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Quantitative Phosphoproteomic Analysis Identifies Targets of the DNA Damage Checkpoint Kinases in Yeast
Author(s) -
Zhou Huilin,
Smolka Marcus B,
Albuquerque Claudio,
Chen Shenghong
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a659-d
Subject(s) - g2 m dna damage checkpoint , biology , microbiology and biotechnology , dna damage , kinase , checkpoint kinase 2 , phosphoproteomics , phosphorylation , chek1 , dna replication , protein phosphorylation , protein serine threonine kinases , dna repair , cell cycle checkpoint , cell cycle , dna , biochemistry , protein kinase a , gene
Cellular response to DNA damage involves an intricate network of proteins with roles in a variety of biological processes, including transcription, DNA repair and cell cycle. In budding yeast, the DNA damage response is regulated by the conserved checkpoint kinases Mec1, Tel1 and Rad53. How these kinases regulate various biological processes remains poorly understood due to a limited known substrates. Here, using a quantitative phosphoproteomic approach, we identified 54 in vivo phosphorylation sites from 48 proteins that are regulated in a Mec1 and Tel1‐dependent manner. Remarkably, the majority of the phosphorylation sites are located in motifs preferentially phosphorylated by Mec1, Tel1 and Rad53. In addition to the identification of 10 known targets, 38 novel targets for DNA damage checkpoint kinases were identified. Most of these targets are involved in DNA replication and repair, mitosis, transcription, RNA metabolism and nuclear transport. We present further evidence to show that Rad53 directly phosphorylates several components of the nuclear pore complex. Taken together, these findings represent the first step towards defining the global network of proteins targeted by the DNA damage checkpoint kinases. This quantitative phosphoproteomic technology should be generally applicable to identify kinase substrates and to study global changes in protein phosphorylation in cells.