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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3694-3702
Interleukin-4 Synergizes With Raf-1 to Promote Long-Term
Proliferation and Activation of c-jun N-terminal Kinase
By
Megan K. Levings,
Darrell C. Bessette, and
John W. Schrader
From The Biomedical Research Centre, University of British Columbia,
Vancouver, British Columbia, Canada.
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ABSTRACT |
This report shows that interleukin-4 (IL-4), which plays a key role
in regulating immune responses, fails to support cellular growth. We
investigated whether this failure of IL-4 to promote growth was because
of its unique inability to activate the Ras/Raf/Erk pathway. Consistent
with other reports, expression in Ba/F3, a factor-dependent
hematopoietic cell line, of either activated Q61KN-Ras or a
hormone-inducible activated Raf-1, resulted in suppression of apoptosis
but not in long-term growth. However, in the presence of IL-4, Ba/F3
cells that expressed either Q61KN-Ras or activated Raf-1
grew continuously at a rate comparable with that stimulated by IL-3.
Investigation of the biochemical events associated with the stimulation
of long-term growth showed that, as expected, the presence of activated
Raf-1 resulted in an increased activity of extracellular signal
regulated kinase (ERK) mitogen-activated protein kinase
(MAPK) but not of c-jun N-terminal kinase/stress-activated
protein kinase (JNK). However, surprisingly, if IL-4 was present, cells
expressing active Raf-1 exhibited increases in JNK activity. These
observations point to a novel mechanism for JNK activation involving
synergy between Raf-1 and pathways activated by IL-4 and suggest that
in hematopoietic cells proliferation is correlated not only with
"mitogen activated" ERK activity, but also with JNK activity.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTERLEUKIN-4 (IL-4) belongs to a
molecular family, which includes hematopoietic growth factors, growth
hormone, and leptin. IL-4 is produced by T cells and mast cells and
acts on immune cells to promote an antibody-mediated immune response to infection. IL-4 was originally termed B-cell growth factor by virtue of its ability to enhance the proliferation of B
cells, as well as their secretion of IgG1 and
IgE.1 Subsequently, IL-4 was shown to also
have growth-promoting effects on T cells and mast cells.2,3
Although IL-4 is frequently classified as a growth factor, there is, in
fact, little evidence that it can itself act as a mitogen. IL-4 was
unable to induce resting B cells to leave G0 and enter the
G1 phase of the cell cycle.4,5 Moreover, the
reported enhancing effects on growth of primary T cells or mast cells
were detected in short-term assays, which measured
[3H]-thymidine incorporation, mitochondrial activity, or
cell numbers at 24 to 48 hours.3,6
The lack of clear biological evidence that IL-4 is a growth factor
correlates with the failure of IL-4 to activate Ras family members7,8 or the downstream kinase cascades. Although all three of the receptor subunits for IL-4 are members of the cytokine receptor superfamily, IL-4 (and the highly related IL-13) are atypical
because they fail to activate the extracellular signal regulated kinase
(ERK) cascade,9 the c-jun N-terminal
kinase/stress-activated protein kinase (JNK) cascade,10 and
the p38 mitogen-activated protein (MAP) kinase
cascade.11 Activation of Ras and the MAP-family cascades
can have varying effects on the cell cycle depending on the strength of
the signal. In general, activation of Raf and the downstream kinases
correlates with progression through the cell cycle.12
Activation of ERK is an absolute requirement for both hematopoietic
cells and fibroblasts to progress from G0 to S-phase,13 and overexpression of a constitutively active
mutant of the upstream activator of ERK, MAP/ERK Kinase 1 (MEK1),
promotes cell-cycle entry.14
IL-4 stimulates the phosphatidlyinositol-3'-kinase (PI3'kinase)
pathway,15 perhaps through stimulation of phosphorylation of the insulin receptor substrates (IRS-1 and IRS-2), which can then
associate with the p85 subunit of PI3'kinase.16,17 Activity of the PI3'-kinase pathway is required for IL-4 to protect cells from
apoptosis18 and to upregulate levels of c-myc mRNA
(Wieler and Schrader, submitted). Stimulation of cells
with IL-4 also leads to activation of JAK-1 and JAK-3 or Tyk-2 and
STAT-6.19 STAT-6 mediates the effects of IL-4 on the
development of TH2 cells and the IgE class of
antibodies20 and some of its anti-inflammatory effects of
IL-4.21
We have confirmed the failure of IL-4 to promote cell-cycle entry and
have hypothesized that this relates to its inability to activate the
Ras/Raf/ERK pathway. We show here that the inability of IL-4 to promote
cell-cycle progression is complemented by expression of activated
mutants of N-Ras or conditionally active mutants of Raf-1.
Surprisingly, we find that IL-4 and Raf not only synergize to promote
proliferation, but also to increase JNK activity.
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MATERIALS AND METHODS |
PKH26 cell labeling.
Ba/F3 cell membranes were labeled with PKH26 according to the
manufacturer's directions (Sigma, Mississauga, Ontario, Canada). An
aliquot of cells was analyzed by flow cytometry at time 0, and the
remaining cells were incubated at 37°C in media supplemented with 2%
(vol/vol) of a 10× concentrate of media conditioned by WEHI-3B (W3)
or 3% (vol/vol) media conditioned by AgXO63 cells that had been
transfected with the murine IL-4 cDNA (IL-4 CM).22 Aliquots
of cells were analyzed by flow cytometry at 24, 48, and 72 hours after
the addition of cytokine.
Recombinant plasmids.
The cDNA for Q61KN-Ras was a gift from Rob Kay (Terry Fox
Labs, Vancouver, Canada). Polymerase chain reaction mutagenesis was used to replace the N-Ras ATG with a BamHI site to allow for
in-frame fusion to a sequence encoding the hemagglutinin (HA) tag
downstream of the cytomegalovirus promoter in pcHA (Giulio
Superti-Furga, EMBL, Heidelberg, Germany). The fidelity of the cDNA was
confirmed by sequencing. The cDNAs encoding  Raf-1:ER or Raf-1:ER
K70W in pBABEpuro23 were a gift from Martin McMahon (UCSF
Cancer Center, San Francisco, CA). The Raf-1:ER protein
product consists of the enhanced green fluorescent protein (EGFP) at
the N-terminus, which is fused to the kinase domain of human c-Raf-1
and, in turn, fused to the hormone binding domain of the human-estrogen
receptor. The Raf-1:ER K70W protein consists of the kinase domain of
human c-Raf-1, which lacks the lysine residue critical for enzymatic activity, and is fused to the hormone binding domain of the estrogen receptor. The glutathione-S-transferase (GST)-JNK1 cDNA in
pEFBOS was a gift from Leonard Zon.24
Cell culture and transfections.
Ba/F3 cells and transfectants were grown at 37°C in humidified
incubators gassed with 5% CO2. Cells were routinely
passaged in RPMI 1640 (GIBCO-BRL, Grand Island, NY) supplemented with
10% fetal calf serum (FCS) (Intergen, Purchase, NY), 50 µmol/L 2-ME, and 2% (vol/vol) W3. Cells expressing Raf-1:ER or Raf-1:ER K70W were maintained as above, but in phenol-red free RPMI 1640 (GIBCO-BRL). For each transfection, cells were mixed with
15 µg of linearized vector and subjected to electroporation by using
a Bio-Rad gene pulser at 960 µF and 270 V. Individual clones of
neomycin-resistant (for Q61KN-Ras and GST-JNK1) or
puromycin-resistant (for Raf-1:ER or Raf-1:ER K70W) cells were
tested for expression of the exogenous cDNA of interest. Expression
of Q61KN-Ras was evaluated by immunoblotting cell lysates
with antibodies against the HA tag (12CA5; Boehringer Mannheim, Laval,
Quebec, Canada). Expression of Raf-1:ER was determined by
flow-cytometric quantitation of EGFP levels. Expression of Raf-1:ER
K70W was determined by immunoprecipitation with antiestrogen receptor
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA; no. sc543) and
immunoblotting with an anti-Raf-1 antibody (Pharmingen, San Diego, CA).
Several positive clones with similar levels of expression of the
exogenous cDNA were derived in each case.
Cell viability and proliferation assays.
Proliferation and viability of cells was assessed by cell counting or
[3H]-thymidine incorporation into de novo synthesized
DNA. For cell counting experiments, the cells were incubated at 1 × 105 cells/mL at 37°C in complete media supplemented as
indicated with 2% (vol/vol) W3, 2 µg/mL synthetic IL-4 (Ian
Clark-Lewis, The Biomedical Research Centre, Vancover, British
Columbia, Canada), or 3% (vol/vol) IL-4 CM, and/or 100 nmol/L
4-hydroxy-tamoxifen (4HT), (Research Biochemicals, Natick, MA). The 4HT
was stored as a 100 µmol/L stock in 100% ethanol at  20°C. An
equivalent volume of ethanol was added to one set of cultures to rule
out solvent effects. Cells that excluded trypan blue were counted at
the indicated times, and cultures were diluted as appropriate to
maintain a consistent density. For [3H]-thymidine uptake
assays, cells were plated at 250 cells per well in a Terasaki
microtitre plate, incubated for 40 hours at 37°C in the
indicated conditions, and then pulsed with 15 µCi/mL for a
further 8 hours. Cells were harvested and incorporation of
[3H]-thymidine assessed in a scintillation counter.
Chemically synthesized IL-3 (Ian Clark-Lewis, The Biomedical Research
Centre) was used at a concentration of 1 µg/mL shown to stimulate
maximal proliferation of Ba/F3 cells. Chemically synthesized IL-4 was
used as indicated at a saturating dose (2 µg/mL).
Immunoprecipitations, kinase assays, and immunoblotting.
To assess biochemical parameters under conditions in which cells were
normally grown, cells were incubated at 37°C for 16 hours at an
initial density of 2 × 105 cells/mL in a 40-mL volume of
complete medium supplemented as indicated with IL-3 (2% W3), IL-4 (2 µg/mL synthetic IL-4 or 3% IL-4 CM), and/or 4HT (100 nmol/L 4HT).
Cells were lysed in lysis buffer10 and the total amount of
protein in each lysate was determined by the BCA protein assay (Pierce,
Rockford, IL). Normalized amounts of protein were subjected to
immunoprecipitation and kinase activity was determined in vitro as
previously described.10 Raf-1:ER was immunoprecipitated
with an antiestrogen receptor antibody (Santa Cruz no. sc543) coupled
to protein A-Sepharose. For assay of Raf activity, the kinase assay
buffer (KAB) consisted of 25 mmol/L HEPES, 10 mmol/L MgCl2,
1 mmol/L MnCl2, and 1 mmol/L DTT. The reaction was
initiated by the addition of 0.5 µg GST-MEK1 (UBI, Lake Placid, NY),
1 µM cold ATP and 10 µCi of [ -32P]ATP, and
incubated for 30 minutes at 30°C. ERK-1 and ERK-2 were immunoprecipitated with agarose-conjugated antibodies (Santa Cruz no.
sc154). To assay ERK activity, the KAB consisted of 20 mmol/L HEPES, 5 mmol/L MgCl2, 5 mmol/L EGTA, 50 mmol/L  -glycerol
phosphate, 2 mmol/L sodium vanadate, and 5 mmol/L 2-ME. The reaction
was initiated by the addition of 15 µg Myelin Basic Protein (MBP) (Sigma) and 10 µCi of [-32P]ATP and incubated for 10 minutes at 30°C. JNK-1 was immunoprecipitated with agarose-conjugated
antibodies (Santa Cruz Biotechnology, catalogue no. sc474). To assay
JNK activity, the KAB consisted of 25 mmol/L HEPES, 25 mmol/L
MgCl2, 2 mmol/L DTT, 50 mmol/L -glycerol phosphate, and
0.5 mmol/L sodium vanadate. The reaction was initiated by addition of 1 µg GST-cJun and 10 µCi of [-32P]ATP and incubated
for 20 minutes at 30°C. Reactions were stopped by addition of sodium
dodecyl sulfate sample buffer. The eluate was subjected to sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and
phosphorylated proteins were detected by autoradiography. To assess
equivalency of loading, membranes were immunoblotted with either an
anti-ERK-2 antibody (Santa Cruz no. sc94), an anti-JNK-1 antibody
(Santa Cruz no. sc1648), an anti-GST antibody (Molecular Probes) or an
anti-GFP antibody (Clontech, Palo Alto, CA).
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RESULTS |
IL-4 is not a true growth factor in Ba/F3 cells.
The addition of IL-4 to cultures of primary mast cells, T cells, B
cells, or factor-dependent hematopoietic cell lines over a period of 24 to 48 hours results in increased survival and incorporation of
[3H]-thymidine.3,6 However, there is no
sustained increase in cell numbers and the cells eventually die. These
observations are compatible with the notions that either IL-4 promoted
short-term survival and cells that had previously entered the cell
cycle were permitted to complete DNA synthesis, or alternatively, that IL-4 was indeed able to stimulate cell-cycle progression and growth in
a fraction of cells, but failed to support their long-term survival. To
distinguish between these possibilities, we exploited a technique based
on labeling the membranes of cells with a fluorescent dye (PKH26) that
cannot be transferred to neighboring cells but is shared between
daughter cells.25 Flow-cytometric analysis of labeled Ba/F3
cells that were growing in IL-3 for increasing periods of time showed
the expected series of peaks corresponding to dilution of the dye by
cell division (Fig 1A). In contrast, analysis of labeled Ba/F3 cells that were cultured for 3 days in IL-4
showed a large peak (66%; Fig 1B) of cells that corresponded to cells
that had completed one cell division with no peaks corresponding to two
or more cell divisions. These data show that IL-4 is unable to
stimulate the repeated entry of cells into cycle.
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| Fig 1.
IL-4 is not a growth factor in Ba/F3 cells. Ba/F3 cells
were labeled with the fluorescent dye PKH26. Immediately after
labeling, incorporation of the dye was analyzed by flow cytometry (time
0). Cells were cultured in complete media supplemented with (A) IL-3 or
(B) IL-4 and aliquots of cells were analyzed by flow cytometry at 24, 48, and 72 hours to determine the amount of dye dilution.
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IL-4 synergizes with Ras to stimulate long-term proliferation.
To test the hypothesis that the failure of IL-4 to promote the
long-term growth of hematopoietic cells rests solely in its inability
to stimulate Ras activity, we generated stable clones of Ba/F3 cells
that expressed an activated mutant of N-Ras (Q61KN-Ras),
fused to an N-terminal HA epitope-tag. We compared the ability of
parental Ba/F3 cells (Fig 2A) and Ba/F3
cells expressing Q61KN-Ras (Q61KN-Ras cells) to
proliferate in the presence or absence of IL-4 by counting cells over a
period of 5 days. Q61KN-Ras cells failed to grow in the
absence of IL-3 (Fig 2B), although, unlike parental Ba/F3 cells, which
were all dead by 96 hours, they did not undergo apoptosis in the
absence of factor over the 5-day duration of the experiment. In
striking contrast to parental Ba/F3, in the presence of IL-4,
Q61KN-Ras cells grew exponentially at a rate comparable
with those of parental or Q61KN-Ras cells in IL-3 (Fig 2B).
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| Fig 2.
Activated Q61KN-Ras in combination with IL-4
stimulates the long-term growth of Ba/F3 cells. (A) Ba/F3 or (B)
Q61KN-Ras cells were washed free of IL-3 and incubated in
IL-3, IL-4, or media alone with no factor (MA) at a density of 1 × 105 cells/mL. Cells were counted in triplicate at the
indicated times and diluted as appropriate to maintain a consistent
density of cells. The results are representative of two independent
experiments, with two independent clones. Error bars represent the SEM
of triplicate samples.
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IL-4 synergizes with Raf-1 to promote long-term proliferation.
There is evidence that Ras is upstream of the three MAP-kinase family
cascades,26 as well as the PI3'-kinase
pathway.27 Because IL-4 activates the PI3'-kinase
pathway,15 but fails to activate Raf-1 or any of the three
MAP-family kinases, ERK, JNK or p38 MAP-kinase,9-11 we
focused on these. As activation of the Raf/ERK pathway has been
strongly correlated with growth, we first asked whether the synergistic
effect of an activated Ras on signals provided by IL-4 could be
replaced by expression of activated Raf-1 protein. We expressed a
fusion protein consisting of an active fragment of human Raf-1 fused to
an enhanced EGFP and the hormone-binding domain of the estrogen
receptor, so that kinase activity could be induced by addition of
estradiol or an analog, 4HT. When expressed in NIH-3T3 cells and
activated by 4HT this protein results in transformation and rapid
activation of MEK1 and ERK.28 We expressed the cDNA in
Ba/F3 cells and individual clones (hereafter referred to as Raf-1:ER
cells) were obtained and screened for EGFP by flow cytometry.
Consistent with observations in NIH-3T3 cells,29 expression
of Raf-1:ER increased 3- to 5-fold after overnight incubation in 100 nmol/L 4HT (data not shown). As documented below, enhanced expression
of Raf1:ER was also accompanied by elevated Raf-1 activity.
We assayed [3H]-thymidine uptake to assess the effects of
activation of Raf-1:ER by increasing concentrations of 4HT (Fig 3A). Even at high
concentrations, 4HT had only a small effect on DNA synthesis. In
contrast, in the presence of IL-4, the addition of 4HT resulted in a
dose dependent, synergistic stimulation of DNA synthesis. The maximal
biological response to 4HT in the presence of saturating levels of IL-4
was observed at 100 nmol/L, and this concentration was used for all
subsequent experiments. Parental Ba/F3 cells failed to respond to 4HT
alone or in combination with IL-4 (Fig 3A).
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| Fig 3.
Activation of  Raf-1:ER in combination with
IL-4 stimulates DNA synthesis and the long-term growth of Ba/F3 cells.
(A) Ba/F3 or Raf-1:ER cells were washed free of IL-3 and plated at
250 cells/well in a Terasaki microtitre plate in medium alone or IL-4,
with increasing concentrations of 4HT. After 40 hours, the cells were
pulsed with [3H]-thymidine for an additional 8 hours,
harvested, and counted in a scintillation counter. (B) Raf-1:ER or
(C) Raf-1:ER K70W cells were washed free of IL-3 and incubated in
IL-3, IL-4, 100 nmol/L 4HT, IL-4 plus 100 nmol/L 4HT or without factor
(MA) at a density of 1 × 105 cells/mL. Cells were counted
in triplicate at the indicated times and diluted as appropriate to
maintain a consistent density. The results are representative of
several independent experiments, with two independent clones. Error
bars represent the SEM of triplicate samples.
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Next, we investigated whether Raf-1 activity and IL-4 synergized to
support long-term proliferation of Raf-1:ER cells by counting cells
over a period of 4 days. As shown in Fig 3B, Raf-1:ER cells cultured in media alone rapidly declined in number, and this
decline could be delayed by the presence of IL-4. The induction of
Raf-1 activity by the presence of 4HT also prevented the decline in
cell numbers seen in media alone, and the number of cells increased slowly. In contrast, the combination of 4HT and IL-4 resulted in a
dramatic, synergistic stimulation of cell growth that closely resembled
that seen with the combination of IL-4 and activated Ras (Fig 2B). To
confirm that the synergistic proliferation in the presence of active
Raf-1 and IL-4 was dependent on Raf-1 kinase activity, we repeated the
experiment with stable clones of Ba/F3 cells that expressed a kinase
inactive form of Raf-1:ER (Raf-1:ER K70W cells). Raf-1:ER K70W
cells in the absence of factor or in IL-4 died at the same rate as
parental Ba/F3 cells whether or not 4HT was present (Fig 3C). Thus, the
synergistic proliferation induced by active Raf-1 and IL-4 was
dependent on Raf-1 kinase activity. Furthermore, as described below,
this synergistic proliferation was dependent on the activity of kinases
downstream of Raf-1 as addition of the MEK1/2 inhibitor PD90859
inhibited the proliferation of Raf-1:ER cultured in IL-4 and 4HT.
Stimulation of Raf-1:ER activity leads to ERK activation.
These results suggested that the relevant pathway downstream of Ras
that synergized with IL-4 was indeed the Raf/MEK/ERK pathway. To
confirm this notion at a biochemical level, we examined the activity of
Raf and ERK in Raf-1:ER cells after the addition of 4HT. We observed
an increase in activity of Raf-1:ER at 2 hours after the addition of
4HT (Fig 4A). However, enzymatic activity of Raf-1:ER continued to increase, peaking 12 to 16 hours after the
addition of 4HT (Fig 4A). Similarly, we detected a
significant increase in ERK activity 2 hours after the addition of 4HT,
and this activity continued to increase with time (Fig 4B). We
performed all subsequent experiments on cells incubated with 4HT for 16 hours to ensure maximal Raf kinase activity. To allow comparison with
parental Ba/F3 cells, which in the absence of IL-3 were only viable
over a 16-hour period if IL-4 were present, we normally assessed the
effect of 4HT in the presence of IL-4. Stimulation of ERK activity in
Raf-1:ER cells by addition of 4HT was dose dependent (Fig
5) and occurred at concentrations of 4HT
above 25 nmol/L. Parental Ba/F3 or Raf-1:ER K70W cells did not show any increase in ERK activity after stimulation with 4HT (data not
shown). Ba/F3 or Raf-1:ER cells that had been stimulated with IL-3
or IL-4 for 16 hours showed no detectable levels of ERK activity (Fig
5; data not shown).
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| Fig 4.
Addition of 4HT activates Raf-1 and ERK kinase activity.
Raf-1:ER cells were stimulated at 37°C in RPMI, 10% FCS and 100 nmol/L 4HT for the indicated time. The cells were lysed and analyzed
for (A) Raf-1 activity by immunoprecipitation with an antiestrogen
receptor antibody followed by an in vitro kinase assay by using
GST-MEK1 as a substrate or (B) ERK activity by immunoprecipitation with
an anti-ERK antibody, followed by an in vitro kinase assay by using MBP
as a substrate. Positive control cells were stimulated with IL-3 for 10 minutes. Phosphorylated proteins were visualized after SDS-PAGE and
autoradiography. A 15 minute and a 1-hour exposure are shown to clearly
demonstrate the activity of Raf-1:ER at both the 16 and 2 hour time
points. The quantity of immunoprecipitated (IP) protein in each lane
was assessed by immunoblotting (IB) with antibodies against GFP
( GFP) or ERK (ERK).
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| Fig 5.
Dose-dependent activation of ERK in Raf-1:ER cells
after stimulation with 4HT for 16 hours. Raf-1:ER cells were
stimulated at 37°C for 16 hours at 2 × 105 cells/mL in
RPMI, 10% FCS plus IL-3, IL-4, or IL-4 plus increasing concentrations
of 4HT. ERK was IP from the cell lysate and kinase activity was
determined in vitro by using MBP as a substrate. Phosphorylated
proteins were visualized after SDS-PAGE and autoradiography. The
quantity of IP protein in each lane was assessed by IB with antibodies
against ERK (ERK).
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To determine whether the activation of ERK that we observed in
Raf-1:ER cells stimulated with 4HT was critical for the
proliferative activity seen in the presence of IL-4, we used an
inhibitor of MEK1/2, PD90859. Raf-1:ER cells were incubated in 4HT
and IL-4 for 16 hours and PD90859 (30 µmol/L) was added 1 hour before
lysis of cell. This resulted in complete inhibition of the activation of ERK (data not shown). Importantly, PD90859 also induced a
dose-dependent inhibition of the proliferation of Raf-1:ER cells
stimulated by 4HT and IL-4 (data not shown).
Stimulation of Raf-1:ER activity leads to JNK activation.
Other work from our laboratory has shown that growth stimuli
like IL-3 or Steel Locus factor induced activation of JNK, although IL-4 did not,10 thus correlating the ability of a growth
factor to stimulate JNK with its ability to stimulate growth.
Therefore, we investigated the activity of JNK in Raf-1:ER cells.
Surprisingly, stimulation of Raf-1:ER cells with 4HT for 16 hours in
the presence of IL-4, resulted in dose-dependent activation of JNK (Fig
6A). Our observation that expression of
active Raf-1 in these conditions resulted in activation of JNK was
unexpected as many investigators have shown JNK to be downstream of
MEK-kinase 1 (MEKK1), but not of Raf-1,30 and IL-4 does not
activate JNK.10 JNK was originally identified as a
stress-activated kinase,31 and we needed to rule out the
possibility that activation of JNK resulted from stress induced by the
addition of 4HT. Therefore, we evaluated the response to 4HT in
parental Ba/F3 cells incubated for 16 hours in the presence of IL-4. In
three independent experiments, we failed to see increased JNK activity
(Fig 6B).
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| Fig 6.
Dose-dependent activation of JNK in Raf-1:ER but not
Ba/F3 cells after stimulation with 4HT for 16 hours. (A) Raf-1:ER
cells were stimulated at 37°C for 16 hours at 2 × 105
cells/mL in RPMI, 10% FCS plus IL-3, IL-4, or IL-4 plus increasing
concentrations of 4HT. (B) Ba/F3 cells were stimulated for 16 hours at
37°C in RPMI supplemented with 10% FCS and IL-3 (3), IL-4 (4), or
IL-4 plus 100 nmol/L 4HT (4 + HT). JNK was IP from the cell lysate,
and kinase activity was determined in vitro by using GST-cJun as a
substrate. Phosphorylated proteins were visualized after SDS-PAGE and
autoradiography. The quantity of immunoprecipitated protein in each
lane was assessed by immunoblotting (IB) with antibodies against JNK
(JNK).
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We were interested to know if MEK1/2 activity was required for
this unexpected activation of JNK. However, in contrast to our
observations on the activation of ERK, the effect of PD90859 on JNK
activation in Raf-1:ER cells cultured overnight in 4HT and IL-4 was
inconsistent. In some experiments PD90859 had no effect on JNK
activation, whereas in others the presence of PD90859 inhibited the
increase JNK activity stimulated by 4HT in the presence of IL-4 (data
not shown). It is possible that the presence of PD90859 over a 16-hour
period had itself a tendency to stress the cells and that this was a
factor in the experiments in which addition of PD98059 did not block
activation of JNK.
Activation of JNK is not due to the production of an autocrine
factor.
Activation of JNK has been reported in NIH-3T3 cells expressing
activated Raf-1:ER and was due to an autocrine production of
heparin-binding epidermal growth factor.30,32,33 Ba/F3 cells do not express receptors for EGF,34 but we were
interested to know whether the activation of JNK we observed was caused
by another autocrine pathway. To address this question, we collected medium from Raf-1:ER cells that had been stimulated with 4HT for 48 hours and concentrated it 10-fold. We incubated cells with this medium,
starting with a concentration of 50% and observed that it had no
ability to suppress apoptosis or promote growth of Ba/F3 cells, either
alone or in combination with IL-4 (data not shown). Moreover,
incubation of Ba/F3 cells for 16 hours in this conditioned medium
failed to result in activation of JNK (data not shown). However, this
approach does not exclude the possibility of an autocrine factor that
was cell-surface bound or extremely labile. To address this
possibility, we first made stable clones of Ba/F3 cells that expressed
JNK-1 that was tagged with GST at the N-terminus so that it was
distinguishable from the endogenous JNK in Ba/F3 cells. We mixed 1 × 106 of these Ba/F3 GST-JNK-1 cells together with 1 × 107 Raf-1:ER cells and cultured the mixture of cells for
16 hours with IL-3, IL-4, or IL-4 plus 4HT. We used a 10:1 ratio of
Raf-1:ER cells to Ba/F3 GST-JNK-1 cells to maximize the possibility
of detecting an autocrine factor. We then lysed the mixture of cells and, using glutathione Sepharose beads, specifically precipitated GST-JNK-1 from the Ba/F3 GST-JNK-1 cells. As shown in Fig
7A, this GST-JNK-1 from the Ba/F3
GST-JNK-1 cells that had been cultured together with a majority of
Raf-1:ER cells stimulated with IL-4 plus 4HT was not activated. In
contrast, analysis of GST-JNK-1 from control mixed cultures that had
been stimulated with IL-3 showed the expected activation of GST-JNK-1.
After depletion of GST-JNK-1 from the lysates, we were able to
determine the activity of the total endogenous JNK-1 in the mixed
lysate by immunoprecipitation with an anti-JNK-1 antibody. These
immunoprecipitates contained JNK from both Raf-1:ER and Ba/F3
GST-JNK-1 cells, but because there was a great excess of Raf-1:ER
cells, the signal from Raf-1:ER cells predominated. As shown in Fig
7B, endogenous JNK was activated in the mixed cultures that were
stimulated with IL4 and 4HT, reflecting the activation of JNK-1 in
Raf-1:ER cells we had previously observed (Fig 6A). The data
presented in Fig 7, thus, provide strong evidence that the activation
of JNK induced in Raf-1:ER cells cultured with IL-4 and 4HT was not
secondary to the production of an autocrine factor.
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| Fig 7.
Failure to detect an autocrine factor that can activate
JNK in a cell-mixing experiment. Cells (1 × 106 Ba/F3)
expressing GST-JNK1 were mixed together with 1 × 107
Raf-1:ER cells and stimulated for 16 hours at 37°C in RMPI, 10%
FCS with either IL-3 (3), IL-4 (4), or IL-4 plus 100 nmol/L 4HT
(4 + HT). (A) GST-JNK1 was affinity purified (AP) from the mixed
cell lysate with glutathione Sepharose beads, and the activity was
determined in vitro assay by using GST-cJun as a substrate. After
SDS-PAGE and autoradiography, the membrane was immunoblotted with
antibodies against GST to assess equivalency of loading (GST). (B)
After the lysate had been cleared with glutathione Sepharose,
endogenous JNK was IP with anti-JNK1 antibodies, and the kinase
activity was determined in vitro by using GST-cJun as a substrate.
After SDS-PAGE and autoradiography, the membrane was immunoblotted with
antibodies against JNK1 (JNK) to assess equivalency of loading.
|
|
IL-4 and Raf-1 synergize to activate JNK.
In the experiments described above, IL-4 was always included on the
basis that in its absence the control, parental Ba/F3 cells died over
the 16-hour period of the experiment. However, as shown in Fig 3B,
Raf-1:ER cells were viable when cultured in 4HT alone. Therefore, we
were able to ask whether 4HT-induced Raf-1 activity alone was
sufficient to induce JNK activity or whether addition of IL-4 was also
necessary. To our surprise, although incubation of Raf-1:ER cells
for 16 hours with 4HT alone induced maximal activity of Raf-1 and Erk
(Fig 4), JNK was only activated when both IL-4 and 4HT were present
(Fig 8A). To confirm that the stimulation
of JNK activity was dependent on Raf-1 kinase activity, we repeated the
experiment in Raf-1:ER K70W cells. The results showed that like the
synergistic stimulation of growth, the synergistic activation of JNK by
Raf-1 and IL-4 was dependent on kinase activity of the Raf-1:ER
protein (Fig 8B). To investigate the kinetics of this synergy, we
stimulated Raf-1:ER cells with 4HT for 16 hours to ensure maximal
levels of Raf-1:ER protein and activity and then acutely stimulated
the cells for 10 minutes with IL-3 or IL-4 (Fig
9A). Stimulation of these cells with IL-4 for 10 minutes failed to induce JNK activity, whereas control cells
stimulated with IL-3 for 10 minutes exhibited activation of JNK (Fig
9B).
View larger version (76K):
[in this window]
[in a new window]
| Fig 8.
Raf-1 and IL-4 synergize to activate JNK. (A) Raf-1:ER
or (B) Raf-1:ER K70W cells were stimulated for 16 hours at 37°C in
RPMI, 10% FCS supplemented with IL-3 (3), IL-4 (4), 100 nmol/L 4HT
(4HT), or both IL-4 and 100 nmol/L 4HT (4 + HT). JNK-1 was IP and
JNK activity was determined in vitro by using GST-cJun as a substrate.
Phosphorylated proteins were visualized after SDS-PAGE and
autoradiography. The membranes were immunoblotted with anti-JNK
antibodies to assess equivalency of loading (JNK).
|
|
View larger version (57K):
[in this window]
[in a new window]
| Fig 9.
Stimulation with IL-4 for 10 minutes in the presence of
activated Raf-1 is not sufficient to activate JNK. Raf-1:ER cells
were stimulated for 16 hours at 37°C in RPMI, 10% FCS with or
without 100 nmol/L 4HT. The cells were then stimulated for 10 minutes
with IL-3 (3) or IL-4 (4) or left untreated as a control (). (A)
Raf-1:ER was IP with antiestrogen receptor antibodies and kinase
activity was determined in vitro by using GST-MEK1 as a substrate. (B)
JNK was IP with anti-JNK1 antibodies and kinase activity was determined
in vitro by using GST-cJun as a substrate. Phosphorylated proteins were
visualized after SDS-PAGE and autoradiography. The membrane was
subsequently immunoblotted with an anti-JNK1 antibody to determine
equivalency of loading.
|
|
| |
DISCUSSION |
Here we show that IL-4 is not a true growth factor in Ba/F3 cells (Fig
1) and present evidence that this is due to its inability to activate
the Ras pathway, and, in particular, the Raf-1 kinase. We have
demonstrated that, whereas neither activated Ras (Fig 2B) nor Raf-1
(Fig 3A and B) alone could stimulate proliferation, either could
synergize with IL-4 and support long-term growth (Figs 2B and 3). Our
data support the notion that activation of Ras and Raf are necessary,
but not sufficient, for cell-cycle progression13 and are
consistent with other reports that expression of activated Ras or Raf
did not lead to factor independent growth of hematopoietic cells, but
were able to inhibit apoptosis induced by withdrawal of
IL-3.35,36
The Raf/MEK/ERK pathway is a key regulator not only of proliferation,
but also of differentiation.12 Recently, it has become clear that the intensity of the Raf signal determines whether a cell
will divide or arrest.23,37 Our results in hematopoietic cells are consistent with this concept, as activation of Raf-1:ER is
clearly necessary for cell division (Fig 3). Moreover, when we
expressed a more highly active version of Raf-1:ER (Raf-1:ER Y340D, Y341D23), which led to a higher level of ERK
activity, we observed cell-cycle arrest, even in the presence of IL-3
or IL-4 (data not shown).
The role of JNK in cell-cycle progression is less clear, but it is
clearly activated not only by stress, but also by growth stimuli.10 In our system, activation of JNK was not
sufficient for cell-cycle progression in the presence of IL-4 as
expression of an activated V12Rac-1, which activates JNK in
Ba/F3 cells,38 did not synergize with IL-4 to support the
long-term growth of Ba/F3 cells (M.K. Levings, R.A. Salmon, Y. Quin,
and J.W. Schrader, unpublished data).
Two signaling pathways activated by IL-4, the PI3'K pathway and the
Jak-STAT pathway, may synergize with Raf-1 activity to promote growth.
STAT6 is required for the downregulation of the cell-cycle inhibitor
p27Kip1 by IL-4.39 The PI3'kinase pathway
promotes cell survival,40 but this effect is unlikely to be
the basis of the synergy with Raf-1 as cells expressing active Raf-1
alone exhibit increased survival. Recently, we have shown that IL-4
upregulates levels of c-myc mRNA through a PI3'kinase-dependent
mechanism (Wieler and Schrader, submitted). This
IL-4-dependent increase in c-myc mRNA is a good candidate for
an IL-4-dependent pathway that could synergize with Raf-1 to promote growth.
We were surprised to find that whereas ERK activity was not detectable
above control levels in cells growing in IL-3 (Fig 5), these cells
exhibited levels of JNK activity equivalent to Raf-1:ER cells that
had been stimulated with IL-4 plus 4HT (Figs 6 through 8). These data
show that, at least in Ba/F3 cells growing in mitogenic concentrations
of IL-3, high levels of JNK activity do not inhibit cell-cycle
progression or induce apoptosis, and, in fact, correlate with
proliferation. However, the high level of JNK activity we observed in
parental Ba/F3 cells is not a general phenomenon. Thus under similar
conditions, other IL-3-dependent cell lines, IL-3-dependent primary
mast cells, or IL-2-dependent primary T cells have undetectable levels
of JNK activity (data not shown). One potential explanation for the
discrepancy in levels of JNK activity may be that Ba/F3 cells do not
express a critical phosphatase such as M3/6 that normally downregulates
JNK activity.41
The activation of JNK after stimulation of Raf-1 activity and addition
of IL-4 was unexpected as neither Raf nor IL-4 alone are able to
activate upstream activators of JNK.10,30,38 We were unable
to find any evidence to suggest that activation of JNK was caused by
stress or the production of an autocrine factor (Figs 6B and 7). We
could not detect a factor in media conditioned by Raf-1:ER cells
that had been activated with 4HT in either a biological assay or a JNK
kinase assay (data not shown). The results of cell-mixing experiments
shown in Fig 7 argue strongly against an indirect mechanism of
activation of JNK through induction of an autocrine factor or
cell-surface bound molecule. There remains, however, the formal
possibility that the activation of JNK is caused by the production of
an autocrine factor that acts on a receptor that is only expressed in
cells expressing activated Raf-1.
Current evidence favors the view that ERK and JNK are activated by
distinct, nonoverlapping mechanisms. Our data suggest that this may not
always be the case, because in the cells studied here, activation of
Raf-1 is clearly able to influence JNK activity (Figs 6 through 8). The
level at which the combination of Raf-1 and IL-4 influences JNK and/or
upstream activators of JNK, such as MKK4&7 and/or MEKK1, remains to be
determined. Nor is it clear whether it is Raf-1 itself or a downstream
kinase (ie, MEK or ERK) that can synergize with IL-4 to activate JNK.
The observation that the synergistic effect of IL-4 and Raf-1 on JNK
activity was not evident after exposure to IL-4 for 10 minutes (Fig 9), argues against an acute effect of IL-4 and suggests that there may be a
requirement for the synthesis of a new protein such as a kinase or
another regulator of kinase cascades. Finally, it is also conceivable
that the observed increase in JNK activity does not result directly
from the activity of Raf and IL-4, but is rather a consequence of
cell-cycle progression.
IL-4 stimulated increases in the lipid products of PI3'kinase activity
could be involved in activation of Rac-1 through the pleckstrin
homology domains in Rho family exchange factors.42,43 However, these phospholipid products alone are not sufficient to
activate JNK or induce Rac-mediated transcription factor
pathways.43 Although Frost et al44 did not find
any evidence for synergy between Raf-1 and V12Rac-1 or
V12Cdc42 in activation of JNK, it is possible that
overexpression of these mutants provided a saturating signal that
masked any potential cooperative effect with Raf-1. It would be
interesting to see if coexpression of an activated PI3'kinase and
activated Raf would lead to activation of JNK, and what effects
addition of PI3'kinase inhibitors would have on JNK activition in our system.
In conclusion, we have demonstrated that the failure of IL-4 to support
long-term cellular growth can be complemented by expression of
activated Raf-1. These data support the notion that Raf-1 activity is
necessary but not sufficient for cell-cycle progression. Our observation that IL-4 and Raf-1 synergize to induce activation of JNK
raises the possibility that JNK activity is also necessary for
cell-cycle progression and that it may be partially regulated downstream of Raf-1. The activation of JNK did not appear to involve early events in IL-4 signal transduction and we could not find evidence
for the involvement of autocrine mechanisms. Further identification of
the pathway(s) activated by IL-4 that interact with the Raf kinase
cascade to activate JNK could provide new insights into the mechanisms
that regulate JNK activity and proliferation.
| |
ACKNOWLEDGMENT |
We thank Martin McMahon for the Raf-1:ER and Raf-1:ER K70W
expression vectors, helpful discussions, and critical reading of the
manuscript. We thank Rob Kay for the Q61KN-Ras cDNA,
Leonard Zon for the GST-JNK-1 cDNA, and Ruth Salmon and Ian Foltz for
critical reading of the manuscript.
| |
FOOTNOTES |
Submitted November 6, 1998; accepted January 20, 1999.
Supported by grants from the Medical Research Council of Canada and The
Canadian Arthritis Society.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to John W. Schrader, MB, PhD, The Biomedical
Research Centre, 2222 Health Sciences Mall, University of British
Columbia, Vancouver, British Columbia, Canada, V6T 1Z3; e-mail:
john{at}brc.ubc.ca.
| |
REFERENCES |
1.
Paul WE:
Interleukin-4: A prototypic Immunoregulatory Lymphokine.
Blood
77:1859, 1991[Free Full Text]
2.
Yokota T, Otsuka T, Mosmann T, Banchereau J, DeFrance T, Blanchard D, De Vries JE, Lee F, Arai K:
Isolation and characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expresses B-cell- and T-cell-stimulating activities.
Proc Natl Acad Sci USA
83:5894, 1986[Abstract/Free Full Text]
3.
Mosmann TR, Bond MW, Coffman RL, Ohara J, Paul WE:
T-cell and mast cell lines respond to B-cell stimulatory factor 1.
Proc Natl Acad Sci USA
83:5654, 1986[Abstract/Free Full Text]
4.
Defrance T, Vanbervliet B, Aubry JP, Takebe Y, Arai N, Miyajima A, Yokota T, Lee F, Arai K, de Vries JE, Banchereau J:
B cell growth-promoting activity of recombinant human interleukin 4.
J Immunol
139:1135, 1987[Abstract]
5.
Oliver K, Noelle RJ, Uhr JW, Krammer PH, Vitetta ES:
B-cell growth factor (B-cell growth factor I or B-cell-stimulating factor, provisional 1) is a differentiation factor for resting B cells and may not induce cell growth.
Proc Natl Acad Sci USA
82:2465, 1985[Abstract/Free Full Text]
6.
Spits H, Yssel H, Takebe Y, Arai N, Yokota T, Lee F, Arai K, Banchereau J, de Vries JE:
Recombinant interleukin 4 promotes the growth of human T cells.
J Immunol
139:1142, 1987[Abstract]
7.
Satoh T, Nakafuku M, Miyajima A, Kaziro Y:
Involvement of ras p21 protein in signal-transduction pathways from interleukin 2, interleukin 3, and granulocyte/macrophage colony-stimulating factor, but not from interleukin 4.
Proc Natl Acad Sci USA
88:3314, 1991[Abstract/Free Full Text]
8.
Duronio V, Welham MJ, Abraham S, Dryden P, Schrader JW:
p21ras activation via hemopoietin receptors and c-kit requires tyrosine kinase activity but not tyrosine phosphorylation of p21ras GTPase- activating protein.
Proc Natl Acad Sci USA
89:1587, 1992[Abstract/Free Full Text]
9.
Welham MJ, Duronio V, Schrader JW:
Interleukin-4-dependent proliferation dissociates p44erk-1, p42erk-2, and p21ras activation from cell growth.
J Biol Chem
269:5865, 1994[Abstract/Free Full Text]
10.
Foltz IN, Schrader JW:
Activation of the stress-activated protein kinases by multiple hematopoietic growth factors with the exception of interleukin-4.
Blood
89:3092, 1997[Abstract/Free Full Text]
11.
Foltz IN, Lee JC, Young PR, Schrader JW:
Hemopoietic growth factors with the exception of interleukin-4 activate the p38 mitogen-activated protein kinase pathway.
J Biol Chem
272:3296, 1997[Abstract/Free Full Text]
12.
Avruch J, Zhang XF, Kyriakis JM:
Raf meets Ras: Completing the framework of a signal transduction pathway.
Trends Biochem Sci
19:279, 1994[Medline]
[Order article via Infotrieve]
13.
Marshall CJ:
Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation.
Cell
80:179, 1995[Medline]
[Order article via Infotrieve]
14.
Mansour SJ, Matten WT, Hermann AS, Candia JM, Rong S, Fukasawa K, Vande Woude GF, Ahn NG:
Transformation of mammalian cells by constitutively active MAP kinase kinase.
Science
265:966, 1994[Abstract/Free Full Text]
15.
Gold MR, Duronio V, Saxena SP, Schrader JW, Aebersold R:
Multiple cytokines activate phosphatidylinositol 3-kinase in hemopoietic cells. Association of the enzyme with various tyrosine- phosphorylated proteins.
J Biol Chem
269:5403, 1994[Abstract/Free Full Text]
16.
Wang LM, Myers MG Jr., Sun XJ, Aaronson SA, White M, Pierce JH:
IRS-1: Essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells.
Science
261:1591, 1993[Abstract/Free Full Text]
17.
Welham MJ, Bone H, Levings M, Learmonth L, Wang LM, Leslie KB, Pierce JH, Schrader JW:
Insulin receptor substrate-2 is the major 170-kDa protein phosphorylated on tyrosine in response to cytokines in murine lymphohemopoietic cells.
J Biol Chem
272:1377, 1997[Abstract/Free Full Text]
18.
Scheid MP, Lauener RW, Duronio V:
Role of phosphatidylinositol 3-OH-kinase activity in the inhibition of apoptosis in haemopoietic cells: Phosphatidylinositol 3-OH-kinase inhibitors reveal a difference in signalling between interleukin-3 and granulocyte-macrophage colony stimulating factor.
Biochem J
312:159, 1995
19.
Watanabe S, Arai K:
Roles of the JAK-STAT system in signal transduction via cytokine receptors.
Curr Opin Genet Dev
6:587, 1996[Medline]
[Order article via Infotrieve]
20.
Kaplan MH, Schindler U, Smiley ST, Grusby MJ:
Stat6 is required for mediating responses to IL-4 and for development of Th2 cells.
Immunity
4:313, 1996[Medline]
[Order article via Infotrieve]
21.
Kuperman D, Schofield B, Wills-Karp M, Grusby MJ:
Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production.
J Exp Med
187:939, 1998[Abstract/Free Full Text]
22.
Karasuyama H, Melchers F:
Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors.
Eur J Immunol
18:97, 1988[Medline]
[Order article via Infotrieve]
23.
Woods D, Parry D, Cherwinski H, Bosch E, Lees E, McMahon M:
Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.
Mol Cell Biol
17:5598, 1997[Abstract]
24.
Sanchez I, Hughes RT, Mayer BJ, Yee K, Woodgett JR, Avruch J, Kyriakis JM, Zon LI:
Role of SAPK/ERK kinase-1 in the stress-activated pathway regulating transcription factor c-Jun.
Nature
372:794, 1994[Medline]
[Order article via Infotrieve]
25.
Slezak SE, Horan PK:
Fluorescent in vivo tracking of hematopoietic cells. Part I. Technical considerations.
Blood
74:2172, 1989[Abstract/Free Full Text]
26.
Cano E, Mahadevan LC:
Parallel signal processing among mammalian MAPKs.
Trends Biochem Sci
20:117, 1995[Medline]
[Order article via Infotrieve]
27.
Rodriguez-Viciana P, Warne PH, Vanhaesebroeck B, Waterfield MD, Downward J:
Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation.
Embo J
15:2442, 1996[Medline]
[Order article via Infotrieve]
28.
Samuels ML, Weber MJ, Bishop JM, McMahon M:
Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase.
Mol Cell Biol
13:6241, 1993[Abstract/Free Full Text]
29.
Pritchard CA, Samuels ML, Bosch E, McMahon M:
Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 Cells.
Mol Cell Biol
15:6430, 1995[Abstract]
30.
Minden A, Lin A, McMahon M, Lange-Carter C, Derijard B, Davis RJ, Johnson GL, Karin M:
Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK.
Science
266:1719, 1994[Abstract/Free Full Text]
31.
Kyriakis JM, Avruch J:
Sounding the alarm: Protein kinase cascades activated by stress and inflammation.
J Biol Chem
271:24313, 1996[Free Full Text]
32.
McCarthy SA, Samuels ML, Pritchard CA, Abraham JA, McMahon M:
Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes.
Genes Dev
9:1953, 1995[Abstract/Free Full Text]
33.
McCarthy SA, Chen D, Yang BS, Garcia Ramirez JJ, Cherwinski H, Chen XR, Klagsbrun M, Hauser CA, Ostrowski MC, McMahon M:
Rapid phosphorylation of Ets-2 accompanies mitogen-activated protein kinase activation and the induction of heparin-binding epidermal growth factor gene expression by oncogenic Raf-1.
Mol Cell Biol
17:2401, 1997[Abstract]
34.
Collins MK, Downward J, Miyajima A, Maruyama K, Arai K, Mulligan RC:
Transfer of functional EGF receptors to an IL3-dependent cell line.
J Cell Physiol
137:293, 1988[Medline]
[Order article via Infotrieve]
35.
Cleveland JL, Troppmair J, Packham G, Askew DS, Lloyd P, Gonzalez-Garcia M, Nunez G, Ihle JN, Rapp UR:
V-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells.
Oncogene
9:2217, 1994[Medline]
[Order article via Infotrieve]
36.
Okuda K, Ernst TJ, Griffin JD:
Inhibition of p21ras activation blocks proliferation but not differentiation of interleukin-3-dependent myeloid cells.
J Biol Chem
269:24602, 1994[Abstract/Free Full Text]
37.
Sewing A, Wiseman B, Lloyd AC, Land H:
High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1.
Mol Cell Biol
17:5588, 1997[Abstract]
38.
Foltz IN, Gerl RE, Wieler JS, Luckach M, Salmon RA, Schrader JW:
Human mitogen-activated protein kinase kinase 7 (MKK7) is a highly conserved c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activated by environmental stresses and physiological stimuli.
J Biol Chem
273:9344, 1998[Abstract/Free Full Text]
39.
Kaplan MH, Daniel C, Schindler U, Grusby MJ:
Stat proteins control lymphocyte proliferation by regulating p27Kip1 expression.
Mol Cell Biol
18:1996, 1998[Abstract/Free Full Text]
40.
Kulik G, Klippel A, Weber MJ:
Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt.
Mol Cell Biol
17:1595, 1997[Abstract]
41.
Smith A, Ramos-Morales F, Ashworth A, Collins M:
A role for JNK/SAPK in proliferation, but not apoptosis, of IL-3-dependent cells.
Curr Biol
7:893, 1997[Medline]
[Order article via Infotrieve]
42.
Han J, Luby-Phelps K, Das B, Shu X, Xia Y, Mosteller RD, Krishna UM, Falck JR, White MA, Broek D:
Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav.
Science
279:558, 1998[Abstract/Free Full Text]
43.
Reif K, Nobes CD, Thomas G, Hall A, Cantrell DA:
Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways.
Curr Biol
6:1445, 1996[Medline]
[Order article via Infotrieve]
44.
Frost JA, Steen H, Shapiro P, Lewis T, Ahn N, Shaw PE, Cobb MH:
Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins.
EMBO J
16:6426, 1997[Medline]
[Order article via Infotrieve]
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ABSTRACT
INTRODUCTION