Low and high doses of ionizing radiation evoke discrete global (phospho)proteome responses

Although cancer risk is assumed to be linear with ionizing radiation (IR) dose, it is unclear to what extent low doses (LD) of IR from medical and occupational exposures pose a cancer risk for humans. Improved mechanistic understanding of the signaling responses to LD may help to clarify this uncertainty. Here, we performed quantitative mass spectrometry-based proteomics and phosphoproteomics experiments, using mouse embryonic stem cells, at 0.5 h and 4 h after exposure to LD (0.1 Gy) and high doses (HD; 1 Gy) of IR.

The proteome remained relatively stable (29; 0.5% proteins responded), whereas the phosphoproteome changed dynamically (819; 7% phosphosites changed) upon irradiation. Dose-dependent alterations of 25 IR-responsive proteins were identified, with only four in common between LD and HD. Mitochondrial metabolic proteins and pathways responded to LD, whereas transporter proteins and mitochondrial uncoupling pathways responded to HD. Congruently, mitochondrial respiration increased after LD exposure but decreased after HD exposure. While the bulk of the phosphoproteome response to LD (76%) occurred already at 0.5 h, an equivalent proportion of the phosphosites responded to HD at both time points. Motif, kinome/phosphatome, kinase-substrate, and pathway analyses revealed a robust DNA damage response (DDR) activation after HD exposure but not after LD exposure. Instead, LD-irradiation induced (de)phosphorylation of kinases, kinase-substrates and phosphatases that predominantly respond to reactive oxygen species (ROS) production.

Our analyses identify discrete global proteome and phosphoproteome responses after LD and HD, uncovering novel proteins and protein (de)phosphorylation events involved in the dose-dependent ionizing radiation responses.