From the Cancer Research Campaign Centre for Cell and Molecular Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Rd., London SW3 6JB, United Kingdom
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ABSTRACT |
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Hemopoietic cytokines such as interleukin-3 and
granulocyte colony-stimulating factor (G-CSF) are potent activators of
hemopoietic cell growth and strongly induce activation of extracellular
signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38
mitogen-activated protein (MAP) kinases. However, the role of these
kinases is unclear. Using specific chemical inhibitors for MEK and p38,
we demonstrate here that both ERK and p38 pathways are critically
involved in the transduction of a proliferative signal and cooperate in
G-CSF-induced cell proliferation. We show that, like ERK and JNK
activation, activation of p38 and its downstream substrate MAP
kinase-activated protein kinase 2 by interleukin-3 or G-CSF requires
Ras activation. We demonstrate that two distinct cytoplasmic regions of
the G-CSF receptor are involved in activation of the p38 pathway: a
region within the 100 membrane-proximal amino acids is sufficient to induce low levels of p38 and MAP kinase-activated protein kinase 2 activation, whereas the membrane-distal phosphorylation site Tyr763 mediates strong activation of these kinases.
The levels of p38 activation correlate closely with those of Ras
activation by G-CSF, suggesting that the degree of Ras activation
is a critical determinant for the extent of p38 activation by
hemopoietic cytokines.
MAP1 kinase pathways are
cellular signaling pathways that enable cells to transduce
extracellular signals into an intracellular response (1). In mammalian
cells, three parallel MAP kinase pathways have been identified. The ERK
pathway is activated in response to signals from cell surface receptors
and has been shown to regulate both cell proliferation and
differentiation, depending on the cell context (2). In contrast, p38
and JNK pathways are primarily activated by cellular stress signals
such as proinflammatory cytokines, heat shock, or UV light and have
therefore also been described as "stress-activated protein kinases"
(3). However, activation of p38 and JNK pathways is not limited to a
cellular stress response, since they can also be potently activated by hemopoietic cytokines (4-7).
Multiple JNK isoforms have been identified that originate from
alternative splicing of three mammalian genes (8-10). Similarly, several isoforms of p38 have been described: p38 MAP kinases (MAPKs) are activated by phosphorylation on Thr and Tyr by
dual-specificity MAPK kinases (MAPKKs), which are themselves activated
by MAPKK kinases (MAPKKKs). In addition, small GTPases acting upstream
of MAPKKKs are critically involved in the regulation of mammalian MAPK
pathways. For example, activation of ERKs requires activation of Ras
and concomitant activation of Raf family kinases such as Raf-1, A-Raf,
or B-Raf, which in turn phosphorylate and activate the dual specificity
kinase MEK. MEK then phosphorylates ERKs on Thr and Tyr, resulting in
ERK activation (22). Dual specificity kinases that activate JNK are
MKK4/SEK-1 (23-25) and MKK7 (26, 27), whereas MKK3 and MKK6 have been
identified as activators of p38, displaying some degree of selectivity
for individual p38 isoforms (15, 28). In addition, various MAPKKKs for
JNK and p38 pathways have been described, such as MEKKs, PAK1, GCK,
MLKs, MUK/DLK, MKK5, and ASK1 (reviewed in Ref. 29), most of which are
capable of activating both pathways. There is strong evidence that the
small GTPases Rac and Cdc42 are involved in the regulation of JNK and
p38 pathways (30-32), although their ultimate relationship with
MAPKKKs remains poorly understood. Experiments with dominant negative
Ras have shown that activation of the JNK pathway by growth factor
receptors and hemopoietic cytokine receptors also requires
signaling via Ras (4, 5, 32).
We and others have previously shown that hemopoietic cytokines act as
potent activators of JNK and p38 pathways (4-7). However, the role of
these pathways in hemopoietic cell signaling is, like that of ERKs,
unclear. Activation of Ras and the ERK pathway had been suggested to
mediate a survival signal rather than cell proliferation (33, 34).
However, experiments with dominant negative and constitutively active
versions of MEK indicated a role for ERK in cell proliferation (35). In
order to gain further insight into the role and regulation of MAP
kinase pathways during hemopoietic cell proliferation, we are studying
signaling via the G-CSF receptor as a model system. G-CSF stimulates
proliferation and survival of early hemopoietic progenitor cells and
induces differentiation of myeloid precursor cells into mature
neutrophils (36). However, it can also act as a strong proliferative
signal on myeloid leukemia cell lines such as NFS-60 (37) and was shown
to be produced as an autocrine growth factor in some types of acute
myeloid leukemia (38). The G-CSF receptor is a 130-kDa transmembrane
protein that forms a homodimer upon ligand binding (39). The
cytoplasmic domain contains four conserved tyrosine residues
(Tyr703, Tyr728, Tyr743, and
Tyr763) that are targets of tyrosine phosphorylation (40).
Although some of these tyrosines have been shown to mediate specific
differentiation pathways (40, 41), none is essential for the induction
of a proliferative response (42). However, we have previously shown that Tyr763 is required for activation of JNK, as well as
for high levels of Shc and Ras activation, during a proliferative
response to G-CSF (4).
We show here that a proliferative response to G-CSF is strongly
associated with activation of p38 and its downstream substrate MAPKAP-K2. We demonstrate that activation of the p38 pathway by G-CSF
is Ras-dependent and involves two regions of the G-CSF
receptor that are also involved in Ras activation: a membrane-proximal region, which is sufficient to induce low levels of p38 activation, and
Tyr763, which is required for strong activation of the p38
pathway. Finally, we present evidence that ERK and p38 pathways
cooperate in G-CSF-induced cell proliferation and that the low level of activation mediated by the membrane-proximal region of the receptor is
sufficient to mediate the proliferative effect of p38.
Cell Culture, Plasmid Construction, and Transfection--
NFS-60
cells were obtained from Dr. James Ihle. BaF3 cells were obtained from
Dr. Mary Collins. N6 cells were a kind gift from Dr. Takaya Satoh and
Dr. Yoshito Kaziro. BaF3 and NFS-60 cells were grown in RPMI 1640 medium, including 10% WEHI3 conditioned medium as a source of IL-3,
10% heat-inactivated fetal calf serum, and 4 mM
L-glutamine. All cells were kept at cell densities below 1 × 106 cells/ml in a humidified atmosphere at
37 °C (5% CO2). Wild-type BaF3 cells are unresponsive
to G-CSF in all of the assays described in this paper.
The cDNA for the murine G-CSF receptor was kindly donated by Dr.
Shigekazu Nagata in the mammalian expression vector pEFBOS (pEFBOS
I62). G-CSF receptor cDNAs harboring C-terminal deletions and Tyr
to Phe substitutions were generated by polymerase chain reaction,
cloned in pEFBOS, and stably transfected into BaF3 cells by
electroporation, and cell lines were selected as described previously
(4). Isolated cell clones were examined for expression of G-CSF
receptors by cross-linking with 125I-labeled G-CSF
(Amersham Pharmacia Biotech) and bis(sulfosuccinimidyl)suberate (Pierce) (4) In all assays described here, at least two independently isolated cell clones were tested for each receptor construct.
Cytokines, Antibodies, and Chemical Inhibitors--
Recombinant
human G-CSF was a generous gift from Roche Pharmaceuticals UK.
Recombinant murine IL-3 was purchased from NBS Biologicals. MAPKAP-K2
antiserum was a gift from Prof. Philip Cohen. ERK2 polyclonal rabbit
antiserum 122 was generated against a C-terminal ERK2 peptide (43).
Phosphospecific p38 antibody was purchased from New England Biolabs,
and monoclonal p38 antibody was purchased from Zymed
Laboratories Inc. SB 203580 p38 inhibitor and SKF 105809 control
compound were a gift from Drs. Peter Young and John Lee (SmithKline
Beecham Pharmaceuticals). PD 98059 MEK inhibitor was a gift from Dr.
Alan Saltiel (Parke Davis Pharmaceuticals).
Cell Stimulation and Cell Lysis--
For kinase assays,
0.5-1 × 107 cells were washed twice with RPMI 1640 to remove WEHI3 conditioned medium and were resuspended in RPMI 1640, 10% heat-inactivated fetal calf serum, 4 mM
L-glutamine at a density of 5 × 106
cells/ml. Cells were then returned to 37 °C (5% CO2)
for 4 h. IL-3, G-CSF, and anisomycin (Sigma) were diluted in RPMI
1640 prior to stimulation and were added to cells to a final
concentration of 20 ng/ml IL-3, 50 ng/ml G-CSF, or 10 µg/ml
anisomycin or as otherwise stated in the figure legends. Stimulation
was stopped by washing the cells once in ice-cold phosphate-buffered
saline, 100 µM Na3VO4. For ERK2
and MAPKAP-K2 assays, cells were lysed in 1 ml of IP buffer (50 mM Tris·HCl, pH 7.0, 1% Nonidet P-40, 150 mM
NaCl, 10 mM sodium pyrophosphate, 5 mM NaF, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). For JNK assays, cells were lysed in 1 ml of JNK lysis buffer (20 mM HEPES, pH 8.0, 75 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 20 mM
For Western blot analysis of tyrosine-phosphorylated p38 MAP kinase,
2 × 106 cells were cytokine-deprived and stimulated
at a cell density of 1 × 106 cells/ml as described
above. Cells were then lysed directly in 200 µl of SDS-sample buffer
(80 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 100 µM dithiothreitol, 0.02% bromphenol blue), sonicated 3 times for 1 min each in a Misonix XL waterbath sonicator to shear the DNA, and analyzed by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
In the case of N6-WT cells, prior to cytokine removal, cells were
induced for 12 h with 5 mM IPTG to allow expression of
Ras mutants (34). Control cells were uninduced. After cytokine removal, induced cells were kept in 5 mM IPTG until lysis. Treatment
with IPTG had no effect on parental BaF3 cells or BaF3 cells expressing G-CSF receptors.
Kinase Assays and Anti-p38 Western Blots--
In order to assay
endogenous MAPKAP-K2 activity, MAPKAP-K2 was immunoprecipitated, and
the immunoprecipitates were subjected to an in vitro kinase
assay. 300 µg of total cell lysate or the amount stated in the figure
legends was immunoprecipitated on a revolving wheel at 4 °C for
1.5 h with 1 µg of MAPKAP-K2 antibody prebound to 20 µl of
protein G-Sepharose. Immunoprecipitates were then washed three times
with HBIB buffer (20 mM HEPES, pH 8.0, 0.05% Triton X-100,
50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 100 µM
Na3VO4) and once with kinase buffer (20 mM HEPES, pH 8.0, 20 mM MgCl2, 20 mM
For ERK2 assays, ERK2 was immunoprecipitated, and kinase activity was
assayed on myelin basic protein as a substrate as described previously
(4). JNK activity was assayed in a "pull-down" kinase assay
employing recombinant GST-Jun-(1-131) as described (4). Phosphorylation of GST-Jun was quantitated on a Molecular Dynamics PhosphorImager, using ImageQuant software.
For p38 Western blot analysis, 40 µl of cell lysate in SDS sample
buffer were analyzed on 10% SDS-polyacrylamide gels. Gels were
subsequently transferred to nitrocellulose, which was then blocked for
at least 2 h in 5% fat-free dried milk in Tris-buffered saline,
0.5% Tween 20 (Sigma) (TBST). The blocked nitrocellulose was probed
with antiphospho-p38 antibody (New England Biolabs, 1:1000) in TBST for
2 h at room temperature, washed in TBST, and further incubated for
1 h with horseradish peroxidase-coupled secondary antibody
(Pierce). Proteins were visualized by ECL (Amersham Pharmacia Biotech).
To detect the total amount of p38 MAP kinase, nitrocellulose membranes
were stripped and reprobed with monoclonal anti-p38 antibody (1:1000,
Zymed Laboratories Inc.).
Cell Proliferation Assays--
Induction of cell proliferation
was assayed by measuring incorporation of [3H]thymidine
during DNA synthesis. Long term proliferation was measured by assaying
cell growth over a 5-day period.
For thymidine uptake experiments, cells were washed once with RPMI 1640 to remove cytokine and were then incubated at a density of 1 × 105 cells/ml in RPMI 1640, 10% heat-inactivated fetal calf
serum, 4 mM L-glutamine at 37 °C for 6 h. Cells were then stimulated with IL-3 to a final concentration of 10 ng/ml or with G-CSF as indicated in the figure legends and plated in
96-well plates at a volume of 200 µl/well. In case of pretreatment of
cells with chemical inhibitors, cells were pretreated with 10 µM SB 203580 or 30 µM PD 98059 or the
concentration stated in the figure legends or with the equivalent
amount of solvent (Me2SO) as a control. 24 h after
cytokine stimulation, 20 µl of RPMI 1640, 10% fetal calf serum
containing 1 µl of [methyl-3H]thymidine (1 mCi/ml; Amersham Pharmacia Biotech) were added, and the cells were
incubated for a further 16 h. Cells were then harvested with a
Dynatech cell harvester on grade 934-AH filter paper (Whatman), and the
filters were dried, mixed with 2 ml of scintillation liquid
(Emulsifier-Safe, Packard), and counted in a scintillation counter.
Long term cell proliferation was examined by assaying the growth of
individual cell clones over 5 days. Cells were counted using a Coulter
Counter (Coulter Electronics Ltd.). Cell proliferation of individual
cell clones in response to G-CSF was compared with cell proliferation
observed in response to IL-3 as an internal standard. To monitor cell
proliferation, cells were plated in 10-ml tissue culture flasks (Nunc)
at 1 × 105 cells/ml. Unless otherwise stated, cells
were maintained at cell densities below 1 × 106
throughout the monitored time. Cells were counted after initial plating
and then every 24 h until the end of the experiment. The increase
in cell numbers was then related to the initial number of cells plated.
G-CSF and IL-3 Induce Rapid Activation of the p38 MAP Kinase
Pathway--
We have previously shown that hemopoietic cytokines such
as G-CSF and IL-3 strongly activate ERK and JNK MAP kinases in
cytokine-deprived hemopoietic cells (4). In order to test whether
cytokine stimulation also results in activation of the p38 MAP kinase
pathway, first we examined G-CSF-induced activation of MAPKAP-K2, a
downstream target of p38 (13), in the pre-B precursor cell line BaF3
(45) (Fig. 1A). MAPKAP-K2
activity in cytokine-deprived BaF3 cells expressing wild-type G-CSF
receptors (BaF3-WT) (4) was low. In contrast, stimulation of these
cells with G-CSF resulted in strong activation of MAPKAP-K2 with a peak
of activity at 5 min poststimulation. MAPKAP-K2 activity remained
sustained on a lower level up to 2 h poststimulation and, in
addition, was found to be elevated in cycling cells.
Activation of p38 requires phosphorylation on Thr180 and
Tyr182 (11, 12), and can be assayed directly using specific
antibodies to the phosphorylated form of p38. Western blotting revealed
that, similar to MAPKAP-K2 activation, p38 phosphorylation peaked at 5 min after G-CSF stimulation (Fig. 1B). Consistent with the
sustained activation of MAPKAP-K2 after G-CSF-treatment,
phosphorylation of p38 persisted for at least 2 h following G-CSF
stimulation, suggesting that the maintenance of MAPKAP-K2 activity
requires concomitant activation of p38. p38 was also found to be more
highly phosphorylated in cycling cells than in cytokine-deprived cells. Stimulation of cells with IL-3 showed a similar time course of MAPKAP-K2 activation to that observed for G-CSF (data not shown). The
duration of p38 activation by G-CSF is very similar to that of ERK
activation but unlike that of JNK, which is switched off completely
within 1 h of stimulation and is inactive in cycling cells
(4).
To confirm that MAPKAP-K2 activation requires p38 function in
vivo, cytokine-deprived BaF3-WT cells were pretreated with a highly specific chemical inhibitor for p38, SB 203580 (11), prior to
stimulation (Fig. 2A). The
results show that MAPKAP-K2 activation by IL-3 or G-CSF, as well as by
a stress-signal such as anisomycin, is totally dependent on p38
function, demonstrating that MAPKAP-K2 activation reflects p38
activation in vivo. SB 203580 treatment had no effect on
cytokine-induced activation of JNK and ERK MAP kinases (data not
shown).
Activation of p38 by G-CSF was also observed in the myeloid leukemia
cell line NFS-60 (Fig. 3B,
top right), indicating that activation by
growth-promoting cytokines is a common event in hemopoietic cells.
Taken together, our experiments demonstrate rapid and sustained
activation of the p38 MAP kinase pathway by hemopoietic cytokines.
Comparison of p38 activation with ERK and JNK activation reveals that
both ERK and p38 MAP kinase pathways are similarly regulated during
cytokine-induced hemopoietic cell proliferation.
G-CSF-induced Activation of the p38 Pathway Involves
Tyr763 and the 100 Membrane-proximal Cytoplasmic Amino
Acids of the G-CSF Receptor--
In order to investigate the potential
role of receptor tyrosine phosphorylation in G-CSF signaling, we have
established a series of BaF3 cell lines stably expressing G-CSF
receptor point and deletion mutants. Experiments with these cells have
shown that activation of JNK required Tyr763 of the
receptor, whereas activation of ERK was independent of this residue
(4). Since many signals that activate JNK also activate p38, we
examined if activation of the p38 pathway also required
Tyr763 by testing MAPKAP-K2 and p38 activation in these
cell lines. Whereas G-CSF receptors harboring point mutations at
Tyr728 and Tyr743 (BaF3-Y728F and BaF3-Y743F,
respectively) induced activation of MAPKAP-K2 and p38 similarly to
wild-type G-CSF receptors, cells expressing Y763F mutant G-CSF
receptors (BaF3-Y763F) showed a strong reduction in p38 and MAPKAP-K2
activation following G-CSF stimulation (Figs. 2 and 3). Similarly, a
receptor truncated at residue 727 (BaF3- Activation of the p38 Pathway by Hemopoietic Cytokines Is Dependent
on Ras--
The finding that high levels of G-CSF-induced p38 activity
are mediated by Tyr763 of the receptor, whereas low levels
of p38 activation do not require this residue, correlates closely with
our previous studies on the levels of Ras activation by mutant G-CSF
receptors. We have previously shown that high levels of Ras activation
(30-35% Ras-GTP) require Tyr763, whereas the 100 membrane-proximal amino acids are sufficient to induce low levels of
Ras activation (10-15% Ras-GTP) (4). These observations suggest that
Ras activation may be linked to p38 activation. In order to see whether
activation of p38 is dependent on Ras, we made use of a BaF3 cell line
harboring an IPTG-inducible dominant-negative N17Ras construct (34),
which we engineered to express wild-type G-CSF receptors (N6-WT cells)
(4). In the absence of IPTG, IL-3- or G-CSF-induced activation of
MAPKAP-K2 and p38 in N6-WT cells was similar to that in BaF3-WT cells
(Fig. 4, A and B).
However, IPTG-induced expression of N17Ras resulted in a complete
inhibition of MAPKAP-K2 activation by IL-3 or G-CSF and strongly
reduced phosphorylation of p38. In contrast, anisomycin-induced activation of MAPKAP-K2 and p38 was not significantly affected by
expression of N17Ras. Thus, our data demonstrate that activation of p38
and its downstream target MAPKAP-K2 by hemopoietic cytokines requires
signaling via Ras. Furthermore, the level of p38 activation closely
correlates with the degree to which Ras is activated.
Cooperation of ERK and p38 MAP Kinase Pathways in G-CSF-induced
Proliferation of BaF3 Cells--
Previous reports based on the use of
dominant-negative Ras have suggested that signaling via Ras and ERK MAP
kinases was not required for proliferation induced by hemopoietic
cytokines such as IL-3 or granulocyte-macrophage colony-stimulating
factor and suggested that in hemopoietic cells the ERK pathway mainly
mediates cell survival (33, 34). In order to investigate the role of Ras-dependent MAP kinase pathways such as ERK and p38
pathways in hemopoietic cell proliferation more closely, we used
specific chemical inhibitors of the ERK and p38 MAP kinase pathways, PD 98059 and SB 203580. PD 98059 is a flavone compound that selectively inhibits MEK and therefore activation of ERK MAP kinases (46), whereas
SB 203580 is a pyridine-imidazole that is highly specific for p38 MAP
kinases and inhibits phosphorylation of their downstream targets (11,
47).
Inhibition of MEK by PD 98059 reduced G-CSF-induced DNA synthesis by
40-50% in both BaF3-WT and BaF3-Y763F cells (Fig.
5, A and B).
Inhibition of p38 by SB 203580 reduced DNA synthesis by a similar
proportion in both cell lines. Moreover, a combination of both
inhibitors resulted in an additive effect on cell proliferation, reducing G-CSF-induced DNA synthesis by 80-90% compared with cells treated with solvent (Me2SO) alone.
In order to study the effect of these inhibitors on DNA synthesis and
kinase activities more carefully, we next examined the effect of PD
98059 and SB 203580 over a range of inhibitor concentrations. The
reduction in DNA synthesis observed by treatment of BaF3-WT cells with
different concentrations of PD 98059 clearly parallels the reduction in
ERK2 activity by the same compound (Fig.
6, A and C).
Similarly, higher doses of SB 203580 cause a parallel reduction of
MAPKAP-K2 activity and DNA synthesis, although lower doses, which lead
to moderately reduced levels of MAPKAP-K2 (and thus p38) activation, do
not inhibit DNA synthesis (Fig. 6, B and D; see
below and "Discussion"). These findings provide strong evidence
that the inhibitory effect of PD 98059 and SB 203580 on DNA synthesis
is mediated by the inhibition of ERK2 and p38, respectively. Moreover,
PD 98059 did not inhibit G-CSF-induced activation of MAPKAP-K2 and JNK,
whereas SB 203580 did not inhibit G-CSF-induced activation of ERK2 and
JNK, further demonstrating the specificity of each inhibitor for
individual MAP kinase pathways (Fig. 7).
Neither inhibitor caused an increase in apoptotic cells in the presence
of cytokine. In order to control further for a potential unspecific
effect of SB 203580 on cell proliferation, cells were also treated with
a structurally related compound that does not inhibit p38 (SKF 105809).
Treatment of both cell lines with SKF 105809 did not affect
G-CSF-induced proliferation (data not shown).
The finding that both BaF3-WT and BaF3-Y763F cell lines were equally
sensitive to treatment with SB 203580 was surprising, since Y763F-G-CSF
receptors activate the p38 pathway only weakly (Figs. 2B and
3). When we compared G-CSF-induced cell proliferation of the two cell
lines, it became clear that both wild-type and Y763F mutant G-CSF
receptors were equally able to induce a proliferative signal as
measured by [3H]thymidine uptake assays (data not shown),
confirming results previously reported by de Koning et al.
(42). Interestingly, suboptimal doses of SB 203580 that only partially
inhibit the p38 pathway have little or no effect on DNA synthesis,
whereas stronger inhibition of this pathway results in a marked
inhibition of DNA synthesis (Fig. 6, B and D).
Taken together, these results strongly indicate that low levels of p38
activation are sufficient to mediate a proliferative p38 signal.
However, when comparing long term cell growth induced by wild-type and
Y763F-mutant G-CSF receptors, we found that BaF3-Y763F cells grew
markedly slower than BaF3-WT cells (Fig.
8). We did not observe an increase in apoptotic cells when comparing long term cell growth of BaF3-Y763F and
BaF3-WT cells (data not shown), indicating a lower proliferative capacity of the Y763F-G-CSF receptor mutant. Although it remains unclear whether the reduced long term proliferation induced by Y763F-G-CSF receptors is due to the lower level of p38 activation, the
absence of JNK activation, or the lower level of Ras activation mediated by this receptor mutant, our experiments clearly demonstrate a
role for MEK and p38 MAP kinase in the control of DNA synthesis and
cell growth and suggest that ERK and p38 MAP kinase pathways cooperate
in G-CSF-induced BaF3 cell proliferation.
We have shown rapid activation of p38 MAP kinase and its
downstream target MAPKAP-K2 in response to hemopoietic cytokines such
as IL-3 and G-CSF. Both p38 and MAPKAP-K2 are activated with similar
kinetics, suggesting that p38 activation is required continuously to
keep MAPKAP-K2 in its active state. In an attempt to elucidate the
mechanism by which hemopoietic cytokine receptors activate p38, we used
the G-CSF receptor as a model receptor for structure-function studies.
Our results identified two distinct regions of the cytoplasmic domain
of the receptor that contribute to p38 activation: the potential
tyrosine phosphorylation site Tyr763 and a region within
the 100 membrane-proximal amino acids. Whereas Tyr763
mediates strong activation of p38, the membrane-proximal region only
induces a low level of p38 activation. It is unclear whether these
different levels of activation reflect distinct subcellular pools of
p38 or represent distinct p38 isoforms. The finding that cytokine-induced activation of MAPKAP-K2 can be inhibited by SB 203580 indicates activation of p38 Activation of the p38 pathway clearly requires signaling via Ras.
Inducible expression of dominant-negative N17Ras blocked activation of
MAPKAP-K2 by IL-3 or G-CSF and strongly reduced detectable tyrosine
phosphorylation of p38. We cannot exclude the possibility that a small
level of p38 activation is independent of Ras. However, the
IPTG-inducible system used in this study still allows a small increase
in Ras-GTP following cytokine stimulation (34), which could be
sufficient to induce some phosphorylation of p38 as observed in Fig.
4B. Interestingly, both Tyr763 and the
membrane-proximal domain of the G-CSF receptor are involved in
G-CSF-induced activation of Ras, and the extent of Ras activation mediated by each region correlates closely with the extent of p38
phosphorylation (4), suggesting that the level of Ras activation is
critical for p38 activation. However, inducible expression of oncogenic
V12Ras alone or together with a Y763F-G-CSF receptor was not sufficient
to induce p38 activation, although the level of activated Ras is very
high in such systems (data not shown). These findings are reminiscent
of activation of JNK by the G-CSF receptor, which is also sensitive to
dominant-negative Ras and is completely dependent on the presence of
Tyr763 (4). These findings suggest that a second signal in
addition to Ras activation is required for activation of JNK and p38
pathways. Alternatively, upstream regulators of Ras such as Shc, Grb2,
or Sos might be involved in activation of JNK and p38 pathways. It has
been reported that Sos not only acts as an exchange factor for Ras but
also can stimulate activation of Rac via its Dbl homology domain (48).
Tyr763 of the G-CSF receptor is required for strong
activation of Shc and the formation of Shc-Grb2 complexes, thus also
affecting the amount of Sos recruited to the membrane in response to
G-CSF stimulation (4). It is possible that the presence of a high
amount of Sos in the vicinity of Rac could lead to activation of Rac,
which could then cooperate with Ras to activate JNK and p38 pathways, whereas low levels of membrane-localized Sos as mediated by the membrane-proximal domain of the G-CSF receptor are insufficient to
stimulate the Rac activation required for activation of JNK and strong
phosphorylation of p38. Importantly, Ras activation mediated by the
membrane-proximal domain alone is totally sufficient for activation of
ERK, demonstrating that G-CSF-induced activation of all three MAP
kinase pathways is Ras-dependent, but distinct levels of
Ras activation are associated with activation of different MAP kinases.
While ERK, JNK, and p38 MAP kinases are all activated strongly in
response to IL-3 or G-CSF, there are differences in the kinetics of
activation. ERK and p38 pathways are activated rapidly, with a peak of
activation between 5 and 10 min and remain active on a lower level for
prolonged times. Moreover, low levels of ERK and p38 activity can also
be found in unsynchronized cycling cells. In contrast, JNK is highly
active 15 min poststimulation but is switched off completely within
1 h and is inactive in cycling cells (4). Thus, there is a stark
contrast between the biphasic activation of ERK and p38 pathways
compared with the short term nature of JNK activation. The exact role
of JNK during a proliferative response to hemopoietic cytokines is
unclear at this point. The finding that growth-promoting cytokines such
as IL-3 and G-CSF activate JNK appears puzzling, since activation of
JNK has been associated with the induction of apoptosis (49, 50).
However, expression of the JNK-specific dual specificity phosphatase
M3/6 in BaF3 cells does not inhibit apoptosis induced by
IL-3-withdrawal or ceramide treatment, suggesting that in hemopoietic
cells, JNKs are not involved in the induction of apoptosis (51).
Furthermore, constitutively active JNK mediates Bcr-Abl-induced
transformation and proliferation of hemopoietic cells (52, 53) and is
involved in tumorigenesis by human T-cell leukemia virus 1 (54). Our own results with BaF3 cells expressing Y763F-mutant G-CSF receptors, which are unable to activate JNK, indicate that G-CSF-induced JNK
activation is not necessary for the stimulation of cell proliferation and confirm those reported by de Koning et al. (42),
although we cannot rule out the possibility that low or basal levels of JNK activation that are not detected by our assays might play a role in
cytokine-induced cell proliferation. However, JNK activation might be
important for the maintenance of long term cell proliferation, since
Y763F-G-CSF receptors are less capable to induce long-term cell growth
than wild-type receptors (Fig. 8).
A controversial issue is the role of Ras and the ERK pathway in
cytokine-driven proliferation of hemopoietic cells. In these cells,
this pathway has previously been implicated mainly in antiapoptotic signaling, and it was questioned whether activation of Ras and ERKs was
directly involved in driving cell cycle progression. This view was
mainly based on experiments with inducible expression systems for a
dominant negative form of Ras, which did not block BaF3 cell
proliferation in response to IL-3 (33, 34). However, IPTG-induced
expression of N17Ras in these cells still allows low levels of Ras
activation by IL-3 (34), and such findings could explain BaF3 cell
proliferation in the presence of N17Ras. Interestingly, the kinetic
profile of ERK activation induced by IL-3 or G-CSF in BaF3 cells is
highly reminiscent of that observed in PC12 cells stimulated by
epidermal growth factor, where this has been shown to mediate cell
proliferation (4, 55, 56). In order to investigate directly whether ERK
and p38 MAP kinases play a similar role in hemopoietic cell
proliferation, we studied the effects of specific chemical inhibitors
for either MEK (PD 98059) or p38 (SB 203580) on BaF3 cell
proliferation. Interestingly, although inhibition of MEK by PD 98059 appeared to accelerate BaF3 apoptosis in cytokine-deprived cells,
neither PD 98059 nor SB 203580 induced apoptosis in the presence of
cytokine (data not shown). Rather, treatment of cells with PD 98059 strongly reduced cell proliferation in response to G-CSF without
causing apoptosis, suggesting that during cytokine-induced cell
proliferation ERKs are critically involved in the induction of DNA
synthesis and cell cycle progression (Figs. 5 and 6). Moreover, the
inhibition of p38 by SB 203580 resulted in a similar reduction of DNA
synthesis. Importantly, SB 203580 treatment of BaF3 cells expressing a
Y763F-mutant G-CSF receptor, which only allows low levels of p38
activation, strongly inhibited G-CSF-induced DNA synthesis, while
suboptimal concentrations of SB 203580, which only partially inhibited
the p38 pathway, had little or no effect on DNA synthesis. Higher doses
of SB 203580, however, resulted in strong inhibition of p38 activity
and DNA synthesis. These results argue for the existence of a minimal
activation threshold for p38 activation that is sufficient to mediate
the proliferative effect of p38 (Figs. 5 and 6). Interestingly, in
T-cells, proliferation in response to IL-2 and IL-7 appears to require
p38 rather than ERK (7). In contrast, we show that in BaF3 cells, which
represent an earlier stage of hemopoietic cell development (45),
combination of both inhibitors reduced DNA synthesis to 90% of that
observed in control cells (Fig. 5), and equivalent results were
obtained for cells proliferating in response to IL-3 (data not shown).
Taken together, these findings demonstrate that in BaF3 cells ERK and
p38 pathways cooperate in induction of DNA synthesis and cell cycle
progression upon stimulation with hemopoietic cytokines.
The targets of cooperative ERK and p38 signaling are unclear. One
possible result could be the induction of c-fos and the AP-1
transcription factor complex. Transcription of c-fos
requires activation of ternary complex factors, which form a ternary
complex with serum response factor and the serum response element (57). The ternary complex factors Elk-1, Sap-1, and Sap-2 can all act as
substrates for ERK, JNK, and p38 MAP kinases (58-63). Cooperation of
ERK and p38 pathways had been shown to be important in the activation
of Elk-1 and Sap-1 and the induction of c-fos transcription during a stress response (61, 64). It has become clear from studies
such as these that parallel MAP kinase cascades can cooperate in the
induction of gene transcription via the serum response element.
Interestingly, c-fos induction in response to cytokines such
as IL-3 has been linked to ERK activation in BaF3 cells (65), and it is
feasible that both ERK and p38 pathways are involved in this response.
Although the immediate targets of cooperative ERK and p38 signaling
remain to be identified, our results suggest that
Ras-dependent ERK and p38 MAP kinase pathways cooperate,
possibly together with other cytokine-induced pathways such as Stat
pathways, to ensure optimal hemopoietic cell proliferation. The high
turnover of hemopoietic cells and the need to respond to extrinsic
challenges such as infections require fast expansion of individual
hemopoietic cell populations. Activation of multiple MAP kinase
pathways may be one of the mechanisms used by hemopoietin receptors to
increase their proliferative capacity and to meet the requirement for
rapid clonal expansion of hemopoietic cells.
INTRODUCTION
Top
Abstract
Introduction
References
, also named CSBP2
or stress-activated protein kinase 2a (3, 11-13); p38
, also named
stress-activated protein kinase 2b (3, 14) and its splice isoform
2
(15, 16), p38
, also termed stress-activated protein kinase 3 or ERK6
(17-19); and p38
, otherwise known as stress-activated protein
kinase 4 (20, 21). p38
and p38
are the most homologous of this
class of MAP kinases and are both inhibited by the pyridine-imidazoles
SB 203580 and SB 202190, whereas the
and
isoforms are
insensitive to these compounds (16, 18, 21).
EXPERIMENTAL PROCEDURES
-glycerophosphate, 0.05% Triton X-100, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin). Cell lysis was performed on a shaking platform at 4 °C
for 30 min. Cell debris and nuclei were removed by centrifugation at
12,000 × g. After protein concentration of cell
lysates was determined by Bradford protein assay (Bio-Rad), lysates
were immediately assayed for kinase activity or stored at
70 °C.
-glycerophosphate). The washed immunoprecipitates were incubated with 30 µl of kinase buffer, including 10 µM ATP (Sigma), 30 µM MAPKAP-K2 peptide
substrate (KKLNRTLSVA) (44), and 66 µCi/ml [
-32P]ATP
(Amersham Pharmacia Biotech) at 30 °C for 30 min. The reaction was
stopped by spotting 20 µl of the reaction onto P81 paper (Whatman), which was subsequently washed five times with 75 mM
orthophosphoric acid. Myelin basic protein kinase activity was
quantified by counting P81 papers in a Cerenkov counter.
RESULTS
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Fig. 1.
G-CSF induces activation of the p38 MAP
kinase pathway. A, time course of MAPKAP-K2 activation.
Cells were deprived of cytokine for 4 h and then restimulated with
G-CSF for the indicated times or with anisomycin for 10 min. Also shown
is MAPKAP-K2 activity in cycling cells. MAPKAP-K2 was
immunoprecipitated from 150 µg of total cell lysate and assayed on
the peptide substrate KKLNRTLSVA. Averages of two independent
experiments are shown. B, time course of p38
phosphorylation. Cells were cytokine-deprived as described in
A and stimulated with G-CSF for the indicated times, and
cell lysates were analyzed by SDS-PAGE. The phosphorylation state of
p38 was determined by Western blotting with an antibody specific for
the phosphorylated form of p38 (pp38, top). To
detect the total amount of p38, the blot was stripped and reprobed with
a monoclonal p38 antibody (p38, bottom).
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Fig. 2.
Activation of MAPKAP-K2 by the G-CSF
receptor. A, activation of MAPKAP-K2 in BaF3 cells
expressing wild-type G-CSF receptors (BaF3-WT). MAPKAP-K2 activation is
dependent on p38 in vivo. MAPKAP-K2 was immunoprecipitated
and assayed as described in the legend to Fig. 1. Prior to stimulation,
cells were pretreated with 10 µM SB 203580 or an
equivalent amount of solvent (Me2SO). Averages from three
independent experiments are shown; error bars
represent S.D. B, activation of MAPKAP-K2 involves
Tyr763 and the 100 membrane-proximal cytoplasmic amino
acids of the G-CSF receptor. Averages from three independent
experiments are shown; error bars represent S.D.
BaF3-Y763F, -Y728F, and -Y743F, BaF3
cells expressing G-CSF receptors with amino acid substitutions at
Tyr763, Tyr728, and Tyr743,
respectively; BaF3- 727, BaF3 cells expressing G-CSF
receptors truncated at amino acid 727.
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Fig. 3.
Induction of p38 phosphorylation by the G-CSF
receptor involves Tyr763 and the 100 membrane-proximal
cytoplasmic amino acids. NFS-60 and BaF3 cells stably expressing
wild-type or mutant G-CSF receptors were assayed for activation of the
p38 pathway. Lysates were subjected to SDS-PAGE and analyzed by
anti-phospho-p38 Western blotting (pp38, upper
panels). Blots were then stripped and reprobed with
monoclonal p38 antibody to control for the total amount of p38
(p38, lower panels).
727), which comprises only
the 100 membrane-proximal amino acids of the cytoplasmic domain,
induced low levels of p38 and MAPKAP-K2 activation (Figs. 2B
and 3), whereas untransfected BaF3 cells did not activate the p38
pathway in response to G-CSF (Fig. 3). These findings show that
Tyr763 is critically involved in G-CSF receptor-mediated
activation of the p38 pathway and is required for maximal activation.
However, unlike JNK activation, a small but significant level of p38
activation is independent of Tyr763, suggesting that two
regions in the G-CSF receptor contribute to activation of the p38
pathway: Tyr763, phosphorylation of which results in strong
activation of p38, and, second, a region within the 100 membrane-proximal amino acids that induces low levels of p38 activation.
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Fig. 4.
Activation of the p38 pathway by hemopoietic
cytokines is dependent on Ras. N6-WT cells were either uninduced
or induced with 5 mM IPTG for 16 h. Cytokine-deprived
cells were stimulated with medium alone ( ) or with IL-3 (
), G-CSF
(
), or anisomycin (
) and subsequently lysed. A,
MAPKAP-K2 immunoprecipitate kinase assays. Averages from three
independent experiments are shown; error bars
represent S.D. B, lysates were subjected to SDS-PAGE and
analyzed by anti-phospho-p38 Western blotting (pp38,
top). The blot was then stripped and reprobed with
monoclonal p38 antibody (p38, bottom).
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Fig. 5.
ERK and p38 MAP kinase pathways cooperate
during G-CSF-induced cell proliferation. Shown is a
[3H]thymidine uptake assay of the effect of chemical
inhibitors for MEK (PD 98059) or p38 (SB 203580) on DNA synthesis of
BaF3-WT (A) and BaF3-Y763F (B) cell lines. Total
counts are shown. Cells were cytokine-deprived for 6 h and
untreated ( ) or pretreated with solvent alone (0.3%
Me2SO) (
), PD 98059 (×), SB 203580 (
), or a
combination of both inhibitors (
) for 30 min and induced to the
indicated concentrations of G-CSF. 24 h poststimulation,
[3H]thymidine was added for 16 h, after which cells
were harvested. Experiments were performed in triplicate, and the
results from three independent experiments are shown; S.E. values were
below 10% and were omitted for presentational reasons.
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Fig. 6.
Dose dependence of MAP kinase inhibition and
inhibition of DNA synthesis by PD 98059 and SB 203580 in BaF3-WT
cells. A and B, -fold activation of ERK2 at
10 min (A) and MAPKAP-K2 at 5 min (B) after G-CSF
stimulation (10 ng/ml) over a range of inhibitor doses. ERK2 was
immunoprecipitated and assayed on myelin basic protein as a substrate,
and MAPKAP-K2 was immunoprecipitated and assayed as described in the
legend to Fig. 1, C and D. A
[3H]thymidine uptake assay of DNA synthesis induced by
G-CSF (10 ng/ml) is shown over a range of inhibitor doses. Total counts
are shown. The assay was performed as described in the legend to Fig.
5. Averages from three independent experiments are shown.
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Fig. 7.
Absence of cross-inhibition of MAP kinase
pathways by PD 98059 and SB 203580. Shown is -fold activation of
ERK2, MAPKAP-K2, and JNK (10 µg/ml) by G-CSF (10 ng/ml) in the
presence of vehicle (Me2SO) alone ( ), PD 98059 (
), or
SB 203580 (
). ERK2 and MAPKAP-K2 were assayed as described in the
legend to Fig. 6. JNK activity 15 min after G-CSF stimulation was
assayed using a GST-Jun pull-down assay. GST-Jun phosphorylation was
quantitated using a PhosphorImager. Averages from three independent
experiments are shown.
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Fig. 8.
Tyr763 of the G-CSF receptor is
required for optimal long term cell proliferation in response to
G-CSF. BaF3-Y763F and BaF3-WT transfectants were cytokine-deprived
for 6 h and were then plated at a density of 10,000 cells/ml at
various concentrations of G-CSF. Cell counts 5 days after plating are
shown. Growth assays were performed in duplicate. Cell clones with
similar growth properties in response to IL-3 were compared, and
results for two independently isolated cell clones for each receptor
are shown (A, BaF3-WT-4 ( ) versus BaF3-Y763F-4
(
); B, BaF3-WT-7 (
) versus BaF3-Y763F-6
(
)). Bars indicate cell proliferation (
) of each
subclone in response to IL-3 (10 ng/ml).
DISCUSSION
or
isoforms by hemopoietic cytokines, since p38
and -
are poor activators of MAPKAP-K2 and
are not inhibited by SB 203580 (21). Further experiments will be
necessary to address the activation state of p38
and
in
cytokine-stimulated cells.
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ACKNOWLEDGEMENTS |
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We are grateful to Takaya Satoh and Yoshito Kaziro for generously providing N6 cells. We also thank Alan Saltiel (Parke Davis Pharmaceuticals) for the PD 98059 compound and Stephen C. Lee and Peter R. Young (SmithKline Beecham Pharmaceuticals) for the SB 203580 compound.
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FOOTNOTES |
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* This work was supported by the Leukaemia Research Fund and the Cancer Research Campaign.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: SmithKline Beecham Pharmaceuticals, Department of
Molecular Neurobiology, New Frontiers Science Park, Harlow, Essex, UK.
§ A Gibb Life fellow of the Cancer Research Campaign. To whom correspondence should be addressed. Tel.: 44-171-352-9772; Fax: 44-171-352-5630; E-mail: chrism{at}icr.ac.uk.
The abbreviations used are:
MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPKAP-K2, MAP kinase-activated protein
kinase 2; IPTG, isopropyl--D-thiogalactopyranoside; G-CSF, granulocyte colony-stimulating factor; IL, interleukin; MAPK, MAP kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; IP, immunoprecipitation; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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