39057_ch14_cellbio.

qxd 8/28/06 5:11 PM Page 589

TRIF/
TICAM-1
TICAM-1

TLR4
TRIF/
TICAM-1

TRAM

MD-2
TRAM

IL-10R
Tyr

Tyr JAK1
Tyr
P

IL-10R

Tyk2 Tyr
Tyr542
Tyr580
P

SHP-2
P
Tyr y Tyr
P

SHP-1
Tyr JAK1 Tyk2 Tyr y Tyr1007

SHP-1
P
y
14 IL-4R
Tyr
Tyr1007
P
P

common
y J

P
3y
P

Tyr542
SHP-2
Tyr580

SHIP
SHP-1
y y Tyr706

Tyr544 Tyr974Tyr921

M-CSFR
Tyr559
Tyr697
Tyr807
Tyr721
Tyr706
P Tyr706
P
P
P

Fc RIa
chain
chain
Tyr
Tyr

chain
Tyr518

Tyr519
Syk

TBK-1 IFN R1
P chainTyr P Tyr
IFN R2

Principles of cell
Tyr759 Tyr915 Tyr915
Tyr759 Tyr440 Tyr
IL-6R
Tyr767 Tyr905 IL-6R TyrJAK1 JAK3Tyr IL-4R common
Tyr767 Tyr905 Tyr
Tyr814 Tyr814
P P
chainTyr
Tyr JAK1 JAK2Tyr1007 SOCS1 Tyr518
IKK Tyr JAK1 Syk
Tyr542 Tyk2 Tyr /JAB Tyr JAK1 JAK3 Tyr
Tyr Gab2 P Tyr519
Tyr580SHP-2
Tyr771 Tyr771
Tyr783
PLC Tyr1254 Tyr783
PLC Tyr1254
P P
IRAK-M Ser
IRAK1 TBK-1 Tyr JAK1
IL-10R
Tyr Tyk2 Tyr Tyr JAK1
Thr Lys Tyr JAK1

P Tyr Gab2 P
Tyr759 Tyr915
Tyr518
IL-6R
Tyr767 Tyr905 IFN R1 JAK2Tyr1007 Syk
Tyr519
Tyr814 Tyr440 common Tyr542SHP-2
Tyr580
P
Tyr
IKK P SOCS1 chain
JAK3 Tyr P
IL-10R IL-10R Tyr542
IL-6R /JAB
PP2A SHP-2 IFN R2
Tyr580 Tyk2 Tyr Tyr
P gp130 P Tyr Gab2 RasGAP RasGAP
P

SOCS1 Tyr STAT5 P

P P SHP-2
Tyr542
Tyr580
P IFN R2 P Tyr Gab2 P
Tyr915 Tyr759 Tyr915 IFN R1

signaling
/JAB P Tyr759 Tyr440 Tyr GDP
Ser385 IL-6R IL-6R
Tyr767 Tyr905 P
Ser385 Tyr767 Tyr905 P Src
IRF-3 IRF-3 P Tyr814 PP Tyr814 P Tyr JAK1 Pi
Ser386 Ser386 P
JAK2 Ras
P P P Tyr JAK1 Tyr1007
Ser Tyk2 Tyr P P
IRAK1 Tyr JAK1 Tyr701 SOCS1
Thr Lys P P STAT1
Ser727
IL-4R
Tyr Tyr542
P
/JAB P P
SHP-2
Tyr580 Tyr341 Ser4
P Tyr STAT5 SOCS3 Thr Tyr GTP
JNK Raf
TAK1Thr187
Thr184
Ser192
Ser TAB1 Thr Ser338 Ser62
Tyr Fyn GTP
SOCS3
Ser
TAB2 TAB2 Tyr
Ser
P
IRS Grb2 SOS Ras
Thr IRAK1 Lys Ser
Thr IRAK1 Lys
P Ub p38MAPK PI3K Ser312(307:R) Thr
TAK1Thr187
Thr184
Ser192
P P
Tyr
P Tyr341 Ser4
Lys63TRAF6 Thr38 Ser473 IRS Ser338
Raf
ys63TRAF6 GDP Ser62
Ub Ser312(307:R)
Ser TAB1 Thr Akt/PKB
Uev1A
Tyr STAT3 P Tyr STAT3 P Tyr Fyn
Ubc13
Grb2
Ser
Thr38 Ser473
Akt/PKB Grb2 SOS
PI3K P Thr
Ser P
P Tyr SOS
Thr Ser
Ser IRAK1
P
Thr Lys
Ub
SOCS3 IRS
P
Ser
P
Ser
P P Grb2 SOS
Thr IRAK1 Lys Thr IRAK1 Lys PSer385 Tyr701
STAT1
Ser312(307:R)
Thr
P Ub P Ub Ser385 Ser727
Lys63TRAF6 Ser386
IRF-3 P P
TRAF2 TRAF1 A20 Ub
P Ser386
Lys63TRAF6 Lys63TRAF6 P PP2B PP2A
Ub Ub
TAB2 P
P
P Ser369
P TAB2 P TAB2 IFN
P Tyr STAT6 P Ser21 P Ser21 R
P
TAK1Thr187 PP
Thr184
Ser192 Ser32 Ser374 Ser32 Ser374
Ser386
IL-4 c-FosSer113 c-Fos Ser113 P
TAK1Thr187
Thr184
Ser192
P
Thr184
TAK1Thr187
Ser192 IL-1ra Ser42 Ser42
P Ser70 P Ser70
P Ser TAB1 Thr
P P P
Ser TAB1 Thr Ser TAB1 Thr ERK1
Thr183
P P Tyr185
P
ERK2 Ser73
Ser63 Ser73 Ser63
0 P c-Jun c-Jun P P
proteasome

Melanie H. Cobb and Elliott M. Ross

P Tyr701 Ser369 Thr577
P
P Tyr STAT3
P Tyr
STAT1
Tyr701
Ser727 P Tyr STAT6 RSK Ser227
P Ser727 STAT6
Tyr P Tyr Ser386
P P P
SEK2/MKK7 SEK2/MKK7
ASK

P P P
Thr Tyr Thr Tyr PI3K
JNK JNK

P MEKK P

The University of Texas Southwestern Medical Center at Dallas

SEK1/MKK4
SOCS3 IL-4R c-fos P
IL-1ra Tyr STAT6 P P Ser383
Tyr
Ser369 Thr577 Ser389
Elk-1
A20 IL-4 RSK Ser227 P
IKK SEK1/MKK4 Thr Tyr Ser386
JNK P
P
Ser176 P
IKK P P Thr183 ERK1
Ser181 Thr183
Ser63 Ser73 c-jun PP2B Tyr185 ERK2
Tyr185
c-Jun P
IKK Lys21 Ser32 P
Lys22
I B Ser36 I B
IFN P Tyr STAT3
p50 p50 P Tyr Ser383
Ser389Elk-1
P Tyr STAT6
P Tyr
NF- B SOCS1/JAB Ser133

Ser276
p65+p50
Ser529
CREB
p60 PIAS3
PKA
LXR
MKP
MKP
NF- B NF- B NF- B

This image represents about 10% of the map of the known signaling interactions and
P P Tyr STAT3
P
P Tyr STAT6 p65+p50 p65+p50 Ser385 P Tyr
p65+p50
Ser276 Ser529 Ser276 Ser529 Ser276 Ser529
Ser385
IRF-3 Tyr701
P
Ser276 Ser529
P P
Ser276 Ser529
P
Ser386
Ser386 P STAT1
Tyr701
Ser727
P P P P P P P Ser727
P Tyr542
PIAS3 SHP-2
Tyr580
Lys21 Ser32 Thr183 ERK1
I B P P Thr183
Lys22 Ser36 Thr183 ERK1 Tyr185 ERK2
CK II Ub P
Tyr185

reactions in the mouse macrophage. Preparing such a map in a computable format is

Lys21 Ser32
PKA Tyr185 ERK2 PAFR I B P
Lys22 Ser36 PTyr701 P
P STAT1
Tyr701
NF- B Ub P
P Tyr STAT5
Ser727 P Ser21 P P
PKA P Tyr P Ser727 Ser32 Ser374 Ser133
P
p65+p50
Ser276 Ser529 NF- B Ub P c-FosSer113 CREB
NF- B Ser42
Lys21 Ser32
Ser70 P RXR LXR
p65+p50 Lys22
I B Ser36 PIAS1
P
IKK Ser276 Ser529
P
p65+p50
Ser276 Ser529
P
Ub
P

the first step in analyzing a large signaling network. This map was prepared by the group
P P
CAPK PKA PTyr701 P P
Ser176 STAT1
Tyr701 P P
Ser181 IKK Ser727
Ser727
Ser63 Ser73
Ser63 Ser73
P
P
SCF TrCP
P PIAS1 p53 c-Jun
IKK P
Ser727
LXR
P
STAT1
Tyr701

NF- B

led by Hiroaki Kitano at the Systems Biology Institute, Tokyo, using their CellDesigner
P PKA P
P Tyr STAT2 SREBP1c
Lys21 Ser32 p65+p50 Lys21 Ser32 UbcH5
I B Ser276 Ser529
I B IRF-2 / bHLH
Lys22 Ser36 Lys22 Ser36 IRF-1
P P IRF-9
CAPK PKA PKA
NF- B NF- B
AP-1
p65+p50 p65+p50

program. Map courtesy of Kanae Oda, Yukiko Matsuoka, and Hiroaki Kitano (The Systems
Ser276 Ser529 Ser276 Ser529
c-Fos+c-Jun 27-hydroxyChol 9
r

TNF TNF acetyl CoA carboxylase LXR

Biology Institute). IL-10

IL-1

IFN-
P

P
Ser484
Ser485
IRF-7
fatty acid synthetase

acyl CoA synthetase

IL-6 IRF-7

IFN-
GM-CSF IRF-9 IRF-2 IRF-1 NOSII/iNOS CPT1
nucleus
IFN-

CHAPTER OUTLINE
IFN- P
Ser484
P Ser485
IRF-7 SREBP1c
PTyr701
STAT1
Tyr701
Ser727
P
/ bHLH
GM-CSF P
P
Ser727

TyrJAK1
P Tyr STAT5
P Tyr IRF-2 Ser484 acetyl CoA
GM-CSFR IRF-1 IRF-7
Ser485 carboxylase
SREBP1c R
GM-CSFR / bHLH
Tyr
P
Ser727
STAT1
GM-CSFR P
Tyr701
?
P Tyr STAT2
JAK2
Tyr1007 Site-2 protease
P Tyr STAT5 IRF-9 acetyl CoA malonyl CoA SCAP

TG TG fatty acid Site

P Tyr STAT3 Golgi

14.1 Introduction 14.21 Heterotrimeric G proteins regulate a wide variety of

lipid droplet synthetase

acyl-CoA fatty acid

IRF-9 HOCl
CoASH MPO
CoASH acyl CoA
malate oxaloacetate
synthetase

effectors
NADPH
Tyr STAT5 Tyr STAT3 citrate carnitine acylcarnitine
malate oxidase

14.2 Cellular signaling is primarily chemical

glycerol 3P citrate
dehydrogenase liase NADPH NADP+ SOD
CPT I Cl-
Thr38 Ser473
CACT
K calpain
Akt/PKB P NOSII/iNOS
Tyr701
STAT1
Ser727
P
e-

14.22 Heterotrimeric G proteins are controlled by a regulatory

malic enzyme PDH kinase PDH kinase
CoASH CPT II
O2 .O -

Receptors sense diverse stimuli but initiate a limited

H2O2

14.3
carnitine 2
P
P P P P
PI3K Thr38 Ser473 pyruvate pyruvate ATP
Akt/PKB ATP F
dehydrogenase dehydrogenase
pyruvate acylcarnitine synthetase

GTPase cycle
carrier ADP hypoxanthine Fe3+
-2 P Tyr STAT2 NAD+ NADH+H+

repertoire of cellular signals P

P
Tyr542
SHP-2
Tyr580
Tyr701
STAT1
Ser727
pyruvate pyruvate

pyruvate
carboxylase
acetyl CoA acyl-CoA H+

O2
H+

e-
e-

xanthine
oxidase
xanthine
LOOH

14.4 Receptors are catalysts and amplifiers 14.23 Small, monomeric GTP-binding proteins are multiuse
switches
14.5 Ligand binding changes receptor conformation
14.24 Protein phosphorylation/dephosphorylation is a major
14.6 Signals are sorted and integrated in signaling pathways
regulatory mechanism in the cell
and networks
14.25 Two-component protein phosphorylation systems are
14.7 Cellular signaling pathways can be thought of as
signaling relays
biochemical logic circuits
14.26 Pharmacological inhibitors of protein kinases may be
14.8 Scaffolds increase signaling efficiency and enhance
used to understand and treat disease
spatial organization of signaling
14.27 Phosphoprotein phosphatases reverse the actions of
14.9 Independent, modular domains specify protein-protein
kinases and are independently regulated
interactions
14.28 Covalent modification by ubiquitin and ubiquitin-like
14.10 Cellular signaling is remarkably adaptive
proteins is another way of regulating protein function
14.11 Signaling proteins are frequently expressed as multiple 14.29 The Wnt pathway regulates cell fate during development
species
and other processes in the adult
14.12 Activating and deactivating reactions are separate and 14.30 Diverse signaling mechanisms are regulated by protein
independently controlled
tyrosine kinases
14.13 Cellular signaling uses both allostery and covalent 14.31 Src family protein kinases cooperate with receptor
modification
protein tyrosine kinases
14.14 Second messengers provide readily diffusible pathways 14.32 MAPKs are central to many signaling pathways
for information transfer
14.33 Cyclin-dependent protein kinases control the cell cycle
14.15 Ca2+ signaling serves diverse purposes in all eukaryotic
cells 14.34 Diverse receptors recruit protein tyrosine kinases to the
plasma membrane
14.16 Lipids and lipid-derived compounds are signaling
molecules 14.35 What’s next?
14.17 PI 3-kinase regulates both cell shape and the activation 14.36 Summary
of essential growth and metabolic functions References
14.18 Signaling through ion channel receptors is very fast
14.19 Nuclear receptors regulate transcription
14.20 G protein signaling modules are widely used and highly
adaptable
589
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 590

nearby), odors, molecules that regulate growth

14.1 Introduction or differentiation, and proteins on the outside
All cells, from prokaryotes through plants and of adjacent cells. A mammalian cell typically
animals, sense and react to stimuli in their en- expresses about fifty distinct receptors that sense
vironments with stereotyped responses that al- different inputs, and, overall, mammals express
low them to survive, adapt, and function in several thousand receptors.
ways appropriate to the needs of the organism. Despite the diversity of cellular lifestyles
These responses are not simply direct physical and the enormous number of substances sensed
or metabolic consequences of changes in the by different cells, the general classes of proteins
local environment. Rather, cells express arrays and mechanisms involved in signal transduc-
of sensing proteins, or receptors, that recognize tion are conserved throughout living cells, as
specific extracellular stimuli. In response to shown in FIGURE 14.1.
these stimuli, receptors regulate the activities • G protein-coupled receptors,
of diverse intracellular regulatory proteins that composed of seven membrane-span-
in turn initiate appropriate responses by the ning helices, promote activation of het-
cell. The process of sensing external stimuli and erotrimeric GTP-binding proteins called
conveying the inherent information to intra- G proteins, which associate with the in-
cellular targets is referred to as cellular signal ner face of the plasma membrane and
transduction. convey signals to multiple intracellular
Cells respond to all sorts of stimuli. Microbes proteins.
respond to nutrients, toxins, heat, light, and • Receptor protein kinases are often
chemical signals secreted by other microbes. dimers of single membrane-spanning
Cells in multicellular organisms express recep- proteins that phosphorylate their in-
tors specific for hormones, neurotransmitters, tracellular substrates and, thus, change
autocrine and paracrine agents (hormone- the shape and function of the target pro-
like compounds from the secreting cell or cells teins. These protein kinases frequently
contain protein interaction domains that
organize complexes of signaling pro-
teins on the inner surface of the plasma
membrane.
• Phosphoprotein phosphatases re-
verse the effect of protein kinases by re-
moving the phosphoryl groups added
Overview of major receptor types in a cell
by protein kinases.
Trans- • Other single membrane-spanning en-
(GPCR) membrane Guanylyl
G protein Receptor Ion scaffold cyclase zymes, such as guanylyl cyclase, have
coupled protein channel an overall architecture similar to the re-
receptor kinase
ceptor protein kinases but different en-
zymatic activities. Guanylyl cyclase
catalyzes the conversion of GTP to 3′:5′-
Two- E1 E1
component E2 E2
cyclic GMP, which is used to propagate
Hetero- complex the signal.
trimeric
G protein Sensor • Ion channel receptors, although di-
( Histidine
kinase (
verse in detailed structure, are usually
oligomers of subunits that each contain
Response
regulator several membrane-spanning segments.
Transcription The subunits change their conforma-
factor
tions and relative orientations to per-
mit ion flux through a central pore.
• Two-component systems may either
NUCLEUS
be membrane spanning or cytosolic. The
number of their subunits is also vari-
able, but each two-component system
contains a histidine kinase domain or
FIGURE 14.1 Receptors form a rather small number of families that share com- subunit that is regulated by a signaling
mon mechanisms of action and overall similar structures. molecule and a response regulator that

590 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 591
contains a phosphorylatable aspartate
(Asp) residue.
• Some receptors are transmembrane
scaffolds that change either the con-
formation or oligomerization of their
intracellular scaffold domains in re-
sponse to extracellular signaling mole-
cules, or ligands, and, thus, recruit
interacting regulatory proteins to a com-
mon site on the membrane.
• Nuclear receptors are transcription
factors, often heterodimers, that may
reside in the cytoplasm until activated
by agonists or may be permanently lo-
cated in the nucleus.
The biochemical processes of signal trans-
duction are strikingly similar among cells.
Bacteria, fungi, plants, and animals use similar
proteins and multiprotein modules to detect
and process signals. For example, evolutionar-
ily conserved heterotrimeric G proteins and G
protein-coupled receptors are found in plants,
fungi, and animals. Similarly, 3′:5′ cyclic AMP
(cAMP) is an intracellular signaling molecule
in bacteria, fungi, and animals; and Ca2+ serves
a similar role in all eukaryotes. Protein kinases
and phosphoprotein phosphatases are used to
regulate enzymes in all cells.
Although the basic biochemical components
and processes of signal transduction are con-
served and reused, they are often used in wildly
divergent patterns and for many different phys-
iological purposes. For example, cAMP is synthe-
sized by distantly related enzymes in bacteria,
fungi, and animals, and acts on different pro-
teins in each organism; it is a pheromone in
some slime molds.
Cells often use the same series of signaling
proteins to regulate a given process, such as
transcription, ion transport, locomotion, and
metabolism. Such signaling pathways are as-
sembled into signaling networks to allow the
cell to coordinate its responses to multiple in-
puts with its ongoing functions. It is now pos-
sible to discern conserved reaction sequences
in and between pathways in signaling networks
that are analogous to devices within the circuits
of analog computers: amplifiers, logic gates,
feedback and feed-forward controls, and mem-
ory.
This chapter discusses the principles and
strategies of cellular signaling first and then dis-
cusses the conserved biochemical components
and reactions of signaling pathways and how
these principles are applied.
14.2 Cellular signaling is
primarily chemical
Key concepts
• Cells can detect both chemical and physical
signals.
• Physical signals are generally converted to
chemical signals at the level of the receptor.
Most signals sensed by cells are chemical, and,
when physical signals are sensed, they are gen-
erally detected as chemical changes at the level
of the receptor. For example, the visual pho-
toreceptor rhodopsin is composed of the pro-
tein opsin, which binds to a second component,
the colored vitamin A derivative cis-retinal (the
chromophore). When cis-retinal absorbs a
photon, it photoisomerizes to trans-retinal,
which is an activating ligand of the opsin pro-
tein. (For more on rhodopsin signaling see 14.20
G protein signaling modules are widely used and
highly adaptable). Similarly, plants sense red and
blue light using the photosensory proteins phy-
tochrome and cryptochrome, which detect pho-
tons that are absorbed by their tetrapyrrole or
flavin chromophores. Cryptochrome homologs
are also expressed in animals, where they prob-
ably mediate adjustment of the diurnal cycle.
A few receptors do respond directly to phys-
ical inputs. Pressure-sensing channels, which ex-
ist in one form or another in all organisms,
mediate responses to pressure or shear by chang-
ing their ionic conductance. In mammals, hear-
ing is mediated indirectly by a mechanically
operated channel in the hair cell of the inner ear.
The extracellular domain of a protein called cad-
herin is pulled in response to acoustic vibration,
generating the force that opens the channel.
Cells sense mechanical strain through a
number of cell surface proteins, including inte-
grins. Integrins provide signals to cells based on
their attachment to other cells and to molecu-
lar complexes in the external milieu.
One major group of physically responsive
receptors is made up of channels that sense elec-
tric fields. Another interesting group are
heat/pain-sensing ion channels; several of these
heat-sensitive ion channels also respond to
chemical compounds, such as capsaicin, the
“hot” lipid irritant in hot peppers.
Whether a signal is physical or chemical, the
receptor initiates the reactions that change the
behavior of the cell. We will discuss how these
effects are generated in the rest of the chapter.
14.2 Cellular signaling is primarily chemical 591
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only a limited number of receptor families, which

14.3 Receptors sense diverse are related by their conserved structures and sig-
stimuli but initiate a naling functions (see Figure 14.1).
There are several useful correlates to the
limited repertoire of two-domain nature of receptors. For example,
cellular signals a cell can control its responsiveness to an extra-
cellular signal by regulating the synthesis or
Key concepts degradation of a receptor or by regulating the
• Receptors contain a ligand-binding domain and an receptor’s activity (see 14.10 Cellular signaling is
effector domain.
remarkably adaptive).
• Receptor modularity allows a wide variety of
signals to use a limited number of regulatory
In addition, the nature of a response is gen-
mechanisms. erally determined by the receptor and its effec-
• Cells may express different receptors for the same tor domain rather than any physicochemical
ligand. property of the ligand. FIGURE 14.2 illustrates the
• The same ligand may have different effects on the concept that a ligand may bind to more than
cell depending on the effector domain of its one kind of receptor and elicit more than one
receptor. type of response, or several different ligands
may all act identically by binding to function-
Receptors mediate responses to amazingly di- ally similar receptors. For example, the neuro-
verse extracellular messenger molecules; hence, transmitter acetylcholine binds to two classes
the cell must express a large number of recep- of receptors. Members of one class are ion chan-
tor varieties, each able to bind its extracellular nels; members of the other regulate G proteins.
ligand. In addition, each receptor must be able Similarly, steroid hormones bind both to nu-
to initiate a cellular response. Receptors, thus, clear receptors, which bind chromatin and reg-
contain two functional domains: a ligand- ulate transcription, and to other receptors in
binding domain and an effector domain, the plasma membrane.
which may or may not correspond to definable Conversely, when multiple ligands bind to
structural domains within the protein. receptors of the same biochemical class, they
The separation of ligand-binding and effec- generate similar intracellular responses. For ex-
tor functions allows receptors for diverse ligands ample, it is not uncommon for a cell to express
to produce a limited number of evolutionarily several distinct receptors that stimulate produc-
conserved intracellular signals through the ac- tion of the intracellular signaling molecule cAMP.
tion of a few effector domains. In fact, there are The effect of the receptor on the cell will also be
determined significantly by the biology of the
cell and its state at any given time.
Ligand binding and effector domains may
evolve independently in response to varied se-
lective pressures. For example, mammalian and
Receptors have a ligand-binding domain and an effector domain invertebrate rhodopsins transduce their signal
CHIMERIC through different effector G proteins (Gt and
Ligand A RECEPTOR Gq, respectively). Another example is calmod-
Ligand A Ligand B Ligand C
ulin, a small calcium-binding regulatory pro-
LBD1 LBD1 LBD1 LBD2 LBD3 tein in animals, which in plants appears as a
distinct domain in larger proteins.
The receptor’s two-domain nature allows
ED1 ED2 ED1 ED1 ED2 the cell to regulate the binding of ligand and
the effect of ligand independently. Covalent
Output Output Output Output Output modification or allosteric regulation can al-
1 2 1 1 2
ter ligand-binding affinity, the ability of the lig-
FIGURE 14.2 Receptors can be thought of as composed of two functional do- and-bound receptor to generate its signal or
mains, a ligand-binding domain (LBD) and an effector domain (ED). The two- both. We will discuss these concepts further in
domain property implies that two receptors that respond to different ligands 14.13 Cellular signaling uses both allostery and co-
(middle) could initiate the same function by activating similar effector do-
mains, or that a cell could express two receptor isoforms (left) that respond to
valent modification.
the same ligand with distinct cellular effects mediated by different effector do- Receptors can be classified either accord-
mains. It also implies that one can create an artificial chimeric receptor with ing to the ligands they bind or the way in which
novel properties. they signal. Signal output, which is character-

592 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 593
istic of the effector domain, usually correlates
best with overall structure and sequence con-
servation. (Receptor families grouped by their
functions are the organizational basis of the sec-
ond half of this chapter.) However, classifying
receptors pharmacologically, according to their
specificity for ligands, is particularly useful for
understanding the organization of endocrine
and neuronal systems and for categorizing the
multiple physiological responses to drugs.
Expression of a receptor that is not nor-
mally expressed in a cell is often sufficient to
confer responsiveness to that receptor’s ligand.
This responsiveness often occurs because the
cell expresses the other components necessary
for propagating the intracellular signal from the
receptor. The precise nature of the response will
reflect the biology of the cell. Experimentally,
responsiveness to a compound can be induced
by introducing the cDNA that encodes the re-
ceptor. For example, mammalian receptors may
be expressed in yeast, such that the yeast re-
spond visibly to receptor ligands, thus provid-
ing a way to screen for new chemicals (drugs)
that activate the receptor.
Finally, it is possible to create chimeric re-
ceptors by fusing the ligand-binding domain
from one receptor with the effector domain
from a different receptor (Figure 14.2). Such
chimeras can mediate novel responses to the
ligand. With genetic modification of the ligand-
binding domain, receptors can be reengineered
to respond to novel ligands. Thus, scientists can
manipulate cell functions with nonbiological
compounds.
14.4 Receptors are catalysts
and amplifiers
Key concepts
• Receptors act by increasing the rates of key
regulatory reactions.
• Receptors act as molecular amplifiers.
Receptors act to accelerate intracellular func-
tions and are, thus, functionally analogous to en-
zymes or other catalysts. Some receptors,
including the protein kinases, protein phos-
phatases, and guanylate cyclases, are themselves
enzymes and thus classical biochemical cata-
lysts. More generally, however, receptors use
the relatively small energy of ligand binding to
accelerate reactions that are driven by alterna-
tive energy sources. For example, receptors that
are ion channels catalyze the movement of ions
across membranes, a process driven by the elec-
trochemical potential developed by distinct ion
pumps. G protein-coupled receptors and other
guanine nucleotide exchange factors catalyze
the exchange of GDP for GTP on the G protein,
an energetically favored process dictated by the
cell’s nucleotide energy balance. Transcription
factors accelerate the formation of the transcrip-
tional initiation complex, but transcription it-
self is energetically driven by multiple steps of
ATP and dNTP hydrolysis.
As catalysts, receptors enhance the rates of
reactions. Most signaling involves kinetic rather
than thermodynamic regulation; that is, sig-
naling events change reaction rates rather than
their equilibria (see the next section). Thus, sig-
naling is similar to metabolic regulation, in
which specific reactions are chosen according to
their rates, with thermodynamic driving forces
playing only a supportive role.
In all signaling reactions, receptors use their
catalytic activities to function as molecular am-
plifiers. Directly or indirectly, a receptor gener-
ates a chemical signal that is huge, both
energetically and with respect to the number
of molecules recruited by a single receptor.
Molecular amplification is a hallmark of recep-
tors and many other steps in cellular signaling
pathways.
14.5 Ligand binding changes
receptor conformation
Key concepts
• Receptors can exist in active or inactive
conformations.
• Ligand binding drives the receptor toward the
active conformation.
A central mechanistic question in receptor func-
tion is how the binding of a signaling molecule
to the ligand-binding domain increases the ac-
tivity of the effector domain. The key to this
question is that receptors can exist in multiple
molecular conformations, some active for sig-
naling and others inactive. Ligands shift the
conformational equilibrium among these con-
formations. The structural changes that occur
during the receptor’s inactive-active isomeriza-
tion and how ligand binding drives these
changes are exciting areas of biophysical re-
search. However, the basic concept can be de-
scribed simply in terms of coupling the
conformational isomerizations of the ligand-
binding and effector domains.
14.5 Ligand binding changes receptor conformation 593
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 594

How do ligands activate (or not activate) a coupled equilibria displays path independence:
receptor? Most of the basic regulatory activities the net free energy difference between two
of receptors can be described by a simple scheme states is independent of which intermediary re-
that considers the receptors as having two in- actions take place. For the receptor, any path
terconvertible conformations, inactive (R) and from R to R*L therefore has the same free en-
active (R*). R and R* are in equilibrium, which ergy change, and the products of the equlib-
is described by the equilibrium constant J. rium constants along each path are equal. For
J the example above, path independence means
R R* that:
Because unliganded receptors are usually J•K* = K•J*
minimally active, J<<1 and an unliganded recep- Therefore, J* / J = K* / K.
tor spends most of its time in the R state. When Thus, if binding to the R* configuration is
a signaling molecule (L) binds, it drives the re- preferred (i.e., K*/K>>1), then ligand binding
ceptor toward the active conformation, R*, in will shift the conformation to the R* state to an
which the effector domain is functional. The lig- equivalent extent (i.e., J*/J>>1). The relative
and-bound receptor thus spends most of its time activation by a saturating concentration of lig-
in the active R* state. and, J*/J, will exactly equal the ligand’s relative
selectivity for the active receptor conformation,
J
R + L R*+ L K*/K. This argument is generally valid for the reg-
ulation of a protein’s activity by any regulatory
K K* ligand.
J* This model explains many properties of re-
R L R* L
ceptors and their ligands both simply and quan-
The mechanism whereby a ligand can ac- titatively.
tivate receptor is a simple consequence of its • First, J must be greater than zero for the
relative affinities for the receptor’s active and equilibrium to exist. Thus, even unli-
inactive conformations. A ligand can bind to ganded receptor has some activity.
the receptor in either of its conformations, de- Overexpressed receptors frequently dis-
scribed here by association constants K for the play their intrinsic low activity.
R state and K* for the R* state. Any ligand that • Because physiological receptors are
binds with higher affinity for the R* conforma- nearly inactive in the absence of ligand,
tion than for R will be an activator. If K* is greater J must be much less than 1 and is prob-
than K, the ligand is an agonist. According to the ably less than 0.01; most receptors are
Second Law of Thermodynamics, a system of less than 1% active without agonist.
• Ligands can vary in their selectivities
between R and R*. Their abilities to ac-
tivate will also vary. Some ligands, re-
ferred to as agonists, can drive formation
of appreciable R*. Others, known as par-
Receptor ligands can vary in their activities and potencies
tial agonists, will promote submaxi-
Fractional activity Fractional activity mal activation. Chemical manipulation
of receptor of receptor
of a ligand’s structure will often alter its
1.0 0.012
activity as an agonist. These relation-
0.8 High affinity 0.010
agonist ships are depicted graphically in FIGURE
0.008
0.6 Lower affinity Inverse 14.3.
agonist 0.006 agonist
0.4 • A ligand that binds equally well to both
Partial 0.004
0.2 agonist the R and R* states will not cause acti-
0.002
vation. However, such a ligand may still
0 0
Log [L] Log [L] occupy the binding site and thereby
competitively inhibit binding of an ac-
FIGURE 14.3 The simple two-state model shown here can describe a wide va- tivating ligand. Such competitive in-
riety of behaviors displayed by receptors and their various regulatory ligands. hibitors, referred to as antagonists, are
The left panel shows fractional activity of a receptor exposed to two agonists frequently used as drugs to block un-
with different affinities and one partial agonist. The right panel shows the ef-
fect of an inverse agonist. If the low fractional activity of unliganded receptor wanted activation of a receptor in var-
is detected as significant biological activity, then its inhibition by the inverse ious disease states.
agonist would be easily detectable. • A ligand that binds preferentially to R

594 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 595
relative to R* will further shift the con-
formational equilibrium to the inactive
state and cause net inhibition. Such lig-
ands are called inverse agonists.
Because J is already low, effects of in-
verse agonists may only be noticeable
if a receptor is overexpressed or if the
receptor is mutated to increase its in-
trinsic activity (i.e., the mutation in-
creases J).
• The extent to which an agonist stimu-
lates a receptor is unrelated to its affin-
ity. Both agonists and antagonists may
bind with either high or low affinity.
Affinity does determine the receptor’s
sensitivity—that is, how low a concen-
tration of ligand can the receptor detect.
Affinities of receptors for natural regu-
latory ligands vary enormously, with
physiologic Kd values ranging from
<10-12 M for some hormones to about
10-3 M for some bacterial chemoattrac-
tants. Another aspect of sensitivity is
how abruptly or gradually the receptor
is activated as the concentration of ag-
onist increases. The above model pre-
dicts that a receptor is activated
significantly at agonist concentrations
between 0.1 and 10 times its Kd. A va-
riety of cellular mechanisms can con-
vert such a conventional response range
of about 100-fold to either a more grad-
ual response or a very steep, switchlike
response.
• This model only describes equilibria. It
makes no predictions about the rates of
ligand binding or release, or of the con-
formational isomerization that leads to
activation.
This model shows how three important as-
pects of receptor action are independently de-
termined. As mentioned above, affinity for
ligand, which determines the concentration
range over which the ligand functions, is inde-
pendent of the ligand’s net effectiveness at driv-
ing receptor activation. The rate of response is
also largely independent of these other two
properties. Each aspect of receptor function can
thus be independently regulated in response to
other incoming signals or by the metabolic or
developmental state of the cell. Such control of
signal input is central to whole-cell coordina-
tion of signal transduction. Examples and mech-
anisms will recur throughout this chapter.
14.6 Signals are sorted and
integrated in signaling
pathways and networks
Key concepts
• Signaling pathways usually have multiple steps
and can diverge and/or converge.
• Divergence allows multiple responses to a single
signal.
• Convergence allows signal integration and
coordination.
Receptors rarely act directly on the intracellu-
lar processes that they ultimately regulate.
Rather, receptors typically initiate a sequence of
regulatory events that involve intermediary
proteins and small molecules. The use of mul-
tistep signaling pathways allows cells to amplify
signals, adjust signaling kinetics, insert control
points, integrate multiple signals, and route sig-
nals to distinct effectors.
Branched pathways give cells the ability to
integrate multiple incoming signals and to di-
rect information to the correct control points.
As FIGURE 14.4 illustrates, branching can be ei-
ther convergent, with multiple signals regulat-
ing common end points, or divergent, with a
single pathway branching to control more than
one process. In multicellular organisms, diver-
gent branching allows a single hormone recep-
tor to initiate distinct cell-appropriate patterns
of responses in different cells and tissues.
Divergent signaling also allows a receptor to
regulate qualitatively different cellular responses
with quantitatively distinct intensities, each de-
pendent on signal amplification in the interme-
diary pathway.
Convergent branching—when several re-
ceptors activate the same pathway to elicit the
same regulatory responses—is also common.
Convergent branching allows multiple incom-
ing signals, both stimulatory and inhibitory, to
be integrated and coordinately regulated at a
common site downstream of the receptors.
Receptors for several different hormones fre-
quently initiate similar or overlapping patterns
of signaling in a single target cell.
Overlapping converging and diverging sig-
naling pathways create signaling networks within
cells that coordinate responses to multiple in-
puts (Figure 14.4). Typically, such pathways are
complex in the number and diversity of their
components and in the topology of their circuit
14.6 Signals are sorted and integrated in signaling pathways and networks 595
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 596
Convergent and divergent signaling pathways
RECEPTORS
TRANSDUCERS
EFFECTORS
Linear, Convergent Divergent Multiply
parallel branched
FIGURE 14.4 Signaling pathways use convergent and divergent branching to co-
ordinate information flow. The diagrams at top show how even a simple, three-
level signaling network can sort information. Convergence or divergence can
take place at multiple points along a signaling pathway. As an example of com-
plexity, the lower portion of the figure shows a small segment (~10%) of the G
protein-mediated signaling network in a mouse macrophage cell line. It omits
several interpathway regulatory mechanisms and completely ignores inputs from
non-G protein-coupled receptors. Pathway map courtesy of Lily Jiang, University
of Texas Southwestern Medical Center.
maps. Signaling networks are also spatially com-
plex. They may include components in various
subcellular locations, with initial receptors and
associated proteins in the plasma membrane, but
with downstream proteins in the cytoplasm or in-
tracellular organelles. Such complexity is neces-
sary to allow the cells to integrate and sort
incoming signals and to regulate multiple intra-
cellular functions simultaneously.
The complexity and adaptability of signal-
ing networks, like the one shown in the lower
half of Figure 14.4, make their dynamics at the
whole-cell level difficult or impossible to grasp
intuitively. Signaling networks resemble large
596 CHAPTER 14 Principles of cell signaling
analog computers, and investigators are increas-
ingly depending on computational tools to un-
derstand cellular information flow and its
regulation. First, many signaling interactions
that include only two or three proteins exert
functions analogous to traditional computa-
tional logic circuits (see the next section). The
theory and experience with such circuits in elec-
tronics facilitate understanding biological sig-
naling functions as well.
The enormous complexity of cellular signal-
ing networks can be simplified by considering
them to be composed of interacting signaling
modules, i.e., groups of proteins that process sig-
nals in well-understood ways. A cellular signal-
ing module is analogous to an integrated circuit
in an electronic instrument that performs a
known function, but whose exact components
could be changed for similar use in another de-
vice. The concept of modular construction facil-
itates both qualitative and quantitative
understanding of signaling networks. We will re-
fer to many standard signaling modules later in
the chapter. Examples include monomeric and
heterotrimeric G protein modules, MAPK cas-
cades, tyrosine (Tyr) kinase receptors and their
binding proteins, and Ca2+ release/uptake mod-
ules. In each case, despite the numerous phylo-
genetic, developmental, and physiologic
variations, understanding the basic function of
that class of module conveys understanding of all
its incarnations. Last, the evolutionary impor-
tance of modules is significant; once the architec-
ture of a module is established it can be reused.
For larger-scale networks, multiplexed,
high-throughput measurements on living cells
have been combined with powerful kinetic mod-
eling strategies to allow an increasingly accurate
quantitative depiction of information flow
within signaling modules or entire networks.
Such models, with sound and experimentally
based parameter sets, can describe signaling
processes in systems too complex for intuitive
or ad hoc analysis. They are also vital as tests of
understanding because they can predict exper-
imental results in ways that can be used to test
the validity of the model. Well-grounded mod-
els can then be used (cautiously) to suggest the
mechanisms of systems for which data sets re-
main unattainable. At even greater levels of
complexity, the theories and tools of computer
science are increasingly giving useful systems-
level analyses of signal flow in cells. Using com-
putational tools to analyze large arrays of
quantitative data allows us to understand cel-
lular information flow and its regulation.
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 597

Developing quantitative models of signaling Simple logic circuits

networks is a frontier in signaling biology. These Logical (Boolean) Quantitative (Analog)
models both help describe network function
A OR B Additive
and pinpoint experiments to clarify mechanism. Response

A Response A + fixed [B]

14.7 Cellular signaling B Response A

B
A + B Response
pathways can be thought
log (agonist concentration)
of as biochemical logic
circuits A AND B
Response
More than additive

Key concepts
• Signaling networks are composed of groups of
biochemical reactions that function as
mathematical logic functions to integrate
information.
• Combinations of such logic functions combine as
signaling networks to process information at more
complex levels.
As introduced in the preceding section, processes
that signaling pathways use to integrate and direct
information to cellular targets are strikingly anal-
ogous to the mathematical logic functions that are
used to design the individual circuits of electronic
computers. Indeed, there are biological equiva-
lents of essentially all of the functional compo-
nents that computer scientists and engineers
consider in the design of computers and electronic
control devices. To understand signaling path-
ways, it is, therefore, useful to consider groups of
reactions within a pathway as constituting logic cir-
cuits of the sort used in electronic computing, as
illustrated in FIGURE 14.5. The simplest example is
when two stimulatory pathways converge. If suf-
ficient input from either is adequate to elicit the
response, the convergence would constitute an
“OR” function. If neither input is sufficient by it-
self but the combination of the two elicits the re-
sponse, then the converging pathways would
create “AND” functions. AND circuits are also re-
ferred to as coincidence detectors—a response
is elicited only when two stimulating pathways
are activated simultaneously.
AND functions can result from the combi-
nation of two similar but quantitatively inade-
quate inputs. Alternatively, two mechanistically
different inputs might both be required to elicit
a response. An example of the latter would be
a target protein that is allosterically activated
only when phosphorylated, or that is activated
by phosphorylation but is only functional when
recruited to a specific subcellular location.
The opposite of an AND circuit is a NOT
function, where one pathway blocks the stim-
A + B
A
B
A + B Response A
B
log (agonist concentration)
A NOT B Less than additi ve
Response
A
A Response
B
A + B
A + B
B
log (agonist concentration)
FIGURE 14.5 Signaling networks use simple logic functions to process
information. Boolean OR, AND, and NOT functions (left) correspond to
the quantitative interactions between converging signals that are shown
on the right.
ulatory effect of another. Simple logic gates are
observed at many locations in cellular signaling
pathways.
We can also think about convergent signal-
ing in quantitative rather than Boolean terms
by considering the additivity of inputs to a dis-
tinct process (see Figure 14.5, right). The OR
function referred to above can be considered to
be the additive positive inputs of two pathways.
Such additivity could represent the ability of
several receptors to stimulate a pool of a partic-
ular G protein or the ability of two protein ki-
nases to phosphorylate a single substrate.
Additivity may be positive, as in the examples
above, or negative, such as when two inhibitory
inputs combine. Inhibition and stimulation may
also combine additively to yield an algebraically
balanced output. Alternatively, multiple inputs
can combine with either more or less than an
additive effect. The NOT function, discussed
above, is analogous to describing a blockade of
stimulation. The AND function describes syn-
ergism, where one input potentiates another
but alone has little effect.
Even simple signaling networks can display
complex patterns of information processing. One

14.7 Cellular signaling pathways can be thought of as biochemical logic circuits 597
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 598
good example is the creation of “memory”: mak-
ing the effect of a transient signal more or less
permanent. Signaling pathways have multiple
ways of setting memories, and of forgetting. One
mechanism, common in protein kinase path-
ways, is the positive feedback loop, illustrated
in the top panel of FIGURE 14.6. In a positive feed-
back loop, the input stimulates a transducer (T),
which in turn stimulates the effector protein (E)
to create the output. If the effector can also ac-
Signal processing circuits
Positive feedback loop : irreversible ON switch
Output
Input T E Output
+ one input to control the reversibility of a sec-
Input strength
Positive feed-forward loop : responds to prolonged input
Output input
Input T E Output
+
Time
Conformational lock - Dual control switch
OH P
Kinase Output
E E
G G
OH P posure to Cdc42 will activate, but exposure to
E OH P E
E E G K G P G the kinase can activate WASP. Phospho-WASP
K P
G G
Time
Phosphatase
FIGURE 14.6 Relatively complex signal processing can be executed by simple
multi-protein modules. The figure depicts three types of signaling modules
(left) and their behavior in response to agonist (right). (top) In a positive
feed-back module, a transducer protein (T) stimulates an effector (E) to pro-
duce a cellular output, but the effector also stimulates the activity of the trans-
ducer. The result can be an all-or-none switch, where input up to a threshold
has little effect, but then becomes committed when feedback from the effec-
tor is sufficient to maintain transducer activity even in the absence of contin-
ued input from the receptor. (center) In a positive feed-forward module, the
effector requires input both from the transducer and from upstream in the path-
way. When stimulation is brief (short horizontal bar under trace at right), sig-
nificant amounts of active transducer do not accumulate and output is minimal.
When stimulation is prolonged (longer bar), signal output is substantial. (bot-
tom) In some dual-control switching modules, the binding of one regulator (G)
can both activate the effector and expose another regulatory site, shown here
as a Ser substrate site (-OH) for a protein kinase. The effector can only be phos-
phorylated or dephosphorylated when G is bound. Therefore, as shown at the
right, addition of G alone will activate but activation of the kinase (K) alone
will not. If kinase is active while G is bound, phosphorylation is resistant to
phosphatase activity unless G is again present to reexpose the phosphoserine
residue (shown on the graph at the right as a bold P).
598 CHAPTER 14 Principles of cell signaling
tivate the transducer, sufficient initial signal can
be fed back to the transducer that it can main-
tain the effector's full signal output even when
input is removed. Such systems typically display
a threshold behavior, as shown on the right.
A positive feed-forward loop can generate
memory of another type (Figure 14.6, middle
panel), indicating the duration of input. In such
circuits, the effector requires simultaneous in-
put from both the receptor and from the inter-
mediary transducer. If the pathway from
receptor through transducer is relatively slow,
or if it requires the accumulation of a substan-
tial amount of transducer, only a prolonged in-
put will trigger a response, as shown in the
time-base output diagram at the right.
A third way to establish memory is to allow
ond regulatory event (Figure 14.6, bottom panel).
WASP, a protein that initiates the polymerization
of actin to drive cellular motion and shape
change, is activated both by phosphorylation
and by the binding of Cdc42, a small GTP-bind-
ing protein (G). However, the phosphorylation
site on WASP is only exposed when WASP is
bound to Cdc42. Phosphorylation thus requires
both activated Cdc42 and activated protein ki-
nase. If Cdc42 dissociates, the phosphorylated
state of WASP persists until another signaling
molecule, whose identity remains uncertain,
binds again to expose the site to a protein phos-
phatase. As shown in the time-base graph, ex-
kinase alone will not. If Cdc42 is present, then
is relatively insensitive to protein phosphatase
(P) alone, but can be dephosphorylated if Cdc42
or another G protein binds to expose the site to
phosphatase.
14.8 Scaffolds increase
signaling efficiency and
enhance spatial
organization of signaling
Key concepts
• Scaffolds organize groups of signaling proteins and
may create pathway specificity by sequestering
components that have multiple partners.
• Scaffolds increase the local concentration of
signaling proteins.
• Scaffolds localize signaling pathways to sites of
action.
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 599

The INAD signaling complex Scaffolds concentrate and insulate signaling proteins

Pheromone
TRP
Rhodopsin GPCR

Cdc42p Cdc42p
Ste20p Ste20p
PKC PKC G protein
PDZ PDZ PDZ PDZ
Z Ste11p
PDZ PDZ
INAD INAD
- - Ste7p
PDZ PDZ PDZ PDZ
Ste5p Ste Fus3p
11p
CaM Ste
CaM 7p
CYTOSOL Fus
3p Scaffold organizes
MAPK cascade Mating
response
FIGURE 14.7 The scaffold InaD organizes proteins that transmit visual
signals in the fly photoreceptor cell. InaD is localized to the photorecep- Pheromone High osmolarity
tor membrane and coordinates light sensing and visual transduction. In
invertebrate eyes, the visual signaling pathway goes from rhodopsin
through Gq to a phospholipase C-, and Ca2+ release triggered by PLC ac-
tion initiates depolarization. This system is specialized for speed, and re- Cdc42p Cdc42p
quires that the relevant proteins are nearby. InaD contains five PDZ
Ste20p Ste20p
domains, each of which binds to the C terminus of a signal transducing
protein. The TRP channel, which mediates Ca2+ entry, PLC-, and a pro- Ste11p Ste11p
tein kinase C isoform that is involved in rapid desensitization all bind con- Ste5p Pbs2p
Ste7p
stitutively to InaD. Rhodopsin and a myosin (NinaC) also bind, and Gq
binds indirectly. Fus3p Hog1p

The proteins in a signaling pathway are fre-
quently colocalized within cells such that their
mutual interactions are favored and their in-
teractions with other proteins are minimized.
Many signaling pathways are organized on scaf-
folds. Scaffolds bind several components of a
signaling pathway in multiprotein complexes
to enhance signaling efficiency. Scaffolds pro-
mote interactions of proteins that have a low
affinity for each other, accelerate activation (and
often inactivation) of the associated compo-
nents, and localize the signaling proteins to ap-
propriate sites of action. Colocalization may be
tonic or regulated, and stimulus-dependent scaf-
folding often determines signaling outputs.
The binding sites on a scaffolding protein
are often localized in distinct modular protein-
binding domains, giving the impression that the
protein is designed simply to hold the compo-
nents of the pathway together. Many scaffold-
ing proteins do lack intrinsic enzymatic activity,
but some signaling enzymes also act as scaffolds.
Binding to a scaffold facilitates signaling by
increasing the local concentrations of the com-
ponents, so that diffusion or transport of mol-
ecules to their sites of action is not necessary. In
the photoreceptor cells of Drosophila, scaffold-
ing of signaling components is critical for rapid
signal transmission. These cells contain the InaD
Scaffold determines
specificity of Ste11p
signaling
Mating Osmo-
response adaptation
FIGURE 14.8 The scaffold Ste5p organizes the components of the MAPK
cascade that mediates the pheromone-induced mating response in
Saccharomyces cerevisiae. In the top left panel, Ste5p brings the compo-
nents of the MAPK cascade to the membrane in response to pheromone. In
the top right panel, binding to the heterotrimeric G protein brings loaded
Ste5p in proximity to the protein kinase Ste20p bound to the activated small
GTP binding protein Cdc42p. Their colocalization facilitates the sequential
activation of the cascade components, resulting in activation of the MAPK
Fus3p and the mating response. The MAP3K Ste11p can regulate not only
the MAPK Fus3p in the mating pathway, but also the MAPK Hog1p in the
high osmolarity pathway, as shown in the bottom two panels. The scaffold
to which Ste11p binds, either Ste5p or Pbs2 (both a scaffold and a MAP2K),
determines which MAPK and downstream events are activated as the out-
put.
scaffolding protein, which has five modular
binding domains, known as PDZ domains. Each
of its PDZ domains binds to a C-terminal motif
of a target protein, thereby facilitating interac-
tions among the associated proteins. FIGURE 14.7
shows a model for how InaD organizes the sig-
naling proteins. The mutational loss of InaD
produces a nearly blind fly, and deletion of a
single PDZ domain can yield a fly with a dis-
tinct visual defect characteristic of the protein
that binds to the missing domain.
A second example is Ste5p, a scaffold for the
pheromone-induced mating response pathway
in S. cerevisiae. FIGURE 14.8 illustrates how Ste5p
binds and organizes components of a mitogen-

14.8 Scaffolds increase signaling efficiency and enhance spatial organization of signaling 599
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 600
activated protein kinase (MAPK) cascade, in-
cluding a MAP3K (Ste11p), a MAP2K (Ste7p)
and a MAPK (Fus3p). (The MAPK cascade will
be discussed in 14.32 MAPKs are central to many
signaling pathways). The function of Ste5p is par-
tially retained even if the positions of its bind-
ing sites for the kinases are shuffled in the linear
sequence of the protein, indicating that a major
role is to bring the enzymes into proximity, rather
than to precisely orient them. Ste5p also binds
to the  subunits of the heterotrimeric G pro-
tein that mediates the actions of mating
pheromones, linking the membrane signal to
the intracellular transducers. Yeast that lack
Ste5p cannot mate, demonstrating that Ste5p is
required for this biological function (but not all
functions) carried out by the pathway.
In addition to facilitating signaling in their
own pathways, scaffolds can enhance signaling
specificity by limiting interactions with other
signaling proteins. Scaffolds thus insulate com-
ponents of a signaling pathway both from acti-
vation by inappropriate signals and from
producing incorrect outputs. For example, the
mating and osmosensing pathways in yeast
share several components, including the MAP3K
Ste11p, but each pathway maintains specificity
because it employs different scaffolds that restrict
signal transmission.
In contrast, the presence of excess scaffold
can inhibit signaling because the individual sig-
naling components will more frequently bind
to distinct scaffold proteins rather than forming
a functional complex. Such dilution among scaf-
folds causes separation rather than concentra-
tion of the components, preventing their
productive interaction.
14.9 Independent, modular
domains specify protein-
protein interactions
Key concepts
• Protein interactions may be mediated by small,
conserved domains.
• Modular interaction domains are essential for
signal transmission.
• Adaptors consist exclusively of binding domains or
motifs.
Modular protein interaction domains or motifs
occur in many signaling proteins and confer the
ability to bind structural motifs in other mole-
cules, including proteins, lipids, and nucleic
600 CHAPTER 14 Principles of cell signaling
acids. Some of these domains are listed in FIGURE
14.9. In contrast to scaffolds, which bind spe-
cific proteins with considerable selectivity, mod-
ular interaction domains generally recognize
not a single molecule but a group of targets that
share related structural features.
Modular interaction domains important for
signal transduction were first discovered in the
protein tyrosine kinase proto-oncogene Src,
which contains a protein tyrosine kinase do-
main and two domains named Src homology
(SH) 2 and 3 domains. The modular SH2 and
SH3 domains were originally identified by com-
parison of Src to two other tyrosine kinases, Fps
and Abl. One or both of these domains appear
in numerous proteins and both are critically in-
volved in protein-protein interactions.
SH3 domains, which consist of approxi-
mately 50 residues, bind to specific short pro-
line-rich sequences. Many cytoskeletal proteins
and proteins found in focal adhesion complexes
contain SH3 domains and proline rich se-
quences, suggesting that this targeting motif
may send proteins with these domains to these
sites of action within cells. In contrast to phos-
photyrosine-SH2 binding, the proline-rich bind-
ing sites for SH3 domains are present in resting
and activated cells. However, SH3-proline inter-
actions may be negatively regulated by phospho-
rylation within the proline-rich motif.
SH2 domains, which consist of approxi-
mately 100 residues, bind to Tyr phosphory-
lated proteins, such as cytoplasmic tyrosine
kinases and receptor tyrosine kinases. Thus, Tyr
phosphorylation regulates the appearance of
SH2 binding sites and, thereby, regulates a set
of protein-protein interactions in a stimulus-
dependent manner.
A clever strategy was used to identify the
binding specificity of SH2 domains. An isolated
recombinant SH2 domain was incubated with
cell lysates and then recovered from the lysates
using a purification tag. The proteins associated
with the SH2 domain were some of the same
proteins that were recognized by antiphospho-
tyrosine antibodies. By this and other methods,
it was discovered that SH2 domains recognize
sequences surrounding Tyr phosphorylation
sites and require phosphorylation of the in-
cluded Tyr for high affinity binding.
Information on specific amino acid se-
quences that recognize and bind to modular
binding domains is being accumulated as these
individual interactions are identified. In addi-
tion, screening programs using cDNA and/or
peptide libraries to assess binding capabilities
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 601

Characteristics of some common modular protein domains

Domain Characteristics Cellular involvement

14-3-3 Binds protein phosphoserine or Protein sequestration

phosphothreonine

Bromo Binds acetylated lysine residues Chromatin-associated

proteins
CARD Dimerization Caspase activation

C1 Binds phorbol esters or diacyl- Recruitment to mem-

glycerol branes
Signal transduction,
C2 Binds phospholipids vesicular trafficking
Calcium-dependent
EF hand Binds calcium
processes
F-Box Binds Skp1 in a ubiquitin-ligase Ubiquitination
complex
FHA Binds protein phosphothreonine Various; DNA damage
or phosphoserine
FYVE Binds to PI(3)P Membrane trafficking,
TGF- signaling
Binds E2 ubiquitin-conjugating
HECT enzymes to transfer ubiquitin to Ubiquitination
the substrate or to ubiquitin
chains
Zinc-binding cysteine-rich motif
Wide variety of
LIM that forms two tandemly
processes
repeated zinc fingers
Binds to the C-terminal 4-5
residues of proteins that have a Scaffolding diverse
PDZ protein complexes
hydrophobic residue at the
terminus; may bind to PIP2 often at the membrane

Binds to specific phosphoinositi-

Recruitment to mem-
PH des, esp. PI-4,5-P2, PI-3,4-P2 or
branes and motility
PI-3,4,5-P3.
RING Binds zinc and may be found in Ubiquitination,
E3 ubiquitin ligases transcription
Homo- and hetero- Wide variety of
SAM
oligomerization processes
Binds to protein phosphotyrosine Tyrosine protein kinase
SH2
(pY) signaling
SH3 Binds to PXXP motifs Various processes
Degenerate sequence of ~34
TPR amino acids with residues Wide variety of
WL/GYAFAP; forms a scaffold processes

Alternative to SH3;
WW Binds proline-rich sequences
vesicular trafficking

FIGURE 14.9 The table describes a subset of known modular protein in-
teraction domains found in many proteins. Interactions mediated by these
domains are essential to controlling cell function. Few if any of these do-
mains exist in prokaryotes. Adapted from the Pawson Lab, Protein Interaction
Domains, Mount Sinai Hospital (http://pawsonlab.mshri.on.ca/).

yield such motifs. Consensus target sequences
for individual domains have been identified
based on the sequence specificity of their bind-
ing to arrayed sequences. These consensus se-
quences can then be used to predict whether
the domain will bind a site in a candidate pro-
tein.
Adaptor proteins, which lack enzymatic
activity, link signaling molecules and target
them in a manner that is responsive to extra-
cellular signals. Adaptor proteins are generally
made up of two or more modular interaction
domains or the complementary recognition
motifs. Unlike scaffolds, adaptors are usually

14.9 Independent, modular domains specify protein-protein interactions 601

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 602

multifunctional because their modular interac-

tion domains and motifs are not as highly spe-
cific. Adaptors bind to two or more other
signaling proteins via their protein-protein in-
teraction domains to colocalize them or to fa-
cilitate additional interactions.
Grb2 is a prototypical adaptor protein that
was identified as a protein that bound to the C-
terminal region of the EGF receptor. Grb2 has
one SH2 and two SH3 domains. It binds consti-
tutively to specific proline-rich segments of pro-
teins through its SH3 domain, although this
binding can be negatively regulated. One target
of Grb2 is SOS, a guanine nucleotide exchange
factor that activates the small GTP-binding pro-
tein Ras in response to EGF signaling. Through
its SH2 domain, Grb2 binds Tyr-phosphorylated
proteins, including the receptors themselves in
a stimulus-dependent manner. Thus, Tyr phos-
phorylation of these receptors in response to
ligand will enable the binding of Grb2 to the re-
ceptors, which, in turn, will recruit SOS to the
membrane-localized receptor. Once at the mem-
brane, SOS can activate its target, Ras.
14.10 Cellular signaling is
remarkably adaptive
Key concepts
• Sensitivity of signaling pathways is regulated to
allow responses to change over a wide range of
signal strengths.
• Feedback mechanisms execute this function in all
signaling pathways.
• Most pathways contain multiple adaptive feedback
loops to cope with signals of various strengths and
durations.
A universal property of cellular signaling pathways
is adaptation to the incoming signal. Cells contin-
uously adjust their sensitivity to signals to main-
tain their ability to detect changes in input. Typically,
when a cell is exposed to a new input, it initiates
a process of desensitization that dampens the cel-
lular response to a new plateau lower than the ini-
tial peak response, as illustrated in FIGURE 14.10.
When the stimulus is removed, the desensitized
state can persist, with sensitivity slowly returning
to normal. Similarly, the removal of a tonic stim-
ulus can hypersensitize signaling systems.

FIGURE 14.10 Top: Upon exposure to Patterns of adaptation in signaling networks

a stimulus, signaling pathways adjust
their sensitivities to adapt to the new R esponse
level of input. Thus, the response de-
cays after initial stimulation. A sec-
Initial
ond similar stimulus will elicit a smaller response
response unless adequate time is al-
lowed for recovery. Bottom: Some adap- Desensitization
tation mechanisms feed back only on
the receptor that is stimulated and do
not alter parallel pathways. Such mech- Agonist Agonist Agonist
anisms are referred to as homologous.
At left, agonist a for receptor R1 can
initiate either of two feedback events Time
that desensitize R1 alone. In other
cases, a stimulus will also cause par- Homologous Heterologous
desensitization desensitization
allel or related systems to desensitize.
At the right, agonist a initiates desen-
sitization of both R1 and R2. The re- a R esponse a R esponse
sponse to agonist b, which binds to
R2, is also desensitized. Such heterol-
ogous desensitization is common. R1 R2 R1 R2
K R1 R2 R1
R 1 or R 2
b
a
X1 X2 Agonist a Reapply X1 X2 Agonist a Reapply
for R1 a or b for R1 a or b

Y Time Y Time

Z Z

602 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 603

Adaptation in signaling is one of the best ex- Multiple adaptation processes occur after a stimulus
amples of biological homeostasis. The adaptabil-
Relative
ity of cellular signaling can be quite impressive. response
Cells commonly regulate their sensitivity to phys- 1 Receptor phosphorylation
iological stimuli over more than a 100-fold range, 2 Arrestin binding
and the mammalian visual response can adapt to
incoming light over a 107-fold range. This re- 3 Receptor
markable ability allows a photoreceptor cell to endocytosis
detect a single photon, and allows a person to
read in both very dim light and intense sunlight. 4 Endosomal receptor
degradation
Adaptability is observed in bacteria, plants, fungi,
5 Receptor transcription
and animals. Many of its properties are conserved inhibited
throughout biology, although the most complex
adaptive mechanisms are found in animals. The
0 1 10 100 1000
general mechanism for adaptation is the nega- Time (seconds)
Agonist
tive feedback loop, which biochemically samples added
the signal and controls the adaptive process.
Adaptation varies with both the intensity and Agonist
binds
the duration of the incoming signal. Stronger or
Agonist
more persistent inputs tend to drive greater adap-
GPCR
tive change and, often, adaptation that persists GRK
for a longer time. Cells can modulate adaptation G protein
1 Receptor
Arrestin
phosphorylation
in this way because adaptation is exerted by a active
G protein 2 Arrestin
succession of independent mechanisms, each with binding
EFFECTORS
its own sensitivity and kinetic parameters.
G protein pathways offer excellent examples 3 Receptor
of adaptation. FIGURE 14.11 shows that the earli- CYTOPLASM
endocytosis
est step in adaptation is receptor phosphoryla-
tion, which is catalyzed by G protein-coupled Receptor
recycling
receptor kinases (GRKs) that selectively recog-
nize the receptor’s ligand-activated conforma- Early
endosome
tion. Phosphorylation inhibits the receptor’s ability
to stimulate G protein activation and also pro- 4 Receptor
degradation
motes binding of arrestin, a protein that further
inhibits G protein activation. Moreover, arrestin
binding primes receptors for endocytosis, which 5 Receptor
transcription
removes them from the cell surface. Endocytosis NUCLEUS inhibited
Lysosome
can also be the first step in receptor proteolysis.
DNA G P C R gene
Along with these direct effects, many receptor
genes display feedback inhibition of transcrip-
tion, such that signaling by a receptor decreases FIGURE 14.11 Multiple adaptation processes are invoked during a stimulus,
its own expression. and multiple nested mechanisms for adaptation are the rule. They are usually
Stimulation thus causes multiple adaptive invoked sequentially according to the duration and intensity of the stimulus.
For GPCRs, at least five desensitizing mechanisms are known, with others act-
processes that range from immediate (phospho- ing on the G protein and effectors.
rylation, arrestin binding) through delayed (tran-
scriptional regulation), and include both reversible these points will alter the kinetics or extent of
and irreversible events. This array of adaptive adaptation (Figure 14.10). In branched pathways,
events has been demonstrated for many G pro- changing these points can determine whether
tein-coupled receptors, and many cells may use adaptation is unique to one input or is exerted
all of them to control output from one receptor. for many similar inputs. If receptor activation trig-
The speed, extent, and reversibility of adaptation gers its desensitization directly, or if an event
are selected by a cell’s developmental program. downstream on an unbranched pathway triggers
Cells can change their patterns of adaptation desensitization, then only signals that initiate with
both qualitatively and quantitatively by altering that receptor will be altered. Receptor-selective
the points in a pathway where feedback is initi- adaptation is referred to as homologous adap-
ated and exerted. In a linear pathway, changing tation (Figure 14.10).

14.10 Cellular signaling is remarkably adaptive 603

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 604
Alternatively, feedback control can initiate
downstream from multiple receptors in a con-
vergent pathway and thus regulate both the
initiating receptor and the others. Such het-
erologous adaptation regulates all the possi-
ble inputs to a given control point. A common
example is the phosphorylation of G protein-
coupled receptors by either protein kinase A or
protein kinase C, which are activated by down-
stream signals cAMP or Ca2+ plus the lipid dia-
cylglycerol, respectively. Like GRK, these kinases
both attenuate receptor activity and promote
arrestin binding.
Cells also alter their responses to incoming
signals for homeostatic reasons. These consid-
erations include phase of the cell cycle, meta-
bolic status, or other aspects of cellular activity.
Again, all these adaptive processes may be dis-
played to a greater or lesser extent in different
cells, different pathways within a cell or differ-
ent situations during the cell’s lifetime.
14.11 Signaling proteins are
frequently expressed as
multiple species
Key concepts
• Distinct species (isoforms) of similar signaling
proteins expand the regulatory mechanisms
possible in signaling pathways.
• Isoforms may differ in function, susceptibility to
regulation or expression.
• Cells may express one or several isoforms to fulfill
their signaling needs.
FIGURE 14.12 Receptors for serotonin have Evolutionary relationship of serotonin receptor isoforms
evolved in mammals as a family of 13 genes that
regulate three of the four major classes of G pro-
teins. While all respond to the natural ligand
serotonin, the binding sites have evolved suf-
ficient differences that drugs have been devel-
oped that specifically target one or more
isoforms. The type 3 serotonin receptors, not
shown here, are ligand-gated ion channels and
are not obviously related to the others.
6 Gs
120 100
Nucleotide substitution distance
604 CHAPTER 14 Principles of cell signaling
Cells increase the richness, adaptability, and
regulation of their signaling pathways by ex-
pressing multiple species of individual signal-
ing proteins that display distinct biochemical
properties. These species may be encoded by
multiple genes or by multiple mRNAs derived
from a single gene by alternative splicing or
mRNA editing. The numerical complexity im-
plicit in these choices is impressive. Consider
the neurotransmitter serotonin: In mammals,
there are thirteen serotonin receptors, each of
which stimulates a distinct spectrum of G pro-
teins of the Gi, Gs, and Gq families. (A four-
teenth serotonin receptor is an ion channel.)
FIGURE 14.12 shows the relationship of serotonin
receptors to these G protein families.
There is also tremendous diversity among
the G proteins and adenylyl cyclases. There are
three genes for Gαi and one each for the closely
related Gαz and Gαo. Furthermore, the Gαo
mRNA is multiply spliced. There are four Gq
members. In addition, there are five genes for
Gβ and twelve for Gγ, and most of the possible
Gβγ dimers are expressed naturally. There are
ten genes for adenylyl cyclases, which are direct
targets of Gs and either direct or indirect targets
of the other G proteins. While all nine mem-
brane-bound adenylyl cyclase isoforms are stim-
ulated by Gαs, they display diverse stimulatory
and inhibitory responses to Gβγ, Gαi, Ca2+,
calmodulin, and several protein kinases, as il-
lustrated in FIGURE 14.13. Thus, stimulation by
serotonin can lead to diverse responses depend-
ing upon the various forms of the proteins that
are engaged at a particular time and location.
Isoforms G protein
1B
1D
1E Gi
1F
1A
5A
5B
Gs
7
4
2A
2C Gq
2B
80 60 40 20 0
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 605

Different isoforms of adenylyl cyclase are regulated differently

Gαs

Gβγ
PKC

Ca2+ NO PKA

Regulators

CaMK inhibit Gαi

activate

CaM

FIGURE 14.13 All of the mammalian membrane-bound adenylyl cyclases are

structurally homologous and catalyze the same reaction, and all are stimulated
by Gs. Their responses to other inputs (protein kinases CaMK, PKA and PKC;
Ca2+; calmodulin (CaM); NO•) are specific to each isoform, allowing a rich com-
binatoric input to cellular cAMP signaling.

Sometimes isoforms of a signaling protein
are subject to quite different kinds of inputs.
For example, all of the members of the phospho-
lipase C family (PLC) hydrolyze phosphatidyli-
nositol-4,5-bisphosphate to form two second
messengers, diacylglycerol and inositol-1,4,5
trisphosphate (see 14.16 Lipids and lipid-derived
compounds are signaling molecules). The distinct
isoforms may be regulated by diverse combina-
tions of Gαq, Gβγ, phosphorylation, monomeric
G proteins, or Ca2+.
Because a cell has multiple options when
expressing a form of a signaling protein, it can
use expression of particular isoforms to alter
how it performs otherwise identical signaling
functions. Different cells express one or more
isoforms to allow appropriate responses, and ex-
pression can vary according to other inputs or
the cell’s metabolic status. In addition, signaling
pathways are remarkably resistant to mutational
or other injuries because loss of a single species
or isoform of a signaling protein can often be
compensated for by increased expression or ac-
tivity of another species. Similarly, engineered
overexpression can result in the reduced expres-
sion of endogenous proteins. The existence of
multiple receptor species can, thus, substantially
add to adaptability and the consequent resist-
ance of signaling networks to damage.
14.12 Activating and
deactivating reactions
are separate and
independently controlled
Key concepts
• Activating and deactivating reactions are usually
executed by different regulatory proteins.
• Separating activation and inactivation allows for
fine-tuned regulation of amplitude and timing.
In signaling networks, individual proteins are
frequently activated and deactivated by distinct
reactions, a feature that facilitates separate reg-
ulation. Common examples include using pro-
tein kinases and phosphoprotein phosphatases

14.12 Activating and deactivating reactions are separate and independently controlled 605
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 606
to catalyze protein phosphorylation and de-
phosphorylation; using adenylyl cyclase to cre-
ate cAMP while using phosphodiesterases to
hydrolyze it or anion transporters to pump it
out of the cell; or using GTP/GDP exchange fac-
tors (GEFs) to activate G proteins and GTPase-
activating proteins (GAPs) to deactivate them.
Depending on stoichiometry and detailed mech-
anism, these strategies can convey either addi-
tive or nonadditive inputs while maintaining
fine control over the kinetics of activation and
deactivation of a signaling pathway. The use of
distinct reactions for activation and deactiva-
tion is analogous to the use of distinct anabolic
and catabolic enzymes in reversible metabolic
pathways.
14.13 Cellular signaling uses
both allostery and
covalent modification
Key concepts
• Allostery refers to the ability of a molecule to alter
the conformation of a target protein when it binds
noncovalently to that protein.
• Modification of a protein’s chemical structure is
also frequently used to regulate its activity.
Cellular signaling uses almost every imagina-
ble mechanism for regulating the activities of
intracellular proteins, but most can be described
as either allosteric or covalent. Individual sig-
naling proteins typically respond to multiple al-
losteric and covalent inputs.
Allostery refers to the ability of a molecule
to alter the conformation of a target protein
when it binds noncovalently to that protein.
Because a protein’s activity reflects its confor-
mation, the binding of any molecule that alters
conformation can change the target protein’s
activity. Any molecule can have allosteric ef-
fects: protons or Ca2+, small organic molecules,
or other proteins. Allosteric regulation can be
both inhibitory or stimulatory.
Covalent modification of a protein’s chem-
ical structure is also frequently used to regulate
its activity. The change in the protein’s chemi-
cal structure alters its conformation and, thus,
its activity. Most regulatory covalent modifica-
tion is reversible. The classic and most common
regulatory covalent event is phosphorylation,
in which a phosphoryl group is transferred from
ATP to the protein, most often to the hydroxyl
group of serine (Ser), threonine(Thr), or tyro-
sine (Tyr). Enzymes that phosphorylate proteins
606 CHAPTER 14 Principles of cell signaling
are known as protein kinases. Their actions are
opposed by phosphoprotein phosphatases, which
catalyze the hydrolysis of the phosphoryl group
to yield free phosphate and restore the unmod-
ified hydroxyl residue. Other forms of covalent
modification are also common and will be ad-
dressed throughout the chapter.
14.14 Second messengers
provide readily diffusible
pathways for information
transfer
Key concepts
• Second messengers can propagate signals between
proteins that are at a distance.
• cAMP and Ca2+ are widely used second messengers.
Signaling pathways make use of both proteins
and small molecules according to their distinc-
tive attributes. A small molecule used as an in-
tracellular signal, or second messenger, has a
number of advantages over a protein as a sig-
naling intermediary. Small molecules can be
synthesized and destroyed quickly. Because they
can be made readily, they can act at high con-
centrations so that their affinities for target pro-
teins can be low. Low affinity permits rapid
dissociation, such that their signals can be ter-
minated promptly when free second messenger
molecules are destroyed or sequestered. Because
second messengers are small, they also can dif-
fuse quickly within the cell, although many cells
have developed mechanisms to spatially restrict
such diffusion. Second messengers are, thus,
superior to proteins in mediating fast responses,
particularly at a distance. Second messengers
are also useful when signals have to be addressed
to large numbers of target proteins simultane-
ously. These advantages often overcome their
lack of catalytic activity and their inability to
bind multiple molecules simultaneously.
FIGURE 14.14 lists intracellular second mes-
sengers developed through evolution. This num-
ber is surprisingly low. Several are nucleotides
synthesized from major metabolic nucleotide
precursors. They include cAMP, cyclic GMP,
ppGppp, and cyclic ADP-ribose. Other soluble
second messengers include a sugar phosphate,
inositol-1,4,5-trisphosphate (IP3), a divalent metal
ion Ca2+, and a free radical gas nitric oxide (NO•).
Lipid second messengers include diacylglycerol
and phosphatidylinositol-3,4,5-trisphosphate,
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 607

phosphatidylinositol-4,5-diphosphate, sphingo- Second messengers

sine-1-phosphate and phosphatidic acid. Second Synthesis/ Pre-
The first signaling compound to be described messenger Targets Release cursor Removal
as a second messenger was cAMP. The name Protein kinase A
Adenylyl
ATP
Phospho-
cyclase diesterase
arose because cAMP is synthesized in animal
Organic
cells as a second, intracellular signal in response Bacterial trans-
anion
cription factors
to numerous extracellular hormones, the first transporter
3':5'-cyclic AMP Cation channel
messengers in the pathway. cAMP is used by (cAMP)
prokaryotes, fungi, and animals to convey in- Cyclic nucleotide
phospho-
formation to a variety of regulatory proteins. diesterase
(Its occurrence in higher plants has still not been Rap GDP/GTP
exchange factor
proved.) (Epac)
Adenylyl cyclases, the enzymes that syn- SpoT-
thesize cAMP from ATP, are regulated in vari- RNA polymerase Rel1A GTP catalyzed
Magic spot hydrolysis
ous ways depending on the organism in which (ppGpp, ppGppp) ObgE trans-
they occur. In animals, adenylyl cyclase is an cription arrest SpoT
integral protein of the plasma membrane whose detector
multiple isoforms are stimulated by diverse Inositol-1,3,5- 2+
agents (see Figure 14.13). In animal cells, adeny- trisphosphate IP3-gated Ca Phospho-
PIP2 Phosphatase
(IP 3) channel lipase C
lyl cyclase is generally stimulated by Gs, which
was originally discovered as an adenylyl cyclase Protein Phospho- PIP2 Diacylglycerol
Diacylglycerol kinase C lipase C kinase
regulator. Some fungal adenylyl cyclases are (DAG)
Trp cation Diacylglycerol
also stimulated by G proteins. Bacterial cyclases channel lipase
are far more diverse in their regulation.
Phosphatidyl- Ion channel Phospho-
cAMP is removed from cells in two ways. inositol-4,5- lipase C
PIP 5-kinase PI-4-P
It may be extruded from cells by an ATP-driven bisphosphate
(PIP 2 ) Transporters Phosphatase
anion pump but is more often hydrolyzed to 5′-
AMP by members of the cyclic nucleotide phos- Protein kinase G
Guanylyl
GTP
Phospho-
cyclase diesterase
phodiesterase family, a large group of proteins
3':5'-Cyclic GMP Cation channel
that are themselves under multiple regulatory
(cGMP) Cyclic nucleotide
controls. phospho-
The prototypical downstream regulator for diesterase
cAMP in animals is the cAMP-dependent pro- ADP-ribose
Cyclic ADP-ribose Ca2+ channel NAD Hydrolysis
tein kinase, but a bacterial cAMP-regulated tran- cyclase
scription factor was discovered shortly thereafter, Cyclic Various two Cyclic di-GMP
Diguanylate
and other effectors are now known (Figure diguanosine- component GTP phospho-
cyclase
monophosphate system proteins diesterase
14.14). The cAMP system remains the proto-
typical eukaryotic signaling pathway in that its Nitric oxide (NO. ) Guanylyl cyclase NO. synthase arginine Reduction
components exemplify almost all of the recog-
Release from
nized varieties of signaling molecules and their storage Reuptake
interactions: hormone, receptor, G protein, Ca2+ Numerous
organelles Stored and
calmodulin or plasma Ca2+ extrusion
adenylyl cyclase, protein kinase, phosphodi- membrane pumps
esterase, and extrusion pump. channels
The second messenger-stimulated protein Akt (protein
kinase PKA is a tetramer composed of two cat- Phosphatidyl- kinase B)
inositol-3,4,5- PI 3-kinase PIP2 Phosphatase
alytic (C) subunits and two regulatory (R) sub- trisphosphate Other PH
domains/proteins
units, as illustrated in FIGURE 14.15. The R subunit
binds to the catalytic subunit in the substrate- FIGURE 14.14 Major second messengers, some of the proteins that they regu-
binding region, maintaining C in an inhibited late, their sources and their disposition.
state. Each R subunit binds two molecules of
cAMP, four cAMP molecules per PKA holoen- ative binding of cAMP generates a very steep
zyme. When these sites are filled, the R subunit activation curve with an apparent threshold be-
dimer dissociates rapidly, leaving two free cat- low which no significant activation of PKA oc-
alytic subunits with high activity. The difference curs, as illustrated in Figure 14.15. PKA activity,
in affinity of R for C in the presence and absence thus, increases dramatically over a narrow range
of cAMP is ~10,000-fold. The strongly cooper- of cAMP concentrations. PKA is also regulated

14.14 Second messengers provide readily diffusible pathways for information transfer 607
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 608

Activation of PKA by cAMP teins throughout the cell ranging from ion chan-
Activated nels to transcription factors, and its conserved
PKA PKA substrate preference frequently permits predic-
C - cAMP C tion of substrates by sequence analysis. The
R - cAMP
(C) R (R) 4 cAMP cAMP response element binding protein CREB
Catalytic Regulatory
subunits R subunits R
- cAMP is phosphorylated by PKA on Ser 133 and is
C - cAMP C
largely responsible for the impact of cAMP on
transcription of numerous genes.
R 2 C 2 + 4 cAMP R2 . cAMP4 + 2C

Kinase activity as a
14.15 Ca2+ signaling serves
function of [cAMP] (%)
100
diverse purposes in all
90% eukaryotic cells
80
Key concepts
• Ca2+ serves as a second messenger and regulatory
60 molecule in essentially all cells.
• Ca2+ acts directly on many target proteins and also
40 regulates the activity of a regulatory protein
calmodulin.
20 • The cytosolic concentration of Ca2+ is controlled by
10% organellar sequestration and release.

2 x 10-9 2 x 10-8 2 x 10-7 Ca2+ is used as a second messenger in all cells,

[cAMP]
FIGURE 14.15 PKA is a heterotetramer composed of two catalytic (C) and
two regulatory (R) subunits. Binding of four molecules of cAMP to the reg-
ulatory subunits induces dissociation of two molecules of C, the active form
of PKA, from the cAMP-bound regulatory subunit dimer. In the bottom panel,
the cooperative binding of four molecules of cAMP generates a steep acti-
vation profile. Activity increases from approximately 10% to 90% as the
cAMP concentration increases only 10-fold. An apparent threshold is intro-
duced because there is little change in activity at low concentrations of
cAMP.
by phosphorylation of its activation loop.
Phosphorylation occurs cotranslationally, and
the activation loop phosphorylation is required
for assembly of the R2C2 tetramer.
The PKAs are mostly cytosolic and are also
targeted to specific locations by binding or-
ganelle-associated scaffolds (A-kinase anchor-
ing proteins, or AKAPs). These AKAPs facilitate
phosphorylation of membrane proteins includ-
ing GPCRs, transporters, and ion channels.
AKAPs can also target PKA to other cellular lo-
cations including mitochondria, the cytoskele-
ton, and the centrosome. AKAPs often harbor
binding sites for other regulatory molecules
such as phosphoprotein phosphatases and ad-
ditional protein kinases, which allows for co-
ordination of multiple signaling pathways and
integration of their outputs.
PKA generally phosphorylates substrates
with a primary consensus motif of Arg-Arg-
Xaa-Ser-Hydrophobic, placing it in a large group
of kinases that recognize basic residues preced-
ing the phosphorylation site. PKA regulates pro-
and is, thus, an even more widespread second
messenger than cAMP. Many proteins bind Ca2+
with consequent allosteric changes in their en-
zymatic activities, subcellular localization, or
interaction with other proteins or with lipids.
Direct targets of Ca2+ regulation include almost
all classes of signaling proteins described in this
chapter, numerous metabolic enzymes, ion
channels and pumps, and contractile proteins.
Most noteworthy may be muscle actomyosin
fibers, which are triggered to contract in re-
sponse to cytosolic Ca2+ (see 8.21 Myosin-II func-
tions in muscle contraction).
Although free Ca2+ is found at concentra-
tions near 1 mM in most extracellular fluids, in-
tracellular Ca2+ concentrations are maintained
near 100 nanomolar levels by the combined ac-
tion of pumps and transporters that either ex-
trude free Ca2+ or sequester it in the endoplasmic
reticulum or mitochondria. Ca2+ signaling is ini-
tiated when Ca2+-selective channels in the en-
doplasmic reticulum or plasma membrane are
opened to allow Ca2+ to enter the cytoplasm.
The most important entrance channels include
electrically gated channels in animal plasma
membranes; a Ca2+ channel in the endoplasmic
reticulum that is opened by another second mes-
senger, inositol 1,4,5-trisphosphate (see below);
and an electrically gated channel in the endo-
plasmic (sarcoplasmic) reticulum of muscle
that opens in response to depolarization of nearby
plasma membrane, a process known as excita-
tion-contraction coupling (see 2.9 Plasma mem-

608 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 609
Calcium binding causes a conformational change in calmodulin
Calcium-free
calmodulin
calmodulin free + 4 Ca 2+
Activation of target
by calmodulin (%)
100
80
60
40
20
10%
3 x 10-8
brane Ca2+ channels activate intracellular functions).
In addition to the proteins that are regulated
by binding Ca2+ directly, many other proteins re-
spond to Ca2+ by binding a widespread Ca2+ sen-
sor, the small, ~17 kDa protein calmodulin.
Calmodulin requires the binding of four mole-
cules of Ca2+ to become fully active, and bind-
ing is highly cooperative, generating a sigmoid
activation profile illustrated in FIGURE 14.16.
Calmodulin generally binds its targets in a Ca2+-
dependent manner, but Ca2+-free calmodulin
may remain bound but inactive in some cases.
For example, calmodulin is a constitutive sub-
unit of phosphorylase kinase that is activated
upon Ca2+ binding. Higher plants again make
major modifications to this paradigm. Calmodulin
is not expressed as a distinct protein but, instead,
is found as a domain in Ca2+-regulated proteins.
In yet another variation, the adenylyl cyclase se-
creted by the pathogenic bacterium Bordetella per-
tussis is inactive outside cells but is activated by
Ca2+-free calmodulin in animal cells, where its
rapid production of cAMP is highly toxic.
FIGURE 14.16 Ribbon diagrams represent-
ing the crystal structures of calmodulin free
Calcium-bound of Ca2+ and bound to four Ca2+ ions reveal
calmodulin bound to
target peptide of CaMK the huge conformational change that
calmodulin undergoes upon Ca2+ binding.
Ca2+-calmodulin causes activity changes in
target proteins. The bottom panel shows
the activation of a target by calmodulin as
a function of the intracellular free Ca2+ con-
centration. The requirement for binding
four Ca2+ ions to induce the conformational
transition results in cooperative activation
of targets. Activity increases from 10% to
Ca 2+ target 90% as the Ca2+ concentration increases
only 10-fold. Structures generated from
Protein Data Bank files 1CFD and 1MXE.
(Ca 2+)4 . calmodulin . active target
90%
3 x 10-7 3 x 10-6
[Ca2+]
14.16 Lipids and lipid-derived
compounds are signaling
molecules
Key concepts
• Multiple lipid-derived second messengers are
produced in membranes.
• Phospholipase Cs release soluble and lipid second
messengers in response to diverse inputs.
• Channels and transporters are modulated by
different lipids in addition to inputs from other
sources.
• PI 3-kinase synthesizes PIP3 to modulate cell
shape and motility.
• PLD and PLA2 create other lipid second
messengers.
Signals that originate at the plasma membrane
may have soluble regulatory targets in the cy-
toplasm or intracellular organelles, but integral
plasma membrane proteins are also subject to
acute controls. For these targets, lipid second
messengers may be primary inputs. Lipids de-
rived from membrane phospholipids or other
14.16 Lipids and lipid-derived compounds are signaling molecules 609
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 610

lipid species play numerous roles in cell signal-
ing. Because their analysis has been more dif-
ficult than for soluble messengers, many
probably remain to be discovered and under-
stood. FIGURE 14.17 shows the structure of some
of these lipids.
Phospholipase Cs (PLCs) are the prototyp-
ical lipid signaling enzymes. PLC isoforms cat-
alyze the hydrolysis of phospholipids between
the 3-sn-hydroxyl and the phosphate group to
yield a diacylglycerol and phosphate ester. In
animals and fungi, PLCs specific for the substrate

Structures of some lipid second messengers

O Phosphatidylinositol (PI)

O
O

O-
O
OH P
O
O OH
6
2 1 4
3
5 OH
HO
OH

O Phosphatidylinositol-3,4,5-trisphosphate (PIP3)

O
O

O-
O
OH P
O
O 6 OH
2 1 4 5 OPO3H-
H-O3PO 3
OPO3H-

O
Diacylglycerol (DAG)

O
O

OH

Inositol trisphosphate (IP3)

OH
OPO3H-
6 OH
2 1 4 5 OPO3H-
HO 3
OPO3H-

O Phosphatidic acid (PA)

O
O

O-
O
P
O
O-

FIGURE 14.17 Structures of some lipid second messengers and the common precursor phosphatidylinositol.
The acyl side chain structures shown here are the most common for mammalian PI lipids. Much of the PA in
cells is derived from PC, and its acyl chains may differ from those shown.

610 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 611
phosphatidylinositol-4,5-bisphosphate (PIP2)
hydrolyze PIP2 to form two second messengers:
1,2-sn-diacylglycerol (DAG) and inositol-1,4,5-
trisphosphate (IP3). The PLC substrate PIP2 is it-
self an important regulatory ligand that
modulates the activity of several ion channels,
transporters, and enzymes. Thus, PLC alters con-
centration of three second messengers; its net
effect depends on the net turnover of the sub-
strate and products.
DAG is probably the best known lipid sec-
ond messenger; its hydrophobicity limits it to ac-
tion in membranes. DAG activates some isoforms
of protein kinase C (PKC), modulates the ac-
tivity of several cation channels and activates at
least one other protein kinase. DAG can be fur-
ther hydrolyzed to release arachidonic acid,
which can regulate some ion channels.
Arachidonic acid is also the precursor of oxida-
tion products, such as prostaglandins and throm-
boxanes, which are potent extracellular signaling
agents. In addition to DAG, PKCs require inter-
action with Ca2+ and an acidic phospholipid,
such as phosphatidylserine, to become activated.
Thus, activation of PKC requires the coincidence
of multiple inputs both to generate DAG and to
increase intracellular Ca2+. There are more than
a dozen PKCs, classified together according to
highly conserved sequences in the catalytic do-
main. Three subgroups of PKCs, also identifiable
by sequence, share different patterns of regu-
lation. Their regulation provides examples of
many ways in which other mammalian protein
kinases are regulated.
The first of these groups, canonical PKCs,
are generally soluble or very loosely associated
with membranes prior to the appearance of
DAG. DAG causes their association with mem-
branes and permits activation upon binding of
other regulators. The second group of PKCs re-
quires similar lipids but not Ca2+, and the third
group requires other lipids but neither DAG nor
Ca2+ for activation.
The N-terminal region of PKCs contains a
pseudosubstrate domain, a sequence that re-
sembles that of a typical substrate except that
the target Ser is replaced with Ala. The pseu-
dosubstrate region binds to the active site to in-
hibit the kinase. Activators cause the
pseudosubstrate domain to flip out of the ac-
tive site. PKCs are also activated by proteoly-
sis, as are many protein kinases with discrete
autoinhibitory domains. Proteases clip a flex-
ible hinge region, which results in loss of the
regulatory domain and consequent activation
of the kinase.
PKC is the major receptor for phorbol esters,
a class of powerful tumor promoters. Phorbol
esters mimic DAG and cause a more massive
and prolonged activation than physiological
stimuli. This massive stimulation can induce
proteolysis of PKC, resulting in downregula-
tion, or loss of the kinase. (For a personal de-
scription on the discovery of protein kinase C
see EXP : 14-0001 )
IP3, the second product of the PLC reaction,
is a soluble second messenger. The most signif-
icant IP3 target is a Ca2+ channel in the endo-
plasmic reticulum. IP3 causes this channel to
open and release stored Ca2+ into the cytoplasm,
thereby rapidly elevating the cytosolic Ca2+ over
100-fold and, in turn, causing the activation of
numerous targets of Ca2+ signaling.
There are at least six families of PIP2-selec-
tive PLC enzymes, defined by their distinct forms
of regulation, domain compositions, and over-
all sequence conservation. Their catalytic do-
mains are all quite similar. The PLC-βs are
stimulated primarily by Gαq and Gβγ (to individ-
ually varying extents). Several are also modu-
lated by phosphorylation. PLC-γ isoforms are
stimulated by phosphorylation on Tyr residues,
frequently by receptor tyrosine kinases. The
PLC-ε isoforms are regulated by small,
monomeric G proteins of the Rho family. The
regulation of the PLC-δs is still incompletely un-
derstood. Two other classes similar to the PLC-
δs, PLC-η and -ζ, have also been defined recently.
(There is no PLC-α.) In addition to their distinct
modes of regulation, all of the PLCs are stimu-
lated by Ca2+, and Ca2+ often acts synergisti-
cally with other stimulatory inputs. This synergy
underlies the intensification and prolongation
of Ca2+ signaling observed in many cells.
Phospholipases A2 and D (PLA2 and PLD)
also hydrolyze glycerol phospholipids in cell
membranes to form important signaling com-
pounds. PLA2 hydrolyzes the fatty acid at the sn-
2 position of multiple phospholipids to produce
the cognate lysophospholipid and the free fatty
acid, which is generally unsaturated. The free
fatty acid is often arachidonic acid, a precursor
of extracellular signals. The biological roles of
free lysophospholipids are not understood in
detail but have been linked to effects on the
structure of the membrane bilayer.
PLD catalyzes a reaction much like that of
PLC but instead hydrolyzes the phosphodiester
on the substituent side of the phosphate group
to form 3-sn-phosphatidic acid. Cellular PLDs act
on multiple glycerol phospholipid substrates,
but phosphatidylcholine is probably the sub-
14.16 Lipids and lipid-derived compounds are signaling molecules 611
39057_ch14_cellbio.qxd 8/28/06
FIGURE 14.18 Activated PI 3-
kinase phosphorylates PIP2 to pro-
duce PIP3. The PH domain-contain-
ing protein kinases PDK1 and Akt
bind to PIP3 at the plasma mem-
brane. Their colocalization facili-
tates the phosphorylation of Akt
by PDK1. A second phosphoryla-
tion within a hydrophobic motif re-
sults in Akt activation by one of
several candidate protein kinases.
The Akt-2 isoform is required to
elicit hallmark actions of insulin.
PIP 3 binding brings Akt and PDK1 to the membrane
PIP 2 phosphorylated
PIP 2
p85 p110
PI 3-kinase
612 CHAPTER 14 Principles of cell signaling
5:11 PM Page 612
strate most relevant to signaling functions. The signaling can be fast and dramatic; it largely ac-
functions of the phosphatidic acid product, counts for directing the mobility of motile mam-
which is also formed by phosphorylation of malian cells.
DAG, remain poorly understood but appear to Lipid mediators are essential in the insulin
include a role in secretion and the fusion of in- signaling pathway. The binding of insulin stimu-
tracellular membranes. lates the Tyr autophosphorylation of its receptor
and the activation of effectors through insulin re-
ceptor substrate (IRS) proteins (see 14.30 Diverse
14.17 PI 3-kinase regulates signaling mechanisms are regulated by protein tyro-
sine kinases). PI 3-kinase is activated when its p85
both cell shape and the subunit binds to IRS1. The PIP3 generated by PI3-
activation of essential kinase binds the protein kinases Akt and phospho-
inositide-dependent kinase-1 (PDK-1) via their PH
growth and metabolic domains. This interaction results in the localiza-
functions tion of Akt to the membrane where it is activated
by PDK1, as illustrated in FIGURE 14.18. Akt phos-
Key concepts
phorylates downstream targets, including pro-
• Phosphorylation of some lipid second messengers
changes their activity.
tein kinases, GAPs, and transcription factors.
• PIP3 is recognized by proteins with a pleckstrin
Activation of Akt, specifically Akt-2, is required
homology domain. for the hallmark actions of insulin including reg-
ulation of glucose transporter translocation, en-
Lipid second messengers may also be modified hanced protein synthesis, and expression of
by phosphorylation. PI 3-kinase phosphorylates gluconeogenic and lipogenic enzymes.
PIP2 on the 3-position of the inositol ring to
form PI 3,4,5-P3, another lipid second messen-
ger. The total activity of PI 3-kinase is too low 14.18 Signaling through ion
to significantly deplete total PIP2, but forma-
tion of small amounts of PIP3 in localized mem-
channel receptors is very
brane domains is vital for altering cell shape fast
and cellular motility.
Key concepts
PIP3 acts by recruiting proteins that con-
• Ion channels allow the passage of ions through a
tain PIP3 binding domains, including pleckstrin pore, resulting in rapid (microsecond) changes in
homology (PH) and FYVE domains, to sites membrane potential.
where they regulate cytoskeletal remodeling, • Channels are selective for particular ions or for
contractile protein function, or other regula- cations or anions.
tory events. These proteins anchor and/or ori- • Channels regulate intracellular concentrations of
regulatory ions, such as Ca2+.
ent the structural or motor proteins involved
in cellular movement and localize signaling pro-
teins to sites of action at the membrane. PIP3 Ligand-gated ion channels are multisubunit,
membrane-spanning proteins that create and
regulate a water-filled pore through the mem-
brane, as illustrated in the X-ray crystal struc-
Akt and PDK1 ture of the nicotinic acetylcholine receptor in
Akt is activated by
bind PIP 3 through
phosphorylation FIGURE 14.19. When stimulated by extracellular
PH domains
PIP 3 agonists, the subunits rearrange their conforma-
tions and orientations to open the pore and, thus,
Akt PDK1
connect the aqueous spaces on either side of the
membrane. The pore has a diameter that allows
PH ions to diffuse freely from one side of the mem-
domains Other
kinase brane to the other, driven by the electrical and
chemical gradients that have been established
Akt PDK1 by ion pumps and transporters. (For more about
channel, pump and transporter mechanics see 2
Glucose uptake
Glycogen synthesis Transport of ions and small molecules across mem-
Antilipolysis branes.) Channels maintain selectivity among
Antiapoptosis
ions by regulating the pore diameter precisely
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 613
and by lining the walls of the pore with appro-
priate hydrophilic residues. Receptor ion chan-
nels can, thus, provide a diffusion path for only
cations or anions, or select among different ions.
Ligand-gated ion channels provide the
fastest signal transduction mechanism found in
biology. Upon binding an agonist ligand, chan-
nels open within microseconds. At synapses,
where neurotransmitters need to diffuse less
than 0.1 micron, a signal in the postsynaptic
cell can be generated in 100 microseconds. In
contrast, receptor-stimulated G proteins require
about 100 milliseconds to exchange GDP for
GTP, and the action of receptor protein kinases
is even slower. Ligand-gated ion channels are
important receptors in many cells in addition
to neurons and muscle, and other ion channels
play equally vital roles in signaling pathways
triggered by other classes of ligands.
Ion channel signaling differs from that of
the other receptors mentioned in this chapter
in that there is no immediate protein target nor,
in most cases, is there a specific second messen-
ger involved. In most cases, channel-mediated
ion flow acts to increase or decrease the cell’s
membrane potential and, thus, modulates all
transport processes for metabolites or ions that
are electrically driven.
Animal cells maintain an inside-negative
membrane potential by pumping out Na+ ions
and pumping in K+ ions (for more on mem-
brane potential see 2.4 Electrochemical gradients
across the cell membrane generate the membrane po-
tential). The opening of a channel selective for
Na+ will thus depolarize cells, and the opening
of a channel for K+ will hyperpolarize cells.
Similarly, because Cl- is primarily extracellular,
opening Cl- channels will also cause hyperpo-
larization. These electrical effects convey infor-
mation to effector proteins that are energetically
coupled to the membrane potential, or to spe-
cific ion gradients, or that bind a specific ion
(such as Ca2+) whose concentration changes
upon channel opening.
The nicotinic acetylcholine receptor is the pro-
totypical receptor ion channel and was the first
receptor that was shown to be a channel. It is a rel-
atively unselective cation channel that causes de-
polarization of the target cell by allowing Na+
influx. It is best known as the excitatory receptor
at the neuromuscular synapse, where it triggers
contraction, but alternative isoforms are also ac-
tive in neurons and many other cells. In muscles,
nicotinic depolarization acts via a voltage-sensitive
Ca2+ channel to allow Ca2+ release from the sar-
coplasmic reticulum into the cytosol. Calcium acts
Nicotinic acetylcholine receptor structure
CLOSED OPEN
Pore Pore
CYTOSOL
FIGURE 14.19 The nicotinic cholinergic receptor is a cation-selective channel
that is composed of five homologous but usually nonidentical subunits that
oligomerize to form a primarily -helical membrane-spanning core. The chan-
nel itself is created within this core, and its opening and closing are executed
by cooperative changes in subunit arrangement. Structure generated from
Protein Data Bank file 2BG9.
as a second (or third) messenger to initiate con-
traction (see 2.13 Cardiac and skeletal muscles are ac-
tivated by excitation-contraction coupling). Nicotinic
receptors promote exocytosis in some secretory
cells by a similar mechanism, where Ca2+ triggers
the exocytic event. In neurons, where nicotinic
stimulation causes an action potential (depo-
larization that is rapidly propagated along the neu-
ron), the initial depolarization is sensed by
voltage-sensitive Na+ channels. Their opening
(along with the action of other channels) propa-
gates the action potential along the neuron.
The nervous system is rich in receptor cation
channels that respond to other neurotransmit-
ters, the most common of which is the amino
acid glutamate (Glu). The three different fam-
ilies of glutamate receptors share the property
of cation conductance, but each family has its
own spectrum of drug responses. All operate as
neuronal activators, with one interesting twist:
The NMDA family of receptors, named for their
response to a selective drug, is permeant to Ca2+
in addition to Na+. A significant component of
its activity is to permit the inward flow of Ca2+,
which acts as a second messenger on a wide va-
riety of targets. Persistant stimulation of NMDA
channels by glutamate released during injury,
or by drugs, can cause toxic amounts of Ca2+ to
enter, resulting in neuronal death.
A second functional group of receptor chan-
nels is selective for anions and, by allowing in-
ward flux of Cl-, hyperpolarizes the target cell.
Anion-selective receptors include those for γ-
aminobutyric acid (GABA) and glycine (Gly). In
neurons, hyperpolarization can inhibit the ini-
tiation of an action potential and/or neurotrans-
mitter release.
14.18 Signaling through ion channel receptors is very fast 613
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 614
Perhaps the most diverse family of ligand-
gated channels is that of the TRP and TRP-like
family, of which about 30 have been found in
mammals. Distinct forms are found in inverte-
brates. The TRP channels are Ca2+-selective
channels that are formed by tetramers of iden-
tical subunits that surround the central chan-
nel. Each subunit is composed of a homologous
bundle of six membrane-spanning helices, but
the N and C termini contain a diverse collec-
tion of regulatory and protein interaction do-
mains, including protein kinase domains (whose
substrates are currently unknown).
All TRP channels allow transmembrane flux
of Ca2+ to permit its action as a second messen-
ger, but different TRP isoforms serve numerous
physiological functions. The prototypical TRP,
found in invertebrate photoreceptors, gates Ca2+
flow from intracellular stores into the cytoplasm
to initiate visual signaling. Others admit Ca2+
from outside the cell, and still others allow Ca2+
to enter the endoplasmic reticulum virtually di-
rectly from the extracellular space because they
form a bridge between the plasma membrane
and channels in the endoplasmic reticulum at
points where the membranes abut each other.
Regulation of TRP channels is perhaps even
more diverse. Various TRP channels respond to
heat, cold, painful stimuli, pressure, and high
or low osmolarity. Many TRPs are regulated ei-
ther positively or negatively by lipids, such as
eicosanoids, diacylglycerol, and PIP2. For ex-
ample, capsaicin, the hot compound in chilis, is
an agonist for some vanilloid receptors (TRPVs).
Still other TRP channels are mechanosensors
that allow cilia to sense fluid flow. The most fa-
mous of these is the sensory channel of the hair
cell of the inner ear. This channel opens when
the apical cilia on the hair cell are bent in re-
sponse to sound-driven fluid flow.
14.19 Nuclear receptors
regulate transcription
Key concepts
• Nuclear receptors modulate transcription by
binding to distinct short sequences in
chromosomal DNA known as response elements.
• Receptor binding to other receptors, inhibitors, or
coactivators leads to complex transcriptional
control circuits.
• Signaling through nuclear receptors is relatively
slow, consistent with their roles in adaptive
responses.
Nuclear receptors are unique among cellular
614 CHAPTER 14 Principles of cell signaling
receptors in that their ligands pass unaided
through the plasma membrane. These recep-
tors, when complexed with their ligands, en-
ter the nucleus and regulate gene transcription.
Ligands for nuclear receptors include sex steroids
(estrogen and testosterone) and other steroid
hormones, vitamins A and D, retinoids and
other fatty acids, oxysterols, and bile acids.
Nuclear receptors are structurally conserved.
They consist of a C-terminal ligand binding do-
main, an N-terminal interaction region that rec-
ognizes components of the transcriptional
machinery and acts as a transactivation domain,
a centrally located zinc finger domain that binds
DNA, and, often, another transactivation do-
main nearer the C-terminus. In the absence of
ligand, these receptors are bound to corepres-
sor proteins that suppress their activity. Upon
hormone binding, corepressors dissociate and
the receptors are assembled in multiprotein
complexes with coactivators that modulate re-
ceptor action and facilitate transcriptional reg-
ulation. As illustrated in FIGURE 14.20, agonists
and antagonists bind to distinct receptor confor-
mations (see 14.5 Ligand binding changes receptor
conformation). Receptor agonists favor the bind-
ing of receptors to coactivators and DNA, and
antagonists favor conformations that block coac-
tivator-receptor binding.
Nuclear receptors bind with high specificity
to hormone response elements in the 5’ un-
transcribed region of regulated genes. Response
elements are typically short direct or inverted
repeat sequences, and a gene may contain re-
sponse elements for several different receptors
in addition to binding sites for other transcrip-
tional regulatory proteins.
The sex steroid estrogen can bind to two
different nuclear receptors, the estrogen recep-
tors ER and ER. Coactivator and corepressor
proteins differentially regulate ER and ER in
transcriptional complexes that are expressed in
specific cell types. Other ligands that bind to
these receptors include valuable therapeutic
agents. For example, 4 hydroxy-tamoxifen is
an estrogen receptor antagonist used in the ther-
apy of estrogen-receptor-positive breast cancer
to inhibit growth of residual cancer cells.
However, unlike its antagonistic effects on the
estrogen receptor in breast, 4 hydroxy-tamox-
ifen displays weak partial agonist activity in
uterus. In the estrogen receptor system, partial
agonists are known as selective estrogen recep-
tor modulators (SERMs). Properties that con-
tribute to partial agonist activity include the
relative expression of the two estrogen recep-
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 615

Estrogen receptor conformation depends on which ligand is bound

Agonist-bound Antagonist-bound
conformation conformation

N N

K362

H11 H5 K362 H5 C
545

H6 H11
538 542
H6
542 H3
545 H3
538

FIGURE 14.20 The estrogen receptor adopts different conformations when

bound to agonists and antagonists. The ligand-binding domain of the estro-
gen receptor is bound to the agonist estradiol on the left and to the antago-
nist raloxifene on the right. Note the marked difference in position of helix 12,
shown in blue in the active structure and green in the inhibited structure.
Reproduced from Brzozowski, A. M., et al. 1997. Molecular basis of agonism and
antagonism in the oestrogen receptor. Nature. 389: 753–758. Photo courtesy
of M. Brzozowski, University of New York.

tors, ER and ER, as well as the expression of
repressors and coactivators that interact with
each receptor type. Thus, the behavior of nu-
clear receptor ligands must be considered in the
tissue, cellular, and signaling context.
14.20 G protein signaling
modules are widely used
and highly adaptable
Key concepts
• The basic module is a receptor, a G protein and an
effector protein.
• Cells express several varieties of each class of
proteins.
• Effectors are heterogeneous and initiate diverse
cellular functions.
Activation of G protein-coupled receptors
(GPCRs) and their associated heterotrimeric G
proteins is one of the most widespread mecha-
nisms of communicating extracellular signals
to the intracellular environment. G protein sig-
naling modules are found in all eukaryotes.
Depending on the species, mammals express
500-1000 GPCRs that respond to hormones,
neurotransmitters, pheromones, metabolites,
local signaling substances, and other regulatory
molecules. Essentially all chemical classes are
represented among the GPCR ligands. In addi-
tion, a roughly equal number of olfactory GPCRs
are expressed in olfactory neurons and work in
combination to screen compounds in the ani-
mal’s environment via the sense of smell.
Because GPCRs are involved in many kinds of
physiologic responses, they are also one of the
most widely used targets for drugs.
A minimal G protein signaling module con-
sists of three proteins: a G protein-coupled re-
ceptor, the heterotrimeric G protein, and an
effector protein, as illustrated in FIGURE 14.21. The
receptor activates the G protein on the inner face
of the plasma membrane in response to an ex-
tracellular ligand. The G protein then activates (or
occasionally inhibits) an effector protein that
propagates a signal within the cell. Thus, signal
conduction in the simplest G protein module is
linear. However, as depicted in FIGURE 14.22, a
typical animal cell may express a dozen GPCRs,
more than six G proteins, and a dozen effectors.
Each GPCR regulates one or more G proteins,
and each G protein regulates several effectors.
Moreover, distinct efficiencies and rates govern
each interaction. Thus, a cell’s G protein network
is actually a signal-integrating computer whose

14.20 G protein signaling modules are widely used and highly adaptable 615
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 616

Heterotrimeric G protein signaling output is a spectrum of cellular signals that is

complex in both amplitude and kinetics. Because
of their conserved parts list, G protein modules
Agonist
are well suited to initiating a wide variety of in-
tracellular signals in response to diverse molec-
GPCR
PIP 2 DAG
ular inputs and can do so over a wide range of
-
time scales (milliseconds to minutes).
Hydrolysis of PIP2 GPCRs are integral plasma membrane pro-
to IP 3 and DAG
Trimeric Activated teins composed of a bundle of seven hydropho-
G protein G protein
dissociates IP 3 bic membrane-spanning helices with an
Receptor
activated extracellular N terminus and cytosolic C termi-
IP 3-gated nus, as depicted in FIGURE 14.23. Based on the
Ca2+ channel
CYTOSOL Release of Ca2+ three-dimensional structure of rhodopsin and
on copious biochemical and genetic data, it is
likely that all GPCRs share the same basic mech-
anism of conformational activation and deacti-
Ca2+
ENDOPLASMIC vation in response to activating ligands (see 14.5
RETICULUM
Ligand binding changes receptor conformation).
FIGURE 14.21 G protein-mediated signal transduction follows a path of ago- Binding of agonist ligand on the extracellular
nist to receptor to heterotrimeric G protein to effector to the effector's out- face of the receptor drives realignment of the he-
put. Both G and G subunits regulate distinct effectors. In the example lices to alter the structure of a binding site for the
shown here, Gq regulates a phospholipase C- to produce two second messen- heterotrimeric G protein on the cytoplasmic face,
gers, diacyglycerol (DAG) and inositol-trisphosphate (IP3). IP3 triggers Ca2+ re-
lease from the endoplasmic reticulum.
and this altered conformation of the G protein-
binding surface promotes G protein activation.

FIGURE 14.22 A portion of the G pro- Partial G protein signaling network in mouse macrophages
tein-mediated signaling network in
macrophages highlights some of the Agonist C5a ISO PGE S1P UDP UTP PAF LPA
complexity of interactions possible in
such systems. Several receptors and G
protein subunits are omitted. Where a GPCR C5aR β 2 AR E2R EDG P2YR P2YR PAFR EDG
named G protein is shown, its signaling
output is probably mediated by its G
subunit. Activation of any G protein also
activates its G subunit, although G-
mediated signaling is usually most promi- G Protein Gi Gs G 12 Gq G 12
nent from Gi trimers. In addition, several
G proteins modulate the activities of
others through poorly understood path-
ways. Only a small sampling of effectors Ad
is shown, and the only adaptive mech- Cyc ??
anism shown is GRK-catalyzed phospho- ATP
rylation of receptors. Data from Paul Effector cAMP PI 3-
Sternweis, Alliance for Cellular Signaling. Kinase PIP 2 PLC- β

PIP 3 DAG + IP 3 IP 2 + P i
Phos-
phatase

Ca 2+ IP 3 R
cAMP
PDE
AMP
Ca 2+
Inactivation pump
mechanisms

GRK

616 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 617

Structure of rhodopsin Heterotrimeric G protein structure

C YTOPLASM

MEMBRAN E

Retinal

FIGURE 14.24 The structure of the nonactivated Gi het-

erotrimer, the G protein that is responsible for inhibition of
FIGURE 14.23 The figure shows the crystal structure adenylyl cyclase and for most G-mediated signaling, is
of the GPCR rhodopsin. Each membrane-spanning he- shown with each subunit colored as shown. GDP is shown
lix is a different color; most structures on the cytoplas- bound to the Gi subunit. Structure generated from Protein
mic face are not shown. The retinal chromophore is Data Bank file 1GP2.
shown within the helix bundle. GPCR sequence similar-
ity separates the mammalian GPCRs into at least four G protein targets
structural families that are so diverse that there may
EFFECTOR PROTEIN
be little sequence similarity among the classes. Within G
a family, similarity is greatest in the membrane-span- protein Stimulated Inhibited
ning helices, less in the interhelical loops, and least in
Gs
the N- and C-terminal domains and in the cytoplasmic Adenylyl cyclase
G olf
loop that connects spans five and six. Regardless, the
generalizations about functional domains in receptors G i (3)
seem to hold true within different families. GPCRs fre- Go K + channel, PI 3-kinase Adenylyl cyclase
Gz
quently form dimers, occasionally heterodimers, and
dimerization can be crucial for function. Structure gen- G gus Other cation channel
erated from Protein Data Bank file 1F88.
Cyclic GMP phospho-
G t (2)
diesterase
The heterotrimeric G proteins to which
G q (4) Phospholipase-Cβ
GPCRs are coupled are composed of a nu-
cleotide-binding Gα subunit and a Gβγ subunit G 12 Rho GEF
G 13
dimer, as illustrated in FIGURE 14.24. The struc-
ture of the trimer and each subunit is known for FIGURE 14.25 G protein-regulated effectors do not share struc-
several states of activation and in complex with tural similarities. They may be ion channels or membrane span-
several interacting proteins. A Gαβγ heterotrimer ning enzymes in the plasma membrane, peripheral proteins on the
inner face of the membrane, or fundamentally soluble proteins
is named according to its α subunit, which largely that can bind to G subunits. The chart shows the major groups
defines the G protein’s selectivity among recep- of G proteins, sorted according to sequence similarity, and some
tors. Each subunit also regulates a distinct group of the effectors that they are known to regulate.
of effector proteins.
Gα subunits are globular, two-domain pro- because of constitutive N-terminal fatty acyla-
teins of 38-44 kDa. The GTP-binding domain tion and because they bind to the membrane-
belongs to the GTP-binding protein superfam- attached Gβγ subunits. Mammals have 16 Gα
ily that includes the small, monomeric G pro- genes that are grouped in subfamilies according
teins (such as Ras, Rho, Arf, Rab; see 14.23 Small, to similar sequence and function (e.g., s, i, q,
monomeric GTP-binding proteins are multiuse and 12). These subfamilies are listed in FIGURE
switches) as well as the GTP-binding translational 14.25.
initiation and elongation factors. A second do- Gβ and Gγ subunits associate irreversibly
main modulates GTP binding and hydrolysis. soon after translation to form stable Gβγ dimers,
Gα subunits are only slightly hydrophobic, but which then associate reversibly with a Gα. Gβ
they are predominantly membrane-associated subunits are 35 kDa proteins composed of seven

14.20 G protein signaling modules are widely used and highly adaptable 617
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 618
-strand repeats that form a cylindrical structure
known as a  propeller. There are five Gβ genes
in mammals. Four encode strikingly similar pro-
teins that naturally dimerize with the twelve Gγ
subunits (Figure 14.24). The fifth, Gβ5, is less
closely related to the others and interacts prima-
rily with a Gγ-like domain in other proteins rather
than with Gγ subunits themselves.
Gγ subunits are smaller (~7 kDa) and far
more diverse in sequence than are the Gβ’s. The
last three amino acid residues of Gγ subunits
are proteolyzed to leave a conserved C-termi-
nal cysteine that is irreversibly S-prenylated
and carboxymethylated, helping to anchor Gβγ
to the membrane. Gβ and Gγ subunits can as-
sociate in most possible combinations. Because
almost all cells express multiple Gβ and Gγ sub-
units, it has been difficult to assign specific roles
to individual Gβγ combinations. The best rec-
ognized interactions of Gβγ subunits occur at
sites on Gβ, although distinct functions of Gγ
have also been supported.
14.21 Heterotrimeric G proteins
regulate a wide variety of
effectors
Key concepts
• G proteins convey signals by regulating the
activities of multiple intracellular signaling
proteins known as effectors.
• Effectors are structurally and functionally diverse.
• A common G-protein binding domain has not been
identified among effector proteins.
• Effector proteins integrate signals from multiple G
protein pathways.
G protein-regulated effectors include enzymes
that create or destroy intracellular second
messengers (adenylyl cyclase, cyclic GMP phos-
phodiesterase, phospholipase C-β, phosphatidyli-
nositol-3-kinase), protein kinases, ion channels
(K+, Ca2+) and possibly membrane transport
proteins (see Figure 14.25). Effectors may be
integral membrane proteins or intrinsically sol-
uble proteins that bind G proteins at the mem-
brane surface. No conserved G protein-binding
domain or sequence motif has been identified
among effector proteins, and most effectors are
related to proteins that have similar functions
but that are not regulated by G proteins.
Sensitivity to G protein regulation, thus, evolved
independently in multiple families of regula-
tory proteins.
618 CHAPTER 14 Principles of cell signaling
Because they can respond to a variety of
Gα and Gβγ subunits, effector proteins can in-
tegrate signals from multiple G protein path-
ways. The different Gα or Gβγ subunits may
have opposite or synergistic effects on a given
effector. For example, some of the membrane-
bound adenylyl cyclases in mammals are stim-
ulated by Gαs and inhibited by Gαi (see Figure
14.13). Many effectors are further regulated by
other allosteric ligands (e.g., lipids, calmodulin)
and by phosphorylation, contributing even more
to integration of information.
Effectors are usually represented as multi-
ple isoforms, and each isoform may be regu-
lated differently, adding to the complexity of G
protein networks. For example, some isoforms
of adenylyl cyclase are stimulated by Gβγ,
whereas others are inhibited. All phospholipase
C-βs are stimulated both by Gαq family mem-
bers and by Gβγ, but the potency and maximal
effect of these two inputs vary dramatically
among the four PLC-β isoforms.
14.22 Heterotrimeric G proteins
are controlled by a
regulatory GTPase cycle
Key concepts
• Heterotrimeric G proteins are activated when the
Gα subunit binds GTP.
• GTP hydrolysis to GDP inactivates the G protein.
• GTP hydrolysis is slow, but is accelerated by
proteins called GAPs.
• Receptors promote activation by allowing GDP
dissociation and GTP association; spontaneous
exchange is very slow.
• RGS proteins and phospholipase C-βs are GAPs for
G proteins.
The key event in heterotrimeric G protein sig-
naling is the binding of GTP to the Gα subunit.
GTP binding activates the Gα subunit, which
allows both it and the Gβγ subunit to bind and
regulate effectors. The Gα subunit remains ac-
tive as long as GTP is bound, but Gα also has
GTPase activity and hydrolyzes bound GTP to
GDP. Gα-GDP is inactive. G proteins thus traverse
a GTPase cycle of GTP binding/activation and hy-
drolysis/deactivation, as depicted in FIGURE 14.26.
Therefore, the control of G protein signaling is
intrinsically kinetic. The relative signal strength,
or amplitude, is proportional to the fraction of
G protein that is in the active, GTP-bound form.
This fraction equals the balance of the rates of
GTP binding and GTP hydrolysis, the activating
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 619
and deactivating arms of the GTPase cycle. Both
limbs are highly regulated over a range of rates
greater than 1000-fold.
Receptors promote G protein activation by
opening the nucleotide-binding site on the G
protein, thus accelerating both GDP dissociation
and GTP association. This process is referred to
as GDP/GTP exchange catalysis. Exchange pro-
ceeds in the direction of activation because the
affinity of G proteins for GTP is much higher than
that for GDP and because the cytosolic concen-
tration of GTP is about 20-fold higher than that
of GDP. Spontaneous GDP/GTP exchange is very
slow for most G proteins (many minutes), which
maintains basal signal output at a low level. In
contrast, receptor-catalyzed exchange can take
place in a few tens of milliseconds, which allows
rapid responses in cells such as visual photore-
ceptors, other neurons, or muscle.
Because receptors are not directly required
for a G protein’s signaling activity, a receptor can
dissociate after GDP/GTP exchange and catalyze
the activation of additional G protein molecules.
In this way, a single receptor may maintain the
activation of multiple G proteins, providing mo-
lecular amplification of the incoming signal.
Other receptors may remain bound to their G
protein targets, which means that they do not
act as amplifiers. However, more tightly bound
receptors can initiate signaling more quickly and
promote G protein reactivation when hydroly-
sis of bound GTP is rapid.
In the absence of stimulus, Gα subunits
hydrolyze bound GTP slowly. The average ac-
tivation lifetime of the G α-GTP complex is
about 10-150 seconds, depending on the G
protein. This rate is far slower than rates of de-
activation often observed in cells when an ag-
onist is removed. For example, visual signaling
terminates in about 10 ms after stimulation by
a photon, and many other G protein systems
are almost as fast. GTP hydrolysis is acceler-
ated by GTPase-activating proteins (GAPs),
which directly bind Gα subunits. In some cases
acceleration exceeds 2000-fold. Such speed is
necessary in systems like vision or neurotrans-
mission, which must respond to quickly chang-
ing stimuli. Because G protein signaling is a
balance of activation and deactivation, GAPs
deplete the pool of GTP-activated G protein
and can thereby also act to inhibit G protein
signaling. GAPs can thus inhibit signaling,
quench output upon signal termination, or
both. What behavior predominates depends
on the GAP’s intrinsic activity and its regula-
tion.
14.22 Heterotrimeric G proteins are controlled by a regulatory GTPase cycle
The regulatory GTPase cycle
Receptor Receptor
+ agonist - agonist
G protein
GDP GTP
Effector protein
G protein-GTP G protein-GTP-
G protein-GDP *ACTIVE* Effector protein
*ACTIVE*
Pi
GAP
FIGURE 14.26 G proteins are activated when GTP binds to the G subunit, such
that both G-GTP and G can bind and regulate the activities of appropriate
effector proteins. G subunits also have intrinsic GTPase activities, and the pri-
mary deactivating reaction is hydrolysis of bound GTP to GDP (rather than GTP
dissociation). Thus, the steady-state signal output from a receptor-G protein
module is the fraction of the G protein in the GTP-bound state, which reflects
the balance of the activation and deactivation rates. Both GTP binding and GTP
hydrolysis are intrinsically slow and highly regulated. GDP binds tightly to G,
such that GDP dissociation is rate-limiting for binding of a new molecule of
GTP and consequent reactivation. Both GDP release and GTP binding are cat-
alyzed by GPCRs. Hydrolysis of bound GTP is accelerated by GTPase-activating
proteins (GAPs). Receptors and GAPs coordinately control both the steady-state
level of signal output and the rates of activation and deactivation of the mod-
ule.
There are two families of GAPs for het-
erotrimeric G proteins. The RGS proteins (reg-
ulators of G protein signaling) are a family of
about 30 proteins, most or all of which have
GAP activity and regulate G protein signaling
rates and amplitudes. The role of RGS proteins
in terminating the G protein signal can be seen
in FIGURE 14.27. Some proteins with RGS do-
mains also act as G protein-regulated effectors.
These include activators of the Rho family of
monomeric GTP-binding proteins (see below)
and GPCR kinases, which are feedback regula-
tors of GPCR function. The second group of G
protein GAPs are phospholipase C-βs. These en-
zymes are effectors that are stimulated by both
Gαq and by Gβγ, but they also act as Gq GAPs,
probably to control output kinetics.
619
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 620
Single photon responses of GAP-deficient mice
Current (pA)
0.50
knockout
heterozygous
wild-type
0.25
0.00
0 2 Time (s) 4 spontaneous nucleotide exchange.
Light flash
FIGURE 14.27 G protein GAPs can accelerate signal termi-
nation upon removal of agonist, and often do not act as in-
hibitors during the response to receptor. The figure shows
the electrical response of a mouse photoreceptor (rod) cell
to a single photon of light. In mice that lack RGS9, the GAP
for the photoreceptor G protein Gt, the signal is prolonged
for many seconds because hydrolysis of GTP bound to Gt is
slow. In wild-type or heterozygous mice, hydrolysis takes
place in about 15 milliseconds, and the decay of the signal
is much faster. Note that the maximal output is similar in
wild-type and mutant mice, indicating that the GAP does
not act as an inhibitor in rod cells. In humans, genetic loss
of RGS9 leads to severe loss of vision that is particularly
marked in bright light. Reproduced from Chen et al. Nature.
2000. 403:557–560. Permission also granted by Ching-Kang
Jason Chen, Virginia Commonwealth University.
While the GTPase cycle described in Figure
14.26 is general, it is highly simplified. Interactions
among receptor, Gα, Gβγ, GAP, and effector are
frequently simultaneous and often demonstrate
complex cooperative interactions. For example,
Gβγ inhibits the release of GDP (to minimize
spontaneous activation), promotes the exchange
catalyst activity of the receptor, inhibits GAP ac-
tivity, and helps initiate receptor phosphoryla-
tion that leads to desensitization. The other
components can be nearly this multifunctional.
In addition, inputs from other proteins can alter
the dynamics of the GTPase cycle at several points.
The core G protein module is, thus, functionally
versatile as a signal processor in addition to be-
ing versatile in the scope of its targets.
620 CHAPTER 14 Principles of cell signaling
14.23 Small, monomeric GTP-
binding proteins are
multiuse switches
Key concepts
• Small GTP-binding proteins are active when bound
to GTP and inactive when bound to GDP.
• GDP/GTP exchange catalysts known as GEFs
(guanine nucleotide exchange factors) promote
activation.
• GAPs accelerate hydrolysis and deactivation.
• GDP dissociation inhibitors (GDIs) slow
Monomeric GTP-binding proteins, which are
encoded by about 150 genes in animals, mod-
ulate a wide variety of cellular processes includ-
ing signal transduction, organellar trafficking,
intra-organellar transport, cytoskeletal assem-
bly, and morphogenesis. The small GTP-bind-
ing proteins that most clearly function in signal
transduction are the Ras and Ras-related pro-
teins (Ral, Rap) and the Rho/Rac/Cdc42 pro-
teins, about 10-15 in all. They are usually about
20-25 kDa in size and are homologous to the
GTP-binding domains of Gα subunits.
The regulatory activities of the small GTP-
binding proteins are controlled by a GTP bind-
ing and hydrolysis cycle like that of the
heterotrimeric G proteins, with similar regula-
tory inputs. They are activated by GTP, and hy-
drolysis of bound GTP to GDP terminates
activation. GDP/GTP exchange catalysts, known
as GEFs (guanine nucleotide exchange factors,
functionally analogous to GPCRs) promote ac-
tivation, and GAPs accelerate hydrolysis and
consequent deactivation. In addition, GDP dis-
sociation inhibitors (GDIs) slow spontaneous
nucleotide exchange and activation to dampen
basal activity, an activity shared by Gβγ subunits
for the heterotrimeric G proteins.
While the underlying biochemical regula-
tory events are essentially identical for
monomeric and heterotrimeric G proteins,
monomeric G proteins use the basic GTPase cy-
cle in additional ways. Signal output by het-
erotrimeric G proteins and many monomeric
G proteins is usually thought to reflect a bal-
ance of their active (GTP-bound) and inactive
(GDP-bound) states in a rapidly turning-over
GTPase cycle. GEFs favor formation of more ac-
tive G protein, and GAPs favor the inactive state.
In contrast, probably an equal number of the
monomeric G proteins behave as acute on-off
switches. Upon binding GTP, they initiate a
process (regulation, recruitment of other pro-
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 621
teins). They then maintain this activity, some-
times for many seconds or minutes, until they
are acted upon by a GAP. For example, the
monomeric G protein Ran regulates nucleocy-
toplasmic trafficking of protein and RNA in both
directions, cooperating with carrier proteins
known as karyopherins (see 5.15 The Ran GTPase
controls the direction of nuclear transport). In the
nucleus, high Ran GEF activity promotes GTP
binding. Nuclear Ran-GTP then binds import
karyopherins to drive dissociation of newly ar-
rived cargo and promote return of the karyo-
pherin to the cytoplasm. It also binds export
karyopherins to permit binding of outgoing
cargo. Outside the nucleus, high Ran GAP ac-
tivity promotes GTP hydrolysis. Cytoplasmic
Ran-GDP dissociates from both the export karyo-
pherins to allow dissociation of outgoing cargo
and from the import karyopherins to allow them
to bind cargo for import. Thus, for monomeric
G proteins such as Ran, each phase of the GTPase
cycle determines a specific, coupled step in a
parallel regulatory cycle.
A second major difference between the
monomeric and the heterotrimeric G proteins
is the structures of the GEFs, GAPs, and GDIs.
Both GEFs and GAPs for monomeric GTP-bind-
ing proteins are structurally heterogeneous (al-
though some clearly related families are
evident). In addition, mechanisms for regulat-
ing these GEFs and GAPs are equally diverse.
They include phosphorylation by protein ki-
nases; allosteric regulation by heterotrimeric
and/or monomeric G proteins, by second mes-
sengers and by other regulatory proteins; sub-
cellular sequestration or recruitment to scaffolds;
and assorted other mechanisms.
The Ras proteins were the first small GTP-
binding proteins to be discovered. They were
identified as oncogene products because they
cause malignant growth if they are either over-
expressed or persistently activated by mutation;
they are among the most commonly mutated
genes in human tumors. Several viral ras genes
figure prominently as oncogenes.
Mammalian cells contain three ras genes (H,
N, and K). They may share inputs and outputs
to varying extents, and they can compensate for
each other in some genetic screens. It has been
difficult to assign unique functions to the indi-
vidual Ras proteins. Inputs to the Ras proteins
are diverse and speak to the importance of Ras
proteins as a crucial node in signaling.
Ras GEFs and GAPS are regulated by both
receptor and nonreceptor Tyr kinases through
direct phosphorylation and by recruitment of
the regulators to the plasma membrane. Other
14.24 Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell
Ras has three main effectors FIGURE 14.28 Ras-GTP binds
Function Effector Target to many proteins. Three well
Protein kinase cascade Raf MAPK
established effectors include
Raf, PI 3-kinase, and RalGDS.
Lipid kinase PI 3-kinase Akt
Activation of these effectors
Exchange factor RalGDS Exocyst activates a MAPK pathway, in-
creases PI 3-kinase activity,
and promotes assembly of a
protein complex involved in
exocytosis of secretory vesi-
cles.
cytoplasmic serine/threonine kinases also con-
verge on Ras activation. Rap1, another mem-
ber of the Ras family, may also fit directly into
this network because it is suspected of compet-
ing with Ras proteins for protein kinase targets;
in vivo it can suppress the oncogenic activity of
Ras. Rap1 is regulated independently, however,
and acts on independent signaling pathways as
well. One of its GAPs is stimulated by the Gi
class of G proteins, for example, and its several
GEFs are stimulated by Ca2+, diacylglycerol, and
cAMP.
Ras proteins generally regulate cell growth,
proliferation, and differentiation by modulat-
ing the activities of multiple effector proteins.
The best known and best studied Ras effector is
the protein kinase Raf, which initiates a MAPK
cascade. FIGURE 14.28 shows well established Ras
effectors.
Rho, Rac, and Cdc42 are related monomeric
GTP-binding proteins that are involved in gen-
erating signals that affect cell morphology. Each
class of proteins regulates its own array of effec-
tors and is controlled by separate groups of GEFs,
GAPs, and GDIs. Effectors regulated by this fam-
ily include phospholipases C and D, multiple
protein and lipid kinases, proteins that nucle-
ate or reorganize actin filaments, and compo-
nents of the neutrophil oxygen activating
system, among others (see 8.14 Small G proteins
regulate actin polymerization).
14.24 Protein phosphorylation/
dephosphorylation is a
major regulatory
mechanism in the cell
Key concepts
• Protein kinases are a large protein family.
• Protein kinases phosphorylate Ser and Thr, or Tyr,
or all three.
• Protein kinases may recognize the primary
sequence surrounding the phosphorylation site.
• Protein kinases may preferentially recognize
phosphorylation sites within folded domains.
621
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 622

Protein phosphorylation is the most common nases involved in two-component signaling are
form of regulatory posttranslational modifica- unrelated to the eukaryotic protein kinase su-
tion. It occurs in all organisms, and it is estimated perfamily and phosphorylate aspartate residues
that about one-third of proteins in animals are rather than serine, threonine, or tyrosine.
at some time phosphorylated. Phosphorylation Protein kinases transfer a phosphoryl group
can stimulate or inhibit the catalytic activity of from ATP to Ser, Thr, and Tyr residues of pro-
an enzyme, the affinity with which a protein tein substrates to form chemically stable phos-
binds other molecules, its subcellular localiza- phate esters, as shown in FIGURE 14.29. In
tion, its ability to be further covalently modified, animals, the distribution of phosphate among
or its stability. Single phosphorylations may cause these three amino acid residues is uneven:
500-fold or greater changes in activity, and pro- ~90%-95% is on Ser, 5%-8% on Thr, and less
teins are often phosphorylated on multiple than 1% on Tyr residues. The human genome
residues in complex and interacting patterns. contains approximately 500 genes that encode
Most protein phosphorylation in eukary- protein kinases, and many protein kinase
otes, and essentially all in animals, is catalyzed mRNAs undergo alternative splicing. This makes
by protein kinases; dephosphorylation is cat- the protein kinase gene superfamily one of the
alyzed by phosphoprotein phosphatases. Both largest functional gene groups. The number and
classes of enzymes are controlled by diverse diversity of these enzymes emphasize the great
mechanisms. In addition, proteins are often and varied uses of protein kinases to regulate cel-
phosphorylated by multiple protein kinases, re- lular functions. Although some protein kinases
sulting in the generation of a range of activity have a limited tissue and/or developmental dis-
states. This complexity allows inputs from dif- tribution, many are ubiquitously expressed.
ferent signaling pathways to be integrated into Protein kinases are grouped according to
the resulting activity of the target. their residue specificity. Protein kinases that
In bacteria, plants, and fungi, an additional phosphorylate Ser will usually also recognize
protein phosphorylating system known as two- Thr, hence the name protein Ser/Thr kinase.
component signaling is vital. The protein ki- Multicellular organisms have protein Tyr ki-
nases, which only recognize Tyr. Dual speci-
ficity protein kinases can phosphorylate Ser,
Thr, and Tyr in the appropriately restricted sub-
strate conformational context and are gener-
Protein kinases are two substrate enzymes ally the most selective of the protein kinases.
SUBSTR A TE 1 SUBSTRATE 2 The analysis of the kinomes of several or-
( Pro te in SER) (Mg 2+ . ATP) ganisms has led to a more elaborate grouping
N H
derived from sequence relationships, shown in
Mg 2+ Adenine FIGURE 14.30, that also reflects to some extent
C O O- O- O-
O- P O
on regulatory mechanisms and substrate speci-
H C CH 2 OH + P O P O CH 2
ficity. For example, the AGC group is named
N H O O O
C O for its founding members, cAMP-dependent
(Mg 2+ . Trip h osp h ate) protein kinase (PKA), cyclic GMP-dependent
Rib ose
protein kinase (PKG), Ca2+, and phospholipid-
dependent protein kinase (PKC). These protein
P ROTEIN KIN ASE kinases are regulated by second messengers and
prefer substrates that contain basic residues near
the phosphorylation site.
PR O DU CT 1 P RODUCT 2 In addition to substrate specificity for amino
( Pho s p ho ryla ted p ro t ein ) (Mg 2+ . ADP) acid residues, most protein kinases are also selec-
N H
tive for local sequence surrounding the substrate
Mg 2+ Adenine site. Screening strategies have resulted in meth-
C O O- O- O-
H C CH 2 O P O- O- P O P O CH 2
ods to predict if proteins contain consensus sub-
N H O O O strate sites for a wide variety of protein kinases.
C O Antibodies can be used to identify and roughly
( Mg 2+ . Dip h osp h ate) quantitate protein phosphorylation at specific
Rib
sites in proteins. Beyond local recognition, pro-
FIGURE 14.29 Protein kinases transfer the -phosphoryl group from ATP to tein kinases may display marked substrate selec-
serine, threonine, or tyrosine residues in protein substrates. tivity among similar proteins based on overall

622 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 623
Human kinome tree
three-dimensional structure, for example, or
among proteins that have been differentially co-
valently modified by phosphorylation or ubiqui-
tination.
In animal cells, some protein kinases are
hormone receptors that span the plasma mem-
brane. Some protein kinase receptors are pro-
tein serine/threonine kinases, such as the
transforming growth factor- (TGF-)receptor,
but the majority are protein tyrosine kinases, in-
cluding receptors for insulin, epidermal growth
factor (EGF), platelet-derived growth factor
(PDGF), and other regulators of cell growth and
differentiation. Other protein kinases are in-
trinsically soluble intracellular enzymes, al-
though they may bind to one or more organellar
membranes.
14.24 Protein phosphorylation/dephosphorylation is a major regulatory mechanism in the cell
FIGURE 14.30 The protein kinases in
the human genome can be grouped ac-
cording to sequence relationships that
reveal seven major branches. The tyro-
sine kinases are contained within one
major branch. The others are Ser/Thr-
specific or dual specificity, and are named
for the best described members: AGC
from PKA, PKG, and PKC; CAMK from the
calcium, calmodulin-dependent kinases;
CMGC from CDKs, MAPKs, GSK3, Clks; CK1
from casein kinase 1; STE from Ste20,
Ste11, and Ste7, the MAP4K, MAP3K,
and MAP2K in the yeast mating path-
way; and TKL, the Tyr kinase-like en-
zymes. Reproduced with permission from
G. Manning, et al. 2002. Science. 298:
1912-1934. © 2002 AAAS. Photo cour-
tesy of Gerard Manning, Salk Institute,
and reprinted with permission of Cell
Signaling Technology, Inc. (www.cellsig-
nal.com).
X-ray crystallographic structures of protein
kinases have revealed a wealth of information
about their mechanism of activation. The con-
served minimum catalytic core of a protein ki-
nase contains about 270 amino acids, yielding
a minimum molecular mass of about 30,000
Da. Within this core, there are two folded do-
mains that form the active site at their inter-
face, as shown in FIGURE 14.31. One or both of
the conserved lysine (Lys) or aspartate (Asp)
residues that are required for phosphoryl trans-
fer are frequently mutated to disrupt kinase ac-
tivity. A sequence near the active site, referred
to as the activation loop, often undergoes a con-
formational rearrangement to generate active
forms of the protein kinases and is the most
common site of regulatory phosphorylation in
623
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 624

ERK2 inactive and active conformations the protein kinase family. There are unique in-
serts on the surface of protein kinases that gen-
INACTIVE (ERK2) ACTIVE (ERK2-P2)
erate specificity in localization, interaction with
N
N terminal
terminal other regulatory molecules, and recognition of
domain
domain
substrates. These landmarks allow both classi-
fication and genetic manipulation of protein ki-
nases.
Thr183
Thr183 Protein kinases have evolved numerous
and diverse regulatory mechanisms to comple-
Thr183
Thr183
ment their number and multiple functions.
Tyr185
Tyr185 Tyr185
Tyr185 These mechanisms include allosteric activation
and inhibition by lipids, soluble small molecules
and other proteins; activating and inhibitory
phosphorylation and other covalent modifica-
C
C terminal
terminal tions, including proteolysis; and binding to scaf-
domain
domain folds and adaptors to enhance activity or limit
nonspecific activities. Many such inputs may
FIGURE 14.31 The structures of unphosphorylated, inactive MAPK ERK2 and
phosphorylated, active ERK2 are compared. ERK2 has a typical protein kinase regulate a single protein kinase in a complex
structure. The smaller N-terminal domain is composed primarily of  strands combinatoric code. Further, multiple protein
and the larger C-terminal domain is primarily -helical. The active site is formed kinases that act sequentially, such as in a pro-
at the interface of the two domains. The activation loop emerges from the ac- tein kinase cascade (see Figure 14.38), can cre-
tive site and is refolded following phosphorylation of the Tyr and Thr residues,
ate uniquely complex signaling patterns.
inducing the repositioning of active site residues. ATP (not shown) binds in
the interior of the active site; productive binding of protein substrates to the
surface of the C-terminal domain is also facilitated by the reorganization of
the activation loop. Structures generated from Protein Data Bank files 1ERK 14.25 Two-component protein
and 2ERK.
phosphorylation systems
are signaling relays
Key concepts
• Two-component signaling systems are composed of
sensor and response regulator components.
• Upon receiving a stimulus, sensor components
undergo autophosphorylation on a histidine (His)
residue.
• Transfer of the phosphate to an aspartyl residue on
the response regulator serves to activate the
Two-component signaling systems regulator.

Ligand
Prokaryotes, plants, and fungi share an alterna-
Sensor/
tive mechanism for regulatory phosphorylation
Histidine His P His P His and dephosphorylation known as two-compo-
kinase
nent signaling. FIGURE 14.32 shows a typical two-
ADP
component system. In this system, the receptor,
ATP referred to as a sensor, responds to a stimulus by
catalyzing its own phosphorylation on a His
Response Asp Asp P Asp P residue. Sensors include chemoattractant recep-
regulator H2O tors in bacteria, a regulator of osmolarity in fungi,
light-sensitive proteins, the receptor for the plant-
P ripening hormone ethylene, and other receptors
His phosphorylation Transfer of phosphate Response regulator for diverse environmental, hormonal, and meta-
to Asp: response deactivated
regulator active bolic signals. The mammalian mitochondrial de-
hydrogenase kinases are related in sequence to
FIGURE 14.32 The basic two-component system is composed of a signal-acti-
the bacterial histidine kinases, although the mam-
vated histidine kinase, referred to as a sensor, and an effector protein, the re-
sponse regulator, that is activated when it is phosphorylated on an aspartate malian enzymes phosphorylate serine or threo-
residue by the sensor. The activity of the response regulator is terminated when nine residues, not histidine. The phosphorylated
the aspartyl-phosphate is hydrolyzed. sensor next transfers its covalently bound phos-

624 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 625
phate to an aspartyl residue on a second protein
known as a response regulator. Response regu-
lators initiate cellular responses, usually by bind-
ing to other cytoplasmic proteins and allosterically
regulating their activities.
Although all two-component systems fol-
low this same general pattern, their structures
and precise reaction pathways vary enormously.
Some two-component systems are composed
of only one protein (sensor and response reg-
ulator in a single polypeptide chain). Others are
composed of a sensor protein and two aspartyl-
phosphorylated proteins, in which the first or
the second may display response regulatory ac-
tivity. Finally, two-component systems usually
lack conventional protein phosphatases.
Hydrolysis of the aspartyl-phosphate bond may
be spontaneous or regulated by the response
regulator itself.
14.26 Pharmacological
inhibitors of protein
kinases may be used to
understand and treat
disease
Key concepts
• Protein kinase inhibitors are useful both for
signaling research and as drugs.
• Protein kinase inhibitors usually bind in the ATP
binding site.
Many inhibitors have been developed for basic
research purposes to explore the functions of
protein kinases. The importance of these en-
zymes in disease processes has also made them
targets of drug screening projects yielding in-
hibitors for many protein kinases. The major-
ity of pharmacological inhibitors of protein
kinases compete with ATP binding. Because of
the huge number of ATP-binding proteins in a
cell, there are inevitable concerns about in-
hibitor specificity not only with respect to the
other protein kinases but also to the other pro-
teins that bind nucleotides. This problem has
been mitigated with variable success through
chemical library screening, structure-based mod-
ification of lead compounds, and inhibitor test-
ing against panels of protein kinases.
Many inhibitors with actions on PKA or
PKCs, for example, have effects on several other
members of the AGC family. Although phar-
macological inhibitors with effects on PKA
abound, the most selective are derived from the
14.27 Phosphoprotein phosphatases reverse the actions of kinases and are independently regulated
naturally occurring small inhibitory protein
known as PKI or the Walsh inhibitor. In vitro
and cell-based screens have identified much
more selective inhibitors for MAP2Ks in the
ERK1/2 pathway. These inhibitors have fewer
known protein kinase cross reactivities, prob-
ably due to the fact that they do not bind in the
ATP site. Among inhibitors that have progressed
in the clinic, compounds developed against the
EGF receptor and certain other protein tyro-
sine kinases have had considerable success.
14.27 Phosphoprotein
phosphatases reverse the
actions of kinases and are
independently regulated
Key concepts
• Phosphoprotein phosphatases reverse the actions
of protein kinases.
• Phosphoprotein phosphatases may
dephosphorylate phosphoserine/threonine,
phosphotyrosine, or all three.
• Phosphoprotein phosphatase specificity is often
achieved through the formation of specific protein
complexes.
Protein phosphorylation is reversed by phospho-
protein phosphatases. These enzymes display dis-
tinct specificities and modes of regulation.
Phosphoprotein phosphatases can be considered
in two broad groups based on their specificity and
sequence relationships: protein-serine/threonine
phosphatases and protein-tyrosine phosphatases.
Most protein-serine/threonine phosphatases
are regulated by association with other proteins.
Targeted localization is the major determinant of
substrate specificity. Phosphoprotein phosphatase
1 (PP1) associates with a variety of regulatory
subunits that specifically direct it to relevant or-
ganelles. One subunit (known as the G subunit),
for example, specifies association with glycogen
particles. The interaction with this subunit is it-
self regulated by phosphorylation. Small protein
inhibitors can suppress PP1 activity.
Phosphoprotein phosphatase 2A (PP2A) is
composed of a catalytic subunit, a scaffolding
subunit, and one of a large number of regulatory
subunits. The regulatory subunit modulates ac-
tivity and localization of the phosphatase. Some
viruses alter the behavior of the cells they infect
by interfering with phosphatase activity. For ex-
ample, cells transformed with the SV40 virus
express a viral protein known as small t anti-
625
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 626
gen. Small t displaces the regulatory subunit
from PP2A and alters the activity and the sub-
cellular localization of the phosphatase. In ad-
dition, natural toxins such as okadaic acid,
calyculin, and microcystin inhibit PP2A and PP1
to varying extents both in vitro and in intact cells.
Another major protein-serine/threonine
phosphatase, called calcineurin (also known as
phosphoprotein phosphatase 2B), is regulated
by Ca2+-calmodulin (see 14.15 Ca2+ signaling
serves diverse purposes in all eukaryotic cells) and
plays essential roles in cardiac development and
T cell activation, among other events. The ma-
jor mechanism of action of the immunosup-
pressants cyclosporin and FK506 is to inhibit
calcineurin.
The protein tyrosine phosphatases (PTPs)
are cysteine-dependent enzymes that utilize a
conserved Cys-Xaa-Arg motif to hydrolyze phos-
phoester bonds in their substrates. The PTPs are
encoded by over 100 genes in humans and are
classified in four subfamilies: the phosphotyro-
sine-specific phosphatases, the Cdc25 phos-
phatases, the dual specificity phosphatases
(DSPs), and the low molecular weight phos-
phatases.
Thirty-eight of the PTPs are highly selec-
tive for phosphotyrosine residues within sub-
strates. Some of the phosphotyrosine-selective
phosphatases are transmembrane proteins,
whereas others are membrane associated. The
most obvious function of the PTPs is to reverse
the functions of tyrosine kinases; however, some
have primary functions in transducing tyrosine
kinase signals. For example, the protein tyro-
sine phosphatase SHP2 (also known as SHPTP2),
binds to certain tyrosine kinase receptors
through its SH2 domain and is itself tyrosine
phosphorylated, thereby creating a binding site
for the SH2 domain-containing adaptor pro-
tein, Grb2, which leads to activation of Ras (see
14.32 MAPKs are central to many signaling path-
ways).
The Cdc25 phosphatases recognize cyclin-
dependent kinase (CDK) family members as
substrates and play a critical role in increasing
CDK activity at key junctures of the cell cycle
(see Figure 14.39 and 11.4 The cell cycle is a cycle
of CDK function). Similar to the dual specificity
kinases, the dual specificity phosphatases are
specific for a restricted number of substrates. A
number of DSPs dephosphorylate MAPKs; these
DSPs are called MAP kinase phosphatases, or
MKPs. Several of these have been implicated
in MAPK nuclear entry and exit. Some MKPs
are encoded by early response genes, whose
626 CHAPTER 14 Principles of cell signaling
products are active near the initiation of the cell
cycle (see 11.7 Entry into cell cycle and S phase is
tightly regulated).
Substrates of other PTP family members,
such as the tumor suppressor PTEN, include
phosphoinositides, which are phosphory-
lated derivatives of the glycerolipid phos-
phatidylinositol that serve as second messengers
(see 14.16 Lipids and lipid-derived compounds are
signaling molecules). Removal of the phosphate
group inactivates the second messenger. It re-
mains unclear whether members of this group
work exclusively on phophoinositides or also
on protein tyrosine phosphate.
14.28 Covalent modification by
ubiquitin and ubiquitin-
like proteins is another
way of regulating protein
function
Key concepts
• Ubiquitin and related small proteins, may be
covalently attached to other proteins as a
targeting signal.
• Ubiquitin is recognized by diverse ubiquitin
binding proteins.
• Ubiquitination can cooperate with other covalent
modifications.
• Ubiquitination regulates signaling in addition to
its role in protein degradation.
An important mechanism for control of protein
function is through covalent modification with
small proteins of the ubiquitin family. Ubiquitin
is one of a family of proteins referred to as ubiq-
uitin-like (Ubl) proteins. Ubiquitin itself is highly
conserved among species, suggesting the func-
tional importance of all of its 76 residues. In ad-
dition to the long-established role of ubiquitin
in initiating protein degradation, ubiquitin mod-
ification also has a variety of functions in signal
transduction.
Ubl proteins are conjugated to the substrate
protein by an isopeptide bond between an amino
group on the substrate, usually from a Lys side
chain, and the C-terminal Gly residue of the
processed Ubl protein. E1, E2, and E3 proteins
are required to catalyze conjugation to Ubl pro-
teins (see Biochem 4.3 Ubiquitin attachment to sub-
strates requires multiple enzymes). Several Ubl
proteins may be attached to one substrate, of-
ten by serial formation of a polyubiquitin chain.
Mono- and polyubiquitination both change the
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 627

protein’s behavior to induce downstream signals. Modification with Ubl proteins plays multiple roles in IL-1 β signaling
Monoubiquitination is a significant regulatory
IL-1 β
modification in vesicular trafficking and DNA re-
pair. For example, the monoubiquitinated form
of the FANCD2 protein becomes associated with
the repair protein BRCA1 at sites of DNA re-
pair. Modification by the Ubl protein SUMO has
roles in nuclear transport, transcription, and
cell cycle progression.
Polyubiquitin chains are formed when the CYTOPLASM
Lys residues of ubiquitin itself, particularly K48
and K63, are ubiquitinated. Addition of polyu-
TRAF6 TRAF6
biquitin with a K48 linkage generally directs pro-
teins to the proteasome for degradation, whereas
conjugation to polyubiquitin chains with a K63
TRAF6 TRAF6
linkage promotes signal transmission, not prote-
olysis. Protein-bound ubiquitin is recognized by
K63 ubiquitination K48 ubiquitination
a variety of ubiquitin binding domains, includ-
ing UIM (ubiquitin-interacting motif), UBA (ubiq-
uitin association), and certain zinc finger domains.
TRAF6 TRAF6 ADP
Such domains have the capacity to act as recep-
tors for ubiquitin within modified proteins. TAB2 TAK1 ATP
Complex formation Degradation
Activation of the transcription factor NF-κB
occurs by a mechanism dependent on modifica-
tion both by the addition of Ubl proteins and phos- ATP

phorylation. This fascinating example of regulation ADP

by ubiquitin is depicted in FIGURE 14.33. Prior to

stimulation, NF-κB is retained in the cytoplasm IKK IKK
in an inactive form by binding to its inhibitor, IκB. Nemo
Phosphorylation of IκB by the IκB kinase (IKK)
complex promotes its recognition by a multisub- NUCLEUS

unit E3 ligase, which directs its ubiquitination

and subsequent proteasomal degradation.
Destruction of IκB allows NF-κB to move to the
nucleus to mediate changes in transcription.
IκB can be stabilized in response to certain
signals through covalent attachment of the Ubl,
SUMO. Sumoylation occurs on the same Lys
residues that must be conjugated to ubiquitin
to achieve IκB degradation. Thus, SUMO at-
tachment stabilizes IκB and attenuates NF-κB ac-
tion. This is one of numerous examples of
crosstalk between Ubl conjugates.
A key regulatory event in NF-κB signaling
is activation of the IKK complex. IKK is itself
regulated by ubiquitination and phosphoryla-
tion. The cytokine interleukin-1β (IL-1) causes
association of adaptor proteins with its recep-
tor to create a receptor activation complex. The
interleukin-1β receptor activation complex re-
cruits another adaptor complex containing
TRAF6. A phosphorylation event releases a
TRAF6 complex from the receptor activation
complex into the cytoplasm.
TRAF6 contains a RING domain, and is an
E3 ubiquitin ligase that catalyzes formation of
DNA
FIGURE 14.33 Activation of NF-B involves steps dependent on the interaction of pro-
teins attached to ubiquitin through ubiquitin-binding proteins, competition by sumoy-
lation, phosphorylation, and ubiquitin-mediated protein degradation.
K63 polyubiquitin chains on the protein kinase
TAK1. Polyubiquitinated TAK1 can then recruit
TAB2 and TAB3, which are adaptor proteins
with conserved zinc finger domains. These par-
ticular zinc finger domains bind to polyubiqui-
tinated TAK1 and enhance its activity. TAK1,
thus activated, phosphorylates and activates
IKK, which then phosphorylates IκB, targeting
it for degradation. Thus, ubiquitin-binding do-
mains, such as the TAB2 and TAB3 zinc fingers,
may selectively recognize K63 polyubiquitin
chains to promote signal transmission.
Naturally occurring small molecules may
control ubiquitin ligase activity directly. Auxin
(indole 3-acetic acid) is a plant hormone that reg-
ulates development by promoting the transcrip-
tion of a large number of genes. Rather than

14.28 Covalent modification by ubiquitin and ubiquitin-like proteins is another way of regulating protein function 627
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 628
stimulating transcription factors, however, auxin
accelerates the degradation of several specific
transcriptional repressors. The auxin receptor is
in fact a ubiquitin ligase complex that targets
the auxin-regulated transcriptional repressors
for proteolysis. F-box proteins account for all
of the auxin binding activity in plant extracts.
14.29 The Wnt pathway
regulates cell fate during
development and other
processes in the adult
Key concepts
• Seven transmembrane-spanning receptors may
control complex differentiation programs.
• Wnts are lipid-modified ligands.
• Wnts signal through multiple distinct receptors.
• Wnts suppress degradation of -catenin, a
multifunctional transcription factor.
Wnt pathways function during embryonic de-
velopment and in the adult in morphogenesis,
body patterning, axis formation, proliferation,
and cell motility. The classical Wnt signaling
mechanism was uncovered largely through
studies of Drosophila and Xenopus development,
as well as by analyzing genetic alterations in
cancer.
Wnt proteins are unusual extracellular lig-
ands. In addition to carbohydrate, they contain
covalently bound palmitate that is essential for
their biological activity. Wnts transduce signals
by binding to multiple distinct receptors. The
most significant are members of the Frizzled
family of seven-transmembrane-spanning recep-
tors.
Wnts regulate the stability of β-catenin,
which either is rapidly degraded or, in response
to Wnt, is stabilized to enter the nucleus and
induce transcription by interacting with TCF
(T-cell factor). Genes induced include c-jun, cy-
clin D1, and many others.
The coordinated activities of the protein ki-
nases glycogen synthase kinase 3 (GSK3) and
casein kinase 1(CK1), the scaffolding proteins
axin and adenomatous polyposis coli (APC), and
the protein disheveled (DSH) are key to β-catenin
stability. In the absence of Wnt, phosphoryla-
tion of β-catenin by CK1 and GSK3 promotes
its ubiquitination and subsequent destruction
by the proteasome. Axin and APC are required
for phosphorylation of β-catenin by GSK3.
In contrast to most seven transmembrane-
628 CHAPTER 14 Principles of cell signaling
spanning receptors, the Frizzled family has not
yet been shown to have significant functions
mediated by a heterotrimeric G protein, and G
proteins may not be central to this pathway.
Instead a proximal step in signaling by Frizzled
involves binding to DSH, which inactivates the
β-catenin destruction mechanism.
Mutations that cause changes in the
amounts of components of the classical pathway
are common in a wide variety of cancers. Both
Wnts and β-catenin may be viewed as proto-
oncogenes. APC is a tumor suppressor and is
mutated in the majority of human colorectal
cancers, for example. Either too little or too
much axin can also disrupt Wnt signaling, and
axin, like APC, is a tumor suppressor.
Wnts utilize additional signaling mecha-
nisms. The receptor proteins Lrp5/6 (which are
related to the low-density lipoprotein receptor)
are Wnt receptors and also bind axin. Wnts bind
to tyrosine kinase receptors to influence axon
guidance and to other proteins that inhibit their
function. Through DSH, Wnts can regulate the
JNK MAPK pathway and Rho family G proteins
to control planar cell polarity. Certain Wnts in-
crease intracellular calcium to activate calcium-
dependent signaling pathways.
14.30 Diverse signaling
mechanisms are regulated
by protein tyrosine kinases
Key concepts
• Many receptor protein tyrosine kinases are
activated by growth factors.
• Mutations in receptor tyrosine kinases can be
oncogenic.
• Ligand binding promotes receptor oligomerization
and autophosphorylation.
• Signaling proteins bind to the phosphotyrosine
residues of the activated receptor.
A large group of protein tyrosine kinases are
receptors that span the plasma membrane and
bind extracellular ligands, as shown in FIGURE
14.34. The receptors are generally activated by
growth factors whose normal physiological func-
tions are to promote growth, proliferation, de-
velopment, or maintenance of differentiated
properties. This group includes receptors for in-
sulin, epidermal growth factor (EGF), and
platelet derived growth factor (PDGF). These
receptors both control the activities of many
other protein kinases of all families and directly
regulate other classes of signaling proteins.
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 629

Because of their physiologic roles as growth Receptor protein tyrosine kinase families
regulators, mutations that activate receptor ty-
rosine kinases are often oncogenic. For exam-
ple, the oncogene erbB results from the
mutational loss of the extracellular ligand-bind-
ing domain of a kinase closely related to the
EGF receptor. This mutation causes constitu-
tive activation of the protein kinase domain.
Point mutations that affect the transmembrane
domain can also cause oncogenic activation, as
is found in the EGF receptor-related neu/HER2
oncogene (see 13.8 Cell growth and proliferation
are activated by growth factors).
KINASE Kinase
Receptor tyrosine kinases are diverse both DOMAINS inserts
in their extracellular ligand-binding domains
EGF Insulin
and, with the exception of a conserved tyrosine receptor receptor
protein kinase domain, their intracellular reg- PDGF FGF
receptor receptor
ulatory regions. These receptors usually have
one membrane span per monomer but some,
such as the insulin receptor, which is a disulfide- FIGURE 14.34 The monomeric tyrosine kinase receptors consist of a
bonded heterotetramer, have two. Ligand bind- globular extracellular domain that binds ligand, a single transmembrane
span, and a globular intracellular region containing the protein kinase
ing to receptor tyrosine kinases favors receptor
domain. The intracellular regions contain additional sequences preced-
oligomerization and enhances kinase activity ing, following, and, in the case of the PDGF and FGF receptor groups,
leading to increased Tyr phosphorylation of the inserted into the protein kinase domain. These regions contain sites of
intracellular domain of the receptor and of as- tyrosine phosphorylation-dependent interactions. The insulin receptor
sociated molecules. These tyrosine-phosphory- is encoded by a single gene. The precursor is proteolyzed into  and 
subunits, which are disulfide bonded to each other. Disulfide bonds also
lated motifs create docking sites for additional
link two  subunits, yielding an obligate heterotetramer.
signal transducers and adaptors.
A comparison of the PDGF and insulin re-
ceptors reveals common themes and a range of
behaviors of receptor tyrosine kinases. The two
PDGF receptors are monomeric receptor tyro- Activation of the PDGF receptor leads to many outputs
sine kinases. The insulin receptor exists in two PDGF
alternatively spliced forms each of which is a
heterotetramer of two  and two  subunits. In
each case, the receptor isoforms utilize some
unique signaling mechanisms.
PDGF
PDGF and insulin each stimulate the kinase receptors
activity of their receptors, causing oligomeriza-
tion and autophosphorylation. Seven or more
sites are phosphorylated on the PDGF receptor,
and each phosphotyrosine residue generates a
binding site for one or more SH2 domain-con-
Src
taining proteins as illustrated in FIGURE 14.35. The
PDGF receptor binds PI 3-kinase, p190 Ras GAP, p190
p85 p110
RasGAP
phospholipase C-, Src (which may catalyze ad- PI 3-kinase
ditional Tyr phosphorylation of the receptor),
Shp2 Grb2
and the SHP2 tyrosine phosphatase which itself CYTOPLASM
binds the adaptor Grb2 (see 14.32 MAPKs are cen- PLC- γ SOS
tral to many signaling pathways). With the excep-
tion of Src, all of these proteins are also receptor
substrates. Thus, substrates are recruited to the
receptor as a consequence of specific interactions FIGURE 14.35 PDGF binds to its receptor and induces receptor au-
tophosphorylation. The autophosphorylated receptor binds target pro-
of substrate SH2 domains with receptor phos- teins that contain SH2 domains.
photyrosine producing changes in activities and
distributions of numerous intracellular signal

14.30 Diverse signaling mechanisms are regulated by protein tyrosine kinases 629
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 630
transducers. This array of signaling events leads
to increased proliferation of connective tissue
during development and in wound healing.
Autophosphorylation also occurs on the in-
sulin receptor to stabilize the active state and to
generate a smaller number of binding sites, as il-
lustrated in FIGURE 14.36. A key event is the Tyr
phosphorylation of insulin receptor substrate
(IRS) proteins, notably IRS1, on as many as a
dozen sites. IRS1 takes over interactions with sev-
eral signaling effectors that, in the case of PDGF,
bind directly to the receptor. Among these tar-
gets is PI 3-kinase which leads to activation of
Akt-2 and several essential metabolic actions of
insulin (see 14.16 Lipids and lipid-derived compounds
are signaling molecules). IRS proteins are also phos-
phorylated by serine/threonine protein kinases to
modulate their signaling capability.
Tyr phosphorylation often enhances the en-
zymatic activity of the associated proteins. Other
proteins gain enhanced function primarily as a
consequence of greater proximity to targets
achieved by binding through their SH2 domains
to phosphotyrosine sites either on the recep-
tors or IRS adaptors. The precise actions of the
many tyrosine kinase receptors are determined
by the overlapping sets of signal transducers
with which they interact, as well as by detailed
differences in amounts of signal transducers,
adaptor accessory proteins and receptor expres-
sion patterns (see Figure 14.43).
Insulin signaling through IRS1
Insulin
Insulin
receptor
PIP 2 PIP 3
p85 p110
PI 3-kinase
IRS1
CYTOPLASM Akt
signaling
FIGURE 14.36 Insulin binding to its receptor causes activation of the
receptor tyrosine protein kinase and autophosphorylation. The receptor
kinase also phosphorylates IRS1, a large adaptor with many potential
phosphorylation sites. IRS1 is an essential intermediate in insulin ac-
tion. PI 3-kinase binds to IRS1 via the SH2 domain within its p85 sub-
unit. Akt and PDK1 bind to PIP3 produced by activated PI 3-kinase so that
PDK1 can phosphorylate and activate Akt (see Figure 14.18).
630 CHAPTER 14 Principles of cell signaling
14.31 Src family protein kinases
cooperate with receptor
protein tyrosine kinases
Key concepts
• Src is activated by release of intrasteric inhibition.
• Activation of Src involves liberation of modular
binding domains for activation-dependent
interactions.
• Src often associates with receptors, including
receptor tyrosine kinases.
The first protein tyrosine kinase to be discovered
was Src, which was identified as the transform-
ing entity in the Rous sarcoma virus. Src is the
prototype of a number of related enzymes, the
Src family kinases. It participates in signaling
pathways regulated by numerous cell surface
receptors, including those that lack their own
kinase domain (see in 14.34 Diverse receptors re-
cruit protein tyrosine kinases to the plasma mem-
brane). Src is bound to the plasma membrane
via an N-terminal myristoyl group. In the in-
active state, Src is phosphorylated on Tyr527, C-
terminal to its catalytic domain, by CSK
(C-terminal Src kinase).
The structure and regulation of Src is de-
picted in FIGURE 14.37. Phosphorylation of Tyr527
causes it to bind to its own SH2 domain. The
SH2 and SH3 domains suppress the kinase ac-
tivity through interactions on the surface of the
protein. The SH3 domain binds to an SH3 bind-
ing site distant from the active site. Activation
of Src by dephosphorylation of Tyr527 causes
its SH2 to dissociate; this causes a conforma-
tional change in the SH3 domain to dissociate
it from the binding site. Viral isolates of Src are
often truncated prior to Tyr527, which increases
their activity.
Conformational changes in the kinase do-
main resulting from dissociation of the SH3 pro-
mote Src autophosphorylation on Tyr416 in its
activation loop and further increase protein ki-
nase activity. An important consequence of the
interaction of Src with its own SH2 and SH3
domains is that these domains cannot bind any-
thing else when in the autoinhibited state; there-
fore, other interactions are promoted when the
SH2 and SH3 domains are released from their
associations with the Src kinase domain. The
heterologous interactions of the SH2 and SH3
domains contribute to Src localization and sig-
naling.
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 631

Structure and regulation of Src

INACTIVE ACTIVE

KINASE
DOMAIN

SH3

SH2 P P

Tyr527

FIGURE 14.37 The structures of inactive and active Src are compared. The in-
active protein is autoinhibited by binding to its own SH2 and SH3 domains.
The SH2 domain binds to phosphorylated Tyr527. The SH3 domain binds to a
noncanonical SH3-binding motif on the opposite side of the kinase domain
active site. In contrast to the steric inhibition of PKA caused by its R subunit,
inhibition of Src by its SH2 and SH3 domains is allosteric. In the active struc-
ture the SH2 and SH3 domains are not bound to the kinase domain and are
available for heterologous interactions. Structures generated from Protein Data
Base files 1FMK and 1Y57.

MAP2Ks are activated by phosphorylation

14.32 MAPKs are central to
many signaling pathways
Key concepts
• MAPKs are activated by Tyr and Thr
phosphorylation.
• The requirement for two phosphorylations creates
a signaling threshold.
• The ERK1/2 MAPK pathway is usually regulated
through Ras.
Mitogen-activated protein kinases (MAPKs) are
present in all eukaryotes. They are among the
most common multifunctional protein kinases
mediating cellular regulatory events in response
to many ligands and other stimuli. MAPKs are
activated by protein kinase cascades consisting
of at least three protein kinases acting sequen-
tially, as illustrated in FIGURE 14.38. Activation
of a MAPK is catalyzed by a MAPK kinase
(MAP2K), which is itself activated by phospho-
rylation by a MAPK kinase kinase (MAP3K).
MAP3Ks are activated by a variety of mecha-
nisms including phosphorylation by MAP4Ks,
oligomerization, and binding to activators such
as small G proteins.
on two Ser/Thr residues; MAP2Ks then acti-
vate MAPKs by dual phosphorylation on Tyr
and Thr residues (Figure 14.30). Each MAP2K
phosphorylates a limited set of MAPKs and few
or no other substrates. The great specificity of
MAP2Ks is one means of insulating MAPKs
from activation by inappropriate signals. Both
Tyr and Thr phosphorylations are required for
maximum MAPK enzymatic activity.
Studies on the MAPK ERK2 led to an un-
derstanding of the events induced by phospho-
rylation that are important for increased activity.
Conformational changes include refolding of
the activation loop to improve substrate posi-
tioning and realignment of catalytic residues;
this is most obvious in the repositioning of 
helix C, which contains a Glu involved in phos-
phoryl transfer.
Amplification occurs moving down the cas-
cade from the MAP3K to the MAP2K step be-
cause the MAP2Ks are much more abundant
than the MAP3Ks. The MAP2K to MAPK step
may also amplify the signal if the MAPK is pres-
ent in excess of the MAP2K. In addition, the
phosphorylation of a MAPK by a MAP2K on a

14.32 MAPKs are central to many signaling pathways 631

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 632

MAPK pathways tional functions. Several scaffolds have been

S. identified that bind to two or more components
G e ne ric cerevisiae Mammals
for each of the three major MAPK cascades, the
G Protein ERK1/2, JNK1-3, and p38 α, β, γ, and δ cascades.
small or Gβγ R as Rac/Cdc42 Rac ? The ERK1/2 pathway is regulated by most
heterotrimer
cell surface receptors, including receptors that
employ tyrosine kinases, GPCRs, and others. The
MAP4K Ste20p PAK/PKC
Ste20 Ste20
?
PDGF receptor, like most receptor systems, acti-
family family
vates the ERK1/2 cascade through Ras. PDGF
stimulates autophosphorylation of its receptor
MAP3K Ste11p R af many many
MEKK2 and the subsequent association of effectors with
MEKK3 its cytoplasmic domain (see 14.30 Diverse signal-
ing mechanisms are regulated by protein tyrosine ki-
MEK1 MEK4 MEK3 nases). In response to PDGF, ERK1/2 promotes cell
MAP2K Ste7p MEK5
MEK2 MEK7 MEK6 proliferation and differentiation by phosphory-
lation of membrane enzymes, proteins involved
in determining cell shape and motility, and also
ERK1 JNK1 p38α by concentrating in the nucleus to phosphory-
MAPK Fus3p ERK5
ERK2 JNK2 p38β
late regulatory factors that control transcription.
JNK3 p38γ
p38δ

Major
targets:
Trans-
cription
c-Jun
Ste12p Elk-1 MEF2 MEF2
factor ATF2
Protein
Rsk MAPKAPK2 Rsk
kinase
Output Mating Proliferation
Development
Differentiation
(and other processes)
FIGURE 14.38 MAPK pathways can be regulated by a diverse group of upstream
regulatory mechanisms that often include adaptors, small G proteins, and MAP4Ks.
These molecules impinge on the activities of MAP3Ks. MAP3Ks regulate one or
more MAP2Ks depending on localization and scaffolding. The MAP2Ks display
great selectivity for a single MAPK type. MAPKs have overlapping and unique
substrates and participate in signaling cascades leading to many cellular responses.
Tyr and a Thr residue creates cooperative acti-
vation of the MAPK; this is another mechanism,
in addition to those described for PKA and
calmodulin, to introduce a threshold and ap-
parently cooperative behavior into the path-
way over a narrow range of input signal. This
multistep cascade provides multiple sites for
modulatory inputs from other pathways.
Stabilized interactions between components
are also important. MAP2Ks, as well as MAPK
substrates and MAPK phosphatases, generally
contain a basic/hydrophobic docking motif that
interacts with acidic residues and binds in a hy-
drophobic groove on the MAPK catalytic do-
main. Additional components including scaffolds
are necessary for the efficient activation of
MAPK cascades in cells and usually have addi-
14.33 Cyclin-dependent protein
kinases control the cell
cycle
Key concepts
• The cell cycle is regulated by cyclin-dependent
protein kinases (CDKs).
• Activation of CDKs involves protein binding,
dephosphorylation, and phosphorylation.
Cell division is regulated positively and nega-
tively by factors that stimulate proliferation and
inputs that monitor cell state. The sum of these
factors is integrated in the regulation of cyclin-
dependent protein kinases (CDKs). CDKs are
protein serine/threonine kinases that are ma-
jor regulators of cell cycle progression. Most
CDKs are regulated both by kinases and phos-
phatases and by association with other proteins
called cyclins. Cyclins are synthesized and de-
graded every cell cycle. Because most CDKs are
dependent upon cyclin binding for activation,
the timing of the synthesis and degradation of
individual cyclins determines when a CDK will
function. The most notable noncycling member
of the CDK family is Cdk5, which is highly ex-
pressed in terminally differentiated neurons.
Cdk5 binds the non-cyclin protein p35 as its ac-
tivating subunit.
We will briefly examine the regulation of
Cdc2, a major CDK in both mammals and yeast.
The first step in regulation of Cdc2 is the asso-
ciation with cyclin. A second step required for
activation of Cdc2 is phosphorylation of a Thr

632 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 633

CDKs require cyclin binding for activation

Tyr15
Tyr15

Lys33
Lys33

Glu51
Glu51

Cdk2
Cdk2

Cyclin
Cyclin A
A

FIGURE 14.39 The view of the crystal structure of CDK2 bound to cyclin A
shows residues in the ATP binding site. The enlargement on the right shows
the interaction between Lys33 and Glu51, catalytic residues that interact with
ATP to promote phosphoryl transfer. Tyr15 is phosphorylated in inactive forms
of CDK2. A phosphoryl group on Tyr15 inhibits CDK activity by interfering with
ATP binding. Structure generated from Protein Data Bank file 1JST.

residue in its activation loop by another CDK
type kinase. In spite of its association with cy-
clin, this form of Cdc2 is not yet active due to
inhibitory phosphorylation of Tyr and Thr
residues in the ATP binding pocket. Release of
inhibition by dephosphorylation of the residues
in the ATP pocket is catalyzed by the Cdc25 fam-
ily of phosphoprotein phosphatases, resulting in
activation of Cdc2. The proximity of the Tyr
residue to catalytic residues is shown in FIGURE
14.39. The complexity of activation of CDKs
makes possible the imposition of cell cycle check-
points. For more on CDKs and cyclins see 11.4
The cell cycle is a cycle of CDK function.
14.34 Diverse receptors recruit
protein tyrosine kinases
to the plasma membrane
Key concepts
• Receptors that bind protein tyrosine kinases use
combinations of effectors similar to those used by
receptor tyrosine kinases.
• These receptors often bind directly to transcription
factors.
Many receptors act through protein tyrosine
kinases, but their cell surface receptors lack ki-
nase activity. Instead, these receptors act by re-
cruiting and activating protein tyrosine kinases
at the plasma membrane. In this group of recep-
tors are integrins, which are key molecules in-
volved in cell adhesion, growth hormone
receptors, and receptors that mediate inflam-
matory and immune responses. While their
structures vary enormously, their mechanisms
of action are related.
Integrins are receptors whose major function
is to attach cells to the extracellular matrix. They
also mediate some interactions with proteins on
other cells, as depicted in FIGURE 14.40. Ligands for
integrins include a number of extracellular ma-
trix proteins, such as fibronectin, as well as cell
surface proteins that cooperate in cell-cell inter-
actions. Integrin ligation provides cells with infor-
mation about their environment that influences
cell behavior. Ligation of integrins initiates signals
that control cell programs, including cell cycle en-
try, proliferation, survival, differentiation, changes
in cell shape, and motility, as well as fine-tuning
responses to other ligands. For more details on in-
tegrins see 15.13 Most integrins are receptors for extra-
cellular matrix proteins and 15.14 Integrin receptors
participate in cell signaling.

14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane 633
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 634
Integrin signaling
ECM
INTEGRINS
Paxillin
Src Talin ILK
Talin
p85 PI 3-
Vinculin Kinase
p110 Vinculin
FAK
Crk CAS
Crk Grb2 Tensin
SOS
Actin
filament
CYTOPLASM
FIGURE 14.40 Integrins bind to an array of cytoplasmic proteins to regulate
the cytoskeleton and intracellular signaling pathways. The associated cytoskele-
tal elements include actin filaments and focal adhesion proteins -actinin, vin-
culin, paxillin, and talin. Signaling molecules include the focal adhesion kinase
FAK; the adaptors Cas, Crk and Grb2; Src and CSK (see 14.31 Src family protein
kinases cooperate with receptor protein tyrosine kinases), PI 3-kinase (see 14.16
Lipids and lipid-derived compounds are signaling molecules); and the Ras ex-
change factor SOS. Stimulation of GTP binding of Ras by SOS leads to activa-
tion of the MAPK pathway (see 14.32 MAPKs are central to many signaling
pathways).
Talin and α-actinin are among cytoskeletal
proteins that interact directly with certain inte-
grin subunits. These cytoskeletal proteins link
integrins to complex cytoskeletal structures
known as focal adhesions.
Focal adhesions connect the cytoskeleton to
signal transduction cascades that communicate
states of cellular attachment to the regulation of
cellular responses. Focal adhesion complexes con-
tain the focal adhesion kinase FAK, which is ac-
tivated by integrin ligation. Autophosphorylation
of FAK recruits signaling proteins containing SH2
domains, especially the p85 subunit of PI 3-ki-
nase and Src family protein kinases. The signal-
ing molecules associated with the integrin-bound
cytoskeletal proteins, whether focal adhesions or
other structural complexes, mediate the diverse
actions of integrins. The association of cytoskele-
tal proteins with integrin receptors also causes
functional changes to the receptors.
Signals that act over a distance, such as hor-
mones, can also employ nonreceptor tyrosine
kinases to transmit their message inside a cell.
Growth hormone (GH) is a protein hormone
secreted by the anterior pituitary gland that reg-
ulates bone growth, fat metabolism, and other
634 CHAPTER 14 Principles of cell signaling
cellular growth phenomena. Absence of growth
hormone results in short stature, whereas hy-
persecretion causes acromegaly, a form of gi-
gantism. The GH receptor is a member of the
cytokine receptor family, which includes recep-
tors for prolactin, erythropoietin, leptin, and
interleukins. All these receptors display similar
biochemical functions, such as association with
members of the JAK/TYK family of protein ty-
rosine kinases, but select for different but over-
lapping sets of cytoplasmic signaling proteins.
Signal transduction by the GH receptor provides
a model for receptors that lack enzymatic func-
tion and act as agonist-promoted scaffolds for
intracellular signaling proteins.
FIGURE 14.41 shows the structure of growth
hormone bound to the extracellular domain of
its receptor. The majority of binding energy
comes from only a small number of residues in
the binding interface. Inside the cell, signaling
by the GH receptor depends significantly on its
association with the cytoplasmic tyrosine pro-
tein kinase Janus kinase 2 (JAK2). FIGURE 14.42
shows that JAK2 binds to a proline-rich region
of the receptor. Ligand binding induces recep-
tor dimerization, which then promotes
activation of JAK2 through intermolecular auto-
phosphorylation.
GH signaling is thus mediated primarily by
inducing Tyr phosphorylation. In addition to
JAK2 autophosphorylation, the receptor itself
becomes Tyr phosphorylated. As is true for recep-
tor tyrosine kinases, Tyr phosphorylation of the
growth hormone receptor creates binding sites
for signaling proteins that contain phosphotyro-
sine-binding domains. Primary targets are tran-
scription factors known as signal transducers and
activators of transcription, or STATs. STATs con-
tain SH2 domains and bind Tyr-phosphorylated
motifs on the growth hormone receptor. While
receptor bound, STATs are Tyr phosphorylated
by JAK2 and then released to travel to the nu-
cleus to mediate changes in transcription.
The growth hormone receptor and the as-
sociated JAK2 also activate other signaling path-
ways. For example, the adaptor Shc is Tyr
phosphorylated by JAK2. Engagement of Shc
leads to activation of Ras and the ERK1/2 MAPK
pathway. Adaptors specialized for insulin-signal-
ing pathways, insulin receptor substrates (IRS)
1, 2, and 3, are also growth hormone targets, per-
haps reflecting the ability of growth hormone
to induce certain insulin-like metabolic actions.
Feedback circuits are also engaged during
GH signaling. The growth hormone receptor
complex binds the adaptor SH2-B, which has a
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 635

Growth hormone structure Growth hormone signaling is transduced by JAK2

Growth
Growth hormone
hormone
receptor
hGH

Dimerization

JAK2 JAK2

JAK2s bind and STAT STAT

phosphorylate
receptor
CYTOPLASM
ΔΔ G (kcal/mol) STATs bind and
> 1.5 are phosphorylated
0.5 to 1.5
-0.5 to 0.5
< -0.5
untested

Phosphorylated
NUCLEUS
STATs bind DNA
FIGURE 14.41 Proteins often interact over a large surface area. Growth
hormone binding to its receptor is an example of the energy of bind-
ing coming primarily from a small number of the contacts between
the two proteins, creating an interaction hot spot. The complex of
growth hormone bound to the growth hormone receptor-binding do-
main determined by crystallography has been peeled apart in this fig-
FIGURE 14.42 The growth hormone receptor binds to JAK2. Many GH signals
ure to show the binding energy associated with residues in the binding
are mediated by Tyr phosphorylation of the receptor by JAK2, which creates
interface from each protein determined by mutagenesis and binding
binding sites for signaling molecules with SH2 domains, notably STATs. STATs
studies. Fewer than half of the residues in the interface contribute
then enter the nucleus to cause changes in gene transcription.
the majority of binding energy. Reproduced with permission from T.
Clackson and J. A. Wells. 1995. Science. 267: 383–386. © AAAS. Photos
courtesy of Tim Clackson, ARIAD Pharmaceuticals, Inc.

stimulatory effect on growth hormone signal-
ing. On the other hand, suppressors of cytokine
signaling (SOCS proteins) are among the genes
whose transcription is induced by growth hor-
mone. As the name indicates, SOCS proteins in-
hibit cytokine signaling in some if not all cases by
inhibiting the activity of JAK2. SOCS proteins
contain an SH2 domain that facilitates their bind-
ing either to phosphorylated JAK2 or cytokine
receptors. The mechanism of signaling inhibi-
tion may differ among SOCS proteins because
some require the GH receptor to interfere with
JAK2 signaling. SOCS-1, on the other hand, binds
directly to the JAK2 activation loop and does not
require a receptor to inhibit JAK2 activity. This
mechanism may be particularly important in GH
signaling because, in contrast to the ligand-in-
duced down regulation mechanisms controlling
many receptors, the GH receptor is degraded in
a ligand-independent manner.
Receptors for cytokines also act by recruit-
ing tyrosine kinases. The cytokines—signaling
proteins that modulate inflammation and cell
growth and differentiation—include interleukins,
leukemia inhibitory factor, oncostatin M, car-
diotrophin-1, cardiotrophin-like cytokine, and
ciliary neurotrophic factor (CNTF). Each cy-
tokine binds a unique receptor, but each recep-
tor binds a transmembrane protein called gp130.
Mechanisms of signaling by gp130 involve in-
teractions with tyrosine kinases of the JAK/TYK
types and transcription factors in the STAT fam-
ily. This mechanism is similar to those employed
by the growth hormone receptor.

14.34 Diverse receptors recruit protein tyrosine kinases to the plasma membrane 635
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 636

Receptor signaling pathways Unlike many cytokine receptors in this class,

Growth
the CNTF receptor does not itself span the mem-
Ligand PDGF Insulin hormone IL-1β TGF-β brane. Instead, it is glycosyl phosphoinosi-
tol (GPI)-linked to the outer face of the plasma
TGF -β membrane. The GPI linkage is a covalent bond,
PDGF Insulin GH IL-1β
Receptor type II
receptor receptor receptor receptor
receptor
and the receptor can be released into the ex-
tracellular fluid by a specific phospholipase. The
Adaptor/
freed receptor may interact with membranes of
subunit SHP2/Grb2 IRS1 gp130 other cells to induce signals.
The use of a common signal transducing
Type I subunit, gp130, suggests that unique mecha-
Transducer SOS/Ras PI 3-kinase JAK JAK
receptor nisms exist to create ligand-specific responses;
under some circumstances competition by the
Kinase
MAPK Akt2
ligand binding subunits for interaction with the
cascade gp130 signal transducer may influence signal-
ing outcomes. FIGURE 14.43 illustrates some par-
Transcription Ternary FOXO STATs STATs SMADs allels in signaling pathways initiated by receptors
factor complex
complex factors with associated or intrinsic protein kinases.
The last receptor type we will discuss takes
FIGURE 14.43 Major signaling cascades controlled by PDGF, insulin, TGF-, IL- the concept of the specific and common sub-
1, and growth hormone are compared. Each receptor either contains or inter- units even further. The complex multiprotein
acts with a protein kinase that associates with or recruits a transducer. The
transducer regulates downstream effectors either directly or through an interme-
T cell receptor (TCR) is found uniquely on T
diate protein kinase cascade. The effectors shown are transcriptional regulators. lymphocytes and is responsible for the ability of
Phosphorylation by the transducer or kinase cascade activates all of the effectors these cells to recognize and respond to specific
except the FOXO proteins, which may be excluded from the nucleus by phospho- antigens. The TCR, illustrated in FIGURE 14.44, is
rylation. The table only shows snapshots of much more complex signaling net- composed of eight subunits that can be described
works controlled by these ligands. Many of these and other intermediates serve
multiple ligands. For example, IRS proteins also contribute to growth hormone
as an assembly of four dimers, αβ, γε, δε, and ζζ.
and IL-1 signaling, and MAPK pathways are regulated by all of these ligands. The specificity of antigen recognition is deter-
mined by the α and β subunits, which are dif-
ferent for each cell. The remaining subunits are
T cell receptor signaling
invariant in TCRs.
The CD3 complex γ, δ, and ε subunits are
similar in sequence to one another. The ζ chain,
unlike the other subunits, appears on certain
MHC other cell types and may be a component of
other receptors, such as the Fc receptor, which
Antigen binds a portion of certain immunoglobulins.
A motif called the immunoreceptor tyro-
TCR sine-based activation motif, or ITAM, which fea-
tures closely spaced pairs of Tyr residues, is key
to signaling by the TCR. The CD3 subunits each
CD3 CD3
contain one ITAM and the ζ chain contains three
Lck
ITAMs, for a total of ten motifs in each TCR.
Engagement of the TCR causes the Src family
ZAP-70 kinases Lck and Fyn to phosphorylate the pairs
of Tyr residues in the ITAMs. The ITAMs then
bind the tandem SH2 domains of the protein ty-
ITAM
After binding of the TCR rosine kinase ζ-chain-associated protein of 70
to the MHC-antigen kDa (ZAP-70), which becomes activated by Src.
complex, Lck
phosphorylates ITAMS Tyr phosphorylation sites on ZAP-70 bind to
other adaptors and signaling molecules, and Tyr
phosphorylation by ZAP-70 activates additional
FIGURE 14.44 The T cell receptor (TCR) is a multisubunit receptor. It is phos- signal transducers. The sum of these events leads
phorylated on activation motifs or ITAMs by Lck, or a related Src family protein to the downstream responses of T cells to anti-
kinase. The phosphorylated residues create binding sites for another tyrosine
protein kinase ZAP-70. ZAP-70 then recruits other signaling molecules to the gen engagement, which include cell cycle pro-
complex including phospholipase C, PI 3-kinase, and a Ras exchange factor to gression and the elaboration of cytokines such
activate downstream signaling pathways. as interleukin-2.

636 CHAPTER 14 Principles of cell signaling

39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 637
14.35 What’s next?
New signaling proteins and new regulatory in-
teractions seem to show up every day. The chal-
lenge now is to understand how cells organize
these proteins and their individual interactions
to create adaptable information-processing net-
works. How do cells use simple chemical reac-
tions to sort and integrate multiple simultaneous
inputs and then direct this information to di-
verse effector machinery? How do they inter-
pret the inputs in the context of their growth and
metabolic activities? In principle, three areas of
research have to contribute to allow us to un-
derstand integrative cellular signaling.
First, we need real-time, noninterfering
biosensors to measure intracellular signaling
reactions. Most current sensors use combina-
tions of fluorescent moieties and signal-bind-
ing protein domains to provide fast optical
readouts. For many pathways, several reactions
can be monitored within cells over subsecond
time scales. We need more, better, and faster
sensors and sensors that can report with single-
cell and subcellular resolution. Genetically en-
coded sensors will be complemented by synthetic
molecules.
Our ability to manipulate signaling net-
works is also improving dramatically but still
falls short. We can manipulate signaling net-
works by overexpression, knockout, and knock-
down of genes, but signaling pathways are
wonderfully adaptive and frequently circum-
vent our best efforts to control them. We still
need chemical regulators that can act promptly
in cells. Structure-based design of such regula-
tory molecules will be vital.
Last, our ability to analyze the behavior of
signaling networks depends on our ability to
measure and interpret signaling quantitatively.
It is ironic but true that really complex systems
cannot be described without explicit quantita-
tive models for how they work. Computational
modeling and simulation of signaling networks
requires both better theoretical understanding
of network dynamics and better algorithmic im-
plementation.
The goal is to understand how cells think.
14.36 Summary
Signal transduction encompasses mechanisms
used by all cells to sense and react to stimuli in
their environment. Cells express receptors that
recognize specific extracellular stimuli, includ-
ing nutrients, hormones, neurotransmitters,
and other cells. Upon receptor binding, signals
are converted to well-defined intracellular chem-
ical or physical reactions that change the activ-
ities and the organization of protein complexes
within cells. The changes directed by the stim-
uli lead to altered cell behavior. The behavior of
the cell is determined then by its intracellular
state and the integrated information from ex-
tracellular stimuli so that the appropriate re-
sponses are achieved.
The basic biochemical components and
processes of signal transduction are conserved
throughout biology. Families of proteins are
used in a variety of ways for many different
physiological purposes. Cells often use the same
series of signaling proteins to regulate multiple
processes, such as transcription, ion transport,
locomotion, and metabolism.
Signaling pathways are assembled into sig-
naling networks to allow the cell to coordinate
its responses to multiple inputs with its ongo-
ing functions. It is now possible to discern con-
served reaction sequences in and between
pathways in signaling networks that are anal-
ogous to devices within the circuits of analog
computers: amplifiers, logic gates, feedback and
feed-forward controls, and memory.
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