Molecular Biology of the Cell Vol. 21, 3475–3486, October 1, 2010 The Rapamycin-sensitive Phosphoproteome Reveals That TOR Controls Protein Kinase A Toward Some But Not All Substrates Alexandre Soulard,* Alessio Cremonesi,* Suzette Moes, Fr e´de´ ric Schu¨ tz, Paul Jeno¨ , and Michael N. Hall Biozentrum, University of Basel, CH-4056 Basel, Switzerland Submitted March 3, 2010; Revised July 6, 2010; Accepted August 4, 2010 Monitoring Editor: Daniel Lew Regulation of cell growth requires extensive coordination of several processes including transcription, ribosome biogen- esis, 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. INTRODUCTION Cells regulate their growth in response to nutrients. To achieve this growth control, cells sense and transduce nutrient signals to coordinate several processes including transcription, ribo- some biogenesis, translation, nutrient transport and metabo- lism, and cell morphogenesis and autophagy. In Saccharomyces cerevisiae, the TOR (Target of Rapamycin) and cAMP-depen- dent protein kinase A (PKA) signaling pathways are the two major pathways that transduce nutrient signals to regulate cell growth (De Virgilio and Loewith, 2006; Santangelo, 2006; Soulard et al., 2009). TOR is found in two highly conserved and functionally distinct complexes corresponding to two effector signaling branches (Wullschleger et al., 2006). Rapamycin-sensitive TOR Complex 1 (TORC1) mediates temporal control of cell growth in response to nutrients by promoting anabolic pro- cesses such as translation and ribosomal protein (RP) gene expression and by antagonizing catabolic processes such as autophagy, ubiquitin-dependent protein degradation, and mRNA degradation (Crespo and Hall, 2002; Loewith et al., This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E10 – 03– 0182) on August 4, 2010. * These authors contributed equally to this work. Address correspondence to: Michael N. Hall (m.hall@unibas.ch). © 2010 A. Soulard et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc- sa/3.0). 2002; Reinke et al., 2004; De Virgilio and Loewith, 2006). In addition, TORC1 regulates lifespan (Kaeberlein et al., 2005). Rapamycin-insensitive TOR Complex 2 (TORC2) mediates spatial control of cell growth by regulating actin cytoskele- ton dynamics, ceramide metabolism, and cell wall integrity (De Virgilio and Loewith, 2006; Aronova et al., 2008; Cybulski and Hall, 2009). PKA in yeast is part of the RAS-cAMP signaling cascade that controls various growth-related processes in response to glucose (Santangelo, 2006). Like TORC1, the PKA path- way regulates translation, ribosome biogenesis, autophagy, stress responses, glucose metabolism, and lifespan (Santan- gelo, 2006). The PKA catalytic subunit is encoded by the three homologous and partly redundant genes TPK1, TPK2, and TPK3. The PKA regulatory subunit that controls PKA in response to cAMP is encoded by BCY1 (Cannon and Tatch- ell, 1987; Toda et al., 1987a,b). In the absence of glucose (i.e., a fermentable carbon source), two BCY1s bind two TPKs to form a catalytically inactive heterotetrameric complex. In the presence of glucose, adenylate cyclase is activated and pro- duces cAMP from ATP. cAMP in turn activates PKA by binding BCY1 and releasing it from TPK (Johnson et al., 1987; Santangelo, 2006). Furthermore, both TPK1 and BCY1 shuttle between the cytoplasm and the nucleus in growing cells, with BCY1 mainly in the nucleus and TPK1 primarily in the cytoplasm (Grifﬁoen et al., 2000, 2001; Schmelzle et al., 2004). TORC1 and PKA regulate common target proteins to ac- tivate or inhibit the same biological processes. For example, both TORC1 and PKA regulate the nuclear localization of SFP1 and CRF1, two transcription cofactors involved in RP gene expression (Jorgensen et al., 2004; Marion et al., 2004; Martin et al., 2004; Schawalder et al., 2004; Wade et al., 2004). Similarly, the stress-inducible transcription factors MSN2 3475