Yeast PP4 Interacts with ATR Homolog Ddc2-Mec1 and Regulates Checkpoint Signaling

• Published in: Molecular cell Vol. 57; no. 2; pp. 273 - 289
• Main Authors: Hustedt, Nicole, Seeber, Andrew, Sack, Ragna, Tsai-Pflugfelder, Monika, Bhullar, Bhupinder, Vlaming, Hanneke, van Leeuwen, Fred, Guénolé, Aude, van Attikum, Haico, Srivas, Rohith, Ideker, Trey, Shimada, Kenji, Gasser, Susan M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.01.2015
• Subjects: Adaptor Proteins, Signal Transducing - metabolism; Cell Cycle Checkpoints; Cell Cycle Proteins - metabolism; Checkpoint Kinase 2 - metabolism; DNA Replication; Epistasis, Genetic; Gene Expression Regulation, Fungal; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins - metabolism; Nuclear Proteins - metabolism; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases - metabolism; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins - metabolism; Signal Transduction
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2014.11.016

Mec1-Ddc2 (ATR-ATRIP) controls the DNA damage checkpoint and shows differential cell-cycle regulation in yeast. To find regulators of Mec1-Ddc2, we exploited a mec1 mutant that retains catalytic activity in G2 and recruitment to stalled replication forks, but which is compromised for the intra-S phase checkpoint. Two screens, one for spontaneous survivors and an E-MAP screen for synthetic growth effects, identified loss of PP4 phosphatase, pph3Δ and psy2Δ, as the strongest suppressors of mec1-100 lethality on HU. Restored Rad53 phosphorylation accounts for part, but not all, of the pph3Δ-mediated survival. Phosphoproteomic analysis confirmed that 94% of the mec1-100-compromised targets on HU are PP4 regulated, including a phosphoacceptor site within Mec1 itself, mutation of which confers damage sensitivity. Physical interaction between Pph3 and Mec1, mediated by cofactors Psy2 and Ddc2, is shown biochemically and through FRET in subnuclear repair foci. This establishes a physical and functional Mec1-PP4 unit for regulating the checkpoint response. [Display omitted] •S phase functions of Mec1 kinase are balanced by phosphatase Pph3-Psy2•Mec1-Ddc2 binds PP4 (Pph3-Psy2) through a Ddc2-Psy2 interaction site•The two complexes colocalize at foci of stalled replication forks•Most mec1-100-sensitive phosphorylation events on HU are PP4 targets The central checkpoint kinase Mec1-Ddc2 kinase (HsATR-ATRIP) is essential for survival of acute replication stress. Hustedt et al. show that the conserved yeast PP4 phosphatase (Psy2-Pph3) binds Mec1-Ddc2 and dephosphorylates S phase targets of Mec1-Ddc2 including Mec1.