p27Kip1 ubiquitination and degradation is regulated by the SCFSkp2 complex through phosphorylated Thr187 in p27

TLDR

p27(Kip1) is a cyclin‑dependent kinase inhibitor whose phosphorylation‑dependent ubiquitination controls the G1‑S transition in the cell cycle. The study aimed to identify the factors that regulate p27 stability by developing a cell‑free extract assay that reproduces its degradation. The assay demonstrated that p27 degradation requires phosphorylation at Thr187 and the SCF(Skp2) E3 ligase complex, as depletion of Skp2, Skp1, or Cul‑1 abolishes degradation and readdition restores it. These results show that SCF(Skp2) specifically recognizes phosphorylated Thr187 on p27, ubiquitinates it, and targets it for proteolysis during cell‑cycle progression.

Abstract

Many tumorigenic processes affect cell-cycle progression by their effects on the levels of the cyclin-dependent kinase inhibitor p27(Kip1) [1,2]. The phosphorylation- and ubiquitination-dependent proteolysis of p27 is implicated 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 on SCF(Skp2), a protein complex implicated in targeting phosphorylated proteins for ubiquitination [7-10]. Immunodepletion of components of the complex - Cul-1, Skp1, or Skp2 - from the extract abolished p27 degradation, while addition of purified SCF(Skp2) to Skp2- depleted extract restored the capacity to degrade p27. A specific association was observed between Skp2 and a p27 carboxy-terminal peptide containing phosphorylated Thr187, but not between Skp2 and the non-phosphorylated peptide. Skp2-dependent associations between Skp1 or Cul-1 and the p27 phosphopeptide were also detected. Isolated SCF(Skp2) contained an E3 ubiquitin ligase activity towards p27. Our data thus suggest that SCF(Skp2) specifically targets p27 for degradation during cell-cycle progression.