Roles of the JAK-STAT system in signal transduction via cytokine receptors

587

Roles of the JAK-STAT system in signal transduction cytokine receptors Sumiko Watanabe* JAK-STAT

signaling

role in the specific Many

cytokines

superfamily evidence

also appear

and Ken-ichi Arai

are known

activation

interacting

indicates

cytokine

pathways

to play an essential

of interferon-inducible with the cytokine

to activate

these

signaling,

including

linked to STATS and general

genes.

receptor

pathways.

that JAKs play an essential

receptor

role(s)

both specific

pathways

Recent

regulating

in

pathways cell growth

and functions.

Addresses Department of Molecular and Developmental Biology, Institute of Medical

Science,

University

of Tokyo, 4-6-l

Shirokane-dai,

Minato-ku,

Tokyo 108, Japan *e-mail: [email protected] Current

Opinion

0 Current

Biology

in Genetics

& Development

Ltd ISSN

1996,

6:587-596

0959-437X

Abbreviations CSFs EGF GM IFN JAK IL MAPK

colony-stimulating epidermal growth granulocyte interferon

factors factor

macrophage

SCF SCID STAT

Janus kinase interleukin mitogen-activated stem cell factor severe combined signal transducers

TGF-P

transforming

protein

kinase

immune deficiency and activators of transcription

growth

factor-p

Introduction play an important role in regulating diverse activities, including the proliferation and differentiation of hematopoietic cells. Each cycokine exhibits a variety of activities on several target cells (pleiotropy)-synergy and cross-talk are often observed among the activities of multiple cytokines. In addition, many cytokines elicit similar and overlapping activities on the same target cells

Cytokines

(redundancy), are non-linear

via

suggesting that cytokine signaling and form a network with multiple

pathways cross-talk

among different cytokines. To date, more than fifty cytokines-including interleukins (ILs), colony-stimulating factors (CSFs). interferons (IFNs), chemokines, the transforming growth factor-p (TGF-P) family, the tumor necrosis factor family, and growth factors -have been identified and are known to form complex cytokine networks. The pleiotropic and redundant natures of the cytokine activities predict possible features of cytokine receptors, namely that, first, a single cytokine has multiple receptors unique to a cell type, second, that different cytokines share a common receptor component, and third, that different cytokine receptors are linked to common signaling pathways.

After cDNA cloning of the receptors, common structural

genes for several cytokine features were found in the

extracellular domain and no sequence motif characteristic of a ryrosine kinase domain was observed. Subsequent studies have revealed that as many as twenty cytokine receptor subunits have similar sequence motifs that define a new cytokine receptor superfamily; this motif was also found in the previously defined growth hormone receptor and prolactin receptor. hloreover, sharing of a subunit by multiple cytokines was demonstrated and this fact partly explains the redundancy of activities of some cytokines as predicted by the second model above. These unique features helped predict several key factors that determine the nature of the signaling system of the cytokine receptor superfamily, namely: the role of shared subunit and redundancy of cytokine actions, the role of the subunit unique to each cytokine and specificity of cytokine action, and the expression pattern of receptor subunit and specificity of cytokine action. It was thought initially that the unique structural features, as well as the rather limited expression in hematopoietic cells, of the cytokine receptor superfamily may account for the cytokine-specific signaling mechanism. Several lines of evidence, however, show that cytokine receptors utilize previously known signaling systems, such as the MAPK/raf pathway which is common to many cell types, rather than utilizing a hematopoietic-specific signaling pathway. Ir thus was something of an enigma how cytokines exerted their specific actions. Phosphorylation of tyrosine residues of several cellular proteins, including the receptor itself, was observed following stimulation with cytokines and efforts have been directed to search for the tyrosine kinase(s) responsible for cytokine receptor signaling. Activation of several tyrosine kinases - such as Src family tyrosine kinases, Syk and Fps-by cytokines has been reported. JAK kinases are now known to play important roles in cytokine signaling but their roles have been rather neglected during the past; they attracted much attention only after rheir roles in the IFN system were recognized. In this article, we review recent findings of JAK involvement

in cytokine

receptor

signaling.

The JAK-STAT system JAK kinase

family

The JAK family of kinases in mammals consists of JAKl, JAKZ, JAK3 and TYKZ. This family was first identified independently either by polymerase chain reaction using a consensus sequence for tyrosine kinases or by low -stringency hybridization. JAKZ and JAKZ are expressed ubiquitously in many tissues whereas JAK3 and TYK2 are expressed in hematopoietic cells and in certain

588

Differentiation and gene regulation

carcinoma

cells

although

their

roles

in

hematopoiesis

remain undetermined. The JAK nomenclature is derived from Janus kinase (or often ‘just another kinase’; in mythology, Janus was the Roman god of gates who had two faces to keep a watch on opposite sides). The family has a common structural feature designated

number of cytokines, such as IL-2 and IL-3, appear to activate Src family kinases and the significance of these observations remained elusive. As a result, on the basis

of roles

of JAK

kinases

in IFN

signaling,

Ihle’s

JAK JH

group [3] have examined the role of JAK kinases in EPO receptor signaling and found that JAK2 is activated

segments JHl to JH7. The kinase domain JHl is located at the most carboxy-terminal portion and the pseudo kinase domain JHZ is at the amino-terminal side of JHl. No SHZ, SH3 or PH-like domains are found in JAK kinases. Interestingly, the dominant mutation of the Drosophila JAK homolog, llopscotc/rlirmofous-~f~a/ resulted in hematopoietic defects. No function of the JAK kinase in mammalian

in response to EPO. They have also shown that JAK2 associates with the region of EPO receptor required for mitogenic action and the tyrosine phosphorylation of cellular proteins induced by EPO [3]. Subsequent studies have revealed that all cytokines which interact with either the type I cytokine receptor family or with the IFN receptor activate member(s) of JAK kinase and STAT proteins (Table 1). In sharp contrast, involvement of the JAK-STAT pathway in tumor necrosis factor, TGF-P or chemokine receptor signaling has not been reported. The role of the JAK-STAT pathway in growth factor receptor signaling remains to be clarified, although it has been described that EGF or SCF activates JAKs and STATS. As expected, receptors sharing the same subunit appear to activate similar sets of JAKs. There is no clear cut one-to-one relationship, however, between the types of JAK kinases and STAT proteins activated in response to cytokines. As we shall discuss, approaches using molecular biology have provided evidence suggesting that the specificity of a STAT is determined by the specific interaction between the STAT and a phosphorylated tyrosine residue of the receptor.

cells was known recognized. Implications

until

their

function

in the interferon

signaling

in IFN

signaling

was

system

Many kinases are known to be activated or phosphorylated by cytokine stimulation but it has been difficult to conclude whether activation of these kinases is essential to elicit cytokine activities. Studies with IFN receptor signaling have revealed that JAK family kinases are involved in IFN-specific gene expression in cooperation with STAT (signal transducers and activators of transcription) proteins [ 11. IFNa specifically activates transcription of a gene termed 6-16 and IFNy activates transcription of the IRFZ gene. With the IFN system, Darnell’s group (e.g. [l]) found that a kinase which interacted with the IFN receptor phosphorylated certain transcription factors directly and resulted in subsequent translocation of the transcription factor to the nucleus.

Table 1 JAKs and STATS activated

The essential roles of JAK family kinases in IFNa/P as well as IFNy signaling were elegantly demonstrated by Stark, Kerr and co-workers [2*]. They established mutant cell lines which were unable to respond to either IFNa@ or IFNy. They subsequently isolated genes that are absent in these mutant cells by a complementation assay using expression cloning. They found that the Ul mutant cell which lacks TYK2 failed to respond to IFNa/P but was able to respond to IFNy. The y-l mutant, which failed to respond to IFNy but retained IFNaIP responsiveness, lacks JAK2. These complementation phenotypes were not substituted by other members of the JAK kinase family. In contrast, the U3 mutant hardly responded to either IFNa/P or IFNy. This unresponsiveness was restored by complementing JAKl. Taken together, these authors showed that JAKl and TYK2 are essential for IFNa/P signaling and that JAKl and JAK2 are essential for IFNy signaling. Other mutants indicated the essential role of STAT1 in IFNa/p/y signaling and of STAT2 in IFNa/P signaling [Z’]. Involvement

of the JAK-STAT

pathway

in cytokine

signaling

Initially, tyrosine were implicated

kinases-such in cytokine

as Src family kinasessignaling. Only a small

Shared subunit

by various cytokines.

Cytokine

JAK

STAT

IL-3 GM-CSF IL-5

JAKl , JAK2 JAKl , JAKP JAKl , JAKP

STAT516 STAT5 STAT5

yc, IL-2p

IL-15 IL-2

JAKl , JAK3 JAKl , JAK3

STAT315 STAT315

YC

IL-7 IL-9

JAKl , JAK3 JAKl, JAKS, TykP

STAT1 STAT1 I3

yc, IL-4

IL-4

JAKl , JAK3

STAT6

IL-4

IL-13

JAKl

STAT6

9P130

IL-6 LIF/CNT OSM

JAKl, JAK2, TykP JAKl, JAKP JAKl, JAKP, TykP

STAT1 13 STAT3 STAT1 13

None

IL-1 2 G-CSF EPO PRL GH TPO IFNa@ IFNy IL-10

JAKP, Tyk2 JAKl , JAKP JAK2 JAKl , JAK2 JAKP JAKP JAKl, Ty2 JAKl , JAK2 JAKl , TykP

STAT314 STAT 1I3 STAT5 STAT5 STAT5 STAT1 I315 STAT1 1213 STAT1 STAT1

Roles of the JAK-STAT system in signal transduction via cytokine receptors Watanabe and Arai

Molecular mechanisms JAK-STAT system

regulating

the

molecules-including

Association with receptor and regulation of JAK activity A JAK protein is known to associate with a cytokine receptor through its amino-terminal domain [4]. Several lines of evidence using receptor mutants have shown that box1 -a conserved motif of the cytokine receptor superfamily located in a membrane proximal region-is required common

for activation or interaction p chain of IL-3, Ghl-CSF

of JAK with PC (the and IL-S receptors),

growth hormone receptor, EPO receptor, and gp130 [3,5,6]. Involvement of a boxl-like motif of the IFNcx receptor 1 for interaction with TYKZ has been reported [7]; box1 is the only region conserved among cytoplasmic regions of cytokine receptor family members. The possibility that JAK is activated by receptor aggregation which induces its own tram-phosphorylation is implicated by analogy with the mode1 proposed for growth factor receptors that have a tyrosine kinase domain. JAK is known to bind constitutively to cytokine receptors such as PC, EPO receptor and gp130. After ligand stimulation, dimerization of cytokine receptor subunits follows, which then induces the dimerization of associated JAKs and results in cross-phosphorylation to activate JAK.

STAT

and

adaptor

589

proteins-to

bind to receptor (Fig. 1). Mutational analyses of receptors has revealed that multiple and distinct signaling pathways exist downstream of cytokine receptors. It appears that JAK is essential for many activities other than STAT activation in EPO and GM-CSF receptor systems. Studies using dominant negative JAKZ have indicated that JAKZ is essential for mitogenesis that is induced by EPO signals [lo]. In the case of PC, there are two distinct signaling pathways, one for activation of c-myc and proliferation, the other for activation of c-fos and c_iun. Experiments carried out using dominant negative JAK2 show that this kinase is involved in all known activities of the IL-3/GM-CSF receptor signaling [ 11’1. For C--OSactivation through MAPK cascade, phosphorylation of the tyrosine residue of PC and SHZ-containing proteins such as She and SHP-2 is likely. In contrast, no tyrosine residue of PC is required for activation of c-myc or cell proliferation. This suggests that substrate(s) other than PC or STAT may be involved in activation of these events and the mechanisms regulating cell proliferation remain uncharacterized.

STATla and STAT2 Only JAKl and STATla, were increased dramatically in IFNa@stimulated

however, SHP-1

Many cytokines activate multiple JAK kinases (Table 1) but it is not clear whether or not there is a cascade of reactions involving heterodimers between different JAKs. Studies using mutant cells lacking a defined JAK kinase have shown that both JAKl and TYKZ are essential for IFNa/P response and JAKl and JAK2 are essential for an IFNy response. Lack of either one of these results in loss of phosphorylation of the other JAKs suggesting that both JAKs are required for an initial event in IFN signaling. Reconstitution studies with mutant cells has shown that complementation of kinase negative JAKl to cells lacking JAKl sustained a substantial expression of IFNy-inducible genes and JAK2 phosphorylation [12”]. This was not the case for JAKZ when kinase negative JAK2 was introduced into JAKZ negative cells. These findings suggest that JAKl is not required for IFNy signals and that JAKZ is responsible for phosphorylation of both JAKl and JAKZ. IL-2 activates both JAKl and JAK3, and JAKl

negative cells derived from ‘moth-eaten’ lack SHP-l), suggesting that the JAK-STAT regulated differentially by SHP-1 [9-l.

mice (that system is

binds to IL-2 receptor p, whereas JAK3 binds to IL-2 receptor y subunit. Mutational analysis of the IL-2 receptor

The next issue that was addressed was the mechanism by which JAK signals generated by cytokines are diminished, leading to the involvement of the SHP-1 phosphatase. SHP-1 is a hematopoietic-specific SHZ-domain-containing tyrosine phosphatase. EPO-induced activation of its receptor negatively regulates JAKZ by SHP-1, which binds co th e phosphorylated tyrosine residue in the carboxy-terminal region of the receptor [WI. A similar mechanism is also predicted for regulation of PC, but it remains to be proven whether or not activities of all JAK family members are regulated negatively by SHP-1. IFNa/P has been known to activate JAKl, Tyk2,

Roles of JAK in cytokine receptor signaling As mentioned earlier, the role of JAKs as activators

and IL-2 receptor y subunits showed that the receptor complex lacking a JAKl binding site transduced signals, whereas the receptor complex lacking a JAK3 binding of

STATS was first demonstrated in the IFN system. In cytokine receptor systems, the function of JAK in signaling is not limited to STAT activation and is responsible for the tyrosine phosphorylation of cytokine receptors as well as of several SE-I&containing signaling molecules. In most cases, phosphorylation of the receptor at tyrosine residues is important for STAT activation. On the binding of a ligand to a cytokine receptor, the first event is likely to be the activation of a JAK(s). Phosphorylation of receptor tyrosine residue(s) by JAK2 allows SH2-containing signaling

site lost all known signals. These there is an essential role of JAK3 enzymatic activity nor a structural

results suggest that but that neither the component of JAKl

is required for IL-2 signaling. The essential role for JAK3 is also demonstrated by reconstitution of JAK3 into fibroblasts which normally lack JAK3 expression [ 131. The high affinity IL-2 receptor, reconstituted by cransfecting a$, and y subunits, was not functional and reconstitution of JAK3 into NIH3T3 cells restored responsiveness to IL-2. This is in contrast to the GM-CSF system where human GM-CSF receptor, reconstituted in fibroblasts, was functional.

Differentiation and gene regulation

590

Figure 1

Unstimulated

receptor

Cvtokine

Cvtokine

MAPK cascade

c 1996 Current Op~mon in Genetics & Development

Model for the activation and inactivation of cytokine type I receptor. Cytokine-induced oligomerization of receptor activates JAK, which is constitutively bound to receptor subunits. Activated JAK phosphorylates the tyrosine residues of the receptor. Several SH2-containing proteins, including STAT, recognize the phosphorylated subunit and bind to it. JAK also phosphorylates these proteins and triggers signaling cascades that transduce to the nucleus. JAK is also responsible for cell proliferation but signaling events between JAK and the initiation of DNA replication are unknown. SHP-1, which binds to the carboxyl terminus of receptor, extinguishes JAK activation.

JAK-STAT pathway of STATS To date, six members of the STAT family with similar structural features have been identified [14]. The existence of an additional STAT-like factor, induced either by lipopolysaccharide or IL-1p stimulation, has also been reported [ 151. The role of each domain of a STAT protein has been revealed by mutational analysis. A DNA-binding domain is located in the amino-terminal half, whereas SH3-like and SH2 domains are in the carboxy-terminal

Structure

end. All STAT binding sites are very similar with the consensus site being TTCCXGGAA. The DNA-binding domains of STAT proteins are highly conserved and the binding specificity is in the carboxy-terminal end, as revealed by chimeric molecules constructed from different STATS [16]. An SH3-like region-the role of which remains undetermined -has weak homology with a typical SH3 domain. In contrast, the SH2 domain of STAT has typical featuresboth structurally and functionally-that are found in other SHZ domains. For example, an essential role of the arginine residue in the SHZ domain in interacting with a phosphorylated tyrosine residue of a target molecule was demonstrated in Src and Abl. A similar observation was also reported with STAT1 and STAT2

[17*].

There is a conserved tyrosine residue within the carboxyterminal region of STAT proteins that is essential for dimerizdtion of STAT proteins in various cell functions

as well as specificity and redundancy of STAT proteins in the signaling of particular cytokines. An SHZ domain is also required for the binding of STAT proteins to receptors via the phosphorylated tyrosine residue. A serine residue, the phosphorylation of which is essential for maximal trans-activation, is also conserved in several STAT proteins whereas, in STAT1 and STAT3, it is dispensable for binding to target DNA (18”]. serine rather than tyrosine In monocytic cell lines, phosphorylation is tightly correlated with GAF activation, concomitant with IFNy-induced differentiation to the macrophage [ 19’1. The surrounding sequence of the serine residue PMSD, is known as a target sequence for blAPK. suggesting a role for MAPK in STAT activation. As IFNs do not activate LIAPK, it was not clear how IFK activates phosphorylation of the serine residue of STAT proteins. It appears that IFNa or IFNP induces binding of MAPK to the a subunit of IFNa/P receptor [ZO”]. In addition, IFNP induces binding of MAPK and STAT1 and dominant negative MAPK inhibits IFNP-induced transcription of target genes. Taken together, these observations suggest, but do not prove, that MAPK is involved in serine phosphorylation of STAT proteins. Even with cytokines that are known to activate the MAPK cascade, the involvement of MAPK in STAT activation is not clear. For example, in PC signaling, the regions of the receptor required for STAT5 activation and MAPK cascade activation are different, suggesting that

Roles of the JAK-STAT system in signal transduction via cytokine receptors Watanabe and Aral

STAT

activation

is independent

of MAPK

(Fig.

2). The

serine residue is conserved in STATla, STAT3, STAT4 and STAT5 but not in STATlB, STAT2, STAT6. It is conceivable that different mechanisms operate in the latter group.

receptor I

5: JAN

4 w PY

Nucleus & Development

The JAK phosphorylated tyrosine residues of a cytokine receptor and the SH2 domain of STAT recognize specific tyrosine residues and bind to them. Recruited STAT is phosphorylated at its carboxy-terminal side tyrosrne (Y) by JAK. Phosphorylated STAT dimerizes through the tyrosine residue and SH2 domain and is further phosphorylated at the carboxy-terminal serine residue. There is a possibility that MAPK, the activation of which is also responsible for phosphorylation.

is also initiated

by JAK,

Interestingly, the IFNa-induced tyrosine phosphorylation of STAT1 depends on phosphorylation of STAT2. In U6A cells that lack STAT2 protein, stimulation of IFNa failed to induce phosphorylation of STAT1 [21]. Further analysis using a series of STAT2 mutants has shown that a STAT2 mutant which is not phosphorylated by IFNa results in the failure of IFNa to induce tyrosine phosphorylation of STAT1 [2*]. The essential role for JAK3 in lymphoid development was also shown in patients with severe combined immune deficiency (SCID) carrying a mutation in JAK3 [22’,23] and in JAK3 knockout mice [24,25]. In all cases, the phenotypes were similar to y-deficient phenotypes.

Association

tyrosine

residues,

attempts

receptor. The essential role of the SH2 domain in binding to the IFN receptor has been characterized by swapping experiments of the SH2 domains of STAT1 and STAT2

inhibit DNA binding of STAT proteins. Direct evidence has been provided by chimeric receptors between the EPO receptor and gp130 [30**].

PY

: 1996 Current Opinion in Genetics

phosphorylated

have been made to elucidate the relationship between STAT binding and phosphorylated tyrosine residues of the

[26]. Experiments using phosphotyrosine peptides derived from a receptor suggest that certain tyrosine residues of the receptor define the specificity of STAT activation. For example, with p91 and the IFNy receptor [27], or with STAT6 and IL-4 receptor [28,29], phosphotyrosine peptides containing certain tyrosine residues of the receptor

Figure 2

Cytokine

to recognize

591

with receptor

Each cytokine activates a different set of STATS and the mechanism that ensures the specificity of this process has now been characterized. Because SH2 is known

Stahl et a/. [30**] have shown that activation of STAT3 by gp130 is determined by tyrosine-based motifs in the cytoplasmic domain of gp130. A similar mechanism has been implicated for other receptors such as STATS and EPO [31], and IFNa receptor 1 and STAT2 [32] by using tyrosine mutated receptor. Analyses of cytokines sharing a common subunit has shown that tyrosine phosphorylated motifs of the receptor determine which STAT proteins will be activated. This information provides a clue as to why cytokines that share a common subunit activate similar STAT proteins [33*]. In PC, a mutant receptor lacking all tyrosine residues in the carboxy-terminal region still activates STATS, suggesting that STATS is activated by a different mechanism. Although it is widely accepted that a STAT is activated by a JAK, activation of a STAT has been reported in Src transformed cells [34*,35’].

Functions

of STATS

As observed initially in IFN systems, STAT proteins are involved in the activation of cytokine-specific genes. hlolecular cloning and characterization of several STAT proteins, such as STAT3 or STATS, clearly shows that these STAT proteins are involved in cytokine-specific gene regulation. STAT3 is the same protein that was originally termed acute phase response factor which governs

STAT5

activation of acute phase genes was cloned initially as encoding

by IL-6 [36,37]. a transcription

factor that plays an essential role in the activation of a milk protein gene in response to prolactin [38]. STAT target sequences lipopolysaccharide

are found in several genes, such binding protein [39], immunoglobulin,

as

and MHC class II, which are induced by certain cytokines. It is often observed that multiple cytokines activate the same STAT, yet different genes are activated as targets: this suggests that STAT is not the sole determinant of specificity of gene activation. It is not known whether or not STAT in cytokine-induced cell proliferation. negative carboxy-terminal truncated pression of IL-3-induced proliferation

proteins are involved By using dominant STATS, partial supis observed [40].

592

On the

Differentiation and gene regulation

other

hand,

mapping

of the

receptor

domain

of

IL-4 receptor or EPO receptor shows a lack of correlation between cell growth as induced by IL-4 and EPO and STAT6/STATS activation [41,42**]. Interestingly, it has also been suggested that STAT1 mediates cell growth arrest signals [43**]. In some cells, EGF or IFNy induces growth arrest in these cells and activation of STAT1 correlates well with these activities. It is interesting to note that activation of the cyclin-dependent kinase inhibitor p2IIVAFIJCIPl gene is regulated by STATl. To date, there are reports that STATl, STAT4 or STAT6 knockout mice display no overt developmental abnormality. A mutant phenotype is observed only when cells derived from the mice are stimulated by cytokines that are known to activate STATS. STAT1 knockout mice are sensitive to infection by microbial pathogens and viruses and fail to respond to IFN but respond normally to other

cytokines

SH2

[44*,45*].

STAT4 knockout mice are viable and fertile but IL-12 functions are impaired, including induction of IFNy, proliferation and cytolytic function of natural killer cells and Thl differentiation [46*,47*]. The phenotypes of STAT6 knockout mice were indistinguishable from those of normal mice but biological response to IL-4 is abrogated [48*-SO’].

?

c-myc proliferation

:’ 1996 Current Opinion in Genetics & Development

Leukemogenesis

and JAK-STAT system

Over time, HTLV-1 infected peripheral blood T cells often grow independently of IL-Z. In such cells, the JAK-STAT pathway is activated constitutively, indicating its participation in HTLV-1 mediated T cell transformation [51-l. Leukemic cells from patients in relapse have constitutively activated JAKZ. Inhibition of JAKZ activity by a specific tyrosine kinase blocker arrests leukemic cells selectively-such as acute lymphoblastic leukaemia cells-from growing both in vitro and in vivo [52]. By using a temperature-sensitive mutant of Bcr/Abl, STAT1 and STATS, but not JAK, were phosphorylated constitutively in several cells. Bcr/Abl bypasses the requirement of JAK and activates STATS directly [53]. On the other hand, in cells derived from Philadelphia chromosome-positive acute lymphoblastic leukemia, lack of constitutive activation of JAK3 or STATS was reported [54] .

Conclusions A cell’s

response

to extracellular

signals

is determined

by the matching of a receptor and a signaling system. Cross-talk may occur between different receptor systems via signaling molecule(s) common to multiple receptor systems. Many cytokines and growth factors activate a cellular program such as proliferation, survival, differentiation, cell death, migration and cell function. They regulate these processes via common signaling pathway(s) such as Ras-raf-MAPK-APl (pathway A) and/or CLN/CDK, c-m~‘c induction, DNA replication (pathway B) or a

Signaling

pathways

of the cytokine

receptor.

There are at least

three distinct signaling pathways and JAK appears to play an essential role in each. Pathway A leads to the activation of c-fos by the phosphorylation of tyrosine residues and the activation of SH2-adaptor proteins and the MAPK cascade. Pathway B leads to c-myc activation and proliferation; the signaling events involved in this pathway are largely unknown. transcriptional activation.

Pathway

C constitutes

STAT-mediated

signaling pathway unique to each (Fig. 3). Biological responses common to cytokines and growth factors may be explained by the fact that pathways A and B are shared between these receptor systems. Some cytokines stimulate only pathway A while others stimulate both A and B. Other than these common activities, unique features of the cytokine system are pleiotropy, redundancy and specificity. The sharing of a common receptor subunit-such as PC, yc, and gpl30-which has the capacity to associate with common as well as unique signaling molecules, accounts for redundancy of the cytokine action. Because the receptor subunits shared by several cytokines activate a unique set of JAKs and STATS, it is possible that the JAK-STAT pathway also regulates the cellular program, including proliferation, survival and differentiation. It should be noted, however, that the JAK-STAT pathway was discovered originally in the IFN system, which protects cells from viral infection and is generally inhibitory for cell growth. The question arises as to

Roles of the JAK-STAT system

in signal transduction

via cytokine

receptors

Watanabe

593

and Arai

Table 2 Signaling

pathways

utilized

PC

F

IL-3

IL-2

IL-4

GM-CSF

IL-1 5

(IL-l 3)

by cytokine

IL-7 IL-9

IL-5

receptors. gpt30

Others

IFN

TNF

GF

TGF

Chemokine

IL-6

G-CSF EPO

IFNa

TNFa

M-CSF

TGFPl

IL-6

LIF

IFNP

TNFP

CNTF

TPO

MlPl a

IL-1 2 Prolactin

FASL CD40L

TGFPP TGFP3

OSM IL-1 1

IFNy IL-10

SCF Flk2 EGF

Activin

MlPl p

PDGF

BMPs(2-7)

PF4

Cardiotropin

MCP-1

GDNF NT3 FGF VEGF HGF

JAKl JAK2

STAT5

JAKl JAKB

STAT3

JAKl JAK3

STAT6

STAT5

JAKl

JAKl

JAK3 TykP

JAKP TykP

STAT1

STAT1

STAT3

STAT3

JAKl JAK2 Tyk2

JAKl JAK2

STAT3 STAT4

STAT1 STAT2 STAT3

TykP

STAT5 A B C Common

A B C pathways

C shared

A

A

A B

C

C

C

with GFR: A, RAS-MAPK-APl

C

; B, c-myc, DNA replication.

whether or not the JAK-STAT pathway is also involved in a common pathway regulating cell growth (pathways A and B) as well as in cytokine-specific functions (these features are summarized in Table 2). Questions remain regarding the mechanisms of action of the JAK-STAT system. First, what is the functional form of JAK and STAT; do they function as a homodimer or form a heterodimer? Second, does JAK directly activate pathway B in regulating the c-myc promoter as well as DNA replication or does it still require a receptor motif beside boxl? Third, do cytokine and growth factor receptors of the tyrosine kinase type activate c-myc promoter and DNA replication by similar mechanisms? Another important issue is the role of the JAK-STAT system in cell differentiation. In cytokine-dependent growth and differentiation of stem cells, an ‘instructive’ model assumes that a cytokine plays role in generating cells of a specific

A B

a deterministic lineage through

signals unique to its receptor. The other (selection) model predicts that a cytokine simply permits the growth of cells expressing a functional receptor. Accumulating evidence is consistent with a selection model for stem cell development, suggesting that lineage-commitment is a fixed process controlled by a cell’s intrinsic program, suggesting that the JAK-STAT system does not play a role in the lineage commitment of stem cells. In contrast, some cytokines appear to act in an instructive manner in the differentiation of the helper T cell subset. For example-in concert with antigen stimulation-IL-12 and IL-4 direct naive cells toward Thl and Th2 respectively, suggesting that functional differentiation of naive T cells is induced

Pathway

unique

to cytokine

receptors:

C, JAK-STAT.

by external signals in a plastic manner. Although the precise role of the JAK-STAT system in this process is as yet undetermined, it is tempting to speculate that it helps to reorganize a cytokine network in reponse to cytokine signals. Further studies on the roles of the JAK-STAT system will shed more light on this biologically important issue. STAT knockout studies demonstrate the importance of these proteins in various cell functions as well as their specificity and redundancy in the signaling of particular cytokines. Until now, knowledge is limited to STATS 1, 4 and 6 but information of other STAT proteins may soon be available. Multiple cytokines show different biological functions, even though these factors activate the same STAT (we have anticipated this phenomenon from cell-type specificity). Knockout studies, along with molecular analyses using cell lines, has revealed the mechanism of determination of cellular response by the combination of STAT and other transcriptional factors.

Acknowledgements l‘hc

authors

thank

John

Allan

Hamilton

for

a critical

reading

of

the

manuscript.

References

and recommended

Papers of particular interest, published have been highlighted as: . l

1.

*

reading

within the annual period of review,

of special interest of outstanding interest Darnell JE Jr, Kerr IM, Stark GR: Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science 1994, 264:1415-l

421.

594

Differentiation and gene regulation

2. .

Qureshi SA, Leung S, Kerr IM, Stark GR, Damell JJE: Function of Stat2 protein in transcriptional activation by alpha interferon. MO/ Cell Biol 1996, 16:288-293. A svstematic analvsis of functional domains of STAT2 bv introducina mutant STAT2 to cells lacking STAT2. Using this approach, tie authors glegantly confirm that, in IFNa stimulation, STAT1 is downstream of STATP. In addition, the importance of the amino terminus for STAT2 phosphorylation and the carboxyl terminus for transcriptional activation are shown. 3.

4.

Witthuhn B, Quelle FW, Silvennoinen 0, Yi T, Tang B, Miura 0, lhle JN: JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated.following EPO stimulation. Cell 1993, 74:227-236. Zhao Y, Wagner F, Franck SJ, Kraft AS: The amino-terminal portion of the JAKP protein kinase is necessary for binding and phosphorylation of the granulocyte-macrophage colonystimulating factor receptor PC chain. I Biol Chem 1995, 270:13814-13818.

5.

Quelle FW, Sate N, Witthuhn BA, lnhorn RC, Eder M, Miyajima A, Griffin J, lhle JN: JAK2 associates with the PC chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. MO/ Cell Biol 1994, 14:4335-4341.

6.

Tanner JW, Chen W, Young RL, Longmore GD, Shaw AS: The conserved box 1 motif of cytokine receptors is required for association with JAK kinases. J Biol Chem 1995, 270:6523-6530. Yan H, Krishnan K, Lim JTE, Contillo LG, Krolewski JJ: Molecular characterization of an alpha interferon receptor 1 subunit (IFNaRl) domain required for TYK2 binding and signal transduction. MO/ Cell Biol 1996, 16:2074-2082.

Klingmuller U, Lorenz U, Cantley LC, Neel BG, Lodish HFSpecific recruitment of SH-PTPI to the erythropoietin receptor causes inactivation of JAK2 and termination of proliferative signals. Cell 1995, 00:729-738. This is the first demonstration that SHP-1 negatively regulates JAK. The authors also demonstrate the association of SHP-I, through its SH2 domain, with EPO receptor via its phosphorylated tyrosine residue. This model has now been extended to other cytokine receptors. David M, Chen H, Goelz S, Lamer AC, Neel BG: Differential regulation of the alpha/beta interferon-stimulated Jak/Stat pathway by the SH2 domain-containing tyrosine phosphatase SHPTPI. MO/ Cell Biol 1995,15:7050-7058. Complementation of SHP-1 to cells derived from an SHP-1 null mouse here shows that SHP-1 increases just the tyrosine phosphorylation of JAKl and STAT1 a but does not affect the activation of TYK2 and STAT2 as induced by IFNa. It appears that SHP-1 regulates the member of JAK and STAT differentially. 9. .

10.

Zhuang H, Pate1 SV, He T-C, Sonsteby SK, Niu Z, Wojchowski DM: Inhibition of erythropoietin-induced mitogenesis by a kinase-deficient form of Jak2. I Biol Chem 1994, 269:21411-21414.

Watanabe S, ltoh T, Arai K: JAKP is essential for activation of c-fos and c-myc promoters and cell proliferation through the human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells. I Biol Chem 1996, 271:12681-l 2686. Experiments using dominant negative JAK2 show that all the known activities centering on c-fos gene activation and cell proliferation as induced by hGM-CSF are mediated by JAKP. JAK2 is also shown to mediate phosphorylation of tyrosine residues of the receptor. 11. .

Briscoe J, Rogers NC, Witthuhn BA, Watling D, Harpur AG, Wilks AF, Stark GR, lhle JN, Kerr IM: Kinase-negative mutants of JAKl can sustain interferon-rinducible gene expression but not an antiviral state. EM60 J 1996, 15:799-809. Bv comolementina kinase neaative JAKl or JAKP to cells lacking either JAKl 0; JAK?, the authors show ihat the kinase activity of JAKI is-dispensable for IFNy activity, even though JAKl protein is essential for IFNy signaling. This indicates that JAKl plays a structural role in the correct assembly and function of receptors. This work provides an insight in the analysis of other cytokines which activate multiple JAKs.

Functional activation of Jakl and Jak3 by selective association with IL-2 receptor subunits. Science 1994, 266:1045-l 047. 14.

lhle JN: STATS: signal transducers and activators of transcription. Cell 1996, 84:331-334.

15.

Tsukada J, Waterman WR, Koyama Y, Webb AC, Auron PE: A novel STAT-like factor mediates lioopolvsaccharide. interleukin 1 (IL-I), and IL-6 signaling and reco&;es a gamma interferon activation site-like element in the /LIB gene. MO/ Cell Biol 1996, 16:2183-2194.

16.

Schindler U, Wu P, Rothe M, Brasseur M, McKnight SL: Components of a Stat recognition code: evidence for two layers of molecular selectivity. lmmuniry 1995, 2:689-697.

1 7. .

Gupta S, Yan H, Wong LH, Ralph S, Krolewski J, Schindler C: The SH2 domains of Stat1 and Stat2 mediate multiple interactions in the transduction of IFN-a signals. EM60 J 1996, 15:1075-l 084. The functions of the SH2 domains of STAT1 and STAT2 here have been analysed in vitro. The authors show that the SH2 domain plays a role in the formation of homodimerization and heterodimerization of STAT through its phosphotyrosine residue. 18. ..

Wen Z, Zhong A, Darnell JE: Maximal activation of transcription by Stat1 and Stat3 requires both tyrosine and serine phosphorylation. Cell 1995, 82:241-250. Serine residue of STAT is phosphorylated following stimulation by a cytokine or growth factor. Reconstitution of STAT1 a-carrying mutation to the serine residue in cells lacking STAT1 shows that serine residues are required for maximal transcriptional activity but not for DNA binding. 19. .

Eilers A, Georgellis D, Klose B, Schindler C, Ziemiecki A, Harpur AG, Wilks AF, Decker T: Differentiation-regulated serine phosphorylation of STAT1 promotes GAF activation in macrophages. MO/ Ceil Biol 1995, 15:3579-3586. Involvement of the JAK-STAT oathwav in the differentiation of hematoooietic cells is an interesting issue. This report indicates that the increask in JAK-STAT activity is coincident with monocytic U937 differentiation. The authors also rep&t that phosphorylation of the serine rather than tyrosine residue is responsible for augmentation of STAT activity. I

David M, Petricoin E Ill, Benjamin C, Pine R, Weber MJ, Lamer AC: Requirement for MAP kinase (ERK2) activity in interferon a- and interferon P-stimulated gene expression through STAT proteins. Science 1995, 269:1721-l 723. The involvement of MAPK is suaaested in ohosohorvlation of the critical serine residue of STAT, in responseto IFNa/P: In abdition, co-precipitation of MAPK with IFNc@ receptor was implicated. Further experiments are needed to r:onfirm this report because activation of the MAPK cascade by IFN has not been described before. 20. ..

21.

Miyazaki T, Kawahara A, Fujii H, Nakagawa Y, Minami Y, Liu ZJ, Oishi I, Silvennoinen 0, Witthuhn BA, lhle JN, Taniguchi T:

Leung S, Qureshi SA, Kerr IM, Darnell JJE, Stark GR: Role of STAT2 in the alpha interferon signaling pathway. MO/ Cell Biol 1995, 15:1312-1317.

Russell SM. Tavebi N. Nakaiima H. Riedv MC. Roberts JL. Aman MJ, Migdne T, ioguchi M, Markeri ML, Buckley RH et a/.: Mutation of lak3 in a patient with SCID: essential role of Jak3 in lymphoid development Science 1995, 270:797-800. Certain mutations within the y subunit cause SCID and JAK3 is known to associate with this; a JAK3 mutation has therefore been expected to cause a similar phenotype. The authors report SCID patients carrying a JAK3 mutation. It appears that a JAK3 mutation now accounts for the SCID phenotype of y subunit mutants. 22.

.

23.

Macchi P, Villa A, Giliani S, Sacco MG, Frattini A, Porta F, Uaazio AG. Johnston JA, Candotti F, O’Shea JJ, Vezzoni P, Notarangelo LD: Mutations of lak-3 gene in patients with autosomal severe combined immune deficiency (SCID). Nature 1995, 377:65-68.

24.

Nosaka T, Van Deursen JMA, Tripp RA, Thierfelder WE, Witthuhn BA, McMickle AP, Doherty PC, Grosveld GC, lhle JN: Defective lymphoid development in mice lacking Jak3. Science 1995, 270:800-802.

25.

Thomis DC, Gurniak CB, Tivol E, Sharpe AH, Berg LJ: Defects in B lymphocyte maturation and T lymphocyte activation in mice lacking Jak3. Science 1995, 270:794-797.

12. ..

13.

1

Roles

of the JAK-STAT

system

26.

Heim MH, Kerr IM, Stark GR, Damell JJE: Contribution of STAT SH2 groups to specific interferon signaling by the Jak-STAT pathway. Science 1995, 267:i 347-l 349.

2 7.

Greenlund AC, Farrar MA, Vivian0 BL, Schreiber RD: Ligandinduced IFNy receptor tyrosine phosphorylation couples the receptor to its signal transduction system (p91). EMBO J 1994, 13:1591-l 600.

28.

Hou J, Schindler U, Henzel WJ, Ho TC, Brasseur M, McKnight SL: An interleukin-4-induced transcription factor: IL-4 Stat Science 1994, 265:1701-l 706.

29.

Yamamoto K, Quelle FW, Thierfelder WE, Kreider BL, Gilbert DJ, Jenkins NA, Copeland NG, Silvennoinen 0, lhle JN: Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation. MO/ Cell Biol 1994, 14:4342-4349.

in signal transduction via cytokine receptors Watanabe and Aral

30 ..

Stahl N, Farruggella TJ, Boulton TG, Zhong 2, Darnell JE Jr, Yancopoulos GD: Choice of STATS and other substrates specified by modular tyrosine-based motifs in cytokine receptors. Science 1995, 267:1349-l 353. This is the first demonstration of how to determine the specificity of STAT as activated by cytokine. Swapping of the region of the receptor containing tyrosine residue shows that choice of STAT for activation was determined by the tyrosine residue and the surrounding motif of the receptor. 31.

Gobert S, Chretien S, Gouilleux F, Muller 0, Pallard C, DusanterFourt I, Groner B, Lacombe C, Gisselbrecht S, Mayeux P: Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation. EM60 J 1996, 15:2434-2441.

32.

Yan H, Krishnan K, RD, Schindler CW, receptor 1 subunit latent form of the 15:1064-1074.

Lin J-X, Migone T-S, Tsang M, Friedmann M, Weatherbee JA, Zhou L, Yamauchi A, Bloom ET, Mietz J, John S, Leonard WJ: The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotropy and redundancy by IL-2, IL-4, IL-7, IL-l 3, and IL-I 5. immunity 1995, 2:331-339. The yc subunit is shared by several cytokine receptors, yet JAK and STAT activated by these cytokines are not the same. After extensive analyses to understand this redundancy and pleiotropy, the authors found that there is a correlation between receptor motifs and activated STATS. This type of study may also provide information regarding redundancy and pleiotropy of the gpl30-related cytokine system. Cao X, Tay A, Guy G, Tan YH: Activation and association of Stat3 with Src in v-%-transformed cell lines. MO/ Cell No/ 1996, 16:1595-1603. The role of JAK on STAT phosphorylation has become a widely accepted dogma. This study demonstrates the possibility that phosphorylation or activation of STAT may occur via Src tyrosine kinase. This study also implicates the role of STAT in transformation. 34. .

35. .

Yu C-L, Meyer DJ, Campbell GS, Larner AC, Carter-Su C, Schwartz J, Jove R: Enhanced DNA-binding activity of a StatSrelated protein in cells transformed by the Src oncoprotein. Science 1995, 269:81-83. This paper has a conclusion similar to that of [34-I. The authors suggest an indirect mechanism for the interaction of Src and STAT because they observe JAK activation in Src transformed cells.

37.

Zhong 2, Wen 2, Darnell JE Jr: Stat3: a stat family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6. Science 1994, 264:95-98.

38.

Wakao Ii. Gouilleux F, Groner B: Mammary gland factor (MGF) is a novel member of the cytokine regulated transcription factor gene family and confers the prolactin response. EMBO 1994, 13:2182-2191.

Schumann RR, Kirschning CJ, Unbehaun A, Aberle H, Knopf H-P, Lamping N, Ulevitch RJ, Herrmann F: The lipopolysaccharidebinding protein is a secretory class 1 acute-phase protein whose gene is transcriptionally activated by APRF/STAT-3 and other cytokine-inducible nuclear proteins. MO/ Cell Biol 1996, 16:3490-3503.

40.

Mui AL-F, Wakao H, Kinoshita T, Kitamura T, Miyajima A: Suppression of interleukin-3-induced gene expression by a Cterminal truncated Stat5: role of Stat5 in proliferation. EMBO J 1996,15:2425-2433.

41.

Quelle FW, Shimoda K, Thierfelder W, Fischer C, Kim A, Ruben SM, Cleveland JL, Pierce JH, Keegan AD, Nelms K et al.: Cloning of murine stat6 and human stat6, stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis. MO/ Cell Biol 1995, 15:3336-3343.

Quelle FW, Wang D, Nosaka T, Thierfelder WE, Stravopodis D, Weinstein Y, lhle JN: Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response. MO/ Cell Biol 1996, 16:1622-l 631. The authors describe the tyrosine residue of Epo receptor, which is required for activation of STAT5 by erythropoietin. As mutation of the tyrosine does not affect cell proliferation induced by Epo, the authors speculate that proliferation and STAT5 activation are separated in Epo signaling.

42. ..

Chin YE, Kitagawa M, Su WCS, You A-H, lwamoto Y, Fu X-Y: Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAFI /CIPl mediated by STATl. Science 1996, 272:719-721. This paper describes a relationship between STAT1 activation and the inhibition of cell growth by EGF and IFNT. Inhibition is caused by recognition of the STAT bindrng site in the promoter region of cyclin-dependent kinase inhibitor p21 by activated STATI. This is the first report describing both negative regulation via the JAK-STAT pathway and the relationship between cell cycle machinery and JAK-STAT signaling.

33. .

Akira S, Nishio Y, lnoue M, Wang X, Wei S, Matsusaka T, Yoshida K, Sudo T, Naruto M, Kishimoto T: Molecular cloning of APRF, a novel ISGF3 p91 -related transcription factor involved in the gp 130-mediated signaling pathway. Cell 1994, 77:63-71.

39.

43. ..

Greenlund AC, Gupta S, Lim JTE, Schreiber Krolewski JJ: Phosphorylated interferon-a (IFNaRi) acts as a docking site for the 113 kDa STAT2 protein. EMBO J 1996,

36.

595

44. .

Meraz MA, White JM, Sheehan KCF Bach EA, Rodig SJ, Dighe AS, Kaplan DH, Riley JK, Greenlund AC, Campbell et al.: Targeted disruption of the Stat7 gene in mice reveals unexpected physiologic specificity in the Jak-Stat signaling pathway. Cell 1996, 84:431-442. The authors here and in [45’] arrive at the same conclusion that STAT1 is essential for IFNa/b and IFNy signaling but not for other cytokines, such as IL-I 0, GH and EGF, which are known to activate STATl. 45. .

Durbin JE, Hackenmiller R, Simon MC, Levy DE: Targeted disruption of the mouse Statl gene results in compromised innate immunity to viral disease. Cell 1996, 84:443-450. The specific requirement of STAT1 for IFN activities but not for other cytokines is unexpected. It is of interest to examine whether or not other STATS, such as STAT3, can substitute STAT1 or has redundant activity of STAT1 in other cytokine signals. 46. .

Kaplan MH, Sun Y-L, Hoey T, Grusby MJ: impaired IL-12 responses and enhanced development of Th2 cells in Statrldeficient mice. Nature 1996, 382:174-l 77. IL-I 2 promotes Thl development but the molecular mechanisms that regulate differentiation of Thl and Th2 cells are largely unknown. IL-1 2 is known to activate STAT4; this study and [47’1 reconfirms this at various levels. STAT4 knockout mice have an impaired development of Thl cells in response to IL-1 2. This report provides an important clue regarding differentiation of Thl cells. 47. .

Thierfelder WE, Van Deursen JM, Yamamoto K, Tripp RA, Sarawar SR, Carson RT, Sangster MY, Vignali DAA, Doherty PC, Grosveld GC, lhle JN: Requirement for Stat4 in interleukin-12 mediated responses of natural killer and T cells. Nature 1996, 382:171-l 74. The conclusion of this paper is the same as that of [46-I and also describes natural killer cell activity induced by IL-1 2. These reports show the primary and specific roles of STAT4 in all the known IL-1 2 activities tested. In additron, knockout of STAT1 and STAT6 implicate STAT protein specificity for particular cytokines. 48. .

J

Takeda K, Tanaka T, Shi W, Matsumoto M. Minami M, Kashiwamura S. Nakanishi K, Yoshida N, Kishimoto T, Akira S: Essential role of Stat6 in IL-4 signalling. Nature 1996, 380:627-630.

596

Differentiation and gene regulation

Mice lacking STAT6 show defects in the IL-4 mediated response, such as expression of cell surface markers and B-cell proliferation co-stimulated by antiIgM. These phenotypes were indistinguishable from those of IL-4-deficient mice. The authors conclude that - between two distinct signaling pathways mediated by STAT6 and 4PS described thus far - STAT6 plays a central role in exerting biological responses mediated by IL-4.

Shimoda K, Van Deursen J, Sangster MY, Sarawar SR, Carson RT, Tripp RA, Chu C, Quelle FW, Nosaka T, Vignali DAA et a/.: Lack of IL-4-induced Th2 response and IgE class switching in mice with disruoted Stat6 gene. Nature 1996. 380:630-633. This report also demonstrates impairment of various IL-4-mediated activities in STAT6 knockout mice. Interestingly, IL-4-mediated proliferation is only partially affected and this observationshould be discussed in terms of involvement of STAT in cell proliferation. 49. .

50. .

Kaplan MH, Schindler U, Smiley ST, Grusby MJ: Stat6 is required for mediating responses to IL-4 and for the development of Th2 cells. lmmuniry 1996, 4:313-319. This paper describes an impairment of IL-13-induced cytokine production in T cells differentiated in virro in addition to impairment of IL-4 activities in STAT6 knockout mice.

51. .

Migone T-S, Lin J-X, Cereseto A, Mulloy JC, O’Shea JJ, Franchini G, Leonard WJ: Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I. Science 1995, 269:79-81. The mechanism of HTLV-1 transformation has long been studied and the role of Tax and other proteins encoded by the pX region is discussed. This report adds a new dimension to the study of the roles of the JAK-STAT pathway in cell transformation and proliferation.

52.

Meydan N, Grunberger T, Dadi H, Shahar M, Arpaia E, Lapidot 2, Leeder JS, Freedman M, Cohen A, Gazit A et al.: Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor. Nafure 1996,379:645-648.

53.

Carlesso N, Frank DA, Griffin JD: Tyrosyl phosphorylation and DNA binding activity of signal transducers and activators of transcription (STAT) proteins in hematopoietic cell lines transformed by Bcr/Abl. I Exp Med 1996, 183:61 l-820.

54.

Kanwar VS, Witthuhn B, Campana D, lhle JN: Lack of constitutive activation of Janus kinases and signal transduction and activation of transcription factors in Philadelphia chromosomepositive acute lymphoblastic leukemia. Blood 1996, 87:491 l-491 2.