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(63), ra13. [DOI: 10.1126/scisignal.2000056] 2Science Signaling
Posas (24 March 2009)
Javier Macia, Sergi Regot, Tom Peeters, Núria Conde, Ricard Solé and Francesc
Transduction
Dynamic Signaling in the Hog1 MAPK Pathway Relies on High Basal Signal`
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COMPUTATIONAL BIOLOGY
Dynamic Signaling in the Hog1 MAPK Pathway
Relies on High Basal Signal Transduction
Javier Macia,
1
* Sergi Regot,
2
* Tom Peeters,
2
Núria Conde,
1,2
Ricard Solé,
1,3 †
Francesc Posas
2
†
(Published 24 March 2009; Volume 2 Issue 63 ra13)
Appropriate regulation of the Hog1 mitogen-activated protein kinase (MAPK) pathway is essential for
cells to survive osmotic stress. Here, we show that the two sensing mechanisms upstream of Hog1
display different signaling properties. The Sho1 branch is an inducible nonbasal system, whereas the
Sln1 branch shows high basal signaling that is restricted by a MAPK-mediated feedback mechanism.
A two-dimensional mathematical model of the Snl1 branch, including high basal signaling and a Hog1-
regulated negative feedback, shows that a system with basal signaling exhibits higher efficiency, with
faster response times and higher sensitivity to variations in external signals, than would systems without
basal signaling. Analysis of two other yeast MAPK pathways, the Fus3 and Kss1 signaling pathways,
indicates that high intrinsic basal signaling may be a general property of MAPK pathways allowing rapid
and sensitive responses to environmental changes.
INTRODUCTION
Appropriate regulation of signaling through mitogen-activated protein
kinase (MAPK) pathways is necessary to maximize cellular responses
to extracellular stimuli (1,2). Exposure of cells to high osmolarity results
in activation of a conserved family of MAPKs—p38 MAPK in mammals
and Hog1 in yeast. In vivo replacement of components of the Hog1
MAPK pathway by their mammalian counterparts showed that there is
a strong functional preservation of these MAPK pathways from yeast to
mammals (3). Exposure of yeast to high osmolarity results in rapid activation
of the Hog1 MAPK signaling pathway, which coordinates the adaptive
program required for cell survival during periods of osmotic stress (3,4).
Activation of the Hog1 MAPK is mediated by two independent up-
stream sensing mechanisms that lead to the activation of either the MAPK
kinase kinase (MAPKKK) Ssk2 (or the functionally redundant Ssk22) or
Ste11 (fig. S1). The Sho1 branch involves the transmembrane protein
Sho1 and the mucin-like transmembrane proteins Hkr1 and Msb2, which
are the potential osmosensors of this branch of the HOG pathway (5–7).
Although additional components still need to be identified to completely
understand signaling from the Sho1 branch, signaling requires the guano-
sine triphosphatase (GTPase) Cdc42, the adaptor protein Ste50, and the
kinases Ste20 and Cla4, which are members of the PAK (p21-activated
protein kinase) family (7–11). When stimulated by osmotic stress, the
Sho1 branch activates the MAPKKK Ste11 and, subsequently, the MAPKK
Pbs2 (12). Although the exact mechanism of Pbs2 activation by the Sho1
module remains unclear, Cdc42, Ste50, and Sho1 act as adaptor proteins
that control the flow of the osmotic stress signal from Ste20 and Cla4 to
Ste11, and then on to Pbs2 (13,14).
A second sensing mechanism involves the “two-component”osmosensor
composed of Sln1, Ssk1, and Ypd1. The Sln1 transmembrane osmosensor
has intrinsic histidine kinase activity and is a homolog of bacterial two-
component signal transducers. Using a phosphorelay mechanism involving
the Ypd1 and Ssk1 proteins, Sln1 inhibits the activity of Ssk1, which con-
trols the activity of the MAPKKKs Ssk2 and Ssk22 (15,16). Therefore,
under normal osmotic conditions, active Sln1 histidine kinase maintains
Ssk1 in its inactive phosphorylated form, whereas, in response to osmotic
stress, the kinase activity of Sln1 is inhibited and then active Ssk1 induces
Ssk2 activity. Signaling from either of the two branches leads to phospho-
rylation of the MAPKK Pbs2 and activation of the MAPK Hog1.
Kinetic analyses of Hog1 phosphorylation using mutants in each of
the two branches of the pathway revealed that they respond differently to
osmotic stress (17). Band-shift analysis with a microfluidic device
showed that, whereas the Sln1 branch of the pathway is capable of fast
signal integration to repeated stimuli, the Sho1 branch does not, suggest-
ing that the signaling properties of the two branches are different (18).
The origin of such differences, as well as their biological relevance, is
still unclear.
Signaling through the MAPK pathway is controlled not only by activity
of the kinases upstream of the MAPK, but also by the activity of protein
phosphatases and various feedback systems. Inactivation of the MAPK is
specifically controlled by direct dephosphorylation by protein phospha-
tases. The type 2C serine/threonine protein phosphatases and the protein
tyrosine phosphatases (Ptp2 and Ptp3) decrease Hog1 and Pbs2 activities
(19). Closure of the Fps1 glycerol channel has been proposed to act as a
feedback mechanism that limits sensor activation in the HOG pathway
(20). In addition, phosphorylation of Sho1 upon Hog1 activation seems
to be important to decrease signaling through this branch (21). Mathemat-
ical modeling and single-cell analyses have shown that inactivation of the
pathway by a mechanism independent of transcription might be important to
modulate acute responses; whereas transcription-dependent mechanisms
might be important for proper adaptation to future stimuli (20,22,23). There-
fore, although most of the components of the signaling pathway have been
defined, the signaling properties of the pathway are still poorly understood
(24,25).
Here, we use a chemical inhibitor of the MAPK Hog1 and extensive
signal quantification to show that the HOG pathway is controlled by high
basal signaling counteracted by a negative feedback regulatory system.
We developed a two-dimensional mathematical model that provided a
1
ICREA-Complex Systems Laboratory, Universitat Pompeu Fabra, E-08003
Barcelona, Spain.
2
Cell Signaling Unit, Departament de Ciències Experimen-
tals i de la Salut, Universitat Pompeu Fabra, E-08003 Barcelona, Spain.
3
Santa
Fe Institute, Santa Fe, NM 87501, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail: ricard.sole@upf.
edu (R.S.) and francesc.posas@upf.edu (F.P.)
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general analytic paradigm based on sensor and target architecture that can
be applied to any signaling pathway and showed that a system displaying
high basal signaling, such as the HOG pathway, exhibits higher efficiency
than systems without basal signaling, in terms of faster response and high-
er sensitivity to small variations in extracellular stimuli. Similar to the
HOG pathway, signaling through the MAPK pathway controlling the
mating response also seems to be controlled by a similar design, indicating
that high basal signaling coupled to negative feedback may be a common
trend in MAPK pathways.
RESULTS
The Sln1 and Sho1 branches of the HOG pathway have
different signaling properties
To understand the underlying properties of the two branches of the HOG
MAPK pathway, we performed several quantitative time-course and dose-
response experiments, monitoring Hog1 phosphorylation in mutants in
which both or only one branch of the pathway was active wild-type yeast,
ssk2/ssk22 double mutant (Sho1 branch active), and ste11 or ste50 mutant
(Sln1 branch active). Cells were subjected to osmotic stress (0.07 to 0.8 M
NaCl), then fixed at various times, and total and phosphorylated Hog1
were detected with specific antibodies by quantitative Western blotting
(see Materials and Methods). By plotting the percentage of phosphoryl-
ated Hog1 relative to the maximum in wild type over time, it is clear that
the two branches contribute differently to Hog1 phosphorylation (Fig. 1).
Cells lacking the Sln1 branch (ssk2 ssk22 mutant) do not respond to low
osmolarity (up to 0.1 M NaCl), display slower responses at each osmolar-
ity, and show a lower maximum response than the responses of the wild
type or ste50 mutant. In contrast, the ste50 mutant cells respond as fast as
wild-type cells, with a maximum response that is similar to that of wild-
type cells, but with a shorter duration (Fig. 1A). At the highest osmolarities
tested, the amplitude of the response of the wild type reached a maximum
and plateaued. At each higher osmolarity, the duration of the maximal
response period was longer. Thus, although both branches seem important
for achieving the wild-type response, the Sln1 branch is critical for setting
the speed and maximum amplitude of the response. Physiologically, these
differences in signaling properties result in mutants lacking signaling through
the Sln1 branch that are more sensitive (reduced growth), both on solid and
in liquid media, to osmotic stress than are mutants lacking signaling through
the Sho1 branch (Fig. 1, B and C).
The fact that the MAPKK Pbs2 receives the signal from two upstream
mechanisms, Sln1 and Sho1, with different dynamic responses to osmotic
stress suggests that Pbs2 may function as a signal integrator involved in
changing the response from one that is “dose encoded,”when the signal is
generated by the Sln1 branch, to one that is “duration encoded,”when the
signal is generated by the Sho1 branch. This would suggest that a reduction
in Pbs2 activity should result in a change on both amplitude and response
duration. Through orthogonal targeting, we created a yeast strain carrying
a mutant allele of PBS2 (pbs2as) that is specifically inhibited by the small-
molecule inhibitor 1NM-PP1 and a cell-permeable analog of 1NM-PP1, SPP86.
After incubation of this pbs2as strain with different concentrations of in-
hibitor and exposure to 0.2 M NaCl, analysis of total and phosphorylated
Hog1 in cell extracts showed that inhibition of Pbs2 reduces the maximum
amplitude of the response (fig. S2); thus, Pbs2 is a limiting factor in Hog1
signaling and suggests that Pbs2 not only transmits information to Hog1 but
might be critical to integrate different kinetic responses from the upstream
branches.
ABC
Fig. 1. Inactivation of any of the two branches of the HOG pathway
results in cells with less efficient signaling and increased sensitivity
to stress. (A) Hog1 phosphorylation is specifically affected in mu-
tants of the Sln1 branch. Different salt concentrations were added
to the indicated strains and quantification of total and phosphory-
lated Hog1 (P-Hog1) was assessed by quantitative Western
blotting. The data represent the percent of P-Hog1 relative to the
maximum in wild type (wt) and are presented as the mean ± SD
from three independent experiments. (B) Both branches of the
HOG pathway contribute differentially to growth upon increasing
osmolarity. The ssk2 ssk22 strain is more sensitive to osmotic
stress than is the ste50 strain. Serial dilutions of indicated strains
were spotted onto YPD and salt plates and growth was scored after
4 days. (C) Cell growth was assessed in liquid media containing
different NaCl concentrations.
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Inhibition of the activity of Hog1 increases
its phosphorylation in both the absence and the
presence of stress
In the absence of stress, catalytically inactive Hog1 shows higher basal
phosphorylation than does wild-type Hog1, and stress causes a prolonged
increase in its phosphorylation compared to wild-type Hog1 (26,27). In
addition, combined deletion of the phosphatases that down-regulate active
Hog1 is lethal unless HOG1 or PBS2 is deleted (28,29). Both results are com-
patible with constant signaling to Hog1, even in the absence of stress, which
must be down-regulated for the cells to maintain viability. If Hog1 is con-
stantly phosphorylated and its activity is required for its dephosphorylation,
then specific inhibition of the kinase activity of Hog1 should result in
increased phosphorylation of Hog1. To study whether Hog1 receives
constant signaling that is silenced by a feedback regulatory loop that de-
pendsonHog1activity,wecreatedaHog1mutant(hog1as)thatissensitive
to the small-molecule inhibitors 1NM-PP1 and SPP86 (30,31). Wild-type
or hog1as cells carrying an empty vector or a vector containing wild-type
HOG1 or a catalytically inactive hog1 (hog1kn) were grown in selective
medium and the inhibitor was added at time 0. Strikingly, addition of the
inhibitor to nonstressed cells resulted in rapid phosphorylation of Hog1
(Fig. 2A). The addition of the inhibitor to cells previously subjected to
stress (10 min) prevented dephosphorylation of hog1as as reported be-
fore (31) (fig. S3). Addition of the inhibitor, once Hog1 was dephosphoryl-
ated after exposure to stress and adaptation, resulted in rephosphorylation
of the kinase (Fig. 2B). Therefore, our results suggest that inhibition of the
kinase activity of the MAPK results in rapid phosphorylation of kinase
even in the absence of stress. Correspondingly, in the absence of stress,
the phosphorylated form of Hog1 does not appear in wild-type cells upon
addition of the inhibitor or in cells containing a wild-type allele of HOG1
together with the hog1as mutant allele (Fig. 2A). Osmotic stress triggered
the accumulation of hog1as fused to green fluorescent protein (GFP) in the
nucleus, whereas in the presence of the inhibitor, hog1as-GFP did not ac-
cumulate in the nucleus (fig. S4).
The Sln1 branch of the HOG pathway is responsible
for high basal signaling to Hog1
Several potential regulatory feedback loops have been described for the
Hog1 pathway. To test whether the control of Hog1 phosphorylation is
exerted by a fast (transcription-independent) or slow (transcription-
dependent) feedback loop, we analyzed phosphorylation of the kinase
upon blockage of transcription or translation. Phosphorylation of Hog1
upon inhibition of its kinase activity is independent of transcription and
translation, because the presence of cycloheximide (a translation inhibitor)
or thiolutin (a transcription inhibitor) did not affect Hog1 phosphorylation
(fig. S5A); these drugs did block the induction of Stl1 protein production
(fig. S5B). The rapid production of phosphorylated Hog1 when Hog1 ac-
tivity is inhibited does not depend on changes in glycerol concentration,
which is known to regulate the HOG pathway. Both hog1as cells with
wild-type FPS1, which encodes a glycerol channel, and hog1as cells with
fps1D1, which encodes a constitutively open glycerol channel, show accu-
mulation of phosphorylated Hog1 in response to inhibition of Hog1 kinase
activity (Fig. 3A). Furthermore, although the amount of phosphorylated
Hog1 produced in response to the inhibitor, as well as in response to osmotic
A
B
Fig. 2. Inhibition of Hog1 activity results in its phosphorylation. (A) Hog1
catalytic activity is required to prevent accumulation of the phosphorylated
form (P-Hog1) in the absence of stress. The indicated strains (wt and hog1as)
containing a vector control or a wild type or catalytically inactive mutant were
grown in SD medium and 5 mM inhibitor (SPP86) was added at time 0. Phos-
phorylated Hog1 (P-Hog1) was assessed as in Fig. 1A. (B) Inhibition of hog1as
after adaptation to stress also induces accumulation of its phosphorylated
form. Wild-type and hog1as strains were stressed with 0.4 M NaCl and in-
hibitor (5 mM SPP86) was added at time 40 min as indicated by the arrow.
Hog1 was detected by chemoluminescence with specific antibodies.
A
B
Fig. 3. Phosphorylation of inactive Hog1 depends on the Sln1 branch and
does not require closure of the glycerol channel. (A)hog1as cells carrying
the wild-type glycerol channel (FPS1) or a constitutively active mutant
(fps1D1) were grown in SD medium and inhibitor (5 mM SPP86) was added
at time 0. Samples were collected at the indicated times and total and
phosphorylated Hog1 were detected by chemoluminescence with specific
antibodies. (B) The indicated strains were grown in YPD, SPP86 (5 mM)
was added at time 0, and phosphorylated Hog1 (P-Hog1) was assessed
as in Fig. 1A.
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stress, was less in cells lacking FPS1, possibly due to increased internal
glycerol content, FPS1 strains still exhibited phosphorylation of hog1as
in the presence of the inhibitor (fig. S6, A and B). Thus, basal signaling
through the HOG pathway is controlled by a fast feedback regulatory mech-
anism that is dependent on the kinase activity of Hog1.
The basal signaling to Hog1 could be produced by either or both of the
upstream sensing mechanisms that converge on Pbs2. To dissect which
branch of the pathway, Sln1 or Sho1 or both, is responsible for high basal
signaling to Hog1, we measured Hog1 phosphorylation after addition of
inhibitor to hog1as cells that also contained ssk2 ssk22 or ste50 mutations.
Phosphorylation of Hog1 upon inhibition of its kinase activity in a ste50
strain showed similar kinetics to those cells in which both branches of the
pathway were active (Fig. 3B). Similar results were obtained in cells
containing sho1 mutation (fig. S7). Thus, loss of signal through the
Sho1 branch does not affect the basal signaling to Hog1. However,
Hog1 phosphorylation in response to inhibition of its kinase activity is
completely absent when the Sln1 branch is inactive (Fig. 3B). Therefore,
the two branches of the HOG pathway display different signaling proper-
ties, with the Sln1 branch mediating high basal signaling to Hog1 and the
Sho1 branch serving as an inducible system.
A mathematical model that includes high basal
signaling and a negative feedback loop describes the
dynamics of the HOG pathway
Previous comprehensive mathematical models for the yeast HOG path-
way included most of the components of the pathway or potential regulatory
loops, but they did not include basal signaling (18,22,23). Therefore, we
developed a new mathematical model that includes a stress-independent,
basal signal for the sensor and the requirement for negative feedback reg-
ulated by the target Hog1, which involves a minimal set of nonlinear in-
teractions (Fig. 4A and Supplementary Materials). The advantage of a model
that involves just the sensor and the target components is that it is amenable
to analytical mathematical analysis without requiring parameter estimation.
Despite its simplicity, this model fully describes the basic mechanisms
behind the observed experimental results (Fig. 4, B to D) and can be studied
analytically by considering only the interactions between the sensor protein
Sln1 (designated A in the model) and the target protein Hog1 (designated B
in the model).
Once cells are subjected to stress, the concentration of signaling-competent
Sln1 (A*) increases as does the active form of Hog1 (B*) (Fig. 4A). Thus,
A*andB* represent the main players immediately after stress, before
cellular adaptation has begun. Assuming that the total concentrations of
the proteins remains constant (22), one can reduce the pathway to a two-
dimensional model, which can be described by a set of two differential
equations, namely, dA*/dt=g(A*, S) (Equation 27 in Supplementary Ma-
terials) and dB*/dt=f(A*,B*) (Equation 28 in Supplementary Materials)
where Srepresents osmotic stress and fand gare nonlinear Hill-like re-
sponse functions. These equations describe the production of active Sln1
in response to stress (designated Sin the model) and the variation in Hog1
phosphorylation that depends on the concentration of active Sln1 and the
Hog1-mediated negative feedback loop (see Supplementary Materials for
a full description of the model and equations).
Fig. 4. A mathematical model of
the Sln1 branch describes the
signaling properties of the path-
way. (A) The minimal backbone
of the model includes the sensor
Sln (A, inactive state; A*, active
state), the MAPK Hog1 (B, in-
active state; B*, active state), os-
molites (G), and a stress-regulated
osmolite channel (C). Activation
of A occurs spontaneously (basal
activation). Deactivation of A* is
stress-dependent, K-a(S), where
Sis osmotic stress. A* stimulates
the formation of B*. B* represses
its own production by either en-
hancing B* deactivation or by
inhibiting B activation (not shown)
and enhances osmolite (G) pro-
duction, which exits through the
stress-regulated channels (C).
(B) The model describes the ex-
perimental data with the dynam-
ics of B* [phosphorylated Hog1
(P-Hog1)] from the model match-
ing the experimental data (colored
dots) (see Supplementary Mate-
rials for parameters) in response
to different Sconditions (concen-
trations of salt). (Cand D) The
model describes that by breaking the B*-dependent feedback, addition of the inhibitor in the experimental condition (arrow) induces phosphorylation of
B after adaptation to stress (C) and in the absence of stress (D).
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With this model we explored two hypothetical scenarios—one in which
the target of the feedback regulatory loop was Hog1 itself and one in which
the target was a component upstream of Hog1. Both cases are biologically
distinct, but the model displays the same qualitative dynamic response (see
Supplementary Materials for the detailed analysis of each scenario). Thus,
this new mathematical model incorporates the basal signaling and the
Hog1-dependent negative feedback loop and allows analysis of the signal
dynamics independently from the specific target of the feedback loop.
The two-dimensional mathematical model demonstrates
why high basal signaling is essential for the stress response
To understand the properties underlying the pathway with the defined
characteristics we studied the geometrical features of the so-called nullclines
of the system (32), which allow one to determine the maximum and
minimum states of the system. In this system, the nullclines are defined
by the two curves g(A*, S)=f(A*, B*) = 0, which allows us to determine
the presence of equilibrium points and their dynamical behavior. Thus, we
can analyze the relationship between active Hog1 and active Sln1 (Fig. 5A).
These curves determine how A* and B* change in time under stress.
Because g= 0 means that A* does not change, and f= 0 means that B*
does not change, the system will move parallel to the Aaxis when approach-
ing for parallel to the Baxis when approaching g. In our model, g=0isa
vertical line and the location of this line depends on the values of the
stress within a range A*
min
<A*<A*
max
, where A*
min
is the nonstress
(basal) value and A*
max
its upper value. Moreover, independently from
the specific set of Hill factors used, the f= 0 nullcline exhibits, for both
theoretical scenarios, generic geometrical features that determine its shape.
The f= 0 nullcline has a horizontal asymptote located at B
v
, crossing the
axis at A*=0,B* = 0 (Fig. 5A), without local maximum or minimum
values within the interval (0, B
v
). Therefore, when cells are adapted to the
environmental osmolarity, the system is in a stable state (A*
min
,B*
min
).
However, when cells are subjected to hyperosmotic stress, the nullcline
g= 0 is abruptly displaced to higher values of A* (that is, A*
max
) and
the previous stable state becomes unstable. After adaptation to the new
osmotic conditions, the nullcline g= 0 moves again to lower values of
A*, and the system evolves toward a new stable state (A
o
*, B
o
*). It is
worth noting that immediately after stress, cells are not adapted and the
production of active Hog1 (B*) is predominantly determined by the tra-
jectory in the A-B plane that depicts the evolution of A* versus B* com-
prised between (A*
min
,B*
min
)and(A*
max
,B*
max
)(Fig.5A).
Duetotheshapeofthenullcline,asystemstartingfromA*
min
=B*
min
=0
(a system without basal signal) will slowly increase, because the vertical
component fof the field has its lowest value exactly where the horizontal
component ghas its highest value. Therefore, the system will display a
delayed response. In contrast, if the system starts with A*
min
>0(inthe
presence of basal signaling), then the response is faster and the delay is
reduced or avoided. Even in cases were the value of A*
min
is not very high
(relatively low basal signaling), the response will be faster because of the
position of the initial point on the region of the nullcline f= 0 with the
steeper slope (Fig 5B). Simulations with different amounts of basal sig-
naling (A*
min
) produce the same relative increase in Hog1 phosphorylation
(B*), showing that the slope of the curve increases with the basal signal,
thus generating faster responses (Fig. 5B).
These results are consistent with the experimental results obtained for
the basal signaling branch Sln1 and for the nonbasal branch Sho1. The
Sln1 branch does not exhibit a delayed response to hyperosmotic stress,
whereas the Sho1 branch does exhibit a delay (Fig. 6A). As a result of the
same geometrical constraints, systems with very low or no basal signal
exhibit less sensitivity to small changes in the external osmolarity. Small
changes in the external osmolarity lead to small changes in the concen-
tration of the activated sensor A*. If the initial point is at (0,0) or near it,
changes in A*willhaveasmalleffectonB* because in this region the
nullcline f= 0 exhibits a lower slope. However, for systems with basal sig-
nal, the initial point will be located in regions of the nullcline where the
slope is steeper, and thus, small changes in A* will trigger large changes in
B*. Eventually, if the basal signal is too high, the initial point is located in
Fig. 5. Nullcline analysis reveals
the need for basal signaling in
the response to osmotic stress.
(A) Phase space A*versusB*.
Concentrations are determined
bytheequationdA*/dt=g(A*, S)
and dB*/dt=f(A*,B*). Nullcline
g= 0 defines a vertical line whose
location depends on the A*val-
ue without stress (basal signal).
Without basal signaling and no
stress, the nullcline is located at
the origin (blue line). If basal sig-
nal is present, then A* > 0 (green
line). The equilibrium state is de-
termined by g(A*,S)=f(A*,B*) =
0 (circle). After stress, nullcline
g= 0 shifts toward higher A*
values (violet line), and the sys-
tem moves to a new state (square).
The trajectory is confined in the gray region. The slope of f= 0 for small A*
values introduces a slowdown in the B* response through time, but once
A* is large enough (A*>A*
min
), B* grows faster. The inset in (A) shows the
ratio of the vertical and horizontal components of the field (f/g)whena
small increase in the concentration of active sensor A*, with respect to
its stable state, is performed. The curve displays an optimum at some A*
p
.
If A*
min
is close to A*
p
, the stress response is faster. (B)SimulationsofB*
versus time for different amounts of basal signaling. By increasing A*(see
trajectories in the inset) higher slopes are observed. (See Supplementary
Materials for details about nullcline analysis.)
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a region of the nullcline f= 0 where the curve is near the horizontal as-
ymptote with a lower slope, and, as a consequence, the pathway will lose
sensitivity. Based on these data, cells adapted to media with different os-
molarities should display different basal signaling. Correspondingly, inhi-
bition of hog1as in different media results in altered phosphorylation
kinetics of Hog1. Thus, Hog1 is phosphorylated faster after inhibition
of Hog1 activity in cells adapted to a medium with higher osmolarity
(Fig.6,BandC).
The overall behavior of the mathematical model is summarized in Fig.
5A, which explains the observed results of reduced sensitivity and slower
response of ssk2 ssk22 cells (Fig. 6A and fig. S8) and reveals that a com-
bination of basal signal, nonlinearity associated with the presence of Hill-
like responses, and a negative feedback provide for the high efficiency
and sensitivity of the pathway and its quick response to even small changes
in osmolarity.
The Fus3 and Kss1 MAPK signaling pathways
are also controlled by a high basal signal
In yeast, other MAPK signaling pathways exist that respond to external
stimuli (2). Both the Fus3 and Kss1 MAPKs are activated by pheromone
(33). We created strains in which only one of the two kinases was present
(fus3 and kss1 strains) and strains in which the remaining kinase could be
inhibited (Fus3as kss1;Kss1as fus3) and tested whether inhibition of the
kinase activity resulted in phosphorylation of the MAPK in the absence
of pheromone. As expected, the addition of pheromone stimulated the phos-
phorylation of wild-type and mutant fus3as, which, in turn, resulted in cell
cyclearrestatG
1
and induction of FUS1 expression (FUS1p:GFP) (Fig. 7).
Interestingly, addition of the inhibitor to the fus3as or kss1as cells triggered
Fus1 or Kss1 phosphorylation (Fig. 7, A and D). However, inhibition of the
kinase activity of the MAPK ( fus3as cells) failed to trigger downstream pro-
cesses, such as arrest in G
1
and FUS1 expression (Fig. 7, B and C). It is
worth noting that, whereas the presence of wild-type Kss1 did not affect the
phosphorylation of fus3as in the presence of inhibitor, the presence of wild-
type Fus3 strongly reduced the phosphorylation of kss1as in the presence of
the inhibitor, indicating that the two kinases might have different input into
the feedback loop(s) in response to pheromone (fig. S9). Both Fus3 and
Kss1 MAPK pathways also seem to include high basal signaling. Thus,
three MAPKs involved in various biological processes, from responding
to osmotic stress to initiating developmental programs, seem to have high
basal signaling that is inhibited by negative feedback regulatory loops con-
trolled by kinase activity of the MAPKs. Intrinsic basal signaling may be a
general property of MAPK pathways, allowing efficient response to envi-
ronmental changes.
DISCUSSION
Signaling through MAPK pathways is essential for cellular response to
extracellular stimuli. Yeast cells activate the Hog1 MAPK to control cel-
lular response and adaptation to osmotic stress and, therefore, precise mod-
ulation of Hog1 activity is necessary to maximize cell survival. Activation
of the MAPK is achieved by two upstream sensing mechanisms with slight-
ly different kinetics of response and sensitivity to osmotic stress [(17)and
our results (Figs. 1 and 6A)], suggesting that both branches may have
different signaling capacity. Partial inhibition of the MAPKK Pbs2 shows
that the kinase not only integrates the signal from the two branches of the
pathway but may also transform the input from a dose-encoded signal to
one that is duration encoded.
The use of a kinase-inhibitable form of Hog1 showed that inactivation
of its kinase activity results in its own rapid and strong phosphorylation,
A
B
C
Fig. 6. Basal signaling is essential for proper dynamic response of the
HOG pathway. (A) Signaling through the Sho1 branch is slower than
signaling through the Sln1 branch. Indicated strains were grown in me-
dium, subjected to 0.8 M NaCl, and phosphorylated Hog1 was assessed
as in Fig. 1A. (B) Reduction of the external osmolarity results in a slower
response. The ste50 strain was grown in YPD medium, cells were centri-
fuged and resuspended in medium or in distilled water as indicated, then
cells were subjected to 0.8 M NaCl stress and Hog1 phosphorylation was
assessed as in (A). (C) The kinetics of phosphorylation of Hog1 after inhi-
bition of Hog1 activity depends on external osmolarity. hog1as cells were
grown in YPD or YPD plus the indicated salt concentrations, spun, and
resuspended in YPD at the same salt concentration, YPD alone, or YPD
diluted in distilled water at the indicated dilution factors. The inhibitor
(5 mM SPP86) was added at time 0 and Hog1 phosphorylation was as-
sessed as described in (A).
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even in the absence of stress, indicating that there is a basal signal into
the MAPK. This is consistent with previous studies that, upon stress, a
catalytically inactive kinase remained phosphorylated longer than did the
wild-type MAPK (26). The use of the inhibitable Hog1 together with spe-
cific mutations in upstream components of the pathway showed that the
basal signal to Hog1 comes from only the Sln1 branch of the pathway,
with the Sho1 branch acting as an inducible system without basal sig-
naling. Thus, the Sln1 branch is possibly the key determinant of the signal-
ing properties of the pathway. Cells with mutations in the Sln1 branch of
the pathway are more sensitive to stress than are cells with mutations in
the Sho1 branch. Our results are consistent with previous bandwidth anal-
ysis with a microfluidic device, which showed that only the Sln1 branch
of the pathway was capable of fast signal integration (18). In some yeast
species, the Sho1 branch is either absent or is not connected to Hog1 MAPK
signaling but instead has a role in morphogenesis rather than osmo-
sensing (34).
Thus, in contrast to the typical assumption that the signal originates in
response to the presence of a stimuli, our experimental studies indicate
that the signaling through several yeast MAPK pathways depends on high
basal signal transduction that must be constantly counteracted by a fast-
acting feedback mechanism that is controlled by the kinase activity of the
MAPK. Although we do not know the exact nature of such a negative
feedback, it does not involve transcription or translation and is indepen-
dent of glycerol accumulation. Cells with a constitutively open glycerol
channel responded in the same manner as the wild type. Protein phospha-
tases are good candidates for mediating the negative feedback. Elimination
of phosphatases results in hyperactivation of the Hog1, and studies on
cells exposed to stress pulses followed by frequency determination showed
the presence of a fast-acting negative feedback regulatory loop and sug-
gested that it could involve protein phosphatases (23). For cells grown in
control or osmotic stress conditions, we can estimate the ratio of the phos-
phorylated and dephosphorylated states of Hog1 in the presence of the
inhibitor by assuming that because the catalytic activity of Hog1 is inhib-
ited and the external osmolarity is maintained constant, the rates of phos-
phorylation and dephosphorylation are constant. Under basal conditions,
the ratio K
phosphorylation
/K
dephosphorylation
≈1.1, whereas under 0.4 M salt
stress conditions, K
phosphorylation
/K
dephosphorylation
≈13.3. These ratios have
been calculated such that they reproduce the different slopes and the rel-
ative difference of phosphorylated Hog1 in the presence of inhibitor (Fig.
6C). We have also analyzed the model to estimate which of the two com-
ponents of the HOG pathway is the target of the negative feedback. The
model shows that regardless of whether the target of the feedback regula-
tory loop is Hog1 or is upstream of Hog1, the qualitative results are simi-
lar. However, the model also indicates that if the target of the feedback
loop is located upstream of Hog1, the system seems to be more efficient
(see the Mathematical Analysis section of the Supplementary Materials).
Feedback regulatory loops have profound implications and, typically,
systems with negative feedback show higher robustness against external
and stochastic noise (35,36), thereby increasing the efficiency in signal trans-
mission. However, the presence of negative feedbacks introduces several con-
straints on the dynamics of the systems because they introduce delays in the
response and reduce the sensitivity. Thus, the high basal signal may function
to counteract the constraints created by the negative feedback loops.
To explore the properties of a system with high basal signal restricted
by a negative feedback loop, we developed a two-dimensional mathemat-
ical model of the HOG pathway based on the minimal backbone of inter-
actions that reproduces the experimental results and allows analytical
analysis of the dynamic properties of the pathway. The analysis of this
model was simplified by considering that just after stress only the sensor-
target protein interaction and the negative feedback are relevant to explain
the basic dynamical behavior. The analysis of the main interactions showed
that the dynamics of the pathway immediately after stress depend on the
balance between the negative feedback and the basal signaling from the sen-
sor. The feedback tends to delay Hog1 phosphorylation; whereas the basal
signal tends to enhance its phosphorylation. Therefore, the sensitivity to
small changes in the external osmolarity depends on the amount of basal
A
BC
D
Fig. 7. The MAPKs Fus3 and Kss1 receive basal signaling in the
absence of stimuli. (A) Inhibition of Fus3 activity leads to its phos-
phorylation. Mating pheromone (a-factor) or inhibitor (SPP86)
was added to the FUS3 kss1 (wt; left panels) or fus3as kss1
mutant (fus3as; right panels) and phosphorylated or total Fus3
was assessed with specific antibodies. (B) Inhibition of fus3as
does not result in cell cycle arrest at G
1
.a-Factor or inhibitor
was added to the fus3as kss1 mutant and DNA was assessed
by fluorescence-activated cell sorting (FACS) analysis. (C)Inhi-
bition of fus3as does not result in induction of FUS1 expression.
Expression of GFP driven by the FUS1 promotor was assessed
in the same strain as in (B) by FACS analysis and cells expres-
sing GFP were quantified. (D) Inhibition of Kss1 activity leads to
its phosphorylation. a-Factor or inhibitor was added to the
KSS1 fus3 (wt; left panels) or kss1as fus3 mutant (kss1as; right
panels) and Kss1 phosphorylation was assessed with Kss1-
specific antibodies.
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signaling, such that this system is much more sensitive than one without
basal signaling. Correspondingly, cells deficient on the Sln1 branch show
reduced sensitivity to small variations in osmolarity, responded more
slowly to osmotic stress, and were more osmosensitive. Because high basal
signaling seems to be a key factor that counteracts the lack of response
and delay caused by the negative feedback, the dynamics of the response
should change upon variations in the basal signal. We showed that cells
that were adapted to a reduced osmolarity exhibited reduced basal signal-
ing, reduced capacity to cope with small variations in stress conditions,
and responded more slowly to osmotic stress (Fig. 6B). Therefore, in this
system with a negative feedback loop, a high basal signal is critical for de-
termining the signaling capacity.
To explore how common basal signaling with negative feedback is,
we extended our studies to the pheromone-responsive Fus3 and Kss1
MAPK pathways. Both kinases became phosphorylated upon inhibition
of their kinase activity, even in the absence of pheromone, suggesting that
these MAPK signaling pathways are also controlled by high basal signal
together with a MAPK-dependent negative feedback. This is consistent
with a study that identified Sst2 as the target of the feedback loop con-
trolledbyFus3(37). Thus, three MAPK pathways appear to have developed
high basal signaling repressed by MAPK-dependent negative feedback loops,
which implies that high intrinsic basal signaling could be a general property
of MAPK pathways, allowing efficient response to environmental changes.
MATERIALS AND METHODS
Quantitative Western blotting
Samples were taken in mid-exponential growth phase and fixed in 20%
trichloroacetic acid for SDS–polyarylamide gel electrophoresis and inmu-
noblotting. Hog1, Fus3, and Pbs2 were detected with antibodies specific
for these proteins (Santa Cruz), phosphorylated Hog1 with an antibody
against phospho p38 (Cell Signaling), and both P-Kss1 and P-Fus3 with
an antibody against phospho p44/42 (Cell Signaling). Quantif ication analysis
was performed by fluorescent detection with the IRDye 800CW donkey
antibody against goat immunoglobulin G (IgG) and the IRDye 680 don-
key antibody against rabbit IgG (LI-COR Biosciences) and the ODISSEY
application software 2.1 (LI-COR Biosciences). All phosphorylated Hog1
values were normalized against the 10-min sample taken from the wild type
stressed with 0.4 M salt.
Flow cytometry
The pheromone pathway was activated with a-factor (2 mg/ml) and DNA
content was assessed by staining with propidium iodide.
Inhibitors
1NM-PP1 was used at 5 mM to inhibit analog-sensitive mutants (38). SPP86,
a cell-permeable adenine analog similar to 1NM-PP1, was used at 5 mM.
Strains and plasmids
W303-1A (wild type) and derivatives ssk2 ssk22,ste50,ste11,sho1,hog1
T100G
(hog1as), ssk2 ssk22 hog1as,ste50 hog1as,fps1 hog1as,bar1 FUS1::GFP,
bar1 kss1 fus3 FUS1::GFP P(FUS3):: fus3
Q93A
(fus3as), bar1 kss1
fus3 FUS1::GFP P(KSS1)::kss1
E94A
(kss1as), bar1 fus3 FUS1::GFP
P(FUS3)::fus3as,bar1 kss1 FUS1::GFP P(KSS1)::kss1as, and pbs2
M435A
(pbs2as) were used in this study. YCPlac111 with HOG1-3HA or hog1
K52S,53N
-
3HA (hog1-KN), YEPlac195 with FPS1 or fps1D13-230 (fps1D1)and
pRS416 with HOG1-GFP or hog1as-GFP plasmids were used in this study.
Growth conditions
Cultures were maintained on YPD (1% yeast extract, 2% Bacto Peptone,
2% glucose) or on the appropriate synthetic dropout (SD) medium [0.17% yeast
nitrogen base without amino acids, 0.5% ammonium sulfate, 2% glucose,
Complete Supplement Mixture quadruple dropout (Qbiogene) (0.6 g/liter),
and histidine, tryptophan, uracil, or leucine combined (40 mg/liter)] to main-
tain the appropriate selection for maintenance of the plasmids. Cells were
grown at 30°C and NaCl was added at various concentrations as indicated.
Solid culture was performed on 1.5% agar plates of YPD or YPD with NaCl
at the indicated concentration. Plates were incubated at 30°C.
SUPPLEMENTARY MATERIALS
www.sciencesignaling.org/cgi/content/full/2/63/ra13/DC1
Mathematical Analysis
Fig. S1. Schematic diagram of the HOG pathway.
Fig. S2. Pbs2 activity is a limiting factor to determine amplitude of Hog1 phosphorylation in
response to osmotic stress.
Fig. S3. Inhibition of Hog1 results in phosphorylation of the MAPK.
Fig. S4. Inhibited Hog1 remains mainly cytoplasmic.
Fig. S5. Inhibition of transcription or translation does not prevent phosphorylation of Hog1.
Fig. S6. Deletion of FPS1 does not abolish hog1as phosphorylation in the presence of inhibitor.
Fig. S7. Neither Sho1 nor Ste50 is essential for phosphorylation of inactive Hog1.
Fig. S8. The Sln1 branch is essential for rapid response to osmotic stress.
Fig. S9. The kinases Fus3 and Kss1 receive basal signaling in the absence of stimuli and
differently control a negative feedback loop.
REFERENCES AND NOTES
1. J. M. Kyriakis, J. Avruch, Mammalian mitogen-activated protein kinase signal transduc-
tion pathways activated by stress and inflammation. Physiol. Rev. 81,807–869 (2001).
2. R. E. Chen, J. Thorner, Function and regulation in MAPK signaling pathways: Lessons
learned from the yeast Sacchar omyces cerevisi ae. Biochim. Biop hys. Acta 1773,
1311–1340 (2007).
3. D. Sheikh-Hamad, M. C. Gustin, MAP kinases and the adaptive response to hyper-
tonicity: Functional preservation from yeast to mammals. Am. J. Physiol. Renal Physiol.
287, F1102–F1110 (2004).
4. E. de Nadal, P. M. Alepuz, F. Posas, Dealing with osmostress through MAP kinase
activation. EMBO Rep. 3, 735–740 (2002).
5. K. Tatebayashi, K. Tanaka, H.-Y. Yang , K. Yamamoto, Y. Matsushita, T. Tomida, M. Imai,
H. Saito, Transmembrane mucins Hkr1 and M sb2 are putative osmosensors in the SHO1
branch of yeast HOG pathway. EMBO J. 26,3521–3533 (2007).
6. E. de Nadal, F. X. Real, F. Posas, Mucins, osmosensors in eukaryotic cells? Trends
Cell Biol. 17,571–574 (2007).
7. I. Dan, N. M. Watanabe, A. Kusumi, The Ste20 group kinases as regulators of MAP
kinase cascades. Trends Cell Biol. 11, 220–230 (2001).
8. V. Reiser, S. M. Salah, G. Ammerer, Polarized localization of yeast Pbs2 depends on
osmostress, the membrane protein Sho1 and Cdc42. Nat. Cell Biol. 2, 620–627
(2000).
9. F. Posas, E. A. Witten, H. Saito, Requirement of STE50 for osmostress-induced activation
of the STE11 mitogen-activated protein kinase kinase kinase in the high-osmolarity
glycerol response pathway. Mol. Cell. Biol. 18, 5788–5796 (1998).
10. D. M. Truckses, J. E. Bloomekatz, J. Thorner, The RA domain of Ste50 adaptor protein
is required for delivery of Ste11 to the plasma membrane in the filamentous growth
signaling pathway of the yeast Saccharomyces cerevisiae.Mol. Cell. Biol. 26,912–928
(2006).
11. M. J. Winters, P. M. Pryciak, Interaction with the SH3 domain protein Bem1 regulates
signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20. Mol. Cell.
Biol. 25, 2177–2190 (2005).
12. F. Posas,H. Saito, Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK:
Scaffold role of Pbs2p MAPKK. Science 276,1702–1705 (1997).
13. K. Tatebayashi, K. Yamamoto, K. Tanaka, T. Tomida, T. Maruoka, E. Kasukawa, H. Saito,
Adaptor functions of Cdc42, Ste50, and Sho1 i n the yeast osmoregulatory HOG MAPK
pathway. EMBO J. 25, 3033–3044 (2006).
14. A. Zarrinpar, R. P. Bhattacharyya, M. P. Nittler, W. A. Lim, Sho1 and Pbs2 act as coscaffolds
linking components in the yeast high osmolarity MAP kinase pathway. Mol. Cell 14,
825–832 (2004).
15. F. Posas, S. M. Wurgler-Murphy, T. Maeda, E. A. Witten, T. C. Thai, H. Saito, Yeast
HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the
SLN1-YPD1-SSK1 “two-component”osmosensor. Cell 86,865–875 (1996).
RESEARCH ARTICLE
www.SCIENCESIGNALING.org 24 March 2009 Vol 2 Issue 63 ra13 8
on May 17, 2013 stke.sciencemag.orgDownloaded from
16. F. Posas, H. Saito, Activation of the yeast SSK2 MAP kinase kinase kinase by the
SSK1 two-component response regulator. EMBO J. 17, 1385–1394 (1998).
17. T. Maeda, M. Takekawa, H. Saito, Activation of yeast PBS2 MAPKK by MAPKKKs or
by binding of an SH3-containing osmosensor. Science 269, 554–558 (1995).
18. P. Hersen, M. N. McClean, L. Mahadevan, S. Ramanathan, Signal processing by the
HOG MAP kinase pathway. Proc. Natl. Acad. Sci. U.S.A. 105, 7165–7170 (2008).
19. H. Saito, K. Tatebayashi, Regulation of the osmoregulatory HOG MAPK cascade in
yeast. J. Biochem. 136, 267–272 (2004).
20. S. Hohmann, M. Krantz, B. Nordl ander, Yeast osmoregulation. Methods Enzymol.
428,29–45 (2007).
21. N. Hao, M. Behar, S. C. Parnell, M. P. Torres, C. H. Borchers, T. C. Elston, H. G. Dohlman,
A systems-biology analysis of feedback inhibition in the Sho1 osmotic-stress-response
pathway. Curr. Biol. 17,659–667 (2007).
22. E. Klipp, B. Nordlander, R. Kruger, P. Gennemark, S. Hohmann, Integrative model of
the response of yeast to osmotic shock. Nat. Biotechnol. 23, 975–982 (2005).
23. J. T. Mettetal , D. Muzzey, C. Gomez -Uribe, A. van Oude naarden, The frequency
dependence of osmo-adaptation in Saccharomyces cerevisiae.Science 319, 482–484
(2008).
24. S. Hohmann, Osmotic stress signaling and osmoadaptation in yeasts. Microbiol. Mol.
Biol. Rev. 66, 300–372 (2002).
25. S. M. O’Rourke, I. Herskowitz, E. K. O’Shea, Yeast go the whole HOG for the hyperosmotic
response . Trends Genet. 18,405–412 ( 2002).
26. S. M. Wurgler-Murphy, T. Maeda, E. A. Witten, H. Saito, Regulation of the Saccharomyces
cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine
phosphatases. Mol. Cell. Biol. 17, 1289–1297 (1997).
27. P. Ferrigno, F. Posas, D. Koepp, H. Saito, P. A. Silver, Regulated nucleo/cytoplasmic
exchange of HOG1 MAPK requires the importin bhomologs NMD5 and XPO1. EMBO
J. 17, 5606–5614 (1998).
28. T. Jacoby, H. Flanagan, A. Faykin, A. G. Seto, C. Mattison, I. Ota, Two protein-tyrosine
phosphatases inactivate the osmotic stress response pathway in yeast by targeting the
mitogen-activated protein kinase, Hog1. J. Biol. Chem. 272, 17749–17755 (1997).
29. M. T akekawa, T. Maeda, H. Saito, Protein phosphatase 2Cainhibits the human
stress-responsive p38 and JNK MAPK pathways. EMBO J. 17, 4744–4752 (1998).
30. S. Kim, K. Shah, Dissecting yeast Hog1 MAP kinase pathway using a chemical genetic
approach. FEBS Lett. 581,1209–1216 (2007).
31. P. J. Westfall, J. Thorner, A nalysis of mitogen-activated protein kinase signaling specificity
in response to hyperosmotic stress: Use of an analog-sensitive HOG1 allele. Eukaryot.
Cell 5,1215–1228 (2006).
32. S. H. Strogatz, Interpreting the human phase response curve to multiple bright-light
exposures. J. Biol. Rhythms 5, 169–174 (1990).
33. H. G. Dohlman, J. E. Slessareva, Pheromone signaling pathways in yeast. Sci. STKE
2006, cm6 (2006).
34. M. Krantz, E. Becit, S. Hohmann, Comparative genomics of the HOG-signalling system
in fungi. Curr. Genet. 49,137–151 (2006).
35. A. Becskei, L. Serrano, Engineering stability in gene networks by autoregulation. Nature
405,590–593 (20 00).
36. T. S. Gardner, J. J. Collins, Neutralizing noise in gene networks. Nature 405,520–521 (2000).
37. R.C.Yu,C.G.Pesce,A.Colman-Lerner,L.Lok,D.Pincus,E.Serra,M.Holl,K.Benjamin,
A. Gordon, R. Brent, Negative feedback that improves information transmission in yeast
signalling. Nature 456,755–761 (2008).
38. A. C. Bishop, J. A. Ubersax, D. T. Petsch, D. P. Matheos, N. S. Gray, J. Blethrow, E. Shimizu,
J. Z. Tsien, P. G. Schultz, M. D. Rose, J. L. Wood, D. O. Morgan, K. M. Shoka t, A chemical
switch for inhibitor-sensitive alleles of any protein kinase. Nature 407,395–401 (2000).
39. We thank E. de Nadal for helpful discussions and support, M. Morillas and A. Vendrell
for strains, and M. Grötli (Sweden) for inhibitor design and supply. This work was
supported by an FPU fellowship to S.R.; grant CSD2007-0015 from Ministerio de Ciencia
y Tecnología, Consolider Ingenio 2010 program; through the European Commission
Directorate General Research, FP6 contract no. ERAS-CT-2003-980409 EURYI (European
Young Investigator Awards) award (www.esf.org/euryi) and QUASI to F.P; and FP6-
2005-NEST-PATH CELLCOMPUT project to F.P. and R.S. The FP laboratory also receives
support from the Fundación Marcelino Botín and Institució Catalana de Recerca i Estudis
Avançats (ICREA) Acadèmia (Generalitat de Catalunya).
Submitted 23 September 2008
Accepted 6 March 2009
Final Publication 24 March 2009
10.1126/scisignal.2000056
Citation: J. Macia, S. Regot, T. Peeters, N. Conde, R. Solé, F. Posas, Dynamic signaling
in the Hog1 MAPK pathway relies on high basal signal transduction. Sci. Signal. 2, ra13
(2009).
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