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

Elisabeth Roitinger, Manuel Hofer, Thomas Köcher, Peter Pichler, Maria Novatchkova, Jianhua Yang, Peter Schlögelhofer, Karl Mechtler

Research output: Contribution to journalArticle

77 Citations (Scopus)

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).

Original languageEnglish
Pages (from-to)556-571
Number of pages16
JournalMolecular & cellular proteomics : MCP
Volume14
Issue number3
DOIs
Publication statusPublished - Mar 2015
Externally publishedYes

Fingerprint

Ataxia Telangiectasia
Arabidopsis
DNA Damage
Phosphopeptides
DNA
Affinity chromatography
Phosphorylation
Repair
DNA Repair
Chromatin
Affinity Chromatography
Metals
Proteins
Protein-Serine-Threonine Kinases
Ionizing radiation
Threonine
Chromatography
Chromatin Assembly and Disassembly
Oxides
Labeling

Keywords

  • Arabidopsis
  • Arabidopsis Proteins
  • Ataxia Telangiectasia Mutated Proteins
  • DNA Repair
  • DNA, Plant
  • Gene Expression Regulation, Plant
  • Mass Spectrometry
  • Mutation
  • Phosphoproteins
  • Proteomics
  • Journal Article
  • Research Support, Non-U.S. Gov't

Cite this

Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana. / Roitinger, Elisabeth; Hofer, Manuel; Köcher, Thomas; Pichler, Peter; Novatchkova, Maria; Yang, Jianhua; Schlögelhofer, Peter; Mechtler, Karl.

In: Molecular & cellular proteomics : MCP, Vol. 14, No. 3, 03.2015, p. 556-571.

Research output: Contribution to journalArticle

Roitinger, Elisabeth ; Hofer, Manuel ; Köcher, Thomas ; Pichler, Peter ; Novatchkova, Maria ; Yang, Jianhua ; Schlögelhofer, Peter ; Mechtler, Karl. / Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana. In: Molecular & cellular proteomics : MCP. 2015 ; Vol. 14, No. 3. pp. 556-571.
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AU - Roitinger, Elisabeth

AU - Hofer, Manuel

AU - Köcher, Thomas

AU - Pichler, Peter

AU - Novatchkova, Maria

AU - Yang, Jianhua

AU - Schlögelhofer, Peter

AU - Mechtler, Karl

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AB - 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).

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KW - Ataxia Telangiectasia Mutated Proteins

KW - DNA Repair

KW - DNA, Plant

KW - Gene Expression Regulation, Plant

KW - Mass Spectrometry

KW - Mutation

KW - Phosphoproteins

KW - Proteomics

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

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JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

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