Abstract

The Cdk2 inhibitor, p27^Kip1, is degraded in a phosphorylation- and ubiquitylation-dependent manner at the G₁–S transition of the cell cycle. Degradation of p27^Kip1 requires import into the nucleus for phosphorylation by Cdk2. Phosphorylated p27^Kip1 is thought to be subsequently re-exported and degraded in the cytosol. Using two-hybrid screens, we now show that p27^Kip1 interacts with a nuclear pore-associated protein, mNPAP60, map the interaction to the 3₁₀ helix of p27 and identify a point mutant in p27^Kip1 that is deficient for interaction (R90G). In vivo and in vitro, the loss-of-interaction mutant is poorly transported into the nucleus, while ubiquitylation of p27R90G occurs normally. In vivo, co-expression of cyclin E and Cdk2 rescues the import defect. However, mutant p27^Kip1 accumulates in a phosphorylated form in the nucleus and is not efficiently degraded, arguing that at least one step in the degradation of phosphorylated p27^Kip1 requires an interaction with the nuclear pore. Our results identify a novel component involved in p27^Kip1 degradation and suggest that degradation of p27^Kip1 is tightly linked to its intracellular transport.