TORC1 Promotes Phosphorylation of Ribosomal Protein S6 via the AGC Kinase Ypk3 in Saccharomyces cerevisiae

González, Asier; Shimobayashi, Mitsugu; Eisenberg, Tobias; Merle, David A.; Pendl, Tobias; Hall, Michael N.; Moustafa, Tarek

doi:10.1371/journal.pone.0120250

Cited by 86 publications

(103 citation statements)

References 34 publications

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“…We shifted WT and set2 Δ cells from nutrient rich (YPD) medium to medium lacking amino acids (SD) and collected samples every 30–60 minutes over a 2-hour time period. We examined the protein and phosphorylation levels of Rps6, the Saccharomyces cerevisiae S6K homolog (Gonzalez et al, 2015), a key downstream target of TORC1. As shown in Figure 3D, the level of Rps6 was much lower in set2 Δ cells compared with their isogenic WT counterparts, and, further, Rps6 had a drastically increased level of phosphorylation at the start of the time course, continuing through 30 minutes after nutrient stress.…”

Section: Resultsmentioning

confidence: 99%

H3K36 Methylation Regulates Nutrient Stress Response in Saccharomyces cerevisiae by Enforcing Transcriptional Fidelity

et al. 2017

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SUMMARY Set2-mediated histone methylation at H3K36 regulates diverse activities including DNA repair, mRNA splicing and suppression of inappropriate (cryptic) transcription. Although failure of Set2 to suppress cryptic transcription has been linked to decreased life span, the extent to which cryptic transcription influences other cellular functions is poorly understood. Here, we uncover a role for H3K36 methylation in the regulation of the nutrient stress response pathway. We found the transcriptional response to nutrient stress was dysregulated in SET2-deleted (set2Δ) cells and was correlated with genome-wide bi-directional cryptic transcription that originated from within gene bodies. Antisense transcripts arising from these cryptic events extended into the promoters of the genes from which they arose and were associated with decreased sense transcription under nutrient stress conditions. These results suggest that Set2-enforced transcriptional fidelity is critical to the proper regulation of inducible and highly regulated transcription programs.

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Section: Resultsmentioning

confidence: 99%

H3K36 Methylation Regulates Nutrient Stress Response in Saccharomyces cerevisiae by Enforcing Transcriptional Fidelity

et al. 2017

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“…Briefly, strains were grown in flasks containing 50 ml of YMM + Pro medium until OD 600 ~ 0.8, and then 700 μl of glutamine (25 mg/ml; 0.5 mg/ml final concentration) was added. Samples were taken at different time points (0, 5, 15, and 30 min) to perform protein extraction and subsequent immunoblot as previously described (Gonzalez et al, ). To evaluate Sch9 phosphorylation, cell extracts were subjected to chemical cleavage with 2‐nitro‐5‐thiocyanatobenzoic acid (Sigma).…”

Section: Methodsmentioning

confidence: 99%

Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains

Kessi-Pérez

Salinas

Molinet

et al. 2018

Yeast

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Saccharomyces cerevisiae is the main species responsible for the alcoholic fermentation in wine production. One of the main problems in this process is the deficiency of nitrogen sources in the grape must, which can lead to stuck or sluggish fermentations. Currently, yeast nitrogen consumption and metabolism are under active inquiry, with emphasis on the study of the TORC1 signalling pathway, given its central role responding to nitrogen availability and influencing growth and cell metabolism. However, the mechanism by which different nitrogen sources activates TORC1 is not completely understood. Existing methods to evaluate TORC1 activation by nitrogen sources are time-consuming, making difficult the analyses of large numbers of strains. In this work, a new indirect method for monitoring TORC1 pathway was developed on the basis of the luciferase reporter gene controlled by the promoter region of RPL26A gene, a gene known to be expressed upon TORC1 activation. The method was tested in strains representative of the clean lineages described so far in S. cerevisiae. The activation of the TORC1 pathway by a proline-to-glutamine upshift was indirectly evaluated using our system and the traditional direct methods based on immunoblot (Sch9 and Rps6 phosphorylation). Regardless of the different molecular readouts obtained with both methodologies, the general results showed a wide phenotypic variation between the representative strains analysed. Altogether, this easy-to-use assay opens the possibility to study the molecular basis for the differential TORC1 pathway activation, allowing to interrogate a larger number of strains in the context of nitrogen metabolism phenotypic differences.

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“…However, recent research showed that Rps6 phosphorylation is completely abolished in cells lacking Ypk3, another AGC kinase, and expression of human S6K in ypk3Δ cells restores Rps6 phosphorylation in a rapamycin‐sensitive manner. The results indicated that Ypk3 is the functional ortholog of mammalian S6K .…”

mentioning

confidence: 94%

Plant S6 kinases do not require hydrophobic motif phosphorylation for activity in yeast lacking Ypk3

Yaguchi

Kozaki

2018

FEBS Letters

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The ribosomal protein S6 kinases (S6K) are among the major substrates and crucial effectors of the target of rapamycin (TOR) kinase, which is an evolutionarily conserved regulator of cell growth and proliferation. Recent research indicates that yeast Ypk3 is an ortholog of mammalian S6Ks. Here, we find that plant S6Ks restore ribosomal protein S6 phosphorylation in a rapamycin-sensitive manner in yeast cells lacking Ypk3. However, phosphorylation of a hydrophobic motif, which is mediated through TOR signaling and essential for mammalian S6K activity, is not detected in plant S6Ks. Furthermore, deletion of the N-terminal region of rice S6Ks shows phosphorylation of the hydrophobic motif and reduced rapamycin sensitivity. Our findings suggest a mechanism of plant S6K activation distinct from that of mammalian S6Ks.

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TORC1 Promotes Phosphorylation of Ribosomal Protein S6 via the AGC Kinase Ypk3 in Saccharomyces cerevisiae

Cited by 86 publications

References 34 publications

H3K36 Methylation Regulates Nutrient Stress Response in Saccharomyces cerevisiae by Enforcing Transcriptional Fidelity

H3K36 Methylation Regulates Nutrient Stress Response in Saccharomyces cerevisiae by Enforcing Transcriptional Fidelity

Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains

Plant S6 kinases do not require hydrophobic motif phosphorylation for activity in yeast lacking Ypk3

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