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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

Ypk3 functions as a Rps6 kinase in vivo.A) Top: Schematic depiction of Ypk3 showing the phosphorylation sites in the T-loop, turn motif (TM) and hydrophobic motif (HM); bottom side: conserved amino acids (phosphorylation sites) within the sequence alignment of human S6K and S. pombe and S. cerevisiae counterparts are highlighted in red. An asterisk (*) indicates positions which have a fully conserved residue. Colon (:) and period (.) indicate conservation between residues of strongly and weakly similar properties, respectively. B) Immunoblot analysis of Rps6 phosphorylation in phosphorylation-deficient Ypk3 mutants. Cells expressing Ypk3-HA or the phosphorylation-deficient mutants were treated with rapamycin (200 ng/ml) for 1 h. Total lysates were analyzed by inmmunoblot. C) Analysis of Rps6 phosphorylation status in cells lacking Pkh1/2. Exponentially growing cells of the strains INA17–4D (WT) and INA106–3B (pkh1tspkh2Δ) at 25°C were shifted to 37°C for 1 h. D) Immunoblot analysis of Rps6 phosphorylation in ypk3Δ cells transformed with the indicated plasmids containing the human S6K1 and S6K2 as well as other AGC family members including RSK2 and RSK3 under the constitutive ADH1 promoter. The asterisk indicates the presence of a non-specific band. E) Rps6 phosphorylation in ypk3Δ cells transformed with the indicated plasmids. Cells were treated with rapamycin (200 ng/ml) for 1 h.
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pone.0120250.g003: Ypk3 functions as a Rps6 kinase in vivo.A) Top: Schematic depiction of Ypk3 showing the phosphorylation sites in the T-loop, turn motif (TM) and hydrophobic motif (HM); bottom side: conserved amino acids (phosphorylation sites) within the sequence alignment of human S6K and S. pombe and S. cerevisiae counterparts are highlighted in red. An asterisk (*) indicates positions which have a fully conserved residue. Colon (:) and period (.) indicate conservation between residues of strongly and weakly similar properties, respectively. B) Immunoblot analysis of Rps6 phosphorylation in phosphorylation-deficient Ypk3 mutants. Cells expressing Ypk3-HA or the phosphorylation-deficient mutants were treated with rapamycin (200 ng/ml) for 1 h. Total lysates were analyzed by inmmunoblot. C) Analysis of Rps6 phosphorylation status in cells lacking Pkh1/2. Exponentially growing cells of the strains INA17–4D (WT) and INA106–3B (pkh1tspkh2Δ) at 25°C were shifted to 37°C for 1 h. D) Immunoblot analysis of Rps6 phosphorylation in ypk3Δ cells transformed with the indicated plasmids containing the human S6K1 and S6K2 as well as other AGC family members including RSK2 and RSK3 under the constitutive ADH1 promoter. The asterisk indicates the presence of a non-specific band. E) Rps6 phosphorylation in ypk3Δ cells transformed with the indicated plasmids. Cells were treated with rapamycin (200 ng/ml) for 1 h.

Mentions: To be active, mammalian S6K requires phosphorylation at three important regulatory motifs: the T- or activation loop (T-loop) which is located in the catalytic domain, the hydrophobic motif (HM) which is located in a non-catalytic region following the kinase domain, and the turn motif (TM), another phosphorylation site that promotes S6K integrity. mTORC1 phosphorylates S6K on the HM and TM whereas PDK1 phosphorylates S6K on the T-loop [21,22]. All three regulatory motifs are present in Ypk3 (Fig. 3A). Interestingly, using a mass-spectrometry-based proteomics approach, it was shown recently that Ypk3 is phosphorylated in vivo at Ser513 (HM) in a TORC1-dependent manner [23]. To investigate whether phosphorylation of the motifs in Ypk3 is important for Ypk3 kinase activity toward Rps6, we generated phosphorylation-deficient mutants by converting the Ser/Thr residues to alanine. In line with mammalian and S. pombe S6K, mutation of Ser321 (T-loop) or Ser513 to alanine abolished Rps6 phosphorylation under normal growth conditions, while mutation of Thr490 in the TM had no impact on Rps6 phosphorylation (Fig. 3B). Altogether, these data suggest that Ypk3 activation requires T-loop and HM phosphorylation to maintain signaling to Rps6. Since Ser321 in the T-loop is required for Rps6 phosphorylation, we sought to investigate if this effect is mediated by the PDK1 orthologs Pkh1 and Pkh2 [24]. Because cells lacking both Pkh1 and Pkh2 are inviable, we assessed phosphorylation of Rps6 in a conditional pkh1Δ pkh2ts mutant. Rps6 phosphorylation was completely abolished in cells shifted to non-permissive temperature (37°C) for 1 h (Fig. 3C). These results indicate that similar to PDK1 in mammals, yeast PDK1 orthologs are required for Rps6 phosphorylation. To further confirm that Ypk3 is equivalent to mammalian S6 kinase, we expressed human S6K1 and S6K2 isoforms as well as other AGC kinase family members including ribosomal protein S6 kinase alpha-3 and 2 (RSK2 and RSK3) under the constitutive ADH1 promoter in cells lacking Ypk3. Heterologous expression of human S6K1 or S6K2 was sufficient to restore Rps6 phosphorylation in ypk3Δ cells (Fig. 3D). In contrast, heterologous expression of RSK2 and RSK3 did not restore Rps6 phosphorylation (Fig. 3D). Interestingly, human S6K-mediated phosphorylation of Rps6 was also sensitive to rapamycin (Fig. 3E), indicating a functional signal transduction pathway. Taken together, our findings demonstrate that Ypk3 is a critical component of the TORC1 pathway and that loss of Ypk3 can be complemented by human S6K.


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 functions as a Rps6 kinase in vivo.A) Top: Schematic depiction of Ypk3 showing the phosphorylation sites in the T-loop, turn motif (TM) and hydrophobic motif (HM); bottom side: conserved amino acids (phosphorylation sites) within the sequence alignment of human S6K and S. pombe and S. cerevisiae counterparts are highlighted in red. An asterisk (*) indicates positions which have a fully conserved residue. Colon (:) and period (.) indicate conservation between residues of strongly and weakly similar properties, respectively. B) Immunoblot analysis of Rps6 phosphorylation in phosphorylation-deficient Ypk3 mutants. Cells expressing Ypk3-HA or the phosphorylation-deficient mutants were treated with rapamycin (200 ng/ml) for 1 h. Total lysates were analyzed by inmmunoblot. C) Analysis of Rps6 phosphorylation status in cells lacking Pkh1/2. Exponentially growing cells of the strains INA17–4D (WT) and INA106–3B (pkh1tspkh2Δ) at 25°C were shifted to 37°C for 1 h. D) Immunoblot analysis of Rps6 phosphorylation in ypk3Δ cells transformed with the indicated plasmids containing the human S6K1 and S6K2 as well as other AGC family members including RSK2 and RSK3 under the constitutive ADH1 promoter. The asterisk indicates the presence of a non-specific band. E) Rps6 phosphorylation in ypk3Δ cells transformed with the indicated plasmids. Cells were treated with rapamycin (200 ng/ml) for 1 h.
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Related In: Results  -  Collection

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pone.0120250.g003: Ypk3 functions as a Rps6 kinase in vivo.A) Top: Schematic depiction of Ypk3 showing the phosphorylation sites in the T-loop, turn motif (TM) and hydrophobic motif (HM); bottom side: conserved amino acids (phosphorylation sites) within the sequence alignment of human S6K and S. pombe and S. cerevisiae counterparts are highlighted in red. An asterisk (*) indicates positions which have a fully conserved residue. Colon (:) and period (.) indicate conservation between residues of strongly and weakly similar properties, respectively. B) Immunoblot analysis of Rps6 phosphorylation in phosphorylation-deficient Ypk3 mutants. Cells expressing Ypk3-HA or the phosphorylation-deficient mutants were treated with rapamycin (200 ng/ml) for 1 h. Total lysates were analyzed by inmmunoblot. C) Analysis of Rps6 phosphorylation status in cells lacking Pkh1/2. Exponentially growing cells of the strains INA17–4D (WT) and INA106–3B (pkh1tspkh2Δ) at 25°C were shifted to 37°C for 1 h. D) Immunoblot analysis of Rps6 phosphorylation in ypk3Δ cells transformed with the indicated plasmids containing the human S6K1 and S6K2 as well as other AGC family members including RSK2 and RSK3 under the constitutive ADH1 promoter. The asterisk indicates the presence of a non-specific band. E) Rps6 phosphorylation in ypk3Δ cells transformed with the indicated plasmids. Cells were treated with rapamycin (200 ng/ml) for 1 h.
Mentions: To be active, mammalian S6K requires phosphorylation at three important regulatory motifs: the T- or activation loop (T-loop) which is located in the catalytic domain, the hydrophobic motif (HM) which is located in a non-catalytic region following the kinase domain, and the turn motif (TM), another phosphorylation site that promotes S6K integrity. mTORC1 phosphorylates S6K on the HM and TM whereas PDK1 phosphorylates S6K on the T-loop [21,22]. All three regulatory motifs are present in Ypk3 (Fig. 3A). Interestingly, using a mass-spectrometry-based proteomics approach, it was shown recently that Ypk3 is phosphorylated in vivo at Ser513 (HM) in a TORC1-dependent manner [23]. To investigate whether phosphorylation of the motifs in Ypk3 is important for Ypk3 kinase activity toward Rps6, we generated phosphorylation-deficient mutants by converting the Ser/Thr residues to alanine. In line with mammalian and S. pombe S6K, mutation of Ser321 (T-loop) or Ser513 to alanine abolished Rps6 phosphorylation under normal growth conditions, while mutation of Thr490 in the TM had no impact on Rps6 phosphorylation (Fig. 3B). Altogether, these data suggest that Ypk3 activation requires T-loop and HM phosphorylation to maintain signaling to Rps6. Since Ser321 in the T-loop is required for Rps6 phosphorylation, we sought to investigate if this effect is mediated by the PDK1 orthologs Pkh1 and Pkh2 [24]. Because cells lacking both Pkh1 and Pkh2 are inviable, we assessed phosphorylation of Rps6 in a conditional pkh1Δ pkh2ts mutant. Rps6 phosphorylation was completely abolished in cells shifted to non-permissive temperature (37°C) for 1 h (Fig. 3C). These results indicate that similar to PDK1 in mammals, yeast PDK1 orthologs are required for Rps6 phosphorylation. To further confirm that Ypk3 is equivalent to mammalian S6 kinase, we expressed human S6K1 and S6K2 isoforms as well as other AGC kinase family members including ribosomal protein S6 kinase alpha-3 and 2 (RSK2 and RSK3) under the constitutive ADH1 promoter in cells lacking Ypk3. Heterologous expression of human S6K1 or S6K2 was sufficient to restore Rps6 phosphorylation in ypk3Δ cells (Fig. 3D). In contrast, heterologous expression of RSK2 and RSK3 did not restore Rps6 phosphorylation (Fig. 3D). Interestingly, human S6K-mediated phosphorylation of Rps6 was also sensitive to rapamycin (Fig. 3E), indicating a functional signal transduction pathway. Taken together, our findings demonstrate that Ypk3 is a critical component of the TORC1 pathway and that loss of Ypk3 can be complemented by human S6K.

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