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Profiling of UV-induced ATM/ATR signaling pathways

Matthew P Stokes1, John Rush, Joan Macneill

  • 1Cell Signaling Technology, 3 Trask Lane, Danvers, MA 01923, USA.

Proceedings of the National Academy of Sciences of the United States of America
|December 14, 2007
PubMed

Insights

Scientists identified hundreds of new phosphorylation sites involved in the DNA damage response. This discovery enhances our understanding of how cells repair genomic damage using key signaling pathways like ATM/ATR.

Area of Science:

  • Molecular Biology
  • Cellular Biology
  • Biochemistry

Background:

  • Cells possess intricate DNA damage response mechanisms, known as the DNA damage checkpoint, crucial for survival against genomic insults.
  • The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) are central to checkpoint signaling, phosphorylating substrates at SQ/TQ motifs.
  • A comprehensive understanding of ATM/ATR signaling is hindered by the incomplete identification of their downstream substrates.

Purpose of the Study:

  • To identify novel protein phosphorylation sites regulated by ATM/ATR signaling in response to UV-induced DNA damage.
  • To expand the known repertoire of DNA damage checkpoint substrates.
  • To provide a resource for deeper investigation into ATM/ATR signaling pathways.

Main Methods:

  • Immunoaffinity phosphopeptide isolation combined with mass spectrometry was employed to identify phosphorylated sites in UV-damaged cells.
  • Semiquantitative analysis was used to determine differentially phosphorylated sites.
  • Validation of identified sites was performed using SILAC, Western blotting, and immunoprecipitation/Western blotting.
  • ATR-specific phosphorylation was assessed using a Seckel syndrome (ATR mutant) cell line.

Main Results:

  • The study identified a total of 570 phosphorylation sites in UV-damaged cells, with 498 being previously undescribed.
  • Semiquantitative analysis revealed 24 known and 192 novel differentially phosphorylated sites upon UV damage.
  • Several identified sites were experimentally confirmed through various biochemical techniques.
  • ATR-specific phosphorylation patterns were investigated in the context of Seckel syndrome cells.

Conclusions:

  • This research provides a significantly expanded list of ATM/ATR signaling targets involved in the DNA damage response.
  • The identified phosphorylation sites offer a valuable resource for future studies aimed at elucidating the complexities of DNA repair pathways.
  • The findings contribute to a more comprehensive understanding of cellular survival mechanisms following genomic insult.

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