Klipp E., Herwig R., Kowald A., Wierling C., Lehrach H. Systems Biology in Practice: Concepts, Implementation and Application

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Signal Transduction

Introduction

Throughout intercellular communication or cellular stress response, the cell senses

extracellular signals. They are commuted to intracellular signals and sequences of

reactions. Different external changes or events may stimulate signaling. Typical sig-

nals are hormones, pheromones, heat, cold, light, osmotic pressure, and appearance

or concentration change of substances such as glucose, K

,Ca

, or cAMP.

On a molecular level, signaling involves the same type of processes as metabo-

lism: production or degradation of substances, molecular modifications (mainly

phosphorylation, but also methylation and acetylation), and activation or inhibition

of reactions. From a modeling point of view, there are some important differences

between signaling and metabolism. First, signaling pathways serve for information

processing and transfer of information, while metabolism provides mainly mass

transfer. Second, the metabolic network is determined by the present set of enzymes

catalyzing the reactions. Signaling pathways involve compounds of different types,

and they may form highly organized complexes and may assemble dynamically

upon occurrence of the signal. Third, the quantity of converted material is high in

metabolism (amounts are usually given in concentrations on the order of µMor

mM) compared to the number of molecules involved in signaling processes (the typi-

cal abundance of proteins in signal cascades is on the order of 10 to 10

molecules

per cell). Finally, the different amounts of components have an effect on the concen-

tration ratio of catalysts and substrates. In metabolism this ratio is usually low; the

enzyme concentration is much lower than the substrate concentration, which gives

rise to the quasi-steady-state assumption used in Michaelis-Menten kinetics (Chapter

5, Section 5.1). In signaling processes, amounts of catalysts and their substrates are

frequently in the same order of magnitude.

Modeling of the dynamic behavior of signaling pathways is often not straightfor-

ward. Knowledge about components of the pathway and their interaction is still lim-

ited and incomplete. The interpretation of experimental data is context- and knowl-

edge-dependent. Furthermore, the effect of a signal often changes the state of the

whole cell, and this implies difficulties for determination of system limits. But in

many cases we may apply the same tools as introduced in Chapter 5.

201

6.1

Function and Structure of Intra- and Intercellular Communication

Cells have a broad spectrum of receiving and processing signals; therefore, not all

of them can be considered here. A typical sequence of events in signaling pathways

is shown in Fig. 6.1 and proceeds as follows. The “signal” (a substance acting as a

ligand or a physical stimulus) approaches the cell surface. Cells have developed two

different modes of importing a signal. First, the stimulus may penetrate the cell

membrane and bind to a respective receptor in the cell interior. Another possibility

is that the signal is perceived by a transmembrane receptor. If the target of the sig-

nal is a receptor, it does not cross the membrane. Instead, the receptor changes its

own state from susceptible to active and then triggers subsequent processes within

the cell. The active receptor stimulates an internal signaling cascade. This cascade

frequently includes a series of changes in protein phosphorylation states. The se-

quence of state changes crosses the nuclear membrane. Eventually, a transcription

factor is activated or deactivated. The transcription factor changes its binding prop-

erties to regulatory regions on the DNA upstream of a set of genes, and the tran-

scription rate of these genes is altered (typically increased). Either the newly pro-

duced proteins or the changes in protein concentration cause the actual response

of the cell to the signal. In addition to this downstream program, signaling path-

202

6 Signal Transduction

Signaling paradigm

Fig. 6.1 Visualization of the signaling paradigm

(for description, see text). The receptor is stimu-

lated by a ligand or another kind of signal, and it

changes its own state from susceptible to active.

The active receptor initiates the internal signal-

ing cascade, including a series of protein phos-

phorylation state changes. Subsequently, tran-

scription factors are activated or deactivated.

The transcription factors regulate the transcrip-

tion rate of a set of genes. The absolute amount

or the relative changes in protein concentrations

alter the state of the cell and trigger the actual

response to the signal.

ways are regulated by a number of control mechanisms including feedback and

feed-forward modulation.

This is the typical picture; however, many pathways may work in a completely dif-

ferent manner. As an example, an overview of signaling pathways that are stimulated

in yeast stress response is given in Fig. 6.2.

6.2

Receptor-Ligand Interactions

Many receptors are transmembrane proteins; they receive the signal and transmit it.

Upon signal sensing, they change their conformation (see Fig. 6.3). In the active

form they are able to initiate a downstream process within the cell. The simplest con-

cept of the interaction between receptor R and ligand L is reversible binding to form

the active complex LR:

L+Rv LR . (6-1)

203

6.2 Receptor-Ligand Interactions

Signaling Pathways in Baker‘s Yeast

Fig. 6.2 Overview of signaling pathways in

yeast: HOG pathway activated by osmotic

shock, pheromone pathway activated by a pher-

omone from cells of opposite mating type, and

pseudohyphal growth pathway stimulated by

starvation conditions. In each case, the signal

interacts with the receptor. The receptor acti-

vates a cascade of intracellular processes in-

cluding complex formations, phosphorylations,

and transport steps. A MAP kinase cascade is a

particular part of many signaling pathways; its

components are indicated by bold border. Even-

tually, transcription factors are activated that

regulate the expression of a set of genes. Be-

sides the indicated connections, further interac-

tions of components are possible. For example,

crosstalk may occur, i.e., the activation of the

downstream part of one pathway by a compo-

nent of another pathway. This is supported by

the frequent incidence of proteins like Ste11 in

the scheme.

The dissociation constant is calculated as



L  R

: (6-2)

Typical values for K

are 10

–12

M…10

–6

Cells have the ability to regulate the number and the activity of specific receptors,

e.g., in order to weaken the signal transmission during long-term stimulation. Bal-

ancing production and degradation regulates the number of receptors. Phosphoryla-

tion of serine/threonine or tyrosine residues of the cytosolic domain by protein ki-

nases mainly regulates the activity. Hence, a more realistic scenario for ligand-recep-

tor interaction is depicted in Fig. 6.4.

We assume that the receptor is present in the inactive state R

or the susceptible

state R

. The susceptible form can interact with the ligand to form the active state

. The inactive or susceptible form is produced from precursors (v

), and all

three forms may be degraded (v

). The rates of production and degradation

processes, as well as the equilibria between the different states, might be influenced

by the cell state, e.g., by the cell cycle stage. In general, the dynamics of this scenario

can be described by the following set of differential equations:

 v

 v

 v

 v

 v

 v

 v

 v

v

 v

 v

: (6-3)

For the production terms, we may assume either constant values or (as mentioned

above) rates that depend on the actual cell state. The degradation terms might be

assumed to be linearly dependent on the concentration of their substrates

). This may also be a first guess for the state changes of the receptor

(e.g., v

). The receptor activation is dependent on the ligand concentration

204

6 Signal Transduction

Fig. 6.3 Schematic representation of receptor activation.

(or any other value related to the signal). A linear approximation of the respective

rate is v

7L. If the receptor is a dimer or oligomer, it might be sensible to

include this information into the rate expression as v

 k

 R



 L

1 K

 L

, where

denotes the binding constant and n the Hill coefficient (Chapter 5, Eq. (5-58)).

Example 6-1

An experimentally confirmed example for the activation of receptor and G protein

of the pheromone pathway has been presented by Yi and colleagues (2003) for the

binding of the pheromone a-factor to the receptor Ste2 in yeast. Concerning the

receptor activation dynamics, they report a susceptible and an active form of the

receptor, but no inactive form (R

= 0, v

= 0). The remaining rates are deter-

mined as follows:

 k

 R

 k

 R

 k

 R

 L

 k

 R

; (6-4)

with the following values for the rate constants: k

 4 (molecules per cell) s

–1

 4  10

4

1

, k

 4  10

3

1

, k

 2  10

1

, and k

 1  10

2

1

The time course of receptor activation is depicted in Fig. 6.5.

205

6.2 Receptor-Ligand Interactions

Fig. 6.4 Schematic representation of processes involved in

receptor activation by a ligand. L = ligand, R

= inactive recep-

tor, R

= susceptible receptor, R

= active receptor. v

= pro-

duction steps, v

= degradation steps, other steps = transition

between inactive, susceptible, and active state of receptor.

Fig. 6.5 Time course of active (solid line)

and susceptible (dashed line) receptor after

stimulation with 1 mM a-factor at t = 0. The

total number of receptors is 10,000. The

concentration of the active receptor in-

creases immediately and then declines

slowly, while the susceptible receptor is

effectively reduced to zero.

6.3

Structural Components of Signaling Pathways

Signaling pathways constitute often highly complex networks, but it has been discov-

ered that they are frequently composed of typical building blocks. These components

include Ras proteins, G protein cycles, phosphorelay systems, and MAP kinase cas-

cades. In this chapter we will discuss their general composition and function as well

as modeling approaches.

6.3.1

G Proteins

G proteins are essential parts of many signaling pathways. The reason for their

name is that they bind the guanine nucleotides GDP and GTP. They are heterotri-

mers, i. e., they consist of three different subunits. G proteins are associated to cell

surface receptors with a heptahelical transmembrane structure, the so-called G pro-

tein–coupled receptors (GPCR). Signal transduction cascades involving (1) such a

transmembrane surface receptor, (2) an associated G protein, and (3) an intracellular

effector that produces a second messenger play an important role in cellular com-

munication and are well studied (Neer 1995; Dohlman 2002). In humans, such G

protein–coupled receptors mediate responses to light, flavors, odors, numerous hor-

mones, neurotransmitters, and other signals (Blumer and Thorner 1991; Dohlman

et al. 1991; Buck 2000). In unicellular eukaryotes, receptors of this type mediate sig-

nals that affect such basic processes as cell division, cell-cell fusion (mating), mor-

phogenesis, and chemotaxis (Blumer and Thorner 1991; Banuett 1998; Dohlman et

al. 1998; Wang and Heitman 1999).

The cycle of G protein activation and inactivation is shown in Fig. 6.6. When GDP

is bound, the G protein a subunit (Ga) is associated with the G protein bg heterodi-

mer (Gbg) and is inactive. Agonist binding to a receptor promotes guanine nucleo-

tide exchange; Ga releases GDP, binds GTP, and dissociates from Gbg. The disso-

ciated subunits Ga or Gbg, or both, are then free to activate target proteins (down-

stream effectors), which initiates signaling. When GTP is hydrolyzed, the subunits

206

6 Signal Transduction

G-Protein Cycle

Fig. 6.6 Activation cycle of G

protein. Without activation, the

heterotrimeric G protein is bound

to GPD. Upon activation by the

activated receptor, an exchange of

GDP with GTP occurs and the G

protein is divided into GTP-bound

Ga and the heterodimer Gbg.Ga-

bound GTP is hydrolyzed, either

slowly in reaction v

or fast in re-

action v

, supported by the RGS

protein. GDP-bound Ga can re-

associate with Gbg (reaction v

are able to re-associate. Gbg antagonizes receptor action by inhibiting guanine nu-

cleotide exchange. RGS (regulator of G protein signaling) proteins bind to Ga, sti-

mulate GTP hydrolysis, and thereby reverse G protein activation. This general

scheme can also be applied to the regulation of small monomeric Ras-like GTPases,

such as Rho. In this case, the receptor, Gbg, and RGS are replaced by GEF and GAP

(see Section 6.3.2).

Direct targets include different types of effectors, such as adenylyl cyclase, phos-

pholipase C, exchange factors for small GTPases, some calcium and potassium

channels, plasma membrane Na

exchangers, and certain protein kinases (Neer

1995; Offermanns 2000; Dohlman and Thorner 2001; Meigs et al. 2001). Typically,

these effectors produce second messengers or other biochemical changes that lead

to stimulation of a protein kinase or a protein kinase cascade (or, as mentioned, are

themselves a protein kinase). Signaling persists until GTP is hydrolyzed to GDP and

the Ga and Gbg subunits re-associate, completing the cycle of activation. The

strength of the G protein–initiated signal depends on (1) the rate of nucleotide ex-

change, (2) the rate of spontaneous GTP hydrolysis, (3) the rate of RGS-supported

GTP hydrolysis, and (4) the rate of subunit re-association. RGS proteins act as

GTPase-activating proteins (GAPs) for a variety of different Ga classes and thereby

shorten the lifetime of the activated state of a G protein and contribute to signal de-

sensitization. Furthermore, they may contain additional modular domains with sig-

naling functions and contribute to diversity and complexity of the cellular signaling

networks (Dohlman and Thorner 1997; Siderovski et al. 1999; Burchett 2000; Ross

and Wilkie 2000).

Example 6-2

The model of the heterotrimeric G protein cycle of the yeast pheromone pathway

was already mentioned in Example 6-1 and is linked to the receptor activation

model via the concentration of the active receptor. The G protein cycle model

comprises two ODEs and two algebraic equations for the mass conservation of

the subunits Ga and Gbg. This gives



v

 v

207

6.3 Structural Components of Signaling Pathways

Fig. 6.7 Time course of G protein activa-

tion. The total number of molecules is

10,000. The concentration of GDP-bound

Ga is low for the whole period due to fast

complex formation with the heterodimer

Gbg.



GTP  v

 v

 G



 G



GTP  G



GDP

total

 G



 G



: (6-5)

The rate equations for the G protein activation, v

, the hydrolysis of G



GTP, v

and v

, and the subunit re-association, v

, follow simple mass action kinetics:

 k

 R

 G



 k

 G



GTP; i  0,1

 k

 G



 G



GDP : (6-6)

The parameters are k

 1  10

5

(molecules per cell)

–1

, k

 0:004 s

1

 0:11 s

1

, and k

 1 (molecules per cell)

–1

. Fig. 6.7 shows the respective

simulation. Note that in the original work two different yeast strains have been

considered. For the strains with a constantly active RGS (SST2

) or with a deletion

of RGS (sst2D), the rate constants k

and k

have been set to zero, respectively.

6.3.2

Ras Proteins

Small G proteins are monomeric G proteins with molecular weight of 20–40 kDa.

Like heterotrimeric G proteins, their activity depends on the binding of GTP. More

than 100 small G proteins have been identified. They belong to five families: Ras,

Rho, Rab, Ran, and Arf. They regulate a wide variety of cell functions as biological

timers that initiate and terminate specific cell functions and determine the periods

of time (Takai et al. 2001).

Ras proteins cycle between active and inactive states (Fig. 6.8). The transition

form GDP-bound to GTP-bound states is catalyzed by a guanine nucleotide ex-

change factor (GEF), which induces exchange between the bound GDP and the cellu-

lar GTP. The reverse process is facilitated by a GTPase-activating protein (GAP),

which induces hydrolysis of the bound GTP (Schmidt and Hall 2002).

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6 Signal Transduction

Ras-Protein

Fig. 6.8 The Ras activation cycle. GEF supports the transition from

GDP-bound to GTP-bound states to activate Ras, while GAP induces

hydrolysis of the bound GTP, resulting in Ras deactivation.

Mutations of the Ras proto-oncogenes (H-Ras,N-Ras,K-Ras) are found in many

human tumors. Most of these mutations result in the abolishment of normal

GTPase activity of Ras. The Ras mutants can still bind to GAP, but they cannot cata-

lyze GTP hydrolysis. Therefore, they stay active for a long time.

6.3.3

Phosphorelay Systems

Most phosphorylation events in signaling pathways take place under consumption

of ATP. Phosphorelay (or phosphotransfer) systems employ another mechanism:

after an initial phosphorylation using ATP (or another phosphate donor), the phos-

phate group is transferred directly from one protein to the next without further con-

sumption of ATP (or external donation of phosphate). Examples are the bacterial

phosphoenolpyruvate:carbohydrate phosphotransferase (Postma et al. 1989, 1993;

Rohwer et al. 2000; Francke et al. 2003), the two-component system of E. coli, or the

Sln1 pathway involved in osmoresponse of yeast (Klipp et al. 2004).

Figure 6.9 shows a scheme of a phosphorelay system from the high osmolarity gly-

cerol (HOG) signaling pathway in yeast. This pathway is organized as follows (Hoh-

mann 2002). It involves the transmembrane protein Sln1,which is present as a dimer.

Under normal conditions, the pathway is active, since Sln1 continuously autopho-

sphorylates at a histidine residue, Sln1H-P, under consumption of ATP. Subsequently,

this phosphate group is transferred to an aspartate group of Sln1 (resulting in Sln1A-

P), then to a histidine residue of Ypd1, and finally to an aspartate residue of Ssk1. Ssk1

is continuously dephosphorylated by a phosphatase. Without stress, the proteins are

present mainly in their phosphorylated form. The pathway is blocked by an increase in

the external osmolarity and a concomitant loss of turgor pressure in the cell. The phos-

phorylation of Sln1 stops, the pathway runs out of transferable phosphate groups, and

the concentration of Ssk1 rises. This constitutes the downstream signal.

209

6.3 Structural Components of Signaling Pathways

Phosphorelay-System

Fig. 6.9 Schematic representation of a

phosphorelay system. (a) Phosphorelay

system belonging to the Sln1-branch of

the HOG pathway in yeast. (b) General

scheme of phosphorylation and depho-

sphorylation.