From the Departments of ¶ Immunology and Pharmacology
and the Divisions of § Radiation Oncology and
Oncology Research, Mayo Foundation,
Rochester, Minnesota 55905
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ABSTRACT |
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Cytokines trigger the rapid assembly of
multimolecular signaling complexes that direct the activation of
downstream protein kinase cascades. Two protein kinases that have been
linked to growth factor-regulated proliferation and survival are
mitogen-activated protein/ERK kinase (MEK) and its downstream target
Erk, a member of the mitogen-activated protein kinase family. Using
complementary pharmacological and genetic approaches, we demonstrate
that MEK and Erk activation requires a phosphatidylinositol 3-kinase
(PI3-K)-generated signal in an interleukin (IL)-3-dependent
myeloid progenitor cell line. Analysis of the upstream pathway leading
to MEK activation revealed that inhibition of PI3-K did not block c-Raf
activation, whereas MEK activation was effectively blocked under these
conditions. Furthermore, agents that elevated cAMP suppressed
IL-3-induced c-Raf activation but did not inhibit MEK activation.
Because c-Raf activation and MEK activation were inversely affected by
PI3-K- and cAMP-dependent pathways, we examined whether
IL-3 activated the alternative Raf isoforms A-Raf and B-Raf. Although
IL-3 did not activate B-Raf, A-Raf was activated by the cytokine.
Moreover, A-Raf activation, like MEK activation, was blocked by
inhibition of PI3-K but was insensitive to cAMP. Experiments with
dominant negative mutants of the Raf isoforms showed that
overexpression of dominant negative c-Raf did not prevent MEK
activation. However, dominant negative A-Raf effectively blocked MEK
activation, suggesting that activation of the MEK-Erk signaling cascade
is mediated through A-Raf. Taken together, these results suggest that
IL-3 receptors engage and activate both c-Raf and A-Raf in hemopoietic
cells. However, these intermediates are differentially regulated by
upstream signaling cascades and selectively coupled to downstream
signaling pathways.
Interleukin-3 (IL-3)1 is
a growth and survival factor for immature and developing myeloid
precursors. IL-3 and the related cytokines granulocyte-macrophage
colony stimulatory factor and IL-5 bind to heterodimeric receptors,
which are composed of unique c-Raf is the most-studied Raf isoform; however, mammalian cells also
express the related Raf isoforms A-Raf and B-Raf (9), which
phosphorylate and activate MEK. Although much is known about the
stimuli that regulate and activate c-Raf and B-Raf, comparatively little is known about A-Raf. Relatively few stimuli have been shown to
activate A-Raf, and these limited studies suggest that A-Raf regulation
is similar to c-Raf regulation (10, 11). Like c-Raf, A-Raf associates
with GTP-bound Ras, suggesting that A-Raf is a downstream Ras effector,
although it currently remains unclear whether A-Raf has unique
functions or is redundant with c-Raf.
In addition to the Raf protein kinases, another Ras effector is the
lipid kinase phosphatidylinositol 3-kinase (PI3-K) (12). Several forms
of PI3-K have been identified, including the heterodimeric PI3-K, which
is composed of a regulatory 85-kDa subunit and a catalytic 110-kDa
subunit (12). PI3-K activation has pleiotropic effects on cell function
and downstream signaling pathways (13). The lipid kinase is required
for growth factor-induced activation of the protein kinases PDK1 (14)
and AKT (15, 16). Both PDK1 and AKT bind 3'-phosphorylated lipids
through their pleckstrin homology domains. PDK1 is required for
p70S6K (17) and AKT activation. AKT participates in
transducing cellular survival signals by phosphorylating and inhibiting
the pro-apoptotic action of BAD, a Bcl-2 family member (18, 19).
However, PI3-K also has functions that are cell type-specific. For
example, PI3-K is essential for receptor-driven activation of the
MEK-Erk signaling cascade in some cell types (20-22) but not in others
(23). In cells that require PI3-K for MEK and Erk activation, analysis of the pathway has demonstrated that in most instances PI3-K was not
required for Ras activation, but the lipid kinase was required for
c-Raf activation. Taken together, these studies suggest that PI3-K may
deliver a cooperative activation signal to c-Raf. However, the nature
of the signal and how it interacts with c-Raf are currently unknown.
Using complementary pharmacologic and genetic strategies, the present
results demonstrate that PI3-K is an essential element of IL-3-induced
activation of the MEK-Erk signaling cascade in primary mouse bone
marrow cells and in the IL-3-dependent cell line FDC-P1.
Additional analyses revealed that agents that elevate cAMP did not
block MEK and Erk activation. These findings prompted us to examine the
upstream signaling pathways that regulate MEK and Erk activation. Our
results demonstrate that, unlike MEK activation, c-Raf activation was
not blocked by PI3-K inhibition. Moreover, MEK activation was not
sensitive to cAMP, whereas c-Raf activation was sensitive to the drug,
suggesting that other MEK kinases activate MEK. Thus, we examined the
Raf isoforms A-Raf and B-Raf. Although B-Raf was not activated by IL-3,
A-Raf activity was induced by the cytokine. Moreover, A-Raf activation,
like MEK activation, required PI3-K and was not blocked by cAMP. Using
dominant negative mutants of both A-Raf and c-Raf, we found that only
kinase-inactive A-Raf blocked MEK activation. Together, these results
document that IL-3 activates both c-Raf and A-Raf and that only A-Raf
activation requires a PI3-K-dependent signal. Furthermore,
even though both isoforms are activated by IL-3, only A-Raf relays MEK-
and Erk-activating signals, suggesting that other molecular mechanisms
determine how activated Raf isoforms engage downstream signaling pathways.
Cell Culture and Preparation--
WEHI-3-conditioned medium was
used as a source of IL-3 for growth of FDC-P1 cells. WEHI-3 cells were
cultured in complete RPMI medium containing additions as described
previously (24). To prepare WEHI-3-conditioned medium, cells were grown
to stationary phase and then incubated for 2 days at 37 °C in a 5%
CO2 atmosphere. The medium was then centrifuged, filtered
though a 0.2 µM filter, and stored at 4 °C. FDC-P1
cells were propagated in RPMI 1640 medium containing 10% fetal calf
serum and supplemented with 10% WEHI-3-conditioned medium. To prepare
primary bone marrow cells, the femurs and tibias from four BALB/c mice
were flushed with FDC-P1 growth medium. The resulting cell suspension
was incubated for 10 min at room temperature in a 50-ml conical tube to
allow large debris to settle. The supernatant was removed and
centrifuged at 600 × g for 8 min, and the cell pellet
was resuspended in FDC-P1 cell growth medium containing 40 µg/ml
gentamycin, 100 units/ml penicillin G, and 100 µg/ml streptomycin.
Cells (1 × 107/ml) were cultured in tissue
culture-treated 100-mm plastic dishes, and fresh medium was added to
the cultures each day. After 3 days, the cells (1-1.5 × 107 per assay point) were deprived of growth factors as
described previously (24) for 4 h.
Cell Transfections--
FDC-P1 cells were transfected by
electroporation. Cells (1 × 107 per transfection)
were mixed with DNA in 400 µl of complete growth medium. Total DNA
was kept constant (40 µg) for all transfections by addition of empty
vector, if required. The DNA-cell suspension was transferred to a 4-mm
cuvette and electroporated with a 10-ms, 350-V pulse using a BTX T820
square wave electroporator (BTX Inc., San Diego, CA).
Reagents--
Recombinant murine IL-3 was purchased from R&D
Systems (Minneapolis, MN). Rabbit polyclonal immunoglobulin G (IgG)
antibodies specific for Erk2 (C-14), c-Raf (C-12), B-Raf (C-19), and
A-Raf (C-20) were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-MEK monoclonal antibody was from Transduction Laboratories (Lexington, KY). Murine monoclonal antibody preparations AU1 and 12CA5
(anti-HA) were purchased from Babco (Berkeley, CA). Monoclonal antibody
9E10 (anti-Myc) hybridoma was from the American Type Culture Collection
(Manassas, VA), and the antibody was purified over protein G-agarose
from ascites fluid from inoculated BALB/c mice. The Erk substrate,
myelin basic protein, was from Upstate Biotechnology (Lake Placid, NY).
The glutathione S-transferase (GST) fusion proteins
GST-ErkKD (MEK substrate) and GST-MEKKD (Raf
substrate) were prepared as described previously (22). The c-Jun
N-terminal kinase substrate was produced in Escherichia coli
using an expression vector generously provided by J. S. Gutkind (National Institute of Dental Research, Bethesda, MD) (25). The c-Raf
cDNA in pUC13 was purchased from American Type Culture Collection.
Plasmid Construction--
All epitope-tagged proteins were
expressed from the elongation factor 1
Epitope-tagged Raf (Myc-Raf) was constructed with PCR by appending a
BamHI site, a Kozak consensus sequence, an initiating Met
codon, and the Myc epitope in frame with the N terminus of c-Raf. A
NotI site was inserted between the Myc epitope and the N
terminus of c-Raf. A C-terminal XbaI site was incorporated
after the stop codon. The digested PCR fragment was ligated into
BamHI- and XbaI-digested pcDNA3 to generate
pcDNA3-Myc-c-Raf. Because expression levels were higher with the
pEF-BOS
Epitope-tagged MEK (Myc-MEK) was constructed by PCR amplification using
primers that fused a NotI site to the N terminus of the MEK1
cDNA, a generous gift of G. Johnson (National Jewish Center for
Immunology and Respiratory Medicine, Denver, CO), and an
XbaI site to the 3'-end. The PCR product was then digested with NotI and XbaI and ligated into the vector
backbone of NotI- and XbaI-digested
pEF-BOS
Epitope-tagged, wild-type, and kinase-inactive c-Raf
(RafKD-HA2) expression constructs were
constructed by PCR amplification of the 5' portion of the gene to
remove all native 5' untranslated sequences. These were replaced with
an EcoRI site and a Kozak consensus sequence. The 3'-end of
c-Raf was modified to remove the stop codon of c-Raf and to append
tandem copies of the HA tag (recognized by the 12CA5 monoclonal
antibody), a new stop codon, and an XbaI site. The
epitope-tagged version was ligated into EcoRI- and
XbaI-digested pEF-BOS
The human A-Raf cDNA was isolated by PCR amplification from a brain
cDNA preparation (CLONTECH) using the Expand
High Fidelity PCR system (Boehringer Mannheim). The 5' PCR primer added
an EcoRI restriction site and a Kozak consensus sequence.
The 3' primer removed the terminal stop codon and was engineered to
provide an in frame fusion (using the XbaI site) with the
same tandem HA tag as was used in construction of the epitope-tagged
c-Raf. The resulting A-Raf PCR product was subcloned into
pEF-BOS-c-RafKD-HA2, replacing the c-Raf coding
sequence with the A-Raf PCR product to yield
pEF-BOS-A-Raf-HA2. To prepare the kinase-inactive A-Raf
expression construct, pEF-BOS-A-RafKD-HA2 was
mutagenized with the GeneEditor kit (Promega, Corp; Madison, WI) to
replace K336 with Trp, an analogous mutation to that present in the
kinase-inactive c-Raf.
The AKT cDNA was obtained by PCR amplification from a commercial
PCR-ready cDNA preparation from human bone marrow
(CLONTECH). The epitope-tagged AKT expression
vector (pEF-BOS-AU1-AKT) was prepared by appending an EcoRI
site, a Kozak consensus site, a translation initiation AUG codon, and
the AU1 epitope tag that was fused in frame to the second codon of AKT.
The 3'-end of the PCR product contained an XbaI site.
pEF-BOS-AU1-AKT was assembled by ligating the epitope-tagged AKT
fragment into EcoRI- and XbaI-digested pEF-BOS
The dominant negative p85 expression vector was prepared from a murine
p85 cDNA clone, which was a generous gift of L. Williams (Chiron
Corp., San Francisco, CA). A portion of the iSH2 domain of p85 was
deleted with the Transformer kit (CLONTECH). The
deletion removed amino acids 479-513 (27) and replaced them with a Glu and Phe residue (an EcoRI site) to generate Protein Kinase Assays--
Raf, MEK, and Erk assays were
performed as described previously (22). c-Jun N-terminal kinase assays
were performed as described by Hibi et al. (28), except that
cells were deprived of IL-3 for 4 h in RPMI 1640 medium
supplemented with 10% fetal calf serum. For all kinase assays, cells
were deprived of growth factors as described previously (22) and
stimulated with 10 or 20 ng/ml IL-3 or 10 ng/ml phorbol myristate
acetate (PMA) for the indicated times. Kinase activities were
quantitated using a Molecular Dynamics PhosphorImager.
DNA Synthesis and Cell Cycle Assays--
FDC-P1 cells were
deprived of growth factors for 12 h in RPMI medium supplemented
with TCM (Celox Laboratory Inc., St. Paul, MN) (24). The cells were
pretreated with either nothing or the indicated concentrations of
wortmannin for 30 min in the same medium and added to 96-well plates
(10,000 cells per well). The cells were stimulated with the indicated
amounts of recombinant IL-3 for 12 h. At the end of this
incubation, [3H]thymidine was added, and the cells were
cultured for an additional 6 h. Cells were then harvested and
processed as described previously (29). For cell cycle analysis, FDC-P1
cells were cotransfected with 1 µg of a green fluorescent protein
(GFP) expression construct (pEGFP-N1, CLONTECH) and
either empty vector or 20 µg of pEF-BOS Wortmannin Blocks IL-3-induced Erk Activation in IL-3-responsive
Primary Bone Marrow Cells and IL-3-dependent FDC-P1
Cells--
Our previous results demonstrated that IL-2-induced Erk
activation in T cells was partially sensitive to wortmannin, suggesting that both wortmannin-sensitive and wortmannin-resistant pathways participated in the Erk activation response in this cell type (22). We
therefore extended our investigation to determine whether Erk
activation induced by other hemopoietic growth factors was also
sensitive to wortmannin. Our initial experiments tested whether the
drug affected IL-3-induced Erk activation in primary bone marrow cells.
Isolated murine bone marrow cells were deprived of exogenous growth
factors for 4 h, pretreated with wortmannin and stimulated with
IL-3. Wortmannin strongly inhibited IL-3-induced Erk activation in
these cells (Fig. 1), but the drug had no
effect on phorbol ester-induced Erk activation. These results
demonstrated that the inhibitory effects of wortmannin on
cytokine-induced Erk activation were not restricted to T cells and that
the wortmannin-sensitive pathway played a major role in IL-3-induced
Erk activation in normal bone marrow cells.
Due to the technical limitations imposed by studies in primary
hemopoietic cells, we determined whether the inhibitory effects of
wortmannin on Erk activation were recapitulated in the
IL-3-dependent cell line FDC-P1 (Fig.
2). Factor-deprived FDC-P1 cells were
treated with wortmannin and stimulated with IL-3. Erk (Fig.
2A) and MEK (Fig. 2B) were immunoprecipitated
from detergent lysates for determination of in vitro kinase
activities. As was observed with the primary bone marrow cells,
wortmannin effectively suppressed both MEK and Erk activation,
indicating that the IL-3 receptor couples to Erk largely through a
wortmannin-sensitive signaling pathway. Because FDC-P1 cells are
amenable to biochemical and genetic analyses, we used this cell line as
a model system to dissect the signaling pathways that regulate
IL-3-induced Erk activation.
Wortmannin Blocks IL-3-induced G1-to-S Phase
Progression--
In many experimental systems PI3-K plays critical
roles in relaying proliferative signals from growth factor receptors.
However, the situation is less clear for hemopoietic cytokine
receptors. Because wortmannin effectively inhibits PI3-K, we asked how
wortmannin would affect IL-3-induced DNA synthesis (Fig.
3). FDC-P1 cells were deprived of growth
factors, which synchronizes them in the G1 phase of the
cell cycle (data not shown). The G1 phase cells were
pretreated with a single addition of the indicated concentrations of
wortmannin and stimulated with IL-3. Because wortmannin is unstable in
solution and PI3-K levels remain suppressed for only 9-12 h after drug
addition (22), cells were pulsed after 12 h with
[3H]thymidine (as cells first enter S phase) and cultured
an additional 6 h before harvesting. Therefore, PI3-K was
maximally inhibited during the progression of the IL-3-stimulated cells
through G1. Wortmannin attenuated IL-3-induced DNA
synthesis at concentrations that inhibited PI3-K and blocked
IL-3-induced Erk activation. Because these experiments were performed
with a single addition of wortmannin, and PI3-K activity would be
recovering during the later times of the experiment, these results
likely do not reflect the full impact of PI3-K inhibition on
G1-to-S phase progression. This inhibition of DNA synthesis
is not the result of cell killing, as FDC-P1 cells cultured in these
concentrations of wortmannin do not undergo apoptosis, even though
PI3-K and AKT activation are fully inhibited (data not shown).
To address the specificity of the effects of wortmannin, we coexpressed
GFP with either empty vector or dominant negative p85 ( Wortmannin and Dominant Negative p85 Block IL-3-induced Erk and AKT
Activation--
Wortmannin blocked MEK and Erk activation by 50%
(IC50) at a concentration of 3 nM (data not
shown), which coincides with the IC50 for PI3-K inhibition
in intact cells (30-32). These results suggested that a
wortmannin-sensitive PI3-K is an upstream regulator of Erk activation
in IL-3-stimulated hemopoietic cells. Therefore, as a complementary
strategy to assess the role of PI3-K in Erk activation, we examined the
effect of dominant negative Dominant Negative p85 Selectively Disrupts
PI3-K-dependent Signaling Pathways--
One potential
complication associated with the dominant negative approach is that
overexpression of mutant proteins that contain abundant protein-protein
interaction domains, as does p85, may nonspecifically disrupt signaling
pathways. We tested this possibility by asking whether Pharmacological Inhibitors Differentially Affect IL-3-induced c-Raf
and MEK Activation--
To further define the signaling pathway that
mediates MEK and Erk activation, we determined whether the MEK
activator, c-Raf, is also inhibited by wortmannin. Fig.
6A shows that IL-3 rapidly activated c-Raf. Surprisingly, however, wortmannin concentrations that
resulted in near-complete inhibition of IL-3-triggered MEK and Erk
activation (see Fig. 2) unexpectedly prolonged and enhanced IL-3-induced c-Raf activation. These results suggest that
wortmannin-sensitive pathways may attenuate or terminate the signals
mediating Raf activation. One such pathway may be the MEK-Erk cascade,
because previous work showed that pharmacological inhibition of MEK
also enhanced platelet-derived growth factor-induced Raf activation (33).
The observation that IL-3-induced c-Raf activation was enhanced in
wortmannin-treated cells, whereas MEK activation was abrogated, suggested one of two possibilities. Either c-Raf does not relay the
MEK-activating signal in FDC-P1 cells, or wortmannin blocks the normal
coupling between c-Raf and MEK in response to IL-3. To discriminate
between these possibilities, we examined what effect blocking c-Raf
activation would have on IL-3-induced Erk activation. Elevation of cAMP
levels activates protein kinase A, which phosphorylates and inhibits
activation of c-Raf (34). Thus, we tested the effect of cAMP on
IL-3-induced c-Raf and MEK activation in FDC-P1 cells by incubating the
cells with either the phosphodiesterase inhibitor IBMX (Fig.
6B) or the nonhydrolyzable cAMP analog
8-chlorophenyl-thio-cAMP (data not shown). Both drugs effectively
blocked IL-3-induced c-Raf activation (see Fig. 6B for IBMX
results). Surprisingly, IBMX and 8-chlorophenyl-thio-cAMP enhanced
IL-3-triggered Erk activation, demonstrating that under conditions
where c-Raf was not activated by IL-3, Erk activation was not impeded.
Thus, these results suggest that c-Raf does not relay the IL-3-induced
MEK-activating signal in this cell type.
Dominant Negative c-Raf Does Not Block IL-3-induced MEK
Activation--
As another method to rule out c-Raf as a mediator of
MEK activation in these cells, we examined the effect of
kinase-inactive c-Raf (c-RafKD-HA2) on IL-3- or
PMA-induced MEK activation (Fig. 7).
Consistent with the results obtained with IBMX-treated cells (see Fig.
6), IL-3-induced MEK activation was not blocked by coexpression of kinase-inactive c-Raf (Fig. 7, A and C). In fact,
when low amounts of kinase-inactive c-Raf are cotransfected,
IL-3-induced MEK activation was reproducibly enhanced. Anti-Myc
immunoblotting suggested that this might be due to increased expression
of the epitope-tagged Myc-MEK, an effect that we observed when cells
were transfected with either 5 or 10 µg of kinase-inactive c-Raf
(Fig. 7B). In contrast, PMA-induced MEK activation was
partially blocked by kinase-inactive c-Raf, even when Myc-MEK
expression was slightly enhanced (5 or 10 µg of kinase-inactive
c-Raf). A potential concern here is that kinase-inactive c-Raf does not
function as an effective dominant negative (although the same construct
blocks MEK activation in other systems; data not shown). However, only
a partial block would be expected because the A-Raf isoform relays
approximately 50% of the MEK-activating signal in PMA-stimulated cells
(see Fig. 9).
IL-3 Does Not Activate B-Raf--
In addition to c-Raf, FDC-P1
cells also express B-Raf. An earlier report indicated that IL-3
activated B-Raf in hemopoietic cells (35); however, in this study,
autophosphorylation of B-Raf was used as a marker for enzyme
activation, rather than using a specific Raf substrate, such as MEK1.
Because Raf isoforms associate with and are phosphorylated by other
protein kinases, autophosphorylation assays may actually reflect the
presence of contaminating transphosphorylating activities.
Consequently, we assayed B-Raf activation in FDC-P1 cells using a
GST-MEKKD fusion protein as substrate (Fig.
8A). As previously reported, B-Raf exhibited high basal activity (7). However, this activity was not
increased by IL-3, nor was it affected by wortmannin and IBMX
treatment, suggesting that B-Raf does not transduce IL-3-triggered MEK-activating signals.
IL-3-induced A-Raf Activation Is Sensitive to
Wortmannin--
Because neither c-Raf nor B-Raf was a candidate as MEK
activators in IL-3-stimulated cells, we also asked whether A-Raf, the third known member of the Raf kinase family, was involved in this response. We found that A-Raf is expressed and is activated by IL-3
(Fig. 8B). Surprisingly, IL-3-induced A-Raf activation was wortmannin-sensitive and IBMX-resistant (Fig. 8B), in
contrast to the c-Raf activation provoked by the same cytokine (Fig.
8C). Also in the same experiment, we confirmed that Erk
activation was sensitive to wortmannin but resistant to IBMX (Fig.
8D). This experiment was repeated three times, and in each
case we observed that wortmannin blocked A-Raf activation. Moreover,
coexpression of dominant negative Dominant Negative A-Raf Blocks IL-3-induced MEK
Activation--
Although IL-3 activated c-Raf, our earlier results
suggested that this protein kinase does not relay an activating signal to MEK. Based on these observations, we genetically blocked A-Raf function to determine its impact on IL-3-induced MEK activation. We
cotransfected FDC-P1 cells with Myc-MEK and increasing amounts of the
kinase-inactive A-Raf expression vector. Factor-deprived cells were
restimulated with IL-3 and were assayed for the activation of the
epitope-tagged Myc-MEK (Fig. 9,
A and C). Coexpression of increasing amounts of
kinase-inactive A-Raf effectively suppressed MEK activation induced by
either IL-3 or PMA. To demonstrate that kinase-inactive A-Raf does not
nonspecifically block IL-3-induced signals, we cotransfected increasing
amounts of dominant negative A-Raf along with AU1-AKT and asked whether
the mutant A-Raf blocked AKT activation (Fig. 9D). In
contrast to the inhibition of IL-3-induced MEK activation, activation
of AKT in cytokine-stimulated cells was not blocked by the
kinase-inactive construct. Thus, these results suggest that A-Raf is a
major transducer of the IL-3-induced MEK-activating signal; however,
other MEK activators may also participate in IL-3-induced MEK
activation.
The present results demonstrate that IL-3-induced activation of
the Erk signaling cascade is dependent on PI3-K. Although the PI3-K
pathway was originally thought to be insulated from the MEK-Erk
signaling cascade, multiple reports have since shown that the pathways
are intertwined in a cell type- and stimulus-specific manner. Some
cells and stimuli, such as IL-3, clearly require PI3-K for MEK
activation, whereas in other cases, the intensity of the stimulus or
the cell type employed determines whether PI3-K participates in MEK and
Erk activation. Efforts have been made to determine at what level in
the signaling cascade PI3-K is required, and several studies
demonstrated that PI3-K is required for activation of c-Raf, a common
MEK activator in growth factor signaling cascades (20, 21). However,
the present results indicate that IL-3 triggers a
PI3-K-dependent pathway in which an alternative Raf isoform, A-Raf, transduces an activating signal to MEK and, in turn,
Erk. Furthermore, these results show that activated Raf isoforms do not
necessarily couple to the expected downstream signaling pathways and
suggest that Raf isoforms have unique functions in cytokine-stimulated cells.
Our findings are in disagreement with the conclusions of Schied and
Duronio (36), who reported that wortmannin inhibited IL-3- and
GM-CSF-induced activation of Erk but that the unrelated PI3-K
inhibitor, LY294002, did not block Erk activation (36). Based on this
finding, they suggested that the effects of wortmannin on
cytokine-induced MEK activation are mediated by a target other than
PI3-K. Although wortmannin is not absolutely specific for PI3-K, we
substantiated our pharmacologic data by genetically blocking PI3-K
activation with a documented dominant negative inhibitor of PI3-K (27).
These results showed that both AKT and MEK activations were equally
sensitive to increasing amounts of the dominant negative PI3-K
construct. Taken together, these results strongly suggest that PI3-K is
an upstream component in IL-3-induced MEK activation. This discrepancy
between the present conclusions and those of Scheid and Duronio (36)
may result from the use of different cell lines. In our studies,
wortmannin had dramatic effects on Erk activation in both primary bone
marrow cells and the myeloid progenitor FDC-P1 cell line. In the
studies of Scheid and Duronio (36), wortmannin had more modest effects on GM-CSF- and IL-3-induced Erk activation in the mast cell-like MC-9
cell line. Possibly, MC-9 cells do not require a
PI3-K-dependent input for MEK activation, and the
inhibition that is observed with wortmannin is indeed due to another
cellular target for wortmannin.
The regulation of all Raf isoforms is a complex multistep process,
which begins with the accumulation of GTP-bound Ras. However, Ras
provides only the initial activating signal, with other cooperating upstream signals required for full Raf activation (4). Thus, the Raf
isoforms serve as a point of convergence for multiple receptor-triggered signaling pathways, one of which may be
PI3-K-dependent. In IL-3-stimulated hemopoietic cells, both
c-Raf and A-Raf are activated. However, in these cells, only A-Raf
activation requires a PI3-K-mediated signal. Furthermore, how PI3-K
relays an activating signal to PI3-K is uncertain. One possibility is
that PI3-K produces a lipid product that can directly affect the Raf
activation process, and previous work has demonstrated that c-Raf binds
lipids (37, 38). Alternatively, a PI3-K-dependent effector
may relay an activating signal to Raf. Several PI3-K effectors have
recently been identified, including the nonclassical PKC isoforms
PKC The present results establish that, like c-Raf and B-Raf, A-Raf is
differentially regulated by extracellular stimuli. Our study also
demonstrates that even under conditions where both c-Raf and A-Raf are
activated, only A-Raf relays activating signals to MEK, which suggests
that A-Raf is selectively scaffolded into the MEK-Erk signaling module
in response to IL-3 stimulation. In the yeast Saccharomyces
cerevisiae, the Ste5 scaffolding protein aligns and segregates the
components of the mating-response mitogen-activated protein kinase
cascade and also serves to insulate one mitogen-activated protein
kinase signaling module from other parallel modules (43). Perhaps PI3-K
participates in the assembly of similar scaffolding complexes in
mammalian cells that permit A-Raf, but not c-Raf, to couple to the
MEK-Erk signaling cascade in IL-3-stimulated cells.
If c-Raf does not mediate activation of MEK and Erk, what is the role
of activated c-Raf in hemopoietic cells? Antisense oligonucleotides directed against c-Raf severely attenuate IL-3-induced FDC-P1 proliferation (44), suggesting that although c-Raf is not required for
MEK and Erk activation, it has other critical functions in these cells.
One possible target of IL-3-activated Raf is the apoptosis-promoting
protein Bad (45). When phosphorylated, Bad is bound to 14-3-3 proteins
in an inactive, cytoplasmic form. When dephosphorylated, Bad
translocates to the mitochondria, where it dimerizes with
Bcl-XL and promotes cell death (46). This function of c-Raf
might explain why c-Raf was required for IL-3-induced FDC-P1
proliferation, even though c-Raf does not relay Erk-activating signals.
Although the Ras-Raf-MEK-Erk signaling cascade has become a central
tenet of modern cell biology, it is becoming increasingly apparent that
considerable plasticity exists with respect to Erk activation pathways.
The present results indicate that PI3-K plays a crucial role in the
activation of Erk by the hemopoietic growth and survival factor IL-3.
Furthermore, the present data also demonstrate the differential
regulation and coupling of c-Raf and A-Raf, suggesting that these two
Raf isoforms perform nonoverlapping and unique functions in
IL-3-responsive cells. These observations suggest that receptors rely
on combinatorial associations among a limited set of signal
transduction intermediates to activate partially redundant downstream
signaling pathways. Although the complexity of the signaling pathways
that converge on Erk seems daunting, the delineation of these pathways
may uncover new cellular targets for the pharmacologic manipulation of
hemopoietic cell functions in immune system dysfunctions and cancer.
INTRODUCTION
Top
Abstract
Introduction
References
subunits and shared
subunits (1).
Ligand-induced receptor subunit heterodimerization activates
receptor-bound protein-tyrosine kinases and initiates a series of
intracellular signaling cascades, including the Erk activation pathway
(2, 3). Although commonly depicted as a simple linear pathway, the
Ras-Erk signaling cascade receives regulatory inputs from several other
receptor-triggered signaling pathways. One key site for signal
integration is at the level of c-Raf. A requisite signal is provided by
the interaction of GTP-bound Ras with c-Raf. Although necessary, Ras
interaction with c-Raf is insufficient for maximal c-Raf activation,
and other signaling pathways impinge on c-Raf (4). The precise
identities of these other pathways remain unclear, but protein kinase C
(PKC) isoforms (5), ceramide-activated protein kinase (6), Src (7), and
JAK family protein-tyrosine kinases (8) have all been implicated in
c-Raf activation.
EXPERIMENTAL PROCEDURES
promoter in a modified
pEF-BOS (26) expression vector (pEF-BOS
RI). The parental vector
contains two EcoRI restriction sites. To generate
pEF-BOS
RI, the EcoRI restriction site on the 3' side of
the polyadenylation sequences was removed. Removal of this restriction
site allowed insertion of cDNA sequences with 5' EcoRI
and 3' XbaI restriction sites. All PCR- and
mutagenesis-derived portions were sequenced to ensure the fidelity of
the amplification and mutagenic procedures, respectively.
RI vector, Myc-c-Raf was transferred to the pEF-BOS
RI
vector using a 5' EcoRI site and a 3' XbaI site.
RI-Myc-c-Raf (removing c-Raf but not the Myc epitope tag) to
generate pEF-BOS
RI-Myc-MEK.
RI to yield
pEF-BOS-c-Raf-HA2. The kinase-inactive version
(pEF-BOS-c-RafKD-HA2) was constructed using the
Transformer mutagenesis kit (CLONTECH, Palo Alto,
CA) to change Lys-375 to Trp.
RI.
p85. The
p85 cDNA was modified on the 3'-end to remove the native stop
codon and append a tandem HA tag and XbaI site to
p85.
The epitope-tagged
p85 cDNA was cloned into pEF-BOS
RI to
yield pEF-BOS
RI-
p85.
RI-
p85-HA2.
Twenty hours later, the cells were incubated in PBS with 20 µg/ml
Hoechst dye 33342 at 37 °C for 20 min. Two-color flow cytometry was
used to analyze transfected cells (using GFP for green fluorescence) for cell cycle (using Hoechst dye for blue fluorescence). Data were
analyzed using Modfit analysis software (Topsham, ME).
RESULTS
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Fig. 1.
Wortmannin blocks IL-3-induced
mitogen-activated protein kinase activation in primary murine bone
marrow cell cultures. Factor-deprived bone marrow cells were
pretreated with Me2SO vehicle (untreated) or 100 nM wortmannin for 30 min. Cells were then restimulated with
IL-3 or PMA for 5 min and lysed. Mitogen-activated protein kinase was
immunoprecipitated, and catalytic activity was assayed. Data are the
average of duplicate determinations. The experiment was repeated three
times with similar results.
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Fig. 2.
Wortmannin blocks IL-3-induced MEK and Erk
activation. Factor-deprived FDC-P1 cells were pretreated (30 min)
with 100 nM wortmannin or left untreated. Cells were
stimulated with IL-3 for the indicated times, Erk (A) or MEK
(B) was immunoprecipitated, and kinase activity was
measured.
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Fig. 3.
Wortmannin blocks IL-3-induced DNA
synthesis. FDC-P1 cells were synchronized into G1 by
IL-3 deprivation, pretreated with nothing (untreated) or the
indicated concentration of wortmannin for 30 min, and restimulated with
the indicated concentrations of IL-3. The cells were pulsed with
[3H]thymidine and were cultured for an additional 6 h. Cells were harvested onto glass mats, and 3H
incorporation into DNA was determined by scintillation counting.
p85) (27).
Flow cytometric analysis of green-fluorescing cells revealed that
transfection with GFP and empty vector produced cells that cycled
normally through the cell cycle (58% G1, 33% S, and 9%
G2/M). In contrast, cells cotransfected with GFP and
p85
were primarily found in G1 (81% G1, 12% S, and 7%
G2/M), suggesting that genetically blocking that pathway
may be more effective than wortmannin in blocking cell cycle
progression. Taken together with the drug study, these results suggest
strongly that PI3-K plays a crucial role in promoting IL-3-induced cell cycle progression.
p85 (
p85-HA2) on
IL-3-induced MEK activation. As a positive control to confirm that
p85-HA2 interrupts PI3-K-mediated signaling, we
cotransfected the cells with an AU1-tagged AKT expression vector.
Expression vectors for all three epitope-tagged proteins were
cotransfected into FDC-P1 cells, with increasing amounts of
p85-HA2 as indicated (Fig.
4A). After factor deprivation,
the transfected cell populations were divided into two equal sets. One
set was stimulated IL-3 (20 min), the detergent lysates were
immunoprecipitated with anti-AU1 antibody, and AKT activity was
determined (Fig. 4A, bottom panel). The other set was
stimulated with IL-3 (5 min), the lysates were immunoprecipitated with
anti-Myc antibody, and MEK kinase assays were performed (see Fig.
6A, top panel). Increasing amounts of cotransfected
p85-HA2 plasmid DNA progressively blocked IL-3-induced
activation of both AKT and MEK with virtually identical potencies (Fig.
4C). However, a small portion of MEK activation was not
blocked by
p85 or wortmannin (see Fig. 2), suggesting that a
PI3-K-independent pathway plays a minor role in IL-3-induced MEK
activation. The alterations in IL-3-induced MEK and AKT activation were
not explained by differences in the expression of the epitope-tagged
protein kinases (Fig. 4B). Because AKT and MEK showed nearly
identical sensitivity to coexpressed
p85-HA2, these
results argue strongly that PI3-K plays a pivotal upstream role in
IL-3-induced MEK activation.
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Fig. 4.
PI3-K mediates MEK and AKT activation in
IL-3-stimulated FDC-P1 cells. FDC-P1 cells were cotransfected with
Myc-MEK (5 µg) and AU1-AKT (5 µg) expression vectors, along with 0, 5, 10, or 20 µg of p85-HA2 expression vector.
Factor-deprived transfected cells were restimulated with IL-3 for 5 min
(MEK assays) or 20 min (AKT assays). A, lysates were
immunoprecipitated with either anti-AU1 (AKT) or anti-Myc (MEK), and
kinase assays were performed. B, to ensure equal expression
of Myc-MEK and AU1-AKT in all transfected cells, a portion of each
lysate was fractionated and immunoblotted with either anti-Myc
(top panel) or anti-AU1 (middle panel). To
demonstrate expression of
p85-HA2, the samples were also
immunoblotted with anti-HA (bottom panel). C,
quantitation of incorporated radiolabel from A. The
experiment was repeated three times with identical results.
p85 blocked
activation of a pathway that is independent of PI3-K. In FDC-P1 cells,
IL-3-induced c-Raf activation is not blocked by wortmannin, indicating
that c-Raf activation is independent of PI3-K-generated signals (Fig.
5). FDC-P1 cells were cotransfected with
AU1-AKT, Myc-c-Raf, and either empty vector or
p85 expression
vector. Factor-deprived cells were then restimulated with IL-3.
Myc-c-Raf and AU1-AKT were immunoprecipitated with anti-epitope
monoclonal antibodies and subjected to kinase assays. IL-3 induced
strong activation of c-Raf that was not blocked by coexpression of
p85. In contrast, coexpression of
p85 effectively blocked AKT
activation. These results indicate that PI3-K is not required for
IL-3-induced c-Raf activation. Moreover, they demonstrate that
p85
does not nonspecifically disrupt all IL-3-triggered mitogenic signals,
suggesting that its effects are specific for the PI3-K pathway.
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Fig. 5.
p85 does not nonspecifically
disrupt IL-3 signal transduction pathways. FDC-P1 cells were
transfected with 10 µg of Myc-c-Raf and 5 µg of AU1-AKT expression
vectors and either empty vector or 20 µg of
p85 expression vector.
Factor-deprived cells were stimulated with IL-3 for 5 min (A-Raf
assays) or 20 min (AKT assays), and kinase assays were performed. To
demonstrate equal expression of transfected Myc-c-Raf and AU1-AKT, the
kinase assay membrane was immunoblotted with
-c-Raf (c-Raf,
second panel from top) or with
-AKT (AKT, bottom
panel).
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Fig. 6.
Wortmannin and IBMX differentially affect
IL-3-induced MEK activation. A, factor-deprived FDC-P1
cells were pretreated with nothing or 100 nM wortmannin for
30 min and stimulated with IL-3 for the indicated times. c-Raf was
immunoprecipitated, and c-Raf kinase activity was determined.
B, factor-deprived FDC-P1 cells were pretreated with nothing
or 400 mM IBMX for 30 min. The cells were then restimulated
with IL-3 for 5 min and lysed. The lysates were split into equal
portions. One portion was immunoprecipitated with anti-Erk serum and
Erk activity was determined (left). The remaining portion of
the lysate was immunoprecipitated with anti-c-Raf serum, and c-Raf
activity was assayed (right).
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Fig. 7.
Kinase-inactive c-Raf suppresses PMA-induced
but not IL-3-induced MEK activation. FDC-P1 cells were
cotransfected with Myc-MEK expression plasmid and either 0, 5, or 10 µg of kinase-inactive c-Raf (c-RafKD-HA2)
expression vector. Factor-deprived cells were restimulated with IL-3 or
PMA for 5 min, and MEK kinase activities were determined (A)
and quantitated (C). To determine expression levels of
transfected genes, a portion of the lysates was immunoblotted with
-Myc to detect Myc-MEK (B, top panel) and
-HA to
detect c-RafKD-HA2 (B, bottom
panel). The results shown are representative of those obtained in
three independent experiments.
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Fig. 8.
IL-3 activates A-Raf but not B-Raf in FDC-P1
cells. Factor-deprived FDC-P1 cells were pretreated with nothing
or 100 nM wortmannin for 30 min and restimulated with IL-3
for 5 min. Detergent lysates were immunoprecipitated with B-Raf
(A, top panel), A-Raf (B, top panel), c-Raf
(C, top panel), or Erk antisera (D, top panel),
and protein kinase activities were assayed. To demonstrate equal
immunoprecipitation and sample loading, the membrane was immunoblotted
with either anti B-Raf (A, bottom panel), anti-A-Raf
(B, bottom panel), anti-c-Raf (C, bottom panel),
or anti-Erk (D, bottom panel). Identical results were
obtained in four independent trials.
p85 also blocked IL-3-induced
activation of epitope-tagged A-Raf (data not shown), thus confirming
that the effects of wortmannin on A-Raf are due to PI3-K inhibition. These results demonstrate that A-Raf and c-Raf are differentially regulated in IL-3-responsive cell lines and suggest that A-Raf, but not
c-Raf, may relay the MEK-activating signal in IL-3-stimulated cells.
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Fig. 9.
Kinase-inactive A-Raf inhibits IL-3-induced
MEK but not AKT activation. FDC-P1 cells were transfected with 5 µg of Myc-MEK expression vector and either 0, 5, 10, or 15 µg of
kinase-inactive A-Raf expression vector. Factor-deprived cells were
restimulated with IL-3 or PMA for 5 min, and MEK catalytic activities
were assayed (A) and quantitated (C). To
demonstrate Myc-MEK expression (B, top panel) and
A-RafKD-HA2 expression (B, bottom
panel), a portion of the cell lysate was immunoblotted with
anti-Myc and anti-HA, respectively. D, AU1-AKT expression
vector was cotransfected with the indicated amounts of kinase-inactive
A-Raf expression vector. AKT activities were assayed and quantitated.
Immunoblotting of kinase assay immunoprecipitates revealed that AU1-AKT
was expressed equally in all samples (data not shown).
DISCUSSION
/
(39) and PKC
(40), the pleckstrin homology
domain-containing kinases AKT (15, 16) and PDK1 (14), and the protein
kinases p70S6K and mTOR (41). Previous work has
demonstrated that rapamycin completely blocks the activation of
p70S6K and mTOR, but the drug does not block MEK and Erk
activation (42), demonstrating that these downstream PI3-K effectors do not relay activating signals to Raf or the MEK-Erk pathway. In the case
of the PKC isoforms, dominant negative PKC
/
and PKC
had no
effect on IL-3-induced MEK activation, suggesting that these PKC
isoforms do not participate in MEK activation in IL-3-stimulated cells.2 Whether the PI3-K
effectors AKT and PDK1 function in MEK activation pathways is less well
studied; however, constitutively active AKT had no effect on Erk
activation in a cell line in which PI3-K was required for MEK
activation, suggesting that in this setting, AKT is not sufficient for
MEK activation (14).
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ACKNOWLEDGEMENTS |
---|
We thank L. Williams, G. J. Johnson, and J. S. Gutkind for generously providing reagents. We also thank Wanda Rhodes for manuscript preparation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM731961 and CA52995 (to R. T. A.) and Grant CA73622 (to L. M. K.) and by the Mayo Foundation.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.
** Leukemia Society of America Scholar.
Leukemia Society of America Special Fellow. To whom
correspondence should be addressed: Div. of Radiation Oncology,
Guggenheim 13, Mayo Clinic, 200 First St. SW, Rochester, MN 55905.
Tel.: 507-284-3124; Fax: 507-284-3906; E-mail:
karnitz.larry{at}mayo.edu.
2 L. M. Karnitz, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: IL, interleukin; PKC, protein kinase C; PI3-K, phosphatidylinositol 3-kinase; mitogen-activated protein, MEK, mitogen-activated protein/ERK kinase; GST, glutathione S-transferase; PMA, phorbol myristate acetate; HA, hemagglutinin; IBMX, isobutylmethylxanthine; PCR, polymerase chain reaction; GFP, green fluorescent protein.
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REFERENCES |
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