N-terminal polyubiquitination and degradation of the Arf tumor suppressor

Unknown mechanisms govern degradation of the p(19)Arf tumor suppressor, an activator of p53 and inhibitor of ribosomal RNA processing. Kinetic metabolic labeling of cells with [H-3]-leucine indicated that p19(Arf) is a relatively stable protein (half-life similar to6 h) whose degradation depends upon the ubiquitin-proteasome pathway. Although plg(Arf) binds to the Mdm2 E3 ubiquitin protein ligase to activate p53, neither of these molecules regulates p19(Arf) turnover. In contrast, the nucleolar protein nucleophosmin/B23, which binds to p19(Arf) with high stoichiometry, retards its turnover, and Arf mutants that do not efficiently associate with nucleophosmin/B23 are unstable and functionally impaired. Mouse p19(Arf) although highly basic (22% arginine content), contains only a single lysine residue absent from human p14(Arf), and substitution of arginine for lysine in mouse p19(Arf) had no effect on its rate of degradation. Mouse p19(Arf) (either wild-type or lacking lysine) and human p14(Arf) undergo N-terminal polyubiquitination, a process that has not as yet been documented in naturally occurring lysine-less proteins. Re-engineering of the p19(Arf) N terminus to provide consensus sequences for N-acetylation limited Arf ubiquitination and decelerated its turnover.