Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response

Abstract

To investigate the temporal regulation of the DNA damage response, we applied quantitative mass spectrometry-based proteomics to measure site-specific phosphorylation changes of nuclear proteins after ionizing radiation. We profiled 5204 phosphorylation sites at five time points following DNA damage of which 594 sites on 209 proteins were observed to be regulated more than 2-fold. Of the 594 sites, 372 are novel phosphorylation sites primarily of nuclear origin. The 594 sites could be classified to distinct temporal profiles. Sites regulated shortly after radiation were enriched in the ataxia telangiectasia mutated (ATM) kinase SQ consensus sequence motif and a novel SXXQ motif. Importantly, in addition to induced phosphorylation, we identified a considerable group of sites that undergo DNA damage-induced dephosphorylation. Together, our data extend the number of known phosphorylation sites regulated by DNA damage, provides so far unprecedented temporal dissection of DNA damage-modified phosphorylation events, and elucidate the cross-talk between different types of post-translational modifications in the dynamic regulation of a multifaceted DNA damage response.

Fig. 1.

Experimental strategy for characterization of protein phosphorylation dynamics. A, three SILAC-encoded cell populations were irradiated with 6 grays (6 Gy) and maintained in culture for different lengths of time before the cells were harvested and mixed in a ratio of 1:1:1. Nuclei were purified from the combined cells followed by protein digestion and affinity enrichment of phosphopeptides by ERLIC and TiO₂ chromatography. The resulting phosphopeptides were analyzed by LC-MS/MS, and the data were processed by MaxQuant software and various bioinformatics methods. B, mass spectra of the regulated Nbs1 peptide TTTPGPSLpSQGVSVDEK and the non-regulated GFGpSEEGSR peptide from RNA-binding protein 8A from two separate experiments showing three isotope clusters representing separate time points (o, 0 min; #, 1 h; *, 20 min; +, 8 h; ×, 5 min). C, time profile for the Nbs1 phosphopeptide determined from isotope ratios. D, summary of the identified phosphopeptides.

Fig. 2.

Validation and clustering of time profiles for phosphosites regulated during DNA damage response. A–C, time profiles and corresponding Western blot analyses of selected phosphosites representing clusters 1, 9, and 3. D, Western blot analyses of phosphosites known to be induced by ionizing radiation. The SMC1 protein was used as loading control. E, clustering of time profiles for 594 regulated phosphosites.

Fig. 3.

Cluster distribution of regulated phosphosites. A, the number of phosphosites associated with each clusters. B, cluster distribution of known DNA damage response proteins and regulated sites associated with more than one cluster. The numbers 1–10 denote the clusters, and the color indicates phosphorylation (green) and dephosphorylation (red). BRCA1 = Breast cancer type 1 susceptibility protein; NBN = Nibrin (NBS1); PRKDC = DNA-dependent protein kinase catalytic subunit (DNAPK_cs); XPC = DNA repair protein complementing XP-C cells.

Fig. 4.

Phosphorylation consensus sequences and kinase motifs. A–C, consensus sequences overrepresented among sites regulated >2-fold (A and B) and 1.5–2-fold (C) as compared with unregulated sites during the DNA damage response. D, cluster distribution of the SQ and SXXQ phosphosite sequence motifs. E, distribution and frequency of best assigned kinase sequence motifs for class I sites derived from the Phosida database. Only motifs with five or more counts among all regulated sites are shown in E. CHK1/2 = Serine/threonine-protein kinase Chk1/Chk2; ERK/MAPK = Extracellular signal-regulated kinase/Mitogen-activated protein kinase; FHA/KAPP = Motif known to bind to Forkhead-associated domain found in kinase-associated protein phosphatase; FHA1/Rad53p = Motif known to bind to Forkhead-associated domain found in Rad53p; PKD = Protein kinase D; PLK1 = Polo-like kinase 1; PKB/AKT = Protein kinase B/alpha serine/threonine-protein kinase; WW/GroupIV = Motif known to bind to Goup4 of WW domains; CAMK2 = Calcium/calmodulin-dependent protein kinase type II; NEK6 = Never in mitosis A-related kinase 6; CDK2 = Cell division protein kinase 2; GSK3 = Glycogen synthase kinase-3; PKA = Protein kinase A; CK1/2 = Casein kinase 1/2.

Fig. 5.

Diversifying the response: functional network of proteins with regulated phosphorylation sites and associated processes. Color code for the nodes are as follows: proteins are red, modifications carried out by the proteins are yellow, and processes related to the proteins are blue. Sumo, small ubiquitin-like modifier. NHEJ = Non-homologous end joining; PNKP = Polynucleotide kinase-3′-phosphatase; DNAPK= DNA-dependent protein kinase d.