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Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
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14-3-3 suppresses the nuclear localization of threonine 157-phosphorylated p27(Kip1)

Toshihiro Sekimoto1, Masahiro Fukumoto, Yoshihiro Yoneda

  • 1Department of Cell Biology and Neuroscience, Graduate School of Medicine, Osaka University, Yamada-oka, Suita, Osaka, Japan.

The EMBO Journal
|April 2, 2004
PubMed

Insights

The p27(Kip1) protein

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • p27(Kip1) (p27) is a cyclin-dependent kinase inhibitor crucial for cell cycle progression.
  • p27 nuclear import is essential for its function in controlling cell cycle.
  • Akt-mediated phosphorylation of p27 at Thr157 inhibits its nuclear import.

Purpose of the Study:

  • To identify the nuclear localization signal (NLS) of p27.
  • To elucidate the mechanism by which Akt phosphorylation affects p27 nuclear import.
  • To understand the role of 14-3-3 proteins in regulating p27 localization.

Main Methods:

  • In vitro nuclear transport assays using importin alpha and beta.
  • Pull-down experiments to identify p27-binding proteins.
  • Analysis of p27 localization in response to Akt phosphorylation and 14-3-3 binding.

Main Results:

  • A bipartite basic amino-acid cluster and downstream residues in p27's C-terminus constitute its NLS.
  • Importin alpha3 and alpha5, with importin beta, mediate p27 nuclear import.
  • 14-3-3 proteins bind to Thr157-phosphorylated p27 and sequester it from importin alpha, causing cytoplasmic retention.
  • This sequestration inhibits p27 nuclear import.

Conclusions:

  • The C-terminal NLS of p27 is critical for its nuclear import.
  • 14-3-3 proteins act as negative regulators of p27 nuclear import by binding to phosphorylated p27.
  • Dysregulation of this mechanism may contribute to cell cycle disorders in cancers with activated Akt.

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