Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.
The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.
Division of Genetics, Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; The Laboratory of Medical Genetics, Harbin Medical University, Harbin 150081, China;
Chunshui
C
Zhou
Division of Genetics, Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA 02114;
Andrew E H
AE
Elia
Division of Genetics, Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;
Maria L
ML
Naylor
Department of Cell Biology, Harvard Medical School, Boston, MA 02115;
Noah
0000-0001-5802-2657
N
Dephoure
Department of Biology, University of Vermont, Burlington, VT 05405;
Bryan A
BA
Ballif
Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114;
Gautam
G
Goel
Division of Genetics, Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;
Qikai
Q
Xu
Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114;
Aylwin
A
Ng
Division of Genetics, Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115;
Danny M
DM
Chou
Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114;
Ramnik J
RJ
Xavier
Department of Cell Biology, Harvard Medical School, Boston, MA 02115; steven_gygi@hms.harvard.edu selledge@genetics.med.harvard.edu.
Steven P
SP
Gygi
Division of Genetics, Department of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115 steven_gygi@hms.harvard.edu selledge@genetics.med.harvard.edu.
Stephen J
SJ
Elledge
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
D018797:Cell Cycle Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae
DNA damage; TOR; checkpoint; phosphatidyl inositol; proteomics