TORC1 Regulates PKA through MPK1 Table 1. Proteins for which phosphorylation decreased at PKA consensus motif RRxS after rapamycin treatment Protein FRA1 GCN2 KCS1 KSP1 LHP1 MAF1 MKS1 SSD1 VTC2 YJL016W Phospho sitea S 56 (0.48 0.16) S 577 (0.18 0.02) S 537 (0.42) S 827 (0.27 0.01) S 883, S 884 (0.11) S 884 (0.40) S 19 (0.51 0.13) S 90 (0.25 0.08) S 209 (0.29) S 518 (0.43 0.18) S 164 (0.56 S 583 (0.24 S 350 (0.50 0.07) 0.03) 0.02) Descriptionb (SGD) Protein involved in negative regulation of transcription of iron regulon eIF2 kinase regulating translation in response to starvation Inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase Ser/thr protein kinase required for haploid ﬁlamentous growth RNA-binding protein required for maturation of tRNA and U6 snRNA precursors Negative regulator of RNA polymerase III in response to nutrients and stress Negative transcriptional regulator involved in Ras-cAMP and lysine biosynthetic pathways, and nitrogen regulation Maintenance of cellular integrity, interacts with components of the TOR pathway Subunit of the vacuolar transporter chaperone (VTC) complex involved in membrane trafﬁcking Putative protein of unknown function a For each protein the coordinate of the regulated phospho-site and the fold-increase the average ratio ( SD) are given in parentheses. No SD indicates that the phospho-sites were identiﬁed in only one experiment. b The description of the function of the protein is given according to the SGD with adaptation. whose phosphorylation was down-regulated upon rapa- mycin treatment, the top scoring target motif was RRxS (the phosphorylated residue is underlined; Figure 1C). This motif was underrepresented in the phosphopeptides whose phosphorylation was up-regulated (Figure 1C). The related motif RxxS appeared in both the up- and down-regulated phosphopeptides (Figures 1C). Finally, the two motifs SP and SxxS were also represented, but only in the up-regulated peptides. Examination of the rapamycin-sensitive phospho- proteome presented by Huber et al. (2009) revealed a similar bias toward RRxS sites for down-regulated phosphopeptides (Supplementary Table 9). Thus, RRxS is a primary target motif for TORC1-dependent phosphorylation. Importantly, RRxS/T is the consensus target motif specif- ically recognized and phosphorylated by PKA (Shabb, 2001; Budovskaya et al., 2005). Of the 55 proteins whose phosphor- ylation was decreased upon rapamycin-treatment, 10 were hypophosphorylated at this PKA target motif. The 10 pro- teins were FRA1, GCN2, KCS1, KSP1, LHP1, MAF1, MKS1, SSD1, VTC2, and YJL016W (Table 1). FRA1, KSP1, LHP1, MAF1, and SSD1 have been shown to be PKA targets in vitro (Budovskaya et al., 2005; Ptacek et al., 2005). MAF1 is phos- phorylated and negatively regulated by PKA and TORC1 in vivo (Moir et al., 2006; Huber et al., 2009; Lee et al., 2009; Wei et al., 2009). The localization of the protein kinase KSP1 is inﬂuenced by PKA (Bharucha et al., 2008), and GCN2, KCS1, KSP1, LHP1, MKS1, and SSD1 have been linked to TORC1 and/or PKA signaling either genetically or biochemically (SGD project; Saccharomyces Genome Database; http://www. yeastgenome.org/; Xie et al., 2005; Huber et al., 2009). Thus, TORC1 appears to mediate phosphorylation of PKA target sites. It is also important to note that many phosphorylated PKA target motifs in our phosphoproteome were not af- fected by rapamycin treatment (Supplementary Table 3). These included the PKA sites in the three well-known PKA targets MSN2, BCY1, and NTH1 (Supplementary Table 3). Thus, TORC1 appears to mediate phosphorylation of only a subset of PKA target sites. TORC1 Activates PKA toward Certain Substrates The above ﬁndings suggested that TORC1 activates PKA at least toward a subset of substrates. To investigate whether Vol. 21, October 1, 2010 TORC1 indeed activates PKA, we performed targeted anal- yses of individual PKA substrates. In particular, we inves- tigated the effect of rapamycin treatment on the in vivo steady-state phosphorylation of the known or suspected PKA substrates MAF1, YPK3, and KSP1. We probed the phosphorylation of these PKA substrates with an antibody that speciﬁcally recognizes a phosphorylated consensus PKA target motif (RRxS/T). This antibody is hereafter re- ferred to anti-RRxS/T. Consistent with our phosphoproteome analysis and as reported previously, we observed that MAF1 phosphoryla- tion at PKA consensus sites was strongly reduced upon rapamycin treatment (Figure 2A). Although this could sug- gest that TORC1 activates PKA toward MAF1, this interpre- tation is complicated by the fact that the TORC1 effector SCH9, an AGC kinase family member like PKA, can also directly phosphorylate PKA sites in MAF1 (Huber et al., 2009; Lee et al., 2009). To further investigate whether TORC1 indeed activates PKA toward MAF1, we used the cell-per- meable phosphodiesterase resistant analogue 8-Bromo- cAMP (Verma et al., 1988) to activate PKA in the absence of TORC1 and SCH9 activity. As expected, PKA inactivation upon glucose starvation resulted in a strong reduction in MAF1 phosphorylation at PKA sites, and addition of 8-Br- cAMP to glucose-starved cells restored MAF1 phosphoryla- tion at PKA sites (Figure 2B and Supplementary Figure 2A). Treatment of cells with 8-Br-cAMP also counteracted the inhibitory effect of rapamycin on the phosphorylation of MAF1 at PKA sites (Figure 2C and Supplementary Figure 2B). These data suggest that TORC1 indeed activates PKA (in addition to SCH9) toward MAF1. We also examined phosphorylation of PKA sites in YPK3 and KSP1. YPK3 is an AGC kinase that is phosphorylated by PKA in vitro (Budovskaya et al., 2005; Ptacek et al., 2005). KSP1 is a serine/threonine kinase required during ﬁlamen- tous growth in response to nutrient limitation (Bharucha et al., 2008). KSP1 localization is inﬂuenced by PKA, and it is also a PKA substrate in vitro (Ptacek et al., 2005; Bharucha et al., 2008). Similar to MAF1 and again consistent with our phosphoproteome analysis, phosphorylation of both KSP1 and YPK3 at consensus PKA site(s) decreased upon rapa- mycin treatment (Figure 2A). Similar to MAF1, but to a lesser extent, activation of PKA with 8-Br-cAMP counter- 3479