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- Bilsland, Elizabeth, 1973, et al.
(author)
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Rck1 and Rck2 MAPKAP kinases and the HOG pathway are required for oxidative stress resistance
- 2004
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In: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 53:6, s. 1743-56
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Journal article (peer-reviewed)abstract
- We demonstrate a role in oxidative and metal stress resistance for the MAPK-activated protein kinases Rck1 and Rck2 in Saccharomyces cerevisiae. We show that Hog1 is robustly phosphorylated in a Pbs2-dependent way during oxidative stress, and that Rck2 also is phosphorylated under these circumstances. Hog1 concentrates in the nucleus in oxidative stress. Hog1 localization is partially dependent on Rck2, as rck2 cells have more nuclear Hog1 than wild-type cells. We find several proteins with a role in oxidative stress resistance using Rck1 or Rck2 as baits in a two-hybrid screen. We identify the transcription factor Yap2 as a putative target for Rck1, and the Zn2+ transporter Zrc1 as a target for Rck2. Yap2 is normally cytoplasmic, but rapidly migrates to the nucleus upon exposure to oxidative stress agents. In a fraction of untreated pbs2 cells, Yap2 is nuclear. Zrc1 co-immunoprecipitates with Rck2, and ZRC1 is genetically downstream of RCK2. These data connect activation of the Hog1 MAPK cascade with effectors having a role in oxidative stress resistance.
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| 2. |
- Bilsland, Elizabeth, 1973, et al.
(author)
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The Bre5/Ubp3 ubiquitin protease complex from budding yeast contributes to the cellular response to DNA damage
- 2007
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In: DNA Repair (Amst). - : Elsevier BV. - 1568-7864. ; 6:10, s. 1471-84
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Journal article (peer-reviewed)abstract
- The ubiquitination status of proteins can control numerous aspects of protein function through targeted destruction or by altering protein-protein interactions, subcellular localization, or enzymatic activity. In addition to enzymes that mediate the conjugation of ubiquitin moieties to target proteins, there are enzymes that catalyze the removal of ubiquitin, termed ubiquitin proteases. One such ubiquitin protease, Ubp3, exists in a complex with a partner protein: Bre5. This complex has been implicated in a variety of cellular activities, and was recently identified in large-scale screens for genetic interactions with known components of the DNA damage response pathway. We found that this complex plays a role in the cellular response to the DNA damaging agent phleomycin and strains lacking the complex have a defect in non-homologous end joining. Although this complex is also important for telomeric silencing, maintenance of the cell wall, and global transcriptional regulation, we present evidence suggesting that the role of this complex in DNA damage responses is distinct from these other roles. First, we found that Ubp3/Bre5 functions antagonistically with Bul1 in DNA damage responses, but not in its other cellular functions. Additionally, we have generated mutants of Bre5 that are specifically defective in DNA damage responses.
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| 3. |
- Bilsland, Elizabeth, 1973
(author)
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The Saccharomyces cerevisiae MAPKAP kinases Rck1 and Rck2 in stress responses
- 2004
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Doctoral thesis (other academic/artistic)abstract
- The Saccharomyces cerevisiae protein kinases Rck1 and Rck2 (Radiation sensitivity Complementing Kinases) were originally cloned as suppressors of Schizosaccharomyces pombe checkpoint mutants. In Saccharomyces cerevisiae we found that overexpression of either kinase has the ability to depress sporulation in mec1 and rad24 mutant backgrounds. Conversely, deletion of RCK1 or RCK2 accelerates the sporulation process. In a parallel study, we found a strong constitutive interaction between the C-terminal portion of Rck2p and the MAPK Hog1p. This interaction allows the in vitro and in vivo phosphorylation of Rck2p by Hog1p upon hyperosmotic stress. Rck2p in vivo phosphorylation was mapped to Ser520, and leads to a hyperactivation of its catalytic activity, measured by the autophosphorylation level of Rck2p. RCK2 overexpression can suppress osmosensitivity of hog1 and pbs2 mutants, and RCK2 deletion can suppress the growth arrest of a hyperactive HOG pathway. Together, these results allowed us to place Rck2p genetically and biochemically downstream of the MAPK Hog1p. We also found that unlike previously stated in the literature, the HOG pathway can be activated by oxidative stress, as measured by Hog1p Tyr/Thr phosphorylation. This activation leads to Hog1 translocation into the nucleus and Rck2p phosphorylation, in an analogous however less pronounced way than observed upon hyperosmotic stress. Using the two-hybrid system, we identify a series of Rck1p/Rck2p interacting proteins with roles in oxidative stress resistance. Two of these putative interactions (Rck1p-Yap2p and Rck2p-Zrc1p), were verified by co-immunoprecipitation and genetic studies, strengthening the possibility of Yap2p and Zrc1p being relevant Rck1p/Rck2p in vivo targets.
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| 4. |
- Cvijovic, Marija, 1977, et al.
(author)
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Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation
- 2007
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In: BMC Bioinformatics. - : Springer Science and Business Media LLC. - 1471-2105. ; 8
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Journal article (peer-reviewed)abstract
- BACKGROUND: The translational efficiency of an mRNA can be modulated by upstream open reading frames (uORFs) present in certain genes. A uORF can attenuate translation of the main ORF by interfering with translational reinitiation at the main start codon. uORFs also occur by chance in the genome, in which case they do not have a regulatory role. Since the sequence determinants for functional uORFs are not understood, it is difficult to discriminate functional from spurious uORFs by sequence analysis. RESULTS: We have used comparative genomics to identify novel uORFs in yeast with a high likelihood of having a translational regulatory role. We examined uORFs, previously shown to play a role in regulation of translation in Saccharomyces cerevisiae, for evolutionary conservation within seven Saccharomyces species. Inspection of the set of conserved uORFs yielded the following three characteristics useful for discrimination of functional from spurious uORFs: a length between 4 and 6 codons, a distance from the start of the main ORF between 50 and 150 nucleotides, and finally a lack of overlap with, and clear separation from, neighbouring uORFs. These derived rules are inherently associated with uORFs with properties similar to the GCN4 locus, and may not detect most uORFs of other types. uORFs with high scores based on these rules showed a much higher evolutionary conservation than randomly selected uORFs. In a genome-wide scan in S. cerevisiae, we found 34 conserved uORFs from 32 genes that we predict to be functional; subsequent analysis showed the majority of these to be located within transcripts. A total of 252 genes were found containing conserved uORFs with properties indicative of a functional role; all but 7 are novel. Functional content analysis of this set identified an overrepresentation of genes involved in transcriptional control and development. CONCLUSION: Evolutionary conservation of uORFs in yeasts can be traced up to 100 million years of separation. The conserved uORFs have certain characteristics with respect to length, distance from each other and from the main start codon, and folding energy of the sequence. These newly found characteristics can be used to facilitate detection of other conserved uORFs.
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