title: 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).
authors.affiliation: From the ‡Institute of Molecular Pathology (IMP), Vienna, Austria; ¶Institute of Molecular Biotechnology (IMBA), Vienna, Austria;
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Roitinger
authors.affiliation: §Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria;
authors.forename: Manuel
authors.initials: M
authors.lastname: Hofer
authors.affiliation: From the ‡Institute of Molecular Pathology (IMP), Vienna, Austria;
authors.forename: Thomas
authors.initials: T
authors.lastname: Köcher
authors.affiliation: From the ‡Institute of Molecular Pathology (IMP), Vienna, Austria; ¶Institute of Molecular Biotechnology (IMBA), Vienna, Austria;
authors.forename: Peter
authors.initials: P
authors.lastname: Pichler
authors.affiliation: From the ‡Institute of Molecular Pathology (IMP), Vienna, Austria; ¶Institute of Molecular Biotechnology (IMBA), Vienna, Austria;
authors.forename: Maria
authors.initials: M
authors.lastname: Novatchkova
authors.affiliation: ‖School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Yang
authors.affiliation: §Department of Chromosome Biology, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria; Karl.Mechtler@imp.ac.at Peter.Schloegelhofer@univie.ac.at.
authors.forename: Peter
authors.initials: P
authors.lastname: Schlögelhofer
authors.affiliation: From the ‡Institute of Molecular Pathology (IMP), Vienna, Austria; ¶Institute of Molecular Biotechnology (IMBA), Vienna, Austria; Karl.Mechtler@imp.ac.at Peter.Schloegelhofer@univie.ac.at.
authors.forename: Karl
authors.initials: K
authors.lastname: Mechtler
mesh_terms: D017360:Arabidopsis / Q000254:growth & development* / Q000378:metabolism / Q000528:radiation effects; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair; D018744:DNA, Plant / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D013058:Mass Spectrometry / Q000379:methods*; D009154:Mutation; D010750:Phosphoproteins / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*
chemical_list: C576589:ATM protein, Arabidopsis; D029681:Arabidopsis Proteins; D018744:DNA, Plant; D010750:Phosphoproteins; C576892:ATR protein, Arabidopsis; D064007:Ataxia Telangiectasia Mutated Proteins