Dynamical and Integrative Cell Signaling:
Challenges for the New Biology
Andre Levchenko
The Whitaker Institute for Biomedical Engineering, The Johns Hopkins
University, 208C Clark Hall, 3400 North Charles Street, Baltimore, Maryland
21218; telephone: 410-516-5584; fax: 410-516-6240;
e-mail: alev@ bme.jhu.edu
Published online 24 November 2003 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.10854
Abstract: Years of careful experimental analysis have
revealed that signaling molecules are organized into
complex networks of biochemical reactions exquisitely
regulated in time and space to provide a cell with high-
fidelity information about an extremely noisy and volatile
environment. A new view of signaling networks as
systems consisting of multiple complex elements interact-
ing in a multifarious fashion is emerging, a view that
conflicts with the single-gene or protein-centric approach
common in biological research. The postgenomic era has
brought about a different, network-centric methodology of
analysis, suddenly forcing researchers toward the opposite
extreme of complexity, where the networks being explored
are, to a certain extent, intractable and uninterpretable.
Both the cartoons of simple pathways and the very large
‘‘hair-ball’’ diagrams of large intracellular networks are
also representations of static worlds, superficially devoid
of dynamics and chemistry. These representations are
often viewed as being analogous to stably linked computer
and neural networks rather than dynamically changing
networks of chemical interactions, where the notions of
concentration, compartmentalization, and diffusion may
be the primary determinants of connectivity. Arguably, the
systems biology approach, relying on computational
modeling coupled with various experimental techniques
and methodologies, will be an essential component of
analysis of the behavior of signal transduction pathways.
Combining the dynamical view of rapidly evolving res-
ponses and the structural view arising from high-through-
put analyses of the interacting species will be the best
approach toward efforts toward greater understanding of
intracellular signaling processes. B 2003 Wiley Periodicals, Inc.
Keywords: signal transduction; computational modeling;
high-throughput data
INTRODUCTION
Over the last decade, exploration of intracellular signal trans-
duction has come of age, firmly establishing the complexity
of biochemical processes in terms of underlying structure
and dynamics. We increasingly consider signal transduction
to involve complex biochemical circuits transducing infor-
mation on the state of the cell and its surroundings to other
Correspondence to: A. Levchenko
B 2003 Wiley Periodicals, Inc.
regulatory networks capable of effecting the appropriate
response, assuming the cell state is other than normal.
Signaling pathways and networks are thus an essential part
of complex decision processes that can result in outcomes as
dramatic and diverse as cell division, differentiation, trans-
formation, or suicide.
As reviewed recently in several excellent literature
surveys (e.g., Bhalla, 2003; Rao and Arkin, 2001; Tyson
et al., 2001, 2003; Weng et al., 1999; Wiley et al., 2003),
postgenomic analysis of signal transduction is increasingly
characterized by reliance on both experiment and computa-
tional investigation to aid our understanding of the signaling
pathway structure and function. The last decade has wit-
nessed the convergence of results from various kinds of
innovative or more ‘‘classic’’ experimental methods, includ-
ing genetic and biochemical studies, use of novel fluores-
cent markers in combination with increasingly precise and
sophisticated microscopy techniques, sequencing of whole
genomes, large-scale DNA array and proteomics experi-
ments, use of microfabrication and microfluidics method-
ologies, etc. Consequently, most pathways can now be fully
traced from the level of receptor to various downstream
targets, and, as such, compared between different species.
This information is further supplemented by a rather diverse,
often still sparse and disorganized array of facts character-
izing pathway members in terms of biochemical constants,
spatial localization, levels of expression, temporal activity
profiles, and biological function. Computational modeling is
increasingly called upon to further integrate the collection of
qualitative facts and disparate measurements into a cohesive
and quantitative description that can be used to guide further
experimentation through verifiable (or, if preferred, falsifi-
able) predictions. These models can be especially useful if
they intend to describe competing mechanisms proposed by
different sources, so that a set of criteria allowing one to
distinguish between different hypotheses can be formulated
based on the underlying computational predictions. Compu-
tational models, by their nature, serve as repositories of the
current knowledge, both established and hypothetical, on
how signaling pathways might operate, providing one with
quantitative codification of this knowledge and with the
ability to simulate the biological processes according to this
codification. As computational models are becoming in-
creasingly mainstream, it is of interest to examine the chal-
lenges present in this emerging area of biological research.
Perhaps paradoxically, some of the challenges have been
brought about by the rapid advent of the postgenomic era
itself, carrying with it an increasing reliance on representa-
tion and analysis of large-scale networks. Although poten-
tially very revealing, the popular ‘‘hair-ball’’ diagrams of
molecular interactions are rarely interpretable and suffer
from many pitfalls characteristic of more classic cartoon
pathway representations (Fig. 1). In particular, the question
of what links between nodes in such diagrams actually
represent in terms of relevant cell responses is not very well
posed. Can the pathways in the networks be identified and
considered separately? What if the network is only partially
active, with multiple ‘‘blind alleys’’ and some signals never
reaching the designated targets? Does such a case represent
extensive or limited crosstalk—the spread of signaling
activity to various pathways not conventionally implicated
in the response to a particular ligand? How does stimulus
timing affect signal propagation? How does one represent
feedback interactions? This list of questions can be expanded
considerably. Another challenge is to understand how repre-
sentations of signaling networks can be expanded to include
other regulatory networks; for example, metabolic, gene
expression, cytoskeletal, etc. Also, above in the complexity
hierarchy, how can cell signaling networks be integrated into
the larger networks of interacting cells, tissues, and possibly
organisms? Although prospects for such integration seem to
be a distant hope, various extensions of the current
simplistic views of cell signaling are inevitable and forth-
coming. In this review, I attempt to analyze the challenges
just mentioned in more detail and outline how some of these
challenges might be approached. Examples from signaling
networks operating in yeast and higher eukaryotes are used
for illustrative purposes only, with no intention of
comprehensive coverage of the signal transduction field.
INTEGRATION OF PATHWAYS INTO NETWORKS
From Pathways to Networks
Signal transduction pathways are often viewed as linear
chains of biochemical reactions leading from the sensor
molecules to intracellular targets. In many instances, such a
view appears both biologically justified and inform-
ative. (Evidence for signal crosstalk discussed herein is
based on various experimental data that should be treated
with caution, especially when the corresponding signaling
proteins are overexpressed.) Indeed, sometimes even the
pathways sharing chemical components may be ‘‘insulated’’
in terms of specificity between the signaling input and the
corresponding output. For instance, several mitogen-acti-
vated protein kinase (MAPK) pathways in yeast S.
cerevisiae share the Ste20 PAK-like kinase and the Ste11
MAPKKK(Posas et al., 1998) (Fig. 2). However, the signal
rarely ‘‘spills over’’ and only genes corresponding to a
specific input are normally expressed. The precise reasons
for this pathway insulation are not known, but several
experimental studies have suggested that the negative
feedbacks present in the pathways may limit crosstalk
through limitation of the duration of signaling. For instance,

Figure 1. High-throughput data representations are often presented as static networks (a), whereas a more realistic approach is to consider them as maps
of potential interactions, in which parts of the network or pathway may be active at any particular time: (b) and (c). Crosstalk between different signaling
pathways and interaction between signal transduction and other regulatory networks can also dynamically activate parts of the network (d). A full activation
of the network (e) occurs rarely, if ever. Dynamical description of network behavior based on quantitative modeling data is advocated here.

774 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 84, NO. 7, DECEMBER 30, 2003
Figure 2. An example of a real biological network composed of signaling pathways in S. cerevisiae. This network demonstrates extensive cross-
activation of various regulatory pathways that can potentially take place based on experimentally found functional interactions. For more details about this
network and its members see text (see also Dohlman and Thorner, 2001; Elion, 2000; Gagiano et al., 2002).

the negative feedback activated by Fus3 does not allow Kss1
to activate the nitrogen-limitation-sensitive genes (Sabbagh
et al., 2001), whereas the negative feedback activated by
Hog1 precludes activation of pheromone-responsive genes
by high osmotic pressure (O’Rourke and Herskowitz, 1998).
This cross-inhibition occurs in spite of substantial activa-
tion of the corresponding kinases (Kss1 and Fus3) by the
inappropriate stimuli. These findings suggest that, although
cross-talk may be quite extensive in parts of the signaling
network, it may not normally percolate to the corresponding
targets. Such instances of ‘‘partial crosstalk’’ may lead to
confusion that can be partly alleviated by a clear definition
of the beginning and endpoints of a pathway and by what is
viewed as the outcome of a signaling event.
Another important factor in establishing the extent of a
signaling crosstalk is the signal duration at various levels of a
signaling pathway, as evidenced, for example, by differ-
ential gene expression in response to transient vs. persistent
signals. For instance, rapid downregulation of a transcription
factor, NFAT, following signal cessation makes it a good
sensor of the duration of rapid calcium transients (Dolmetsch
et al., 1997; Feske et al., 2001). Depending on the duration of
its activity, NFAT can induce expression of different groups
of genes, leading to activation of increasing subsets of
signaling networks (Feske et al., 2000). The importance of
the duration of MAPK signaling in determining signaling
outcomes, as diverse as cell division versus cell differ-
entiation, is also well established (see Marshall [1995] for a
seminal review). This is further in evidence in activation of
the nuclear factor NF-nB pathway, where increasing signal
duration entails expression of progressively larger groups of
genes, including ligands capable of autocrine signal trans-
duction (Hoffmann et al., 2002).
In addition, signaling crosstalk can be affected by
the signal strength. Different MAPK pathways in T cells
have disparate activation thresholds, so that only the
mitogenic Erk kinase is activated in response to both low
and high signaling inputs, whereas stress-induced JNK and
p38 kinases are activated only at high input values (Gong
et al., 2001). Another example of signal-amplitude-depend-
ent crosstalk is the bell-shaped kinetics of cAMP accumu-
lation in brown adipocytes in response to norepinephrine
(NE) (Bronnikov et al., 1999). A detailed analysis revealed
that the adenylyl cyclase-mediated production of cAMP was
upregulated at low NE concentrations through G-protein
signaling, as expected in the h-adrenergic response. How-
ever, higher NE concentrations led to increasing cytosolic
Ca2+ concentration, which stimulated a calmodulin-con-
trolled phosphodiesterase, possibly PDE-1. Activation of the
phosphodiesterase, in turn suppressed the adenylylcyclase,
leading to lower cAMP production. In EGF signaling it has
been demonstrated that a positive effect of PI3K on MAPK
activity is present at low, but not high, ligand concentrations
(Wennstrom and Downward, 1999).
The present examples demonstrate that signaling cross-
talk, in addition to being organism- and cell-type-specific,
can also be context-specific, which may further complicate
dealing with the ‘‘hair-ball’’ diagrams representing the maps
of potential signaling pathway interactions. It is crucially
important to clearly designate the beginning and endpoints
of a pathway and specify the amplitude and duration of the
input signal. It is likely that considering only experimentally

LEVCHENKO: CELL SIGNALING IN THE NEW BIOLOGY 775

validated computational models of coupled signaling path-
ways with an explicit emphasis on the kinetics of signaling
will result in both a clear definition and accurate description
of the interaction between signaling pathways.
From Signaling Networks to Cell Regulation
Networks
In addition to sensing and transmitting information about
extracellular and intracellular stimuli, signaling path-
ways serve to distribute the information to various response
elements within the cell, which might in turn affect the
signaling pathways in a feedback fashion. For instance, the
yeast pheromone pathway (Fig. 2) transmits signals to actin
cytoskeleton-regulating cell chemotropism toward the
pheromone source, and to the nucleus, resulting in changes
in expression of multiple genes, including those regulating
the cell cycle (Ayscough and Drubin, 1998; Cherkasova
et al., 1999; Lyons et al., 1996; Peter et al., 1993). The yeast
protein kinase A pathway, involved in sensing of ambient
glucose levels (the ‘‘starvation’’ pathway), signals both to
the metabolic pathways and to a range of genes mediating
exploratory cell behavior (Muller et al., 2003; Pan et al.,
2000; Pan and Heitman, 2002). PKA signaling to the
metabolic network proceeds through phosphorylation and
activation of phosphofructokinase-2 (PFK2), resulting in an
increase in the fructose-2,6-bisphosphate level. This meta-
bolic messenger in turn activates the key regulatory enzyme,
phosphofructokinase-1 (PFK1), and inhibits the glyconeo-
genetic enzyme, fructose-bisphosphatase-1 (FBpase1), thus
affecting glycolytic flux. The genes affected by the PKA
pathway include FLO11, which has a product (Pan and
Heitman, 2002) that regulates cell – cell and cell –matrix
adhesion and may affect the efficiency of colony growth into
the regions with a better nutrient supply. In both cases, the
signal transduction pathways are integrated with other
extensive biochemical networks, feeding back by affecting
the expression of various members of the pathway (the
pheromone response) or the levels of intermediate compo-
nents (e.g., regulation of the particular way of ATP synthesis
by the glucose-responsive PKA pathway). One of the
challenges in development of computational models of
signal transduction is thus to present an integrative view, in
which signaling networks affect and are affected by other
regulatory networks.
Clearly, detailed coupling of models of signaling path-
ways and models of other regulatory networks is a tremen-
dous task. The ‘‘lumped’’ models can rapidly increase in
complexity and lose their predictive power. Thus, retaining a
modular view of a network, in which parts may be separable
both theoretically and experimentally, might be a wiser
strategy. For instance, networks can be decomposed based
on structural or functional module identification, classifica-
tion of processes occurring on different time scales, or
localization information. Note that these network segrega-
tion techniques may or may not reproduce the initial
separation into a signal transduction and ‘‘nonsignaling’’
776 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 84, NO. 7, DECEMBER 30, 2003
components. For instance, phosphorylation events in signal-
ing and metabolic reactions may be decoupled from slower
processes corresponding to gene transcription or cytoskele-
ton rearrangement. In this time-scale separation, depending
on the rates of corresponding processes, concentrations of
some molecules can be viewed as almost constant, effec-
tively turning the corresponding differential equations into
algebraic ones and simplifying the underlying model. On the
other hand, a functional module, such as a MAPK cascade
with a positive feedback serving to convert a graded input
into an all-or-none output, can involve gene transcription as
an essential feedback mechanism (Ferrell, 2002).
The hypothesis that various intracellular processes may
rely on molecular networks that are hierarchical and modular
in structure is experiencing a revival, now that many relevant
regulatory networks are being identified and charac-
terized (Hartwell et al., 1999; Thieffry and Romero, 1999).
However, so far, the definition of a module has been at best
vague and no standard methodology for module identi-
fication exists. There has been some progress in the
demonstration that cellular regulatory networks are in fact
hierarchical. Indeed, various clustering techniques and
searches for predetermined connectivity patterns have
provided considerable evidence for nonrandomness of
metabolic, transcriptional, and protein interaction networks
(Holme et al., 2003; Lee et al., 2002; Milo et al., 2002;
Ravasz et al., 2002; Rives and Galitski, 2003). The presence
of recurring topological connectivity motifs was an espe-
cially surprising feature of transcriptional networks. Despite
these successes, it is at present not clear whether the highly
connected parts of networks can indeed be viewed as
functional modules, in the sense of being semiautonomic and
replaceable parts, or perhaps reflections of the inherent
control requirements for the regulatory processes involved.
Again, use of computational models allowing dynamical
simulation of the underlying systems has a considerable
potential for revealing further properties of the identified
motifs and for definition of what a functional module might
be. Initial attempts in this interesting area of research are
being made (Bhalla, 2002; Bruggeman et al., 2002; Thieffry
and Romero, 1999).
From Cell Regulation Networks to Cell –Cell
Communication Networks in Tissues
The stimuli transduced by intracellular signaling pathways
often reflect the state of the ambient medium, which may it-
self be affected by other cells. Thus, signals can be ex-
changed between cells directly (e.g., by means of a secreted
ligand) or indirectly (e.g., by influencing the concentrations
of nutrients and metabolites in the extracellular milieu). The
overall result of such interaction may be a coordinated re-
sponse of groups of cells that is adaptive and self-organizing.
The exact nature of such a response is determined, at least
in part, by the identity of the signal exchanged and the
pathways involved in the signal transduction. However,
advanced knowledge of the signals and pathways may be
insufficient for a straightforward prediction of the outcome.
As espoused by the still-developing theory of complex
systems, a combined evolution of interacting ‘‘agents’’ can
lead to emerging complex, often unpredictable behavior pat-
terns, even if the agents are identical and rules of interaction
are relatively simple. The rich behavior of various classes of
cell automata is an often-cited example of such unpredict-
ability (Wolfram, 2002).
We can get a glimpse of the complexity stemming from
cell – cell interactions by analyzing the growth of bacterial
and yeast colonies. The coordinated cell behavior can lead to
very complex shapes assumed by these extremely large
groups of cells over prolonged periods. For instance, micro-
bial colonies can form what is referred to as Eden patterns,
characterized by unstable growth at the edge of the colony
(growth front instabilities), leading to characteristic petal-
like formations (Ben-Jacob et al., 1994; Lacasta et al., 1999;
Reynolds and Fink, 2001). These petals can potentially
structure the colony by allowing it to concentrate growth to
specific zones or form channels for nutrient excess and
metabolite removal. A detailed analysis of this and more
complex morphologies in giant yeast colonies has revealed
that the likely determinants of the ultimate shape assumed
include the nutrient and metabolite concentrations, the
mechanical interaction with the underlying substratum, and
potentially secreted ligands (Sams, 1997; A. Levchenko,
unpublished results). The nutrient-sensitive signaling path-
ways are likely to be involved, feeding into the individual
cell responses that regulate the hydrophobicity of the cell
surface or the rate of cell division. For instance, it has been
reported that PKA and a MAPK pathway (the Kss1 pathway;
see Fig. 2) are crucially important for both complex colony
geometries and gel invasion by growing cells. These
pathways can regulate both cell – cell adhesion via the
expression of FLO11, an extracellular glucan glucosidase,
and cell cycle via interaction with cyclins and other compo-
nents of the cell-cycle ‘‘engine’’ (Muller et al., 2003). These
pathways, in turn, can be regulated by the presence of
nutrients in the extracellular medium, which has concen-
trations that are regulated by the presence of cells in a
feedback fashion (Gagiano et al., 2002; Rolland et al., 2002).
How can one deal with the increasing complexity present
at the cell – cell communication level? One promising
possibility is to formulate the relevant input – output func-
tional relationships for the signaling pathways involved and
use them to establish the ‘‘rules’’ of cell –cell interactions.
The signals received by a given cell from other cells would
be inferred from the corresponding transport (diffusion)
terms and from the principle of linear signal superposition.
The signal input – output relationship determining the cell
response to the signal received can be quite complex and
time-dependent. The updates of the signaling states for
individual cells determine the evolution of the whole cell
community. Although a way to deal with this problem
analytically has been recently proposed (Pribyl et al., 2003),
the complexity of the underlying geometry and signaling
LEVCHENKO: CELL SIGNALING IN THE NEW BIOLOGY
response implies that this problem should be handled
primarily though computational simulation.
THE IMPORTANCE OF NUMBERS
In addition to obscuring the importance of the underlying
activation dynamics, the cartoon representations of sig-
naling pathways may suggest that the concentrations of
the signaling components can have, at most, only minor
quantitative influence on the outcome of a stimulus trans-
duction. This view is rooted in the kind of logical approach in
which a link in a network or an arrow in a cartoon diagram
are viewed as defining whether the effect is activating and
inhibitory, with the actual ‘‘strengths’’ of interactions
often of little consequence. However, this outlook is more
reflective of the genetic rather than biochemical approach to
the problem. Indeed, genetic epistasis analysis often
involves logical arguments on binary functions, such as
‘‘if A is absent, is B still present?’’ On biochemical grounds,
however, one is led to consider potentially much more
complex types of relationships between the same A and
B. Hence, not surprisingly, the concentrations of various
‘‘second messengers’’ are predicted and often observed to
have dramatic qualitative effects on how a certain signal is
received, transferred, and acted upon by a cell.
There is ample evidence that the extent of receptor ex-
pression can determine whether a cell enters into a cell cycle,
arrests growth, or undergoes apoptosis (see, e.g., Bauer et al.,
1998; Hendriks et al., 2003; Lebel et al., 1994). Clearly, it is
vitally important for the cell to make such a decision
correctly. However, it is not at all clear how the functionally
acceptable range of receptor concentration values is main-
tained by a cell. It is possible, but not certain, that the cell
monitors the baseline levels of activity through continuous,
low-level activation of signaling pathways and uses nega-
tive-feedback-mediated adaptation to adjust the baseline
signaling to a certain tolerable strength. Such an adjustment
may include regulation of the receptor concentration.
Concentrations of members of signaling pathways down-
stream of the receptor can also have a profound qualitative
affect on signal transduction. In the case of MAPK cascades
(see the earlier examples for yeast), it can be shown that
overexpression of MAPKK, MAPK, or a scaffold molecule
binding the members of the cascade together, when present
above a certain optimal level, can lead to signal inhibition
rather than signal enhancement (Bell et al., 1999; Burack and
Sturgill, 1997; Levchenko et al., 2000; Sugiura et al., 1999).
In yeast and other organisms, concentration and localization
of the members of MAPK pathways is actively regulated by
enhanced gene transcription and degradation as well as
anchoring to the sites of cell polarization (for literature
pertaining to yeast see Dohlman and Thorner [2001] and
Elion [2000]). Thus, we might expect that signal strength is
regulated both as a function of the stimulus and the local
concentration of signaling components, with signal down-
regulation possible even in the absence of traditional
negative regulators.
777
 The concentrations of MAPK phosphatases and other
negative signaling pathway components may affect whether
the MAPK pathway activity exhibits graded or switch-like
responses. For example, in prostaglandin-derived growth
factor (PDGF)-stimulated MAPK activation of fibroblasts,
prepulsing of cells with PDGF prior to persistent stimulation
may cause expression of the inducible MAPK phophatase
MKP-1, which is predicted (based on a computational mo-
del) and observed to make the resultant MAPK activation
proportional to the PDGF concentration (Bhalla et al., 2002).
This signaling response is in contrast to all-or-none MAPK
activation profiles in the absence of the initial prepulse,
suggesting that the history of cell exposure to a ligand may
be important for signaling outcome. It is worth noticing that
other pathways, including the glucocorticoid (Metzler et al.,
1998), calcium calcineurin (Lim et al., 2001), and arach-
idonic acid (Metzler et al., 1998) pathways, may also affect
MKP-1 expression, suggesting interesting possibilities for
signaling crosstalk.
Variations in relative concentrations of different isoforms
of a signaling molecule may also affect signaling outcome.
For instance, in the NF-nB signaling pathway, depending on
the concentration of promiscuous isoforms of the negative
regulator of the pathway, InB, a transient stimulus can lead
to either transient or sustained responses, with differential
effects on gene expression (Hoffmann et al., 2002). Changes
in isoform composition of various composite transcription
factors, including AP-1 (Bakiri et al., 2002; Lallemand
et al.,1997) and NF-nB (Miyamoto et al., 1994), can also
define which genes are expressed and with what efficiency.
Different isoforms of the somatostatin type 2 receptor can
lead to opposite effects on cell proliferation, depending on
their relative amounts (Sellers et al., 2000). These and mul-
tiple other examples underscore the importance of account-
ing for the relative concentrations of signaling cascade
members coexpressed in different forms.
Another example of the importance of precise concen-
tration regulation for the outcome of a signaling process
involves dependence of various transcription factors, the
endpoints of multiple signaling pathways, on the same
coactivator. For instance, CBP/p300 coactivator family
members are recruited by a variety of transcription factors,
including p53 (Chan and La Thangue, 2001), NF-nB (Ravi
et al., 1998), C/EBP (Mink et al., 1997), AP-1 (Albanese
et al., 1999), Ets-1 (Yang et al., 1998), MyoD (Yuan et al.,
1996), STAT1 (Bhattacharya et al., 1996), and others. As
CBP/p300 is present in limiting quantities, inhibitory
crosstalk between different pathways can result, because
a transcription factor activated by one pathway might
sequester CBP/p300 away from interaction with another
factor activated by a different pathway. Clearly, upregula-
tion of expression of CBP/p300 may abolish such negative
pathway cross-regulation, with a significant influence on the
resulting cell behavior.
These findings raise both the practical problem of accurate
accounting of the concentrations of the interacting molecular
species and the scientific problem of understanding how the
778 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 84, NO. 7, DECEMBER 30, 2003
concentrations of molecules appropriate for the correct
signal transduction are maintained through homeostatic
processes in the absence of stimuli. One important and
common change often observed in human malignancies, for
instance, is different patterns of gene expression leading to
up- or downregulation of expression of signaling network
components (Guo, 2003).
SYNEXPRESSION GROUPS OF SIGNALING
PROTEINS: A CROSS BETWEEN GENOMICS AND
BIOCHEMISTRY
Because, as discussed in the previous section, the concen-
trations of pathway components can be a determining factor
in the outcome of signal transduction, it is important to
know how these concentrations may vary before or during
signaling. Although their regulation is probably multi-
factorial and gene-specific, there is one noteworthy observa-
tion suggesting that there is coordination in how signaling
components are coexpressed. This observation relates to the
notion of the operon, which is a set of genes in prokaryotes
that are colocalized and coregulated by the same tran-
scription factor(s). Similarly, in eukaryotes, sets of coregu-
lated genes coding for members of the same signaling
pathway have been identified recently (Gawantka et al.,
1998; Niehrs and Pollet, 1999). These so-called ‘‘synex-
pression groups’’ include both ostensibly positive regula-
tors—for example, receptors as well as negative regulators
of pathways. Examples are the bone morphogenetic protein
(BMP)-4 synexpression group, including the ligands BMP-4
and BMP-7, and inhibitory components, such as SMAD-6
and SMAD-7 as well as BAMBI (Gawantka et al., 1998;
Grotewold et al., 2001). It is not clear if the components of
the signaling pathway downstream of the receptor (SMAD-1
and SMAD-4) are upregulated during signaling, but differ-
ent spatial localization patterns within developing tissues
suggest that they are not members of the synexpres-
sion group. The same organizational principle of receptors
and specific negative regulators, but not necessarily
intermediate components, belonging to the same synexpres-
sion group is also valid for the fibroblast growth factor (FGF)
and Notch pathways (Furthauer et al., 2002; Lahaye et al.,
2002; Lamar et al., 2001; Tsang et al., 2002). In the yeast
pheromone-sensing pathway, however, most components of
the pathway, including the MAPK itself, seem to be
upregulated in a coordinated fashion following signal
reception (Steffen et al., 2002).
The existence of synexpression groups can be useful in the
identification of novel pathway components. As demon-
strated recently for yeast MAPK pathways (Steffen et al.,
2002), one can reconstruct, with relatively high fidelity, a
signaling pathway by performing coexpression clustering
analysis of large-scale gene expression data in combination
with information on pairwise protein –protein interaction.
As mentioned earlier, the applicability of this technique to
pathways other than the MAPK pathways in yeast is still in
doubt, due to a potentially limited membership in synex-
pression groups. However, the cross-correlation approaches
to large-scale data collection, perhaps complemented with
information on chemical modification status as a function of
time, seems to be a promising avenue in postgenomic signal-
ing research.
In addition to potentially facilitating mapping of signal-
ingpathways, synexpression has notable implications
for modeling signal transduction. Indeed, members of
synexpression groups may indicate where points of specific-
ity of a pathway may be. As noted previously, synexpression
groups consistently include signal receptors and negative
regulators. It is not surprising that receptors are among the
factors determining specificity, but the nature of how
negative regulators determine specificity is not entirely
certain. In this situation modeling can be very revealing and,
in combination with further experimentation, may shed light
on the mechanisms of crosstalk and pathway specificity.
HOW TO HANDLE INFORMATION
As outlined earlier, signaling pathways are essentially dy-
namic and, as such, should be studied in a quantitative
fashion. If there is any hope for a scalable computational
analysis of signaling in the context of large-scale interaction
networks, databases summarizing vast amounts of experi-
mental and computational data should be assembled and
maintained in a manner that is both intuitive (investigator-
friendly) and powerful. Although multiple attempts have
been made at construction of signal transduction databases,
very few propose a marriage of experimental and computa-
tional approaches. In what follows I briefly review some of
the existent and developing database environments of
potential use for signal transduction research.
Most of the existing databases aim at summarizing
various kinds of experimental findings, providing links to
gene otology databases, literature references, and other
kinds of descriptive information, but lack the ability to
convey the dynamical characteristics of signaling activity.
Examples of such databases include: the Signaling Path-
ways Database, SPAD ( http://www.grt.kyushu-u.ac.jp/
spad/); the Alliance for Cell Signaling, AfCS databases
(http://www.cellularsignaling.org/); the database resource
for the analysis of signal transduction in cells (primarily
plant-oriented,http://www.drastic.org.uk/), Transpath
( http://transpath.gbf.de/); the Cell Signaling Network
Database, CSNDB (http://geo.nihs.go.jp/csndb/); the Bio-
molecular Interactions Network Database, BIND (http://
www.bind.ca/); the Database of Interacting Proteins, DIP
(http://dip.doe-mbi.ucla.edu/); the Protein Kinase Resource,
PKR ( http://pkr.sdsc.edu/html/index.shtml); the Data-
base of Sensory signal transduction proteins, SENTRA
(only prokaryotic,http://www-wit.mcs.anl.gov/sentra/); the
Kyoto Encyclopedia of Genes and Genomes, KEGG (http://
www.genome.ad.jp/kegg/regulation.html); and aMAZE
(http://www.ebi.ac.uk/research/pfmp/), the two latter data-
bases being oriented primarily toward metabolic pathways.
These databases can be queried to obtain a very useful,
detailed look at a fragment of a pathway, both in terms of
LEVCHENKO: CELL SIGNALING IN THE NEW BIOLOGY
its structure (e.g., the nucleotide and amino acid sequence
for a protein member of a pathway, the list of mutants, the
domain composition, etc.) and function (e.g., the enzymatic
nature, localization pattern, etc.).
In some databases, detailed information on the compo-
nents of signaling pathways is assembled into clickable maps
showing how the components described might be fitting
together. Science’s Signal Transduction Knowledge Envi-
ronment, STKE ( http://stke.sciencemag.org), especially
its connections maps subsection, is one example of such a
database aimed at a more comprehensive analysis of
signaling pathways. This database has an ever-expanding
collection of pathways contributed by experts in the
corresponding fields, which is a definite advantage com-
pared with other databases whose curation is often done by
nonbiologists. Currently, the connection maps provide
information on localization and functionality of path-
way components along with examples of ‘‘canonical’’ and
specific pathways in which the component participates. This
database structure easily lends itself to development of
further analytical tools allowing for analysis of networks and
construction of novel maps by the user.
To move from purely graphical representation of signal-
ing networks to the description of the underlying dynamics, a
way needs to be found to assemble a database containing
computational and mathematical models corresponding to
the pathways of interest. This task is very challenging in
terms of both establishing a standard representation
of modeling information and mapping between graphical
and modeling information, which must be developed in
a consistent and comprehensive manner. One recently
introduced example of a prototype database of models of
signal transduction is the Database of Quantitative Cell
Signaling, DOQCS ( http://doqcs.ncbs.res.in/index.html).
This database is an extension and generalization of a library
of models of signal transduction implemented in the
KINETIKIT software package, accumulated by the developers
over several years of research. The pathway components are
characterized by a description of the reactions, in which each
component participates, represented by both a verbal and
mathematical description. There is also some information on
the parameter values with corresponding literature refer-
ences and a link-out to implementation in KINETIKIT and
other formats. Another interesting feature of DOQCS is its
ability to compare different models, with the comparison
metric (the so-called similarity matrix), allowing the user to
arrange the models into a kind of phylogenetic tree. One
important deficiency of DOQCS is its rather limited toolkit
available to the user for trying to modify existent models to
describe novel pathways or those not present in the database.
Effectively, it is assumed that the user is either a practicing
modeler or is willing to invest a considerable amount of time
into learning modeling. Similar drawbacks are also present
in other attempts at creating modeling databases.
An even more recent development of a modeling database
is SIGPATH (http://icb.mssm.edu/crt/SigPath/index.xml). A
distinguishing feature of this database is the existence of a
779
tool for building a model by the user based on the models and
parameter values stored in the database and exporting this
model into independent modeling software platforms. This
added functionality is likely to be quite useful for making the
database an analysis tool in addition to a knowledge storage
environment. In this respect SIGPATH is paralleled by
another attempt at creating a model-building and simulation
tool (SIGTRAN) at the Cell Systems Initiative, University of
Washington (www.csi.washington.edu). SIGTRAN, how-
ever, does not specifically claim to be aimed at building a
large-scale model storage environment.
Another attempt being made to combine the database and
model building, storage, and analysis tools in a single inter-
active environment is a result of a consortium of experimen-
tal biologists, computer scientists, and biomedical engineers.
This software, the Signal Transduction Modeling Integ-
rated Database, SIGMOID ( http://sigmoid.sourceforge.net),
is envisioned as a database that could combine the graph
analysis functionality inherent in STKE-like connection
maps databases with a simulation tool based on a database of
computational models containing the results of simulations
and the ability to compare experimental and simulation data.
This database is intended for queries about the simulation
data with the emphasis of matching specific model types to
the experimental data obtained by the user independently.
The ability to build new models and explore and change
existing ones, to perform detailed analyses of the simulation
results, and to connect to other essential databases is likely to
make this tool particularly useful.
Most of the examples discussed in this study show that
the current databases relevant to cell signaling are very
much a work in progress. They suffer from the common
problems of ad hoc curation, lack of quantitative parameter
values, slow and spurious database population, limited
scalability, and lack of standard of model representation.
Some of these problems are alleviated by the advent of a
mark-up representation of modeling information (SBML,
http://www.sbw-sbml.org/index.html; CellML, http://
www.cellml.org). These markup languages will simplify
encoding modeling information in a standardized way, thus
enabling convenient storage and retrieval of computational
signaling models. Other problems, such as reliance on the
‘‘known’’ parameters from the literature that are based on
measurements of chemical reactions in solution using
recombinant proteins or domains thereof, and thus inappro-
priate for the intracellular environment, may present a more
significant problem for the ‘‘pure’’ modelers. There is little
doubt, however, that the obstacles in signaling database
development will be ultimately overcome, and that
experimental biologists will be able to witness and make
use of the dynamical and quantitative nature of signaling
networks provided by modeling analysis.
CONCLUSIONS
It is a truism nowadays that biology is destined to become an
essentially quantitative science. However, on the road to full
780 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 84, NO. 7, DECEMBER 30, 2003
quantification of biological phenomena there are many
obstacles. Many of these stem from the multilevel complexi-
ty displayed by living systems and by the enormous
difficulty in obtaining detailed dynamical and quanti-
tative experimental information. Often a deliberately
agnostic view is taken that regards any quantitative argument
as highly suspect within the framework of highly complex
biological systems. This view is often accompanied by an
equally ardent adherence to purely discovery-driven rather
than hypothesis- or model-driven research. However, these
anti-reductionist positions have only a limited time span. As
the problems addressed become more sophisticated, simple
logical arguments are likely to fail to shed any significant
light on the underlying mechanisms and their regulation.
Moreover, biochemical processes are hard to abstract into a
purely logical or graphical description. Rather they demand
a detailed description of timing, concentrations, compart-
mentalization, and other essentially quantitative character-
istics, combined into an integrative modeling framework.
This is especially true in the analysis of signaling processes
characterized by transient dynamics and tight cross-regu-
lation. One of the important challenges faced by students of
cell signaling is how to extend computational modeling to
the mainstream of research and conduct it on the par with an
increasingly automated experimental analysis. This chal-
lenge is likely to change the way biological exploration will
be done in the 21st century.
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