Transcript Signal Transduction Pathways • Signal Transduction

Chapter 14
Signal-Transduction
Pathways
G proteins
Signal-transduction circuits in biological systems have molecular on-off switches that, like those in a
computer chip, transmit information when “on”. Common among these circuits are those including
G proteins, which transmit a signal when bound to GTP and are silent when bound to GDP
Outline
14.1 Heterotrimeric G proteins transmit signals and reset
themselves
14.2 Insulin signaling: Phosphorylation cascades are
central to many signal-transduction processes
14.3 EGF signaling: signal-transduction systems are
poised to respond
14.4 Many elements recur with variation in different
signal-transduction pathways
14.5 Defects in signal-transduction pathways can lead to
cancer and other diseases
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Signal Transduction Pathways
• Signal Transduction – chain of events that converts the
message “this molecule is present” to a physiological response
β-cells in pancreas
Adrenal glands
Epinephrine腎上腺素
Epinephrine
+
β-Adrenergic receptor
Energy-store
mobilization
insulin胰島素
insulin
+
Insulin receptor
Epidermal growth factor (EGF)
+
EGF receptor
Increased glucose
Uptake/ glycogen storage
Expression of growth
promoting gene
(wound repair)
Fig 14.1 Three signal –transduction pathways. The binding of signaling molecules
to their receptors initiates pathways that lead to important physiological 3
response
Key Steps in Signal Transduction
•Release of the primary messenger
Hormone
•Reception of the primary messenger
(ligand)
Feedback pathways •Delivery of the message inside the
cell by the second messenger
Reception
cell-surface receptor
•Activation of effectors that directly
alter the physiological response
Amplification
•Termination of the Signal
Signal
Transduction
Response(s)
Fig 14.2 Principles of signal transduction
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Common Second Messengers
•Second messengers are intracellular molecules
– relay information from the receptor-ligand complex
– change in concentration in response to environmental
signals
Cyclic AMP
– Mediate the next step in the molecular information circuit
•Signal may be amplified
•Often free to diffuse throughout cell
•Common second messengers may create “cross talk”
Fig 14.3 common second messenger
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14.1 Heterotrimeric G Proteins Transmit
Signals and Reset Themselves
•Epinephrine
–A hormone secreted by the adrenal
glands of mammals in response to
internal and external stressor
–Signaling begins with ligand
(Epinephrine) bind to β-adrenergic
receptor (β-AR)
•A member of seven-transmembrane-helix
(7TM) receptors >20,000 receptors
serpentine (蜿蜒的,蛇的) receptors: the
single polypeptide chain "snakes"
Fig 14.4 The 7TM receptor through the membrane seven times
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14.1 Heterotrimeric G Proteins Transmit
Signals and Reset Themselves
•Rhodopsin 視紫質(Chapter 33)
–A well-characterized member of the 7TM receptor
family
–retina (視網膜) protein
•senses the presence of photons (ligand)
•initiate the signaling cascade responsible for visual
sensation
–lysine residue within rhodopsin is covalently
modified by a form of vitamin A 11-cis-retinal
•exposure to light induces the isomerization of 11-cisretinal to its all trans form, producing structural
changes in the receptor
Fig 14.5 (A) Structures of
•initiation of an action potential as visual stimulusrhodopsin
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•β2-adrenergic receptor
–Identified the structure by the inhibitor,
carazolol, competes with epinephrine
–Similarities with rhodopsin
–Mechanism
•Binding of a ligand from outside the cell
induces a structural rearrangement in the
part of the 7TM receptor that is positioned
inside the cell
Fig 14.5 (B) Structures of β2adrenergic receptor
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Ligand Binding to 7TM Receptors Leads to the
Activation of heterotrimeric G Proteins
G proteins, also known as guanine
•Epinephrine bind to β2nucleotide-binding proteins
adrenergic receptor
conformational change of
cytoplasmic domain
activated G protein
activate adenylate cyclase
catalyzed the conversion of
ATP into cyclic AMP (cAMP)
activate Protein kinase A
Fig 14.6 Activation of protein kinase A by9a
G-protein pathway
G Proteins Cycle Between GDP- and GTPBound Forms
•G protein
–Unactivated state: G protein is bound to GDP
•Heterotrimer consisting of α, β, and γ subunits
– α Subunit (Gα) binds the nucleotide
» A member of the P-loop NTPase family
» Participate in nucleotide binding
– α and γ subunit anchor to the membrane
Fig 14.7 A heterrtrimeric G
protein
–Hormone-bound receptor is catalyze the exchange of GTP
for bound GDP
•GTP binding induces conformational change
•Decreases Gα affinity for Gβγ Gα dissociation
• Gα can now bind to other proteins (adenylate cyclase)
 Gαs (s =stimulatory)
Because signal through G protein
7TM receptors are called G-protein coupled
receptors (GPCRs)
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•Gα binding to adenylate cyclase
–a membrane protein that contains 12
membrane-spanning helices
•two large cytoplasmic domains form
the catalytic part of the enzyme
–Converts ATP into cAMP
Fig 14.8 Adenylate cyclase
activation
Catalytic fragment
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What does the cAMP do?
•Increase concentration of cAMP can effect a wild range
of cellular processes:
–cAMP stimulates production of ATP for muscle
contraction
–Enhance the degradation of fuel stores
–Increase the secretion of acid by gastric mucosa
–Leads to the dispersion of melanin pigment granules
–Diminishes the aggregation of blood platelets
–Induces the opening of chloride channels
Most effect of cAMP in eukaryotic cells are mediated by the
activation of a single protein kinase: protein kinase A (PKA)
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•Protein kinase A
–Consists two regulatory (R) chain and two catalytic (C) chain
(Ch10)
•In the absence of cAMP inactive
•cAMP binds to the R chains releases the catalytic chains
 active
–Activated PKA then phosphorylates specific serine and
threonine in many targets to alter their activity
• PKA phosphorylates two enzymes that lead to the breakdown
of glycogen (Ch21)
•PKA stimulates the expression of specific genes by
phosphorylating a transcriptional activator called cAMP
response element binding (CREB) protein  gene expression
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Epinephrine signaling pathway
–On binding of ligand, the receptor
activates a G protein that in turn
activates the enzyme adenylate
cyclase.
–Adenylate cyclase generates the
second messenger cAMP.
– The increase in cAMP results in a
biochemical response to the initial
signal
Fig 14.9 Epinephrine signaling
pathway
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• How is the signal initiated by epinephrine switched off?
1. G proteins spontaneously reset themselves through GTP
Hydrolysis
• Intrinsic GTPase activity of Gα -- hydrolyze bound GTP to GDP and
Pi.
– the bound GTP acts as a built-in clock that spontaneously resets the
Gα subunit after a short time period.
– After GTP hydrolysis and release the Pi, the GDP-bound form of Gα
then reassociates with Gβγ to re-form the inactive heterotrimeric
protein.
Fig 14.10 Reseting Gα.
Intrinsic GTPase activity
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heterotrimeric protein
• How is the signal initiated by epinephrine switched off?
1. G proteins spontaneously reset themselves through GTP
Hydrolysis
2. Signal termination
1. Hormone dissociates, returning the receptor to its initial,
unactivated state
2. the hormone-receptor complex activates a kinase that
phosphorylates serine and threonine residues in the carboxylterminal tail of the receptor.
– Diminishes the ability to activate G protein
β-adrenergic receptor kinase
Fig 14.11 Signal termination. (G-protein receptor kinase 2, GRK2)
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Some 7TM receptors activate the
phosphoinositide cascade
•phosphoinositide cascade
–angiotensin II (血管緊縮素) (ligand, peptide hormone,
control blood pressure) -- angiotensin II receptor
–angiotensin II receptor activates Gαq protein
–GTP-form Gαq binds to activate the β isoform of
phospholipase C
•Catalyzes the cleavage of PIP2 into inositol 1,4,5-triphosphate
(IP3) and Diacylglycerol (DAG) which stays in the membrane
Fig 14.12 phospholipase C
reaction.
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•inositol 1,4,5-triphosphate (IP3)
– soluble and diffuses away from the
membrane
– causes the rapid release of Ca2+ from
intracellular stores in the
endoplasmic reticulum
•specific IP3 -gated Ca2+ -channel
proteins in the ER membrane open to
allow calcium ions to flow from the
ER into the cytoplasm
– Elevated level of cytoplasmic Ca2+
triggers smooth muscle contraction,
glycogen breakdown, and vesicle
release
Calcium is also a signal molecule: it can
bind proteins called calmodulin and
enzymes such as protein kinase C
Fig 14.13 phosphoinositide
cascade
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.
•Diacylglycerol (DAG)
–remains in the plasma membrane
– activates protein kinase C (PKC)
•phosphorylates serine and threonine
residues in many target proteins.
•the specialized DAG binding domains
of this kinase require bound calcium.
IP3 increases the Ca2+ concentration,
and Ca2+ facilitates the DAG-mediated
activation of protein kinase C.
Fig 14.13 phosphoinositide
cascade
Both IP3 and DAG act transiently because they
are
.
converted into other species by phosphorylation
or other processes.
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Ca2+ is a widely used second messenger
•Ca2+ participates in many signaling processes, the
properties:
1. Fleeting changes in [Ca2+] are readily detected
•intracellular levels are low to prevent the precipitation
of carboxylated and phosphorylated compounds
2. can bind tightly to proteins and induce substantial
structural rearrangements
•bind well to negatively charged oxygen atoms (side
chains of glutamate and aspartate) and uncharged
oxygen atoms (main-chain carbonyl groups and sidechain oxygen atoms from glutamine and asparagine).
•Ca2+ can coordinated multiple ligands -from six to eight
oxygen atoms- crosslink different protein segments and
induce significant conformational changes
Fig 14.14 calcium-binding site.
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•Scientists can monitor cellular [Ca2+] in real
–Fura-2 (molecular-imaging agents) can bind Ca2+ and change
their fluorescent properties on Ca2+ binding
•Fura-2 binds Ca2+ through appropriately positioned oxygen
atoms (in red) within its structure
•When Fura-2 introduced into cells, change in available Ca2+ by
detection changes in fluorescence
Fig 14.15 calcium imaging
Red: high Ca2+
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Calcium ion often activates the
regulatory protein calmodulin
•Calmodulin (CaM)
–a 17-kd protein with four Ca2+-binding sites
•Member of EF-hand protein family
– EF-hand is a Ca2+ binding motif: Helix, loop, helix motif
Fig 14.16 EF hands
2+
– 7 seven oxygen are coordinated to each Ca
–serves as a calcium sensor in nearly all eukaryotic cells
–At cytoplasmic concentrations above about 500 nM, Ca2+
binds to and activates calmodulin
•Conformational changes expose hydrophobic residues
•Newly exposed surfaces can bind other proteins, eg, CaM
kinase I, and stimulating them
 phosphorylate proteins
Calmodulin-dependent
protein kinase
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Fig 14.17 calmodulin binds to the α helices.
•Signal transduction pathway:
–Secondary messenger is increased : Ca2+
–The signal is sensed by a second-messenger-binding
protein: calmodulin
–Second-messenger-binding protein acts to generate
changes in enzyme : calmodulin-dependent kinases
•Phosphorylate many different proteins
– Regulate fuel metabolism (調節代謝)
– Ionic permeability (改變離子通透)
– Neurotransmitter synthesis (神經傳導物質的合成)
– Neurotransmitter release (神經傳導物質的釋放)
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14.2 Insulin signaling: Phosphorylation
cascades are central to many signaltransduction processes
•Signal transduction pathways initiated by
receptors
kinases as part of the receptor structures
Insulin signaling
Focus on “ leads to the mobilization of glucose
transporters to the cell surface”
•Insulin
–The hormone released in response to
increased blood- glucose levels
–a peptide hormone that consists of two
chains, linked by three disulfide bonds
Fig 14.18 insulin structure
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Insulin signaling
•Insulin receptor Receptor tyrosine kinase
–Dimer of two identical subunits (homodimer)
•Each unit consists of one chain and one chain linked
to one another by disulfide bond
–Two α subunits move together around a insulin
Fig 14.19 The insulin
–Each β subunit has a kinase domain similar to PKA
receptor
– Differs from PKA (protein kinase A)
• Is a tyrosine kinase
•The kinase is in an inactive conformation when the
domain is not covalently modified
– Requires tyr in activation loop be phosphorylated
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Insulin binding results in the cross-phosphorylation
and activation of the insulin receptor
•How is the activation loop phosphorylated?
–the two α subunits move together to surround one insulin
molecule, the kinase domains also draw closer together
–the two β subunits forced together, the kinase domain
catalyze the phosphoryl groups from ATP to tyrosine
residues in the activation loops -- conformational
change takes place – active conformation
Fig 14.20 Activation of the insulin
receptor by phosphorylation
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Activated insulin-receptor kinase
initiates a kinase cascade
•Insulin-receptor kinase phosphorylation sites act as
docking sites for other substrates, including insulinreceptor substrates (IRS)
–IRS-1 and IRS-2 are two homologous proteins with a
common modular structure
•pleckstrin homology domain: binds phosphoinositide lipids
•phosphotyrosine-binding domain
anchoring the IRS protein to the insulin receptor and the
membrane
• Tyr-X-X-Met (YXXM): four sequences are phosphorylated by
the insulin-receptor tyrosine kinase
Fig 14.22 the modular structure of insulinreceptor substrates IRS-1 and IRS-2
27
IRS act as adaptor protein!!
(not enzyme but serve to tether the
downstream components of this
signaling pathway to the membrane,
eg., phosphoinositide 3-kinase)
Fig 14.21 insulin signaling
Fig 14.25 insulin signaling pathway
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•lipid kinases have Src homology 2 (SH2)
domains
–Src homology 2 (SH2) domains bind to
specific phosphotyrosine domains in the IRS
protein
–Lipid kinases (also called phosphoinositide
3-kinase; PI3Ks ) function on phosphorylate
PIP2 to PIP3
Fig 14.23 Structure of the
SH2 domain
Fig 14.24 Action of a lipid kinase in insulin signaling
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•PIP3 activates a protein kinase PDK1 (with a pleckstrin
homology domain that specific for PIP3)
•PDK1 activates another protein kinase Akt
–Akt not membrane anchored, and moves through the cell
to phosphorylates targets
•control the trafficking of the glucose receptor GLUT4 to the
cell surface
•target enzymes that stimulate glycogen synthesis
30
Insulin signaling pathway
31
Effect of insulin on glucose uptake and
metabolism
•Insulin binds to its receptor, which in turn starts many
protein activation cascades, include:
– translocation of Glut-4 transporter to the plasma
membrane and influx of glucose
–Glycogen synthesis
–Glycolysis
–fatty acid synthesis
32
http://en.wikipedia.org/wiki/GLUT4
Insulin signaling is terminated by the
action of phosphatases
•In insulin signaling, three classes of enzymes are of
particular importance in shutting off the signaling
pathway:
–Protein tyrosine phosphatases: remove phosphoryl
groups from tyrosine residues on the insulin receptor and
the IRS adaptor proteins
–Lipid phosphatases: hydrolyze PIP3 to PIP2
–Protein serine phosphatases: remove phosphoryl groups
from activated protein kinases such as Akt
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14.3 EGF signaling: signal-transduction
systems are poised to respond
•EGF Signaling
–Signal molecule epidermal growth factor (EGF) binds to a
receptor tyrosine kinase (EGF receptor; EGFR) that
participates in cross-phosphorylation reactions
•Epidermal growth factor (EGF)
–a 6-kd polypeptide that stimulates the growth of
epidermal and epithelial cells
Fig 14.26 Structure of epidermal growth factor
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•EGF receptor
–A dimer of two identical subunit
•Exits as monomers until they bind EGF
–EGF-binding domain that lies outside the cell
•Dimerization is mediated by a dimerization arm
•the dimer binds two ligand molecules
–a single transmembrane helix-forming region
–the intracellular tyrosine kinase domain
•participates in cross-phosphorylation reactions
– One unit phosphorylated by another unit within a dimer
•Not within the activation loop of kinase
–the tyrosine-rich domain at the carboxyl terminus
•5 tyrosines are phosphorylated
Binding of EGF to the extracellular
domain causes the receptor to dimerize
and undergo cross-phosphorylation
and activation
Fig 14.27 Molecular structure of EGF receptor
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Fig 14.28 EGF receptor dimerization
•Why doesn’t the receptor dimerized
and signal in the absence of EGF?
–Conformational different
•The dimerization arm binds to a domain
within the same monomer
•Hold the receptor in a closed
configuration
•makes it unavailable for interaction with
the other receptor
Fig 14.29 Structure of the
unactivated EGF receptor
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EGF signaling leads to the activation of
Ras, a small G protein
•After EGF receptor phosphorylation
–The SH2 domain of an adaptor protein, Grb-2, binds to the
phosphotyrosine residues of the EGF receptor
–Grb-2 binds Sos using two Src homology 3 (SH3) domains
• SH3 domains bind proline-rich polypeptides
– Sos, in turn, binds to Ras and activates it
• Ras in a class of proteins called “small G proteins”
– GDP-Ras  GTP-Ras
•Sos as a guanine-nucleotide-exchange factor (GEF)
Structure of Grb-2,
an Adaptor Protein
Fig 14.30 Ras activation
37
mechanism
Activated Ras initiates a protein kinase
cascade
•GDP-Ras  GTP-Ras change conformation
–Binds other proteins, including Raf, a protein kinase
(protein-protein interaction)
–Raf then undergoes a conformational change that
activates its kinase domain
•Ras and Raf are anchored to membrane, through a
covalently bound isoprene lipid
–Raf phosphorylates other proteins, including the kinases
termed MEKs
–MEKs activate “extracellular signal-regulated kinases”
(ERKs)
–ERKS phosphorylate many substrates, including other
kinases and transcription factors
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Ras and Raf Signal Transduction
(or other extracellular signaling molecule)
Protein tyrosine
kinase domain
Dimer
activated
adapter
phosphorylation
MAPK/ERK kinase
phosphorylation
phosphorylation
Extracellular-signalregulated kinase
phosphorylation
Fig 12.36
Enhanced transcription
12-39
More cell division
EGF signaling
pathway
Fig 14.31 EGF signaling pathway
EGF signaling is terminated by protein
phosphatases and the intrinsic GTPase activity of
Ras
•protein phosphatases play key roles in the
termination of EGF signaling
•Signal activation also initiates signal termination
–Ras possesses intrinsic GTPase activity
–The activated GTP form of Ras spontaneously converts
into the inactive GDP form
–GTPase-activating proteins (GAPs) interact with small G
proteins in the GTP-bound form and facilitate GTP
hydrolysis
41
Small G proteins or small GTPases
•Difference between small G proteins and heterotrimeric G proteins
Size
small G proteins
heterotrimeric G proteins
20-25 kd
30-35 kd
Monomer
trimer
• Small G proteins have many key mechanistic and structural motifs
in common with the Gα subunit of the heterotrimeric G proteins 42
proteins are low-molecularThe GTPase cycle •RAS-family
weight guanine-nucleotide-binding proteins.
– inactive when bound to GDP
– active when bound to GTP
•Regulation of this molecular: through a
GDP-GTP cycle
– guanine nucleotide-exchange factors (GEFs)
• catalyse the exchange of GDP for GTP
Inactive form
– GTPase-activating proteins (GAPs)
• increase the rate of GTP hydrolysis to GDP
Active form
•In the case of RHO proteins, another layer
of regulation is provided by RHO–GDPdissociation inhibitors (RHOGDIs), which
sequester RHO away from the GDP–GTP
cycle.
•GTPases interact with various effector
proteins, which influence the activity
and/or localization of these effectors; this
ultimately influences cell-cycle progression
43
Nature Reviews Molecular Cell Biology 5, 355-366 (May 2004)
RAS signaling
44
Nature Reviews Molecular Cell Biology 13, 39-51 (January 2012)
14.4 Many elements recur with variation in
different signal-transduction pathways
• Signal transduction has many common themes
–Protein kinases are central to many signal-transduction
pathways
•protein kinases often phosphorylate multiple substrates and
able to generate a diversity of responses
–Second messengers participate in many signaltransduction pathways
–Specialized domains that mediate specific interactions are
present in many signaling proteins
•pleckstrin homology domain: facilitate protein interactions
with the lipid PIP3
•SH2 domain: mediate interactions with polypeptides
containing phosphorylated tyrosine residues
•SH3 domain: interact with peptide sequences that contain
multiple proline residues
45
14.5 Defects in signal-transduction pathways
can lead to cancer and other diseases
•Signal transduction pathways can malfunction, leading
to disease such as cancer
•For example:
–Rous sarcoma virus is a retrovirus that cause sarcoma in
chicken
•v-src genes is necessary for viral replication
– an oncogene and v-Src is a protein tyrosine kinase
–Normal chicken muscle cell: c-src
•Not induce cell transform proto-oncogene
•Encoded a signal transduction protein: regulates cell growth
46
•c-Src
–Tyrosine residue near the C-terminal
•When phosphorylated, it bound intramolecularly by the SH2
–the linker between the SH2 domain and the protein kinase
domain is bound by the SH3 domain
–These interactions hold the kinase domain in an inactive
conformation
•v-Src
–C terminal 19 amino acids are replaced (lacks tyrosine)
–Always active and promote unregulated cell growth
47
When Things Go Wrong…
•Not all oncogenes caused by viruses
•A mutated form of Ras can cause cancer
– Usually mutations lead to loss of hydrolysis activity
–If GTP cannot be hydrolyzed to GDP, stuck in the “on”
position, stimulating cell growth
•Mutations of “tumor suppressor genes” can cause
cancer
– E.g., mutations in genes that code for phosphatases
involved in EGF signal termination
EGF signaling persists initiated, stimulating inappropriate
cell growth
48
Monoclonal antibodies can be used to inhibit
signal-transduction pathways activated in tumors
•Mutated/overexpessed receptor tyrosine kinases often
observed in tumors
–Epidermal-growth-factor receptor (EGFR) is
overexpressed in some human epithelial cancers,
including breast, ovarian, and colorectal cancer
–Some EGFR can dimerize and send growth signal even in
absence of EGF
• Cetuximab (Erbitux) an antibody used therapeutically to
prevent dimerization in colorectal cancer
–EGFR family member, Her2 overexpressed in~30% of
breast cancers
•Trastuzumab (Herceptin) an antibody used to inhibit breast
cancer
49
Protein kinase inhibitors may be
effective anticancer drugs
•Protein kinase inhibitors as anticancer
drugs
– Chronic myelogenous leukemia (CML,慢
性骨髓性白血病) often due to
chromosomal defect where parts of
chromosomes 9 and 22 are translocated,
causing overexpression of a kinase (BcrAbl kinase)
• An inhibitor specific for this kinase,
Gleevec (STI-571, imatinib mesylate) very
effective treatment
Fig 14.33 The formation of the bcr-abl
gene by translocation
50
DNA Damage and Repair
慢性骨髓性白血病 (chronic
myelogenous leukemia) 的病生理機轉最
重要的就是第9對以及第22對染色體轉位，
t(9:22)，又稱為費城染色體(Philadelphia
Chromosome)。
這種染色體轉位會造成原來位在第9對染色
體的Abelson (ABL) proto-oncogene接到
第22對染色體的breakpoint cluster
region (BCR) 基因上，形成BCR-ABL
chimeric 基因。正常的ABL 基因在轉錄轉
譯後產生的tyrosine kinase會受到嚴密的
調控；但發生費城染色體所形成的BCR-ABL
fusion基因則失去正常的調控機轉，造成
tyrosine kinase過度表現的情形。在慢性
骨髓性白血病患者，有大於 90% 的患者會
有費城染色體，有大於 95% 的患者可以利
用PCR的方式找到BCR-ABL 基因。
51
Cholera霍亂and whooping cough百日咳are
due to altered G-protein activity
•Chloera toxin- Chloeragen is secreted by the intestinal
bacterium Vibrio cholerae
–Two functional unit– α β subunit that binds to GM1
ganglisoides of the intestinal epithelium
–Catalytic A subunit that enters the cells
•A subunits catalyzes the covalent modification of a Gαs protein
– The α subunit is modified by the attachment of an ADP-ribose to an
arginine
– Stabilizes the GTP-bound form of Gαs  active form
continuously activates protein kinase A
opens a chloride channel and inhibit sodium absorption
• whooping cough – pertussis toxin from Bordetella
pertussis
– Adds an ADP-ribose moiety to Gαi, inhibit adenylate cyclase,
close Ca2+ channel, open K+ channel, function “off”
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