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14-3-3 proteins play a role in the cell cycle by shielding cdt2 from ubiquitin-mediated degradation

Ashraf Dar1, David Wu1, Nicholas Lee1

  • 1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Molecular and Cellular Biology
|August 27, 2014
PubMed

Insights

14-3-3 proteins stabilize Cdt2 by shielding it from degradation. This interaction is crucial for cell cycle progression and prevents the accumulation of key cell cycle regulators like Set8.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cdt2 is a key component of the CRL4(Cdt2) E3 ubiquitin ligase complex, regulating cell cycle progression through proteasomal degradation of substrates like Cdt1, p21, and Set8.
  • Cdt2 stability is tightly controlled, with SCF(FbxO11) mediating its proteasomal degradation.

Purpose of the Study:

  • To investigate the role of 14-3-3 proteins in regulating Cdt2 stability and its impact on cell cycle progression.
  • To elucidate the mechanism by which 14-3-3 proteins interact with Cdt2.

Main Methods:

  • Co-immunoprecipitation to study protein interactions.
  • Western blotting to assess protein levels and ubiquitination.
  • Site-directed mutagenesis to analyze the role of T464 phosphorylation.
  • Depletion and overexpression studies of 14-3-3 proteins and FbxO11.
  • Cell cycle analysis and proliferation assays.

Main Results:

  • 14-3-3 proteins bind to phosphorylated Cdt2 (T464), protecting it from polyubiquitination and proteasomal degradation.
  • Depletion of 14-3-3 proteins enhances Cdt2 interaction with FbxO11, leading to Cdt2 degradation.
  • Overexpression of 14-3-3 proteins protects phospho-mimicking Cdt2 (T464D) but not non-phosphorylatable Cdt2 (T464A) from ubiquitination.
  • 14-3-3 depletion causes G2/M cell cycle delay and reduced proliferation, partly due to Set8 accumulation.

Conclusions:

  • 14-3-3 proteins act as crucial regulators of Cdt2 stability through phosphorylation-dependent interactions.
  • Stabilization of Cdt2 by 14-3-3 proteins is essential for proper cell cycle progression and proliferation.
  • The findings reveal a novel regulatory mechanism for CRL4(Cdt2) activity impacting key cell cycle substrates.

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