N-terminal polyubiquitination and degradation of the Arf tumor suppressor

Abstract

Unknown mechanisms govern degradation of the p19^Arf tumor suppressor, an activator of p53 and inhibitor of ribosomal RNA processing. Kinetic metabolic labeling of cells with [³H]-leucine indicated that p19^Arf is a relatively stable protein (half-life ∼6 h) whose degradation depends upon the ubiquitin–proteasome pathway. Although p19^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.