TORC2 and the AGC kinase Gad8 regulate phosphorylation of the ribosomal protein S6 in fission yeast

Abstract

TOR (Target Of Rapamycin) signalling coordinates cell growth and division in response to changes in the nutritional environment of the cell. TOR kinases form two distinct complexes: TORC1 and TORC2. In mammals, the TORC1 controlled S6K1 kinase phosphorylates the ribosomal protein S6 thereby co-ordinating cell size and nutritional status. We show that the Schizosaccharomyces pombe AGC kinase Gad8 co-immunoprecipitates with the ribosomal protein S6 (Rps6) and regulates its phosphorylation status. It has previously been shown that Gad8 is phosphorylated by TORC2. Consistent with this, we find that TORC2 as well as TORC1 modulates Rps6 phosphorylation. Therefore, S6 phosphorylation in fission yeast actually represents a read-out of the combined activities of TORC1 and TORC2. In contrast, we find that the in vivo phosphorylation status of Maf1 (a repressor of RNA polymerase III) specifically correlates with TORC1 activity.

Keywords: Gad8; Maf1; S. pombe; S6; S6K; TOR.

Fig. 1.. Rps6 co-immunoprecipitates with Gad8.

(A) Immunoprecipitation of Gad8.HA and wild type control cultures. A western blot probed with anti-HA antibody shows the specific Gad8.HA immunoprecipitation (left). The Brilliant blue stained NUPAGE gel used for analysis by mass spectrometry (right). Intense band indicated by arrow shows Gad8.HA immunoprecipitate. (B) Mass spectrometry data analysed by Scaffold^TM 3. The number of unweighted spectra and unique peptides for each protein are shown.

Fig. 2.. Phosphorylation of Rps6 is Gad8 and Tor1 dependent.

(A) WT, gad8.KD and gad8Δ were harvested at early exponential phase in minimal medium using glutamate or ammonium as the nitrogen source. The phosphorylated Rps6 was probed by anti-PAS antibody. Total Rps6 was detected by anti-S6 antibody, as a loading control. (B) Deletion of tor1 or the Rictor homologue ste20 diminished the phosphorylation of Gad8. WT, tor1Δ and ste20Δ strains were cultured in EMMG to early exponential phase. Gad8 was probed with anti-Gad8 antibodies and tubulin was used as loading control. (C) gad8Δ and tor1Δ down-regulates Rps6 phosphorylation. Cells were cultured in EMMG medium and the blots were probed as in (A). (D) Rps6 phosphorylation levels in WT cells were compared in glutamate and ammonium containing medium. (E) Psk1, but not Sck1 or Sck2 regulate Rps6 phosphorylation. (F) gad8Δ and psk1Δ were grown in EMMG and EMM2 and the Rps6 phosphorylation levels were compared. (G) Rapamycin inhibits Rps6 phosphorylation. WT and tor1Δ were grown in EMMG to early exponential phase and treated with 0.3 µg/ml rapamycin for 30 min.

Fig. 3.. Both TORC1 and TORC2 regulate Rps6 phosphorylation while Maf1 phosphorylation is TORC1 specific.

All cells were grown in EMMG (A) Phosphorylation of Rps6 is TORC1 and TORC2 dependent. WT and temperature sensitive TORC1 mutants mip1.310 (Raptor) and tor2.51 were cultured to exponential phase at permissive temperature (28°C). The cells were heat shocked at 37°C for 2 hr (left). Deletion of tor1, ste20 (Rictor) or sin1 reduced the phosphorylation level of Rps6 (right). (B) Gad8 S546 phosphorylation is TORC2 specific. All strains were treated as in (A) and the phosphorylated protein was detected by P-Gad8.S546 specific antibodies and total Gad8 by anti-Gad8 antibodies. (C) Maf1 phosphorylation is TORC1 but not TORC2 dependent. TORC1 and TORC2 mutants expressing Maf1.pk were treated as in (A). Maf1.pk was detected by anti-pk antibodies. (D) Rapamycin inhibits Maf1.pk phosphorylation. Cells were grown and treated with rapamycin, and Maf1.pk was detected as previously described. (E) Gad8 is not required for Maf1 phosphorylation. (F) Slower migrating Maf1.pk bands represent phosphorylation. When λ phosphatase was added to immunoprecipitated Maf1.pk, only the faster migrating non-phosphorylated Maf1.pk was observed.

Fig. 4.. TORC dependent substrates.

A schematic model of TOR signalling in fission yeast. TORC1 specific control of Maf1 phosphorylation is shown in green. TORC2 specific phosphorylation of Gad8 is shown in red. The ribosomal protein S6 is regulated by both TORC1 and TORC2 and the AGC kinases Gad8 and Psk1.