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



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



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



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



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×ËáÃ¸

Mg++ ATP

ÉúÎïÐ§Ó¦

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

×ªÂ¼»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×Ëá¼¡´¼ IP3

Á×Ö¬Ã¸C 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


G

effectors

P P

SRC

generalm echanism s

Arr


These can be considered general m echanism s.

Www.ggene.Cn 44

N

C


G

effectors

P P

SRC

generalm echanism s

pPRO

hom

er

SH3

h o m e r

IP3R

Ca 2+

Arr


Www.ggene.Cn 45

N

C


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


M APK

Www.ggene.Cn 46

N

C


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


M APK

Www.ggene.Cn 47

N

C


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


Na+/H+ exchanger

M APK

Www.ggene.Cn 48

N

C


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


Na+/H+ exchanger

M APK

Www.ggene.Cn 49

N

C


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

?


Na+/H+ exchanger

M APK

Www.ggene.Cn 50

N

C



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



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 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.


• 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


• Autophosphorylation on tyrosine

P

P

P

P

RAS-RAF-MEK-MAPK

Www.ggene.Cn 67



• 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



• 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



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



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


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.

cellular signal transduction pathway

Documents

camp dependent protein

unknowna protein kinase

hormone signal

gdp pi

camp r2camp4

substitution of gtp

synthesis of camp

complex signal cascades