Profiling of UV-induced ATM/ATR signaling pathways

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.

Fig. 1.

Semiquantitative analysis of control vs. UV-damaged M059K cells reveals diversity of known/previously undescribed substrates. Pie chart shows protein classes identified in the semiquantitative analysis. All classes not shown have been collapsed into the “miscellaneous” category. The percentage of the total for each protein class is shown.

Fig. 2.

Western blotting and IP/Western blotting confirm phosphorylation of known/previously undescribed substrates of ATM/R seen in immunoaffinity purification/LC-MS/MS analysis. (A) Untreated (C) or UV-treated (UV) M059K lysates were subjected to Western blotting with the antibody indicated. (B) Proteins were immunoprecipitated from untreated or UV-treated M059K cell lysates with ATM/R substrate motif antibody-1 (53BP1, RPA1, and Mre11), or antibody-2 (BAP1) and blotted with the indicated antibody. “Supe” denotes post-IP supernatants; “Eluate” denotes IP elutions. For clarity, an arrow is shown to denote the band of interest in the Mre11 blot, whereas the asterisk (*) denotes a contaminating band.

Fig. 3.

Characterization of the DNA damage response in a Seckel cell line compared with a matched control/confirmation of immunoaffinity purification/LC-MS/MS results. (A) GM00200-matched control cells (Control) or GM18366 Seckel syndrome cells (Seckel) were untreated (C) or UV-treated (UV) and blotted with the indicated antibody. (B) Control or Seckel cells were untreated (C) or UV-treated (UV) and blotted with the indicated antibody against sites found in the semiquantitative analysis. ATM/R motif antibody-2 was shown to detect phosphorylated DNA-PK and valosin-containing protein (VCP) (17).