Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions

Abstract

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database.Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments.In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se Further, our data will have a significant impact on the ongoing debate about patient treatment modalities.

© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

Fig. 1.

Determination and validation of radiobiological equivalent doses.A, Clonogenic survival assay for A549 cells exposed to dose series of X-rays, protons and carbon ions. Doses were set to achieve 30% survival fraction leading to the following doses used throughout the study: 6 Gy X-ray, 3.5 Gy protons and 2 Gy carbon ions. Data represents mean ± S.D. of three independent experiments. B, Representative images of TP53BP1 foci (red) initial (1h) and residual (24h) after irradiation with the different radiation qualities. Cell nuclei were counterstained with DAPI (blue). Selected nuclei are magnified to emphasize the foci pattern following the individual treatments. C, Percentage of cells containing none, one or more residual TP53BP1 foci per nucleus 24h after irradiation. Foci were counted in two independent experiments (n>2000 cell nuclei per condition and experiment) using ImageJ and mean ± S.D. is illustrated. D, Western blot analysis of phosphorylation sites and their basal protein expression in response to different radiation qualities 2h after irradiation. Selected sites represent key player in the DNA damage signaling and are known to respond to ionizing radiation dependent DNA damage. GAPDH was used as loading control. E, DNA damage response network, illustrating the quantitative Western blot results using Cytoscape plugin PhosphoPath (34). Additional proteins were added to complement the network. Functional analysis of the network using the ClueGO plugin within Cytoscape revealed three predominant biological processes demonstrating their affiliation to the DNA damage response as well as equal regulation by the different radiation qualities.

Fig. 2.

Experimental workflow for the analysis of radiation induced changes of proteome and phosphoproteome.A, SILAC labeled A549 cells were irradiated with radiobiological equivalent doses and harvested 2h after treatment. Heavy labeled samples irradiated with X-rays, protons and carbon ions respectively, were mixed with light labeled control samples for accurate quantification. SDS-PAGE with subsequent in-gel tryptic digestion was performed for proteome analysis and a combination of IMAC and TiO2 phosphopeptide enrichment after in-solution digestion for phosphoproteome analysis. Resulting samples were analyzed by LC-MS/MS and data processing was conducted with MaxQuant. Selected phosphorylation sites were subsequently validated using synthetic medium isotope labeled phosphopeptides in a targeted spike-in approach. B, Descriptive results of the proteome and phosphoproteome profiling of irradiated A549 cells (*Regulation between irradiated and control samples; §According to PhosphoSitePlus). C, Distribution of phosphorylated serine, threonine, and tyrosine residues among quantified phosphosites. D, Distribution of singly and doubly phosphorylated peptides among quantified phosphosites.

Fig. 3.

Clustering and GO enrichment analysis of radiation regulated phosphorylation sites.A, Hierarchical clustering of the 181 regulated phosphosites following treatment with different radiation qualities in three biological replicates each (X: X-rays, 1H: Proton, 12C:Carbon). Values represent the normalized log2-transformed ratios for the change in phosphorylation status between irradiated and control samples. B, Functional analysis using the ClueGO plugin within Cytoscape to identify enriched GO terms in the data set of regulated phosphoproteins. Enriched GO terms were grouped according to GO hierarchy and displayed in pie charts representing the number of associated genes per group term. Detailed information about the enrichment analysis can be found in supplemental Table S4.

Fig. 4.

Phosphorylation consensus sequences and putative kinases of radiation regulated phosphorylation sites. Six consensus sequences were identified using the Motif-X software to be overrepresented in the set of 181 regulated phosphorylation sites compared with nonregulated sites during the response to ionizing radiation. Putative kinases were assigned and the heatmaps show the phosphorylation sites belonging to the different motifs (GSK-3: glycogen synthase kinase 3, ERK1/2: mitogen-activated protein kinases, CDK5: cyclin-dependent kinase 5, PKA: cAMP-dependent protein kinase, CaMKII: Ca2+/calmodulin-dependent protein kinase II, Akt: RAC-alpha serine/threonine-protein kinase, ATM: ataxia telangiectasia mutated).

Fig. 5.

Differential regulation of phosphorylation sites between radiation qualities.A, Unsupervised principle component analysis for confirmation of differences between the radiations' mode of action. Of note, clear separation between X-rays and particle based radiations in the first component. B, Volcano plots showing the phosphorylation sites being differentially regulated between the radiation qualities. Phosphosites with p value < 0.01 and 0.67< fold change > 1.5 are colored in blue; sites with p value < 0.001 and 0.5< fold change > 2 are colored in orange and labeled with their corresponding gene name. C, Western blot of the differentially regulated phosphorylation site S75 on SRC indicating strong dephosphorylation by both particle radiations whereas X-rays induce no significant regulation. Basal protein expression level shows no alterations. GAPDH was used as loading control. D, Bar charts illustrating quantitative Western blot results as well as SILAC based LC-MS/MS results, both at phosphorylation and basal protein expression level for three independent phosphorylation sites: RAD50 (S635), NUMA1 (S395) and SRC (S75) revealing excellent correlation between the two orthogonal analyses.

Fig. 6.

Validation of phosphorylation sites using synthetic isotope labeled phosphopeptides.A, Validation of the differentially regulated phosphorylation sites exemplified by S56 on VIM. Quantification of the depicted phosphorylation site after irradiation shows differential regulation between X-rays and the two particle treatments as seen in the left panel (mean ± S.E., n = 3). Identification of the phosphosite is verified by comparing selected transitions (central panel) and comparing the retention time in the discovery data set with the retention time of the synthetic peptide (right panel). B, MS1 spectra of VIM S56 peptides for the three different treatments (X-ray, 1H: protons, 12C: carbon ions). C, MS2 spectra of VIM S56 peptides for the three different isotopic states (§light peptide (sham), $medium peptide (synthetic); #heavy peptide (irradiated)). Zoom demonstrates exemplarily the mass shift between the isotopic states for the y8-ion of the depicted peptide.

Fig. 7.

Validated response to different radiation qualities.A, Phosphosite regulation following irradiation with X-rays, protons (1H) and carbon ions (12C) plotted as heatmap for 28 phosphopeptides validated using synthetic isotope labeled phosphopeptides in a spike-in experiment. Phosphopeptides are grouped into two clusters. Cluster 1 contains peptides of the p(S/T)Q and nonassigned motifs. Cluster 2 contains the peptides of the p(S/T)P motif. Values represent the normalized log2-transformed ratios for the change in phosphorylation status between irradiated and control samples. B, Functional analysis using the ClueGO plugin within Cytoscape to identify enriched GO terms in the set of validated phosphopeptides represented by their corresponding protein. Analysis was performed in the two illustrated clusters separately to uncover their particular characteristics concerning cellular localization and biological function. Illustrated are the five most significant terms in each cluster by their corresponding p value. C, NetworKIN algorithm in KinomeXplorer (32, 33) was used to predict associated kinases in order to estimate potential upstream regulators of the identified alterations. Kinase prediction for phosphorylation sites being differentially regulated between the radiation qualities. Regulation of the phosphosites as well as predicted kinases are illustrated using Cytoscape plugin PhosphoPath (34) D, Sites on TP53BP1 show likewise up- and downregulation. Upregulations are conducted by the phosphoinositide-3-like kinases (PIKK); ATM, ATR and PRKDC. Concurrently, three phosphorylation sites are downregulated by decreased CDK1 activity. Regulation of the phosphosites as well as predicted kinases are illustrated using Cytoscape plugin PhosphoPath (34) E, Schematic graph of TP53BP1 (1,972 amino acids) including two noteworthy structural features containing radiation regulated phosphorylation sites identified in this study: N-Terminal clusters of the p(S/T)-Q motif and two C-terminal BRCT domains. Domains not affected by our results are left out. The two depicted domains are responsible for TP53BP1 interaction with RIF1/PTIP and p53/EXPAND1 respectively (40).