Abstract B09: DNA damage inducible phosphorylation of ATR at threonine 1989 and quantitative analysis of the effect of ATR inhibition on DNA damage signaling using PTMScan
Authors: Roberts Hayley J. , Stokes Matthew P. , Jia Xiaoying , Lee Kimberly A. , Keezer Susan M. 
Published: April 2017

DNA repair pathways and checkpoint control are crucial in the maintenance of genome integrity. Agents that cause DNA damage, and agents that perturb DNA repair pathways, have been used successfully in the treatment of human cancer.

The PI3K-like protein kinases ATR (ATM and Rad3-related) and ATM (ataxia telangiectasia mutated) are critical regulators of the DNA damage response (DDR), signaling to downstream effector molecules that in turn regulate cellular responses such as DNA repair, cell cycle arrest, and cell death.

ATM is activated in response to DNA damage in part via autophosphorylation at serine 1981. ATR was long thought to exist in a constitutively active state in cells, with DNA damage-induced signaling occurring via recruitment of ATR to single stranded DNA and sites of replication stress. Recent work, however, has shown autophosphorylation of ATR at threonine 1989, a homologous site to ATM Ser1981. Like ATM Ser1981, phosphorylation of ATR Thr1989 occurs in response to DNA damage, indicating that phosphorylation at this site is important in ATR-mediated signaling.

We have generated highly specific antibodies to both ATM Ser1981 and ATR Thr1989. Using these antibodies, we have identified conditions under which these sites are phosphorylated in cultured human cell lines. While phosphorylation of ATM was induced in response to a wide variety of DNA damage-inducing agents, phosphorylation of ATR was induced under a small subset of these conditions, most robustly with the alkylating agent mitomycin C, the ribonucleotide reductase inhibitor hydroxyurea and the topoisomerase II inhibitor etoposide. All of these agents inhibit DNA synthesis, albeit through different mechanisms.

We then identified changes downstream of ATM and ATR under conditions of ATR Thr1989 phosphorylation. Checkpoint kinases Chk1 and Chk2 are phosphorylated and activated by ATR and ATM, respectively, though there is crosstalk between the two signaling pathways. Phosphorylation of ATR Thr1989, as well as phosphorylation of the ATR substrate Chk1 Ser317, but not phosphorylation of ATM Ser1981 or Chk2 Thr68, was abrogated by treatment with the ATR inhibitor VE-821. Using western blotting, we also detected a DNA damage-induced electrophoretic mobility shift of CtIP (CtBP-interacting protein), a protein involved in the generation of single stranded DNA. The change in CtIP was partially abrogated by the ATR inhibitor, indicating that posttranslational modification of CtIP may be related to ATR Thr1989 phosphorylation.

Using cell lines with and without DNA damage, and in the presence or absence of kinase inhibitor, we performed PTMScan proteomic analysis to identify and quantify changes in phosphorylation levels of proteins and sites containing a consensus ATM/ATR substrate motif (S*Q/T*Q). Changes in phosphopeptide abundance between samples monitored by PTMScan provides insight into signaling components regulated by the ATM and ATR DNA damage response.

Citation Format: Hayley J. Roberts, Matthew P. Stokes, Xiaoying Jia, Kimberly A. Lee, Susan M. Keezer. DNA damage inducible phosphorylation of ATR at threonine 1989 and quantitative analysis of the effect of ATR inhibition on DNA damage signaling using PTMScan [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B09.