Signal transduction. Part 2.
Second messengers.
 Second messengers are diffusible small molecules that
amplify and spread intracellularly an incoming signal
cAMP – amplifies GPCR-perceiving signals
PI3P and DAG – amplifies GPCR and some membrane PK
(e.g. EGFR-perceiving) signals
cGMP – amplifies signals perceived by membrane guanylate
cyclases
cAMP signaling cAMP amplifyes GPCR signals
 cAMP is syntehsized by adenylyl cyclase
and destroyed by cAMP-phosphodiesterase
 Intracellular concentration of cAMP can be
rapidly changed
 PKA is a major target of cAMP signaling
Glycogen breakdown in skeletal
muscles in response to adrenalin
 The cascade includes
changes in protein activity
and does not include
changes in gene
expression, that is why it
is very fast
 Major participants:
Adrenalin
GPCR (The β2 adrenergic
receptor=ADRB2)
G-protein
Adenylyl cyclase
PKA
Phosphorylase kinase
Glycogen phosphorylase
glycogen(n residues) + Pi glycogen(n-1 residues) + glucose-1-phosphate
Enzymes that catalyze reactions with
phosphate groups
 Kinases - transferases
 G-proteins- hydrolases
 Phosphatases - hydrolases
 Glycogen posphorylase –transferase and hydrolase
Other signaling pathways
mediated by cAMP
 cAMP-regulated channels
 PKA-regulated channels
 Transcriptional factor CREB
(cAMP response element
(CRE)-binding) – in nucleus!
 The alternative signaling
Phospholipid signaling
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pathway involves PI(4,5)P2
degradation by
Phosphoinositidephospholipase C(PLC )
Inositol 1,4,5-triphosphate
(IP3) is soluble
Diacylglycerol (DAG) is
membrane bound
There are several PLC
isoforms
PLCβ is activated by Gq
PLCγ is activated by
polypeptide growth factor
receptors, such as plateletderived growth factor
(PDGFR), epidermal
growth factor (EGFR)
Phospholipid signaling
 PLCβ is activated by Gq
 Inositol 1,4,5-triphosphate
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(IP3) is soluble
Diacylglycaerol (DAG) is
membrane bound
DAG activates PKC
IP3 rises intracellular Ca2+
IP3 can be broken down by
phosphatases or turned into
inositol.
DAG is metabolized via
hydrolysis to yield glycerol
and fatty acids or
phosphorylation to form
phosphatidic acid.
Ca2+ signaling
 Ca2+ is a second messenger
 S. Ringer found that in the presence of Ca2+ isolated frog
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heart maintained activity for hours,
Locke described that absence of Ca2+ inhibited neuromuscular
transmission.
Kamada and Kimoshita discovered in 1943 that introduction of
Ca2+ into muscle fibers caused their contraction.
Intracytosolic concentration ≈50-100 nM
Extracellular concentration ≈ 1-2 mM
Intracellular stores ≈ 30-300 mM
 Without special signaling
intracellular Ca2+
concentration is low due to
ion pumps Ca2+-ATPases in
the plasma membrane and ER
(SERCA), and Na+/Ca2+
exchanger in the plasma
membrane.
 When cytoplasmic Ca2+
rises, neighbouring Ca2+
channels are activated
progressively. Their opening
leads to a Ca2+ “wave”. This
is an example of positive
feedback.
Calmodulin is the best studied target of
Ca2+
 Calmodulin changes
conformation upon
Ca2+ binding
 CAMKs are the best
studied target of
calmodulin
 Look target of CAMKs
in the previous lecture
cGMP
 The cyclic GMP signaling
Sharma and Duda 2014
system consists of a single
protein - Membrane
guanylate cyclase
 The inhibiting enzyme is
cGMP-phosphodiestrase
 The effector protein is
PKG
 cGMP serves as a second
messenger in a vertebrate
eye. It converts the visual
signals (photons) to nerve
impulses.
Both GPCR and MGC input in the
phosphotransduction system
 Here, GPCR (rodopsin) is a light acceptor. It plays an activating role
via interactions with G-protein (transducin)
 Transducin activates cGMP-phosphodiesterase
 Reduction in cGMP concentration results in closing of on channels
 MGC plays an inhibitory role via opening ion channels and
stimulation of Na/Ca influx
Phototransduction
 It takes about 20 ms for
production of a
neurotransmitting signal
upon photon/rodopsin
interaction
Alberts Ecb
Receptors and their regulation
We consider three types of receptors
G-protein coupled receptors (GPCRs)
Ion-channel coupled receptors
Enzyme-coupled receptors
Ion channel-coupled receptors
 Convert chemical signals
into electrical signals
 Binding of a ligand opens
the channel
Ion channel-coupled receptors
 Some ICCR are GPCR
coupled with ion channels
 When the GPCR binds a
ligand and changes
conformation, this change is
directly transmitted to the
channel and results in a
change in gating and in the
ionic current through the
channel.
 Moreau, C. J. et al. Coupling
ion channels to receptors for
biomolecule sensing. Nature
Nanotechnology 3, 620-625
(2008)
Enzyme-coupled receptors
 Instead of association with G-protein, the cytoplasmatic
domain of ECRs has enzymatic activity or recruits an
enzyme
 Growth factor receptors (including EGFR, PDGFR,
HGFR and other receptors of Tyropsine kinase Family,
TKRs) are the most important class of ECRs that regulates
cell proliferation, differentiation and growth as well as
cytosceleton rearrangements
 TKRs has one membrane spanning domain which is an
alfa-helix.
TKRs
 Ligand binding causes dimerization
(oligomerization)
 Dimerization results in phosphorylation
of each subunit by another subunit – we
speak about self-phosphorylation
 Self-phosphorylation of intracellular
domains causes an assembly of a
transient intracellular signaling
complex
 Inhibition of TKRs via action of
phosphatases and endocytosis
TKR signaling
 The process of adaptor and effector proteins binding with
activated ECR is known as docking
 Several independent signaling pathways might be induced by
proteins of intracellular signaling complex
 The most important effector proteins of the RTK-associated
signaling complex:
PLCγ
PKC
Ras (small GTPase)
PI3K
Ras signaling cascade
 Ras is activated by almost
all TKRs
 Activation is via activation
of Ras-GEF by activated
TKR
 The TKR-Ras specific GEF
is Son of Sevenless (SOS)
There are three pathways that are activated by Ras
 IP3/DAG
 Raf/MEK/Erk MAPK signaling pathway
 PI3K/Akt pathway
Raf/Mek/Erk MAPK signaling pathway
 Ras initiates the MAPK signaling pathways when is bound with Raf
 In this pathway each upstream molecule acts as a kinase to
phosphorylate a subsequent downstream molecule. An eventual
result is the modulation of transcription via the phosphorylation
of a number of transcription factors.
 This pathways involved in regulation of cell proliferation,
differentitation and survival
It can inhibit Fas-mediated apoptosis in T cells
Controls production of IL-2 and some chemokines
Involved in Fcgamma-mediated phagocysotosis
 About 30 % of cancer cells carry a mutation that locks Ras in an
active form
Raf/Mek/Erk MAPK signaling pathway
• Raf =MAPKK kinase
= MAPKKK
• MAPK/ERK kinase =
MEK=MAPK kinase=
MAPKK
• MAPK = mitogenactivated protein kinase
= extracellular signal
regulated kinase =
ERK
Alberts et al., ECCB
PI3K pathway
 Phosphoinositide 3 kinases
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 Akt=PKB is a serin-threonine
PK
 Akt acts as an anti-apoptotic
and cell survival promoting
factor
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(PI3K) are composed of two
subunits, regulatory (p85) and
catalytic (p110).
p85 is activated by Ras-GTP
PI3K converts PtdIns(4,5)P2
into PtdIns(3,4,5)P3.
PtdIns(3,4,5)P3 serves as a
docking site (a transient lipid
anchor) for mane proteins
The most important
downstream pathway is known
as an Akt pathway
PI3K/Akt-controlled pathways
Anti-apoptotic
pathway via
inhibition of
 Bad/BAX
 Caspase-9
 The NF-kB
inhibitor IKK
Cell-growth
promoting
pathway via
 Tor
NF-kB
 NF-kB is a transcription
NF-kB
NF-kB
factor
 NF-κB regulates the
expression of cytokines,
growth factors and inhibitors
of apoptosis, receptors
involved in immunity
including.
 Moreover, pathological
dysregulation of NF-κB is
linked to inflammatory and
autoimmune diseases as well
as cancer.
 NF-κB is not synthesized de
novo; its transcriptional
activity is silenced by
interactions with inhibitory
IκB proteins present in the
cytoplasm.
Ubiquitin-proteasome pathway
 The Ubiquitin Proteasome
Pathway (UPP) is the principal
mechanism for protein
catabolism in the mammalian
cells.
 Two steps: tagging of the
substrate protein by the
covalent attachment of
multiple ubiquitin molecules
(Conjugation); and
the subsequent degradation of
the tagged protein by the 26S
proteasome, composed of the
catalytic 20S core and the 19S
regulator (Degradation).
PI3K/Akt-controlled cell growth
promoting pathway
 Tor (target of rapamycin) is a
serine-threonin PK
 Activated Tor inhibits proteins
degradation and stimulates
protein synthesis
EGFR is a prototype ECR
EGFR inhibition
 There two major
pathways for a fast EGFR
inhibition
 Dephosphrylase by PTPs
protein tyrosine
phosphatases )
 Clathrin mediated
endocytosis
Signaling cascades activating by GPCRs/TKRs
The Notch receptor – Delta signal
protein interactions
 Here there is an example
of contact-depedendent
signaling
 DSL binding to the Notch
receptor triggers signaling
through successive
proteolytic cleavages by
ADAM protease.
 The intracellular Notch
domain acts as
atranscriptional regulator
Extracellular ligands: hormones
 Adrenalin: produced by
adrenal glands
 Affect multiple organs
 The receptor - β2
adrenergic
receptor=ADRB2
 The effect depends on a
cell type and particularly
on G-protein type
Acetylcholine
 Acetylcholine binds muscarinic
GPCR-type receptors
The response can be fast or slow
Ligands that act without receptors
 Steroid hormones:
Cortisol
Estradiol, testosterone
 Thyroid hormones: thyroxine
Transcriptional factors are targets of
 Gases
NO (nitric oxide) is produced by
endothelial cells in response to
neurotransmitters and affects
muscle cells. NO targets guanylyl
cyclase.
NO signaling
Animals vs plants
 Plant PKs are structurally
different from
mammalians
 The binding of a ligand
(ethylene) switch off the
receptor
 Still, the effect is the
same: interactions with a
ligand switch on gene
expression
Integrative cell response
 The outcome
depends on
balancing of
different signals
affecting the cell