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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.

Pubmed ID: 18570878

Authors

  • Lee H
  • Yuan C
  • Hammet A
  • Mahajan A
  • Chen ES
  • Wu MR
  • Su MI
  • Heierhorst J
  • Tsai MD

Journal

Molecular cell

Publication Data

June 20, 2008

Associated Grants

  • Agency: NCI NIH HHS, Id: CA69472

Mesh Terms

  • Binding Sites
  • Cell Cycle Proteins
  • Checkpoint Kinase 2
  • DNA Damage
  • DNA, Fungal
  • Enzyme Activation
  • Kinetics
  • Ligands
  • Phosphothreonine
  • Protein Binding
  • Protein Kinases
  • Protein Processing, Post-Translational
  • Protein-Serine-Threonine Kinases
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Sensitivity and Specificity