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The cyclin dependent kinase inhibitor p27 is a key regulator of the cell cycle, and diminished expression of this inhibitor has been associated with poor prognosis in cancers of the breast, lung and colon. p27 is regulated through several mechanisms, including phosphorylation, which controls p27 degradation and subcellular localization. In human cells, phosphorylation at threonine 157 has been implicated in the cytoplasmic localization of p27, which is mediated by binding to 14-3-3 proteins. However, this residue is not conserved in murine p27. We therefore examined other potential phosphorylation sites as determinants of p27 localization and binding to 14-3-3 proteins in our model, the Eker rat, and a tuberin-null cell line derived from an Eker Leiomyoma Tumor (ELT-3). In tuberin-null tumors and ELT-3 cells p27 was stabilized, but was sequestered in the cytoplasm. Re-introduction of tuberin into ELT-3 cells resulted in destabilization of p27 as shown by diminished protein levels. To determine the mechanism of p27 stabilization and localization, we used a protein chip array assay to investigate sites of possible 14-3-3 binding to p27. We determined that 14-3-3 binds murine p27 despite the absence of a T157 residue and that the observed binding was dependent on phosphorylation of p27 at T197. Flag-tagged p27 and p27-T197A were generated to further investigate the role of T197 on the stability and regulation of murine p27. Using GST-14-3-3 fusion proteins, 14-3-3 was shown to bind p27-T197A with lower affinity than wild-type p27. Furthermore, pulse-chase experiments demonstrated that the p27-T197A mutant had a decreased half-life when compared to wild-type p27. These results demonstrate that cytoplasmic sequestration and potential inactivation of murine p27 occurs via 14-3-3 binding to p27 upon phosphorylation of T197. Therefore, while phosphorylation of p27 is an important mechanism for regulating localization and function, T157 is not required for cytoplasmic sequestration, and in murine systems, T197 is a critical residue regulating p27 stability and localization within the cell.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]