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IFNs,Cytokines & the JAKSTAT Signaling Cascade
March 10, 2005
Chris Schindler
Innate Viral Interfering Activity is
Discovered in Cultured Cells (1950s)
1960s
1970s
1980s
Initial and intermittent characterization of IFN activity.
Purification of “Immune”,
“Leukocyte” & “Fibroblast” IFNs
yields >10 type I IFNs (a & b) and
one type II IFN (IFN-g).
Cloned IFNs, the first of many
disappointments for biotech
industry, is a boon to molecular
biologists.
An Evolving IFN-a Signaling Paradigm
IFN-a
PTK-x
P
P
Stat2
IRF-9
Stat1
P
P
P
P
antiviral*
antiproliferative*
chemokine*
PKR
OAS
IP10
iNOS
Interferon Stimulated Response Elements
(ISREs)
ISG54
ISG15
GBP
IP10
OAS
HLA-A
MX
TAGTTTCACTTTCCC
CAGTTTCGGTTTCCC
TACTTTCAGTTTCAT
AGGTTTCACTTTCCA
TGGTTTCGTTTCCTC
CAGTTTCTTTTCTCC
GAGTTTCGTTTCTGA
Identification of “High IFN Producing Cells”
(HIPCs) in vivo
A small
subset of
WBCs
produce
most of the
circulating
IFN-Is
The IFN-I Autocrine Loop and Toll Like Receptors (TLRs)
dsRNA
TLR3
IFN-b
LPS
TLR4
I FN-b
ssDNA
I FNAR
NFkB
TLR7
IKKa/b/e
CpG
IRF-8 ?
?
IKKa/b/e
IRF-3
St1/St2
IFN-b
IRF-7
danger
IFN-a’s
P
TLR9
IRF-7
IRF-7
IFN-a’s
danger
IFN Type II (a.k.a. IFN-g) Signaling
Paradigm
IFN-g
IFNgR
Jak2
Jak1
Y
Stat1
Stat1
P
P
inflammation*
macrophage act.*
chemokines*
IRF1
MIG
GBP
iNOS
CIITA
GAS Binding Elements
Gene
GBP
FcgRI
ICSBP
ICAM-1
c-fos
IFP53
IRF-1
IRF-1
Ly6E
mig
Species
human
human
human
human
human
human
human
mouse
mouse
mouse
Element
ATT AC T CT AAA
TTT CC C AG AAA
TTT CT C GG AAA
TTT CC C GG AAA
GTT CC C GT CAA
ATT CT C AG AAA
TTT CC C CG AAA
TTT CC C CG AAA
ATT CC T GT AAG
CTT AC T AT AAA
Tyk2: Member of the Unusual
Janus Kinase Family
JH7
0
100
JH6
200
JH5
300
400
JH4
500
JH3
600
700
Tyk2
Jak1
Jak2
Jak3
JH2/yK
800
900
JH1/TK
1000
1100
Expressed in all tissues
Expressed in hematopoietic tissues
1200
STATs & Four Helix Bundle Cytokines
Stat1
Stat2
Stat3
Stat4
Stat5a/b
Stat6
IFN-I(a,b,,) & IFN-g
IFN-I(a,b,,)
IL-10 and IL-6 families
IL-12, IL-23, IL-27
Single Chain, IL-2 & IL-3 families
IL-4, IL-13
Conserved STAT Domains
NH2
0
100
Coil-Coil
200
300
DNA
400
LK
500
SH2
600
Y TAD
700
800
STAT Evolution
•
STAT homologues have been identified in most model metazoans, including
Dictyostelium, C. elegans, Drosophila, Anopheles, Xenopus and zebrafish.
•
STAT homologues in C. elegans are very distantly related.
•
The three STAT Dictyostelium homologues are structurally and functionally
quite divergent. The Dictyostelium STATs, most closely related to Stat3 &
Stat5 may encode the primordial SH2 domain.
•
Homologues of a STAT (37 % identity to Stat5), JAK (27 % identity to Jak2),
cytokine and cytokine receptor have each been identified in Drosophila. This
pathway play several important developmental roles.
•
Zebra fish encodes a Stat1 and Stat3 homologue. The Stat3 homologue is
developmentally expressed and syntenic with both murine and human Stat3.
The Stat1 homologue is more distantly related and expressed later in life.
•
Sequence analysis suggests that mammalian STATs evolved from a single
syntenic Stat3-Stat5 (murine chromosome 17) tandem cluster that was
duplicated to form the Stat1-Stat4 (murine chromosome 2) and Stat2-Stat6
(murine chromosome 12) tandem clusters. This was followed by the
duplication of Stat5 into Stat5a and Stat5b.
Stat1
•
Transduces critical signals for all three classes of IFNs.
•
Stat1 is activated by several other ligands, but its role appears to be more
secondary.
•
There are two isoforms - Stat1b, which is carboxy terminally truncated before
the TAD, is assumed to function as an antagonist of Stat1a.
•
Generic biological activities include:
– Promoting the expression of inflammatory or “danger” signals.
– Antagonizing proliferation and/or promoting apoptosis.
– Stat3 appears to antagonize both of these activities.
•
Stat1 is tyrosine phosphorylated on Y701 and serine phosphorylated on S727 (in
the TAD). This latter phosphorylation is required for maximal transcription of
some genes.
•
Stat1 has also been reported to be acetylated at K785 and SUMOylated at K703.
•
Stat1 activity is antagonized by phosphatases directed at the receptor and
JAKs, and directly by one or more nuclear phosphatases.
•
Suppressor of Cytokine Signaling 1 (SOCS-1), a Stat1 target gene, directs an
important negative feed back loop to suppress IFNg-Stat1 dependent signals.
Type I IFN Signaling
Tyk2
P
Jak1
Y
Y
Y
P
Stat2
IRF-9
Stat1
P
P
P
P
P
P
P
P
Stat2
• Transduces critical signals for type I IFNs and -IFNs.
• Murine and Human Stat2 are functionally analogous but
uncharacteristically divergent in sequence, especially in the TAD.
• Human Stat2 is tyrosine phosphorylated on Y690 and murine Stat2 on
Y689. Other modifications have not been reported, although it is likely
to be serine phosphorylated.
• Stat2 is the only STAT that does not appear to homodimerize and bind
DNA directly.
• Stat2 activity is antagonized by phosphatases directed at the receptor
and JAKs, and directly by one or more nuclear phosphatases.
• In contrast to Stat1 and Stat3, the distribution of Stat2 is unstimulated
cells is almost exclusively cytoplasmic.
Murine
Coil-Coil
DNA
SH2
Y689
TAD
923 aa
76 % Homology
Human
851 aa
Coil-Coil
DNA
Divergent
SH2
Y690
TAD
Stat3
•
Transduces signals for type I IFNs,-IFNs, the critical signals for the extended
IL-10 family and the extended IL-6 families.
•
There are two isoforms, Stat3a & Stat3b. Like Stat1b, Stat3b is carboxy
terminally truncated before the TAD, but has been shown to direct the
expression of a unique set of genes through association with other
transcription factors.
Generic biological activities include:
•
– Promoting cell growth associated with cell transformation.
– Antagonizing the expression of inflammatory or “danger” signals.
– Stat1 antagonizes both of these activities (a ying-yang relationship).
•
Stat3 is the only STAT that is essential for development (i.e., the knockout is
embryonic lethal). Tissues specific knockouts reveal an important roles in
protecting tissues form inflammation and suppressing transformation.
•
Stat3 is tyrosine phosphorylated at Y705, serine phosphorylated at S727 and
acetylated at K785.
•
Stat3 activity is antagonized by phosphatases directed at the receptor and
JAKs, and directly by one or more nuclear phosphatases.
•
In response to stimulation by the IL-6 family of ligands, Stat3 activity is directly
suppressed by SOCS-3, a Stat3 target gene.
Endocytosis
IL-6 Signaling
Cytoplasm
IL-6
IL-6Ra
gp130
Jak2
Cytoplasm
Y
Y
Y
Stat3
P
Jak1
P
Jak2 Jak1
Stat3
SHP-2
P
P
MAPK
ERK
Cytoplasm
Endosome
Nucleus
P
P
Gene
Expression
Jak2 Jak1
Stat5a and Stat5b
• Transduce critical signals for all members of the IL-2, IL-3 and single
chain cytokine receptor families, i.e. is Stat5 is functionally pleiotropic
like its ancestral partner Stat3.
• Consistent with their significant level of homology, Stat5a & Stat5b can
functionally overlap, especially in hematopoietic tissues (e.g., in
response to IL-2 and IL-3 family ligands).
• However, gene targeting studies have underscored a specificity for
Stat5a in prolactin response and a specificity for Stat5b in GH
response.
• Stat5 has been implicated in the development of some hematopoietic
tumors and the regulation of some stem cell populations.
• SOCS-2 specifically antagonizes Stat5b activation in response to GH
(i.e., SOCS-2 knockout mice exhibit gigantism).
Stat4 & Stat6
• Stat4 and Stat6 exhibit a the most ligand specificity.
– Stat4 transduces signals for members of the IL-12 family. (IFN-Is can also
promote a partial activation of Stat4).
– Stat6 transduces signals for closely related IL-4 and IL-13.
• Stat4, Stat6 and their activating ligands play an essential role in the
development of effector CD4+ T-helper cells.
– Stat4 directs the development of Th1 cells.
– Stat6 directs the development of Th2 cells.
• Full Stat4 activation requires both tyrosine phosphorylation at Y689 and
serine phosphorylation at S721.
• The ability of IL-18 to direct Stat4 serine phosphorylation accounts for
its ability to synergize with some Stat4 activating ligands (e.g., IFN-Is)
to direct the activation to Th1 cells.
Cytokines and the Th1-Th2 Paradigm
Ag
iDC
Flt-3L
GM-CSF
IFN-a
DC
IFN-a
IL-12
Th1
Stat4
Stat6
Th2
Th0
IFN-g
IL-2
TNF
IL-4
IL-5
IL-6
IL-10
TGF-b
The Four (a)-Helix Bundle Family of Cytokines
-
There are ~50 members; a.k.a - hematopoietins
They play important roles in development/activation of lymphocytes & myelocytes.
They all transduce pivotal signals through JAKs and STATs
They can be divided into two families based on the receptors they bind:
Cytokines that bind type I cytokine receptors:
The gC Family IL-2 family includes IL-2, IL-7, TSLP, IL-9, IL-15, IL-21 (Stat5)
IL-4 family includes IL-4 and IL-13 (Stat6)
The IL-3 family includes IL-3, IL-5 and GM-CSF (Stat5)
The Extended gp130 Family- (Stat3)
IL6-gp130 family includes IL-6, IL-11, CNTF, LIF, OSM, CT-1, NP,
NNT1/BSF3 (Stat3)
gp130-like family includes OSM, IL-31, G-CSF, Leptin
IL-12 family includes IL-12, IL-23, IL-27, IL-30 (Stat4*)
The single chain family includes Epo, GH, PRL, Tpo (Stat5)
Cytokines that bind type II cytokine receptors:
The human Type I IFN Family includes 12 a-IFNs, IFN-b, IFN-, limitin(Stat1-3)
Type II IFN Family includes only IFN-g (Stat1)
-IFN family includes IL-28a, IL-28b, IL-29 (Stat1-3)
IL-10 family includes IL-10, IL-19, IL-20, IL-22, IL-24, IL-26 (Stat3)