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

Abstract

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 cross talks 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.

Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition

“…In this work we have used motif-specific IAE and MS to assess known DDR phosphorylation events (13,14,29,30) while simultaneously interrogating signals unique to this model in which coadministration of GDC-0973 with GDC-0941 has proven efficacious (Fig. 4D and Dataset S3) (17,18).…”

Identification of Proteins at Active, Stalled, and Collapsed Replication Forks Using Isolation of Proteins on Nascent DNA (iPOND) Coupled with Mass Spectrometry

“…The centrosomal protein CP110, the DNMT1 recruiting protein UHRF1, and the euchromatic histone methyltransferase EHMT1 have predicted AlkB homologue 2 PCNA-interacting motifs (24). To further analyze the proteins, the dataset was compared with two published proteomics screens that identify substrates of the ATM or ATR checkpoint kinase substrates (25,26). At least 19 of the iPOND-MS enriched proteins are putative ATM/ ATR substrates that were identified in these proteomic screens (Fig.…”

“…Gene not in query refers to proteins known to physically interact with other proteins in the depicted physical interaction network but that were not identified in the iPOND-MS screen. C, proteins that contain PCNA-interacting motifs (24) or ATM/ATR phosphorylation sites (25,26) or that cause increased DNA damage signaling when silenced by siRNA (27,28) are listed.…”

ATM-dependent Phosphorylation of the Fanconi Anemia Protein PALB2 Promotes the DNA Damage Response

“…Indeed, proteome-wideprofilingofDNAdamage-inducedproteinphosphorylation events revealed hundreds of protein factors that become post-translationally modified in response to IR (23,30,31). Although many of the IR-induced phosphorylation events targeted consensus sequences that are recognized by the master DDR kinases ATM and ATR, regulation and functional significance of most of these modifications remain undefined.…”