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Potential Regulation of Ste20 Function by the Cln1-Cdc28 and Cln2-Cdc28 Cyclin-dependent Protein Kinases

1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês

10.1074/jbc.273.39.25089

ISSN

1083-351X

Autores

L. J. W. M. Oehlen, Frederick R. Cross,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

The activity of the Saccharomyces cerevisiae pheromone signal transduction pathway is regulated by Cln1/2-Cdc28 cyclin-dependent kinase. High level expression of CLN2 can repress activation of the pathway by mating factor or by deletion of the α-subunit of the heterotrimeric G-protein. We now show that CLN2 overexpression can also repress FUS1 induction if the signaling pathway is activated at the level of the β-subunit of the G-protein (STE4) but not when activated at the level of downstream kinases (STE20 and STE11) or at the level of the transcription factor STE12. This epistatic analysis indicates that repression of pheromone signaling pathway by Cln2-Cdc28 kinase takes place at a level around STE20. In agreement with this, a marked reduction in the electrophoretic mobility of the Ste20 protein is observed at the time in the cell cycle of maximal expression of CLN2. This mobility change is constitutive in cells overexpressing CLN2 and absent in cells lackingCLN1 and CLN2. These changes in electrophoretic mobility correlate with repression of pheromone signaling and suggest Ste20 as a target for repression of signaling by G1cyclins. Two morphogenic pathways for which Ste20 is essential, pseudohyphal differentiation and haploid-invasive growth, also requireCLN1 and CLN2. Together with the previous observation that Cln1 and Cln2 are required for the function of Ste20 in cytokinesis, this suggests that Cln1 and Cln2 regulate the biological activity of Ste20 by promoting morphogenic functions, while inhibiting the mating factor signal transduction function. The activity of the Saccharomyces cerevisiae pheromone signal transduction pathway is regulated by Cln1/2-Cdc28 cyclin-dependent kinase. High level expression of CLN2 can repress activation of the pathway by mating factor or by deletion of the α-subunit of the heterotrimeric G-protein. We now show that CLN2 overexpression can also repress FUS1 induction if the signaling pathway is activated at the level of the β-subunit of the G-protein (STE4) but not when activated at the level of downstream kinases (STE20 and STE11) or at the level of the transcription factor STE12. This epistatic analysis indicates that repression of pheromone signaling pathway by Cln2-Cdc28 kinase takes place at a level around STE20. In agreement with this, a marked reduction in the electrophoretic mobility of the Ste20 protein is observed at the time in the cell cycle of maximal expression of CLN2. This mobility change is constitutive in cells overexpressing CLN2 and absent in cells lackingCLN1 and CLN2. These changes in electrophoretic mobility correlate with repression of pheromone signaling and suggest Ste20 as a target for repression of signaling by G1cyclins. Two morphogenic pathways for which Ste20 is essential, pseudohyphal differentiation and haploid-invasive growth, also requireCLN1 and CLN2. Together with the previous observation that Cln1 and Cln2 are required for the function of Ste20 in cytokinesis, this suggests that Cln1 and Cln2 regulate the biological activity of Ste20 by promoting morphogenic functions, while inhibiting the mating factor signal transduction function. p21-activated kinases cyclin-dependent kinase polyacrylamide gel electrophoresis mitogen-activated protein. Binding of mating factor to a specific receptor in haploidSaccharomyces cerevisiae cells activates a signal transduction pathway that prepares for conjugation with cells of the opposite mating type. The transduction of the signal starts with binding of the peptide mating factor to a seven-transmembrane domain receptor (Ste2 in a mating type cells and Ste3 in α-cells), which then activates a heterotrimeric G-protein by releasing an active β-γ complex from the inhibitory α-subunit (α-, β-, and γ-subunits are encoded, respectively, by theGPA1, STE4, and STE18 genes). The activated G-protein transmits the signal to a set of serine/threonine protein kinases that are activated in a sequential order. The first of these is Ste20, a member of the family of p21-activated kinases (PAKs),1 and then Ste11 (a MAP kinase kinase kinase), Ste7 (a MAP kinase kinase), and finally a MAP kinase (Fus3 or in some cases Kss1) are activated. Activation of the MAP kinase (i) stimulates the transcription of many genes involved in the conjugation process through the transcription factor Ste12, (ii) results in arrest in G1-phase of the cell cycle through the Far1 protein, and (iii) leads to specific morphological changes that are required for efficient cell fusion. Several review articles (1Sprague G.F. Thorner J.W. Jones E.W. Pringle J.R. Broach J.R. The Molecular and Cellular Biology of the Yeast Saccharomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1992: 657-744Google Scholar, 2Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar, 3Bardwell L. Cook J.G. Inouye C.J. Thorner J. Dev. Biol. 1994; 166: 363-379Crossref PubMed Scopus (142) Google Scholar, 4Leberer E. Thomas D.Y. Whiteway M. Curr. Opin. Genet. & Dev. 1997; 7: 59-66Crossref PubMed Scopus (189) Google Scholar) describe the mating factor signal transduction pathway and other MAP kinase-based pathways in more detail. Some of the components of the mating factor signal transduction pathway are required for functions other than sexual differentiation. Agar-invasive growth of haploid cells (haploid-invasive growth) and pseudohyphal growth of diploid cells both require Ste20, Ste11, Ste7, and Ste12 (5Gimeno C.J. Ljungdahl P.O. Styles C.A. Fink G.R. Cell. 1992; 68: 1077-1090Abstract Full Text PDF PubMed Scopus (990) Google Scholar, 6Liu H. Styles C.A. Fink G.R. Science. 1993; 262: 1741-1744Crossref PubMed Scopus (428) Google Scholar, 7Roberts R.L. Fink G.R. Genes Dev. 1994; 8: 2974-2985Crossref PubMed Scopus (526) Google Scholar). In addition, from the lethal phenotype of cells that are deleted for both Ste20 and Cla4, a related PAK family member, Ste20, appears to share a function in the budding/cytokinesis cycle with Cla4 (8Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (310) Google Scholar). The overlap in function is only partial, as Cla4 has no known function in mating factor signal transduction. Therefore, Ste20 is not only critical for sexual differentiation in response to mating factor but can also play a role in morphogenesis during the vegetative cell cycle. The small G-protein Cdc42 can interact with a specific domain in the N terminus of Ste20 that is conserved among PAK family members (2Sells M.A. Chernoff J. Trends Cell Biol. 1997; 7: 162-167Abstract Full Text PDF PubMed Scopus (265) Google Scholar). This interaction of Cdc42 with Ste20 is dispensable forin vitro kinase activity and the mating factor signal transduction functions of Ste20 but appears critical for the vegetative morphological roles of Ste20 (9Peter M. Neiman A.M. Park H.O. Van Lohuizen M. Herskowitz I. EMBO J. 1996; 15: 7046-7059Crossref PubMed Scopus (191) Google Scholar, 10Leberer E. Wu C.L. Leeuw T. Fourest-Lieuvin A. Segall J.E. Thomas D.Y. EMBO J. 1997; 16: 83-97Crossref PubMed Scopus (166) Google Scholar, 11Oehlen L.J.W.M. Cross F.R. J. Biol. Chem. 1998; 273: 8556-8559Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Similarly, full morphogenic function of Cla4 also requires interaction of Cla4 with Cdc42 (12Benton B.K. Tinkelenberg A. Gonzalez I. Cross F.R. Mol. Cell. Biol. 1997; 17: 5067-5076Crossref PubMed Scopus (101) Google Scholar). Both the basal activity (in absence of ligand stimulation) and the mating factor-induced activity of the mating factor signal transduction pathway are cell cycle-regulated (13Zanolari B. Riezman H. Mol. Cell. Biol. 1991; 11: 5251-5258Crossref PubMed Google Scholar, 14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar, 15Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar). In the absence of mating factor stimulation, fluctuations are observed for transcripts of many genes that are involved in the mating reaction and whose transcription involves Ste12 (13Zanolari B. Riezman H. Mol. Cell. Biol. 1991; 11: 5251-5258Crossref PubMed Google Scholar, 14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar, 15Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar, 16McKinney J.D. Chang F. Heintz N. Cross F.R. Genes Dev. 1993; 7: 833-843Crossref PubMed Scopus (123) Google Scholar). A common pattern for transcription of such genes is that transcription is high in G1-phase and then declines as cells enter S-phase (15Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar). The activity of the Fus3 protein kinase shows a similar cell cycle pattern (17Wassmann K. Ammerer G. J. Biol. Chem. 1997; 272: 13180-13188Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), and this regulation is likely to be required for the cell cycle regulation of basal transcription. The mating factor-induced signal transduction activity is also strictly regulated, with maximal activation during M/G1-phase, and reduced activation in S-phase (13Zanolari B. Riezman H. Mol. Cell. Biol. 1991; 11: 5251-5258Crossref PubMed Google Scholar, 14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). This regulation of the induced signal transduction activity depends specifically on the G1 cyclins CLN1 and CLN2 (14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). These cyclins are expressed in late G1-phase, and when Cln1 and Cln2 associate with the cyclin-dependent kinase (CDK) Cdc28, they help to promote the transition of cells from G1- to S-phase (18Cross F.R. Curr. Opin. Cell Biol. 1995; 7: 790-797Crossref PubMed Scopus (105) Google Scholar, 19Nasmyth K. Trends Genet. 1996; 12: 405-412Abstract Full Text PDF PubMed Scopus (295) Google Scholar). High level expression of CLN2 strongly reduces induction of mating specific genes by mating factor or by deletion of the α-subunit of the heterotrimeric G-protein (14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). This suggest that regulation of mating factor signal transduction activity by Cln1/2-Cdc28 takes place at a level downstream of the mating factor receptor and the α-subunit. Here we present a more detailed analysis of the regulation of the mating factor signal transduction pathway by the G1 cyclinsCLN1 and CLN2. A combination of genetic, biochemical, and cell biological observations suggests that Cln1/2-Cdc28 regulate the function of the Ste20 protein kinase. The genotypes of the strains used in this study are given in Table I. Strains were isogenic to BF264-15D (trp1-1a leu2-3, 112 ura3 ade1 his2) except where indicated. Strains were constructed by standard techniques for crossing and gene replacement (20Ausubel 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 York1987Google Scholar). Plasmids that provided fragments for the creation of disruption alleles are as follows: pAB506 (ste2::LEU2 (21Konopka J.B. Jenness D.D. Hartwell L.H. Cell. 1988; 54: 609-620Abstract Full Text PDF PubMed Scopus (166) Google Scholar)), pM59p7 (ste18::URA3 (22Whiteway 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 (331) Google Scholar)), p4-121 (ste4::LEU2 (provided by V. MacKay, Seattle)), pSF32 (ste5::URA3 (provided by V. MacKay, Seattle)), pEL45 (ste20::URA3 (23Leberer E. Dignard D. Hougan L. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4805-4813Crossref PubMed Scopus (76) Google Scholar)), pSL1094 (ste11::URA3 (24Stevenson B.J. Rhodes N. Errede B. Sprague Jr., G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar)), pNC113 (ste7::LEU2 (25Company M. Errede B. Mol. Cell. Biol. 1988; 8: 5299-5309Crossref PubMed Scopus (24) Google Scholar)), pBC65 (kss1::URA3 (26Courchesne W.E. Kunisawa R. Thorner J. Cell. 1989; 58: 1107-1119Abstract Full Text PDF PubMed Scopus (191) Google Scholar)), pYEE98 (fus3::LEU2 (27Elion E.A. Grisafi P.L. Fink G.R. Cell. 1990; 60: 649-664Abstract Full Text PDF PubMed Scopus (308) Google Scholar)), pSUL16 (ste12::LEU2 (28Fields S. Herskowitz I. Mol. Cell. Biol. 1987; 7: 3818-3821Crossref PubMed Scopus (48) Google Scholar)), pBB119 (cla4::TRP1 (12Benton B.K. Tinkelenberg A. Gonzalez I. Cross F.R. Mol. Cell. Biol. 1997; 17: 5067-5076Crossref PubMed Scopus (101) Google Scholar)), pPB590 (akr1::URA3 (29Kao L.R. Peterson J. Ji R. Bender L. Bender A. Mol. Cell. Biol. 1996; 16: 168-178Crossref PubMed Scopus (52) Google Scholar), pKO2 (= pPB642,bem1::LEU2 (30Chenevert J. Corrado K. Bender A. Pringle J. Herskowitz I. Nature. 1992; 356: 77-79Crossref PubMed Scopus (158) Google Scholar)). Gene disruptions were made by one-step gene replacement with appropriately digested DNA. In some cases the original auxotrophic markers on disruption cassettes were altered using "marker swap" plasmids (31Cross F.R. Yeast. 1997; 13: 647-653Crossref PubMed Scopus (140) Google Scholar). Deletion alleles forCLN genes and CLN expression constructs were as described previously (14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Other plasmids used were as follows: pL19 (pURA3-GAL1::STE4 (32Whiteway M. Hougan L. Thomas D.Y. Mol. Cell. Biol. 1990; 10: 217-222Crossref PubMed Scopus (107) Google Scholar)),pURA3-GAL1::STE20ΔN (33Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar), pVTU-STE20 (pURA3-ADH-STE20 (23Leberer E. Dignard D. Hougan L. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4805-4813Crossref PubMed Scopus (76) Google Scholar)), pGA2013 (pTRP1-GAL1::STE5-myc (provided by G. Ammerer, Vienna)), pGU-STE11ΔN (pURA3- GAL1::STE11ΔN (34Cairns B.R. Ramer S.W. Kornberg R.D. Genes Dev. 1992; 6: 1305-1318Crossref PubMed Scopus (143) Google Scholar)), pSL1508 and pSL1509 (respectivelypURA3-STE11.1 and pURA3-STE11.4 (24Stevenson B.J. Rhodes N. Errede B. Sprague Jr., G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar)), and pGK40 (pURA3-GAL1::STE12 (35Dolan J.W. Fields S. Genes Dev. 1990; 4: 492-502Crossref PubMed Scopus (96) Google Scholar)).Table IStrains1255–5CMATa bar1BOY391MATa bar1 his3 HIS2BOY1037MATa bar1 LEU2::GAL1::CLN2BOY389MATa bar1 his3 HIS2 TRP1::GAL1::CLN2BOY575MATa bar1 ste2::LEU2BOY1151MATa bar1 ste18::LEU2BOY527MATa bar1 ste4::LEU2BOY1149MATa bar1 ste5::LEU2BOY594MATa bar1 ste20::URA3BOY1370MATa bar1 ste20::LEU2BOY1277MATa bar1 ste11::URA3BOY1289MATa bar1 ste11::TRP1BOY763MATa bar1 ste7::LEU2BOY906MATa bar1 kss1::LEU2 fus3::TRP1BOY522MATa bar1 kss1::LEU2 fus3::URA3BOY529MATa bar1 ste12::LEU2BOY501MATa bar1 cdc15–2BOY443MATaBOY1427MATa LEU2::GAL1::CLN2BOY445MATa ste20::LEU2BOY743MATa bar1 cdc28–13BOY1143MATa bar1 cdc28–13 cln1 cln2 4×CLN3BOY836MATa bar1 cln1 cln2 cln3 pTRP1::GAL1::CLN1BOY183MATa bar1 cln1 cln2 cln3 LEU2::GAL1::CLN2BOY747MATa bar1 cln1 cln2 cln3 LEU2::GAL1::CLN3BOY796MATalpha bar1 cla4::TRP1BOY1113MATa bar1 akr1::URA3 his3BOY1162MATalpha bar1 akr1::URA3BOY489MATa bar1 ste20::TRP1BOY1138MATa bar1 bem1::LEU2L5976MATa/MATαBOY1565MATa/MATα cln1::URA3/cln1::URA3 cln2::TRP1/ cln2::LEU210560–4DMATaBOY1452MATa cln1::URA3BOY1459MATa cln2::LEU2BOY1451MATa cln1::URA3 cln2::LEU2Most strains were isogenic to BF264–15D (trp1–1a leu2–3, 112 ura3 ade1 his2).Strain isogenic to W303.Strains are ura3–52 trp1::hisG leu2::hisG and congenic to Σ1278b (6Liu H. Styles C.A. Fink G.R. Science. 1993; 262: 1741-1744Crossref PubMed Scopus (428) Google Scholar). Open table in a new tab Most strains were isogenic to BF264–15D (trp1–1a leu2–3, 112 ura3 ade1 his2). Strain isogenic to W303. Strains are ura3–52 trp1::hisG leu2::hisG and congenic to Σ1278b (6Liu H. Styles C.A. Fink G.R. Science. 1993; 262: 1741-1744Crossref PubMed Scopus (428) Google Scholar). Cells were grown in YEP medium or synthetic dropout medium with raffinose, galactose, or dextrose as described (14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Assays for pseudohyphal growth in diploid cells and agar-invasive growth in haploid cells were performed as described (5Gimeno C.J. Ljungdahl P.O. Styles C.A. Fink G.R. Cell. 1992; 68: 1077-1090Abstract Full Text PDF PubMed Scopus (990) Google Scholar, 7Roberts R.L. Fink G.R. Genes Dev. 1994; 8: 2974-2985Crossref PubMed Scopus (526) Google Scholar). Cell cycle synchronization of cln1−, cln2−, and cln3− cells by conditional cyclin expression from theGAL1 promoter and synchronization protocols for strains with thermosensitive cdc15-2 and cdc28-13 alleles were as described (14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Cell cycle progression was followed by determining the fraction of unbudded cells or by analysis of transcripts with known patterns of cell cycle regulation. Procedures for RNA hybridization ("Northern") mRNA analysis were as described previously (16McKinney J.D. Chang F. Heintz N. Cross F.R. Genes Dev. 1993; 7: 833-843Crossref PubMed Scopus (123) Google Scholar). FUS1 and CLN2 DNA restriction fragments were excised from low melting point agarose gels, and SST2, TCM1, and H2A fragments were generated by polymerase chain reaction as described (15Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar). DNA fragments were radiolabeled by random-prime labeling using a Prime-It kit (Stratagene), and transcript levels were visualized and quantitated using a Molecular Dynamics STORM PhosphorImager system. Immunoblot ("Western") protein analysis by enhanced chemiluminescence was essentially as described (15Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar). Polyclonal rabbit antibodies, which were raised against residues in kinase-domains VI or XI of Ste20 (Kinetek, Richmond, British Columbia, Canada), were used at a dilution 1:2000. We have previously shown that constitutive expression of CLN2 from the strong GAL1 promoter can effectively block the response to mating factor (14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). Activation of the mating factor pathway by deletion of the α-subunit of the heterotrimeric G-protein can also be repressed by overexpression of CLN2 (14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar). This latter observation suggests that inhibition of the mating factor pathway by CLN2 takes place at a level downstream of the mating factor receptor and the α-subunit of the G-protein. We wanted to determine the site of action of Cln2 on the mating factor pathway more precisely. For this epistatic analysis, we used expression constructs of particular genes, whose high level expression has been shown to induce the mating factor response pathway. Among these are STE4 (the β-subunit of the G-protein) (32Whiteway M. Hougan L. Thomas D.Y. Mol. Cell. Biol. 1990; 10: 217-222Crossref PubMed Scopus (107) Google Scholar), activated alleles of the STE20 and STE11kinases (24Stevenson B.J. Rhodes N. Errede B. Sprague Jr., G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar, 33Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar, 34Cairns B.R. Ramer S.W. Kornberg R.D. Genes Dev. 1992; 6: 1305-1318Crossref PubMed Scopus (143) Google Scholar), the "scaffolding" protein STE5(36Akada R. Kallal L. Johnson D.I. Kurjan J. Genetics. 1996; 143: 103-117Crossref PubMed Google Scholar, 37Hasson M.S. Blinder D. Thorner J. Jenness D.D. Mol. Cell. Biol. 1994; 14: 1054-1065Crossref PubMed Google Scholar), and the transcription factor STE12 (35Dolan J.W. Fields S. Genes Dev. 1990; 4: 492-502Crossref PubMed Scopus (96) Google Scholar). We studied the effect of simultaneous expression of CLN2 and these activators of the mating factor response pathway (Fig. 1). In all cases the signal transduction activity of wild type cells and GAL1::CLN2 cells without the expression construct served as a control. As shown in Fig. 1 A, overexpression of CLN2 from theGAL1 promoter can prevent the induction of FUS1transcription caused by overexpression of STE4. Even whenSTE4-overexpressing cells were treated with mating factor, simultaneous overexpression of CLN2 could prevent the induction of FUS1. In contrast, overexpression of a truncated allele of STE20 (STE20ΔN, Fig. 1 B), a truncated allele of STE11(STE11ΔN, Fig. 1 D) and STE12 (Fig. 1 E), resulted in elevated levels of FUS1transcription in the presence of high levels of CLN2. In all these cases, expression of CLN2 from the GAL1promoter also failed to prevent the additional elevation of FUS1 transcript levels by addition of mating factor (Fig. 1,B, D, and E). (It should be noted that, in a previous publication (14Oehlen L.J.W.M. Cross F.R. Genes Dev. 1994; 8: 1058-1070Crossref PubMed Scopus (105) Google Scholar), we have referred to preliminary results that appeared to show epistasis of GAL1::STE4 to GAL1::CLN2. These data now turn out to be incorrect and were probably due to a mix-up of plasmids. We wish to apologize for any problem that this may have caused.). We found that high level CLN2 expression not only failed to down-regulate elevated FUS1 transcription induced byGAL1::STE11ΔN (Fig. 1 D) but alsoSST2 transcription (the transcript is regulated similarly toFUS1 (38Dietzel C. Kurjan J. Mol. Cell. Biol. 1987; 7: 4169-4177Crossref PubMed Scopus (161) Google Scholar)) induced by activated STE11 alleles (STE11-1 and STE11-4 (24Stevenson B.J. Rhodes N. Errede B. Sprague Jr., G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar)) expressed from their own promoter (Fig. 2 D). In fact, high level CLN2 expression may somewhat enhance the effect of the STE11-4 allele on SST2transcription. This finding is in contrast to results that were recently reported by others (17Wassmann K. Ammerer G. J. Biol. Chem. 1997; 272: 13180-13188Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Even using the same strains that were used in that study (17Wassmann K. Ammerer G. J. Biol. Chem. 1997; 272: 13180-13188Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), we have been unable to reproduce the result that high level CLN2 expression represses the elevation of FUS1 transcription caused by the activated allelesSTE11-1 and STE11-4 (data not shown). The results shown in Fig. 2 D cannot be explained by ineffective expression of CLN2, as mating factor-induced transcription of SST2 is effectively blocked by high level CLN2expression in these cells (Fig. 2 E). We have no explanation at present for this discrepancy. The GAL1::STE20ΔN construct fails to complement the mating defect of ste4, ste5, ste11, ste7, and ste12 cells (33Ramer S.W. Davis R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 452-456Crossref PubMed Scopus (169) Google Scholar). To provide additional epistatic information, we determined the effects of overexpression of GAL1::STE20ΔN onFUS1 transcription in strains that were deleted for various components of the mating factor signal transduction pathway (Fig. 2 A). Induction of FUS1 byGAL1::STE20ΔN was observed in cells lacking the mating factor receptor (STE2), components of the heterotrimeric G-protein (STE4 and STE18), and STE5. However, other components of the signal transduction pathway were required for GAL1::STE20ΔN-inducedFUS1 transcription. These data are consistent with the established epistatic position of STE20 downstream of the G-protein and upstream of the STE11-STE7-MAP kinase cassette (23Leberer E. Dignard D. Hougan L. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4805-4813Crossref PubMed Scopus (76) Google Scholar) and thus demonstrate the usefulness of theGAL1::STE20ΔN construct for epistatic analysis. The epistatic position of STE5 in the mating factor response pathway is complicated, possibly because of the many proteins with which Ste5 interacts (39Elion E.A. Trends Cell Biol. 1995; 5: 322-327Abstract Full Text PDF PubMed Scopus (77) Google Scholar). A GAL1::STE5 construct has been used previously in epistatic experiments, but when plating efficiency of cells containing a GAL1::STE5plasmid was monitored, this yielded rather complex results (36Akada R. Kallal L. Johnson D.I. Kurjan J. Genetics. 1996; 143: 103-117Crossref PubMed Google Scholar). We tested the FUS1 induction by theGAL1::STE5 construct in strains deleted for various components of the signal transduction pathway (Fig. 2 B). FUS1 induction by overexpression of STE5 required, with the exception of STE2, the presence of all the tested components of the mating factor signal transduction pathway. Activated alleles of STE5 were previously shown to partially complement the mating defect of strains deleted for the mating factor receptor, components of the heterotrimeric G-protein or Ste20, but not deletion of components of the STE11-STE7-MAP kinase cassette (23Leberer E. Dignard D. Hougan L. Thomas D.Y. Whiteway M. EMBO J. 1992; 11: 4805-4813Crossref PubMed Scopus (76) Google Scholar, 37Hasson M.S. Blinder D. Thorner J. Jenness D.D. Mol. Cell. Biol. 1994; 14: 1054-1065Crossref PubMed Google Scholar). Our data confirm the findings of Hasson et al. (37Hasson M.S. Blinder D. Thorner J. Jenness D.D. Mol. Cell. Biol. 1994; 14: 1054-1065Crossref PubMed Google Scholar) and extend previous analyses (37Hasson M.S. Blinder D. Thorner J. Jenness D.D. Mol. Cell. Biol. 1994; 14: 1054-1065Crossref PubMed Google Scholar, 40Leberer E. Dignard D. Harcus D. Hougan L. Whiteway M. Thomas D.Y. Mol. Gen. Genet. 1993; 241: 241-254Crossref PubMed Scopus (49) Google Scholar) by showing that induction of FUS1by high level expression of STE5 also requiresSTE20. The failure of induction of FUS1 byGAL1::STE5 in ste20− cells suggests that slow growth of such cells that was observed by Akada et al. (36Akada R. Kallal L. Johnson D.I. Kurjan J. Genetics. 1996; 143: 103-117Crossref PubMed Google Scholar) is not due to increased transcriptional activity of the mating factor response pathway. On the whole, the data obtained withGAL1::STE5 constructs yield rather complex results that are difficult to place in an epistatic series and therefore are of limited use in establishing the position of the negative effect of Cln2-Cdc28 on the mating factor signal transduction pathway. Because of this, the observation that GAL1::CLN2 expression represses the signal generated by GAL1::STE5 only in the absence, and not in the presence of mating factor (Fig. 1 C), is also difficult to interpret. The epistatic position of Ste11 in relation to most other components of the mating factor signal transduction pathway is fairly well established by transcriptional-induction and mating-complementation assays using STE11-1 and STE11-4 alleles (24Stevenson B.J. Rhodes N. Errede B. Sprague Jr., G.F. Genes Dev. 1992; 6: 1293-1304Crossref PubMed Scopus (241) Google Scholar) or the GAL1::STE11ΔN construct (34Cairns B.R. Ramer S.W. Kornberg R.D. Genes Dev. 1992; 6: 1305-1318Crossref PubMed Scopus (143) Google Scholar). The observation that STE11-1 and STE11-4 also induce significant levels of SST2 in ste20 cells (Fig. 2 C) is consistent with the general notion that Ste11 acts downstream of Ste20. Taken together, the data presented in Figs. 1 and 2 show thatGAL1::CLN2 represses the mating factor pathway at a level which is at or downstream of STE4 and at or upstream of STE20. The observations that mating factor-induced hyperphosphorylation of Ste7 (41Zhou Z. Gartner A. Cade R. Ammerer G. Errede B. Mol. Cell. Biol. 1993; 13: 2069-2080Crossref PubMed Scopus (120) Google Scholar), tyrosine phosphorylation of Fus3 (42Errede B. Gartner A. Zhou Z. Nasmyth K. Ammerer G. Nature. 1993; 362: 261-264Crossref PubMed Scopus (145) Google Scholar) (assayed with anti-phosphotyrosine antibodies in immunoprecipitates of Fus3), and Fus3 kinase activation can be prevented by high level expression of CLN2 (data not shown (15Oehlen L.J.W.M. McKinney J.D. Cross F.R. Mol. Cell. Biol. 1996; 16: 2830-2837Crossref PubMed Scopus (94) Google Scholar)) are consistent with this epistatic placement of the negative effect of Cln2-Cdc28 on the mating factor response pathway. Since the Ste20 protein is one of the potential targets suggested by epistatic analysis and since studies of the Ste20 homolog Cla4 suggest a potential genetic interaction between Ste20 and the G1cyclins CLN1 and CLN2 (8Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (310) Google Scholar, 12Benton B.K. Tinkelenberg A. Gonzalez I. Cross F.R. Mol. Cell. Biol. 1997; 17: 5067-5076Crossref PubMed Scopus (101) Google Scholar), we focused on Ste20 as a potential site for repression of mating factor signal transduction. We first looked at the abundance of the Ste20 protein at various cell cycle stages. For this purpose, temperature-sensitivecdc15-2 cells were arrested in late M-phase at restrictive temperature, and synchronous cell cycle progression was then initiated by lowering the temperature. Ste20 appears to be present at all cell cycle positions, but there are marked changes in the mobility of the protein when cells progress through the cell cycle (Fig.

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