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Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization
Naoya Fujita1, Saori Sato, Kazuhiro Katayama
1Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan.
Abstract:
In many human cancers, the cyclin-dependent kinase inhibitor p27(Kip1) is expressed at low or undetectable levels. The decreased p27(Kip1) expression allows cyclin-dependent kinase activity to cause cells to enter into S phase and correlates with poor patient survival. Inhibition of serine/threonine kinase Akt signaling by some pharmacological agents or by PTEN induces G(1) arrest, in part by up-regulating p27(Kip1). However, the role of Akt-dependent phosphorylation in p27(Kip1) regulation is not clear. Here, we show that Akt bound directly to and phosphorylated p27(Kip1). Screening p27(Kip1) phosphorylation sites identified the COOH-terminal Thr(198) residue as a novel site. Further analysis revealed that 14-3-3 proteins bound to p27(Kip1) through Thr(198) only when it was phosphorylated by Akt. Although Akt also phosphorylated p27(Kip1) at Ser(10) and Thr(187), these two sites were not involved in the binding to 14-3-3 proteins. p27(Kip1) phosphorylated at Thr(198) exists only in the cytoplasm. Therefore, Akt promotes cell-cycle progression through the mechanisms of phosphorylation-dependent 14-3-3 binding to p27(Kip1) and cytoplasmic localization.
Insights
The Akt kinase phosphorylates p27(Kip1) at Thr198, promoting its binding to 14-3-3 proteins. This interaction leads to p27(Kip1) cytoplasmic localization, driving cell-cycle progression in human cancers.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Low p27(Kip1) expression in human cancers correlates with poor survival.
- Akt signaling inhibition can induce cell-cycle arrest via p27(Kip1) upregulation.
- The precise role of Akt-dependent phosphorylation in p27(Kip1) regulation remains unclear.
Purpose of the Study:
- To elucidate the role of Akt-dependent phosphorylation in p27(Kip1) regulation.
- To identify specific phosphorylation sites on p27(Kip1) targeted by Akt.
- To investigate the functional consequences of Akt-mediated p27(Kip1) phosphorylation.
Main Methods:
- Direct binding assays between Akt and p27(Kip1).
- Phosphorylation site mapping of p27(Kip1) by Akt.
- Analysis of 14-3-3 protein binding to phosphorylated p27(Kip1).
- Cellular localization studies of p27(Kip1).
Main Results:
- Akt directly binds and phosphorylates p27(Kip1).
- Thr198 is identified as a novel Akt phosphorylation site on p27(Kip1).
- Akt-mediated phosphorylation of Thr198 facilitates 14-3-3 protein binding.
- Phosphorylated p27(Kip1) at Thr198 is localized to the cytoplasm, unlike other sites.
- Akt promotes cell-cycle progression through p27(Kip1) phosphorylation and cytoplasmic retention.
Conclusions:
- Akt-dependent phosphorylation of p27(Kip1) at Thr198 is a key regulatory mechanism.
- This phosphorylation event drives 14-3-3 binding and cytoplasmic localization of p27(Kip1).
- These findings reveal a novel pathway by which Akt promotes cancer cell proliferation.