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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.


Ypk3, but not Sch9, is required for Rps6 phosphorylation at Ser232/233.A) The indicated strains in the BY4742 background were grown in synthetic complete medium for 30 h (stationary phase) and then shifted to fresh medium (+) for 1 h. Total lysates were analyzed by Immunoblot using anti-phospho-S6 antibody. B) Immunoblot analysis of Rps6 phosphorylation in WT and Sch9-deficient cells in the TB50a background. Cells were shifted from nitrogen-free medium to SC medium as in Fig. 1E and aliquots were taken at the indicated time points. C) Immunoblot analysis of Rps6 phosphorylation in WT and ypk3Δ cells. Cells were shifted from nitrogen-free medium to SC medium as in Fig. 1E and aliquots were taken at the indicated time points.
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pone.0120250.g002: Ypk3, but not Sch9, is required for Rps6 phosphorylation at Ser232/233.A) The indicated strains in the BY4742 background were grown in synthetic complete medium for 30 h (stationary phase) and then shifted to fresh medium (+) for 1 h. Total lysates were analyzed by Immunoblot using anti-phospho-S6 antibody. B) Immunoblot analysis of Rps6 phosphorylation in WT and Sch9-deficient cells in the TB50a background. Cells were shifted from nitrogen-free medium to SC medium as in Fig. 1E and aliquots were taken at the indicated time points. C) Immunoblot analysis of Rps6 phosphorylation in WT and ypk3Δ cells. Cells were shifted from nitrogen-free medium to SC medium as in Fig. 1E and aliquots were taken at the indicated time points.

Mentions: We next examined the role of Sch9 in Rps6 phosphorylation in vivo. Sch9 is a downstream target of TORC1 and was previously been shown to phosphorylate Rps6 in vitro. Unexpectedly, sch9Δ cells, similar to WT cells, were able to phosphorylate Rps6 after shifting cells from deplete medium (stationary phase cells) into fresh, complete medium (Fig. 2A). To identify the kinase that is requisite for Rps6 phosphorylation, we screened several mutants known to be involved in TORC1 signaling or that resemble features of the human S6K. We identified Ypk3, an AGC kinase that is homologous to the TORC2-targeted kinases Ypk1 and Ypk2 [19], as a promising candidate. Cells lacking Ypk3, but not Ypk1 or Ypk2, were defective for Rps6 phosphorylation under nutrient-rich conditions (Fig. 2A). We further validated, in a different genetic background, our results demonstrating that Sch9 plays little-to-no role in Rps6 phosphorylation. Rps6 phosphorylation upon stimulation of nitrogen-starved sch9Δ cells with fresh medium was comparable to that observed in WT cells (Fig. 2B). However, cells lacking Sch9 expressed lower amounts of Rps6 protein (Fig. 2B), an observation that is consistent with the role of Sch9 in promoting ribosomal protein gene expression [9,20]. Next, we examined the kinetics of Rps6 phosphorylation upon stimulation of nitrogen-starved WT or ypk3Δ cells with fresh medium. Rps6 phosphorylation in WT cells increased during the 15 min after stimulation and thereafter remained constant up to 60 min, whereas in ypk3Δ cells Rps6 phosphorylation was completely absent (Fig. 2C). Together our data demonstrate that Ypk3 is rate-limiting for phosphorylation of Rps6 at Ser232/233.


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)

Ypk3, but not Sch9, is required for Rps6 phosphorylation at Ser232/233.A) The indicated strains in the BY4742 background were grown in synthetic complete medium for 30 h (stationary phase) and then shifted to fresh medium (+) for 1 h. Total lysates were analyzed by Immunoblot using anti-phospho-S6 antibody. B) Immunoblot analysis of Rps6 phosphorylation in WT and Sch9-deficient cells in the TB50a background. Cells were shifted from nitrogen-free medium to SC medium as in Fig. 1E and aliquots were taken at the indicated time points. C) Immunoblot analysis of Rps6 phosphorylation in WT and ypk3Δ cells. Cells were shifted from nitrogen-free medium to SC medium as in Fig. 1E and aliquots were taken at the indicated time points.
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Related In: Results  -  Collection

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pone.0120250.g002: Ypk3, but not Sch9, is required for Rps6 phosphorylation at Ser232/233.A) The indicated strains in the BY4742 background were grown in synthetic complete medium for 30 h (stationary phase) and then shifted to fresh medium (+) for 1 h. Total lysates were analyzed by Immunoblot using anti-phospho-S6 antibody. B) Immunoblot analysis of Rps6 phosphorylation in WT and Sch9-deficient cells in the TB50a background. Cells were shifted from nitrogen-free medium to SC medium as in Fig. 1E and aliquots were taken at the indicated time points. C) Immunoblot analysis of Rps6 phosphorylation in WT and ypk3Δ cells. Cells were shifted from nitrogen-free medium to SC medium as in Fig. 1E and aliquots were taken at the indicated time points.
Mentions: We next examined the role of Sch9 in Rps6 phosphorylation in vivo. Sch9 is a downstream target of TORC1 and was previously been shown to phosphorylate Rps6 in vitro. Unexpectedly, sch9Δ cells, similar to WT cells, were able to phosphorylate Rps6 after shifting cells from deplete medium (stationary phase cells) into fresh, complete medium (Fig. 2A). To identify the kinase that is requisite for Rps6 phosphorylation, we screened several mutants known to be involved in TORC1 signaling or that resemble features of the human S6K. We identified Ypk3, an AGC kinase that is homologous to the TORC2-targeted kinases Ypk1 and Ypk2 [19], as a promising candidate. Cells lacking Ypk3, but not Ypk1 or Ypk2, were defective for Rps6 phosphorylation under nutrient-rich conditions (Fig. 2A). We further validated, in a different genetic background, our results demonstrating that Sch9 plays little-to-no role in Rps6 phosphorylation. Rps6 phosphorylation upon stimulation of nitrogen-starved sch9Δ cells with fresh medium was comparable to that observed in WT cells (Fig. 2B). However, cells lacking Sch9 expressed lower amounts of Rps6 protein (Fig. 2B), an observation that is consistent with the role of Sch9 in promoting ribosomal protein gene expression [9,20]. Next, we examined the kinetics of Rps6 phosphorylation upon stimulation of nitrogen-starved WT or ypk3Δ cells with fresh medium. Rps6 phosphorylation in WT cells increased during the 15 min after stimulation and thereafter remained constant up to 60 min, whereas in ypk3Δ cells Rps6 phosphorylation was completely absent (Fig. 2C). Together our data demonstrate that Ypk3 is rate-limiting for phosphorylation of Rps6 at Ser232/233.

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.