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TORC1 promotes phosphorylation of ribosomal protein S6 via the AGC kinase Ypk3 in Saccharomyces cerevisiae.

González A, Shimobayashi M, Eisenberg T, Merle DA, Pendl T, Hall MN, Moustafa T - PLoS ONE (2015)

Bottom Line: Using a highly specific antibody that recognizes phosphorylation of the bona fide TORC1 target ribosomal protein S6 (Rps6) in yeast, we found that nutrients rapidly induce Rps6 phosphorylation in a TORC1-dependent manner.Phosphorylation-deficient mutations in regulatory motifs of Ypk3 abrogate Rps6 phosphorylation, and complementation of ypk3Δ cells with human S6 kinase restores Rps6 phosphorylation in a rapamycin-sensitive manner.Our findings demonstrate that Ypk3 is a critical component of the TORC1 pathway and that the use of a phospho-S6 specific antibody offers a valuable tool to identify new nutrient-dependent and rapamycin-sensitive targets in vivo.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Basel, Switzerland.

ABSTRACT
The target of rapamycin complex 1 (TORC1) is an evolutionarily conserved sensor of nutrient availability. Genetic and pharmacological studies in the yeast Saccharomyces cerevisiae have provided mechanistic insights on the regulation of TORC1 signaling in response to nutrients. Using a highly specific antibody that recognizes phosphorylation of the bona fide TORC1 target ribosomal protein S6 (Rps6) in yeast, we found that nutrients rapidly induce Rps6 phosphorylation in a TORC1-dependent manner. Moreover, we demonstrate that Ypk3, an AGC kinase which exhibits high homology to human S6 kinase (S6K), is required for the phosphorylation of Rps6 in vivo. Rps6 phosphorylation is completely abolished in cells lacking Ypk3 (ypk3Δ), whereas Sch9, previously reported to be the yeast ortholog of S6K, is dispensable for Rps6 phosphorylation. Phosphorylation-deficient mutations in regulatory motifs of Ypk3 abrogate Rps6 phosphorylation, and complementation of ypk3Δ cells with human S6 kinase restores Rps6 phosphorylation in a rapamycin-sensitive manner. Our findings demonstrate that Ypk3 is a critical component of the TORC1 pathway and that the use of a phospho-S6 specific antibody offers a valuable tool to identify new nutrient-dependent and rapamycin-sensitive targets in vivo.

No MeSH data available.


Related in: MedlinePlus

Phenotypic characterization of the Rps6 phosphorylation deficiency.A) WT strain BY4742 and the indicated mutants were tested for growth in YPD medium. Growth was monitored at the indicated times. Data are presented as the mean ± S.E.M. of triplicate determinations and correspond to a representative experiment among three. Statistical analysis was performed by using two-way ANOVA. WT vs. rp6bΔ (significant: p < 0.001), WT vs. rps6aS232A S233Arp6bΔ (significant: p = 0.001), rp6bΔ vs. rps6aS232A S233Arp6bΔ (not significant: p = 0.153). B) Cell size of the indicated strains was measured. Two-way ANOVA analysis was performed. **, p<0.01; ns, not significant. C) WT strain BY4742 and the indicated mutants were spotted onto YPD plates containing either 4 ng/ml of rapamycin or drug vehicle alone. Plates were grown for 3 days at 30°C. Pictures correspond to a representative experiment among three. D) Model of regulation of Rps6 phosphorylation by TORC1-Ypk3.
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pone.0120250.g004: Phenotypic characterization of the Rps6 phosphorylation deficiency.A) WT strain BY4742 and the indicated mutants were tested for growth in YPD medium. Growth was monitored at the indicated times. Data are presented as the mean ± S.E.M. of triplicate determinations and correspond to a representative experiment among three. Statistical analysis was performed by using two-way ANOVA. WT vs. rp6bΔ (significant: p < 0.001), WT vs. rps6aS232A S233Arp6bΔ (significant: p = 0.001), rp6bΔ vs. rps6aS232A S233Arp6bΔ (not significant: p = 0.153). B) Cell size of the indicated strains was measured. Two-way ANOVA analysis was performed. **, p<0.01; ns, not significant. C) WT strain BY4742 and the indicated mutants were spotted onto YPD plates containing either 4 ng/ml of rapamycin or drug vehicle alone. Plates were grown for 3 days at 30°C. Pictures correspond to a representative experiment among three. D) Model of regulation of Rps6 phosphorylation by TORC1-Ypk3.

Mentions: The biological role of S6 phosphorylation in yeast is not well understood. Here we compared cell proliferation, cell size and rapamycin sensitivity of WT, rps6bΔ, and phosphorylation-deficient rps6aS232A S233Arps6bΔ cells. In agreement with previous observations [10,11,25], rps6bΔ cells proliferated slower than WT cells, indicating that Rps6b is important for growth. However, the phosphorylation-deficient mutant resembled rps6bΔ cells (Fig. 4A). Cell size was reduced by 6% in rps6bΔ mutants and rps6aS232A S233Arps6bΔ cells showed a similar decrease in size (Fig. 4B). When treated with rapamycin, rps6aS232A S233Arps6bΔ cells exhibited additional mild hypersensitivity compared to rps6bΔ cells (Fig. 4B). Thus, our data indicate that Rps6a phosphorylation is not important for cellular proliferation in rps6bΔ cells.


TORC1 promotes phosphorylation of ribosomal protein S6 via the AGC kinase Ypk3 in Saccharomyces cerevisiae.

González A, Shimobayashi M, Eisenberg T, Merle DA, Pendl T, Hall MN, Moustafa T - PLoS ONE (2015)

Phenotypic characterization of the Rps6 phosphorylation deficiency.A) WT strain BY4742 and the indicated mutants were tested for growth in YPD medium. Growth was monitored at the indicated times. Data are presented as the mean ± S.E.M. of triplicate determinations and correspond to a representative experiment among three. Statistical analysis was performed by using two-way ANOVA. WT vs. rp6bΔ (significant: p < 0.001), WT vs. rps6aS232A S233Arp6bΔ (significant: p = 0.001), rp6bΔ vs. rps6aS232A S233Arp6bΔ (not significant: p = 0.153). B) Cell size of the indicated strains was measured. Two-way ANOVA analysis was performed. **, p<0.01; ns, not significant. C) WT strain BY4742 and the indicated mutants were spotted onto YPD plates containing either 4 ng/ml of rapamycin or drug vehicle alone. Plates were grown for 3 days at 30°C. Pictures correspond to a representative experiment among three. D) Model of regulation of Rps6 phosphorylation by TORC1-Ypk3.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359079&req=5

pone.0120250.g004: Phenotypic characterization of the Rps6 phosphorylation deficiency.A) WT strain BY4742 and the indicated mutants were tested for growth in YPD medium. Growth was monitored at the indicated times. Data are presented as the mean ± S.E.M. of triplicate determinations and correspond to a representative experiment among three. Statistical analysis was performed by using two-way ANOVA. WT vs. rp6bΔ (significant: p < 0.001), WT vs. rps6aS232A S233Arp6bΔ (significant: p = 0.001), rp6bΔ vs. rps6aS232A S233Arp6bΔ (not significant: p = 0.153). B) Cell size of the indicated strains was measured. Two-way ANOVA analysis was performed. **, p<0.01; ns, not significant. C) WT strain BY4742 and the indicated mutants were spotted onto YPD plates containing either 4 ng/ml of rapamycin or drug vehicle alone. Plates were grown for 3 days at 30°C. Pictures correspond to a representative experiment among three. D) Model of regulation of Rps6 phosphorylation by TORC1-Ypk3.
Mentions: The biological role of S6 phosphorylation in yeast is not well understood. Here we compared cell proliferation, cell size and rapamycin sensitivity of WT, rps6bΔ, and phosphorylation-deficient rps6aS232A S233Arps6bΔ cells. In agreement with previous observations [10,11,25], rps6bΔ cells proliferated slower than WT cells, indicating that Rps6b is important for growth. However, the phosphorylation-deficient mutant resembled rps6bΔ cells (Fig. 4A). Cell size was reduced by 6% in rps6bΔ mutants and rps6aS232A S233Arps6bΔ cells showed a similar decrease in size (Fig. 4B). When treated with rapamycin, rps6aS232A S233Arps6bΔ cells exhibited additional mild hypersensitivity compared to rps6bΔ cells (Fig. 4B). Thus, our data indicate that Rps6a phosphorylation is not important for cellular proliferation in rps6bΔ cells.

Bottom Line: Using a highly specific antibody that recognizes phosphorylation of the bona fide TORC1 target ribosomal protein S6 (Rps6) in yeast, we found that nutrients rapidly induce Rps6 phosphorylation in a TORC1-dependent manner.Phosphorylation-deficient mutations in regulatory motifs of Ypk3 abrogate Rps6 phosphorylation, and complementation of ypk3Δ cells with human S6 kinase restores Rps6 phosphorylation in a rapamycin-sensitive manner.Our findings demonstrate that Ypk3 is a critical component of the TORC1 pathway and that the use of a phospho-S6 specific antibody offers a valuable tool to identify new nutrient-dependent and rapamycin-sensitive targets in vivo.

View Article: PubMed Central - PubMed

Affiliation: Biozentrum, University of Basel, Basel, Switzerland.

ABSTRACT
The target of rapamycin complex 1 (TORC1) is an evolutionarily conserved sensor of nutrient availability. Genetic and pharmacological studies in the yeast Saccharomyces cerevisiae have provided mechanistic insights on the regulation of TORC1 signaling in response to nutrients. Using a highly specific antibody that recognizes phosphorylation of the bona fide TORC1 target ribosomal protein S6 (Rps6) in yeast, we found that nutrients rapidly induce Rps6 phosphorylation in a TORC1-dependent manner. Moreover, we demonstrate that Ypk3, an AGC kinase which exhibits high homology to human S6 kinase (S6K), is required for the phosphorylation of Rps6 in vivo. Rps6 phosphorylation is completely abolished in cells lacking Ypk3 (ypk3Δ), whereas Sch9, previously reported to be the yeast ortholog of S6K, is dispensable for Rps6 phosphorylation. Phosphorylation-deficient mutations in regulatory motifs of Ypk3 abrogate Rps6 phosphorylation, and complementation of ypk3Δ cells with human S6 kinase restores Rps6 phosphorylation in a rapamycin-sensitive manner. Our findings demonstrate that Ypk3 is a critical component of the TORC1 pathway and that the use of a phospho-S6 specific antibody offers a valuable tool to identify new nutrient-dependent and rapamycin-sensitive targets in vivo.

No MeSH data available.


Related in: MedlinePlus