Research ResourcePhosphoproteomics

ATM-Dependent and -Independent Dynamics of the Nuclear Phosphoproteome After DNA Damage

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Science Signaling  07 Dec 2010:
Vol. 3, Issue 151, pp. rs3
DOI: 10.1126/scisignal.2001034

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Beyond ATM in the DNA Damage Response

Because genome stability is essential to cellular and organismal survival, cells have evolved elaborate mechanisms to respond to DNA damage. A key protein involved in the initiation of the cellular response to DNA double-strand breaks, which can be caused by chemicals or radiation, is the protein kinase ATM. Bensimon et al. performed a quantitative analysis of the phosphoproteomic changes that occurred in response to double-strand breaks and found that a large proportion of the changes in phosphorylation were not attributable to ATM activity, suggesting a much larger kinase-mediated phosphorylation network in this critical response. Bioinformatic analysis suggested several candidate kinases that carry out the ATM-independent phosphorylations. Mapping sites that showed decreased phosphorylation in response to DNA damage suggested kinases that might be inhibited or whose action might be reversed by phosphatases activated during the DNA damage response. In addition to revealing a previously unknown phosphorylation site on ATM implicated in its retention at sites of damage, this study also provides a plethora of opportunities for deeper investigation into the phosphorylation network involved in maintaining genome stability.


The double-strand break (DSB) is a cytotoxic DNA lesion caused by oxygen radicals, ionizing radiation, and radiomimetic chemicals. Cells cope with DNA damage by activating the DNA damage response (DDR), which leads either to damage repair and cellular survival or to programmed cell death. The main transducer of the DSB response is the nuclear protein kinase ataxia telangiectasia mutated (ATM). We applied label-free quantitative mass spectrometry to follow the dynamics of DSB-induced phosphoproteome in nuclear fractions of the human melanoma G361 cells after radiomimetic treatment. We found that these dynamics are complex, including both phosphorylation and dephosphorylation events. In addition to identifying previously unknown ATM-dependent phosphorylation and dephosphorylation events, we found that about 40% of DSB-induced phosphorylations were ATM-independent and that several other kinases are potentially involved. Sustained activity of ATM was required to maintain many ATM-dependent phosphorylations. We identified an ATM-dependent phosphorylation site on ATM itself that played a role in its retention on damaged chromatin. By connecting many of the phosphorylated and dephosphorylated proteins into functional networks, we highlight putative crosstalks between proteins pertaining to several cellular biological processes. Our study expands the DDR phosphorylation landscape and identifies previously unknown ATM-dependent and -independent branches. It reveals insights into the breadth and complexity of the cellular responses involved in the coordination of many DDR pathways, which is in line with the critical importance of genomic stability in maintenance of cellular homeostasis.

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