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.
Slide 4
Nature Reviews Immunology 10, 236-247 TH1 like response TH2
like response Antigen Cytokines secreted by immune cells instruct
T-cells Effector B cells ('Be1' and 'Be2' cells) can secrete
cytokines, such as interferon- (IFN), interleukin-12 (IL- 12), 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.
Slide 5
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, 154163 (2013)
Slide 6
3 General types of cytokine receptors Q: why did nature evolve
multiple chain receptors? Epo, GH, Prl, GM-CSF Class I Single chain
(dimer) Class II Class III IL-2, Interferons Multiple unique chains
(2 or 3) IL-6, LIF, IL-11, CNTF), CT-1, CLC, OSM, IL-27 and IL-31.
Unique + Common chains (tetramer) (gp130)
Slide 7
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 Canonical
JAKSTAT pathway Activating cross Tyr PO 4 of JAK PO 4 of JAK
Binding & PO 4 of STAT THM: 3 Tyr-P required; catalyzed by a
kinase that is NOT a part of the receptor Dimerization of
STATs
Slide 8
So, lets go into more detail about each of these players
Slide 9
8 Structural organization 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); TAD:
Transcription activation domain; FERM ( band4.1, ezrin,radixin,
& moesin) binds to STATS and other proteins FERM DBD SH3 SH2
TAD Stat2/p300(CBP) Stat1/p300(CBP) Stat5/ERK Stat1/MCM5 Stat5/Nmi
Stat2/p48 Stat1/p300(CBP) Stat1/PIAS1 Stat1/p48 Stat3/c-Jun YPYP
SPSP Signal Transducers and Activators of Transcription Different
regions have different functions or bind different transcriptional
regulators At least 6 families of STATS
Slide 10
Tyr SH2 STAT monomers STAT dimer (binds DNA) Phosphorylation
and SH2-phosphotyrosine binding How do SH2 and DNB domains work?
FERM DBD SH3 SH2 TAD YPYP SPSP P
Slide 11
(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).
SH2 domain Linker domain DNA-binding domain Coiled coil domain
Structure of STAT bound to DNA Nutcracker Model
Slide 12
11 The core structure (amino acids 130712) shows binding of a
STAT1 dimer to DNA and the location of binding sites of various
proteins in various domains. The amino- terminal 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, N- Myc interactor; PIAS, protein
inhibitor of activated STAT. Nature Reviews Molecular Cell Biology
3; 651-662 STAT domain structure and protein binding sites
Slide 13
12 Structure & number of Jaks 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 whose
precise function has not yet been determined. Pseudokinase Domain
Kinase Domain N C JH7 JH6 JH5 JH4 JH3 JH2 JH1 Tyk2140 kDa
36%19p13.2Ubquitous Jak1135 kDa36% 1p31.2Ubiquitous Jak2130
kDa47%10p23Ubiquitous Jak3120 kDa - 4q31Myeloid/Lymphoid Size
Identity Chromosome Expression JANUS KINASES Q: What does acronym,
JAK stand for? FERM
Slide 14
13 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 of the heavens.
Slide 15
14 Different cytokine receptors bind different combinations of
Jaks TYPE HORMONE PHOSPHORYLATION Single specificity Growth Hormone
Homo-phosphorylation Promiscuous IL-6Multi- phosphorylation (all
JAKs) Obligate Hetero INF , INF Hetero-phosphorylation (2 different
JAKs) INF Jak1 Jak2 INF Jak1 Tyk2 (Mutate Jak1 ---> no Tyk2 PO 4
(Mutate Jak1 ---> no Jak2 PO 4 PO 4 O How determined? Why
important? Different Jaks PO 4 different STATS
Slide 16
15 Interferon receptors and activation of classical JAKSTAT
pathways by type I and type II interferons From Nat Rev Immunol 376
| MAY 2005 All type I interferons (IFNs) bind a common receptor
which is known as the type I IFN receptor. The type I IFN receptor
is composed of two subunits, IFNAR1 and IFNAR2, which are
associated with the Janus activated kinases (JAKs), tyrosine kinase
2 (TYK2) and JAK1, respectively. A single type II IFN, IFN-g, binds
a distinct cell-surface receptor, 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 STAT1STAT2IRF9 (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. Example of how one can get specificity of function by
different interferons by expressing and utilizing different
combinations of cytokine receptors, Jaks and STATS
Slide 17
How is Cytokine Function Regulated? A: Several types of
negative feedback
Slide 18
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).
Rico-Bautista et al 2006 Other SOCS Box containing proteins CIS =
Cytokine-Induced SH2 protein; SOCS = Suppressor of Cytokine
Signaling SSI = STAT-induced STAT Inhibitor Kinase domain binding
(Kinase Inhibitor) P-tyrosine binding (STAT competitor Elongin B/C
binding (ubiquitination)
Slide 19
18 (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. 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. Negative regulation of cytokine
signaling: STAT- induced STAT inhibitor Naka et al.,TiBs,
24:394-398 SSI-1 type inhibition CIS-1 type inhibition THM - 2
sites of inhibition (Jaks or STAT binding to Receptor) = SOCS1 SOCS
bind to and inhibit JAKs CIS inhibits STAT binding
Slide 20
19 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
dominant- negative proteins by occupying DNA as non- functional
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 Nature Reviews Molecular Cell
Biology 3; 651-662 (2002); STATS: Transcriptional control and
biological impact Other negative regulators of STAT proteins Why so
many different mechanisms for controlling STATS?
Slide 21
20 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 co- repressor proteins
such as histone deacetylase (HDAC). c | PIAS proteins can promote
the conjugation of small ubiquitin- related modifier (SUMO) to
STAT1. The significance of STAT1 sumoylation in regulating STAT1
activity is controversial and needs to be clarified Nature Reviews
Immunology 3; 900-911 Proposed mechanisms for inhibiting the JAK
STAT pathway by PIAS proteins Block DNA binding Act as co-
repressors Promote sumoylation
Slide 22
21 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. STAT1 can be modified by SUMO at lysine residue
703. Direct interactions between PIAS1 and STAT1 may interfere with
the STAT1 ability to bind DNA. Biochemical Pharmacology 70 (2005)
649657
Slide 23
Example of Feedback Regulation by SOCS
Slide 24
23 Leptin induces SOCS-3, but not CIS, SOCS-1, or SOCS-2, mRNA
in CHO cells expressing the long form of the leptin receptor. The
role of SOCS-3 in Leptin signaling and resistance J Flier lab JBC
274:30059 99 CIS mRNA SOCS-1 mRNA SOCS-2 mRNA SOCS-3 mRNA CHO-OBRI
O hr 1hr 2 hr 4hr stimulation with leptin THM - specificity of
induction and different time courses THM - induction of SOCS-3
causes decreased cytokine coupling
Slide 25
How important are SSI proteins? Studies with SOCS1 KO mice
Slide 26
25 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. Abbreviations: JAK, Janus tyrosine kinase, SSI,
STAT-induced STAT inhibitor; STAT, signal transducers and
activators of transcription. Naka et al.,Trends in Biochemical
Sciences, 24:394-398 SOCS-1 KO ---> Post-Embryonic Lethal
Rescued by cross of heterozygotes to INF- KO
Slide 27
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
Slide 28
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 form that constitutively associates with the
guanine-nucleotide- exchange factor (GEF) C3G. A member of the STAT
(signal transducer and activator of transcription) family of
proteins, STAT5, is associated with tyrosine kinase 2 (TYK2) that
is bound to the type I interferon (IFN) receptor 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
CRKLSTAT5 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
CRKLSTAT5 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 | Jak2 can also phosphorylate CRKL allowing it to be active
itself just like a STAT and form a heterodimer It can also scaffold
and activate C3G, (GEF) leading to increased RAP1 activity.
Slide 29
28 Interferon (IFN)-activated JAKs regulate the phosphorylation
(activation) of VAV or other 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. Mechanisms of activation of MAP kinase, p38 and
its downstream effectors by type I interferons Nat Rev Immunol 376
MAY 2005 THM: Tyk2 and Jak1 can also directly activate GEFs
Slide 30
29 JAK2-mediated activation of STATs and ERK/MAPK by GH or a
growth factor (GF) Current Biology Linda A. Winston, Tony Hunter
1996, 6:668-671 THM: Just because effect is due to a cytokine,
doesnt mean that it has to be STAT pathway PI3K AKT
Slide 31
MSH 5HT JAK-STAT 1:4, 250256; 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. Activation of the JAK-STAT pathway via AT1R
Slide 32
31 Please - Dont 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??
Slide 33
32 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,
phosphatidylinositol (4,5)- bisphosphate; PI(3,4,5)P3,
phosphatidylinositol (3,4,5)-trisphosphate); Shr, C-terminal SH2
domain- containing adaptor protein. SOCS can regulate insulin
pathway at several points Flier 06
Slide 34
33 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. Kramer & Heinzel, Molecular and Cellular Endocrinology
(2009) THM: One more mechanism of regulation HAT Tyr Ptase TCP45 is
a tyrosine phosphatase CBP is a HAT and CBP is a CREB binding
protein
Slide 35
34 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. Kramer & Heinzel, Molecular and
Cellular Endocrinology (2009) 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.
Slide 36
Intracellular sensors in innate immunity to viruses: a
mechanism for control of cytokine synthesis 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 After induction many
cytokines need to be activated by proteolytic clipping of the
prohormone
Slide 37
36 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 1 (IL 1) and IL 18 through cleavage of pro-IL 1 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 5 -triphosphate moiety of single-stranded (ss)RNA
virus genomes and then signals through CARDCARD 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 (IFN/) genes. RIG I also regulates IL 1
production transcriptionally and post-translationally following
recognition of 5 -triphosphate double-stranded (ds)RNA. Whereas RIG
I-triggered transcription of pro-IL 1 depends on nuclear factor-B
(NF- B) activation and is mediated by MAVS, inflammasome formation,
caspase 1 activation, and IL 1 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 primary- response protein 88; RIPK2,
receptor-interacting serine-threonine protein kinase 2; ROS,
reactive oxygen species; TLR, Toll-like receptor; TNF, tumour
necrosis factor; TRIF, TIR-domain- containing adaptor protein
inducing IFN.