A k c om SCH9 sch9 Rap 45’ - - + - + BCY1-P (T129) B BCY1 MPK1-P MPK1 C TPK1-HA DAPI Rap IP total MAF1-P (PKA) MAF1 (tot) k co SCH9 sch9 m Rap 30’ - - + - + Phos/tot (%) 100 20 37 29 SCH9 sch9 sch9-5A A. Soulard et al. and T129 to alanine or to phosphomimetic aspartate. Each mutant was then examined for rapamycin sensitivity and rapamycin-induced glycogen accumulation, two pheno- types that reﬂect PKA inactivity (Schmelzle et al., 2004; Zurita-Martinez and Cardenas, 2005). We note that mutation of T129 reduced recognition by anti-RxxS/T, whereas mu- tation of S74 and S83 had no effect on recognition by this antibody (Figure 4A and data not shown). Thus, the region containing T129 was the main epitope in BCY1 recognized by anti-RxxS/T (Figure 4A). The phosphomimetic mutation BCY1T129D conferred increased sensitivity to rapamycin and enhanced accumulation of glycogen upon TORC1 inhibition (Figure 4, B and C), suggesting that the BCY1T129D protein is a constitutive inhibitor of PKA and that T129 phosphoryla- tion activates BCY1 to inhibit PKA (at least toward some substrates). However, BCY1T129A behaved like wild-type BCY1, as determined by rapamycin sensitivity and glycogen accumulation. Furthermore, mutation of T129 to alanine or aspartate did not affect nuclear localization of BCY1, rapa- mycin-dependent nuclear localization of TPK1, BCY1-TPK1 interaction, or PKA-dependent phosphorylation of MAF1 (data not shown). These results suggest that phosphoryla- tion of a site(s) other than T129 can also activate BCY1. This other site is likely not S74, S83, or T131, as mutation of any one of these sites had no phenotypic consequence in our assays (data not shown). Taken together, our results suggest that TORC1 controls PKA (at least toward some substrates) by inhibiting phosphorylation of T129 and possibly other so far unknown sites in BCY1. TORC1 Controls PKA via SCH9 As reported recently, TORC1 directly phosphorylates and activates the serine/threonine kinase and S6K homolog SCH9 (Urban et al., 2007). Several reports have suggested that SCH9 is linked to PKA signaling. For example, the SCH9 gene was initially identiﬁed as a multicopy suppressor of mutations that reduce PKA activity, and SCH9 prevents glycogen accumulation and activates ribosomal protein gene expression similar to PKA (Toda et al., 1988; Urban et al., 2007). To determine whether TORC1 controls BCY1 (and PKA) via SCH9, we examined BCY1 T129 phosphorylation in SCH9-deleted cells. As shown in Figure 5A, BCY1 was hyperphosphorylated at T129 in the sch9 deletion mutant, even when TORC1 was active (absence of rapamycin). Con- sistent with this observation, PKA-dependent phosphoryla- tion of MAF1 was decreased in the sch9 mutant (Figure 5B). Cells deleted for SCH9 also displayed constitutive nuclear localization of the catalytic subunit TPK1 (Figure 5C). sch95A mutant cells, in which all residues required for SCH9 phos- phorylation and activation by TORC1 are mutated to ala- nine, also displayed constitutive nuclear accumulation of TPK1 (Figure 5C). Furthermore, in normal growth condi- tions, sch9 deletion and sch95A mutants displayed constitu- tive nuclear accumulation of the PKA target YAK1, similar to what is observed in wild-type cells treated with rapamy- cin (data not shown and Schmelzle et al., 2004). Together, these observations suggest that TORC1 inhibits BCY1 T129 phosphorylation and TPK1 nuclear accumulation through SCH9. The MAP Kinase MPK1 Directly Phosphorylates BCY1 upon TORC1 Inhibition The results described above suggested that, upon TORC1 inhibition, BCY1 is phosphorylated on T129 and thereby activated. What is the T129 kinase that is inhibited by TORC1? Two serine/threonine kinases, YAK1 and MCK1, have been shown to mediate BCY1 phosphorylation upon 3482 - + - + - + 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 expo- nential phase and treated 45 min with rapamycin ( ) or drug vehicle ( ) before total protein extraction. HA-BCY1 was immuno- precipitated (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 phosphoryla- tion 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 immu- noﬂuorescence 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). nutrient limitation or other stress (Werner-Washburne et al., 1991; Grifﬁoen et al., 2001). YAK1 is activated upon TORC1 inhibition and antagonizes the PKA pathway (Zappacosta et al., 2002; Martin et al., 2004; Deminoff et al., 2006). MCK1 is a glycogen synthase kinase 3 (GSK3) homologue involved in meiosis (Neigeborn and Mitchell, 1991; Kassir et al., 2006). In mutants deleted for YAK1 or MCK1, rapamycin-induced phosphorylation of BCY1 T129 was reduced but not abolished (Supplementary Figures 5, A and B). In addition, YAK1 and MCK1 puriﬁed from rapamycin-treated or -untreated yeast cells failed to phosphorylate recombinant GST-BCY1 in vitro (data not shown). These ﬁndings suggest that YAK1 and MCK1 may affect BCY1 phosphorylation but are not the kinases that directly phosphorylate BCY1 T129 upon TORC1 inhibition. Next, we examined BCY1 T129 phosphorylation in cells lacking either NPR1 or SLT2/MPK1, two serine/threonine kinases that are activated upon TORC1 inhibition (Schmidt et al., 1998; Krause and Gray, 2002; Torres et al., 2002). NPR1 controls the sorting of several amino acid transporters in Molecular Biology of the Cell