Sit4 Is Required for Proper Modulation of the Biological Functions Mediated by Pkc1 and the Cell Integrity Pathway inSaccharomyces cerevisiae
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.m203515200
ISSN1083-351X
AutoresMaría Ángeles de la Torre-Ruiz, Jordi Torres‐Rosell, Joaquı́n Ariño, Enrique Herrero,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMaintenance of cellular integrity inSaccharomyces cerevisiae is carried out by the activation of the protein kinase C-mediated mitogen-activated protein kinase (PKC1-MAPK) pathway. Here we report that correct down-regulation of both basal and induced activity of the PKC1-MAPK pathway requires the SIT4 function. Sit4 is a protein phosphatase also required for a proper cell cycle progression. We present evidence demonstrating that the G1 to S delay in the cell cycle, which occurs as a consequence of the absence of Sit4, is mediated by up-regulation of Pkc1 activity. Sit4 operates downstream of the plasma membrane sensors Mid2, Wsc1, and Wsc2 and upstream of Pkc1. Sit4 affects all known biological functions involving Pkc1, namely Mpk1 activity and cell wall integrity, actin cytoskeleton organization, and ribosomal gene transcription. Maintenance of cellular integrity inSaccharomyces cerevisiae is carried out by the activation of the protein kinase C-mediated mitogen-activated protein kinase (PKC1-MAPK) pathway. Here we report that correct down-regulation of both basal and induced activity of the PKC1-MAPK pathway requires the SIT4 function. Sit4 is a protein phosphatase also required for a proper cell cycle progression. We present evidence demonstrating that the G1 to S delay in the cell cycle, which occurs as a consequence of the absence of Sit4, is mediated by up-regulation of Pkc1 activity. Sit4 operates downstream of the plasma membrane sensors Mid2, Wsc1, and Wsc2 and upstream of Pkc1. Sit4 affects all known biological functions involving Pkc1, namely Mpk1 activity and cell wall integrity, actin cytoskeleton organization, and ribosomal gene transcription. mitogen-activated protein kinase hemagglutinin fluorescence activated cell sorter ribosomal proteins The Saccharomyces cerevisiae gene SIT4 codes for a Ser/Thr protein phosphatase member of the PPP phosphatase family that is closely related to the PP2A family (1Arndt K.T. Styles C.A. Fink G.R. Cell. 1989; 56: 527-5371Abstract Full Text PDF PubMed Scopus (177) Google Scholar, 2Doseff A.I. Arndt K.T. Genetics. 1995; 141: 857-871Crossref PubMed Google Scholar). Sit4 displays a high level of identity to both the fission yeast phosphatase ppe1and the human protein phosphatase 6, which are involved in cell cycle regulation (3Shimanuki M. Kinoshita N. Ohkura H. Yoshida T. Toda T. Yanagida M. Mol. Biol. Cell. 1993; 4: 303-313Crossref PubMed Scopus (69) Google Scholar, 4Bastians H. Ponsting H. J. Cell Sci. 1996; 109: 2865-2874Crossref PubMed Google Scholar). Sit4 participates in a number of cellular processes such as the Tor pathway-mediated response to nutrients (5Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (437) Google Scholar, 6Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (789) Google Scholar, 7Jiang Y. Broach J.R. EMBO J. 1999; 18: 2782-2792Crossref PubMed Scopus (272) Google Scholar) and the regulation of monovalent ion homeostasis and intracellular pH (8Masuda C.A. Ramıárez J. Peña A. Montero-Lomelıá M. J. Biol. Chem. 2000; 40: 30957-30961Abstract Full Text Full Text PDF Scopus (41) Google Scholar). Sit4 also plays an important role in cell cycle regulation, as it is required for the proper G1 to S phase transition (9Sutton A. Inmanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (270) Google Scholar, 10Mann D.J. Dombradi V. Cohen P.T. EMBO J. 1993; 12: 4833-4842Crossref PubMed Scopus (45) Google Scholar). Cells deleted for SIT4 are either nonviable or display slow growth because of an expanded passage through G1 (9Sutton A. Inmanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (270) Google Scholar,11Di Como C.J. Bose R. Arndt K.T. Genetics. 1995; 139: 95-107Crossref PubMed Google Scholar, 12Sutton A. Freiman R. Genetics. 1997; 147: 57-71Crossref PubMed Google Scholar, 13Clotet J. Garıá E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google Scholar). This delay is partly because of the role of SIT4 in the normal transcription control of the G1 cyclin genesCLN1 and CLN2, and also in the control ofSWI4, coding for a DNA-binding protein required for transcriptional modulation of CLN1/CLN2 (14Fernandez-Sarabia M.J. Sutton A. Zhong T. Arndt K.T. Genes Dev. 1992; 6: 2417-2428Crossref PubMed Scopus (114) Google Scholar, 11Di Como C.J. Bose R. Arndt K.T. Genetics. 1995; 139: 95-107Crossref PubMed Google Scholar). In addition, SIT4 is believed to function in a pathway parallel to CLN3 for the activation of CLN1 andCLN2 expression through BCK2 (15Di Como C.J. Chang H. Arndt K.T. Mol. Cell. Biol. 1995; 15: 1835-1846Crossref PubMed Scopus (92) Google Scholar). Ppz1 and Ppz2 (16Posas F. Casamayor A. Morral N. Ariño J. J. Biol. Chem. 1992; 267: 11734-11740Abstract Full Text PDF PubMed Google Scholar, 17Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar) represent another subset of Ser/Thr protein phosphatases, which play an opposite role to Sit4 in cell cycle regulation (13Clotet J. Garıá E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google Scholar). The absence of PPZ1 compensates for the delay in cyclin accumulation and also alleviates the budding defect observed in a sit4Δ mutant (13Clotet J. Garıá E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google Scholar). PPZ1 has been reportedly involved in the maintenance of cell integrity in cooperation with the PKC1-mitogen activated protein kinase (MAPK)1 pathway (17Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar). Overproduction of Ppz1 suppresses the lysis phenotype of null mutants in PKC1 and MPK1 (17Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar). The PKC1-MAPK pathway is a phosphorylation cascade that responds to signals related to yeast cell integrity, such as: mating pheromone (18Zarzov P. Mazzoni C. Mann C. EMBO J. 1996; 15: 83-91Crossref PubMed Scopus (197) Google Scholar), low osmolarity (19Davenport 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), and high temperatures (20Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (419) Google Scholar). Mpk1/Slt2 is the last kinase member of the pathway. Simultaneous deletion of MPK1 and PPZ1is lethal for the cell (17Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar). Cell wall stress is detected by the plasma membrane sensors Mid2 (21Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar), Wsc1/Hsc77/Slg1, Wsc2, and Wsc3 (22Delley P.A. Hall M.N. J. Cell Biol. 1999; 147: 163-174Crossref PubMed Scopus (241) Google Scholar, 23Verna 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), and the signal is transmitted downstream via the GTP-binding protein Rho1 that activates the PKC1-MAPK module (21Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar, 22Delley P.A. Hall M.N. J. Cell Biol. 1999; 147: 163-174Crossref PubMed Scopus (241) Google Scholar). Pkc1 phosphorylates the MAPK kinase kinase Bck1 (24Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (269) Google Scholar), which in turn, transmits the signal to the redundant MAPK kinases: Mkk1 and Mkk2 (25Irie 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). These finally phosphorylate the Slt2/Mpk1 MAPK (26Torres L. Martin H. Garcia-Saez M.I. Arroyo J. Molina M. Sánchez M. Nombela C. Mol. Microbiol. 1991; 5: 2845-2854Crossref PubMed Scopus (144) Google Scholar) on both Tyr192 and Thr190 residues (19Davenport 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, 27Lee 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, 28Lim Y.M. Tsuda L. Inoue Y.H. Irie K. Adachi-Yamada T. Hata M. Nishi Y. Matsumoto K. Nishida Y. Genetics. 1997; 146: 263-273Crossref PubMed Google Scholar) causing the activation of the kinase. Phosphorylation and activation of Mpk1 leads to a number of cellular responses. Thus, activation of Mpk1 results in phosphorylation of the transcriptional factor Swi6 through which the pathway is linked to the cell cycle regulatory machinery (29Sidorova J.M. Mikesell G.E. Breeden L.L. Mol. Biol. Cell. 1995; 6: 1641-1658Crossref PubMed Scopus (85) Google Scholar, 30Madden K. Sheu Y.J. Baetz K. Andrews B. Snyder M. Science. 1997; 275: 1781-1784Crossref PubMed Scopus (206) Google Scholar). The PKC1 pathway is also involved in budding control (18Zarzov P. Mazzoni C. Mann C. EMBO J. 1996; 15: 83-91Crossref PubMed Scopus (197) Google Scholar) and cell wall synthesis (21Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar), by regulating (i) the expression (often in a cell cycle-dependent fashion) of several genes coding for proteins related to cell wall synthesis and structure (31Igual J.C. Johnson A.L. Johnston L.H. EMBO J. 1996; 15: 5001-5013Crossref PubMed Scopus (221) Google Scholar, 32Zhao C. Jung U.S. Garret-Engele P. Roe T. Cyert M.S. Levin D.E. Mol. Cell. Biol. 1998; 18: 1013-1022Crossref PubMed Google Scholar, 33Jung U.S. Levin D.E. Mol. Microbiol. 1999; 34: 1049-1057Crossref PubMed Scopus (351) Google Scholar, 34de Nobel H. Ruiz C. Martin H. Morris W. Bru S. Molina M. Klis F.M. Microbiology. 2000; 146: 2121-2132Crossref PubMed Scopus (221) Google Scholar), and (ii) the organization of the actin cytoskeleton (35Helliwell S.B. Schmidt A. Ohya Y. Hall M.N. Curr. Biol. 1998; 8: 1211-1214Abstract Full Text Full Text PDF PubMed Google Scholar). Genetic evidence indicates that Ppz1/Ppz2 phosphatases act independently of the PKC1-MAPK pathway (17Lee K.S. Hines L.K. Levin D.E. Mol. Cell. Biol. 1993; 13: 5843-5853Crossref PubMed Scopus (115) Google Scholar). Their role therefore seems to be different from that of other phosphatases, such as Ptp2/Ptp3 (36Mattison C.P. Spencer S.S. Kresge K.A. Lee J. Ota I.M. Mol. Cell. Biol. 1999; 19: 7651-7660Crossref PubMed Scopus (86) Google Scholar) or Msg5 (37Martıán H. Rodriguez-Pachoán J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 14: 1511-1519Abstract Full Text Full Text PDF Scopus (291) Google Scholar), which are known to dephosphorylate and negatively regulate certain components of the pathway. The observation that Ppz1 and Sit4 exhibit a functional antagonism in cell cycle regulation (13Clotet J. Garıá E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google Scholar) prompted us to investigate whether this antagonism could also be extended to the functional connection with the PKC1 pathway. Here we demonstrate that Sit4 is required for down-regulation of Pkc1 activity, and is consequently needed for a number of functions that depend on this kinase, such as Mpk1 activity, cytoskeleton organization, ribosomal gene expression, and cell cycle progression. The S. cerevisiae strains used in this work are listed in Table I. Unless otherwise stated, they are derived from either CML125 or CML128 wild type strains (38Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). The following strains were obtained from their corresponding diploids by tetrad analysis: MML200 and MML203 from MML182, MML216 from MML185, MML231 and MML233 from MML230, and MML344 and MML345 from MML282.Table IYeast strains used in this workStrainRelevant genotypeRef.CML125MATα leu2–3,112 ura3–52 trp1 his4 can1rThis workCML128MATa as CML125Gallego et al. (39Gallego C. Garıá E. Colomina N. Herrero E. Aldea M. EMBO J. 1997; 16: 7196-7206Crossref PubMed Scopus (136) Google Scholar)JA-110MATasit4:TRP1Clotet et al.(13Clotet J. Garıá E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google Scholar)JA-117MATasit4:kanMX4Clotet et al.(13Clotet J. Garıá E. Aldea M. Ariño J. Mol. Cell. Biol. 1999; 19: 2408-2415Crossref PubMed Scopus (67) Google Scholar)CML399MATampk1:URA3This workMML182MATa/αSIT4/sit4:TRP1 BCK1/bck1:kanMX4This workMML200MATαbck1:kanMX4This workMML203MATasit4:TRP1 bck1:kanMX4This workMML282MATa/αPKC1/pkc1:LEU2, SIT4/sit4:kanMX4This workMML302MATα pkc1:LEU2 sit4:kanMX4(pBCK1–20)This workMML304MATαpkc1:LEU2(pBCK1–20)This workMML344MATapkc1:LEU2This workMML345MATα pkc1:LEU2 sit4:kanMX4This workMML382MATawsc1:CaURA3This workMML384MATawsc2:natMX4This workMML387MATamid2:kanMX4This workMML388MATamid2:kanMX4sit4:TRP1This workMML392MATawsc1:CaURA3 wsc2:natMX4This workMML393MATawsc1:CaURA3 mid2:kanMX4This workMML398MATawsc2:natMX4sit4:TRP1This workMML400MATawsc1:CaURA3 wsc2:natMX4 sit4:TRP1This workMML400MATawsc1:CaURA3 mid2:kanMX4 sit4:TRP1This workMML402MATawsc1:CaURA3 sit4:TRP1This work Open table in a new tab Yeast cells were usually grown in YPD medium (2% yeast extract, 1% peptone, 2% glucose) or in the selective glucose minimal medium, SD (0.67% yeast nitrogen base, 2% glucose, and the required amino acids) (39Gallego C. Garıá E. Colomina N. Herrero E. Aldea M. EMBO J. 1997; 16: 7196-7206Crossref PubMed Scopus (136) Google Scholar). Where indicated, d-sorbitol was added to a final concentration of 1 m. To repress expression of thePKC1 gene under the tetO7 promoter (40Garıá E. Piedrafita L. Aldea M. Herrero E. Yeast. 1997; 13: 837-848Crossref PubMed Scopus (500) Google Scholar), cells were, respectively, grown in SD plus 10 μg/ml doxycycline until early log phase, then filtered and washed in the same medium without doxycycline. Cells were then resuspended in YPD and incubated for 6 h at 25 °C. After that, cultures were split in two. One-half was kept at the same temperature and the other was shifted to 37 °C for 30 min. Cells were subsequently collected by filtration and treated for total protein extraction as described in Ref. 41Watanabe M. Chen C.Y. Levin D.E. J. Biol. Chem. 1994; 269: 16829-16836Abstract Full Text PDF PubMed Google Scholar. Yeast transformations were performed as described in Ref. 42Gietz R.D., St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 220: 1425Crossref Scopus (2863) Google Scholar. TheMPK1 gene was disrupted using a URA3 cassette (26Torres L. Martin H. Garcia-Saez M.I. Arroyo J. Molina M. Sánchez M. Nombela C. Mol. Microbiol. 1991; 5: 2845-2854Crossref PubMed Scopus (144) Google Scholar). The URA3 marker from Candida albicans (43Goldstein A.L. Pan X. McCusker J.H. Yeast. 1999; 15: 507-511Crossref PubMed Scopus (102) Google Scholar) was used to disrupt the WSC1 gene according to the one-step disruption method (44Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2217) Google Scholar). This method was also employed to disrupt theMID2, BCK1, and MSG5 genes with thekanMX4 module and the WSC2 gene with thenatMX4 module (45Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1340) Google Scholar). DNA manipulation, plasmid recovery, and bacterial transformation were performed according to standard methods (46Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1989Google Scholar). Escherichia coli DH5α (Invitrogen) was used for plasmid amplification. YEplac195-SIT4 (TRP2μ) plasmid harbors a genomic 2.65-kb SnaBI-NheI fragment containing the SIT4 gene cloned in the SmaI-XbaI restriction site of YEplac195 (47Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2500) Google Scholar). Plasmid pMM66 is a YEplac195 derivative (URA3/2μ) (48Johnston L.H. Hohnson A.L. Methods Enzymol. 1997; 283: 342-350Crossref PubMed Scopus (21) Google Scholar) containing MSG5 under its own promoter cloned at the SmaI vector site.MSG5 was amplified by PCR from yeast genomic DNA. Plasmid pMM69 is a YEplac195 derivative harboring (PCR-amplified)PTP2 under its own promoter and cloned into theKpnI and HindIII vector sites. Plasmid pMM126 is a pCM265 derivative (URA3/ CEN) (40Garıá E. Piedrafita L. Aldea M. Herrero E. Yeast. 1997; 13: 837-848Crossref PubMed Scopus (500) Google Scholar) that containsPCK1 under the tetO7 promoter and is tagged with three copies of the HA epitope at the N terminus of the protein. The PCK1-coding sequence was obtained from genomic DNA by PCR and directionally cloned into PmeI andPstI vector sites (47Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2500) Google Scholar). The pRS413-BCK1–20plasmid is described in Ref. 24Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (269) Google Scholar. The pMpk1-HA plasmid is a YEP352 derivative, Mpk1 ORF is cloned under its own promoter and HA-tagged in C-terminal (a gift from Maria Molina, University Complutense, Madrid, Spain). For synchronization experiments, cells were exponentially grown to 107 cells/ml. S and G2 arrests were performed with hydroxyurea and nocodazole, respectively, at the concentrations indicated in the text. G1 arrests were performed either by α-factor treatment (10 μg/ml) or by elutriation. All cell cycle arrests were performed at 25 °C for 2 h in the case of the wild type strain and for 4 h in the case of the sit4Δ mutant. Cells were elutriated according to the protocol described in Ref. 48Johnston L.H. Hohnson A.L. Methods Enzymol. 1997; 283: 342-350Crossref PubMed Scopus (21) Google Scholar. Fluorescence-activated cell sorting (FACS) sample analysis (49Nash R. Tokiwa G. Anand S. Erickson K. Futcher A.B. EMBO J. 1988; 7: 4335-4346Crossref PubMed Scopus (378) Google Scholar) was used to confirm correct synchronization. Cells were stained with rhodamine-phalloidin as described in Ref. 50Pringle J.R. Adams A.E.M. Drubin D.G. Haarer B.K. Methods Enzymol. 1991; 194: 565-602Crossref PubMed Scopus (598) Google Scholar. For Western analysis, cells were collected on ice, filtered through 0.22-μ Millipore membranes, washed with ice-cold medium, transferred to Eppendorf tubes with 1 ml of ice-cold medium, and then centrifuged for 30 s at 13,000 rpm. Total yeast protein extracts were prepared in ice-cold lysis buffer (75 mm Tris-HCl, pH 7.5, 0.45m KCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 1 mml-1-tosylamido-2-phenylethyl chloromethyl ketone, 1 mm pepstatin, 1 mm β-glycerophosphate, 1 mm EGTA, 5 mm sodium pyrophosphate, and the protease inhibitors chymostatin, leupeptin, and antipain each at 5 nmol/μl). After the addition of SDS to a final concentration of 1%, lysates were then boiled at 95 °C for 5 min. Equivalent amounts of total protein extracts were run on SDS-PAGE gels with 10% acrylamide. The anti-phospho-p44/p42 antibody (New England Biolabs) was used at a final dilution of 1:2000 in TBST buffer. The anti-Swi6 antibody was used at a dilution of 1:10,000 in the same buffer, and the anti-GST-Mpk1 antibody (37Martıán H. Rodriguez-Pachoán J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 14: 1511-1519Abstract Full Text Full Text PDF Scopus (291) Google Scholar, 51Martıán H. Arroyo J. Sánchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (111) Google Scholar) at a dilution of 1:1000 in the presence of 5% fat milk (51Martıán H. Arroyo J. Sánchez M. Molina M. Nombela C. Mol. Gen. Genet. 1993; 241: 177-184Crossref PubMed Scopus (111) Google Scholar). Horseradish peroxidase-linked secondary antibodies, anti-rabbit, or anti-mouse (NA931 and NA934, AmershamBiosciences), were used at a 1:10000 dilution and incubated in TBST buffer containing 2% fat milk for the anti-phospho-Mpk1 and 0.25% fat milk for the other two primary antibodies. Chemiluminescent detection was performed using the Supersignal substrate (Pierce) in a Lumi-Imager (Roche Molecular Biochemicals). Immunoprecipitation of Pkc1 and in vitro protein kinase assays were performed using either myelin basic protein (fragment 4-14, Sigma) or a Bck1-Ser939 synthetic peptide according to Ref. 41Watanabe M. Chen C.Y. Levin D.E. J. Biol. Chem. 1994; 269: 16829-16836Abstract Full Text PDF PubMed Google Scholar. To inhibit Pkc1 activity, 4 × 10−9m staurosporine was added to the kinase reaction (52Antonsson B. Montessuit S. Friedli L. Payton M.A. Paravicini G. J. Cell Biol. 1994; 269: 16821-16828Google Scholar). Mpk1 immunoprecipitation and protein kinase assay were conducted following the protocol described in Ref. 20Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (419) Google Scholar. We had previously tested Mpk1 basal and heat shock-induced levels of activity in a ppz1Δ mutant and observed that they were severely impaired (data not shown). 2M. A. de la Torre-Ruiz, J. Torres, J. Ariño, and E. Herrero, unpublished observations. To examine whether Mpk1 phosphorylation levels were higher in the absence in Sit4 than in wild type cells (antagonically to that observed in theppz1Δ mutant), we used the anti-phospho-p44/42 MAPK antibodies raised against dually phosphorylated (Thr202/Tyr204)-p44/42 MAPK. These antibodies allow accurate monitoring of Mpk1 activity (23Verna 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, 37Martıán H. Rodriguez-Pachoán J.M. Ruiz C. Nombela C. Molina M. J. Biol. Chem. 2000; 14: 1511-1519Abstract Full Text Full Text PDF Scopus (291) Google Scholar). Wild type andsit4Δ cells were grown at 25 °C and then shifted at 37 °C for various time periods (Fig.1A). sit4Δ cells showed increased basal phosphorylation levels as compared with wild type cells (Fig. 1A). Upon heat shock, Mpk1 phosphorylation was much more intense in mutant than wild type cells, and remained higher for longer periods. These changes could not be ascribed to variations in the total amount of Mpk1 protein, as their levels remained constant in all cases, a fact deduced by probing the same protein samples with an anti-GST-Mpk1 antibody (Fig. 1A). Quantification and normalization of phosphorylation levels revealed that after 30 min of heat shock, Mpk1 phosphorylation levels in thesit4Δ mutant were 10 times higher than in wild type cells (Fig. 1B). We performed an in vitro Mpk1 kinase assay in order confirm that the dual Mpk1 phosphorylation detected with the p44/42 antibody correlated to Mpk1 activity. As expected, we were able to detect greater Mpk1 kinase activity levels insit4Δ exponentially growing cells, both at 25 °C or after 30 min of shifting cells at 37 °C compared with the wild type (Fig. 1C). To prove that the increase in Mpk1 activity observed in a sit4Δ strain was not because of an indirect effect caused by the lack of this gene, we performed overexpression analyses by using a multicopy plasmid carrying Sit4 under its own promoter. Sit4 overexpression provoked a dramatic decrease in both the basal and induced phosphorylation state of Mpk1 in wild type cells (Fig. 1D), which demonstrates that Sit4 exercises specific regulatory control over Mpk1 activity. We reasoned that if Mpk1 was hyperphosphorylated in cells lacking Sit4, this could result in biological changes derived from kinase activation. To test this we examined the phosphorylation of Swi6 (a transcription factor whose phosphorylation after heat shock depends on Pkc1-mediated Mpk1 activation (29Sidorova J.M. Mikesell G.E. Breeden L.L. Mol. Biol. Cell. 1995; 6: 1641-1658Crossref PubMed Scopus (85) Google Scholar, 30Madden K. Sheu Y.J. Baetz K. Andrews B. Snyder M. Science. 1997; 275: 1781-1784Crossref PubMed Scopus (206) Google Scholar, 53Sheu Y.J. Santos B. Fortin N. Costigan C. Snyder M. Mol. Cell. Biol. 1998; 18: 4053-4059Crossref PubMed Scopus (195) Google Scholar)). We monitored Swi6 phosphorylation with polyclonal antibodies that detect two forms of the protein in wild type cells: a faster migrating band corresponding to the hypophosphorylated Swi6 state and a slower mobility hyperphosphorylated Swi6 band. In nonstressed wild type cells only the hypophosphorylated form was detected, and shifting the cells to 37 °C resulted in the appearance of hyperphosphorylated Swi6 (Fig. 1A). In contrast, in sit4Δ cell extracts hyperphosphorylated Swi6 was readily observed at 25 °C. Moreover, after heat shock the proportion of this form dramatically increased with respect to wild type cells, and remained higher throughout the experiment (Fig. 1A). Hyperphosphorylation of Swi6 insit4Δ cells after heat shock was fully mediated by Mpk1, as no changes in mobility were observed in a sit4Δ-mpk1Δdouble mutant (data not shown). In conclusion, the above results point to Sit4 being necessary for negative modulation of Mpk1 activity and, consequently also for downstream processes dependent on this activity. It could be hypothesized that the increased activation of Mpk1 found in sit4Δ cells derives from intrinsic cell wall defects in the mutant that would lead to constitutive hyperactivation of the PKC1-MAPK pathway. To test this possibility, we monitored Mpk1 phosphorylation in cells growing in the presence of 1 msorbitol used as an osmotic stabilizer. This condition prevented Mpk1 phosphorylation when the cell wall was severely stressed (34de Nobel H. Ruiz C. Martin H. Morris W. Bru S. Molina M. Klis F.M. Microbiology. 2000; 146: 2121-2132Crossref PubMed Scopus (221) Google Scholar). Growth in the presence of sorbitol resulted in reduced Mpk1 phosphorylation after the shift to 37 °C (Fig.2A). However, the presence of the stabilizer did not prevent Mpk1 basal phosphorylation in asit4Δ mutant with respect to other wild type cells. Therefore, hyperactivation of Mpk1 in the absence of Sit4 is not a consequence of hypothetical cell wall defects derived from the lack of Sit4. In fact, a sit4Δ mutant showed increased tolerance to treatment with zymolyase (a combination of 1,3-β-glucanase and protease enzymes that degrade the yeast cell wall) with respect to wild type cells (Fig. 2B). In contrast, as previously described (34de Nobel H. Ruiz C. Martin H. Morris W. Bru S. Molina M. Klis F.M. Microbiology. 2000; 146: 2121-2132Crossref PubMed Scopus (221) Google Scholar) the mpk1Δ mutant was hypersensitive to enzymatic digestion. Exponentially growing cultures of sit4Δ are enriched in G1 cells, and it has been reported that Mpk1 becomes phosphorylated at the G1/S transition in a cell cycle-dependent manner (18Zarzov P. Mazzoni C. Mann C. EMBO J. 1996; 15: 83-91Crossref PubMed Scopus (197) Google Scholar). Increased Mpk1 dual-phosphorylation in a sit4Δ mutant could therefore result from the presence of a higher proportion of cells in G1 in asynchronous cultures. To discard this possibility, we performed heat-shock experiments with cells synchronized with α-factor in G1, in S with hydroxyurea, and in G2 with nocodazole. In all three cases basal and heat-induced Mpk1 phosphorylation were higher in sit4Δthan in wild type cells (Fig. 2C). As these treatments are somewhat stressful for cells and might affect Mpk1 phosphorylation, G1 small daughter cells were recovered by elutriation, and allowed to progress through the cell cycle, to monitor Mpk1 phosphorylation. At all tested time points, Mpk1 phosphorylation insit4Δ cells was more intense than in wild type cells (Fig.2D). It can therefore be concluded that the absence of Sit4 affects Mpk1 phosphorylation regardless of its position in the cycle. This also demonstrates that the higher activity detected insit4Δ cells is not merely a circumstantial effect caused by the partial synchronization in G1 in this mutant. We constructed pkc1Δ-sit4Δ andbck1Δ-sit4Δ double mutants to investigate whether the induction of Mpk1 activity that occurs in the absence of Sit4 was dependent on Pkc1. However, neither Mpk1 phosphorylation nor Swi6 hyperphosphorylation were observed in pkc1Δ-sit4Δ (Fig.3A, and data not shown) orbck1Δ,sit4Δ (not shown) double mutants at 25 or 37 °C. We conclude that an intact PKC1-MAPK module is required for the Mpk1 activation caused by the absence of Sit4 function. This points against Sit4 defining a Mpk1-inactivating pathway independent from Pkc1. We followed two different approaches to functionally situate Sit4 with respect to Pkc1. First, we used a BCK1–20 allele that constitutively activates Mpk1 (24Lee K.S. Levin D.E. Mol. Cell. Biol. 1992; 12: 172-182Crossref PubMed Scopus (269) Google Scholar). We transformed thePKC1/pkc1ΔSIT4/sit4Δ diploid strain using a plasmid bearing the BCK1–20 allele. After sporulation, thepkc1Δ,sit4Δ, pkc1Δ, and sit4Δstrains (bearing the pBCK1–20 plasmid) were isolated and Mpk1 phosphorylation was subsequently analyzed. It is important to stress that after heat shock Mpk1 still became hyperactivated in the presence of a BCK1–20 allele (not shown). Our reasoning was therefore as follows: if Sit4 acts downstream of Pkc1 as a negative regulator for the pathway, then both sit4Δ/pBCK1–20 andpkc1Δ-sit4Δ/pBCK1-20 cells would exhibit higher levels of Mpk1 activity than wild type and pkc1Δ cells transformed with pBCK1–20, respectively. Alternatively, if Sit4 acts upstream of Pkc1 we would first expect the constitutive phosphorylation level of Mpk1 in sit4Δ/pBCK1–20 cells to be higher than in any of the other strains tested, and second we would expectpkc1Δ-sit4Δ/pBCK1-20 and pkc1Δ/pBCK1–20 c
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