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Localized Feedback Phosphorylation of Ste5p Scaffold by Associated MAPK Cascade

2004; Elsevier BV; Volume: 279; Issue: 45 Linguagem: Inglês

10.1074/jbc.m405681200

1083-351X

Annette Flotho, David Simpson, Maosong Qi, Elaine A. Elion,

Ubiquitin and proteasome pathways

Scaffold proteins play pivotal roles during signal transduction. In Saccharomyces cerevisiae, the Ste5p scaffold protein is required for activation of the mating MAPK cascade in response to mating pheromone and assembles a G protein-MAPK cascade complex at the plasma membrane. To serve this function, Ste5p undergoes a regulated localization event involving nuclear shuttling and recruitment to the cell cortex. Here, we show that Ste5p is also subject to two types of phosphorylation and increases in abundance as a result of MAPK activation. During vegetative growth, Ste5p is basally phosphorylated through a process regulated by the CDK Cdc28p. During mating pheromone signaling, Ste5p undergoes increased phosphorylation by the mating MAPK cascade. Multiple kinases of the mating MAPK cascade contribute to pheromone-induced phosphorylation of Ste5p, with the mating MAPKs contributing the most. Pheromone induction or overexpression of the Ste4p Gβ subunit increases the abundance of Ste5p at a post-translational step, as long as the mating MAPKs are present. Increasing the level of MAPK activation increases the amount of Ste5p at the cell cortex. Analysis of Ste5p localization mutants reveals a strict requirement for Ste5p recruitment to the plasma membrane for the pheromone-induced phosphorylation. These results suggest that the pool of Ste5p that is recruited to the plasma membrane selectively undergoes feedback phosphorylation by the associated MAPKs, leading to an increased pool of Ste5p at the site of polarized growth. These findings provide evidence of a spatially regulated mechanism for post-activation control of a signaling scaffold that potentiates pathway activation. Scaffold proteins play pivotal roles during signal transduction. In Saccharomyces cerevisiae, the Ste5p scaffold protein is required for activation of the mating MAPK cascade in response to mating pheromone and assembles a G protein-MAPK cascade complex at the plasma membrane. To serve this function, Ste5p undergoes a regulated localization event involving nuclear shuttling and recruitment to the cell cortex. Here, we show that Ste5p is also subject to two types of phosphorylation and increases in abundance as a result of MAPK activation. During vegetative growth, Ste5p is basally phosphorylated through a process regulated by the CDK Cdc28p. During mating pheromone signaling, Ste5p undergoes increased phosphorylation by the mating MAPK cascade. Multiple kinases of the mating MAPK cascade contribute to pheromone-induced phosphorylation of Ste5p, with the mating MAPKs contributing the most. Pheromone induction or overexpression of the Ste4p Gβ subunit increases the abundance of Ste5p at a post-translational step, as long as the mating MAPKs are present. Increasing the level of MAPK activation increases the amount of Ste5p at the cell cortex. Analysis of Ste5p localization mutants reveals a strict requirement for Ste5p recruitment to the plasma membrane for the pheromone-induced phosphorylation. These results suggest that the pool of Ste5p that is recruited to the plasma membrane selectively undergoes feedback phosphorylation by the associated MAPKs, leading to an increased pool of Ste5p at the site of polarized growth. These findings provide evidence of a spatially regulated mechanism for post-activation control of a signaling scaffold that potentiates pathway activation. Cells employ complex signal transduction networks to properly adapt to environmental stimuli and integrate different external cues with the physiological state of the cell. Scaffold and adapter proteins play crucial roles in mediating the temporal and spatial organization of the networks of signal transduction enzymes that mediate responses to stimuli (1Elion E.A. Trends Cell Biol. 1995; 5: 332-337Abstract Full Text PDF Scopus (77) Google Scholar, 2Pawson T. Scott D.J. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1887) Google Scholar, 3Whitmarsh A.J. Davis R.J. Trends Biochem. Sci. 1998; 23: 481-485Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 4Tsunoda S. Zuker C.S. Cell Calcium. 1999; 26: 165-171Crossref PubMed Scopus (82) Google Scholar, 5Schaeffer M.J. Weber H.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1397) Google Scholar, 6Burack W.R. Shaw A.S. Curr. Opin. Cell Biol. 2000; 12: 211-216Crossref PubMed Scopus (278) Google Scholar, 7Miller W.E. Lefkowitz R.J. Curr. Opin. Cell Biol. 2001; 13: 139-145Crossref PubMed Scopus (278) Google Scholar, 8Raabe T. Rapp U.R. Science's STKE. 2002; (http://www.stke.sciencemag.org/cgi/content/full/sigtrans;2002/136/pe28)PubMed Google Scholar). To fulfill these tasks, scaffold and adapter proteins are regulated at the level of phosphorylation, oligomerization, and cellular distribution, which may influence the activity and location of the scaffold and its binding partners (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 10Blondel M. Galan J.M. Chi Y. Lafourcade C. Longaretti C. Deshaies R.J. Peter M. EMBO J. 2000; 19: 6085-6097Crossref PubMed Scopus (102) Google Scholar, 11Brennan J.A. Volle D.J. Chaika O.V. Lewis R.E. J. Biol. Chem. 2002; 277: 5369-5377Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 12Roy F. Laberge G. Douziech M. Ferland-McCollough D. Therrien M. Genes Dev. 2002; 16: 427-438Crossref PubMed Scopus (171) Google Scholar, 13Wang Y. Elion E.A. Mol. Biol. Cell. 2003; 14: 2543-2558Crossref PubMed Scopus (31) Google Scholar, 14Matsuura H. Nishitoh H. Takeda K. Matsuzawa A. Amagasa T. Ito M. Yoshioka K. Ichijo H. J. Biol. Chem. 2002; 277: 40703-40709Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar).The mating response of haploid Saccharomyces cerevisiae cells provides one of the best studied examples of a signaling pathway that is regulated by a scaffold protein (15Elion E.A. Curr. Opin. Microbiol. 2000; 3: 573-581Crossref PubMed Scopus (223) Google Scholar, 16Elion E.A. J. Cell Sci. 2001; 114: 3967-3978Crossref PubMed Google Scholar, 17Dohlman H.G. Thorner J.W. Annu. Rev. Biochem. 2001; 70: 703-754Crossref PubMed Scopus (352) Google Scholar). Upon binding of mating pheromone to a G protein-coupled receptor of the serpentine family, the Gβγ (Ste4p/Ste18p) dimer of the G protein is released from an inhibitory Gα subunit (Gpa1p) and activates a mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; NLS, nuclear localization signal; HA, hemagglutinin.1The abbreviations used are: MAPK, mitogen-activated protein kinase; PIPES, 1,4-piperazinediethanesulfonic acid; GST, glutathione S-transferase; NLS, nuclear localization signal; HA, hemagglutinin. cascade. The MAPK cascade consists of a MAPKKK Ste11p, a MAPKK Ste7p, and two MAPKs, Fus3p and Kss1p, of which Fus3p is the major MAPK. The relay of the signal through the MAPK cascade is achieved through sequential phosphorylation of each kinase. Previous work has established that the Ste5p scaffold is essential for this signal relay and plays two distinct roles: Ste5p binds to Ste11p, Ste7p, and Fus3p and tethers them into an active complex. In addition, Ste5p binds to the Gβ subunit of the activated G protein and enables Ste11p to be activated by Ste20p, a p21-activated protein kinase that is enriched at the plasma membrane through its association with Cdc42p, a Rhotype GTPase.A variety of evidence argues that it is the interaction between a RING-H2 domain in Ste5p and the Gβ subunit (Ste4p) that allows for the assembly of the associated MAPK cascade near Ste20p at the plasma membrane (18Inouye C. Dhillon N. Thorner J. Science. 1997; 278: 103-106Crossref PubMed Scopus (140) Google Scholar, 19Feng Y. Song L.Y. Kincaid E. Mahanty S.K. Elion E.A. Curr. Biol. 1998; 8: 267-278Abstract Full Text Full Text PDF PubMed Google Scholar, 20Pryciak P.M. Huntress F.A. Genes Dev. 1998; 12: 2684-2697Crossref PubMed Scopus (194) Google Scholar, 21Van Drogen F. Stucke V.M. Jorritsma G. Peter M. Nat. Cell Biol. 2001; 3: 1051-1059Crossref PubMed Scopus (105) Google Scholar). Localization studies indicate that Ste5p undergoes an elaborate recruitment process to be functional (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). During vegetative growth, Ste5p continuously shuttles between cytoplasm and nucleus. In response to mating pheromone, a pool of Ste5p that is derived from the nucleus is recruited to Ste4p at the plasma membrane.Despite the pivotal role of Ste5p in regulating the mating MAPK cascade, little is known about how it is regulated at a molecular level. The active form of Ste5p is an oligomer, which may also shuttle and be recruited to the plasma membrane (13Wang Y. Elion E.A. Mol. Biol. Cell. 2003; 14: 2543-2558Crossref PubMed Scopus (31) Google Scholar, 22Yablonski D. Marbach I. Levitzki A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13864-13869Crossref PubMed Scopus (67) Google Scholar). Ste5p may also undergo conformational changes to mediate activation of the MAPK cascade (13Wang Y. Elion E.A. Mol. Biol. Cell. 2003; 14: 2543-2558Crossref PubMed Scopus (31) Google Scholar, 23Sette C. Inouye C.J. Stroschein S.L. Iaquinta P.J. Thorner J. Mol. Biol. Cell. 2000; 11: 4033-4049Crossref PubMed Scopus (50) Google Scholar). In vitro evidence suggests that Fus3p phosphorylates Ste5p (24Kranz J.E. Satterberg B. Elion E.A. Genes Dev. 1994; 8: 313-327Crossref PubMed Scopus (108) Google Scholar), however, in vivo evidence in support of such a feedback regulatory mechanism has been lacking. Previous work suggests that the bulk pool of Ste5p is phosphorylated on at least 15 serine and threonine residues during vegetative growth (25.Hasson, M. S. (1992) Analysis of Saccharomyces cerevisiae Pheromone Response: Biochemical and Genetic Characterization of the Ste5 Protein. Ph.D. thesis, University of California, Berkeley, CAGoogle Scholar), however, phosphorylation as a result of mating pheromone was not detected. To better understand how Ste5p phosphorylation is regulated, we devised a methodology that allows reproducible detection of phosphorylated forms of Ste5p expressed at native levels in vivo both during vegetative growth and pheromone signaling. Using this methodology, we find that Ste5p is phosphorylated by two distinct sets of kinases during vegetative growth and in response to mating pheromone. Pheromone-induced phosphorylation requires plasma membrane localization of Ste5p and is primarily regulated by the mating MAPKs with additional input by upstream kinases. Moreover, the mating MAPKs positively regulate the abundance of Ste5p at a post-translational step during pheromone stimulation, suggesting a potential level of feedback control that could be regulated by phosphorylation.MATERIALS AND METHODSStrains and Plasmids—See Table I for a list of yeast strains and plasmids used in this study. Yeast strains were grown in standard selective synthetic complete (SC) media. Strains transformed with PGAL-driven genes were pre-grown in 2% raffinose medium, and then switched to 2% galactose medium to induce transcription. MAPK cascade kinase deletion strains carrying an additional copy of the STE12 gene under control of a leaky GAL1 promoter (pNC252) were grown in 2% dextrose medium, which permitted low-level transcription of STE12. The leaky GAL1 promoter was confirmed in a growth test using a PFUS1-HIS3 reporter gene. Pheromone induction was performed at a cell density of ∼A600 1.0 for 1 h with 250 nm α-factor (C. Dahl, Harvard Medical School, Boston, MA) for bar1Δ cells and 5 μm for BAR1 cells, unless indicated otherwise. In some instances, mating pathway activation employed overexpression of the STE4 genes as follows: cells carrying PGAL-STE4 (pL19) were grown in 2% raffinose medium to a cell density of ∼A600 0.8, then switched to 2% galactose medium for 4 h prior to a 1-h α-factor exposure. Cells carrying temperature-sensitive mutations were grown at room temperature, and then shifted to 37 °C for 4 h. Transformation of yeast was performed as described (26Baker R. Nucleic Acids Res. 1991; 19: 1945Crossref PubMed Scopus (11) Google Scholar) with the addition of 40 mm dithiothreitol upon plasmid DNA incubation. Standard cloning techniques were used to construct all plasmids. pCU-NLSK128T-S5-M9 was made by swapping a 1.2-kb AFLII-SphI fragment of pSKM96 (S. Mahanty) into pSKM12 (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). pCL-S5C180A-M9 was made by swapping a 4.1-kb SacI-SphI fragment of pSKM88 (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) into pSKM49 (S. Mahanty). Gene replacements were carried out by homologous recombination using EY957/pNC113 to create AFY112 and EY1110/pSURE11 to create AFY335. Gene replacements were confirmed by mating assays. AFY49, AFY104, and AFY274 are ura3- derivatives of EY1881 (E. Elion), K4580 (27Cvrckova F. De Virgilio C. Manser E. Pringle J.R. Nasmyth K. Genes Dev. 1995; 9: 1817-1830Crossref PubMed Scopus (307) Google Scholar) and EY1883 (28Lyons D.M. Mahanty S.K. Choi K.Y. Manandhar M. Elion E.A. Mol. Cell. Biol. 1996; 16: 4095-4106Crossref PubMed Scopus (78) Google Scholar), respectively; obtained by selection with 5-fluoro-orotic acid.Table IYeast strains and plasmids used in this study All yeast strains are MATa and are derivatives of W303, if not noted otherwise.Strains/plasmidsGenotype/descriptionSourceEY699MATa ura3-1 leu2-3, 112 trp1-1 his3-11,15 ade2-1 can1–100R. RothsteinEY700fus3–6::LEU2Elion lab collectionEY705ste5ΔH3::TRP1Elion lab collectionEY718ste12Δ::URA3Elion lab collectionEY723fus3–6::LEU2 kss1Δ::URA3Elion lab collectionEY725kss1Δ::URA3Elion lab collectionEYL357msn5::HIS3Elion lab collectionEY957bar1ΔElion lab collectionEY940bar1Δ fus3–6::LEU2Elion lab collectionEY1110bar1Δ fus3–6::LEU2 kss1::ADE2Elion lab collectionEY1119bar1Δ kss1Δ::HIS3Elion lab collectionEY1775bar1Δ ste5Δ::TRP1Elion lab collectionEY2786bar1Δ ste20::TRP1 lys2::PFUS1-HIS3 his3Δ200Elion lab collectionAFY49bar1Δ ste11Δ::ura3 ste5::TRP1This studyAFY112bar1Δ ste7::LEU2This studyAFY335bar1Δ ste11Δ fus3–6::LEU2 kss1Δ::ADE2This studyEY1262bar1Δ far1Δ lys2::PFUS1-HIS3 his3Δ200Elion lab collectionEY1298bar1Δ STE11-4 far1Δ lys2::PFUS1-HIS3 his3Δ200Elion lab collectionAFY274bar1Δ STE11-4 ste4Δ::ura3 far1Δ lys2::PFUS1-HIS3 his3Δ200This studyBY819bar1Δ fus3–6::LEU2 ste5ΔH3::TRP1Elion lab collectionK1950ura3-1 leu2-3,112 trp1-1 his3-11,15 ade2-1 can1-100K. NasmythAFY104ste20Δ::ura3 cla4::LEU2 + cla4–75This studyL4842ura3-1 leu2-3,112 trp1-1 his3-11,15 ade2-1 can1-100K. NasmythPY1236cdc28-4D. PellmanFLY93ura3-52 leu2-3,112 trp1Δ1 his3Δ200 (S288C derivative)D. DrubinpSKM12STE5-MYC9CENURA3S. MahantyPSKM30PGAL-STE5-MYC9CENURA3S. MahantypSKM49STE5-MYC9CENLEU2S. MahantypSKM92STE5-MYC9CENHIS3S. MahantypSKM42STE5Δ49–66-MYC9CENURA3S. MahantypSKM46TagNLS-STE5-MYC9CENURA3S. MahantypSKM88STE5C180A-MYC9CENURA3S. MahantypCL-S5C180A-M9STE5C180A-MYC9CENLEU2This StudypSKM21STE5-GFPCENURA3S. MahantypCU-NLSK128T-S5-M9TagNLSK128T-STE5-MYC9CENURA3This studypH-GS5-CTMPGAL-STE5-CTMCENHIS3P. PryciakpNC252PGAL-STE122 μURA3P. PryciakpL19PGAL-STE4CENURA3M. WhitewaypYEE121FUS3-HACENURA3E. ElionpYEE128FUS3R42-HACENURA3E. ElionpEMBL-GSTPGAL-GST2 μURA3C. ChanpYBS186PGAL-GST-STE52 μURA3B. SatterbergpSURE11ste11::hisG-URA3-hisGM. HassonpNC113ste7::LEU2B. Errede Open table in a new tab Assessment of Ste5p Abundance—Cells harboring GAL1-STE4 (pL19) and STE5-MYC9 (pSKM49 or pSKM92) were grown in SC selective medium containing 2% raffinose to an A600 of ∼0.75, then pelleted and resuspended in fresh medium containing 2% galactose and induced for 4 h with shaking at 30 °C, followed by treatment with α factor for the indicated times. The cycloheximide experiments were done by growing cells in SC selective medium containing 2% dextrose or 2% galactose to an A600 ∼ 0.75, then treated with 10 mg/ml cycloheximide and 50 nm α factor in the indicated order for the indicated lengths of time. Whole cell extracts were prepared as previously described by glass bead breakage (29Elion E.A. Satterberg B. Kranz J.E. Mol. Biol. Cell. 1993; 4: 495-510Crossref PubMed Scopus (213) Google Scholar) in the described buffer with addition of 150 mm NaCl, 2 mm benzamidine, 4 mm 1,10-phenanthroline, 50 mm NaF, 1:100 dilution of phosphatase inhibitor mixture (Sigma P2850). Decreased total protein recovered from cycloheximide-treated cells provided evidence that translation had been inhibited. The pulse expression experiments were done by expressing STE5-MYC9 from the GAL1 promoter for 1.5 h in SC selective medium containing 2% galactose to an A600 ∼ 0.5, removing an aliquot, then pelleting the cells at room temperature and resuspending them in prewarmed SC selective medium at pH 4 containing 2% glucose, with or without 2 μm α factor. The inclusion of 150 mm NaCl or greater in the breaking buffer was essential for efficient recovery of Ste5 protein and detection of increases in abundance after α factor treatment.Northern Analysis—Northern analysis was performed as described (30Elion E.A. Warner J.R. Cell. 1984; 39: 663-673Abstract Full Text PDF PubMed Scopus (152) Google Scholar). STE5 mRNA was detected with an internal 1.5-kb KpnI-SalI fragment from STE5, and FUS3 mRNA was detected with a 3.0-kb EcoRI-EcoRI fragment from pYEE93 (31Elion E.A. Grisafi P.A. Fink G.R. Cell. 1990; 60: 649-664Abstract Full Text PDF PubMed Scopus (308) Google Scholar). DNA fragments were purified from low melting agarose gels, radiolabeled by the hexamer labeling method, then purified by ethanol precipitation with carrier tRNA.Gel-shift Assays—A Myc-tagged Ste5p (Ste5-Myc9p (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar)) that is fully functional and expressed at native levels was used for the analysis. For whole cell extractions, equal numbers of cells were normalized by cell density, washed in ice-cold H2O containing 1 mm phenylmethylsulfonyl fluoride, resuspended in SDS-loading buffer containing 60 mm Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol and bromphenol blue, and then broken by vortexing with acid-washed glass beads (Sigma). The lysates were boiled then electrophoresed on 7.5% SDS-PAGE gels. Immunoblots were probed with 9E10 mAb (Harvard University Monoclonal Antibody facility; cell supernatant, 1:25 dilution) and αTcm1 mAb (J. Warner, Albert Einstein College of Medicine, Bronx, NY; 1:10,000 dilution). Immunoreactivity was detected with horseradish peroxidase-conjugated secondary antibody (Bio-Rad) using the enhanced chemiluminescence system (Amersham Biosciences).Phosphatase Treatment of Ste5-MYC9p Immune Complexes—Whole cell extracts were prepared exactly as described above except the extraction buffer contained 0.14% β-mercaptoethanol and no bromphenol blue. 150 μl of protein extract from 20 A600 units worth of cells was diluted 1:20 in modified H buffer (29Elion E.A. Satterberg B. Kranz J.E. Mol. Biol. Cell. 1993; 4: 495-510Crossref PubMed Scopus (213) Google Scholar) that also contained 150 mm NaCl, 50 mm NaF, 5 mm benzamidine, and 1 mm EDTA. Immunoprecipitations of Ste5-MYC9p were carried out as described (29Elion E.A. Satterberg B. Kranz J.E. Mol. Biol. Cell. 1993; 4: 495-510Crossref PubMed Scopus (213) Google Scholar) using 60 μl of 9E10 cell supernatant and 50 μl of Protein A-Sepharose beads (Sigma). Ste5-MYC9p immunoprecipitates were washed in dephosphorylation buffer (40 mm PIPES, pH 6.0, 1 mm dithiothreitol, 2 mm phenylmethylsulfonyl fluoride and protease inhibitors (leustatin, pepstatin, chymostatin, papain, 10 μg/ml each)). Each reaction used ⅓ of the immune complexes and 0.35 unit of acid phosphatase from potato (Fluka) dissolved in storage buffer containing 100 mm HEPES, pH 7.4, 0.5 mm MgCl2, 0.5 mm dithiothreitol, 50% glycerol. In addition, the phosphatase inhibitors, 50 mm NaF and 10 mm orthovanadate, were added where indicated. The reactions were performed at 4 °C and inactivated after 1 h by boiling the immune complexes in SDS loading buffer at 95 °C for 10 min. Immunoblot analysis was performed as described above. Bacterial alkaline phosphatase and protein phosphatase 2 were less effective at dephosphorylating Ste5-MYC9p in other experiments. 2H. Sadhegi and E. Elion, unpublished data.Kinase Assays—Fus3-HAp, Fus3K42R-HAp, and GST-Ste5p immune complexes were prepared from whole cell extracts as described (24Kranz J.E. Satterberg B. Elion E.A. Genes Dev. 1994; 8: 313-327Crossref PubMed Scopus (108) Google Scholar, 28Lyons D.M. Mahanty S.K. Choi K.Y. Manandhar M. Elion E.A. Mol. Cell. Biol. 1996; 16: 4095-4106Crossref PubMed Scopus (78) Google Scholar). Kinase assays were performed as described (29Elion E.A. Satterberg B. Kranz J.E. Mol. Biol. Cell. 1993; 4: 495-510Crossref PubMed Scopus (213) Google Scholar). Duplicate immune complexes were analyzed by immunoblot analysis using 12CA5 monoclonal antibody to detect Fus3-HAp and Fus3K42R-HAp and anti-GST antiserum to detect GST and GST-Ste5p.Visualization of Ste5-GFP and Ste5-Myc9p—WT and STE11-4 cells harboring either Ste5-GFP (expressed at native levels from the CUP1 promoter) (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) or Ste5-Myc9p (expressed from a multicopy plasmid) (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) were grown to logarithmic phase, then induced with 50 nm α factor. Ste5-GFP samples were visualized by live cell microscopy, whereas Ste5-Myc9p samples were fixed and prepared for indirect immunofluoresence with 9E10 monoclonal antibody (1:1000 dilution of ascites fluid) and a rabbit anti-mouse secondary antibody conjugated to CY3 (1:1000 dilution) essentially as described (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). 3M. Qi and E. Elion, submitted for publication. Samples were observed on an Axioskop 2 microscope (Carl Zeiss, Thornwood, NY) linked to a digital camera (C4742–95; Hamamatsu, Bridgewater, NJ).RESULTSThe Mating MAPKs Positively Regulate the Abundance of Ste5p Post-translationally—To study potential post-translational modification of Ste5p we expressed a fully functional tagged derivative of Ste5p (Ste5-MYC9p) (9Mahanty S.K. Wang Y. Farley F.W. Elion E.A. Cell. 1999; 98: 501-512Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) at native levels from a centromeric plasmid in a ste5Δ strain. Immunoblot analysis showed that the abundance of Ste5p increases as a result of mating pheromone stimulation, with the increase most apparent 1 h after α factor addition, as confirmed by reprobing for the ribosomal protein Tcm1p (Fig. 1A). Overexpression of the Ste4p Gβ subunit, which binds Ste5p, further increased the abundance of Ste5p in the presence of α factor (Fig. 1A) and was sufficient to increase the abundance of Ste5p in the absence of α factor (Fig. 1B). Similar increases in abundance were found for Fus3p (Fig. 1, A and B). The increase in Ste5p abundance was dependent on the mating MAPKs and was blocked in a fus3Δ kss1Δ double mutant (Fig. 1B). In contrast, the level of STE5 mRNA did not increase as a result of activation of the mating pathway, and was not affected by mutations in the mating MAPKs or STE12, whereas the level of FUS3 mRNA was increased by mating pheromone and decreased by mutations in STE5 and STE12 (Fig. 1C). These findings suggested that the increase in Ste5p protein was post-transcriptional.To determine whether the increase in Ste5p abundance might be post-translational, we treated cells with cycloheximide to block translation of Ste5p and then added α factor. Prior analysis has shown that α factor activation of the mating MAPKs still occurs in the presence of cycloheximide (32Farley F. Satterberg B. Goldsmith E.A. Elion E.A. Genetics. 1999; 151: 1425-1444PubMed Google Scholar). Immunoblot analysis of the cycloheximide-treated cells revealed an increase in Ste5p abundance after α factor treatment (Fig. 1D), demonstrating that the increase is post-translational. To circumvent secondary effects of cycloheximide on the level of components in the pathway that activate the MAPKs, we also induced with α factor for 1 h before adding cycloheximide for another hour in wild type and fus3Δ kss1Δ cells. The level of Ste5p was still greater as a result of α factor induction with no increase in the absence of Fus3p and Kss1p (Fig. 1E). A pulse expression method was next used to determine whether Ste5p is stabilized during α factor induction (33Zhou P. Methods Mol. Biol. 2004; 284: 67-78PubMed Google Scholar). The transcription of the STE5-MYC9 gene was induced for 90 min with the GAL1 promoter. Further expression was repressed by the addition of dextrose and the abundance of Ste5-Myc9p was monitored for 150 min in cells treated with or without α factor. The level of Ste5-Myc9p fell to 24% of the initial level in the cells that had not been treated with α factor compared with a much smaller decline to ∼85% in the α factor-treated cells (Fig. 1, F and G). Thus, Ste5p abundance increases at a post-translational step as a result of activation of the mating MAPKs, possibly as a result of stabilization of Ste5p from degradation. However, these findings do not rule out the possibility that the α factor-induced increase in Ste5p abundance may also involve enhanced translation of STE5 mRNA, because the level of Ste5p increases at the earliest (10 min) time point after the shift to glucose in α factor-treated cells (Fig. 1F).Ste5p Is Rapidly and Specifically Modified in Response to Pheromone Signaling—Previous unpublished work in this laboratory suggested that Ste5p is modified in vivo in response to the α factor mating pheromone, but it was difficult to reproducibly detect the modified forms using conventional methods of extract preparation (34.Sadeghi, H. (1999) Detection of Possible Differences in Phosphorylation of Nuclear Versus Cytoplasmic Ste5 and Possible Physical Interactions with the CDKI Far1. Master thesis, Harvard UniversityGoogle Scholar) (note broad mobility of Ste5p in Fig. 1). To better capture the modification status of Ste5p we lysed cells directly in SDS-loading buffer and separated these lysates on SDS-PAGE gels. Under these conditions, we detected a pattern of at least two differently migrating species of Ste5p during vegetative growth, and this pattern shifted toward a slower migrating species in the presence of mating pheromone in addition to greater abundance (Fig. 2A). The difference in the migration pattern in the absence and presence of mating pheromone was highly reproducible and not attributed to differences in loading, as shown by the relative levels of Tcm1p.Fig. 2Modification of Ste5p during vegetative growth and in response to pheromone signaling. A, Ste5-MYC9p modification in the absence and presence of α factor is highly reproducible. Ste5-MYC9p was expressed from its native promoter on a CEN plasmid (pSKM12) in a MATa bar1Δ ste5Δ strain (EY1775) in the absence or presence of α-factor. B, α factor time course in the presence of cycloheximide. Vegetatively dividing cells were exposed to cycloheximide for 10 min, the culture was then split into aliquots and induced with α factor for the indicated times. C, effect of osmotic shock on Ste5p modification. Note: lane 1 is overloaded compared with the other lanes based on Ponceau S staining of the immunoblot. Vegetatively dividing ste5Δ cells (EY1775) expressing Ste5-MYC9p (pSKM12) were exposed to 0.4 m NaCl for the indicated times. D, Ste5-MYC9p modification in the S288C strain background. S288C cells (FLY93) expressing Ste5-MYC9p (pSKM49) were induced with 5 μm α factor for 10 min. Samples were prepared by vortexing, heating, and sonicating in loading buffer as described under "Materials and Methods."View Large Image Figure ViewerDownload (PPT)To determine whether the modification on Ste5p was the result of an initial signaling event, we compared its mobility in an α factor time course of cells that had been pretreated with cycloheximide. An optimal shift in the Ste5p migration pattern was detected as early as 5 min after pheromone exposure, and this migration pattern was independent of the presence of cycloheximide (Fig. 2B, only +CHX data shown). 15 to 30 min after induction, the level of slower migrating Ste5p species declined slightly, but still remained detectably elevated after 60 min of α-factor exposure. These findings indicate that Ste5p modification occurs early in the signaling process and is not dependent upon transcriptional induction.To determine whether the Ste5p modification was specific to the mating pheromone stimulus, we tested whether stimuli that activate signaling pathways other than the mating MAPK cascade would affect Ste5p modification. When cells were exposed to osmotic stress conditions known to activate the high osmolarity growth pathway (i.e. 0.4 m NaCl), Ste5p did not undergo a mobility shift (Fig. 2C, although the abundance declined slightly at the 60-min time point). Similar results were obtained when cells were exposed to heat shock, which activates the protein kinase C pathway (data not shown). Therefore, the modification of Ste5p that occurs in the presence of mating pheromone is a specific consequ