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

摘要

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. Author Summary Human cells activate the DNA damage response (DDR) to repair DNA damage and to prevent cells with DNA damage from proliferating. Alterations to the DDR are strongly implicated in the development of cancer. Using the budding yeast model system, we have studied how the regulation of the key DDR component Rad9 is integrated into cell cycle control. The cyclin-dependent kinase Cdc28 that regulates the yeast cell cycle also extensively phosphorylates Rad9 during cell cycle progression. We show here that Cdc28 controls Rad9 function in the activation of the important downstream DNA damage effector kinase Chk1. Two sites of phosphorylation in the N-terminus of Rad9 are crucial for the physical interaction between Rad9 and Chk1 regulated by Cdc28. We propose a novel model for Chk1 activation whereby a subset of Rad9 and Chk1 interacts constitutively in the absence of DNA damage. The Rad9/Chk1 complex is recruited to sites of DNA damage where activation of Chk1 involves additional DDR–specific protein kinases. Human cells contain multiple Rad9-like proteins that are also known to be cell cycle phosphorylated in the absence of exogenous DNA damage, suggesting that our observations may have important implications for DDR regulation in human cells.