Phosphorylation of p27^Kip1 on serine 10 is required for its binding to CRM1 and nuclear export

Phosphorylation of the cyclin-dependent kinase inhibitor p27^Kip1 has been thought to regulate its stability. Ser¹⁰ is the major phosphorylation site of p27^Kip1, and phosphorylation of this residue affects protein stability. Phosphorylation of p27^Kip1 on Ser¹⁰ has now been shown to be required for the binding of CRM1, a carrier protein for nuclear export. The p27^Kip1 protein was translocated from the nucleus to the cytoplasm at the G₀-G₁ transition of the cell cycle, and this export was inhibited by leptomycin B, a specific inhibitor of CRM1-dependent nuclear export. The nuclear export and subsequent degradation of p27^Kip at the G₀-G₁ transition were observed in cells lacking Skp2, the F-box protein component of an SCF ubiquitin ligase complex, indicating that these early events are independent of Skp2-mediated proteolysis. Substitution of Ser¹⁰ with Ala (S10A) markedly reduced the extent of p27^Kip1 export, whereas substitution of Ser¹⁰ with Asp (S10D) or Glu (S10E) promoted export. Co-immunoprecipitation analysis showed that CRM1 preferentially interacted with S10D and S10E but not with S10A, suggesting that the phosphorylation of p27^Kip1 on Ser¹⁰ is required for its binding to CRM1 and for its subsequent nuclear export.