Phosphorylation of p27Kip1 at threonine 198 by p90 ribosomal protein S6 kinases promotes its binding to 14-3-3 and cytoplasmic localization

The cyclin-dependent kinase inhibitor p27(Kip1) plays an important role in cell cycle regulation. The cyclin-dependent kinase-inhibitory activity of p27(Kip1) is regulated by changes in its concentration and its subcellular localization. Several reports suggest that phosphorylation of p27(Kip1) at serine 10, threonine 157, and threonine 187 regulate its localization. We have previously identified that carboxyl-terminal threonine 198 (Thr(198)) in p27(Kip1) is a novel phosphorylation site and that Akt is associated with the phosphorylation at the site (Fujita, N., Sato, S., Katayama, K., and Tsuruo, T. (2002) J. Biol. Chem. 277, 28706 - 28713). We show herein that activation of the Ras/Raf/mitogen-activated protein kinase kinase (MAPK kinase/MEK) pathway also regulates phosphorylation of p27(Kip1) at Thr(198). MAPKs were not directly associated with p27(Kip1) phosphorylation at Thr198, but the p90 ribosomal protein S6 kinases (RSKs) could bind to and directly phosphorylate p27(Kip1) at Thr(198) in a Ras/ Raf/MEK-dependent manner. RSK-dependent phosphorylation promoted the p27(Kip1) binding to 14-3-3 and its cytoplasmic localization. To prove the direct relationship between 14-3-3 binding and cytoplasmic localization, we constructed a p27(Kip1)-R18 fusion protein in which the R18 peptide was fused to the carboxyl-terminal region of p27(Kip1). The R18 peptide is known to interact with 14-3-3 independent of phosphorylation. The p27(Kip1)-R18 distributed mainly in the cytosol, whereas mutant p27(Kip1)-R18 (p27(Kip1)- R18-K2) that had no 14-3-3 binding capability existed mainly in the nucleus. These results indicate that RSKs play a crucial role in cell cycle progression through translocation of p27(Kip1), in addition to Akt, to the cytoplasm in a phosphorylation- and 14-3-3 binding-dependent manner.