Activation of the Saccharomyces Cerevisiae Filamentation/Invasion Pathway by Osmotic Stress in High-Osmolarity Glycogen Pathway Mutants

Activation of the Saccharomyces cerevisiae Filamentation/Invasion Pathway by Osmotic Stress in High-Osmolarity Glycogen Pathway Mutants

K. D. Davenport, K. E. Williams, B. D. Ullmann and M. C. Gustin Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892 Manuscript received January 7, 1999 Accepted for publication July 6, 1999

ABSTRACT Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1⌬ could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.

EAST cells, like many other eukaryotes, utilize mito- rate activating signal and physiological response (for Y gen-activated protein kinase (MAPK) cascades to review see Gustin et al. 1998), it is becoming increas- transmit signals from plasma-membrane-associated sen- ingly clear that the MAPK pathways interact with each sory complexes to the nucleus, where transcriptional other. For example, the HOG pathway MAPK Hog1p is responses are elicited (for review see Banuett 1998; rapidly dephosphorylated in response to decreases in Gustin et al. 1998). MAPK cascades are composed of extracellular osmolarity in a Slt2p-dependent manner three conserved families of protein kinases: the MAPK, (Davenport et al. 1995), suggesting that the HOG path- the MAPK and ERK kinase (MEK), and the MEK kinase way is negatively regulated by the cell integrity pathway. (MEKK; for review see Davis 1993). The MEKK receives The phosphorylation of the pheromone response path- a signal from an upstream pathway component and way MAPK Fus3p in response to hypertonic stress is phosphorylates a conserved threonine and serine resi- enhanced by the deletion of HOG1 or the HOG pathway due within the MEK’s activation domain (Kyriakis et al. MEK gene PBS2 (Hall et al. 1996). The observation 1992; Lange-Carter et al. 1993). Once phosphorylated, that the pheromone response pathway is activated in the activated MEK phosphorylates a threonine and a response to hypertonic stress in the absence of Hog1p tyrosine residue within the activation domain in the is further supported by evidence that a transcriptional MAPK (Crews and Erikson 1992). The phosphorylated target of the pheromone response pathway, FUS1, is also MAPK is then able to phosphorylate the targets of the induced by osmotic stress in the absence of catalytically MAPK cascade, including transcription factors (Gille et active Hog1p (Hall et al. 1996; O’Rourke and Hers- al. 1992; Seth et al. 1992) and other regulatory proteins kowitz 1998). It has been suggested that these data (Cook et al. 1996). indicate that Hog1p prevents the inappropriate activa- The budding yeast Saccharomyces cerevisiae contains five tion of the pheromone response pathway by osmotic MAPK cascades, each with its own unique MAPK: Fus3p stress. in the pheromone response pathway, Kss1p in the fila- These cross-pathway interactions may provide a mech- mentation/invasion pathway, Hog1p in the high-osmo- anism for establishing and maintaining signaling speci- larity-growth (HOG) pathway, Slt2p in the cell integrity ficity. The question is whether these interactions are pathway (Figure 1), and Smk1p in the spore wall assem- physiologically significant or merely introduced by the bly pathway. Although these pathways each have a sepa- genetic manipulations of the organism. For example, the activation of Fus3p by high osmolarity in the absence of Hog1p (Hall et al. 1996) may indicate an important Corresponding author: Mike Gustin, Department of Biochemistry and Cell Biology, MS140, Rice University, 6100 S. Main, Houston, TX role for Hog1p in the maintenance of signal specificity, 77005-1892. E-mail: [email protected] or it may be an artifact of the experimental overexpres-

Genetics 153: 1091–1103 (November 1999) 1092 K. D. Davenport et al.

mutant, pbs2-3. Prevention of Kss1p phosphorylation by the overexpression of the MAPK phosphatase MSG5, the deletion of KSS1, or the deletion of upstream filamentation/invasion pathway MAPK cascade genes is suf- ficient to suppress not only the osmosensitivity of pbs2-3, but also one of the morphological phenotypes associated with HOG pathway mutants on high-osmolarity media: the formation of long projections. These data indicate that the filamentation/invasion pathway is in- appropriately activated by hyperosmotic stress in cells lacking a functional HOG pathway.

MATERIALS AND METHODS

Strains, media, and general methods: The yeast strains and plasmids used in this study are listed in Table 1. The bacterial strain DH5␣ was used for all plasmid amplifications and isola- tions. Growth media (YEPD, supplemented SD, and LB) were prepared as described (Kaiser et al. 1994). Common proce- dures (DNA manipulations, bacterial propagation, etc.) were followed as described (Sambrook et al. 1989). Techniques used for genetic crosses, sporulation, dissection, and propagation of S. cerevisiae are described elsewhere (Kaiser et al. 1994). Yeast were transformed by the one-step method (Chen et al. Figure 1.—MAPK cascades in yeast. 1992). Isolation, cloning, and sequencing of pbs2-3: Wild-type strain MG159B was mutagenized with ethyl methanesulfonate (EMS) sion of FUS3. Other examples, such as the activity of and screened for colonies that failed to grow on YEPD supplemented with 900 mm NaCl or 1.5 m sorbitol and that showed Fus3p in the repression of the filamentation/invasion a reduced production of glycerol in high-osmolarity media. pathway and the maintenance of pheromone response Four complementation groups of recessive mutations were pathway signal specificity (Madhani et al. 1997), seem identified and designated hog1–hog4 (Brewster et al. 1993). to indicate that some interactions are physiologically The mutants in the hog4 complementation group (alleles of significant. However, in general, the prevalence and PBS2; Brewster et al. 1993) were then screened for the ability to grow at an intermediate osmolarity of 400 mm NaCl (YEPD physiological significance of these cross-pathway interac- plus 400 mm NaCl). Only one mutant, hog4-3 (renamed pbs2- tions are not yet well known. 3), was able to grow at this osmolarity. Thus, the phenotype The HOG pathway mutants hog1 and pbs2 were first of pbs2-3 was intermediate between a pbs2⌬ strain and the wild- isolated in a screen for yeast unable to grow or produce type strain. pbs2-3 was backcrossed to W303-1A four times to glycerol in high-osmolarity media (Brewster et al. produce strain KDY1. Genomic DNA was extracted (Hoffman 1993). Additionally, budding and growth defects have and Winston 1987) from strain KDY1 and used as a template to amplify the PBS2 locus by PCR. PCR products from three also been described for these mutants (Brewster and independent reactions were cloned using the TA cloning kit Gustin 1994). In high-osmolarity media, hog1⌬ or pbs2⌬ (Invitrogen, San Diego) to produce pKD10, pKD11, and cells will abandon a small bud and grow a new bud. pKD12. One clone, pKD10, was sequenced (Seqwrite) and This double-budded phenotype may indicate a defect compared to the Saccharomyces Genome Database and pre- in cell cycle regulation in HOG pathway mutants that viously published sequences (Boguslawski and Polazzi 1987). Discrepancies between the pbs2-3 sequence and published se- are exposed to high-osmolarity media (Brewster and quences of PBS2 were verified by sequencing portions of Gustin 1994). A second growth defect observed in pKD11 and pKD12 to ensure that the mutations identified in HOG pathway mutants grown in high-osmolarity media pKD10 were not introduced by PCR. Two mutations were is the production of long cellular projections (Brew- identified, which are predicted to code for the following substi- ster and Gustin 1994), indicating stimulated or unreg- tutions in the polypeptide sequence: proline for serine at position 168 (S168P) and aspartate for glycine at position 509 ulated polarized growth in HOG mutants exposed to (G509D). Restriction fragments from pbs2-3 containing either high osmolarity. However, the precise cause of these one or both of the identified mutations were used to replace morphological defects is not known. the corresponding restriction fragments of a plasmid con- In this article, we provide evidence that part of the taining PBS2 to produce pKD13, pKD14, and pKD15. A strain HOG pathway mutant phenotype is a consequence of deleted for PBS2 (KDY9) was transformed with these plasmids the loss of HOG-pathway-dependent inhibition of a sec- and assayed for growth on high-osmolarity media (YEPD plus 400 or 900 mm NaCl) to identify which of the mutations were ond MAPK pathway, the filamentation/invasion path- responsible for the pbs2-3 growth phenotype. way. Kss1p is shown here to be phosphorylated in re- High-copy suppressor screen: A pbs2-3 strain (KDY1) was sponse to hyperosmotic shock in a HOG pathway MEK transformed with two high-copy plasmid yeast genomic DNA Osmotic Activation of the MAPK Kss1 1093

TABLE 1 Strains and Plasmids

Genotype Reference or source Strain MG159B MAT␣ ura3-52 Brewster et al. (1993) hog4-3 MATa pbs2-3 (congenic with MG159B) Brewster et al. (1993) W303-1A MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 Laboratory stock KDY1 pbs2-3 This work KDY2 pbs2-3 kss1⌬::URA3 This work KDY3 pbs2-3 fus3⌬::LEU2 This work KDY4 pbs2-3 slt2⌬::TRP1 This work KDY5 pbs2-3 ste12⌬::URA3 This work KDY6 pbs2-3 ste7⌬::URA3 This work KDY7 pbs2-3 ste11⌬::URA3 This work KDY8 pbs2-3 sho1⌬::URA3 This work KDY9 pbs2⌬::LEU2 This work KDY10 hog1⌬::TRP1 This work KDY11 pbs2⌬::LEU2 kss1⌬::URA3 This work KDY12 pbs2⌬::LEU2 ste7⌬::URA3 This work KDY13 hog1⌬::TRP1 kss1⌬::URA3 This work KDY14 hog1⌬::TRP1ste7⌬::URA3 This work KDY15 pbs2-3 kss1⌬::URA3 fus3⌬::LEU2 This work KDY16 pbs2-3 kss1⌬::URA3 slt2⌬::TRP This work KDY17 pbs2-3 fus3⌬::LEU2 slt2⌬::TRP This work KDY18 pbs2-3 kss1⌬::URA3 fus3⌬::LEU2 slt2⌬::TRP This work Plasmid pRS426 URA3 2␮ Christianson et al. (1992) pRS423 HIS3 2␮ Christianson et al. (1992) pKD1 URA3 2␮ MSG5 This work pKD2 URA3 2␮ TRX2 This work pKD3 URA3 2␮ CAK1 This work pKD4 HIS3 2␮ MSG5 This work pKD5 HIS3 2␮ TRX1 This work pKD6 HIS3 2␮ TRX2 This work pKD7 HIS3 2␮ CAK1 This work pKD8 pGEM-7ZFϩ KSS1 This work pKD9 pGEM-7ZFϩ kss1⌬::URA3 This work pSL 1077 pBR322 Ste7⌬::URA3 Fields et al. (1988) pSL 1094 pBluescript KSϩ ste11⌬::URA3 Fields et al. (1988) pSL 1311 pSPGS ste12⌬::URA3 Fields et al. (1988) pYEE98 pUC119 fus3-6⌬::LEU2 Elion et al. (1990) YEpT-KSS1 TRP1 2␮ KSS1 Bardwell et al. (1996) YEpT-kss1 TRP1 2␮ kss1 Y-F, T-A Bardwell et al. (1996) YEpL-FTyZ LEU2 2␮ Ty1::lacZ Cook et al. (1997) YEpU-FTyZ URA3 2␮ Ty1::lacZ Cook et al. (1997) All strains used in this study are congenic with W303-1A, with the exception of MG159B and hog4-3. libraries (M. F. Hoekstra, unpublished results; C. J. Con- Plasmids: pKD2, pKD3, and pKD5–pKD7 were constructed nelly and P. Hieter, unpublished results). The transformants by first amplifying the appropriate open reading frame (TRX2, per library) were screened for the ability to grow TRX1,orCAK1) and 1-kb flanking DNA from wild-type (W303- 30,000ف) on YEPD supplemented with either 1 m KCl or 1 m sorbitol. 1A) genomic DNA using primers designed to add XhoI sites Plasmid DNA was isolated from osmoresistant colonies, ampli- to the ends of the PCR product. The PCR products were fied in bacteria, and transformed into KDY1 to test for suppres- digested with XhoI and ligated to SalI-digested pRS426 (2␮ sion of pbs2-3 osmosensitivity. Twelve colonies that demon- URA3) or pRS423 (2␮ HIS3; Christianson et al. 1992). Plas- strated plasmid-dependent osmotic resistance were isolated. mids pKD1 and pKD4 were constructed by subcloning an kb XhoI-NotI fragment of the library isolate containing-2.2ف -Plasmids were isolated and divided into five classes by restric tion mapping. The largest class (five clones) consisted of plas- MSG5 to XhoI-NotI-digested pRS426 or pRS423, respectively. mids containing PBS2. Two plasmids contained HOG1. The Primers designed to add an XhoI site on both ends of the remaining three classes of high-copy suppressors contained resulting PCR product were used to amplify a fragment con- MSG5 (two clones), CAK1 (two clones), or TRX1 (one clone), taining the KSS1 open reading frame and 500 bp of upstream- as determined by the comparison of the plasmid DNA se- and downstream-flanking DNA from wild-type (W303-1A) ge- quence (Seqwrite) to the Saccharomyces Genome Database. nomic DNA. An XhoI-digested PCR fragment containing the 1094 K. D. Davenport et al.

KSS1-coding sequence and flanking DNA was ligated to XhoI- primed labeling kit (Ambion, Austin, TX). The membrane digested pGEM-7ZF(ϩ) (Promega, Madison, WI) to produce and probe were then incubated overnight at 65Њ, washed, pKD8. pKD8 was used as a template to amplify the 500-bp dried briefly, and exposed to a phosphoimager plate. The regions flanking the KSS1 open reading frame and the vector, phosphoimaging plate was then processed using a Fujix but not the KSS1-coding sequence, using primers that add a BAS100 phosphoimager. NotI site to the ends of the resulting PCR product. Primers ␤-Galactosidase assays: Cells were grown to saturation over- -in YEPD, and grown for an addi 0.4ف containing NotI sites were also used to amplify the URA3 gene night, diluted to OD600 from YDp-U (Berben et al. 1991). These two PCR products tional 2 hr prior to the addition of solutes for the desired were digested with NotI and ligated to produce pKD9. pKD9 stress. After the desired time had elapsed, cells were pelleted, was used as a template to amplify the URA3 and flanking DNA transferred in 500 ␮l of ice-cold Z buffer (16.1 g/liter Na2 by PCR, and the product was used to replace the genomic HPO4·7H2O, 5.5 g/liter NaH2PO4·H2O, 0.75 g/liter KCl, 0.246 copy of KSS1 in yeast. The deletion of KSS1 was confirmed in g/liter MgSO4·7H2O, pH 7.0) to a 2-ml tube, pelleted, and uracil prototrophs by PCR and by sterility in combination with frozen on dry ice. To isolate the protein, 250 ␮l Z buffer and fus3⌬. 12.5 ␮l40mm PMSF were added to the pellet prior to four The plasmids used in the disruption of STE11, STE7, and freeze/thaw cycles (10-sec incubation in liquid nitrogen and STE12 were a gift from George Sprague. pYEE98 (pUC119 90-sec incubation in a 37Њ water bath). Cell debris was removed fus3-6::LEU2) was a gift from the Elion lab. Lee Bardwell pro- by centrifugation, and the protein content of the supernatant vided YEpT-KSS1 and YEpT-kss1. YEp352-CAK1, provided by was determined (Bio-Rad, Richmond, CA). Supernatant (100 Ed Winter, was used to further test whether overexpressed ␮l) was added to 900 ␮l Z buffer with 200 ␮l 4 mg/ml CAK1 can suppress pbs2-3 osmosensitivity. o-nitrophenyl-␤-d-galactopyranoside (Sigma). After a 5- to 20- Growth analysis: Overnight cultures of the desired strains min incubation, 1 ml 1 m Na2CO3 was added to stop the -in YEPD and grown for 2–3 hr at reaction, and ␤-galactosidase activity was determined by mea 0.1ف were diluted to OD600 30Њ. Cell densities were again adjusted to OD600 ϭ 0.1 in YEPD suring the OD420. and used to make serial dilutions in a 96-well plate. Equal Microscopy: Cells were grown to log phase, stressed with volumes of these cultures were transferred to YEPD plates the addition of solute (1 m KCl, 1 m sorbitol, or 900 mm NaCl), containing various solutes by use of a 48-prong replicating and fixed by addition of formaldehyde to a final concentration tool. Plates were incubated at 30Њ for various lengths of time, of 3.7%. After incubation for at least 1 hr, cells were pelleted, as indicated in the figure legends. washed once with PBS (8 g/liter NaCl, 0.2 g/liter KCl, 1.44 Immunoblots: Protein extracts were prepared from treated g/liter Na2HPO4, 0.24 g/liter KH2PO4, pH 7.4), and resus- and untreated cells, as described previously (Brewster et al. pended in PBS for storage. Cells were then diluted, sonicated 1993). A total of 20 ␮g of each protein sample was separated briefly to disrupt cell clumps, and spotted onto Superfrost by SDS-PAGE and transferred to Protran nitrocellulose filters Plus slides (Fisher Scientific, Pittsburgh). After allowing the (Schleicher & Schuell, Keene, NH). Molecular weight markers cells to adhere to the slide, the remaining liquid was removed (10 kD; GIBCO BRL, Gaithersburg, MD) were visualized by by aspiration. Five microliters of mounting media (Vecta- staining the membrane with Ponceau S (Sigma, St. Louis) and shield; Vector Laboratories, Burlingame, CA) was added di- marking the location with a pencil. The membrane was rinsed rectly to the slide, covered with a coverslip, sealed using nail and incubated with blocking buffer [3% BSA in TBS-T (6.056 polish, and stored at 4Њ. The yeast cells were visualized using g/liter Tris, 8.766 g/liter NaCl, 0.05% Tween 20, pH 8.0)]. an Axioskop (Zeiss, Thornwood, NY) set for differential inter- Monoclonal antibodies [antiphosphotyrosine (Upstate Bio- ference contrast (DIC) microscopy. technology, Lake Placid, NY) or anti-phosphoERK (Sigma)] were diluted 1:1000 in blocking buffer, added to the membrane, and incubated at room temperature for at least 90 RESULTS min. Following incubation, the membranes were washed three times for 10 min in TBS-T. Secondary antibody (HRP-conju- Isolation of a partially functional allele of PBS2: In gated anti-mouse; Boehringer Mannheim, Indianapolis) was a screen for osmosensitive mutants defective in HOG diluted 1:5000 in blocking buffer and incubated with the mem- pathway function (see materials and methods), a par- branes for at least 40 min. The membranes were then washed four times for 10 min in TBS-T prior to the addition of ECL tially functional allele of PBS2, pbs2-3, was isolated. reagent (Amersham, Arlington Heights, IL) and exposure to pbs2-3 exhibits an intermediate osmosensitivity com- Hyperfilm ECL (Amersham). pared to wild-type and pbs2⌬ strains. A pbs2-3 strain, Northern analysis: Cells were grown overnight, diluted to unlike a pbs2⌬ strain, can grow on media supplemented in YEPD, and grown for an additional 2 hr prior with 400 mm NaCl (Figure 2A). However, a pbs2-3 strain 0.4ف OD600 to the addition of solute (NaCl, KCl, or sorbitol) to produce a hyperosmotic shock. Cells were pelleted and then lysed by cannot grow on media supplemented with 900 mm vortexing with glass beads in the presence of phenol and RNA NaCl, though a wild-type strain can grow under these lysis buffer (0.5 m NaCl, 10 mm EDTA, 50 mm Tris, pH 8.0). conditions (Figure 2A). The RNA in the aqueous phase was then precipitated, dried Hypertonic-stress-induced changes in Hog1p phos- briefly, and then resuspended in 50 ␮l diethyl pyrocarbonate- phorylation and GPD1 transcription were analyzed to treated water. A total of 2 ␮l10ϫ NBC (0.5 m boric acid, 10 mm sodium citrate, 50 mm NaOH, pH 7.5), 3 ␮l formaldehyde, determine if the pbs2-3 mutation affected signal trans- 10 ␮l formamide, 2 ␮l10ϫ loading buffer (15% Ficoll, 0.1 m duction through the HOG pathway. As a MAPK, Hog1p EDTA, 0.25% bromophenol blue), and 1 ␮l ethidium bromide is activated by phosphorylation on a conserved tyrosine (1 mg/ml) were added to 4 ␮g RNA. Following electrophoretic and threonine residue, allowing detection of activated ϩ separation, the RNA was transferred to a Hybond N mem- Hog1p using commercially available antiphosphotyro- brane (Amersham), UV cross-linked, and incubated for at sine antibodies. There is increased phosphorylation of least 1 hr at 65Њ with Church buffer (7% SDS, 250 mm Na2PO4, pH 7.5; Shifman and Stein 1995). Radiolabeled probes were Hog1p and subsequent induction of GPD1 mRNA accu- prepared from gel-purified PCR products using a random- mulation in wild-type strains following osmotic stress Osmotic Activation of the MAPK Kss1 1095

(Figure 2, B and C), consistent with previous observa- tions (Brewster et al. 1993; Albertyn et al. 1994). These responses to increases in osmolarity are absent in pbs2⌬ strains. In a pbs2-3 strain, the levels of Hog1p phosphorylation and GPD1 mRNA following osmotic stress were intermediate between the wild-type and deletion strains. These data show that the partial osmosensitivity of the pbs2-3 strain is correlated with a partial loss of signaling through the HOG pathway. To identify the mutations responsible for the pbs2-3 phenotype, the pbs2-3 locus was cloned and sequenced. Two mutations were identified by comparison of the pbs2-3 sequence to published PBS2 sequences. The pbs2-3 mutations are predicted to result in a substitution of proline for serine at position 168 (S168P) and a substitution of aspartate for glycine at position 509 (G509D) in the polypeptide chain of this MEK. Plasmids were constructed in which only one of the two mutations was present in an otherwise wild-type PBS2 gene. When the single mutant plasmids were introduced into pbs2⌬ strains, the plasmid containing the S168P PBS2 mutation appeared to complement fully while the plasmid coding for the G509D PBS2 mutation gave a phenotype intermediate between wild type and pbs2⌬, similar to that seen in a pbs2-3 strain. Thus, the G509D substitution appears to be responsible for the phenotype of a pbs2-3 strain. The G509D substitution occurs near the residues (S514 and T518) predicted to be phosphorylated by the MEKKs of the HOG pathway (Maeda et al. 1995). The substitution of alanine for either S514 or T518 in Pbs2p prevents signaling through the HOG pathway in response to osmotic stress (Maeda et al. 1995). Although Figure 2.—Description of the pbs2-3 phenotype and the the exact effect of the G509D substitution on Pbs2p three high-copy suppressors of pbs2-3 osmosensitivity. (A) The activity is unknown, it may be that the perturbation of indicated strains were grown as described in materials and the activation site by the G509D substitution in pbs2-3p methods, spotted on YEPD plates supplemented with the indicated osmolytes, and grown for 2 days (control and 400 could interfere with the efficient activation of pbs2-3p. mm NaCl) or 5 days (1 m sorbitol, 1 m KCl, 900 mm NaCl). The G509D substitution is also within the kinase domain (B) Protein was isolated from W303-1A pRS426 (WT 2␮), of Pbs2p and, therefore, could also affect the catalytic KDY1 pRS426 (pbs2-3 2␮), KDY9 pRS426 (pbs2⌬ 2␮), and activity of Pbs2p independently of any effect on activa- KDY1 pKD1 (pbs2-3 2␮ MSG5) before and after the addition tion of the MEK. of NaCl (400 mm). The protein samples were used to prepare antiphosphotyrosine immuoblots, as described in materials High-copy suppressor screen: The partially osmosen- and methods. (C) RNA was isolated from the indicated strains sitive pbs2-3 strain (KDY1) was used in a screen to identify grown in YEPD followed by growth in YEPD supplemented proteins that affect growth on high-osmolarity media. with 1 m sorbitol for 1 or 3 hr. Total RNA (4 ␮g) was used pbs2-3 was transformed with two high-copy yeast geno- for each sample in the RNA blot, as described in materials mic libraries and screened for plasmid-dependent and methods. growth on high-osmolarity media (YEPD plus 900 mm NaCl). The HOG pathway genes PBS2 and HOG1 both strains (data not shown). Msg5p is a dual-specificity (ty- suppressed the osmosensitivity of the pbs2-3 strain (data rosine and threonine) phosphoprotein phosphatase not shown). Three additional genes were also identified that has been implicated previously in the downregula- as high-copy extragenic suppressors of pbs2-3: CAK1, tion of the pheromone response pathway and cell integ- TRX1, and MSG5 (Figure 2A). Cak1p is the cyclin-depen- rity pathway MAPKs, Fus3p, and Slt2p, respectively (Doi dent, kinase-activating kinase in yeast (Espinoza et al. et al. 1994; Watanabe et al. 1995). The suppression of 1996; Kaldis et al. 1996; Thuret et al. 1996). TRX1 is a MAPK cascade mutant by the overexpression of a one of the two thioredoxin genes in yeast (Gan 1991). MAPK phosphatase was intriguing, so MSG5 was selected After obtaining TRX1 in the suppressor screen, a high- for further study. copy plasmid containing TRX2 was constructed and that Suppression of pbs2-3 osmosensitivity by high-copy plasmid also suppresses the osmosensitivity of pbs2-3 MSG5 is not due to increased signaling through the 1096 K. D. Davenport et al.

HOG pathway: One possible explanation for the suppression of pbs2-3 by high-copy MSG5 is that the overexpression of Msg5p increases the signaling efficiency of the impaired HOG pathway. However, neither Hog1p tyrosine phosphorylation nor GPD1 mRNA levels are altered by MSG5 overexpression in a pbs2-3 strain (Fig- ure 2, B and C). Additionally, high-copy MSG5 also weakly suppresses the osmosensitivity of a pbs2⌬ strain (data not shown). One hypothesis to explain these data is that the activity of one or more Msg5p-regulated MAPK pathways is/are deleterious to growth at high osmolarity. Kss1p is the relevant target of Msg5p in the suppression of pbs2-3 osmosensitivity: Msg5p is a phosphoprotein phosphatase that catalyzes the dephosphorylation of tyrosine and threonine residues of a subset of acti- Figure 3.—Suppression of pbs2-3 osmosensitivity by dele- vated MAPKs, shifting them to a catalytically inactive tion of KSS1. The indicated strains were grown and spotted state. Previous work identified Msg5p as a negative regu- on YEPD supplemented with the indicated osmolytes, as described in materials and methods. The plates were incu- lator of Fus3p and Slt2p, MAPKs in the pheromone bated at 30Њ for 2 days (YEPD) or 5 days (YEPD ϩ osmolyte). response and cell integrity pathways, respectively, but not as a negative regulator of the MAPK Hog1p (Doi et al. 1994; Watanabe et al. 1995). Interestingly, both fus3⌬ strains have significantly higher levels of induction Slt2p and Fus3p have a threonine-glutamate-tyrosine of the filamentation/invasion pathway reporter gene (TEY) activation sequence similar to the mammalian FRE::lacZ, and they show enhanced invasion. Thus, the Erk1p (Robinson and Cobb 1997), while Hog1p has enhancement of pbs2-3 osmosensitivity by fus3⌬ could a threonine-glycine-tyrosine (TGY) activation sequence be due to either the loss of a pheromone response similar to mammalian stress-activated protein kinases pathway function or an increased activity of the fila- such as p38 (Kyriakis and Avruch 1996). Of the two mentation/invasion pathway. This latter possibility is other MAPKs in yeast, Kss1p also has a TEY activation further supported by the following results. sequence (Courchesne et al. 1989) and is a possible In contrast to the results with slt2⌬ and fus3⌬, deletion target of Msg5p. Smk1p, however, has a threonine-gluta- of KSS1 suppressed the osmosensitivity of a pbs2-3 strain mine-tyrosine (TNY) activation sequence (Krisak et al. on media containing 1 m sorbitol or 1 m KCl (Figure 1994), is expressed only during sporulation (Pierce et 3). Although interaction between Msg5p and Kss1p had al. 1998), and is therefore an unlikely candidate. To not been described previously, the growth of the pbs2-3 identify which of the MAPKs is the relevant target of strain lacking KSS1 on high-osmolarity media suggests Msg5p with regard to the suppression of pbs2-3 osmosen- that Kss1p is a target of Msg5p. This assertion is sup- sitivity, deletions of FUS3, SLT2, and KSS1 were con- ported by the observation that there is no additional structed in pbs2-3 and PBS2 backgrounds and tested for suppression of osmosensitivity by overexpressing MSG5 growth at high osmolarity. in a pbs2-3 kss1⌬ strain (data not shown). These data pbs2-3 strains lacking SLT2 did not grow noticeably indicate that activated Kss1p and, by extension, activated different than pbs2-3 alone on high-osmolarity media filamentation/invasion pathway may be deleterious for (Figure 3). In addition, the osmosensitivity of a pbs2-3 growth at high-osmolarity media in a strain with a defec- slt2⌬ strain is still strongly suppressed by MSG5 overex- tive HOG pathway. pression (data not shown). These data suggest that Slt2p Double and triple MAPK deletions were also made is not part of the putative MAPK pathway predicted to in pbs2-3 in an effort to unmask additional interactions be downregulated by Msg5p as part of the suppression between the various pathways analyzed. A consistent of pbs2-3 osmosensitivity. pattern of suppression by strains lacking KSS1 and sensi- pbs2-3 strains lacking FUS3 grew very poorly on the tivity in strains with a KSS1 was observed regardless of high-osmolarity media tested, though growth on YEPD the other deletions in the pbs2-3 strain. These data again alone was unaffected. This indicates that Fus3p is not support a model where Kss1p is the only relevant MAPK the critical target of Msg5p in the suppression of pbs2-3 downregulated by Msg5p in the suppression of pbs2-3 osmosensitivity. Surprisingly, the deletion of FUS3 not osmosensitivity. only failed to suppress the osmosensitivity of a pbs2-3 It has been demonstrated recently that Kss1p in its strain, it enhanced the osmosensitivity of this stain rela- nonphosphorylated (nonactivated) state is an inhibitor tive to pbs2-3 alone (Figure 3). Fus3p mediates mating of the filamentation/invasion pathway (Cook et al. 1997; responses (Elion et al. 1991; Gartner et al. 1992), and Madhani et al. 1997; Bardwell et al. 1998). When a it appears to negatively regulate the filamentation/inva- Kss1p mutant lacking the threonine and tyrosine phos- sion pathway (Madhani and Fink 1997). For example, phorylation sites in the activation loop of the enzyme Osmotic Activation of the MAPK Kss1 1097 was expressed in a pbs2-3 strain, the suppression of the tion/invasion pathway are activated in response to hy- pbs2-3 osmosensitivity was even stronger than that seen pertonic stress in pbs2-3 mutants: The increased phos- in a pbs2-3 kss1⌬ strain (data not shown). This further phorylation of Kss1p observed in a pbs2-3 strain suggests supports the assertion that activated Kss1p contributes the possibility that the filamentation/invasion pathway to the growth defect of a pbs2-3 strain on high osmolarity. is activated under hyperosmotic conditions. We further Kss1p is phosphorylated in response to osmotic shock investigated this possibility by measuring the induction in the absence of a functional HOG pathway: If Kss1p of downstream gene targets of the filamentation/inva- dephosphorylation by overexpressed Msg5p suppresses sion pathway. Normally, when the filamentation/inva- the osmosensitivity of a pbs2-3 strain, then the level of sion pathway phosphorylates Kss1p, Kss1p activates the Kss1p phosphorylation may be an important determi- transcription factors Ste12p and Tec1p, which in turn nant for growth at high osmolarity in HOG pathway activate transcription from the filamentation/invasion mutants. To investigate the phosphorylation state of response elements (FREs) present in the promoters of Kss1p in pbs2-3 strains exposed to high osmolarity, im- genes required for filamentous growth (Madhani and munoblots of proteins extracted from various strains Fink 1997). before and after hyperosmotic shock were probed with Two methods have been used to analyze the transcrip- a commercially available monoclonal antibody raised tion of filamentation/invasion-pathway-responsive genes against a phosphorylated ERK1 activation loop. ERK1 under a variety of conditions. The promoter for TEC1 is a mammalian MAPK with the same TEY activation has a FRE, providing a positive feedback loop for the sequence as the yeast MAPKs Kss1p, Fus3p, and Slt2p. filamentation/invasion pathway and making the accu- Due to the sequence similarity in this region, the anti- mulation of TEC1 mRNA a sensitive, endogenous indica- phosphoERK antibody also recognizes the phosphory- tor of pathway activation (Madhani and Fink 1997). lated forms of Kss1p, Fus3p, and Slt2p, albeit with differ- However, a recent publication has indicated that TEC1 is ent sensitivities (data not shown). not under the sole control of the filamentation/invasion An antiphosphoERK immunoreactive band at the ex- pathway, as it is also activated by the pheromone re- kD) appears in the lane sponse pathway (Oehlen 1998), a result independently 43ف) pected mobility for Kss1p corresponding to pbs2-3 cells 1 hr following hypertonic confirmed by our lab (data not shown). An alternative shock, but not in wild-type cells nor in cells overexpress- assay for filamentous pathway activation is the use of a ing MSG5 under the same conditions (Figure 4A). This FRE::lacZ construct (Madhani and Fink 1997). This 43-kD band is absent in extracts from cells lacking a KSS1 construct contains a ␤-galactosidase gene under the con- gene and is much darker in the lane corresponding to trol of the filamentous pathway responsive elements. cells overexpressing KSS1 than in control cells (Figure This construct appears to be very specific for filamenta- 4B). In contrast, there was no evidence of increased tion/invasion pathway signals (Madhani and Fink phosphorylation of Fus3p or Mpk1p following osmotic 1997). We have analyzed both TEC1 and FRE::lacZ ex- stress (Figure 4, A and B). This evidence strongly sug- pression to assay the activation of the filamentation/ gests that Kss1p at its normal physiological concentration invasion pathway transcriptional targets. within the cell is phosphorylated in pbs2-3 cells following Wild-type cells had a low basal level of TEC1 mRNA osmotic stress. that did not increase appreciably following hyperos- Downstream transcriptional targets of the filamenta- motic shock (Figure 5A). In a pbs2-3 strain, there was a

Figure 4.—Kss1p is phosphorylated in response to hypertonic stress in a pbs2-3 strain. Protein was extracted from (A) W303- 1A pRS426 (WT 2␮), KDY1 pRS426 (pbs2-3 2␮), and KDY1 pKD1 (pbs2-3 2␮ MSG5), or (B) W303 1A (WT), KDY1 (pbs2-3), KDY2 (pbs2-3 kss1⌬), or KDY2 YEpT-KSS1 (pbs2-3 kss1⌬ 2␮ KSS1) before and after the addition of 1 m KCl. The protein samples were separated by SDS-PAGE, blotted to nitrocellulose, and probed with antiphosphoERK antibodies, as described in materials and methods. 1098 K. D. Davenport et al. fold over wild pathway mutants following osmotic shock, deletions of-1.6ف) high basal level of TEC1 expression type). Exposure of the pbs2-3 strain to hyperosmotic the upstream components of the filamentation/inva- stress induced a two- to threefold increase in the TEC1 sion pathway were constructed in the pbs2-3 back- mRNA expression over the elevated basal levels and ground. These strains were assayed for growth (Figure remained high. In contrast, pbs2-3 strains overexpress- 6A), FRE::lacZ-dependent ␤-galactosidase activity (Fig- ing MSG5 had a slightly elevated basal level of TEC1 ure 6B), and expression of TEC1 (data not shown) fol- .fold over wild type) that increased moder- lowing osmotic stress-1.3ف) mRNA ately (1.4-fold over basal levels) 1 hr after hyperosmotic The deletion of filamentation/invasion pathway MAPK shock and decreased steadily thereafter. cascade components (STE11, STE7,orKSS1) suppresses Similar results were obtained using an FRE::lacZ re- the osmosensitivity of a pbs2-3 strain (Figure 6A). The porter construct to assay filamentation/invasion path- extent of suppression by these is roughly equivalent to way activity (Figure 5B). There is an increased basal that seen in a pbs2-3 strain overexpressing MSG5 (Figure level of FRE::lacZ expression in a pbs2-3 strain (1.6-fold) 6A). Interestingly, the deletion of STE7 has a greater compared to a wild-type strain. In the pbs2-3 strain, a two- suppressive effect on a pbs2-3 strain than the deletion to threefold increase in FRE::lacZ was observed following of either KSS1 or STE11. osmotic stress, while there is a modest 1.6-fold increase The deletions of filamentation/invasion pathway in the wild-type strain with the same treatment. These MAPK signaling cascade components also inhibit the data suggest that the filamentation/invasion-pathway- expression of FRE::lacZ activity in pbs2-3 cells (Figure responsive genes are activated following osmotic stress 6B). The basal levels of FRE::lacZ expression are consid- in a pbs3-2 strain. erably diminished in pbs2-3 strains lacking the upstream The requirement for KSS1 in the activation of kinases of the filamentation/invasion pathway. Also, FRE::lacZ was investigated. Interestingly, the deletion strains lacking a functional filamentation/invasion of KSS1 results in a reduced basal level of FRE::lacZ pathway MAPK cascade no longer have an increase in expression in the pbs2-3 strain back to the level observed FRE::lacZ-dependent ␤-galactosidase expression follow- in wild type. This low basal level does not increase following osmotic stress. Similar results were obtained when ing osmotic stress, consistent with the requirement for assaying for TEC1 expression following hypertonic stress Kss1p in the osmotic induction of the filamentation/ in pbs2-3 strains deleted for filamentation/invasion invasion pathway in strains lacking a functional HOG pathway MAPK cascade components (data not shown). pathway. These data suggest that the transcriptional tar- The loss of filamentous-pathway-responsive gene induc- gets of the filamentation/invasion pathway are activated tion following osmotic stress in strains with deletions in in response to osmotic stress in strains lacking a fully STE11, STE7,orKSS1 indicates that the entire MAPK functional HOG pathway in a Kss1p-dependent manner. module must be present for efficient activation of the Ste7p and Ste11p are required for growth inhibition filamentation/invasion pathway in strains lacking a fully and transcriptional activation of filamentation/invasion- functional HOG pathway. pathway-responsive genes: To determine if other com- Deletions of filamentation/invasion pathway compo- ponents of the filamentation/invasion pathway are re- nents suppress the morphological defects of pbs2-3 on quired for the inappropriate activation of Kss1p in HOG high osmolarity: HOG pathway mutants exhibit severe

Figure 5.—TEC1 transcription and FRE::lacZ expression is activated in response to hypertonic stress in a pbs2-3 strain. (A) RNA was isolated from W303-1A pRS426 (WT 2␮), KDY1 pRS426 (pbs2-3 2␮), or KDY1 pKD1 (pbs2-3 2␮ MSG5) grown in YEPD (0) followed by growth in YEPD supplemented with 1 m KCl for the indicated lengths of time. (B) Protein isolated from W303- 1A, KDY1, and KDY2 was transformed with YEpL-FTyZ before (0) or 3 hr following (3) exposure to 1 m sorbitol. The protein was then assayed for ␤-galactosidase activity, as described in materials and methods. Osmotic Activation of the MAPK Kss1 1099

Figure 6.—Upstream components of the filamentation/invasion pathway are required for the inhibition of growth on high- osmolarity and FRE::lacZ expression by high osmolarity in a pbs2-3 strain. (A) Strains deleted for various components of the filamentation/invasion pathway were grown in YEPD and spotted to plates supplemented with various solutes, as described in materials and methods. (B) Protein was isolated from the indicated strains containing YEpL-FTyZ (Cook et al. 1997) before (0) or 3 hr following (3) exposure to 1 m sorbitol. The protein was then assayed for ␤-galactosidase activity, as described in materials and methods. morphological defects following prolonged exposure tion of the filamentation/invasion pathway by hyperos- to high-osmolarity conditions (Brewster and Gustin motic shock. One high-copy suppressor of the HOG 1994). One such defect is the presence of multiple buds, pathway MEK mutant pbs2-3 identified in this study was a possible indication of a cell cycle defect in HOG path- MSG5 (Figure 2A), a MAPK phosphatase. The critical way mutants on high osmolarity. A second form of aber- target of Msg5p in the suppression of pbs2-3 osmosensi- rant morphology is the production of long projections tivity, Kss1p, was identified on the basis of suppression reminiscent of the hyphae of pathogenic yeast during of pbs2-3 by kss1⌬ (Figure 3) and the lack of effect of virulent growth phase (Lo et al. 1997) and the pseudo- high-copy MSG5 in a pbs2-3 kss1⌬ strain (data not hyphae of S. cerevisiae (Gimeno et al. 1992). To deter- shown). In strains lacking a functional HOG pathway, mine if these morphological defects are dependent on Kss1p is phosphorylated (Figure 4) and filamentation/ the filamentation/invasion pathway, wild-type, pbs2-3, invasion pathway gene targets are transcriptionally acti- and pbs2-3 strains deleted for STE7 and KSS1 were grown vated (Figure 5) following osmotic stress. The deletion in YEPD or YEPD supplemented with 1 m KCl and ana- of the filamentation/invasion pathway MAPK module lyzed by DIC microscopy (Figure 7). components STE7 or STE11 inhibited hyperosmolarity- pbs2-3 strains exposed to high osmolarity produce induced filamentation/invasion pathway activation long projections (Figure 7) similar to those observed in (Figure 6B) and restored high-osmolarity growth in hog1⌬ and pbs2⌬ strains on high osmolarity (Brewster pbs2-3 strains (Figure 6A). Finally, one of the morpho- et al. 1993). The projections frequently have striations logical defects of HOG pathway mutants on high osmo- that are strikingly similar to those produced by the in- larity, the presence of long projections, was demon- complete septation of pseudohyphae (Gimeno et al. strated to be dependent on the presence of an intact 1992). The long projections were not seen in pbs2-3 filamentation/invasion pathway (Figure 7). strains lacking KSS1 or STE7. However, several cells with The HOG pathway negatively regulates the filamenta- multiple buds were observed in pbs2-3 strains lacking tion/invasion pathway: Our data demonstrate that the KSS1 or STE7 on high osmolarity (white arrows in Figure filamentation/invasion pathway is activated in response 7). These data indicate that the aberrant polarized to osmotic stress in strains with a compromised HOG growth morphology of HOG pathway mutants requires pathway, but that it is not activated in wild-type strains. the filamentation/invasion pathway even though the Kss1p phosphorylation (Figure 4), TEC1 mRNA levels multiple-bud phenotype does not. (Figure 5A), and FRE::lacZ expression (Figure 5B) are all increased following osmotic stress in strains lacking a fully functional HOG pathway. In strains with an intact DISCUSSION HOG pathway, there is no detectable change in Kss1p In this article, we describe a previously unknown con- phosphorylation (Figure 4) or TEC1 mRNA (Figure 5A) sequence of disrupting the HOG pathway—the activa- levels following an increase in osmolarity. Though a 1100 K. D. Davenport et al.

Other results are consistent with a model in which the HOG pathway negatively regulates the filamentation/ invasion pathway. Diploid hog1⌬/hog1⌬ yeast of the R strain background show more active pseudohyphal development on low-nitrogen media than does a wild-type R strain, indicating an increased filamentation/invasion pathway activity (Madhani et al. 1997; O’Rourke and Herskowitz 1998). Our data may provide a biochemi- cal explanation for the hyperpseudohyphal phenotype of hog1⌬ cells, as we have demonstrated that the basal filamentation/invasion pathway activity, as measured by TEC1 mRNA and FRE::lacZ activity, is increased in HOG pathway mutants. We observed that the elevated basal levels of TEC1 and FRE::lacZ could be eliminated by the deletion of the components of the filamentation/ invasion pathway MAPK cascade. Together, these data suggest that it is the loss of filamentation/invasion pathway regulation by the HOG pathway that causes the hyperpseudohyphal phenotype of HOG pathway mutants in the R background. The activation of the filamentation/invasion pathway is correlated with growth inhibition on high-osmolarity media: The activation of the filamentation/invasion pathway has a physiological effect on yeast—growth inhibition on high osmolarity. The level of FRE-dependent transcription and the amount of pbs2-3 strain growth on high-osmolarity media appear to be inversely correlated with each other. pbs2-3 strains have increased FRE::lacZ and TEC1 expression, and they have a growth defect on high-osmolarity media (Figures 5, B and A, and 2A, respectively). A pbs2-3 ste7⌬ strain has a much lower level of FRE::lacZ expression than does a pbs2-3 strain, and it grows well on high-osmolarity media (Figures 6, B and A, respectively). The growth inhibition of a pbs2-3 kss1⌬ strain is intermediate between that of a pbs2-3 Figure 7.—The formation of long projections by pbs2-3 strain and a pbs2-3 ste7⌬ strain (Figure 6A), correlated strains on high osmolarity is prevented by deletion of fila- with the intermediate levels of TEC1 mRNA accumula- mentation/invasion pathway genes. The indicated strains were tion (data not shown) and FRE::lacZ expression (Figure grown to log phase, and samples were removed before (control) or after continued growth in 1 m KCl for 12 hr. Cells were 6B). These data suggest that an activated filamentation/ fixed in 3.7% formaldehyde and mounted for microscopic invasion pathway contributes to the decreased growth analysis, as described in materials and methods. Cells were of a pbs2-3 strain on high-osmolarity media. visualized by DIC microscopy using a Zeiss Axioskop fitted A pbs2-3 strain with a deletion in STE11 does not seem with a digital camera. White arrows indicate the presence of to fit such a model, though the complex roles of Ste11p a second bud. may explain the discrepancy. A pbs2-3 ste11⌬ strain and a pbs2-3 ste7⌬ strain have equally low levels of TEC1 (data small increase in FRE::lacZ expression could be detected not shown) and FRE::lacZ expression following osmotic in wild-type strains following osmotic stress, the magni- stress (Figure 6B). However, the deletion of STE11 does tude of the increase was lower than in HOG mutants not suppress the osmosensitivity of pbs2-3 as well as the (Figure 5B). These data are consistent with either the deletion of STE7 does (Figure 6A). The loss of Ste11p inappropriate activation or the loss of repression of the results in both a loss of filamentation/invasion pathway filamentation/invasion pathway in HOG mutants dur- activation (increased osmotolerance) and the loss of ing osmotic stress. However, the increased basal levels one upstream branch of the HOG pathway (possibly of TEC1 mRNA (Figure 5A) and FRE::lacZ expression decreased osmotolerance). Though the loss of the (Figure 5B) in pbs2-3, hog1⌬,orpbs2⌬ strains are more Sho1p branch of the HOG pathway did not affect high- consistent with the latter model. Together, these data osmolarity growth in an otherwise wild-type strain support a role for the HOG pathway in negatively regu- (Posas and Saito 1997), the increased sensitivity of the lating the filamentation/invasion pathway. pbs2-3 strain to perturbations in signaling may account Osmotic Activation of the MAPK Kss1 1101 for the reduced growth on high-osmolarity in a ste11⌬ example, if cells encounter both increased osmolarity strain relative to a ste7⌬ strain. and nutrient loss, it may be more advantageous for the The correlation of filamentation/invasion pathway yeast cell to move the yeast (or its progeny) to an envi- activity and decreased growth on high osmolarity is ronment more conducive to growth via filamentation, found not only in pbs2-3 cells, but also in other HOG e.g., the inside of a grape vs. the skin. This response pathway mutants. On high-osmolarity media, a pbs2⌬ could be selected at the level of intracellular signaling strain has higher levels of TEC1 mRNA and FRE::lacZ if the filamentation/invasion pathway activation by both expression than does a pbs2-3 strain under the same osmolarity and nutrient deprivation overcomes the conditions (data not shown). The higher activity of the HOG-pathway-mediated repression. However, if the cell filamentation/invasion pathway in pbs2⌬ relative to is simply being dehydrated but has a nutrient-rich envi- pbs2-3 is correlated with greater growth inhibition of ronment, the HOG pathway repression of the fila- pbs2⌬ relative to pbs2-3 (Figure 2A). Likewise, a hog1⌬ mentation pathway will prevent the expenditure of cel- strain has a greater accumulation of TEC1 mRNA follow- lular energy on filamentation and focus its energy on ing hyperosmotic stress than does a pbs2-3 strain, and overcoming the osmotic challenge. it is also less osmotolerant than a pbs2-3 strain (data not Cross-talk between MAPK cascades in yeast: One pos- shown). sible mechanism for cross-pathway regulation is the acti- The deletion of filamentation/invasion pathway vation by one MAPK pathway of a phosphatase with a genes failed to fully complement a pbs2-3 strain (Figure specificity for components of a second MAPK pathway. 6A) or cells with a deletion of either HOG1 or PBS2 (data In HeLa cells, the MAPK ERK2 induces the expression not shown). These data indicate that the deactivation of the tyrosine/threonine phosphatase MKP1-1, which of the filamentation/invasion pathway is not the sole is more active against the SAPK and p38 than ERK2 determinant of growth on high-osmolarity media. This itself (Franklin and Kraft 1997). Thus, a mechanism is not unexpected, as previous work demonstrated the exists for the downregulation of p38 and SAPK by ERK2 importance of GPD1 expression in the growth of yeast through the transcription of a phosphatase. MAPKs may on high-osmolarity media (Albertyn et al. 1994). The also directly activate MAPK phosphatases, as is the case expression of GPD1 in a pbs2-3 strain is not altered by with ERK2 and the phosphatase MPK3-1 (Camps et al. the overexpression of MSG5 (Figure 2C) nor by the 1998). However, it is not yet known if the direct activa- deletion of filamentation/invasion pathway genes (data tion of phosphatases by MAPKs is part of the cross- not shown). These data indicate that the HOG pathway pathway regulation mechanisms. coordinates responses to osmotic stress that affect There are a number of phosphatases in yeast that growth on high-osmolarity media independently of the could act in an analogous manner to the mammalian repression of the filamentation/invasion pathway. phosphatases described above. Ptp2p is transcriptionally Mechanism of filamentation/invasion pathway activa- regulated by the HOG pathway (Jacoby et al. 1997; tion after osmotic stress: The research presented here Wurgler-Murphy et al. 1997), but is more specific for demonstrates that the filamentation/invasion pathway Hog1p than for the ERK-like MAPKs (Zhan et al. 1997). is activated following hypertonic stress in HOG pathway Ptp3p is perhaps a more likely candidate, as it is activated mutants. It is perhaps significant that two pathways share by the HOG pathway (Jacoby et al. 1997; Wurgler- a MEKK, Ste11p. It is therefore possible that the activa- Murphy et al. 1997) and has a higher specificity for the tion of the filamentation/invasion pathway following ERK-like MAPKs than for Hog1p (Zhan et al. 1997). hypertonic stress occurs because of a leakage of signal However, neither the overexpression of Ptp2p nor through this common component. It is unclear whether Ptp3p suppressed the osmosensitivity of a pbs2-3 strain Ste11p freely diffuses between pathways or is bound (data not shown), though a dependence on a wild-type tightly in a signaling complex within the individual path- HOG pathway for full phosphatase activity could ac- ways. In the case of the pheromone response pathway, count for the lack of suppression. Msg5p is specific for it appears that Ste11p is tightly bound to the scaffold the ERK family of MAPKs, including Slt2p (Watanabe protein Ste5p. Though a role for Pbs2p as a similar et al. 1995), Fus3p (Doi et al. 1994), and Kss1p (this scaffold has been suggested (Posas and Saito 1997), study) in yeast, and suppresses pbs2-3 osmosensitivity it is unclear how tightly the signaling components of when overexpressed. However, the transcription of the HOG pathway are bound. Thus, the activation of the Msg5p is not Hog1p dependent (data not shown), filamentation/invasion pathway may be an unfortunate though post-transcriptional activation of Msg5p by the consequence of the sharing of Ste11p between the two HOG pathway is still possible. pathways. MAPK phosphatase specificity: MSG5 had been iden- Alternatively, the simultaneous activation of the fila- tified previously as a high-copy suppressor of constitu- mentation/invasion pathway by osmotic stress and the tively active mutants in pheromone response (Doi et al. repression of the filamentation/invasion pathway by the 1994) and cell integrity pathways (Watanabe et al. HOG pathway may be part of a mechanism providing 1995), presumably by downregulating the MAPKs of the most advantageous response to a given stimuli. For these pathways, Fus3p and Slt2p. Here, we have pro- 1102 K. D. Davenport et al. vided evidence that is consistent with the hypothesis and division after osmotic stress requires a MAP kinase pathway. Yeast 10: 425–439. that Msg5p acts on the filamentation/invasion pathway Brewster, J. L., T. de Valoir, N. D. Dwyer, E. Winter and M. C. MAPK Kss1p. It is perhaps significant that the three Gustin, 1993 An osmosensing signal transduction pathway in MAPKs that are targets of Msg5p have identical TEY yeast. Science 259: 1760–1763. Brunet, A., and J. Pouyssegur, 1996 Identification of MAP kinase activation sequences, while Hog1p and Smk1p have TGY domains by redirecting stress signals into growth factor responses. and TQY sequences, respectively (Robinson and Cobb Science 272: 1652–1655. 1997; Gustin et al. 1998). This may indicate that Msg5p Camps, M., A. Nichols, C. Gillieron, B. Antonsson, M. Muda et al., 1998 Catalytic activation of the phosphatase MKP-3 by ERK2 exhibits specificity for the ERK1 family of MAPKs. How- mitogen-activated protein kinase. Science 280: 1262–1265. ever, additional regions of the MAPK are believed to Chen, D. C., B. C. Yang and T. T. Kuo, 1992 One-step transforma- affect its interactions with upstream MAPK cascade com- tion of yeast in stationary phase. Curr. Genet. 21: 83–84. Christianson, T. W., R. S. Sikorski, M. Dante, J. H. Shero and P. ponents (Brunet and Pouyssegur 1996) and may af- Hieter, 1992 Multifunctional yeast high-copy-number shuttle fect the specificities of MAPK phosphatases as well. vectors. Gene 110: 119–122. The multiple responsibilities of the HOG pathway in Cook, J. G., L. Bardwell, S. J. Kron and J. Thorner, 1996 Two novel targets of the MAP kinase Kss1p are negative regulators of the response to hyperosmotic shock are complicated: invasive growth in the yeast Saccharomyces cerevisiae. Genes Dev. Hyperosmotic stress is a serious challenge to a cell’s 10: 2831–2848. survival. As such, hyperosmotic stress response requires Cook, J. G., L. Bardwell and J. Thorner, 1997 Inhibitory and activating functions for MAPK Kss1p in the S. cerevisiae filamen- the simultaneous regulation of several components. In tous-growth signalling pathway. Nature 390: 85–88. addition to producing a proper response to the stimulus Courchesne, W. E., R. Kunisawa and J. Thorner, 1989 A putative of osmotic stress, the HOG pathway has an important protein kinase overcomes pheromone-induced arrest of cell cy- cling in S. cerevisiae. Cell 58: 1107–1119. role in preventing inappropriate signaling through Crews, C. M., and R. L. Erikson, 1992 Purification of a murine other MAPK pathways. The mechanism of HOG-medi- protein-tyrosine/threonine kinase that phosphorylates and acti- ated regulation of other MAPK pathways in yeast is as vates the Erk-1 gene product: relationship to the fission yeast byr1 gene product. Proc. Natl. Acad. Sci. USA 89: 8205–8209. yet unknown, but it will prove to be an exciting area of Davenport, K. R., M. Sohaskey, Y. Kamada, D. E. Levin and M. C. study in the future. Gustin, 1995 A second osmosensing signal transduction pathway in yeast. Hypotonic shock activates the PKC1 protein kinase- We thank Ed Winter, Elaine Elion, Jeremy Thorner, and Gerald regulated cell integrity pathway. J. Biol. Chem. 270: 30157–30161. Fink for their generous donation of several plasmids used in this Davis, R. J., 1993 The mitogen-activated protein kinase signal trans- study. United States Biologicals supplied many chemicals used in this duction pathway. J. Biol. Chem. 268: 14553–14556. research. We also thank Jacobus Albertyn, Matt Alexander, and Sue Doi, K., A. Gartner, G. Ammerer, B. Errede, H. Shinkawa et al., Gibson for their critique of this manuscript. Research was supported by 1994 Msg5p, a novel protein phosphatase, promotes adaptation the National Science Foundation (grant MCB 9506987), the American to pheromone response in S. cerevisiae. EMBO J. 13: 61–70. Cancer Society (grant BE-224), and the National Aeronautics and Elion, E. A., P. L. Grisafi and G. R. Fink, 1990 FUS3 encodes ϩ Space Administration (grant NAGS-4072). K.D.D. was supported by a cdc2 /CDC28-related kinase required for the transition from mitosis into conjugation. Cell 60: 649–664. the National Institutes of Health (grant GMO-8362-7) and by Quality Elion, E. A., J. A. Brill and G. R. Fink, 1991 Fus3p represses CLN1 Bioresources, Inc. and CLN2 and in concert with Kss1p promotes signal transduction. Proc. Natl. Acad. Sci. USA 88: 9392–9396. Espinoza, F. H., A. Farrell, H. Erdjument-Bromage, P. Tempst and D. O. 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