A. Soulard et al. was conﬁrmed by the observation that MPK1 deletion abol- ished the hyperaccumulation of glycogen provoked by a TOR1 deletion (Figure 6D). Conversely, and similar to rapa- mycin treatment, activation of the cell wall integrity path- way by SDS or caffeine (Levin, 2005) stimulated glycogen accumulation (Supplementary Figure 6D). Furthermore, in MPK1-deleted cells, phosphorylation of PKA sites in MAF1 and YPK3 was increased and rapamycin-induced dephos- phorylation of these sites was reduced (Figure 6, E and F). Thus, MPK1 appears to have a physiological role in TORC1- mediated control of PKA. Is SCH9 upstream of MPK1 in controlling phosphoryla- tion of BCY1 T129, as expected from the above ﬁndings? MPK1 was hyperphosphorylated in sch9 deletion and sch95A mutants, even when TORC1 was active (absence of rapamy- cin; Figure 5A and Supplementary Figure 6A). Furthermore, sch9 deletion mutants displayed enhanced resistance to zy- molyase, a cell wall– damaging agent (Supplementary Fig- ure 6B), suggesting that the cell wall integrity pathway was hyperactive in cells lacking SCH9. Thus, SCH9 appears to negatively control both BCY1 T129 phosphorylation and MPK1 activity, suggesting that SCH9 is indeed upstream of MPK1 in controlling BCY1 T129 phosphorylation. The above results taken together suggest that TORC1-SCH9 signaling activates PKA (at least toward some substrates) by inhibit- ing MPK1 direct phosphorylation of BCY1 T129. DISCUSSION To obtain insight into the shared regulation by PKA and TORC1 (see Introduction), we investigated the TORC1 phos- phoproteome. This revealed that PKA phosphorylation sites are a primary target of TORC1, suggesting that TORC1 is an upstream activator of PKA. However, many PKA sites were not affected by TORC1, suggesting that TORC1 activates PKA selectively rather than globally (see below). Thus, both models that account for the shared regulation by TORC1 and PKA appear to be valid. First, TORC1 is an upstream regulator of PKA toward some substrates, such as MAF1, YPK3, and KSP1, whose phosphorylation by PKA is TORC1- dependent. Second, TORC1 and PKA are in distinct but parallel pathways that converge on common target proteins or processes. This latter case includes proteins whose phos- phorylation by PKA is not TORC1 dependent. To investigate the molecular mechanism by which TORC1 controls PKA, we focused on the PKA negative regulatory subunit BCY1. We found that TORC1 inhibits phosphoryla- tion of T129 (and other sites) in BCY1. Phenotypes conferred by mutation of T129 to phosphomimetic aspartate suggest that T129 phosphorylation activates BCY1 and thereby inac- tivates PKA. Furthermore, we found that TORC1 inhibits BCY1 T129 phosphorylation by inhibiting the MAP kinase MPK1. TORC1 inhibits MPK1 via the S6K ortholog SCH9. Thus, we have deﬁned a pathway, consisting of TORC1- SCH9-MPK1-BCY1-PKA, by which TORC1 prevents inacti- vation of PKA. Although TORC1 phoshorylates and acti- vates SCH9 directly and MPK1 phosphorylates (T129) and activates BCY1 directly, it remains to be determined how SCH9 inhibits phosphorylation and activation of MPK1 (see below). How could regulation of BCY1 by TORC1 control PKA activity toward some but not all PKA substrates? On rapa- mycin treatment, BCY1 sequesters PKA in the nucleus (Schmelzle et al., 2004). This and our above ﬁndings suggest that phosphorylated BCY1 (at T129 and likely other sites) maintains PKA in the nucleus upon TORC1 inhibition and may thus control access of PKA to speciﬁc substrates. This 3484 control of PKA by BCY1 phosphorylation would be inde- pendent of PKA control by binding of cAMP to BCY1. This model is also supported by our observation that any mutation that disrupts the BCY1-TPK1 interaction, e.g., BCY1S145D or TPK1T241A/D/E, prevents nuclear accumula- tion of TPK1 upon rapamycin treatment (Supplementary Figure 7 and data not shown). Finally, this model is similar to a model proposed by Grifﬁoen et al. (2001, 2003), suggest- ing that BCY1 phosphorylation upon glucose limitation con- trols PKA localization and accessibility to substrates. An alternative model is that T129-phosphorylated BCY1 binds and inhibits only a speciﬁc pool of PKA. This separate pool of PKA could be due to differential modiﬁcation or localiza- tion of PKA by unknown factors. Different pools of PKA might be in part explained by the fact that there are three catalytic subunits of PKA (TPK1, TPK2, and TPK3) in yeast. Conﬁrmation of the above or other possible models will require further study. Grifﬁoen et al. (2001, 2003) showed that BCY1 is phosphor- ylated on two clusters of serines (CI and CII) in response to stress. Here we identiﬁed, in addition to CI and CII, a third cluster of serine and threonine residues (CIII) that are phos- phorylated upon rapamycin treatment. T129 in CIII is an important residue in the regulation of BCY1 and ultimately PKA. However, the mild phenotypes conferred by mutation of T129 to alanine or aspartate suggest that phosphorylation at this residue is not the sole event regulating BCY1 and PKA upon TORC1 inhibition. BCY1 is phosphorylated on at least 16 serine or threonine residues other than T129, includ- ing S74, S79, S83, T131, and S145. However, individual mu- tation of S74, S79, and S83 and T131 had no phenotypic consequence (data not shown). A phosphomimetic mutation of S145 resulted in BCY1 inactivation, suggesting that phos- phorylation of this site regulates BCY1, but the phosphory- lation of S145 was independent of TORC1 (Supplementary Figure 7). Further study will be required to determine the roles and kinase(s) for all the phosphorylation sites in BCY1. MPK1 is a MAP kinase in the PKC1/cell wall integrity pathway that is activated in response to several stresses including TORC1 inhibition (Krause and Gray, 2002; Torres et al., 2002; Levin, 2005). Earlier studies have reported ge- netic interactions between the cell wall integrity pathway and the PKA pathway. For example, activation of the cell wall integrity pathway suppresses the heat-shock sensitivity caused by hyperactive RAS-cAMP signaling, and the RHO1 GDP/GTP exchange factor ROM2, which acts early in the cell integrity pathway, is required for PKA-mediated stress response (Verna et al., 1997; Park et al., 2005; Kuranda et al., 2006). Our observation that MPK1 phosphorylates and acti- vates BCY1 is consistent with the above genetic interactions. Finally, we note that MPK1 is likely not the only kinase involved in PKA regulation by TORC1, as rapamycin-in- duced nuclear accumulation of PKA is not altered by loss of MPK1 (whereas BCY1 phosphorylation is) or presence of BCK1-20, a hyperactive MAPKKK BCK1 upstream of MPK1 (data not shown). Furthermore MPK1 deletion reduces but does not abolish rapamycin-induced dephosphorylation of MAF1 and YPK3. TORC1 directly phosphorylates and activates the protein kinase SCH9 (Urban et al., 2007). SCH9 was originally iden- tiﬁed as a multicopy suppressor of a PKA defect (Toda et al., 1988). Furthermore, SCH9 deletion severely decreases PKA- dependent phosphorylation of MSN2 and MAF1 (Trott et al., 2005; Huber et al., 2009; Lee et al., 2009). In agreement with these previous observations, we ﬁnd that SCH9 acts down- stream of TORC1 to maintain MPK1 inactive and PKA ac- tive. The molecular link between SCH9 and MPK1 is likely Molecular Biology of the Cell