Overexpression of the G1-cyclin Gene CLN2Represses the Mating Pathway in Saccharomyces cerevisiaeat the Level of the MEKK Ste11
1997; Elsevier BV; Volume: 272; Issue: 20 Linguagem: Inglês
10.1074/jbc.272.20.13180
ISSN1083-351X
AutoresKatja Wassmann, Gustav Ammerer,
Tópico(s)Plant Gene Expression Analysis
ResumoBasal and induced transcription of pheromone-dependent genes is regulated in a cell cycle-dependent way. FUS1, a gene strongly induced after pheromone treatment, shows high mRNA levels in mitosis and early G1 phase of the cell cycle, a decrease in G1after START and again an increase in S phase. Overexpression ofCLN2 was shown to repress the transcript number of pheromone-dependent genes (1). We asked whether the activities of components of the mating pathway fluctuate during the cell cycle. We were also interested in determining at what level Cln2 represses the signal transduction machinery. Here we show that the activity of the mitogen-activated protein kinase Fus3 indeed fluctuates during the cell cycle, reflecting the oscillations of the gene transcripts. CLN2 overexpression represses Fus3 kinase activity, independently of the phosphatase Msg5. Additionally, we show that the activity of the MEK Ste7 also fluctuates during the cell cycle. Increased Cln2 levels repress the ability of hyperactiveSTE11 alleles to induce the pathway. G protein-independent activation of Ste11 caused by an rga1 pbs2 mutation is resistant to high levels of Cln2 kinase. Therefore our results suggest that Cln2-dependent repression of the mating pathway occurs at the level of Ste11. Basal and induced transcription of pheromone-dependent genes is regulated in a cell cycle-dependent way. FUS1, a gene strongly induced after pheromone treatment, shows high mRNA levels in mitosis and early G1 phase of the cell cycle, a decrease in G1after START and again an increase in S phase. Overexpression ofCLN2 was shown to repress the transcript number of pheromone-dependent genes (1). We asked whether the activities of components of the mating pathway fluctuate during the cell cycle. We were also interested in determining at what level Cln2 represses the signal transduction machinery. Here we show that the activity of the mitogen-activated protein kinase Fus3 indeed fluctuates during the cell cycle, reflecting the oscillations of the gene transcripts. CLN2 overexpression represses Fus3 kinase activity, independently of the phosphatase Msg5. Additionally, we show that the activity of the MEK Ste7 also fluctuates during the cell cycle. Increased Cln2 levels repress the ability of hyperactiveSTE11 alleles to induce the pathway. G protein-independent activation of Ste11 caused by an rga1 pbs2 mutation is resistant to high levels of Cln2 kinase. Therefore our results suggest that Cln2-dependent repression of the mating pathway occurs at the level of Ste11. Mating between cells of the two opposite haploid cell types ofSaccharomyces cerevisiae requires recognition of the mating partner through secreted pheromones. In response to pheromone both mating partners arrest their cell cycle in G1 (2Hartwell L.H. Bacteriol. Rev. 1974; 38: 164-198Crossref PubMed Google Scholar, 3Pringle J.R. Hartwell L.H. Strathern J. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1981: 97-142Google Scholar) and induce a set of pheromone-dependent genes (4Sprague G.F. Thorner J.W. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces , Gene Expression. 1992: 657-744Google Scholar). They undergo morphological changes (shmoo formation), which enables two cells of opposite mating type to fuse and form a diploid zygote. Mating is restricted to a short period in the G1 phase of the cell cycle (5Cross F. Hartwell L.H. Jackson C. Konopka J.B. Annu. Rev. Cell Biol. 1988; 4: 429-457Crossref PubMed Scopus (150) Google Scholar, 6Dolan J.W. Fields S.I. Biochim. Biophys. Acta. 1991; 1008: 155-169Crossref Scopus (83) Google Scholar, 7Marsh L. Neiman A.M. Herskowitz I. Annu. Rev. Cell Biol. 1991; 7: 699-728Crossref PubMed Scopus (154) Google Scholar, 8Sprague G.F. Trends Genet. 1991; 11: 393-398Crossref Google Scholar). Cells which have passed a point called START in G1 are committed to undergo a new cell cycle and cannot respond to mating pheromone until they reach G1 again (9Reid B.J. Hartwell L.H. J. Cell Biol. 1977; 75: 355-365Crossref PubMed Scopus (90) Google Scholar,10McKinney J.D. Chang F. Heintz N. Cross F.R. Genes Dev. 1993; 7: 833-843Crossref PubMed Scopus (123) Google Scholar). Transition through START is mediated by the function of the G1 cyclins Cln1, Cln2, and Cln3, which are the regulatory subunits of the Cdc28 kinase (11Richardson H.E. Wittenberg C. Cross F. Reed S.I. Cell. 1989; 59: 1127-1133Abstract Full Text PDF PubMed Scopus (376) Google Scholar, 12Wittenberg C. Sugimoto K. Reed S.I. Cell. 1990; 62: 225-237Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 13Reed S.I. Annu. Rev. Cell Biol. 1992; 8: 529-561Crossref PubMed Scopus (266) Google Scholar, 14Futcher B.A. Semin. Cell Biol. 1991; 2: 205-212PubMed Google Scholar, 15Nasmyth K. The Harvey Lectures. 1995; 88: 141-171Google Scholar). In response to mating pheromone the activity of the Cdc28-Cln kinase gets inhibited, so that the cell cannot pass START (16Peter M. Herskowitz I. Science. 1994; 265: 1228-1231Crossref PubMed Scopus (196) Google Scholar). Mating pheromone induces a MAPK 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, MAP kinase/ERK kinase; MEKK, MEK kinase; MOPS, 4-morpholinepropanesulfonic acid. 1The abbreviations used are: MAPK, mitogen-activated protein kinase; MEK, MAP kinase/ERK kinase; MEKK, MEK kinase; MOPS, 4-morpholinepropanesulfonic acid. pathway which ultimately results in the phosphorylation of the putative Cdk inhibitor (cyclin-dependent kinase inhibitor) Far1 (17Peter M. Gartner A. Horecka J. Ammerer G. Herskowitz I. Cell. 1993; 73: 747-760Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 18Tyers M. Futcher B. Mol. Cell. Biol. 1993; 13: 5659-5669Crossref PubMed Scopus (140) Google Scholar). Phosphorylation of Far1 causes its association with the Cdc28-Cln1 and Cdc28-Cln2 kinases and thereby inhibits their ability to drive the cell through START (17Peter M. Gartner A. Horecka J. Ammerer G. Herskowitz I. Cell. 1993; 73: 747-760Abstract Full Text PDF PubMed Scopus (273) Google Scholar, 18Tyers M. Futcher B. Mol. Cell. Biol. 1993; 13: 5659-5669Crossref PubMed Scopus (140) Google Scholar, 19Zanolari B. Riezman H. Mol. Cell. Biol. 1991; 11: 5251-5258Crossref PubMed Google Scholar). Both mating partners are arrested in the same stage of the cell cycle when they fuse. This mechanism ensures the correct ploidy of the zygote. Transcription of pheromone-inducible genes such as FUS1, STE2 (1Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar, 19Zanolari B. Riezman H. Mol. Cell. Biol. 1991; 11: 5251-5258Crossref PubMed Google Scholar), SST2, STE12, andMFA2 (20Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar) has been shown to fluctuate during the cell cycle. It reaches its maximum during late mitosis and early G1, decreases drastically in late G1 around START, and increases again in the G2 phase of the cell cycle (1Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar, 20Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar). The transcript of FAR1, which encodes a putative cyclin-dependent kinase inhibitor, also fluctuates during the cell cycle, but in contrast to other pheromone-inducible genes, its transcription in G2/M is Mcm1-dependent (20Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar). The stability of the Far1 protein is also regulated during the cell cycle. Far1 protein can be detected only in early G1 cells and is rapidly degraded in other stages of the cell cycle (10McKinney J.D. Chang F. Heintz N. Cross F.R. Genes Dev. 1993; 7: 833-843Crossref PubMed Scopus (123) Google Scholar). This ensures that the cell cycle arrest in response to pheromone occurs in early G1 only. The cell cycle regulated restriction of pheromone-dependent gene transcription may reflect an important mechanism ensuring that the responsiveness of the cell to mating pheromone is maximal in early G1. The decrease of pheromone-dependent gene transcription in late G1 correlates with an increase in the appearance of G1 cyclin. Overexpression of CLN2represses FUS1 transcription (1Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Preliminary epistasis experiments demonstrated that the repression occurs downstream of the receptor (1Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar) and that it involves neither Sst2 (implicated in recovery from pheromone-induced G1 arrest) nor the carboxyl-terminal part of the pheromone receptor (implicated in desensitization). Potential targets of Cln2-mediated repression are the components of the pheromone-induced MAPK pathway. This signal transduction pathway has been the focus of several reviews (4Sprague G.F. Thorner J.W. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces , Gene Expression. 1992: 657-744Google Scholar, 21Ammerer G. Curr. Opin. Genet. Dev. 1994; 4: 90-95Crossref PubMed Scopus (69) Google Scholar, 22Bardwell L. Cook J.G. Inouye C.J. Thorner J. Dev. Biol. 1994; 166: 363-379Crossref PubMed Scopus (142) Google Scholar). In short, activation is mediated by the binding of pheromone to a seven-transmembrane receptor coupled to a heterotrimeric G protein. Upon pheromone induction Gα dissociates and releases Gβγ (23Dietzel C. Kurjan J. Cell. 1987; 50: 1001-1010Abstract Full Text PDF PubMed Scopus (264) Google Scholar, 24Miyajima I. Nakafuku M. Nakayama N. Brenner C. Miyajima A. Kaibuchi K. Arai K. Kaziro Y. Matsumoto K. Cell. 1987; 50: 1011-1019Abstract Full Text PDF PubMed Scopus (208) Google Scholar, 25Jahng K. Ferguson J. Reed S.I. Mol. Cell. Biol. 1988; 8: 2484-2493Crossref PubMed Scopus (80) Google Scholar, 26Blinder D. Jenness D.D. Mol. Cell. Biol. 1989; 9: 3720-3726Crossref PubMed Scopus (18) Google Scholar, 27Whiteway M. Hougan L. Dignard D. Thomas D.Y. Bell L. Saari G.C. Grant F.J. O'Hara P. MacKay V.L. Cell. 1989; 56: 467-477Abstract Full Text PDF PubMed Scopus (329) Google Scholar). Gβγ transmits the signal to the Ste20 kinase (28Leberer E. Dignard D. Harcus D. Hougan L. Whiteway M. Thomas D.Y. Mol. Gen. Genet. 1993; 241: 241-254Crossref PubMed Scopus (49) Google Scholar, 29Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar) by an as yet unknown mechanism involving the GTPase Cdc42, the guanine nucleotide exchange factor Cdc24 (30Simon M.-N. De Virgilio C. Souza B. Pringle J.R. Abo A. Reed S.I. Nature. 1995; 376: 702-705Crossref PubMed Scopus (179) Google Scholar, 31Zhao Z.-S. Leung T. Manser E. Lim L. Mol. Cell. Biol. 1995; 15: 5246-5257Crossref PubMed Scopus (160) Google Scholar) and the GTPase-activating protein Rga1 (32Stevenson B. Ferguson B. De Virgilio C. Bi E. Pringle J.R. Ammerer G. Sprague G.F. Genes Dev. 1995; 9: 2949-2963Crossref PubMed Scopus (102) Google Scholar). Downstream of Ste20 the sequential activation of several protein kinases, tethered together by the scaffold protein Ste5, propagates the signal (28Leberer E. Dignard D. Harcus D. Hougan L. Whiteway M. Thomas D.Y. Mol. Gen. Genet. 1993; 241: 241-254Crossref PubMed Scopus (49) Google Scholar). These kinases belong to the family of highly conserved MAPK pathways (21Ammerer G. Curr. Opin. Genet. Dev. 1994; 4: 90-95Crossref PubMed Scopus (69) Google Scholar) and function in the linear order Ste11, Ste7, and Fus3/Kss1. The MAPKs Fus3 and Kss1 are thought to activate the transcription factor Ste12, which mediates the transcriptional induction of pheromone-dependent genes (33Dolan J.W. Kirkman C. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5703-5707Crossref PubMed Scopus (179) Google Scholar, 34Errede B. Ammerer G. Genes Dev. 1989; 3: 1349-1361Crossref PubMed Scopus (186) Google Scholar, 35Elion E.A. Satterberg B. Kranz J.E. Mol. Biol. Cell. 1993; 4: 495-510Crossref PubMed Scopus (212) Google Scholar). In this work we investigate which components of the pheromone-induced signal transduction pathway are regulated in a cell cycle-dependent way. We show that the activity of the MAPK Fus3 fluctuates during the cell cycle. Furthermore, Fus3 kinase activity is repressed by overexpression of CLN2. The repression occurs independent of the function of the phosphataseMSG5, which dephosphorylates Fus3 (36Doi K. Gartner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (204) Google Scholar). The activity of the MEK Ste7 was shown to respond to the cell cycle as well. By the use of hyperactive alleles of STE11 and a deletion ofRGA1, we conclude that CLN2 represses the mating pathway at the level of Ste11. The studies in this work represent an important contribution to the understanding of the regulation of a signaling cascade by the cell cycle and the phenomenon described here may be relevant in the regulation of MAPK pathways in higher eucaryotic cells. The S. cerevisiae strains utilized in this study are listed in Table I. Standard yeast techniques were used as described in Sherman et al. (37Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). YEP (complete) medium, minimal synthetic medium, and supplements are described by Hicks and Herskowitz (38Hicks J.B. Herskowitz I. Genetics. 1976; 83: 245-258Crossref PubMed Google Scholar). Yeast transformations were performed either as described by Ito et al. (39Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) and Beggs (40Beggs J.D. Nature. 1978; 275: 104-109Crossref PubMed Scopus (792) Google Scholar), or by one-step transformation (41Chen D.-C. Yang B.-C. Kuo T.-T. Curr. Genet. 1992; 21: 83-84Crossref PubMed Scopus (576) Google Scholar). Induction of S. cerevisiae cells with pheromone required 0.3 μg/ml α-factor for bar1 strains and 1 μg/ml for BAR1 strains. Unless otherwise indicated cells were induced for 10 min with pheromone. For the expression of the galactose inducible GAL1–10 CLN2 construct cells were pregrown on YEP medium plus 2% raffinose. The GAL1–10 promoter was induced for 2.5 h with galactose, the control was either grown in YEP plus 2% raffinose, or, where indicated, glucose was added to a final concentration of 2%.Table IYeast strainsStrainRelevant genotypeSourceSY1390MATαSTE + FSU1::HIS3 leu2 ura3 trp1 his3200::ura3 pep4::ura3 can1Stevensonet al. (51Stevenson B.J. Rhodes N. Errede B. Sprague G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar)SY1865SY1390 exceptSTE11–1Stevenson et al. (51Stevenson B.J. Rhodes N. Errede B. Sprague G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar)SY1866SY1390 except STE11–4Stevenson et al. (51Stevenson B.J. Rhodes N. Errede B. Sprague G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar)SY1969SY1866 exceptste4::LEU2Stevenson et al.(51Stevenson B.J. Rhodes N. Errede B. Sprague G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar)BSY179MATα STE+ FUS1::HIS3 his3 mfa2▵-1::FUS1-lac-Z ade1 leu2 trp1 ura3Stevenson et al. (32Stevenson B. Ferguson B. De Virgilio C. Bi E. Pringle J.R. Ammerer G. Sprague G.F. Genes Dev. 1995; 9: 2949-2963Crossref PubMed Scopus (102) Google Scholar)BSY193BSY179 except ste4::LEU2 rgal1-1 pbs2-99Stevenson et al. (32Stevenson B. Ferguson B. De Virgilio C. Bi E. Pringle J.R. Ammerer G. Sprague G.F. Genes Dev. 1995; 9: 2949-2963Crossref PubMed Scopus (102) Google Scholar)KW19SY1390 except GAL1-10 CLN2::URA3This studyKW20SY1866 except GAL1-10 CLN2::URA3This studyKW21SY1865 except GAL1-10 CLN2::URA3This studyKW22BSY193 except GAL1-10 CLN2::URA3This studyKW24SY1969 except GAL1-10 CLN2::URA3This studyK1107MATa HMLa HMRa ho-ßgal ura3 HIS4 ade2-1 can1-100 met his3 leu2 trp1-1 SSD1Cvrckova and Nasmyth (55Cvrcková F. Nasmyth K. EMBO J. 1993; 12: 5277-5286Crossref PubMed Scopus (140) Google Scholar)K3735K1107 except cla2▵::LEU2Cvrckova and Nasmyth (55Cvrcková F. Nasmyth K. EMBO J. 1993; 12: 5277-5286Crossref PubMed Scopus (140) Google Scholar)GA526K1107 except msg5::LEU2 bar1Doi et al. (36Doi K. Gartner A. Ammerer G. Errede B. Shinkawa H. Sugimoto K. Matsumoto K. EMBO J. 1994; 13: 61-70Crossref PubMed Scopus (204) Google Scholar)KW15GA526 except GAL1-10 CLN2::URA3This studyK699MATa ho ade2-1 trp1-1 can1-100 leu2-3,112 his3-11,15 ura3 GAL psi+ssd1-d2W303-1AGA246MATa cdc4-1 ura3 leu2 trp1K. Nasmyth (Institute for Molecular Pathology, Vienna)GA248MATa cdc15-2 ura3 leu2 trp1K. NasmythKW8GA248 except GAL1-10 CLN2::URA3This studyKW38K699 except GAL1-10 CLN2::URA3This studyKS114MATa trp1 leu2 ura3 his4J. Pringle (University of North Carolina, Chapel Hill)KS116KS114 exceptbem1▵::LEU2J. Pringle Open table in a new tab Standard DNA manipulations were performed according to Sambrook et al. (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) or Ausubelet al. (43Ausubel 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 and Green Publishing Associates, New York1989Google Scholar). In Table II the plasmids used in this study are listed. The GAL1–10 CLN2 plasmid K2573 was cut with EcoRV and integrated into the genome. The integration was checked by Southern blotting.Table IIPlasmidsPlasmidCharacteristics and sourceGA1855CLN2 coding sequence in a modified pNC160 vector as aNdeI-BgIII fragment under the control of theTP11 promoter as a BamHI-EcoRI fragmentGA1896NH2-terminal Far1 (aa 50–292) fragment under the control of the T7 promoter for expression inE. coli, described in Peter et al. (17Peter M. Gartner A. Horecka J. Ammerer G. Herskowitz I. Cell. 1993; 73: 747-760Abstract Full Text PDF PubMed Scopus (273) Google Scholar)GA1944FUS3 R42 as a GST fusion under the control of the T7 promoter for expression in E. coli, described in Errede et al. (44Errede B. Gartner A. Zhou A. Nasmyth K. Ammerer G. Nature. 1993; 362: 261-264Crossref PubMed Scopus (144) Google Scholar)GA1903Contains the Myc epitope-tagged FUS3 sequence under the control of the TP11 promoter, described in Zhouet al. (45Zhou Z. Gartner A. Cade R. Ammerer G. Errede B. Mol. Cell. Biol. 1993; 13: 2069-2080Crossref PubMed Scopus (120) Google Scholar)GA1905As GA1903, but expressing the catalytic mutant protein (FUS3M-R 42), described in Zhou et al. (45Zhou Z. Gartner A. Cade R. Ammerer G. Errede B. Mol. Cell. Biol. 1993; 13: 2069-2080Crossref PubMed Scopus (120) Google Scholar)pNC318Myc epitope-taggedSTE7 sequence under the control of the CYC1promoter, described in Zhou et al. (45Zhou Z. Gartner A. Cade R. Ammerer G. Errede B. Mol. Cell. Biol. 1993; 13: 2069-2080Crossref PubMed Scopus (120) Google Scholar)pNC318-R220As pNC318, but expressing the catalytical mutant protein (STE7M-R 220), described in Zhou et al. (45Zhou Z. Gartner A. Cade R. Ammerer G. Errede B. Mol. Cell. Biol. 1993; 13: 2069-2080Crossref PubMed Scopus (120) Google Scholar)C2573Yiplac211 containing a GAL1–10 CLN2 fusion, described by Amon et al. (69Amon A. Tyers M. Futcher B. Nasmyth K. Cell. 1993; 74: 993-1007Abstract Full Text PDF PubMed Scopus (294) Google Scholar) Open table in a new tab Far1 was expressed in E. coli from the plasmid GA1896, which contains the NH2-terminal fragment of Far1 (amino acids 50–301) under the control of the isopropyl β-d-thiogalactopyranoside-inducible T7 promoter. Expression and purification of the protein are described in Peter et al. (17Peter M. Gartner A. Horecka J. Ammerer G. Herskowitz I. Cell. 1993; 73: 747-760Abstract Full Text PDF PubMed Scopus (273) Google Scholar). Fus3-R42 was expressed as a glutathione S-transferase fusion protein from the plasmid GA1944 in E. coli and purified as described in Erredeet al. (44Errede B. Gartner A. Zhou A. Nasmyth K. Ammerer G. Nature. 1993; 362: 261-264Crossref PubMed Scopus (144) Google Scholar). Fus3 kinase assays were done as described in Peter et al. (17Peter M. Gartner A. Horecka J. Ammerer G. Herskowitz I. Cell. 1993; 73: 747-760Abstract Full Text PDF PubMed Scopus (273) Google Scholar), except that the reactions were done in 6 μl of HBII buffer, where 3 μCi of [γ-32P]ATP (6000Ci/mmol), 0.5–2 μl (0.1 μg/μl) of Far1 substrate, and 25 mm MOPS, pH 7.2, up to a final volume of 16 μl were added. The reactions were carried out at 30 °C for 30 min. The strains used contain the Myc epitope-tagged Ste7 wild-type protein (pNC318) or an inactivated version of Ste7 (pNC318-R220) on a centromeric plasmid under the control of the CYC1 promoter (45Zhou Z. Gartner A. Cade R. Ammerer G. Errede B. Mol. Cell. Biol. 1993; 13: 2069-2080Crossref PubMed Scopus (120) Google Scholar). 70-ml cultures were grown to an OD600 around 0.8 in YEP plus 2% sucrose.cdc15–2 and cdc4–1 cells were arrested as described. Unless otherwise indicated everything was done at 4 °C. Cells were harvested by centrifugation, washed with kinase extract buffer (KEB) (50 mm Tris, pH 7.5, 150 mm NaCl, 5 mm EDTA, O.1% Nonidet P-40, 50 mm sodium fluoride, 30 mmNa2H2P2O7, 15 mm 4-nitrophenyl phosphate, O.1 mmorthovanadate, 1 mm phenylmethylsulfonyl fluoride, 40 μg/ml aprotinin, and 20 μg/ml leupeptin), and resuspended in 200 μl of breakage buffer (KEB without sodium fluoride and Na2H2P2O7). Cells were lysed by vortexing them with glass beads 3 times for 4 min. The extracts were cleared by centrifugation once for 5 min and twice for 15 min at 14,000 rpm. The protein concentration was determined using the Bio-Rad protein assay as recommended by the manufacturer. 300 μg of protein extract were used for the immunoprecipitation. Ste7 was precipitated with approximately 10 μg of anti-Myc 9E10 antibody for 1.5 h. 20 μl of protein A-Sepharose beads in a 1:1 suspension (preincubated in breakage buffer) were added. After 1 h incubation the beads were washed 5 times in breakage buffer, and 3 times in HEPES, 25 mm, pH 7.5. For the kinase assay, 6 μl of kinase assay buffer (25 mm HEPES, pH 7.5, 15 mmMgCl2, 5 mm EGTA, 1 mmdithiothreitol, 0.1 mm orthovanadate, 15 mmphenylmethylsulfonyl fluoride, 15 mm 4-nitrophenyl phosphate, 40 μg/ml aprotinin, and 20 μg/ml leupeptin) were added to the beads. 0.5 μg of E. coli purified Fus3-R42 as a substrate, 0.2 μl of [γ-32P]ATP (10 μCi/μl), 1 μl of HEPES, 250 mm, 2 μl of 1 mm ATP, and H2O up to a final volume of 20 μl were added. The kinase assay was incubated at 30 °C for 30 min. The reaction was stopped by adding SDS sample buffer (46Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar) and boiling the extracts for 4 min. Phosphorylation of the substrate was detected by SDS-polyacrylamide gel electrophoresis analysis on a 9.5% polyacrylamide (28:2, acrylamide-bisacrylamide) gel (46Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205523) Google Scholar). Yeast total RNA preparation was performed as described by Cross and Tinkelenberg (47Cross F.R. Tinkelenberg A.H. Cell. 1991; 65: 875-883Abstract Full Text PDF PubMed Scopus (249) Google Scholar). RNA was separated on formaldehyde-agarose gels and transferred to Boehringer Mannheim nylon membranes (as recommended by the manufacturer). Northern blots were processed as described by Priceet al. (48Price C. Nasmyth K. Schuster T. J. Mol. Biol. 1991; 218: 543-556Crossref PubMed Scopus (61) Google Scholar). The DNA fragments used as probes were labeled by random priming and isolated as follows: SST2, aClaI/HapI fragment was cut out from theSST2 coding region; CMD1, with the oligos 5′-GTCCTCCAATCTTACCGAAG-3′ and 5′-TTACAAGTAGAATCCATTTAGATAACAAAGCAGCG-3′ the CMD1 coding region was isolated by PCR from genomic DNA; CLN2, aSalI/ClaI fragment was cut out from theCLN2 coding region; CDC28, anEcoRI/PstI fragment from the plasmid K2354, containing a GAL1–10 CDC28 construct (by A. Amon) was isolated; FUS1, the HindIII/SalI fragment of the FUS1 coding region was isolated;STE3, a HindIII fragment from the STE3coding region was isolated; ACT1 was isolated as aXhoI/KpnI fragment from the ACT1coding sequence. DNA content of propridium iodide-stained cells was measured on a Becton-Dickinson FACScan as described by Lewet al. (49Lew D.J. Marini N.J. Reed S.I. Cell. 1992; 69: 317-327Abstract Full Text PDF PubMed Scopus (77) Google Scholar) and Epstein and Cross (50Epstein C.B. Cross F.R. Genes Dev. 1992; 6: 1695-1706Crossref PubMed Scopus (299) Google Scholar). Cells were grown at permissive temperature at 24 °C in the appropriate medium from OD600 0.2 to 0.8. The cultures were diluted to OD600 0.4 and arrested for 3–4 h at 37 °C. The arrest was checked by microscopic examination and determination of the budding index. cdc15–2 cells were arrested as described above. The cultures were released by diluting them with ice-cold YEPD to OD600 O.5 and further cooling them down on ice till the temperature was 24 °C. They were released at 24 °C. Aliquots were taken at the indicated time points for kinase assays, FACS analysis, RNA preparation, protein extracts for Western blots, and for the determination of the budding index. The transcripts of pheromone-dependent genes fluctuate during the cell cycle (1Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar, 19Zanolari B. Riezman H. Mol. Cell. Biol. 1991; 11: 5251-5258Crossref PubMed Google Scholar, 20Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar). To determine whether the transcriptional fluctuations are the consequence of differences in the activity of the MAPK Fus3, we expressed FUS3 constitutively from theTPI1 promoter to examine kinase activity independently of its transcriptional regulation during the cell cycle. Kinase assays from distinct cell cycle arrested cells were performed with a Myc epitope-tagged Fus3. Fus3 was immunoprecipitated with 9E10 anti-Myc monoclonal antibody. Cells harboring a temperature-sensitive mutation in the CDC15 or the CDC4 gene were arrested at the restrictive temperature in late mitosis or late G1, respectively. In vitro Fus3 kinase assays using bacterial recombinant Far1 as a substrate were performed under pheromone-induced conditions (Fig. 1 A) and uninduced conditions (data not shown). According to the fluctuations observed for pheromone-dependent genes, Fus3 MAPK activity was expected to be high in cells arrested in late mitosis, and low in cells arrested in late G1. As can be seen in Fig. 1 A, Fus3 kinase activity shows the same cell cycle-dependent pattern as described for the FUS1 transcript (1Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Fus3 activity is high in cdc15–2 arrested cells (lane 3), and low in cdc4–1 arrested cells (lane 5). To further analyze the cell cycle-dependent regulation of basal Fus3 kinase activity we performed cdc15–2 release experiments in the absence of pheromone. The cultures were arrested at restrictive temperature in late mitosis and released from the block at permissive temperature. The arrest was checked by microscopic examination and determination of the budding index (data not shown). At the indicated time points, samples were taken for kinase assays (Fig.1 B) and Northern analysis (Fig. 1 C). The activity of Fus3 was examined by in vitro kinase assays. As seen in Fig. 1 B the activity of Fus3 shows the same cell cycle-dependent regulation which had been observed for the transcripts of mating dependent genes. Differences in the activity of Fus3 could have been due to differences in Fus3 protein levels. To rule out this possibility, Western blot analysis was performed. Aliquots from the cdc15–2 release experiment in Fig. 1, B and C, were used for the preparation of cell extracts. The Western blot was probed with anti-Fus3 and anti-Cdc28 polyclonal antibodies. The Fus3 protein concentration stays constant throughout the cell cycle and is not responsible for the fluctuations in kinase activity or in expression of pheromone-dependent genes (data not shown). A second release experiment to study the timing of Fus3 activity during the cell cycle was performed. The release was checked by FACScan analysis (Fig. 1 E). Fus3 activity and CLN2mRNA fluctuate antagonistically (Fig. 1 D). These data are in accordance with previous studies showing that theFUS1 and CLN2 transcripts fluctuate antagonistically (1Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Basal and pheromone-induced activity of the MAPK Fus3 are cell cycle- regulated, but the protein levels of Fus3 stay constant. The activity of the MAPK Fus3 reaches its maximum in late mitosis and early G1, drops after START, is low in late G1, and increases again in S phase. The pattern of its cell cycle regulation is the same as observed for the FUS1transcript and reaches its maximum when CLN2 levels are low. Overexpression of the G1 cyclin genesCLN1 and CLN2 represses transcription ofFUS1 (1Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). To see whether CLN2 overexpression reduces Fus3 kinase activity, CLN2 was overexpressed from the GAL1–10 promoter in cycling cells and Fus3 activity was examined by in vitro kinase assays. Myc epitope-tagged Fus3, expressed from the TPI1 prom
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