A. Soulard et al. and MSN4 are dephosphorylated and rapidly accumulate in the nucleus upon TORC1 or PKA inhibition (Gorner et al., 1998; Beck and Hall, 1999). Inhibition of either TORC1 or PKA also leads to the dephosphorylation, nuclear accumu- lation, and activation of MAF1, a repressor of RNA poly- merase III (Moir et al., 2006; Huber et al., 2009; Lee et al., 2009). Seemingly conﬂicting models have been proposed to account for the overlapping regulation by TORC1 and PKA. According to one model, TORC1 and PKA are in distinct but parallel pathways that converge on common target proteins or processes. For example, TORC1 and PKA independently regulate MSN2 and ATG13 phosphorylation (Santhanam et al., 2004; Stephan et al., 2009). Alternatively, several ﬁndings suggest that TORC1 is an activator of PKA, placing the two kinases in the same signaling pathway. First, hyperactiva- tion of the RAS-cAMP-PKA pathway suppresses a TORC1 deﬁciency, whereas down-regulation of the PKA pathway leads to increased rapamycin sensitivity (Schmelzle et al., 2004; Zurita-Martinez and Cardenas, 2005). Second, TORC1 controls RP gene expression via PKA (Martin et al., 2004; Schmelzle et al., 2004). Third, the AGC kinase SCH9, which is a direct substrate of TORC1, was initially identiﬁed as a multicopy suppressor of mutations that reduce PKA activity (Toda et al., 1988; Urban et al., 2007). Fourth, inactivation of TORC1 by rapamycin causes a rapid accumulation of the PKA catalytic subunit TPK1 in the nucleus (Schmelzle et al., 2004). All together, the above ﬁndings are inconsistent with a single model to explain the common regulation by TORC1 and PKA. The cell wall integrity pathway consists of the RHO1- PKC1-BCK1-MKK1/2-MPK1 signaling cascade. This path- way allows yeast cells to adapt their growth and shape in response to cell wall stress (Levin, 2005). Recently, several reports have linked the cell wall integrity pathway to both TORC1 and PKA signaling. For example, inactivation of TORC1 leads to the phosphorylation and activation of the mitogen-activated protein (MAP) kinase MPK1, and several components of the cell wall integrity pathway show genetic interactions with components of the PKA pathway (Verna et al., 1997; Krause and Gray, 2002; Torres et al., 2002; Park et al., 2005; Kuranda et al., 2006). However, a molecular link between TORC1 and PKA via the cell wall integrity pathway is unknown. To better understand the relationship between TORC1 and PKA, we further investigated the effect of TORC1 inhi- bition on PKA. We observed, by large-scale quantitative phosphoproteome analysis and targeted experiments, that a distinct subset of PKA target sites become dephosphorylated upon rapamycin treatment. To investigate the mechanism by which TORC1 controls PKA, we focused on the PKA regulatory subunit BCY1. TORC1 regulates PKA by inhibit- ing phosphorylation of many sites in BCY1 between a dimerization domain and the cAMP-binding domain. TORC1 inhibits BCY1 phosphorylation via SCH9 and the MAP kinase MPK1. Thus, we deﬁne a signaling cascade in which TORC1 is upstream of PKA toward at least some substrates. MATERIALS AND METHODS Strains, Plasmids, Media, and Genetic Manipulations The S. cerevisiae strains and plasmids used in this study are listed in Supple- mentary Tables 1 and 2, respectively. All strains from our laboratory are isogenic with TB50. Yeast manipulations, including cell cultures, sporulation, tetrad dissections, and genetic techniques, were carried out essentially as described by Guthrie and Fink (1991). The media were YPD (1% yeast extract, 1% peptone, 2% dextrose, plus 2% agar for solid media) and minimal syn- thetic medium (SD; yeast nitrogen base at 6.7 g/l, 2% dextrose, relevant amino 3476 acids and 2% agar for plates). YP medium was used for the glucose depletion experiment. SDS in YPD was 0.01%. Cells were treated with rapamycin at 200 ng/ml ﬁnal concentration (added from a 1 mg/ml stock solution in 90% ethanol–10% Tween20) and/or 8-Bromo-cAMP at 5 mM ﬁnal concentration (from 250 mM stock solution in water). Before 8-Bromo-cAMP treatment, cells were centrifuged and resuspended in 5 ml of the required medium. In most experiments, yeast strains carrying a plasmid were precultured in SD medium lacking the corresponding amino acids for plasmid maintenance and subse- quently diluted into YPD medium. Cells were then grown for 4 –5 h (to OD600 about 0.8) before treatment. For SILAC labeling, yeast cells were grown in SD medium containing 13C6-arginine and 13C6,15N2-l-lysine (Cambridge Isotope Laboratories, Andover, MA). Transformations of S. cerevisiae cells were ac- cording to the lithium acetate method with single-strand carrier DNA and dimethyl sulfoxide (DMSO; Hill et al., 1991). All deletion and genomically tagged strains were constructed by PCR-based gene targeting (Wach et al., 1994). One-step site directed mutagenesis was performed as described previ- ously (Zheng et al., 2004). Fluorescence Microscopy Fluorescence microscopy and indirect immunoﬂuorescence on whole ﬁxed cells was performed as described previously (Schmelzle et al., 2004). Protein Extraction, Immunoprecipitation, Western Blotting, and Phosphatase Treatment Protein extracts were prepared from exponentially growing cells. At least 25 OD cells were broken in a bead beater in lysis buffer (PBS containing 0.5% Tween 20 and 10% glycerol) containing 1 Roche protease inhibitor cocktail, 1 mM PMSF, and phosphatase inhibitors (10 mM NaF, 10 mM NaN3, 10 mM p-nitrophenyl phosphate, 10 mM sodium pyrophosphate, and 10 mM -glyc- erophosphate). hemagglutinin (HA)-, MYC-, or tandem afﬁnity puriﬁcation (TAP)-tagged proteins were immunoprecipitated from cell extracts with pro- tein A-Sepharose beads coupled to anti-HA antibody (12C2A), protein G- Sepharose beads coupled to anti-MYC antibody (1-9E10.2), or human IgG- Sepharose beads (GE Healthcare, Waukesha, WI), respectively. For the phosphatase experiment, beads were further washed with phosphatase buffer (50 mM Tris-Cl, pH 7.5, 1 mM MgCl2), resuspended in 100 l phosphatase buffer, and incubated for 10 min at 30°C. Twenty units of calf intestine alkaline phosphatase was added, and the reaction was incubated for 15 min 30°C. For the gel-shift assay, the acrylamide-bisacrylamide ratio for SDS- PAGE was changed to 172:1. The primary antibodies for Western blotting were as follows: anti-HA (mouse monoclonal, 12C2A), anti-MYC (mouse monoclonal, 1-9E10.2), anti-protein A (rabbit polyclonal, Sigma-Aldrich, St. Louis, MO), anti-BCY1 (goat polyclonal, Bcy1 [yN19] sc-6765, Santa Cruz Biotechnology, Santa Cruz, CA), anti-RxxS/T (rabbit polyclonal phospho- [Ser/Thr] PKA substrate antibody no. 9621, Cell Signaling, Beverly, MA), anti-RRxS/T (phospho-PKA Substrate [RRXS/T; 100G7E], rabbit mAb no. 9624, Cell Signaling), anti-MPK1 (goat polyclonal, Mpk1 [yC-20] sc-6803, Santa Cruz Biotechnology), anti-phospho MPK1 (rabbit polyclonal, phospho- p44/42 MAPK [Thr202/Tyr204] antibody no. 9101, Cell Signaling). (The phosphorylated residues are underlined.) Identiﬁcation of Phosphorylation Sites in BCY1 Wild-type TB50a cells or cells expressing N-terminally H- tagged BCY1 (SA094) were grown in 500 ml YPD to OD600 nm of 1 and treated with rapamycin (200 ng/ml) or drug vehicle for 90 min. Cells were broken with glass beads with RIPA buffer (25 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Nonidet NP-40, 1% Na-deoxycholate) supplemented with 1 Roche protease inhibitor cocktail and phosphatase inhibitors (10 mM NaF, 0.2 M okadaic acid, and 20 nM calyculin A). HA-tagged BCY1 was immunoprecipitated from 40 mg of total protein extract with 1 l of monoclonal anti-HA antibody (HA.11; Covance, Princeton, NJ) at 4°C for 18 h followed by incubation with 100 l of Dynabeads protein G (Invitrogen, Carlsbad, CA) for 4 h at 4°C. The beads were washed and HA-BCY1 was eluted from the beads with 10 l 3 M urea, and 20 mM DTT for 10 min at room temperature. HA-BCY1 was digested with trypsin for 18 h at 37°C. Proteolysis was stopped with 8.4 l acetic acid, and the peptides were dried in a Speed Vac. Phosphopeptides were enriched with TiO2-magnetic beads (Perkin Elmer-Cetus, Waltham, MA) according to the man- ufacturer’s instructions. The ﬁnal peptides were analyzed by capillary liquid chromatography tandem MS (LC/MS/MS) using a 300SB C-18 column (0.3 50 mm; Agilent Technologies, Basel, Switzerland) connected on line to an LTQ-Orbitrap hybrid instrument (Thermo Finnigan, San Jose, CA; see below). Glycogen Staining Staining of intracellular glycogen was performed with iodine vapor as pre- viously described (Barbet et al., 1996). MPK1 Kinase Assay Yeast cells expressing HA-tagged MPK1 were grown in 500 ml YPD to an OD600 nm of 1 and then treated for 30 min with rapamycin or drug vehicle. Protein extraction, immunoprecipitation, and in vitro kinase assays were Molecular Biology of the Cell