Mechanism of Interleukin 2-Induced Signal Transduction...multiple kinases trigger multiple signaling...
Transcript of Mechanism of Interleukin 2-Induced Signal Transduction...multiple kinases trigger multiple signaling...
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Mechanism of Interleukin 2-Induced
Signal Transduction
Hironobu Asao
Department of Immunology, Yamagata University School of Medicine,
Yamagata, Japan(Accepted April 2, 2003 )
ABSTRACT
Address for Correspondence : Hironobu Asao, Department of Immunology, Yamagata Univer-sity School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan
Extensive studies on the IL-2 and IL-2 receptor system have established fundamental
principles of immune responses and signal transducing mechanisms of hematopoietic
cell proliferation and survival. IL-2Rb and IL-2Rg chains are associated with multiple
tyrosine kinases including JAK1, JAK3 and other tyrosine kinases such a p56lck. IL-2
binding to these receptors leads to conformational changes of the receptors followed by
the recruitment of several kinases leading to their activation. The JAKs and activated
tyrosine kinases appear to be a trigger for the downstream signal cascades. Other
downstream messenger molecules such as the STATs, Shc, PI3K-Akt, Ras-ERK1/2 and
so on, cross talk with this cascades. The interplay however leading to the regulation of
target genes still remain largely obscure. For example, although phosphorylation of
tyrosine 338 of IL-2Rb is required for both signal transduction to Shc and PI3K, how it
regulates further signals from Shc to the Grb2-Sos-Ras pathway or to the PI3K-Akt
pathway is unknown. There are also conflicting reports on the functional roles of
STAT5, STAT1 and STAT3 on IL-2 signaling. Further work is needed to clarify how the
multiple kinases trigger multiple signaling cascades and cooperate with each other in
the regulation of gene induction for cell growth, survival or differentiation in different
cell types and conditions in vitro and also in vivo.
Key words:IL-2, tyrosine kinase, signal transduction, cytokine
Yamagata Med J 2003;21(2):141-154
many other soluble mediators, play a pivotal
role in the development and regulation of the
immune system. Interleukin 2 (IL-2) is one of
the earlier discovered cytokines secreted by
INTRODUCTION
Cytokines, which include interleukins and
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activated peripheral blood lymphocytes and
shown to promote T lymphocyte expansion1).
IL-2 is also known to activate lymphokine-
activated killer (LAK), cytotoxic T lymphocyte
(CTL) activity, and to promote B lymphocyte
proliferation and immunoglobulin secretion2).
IL-2 binds to target cells through its
receptors expressed on the cell surface and
transduces its signals into the cells. IL-2
receptor (IL-2R) consists of three distinct
subunits, IL-2Rα chain (IL-2Rα), IL-2Rβ
chain (IL-2Rβ) and IL-2Rγ chain (IL-2Rγ)3)-7).
IL-2 forms a heterotrimer with these receptors
and this heteromerization provokes tyrosine
phosphorylation of these receptors8)-11), which is
thought to be a first event in the intracellular
signal transduction. Importantly these recep-
tors have no intrinsic kinase activity, unlike
classical growth factor receptors such as
epidermal growth factor receptor (EGF-R) and
pletelet derived growth factor receptor (PDGF-
R). Because of vigorous studies to identify the
kinases catalyzing such protein phosphory-
lation especially on tyrosine residues, many
kinds of tyrosine kinases were found to be
involved in the IL-2 signaling system12).
Here we will discuss the mechanisms of
signal transduction from the IL-2Rs to the
nucleus through multiple tyrosine kinase
concerted systems.
IL-2 receptors
IL-2Rα (CD25) is a type I membrane
protein whose molecular weight is 55 kD. IL-
2Rα has only 13 amino acids in its cytoplasmic
region. However IL-2 binds to IL-2Rα with
low affinity (Kd: 10 nM), and is thought to
have no function on the intracellular signal
transduction13). The expression of IL-2Rα is
tightly restricted on peripheral activated T and
B cell, triple negative thymocytes and bone
marrow pre-B cells. The most important
function of IL-2Rα is forming a functional
high affinity (Kd: 10 pM) receptor on these
cells together with IL-2Rβ and IL-2Rγ2)
(Fig.1). IL-2Rβ (CD122) and IL-2Rγ (CD132)
are also type I membrane proteins whose
molecular weight is 75 kD and 64 kD
respectively, and belong to the cytokine
receptor family which is characterized by the
presence of a cytokine receptor domain of
about 210 amino acids in the extracellular
region14),15). This domain is separated by two
homologous type III fibronectin subdomains.
The first subdomain is characterized by the
presence of two pairs of cysteine residues at
the N-terminal part, and the second subdo-
main has a consensus tryptophan-serine-X-
tryptophan-serine motif (WS motif). The
heterodimer composed of IL-2Rβ and IL-2Rγ
can bind to IL-2 with intermediate affinity
(1nM) and both receptors are necessary for a
functional IL-2 receptor complex16)-20). Cytokine
receptor family members are also character-
ized by the absence of intrinsic tyrosine kinase
activity. Which kinases phosphorylate the
different intracellular proteins and transduce
downstream signals after IL-2 binding to its
receptor?
The JAK (Janus tyrosine kinase) family
protein tyrosine kinases
Four members of the JAK family kinases,
JAK1, JAK2, JAK3 and Tyk2, were found as
non-receptor type tyrosine kinases in the
mammalian system. Many reports showed that
these family members are deeply involved in
the signal transduction from all cytokine
receptors21). JAK1 and JAK3 were phosphory-
lated on tyrosine residue after IL-2 treatment,
and were reported to be associated with IL-
2R β and IL-2Rγ respectively in an IL-2-inde-
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pendent manner22)-27) (Fig.1). The JAK associa-
tion sites on these receptors are the S-region,
including the Box1 of IL-2Rβ , and the portion
from amino acid 38 through 56 of the
intracytoplasmic region of IL-2Rγ.
The indispensability of the JAK-associated
region for IL-2-induced cell proliferation
signaling, was shown with experiments using
deletion mutants of IL-2Rβ and IL-2Rγ.
However, a mutation of Box1 in the IL-2Rβ
can transduce IL-2-induced cell proliferation
signals together with wild type IL-2Rγ in a T
cell line, although the mutant IL-2Rβ cannot
bind nor activate JAK128). Migone, et al.
reported that JAK1 is important for the
efficient recruitment of the p85 molecule of
phosphatidylinositol 3-kinase (PI3Kp85) to IL-
2Rβ29). In addition PMA-stimulated thymo-
cytes from JAK1-deficient mice showed a
reduced response to IL-2 30). Taken together
these results indicate that there may be two
pathways of IL-2-induced signaling; JAK1
dependent and JAK1 independent.
Miyazaki, et al. reported JAK3 is required
for the IL-2-induced DNA synthesis in NIH-
3T3 cell expressing IL-2Ra, β and γ, since
JAK3 is not expressed in the parent cells27).
Furthermore a dominant negative form of
JAK3, whose kinase domain was deleted,
inhibited IL-2-induced JAK1 activation, c-fos
and c-myc gene induction, and IL-2-dependent
cell growth, but not bcl2 induction31). On the
other hand, Nelson, et al. demonstrated that
not only JAK3 activation, but also the
membrane proximal domain of IL-2Rγ is
indispensable for IL-2-induced T cell growth
Fig.1. IL-2-induced tyrosine phosphorylation of IL-2 receptor β chain.IL-2 binding to IL-2 receptor complex, induces activation of many types of tyrosine kinases followed by tyrosine phosphorylation of the receptors. Phosphorylated tyrosine residues recruit second messengers like Shc, PI-3K and STATs. Cysteine repeat and WS motif on type III fibronectin subdomain homology of IL-2Rβ are indicated as c-c and WS, respectively. S, A and H regions of IL-2Rβ are indicated as S, A and H, respectively.
IL-2 signaling
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signals32). Tsujino, et al. showed that a C-
terminal deletion mutant of IL-2Rγ, which
cannot associate with JAK3, still activated
JAK1 and STATs, and phosphorylated
PI3Kp8533). These results suggest the existence
of a JAK3 independent signaling pathways.
JAK3 gene targeted mice had no NK cells, a
drastic reduction in thymocytes and showed a
severe block in B cell development at the pre-B
stage in the bone marrow, in addition to having
functionally deficient residual T and B
cells34)-36). Although these phenotypes were
similar to that of IL-2Rγ-deficient mice 37)-39),
they are not thought to be due to the deficiency
of IL-2, but rather due to IL-7, IL-15 or IL-21
signaling that are also using the IL-2Rγ-JAK3
system40)-43). It is clear that these JAKs are the
key enzymes for the downstream signaling
events. After IL-2 binding to its receptor, the
conformation of heterodimer or heterotrimer
receptor is changed and these JAKs might be
close to each other and become cross-activated.
How do activated JAKs transduce signals?
STAT (Signal Transducer and Activator
of Transcription) family
All the cytokine receptor family members
activate JAKs after cytokine binding. STATs
are thought to be major signaling molecules
which are in the downstream of activated
JAKs44). Activated JAKs phosphorylate tyrosine
residues on its receptor itself. STATs are
thought to recognize phosphotyrosine on the
receptor through its SH2 domain. Recruited
STATs on the receptor are tyrosine phosphory-
lated on the C-terminal part by JAKs then
separate from the receptor and form a homo or
heterodimer complex via reciprocal interac-
tions between their SH2 domains. Dimerized
STATs are further phosphorylated on serine
residue by serine/threonine kinases and
become maximally activated. Finally, activated
STATs translocate to the nucleus and function
as transcriptional factors.
In the IL-2 system, STAT5 is mainly
activated after stimulation45)-54) (Fig.1). Fried-
mann, et al. reported tyrosine 338, 392 and 510
of IL-2Rβ were phosphorylated after IL-2
binding55). IL-2Rβ mutants in which tyrosine
392 and 510 were replaced by phenylalanine,
could not activate STAT5 and reduced IL-2-
induced 32D mouse myeloid cells proliferation.
In contrast to this, BaF-B03 mouse pro-B cells
expressing the IL-2Rβ mutant deleted of the
C-terminal half including STAT5 binding sites
(tyrosine 392 and 510), proliferate in response
to IL-2 without activation of STAT5 just as
well as IL-2Rβ wild type expressing cells47). By
using dominant-negative forms of STAT5,
STAT5 was shown to participate in IL-3-
dependent proliferation56), 57). They also showed
cis, osm, pim-1 and cyclin D1 as the target
genes of STAT5. To investigate the function of
STAT5 directly, Onishi, et al. screened
randomly mutated STAT5 which can maintain
IL-3-dependent Ba/F3 cells without IL-3 58).
They identified constitutive active types of
STAT5 which induce cytokine independent
growth of Ba/F3. In this active form of STAT5
expressing cells, pim-1, bcl-x and c-myc are
upregulated without IL-3 59). Interestingly, IL-3
treatment to these cells leads to differentiation
and apoptosis accompanied with strong
induction of JAB, CIS and p21WAF1/Cip1.
These results suggest STAT5 activity may
regulate cell fate through the control of the
target genes expressions (Fig.2).
There are two genes highly conserved for
STAT5, STAT5a and STAT5b. Teglund, et al.
established STAT5a and b-double-deficient
mice, and noticed that the lymphoid develop-
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ment is normal 60). Anti-CD3 antibody-induced
cell proliferation of peripheral T cell from
STAT5a/b deficient mice is profoundly reduced
in the presence or absence of IL-2 or IL-4 61), 62).
So STAT5 seems to have an essential role in IL-
2 and IL-4-induced T cell proliferation through
some cell cycle regulating genes like p27Kip1,
cyclin D2, D3, E, A and cdk6. However, there
are also conflicting results with the phenotypes
of STAT5a/b deficient mice. Fujii, et al.
established mutants IL-2Rb transgenic mice
on the IL-2Rβ null background 63). When IL-
2Rβ mutants lacking STAT5 binding sites (H-
region) were transgened, spleen cells could not
induce IL-2R α expression, but however
responded to IL-2 normally without activation
of STAT5 after TCR cross-linking with anti-
CD3 antibody. Furthermore, by using a
retroviral transfection system, Van Parijs, et al.
reconstituted mutant IL-2Rβ into the periph-
eral T cells of IL-2Rβ deficient mice, and
clearly demonstrated either H-region or
tyrosine 338 where is in A-region of IL-2Rβ is
required for the IL-2-induced T cell prolifera-
tion after TCR cross-linking 64)(Fig.1). The
functional role of STAT5 for cell proliferation
seems to be important but not indispensable
and they show STAT5 is not required for cell
survival. Taken together, it is unclear why T
cells from STAT5a/b deficient mice cannot
respond to IL-2 at all. One possible explanation
is that STAT5 may have some indispensable
role in the maintenance of essential compo-
nents of the IL-2/IL-2R system. For example,
in the case of STAT1, basal expression of
STAT1 is required for the maintenance of
caspase expression65). Another explanation is
the impairment of not only IL-2/IL-2R system,
but also TCR signaling in the STAT5a/b
deficient mouse because STAT5 is reported to
be involved in TCR signaling in mouse D10 cell
line 66). The duration of transient expression of
IL-2R α in TCR-stimulated T cells from
STAT5a/b deficient mice is much shorter than
that of T cells from wild type mice 62). However,
this is not the reason why STAT5a/b deficient
T cells lose their response to IL-2, as IL-2Rα
overexpressed STAT5a/b deficient T cells still
show no proliferative response to IL-2 64).
Further analysis seems to be necessary to
explain the functional significance of STAT5 in
the T cell proliferation through the TCR and
IL-2/IL-2R system.
Unique functions of IL-2 in vivo are the
termination of T cell activation and mainte-
nance of tolerance. Fas/Fas L system is though
to be the main machinery of activation-induced
cell death of activated T cells. Van Parijs, et al.
also showed STAT5 activation is necessary for
the induction of Fas L and activation-induced
cell death64). These results raise another
question of how IL-2 regulates T cell activation
and T cell death through the same pathway.
Besides STAT5, IL-2 activates STAT1 and
STAT3, and recent reports showed that STAT1
and STAT3 are associated through the acidic
region (A-region) of IL-2Rβ which is a
different portion from the STAT5 binding
site67) (Fig.2). Akaishi, et al. demonstrated that
IL-2-induced IL-2Rα induction was impaired
in peripheral T cell from T cell specific STAT3
deficient mice 68). The functional roles of STAT1
in IL-2/IL-2R system are unclear.
STATs activities are secondary regulated
with serine phosphorylation44). Some reports
show serine/threonine kinases as the candi-
dates which catalyze this phosphorylation;
ERK1/2 for STAT5 69), mTOR for STAT3 70) and
p38MAPK for STAT1 71). ERK1/2 is also
reported to be an inhibitor of STAT3 72), 73). In
IL-2 signaling
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Fig.2. IL-2-induced signaling network.Molecules thought to be involved in the IL-2 signaling network and some regulated genes which are related to cell proliferation and survival are shown.
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contrast with this, another report suggests
ERK1/2 is not a kinase for STAT5 74). The
entire network of STATs activation is still
obscure.
p52shc
Several cytokines including EGF and PDGF
activate Ras and its downstream signal
transducer ERK1/2. Burns, et al. showed IL-2
induces tyrosine phosphorylation of p52shc
adaptor protein in T cells 75). Later Shc is
demonstrated to be associated with IL-2Rβ
through phosphorylated tyrosine 338 and PTB
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domain of Shc 55), 76) (Fig.1). Phosphorylated Shc
is thought to recruit Grb2 and induces a
downstream cascade involving Sos, Ras, Raf
and ERK1/2 77), 78). Finally ERK1/2 translocates
to the nucleus and participates in the induction
of genes such as c-fos. p56lck is the first
identified tyrosine kinase associated with the
IL-2R 79). p56lck is associated with the A-region
of IL-2Rβ and p56lck activation leads to the
induction of c-fos and c-jun 80), 81). However, the
relationship between the Shc-Grb2 pathway
and p56lck pathway is unclear. Lord, et al.
reported that Shc activation is enough to
activate MAP kinase, induce c-fos and c-myc
genes and promote cell proliferation with
chimeric receptor of IL-2Rβ proximal region
and Sch fusion protein 82). They also showed
that the chimera receptor can induce cell
survival regulator genes, bcl-2 and bcl-x, but
this activity is not sufficient to sustain the long-
term cell viability. What molecules besides the
Ras-ERK1/2 pathway, are in the downstream
of Shc to regulate the bcl-2 and bcl-x should be
clear.
Phosphatidylinositol 3-kinase (PI3K)-Akt
pathway
IL-2 is well known to activatePI3K 83)-85).
Later Akt (serine/threonine protein kinase B
(PKB)) was reported to be at the downstream
of PI3K 86). Akt activity is thought to play
important roles in many cytokine and growth
factor-induced cell survival. Van Parijs, et al.
showed Akt phosphorylation is dependent on
tyrosine 338 of IL-2Rβ which is the same
interaction site of Shc, and signal through
tyrosine 338 of IL-2Rβ is sufficient for T cell
growth and survival 63). PI3K-Akt cascade may
interact with the tyrosine 338 directly and
transduce T cell survival signals instead of Shc.
This model is supported by the data that a
catalytically active Akt partially protects cells
from IL-3-withdrawal-induced apoptosis and
promotes cell cycle progression87). They also
showed c-myc and bcl2 are constitutively
upregulated in the active Akt expressing cells.
On the other hand, E2F, the key molecule in
the cell cycle, is activated with IL-2 88). By using
a transient reporter assay, they showed IL-2-
induced E2F activation is blocked with
dominant negative mutant forms of PI3K or
the PI3K inhibitor, LY294002. LY294002 also
inhibited IL-2-mediated induction of cyclin D3
gene and down-regulation of p27kip1. In this
way, IL-2 may induce phosphorylation of Rb
and p130, activate E2F and progress cell cycle
at least through PI3K-Akt pathway.
Another pathway for cell survival through
Akt is that involving BAD phosphorylation.
Akt is reported to phosphorylate BAD, which
belongs to Bcl2 family and induces apopto-
sis89),90). Phosphorylated BAD by Akt is inac-
tivated and cells are protected from apoptosis.
However the conclusion of Akt as a BAD
kinase is arguable because BAD kinase could
be different from Akt, for example MEK-
ERK1/2 pathway, and responsible kinases in
BAD phosphorylation may be dependent on
individual cytokines 91). These results suggest
PI3K-Akt pathway may contribute to IL-2-
induced cell cycle progression, cell proliferation
and survival (Fig.2).
Pyk2 pathway
Pyk2 is a member of the FAK (focal adhesion
kinase) family protein tyrosine kinase. Mi-
yazaki, et al. reported that IL-2 activates Pyk2
which is associated and activated with JAK3 92).
The kinase domain deleted mutant of Pyk2,
blocked IL-2-mediated cell proliferation. How-
ever the downstream molecules of Pyk2 are
still unknown.
IL-2 signaling
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Syk pathway
Syk is a member of the ZAP70 family protein
tyrosine kinases which are characterized by
the presence of two SH2 domains. Generally
Syk and ZAP70 are involved and have pivotal
roles in BCR and TCR signaling pathway
respectively. Syk is also associated with the S-
region of IL-2Rβ and activated by IL-2
stimulation 93). Cross-linking of membrane an-
choring type of Syk mutant leads to c-myc gene
induction.
mTOR (the mammalian target of rapamycin)
Rapamycin, a lipophilic macrolide antibiot-
ics, inhibits IL-2-induced T cell proliferation in
the late G1 phase. Rapamycin works on a
serine/threonine kinase, mTOR as a complex
with FKBP12 and inhibits mTOR activity. One
possible target of mTOR is PHAS-1 which is
involved in the regulation of protein transla-
tion94). Another molecule that is activated
through mTOR is p70S6 kinase which
phosphorylates S6 ribosomal protein and
regulates the translation95). These findings
suggest that rapamycin interacts with the
translational regulator protein and inhibits its
function. So rapamycin may inhibit IL-2-
mediated signal transduction at the transla-
tional level.
STAM (signal transducing adaptor mole-
cule) and Hgs/Hrs (HGF-regulated tyro-
sine kinase substrate)
In the IL-2-induced signaling, several protein
are phosphorylated on tyrosine residues.
Among these phosphoproteins, STAM and Hgs
were purified and their cDNA were cloned as
novel molecules 96)-98). STAM has an SH3
domain and a tyrosine cluster region with an
ITAM (immunoreceptor tyrosine-based activa-
tion motif). STAM is found to be associated
with JAK2 and JAK3 and phosphorylated with
not only IL-2, but also many other cytokines
which activate JAK2 or JAK3 99)(Fig.2).
Hgs/Hrs has FYVE finger domain, localizes to
early endosomes and thought to be implicated
in vesicular trafficking 100)-103). Because STAM
and Hgs/Hrs are associated with their coiled-
coil motifs to each other 98), STAM may play
some roles not only in the downstream of
JAKs, but also in vesicular transport.
CONCLUSIONS
IL-2-binding to the receptors causes confor-
mational change of the receptors followed by
the kinases recruitment including JAK1, JAK3
and other tyrosine kinases such p56lck, Pyk2
and Syk. These tyrosine kinases are activated
by each other and conduct many orchestrated
downstream signals. Activated kinases phos-
phorylate tyrosine residues on the receptors as
a trigger of downstream signal cascades. Many
downstream messenger molecules like STATs,
Shc, PI3K, which possess SH2 domain, bind on
the tyrosine-phosphorylated receptors and are
activated, and then transduce signals to
nucleus directly, or through Ras-ERK1/2
kinases or Akt kinase, respectively. However,
further work is needed to clear how multiple
kinase triggered-multiple signaling cascades
cooperate with each other and regulate gene
induction for cell growth, survival, differentia-
tion or cell death.
We thank Lishomwa C. Ndhlovu for reading
the manuscript and making many helpful
suggestions.
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