By
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From the * Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics, § Department of Medicine and
Department of Microbiology, and ¶ Integrated Program in Molecular,
Cellular, and Biophysical Studies, Columbia University, College of Physicians and Surgeons,
New York 10032
Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.
The proto-oncogene c-mpl (1, 2) is the receptor for
thrombopoietin (TPO)1, a cytokine which has been
shown to be the major regulator of megakaryopoiesis and
platelet formation (3). C-mpl was originally isolated as
the cellular homologue of the transforming oncogene v-mpl
of the myeloproliferative leukemia virus (MPLV) (1). Like
many cytokine receptors, c-mpl is a member of the hematopoietic receptor superfamily (6). This family is characterized by conserved cysteine residues and a common amino
acid motif -WSXWS- in the extracellular domain, and by
the lack of intrinsic tyrosine kinase activity in the intracellular domain (6). Nevertheless, tyrosine phosphorylation
plays an important role for the intracellular signaling events
initiated by these receptors. It has become apparent that
nonreceptor tyrosine kinases, such as Jak and Src family members, are recruited by these receptors and mediate the
tyrosine phosphorylation of cellular target proteins (6, 7).
The signal transduction of cytokine receptors has been extensively studied over the last several years and numerous
proteins have been identified which are involved in the signaling pathways leading from the membrane to the nucleus. The Jak kinases seem to function very early on in this
process (6, 7). They bind to the intracellular part of cytokine receptors either constitutively or after ligand stimulation and their kinase activities are upregulated after receptor activation. This is believed to result in tyrosine
phosphorylation of the receptor itself and of the STAT
proteins, a novel class of SH2 domain-containing transcription factors. The STATs become activated upon phosphorylation and translocate from the cytoplasm to the nucleus
where they bind to specific DNA motifs. To date, four Jak
kinases, Jak-1, Jak-2, Jak-3 and Tyk-2, and at least six different STAT proteins (STAT 1-6) have been described (6, 7). Different cytokine receptors activate distinct but overlapping sets of Jaks and STATs.
Ligand stimulation of c-mpl has been shown to result in
the phosphorylation and activation of Jak-2, Tyk-2 and
STAT1, STAT3, and STAT5 (8). Furthermore, TPO-induced phosphorylation of Shc, MAPK, Raf-1, Cbl, Vav,
SHPTP-1, and SHPTP-2 has been described (8). It is
not clear to what extent the Jak kinases are responsible for
phosphorylation of proteins other than STATs.
The intracellular domains of receptors of the hematopoietic receptor superfamily share two membrane-proximal regions of weak homology, designated box1 and box2 (6).
Both motifs have been shown to be crucial for ligand-induced
cell proliferation and activation of Jaks (13). Box1 is required for binding of Jaks (14, 18, 19). Previous studies of
c-mpl and various other cytokine receptors with mutations
in the box1/box2 region have shown a correlation between Jak activation and cell proliferation (13, 14, 16, 20,
21), suggesting that Jak activation might be essential. Here
we describe a deletion mutant of the thrombopoietin receptor c-mpl which reveals that proliferation can be induced without activating Jaks.
Antibodies.
Polyclonal rabbit antisera against Jak-1, Jak-2,
Jak-3, and Shc were purchased from Upstate Biotechnology (Lake
Placid, NY). Polyclonal antibodies against Vav, Raf-1, STAT3,
STAT5a, STAT5b, and c-myc, and a monoclonal anti-Erk2 antibody were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-Tyk-2 antibodies were kindly provided by Dr. John
Krolewski (Columbia University, New York). Horseradish peroxidase-conjugated anti-phosphotyrosine mAb RC20 (clone
PY20) was purchased from Transduction Laboratories (Lexington,
KY). Anti-active MAPK polyclonal antibodies were obtained from
Promega (Madison, WI). Polyclonal anti-c-fos antibodies were purchased from Oncogene Sciences. Anti-STAT1 antbodies
(29130) were kindly provided by Dr. Christian Schindler (Columbia University).
Expression Constructs.
c-mpl deletion mutants were constructed by sequential PCR using the murine c-mpl cDNA (plasmid pSK-c-mpl, provided by Dr. Philip Leder, Harvard Medical
School, Cambridge, MA; reference 2) as a template and cloned
into the mammalian expression vector MT21myc (22) in frame with
a myc-epitope at the 3 Proliferation Assay.
Cells were cultured at a density of 5 × 104
per 200 µl in a 96-well round-bottom microtiter plate with varying concentrations of recombinant TPO in culture medium for
48 h. During the last 6 h of culture, cells were pulse-labeled with
0.5 µCi of [3H]thymidine (specific activity 5 Ci/mmol; Amersham), and [3H]thymidine incorporation was quantified by scintillation counting as described (24).
Cell Culture and Transfections.
BAF/3 cells were cultured in
RPMI medium supplemented with 10% FCS, 2 mM L-glutamine, antibiotics and 10% WEHI-3 supernatant as a source of IL-3.
COS cells were maintained in DMEM containing 10% FCS, 2 mM
L-glutamine and antibiotics. BAF/3 cells were cotransfected with
pSV2neo (1 µg) and the receptor expression plasmids (10 µg) by
electroporation using a Gene-Pulser (Bio-Rad Laboratories, Richmond, CA). 2 × 107 cells in 0.5 ml PBS were pulsed in a 0.4-cm
cuvette with 250 V, 960 µF. Stable transfectants were selected sequentially in 2 mg/ml G418 and 25 ng/ml TPO. Receptor expression was confirmed by Western blot analysis with a monoclonal antibody against the myc-epitope (anti-human myc mAb,
clone 9E10; Oncogene Science). COS cells were transfected with
5 µg of DNA per 2 × 106 to 4 × 106 cells by the chloroquine
DEAE-dextran method (25). Stimulation experiments with COS
cells were performed 48 h after transfection.
Growth Factor Stimulation, Western Blot Analysis and Immunoprecipitation.
BAF/3 transfectants were growth factor-starved for 8-12 h
in RPMI supplemented with 10% FCS. Stimulation was performed
at a concentration of 1 × 108 cells/ml with 200 ng/ml recombinant human TPO (generously provided by Amgen, Thousand
Oaks, CA) or 50 ng/ml recombinant murine IL-3 (Sigma Chem.
Co., St. Louis, MO). Stimulation was stopped and cell extracts
were prepared with lysis buffer (20 mM Tris-HCl, pH 8, 138 mM NaCl, 10% glycerol, 1% NP-40, 0.025 mM p-nitrophenyl guanidinobenzoate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM Na3VO4, 2 mM EDTA, 10 mM NaF) at 1 × 107 cells/250 µl as
described (8). Proteins were resolved by SDS-PAGE (7.5% gel).
Western blot analysis and immunoprecipitations were performed
as described (8). Kinase activity of Jak-2 immunoprecipitates was
analyzed in an in vitro kinase assay (26). In brief, immunoprecipitates were washed twice in kinase buffer (10 mM Hepes, pH 7.4, 2 mM MnCl2, 10 mM MgCl2, 150 mM NaCl, 1 mM DTT, 0.1 mM PMSF, 0.1 mM Na3VO4) and resuspended in 40 µl kinase
buffer. 20 µCi [32P]ATP (specific activity 3000 Ci/mmol) were
added and the kinase reactions were incubated for 30 min at
room temperature. Reactions were terminated with 2× Laemmli
buffer and analyzed by SDS-PAGE.
Electrophoretic Mobility-Shift Assay.
Whole cell extracts and
shift reactions were prepared as described previously (26). The
probe used was from the IRF-1 GAS element; 5 PI 3-Kinase Assay.
PI 3-kinase activity was measured as described (27). Cell lysates were immunoprecipitated with anti-phosphotyrosine antibodies (PY20; Transduction Laboratories) and
the immunoprecipitates were washed three times with lysis buffer, twice with LiCl buffer (0.5 M LiCl in 0.1 M Tris, pH 7.5),
twice with kinase buffer (20 mM Hepes, pH 7.4, 15 mM MgCl2,
1 mM Na3VO4, 0.5 mM PMSF), and subsequently resupended in
45 µl of kinase buffer containing 10 µCi A series of deletion mutants of c-mpl was constructed; two
selected mutants are depicted in Fig. 1 a. Mutant c-mpl
Stimulation of c-mpl by its ligand results in tyrosine phosphorylation and activation of Jak-2
(references 8, 13). As expected, tyrosine phosphorylation of Jak-2 was observed as early as 5 min after stimulation and was still visible after 30 min in cells expressing the
wild-type receptor (Fig. 2 a). Jak-2 phosphorylation was not
induced in TPO-stimulated BAF-mpl
The unexpected inability of c-mpl Tyrosine phosphorylation of Jaks leads to activation of
their kinase function (6). To monitor Jak-2 activation, the
kinase activity of Jak-2 immunoprecipitates from TPO-stimulated BAF-mplwt and BAF-mpl Tyk-2, another member of the Jak family, has recently
been reported to be tyrosine phosphorylated after TPO receptor stimulation (11). To determine whether Tyk-2 is
activated and might compensate for the lack of Jak-2 activation in BAF-mpl We next analyzed whether the failure of c-mpl
Our results demonstrate that c-mpl
c-mpl
Previous studies have implicated PI 3-kinase in the mitogenic response induced by a number of cytokines (6, 30,
31). To study this pathway we analyzed PI 3-kinase activity
in anti-phosphotyrosine immunoprecipitates from BAF-mplwt, BAF-mpl
c-mpl Previous analyses of various cytokine receptors with mutations in the box1/box2 region suggested that the inability
to activate Jaks always correlates with the complete loss of
the mitogenic response (13, 14, 16, 20, 21) and all major
downstream signaling events (13, 20, 21, 32). Unlike our
findings, these results suggested an absolute requirement of
Jak activation for receptor activity. However, none of these
mutants included an internal deletion of the region membrane-proximal to box1 that left box1 intact. The discrepancy between the rather specific effect of the deletion
proximal to box1 and the obliterative effects of previous
deletions in the box1/box2 region may be explained by the
presence of binding or activation domains in box1/box2 for PI 3-kinase and other as yet undefined kinases or signaling molecules. Alternatively, this region may be structurally
important for the proper positioning of remaining domains.
Nevertheless, our results virtually rule out the possibility
that the drastic effects of deletions in the box1/box2 region
are solely due to the absence of Jak activation.
Another approach to disrupting the Jak-STAT pathway
has been the use of kinase-deficient forms of Jaks as dominant-negative inhibitors of endogenous Jak activity (33, 34).
Expression in factor-dependent cells of kinase-deficient Jak-2
decreased IL-3- or GM-CSF-induced cell proliferation and
abrogated erythropoietin-induced proliferation (33, 34). The
mechanism of inhibition, however, is uncertain. Notably,
in one case the Jak-2 mutant suppressed IL-2 signals that do
not involve Jak-2 (33), suggesting that the effects of overexpression of such mutants are not restricted to the inhibition
of Jak-2 but may also interfere with other signaling events.
The molecular mechanism of the phosphorylation of
Shc, Vav and the receptor itself in the absence of Jak activation
remains to be elucidated. Src family kinases as well as c-fes,
btk, tec, syk (6), and c-kit (35) have all been shown to be
activated by various cytokine receptors. However, activation of these kinases is not as universal as activation of the
Jaks. To date none of these kinases has been linked to the
TPO receptor. In our hands, TPO does not activate lyn,
fyn, fes, tec, or syk in BAF-mplwt and BAF-mpl Our results demonstrate that the Jak-STAT pathway is
not essential to all cytokine receptor systems for stimulation
of a mitogenic response. Thus, other signaling pathways
must be sufficient to mediate this response and PI 3-kinase
appears to be at least one essential part of that signal. Nevertheless, we emphasize that our results do not rule out that
under physiological conditions the Jak-STAT pathway may
contribute to proliferation. The selective effect of the described mutation on Jak-STAT signaling should prove useful in defining the role of this pathway in TPO-mediated differentiation. Finally, it will be important to test whether analogous mutations in other cytokine receptors may have
similarly selective effects.
end of the cloning site. Deletion mutants
were generated with the help of overlapping oligonucleotides by
standard methods (23). To delete aa 505-514 in c-mpl
7, internal
oligonucleotides were: 5
-ATGCCTCAGTAGCAGCAGTAGGCCCAG-3
and 5
-CTGCTGCTACTGAGGCATGCTTTTGTGG-3
; to delete aa 515-522 in c-mpl
8: 5
-GTCTGGAAGTCTCCTGTAGTGCGCAGG-3
and 5
-TACAGGAGACTTCCAGACCTACACCGG-3
. The deletions were introduced
into a 850-bp fragment of c-mpl extending from the BamHI site
(bp 1124) to the stop codon; the flanking oligonucleotides used
to amplify the region were: 5
-TTTTGGATCCACCAGGCTGTGCTCC-3
and 5
-GACTGCGTCGACGGCTGCTGCCAATAGCTTAG-3
. The amplified fragments were digested with
BamHI and SalI and cloned into SH-mpl-N (plasmid SH2-1 containing the EcoRI-BamHI fragment of c-mpl). The resulting EcoRI-SalI fragment encoding for the full-length c-mpl cDNA
or the different deletion mutants was isolated and cloned into the plasmid MT21myc in frame with the myc epitope. In the double
mutant c-mpl-
7
C the COOH-terminal 25 amino acids were
deleted using an internal NcoI site (bp 1796). The deletions were
confirmed by sequence analysis.
-gatc-GATTTCCCCGAAAT-3
(reference 7). For supershift assays, standard shift reactions were incubated with pre-immune antibodies
or antibodies to STAT1, STAT3, and STAT5 (1:20 dilution) for
30 min at 4°C.
-[32P]ATP and 25 µM
cold ATP. 5 µl PI (4 mg/ml in DMSO; Avanti Polar Lipids, Alabaster, Alabama) were added and the reaction was incubated for
15 min at RT. The kinase reaction was stopped by adding 40 µl
of 1 N HCl and the lipids were extracted with 80 µl of CH3CL/ MeOH (1:1) and analyzed by thin layer chromatography. Unlabeled PI3-P (Sigma) detected by iodine staining was used as a standard. Labeled PI3-P was visualized and quantified using a PhosphorImager.
Mitogenic Response Mediated by c-mpl Deletion Mutants.
7
lacks the first 10 amino acids (aa) (KWQFPAHYRR, aa
505-514; reference 2) of the cytoplasmic domain but retains an intact box1, whereas mutant c-mpl
8 retains the
juxtamembrane region but lacks the NH2-terminal half of
box1 (LRHALWPS, aa 515-522). Cell lines stably expressing the wild-type and mutant receptors were established by
transfection of the IL-3-dependent cell line BAF/3. Comparable levels of receptor expression were detected in cells
expressing c-mplwt (BAF-mplwt), c-mpl
7 (BAF-mpl
7),
and c-mpl
8 (BAF-mpl
8) (Fig. 1 b). The transfected cells
were then analyzed for their mitogenic response to TPO
(Fig. 1 c). Expression of c-mplwt conferred responsiveness
to TPO as shown previously (4, 5) (Fig. 1 c). BAF-mpl
7
cells also showed a strong proliferative response to TPO,
though higher levels of TPO were required when compared to BAF-mplwt. BAF-mpl
8 cells were completely
unresponsive to TPO (Fig. 1 C) and parental BAF/3 cells
(not shown), demonstrating that TPO-responsiveness required expression of a functional receptor in these cells.
These results indicate that the first 10 aa of the c-mpl cytoplasmic domain are dispensable for a mitogenic response, whereas an intact NH2-terminal half of box1 is absolutely
required. BAF-mpl
7 cells retained their proliferative capacity in TPO for a prolonged period of time (>3 mo, data
not shown), suggesting that the mutant receptor provides
the signals necessary for long-term survival.
Fig. 1.
Mitogenic response of BAF/3 cells expressing c-mpl mutants.
(a) Schematic representation of c-mplwt and deletion mutants c-mpl7 and
c-mpl
8. (b) Stable expression of c-mplwt, c-mpl
7 or c-mpl
8 in BAF/
3 transfectants. Cell lysates prepared from 2 × 106 cells were resolved on a
7.5% SDS-PAGE gel, transferred to a nitrocellulose membrane and immunoblotted with a mAb against the myc-epitope. (c) TPO-induced proliferation of BAF-mplwt, BAF-mpl
7, and BAF-mpl
8 cells. [3H]thymidine incorporation as an indicator of cellular proliferation was measured at
different TPO concentrations. The mean of triplicate counts for each data
point is shown.
[View Larger Versions of these Images (13 + 42 + 16K GIF file)]
8 cells (not shown). Surprisingly, stimulation of BAF-mpl
7 with TPO also failed
to induce tyrosine phosphorylation of Jak-2 (Fig. 2 a), even
after increasing the TPO concentration ten-fold (data not
shown). IL-3 was able to induce tyrosine phosphorylation
of Jak-2 in both BAF-mplwt and BAF-mpl
7 cells at comparable levels, demonstrating that Jak-2 is intact in BAF-mpl
7 cells (Fig. 2 a). Stable expression of c-mpl
7 in the
IL-3-dependent myeloid cell line 32D further confirmed the ability of this mutant to induce a mitogenic response
(not shown) in the absence of Jak-2 phosphorylation (Fig. 2 b).
Fig. 2.
C-mpl7 does not
activate Jaks. (a) TPO stimulates
tyrosine phosphorylation of Jak-2
in BAF-mplwt but not in BAF-mpl
7 cells. Growth factor-deprived cells were left untreated
or were stimulated with TPO or
IL-3 for the indicated times and
lysates were prepared. Jak-2 was
immunoprecipitated with anti-
Jak-2 antiserum and subsequently immunoblotted with anti-phosphotyrosine antibodies. Membranes were stripped and reprobed
with anti-Jak-2 antiserum to confirm equal loading of protein in
all lanes. (b) TPO stimulates tyrosine phosphorylation of Jak-2 in
32Dmplwt but not 32Dmpl
7
cells. (c) Jak-2 is tyrosine phosphorylated in TPO-stimulated
(15 min) COS cells transiently expressing c-mplwt but not in cells
expressing c-mpl
7. Lysates were
prepared and probed with anti-myc antibodies to confirm equal
levels of receptor expression in all
samples. An antiphosphotyrosine
immunoblot of Jak-2 immunoprecipitates was performed. (d) Activation of Jak-2 kinase in TPO-stimulated BAF-mplwt but not in
BAF-mpl
7 cells. Kinase activity
of Jak-2 immunoprecipitates was
measured as autophosphorylation
in an in vitro kinase assay. (e) TPO-stimulated tyrosine phosphorylation of Tyk-2 in BAF-mplwt but
not in BAF-mpl
7 cells. Tyk-2
was immunoprecipitated with anti-
Tyk-2 antibodies and subsequently
blotted with antiphosphotyrosine antibodies. Membranes were
stripped and reprobed with anti-
Tyk-2 antibodies to confirm equal
protein loading. (f) Antiphosphotyrosine blot of Jak-1 and Jak-3
immunoprecipitates. IP, immunoprecipitation; WB, Western Blot.
[View Larger Version of this Image (52K GIF file)]
7 to mediate phosphorylation of Jak-2 was also confirmed in COS cells transiently transfected with the deletion mutants (Fig. 2 c).
COS cells transfected with c-mplwt or c-mpl
7 were stimulated with TPO and tyrosine phosphorylation of Jak-2 was
analyzed (Fig. 2 c). Tyrosine phosphorylation of Jak-2 was
detected in cells transfected with c-mplwt but not with c-mpl
7, although similar amounts of receptor were expressed in both transfectants (Fig. 2 c).
7 cells was measured in an in vitro kinase assay (26). Jak-2 kinase activity
was strongly activated in stimulated BAF-mplwt but not in
BAF-mpl
7 cells (Fig. 2 d).
7 cells, we analyzed tyrosine phosphorylation of Tyk-2 in TPO-stimulated BAF-mplwt and
BAF-mpl
7 cells. Phosphorylation of Tyk-2 was detected
at 5 min and 15 min after stimulation of the wild-type receptor but not after stimulation of c-mpl
7 (Fig. 2 e). Furthermore, neither Jak-1 nor Jak-3 were tyrosine phosphorylated in TPO-stimulated BAF-mplwt and BAF-mpl
7
cells (Fig. 2 f). Thus, c-mpl
7 mediates a mitogenic response without detectable phosphorylation of any of the
known Jaks.
7 to activate Jaks was also reflected in a lack of activation of their
major targets, the STAT proteins. Tyrosine phosphorylation of STATs by Jaks leads to activation of their DNA-binding activity (6, 7). Stimulation of the TPO receptor has
been described to activate STAT1, 3, and 5 (10). Using
a DNA probe (GAS-element) (7) which can detect several
activated STATs (including STAT1, 3, and 5), we measured STAT DNA-binding activity in lysates prepared from
TPO-stimulated BAF-mplwt and BAF-mpl
7 cells (Fig. 3
a), and also 32D-mplwt and 32D-mpl
7 cells (data not
shown) in an electrophoretic mobility-shift assay (EMSA).
Complex formation was detected in cells expressing the wild-type receptor but not in cells expressing the mutant receptor. The GAS-binding activity was seen as early as 5 min
after TPO stimulation and was still present after 1 h of
stimulation (Fig. 3 a) whereas no GAS-binding activity was
detected in BAF-mpl
7 cells at any of the time points analyzed. Increasing the concentration of TPO up to 500 or
1,000 ng/ml did not enhance the GAS-binding activity in
BAF-mplwt cells and did not result in any detectable activity in BAF-mpl
7 cells (Fig. 3 a). The identity of the
STATs present in the different complexes detected in
TPO-stimulated BAF-mplwt cells was analyzed by supershift assays with antibodies to STAT1, 3, and 5 (Fig. 3 b).
The complex with the lowest mobility was supershifted
with anti-STAT5 antibodies. Antibodies to STAT1 supershifted the complex with the highest mobility. The complex
with intermediate mobility was diminished by anti-STAT1
and anti-STAT3 antibodies indicating that the complex probably consists of STAT1/STAT3 heterodimers. To confirm
the activation of STAT3, an anti-phosphotyrosine immunoblot of STAT3 immunoprecipitates was performed showing phosphorylation of STAT3 by the wild-type receptor
but not by the mutant receptor (Fig. 3 c). The inability of
c-mpl
7 to induce a STAT-DNA complex is consistent
with the observed lack of Jak activation in TPO-stimulated BAF-mpl
7 cells and 32D-mpl
7 cells. Moreover, this result excludes the possibility that another, as yet unidentified
Jak kinase is activated by the mutant receptor to induce
STAT DNA-binding activity.
Fig. 3.
Induction of GAS-binding activity in BAF-mplwt but not
BAF-mpl7 cells. (a) Growth factor-deprived cells were left untreated or
were stimulated with TPO. The time points and the TPO concentrations analyzed are indicated. Cell extracts were prepared and analysed by
EMSA using the IRF-1 GAS probe. GAS-binding activity was detected in BAF-mplwt but not BAF-mpl
7 cells. (b) The identity of the GAS-binding complexes in BAF-mplwt cells (5
stimulation) was examined in
supershift assays with antibodies specific for STAT1, 3, and 5 ( STAT5a
and STAT5b antibodies were pooled). (c) Antiphosphotyrosine blot of
STAT3 immunoprecipitates shows that STAT3 is tyrosine phosphorylated after TPO-stimulation of the wild-type but not the mutant receptor.
Membrane was stripped and reprobed with anti-STAT3 antibodies to
confirm equal protein loading.
[View Larger Versions of these Images (85 + 73 + 31K GIF file)]
7 is able
to mediate TPO-stimulated proliferation without activation
of the Jak-STAT pathway. We therefore asked if other signaling pathways previously described for c-mpl (8) were
activated in TPO-stimulated BAF-mpl
7 or BAF-mpl
8
cells. As shown in Fig. 4, stimulation of both c-mplwt and
c-mpl
7 induced tyrosine phosphorylation of Shc (a), Vav
(b) and c-mpl (c). In constrast, c-mpl
8 was completely inactive (data not shown). Phosphorylation of Shc and Vav
was slightly reduced and phosphorylation of the receptor
itself was markedly reduced in BAF-mpl
7 cells as compared to BAF-mplwt cells. A phosphotyrosine blot of total
cell lysates after TPO stimulation (Fig. 4 d) was in agreement with the above observations: protein tyrosine phosphorylation was still detectable in BAF-mpl
7 cells but the
number of proteins phosphorylated and the degree of phosphorylation was reduced compared to BAF-mplwt cells.
No tyrosine phosphorylated proteins were detected in lysates from TPO-stimulated BAF-mpl
8 cells (data not
shown). These results suggest that c-mpl
7 mediates activation of tyrosine kinase(s) other than Jaks. The mutation
in box1 in c-mpl
8 appears to disrupt activation of not
only the Jaks but also the additional or alternative tyrosine
kinase(s) active in BAF-mpl
7 cells.
Fig. 4.
Effect of TPO stimulation on Shc, Vav, the receptor
itself, Raf-1, and MAPK.
Growth factor-deprived BAF-mplwt and BAF-mpl7 cells
were either left untreated or
stimulated with TPO for the indicated times and cell extracts
were prepared. Immunoprecipitations were performed with antibodies to Shc (a), Vav (b), and
myc (c) and the immunoprecipitates were blotted with antiphosphotyrosine antibodies (a-c). To
confirm equal loading of protein,
membranes were stripped and
reprobed with the antibodies
used for immunoprecipitations (lower panel of a-c). In (c) a
higher amount of c-mpl
7 protein was immunoprecipitated. (d)
Antiphosphotyrosine immunoblot of total cell lysates. (e) Cell lysates were immunoblotted with
an antibody to Raf-1. The lower
mobility of Raf-1 seen after
stimulation with TPO in BAF-mplwt and BAF-mpl
7 reflects
the increased phosphorylation of
Raf-1 on serine. (f) Cell lysates
were immunoblotted with anti-active MAPK antibodies which
recognize the active forms of
Erk-1 and Erk-2 (different exposures of the same membrane are
shown in the upper and middle
panel). Membranes were stripped
and reprobed with anti-Erk2 antibodies to confirm equal protein
loading.
[View Larger Version of this Image (61K GIF file)]
7 also retained the ability of the wild-type receptor (28, 29) to induce phosphorylation of the serine-threonine kinases Raf-1 (Fig. 4 e) and MAPK (Fig. 4 f), and
upregulation of c-fos and c-myc expression (Fig. 5). While
the c-mpl
7-mediated effect on Raf-1 was comparable to
the wild-type receptor, the phosphorylation of MAPK induced by the mutant receptor was reduced in its intensity
and duration (Fig. 4 f). Induction of c-fos and c-myc protein synthesis was reduced approximatively threefold in
BAF-mpl
7 cells as compared to BAF-mplwt cells. In an
effort to further investigate the importance of these signals
for Jak-independent proliferation, we generated the mutant
c-mpl
7
C by introducing an additional COOH-terminal
truncation (aa 601-625) in the c-mpl
7 mutant; it has been
previously shown that this region is required for both Shc
activation and receptor phosphorylation (13, 21). This
double mutant failed to induce tyrosine phosphorylation of Jak and Shc and phosphorylation of Raf-1 but nevertheless
was sufficient to mediate proliferation in BAF/3 cells although maximal proliferation was reduced about twofold
when compared with the c-mpl
7 mutant (data not
shown). These data suggest that the mitogenic signal required neither Jak activation nor Shc or Raf-1 phosphorylation.
Fig. 5.
TPO stimulates c-fos and c-myc synthesis in BAF-mplwt and
BAF-mpl7 cells. Growth factor-deprived cells were washed twice and
incubated for 30 min at a density of 107 per ml in RPMI 1640 deficient in
methionine and cysteine (ICN). Cells were metabolically labeled as described (36) by adding 0.5 mCi of [35S]methionine (Translabel; ICN) per
ml to the cell suspension. TPO (200 ng/ml) was added simultaneously
and cells were incubated for the indicated times. Unstimulated (U) cells
were incubated with [35S]methionine in the absence of TPO for 1 h. Cell
extracts were prepared and c-fos and c-myc were immunoprecipitated
with antibodies to c-fos (top) or c-myc (bottom). Immunoprecipitates were
resolved by SDS-PAGE (7.5% gel) and analyzed by fluorography. Signals
were quantified with a PhosphorImager.
[View Larger Version of this Image (78K GIF file)]
7 cells and BAF-mpl
8 cells before and
after TPO stimulation. c-mpl
7 mediated an increase in PI
3-kinase activity comparable to the wild-type receptor
(Fig. 6), indicating that Jak activation is not a prerequisite
for PI 3-kinase activation. Mutant c-mpl
8 showed no increase in PI 3-kinase activity. Incubation of BAF-mplwt
and BAF-mplD7 cells with increasing concentrations of the
PI 3-kinase inhibitor wortmannin (1, 10, 100, and 1,000 nM)
resulted in a concentration-dependent decrease in TPO-dependent proliferation as monitored by [3H]thymidine incorporation after 48 h. Approximatively 50% inhibition of
maximal proliferation was observed at a concentration of
100 nM wortmannin, similar to results obtained by others
for erythropoietin- or IL-7-induced proliferation (30, 31);
proliferation was completely abolished at 1 µM (data not
shown). These results suggest that PI 3-kinase may be an
essential player in the generation of the mitogenic response
by TPO. In this context it is of interest that the proliferation-defective mutant c-mpl
8 does not activate PI 3-kinase
(Fig. 6) but that the proliferation-competent C-terminal truncation mutant c-mpl
7
C still mediates PI 3-kinase
activation (M. Dorsch, and S.P. Goff, unpublished observation).
Fig. 6.
Activation of PI 3-kinase in TPO-stimulated BAF-mplwt
and BAF-mpl7 cells. Growth factor-deprived cells were left untreated or were stimulated with TPO for 5 min and cell extracts were prepared. Immunoprecipitations were performed with anti-phosphotyrosine antibodies and the immunoprecipitates were analyzed for PI 3-kinase activity.
Formation of PI 3-P was inhibited by inclusion of 100 nM wortmannin
(+ Wort.) in the kinase reaction. The ratios of labeled PI 3-P in stimulated samples/unstimulated samples are shown as fold activation. The results shown represent one out of three experiments with similar outcomes.
[View Larger Version of this Image (13K GIF file)]
7 is the first cytokine receptor mutation that disrupts Jak activation while preserving other cytokine-stimulated events. Our results indicate that neither proliferation
nor phosphorylation of Shc, Vav, Raf-1, and c-mpl, nor
induction of PI 3-kinase activity requires the activation of
Jaks. However, the reduction of some of these responses
for c-mpl
7 relative to c-mplwt suggests that the full induction of these events depends upon the cooperation of
an intact Jak-STAT pathway with other signaling pathways.
7 cells
(M. Dorsch and S.P. Goff, unpublished observations).
Address correspondence to Stephen P. Goff, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons, 701 W 168th Street, HHSC Rm 1127A, New York, NY 10032. Phone: (212) 305-3794; Fax: (212) 305-8692; E-mail: goff{at}cuccfa.ccc.columbia.edu
Received for publication 13 June 1997 and in revised form 5 September 1997.
M. Dorsch is a fellow of the Howard Hughes Medical Institute. S.P. Goff is a Howard Hughes Medical Institute investigator.We thank Dr. Steven Greenberg for help with the PI 3-kinase assay.
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