Regulation of the Cell Integrity Pathway by Rapamycin-sensitive TOR Function in Budding Yeast
2002; Elsevier BV; Volume: 277; Issue: 45 Linguagem: Inglês
10.1074/jbc.m205408200
ISSN1083-351X
AutoresJordi Torres‐Rosell, Charles J. Di Como, Enrique Herrero, María Ángeles de la Torre-Ruiz,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that inSaccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G0. The TOR (target of rapamycin) pathway controls cell growth in response to nutrient availability in eukaryotic cells. Inactivation of TOR function by rapamycin or nutrient exhaustion is accompanied by triggering various cellular mechanisms aimed at overcoming the nutrient stress. Here we report that inSaccharomyces cerevisiae the protein kinase C (PKC)-mediated mitogen-activated protein kinase pathway is regulated by TOR function because upon specific Tor1 and Tor2 inhibition by rapamycin, Mpk1 is activated rapidly in a process mediated by Sit4 and Tap42. Osmotic stabilization of the plasma membrane prevents both Mpk1 activation by rapamycin and the growth defect that occurs upon the simultaneous absence of Tor1 and Mpk1 function, suggesting that, at least partially, TOR inhibition is sensed by the PKC pathway at the cell envelope. This process involves activation of cell surface sensors, Rom2, and downstream elements of the mitogen-activated protein kinase cascade. Rapamycin also induces depolarization of the actin cytoskeleton through the TOR proteins, Sit4 and Tap42, in an osmotically suppressible manner. Finally, we show that entry into stationary phase, a physiological situation of nutrient depletion, also leads to the activation of the PKC pathway, and we provide further evidence demonstrating that Mpk1 is essential for viability once cells enter G0. FK506-binding protein glutathione S-transferase mitogen-activated protein kinase protein kinase C Rapamycin is an antibiotic macrolide, with a strong antiproliferative action in eukaryotic cells. Its target is FK506-binding protein (FKBP)112, a small protein belonging to the FKBP family of peptidylprolyl isomerases (1Harding M.W. Galat A. Uehling D.E. Schreiber S.L. Nature. 1989; 341: 758-760Crossref PubMed Scopus (1197) Google Scholar, 2Siekierka J.J. Hung S.H. Poe M. Lin C.S. Sigal N.H. Nature. 1989; 341: 755-757Crossref PubMed Scopus (883) Google Scholar). An FKBP12-rapamycin complex is able to bind the TOR proteins (target of rapamycin, also known as FRAP, RAFT, RAPT, or mTOR (3Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1562) Google Scholar)) and to block TOR signaling to downstream effectors. The TOR proteins are members of the phosphatidylinositol kinase-related kinase family, and despite displaying significant homology to lipid kinases (4Kunz J. Henriquez R. Schneider U. Deuter-Reinhard M. Movva N.R. Hall M.N. Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (729) Google Scholar), they have been shown to be Ser/Thr protein kinases (5Alarcon C.M. Heitman J. Cardenas M.E. Mol. Biol. Cell. 1999; 10: 2531-2546Crossref PubMed Scopus (65) Google Scholar). In Saccharomyces cerevisiae cells, the TOR proteins promote association between the Sit4 and Tap42 proteins under favorable nutrient conditions (6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar). Two other 2A protein phosphatases, Pph21p and Pph22p, also associate with Tap42 in a TOR-dependent rapamycin-sensitive manner (6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar). The SIT4 gene codes for a Ser/Thr protein phosphatase closely related to the protein phosphatase 2A family (7Arndt K.T. Styles C.A. Fink G.R. Cell. 1989; 56: 527-537Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 8Doseff A.I. Arndt K.T. Genetics. 1995; 141: 857-871Crossref PubMed Google Scholar) and displays a high level of identity to human protein phosphatase 6. Tap42 shows sequence homology to the mammalian α4 protein, which in turn is able to associate with protein phosphatase 6 (9Murata K., Wu, J. Brautigan D.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10624-10629Crossref PubMed Scopus (195) Google Scholar, 10Chen J. Peterson R.T. Schreiber S.L. Biochem. Biophys. Res. Commun. 1998; 247: 827-832Crossref PubMed Scopus (159) Google Scholar). Tap42 can be phosphorylated directly by TOR, and this phosphorylation increases Tap42 affinity for the phosphatases (11Jiang Y. Broach J.R. EMBO J. 1999; 18: 2782-2792Crossref PubMed Scopus (276) Google Scholar). In yeast cells, inhibition of TOR function by rapamycin results in dissociation of the Sit4-Tap42 complex (6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar) and in cellular responses similar to those exhibited in nutrient-starved cells. These include down-regulation of translation initiation (6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar), repression of ribosome biogenesis (12Powers T. Walter P. Mol. Biol. Cell. 1999; 10: 987-1000Crossref PubMed Scopus (329) Google Scholar), cell cycle arrest (13Barbet N.C. Schneider U. Helliwell S.B. Stansfield I. Tuite M.F. Hall M.N. Mol. Biol. Cell. 1996; 7: 25-42Crossref PubMed Scopus (603) Google Scholar), induction of autophagy (14Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar), and acquisition of thermotolerance (6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar). Both TOR and the Tap42-phosphatase complex are also involved in the repression of the starvation transcriptional program (15Cardenas M.E. Cutler N.S. Lorenz M.C., Di Como C.J. Heitman J. Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (484) Google Scholar, 16Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (470) Google Scholar), which is achieved by preventing the nuclear translocation of specific transcription factors (17Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (803) Google Scholar), and in the Tap42-mediated stabilization of amino acid permeases (18Schmidt A. Bickle M. Beck T. Hall M.N. Cell. 1997; 88: 531-542Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). Recently, TOR signaling has been shown to control autophagy via an Apg1 protein kinase complex, although Tap42 has been proposed not to be involved in this specific signaling (19Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (914) Google Scholar). Thus, most of TOR cellular functions imply the regulation of the Tap42-phosphatase complexes. Based on correlations established between the Sit4-Tap42 association state and the phosphorylation of downstream effectors, it has been proposed that Tap42 may inhibit Sit4 phosphatase activity (17Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (803) Google Scholar, 18Schmidt A. Bickle M. Beck T. Hall M.N. Cell. 1997; 88: 531-542Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), although no direct target of the phosphatase has been described yet. The TOR genes were originally identified in yeast by the fact that certain mutations in them conferred resistance to growth inhibition by rapamycin (3Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1562) Google Scholar). The two yeast Tor proteins termed Tor1 and Tor2, can bind to the FKBP12 homolog (Fpr1)-rapamycin complex in budding yeast. TOR1 and TOR2 display a high degree of sequence homology. However, although both regulate the Sit4-Tap42 complex in response to nutrients, TOR2 plays an additional essential function that is not shared by TOR1(20Zheng X.F. Florentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (248) Google Scholar). The TOR2 essential function has been related to the organization of the actin cytoskeleton (21Schmidt A. Kunz J. Hall M.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13780-13785Crossref PubMed Scopus (214) Google Scholar). A temperature-sensitivetor2ts mutant displays lower activity levels of the GTPase exchange factor Rom2 (18Schmidt A. Bickle M. Beck T. Hall M.N. Cell. 1997; 88: 531-542Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar), which in turn is needed to activate the essential Rho1 small GTPase (22Bickle M. Delley P.A. Schmidt A. Hall M.N. EMBO J. 1998; 17: 2235-2245Crossref PubMed Scopus (162) Google Scholar). Growth and actin polarization defects of tor2ts alleles are rescued by high copy expression of Rho1 or any of the members of the protein kinase C (PKC)/cell integrity pathway (23Helliwell S.B. Schmidt A. Ohya Y. Hall M.N. Curr. Biol. 1998; 8: 1211-1214Abstract Full Text Full Text PDF PubMed Google Scholar), as well as by cell wall damage-mediated activation of the pathway (22Bickle M. Delley P.A. Schmidt A. Hall M.N. EMBO J. 1998; 17: 2235-2245Crossref PubMed Scopus (162) Google Scholar). 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EMBO J. 1997; 16: 7196-7206Crossref PubMed Scopus (137) Google ScholarCML125MATα As CML12833de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (58) Google ScholarJA-110CML128 sit4∷trp175Clotet J. Garı́ E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (70) Google ScholarMML382CML128 wsc1∷caURA333de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (58) Google ScholarMML384CML128 wsc2∷natMX433de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (58) Google ScholarMML387CML128 mid2∷kanMX433de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (58) Google ScholarMML392CML128 wsc1∷caURA3 wsc2∷natMX433de la Torre-Ruiz M.A. Torres J. 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Science. 1991; 253: 905-909Crossref PubMed Scopus (1562) Google ScholarJH11–1cJK9–3da TOR1–13Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1562) Google ScholarJH12–17bJK9–3da TOR2–13Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1562) Google ScholarMML304MATα pkc1∷LEU2(pBCK1–20)33de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (58) Google ScholarMML378JK9–3da tor1∷kanMX4This studyMML380JK9–3da TOR2–1 tor1∷kanMX4This studyMML447CML128 tor1∷kanMX4 mpk1∷URAThis studyMML448CML128 tor1∷kanMX4This studyCY4907W303 MATa ura3–1 leu2–3,112 his3–11,15 trp1–1 ade2–1 can1–100 tap42∷TRP1 SSD1-ν {TAP42 onLEU2/CEN}6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google ScholarCY4908W303MATa ura3–1 leu2–3,112 his3–11,15 trp1–1 ade2–1 can1–100 tap42∷TRP1 SSD1-ν {tap42–11 onLEU2/CEN}6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar Open table in a new tab Yeast strains were grown in YPD medium (2% yeast extract, 1% peptone, 2% glucose). To monitor entry into stationary phase, cells were grown in selective glucose minimal medium, SD (0.67% yeast nitrogen base, 2% glucose, and the required amino acids) (38Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). Osmotic stabilization was provided where indicated by adding sorbitol or KCl to a final concentration of 0.8 and 0.5m, respectively. Except where stated, cells were grown at 25 °C. To inactivate the temperature-sensitive rho1-104allele, cells were shifted from 25 to 39 °C for 45 min (39Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Rapamycin (from Sigma) was stored at −20 °C as a 1 mg/ml stock solution (90% ethanol and 10% Tween) and used at a final concentration of 200 ng/ml. Tunicamycin was stored at −20 °C as a 5 mg/ml stock solution (75% methanol) and used at a final concentration of 2.5 μg/ml. Raw data (16Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (470) Google Scholar, 40Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3763) Google Scholar) corresponding to Mpk1-regulated genes were plotted as a function of time in a Microsoft Excel worksheet. To maximize inductions and to avoid punctual deviations, data were plotted as a stacked area profile. For Western analysis, cultures were grown overnight, and cells were harvested by filtration through 0.22-μm Millipore membranes, washed with ice-cold medium, transferred to Eppendorf tubes, and centrifuged for 15 s at 14,000 rpm. Total yeast protein extracts were prepared as described by Gallego et al. (41Gallego C. Garı́ E. Colomina N. Herrero E. Aldea M. EMBO J. 1997; 16: 7196-7206Crossref PubMed Scopus (137) Google Scholar). The protein concentration in the supernatants was determined by a Micro DC protein assay (Bio-Rad). Equivalent amounts of total protein extracts were run on 10% SDS-polyacrylamide gels. The anti-phospho-p44/p42 antibody (New England Biolabs) was used at a final dilution of 1:5,000 in TBST buffer, and the anti-GST-Mpk1 antibody (39Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 42Martin H. Arroyo J. Sanchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (115) Google Scholar) at a 1:2,000 dilution in the presence of 5% fat milk. Horseradish peroxidase-linked anti-rabbit secondary antibody (NA931, Amersham Biosciences) was used at a 1:10,000 dilution and incubated in TBST buffer containing 1% fat milk for the anti-phospho-Mpk1 and 0.25% fat milk for the anti-GST-Mpk1 primary antibody. Chemoluminescent detection was performed using the Supersignal Substrate (Pierce) in a Lumi-Imager equipment (Roche Molecular Biochemicals). Cells were fixed in 4% formaldehyde for 10 min, centrifuged at 3,000 rpm 5 min, and fixed overnight in phosphate-buffered saline plus 4% formaldehyde. Cells were washed once with phosphate-buffered saline containing 10 mmethanolamine, and once more with phosphate-buffered saline. For F-actin staining, rhodamine-phalloidin (from Sigma; stored as a 6.6 μm solution at −20 °C in methanol) was used at a final concentration of 0.6 μm. Cells were stained for at least 2 h in the dark and washed five times with phosphate-buffered saline before resuspending in mounting solution. All centrifugations were performed at 3,000 rpm. To test whether rapamycin had any effect on the activity of the PKC pathway, we performed Western blot analysis of total cell extracts using anti-phospho-p44/42 MAPK antibodies. These antibodies specifically recognize the doubly phosphorylated form of Mpk1 and allow accurate monitoring of Mpk1 activity (26Verna J. Lodder A. Lee K. Vagts A. Ballester R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13804-13809Crossref PubMed Scopus (318) Google Scholar, 39Martin H. Rodriguez-Pachon J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 275: 1511-1519Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Rapamycin was added to wild type cultures growing exponentially at 25 °C in rich medium (YPD), and samples were taken at the indicated times (Fig.1 A). We observed a very rapid increase in the amount of the active Mpk1 form in response to rapamycin, already detectable after 15 min of treatment. Maximum levels of Mpk1 activity were reached after 45 min and remained high for the duration of the experiment (60 min). The induction of Mpk1 activity was not the result of an increase in the Mpk1 protein levels, which remained constant throughout the experiment (Fig. 1 A), as observed after probing the same extracts with anti-Mpk1 polyclonal antibodies. Two related MAPKs, Fus3 and Kss1, are also recognized in their active, doubly phosphorylated state by the same antibody (43Bardwell L. Cook J.G. Voora D. Baggott D.M. Martinez A.R. Thorner J. Genes Dev. 1998; 12: 2887-2898Crossref PubMed Scopus (143) Google Scholar, 44Sabbagh Jr., W. Flatauer L.J. Bardwell A.J. Bardwell L. Mol. Cell. 2001; 8: 683-691Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). However, no significant change in their activity was detected after the addition of rapamycin (Fig. 1, A and B). The latter suggests that rapamycin-mediated MAPK activation is specific for Mpk1. The transcripts up-regulated by Mpk1 activation have been described recently using whole-genome approaches and comprise mostly cell wall and stress-responsive genes (45Jung U.S. Levin D.E. Mol. Microbiol. 1999; 34: 1049-1057Crossref PubMed Scopus (359) Google Scholar). The induction of all of them is expected to occur under any stress that activates Mpk1, including rapamycin treatment. To check the validity of this hypothesis, we carried out an in silico study of the behavior of Mpk1-regulated genes under different environmental perturbations that either induce or do not affect Mpk1 activity. Data on Mpk1-regulated genes was extracted from previously published works (40Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3763) Google Scholar) and plotted as a function of time (Fig. 1 C). As expected, Mpk1-regulated genes are induced by heat shock and hypotonic shock, situations that give rise to Mpk1 activation, but not by hydrogen peroxide or menadione treatment (Fig. 1 C and data not shown). In a further step, we carried out an analogous study on other published data (16Hardwick J.S. Kuruvilla F.G. Tong J.K. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (470) Google Scholar) to check whether the same group of genes was also induced upon rapamycin treatment. As shown in Fig. 1 C, expression of Mpk1-regulated genes is induced by rapamycin treatment in a fashion that correlates with the increase in Mpk1 activity (Fig. 1, A andC), although different concentrations of the inhibitor were used in each experiment. Overall, these observations evidence that the pattern of Mpk1-regulated gene expression serves as a good marker of Mpk1 activity. Moreover, because all of those genes are up-regulated by rapamycin, it shows that the signal from rapamycin to Mpk1 is transmitted to substrates downstream from this kinase, leading to the transcriptional induction of Mpk1-regulated genes. The TOR proteins have been proposed to be central sensors of the quality of carbon and nitrogen sources (46Shamji A.F. Kuruvilla F.G. Schreiber S.L. Curr. Biol. 2000; 10: 1574-1581Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar), and rapamycin has been reported to cause effects similar to those exhibited by nutrient-starved cells. Besides, cells in stationary phase display phenotypes similar to those caused by rapamycin (6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar). Thus, we reasoned that cells entering stationary phase could also induce Mpk1 activity, as observed when TOR proteins are blocked by rapamycin (Fig.1 A). Wild type cells were inoculated in fresh minimal medium, and samples were collected at the times indicated. As shown in Fig. 1 D, Mpk1 became strongly activated as cells progressively entered into stationary phase (2–4 days) and became less active at later time points. The increase in Mpk1 activity was not caused by changes in the levels of the Mpk1 protein, as checked with anti-Mpk1 polyclonal antibodies (Fig. 1 D). Moreover, although entry into stationary phase does not affect viability in wild type cultures, mpk1Δ mutant cells rapidly lost viability upon exit from exponential growth (Fig. 1 E). In a genomewide screen for genes whose deletion results in alterations on the growth response to rapamycin, it has been reported that both MPK1and SWI6 (whose product is a Mpk1 target) confer rapamycin hypersensitivity when deleted (47Chan T.F. Carvalho J. Riles L. Zheng X.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13227-13232Crossref PubMed Scopus (158) Google Scholar). Other authors have shown recently that an intact PKC pathway is needed to maintain viability upon nutritional deprivation (48Krause S.A. Gray J.V. Curr. Biol. 2002; 12: 588-593Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). These results, together with ours, suggest that Mpk1 activation, and the subsequent induction of its target genes, is essential for cells to remain viable once they enter stationary phase or are treated with rapamycin. To prove the direct involvement of members of the TOR pathway in mediating rapamycin signaling to Mpk1, we checked the behavior of rapamycin-resistant TOR mutants.TOR1-1 and TOR2-1 bear, respectively, chromosomal alleles of TOR1 and TOR2 which are insensitive to the immunosuppressant drug because their products cannot interact with the Fpr1-rapamycin complex. The JK9-3da wild type strain induced Mpk1 phosphorylation with kinetics identical to those of the CML128 wild type strain used above (Figs. 1 A and2 A). However TOR1-1cells displayed no change in Mpk1 activity in response to rapamycin treatment, whereas in TOR2-1 cells a mild activation was still detected (Fig. 2 A). The results clearly indicate that Tor1 inhibition mediated by rapamycin signals to Mpk1 inducing its activity. Therefore, activation of the MAPK is not the result of a direct cell wall damage effect caused by rapamycin. We hypothesized that the slight Mpk1 activation still observed in TOR2-1cells could be the result of the rapamycin-mediated blockage of a fully inhibitable Tor1 protein. Thus, inhibition of Tor2 in wild type cells could still contribute to rapamycin signaling to Mpk1. Support for this hypothesis is the observation that Fpr1-rapamycin binding to Tor1 occurs at 10-fold lower rapamycin concentrations compared with those necessary to bind to Tor2 (49Lorenz M.C. Heitman J. J. Biol. Chem. 1995; 270: 27531-27537Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar); hence, the inhibitory complex has more affinity for Tor1 than for Tor2. To test whether Tor2 inhibition by rapamycin is also involved in the induction of the PKC pathway, we deleted the TOR1 gene in both the wild type and the TOR2-1 strains, and the levels of Mpk1 activation were checked in the single and double mutants. Thetor1Δ mutant displayed activation of Mpk1 in response to rapamycin treatment (Fig. 2 A), although the induction was less pronounced than in wild type cells. This result clearly indicates that in these conditions Tor2 inhibition by rapamycin mediates induction of Mpk1 activity. Furthermore, no increase in the activation of the MAPK was detectable in tor1Δ TOR2-1 double mutant cells after addition of the drug (Fig. 2 A). In light of these results, we propose that either Tor1 or Tor2 inhibition by rapamycin can mediate Mpk1 activation. Interestingly,tor1Δ mutant cells, which may be compromised for TOR function (because they rely on the single TOR2 gene), displayed higher levels of both basal and induced Mpk1 activity than wild type cells, which suggests that partial elimination of TOR function leads to a constitutive increased activation of Mpk1 (as rapamycin does). Taken together, these data show that a block in TOR function may act by up-regulating the PKC pathway. An active TOR pathway promotes the association of the Tap42 subunit with the protein phosphatase 2A and Sit4 (6Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Schola
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