Nedd8 modification of Cul-1 activates SCFβTrCp-dependent ubiquitination of IκBα

Regulation of NF-kappa B occurs through phosphorylation-dependent ubiquitination of I kappa B alpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of I kappa B alpha is carried out by a ubiquitin-ligase enzyme complex called SCFbeta TrCP. Here we show that Nedd8 modification of the Cul-1 component of SCFbeta TrCP is important for function of SCFbeta TrCP in ubiquitination of I kappa B alpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrate of SCFbeta TrCP phosphorylated I kappa B alpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCFbeta TrCP in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed beta TrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of I kappa B alpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCFbeta TrCP containing a K720R mutant of Cul-1 only weakly supported I kappa B alpha ubiquitination compared to SCFbeta TrCP containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCFbeta 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 I kappa B alpha.