A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints

J Biol Chem. 2007 Jun 15;282(24):17330-4. doi: 10.1074/jbc.C700079200. Epub 2007 May 3.

Abstract

ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Phospho-Specific / metabolism
  • Ataxia Telangiectasia Mutated Proteins
  • Cell Cycle / physiology
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • DNA Damage
  • DNA Repair
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proteome / analysis*
  • Reproducibility of Results
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / metabolism*
  • Ubiquitin / metabolism*

Substances

  • Antibodies, Phospho-Specific
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Proteome
  • Tumor Suppressor Proteins
  • Ubiquitin
  • ATM protein, human
  • ATR protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Protein-Serine-Threonine Kinases
  • Proteasome Endopeptidase Complex