Cytoplasmic localization and ubiquitination of p21^Cip1 by reactive oxygen species

Reactive oxygen species were previously shown to trigger p21^Cip1 protein degradation through a proteasome-dependent pathway, however the detailed mechanism of degradation remains to be elucidated. In this report, we showed that p21^Cip1 was degraded at an early phase after low dose H₂O₂ treatment of a variety of cell types and that preincubation of cells with the antioxidant, N-acetylcysteine, prolonged p21^Cip1 half-life. A mutant p21^Cip1 in which all six lysines were changed to arginines was protected against H₂O₂ treatment. Direct interaction between p21^Cip1 and Skp2 was elevated in the H₂O₂-treated cells. Disruption of the two nuclear export signal (NES) sequences in p21^Cip1, or treatment with leptomycin B blocked H₂O₂-induced p21^Cip1 degradation. Altogether, these results demonstrate that reactive oxygen species induce p21^Cip1 degradation through an NES-, Skp2-, and ubiquitin-dependent pathway.