Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Aug 19;286(33):28707-28714.
doi: 10.1074/jbc.M111.248914. Epub 2011 Jun 24.

Thr-1989 phosphorylation is a marker of active ataxia telangiectasia-mutated and Rad3-related (ATR) kinase

Edward A Nam  1 , Runxiang Zhao  2 , Gloria G Glick  2 , Carol E Bansbach  2 , David B Friedman  2 , David Cortez  3
Affiliations

Thr-1989 phosphorylation is a marker of active ataxia telangiectasia-mutated and Rad3-related (ATR) kinase

Edward A Nam et al. J Biol Chem. .

Abstract

The DNA damage response kinases ataxia telangiectasia-mutated (ATM), DNA-dependent protein kinase (DNA-PK), and ataxia telangiectasia-mutated and Rad3-related (ATR) signal through multiple pathways to promote genome maintenance. These related kinases share similar methods of regulation, including recruitment to specific nucleic acid structures and association with protein activators. ATM and DNA-PK also are regulated via phosphorylation, which provides a convenient biomarker for their activity. Whether phosphorylation regulates ATR is unknown. Here we identify ATR Thr-1989 as a DNA damage-regulated phosphorylation site. Selective inhibition of ATR prevents Thr-1989 phosphorylation, and phosphorylation requires ATR activation. Cells engineered to express only a non-phosphorylatable T1989A mutant exhibit a modest ATR functional defect. Our results suggest that, like ATM and DNA-PK, phosphorylation regulates ATR, and phospho-peptide specific antibodies to Thr-1989 provide a proximal marker of ATR activation.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
ATR is phosphorylated on Thr-1989. A, annotated tandem mass spectrum of a doubly charged peptide ion (m/z 942.10) consistent with the ATR sequence GVELCFPENETPPEGK with the threonine phosphorylated at position 1989 (Xcorr = 3.63). Labeled b ions and y ions are denoted by cleavage brackets above and below the sequence, respectively. B, sequence alignment of ATR orthologues. Black-shaded boxes indicate conserved residues, and gray boxes denote residues with similar characteristics.
FIGURE 2.
FIGURE 2.
DNA damage induces ATR Thr-1989 phosphorylation. A, ATRflox/−TR cells were infected with adenovirus encoding either GFP or Cre. ATR was immunoprecipitated (IP), and immune complexes were separated by SDS-PAGE prior to immunoblotting with phospho-specific (pATR) or total (N19) ATR antibodies. B–D, anti-ATR or control IgG immunoprecipitates from cells treated with HU, UV, or IR for the indicated times were separated by SDS-PAGE and immunoblotted as indicated. CHK1 phosphorylation was examined in total cell lysates. D, quantification of pATR/ATR 1 h post-HU treatment over three independent experiments. *, p < 0.01. E–G, cells were mock- or HU-treated for 6 h. E, anti-HA immunoprecipitates from control ATRflox/−TR (Ctl) or ATRdel/−TR cells expressing FLAG-HA-ATR were incubated with or without λ phosphatase prior to immunoblotting. F, FLAG-ATR WT or T1989A were expressed in 293T cells. FLAG immunoprecipitates were mock-treated or treated with phosphatase prior to immunoblotting. G, FLAG immunoprecipitates from ATRflox/−TR (Ctl), FLAG-HA-ATR WT, or FLAG-HA-ATR T1989A expressing ATRflox/−TR cells were immunoblotted.
FIGURE 3.
FIGURE 3.
Thr-1989 phosphorylation depends on ATR kinase activity. A–D, immunoprecipitates (IP) or total cell lysates were analyzed by immunoblotting with the indicated antibodies. A, FLAG-ATR (WT) or FLAG-ATR kinase dead (KD) expressing 293T cells were treated with HU for 0 or 6 h. Ctl, cells transfected with an empty vector. B, 293T cells were treated with the following kinase inhibitors in the presence or absence of HU for 6 h: 10 mm caffeine (Caf), 10 μm KU55933 (KU), or 1 μm NU7441 (NU). C, ATRflox/−TR (Ctl) or ATRdel/−TR cells expressing FLAG-HA-ATR were treated with 2 mm HU and 5 μm AZ20 for 6 h, as indicated. D, 293T cells were treated with HU and 3 μm AZ20 for 6 h.
FIGURE 4.
FIGURE 4.
Thr-1989 phosphorylation requires ATR activation via the PRD. A, empty vector (Ctl), FLAG-ATR-WT, or FLAG-ATR-PRD expression vectors were transfected into 293T cells. Cells were HU-treated or untreated for 6 h, lysed, and FLAG-immunoprecipitates (IP) immunoblotted to detect phosphorylated and total ATR. B and C, wild-type ATR (WT), ATR T1989A, or ATR T1989E proteins complexed with ATRIP were isolated from transfected 293T cells and incubated with MCM2 substrate, [γ-32P]ATP, and increasing concentrations of recombinant GST-TOPBP1-AAD or GST (B) or GST-TOPBP1-BRCT7&8 or GST-TOPBP1-AAD+BRCT7&8 (C), as indicated. Kinase reactions were separated by SDS-PAGE and detected by phosphorimaging ([32P]ATR and [32P]MCM2). The amount of ATR, ATRIP, TOPBP1, MCM2, and GST proteins in each reaction was detected by staining with Coomassie blue.
FIGURE 5.
FIGURE 5.
ATR Thr-1989 phosphorylation is dispensable for cellular recovery from replication stress. A, ATRflox/−TR cells with an integrated tetracycline-responsive expression vector encoding FLAG-HA-ATR (WT) or FLAG-HA-T1989A proteins were analyzed by immunofluorescence after a transient induction of protein expression. B, localization of wild-type or T1989A ATR in HU-treated cells. C and D, ATRflox/−TR, WT-ATRflox/−TR, or two independent clones of T1989A-ATRflox/−TR cells were cultured in tetracycline media and infected with control (AdGFP) or Cre-expressing adenovirus (AdCre) to delete the floxed ATR allele. C, four days after infection, cells were treated with 0 or 2 mm HU for 6 h. Lysates were separated by SDS-PAGE and immunoblotted with the indicated antibodies. D, AdCre-infected cells were treated with HU for 24 h, washed once, and released into growth media containing nocodazole for 0, 4, or 10 h. Cells were fixed, stained with propidium iodide, and examined for DNA content by flow cytometry. Untreated (Asyn) cells were also analyzed.
FIGURE 6.
FIGURE 6.
Functional analysis of Thr-1989 phosphorylation. A–C, ATRflox/−TR, WT-ATRflox/−TR, or two independent clones of T1989A-ATRflox/−TR cells were cultured in tetracycline media and infected with control (AdGFP) or Cre-expressing adenovirus to delete the floxed ATR allele. The infected cells were plated at low density and then cultured for 14 days in the presence of tetracycline. Surviving colonies were stained (A), scored (B), and PCR-genotyped (C). D and E, surviving WT-ATRdel/−TR and T1989A-ATRdel/−TR clones were challenged with 2 mm HU for 0, 15, or 30 min. Cell lysates were separated by SDS-PAGE and analyzed by quantitative immunoblotting. E, the amount of T1989A ATR in each clone (expressed as a percentage of the level in the parental ATRflox/−TR cells) was compared with the amount of CHK1 phosphorylation following the 15- or 30-min HU treatment.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Perry J., Kleckner N. (2003) Cell 112, 151–155 - PubMed
    1. Bosotti R., Isacchi A., Sonnhammer E. L. (2000) Trends Biochem. Sci 25, 225–227 - PubMed
    1. Mordes D. A., Glick G. G., Zhao R., Cortez D. (2008) Genes Dev. 22, 1478–1489 - PMC - PubMed
    1. Cortez D., Guntuku S., Qin J., Elledge S. J. (2001) Science 294, 1713–1716 - PubMed
    1. Brown E. J., Baltimore D. (2000) Genes Dev. 14, 397–402 - PMC - PubMed

Publication types

MeSH terms