Site-Specific Phosphorylation of the DNA Damage Response Mediator Rad9 by Cyclin-Dependent Kinases Regulates Activation of Checkpoint Kinase 1

Carla Manuela Abreu,Jeremy Lynn Balsbaugh,Patrick A Kiely,Rosemary O'Connor,Jeffrey Shabanowitz,Andrew William Dawdy,Sarah Eivers,Noel Francis Lowndes,Ramesh Kumar,Karen Finn,Kevin Creavin,Donald F Hunt,Muriel Grenon,Danielle Hamilton

doi:10.1371/journal.pgen.1003310

Abstract

The mediators of the DNA damage response (DDR) are highly phosphorylated by kinases that control cell proliferation, but little is known about the role of this regulation. Here we show that cell cycle phosphorylation of the prototypical DDR mediator Saccharomyces cerevisiae Rad9 depends on cyclin-dependent kinase (CDK) complexes. We find that a specific G2/M form of Cdc28 can phosphorylate in vitro the N-terminal region of Rad9 on nine consensus CDK phosphorylation sites. We show that the integrity of CDK consensus sites and the activity of Cdc28 are required for both the activation of the Chk1 checkpoint kinase and its interaction with Rad9. We have identified T125 and T143 as important residues in Rad9 for this Rad9/Chk1 interaction. Phosphorylation of T143 is the most important feature promoting Rad9/Chk1 interaction, while the much more abundant phosphorylation of the neighbouring T125 residue impedes the Rad9/Chk1 interaction. We suggest a novel model for Chk1 activation where Cdc28 regulates the constitutive interaction of Rad9 and Chk1. The Rad9/Chk1 complex is then recruited at sites of DNA damage where activation of Chk1 requires additional DDR–specific protein kinases.

Highlights

Eukaryotic cells have developed highly conserved surveillance pathways known as the DNA damage response (DDR) to preserve genome integrity after genotoxic insult
Using the budding yeast model system, we have studied how the regulation of the key DDR component Rad9 is integrated into cell cycle control
We propose a novel model for Chk1 activation whereby a subset of Rad9 and Chk1 interacts constitutively in the absence of DNA damage

Summary

Introduction

Eukaryotic cells have developed highly conserved surveillance pathways known as the DNA damage response (DDR) to preserve genome integrity after genotoxic insult. These pathways inhibit replication and segregation of damaged DNA by activating checkpoints and regulating transcription, replication and repair [1]. Central to the DDR are protein kinases that are activated by DNA lesions. The human phosphatidylinositol 3-kinase-like kinases (PIKKs), ATM, ATR and DNA-PK occupy central points in the DNA damage-induced signalling pathways [1,3]. Once activated by DNAprotein structures generated in response to lesions, PIKKs regulate numerous DDR proteins including the downstream ‘checkpoint’

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