From the Department of Neurology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115 and § DeCode Genetics, Lynghals 1, Reykjavik, Iceland
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ABSTRACT |
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Receptor tyrosine kinases are classified into
subfamilies, which are believed to function independently, with
heterodimerization occurring only within the same subfamily. In this
study, we present evidence suggesting a direct interaction between the
epidermal growth factor (EGF) receptor (EGFR) and the platelet-derived
growth factor (PDGF
) receptor (PDGF
R), members of different
receptor tyrosine kinase subfamilies. We find that the addition of EGF to COS-7 cells and to human foreskin Hs27 fibroblasts results in a
rapid tyrosine phosphorylation of the PDGF
R and results in the
recruitment of phosphatidylinositol 3-kinase to the PDGF
R. In R1hER
cells, which overexpress the EGFR, we find ligand-independent tyrosine
phosphorylation of the PDGF
R and the constitutive binding of a
substantial amount of PI-3 kinase activity to it, mimicking the effect
of ligand in untransfected cells. In support of the possibility that
this may be a direct interaction, we show that the two receptors can be
coimmunoprecipitated from untransfected Hs27 fibroblasts and from COS-7
cells. This association can be reconstituted by introducing the two
receptors into 293 EBNA cells. The EGFR/PDGF
R association is
ligand-independent in all cell lines tested. We also demonstrate that
the fraction of PDGF
R bound to the EGFR in R1hER cells undergoes an
EGF-induced mobility shift on Western blots indicative of
phosphorylation. Our findings indicate that direct interactions
between receptor tyrosine kinases classified under different
subfamilies may be more widespread than previously believed.
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INTRODUCTION |
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Receptor tyrosine kinases have been divided into subfamilies based
on common structural features (1). The epidermal growth factor
(EGF)1 subfamily includes the
ErbB1/EGFR, ErbB2/HER2/Neu, ErbB3/HER3, and ErbB4/HER4 receptors and is
characterized by the presence of two extracellular cysteine-rich
domains and an uninterrupted kinase domain (2). The PDGF receptor
subfamily includes the PDGFR, PDGF
R, stem cell factor receptor
(Kit), colony-stimulating factor receptor (Fms), and Flk2 receptors and
is characterized by the presence of five extracellular
immunoglobulin-like domains. Members of this subfamily contain a kinase
insert in which a regulatory region has been inserted into the
conserved kinase domain.
It is established that growth factors bind to specific receptors. Binding of the growth factor to its receptor may result in homodimerization or heterodimerization between members of a receptor subfamily (3, 4). For example, heterodimerization has been well described between members of the EGFR subfamily (5). The addition of EGF to a number of cell lines results in EGFR-dependent tyrosine phosphorylation of ErbB2 (6-9). EGF can induce dimerization of EGF receptors and ErbB2 in transfected NR6 and NIH3T3 cells (10, 11) and also in SKBR-3 cells, which overexpress the ErbB2 receptor (12). Similarly, heregulin may induce heterodimeric complexes between ErbB2 and ErbB3 or ErbB4 (13, 14). A number of heterodimeric combinations of these receptors have been described, and some combinations are favored over others (15). It is important to note that heterodimerization between the EGFR and ErbB2 proteins can be detected even in the absence of ligand (10, 16).
Heterodimerization may have important functional consequences.
Phosphatidylinositol (PI) 3-kinase has been shown to bind directly to
activated receptors at domains that are autophosphorylated on tyrosine
and contain a Tyr-X-X-Met motif (17). The EGFR
lacks the binding motifs for the SH2 domains of the PI 3'-kinase, while the ErbB3 receptor may have little or no kinase activity but has multiple copies of the binding motif for PI 3-kinase (18-20).
Stimulation of cells with EGF is known to increase the activity of PI
3-kinase. Although some studies have shown an increase in
EGFR-associated PI 3-kinase activity upon ligand stimulation, this is
considerably less than the increase associated with other activated
receptors (20, 21). This suggested that the mechanism of this increase may be a recruitment by the activated EGFR of other members of the EGFR
subfamily, such as ErbB3. This has been demonstrated in A431 cells,
where stimulation of cells with EGF increases ErbB3-associated PI
3-kinase activity (20, 22), or by using chimeric receptors in
fibroblasts (18). It should be noted that EGF does not bind to ErbB3.
In addition, different receptor heterodimers may alter ligand binding
kinetics, with resultant variations in signal characteristics (23).
Recently, transactivation of the EGFR has also been described in
response to stimulation of G-protein-coupled receptors (24). In the
PDGFR subfamily, the different isoforms of PDGF induce different
dimeric forms of the receptors; e.g. PDGF-AA induces
homodimers only while PDGF-BB induces all three combinations of
receptors (i.e.
,
, and
(25)). However,
for polypeptide growth factors, heterodimerization is generally
believed to be limited to members of the same subfamily of receptor
tyrosine kinases. Thus, although receptors from different subfamilies
may use common substrates, they are believed to function independently, without directly influencing each other and directly interacting only
with members of the same subfamily.
The engagement of a receptor tyrosine kinase by its ligand results in
dimerization, activation of the kinase activity of the receptor, and
autophosphorylation (26). Autophosphorylation of an activated receptor
results in the creation of docking sites for SH2 and
phosphotyrosine-binding domain-containing proteins that associate with
the receptor (27). These include proteins believed to have adaptor
functions, such as Shc and Grb2. Another class of SH2 domain-containing
proteins that bind to autophosphorylated receptor tyrosine kinases have
intrinsic catalytic activity. These include proteins such as
phospholipase C-1 and phosphatidylinositol 3-kinase, which associate
with the activated receptor. PI 3-kinase is composed of two subunits,
an 85-kDa regulatory or adaptor subunit and a 110-kDa catalytic subunit
(28). The p85 subunit has two SH2 domains in its carboxyl-terminal
half. The enzyme is capable of phosphorylating the D-3 position on
phosphatidylinositol, phosphatidylinositol 4-phosphate, or
phosphatidylinositol 4,5-biphosphate. PI 3-kinase may be an important
mediator of mitogenic signaling in certain cell types. Studies with
mutant PDGF receptors lacking association sites for PI 3-kinase show a
decrease in DNA synthesis upon PDGF stimulation (29, 30). Restoring the
PI 3-kinase binding site to a mutant PDGFR deficient in mitogenic
signaling restores the ability of the receptor to initiate DNA
synthesis (31). Other functions of PI 3-kinase (reviewed in Ref. 32)
include cellular trafficking and cytoskeletal alterations induced by
growth factor stimulation and a role in cell survival.
PDGF induces a heterologous down-regulation of EGF receptors (33).
Stimulation by PDGF or phorbol esters results in a decrease in the
affinity of the EGF receptor for its ligand without influencing the
number of receptors and results in a decrease in the kinase activity of
the EGF receptor (34-36). Stimulation of fibroblasts with PDGF or with
phorbol esters leads to phosphorylation of the EGF receptor at
threonine 654, which is a site of protein kinase C phosphorylation.
This led to the suggestion that protein kinase C activation mediated by
the PDGFR leads to phosphorylation of the EGFR at threonine 654, and
this phosphorylation is responsible for transmodulation. However,
subsequent studies have suggested that neither activation of protein
kinase C nor phosphorylation at threonine 654 are required for the
transmodulation induced by PDGF (37, 38). The mechanism of
PDGFR-mediated transmodulation remains unknown. An influence of the
EGFR on PDGFR receptor signaling, to our knowledge, has not previously
been described. In this study, we present evidence suggesting that the
EGFR associates with and directly influences signaling through the
PDGF receptor.
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EXPERIMENTAL PROCEDURES |
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Cell Lines, Growth Factors, and Transfection--
Hs27, COS-7,
Rat-1 R1hER, B82L, and 293 EBNA cells (Invitrogen) were maintained in
Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. R1hER cells are Rat-1 fibroblasts transfected with a
human EGFR construct. Human recombinant PDGF-BB and human recombinant
EGF were used at a concentration of 50 and 100 ng/ml, respectively, for
5 min unless specified otherwise. A human PDGF receptor construct
was cloned into pcDNA 3.1 (+) vector using standard molecular
cloning techniques. A human EGFR construct was cloned into pcDNA
3.1 (
) vector. The empty vectors were purchased from Invitrogen.
Transient transfections were performed using the calcium phosphate
method (39). Expression of transfected genes was confirmed by Western
blotting. For transient transfection experiments, cells were harvested
24 h after transfection.
Western Blotting, Immunoprecipitation, and Antibodies-- Standard protocols were used for immunoprecipitation and Western blotting in different experiments (39). Quantitation of proteins was performed by using a Bio-Rad detergent-compatible protein assay kit and confirmed by Coomassie staining of aliquots subjected to SDS-polyacrylamide gel electrophoresis. For immunoprecipitation, cells were serum-starved and exposed to growth factor for the times indicated. Cells were subsequently lysed in a modified radioimmune precipitation buffer (150 mM NaCl, 1% Nonidet P-40, 0.25% deoxycholate, 1 mM EGTA, 1 mM NaF, 50 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate), and equal amounts of protein were incubated with the primary antibody for 90 min. Protein A-Sepharose or Protein G-agarose beads were subsequently added to the lysates and incubated overnight at 4 °C. The beads were subsequently washed and solubilized in SDS sample buffer and then boiled and analyzed by SDS-polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose. Western blots were developed with ECL reagents (Amersham Corp.). In experiments where cell lysates were examined directly, cells were lysed in SDS sample buffer, boiled, and subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting. The phosphotyrosine antibody used in all of the experiments described in this study was PY-20, obtained from Transduction Laboratories (Lexington, KY). Other antibodies used for the different experiments were as follows.
For detecting tyrosine phosphorylation of the PDGFPI 3-Kinase Assays--
PI 3-kinase assays were done as follows
(42). Cells were serum-starved for 48 h prior to treatment with
PDGF-BB or EGF as indicated. The cells were incubated with growth
factors at 37 °C for 5 min and then washed with ice-cold buffer A
(137 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1 mM CaCl2, 1 mM MgCl2)
and 100 µM NaVO4 and lysed with in 1 ml of
buffer A containing 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, and 100 µM
NaVO4. 2 µl of anti-PDGF-R kinase insert antibody
(Pharmingen, 15756E; Ref. 43) was added and incubated for 90 min on
ice, after which protein A-Sepharose beads were added. Following a 1-h
incubation at 4 °C, the beads were washed three times in buffer A
containing 1% Nonidet P-40; twice in 100 mM Tris-HCl, pH
7.4, 500 mM LiCl; and twice in TNE (100 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA), all containing 100 µM NaVO4. The
immunoprecipitates were resuspended in 50 µl of TNE, 10 µl of 100 mM MgCl2, and 10 µl of sonicated 1 mg/ml PI
(Avanti Polar Lipids), dried under Argon, and resuspended in 10 mM HEPES, 1 mM EGTA (pH 7.0). The reaction
volume was incubated with a 10-µl volume of ATP (10 µCi of
-32P, 100 mM MgCl2, 5 mM HEPES, and 0.25 µM unlabeled ATP) at
37 °C for 10 min and was stopped by adding 20 µl of 6 N HCl and 160 µl of chloroform/methanol (1:1, v/v). The
aqueous and lipophilic phases were separated by centrifugation for 10 min at 14,000 rpm, and 40 µl of the lower phase was spotted onto a
silica gel 60 plate (Merck), previously immersed in 1% potassium
oxalate and heat-activated. After developing the plate in
chloroform/methanol/H2O/NH4OH (60:47:11:1.8,
v/v), the radioactive phosphate spots were detected by autoradiography,
identified by comparison with phospholipid standards, and quantitated
by liquid scintillation counting.
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RESULTS |
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The EGF Receptor Induces Tyrosine Phosphorylation of the PDGF
Receptor--
The Hs27 fibroblast cell line and COS-7 cells express
receptors for both PDGF and EGF. In this study, we have examined
signaling through the PDGFR. Stimulation of quiescent cells with
PDGF results in a rapid tyrosine phosphorylation of the PDGF receptor
(Fig. 2B). We find that stimulation of cells with EGF also
results in an increase in the phosphotyrosine content of the PDGFR
(Figs. 1 and
2). This increase can be seen within 5 min in COS-7 cells and can be detected by immunoprecipitating cell
lysates with phosphotyrosine antibodies followed by immunoblotting with
a PDGF
R antibody (Pharmingen, 15746E), as shown in Fig.
1B. Also, if PDGF
R antibodies are used for
immunoprecipitation of denatured cell lysates and this is followed by
immunoblotting with phosphotyrosine antibodies, an EGF-induced increase
in tyrosine phosphorylation of the PDGFR can be detected (Fig.
1A). Note that this antibody fails to immunoprecipitate the
denatured EGFR from EGF-stimulated A431 cells (Fig. 1A).
Also, stripping this blot and reprobing it with EGFR antibodies showed no staining (not shown). EGF-induced tyrosine phosphorylation of the
PDGF
R is also noted when samples are not denatured prior to
immunoprecipitation. The same results were obtained with Hs27 cells
(Fig. 2, A and B). We estimate that about 5% of
the PDGF
receptors undergo tyrosine phosphorylation in these cells
in response to EGF (Fig. 2B). We are unable to detect any
increase in tyrosine phosphorylation of the PDGFR in response to EGF in
B82L cells, which lack the EGFR. We do not detect tyrosine
phosphorylation of the EGFR in response to PDGF (not shown).
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The EGFR Recruits PI 3-Kinase to the PDGFR--
Activation of a
number of receptor tyrosine kinases results in the association of PI
3-kinase with the receptor and an increase in receptor-associated PI
3-kinase activity. Although stimulation of cells with EGF leads to an
increase in PI 3-kinase activity in phosphotyrosine immunoprecipitates,
there is little increase in EGFR-associated activity. The mechanism of
this increase may be a recruitment by the activated EGFR of other
members of the EGFR subfamily, such as ErbB3. To study whether the
PDGFR could play a similar role to ErbB3, we examined whether
exposure of cells to EGF would lead to an increase in the PI 3-kinase
activity associated with the PDGF
R. We find that in Hs27 cells EGF
induces a 2-fold increase in the PI 3-kinase activity associated with the PDGF
R, which is similar to the increase seen in phosphotyrosine immunoprecipitates (Fig. 3A).
Also, when cells are preincubated with a low concentration of PDGF (5 ng/ml) for 30 min and this is followed by stimulation with EGF, we can
detect a significant further increase in the PI 3-kinase activity
associated with the PDGF
R (Fig. 3A), over PDGF alone.
Treatment with 5 ng/ml PDGF alone for 30 min leads to about a 20-fold
increase, while further stimulation with EGF increases the PI 3-kinase
activity to about 30-fold compared with unstimulated cells. Similar
results were seen with COS-7 cells (Fig. 3B).
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The EGFR Coimmunoprecipitates with the PDGFR--
Although the
EGFR has been shown to heterodimerize with other members of the EGFR
subfamily, heterodimerization with the PDGFR has not been described. To
determine whether the tyrosine phosphorylation of the PDGFR in
response to EGF was mediated directly by the EGFR, we looked for a
physical association between the two receptors. Surprisingly, we found
that the two receptors can be coimmunoprecipitated from Hs27 and COS-7
cells in the absence of ligand. This association was detected by
immunoprecipitating the EGFR and staining Western blots with the
PDGF
R (Fig. 5A). To confirm
this association immunoprecipitation was then performed with PDGF
R
antibodies followed by blotting with EGFR antibodies with the same
result (Fig. 5B). About 5% of the PDGF receptors exist in a
complex with the EGFR in these cells (Fig. 5C). These
results were consistently obtained using a number of antibodies
recognizing different epitopes on the receptors (described under
"Experimental Procedures"), isotype-matched negative controls, and
peptide inhibition of immunoprecipitation.
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The PDGFR and EGFR Associate in Transiently Transfected 293 Cells--
We further explored the association between the two
receptors in 293EBNA cells, which normally express little or no
endogenous EGFR (45). In addition, we were unable to detect the
PDGF
R in these cells by immunoblotting. Both receptors were
introduced into these cells by calcium phosphate transfection. A human
PDGF
R cDNA construct was cloned into the PCDNA 3.1 vector.
Expression in 293 cells was confirmed by immunoblotting with PDGF
R
antibodies. A human EGFR construct was also cloned into a PCDNA 3.1 vector (Invitrogen). Expression in 293 cells was confirmed by
immunoblotting with EGFR antibodies. We coexpressed the receptors using
the empty vector as a control, and 24 h after transfection we
immunoprecipitated cell lysates with PDGF
R antibodies, followed by
Western blotting and staining with EGFR antibodies. The EGFR can be
detected in cells where both receptors were introduced but not in cells
where the the PDGF
R was transfected with a control vector (Fig.
6A). The reverse experiment
was done by immunoprecipitating the EGFR and staining Western blots
with PDGF
R antibodies. This again confirmed the association between
the two receptors (Fig. 6B). As is the case in untransfected
cells, the addition of either PDGF or EGF did not result in any
increase in the amount of coimmunoprecipitating receptor.
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DISCUSSION |
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In this study, we describe interactions between the EGF and the
PDGF receptors, which are members of different receptor tyrosine kinase subfamilies. The interactions observed between the two receptors
include a physical association between the two receptors and tyrosine
phosphorylation of the PDGF receptor by the activated EGFR and
EGF-induced recruitment of PI 3-kinase to the PDGFR. These studies were
done in Hs27 human foreskin fibroblasts and in R1hER fibroblasts cells,
which overexpress the human EGFR. These effects are also seen in the
COS-7 and in 293 cells, demonstrating that these interactions extend to
other cell types.
Stimulation of Hs27 and COS-7 cells with EGF results in tyrosine
phosphorylation of PDGF receptors. EGF has previously been demonstrated to induce tyrosine phosphorylation of other members of the
EGFR subfamily. As discussed earlier, the addition of EGF to certain
cell lines results in tyrosine phosphorylation of the ErbB2 and ErbB3
receptors, although EGF does not bind to ErbB2 or ErbB3. Similarly, EGF
does not bind to the PDGFR, and the tyrosine phosphorylation of the
PDGF
R in response to EGF seen in Hs27 and COS-7 cells is likely to
result from activation of the EGFR; also, these effects are not seen in
B82L cells, where the EGFR is not expressed. Furthermore,
overexpression of the EGFR in Rat-1 fibroblasts causes a substantial
ligand-independent tyrosine phosphorylation of the PDGF
R. Tyrosine
phosphorylation of the EGFR in response to PDGF, however, was not
observed in any of the cell lines we tested.
The EGFR-mediated tyrosine phosphorylation of the PDGF receptor could
have a number of functional consequences. It could influence the kinase
activity of the PDGF receptor, leading to tyrosine phosphorylation of
downstream substrates and/or result in recruitment of SH2
domain-containing proteins to the receptor. At least one of these
outcomes is seen in untransfected cells, namely the association of PI
3-kinase with the PDGFR following EGF stimulation. This suggests the
following order of events. Activation of the EGFR leads to tyrosine
phosphorylation and activation of the PDGF
R. This leads to the
association of PI 3-kinase with the PDGF
R. Although the EGF-induced
increase in PI 3-kinase activity associated with the PDGF
R is small
(a 2-fold increase), even small increases in PI 3-kinase activity may
be functionally significant. For example, PDGF-induced cytoskeletal
changes such as membrane ruffling, which is dependent on PI 3-kinase
activity, have been observed with concentrations of PDGF as low as 3 ng/ml (46). It should be noted that the activation of PI 3-kinase by
PDGF is dose-dependent over a certain range. If cells are
preincubated with low doses of PDGF, EGF induces a substantial further
increase in the PI 3-kinase activity associated with the PDGF
R. We
looked for a similar association between the PDGF
R and other SH2
domain-containing proteins such as phospholipase C-
1,
p120GAP, and SHP-2 in cells exposed to EGF. No association
was found.
Overexpression of the EGFR in Rat-1 cells results in a constitutive
tyrosine phosphorylation of the PDGFR, mimicking the effect of EGF
stimulation in untransfected cells. In addition, there is a substantial
constitutive association of the PDGF
R with PI 3-kinase in R1hER
cells, again consistent with results seen in untransfected cells upon
the addition of EGF. The PI 3-kinase activity in phosphotyrosine
immunoprecipitates is significantly increased in R1hER cells, in the
absence of ligand. Almost all of this activity may be associated with
the PDGF
R in these cells. It should also be noted that PI 3-kinase
is the only SH2 domain-containing protein we can detect that binds
constitutively to the PDGF
R in R1hER cells. These observations lead
us to infer the following. First, that EGF-mediated increases in PI
3-kinase activity may involve the recruitment of the PDGF
R in
certain cells. As noted before, the addition of EGF to A431 cells leads
to an increase in PI 3-kinase activity associated with the ErbB3
receptor, another member of the EGFR subfamily. The PDGF
R may serve
a similar function. Secondly, overexpression of the EGFR in R1hER cells
mimics the effect of EGF stimulation on the PDGF
R in untransfected
cells in a ligand-independent fashion. This suggests that although
R1hER cells express high levels of the EGFR, observations made in these cells may provide clues to the interactions between the two receptors under physiologic conditions. R1hER cells may also serve as a model for
interactions between the receptors in tumors that overexpress the EGFR
and also express the PDGF
R.
What is the mechanistic basis for this influence of the EGFR on PDGFR
signaling? We have shown that ligand-dependent activation of the EGFR results in tyrosine phosphorylation of the PDGFR in
untransfected cells, while overexpressing the EGFR leads to such an
effect in the absence of ligand. This effect could be mediated directly
by the EGFR or by intermediate kinases. Two observations suggest that
this may be a direct interaction. First, the EGFR binds to the PDGF
R
as detected by coimmunoprecipitation experiments. Although this
association is ligand-independent, there is precedent for this. It has
previously been shown that heterodimerization may occur between EGFR
and ErbB2 proteins even in the absence of ligand (10, 16). Secondly, in
R1hER cells the fraction of PDGF
receptors associated with the EGFR
undergoes an EGF-induced mobility shift suggestive of phosphorylation.
This again supports a direct interaction between the two receptors.
From the studies presented here, we conclude that direct interactions between receptor tyrosine kinases classified under different subfamilies may be more widespread than previously believed. This may include heterodimerization or oligomerization and/or transphosphorylation with resultant recruitment of SH2 domain-containing proteins to the activated receptor. Such a scheme would alter the signaling repertoire of the receptor depending on other receptors expressed in the same cell and has obvious implications for specificity in cellular signaling. The ability of different receptor tyrosine kinases to directly influence each other is also relevant to a better understanding of coordination of signals generated by multiple cytokines acting on the same cell.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Michael Weber and Gordon Gill for generous gifts of R1hER and B82L cells. We thank Dr. Axel Ullrich for an EGFR cDNA construct. We thank Dr. Stephen Soltoff for critically reading this manuscript. We thank Drs. Stephen Soltoff, Alex Toker, Geraint Thomas, and Anthony Couvillon for help with PI 3-kinase assays.
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FOOTNOTES |
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* This work was supported in part with National Institutes of Health Grant ROINS32977.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.
To whom correspondence should be addressed: The Harvard
Institutes of Medicine, 77 Avenue Louis Pasteur, Room 807, Boston, MA 02115. Tel.: 617-667-0837; Fax: 617-667-0811; E-mail:
ahabib{at}bidmc.harvard.edu.
1
The abbreviations used are: EGF, epidermal
growth factor; EGFR, EGF receptor; PDGF, platelet-derived growth
factor; PDGFR, PDGF receptor; PDGFR, PDGF
receptor; PI,
phosphatidylinositol; SH2, Src homology 2; IP,
immunoprecipitation.
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REFERENCES |
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