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Phosphorylation of FANCD2 on two novel sites is required for mitomycin C resistance

Gary P H Ho1, Steven Margossian, Toshiyasu Taniguchi

  • 1Dana-Farber Cancer Institute, Department of Radiation Oncology, Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA.

Molecular and Cellular Biology
|September 1, 2006
PubMed

Insights

The Fanconi anemia (FA) pathway

Area of Science:

  • DNA damage response
  • Cell cycle regulation
  • Molecular biology

Background:

  • The Fanconi anemia (FA) pathway is crucial for DNA cross-linking resistance.
  • Monoubiquitination of FANCD2 is a key downstream event in the FA pathway.
  • ATM and ATR kinases play roles in FANCD2 modification and cell cycle checkpoints.

Purpose of the Study:

  • Identify novel DNA damage-inducible phosphorylation sites on FANCD2.
  • Investigate the role of ATR in FANCD2 monoubiquitination and cellular resistance.
  • Elucidate the involvement of ATM and ATR in FANCD2 phosphorylation and intra-S-phase checkpoint control.

Main Methods:

  • Site-directed mutagenesis to identify phosphorylation sites.
  • Western blotting to detect FANCD2 monoubiquitination.
  • siRNA-mediated knockdown of ATM and ATR kinases.
  • Analysis of intra-S-phase checkpoint activation.

Main Results:

  • Two novel DNA damage-inducible phosphorylation sites on FANCD2 identified: threonine 691 and serine 717.
  • ATR-mediated phosphorylation of Thr691 and Ser717 promotes FANCD2 monoubiquitination and resistance to cross-linking agents.
  • Phosphorylation of these sites is essential for the intra-S-phase checkpoint response.
  • ATM and ATR kinases collaborate in the IR-inducible phosphorylation of FANCD2.

Conclusions:

  • FANCD2 phosphorylation by ATR is critical for DNA damage resistance and cell cycle checkpoint control.
  • The FA pathway, through FANCD2, integrates DNA damage signaling with S-phase progression.
  • ATM and ATR kinases are key regulators of FANCD2 phosphorylation in response to DNA damage.

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