Psk1, an AGC kinase family member in fission yeast, is directly phosphorylated and controlled by TORC1 and functions as S6 kinase

Abstract

Target of rapamycin (TOR), an evolutionarily conserved serine/threonine protein kinase, plays pivotal roles in several important cellular processes in eukaryotes. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1), which includes Tor2 as a catalytic subunit, manages the switch between cell proliferation and differentiation by sensing nutrient availability. However, little is known about the direct target of TORC1 that plays key roles in nutrient-dependent TORC1 signaling in fission yeast. Here we report that in fission yeast, three AGC kinase family members, named Psk1, Sck1 and Sck2, which exhibit high homology with human S6K1, are phosphorylated under nutrient-rich conditions and are dephosphorylated by starvation conditions. Among these, Psk1 is necessary for phosphorylation of ribosomal protein S6. Furthermore, Psk1 phosphorylation is regulated by TORC1 in nutrient-dependent and rapamycin-sensitive manners in vivo. Three conserved regulatory motifs (the activation loop, the hydrophobic and the turn motifs) in Psk1 are phosphorylated and these modifications are required for Psk1 activity. In particular, phosphorylation of the hydrophobic motif is catalyzed by TORC1 in vivo and in vitro. Ksg1, a homolog of PDK1, is also important for Psk1 phosphorylation in the activation loop and for its activity. The TORC1 components Pop3, Toc1 and Tco89, are dispensable for Psk1 regulation, but disruption of pop3(+) causes an increase in the sensitivity of TORC1 to rapamycin. Taken together, these results provide convincing evidence that TORC1/Psk1/Rps6 constitutes a nutrient-dependent signaling pathway in fission yeast.

Fig. 1.

Nitrogen source-dependent phosphorylation of S6K homologs in fission yeast. (A) A phylogenetic tree of fission yeast homologs of human S6K1 was generated using the ClustalW program. (B) Cells of strains JUp1204 (non-tagged), AN0151 (Sck1-3HA), AN0176 (Gad8-3HA), AN0153 (Sck2-3HA) and AN0180 (Psk1-13myc) were cultured in EMM, then washed and starved in EMM-N for 1 hour. After the starvation, ammonium (final concentration, 0.5%; +) or water (−) was added to cells and they were incubated for 30 minutes. (C) Protein extracts from ammonium-stimulated cells as described in B were incubated with λ-phosphatase in the presence or absence of its inhibitors. (B,C) Protein extracts were subjected to immunoblotting with the indicated antibodies.

Fig. 2.

Identification of Psk1 as the major kinase to phosphorylate Rps6. (A–C) Proteins were probed with the indicated antibodies. (A) JUp1204 (WT), AN0170 (sck1Δ), AN0203 (sck2Δ) and AN0133 (psk1Δ) cells were cultured in EMM (+), washed, and then incubated in EMM-N for 30 minutes (−). (B) JUp1204 (WT), AN0129 (rps601Δmyc-rps602) and AN0168 (psk1Δrps601Δmyc-rps602) cells were washed and incubated in EMM with (+) or without (−) ammonium for 20 minutes. (C) AN0216 (T415E) and AN0219 (K120A) cells were grown in EMM and harvested. Myc-tagged Psk1 was immunoprecipitated and an in vitro Psk1 kinase assay was carried out using GST–Rps602 proteins as substrates, as described in Materials and Methods. Phosphorylation of Rps602 was evaluated by immunoblotting. The arrowhead denotes a band corresponding to phosphorylated Rps602 probed with the PAS antibody.

Fig. 3.

Psk1 is a downstream target of TORC1. (A–C) Proteins were probed with the indicated antibodies. (A) AN0182 (tor2⁺) and AN0184 (tor2^L1310P) cells (0 minute) were washed and incubated in EMM-N for the indicated times. (B) AN0180 (WT), AN0217 (tor2-ts6) and AN0218 (tor2-ts10) cells were grown in EMM at 25°C. For treatment at the non-permissive temperature, cell culture was shifted to 35°C for 1 hour before cells were transferred to pre-warmed EMM-N. After incubation in EMM-N for 1 hour at either 25°C or 35°C, ammonium was added (0 minutes) to cells to 0.5% and incubated for 15 minutes. (C) AN0181 (tor2⁺) and AN0185 (tor2^S1837E) cells were treated with 200 nM rapamycin for the indicated times. (D) In vitro kinase assay of Tor2 was carried out as described in Materials and Methods. The gel was dried and autoradiographed (the right panel). The left panel (input) shows the Coomassie Brilliant Blue staining. Flag-Tor2 represents the immunoblotting of the immunoprecipitated Flag-Tor2 protein.

Fig. 4.

Effect of gene disruptions of the TORC1 components on phosphorylation of the TORC1 downstream factors. (A–C) Proteins were probed with the indicated antibodies. (A) AN0179 (WT), AN0233 (pop3Δ), AN0237 (toc1Δ) and AN0238 (tco89Δ) cells in EMM (+) were washed and cultured in EMM-N for 15 minutes (−). (B) Cells as described in A were treated with 150 nM rapamycin (rapa) for the indicated times. (C) AN0179 (WT) and AN0233 (pop3Δ) cells were cultured with the indicated concentrations of rapamycin for 1 hour. For B and C the relative reduction of Psk1 phosphorylation (%) was estimated by densitometry using ImageJ software and normalized to tubulin expression. The amount of Psk1 phosphorylation in wild type before addition of rapamycin (0 minutes) was set as 100.

Fig. 5.

Role of the predicted phosphorylation sites of Psk1 in its kinase activity. (A) Schematic diagram of Psk1 that has a kinase domain in the middle region and an AGC-kinase C-terminal domain. The predicted amino acid sequence indicates Lys120 at the ATP-binding site, Ser248 in the activating loop (T-loop), Thr392 in the turn motif (TM) and Thr415 in the hydrophobic motif (HM), as shown in supplementary material Fig. S1. (B–F) Proteins were probed with the indicated antibodies. (B) AN0179 (WT), AN0219 (K120A), AN210 (S248A), AN211 (T392A) and AN0212 (T415A) cells were cultured in EMM (+). Another portion of the AN0179 culture was washed and cultured in EMM-N for 20 minutes. (C) AN0179 (WT), AN0213 (T392E) and AN0216 (T415E) cells in EMM (+) were washed and cultured in EMM-N for 20 minutes (−). (D) The left panels: AN0182 (WT) and AN0184 (L1310P) cells in EMM (+) were washed and cultured in EMM-N for 30 minutes. The right panels: AN0181 (WT) and AN0185 (S1837E) in EMM were treated with DMSO (−) or 200 nM rapamycin (+) and incubated for 1 hour. Psk1-13myc was immunoprecipitated and subjected to immunoblotting. (E) In vitro phosphorylations of GST–Psk1 proteins as indicated by Tor2 was carried out as described in Fig. 3D. (F) AN0179 (WT), AN210 (S248A), AN211 (T392A) and AN0212 (T415A) cells were cultured in EMM (+). Another portion of the AN0179 culture was washed and cultured in EMM-N for 20 minutes.

Fig. 6.

Ksg1, a PDK1 homolog, is required for phosphorylation and activity of Psk1. (A) Thermal treatment of AN0180 (WT), AN0243 (ksg1-ts208) and AN245 (ksg1-ts358) cells was carried out as described in Fig. 3B. After incubation of cells in EMM-N for 1 hour at 25°C or 35°C (0 minute), ammonium was added to 0.5% and the cells were incubated for a further 15 minutes. Proteins were probed with the indicated antibodies. (B) In vitro phosphorylation of GST–Psk1 proteins, as indicated, by GST–Ksg1 was carried out as described in the Materials and Methods. The gel was dried and autoradiographed (upper panel). The lower panel (input) shows the Coomassie Brilliant Blue staining.

Fig. 7.

The TORC1 signaling responds to the availability of glucose and glutamine. (A–D) Proteins were probed with the indicated antibodies. (A) AN0179 cells were washed and incubated in EMM, EMM low glucose (Low G), or EMM+2-DG for 15 minutes. (B) AN0180 (0 minute) cells were washed and incubated with either EMM low glucose (LowG) or EMM-N with DMSO or cycloheximide (CHX; 50 µg/ml) for the indicated times. (C) After incubation in EMM-N for 1 hour (−), AN0179 cells were incubated in EMM (+) or EMM containing either 20 mM glutamic acid (Glu), glutamine (Gln), proline (Pro) or leucine (Leu) instead of ammonium for 15 minutes. (D) Left panel: AN0179 cells were incubated in EMM in the absence or presence of 5 or 10 mM L-methionine sulfoximine (MSX) for 30 minutes or in EMM plus 20 mM glutamine instead of ammonium (Gln) with 5 mM MSX. Right panel: AN0179 cells were transferred to EMM (+NH₄) or EMM plus 20 mM glutamine (Gln) with 5 mM MSX and incubated for the indicated times.

Fig. 8.

Sck1 and Sck2 might also be partly involved in TORC1 signaling. (A) In vitro phosphorylation of Sck1 and Sck2 by Tor2 carried out as described in Fig. 3D. (B) AN0163 (tor2⁺), AN0164 (tor2⁺), AN0166 (tor2^L1310P), AN0167 (tor2^S1837E) cells were nitrogen-starved and treated with rapamycin as described in Fig. 3A,C. Proteins were probed with the indicated antibodies. The relative reduction of Sck1 phosphorylation (%) was estimated by densitometry using ImageJ software, and normalized to tubulin expression. The amount of Sck1 phosphorylation in wild type before nitrogen starvation or treatment with rapamycin (0 minutes) was set as 100 (B).