Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling.
Budding yeast Rad53 is an essential protein kinase that is phosphorylated and activated in a MEC1- and TEL1-dependent manner in response to DNA damage. We studied the role of Rad53 phosphorylation through mutation of consensus phosphorylation sites for upstream kinases Mec1 and Tel1. Alanine substitution of the Rad53 amino-terminal TQ cluster region reduced viability and impaired checkpoint functions. These substitution mutations spared the basal interaction with Asf1 and the DNA damage-induced interactions with Rad9. However, they caused a decrease in DNA damage-induced Rad53 kinase activity and an impaired interaction with the protein kinase Dun1. The Dun1 FHA (Forkhead-associated) domain recognized the amino-terminal TQ cluster of Rad53 after DNA damage or replication blockade. Thus, the phosphorylation of Rad53 by upstream kinases is important not only for Rad53 activation but also for creation of an interface between Rad53 and Dun1.
Lee
Soo-Jung
SJ
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510,USA.
Schwartz
Marc F
MF
Duong
Jimmy K
JK
Stern
David F
DF
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2; D004249:DNA Damage / Q000502:physiology; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D015398:Signal Transduction / Q000502:physiology*
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C069930:rad9 protein; D011494:Protein Kinases; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; C119549:Mek1 protein, S pombe; D017346:Protein Serine-Threonine Kinases; C513623:TEL1 protein, S cerevisiae; C513624:tel1 protein, S pombe; C067022:RAD53 protein, S cerevisiae; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D000409:Alanine