Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases.
Understanding the role of DNA damage checkpoint kinases in the cellular response to genotoxic stress requires the knowledge of their substrates. Here, we report the use of quantitative phosphoproteomics to identify in vivo kinase substrates of the yeast DNA damage checkpoint kinases Mec1, Tel1, and Rad53 (orthologs of human ATR, ATM, and CHK2, respectively). By analyzing 2,689 phosphorylation sites in wild-type and various kinase-null cells, 62 phosphorylation sites from 55 proteins were found to be controlled by the DNA damage checkpoint. Examination of the dependency of each phosphorylation on Mec1 and Tel1 or Rad53, combined with sequence and biochemical analysis, revealed that many of the identified targets are likely direct substrates of these kinases. In addition to several known targets, 50 previously undescribed targets of the DNA damage checkpoint were identified, suggesting that a wide range of cellular processes is likely regulated by Mec1, Tel1, and Rad53.
Smolka
Marcus B
MB
Ludwig Institute for Cancer Research, La Jolla, CA 92093-0653, USA.
Albuquerque
Claudio P
CP
Chen
Sheng-hong
SH
Zhou
Huilin
H
D018797:Cell Cycle Proteins / Q000378:metabolism; D064447:Checkpoint Kinase 2; D002853:Chromatography, Liquid; D004249:DNA Damage; D005656:Fungal Proteins / Q000032:analysis* / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D009153:Mutagens / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009153:Mutagens; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C513623:TEL1 protein, S cerevisiae; C067022:RAD53 protein, S cerevisiae