Transcript Conj532 - Cytokine - Jak/STAT Pathways T-helper cell subsets and cytokine profiles Th1, Th2 and Th17 cells are a separate lineage.

Conj532 - Cytokine - Jak/STAT Pathways
T-helper cell subsets and cytokine profiles
Th1, Th2 and Th17 cells are a separate lineage of CD4+ T cells, distinct from other T cell subsets. Every
specific T helper cells produce its specific cytokines. T-bet, T-box expressed in T cells; FoxP3, forkhead box
P3; ROR, retinoid-related orphan receptor. Makoto Kudo, et al. Front Microbiol. 2013;4:263.
Cytokines secreted by immune cells “instruct” T-cells
TH1 like
response
Antigen
Nature Reviews Immunology 10, 236-247
TH2 like
response
Effector B cells ('Be1' and 'Be2' cells) can secrete
cytokines, such as interferon-γ (IFNγ), interleukin-12 (IL12), IL-4 and IL-2, that reinforce and stabilize the cytokine
profile of effector T helper 1 (TH1) and TH2 cells. In
addition, the effector B cells can recruit additional naive T
cells into the inflammatory response.
Cellular Mechanisms in Rheumatoid Arthritis
Pathogenesis of RA: synovial and systemic inflammation. Inflammation in RA is caused by activation of T cells, B cells
and macrophages, which releases cytokines such as IL-1, IL-6 and TNF. These cytokines cause local joint damage through
increased production of metalloproteinases and activation of osteoclasts. IL-1, IL-6 and TNF also leak out to the blood
stream resulting in systemic inflammation: anaemia, thrombocytosis, fatigue, osteoporosis and the acute-phase response.
Abbreviations: IL, interleukin; RA, rheumatoid arthritis. Choy, E. H. et al. Nat. Rev. Rheumatol. 9, 154–163 (2013)
3 General types of cytokine receptors
Class I
a a
Single chain
(dimer)
Epo, GH, Prl, GM-CSF
Class II
a b (gp130)
Unique + Common
chains (tetramer)
IL-6, LIF, IL-11,
CNTF), CT-1,
CLC, OSM, IL-27
and IL-31.
Class III
a
b
g
Multiple unique
chains (2 or 3)
IL-2, Interferons
Q: why did nature evolve
multiple chain receptors?
“Canonical” JAK–STAT pathway
Activating cross
Tyr PO4 of JAK
PO4 of JAK
THM: 3 Tyr-P required; catalyzed by a kinase
that is NOT a part of the receptor
Three sequential tyrosine phosphorylations triggered by cytokine–
receptor interaction. Receptor dimerization allows
transphosphorylation and activation of Janus kinases (JAKs). This
is followed by phosphorylation of receptor tails and the recruitment
of the Signal Transducers and Activators of Transcription (STAT)
proteins through their Src-homology-2 domains. STAT tyrosine
phosphorylation then occurs. Dimerization of activated (tyrosine
phosphorylated) STAT is followed by nuclear entry.
Nature Reviews Molecular Cell Biology 3; 651-662
Binding & PO4 of STAT
Dimerization of STATs
So, lets go into more detail about
each of these players
Structural organization of STATs
Signal Transducers and Activators of Transcription
Different regions have different functions or bind different
transcriptional regulators
FERM
DBD
Stat2/p48
Stat1/p300(CBP)
Stat1/PIAS1
Stat3/c-Jun
SH3
SH2
SP
YP
TAD
Stat2/p300(CBP)
Stat1/p300(CBP)
Stat5/ERK
Stat1/MCM5
Stat1/p48
Stat5/Nmi
At least 6 families of STATS
The domain structure of STAT. The contact regions of STAT-interacting proteins are indicated by red lines.
DBD: DNA binding domain; SH3: Src homology 3 domain (poly Pro); SH2: Src homology 2 domain (pY);8TAD:
Transcription activation domain; FERM ( band4.1, ezrin,radixin, & moesin) binds to STATS and other proteins
How do SH2 and DNB domains work?
FERM
Tyr
DBD
SH3
YP
SP
SH2
TAD
Tyr
P
SH2
Phosphorylation and
SH2-phosphotyrosine
binding
STAT monomers
STAT dimer
(binds DNA)
Structure of STAT bound to DNA
SH2 domain
Linker domain
DNA-binding domain
Coiled coil
domain
Nutcracker Model
(a) Crystal
structure of an N
and C-terminally
truncated Stat1
molecule bound to
DNA. The structure
of truncated Stat3
is virtually
superimposible
with that of Stat1
(Chen et al., 1998).
STAT domain structure and protein binding sites
The core structure
(amino acids 130–712)
shows binding of a
STAT1 dimer to DNA
and the location of
binding sites of various
proteins in various
domains. The aminoterminal structure, the
placement of which in
the intact structure is
undefined, also
interacts with various
partners, as does the
carboxy-terminal
transactivation domain,
the structure of which is
unknown. CBP, CREB
binding protein; IRF,
interferon regulatory
factor; Mcm, mini
chromosome
maintenance; Nmi, NMyc interactor; PIAS,
protein inhibitor of
activated STAT.
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Nature Reviews Molecular Cell Biology 3; 651-662
Structure & number of Jaks
JH7 JH6 JH5
N
JH4
JH3
JH2
JH1
C
FERM
Pseudokinase
Domain
JANUS KINASES
Size
Identity
Chromosome
Kinase
Domain
Expression
Tyk2
140 kDa
36%
19p13.2
Ubquitous
Jak1
135 kDa
36%
1p31.2
Ubiquitous
Jak2
130 kDa
47%
10p23
Jak3
120 kDa
-
4q31
Ubiquitous
Myeloid/Lymphoid
Seven domains, termed Jak homology (JH) domains 1-7 are shared among Jaks.
The JH1 domain is the kinase domain and the JH2 domain is a pseudokinase domain
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whose precise function has not yet been determined.
Q: What does acronym, JAK stand for?
Janus - the “two-faced” god,
keeper of the gate
The Janus kinases, were thought to contain 2 types of phosphate-transferring
domains. Thus, it is named after “Janus”, the Roman two-faced gatekeeper
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of the heavens.
Different cytokine receptors bind
different combinations of Jaks
TYPE
HORMONE
PHOSPHORYLATION
Single specificity
Growth Hormone
Homo-phosphorylation
Promiscuous
(all JAKs)
IL-6
Multi- phosphorylation
Obligate Hetero
(2 different JAKs)
INFa, INFg
Hetero-phosphorylation
How determined?
INFa
Jak1
Tyk2
INFg
Jak1
Jak2
Why important?
(Mutate Jak1 ---> no Jak2 PO4
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PO
O
4
Different
Jaks PO4 different STATS
(Mutate Jak1 ---> no Tyk2 PO4
Interferon receptors and
activation of classical
JAK–STAT pathways by
type I and type II interferons
All
type I interferons
(IFNs)
bind
a common
receptor
Example
of how
one
can
get specificity
which is known as the type I IFN receptor. The type I
of function by different interferons by
IFN receptor is composed of two subunits, IFNAR1 and
expressing
and
utilizing
IFNAR2,
which are
associated
withdifferent
the Janus activated
kinases
(JAKs), tyrosine
kinase 2 (TYK2)
and JAK1,
combinations
of cytokine
receptors,
respectively. A single type II IFN, IFN-g, binds a distinct
Jaks andreceptor,
STATS
cell-surface
which is known as the type II IFN
receptor. This receptor is also composed of 2 subunits,
IFNGR1 and IFNGR2, which are associated with JAK1
and JAK2, respectively. Activation of the JAKs that are
associated with the type I IFN receptor results in tyrosine
phosphorylation of STAT2 (signal transducer and
activator of transcription 2) and STAT1; this leads to the
formation of STAT1–STAT2–IRF9 (IFN-regulatory factor
9) complexes, which are known as ISGF3 (IFN-stimulated
gene (ISG) factor 3) complexes. These complexes
translocate to the nucleus and bind IFN-stimulated
response elements (ISREs) to initiate gene transcription.
Both type I and II IFNs also induce formation of STAT1–
STAT1 homodimers that translocate to the nucleus and
bind GAS elements in the promoter of some ISGs,
thereby initiating transcription of these genes. The GAS
element and ISRE sequences are shown.
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From Nat Rev Immunol 376 | MAY 2005
How is Cytokine
Function Regulated?
A: Several types of negative feedback
Diagram of domains in STAT-induced STAT
Inhibitors, SOCS & CIS proteins
At least eight proteins belong
to the SOCS family of
proteins are shown (upper
panel). They are
characterized by the
presence of an SH2 central
domain and the SOCS box
domain at the C-terminus. A
small domain called kinase
inhibitory region (KIR), only
found in SOCS1 and
SOCS3, is shown as a small
box at the N-terminal region.
SOCS proteins can interact
with phosphotyrosine
phosphorylated proteins
through their SH2 domain
and with Elongin BC through
their SOCS box domain.
Other proteins containing a
SOCS box domain but
lacking a SH2 domain are
also shown (lower panel).
Kinase domain binding
(Kinase Inhibitor)
P-tyrosine binding
(STAT competitor
Elongin B/C
binding
(ubiquitination)
Other SOCS Box containing proteins
CIS = Cytokine-Induced SH2 protein;
SOCS = Suppressor of Cytokine Signaling
SSI = STAT-induced STAT Inhibitor
Rico-Bautista et al 2006
Negative regulation of cytokine signaling: STATinduced STAT inhibitor
Naka et al.,TiBs, 24:394-398
= SOCS1
SOCS bind to
and inhibit
JAKs
CIS inhibits
STAT binding
SSI-1 type inhibition
CIS-1 type inhibition
THM - 2 sites of inhibition (Jaks or STAT binding to Receptor)
(a) Binding of JAK to cytokine receptors and activation of STAT. (b) SSI-type inhibition of cytokine signaling. The
gene encoding SSI-1 is induced by STAT dimers, resulting in the production of SSI-1 and inhibition of cytokine
signaling by binding of SSI-1 to the kinase domain of the JAK family. (c) CIS1-type inhibition of cytokine
signaling. The gene encoding CIS1 is induced by STAT5 dimers, resulting in the production of CIS1 and
inhibition of cytokine signaling by binding of CIS1 to the STAT binding site of cytokine receptors.
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Abbreviations used: CIS1, cytokine-inducible SH2 protein 1; GAS motif, -Stat activated site; JAK, Janus
tyrosine kinase, SSI, STAT-induced STAT inhibitor; STAT, signal transducers and activators of transcription.
Other negative
regulators of
STAT proteins
Phosphatases (a) and suppressors of cytokine
signalling (SOCS proteins) (b) block further STAT
activation in the cell cytoplasm. In the nucleus,
nuclear phosphatases (c) can mediate STAT
dephosphorylation, and interactions with proteins
that inhibit activated STAT proteins (PIAS) (d) can
also occur. In addition, naturally occurring short
forms of STATs can potentially act as dominantnegative proteins by occupying DNA as nonfunctional protein or by binding to a wild-type STAT
protein (e). JAK, Janus kinase; STAT, signal
transducers and activators of transcription. Note,
also shown in green is a likely regulation of JAKs
by ubiquitination/phosphorylation
Why so many different mechanisms
for controlling STATS?
Nature Reviews Molecular Cell Biology 3; 651-662 (2002);
STATS: Transcriptional control and biological impact
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Proposed mechanisms for inhibiting the JAK–
STAT pathway by PIAS proteins
Fig 4 | Different PIAS (protein
inhibitor of activated STAT)
proteins can inhibit the Janus
kinase (JAK)–signal transducer
and activator of transcription
(STAT) pathway through distinct
mechanisms.
a | PIAS1 and PIAS3 block the
DNA-binding activity of STAT
dimers.
b | PIASX and PIASY might act
as transcriptional co-repressors
of STAT by recruiting other corepressor proteins such as
histone deacetylase (HDAC). c |
PIAS proteins can promote the
conjugation of small ubiquitinrelated modifier (SUMO) to
STAT1. The significance of
STAT1 sumoylation in regulating
STAT1 activity is controversial
and needs to be clarified
Block DNA binding
Act as corepressors
Promote sumoylation
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Nature Reviews Immunology 3; 900-911
Pias Proteins act as E3 ligases for
SUMO
Left - Ubiquitin is coupled to E-1 ubiquitin-activating enzyme and in turn transferred to E-2 ubiquitin-conjugating
enzyme. E3 ubiquitin ligase combines with the charged E2 and forms an isopeptide bond between ubiquitin
and the target protein. PIAS proteins act as E3 ligases for SUMO. SUMO shares 18% homology with
ubiquitin.
Right - PIAS1, PIAS3 and PIASx sumoylate STAT1 at Lys-703- close to Tyr-701 where JAK is phosphorylated.
21may
STAT1 can be modified by SUMO at lysine residue 703. Direct interactions between PIAS1 and STAT1
interfere with the STAT1 ability to bind DNA.
Biochemical Pharmacology 70 (2005) 649–657
Example of Feedback Regulation by
SOCS
The role of SOCS-3 in
Leptin signaling and
resistance
THM - induction of SOCS-3 causes
decreased cytokine coupling
O hr 1hr 2 hr 4hr stimulation with leptin
CHO-OBRI
CIS mRNA
SOCS-1 mRNA
SOCS-2 mRNA
SOCS-3 mRNA
Leptin induces SOCS-3, but not CIS, SOCS-1,
THM - specificity of induction
or SOCS-2, mRNA in CHO cells expressing
and
different
the
long
form of thetime
leptin courses
receptor.
23
J Flier lab JBC 274:30059 ‘99
How important are SSI proteins?
Studies with SOCS1 KO mice
In SOCS1 knockout
mice, negative
regulation of cytokine
signaling is
diminished
Lack of binding of
SSI-1 to JAK leads to
prolonged activation
of the JAK/STAT
pathway and
prolonged action of
cytokines.
SOCS-1 KO --->
Post-Embryonic
Lethal
Rescued by cross
of heterozygotes
to INF-g KO
Abbreviations: JAK,
Janus tyrosine kinase,
SSI, STAT-induced STAT
inhibitor; STAT, signal
transducers and
activators of
transcription.
25
Naka et al.,Trends in Biochemical Sciences, 24:394-398
“Cross talk” between Jak/STAT and
other signaling pathways
THM1: Other pathways can be activated by
ligand binding to "cytokine" receptors
THM2: Jaks can bind & activate other tyrosine
kinase pathways
THM3: Other serine kinase pathways can
modulate Jak/STAT function
THM4: SOCS proteins can modulate other
pathways
Activation of CRKL by the
type I INF receptor, and
role of CRKL in type-I
INF-mediated signaling
Fig
2 CRKL
is present
as a latent cytoplasmic
Jak2
can also
phosphorylate
CRKL form that
constitutively
associates
withitself
the guanine-nucleotideallowing
it to
be active
just like a STAT
exchange factor (GEF) C3G. A member of the STAT
and
form
a heterodimer
(signal
transducer
and activator of transcription) family
proteins,
is associated
with tyrosine
Itofcan
alsoSTAT5,
scaffold
and activate
C3G,kinase 2
(TYK2) that is bound to the type I interferon (IFN)
(GEF)
to increased
RAP1 activity.
receptorleading
subunit IFNAR1.
After engagement
of the type
I IFN receptor by an IFN, CRKL associates with TYK2
and undergoes rapid tyrosine phosphorylation. The
activated form of CRKL forms a signaling complex with
STAT5, which also undergoes TYK2-dependent
tyrosine phosphorylation. The CRKL–STAT5 complex
translocates to the nucleus and binds specific GAS
(IFN-activated site) elements that are present in the
promoters of certain IFN-stimulated genes (ISGs),
which initiates transcription of these genes. The specific
GAS sequence bound by CRKL–STAT5 is shown. The
IFN-dependent phosphorylation (activation) of CRKL
also results in induction of the GEF activity of C3G.
C3G subsequently regulates the small G-protein RAP1,
resulting in activation of this GTPase, which may then
promote growth-inhibitory responses JAK, Janus
activated kinase.
Nat Rev Immunol 376 | MAY 2005 |
Mechanisms of
activation of MAP
kinase, p38 and its
downstream effectors
by type I interferons
THM: Tyk2 and Jak1 can also
Interferon (IFN)-activated JAKs regulate the
directly activate
GEFsof VAV or other
phosphorylation
(activation)
guanine-nucleotide-exchange factors (GEFs),
resulting in downstream activation of RAC1
and, possibly, other small G proteins (SGPs)
that can regulate the signaling pathway of the
mitogenactivated protein kinase (MAPK) p38. A
MAPK kinase kinase (MAPKKK) is
subsequently activated and regulates
downstream activation of the MAPK kinases
MAPKK3 and MAPKK6, which directly
phosphorylate p38, resulting in its activation.
Activated p38 subsequently regulates activation
of multiple downstream effectors, including
MAPK-activated protein kinase 2 (MAPKAPK2),
MAPKAPK3, mitogen- and stress-activated
kinase 1 (MSK1) and MAPK-interacting protein
kinase 1 (MNK1). IFNAR1, type I IFN receptor
subunit 1; IFNAR2, type I IFN receptor subunit
2; TYK2, tyrosine kinase 2.
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Nat Rev Immunol 376 MAY 2005
JAK2-mediated activation of STATs and ERK/MAPK by
GH or a growth factor (GF)
PI3K
AKT
THM: Just because effect
is due to a cytokine,
doesn’t mean that it has
to be STAT pathway
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Current Biology Linda A. Winston, Tony Hunter 1996, 6:668-671
Activation of the JAK-STAT pathway via AT1R
MSH
5HT
Ang II activates JAK2 via the G protein-dependent and -independent mechanisms leading to gene transcription
and vasoconstriction. Various second messengers including PKC, Pyk2, Arhgef1 and SHP2 are involved in
these pathways.
JAK-STAT 1:4, 250–256;
Please - Don’t get behind on
reading. You can be sure that
some of exam questions will
come from the readings.
For example, in the research paper I assigned that
came out just last week,
Do you think that the authors are correct when they
say that these inflamasomes that contain NLRP3 are
direct binders and effectors for cAMP action on
immune system??
31
SOCS can regulate insulin pathway at several points
Flier 06
SOCS proteins inhibit insulin receptor signaling by binding to the insulin receptor, thereby blocking access of signaling
intermediates and inhibiting insulin receptor tyrosine kinase activity, leading to a reduction in insulin-receptor directed
phosphorylation of IRS-1 and its downstream events, and by targeting IRS-1 and IRS-2 for proteosomal degradation.
Abbreviations: PKB, protein kinase B (also known as Akt); PDK1 and 2, phosphoinositide-dependent kinase 1 and 2; PI(4,5)P2,
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phosphatidylinositol (4,5)- bisphosphate; PI(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate); Shr, C-terminal SH2 domaincontaining adaptor protein.
Termination of
STAT1 signaling via
acetylation
IFNs induce STAT1 signaling. The
nuclear HAT CBP catalyzes
acetylation of phosphorylated
nuclear STAT1. Subsequently,
TCP45 is recruited and STAT1
becomes dephosphorylated, exits
the nucleus, and acquires latency.
THM: One more mechanism
of regulation
HAT
TCP45 is a tyrosine phosphatase
CBP is a HAT and
CBP is a CREB binding protein
Kramer & Heinzel, Molecular and Cellular
Endocrinology (2009)
Tyr Ptase
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A phospho-acetyl switch
controls STAT1 signaling
A phospho-acetyl switch controls STAT1 signaling.
Modifications of STAT1 are dynamically regulated. A
phospho-acetyl switch controls STAT1 upon activation by
IFN. Serine phosphorylation of STAT1 regulates repressive
sumoylation of STAT1 (pY, tyrosine phosphorylation; Ac,
lysine acetylation; pS, serine phosphorylation;
Su,sumoylation; MAPK,MAPkinases). STAT1/STAT2
heterodimers serve as example.
Acetylation of STAT1 antagonizes its IFN-induced
phosphorylation. The balance between STAT1 acetylation
and phosphorylation determines STAT1 activity and IFN
signaling. STAT1 homodimers serve as an example.
34
Kramer & Heinzel, Molecular and Cellular
Endocrinology (2009)
Intracellular sensors in innate immunity to viruses:
a mechanism for control of cytokine synthesis
After induction many
cytokines need to be
activated by proteolytic
clipping of the
prohormone
PRRs = pattern recognition receptors
TLRs ( Toll-like), RLRs (RIG-1-like), CLRs (C-type lectin), &
NLRs (nucleotide binding domain leucine rich repeats
CARD = caspase recruitment domain
Figure 1 | Intracellular sensors in innate immunity to viruses. Viral pathogen-associated
molecular patterns (PAMPs) activate nucleotide-binding oligomerization domain (NOD)-like
receptors (NLRs) and inflammasomes to initiate signalling cascades that lead to the production
of pro-inflammatory cytokines, thereby amplifying antiviral innate immune responses. In the
presence of viral PAMPs, NLR family PYD-containing protein 3 (NLRP3) and absent in
melanoma 2 (AIM2) oligomerize and recruit the adaptor protein apoptosis-associated speck-like
protein containing a CARD (ASC) through homotypic pyrin domain (PYD) interactions. The
caspase-recruitment domain (CARD) of ASC binds the CARD of pro-caspase-1, leading to
caspase-1 activation and the production of interleukin-1β (IL-1β) and IL-18 through cleavage of
pro-IL-1β and pro-IL-18. Retinoic acid inducible gene-I (RIG-I) contains an RNA helicase domain
and an amino-terminal CARD. The helicase domain of RIG-I senses the 5ʹ-triphosphate moiety
of single-stranded (ss)RNA virus genomes and then signals through CARD–CARD interactions
with the adaptor molecule mitochondrial antiviral signalling protein (MAVS). This results in the
phosphorylation and activation of interferon (IFN) response factor 3 (IRF3) and IRF7 to turn on
the transcription of type I IFN (IFNα/β) genes. RIG-I also regulates IL-1β production
transcriptionally and post-translationally following recognition of 5ʹ-triphosphate double-stranded
(ds)RNA. Whereas RIG-I-triggered transcription of pro-IL-1β depends on nuclear factor-κB (NFκB) activation and is mediated by MAVS, inflammasome formation, caspase-1 activation, and
IL-1β and IL-18 production in response to RIG-I activation involve ASC. The NLRs NOD2, NLR
family member X1 (NLRX1) and NLR family CARD-containing protein 5 (NLRC5) associate with
MAVS. Whereas NOD2 mediates the induction of type I IFNs, NLRX1 and NLRC5 inhibit RIG-I–
MAVS interactions and thereby negatively regulate type I IFN production. LRR, leucine-rich
repeat; MAPK, mitogen-activated protein kinase; MYD88, myeloid differentiation primaryresponse protein 88; RIPK2, receptor-interacting serine-threonine protein kinase 2; ROS,
reactive oxygen species; TLR, Toll-like receptor; TNF, tumour necrosis factor; TRIF, TIR-domain36
containing adaptor protein inducing IFNβ.