High osmolarity glycerol (HOG) pathway-induced phosphorylation and activation of 6-phosphofructo-2-kinase are essential for glycerol accumulation and yeast cell proliferation under hyperosmotic stress.

In response to changes in the environment, yeast cells coordinate intracellular activities to optimize survival and proliferation. The transductions of diverse extracellular stimuli are exerted through multiple mitogen-activated protein kinase (MAPK) cascades. The high osmolarity glycerol (HOG) MAPK pathway is activated by increased environmental osmolarity and results in a rise of the cellular glycerol concentration to adapt the intracellular osmotic pressure. We studied the importance of the short time regulation of glycolysis under hyperosmotic stress for the survival and proliferation of yeast cells. A stimulation of the HOG-MAPK pathway by increasing the medium osmolarity through addition of salt or glucose to cultivated yeast leads to an activation of 6-phosphofructo-2-kinase (PFK2), which is accompanied by a complex phosphorylation pattern of the enzyme. An increase in medium osmolarity with 5% NaCl activates PFK2 3-fold over the initial value. This change in the activity is the result of a 4-fold phosphorylation of the enzyme mediated by protein kinases from the HOG-MAPK pathway. In the case of hyperosmolar glucose a 5-fold PFK2 activation was achieved by a single phosphorylation with protein kinase A near the carboxyl terminus of the protein on Ser(644) and an additional 5-fold phosphorylation within the same amino-terminal fragment as in the presence of salt. The effect of hyperosmolar glucose is the result of an activation of the Ras-cAMP pathway together with the HOG-MAPK pathway. The activation of PFK2 leads to an activation of the upper part of glycolysis, which is a precondition for glycerol accumulation. Yeast cells containing PFK2 accumulate three times more glycerol than cells lacking PFK2, which are not able to grow under hypertonic stress.

In response to changes in the environment, yeast cells coordinate intracellular activities to optimize survival and proliferation. The transductions of diverse extracellular stimuli are exerted through multiple mitogen-activated protein kinase (MAPK) cascades. The high osmolarity glycerol (HOG) MAPK pathway is activated by increased environmental osmolarity and results in a rise of the cellular glycerol concentration to adapt the intracellular osmotic pressure. We studied the importance of the short time regulation of glycolysis under hyperosmotic stress for the survival and proliferation of yeast cells. A stimulation of the HOG-MAPK pathway by increasing the medium osmolarity through addition of salt or glucose to cultivated yeast leads to an activation of 6-phosphofructo-2-kinase (PFK2), which is accompanied by a complex phosphorylation pattern of the enzyme. An increase in medium osmolarity with 5% NaCl activates PFK2 3-fold over the initial value. This change in the activity is the result of a 4-fold phosphorylation of the enzyme mediated by protein kinases from the HOG-MAPK pathway. In the case of hyperosmolar glucose a 5-fold PFK2 activation was achieved by a single phosphorylation with protein kinase A near the carboxyl terminus of the protein on Ser 644 and an additional 5-fold phosphorylation within the same amino-terminal fragment as in the presence of salt. The effect of hyperosmolar glucose is the result of an activation of the Ras-cAMP pathway together with the HOG-MAPK pathway. The activation of PFK2 leads to an activation of the upper part of glycolysis, which is a precondition for glycerol accumulation. Yeast cells containing PFK2 accumulate three times more glycerol than cells lacking PFK2, which are not able to grow under hypertonic stress.
Cells of the yeast Saccharomyces cerevisiae possess rapidly responding, highly complex signaling pathways. These pathways allow the cells to quickly adapt to a changing environment (1). Prominent among yeast signaling pathways are the mitogen-activated protein kinase (MAPK) 1 cascades.
In the yeast S. cerevisiae, a variety of external stimuli activate the MAPK pathways which convert these signals into appropriate metabolic responses (1). Five of these MAPK cascades have been characterized that respond to such diverse environmental conditions as the presence of mating pheromones, changes in osmotic pressure, heat stress, and nutrient availability (2). The yeast S. cerevisiae adapts to growth under conditions of increased external osmolarity through activation of the high osmolarity glycerol (HOG) MAPK pathway (3). The activation of this pathway ensures the accumulation of a high intracellular concentration of glycerol to reduce the transmembrane difference of osmotic pressure and to prevent the loss of water (4). The stimulation of glycerol synthesis is achieved by activating transcription of genes required for glycerol synthesis such as GPD1 encoding glycerol-3-phosphate dehydrogenase (5,6).
The yeast monofunctional 6-phosphofructo-2-kinase (PFK2) catalyzes the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P 2 ), a signal molecule connecting environmental changes with glycolysis (7,8). Fru-2,6-P 2 is the most powerful activator of 6-phosphofructo-1-kinase, a key regulatory enzyme of glycolysis (9). A stimulation of the Ras-cAMP pathway by glucose addition to cultivated yeast cells leads to an in vivo activation of PFK2, which is accompanied by a rather complex phosphorylation pattern of the enzyme (10). The phosphorylation of the protein at Ser 644 is the result of PKA stimulation, while the protein kinase(s) catalyzing the 5-fold phosphorylation of the peptide fragment T 67-101 is (are) still unknown. PFK2 lacking this peptide T 67-101 is inactive (10).
Recently we reported that under hyposmotic stress yeast PFK2 was found to be inhibited by in vivo phosphorylation on Ser 652 . The phosphorylation and hence inactivation of PFK2 are under control of the PKC MAPK pathway and reduce the rate of glycolysis, leading to an accumulaton of glucose 6-phosphate (G-6-P) (11).
The present study examines the regulation of the glycolysis under hyperosmotic stress with the focus on the phosphorylation and activity change of PFK2. We also investigate the role of PFK2 in the control of the cellular glycerol concentration and survival of yeast cells under hyperosmotic stress.

EXPERIMENTAL PROCEDURES
Materials-Yeast nitrogen base and casamino acids were from Difco. The expression vector pMK11PFK2 was a gift from M. Kretschmer. [ 32 P]Orthophosphoric acid, HiTrap affinity columns, and acetonitrile were from Amersham Biosciences. The Expand High Fidelity PCR System was from Roche Diagnostics. Restriction endonucleases were purchased from MBI Fermentas. T4 ligase and phosphoserine/phosphothreonine-specific protein phosphatase-2A (PP2A) were obtained from Promega. The BigDye Terminator Cycle Sequencing Kit was from PE Applied Biosystems. Trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) and high performance liquid chromatographygrade water were from Sigma. ␣-Cyano-4-hydroxycinnamic acid was from Fluka, and trifluoroacetic acid was from J. T. Baker. The Super Signal West Pico Substrate was from Pierce. The HOG-MAPK pathway mutants were a gift from Dr. S. M. O'Rourke and Dr. I. Herskowitz.
Yeast Expression Vector-The plasmid pMK11PFK2 using uracil as selection marker contains the open reading frame of yeast PFK2 fused to the Gal1 promoter. It was modified with a His 6 tag linker at the NH 2 terminus of the PFK2 cDNA, resulting in pMK11PFK2His. The introduced histidine residues are excluded from the numbering of amino acid residues in the primary structure of PFK2his. The plasmid YEP352Gal1 was used as control vector.
Site-directed Mutagenesis of PFK2-The point mutations Ser 644 3 Ala and Ser 652 3 Ala were introduced into the plasmid pMK11PFK2His described in Refs. 17 and 11, respectively. The deletion mutant for the peptide T 67-101 was constructed according to Ref. 10.
Protein Expression and Purification-The S. cerevisiae strain DFY658 was transformed with pMK11PFK2His. Fresh culture medium (YNB-P) supplemented with 2% galactose was inoculated from overnight cultures and incubated at 30°C for 48 h. Yeast cells were harvested by centrifugation and disrupted with glass beads. The purification procedure of the wild-type PFK2 and the mutant proteins was performed at room temperature using a HiTrap affinity column according to Ref. 18. The in vivo phosphorylated proteins were purified at 4°C.
Western Blot Analysis-SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described previously (18) using a 10% acrylamide gel. After electrophoresis, proteins were transferred to nitrocelulose membranes. The blots were probed with a rabbit antiserum raised against PFK2 and then incubated with horseradish peroxidaseconjugated sheep anti-rabbit IgG. Proteins were visualized by chemiluminescence detection with Super Signal West Pico Substrate.
High Osmolarity Stress Experiments-The induction of hyperosmotic shock to yeast cells cultured in YNB-P was performed according to Luyten et al. (21). After reaching the log phase the cells were centrifuged, and the pellet was resuspended either in YNB-P (control) or in hyperosmotic medium (YNB-P supplied with 5% NaCl or 1 M glucose). After 30-min incubation time at 30°C, yeast cells were quickly chilled and collected by centrifugation. Ice-cold buffer containing protease and phosphatase inhibitors (100 mM sodium phosphate, pH 7.4, 600 mM NaCl, 10 mM imidazole, 5 mM mercaptoethanol, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 0.1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 0.01 g/l leupeptin, 0.01 g/l pepstatin) was added to the cell pellet, and the cells were disrupted using glass bead agitation. The change of the PFK2 activity under hyperosmotic stress was followed.
To determine 32 P incorporation during the hyperosmotic stress, yeast cells were grown on YNB-LP. After reaching the log phase the cells were gently centrifuged, resuspended in hyperosmotic medium (YNB-LP supplied with 5% NaCl or 1 M glucose) with [ 32 P]inorganic phosphate, and incubated at 30°C for different times. After rapid cooling PFK2 was prepared, denaturated in loading buffer, and electrophoresed (SDS-PAGE). After the gels were stained and dried, the phosphate incorporation was analyzed on a PhosphorImager (Amersham Biosciences).
For the identification of the PFK2 in vivo phosphorylation sites by MALDI-TOF MS analysis, yeast cells were cultured to log phase in YNB-P and then transferred into YNB-P supplied with 5% NaCl or 1 M glucose. After 30 min of incubation at 30°C under shaking, the cells were harvested, and the PFK2 was purified as described above. After SDS-PAGE and in-gel digestion the peptide fragments were analyzed with MALDI-TOF MS.
Dephosphorylation by PP2A-After each phosphorylation experiment an aliquot of the modified protein was dephosphorylated. The protein was equilibrated with 50 mM Tris-HCl buffer, pH 7.4, 18 mM MgCl 2 , 1 mM dithiothreitol, 0.01 mM EGTA, 0.05% mercaptoethanol, 0.1 mg/ml bovine serum albumin. Treatment with phosphoserine/phosphothreonine-specific protein phosphatase-2A (2.5 units/ml) was carried out for 30 min at 37°C according to Ref. 22. The reaction was terminated by addition of loading buffer. After SDS-PAGE the PFK2 was prepared for MALDI-TOF MS as described below. To analyze the change of PFK2 activity after dephosphorylation the enzyme activity assays were carried out as described above.
In-gel Digestion and MALDI-TOF MS Analysis-The in-gel digestion of PFK2 was performed according to Ref. 18. The extraction of the tryptic digest from the gel pieces was carried out with 50% acetonitrile and 0.1% trifluoroacetic acid. The tryptic digest was crystallized with a matrix solution containing ␣-cyano-4-hydroxycinnamic acid. The matrix solution was prepared according to Ref. 23, and 20 mM diammonium citrate was added to increase the detection efficiency of the phosphopeptide fragments. The mixture was thoroughly vortexed and centrifuged, leaving a clear matrix solution. The sample matrix solution was deposited onto the target as two-layer sample preparation (24). All mass spectra were obtained on a Bruker BiflexIII mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) operated in the linear or reflector mode. System calibration was carried out according to Ref. 18.
Determination of Glycerol and Glucose 6-Phosphate-Hyperosmotic stressed cells were prepared as described above. Samples were withdrawn by filtration, and the filters carrying the cells were quickly frozen in liquid nitrogen. After thawing the cells were disrupted in 0.5 M perchloric acid by shaking with an equal volume of glass beads (0.5-mm diameter) using a vortex mixer (5 ϫ 30 s with a 1-min cooling period between each mixing). After centrifugation the supernatant was neutralized with KOH. Glycerol determination was carried out in the supernatant by absorbance measurement at 340 nm using glycerokinase and glycerol-3-phosphate dehydrogenase as described by Albertyn et al. (5). G-6-P was determined according to Ref. 25 using glucose-6phosphate dehydrogenase. Glycerol and G-6-P concentrations were calculated per gram dry weight of cells.
Determination of Fructose 2,6-Bisphosphate-The extracts were prepared according to Ref. 26. The Fru-2,6-P 2 concentration was measured according to Ref. 27 and related to the cell dry weight.

Hyperosmolarity Induced in Vivo Phosphorylation and Activation of the PFK2
Hyperosmolarity with NaCl-To study the effect of hyperosmolarity on the regulation of glycolysis and the role of PFK2 in cell adaptation to the osmotic stress yeast cells were exposed to hypertonic shock as described under "Experimental Procedures." Both the activity change and the phosphorylation status of PFK2 were monitored. Under hypertonic stress with 5% NaCl and in the presence of 32 P-labeled inorganic phosphate the PFK2 was in vivo phosphorylated (Fig. 1A, i). 32 P incorporation increased within the time of incubation without any augmentation of the PFK2 protein concentration (Fig. 1A, ii). Parallel to the phosphorylation an activation of the PFK2 was observed. The PFK2 activity increased about 3-fold compared with isosmotic conditions (Fig. 2A). The increase of the PFK2 activity after in vivo phosphorylation was reversed by dephosphorylation with PP2A (Table I).

6-Phosphofructo-2-kinase and HOG-MAPK Pathway
Hyperosmolarity with Glucose-In the case of hyperosmolar extracellular glucose (1 M) the effects were similar to those of NaCl. PFK2 was phosphorylated (Fig. 1B, i) and activated (Fig.  2) at a constant level of PFK2 protein (Fig. 1B, ii). PFK2 activation is accompanied by a 20-fold increase of the Fru-2,6-P 2 concentration (Fig. 3). Although the activation of PFK2 was more pronounced in the case of hyperosmolar glucose than with hyperosmolar NaCl, there was no significant difference in the amounts of Fru-2,6-P 2 synthesized. The PFK2 activation under hyperosmotic shock with glucose was reduced to the isosmotic level after dephosphorylation (Table I).
The Role of PFK2 in Glycerol Synthesis and for the Control of Yeast Glycolysis under Hypertonic Stress-We studied the effects of PFK2 on the intracellular glycerol and G-6-P concen-trations after addition of NaCl to 5% in the medium. Cells of the PFK2 deficient strain DFY658 transformed with pMK11 (overexpressing PFK2) or with YEP352/Gal1 (no PFK2) were exposed to the hyperosmotic stress. When NaCl was added the intracellular glycerol concentration in the cells containing PFK2 increased rapidly to reach a maximum concentration of about 200 mmol/g dry weight (Fig. 4A). In parallel, the concentration of G-6-P, a key metabolite of the upper part of glycolysis, decreased (Fig. 4B). In contrast, in the cells lacking PFK2 glycerol accumulated very slowly reaching a maximum of only 60 mmol/g dry weight, about a third of the concentration reached in the presence of PFK2 (Fig. 4A). Only a slight decrease of the G-6-P concentration could be observed under these conditions. The effects of hyperosmolarity induced by high glucose concentration in the medium on glycerol and G-6-P contents of cells containing PFK2 and lacking PFK2 are nearly identical to those observed with hyperosmolar NaCl (data not shown).
Glycerol accumulation under hyperosmotic stress influences cell growth. Fig. 5 shows the time course of cell density in hyperosmotic medium (5% NaCl). DFY658 cells overexpressing PFK2 grow almost normal after a short adaptation pause. In contrast, the slow glycerol accumulation in the cells without PFK2 is accompanied by a stop of cell growth under hyperosmotic stress.
Hyperosmolarity Tests with Yeast Mutants of the HOG-MAPK Pathway-Hyperosmolarity activates both the HOG-MAPK pathway (1, 3) and PFK2 (see above). To prove that the phosphorylation and activation of the PFK2 is the result of a stimulation of the HOG-MAPK pathway, yeast mutants lacking key enzymes of the HOG-MAPK pathway were exposed to hyperosmotic shock with NaCl or glucose and PFK2 activity was monitored. In isosmotic medium PFK2 activity is almost the same in the wild-type strain and the HOG-MAPK pathway mutant strains (Fig. 6). In the case of treatment with NaCl only PFK2 from the wild-type strain was activated, whereas the activity of the PFK2 from mutant strains was not affected. In the case of hyperosmotic shock with glucose the PFK2 was activated in all yeast strains (Fig. 6).

MALDI-TOF MS Analysis of the Tryptic Digest of in Vivo Phosphorylated PFK2 after Hypertonic Shock
Hyperosmolarity with NaCl-Cells of the yeast strain DFY658 transformed with the plasmid pMK11PFK2His6 overexpressing His-tagged PFK2 were exposed to 5% NaCl. The identification of the in vivo phosphorylation site(s) of PFK2 was achieved by comparing the results of MALDI-TOF MS peptide mass fingerprinting of the tryptic digests of purified in vivo phosphorylated and in vitro dephosphorylated PFK2. The superposition of the mass spectra resulting from the two tryptic digests confirmed that the PFK2 was in vivo phosphorylated. This posttranslational modification affects the peptide fragment T 67-101 with m/z 3755.8, which was phosphorylated up to four times. From mono-to tetraphosphorylation all phosphorylation states coexist (Fig. 7A). All phosphates could be removed by treatment with PP2A (Fig. 7B).
Hyperosmolarity with Glucose-Similar to hypertonic NaCl also osmotic stress exerted by hyperosmolar glucose causes a phosphorylation of the peptide fragment T 67-101 (m/z 3755.8, Fig. 8). However, with glucose this fragment was phosphorylated up the five times. An additional phosphorylation affects the fragment T 642-654 carrying Ser 644 (data not shown). To identify the phosphorylation site hyperosmolarity experiments with the PFK2 Ser 652 3 Ala mutant were performed. In the mass spectrum of the tryptic digest obtained from the phosphorylated enzyme an additional peak ([MH ϩ ] ϩ P: m/z 1477.2) 80

6-Phosphofructo-2-kinase and HOG-MAPK Pathway
Da larger than that of the unphosphorylated peptide T 642-654 (m/z 1397.2) was observed (Fig. 9A). The absence of this peak in the spectrum of dephosphorylated PFK2 (Fig. 9B) confirms that it results from the phosphorylation of Ser 644 . In control experiments using the PFK2 Ser 644 3 Ala mutant no phosphorylation of the peptide T 642-654 was observed (data not shown).
Effect of Hyperosmolarity Stress with Glucose on PFK2 from RS13-58A-Cells of the yeast strain RS13-58A, which lacks two catalytic subunits of PKA and is attenuated in the remaining catalytic subunit, were transformed with pMK11PFK2His6 and exposed to 1 M glucose. Under this hypertonic stress and in the presence of 32 P-labeled inorganic phosphate PFK2 was in vivo phosphorylated (Fig. 10). The incorporation of 32 P increased with the incubation time up to 20 min (Fig. 10A). The purified PFK2 was analyzed by SDS-PAGE before in-gel digestion with trypsin. MALDI-TOF MS analysis showed that the PFK2 was phosphorylated 4-fold on the peptide fragment T 67-101 (Fig. 10B), while the fragment carrying Ser 644 was not modified (data not shown).
Effect of Hyperosmolarity on the NH 2 -terminal Deletion Mutant of PFK2-To prove the functional significance of the peptide T 67-101 and its phosphorylation, a PFK2 mutant lacking this peptide was constructed, expressed in, and purified from, yeast strain DFY658. The in-gel digestion of the purified mutant protein followed by MALDI-TOF MS fingerprinting confirmed its identity (10). Activity tests in extracts from cells grown under isosmotic conditions showed that this mutant was catalytically inactive. Also, after hyperosmolar stress with either NaCl or glucose no activity could be detected in cell-free extracts (data not shown).

DISCUSSION
Osmotic Stress Induced in Vivo Phosphorylation and Activation of the PFK2-An increase of the environmental osmolarity requires cellular reactions to counteract this stress. In the yeast S. cerevisiae, the membrane proteins Sln1p and Sho1p have been described as sensors of the two upstream branches controlling the HOG-MAPK pathway (3,28,29). The activated MAPK cascade stimulates different processes, which act together to counterbalance the loss of water. The intracellular glycerol accumulation is one of the best known and well understood reactions of yeast cells on increased extracellular osmolarity (1,5,30). It occurs in a two-step process: immediate closure of plasma membrane channels to ensure retention of glycerol whose production is thereafter increased in the second phase (31). In our work we focus on the importance of the short time regulation of glycolysis for the glycerol production compensating hyperosmotic stress. Yeast PFK2 catalyzes the synthesis of Fru-2,6-P 2 , which is  3. Fructose 2,6-bisphosphate concentration changes in the yeast strain DFY658 exposed to high osmolarity. Cells were exposed to hyperosmolarity with 1 M glucose (q) or 5% NaCl (E) for 30 min before determination of Fru-2,6-P 2 concentration.

6-Phosphofructo-2-kinase and HOG-MAPK Pathway
the most powerful activator of glycolysis (9). The monofunctional PFK2 can be either activated or inactivated by in vivo phosphorylation (7,11). We have shown earlier that phosphorylation at Ser 652 by PKC under hyposmotic stress inactivates PFK2 (11). On the other hand, glucose induction of yeast cells activates PFK2 by a single phosphorylation at Ser 644 and a multiple phosphorylation within the peptide T 67-101 (10). In this work, yeast cells were exposed to high osmolarity by adding either NaCl or glucose to the medium. In both cases PFK2 was phosphorylated and in consequence activated. The activation observed after glucose was more pronounced (five times) than after NaCl (three times). The activation of PFK2 resulted in an increase of the Fru-2,6-P 2 concentration in both cases. As a consequence of the activation of glycolysis the glycerol concentration increases, whereas the G-6-P concentration decreases. Yeast cells expressing PFK2 were able to increase the glycerol concentration rapidly up to 5-fold in reaction to the external osmotic stimulus. In contrast, cells lacking PFK2 were unable to produce enough glycerol to counteract the osmotic challenge. This led to a slow cell growth. The same effect was observed by others (3) with yeast strains carrying deletions of the yeast homolog of the mammalian MAPK p38 (HOG1) or the MAPK kinase PBS2. These cells produce less glycerol and do not grow at high osmolarity (3). For glycerol synthesis during hyperosmotic stress the overexpression of the glycerol-3phosphate dehydrogenase gene GPD1 is known to play an important role (5). Glycerol is produced from the glycolytic intermediate dihydroxyacetone phosphate in two steps catalyzed by NAD ϩ -dependent glycerol-3-phosphate dehydrogenase and glycerol-3-phosphatase. This response is supported by a stimulation of glycolysis through activation of the PFK2 to reinforce the synthesis of dihydroxyacetone phosphate. Although the function of the glycerol-3-phosphate dehydrogenase is intact and the glycerol facilitator Fssp, which is responsible for glycerol uptake from the medium is present, the yeast mutant lacking PFK2 was found incapable of glycerol accumulation and did not grow in hyperosmotic medium. This finding supports the importance of PFK2 and the upper part of glycolysis for glycerol accumulation.
Yeast strains carrying mutations in key enzymes of HOG-MAPK pathway showed different reactions to hyperosmolarity. In the case of hyperosmotic NaCl the PFK2 from HOG-MAPK mutant strains was not activated. This proves that the phosphorylation and activation of PFK2 under high osmolarity with NaCl is mediated by protein kinases from the HOG-MAPK pathway. In the presence of hyperosmolar glucose a clear increase of the activity was measured. However, the cells do not grow at high osmolarity (14). This shows that PFK2 activation alone without an intact HOG-MAPK pathway and without increased expression of glycerol-3-phosphate dehydrogenase is not sufficient to balance the increased osmotic pressure.
Characterization of the in Vivo Phosphorylation of PFK2-The determination of protein phosphorylation sites is an essential step in the analysis of protein kinase-mediated signaling pathways. While a direct determination of individual phosphorylation sites of phosphoproteins in vivo has been difficult up to now, the combination of MALDI-TOF MS, radioactive labeling, and site-directed mutagenesis represents a reliable strategy for the identification of protein phosphorylation sites. MALDI-TOF MS analysis of the tryptic digest of PFK2 purified from yeast cells exposed to hypertonic stress showed a 4-fold (NaCl) or 6-fold (glucose) phosphorylation of the enzyme. Glucose causes a single phosphorylation of the Ser 644 on the peptide fragment T 642-654 and a 5-fold phosphorylation of the peptide T 67-101 . This peptide is 4-fold phosphorylated after hyperosmolar NaCl. An examination of the primary structure of PFK2 shows that the peptide T 67-101 comprises several potential phosphorylation sites with different consensus sequences. Ser 70 is a potential site for calmodulin-dependent protein kinase and PKA; both proteins are known to be involved in the regulation of glycolysis (9,(32)(33)(34). The yeast RcK2 protein kinase is a member of the family of calmodulin-dependent protein kinases. Its phosphorylation and activation through the HOG-MAPK pathway has been reported (35). This knowledge supports the assumption that PFK2 could be a substrate of RcK2. Ser 70 , Thr 72 , Ser 90 , and Ser 92 are surrounded by proline residues. This kind of consensus sequence (Pro-X-Ser/ Thr-Pro (36) or Pro-Leu-Ser/Thr-Pro (37)) is typical for MAPK. Thr 88 is a potential substrate for casein kinase II, which is involved in the salt tolerance in bakers' yeast (38).
It has been reported that PFK2 is activated by fermentable sugars via the RAS/cAMP pathway and that this activation is a result of the phosphorylation of the PFK2 by PKA and other protein kinases from the Ras/cAMP pathway (10). The phosphorylation pattern of PFK2 observed after hyperosmolar glucose is identical with the one we observed in earlier work after FIG. 5. Growth of yeast cells under hyperosmotic stress with 5% NaCl. q, DFY658 cells overexpressing PFK2 after transfer to hyperosmotic medium; E, DFY658 cells (lacking PFK2) after transfer to hypertonic medium.
FIG. 6. Effect of hyperosmolarity on PFK2 activity in mutant yeast strains deficient in key enzymes of the HOG-MAPK pathway. White bars, activity of the PFK2 in isosmotic medium; gray bars, activity of the PFK2 after exposing the mutants to high osmolarity with 1 M glucose; black bars, activity of the PFK2 after exposing the mutants to high osmolarity with 5% NaCl. Values represent the mean Ϯ S.D. of three independent experiments.

6-Phosphofructo-2-kinase and HOG-MAPK Pathway
induction of the Ras-cAMP pathway with 2% glucose (10). The results of the experiment with hyperosmolar glucose and the PKA deficient yeast strain (RS13-58A) support the notion that the 4-fold phosphorylation of the peptide fragment T 67-101 and in consequence the activation of the PFK2 are mediated by protein kinases from the HOG-MAPK pathway and not from Ras-cAMP pathway. Only the phosphorylation of the Ser 644 and the phosphorylation of one residue on the T 67-101 are presumably mediated by the Ras-cAMP pathway. In yeast cells a decrease in cAMP concentration was measured under hyper-

6-Phosphofructo-2-kinase and HOG-MAPK Pathway
tonic conditions induced by NaCl (39) leading to an inhibition of PKA (40). This may explain the missing phosphorylation of Ser 644 in PFK2. At the peptide T 67-101 Ser 70 is also a potential phosphorylation site of the PKA. The missing fifth phosphoryl-ation after hyperosmolar NaCl could also result from the inhibition of the PKA.
Depending on its concentration in the culture medium glucose can activate different pathways (41,42). First, the glucose

6-Phosphofructo-2-kinase and HOG-MAPK Pathway
induces the activation of the PKA through the membrane receptor Gpr1 of the Ras-cAMP pathway and adenylate cyclase. The activated PKA can then phosphorylate PFK2 at Ser 644 and probably at Ser 70 . Second, glucose at high concentration activates the HOG-MAPK pathway and leads to the phosphorylation of the PFK2 by protein kinases of this pathway.