Cells maintain their genome integrity via a surveillance network known as the DNA damage response (DDR). The importance of the DDR is revealed by physiological abnormalities that manifest as a result of genetic defects in specific signaling components (nodes) in the DDR network (Matsuoka et al., 2007
). Research into DDR pathways has thus far focused on elucidating the hierarchy of signals that propagate from sites of DNA damage to drive cellular outcomes such as cell-cycle arrest, DNA repair, and apoptosis. Key to these signaling cascades are the major transducers that link the DNA damage sensors to downstream effectors; the most studied are the ATM (Ataxia-telangiectasia mutated) and ATR (ATM and Rad3-related) serine/threonine kinases. In response to specific forms of DNA damage, ATM and ATR phosphorylate substrates (e.g., p53, Chk2, and BRCA1) bearing the SQ/TQ motif, thereby initiating the DDR. Despite the importance of these kinases, ATM and ATR substrate elucidation has progressed slowly, with just over 20 substrates identified over the past decade. In addition, connecting diverse DNA damaging agents with the activation of networks which coordinate DNA repair mechanisms and cellular outcomes remains a significant challenge (Matsuoka et al., 2007
Several mass spectrometry (MS) studies focused on the largescale elucidation of potential substrates for ATM and ATR kinases have now revealed the striking complexity of the DDR. In a pioneering study in which phosphospecific pS/pT-Q antibodies were used to enrich for phosphoproteins prior to SILAC-based MS quantification (), Elledge and colleagues identified over 900 phosphorylation sites on 700 proteins that were upregulated at least 4-fold upon induction of double-strand breaks using ionizing radiation (IR) (Matsuoka et al., 2007
). These phosphorylation sites were found on proteins that perform a remarkable diversity of functions, including previously unidentified sites on known DDR proteins such as ATM itself, FANCD2, and BRCA1, as well as proteins that previously were not associated with the DDR, including RNA-splicing factors. By superimposing this list of proteins on known protein-protein interactions, the authors have generated multiple DDR modules that interact to form a larger network. Owing to the remarkable number of new DDR responsive SQ/TQ substrates identified in this study, it is not surprising that many of the modules identified were previously unknown DDR network components; indeed, some might account for previously enigmatic cellular processes observed in response to DNA damage. Most intriguing of these observations was the suggestion that the DDR network intimately intersects with multiple components of the insulin signaling network, including the PI3K-AKT pathway. It is tempting to speculate that the DDR co-opts this network to inhibit or delay apoptosis in response to DNA damage, thereby providing the necessary time for the DNA repair pathways to respond. Understanding the interactions between these two networks might provide clues regarding how cells decide between cell survival and apoptosis following DNA damage.
Overview of Phosphoproteomic Approaches
As is the case with all large-scale studies, despite the generation of massive amounts of data, many fundamental questions remain. For instance, what is the functional significance of the multiple phosphorylation sites identified on ATM and other DDR proteins? Are responsive phosphorylation sites ATM or ATR substrates, or might other PI3K-like protein kinase (PIKK) family members, known to respond to DNA damage (e.g., DNA-PK and SMG-1), be responsible (Matsuoka et al., 2007
)? It is worth noting that the study performed by Elledge and coworkers provides only a glimpse into the complexity of the phosphorylation events involved in the DDR. This point is highlighted by a recent study in which phospho-motif specific antibodies were used to enrich for peptides containing pS/pT-Q, with the goal of identifying substrates of SQ/TQ-directed kinases (e.g., ATR) activated by ultraviolet (UV) irradiation, which causes single-strand breaks (Stokes et al., 2007
). Despite the identification of over 200 UV-responsive phosphorylation sites with the proper SQ/TQ motif, only 46% overlapped with those identified in the IR study. This lack of overlap between the two data sets could result from biochemical differences in the DDR network in response to double-strand (IR) versus single-strand (UV) breaks or could be caused by experimental variation between the two studies, including cell line and phosphospecific-versus-phosphomotif antibody differences.
The phosphorylation sites identified in both data sets are of greater interest, as they might be associated with common pathways that respond to multiple DNA insults, or they could represent an activation cascade of multiple members of the PIKK family in response to specific DNA-damage insults. For instance, in response to double-strand breaks, ATM and DNA-PK activation is followed by subsequent ATR activation via both ATM-dependent and -independent mechanisms (Matsuoka et al., 2007
). To unravel the kinase dependency of selected phosphorylation sites, biochemical perturbation was combined with quantitative MS to identify common nodes and potential PIKK-specific substrate candidates from these two studies. Specifically, to identify ATM-dependent phosphorylation events, cells were treated with IR in the presence of KU-55933, a specific ATM inhibitor; to identify ATM-dependent phosphorylation events, ATR-deficient Seckel cells were treated with UV. Approximately 70% of the sites that were diminished in the UV-irradiated Seckel cells were also upregulated in the general cellular response to IR and, therefore, likely represent ATR-dependent sites phosphorylated in response to either UV- or IR-induced DNA damage, possibly through the sequential activation of ATR by ATM in the latter case. Interestingly, two ATR-dependent sites, one on EYA3, a phosphatase involved in organogenesis, and one on SMC1, a member of the cohesin complex, are also ATM-dependent. This finding suggests that these proteins might be shared nodes in the cellular response to different forms of DNA damage, activated by both ATM and ATR. Although SMC1 was previously implicated as a substrate for both ATM and ATR kinases, with kinase dependency linked to the extent of DNA damage (Wakeman and Xu, 2006
), EYA3 is a newly defined common node whose role in regulating the DDR remains undetermined. In addition to these common nodes, the splicing factor SFRS14 was found to be ATR dependent in the UV study and ATM independent in the IR study. SFRS14 is, therefore, likely to be an ATR-specific substrate involved in both UV- and IR-activated DDR in an ATM-independent fashion.
Unfortunately neither of these phosphoproteomic analyses were performed to saturation (only 18 SQ/TQ phosphorylation sites were quantified from UV-treated Seckel cells), and therefore, the mechanistic insights from these studies are limited. However, this small amount of data already highlights the ability of phosphoproteomic screening, when combined with multiple biological perturbations, to identify well-characterized mechanistic relationships and to define novel linkages. Subsequent directed biological experiments should clarify the mechanistic role(s) for such proteins. Likewise, more extensive analysis of these biological systems will identify additional nodes that are common or distinct to the various PIKK family members. In this manner, functional characterization of the hundreds of potential substrates can facilitate greater understanding of how this complex network is regulated. This information will be critical in understanding the contribution of the DDR to cancer etiology and for selecting potential targets to overcome resistance to DNA-damaging chemotherapeutics in cancer patients.