Proteomic investigations reveal a role for RNA processing factor THRAP3 in the DNA damage response

Abstract

The regulatory networks of the DNA damage response (DDR) encompass many proteins and posttranslational modifications. Here, we use mass spectrometry-based proteomics to analyze the systems-wide response to DNA damage by parallel quantification of the DDR-regulated phosphoproteome, acetylome, and proteome. We show that phosphorylation-dependent signaling networks are regulated more strongly compared to acetylation. Among the phosphorylated proteins identified are many putative substrates of DNA-PK, ATM, and ATR kinases, but a majority of phosphorylated proteins do not share the ATM/ATR/DNA-PK target consensus motif, suggesting an important role of downstream kinases in amplifying DDR signals. We show that the splicing-regulator phosphatase PPM1G is recruited to sites of DNA damage, while the splicing-associated protein THRAP3 is excluded from these regions. Moreover, THRAP3 depletion causes cellular hypersensitivity to DNA-damaging agents. Collectively, these data broaden our knowledge of DNA damage signaling networks and highlight an important link between RNA metabolism and DNA repair.

Figure 1

Proteome-wide investigation of the DNA damage response. (A) Schematic presentation of the SILAC-based quantitative strategy. Phosphoproteome, acetylome and proteome were measured from SILAC-labeled U2OS cells treated with DNA damage inducing agents as outlined. (B) Cellular distribution of DDR-regulated phosphorylation and acetylation sites. The DDR-upregulated sites are significantly more localized to the nucleus compared to non-regulated sites. (C and D) Cellular distribution of DNA damage-upregulated phosphorylation sites that do or do not contain the S/TQ motif. The bar plots show nuclear or non-nuclear distribution of upregulated sites compared to non-regulated sites. p-values were calculated using Fisher exact test. (See also Figure S1)

Figure 2

Sequence properties of DNA damage-upregulated phosphorylation sites. (A) The ice-Logo plot shows frequency of 6 amino acids flanking each side of phosphorylated residue. The amino acid sequences of DDR-regulated sites (both from etoposide and IR) were compared with the phosphorylation sites that do not change under these conditions. The numbers in the middle indicate the position of amino acid in peptide relative to the central, phosphorylated amino acid. Amino acids that are more frequently observed near the DDR-upregulated sites are indicated over the middle line, whereas the amino acids with lower frequency at these positions are indicated below the line. A significantly increased phosphorylation of sites conforming to the ATM/ATR/DNA-PK kinase motif (S/TQ) is observed upon DDR. The corresponding plot (right panel) shows distribution of SILAC ratios for phosphorylation sites conforming to the indicated phosphorylation motif. (B) DDR-regulated S/TQ sites disfavor arginine at positions preceding the phosphorylation sites. (C) Amino acid preference for non-S/TQ sites upregulated by DNA damage. (See also Figure S2 and S3)

Figure 3

Functional modules of DDR-regulated phosphoroteins. (A) Network analysis of DDR-upregulated phosphoproteins using STRING (Szklarczyk et al.) showing connectivity among the proteins with increased phosphorylation upon DNA damage (STRING confidence score 0.7). Two of the highly interconnected subnetworks identified by MCODE are shown. (B) Schematic overview of cellular processes and involved proteins that show increased phosphorylation after DNA damage. Proteins with increased phosphorylation on S/TQ sites are colored in red whereas proteins without color show increased phosphorylation on non-S/TQ sites. (See also Figure S4)

Figure 4

CYLD regulates DNA damage-induced activation of NF-κB signaling. (A) Phosphorylation of CYLD Ser-418 by DNA damage inducing agents. Cells were treated with TNF-α, IR, or etoposide (Etp) and phosphorylation was analyzed using Ser-418 phospho-specific CYLD antibody. (B) Dynamics of DDR-induced CYLD phosphorylation. Cells were irradiated and allowed to recover for the indicated time points, and phosphorylation was detected as described above. (C) CYLD inhibits nuclear translocation of the p65 subunit of NF-κB. Cells were transfected with a plasmid encoding CYLD and nuclear translocation of p65 was analyzed by immunofluorescence microscopy after treatment with TNF-α or IR. (D and E) CYLD inhibits DNA damage-induced NF-κB transcriptional activity. Cells were transfected with the indicated CYLD constructs or empty vector and NF-κB activation was monitored with luciferase-based reporter assays. Error bars specify the standard deviation of the three independently performed experiments. p**<0.05. (See also Figure S5)

Figure 5

PPM1G is phosphorylated in response to DNA damage and is recruited to DNA damage sites. (A) Detection of PPM1G phosphorylation by Ser-61 phospho-specific Bid antibody (α-phospho) in GFP immunoprecipitates from U2OS cells transfected with GFP or GFP-PPM1G plasmids and treated with 3 μM etoposide (Etp) for 2 hours or 10 Gy IR (1 hour recovery). Position of GFP-PPM1G and GFP is indicated by arrow. (B) Sequence alignment of the region encompassing Ser-183 and Ser-61 on PPM1G and Bid, respectively. (C) Western blotting analysis of GFP immunoprecipates and total cell extracts from U2OS cells transfected with wild-type GFP-PPM1G or indicated phospho-mutants and treated with etoposide (Etp) where indicated. (D) Recruitment of PPM1G to sites of DNA damage in U2OS cells transfected with GFP-PPM1G plasmid and fixed at indicated times following micro-irradiation.

Figure 6

THRAP3 is phosphorylated in response to DNA damage and is excluded from DNA damage sites. (A) and (B) Phosphorylation of THRAP3 in cell extracts untreated or treated with etoposide (Etp; 3 μM, 2h), HU or 4NQO, which were treated with λ phosphatase (Ptase) where indicated. (C) Phosphorylation of wild-type HA-THRAP3 or indicated THRAP3 phospho-mutants in U2OS cells treated with 4NQO where indicated. (Note that antibody detection was not reduced further by mutating all 6 phosphorylation sites to Ala; 6×S/A). (D) Phosphorylation of THRAP3 in cells treated with inhibitors of ATM (ATMi), DNA-PK (D-PKi), ATR (ATRi) or wortmannin (Wort) for 1 hour, then treated with Etp (3 μM, 2h). (E) Clonogenic survival of cells transfected with siluci or siTHRAP3-1 and treated with HU at the indicated doses. Results are presented as an average from 3 experiments −/+ SEM. Depletion of THRAP3 is shown by Western blotting. (F) Exclusion of THRAP3 from sites of laser-induced DNA damage in HeLa-GFP-THRAP3 cells. Quantification of green (GFP-THRAP3) and red (γH2AX) was performed by Volocity software. The graph shows the intensity of fluorescence (y-axis) along the white line (x-axis) indicated by arrow. (G) Exclusion of THRAP3 from laser-damage in cells fixed at indicated times. (H) Exclusion of THRAP3 from DNA damage sites is inhibited in cells treated with inhibitors of ATM (ATMi), DNA-PK (D-PKi) and ATR (ATRi) for 1 hour prior to micro-irradiation. (untr = untreated; p = phospho). (See also Figure S6)

Figure 7

Exclusion of THRAP3 is linked to transcriptional inhibition. (A) Incorporation of EU in U2OS cells is inhibited at sites of laser-induced DNA damage. (B) Exclusion of RNA PolII from sites of micro-irradiation (cells stained with phospho-Ser2-C-terminal domain specific antibody). (C) Exclusion of THRAP3 from laser-damage is reduced upon depletion of RNF8 and RNF168 (the efficiency of RNF8/RNF168 depletion is shown by abrogation of 53BP1 recruitment). Shown are representative fields of cells derived from three independent experiments. For the boxed cells, fluorescence intensity along the white line was measured by Volocity software and the resulting graphs are provided on the right. Quantifications for all cells in the fields are provided in Figure S7B