From the Ottawa Regional Cancer Centre, Centre for
Cancer Therapeutics, Ottawa, Ontario K1H 1C4, Canada, the
§ Department of Biochemistry, Microbiology and Immunology,
Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5,
Canada, and the
Institute of Biological Sciences, National
Research Council of Canada, Ottawa, Ontario K1A OR6, Canada
Received for publication, September 16, 2002, and in revised form, December 1, 2002
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ABSTRACT |
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EGFRvIII is a mutant epidermal growth
factor that promotes aggressive growth of glioblastomas. We made a
plasmid that directed the expression of an EGFRvIII with three copies
of the Flag epitope at its amino terminus. Flag-tagged EGFRvIII was
expressed at the same levels as unmodified EGFRvIII, and showed the
same subcellular localization. However, the Flag epitope could only be
detected on EGFRvIII present in the endoplasmic reticulum; the epitope was covalently modified during trafficking of the receptor through the
Golgi so that it was no longer recognized by anti-Flag antibody. This
property was exploited to selectively purify nascent EGFRvIII from
glioblastoma cells. Nascent EGFRvIII was found to copurify with a set
of other proteins, identified by mass spectrometry as the two
endoplasmic reticulum chaperones Grp94 and BiP, and the two cytosolic
chaperones Hsc70 and Hsp90. The Hsp90-associated chaperone Cdc37 also
co-purified with EGFRvIII, suggesting that Hsp90 binds EGFRvIII as a
complex with this protein. Geldanamycin and radicicol, two chemically
unrelated inhibitors of Hsp90, decreased the expression of EGFRvIII in
glioblastoma cells. These studies show that nascent EGFRvIII in the
endoplasmic reticulum associates with Hsp90 and Cdc37, and that the
Hsp90 association is necessary to maintain expression of
EGFRvIII.
Glioblastoma multiforme is an incurable disease with a median
survival time that is typically less than 1 year. Surgery and radiation
have been shown to increase survival times, but the disease responds
poorly to chemotherapy. One of the most common genetic abnormalities
found in glioblastoma is amplification of the gene for
EGFR.1 Along with
amplification, the EGFR gene is frequently mutated. The most common
mutation is a deletion of exons 2-7. This deletion is in-frame and
results in the expression of a truncated EGFR, known as EGFRvIII, in
which amino acids 6-273 of the extracellular domain are replaced by a
single glycine residue (1-4). Multiple studies have demonstrated that
EGFRvIII is expressed in approximately half of all glioblastomas
(5-8), and there is evidence that its expression is associated with a
poorer prognosis (9). Consistent with this, EGFRvIII has been shown to
greatly enhance the tumorigenicity of human glioblastoma cells grown as
xenografts in nude mice (10).
Studies in mouse models of glioblastoma show that EGFRvIII cooperates
with mutations at the INK4a/ARF locus to promote the formation of
glioblastoma (11). Thus, whereas EGFRvIII expression in normal mice
does not give rise to tumors, expression of EGFRvIII in
INK4a/ARF( The mutation in EGFRvIII has been shown to generate a receptor with
many properties that are distinct from those of normal EGFR (reviewed
in Refs. 14-16). EGFRvIII is unable to bind epidermal growth factor
and transforming growth factor To better characterize the properties of EGFRvIII, we have generated an
epitope-tagged version of the receptor that can be purified rapidly and
efficiently. Here we describe the characterization of the
epitope-tagged receptor, its purification, and the identification of
proteins that co-purify with the receptor.
Plasmid Constructs--
Plasmid containing the cDNA for the
Moloney murine leukemia virus ecotropic receptor was from Dr. L. Albritton, University of Tennessee, Memphis, TN. The insert was removed
by digestion with BamHI and SalI and subcloned
into the retroviral vector pWZL-Hygro (obtained from Dr. S. Lowe, Cold
Spring Harbor Laboratory, Cold Spring Harbor, NY). To construct the
vector expressing triple Flag-tagged EGFRvIII, the cDNA for
EGFRvIII was excised from pLERNL (10) (obtained from Dr. W. Cavenee,
Ludwig Institute for Cancer Research, La Jolla, CA) by SalI
digestion and subcloned into the SalI site of a modified
version of the vector T7-blue (Novagen, Inc., Madison, WI) in which the
unique SphI site had been removed. Site-directed mutagenesis
was then used to create a silent SphI restriction site at
codons 43-44 of the mutant receptor, using the oligonucleotide
SPHV3 (CGCATGCTCGGACGCACGAGCCGTGATC). This plasmid was then used as a
template for two PCRs, using two primer pairs: KPNV3
(CGGTACCACGCTGCAGACGCG) and TF1
(GATGTCATGATCTTTATAATCACCGTCATGGTCTTTGTAGTCCAGAGCCCGACTCGCCGGGCA); and
TF2 (GATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGCTGGAGGAAAAGAAAGGT) and SPHV3 (see above). The two PCR products were gel-purified and
joined in a third PCR using the KPNV3 and SPHV3 primer pair and the two
initial PCR products as templates. The final PCR product was subcloned
and sequenced. The KpnI/SphI fragment was excised and ligated into T7-blue containing EGFRvIII cDNA with the silent SphI site, which had also been digested with KpnI
and SphI. The full-length cDNA for EGFRvIII containing
the triple Flag tag sequence was then excised with SalI and
ligated into SalI digested pLERNL. Clones showing the
correct orientation by restriction digest were designated
pLRNLtft Antibodies--
Anti-Flag M2 antibody and anti-Flag M2 affinity
gel were from Sigma. Antiphosphotyrosine antibody was from Transduction
Labs (Lexington, KY). Antibody to the carboxyl terminus of EGFR was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Golgin 97 antibody
was from Molecular Probes, Inc. (Eugene, OR). Anti-Sec61 Cell Lines--
U87MG and U87MG Transfections and Transductions--
Replication-incompetent
retroviruses were made using the three plasmid transient transfection
systems described by Soneoka et al. (24). Transduction of
U87MG glioblastoma cells was done by incubating the cells with virus in
the presence of 8 µg/ml Polybrene for 2 h. U87MGecoR cells,
which express the ecotropic receptor for Moloney leukemia virus, were
made by transduction with amphotropic retrovirus followed by selection
in 75 µg/ml hygromycin B. U87MGtft Flow Cytometry--
Cells growing in tissue culture flasks were
detached using Cell Dissociation buffer (Invitrogen), pelleted by
centrifugation, and resuspended in PBS containing 1% bovine serum
albumin at a concentration of 4 × 106 cells per ml.
200-µl aliquots of cells were then incubated on ice with the
indicated antibodies for 20 min. Cells were pelleted again, resuspended
in 100 µl of PBS containing 1% bovine serum albumin, and incubated
with secondary antibody (either fluorescein isothiocyanate-labeled goat
anti-mouse IgG antibody, or fluorescein isothiocyanate-labeled goat
anti-rabbit IgG antibody, both from Jackson Laboratories, Bar Harbor,
ME) for 20 min on ice. Cells were pelleted again and resuspended in 500 µl of PBS containing 1% bovine serum albumin. Flow cytometry was
performed using a BD LSR flow cytometer (BD Biosciences, San Jose, CA).
Immunofluorescence--
Cells were grown on gelatin-subbed
coverslips in 35-mm dishes. Cells were then washed with cold PBS and
fixed with 4% paraformaldehyde in PBS for 1 h. Where indicated,
cells were permeabilized with 0.2% Triton X-100 in PBS for 10 min.
Cells were blocked with 5% normal goat serum for 30 min and then
incubated with primary antibody for 1 h at room temperature. Cells
were washed three times with PBS and incubated with secondary antibody
(either Cy3-labeled goat anti-rabbit antibody used at 3.75 µg/ml or
Cy3-labeled goat anti-mouse antibody used at 2.8 µg/ml, both from
Jackson Laboratories) for 1 h at room temperature. Cells were
washed again, counterstained with Hoechst 33258, and mounted on slides.
Immunofluorescence was performed using a Zeiss Axioskop2 fluorescence
microscope and images were captured with a CCD camera. Images were
deconvolved using Axiovision 3.0 software (Carl Zeiss, Inc., Thornwood,
NY). For double labeling, immunofluorescence was performed as above using anti-Sec61 rabbit polyclonal antibody as an endoplasmic reticulum
marker, or anti-Golgin 97 mouse monoclonal antibody as a Golgi marker.
After incubation with first and second antibodies, cells were washed
and incubated for 1 h at room temperature with anti-Flag M2
antibody that had been labeled with Zenon One Alexa Fluor 488 IgG1 labeling reagent (Molecular Probes, Inc.). Cells were
then washed, fixed again with 4% para-formaldehyde for 15 min at room temperature, washed, and mounted.
Immunopurification of EGFRvIII--
U87MGtft Mass Spectrometry--
Dithiothreitol was added to the affinity
gel eluates to a final concentration of 10 mM, and the
eluates were then boiled for 5 min. Acrylamide was then added to a
final concentration of 1% (w/v) and eluates were incubated for 1 h at room temperature. Samples (40 µl per lane) were then loaded onto
10% SDS-polyacrylamide gels and electrophoresed. Gels were stained
with GelCode Blue Stain reagent (Pierce), destained with
H2O, and photographed. Stained bands were cut of gels and
stored in aqueous 1% acetic acid. For mass spectrometry analysis,
bands were then washed extensively with deionized water, shrunk with
acetonitrile, re-swollen with 50 mM ammonium bicarbonate
containing sequencing grade, modified trypsin (10 ng/µl) from Promega
(Madison, WI). Sufficient 50 mM ammonium bicarbonate was
added to cover the gel pieces (typically 30 µl). The tubes were
sealed and incubated overnight at 37 °C. The digest solutions were
removed and the gel pieces were extracted with 50 µl of 5% acetic
acid and then with 50 µl of 5% acetic acid in 50% aqueous
methanol. The extracts were pooled with the digest solutions and
concentrated to ~10 µl.
The extracts were analyzed by rapid LC-MS/MS using a CapLC high
performance liquid chromatography pump (Waters Associates, Millford,
MA) coupled to a Q-TOF2 hybrid quadrupole time-of-flight mass
spectrometer (Micromass, Manchester, UK). The entire samples were
injected onto a 0.3 × 5-mm C18 micro precolumn
cartridge (Dionex/LC-Packings, San Francisco, CA). The peptides were
retained while the sample solution was washed to waste. The trap was
then brought online with the mass spectrometer and the peptides were eluted with a rapid gradient supplied by the CapLC pump (5-80% acetonitrile, 0.2% formic acid in 6 min, 1 µl/min flow rate). The
mass spectrometer was set to operate in automatic MS/MS acquisition mode and tandem mass spectra were acquired on doubly and triply protonated precursor ions only. These spectra were then searched against the Swiss-Prot/TrEMBL and NCBI nr protein sequence data bases
using Mascot DaemonTM (Matrix Science Ltd., London, UK), an
algorithm that uses mass spectrometry data to identify proteins in
primary sequence data bases.
Immunoprecipitations--
Small scale immunoprecipitations were
carried out using the same basic procedure as described above for
immunopurification of EGFRvIII, with the following changes: cells
growing in two 10-cm tissue culture dishes were washed once with cold
PBS and then scraped directly into 0.5 ml of lysis buffer; cleared
extracts were incubated for 45 min at 4 °C with 5 µg of His-tagged
MR1dsFv antibody fragment (25); 100 µl of Ni-NTA Superflow (Qiagen, Inc., Mississauga, Ontario, Canada) was then added and tubes were incubated with rocking at 4 °C for 1 h; pellets were washed
5-7 times with 750 µl of lysis buffer before adding 50 µl of 2×
Laemmli buffer.
Western Blotting--
Western blotting was performed as
described previously (26). After electrophoretic transfer from the gel,
blots were stained with Amido Black to check that equal sample loading
and transfer was achieved.
Construction and Expression of Flag-tagged EGFRvIII--
To allow
efficient purification of EGFRvIII, we constructed a version in which a
triple Flag tag sequence was inserted between the leader sequence and
the start of the mature protein (Fig. 1).
The triple Flag tag amino acid sequence has previously been shown to
bind with high affinity to the anti-Flag M2 monoclonal antibody (27). A
codon for a single leucine residue was included at the amino terminus
of the triple Flag tag sequence to preserve the same signal peptide
cleavage site as in normal EGFRvIII. The sequence for tagged EGFRvIII
(hereafter designated tftEGFRvIII) was subcloned into a retroviral
vector. Initially expression was tested by transient transfection into
293T cells (Fig. 2A). Cell lysates were prepared and analyzed by Western blotting, using polyclonal antibody to the carboxyl terminus of EGFR. Unmodified EGFRvIII migrated as a doublet, in agreement with previous results (21). TftEGFRvIII was expressed at the same levels as unmodified EGFRvIII, and also migrated as a doublet, with both bands migrating at
a slightly higher apparent molecular weight, as expected given the
additional 23 amino acids added at the amino terminus. From this result
it can also be inferred that tftEGFRvIII is glycosylated to the same
extent as unmodified EGFRvIII. TftEGFRvIII was also found to be
autophosphorylated to the same extent as EGFRvIII, showing that the
addition of the epitope tag did not affect its constitutive tyrosine
kinase activity. As expected tftEGFRvIII, but not EGFRvIII, was
detected by the anti-Flag antibody M2. We also performed a test
immunoprecipitation of tftEGFRvIII expressed by transient transfection
in 293T cells. TftEGFRvIII could be reduced to undetectable levels in
cell lysates by incubation with M2 antibody affinity gel, showing that
the binding was very efficient (not shown).
We next made ecotropic replication-incompetent retrovirus containing
tftEGFRvIII, and used these to infect U87MG human glioblastoma cells
that had been modified to express the ecotropic receptor for Moloney
murine leukemia virus. Infected cells were selected for resistance to
G418 and drug-resistant populations of cells (designated U87MGtft
Immunofluorescence on permeabilized and nonpermeabilized cells using
the antibody to the amino terminus of EGFRvIII showed that the
subcellular distribution of tftEGFRvIII and EGFRvIII were the same (not
shown). As with the flow cytometry, we were unable to detect Flag
epitope on the surface of nonpermeabilized U87MGtft
Western blot analysis of tftEGFRvIII expressed in either 293T cells or
glioblastoma cells showed that antibody to the carboxyl terminus of
EGFR recognized two bands (Figs. 2 and
5). In contrast, antibody to the Flag
epitope only recognized a single band. Careful comparison of blots that
were run on the same gel showed that the Flag antibody recognized the
lower of the two bands recognized by the EGFR antibody (Fig.
5A). The different EGFRvIII bands likely represent receptors
with different glycosylation status. (EGFRvIII contains eight of the 12 potential N-linked glycosylation sites present in EGFR.) To
investigate this further, we treated U87MGtft
The results from flow cytometry, immunofluorescence, and Western blot
analysis show that tftEGFRvIII is expressed at the same levels and in
the same localization pattern as nontagged EGFRvIII, but that the
triple Flag epitope is covalently modified (by an unknown mechanism) in
the Golgi apparatus so that it is no longer recognized by the anti-Flag
antibody. This provides a system in which nascent EGFRvIII in the
endoplasmic reticulum can be selectively purified for characterization.
Immunopurification of Flag-tagged EGFRvIII--
TftEGFRvIII was
purified from ~5 × 107 U87MGtft Mass Spectrometry Identification of Bands Copurifying with
EGFRvIII--
Bands from the one-dimensional gel lane of purified
tftEGFRvIII were excised and the proteins were identified by in-gel
tryptic digestion and mass spectrometric analysis (Fig. 6). The major band was identified as tftEGFRvIII, as were the two bands immediately below it. However, the lower band in this pair also contained as second
protein, identified as the endoplasmic reticulum chaperone Grp94. The
three other unique protein bands were identified as: 1) a mixture of
the closely related cytosolic heat shock/chaperone proteins Hsp90
In this paper, we have chosen to focus on the interaction of EGFRvIII
with Hsp90. To confirm the mass spectrometry identification of Hsp90,
we analyzed immunopurified EGFRvIII by Western blots, using an antibody
that recognizes Hsp90
To confirm that the interaction of EGFRvIII with Hsp90 was not an
artifact caused by the addition of the epitope tag, we also performed
small scale immunoprecipitations of nontagged EGFRvIII and probed these
by Western blotting. These immunoprecipitations were done using an
engineered antibody fragment that is specific for EGFRvIII. (These
immunoprecipitations are specific but relatively inefficient, and so
were not used for large scale immunopurifications.) For these
experiments, we used U87MG and U87MG
Hsp90 does not interact with "client" proteins on its own, but
rather forms a set of different complexes with other proteins, some of
which also have chaperone-like activity (reviewed in Ref. 29). One of
these is the protein Cdc37, which appears to promote the interaction of
Hsp90 with protein kinases (30). We probed Western blots of
immunopurified EGFRvIII with antibody to Cdc37 and detected a single
band of the expected size in immunopurified EGFRvIII, which was absent
in the control lane (Fig. 7C). This suggests that Hsp90
binds EGFRvIII in association with Cdc37. In gels stained with
colloidal Coomassie Blue this region is obscured by IgG heavy chain
that is present in affinity gel eluates (see Fig. 6), which explains
why Cdc37 was not detected in our original screen for proteins binding
to EGFRvIII. Cdc37 expression levels were the same in U87MGecoR and
U87MGtft
A second protein with chaperone-like activity that associates with
Hsp90 is p60/Hop (31, 32). We also probed immunopurified tftEGFRvIII
with antibody to p60/Hop; although this antibody readily recognized a
single protein in U87MGecoR and U87MGtft Effects of the Hsp90 Inhibitors on EGFRvIII--
The natural
product geldanamycin binds in the amino-terminal ATP-binding domain of
Hsp90, blocking many of the activities of this protein within the cell
(33). To assess the role of the association of Hsp90 with EGFRvIII, we
treated U87MG
The natural product radicicol also binds Hsp90 at its amino-terminal
ATP-binding site, although it is chemically unrelated to geldanamycin
(37). Radicicol was able to decrease levels of phosphorylated EGFRvIII
and EGFRvIII protein at concentrations of 300 nM or greater
(Fig. 8B). Radicicol also down-regulated expression of cdk4
at a similar concentration without affecting ERK expression. Radicicol
has a 5-fold lower affinity for Grp94 than for Hsp90 (37); because the
same concentration of radicicol decreases the expression of both
EGFRvIII and cdk4 (which interacts with Hsp90 but not Grp94), this
indicates that the effects of radicocol on EGFRvIII are because of
inhibition of Hsp90, rather than Grp94.
We have made and characterized a version of EGFRvIII that contains
three copies of the Flag epitope at the amino terminus of the receptor
after removal of the leader sequence. Surprisingly, we found that the
immunoreactivity of this epitope tag was lost during EGFRvIII
maturation, most likely because of a covalent modification taking place
in the Golgi apparatus. There were two main lines of evidence for this:
first, immunofluoresence localization showed loss of Flag epitope
immunoreactivity in the Golgi; second, the Flag epitope could only be
detected on EGFRvIII that had not undergone conversion of its
carbohydrate into complex oligosaccharides, a process that occurs in
the Golgi. We do not know what the covalent modification is at this
time. It is very unlikely that it is glycosylation, as the Flag epitope
does not contain sites for either N-linked or
O-linked glycosylation (see Fig. 1). The Flag epitope is
rich in aspartate residues; one possibility is that the loss of
immunoreactivity is because of The loss of Flag epitope immunoreactivity as EGFRvIII passes through
the Golgi provides a convenient method for selectively purifying newly
synthesized EGFRvIII. We were able to purify the nascent receptor in
microgram quantities, and showed that a set of other proteins
reproducibly co-purified with it. These were identified by mass
spectrometry as the two endoplasmic chaperones Grp94 and Grp78/Bip, and
the two cytosolic chaperones Hsc70 and Hsp90.
In this paper we have focused on the association of EGFRvIII with
Hsp90, a finding that is of interest from both basic science and
clinical perspectives. Hsp90 is a member of the heat shock protein/chaperone family: the major role of this family is to assist in
the folding of newly synthesized proteins in the cell, and to assist in
protein refolding after environmental insults (40, 41). Hsp90 function
has been studied in most detail with respect to its role in steroid
receptor function, where it has a clear role both in receptor assembly
and in maintenance of the high affinity hormone binding of the mature
receptor (29). Hsp90 has also been shown to interact with a number of
tyrosine kinases including the Src family members v-Src (42) and
Hck (43), erbB2 (34), and with the serine/threonine kinases Raf (44) and cdk4 (35). These studies have led to the idea that Hsp90 may have a
general role in supporting signaling by oncogenic protein kinases.
Whereas we clearly detect an interaction between EGFRvIII and Hsp90, Xu
et al. (34) have reported that normal EGFR does not interact
with Hsp90. This may indicate that there is a greater degree of
association of Hsp90 with EGFRvIII than with normal EGFR. A
preferential association of EGFRvIII with Hsp90 would parallel previous
studies showing that oncogenic, constitutively active v-Src
preferentially interacts with Hsp90 compared with normal cellular Src
(42).
In systems where Hsp90 function has been studied in detail, Hsp90
interacts with client proteins as part of a complex of other proteins that often also have chaperone-like activity. The
immunophilins FKBP51, FKBP52, and CyP-40, the protein phosphatase PP5,
and p60/Hop all appear to bind at a common site on Hsp90 (45). These
proteins bind Hsp90 via a domain known as the tetratricopeptide repeat (46). Another Hsp90-interacting protein, Cdc37, binds Hsp90 at a site
that may overlap with the tetratricopeptide repeat domain-binding site
(30). We found that Cdc37 copurified with nascent EGFRvIII, suggesting
that some or all of the Hsp90 associated with EGFRvIII is present as a
complex with Cdc37. The binding of Cdc37 and p60/Hop to Hsp90 are
mutually exclusive (30). We did not detect p60/Hop in association with
immunopurified EGFRvIII, suggesting that the nascent receptor is
selective for the type of Hsp90 complex that it interacts with.
Cdc37 was first identified as the product of a cell division cycle
start gene in yeast (47). A Drosophila homologue of Cdc37 was also identified in genetic screens for mutations that impair signaling by the sevenless receptor tyrosine kinase, which is required
for differentiation of the R7 photoreceptor neuron (48). After this,
several groups showed that the 50-kDa protein that copurified with
v-Src and Hsp90 was the mammalian homologue of Cdc37 (35, 49). Cdc37
also interacts with several other kinases including Raf and cdk4 (30,
35). There is evidence that Cdc37 plays a role in tumor development, as
mice engineered to overexpress Cdc37 under control of either the
Moloney murine leukemia virus promoter (50) or a probasin promoter (51)
develop tumors at a high frequency. Cdc37 is also overexpressed in
prostate cancer (51). Biochemically, Cdc37 has been shown to have
chaperone activity and, to a limited extent, can substitute for the
chaperone activity of Hsp90 (52). Cdc37 is able to rescue expression of a mutant version of the Zap70 kinase that is found in one form of
severe combined immunodeficiency disease (53); this may be a parallel
to our own work, with Cdc37 helping to rescue the function of a mutant
EGFR. Although an earlier study in Drosophila suggested a
link between Cdc37 and receptor tyrosine kinases (48), the work
presented here is the first demonstration of a physical interaction between Cdc37 and a receptor tyrosine kinase.
The Hsp90 inhibitor geldanamycin was able to decrease expression of
EGFRvIII at a concentration similar to its reported affinity for Hsp90
(34). The interaction with Hsp90 therefore appears to be essential to
maintain high level expression of EGFRvIII. Geldanamycin treatment also
reduced levels of cdk4 in glioblastoma cells, in agreement with results
seen in other cell types. EGFRvIII cooperates with cdk4-activating
mutations (either mutation of the cdk4 repressor Ink4a, or
overexpression of cdk4) to induce the formation of glioma (11). Hsp90
inhibition is therefore a means to simultaneously inactivate two key
signaling pathways that are aberrantly activated in glioblastoma.
Inhibitors of Hsp90 are currently under evaluation as cancer
therapeutics, and our study suggests that EGFRvIII may be a novel
target for the antitumor activity of these drugs (54). The ability of
Hsp90 inhibitors to block two key signaling pathways in glioblastoma
may give them a therapeutic advantage over agents such as EGFR tyrosine
kinase inhibitors, which target a single pathway.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) mice does give rise to tumors with a high frequency. The INK4a/ARF locus contains genes coding for two different proteins (12). One of these is Ink4a, a repressor of
cyclin-dependent kinase 4 (cdk4); the other is Arf, which
has a role in regulating p53 levels. EGFRvIII will also form tumors in
mice if cdk4 is overexpressed at the same time, indicating that these
two pathways cooperate in the formation of glioblastoma. It is likely
that these two pathways also cooperate in the formation of human
glioblastoma, as there is a high concurrence of EGFR overexpression and
mutations at the INK4a/ARF locus in these tumors (13).
(two activating ligands for
EGFR) (10, 17, 18), and has a constitutive, ligand-independent tyrosine
kinase activity (19, 20). In addition, EGFRvIII is internalized slowly
compared with normal EGFR after ligand activation (19). EGFRvIII
appears to preferentially use a different subset of downstream
signaling pathways compared with normal EGFR (18, 21), and shows
differential sensitivity to EGFR tyrosine kinase inhibitors (22).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
.
antibody
was from Upstate Biotechnology (Lake Placid, NY). Anti-cdk4 (Ab-6) and
anti-Cdc37 antibodies were from Neomarkers (Fremont, CA). Anti-Hsp90
and anti-p60/Hop antibodies were from StressGen Biotechnologies Corp.
(Victoria, British Columbia, Canada). To make rabbit polyclonal
antibodies specific for EGFRvIII, a multiple antigenic peptide
containing four copies of the sequence LEEKKGNYVVTDHG (the amino
terminus of EGFRvIII plus a glycine spacer) was synthesized and
purified on a Superdex peptide column (Amersham Biosciences). This was then used to immunize rabbits. Polyclonal antibodies were
purified from rabbit serum using the peptide LEEKKGNYVVTDH-biotin (23)
bound to Ultralink immobilized streptavidin gel (BioLynx, Inc.,
Brockville, ON).
EGFR cell lines were obtained
from Dr. W. Cavenee, Ludwig Institute for Cancer Research, La Jolla,
CA. 293T cells were obtained from Dr. J. Bell, Ottawa Regional Cancer
Centre, Ottawa, Canada. All cells were grown in Dulbecco's modified
Eagle's medium supplemented with 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum.
cells, which express triple
Flag-tagged EGFRvIII, were made by transduction of U87MGecoR cells with
ecotropic retrovirus followed by selection in 400 µg/ml G418.
cells or control
U87MGecoR cells were plated at 2.5 × 106 in 15-cm
tissue culture dishes (five dishes per cell line). After 3 days, media
in the dishes was changed to media containing 0.5% fetal calf serum.
The following day, cells were washed once with ice-cold PBS and scraped
into the same buffer (50 ml total for each cell line). Cells were then
pelleted and resuspended in 1 ml of lysis buffer (20 mM
Tris-HCl, 150 mM NaCl, 1% Triton X-100, 20 mM
NaF, 1 mM Na3VO4, 1 mM
para-nitrophenyl phosphate, 1 µg/ml each of leupeptin,
pepstatin, and aprotinin, 5 mM benzamidine, 1 mM
-mercaptoethanol, pH 7.5). Cells were homogenized by
passage once through a 27-gauge needle, and insoluble debris was
removed by centrifugation at 12,000 rpm (15,000 × g)
in an Eppendorf 5417R centrifuge for 10 min at 4 °C. The supernatant
was transferred to a fresh tube and assayed for total protein content
using Coomassie Plus Protein Assay Reagent (Pierce). Equal amounts of
total protein from the two cell lines were then made up to the same
volume with lysis buffer (~1 ml). 50 µl of the original stock
suspension of anti-Flag M2 affinity gel was washed three times with
lysis buffer and added to each tube. The tubes were then mixed with
gentle rocking at 4 °C for 2 h. The resin was then pelleted and
washed four times with 1 ml of lysis buffer. Bound proteins were eluted from the affinity gel by the addition of 50 µl of 2× Laemmli buffer without reducing agent (4% (w/v) sodium dodecyl sulfate, 0.1% (v/v)
glycerol, 125 mM Tris-HCl, pH 6.8) followed by heating at 90 °C for 5 min. The affinity gel was then removed by centrifugation and the supernatant was transferred to a fresh tube.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
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Fig. 1.
Nucleotide and amino acid sequence of the
amino terminus of triple Flag-tagged EGFRvIII. The last six codons
of the EGFRvIII leader sequence are underlined with a
solid line. The inserted triple Flag tag sequence is shown
in bold. The normal amino terminus sequence of mature
EGFRvIII is underlined with a dashed line.
View larger version (41K):
[in a new window]
Fig. 2.
Expression of tftEGFRvIII in 293T cells and
glioblastoma cells. A, 293T cells were transiently
transfected with: lane a, no DNA; lanes b and
d, pLERNL, which expresses EGFRvIII; lane c,
pLRNLtft , which expresses triple Flag-tagged EGFRvIII. Total cell
lysates were analyzed by Western blotting with antibodies to EGFR,
phosphotyrosine, and Flag epitope. B, U87MGecoR glioblastoma
cells (lane a) were transduced with retroviral vectors
containing cDNA for either triple Flag-tagged EGFRvIII (lane
b) or unmodified EGFRvIII (lane c). Total cell lysates
were then analyzed by Western blotting using antibodies to EGFR and
phosphotyrosine. We have shown previously that the upper
phosphotyrosine band, seen in lanes b and c,
represents autophosphorylated EGFRvIII (21).
cells) were used for further experiments. Western blot analysis showed
that these cells also expressed tftEGFRvIII and autophosphorylated
tftEGFRvIII at levels similar to those seen in U87MG cells infected
with a retrovirus containing the cDNA for unmodified EGFRvIII (Fig.
2B). Flow cytometry, using an antibody that recognizes the
amino terminus of EGFRvIII, showed that these two cell populations also
expressed similar amounts of cell-surface EGFRvIII (Fig.
3, panels c and d).
However, we were unable to detect the triple Flag epitope on the
surface of U87MGtft
cells by flow cytometry (Fig. 3, panels
e and f).
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Fig. 3.
Analysis of U87MGtft
cells by flow cytometry. a, U87MGecoR cells
labeled with affinity purified rabbit IgG specific for EGFRvIII;
b, U87MGtft
cells labeled with nonimmune rabbit IgG;
c, U87MGtft
cells labeled with affinity purified rabbit
IgG specific for EGFRvIII; d, U87MGecoR cells transduced
with unmodified EGFRvIII and labeled with affinity purified rabbit IgG
specific for EGFRvIII; e, U87MGtft
cells labeled with
nonimmune mouse IgG1; f, U87MGtft
cells
labeled with anti-Flag mouse monoclonal IgG1
antibody.
cells.
Immunofluorescence of permeabilized cells shows that the triple Flag
epitope is detectable within the cell (Fig. 4). The pattern of staining suggested
that the localization was mainly in the endoplasmic reticulum. This was
confirmed by double immunofluorescence with anti-Flag antibody and an
antibody to the endoplasmic reticulum protein Sec 61
, which showed a
clear overlap in the staining pattern (Fig. 4, panels d-f).
Double immunofluorescence with an antibody to the Golgi marker protein
Golgin 97 showed that while some of the triple Flag epitope was present
in the Golgi, it was not present throughout the Golgi (Fig. 4,
a-c); this suggests that Flag epitope immunoreactivity is
lost during the passage of tftEGFRvIII through this organelle.
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Fig. 4.
Analysis of tftEGFRvIII expression by
immunofluorescence. All images are of U87MGtft cells and
have been deconvolved as described under "Materials and Methods."
a, cells stained with anti-Golgin 97 antibody, a Golgi
marker, and Cy3-labeled secondary antibody; b, the same
cells stained with Alexa Fluor 488-labeled anti-Flag antibody;
c, merged images from a and b. The
presence of some yellow color shows that there is some
colocalization of the two proteins. However, Flag epitope is not
detected throughout the Golgi, as red color is still
evident; d, cells stained with anti-Sec61 antibody, an
endoplasmic reticulum marker, and Cy3-labeled secondary antibody;
e, the same cells stained with Alexa Fluor 488-labeled
anti-Flag antibody; f, merged images from d and
e. The absence of any red color in the merged
images shows that the Flag epitope is present in its immunoreactive
form throughout the endoplasmic reticulum.
cells with
tunicamycin, which blocks the core N-linked
glycosylation of proteins that occurs in the endoplasmic reticulum.
Western blot analysis showed that tunicamycin-treated cells contained a
low molecular weight form of tftEGFRvIII that is probably
nonglycosylated receptor (Fig. 5B). This form was smaller
than the lower band of the tftEGFRvIII doublet, suggesting that the
lower doublet band is at least partly glycosylated. The lower band of
the tftEGFRvIII doublet was absent after tunicamycin treatment, whereas
the upper band was still present. A likely explanation for this
was that the lower band represented core-glycosylated
tftEGFRvIII, whereas the upper band represented tftEGFRvIII in which
the carbohydrate had been converted to complex oligosaccharides, a
process that occurs in the Golgi. After tunicamycin treatment, no new
core-glycosylated tftEGFRvIII would form, but any that was present at
the initiation of tunicamycin treatment would be converted into
tftEGFRvIII containing complex oligosaccharide. We also treated
U87MGtft
cells with the
-mannosidase I inhibitor
deoxymannojirimycin (28), which inhibits the formation of complex
oligosaccharides (Fig. 5C). A 24-h treatment with
deoxymannojirimycin resulted in loss of the upper tftEGFRvIII band,
whereas levels of the lower band were enhanced. This supports the idea
that the lower tftEGFRvIII band, which is recognized by the anti-Flag
antibody, is tftEGFRvIII that has not yet undergone conversion of its
carbohydrate residues to complex oligosaccharides in the Golgi.
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Fig. 5.
Western blot analysis of tftEGFRvIII from
cells treated with glycosylation inhibitors. A, total
cell extract from U87MGtft cells probed with antibody to the
carboxyl terminus of EGFR (left panel) or with anti-Flag
antibody (right panel). Molecular weight markers are shown
on the left. The migration position of the EGFRvIII doublet
is marked with a pair of arrows on the
right in A-C. B, total cell extracts
from U87MGtft
cells either treated with vehicle alone (left
lane) or treated for 24 h with 1 µg/ml tunicamycin
(right lane), and probed with antibody to the carboxyl
terminus of EGFR; C, total cell extracts from U87MGtft
cells either treated with vehicle alone (left lane) or
treated for 24 h with 2.5 mM deoxymannojirimycin
(right lane), and probed with antibody to the carboxyl
terminus of EGFR.
cells. As a
control, the same purification procedure was performed on the parent
glioblastoma cell line that does not express tftEGFRvIII. Immunopurified tftEGFRvIII was analyzed by one-dimensional gel electrophoresis and staining with colloidal Coomassie Blue (Fig. 6). A prominent band of the expected
molecular weight for tftEGFRvIII was evident. Based on comparison with
bovine serum albumin standards run on the same gel, 1-2 µg of
tftEGFRvIII were purified from 5 × 107 cells. Seven
other bands were also found to reproducibly copurify with tftEGFRvIII.
(Additional bands were also detected in some preparations.) Two of
these were present in the control lane, and were not studied further.
The five other bands were absent in the control lane. Based on
comparison with bovine serum albumin standards, these were present at
about 1/10th the amount of the tftEGFRvIII band.
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Fig. 6.
Immunopurification of tftEGFRvIII and mass
spectrometry identification of associated proteins. TftEGFRvIII
was purified from U87MGtft cells as described under "Materials and
Methods." As a control, the same purification procedure was also
performed on U87MGecoR cells (left lane).
Immunopurifications were analyzed by SDS-polyacrylamide gel
electrophoresis, followed by staining with colloidal Coomassie Blue.
Identities of the proteins present in specific bands, determined by
mass spectrometry, are shown on the right.
and Hsp90
; 2) the endoplasmic reticulum heat shock/chaperone protein
Grp78, also known as BiP; and 3) the cytosolic heat shock/chaperone
protein Hsc70.
and weakly cross-reacts with Hsp90
; a band
of the expected size was labeled in the EGFRvIII immunoprecipitate and
was absent in the control lane (Fig.
7A). To determine whether
EGFRvIII had any effects on overall expression levels of Hsp90, Western
blots of total cell lysates from U87MGecoR and U87MGtft
cells were
probed with antibody to Hsp90. Expression levels were the same in the
two cell lines (Fig. 7B). Thus the presence of Hsp90 in
EGFRvIII immunoprecipitates is not because of higher levels of these
proteins in cells expressing EGFRvIII, but rather is because of a
physical association between the proteins.
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Fig. 7.
Association of Hsp90 and Cdc37 with
EGFRvIII. A, Western blot of EGFRvIII immunopurified
from U87MGtft cells (lane b) or mock immunopurified from
U87MGecoR cells (lane a) probed with antibody to Hsp90.
B, Western blots of total cell extracts from either
U87MGecoR cells (lane a) or U87MGtft
cells (lane
b), probed with antibody to Hsp90 (top), Cdc37
(middle), or p60/Hop (bottom). C,
Western blot of EGFRvIII immunopurified from U87MGtft
cells
(lane b) or mock immunopurified from U87MGecoR cells
(lane a) probed with antibody to Cdc37. D,
immunoprecipitations of nontagged EGFRvIII. Immunoprecipitations were
performed with a His-tagged version of a Fv antibody fragment specific
for EGFRvIII (MR1dsFv) and immobilized Ni2+ resin, as
described under "Materials and Methods." Lanes a and
b show an immunoprecipitation in which U87MG
EGFR cell
extracts were incubated without MR1dsFv (lane a) or with
MR1dsFv (lane b) followed by incubation with immobilized
Ni2+ resin (both lanes); lanes c and
d show a second, independent immunoprecipitation in which
U87MG
EGFR cell extract (lane c) or U87MG cell extract
(lane d) were incubated with MR1dsFv and immobilized
Ni2+ resin. Immunoprecipitates were analyzed by Western
blotting for the presence of EGFRvIII and Hsp90. E,
nontagged EGFRvIII was immunoprecipitated as in D and
analyzed by Western blotting for the presence of EGFRvIII and Cdc37.
Lane a, U87MG
EGFR cell extract incubated in the absence
of MR1dsFv; lane b, U87MG
EGFR cell extract incubated in
the presence of MR1dsFv.
EGFR cells (obtained from Dr. W. Cavenee), which have been described previously (10). Western blot
analysis of EGFRvIII immunoprecipitates showed that Hsp90 was present
(Fig. 7D). Hsp90 was absent in control immunoprecipitations from U87MG
EGFR cells in which the primary antibody was omitted, and
in control immunoprecipitations done with U87MG cells. This shows that
Hsp90 also interacts with nontagged EGFRvIII.
cells (Fig. 7B, middle panel),
showing that the presence of Cdc37 in EGFRvIII immunopurifications is
not because of overexpression in the latter cell line. Cdc37 was also
present in small-scale immunoprecipitations of nontagged EGFRvIII,
performed as described above (Fig. 7E).
cells (Fig. 7B, bottom panel), we did not detect this protein
in immunopurified tftEGFRvIII preparations.
EGFR cells with different concentrations of
geldanamycin. Overnight treatment of these cells with 1 µM geldanamycin did not induce apoptosis in these cells,
as judged by morphological criteria, but did cause the cells to adopt a
more flattened appearance with less refractile edges that is more
typical of nontransformed cells. This treatment did not induce any
gross changes in protein expression patterns, but did induce
significant decreases in EGFRvIII protein levels (Fig.
8A). As well as inhibiting
Hsp90, geldanamycin also is able to inhibit Grp94. Xu et al.
(34) have reported relative drug binding affinities of 0.3 and 1 µM for geldanamycin binding to Hsp90 and Grp94,
respectively. Geldanamycin was able to induce EGFRvIII degradation at
concentrations as low as 100 nM, suggesting that its
effects are mediated by inhibition of Hsp90, rather than Grp94. This
result is consistent with EGFRvIII being a client protein for Hsp90. In
agreement with studies in other cell types, geldanamycin also decreased
levels of the Hsp90 client protein cdk4 in glioblastoma cells (35);
this occurred at the same concentration that decreased expression of
EGFRvIII (Fig. 8A). Also in agreement with studies in other
cell types, geldanamycin did not affect the expression of ERK (36).
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Fig. 8.
Effects of geldanamycin and radicicol on
EGFRvIII expression. A, U87MG EGFR cells were treated for
24 h with geldanamycin at the following concentrations:
a, 0 nM; b, 10 nM;
c, 30 nM; d, 100 nM;
e, 300 nM; f, 1000 nM.
Total cell extracts were then analyzed by Western blotting. The
top panel shows a Western blot stained for total protein
with Amido Black. The lower three panels show Western blots
probed with antibody to EGFR, cdk4, and ERK, as indicated.
B, U87MGtft
cells were treated with radicicol for
24 h at the following concentrations: a, 0 nM; b, 100 nM; c, 300 nM, d, 1000 nM; e, 3000 nM. Total cell extracts were then analyzed by Western
blotting for EGFR, phosphotyrosine, cdk4, and ERK.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-hydroxylation of aspartate residues,
a post-translational modification that has been shown to occur in some
secreted proteins (38, 39).
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ACKNOWLEDGEMENT |
---|
We thank Ricardo Marius in Dr. John Bell's lab, Ottawa Regional Cancer Centre, for synthesizing the multiple antigenic peptide.
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FOOTNOTES |
---|
* This work was supported in part by the National Cancer Institute of Canada with funds from the Terry Fox Run.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.
¶ Supported by a Government of Ontario-Zelda Adessky Graduate Scholarship in Science and Technology. Present address: Dept. of Biochemistry and Molecular Biology, University of Calgary, Room 357, Heritage Medical Research Building, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada.
** To whom correspondence should be addressed: Ottawa Regional Cancer Centre, Centre for Cancer Therapeutics, 3rd Floor, 503 Smyth Rd., Ottawa, Ontario K1H 1C4, Canada. Tel.: 613-737-7700 (ext. 6772); Fax: 613-247-3524; E-mail: Ian.Lorimer@orcc.on.ca.
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M209494200
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ABBREVIATIONS |
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The abbreviations used are: EGFR, epidermal growth factor receptor; EGFRvIII, mutant epidermal growth factor receptor lacking amino acids 6-273 of the extracellular domain; PBS, phosphate-buffered saline; MS, mass spectroscopy; cdk4, cyclin-dependent kinase 4.
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REFERENCES |
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