Profiling of UV-induced ATM/ATR signaling pathways

Matthew P. Stokes, John Rush, Joan MacNeill, Min Ren Jian, Kam Sprott, Julie Nardone, Vicky Yang, Sean A. Beausoleil, Steven P. Gygi, Mark Livingstone, Hui Zhang, Roberto D. Polakiewicz, Michael J. Comb

Research output: Contribution to journalArticle

Abstract

To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.

Original languageEnglish (US)
Pages (from-to)19855-19860
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number50
DOIs
StatePublished - Dec 11 2007

Keywords

  • DNA damage
  • Mass spectrometry
  • Phosphorylation

ASJC Scopus subject areas

  • General

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  • Cite this

    Stokes, M. P., Rush, J., MacNeill, J., Jian, M. R., Sprott, K., Nardone, J., Yang, V., Beausoleil, S. A., Gygi, S. P., Livingstone, M., Zhang, H., Polakiewicz, R. D., & Comb, M. J. (2007). Profiling of UV-induced ATM/ATR signaling pathways. Proceedings of the National Academy of Sciences of the United States of America, 104(50), 19855-19860. https://doi.org/10.1073/pnas.0707579104