Osmotic stress induces specific cellular responses that include
changes in the activity of solute transporters (1, 2) and enzymes involved in solute
accumulation(3, 4) , the expression of genes encoding
enzymes required for solute
synthesis(5, 6, 7, 8) , stress
resistance(5, 9) , and cell wall
structure(10) . Despite their importance for cell growth and
survival, the signaling mechanisms responsible for mediating osmotic
stress-specific responses are not nearly as well understood as those
which mediate responses to ligands such as growth factors or hormones.
Our understanding of how eukaryotic cells sense and respond to changes
in osmolarity has been helped recently by studies of this problem in
the budding yeast Saccharomyces cerevisiae.
In yeast, a
protein kinase cascade called the HOG (
)pathway (11) plays a central role in mediating cellular responses to an
increase in external osmolarity. This pathway is defined by the HOG1(11) and PBS2(11, 12, 13) genes encoding members of
the MAPK (mitogen-activated protein kinase) and MAPKK (MAP kinase
kinase) family, respectively(14, 15) . Addition of
NaCl or sorbitol to increase the osmolarity of the medium induces yeast
to accumulate glycerol (6) and thereby restore the osmotic
gradient across the cell membrane. This response, which involves
increased expression of the glycerol-3-phosphate dehydrogenase gene GPD1(5, 6, 7) , is blocked in a hog1
mutant(7) . Other responses to an increase
in osmolarity such as reorientation of cell growth and division (16) and induction of gene expression (17) are also
defective in hog1
and pbs2
mutants. HOG
pathway activation involves increased phosphorylation of a Hog1p
tyrosine residue conserved among all MAP kinases which is required for
growth at high osmolarity(17) . Mammalian cells contain
structural and functional homologs of Hog1p, suggesting that the HOG
pathway is conserved among
eukaryotes(18, 19, 20, 21) .
In
its natural environment, yeast cells are exposed to not only increases
but also decreases in osmolarity. Although the HOG pathway has a clear
role in mediating cell responses to increases in osmolarity, little is
known about how yeast sense and respond to decreases in osmolarity.
Yeast cells contain four known MAP kinase
cascades(22, 23, 24) . One of these, referred
to here as the PKC1 pathway, is mediated by a protein kinase C-like
protein encoded by the gene PKC1(25) . Other protein
kinases on the PKC1 pathway have been identified using different
genetic approaches and placed into a linear pathway that proceeds
downward from Pkc1p to MAPKKK (called Bck1p or Slk1p) (26, 27, 28) to two MAPKK (Mkk1p and Mkk2p) (29) to MAPK (called Mpk1p or Slt2p)(30, 31) .
A comparison of deletion mutants in the HOG pathway to those in the
PKC1 pathway reveal opposite phenotypes. For example, a hog1
MAPK mutant grows in low but not high osmolarity
medium while a mpk1
MAPK mutant grows in high but not low
osmolarity medium(30) , a phenotype exacerbated by growth at
elevated temperature, i.e. 37 °C. Mutants in other genes
of the PKC1 pathway show a phenotype similar to that of mpk1
(26, 29, 31, 32, 33) .
Although there are other possible explanations, this observation is
consistent with a model in which the PKC1 pathway, like the HOG
pathway, mediates an osmotic signal and induces cellular responses
required for growth at the new (lower) osmolarity. At the time this
work was initiated there was no known activating signal for this kinase
cascade. In this report we test the hypothesis that the PKC1 kinase
cascade is an osmosensing signal transduction pathway which responds to
hypotonic shock as an activating signal.
EXPERIMENTAL PROCEDURES
Materials
The yeast strain used for most
experiments was YPH102 (MATaura3 leu2 his3 ade2
lys2)(34) , into which different plasmids were introduced
by LiAc-based transformation (35) with selection on
uracil-deficient medium for URA3 carried on the plasmid. Other
strains used in experiments are described in the legends of the figures
in which they were used. Plasmids and PKC1 pathway mutant strains were
obtained from Michael Snyder (Yale), Kunihiro Matsumoto (Nagoya
University), and Carl Mann (Centre d'Etudes de Saclay,
Gif-sur-Yvette). The MPK1-hemagglutinin (HA) plasmid (36) rescued the mpk1
mutant phenotype of reduced
growth in low osmolarity medium indicating that addition of the HA
epitope did not interfere with the normal function of the Mpk1p. A
similar plasmid carrying a mutation which codes for a substitution of
phenylalanine for the conserved tyrosine in the MPK1 gene (pMPK1-HA
Y192F) (36) did not rescue the mpk1
phenotype and
was deleterious to a wild-type strain transformed with the plasmid
(data not shown).
Growth Conditions
Cultures of plasmid-bearing
yeast were grown overnight in uracil-deficient medium and then grown to
log phase on the day of the experiment in YEPD (2% Bacto-peptone, 2%
glucose, 1% yeast extract), 20% YEPD (0.4% Bacto-peptone, 0.4% glucose,
0.2% yeast extract), or 20% YEPD containing additional solute (NaCl,
sorbitol, or glucose) to raise the osmolarity. In some experiments, the
osmolarity was increased by the addition of a concentrated solution of
solute or decreased by addition of either water or conditioned medium
with a lower osmolarity. For the latter type of experiment, two log
phase cultures were grown to the same cell density, one grown in 20%
YEPD, the other in 20% YEPD plus 1 M sorbitol. Cells were
removed from the former culture by centrifugation and the resulting
supernatant added to the latter culture to lower osmolarity without
changing the levels of nutrients or other components in the growth
medium. As a control for the effects of dilution, conditioned medium
from a culture grown in 20% YEPD + 1 M sorbitol was added
to a 20% YEPD + 1 M sorbitol culture.
Preparation of Cell Extracts and Immunoblot
Analysis
Activation of the PKC1 pathway was detected using a
previously described procedure for immunoblot analysis of MAPK (Mpk1p)
tyrosine phosphorylation(37) . Briefly, after different
experimental manipulations to change the external osmolarity, yeast
cell cultures were quickly chilled, and cells were collected by rapid
centrifugation. Ice-cold buffer containing protease and phosphatase
inhibitors (50 mM Tris HCl, pH 7.5, 1% sodium deoxycholate, 1%
Triton X-100, 0.1% sodium dodecyl sulfate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.1 mM sodium vanadate, 0.05%
phenylmethylsulfonyl fluoride, 0.05 µg/µl aprotinin, 0.01
µg/µl leupeptin, and 0.01 µg/µl pepstatin) was added to
the cell pellet and the cells lysed using glass bead agitation in a
mini-beadbeater. The resulting homogenate was centrifuged for 15 min in
a microcentrifuge, and proteins from the high speed supernatant were
resolved by SDS-polyacrylamide gel electrophoresis, loading the same
amount of protein (usually 20 µg) for each sample. Protein
concentrations were measured by the Bradford (38) method with
bovine serum albumin as a standard. After transfer of proteins to
nitrocellulose (0.2 µm, Schleicher & Schuell) using a semidry
blotting apparatus (Bio-Rad), tyrosine phosphorylation of different
yeast proteins was detected by incubation of the membrane blot with an
anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology, Inc.),
followed by an alkaline phosphatase-conjugated goat anti-mouse IgG
antibody (Promega), and visualization of immune complexes with the
chromogenic alkaline phosphatase substrate 5-bromo-4-chloro-3-indoyl
phosphate/nitro blue tetrazolium. To detect the Mpk1p MAPK containing
the HA epitope(39) , immunoblots were probed with the 12CA5
anti-HA monoclonal antibody (Babco or Boehringer Mannheim) and then a
horseradish peroxidase-conjugated sheep anti-mouse IgG antibody
(Amersham Corp.). Immune complexes were detected using an enhanced
chemiluminescence procedure for detecting peroxidase activity (Amersham
Corp.).
RESULTS
Hypotonic Shock Induces the Tyrosine Phosphorylation of
Mpk1p
Specific extracellular signals activate MAP kinases by
inducing their phosphorylation on a single conserved threonine and a
nearby tyrosine(40, 41) . For Hog1p and the PKC1
pathway MAPK Mpk1p, like other MAP kinases, phosphorylation of this
conserved tyrosine residue is required for pathway function because
substitution of the non-phosphorylatable residue phenylalanine for the
conserved tyrosine blocks pathway-specific
responses(17, 31) . Therefore, to determine whether
hypotonic shock activates the PKC1 pathway, we exposed the yeast strain
YPH102 and YPH102 mpk1::HIS3 (mpk1
) to decreases in
external osmolarity and then assayed cell proteins for tyrosine
phosphorylation by immunoblot analysis with an antibody to
phosphotyrosine. One minute after the osmolarity of the medium was
lowered by reducing the concentration of sorbitol from 1 M to
0.2 M, an increase in tyrosine phosphorylation of a single
band with an apparent size of 68 kDa was observed in the wild type but
not in the mpk1
strain ( Fig. 1(left)). A
gene fusion coding for Mpk1p tagged at the COOH terminus with a HA
epitope (38) was introduced on a high copy 2µ plasmid into
an mpk1
strain. Decreasing the osmolarity stimulated the
tyrosine phosphorylation of a band that migrated more slowly than that
in the wild type strain (YPH102). In the mpk1
strain
transformed with a plasmid identical to the MPK1-HA-containing
plasmid with the exception of a point mutation in MPK1 which
substitutes a phenylalanine for the conserved tyrosine, no band is seen
in the anti-phosphotyrosine immunoblot. An immunoblot of the same cell
extracts with an anti-HA epitope antibody ( Fig. 1(right)) revealed an immunoreactive protein found
in the MPK1-HA and mutant MPK1-HA containing strains,
which had the same mobility as the tyrosine phosphorylated band. We
interpret these observations to mean that the PKC1 pathway MAPK Mpk1p
is tyrosine phosphorylated in response to hypotonic shock.
Figure 1:
Hypotonic shock-induced tyrosine
phosphorylation of the PKC1 pathway MAP kinase Mpk1p. Left,
anti-phosphotyrosine immunoblot analysis of phosphorylation in response
to hypotonic shock. Cells were exposed for 1 min to either no change in
osmolarity (Iso) or a decrease in osmolarity (Hypo)
before rapid cooling and preparation of cell extracts (see
``Experimental Procedures''). The four strains tested were
YPH102 with the control 2µ plasmid pRS426 (34) (lanes 1 and 2), YPH102 mpk::HIS3 (mpk1
) with the
control plasmid pRS426 (lanes 3 and 4), pMPK1-HA (lanes 5 and 6), and pMPK1-HA Y192F (lanes 7 and 8). Right, anti-HA immunoblot analysis of
Mpk1p-HA in cell extracts. Shown are the immunoblots of samples
identical to those in lanes
5-8.
Time Course of Mpk1p Phosphorylation
MAP kinase
pathways induce rapid (<1 min) changes in MAPK tyrosine
phosphorylation in response to specific signals. As shown in Fig. 2, lowering external osmolarity induced an increase in
Mpk1p tyrosine phosphorylation that occurred within 15 s of the
stimulus and persisted for 10-15 min. After 30 min when the
tyrosine phosphorylation of Mpk1p had dropped to nearly prestimulus
level, a further reduction in the osmolarity of the medium caused by
addition of water induced a second rapid increase in Mpk1p tyrosine
phosphorylation. Because cells containing a single chromosomal copy of
MPK1 produce a small amount of Mpk1p which makes detection of
phosphotyrosine difficult, this and following experiments were carried
out with cells containing multiple copies of MPK1-HA.
Figure 2:
Transient induction of Mpk1p
phosphorylation by a hypotonic shock. Anti-phosphotyrosine immunoblot
analysis of Mpk1p phosphorylation at different times after hypotonic
shock of YPH102 containing the 2µ MPK1-HA plasmid. Aliquots of a
single culture were withdrawn before (0) and at the indicated
times following a change in the sorbitol concentration of the medium
from 1 M to 0.2 M (see ``Experimental
Procedures'') and 1 min after a final dilution of the medium in a
ratio of 1 part culture to 3 parts water (30` + 1`). These aliquots were rapidly cooled, and cell extracts
were prepared and assayed as described under ``Experimental
Procedures.''
Osmotic Dependence of MAP Kinase
Phosphorylation
To extend and confirm the observation that Mpk1p
and Hog1p respond in opposite fashion to osmotic changes, the in
vivo tyrosine phosphorylation of both kinases was measured after
changing the external osmolarity to a range of higher and lower levels.
Specifically, a culture was grown to log phase in medium containing 1 M sorbitol, and then the osmolarity decreased or increased by
addition of water with varying concentrations of sorbitol. Cells were
collected 1 min after the osmotic change and MAP kinase tyrosine
phosphorylation measured as before using an immunoblot procedure.
Compared to the control cells (Fig. 3, marked by an asterisk (*)) where external osmolarity was unchanged, decreasing
osmolarity induced an increase in Mpk1p phosphorylation that was
proportional to the magnitude of the osmotic shock. Increasing
osmolarity induced an increase in tyrosine phosphorylation of a band
that we has the same mobility relative to molecular weight standards as
that which was previously identified as Hog1p and, as expected from
previous results, was absent in hog1
cells (not shown).
We noted that it was easier to detect both basal and high
osmolarity-induced increases in tyrosine phosphorylation in Hog1p in
cells containing a high copy Mpk1p (or Mpk1p-HA) plasmid, although
anti-Hog1p immunoblot analysis (17) showed that the amount of
Hog1p was unchanged under these different conditions (data not shown).
This Mpk1p overexpression-induced increase in the amount of tyrosine
phosphorylated Hog1p is blocked in mutants lacking protein kinases
upstream of Mpk1p on the PKC pathway (see below). The physiological
significance and explanation of this phenomenon is unknown.
Figure 3:
Osmotic dependence of Mpk1p and Hog1p
phosphorylation. Strain used was YPH102 containing the 2µ MPK1-HA
plasmid. Time of incubation in different osmotic medium before chilling
cells and extract preparation was 1 min. Fraction of initial osmolarity
refers to the ratio of final sorbitol concentration to that present in
the initial culture medium (20% YEPD plus 1 M sorbitol).
Osmolarity was changed by diluting 10 ml of cell culture into 40 ml of
water containing different concentrations of
sorbitol.
Solute Independence of the Hypotonic Response
In
experiments decribed above, external osmolarity was changed by altering
the concentration of sorbitol. To determine if the phosphorylation of
Mpk1p was due to osmotic changes or a sorbitol specific response, we
tested whether changes in the concentration of other solutes, namely
glucose or NaCl, would also activate Mpk1p phosphorylation. As shown in Fig. 4, cells were grown to log phase in 20% YEPD plus 1 M glucose (or 0.5 M NaCl) and then shifted to the medium
with the same (Iso), lower (Hypo), or higher (Hyper) concentration of glucose (or NaCl). In both cases,
Mpk1p and Hog1p were tyrosine-phosphorylated in response to hypotonic
(Hypo) and hypertonic (Hyper) shock, respectively. Therefore, the Mpk1p
phosphorylation responses are independent of the varied solute.
Figure 4:
Solute-independent osmotic shock-induced
MAP kinase phosphorylation. Same strain and incubation time as in Fig. 3. Cultures were grown in 20% YEPD with 0.95 M glucose for 3 h and then diluted 1:4 with: H
O, 1 M glucose, or 2 M glucose. The experiment was then repeated
substituting NaCl for glucose at half the stated molarities (roughly
the same osmolarity).
Osmotic Regulation of Mpk1p Phosphorylation Involves
Upstream Kinases in the PKC Pathway
To determine whether Mpk1p
phosphorylation by hypotonic shock is mediated through the PKC1
pathway, we measured this response in different mutant strains. As
shown in Fig. 5(top), hypotonic shock-induced tyrosine
phosphorylation of Mpk1p-HA was not detectable in strains containing
deletions in the genes that encode protein kinases upstream of Mpk1p on
the PKC1 pathway. These include a protein kinase C mutant (pkc1
)(25) , a MAPKKK mutant (bck1
)(27) , and a MAPKK mutant (mkk1
mkk2
)(29) . The failure to
detect Mpk1p-HA tyrosine phosphorylation could be explained by PKC1
pathway-dependent expression of Mpk1p-HA. However, immunoblot analysis
with the anti-HA antibody (Fig. 5, bottom) showed that
the amount of Mpk1p-HA was independent of the upstream kinases in the
PKC1 pathway.
Figure 5:
Hypotonic shock-induced Mpk1p
phosphorylation in PKC1 pathway mutants. Strains containing the
indicated deletion mutations plus a high copy 2µ MPK1-HA
plasmid were grown to log phase in 20% YEPD plus 1 M sorbitol.
These cultures were then diluted 1:34 with water and the cells
collected 1 min later for preparation of cell extracts and immunoblot
analysis of tyrosine phosphorylation (top) and Mpk1-HAp (bottom). Strains were the following: wild-type (YPH102) (34) , mkk1,2
(3233-1B)(29) , bck1
(Y782) (27) , and pkc1
(DL376)(33) .
Tyrosine phosphorylation of Mpk1p was inhibited in
cells exposed to an increase in osmolarity of the medium while Hog1p
tyrosine phosphorylation increased (Fig. 6). To test whether
these responses involved the PKC1 pathway, this experiment was repeated
using a BCK1-20 mutant which is reported to encode a
constitutively active form of the Bck1p protein kinase(26) .
This strain no longer shows the high osmolarity-induced decrease in
Mpk1p phosphorylation. Note that the high osmolarity-induced increase
in Hog1p phosphorylation was relatively unaffected by this mutation.
Figure 6:
Hypertonic shock induces a BCK1-dependent
decrease in Mpk1p phosphorylation. YPH102 strains containing either a
high copy 2µ MPK1 plasmid (left) or a low copy
CEN BCK1-20 plasmid (26) (right) were grown
separately to log phase in YEPD. The cultures were then split, and 0.4 M NaCl was added to one of the two cultures. Cells were then
collected after 1 and 10 min, and protein tyrosine phosphorylation was
analyzed by immunoblot.
DISCUSSION
Osmosensing MAP Kinase Pathways in Yeast
Our
results show that there are two osmosensing signal transduction
pathways in yeast, each containing structurally similar protein
kinases(22, 24) . The symmetry between the pathways is
striking. The HOG pathway genes HOG1 and PBS2 are
required for cell growth at high osmolarity and high osmolarity rapidly
induces a transient, PBS2-dependent hyperphosphorylation of
the Hog1p MAP kinase(11) . The PKC1 pathway genes PKC1, BCK1 (SLK1), MKK1/MKK2, and MPK1 (SLT2) are required for cell growth at low
osmolarity(26, 29, 30, 31, 32, 33) ,
and low osmolarity rapidly induces a transient, MKK1/MKK2-dependent hyperphosphorylation of the Mpk1p MAP
kinase (this study). Mpk1p kinase activity is rapidly elevated in cells
exposed to a hypotonic shock (36) . The HOG pathway has a
fairly well defined role in the cellular response to an increase in
osmolarity. Based on the phenotypes of hog1
and pbs2
mutants, the HOG pathway is required for high
osmolarity-stimulated transcription of specific genes (7, 17) leading to increased synthesis of the
principal osmolyte glycerol (11) and general stress
resistance(17) . The PKC1 pathway is required for constructing
a cell wall. Cells without PKC1 die by cell
lysis(32, 33) . Deletion mutations in BCK1 (SLK1), MKK1/MKK2, or MPK1 (SLT2) have similar phenotypes: cell lysis that is
accentuated by growth at higher temperatures. This
temperature-sensitive cell lysis phenotype is suppressed by growth on
high osmolarity medium and is correlated with a decrease in glucan
content of the cell wall(10, 42) . The mechanism
responsible for the weakened cell walls in PKC1 pathway mutants is not
known with any certainty but may involve defects in polarized vesicle
secretion/cell growth (43) or changes in glucan
content(10) . Therefore, one possible role of the PKC1 pathway
is to regulate cell wall properties in response to changes in external
osmolarity. This type of physiological response has been observed in
fungi. The constant growth rate of the fungus Achyla bisexualis in medium of different osmolarity is correlated with changes in
the mechanical properties of their cell wall with a stronger wall at
low osmolarity than at high osmolarity(44) .The complex
phenotype of PKC1 pathway mutants suggests that this pathway responds
to physiological signals beside changes in external osmolarity. Besides
the sensitivity to low osmolarity, such mutants are altered in cell
morphogenesis(27, 45) . Mutants lacking the PKC1
pathway MAP kinase kinase kinase BCK1 (SLK1) are
sensitive to starvation with defects indicative of a failure to exit
the vegetative growth cycle(27) . Compared to wild-type (BCK1
(SLK1
))
cells, bck1
(slk1
) mutants do not
accumulate glycogen, fail to undergo meiosis, are heat shock-sensitive,
continue to form buds in stationary phase cultures, and lose viability
in nutrient-poor medium(45) . These phenotypes are independent
of the osmolarity of the medium(45) . These defects in growth
control suggest that the PKC1 pathway has a role in nutrient sensing.
The PKC1 pathway is required for growth at elevated temperature and the
Mpk1p kinase activity is strongly activated by exposure of cells to
higher temperature(36) . How functions of the PKC1 pathway such
as osmosensing, temperature-sensing, and nutrient sensing are
coordinated with each other remains to be determined.
An important
aspect of the two yeast osmosensing MAP kinase pathways is that similar
pathways appear to exist in cells from other eukaryotes including
mammals. In the case of the HOG pathway, Hog1p shows a high degree of
similarity in amino acid sequence to a subgroup of MAP kinases, several
members of which show increased tyrosine phosphorylation in cells
exposed to an increase in osmolarity. Two mammalian members of this
subgroup, p38 (18) and JNK1(19) , have been
expressed in a yeast hog1
strain and shown to complement
the high osmolarity-sensitive growth phenotype of this mutant. The PKC1
pathway Mpk1p is closely related in amino acid sequence to a second
subgroup of MAP kinases that includes the mammalian ERK2 (p44
) and ERK1 (p42
)(15) . Strikingly, studies in a human
intestinal cell line show that both of these MAP kinases show increased
tyrosine phosphorylation after exposure of cells to a decrease in
external osmolarity(46) . The low osmolarity-sensitive growth
phenotype of an mpk1
mutant and a bck1
(slk1
) mutant are complemented by expression in yeast of
a Xenopus ERK2 MAP kinase and a mammalian MAP kinase kinase
kinase (MEKK), respectively(31, 47) . Although studies
of osmosensing pathways in mammals and yeast are just beginning, these
similarities encourage the idea that other functions of such pathways (48, 49) will also be conserved.
Cross-regulation between Osmosensing Pathways
The
presence in a single cell of two different signaling pathways (HOG and
PKC1) that are regulated in opposite directions by changes in external
osmolarity raises several questions. For example, do these pathways
regulate each other? We have used a genetic approach to address this
question and found no evidence of synergy or suppression in the growth
phenotype of double mutants nor was the growth phenotype of mutants in
one pathway affected by overexpression of a protein kinase in the other
pathway (data not shown). These data suggest that the two osmosensing
pathways act independently of the other in supporting growth at
different osmolarity. In wild-type cells with an intact PKC1 pathway (Fig. 5, left two lanes), decreasing external
osmolarity caused a decrease in Hog1p tyrosine phosphorylation. PKC1
pathway mutants started out with a lower basal level of tyrosine
phosphorylation of Hog1p. For each of the mutants examined, this amount
of Hog1p phosphorylation did not change after a decrease in the
osmolarity of the medium. These data suggest that the PKC1 pathway
affects signaling through the HOG pathway when cells are exposed to a
decrease in external osmolarity. The physiological significance of this
apparent cross-talk between osmosensing pathways will require more
information about the currently unknown targets of the PKC1 pathway.