Regulation of the SCF-ROC1 ubiquitin ligase activity

This study focuses on the regulation of the SCF-ROC1 E3 ligase, the prototypical cullin family E3 ubiquitin ligase. Deletion analysis reveals that the N-terminus of CUL1 is necessary and sufficient for binding Skp1, but not ROC1. In contrast, the C-terminus of CUL1 binds to ROC1 through the cullin homology domain, to form a ligase complex active in ubiquitin polymerization. These results show that CUL1 is a dual-function protein that forms a substrate targeting module through the adapter subunit Skp1 at its N-terminus, while the C-terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.; Linkage of ubiquitin-like protein, Nedd8, to the CUL1 subunit of the ROC1-CUL1 sub complex is both necessary and sufficient to activate the ROC1-CUL1 core ligase ubiquitin polymerization activity. Therefore, Nedd8 is a novel regulator the SCF by enhancing its ability to promote polyubiquitin chain synthesis and, hence, the rapid turnover of protein substrates.; The conjugation of Nedd8 to ROC1-CUL1 selectively activates Cdc34 catalyzed lysine 48-linked ubiquitin chain synthesis, the predominant signal proteasome targeting signal. Distinct regions within the human Cdc34 C-terminal tail are responsible for multi-ubiquitin chain assembly and for physical interactions with the Nedd8-conjugated ROC1-CUL1. Six charged residues, uniquely present on the surface of Nedd8, are required to activate the ubiquitin ligase activity of ROC1-CUL1. These findings indicate that Nedd8 charged surface residues participate in the activation of ROC1-CUL1 to specifically support Cdc34 catalyzed ubiquitin polymerization.; DEN1, a novel protease belonging to the Ulp1 SUMO protease family was also characterized. DEN1 selectively binds to Nedd8 and hydrolyzes the C-terminus of Nedd8 precursor. At low concentrations, DEN1 processes hyper-neddylated CUL1 to generate predominantly mono-neddylated form. At elevated concentrations, DEN1 is able to completely remove Nedd8 from CUL1. In contrast the COP9 signalosome efficiently cleaves the mononeddylated CUL1 conjugate, poorly processes hyper-neddylated CUL1, and lacks Nedd8 C-terminal hydrolase activity. These results suggest DEN1 is a novel regulator of the Nedd8 pathway.