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14-3-3 interacts with the tumor suppressor tuberin at Akt phosphorylation site(s)

Matt Y Liu1, Shengli Cai, Alexsandra Espejo

  • 1Department of Carcinogenesis, Science Park-Research Division, The University of Texas M. D. Anderson Cancer Center, Smithville, Texas 78957, USA.

Cancer Research
|November 20, 2002
PubMed

Insights

Tuberin, a tumor suppressor protein, interacts with 14-3-3 proteins at sites phosphorylated by Akt. This interaction, crucial for regulating cell signaling, involves all seven 14-3-3 isoforms.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Oncogenes

Background:

  • Tuberin, encoded by the tuberous sclerosis complex 2 (TSC2) gene, is a tumor suppressor protein.
  • Tuberin negatively regulates the Akt signaling pathway, a key pathway in cell growth and survival.
  • The interaction between tuberin and 14-3-3 proteins is implicated in TSC2 function, but the precise nature of this interaction is not fully understood.

Purpose of the Study:

  • To investigate the functional interaction between tuberin and 14-3-3 proteins.
  • To identify specific binding sites and conditions governing tuberin-14-3-3 complex formation.
  • To elucidate the role of Akt phosphorylation in mediating this interaction.

Main Methods:

  • In silico analysis to identify potential 14-3-3 binding sites in tuberin.
  • Western blotting using phospho-specific antibodies against Akt phosphorylation sites and 14-3-3 binding sites.
  • Inhibition of phosphatidylinositol 3'-kinase (PI3K) activity to assess its effect on tuberin phosphorylation and 14-3-3 binding.
  • Protein domain microarray to identify phospho-specific binding of tuberin peptides to 14-3-3.
  • Glutathione S-transferase (GST) pull-down assays using 14-3-3 fusion proteins and phosphorylated/unphosphorylated tuberin peptides.
  • Coimmunoprecipitation assays to confirm endogenous tuberin-14-3-3 complex formation.

Main Results:

  • Multiple high-stringency 14-3-3 binding sites in tuberin were identified in silico, overlapping with Akt phosphorylation sites.
  • Mitogen-induced tuberin phosphorylation correlated with 14-3-3 binding, as detected by specific antibodies.
  • Inhibition of PI3K blocked tuberin recognition by both Akt and 14-3-3 antibodies.
  • A tuberin peptide containing Ser(939) showed phospho-specific binding to 14-3-3.
  • All seven known 14-3-3 isoforms bound to tuberin in GST pull-down assays.
  • This binding was dependent on the phosphorylation of Ser(939) in tuberin.
  • Coimmunoprecipitation confirmed the interaction between endogenous tuberin and 14-3-3 proteins.

Conclusions:

  • Tuberin possesses functional and overlapping recognition sites for 14-3-3 proteins and Akt.
  • Akt-mediated phosphorylation of tuberin is critical for its interaction with 14-3-3 proteins.
  • This interaction likely plays a significant role in the tumor suppressor function of tuberin by modulating PI3K/Akt signaling.

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