From the Department of Microbiology and Cancer Center, Box 441, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
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ABSTRACT |
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Accumulating evidence indicates that interactions
between the epidermal growth factor receptor (EGFR) and the nonreceptor tyrosine kinase c-Src may contribute to an aggressive phenotype in
multiple human tumors. Previous work from our laboratory demonstrated that murine fibroblasts which overexpress both these tyrosine kinases
display synergistic increases in DNA synthesis, soft agar growth, and
tumor formation in nude mice, and increased phosphorylation of the
receptor substrates Shc and phospholipase The epidermal growth factor receptor
(EGFR)1 is a 170-kDa
single-pass transmembrane tyrosine kinase that undergoes homo- or heterodimerization and enzymatic activation following ligand binding (1, 2). These events result in the trans-(auto)-phosphorylation of
multiple Tyr residues in the COOH-terminal tail of the molecule that
serve as binding sites for cytosolic signaling proteins containing Src
homology 2 (SH2) domains (3). Five sites of in vivo
autophosphorylation have been identified in the EGFR: three major
(Tyr1068, Tyr1148, and Tyr1173) and
two minor (Tyr992 and Tyr1086) (4-7). These
sites bind a variety of downstream signaling proteins which contain SH2
domains, including Shc (8) and PLC c-Src is a nonreceptor tyrosine kinase that functions as a
co-transducer of transmembrane signals emanating from a variety of
polypeptide growth factor receptors, including the EGFR (see Refs. 10
and 11, and reviewed in Ref. 12). Overexpression of wild type (wt) and
dominant negative forms of c-Src in murine C3H10T1/2 fibroblasts that
express normal levels of receptor, as well as experiments involving the
microinjection of antibodies specific for Src family members, have
revealed that c-Src is a critical component of EGF-induced mitogenesis
(10, 11, 13). Cells which express high levels of EGFR become
transformed upon continual exposure to EGF (14), and co-overexpression
of c-Src in these cells dramatically potentiates their growth and
malignant properties (15). Together, these findings indicate that c-Src co-operates with the EGFR in the processes of both mitogenesis and transformation.
Subsequent studies in 10T1/2 cells revealed that potentiation of
EGF-induced growth and tumorigenesis by c-Src, which is observed only
in cells overexpressing both c-Src and the receptor, correlates with
the EGF-dependent formation of a heterocomplex containing c-Src and activated EGFR, the appearance of two unique in
vitro non-autophosphorylation sites on receptors in complex with
c-Src, and enhanced in vivo tyrosyl phosphorylation of the
receptor substrates, PLC Cell Lines--
The derivation and characterization of the
clonal C3H10T1/2 murine fibroblast cell lines used in this study, Neo
(control), 5H (c-Src overexpressor), NeoR1 (human EGFR overexpressor),
and 5HR11 (c-Src/EGFR double overexpressor) have been described
previously (10, 11, 13). 5H and 5HR11 express equal levels of c-Src (~25-fold over endogenous), and NeoR and 5HR11 express nearly equal
levels of cell surface receptors (~2 × 105
receptors/cell or ~40-fold over endogenous). Cells were maintained in
Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.,
Gaithersburg, MD), containing 10% fetal calf serum, antibiotics, and
G418 (400 µg/ml). When indicated, confluent cultures were starved of
serum overnight, prior to stimulation with 100 ng/ml purified mouse EGF (Sigma).
To create cells transiently overexpressing HER1 which contained a
Tyr to Phe mutation at position 845, a
DraIII-BstEII fragment from pCO11 (gift of Laura
Beguinot), including the mutation at position 845, was subcloned into a
pcDNA vector containing wild type human HER1 (gift of Dr. Stuart
Decker, Parke Davis, Ann Arbor, MI). Neo control 10T1/2 fibroblasts
were transiently transfected with 30 µg of Superfect (Qiagen,
Chatsworth CA) and 4 µg of vector, wt HER1, or Y845F HER1 plasmid DNA
according to the manufacturers' directions and incubated for 48 h.
For overexpression of c-Src in breast cancer cells, pcDNAc-Src was
constructed by inserting the c-Src XhoI fragment from an existing pVZneo vector into the multicloning site of pcDNA3
(Invitrogen, San Diego, CA). MDA468 cells, obtained from N. Rosen
(Sloan Kettering Cancer Center, New York), were maintained in DMEM plus
5% serum. MDA468 cells stably overexpressing chicken c-Src (clone
MDA468c-Src) were generated by LipofectinTM (Life
Technologies, Inc.)-mediated gene transfer of pcDNAc-Src into
parental MDA468 cells and selection with 400 µg/ml G418. Parental
MDA468 cells overexpress c-Src approximately 5-fold, as compared with
Hs578Bst normal breast epithelial cells, and contain approximately
106 receptors/cell (Ref.
16),2 while MDA468c-Src cells
overexpress c-Src approximately 25-fold over levels found in normal
breast epithelial cells.
Antibodies--
EGFR-specific mouse monoclonal antibodies (mAbs)
3A and 4A were provided by D. McCarley and R. Schatzman of Syntex
Research, Palo Alto, CA. Their derivation has been described previously and their epitopes have been mapped to residues 889-944 and
1052-1134, respectively. EGFR-specific mAb F4, directed against amino
acids 985-996, was obtained from Sigma. GD11 antibody is directed
against the SH3 domain of c-Src and was characterized previously in our laboratory (17, 18). Q9 antibody was raised in rabbits against the
COOH-terminal peptide of c-Src (residues 522-533) and exhibits a
higher affinity for c-Src than for other Src family members (19, 20).
Antiphosphotyrosine (Tyr(P)) antibody (4G10) was purchased from UBI
(Lake Placid, NY). Negative control antibodies included pooled and
purified normal rabbit or mouse immunoglobulin.
Immunoprecipitation, Western Immunoblotting, and in Vitro Kinase
Assays--
Methods for immunoprecipitation, Western immunoblotting,
and in vitro kinase assays have been described previously
(10, 11, 15). Cells were lysed either in CHAPS detergent buffer (10 mM CHAPS, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM sodium
orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptin, and 0.5% aprotinin), or in RIPA detergent buffer
(0.25% sodium deoxycholate, 1% Nonidet P-40, 50 mM
Tris-HCl, pH 7.2, 150 mM NaCl, 2 mM EDTA, 1 mM sodium orthovanadate, 50 µg/ml leupeptin, and 0.5%
aprotinin). Protein concentrations of detergent lysates were determined
by the BCA protein assay (Pierce, Rockford, IL). 500 µg of cell
lysate was used for immunoprecipitations, and 50 µg was used for
Western blotting. For Western immunoblotting, binding of primary murine
or rabbit antibodies to Immobilon membranes was detected with either
125I-labeled goat anti-mouse IgG (NEN Life Science Products
Inc.) or 125I-protein A (NEN) used at 1 µCi/ml, specific
activity 100 µCi/ml. For kinase assays, immunoprecipitates were
prepared in and washed twice in CHAPS buffer, then washed twice
with HBS buffer (150 mM NaCl, 20 mM HEPES, pH
7.4). Each kinase reaction was conducted in 20-µl volumes containing
20 mM PIPES, pH 7.5, 10 mM MnCl2, and 10 µCi of [ In Vitro Binding and Far Western Analysis Using
GSTc-SrcSH2--
The construction and preparation of GST fusion
proteins containing the SH2 domain of c-Src was described previously
(21). To reconstitute binding between tyrosyl-phosphorylated EGFR and the SH2 domain of c-Src, 2 µg of immobilized GST-c-SrcSH2 fusion protein was incubated with 100 µg of 10T1/2 cell lysate protein prepared in RIPA buffer. After 3 h gentle mixing at 4 °C, beads were washed three times with RIPA buffer, resuspended in SDS sample buffer, and boiled. Eluted proteins were separated by SDS-PAGE, transferred to Immobilon, and immunoblotted with either the Tyr(P) or
the 3A/4A or F4 (Sigma) monoclonal EGFR antibodies.
To assess direct binding of GST-c-SrcSH2 to the EGFR, receptor
from 500 µg of cell lysate protein in RIPA buffer was
immunoprecipitated with 3A/4A mAbs. The resulting EGFR
immunoprecipitates were resolved by SDS-PAGE, transferred to Immobilon
membranes, and incubated with 1 mg/ml purified GST-c-SrcSH2 fusion
protein in blocking buffer at 4 °C overnight. The membrane was then
probed with 1 µg/ml affinity purified, polyclonal rabbit anti-GST
antiserum in blocking
buffer,3 and immunoglobulin
binding was detected by 125I-protein A.
Metabolic Labeling--
NeoR1 or 5HR11 cells were grown to
50-75% confluency in 150-mm dishes, washed with phosphate-free DMEM,
and incubated for 18 h in phosphate-free DMEM containing 0.1%
dialyzed fetal bovine serum and 1 mCi/ml
[32P]orthophosphate (NEN Life Science Products Inc.) in a
final volume of 10 ml. For pervanadate treatment, labeling medium was
adjusted to a concentration of 3 mM
H2O2 and 5 µM
Na3VO4 just prior to EGF stimulation. Cells
were stimulated in the presence of pervanadate by addition of 100 ng/ml
EGF to the labeling medium for 5 min, washed twice with phosphate-free
DMEM, and lysed in CHAPS detergent buffer. Extract from an entire plate
(approximately 1-2 mg of protein) was immunoprecipitated with c-Src or
EGFR-specific antibodies as described above.
Two-dimensional Tryptic Phosphopeptide
Analysis--
Immunoprecipitates of in vitro or in
vivo 32P-labeled EGFR were resolved by SDS-PAGE. The
EGFR was localized by autoradiography, excised from the gel, and
digested with trypsin as described by Boyle et al. (22).
Phosphotryptic peptides were separated by electrophoresis at pH 1.9 in
the first dimension and ascending chromatography in the second
dimension on cellulose thin layer chromatography (TLC) plates.
Chromatography buffer contained isobutyric acid, 1-butanol, pyridine,
acetic acid, H2O (125:3.8:9.6:5.8:55.8). Migration of
synthetic phosphopeptides was detected by spraying the dried TLC plate
with a hypochlorite solution consisting of sequential sprays with 10%
commercial Clorox, 95% ethanol, 1% potassium iodide, and saturated
o-tolidine in 1.5 M acetic acid, as described in
Stewart and Young (23).
High Performance Liquid Chromatography (HPLC)--
For HPLC
analysis of peptides derived from the EGFR associated with c-Src,
32P-labeled phosphotryptic peptides were prepared as above
and suspended in 0.05% trifluoroacetic acid. Peptides were injected
into a Perkin-Elmer Series 4 Liquid Chromatograph equipped with a Vydac
C18 column (4.6 × 250 mm) and eluted with increasing
concentrations of acetonitrile (0 to 100%) at a flow rate of 1 ml/min,
as described by Wasilenko et al. (24). 500-µl fractions
were collected, and Cerenkov counts of each fraction were determined.
Fractions containing peptides "0" and "3" were identified by
two-dimensional TLC analysis for their ability to co-migrate with the
appropriate peptide in a mixture of total in vitro
phosphorylated receptor peptides. Appropriate fractions were then
lyophilized and subjected to Edman degradation.
Edman Degradation--
HPLC fractions of 32P-labeled
EGFR phosphotryptic peptides or spots eluted from TLC plates were
subjected to automated Edman degradation, as performed by the
University of Virginia Biomolecular Research Facility. Briefly,
phosphorylated peptides were coupled to a Sequelon aryl amine membrane
(25), washed with 4 × 1 ml of 27% acetonitrile, 9%
trifluoroacetic acid, and 2 × 1 ml of 50% methanol, and
transferred to an applied Biosystems 470A sequenator using the
cartridge inverted as suggested by Stokoe et al. (26). The
cycle used for sequencing was based on that of Meyer et al. (27), but modified by direct collection of anilinothiazolinone amino
acids in neet trifluoroacetic acid as described by Russo et
al. (28). Radioactivity was measured by Cerenkov counting.
Identification of Peptides 0 and 3--
Phosphorylated peptides
(corresponding to residues GMN(Y-P)LEDR, candidate for peptide 3; or
E(Y-P)HAEGGK, candidate for peptide 0) were synthesized by the
University of Virginia Biomolecular Research Facility. Synthetic
peptides were mixed with oxidized in vitro labeled
phosphotryptic peptides from c-Src-associated EGFR, separated on
cellulose TLC plates, and visualized by spraying with the hypochlorite
solution as described above. One candidate for peptide 3 (GMNYLEDR) was
synthesized as a phosphopeptide and tested for comigration as above.
Another candidate for peptide 3 (DPHY1101QDPHSTAVGNPEYLNTVQPTCVNSTF DSPAHWAQK), which was
too large to chemically synthesize, was tested by further digestion of
in vitro labeled peptide 3 with a proline-directed protease
(Seikagaku, Rockville, MD), according to the method of Boyle et
al. (22). In brief, the spot corresponding to peptide 3 was
scraped off the TLC plate, eluted with pH 1.9 buffer, and digested with
5 units of proline-directed protease in 50 mM ammonium
bicarbonate at pH 7.6 at 37 °C for 1 h. Peptides were separated
by two-dimensional electrophoresis as described above.
BrdUrd Incorporation--
Neo control cells, which had been
transfected with cDNAs encoding wild type EGFR, Y845F EGFR, or
vector alone were cultured for 48 h, then serum starved for an
additional 30 h prior to the administration of 100 µM BrdUrd and either 100 ng/ml EGF or 10% fetal calf
serum in fresh growth medium. Treated cells were incubated for 18 h and co-stained for HER1 expression and BrdUrd incorporation as
described by the manufacturer of the BrdUrd-specific antibody (Boehringer Mannheim). Briefly, fixed cells were treated with 2 N HCl for 1 h at 37 °C and incubated with a mixture
of primary antibodies (1:100 dilution of the HER1-specific Ab-4, and a
1:15 dilution of anti-BrdUrd mouse antibody in serum-free medium for 1 h at 37 °C), followed by incubation with a mixture of
secondary antibodies (75 µg/ml fluorescein isothiocyanate-conjugated
goat anti-rabbit IgG and 4 µg/ml Texas Red-conjugated goat anti-mouse IgG) for 1 h at 37 °C. Both secondary reagents were obtained
from Jackson Immunoresearch Laboratories, West Grove, PA.
Direct Binding of c-Src SH2 Domain to the EGFR--
Previous work
from our laboratory demonstrated a synergistic interaction between
c-Src and the EGFR which led to increased cell growth and tumor
development (10, 11, 15). This functional synergism was most striking
when cells overexpressed both c-Src and the EGFR (5HR cells) and
correlated with the ability of c-Src and the EGFR to form specific,
EGF-dependent heterocomplexes in vivo. The
formation of this c-Src·EGFR complex raises the question of whether
binding between c-Src and the EGFR occurs directly, or is mediated by
another protein present in the complex. To test whether association
could be mediated by a Tyr(P)-SH2 interaction, lysates from
unstimulated and stimulated Neo, 5H, NeoR, or 5HR cells were incubated
with a GST-c-SrcSH2 bacterial fusion protein linked to agarose beads,
and precipitated proteins were probed with Tyr(P) antibody. Fig.
1, panel A, lanes 4 and 8, show that a tyrosyl-phosphorylated protein of 170 kDa
was precipitated by GST-c-SrcSH2 from extracts of cells overexpressing
the EGFR after activation of the receptor with EGF. This 170-kDa
protein co-migrated with the EGFR precipitated with receptor-specific
mAbs 3A/4A (data not shown). Other proteins that bound c-SrcSH2
included p125FAK (21), which was detected in all the cell
lysates, a 75-80-kDa protein, cortactin, which was most prominent in
5H cells (30), and a 62-kDa protein, presumed to be related to the
62-kDa "DOK" protein associated with p120Ras-GAP
(31-34). These results suggest that in vivo, multiple
Tyr(P)-containing proteins in addition to the EGFR are capable of
interacting with c-Src via its SH2 domain and contribute to the highly
tumorigenic phenotype of the double overexpressing cells. Incubation of
cell extracts with GST-beads alone resulted in no detectable binding of
Tyr(P)-containing proteins (data not shown).
To confirm that the 170-kDa protein was indeed the EGFR, lysates
prepared from unstimulated and stimulated NeoR and 5HR cells were
precipitated with immobilized GST-c-SrcSH2, and bound proteins were
immunoblotted with EGFR-specific mAbs 3A/4A. Fig. 1, Panel B, demonstrates that receptor antibody detected the 170-kDa
protein only in stimulated cells, as in Panel A, confirming
its identity as the EGFR. To test if the interaction between the
activated EGFR and c-SrcSH2 could be direct, receptor
immunoprecipitates were subjected to a "Far Western" overlay
experiment, using GST-c-SrcSH2, GST-specific antibody, and
125I-protein A. Fig. 1, Panel C, lanes 2 and
4, shows that GST-c-SrcSH2 bound the EGFR and, as predicted,
the interaction required activation by EGF. GST alone exhibited no
binding (data not shown). Panel D verified that nearly equal
amounts of receptor were present in all immunoprecipitates. These
results provide evidence for the involvement of SH2-Tyr(P) interactions
in the formation of the EGFR·c-Src complex.
In Vivo and in Vitro Phosphorylation of Novel,
Non-autophosphorylation Sites on the EGFR in Complex with
c-Src--
Overexpression of both EGFR and c-Src in 10T1/2 cells
results in increased tyrosyl phosphorylation of receptor substrates, PLC
Initial attempts to detect peptides 0 and 3 in receptor
immunoprecipitations from 32P-labeled NeoR or 5HR cells
yielded phosphopeptide maps that contained peptide 3 but no or barely
detectable levels of peptide 0 (Fig. 3,
Panels A and C). Neither could peptide 3 nor
peptide 0 be detected reproducibly in receptor that was associated with
c-Src from 5HR cells (data not shown). Furthermore, in receptor
immunoprecipitations, the levels of peptide 3 derived from NeoR
versus 5HR cells appeared nearly equal (compare Panels
A and C), suggesting that peptide 3 may not be an
in vivo, c-Src-dependent site of
phosphorylation. In these experiments, lysates were prepared in CHAPS
buffer containing a mixture of conventional protease and phosphatase
inhibitors, including orthovanadate (see "Materials and Methods").
However, modification of the EGF treatment regimen to include
pervanadate during stimulation allowed us to detect peptide 0 in
receptor immunoprecipitates from NeoR (Panel B) and 5HR
(Panel D) cells. These conditions revealed more peptide 0 in
receptor from 5HR than from NeoR cells, confirming the ability of c-Src
to modulate the phosphorylation of this peptide. Of special note was
the finding that peptide 0 was the only peptide seen to increase in
phosphorylation in response to pervanadate treatment, suggesting that
its phosphorylation is more labile than that of peptide 3 or the other
phosphorylations on the receptor, which presumably correspond to
autophosphorylation sites. Together with the in vitro
studies depicted in Fig. 2, the results from the in vivo
experiments indicate that peptide 0 is an in vitro and
in vivo site of receptor phosphorylation that is regulatable
by c-Src. Following this line of reasoning, the low level of peptide 0 phosphorylation seen in receptor immunoprecipitates from NeoR cells
(Fig. 3, Panel B) could be due to endogenous c-Src. However,
the involvement of other tyrosine kinases in the in vivo phosphorylation of peptide 0 cannot be ruled out.
Whether c-Src alone plays a role in regulating the phosphorylation of
peptide 3 in vivo is less clear. In vitro,
peptide 3 phosphorylation appears to be unique to the receptor
associated with c-Src (compare Panels A and B of
Fig. 2), and HPLC analysis corroborates this, where phosphorylation of
the peak corresponding to peptide 3 was found to be ~3.5-fold greater
when the receptor was associated with c-Src versus free
receptor (data not shown). Furthermore, the level of in vivo
phosphorylation of peptide 3 in the c-Src-associated receptor is
greater than that found in the "free" receptor (compare Fig. 2,
Panel C, with Fig. 3, Panel D). However, peptide
3 is readily detected in free receptor labeled in vivo, and
its level of phosphorylation does not appear to increase to any great
extent in 5HR versus NeoR cells (Fig. 3, Panels B and D). These data can be interpreted to mean either that
peptide 3 contains a non-labile site of phosphorylation, regulatable by c-Src (in contrast to peptide 0), or that phosphorylation of peptide 3 may be regulated by an additional tyrosine kinase in
vivo.
To identify the amino acids phosphorylated in vitro in a
c-Src-dependent manner, fractions containing peptides 0 and
3 were isolated by HPLC. Peptide 0 eluted at 8.5% acetonitrile, while peptide 3 eluted at 10.5% acetonitrile (not shown). These HPLC fractions, which were of greater than 95% purity, were subjected to
sequential Edman degradation to determine the cycle number at which
radioactivity was released. Results from these analyses indicated that
a phosphoamino acid residue was located at the second position of
peptide 0 (Fig. 4, Panel A)
and at the fourth position of peptide 3 (Fig. 4, Panel B).
Of the tryptic peptides generated from the intracellular domain of the
EGFR which contain Tyr residues, those peptides containing
Tyr845, Tyr867, or Tyr891 were
potential candidates for peptide 0, while those peptides containing
Tyr803 or Tyr1101 were potential candidates for
peptide 3 (see Table I).
The Tyr845-containing peptide was selected for further
study as a candidate for peptide 0, since it showed 50% homology to
sequences contained within the autophosphorylation site of Src
(Tyr416), indicating that it could be a potential c-Src
target. The octamer composed of E(P-Y845)HAEGGK (peptide 0)
was chemically synthesized to include a phosphorylated Tyr845 and analyzed either alone (Fig.
5, Panel A) or in a mixture
with total peptides from in vitro labeled, c-Src-associated
receptor by two-dimensional TLC (Panel C). The synthetic
octamer comigrated with peptide 0 in the mixture, thereby identifying
Tyr845 as the phosphorylated residue in peptide 0.
Since peptides 0 and 3 migrated similarly in the two-dimensional
chromatography, it was expected that they would share similar isoelectric points and hydrophobicities. Both candidates for
peptide 3 (GMNY803LEDR or DPHY1101
QDPHSTAVGNPEYLNTVQPTCVNSTFDSPAHWAQK, see Table I) had theoretical isoelectric points and calculated hydrophobic indices (22) similar to
those of the Tyr845-containing peptide, indicating that
both were potential candidates. The Tyr803-containing
peptide was selected first for further study, since it was smaller and
more easily synthesized. However, this synthetic phosphopeptide did not
co-migrate with peptide 3 nor with any of the other EGFR
phosphopeptides (data not shown), indicating that the
Tyr1101-containing peptide was the preferred candidate. To
verify the identity of peptide 3, in vitro labeled peptide 3 was scraped off the TLC plate, eluted with pH 1.9 buffer, and subjected
to further digestion with a proline-directed protease as described under "Materials and Methods." Since, of the two candidate
peptides, only the Tyr1101-containing peptide contains
proline residues, any change in mobility resulting from digestion with
this protease would confirm its identity as peptide 3. As a control,
peptide 0, which does not contain any proline residues, was digested
with proline-directed protease and no change in mobility was observed
(data not shown). Fig. 6 shows that
digestion of spot 3 with the proline-directed protease resulted in a
change of migration primarily in the first dimension (compare
Panel A with Panel B). To confirm that a mobility shift was indeed occurring, digested and undigested peptide 3 were
mixed (Panel C). The results identify peptide 3 as
Tyr1101.
Phosphorylation of Tyr845 and Tyr1101 in
HER1 from Breast Tumor Cells--
Our laboratory has previously
demonstrated the presence of EGF- dependent c-Src·EGFR
heterocomplexes in several human breast tumor cell lines including
MDA468, which overexpresses both c-Src and HER1 (16). Since the
presence of this heterocomplex is correlated with general increases in
downstream receptor-mediated signaling and tumorigenicity in these
cells, as compared with cell lines which do not overexpress the EGFR,
we wished to investigate whether Tyr845 and/or
Tyr1101 were phosphorylated in c-Src-associated EGFR
derived from breast tumor cells. Fig. 7
demonstrates that phosphopeptides 0 and 3 are both present in in
vitro labeled, c-Src-associated EGFR from EGF-stimulated MDA468
cells, although peptide 0 is weakly detected in the absence of
pervanadate treatment. To further investigate the role of c-Src in
mediating the phosphorylation of these sites, an MDA468 derivative cell
line which stably overexpresses c-Src approximately 25-fold over levels
in normal breast epithelial cells (MDA468c-Src cells, Panel
B) was created. In these cells, the phosphorylation of peptide 0 (Tyr845) was greatly enhanced, while the phosphorylation of
peptide 3 (Tyr1101) was unchanged (Panel C).
Role of Tyr845 in EGF-dependent
Mitogenesis--
A tyrosyl residue homologous to Tyr845 is
conserved in many other receptor tyrosine kinases, and mutation of
these conserved tyrosines to phenylalanine results in a reduced ability
of the receptors to signal downstream events (35-37). Thus, it is
possible that mutation of Tyr845 to phenylalanine would
likewise decrease EGF-dependent signaling through the EGFR.
To directly test the requirement of Tyr845 phosphorylation
for receptor function, a variant receptor bearing a Y845F mutation was
transiently transfected into Neo cells, and the effects on DNA
synthesis were assayed by measuring bromodeoxyuridine (BrdUrd) incorporation in response to EGF (Fig.
8). The level of BrdUrd incorporation in
cells expressing the Y845F mutant EGFR was reduced to approximately
30% of that induced by the wild type receptor, indicating that the
mutant EGFR could interfere with the function of endogenous receptor
and was thus acting in a dominant negative manner. Similar results were
obtained when Y845F receptor was expressed in cells which overexpress
c-Src (38). These findings suggest that phosphorylation of
Tyr845 is necessary for the mitogenic function of the
receptor.
Previous studies from our laboratory using the C3H10T1/2 murine
fibroblast model demonstrated that simultaneous overexpression of c-Src
and EGFR potentiates EGF-dependent mitogenesis,
transformation, and tumorigenesis, as well as EGF-dependent
association of c-Src with the receptor and increases in tyrosyl
phosphorylation of the receptor substrates Shc and PLC Here we identify these c-Src dependent sites as Tyr845 and
Tyr1101 and demonstrate that they become phosphorylated in
murine fibroblasts both in vitro and in vivo in
c-Src/EGFR double overexpressing cells in an EGF-dependent
manner. Enhanced phosphorylation of Tyr845 was also
observed in MDA468 human breast cancer cells when c-Src was
overexpressed, indicating that such phosphorylations can occur in cells
of both mesodermal and epithelial origin. More importantly, the fact
that cells expressing a Y845F variant of the EGFR are impaired in their
ability to synthesize DNA in response to EGF treatment provides direct
evidence for the importance of this phosphorylation. Together, these
findings support the hypothesis that the c-Src-mediated phosphorylation
of Tyr845 is a critical event for EGFR function, and
in certain situations where overexpression of these molecules exists
(such as in certain breast tumors), the increased receptor signaling
resulting from this phosphorylation could lead to enhanced tumorigenesis.
Tyr845 resides in an intriguing position on the receptor,
namely in the activation lip of the kinase domain (39, 40). Amino acid
sequences in this lip are highly conserved among tyrosine kinases (41).
Crystallographic studies indicate that phosphorylation of
Tyr845 homologues stabilizes the activation lip, maintains
the enzyme in an active state, and provides a binding surface for
substrate proteins; while mutation of these sites in their respective
receptors results in decreases in cell growth and transformation (37, 40-43). A similar situation appears to exist for the EGFR, as cells expressing the Y845F variant receptor showed decreases in their ability
to respond mitogenically to EGF. This impairment of DNA synthesis
occurred both in a background of endogenous levels of c-Src, as shown
here, as well as in cells where c-Src was overexpressed (38). This
finding argues that endogenous levels of c-Src are capable of mediating
the phosphorylation of Tyr845 and that the Y845F form of
the receptor acts in a dominant negative fashion. Which downstream
targets of the receptor are affected in various cell types by the Y845F
mutation is not known. Other studies from our laboratory demonstrate
that EGF-induced increases in Shc and mitogen-activated protein kinase
tyrosyl phosphorylation occur normally when the Y845F receptor is
transiently co-expressed in COS cells (38). This finding suggests that
a mitogen-activated protein kinase-independent pathway plays a
more dominant role in mitogenic signaling emanating from the receptor
when it is phosphorylated on Tyr845.
That phosphorylation of this Tyr845 residue may regulate
receptor activity is consistent with the observation that a
Tyr845 homologue is not found in the EGFR family member
erbB3/HER3, which is known to lack kinase activity (44). However,
unlike the situation resulting from mutation of the analogous site in other receptor tyrosine kinases, mutation of Tyr845 does
not appear to alter the EGF receptor's ability to autophosphorylate or
to phosphorylate the downstream substrate, Shc (38). In many tyrosine
kinases, including Src, JAK 2, and receptors for colony stimulating
factor-1, platelet-derived growth factor, insulin, and fibroblast
growth factor, the Tyr845 homologue is an
autophosphorylated residue (35, 36, 45-48). However, to date
Tyr845 has not been identified as an autophosphorylation
site for the EGF receptor. This could be due to the highly labile
nature of the phosphorylation and/or to the fact that c-Src appears to
regulate its phosphorylation (see Figs. 2, 3, and 7). Together these
findings raise a number of questions: namely, whether c-Src
phosphorylates Tyr845 directly, whether binding of c-Src to
the receptor causes the receptor to phosphorylate itself, or whether
another tyrosine kinase which mediates the phosphorylation is recruited
into the complex or activated by c-Src.
Several pieces of evidence support the hypothesis that c-Src
phosphorylates the receptor directly. First, Tyr845 is
homologous to Tyr416 in Src, which is an
autophosphorylation site for Src (39). Additional evidence comes from
our studies with both 10T1/2 murine fibroblasts and MDA468 breast
cancer cells overexpressing c-Src, where an enhanced phosphorylation of
Tyr845 is observed. Moreover, other studies from our
laboratory demonstrate that overexpression of a kinase inactive form of
c-Src in 10T1/2 cells or in MDA468 cells results in a striking decrease
in Tyr845 phosphorylation
(38).4 These latter findings
indicate that c-Src kinase activity is necessary for the
phosphorylation of Tyr845 and strongly argue that
Tyr845 is a direct substrate of c-Src. Last, in
vitro affinity precipitation and Far Western analyses (Fig. 1,
this report, and Refs. 29, 49, and 50) demonstrate that the c-Src SH2
domain can bind activated EGFR specifically and directly, suggesting
that recruitment of other tyrosine kinases is not necessary to mediate
the phosphorylation of Tyr845. However, other EGFR family
members (including HER2/neu) (2, 51, 52) and several
cytosolic tyrosine kinases, such as other c-Src family members (13) and
JAK kinases (53, 54), have been reported to be involved in
receptor-mediated signaling, and we cannot exclude their possible
involvement in phosphorylation of Tyr845 or of
Tyr1101. Whether simple binding of c-Src induces a
conformational change in the receptor so that it can autophosphorylate
is a much more difficult question to address, a question that minimally
awaits identification of the c-Src-binding site.
Other investigators have also described Src-mediated phosphorylations
on the EGFR, and Wasilenko et al. (24) demonstrated that in
NIH3T3 cells co-expressing the transforming oncoprotein v-Src along
with EGFR, the receptor contained several novel sites of tyrosine
phosphorylation, one of which they postulated might be
Tyr845 (SPY1). Sato et al. (55) provide
additional evidence for phosphorylation of Tyr845 in A431
cells in a c-Src-dependent fashion, while Stover et
al. (56) showed that Tyr891 and Tyr920
were phosphorylated in the c-Src-associated EGFR derived from MCF7
cells. However, neither we nor Sato et al. (55) have been able to detect phosphorylation of Tyr891 or
Tyr920, and none of these reports have linked the various
phosphorylations to biological changes in receptor activity
(e.g. mitogenesis, tumorigenesis). Thus, while there is some
discrepancy among the different cell systems, our data and those of
others indicate that Tyr845 is a major
c-Src-dependent phosphorylation site on the EGFR, and that
it is associated with increases in receptor function. These findings
suggest that multiple tyrosine phopshorylations may be regulated by
c-Src.
A potential role for Tyr1101 is more unclear, as this
residue is not conserved among EGFR family members and its
phosphorylation level in vivo is not as noticeably altered
upon c-Src overexpression as is that of Tyr845 (see Fig.
3). However, Tyr1101 may function as a docking site for
novel or known signaling proteins, perhaps in an
SH2-dependent manner similar to that of the other autophosphorylation sites in the COOH terminus. One of the candidate binding proteins is c-Src itself. In peptide inhibition experiments using synthetic peptides to inhibit the binding between the EGFR and
the SH2 domain of c-Src, the SH2 domain of c-Src was shown to bind
Tyr992 (49, 55) and Tyr1101 (50)
preferentially. Thus, c-Src could bind one of these sites, which could
position it to phosphorylate Tyr845. In MDA468 breast
cancer cells, Tyr845 appeared to be the site most affected
by c-Src. While the data from the 10T1/2 system suggests that the
phosphorylation of both Tyr1101 and Tyr845 is
dependent on c-Src, it may be that the phosphorylation of each peptide
turns over at different rates in different cell types. Also, the
endogenous levels of c-Src in the parental MDA468 cells may be capable
of phosphorylating Tyr1101 to a maximal extent, and no
further phosphorylation could result from overexpression. In this
regard, overexpression of c-Src may allow for maximal phosphorylation
of Tyr845 if this phosphorylation turns over at a faster
rate, which appears to be the case as the results from Fig. 4 indicate.
Our data show that phosphorylation on Tyr845 appears to be
critical for EGFR-mediated mitogenesis. Moreover, our results (Figs. 3
and 7) suggest that basal levels of c-Src are able to mediate phosphorylation of Tyr845 to some extent, and that this
phosphorylation is important to receptor function. In a cell where
overexpression and/or activation of c-Src has occurred, as is found in
breast cancer, the proper negative regulation of this phosphorylation
may be lost, resulting in the increased EGF-dependent
signaling and tumorigenicity. We speculate that c-Src and EGFR act
synergistically (via phosphorylation of the receptor by c-Src) to
induce enhanced signaling in cells which overexpress both these kinases.
as compared with single
overexpressors. These parameters correlated with the ability of c-Src
and EGFR to form an EGF-dependent heterocomplex in
vivo. Here we provide evidence that association between c-Src and
EGFR can occur directly, as shown by receptor overlay experiments, and
that it results in the appearance of two novel tyrosine
phosphorylations on the receptor that are seen both in
vitro and in vivo following EGF stimulation. Edman
degradation analyses and co-migration of synthetic peptides with
EGFR-derived tryptic phosphopeptides identify these sites as
Tyr845 and Tyr1101. Tyr1101 lies
within the carboxyl-terminal region of the EGFR among sites of receptor
autophosphorylation, while Tyr845 resides in the catalytic
domain, in a position analogous to Tyr416 of c-Src.
Phosphorylation of Tyr416 and homologous residues in other
tyrosine kinase receptors has been shown to be required for or to
increase catalytic activity, suggesting that c-Src can influence EGFR
activity by mediating phosphorylation of Tyr845. Indeed,
EGF-induced phosphorylation of Tyr845 was increased in
MDA468 human breast cancer cells engineered to overexpress c-Src as
compared with parental MDA 468 cells. Furthermore, transient expression
of a Y845F variant EGFR in murine fibroblasts resulted in an ablation
of EGF-induced DNA synthesis to nonstimulated levels. Together, these
data support the hypothesis that c-Src-mediated phosphorylation of EGFR
Tyr845 is involved in regulation of receptor function, as
well as in tumor progression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
(9). Binding of these or other
signaling proteins to the receptor and/or their phosphorylation results
in transmission of subsequent signaling events that culminate in DNA
synthesis and cell division.
and Shc (15). These findings suggested that
c-Src-dependent phosphorylations on the EGFR can result in
hyperactivation of receptor kinase activity, as measured by the
enhanced ability of the receptor to phosphorylate its cognate
substrates. This report identifies Tyr845 and
Tyr1101 as c-Src-dependent sites of
phosphorylation, which are present both in vitro and
in vivo in receptor from 10T1/2 double overexpressing fibroblasts and from MDA468 human breast cancer cells. In the MDA468
cells, overexpression of c-Src results in a further increase in the
phosphorylation of Tyr845, indicating that c-Src either
phosphorylates this site directly or activates a secondary kinase which
is responsible. Moreover, cells which transiently express EGFR bearing
a Tyr to Phe mutation at Tyr845 are impaired in their
ability to synthesize DNA in response to EGF, suggesting that this
c-Src mediated phosphorylation site is important for receptor function.
MATERIALS AND METHODS
-32P]ATP (6000 Ci/mmol, NEN) for 10 min at room temperature. Incubations were terminated by addition of
sample buffer, and labeled products were resolved by SDS-PAGE and
visualized by autoradiography.
RESULTS
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Fig. 1.
In vitro association between
activated EGFR and c-Src SH2 domain. 500 µg of lysate protein
from the indicated nonstimulated cells or cells stimulated with 100 ng/ml EGF for 2 min was incubated with GST-c-SrcSH2 fusion protein
immobilized on glutathione-agarose beads (Panels A and
B) or EGFR mAbs 3A/4A bound to Protein A (Panels
C and D), as described under "Materials and
Methods." Affinity-precipitated proteins were washed and subjected to
SDS-PAGE, transferred to Immobilon membranes, and probed with:
A, Tyr(P) mAb 4G10; B, EGFR mAbs 3A/4A;
C, GST-c-Src SH2 fusion protein; and D, EGFR mAb
3A/4A. Binding of primary antibody was visualized by incubating
membranes with 125I-labeled goat anti-mouse IgG
(Panels A, B, and D), and binding of GST-c-SrcSH2
fusion protein was detected by rabbit anti-GST and
125I-protein A (Panel C). GST-c-SrcSH2 fusion
protein is shown to bind directly to activated EGFR.
and Shc, following EGF treatment (15). These findings suggest that the c-Src-associated receptor is modified in some manner as to
increase its kinase activity. To examine the receptor for novel
phosphorylations, the in vitro phosphorylated,
c-Src-associated 170-kDa protein was excised from the gel and subjected
to two-dimensional phosphotryptic peptide analysis. The phosphopeptide
map of c-Src-associated receptor was then compared with the map of the
free receptor, immunoprecipitated with receptor antibody. Fig.
2, Panels A and B,
demonstrate that the maps are nearly identical; however, two additional
phosphorylations (designated peptides 0 and 3) were seen in the map of
the EGFR complexed with c-Src, suggesting that c-Src was responsible
for their phosphorylation. Consistent with this notion, two-dimensional
phosphoamino acid analysis of the in vitro labeled EGFR
demonstrated that peptides 0 and 3 contained only phosphotyrosine (data
not shown). Panel C shows that the two novel phosphopeptides
were also detected in the receptor found in complex with c-Src from
32P metabolically labeled 5HR cells that had been treated
with pervanadate and EGF for 5 min. These data indicate that two
phosphorylations occur on the EGFR both in vitro and
in vivo when c-Src becomes physically associated with the
receptor following EGF stimulation.
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Fig. 2.
EGFR phosphotryptic peptides radiolabeled
in vitro or in vivo. For
in vitro labeling (Panels A and B),
5HR and NeoR cells were stimulated with 100 ng/ml EGF for 30 min,
followed by lysis in CHAPS buffer and immunoprecipitation of extract
proteins with either c-Src-specific (GD11) or EGFR-specific (3A/4A)
antibody. Precipitated proteins were then subjected to an in
vitro kinase reaction, and products were analyzed by SDS-PAGE and
autoradiography. For in vivo experiments (Panel
C), cells were labeled for 18 h in phosphate-free media
containing [32P]orthophosphate, stimulated with 100 ng/ml
EGF for 5 min in the presence of pervanadate, and lysed in CHAPS
buffer. Extracts were immunoprecipitated with GD11 antibody, and
precipitated proteins were analyzed by SDS-PAGE and autoradiography.
c-Src-associated, 32P-labeled EGFR was eluted
from gel slices, and samples were trypsinized and analyzed by
two-dimensional TLC as described previously (17). Labeled peptides were
visualized by autoradiography. Panel A, in vitro
labeled EGFR immunocomplexes from NeoR cells (2000 cpm); Panel
B, in vitro labeled c-Src-associated EGFR from 5HR
cells (2000 cpm); Panel C, c-Src-associated EGFR from 5HR
cells labeled in vivo (3000 cpm). Tryptic maps were exposed
to Pegasus Blue film (Pegasus, Burtonsville, MD) for 18 h.
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Fig. 3.
Phosphorylation of peptides 0 and 3 in
metabolically labeled pervanadate-treated cells. NeoR and 5HR
cells were incubated for 18 h with
[32P]orthophosphate as above. Pervanadate (3 mM H2O2 and 5 µM
Na3VO4) was added (Panels B and
D) or not (Panels A and C) along with
100 ng/ml EGF for 5 min prior to lysis in RIPA detergent buffer. EGFR
was immunoprecipitated with mAbs 3A/4A, and the receptor was processed
for phosphotryptic analysis as described in the legend to Fig. 3.
Panel A, EGFR from NeoR cells; Panel B, EGFR from
pervanadate-treated NeoR cells; Panel C, EGFR from 5HR
cells; Panel D, EGFR from pervanadate-treated 5HR cells.
~3000 cpm were loaded per TLC plate. TLC plates were exposed to
Pegasus blue film for 18 h.
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Fig. 4.
Edman degradation of peptides 0 and 3. Peptides 0 and 3 were isolated by HPLC and subjected to automated Edman
analysis. A, 32P from peptide 0 was released at
the second cycle, indicating a phosphorylated tyrosine at position 2;
B, 32P from peptide 3 was released at the fourth
cycle, indicating a phosphorylated tyrosine at position 4.
Candidates for peptides 0 and 3
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Fig. 5.
Identification of peptide 0. The
octapeptide E(Y-P)HAEGGK was synthesized to contain phosphorylated
Tyr845 and analyzed by two-dimensional
electrophoresis/chromatography on TLC plates, either alone (Panel
A) or in a mixture with total in vitro labeled tryptic
phosphopeptides derived from the receptor which co-precipitated with
c-Src (Panel C). The synthetic phosphopeptide, detected by
hypochlorite spraying, co-migrated with tryptic peptide 0, verifying
Tyr845 as the site on the receptor whose phosphorylation is
dependent on c-Src. Panel B, total phosphopeptides from
c-Src-associated receptor alone. Panel D, sequence homology
between the peptide containing Tyr416 of c-Src and the
peptide containing Tyr845 of the EGFR. 3000 cpm of in
vitro labeled tryptic phosphopeptides were loaded along with 2 µg of synthetic phosphopeptide.
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Fig. 6.
Identification of peptide 3. In
vitro phosphorylated peptide 3 (as in Fig. 5B) was
scraped and eluted from the TLC plate and subjected to digestion with
proline-directed protease. Undigested or digested, eluted peptide 3 was
then analyzed by two-dimensional TLC either alone (Panels A
and B, respectively) or mixed (Panel C). The
altered mobility of digested peptide 3 indicates the presence of a
proline in the sequence and identifies the peptide as containing
Tyr1101. 100 cpm of either digested or undigested peptide 3 were loaded on each TLC plate.
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Fig. 7.
Phosphorylation of Tyr845 and
Tyr1101 in MDA468 breast tumor cells. MDA468 or
MDA468c-Src cells were stimulated with 100 ng/ml EGF for 30 min,
followed by lysis in CHAPS buffer and immunoprecipitation of extract
proteins with either c-Src-specific (GD11) or EGFR-specific (F4)
antibody. Precipitated proteins were then subjected to an in
vitro kinase reaction. The labeled EGFR was eluted from gel
slices, and samples were trypsinized and processed as described
previously in the legend to Fig. 3. Labeled peptides were visualized by
autoradiography. Panel A, phosphotryptic peptides from
in vitro labeled EGFR immunocomplexes from MDA468 cells
(4000 cpm). Panel B, protein extracts (50 µg) from MDA468
parental, 5HR, or MDA468c-Src cells which overexpress c-Src, were
separated by SDS-PAGE and subjected to immunoblotting with GD11
antibody. Panel C, phosphotryptic peptides from in
vitro labeled, c-Src-associated EGFR from MDA468c-Src cells (4000 cpm).
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Fig. 8.
Phosphorylation of Tyr845 is
required for EGF-induced DNA synthesis. Neo control cells were
transfected with plasmid DNA encoding Y845F or wild type EGFR, cultured
for 2 days, serum starved for 30 h, and left untreated or treated
with 40 ng/ml EGF for an additional 18 h. Cells were fixed and
co-stained for EGFR expression and BrdUrd incorporation. Results are
expressed as the mean percent ± S.E. of cells expressing EGFR
that were positive for BrdUrd incorporation. Thirty-five to 75 cells
were analyzed for each variable in three independent experiments.
DISCUSSION
(15). These
events correlated with the appearance of two novel tyrosine
phosphorylation sites on the receptor, suggesting that one mechanism by
which c-Src could synergize with the EGFR is by physically complexing
with it and mediating the phosphorylation of novel
non-autophosphorylation tyrosine residues, which in turn may result in
hyperactivation of the receptor and enhanced phosphorylation of
receptor substrates. This increased signaling would then culminate in
augmented cell division and tumor growth. Such a model was
recapitulated in breast cancer cell lines of epithelial origin, wherein
cell lines that express high levels of c-Src and EGFR exhibit
EGF-dependent association between c-Src and the receptor,
augmented signaling through Shc and MAP kinase, and enhanced tumor
formation, as compared with breast tumor cell lines which do not
overexpress both c-Src and the EGFR (16). Because these and other
studies link c-Src and the EGFR etiologically to tumorigenesis and
malignant progression in many human tumors (reviewed in Ref. 12),
identification of the two novel c-Src-dependent
phosphorylations on the receptor and determination of their functions
has taken on added importance, as they represent possible sites
for therapeutic intervention.
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ACKNOWLEDGEMENTS |
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We thank Drs. John Shannon and Jay Fox of the Biomolecular Research Facility for Edman analysis, synthetic peptide production, and helpful advice in identification of peptide 3; Dr. Michael Weber for directing us toward comparisons of peptide 0 and "SPY1," and members of the Parsons-Weber-Parsons research group for critical discussion.
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FOOTNOTES |
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* This work was supported by United States Department of Health and Human Services Grants CA3948 and CA71449 (to S. J. P.), Council for Tobacco Research Grant 4621 (to S. J. P.), and Department of Defense Grants DAMD17-97-1-7329 (to J. S. B.) and DAMD17-96-1-6126 (to D. A. T.).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. Tel.: 804-924-2352;
Fax: 804-982-0689; E-mail: sap{at}virginia.edu.
2 N. Rosen, personal communication.
3 J. H. Chang and S. Parsons, unpublished data.
4 J. S. Biscardi and D. A. Tice, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
EGFR, epidermal
growth factor receptor;
DMEM, Dulbecco's modified Eagle's medium;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PLC, phospholipase C
;
PIPES, 1,4-piperazinediethanesulfonic acid;
PAGE, polyacrylamide gel electrophoresis;
HPLC, high performance liquid
chromatography;
BrdUrd, bromodeoxyuridine;
GST, glutathione
S- transferase;
mAb, monoclonal antibody.
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REFERENCES |
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