Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana

Abstract

The reversible phosphorylation of proteins on serine, threonine, and tyrosine residues is an important biological regulatory mechanism. In the context of genome integrity, signaling cascades driven by phosphorylation are crucial for the coordination and regulation of DNA repair. The two serine/threonine protein kinases ataxia telangiectasia-mutated (ATM) and Ataxia telangiectasia-mutated and Rad3-related (ATR) are key factors in this process, each specific for different kinds of DNA lesions. They are conserved across eukaryotes, mediating the activation of cell-cycle checkpoints, chromatin modifications, and regulation of DNA repair proteins. We designed a novel mass spectrometry-based phosphoproteomics approach to study DNA damage repair in Arabidopsis thaliana. The protocol combines filter aided sample preparation, immobilized metal affinity chromatography, metal oxide affinity chromatography, and strong cation exchange chromatography for phosphopeptide generation, enrichment, and separation. Isobaric labeling employing iTRAQ (isobaric tags for relative and absolute quantitation) was used for profiling the phosphoproteome of atm atr double mutants and wild type plants under either regular growth conditions or challenged by irradiation. A total of 10,831 proteins were identified and 15,445 unique phosphopeptides were quantified, containing 134 up- and 38 down-regulated ATM/ATR dependent phosphopeptides. We identified known and novel ATM/ATR targets such as LIG4 and MRE11 (needed for resistance against ionizing radiation), PIE1 and SDG26 (implicated in chromatin remodeling), PCNA1, WAPL, and PDS5 (implicated in DNA replication), and ASK1 and HTA10 (involved in meiosis).

Fig. 1.

Workflow for identification of ATM/ATR dependent and independent phosphorylations. Wild type and atm atr double mutant plants were either exposed to irradiation or grown under regular conditions. Extracted proteins were purified via FASP and labeled with iTRAQ. Phosphopeptides were enriched by consecutive application of IMAC and TiO₂. Both, the phosphopeptide-enriched fraction and the flow-through of the TiO₂ chromatography, were separated by SCX chromatography and fractions were analyzed by reversed phase LC-MS/MS.

Fig. 2.

Numbers of peptides identified after phosphopeptide enrichment. Following FASP and phosphopeptide enrichment samples were fractionated by SCX chromatography and analyzed by LC-MS/MS. A, Diagram showing the distribution of nonphosphorylated, singly and higher phosphorylated, and N-terminally acetylated peptides within each of the 94 collected fractions. The combined salt- and pH-gradient is indicated as a solid and dashed line, respectively. B, Pie chart diagram depicting the number of fractions in which a unique peptide was found. C, The phosphorylation status of the identified peptides of all combined fractions is depicted. D, The unique phosphopeptides identified with the two different approaches (FASP and Wessel/Fluegge (W/F)) and with unambiguously localized phospho-sites are compared with those deposited in the PhosPhAt 4.0 database.

Fig. 3.

Overview of relative phosphopeptide abundances. Normalized and quantified phosphopeptides were grouped into five different categories depending on the relative abundance in the four different samples as measured by their iTRAQ ratios. The first row depicts phosphopeptides that are up-regulated following exposure to IR and dependent on ATM/ATR. The second row depicts phosphopeptides enriched after exposure to IR, but independent of ATM/ATR. Phosphorylated S/T-Q motifs are only present in peptides with higher abundance depending on both, IR exposure and ATM and ATR (first row).

Fig. 4.

Motif X analysis of significantly up-regulated phosphopeptides. A, Three consensus motifs are significantly enriched in the dataset of irradiation induced and ATM/ATR dependent up-regulated phosphopeptides. Five motifs are found in the dataset corresponding to ATM/ATR independent up-regulated phosphopeptides. The enrichment was calculated against the background of all quantified phosphopeptides with localized sites and is shown in the last column of the table. (*** - Bonferroni corrected p value = 0.0004) B, Graphical representation of the amino acid environment of the identified motifs shown in panel A.

Fig. 5.

Graphical representation of ATM/ATR dependent phosphorylation targets clustered according to GO-annotation terms. Selected phosphoproteins carrying irradiation induced and ATM/ATR dependent up-regulated phosphosites are grouped according to their biological function and color-coded according to their regulatory ratio.

Fig. 6.

Many plant ATM/ATR targets are functionally interconnected and are related to human orthologs. The 108 proteins identified as direct and indirect targets of ATM and ATR were entered to STRING version 9.1 to identify known and predicted protein–protein interactions. About half of the proteins could be connected by either experimental evidence (red lines), by interactions based on curated databases (orange lines) or by text-mining (gray lines). The node colors indicate, with how many out of five used programs a human ortholog could be identified. No color: 0, light yellow: 1, dark yellow: 2, orange: 3, brown: 4, dark red: 5. Candidates with orthologs being affected by differential phosphorylation in response to DNA damage in other screens are marked with dark gray edges (see also supplemental Table S9).