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. 2010 Oct 1;21(19):3475-86.
doi: 10.1091/mbc.E10-03-0182. Epub 2010 Aug 11.

The Rapamycin-Sensitive Phosphoproteome Reveals That TOR Controls Protein Kinase A Toward Some but Not All Substrates

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Free PMC article

The Rapamycin-Sensitive Phosphoproteome Reveals That TOR Controls Protein Kinase A Toward Some but Not All Substrates

Alexandre Soulard et al. Mol Biol Cell. .
Free PMC article

Abstract

Regulation of cell growth requires extensive coordination of several processes including transcription, ribosome biogenesis, translation, nutrient metabolism, and autophagy. In yeast, the protein kinases Target of Rapamycin (TOR) and protein kinase A (PKA) regulate these processes and are thereby the main activators of cell growth in response to nutrients. How TOR, PKA, and their corresponding signaling pathways are coordinated to control the same cellular processes is not understood. Quantitative analysis of the rapamycin-sensitive phosphoproteome combined with targeted analysis of PKA substrates suggests that TOR complex 1 (TORC1) activates PKA but only toward a subset of substrates. Furthermore, we show that TORC1 signaling impinges on BCY1, the negative regulatory subunit of PKA. Inhibition of TORC1 with rapamycin leads to BCY1 phosphorylation on several sites including T129. Phosphorylation of BCY1 T129 results in BCY1 activation and inhibition of PKA. TORC1 inhibits BCY1 T129 phosphorylation by phosphorylating and activating the S6K homolog SCH9 that in turn inhibits the MAP kinase MPK1. MPK1 phosphorylates BCY1 T129 directly. Thus, TORC1 activates PKA toward some substrates by preventing MPK1-mediated activation of BCY1.

Figures

Figure 1.
Figure 1.
Quantitative analysis of the rapamycin-sensitive phosphoproteome by SILAC. (A) Schematic overview of the experimental approach for the identification of TORC1 regulated phosphorylation sites in S. cerevisiae. Two yeast cultures (strain YPJ2) were metabolically labeled with 12C6-14N2-lysine/12C6-arginine (light) or 13C6-15N2-lysine/13C6-arginine (heavy). The heavy culture was treated for 15 min with rapamycin. Extracts were mixed in a 1:1 ratio and separated by preparative SDS-PAGE. The gel was horizontally sliced into 16 sections and in-gel–digested, and phosphopeptides were IMAC-enriched and analyzed in an LTQ-Orbitrap. (B) Merged data from the four experiments revealed 972 phosphoproteins, corresponding to 2487 unique phosphopeptides and 2607 unique phosphosites. (C) Analysis by Motif-X of all regulated phosphopeptides.
Figure 2.
Figure 2.
Influence of TORC1 inhibition on PKA substrate phosphorylation in vivo. (A) Rapamycin affects the phosphorylation state of different PKA substrates. Yeast strains expressing MAF1-HA (SA148), YPK3-HA (SA216), or KSP1-HA (SA219) were grown to exponential phase and treated for the indicated time with 200 ng/ml rapamycin (+) or drug vehicle (−). After protein extraction each tagged protein was immunoprecipitated and analyzed by Western blot for total protein (tot) with anti-HA antibody and for phosphorylation at PKA site (PKA) with anti-RRxS/T antibody. TB50a was used for mock control. (B) The yeast strain SA148 expressing MAF1-HA was grown to exponential phase in YPD and then shifted or not to YP for 2 h. Aliquots of cells were then treated for 30 min with 5 mM of 8-Br-cAMP. MAF1-HA was immunoprecipitated from total cell lysate and analyzed as described in A. TB50a was used as mock control. For each condition, the phosphorylation at PKA sites was quantified and normalized against the level of total protein and expressed as a percentage [Phos/tot (%)]. (C) The yeast strain SA148 expressing MAF1-HA was grown to exponential phase in YPD and then pretreated or not with 5 mM of 8-Br-cAMP during 5 min. Aliquots of cells were then treated for 30 min with 200 ng/ml rapamycin. Phosphorylation of MAF1-HA was analyzed as in A and B. TB50a was used for mock control. Phosphorylation was quantified as in B [Phos/tot (%)]. (D) The yeast strain SA216 expressing YPK3-HA was treated and analyzed as in C.
Figure 3.
Figure 3.
The PKA regulatory subunit BCY1 is highly phosphorylated upon TORC1 inhibition. (A) Exponentially growing cells expressing HA-BCY1 from a plasmid (SA094) or not (TB50a) were treated for 2 h with drug vehicle or rapamycin at 200 ng/ml. Total proteins were extracted and HA-BCY1 was immunoprecipitated. Aliquots of IPs belonging to the rapamycin treated samples were further treated with phosphatase (PPase) or not, in presence of phosphatase inhibitors (PPi) or not. BCY1 phosphorylation was observed by gel-shift assay followed by Western blotting with an anti-HA antibody. (B) Graphical representation of the phosphorylated amino acids identified in BCY1 by phosphopeptide enrichment and MS. CII/III, cluster II/III. (C) Similar to A except that yeast cells were treated with rapamycin for 45 min (Rap). Total BCY1 was followed by Western blot with anti-BCY1 (BCY1) antibody and the different phosphorylated form of BCY1 with anti-RRxS/T antibody [(BCY1-P (S145)] and anti-RxxS/T antibody [BCY1-P (T129)], respectively. (D) The procedure used in B was used to analyze rapamycin-induced phosphorylation of HA-BCY1 in wild-type (WT = TB50a), TOR1 deletion (tor1Δ = AN9-2a), and rapamycin resistant TORs (TORRR = TOR1-1 TOR2-1 = RL206-4D) yeast strain carrying pTS137 (panel IP). In parallel, total (MPK1) and activated MPK1 (MPK1-P) were visualized by Western blot on the total cell lysates used for the HA-BCY1 IPs (panel total). (E) Wild-type cells (WT = SA094) expressing HA-BCY1 (pTS137) were treated with 200 ng/ml rapamycin (Rap) or shifted to YP medium lacking glucose (−Glu) for the indicated time. Total (HA-BCY1) and phosphorylated BCY1 [BCY1-P (T129)] were followed as in D.
Figure 4.
Figure 4.
BCY1 T129 phosphorylation after rapamycin treatment lowers PKA activity. (A) The yeast strains expressing wild-type (WT) or different mutants of HA-BCY1 at threonine 129 (T129A, T129D) were grown to exponential phase and treated or not with rapamycin (200 ng/ml) for 45 min. After total protein extraction, HA-BCY1 was immunoprecipitated. Total BCY1 and phosphorylated BCY1 at S145 and T129 were detected by Western blot with the corresponding antibodies. (B) The yeast strains used in A were grown to exponential phase. A cell quantity corresponding to 1.0 OD600 nm was serially 10-fold diluted and spotted on YPD plates containing DMSO or 5 ng/ml rapamycin. Plates were then incubated at 30°C for 3–5 d. (C) Glycogen accumulation was tested by iodine staining after 5-h rapamycin or drug vehicle treatment of yeast strains expressing the wild-type (BCY1 = SA094) and mutated form of BCY1 at threonine 129 (T129A and T129D).
Figure 5.
Figure 5.
TORC1 inhibits MPK1 and BCY1 T129 phosphorylation through the AGC kinase SCH9. (A) To follow the influence of SCH9 on BCY1 T129 and MPK1 phosphorylation, the yeast strains SA094 (WT) and SA135 (sch9) expressing HA-BCY1 were grown to exponential phase and treated 45 min with rapamycin (+) or drug vehicle (−) before total protein extraction. HA-BCY1 was immunoprecipitated (IP) and analyzed by Western blot with anti-HA (HA-BCY1) and anti-RxxS/T [BCY1-P (T129)] antibodies (IP panel). Total (MPK1) and activated MPK1 (MPK1-P) were visualized in total cell lysate by Western blot with the corresponding antibodies (panel total). (B) Effect of SCH9 deletion on PKA-dependent phosphorylation of MAF1. The yeast strains SA148 (MAF1-HA SCH9) and SA232 (MAF1-HA sch9) were grown to exponential phase and treated for 30 min with rapamycin (+) or drug vehicle (−) before total protein extraction. MAF1-HA was immunoprecipitated and analyzed by Western blot with anti-HA [MAF1 (tot)] and anti-RRxS/T [MAF1-P (PKA)] antibodies. For each condition, the phosphorylation at PKA sites was quantified, normalized against the level of total protein, and expressed as percentage [Phos/tot (%)]. (C) The rapamycin-dependent localization of TPK1-HA (TPK1) was followed by immunofluorescence of yeast strain SA20X carrying a plasmid expressing wild-type SCH9 (WT = pJU675), no SCH9 (sch9 = pRS416), or the mutant sch9-5A (sch9-5A = pJU822). Cells were treated 30 min with rapamycin (+) or drug vehicle (−). TPK1-HA was localized by the use of an anti-HA antibody. The nucleus was observed by DAPI staining (DNA).
Figure 6.
Figure 6.
BCY1 T129 phosphorylation is mediated by activation of the MAP kinase MPK1. (A) Yeast strains wild type (MPK1 = TB50a) or deleted (mpk1 = TS45-1a) for MPK1 and expressing HA-BCY1 from a plasmid (pTS137) were treated for 45 min with 200 ng/ml rapamycin (+) or drug vehicle (−). After protein extraction, HA-BCY1 was immunoprecipitated and analyzed by Western blot using anti-HA (HA-BCY1), anti-RRxS/T [BCY1-P (S145)], and anti-RxxS/T (BCY1-P (T129)) antibodies. TB50a was used as a mock control. (B) Total (HA-BCY1) and phospho-T129 BCY1 [BCY1-P (T129)] were analyzed as in A after 45-min treatment with rapamycin (200 ng/ml), SDS (0.01%), or drug vehicle of wild-type strain (WT = TB50a) or of a strain expressing an hyperactive form of BCK1 (BCK1-20) from a plasmid (pRS316::BCK1-20; panel IP). In parallel, total MPK1 (MPK1) and active phosphorylated MPK1 (P-MPK1) were analyzed by Western blot on the total extracts used for HA-BCY1 IPs with the corresponding antibodies (panel total). (C) In vitro kinase assay. MPK1-HA was immunoprecipitated from the yeast strain TS99-5c treated with rapamycin (+) or drug vehicle (−) for 45 min. The assay was performed as describe in Materials and Methods in presence of 1 μg of recombinant GST-BCY1 expressed from E. coli. Total and phosphorylated proteins were visualized by Coomassie staining (Coomassie) and autoradiography (32P), respectively. An aliquot of purified MPK1-HA was conserved for analysis of total (MPK1) and activated MPK1 (MPK1-P) by Western blot as above (WB). The level of BCY1 phosphorylation was quantified by densitometric analysis (below panel 32P). MBP, a commonly used MPK1 in vitro substrate, was used as a positive control (data not shown) (D) Glycogen accumulation was tested by iodine staining after 5-h rapamycin or drug vehicle treatment of yeast strains wild type (WT = SA180) or deleted for MPK1 (mpk1 = SA183), for TOR1 (tor1 = SA181), or for both genes (tor1 mpk1 = SA185). (E) Effect of MPK1 deletion on PKA-dependent phosphorylation of MAF1. Yeast strains containing MAF1-HA and expressing (MPK1; strain SA148) or lacking MPK1 (mpk1; strain SA233) were grown to exponential phase and treated for the indicated time with rapamycin or drug vehicle before total protein extraction. MAF1-HA was immunoprecipitated and analyzed by Western blot with anti-HA [MAF1 (tot)] and anti-RRxS/T (MAF1-P (PKA)) antibodies. MAF1 phosphorylation at PKA sites was quantified as described in Figure 1 [Phos/tot (%)]. (F) Effect of MPK1 deletion on PKA-dependent phosphorylation of YPK3. Yeast strains containing YPK3-HA and expressing (MPK1; strain SA234) or lacking MPK1 (mpk1; SA235) were analyzed as in E except that the cells were treated with rapamycin (+) or drug vehicle (−) for 45 min.

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References

    1. Aronova S., Wedaman K., Aronov P. A., Fontes K., Ramos K., Hammock B. D., Powers T. Regulation of ceramide biosynthesis by TOR complex 2. Cell Metab. 2008;7:148–158. - PMC - PubMed
    1. Barbet N. C., Schneider U., Helliwell S. B., Stansfield I., Tuite M. F., Hall M. N. TOR controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell. 1996;7:25–42. - PMC - PubMed
    1. Beck T., Hall M. N. The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature. 1999;402:689–692. - PubMed
    1. Bharucha N., Ma J., Dobry C. J., Lawson S. K., Yang Z., Kumar A. Analysis of the yeast kinome reveals a network of regulated protein localization during filamentous growth. Mol. Biol. Cell. 2008;19:2708–2717. - PMC - PubMed
    1. Budovskaya Y. V., Stephan J. S., Deminoff S. J., Herman P. K. An evolutionary proteomics approach identifies substrates of the cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA. 2005;102:13933–13938. - PMC - PubMed

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