Regulatory Mechanisms for Modulation of Signaling through the Cell Integrity Slt2-mediated Pathway in Saccharomyces cerevisiae
2000; Elsevier BV; Volume: 275; Issue: 2 Linguagem: Inglês
10.1074/jbc.275.2.1511
ISSN1083-351X
AutoresHumberto Martı́n, José M. Rodrı́guez-Pachón, Cristina Ruíz‐Romero, César Nombela, Marı́a Molina,
Tópico(s)Plant-Microbe Interactions and Immunity
ResumoSignal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs. We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway. We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization. The lack of the putative cell surface sensor Mid2 prevents vanadate- but not caffeine-induced Slt2 phosphorylation. Disruption of the Rho1-GTPase-activating protein genes SAC7and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway. MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2. Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression ofMSG5 in a sac7Δ mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels. These data present the first evidence for a negative regulation of the cell integrity pathway. Signal transduction mediated by the mitogen-activated protein kinase (MAPK) Slt2 pathway is essential to maintain the cell wall integrity in Saccharomyces cerevisiae. Stimulation of MAPK pathways results in activation by phosphorylation of conserved threonine and tyrosine residues of MAPKs. We have used an antibody that specifically recognizes dually phosphorylated Slt2 to gain insight into the activation and modulation of signaling through the cell integrity pathway. We show that caffeine and vanadate activate this pathway in the absence of osmotic stabilization. The lack of the putative cell surface sensor Mid2 prevents vanadate- but not caffeine-induced Slt2 phosphorylation. Disruption of the Rho1-GTPase-activating protein genes SAC7and BEM2 leads to constitutive Slt2 activation, indicating their involvement as negative regulators of the pathway. MAPK kinases also seem to participate in signaling regulation, Mkk1 playing a greater role than Mkk2 in signal transmission to Slt2. Additionally, one of the phosphatases involved in Slt2 dephosphorylation is likely to be the dual specificity phosphatase Msg5, since overexpression ofMSG5 in a sac7Δ mutant eliminates the high Slt2 phosphorylation, and disruption of MSG5 in wild type cells results in increased phospho-Slt2 levels. These data present the first evidence for a negative regulation of the cell integrity pathway. mitogen-activated protein kinase MAPK kinase glutathioneS-transferase GTPase-activating protein The PKC1-mediated MAPK1pathway is one of the signal transduction pathways that operates inSaccharomyces cerevisiae to control yeast cell biology. This signaling pathway is essential for the maintenance of cell integrity in a variety of environmental conditions and during morphogenetic events, through the regulation of cell wall and actin cytoskeleton dynamics (1.Heinisch J.J. Lorberg A. Schmitz H.P. Jacoby J.J. Mol. Microbiol. 1999; 32: 671-680Crossref PubMed Scopus (287) Google Scholar,2.Schmidt A. Hall M.N. Annu. Rev. Cell Dev. Biol. 1998; 14: 305-338Crossref PubMed Scopus (368) Google Scholar). The so-called cell integrity pathway is induced in periods of polarized growth during budding and mating (3.Zarzov P. Mazzoni C. Mann C. EMBO J. 1996; 15: 83-91Crossref PubMed Scopus (197) Google Scholar) and in response to environmental conditions that jeopardize cell wall stability, such as high temperature (4.Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (419) Google Scholar), hypotonic shock (5.Davenport K.R. Sohaskey M. Kamada Y. Levin D.E. Gustin M.C. J. Biol. Chem. 1995; 270: 30157-30161Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), or interference with wall synthesis (6.Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar). Accordingly, lack of functionality of the pathway leads to cell lysis when yeast cells are exposed to these inducing conditions. This lysis defect can be prevented by osmotic stabilization, indicating a failure in the maintenance of a functional cell wall structure (7.Torres L. Martin H. Garcia-Saez M.I. Arroyo J. Molina M. Sanchez M. Nombela C. Mol. Microbiol. 1991; 5: 2845-2854Crossref PubMed Scopus (144) Google Scholar, 8.Levin D.E. Bartlett-Heubusch E. J. Cell Biol. 1992; 116: 1221-1229Crossref PubMed Scopus (302) Google Scholar). Recently, two different types of putative cell surface sensors for cell integrity signaling have been described. Hcs77/Slg1/Wsc1 belongs to the family of four transmembrane proteins encoded by WSC genes, and their involvement in heat shock activation of the Pkc1-mediated pathway has been reported (9.Gray J.V. Ogas J.P. Kamada Y. Stone M. Levin D.E. Herskowitz I. EMBO J. 1997; 16: 4924-4937Crossref PubMed Scopus (203) Google Scholar, 10.Verna J. Lodder A. Lee K. Vagts A. Ballester R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13804-13809Crossref PubMed Scopus (315) Google Scholar, 11.Jacoby J.J. Nilius S.M. Heinisch J.J. Mol. Gen. Genet. 1998; 258: 148-155Crossref PubMed Scopus (97) Google Scholar). Mid2 has been described as another putative sensor for this pathway in response to pheromone, high temperature, and cell wall disturbances (6.Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar,12.Rajavel M. Philip B. Buehrer B.M. Errede B. Levin D.E. Mol. Cell. Biol. 1999; 19: 3969-3976Crossref PubMed Scopus (173) Google Scholar). Mtl1 seems to have a partially redundant function with its homologue Mid2 (6.Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar, 12.Rajavel M. Philip B. Buehrer B.M. Errede B. Levin D.E. Mol. Cell. Biol. 1999; 19: 3969-3976Crossref PubMed Scopus (173) Google Scholar). Although the molecular mechanisms by which these sensors transmit the signal to downstream components are unknown, the Rho1-GDP/GTP exchange factor Rom2 has been reported to mediate the activation of Rho1 by cell wall alterations (13.Bickle M. Delley P.A. Schmidt A. Hall M.N. EMBO J. 1998; 17: 2235-2245Crossref PubMed Scopus (158) Google Scholar). Rho1 is a small GTPase that functions as a binary switch between two interconvertible GTP-bound active and GDP-bound inactive forms. The switch is up-regulated by the GDP/GTP exchange factors Rom1 and Rom2 (14.Ozaki K. Tanaka K. Imamura H. Hihara T. Kameyama T. Nonaka H. Hirano H. Matsuura Y. Takai Y. EMBO J. 1996; 15: 2196-2207Crossref PubMed Scopus (181) Google Scholar) and down-regulated by the GTPase-activating proteins (GAPs) Sac7 and Bem2 (15.Peterson J. Zheng Y. Bender L. Myers A. Cerione R. Bender A. J. Cell Biol. 1994; 127: 1395-1406Crossref PubMed Scopus (159) Google Scholar, 16.Schmidt A. Bickle M. Beck T. Hall M.N. Cell. 1997; 88: 531-542Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Among other functions, Rho1 is known to bind and activate Pkc1 (17.Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (302) Google Scholar, 18.Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). This protein kinase in turn activates a MAPK cascade that is composed of the MAPKK kinase Bck1/Slk1 (19.Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (269) Google Scholar, 20.Costigan C. Gehrung S. Snyder M. Mol. Cell. Biol. 1992; 12: 1162-1178Crossref PubMed Scopus (200) Google Scholar), the redundant MAPKKs Mkk1 and Mkk2 (21.Irie K. Takase M. Lee K.S. Levin D.E. Araki H. Matsumoto K. Oshima Y. Mol. Cell. Biol. 1993; 13: 3076-3083Crossref PubMed Scopus (258) Google Scholar), and the MAPK Slt2/Mpk1 (7.Torres L. Martin H. Garcia-Saez M.I. Arroyo J. Molina M. Sanchez M. Nombela C. Mol. Microbiol. 1991; 5: 2845-2854Crossref PubMed Scopus (144) Google Scholar, 22.Lee K.S. Irie K. Gotoh Y. Watanabe Y. Araki H. Nishida E. Matsumoto K. Levin D.E. Mol. Cell. Biol. 1993; 13: 3067-3075Crossref PubMed Scopus (308) Google Scholar). Signaling is transmitted through this protein kinase cascade by sequential phosphorylation. MAPKKs are dual specificity protein kinases that catalyze the phosphorylation of the MAPK on both tyrosine and threonine conserved residues in subdomain VIII, leading to the activation of this last element of the cascade. Transcription of a number of genes involved in cell wall biosynthesis has been shown to be dependent on this pathway (23.Igual J.C. Johnson A.L. Johnston L.H. EMBO J. 1996; 15: 5001-5013Crossref PubMed Scopus (221) Google Scholar, 24.Zhao C. Jung U.S. Garrett-Engele P. Roe T. Cyert M.S. Levin D.E. Mol. Cell. Biol. 1998; 18: 1013-1022Crossref PubMed Google Scholar), indicating the importance of a functional pathway to ensure cell integrity. Hyperactivation of MAPK pathways is known to lead to severe growth defects. Therefore, activation of MAPKs needs to be tightly controlled by mechanisms that inhibit the activity of the pathways when there is no activating input and that rapidly down-regulate this activity following stimulation. Protein phosphatases that negatively regulate the HOG and the mating pathways have been characterized. The tyrosine phosphatases Ptp2 and Ptp3 operate in both pathways, playing a role in maintaining a low basal level of tyrosine-phosphorylated Hog1 and Fus3 and in the adaptation to osmotic shock and pheromone treatment (25.Jacoby T. Flanagan H. Faykin A. Seto A.G. Mattison C. Ota I. J. Biol. Chem. 1997; 272: 17749-17755Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 26.Wurgler-Murphy S.M. Maeda T. Witten E.A. Saito H. Mol. Cell. Biol. 1997; 17: 1289-1297Crossref PubMed Scopus (220) Google Scholar, 27.Zhan X.L. Deschenes R.J. Guan K.L. Genes Dev. 1997; 11: 1690-1702Crossref PubMed Scopus (125) Google Scholar). An additional phosphatase has been shown to act in the mating pathway, the dual specificity phosphatase Msg5, but it only participates in adaptation by inactivating Fus3 following pheromone stimulation (27.Zhan X.L. Deschenes R.J. Guan K.L. Genes Dev. 1997; 11: 1690-1702Crossref PubMed Scopus (125) Google Scholar, 28.Doi K. Gartner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (204) Google Scholar). MSG5 has also been identified as a multicopy suppressor of a hyperactive MKK1 allele (29.Watanabe Y. Irie K. Matsumoto K. Mol. Cell. Biol. 1995; 15: 5740-5749Crossref PubMed Scopus (170) Google Scholar). Additionally, a genetic interaction of the type 2C serine threonine phosphatase gene PTC1 with the PKC1 pathway has been reported (30.Huang K.N. Symington L.S. Genetics. 1995; 141: 1275-1285Crossref PubMed Google Scholar). However, mechanisms responsible for the negative modulation of Slt2 remain largely unknown. In this study, we have used an anti-active MAPK antibody raised against the dually phosphorylated region (Thr202/Tyr204) within the catalytic core of p44/42 MAPKs to specifically detect the active form of the MAPK Slt2. Using this "read-out" for signaling through the cell integrity pathway, we have identified caffeine and vanadate as novel compounds that lead to Slt2 activation and have studied how cells sense these stimuli. We report that Sac7 and Bem2 are negative regulators of this pathway. Additionally, we found that Mkk1 plays a more important role than Mkk2 in signaling to Slt2. Finally, evidence for a role of Msg5 in Slt2 dephosphorylation is also presented. S. cerevisiaestrains used in this study are listed in TableI. Standard procedures were employed for yeast genetic manipulations (31.Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2526) Google Scholar). For strain BJ5464-DK, SLT2was disrupted with URA3 as described previously (7.Torres L. Martin H. Garcia-Saez M.I. Arroyo J. Molina M. Sanchez M. Nombela C. Mol. Microbiol. 1991; 5: 2845-2854Crossref PubMed Scopus (144) Google Scholar). Yeast transformations were performed by the lithium acetate method (32.Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar).Table IStrains used in this workStrainRelevant genotypeSource or referenceBJ5464MATa, ura3–52, leu2Δ1, trp1, his3Δ200 pep4::HIS2, pbr1Δ1.6R, can1Yeast Genetic Stock CenterBJ5464-DKMATa, ura3–52, leu2Δ1, trp1, his3Δ200 pep4::HIS2, pbr1Δ1.6R, can1, slt2Δ::URA3This workTD28MATa, ura3–52, inos1–151, can RRef. 7.Torres L. Martin H. Garcia-Saez M.I. Arroyo J. Molina M. Sanchez M. Nombela C. Mol. Microbiol. 1991; 5: 2845-2854Crossref PubMed Scopus (144) Google ScholarTD28-F54MATa, ura3–52, inos1–151, can R, slt2-F54Ref. 36.Martin H. Arroyo J. Sanchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (111) Google Scholar1783MATa, ura3–52, his4, trp1–1, leu2–3,112, can RRef. 22.Lee K.S. Irie K. Gotoh Y. Watanabe Y. Araki H. Nishida E. Matsumoto K. Levin D.E. Mol. Cell. Biol. 1993; 13: 3067-3075Crossref PubMed Scopus (308) Google ScholarDL454MATa, ura3–52, his4, trp1–1, leu2–3,112, can R slt2Δ::TRP1Ref. 22.Lee K.S. Irie K. Gotoh Y. Watanabe Y. Araki H. Nishida E. Matsumoto K. Levin D.E. Mol. Cell. Biol. 1993; 13: 3067-3075Crossref PubMed Scopus (308) Google ScholarDL443MATa, slt2D35, trp1–1, leu2–3,112, ura3–52Ref. 22.Lee K.S. Irie K. Gotoh Y. Watanabe Y. Araki H. Nishida E. Matsumoto K. Levin D.E. Mol. Cell. Biol. 1993; 13: 3067-3075Crossref PubMed Scopus (308) Google ScholarOHNY1MATa, ura3, his3, trp1, leu2, ade2Ref. 17.Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (302) Google ScholarHNY21MATa, ura3, his3, trp1, leu2, ade2, rho1–104Ref. 52.Yamochi W. Tanaka K. Nonaka H. Maeda A. Musha T. Takai Y. J. 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Acta. 1995; 1260: 94-96Crossref PubMed Scopus (14) Google ScholarW303–1Asms1(mid2)ΔMATa ade2–1 can1–100 trp1–1 ura3–1 his3–11,15 leu2–3,112 sms1Δ::URA3Ref. 39.Takeuchi J. Okada M. Toh Kikuchi Y. Biochim. Biophys. Acta. 1995; 1260: 94-96Crossref PubMed Scopus (14) Google Scholar Open table in a new tab General DNA methods were employed using standard techniques (33.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Plasmids used in this study are as follows: pEG-KG-MKK1/2 are pEG-KG (URA3 GAL1-GST leu2-d 2μ) (34.Mitchell D.A. Marshall T.K. Deschenes R.J. Yeast. 1993; 9: 715-722Crossref PubMed Scopus (265) Google Scholar) -based plasmids carrying the MKK1 andMKK2 structural genes. Details of the construction and structure of these plasmids are described by Soler et al. (35.Soler M. Plovins A. Martin H. Molina M. Nombela C. Mol. Microbiol. 1995; 17: 833-842Crossref PubMed Scopus (35) Google Scholar). pHR0 is pHR70-HIS3 (URA3 HIS3 CEN) containingSLT2 (36.Martin H. Arroyo J. Sanchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (111) Google Scholar). YEplacMSG5 (28.Doi K. Gartner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (204) Google Scholar) is YEplac181 (LEU2 2μ) (37.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2500) Google Scholar) harboringMSG5. YCpMSG5 was constructed by inserting a 4-kilobase pairXbaI–PstI fragment of YEpMSG5 containingMSG5 into the vector YCplac111 (LEU2 CEN) (37.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2500) Google Scholar) and then inserting into the XbaI site of this plasmid a 1.2-kilobase pair XbaI–XbaI fragment bearingURA3. pRS316-BCK1–20 is pRS316 (URA3 CEN) (38.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) containing BCK1–20 (19.Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (269) Google Scholar). pYES2-SLT2 and pYES2-SNQ2 are pYES2 (URA3 GAL1 2μ) (Invitrogen) containing the cDNA sequences encoding Slt2 and Snq2, respectively. YEPD (1% yeast extract, 2% peptone, and 2% glucose) broth or agar was the complete medium used for growing yeast strains. YEPG was YEPD with 2% galactose instead of glucose. Synthetic minimal medium (SD) contained 0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% ammonium sulfate, and 2% glucose and was supplemented with appropriate amino acids and nucleic acid bases (31.Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2526) Google Scholar). SG and SR were SD with 2% galactose or raffinose, respectively, instead of glucose. Where indicated,d-sorbitol was added to the media to a final concentration of 1 m. When needed, caffeine and sodium orthovanadate (Sigma) were dissolved in sterile water just before use and added to the medium to the desired concentration. Galactose induction experiments in liquid were performed by growing cells in SR minimal selective medium to log phase and then by adding galactose to 2% for 8 h. Yeast cells were grown overnight to mid-log phase in the appropriate medium. The cultures were then diluted to A 600 = 0.2 and grown for one generation prior to collection, shifting to 39 °C, or treatment with the desired compound for the duration of the experiment. Cells were collected on ice by adding 20 ml of the culture to an equal volume of ice in a Falcon centrifuge tube and pelleted in a refrigerated centrifuge. Cells were then transferred with 1 ml of ice-cold water to an Eppendorf tube, pelleted, and immediately broken or frozen in dry ice. Cells were lysed in 120 μl of cold lysis buffer (50 mm Tris-HCl (pH 7.5), 10% glycerol, 1% Triton X-100, 0.1% SDS, 150 mm NaCl, 50 mm NaF, 1 mm sodium orthovanadate, 50 mm β-glycerol phosphate, 5 mm sodium pyrophosphate, 5 mmEDTA, 1 mm phenylmethylsulfonylfluoride, and the protease inhibitors tosylphenylalanine chloromethyl ketone, tosyllysine chloromethyl ketone, leupeptin, pepstatin A, antipain, and aprotinin each at 25 μg/ml) by vigorous shaking with 0.45-mm glass beads in a fast prep cell breaker (Bio 101; level of 5.5 for 20 s). Cell extracts were separated from glass beads and cell debris, collected in a new Eppendorf tube by centrifugation, and further clarified by a 13,000 × g spin for 15 min at 4 °C. The protein concentration of the supernatants was measured at 280 nm and normalized with lysis buffer. Then 2× SDS-polyacrylamide gel electrophoresis sample loading buffer was added, and samples were boiled for 5 min. Protein samples (50 μg) were fractionated by SDS-polyacrylamide gel electrophoresis using 8% polyacrylamide gels and transferred to nitrocellulose membranes (Hybond; Amersham Pharmacia Biotech). Membranes were probed with either anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody (New England Biolabs) at 1:2000 dilution to detect dually phosphorylated Slt2 or with anti-GST-Slt2 antibody (36.Martin H. Arroyo J. Sanchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (111) Google Scholar) at 1:1000 dilution to detect Slt2 or GST fusion proteins in the presence of 5% nonfat milk for 2 h at room temperature. The primary antibody was detected using a horseradish peroxidase-conjugated anti-rabbit antibody with the ECL detection system. Alternatively, to monitor the amount of Slt2, blots were then stripped and reprobed with anti-GST-Slt2 antibody. Then immunoreactivity was localized again as described above. To gain further insight into the function and regulation of the cell integrity pathway, we used a commercial specific antibody raised against dually phosphorylated (Thr202/Tyr204)-p44/42 MAPK (New England Biolabs) as a tool to easily monitor the phosphorylation in Thr190 and Tyr192 of Slt2. The catalytic kinase activity of this family of proteins depends on phosphorylation of both conserved residues (40.Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1653) Google Scholar), and thereby this antibody can be used to detect the activation status of the Slt2 MAPK. Slt2 kinase activation has been reported to occur after a mild heat shock (4.Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (419) Google Scholar). Therefore, we used this antibody to perform immunoblots with extracts from the strain BJ5464 and the isogenic slt2 deleted mutant BJ5464-DK growing at 24 °C and at 39 °C. This background was chosen for these experiments because of the low level of protease activity. As shown in Fig. 1, whereas extracts from wild type cells growing at 24 °C presented a weak band corresponding to the basal level of the dually phosphorylated form of Slt2, the shift of growing cells to 39 °C for 2 h resulted in a strong increase of the Slt2 signal intensity. This band was absent in the control lane from the slt2Δ strain. Osmotic stabilization of the cells at 39 °C prevented the rise in the corresponding phospho-Slt2 signal. Immunoblot analysis with anti-GST-Slt2 antibody (36.Martin H. Arroyo J. Sanchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (111) Google Scholar) showed that the Slt2 levels were approximately constant throughout the experiment, indicating that the anti-phospho-MAPK antibody recognizes the Slt2 protein, which is phosphorylated as a consequence of the pathway activation. This conclusion is in agreement with previous data (10.Verna J. Lodder A. Lee K. Vagts A. Ballester R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13804-13809Crossref PubMed Scopus (315) Google Scholar) using the same antibody on extracts from cells overexpressing Slt2-HA. To reinforce the evidence that indicates that this method is accurate at detecting signaling through the pathway, we tested whether Pkc1 hyperactivation resulted in an increased phospho-Slt2 signal in Western blots. The substitution of amino acid 398 of Pkc1, located in the pseudosubstrate site, leads to the incapacitation of this site and thereby to the constitutive activation of the kinase (17.Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (302) Google Scholar, 41.Watanabe M. Chen C.Y. Levin D.E. J. Biol. Chem. 1994; 269: 16829-16836Abstract Full Text PDF PubMed Google Scholar). We used the plasmid pEX-PKC1 A398A405A406, bearing a dominant constitutively activated allele of PKC1 in which the arginine 398 and the basic conserved residues of the pseudosubstrate site arginine 405 and lysine 406 have been changed to alanine (PKC1 A398A405A406), placed under the control of the strong GAL1 promoter in the multicopy vector Yeplac112. 2A. Mendoza and J. F. Garcı́a-Bustos, unpublished results. As shown in Fig.1, induction of PKC1 A398A405A406 expression in the strain BJ5464 led to a dramatic increase in the phospho-Slt2-dependent signal in Western blots. These results confirmed the effectiveness of the assay to monitor the level of activation of the pathway and indicated that only a fraction of cellular Slt2 is phosphorylated under mild heat shock treatment. The presence of 1 m sorbitol in the medium did not reduce the level of Slt2 phosphorylation in cells overexpressing the hyperactive PKC1 allele (Fig. 1). This is consistent with the currently accepted model in which the osmotic stabilization of the medium prevents Slt2 activation by physical stabilization of the cell surface. In this case, activation of the pathway would not be triggered from the cell surface but as a consequence of the constitutive activation of an upstream element of the MAPK cascade. In accordance with a previous work using other hyperactivePKC1 alleles (41.Watanabe M. Chen C.Y. Levin D.E. J. Biol. Chem. 1994; 269: 16829-16836Abstract Full Text PDF PubMed Google Scholar), overproduction of Pkc1A398A405A406 completely inhibited the growth of wild type strains. However, the deleterious effect on growth does not seem to be only a consequence of the strong Slt2 activation, since overexpression of this PKC1 allele in a slt2Δmutant also led to cell lethality (data not shown). This result provides additional evidence that Pkc1 acts through a bifurcated pathway. We exploited the anti-phospho-p44/42 MAPK antibody in an attempt to determine conditions that lead to Slt2 activation. Because sensitivity to caffeine and vanadate is a characteristic phenotype in mutants affected in the Pkc1 pathway (20.Costigan C. Gehrung S. Snyder M. Mol. Cell. Biol. 1992; 12: 1162-1178Crossref PubMed Scopus (200) Google Scholar, 42.Martin H. Castellanos M.C. Cenamor R. Sanchez M. Molina M. Nombela C. Curr. Genet. 1996; 29: 516-522Crossref PubMed Scopus (45) Google Scholar, 43.Nakamura T. Ohmoto T. Hirata D. Tsuchiya E. Miyakawa T. Mol. Gen. Genet. 1997; 256: 481-487Crossref PubMed Scopus (9) Google Scholar), it was tempting to speculate that activation of this pathway is essential for resistance to these compounds. Therefore, we analyzed the effect of caffeine and vanadate on Slt2 phosphorylation and compared it with that of thermal stress. Wild type cells growing at 24 °C were either shifted to 39 °C or treated with these chemicals, and the amount of phospho-Slt2 was observed at different time points. As shown in Fig.2 A, 5 min after both the temperature shift and the addition of 5 mm vanadate, a significant increase in Slt2 phosphorylation was observed. The amount of phospho-Slt2 increased with time, peaking at 20–30 min in both cases and remaining at this level at the later time points. The addition of 12 mm caffeine in the medium resulted in a slower time course of Slt2 phosphorylation. No further variation in the phospho-Slt2 signal induced by the three stimuli was detected in the following 4 h (data not shown). The presence of 1 m sorbitol in the medium abolished Slt2 activation in response to both compounds as happened under thermal stress (Fig. 2 B). Kamada et al. (4.Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (419) Google Scholar) have shown that prevention of heat-induced Slt2 activation by osmotic stabilization was not a consequence of activating the HOG pathway. Therefore, we determined the level of phospho-Slt2 in thehog1 mutant JBY10 under caffeine and vanadate exposure. Fig.2 C shows that in this mutant the induced Slt2 phosphorylation was also inhibited by 1 m sorbitol, indicating that high osmolarity prevents the pathway activation in response to both agents by mechanisms independent of Hog1. These data suggest that all of these stimuli trigger the activation of the cell integrity pathway by a common mechanism that can be inhibited by osmotic stabilization. The ability of sorbitol to rescue the sensitivity of mutants affected in the cell integrity pathway to caffeine (42.Martin H. Castellanos M.C. Cenamor R. Sanchez M. Molina M. Nombela C. Curr. Genet. 1996; 29: 516-522Crossref PubMed Scopus (45) Google Scholar) and vanadate (data not shown) and to prevent Slt2 phosphorylation induced by these agents suggests that these compounds could be perturbing cell wall stability. In this case, activation of the pathway in response to these stimuli would be mediated by the previously identified cell surface sensors. Mid2 has been shown to be required for induction of Slt2 tyrosine phosphorylation during exposure to high temperature, mating pheromones, and Calcofluor White (6.Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar). Calcofluor White interferes with cell wall assembly; therefore, a role fo
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