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Interplay between lysine methylation and Cdk phosphorylation in growth control by the retinoblastoma protein

Simon M Carr1, Shonagh Munro, Benedikt Kessler

  • 1Department of Clinical Pharmacology, Laboratory of Cancer Biology, University of Oxford, Oxford, UK.

The EMBO Journal
|December 2, 2010
PubMed

Insights

Methylation of lysine 810 in the retinoblastoma protein (pRb) by Set7/9 controls cell cycle progression. This methylation prevents cyclin-dependent kinase (Cdk) binding, maintaining pRb

Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • The retinoblastoma protein (pRb) is a key regulator of cell cycle progression, acting as a target for cyclin-dependent kinases (Cdks).
  • Cdk-mediated phosphorylation of pRb controls its activity and is crucial for cell cycle advancement.
  • Understanding the regulation of pRb phosphorylation is vital for comprehending cell cycle control and cancer development.

Purpose of the Study:

  • To investigate a novel regulatory mechanism influencing Cdk recognition and phosphorylation of substrate proteins.
  • To elucidate the role of lysine methylation in modulating pRb function and cell cycle control.
  • To determine the specific contribution of Set7/9-mediated methylation at K810 of pRb.

Main Methods:

  • Site-directed mutagenesis to create a non-methylatable pRb mutant (K810A).
  • In vitro binding assays to assess Cdk-cyclin interaction with pRb.
  • Western blotting and immunofluorescence to analyze pRb phosphorylation status and cell cycle arrest.
  • Enzyme assays to confirm methylation activity of Set7/9 on pRb K810.

Main Results:

  • Methylation of lysine (K) 810 in pRb by the methyltransferase Set7/9 inhibits Cdk binding and subsequent phosphorylation.
  • K810 methylation retains pRb in its hypophosphorylated, growth-suppressive state.
  • Set7/9 is essential for efficient DNA damage-induced cell cycle arrest, and a non-methylatable pRb mutant shows impaired cell cycle arrest.
  • Methylation at K810 influences phosphorylation at other sites within pRb.

Conclusions:

  • Targeted methylation of a lysine residue within a Cdk-consensus site represents a novel regulatory mechanism for pRb.
  • Methylation acts as a critical checkpoint, integrating DNA damage signals to control cell cycle progression via pRb.
  • The interplay between methylation and phosphorylation provides a sophisticated layer of control over cell cycle progression.

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