Abstract
To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated by DNA damage, provides so far unprecedented temporal dissection of DNA damage-modified phosphorylation events, and elucidate the cross-talk between different types of post-translational modifications in the dynamic regulation of a multifaceted DNA damage response.
Highlights
To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometrybased proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation
The direct targets of ataxia telangiectasia mutated (ATM) include proteins ranging from the initial sensing and enzymatic processing of double strand breaks (DSBs), histones (H2AX), DNA damage response (DDR) signaling mediators, repair factors (e.g. BRCA1 and TopBP1), checkpoint transducers (e.g. CHK2), chromatin modifiers (e.g. KAP1), transcription factors and tumor suppressors, up to ATM itself
A large scale proteomics analysis identified several hundred proteins targeted by ATM or ATMrelated kinases [7], and the list of the DSB-induced phosphorylations will inevitably expand after including kinases such as CHK1 and CHK2 operating downstream of ATM
Summary
To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometrybased proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. Proteins in nuclear lysates (supplemental Fig. S2) were digested with trypsin, and the resulting peptide mixtures were separated and enriched for phosphopeptides by ERLIC [10] and TiO2 chromatography [11]. The mass spectrometry-derived time profiles were compared with Western blot analyses for selected regulated phosphorylation sites known to be involved in the DDR (Fig. 2, A–C).
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