From the Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel
Received for publication, February 28, 2001, and in revised form, April 13, 2001
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ABSTRACT |
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Mitogen-activated protein kinases (MAPKs) play
pivotal roles in growth, development, differentiation, and apoptosis.
The exact role of a given MAPK in these processes is not fully
understood. This question could be addressed using active forms of
these enzymes that are independent of external stimulation and upstream
regulation. Yet, such molecules are not available. MAPK activation
requires dual phosphorylation, on neighboring Tyr and Thr residues,
catalyzed by MAPK kinases (MAPKKs). It is not known how to force
MAPK activation independent of MAPKK phosphorylation. Here we describe
a series of nine hyperactive (catalytically and biologically),
MAPKK-independent variants of the MAPK Hog1. Each of the active
molecules contains just a single point mutation. Six mutations are in
the conserved L16 domain of the protein. The active Hog1 mutants were
obtained through a novel genetic screen that could be applied for
isolation of active MAPKs of other families. Equivalent mutations,
introduced to the human p38 MAPK1 is a generic term
for a large family of enzymes, which function in a variety of signal
transduction pathways. Mammalian MAPKs are divided into at least three
subfamilies (ERKs, p38s, and JNKs) based on degree of homology,
biological activities, and phosphorylation motif (1-6). Although
highly homologous in structure and in pattern of activation, each MAPK
is activated in response to a specific battery of signals and in turn
phosphorylates a particular array of substrates. As a result, each MAPK
imposes specific effects on the cell. For example, in many cell lines (e.g. fibroblasts), the ERK MAPKs are activated when cells
are exposed to growth factors, and their activation is important for enhancement of cell proliferation (7, 8). In other cells (neuronal and
myogenic cell lines), however, activation of ERKs is associated with
growth arrest and differentiation (7, 9). In contrast to the ERK
enzymes, the activity of p38 and JNK MAPKs is only slightly induced by
growth factors. These enzymes are strongly activated in response to
stress signals such as heat shock, osmotic shock, UV radiation,
cytokines, and metabolic inhibitors. JNK and p38 MAPKs seem to be
responsible mainly for protective responses,
stress-dependent apoptosis, and inflammation (3, 10). In
some cell types, however, p38 and JNK may play a role in
differentiation and development (3, 10, 11). Studies with knockout mice
and knockout cell lines revealed essential roles for MAPKs in various
aspects of embryonal development (12-17).
Although many aspects of MAPK biology have been revealed, the exact
role of each MAPK in a given biological system is not fully understood
and is difficult to study. The main reason for this difficulty is our
inability to activate a given MAPK in vivo and to follow the
biochemical and physiological consequences. Currently, a MAPK is
experimentally activated in vivo using extracellular stimuli
or through expression of an active form of a component that functions
upstream to that MAPK (7, 8). Each of these treatments activates more
than one MAPK and evokes many cellular responses. Activation of a given
MAPK per se could be obtained theoretically by expressing an
active form of this kinase, which would be active independently of
external signals and upstream components. However, the catalytic
activity of MAPKs is tightly regulated and strictly dependent on
upstream activation (2, 18, 19). Although the mechanisms of MAPK
activation have been revealed (2, 20, 21; see below), this knowledge
could not be applied for the production of active forms of MAPKs. Here
we describe a novel genetic screening system, which produces and
isolates such active kinases.
Following exposure of cells to an extracellular ligand, the activity of
the relevant MAPK increases ~1000-fold (18). This activation is
mediated through a complex signal transduction net that culminates in
phosphorylation of the MAPK by a MAPK kinase (MAPKK). MAPKKs are dual
specificity kinases that phosphorylate MAPKs on particular Thr and Tyr
residues. This dual phosphorylation is the basis for the dramatic
increase in MAPK activity. MAPKs mutated in either the Thr or Tyr
phosphoacceptors cannot be activated (18, 22). The unusual mode of MAPK
activation (through dual phosphorylation) underlies the difficulties in
producing active forms of these enzymes, because a
PO4-Thr-X-PO4-Tyr structure is
difficult or impossible to mimic by mutagenesis (18).
Comparison of the crystal structure of ERK2 with that of dually
phosphorylated ERK2 (21) shows that phosphorylation of the activation
loop induces conformational changes in both the activation loop itself
and in another domain at the COOH-terminal extension (Pro309-Arg358) known as L16. These
conformational changes induce tight interactions between the
phosphorylation lip and the L16 domain. A recent report suggested that
these interactions create an interface for homodimerization of the
kinase molecules. The putative dimerization is stabilized by
hydrophobic contacts involving mainly leucine residues located at L16
of the two monomers and by an ion pair involving Phe329 and
Glu343 (located in L16) of one monomer and
His176 of the other monomer (20). It is not known
whether other MAPKs (e.g. JNKs and p38s) are dimerized
upon phosphorylation and activation. Although the crystal structures of
ERK2 and phospho-ERK2 revealed the conformational changes that activate
the enzyme, they did not suggest a strategy for producing a
constitutively active MAPK by mutagenesis.
We have devised a novel protocol for isolation of active forms of MAPKs
using a genetic screen in yeast. The yeast Saccharomyces cerevisiae possesses five different MAPKs, which are highly
homologous to their mammalian counterparts (1, 23). The yeast MAPKs Fus3, Kss1, and Mpk1 are close to the ERK subfamily. The yeast Hog1
MAPK has a QM*TG*YVSTR phosphorylation motif (almost identical to that
of p38) and is functionally replaced by either JNKs (24) or p38s (25).
Hog1 is phosphorylated and activated by the MAPKK Pbs2 (26), a
functional homolog of JNKK1/MKK4 (27). The Pbs2/Hog1 MAPK cascade is
essential for survival of yeast cells under high osmotic conditions.
Yeast cells lacking either PBS2 or HOG1 cannot grow on media supplemented with high concentrations of sugar or salt (26). Our effort to produce active forms of MAPKs took advantage
of the Pbs2/Hog1 pathway. We reasoned that it should be possible to
screen a library of randomly mutated HOG1 genes in cells
lacking Pbs2 activity. The premise is that a HOG1 clone that
allows pbs2 Previous efforts to obtain active forms of MAPKs were only partially
successful. Several gain-of-function mutations that were identified in
the S. cerevisiae FUS3 (28, 29) and in the Rolled MAPK of Drosophila melanogaster (30, 31) did not render the kinases catalytically hyperactive. The use of MAPKK-MAPK hybrids (32,
33) seems more useful. Yet, in the in vivo situation, MAPKKs
and MAPKs are not colocalized in the cell and are differently controlled; thus, the use of the chimeric proteins might be
problematic for physiological studies. Hence, despite efforts through
various approaches, active forms of MAPKs, which are independent of
MAPKK activity, are not available.
The approach taken in this study is most stringent, because it screens
for MAPKs that are active in the complete absence of their relevant
MAPKK. In addition, the design of the screen forces just minor changes
in the MAPK (point mutations). We describe the isolation of nine
different point mutations in the Hog1 kinase. Each mutation is
sufficient to render Hog1 catalytically and biologically active,
independent of upstream regulation. Remarkably, insertion of equivalent
mutations to the human p38 Yeast Strains and Media--
The S. cerevisiae
strains used in this study were the pbs2 Mutagenesis Procedures--
The library of HOG1
mutants was produced in bacterial strain LE30 according to Silhavy
et al. (34). A plasmid carrying the HOG1 and
URA3 genes (pRS426-HOG1 obtained from M. Gustin)
was introduced into LE30 cells. About 50,000 colonies were obtained and
allowed to grow on LB ampicillin plates for 24 h. All
colonies were collected in a pool into 1 liter of LB medium (containing ampicillin) and further grown for 12 h. Then, the culture was diluted 1:5 and further grown for 15 h prior to plasmid preparation.
Site-directed mutagenesis was performed by polymerase chain reaction or
with the QuickChange kit (Stratagene) according to the recommendations
of the manufacturer. The sequences of the primers used for
mutagenesis are listed in Table
I.
Screening of the HOG1 Mutant Library--
Transformation of the
library of HOG1 mutants into yeast was performed as
described by Schiestl and Gietz (35). Transformed cells were plated on
selective YNB-URA plates. Colonies that grew (about 10,000 per 100-mm
plate) were replica-plated onto YPD plates containing different
concentrations of NaCl (0.9, 1.1, and 1.3 M).
Plasmid loss assay, for positive colonies, was performed by
streaking patches of positive colonies on YPD plates, allowing the colonies to grow for 24 h, and isolating single
colonies. These single colonies were replica-plated onto YNB-URA plates as well as onto NaCl-containing YPD plates.
RNA Preparation and Analysis--
Cells were grown in 100 ml of
YNB-URA to an A600 of 0.5-0.9, split, and
collected by centrifugation. Half of the cells were induced for 90 min
with YNB-URA containing 1 M NaCl, whereas the other half of
the cells were resuspended in YNB-URA. After induction, cells were
centrifuged and frozen in liquid nitrogen. RNA preparation, primer
extension analysis, and sequences of the primers were previously described (36, 37).
Preparation of Cell Lysates and Western Blot
Analysis--
50-ml cell cultures were grown to an
A600 of 0.5-1.0. Half of the culture was
induced with 1 M NaCl for 10 min, as described above.
Cultures were pelleted and resuspended in 8 ml of water and 10 ml of 20% trichloroacetic acid. After the samples were repelleted,
they were resuspended in 200 µl of 20% trichloroacetic acid
at room temperature, and 650-mg of glass beads were added. Each sample
was vortexed twice for 4 min each time. Supernatants were
transferred to new Eppendorf tubes, and glass beads were rinsed twice
with 200 µl of 5% trichloroacetic acid (the final concentration of
trichloroacetic acid was 10%). Following centrifugation, pellets were
resuspended in 200 µl of 2× Laemmli sample buffer followed by
addition of 100 µl of 1 M Tris base. Samples were vortexed for 30 s and boiled for 3 min prior to centrifugation. Supernatant was used.
The SDS-polyacrylamide gel electrophoresis, Western blot, and ECL
reaction for identification of the HA-Hog1 protein and the phosphorylated Hog1 were performed as described in Sambrook et al. (38). The antibody for the HA tag, 12CA5, is monoclonal. A
dilution of 1:1000 was used. For identification of the double phosphorylated Hog1, Preparation of Native Cell Lysates and in Vitro Kinase
Assay--
200-ml cell cultures were grown to an
A600 of 0.5-1.0. After a 10-min induction with
1 M NaCl, as described above, cells were centrifuged at
4 °C, and the pellet was washed with 50 ml of ice-cold water.
Following centrifugation the pellet was resuspended in 1-2 volumes of
ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 0.25 M NaCl, 0.1% Nonidet P-40, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml trypsin inhibitor, 10 µg/ml pepstatin A, 313 µg/ml
benzamidine, 1 mM sodium vanadate, 10 mM NaF, 1 mM p-nitrophenyl phosphate, 10 mM
For immunoprecipitation of the Hog1 protein, 40 µl of 50% protein A
were washed with ice-cold phosphate-buffered saline buffer and
preconjugated with 5 µl of 12CA5 antibody overnight at 4 °C. Following two washes with phosphate-buffered saline buffer and then one
with lysis buffer, the beads were resuspended in lysis buffer, and 300 µg of the lysate were added. Samples were incubated for 1 h at
4 °C. Then, three washes with lysis buffer followed by three washes
with kinase buffer (25 mM HEPES, pH 7.5, 20 mM MgCl2, 20 mM Expression, Purification, and Analysis of Human p38 Rationale and Design of the Genetic Screen for Active Forms of
Hog1--
Our screen for active forms of MAPKs takes advantage of the
pbs2 Isolation of Point Mutations in HOG1, Which Render It Independent
of Pbs2 Activation--
A library of HOG1 mutants was
produced (34, 41) and introduced into a pbs2
The 41 true positive clones were
sequenced. Each clone was found to contain a single point mutation in
the coding sequence of Hog1. Many of the clones harbor an identical
mutation (Table II), suggesting that the screen was saturated.
Altogether, nine different point mutations were identified (Table II;
Figs. 2A and 3). To show
unequivocally that each of these point mutations is sufficient to
render Hog1 independent of Pbs2, we introduced, through site-directed
mutagenesis, each mutation to a wild type HOG1 gene and
expressed the resulting mutants in pbs2
The results shown so far demonstrate that the HOG1 variants
isolated in the screen are independent of Pbs2. Because MAPKs may be
active as dimers (2, 20), the possibility remains that the mutants are
not exclusively independent but dimerize with the endogenous Hog1 that
is expressed in the pbs2
To verify that the active Hog1 mutants rescue pbs2 Six of the Activating Mutations Are Located in the Dimerization
Domain--
Fig. 3A shows the location of the mutations
within the HOG1 linear sequence. Strikingly, six of the nine
mutations are located in a short stretch of residues between amino
acids 314 and 332, which is part of the L16 domain. These mutated
residues appear to be in close proximity to (but not within) the
docking domain suggested recently by Nishida and co-workers (42). The
other two mutations are located at the NH2 terminus. One
mutation (D170A) is located just four amino acids from the
phosphoacceptor Thr174. The second mutation in the
NH2 terminus is Y68H. Alignment of the HOG1
sequence with the sequences of mammalian MAPKs (Fig. 3B)
reveals that all mutations but one, W320R, occur in residues that are
conserved in at least one subfamily.
Hog1 Variants Are Catalytically Active in Vivo in the Absence of
Salt Induction--
Having isolated Hog1 variants that function
biologically independently of Pbs2, we wished to reveal the biochemical
basis of their unusual property. The most attractive explanation would be that these molecules have acquired intrinsic catalytic activity. To
directly test its kinase activity, each mutant was expressed in
hog
In summary, all Hog1 mutants isolated in the screen are hyperactive
independently of any induction. Their specific activity is way above
the maximal activity of wild type Hog1, but the activity of some
mutants could be even further induced by salt via an unknown mechanism.
Insertion of Equivalent Mutations into the Human p38
Analyzing the results of the in vitro kinase assay (Fig.
5A), we noticed a phosphoprotein of 38 kDa. This
phosphoprotein appeared only when active p38 mutants were used (Fig.
5A). Because this protein is almost certainly an
autophosphorylated p38, we examined the autophosphorylation activity of
the mutant enzymes. We found that p38Phe327Leu and
p38Phe327Ser have acquired the intrinsic capability for
autophosphorylation (Fig. 5B). The autophosphorylation
activity of the mutants is directed mainly toward Thr180,
as determined by Western blot analysis (data not shown). Some phosphorylation on Tyr182 was detected on both wild type
and mutant recombinant p38. Thus, an increase in Thr180
autophosphorylation is most probably the mechanism responsible for the
independence of the active variants of upstream MAPKKs. Autophosphorylation activity of wild type p38 was barely detectable (Fig. 5B).
Most of the Active Hog1 Mutants Are Not Phosphorylated on
Thr174 and Tyr176 in pbs2
Although it seems that the mutants have acquired intrinsic activity, we
wished to rule out the possibility that the Hog1 mutants are active
in vivo because they acquired an affinity to another MAPKK,
which phosphorylates and activates them. Because at least five MAPK
cascades function in yeast (1, 23), there should be at least four
intact MAPKKs in the pbs2
Strikingly, mutants F318L and F318S expressed in hog1 The Activity of the Active Hog1 Variants Depends on
Thr174 but Not on Tyr176--
The Western blot
analysis strongly suggests that the active Hog1 mutants do not require
phosphorylation for their activity. Yet, because some studies suggested
that very low phosphorylation, which may not be detected in the Western
analysis, could induce significant activation (44), we decided to prove
this unequivocally. To this end we mutated in each of the active
variants the phosphoacceptors Thr174 and
Tyr176. We expected that all mutants (and in particular
F318S, W320R, and W332R, which are not phosphorylated at all in
pbs2
Unexpectedly, when we mutated Thr174 to Ala the mutants
lost their ability to rescue either hog1 Active Hog1 Molecules Reveal Novel Biological Effects of
Hog1--
Because the active Hog1 mutants were isolated as molecules
that allow pbs2 Although MAPKs are involved in pivotal biological processes and
are therefore extensively studied, it has been difficult to address the
exact role of a given MAPK in a particular biological system. Such a
question could be approached using active forms of MAPKs that were
hitherto not available. This study describes a novel genetic screen in
yeast that provides active MAPK molecules that function independently
of their MAPKK.
The basic rationale behind this screen is that only an active form of a
MAPK would induce the appropriate respective phenotype in a MAPKK null
strain. This rationale was applied here for isolation of active Hog1
molecules. The mutants isolated could execute all Hog1 biochemical and
biological activities independently of Pbs2 activity. Furthermore, the
active Hog1 variants dramatically affected the growth rate and other
properties of the cell, disclosing novel biological activities of Hog1.
Their ability to perform all these functions stems from the intrinsic
catalytic activity they acquired. The importance of the mutants for the
studies of MAPKs in general is manifested by the fact that insertion of
similar mutations into the human p38 Thus, the results with p38 Some of the mutants isolated (e.g. Hog1F318S and
Hog1F318L) are stronger than others and show very high
basal activity in vivo. Strikingly, the activity of other
mutants was further increased by salt. What mechanism could be
responsible for this increase in activity of some mutants when salt is
provided? It may be that activation of Hog1 requires the removal of an
inhibitory component, in addition to MAPKK-dependent
phosphorylation. In such a case, the salt-dependent increase in the activity of the mutants is a result of this molecular event. This possibility may be supported by studies that identified several inhibitors of JNK1, including JNK-interacting protein (45) and GST (46). It may be that the mechanism of action of some
active mutants involves their release from an inhibitory complex in
combination with an increase in their catalytic activity.
We thus believe that more than one mechanism underlies the activity of
the mutants. Some mutants may form spontaneous dimers, whereas others
may use another mechanism (escaping inhibition?). The differences in
the intrinsic catalytic activities of the various mutants support the
idea of different mechanisms. Clearly, many mechanistic studies are
required to fully explain the mechanism of action of each mutant. In
addition, structural studies, which are in progress, will reveal the
changes in protein folding induced by each mutation.
We may already speculate, however, that combining, on the same Hog1
molecule, a mutation that uses one mechanism with a mutation that uses
another may generate an enzyme molecule that is even more active
in vivo than the molecules described here. As already stated, our preliminary results in this direction support this notion.
Finally, the rationale that was successfully applied here for isolation
of active Hog1 molecules could be used to screen for other active
MAPKs. For example, active forms of Fus3 could be isolated in cells
lacking the MAPKK Ste7, using the mating phenotype as the biological
assay. Analogously, constitutively active forms of Kss1 could induce a
pseudohyphae phenotype in ste7, rendered the enzyme active even when
produced in Escherichia coli, showing that the mutations
increased the intrinsic catalytic activity of p38. It implies
that the activating mutations could be directly used for production of
active forms of MAPKs from yeasts to humans and could open the way to
revealing their biological functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
cells to grow on salt should encode an
active, MAPKK-independent Hog1 (see Fig. 1).
rendered this enzyme catalytically active also.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain
MAY1 (MATa, ura3-52,
lys2-801amber,
ade2-101ochre, leu2-
1,
his3-
200,
pbs2-
2::LEU2), the
hog1
strain JBY13 (MATa, leu2, ura3, his3, trp1,
ade2, lys2,
hog1::TRP1), and
hog1
pbs2
(JBY13, in which
the PBS2 gene was disrupted). The JBY13 and
MAY1 strains were obtained from M. Gustin (Rice University,
Houston, TX). The hog1
pbs2
strain was
produced in this study. Cultures were maintained on YPD (1% yeast
extract, 2% Bacto Peptone, 2% glucose) or on the synthetic
medium YNB-URA (0.17% yeast nitrogen base without amino acids
and NH4(SO4)2, 0.5%
ammonium sulfate, 2% glucose, and 40 mg/liter adenine, histidine,
tryptophan, lysine, leucine, and methionine). The ability of cells to
grow under osmotic shock was tested on YPD plates supplemented with
NaCl (salt concentrations are described for each experiment). To induce
osmotic shock in liquid media, cultures were grown to
logarithmic phase (A600 of 0.5-1.0) at
30 °C. Cells were split in half, collected by centrifugation, and
resuspended in the same media or in media containing 1 M NaCl. Cells were collected 10 min later.
Sequences of primers used in polymerase chain reaction and
site-directed mutagenesis reactions
-P-p38 antibodies (New England Biolabs) were
used (diluted 1:2,500). The secondary antibodies, anti-mouse and
anti-rat, respectively, were diluted 1:10,000.
-glycerol-P). 600 mg of glass beads were added, and the pellet was
vortexed eight times for 1 min each time. Samples were
centrifuged at 800 × g for 5 min, and the supernatant
was centrifuged again at 15,000 × g for 15 min at
4 °C. Samples were aliquoted to small volumes (~200 µl) and
frozen immediately in liquid nitrogen.
-glycerol-P, 10 mM
p-nitrophenyl phosphate, 0.5 mM sodium vanadate,
1 mM dithiothreitol) were performed. The kinase reaction
was performed by resuspending the beads in 30 µl of kinase buffer
containing 20 µM ATP, 5 µCi of
[
-32P]ATP, and 9 µg of GST-ATF2 as a substrate. The
experimental details on optimizing the conditions for the Hog1 in
vitro kinase assay, kinetic parameters, and affinities to
substrates are described elsewhere. The reaction took place at 30 °C
for 30 min and was stopped by transferring the samples to ice and
adding 10 µl of Laemmli loading buffer ×4 prior to boiling.
Kinase reactions were separated through SDS-polyacrylamide gel
electrophoresis. The gels were dried under vacuum, and exposed to x-ray film.
--
Wild
type and active mutants of p38
were expressed in Escherichia
coli using the pET15b expression vector (Novagen) so that all
proteins contained a polyhistidine tag in their NH2
terminus. Growth conditions, induction of expression, and protein
purification were performed according to Wang et al. (39).
Purified proteins were assayed as previously described (40) using
GST-ATF2 (20 µg) as a substrate.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain, which lacks the MAPKK Pbs2 and cannot grow
under high osmotic conditions. Overexpression of the MAPK Hog1 (the Pbs2 target) did not enable pbs2
cells to grow on
salt (see Fig. 2A; second row in each plate),
demonstrating the absolute dependence of Hog1 activity on Pbs2-mediated
phosphorylation. This result forms the basis for our premise that only
an active, independent form of Hog1 might enable pbs2
cells to grow on salt. The idea is therefore to produce a library of
HOG1 mutants, hoping that one mutant or a few mutants would
gain intrinsic catalytic activity, which is independent of Pbs2
activation. The rare active mutants will be identified by introducing
the library to pbs2
cells and selecting colonies on salt.
Each colony that grows should harbor an active, Pbs2-independent Hog1
kinase (Fig. 1).
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Fig. 1.
General scheme and rationale of the genetic
screen. The asterisk indicates a Pbs2-independent
active enzyme.
strain (26).
5 × 105 transformants were obtained and allowed to
grow on selective media (YNB with no uracil) with no salt. Then
colonies were replica-plated to plates supplemented with NaCl (0.9-1.3
M). 150 positive colonies (colonies that grew on salt
concentrations of 1.1 M NaCl or higher) were collected. The
linkage between growth on salt and the library plasmid was verified
through a plasmid loss assay and then by retransfection (following
purification from yeast) to pbs2
cells. 41 clones passed
these tests (data not shown) and were considered true positives
(Table II). Fig.
2A shows that the Hog1
mutants isolated, but not wild type Hog1, enable pbs2
cells to grow on salt.
Screen results
cells. All nine
mutations allowed growth on salt, showing that indeed each point
mutation is sufficient to make Hog1 independent of Pbs2.
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Fig. 2.
HOG1 active mutants, but not
HOG1 wild type, are able to rescue pbs2
cells and hog1
pbs2
cells from
high salt concentrations. A, Two rows of plates are
shown. On the left-hand side of each row is a master plate
(not containing salt), and the other two plates are replicas
(containing salt). Each plate is divided into two parts; the
right side contains hog1
cells (used as
controls (22)), and the left side contains
pbs2
cells (26). Note that replica plates are
supplemented with two different concentrations of NaCl. PBS2
and vector, written in white on the plates,
denote the plasmid identity in the cells of the upper
row. In all other rows, the same plasmids (wild type
HOG1 or mutants) are expressed in both pbs2
and hog1
cells. B, the upper panel
shows the master plate on the left. Plasmids are
indicated in the scheme on the right; the lower
panel shows replica plating on two different salt
concentrations.
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Fig. 3.
Many of the Hog1-activating mutations
occurred in amino acids that are conserved in other MAPKs.
A, Hog1 protein sequence. Mutation sites are shown in color.
B, sequence alignment of yeast and mammalian MAPKs, showing
mutation sites in blue. Phosphoacceptors Thr and Tyr are
shown in red. Note that numbers in B do not
reflect actual locations of amino acids in the proteins but the
alignment positions.
strain. We therefore tested the
ability of the active mutants to allow the
pbs2
hog1
double knockout strain to grow on
salt. Obviously, this strain is not rescued by overexpression of either
Hog1 or Pbs2 (Fig. 2B). Yet, it is rescued by the Hog1
mutants (Fig. 2B), showing that they are active
biologically, functioning alone, independently of Pbs2 and endogenous Hog1.
cells
by activating the authentic biochemical pathway downstream to Hog1, we
measured mRNA levels of GPD1 and GPP2 genes.
The products of these genes are involved in glycerol biosynthesis, and
their expression is Hog1-dependent (1). Our analysis
revealed that all the Hog1 active mutants, but not wild type Hog1,
induced transcription of these genes in pbs2
cells (data
not shown).
cells, immunoprecipitated, and assayed in
vitro for its ability to phosphorylate ATF2 (Fig.
4). Strikingly, most mutants exhibited
very high catalytic activity in the absence of any stimulation (Fig.
4). Under these conditions, the activity of wild type Hog1 molecules
was barely detectable (Fig. 4, upper panel, lane
3). Particularly high basal kinase activity was measured for
Hog1F318L and Hog1F318S. The activity of these
molecules did not change when cells were treated with salt (Fig. 4).
Thus, Hog1F318L and Hog1F318S manifest their
maximal catalytic activity under any growth conditions and could be
regarded as constitutively active molecules. Because equal amounts of
Hog1 proteins were used in all assays (Fig. 4), it is clear that the
specific activity of the mutants is much higher than that of wild type
Hog1. Not only Hog1F318L and Hog1F318S but also
all the other mutants showed very high catalytic activity in the
absence of any stimulation. Yet, the activity of the other mutants
further increased when salt was added to the culture. Namely,
Hog1Y68H, Hog1D170A, Hog1W320R,
Hog1F322L, and Hog1W332R mutants are also
hyperactive under any growth conditions but manifest their maximal
activity after salt induction. We believe that the further increase in
activity of some mutants reveals a new mechanism of control of Hog1
(perhaps a removal of a repressor; see "Discussion").
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Fig. 4.
Hog1 mutants are catalytically active in the
absence of salt induction. Hog1 wild type or active Hog1 mutants
were expressed and immunoprecipitated from hog1 cells and
assayed in vitro using GST-ATF2 as a substrate. Cells were
either exposed or not exposed to 1 M NaCl. The upper
panel of each image shows the autoradiogram of the in
vitro kinase assay. The negative control is Hog1 activity measured
in pbs2
cells (left lanes of upper image). The
lower panel of each image shows Western blots of the
lysates, displaying the Hog1 protein levels. A negative control for the
Western blot is a lysate prepared from hog1
cells
harboring an empty vector (first two lanes from the
left of the upper image).
Renders the
Kinase Catalytically Active--
To obtain another indication that the
mutants have acquired intrinsic, independent catalytic activity, we
attempted to produce them as recombinant proteins in E. coli
and to measure their activity. Yet, we were not able to produce soluble
Hog1 proteins. Because soluble recombinant versions of Hog1 are
difficult to obtain (other laboratories were also not successful in
producing recombinant Hog1), we decided to test the activating
mutations in the human p38
protein, which is known to be soluble
when expressed in bacteria. This approach is validated by the fact that
the activating mutations of HOG1 occur in residues that are
conserved in mammalian MAPKs (Fig. 3B). Hence, we replaced
Phe327 of the human p38
gene (homolog of
Phe322 in HOG1) with Leu or Ser. The
p38Phe327Leu and p38Phe327Ser, as well as a
wild type p38 protein, were expressed in E. coli utilizing
the pET15 vector and purified using Ni2+-agarose. The
recombinant purified proteins were tested for their kinase activity
in vitro (Fig. 5A).
As expected, wild type p38 exhibited very low catalytic activity. The
mutants, however, exhibited significant kinase activity (at least
70-fold higher than wild type; Fig. 5A). Because the mutated
p38 enzymes are active as recombinant proteins expressed in bacteria,
it is clear that they have acquired intrinsic catalytic activity, which
is independent of activation by any upstream MAPKK. Furthermore, the
result with the p38 mutants validates the notion that mutations
identified in the HOG1 screen could be used to produce
active forms of mammalian MAPKs.
View larger version (42K):
[in a new window]
Fig. 5.
Human p38 proteins
carrying mutations equivalent to those that activate Hog1 possess an
intrinsic catalytic activity and the capability to
autophosphorylate. Wild type (w.t.) p38 protein
and mutants p38F327L and p38F327S were
expressed in E. coli and purified to near homogeneity.
Purified proteins were tested for their kinase activity in
vitro using GST-ATF2 as a substrate (A) and for
autophosphorylation activity (B). Assay mixtures were
separated through SDS-polyacrylamide gel electrophoresis. Gels were
stained with Coomassie Brilliant Blue (upper panels) and
exposed to x-ray film (lower panels).
Cells--
The
results shown above suggest that the Hog1 active mutants acquired an
increased intrinsic catalytic activity. This idea is most strongly
supported by the results with the active recombinant p38 proteins,
which certainly were not activated by E. coli proteins.
cells. To address this
possibility, we measured, by Western blot analysis, the phosphorylation status of the Hog1 mutants. We used anti-phospho-p38 antibodies that
recognize dually phosphorylated Hog1 (43). Whole cell extracts were
prepared from pbs2
and hog1
cells
expressing wild type Hog1 or the various mutants. Extracts were
prepared from cells exposed, or not exposed, to salt. The Western blot
analysis revealed several phosphorylation patterns (Fig.
6). Most significantly, when expressed in
pbs2
cells, the active Hog1 mutants F318S, W320R, and
W332R show no detectable phosphorylation (Fig. 6A). Hog1
mutants Y68H, D170A, and F318L show low levels of phosphorylation in
pbs2
cells. This level of phosphorylation is even below
the level measured in wild type Hog1 expressed in pbs2
cells exposed to osmotic shock (Fig. 6A, upper
panel, lane 8). A possible explanation for the low
level of Hog1 phosphorylation in pbs2
cells could be
autophosphorylation activity (28, 40). It should be stressed that this
low phosphorylation of Hog1 in pbs2
cells was
insufficient to allow growth on salt (Fig. 2A). Taken
together, these results suggest that the dual phosphorylation state of
the active mutants is not related to their intrinsic catalytic
activity. This notion has been further addressed by mutagenizing the
phosphoacceptors Thr174 and Tyr176 in all the
mutants (see below). Hog1 mutants showed a different pattern of
phosphorylation when expressed in hog1
cells (Fig. 6B). First, mutants Y68H, D170A, A314T, and W320R are
phosphorylated in the absence of salt stimulation, at a significantly
higher level than that of the wild type Hog1. Second, the level of
their phosphorylation following induction with salt is similar, if not higher, than that of wild type. Thus, the presence of Pbs2 was important for increased phosphorylation of these mutants (compare Fig.
6, A and B), although Pbs2 was not required for
their biological activity (Fig. 2).
View larger version (67K):
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Fig. 6.
Many active forms of Hog1 do not require
phosphorylation for their activity. Analysis of the
phosphorylation state of the different Hog1 mutants expressed in
pbs2 cells (A) and in hog1
cells
(B). All Hog1 proteins used were fused to an HA epitope.
Western blot analyses are shown. The upper panel of each
image shows the levels of dually phosphorylated Hog1 using
anti-phosph-p38 antibodies; the lower panel shows total Hog1
protein levels using anti-HA antibodies.
seem to be labile. As shown in Fig. 6B (upper
panel, lanes 11 and 12 and lower
panel, lanes 5 and 6), in lysates prepared
from hog1
cells expressing Hog1F318L or
Hog1F318S, a 35-kDa protein reacted with the anti-HA
antibodies. Normally, the apparent molecular mass of native Hog1 is
about 50 kDa. The 35-kDa peptide appeared only when some lysis
protocols were used (trichloroacetic acid protocol) and not others
(native lysis; see Fig. 4, for example; for protocols, see "Material
and Methods"). Thus, Hog1F318L and Hog1F318S
are intact proteins in vivo but are susceptible to cleavage
when vigorous lysis procedures are used. It is clear that these
mutations induced dramatic structural changes, which are probably
responsible for the high catalytic activity of these two mutants. Note
that Hog1F318L and Hog1F318S molecules are
fully functional in hog1
cells and allow growth on salt
(Fig. 2). In summary, the Western blot analysis suggests that
in pbs2
cells most mutants are not activated through dual phosphorylation, supporting the results above (Figs. 4 and 5) that
strongly suggested that the mutants acquired an intrinsic independent
catalytic activity.
cells) would tolerate replacement of
Thr174 with Ala and Tyr176 with Phe and would
remain active. As can be seen in Fig. 7,
replacement of Tyr176 with Phe had no effect on the ability
of most mutants to allow growth of both hog1
cells and
pbs2
cells (see Fig. 8 for
Hog1D170AY176F). Only Hog1A314T and
Hog1W320R were affected by the Y176F mutation. This result
suggests that phosphorylation of Tyr176 is dispensable for
the activity of the mutants and further supports the notion
that they are independent of any MAPKK.
View larger version (48K):
[in a new window]
Fig. 7.
Active Hog1 mutants containing the mutation
Y176F can rescue yeast from high salt concentrations but cannot when
containing the T174A mutation. Each plate is divided into two.
Hog1 wild type and mutants on the left side contained the
mutation T174A, and on the right side they contained the
mutation Y176F. A, Hog1 molecules were expressed in
hog1 cells. The positive control (first row,
left side) was a wild type Hog1 expressed in
hog1
cells; the negative control (first row,
right side) was a wild type Hog1 expressed in
pbs2
cells. B, proteins were expressed in
pbs2
cells. The positive control was
Hog1F318S expressed in pbs2
cells
(first row, right side); the negative control was
wild type Hog1 expressed in pbs2
cells (first
row, left side).
View larger version (53K):
[in a new window]
Fig. 8.
Elimination of both phosphorylation sites
(indicated by two asterisks) in Hog1 wild type and
mutants abolishes their activity in both pbs2 cells
(A) and in hog1
cells
(B). Replacement of only Tyr176 with
Phe does not abolish the activity of Hog1D170A (indicated
with one asterisk; A, left plate,
row 5). The upper plates are master plates
containing YNB-URA media that were replica-plated onto YPD + 1.3 M NaCl plates.
or
pbs2
cells (Fig. 7). Similarly, when both
Thr174 and Tyr176 were changed to Ala
and Phe, respectively, none of the mutants could support growth on salt
(Fig. 8). These findings demonstrate that most Hog1 mutants do not
require dual phosphorylation for their activity (Fig. 6A)
but depend on Thr174. It is possible that phosphorylation
of Thr174 is not required, but this residue plays an
essential role in maintaining the catalytic core of the enzyme. Indeed,
not only did Robbins et al. (18), who replaced the
Thr183 phosphoacceptor in ERK2 with Glu, not observe an
increase in activity, but in fact they measured a dramatic reduction in
the maximal kinase activity. Alternatively, it is possible that the Hog1 mutants acquired autophosphorylation activity that selectively phosphorylates Thr174.
cells to grow on salt, they are by
definition, biologically active. In addition to their ability to allow
growth under osmotic stress, we noticed that expression of the active forms in wild type cells significantly affected important biological properties of the cells. The growth rate under optimal growth conditions of cells expressing the mutants was dramatically reduced. The generation time of these cultures was about 2 times longer than
that of cells expressing wild type Hog1. In addition, the active
mutants increased cell aggregation and flocculation (data not shown). A
detailed description of the phenotypes induced by the mutants will be
provided elsewhere.2 These
effects disclose novel roles for Hog1 in growth arrest and cell-cell
interaction and provide strong indications for the potential uses of
the active molecules for biological studies.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
kinase rendered it
catalytically active. Preliminary studies with mammalian cells in
culture show that the active p38 mutants are highly active in
vivo, similar to the Hog1 mutants in yeast.
open the way to producing more active
forms of various MAPKs based on the mutations identified in Hog1.
Insertion of appropriate mutations into JNK (equivalent to the
HOG1 mutations Y68H and W322R (Fig. 3)) and into ERK2
(equivalent to the HOG1 mutations D170A, A314T, F318S, and
F318L (Fig. 3)) may render these MAPKs active. A systematic effort that
will test these mutations in a variety of MAPKs could provide a useful
battery of active forms of each MAPK. In addition to suggesting
particular mutations that may render MAPKs constitutively active, the
mutations identified in our study point out the domains that should be
mutated in an effort to activate MAPKs. Six of the nine mutations were found in L16 between residues 314 and 332. This domain is equivalent to
residues 323-341 in ERK2, which was suggested to be important in
forming the interface for dimerization (20). It is currently not known
whether Hog1 or p38 are active as dimers. It could be, however, that
the activating mutations (in particular, mutations that replaced a
charged amino acid with a hydrophobic residue) support formation of
intermolecular contacts and consequently the formation of an active
dimer. Additional support for this notion comes from the D170A
mutation. Asp170 is homologous to
Asp173 in ERK2, which resides near His176,
which forms an ion pair with Glu343 of another monomer
(20). The possibility that each mutant stabilizes a dimer is exciting,
because it suggests that combining several mutations on the same MAPK
molecule would generate an even more stable and more active dimer. We
are currently combining mutations on the same Hog1 molecule, and
preliminary results suggest that indeed certain combinations result in
superactive kinases. Another possibility to explain the intrinsic
activity of the mutants is that the mutations strengthen the contacts
between L16 and the phosphorylation lip of the same molecule. Such
contacts may result in refolding the phosphorylation lip from the
closed conformation to the open structure (21).
diploid strains.
Furthermore, one can envisage a screen for active forms of mammalian
MAPKs in yeast. For example, because p38 and JNK are biologically
functional in yeast (24, 25), it should be possible to screen for
active forms of these enzymes directly in pbs2
cells.
However, the results with p38
(Fig. 5) suggest that screening for
active forms of other MAPKs may not be necessary. The activating
mutations identified in Hog1 could be applied directly in many other MAPKs.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Gustin for plasmids and yeast
strains, Melanie Cobb for most useful advice on p38 expression and
purification, Irit Marbach for constructing the
hog1pbs2
strain, Ariel Stanhill for
fruitful discussions and ideas throughout the project, and Ariel
Stanhill, Sergei Braun, Roger Kornberg, Moran Benhar, and Melanie
Grably for critical comments about the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Israel Cancer Association (to D. E.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 972 2 6584718;
Fax: 972 2 6586448; E-mail: Engelber@vms.huji.ac.il.
Published, JBC Papers in Press, April 17, 2001, DOI 10.1074/jbc.M101818200
2 M. Bell et al., manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MAPKK, MAPK kinase; HA, hemagglutinin; GST, glutathione S-transferase.
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