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Blood, Vol. 95 No. 6 (March 15), 2000:
pp. 2076-2083
NEOPLASIA
From U434 INSERM, Centre d'Etude du Polymorphisme Humain (CEPH),
Paris, France; UMR 146 CNRS, Institut Curie-Section
Recherche, Centre Universitaire, Orsay, France; U363
INSERM, Institut Cochin de Génétique Moléculaire
(ICGM), Université René Descartes, Paris, France.
The involvement of the cytokine signaling pathway in oncogenesis has
long been postulated. Recently, rearrangements of the gene encoding the
tyrosine Janus kinase 2 (JAK2) have been reported in human leukemias
indicating a direct JAK-signal transduction and activator of
transcription (STAT)-mediated leukemic process. The leukemia-associated
TEL-JAK2 fusion protein is formed by the oligomerization domain of the
translocated ets leukemia (TEL) protein fused to the catalytic domain
of JAK2. TEL-mediated oligomerization results in a constitutive
tyrosine kinase activity that, in turn, is able to confer growth factor
independence to the murine hematopoietic interleukin-3 (IL-3)-dependent
Ba/F3 cell line. Results of the present study indicate that fusion
proteins containing the oligomerization domain of TEL and the tyrosine
kinase domains of Jak1, Jak2, JAK3, or TYK2 share similar properties
and are able to efficiently substitute for the survival and mitogenic
signals controlled by IL-3, without concomitant activation of the IL-3
receptor. Electrophoretic mobility shift assays demonstrated Stat5 as
the only activated Stat factor in TEL-Jak2- and TEL-Jak1-expressing
cells, whereas other Stats, namely Stat1 and Stat3, could be detected
in TEL-JAK3-, TEL-TYK2-, and also in TEL-ABL-expressing Ba/F3 cells.
High levels of expression of the Stat5-target genes pim-1, osm, and Cis
were observed in all the cytokine-independent cell lines. Furthermore,
the expression of a dominant negative form of Stat5A markedly
interfered with the growth factor independence process mediated by
TEL-Jak2 in Ba/F3 cells. Because the BCR-ABL and TEL-PDGF
The Janus kinases (JAK) are receptor-associated
tyrosine kinases involved in intracellular signaling pathways of a wide
variety of cytokine and growth factor receptors (for a review, see Ihle et al1). Four members of the JAK kinase family have been
identified in mammals: JAK1, JAK2, and TYK2, which are ubiquitously
expressed, and JAK3 whose expression is predominant in hematopoietic
cells. Structural similarities shared by the JAK proteins define 7 JAK homology regions (JH1-7, Figure 1). Among
these, the JH2 region is a pseudocatalytic domain, lacking enzymatic
activity but assumed to serve as a regulatory region for the JAK
activity and as an anchoring region for substrates and regulatory
proteins. The JH1 region located at the carboxy-terminus is endowed
with tyrosine kinase activity. JAKs are transiently activated following
ligand binding and they subsequently phosphorylate multiple substrates, including the JAKs themselves, the cytoplasmic domain of
cytokine-receptors, adapter proteins, tyrosine phosphatases, and the
signal transducers and activators of transcription (STAT). STATs form a
family of cytoplasmic transcription factors that comprise 7 proteins in mammals (Stat 1-4, 5A, 5B, and 6). STATs are activated by the JAKs,
form homodimers or heterodimers, and translocate to the nucleus where
they participate to transcriptional regulation (for reviews, see
Darnell2 and Pellegrini and Dusanter-Fourt3).
Deregulation of the JAK-STAT signaling pathway has often been
associated with cellular transformation, although its relevance to
oncogenic processes was not clearly established. For example, constitutive activation of STATs and JAKs is a recurrent observation in
cell lines transformed by viruses and oncogenes and in human leukemic
blood samples (reviewed in Garcia and Jove4). Furthermore, Stat3 is required in v-src-mediated transformation of
fibroblasts.5,6 A direct link between JAK deregulation and
malignant transformation has recently been established by the
rearrangement of the JAK2 gene as the result of specific chromosomal
translocations in human leukemias. These translocations result in the
fusion of various parts of JAK2 to the amino-terminal region of the
Translocated Ets Leukemia (TEL) protein, whose gene is frequently
rearranged in human malignancies.7,8 The resulting fusion
proteins always include the JAK2 catalytic domain. The NH2-terminal
Conserved Region (NCR) of TEL has been shown to mediate homotypic
oligomerization of the TEL-Jak2 protein leading to constitutive
tyrosine kinase activity. The fusion protein exhibits transforming
properties, as judged by its ability to relieve the growth factor
dependency of murine hematopoietic Ba/F3 cells.7 Similar
properties have been shown to be critical for the transforming
properties of several neoplasia-associated tyrosine kinases fused to
TEL, such as TEL-ABL9 and TEL-PDGF We studied TEL-JAK chimeric proteins consisting of the catalytic
domains of Jak1, Jak2, JAK3, and TYK2 fused to the NCR domain of TEL.
Oligomerization of the JAK kinase domains through the NCR domain
resulted in constitutive tyrosine kinase activity of TEL-JAK chimeras
and in the activation of their transforming properties, similar to
those of the TEL-Jak2 fusion. Stat5 activation was observed in cells
transformed by all TEL-JAK kinases and shown to be essential to the
mitogenic properties of TEL-Jak2.
Plasmids
Cell culture, interleukin-3 independence, and transfections
Nuclear extracts and electrophoretic mobility shift assays Nuclear extracts were made as reported previously.13 For electrophoretic mobility shift assays (EMSA), 20 µL of nuclear extracts from TEL-ABL and TEL-JAK-transformed Ba/F3 cells were incubated with the 32P-labeled m67SIE 5'-CATTTCCCGTAAATC-3' and bovine -casein
5'-AGATTTCTAGGAATTCAAATC-3' STAT binding sites. Eight
microliters of nuclear extracts from murine IL-3-stimulated parental
Ba/F3 cells were used as controls. Supershift assays were performed by
using anti-Stat1 (Transduction Laboratories, Lexington, KY), anti-Stat3
(Santa Cruz Biotechnology, Inc, Le Perray-en Yvelines, France) and
anti-Stat5 antibodies.14
Northern blots Northern blotting analysis was performed by using 10 µg of total RNA and probes corresponding to murine cDNA fragments for pim-1, osm, cis, c-myc, c-jun, and c-fos labeled with 32P- CTP by
random-priming. Northern blots were normalized by hybridization with a
GAPDH probe. Radioactivity was quantified using a Molecular Imager
(Bio-Rad).
Cell lysates, immunoprecipitates, and Western blot analyses For Western blotting of total cell lysates, Ba/F3 cells were lysed in 1% Brij 96, 10 mM Tris/Hcl pH 7.4, 150 mM NaCl, 5mM EDTA, 10% glycerol, 0.02% NaN3, and 1 mM Na3VO4. Whole cell lysates were cleared by centrifugation at 13 000g for 20 minutes at 4°C and 50 µg of cellular protein was separated through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to nitrocellulose, and blotted with an antiphosphotyrosine antibody 4G10 (Upstate Biotechnology, Inc, Souffelweyersheim, France). For IL-3 receptor subunit (IL-3R) immunoprecipitation analysis, 20 × 106
Ba/F3 cells were lysed in the above buffer and supernatants were incubated 2 hours at 4°C with a rabbit polyclonal anti-IL-3R antibody (Santa Cruz Biotechnology, Inc.). Immunoprecipitates were
separated by 8% SDS-polyacrylamide gels, transferred to
nitrocellulose, and blotted with antiphosphotyrosine and anti-IL-3R
subunit antibodies. Expression of stably transfected TEL-JAK and
TEL-ABL fusions in Ba/F3 cells was evaluated by immunoprecipitation
followed by Western blot analysis, using a rabbit polyclonal
immunoserum raised against the amino-terminal part of the human TEL
protein ( N-TEL)15 present in all the chimeras.
Immunocomplexes were detected using the ECL detection kit (Amersham,
Orsay, France). Stripping and probing were performed according to the
manufacturer's instructions.
TEL-JAK fusion proteins exhibit constitutive tyrosine kinase activity and confer factor-independent growth to IL-3-dependent Ba/F3 cells To investigate for potential biologic differences between the tyrosine kinase domains (JH1) of JAK family members, we generated chimeric proteins modeled on the structure of the oncogenic TEL-Jak2 fusion in which the JH1 domains of JAK1, JAK3, and TYK2 were fused to TEL sequences. The schematic structures of these TEL-JAK proteins are summarized in Figure 1. All TEL-JAK fusions include the NCR region of TEL responsible for the homotypic oligomerization of TEL and TEL-derived fusion proteins. To determine if TEL sequences outside of the NCR contribute to transformation, a truncated mTEL-Jak2 fusion was constructed, directly linking the NCR region to the JH1 of Jak2. The murine TEL-TYK2 chimeric protein was constructed similarly to the above fusion. Western blotting analysis using antiphosphotyrosine antibody showed the constitutive phosphorylation of in vitro produced TEL-JAK chimeras (data not shown), a property known to reflect the intrinsic tyrosine kinase activity of the chimeric proteins.7,10 These results confirm that oligomerization of the catalytic domains of JAKs through the NCR domain of TEL is sufficient to allow their constitutive activation.
The TEL-JAK proteins do not significantly phosphorylate tyrosine
residues of the IL-3 All TEL-JAK fusion proteins induce functional activation of Stat5 factors To determine if the different TEL-JAK fusions selectively activate members of the Stat family, Stat DNA binding activities were analyzed by EMSA. Nuclear extracts from the TEL-JAK- and TEL-ABL-expressing lines were incubated with probes containing various Stat binding sequences, known to have different affinities for activated Stats. The-casein probe contains Stat5 binding sites and binds the 2 highly
homologous Stat5A and Stat5B factors.16 As a positive
control, parental Ba/F3 cells were stimulated with recombinant murine
IL-3 (mIL-3). As shown in Figure 3A,
nuclear extracts from all TEL-JAK-transformed cell lines showed a
specific Stat5 DNA binding activity, consisting of a major protein-DNA complex, which can be supershifted with anti-Stat5 antibodies (Figure
3A, compare lanes 4, 7, 10, 13, and 16 to lanes 6, 9, 12, 15, and 18, respectively). Specificity of Stat5--casein complexes was confirmed by competitive experiments using unlabeled -casein probe (Figure 3A, lanes 5, 8, 11, 14, and 17). Nonrelevant competitors did not interfere with the shifted complexes (data not shown). Both
Stat5A and Stat5B factors are expressed in Ba/F3 cells but a more
pronounced Stat5A DNA binding activity was observed in all transformed
Ba/F3 cells. In nuclear extracts from TEL-JAK3 (Figure 3A, lane 13) and
mTEL-TYK2 (Figure 3A, lane 16), a faint signal corresponding to a
higher mobility complex was observed, similar to the one seen after
mIL-3-stimulation (Figure 3A, lane 1). This complex corresponds to
Stat1, because the -casein probe is known to exhibit low affinity
for Stat1 factors, which are activated in Ba/F3 cells on IL-3
stimulation.17 Furthermore, supershifting experiments with
an anti-Stat1 antibody confirmed the specific interactions of Stat1
with the -casein probe with these extracts (data not shown). A
constitutive activation of Stat5 factors was also seen in
TEL-ABL-transformed cells (Figure 3A, lanes 19-21). In addition, Stat5
is activated in TEL-PDGFR-transformed cells (A.B., unpublished
data). Collectively, these results establish the recurrent activation
of Stat5 DNA binding activity in Ba/F3 cells for all TEL-fusion
proteins studied to date.
Stat1 and Stat3 are activated in Ba/F3 cells transformed by the TEL-JAK3, mTEL-TYK2, and TEL-ABL fusions Specific activation of Stat1 and Stat3 factors was assessed by using the high affinity m67SIE sequence, derived from the STAT binding motif of the c-fos promoter18 (Figure 3B). This probe allows the formation of 3 DNA-protein species that correspond to homodimers of Stat1, homodimers of Stat3, and heterodimers of Stat1 and Stat3. Constitutive activation of both factors was observed in the TEL-JAK3-, mTEL-TYK2-, and TEL-ABL-expressing cells. Nuclear extracts from TEL-JAK3-transformed Ba/F3 cells showed patterns of protein-DNA complexes and of supershifts with anti-Stat1 and anti-Stat3 antibodies similar to those observed with mIL-3-stimulated cells extracts used as control (Figure 3B, compare lanes 1-4 to 5-8, respectively). Nuclear extracts from the mTEL-TYK2-expressing cells only formed 2 shifted DNA-protein complexes with a high degree of specificity as shown by competition with excess cold m67SIE probe (Figure 3B, compare lanes 9 and 10) and incubation with an anti-Stat1 antibody confirmed the presence of Stat1 factors in the low migrating DNA-protein complex. The presence of 2 shifted complexes indicated that some Stat3 was activated in these extracts and preferentially associated with Stat1 to form Stat1-Stat3 heterodimers, although the use of an anti-Stat3 antibody did not clearly reveal a Stat3 DNA binding activity (Figure 3B, lane 11). It is noteworthy that lower levels of activated Stat1 factors were observed in TEL-JAK3-expressing cells, as compared to mTEL-TYK2-expressing cells (Figure 3B, compare lanes 5 and 9). Minor Stat1 and Stat3 activations were also evidenced in nuclear extracts from Ba/F3 cells expressing the TEL-ABL fusion (data not shown).
A specific set of cytokine-responsive genes is induced in the TEL-JAK-transformed Ba/F3 cell lines To further investigate the molecular events leading to growth factor independence conferred by TEL-JAK fusions in the Ba/F3 cellular context, we examined by Northern blot the induction of a series of cytokine-responsive genes, including genes whose products are involved in the control of cellular proliferation. Parental Ba/F3 cells cultured in the presence of WEHI conditioned medium, as a source of growth factors, will be referred to as proliferating cells. The c-myc, c-jun, and c-fos genes are immediate early genes induced in response to IL-3 in Ba/F3 cells19 and the cytokine-driven activation of both c-myc and c-fos transcription has been directly linked to the activation of JAK pathways.20,21 All TEL-JAK-expressing cells exhibited elevated levels of c-myc, c-jun, and c-fos expression, equivalent to those found in proliferating parental cells, indicating that expression of chimeric proteins efficiently substitute for the normal IL-3-mediated mitogenic signals. Thus, it appears that signaling pathways activated by the leukemogenic TEL-Jak2 protein and by the chimeric TEL-JAK fusions studied here can mimic at least some of the events triggered by the cytokine-receptor interaction. In contrast, the expression of TEL-ABL resulted in high expression levels of c-fos, c-jun, and c-myc, because messenger RNA (mRNA) levels were found to be 2, 2.5, and 3 times higher, respectively, as compared to those seen in proliferating parental cells. These findings are in agreement with previous reports indicating that c-myc expression is up-regulated in Ba/F3 cells transformed by the TEL-ABL fusion.22
Constitutive expression of a dominant negative form of Stat5A in
Ba/F3 cells significantly interferes with the growth factor
independence process mediated by TEL-Jak2
Chromosomal translocations resulting in TEL-JAK2 fusion proteins
have been reported in several human leukemias.7,8 The TEL-JAK2 fusion proteins described to date transform growth
factor-dependent cell lines and are leukemogenic in mouse
models7,28 (and our unpublished data). Several examples of
tyrosine kinases constitutively activated through ectopic
oligomerization have been reported in human hematopoietic malignancies,
one of the most extensively studied being the BCR-ABL fusion protein in
chronic myelogenous leukemia with the Philadelphia
chromosome.29 Thus, the identification of signaling
molecules regulated by these kinases is essential to elucidate the
mechanisms underlying their transforming and leukemogenic properties.
The authors thank N. Perrimon for providing the Hopscotch cDNA and the
LPH from the Hôpital Saint-Louis (Paris, France) for artwork.
Supported in part by grants from the Association contre le
Cancer (ARC), the Ligue Nationale contre le Cancer (LNCC), and the
Comité de Paris of the LNCC. R.M. and S.D. are supported by a
Ministère de l'Education Nationale, de la Recherche et des Technologies (MENRT) fellowship.
Submitted May 27, 1999; accepted October 28, 1999.
Reprints: O. A. Bernard, U434 INSERM-CEPH, 27 rue
Juliette Dodu, 75010 Paris, France; e-mail:
olivier.bernard{at}cephb.fr.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
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Abstract
Introduction
749) from the
pRSVNeo vector has been previously reported.
RI
5'-AGCATGTTTCAAGGATTTGAGATGTATTTCCCAGAAAAG-3') and I
5'-GTCAA CTTCCCAAGAACAGAA-3') probes. Extracts of COS7 cells co-transfected with TEL-Jak2 and Stat4 or Stat6 expression vectors (a kind gift of J. R. Darnell) were used as positive controls.
),
starved (starved), and stimulated by mIL-3 (stim) were loaded as
controls. The indicated values of Cis induction are expressed relative
to the values obtained by proliferating Ba/F3 cells. A GAPDH probe was
used as internal standard.
