Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases

Abstract

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. mass spectrometry ͉ Mec1 ͉ N-isotag ͉ phosphorylation ͉ Rad53

Dpb11 activates the Mec1–Ddc2 complex

“…Mec1 targets its substrates at SQ/TQ motifs (3,25). In the course of our kinase assays, we noticed that Dpb11-C itself served as a Mec1 substrate.…”

“…In Saccharomyces cerevisiae, the PIKK (Phosphatidylinositide 3-kinase-related kinase) kinase Mec1, the mammalian ATR ortholog, senses DNA damage and replication stress and initiates the DNA damage response (2). Mec1 phosphorylates substrates involved in DNA replication and repair, cell cycle checkpoints, RNA metabolism, and transcription (3).…”

RING Finger and WD Repeat Domain 3 (RFWD3) Associates with Replication Protein A (RPA) and Facilitates RPA-mediated DNA Damage Response

“…Identification of key players and the characterization of their molecular functions enable a more thorough understanding of the DDR pathways, which are critical for maintaining genomic integrity. Several recent proteomic screens have drastically expanded the landscape of DDR pathways with the identification of hundreds of putative ATM/ATR substrates (5)(6)(7). Recently, we investigated the role of a previously uncharacterized protein, RING finger and WD repeat domain 3 (RFWD3, also known as RNF201 and FLJ10520; GenBank TM number 55159) in DDR (5,8).…”

Clinical implications of the basic defects in Cockayne syndrome and xeroderma pigmentosum and the DNA lesions responsible for cancer, neurodegeneration and aging

“…Consideration of the large number of potential protein targets of the ATM, ATR and CHK2 kinases, the possibility of identifying clinically relevant targets is daunting (Smolka et al, 2007). Several potential points of attack are conceivable: premature entry into the cell cycle, the proteasomal pathway, apoptosis and ROS.…”