A Proteome-wide Analysis of Kinase-Substrate Network in the DNA Damage Response

Sheng-Hong Chen,Claudio P Albuquerque,Jason Liang,Raymond T Suhandynata,Huilin Zhou

doi:10.1074/jbc.m110.106989

Sheng-Hong Chen, Claudio P Albuquerque + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m110.106989

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Abstract

The DNA damage checkpoint, consisting of an evolutionarily conserved protein kinase cascade, controls the DNA damage response in eukaryotes. Knowledge of the in vivo substrates of the checkpoint kinases is essential toward understanding their functions. Here we used quantitative mass spectrometry to identify 53 new and 34 previously known targets of Mec1/Tel1, Rad53, and Dun1 in Saccharomyces cerevisiae. Analysis of replication protein A (RPA)-associated proteins reveals extensive physical interactions between RPA-associated proteins and Mec1/Tel1-specific substrates. Among them, multiple subunits of the chromatin remodeling complexes including ISW1, ISW2, INO80, SWR1, RSC, and SWI/SNF are identified and they undergo DNA damage-induced phosphorylation by Mec1 and Tel1. Taken together, this study greatly expands the existing knowledge of the targets of DNA damage checkpoint kinases and provides insights into the role of RPA-associated chromatins in mediating Mec1 and Tel1 substrate phosphorylation in vivo.

Highlights

IntroductionMec is primarily responsible for the activation of downstream checkpoint kinases including Rad53 [11, 12], whereas Tel has a more prominent role in regulating telomere length [13]
Grant GM080463 and the Ludwig Institute for Cancer Research. □S The on-line version of this article contains supplemental Tables S1–S7 and Fig. S1. 1 Both authors contributed to this work. 2 To whom correspondence should be addressed: CMM-East, Rm. 2070, 9500
Because Mec1 and Tel1 control the activity of Rad53, which in turn controls the activity of Dun1, we examined the overlap among the kinase-dependent phosphopeptides. 17 phosphopeptides are dependent on Mec1/Tel1, Rad53, and Dun1; they are considered as candidates for being Dun1specific targets. 42 phosphopeptides are dependent on both Mec1/Tel1 and Rad53, yet independent of Dun1; they are considered as candidate Rad53 targets

Summary

Introduction

Mec is primarily responsible for the activation of downstream checkpoint kinases including Rad53 [11, 12], whereas Tel has a more prominent role in regulating telomere length [13]. Dun phosphorylates Sml, a ribonucleotide reductase (RNR) inhibitor, leading to Sml degradation and increased dNTP levels in cells [41, 42]. Both Rad and Dun appear to control the phosphorylation of Rfx1/Crt1 [43], which regulates the transcriptional induction of RNR genes.

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