cellular signal transduction pathway
description
Transcript of cellular signal transduction pathway
Www.ggene.Cn 1
Www.ggene.Cn 2
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
Www.ggene.Cn 3
GG
Effector
Signal
G Protein Signal Cascade
Www.ggene.Cn 4
Extracellular
Cytoplasmic
COOH-
-NH2
i1
i2
i3
e1e2 e3
TM1 TM2 TM3 TM4 TM5 TM6 TM7
G-protein-coupled G-protein-coupled receptorsreceptors
-S-S-
Binding of ligands
Location of G
Www.ggene.Cn 5
4 superfamilies binds GTP
G-protein subunits
• Three subunits
Www.ggene.Cn 6
Diversity of G Protein-Coupled Receptor Signal Transduction Pathways
Www.ggene.Cn 7
Www.ggene.Cn 8
Www.ggene.Cn 9
AC
hormone signal outside GPCR plasma membrane
GTP GDP ATP cAMP + PPi
cytosol
GDP GTP
A G-protein that is part of a pathway that stimulates Adenylate Cyclase is called Gs & its a subunit Gsa.
Www.ggene.Cn 11
The sequence of events by which a hormone activates cAMP signaling:
1. Initially Ga has bound GDP, and αβ& γ subunits are complexed together.
AC
hormone signal outside GPCR plasma membrane
GTP GDP ATP cAMP + PPi
cytosol
GDP GTP
Www.ggene.Cn 12
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).
AC
hormone signal outside GPCR plasma membrane
GTP GDP ATP cAMP + PPi
cytosol
GDP GTP
Www.ggene.Cn 13
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.
AC
hormone signal outside GPCR plasma membrane
GTP GDP ATP cAMP + PPi
cytosol
GDP GTP
Www.ggene.Cn 14
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.
AC
hormone signal outside GPCR plasma membrane
GTP GDP ATP cAMP + PPi
cytosol
GDP GTP
Www.ggene.Cn 15
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). R2C2
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 cAMP( )R
RR
C
C C
µ°°×ÖÊ µ°°×ÖÊ P
Á×Ëáø
Mg++ ATP
ÉúÎïЧӦ
PKA
Www.ggene.Cn 18
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
Www.ggene.Cn 19
N CDBD
ÅäÌå
GsAC
ATPcAMP
PKA
P
121£¬133£¬156CREB
ת¼»î»¯Óò
ÊÜÌå
Www.ggene.Cn 20
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).
H3N+ C COO
CH OH
CH3
H
threonine (Thr)
H3N+ C COO
CH2
OH
H
serine (Ser)
Www.ggene.Cn 21
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.
P r o te in O H + A T P P r o te in O P
O
O
O
+ A D P
P i H 2 O
P r o te in K in a s e
P r o te in P h o s p h a ta s e
Www.ggene.Cn 22
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).
P r o te in O H + A T P P r o te in O P
O
O
O
+ A D P
P i H 2 O
P r o te in K in a s e
P r o te in P h o s p h a ta s e
Www.ggene.Cn 23
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.
Www.ggene.Cn 24
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.
Www.ggene.Cn 25
Cyclic Nucleotide Metabolism - cAMP
Turn off of the signal :
3. Hormone receptor desensitization occurs.
This process varies with the hormone. Some receptors are phosphorylated via G-
protein-coupled 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.
Www.ggene.Cn 27
Www.ggene.Cn 28
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.
Www.ggene.Cn 29
A GPCR (receptor) is activated. GTP exchanges for GDP.
Gqa-GTP activates Phospholipase C.
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.
Www.ggene.Cn 30
Cleavage of PIP2, catalyzed by Phospholipase C, yields two second messengers:
inositol-1,4,5-trisphosphate (IP3)
diacylglycerol (DG).
Www.ggene.Cn 31
O
OHOH
OH
O
OP
PP
CH2O
CHO OCR2
CH2O OCR1
CH2OH
CHO OCR2
CH2O OCR1
O
OHOH
OH
O
OP
PP
Á×Ö¬õ£¼¡´¼ PI
Á×Ö¬õ£¼¡´¼-4,5-Ë«Á×Ëá PI2P
¶þÖ¬õ£¸ÊÓÍ DG
ÈýÁ×Ëἡ´¼ IP3
Á×֬øC PLC
Ë®½âH + R
Gµ°°× ½éµ¼
¼¤»î
Www.ggene.Cn 32
Diacylglycerol, with Ca++, activates Protein Kinase C,
which catalyzes phosphorylation of several cellular proteins, altering their activity.
Www.ggene.Cn 33
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
Www.ggene.Cn 34
Www.ggene.Cn 35
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.
Ca++
ATP ADP + Pi
Ca++
IP3
calmodulin
endoplasmic reticulum
Ca++
Ca++-ATPase
Ca++-release channel
Www.ggene.Cn 36
Ca2+-Calmodulin dependent protein kinases (CaM kinase)
Extensive substrate for CaM kinasePhosphorylation of Ser/Thr residues
Activate AC, PDE, Nos, Ca2+-ATPase IP3 receptor
PTP
Www.ggene.Cn 37
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.
Sequential dephosphorylation of IP3 by enzyme-catalyzed 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.
Www.ggene.Cn 39
Www.ggene.Cn 40
GGi/oi/o
GGss
GGqq
GG12/1312/13
inhibition of cAMP production
inhibition of Ca2+ channels
activation of GIRK K+ channels
G-protein subtypes
increased synthesis of cAMP
activation of Ca2+ and K+
channels
activation of PLC leading to
activation of PKC (DAG)
intracellular Ca2+ release (IP3)
mediates signalling between GPCRs and RhoA (GTPase)
function under investigation
Www.ggene.Cn 41
GPCR signallingGPCR signalling
Www.ggene.Cn 42
N
C
I II III IV V VI VII
the signal transduction m achine
Www.ggene.Cn 43
N
C
I II III IV V VI VII
G
effectors
P P
SRC
generalm echanism s
Arr
the signal transduction m achine
These can be considered general m echanism s.
Www.ggene.Cn 44
N
C
I II III IV V VI VII
G
effectors
P P
SRC
generalm echanism s
pPRO
hom
er
SH3
h o m e r
IP3R
Ca 2+
Arr
the signal transduction m achine
Www.ggene.Cn 45
N
C
I II III IV V VI VII
G
effectors
P P
SRC
generalm echanism s
pPRO
hom
er
SH3
h o m e r
IP3R
Ca 2+
pPRO
G rh2
Nck
SH3Arr
the signal transduction m achine
M APK
Www.ggene.Cn 46
N
C
I II III IV V VI VII
G
effectors
P P
SRC
generalm echanism s
pPRO
hom
er
SH3
h o m e r
IP3R
Ca 2+
pPRO
G rh2
Nck
SH3
InaDPDZ
PLC PKCTRP
Arr
the signal transduction m achine
M APK
Www.ggene.Cn 47
N
C
I II III IV V VI VII
G
effectors
P P
SRC
generalm echanism s
pPRO
hom
er
SH3
h o m e r
IP3R
Ca 2+
pPRO
G rh2
Nck
SH3
InaDPDZ
PLC PKCTRP
CNHERF
Arr
the signal transduction m achine
Na+/H+ exchanger
M APK
Www.ggene.Cn 48
N
C
I II III IV V VI VII
G
effectors
P P
SRC
generalm echanism s
pPRO
hom
er
SH3
h o m e r
IP3R
Ca 2+
pPRO
G rh2
Nck
SH3
InaDPDZ
PLC PKCTRP
CNHERF
Arr
CcNOS
arginine
NO
the signal transduction m achine
Na+/H+ exchanger
M APK
Www.ggene.Cn 49
N
C
I II III IV V VI VII
G
effectors
P P
SRC
generalm echanism s
pPRO
hom
er
SH3
h o m e r
IP3R
Ca 2+
pPRO
G rh2
Nck
SH3
InaDPDZ
PLC PKCTRP
CNHERF
Arr
CcNOS
arginine
NO
eNOS
?
the signal transduction m achine
Na+/H+ exchanger
M APK
Www.ggene.Cn 50
N
C
I II III IV V VI VII
the signal transduction m achine
G
effectors
P P
SRC
generalm echanism s
pPRO
hom
er
SH3
h o m e r
IP3R
Ca 2+
pPRO
G rh2
Nck
SH3
InaDPDZ
PLC PKCTRP
CNHERF
Arr
CcNOS
arginine
NO
eNOS
?
NPxxY
ARF
RhoPLD
Na+/H+ exchanger
M APK
Www.ggene.Cn 51
N
C
I II III IV V VI VII
the signal transduction m achine
G
effectors
P P
SRC
generalm echanism s
pPRO
hom
er
SH3
h o m e r
IP3R
Ca 2+
pPRO
G rh2
Nck
SH3
InaDPDZ
PLC PKCTRP
CNHERF
Arr
CcNOS
arginine
NO
eNOS
?
NPxxY
ARF
RhoPLD
Na+/H+ exchanger
M APK
GPC Rs are truly rem arkable signal transduction m achines.
In sum m ary.....
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.
Www.ggene.Cn 53
Most GTP-binding proteins depend on helper proteins
GAPsGTPase Activating Proteins, promote GTP hydrolysis.
protein-GTP (active) GDP GEF GAP GTP Pi protein-GDP (inactive)
Www.ggene.Cn 54
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 GAP GTP Pi protein-GDP (inactive)
cGMP- 蛋白激酶途径鸟苷酸环化酶( GC )GC 的激活间接地依靠 Ca2+
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
H
RGC
GTP
cGMPcGMP-依赖性蛋白激酶
蛋白激酶 G (一个 cGMP结合位点)
Www.ggene.Cn 56
Cyclic Nucleotide Metabolism - cGMP
Cyclic Nucleotide Metabolism – cGMPCyclic 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
Www.ggene.Cn 59
• Signal transduction through the MAPK pathways
• Protein-protein interactions
• Protein kinase cascades
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.
Www.ggene.Cn 61
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)
Www.ggene.Cn 62
Www.ggene.Cn 63
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.
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
Www.ggene.Cn 65
RAS-RAF-MEK-MAPK
• Ligand binds receptor PTK
Www.ggene.Cn 66
• Ligand binds receptor PTK
• Autophosphorylation on tyrosine
P
P
P
P
RAS-RAF-MEK-MAPK
Www.ggene.Cn 67
• Ligand binds receptor PTK
• Autophosphorylation on tyrosine
• GRB2 (a SH2- and SH3-containing protein) binds to the receptor phosphotyrosine motif Y-V/L-N-X via its SH2 domain
P
P
P
P
RAS-RAF-MEK-MAPK
SH2 SH
3
GRB2
SOS
Www.ggene.Cn 68
• Ligand binds receptor PTK
• Autophosphorylation on tyrosine
• GRB2 (a SH2- and SH3-containing protein) binds to the receptor phosphotyrosine motif Y-V/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
P
P SH2 SH
3
GRB2
SOS
RAS-RAF-MEK-MAPK
Www.ggene.Cn 69
P
P
P
P SH2 SH
3
GRB2
SOS
RAS-RAF-MEK-MAPK
GRB2• 24 kDa adaptor molecule.• Only contains an SH2 domain between two SH3 domains
Www.ggene.Cn 70
• Recruitment of SOS to the close proximity of RAS in the membrane
P
P
P
P SH2 SH
3
GRB2
SOS
RAS-RAF-MEK-MAPK
GRB2• The SH2 of GRB2 binds to phosphotyrosine in a number of receptor PTK: for EGF, PDGF, colony-stimulating factor-1 (CSF-1), 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.
Www.ggene.Cn 71
P
P
P
P SH2 SH
3
GRB2
SOS
GDPRAS
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.
Www.ggene.Cn 72
• RAS becomes activated by exchanging GDP for GTP
P
P
P
P SH2 SH
3
GRB2
SOS
GDPGTPRAS
RAS-RAF-MEK-MAPK
Www.ggene.Cn 73
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
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.
Www.ggene.Cn 74
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
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 anti-RAS 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.
Www.ggene.Cn 75
• 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
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
RAS-RAF-MEK-MAPK
Www.ggene.Cn 76
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
RAS-RAF-MEK-MAPK
RAF• RAF is a serine/ threonine protein kinase
• RAF is a MAPK kinase kinase (MAPKKK).
• Can transform cells when constitutively active or when overexpressed.
Www.ggene.Cn 77
14-3-3P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
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.
Www.ggene.Cn 78
14-3-3P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
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).
Www.ggene.Cn 79
14-3-3
• Activation of RAF (most likely by phosphorylation of RAF and binding to the scaffold protein 14-3-3)
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
RAS-RAF-MEK-MAPK
Www.ggene.Cn 80
14-3-3• Activated RAF in turn activates MEK (also called MAPK kinase) by phosphorylation on two conserved serine residues in MEK.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
RAS-RAF-MEK-MAPK
Www.ggene.Cn 81
14-3-3
• Activated RAF in turn activates MEK (also called MAPK kinase) by phosphorylation on two conserved serine residues in MEK.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
RAS-RAF-MEK-MAPK
Www.ggene.Cn 82
14-3-3P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
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.
Www.ggene.Cn 83
14-3-3
• Activated MEK activates MAPK (a serine/threonine protein kinase) by phosphorylation of conserved threonine and tyrosine residues.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
MAPK
P P
RAS-RAF-MEK-MAPK
Www.ggene.Cn 84
14-3-3
• Activated MEK activates MAPK (a serine/threonine protein kinase) by phosphorylation of conserved threonine and tyrosine residues.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
MAPK
P P
RAS-RAF-MEK-MAPK
Www.ggene.Cn 85
14-3-3P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
MAPK
P P
RAS-RAF-MEK-MAPK
MAPK• Five isoforms of ERK have been identified, but ERK1 and ERK2 have been most studied.
Www.ggene.Cn 86
14-3-3P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
MAPK
P P
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).
Www.ggene.Cn 87
14-3-3P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
MAPK
P P
RAS-RAF-MEK-MAPK
MAPK• Inactivation of MAPK is achieved by several phosphatases: the serine/threonine phosphatase PP2A
Www.ggene.Cn 88
14-3-3• Activated MAPK phosphorylates a number of substrates in the cytoplasm
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
MAPK
P PSubstrates
Substrates
P
P
RAS-RAF-MEK-MAPK
14-3-3
• Activated MAPK phosphorylates a number of substrates in the cytoplasm
it also translocated into the nucleus(within min) where it phosphorylates nuclear transcription factors.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
MAPK
P P
Substrates
RAS-RAF-MEK-MAPK
MAPK
P P
14-3-3
• Activated MAPK phosphorylates a number of substrates in the cytoplasm
it also translocated into the nucleus(within min) where it phosphorylates nuclear transcription factors.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
Substrates
RAS-RAF-MEK-MAPK
MAPK
P P
MAPK
P P
14-3-3
• Activated MAPK phosphorylates a number of substrates in the cytoplasm
it also translocated into the nucleus(within min) where it phosphorylates nuclear transcription factors.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
Substrates
RAS-RAF-MEK-MAPK
MAPK
P P
MAPK
P P
14-3-3
• Activated MAPK phosphorylates a number of substrates in the cytoplasm
it also translocated into the nucleus(within min) where it phosphorylates nuclear transcription factors.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
Substrates
RAS-RAF-MEK-MAPK
MAPK
P P
MAPK
P P
P
14-3-3
• Activated MAPK phosphorylates a number of substrates in the cytoplasm
it also translocated into the nucleus(within minutes) where it phosphorylates nuclear transcription factors.
Transcription of genes important for cell proliferation.
P
P
P
P SH2 SH
3
GRB2
SOS
RAS GTP
RAF
MEK
P P
RAS-RAF-MEK-MAPK
SubstratesMAPK
P P
MAPK
P P
P
Www.ggene.Cn 94
Transcription Factor Regulation (CREB, Elk-1 and c-Fos) SIGMA-ALDRICH
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.
Www.ggene.Cn 96
•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).
Substrates of MAPK:
JNK pathway
• JNK translocation into the nucleus phosphorylation of the transcription factor c-JUN at the N-terminal residues (Ser63 and Ser73)
activation of transcription by c-JUN
Rac1/Cdc42 GTP
STRESS
PAK
MEKK1-3P P
MEK4P P
JNKP P
c-JUNP
Www.ggene.Cn 98
Rac1/Cdc42 GTP
STRESS
PAKMEKK1-3P P
14-3-3P
P
P
P SH2 SH
3
Grb2
Sos
Ras GTP
Raf
MEK4P P
MEK
P P
JNKP P
MAPK
P P
Www.ggene.Cn 99
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.
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
Www.ggene.Cn 101
The JAK/STAT Signaling Pathway
The JAK/STAT Signaling PathwayA 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). Ligand-induced 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 Homology-2 (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.
Www.ggene.Cn 103
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
Www.ggene.Cn 104
Glucocorticoid Receptor Signaling
Www.ggene.Cn 105
Glucocorticoid Receptor Signaling
The glucocorticoid hormone, cortisol, passes through the plasma membrane into the cytoplasm where it binds to the specific, high-affinity 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 DNA-binding 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.