DOI: 10.1126/science.1068873
, 1018 (2002);297Science
et al.Upinder S. Bhalla
Mitogen-Activated Protein Kinase Signaling Network
MAP Kinase Phosphatase As a Locus of Flexibility in a
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mice, resembling the disease produced by wt
PV1(M) (13). However, a larger inoculum of
sPV1(M) than PV1(M) was necessary to paralyze
or kill the animals (Table 1). The increase
in the magnitude of attenuation was unexpected,
because all nucleotide substitutions introduced
into sPV1(M) resulted in silent mutations
in the ORF, except for the newly created Xma
I and Stu I sites in the 5Ј nontranslated region
(NTR) and 2B region, respectively. These latter
changes had been shown previously to have no
influence on viral replication in tissue culture
(20, 21). However, the silent mutations that we
introduced into the poliovirus genome may exert
a strong influence on pathogenesis by hitherto
unknown mechanisms.
The presence or absence of genetic markers
in the inoculated virus and the virus isolated
from the spinal cords of paralyzed mice
was confirmed by amplification of the viral
RNA by RT-PCR and restriction enzyme
analysis. Our results show that the viruses
isolated from the spinal cords of paralyzed
mice resembled the inoculated virus (fig. S1).
Our data also confirm that the synthetic virus
was the causative agent of the flaccid paralysis
observed in the sPV1(M)-infected mice.
The chemical synthesis of the viral genome,
combined with de novo cell-free synthesis, has
yielded a synthetic virus with biochemical and
pathogenic characteristics of poliovirus. In
1828, when Wo¨hler synthesized urea, the theory
of vitalism was shattered (22). If the ability to
replicate is an attribute of life, then poliovirus is
a chemical
[C332,652H492,388N98,245O131,196- P7501S2340,
see (2)] with a life cycle.
As a result of the World Health Organization’s
vaccination campaign to eradicate
poliovirus (23), the global population is better
protected against poliomyelitis than ever
before. Any threat from bioterrorism will
arise only if mass vaccination stops (23) and
herd immunity against poliomyelitis is lost.
There is no doubt that technical advances will
permit the rapid synthesis of the poliovirus
genome, given access to sophisticated resources.
The potential for virus synthesis is
an important additional factor for consideration
in designing the closing strategies of the
poliovirus eradication campaign.
References and Notes
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of Virology, R. G. Webster, A. Granoff, Eds. (Academic
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1348.
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(1985).
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27, 353 (1993).
8. C. L. Mendelsohn, E. Wimmer, V. R. Racaniello, Cell
56, 855 (1989).
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10. S. K. Jang et al., J. Virol. 62, 2636 (1988).
11. J. Pelletier, N. Sonenberg, Nature 334, 320 (1988).
12. W. K. Xiang, A.V. Paul, E. Wimmer, Semin. Virol. 8,
256 (1987).
13. Materials and methods are available as supporting
material on Science Online.
14. S. van der Werf, J. Bradley, E. Wimmer, F. W. Studier,
J. J. Dunn, Proc. Natl. Acad. Sci. U.S.A. 82, 2330
(1986).
15. J. Cello, A. V. Paul, E. Wimmer, unpublished data.
16. P. Nobis et al., J. Gen. Virol. 66, 2563 (1985).
17. S. Koike et al., Proc. Natl. Acad. Sci. U.S.A. 88, 951
(1991).
18. H. Horie et al., J. Virol. 68, 681 (1994).
19. M. Gromeier, H.-H. Lu, E. Wimmer, Microb. Pathog.
18, 253 (1995).
20. C. Mirzayan, E. Wimmer, Virology 189, 547 (1992).
21. W. Xiang, K. S. Harris, L. Alexander, E. Wimmer,
J. Virol. 69, 3658 (1995).
22. F. Wo¨hler, Ann. Phys. Chem. 88, 253 (1828).
23. A. Nomoto, I. Arita, Nature Immunol. 3, 205 (2002).
24. We thank A. Wimmer and J. Benach for valuable
comments on the manuscript. We are indebted to
B. L. Semler for a sample of cell-free HeLa cell extract.
Supported by Contracts N65236-99-C-5835 and
N65236-00-M-3707 from the Defense Advanced Research
Project Agency.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1072266/DC1
Materials and Methods
Fig. S1
References and Notes
26 March 2002; accepted 25 June 2002
Published online 11 July 2002;
10.1126/science.1072266
Include this information when citing this paper.
MAP Kinase Phosphatase As a
Locus of Flexibility in a
Mitogen-Activated Protein
Kinase Signaling Network
Upinder S. Bhalla,1
*† Prahlad T. Ram,2
*† Ravi Iyengar2
Intracellular signaling networks receive and process information to control cellular
machines. The mitogen-activated protein kinase (MAPK) 1,2/protein kinase C (PKC)
system is one such network that regulates many cellular machines, including the
cell cycle machinery and autocrine/paracrine factor synthesizing machinery. We
used a combination of computational analysis and experiments in mouse NIH-3T3
fibroblasts to understand the design principles of this controller network. We find
thatthegrowthfactor–stimulatedsignalingnetworkcontainingMAPK1,2/PKCcan
operate with one (monostable) or two (bistable) stable states. At low concentrations
of MAPK phosphatase, the system exhibits bistable behavior, such that brief
stimulus results in sustained MAPK activation. The MAPK-induced increase in the
amounts of MAPK phosphatase eliminates the prolonged response capability and
moves the network to a monostable state, in which it behaves as a proportional
response system responding acutely to stimulus. Thus, the MAPK 1, 2/PKC controller
network is flexibly designed, and MAPK phosphatase may be critical for this
flexible response.
Intracellular signaling pathways communicate
extracellular information to modulate
cellular functions in response to external
stimuli. Signaling pathways function not only
to transmit information but also to process the
information as it is being transmitted. Such
processing occurs because signaling pathways
interact with one another to form networks
(1–3). The processing occurs both
through summation of inputs and through the
temporal characteristics of pathways. For instance,
the MAPK cascade communicates
signals from growth factors that bind to receptor
tyrosine kinases to the transcriptional
Table 1. Biological characterization of sPV1(M). Plaque reduction assay in the presence (ϩ) and absence
(Ϫ) of antibodies as described in (13). Anti-PV1(M) and anti-PV2(L) are neutralizing polyclonal antibodies
specific for types 1 and 2 poliovirus, respectively. Neuropathogenicity of sPV1(M) and wt PV1(M) was
assayed in hPVR-tg mice as described in (13). PLD50 is defined as the amount of virus that caused paralysis
or death in 50% of the inoculated mice.
Virus
PFU
PLD50
(log10 PFU)
Mab D171 Anti-PV1(M) Anti-PV2(L)
– ϩ – ϩ – ϩ
sPV1(M) 83 0 91 0 88 92 6.2
wt PV1(M) 89 0 86 0 90 87 2.0
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machinery and other cellular effectors (4, 5).
However, receptor tyrosine kinases activate
other effectors such as phospholipase C–␥
(PLC-␥), which leads to the activation of
PKC (6). PKC in turn stimulates MAPK 1, 2
through activation of the protein kinase c-Raf
(7). MAPKs activate cytoplasmic phospholipase-A2
(cPLA2) (8, 9). The arachidonic
acid produced by cPLA2 also stimulates PKC
(10, 11). These pairwise connections create a
potential positive-feedback loop in the
MAPK network (Fig. 1A). Such a simple
network can function as a bistable switch
wherein a brief extracellular stimulus results
in sustained MAPK activation (12).
Signal flow through this MAPK network
reflects a balance between the positive phosphorylation
and the reverse dephosphorylation
reactions. We therefore considered two
key phosphatases in this system: protein
phosphatase 2A (PP2A), a broad-specificity
Ser-Thr phosphatase that dephosphorylates
both Raf and MAP/ERK kinase (MEK) (13,
14); and MAPK phosphatase (MKP), a dualspecificity
phosphatase that dephosphorylates
both Tyr and Thr residues on MAPKs and
whose expression is transcriptionally regulated
by MAPK (15). MAPK also phosphorylates
MKP, which reduces ubiquitination and
degradation of MKP (16). The increase in
amounts of MKP constitutes a negative feedback.
Computational analysis shows that
MKP activity at specified levels and duration
can limit the activated steady state of MAPK
(12). Coupling the positive- and negativefeedback
loop within the system could lead to
oscillations. However, we had found although
the system could move between regimes
where it could exist at two (basal and
activated) stable states (bistable) and one
(basal) stable state, the system was not oscillatory
(17). What then might be the systems
function of MKP induction? Computational
analysis suggests that at higher amounts of
MKP, the network would function as a proportional-response
system yielding differing
amounts of activated MAPK for varying concentrations
of growth factors, whereas at lower
concentrations of MKP the system might
function as an all-or-none switch at steady
state. This hypothesis would suggest that the
MAPK network is designed with the capability
of mounting several types of responses,
i.e., in a flexible manner. Indeed, one important
question in biological systems is whether
regulatory networks are flexibly designed.
We integrated computational analysis with
experiments in NIH-3T3 fibroblasts to determine
whether the MAPK controller network
is flexibly designed.
We modeled the MAPK network with a
system of coupled ordinary differential equations
in the general neural simulator GENESIS
with the Kinetikit interface (18). The
detailed equations and constants have been
described (12, 17) and are provided in the
supporting text (19). Initial simulations
showed that after a brief (5 min) stimulation
by a growth factor, MAPK activity
remained elevated for an extended (30 to 45
min) period (Fig. 1B). As a result of system
complexity related to MAPK-induced increases
in the amounts of its negative regulator
MKP, the activated state is temporally
constrained, i.e., not maintained
indefinitely. There was minimal decrease in
the MAPK 1, 2 activity for up to 40 min,
followed by a steady decline (Fig. 1B). We
have operationally defined the activated
steady state as a period that is at least five
times longer than the initial stimulation (at
least 25 min of high activity for 5-min
stimulation by growth factor).
Under this temporal constraint, we computed
the dependence of activation of cPLA2
on MAPK and vice versa at steady state (25
to 40 min) after 5 min of stimulation (Fig.
1C). The system exhibits bistable behavior as
seen by the three intersection points between
the two computed concentration-effect curves
(Fig. 1C). Initial stimulation by PDGF that
raised MAPK or cPLA2 activity above the
levels defined by the middle metastable intersection
point (denoted by number 2 in Fig.
1C) allows the system to come to an activated
steady state (uppermost intersection point,
number 3 in Fig. 1C) whereas the low-level
stimulation results in the system relaxing to
the basal state (lowermost intersection point,
number 1 in Fig. 1C) as soon as the stimulus
was withdrawn. We examined whether we
could observe a similar profile experimentally
(19). NIH-3T3 cells deprived of serum
were stimulated with PDGF for 5 min. The
cells were washed and the activation state of
MAPK was determined by immunoblotting
1
National Center for Biological Sciences, Bangalore
560065 India. 2
Department of Pharmacology and Biological
Chemistry, Mount Sinai School of Medicine,
New York, NY 10029, USA.
*These authors contributed equally to this work.
†Queries regarding computational analysis should
be addressed to U.S.B. (e-mail: bhalla@ncbs.res.in)
and experimental work to P.T.R. (e-mail: prahlad.
ram@mssm.edu
Fig. 1. The receptor tyrosine kinase, MAP kinase,
and protein kinase C signaling network and feedback
loop. (A) Block diagram of network. The
components highlighted in green form the positive-feedback
loop. The pathways highlighted in
orange are inhibitory. This block diagram abstracts a complex network that is described in detail in
the supplementary materials. Abbreviations: PDGFR, platelet-derived growth factor receptor; PKC,
protein kinase C; AA, arachidonic acid; PP2A, protein phosphatase 2A; MEK, MAPK or ERK kinase;
MAPK, mitogen-activated protein kinase 1, 2; cPLA2, cytoplasmic phospholipase A2; MKP, MAP
kinase phosphatase; DAG, diacylglycerol. (B) Simulation of the levels of active MAPK at various time
points after activation with 50 ng/ml PDGF for 5 min; the stimulus period is indicated by the bar
at the bottom of the graph. (C) Intersecting concentration-effect curves for steady-state MAPK
activation by cPLA2 (ᮀ), and cPLA2 activation by MAPK (), plotted on the same axes. cPLA2
activity is measured in terms of concentrations of AA, its product. MKP activity in these simulations
is fixed at the initial levels of 2.4 nM total MKP (MKP-1 ϩ MKP-2). The three intersection points
define stable points of the system. Point 1 is the basal level of activity, point 2 is a metastable
point, and point 3 is the high stable state of activity. (D) Activation state of MAPK in NIH-3T3 cells
after stimulation with PDGF. Serum-starved cells were stimulated with 50 ng/ml PDGF for 5 min
and then washed with serum-free medium. The cells were then reincubated in serum-free medium
for various lengths of time. Protein from soluble cell lysate was probed with antibody specific for
dual-phosphorylated MAPK (Phospho-MAPK 2).
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with antibody to the phosphophorylated
forms of MAPK 1 and 2. MAPK activity
increased within 5 min and stayed active for
more than 40 min after removal of PDGF
(Fig. 1D). Thus, for approximately eightfold
duration of the initial stimulus the system
stays stably active. Because the parameters
used for the modeling are largely derived
from in vitro measurements, often with purified
proteins, there is no quantitative congruence
between the simulations and the experiments
conducted in the intact cell. However,
the qualitative features predicted by the simulation
were observable experimentally in an
intact cell system.
If the persistently increased MAPK activity
is due to the presence of a feedback loop,
then inhibition of cPLA2, a target of MAPK,
should block the persistent activation of
MAPK while allowing acute stimulation.
Computational analysis indicated that this is
indeed the case (Fig. 2A). Experimentally,
NIH-3T3 cells were treated without or with
arachidonyltrifluoromethyl ketone (AACOF3),
a selective inhibitor of cPLA2 (19),
then stimulated with a 5-min pulse of PDGF,
washed, and assayed for activation of MAPK
1, 2. Treatment with the cPLA2 inhibitor did
not inhibit acute stimulation, but MAPK 1, 2
was not persistently activated (Fig. 2B).
These results indicate that PKC may have a
pivotal role in maintaining the feedback, because
the arachidonic acid produced by
cPLA2 in conjunction with basal concentrations
of diacylglycerol results in the stimulation
of PKC at low Ca2ϩ
(20). Hence, inhibition
of PKC should block the persistent
activation of MAPK 1, 2 and did so in computational
experiments (Fig. 2C). In experiments,
we used bisindolylmaleimide 1
(BIM1), an inhibitor of the typical forms of
PKC, to determine if it blocked the persistent
activation of MAPK. BIM1 does not affect
the initial activation of MAPK 1, 2, but the
activated MAPK 1, 2 returns to basal state
within 30 min (19). Similar results were obtained
when the levels of PKC were decreased
by pretreatment with phorbol esters
(19) (fig. S1). Thus, we observed a network
of MAPK 1, 2, cPLA2, and PKC in NIH-3T3
cells that behaves as a bistable system, i.e.,
moves between two stable states in a stimulus-dependent
manner.
We next analyzed the role of MKP or any
other protein phosphatase in regulating this
bistable behavior. Because dephosphorylation
of Raf and MEK by PP2A can also
regulate bistability, we determined the ability
of the network to exhibit bistable behavior at
fixed varying concentrations of PP2A and
MKP. At each paired concentration of phosphatases,
we used concentration-effect analysis
at steady state to obtain stable and metastable
points (Figs. 1C and 3C). We obtained
2200 such concentration-effect curves. If a
single stable point was obtained, then the
system existed in a monostable state. If two
stable points and one metastable point were
obtained, the system was in a bistable state.
Figure 3A displays the results of these simulations
in a perspective view in three dimensions.
The breaking-wave–like bistable
region in the center represents the bistable
region. The same data viewed from above in
two dimensions are shown in Fig. 3B. The
bistable behavior denoted by diamonds occupies
a defined region of parameter space, i.e.,
at paired ratios of PP2A to MKP. From the
simulation in Fig. 3B, it can be seen that
increasing concentrations of MKP at fixed
concentrations of PP2A decreases the bistable
region and would move the system into a
monostable regime. To ascertain that at elevated
MKP levels the system exhibited
monostable behavior, we analyzed the effect
of varying concentrations of active MAPK on
activated cPLA2 and vice versa. This computation
is identical to that shown in Fig. 1C
except for the higher MKP concentration (12
nM). There is a single intersection point at the
basal state of MAPK and cPLA2 when MKP
levels are increased by about fivefold (Fig.
3C). Because PP2A levels are very tightly
controlled within the cell (21), we focused on
MKP, which is induced by MAPK activation.
Thus, with everything else in the system staying
the same, the elevation of MKP levels
would move the system into a monostable
regime.
We tested the temporal effect of increasing
MKP (concentrations and hence activity)
on sustained MAPK activity (Fig. 3D). Here,
a 5-min stimulatory pulse (e.g., PDGF stimulation)
was applied and MAPK 1, 2 activity
was followed for 90 min with different rates
of MKP synthesis, thereby resulting in different
amounts of steady-state MKP. At low
concentrations of MKP, persistent activation
is obtained, but at higher concentrations of
MKP, upon withdrawal of the stimulus, the
activated MAPK 1, 2 returns to the basal
state. To provide an experimental frame of
reference, we measured the amounts of MKP
induced by MAPK activation. Treatment
with PDGF results in a rapid increase in MKP
Fig. 2. Role of cPLA2 and PKC activity in sustained MAPK activation. (A) Simulated values of MAPK
activity at various time points after stimulation with 50 ng/ml PDGF for 5 min, with (dashed line)
or without (solid line) cPLA2 activity. The bar indicates the duration of stimulus, and the vertical
dashed line corresponds to the first experimental time point as shown in (B). Simulated traces were
independently scaled. (B) Effects of the cPLA2 inhibitor on MAPK activity in NIH-3T3 cells after
stimulation with PDGF and with (E) or without (ᮀ) the cPLA2 inhibitor. Serum-starved cells were
incubated with 10 ␮M AACOF3 for 2 hours before stimulation with PDGF (50 ng/ml) for 5 min. The
cells were washed with serum-free medium and reincubated for the times indicated. Proteins from
soluble cell lysate were probed with antibody specific for dual-phosphorylated MAPK (PhosphoMAPK
2). (C) Simulated values of MAPK activity at various time points after stimulation with 50
ng/ml PDGF for 5 min, with normal (dashed line) or 80% blocked (solid line) PKC activity. The bar
indicates the stimulus, and the vertical dashed line corresponds to the first experimental time point
as shown in (B). (D) MAPK activity in NIH-3T3 cells after stimulation with PDGF and with (E) or
without (ᮀ) PKC inhibitor. Serum-starved cells were incubated with 10 ␮M BIM1 for 30 min before
stimulation with PDGF (50 ng/ml) for 5 min. The cells were washed with serum-free medium and
reincubated for the times indicated. Proteins from soluble cell lysate were probed with antibody
specific for dual-phosphorylated MAPK (Phospho-MAPK 2).
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protein by about 30 min, and MKP stays
elevated for over 2 hours (Fig. 3E). In cells
containing threefold elevated levels of MKP
(19), a 5-minute stimulation with PDGF increases
activated MAPK 1, 2 briefly; removal
of the stimulus results in a return to basal
state within 30 min (Fig. 3F). To ascertain
that this lack of prolonged activation was due
to elevated MKP, we determined that transfection
with MKP1 produced a similar effect
(19) (fig. S2). Thus, it appears that elevation
of MKP can push the MAPK 1, 2 system into
a monostable regime in NIH-3T3 fibroblasts.
We next analyzed how the bistable and
monostable MAPK 1, 2 system would respond
to varying concentrations of PDGF.
Computational analysis showed that when
the system was capable of bistable behavior,
a sharp threshold was observed when
the effect of varying concentrations of
PDGF on sustained activation of MAPK
was measured (Fig. 4A). We determined if
this could be observed in NIH-3T3 cells.
We treated cells with varying concentrations
of PDGF for 5 min. Cells were
washed and incubated for 30 min and the
activation state of MAPK 1, 2 was measured.
A sharp transition between 5 and 10
ng/ml PDGF was observed (Fig. 4B). Thus,
the bistable response works in a switchlike
manner. When the amount of MKP is increased,
the sustained switching response
does not occur and the computational analysis
shows an acute MAPK 1, 2 response
that is proportional over at least a 10-fold
concentration range of added growth factor
(Fig. 4C). Thus, at high MKP concentrations
the system behaves as a proportionalresponse
system. To investigate whether
this could be observed in the NIH-3T3
cells, we stimulated NIH-3T3 cells with
PDGF for 5 min to increase MKP concentrations.
The cells were washed, incubated
for 60 min, and restimulated with varying
concentrations of PDGF for 5 min, and the
Fig. 3. Role of MAPK
phosphatase in regulating
the bistable behavior.
(A) Three-dimensional
plot of the bistable
behavior of MAPK/
cPLA2/PKC network as a
function of regulation
by PP2A and MKP. MKP
feedback activation is
disabled, and only
steady-state MKP is applied
at the plotted levels.
The vertical axis
measures feedback loop
activity as MAPK activity.
The upper region
represents a state of
high activity, and the
lower plane one of low
activity. In the intermediate
“breaking wave”
region, the curves fold
around from the upper
to the lower state, and
thus this comprises a
bistable region where
both states can exist.
The white curve represents
the system response
at varying levels
of MKP at the reference
level of 0.225 ␮M PP2A.
The white arrow indicates
the trajectory of
system upon PDGF
stimulation and elevation
to a state of high
activity. The cyan arrow
indicates the trajectory
as MKP-1
builds up, and eventually
turns off the
feedback-loop activity. (B) Regions of monostable (dashes) and bistable
(diamonds) behavior of the feedback system. This is equivalent to looking at
plot B from above. There is a large range of combinations of PP2A and MKP
activities over which bistability will occur. (C) Concentration-effect curves
for steady-state MAPK activation by PLA2 (E), and PLA2 activation by MAPK
(‚), plotted on the same axes. cPLA2 activity is measured in terms of
concentrations of AA, its product. MKP activity in these simulations is fixed
at the induced levels of 12 nM total MKP (about five times basal level). The
intersection point (arrow) defines the one low-activity stable point of the
system. (D) Sustained MAPK activity is dependent on the rates of MKP
synthesis and the resultant steady-state MKP levels. In this simulation a
5-min activation with PDGF is followed with varying rates of MKP synthesis.
MAPK activity is plotted as a function of time as MKP is being
synthesized. (E) MKP levels following PDGF stimulus in NIH-3T3 cells.
Serum-starved cells were stimulated with PDGF (50 ng/ml) for 5 min,
washed, and reincubated in serum-free medium for the times indicated.
Proteins from soluble cell lysate were probed with antibody for MKP. (F)
Effect of MKP levels on sustained activation of MAPK. Serum-starved
NIH-3T3 cells were stimulated with PDGF (50 ng/ml) for 5 min (E) or
vehicle (ᮀ), washed, and reincubated for 60 mins in serum-free medium.
The cells were restimulated with PDGF (50 ng/ml) for 5 min, washed, and
reincubated in serum-free medium for the times indicated. Proteins from
soluble cell lysate were probed with antibody specific for dual-phosphorylated
MAPK (Phospho-MAPK2). (Inset) Lysate probed for MKP, showing
that levels of MKP are higher in the cells after the prepulse of PDGF
followed by incubation for 60 min.
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activity state of MAPK was determined.
Increasing concentrations of PDGF gave
correspondingly increasing amounts of
MAPK 1, 2 activity from 0.5 to 10 ng/ml of
PDGF (Fig. 4D). Thus, at elevated concentrations
of MKP, the MAPK network is
monostable and behaves only as a proportional-response
system. MKP does not appear
to have a role in the acute deactivation
of MAPK (22) (Figs. 2 and 3). Knockout of
MKP1 also does not yield an acute phenotype
(23). The acute deactivation is likely
to be controlled by PP2A dephosphorylating
Raf and MEK and by other phosphatases
working at the level of MAPK 1, 2.
Thus, the role of the increased MKP expression
appears to be to determine the
system response capabilities to subsequent
stimuli as shown below.
One of the functions of signaling networks
is to confer adaptability to cellular
responses. We examined whether a previous
stimulus-and-response history could influence
subsequent responses of the system
to a new stimulus. To analyze this computationally,
we stimulated the network with
varying concentrations of PDGF for 5 min;
the stimulus was then removed, and after
60 min, a saturating stimulus of 50 ng/ml
PDGF was delivered for 5 min followed by
removal of the stimulus. MAPK activity
was followed as a function of time (Fig.
5A). This dual stimulus protocol illustrates
the effects of prior history of stimulation on
later responses to stimuli. Computationally,
we found that the final sustained MAPK
activity depends on the concentration of the
PDGF prepulse. Only concentrations of
PDGF that were initially below the threshold
elicited a switchlike response to the
second stimulus (Fig. 5B). The observed
response shows a sharp threshold with the
same half-maximum as for turn-on of the
MAPK bistable state, but with the opposite
slope. We experimentally measured MAPK
activity at the final time point, 30 min after
the second stimulus (Fig. 5C) (19). Cells
that were initially stimulated with concentrations
of PDGF that are above the threshold,
to elicit a prolonged MAPK response
and therefore an increase in MKP, are no
longer able to sustain a prolonged MAPK
response upon restimulation with a suprathreshold
stimulus. Thus, it appears that the
network adapts to a stimulus such that if the
stimulus is greater than the threshold and
elicits a switchlike response, a subsequent
suprathreshold stimulus does not elicit a
second switchlike response. Conversely, if
the initial stimulus is below threshold and
does not elicit a sustained response, a subsequent
suprathreshold stimulus is now capable
of eliciting a switchlike response.
This flexibility of the network confers upon
it the ability to respond to a series of stimuli
in different ways depending upon the
prior history of stimulus.
Our integrated computational and experimental
analyses show that the MAPK system
in some mammalian cells is flexibly
designed such that it can move between
bistable and monostable regimes in a selfregulated
manner because activation of
MAPK increases the amounts of MKP both
by de novo synthesis and by direct phosphorylation
to block degradation. Such
self-regulated flexibility is likely to be an
important characteristic of biological devices,
necessary for biological systems to
adapt and survive. The MAPK 1, 2 pathway
is capable of regulating multiple cellular
machines, including the transcriptional machinery,
to stimulate the expression of cyclins
that trigger entry into the cell cycle
and the synthesis of arachidonic acid, a
precursor for many autocrine and paracrine
factors. It would not be advantageous to the
cell if, every time an autocrine or paracrine
factor needed to be synthesized, the cell
were pushed into the cell cycle. Because
sustained MAPK activity is required to reenter
the cell cycle (24), it is possible that
increased MKP concentrations allow the
MAPK network to respond to external
stimulation to control the biochemical machinery
without engaging the cell-cycle
machinery.
It is paradoxical that although the bistable
behavior is a systems property as a whole,
MKP appears to be the locus of flexibility.
Thus, MKP can be thought to have distinct
properties at two levels: At the molecular
level, MKP is a dual-specificity phosphatase;
at the systems level, MKP functions as the
locus of flexibility that determines whether or
not the system exhibits bistable behavior.
This is the second phosphatase that we have
found to play a determinant role in systems
behavior. In the adenosine 3,5-monophosphate–␣-calcium-calmodulin–dependent
kinase
II (cAMP-CAM-kinase II) network, protein
phosphatase–1 gates signal flow through
the CAM-kinase II pathway during the induction
of long-term potentiation of synaptic
responses (25). Perhaps other negative regulators
of signaling such as phosphatases,
phosphodiesterases, and GTPase-activating
proteins also endow signal networks with
unique systems capabilities.
Fig. 4. Effects of varying concentrations of PDGF on MAPK activity at low and high MKP levels. (A)
Simulation of MAPK response to varying concentrations of PDGF at basal MKP levels. (Inset)
Enlargement of the same curve from 0 to 10 ng/ml. (B) PDGF concentration-effect curve in
NIH-3T3 cells. Serum-starved NIH-3T3 cells were stimulated with various concentrations of PDGF
for 5 min, washed, and reincubated for 30 min in serum-free medium. Proteins from soluble cell
lysate were probed with antibody specific for dual-phosphorylated MAPK (Phospho-MAPK 2).
(Inset) Enlargement of the curve from 0 to 10 ng/ml PDGF. The threshold is within experimental
accuracy given the conversion 1 ng/ml ϭ 30 pM PDGF. (C) Simulation of MAPK response to varying
concentrations of PDGF at elevated MKP levels (0.01 ␮M, about four to five times basal). (D) Effects
of varying concentrations of PDGF on MAPK activity in NIH-3T3 cells at high MKP levels.
Serum-starved NIH-3T3 cells were stimulated with 50 ng/ml PDGF for 5 min, washed, and
reincubated for 60 min in serum-free medium. The cells were then restimulated with various
concentrations of PDGF for 5 min. Proteins from soluble cell lysate were probed with antibody
specific for dual-phosphorylated MAPK (Phospho-MAPK 2). (Inset) Enlargement of the concentration-effect
curve from 0 to 10 ng/ml PDGF.
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Current experimental techniques allow
us to observe the predicted systems behavior
only in a qualitative manner. To determine
quantitative accuracy of our models,
techniques that quantitatively and selectively
measure biochemical reactions within
the cell must be developed. Nevertheless,
this first glimpse into the systems
capabilities of a well-understood and widely
used cell signaling system allows us to start
unraveling the underlying complexity in nature’s
design of this controller network.
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26. We thank R. Blitzer for critical reading of the manuscript.
We also thank the anonymous reviewers for
their incisive comments that were most useful in
revising this paper. This research was supported by
NIH grants GM-54508 and CA-81050 (R.I.) and the
Wellcome Trust and NCBS funds (U.S.B.). P.T.R. was
supported by an NIH postdoctoral fellowship (grant
CA-79134).
Supporting Online Material
www.sciencemag.org/cgi/content/full/297/5583/1018/
DC1
Supporting text
10 December 2001; accepted 13 June 2002
Est1p As a Cell Cycle–Regulated
Activator of Telomere-Bound
Telomerase
Andrew K. P. Taggart, Shu-Chun Teng,* Virginia A. Zakian†
In Saccharomyces cerevisiae, the telomerase components Est2p, TLC1 RNA,
Est1p, and Est3p are thought to form a complex that acts late during chromosome
replication (S phase) upon recruitment by Cdc13p, a telomeric DNA
binding protein. Consistent with this model, we show that Est1p, Est2p, and
Cdc13p are telomere-associated at this time. However, Est2p, but not Est1p,
also binds telomeres before late S phase. The cdc13-2 allele has been proposed
to be defective in recruitment, yet Est1p and Est2p telomere association persists
in cdc13-2 cells. These findings suggest a model in which Est1p binds telomeres
late in S phase and interacts with Cdc13p to convert inactive, telomere-bound
Est2p to an active form.
In S. cerevisiae, telomeric DNA consists of
irregular C1-3A/TG1-3 double stranded repeats
as well as the G tail, a Ͼ30 nucleotide
single-stranded overhang of TG1-3 DNA that
is detectable only in late S phase (1, 2).
Telomere length is maintained by telomerase,
a specialized reverse transcriptase with an
integral RNA template. In yeast, telomerase
action requires at least five genes, deficiencies
in any or all of which lead to the same est
(ever shortening telomeres) phenotype, a progressive
telomere shortening leading to eventual
cell death (3). EST2 and TLC1 encode the
reverse transcriptase and RNA subunit, respectively.
Telomerase action in vivo also
requires Est1p, Est3p, and Cdc13p, a protein
that binds single-stranded TG1-3 DNA. A
current model for telomerase regulation is
that Cdc13p binds G tails in late S phase to
recruit a telomerase holoenzyme consisting
of Est1p, Est2p, Est3p, and TLC1 RNA (4–
Department of Molecular Biology, Princeton University,
Princeton, NJ 08544, USA.
*Present address: Department of Microbiology, College
of Medicine, National Taiwan University, Taiwan.
†To whom correspondence should be addressed. Email:
vzakian@molbio.princeton.edu
Fig. 5. History-dependent responses of the MAPK system. A varying concentration of PDGF was
delivered for 5 min, followed by a wash and reincubation. After 60 min a second stimulation was
delivered at 50 ng/ml, which is normally saturating to elicit prolonged MAPK activation. (A) Computational
traces show range of response time-courses. Initial and second stimulation durations are
indicated by horizontal bars. Concentrations of PDGF during the initial stimulation are 20 pM (0.67
ng/ml), 100 pM (3.33 ng/ml), 200 pM (6.67 ng/ml), 500 pM (16.67 ng/ml), 1 nM (33.33 ng/ml), and 100
nM (333.33 ng/ml). (B) Computationally derived MAPK activities at the final time point, 100 min after
start. (C) Effects of pretreatment of NIH-3T3 cells with varying concentrations of PDGF on subsequent
stimulation by a saturating concentration. Serum-starved NIH-3T3 cells were stimulated with various
concentrations of PDGF as indicated for 5 min, washed, and reincubated for 60 min in serum-free
medium. The cells were then restimulated with 50 ng/ml PDGF for 5 min, washed, and incubated for
30 min. Proteins from soluble cell lysate were probed with antibody specific for dual-phosphorylated
MAPK (Phospho-MAPK 2).
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