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The Saccharomyces cerevisiae 14-3-3 proteins Bmh1 and Bmh2 directly influence the DNA damage-dependent functions of Rad53

Laboratory of Chromosome Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 03/2007; 104(8):2797-802. DOI: 10.1073/pnas.0611259104
Source: PubMed

ABSTRACT In this study, we mutated autophosphorylation sites in Rad53 based on their conservation with previously identified autophosphorylation sites in the mammalian Rad53 ortholog, Chk2. As with wild-type Rad53, the autophosphorylation mutant, rad53-TA, undergoes Mec1/Tel1-dependent interactions with Rad9 and Dun1 in response to genotoxic stress. Whereas rad53-TA in vitro kinase activity is severely impaired, the rad53-TA strains are not completely deficient for cell-cycle checkpoint functions, indicating that the mutant kinase retains a basal level of function. We describe a genetic interaction among Rad53, Dun1, and the 14-3-3 proteins Bmh1 and Bmh2 and present evidence that 14-3-3 proteins directly facilitate Rad53 function in vivo. The data presented account for the previously observed checkpoint defects associated with 14-3-3 mutants in Saccharomyces pombe and Saccharomyces cerevisiae. The 14-3-3 functional interaction appears to modulate Rad53 activity, reminiscent of 14-3-3's effect on human Raf1 kinase and distinct from the indirect mode of regulation by 14-3-3 observed for Chk1 or Cdc25.

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    • "Phosphorylation of the SCDs is closely associated with protein function and cell viability. For instance, phosphorylation at ScRad53-T354 and ScRad53-T358 in the activation loop is required for kinase activity [64]–[66]. Trans-phosphorylation of the ScRad53 N-terminal SCD is crucial for interaction with Dun1, the complex of which is involved in G2/M checkpoint [67]. Chk2-T68 phosphorylation is dependent on ATM/ATR and triggers Chk2 oligomerization, which led to PIKK-independent kinase activation [66], [68]. "
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    • "Along with the previously well-characterized proteins above, several additional DNA damage-associated proteins were identified as differentially expressed on MMS treatment including Bmh1, Pst2 Vma2, and Vma4 (see Table 2). Bmh1 is a 14-3-3 protein family member, which has been shown to directly modulate Rad53 activity [33]. Pst2, a predicted oxidative response protein, has also been implicated in DNA damage responses [29]. "
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    • "After selecting Rad53 in the view, we can learn from its description field that Rad53 mediates the activation of the cell-cycle checkpoint (Schwartz et al., 2002), thus interacting with the cdc13-1 phenotype. Examination of the interaction between Bmh2 and Rad53 reveals an annotation indicating that this interaction was originally reported by Usui and Petrini (2007). These authors showed that both Bmh1 and Bmh2 directly bind to the active (phosphorylated) Rad53 protein, thus enhancing its signalling effect. "
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