Signal transduction
⇌
Whenever I've had to resort to adrenaline to the heart it has never worked, Hema said to
herself. Not once. Maybe I do it as a way to signal to myself that the patient is dead. But
surely it must have worked, somewhere, with someone. Why else was it taught to us?
Cutting for stone. Abraham verghese
1
The Structure and Function of Signal Pathways
• The enormous structural variety and functional capacity of
multicellular organisms is due to their ability to coordinate the
biochemical reactions of the various cells of the total organism
• The basis for this coordination is the intercellular
communication, which allows a single cell to influence the
behavior of other cells in a specific manner
• Cells can communicate in different ways:
1. Chemical Messengers: Cells send out signals in the form of
specific chemical messengers that the target cell converts
into a biochemical reaction
2. Gap Junctions: Communication between bordering cells is
possible via direct contact in the form of “gap junctions”
3. Cell-cell interaction via cell surface proteins: A cell surface
protein of one cell binds a specific complementary protein
(or carbohydrate chain) on another cell
2
• A further intercellular
communication mechanism relies
on electrical processes. The
conduction of electrical impulses
by nerve cells is based on changes
in the membrane potential
• Signal transduction is the process
by which cells of a particular type
receive and transform a
biochemical signal into a
physiological reaction
• Signaling pathways are involved in
the coordination of metabolite
flux, the regulation of cell division,
differentiation and development of
an organism, processing of sensory
3
information
• In the communication between cells of an organism, the signals
(chemical messengers or electrical signals) are produced in
specialized cells
• The signal-producing function of these cells is itself regulated,
so that the signal is only produced upon a particular stimulus
• The following steps are involved in intercellular communication:
 Formation of a signal in the signal-producing cell as a result
of an external trigger
 Transport of the signal to the target cell
 Registration of the signal in the target cell
 Further transmission of the signal into the target cell
 Transformation of the signal into a biochemical or electrical
reaction in the target cell
 Termination of the signal
• Specialized proteins, termed receptors, are utilized for the
reception of signals
4
• There are two principal ways
by which target cells can
process incoming signals:
a) Cell surface receptors
receive the signal at the
outside of the cell, become
activated and initiate a
signaling chain in the
interior of the cell.
• In such signaling pathways
the membrane-bound
receptor transduces the
signal at the cell membrane
so that it is not necessary for
the signal to actually enter
the cell
5
b) The messenger enters into the target cell and binds and
activates the receptor localized in the cytosol or nucleus
• Upon receiving a signal, a receptor becomes activated to
transmit the signal further
• The activated receptor passes the signal onto components,
usually proteins, further downstream in the signaling pathway,
which then become activated themselves for further signal
transmission
• A chain of serially operating, intracellular signal transduction
processes results
• Finally, a specific biochemical process is triggered in the cell,
which represents the endpoint of the signaling pathway
• Cells possess multiple mechanisms to regulate the intercellular
communication as well as the intracellular signal transduction
• This allows a specific termination of communication between
cells. Often feedback mechanisms are used to adapt the cellular
6
response to the needs of the organism
• The key components of intracellular signal transduction
include receptors, protein kinases and protein phosphatases,
regulatory GTPases, adaptor proteins and second messengers
• The regulatory GTPases function as switches that can exist in
an active or inactive form
• Adaptor proteins mediate the signal transmission between
proteins of a signaling chain by bringing these proteins
together
• They function as clamps to co-localize proteins for an effective
and specific signaling
• Furthermore, adaptor proteins help to target signaling
proteins to specific sub-cellular sites
• Second messengers are chemical signaling substances
produced or released due to the intracellular activation of
enzymes in a signaling chain
• Extracellular signaling molecules are released either by
7
exocytosis or passive diffusion into the extracellular space
• In special cases, membrane-bound proteins are also used as
signaling molecules
• Signaling molecules for the communication between cells are
known as hormones
• Hormones that are proteins and regulate cell proliferation are
known as growth factors
• The chemical nature of hormones is extremely variable
• Hormones can be proteins, peptides, amino acids and amino
acid derivatives, derivatives of fatty acids, nucleotides, steroids,
retinoids, small inorganic molecules, such as NO
• The modification of hormones can lead to compounds that are
known as agonists or antagonists
• Antagonists are hormone derivatives that bind to a receptor but
do not initiate signal transduction
• Antagonists block the receptor and thus terminate signal
transduction. They have broad medical application since they
8
specifically interfere with certain signal transduction pathways
• Various forms of intercellular communication can be
discerned based on the range of the signal transmission
• In endocrine signaling, the hormone is synthesized in specific
signaling, or endocrine cells and exported via exocytosis into
the extracellular medium (e.g., blood or lymphatic fluid)
• The hormone is then distributed throughout the entire body
via the circulatory system so that remote regions of an
organism can be reached
• Paracrine signal transduction occurs over medium range.
• The producing cell must be found in the vicinity of the
receiving cells for this type of communication
• The signaling is local, and the participating signaling
molecules are termed tissue hormones or local mediators
• A special case of paracrine signal transduction is synaptic
neurotransmission in which a nerve cell communicates with
either another nerve cell or with a muscle cell
9
• In autocrine signaling, cells of the same type communicate with
one another
• If an autocrine hormone is secreted simultaneously by many
cells then a strong response occurs in the cells
• Autocrine mechanisms are of particular importance in the
immune response
• A special case of signal transduction is represented by a class of
small, reactive signaling molecules, such as NO
• NO is synthesized in a cell in response to an external signal and
is delivered to the extracellular fluid
• Either by diffusion or in a protein-bound form, the NO reaches
neighboring cells, and modification of target enzymes ensues,
resulting in a change in the activity of these enzymes
• NO is characterized as a mediator that lacks a receptor in the
classical sense
o Signal pathways commonly amplify the initial signal received by
10
the receptor during the course of the signal transduction
SIGNALING BY NUCLEAR
RECEPTORS
• Nuclear receptors regulate
gene expression in response to
binding lipophilic molecules
and are thereby involved in
the control of a diversity of
cellular processes
• These proteins are ligandactivated transcription factors
that are localized in the
cytoplasm and/or in the
nucleus
• The ligands pass the cell
membrane by simple diffusion
and bind to the receptors 11
• By binding to DNA elements in the control regions of target
genes the ligand-bound receptor influences the transcription of
these genes and thus transmits hormonal signals into a change
of gene expression
• The naturally occurring ligands of nuclear receptors are
lipophilic hormones, among which the steroid hormones, the
thyroid hormone T3, and derivatives of vitamin A and D have
long been known as central regulators
• In recent years it has been recognized that intracellularly formed
lipophilic metabolites can also serve as ligands for nuclear
receptors and can regulate gene expression through their
binding to nuclear receptors
• These compounds include prostaglandins and leukotrienes
• They also include other molecules synthesized intracellularly
as normal metabolites such as fatty acids and bile acids and
substances derived from foreign lipophilic substances like drugs
12
13
• The receptors such as PPAR are quite promiscuous with
respect to the nature of the ligand and can bind a broad
range of lipophilic ligands
• This type of receptors is thought to be involved in the
regulation of metabolism and in detoxification
• In comparison to signaling pathways which utilize
transmembrane receptors , signaling via nuclear receptors is
of relatively simple structure
• The pathways lead directly, with only a few participating
protein components, from the extracellular space to the
level of transcription in the nucleus
• Nuclear receptors have got separate ligand-binding and
DNA-binding domains
• That part of the DNA bound specifically by nuclear
receptors is known as hormone response element (HRE)14
• Based on the receptor activation mechanism, the nuclear
receptors may be divided into two basic groups
• In the first group (those including most of the steroid hormone
receptors), the receptors can be localized in the nucleus or in
the cytoplasm
• The receptors of the other group are always localized in the
nucleus. Representative ligands of these receptors are the
derivatives of retinoic acid, the T3 hormone and Vit D
• The transport of steroid hormones occurs in the form of a
complex with a specific binding protein
• An example of such a binding protein is transcortin, which is
responsible for the transport of the corticosteroids
• The steroid hormones enter the cell by diffusion and activate
the cytosolic receptors. In the absence of steroid hormones, the
receptors remain in an inactive complex- aporeceptor complex
• In the aporeceptor complex the receptor is bound to chaperones
15
Signal Transduction by Steroid Hormone
Receptors
• The binding of the hormone to the aporeceptor complex leads
to activation of the receptor and initiates the translocation of
the receptor into the nucleus where it binds its HRE
16
• In contrast to signal transduction by the steroid hormone
receptors, there are multiple pathways by which the ligands of
retinoic acid group are made available for receptor activation
• They can follow the classical endocrinological pathway (like vit
D), be synthesized inside the cell from inactive precursors (like
retinoic acid) or their full synthesis could take place inside a
cell (like prostaglandin J2)
• In addition, the receptors in this group are found bound with
the corresponding HRE in the absence of hormone, acting as
repressors of gene activity
• In the presence of the hormone an activation of gene
expression is usually observed
• In general, HREs are composed primarily of two copies of a
hexamer DNA sequence
• The hexamers can be inverted (palindromic), everted or direct
repeats
17
Inverted
Everted
Direct
Signal Transduction by RXR
Heterodimers
18
• The receptors bind to the cognate HRE mainly as dimers,
allowing the formation of homodimers as well as
heterodimers between various receptor monomers
• Very few nuclear receptors are known whose HRE contains
only a single copy of the recognition sequence. These
receptors bind as monomers to the HRE
• The HREs of the steroid hormone receptors possess a
palindromic structure; homodimers of receptors are
formed
• The HRE of the nuclear receptors for all-trans retinoic acid,
9-cis retinoic acid, the T3 hormone and vitamin D usually
exhibit a direct repeat of the recognition sequence,
resulting in the formation of heterodimers on the DNA
• One of the partners in the heterodimer is always the
receptor for 9-cis retinoic acid, RXR
19
SIGNAL TRANSMISSION THROUGH TRANSMEMBRANE
RECEPTORS
• Another way of transducing signals into the interior of the cell
is through transmembrane receptors
• Transmembrane receptors are integral membrane proteins, i.e.,
they possess a structural portion that spans the membrane
• An extracellular domain, a transmembrane domain and an
intracellular or cytosolic domain can be differentiated within the
20
structure
• In many receptors, the extracellular domain contains the
ligand-binding site
• The function of the transmembrane domain is to pass the signal
on to the cytosolic domain of the receptor
• Two basic mechanisms are used for conduction of the signal to
the cytosol:
1. Specific protein-protein interactions –the next protein
component in the signal transmission pathway, the effector
protein, is activated
• The conformational change that accompanies the perception
of the signal by the receptor creates a new interaction surface
for proteins that are located downstream of the receptor
2. Activation of Enzymes –the arrival of the signal triggers
enzyme activity in the cytosolic domain of the receptor,
which, in turn, pulls other reactions along with it
• The enzyme usually has Tyrosine kinase activity
21
• However, there are other examples where tyrosine
phosphatase or Ser/Thr-specific protein kinase activity is
activated
• The enzyme activity may be an integral part of the receptor,
or it may also be a separate enzyme associated with the
receptor on the inner side of the membrane
G-Protein Coupled Receptors (GPCR)
• Of the transmembrane receptors, the G protein-coupled
receptors form the largest single family
• GPCR can be activated by extracellular ligands or sensory
signals
• Extracellular ligands include biogenic amines, such as
adrenaline and noradrenaline, histamine, serotonin, lipid
derivatives, nucleotides, retinal derivatives, peptides such as
bradykinin and large glycoproteins such as luteinizing
hormone, and parathormone
22
• Physical stimuli such as light signals are registered and
converted into intracellular signals by GPCR; they are also
involved in perception of taste and smell
• A characteristic structural feature of the GPCR is the presence
of 7-transmembrane helices
• For the vast majority of 7-helix transmembrane receptors the
next downstream located signaling protein is a heterotrimeric
GTP/GDP binding protein (G-protein)
23
• When a ligand binds to a GPCR, the structure of the
transmembrane part is altered and this change is passed on to
the cytoplasmic loops of the receptor
• As a consequence, a high-affinity surface is created for binding
of the G protein
• The G protein, which exists as the inactive GDP form, now binds
to the activated receptor and is itself activated
• An exchange of GDP for GTP takes place, and the βγ-subunit of
the G protein dissociates
• Once the G protein is activated, it frees itself from the complex
with the receptor, which either returns to its inactive ground
state or activates further G proteins
• A phenomenon often seen in transmembrane receptors in
general, and in G protein-coupled receptors in particular, is
desensitization
• Desensitization means a weakening of the signal transmission
24
under conditions of long-lasting stimulation
• Despite the persistent effect of extracellular stimuli, the
signal is no longer passed into the cell interior, or only in a
weakened form, during desensitizing conditions
• A common way of desensitization is the phosphorylation
of the receptor at the cytoplasmic side by specific protein
kinases
• Phosphorylation of the receptor can be carried out by
protein kinase A or protein kinase C
• This is a feedback mechanism since PKA and PKC are
activated by GPCR
• Another way of phosphorylation is through G protein
coupled receptor protein kinases (GRK)
• The phosphate residues introduced by GRK serve as
attachment sites for arrestin which serves as a trigger for
internalization of the receptor to endosomes
25
26
• The superfamily of GTPases includes the heterotrimeric G
proteins, the Ras family of small GTPases and the family of
initiation and elongation factors
• The defining feature of this group is that its members have got
a “switch function”
• The binding of GTP brings about the transition into the active
form (turned on)
• Hydrolysis of the bound GTP by the intrinsic GTPase activity
converts the protein into the inactive, GDP-bound form (turned
off)
• Thus, the GTPase activity is one way of terminating signaling
• In the case of heterotrimeric G-proteins, the α-subunit has a
binding site for GTP or GDP and carries the GTPase activity
• Based on comparison of the amino acid sequences, the
heterotrimeric G-proteins are divided into four families
• The members of the Gs subfamily are activated by hormone
27
receptors, by odor receptors and by taste receptors
• Examples include signal transmission by type β-adrenaline
receptors and by glucagon receptors
• During perception of smell, the smell receptors are activated,
and these then pass the signal on via the olfactory G protein Golf
• Perception of sweet taste is also mediated via a Gs protein
• Transmission of the signal further involves an adenylyl cyclase
in all cases, the activity of which is stimulated by the Gsproteins
• The first members of the Gi subfamily to be discovered
displayed an inhibitory effect on adenylyl cyclase, hence the
name Gi, for inhibitory G proteins
• Other members of the Gi subfamily have phospholipase C as the
corresponding effector molecule
• Type α-2 adrenergic receptors fall into this group
• Signal transmission in the vision process is mediated via G
proteins known as transducins (Gt)
28
• Perception of bitter taste also takes place via Gi
• The Gq subfamily includes G-proteins associated with type α-1
adrenergic receptors
• There is also a fourth subfamily known as G12
• Two bacterial toxins, namely pertussis toxin and cholera toxin,
were of great importance in determining the function of Gproteins
• Both toxins catalyze ADP ribosylation of proteins. During ADP
ribosylation, an ADP-ribose residue is transferred from NAD+ to
an amino acid residue of a substrate protein
• Cholera toxin is an enterotoxin made up of one A subunit
(composed of one A1 and one A2 peptide joined by a disulfide
link) and five B subunits and has a molecular mass of
approximately 84 kDa
• In the small intestine, the toxin attaches by means of the B
subunits binding to the ganglioside GM1 present in the plasma
29
membrane of mucosal cells
• The A subunit then dissociates, and the A1 peptide passes
across to the inner aspect of the plasma membrane
• It then catalyzes the ADP-ribosylation of the GTP-binding
regulatory component (Gs) of adenylyl cyclase, upregulating
the activity of this enzyme
• Thus, adenylyl cyclase becomes chronically activated resulting
in an elevation of cAMP
30
• PKA then phosphorylates the regulatory domains of the cystic
fibrosis transmembrane conductance regulator (CFTR) and the
Na+-H+ exchanger
• This leads to the inhibition of Na+ absorption and the
enhancement of the secretion of Cl• Thus, massive amounts of NaCl accumulate inside the lumen of
the intestine, attracting water by osmosis and contributing to
the liquid stools characteristic of cholera
• Pertussis toxin is a protein secreted by the bacterium Bordetella
pertussis which causes whooping cough
• Pertussis toxin carries out an ADP-ribosylation at a cysteine
residue of a Gi protein that inhibits adenylyl cyclase, closes Ca2+
channels, and opens K+ channels
• The effect of this modification, however, is to lower the G
protein's affinity for GTP, effectively trapping it in the "off"
conformation. The pulmonary symptoms have not yet been
31
traced to a particular target of the Gi protein
Mechanism of Action of
Cholera Toxin
32
Effector Molecules of G-Proteins
• Activated G proteins pass the signal on to subsequent
effector molecules that have enzyme activity or function
as ion channels
• Important effector molecules are adenylyl cyclase,
phospolipases, and cGMP-specific phosphodiesterases
• The activation of these enzymes leads to concentration
changes of diffusible signal molecules such as cAMP,
cGMP, diacylglycerol or inositol triphosphate (IP3), and Ca2+
, which trigger further specific reactions
cAMP
• 3’-5’-cyclic AMP is a central intracellular second messenger
that influences many cellular functions, such as
gluconeogenesis, glycolysis, lipogenesis, muscle
contraction, membrane secretion, learning processes, ion
33
transport, differentiation,…
• Concentration of cAMP is controlled primarily by two means,
namely via new synthesis by adenylyl cyclase and degradation
by phosphodiesterases
• Cyclic AMP binds to and activates different signaling proteins
• It can regulate ion passage through cAMP-gated ion channels
• The majority of the biological effects of cAMP are mediated by
the activation of protein kinases. Protein kinases regulated by
cAMP are classified as protein kinase A
• In the absence of cAMP, protein kinase A exists as a tetramer,
composed of two regulatory (R) and two catalytic (C) subunits
• In the tetrameric R2C2 form, protein kinase A is inactive since
the catalytic center of the C subunit is blocked by the R subunit
• Upon binding of four molecules of cAMP, the enzyme
dissociates into an R subunit dimer with four molecules of
cAMP bound and two free C subunits which are now released
from inhibition by the regulatory subunits and can thus
34
phosphorylate Ser/Thr residues on specific substrate proteins
Cyclic-GMP
• Like cAMP, 3’-5’-cGMP is a
widespread second messsenger
• Analogous to cAMP, cGMP is formed
by catalysis via guanylyl cyclase from
GTP
• While adenylyl cyclase is an integral
membrane protein, guanylyl cyclase
can be found either associated with
membranes or as a soluble cytosolic
form
35
Cytosolic Signaling via cAMP
36
*cAMP and Gene Transcription
• Whereas some responses mediated by cyclic AMP occur
within seconds and do not depend on changes in gene
transcription, others do depend on changes in the
transcription of specific genes
• When PKA is activated by cAMP, it enters into the nucleus
and phosphorylates a specific gene regulatory protein called
cyclic AMP response element-binding (CREB) protein
• Phosphorylated CREB then recruits a transcriptional
coactivator called CREB-binding protein (CBP)
• The CREB/CBP complex binds to CRE on specific genes and
activates transcription
• This signaling pathway controls many processes in cells,
ranging from hormone synthesis (e.g. somatostatin) in
endocrine cells to the production of proteins required for the
induction of long-term memory in the brain
37
38
*Signaling through the βγ –subunit
• In some other cases, G proteins directly activate or inactivate
ion channels in the plasma membrane of the target cell
• Acetylcholine reduces both the rate and strength of heart
muscle cell contraction
• This effect is mediated by a special class of acetylcholine
receptors that activate the Gi protein
• Once activated, the α subunit of Gi inhibits adenylyl cyclase
while the βγ subunits bind to K+ channels in the heart muscle
cell plasma membrane and open them
• The opening of these K+ channels makes it harder to depolarize
the cell and thereby contributes to the inhibitory effect of
acetylcholine on the heart
• These acetylcholine receptors, which can be activated by the
fungal alkaloid muscarine, are called muscarinic acetylcholine
receptors to distinguish them from nicotinic acetylcholine
39
receptors
• Nicotinic receptors are ion-channel-coupled receptors on
skeletal muscle and nerve cells that can be activated by the
binding of nicotine, as well as by acetylcholine
40
• The second messenger function of cGMP is directed towards
three targets: cGMP-dependent protein kinases (protein kinase
G, PKG), ion channels and cAMP phosphodiesterases
• Cyclic GMP carries different messages in different tissues. In
the kidney and intestine it triggers changes in ion transport and
water retention; in cardiac muscle, it signals relaxation; in the
brain it may be involved both in development and in adult brain
41
function
• Guanylyl cyclase in the kidney is activated by the hormone
atrial natriuretic factor (ANF), which is released by cells in
the atrium of the heart when the heart is stretched by
increased blood volume
• Carried in the blood to the kidney, ANF activates guanylyl
cyclase in cells of the collecting ducts
• The resulting rise in [cGMP] triggers increased renal
excretion of Na+ and, consequently, of water, driven by the
change in osmotic pressure
• Water loss reduces the blood volume, countering the
stimulus that initially led to ANF secretion
• Vascular smooth muscle also has an ANF receptor—
guanylyl cyclase; on binding to this receptor, ANF causes
vasodilation, which increases blood flow while decreasing
blood pressure
42
• A similar receptor guanylyl cyclase in the plasma membrane of
intestinal epithelial cells is activated by an intestinal peptide,
guanylin, which regulates Cl- secretion in the intestine
• This receptor is also the target of a heat-stable peptide
endotoxin produced by E. coli and other gram-negative
bacteria
• The elevation in [cGMP] caused by the endotoxin increases Clsecretion and consequently decreases reabsorption of water by
the intestinal epithelium, producing diarrhea
• The soluble guanylyl cyclases are regulated by the second
messenger NO
• They have a heme group that confers NO-sensitivity. NO
binding to the heme group results in activation of the guanylyl
cyclase activity
• Acetylcholine is a vasodilator that acts by causing relaxation of
the smooth muscle of blood vessels
43
• However, it does not act directly on smooth muscle
• If endothelial cells are stripped away from underlying smooth
muscle cells, acetylcholine no longer exerts its vasodilator
effect
• This indicates that vasodilators such as acetylcholine initially
interact with the endothelial cells of small blood vessels via
receptors
• The receptors are coupled to the phosphoinositide cycle,
leading to the intracellular release of Ca2+ through the action of
inositol trisphosphate
• In turn, the elevation of Ca2+ leads to the liberation of NO also
known as endothelium-derived relaxing factor (EDRF), which
diffuses into the adjacent smooth muscle
• This leads to the elevation of intracellular levels of cGMP which
in turn stimulates the activities of certain PKG, which probably
phosphorylate specific muscle proteins, causing relaxation;
however, the details are still being clarified
44
• In the heart, cGMP reduces the forcefulness of contractions by
stimulating the ion pump(s) that expel Ca+2 from the cytosol
• This NO-induced relaxation of cardiac muscle is the same
response brought about by nitroglycerin tablets and other
nitrovasodilators taken to relieve angina, the pain caused by
contraction of a heart deprived of O2 because of blocked
coronary arteries
• Another important cardiovascular effect of NO is that by
increasing synthesis of cGMP, it acts as an inhibitor of platelet
aggregation
• NO is unstable and its action is brief; within seconds of its
formation, it undergoes oxidation to nitrite, nitrate or
peroxynitrite
• Nitrovasodilators produce long-lasting relaxation of cardiac
muscle because they break down over several hours, yielding a
steady stream of NO
45
• The effects of increased cGMP synthesis diminish after the
stimulus ceases, because a specific phosphodiesterase (cGMP
PDE) converts cGMP to the inactive 5-GMP
• Humans have several isoforms of cGMP PDE, with different
tissue distributions
• The isoform in the blood vessels of the penis is inhibited by the
drug sildenafil citrate (Viagra), which therefore causes cGMP
levels to remain elevated once raised by an appropriate
stimulus, accounting for the usefulness of this drug in the
treatment of erectile dysfunction
• NO is inhibited by hemoglobin and other heme proteins, which
bind it tightly
• Administration of NO synthase inhibitors to animals and
humans leads to vasoconstriction and a marked elevation of
blood pressure, indicating that NO is of major importance in
the maintenance of blood pressure in vivo
46
47
The Synthesis and (One) Action of NO
Inositol phospholipids and inositol phosphates
• Inositol-containing phospholipids of the plasma membrane are
the starting compounds for the formation of various inositol
messengers in response to various signals
• These messengers include the central second messengers
diacylglycerol (DAG) and inositol trisphosphate (IP3) as well as
membrane-bound phosphatidyl inositol phosphates (e.g. PIP3)
• Phosphatidylinositol is first phosphorylated by specific kinases
at the 4’ and 5’ positions of the inositol residue, leading to the
formation of phosphatidyl inositol-4,5-bisphosphate (PIP2)
• From PIP2, two paths lead to physiologically important
messenger substances
• One path is phosphorylation to yield PIP3 , which functions as a
membrane-localized messenger
• The other option is cleavage by phospholipase C, forming the
second messengers DAG and IP3
48
Phosphoinositide Signaling
49
• IP3 activates the release of Ca2+ , while DAG acts primarily by
stimulation of protein kinase C (PKC)
• Phospholipase C can be activated by G-proteins or by
transmembrane receptors with intrinsic or associated
enzymatic activity
• Ca2+ is a ubiquitous signaling molecule whose signaling
function is activated by its release from intracellular stores or
through Ca2+ entry channels from the extracellular side
• The concentration of free Ca2+ in the cytosol of resting cells is
very low, about 10–7 M
• One reason that the cell tries to keep the free Ca2+
concentration low is the ability of these ions to form poorly
soluble complexes with inorganic phosphate
• The low concentration of free cytosolic Ca2+ is opposed by a
large storage capacity for Ca2+ in specific organelles and
vesicles and by a high concentration in the extracellular region
50
where Ca2+ is present at millimolar concentration
• Storage sites include endoplasmic reticulum, mitochondria and
calciosomes
• In the endoplasmic reticulum, Ca2+ exists in complex with the
storage protein calreticulin
• In the protein-bound and compartmentalized form, Ca2+ is not
freely available but may be released in the process of signal
transduction
• In muscle cells, Ca2+ is stored in the sarcoplasmic reticulum
• The storage takes place particularly by binding to the storage
protein calsequestrin. Ca2+ is released from storage by a neural
stimulus and initiates muscle contraction
• Mobilization of Ca2+ from the Ca2+ stores of the endoplasmic
reticulum takes place with the help of Ca2+ channels, of which
two types stand out: the IP3 receptors and the ryanodine
receptors
• Ca2+ enters from the extracellular space through either
51
voltage-gated or ligand-gated channels
• One of the primary functions of Ca2+ entry into cells is to charge
up the internal stores, which can then release an internal Ca2+
signal
• Ca+2 that has entered from the extracellular space plays a role
in the opening of IP3 and ryanodine receptors
• Overall, multiple pathways can be used for mobilizing Ca2+
from the internal stores
• A Ca2+ signaling ‘toolkit’ is available from which cells can select
specific components to activate the internal Ca2+ stores and to
generate a variety of different Ca2+ signals that suit their
physiology
• The cytosolic Ca2+ concentration is generally increased only
temporarily and is often only locally increased during
stimulation of cells
• The cell possesses efficient Ca2+ transport systems, which can
rapidly transport Ca2+ back into the extracellular region or into
52
the storage organelles
Tools for Ca2+ Release
53
Mechanisms for Ca2+ Increase and
Decrease
• Ca2+ -ATPases, in particular,
are involved in draining the
cytosol of Ca2+ back into the
extracellular region
• The Ca2+ -ATPases perform
active transport of Ca2+
against its concentration
gradient, using the hydrolysis
of ATP as an energy source
• Other transport systems in
the plasma membrane
exchange Na+ ions for Ca2+
(use the energy of the Na+
gradient)
• These Na+-Ca2+ exchange
proteins are located especially
in muscle cells and in neurons
54
• Examples of Ca2+ -dependent reactions are numerous and
affect many important processes of the organism, including
muscle contraction, vision process, cell proliferation, secretion,
cell motility, formation of the cytoskeleton, exocytosis, gene
expression, reactions of intermediary metabolism,…
• The information encoded in transient Ca2+ signals is deciphered
by various intracellular Ca2+-binding proteins that convert the
signal into a wide variety of biochemical changes
• There are two principle mechanisms by which Ca2+ can perform
a regulatory function:
1. Direct Activation of Proteins
• There are many enzymes that have a specific Ca2+ -binding
center in the active site and for which Ca2+ has an essential
role in catalysis
• Examples of Ca2+ -dependent enzymes are phospholipase A2
and PKC (which is activated by the joint action of DAG and
55
Ca2+ )
2. Binding of Ca2+ Receptors
• The Ca2+ receptors are Ca2+ sensing proteins that activate
target proteins in response to changes in Ca2+ concentration
• The most widespread Ca2+ receptor is calmodulin
• Calmodulin is a small protein of ca. 150 amino acids. It has got
two globular domains each capable of binding 2 calcium ions
• The Ca2+/calmodulin complex is involved in in regulation of
mitosis, neuronal signal transduction, muscle contraction,
glucose metabolism,….
• Ca2+ receptors related to calmodulin include troponin C (which
can only bind 2 Ca2+ ) and recoverin (which is involved in vision)
The Mechanism of Vision
• The molecule that absorbs light in the eyes is cis-retinal, which
is linked covalently (via a lysine residue) to a protein, opsin
• In the rods, the complex of retinal and opsin is known as
rhodopsin, which is also referred to as the photopigment
56
• There are also red, blue and green sensitive opsins in the cons
Ca2+/Calmodulin Activation
of Different Proteins
57
• The absorption of photons by 11-cis-retinal converts it to the all
trans-form which induces a conformational change in opsin
• Upon this change, the retinal dissociates from the opsin. Opsin
is now free to initiate the sequence of events that leads to the
detection of light
• The opsin interacts with a membrane trimeric G-protein, known
as transducin, which results in an exchange GDP for GTP on the
transducin
• This activates the trimeric G-protein in the usual way, i.e. by
dissociation of the α-β-γ complex, which releases the α-subunit
• The α-subunit activates an enzyme, cyclic GMP
phosphodiesterase, which decreases the concentration of cGMP
• In the dark, cGMP binds to a channel that allows the entry of
Na+ and Ca+2 and leads to depolarization
• When the level of cGMP falls, the channels are closed and the
membrane is hyperpolarized
58
Light-Induced Hyperpolarization of
Rod Cells
59
• Hyperpolarization decreases the release of the
neurotransmitter glutamate into the synapse that connects
the photoreceptor cell to the bipolar neurons
• The decreased release of glutamate leads to the
depolarization of the bipolar neurons
• The action potential created in the bipolar neurons is relayed
to the visual cortex
• The visual signal is terminated through a combination of ways
 The intrinsic GTPase activity of transducin means the cGMP
phosphodiesterase is no longer activated; cGMP levels rise
 When the levels of Ca2+ fall, the inhibition on guanylyl cyclase
is relieved while cGMP PDE is inhibited
 Rhodopsin is desensitized by rhodopsin kinase. The
phosphorylated rhodopsin is bound by the protein arrestin
 Recoverin inhibits rhodopsin kinase at high [Ca2+ ], but the
inhibition is relieved when [Ca2+ ] drops after illumination
60
61
The Cis/ Trans Cycle
• On a relatively long time scale (seconds to minutes), the all-transretinal of an excited rhodopsin molecule is removed and replaced
by 11-cis-retinal, to produce rhodopsin that is ready for another
round of excitation
• If properly dark adapted, the eye can detect a single photon
• Vitamin A deficiency reduces the amount of rhodopsin in the
retina, increasing the minimum amount of illumination that can
be detected (the visual threshold) and causing night blindness
62
•
•
•
•
•
Transmembrane Receptors and Tyrosine Kinase Activity
PKA, PKC, PKG, Ca2+/calmodulin-dependent kinases, MLCK,
glycogen phosphorylase kinase, rhodopsin kinase and
numerous other kinases are Ser/Thr kinases
In addition, multicellular organisms communicate through
the binding of protein ligands to transmembrane receptors
that are dependent of Tyr kinase activity
Some transmembrane receptors possess intrinsic tyrosine
kinase activity; these receptors are known as receptor
tyrosine kinases
Ligand binding to an extracellular domain of the receptor is
coupled to the stimulation of tyrosine kinase activity
localized on a cytoplasmic receptor domain
The ligand-binding domain and the tyrosine kinase domain
are part of one and the same protein
63
Associated
Intrinsic
• Another type of transmembrane receptor is associated, on the
cytoplasmic side, with a tyrosine kinase that is activated when a
ligand binds to the extracellular receptor
• The tyrosine kinase and the receptor are not located on the
same protein in this case
64
• Activation of tyrosine-specific protein kinase activity is
triggered, in particular, by signals that control cell growth and
differentiation
• Ligand binding to the extracellular portions of the receptor
tyrosine kinase induces the non-covalent oligomerization –
mostly dimerization – of monomeric receptors, or it induces a
structural rearrangement in a preassembled oligomeric
receptor facilitating tyrosine autophosphorylation
• For ligand-induced dimerization, two pathways have been
described
• In the first pathway, which applies for the growth hormone
receptor and the erythropoietin receptor, the ligand has two
binding sites for the receptor molecule and brings about a
dimerization of the receptor
• In the absence of the ligand, the receptor exists in a monomeric
form
65
• In another pathway, two ligands are needed (e.g. EGF)
Ligand-Induced
Dimerization
• In the case of the insulin receptor, the bound ligand appears to
stabilize a distinct conformation of the pre-assembled
oligomeric form of the receptor, fixing the receptor in a
catalytically active state
• The insulin receptor is a heterotetrameric protein composed of
two αβ-units linked by disulfide bridges
• The binding of insulin brings about a change in the relative
configuration of the two tyrosine kinase domains, in such a way
that mutual Tyr phosphorylation is enabled
66
Autophosphorylation
• In the absence of the ligand, the two active sites are thought to
be too distant for this trans-phosphorylation
Effector Proteins of Receptor Tyrosine Kinases
• Autophosphorylation of receptor tyrosine kinases has a double
effect
• The tyrosine kinase activity undergoes autoactivation by
phosphorylation of Tyr residues localized in the catalytic
domain
• In addition, Tyr residues that lie outside the active center are
67
phosphorylated
• The phosphotyrosine residues thereby created serve as
binding sites for effector molecules next in the sequence
of the signal transduction pathway
• The phosphotyrosine residues of the activated receptors
are attachment points for effector proteins that possess a
phosphotyrosine-binding domain, such as the SH2 domain
(the Src homology 2 domain, named for the first protein in
which it was found, the src protein of the Rous sarcoma
virus)
• The docking of signaling proteins to autophosphorylation
sites provides a mechanism for assembly and recruitment
of signaling complexes
• Many of the signaling pathways activated by receptor
tyrosine kinases ultimately lead to the activation of
transcription factors, influencing central differentiation and
68
developmental programs of the cell
Insulin Signaling- example of intrinsic tyrosine kinase activity
• The activated phosphorylated insulin receptor binds a protein
called IRS-1 (insulin receptor substrate-1)
• The activated receptor kinase phosphorylates IRS-1 at multiple
sites, creating multiple binding sites for different proteins with
SH2 domains
• One of the phosphotyrosine sites of IRS binds the SH2 domain
of Grb2
• Grb2 is also anchored to PIP3 in the plasma membrane through
its PH (pleckstrin homology) domain
• Grb 2 has got a third domain (SH3) which binds with Sos
• Sos catalyzes the replacement of bound GDP by GTP on Ras, a
G protein
• When GTP is bound, Ras can activate a protein kinase, Raf-1 ,
the first of three protein kinases—Raf-1, MEK, and ERK—that
form a cascade in which each kinase activates the next by
69
phosphorylation
70
• When Erk is activated, it mediates some of the biological effects
of insulin by entering the nucleus and phosphorylating proteins
such as Elk1, which modulates the transcription of about 100
insulin-regulated genes
• ERK is a member of the MAPK family (mitogen-activated
protein kinases; mitogens are signals that act from outside the
cell to induce mitosis and cell growth)
• The above is the pathway insulin follows to exert its effects on
gene transcription
• Below are the ways insulin affects cytosolic processes
• At other phosphotyrosine sites on IRS, phospholipase C and
PI 3-kinase bind using their SH2 domains
• Phospholipase C leads to Ca2+ signaling
• The signal pathway initiated by the insulin receptor complex
involving PI 3-kinase leads to activation of protein kinase B
(PKB), a serine-threonine kinase that mediates many of the 71
downstream effects of insulin
Cytosolic Effects of Insulin
72
• PI 3-kinase synthesizes PIP3 which serves as a docking site for
PKB and PDK1 (phosphoinositide-dependent kinase-1)
• PDK1 phosphorylates and activates PKB
• PKB phosphorylates Ser and Thr residues on target proteins like
glycogen synthase kinase (GSK ) 3, and Glut 4; this favors
glycogen synthesis and entry of glucose into the cells
• PKB also functions in several other signaling pathways,
including that triggered by 9-tetrahydrocannabinol (THC), the
active ingredient of marijuana and hashish
• THC activates the CB1 receptor in the plasma membrane of
neurons in the brain
• One consequence of CB1 activation is the stimulation of
appetite, one of the well-established effects of marijuana use
• The normal ligands for the CB1 receptor are endocannabinoids
such as anandamide, which serve to protect the brain from the
toxicity of excessive neuronal activity-as in an epileptic seizure
73
The Cytokine Receptors -examples of associated tyrosine kinase
activity
• The large family of cytokine receptors includes receptors for
many kinds of local mediators (collectively called cytokines), as
well as receptors for some hormones, such as growth hormone
and prolactin
• These receptors are stably associated with cytoplasmic tyrosine
kinases called Janus kinases (JAKs) (after the two-faced Roman
god), which phosphorylate and activate gene regulatory proteins
called STATs (signal transducers and activators of
74
transcription)
• STAT proteins are located in the cytosol and are referred to as
latent gene regulatory proteins because they only migrate into
the nucleus and regulate gene transcription after they are
activated
• Although many intracellular signaling pathways lead from cellsurface receptors to the nucleus, where they alter gene
transcription, the JAK–STAT signaling pathway provides one of
the more direct routes
• Cytokine receptors are dimers or trimers and are stably
associated with one or two of the four known JAKs (JAK1, JAK2,
JAK3, and Tyk2)
• Cytokine binding alters the arrangement so as to bring two
JAKs into close proximity so that they transphosphorylate each
other, thereby increasing the activity of their tyrosine kinase
domains
• The JAKs then phosphorylate tyrosines on the cytokine
receptors, creating phosphotyrosine docking sites for STATs75
• Some adaptor proteins can also bind to some of these sites
and couple cytokine receptors to the MAPK pathway
• There are at least six STATs in mammals. Each has an SH2
domain that performs two functions
• First, it mediates the binding of the STAT protein to a
phosphotyrosine docking site on an activated cytokine
receptor
• Once bound, the JAKs phosphorylate the STAT on tyrosines,
causing the STAT to dissociate from the receptor
• Second, the SH2 domain on the released STAT now mediates
its binding to a phosphotyrosine on another STAT molecule,
forming either a STAT homodimer or a heterodimer
• The STAT dimer then translocates to the nucleus, where, in
combination with other gene regulatory proteins, it binds to
a specific DNA response element in various genes and
stimulates their transcription
76
77
The JAK-STAT
Signaling Pathway
•
•
•
•
•
Receptor Serine/Threonine Kinases
The transforming growth factor-β (TGF β) superfamily
consists of a large number of structurally related, secreted,
dimeric proteins
They act either as hormones or, more commonly, as local
mediators to regulate a wide range of biological functions in
all animals
During development, they regulate pattern formation and
influence various cell behaviors, including proliferation,
differentiation, extracellular matrix production, and cell
death
In adults, they are involved in tissue repair and in immune
regulation, as well as in many other processes
All of these proteins act through enzyme-coupled receptors
that have a serine/threonine kinase domain on the cytosolic
78
side of the plasma membrane
• There are two classes of these receptor serine/threonine
kinases—type I and type II—which are structurally similar
homodimers
• Each member of the TGFβ superfamily binds to a characteristic
combination of type-I and type-II receptor dimers, bringing the
kinase domains together so that the type-II receptor can
phosphorylate and activate the type-I receptor, forming an
active tetrameric receptor complex
• Once activated, the receptor complex uses a strategy for
rapidly relaying the signal to the nucleus that is very similar to
the JAK–STAT strategy used by cytokine receptors
• The activated type-I receptor directly binds and phosphorylates
a latent gene regulatory protein of the Smad family (named
after the first two identified, Sma in C. elegans and Mad in
Drosophila)
• The Smads bound by receptors are Smads 1, 2, 3, 5 and 8 and
79
are known as receptor Smads (R-Smads)
The Smad Signaling
Pathway
80
• Once one of these R-Smads has been phosphorylated, it
dissociates from the receptor and binds to Smad4 (called a
co-Smad), which can form a complex with any of the five RSmads
• The Smad complex then translocates into the nucleus, where
it associates with other gene regulatory proteins and
regulates the transcription of specific target genes
SIGNAL TRANSDUCTION IN CELL BIRTH, DEATH AND CANCER
• Cells execute their reproduction in a cyclic process, in which at
least two phases, S phase and M phase, can be differentiated
on the basis of biochemical and morphological features
• The biochemical characteristic of S (synthesis) phase is the
replication of nuclear DNA and thus doubling of the genetic
information
• In M (mitosis) phase, division of the chromosomes between the
81
daughter cells is prepared and carried out
• In most cell types, two further phases can be
distinguished, G1 and G2 phase
• G1 phase covers the period between M phase and S phase
while G2 phase covers the period between S phase and M
phase
• From G1 phase, the cell may transfer into a quiescent state
known as G0 phase
• Appropriate signals (e. g., addition of growth factors) can
induce the cell to return from G0 into G1 phase and proceed
with the cell cycle
• Rapidly dividing cells in mammals require 12–24 h for
completion of a cell cycle
• In some cell types, such as early embryonic cells, the
period between the S and the M phases is reduced to the
extent that discrete G1 and G2 phases cannot be identified.
82
The duration of the cell cycle is then only 8–60 min
• The cell-cycle control system is based on a connected series
of biochemical switches, each of which initiates a specific
cell-cycle event
• The cell cycle has three major regulatory transition points
known as checkpoints
• The first checkpoint is Start (or the restriction point) in late
G1, where the cell commits to cell-cycle entry and
chromosome duplication
• The second is the G2/M checkpoint, where the control system
triggers the early mitotic events that lead to chromosome
alignment on the spindle in metaphase
• The third is the metaphase-to-anaphase transition, where
the control system stimulates sister-chromatid separation,
leading to the completion of mitosis and cytokinesis
• The control system blocks progression through each of these
checkpoints if it detects problems inside or outside the cell83
• If the control system senses problems in the completion of
DNA replication, for example, it will hold the cell at the
G2/M checkpoint until those problems are solved
• Similarly, if extracellular conditions are not appropriate for
cell proliferation, the control system blocks progression
through Start, thereby preventing cell division until
conditions become favorable
The Components of The Cell Cycle Control System
• The central components of the cell-cycle control system
are members of a family of protein kinases known as
cyclin-dependent kinases (Cdks)
• The activities of these kinases rise and fall as the cell
progresses through the cycle, leading to cyclical changes in
the phosphorylation of intracellular proteins that initiate
or regulate the major events of the cell cycle
84
• An increase in Cdk activity at the G2/M checkpoint, for example,
increases the phosphorylation of proteins that control
chromosome condensation, nuclear envelope breakdown,
spindle assembly, and other events that occur at the onset of
mitosis
• Cyclical changes in Cdk activity are controlled by a complex
array of enzymes and other proteins that regulate these kinases
• The most important of these Cdk regulators are proteins known
as cyclins
• Cdks, as their name implies, are dependent on cyclins for their
activity: unless they are tightly bound to a cyclin, they have no
protein kinase activity
• Cyclins were originally named because they undergo a cycle of
synthesis and degradation in each cell cycle
• The levels of the Cdk , by contrast, are relatively constant
• There are four classes of cyclins, each defined by the stage of
85
the cell cycle at which they bind Cdks and function
• All eukaryotic cells require three of these classes:
1. G1/S-cyclins activate Cdks in late G1and thereby help trigger
progression through Start, resulting in a commitment to cellcycle entry. Their levels fall in S phase
2. S-cyclins bind Cdks soon after progression through Start and
help stimulate chromosome duplication. S-cyclin levels
remain elevated until mitosis, and these cyclins also
contribute to the control of some early mitotic events
3. M-cyclins activate Cdks that stimulate entry into mitosis at
the G2/M checkpoint. M-cyclins are destroyed in mid-mitosis
• In most cells, a fourth class of cyclins, the G1-cyclins, helps
govern the activities of the G1/S cyclins
• There are four Cdks. Two interact with G1 cyclins, one with G1/Sand S-cyclins, and one with M-cyclins
• In the absence of cyclin, the active site in the Cdk protein is
partly obscured by a slab of protein, like a stone blocking the
86
entrance to a cave
• Cyclin binding causes the slab to move away from the active
site, resulting in partial activation of the Cdk
• Full activation of the cyclin-Cdk complex then occurs when a
separate kinase, the Cdk-activating kinase (CAK),
phosphorylates an amino acid near the entrance of the Cdk
active site
• This causes a small conformational change that further
increases the activity of the Cdk, allowing the kinase to
phosphorylate its target proteins effectively and thereby induce
specific cell-cycle events
87
• Several additional mechanisms fine-tune Cdk activity at specific
stages of the cycle
• Phosphorylation at a pair of amino acids in the roof of the
kinase active site inhibits the activity of a cyclin-Cdk complex
while dephosphorylation of these sites increases the activity
• The binding of Cdk inhibitor proteins (CKIs) also regulates
cyclin-Cdk complexes
• CKI binding stimulates a large rearrangement in the structure of
the Cdk active site, rendering it inactive
The Inhibition of Cdk
88
Summary of The Cell Cycle Progression
G1 progression
• Following exit from mitosis, cells can enter a quiescent state
or they can continue in G1, which requires the presence of
mitogenic signals in the form of growth hormones
• Signaling by growth hormones increases the level of D-type
cyclins because of increased transcription
• The increase in D-type cyclins and the formation of cyclin DCDK4/6 complexes has at least a twofold effect
• The metabolism and growth of the cells are stimulated and
the cells are able to reach the critical size required for
crossing of the restriction point
• Furthermore, the pRb (retinoblastoma protein) becomes
initially phosphorylated by the cyclin D-CDK4/6 complexes,
and cells are thus prepared to cross the restriction point
89
• In quiescent cells, Rb is complexed with E2F (a class of
transcription factors), resulting in inhibition of these
transcription factors
• Phosphorylation of Rb releases it from E2F, and E2F is then free
to activate the transcription of genes required for entry into S
Activation of cyclin E/CDK2 and restriction point crossing
• As a consequence of the increased formation of cyclin DCDK4/6 complexes, the inhibitor p27 is sequestered from
complex formation with (and inhibition of) cyclin E-CDK2
• The now active cyclin E-CDK2 continues phosphorylation of
pRb and thereby initiates transcription of E2F-responsive
genes, among which is the gene for cyclin E
• Activation of cyclin E-CDK2 also requires dephosphorylation
• Now the requirements for restriction point crossing are fulfilled
and the continued action of the E2F transcription factors
provides for the enzymes that are necessary for entry into and
90
progress through S phase
Control of the G1/S Transition
in the Cell Cycle
91
S phase progression
• Among the target genes of the E2F transcription factors is the
gene for cyclin A, which increases at the beginning of S phase
• The cyclin A-CDK2 and the cyclin E-CDK2 complexes are
thought to phosphorylate important components of initiation
complexes of DNA replication and thereby induce the transition
of pre-replication complexes to the post-replicative state
• Shortly after entry into S phase, the cyclin E is targeted for
degradation in the ubiquitin-proteasome pathway, and the
activity of the cyclin E-CDK2 is shut off
• Further progress through S phase requires the continued action
of cyclin A-CDK2 complexes
Cyclins and Corresponding
Phases of the Cell Cycle
92
G2/M transition and progress through M Phase
• During S phase and G2 phase, the cyclin B-CDK1 complex (also
known as the MPF, mitosis-promoting factor) accumulates in
an inhibited phosphorylated state and is activated by the
action of phosphatases at the G2/M transition
• The active cyclin B-CDK1 complex phosphorylates numerous
substrates and is inactivated by proteolysis only at the end of M
phase and during G1 phase
The Cell Cycle and the DNA Damage Response
• Progression through the cell cycle, and thus the rate of cell
proliferation, is controlled not only by extracellular mitogens
but also by other extracellular and intracellular mechanisms
• One of the most important influences is DNA damage, which
can occur as a result of spontaneous chemical reactions in DNA,
errors in DNA replication, or exposure to radiation or certain
chemicals
93
• It is essential that the cell repair damaged chromosomes before
attempting to duplicate or segregate them
• The cell-cycle control system can readily detect DNA damage
and arrest the cycle at either of two checkpoints-one at Start in
late G1, which prevents entry into the cell cycle and into S
phase, and one at the G2/M checkpoint, which prevents entry
into mitosis
• DNA damage initiates a signaling pathway by activating one of
a pair of related protein kinases called ATM and ATR, which
associate with the site of damage and phosphorylate various
target proteins, including two other protein kinases
• Together these various kinases phosphorylate other target
proteins that lead to cell-cycle arrest
• A major target is the gene regulatory protein p53, which
stimulates transcription of the gene encoding a Cdk inhibitory
protein called p21
94
• p21 binds to G1/S-Cdk and S-Cdk complexes and inhibits
their activities, thereby helping to block entry into the cell
cycle
• DNA damage activates p53 by an indirect mechanism
• In undamaged cells, p53 is highly unstable and is present at
very low concentrations
• This is largely because it interacts with another protein,
Mdm2, which acts as a ubiquitin ligase that targets p53 for
destruction by proteasomes
• Phosphorylation of p53 after DNA damage reduces its
binding to Mdm2
• This decreases p53 degradation, which results in a marked
increase in p53 concentration in the cell
• In addition, the decreased binding to Mdm2 enhances the
ability of p53 to stimulate gene transcription
95
How DNA damage arrests
the Cell Cycle in G1
96
• A low level of DNA damage occurs in the normal life of any cell
and this damage accumulates in the cell’s progeny if the
damage response is not functioning
• Over the long term, the accumulation of genetic damage in
cells lacking the DNA damage response leads to an increase in
the frequency of cancer-promoting mutations
• Indeed, mutations in the p53 gene occur in at least half of all
human cancers
• This loss of p53 function allows the cancer cell to accumulate
mutations more readily
o What happens if DNA damage is so severe that it cannot be
repaired?
• Animal cells with severe DNA damage do not attempt to
continue division, but instead commit suicide by undergoing
apoptosis
• Thus, unless the DNA damage is repaired, the DNA damage
response can lead to either cell-cycle arrest or cell death 97
• Many of the components of mitogenic signaling pathways are
encoded by genes that were originally identified as cancerpromoting genes, or oncogenes because mutations in them
contribute to the development of cancer
• The mutation of a single amino acid in the small GTPase Ras,
for example, causes the protein to become permanently
overactive, leading to constant stimulation of Ras-dependent
signaling pathways, even in the absence of mitogenic
stimulation
• However, when a hyperactivated form of Ras or Myc is
experimentally overproduced in most normal cells, the result is
not excessive proliferation but the opposite: the cells undergo
either cell-cycle arrest or apoptosis.
• The normal cell seems able to detect abnormal mitogenic
stimulation, and it responds by preventing further division
• Excessive mitogenic stimulation, often leads to the production
98
of a cell-cycle inhibitor protein called Arf
• Arf binds and inhibits Mdm2 –p53 will be activated
• With this protective mechanisms in place, it seems hard for
cancer cells to arise
• But the protective system is often inactivated in cancer cells by
mutations in the genes that encode essential components of
the checkpoint responses, such as Arf or p53 or the proteins
that help activate them
Excessive Mitogenic
Stimulation and Cell Cycle
Arrest
99
Apoptosis
• Apoptosis depends on a family of proteases that have a
cysteine at their active site and cleave their target proteins at
specific aspartic acids
• They are therefore called caspases (c for cysteine and asp for
aspartic acid)
• Caspases are synthesized in the cell as inactive precursors, or
procaspases, which are typically activated by proteolytic
cleavage
• Procaspase cleavage is catalyzed by other already active
caspases
• Once activated, caspases cleave, and thereby activate, other
procaspases, resulting in an amplifying proteolytic cascade
• Some of the procaspases that operate in apoptosis act at the
start of the proteolytic cascade and are called initiator
procaspases; when activated, they cleave and activate
100
downstream executioner procaspases
• Executioner procaspases then cleave and activate other
executioner procaspases as well as specific target proteins in
the cell
• Among the many target proteins cleaved by executioner
caspases are the nuclear lamins, the cleavage of which causes
the irreversible breakdown of the nuclear lamina
• Another target is a protein that normally holds the DNAdegrading enzyme , endonuclease in an inactive form; its
cleavage frees the endonuclease to cut up the DNA in the cell
nucleus
• Other target proteins include components of the cytoskeleton
and cell-cell adhesion proteins that attach cells to their
neighbors
• The cleavage of these proteins helps the apoptotic cell to round
up and detach from its neighbors, making it easier for a healthy
neighboring cell to engulf it, or, in the case of an epithelial cell,
101
for the neighbors to extrude it from the cell sheet
Activation of Procaspases
During Apoptosis
102
• The two best understood signaling pathways that can activate a
caspase cascade leading to apoptosis in mammalian cells are
called the extrinsic pathway and the intrinsic pathway
The Intrinsic Pathway of Apoptosis
• Cells can activate their apoptosis program from inside the cell,
usually in response to injury or other stresses, such as DNA
damage or lack of oxygen, nutrients, or extracellular survival
signals
• This intrinsic pathway depends on the release into the cytosol
of mitochondrial proteins that normally reside in the
intermembrane space of these organelles
• A crucial protein released from mitochondria in the intrinsic
pathway is cytochrome c, a water-soluble component of the
mitochondrial electron-transport chain
• When released into the cytosol, it has an entirely different
function: it binds to a procaspase-activating adaptor protein
103
called Apaf1 (apoptotic protease activating factor-1)
• Apaf1 oligomerizes into a wheel-like heptamer called an
apoptosome
• The Apaf1 proteins in the apoptosome then recruit initiator
procaspase proteins (procaspase-9) which are activated by
proximity in the apoptosome
• The activated caspase-9 molecules then activate downstream
executioner procaspases to induce apoptosis
• A major class of intracellular regulators of apoptosis is the Bcl2
family of proteins
• Bcl2 proteins regulate the intrinsic pathway of apoptosis mainly
by controlling the release of cytochrome c and other
intermembrane mitochondrial proteins into the cytosol
• Some Bcl2 proteins are pro-apoptotic and promote apoptosis by
enhancing the release, whereas others are anti-apoptotic and
inhibit apoptosis by blocking the release
• p53 accumulated in response to irreparable DNA damage
activates the transcription of genes for pro-apoptotic Bcl2 104
The Intrinsic Pathway of Apoptosis
105
The Extrinsic Pathway of Apoptosis
• Extracellular signal proteins binding to cell-surface death
receptors trigger the extrinsic pathway of apoptosis
• Death receptors are transmembrane proteins containing an
extracellular ligand-binding domain, a single transmembrane
domain, and an intracellular death domain, which is required by
the receptors to activate the apoptotic program
• The receptors are homotrimers and belong to the tumor
necrosis factor (TNF) receptor family, which includes a receptor
for TNF itself and the Fas death receptor
• The ligands that activate the death receptors are also
homotrimers; they are structurally related to one another and
belong to the TNF family of signal proteins
• A well-understood example of how death receptors trigger the
extrinsic pathway of apoptosis is the activation of Fas on the
surface of a target cell by Fas ligand on the surface of a killer
106
(cytotoxic) lymphocyte
• Fas has a central role in the physiological regulation of
programmed cell death in the immune system, where it is
mainly used to instruct lymphocytes to die during immune
responses
• When activated by the binding of Fas ligand, the death domains
on the cytosolic tails of the Fas death receptors recruit
intracellular adaptor proteins, which in turn recruit initiator
procaspases (procaspase-8, procaspase-10, or both), forming a
death-inducing signaling complex (DISC)
• Once activated in the DISC, the initiator caspases activate
downstream executioner procaspases to induce apoptosis
• In some circumstances, death receptors activate other
intracellular signaling pathways that do not lead to apoptosis.
• TNF receptors, for example, can also activate the NFκ B
pathway, which can promote cell survival and activate genes
involved in inflammatory responses. Which responses dominate
107
depends on the type of the cell and other signals acting on it
The Extrinsic Pathway of Apoptosis
108
The Molecular Basis of Cancer
• Tumor cells have special features compared to normal cells
• The phenotype of a tumor cell is characterized by the following
characteristics:
 increased rate of cell division, loss of normal growth control
 loss of ability to differentiate
 loss of contact inhibition
increased capability for invasion of neighboring tissue
(metastasis)
immortalization
• The cells of a fully grown, aggressive tumor have acquired these
properties in a slow, multi-step process with the characteristics
of cellular evolution
• This development is associated with a selection process, in the
course of which, cells that have lost their growth-regulating
mechanisms predominate
109
• Tumor cells differ from their progenitor cells in having acquired
a large number of genetic changes
• It is estimated that tumor cells accumulate several thousand to
several hundred thousand changes in DNA sequence
• Two types of gene are associated with development of a tumor
cell: oncogenes (onkos Greek for mass or bulk) and tumor
suppressor genes
• Oncogenes encode proteins involved in the process of
proliferation; that is, those involved in the cell cycle
• These proteins are synthesized in amounts or with an activity
that accelerates progression through the cycle
• In contrast, tumor suppressor genes encode proteins that can
decelerate or arrest progression through the cycle
• An oncogene has been compared with a car with a stuck
accelerator: the car moves even if you take your foot off the
accelerator. However, the car will stop if the brake is pressed
110
(this is the effect of the tumor suppressor gene)
• A mutation in or the loss of a tumor suppressor gene is
analogous to a faulty brake, i.e. it no longer halts proliferation
of the abnormal cell
Tumor Suppressors
• There are two classes of tumor suppressor genes: gatekeeper
genes and caretaker genes
• Gatekeeper genes directly prevent growth of tumors by
inhibiting cell division or promoting apoptosis. Examples are
pRb and p53
• Caretakers have an indirect influence on tumor formation.
These are susceptibility genes that indirectly suppress tumor
formation by maintaining the integrity of the genome
• Impairment of the caretaker function will enhance the
accumulation of mutations
• An important class of caretakers includes DNA repair enzymes
• Loss of tumour suppressor activity leads to production of a
111
tumour in a homozygous recessive manner
• This means that both alleles must be mutated for loss of
control of the cell cycle to be lost and uncontrolled
proliferation to occur
• At least 30 different tumour suppressor genes have been
identified
• The mutations that affect tumor suppressor genes are
known as “loss-of-function” mutations
Oncogenes
• Several genes are involved in the stimulation of
proliferation in a normal cell; that is, they express proteins
that directly or indirectly regulate this process
• These proteins include growth factors, growth factor
receptors, signaling proteins and transcription factors,
which are organized into a sequence that links a growth
factor to the process of proliferation
112
• If a mutation occurs in any one of these genes which results in
an increase in the activity of one of the proteins in this
sequence, the risk of development of a tumour increases
• Such a mutated gene is termed an oncogene. The precursor
gene for the oncogene is termed a proto-oncogene
• An oncogene is defined by its ability to transform cells in
culture
• The proteins expressed by oncogenes are similar to those
expressed by proto-oncogenes but are more active or less well
controlled than the normal protein
• This means, the mutation in oncogenes is a “gain-of-function”
mutation
• Oncogenes generally have dominant character
• The mutation of a proto-oncogene to an oncogene is
phenotypically visible when only one of the two copies of the
gene in a diploid chromosome set is affected by the mutation
113
• Oncogenes were not originally discovered in tumour cells
but, as an integral part of the genome of some retroviruses.
When RNA, isolated from a retrovirus, was transfected into
cells in culture, the cells were transformed
• During the process of transcription and production of viral
genomic RNA, a portion of the host DNA sequence is
sometimes incorporated into the viral genome
• This host genetic sequence may contain a proto-oncogene
(c-onc), which is then subject to a mutation that accompanies
viral replication
• During a subsequent round of viral infection, the mutated
proto-oncogene sequence (now an oncogene, v-onc) will be
inserted into the DNA of the host cell
• Proto-oncogenes can also be converted, via activating
mutations, into oncogenes, without the involvement of
114
viruses
• Radiation and chemical carcinogens act
by causing a mutation in the regulatory region of a gene,
increasing the rate of production of the proto-oncogene
protein, or
producing a mutation in the coding portion of the protooncogene that results in the synthesis of a protein of slightly
different amino acid composition capable of transforming the
cell
• The entire proto-oncogene or a portion of it may be transposed
or translocated, that is, moved from one position in the
genome to another
• In its new location, the proto-oncogene may be controlled by a
more active promoter and, therefore, overexpressed (increased
amounts of the protein product may be produced)
• If only a portion of the proto-oncogene is translocated, it may
be expressed as a truncated protein with altered properties, or
it may fuse with another gene and produce a fusion protein115
Transforming Mutations in
Proto-Oncogenes
116
• The truncated or fusion protein would be hyperactive and cause
inappropriate cell growth
• The proto-oncogene may be amplified, so that multiple copies
of the gene are produced in a single cell
• If more genes are active, more proto-oncogene protein will be
produced, increasing the growth rate of the cells
Groups of Proto-Oncogenes
Growth factors and growth factor receptors
• If too much of a growth factor or a growth factor receptor is
produced, the target cells may respond by proliferating
inappropriately
• Mutations may also lead to a receptor being stuck in the “on”
position even in the absence of ligand
Signal transduction proteins
• A common example is the gene for the GTPase switch protein
Ras. Ras is involved in the MAPK pathway
117
• If the GTPase activity of Ras is blocked, downstream mitogenic
signaling would continue unabated
Transcription factors
• The MAPK pathway leads to the transcription of Myc, Fos, Jun,
that in turn activate the transcription of many proteins needed
for cell division
• The ways in which transcription factors could be converted to
oncogenes include stimulation of transcription of the gene
encoding the transcription factor; increasing the rate of its
translocation from the cytosol to the nucleus; activation of the
transcription factor by phosphorylation or by increasing its
affinity for DNA
Cyclins
• Oncogenic activation of cyclins is mostly observed for the
D-type cyclins, which play a central role in the transition from
Go to G1 and for G1 progression. Increased levels of D-type
118
cyclins and of CDK4/6 activity are frequently found in tumors
119
The Development of Colorectal Cancer and
its Genetic Basis
120