From the Department of Cell and Cancer Biology,
Medicine Branch, NCI, National Institutes of Health, Rockville,
Maryland 20850, the § Department of Cell Biology, Duke
University Medical Center, Durham, North Carolina 27710, and the
¶ Department of Biological Regulation, Weizmann Institute of
Science, Rehovot 76100, Israel
Received for publication, July 31, 2000, and in revised form, October 27, 2000
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ABSTRACT |
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ErbB receptors are a family of
ligand-activated tyrosine kinases that play a central role in
proliferation, differentiation, and oncogenesis. ErbB2 is overexpressed
in >25% of breast and ovarian cancers and is correlated with poor
prognosis. Although ErbB2 and ErbB1 are highly homologous, they respond
quite differently to geldanamycin (GA), an antibiotic that is a
specific inhibitor of the chaperone protein Hsp90. Thus, although both
mature and nascent ErbB2 proteins are down-regulated by GA, only
nascent ErbB1 is sensitive to the drug. To reveal the underlying
mechanism behind these divergent responses, we made a chimeric receptor (ErbB1/2) composed of the extracellular and transmembrane domains of
ErbB1 and the intracellular domain of ErbB2. The ErbB1/2 protein is
functional since its kinase activity was stimulated by epidermal growth
factor. The sensitivity of ErbB1/2 to GA was similar to that of ErbB2
and unlike that of ErbB1, indicating that the intracellular domain of
the chimera confers GA sensitivity. This finding also suggests that the
GA sensitivity of mature ErbB2 depends on cytosolic Hsp90, rather than
Grp94, a homolog of Hsp90 that is restricted to the lumen of the
endoplasmic reticulum, although both chaperones bind to and are
inhibited by GA. Lack of Grp94 involvement in mediating ErbB2
sensitivity to GA is further suggested by the fact that a GA derivative
with low affinity for Grp94 efficiently depleted ErbB2 protein in
treated cells. To localize the specific region of ErbB2 that confers GA
sensitivity, we made truncated receptors with progressive deletions of
the cytoplasmic domain and tested the GA sensitivity of these
molecules. We found that ErbB2 constructs containing an intact kinase
domain retained GA sensitivity, whereas those lacking the kinase domain
(ErbB2/DK) lost responsiveness to GA completely. Hsp90
co-immunoprecipitated with all ErbB2 constructs that were sensitive to
GA, but not with ErbB2/DK or ErbB1. Both tyrosine-phosphorylated and
non-phosphorylated ErbB2 proteins were similarly sensitive to GA, as
was a kinase-dead ErbB2 mutant. These data suggest that Hsp90 uniquely
stabilizes ErbB2 via interaction with its kinase domain and that GA
stimulates ErbB2 degradation secondary to disruption of ErbB2/Hsp90 association.
The ErbB2 gene (also known as Her2/neu), a
homolog of the rat neu gene, encodes a 185-kDa receptor-like
glycoprotein that is a member of the ErbB family of receptor tyrosine
kinases that also include the epidermal growth factor
(EGF)1 receptor (ErbB1) (1),
ErbB3 (2), and ErbB4 (3). ErbB receptors are single transmembrane
proteins with an extracellular domain (ECD) that bears two
cysteine-rich clusters and is responsible for interaction with
polypeptide ligands and an intracellular domain (ICD) that contains a
tyrosine kinase motif and a long hydrophilic segment at the C-terminal
end (4). Binding of the ECD with ligands causes hetero- and/or
homodimerization of ErbB proteins, followed by stimulation of their
intrinsic kinase activity, leading in turn to the phosphorylation of
tyrosine residues in the C-terminal tail (5). In the case of
EGF-stimulated ErbB1, the E3 ubiquitin ligase Cbl binds to the
tyrosine-phosphorylated C terminus and mediates ubiquitination and
down-regulation of the receptor (6, 7). Although no ligand has been
found for ErbB2, the protein seems to be the preferred dimerization
partner for the other ErbB receptors, perhaps because it is not
normally a target of Cbl and may thus protect the other ErbB members
with which it dimerizes from Cbl-mediated down-regulation (8).
The ErbB2 gene was first isolated in the rat from chemically
induced neuroblastomas based on its ability to transform NIH 3T3 cells
(9, 10). It has been shown that overexpression of ErbB2 causes cell
transformation and tumorigenesis (11), and recent in vitro
experiments have shown that ErbB2 is required for induction of
carcinoma cell invasion by other members of the ErbB family (12).
Moreover, overexpression of ErbB2 in cells devoid of other ErbB
proteins potentiates cell migration. These results suggest that ErbB2
expression is related to the malignancy of tumor cells. In fact,
ErbB2 is often amplified in various solid tumors, and the
clinical implications of its overexpression in breast and ovarian
cancers have been well described (5).
Geldanamycin (GA), a benzoquinone ansamycin antibiotic, is a specific
inhibitor of the 90-kDa chaperone protein family, which includes the
cytosolic heat shock protein Hsp90 (13) and the endoplasmic reticulum
(ER)-localized glucose-regulated protein Grp94 (14, 15). GA has
antitumor activity in vivo and has been shown to cause rapid
depletion of the ErbB2 protein mediated by its
ubiquitin-dependent proteasomal degradation (16). Although ErbB2 is highly homologous to ErbB1 in amino acid sequence, its sensitivity to GA is quite different. Whereas only newly synthesized ErbB1 is sensitive to GA, both mature and nascent ErbB2 proteins are
efficiently down-regulated by the drug (14, 17). Although association
of a number of cytosolic kinases with Hsp90 is necessary to maintain
their solubility, intracellular trafficking, and correct response to an
activating signal (15), a requirement for chaperone association with
transmembrane kinases is unclear. Likewise, although Grp94 is thought
to be part of a chaperone cascade that participates in the maturation
process undergone by transmembrane and secreted proteins as they pass
through the ER, its direct requirement for or participation in ErbB
maturation has not been demonstrated. Because a clinically tolerated GA
derivative is currently in phase I trial, it is important to fully
understand how it targets the ErbB2 protein for degradation and to
uncover the biologic rationale for the divergent responses of ErbB1 and
ErbB2 to this drug.
ErbB proteins are type I transmembrane proteins whose extracellular
domain is glycosylated while in the ER; and thus, these proteins may
come in contact with both Hsp90 and Grp94 during their synthesis and
maturation. Therefore, the mechanism by which GA destabilizes mature
ErbB2 (but not mature ErbB1) might be related to drug effects on ErbB
interaction with either or both chaperone proteins. Although initial
reports implicated GA interference with ErbB/Grp94 association as the
drug's primary mechanism of action with respect to ErbB2 (14,
16), at least one study has suggested an involvement of the
protein's kinase domain, which never contacts Grp94, in this process
(18). If Grp94 association with ErbB2 at the plasma membrane were
responsible for ErbB2 sensitivity to GA, the chaperone would have to
remain associated with the extracellular domain of the kinase at the
cell surface even though Grp94 is thought to be restricted to the ER.
Conversely, if the kinase domain of ErbB2 determines the protein's GA
sensitivity, mature ErbB2 should associate with Hsp90.
In this study, we demonstrate that a functional chimeric protein
containing the ErbB1 extracellular and transmembrane domains and the
ErbB2 intracellular domain displays GA sensitivity that is unlike that
of ErbB1, but indistinguishable from that of ErbB2. Furthermore, we
conclusively show that the GA sensitivity of the mature ErbB2 protein
is conferred by its intracellular domain, secondary to GA-mediated
disruption of Hsp90 association with this region. Lack of interaction
of mature ErbB1 and certain C-terminal ErbB2 truncation mutants with
Hsp90 is consistent with the relative insensitivity of these constructs
to GA. Finally, a GA derivative with similar affinity for Hsp90, but
with a 90-fold weaker affinity for Grp94, depletes ErbB2 protein from
tumor cells at a concentration similar to that of GA. Taken together,
these data confirm that the stability of the mature ErbB2 protein, in
contrast to ErbB1, requires association of its kinase domain with Hsp90.
Cells and Antibodies--
SKBR3, A431, and COS-7 cells were
purchased from American Type Culture Collection. Anti-ErbB1
immunoprecipitation antibody was purchased from Oncogene Science Inc.
(Ab-1), and Western blot antibody was from Upstate Biotechnology, Inc.
(LA1). Anti-ErbB2 immunoprecipitation antibody was from Oncogene
Science Inc. (Ab-5), and Western blot antibodies were from Oncogene
Science Inc. (Ab-3 for the ICD) and Transduction Laboratories (clone 42 for the ECD). Rat anti-Hsp90 monoclonal antibody was from Stressgen
Biotech Corp. (SPA-835), and goat anti-Hsp70 and anti-Hsc70 polyclonal antibodies were from Santa Cruz Biotechnology. Anti-phosphotyrosine monoclonal antibody was from Oncogene Science Inc. (Ab-2). Recombinant human EGF was purchased from Life Technologies, Inc.
Preparation of Plasmid
Constructs--
pcDNA3-ErbB1 and
pcDNA3ErbB2 were described previously (19).
Chimeric ErbB1/2 was made by joining three fragments. The first fragment was cut out of pcDNA3-ErbB1 with
Acc65I and BsmI. The second fragment was
amplified from ErbB1 by polymerase chain reaction using the
5'-end primer 5'-CCGGAGCCCAGGGACTGCGTCTCT-3' and the 3'-end primer
5'-CGCTTCCGGACGATGTGGCGCCTTCGCA-3', which contains a silent mutation of
T to C at position 2208 of ErbB1 to make a BspEI
cutting site. The third fragment was excised from
pcDNA3-ErbB2 using BspEI and XbaI.
The three fragments were ligated and inserted into the pcDNA3
vector, and the resulting construct was verified by restriction enzyme
mapping and sequencing. Truncated ErbB2 proteins were made by joining a
fragment cut out of pcDNA3-ErbB2 with
HindIII/SphI with another fragment amplified by
polymerase chain reaction. The polymerase chain reaction was performed
with the shared 5'-end primer 5'-GATGAGGAGGGCGCATGCCAGCCTT-3' and the 3'-end primer 5'-GCGCTCGAGTTACTCAGAGGGCAGGGGTACTG-3' for
ErbB2/DT, 5'-GCGCTCGAGTTAGAAGAAGCCCTGCTGGGGTA-3' for ErbB2/DHC, or
5'-GCGCTCGAGTTATCTCCGCATCGTGTACTTCC-3' for ErbB2/DK. The products
of polymerase chain reactions were digested with SphI
and XhoI and, together with the first fragment, were ligated
into the pcDNA3 vector. Kinase-deficient ErbB2 (ErbB2/K753A) was
made by mutating lysine 753 of wild-type ErbB2 to alanine using the
GeneEditor in vitro site-directed mutagenesis system (Promega) with the ErbB2-specific primer
5'-ATTCCAGTGGCCATCGCAGTGTTGAGGGAAAA-3'.
Cell Culture and Transient Transfection--
SKBR3 cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
fetal calf serum, 2 mM glutamine, 1 mM Hepes,
and 100 units of penicillin and streptomycin. COS-7 cell culture medium
contained 90% Dulbecco's modified Eagle's medium, 10% fetal calf
serum, 2 mM glutamine, 1 mM Hepes, and 1 mM sodium pyruvate. For transient transfections, each
plasmid, which was premixed with FuGene 6 (Roche Molecular
Biochemicals), was added to cells at 50-70% confluency. Cells were
continually cultured in the same medium for 24-48 h until lysis. For
EGF stimulation, cells were first incubated with Opti-MEM (Life
Technologies, Inc.) for 24 h; then EGF was added at 100 ng/ml; and
incubations were continued at 37 °C for 10 min.
Immunoprecipitation and Western Blotting--
Cells were washed
once with cold phosphate-buffered saline (pH 7.0) and lysed by scraping
in TMNSV buffer (50 mM Tris-HCl (pH 7.5), 20 mM
Na2MoO4, 0.09% Nonidet P-40, 150 mM NaCl, and 1 mM sodium orthovanadate)
supplemented with CompleteTM proteinase inhibitors (Roche
Molecular Biochemicals). Cell lysates were clarified by centrifugation
at 14,000 rpm (4 °C) for 15 min, and protein concentration was
determined by the BCA method (Pierce). For immunoprecipitation, 1 mg of
lysate protein was incubated with 4 µg of mouse monoclonal antibodies
at 4 °C for 2 h, followed by the addition of protein
A-Sepharose beads (Amersham Pharmacia Biotech), which were precoated
with rabbit anti-mouse IgG, and rotation at 4 °C overnight. The
beads were washed five times with lysis buffer, resuspended in 1× SDS
sample buffer (80 mM Tris-HCl (pH 6.8), 2% SDS, 10%
glycerol, 100 mM dithiothreitol, and 0.0005% bromphenol
blue), and boiled for 5 min. Immunoprecipitated proteins (or cell
lysates mixed with 5× SDS sample buffer) were separated by 8%
SDS-PAGE. Western blotting was performed as described previously (14).
Preparation and Use of Immobilized GA--
GA was derivatized to
Affi-Gel resin (Bio-Rad) as described (13). Briefly, GA was dissolved
in chloroform and allowed to react with 1,6-hexanediamine for 2 h
at room temperature. After aqueous extraction, the resulting
17-hexamethylenediamine-17-demethoxygeldanamycin was dried under
vacuum, redissolved in dimethyl sulfoxide, and reacted with Affi-Gel 10 resin for 4 h at room temperature. After several washes, beads
were stored in cell lysis buffer and used within 10 days for
competition assays. For competition assays, increasing concentrations
of soluble test drugs were incubated on ice with cell lysates for 30 min; 100 µl of a 50% suspension of GA resin was added; the volume
was adjusted to 500 µl with lysis buffer; and samples were rotated at
4 °C for 2 h. The GA resin was washed four times with lysis
buffer; 100 µl of 1× SDS sample buffer was added; and the samples
were processed as described above. After SDS-PAGE and transfer to
nitrocellulose, blots were probed for Hsp90, and band densities were
obtained by densitometry using Adobe Photoshop and NIH Image software.
Analysis of GA and WX514 Binding to Grp94--
250-µl binding
reactions consisting of 5 µg of Grp94, 20 nM
N-[3H]ethylcarboxamidoadenosine (NECA), 50 mM Tris (pH 7.5), and various concentrations of competitor
were incubated on ice for 1 h. Grp94 was then collected on
polyethyleneimine-treated glass filters, washed twice with 4 ml of
ice-cold 50 mM Tris (pH 7.5), dried, and counted in a
scintillation counter to determine the amount of Grp94-bound
[3H]NECA (20). All assays were performed in triplicate
and corrected for nonspecific ligand binding.
Sensitivity of Mature ErbB2 to GA Is Conferred by Its
ICD--
ErbB1 and ErbB2 proteins are differentially sensitive to GA.
A431 cells, which overexpress ErbB1, and SKBR3 cells, which overexpress ErbB2, were both exposed to GA for varying times, and ErbB proteins in
whole cell extracts were detected by Western blotting (Fig. 1). Whereas the ErbB2 protein level
declined by >80% within 4 h and was nearly undetectable by
6 h, the ErbB1 level did not decline noticeably until 16 h
after drug exposure. ErbB1 responded similarly to cycloheximide (data
not shown), suggesting that GA effects on ErbB1 were mediated solely by
its ability to destabilize newly synthesized ErbB1 protein, as
suggested by Sakagami et al. (17), whereas mature ErbB2
retained GA sensitivity (14).
Since ErbB1 and ErbB2 proteins respond differently to GA, we
constructed a chimeric protein (ErbB1/2) consisting of the ECD and
transmembrane domain of ErbB1 and the ICD of ErbB2 to determine whether
the ECD or ICD of ErbB2 determines sensitivity of the mature protein to
GA (Fig. 2A). We ascertained
that the chimeric protein was functional by testing its responsiveness
to EGF after transfection into COS-7 cells. As is evident from Fig.
2B, EGF efficiently stimulated the autophosphorylation of
ErbB1/2, in contrast to its lack of effect on the autophosphorylation
of transfected ErbB2 (Fig. 2B, upper panel). The
EGF responsiveness of ErbB1/2 suggests that the protein attained a
native conformation in the transfected cells. Next, we compared the GA
sensitivity of ErbB1/2 with that of both ErbB1 and ErbB2 following
transient transfection into COS-7 cells. Treatment of cells with 1 µM GA for 6 h almost totally depleted ErbB1/2 and
ErbB2 proteins, whereas only a portion of ErbB1 was degraded by 6 h (Fig. 2B, lower panel). These data indicate
that mature ErbB1/2 retains ErbB2-like GA sensitivity, thus implying
that the ICD of ErbB2, rather than its ECD or transmembrane domain,
mediates the responsiveness of the mature protein to GA.
Depletion of the ErbB2 Protein by GA Requires the Presence of Its
Kinase Domain--
The ICD of ErbB2 is 580 amino acids long and is
composed of a 14-amino acid juxtamembrane segment, a 343-amino acid
kinase domain, and a long hydrophilic C-terminal tail (Fig.
3A). The juxtamembrane segment
and the kinase domain are highly conserved between ErbB1 and ErbB2,
whereas the C-terminal tail is moderately homologous, albeit a short
stretch (amino acids 1097-1123) within this region shows increased
homology. To further localize the region of the ICD that mediates the
GA response, we progressively truncated ErbB2 from its C terminus (Fig.
3A) and tested the GA sensitivity of these truncations in
transiently transfected COS-7 cells. Our data show that deletion of the
entire hydrophilic C-terminal tail, including the autophosphorylation
sites, did not affect the GA sensitivity of ErbB2 since both
ErbB2/DT (deletion of 132 amino acids at the C terminus) and ErbB2/DHC
(deletion of the last 224 C-terminal amino acids) were effectively
depleted by GA (Fig. 3B). However, when the C-terminal
deletion included the kinase domain (ErbB2/DK), the truncated protein
lost its GA sensitivity completely. Thus, in cells exposed to 1 µM GA for up to 31 h, the ErbB2/DK protein level
remained identical to that in untreated cells (Fig. 3B),
suggesting that neither mature nor nascent ErbB2/DK is sensitive to GA
and indicating that the ErbB2 kinase domain is obligatory for
GA-induced down-regulation of the protein.
ErbB2 Kinase Activity Is Not Required for GA
Sensitivity--
Since we have shown that the ErbB2 kinase domain
confers sensitivity to GA, we next asked whether an active kinase is
required for such sensitivity, as is apparent for Cbl-mediated
down-regulation of ErbB1 (7). This is of particular interest
considering that ErbB3, a member of the ErbB family containing an
inactive kinase domain, is insensitive to GA (21). To answer this
question, we created a kinase-deficient ErbB2 construct by mutating
lysine 753 to alanine, and we tested its sensitivity to GA. ErbB2/K753A was not autophosphorylated on tyrosine in COS-7 cells, in contrast with
wild-type ErbB2, confirming its lack of kinase activity (Fig. 4, lower panel). However,
ErbB2/K753A was depleted from cells by treatment with GA to a degree
comparable to wild-type ErbB2 (Fig. 4, upper panel),
confirming that the functional activity of the kinase domain is not
important for GA-induced down-regulation of ErbB2 and suggesting
instead that a structural motif contained within this region plays a
major role in conferring GA sensitivity.
Involvement of the Chaperone Protein Hsp90 in GA-induced
Down-regulation of ErbB2--
The chaperone protein Hsp90 is the
molecular target of GA in the cytosol (22-24). GA exerts its
destabilizing effects on soluble kinases by altering their association
with Hsp90-containing multiprotein complexes, thereby targeting them
for proteolytic degradation (15). Although it has not been shown
previously, we reasoned that, in analogy to the soluble kinases, ErbB2
must also interact with Hsp90. We first examined ErbB2/Hsp90
association in ErbB2-overexpressing SKBR3 cells. We immunoprecipitated
ErbB2 from SKBR3 cells using an antibody recognizing the ErbB2 ECD, and
coprecipitation of Hsp90 was examined by Western blotting. We found
that Hsp90 coprecipitated with ErbB2 proteins from untreated SKBR3
cells (Fig. 5A). In contrast, when cells were treated with GA for 1 h, Hsp90 disappeared from ErbB2 immunoprecipitates, even though at this time we detected no
change in the amount of ErbB2 protein immunoprecipitated. As seen with
Hsp90-dependent soluble kinases (13), an increased association of Hsp70 with ErbB2 coincided with the loss of
coprecipitated Hsp90 after GA treatment (Fig. 5A). GA did
not affect the level of either chaperone protein measured in total cell
lysate (data not shown).
We next examined whether endogenous Hsp90 associated with ErbB1, the
ErbB1/2 chimeric protein, and ErbB2 in transiently transfected COS-7
cells. Although Hsp90 coprecipitated with wild-type ErbB2 and with the
ErbB1/2 chimera, the chaperone was not found in ErbB1 immunoprecipitates, even though all three ErbB constructs were expressed to a similar degree (Fig. 5B). Thus, Hsp90
association (or its lack of) with these transfected proteins correlates
with their sensitivity profiles for GA. EGF did not affect the binding of Hsp90 to the EGF-responsive ErbB1/2 chimera, suggesting that Hsp90
association is not regulated by receptor tyrosine phosphorylation (Fig.
5B) and supporting our earlier data (see Fig. 4) that the tyrosine phosphorylation state of ErbB2 does not affect its GA sensitivity. Last, we examined endogenous Hsp90 association with the panel of C-terminal ErbB2 truncation mutants expressed in COS-7
cells, and we found that Hsp90 coprecipitated with all of the ErbB2
truncations, except ErbB2/DK (Fig. 5C). Again, this correlates perfectly with the respective GA sensitivities of these truncated ErbB2 proteins and indicates the importance of Hsp90 association in modulating ErbB2 stability.
Binding to Hsp90, but Not Grp94, Determines Benzoquinone Ansamycin
Activity for ErbB2--
To determine whether GA binding to Grp94 may
also contribute to drug-stimulated ErbB2 destabilization, we utilized a
GA derivative, WX514, with 3-fold less affinity than GA for Hsp90, but
with 90-fold weaker affinity for Grp94. Relative drug binding
affinities for Hsp90 were determined by competition of soluble drugs
with Hsp90 binding to immobilized GA, as described previously (13).
Relative binding affinities for Grp94 were determined by drug
competition with the adenosine nucleotide analog [3H]NECA
binding to soluble Grp94, as NECA and GA share the same binding site on
Grp94 (20). As shown in Fig. 6
(A and B), soluble GA competed with immobilized
GA for Hsp90 binding with an IC50 ~0.3 µM,
whereas the IC50 for WX514 was ~1 µM. In
contrast, the IC50 for GA competition with
[3H]NECA binding to Grp94 was ~1 µM,
whereas that of WX514 was 90 µM. With these data in hand,
we examined the ability of WX514 to deplete the mature ErbB2 protein
from SKBR3 cells (Fig. 6C). Corresponding data obtained with
GA are shown for comparison. The concentration of WX514 that caused
50% ErbB2 depletion was ~3.5 µM, whereas at 10 µM WX514, ErbB2 depletion was complete. These
concentrations are 3- and 10-fold, respectively, the relative affinity
of WX514 for Hsp90, but they are one-twenty-fifth and one-ninth the
relative affinity of WX514 for Grp94. Thus, these data support the
hypothesis that inhibition of Hsp90, but not Grp94, is responsible for
ErbB2 destabilization.
ErbB1 and ErbB2 are highly homologous type I transmembrane
receptor tyrosine kinases whose overexpression in various solid tumors
correlates with poor clinical prognosis. Mature ErbB1 and ErbB2
proteins are differentially sensitive to GA. Although in their nascent
form, both ErbB proteins are destabilized by this drug, only mature
ErbB2 retains GA sensitivity. The reason for this discrepancy is not
known. GA binds specifically to the cytosolic chaperone protein
Hsp90(13) and to its ER-restricted homolog Grp94(14). Although GA
binding to Grp94 probably inhibits its chaperone activity in the ER
(20), the nature of Grp94 function is still not well understood,
although along with other chaperones, it is thought to participate in
the maturation of transmembrane and secreted proteins (25). The
function of Hsp90 is somewhat clearer. It forms two multiprotein
complexes, each composed of a set of distinct co-chaperone proteins.
These Hsp90-containing multi-chaperone complexes in turn associate with
a distinct set of client proteins (e.g. many soluble kinases
and steroid receptors). The nature of the particular multi-chaperone
complex in which Hsp90 participates is determined by its conformation,
which is regulated by occupation of an amino-terminal
nucleotide-binding pocket by either ATP or ADP. When ATP-bound, Hsp90
forms a multi-chaperone complex that stabilizes its client protein
and permits its activation. In contrast, when ADP-bound, Hsp90 forms a
multi-chaperone complex that targets its client protein to the
proteasome for degradation. The formation of both types of
Hsp90-containing multi-chaperone complexes and their association with
client proteins are labile and reversible and depend primarily upon
intracellular ATP concentration. GA binds in the nucleotide pocket of
Hsp90 with higher affinity than either ATP or ADP, but the conformation
of GA-bound Hsp90 mimics that of the ADP-bound chaperone. Thus, GA
promotes accumulation of the destabilizing Hsp90-containing
multi-chaperone complex, leading to client protein degradation (for
review, see Ref. 15).
Although a requirement for Hsp90 interaction has been demonstrated for
several non-receptor tyrosine and serine/threonine kinases, including
RIP, p60v-src, and p210bcr/abl, and
for progesterone and glucocorticoid receptors, similar data showing
dependence of mature transmembrane kinases on Hsp90 and the effects of
GA on such client protein-chaperone complexes are lacking. Thus,
although the rapid down-regulation of mature ErbB2 by GA is well
established, a direct involvement of Hsp90 in this phenomenon has not
been reported.
In this study, we have shown that endogenous Hsp90 and ErbB2 do indeed
associate in ErbB2-overexpressing SKBR3 cells and in transiently
transfected COS-7 cells. As is the case with non-receptor kinases,
shortly after GA addition, the nature of the ErbB2-chaperone complex
shifts from one that is stabilizing (containing Hsp90, lacking Hsp70)
to one that is destabilizing (reduced Hsp90, elevated Hsp70), and this
is followed by rapid degradation of the kinase. Our data demonstrate
that Hsp90 association is specific for ErbB2 and is not seen with
ErbB1. Furthermore, the presence of the ErbB2 kinase domain is
minimally required to observe Hsp90 interaction and GA sensitivity. The
ErbB2-like GA sensitivity of an ErbB1/2 chimeric protein containing the
ECD and transmembrane region of ErbB1 and the ICD of ErbB2 confirms the
importance of the ICD in mediating the sensitivity of ErbB2 to GA.
Interestingly, EGF-induced autophosphorylation of the ErbB1/2 chimera
does not affect ErbB2/Hsp90 association. Thus, Hsp90 binding to the
kinase domain of ErbB2 is not intrinsically inhibitory, as it is for
v-Src (26). The stability of kinase domain-deleted ErbB2 (ErbB2/DK) in
the presence of GA and our observation that WX514, an Hsp90-binding GA
derivative, is able to deplete mature ErbB2 at concentrations far below
its affinity for Grp94 both supply conclusive evidence that GA-induced destabilization of ErbB2 is secondary to disruption of Hsp90
association with the ErbB2 kinase domain.
Unlike endogenous ErbB1 expressed in A431 cells, on which GA has almost
no effect within 6-8 h, ErbB1 expressed in transiently transfected
cells is discernibly down-regulated in response to GA, although much
less so than ErbB2. The apparently increased GA sensitivity of ErbB1 in
COS-7 cells may be related to its higher basal level of
autophosphorylation. Whereas in the absence of EGF, ErbB1 in A431 cells
is not detectably phosphorylated (data not shown), transiently
transfected ErbB1 in COS-7 cells is moderately autophosphorylated in
the absence of ligand. EGF-stimulated ErbB1 phosphorylation results in
recruitment of the E3 ubiquitin ligase c-Cbl, which mediates
ubiquitination-dependent degradation of the phosphorylated
receptor (7, 27). When added to the enhanced instability of newly
synthesized ErbB1 in the presence of GA, this phenomenon may contribute
to the decrease in the ErbB1 steady-state level observed in
transfected, drug-treated cells.
Although ErbB2 is not normally efficiently ubiquitinated by c-Cbl, this
ubiquitin ligase has been implicated in mediating antibody-induced
ErbB2 down-regulation (28). However, c-Cbl-mediated down-regulation
requires tyrosine phosphorylation of C-terminal ErbB2 residues, whereas
GA-mediated down-regulation of ErbB2 requires no motifs C-terminal to
the kinase domain. In addition, neither ErbB2 phosphorylation nor
kinase activity is required for GA sensitivity. Thus, an ErbB1/2
chimeric protein unstimulated by EGF is sensitive to GA, as are
autophosphorylated ErbB2 and a kinase-dead ErbB2 point mutant. Although
GA-induced ErbB2 polyubiquitination is observed prior to degradation of
the protein, the ubiquitin ligase responsible for mediating the GA
effect remains elusive. Recently, a novel GA-inducible ubiquitinating
activity was identified in cell
lysates,2 and
characterization of this activity is in progress.
Our data definitively show that Hsp90 interacts with the kinase domain
of ErbB2 and that GA-stimulated destabilization of ErbB2 is preceded by
disruption of ErbB2/Hsp90 association, yet it remains unclear why ErbB2
stability, and not that of ErbB1, requires chaperone binding. Hsp90
association with several non-receptor kinases is necessary to maintain
their solubility and to permit their correct intracellular trafficking
following ligand stimulation (15). However, the location of mature
ErbB2 is fixed in the plasma membrane, and this kinase has no known
ligand. Although ErbB2 is the only ligandless ErbB family member
(5), it is the preferred partner in ErbB heterodimers (19, 29). ErbB2 is recruited to an ErbB heterodimer in response to ligand binding to
its ErbB partner (5). We therefore currently favor the hypothesis that
mature ErbB2 requires Hsp90 association with its kinase domain to
maintain the conformation necessary to heterodimerize with other
ligand-activated ErbB proteins. In fact, a motif within the C-terminal
region of the ErbB2 kinase domain has been suggested to play a role in
dimerization (30). Studies to investigate the possible involvement of
this motif in Hsp90 binding to ErbB2 are in progress.
How did ErbB2 acquire its unique dependence on Hsp90? ErbB2
is likely to have arisen from ErbB1 through a gene
duplication event since invertebrates contain only an ErbB1-like
ligand-activated protein (30). Analysis of the ErbB2 amino acid
sequence reveals that it contains an insert not found in the other ErbB
proteins, and this insert occurs near a residue in ErbB1 shown to be in close proximity to bound EGF (31). It has been speculated that this
altered sequence in the ErbB2 ECD may prevent it from binding ligand
(30). The appearance of such a protein must have surely been considered
a mutational event by the vertebrate organism in which it arose.
Rutherford and Lindquist (32) recently proposed that Hsp90 binding to
mutated proteins may stabilize them while masking their phenotypic
expression, thus allowing accumulation of multiple silent mutations
during evolution and providing the organism with a greater diversity of
responses when faced with unexpected environmental stress. If
ErbB2 evolved by mutation from ErbB1 to become a
ligandless heterodimerization partner, it may have simultaneously
acquired dependence on Hsp90 for its stability. The growth and survival
advantage conferred by ErbB2 would certainly favor its ultimate
evolutionary selection.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ErbB2, but not ErbB1, is sensitive to brief
exposure to GA. A431 and SKBR3 cells were treated with 3 µM GA for increasing times. Cell lysates were separated
by SDS-PAGE and Western-blotted for either ErbB1 (A431 lysates) or
ErbB2 (SKBR3 lysates). Equal loading of total proteins in each lane was
confirmed by Ponceau red staining of the nitrocellulose membranes prior
to blocking and antibody probing.
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Fig. 2.
Construction of chimeric ErbB1/2 and
comparison of its sensitivity to GA with that of ErbB1 and ErbB2.
A, shown is a schematic diagram showing the construction of
ErbB1/2. ErbB1/2 is composed of the ECD and transmembrane domain of
ErbB1 and the ICD of ErbB2, including its kinase domain and the
hydrophilic C-terminal tail. B, COS-7 cells, transiently
transfected with 1 µg of ErbB1, ErbB2, or
ErbB1/2 plasmid DNA, were cultured in Opti-MEM for 24 h
and then treated with recombinant human EGF (upper panel) or
GA (lower panel) and lysed in TMNSV buffer. Lysates were
separated by SDS-PAGE, transferred to nitrocellulose, and probed with
antibodies directed against phosphotyrosine (upper panel) or
against ErbB1 and ErbB2 (lower panel). PM, plasma
membrane; CRD, cysteine-rich domain; IB,
immunoblot.
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Fig. 3.
Sensitivity of truncated ErbB2 proteins to
GA. Truncated ErbB2 proteins were made by introducing a stop codon
at the locations indicated. COS-7 cells (6-well plate) were transfected
with 1 µg of plasmid DNA for each construct. 16 h after
transfection, cells were treated with or without 1 µM GA
for 6 or 31 h and lysed in TMNSV buffer. ErbB2 protein levels were
detected by Western blotting using a monoclonal antibody against the
ErbB2 ECD (clone 42).
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Fig. 4.
Sensitivity of kinase-deficient ErbB2 to
GA. Kinase-deficient ErbB2 was made by mutating lysine 753 in
wild-type ErbB2 to alanine. ErbB2/K753A (1 µg) was transiently
transfected into COS-7 cells, which were then treated with 1 µM GA for 6 h and lysed in TMNSV buffer. The kinase
activity of the ErbB2 mutant was monitored by its autophosphorylation
using anti-phosphotyrosine antibody, and its sensitivity to GA was
detected by Western blotting as described in the legend to Fig.
3B. IB, immunoblot.
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Fig. 5.
Association of Hsp90 with ErbB proteins
detected by immunoprecipitation. A, SKBR3 cells,
treated for 1 h with or without GA (1 µM), were
lysed in TMNSV buffer. ErbB2 proteins were immunoprecipitated
(IP) by first incubating the clarified cell lysate with
monoclonal antibody Ab-5, followed by the addition of protein
A-Sepharose beads coated with rabbit anti-mouse IgG. Immunoprecipitated
proteins were solubilized in SDS sample buffer and separated by
SDS-PAGE. Blots were probed with anti-Hsp90, anti-Hsp70/Hsc70, or
anti-ErbB2 antibodies. B, COS-7 cells, cultured in 200 × 15-mm plates, were transfected with 10 µg of ErbB1,
ErbB2, or ErbB1/2 plasmid DNA. 24 h after
transfection, cells were treated with 100 ng/ml EGF for 10 min at
37 °C and lysed in TMNSV buffer. For immunoprecipitation, anti-ErbB2
antibody (Ab-5) was used for ErbB2-transfected cell lysate,
and anti-ErbB1 antibody (Ab-1) for ErbB1- and
ErbB1/2-transfected cell lysate. Membranes were
Western-blotted for Hsp90 (upper panel) or with a mixture of
anti-ErbB1 and anti-ErbB2 antibodies (lower panel).
C, COS-7 cells were transfected with 10 µg of full-length
or truncated ErbB2 DNA. Cell lysis, immunoprecipitation
(with anti-ErbB2 antibody only), and Western blotting were performed as
described above. IB, immunoblot.
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Fig. 6.
Depletion of the ErbB2 protein correlates
with drug binding to Hsp90 but not to Grp94. A,
increasing concentrations of either geldanamycin or WX514 were added to
cell lysates and allowed to incubate for 30 min at 4 °C prior to the
addition of immobilized GA. Hsp90 bound to immobilized GA was analyzed
as described under "Materials and Methods." Data are expressed as a
percentage of Hsp90 bound to immobilized GA in the absence of
competitor. B, increasing concentrations of geldanamycin or
WX514 were incubated together with purified Grp94 and
[3H]NECA on ice for 1 h. The amount of
[3H]NECA bound to Grp94 was determined by filtration as
described under "Materials and Methods." C, SKBR3 cells
were incubated for 6 h with increasing concentrations of GA or
WX514. Cells were lysed, and the ErbB2 content of total lysates was
determined as described in the legend to Fig. 1. Ponceau red staining
was used to confirm equal loading of all samples.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* 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: Dept. of Cell and
Cancer Biology, Medicine Branch, NCI, NIH, 9610 Medical Center Dr.,
Suite 300, Rockville, MD 20850. Tel.: 301-402-3128 (ext. 318); Fax:
301-402-4422; E-mail: len@helix.nih.gov.
Published, JBC Papers in Press, November 8, 2000, DOI 10.1074/jbc.M006864200
2 I. Alroy and Y. Yarden, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: EGF, epidermal growth factor; ECD, extracellular domain; ICD, intracellular domain; E3, ubiquitin-protein isopeptide ligase; GA, geldanamycin; ER, endoplasmic reticulum; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; NECA, N-ethylcarboxamidoadenosine.
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