Targeting of the cyclin-dependent kinase inhibitor p27Kip1 for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27Kip1 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 p27Kip1 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 p27Kip1 accumulation during S-G2 phases of the first cell cycle. Polyubiquitination of p27 Kip1 in the nucleus is dependent on Skp2 and phosphorylation of p27Kip1 on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2-/- cells, even with a threonine 187 → alanine mutant of p27Kip1 as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27Kip1 degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G0 to G 1.
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