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

Cornell University, Итак, New York, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2007; 104(8):2797-802. DOI: 10.1073/pnas.0611259104
Source: PubMed


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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    ABSTRACT: The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role. Here we report that the Pph3/Psy2 phosphatase complex, known to be involved in Rad53 dephosphorylation, is required for cellular responses to the DNA-damaging agent methyl methanesulfonate (MMS) but not the DNA replication inhibitor hydroxyurea (HU) in C. albicans. Deletion of either PPH3 or PSY2 resulted in enhanced filamentous growth during MMS treatment and continuous filamentous growth even after MMS removal. Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated. We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS. Therefore, our studies have identified a novel molecular mechanism mediating DNA damage response to MMS in C. albicans.
    Full-text · Article · May 2012 · PLoS ONE
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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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    ABSTRACT: ABSTRACT: Protein enrichment by sub-cellular fractionation was combined with differential-in-gel-electrophoresis (DIGE) to address the detection of the low abundance chromatin proteins in the budding yeast proteome. Comparisons of whole-cell extracts and chromatin fractions were used to provide a measure of the degree of chromatin association for individual proteins, which could be compared across sample treatments. The method was applied to analyze the effect of the DNA damaging agent methyl methanesulfonate (MMS) on levels of chromatin-associated proteins. Up-regulation of several previously characterized DNA damage checkpoint-regulated proteins, such as Rnr4, Rpa1 and Rpa2, was observed. In addition, several novel DNA damage responsive proteins were identified and assessed for genotoxic sensitivity using either DAmP (decreased abundance by mRNA perturbation) or knockout strains, including Acf2, Arp3, Bmh1, Hsp31, Lsp1, Pst2, Rnr4, Rpa1, Rpa2, Ste4, Ycp4 and Yrb1. A strain in which the expression of the Ran-GTPase binding protein Yrb1 was reduced was found to be hypersensitive to genotoxic stress. The described method was effective at unveiling chromatin-associated proteins that are less likely to be detected in the absence of fractionation. Several novel proteins with altered chromatin abundance were identified including Yrb1, pointing to a role for this nuclear import associated protein in DNA damage response.
    Full-text · Article · Oct 2011 · Proteome Science
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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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    ABSTRACT: The rise of high-throughput technologies in the post-genomic era has led to the production of large amounts of biological data. Many of these datasets are freely available on the Internet. Making optimal use of these data is a significant challenge for bioinformaticians. Various strategies for integrating data have been proposed to address this challenge. One of the most promising approaches is the development of semantically rich integrated datasets. Although well suited to computational manipulation, such integrated datasets are typically too large and complex for easy visualization and interactive exploration. We have created an integrated dataset for Saccharomyces cerevisiae using the semantic data integration tool Ondex, and have developed a view-based visualization technique that allows for concise graphical representations of the integrated data. The technique was implemented in a plug-in for Cytoscape, called OndexView. We used OndexView to investigate telomere maintenance in S. cerevisiae. The Ondex yeast dataset and the OndexView plug-in for Cytoscape are accessible at
    Full-text · Article · Mar 2011 · Bioinformatics
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