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Molecular and Cellular Biology, September 2008, p. 5172-5183, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00589-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Phosphorylated Ssk1 Prevents Unphosphorylated Ssk1 from Activating the Ssk2 Mitogen-Activated Protein Kinase Kinase Kinase in the Yeast High-Osmolarity Glycerol Osmoregulatory Pathway{triangledown}

Tetsuro Horie,{dagger} Kazuo Tatebayashi,{dagger} Rika Yamada, and Haruo Saito*

Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

Received 11 April 2008/ Returned for modification 12 May 2008/ Accepted 16 June 2008


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ABSTRACT
 
In Saccharomyces cerevisiae, external high osmolarity activates the Hog1 mitogen-activated protein kinase (MAPK), which controls various aspects of osmoadaptation. Ssk1 is a homolog of bacterial two-component response regulators and activates the Ssk2 MAPK kinase kinase upstream of Hog1. It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) is the active form and that Ssk1 phosphorylated (Ssk1~P) at Asp554 by the Sln1-Ypd1-Ssk1 multistep phosphorelay mechanism is the inactive form. In this study, we show that constitutive activation of Ssk2 occurs when Ssk1 phosphorylation is blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits its interaction with Ypd1, the donor of phosphate to Ssk1. Thus, Ssk1-OH is indeed necessary for Ssk2 activation. However, overexpression of wild-type Ssk1 or of an Ssk1 mutant that cannot bind Ssk2 prevents constitutively active Ssk1 mutants from activating Ssk2. Therefore, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself. We also found that Ssk1 exists mostly as a dimer within cells. From mutant phenotypes, we deduce that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. Hence, because Ssk1~P binds to and inhibits Ssk1-OH, moderate fluctuation of the level of Ssk1-OH does not lead to nonphysiological and detrimental activation of Hog1.


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INTRODUCTION
 
The budding yeast Saccharomyces cerevisiae survives widely fluctuating osmotic conditions in its natural habitat. To cope with an increased external osmolarity, yeast cells synthesize and retain, intracellularly, the compatible osmolyte glycerol (8, 11). Osmostressed cells also temporarily arrest cell cycle progression and halt protein synthesis, during which they readjust to the changed environment (2, 5, 39). These events are governed by the high-osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 mitogen-activated protein kinase (MAPK) cascade. Defects in the HOG pathway cause severe osmosensitivity in cell growth.

The Hog1 MAPK cascade is regulated by the functionally redundant but mechanistically distinct upstream signaling pathways, termed the SHO1 branch and the SLN1 branch. A signal emanating from either branch converges on a common MAPK kinase (MAPKK), termed Pbs2, which is the specific activator of the Hog1 MAPK (3, 15, 16). In the SHO1 branch, two putative transmembrane (TM) osmosensors, Msb2 and Hkr1, detect osmotic stress and, together with the membrane anchor protein Sho1, generate an intracellular signal that leads to activation of the Ste11 MAPKK kinase (MAPKKK), which activates the Pbs2 MAPKK (37, 38). In the SLN1 branch, the TM histidine kinase Sln1 detects turgor changes and transmits a signal via the Sln1-Ypd1-Ssk1 multistep phosphorelay system (16, 26, 27). Ssk1 is an activator of the redundant Ssk2 and Ssk22 MAPKKKs, which activate the Pbs2 MAPKK (36). An outline of signal transduction through the SLN1 branch is schematically shown in Fig. 1; for the structure of Ssk1, see Fig. 2A.


Figure 1
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  		FIG. 1. A schematic model of the SLN1 branch of the yeast HOG pathway. The flow and regulation of signal through the SLN1 branch is shown. Thin arrows indicate the transfer of a phosphoryl group through the Sln1-Ypd1-Ssk1 phosphorelay, thick arrows indicate positive signal flow, and T-shaped bars indicate negative regulation. The encircled P's represent phosphoryl groups.


Figure 2
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  		FIG. 2. Constitutive activation of the HOG pathway by nonphosphorylatable Ssk1 mutants. (A) A diagram of the Ssk1 response regulator. The locations of the conserved receiver domain, the Ssk2-binding domain (BD) (25), and the phosphorylatable Asp554 are shown. (B) Characterization of Ssk1 Asp554 mutants. The yeast strain TM198 (ssk1{Delta}) was transformed with a single-copy plasmid carrying either wild-type (WT) or the indicated Asp554 mutant SSK1 gene under the control of the GAL1 promoter. Each transformant was spotted on SC (Glu) and SGal (Gal) plates at the same concentrations. Galactose-induced expression of a constitutively active Ssk1 mutant protein is lethal to cells. (C) Constitutively active Ssk1 mutants induce the HOG-specific reporter 8xCRE-lacZ. The yeast strain TM198 (ssk1{Delta}) was cotransformed with an 8xCRE-lacZ reporter plasmid and the indicated SSK1 expression plasmid under the control of the GAL1 promoter. Expression of wild-type or mutant Ssk1 was induced by galactose for 140 min before assay of the 8xCRE-lacZ reporter. Expression levels of the Ssk1 mutant proteins and the endogenous Hog1 protein (loading control) are shown in the lower panels.

Signaling through the Sln1-Ypd1-Ssk1 multistep phosphorelay is a complex variation of so-called prokaryotic two-component signaling systems. In its simplest form, a two-component system is composed of a sensor histidine kinase (HK) that detects environmental changes and an effector molecule, called a response regulator (RR) (7, 33). In canonical two-component systems, HK is activated upon sensing an environmental change and phosphorylates a His residue (His~P) within the same molecule. The high-energy phosphoryl group (His~P) is then transferred to an Asp residue in the conserved receiver (Rec) domain of a cognate RR. Asp phosphorylation induces a conformation change in the Rec domain and modulates the activity of an associated output domain in the RR molecule. Numerous variations on the common theme of the His-Asp phosphorelay exist even within a single bacterial species (19).

In the yeast Sln1-Ypd1-Ssk1 multistep phosphorelay, the basic His-Asp phosphorelay reaction is duplicated so that a phosphoryl group is carried in the sequence His-Asp-His-Asp (26). Sln1 is structurally similar to many bacterial sensor HKs in that it has two TM domains and a cytoplasmic HK domain (22). It differs, however, from simpler HKs in that it also contains a C-terminal Rec domain within the molecule. Sln1 also differs from most other HKs in that it is catalytically active in the absence of stimulus: its kinase activity is repressed when cells are placed under hyperosmotic stress. In unstressed cells Sln1 auto-phosphorylates at His576 and then transfers the phosphoryl group to Asp1144 (Asp1144~P) in its C-terminal Rec domain. The phosphate in Sln1-Asp1144~P is then transferred to His64 of the intermediary protein Ypd1 and finally to Asp554 of another Rec domain protein, Ssk1 (14, 26).

Ssk1 is a molecular switch whose activity is controlled by its phosphorylation state. Specifically, it is believed that unphosphorylated Ssk1 (Ssk1-OH), which accumulates in osmostressed cells, activates the Ssk2/Ssk22 MAPKKKs. This model is based on the following observations. An ssk1{Delta} mutant is osmosensitive if it is also defective in the redundant SHO1 branch, indicating that Ssk1 is a positive regulator in the SLN1 branch (21). Ssk1 binds to an N-terminal regulatory domain of the Ssk2/Ssk22 MAPKKKs, as demonstrated by both two-hybrid analyses and coprecipitation assays (25), indicating that Ssk1 directly regulates Ssk2/Ssk22 activity. In contrast, sln1{Delta} or ypd1{Delta} mutant cells, in which Ssk1 is not phosphorylated, constitutively activate the Ssk2-Pbs2-Hog1 pathway, indicating that Ssk1-OH, rather than phosphorylated Ssk1 (Ssk1~P), is the active form (16, 26). Mutations that disrupt the phosphorelay, such as Sln1-H576Q, Sln1-D1144N, or Ypd1-H64Q, constitutively activate the HOG pathway, as do sln1{Delta} or ypd1{Delta} mutations (16, 26). Neither sln1{Delta} nor ypd1{Delta} can activate the HOG pathway in ssk1{Delta} mutant cells, supporting an activator role for Ssk1 that acts downstream of Sln1 and Ypd1. Finally, Ypd1 is phosphorylated at His64 in the absence of osmostress but dephosphorylated in osmostressed cells, indicating that the Sln1 HK activity is downregulated by external high osmolarity (26).

Thus, the general signal flow in the SLN1 branch has been established. Nonetheless, the prevailing model of Ssk2 activation, namely, that Ssk1-OH binds and activates Ssk2, seems incomplete. In particular, such a mechanism would not offer any safeguard against nonphysiological activation of the Hog1 MAPK cascade by fluctuations in the Ssk1-OH concentration. Such fluctuation might be expected as phosphoaspartate is intrinsically unstable and subject to spontaneous hydrolysis. Thus, it seems unavoidable that a certain level of Ssk1-OH must be present in unstressed cells. How, then, is an unprovoked nonphysiological activation of the HOG pathway averted? In this article, we intend to answer this question.


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MATERIALS AND METHODS
 
Yeast strains. The S. cerevisiae strains used in this study are listed in Table 1.


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  		TABLE 1. Yeast strains used in this study

Buffers and media. Standard yeast medium and genetic procedures were as described previously (29). The following media were used: YPD (10 g/liter yeast extract, 20 g/liter tryptone, and 20 g/liter glucose), YPGal (10 g/liter yeast extract, 20 g/liter tryptone, and 20 g/liter galactose), SC (6.7 g/liter yeast nitrogen base, 20 g/liter glucose, and the appropriate yeast synthetic dropout medium supplements), SGal (6.7 g/liter yeast nitrogen base, 20 g/liter galactose, and the appropriate yeast synthetic dropout medium supplements), SRaf (6.7 g/liter yeast nitrogen base, 20 g/liter raffinose, with the appropriate yeast synthetic dropout medium supplements), CAD (6.7 g/liter yeast nitrogen base, 5 g/liter Casamino Acids, 20 g/liter glucose, and, when required, 40 mg/liter adenine, 40 mg/liter tryptophan, and/or 20 mg/liter uracil), CAGal (6.7 g/liter yeast nitrogen base, 5 g/liter Casamino Acids, 20 g/liter galactose, and, when required, 40 mg/liter adenine, 40 mg/liter tryptophan, and/or 20 mg/liter uracil), and CARaf (6.7 g/liter yeast nitrogen base, 5 g/liter Casamino Acids, 20 g/liter raffinose, and, when required, 40 mg/liter adenine, 40 mg/liter tryptophan, and/or 20 mg/liter uracil). Buffer A contains 50 mM Tris-HCl (pH 7.5), 15 mM EDTA, 15 mM EGTA, 2 mM dithiothreitol, 0.2% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 µg/ml leupeptin, 50 mM NaF, 25 mM β-glycerophosphate, and 150 mM NaCl. Buffer B contains 50 mM Tris-HCl (pH 7.5), 2 mM dithiothreitol, 0.2% TritonX-100, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 5 µg/ml leupeptin, 10 mM MgCl2, and 150 mM NaCl. Buffer Z contains 50 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, and 1 mM MgSO4, adjusted to pH 7.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (1x) contains 60 mM Tris-HCl (pH 6.8), 2% SDS, 700 mM 2-mercaptoethanol, 10% glycerol, and 25 µg/ml bromophenol blue.

Vector plasmids. p426TEG1 (PTEF2-GST URA3+ 2µm) is a multicopy vector that allows a constitutive expression of glutathione S-transferase (GST) fusion proteins using the strong TEF2 promoter (26). YCpIF16 (PGAL1-HA TRP1+ CEN4) is a single-copy vector that allows galactose-inducible expression of hemagglutinin (HA)-tagged proteins using the GAL1 promoter (6). p416GALS (PGALS URA3+ CEN6) is a single-copy vector with a truncated version of the GAL1 promoter that allows low-level galactose-inducible expression (20).

Plasmid constructs. The expression plasmids pGSS1 (GAL1::SSK1) and pGSSN1 (GAL1::SSK1-D554N); the two-hybrid plasmids pLexA-SSK1, pACT-SSK2(50-1147), and pACT-YPD1; and the HOG reporter plasmid pRS414-8xCRE-lacZ have been described previously (16, 25, 26, 38). Missense mutant derivatives of pGSS1 were either isolated by mutant screening or made by oligonucleotide-based mutagenesis. Other deletion or missense mutant derivatives were made by oligonucleotide-based mutagenesis. All mutations were confirmed by nucleotide sequence determination.

Isolation of constitutively active Ssk1 mutants. A DNA segment including the entire SSK1 coding region was mutagenized by error-prone PCR in the presence of 0.1 mM MnCl2. TM198 (ssk1{Delta}::LEU2) was cotransformed with the PCR products and linearized pGSSN1 (PGAL1-SSK1 D554N HIS3) from which the SSK1 codons 18 to 602 had been removed. Gap-repaired plasmids were selected on SC (without His) plates. Colonies were then replica plated onto an SC (without His) plate and an SGal (without His) plate. After a 1- to 2-day incubation at 30°C, colonies that formed only on the SC plate were recovered. Those colonies contained candidates of constitutively active SSK1 mutants, which would ectopically induce Hog1 activity and render the host cells nonviable.

Isolation of dominantly inhibitory Ssk1 mutants. The SSK1 DNA segment was mutagenized by error-prone PCR. The yeast strain TM229 with the temperature-sensitive mutation sln1-ts4 was cotransformed with the PCR products and linearized pGSS1 (PGAL1-SSK1 HIS3) in which SSK1 codons 18 to 602 had been removed. Gap-repaired plasmids were selected on SC (without His) plates at 28°C (a permissive temperature for sln1-ts4). Colonies were then replica plated onto two YPGal plates and incubated for 1 to 2 days at 25°C and 37°C (a nonpermissive temperature for sln1-ts4). Colonies that were viable on both plates were recovered. These colonies contained candidates of dominantly inhibitory SSK1 mutants, which inhibit the lethal activation of the HOG pathway by sln1-ts4 at a nonpermissive temperature.

Yeast two-hybrid analyses. Yeast two-hybrid analysis was performed as described previously (25), except that the yeast strain FP12, a pbs2{Delta} derivative of L40, was used to prevent the lethality caused by expression of hyperactive Ssk1 mutant proteins. β-Galactosidase activity (Miller units) was calculated using the following formula: β-galactosidase units = 1,000 x the optical density at 420 nm (OD420)/[incubation time (min) x volume of the culture (ml) x OD600 of the culture] (17).

The HOG-specific 8xCRE-lacZ reporter assay. At least three independent single colonies were freshly grown in CAD medium until an OD600 of ~0.5 was attained. Cells were collected following pretreatment with (or without) 0.4 M NaCl for 30 min, washed, suspended in buffer Z, and placed in liquid nitrogen. Cell suspensions were thawed in a 37°C water bath and immediately frozen again in order to permeabilize the cells. For the assay of β-galactosidase activity, the chromogenic substrate o-nitrophenyl-β-D-galactoside was added in excess, and the extracts were incubated at 37°C until a mid-yellow color had developed. Reactions were stopped by the addition of 1 M Na2CO3. Cell supernatants were collected by centrifugation at 18,000 x g for 10 min at 4°C. β-Galactosidase activity was assayed as above.

Generation of the anti-Ssk1 polyclonal antibody. A bacterial expression plasmid (pFP31) for His6-tagged full-length Ssk1 (His6-Ssk1-FL) was constructed in the pRSET B vector (Invitrogen). His6-Ssk1-FL was expressed in Escherichia coli BL21 Star(DE3) pLysS transformed with pFP31 and purified using a Ni2+ column, essentially as previously described (25, 34). A rabbit was immunized with 20 µg of the purified His6-Ssk1-FL protein in Freund's complete adjuvant, followed by five rounds of booster injections of 20 µg of the purified protein in Freund's incomplete adjuvant. The anti-Ssk1 antiserum was affinity purified using a HiTrap N-hydroxysuccinimide-activated HP column conjugated with the purified His6-Ssk1-FL protein, according to the manufacturer's instructions (GE Healthcare Bioscience). No material that cross-reacted with the affinity-purified antibody was found in the extract of ssk1{Delta} cells corresponding to the molecular mass of Ssk1 (~82 kDa).

In vivo coprecipitation assay. Cells were grown in SRaf medium until an OD600 of ~0.5 was attained; galactose was then added to a final concentration of 2%, and the incubation was continued for an additional 3 h. Cells were collected, suspended in ice-cold buffer A, and immediately frozen in liquid nitrogen. Cell extracts were prepared at 4°C by vortexing cell suspensions vigorously with glass beads and were collected by centrifugation at 9,000 x g for 10 min. For in vivo coprecipitation assays using a GST fusion protein as bait, cell extracts were incubated with glutathione-Sepharose beads (GE Healthcare) in buffer A for 3 h with gentle rotation at 4°C. The precipitates were washed five times with ice-cold buffer A, resuspended in SDS loading buffer, boiled for 5 min, and subjected to SDS-PAGE. For Ssk1-Ssk2 binding experiments, buffer B, which contains 10 mM MgCl2, was used in place of buffer A.

Immunoblotting analyses. Immunoblotting analyses were carried out as described previously (35). The anti-Hog1 antibody yC20 (Santa Cruz), a custom-made anti-Ssk1 rabbit antibody, anti-GST monoclonal antibody (MAb) SC-138 (Santa Cruz Biotechnology), anti-HA MAbs SC-7392 (Santa Cruz Biotechnology) and 12CA5 (Roche), and anti-pan actin MAb Ab-5 (Lab Vision Corp.) were used as primary antibodies for detection by immunoblotting.

Expression and purification of GST-tagged Ssk1. Yeast cells expressing GST-tagged Ssk1 (residues 426 to 713) were grown in 3 liters of CAD (without Ura) medium overnight at 30°C. Cells were harvested by centrifugation at 3,000 rpm for 15 min, resuspended in 10 ml of buffer A, and lysed with glass beads in a bead beater. Cell lysate was centrifuged at 20,000 rpm for 60 min at 4°C. The cleared lysate was loaded to a 1-ml GSTrap HP column (GE Healthcare) and eluted with 50 mM Tris-HCl (pH 8.0) and 20 mM reduced glutathione. Fractions containing GST-tagged Ssk1 were pooled and digested with 0.1 unit/µl of thrombin at 22°C for 6 h to cleave a thrombin substrate site between the GST and the Ssk1 domains.

Gel filtration analysis for Ssk1 dimerization. Size exclusion chromatography was performed on a fast-performance liquid chromatography AKTA Explorer 10S system with a HiLoad 16/60 Superdex 75 prep grade column (GE Healthcare). Chromatography was run at a flow rate of 1.0 ml/min using 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 10 mM MgCl2 as the mobile phase at 4°C, and 1.5-ml fractions were colleted. Ssk1 in each fraction was determined by immunoblotting using an anti-Ssk1 polyclonal antibody. Molecular mass standards were RNase (13.7 kDa), chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), and albumin (67 kDa).


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RESULTS
 
Mutation of Asp554 to a variety of other amino acids constitutively activates Ssk1. Initially, we tested the prediction that Ssk1 Asp554 mutants that cannot be phosphorylated would constitutively activate the HOG pathway. We first utilized an assay based on the fact that constitutive activation of the HOG pathway is toxic to cell growth (5, 16, 26). Overexpression of wild-type Ssk1 using the inducible GAL1 promoter in a single-copy vector is nontoxic, as shown in Fig. 2B. However, overexpression of Ssk1 mutants locked in the active form should be lethal. Contrary to the prediction, however, overexpression of Ssk1-D554N, in which Asp554 was mutated to the unphosphorylatable Asn, was not lethal (Fig. 2B). To test if this phenotype is specific to the Asp-to-Asn mutation, we mutated the Asp554 residue to several other amino acids. We thus found that expression of Ssk1 mutants in which the Asp554 residue is replaced by Glu, Gln, Lys, Arg, Ala, Ser, Cys, His, Pro, or Val but not by Asn or Gly was lethal. To more quantitatively examine HOG pathway activation by these mutants, we assayed induction of the HOG-specific reporter gene, 8xCRE-lacZ (38). As seen in Fig. 2C (upper panel), overexpression of wild-type Ssk1 only weakly induced the 8xCRE-lacZ reporter. In contrast, those mutants whose overexpression was lethal, such as D554S, very strongly induced 8xCRE-lacZ. More important, even the nonlethal mutants D554N and D554G induced the 8xCRE-lacZ reporter, although somewhat less strongly than others. It was potentially possible, however, that the elevated activities of these Ssk1-D554 mutants were due, at least partly, to expression of the mutant proteins at levels higher than the wild-type Ssk1. To test this possibility, we assessed the expression level of Ssk1 mutant proteins by immunoblotting using a polyclonal anti-Ssk1 antibody. As also seen in Fig. 2C (lower panel), however, the expression level of all Ssk1-D554 mutants was comparable to that of wild-type Ssk1. Thus, we conclude that all 12 different substitution mutations at Asp554 converted Ssk1 to a constitutively active form.

Expression of wild-type Ssk1 at very high levels can activate the HOG pathway. In the above experiments, we showed that overexpression of wild-type Ssk1 using a single-copy PGAL1 vector only very weakly activated the HOG pathway. To test the possibility that higher levels of Ssk1 might more vigorously activate the HOG pathway, we assayed the effect of Ssk1 expression from a multicopy PGAL1 vector. As shown in Fig. 3A, the expression level of Ssk1 is significantly higher when Ssk1 is expressed from a multicopy PGAL1-SSK1 plasmid than when it is expressed from a single-copy PGAL1-SSK1 plasmid. More important, hyperexpression of wild-type SSK1 was moderately toxic and induced relatively strong expression of the HOG reporter gene (Fig. 3B and C). Therefore, under these conditions, it is likely that the amount of Ssk1 is so high that the capacity of the Sln1-Ypd1-Ssk1 phosphorelay is insufficient to convert the majority of Ssk1 to Ssk1~P. This observation is consistent with the result of a systematic overexpression survey, in which Ssk1 overexpression using a multicopy PGAL1 vector was scored as a toxic level of 1.5 (on a scale where 1 is death, and 5 is normal growth) (32). Nonetheless, it should also be noted that the level of HOG pathway activation by a multicopy wild-type Ssk1 protein is still substantially weaker than that by single-copy Ssk1-D554S (Fig. 3C). Furthermore, the endogenous Ssk1, which is expressed at a much lower level than Ssk1 expressed from either PGAL1-SSK1 vector, can support a robust HOG pathway activation upon osmostress (Fig. 4B). These observations suggest that either the concentration of Ssk1-OH does not reach a very high level even when Ssk1 is expressed from the multicopy PGAL1-SSK1 vector or, more likely, that there is a mechanism that retards Ssk2 activation by Ssk1-OH, especially when Ssk1~P coexists with Ssk1-OH.


Figure 3
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  		FIG. 3. Hyperexpression of wild-type Ssk1 activates the HOG pathway. (A) Expression level of Ssk1 under various conditions. For lanes 3 and 4, the yeast strain TM198 (ssk1{Delta}) was transformed with a pRS416-based single-copy vector and a pRS426-based multicopy vector, respectively, each encoding the identical PGAL1-SSK1 fusion gene. Expression of Ssk1 was induced by 2% galactose for 120 min. In lane 2, the endogenous level of Ssk1 expression was measured for comparison using the yeast strain TH075 (SSK1+). As a loading control, the expression level of the endogenous Hog1 protein is shown. (B) Galactose-induced hyperexpression of wild-type Ssk1 is moderately toxic. The yeast strain TM198 (ssk1{Delta}) was transformed with a single-copy or a multicopy vector encoding the PGAL1-SSK1 fusion gene, as indicated. Glu, CAD plate; Gal, CAGal plate. (C) Hyperexpression of wild-type (WT) Ssk1 induces the HOG-specific reporter 8xCRE-lacZ. The yeast strain TM198 (ssk1{Delta}) was cotransformed with an 8xCRE-lacZ reporter plasmid and the indicated SSK1 expression plasmid under the control of the GAL1 promoter. Expression of the wild-type or mutant Ssk1 was induced by galactose for 120 min before assay of the 8xCRE-lacZ reporter.


Figure 4
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  		FIG. 4. The Ssk1-binding domain is required for activation of the Ssk2 MAPKKK. (A) A schematic diagram of Ssk2. The Ssk1-binding domain (BD) defined previously by two-hybrid analyses (25), a region necessary for activation defined in this study, and the conserved kinase domain are indicated. (B and C) Role of Ssk2 N-terminal region in activation of the HOG pathway. The yeast strain TH075 (ssk2{Delta} ssk22{Delta} ste11{Delta}) was cotransformed with a single-copy plasmid carrying either wild type (WT) or one of the indicated SSK2 deletion constructs and a second plasmid encoding the 8xCRE-lacZ reporter gene. Cells were stimulated with (or without) 0.4 M NaCl for 30 min before the reporter assay. In panel C, the HOG pathway was also stimulated by expression of the constitutively active Ssk1-D554S mutant. (D) Osmosensitivity of SSK2 mutant cells. The yeast strain TH075 was transformed with a single-copy plasmid carrying either wild-type SSK2 or one of the indicated SSK2 deletion constructs, and their growth on high-osmolarity (1.0 M sorbitol or 0.8 M NaCl) plates was compared.

The Ssk1-binding domain in Ssk2 is essential for Ssk2 activation either by Ssk1-D554S or by osmotic stress. To confirm the prediction that constitutively active Ssk1 mutants activate the HOG pathway through binding to Ssk2, we made a series of ~50-amino-acid deletion constructs in the Ssk2 N-terminal region (residues 1 through 486). Previously, the Ssk1-binding domain was localized between Ssk2 residues 294 and 413 by two-hybrid and coprecipitation assays (Fig. 4A) (25). The SSK2 mutant constructs were individually expressed in an ssk2{Delta} ssk22{Delta} ste11{Delta} host strain, and induction of the 8xCRE-lacZ reporter by osmostress was assayed. In this triple MAPKKK disruption mutant, 8xCRE-lacZ expression is absolutely dependent on the plasmid-borne SSK2 gene. All the SSK2 mutants that have a deletion within the region between amino acids (aa) 354 and 486 are defective in osmostress induction of the reporter, whereas no mutants between the N terminus and aa 368 were defective (Fig. 4B). To further define the region necessary for Ssk2 activation, additional deletion constructs were constructed, in which 20 consecutive amino acids were deleted in the region between aa 321 and 440. The SSK2 deletion mutants within the region between aa 381 and 440, namely, deletions of aa 381 to 400 [{Delta}(381-400)], {Delta}(401-420), and {Delta}(421-440), were defective in osmotic induction of the 8xCRE-lacZ reporter, while deletion mutants within the region between aa 321 and 380 were practically identical to the wild-type SSK2 (Fig. 4C). Consistent with the effects on reporter induction, yeast cells harboring the SSK2 {Delta}(381-400), {Delta}(401-420), or {Delta}(421-440) mutation are osmosensitive, although SSK2 {Delta}(421-440) is slightly less osmosensitive than the other two mutants (Fig. 4D). These results indicate that the Ssk2 region between aa 381 and 440 is essential for Ssk2 activation by osmostress. This region overlaps with the Ssk1-binding domain (aa 294 to 413) previously determined (Fig. 4A), indicating that binding of Ssk1 is a necessary step in Ssk2 activation. It should be noted, however, that because this region also partially overlaps with the actin-interacting domain (45), some of the observed effects might be due to a lack of interaction with actin. We found, however, that precisely the same region is required for reporter induction by the constitutively active Ssk1-D554S mutant (Fig. 4C). Thus, the unphosphorylatable Ssk1 mutant appears to activate the HOG pathway by the same mechanism by which osmostress activates the pathway.

Ypd1-nonbinding Ssk1 mutants are hyperactive. To gain further insight into the mechanism of Ssk2 activation by Ssk1, we isolated additional hyperactive Ssk1 mutants using a screening strategy based on the observation that expression of hyperactive Ssk1 is toxic (see Materials and Methods). Of the 21 mutants that we isolated, two had only one mutation at the phosphorylation site (D554S); other mutants contained more than one mutation. To identify which of the mutations were responsible for Ssk1 hyperactivity, we constructed and tested SSK1 mutants that contained an individual mutation. Several of these individual mutations were found to be insufficient to activate Ssk2 in the absence of a second mutation and were not characterized further. For the other SSK1 mutants a single amino acid substitution could be identified that was responsible for hyperactivity. These mutations were E510G, N512T, I514T, and I518T. Expression of these Ssk1 mutant proteins using the GAL1 promoter in a single-copy vector was lethal, suggesting that they strongly activate the HOG pathway (Fig. 5A). Indeed, their expression induced the HOG-specific 8xCRE-lacZ reporter (Fig. 5B). These Ssk1 mutants required only the receiver domain for Ssk2 activation, as deletion of the Ssk1 N-terminal region (Ssk1-{Delta}N) did not abolish their lethality (Fig. 5C and data not shown).


Figure 5
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  		FIG. 5. Ypd1-nonbinding Ssk1 mutants are hyperactive. (A) Galactose-induced overexpression of hyperactive Ssk1 mutant proteins is lethal. (B) Expression of hyperactive Ssk1 mutant proteins induced the HOG-specific 8xCRE-lacZ reporter. (C) Overexpression of the receiver domain of the hyperactive Ssk1-I514T mutant was lethal. In panels A to C, the yeast strain TM198 (ssk1{Delta}) was used. Glu, SC plate with glucose; Gal, SGal plate. (D) Two-hybrid analysis of Ssk1 interactors. FP12, a pbs2{Delta} host strain for yeast two-hybrid assays, was cotransformed with an activation domain (AD) fusion construct, AD-Ypd1 (left) or AD-Ssk2-N (right), and a DNA binding domain (DB)-Ssk1 fusion construct with the indicated mutation. Values are the average of three independent cultures. (E) The predicted location of the Ypd1-nonbinding Ssk1 mutations in a structure model of the conserved receiver domain. Because the structure of Ssk1 has not yet been determined, the crystallographic structure of the E. coli CheY protein (Protein Data Bank code 3CHY) was used as a homologous model. WT, wild type.

To identify possible mechanism(s) by which these Ssk1 mutants become hyperactive, we examined their capacity to bind the two known interactors of Ssk1, namely, Ypd1 and Ssk2, by two-hybrid analyses. As shown in Fig. 5D, all four of the Ssk1 mutants are completely incapable of binding to Ypd1, whereas they can bind to Ssk2. Thus, it is likely that these Ssk1 mutants are hyperactive because they cannot be phosphorylated by Ypd1. When the four mutations are placed on the three-dimensional (3D) structure of the homologous receiver domain of the bacterial CheY protein (40), they are all clustered in the β1-{alpha}1 turn and the {alpha}1 helix, which are likely to be the Ypd1-interacting surface of the molecule (Fig. 5E). Based on these findings, we conclude that Ssk1 mutants are hyperactive if they do not have the phospho-accepting Asp554 residue or if they cannot interact with Ypd1. In either case, unphosphorylated Ssk1 accumulates to activate Ssk2.

Ssk1~P has a negative regulatory role. In the absence of osmostress, no activation of Ssk2 occurs. That Ssk1-OH activates Ssk2 implies that almost all Ssk1 molecules in the cell must be maintained in the phosphorylated state (Ssk1~P). It would be difficult, however, to sustain such a condition, especially when Ssk1 is overexpressed, because Ssk1~P can spontaneously hydrolyze to Ssk1-OH. Nonetheless, overexpression of Ssk1 (using a single-copy vector) did not appreciably induce the 8xCRE-lacZ reporter (Fig. 5B). This led us to hypothesize that there might be a negative regulator that prevents Ssk1-OH from activating Ssk2 in the absence of osmostress. To determine if such a regulator exists, we screened for multicopy suppressor genes of hyperactive Ssk1 mutants. As shown earlier (Fig. 5A), expression of the hyperactive Ssk1-I514T from the GALS promoter is lethal. Using a YEp13 multicopy yeast DNA library, we isolated 20 genomic DNA clones that could suppress the lethality of Ssk1-I514T. Seven of them encoded a protein tyrosine phosphatase, either PTP2 (four clones) or PTP3 (three clones), which deactivate the Hog1 MAPK (12, 41). These phosphatases have been shown to suppress Hog1 activation in sln1 or ypd1 mutants, in which Ssk1-OH accumulates (16, 26). Unexpectedly, however, the remaining 13 clones contained DNA fragments including the entire SSK1 gene. A subcloning experiment confirmed that the SSK1 gene itself was responsible for the suppression of Ssk1-I514T lethality (Fig. 6A). In the wild-type host cells (used in the experiment shown in Fig. 6A), at least some Ssk1 molecules are expected to be in the phosphorylated (Ssk1~P) form. Thus, these results imply that Ssk1~P is not merely inert but, rather, inhibitory.


Figure 6
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  		FIG. 6. Ssk1 can inhibit Ssk2 activation. (A) Identification of multicopy suppressor genes of the lethality of hyperactive Ssk1. The yeast strain TM325 (ssk1{Delta}) was cotransformed with a plasmid encoding the hyperactive Ssk1-I514T mutant under the control of the moderately strong PGALS promoter (20) and a multicopy (YEp13) plasmid that encodes the indicated gene expressed from the own promoter. Cells were spread on a CAGal (without Ura) plate. (B) An Ssk1 mutant that suppresses the lethality of the temperature-sensitive sln1-ts4 mutation. The yeast strain TM229 (sln1-ts4) was transformed with a plasmid that expresses either wild-type SSK1 or the indicated mutant SSK1 under the control of the strong PGAL1 promoter. Transformed cells were streaked on YPGal plates and incubated at 25°C or 37°C. (C) A dominant inhibitory effect of the Ssk1-T/S D/G double mutant. The yeast strain TH074 (ssk2{Delta} ssk22{Delta} ssk1{Delta}) was first transformed with two plasmids, one encoding the HOG reporter gene 8xCRE-lacZ and the second encoding the wild-type SSK2 gene. The cells were further transformed with two additional plasmids encoding the indicated SSK1 mutant genes (or the empty vector). Ssk1 protein expression was induced by galactose for 2 h, following which reporter expression was assayed. (D) Ssk1-T213S mutant protein is more stable than wild-type Ssk1. Yeast strain TM198 (ssk1{Delta}) was transformed with a single-copy plasmid carrying either wild-type or the indicated mutant SSK1 gene under the control of the GAL1 promoter. Expression of the cloned SSK1 gene was induced by galactose for 3 h, following which cell lysates were subjected to SDS-PAGE and immunoblotting with an anti-Ssk1 rabbit antibody (top) or with an anti-actin antibody (bottom). (E) Ssk1 turnover. The yeast strain TH074 (ssk2{Delta} ssk22{Delta} ssk1{Delta}) was transformed with single-copy plasmids encoding the PGAL1-SSK1 promoter fusion gene with the indicated mutation(s). Ssk1 expression was induced with 2% galactose for 3 h. At time zero, the medium was changed to SC medium with 2% glucose and 500 µg/ml cycloheximide, with or without 0.4 M NaCl. Ssk1 was detected by immunoblotting as described above. WT, wild type; T/S D/G, T213S D628G.

A dominant inhibitory Ssk1 mutant. It is difficult to study the inhibitory activity of Ssk1~P in the complete absence of Ssk1-OH as these two forms always coexist. Therefore, we screened for Ssk1 mutants that might mimic Ssk1~P even in the absence of phosphorylation. In practice, this was done by isolating SSK1 mutants that could suppress, when overexpressed, the lethality of sln1-ts4 at the nonpermissive temperature. An SSK1 mutant thus isolated contained two mutations: T213S in the N-terminal region and D628G in the receiver domain. To determine if both mutations are necessary for suppression, we compared the suppressive effect of the single mutations (T213S or D628G) to that of the double mutation (T213S D628G) on sln1-ts4 lethality. Expression of the Ssk1-D628G single mutant protein from the GAL1 promoter only marginally suppressed sln1-ts4 lethality, and expression of Ssk1-T213S did not suppress it at all (Fig. 6B). Maximum suppression was attained only when both T213S and D628G existed in the same Ssk1 molecule (Ssk1-T/S D/G).

We also found that overexpression of Ssk1-T/S D/G, or, to a lesser extent, even overexpression of wild-type Ssk1, could suppress Ssk2 activation induced by the hyperactive Ssk1 mutants. When hyperactive Ssk1 mutants such as D554S or I514T were expressed, strong induction of the 8xCRE-lacZ reporter gene was observed (Fig. 6C). When in the same cells either wild-type Ssk1 or the inhibitory mutant Ssk1-T/S D/G was also expressed, there was a significant reduction in the reporter expression.

The synergistic effect of the T213S and D628G mutations, as well as their locations in two different domains in Ssk1, suggested that they might contribute differently to inhibition of Ssk2 activation. Thus, we examined their individual roles in Ssk2 inhibition. When the steady-state expression level of these mutant Ssk1 proteins was examined, we found that Ssk1-T213S and Ssk1-T213S D628G are present at much higher levels than wild-type Ssk1 or Ssk1-D628G (Fig. 6D). The higher expression level of Ssk1-T213S might be related to the previously reported observation that unphosphorylated Ssk1 is degraded by the ubiquitin-proteasome system (30). We therefore examined the time course of Ssk1 degradation in the presence of the translation inhibitor cycloheximide (Fig. 6E). There was, however, no significant degradation of any protein either in the presence or absence of osmostress. Nonetheless, the expression levels of Ssk1-T213S and Ssk1-T213S D628G were significantly higher than those of wild-type Ssk1 and Ssk1-D628G. Thus, the mechanism by which a higher Ssk1 expression level is achieved by the T213S mutation remains unclear. However, regardless of the mechanism by which a higher expression level is attained, the effect of the T213S mutation is likely to increase the amount of Ssk1-D628G to a level at which it can effectively inhibit Ssk2 activation.

Ssk1-D628G is incapable of activating Ssk2. We then examined whether Ssk1-D628G can support HOG pathway activation induced either by osmostress or by inactivation of the Sln1 histidine kinase. The sho1{Delta} ssk1{Delta} double mutant cells are defective in both the SHO1 and the SLN1 branches and are thus osmosensitive and incapable of inducing the HOG reporter gene. Expression of wild-type Ssk1 can complement the osmosensitive defect of sho1{Delta} ssk1{Delta} and support osmostress induction of the HOG pathway reporter (Fig. 7A and B). In contrast, expression of Ssk1-D628G neither complemented the osmosensitive defect nor supported the osmostress induction of the HOG reporter.


Figure 7
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  		FIG. 7. The Ssk1 D628G mutant cannot activate Ssk2. (A and B) The yeast strain TM288 (sho1{Delta} ssk1{Delta}) was transformed with a single-copy plasmid carrying either the wild-type or a D628G mutant SSK1 gene (with the SSK1 promoter) together with an 8xCRE-lacZ reporter plasmid. (A) Osmosensitivity of the mutant cells. The transformed cells were streaked on SC (without His) plates with or without 0.8 M NaCl. (B) Osmostress activation of the HOG pathway. The transformed cells were stimulated with 0.4 M NaCl for 30 min, and induction of the HOG-specific reporter gene was assayed. (C and D) The yeast strain TH101 (sln1-ts4 ssk1{Delta}) was transformed as described in panel A. (C) Suppression of the sln1-ts4 lethality. The transformed cells were streaked on SC (without His) plates and incubated at 30°C or 37°C. (D) Activation of the HOG pathway by sln1-ts4. The transformed cells that were growing at 30°C were shifted to 37°C for 30 min, and induction of the HOG-specific reporter gene was assayed. WT, wild type.

At nonpermissive temperatures, sln1-ts4 mutant cells are lethal because lack of Ssk1 phosphorylation leads to a continuous activation of the HOG pathway, which is toxic. The sln1-ts4 ssk1{Delta} double mutant cells are viable, but expression of the wild-type SSK1 gene in these cells restored both the lethality (Fig. 7C) and induction of the HOG reporter gene (Fig. 7D). In contrast, expression of Ssk1-D628G neither restored the lethality nor induced the HOG reporter gene, indicating that Ssk1-D628G cannot activate Ssk2 even if it is not phosphorylated by the phosphorelay.

Ssk1-D628G cannot bind Ssk2. A potential mechanism by which high levels of Ssk1-D628G inhibit Ssk2 activation is through competition with wild-type Ssk1 for an interacting protein. Therefore, we examined the binding of Ssk1-D628G to known Ssk1 interactors, namely Ypd1 and Ssk2, by two-hybrid assays. As shown in Fig. 8A, both Ssk1-T213S and Ssk1-D628G bind to Ypd1 with a similar efficiency as the wild-type Ssk1 protein. Thus, neither mutation affects the Ssk1-Ypd1 interaction. In contrast, Ssk1-D628G could not bind to Ssk2 at all, whereas wild-type Ssk1 and the Ssk1-T213S mutant could efficiently bind Ssk2 (Fig. 8B). In vivo coprecipitation assays corroborated the finding that Ssk1-D628G does not bind to Ssk2 (Fig. 8C, lane 6), whereas the hyperactive Ssk1-D554S strongly binds to Ssk2 (lane 5). It is probably significant that wild-type Ssk1 weakly binds to Ssk2 (lane 4), hinting that a small fraction of Ssk1 is indeed dephosphorylated. These findings are consistent with the model that wild-type Ssk1 does not activate Ssk2 in the absence of stimulation, not merely because Ssk1~P does not bind to Ssk2 but also because Ssk1~P prevents any residual Ssk1-OH from activating Ssk2.


Figure 8
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  		FIG. 8. The Ssk1-D628G mutant cannot bind Ssk2. (A and B) Binding of various Ssk1 mutants to a fusion construct of the activation domain (AD) with Ypd1 (A) and the N terminus of Ssk2 (AD-Ssk2-N) (B) was examined by two-hybrid analyses as described in the legend of Fig. 5D. DB, DNA binding domain. (C) In vivo coprecipitation assay demonstrating Ssk1-Ssk2 binding. The yeast strain TH074 (ssk2{Delta} ssk22{Delta} ssk1{Delta}) was cotransformed with an expression plasmid for HA-tagged Ssk1-R (wild type or the indicated mutants) and a second plasmid for expression of GST-Ssk2-K/N (K1295N, a kinase-dead mutant, was used to prevent toxicity) or GST alone. GST-Ssk2-K/N and control GST were affinity purified on glutathione beads, and coprecipitated HA-Ssk1-R was detected by anti-HA immunoblotting (top row). The lower rows show the expression level of each protein. WT, wild type; IP, immunoprecipitation; IB, immunoblotting.

Dimerization of Ssk1. How do Ssk1~P and Ssk1-D628G prevent Ssk1-OH from activating Ssk2? We considered three possibilities. The first possibility is that Ssk1~P and Ssk1-D628G abortively bind to Ssk2, thus competitively inhibiting binding of Ssk1-OH to Ssk2. This idea is untenable, however, because the results shown in Fig. 8C clearly show that Ssk1-D628G does not bind to Ssk2 at all, and wild-type Ssk1 binds to Ssk2 only weakly. The second possibility, that Ssk1~P and Ssk1-D628G somehow modify Ypd1, is also unlikely because Ssk1-T/S D/G can inhibit even the Ypd1-nonbinding Ssk1-I514T hyperactive mutant. In any case, inhibition of Ypd1 should activate Ssk2 rather than inhibit it. The third possibility is that Ssk1~P and Ssk1-D628G directly inhibit Ssk1-OH. This model would require that Ssk1 can interact with a second molecule of Ssk1. To test this idea, we expressed an HA-tagged Ssk1 receiver domain (HA-Ssk1-R) and a GST-tagged Ssk1-R domain (GST-Ssk1-R), either individually or in combination, in yeast and determined if they could coprecipitate. As shown in Fig. 9A, Ssk1-R could bind to another Ssk1-R, regardless of whether the Ssk1-R was wild-type, the D554S, or the D628G mutant.


Figure 9
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  		FIG. 9. Dimerization of Ssk1. (A) In vivo coprecipitation assay demonstrating Ssk1 dimerization. The yeast strain TH074 (ssk2{Delta} ssk22{Delta} ssk1{Delta}) was cotransformed with an expression plasmid for HA-tagged Ssk1-R and another for GST-tagged Ssk1-R. Each construct was either wild-type (WT) or carried a mutation as indicated. GST-Ssk1-R or control GST was affinity purified on glutathione beads, and coprecipitated HA-Ssk1-R was detected by anti-HA immunoblotting (top row). The lower rows show the expression level of each protein. (B) Purification of GST-Ssk1-R and proteolytic cleavage in vitro. The GST-Ssk1-R fusion protein was expressed from the inducible GAL1 promoter in the yeast strain TH074. After glutathione column affinity purification of GST-Ssk1-R, the linker between GST and Ssk1-R was cleaved by thrombin digestion. The cleavage products were separated by SDS-PAGE and stained with Coomassie brilliant blue. Lane 1, purified GST-Ssk1-R; lane 2, mock digestion; lane 3, thrombin digestion. (C) Gel filtration analysis of Ssk1-R. Size exclusion chromatography was performed with the cleaved Ssk1-R on a Superdex 75 column. Aliquots from each fraction were electrophoresed through SDS-PAGE, and a blot was probed with an anti-Ssk1 rabbit antibody. Vo, void volume; M, molecular mass marker; IP, immunoprecipitation; IB, immunoblotting.

We next examined whether Ssk1 forms a dimeric structure or a higher oligomeric structure. For this purpose, GST-Ssk1-R (aa 426 to 713) was expressed in yeast, purified, and proteolytically cleaved by thrombin at the junction between GST and Ssk1-R. After thrombin digestion, Ssk1-R migrates at the expected monomer molecular mass of 34 kDa in SDS-PAGE gels (Fig. 9B). On a gel filtration column, however, Ssk1-R eluted at a calculated molecular mass of about 68 kDa, which is consistent with a dimeric structure (Fig. 9C). No Ssk1-R was detected at the monomer (34 kDa) position. Thus, we conclude that the native form of Ssk1 in yeast cells is mostly dimeric. In the Discussion section, we will develop a model of how Ssk2 activation is regulated by an interplay between inhibitory Ssk1~P and activating Ssk1-OH.


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DISCUSSION
 
It has been proposed that unphosphorylated Ssk1 (Ssk1-OH) activates the Ssk2-Pbs2-Hog1 MAPK cascade, based on the finding that the Hog1 MAPK is constitutively activated in sln1{Delta} or ypd1{Delta} mutant cells (16, 26). In this study, we presented further evidence that it is the Ssk1-OH form that activates the Ssk2-Pbs2-Hog1 MAPK cascade. Thus, constitutive activation of Ssk2 occurred when Ssk1 phosphorylation was blocked by either an Ssk1 mutation at the phosphorylation site or an Ssk1 mutation that inhibits the interaction between Ssk1 and Ypd1~P. Therefore, Ssk1-OH is indeed necessary for Ssk2 activation. However, if Ssk1-OH activates Ssk2, then in order to prevent ectopic activation of the HOG pathway, it is necessary to maintain a very low level of Ssk1-OH when no osmostress is applied. This is a difficult condition to meet as Asp~P is typically unstable and rapidly hydrolyzes to Asp-OH. Furthermore, we would expect that when Ssk1 is overexpressed, the concentration of Ssk1-OH would rise as the phosphotransfer capacity of the Sln1-Ypd1 system becomes saturated, leading to activation of the HOG pathway. Indeed, this occurs but only when Ssk1 is massively overexpressed from the GAL1 promoter in a multicopy vector (Fig. 3C). Overexpression of Ssk1 using a single-copy vector does not significantly activate the HOG pathway.

These observations raised the possibility that there might be a negative regulatory mechanism that somehow inhibits Ssk2 from being activated by low levels of Ssk1-OH. One candidate for such a negative regulator was Ypd1. It was possible that in addition to transferring the phosphoryl group to Ssk1, Ypd1~P might also directly inhibit Ssk2. In this study, however, we found that Ssk1-D554S or Ssk1-I514T could fully activate the HOG pathway under the conditions at which Ypd1 was phosphorylated, excluding a role for Ypd1~P as a negative regulator. We thus searched for a negative regulator of Ssk2 activation by screening for a multicopy suppressor of constitutively active Ssk1 mutants. This led to the unexpected finding that Ssk1~P inhibits activation of Ssk2 by Ssk1-OH. In other words, Ssk1 has a dual function as both an activator of Ssk2 and an inhibitor of Ssk1 itself.

What is the basis of this dual regulatory role of Ssk1? Two observations indicate that the target of negative regulation by Ssk1~P is Ssk1-OH rather than Ssk2. The first is that Ssk1-D628G, which cannot interact with Ssk2, inhibits Ssk2 activation by Ssk1-OH, excluding Ssk2 as the direct target of inhibition by Ssk1-D628G (and by inference, Ssk1~P). The second is that Ssk1 forms a stable dimer, suggesting that Ssk1~P inhibits Ssk1-OH by forming a heterologous dimer composed of Ssk1-OH and Ssk1~P (Fig. 10). If 10% of Ssk1 is dephosphorylated, only 1% of the Ssk1 dimer is in the fully active configuration, namely, Ssk1-OH/Ssk1-OH. This will prevent a nonphysiological activation of the HOG pathway by spontaneous dephosphorylation of Ssk1~P in osmotically unstressed cells.


Figure 10
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  		FIG. 10. Model of Ssk2 activation by an Ssk1 dimer. Ssk1 molecules exist mostly as a dimer within cells. We deduce from mutant phenotypes that only the Ssk1-OH/Ssk1-OH dimer can activate Ssk2 efficiently. In unstressed cells, where most Ssk1 is phosphorylated at Asp554, low levels of Ssk1-OH are retained mostly in an Ssk1~P/Ssk1-OH dimer that is ineffective for Ssk2 activation. Therefore, little activation of the HOG pathway occurs. Significant Ssk2 activation takes place only when a sufficient amount of Ssk1 is dephosphorylated. Thus, not only Ssk1 phosphorylation but also Ssk1~P/Ssk1-OH dimerization prevents spontaneous activation of the HOG pathway. Pi, inorganic phosphate.

Because the Ssk1-OH/Ssk1-OH dimer can bind two molecules of Ssk2, it will indirectly dimerize Ssk2. In contrast, Ssk1-OH/Ssk1~P binds only one molecule of Ssk2. Very often, activation of an enzyme of the MAPKKK family involves its dimerization although the specific mechanism of dimerization might be distinct for each MAPKKK (18). Thus, our current data are consistent with the model of Ssk2 activation in which an Ssk1-mediated Ssk2 dimerization is an essential step. A direct demonstration of this model has so far been unsuccessful, due in part to the difficulty of detecting Ssk2 dimerization at physiologically relevant low levels of Ssk2 expression.

How does the dephosphorylation of Ssk1 at Asp554 induce its binding to Ssk2? The Ssk2-nonbinding mutant isolated in this study shed some light on the Ssk1-Ssk2 interaction. Although the 3D structure of the Ssk1 receiver domain is yet unknown, the structure of the homologous E. coli receiver domain protein CheY offers an excellent model. Previous genetic and nuclear magnetic resonance studies have indicated that it is the {alpha}4-β5-{alpha}5 face of CheY that binds to its target protein, FilM (4). When high-resolution structures of active and inactive CheY were compared, a conserved residue in this face, CheY Tyr106, was found to flip between a solvent-exposed inactive conformation and a buried active conformation (9). Similar conformation changes of the conserved Tyr (or Phe) were observed in other receiver domain structures (28). The Ssk2-nonbinding mutation, Ssk1-D628G, occurs at the position equivalent to CheY Gly105, i.e., right next to CheY Tyr106. Thus, it is likely that Ssk1 also uses the same {alpha}4-β5-{alpha}5 face as its Ssk2 binding site and that the D628G mutation locks the mutant protein into the nonbinding conformation. Phosphorylation of Ssk1 at Asp554 may also induce a similar conformation change that disallows its binding to Ssk2. Thus, we predict that the conformation of Ssk1-D628G mimics that of Ssk1~P.

There are additional mechanisms that might prevent excessive or prolonged activation of the HOG pathway by the SLN1 branch. First, it has been reported that in the presence of a substoichiometric amount, Ypd1 extends the half-life of Ssk1~P dephosphorylation to ~42 h in vitro (13). However, how Ypd1 stabilizes a 10-fold excess of Ssk1~P or if a similar stabilization occurs in vivo is unknown. With such a long half-life, it would be difficult to accumulate a sufficient level of Ssk1-OH to activate the Hog1 MAPK cascade following osmostress. The half-life of Ssk1~P dephosphorylation in the absence of Ypd1 is ~13 min (13), which is closer to but still significantly longer than the observed activation time of the SLN1 branch (1 to 2 min following osmostress) (10, 15). Thus, there might be a mechanism that accelerates Ssk1 dephosphorylation upon osmostress. Second, it has also been reported that Ssk1-OH is preferentially degraded by a ubiquitin/proteasome-dependent mechanism (30). The degradation, however, occurs over a time scale of an hour, suggesting that it is more relevant as a mechanism of downregulation after prolonged osmostress rather than as a mechanism to suppress noisy activation. Thus, neither of these mechanisms can provide an adequate explanation for both low basal activation of the pathway in the absence of osmostress and rapid activation upon exposure to osmostress. Finally, it should be noted that there is an Ssk1-independent Ssk2 activation mechanism as actin repolarization after osmostress is dependent on the catalytic activity of Ssk2 but not on Ssk1 (1, 44). Indeed, ssk1{Delta} ste11{Delta} mutant cells are consistently less osmosensitive than ssk2/22{Delta} ste11{Delta} mutant cells (our unpublished observation), supporting the notion that some activation of Ssk2/Ssk22 occurs in the absence of Ssk1. However, because this alternative Ssk2 activation is relatively modest in intensity, it does not affect the interpretation of the results reported here.

Although it was not the main purpose of this study, our results also shed some light on the mechanism of the Ssk1-Ypd1 interaction. The structure of Ypd1 is an all-helical tertiary fold with a four-helix bundle core that is composed of helices {alpha}B, {alpha}C, {alpha}D, and {alpha}G (31, 43). The phosphorylatable histidine (His64), located in the center of helix {alpha}C, protrudes out from the surface of the helical bundle. Although a 3D structure of the Ssk1-R domain is yet to be determined, the structure of the isolated Sln1 receiver domain (Sln1-R) and that of Sln1-R complexed with Ypd1, have been solved (42). Because Sln1-R and Ssk1-R are highly homologous, it is reasonable to assume that the essential features of the Ypd1/Sln1-R interaction are recapitulated in the Ypd1/Ssk1-R interaction. In the Ypd1/Sln1-R structure, helices {alpha}A, {alpha}B, and {alpha}C of Ypd1 interact mainly with helix {alpha}1 of Sln1-R and also with several loops surrounding the active site (the phospho-accepting aspartate) of Sln1-R. Surface residues from the {alpha}1 helix of Sln1-R are mainly hydrophobic and contribute a large part of the binding surface with helices {alpha}A, {alpha}B, and {alpha}C of Ypd1. Two of the four Ypd1-nonbinding Ssk1 mutants we isolated affect hydrophobic residues in helix {alpha}1 (Ile514 and Ile518). These residues correspond to Val1098 and Val1102 of Sln1, which, respectively, contact with Ypd1 Ser69 and Leu31 (42). Other Ypd1-nonbinding Ssk1 mutants are within the β1-{alpha}1 loop (Glu510 and Asn512). Ssk1 Asn512 corresponds to Sln1 Asn1096, which forms several hydrogen bonds with Ypd1 Gln38 and Phe65. Ala-substitution mutations at these Ypd1 residues (S69A, L31A, Q38A, and F65A) disrupt binding of Ypd1 to both Sln1-R and Ssk1-R (23). Thus, our analyses of the Ypd1-nonbinding Ssk1 mutants lend strong support to the theoretical prediction that the N-terminal {alpha}1 helix of Ssk1-R is the most important part of Ssk1-R for interaction with Ypd1 (24).

In this study, we uncovered a novel role for Ssk1 as an inhibitor of Ssk2 activation in addition to its role as an Ssk2 activator. We have also presented a possible mechanism by which Ssk1 might mediate both functions. There still remain, of course, unsolved questions concerning Ssk2 activation and regulation. For example, how Ssk2 is activated by dimerization is unknown, apart from the fact that auto-phosphorylation is somehow involved (25). Also, whether the apparently redundant Ssk22 has any specific function is unknown. The data presented here will facilitate research to answer these questions. Furthermore, the mechanism by which Ssk1 modulates Ssk2 activity might also provide a model relevant to other members of the MAPKKK family in both yeast and mammalian cells.


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ACKNOWLEDGMENTS
 
We thank P. O'Grady for critical reading of the manuscript, T. Maeda for unpublished yeast strains, and E. Kasukawa for excellent technical assistance.

This work was supported in part by several grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan to H.S. and K.T. and by a grant from the Salt Science Research Foundation (number 0715) to K.T.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Phone: 81 3 5449 5505. Fax: 81 3 5449 5701. E-mail: h-saito{at}ims.u-tokyo.ac.jp Back

{triangledown} Published ahead of print on 23 June 2008. Back

{dagger} T.H. and K.T. contributed equally to this work. Back


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REFERENCES
 
    1
  1. Bettinger, B. T., M. G. Clark, and D. C. Amberg. 2007. Requirement for the polarisome and formin function in Ssk2p-mediated actin recovery from osmotic stress in Saccharomyces cerevisiae. Genetics 175:1637-1648.[Abstract/Free Full Text]
    2. 2
  2. Bilsland-Marchesan, E., J. Ariño, H. Saito, P. Sunnerhagen, and F. Posas. 2000. Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1. Mol. Cell. Biol. 20:3887-3895.[Abstract/Free Full Text]
    3. 3
  3. Brewster, J. L., T. de Valoir, N. D. Dwyer, E. Winter, and M. C. Gustin. 1993. An osmosensing signal transduction pathway in yeast. Science 259:1760-1763.[Abstract/Free Full Text]
    4. 4
  4. Cho, H. S., J. G. Pelton, D. Yan, S. Kustu, and D. E. Wemmer. 2001. Phosphoaspartates in bacterial signal transduction. Curr. Opin. Struct. Biol. 11:679-684.[CrossRef][Medline]
    5. 5
  5. Escote, X., M. Zapater, J. Clotet, and F. Posas. 2004. Hog1 mediates cell-cycle arrest in G1 phase by the dual targeting of Sic1. Nat. Cell Biol. 6:997-1002.[CrossRef][Medline]
    6. 6
  6. Foreman, P. K., and R. W. Davis. 1994. Cloning vectors for the synthesis of epitope-tagged, truncated and chimeric proteins in Saccharomyces cerevisiae. Gene 144:63-68.[CrossRef][Medline]
    7. 7
  7. Gao, R., T. R. Mack, and A. M. Stock. 2007. Bacterial response regulators: versatile regulatory strategies from common domains. Trends Biochem. Sci. 32:225-234.[CrossRef][Medline]
    8. 8
  8. Gustin, M. C., J. Albertyn, M. Alexander, and K. Davenport. 1998. MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62:1264-1300.[Abstract/Free Full Text]
    9. 9
  9. Halkides, C. J., M. M. McEvoy, E. Casper, P. Matsumura, K. Volz, and F. W. Dahlquist. 2000. The 1.9 A resolution crystal structure of phosphono-CheY, an analogue of the active form of the response regulator, CheY. Biochemistry 39:5280-5286.[CrossRef][Medline]
    10. 10
  10. Hersen, P., M. N. McClean, L. Mahadevan, and S. Ramanathan. 2008. Signal processing by the HOG MAP kinase pathway. Proc. Natl. Acad. Sci. USA 105:7165-7170.[Abstract/Free Full Text]
    11. 11
  11. Hohmann, S. 2002. Osmotic stress signaling and osmoadaptation in yeasts. Microbiol. Mol. Biol. Rev. 66:300-372.[Abstract/Free Full Text]
    12. 12
  12. Jacoby, T., H. Flanagan, A. Faykin, A. G. Seto, C. Mattison, and I. Ota. 1997. Two protein-tyrosine phosphatases inactivate the osmotic stress response pathway in yeast by targeting the mitogen-activated protein kinase, Hog1. J. Biol. Chem. 272:17749-17755.[Abstract/Free Full Text]
    13. 13
  13. Janiak-Spens, F., D. P. Sparling, and A. H. West. 2000. Novel role for an HPt domain in stabilizing the phosphorylated state of a response regulator domain. J. Bacteriol. 182:6673-6678.[Abstract/Free Full Text]
    14. 14
  14. Janiak-Spens, F., J. M. Sparling, M. Gurfinkel, and A. H. West. 1999. Differential stabilities of phosphorylated response regulator domains reflect functional roles of the yeast osmoregulatory SLN1 and SSK1 proteins. J. Bacteriol. 181:411-417.[Abstract/Free Full Text]
    15. 15
  15. Maeda, T., M. Takekawa, and H. Saito. 1995. Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. Science 269:554-558.[Abstract/Free Full Text]
    16. 16
  16. Maeda, T., S. M. Wurgler-Murphy, and H. Saito. 1994. A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369:242-245.[CrossRef][Medline]
    17. 17
  17. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    18. 18
  18. Miyake, Z., M. Takekawa, Q. Ge, and H. Saito. 2007. Activation of MTK1/MEKK4 by GADD45 through induced N-C dissociation and dimerization-mediated trans autophosphorylation of the MTK1 kinase domain. Mol. Cell. Biol. 27:2765-2776.[Abstract/Free Full Text]
    19. 19
  19. Mizuno, T. 1998. His-Asp phosphotransfer signal transduction. J. Biochem. 123:555-563.[Abstract/Free Full Text]
    20. 20
  20. Mumberg, D., R. Müller, and M. Funk. 1994. Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res. 22:5767-5768.[Free Full Text]
    21. 21
  21. O'Rourke, S. M., and I. Herskowitz. 1998. The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev. 12:2874-2886.[Abstract/Free Full Text]
    22. 22
  22. Ota, I. M., and A. Varshavsky. 1993. A yeast protein similar to bacterial two-component regulators. Science 262:566-569.[Abstract/Free Full Text]
    23. 23
  23. Porter, S. W., and A. H. West. 2005. A common docking site for response regulators on the yeast phosphorelay protein YPD1. Biochem. Biophys. Res. Commun. 1748:138-145.
    24. 24
  24. Porter, S. W., Q. Xu, and A. H. West. 2003. Ssk1p response regulator binding surface on histidine-containing phosphotransfer protein Ypd1p. Eukaryot. Cell 2:27-33.[Abstract/Free Full Text]
    25. 25
  25. Posas, F., and H. Saito. 1998. Activation of the yeast SSK2 MAP kinase kinase kinase by the SSK1 two-component response regulator. EMBO J. 17:1385-1394.[CrossRef][Medline]
    26. 26
  26. Posas, F., S. M. Wurgler-Murphy, T. Maeda, E. A. Witten, T. C. Thai, and H. Saito. 1996. Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor. Cell 86:865-875.[CrossRef][Medline]
    27. 27
  27. Reiser, V., D. C. Raitt, and H. Saito. 2003. Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure. J. Cell Biol. 161:1035-1040.[Abstract/Free Full Text]
    28. 28
  28. Robinson, V. L., D. R. Buckler, and A. M. Stock. 2000. A tale of two components: a novel kinase and a regulatory switch. Nat. Struct. Biol. 7:626-633.[CrossRef][Medline]
    29. 29
  29. Rose, M. D., F. Winston, and P. Hieter. 1990. Methods in yeast genetics: a laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
    30. 30
  30. Sato, N., H. Kawahara, A. Toh-e, and T. Maeda. 2003. Phosphorelay-regulated degradation of the yeast Ssk1p response regulator by the ubiquitin-proteasome system. Mol. Cell. Biol. 23:6662-6671.[Abstract/Free Full Text]
    31. 31
  31. Song, H. K., J. Y. Lee, M. G. Lee, J. Moon, K. Min, J. K. Yang, and S. W. Suh. 1999. Insights into eukaryotic multistep phosphorelay signal transduction revealed by the crystal structure of Ypd1 from Saccharomyces cerevisiae. J. Mol. Biol. 293:753-761.[CrossRef][Medline]
    32. 32
  32. Sopko, R., D. Huang, N. Preston, G. Chua, B. Rapp, K. Kafadar, M. Snyder, S. G. Oliver, M. Cyert, T. R. Hughes, C. Boone, and B. Andrews. 2006. Mapping pathways and phenotypes by systematic gene overexpression. Mol. Cell 21:319-330.[CrossRef][Medline]
    33. 33
  33. Stock, A. M., V. L. Robinson, and P. N. Goudreau. 2000. Two-component signal transduction. Annu. Rev. Biochem. 69:183-215.[CrossRef][Medline]
    34. 34
  34. Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189:113-130.[CrossRef][Medline]
    35. 35
  35. Takekawa, M., and H. Saito. 1998. A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 95:521-530.[CrossRef][Medline]
    36. 36
  36. Tatebayashi, K., M. Takekawa, and H. Saito. 2003. A docking site determining specificity of Pbs2 MAPKK for Ssk2/Ssk22 MAPKKKs in the yeast HOG pathway. EMBO J. 22:3624-3634.[CrossRef][Medline]
    37. 37
  37. Tatebayashi, K., K. Tanaka, H.-Y. Yang, K. Yamamoto, Y. Matsushita, T. Tomida, M. Imai, and H. Saito. 2007. Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway. EMBO J. 26:3521-3533.[CrossRef][Medline]
    38. 38
  38. Tatebayashi, K., K. Yamamoto, K. Tanaka, T. Tomida, T. Maruoka, E. Kasukawa, and H. Saito. 2006. Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway. EMBO J. 25:3033-3044.[CrossRef][Medline]
    39. 39
  39. Teige, M., E. Scheikl, V. Reiser, H. Ruis, and G. Ammerer. 2001. Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast. Proc. Natl. Acad. Sci. USA 98:5625-5630.[Abstract/Free Full Text]
    40. 40
  40. Volz, K., and P. Matsumura. 1991. Crystal structure of Escherichia coli CheY refined at 1.7-angstrom resolution. J. Biol. Chem. 266:15511-15519.[Abstract/Free Full Text]
    41. 41
  41. Wurgler-Murphy, S. M., T. Maeda, E. A. Witten, and H. Saito. 1997. Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases. Mol. Cell. Biol. 17:1289-1297.[Abstract]
    42. 42
  42. Xu, Q., S. W. Porter, and A. H. West. 2003. The yeast YPD1/SLN1 complex: insights into molecular recognition in two-component signaling systems. Structure 11:1569-1581.[Medline]
    43. 43
  43. Xu, Q., and A. H. West. 1999. Conservation of structure and function among histidine-containing phosphotransfer (HPt) domains as revealed by the crystal structure of YPD1. J. Mol. Biol. 292:1039-1050.[CrossRef][Medline]
    44. 44
  44. Yuzyuk, T., and D. C. Amberg. 2003. Actin recovery and bud emergence in osmotically stressed cells requires the conserved actin interacting mitogen-activated protein kinase kinase kinase Ssk2p/MTK1 and the scaffold protein Spa2p. Mol. Biol. Cell 14:3013-3026.[Abstract/Free Full Text]
    45. 45
  45. Yuzyuk, T., M. Foehr, and D. C. Amberg. 2002. The MEK kinase Ssk2p promotes actin cytoskeleton recovery after osmotic stress. Mol. Biol. Cell 13:2869-2880.[Abstract/Free Full Text]

Molecular and Cellular Biology, September 2008, p. 5172-5183, Vol. 28, No. 17
0270-7306/08/$08.00+0     doi:10.1128/MCB.00589-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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