The kinases Rad53 and Dun1 are important components of a checkpoint kinase cascade activated in response to DNA damage in Saccharomyces cerevisiae. Both enzymes contain forkhead-associated (FHA) domains, which bind specifically to phosphothreonine (pThr) residues. Rad53 has four Thr residues clustered in its N-terminal SCD1 domain. Upon phosphorylation by upstream kinases, Rad53 interacts with Dun1 through the Dun1-FHA domain to activate Dun1; however, Rad53 also mediates Dun1-independent responses to DNA damage. Although mutant Rad53 proteins that contain only one of the four SCD1 Thr residues are readily activated by upstream kinases, they cannot activate Dun1, which led Lee et al. to investigate the pThr requirements for Dun1 activation. Surface plasmon resonance assays showed that the recombinant Dun1-FHA domain bound with greater affinity to Rad53-SCD1-derived phosphopeptides containing both pThr5 and pThr8 than to phosphopeptides that did not have both of these residues. Nuclear magnetic resonance (NMR) studies and other structural analyses showed that the Dun1-FHA domain differed from that of Rad53 in having not one, but two high-affinity pThr-binding sites. The authors then generated four new rad53 alleles to express Rad53 proteins with various combinations of Thr or Ala residues. Treatment of yeast strains expressing each mutant rad53 allele with a DNA-damage-inducing agent, followed by in vitro analysis of the kinase activity of immunoprecipitated Dun1, showed that the presence of both Thr5 and Thr8 in the Rad53-SCD1 was required for optimal Dun1 activity. Mass spectrometry studies showed the presence of monophosphorylated and pThr5-pThr8 diphosphorylated Rad53 proteins in response to DNA damage in vivo. Together these data suggest that whereas monophosphorylation activates Rad53, diphosphorylation of Rad53 is required to activate the Dun1-dependent arm of the DNA-damage response.
H. Lee, C. Yuan, A. Hammet, A. Mahajan, E. S.-W. Chen, M.-R. Wu, M.-I. Su, J. Heierhorst, M.-D. Tsai, Diphosphothreonine-specific interaction between an SQ/TQ cluster and an FHA domain in the Rad53-Dun1 kinase cascade. Mol. Cell 30, 767-778 (2008). [PubMed]