Degradation of p27^Kip1 at the G₀-G₁ Transition Mediated by a Skp2-independent Ubiquitination Pathway

Targeting of the cyclin-dependent kinase inhibitor p27^Kip1 for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27^Kip1 at the G₀-G₁ transition of the cell cycle has now been shown to proceed normally in Skp2^-/- lymphocytes, whereas p27 ^Kip1 proteolysis during S-G₂ phases is impaired in these Skp2-deficient cells. Degradation of p27^Kip1 at the G ₀-G₁ transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Skp2 ^-/- lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of p27^Kip1 accumulation during S-G₂ phases of the first cell cycle. Polyubiquitination of p27 ^Kip1 in the nucleus is dependent on Skp2 and phosphorylation of p27^Kip1 on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2^-/- cells, even with a threonine 187 → alanine mutant of p27^Kip1 as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27^Kip1 degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G₀ to G ₁.