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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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