was significantly elevated in muscles of cachectic mice. Furthermore, the proteasome inhibitor lactacystin blocked the increase in protein breakdown but had no significant effect on proteolysis. Several markers of the ubiquitin-proteasome pathway, E2(14k) mRNA and E2(14k) protein and ubiquitin-protein conjugates, were not elevated. Future investigations with this new model should gain further insights into the mechanisms of cachexia and provide a background to evaluate novel and more efficacious therapies.