title: Site-specific phosphorylation of the DNA damage response mediator rad9 by cyclin-dependent kinases regulates activation of checkpoint kinase 1.
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.
authors.affiliation: Centre for Chromosome Biology, School of Natural Science, National University of Ireland Galway, Galway, Ireland.
authors.forename: Carla Manuela
authors.initials: CM
authors.lastname: Abreu
authors.forename: Ramesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Danielle
authors.initials: D
authors.lastname: Hamilton
authors.forename: Andrew William
authors.initials: AW
authors.lastname: Dawdy
authors.forename: Kevin
authors.initials: K
authors.lastname: Creavin
authors.forename: Sarah
authors.initials: S
authors.lastname: Eivers
authors.forename: Karen
authors.initials: K
authors.lastname: Finn
authors.forename: Jeremy Lynn
authors.initials: JL
authors.lastname: Balsbaugh
authors.forename: Rosemary
authors.initials: R
authors.lastname: O'Connor
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Kiely
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Shabanowitz
authors.forename: Donald F
authors.initials: DF
authors.lastname: Hunt
authors.forename: Muriel
authors.initials: M
authors.lastname: Grenon
authors.forename: Noel Francis
authors.initials: NF
authors.lastname: Lowndes
mesh_terms: D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004789:Enzyme Activation; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D016196:S Phase / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000502:physiology*
chemical_list: D018797:Cell Cycle Proteins; C069930:rad9 protein; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; D017489:CDC28 Protein Kinase, S cerevisiae