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.
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.
Mu
Jung-Jung
JJ
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Wang
Yi
Y
Luo
Hao
H
Leng
Mei
M
Zhang
Jinglan
J
Yang
Tao
T
Besusso
Dario
D
Jung
Sung Yun
SY
Qin
Jun
J
D000595:Amino Acid Sequence; D045163:Antibodies, Phospho-Specific / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D020543:Proteome / Q000032:analysis*; D015203:Reproducibility of Results; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D045163:Antibodies, Phospho-Specific; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D020543:Proteome; D025521:Tumor Suppressor Proteins; D025801:Ubiquitin; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D046988:Proteasome Endopeptidase Complex