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Volume 272, Number 44, Issue of October 31, 1997 pp. 27665-27670
(Received for publication, April 16, 1997, and in revised form, August 27, 1997)
§¶ and
From the 
Department of Medicine, Division of Signal
Transduction, Beth Israel Hospital and the Department of Cell Biology,
Harvard Medical School, Boston, Massachusetts 02115 and the
§ Department of Physiology, Tufts University School of
Medicine, Boston, Massachusetts 02114
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT 
Phosphoinositide (PI) 3-kinase and the
mitogen-activated protein (MAP) kinase cascades are activated by many
of the same ligands. Several groups have reported involvement of PI
3-kinase in the activation of Erk1 and Erk2, whereas many other groups
have shown that activation of Erk1 and Erk2 is not sensitive to
inhibitors of PI 3-kinase such as wortmannin. Here we show that
wortmannin inhibition of the MAP kinase pathway is cell type- and
ligand-specific. Wortmannin blocks platelet-derived growth factor
(PDGF)-dependent activation of Raf-1 and the MAP kinase
cascade in Chinese hamster ovary cells, which have few PDGF receptors,
but has no significant effect on Erk activation in Swiss 3T3 cells,
which have high levels of PDGF receptors. However, wortmannin blocks
activation of Erk proteins if Swiss 3T3 cells are stimulated with
lower, physiological levels of PDGF. These results suggest that PI
3-kinase is in an efficient pathway for activation of MAP kinase, but
that MAP kinase can be stimulated by a redundant pathway when a large
number of receptors are activated. We present evidence that a protein
kinase C family member downstream of phospholipase C
is involved in the redundant pathway.
INTRODUCTION
PI1 3-kinase has been
implicated as being involved in the signal transduction of virtually
all growth factors studied and in the transformation of cells by
several oncoproteins (1, 2). Activation of PI 3-kinase with growth
factors results in the appearance of the lipid products of this enzyme,
PtdIns-3,4-P2 and PtdIns-3,4,5-P3, within
seconds to minutes. There is also a correlation between the elevation
in PtdIns-3,4-P2 and PtdIns-3,4,5-P3 levels and cell transformation. These correlations have suggested an important role for PI 3-kinase in signal transduction pathways leading to cell growth and transformation. Indeed, the catalytic subunit of PI
3-kinase (p110
) has recently been identified as a retroviral oncogene (3). Overexpression of p110 in chick embryo
fibroblasts results in constitutive elevation of
PtdIns-3,4-P2 and PtdIns-3,4,5-P3 and cell
transformation.
The MAP kinase pathway is another key component in the transduction of signals leading to growth and transformation. This pathway consists of a linear cascade of the protein kinases Raf, MEK, and MAP kinase/Erk; like PI 3-kinase, Erk1 and Erk2 are acutely activated by growth factors and are found constitutively activated in many transformed cell lines. The Erk proteins are phosphorylated and activated by the dual specificity kinase MEK (MAP kinase/Erk kinase), which is phosphorylated and activated by the serine/threonine kinase Raf. Raf is recruited to the membrane of activated cells by direct binding to Ras-GTP. This recruitment by activated Ras is necessary but not sufficient for full activation of Raf (4); therefore, other factors that are necessary for activation of the MAP kinase pathway may feed into the pathway at Raf. A search for other sources that feed into the MAP kinase pathway has yielded several candidates including Src, members of the PKC family, and PI 3-kinase (45).
The relevance of PI 3-kinase for activation of Erk has been
controversial. Expression of activated forms of p110 PI 3-kinase has
been reported to stimulate the MAP kinase pathway in one case (5), but
not in others (6-9). A recent study found that overexpression of the
p110 type of PI 3-kinase resulted in the activation of Erk, but that
p110 did not (9). Inhibition of PI 3-kinase with wortmannin (10-16)
or dominant-negative PI 3-kinase (9) has been shown to block activation
of MAP kinase in some but not all cells (17-19).
Several direct targets of the lipid products of PI 3-kinase have been
identified that could act as intermediates between PI 3-kinase and the
MAP kinase pathway. Members of the novel PKC family
(calcium-independent), PKC
, PKC
, and PKC
(20), have been shown
to be activated in vitro by two lipid products of PI 3-kinase, PtdIns-3,4-P2 and PtdIns-3,4,5-P3.
Inhibition of thrombin-dependent phosphorylation of
pleckstrin by wortmannin, combined with the ability of these lipids to
stimulate phosphorylation of pleckstrin when added to permeabilized
platelets (21, 22), suggests that they activate PKC family members
in vivo. PDGF activates PKC (23) and PKC
(24) by a
mechanism that requires PI 3-kinase. Several PKC family members
(25-27) have already been implicated in the activation of the MAP
kinase pathway via phosphorylation of Raf. Another potential
intermediate is the serine/threonine protein kinase Akt/PKB, which
binds to and is activated by PtdIns-3,4-P2 in
vitro (28, 29), and PDGF stimulates this enzyme via activation of
PI 3-kinase (30). The serine/threonine kinase p70S6K has
been shown to be downstream of PI 3-kinase (31). Thus, a potential
means for PI 3-kinase to feed into the MAP kinase pathway could be via
activation of one (or more) of these targets of PI 3-kinase.
We have attempted to better understand the role that PI 3-kinase plays in the activation of the MAP kinase pathway. We show here that the effect of wortmannin on the activation of Erk varies not only between cell lines, but within a single cell line depending on both the kind and concentration of the growth factor used. We have found that Erk activity is sensitive to wortmannin when relatively few receptors are activated, but resistant if a large number of receptors are activated. We have also found that a down-regulatable PKC contributes to wortmannin-resistant MAP kinase activation.
MATERIALS AND METHODS
CHO/HIR cells are a CHO clone expressing the human insulin receptor and were maintained in RPMI 1640 medium and 10% fetal calf serum. Balb/c/3T3 clone A31 fibroblasts and NIH/3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium and 10% fetal calf serum. Quiescent cell cultures were established by changing the medium to 0.1% (A31 and NIH/3T3 cells) or 0% (CHO cells) fetal calf serum overnight.
Treatment with WortmanninWortmannin was diluted
immediately prior to use in Me2SO from a 10 mM
stock kept in Me2SO at 
70 °C. Wortmannin or the
Me2SO carrier control was added to the cells for 15-20 min
before the addition of growth factors.
Anti-Erk2 (C-14), anti-Raf-1 (C-12), and
anti-PLC1 (530) affinity-purified polyclonal antibodies were
purchased form Santa Cruz Biotechnology. Anti-p85 polyclonal antibody
was raised against the amino-terminal SH2 domain of p85.
Dishes of cells were washed two times with ice-cold phosphate-buffered saline and placed on ice. Cells were immediately lysed in 1 ml of radioimmune precipitation assay buffer (50 mM Tris (pH 7.2), 150 mM NaCl, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 0.5 mM EDTA, and 10% glycerol) collected in an Eppendorf tube, and rocked for 10 min at 4 °C. The cell lysate was then spun at 12,000 × g for 5 min at 4 °C, and the supernatant was assayed for protein concentration and transferred to another tube. The immunoprecipitating antibody and 30 µl of a protein A-Sepharose bead slurry (50%) were added; the suspension was then rocked for 2-3 h. Beads were collected and washed three times with radioimmune precipitation assay buffer and twice with TNE buffer (10 mM Tris (pH 7.2), 100 mM NaCl, 1 mM EDTA) or protein kinase buffer (20 mM Tris (pH 7.0) and 5 mM MgCl2). Radioimmune precipitation assay buffer also contained 1 mM dithiothreitol, 1 mM ZnCl2, 1 mM sodium vanadate, 25 mM NaF, 10 mM sodium pyrophosphate, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 200 µM 4-(2-aminoethyl)benzenesulfonyl fluoride added fresh. TNE buffer and protein kinase buffer contained the same concentrations of dithiothreitol, vanadate, NaF, and leupeptin.
ImmunoblottingImmunoblots were developed either by ECL (Renaissance, NEN Life Science Products) followed by exposure to film or by Vistra ECF Western blotting reagent (Amersham Corp.) followed by quantitation on a Molecular Dynamics Storm imaging system.
Immune Complex Protein Kinase AssaysImmunoprecipitated and
washed proteins were assayed on the beads. For MAP kinase assays, 40 µl of protein kinase buffer with 20 µg/ml myelin basic protein were
added to the beads, followed by 5 µl of a 200 µM ATP
solution containing 10 µCi of [-32P]ATP. The
reaction was incubated at room temperature for 5 min and stopped by
adding 12.5 µl of 5 × SDS-polyacrylamide gel loading buffer.
The mixture was then separated by SDS-polyacrylamide gel electrophoresis, and the wet gel (after fixing) was quantitated on a
Bio-Rad Molecular Imager. Results obtained with the Bio-Rad Molecular
Imager were linear with results obtained by cutting out radioactive
bands and quantitating in a scintillation counter. For Raf kinase
assays, 40 µl of protein kinase buffer containing 100 mM
NaCl, 1 µg of glutathione S-transferase-K97A MEK (Upstate Biotechnology, Inc.; eluted from beads with 20 mM
glutathione), 15 µM ATP, and 10 µCi of
[-32P]ATP were added to the washed beads. Reactions
were incubated at 30 °C for 30 min with intermittent agitation and
stopped by the addition of 12.5 µl of 5 × SDS-polyacrylamide
gel loading buffer. Samples were separated on 8% SDS-polyacrylamide
gels, transferred to nitrocellulose, and exposed on a Bio-Rad Molecular Imager.
Lipid kinase assays were performed
essentially as described (32). Briefly, 20 µl of 0.5 mg/ml sonicated
crude bovine brain phosphoinositides (sonicated in 20 mM
HEPES (pH 7.0) and 0.1 mM EGTA) were added to
immunoprecipitated enzyme (~20-µl volume), followed by 10 µl of
5 × ATP/MgCl2 mixture (250 M ATP and 25 mM MgCl2 in 20 mM HEPES (pH 7.0))
containing 0.5-20 µCi of [-32P]ATP/reaction.
Reactions were left at room temperature for 5 min before stopping with
60 µl of 2 M HCl. Lipids were extracted by the addition
of 160 µl of chloroform/methanol (1:1), and the organic phase was
collected and analyzed by TLC. The TLC solvent was 65% 1-propanol and
35% acetic acid (1 M). The radioactivity on the TLC plate
was imaged and quantified using the Bio-Rad Molecular Imager.
RESULTS
The effect of wortmannin on PDGF-dependent
activation of MAP kinase was examined in three cell lines (Fig.
1). In CHO/HIR cells, wortmannin (100 nM) almost completely blocked PDGF-dependent activation of Erk2. In A31 cells, ~70% inhibition of MAP kinase was
observed. Inhibition of PtdIns-3,4-P2 and
PtdIns-3,4,5-P3 in vivo production correlated
with inhibition of MAP kinase activity in A31 cells (Fig.
2). In contrast, wortmannin had little
effect on PDGF-dependent activation of Erk proteins in
Swiss 3T3 cells (Fig. 1). A trivial explanation for this could be that
wortmannin is more effective in inhibiting PI 3-kinase in CHO and A31
cells than in Swiss 3T3 cells. However, we find that 100 nM
wortmannin is an effective inhibitor of PI 3-kinase activity in all
three cell lines as judged by inhibition of PtdIns-3,4-P2
and PtdIns-3,4,5-P3 production in intact cells (A31 and
CHO/HIR cells) or immune complex kinase assays using 4G10
anti-phosphotyrosine antibody (all cell lines) (data not shown).
Consistent with this result, insulin-stimulated MAP kinase activation
in Swiss 3T3 cells is almost completely inhibited by wortmannin. In
contrast, in CHO/HIR cells, where the insulin receptor is
overexpressed, wortmannin is less effective in blocking
insulin-dependent activation of MAP kinase (Fig. 1). These
results suggest that PI 3-kinase is necessary for
PDGF-dependent activation of the MAP kinase pathway in CHO
and A31 cells, but that in Swiss 3T3 cells, other pathways are utilized
for its activation.
[View Larger Version of this Image (20K GIF file)]
[View Larger Version of this Image (24K GIF file)]
These surprising results might be explained by the different levels of expression of receptors for PDGF in the three cell types. CHO cells express low levels of PDGF receptors (<20,000/cell) (33), and wortmannin is an effective inhibitor of the PDGF response in these cells. Swiss 3T3 cells express high levels of PDGF receptors (400,000/cell) (34), and the PDGF response is resistant to wortmannin in these cells. A31 cells have intermediate levels of PDGF receptors (150,000/cell) (35), and MAP kinase activation is partially inhibited by wortmannin. Likewise, the relative resistance of CHO/HIR cells to wortmannin inhibition of insulin-stimulated MAP kinase activity could be explained by the high level of insulin receptor expression in these cells.
Wortmannin Inhibits MAP Kinase Activation in Swiss 3T3 Cells at Low but Not High PDGF ConcentrationsAn alternative explanation of
the results in Fig. 1 is that the cell lines differ in their
composition of PDGF - and 
-receptors, and this could account for
differences in wortmannin sensitivity. On the basis of activation with
PDGF-AA versus PDGF-BB and immunoprecipitation of
Tyr-phosphorylated PDGF receptors with isoform-specific antibodies, CHO
cells contain mainly PDGF -receptors, and Swiss 3T3 cells contain
mainly PDGF -receptors (data not shown). To test the idea that
wortmannin sensitivity of MAP kinase activation depends on the number
and not the type of receptors that become stimulated, we reexamined MAP
kinase activation in Swiss 3T3 cells at suboptimal concentrations of
PDGF. This approach varies the number of receptors stimulated, but not
the type. As shown in Fig. 3, when low
concentrations of PDGF are used to stimulate the cells, MAP kinase
activation is sensitive to wortmannin inhibition. These results suggest
that to efficiently activate MAP kinase at low concentrations of growth factor or in cells that have few receptors, PI 3-kinase must be activated. However, PI 3-kinase becomes less important for this pathway
when a large number of receptors become activated, presumably because a
second pathway that requires more receptors provides a redundant
signal.
[View Larger Version of this Image (32K GIF file)]
PI 3-Kinase Is Activated in Swiss 3T3 Cells at Low PDGF Concentrations
To test this model, we examined whether PI
3-kinase was activated in Swiss 3T3 cells at the low PDGF
concentrations at which wortmannin inhibition of MAP kinase is
observed. As shown in Fig. 4, recruitment
of PI 3-kinase to an anti-Tyr(P)-precipitable complex occurs at very
low concentrations of PDGF (half-maximal at 2-3 ng/ml), consistent
with a role for PI 3-kinase in the activation of MAP kinase at low PDGF
concentrations. In contrast, anti-Tyr(P) precipitation of PLC1
saturates at higher concentrations of PDGF (half-maximal at 10 ng/ml).
It is possible that PLC1 is the redundant signal, parallel to PI
3-kinase, leading to wortmannin-resistant MAP kinase activation at
higher PDGF concentrations. This idea is consistent with reports that
some PKC family members, which are downstream of PLC, can feed into
the MAP kinase pathway at Raf (25-27).
. As described in the legend to Fig.
3, Swiss 3T3 cells were stimulated with various concentrations of PDGF
for 5 min before being lysed. Anti-phosphotyrosine immunoprecipitations were performed; resolved by SDS-polyacrylamide electrophoresis; and
blotted with antibodies against p85, the regulatory subunit of PI
3-kinase, and PLC1 (PLCg1). The blots were quantitated using a Molecular Dynamics Storm imaging system.
[View Larger Version of this Image (14K GIF file)]
Inhibition of PKC and PI 3-Kinase Together, but Neither Alone, Leads to Inhibition of MAP Kinase in Swiss 3T3 Cells
The
possibility that a PKC family member contributes to
PDGF-dependent activation of MAP kinase was investigated.
Prolonged treatment of cells with the phorbol ester
12-O-tetradecanoylphorbol-13-acetate results in degradation
of PKC family members. Under the conditions used here, PKC is
down-regulated >95%, and PKC is down-regulated 80%. PKC
down-regulation alone had little effect on PDGF-dependent activation of MAP kinase in Swiss 3T3 cells (Fig.
5). However, the MAP kinase activity in
the 12-O-tetradecanoylphorbol-13-acetate-treated cells was
inhibited by wortmannin. These results are consistent with two
redundant pathways for activation of MAP kinase in Swiss 3T3 cells at
high PDGF concentrations: a PI 3-kinase-dependent pathway
and a PKC-dependent pathway that is likely downstream of
PLC.
[View Larger Version of this Image (20K GIF file)]
Wortmannin Blocks Activation of Raf Kinase
To better define
the contribution of PI 3-kinase to the activation of the MAP kinase
pathway, the effect of wortmannin on Raf activation was examined in CHO
cells. Raf kinase activity peaked within 2 min of PDGF addition (Fig.
6), and wortmannin almost completely
inhibited the activation.
[View Larger Version of this Image (24K GIF file)]
DISCUSSION
The MAP kinase pathway plays a central role in the transduction of signals for growth, differentiation, and other cellular responses. This modular cascade of kinases is a link between receptor signaling events and nuclear events. One of the important links in this pathway to be established was the connection between receptor signaling and the MAP kinase cascade. This link appeared to be found when studies showed that receptor activation of Ras results in Ras-GTP binding to Raf, recruiting it to the membrane (36-39). While subsequent studies have shown that recruitment of Raf to the membrane is sufficient for its activation (40), other studies have shown that once recruited to the membrane, additional steps are required for full activation of Raf (4). Thus, Ras probably contributes to the activation of Raf by recruiting it to the membrane and inducing a conformational change that facilitates activation by other factors. One of the most pressing challenges in signal transduction is to identify the other factors that contribute to the activation of Raf and the MAP kinase cascade.
Many studies have used wortmannin or dominant-negative forms of PI 3-kinase to address the involvement of this enzyme in the activation of the MAP kinase pathway. While several studies have found that wortmannin (10-15) or dominant-negative PI 3-kinase (9) effectively inhibits activation of the MAP kinase cascade, many other studies have found that inhibition of PI 3-kinase has no effect on the activation of Erk proteins (17-19). The results in Fig. 1 address this apparent contradiction. We show that wortmannin sensitivity is clearly dependent on the system under study. The agreement between the results with wortmannin and those with dominant-negative PI 3-kinase have firmly established that PI 3-kinase is required for MAP kinase activation by growth factors in certain cells. However, as we show here, in some cells, inhibition of PI 3-kinase does not affect activation of MAP kinase.
The results that we present are consistent with a model in which PI 3-kinase is required for activation of the MAP kinase cascade under conditions in which relatively few receptors are activated, but is redundant with a parallel pathway when many receptors become activated. This model is supported by comparison of cells with different numbers of PDGF and insulin receptors (Fig. 1) and is directly addressed by varying the number of activated receptors in a single cell type (Fig. 3).
Wortmannin blocks acute (2 min) PDGF-dependent activation of Raf in CHO cells, indicating that, in these cells, PI 3-kinase is required for an early step in the cascade. This result is in agreement with a previous study showing that wortmannin blocks Raf kinase activation by nerve growth factor (10), but not Ras activation by nerve growth factor (41), insulin-like growth factor-1 (10), or insulin (42).
The redundant signal for MAP kinase activation that emerges when a
large number of receptors are activated apparently involves a PKC
family member. Down-regulation of PKCs did not block
PDGF-dependent activation of MAP kinase, unless PI 3-kinase
was also inhibited. Thus, PI 3-kinase contributes to Raf kinase and MAP
kinase activation by a pathway that does not require a down-regulatable
PKC family member. At high levels of stimulated PDGF receptors, a
second pathway involving PKC family members circumvents the need for PI
3-kinase. This second pathway is probably mediated by
PDGF-dependent activation of PLC, resulting in elevated
Ca2+ and diacylglycerol levels. Phorbol esters are known to
activate Raf and MAP kinases in intact cells, and various PKC family
members have been shown to activate Raf kinase in vitro
(25-27). Although the result in Fig. 5 indicates that PI
3-kinase-dependent activation of Raf kinase does not
involve a down-regulatable PKC, they do not rule out the possibility
that a nonconventional PKC or an atypical PKC that is not efficiently
down-regulated is involved. Both PtdIns-3,4-P2 and
PtdIns-3,4,5-P3 activate nonconventional PKCs in
vitro (20), and PI 3-kinase has been shown to be required for
PDGF-dependent recruitment of PKC (23) and PKC (24) to the membrane. PKC is partially down-regulated under the
conditions of Fig. 5 (80%), but could still be contributing to the
wortmannin-sensitive pathway. Alternatively, other known or unknown
targets of PI 3-kinase could feed into the MAP kinase pathway. Thus, an
attractive model is that stimulation of PI 3-kinase provides an
efficient pathway for Raf kinase activation at low PDGF levels via
stimulation of one of its effectors, whereas activation of PLC at
higher PDGF concentration provides a redundant signal via activation of
a down-regulatable PKC family member.
The apparent redundancy between PI 3-kinase- and
PLC-dependent pathways for Raf kinase activation is
further complicated by evidence that a product of PI 3-kinase is
required for maximal activation of PLC. Wortmannin has been shown to
partially block inositol-3,4,5-P3 production in several
cell types when the pathway stimulated involves PLC (43, 44). While
these results could be explained by wortmannin inhibition of targets
other than PI 3-kinase, Rhee and
co-workers2 has recently
found that PtdIns-3,4,5-P3 activates PLC in
vitro by binding to the SH2 domains of this enzyme. Thus,
inhibiting PI 3-kinase could prevent direct activation of PKCs by D3
phosphoinositides and also inhibit activation of conventional PKCs by
reducing the magnitude of PLC activation. This latter effect is
unlikely to explain the wortmannin inhibition of insulin-stimulated MAP
kinase since insulin does not activate PLC.
Finally, although a requirement for PI 3-kinase in MAP kinase
activation is now firmly established in several systems, our results do
not imply that activation of PI 3-kinase alone is sufficient to fully
activate Raf or MAP kinase. In fact, we do not find significant elevation of MAP kinase activity in chick embryo fibroblasts stably transformed with viral or cellular forms of p110 PI
3-kinase,3 even though these
cells have significantly elevated levels of PtdIns-3,4-P2
and PtdIns-3,4,5-P3 (3) and increased Akt/PKB protein
kinase activities (3). This result is in contrast to the recent report
that overexpression of p110 PI 3-kinase stimulates MAP kinase in
COS-7 cells (9). There are several problems with interpreting these
types of experiments. It is possible that acute activation of PI
3-kinase is sufficient to activate MAP kinase, but that continual
elevation of D3 lipids results in a feedback pathway that inhibits MAP
kinase. Conversely, it is possible that the lipid products of PI
3-kinase alone are not sufficient to activate MAP kinase, but that
certain cells transformed with PI 3-kinase secrete factors that
stimulate MAP kinase in an autocrine manner. Further work will be
necessary to address the sufficiency of PI 3-kinase for MAP kinase
activation.
FOOTNOTES
REFERENCES
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D. J. Levinthal and D. B. DeFranco Transient Phosphatidylinositol 3-Kinase Inhibition Protects Immature Primary Cortical Neurons from Oxidative Toxicity via Suppression of Extracellular Signal-regulated Kinase Activation J. Biol. Chem., March 19, 2004; 279(12): 11206 - 11213. [Abstract] [Full Text] [PDF]
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C. E. Runyan, H. W. Schnaper, and A.-C. Poncelet The Phosphatidylinositol 3-Kinase/Akt Pathway Enhances Smad3-stimulated Mesangial Cell Collagen I Expression in Response to Transforming Growth Factor-{beta}1 J. Biol. Chem., January 23, 2004; 279(4): 2632 - 2639. [Abstract] [Full Text] [PDF]
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L. O. Murphy, J. P. MacKeigan, and J. Blenis A Network of Immediate Early Gene Products Propagates Subtle Differences in Mitogen-Activated Protein Kinase Signal Amplitude and Duration Mol. Cell. Biol., January 1, 2004; 24(1): 144 - 153. [Abstract] [Full Text] [PDF]
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T. Araki, H. Nawa, and B. G. Neel Tyrosyl Phosphorylation of Shp2 Is Required for Normal ERK Activation in Response to Some, but Not All, Growth Factors J. Biol. Chem., October 24, 2003; 278(43): 41677 - 41684. [Abstract] [Full Text] [PDF]
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