A B D BCY1 1 BCY1-P (T129) BCY1 MPK1-P MPK1 IP total Rapamycin - - + + + PPase - - - + + PPi - - - - + BCY1 CII S68; S70 S74; S77 S79; S81 S83; S84 CIII T129 S130 T131 T144 S145 S147 T150 T160 Dimerization domain Autoinhibitory domain mock WT tor1∆ TORRR Rap 45’ - + - + - + - + C E ck HA- o BCY1 m Rap 45’ - - + BCY1-P (T129) BCY1-P (S145) BCY1 S286 cAMP binding domains 416 k co HA-BCY1 m Rap (min) 0 0 45 120 0 -Glu (min) 0 0 0 0 120 BCY1-P (T129) BCY1 Figure 3. The PKA regulatory subunit BCY1 is highly phosphor- ylated upon TORC1 inhibition. (A) Exponentially growing cells expressing HA-BCY1 from a plasmid (SA094) or not (TB50a) were treated for 2 h with drug vehicle or rapamycin at 200 ng/ml. Total proteins were extracted and HA-BCY1 was immunoprecipitated. Aliquots of IPs belonging to the rapamycin treated samples were further treated with phosphatase (PPase) or not, in presence of phosphatase inhibitors (PPi) or not. BCY1 phosphorylation was observed by gel-shift assay followed by Western blotting with an anti-HA antibody. (B) Graphical representation of the phosphory- lated amino acids identiﬁed in BCY1 by phosphopeptide enrich- ment and MS. CII/III, cluster II/III. (C) Similar to A except that yeast cells were treated with rapamycin for 45 min (Rap). Total BCY1 was followed by Western blot with anti-BCY1 (BCY1) anti- body and the different phosphorylated form of BCY1 with anti- RRxS/T antibody [(BCY1-P (S145)] and anti-RxxS/T antibody [BCY1-P (T129)], respectively. (D) The procedure used in B was used to analyze rapamycin-induced phosphorylation of HA-BCY1 in wild-type (WT TB50a), TOR1 deletion (tor1 AN9-2a), and rapamycin resistant TORs (TORRR TOR1-1 TOR2-1 RL206-4D) yeast strain carrying pTS137 (panel IP). In parallel, total (MPK1) and activated MPK1 (MPK1-P) were visualized by Western blot on the total cell lysates used for the HA-BCY1 IPs (panel total). (E) Wild- type cells (WT SA094) expressing HA-BCY1 (pTS137) were treated with 200 ng/ml rapamycin (Rap) or shifted to YP medium lacking glucose ( Glu) for the indicated time. Total (HA-BCY1) and phosphorylated BCY1 [BCY1-P (T129)] were followed as in D. S145 is part of an RRxS motif, whereas S74, S83, and T129 are part of RxxS/T sequences. To further characterize the rapamycin-induced phosphorylation sites in BCY1, we took advantage of two antibodies, the previously described anti- RRxS/T that speciﬁcally recognizes a phosphorylated PKA site (e.g., phosphorylated S145) and a second antibody (anti- RxxS/T) that recognizes the related, phosphorylated motif RxxS/T (e.g., phosphorylated T129). As described below and in supplementary information, the speciﬁcity of the two antibodies was conﬁrmed by mutagenesis of their recogni- tion sites in BCY1 (Figure 4A and Supplementary Figure 7). As shown in Figure 3C, HA-BCY1 immunoprecipitated with anti-HA was detected with both anti-RRxS/T and anti- RxxS/T. Although the signal obtained with anti-RRxS/T did Vol. 21, October 1, 2010 TORC1 Regulates PKA through MPK1 BCY1 T129A T129D Rap 45’ - + - + - + YPD DMSO YPD Rap 5ng/ml WT T129A T129D A B C BCY1-P (T129) BCY1-P (S145) BCY1 BCY1 T129A T129D - Rap +Rap Figure 4. BCY1 T129 phosphorylation after rapamycin treatment lowers PKA activity. (A) The yeast strains expressing wild-type (WT) or different mutants of HA-BCY1 at threonine 129 (T129A, T129D) were grown to exponential phase and treated or not with rapamycin (200 ng/ml) for 45 min. After total protein extraction, HA-BCY1 was immunoprecipitated. Total BCY1 and phosphory- lated BCY1 at S145 and T129 were detected by Western blot with the corresponding antibodies. (B) The yeast strains used in A were grown to exponential phase. A cell quantity corresponding to 1.0 OD600 nm was serially 10-fold diluted and spotted on YPD plates containing DMSO or 5 ng/ml rapamycin. Plates were then incu- bated at 30°C for 3–5 d. (C) Glycogen accumulation was tested by iodine staining after 5-h rapamycin or drug vehicle treatment of yeast strains expressing the wild-type (BCY1 SA094) and mutated form of BCY1 at threonine 129 (T129A and T129D). not change signiﬁcantly upon rapamycin treatment [Figure 3C, see P-BCY1 (S145)], the signal observed with anti- RxxS/T increased upon TORC1 inhibition [Figure 3C, see P-BCY1(T129)]. Thus, consistent with BCY1 phosphosite- mapping experiments described above, TORC1 inhibits phosphorylation of RxxS/T sites in BCY1 and has no effect on RRxS145 phosphorylation. To conﬁrm that the effect of rapamycin on BCY1 phos- phorylation was indeed due to inhibition of TORC1, we examined BCY1 phosphorylation in TOR mutant strains. In a TOR1 deletion strain, BCY1 was hyperphosphorylated at the RxxS/T site(s). Conversely, this site(s) remained insen- sitive to rapamycin in the rapamycin resistant (TORRR) TOR1-1 TOR2-1 strain (Figure 3D). Thus, the effect of rapa- mycin on phosphorylation of BCY1 at the RxxS/T motif was due to TORC1 inhibition. Furthermore, phosphorylation of the RxxS/T motif in BCY1 increased with increasing time of rapamycin treatment, reaching a level after 2 h comparable to that of glucose starvation for 2 h (Figure 3E). Taken together, the above results suggest that TORC1 (and glu- cose) inhibits phosphorylation of multiple sites in BCY1, some if not all of which are part of an RxxS/T motif (e.g., S74, S83, and T129). TORC1-sensitive BCY1 T129 Phosphorylation Inactivates PKA To determine the effect of TORC1-sensitive BCY1 phosphor- ylation on PKA function, we mutated individually S74, S83, 3481