Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade.
Forkhead-associated (FHA) domains recognize phosphothreonines, and SQ/TQ cluster domains (SCDs) contain concentrated phosphorylation sites for ATM/ATR-like DNA-damage-response kinases. The Rad53-SCD1 has dual functions in regulating the activation of the Rad53-Dun1 checkpoint kinase cascade but with unknown molecular mechanisms. Here we present structural, biochemical, and genetic evidence that Dun1-FHA possesses an unprecedented diphosphothreonine-binding specificity. The Dun1-FHA has >100-fold increased affinity for diphosphorylated relative to monophosphorylated Rad53-SCD1 due to the presence of two separate phosphothreonine-binding pockets. In vivo, any single threonine of Rad53-SCD1 is sufficient for Rad53 activation and RAD53-dependent survival of DNA damage, but two adjacent phosphothreonines in the Rad53-SCD1 and two phosphothreonine-binding sites in the Dun1-FHA are necessary for Dun1 activation and DUN1-dependent transcriptional responses to DNA damage. The results uncover a phospho-counting mechanism that regulates the specificity of SCD, and provide mechanistic insight into a role of multisite phosphorylation in DNA-damage signaling.
Lee
Hyun
H
Genomics Research Center, Academia Sinica, Taipei 115, Taiwan.
Yuan
Chunhua
C
Hammet
Andrew
A
Mahajan
Anjali
A
Chen
Eric S-W
ES
Wu
Ming-Ru
MR
Su
Mei-I
MI
Heierhorst
Jörg
J
Tsai
Ming-Daw
MD
D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004271:DNA, Fungal / Q000235:genetics; D004789:Enzyme Activation; D007700:Kinetics; D008024:Ligands; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D008024:Ligands; D029701:Saccharomyces cerevisiae Proteins; D010769:Phosphothreonine; D011494:Protein Kinases; C104169:DUN1 protein, S cerevisiae; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae