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From the
Following ligand binding, the epidermal growth factor receptor
(EGF-R) autophosphorylates itself on tyrosine residues located in its
carboxyl terminus; in vitro, three sites are highly
phosphorylated, while two other sites are phosphorylated to lesser
extents. In the presence of the Src protein-tyrosine kinase, in
vitro phosphorylation of the minor autophosphorylation sites was
increased, and four additional residues were phosphorylated. Following
EGF stimulation, two (Tyr-891 and Tyr-920) were found to be
phosphorylated in a colorectal cell line (DLD-1) and in a breast tumor
cell line (MCF7). The remaining in vitro sites were not found
to be highly phosphorylated in vivo. The sequences surrounding
Tyr-891 and Tyr-920 match the reported consensus binding sequences for
the SH2 domains of Src and the regulatory domain of
phosphatidylinositol 3-kinase (p85
The cellular events that result from extracellular signaling by
growth factors are initiated primarily by members of the transmembrane
protein-tyrosine kinase family (for review, see Ullrich and
Schlessinger(1990)). Typically, these receptor kinases dimerize upon
binding ligand, which results in enzyme activation and the subsequent
inter- and/or intra-molecular autophosphorylation on tyrosine residues.
These receptor autophosphorylation sites act as docking sites for a
wide range of regulatory molecules, each of which contain at least one
Src homology 2 (SH2) domain that mediates interaction with the receptor
(for reviews, see Koch et al.(1991) and Cantley et
al.(1991)). Facilitation of substrate phosphorylation, regulation
of enzymatic activity, and alterations in subcellular localization are
three documented effects of SH2-mediated docking (Sugimoto et
al., 1994; Uchida et al., 1994; Moran et al.,
1991; Vega et al., 1992; and Pronk et al., 1993).
Five sites of autophosphorylation have been identified in the
EGF-R,
Other proteins have
been reported to bind to the EGF-R, but the site or sites of binding
have not been identified. Recently, the prototype SH2-containing
protein, pp60
While
we do not discount the possibility that the first two explanations may
be valid for some of the confusion in the literature, in this report we
show evidence that Src can phosphorylate the EGF-R on novel sites in vitro, that Src and P85
Src, containing a glycine to alanine substitution at position 2 to
prevent myristylation, was expressed using the baculovirus-Sf9 cell
system and purified essentially as previously described (Lydon et
al., 1992). The purified enzyme was dephosphorylated by addition
of calf intestinal alkaline phosphatase (Boehringer Mannheim, 100
units/mg of Src). The reaction proceeded for 4 h at room temperature or
overnight at 4 °C in 25 mM Tris, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, 10 mM MgCl
Csk-inactivated Src was produced by phosphorylating Src with Csk (a
kind gift from Dr. Maria Ruzzene and Prof. L. A. Pinna, Padova, Italy),
a kinase known to specifically phosphorylate Tyr-527 of human Src. The
conditions for this reaction (50 mM Tris, pH 7.5, 6 mM MnCl
To determine
phosphorylation of the receptor protein-tyrosine kinases (RPTKs) by Src
and of Src by the RPTKs, specific inhibitors of the target kinase being
phosphorylated were used. 10 µM of CGP 46251 or CGP 47130
were used to abolish autophosphorylation of the EGF-R or ErbB2,
respectively, without effecting phosphorylation by Src ().
Similarly, 10 µM CGP 59272 completely inhibited Src
without effecting phosphorylation by EGF-R or ErbB2 kinases ().
Cell lysates and immunoprecipitated
proteins were separated in a 12% SDS-polyacrylamide mini-gel and then
transferred to polyvinylidene difluoride membrane using a horizontal
semi-dry electroblotter (JKA-Biotech). An alkaline phosphatase
detection system using the fast red substrate (Pierce) or a
peroxidase-catalyzed enhanced chemiluminescence (ECL, Amersham, United
Kingdom) detection system were used for immunodetection.
Mapping of phosphopeptides was performed according to the protocol
supplied with the HTLE-7000 peptide mapping system (CBS Scientific
Co.), with the exception that
Because in vitro phosphorylation is not always an indicator
of physiologically relevant events, we examined the phosphorylation of
the EGF-R in its cellular context. Immunoprecipitated EGF-R from a
breast tumor cell line (MCF7, Fig. 2) and a colorectal tumor cell
line (DLD-1, data not shown) was phosphorylated on Tyr-891 and Tyr-920,
as well as all of the autophosphorylation sites. Moreover, the
phosphorylation of these sites was to a similar extent as the
previously reported autophosphorylation sites. Closer analysis of the
sequences surrounding these sites revealed that Tyr-891 is similar to
the consensus binding sequence of the Src SH2 domain (),
and Tyr-920 fits the consensus binding sequence of the SH2 domain of
the P85
Because Src only bound to the Src-phosphorylated
form of ErbB2, we looked to see if Src phosphorylates
non-autophosphorylation sites in this closely related receptor (Fig. 4). Based on similarity to the EGF-R, the sites of
autophosphorylation of ErbB2 can be predicted. The three major sites
and one of the minor sites would all be found on only two tryptic
peptides, with the fifth minor site comprising a third phosphopeptide.
Our data suggest these predictions are correct, since phosphopeptide
mapping revealed two major phosphopeptides and one minor form (Fig. 4A). At least four or five additional
phosphopeptides are observed after ErbB2 phosphorylation with Src,
indicating at least that many additional tyrosine residues are
phosphorylated. The occurrence of these phosphorylations in vitro does not mean they would all occur in vivo. However, the
three tyrosines that are phosphorylated by EGF-R in vivo are
conserved in ErbB2. Thus, sites for binding of Src and P85
The EGF receptor and ErbB2 have each been demonstrated to
autophosphorylate multiple sites in vitro (Fig. 8).
Kinase inactive mutants fail to phosphorylate these sites in response
to ligand stimulation, suggesting that autophosphorylation also takes
place in vivo. In this report, we demonstrate that a
non-receptor tyrosine kinase, Src, is capable of phosphorylating each
of these receptors to a high level. In vitro, Src
phosphorylates all of the EGF-R autophosphorylation sites plus an
additional five tyrosine residues. Two of these additional sites
(Tyr-891 and Tyr-920) were observed to be phosphorylated in a
colorectal carcinoma cell line (DLD-1) and in a breast tumor cell line
(MCF-7). In cells that overexpress EGF-R (i.e. A431)
autophosphorylation may predominate, thus explaining why these
additional sites have been missed in the few previous studies that have
mapped in vivo phosphotyrosine sites. Although EGF-R is
overexpressed in several of the carcinoma cell lines used in this
study, Src is also highly expressed (Masanori et al., 1990).
The correlation of the overexpression of Src with phosphorylation of
these non-autophosphorylated sites indicates that Src may be the
endogenous kinase responsible for this phosphorylation. This conclusion
is further supported by the observation that phosphorylation of these
new sites is only observed under conditions where Src is found to
associate with the EGF-R.
Both Src and P85
It has recently been reported that the a carboxyl-terminal truncated
EGF-R (that has its carboxyl-terminal tail including all known
autophosphorylation sites removed) can still mediate EGF-induced
proliferation and growth. However, when each of the individual
autophosphorylation sites are mutated to phenylalanine, the EGF-R is
unable to transduce a proliferative signal. Since truncation is a
drastic alteration, it was surprising that it could retain more of its
functionality. Our studies may suggest an explanation for this
observation. Perhaps the three tyrosine residues phosphorylated by Src
in the carboxyl terminus of the kinase domain are sufficient to mediate
these effects of EGF. However, Src may not be able to phosphorylate
these sites before the EGF-R tail has been autophosphorylated (i.e. the tail may fold over and block these sites, a suggestion for
which there is some evidence (Gill et al., 1988)). Therefore,
if the autophosphorylation sites are mutated, the tail cannot be
phosphorylated; so, the remaining sites are blocked from being
phosphorylated. On the other hand, truncation would completely remove
the tail, leaving these sites open for phosphorylation by Src, and
signaling from SH2 proteins that interact with these sites could still
occur.
Coker et al.(1994) have recently reported that a
kinase negative mutant of EGF-R retained the capacity to stimulate DNA
synthesis upon EGF stimulation. This inactive kinase was phosphorylated
on tyrosine in response to EGF by what the authors described as an
associated tyrosine kinase. Thus, there is ample evidence that
additional tyrosine kinases assist in mediating the response to growth
factors such as EGF.
EGF signaling has been demonstrated to be
enhanced by overexpression of Src and may cause the activation of Src
(Luttrell et al., 1988). Our findings that Src creates
additional SH2 docking sites on EGF-R and that one or more of these is
a docking site for Src suggests an explanation of how these effects may
occur. Activation of Src has been measured directly in EGF-stimulated
cells, but it has also been observed that certain physiological Src
substrates become phosphorylated in response to EGF stimulation. The
role of Src in EGF signaling is probably not limited to the creation of
additional SH2 docking sites on the receptor and is probably involved
in the phosphorylation of downstream regulatory molecules. It is also
possible that some of the proteins that have been thought to be
substrates of EGF-R may actually be phosphorylated by Src bound to the
EGF-R. Src has been demonstrated to associate with EGF-R, ErbB2,
PDGF-R, colony-stimulating factor-1 receptor, c-Kit, and the fibroblast
growth factor receptor (Mohammadi et al., 1991; Luttrell et al., 1994; Courtneidge et al., 1993; Zhan et
al., 1994). These interactions support the hypothesis that Src is
directly involved with many growth factor signaling pathways.
Furthermore, mice with homozygous deletions of the src gene
die within the first few weeks after birth (Soriano et al.,
1991). Surprisingly, they do not have detectable abnormalities in the
brain or platelets (tissues rich in Src). However, they are deficient
in bone remodeling. Although Src is necessary for survival after the
first few weeks of life, it is not essential for cell viability,
suggesting that its function is nonessential or that redundant pathways
exist that would ensure cell viability even in the absence of Src.
As just implied, EGF is not the only growth factor that seems to
involve Src in its signaling. PDGF stimulation of cells results in
activation and phosphorylation of Src, and the PDGF-R directly
associates with Src via a PDGF-R autophosphorylated site. ErbB2 has
been reported to associate with Src in mammary tumor cells (Luttrell et al., 1994). We now report that the EGF-R and ErbB2 are also
phosphorylated by Src on non-autophosphorylation sites, and it is these
additional sites that are required for Src binding. Therefore, Src may
be an intrinsic part of many growth factor signaling pathways. This
view is consistent with the ubiquitous expression of Src in all cell
types.
While we present strong evidence that these mechanisms occur
in at least some tumor cell lines, it is unclear whether they
contribute in any way to tumorigenesis or if they are involved in EGF
signal transduction in non-transformed cells. Future studies, with
EGF-R mutants lacking the sites phosphorylated by Src, should help to
elucidate the role of these sites in EGF signaling.
The
IC
It is possible
that the nonautophosphorylated sites are phosphorylated by a kinase
other than Src; however, these sites are phosphorylated by Src in
vitro and in tumor cells that overexpress Src (Fig. 2). Src and
P85
We are grateful to M. Becker for supplying EGF
receptor and ErbB2 and P. Haberthuer for assistance in large scale
expression of pp60
Volume 270,
Number 26,
Issue of June 30, pp. 15591-15597, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
(*)
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
), respectively. In
vitro, both of these proteins were found to bind to
Src-phosphorylated EGF-R with 100-fold greater affinity than to
autophosphorylated EGF-R, demonstrating that Src creates new sites for
SH2 binding. Furthermore, Csk-inactivated Src was activated by
interaction with Src-phosphorylated EGF-R but not by autophosphorylated
EGF-R. Upon EGF treatment of MCF7 or three colorectal carcinoma cell
lines (WiDr, DLD-1, and LS174T), the EGF-R coimmunoprecipitated with
both p85
and Src. Evidence is also presented that suggests that an
EGF-R-related protein, ErbB2, may be involved in similar Src-mediated
interactions. These data demonstrate that EGF-R is phosphorylated in vivo at non-autophosphorylation sites and that these novel
sites can act as docking sites for Src, P85, and potentially other
SH2-containing proteins. In addition, the data suggest a tyrosine
phosphatase-independent mechanism for the elevation of Src activity in
cells exposed to growth factors. Overexpression of Src, EGF-R, and/or
ErbB2 in breast and colorectal tumor cells suggests the potential that
such interactions may contribute to the transformed phenotype of these
carcinomas.
()three major (Tyr-1068, Tyr-1148, and
Tyr-1173) and two minor (Tyr-992 and Tyr-1086) (Downward et
al., 1984; Hsuan et al., 1989; Margolis et al.,
1989; and Walton et al., 1990). Several SH2-containing
proteins have been demonstrated to bind to one or more of these sites
both in vitro and in vivo (Grb2, phospholipase
C-, SHC, Syp, and GTPase-activating protein) (Batzer et
al., 1994; Margolis et al., 1990; Rotin et al.,
1992; Feng et al., 1994; Xiao et al., 1994).
Recently, an unprecedented collaboration of laboratories has undertaken
a thorough survey of the consensus binding sequences for many of the
SH2 domains (Songyang et al., 1993, 1994). They have found
that the SH2 domains can generally be placed into families of related
binding specificities, with the three residues on the carboxy side of
the phosphotyrosine having the greatest influence on affinity. However,
binding of SH2-containing proteins to whole proteins appears to be less
predictable than the interactions between isolated SH2 domains and
simple peptides (Soler et al., 1994).
(Src) was reported to
associate with the EGF-R (also known as ErbB1) and a related protein,
ErbB2, in a breast tumor cell line (Luttrell et al., 1994).
This correlates with evidence that overexpression of Src enhances the
mitogenic response of cells to EGF (Chang et al., 1993).
However, none of the autophosphorylation sites in the EGF-R are similar
to the reported consensus binding sequence for Src-SH2
(pTyr-acidic-acidic-hydrophobic) (Songyang et al., 1993).
Similarly, the regulatory domain of phosphatidylinositol 3-kinase
(P85
) has also been reported to bind EGF-R, but none of the
autophosphorylated sites match its consensus sequence either
(pYXXM) (Songyang et al., 1993; Hu et al.,
1992; McGlade et al., 1992). There would seem to be three
possible explanations for these findings: 1) these observed bindings
are artifacts and are not physiologically important, 2) consensus
sequences based on peptides are not reliable predictors of protein
binding or, 3) there may be other tyrosine residues within the EGF-R
that are phosphorylated and can bind SH2-containing proteins.
preferentially associate with
the EGF-R after it has been phosphorylated by Src, that these
Src-phosphorylated sites (including consensus binding sites for Src and
P85) are phosphorylated in cells in response to EGF, that P85
and Src bind to the EGF-R in these cells in an EGF-dependent manner,
and that Csk-inactivated Src is reactivated upon binding to the Src
phosphorylated EGF-R. In addition, ErbB2, a closely related receptor
kinase, is also phosphorylated by Src in vitro. The P85
and Src consensus binding sites found in the EGF-R are highly conserved
in ErbB2, suggesting similar SH2 interactions may exist with the EGF-R
growth factor receptor family. This evidence suggests the possible
involvement of these interactions in breast and colorectal carcinomas.
Cell Culture
MCF7 breast tumor cells
were a gift from Dr. N. Hynes (Friedrich Miescher Institute, Basel,
Switzerland), while DLD-1, WiDr, and LS174T colorectal tumor cells were
a gift from Dr. T. Hall (Ciba-Geigy Ltd., Basel, Switzerland). These
cells were grown at 37 °C with 6% CO
in
Dulbecco's modified essential medium containing 10% fetal calf
serum. All tissue culture reagents were from Life Technologies, Inc.
For radiolabeling cells, 0.5 mCi of inorganic P was added
per 10-cm plate of cells at
70% confluence after 8 h of serum
starvation. After 4 h, the cells were treated with 50 ng/ml EGF and 10
min later lysed in RIPA buffer (50 mM Tris, pH 7.5, 150
mM NaCl, 5 mM EDTA, 200 mM
Na
VO
, 0.5% sodium deoxycholate, 0.2% Nonidet
P-40, 0.05% SDS, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1
mM phenylmethylsulfonyl fluoride). The same procedure was used
for non-radioactive immunoprecipitations, excluding the addition of P.
Preparation of ErbB2, EGF-R, p60
EGF-R and ErbB2 truncation mutants,
including only the cytoplasmic portion of the proteins, were expressed
in the baculovirus-Sf9 insect cell system and purified as previously
described (McGlynn et al., 1992; Guy et al., 1992).
, and
Csk-inactivated Src
, 1 mM ZnCl. Dephosphorylated
Src was isolated by separation on a 5/5 Mono-Q column (Pharmacia).
, 100 µM ATP, 0.02% Nonidet P-40, 1
mM dithiothreitol) also allow extensive autophosphorylation to
occur. The heterogeneous mixture of phosphorylated forms was separated
on a Mono-Q column as described (Stover et al., 1994). A
homogeneous peak containing inactive, tyrosine-phosphorylated Src was
designated Csk-inactivated Src.
Kinase Reactions
To phosphorylate
synthetic peptides, 1 nmol of peptide was phosphorylated with 1 µg
of Src for 1 h at room temperature in 50 µl of kinase buffer (20
mM Tris, pH 7.5, 1 mM EDTA, 10 mM MgCl, 1 mM dithiothreitol, and 200 µM [P]ATP). In vitro autophosphorylation and Src phosphorylation of the EGF-R were
performed under the same conditions except that the proportion of Src
to EGF-R was approximately 1:10 by weight.
Immunoprecipitation and Western
Blots
Cell lysates were precleared by centrifugation at
10,000 rpm in a Sorval SS-34 rotor. ErbB2, EGF-R, or Src were
immunoprecipitated using 5 µg of -EGF-R antibody (monoclonal
antibody F4, Boehringer Mannheim, Germany) or -Src antibody
(monoclonal antibody 327, Oncogene Science) and 50 µl of a slurry
of protein G-Sepharose (Pharmacia, Upsala, Sweden). Immune complexes
were washed three times in 10 ml of RIPA buffer at room temperature for
10 min on a revolving disk.
Phosphopeptide Mapping
Seven sites of
phosphorylation on the EGF-R, following treatment with Src, were
identified by mass spectrometry and synthetic peptide mapping (data not
shown). These seven sites were then verified to be phosphorylated in vivo by demonstrating that a mixed pool of these seven
phosphopeptides comigrated with peptides generated from EGF-R from
EGF-stimulated MCF7 cells. EGF-R or ErbB2 were phosphorylated in the
presence of [P]ATP (as above) or were
immunoprecipitated from cells as described above, and the proteins were
then isolated by SDS-polyacrylamide gel electrophoresis, excised from
the gel, and washed in 50% acetonitrile for 4 h at room temperature.
Peptides generated from in vivo labeled proteins were then
treated with protein phosphatase 2
to remove phosphate from serine
and threonine residues. The completion of this reaction was verified by
phosphoamino acid analysis (not shown). After air drying, the gel slice
was rehydrated in 100 mM NHCO containing 5 µg of trypsin and 1 µg of endoproteinase
Lys-C. The digestion was left to proceed overnight at 30 °C, and
the peptides were then eluted by rocking in 50% acetonitrile for 4 h.
Synthetic peptides were phosphorylated as described above, separated
from the other reaction ingredients by reverse phase high pressure
liquid chromatography on a 2.2 100-mm C18 column (Vydac).
P was used in place of
P. In brief, after lyophilization, the peptides were
resuspended in 10 µl of pH 1.9 buffer (2.2% formic acid and 7.8%
acetic acid). 1-5 µl (
5000 cpm) of the sample were
spotted on cellulose TLC plates (Merck), and electrophoresis at pH 1.9
and 1000 V proceeded for 50 min. After drying, the plate was then
placed in a chromatography tank containing phospho-chromatography
buffer (37.5% n-butanol, 25% pyridine, and 7.5% acetic acid)
for 12-16 h for separation in the second dimension.
Phosphopeptides were detected by autoradiography and with a
phosphorImager (Molecular Dynamics). Synthetic peptides were purchased
from Genosys Biotechnologies, Inc.
Src Kinase Assay
To determine the effect
on Src activity of association with the EGF-R, Src was incubated with
autophosphorylated EGF-R or Src-phosphorylated EGF-R. 10 µM CGP 46251 was then added to block EGF-R from interfering with the
assay of Src kinase activity. Src was added to 50% the
concentration of EGF-R (50 nM based on a Bradford protein
assay). The activity of Src was then assessed using 1 mM angiotensin II or 1 mg/ml enolase as a substrate in kinase buffer
including 10 µM [
P]ATP. Both assays
yielded similar results; however, the data presented are using enolase
as a substrate. After 10 min, the reaction was stopped by addition of
10 µl of acetic acid. Phosphate incorporation into angiotensin II
was then measured, by spotting 10 µl of the reaction mix onto P81
paper (Whatman), washing 3 times in 0.5% phosphoric acid, and measuring
radioactivity in 1 ml of scintillation fluid.
Determination of Relative SH2 Binding
Affinities
A GST-P85-SH2 fusion protein was obtained
from Santa Cruz Biotechnology, Inc. The relative binding affinities of
this protein and of the whole Src protein to differentially
phosphorylated forms of the EGF-R were determined as follows: EGF-R was
autophosphorylated or phosphorylated by Src as described above except
Src was only present at 1/50th the concentration of the EGF-R. The
kinase reactions were stopped by the addition of EDTA to five times the
concentration of divalent metal (50 mM). Then, the reactions
were diluted to one of three concentrations, 1, 10, or 100 nM,
simply by adjusting the volume to 50 µl, 500 µl, or 5 ml. An
equal amount of Src or P85-SH2 was added to each tube, making the
final concentration of these proteins 1, 10, or 100 nM as
well. After a 30-min incubation at 4 °C, the volume of each tube
was adjusted to 10 ml, and EGF-R was immunoprecipitated (20 µg of
-EGF-R antibody, clone F4 (Boehringer Mannheim), and 100 µl of
protein G-Sepharose). The immune complexes were boiled in SDS-sample
buffer, and Western blots were incubated with either -Src
(monoclonal antibody 327) or -GST (Santa Cruz Biotechnology, Inc.)
antibodies as appropriate.
Src Phosphorylates EGF-R and ErbB2 at
Nonautophosphorylation Sites
Recent reports that Src can
associate with EGF-R and ErbB2 in mammary tumor cell lines (Luttrell et al., 1994) stimulated us to study this interaction in
greater depth in vitro. However, we found that Src did not
bind to autophosphorylated EGF-R or ErbB2 proteins (Fig. 1).
Since the recombinant EGF-R we used was a truncated protein (lacking
the extracellular and transmembrane domains), we postulated that
perhaps our protein had an altered autophosphorylation pattern.
Therefore, two-dimensional phosphopeptide mapping of trypsin and
endoproteinase Lys-C cleaved, autophosphorylated recombinant EGF-R was
performed and compared with peptide maps from the holoenzyme. Three
dominant spots were observed, while two additional spots were visible
after extended exposure (Fig. 2). Three dominant spots were
identified as phosphopeptides that contain pTyr-1068, pTyr-1148, and
pTyr-1173 by direct comigration of P-labeled synthetic
peptides. The doublet where the pTyr-1068 phosphopeptide migrated is
probably due to differential digestion by trypsin since a lysine and
arginine are adjacent on the amino-terminal end of this peptide.
Because trypsin does not cleave amino-terminal residues efficiently, a
mixture of two cleavage products would be produced. Therefore, the
major sites identified in vitro are the same sites reported to
be the major sites of EGF-R autophosphorylation, both in vitro and in vivo, indicating that the low binding affinity of
Src was not due to a non-physiological in vitro autophosphorylation pattern.
Figure 1:
Association of Src with EGF-R and
ErbB2. 5 µg of each RPTK were either allowed to autophosphorylate
or were incubated with 100 ng of Src in the presence of an inhibitor
for the appropriate RPTK (see Table I). 20 mM EDTA was added
to stop the phosphorylation reactions, and 5 µg of Src was added to
each. After rocking for 15 min at 4 °C, the RPTKs were
immunoprecipitated and washed three times for 20 min each time in Tris
buffer, containing 1% Nonidet P-40 and 200 mM NaCl, and once
in 20 mM Tris, pH 7.5. Immunoblotting, using an anti-Src
monoclonal antibody 327, determined the presence of Src in the immune
complexes. As controls, the total amount of Src and 1/10th of the Src
added to each reaction were loaded into the first two lanes, as
indicated.
Figure 2:
Phosphopeptide maps of EGF-R
autophosphorylated or phosphorylated by Src (A and B,
respectively). EGF-R was phosphorylated, and tryptic peptides were
generated as described under ``Materials and Methods.'' Seven P-phosphorylated peptides, specifically synthesized to be
identical to tryptic fragments of EGF-R that include the tyrosines
reported to be or identified to be phosphorylated during Src treatment
of EGF-R, were pooled and mapped in a similar fashion. The identity of
each peptide was determined by mixing a pool of cold phosphorylated
peptides with a single
P-phosphorylated peptide and
matching the radioactivity with a particular ninhydrin-stained spot. D shows the phosphopeptide map of trypsin-cleaved EGF-R
immunoprecipitated from MCF7. Peptides from C and D were pooled and run together (E) to verify that the seven
synthetic peptides comigrate with the in vivo generated
peptides, indicating that they are indeed the same. The only difference
in the preparation of EGF-R immunoprecipitated from cells was that it
was treated with the serine/threonine phosphatase PP2a to remove any
seryl- or threonyl-attached phosphate. All the maps were exposed to a
PhosphorImager plate for 15 h and then scanned by a PhosphorImager
(Molecular Dynamics). The intensity range for all of the samples is set
to 0.10-999.87. The origin of each map is marked by a +
sign.
The platelet-derived growth factor
receptor (PDGF-R) has been demonstrated to phosphorylate Src (Gould and
Hunter, 1988). We therefore tested the ability of EGF-R and ErbB2 to
phosphorylate Src. Neither kinase was able to do this (Fig. 3B); however, Src was found to strongly
phosphorylate both the EGF-R and ErbB2 proteins (Fig. 3A). This finding opened up the possibility that
Src creates its own binding site on these receptor kinases. This
appears to be the case as Src was able to bind to Src-phosphorylated
EGF-R and ErbB2 but not to these proteins after simple
autophosphorylation (Fig. 1). In these experiments, EGF-R and
ErbB2 enzyme activities were inhibited by selective protein tyrosine
kinase inhibitors that completely abolish their activity at
concentrations that do not effect phosphorylation by Src, ensuring that
the phosphorylation of these RPTKs is due to Src and not to a change in
autophosphorylation caused by the presence of Src.
Figure 3:
A, the
ability of Src to phosphorylate EGF-R and ErbB2 was determined. 5
µg of receptor were incubated with [P]ATP
and, where indicated, an appropriate inhibitor (10 µM CGP
46251 or 10 µM CGP 47130) and 100 ng of Src. The proteins
were separated on a 12% SDS-polyacrylamide gel and visualized by x-ray
autoradiography. B, the ability of each of the RPTKs to
phosphorylate Src was determined in a similar fashion. 5 µg of Src
were incubated with [
P]ATP and, where indicated,
a specific inhibitor of Src (10 µM CGP 59272) and 100 ng
of the indicated receptor tyrosine kinase. The proteins were separated
by gel electrophoresis and visualized by autoradiography. PTK,
protein-tyrosine kinase.
Two-dimensional
phosphopeptide mapping of Src-phosphorylated EGF-R revealed that Src
phosphorylates the three major EGF-R autophosphorylation sites, two
minor sites, which have previously been reported to be
autophosphorylated (Tyr-992 and Tyr-1086), and five novel sites. In our
hands, autophosphorylation resulted in a phosphate to protein ratio of
2.8, while Src phosphorylation of EGF-R resulted in a ratio of 6.4.
Three of these additional sites were identified as (Tyr-845, Tyr-891,
and Tyr-920) by mass spectrometry and subsequently by comigration of
phosphorylated synthetic peptides to the phosphopeptides from EGF-R
(data not shown). These three sites are located within the
carboxyl-terminal portion of the kinase domain rather than in the tail
where the autophosphorylation sites are found. The remaining two spots
were not identified but are located within the carboxyl-terminal tail,
since a GST-EGF-R tail fusion protein (amino acids 952-1210 of the
EGF-R) phosphorylated by Src also yielded these phosphopeptides (data
not shown). There are only three tyrosines in the tail not already
known to be autophosphorylated; therefore, these two sites represent at
least two out of the three remaining tyrosines within the tail.
regulatory domain of PI3 kinase (Songyang et al.,
1993). Since Src and P85 have both been reported to bind to EGF-R
and none of the EGF-R autophosphorylation sites fit either of their
consensus binding sequences, these new sites may be responsible for
these interactions.
should
be created if these sites are phosphorylated in vivo.
Figure 4:
Phosphopeptide maps of autophosphorylated (A) or Src-phosphorylated (B)
ErbB2.
Src and P85
To assess the importance of the
nonautophosphorylation sites, the EGF-R was allowed to
autophosphorylate or was phosphorylated by Src (in the presence of CGP
46251 to block EGF-R autophosphorylation, ). The relative
binding affinities of Src and P85 Preferentially Bind to
Src-phosphorylated EGF-R to each of these forms of EGF-R
was then assayed (Fig. 5). This assay depended upon dilution of
equal quantities of proteins into different volumes to adjust the
concentrations. Thus, 5 pmol of EGF-R and an equal amount of Src or
P85 were diluted to 50 µl, 500 µl, and 5 ml to make
concentrations of 100, 10, and 1 nM, respectively. Because
this technique changes the concentration of both proteins
simultaneously, it is not amenable to Michaelis-Menten kinetics;
however, it effectively reveals differences in relative binding
affinities. Src bound to Src-phosphorylated EGF-R (1 nM) at
concentrations approximately 100-fold more dilute than it bound to
autophosphorylated EGF-R (100 nM). P85 had a similar
preference for Src-phosphorylated EGF-R (Fig. 5).
Figure 5:
The
relative affinity of full-length Src (A) or the amino-terminal
SH2 domain of P85 (B) for either autophosphorylated (A and B, lanes 1-3) or
Src-phosphorylated (A and B, lanes
4-6) EGF-R were examined.
Binding of
Src and P85 to EGF-R was competed for by addition of
phosphorylated synthetic peptides corresponding to phosphorylation
sites on EGF-R. The two novel sites (Tyr-891 and Tyr-920), as well as
the two minor autophosphorylation sites that appear to be enhanced in vivo (Tyr-992 and Tyr-1086), were used to try to compete
for Src and P85 binding. The same type of binding assay as in Fig. 5was done in the presence of increasing amounts of one of
the four peptides. P85 was preferentially competed away by the
Tyr-920 site (LPQPPICTIDVpYMIMVK), K = 35 µM, while the other peptides did
not inhibit binding up to 100 µM. Src was slightly
preferentially competed by Tyr-891 (PpYDGIPASEISSILEK), K
= 60 µM; however,
Tyr-992 (K
= 75 µM)
and Tyr-920 (K
= 80
µM) had similar competition kinetics. To test if Src and
P85
are associated with EGF-R in vivo, cells were
stimulated with EGF for 10 min or were left untreated, and then EGF-R
was immunoprecipitated. Western blots of the immune complexes with
monoclonal antibodies against Src or P85 revealed that they were
associated with EGF-R in MCF7 cells in three colorectal carcinoma cell
lines (DLD-1, WiDr, LS174T) and to a lesser extent in NIH 3T3 cells (Fig. 6A). In each case, the interactions were only
observed after treatment with EGF. In contrast, no Src or P85 were
observed in EGF-R immune complexes from A431 cells. Because EGF-R is
highly overexpressed in A431 cells, the proportion of EGF-R
phosphorylated by Src may be too small to allow detection of
associating proteins that are dependent on these non-autophosphorylated
sites. Alternatively, this mechanism may be regulated in a cell
type-specific manner.
Figure 6:
A, association of Src with EGF-R was
assessed in cells stimulated with EGF. EGF-R was immunoprecipitated
from the indicated cells with a monoclonal antibody specific for a
sequence within the cytoplasmic region of EGF-R (clone F4). The washed
immune complexes were analyzed for Src association by Western blot
using monoclonal antibody 327. B, similarly, the interaction
of P85 with EGF-R, in the same cells, was analyzed by washing the
polyvinylidene difluoride membranes from A in 10% acetic acid
and then reimmunostaining with anti-P85
antibody.
Binding of proteins containing enzymatic
domains to growth factor receptors by their SH2 domains has been
observed to effect the activity of several enzymes (Stover et
al., 1994; Lechleider et al., 1993; Moran et
al., 1991). Therefore, the activity of Src was assayed in the
presence of autophosphorylated or Src-phosphorylated EGF-R (Fig. 7). In this experiment, a selective EGF-R kinase inhibitor
was used to block autophosphorylation of the Src-phosphorylated EGF-R
and to block both autophosphorylated and Src-phosphorylated EGF-R from
interfering with the assay of Src activity. Neither phosphorylated
forms of the EGF-R had any impact on Src activity.
Figure 7:
Graph demonstrating activation of
Csk-inactivated Src in the presence of RPTKs after being either
autophosphorylated or phosphorylated by Src. The activities of
dephosphorylated Src and Csk-inactivated Src (pY527-Src) in the absence
of receptors (lanes 1 and 2, respectively) are shown
as references. The subsequent bars represent the activity of
Csk-inactivated Src in the presence of the indicated RPTK. Appropriate
RPTK inhibitors were included during the Src assays, so the
contribution by the RPTKs was negligible (data not shown). Enolase was
used as the substrate at a concentration of 1 mg/ml, and cpm
incorporated was measured by spotting 10 µl of each reaction on P81
paper, followed by washing and scintillation counting. It should be
noted that only approximately one-fifth of the total amount of Src was
found to be associated with the receptors in immune complexes under
similar conditions as those used here (see Fig. 1); therefore, the
amount of reactivation of the bound fraction of Src may be much greater
than that indicated.
We have recently
demonstrated that dephosphorylated Src is not affected by addition of
phosphopeptides that mimic the carboxyl-terminal regulatory tail of
Src, but Csk-inactivated Src (phosphorylated at Tyr-527) is activated
in the presence of such phosphotyrosine-containing peptides (Stover et al., 1994). Csk-inactivated Src was found to be activated
7-8-fold in the presence of Src-phosphorylated EGF-R, while it
was unaffected by autophosphorylated EGF-R (Fig. 7).
Figure 8:
Schematic diagram of EGF-R and its
EGF-induced phosphorylation sites. Five sites of autophosphorylation (Auto-P) within the carboxyl-terminal tail are indicated, as
are two sites that are found to be phosphorylated in DLD-1 cells.
Because these two sites are phosphorylated by Src but are not
autophosphorylated in vitro, they are labeled as Src sites.
Note that Src is capable of phosphorylating all of the indicated sites in vitro and increases the level of phosphorylation on the
minor autophosphorylated sites (Tyr-992 and Tyr-1086) to the level of
the major sites.
Wasilenko et al.(1991) have
demonstrated that in cells transformed by
pp60, the EGF-R is constitutively
phosphorylated at nonautophosphorylation sites. Furthermore,
phospholipase C-
is also constitutively phosphorylated on tyrosine
in these cells. Because phospholipase C-
is an in vivo substrate of the EGF-R kinase, the authors concluded that the
basal activity of EGF-R had been activated by
pp60
phosphorylation. However, in
our hands, phosphorylation of EGF-R by Src has no effect on EGF-R
activity in vitro (data not shown). Therefore, we suggest that
the constitutive phosphorylation of phospholipase C-
in
Src-transformed cells may be due to phosphorylation by Src itself, or
phospholipase C-
may be capable of binding to one of the sites of
phosphorylation by Src; thus, it becomes a substrate for EGF-R in the
absence of dimerization and autophosphorylation. Phospholipase C-
has been shown to specifically bind to pTyr-992, a minor
autophosphorylated site. In our hands, this site was a very minor site,
barely visible above background on an overexposed film (not shown).
However, after Src phosphorylation, this site was as highly
phosphorylated as the major sites of autophosphorylation, correlating
with our observations of EGF-R phosphorylation in vivo following EGF stimulation. These data suggest that phospholipase
C-
may actually bing to a predominantly non-autophosphorylated
site. Whether this site is actually phosphorylated by Src or another
kinase in vivo is still not certain.
have been found to bind to the EGF-R in previous reports, and our
results add further evidence that this is indeed the case in the cell
types examined in this study. However, none of the autophosphorylation
sites of EGF-R fit the consensus binding sites for either Src or the
P85 SH2 domains, and we observe only weak binding of these
proteins to the autophosphorylated EGF-R. It would appear likely that
it is more than an artifactual coincidence that, of the three
non-autophosphorylated sites observed to be phosphorylated in cells,
one is a text book example of a P85 binding site while another
matches the binding requirements of Src-SH2 quite well. Furthermore,
the sequences surrounding these two sites are far more conserved
between EGF-R and ErbB2 than are any of the major autophosphorylation
sites. Taken together, these observations make a strong argument for a
physiological role for Src phosphorylation of these tyrosine residues.
Table: Characteristics of PTK inhibitors used
values (concentration resulting in a 50% reduction in
phosphotransfer from ATP to substrate) of the compounds used in this
study toward the relevant kinases are tabulated below. The IC
values for EGF-R and Src were determined using exogenous
substrates as previously described (Buchdunger et al., 1994).
The IC
values for ErbB2 were estimated from inhibition of
autophosphorylation.
Table: EGF induced
tyrosine phosphorylations found on the EGF receptor
are tentatively indicated to bind to specific sites since they
only bind to Src-phosphorylated EGF-R, and only one of these sites fits
the binding specificity for each protein.
.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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M. F. Denning, A. A. Dlugosz, D. W. Threadgill, T. Magnuson, and S. H. Yuspa Activation of the Epidermal Growth Factor Receptor Signal Transduction Pathway Stimulates Tyrosine Phosphorylation of Protein Kinase C [IMAGE] J. Biol. Chem., March 8, 1996; 271(10): 5325 - 5331. [Abstract] [Full Text] [PDF]
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M. Jost, T. M. Huggett, C. Kari, L. H. Boise, and U. Rodeck Epidermal Growth Factor Receptor-dependent Control of Keratinocyte Survival and Bcl-xL Expression through a MEK-dependent Pathway J. Biol. Chem., February 23, 2001; 276(9): 6320 - 6326. [Abstract] [Full Text] [PDF]
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