P27(Kip1) ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27

Many tumorigenic processes affect cell-cycle progression by their effects o n the levels of the cyclin-dependent kinase inhibitor p27(Kip1) [1,2]. The phosphorylation- and ubiquitination-dependent proteolysis of p27 is implica ted in control of the G1-S transition in the cell cycle [3-6]. To determine the factors that control p27 stability, we established a cell-free extract assay that recapitulates the degradation of p27, Phosphorylation of p27 at Thr187 was essential for its degradation. Degradation was also dependent o n SCFSkp2, a protein complex implicated in targeting phosphorylated protein s for ubiquitination [7-10]. Immunodepletion of components of the complex C ul-1, Skp1, or Skp2 - from the extract abolished p27 degradation, while add ition of purified SCFSkp2 to Skp2-depleted extract restored the capacity to degrade p27, A specific association was observed between Skp2 and a p27 ca rboxy-terminal peptide containing phosphorylated Thr187, but not between Sk p2 and the nonphosphorylated peptide. Skp2-dependent associations between S kp1 or Cul-1 and the p27 phosphopeptide were also detected. Isolated SCFSkp 2 contained an E3 ubiquitin ligase activity towards p27, Our data thus sugg est that SCFSkp2 specifically targets p27 for degradation during cell-cycle progression. (C) Elsevier Science Ltd ISSN 0960-9822.