INTRODUCTION

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Mitogen-activated protein kinase (MAPK) pathways comprise three sequentially acting kinases, MEKK (or Raf), MEK, and MAPK, known as the MAPK module 16, 22, 48. MEKKs can be activated by interaction with upstream components and phosphorylation on Ser, Thr, and Tyr residues. Activated MEKKs then activate MEK by phosphorylating Ser/Thr residues. Activated MEK activates MAPK by phosphorylating a Thr and a Tyr residue in the phosphorylation lip. Phosphorylation of both residues is required for full MAPK activation. There are three groups of MAPKs, classified by the signals that activate them and their phosphorylation lip sequence. One group, including vertebrate ERK1 and ERK2, contains the phosphorylation lip sequence TEY and is activated by growth factors, mitogens, and cytokines (22, 48). A second group, vertebrate c-Jun N-terminal kinase (JNK)-stress-activated protein kinase, contains the phosphorylation lip sequence TPY and is activated by UV light, osmotic stress, tumor necrosis factor, and interleukin-1 (22, 48). A third group, including Saccharomyces cerevisiae Hog1, Schizosaccharomyces pombe Spc1, and vertebrate p38, contains the phosphorylation lip sequence TGY and is activated by osmotic stress and other environmental stresses (16, 22, 48).

At least six MAPK cascades operate in S. cerevisiae to regulate physiologically distinct responses (16). The HOG pathway allows yeast to grow in high-osmolarity environments by inducing the expression of osmoprotectants. The upstream portion of this pathway has two branches (Fig. 1). One branch is the two-component signaling system comprising Sln1, Ypd1, and Ssk1 (25, 33, 37). Genetic and biochemical data support the following model for activation of this pathway. Sln1 is a plasma membrane-bound His/Asp kinase that is phosphorylated in the absence of stress. Osmotic stress causes its dephosphorylation, leading to dephosphorylation of Ypd1, a His kinase, and Ssk1, an Asp kinase (37). Unphosphorylated Ssk1 activates the MEKKs, Ssk2 and Ssk22, by binding to their N-terminal inhibitory domains (23). For Ssk2, this has been shown to result in autophosphorylation of a Thr residue and kinase activation (34). Activated Ssk2 and Ssk22 then activate the MEK, Pbs2, by phosphorylating a Ser and a Thr residue in the T loop. Activated Pbs2 activates the MAPK, Hog1, by phosphorylation of a Thr and Tyr residue in the phosphorylation lip (4, 42). A second branch of the HOG pathway is activated by the osmosensor Sho1 (23), which signals to Ste20, Ste50, Ste11, and then Pbs2 (32, 35, 36, 38, 50).

View larger version (27K): [in this window] [in a new window]   FIG. 1.   Protein phosphatase inactivation of the HOG pathway. The HOG pathway is regulated by two membrane-bound osmosensors, Sln1 and Sho1. Both Sln1 and Sho1 activate the MAPK cascade (boxed). The two-component regulators, Sln1, Ypd1, and Ssk1, negatively regulate the two MEKKs, Ssk2 and Ssk22, while Sho1, Ste20, and Ste50 positively regulate the MEKK, Ste11. The MEKKs activate a MEK, Pbs2, and the MAPK, Hog1. Two protein tyrosine phosphatases, Ptp2 and Ptp3, inactivate Hog1 by dephosphorylating the pY residue in the phosphorylation lip, while the type 2C Ser/Thr phosphatase, Ptc1, dephosphorylates the pT residue in the phosphorylation lip.

The identity of physiologically relevant protein phosphatases that inactivate MAPK pathways is less well established. Protein phosphatases can inactivate MEKK, MEK, and MAPK, since they require phosphorylation on Ser, Thr, and Tyr residues for activity (21, 22). Vertebrate MEKK and MEK are inactivated by the type 2A Ser/Thr phosphatase (PP2A) in vitro and may be inactivated by PP2A in vivo (1, 2, 17, 45). Stress-activated vertebrate MEKs, MKK6 and SEK1 (MKK4/JNKK1), are also inactivated by the type 2C Ser/Thr-specific phosphatase (PP2C) in vivo and in vitro (46). Since MAPKs require phosphorylation of a Thr and Tyr residue in the phosphorylation lip to be active, they can be inactivated by Ser/Thr phosphatases, protein tyrosine phosphatases (PTPs) specific for phosphotyrosine (pY), and dual-specificity phosphatases, capable of dephosphorylating both the phosphorylation lip phosphothreonine (pT) and pY residues (21, 22). Among the Ser/Thr phosphatases, PP2A has been shown to inactivate MAPK (1, 15, 45), and PP2C has been found to inactivate the stress-activated MAPK, p38 (46). In S. pombe, there are conflicting reports regarding the activity of PP2C on the stress-activated MAPK, Spc1/Sty1. Two PP2C phosphatases, Ptc1 and Ptc3, were shown to be important for inactivating Spc1/Sty1 activated by heat stress (31), while Ptc1 was shown to be unimportant for inactivating Spc1/Sty1 activated by osmotic stress (13).

In S. cerevisiae, MAPKs have been shown to be inactivated by a dual-specificity phosphatase, PTPs, and potentially PP2Cs. The dual-specificity phosphatase Msg5 inactivates Fus3 (11) but has not been shown to inactivate other MAPKs. The PTPs Ptp2 and Ptp3 inactivate Hog1, Fus3, and Mpk1 with different specificities (Fig. 1) (19, 27, 51, 52). The identity of Ser/Thr phosphatases that inactivate S. cerevisiae MAPKs has not been established. Two PP2Cs, Ptc1 and Ptc3, have been implicated as negative regulators of the HOG pathway by genetic means, but their substrates have not been identified. Initially, Maeda et al. identified PTC1 as a gene whose mutation produced a synthetic growth defect with deletion of PTP2 (24). We showed that this growth defect was suppressed by deletion of HOG1 (19), further suggesting that Ptc1 negatively regulates the HOG pathway. Previous studies also showed that overexpression of PTC1 or PTC3 suppressed lethality conferred by deletion of SLN1, which is known to hyperactivate the HOG pathway (25). In this work, we show that PTC2, encoding a type 2C Ser/Thr phosphatase closely related to Ptc3, also inactivates this pathway, and that Ptc1 inactivates the HOG pathway by dephosphorylating the MAPK, Hog1. Unlike vertebrate PP2C, Ptc1 does not have a strong effect on the MEK, Pbs2.

	MATERIALS AND METHODS

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Strains, media, and genetic techniques. Yeast strains (Table 1) were derived from BBY45 (MATa leu2-3,112 trp1-1 his3-200 ura3-52 lys2-801) (3) except where noted. Isolation of PTC genes and examination of PTC mutants was performed using IMY101, a sln1::HIS3 strain carrying a low-copy-number centromere (CEN)-based plasmid containing the wild-type SLN1 gene and the URA3 gene (19). Hog1 kinase assays were performed using IMY102, a hog1::TRP1 strain carrying pHOG1-ha2, and IMY104, a hog1::TRP1 ptc1::LEU2 strain carrying pHOG1-ha2. The hog1::TRP1 strain IMY100 (19) and the ptc1::LEU2 strain ASY1 (19) were crossed, sporulated, and dissected to produce the ptc1::LEU2 hog1::TRP1 strain IMY103. PTC1 and HOG1 were overexpressed from the GAL1 promoter in IMY105, a hog1 strain produced by transformation of the galactose-inducible strain 334 (Table 1) (18) with the hog1::hisG allele (32). Hog1 kinase assays in strains overexpressing PTC1 were performed using IMY105, carrying pHOG1-ha2 and pKT-PTC1, a plasmid overexpressing PTC1 (see below). The control strain was IMY105 carrying pHOG1-ha2 and pEG(KT) (29). Glutathione S-transferase (GST)-Hog1 was isolated from JWY1, which is IMY105 carrying pKT-HOG1. Growth of strains expressing the hyperactive SSK2N allele was assessed in the wild type, JD53 (Table 1) (10), and IMY107, a ptc1::LEU2 strain produced by transforming JD53 with the tpd1::LEU2-3 allele (40).

PTC3 isolation. PTC3 was isolated as a negative regulator of the HOG pathway by selecting for plasmids from a yeast genomic library that suppressed lethality of the sln1 strain in the previously described 5-fluoro-orotic acid (5-FOA) assay (19). Strain IMY101, a sln1 strain carrying pSLN1-URA3, is necessarily Ura⁺ since it requires the SLN1 gene for viability. IMY101 is inviable on media containing the drug 5-FOA, since it is toxic to strains that are Ura⁺. IMY101 was transformed with a multicopy yeast genomic library based in the vector YEp13 (30) (American Type Culture Collection). Transformants capable of vigorous growth on 5-FOA were identified, and the plasmid DNA was isolated using standard methods. For further study, PTC3 was subcloned as a 4.6-kb BamHI fragment into the multicopy vector YEplac181 (14).

Plasmids. PTC1, previously identified as negative regulator of this pathway, and PTC2, a gene closely related to PTC3, were isolated by PCR. A 1.5-kb fragment of PTC1, containing 304 bp of 5' and 308 bp of 3' flanking sequence, and a 2-kb fragment of PTC2, containing 275 bp of 5' and 325 bp of 3' flanking sequence, were subcloned into YEplac181 (14). Mutants Ptc1D58N, Ptc2D62N, and Ptc3D62N were produced by site-directed mutagenesis using the oligonucleotides containing the underlined mutated codons, 5'-GCGGTGTTTAATGGACATGCTGGG-3', 5'-TTTATGGTATATTTAACGGTCATGGTG-3', and 5'-TTTACGGTATATTCAATGGTCATGGTGGC-3', respectively. PTC1 was overexpressed as a fusion to GST from the GAL1 promoter in the vector pEG(KT) (29).

ptc2::TRP1

ptc3::HIS3

ptc2::TRP1

ptc3::HIS3

Immunoblot analysis. Levels of singly (Hog1-pY) and dually (Hog1-pT,pY) phosphorylated Hog1 were examined by immunoblotting with anti-pY antibody (PY20; Santa Cruz Biotechnology) and antibody specific for dually phosphorylated p38 (phospho-p38 antibody; New England Biolabs), respectively. Cells from 4.5 ml of culture at 1 U (A₆₀₀) were harvested by centrifugation, boiled in L1 buffer, and diluted in L2 buffer as described previously (27). Protein samples, 82 µg for the Hog1-pY blot and 113 µg for the phospho-p38 blot, were boiled in sample buffer, and Western analysis was performed (19). Hog1-HA and Hog1-K52M-HA were detected by immunoblotting with antibody 12CA5 (BAbCo), and GST and GST-Ptc1 were detected using anti-GST antibody (Pharmacia). For all immunoblots, alkaline phosphatase-conjugated secondary antibodies were used, and the blots were visualized with 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium (Promega).

Hog1 kinase assay. Hog1 kinase activity in unstressed and osmotically stressed cells was examined as follows. Hog1-HA was isolated from strains grown to exponential phase, ~1 U (A₆₀₀), that were untreated or exposed to 0.4 M NaCl for various times. Cells were harvested by centrifugation and disrupted by glass bead lysis in buffer A (50 mM Tris-HCl [pH 7.5], 50 mM NaCl, 5 mM EGTA, 5 mM MgCl₂, 0.1% TX-100, and 1 mM dithiothreitol [DTT], containing protein phosphatase inhibitors (50 mM -glycerophosphate and 1 mM sodium vanadate) and protease inhibitors (aprotinin, leupeptin, antipain, and chymostatin [each at 20 µg/ml] and 1 mM phenyl methylsulfonyl fluoride [PMSF]). The lysates were clarified by centrifugation, and Hog1-HA was immunoprecipitated by incubation with 2.5 µl of anti-HA antibody 12CA5 (1 mg/ml; BAbCo) for 1 h at 4°C and 12.5 µl of protein A-Sepharose for 0.5 h at 4°C. The immunoprecipitates were washed once with buffer A, twice with buffer B (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM EGTA, 5 mM MgCl₂, 1 mM DTT, 50 mM -glycerophosphate, 1 mM sodium vanadate, 1 mM PMSF), and once with kinase assay buffer (50 mM Tris-HCl [pH 7.5], 50 mM NaCl, 2 mM EGTA, 10 mM MgCl₂, 50 mM -glycerophosphate, 0.1 mM sodium vanadate, 1 mM DTT). To assay Hog1 kinase activity, Hog1-HA bound to protein A-Sepharose was incubated with [-³²P]ATP (0.1 mM, 8,000 cpm/pmol) and myelin basic protein (MBP; 1 µM; Sigma) for 30 min at 30°C. Incorporation of ³²P into MBP was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantified by PhosphorImager (Molecular Dynamics) analysis.

Ptc1 inactivation of Hog1 kinase in vitro. GST-Hog1 was isolated from strain JWY1 grown to exponential phase in synthetic medium lacking uracil and containing 2% galactose. To activate Hog1, cells in exponential phase (~1 U [A₆₀₀]) were exposed to 0.4 M NaCl for 3 min at 30°C. Cells were isolated by centrifugation, and ~1.5 × 10⁸ cells were lysed by glass beading in 1 ml of buffer A. The lysate was clarified by centrifugation and mixed with 180 µl of a 1:1 slurry of glutathione-Sepharose (Pharmacia) for 1 h at 4°C. The resin was washed twice with 600 µl of buffer A, twice with 600 µl of buffer C (50 mM Tris-HCl [pH 7.5], 250 mM NaCl, 5 mM EGTA, 5 mM MgCl₂, 1 mM DTT, 50 mM -glycerophosphate, 1 mM sodium vanadate, 1 mM PMSF), and twice with 600 µl of protein phosphatase buffer (50 mM Tris-HCl [pH 7], 0.1 mM EGTA, 5 mM MgCl₂, 0.1% 2-mercaptoethanol). GST-Hog1 bound to resin was resuspended in protein phosphatase buffer and aliquoted into eight 15-µl fractions, each containing ~170 ng of GST-Hog1. The bound GST-Hog1 was incubated with Ptc1 or Ptc1D58N (0 to 0.3 µg/µl) for 30 min at 37°C in phosphatase buffer. The reaction was terminated by addition of 300 µl of ice-cold buffer B, centrifugation, and washing as described below. Ptc1 inactivation assays were also done in the presence of Mn²⁺. For these assays, MgCl₂ in buffers A, B, and C was replaced by 5 mM MnCl₂ and phosphatase buffer contained 20 mM MnCl₂. To assay Hog1 kinase activity, the resin was washed twice with kinase assay buffer and incubated with MBP and [-³²P]ATP as described above. Samples were examined by SDS-PAGE and PhosphorImager analysis.

Purification of Ptc1, Ptc1D58N, and Pbs2EE from E. coli. His₆-Ptc1, His₆-Ptc1D58N, and His₆-Pbs2EE were expressed from the vector pRSETa in E. coli BL21(DE3)pLysS. The strains were grown in 2×YT medium at 37°C to early log phase, (~0.5 U [A₆₀₀]), cooled to 23°C, and induced with 0.5 mM isopropyl--D-thiogalactopyranoside until the culture reached 1.0 unit (A₆₀₀). To isolate His₆-tagged proteins, cells were lysed in sonication buffer (20 mM Tris-HCl [pH 8], 100 mM NaCl) and centrifuged at 17,000 × g for 15 min to clarify the supernatant, and 25 ml of lysate was incubated with 1.25 ml of Co²⁺-immobilized metal affinity resin (Talon; Clontech) for 1 h at 4°C. The resin was transferred to a column and washed with 20 mM Tris-HCl (pH 8), 100 mM NaCl, and 10 mM imidazole, and the His₆-tagged proteins were eluted with the same buffer containing 75 mM imidazole. Fractions containing His₆-tagged proteins were identified by immunoblotting using anti-His₆ antibody (BAbCo), and the amount of His₆-tagged protein was quantified using the Pierce bicinchoninic acid protein assay.

In vitro phosphorylation and dephosphorylation of Hog1. GST-Hog1 was phosphorylated with Pbs2EE and [-³²P]ATP in vitro. GST-Hog1 was isolated from ~1.5 × 10⁸ JWY1 cells grown to exponential phase in synthetic medium lacking uracil and containing 2% galactose. Cells were isolated by centrifugation and lysed in 600 µl of buffer A. The lysate was clarified by centrifugation and mixed with 180 µl of a 1:1 slurry of glutathione-Sepharose (Pharmacia) for 1 h at 4°C. The beads were washed twice with 600 µl of buffer A, twice with 600 µl of buffer C, and twice with 100 µl of kinase assay buffer. GST-Hog1 bound to resin was resuspended in kinase assay buffer and incubated with His₆-Pbs2EE (0.8 µg/µl) and [-³²P]ATP (0.1 mM, 80,000 cpm/pmol) for 90 min at 30°C. To dephosphorylate Hog1, bead-bound GST-Hog1 was washed twice with 100 µl of phosphatase assay buffer and incubated with Ptc1 (0 or 1.2 µg/µl) for 30 min at 37°C. GST-Hog1 was isolated for phosphoamino acid analysis by SDS-PAGE and transferred to a polyvinylidene difluoride membrane.

Phosphoamino acid analysis. ³²P-labeled GST-Hog1, untreated or incubated with Ptc1, was examined by phosphoamino acid analysis (20). The polyvinylidene difluoride membrane with bound GST-Hog1 was rinsed in methanol and then water and hydrolyzed in 200 µl of 5.7 N HCl at 110°C for 1 h. The filter was removed, and the sample was lyophilized and resuspended in 10 µl of pH 1.9 buffer (2.5% formic acid, 7.8% glacial acetic acid) containing 2.5 nmol of each of the phosphoamino acid standards, pT, pY, and phosphoserine (Sigma). The sample was applied to a cellulose thin-layer chromatography plate and electrophoresed in the first dimension in pH 1.9 buffer and in the second dimension in pH 3.5 buffer (0.5% pyridine, 5.0% glacial acetic acid). The plate was treated with ninhydrin to visualize phosphoamino acid standards, and PhosphorImager analysis was used to examine the radiolabeled amino acids.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Ptc1 inactivates the HOG pathway. Yeast strains that constitutively activate the HOG pathway exhibit growth defects due to hyperactivation of the MAPK module comprising the MEKKs, Ssk2 and Ssk22, the MEK, Pbs2, and the MAPK, Hog1 (Fig. 1). A strain that lacks the receptor histidine kinase response regulator, SLN1, hyperactivates the downstream MAPK cascade and is lethal (25, 33). Overexpression of protein phosphatases restores viability by inactivating the MAPK cascade. For example, overexpression of PTP2 or PTP3, encoding protein tyrosine phosphatases that dephosphorylate Hog1, suppresses lethality of the sln1 strain by inactivating Hog1 (19, 51). Three genes, PTC1, PTC2, and PTC3, suppressed sln1 lethality from multicopy (Fig. 2) but not low-copy-number CEN-based plasmid (data not shown).

View larger version (101K): [in this window] [in a new window]   FIG. 2.   PTC1, PTC2, and PTC3 suppress growth defects due to Hog1 hyperactivation. Deletion of SLN1 hyperactivates the downstream MAPK cascade and is lethal. Expression of PTC1, PTC2, and PTC3 from multicopy plasmids suppressed lethality of the sln1 strain, while overexpression of the metal binding site mutations ptc1D58N, ptc2D62N, and ptc3D62N did not. The ability of PTCs and their mutant counterparts to suppress sln1 lethality was examined using the 5-FOA assay. IMY101, the sln1 strain bearing pSLN1(URA3), a CEN-based plasmid expressing the SLN1 and URA3 genes, was transformed with multicopy plasmids bearing wild-type (wt) PTC genes (pPTC1, pPTC2, and pPTC3), mutant PTCs (pPTC1D58N, pPTC2D62N, and pPTC3D62N), or empty vector (YEplac181). The transformants were patched on synthetic medium lacking leucine and containing 5-FOA and then examined for growth after 3 days at 30°C.

ptc1 hog1

View this table: [in this window] [in a new window]   TABLE 2.   Growth of ptc and ptc ptp mutant strainsa

ptc1 ptp2

Ptc1 can function downstream of the MEKK Ssk2. Since overexpressing PTC1 suppressed lethality of the sln1 strain, Ptc1 could conceivably inactivate any component downstream of Sln1 that requires Ser or Thr phosphorylation for activity. The MEKKs, Ssk2 and Ssk22, the MEK, Pbs2, and the MAPK, Hog1, are all potential candidates for Ptc1 inactivation. In an attempt to delineate the point of action of Ptc1 in this pathway, we examined the effect of overexpressing or deleting PTC1 in a strain expressing a hyperactivated MEKK allele, SSK2N, that lacks the amino-terminal domain inhibitory for kinase activity. Overexpression of this allele was previously shown by Maeda et al. (23) to be lethal due to hyperactivation of Pbs2 and Hog1. In our strain background, however, overexpressing SSK2N from the GAL1 promoter produced only a slight growth defect (Fig. 3). Overexpression of PTC1 in this strain did not improve growth (data not shown). Such an improvement may be difficult to detect since SSK2N conferred only a mild growth defect. In contrast, deletion of PTC1 in the SSK2N overexpressor was lethal (Fig. 3). Therefore, Ptc1 may inactivate the HOG pathway by acting downstream of MEKK, although this experiment does not rule out the possibility that Ptc1 acts on MEKKs or upstream of them.

View larger version (76K): [in this window] [in a new window]   FIG. 3.   Ptc1 acts downstream of the MEKK, Ssk2. Deletion of PTC1 exacerbated growth defects due to overexpression of SSK2N, consistent with its role as a negative regulator. The wild type and a ptc1 strain were transformed with pSSK2N, a multicopy plasmid expressing the hyperactive MEKK allele, SSK2N, under regulation of the GAL1 promoter, or the empty vector pYES2. Growth on galactose conferred only a slight defect in the wild type but was lethal in a ptc1 strain. Growth of strains on selective medium (SM) containing galactose was assessed after 3 days at 30°C.

Ptc1 inactivates Hog1 but has little effect on Pbs2 in vivo. We next used another assay to examine the point of action of Ptc1 in this pathway. If Ptc1 inactivates upstream kinases that activate Hog1, such as Pbs2, then overexpression of PTC1 should inhibit activation of Hog1. To assess Hog1 activation, we examined the level of Tyr176 phosphorylation in the phosphorylation lip. We chose this assay, since it should be unaffected by potential Ptc1-dependent dephosphorylation of Hog1-pT. Overexpression of PTC1 did not reduce the level of Hog1-pY induced by osmotic stress (Fig. 4A), suggesting that Ptc1 does not inactivate components upstream of Hog1. Since Hog1-pY could be due to autophosphorylation, we also performed this experiment with the catalytically inactive mutant Hog1-K52M (26). Osmotic stress also induced phosphorylation of Hog1-K52M in the PTC1 overexpressor (data not shown), suggesting that Ptc1 does not strongly affect Pbs2.

View larger version (56K): [in this window] [in a new window]   FIG. 4.   Ptc1 inhibits the activity of Hog1 but not Pbs2 in vivo. (A) The effect of PTC1 overexpression on Pbs2 activity was examined by monitoring the level of Hog1-pY, using anti-PY antibody. Hog1-pY was examined in the wild-type strain [334 carrying empty vector pEG(KT)] and in a strain overexpressing PTC1 (334 carrying pKT-PTC1). Levels of GST and GST-Ptc1 were examined using anti-GST antibody. (B) Hog1 kinase activity was examined in the wild type, IMY105 carrying pHOG1-ha and empty vector pEG(KT), and in a strain overexpressing PTC1, IMY105 carrying pHOG1-ha and pKT-PTC1. Hog1-HA was isolated from yeast that were untreated or exposed to 0.4 M sodium chloride for the indicated times. Immunoprecipitated Hog1-HA was incubated with MBP and [-32P]ATP, and the radiolabel incorporated into MBP was quantified by PhosphorImager analysis. The level of Hog1-HA in lysates was assessed by immunoblotting with anti-HA antibody.

View larger version (57K): [in this window] [in a new window]   FIG. 5.   A strain lacking PTC1 shows defects in Hog1 activation and inactivation. (A) Hog1 kinase activity and the level of Hog1 protein were examined in the wild-type strain IMY102 and in ptc1 strain IMY103. Hog1-HA was isolated from yeast cells that were untreated or exposed to 0.4 M sodium chloride for the indicated times, and Hog1 kinase activity was assessed as described in the legend to Fig. 4. The level of Hog1-HA was assessed using anti-HA antibody. (B) Dual phosphorylation of kinase-inactive Hog1-K52M is rapid in both wild-type ptc1 strains. PTC1 hog1 and ptc1 hog1 strains expressing Hog1-K52M-HA were exposed to 0.4 M NaCl for the indicated times. Activation of Hog1-K52M-HA was monitored using antibody specific for dually phosphorylated Hog1, and its relative level of expression was assessed using anti-HA antibody.

Ptc1 inactivates Hog1 in vitro. Since Ptc1 inactivates Hog1 in vivo, we asked whether Ptc1 could dephosphorylate Hog1 in vitro. Activated GST-Hog1 was isolated from yeast and treated with Ptc1 or the metal binding site mutant Ptc1D58N, purified from E. coli. GST-Hog1 kinase activity was then examined by incubation with MBP and [-³²P]ATP. Treatment of GST-Hog1 with Ptc1 inactivated Hog1, while treatment with Ptc1D58N did not (Fig. 6A). We also examined the divalent cation requirement of Ptc1 for catalytic activity. Previously, a partially purified preparation of Ptc1 expressed in E. coli was shown to have greater activity with Mn²⁺ than with Mg²⁺, using phosphorylated casein as a substrate (40, 41). Using GST-Hog1 as a substrate, we found that Ptc1 had at least 10-fold-greater activity with Mn²⁺ than with Mg²⁺ (Fig. 6A).

View larger version (85K): [in this window] [in a new window]   FIG. 6.   Ptc1 inactivates Hog1 kinase activity in vitro by dephosphorylating pT. (A) Ptc1 inactivated Hog1 kinase activity in vitro. GST-Hog1 was isolated from osmotically stressed yeast strain JWY1 and incubated in the absence or presence of wild-type Ptc1 or mutant Ptc1D58N for 30 min at 30°C. Ptc1 or PtcD58N in the amounts specified (0 to 9 µg) was incubated in the presence of 5 mM Mg2+ or 20 mM Mn2+. The bead-bound GST-Hog1 was washed extensively and incubated with MBP and [-32P]ATP. Radiolabel incorporated into MBP was examined by PhosphorImager analysis. (B) Hog1 was phosphorylated at Thr and Tyr by the hyperactive MEK mutant, Pbs2EE, in vitro. GST-Hog1 was isolated from untreated yeast using glutathione-Sepharose. The bead-bound GST-Hog1 was phosphorylated by incubation with Pbs2EE purified from E. coli and [-32P]ATP. Phosphoamino acid analysis was performed to examine the level of pT and pY. Arrows indicate the position of phosphoamino acid standards as revealed by ninhydrin staining. pS, phosphoserine. (C) Ptc1 specifically dephosphorylates Hog1-pThr in vitro. 32P-phosphorylated GST-Hog1 was treated with Ptc1 and examined as above.

Subcellular localization of Ptc1. Since Hog1 accumulates in the nucleus upon osmotic stress (12, 26, 39), we predicted that at least a fraction of Ptc1 would be present in the nucleus. To address this question, GFP was fused to Ptc1 and expressed from either low-copy-number or multicopy plasmids in a ptc1 strain. In most cells, GFP-Ptc1 was found evenly distributed between the cytoplasm and the nucleus; in some cells, it was concentrated in the nucleus (Fig. 7). Osmotic stress did not alter the localization of Ptc1 (data not shown). Thus, the localization of Ptc1 is consistent with its ability to regulate the basal activity of Hog1, as well as dephosphorylating activated Hog1 found concentrated in the nucleus.

View larger version (87K): [in this window] [in a new window]   FIG. 7.   Ptc1 is both cytoplasmic and nuclear. The subcellular localization of a GFP-Ptc1 fusion protein expressed from a multicopy plasmid was examined in a ptc1 strain. Cells in exponential growth phase were visualized using a Zeiss fluorescence microscope with a charge-coupled device camera. 4',6-Diamidino-2-phenylindole (DAPI) was used to visualize the nucleus.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we showed that Ptc1 inactivates the Hog1 MAPK in vivo and in vitro, establishing the importance of a PP2C in regulation of MAPK signaling in S. cerevisiae. The phosphatase activity of Ptc1 and of two closely related phosphatases, Ptc2 and Ptc3, was required for pathway inactivation since metal binding site mutants could not suppress the severe growth defect of the sln1 strain due to Hog1 hyperactivation. Ptc1 inhibited the HOG pathway by inactivating Hog1 but not Pbs2. Overexpression of PTC1 did not inhibit Tyr phosphorylation of Hog1, suggesting it does not act on Pbs2 (Fig. 4A). Similarly, overexpression of S. pombe Ptc1 and Ptc3 did not inhibit Tyr phosphorylation of Spc1/Sty1 (13, 31). Thus yeast PP2C appears to differ from mammalian PP2C, which inhibits the stress-activated MEKs, MKK6 and SEK1 (46). Further study of S. cerevisiae PTC1 showed that its overexpression inhibited osmotic stress-induced Hog1 kinase activation, while its deletion led to a defect in adaptation, elevated basal Hog1 kinase activity, and an inability to rapidly activate Hog1. The biochemical basis for Ptc1 effects on Hog1 activity was specific dephosphorylation of the pT but not the pY residue in the phosphorylation lip.

Examination of Hog1 kinase activity before and during treatment with osmotic stress showed that Ptc1 plays a role in adaptation. In the wild type, Hog1 reached a peak of activity at 3 min and declined rapidly thereafter, whereas in the ptc1 strain, Hog1 kinase activity did not decrease significantly for up to 1 h. In this respect, the role of Ptc1 is similar to that of the PTPs that dephosphorylate Hog1-pY. Previously, we and others showed that strains lacking the PTPs poorly dephosphorylated Hog1-pY during continuous exposure to osmotic stress (19, 51, 53). PTP-mediated adaptation may require their upregulation, since osmotic stress induces PTP2 and PTP3 transcripts in a Hog1-dependent manner (19). Unlike PTP transcripts, however, the PTC1 transcript did not increase in response to osmotic stress (data not shown).

A second role for Ptc1 is to maintain a low basal level of Hog1 activity. In the absence of osmotic stress, Hog1 kinase activity was higher in the ptc1 strain than in the wild type. We believe that increased Hog1 activity in the ptc1 strain is due to increased phosphorylation of Thr174, and not Thr174 and Tyr176, as Ptc1 is specific for the pT residue (Fig. 6C). That Thr174-phosphorylated Hog1 is active is supported by in vivo studies, where Hog1-Y176F, but not Hog1-T174A, allows growth on high-osmolarity media (42; S. S. Spencer and I. M. Ota, unpublished data).

Unexpectedly, maintaining a low basal Hog1 kinase activity was critical for its rapid signal-induced activation. Deletion of PTC1 led to a significant delay in Hog1 activation (Fig. 5A). One factor necessary for this delay was Hog1 kinase activity. Substitution of the wild-type Hog1 with the kinase-inactive Hog1-K52M led to a similar rapid activation in the ptc1 and wild-type PTC1 strains (Fig. 5B). One possible explanation for this delay could be Hog1 kinase-dependent activation of negative regulators. For example, PTP2 and PTP3 transcripts are upregulated by osmotic stress in a Hog1-dependent manner, and their constitutive overexpression in the ptc1 strain could inhibit Hog1 activation (19). Other mechanisms independent of gene expression could also come into play. For example, the level of negative regulators can be increased through their stabilization. Phosphorylation of the dual-specificity phosphatase MKP-1 by its substrate, ERK, was shown to increase MKP-1 activity by inhibiting its proteolysis (5).

An alternative explanation for delayed Hog1 activation in the ptc1 strain is that Ptc1 also acts as a positive regulator in the pathway. For example, MEK retrophosphorylated by MAPK at specific Thr residues is inhibitory for MEK activity (6). A Ser/Thr phosphatase that specifically dephosphorylates these sites could activate MEK. Whether Ptc1 could be such a phosphatase remains to be answered. A precedent for a Ser/Thr phosphatase acting as both a positive and a negative regulator has been observed in the Drosophila MAPK pathway regulating photoreceptor development. PP2A acts as a negative regulator downstream of Ras and a positive regulator downstream of Raf (47). Similarly, in Caenorhabditis elegans, PP2A acts as a positive regulator downstream of Ras (44). Further examination of these phosphatases will be required to determine the biochemical basis for their positive regulatory effect on MAPK pathways.

Genetic and biochemical evidence now show that Ptc1 and Ptp2 act together to regulate Hog1 activity in vivo. The synthetic growth defect of the ptc1 ptp2 strain due to Hog1 hyperactivation (19, 24) can be explained by increased phosphorylation of Hog1 at the phosphorylation lip residues. Increased phosphorylation of Thr174 in the ptc1 strain, or Tyr176 in the ptp2 strain, is not sufficient to produce a growth defect, but hyperphosphorylation of both residues in the ptc1 ptp2 strain is nearly lethal. The function of Ptc1 and PTPs separately regulating Hog1 activity via dephosphorylating pT or pY is not known. The S. pombe stress-activated MAPK, Spc1, is also regulated by PTPs and PP2Cs (28, 31, 43). Interestingly, Spc1-pY dephosphorylation is delayed compared to pT during adaptation to heat stress (31), but the biological function of this regulation is not known. Continued examination of the role of PTCs and how they act together with PTPs to regulate the HOG pathway should provide further insights into the regulation of MAPK pathways by protein phosphatases.