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EUKARYOTIC CELL SIGNALING IV & V
The JAK-STAT Pathways
Dr. Ke Shuai
Office: 9-240M Factor
Tel: X69168
E-Mail: [email protected]
The discovery of the JAK-STAT pathway
1. Using interferons (IFNs) as the model system.
IFNs consist of two types: type I (IFN-a, IFN-b) and type II (IFN-g). Type I and II
IFNs bind to different receptors and activate overlapping but distinct genes.
2. Identification of genes which are specifically activated by IFN-a and IFN-g,
characterizing the specific DNA sequences responsible for IFN stimulation in the
promoters of these genes.
ISRE: Interferon-a stimulated responsive element
GAS: gamma-activation sequence
3. Identification of STATs (signal transducer and activator of transcription)
Biochemical purification
4. A genetic approach led to the discovery of the role of JAKs in IFN signaling as well
as the importance of STATs in IFN-mediated gene activation.
The JAK (Janus Kinase) family
JAK1
JAK2
JAK3
Tyk2
STAT: Signal Transducer and Activator of Transcription
N-terminal
Domain
Coiled Coil
Domain
DNA Binding
Domain
SH2
Domain
Linker
Domain
Y701
N1
Transact.
Domain
-C
136
317
488
576
683 712
750 aa
Chen et al. (1998) Cell 93:827-839
Ligand
Stat1 (a, b
Stat2
Stat3 (a, b
Stat4
Stat5A
Stat5B
Stat6
IFN-a , -b, g
IFN-a , -b
IFN-a , IL-6, EGF
IL-12, IFN-a
IL-2, EPO, PRL
IL-2, EPO, PRL, GH
IL-4, IL-13
In vivo Function
Innate immunity
IFN-a response
Embryonic development
Th1 response
Mammary gland development, lactation
Sexual dimorphism, T cell/ NK cell growth
Th2 response
STAT Structure and Protein Binding Sites
The JAK-STAT Pathway
. Activated by a variety of cytokines and growth factors
Interferons: IFN-a, -b, g
Interleukins: IL-2, 3, 4, 5, 6, 7, 9, 10, 12, 13, 15
Growth hormones: EPO, GH, PRL
Growth factors: EGF, PDGF, CSF-1, G-CSF
.
Involved in a wide spectrum of physiological settings
Cell proliferation
Cell differentiation
Apoptosis
Immunity
Development
Hematopoiesis
The IFN-activated JAK-STAT pathways
Nucleus
Cytoplasm
48
Tyk2
p
1
48
p
IFN-a
1
Jak1
p
1
2
p
2
ISRE
2
p
1a
p
1a
Jak1
IFN-g
1a/b
Jak2
1a/b
p
GAS
p
1a/b
p
1b
p
1b
GAS
X
The JAK-STAT Pathway
Ligand
JAK
JAK
JAK
JAK
Y
JAK
P
SH2
SH2-P
Y
SH2
Y
SH2
Nucleus
STAT
Cytoplasm
SH2-P
Transcription
Canonical JAK-STAT Pathway
Variations in Mechanisms of STAT Activation
Characteristics of STAT signaling
1. JAKs are constitutively associated with the receptor and are activated
upon receptor dimerization.
2. STAT pre-exists in the cytoplasm and is activated by tyrosine
phosphorylation and then translocates into the nucleus to activate genes.
3. The STAT SH2 domain has dual roles: 1) binding to the receptor
2) forming homo- or heterodimers
4. STAT dimerization is mediated through SH2-phosphotyrosine
interactions. Dimerization is required for STAT to bind to DNA.
5. Specificity of the STAT signaling pathways can be achieved at multiple
steps.
Negative Regulation of The JAK-STAT Pathway
JAK
JAK
SOCS/JAB/SSI
Starr et al. (1997) Nature 387:917
Endo et al. (1997) Nature 387:921
Naka et al. (1997) Nature 387:924
Y
SH2-P
STAT
SH2-P
PIAS
Chung et al. (1997) Science 278:1803
The Structure of SOCS (Suppressor Of Cytokine
Signaling) Proteins
N
C
SOCS/JAB/SSI
SH2
SOCS Box
CIS: cytokine-inducible SH2-containing protein
SOCS: 1-7
The identification of SOCS-1 in IL-6 signaling
Three different approaches were used:
1. Expression cloning
2. Yeast two-hybrid cloning
3. Screening cDNA library with a specific antibody
Expression of SOCS-1 suppresses IL-6-induced macrophage differentiation
SOCS-1 inhibits the activation of Stat3 by IL-6
Inhibition of IFN-induced Stat1 Activation by SOCS1 and SOCS3
HeLa-hyg
IFN : -
a
g
HeLa-SOCS1
-
a
g
HeLa-SOCS2
-
A
a
g
HeLa-SOCS3
-
a
g
pStat1
a-pStat1
B
Stat1
a-Stat1
C
SOCS3
SOCS2
SOCS1
a-Flag
Negative Regulation of JAK/STAT Signaling by SOCS Proteins
JAK
JAK
SOCS
P-Y
SH2
Y
SH2-P
SH2
STAT
SH2-P
P-Y
SOCS
PIAS: protein inhibitor of activated STAT
LXXLL
Zinc finger
region
Acidic
domain
Serine-threoninerich region
PIAS1
650 aa
PIAS3
583 aa
PIASxa
572 aa
PIASxb
621 aa
PIASy
510 aa
The Strategy to Characterize PIAS Proteins in STAT Signaling
1. Preparing specific anti-PIAS antibodies
2. In vivo co-immunoprecipitation analysis
3. Analysis of PIAS effects on STAT-mediated gene activation
4. Study of the molecular mechanisms of PIAS action
Blot:
IFN
IL-6
Unt
IFN
IL-6
B
Unt
IFN
A
IL-6
Unt
PIAS3 Interacts with Stat3 but not Stat1
a-pStat1
Stat3
Blot:
a-Stat1
Blot:
a-pStat3
IP:
a-PIAS3c
Blot: a-Stat3
Blot:
a-Stat3
Blot: a-PIAS3c
IP: a-PIAS3c
Blot: a-Stat1
IFN
IL-6
Unt
IFN
IL-6
B
Unt
IFN
IL-6
A
Unt
PIAS1 Interacts With Activated Stat1 But Not Stat3
Blot: a-pStat1
Stat1
Blot: a-Stat1
Blot: a-pStat3
Blot: a-Stat3
IP: a-PIAS1n
Blot: a-Stat1
Blot: a-PIAS1n
IP: a-PIAS1n
Blot: a-Stat3
PIAS3 Inhibits Stat3-mediated Gene Activation
A
Luciferase Activity
(relative units)
9
8
7
6
- IL-6
+ IL-6
5
4
3
2
1
0
Stat3(g)
PIAS3(g)
-
1.0
-
1.0
0.5
1.0
1.0
PIAS1 Inhibits Stat1-mediated Gene Activation
Fold Induction
40
30
20
10
0
Stat1 (g)
PIAS1 (g)
0
0
1
0
0
2
1
0.2
1
0.5
1
1
Flag-Stat1
Flag-PIAS1
IFN-g: - +
-
+ -
+
-
aFLAG
+
-
+ -
+
PIAS3 Inhibits the DNA Binding Activity of Stat3
A
GST
(ng) 0
IL-6 -
0
+
GST-PIAS3
20 50 100 200 20 50 100 200
+ +
+ +
+ +
+ +
Stat3/3
Stat1/3
Stat1/1
B
(ng)
0
TNF-aNFB
GST
0
+
20
+
GST-PIAS3
50 100 200 20 50 100 200
+ +
+
+ + +
+
Inhibition of STAT signaling by PIAS
1. PIAS1 and PIAS3 specifically interact with phosphorylated
Stat1 and Stat3, respectively, in a ligand-dependent manner.
2. PIAS inhibits STAT-mediated gene activation by blocking
the DNA binding activity of STAT.
JAK
JAK
P-Y
SH2
SH2-P
Y
SH2
STAT
SH2-P
SH2-P
P-
SH2-P
P-Y
PIAS
The Negative Regulation of STAT Signaling Pathways
Summary of techniques used in cell signaling studies
A. Identify signaling components
1. Yeast two-hybrid assays
2. Expression cloning
3. Searching for family members: computer search; degeneraged
PCR strategy as well as low stringency cDNA library screening
4. Biochemical purification
5. Protein complex pull-down by immunoprecipitation followed
by mass spectrometry analysis
6. Genetics
B. Protein-protein interactions
1. Yeast two-hybrid assays
2. Co-immunoprecipitation analysis: endogenous or cooverexpression
3. In vitro binding : GST-pull down, etc.
4. Far Western analysis
C. Functional assays
1. Reporter gene assays, Northern blot analysis
2. EMSA (gel shift)
3. Modification analysis: labeling, immunoprecipitation followed
by specific antibody, identify site and mutate and test its
importance
4. Dominant negative or constitutive active mutants
5. Antisense
6. Knock-outs