Post-translational modification of nucleolin by monoubiquitylation in ovarian carcinoma cells Free

2803

Ubiquitin is a small eukaryotic protein that is conjugated to lysine residues on target proteins. Proteins with polyubiquitin chains, in which lysine 48 of ubiquitin binds to the C-terminal glycine of the next ubiquitin molecule, are degraded by the 26S proteasome complex. Monoubiquitylation, single ubiquitin conjugation without polymer chain formation, does not result in protein degradation. Recent studies on histones, plasma membrane proteins in endocytosis, and Gag proteins involved in retrovirus budding suggest that monoubiquitiylation may regulate the location and function of its conjugating proteins. Sequencing of proteins differentially ubiquitylated in benign and malignant ovarian epithelial tumor cells led to the discovery that nucleolin, a major eukaryotic phosphoprotein responsible for ribosome biogenesis and maturation, is monoubiquitylated in various human ovarian carcinoma cell lines. Proteins from nuclear and cytoplasmic fractions of HEY ovarian carcinoma cells were immunoprecipitated with an antibody against ubiquitin. Subsequent immunoblotting with an antibody against nucleolin showed that the majority of monoubiquitylated nucleolin existed in the nuclear compartment. Since nucleolin is regulated by various kinases, we sought to determine whether monoubiquitylation of nucleolin is related to its state of phosphorylation. Phosphorylated and non-phosphorylated proteins from HEY cells were separated by column chromatography and immunoprecipitated with an antibody against ubiquitin. The only form of monoubiquitylated nucleolin found in the cytoplasm was phosphorylated while phosphorylated and non-phosphorylated forms were both present in the nucleus, with a predominance of the non-phosphorylated form. A unique 70kDa fragment of nucleolin that was both monoubiquitylated and phosphorylated was observed in the cytoplasm. Other authors have suggested that this 70kDa fragment of nucleolin triggers initiation of apoptosis by activating endonucleases. We conclude that monoubiquitylation is an important mechanism of regulation of nucleolin, which may cooperate with phosphorylation to control its cellular localization as well as its effect on apoptosis and, possibly, malignant transformation.