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cellular signal transduction pathway

G protein-mediated signal transduction

Tyrosine protein kinase-mediated signal
transduction

Guanylate cyclase signal transduction pathway

Nuclear receptor-mediated signal transduction
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G Protein Signal Cascade
G


Effector
Signal
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3
G-protein-coupled receptors
Binding of ligands
Extracellular
-NH2
-S-S-
e1
TM1
TM2
e3
e2
TM3
TM4
TM5
TM6
TM7
i2
i1
Cytoplasmic
i3
Location of G
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COOH
-
4
G-protein subunits
• Three subunits




4 superfamilies
 binds GTP



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Diversity of G Protein-Coupled Receptor Signal
Transduction Pathways
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9
A G-protein that
is part of a
pathway that
stimulates
Adenylate
Cyclase is called
Gs & its a
subunit Gsa.
hormone
signal
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
ATP cAMP + PPi
hormone
signal
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
ATP cAMP + PPi
The sequence of events by which a hormone
activates cAMP signaling:
1. Initially Ga has bound GDP, and αβ& γ
subunits are complexed together.
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11
hormone
signal
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
ATP cAMP + PPi
2. Hormone binding to a 7-helix receptor (GPCR) causes a
conformational change in the receptor that is
transmitted to the G protein.
The nucleotide-binding site on Ga becomes more
accessible to the cytosol, where [GTP] > [GDP].
Ga releases GDP & binds GTP (GDP-GTP exchange).
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hormone
signal
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
ATP cAMP + PPi
3. Substitution of GTP for GDP causes another
conformational change in Ga.
Ga-GTP dissociates from the inhibitory βγ complex & can
now bind to and activate Adenylate Cyclase.
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hormone
signal
outside
GPCR
plasma
membrane

AC
GDP GTP
GTP
GDP
cytosol
ATP cAMP + PPi
4. Adenylate Cyclase, activated by Ga-GTP,
catalyzes synthesis of cAMP.
5. Protein Kinase A (cAMP Dependent Protein
Kinase) catalyzes phosphorylation of various
cellular proteins, altering their activity.
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Protein Kinase A
(cAMP-Dependent Protein Kinase)
transfers Pi from ATP to OH of a Ser or Thr in
a particular 5-amino acid sequence.
Protein Kinase A in the resting state is a
complex of:
•
2 catalytic subunits (C)
•
2 regulatory subunits (R).
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R2C2
15
The domain of (R) binds to the active site of
(C), blocking its activity.
When each (R) binds 2 cAMP, a conformational
change causes (R) to release (C).
Each catalytic subunit can then catalyze
phosphorylation of Ser or Thr on target
proteins.
R2C2 + 4 cAMP  R2cAMP4 + 2 C
R
R
C
R
R
C
PKA
cAMP(
)
+
C
ATP
++
Mg
µ°°×ÖÊ
µ°°×ÖÊ
Á×Ëáø
P
ÉúÎïЧӦ
Effects of PKA
Ser/Thr残基磷酸化
（1）对代谢的调节作用
肾上腺素调节糖原分解
（2）对基因表达的调节作用
cAMP应答元件（CRE）基因调控区
cAMP应答元件结合蛋白（CREB）反式作用因子
PKA的催化亚基进入细胞后使CERB特定的Ser/Thr磷
酸化，形成同源二聚体，结合DNA，激活转录
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ÅäÌå
ÊÜÌå
AC
Gs
ATP
cAMP
PKA
P
N
DBD
ת¼»î»¯Óò
C
121£¬133£¬
156
CREB
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serine (Ser)
threonine (Thr)
H
H
H3N+
C
COO
H3N+
C
COO
CH2
CH OH
OH
CH3
Many enzymes are regulated by covalent
attachment of phosphate, in ester linkage, to
the side-chain hydroxyl group of a particular
amino acid residue (serine, threonine, or
tyrosine).
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O
Protein Kinase
OH + ATP
Protein
Protein
O
P
O + ADP
O
Pi
H2O
Protein Phosphatase


A protein kinase transfers the terminal
phosphate of ATP to a hydroxyl group on a
protein.
A protein phosphatase catalyzes removal of
the Pi by hydrolysis.
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O
Protein Kinase
OH + ATP
Protein
Protein
O
P
O + ADP
O
Pi
H2O
Protein Phosphatase
Protein kinases and phosphatases are themselves
regulated by complex signal cascades. For
example:


Some protein kinases are activated by Ca2+calmodulin.
Protein Kinase A is activated by cyclic-AMP (cAMP).
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Turn off of the signal:
1. Ga hydrolyzes GTP to GDP + Pi. (GTPase).
The presence of GDP on Ga causes it to rebind
to the inhibitory βγ complex.
Adenylate Cyclase is no longer activated.
2. Phosphodiesterase catalyzes hydrolysis of
cAMP  AMP.
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Phosphodiesterase enzymes catalyze:
cAMP + H2O  AMP
The phosphodiesterase that cleaves
cAMP is activated by phosphorylation
catalyzed by Protein Kinase A.
Thus cAMP stimulates its own
degradation, leading to rapid turn off of
a cAMP signal.
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Cyclic Nucleotide Metabolism - cAMP
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Turn off of the signal :
3. Hormone receptor desensitization occurs.
This process varies with the hormone.


Some receptors are phosphorylated via G-proteincoupled receptor kinases.
The phosphorylated receptor may then bind to a
protein arrestin that blocks receptor-G-protein
activation & promotes removal of the receptor from
the membrane by clathrin-mediated endocytosis.
4. Protein Phosphatase catalyzes removal by
hydrolysis of phosphates that were attached to
proteins via Protein Kinase A.
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Phosphatidylinositol Signal Cascades
Some hormones activate a signal cascade based
on the membrane lipid phosphatidylinositol.
phosphatidylinositol-4,5-bisphosphate
(PIP2).
PIP2 is cleaved by
the enzyme Phospholipase C.
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Different isoforms of PLC have different
regulatory domains, & thus respond to
different signals.
One form of Phospholipase C is activated by a
G-protein, Gq.
A GPCR (receptor) is activated. GTP
exchanges for GDP.
Gqa-GTP activates Phospholipase C.
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Cleavage of PIP2, catalyzed by Phospholipase
C, yields two second messengers:
inositol-1,4,5-trisphosphate (IP3)
diacylglycerol (DG).
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Á×Ö¬õ£¼¡´¼
PI
H + R
Á×Ö¬õ£¼¡´¼-4,5-Ë«Á×Ëá
Gµ°°×
½éµ¼
¼¤»î
PI2P
CH2O OCR1
CHO OCR2
Á×֬øC Ë®½â CH2O P O
PLC
OH OH
PO
O P
OH
CH2O OCR1
CHO OCR2
¶þÖ¬õ£¸ÊÓÍ
DG
ÈýÁ×Ëἡ´¼
IP3
P
CH2OH
O
OH OH
PO
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OH
O P
31
Diacylglycerol,
with Ca++,
activates Protein
Kinase C,
which catalyzes
phosphorylation of
several cellular
proteins, altering
their activity.
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Functions of PKC
1 对代谢的调节作用
Ser/Thr残基磷酸化
靶蛋白：质膜受体、膜蛋白和多种酶
2 对基因表达的调节作用
早期反应: expression of immediate-early gene
晚期反应
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Ca++
Ca++-release channel
IP3
Ca
ATP
calmodulin
Ca
++
endoplasmic
reticulum
Ca++-ATPase
ADP + Pi
++
IP3 activates Ca++-release channels in ER membranes.
Ca++ stored in the ER is released to the cytosol, where it
may bind calmodulin, or help activate Protein Kinase C.
Signal turn-off includes removal of Ca++ from the cytosol
via Ca++-ATPase pumps, & degradation of IP3.
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Ca2+-Calmodulin dependent protein
kinases (CaM kinase)
Extensive substrate for CaM kinase
Phosphorylation of Ser/Thr residues
Activate AC, PDE, Nos, Ca2+-ATPase
IP3 receptor
PTP
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IP3 may instead be phosphorylated via specific
kinases, to IP4, IP5 or IP6. Some of these have
signal roles.
E.g., the IP4 inositol-1,3,4,5-tetraphosphate in
some cells activates plasma membrane Ca++
channels.
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Sequential dephosphorylation of IP3 by enzymecatalyzed hydrolysis yields inositol, a substrate
for synthesis of PI..
PI 3-Kinases instead catalyze phosphorylation
of phosphatidylinositol at the 3 position of the
inositol ring.
PI-3-P, PI-3,4-P2, PI-3,4,5-P3, & PI-4,5-P2 have
signaling roles.
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G-protein subtypes
Gi/o


Gs


Gq

G12/13



 inhibition
of cAMP
production
 mediates
signalling
 increased
activation
synthesis
of PLC
cAMPleading
to between GPCR
and RhoA (GTPase)
2+ channels
 inhibition
of
Ca
activation
activation
of Ca2+ andofK+PKC
channels
(DAG)
 function under investigation
+ channels
activation of GIRK
K
intracellular Ca2+ release (IP )
3
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GPCR signalling
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N
I
II
III
IV
V
VI
the signal transduction machine
VII
C
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N
I
II
III
IV
V
VI
the signal transduction machine
VII
 
G
P P
A
rr
SRC
effec
to
rs
C
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
T
h
e
s
e
c
a
n
b
e
c
o
n
s
i
d
e
r
e
d
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
.
Www.ggene.Cn
43
N
I
II
III
IV
V
VI
the signal transduction machine
VII
 
G
p
P
R
O
ho
m
er
S
H
3
P P
A
rr
r
me
o
h
IP
3
R
SRC
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
C
effec
to
rs
2
+
C
a
Www.ggene.Cn
44
N
I
II
III
IV
V
VI
the signal transduction machine
VII
 
G
p
P
R
O
p
P
R
O
S
H
3
S
H
3
ho
m
er
Nck
2
Grh
P P
A
rr
r
me
o
h
M
A
P
K
IP
3
R
SRC
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
C
effec
to
rs
2
+
C
a
Www.ggene.Cn
45
N
I
II
III
IV
V
VI
the signal transduction machine
VII
 
G
PLC
T
R
P
p
P
R
O
p
P
R
O
S
H
3
S
H
3
ho
m
er
Nck
2
Grh
PKC
P P
A
rr
r
me
o
h
M
A
P
K
IP
3
R
SRC
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
C
P
D
Z
effec
to
rs
2
+
C
a
In
aD
Www.ggene.Cn
46
N
the signal transduction machine
+
+
N
a
/H
e
x
c
h
a
n
g
e
r
I
II
III
IV
V
VI
VII
 
G
PLC
T
R
P
p
P
R
O
p
P
R
O
S
H
3
S
H
3
ho
m
er
Nck
2
Grh
PKC
P P
A
rr
r
me
o
h
M
A
P
K
N
F
IP
3
R
SRC
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
C
R
HE
C
P
D
Z
effec
to
rs
2
+
C
a
In
aD
Www.ggene.Cn
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N
the signal transduction machine
+
+
N
a
/H
e
x
c
h
a
n
g
e
r
I
II
III
IV
V
VI
VII
 
G
PLC
T
R
P
p
P
R
O
p
P
R
O
S
H
3
S
H
3
ho
m
er
Nck
2
Grh
PKC
P P
A
rr
r
me
o
h
M
A
P
K
N
F
IP
3
R
SRC
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
C
R
HE
C
P
D
Z
effec
to
rs
2
+
C
a
C
In
aD
cN
OS
NO
Www.ggene.Cn
a
r
g
i
n
i
n
e
48
N
the signal transduction machine
+
+
N
a
/H
e
x
c
h
a
n
g
e
r
I
II
III
IV
V
VI
VII
 
G
PLC
?
p
P
R
O
S
O
N
e
S
H
3
r
me
o
h
C
N
R
HE
F
IP
3
R
SRC
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
S
H
3
P P
A
rr
M
A
P
K
T
R
P
p
P
R
O
ho
m
er
Nck
2
Grh
PKC
C
P
D
Z
effec
to
rs
2
+
C
a
C
In
aD
cN
OS
NO
Www.ggene.Cn
a
r
g
i
n
i
n
e
49
N
the signal transduction machine
+
+
N
a
/H
e
x
c
h
a
n
g
e
r
I
II
III
IV
V
VI
VII
 
G
N
P
x
x
Y
ARF
?
ho
m
er
r
me
o
h
C
N
R
HE
F
IP
3
R
SRC
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
S
H
3
P P
A
rr
M
A
P
K
T
R
P
p
P
R
O
S
O
N
e
S
H
3
PKC
Rho
p
P
R
O
Nck
2
Grh
PLC
PLD
C
P
D
Z
effec
to
rs
2
+
C
a
C
In
aD
cN
OS
NO
Www.ggene.Cn
a
r
g
i
n
i
n
e
50
N
the signal transduction machine
+
+
N
a
/H
e
x
c
h
a
n
g
e
r
I
II
III
IV
V
VI
VII
 
G
N
P
x
x
Y
ARF
?
ho
m
er
S
H
3
P P
A
rr
r
me
o
h
M
A
P
K
T
R
P
p
P
R
O
S
O
N
e
S
H
3
PKC
Rho
p
P
R
O
Nck
2
Grh
PLC
PLD
C
N
R
HE
F
IP
3
R
SRC
g
e
n
e
r
a
l
m
e
c
h
a
n
i
s
m
s
C
P
D
Z
effec
to
rs
2
+
C
a
C
In
aD
cN
OS
I
n
s
u
m
m
a
r
y
.
.
.
.
.
GPCRs are truly remarkable signal transduction machines.
Www.ggene.Cn
NO
a
r
g
i
n
i
n
e
51
Small GTP-binding proteins include

Ras (growth factor signal cascades).
Rho (regulation of actin cytoskeleton)

Rab (vesicle targeting and fusion).



ARF (forming vesicle coatomer coats).
Ran (transport of proteins into & out of the
nucleus).
All GTP-binding proteins differ in conformation
depending on whether GDP or GTP is present at their
nucleotide binding site.
Generally, GTP binding induces the active state.
Most GTP-binding
proteins depend on
helper proteins
GAPs
GTPase Activating
Proteins, promote
GTP hydrolysis.
protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
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GEFs, Guanine
Nucleotide Exchange
Factors, promote
GDP/GTP exchange.
The activated receptor
(GPCR) serves as GEF
for a heterotrimeric G
protein.
protein-GTP (active)
GDP
GEF
GTP
GAP
Pi
protein-GDP (inactive)
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54
cGMP-蛋白激酶途径
鸟苷酸环化酶（GC）
GC的激活间接地依靠Ca2+
GTP
H
GC
R
cGMP
cGMP-依赖性蛋白激酶
蛋白激酶G
（一个cGMP结合位点）
效应蛋白磷酸化
NO作用机理：肌肉细胞中激活GC→蛋白激
酶G
Cyclic Nucleotide Metabolism - cGMP
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Cyclic Nucleotide Metabolism – cGMP
Cyclic nucleotides have been extensively studied as second messengers of
intracellular events initiated by activation of many types of hormone and
neurotransmitter receptors. Cyclic guanosine monophosphate (cGMP) serves
as a second messenger in a manner similar to that observed with cAMP.
Peptide hormones, such as the natriuretic factors, activate receptors that
are associated with membrane-bound guanylate cyclase (GC). Receptor
activation of GC leads to the conversion of GTP to cGMP. Nitric oxide (NO)
also stimulates cGMP production by activating soluble GC, perhaps by binding
to the heme moiety of the enzyme. Similar to cAMP, cGMP mediates most of
its intracellular effects through the activation of specific cGMP dependent
protein kinases (PKG). Several families of phosphodiesterases (PDE-I-VI)
act as regulatory switches by catalyzing the degradation of cGMP to
guanosine-5’-monophosphate (5’-GMP). PDE I is stimulated by Ca2+ calmodulin, perhaps through phosphorylation by PKA. PDE II is a low affinity
PDE that can cleave both cAMP and cGMP. The activity of PDE II is
stimulated by cGMP. PDE III is inhibited by cGMP while PED V binds to
cGMP and plays a role in regulating smooth muscle contraction. PDE VI is a
high affinity PDE specifically localized in photoreceptors that is selective
for cGMP.
TPK Pathways
酪氨酸蛋白激酶 TPK
受体型TPK (TPK-Ras-MAPK)
细胞质膜 催化型受体
胰岛素、表皮生长因子、某些原癌基因
非受体型TPK 胞浆
配体与单跨膜螺旋受体结合
催化型受体二聚化 ，二聚体的TPK被激活（自
身磷酸化）
非催化型受体的酪氨酸残基被非受体型TPK磷
酸化
• Signal transduction through the MAPK
pathways
• Protein-protein interactions
• Protein kinase cascades
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Signal Transduction by the Mitogenic Pathways
• Cells in an organism receive a variety of
extracellular stimuli for cell proliferation.
• Signal transduction is the intracellular
events (protein-protein interaction and
phosphorylation) that convey extracellular
stimuli into specific cellular responses.
• The best characterized mitogenic pathway is
the mitogen-activated protein kinase (MAPK)
cascades, involved in mitogenic and stress
response signalling.
The MAPK signalling pathways
• The MAPK pathway:
MAPK/ERK (external-signal regulated kinase)
JNK (C-Jun N-terminal kinase)/SAPK (stress
activated protein kinase)
P38MAPK
ERK5/BMK1 (big mitogen-activated protein kinase)
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The MAPK signalling pathways
• The RAS-activated MAPK pathway:
RAS-RAF-MEK-MAPK
represents the first example where all the
steps in a complete signalling cascade from
the cell surface receptor PTK, to the
nuclear transcription is known.
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The MAPK signalling pathways
• Many growth stimulation converges on the
kinase cascade that activates the MAPK
(also called ERK1 and ERK2 = extracellular
signal-regulated kinase 1 and 2).
• Upon activation, MAPK then translocate
into the nucleus and induce the transcription
of the immediate early genes (e.g. MYC).
Note:
Immediate early genes: genes that are expressed early during
stimulation. They are usually transcription factors
RAS-RAF-MEK-MAPK
• Ligand binds receptor PTK
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RAS-RAF-MEK-MAPK
• Ligand binds receptor PTK
• Autophosphorylation on tyrosine
P
P
P
P
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RAS-RAF-MEK-MAPK
• Ligand binds receptor PTK
• Autophosphorylation on tyrosine
• GRB2 (a SH2- and SH3containing protein) binds to the
receptor phosphotyrosine motif YV/L-N-X via its SH2 domain
P
P
SOS
P
P
GRB2
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RAS-RAF-MEK-MAPK
• Ligand binds receptor PTK
• Autophosphorylation on tyrosine
• GRB2 (a SH2- and SH3containing protein) binds to the
receptor phosphotyrosine motif YV/L-N-X via its SH2 domain
• The SH3 of GRB2 binds
constitutively to the proline-rich
sequence in the C-terminus of SOS
(a guanine nucleotide exchange
factor for RAS).
P
P
SOS
P
P
GRB2
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RAS-RAF-MEK-MAPK
GRB2
• 24 kDa adaptor molecule.
• Only contains an SH2
domain between two SH3
domains
P
P
SOS
P
P
GRB2
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RAS-RAF-MEK-MAPK
GRB2
• The SH2 of GRB2 binds
to phosphotyrosine in a
number of receptor PTK:
for EGF, PDGF, colonystimulating factor-1 (CSF1), stem cell factor (SCF);
 all lead to the activation
of RAS pathway.
i.e. GRB2 functions as a
point of convergence for
Ras-activated signaling
pathways.
P
P
SOS
P
P
GRB2
• Recruitment of SOS to the
close proximity of RAS in the
membrane
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RAS-RAF-MEK-MAPK
SOS
• 180 kDa guanine
nucleotide exchange
factor for RAS; stimulate
the formation of active
GTP-bound RAS.
• SOS belongs to the DBL
family of oncogenes (14
members). Most of these
activate the RHO family of
GTP-binding proteins
involved in cytoskeletal
reorganization. SOS is
one of the few known
member of the family for
RAS.
P
RAS
P
GDP
SOS
P
P
GRB2
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RAS-RAF-MEK-MAPK
P
• RAS becomes activated by
exchanging GDP for GTP
RAS
P
GTP
GDP
SOS
P
P
GRB2
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RAS-RAF-MEK-MAPK
RAS
• 21 kDa GTPase protein.
• A special case of G protein
monomer
• GTP-bound form: active
GDP-bound form: inactive.
• originally identified as oncogenes
in several retroviruses, and are
activated in a variety of human
tumors.
P
RAS
P
GTP
SOS
P
P
GRB2
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RAS-RAF-MEK-MAPK
RAS
• Oncogenic forms of RAS
often have point mutations
that lock RAS in the active
GTP-bound form.
Evidence: Cells were induced
to proliferate by + PDGF and
EGF. Microinjection of antiRAS antibodies into the cells
blocked the cell proliferation.
Microinjection of a
constitutively active mutant of
RAS caused cells to proliferate
in the absence of PDGF and
EGF.
P
RAS
P
GTP
SOS
P
P
GRB2
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RAS-RAF-MEK-MAPK
P
• The RAS-GTP effector domain
interacts with the N-terminal
regulatory region of the RAF
(serine/threonine protein kinase),
hence recruiting RAF to the
membrane
RAS
P
SOS
P
GTP
RAF
P
GRB2
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RAS-RAF-MEK-MAPK
RAF
• RAF is a serine/
threonine protein kinase
• RAF is a MAPK kinase
kinase (MAPKKK).
P
• Can transform cells when
constitutively active or
when overexpressed.
P
RAS
P
SOS
GTP
RAF
P
GRB2
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RAS-RAF-MEK-MAPK
RAF
• The 14-3-3 family of
scaffold proteins interacts
constitutively with RAF via
the phosphorylated
Ser621 in RAF
Both 14-3-3 and RAS may
be required for activation
of RAF.
P
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
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RAS-RAF-MEK-MAPK
RAF
• RAS recruits RAF to the
membrane. Membrane
targeting of RAF is
necessary to fully activate
RAF.
(Expression of mutant RAF
that cannot bind RAS  no
stimulation of cell
proliferation by a
constitutively active RAS).
P
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
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RAS-RAF-MEK-MAPK
P
• Activation of RAF
(most likely by phosphorylation of
RAF and binding to the scaffold
protein 14-3-3)
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
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RAS-RAF-MEK-MAPK
P
• Activated RAF in turn activates
MEK (also called MAPK kinase) by
phosphorylation on two conserved
serine residues in MEK.
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
P P
MEK
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RAS-RAF-MEK-MAPK
P
• Activated RAF in turn activates
MEK (also called MAPK kinase) by
phosphorylation on two conserved
serine residues in MEK.
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
P P
MEK
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RAS-RAF-MEK-MAPK
MEK
• Also called MAPK kinase
(MAPKK).
• Phosphorylated on Ser218
and Ser222 by activated
RAF.
• Mutation of MEK that
leads to constitutive
activity (by replacing the
two Ser with glutamic acids
or aspartic acids - by mimic
phosphorylation)  MAPK
activation, mitogenicity, and
cellular transformation.
P
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
P P
MEK
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RAS-RAF-MEK-MAPK
P
• Activated MEK activates MAPK (a
serine/threonine protein kinase) by P
phosphorylation of conserved
threonine and tyrosine residues.
RAS
P
GTP
14-3-3
SOS
RAF
P
GRB2
P P
MEK
P P
MAPK
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RAS-RAF-MEK-MAPK
P
• Activated MEK activates MAPK (a
serine/threonine protein kinase) by P
phosphorylation of conserved
threonine and tyrosine residues.
RAS
P
GTP
14-3-3
SOS
RAF
P
GRB2
P P
MEK
P P
MAPK
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RAS-RAF-MEK-MAPK
MAPK
• Five isoforms of ERK have
been identified, but ERK1
and ERK2 have been most
studied.
P
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
P P
MEK
P P
MAPK
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RAS-RAF-MEK-MAPK
MAPK
• MAPK activation is biphasic:
a transient peak within 5-10
min, and a sustained peak
lasting several hours. The
different activation kinetics
 different cellular response
(EGF induces only the
transient response and
stimulate cell growth; but
NGF (nerve growth factor)
induces the sustained
response and stimulate
differentiation).
P
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
P P
MEK
P P
MAPK
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RAS-RAF-MEK-MAPK
MAPK
• Inactivation of MAPK is
achieved by several
phosphatases: the
serine/threonine
phosphatase PP2A
P
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
P P
MEK
P P
MAPK
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RAS-RAF-MEK-MAPK
• Activated MAPK phosphorylates a
number of substrates in the
cytoplasm
P
RAS
P
14-3-3
SOS
P
GTP
RAF
P
GRB2
Substrates P
P P
MEK
P P
MAPK
Substrates P
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RAS-RAF-MEK-MAPK
• Activated MAPK phosphorylates a
number of substrates in the cytoplasm
it also translocated into the
nucleus(within min) where it
phosphorylates nuclear transcription P
factors.
P
P
P
RAS
GTP
14-3-3
SOS
GRB2
RAF
P P
MEK
P P
MAPK
Substrates
RAS-RAF-MEK-MAPK
• Activated MAPK phosphorylates a
number of substrates in the cytoplasm
it also translocated into the
nucleus(within min) where it
phosphorylates nuclear transcription P
factors.
P
P
P
RAS
GTP
14-3-3
SOS
GRB2
RAF
P P
MEK
P P
MAPK
Substrates
RAS-RAF-MEK-MAPK
• Activated MAPK phosphorylates a
number of substrates in the cytoplasm
it also translocated into the
nucleus(within min) where it
phosphorylates nuclear transcription P
factors.
P
P
P
RAS
GTP
14-3-3
SOS
GRB2
RAF
P P
MEK
P P
MAPK
Substrates
RAS-RAF-MEK-MAPK
• Activated MAPK phosphorylates a
number of substrates in the cytoplasm
it also translocated into the
nucleus(within min) where it
phosphorylates nuclear transcription P
factors.
P
P
P
RAS
GTP
14-3-3
SOS
GRB2
RAF
P P
MEK
P P
Substrates P
MAPK
RAS-RAF-MEK-MAPK
• Activated MAPK phosphorylates a
number of substrates in the cytoplasm
it also translocated into the
nucleus(within minutes) where it
phosphorylates nuclear transcription P
factors.
P
P
P
 Transcription of genes important for
cell proliferation.
RAS
GTP
14-3-3
SOS
GRB2
P P
MEK
P P
Substrates P
RAF
MAPK
Transcription Factor Regulation (CREB, Elk-1 and c-Fos)
SIGMA-ALDRICH
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Transcription Factor Regulation (CREB, Elk-1 and c-Fos)
The transcription factor CREB binds to the cAMP response element
(CRE) and activates gene transcription in response to a wide variety of
extracellular signals (including growth factors, hormones, and
neurotransmitters). Transcriptional activation of CREB is controlled
through phosphorylation at Ser133 by p90Rsk and the p44/42 MAP kinase.
The transcriptional activity of the proto-oncogene c-Fos has been
implicated in cell growth, differentiation, and development. Fos is induced
by many stimuli, ranging from mitogens to pharmacological agents. c-Fos
has been shown to be associated with another proto-oncogene, c-Jun,
and together they bind to the AP-1 binding site to regulate gene
transcription. Like CREB, c-Fos is regulated by p90Rsk. Elk-1 is a
transcription factor that is activated by the MAP kinase-mediated
phosphorylation of a Ser/Thr cluster at the carboxyl terminus. Activated
Elk-1 binds to the serum response element (SRE) to induce gene
transcription in response to serum and growth factors. Recent studies
have also demonstrated that Elk-1 is a target for the stress-activated
kinase SAPK/JNK.
Substrates of MAPK:
•MAPK phosphorylates certain motif.
•In the cytoplasm, MAPK phosphorylates its upstream
components in a negative feedback loop - MAPK
phosphorylates SOS, RAF, MEK  inhibition of MAP
kinase pathway.
•In the nucleus, MAPK phosphorylates a number of
transcription factors (e.g. Elk1) increase transcription
(e.g. of c-Fos mRNA).
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JNK pathway
STRESS
• JNK translocation into the
nucleus
 phosphorylation of the
transcription factor c-JUN at
the N-terminal residues (Ser63
and Ser73)
Rac1/Cdc42 GTP
PAK
 activation of transcription
by c-JUN
P P
MEKK1-3
P P
MEK4
P P
P
c-JUN
JNK
STRESS
P
Ras
P
Rac1/Cdc42 GTP
14-3-3
Sos
P
GTP
Raf
P
P P
MEKK1-3
PAK
Grb2
P P
P P
MEK
MEK4
P P
P P
MAPK
JNK
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Specificity of MAP kinase pathways
• When cells are treated with mitogenic agents (e.g.
growth factors), MAPK (ERK) become strongly
activated but JNK is poorly activated.
• Conversely, when cells are challenged with stress,
JNK is activated but MAPK (ERK) is only weakly
activated.
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JAK(just another kinase, Janus protein tyrosine kinase)
-STAT(signal transducer and activator of transcription)
GF、IFN、EPO、G-CSF、IL-2、IL-6
其受体分子缺乏酪氨酸蛋白激酶活性，借助细胞内的一类具有
激酶结构的连接蛋白JAKs完成信息转导
配体与非催化型受体结合后，能活化各自的JAKs，再通过激活
信号转导子和转录激动子（STAT）而影响基因的转录调节
The JAK/STAT Signaling Pathway
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The JAK/STAT Signaling Pathway
A wide variety of extracellular signals activate the STAT (signal
transducers and activators of transcription) class of transcription
factors. Many cytokines, lymphokines, and growth factors signal
through a related superfamily of cell surface receptor tyrosine kinases
that are associated with and activate Janus kinases (JAKs). Ligandinduced dimerization of the receptor induces the reciprocal tyrosine
phosphorylation of the associated JAKs, which, in turn, phosphorylates
tyrosine residues on the cytoplasmic tail of the receptor. These
phosphorylated tyrosines serve as docking sites for the Src Homology2 (SH-2) domain of the STAT protein, and JAK catalyzes the tyrosine
phosphorylation of the receptor-bound STAT. Phosphorylation of STAT
at a conserved tyrosine residue induces SH-2-mediated homo- or
heterodimerization, followed by translocation of the STAT dimer to the
nucleus. STAT dimers bind to specific DNA response elements in the
promoter region of target genes to activate gene expression. APS
(adaptor molecule containing pleckstrin homology and SH-2 domains)
can inhibit the JAK- STAT pathway by binding to the cytoplasmic
domain of the receptor where it is phosphorylated (activated) by JAK.
Activated APS binds to c-Cbl and blocks STAT activation.
Nuclear Receptor Superfamily
信息分子：糖皮质激素、盐皮质激素、雄
激素、孕激素、雌激素、甲状腺素、
1，25-(OH)2-D3
核内受体：雄激素、孕激素、雌激素、甲
状腺素
胞浆受体：糖皮质激素
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Glucocorticoid Receptor Signaling
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Glucocorticoid Receptor Signaling
The glucocorticoid hormone, cortisol, passes through the plasma
membrane into the cytoplasm where it binds to the specific, highaffinity glucocorticoid receptor (GR). The resulting complex is the
non-DNA-binding oligomer of the GR, in which the receptor is
complexed with other proteins. In this complex, the DNA-binding
domain of the receptor is bound by the heat shock protein 90
(hsp90) dimer. Other proteins in this complex include heat shock
protein 70 (hsp70) and FKBP52. Dissociation of the oligomeric
complex yields the free cortisol-receptor subunit in the DNAbinding form. The activated receptor forms a homodimer and is
translocated to the nucleus through the nucleopore. Inside the
nucleus, the receptor complex binds to specific DNA responsive
elements (GRE) to activate gene transcription.
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