Fission yeast TORC1 regulates phosphorylation of ribosomal S6 proteins in response to nutrients and its activity is inhibited by rapamycin

Abstract

Cellular activities are regulated by environmental stimuli through protein phosphorylation. Target of rapamycin (TOR), a serine/threonine kinase, plays pivotal roles in cell proliferation and cell growth in response to nutrient status. In Schizosaccharomyces pombe, TORC1, which contains Tor2, plays crucial roles in nutrient response. Here we find a nitrogen-regulated phosphoprotein, p27, in S. pombe using the phospho-Akt substrate antibody. Response of p27 phosphorylation to nitrogen availability is mediated by TORC1 and the TSC-Rhb1 signaling, but not by TORC2 or other nutrient stress-related pathways. Database and biochemical analyses indicate that p27 is identical to ribosomal protein S6 (Rps6). Ser235 and Ser236 in Rps6 are necessary for Rps6 phosphorylation by TORC1. These Rps6 phosphorylations are dispensable for cell viability. Rps6 phosphorylation by TORC1 also responds to availability of glucose and is inhibited by osmotic and oxidative stresses. Rapamycin inhibits the ability of TORC1 to phosphorylate Rps6, owing to interaction of the rapamycin-FKBP12 complex with the FRB domain in Tor2. Rapamycin also leads to a decrease in cell size in a TORC1-dependent manner. Our findings demonstrate that the nutrient-responsive and rapamycin-sensitive TORC1-S6 signaling exists in S. pombe, and that this pathway plays a role in cell size control.

Fig. 1.

Phosphorylation of p27 in response to nitrogen availability is recognized by the anti-PAS antibody. (A) Cells of JUp1204 were cultured in EMM medium at 30°C to exponential phase, then washed and incubated in EMM with (+) or without (−) ammonium for the indicated time points. Cell extracts were probed with anti-PAS antibody or with anti-phospho-eIF2α (Ser51) antibody to detect phosphorylation of eIF2α at Ser52. In the top panel, positions of predicted phosphoproteins (p27, p70, p90 and p150) and molecular size markers are indicated. Tubulin is shown as a loading control (bottom). (B) Exponentially growing cells of JUp1204 were starved in EMM-N (a nitrogen-free version of EMM) for 1 hour, and then water (no addition) or 0.5% ammonium chloride (final concentration) was added. The cells were taken at the indicated time points, and extracts were probed with the anti-PAS antibody for phosphorylation of p27 or anti-tubulin antibody. (C) Extracts from exponentially growing cells of JUp1204 were treated with λ-phosphatase in the presence or absence of its inhibitors and probed for phosphorylation of p27 or tubulin.

Fig. 2.

TORC1 regulates p27 phosphorylation. Extracts were subjected to immunoblotting with the indicated antibodies. (A) Cells of JUp1348 (WT), JUp1350 (tor2^L1310P) and JUp1352 (tor2^E2221K) were grown in EMM at 30°C to exponential phase, then washed and treated with EMM with (+) or without (−) ammonium for 15 minutes. (B) Exponentially growing cells of JUp1348 (WT) and JUp1350 (tor2^L1310P) (0 minutes) were washed and incubated in EMM-N (a nitrogen-free version of EMM). The cells were harvested for the indicated time points. (C) Cells of JUp1246 (WT), JUp1247 (tor2-ts6) and JUp1252 (tor2-ts10) were grown in EMM at 25°C to exponential phase. When cells were treated at a non-permissive temperature, cell culture was shifted to 34°C for 1 hour before the cells were transferred to pre-warmed EMM-N. After incubation in EMM-N for 1 hour at either 25°C or 34°C, the cells were added with ammonium or water and then incubated for 15 minutes. (D) Exponentially growing cells of JY450 (WT), TA99 (tor1Δ) and JT293 (ste20Δ) carrying pREP41 (vector) in EMM+Adenine were treated with EMM+Adenine (+) or EMM-N (−) for 15 minutes at 30°C. (E) JY450 (WT), FY13927 (phh1Δ) and JZ858 (cgs1Δ) carrying pREP41 (vector) were grown and treated as described in D.

Fig. 3.

The Tsc1-Tsc2 complex and Rhb1 are involved in the regulation of p27 phosphorylation. Extracts were subjected to immunoblotting with the indicated antibodies. (A) JUp1204 (WT) and PJ001 (tsc2Δ) were grown and treated as described in Fig. 2A. (B) Cells of 972 (WT) and PJ001 (tsc2Δ) were treated with EMM-N (a nitrogen-free version of EMM) as described in Fig. 2B. (C) JUp1188 (WT), JUp1190 (rhb1^V17A) and JUp1194 (rhb1^K120R) were grown and treated as described in Fig. 2A. The amount of myc-Rhb1 was examined by anti-myc antibody. (D) JUp1188 (WT) and JUp1190 (rhb1^V17A) were treated with EMM-N as described in Fig. 2B.

Fig. 4.

Identification of ribosomal protein S6 as p27. (A,B,D) Extracts were subjected to immunoblotting with the indicated antibodies. (A) Cells of 972 (WT), AN0094 (rps601Δ) and AN0097 (rps602Δ) were grown and treated as described in Fig. 2A. (B) JUp1204 (WT) and AN0138 (rps601Δ myc-rps602⁺) were grown and treated as described in Fig. 2A. The amount of myc-Rps602 was examined by anti-myc antibody. (C) AN0098 (rps602Δ) was transformed with pREP1-myc (vector), pREP1-myc-rps601⁺(rps601⁺) or pREP1-myc-rps602⁺(rps602⁺), and the transformants were treated as described in Fig. 2A. The immunoprecipitates with anti-myc antibody were subjected to immunoblotting with the indicated antibodies. LC denotes light chain of immunoglobulin caused by immunoprecipitation. (D) AN0138 (rps601Δ myc-rps602⁺) (0 minutes) was transferred to EMM-N or added with 200 μM rapamycin (final concentration). The cells were incubated for the indicated time.

Fig. 5.

Identification of phosphorylation sites in Rps6. Extracts were subjected to immunoblotting with the indicated antibodies. (A) Ser235 and Ser236 in Rps6 are necessary for phosphorylation of the ribosomal protein. AN0098 (rps602Δ) cells harboring pREP1-myc (vector), pREP1-myc-rps601⁺ (rps601⁺), pREP1-myc-rps601^S235A,S236A (rps601SSAA), pREP1-myc-rps602⁺ (rps602⁺) or pREP1-myc-rps602^S235A,S236A (rps602SSAA) were treated as described in Fig. 2A. (B) AN0098 (rps602Δ) cells harboring pREP1-myc (vector), pREP1-myc-rps601⁺ (rps601⁺), pREP1-myc-rps601^S235A (rps601S235A), pREP1-myc-rps601^S236A (rps601S236A), pREP1-myc-rps602⁺ (rps602⁺), pREP1-myc-rps602^S235A (rps602S235A) or pREP1-myc-rps602^S236A (rps602S236A) were treated as described in Fig. 2A. (C) Cells of 972 (WT), AN0228 (rps601Δ rps602SSAA; #1) and AN0229 (rps601Δrps602SSAA; #2) were grown in YES at 30°C to exponential phase and harvested as +N (+). A half volume of 972 was washed and treated with EMM-N (a nitrogen-free version of EMM; −) for 30 minutes.

Fig. 6.

Characterization of the TORC1-dependent Rps6 phosphorylation. Extracts were subjected to immunoblotting with the indicated antibodies. (A) TORC1 activity is diminished by glucose depletion. JUp1204 (WT) and JUp1350 (tor2^L1310) were treated with EMM, EMM-N (a nitrogen-free version of EMM) or EMM-C (a carbon-depletion version of EMM) for 15 minutes at 30°C. (B,C) TORC1 activity is inhibited by oxidative and osmotic stresses. JUp1204 (WT) and JUp1350 (tor2^L1310P) were incubated in EMM containing 1 mM H₂O₂ (B) or 1 M KCl (C) for the indicated time points.

Fig. 7.

Rapamycin inhibits Rps6 phosphorylation by blocking TORC1. Extracts were subjected to immunoblotting with the indicated antibodies. (A) Cells of JUp1204 (0 minutes) were added with either 200 nM rapamycin or DMSO (vehicle) and incubated for the indicated time points. (B) JUp1204 was added with the indicated concentrations of rapamycin (rapa) and incubated for 40 minutes at 30°C. (C) Cells of 972 (WT), AN0066 (fkh1Δ) and AN0083 (tor2^S1837E) were added with 200 nM rapamycin and incubated for 40 minutes at 30°C.

Fig. 8.

Rapamycin leads to reduction of cell size in wild-type but not tor2^S1837E cells. Exponentially growing cells of 972 (WT) and AN0083 (tor2^S1837E) were diluted to OD₅₉₅=0.06 and then incubated in EMM with either DMSO (control) or 200 nM rapamycin at 30°C. After 14 hours incubation, the cells were stained with propidium iodide to analyze cellular DNA content and cell size (measured as forward scatter) by flow cytometry. The upper half shows a representative experiment. The green line overlaid on the profile (purple) of cell size from the rapamycin-treated cells indicates the profile outline for control DMSO-treated cells. Profile of DNA content from the wild-type cells that were starved for nitrogen source for 14 hours is shown at the bottom. The bar graph shows the average size as a mean FSC-H ± s.e.m. (n=4). **P<0.01.