A. Soulard et al. A MAF1-P (PKA) MAF1 (tot) YPK3-P (PKA) YPK3 (tot) KSP1-P (PKA) KSP1 (tot) k co YPK3-HA m Rap 45’ - - + k co KSP1-HA m Rap 45’ - - + MAF1-HA Rap 45’ - + - k c o m k c o MAF1-HA m YPD YP 100 112 3 43 B C D MAF1-P (PKA) MAF1 (tot) Phos/tot (%) MAF1-P (PKA) MAF1 (tot) Phos/tot (%) YPK3-P (PKA) YPK3 (tot) 8-Br-cAMP - - + - + k c o MAF1-HA m 8-Br-cAMP - - - + + Rap - - + - + 100 14 142 56 k co YPK3-HA m 8-Br-cAMP - - - + + Rap - - + - + Phos/tot (%) 100 4 85 36 Figure 2. Inﬂuence of TORC1 inhibition on PKA substrate phosphor- ylation in vivo. (A) Rapamycin affects the phosphorylation state of different PKA substrates. Yeast strains expressing MAF1-HA (SA148), YPK3-HA (SA216), or KSP1-HA (SA219) were grown to exponential phase and treated for the indicated time with 200 ng/ml rapamycin ( ) or drug vehicle ( ). After protein extraction each tagged protein was immunoprecipitated and analyzed by Western blot for total protein (tot) with anti-HA antibody and for phosphorylation at PKA site (PKA) with anti-RRxS/T antibody. TB50a was used for mock control. (B) The yeast strain SA148 expressing MAF1-HA was grown to exponential phase in YPD and then shifted or not to YP for 2 h. Aliquots of cells were then treated for 30 min with 5 mM of 8-Br-cAMP. MAF1-HA was immunoprecipitated from total cell ly- sate and analyzed as described in A. TB50a was used as mock control. For each condition, the phosphorylation at PKA sites was quantiﬁed and normalized against the level of total protein and expressed as a percentage [Phos/tot (%)]. (C) The yeast strain SA148 expressing MAF1-HA was grown to exponential phase in YPD and then pretreated or not with 5 mM of 8-Br-cAMP during 5 min. Aliquots of cells were then treated for 30 min with 200 ng/ml rapa- mycin. Phosphorylation of MAF1-HA was analyzed as in A and B. TB50a was used for mock control. Phosphorylation was quantiﬁed as in B [Phos/tot (%)]. (D) The yeast strain SA216 expressing YPK3-HA was treated and analyzed as in C. acted the inhibitory effect of rapamycin on the phosphory- lation of YPK3 at PKA sites (Figure 2D). We did not examine the effect of 8-Br-cAMP on KSP1 for technical reasons. The above taken together indicates that MAF1, YPK3, and likely KSP1 are phosphorylated by PKA in a TORC1-dependent manner. Type 2A and type 2A-like phosphatases are well-known downstream effectors of TORC1 and are activated upon TORC1 inactivation (Jiang and Broach, 1999; Jacinto et al., 2001; Crespo and Hall, 2002). We have reported previously that the effect of TORC1 inhibition on several PKA-depen- dent processes is independent of both PP2A and the PP2A- like phosphatase SIT4 (Martin et al., 2004; Schmelzle et al., 2004). The above results with 8-Br-cAMP also suggest that the rapamycin-induced decrease in PKA target site phos- 3480 phorylation is due to loss of PKA activity rather than to activation of a phosphatase. We also used anti-RRxS/T to probe MSN2, CDC25, YAK1, and CKI1, all PKA substrates but whose PKA site phosphor- ylation was either not detected (CDC25, YAK1, and CKI1) or not altered in response to rapamycin (MSN2) in our phos- phoproteome analysis. Probing with anti-RRxS/T detected all four proteins but failed to detect a change in their phos- phorylation upon rapamycin treatment (see Supplementary Figure 3). Thus, large scale and targeted analyses of PKA site phosphorylation suggest that TORC1, rather than activating PKA globally, selectively activates PKA toward a subset of substrates. BCY1 Is Highly Phosphorylated upon TORC1 Inactivation To obtain insight into the molecular mechanism by which TORC1 might regulate PKA, we focused on BCY1. After conﬁrming earlier observations that physiologically relevant TPK1–BCY1 interaction cannot be monitored in vitro or in vivo (Supplementary Figure 4 and data not shown), we examined BCY1 phosphorylation. As shown previously, BCY1 is highly phosphorylated upon glucose starvation, and this phosphorylation appears to be important for PKA nuclear localization, suggesting that BCY1 phosphorylation leads to BCY1 activation and PKA inactivation (Werner- Washburne et al., 1991; Grifﬁoen et al., 2001). We observed that rapamycin treatment resulted in a slower migrating, phosphatase-sensitive form of BCY1 (Figure 3A), suggesting that TORC1 might signal to PKA via inhibition of BCY1 phosphorylation. To investigate further whether TORC1 signals to PKA via BCY1 phosphorylation, we identiﬁed the phosphorylation sites in HA-tagged BCY1 isolated from rapamycin-treated and untreated cells (see Materials and Methods). In total, 17 phosphorylation sites were identiﬁed (Figure 3B and Sup- plementary Table 10). The 17 sites fell into three clusters, all in the N-terminal part of BCY1 (Figure 3B). The ﬁrst cluster was S68, S70, S74, S77, S79, S81, S83, and S84. This cluster of phosphorylation sites, termed cluster II (CII), was described previously and was shown to be important for BCY1 func- tion (Grifﬁoen et al., 2001). The second cluster of phosphor- ylated residues consisted of T144, S145, S147, T150, and T160. This cluster falls within or near the so-called autoin- hibitory domain where the catalytic subunit of PKA “auto- phosphorylates” the highly conserved S145 (RRxS145) to in- hibit BCY1 (Kuret et al., 1988; Werner-Washburne et al., 1991). The third cluster of phosphorylated residues included T129, S130, and T131. This cluster was novel and was termed cluster III (CIII). We note that, in addition to the above 17 phosphorylation sites, we also detected phosphorylation in an extreme N-terminal, HA-containing fragment of BCY1, but were unable to pinpoint the phosphorylated residue(s) (data not shown). This phosphorylation is presumably re- lated to the previously identiﬁed cluster I (CI; Grifﬁoen et al., 2001). Consistent with the above observation that rapamycin treatment induced BCY1 phosphorylation as measured by altered electrophoretic mobility (Figure 3A), we detected more BCY1 phosphopeptides in extracts from rapamycin- treated cells compared with untreated cells. In particular, sites in CIII (and to a lesser extend in CII) were hyperphos- phorylated in response to rapamycin (Supplementary Table 10). These results suggest that there are many TORC1-sen- sitive phosphorylation sites in BCY1, mainly in CIII. This may explain why previous studies, which did not examine rapamycin-treated cells, did not detect the phosphorylation sites in CIII. Molecular Biology of the Cell