Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability

The association of the p27(kip1) protein with cyclin and cyclin-dependent kinase complexes inhibits their kinase activities and contributes to the control of cell proliferation. The p27(kip1) protein has now been shown to be phosphorylated in vivo, and this phosphorylation reduces the electrophoretic mobility of the protein. Substitution of Ser¹⁰ with Ala (S10A) markedly reduced the extent of p27(Kip1) phosphorylation and prevented the shift in electrophoretic mobility. Phosphopeptide mapping and phosphoamino acid analysis revealed that phosphorylation at Ser¹⁰ accounted for ≃70% of the total phosphorylation of p27(Kip1), and the extent of phosphorylation at this site was ≃25- and 75-fold greater than that at Ser¹⁷⁸ and Thr¹⁸⁷, respectively. The phosphorylation of p27(kip1) was markedly reduced when the positions of Ser¹⁰ and Pro¹¹ were reversed, suggesting that a proline-directed kinase is responsible for the phosphorylation of Ser¹⁰. The extent of Ser¹⁰ phosphorylation was markedly increased in cells in the G₀-G₁ phase of the cell cycle compared with that apparent for cells in S or M phase. The p27(Kip1) protein phosphorylated at Ser¹⁰ was significantly more stable than the unphosphorylated form. Furthermore, a mutant p27(kip1) in which Ser¹⁰ was replaced with glutamic acid in order to mimic the effect of Ser¹⁰ phosphorylation exhibited a marked increase in stability both in vivo and in vitro compared with the wild-type or S10A mutant proteins. These results suggest that Ser¹⁰ is the major site of phosphorylation of p27(Kip1) and that phosphorylation at this site, like that at Thr¹⁸⁷, contributes to regulation of p27(Kip1) stability.