Phosphoproteomics reveals novel modes of function and inter-relationships among PIKKs in response to genotoxic stress

Abstract

The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR, and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR.

Keywords: ATM; DNA damage response; PIKKs; ataxia-telangiectasia; phosphoproteomics.

Figure 1. Modulation of the cellular phosphoproteome following NCS treatment

1. A

   Schematic workflow of the phosphoproteomic experiment. Cells were treated with 20 ng/ml NCS in the presence of selective inhibitors of ATM (KU60019, 5 µM), ATR (AZ20, 0.5 µM), DNA‐PK (NU7441, 5 µM), or DMSO (inhibitor solvent). Inhibitors were added 30 min prior to NCS treatment, and samples were collected 20, 60, and 240 min following NCS addition. Protein extracts were digested into peptides and subsequently enriched for phosphopeptides, which were measured using LC‐MS/MS and subjected to label‐free quantification and subsequent data processing and analysis using the MaxQuant (Cox & Mann, 2008; Tyanova et al, 2016a) and the Perseus (Tyanova et al, 2016b) software. An FDR of 0.05 together with a minimal fold change of twofold was applied to determine regulation in response to NCS and PIKK inhibitors.

2. B

   Enriched GO cellular compartments (gray) among NCS‐induced phosphorylations and dephosphorylations, and GO biological processes (purple‐red) among NCS‐induced phosphorylations, in WT cells. Enrichment was tested using the Fisher exact test implemented in Perseus (Tyanova et al, 2016b).

3. C

   Enriched motifs among NCS‐induced phosphorylations (yellow) and dephosphorylations (gray) in WT cells. Enrichment was tested using the Fisher exact test implemented in Perseus (Tyanova et al, 2016b).

Figure EV1. Dose‐dependent action of PIKK inhibitors

Western blots of WT (NL‐550) cells treated for 1 h with neocarzinostatin (NCS; 20 ng/ml) or hydroxyurea (HU; 1 mM) in the presence of the indicated inhibitors. The inhibitors were added to the cultures 30 min prior to the addition of NCS or HU.

1. A

   ATMi inhibited ATM in a dose‐dependent manner, as demonstrated by the phosphorylation of S824/KAP‐1.

2. B

   DNA‐PKi inhibited DNA‐PK in a dose‐dependent manner, as demonstrated by the autophosphorylation of S2056/DNA‐PKcs.

3. C, D

   ATRi inhibited ATR in a dose‐dependent manner, as demonstrated by HU‐induced phosphorylation of S345/CHK1.

Figure 2. PIKK dependencies of DNA damage‐induced dynamics of the phosphoproteome in WT cells

1. A

   Relative share of the three PIKKs in NCS‐responsive phosphorylations.

2. B

   Venn diagram depicting the relative share of the three PIKKs in NCS‐induced phosphorylations occurring on S/TQ sites.

3. C

   The profile of a group of substrates targeted by ATM 20 min after neocarzinostatin (NCS) addition and by ATR 1 h after hydroxyurea (HU) addition. Box plots depict 20 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

4. D

   Western blotting analysis confirming that the ATM substrate, pS824/KAP1 is targeted by ATR in response to HU. pS345/CHK1—an established ATR substrate—served as a positive control for ATR activation.

Source data are available online for this figure.

Figure 3. Chemical inhibition of DNA‐PK enhances ATM‐mediated phosphorylations in WT cells

1. A

   A cluster of 71 neocarzinostatin (NCS)‐induced phosphorylations that were enhanced upon continuous inhibition of DNA‐PK. Untreated cells are marked UT. This cluster breaks down into two distinct groups according to their ATM dependence 240 min after NCS addition. Clusters were obtained using K‐means algorithm implemented in Perseus on Z‐scored intensities. Box plots depict 71 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

2. B

   Depicted are phosphopeptides from (A) that responded to NCS treatment in A‐T cells. No significant elevation was observed in these phosphorylations in A‐T cells following continuous inhibition of DNA‐PK. Box plots depict 19 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 time the interquartile range (IQR).

3. C

   The cluster depicted in (A) was enriched for ATM‐dependent phosphorylations and the S/TQ phosphorylation motifs. Enrichment was tested using the Fisher exact test implemented in Perseus (Tyanova et al, 2016b).

4. D

   STRING‐Network representation of the proteins in the same cluster. Proteins phosphorylated on the S/TQ motif are marked with a yellow border, and the thickness of connecting lines represents the combined score for interaction confidence according to STRING.

5. E

   Western blotting analysis of selected phosphorylations in the above cluster showing DNA‐PKi‐induced elevation.

6. F

   Temporal dynamics of DNA‐PK‐dependent attenuation of pS824/KAP‐1.

Source data are available online for this figure.

Figure EV2. DNA‐PKi elevates the ATM response in U2‐OS cells

Western blotting analysis of selected phosphorylations showing their DNA‐PKi‐dependent elevation 4 h after neocarzinostatin (NCS) addition.

Figure 4. PIKK‐dependent compensation for ATM absence in A‐T cells

1. A

   A subset of exclusive ATM‐dependent sites in WT cells, which responded to neocarzinostatin (NCS) also in A‐T cells. Their dependencies in A‐T cells are shown.

2. B

   Dependencies of S/TQ phosphorylations within the same subset.

3. C

   The first time points at which “compensated sites” from (A) responded to NCS in WT and A‐T cells are presented.

4. D

   Temporal kinase dependencies of the “compensated sites” in A‐T cells. Shown are the numbers of "compensated sites" in A‐T cells, which depended on each kinase at the 20 and 240 min time points. The early ones depend mainly on DNA‐PK, while the later ones—on ATR.

5. E

   STRING‐Network representation of the proteins that were included in the early, DNA‐PK‐dependent compensation (purple) or the late, ATR‐dependent compensation (green). Proteins phosphorylated on the S/TQ motif are highlighted by yellow margins. The thickness of connecting lines represents the combined score for interaction confidence according to STRING.

6. F

   Western blotting analysis confirming high‐throughput results for selected ATM substrates.

Figure 5. Compensation for chemical inhibition of ATM in WT cells

1. A

   A group of exclusively ATM‐dependent sites that were ATM‐dependent 1 hr after NCS addition and were still phosphorylated or dephosphorylated 4 h after treatment, but were not affected by ATMi at that time point. Box plots depict 18 phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

2. B

   This group was enriched for sites that were compensated in an ATR‐dependent manner in A‐T cells. Box plots depict nine phosphopeptides measured in two independent biological replicates. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

3. C

   Western blotting analysis of pS343/NBS1, which is compensated upon ATM inhibition, and pS824/ KAP‐1 and pS395/NUMA, which are not compensated under these conditions.

Figure 6. Representative SRM measurements of specific phosphorylation sites

Cells were either untreated (UT) or treated with neocarzinostatin (NCS) in the presence or absence of PIKK inhibitors. Intensities from three independent biological replicates were normalized against reference synthetic standards and are depicted as mean ± SEM.

Figure EV3. SRM measurements with KU55933

HeLa cells were either untreated (UT) or treated with 10 µM KU55933 and 50 ng/ml neocarzinostatin (NCS) and harvested 1 h following NCS treatment. Depicted are the z‐scored intensities of 18 phosphopeptides that were elevated at least twofold following NCS. Box plots depict 18 phosphopeptides measured in one replicate. The box indicates the range from first to third quartiles, and the central band represents the median. Upper and lower whiskers extend from the box to the maximum and minimum values which are not farther than 1.5 times the interquartile range (IQR).

Figure 7. A model of PIKK collaboration in the cellular response to genotoxic stress, based on the results of this study

ATM‐proficient cells respond to DSBs with initial, robust ATM‐mediated phosphorylation of numerous substrates, after which ATM’s activity is attenuated in a DNA‐PK‐dependent manner. In ATM‐deficient cells, some ATM substrates are targeted by DNA‐PK at the early phase of the response, or by ATR in the later phase, but to a lesser extent than in WT cells. ATR‐dependent compensation for ATM absence in A‐T cells includes significantly more substrates than that of DNA‐PK and involves sites that ATR usually targets in response to genotoxic stresses other than DSBs. A few of these can be targeted by ATR also when ATM is chemically inhibited in WT cells.