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.
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