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A dynamic equilibrium between CDKs and PP2A modulates phosphorylation of pRB, p107 and p130

Judit Garriga1, Arun L Jayaraman, Ana Limón

  • 1Fels Institute for Cancer Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.

Cell Cycle (Georgetown, Tex.)
|October 7, 2004
PubMed

Insights

Cellular protein synthesis inhibition rapidly dephosphorylates pocket proteins by inactivating cyclin-dependent kinases (CDKs). This unmasks a phosphatase activity, primarily Protein Phosphatase 2A (PP2A), which regulates pocket protein phosphorylation throughout the cell cycle.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Pocket proteins are phosphorylated by G(1) cyclin/CDK complexes from mid-G(1) through mitosis.
  • Dephosphorylation of pocket proteins typically occurs during mitosis.
  • The mechanisms driving rapid dephosphorylation under specific cellular conditions were not fully understood.

Purpose of the Study:

  • To investigate the mechanisms behind the rapid dephosphorylation of pocket proteins induced by cycloheximide (CHX) and flavopiridol.
  • To identify the specific phosphatase involved in this dephosphorylation process.
  • To elucidate the dynamic interplay between CDKs and phosphatases in regulating pocket protein phosphorylation.

Main Methods:

  • Inhibition of cellular protein synthesis using cycloheximide (CHX).
  • Direct inhibition of cyclin-dependent kinases (CDKs) using flavopiridol.
  • Treatment with specific phosphatase inhibitors (okadaic acid) and analysis of SV40 small t antigen effects.
  • Biochemical assays including co-immunoprecipitation to detect protein interactions.

Main Results:

  • CHX and flavopiridol induced rapid dephosphorylation of pocket proteins by inactivating D-type and G(1)/S CDKs, respectively.
  • This inactivation unmasked a phosphatase activity targeting pocket proteins throughout the cell cycle.
  • Protein Phosphatase 2A (PP2A) was identified as the key enzyme responsible, as its inhibition prevented dephosphorylation.
  • PP2A catalytic subunit directly interacted with pocket proteins p130 and p107.

Conclusions:

  • Pocket protein phosphorylation is regulated by a dynamic balance between CDK activity and PP2A activity.
  • Inhibition of CDK activity, even transiently, can lead to significant dephosphorylation mediated by PP2A.
  • These findings have implications for understanding cell cycle control and checkpoint mechanisms affecting pocket protein function.

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