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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

Characterization of the phospho-specific Rps6 antibody.A) C-terminal peptide sequence of human S6 and its orthologs in S. pombe and S. cerevisiae. The residues phosphorylated by S6K are highlighted in red. B) Immunoblot analysis of S. cerevisiae Rps6 phosphorylation in several rps6 mutants in the BY4742 background. Samples from exponentially growing cultures of the indicated strains were taken. Total lysates were resolved by SDS—PAGE on 10% polyacrylamide gels and analyzed by immunoblot. Actin was used as loading control. C) Samples of exponentially growing TB50a cells were taken. Cells were lysed in lysis buffer either in the absence (lanes 1 and 2) or presence (lane 3) of phosphatase inhibitor mixture (PPi). Total protein lysates were incubated with 2 U of alkaline phosphatase (Roche) for 30 min at 37°C and analyzed by immunoblot as in B. Ponceau S staining of the membrane indicates the presence of alkaline phosphatase. D) Immunoblot analysis of Rps6 phosphorylation in WT and rapamycin-resistant TOR1–1 TOR2–1 cells. Cells were treated with rapamycin (200 ng/ml) for the indicated times. Total lysates were analyzed as in B. E) Immunoblot analysis of S. cerevisiae Rps6 phosphorylation in WT cells. Exponentially growing cells in SC medium were shifted to nitrogen-free medium and cultured for 1 h. Then, SC medium containing 200 ng/ml rapamycin or drug vehicle was added and growth was resumed for 1 h. Total lysates were analyzed as in B. F) Immunoblot analysis of Sch9 and Rps6 phosphorylation upon glutamine stimulation. YSBN9 cells expressing Sch9–3xHA were grown in YMM with proline as the only nitrogen source. Then, glutamine (final concentration of 0.5 g/l) was added and samples were taken at the indicated times. To assess the Sch9 C-terminal phosphorylation state, protein samples were chemically cleaved with NTCB.
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pone.0120250.g001: Characterization of the phospho-specific Rps6 antibody.A) C-terminal peptide sequence of human S6 and its orthologs in S. pombe and S. cerevisiae. The residues phosphorylated by S6K are highlighted in red. B) Immunoblot analysis of S. cerevisiae Rps6 phosphorylation in several rps6 mutants in the BY4742 background. Samples from exponentially growing cultures of the indicated strains were taken. Total lysates were resolved by SDS—PAGE on 10% polyacrylamide gels and analyzed by immunoblot. Actin was used as loading control. C) Samples of exponentially growing TB50a cells were taken. Cells were lysed in lysis buffer either in the absence (lanes 1 and 2) or presence (lane 3) of phosphatase inhibitor mixture (PPi). Total protein lysates were incubated with 2 U of alkaline phosphatase (Roche) for 30 min at 37°C and analyzed by immunoblot as in B. Ponceau S staining of the membrane indicates the presence of alkaline phosphatase. D) Immunoblot analysis of Rps6 phosphorylation in WT and rapamycin-resistant TOR1–1 TOR2–1 cells. Cells were treated with rapamycin (200 ng/ml) for the indicated times. Total lysates were analyzed as in B. E) Immunoblot analysis of S. cerevisiae Rps6 phosphorylation in WT cells. Exponentially growing cells in SC medium were shifted to nitrogen-free medium and cultured for 1 h. Then, SC medium containing 200 ng/ml rapamycin or drug vehicle was added and growth was resumed for 1 h. Total lysates were analyzed as in B. F) Immunoblot analysis of Sch9 and Rps6 phosphorylation upon glutamine stimulation. YSBN9 cells expressing Sch9–3xHA were grown in YMM with proline as the only nitrogen source. Then, glutamine (final concentration of 0.5 g/l) was added and samples were taken at the indicated times. To assess the Sch9 C-terminal phosphorylation state, protein samples were chemically cleaved with NTCB.

Mentions: TORC1-mediated phosphorylation sites in S6 are conserved from yeast to human. In S. cerevisiae, Ser232 and Ser233 correspond to Ser235 and Ser236 in human S6 (Fig. 1A). S6 phosphorylation is widely used to monitor mTORC1 activity. Using a highly specific commercial anti-phospho-S6 (Ser235/236) antibody, we tested whether we could detect Rps6 phosphorylation in S. cerevisiae. Under nutrient rich conditions, we detected a 27 kDa protein that corresponds in size to Rps6 (Fig. 1B). To demonstrate that the antibody indeed recognizes only phosphorylated Rps6, we tested the phospho-specific antibody using rps6 mutants. Single deletion of RPS6A or RPS6B did not affect Rps6 phosphorylation. Serine-to-alanine mutation of the two serine residues in Rps6a combined with RPS6B deletion (rps6aS232A S233Arps6bΔ) abolished Rps6 phosphorylation (Fig. 1B). Moreover, treatment of protein lysates with alkaline phosphatase reduced Rps6 phosphorylation (Fig. 1C). These findings confirm that Rps6 phosphorylation sites are present in S. cerevisiae and validate the specificity of the anti-phospho-S6 antibody. We next asked whether Rps6 phosphorylation is regulated by TORC1. Inhibition of TORC1 by rapamycin treatment decreased Rps6 phosphorylation in wild-type (WT) cells but failed to decrease Rps6 phosphorylation in a rapamycin-resistant TOR1–1 TOR2–1 strain (Fig. 1D). Since TORC1 activity is controlled by nutrients, in particular by nitrogen source availability [14,17,18], we next investigated if Rps6 phosphorylation is also regulated by the nitrogen source. Nitrogen starvation resulted in decreased Rps6 phosphorylation, and re-stimulation of nitrogen-starved cells increased Rps6 phosphorylation (Fig. 1E). Thus, Rps6 phosphorylation is regulated by TORC1. We recently demonstrated that TORC1 activity is stimulated by glutamine using the phosphorylation state of Sch9 as a readout [14]. Thus, we next compared the kinetics of Rps6 and Sch9 phosphorylation in cells grown in the presence of the less-preferred nitrogen source proline upon stimulation with glutamine. Both Sch9 and Rps6 phosphorylation increased within 2 minutes after the addition of glutamine (Fig. 1F). After this initial increase, Sch9 phosphorylation decreased at 10 min to rise again at 30 min. Rps6 phosphorylation increased until 10 min after glutamine addition and then remained constant (Fig. 1F). Overall, these results underscore the validity of Rps6 phosphorylation as a specific readout for TORC1 activity in vivo.


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)

Characterization of the phospho-specific Rps6 antibody.A) C-terminal peptide sequence of human S6 and its orthologs in S. pombe and S. cerevisiae. The residues phosphorylated by S6K are highlighted in red. B) Immunoblot analysis of S. cerevisiae Rps6 phosphorylation in several rps6 mutants in the BY4742 background. Samples from exponentially growing cultures of the indicated strains were taken. Total lysates were resolved by SDS—PAGE on 10% polyacrylamide gels and analyzed by immunoblot. Actin was used as loading control. C) Samples of exponentially growing TB50a cells were taken. Cells were lysed in lysis buffer either in the absence (lanes 1 and 2) or presence (lane 3) of phosphatase inhibitor mixture (PPi). Total protein lysates were incubated with 2 U of alkaline phosphatase (Roche) for 30 min at 37°C and analyzed by immunoblot as in B. Ponceau S staining of the membrane indicates the presence of alkaline phosphatase. D) Immunoblot analysis of Rps6 phosphorylation in WT and rapamycin-resistant TOR1–1 TOR2–1 cells. Cells were treated with rapamycin (200 ng/ml) for the indicated times. Total lysates were analyzed as in B. E) Immunoblot analysis of S. cerevisiae Rps6 phosphorylation in WT cells. Exponentially growing cells in SC medium were shifted to nitrogen-free medium and cultured for 1 h. Then, SC medium containing 200 ng/ml rapamycin or drug vehicle was added and growth was resumed for 1 h. Total lysates were analyzed as in B. F) Immunoblot analysis of Sch9 and Rps6 phosphorylation upon glutamine stimulation. YSBN9 cells expressing Sch9–3xHA were grown in YMM with proline as the only nitrogen source. Then, glutamine (final concentration of 0.5 g/l) was added and samples were taken at the indicated times. To assess the Sch9 C-terminal phosphorylation state, protein samples were chemically cleaved with NTCB.
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Related In: Results  -  Collection

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pone.0120250.g001: Characterization of the phospho-specific Rps6 antibody.A) C-terminal peptide sequence of human S6 and its orthologs in S. pombe and S. cerevisiae. The residues phosphorylated by S6K are highlighted in red. B) Immunoblot analysis of S. cerevisiae Rps6 phosphorylation in several rps6 mutants in the BY4742 background. Samples from exponentially growing cultures of the indicated strains were taken. Total lysates were resolved by SDS—PAGE on 10% polyacrylamide gels and analyzed by immunoblot. Actin was used as loading control. C) Samples of exponentially growing TB50a cells were taken. Cells were lysed in lysis buffer either in the absence (lanes 1 and 2) or presence (lane 3) of phosphatase inhibitor mixture (PPi). Total protein lysates were incubated with 2 U of alkaline phosphatase (Roche) for 30 min at 37°C and analyzed by immunoblot as in B. Ponceau S staining of the membrane indicates the presence of alkaline phosphatase. D) Immunoblot analysis of Rps6 phosphorylation in WT and rapamycin-resistant TOR1–1 TOR2–1 cells. Cells were treated with rapamycin (200 ng/ml) for the indicated times. Total lysates were analyzed as in B. E) Immunoblot analysis of S. cerevisiae Rps6 phosphorylation in WT cells. Exponentially growing cells in SC medium were shifted to nitrogen-free medium and cultured for 1 h. Then, SC medium containing 200 ng/ml rapamycin or drug vehicle was added and growth was resumed for 1 h. Total lysates were analyzed as in B. F) Immunoblot analysis of Sch9 and Rps6 phosphorylation upon glutamine stimulation. YSBN9 cells expressing Sch9–3xHA were grown in YMM with proline as the only nitrogen source. Then, glutamine (final concentration of 0.5 g/l) was added and samples were taken at the indicated times. To assess the Sch9 C-terminal phosphorylation state, protein samples were chemically cleaved with NTCB.
Mentions: TORC1-mediated phosphorylation sites in S6 are conserved from yeast to human. In S. cerevisiae, Ser232 and Ser233 correspond to Ser235 and Ser236 in human S6 (Fig. 1A). S6 phosphorylation is widely used to monitor mTORC1 activity. Using a highly specific commercial anti-phospho-S6 (Ser235/236) antibody, we tested whether we could detect Rps6 phosphorylation in S. cerevisiae. Under nutrient rich conditions, we detected a 27 kDa protein that corresponds in size to Rps6 (Fig. 1B). To demonstrate that the antibody indeed recognizes only phosphorylated Rps6, we tested the phospho-specific antibody using rps6 mutants. Single deletion of RPS6A or RPS6B did not affect Rps6 phosphorylation. Serine-to-alanine mutation of the two serine residues in Rps6a combined with RPS6B deletion (rps6aS232A S233Arps6bΔ) abolished Rps6 phosphorylation (Fig. 1B). Moreover, treatment of protein lysates with alkaline phosphatase reduced Rps6 phosphorylation (Fig. 1C). These findings confirm that Rps6 phosphorylation sites are present in S. cerevisiae and validate the specificity of the anti-phospho-S6 antibody. We next asked whether Rps6 phosphorylation is regulated by TORC1. Inhibition of TORC1 by rapamycin treatment decreased Rps6 phosphorylation in wild-type (WT) cells but failed to decrease Rps6 phosphorylation in a rapamycin-resistant TOR1–1 TOR2–1 strain (Fig. 1D). Since TORC1 activity is controlled by nutrients, in particular by nitrogen source availability [14,17,18], we next investigated if Rps6 phosphorylation is also regulated by the nitrogen source. Nitrogen starvation resulted in decreased Rps6 phosphorylation, and re-stimulation of nitrogen-starved cells increased Rps6 phosphorylation (Fig. 1E). Thus, Rps6 phosphorylation is regulated by TORC1. We recently demonstrated that TORC1 activity is stimulated by glutamine using the phosphorylation state of Sch9 as a readout [14]. Thus, we next compared the kinetics of Rps6 and Sch9 phosphorylation in cells grown in the presence of the less-preferred nitrogen source proline upon stimulation with glutamine. Both Sch9 and Rps6 phosphorylation increased within 2 minutes after the addition of glutamine (Fig. 1F). After this initial increase, Sch9 phosphorylation decreased at 10 min to rise again at 30 min. Rps6 phosphorylation increased until 10 min after glutamine addition and then remained constant (Fig. 1F). Overall, these results underscore the validity of Rps6 phosphorylation as a specific readout for TORC1 activity in vivo.

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