TORC1 Regulates PKA through MPK1 Figure 6. BCY1 T129 phosphorylation is me- A B dTMiBaP5tK0eda1). o(bAryd)eYaleecatteisvdtas(tmitorapnikn1sofwTitlShd4e5ty-M1paeA)f(PoMrPkMKinP1aKs1e Rap 45’ m- oc+k M- PK+1 m-pk+1 ckom WT B-C2K01 WT and expressing HA-BCY1 from a plasmid BCY1-P (T129) Rap 45’ - - + - + SDS (pTS137) were treated for 45 min with 200 ng/ml rapamycin ( ) or drug vehicle ( ). BCY1-P (T129) After protein extraction, HA-BCY1 was im- BCY1-P (S145) munoprecipitated and analyzed by Western BCY1 [bBloCtYu1s-iPng(Sa1n4t5i-)H], Aan(HdAa-nBtCi-RYx1x),Sa/nTti-(RBRCxYS1/-TP BCY1 IP (T129)) antibodies. TB50a was used as a mock control. (B) Total (HA-BCY1) and phospho- C k MPK1-P T129 BCY1 [BCY1-P (T129)] were analyzed as co MPK1-HA i(n20A0 nagft/emrl4)5,-SmDiSn (t0r.e0a1t%m)e,notrwdirtuhgrvapehamiclyecoinf Rap 45’ - m - + MPK1 wild-type strain (WT TB50a) or of a strain total expressing an hyperactive form of BCK1 GST-BCY1 (BCK1-20) from a plasmid (pRS316::BCK1-20; MPK1 apcatniveel IpPh).oIsnphpoarraylllaetle,dtoMtaPlMK1PK(P1- M(MPPKK1)1)waenrde 32P 1x 1,6x 2,3x D WT analyzed by Western blot on the total extracts used for HA-BCY1 IPs with the correspond- GST-BCY1 mpk1 ing antibodies (panel total). (C) In vitro kinase MPK1 assay. MPK1-HA was immunoprecipitated from the yeast strain TS99-5c treated with coomasie tor1 rapamycin ( ) or drug vehicle ( ) for 45 min. The assay was performed as describe in Ma- MPK1-P tor1 terials and Methods in presence of 1 g of mpk1 rceolcio.mTobtianlaanntdGpShTo-sBpChYo1ryleaxtpedrespsreodteifnrsomwerEe. MPK1 - Rap + visualized by Coomassie staining (Coomas- WB sie) and autoradiography (32P), respectively. svAeanrtveaeddliqMfuoPorKta1onfa(lMpysuPirsKiﬁo1e-fPdt)oMtbaylPK(MW1-PeHsKtAe1r)nwanabdslocatoctanis-- E ckom MPK1 mpk1 F ckomMPK1 mpk1 above (WB). The level of BCY1 phosphoryla- Rap (min) - - 5 10 - 5 10 Rap 45’ - - + - + tion was quantiﬁed by densitometric analysis (below panel 32P). MBP, a commonly used MAF1-P (PKA) YPK3-P (PKA) MPK1 in vitro substrate, was used as a posi- taicvceu mcounltartoioln(dwataas ntoesttesdhobwyn)io(dDin)eGslytaciongineng MAF1 (tot) YPK3 (tot) after 5-h rapamycin or drug vehicle treatment Phos/tot (%) 100 76 33 124 102 61 Phos/tot (%) 100 20 126 52 of yeast strains wild type (WT SA180) or deleted for MPK1 (mpk1 SA183), for TOR1 (tor1 SA181), or for both genes (tor1 mpk1 SA185). (E) Effect of MPK1 deletion on PKA-dependent phosphorylation of MAF1. Yeast strains containing MAF1-HA and expressing (MPK1; strain SA148) or lacking MPK1 (mpk1; strain SA233) were grown to exponential phase and treated for the indicated time 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. MAF1 phosphorylation at PKA sites was quantiﬁed as described in Figure 1 [Phos/tot (%)]. (F) Effect of MPK1 deletion on PKA-dependent phosphorylation of YPK3. Yeast strains containing YPK3-HA and expressing (MPK1; strain SA234) or lacking MPK1 (mpk1; SA235) were analyzed as in E except that the cells were treated with rapamycin ( ) or drug vehicle ( ) for 45 min. response to nutrients (Schmidt et al., 1998). Deletion of NPR1 had no effect on T129 phosphorylation upon rapamycin treatment (Supplementary Figure 5C). The MAP kinase MPK1 is part of the PKC1-BCK1-MKK1/2-MPK1 cascade that regulates cell wall integrity and entry into quiescence in response to stress (Levin, 2005). Deletion of MPK1 com- pletely abolished rapamycin-induced T129 phosphorylation (Figure 6A). To further investigate a role of MPK1 in BCY1 phosphorylation, we examined T129 phosphorylation in re- sponse to conditions that activate the cell wall integrity pathway. We activated the cell wall integrity pathway with a genetically activated MAPKKK (BCK1-20 mutation) or with chemicals such as SDS, Congo Red, and sodium or- thovanadate that disrupt the cell wall. MPK1 activation was monitored with an antibody that recognizes phosphorylated p44/42 ERK MAPK but that also recognizes phosphorylated MPK1 in yeast (Verna et al., 1997; Delley and Hall, 1999). Like rapamycin treatment (Krause and Gray, 2002; Torres et Vol. 21, October 1, 2010 al., 2002 and Figure 6B), activation of the cell wall integrity pathway stimulated both MPK1 and BCY1 T129 phosphor- ylation (Figure 6B and Supplementary Figure 6C). We next examined whether MPK1 immunopuriﬁed from rapamycin- treated cells phosphorylates recombinant BCY1 in vitro. As shown in Figure 6C, MPK1 puriﬁed from rapamycin-treated cells indeed phosphorylated recombinant BCY1. Recombinant BCY1 in which T129 was mutated to alanine was less phos- phorylated compared with wild-type BCY1 (Supplementary Figure 6E). These results suggest that MPK1 directly phosphor- ylates BCY1 T129 upon TORC1 inhibition in vivo. To investigate the physiological signiﬁcance of MPK1- dependent BCY1 phosphorylation upon TORC1 inhibition, we examined rapamycin-induced glycogen accumulation and PKA-dependent phosphorylation of MAF1 and YPK3 in cells lacking MPK1. Rapamycin-treated, MPK1-deleted cells accumulated less glycogen than wild-type cells (Figure 6D). A role for MPK1 in TORC1-sensitive glycogen accumulation 3483