From the Division of Rheumatology, Immunology, and Allergy,
Department of Medicine, Brigham and Women's Hospital, Harvard Medical
School, Boston, Massachusetts 02115, ¶ Brown University Oncology
Group, The Miriam Hospital, Providence, Rhode Island 02906, and
Department of Radiation Oncology, Tufts University, New England
Medical Center, Boston, Massachusetts 02111
Received for publication, September 19, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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ErbB2 overexpression contributes to the evolution
of a substantial group of human cancers and signifies a poor clinical
prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a
new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB
receptor down-regulation, and this mechanism appears to contribute to
ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies.
Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce
rapid ubiquitinylation and down-regulation of ErbB2. However, the
ubiquitin ligase(s) involved has not been identified. Here, we show
that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of
wild type CHIP, but not its U-box mutant H260Q, induced
ubiquitinylation and reduction in both cell surface and total levels of
ectopically expressed or endogenous ErbB2 in vivo, and this
effect was additive with that of 17-allylamino-geldanamycin (17-AAG).
The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2
ubiquitinylation. Wild type ErbB2 and a mutant incapable of association
with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG,
implying that Cbl does not play a major role in geldanamycin-induced
ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP
and ErbB2 coimmunoprecipitated with each other, and this association
was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic
elevation of ErbB2 association with Hsp70 and prevented the
17-AAG-induced dissociation of Hsp90. Our results demonstrate that
ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role
for CHIP E3 activity in controlling both the association of Hsp70/Hsp90
chaperones with ErbB2 and the down-regulation of ErbB2 induced by
inhibitors of Hsp90.
ErbB2 is a member of the ErbB receptor tyrosine
kinase family, which also includes the epidermal growth factor
receptor (EGFR1/ErbB1),
ErbB3, and ErbB4. The members of this receptor tyrosine kinase family
play key roles in normal cell proliferation, differentiation, survival,
and migration and have emerged as important contributing factors in
tumorigenesis (1). ErbB2 overexpression, found in nearly a third of
breast cancer patients as well as in other cancers, is associated with
poor responsiveness to conventional therapy and shorter relapse-free
survival, validating ErbB2 as a molecular target for therapy through
attenuation of ErbB2 signaling (2).
A crucial physiological mechanism of ErbB receptor signal attenuation
involves the down-regulation of receptors from the cell surface, a
process that includes initial ligand-induced endocytosis and subsequent
sorting to the lysosome for degradation (3). Recent studies have
focused on the role of ErbB receptor ubiquitinylation, mediated by the
ubiquitin ligase Cbl, as a crucial mechanism to control ErbB receptor
sorting to the lysosome. Upon EGF stimulation, EGFR associates with Cbl
and undergoes ubiquitinylation, which facilitates its lysosomal sorting
and eventual degradation (3-8). In contrast, ErbB2 activated by
heregulin (through heterodimer formation with ErbB3/4) fails to
interact with Cbl and is inefficiently ubiquitinylated (9-12). As a
result, ErbB2 avoids the lysosomal fate and recycles to the cell
surface instead. Furthermore, ErbB2 heterodimerized with other ErbB
family members also avoids the lysosomal fate and is preferentially
recycled, accounting for its enhanced signaling potency (9-13).
Importantly, the therapeutic anti-ErbB2 antibody Trastuzumab
(HerceptinTM) induces an ErbB2-Cbl interaction, which results in ErbB2
ubiquitinylation and degradation (14, 15). These findings highlight the
potential use of ubiquitin ligase-mediated ErbB2 down-regulation as a
therapeutic strategy in ErbB2-overexpressing cancers.
It has been demonstrated that treatment of breast and other cancer cell
lines with ansamycin antibiotics such as geldanamycin (GA) depletes the
cell surface ErbB2 via ubiquitinylation and subsequent degradation (16,
17). The effect of GA on ErbB2 is apparently not mediated by Cbl, since
EGFR, a Cbl target, is GA-resistant. Furthermore, ErbB2 kinase activity
is dispensable for GA action, whereas the kinase activity is required
for Cbl-mediated EGFR or ErbB2 ubiquitinylation (16-21). These
findings point to the existence of a novel ubiquitin ligase machinery
that could be recruited to attenuate ErbB2 signaling. To date, however,
the identity of such an ubiquitin ligase remains unknown.
Previous studies reveal that GA is a specific inhibitor of the
molecular chaperone Hsp90, suggesting that Hsp90 is required for mature
ErbB2 stability (21). Indeed, mature ErbB2 is associated with Hsp90,
and GA treatment leads to the disassociation of Hsp90 from ErbB2, with
concomitant recruitment of Hsp70 (21). It is, therefore, likely that GA
treatment leads to remodeling of the ErbB2-associated chaperone
complex, resulting in the recruitment of a ubiquitin ligase for the
ubiquitinylation of ErbB2.
Recent studies identify a co-chaperone protein, CHIP
(carboxyl terminus
Hsc70-interacting protein) (Fig.
2A), whose three tetratricopeptide repeats bind to
Hsp70/Hsc70 and Hsp90, whereas its carboxyl-terminal U-box domain
associates with ubiquitin-conjugating enzymes (Ubc), thus satisfying
the requirements of a chaperone-associated ubiquitin ligase (22-26).
Importantly, CHIP has been demonstrated to negatively regulate Hsp70
and Hsp90 function and convert Hsp90 complexes from a chaperone
function to one that promotes the ubiquitinylation and degradation of
client proteins, such as the glucocorticoid receptor (23), the cystic
fibrosis transmembrane conductance regulator (24), and c-Raf kinase
(26). Here we investigated if CHIP serves as an ErbB2-directed
ubiquitin ligase. We demonstrate that CHIP associates with ErbB2
in vivo and that wild type CHIP, but not its U-box mutant,
induces ErbB2 ubiquitinylation and down-regulation. CHIP also enhances
the effects of GA treatment, and the U-box mutant of CHIP inhibits
GA-induced ErbB2 ubiquitinylation. Together with results published by
Neckers and co-workers while this paper was under review (27) our
studies support the idea that CHIP ubiquitin ligase provides a novel
mechanism to down-regulate ErbB2 via the ubiquitin pathway.
Cell Lines and Reagents--
The human breast cancer cell line
SKBR-3 and the embryonic kidney epithelial cell line 293T were obtained
from ATCC (Manassas, VA). 17-Allylamino-geldanamycin (17-AAG, NSC
330507, provided by the Drug Synthesis and Chemistry Branch, Division
of Cancer Treatment and Diagnosis of the NCI, National Institutes of
Health, Bethesda, MD) and MG-132 (Calbiochem) were dissolved in
Me2SO at 1 mM and 50 mM,
respectively. Cycloheximide (Calbiochem) was dissolved in
ethanol at 100 mM.
Antibodies--
The rabbit polyclonal antibody (Ab) neu C-18
(anti-ErbB2) and mouse monoclonal antibodies W27 (anti-Hsp70), F-8
(anti-Hsp90), and D-10 (anti- Plasmids--
A pcDNA3 expression construct encoding the Myc
epitope-tagged full-length CHIP protein (amino acids 1-303) was
generated from a human CHIP IMAGE clone (ID 3847704, ATCC,
Manassas, VA) by PCR (primer sequences are available upon request). The
pGEX4T-2 bacterial expression constructs encoding the full-length or
Transient Transfection, Cell Lysis, Immunoprecipitation, and
Immunoblotting--
SKBR-3 or HEK 293T cells were plated in 100-mm
dishes and transfected with the indicated expression plasmids using the
FuGENE 6 reagent (Roche Molecular Biochemicals) according to the
manufacturer's instructions. Cell lysates were prepared 36 h
after transfection in a buffer consisting of 50 mM Tris, pH
7.5, 150 mM sodium chloride, 1% Triton X-100, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each
leupeptin, pepstatin, chymostatin, antipain, and aprotinin (Sigma).
Where indicated, the transfected cells were treated with 17-AAG or
MG-132 or an equivalent amount of vehicle (Me2SO) for
various time periods before harvesting. Immunoprecipitations were
carried out as described previously (29, 30). Immune complexes were
washed with lysis buffer or RIPA buffer (lysis buffer supplemented with
0.5% deoxycholate and 0.1% SDS) as indicated in the figure legends,
resolved by SDS-PAGE, and transferred to polyvinylidene difluoride
membranes (Invitrogen). Immunoblotting, membrane stripping, and
reprobing were carried out as described previously (29, 30).
GST Pull-down Assay--
One-mg aliquots of cell lysate proteins
were incubated with 50 µg of GST or GST-Cbl-N fusion protein
immobilized on glutathione-Sepharose beads (Amersham Biosciences) at
4 °C for 3 h. The beads were washed 5 times with cold lysis
buffer, and the bound proteins were detected by Western blotting.
Biotin Labeling of Cell Surface ErbB2 and Assessment of ErbB2
Down-regulation--
CHIP- or vector-transfected SKBR-3 cells were
washed 3 times with ice-cold phosphate-buffered saline containing 20 mM HEPES, pH 7.5, and then incubated in the same buffer
with 400 µg/ml sulfo-N-hydroxysulfosuccinimide-biotin (Pierce) for 40 min at 4 °C. The cells were washed 3 times with ice-cold phosphate-buffered saline and incubated in pre-warmed medium
with 100 nM 17-AAG or Me2SO control for the
indicated times before lysis. Anti-ErbB2 immunoprecipitates (IPs) of
the lysates were resolved by SDS-PAGE and blotted with HRP-conjugated
streptavidin. Densitometry of ErbB2 bands was carried out using the
Scion Images for WindowsTM software (Version beta3b,
Frederick, MD), and the data were expressed as a percentage of the
signals obtained with untreated cells.
In Vitro Ubiquitinylation Assay--
The GST-CHIP and GST-Cbl
fusion proteins were affinity-purified from Escherichia coli
lysates using glutathione-Sepharose beads, eluted with reduced
glutathione, and stored at Ubiquitinylation and Down-regulation of Both Endogenous and
Ectopically Expressed ErbB2 by 17-AAG--
Ansamycin antibiotics, such
as GA, specifically bind to Hsp90 and inhibit its ATPase activity (18).
Exposure of cells to GA induces the dissociation of Hsp90 from ErbB2
and other Hsp90-associated signaling proteins, resulting in their
degradation (19-21). A more potent GA derivative, 17-AAG, is
undergoing clinical assessment as a potential anti-cancer agent (31).
We examined whether 17-AAG, like its parent compound, induces the
down-regulation of ErbB2 via the ubiquitin pathway. As anticipated
(19-21), 17-AAG treatment led to a significant loss of ErbB2 in both
SKBR-3 cells (endogenous ErbB2) and 293T cells (exogenous ErbB2) (Fig.
1). Treatment of cells with the
proteasome inhibitor MG-132 prevented 17-AAG-induced loss of ErbB2
(Fig. 1, middle and bottom panels). Furthermore, 17-AAG treatment induced the ubiquitinylation of ErbB2, as revealed by
anti-ubiquitin immunoblotting of ErbB2 IPs, and MG-132 treatment markedly enhanced the ubiquitinylation signal (Fig. 1, top left panels, compare lane 1 with lane 2 and
lane 3 with lane 4; top right panel,
compare lane 1 with lane 2 and lane 3 with lane 4). Thus, 17-AAG, like GA (19-21), induces ErbB2
down-regulation via the ubiquitin pathway.
The Co-chaperone Protein CHIP Functions as a Ubiquitin Ligase
toward ErbB2 in Vitro--
Given the linkage of GA-induced ErbB2
degradation to inhibition of Hsp90 (21) and the recent observations
that the Hsp70/Hsp90 co-chaperone CHIP functions as a ubiquitin ligase
toward several Hsp70/Hsp90 client proteins (22-26,
32-36), we asked if CHIP functions as a ubiquitin ligase toward ErbB2,
using an in vitro ubiquitinylation assay with purified
GST-CHIP fusion proteins (Fig.
2A). Purified CHIP could readily auto-ubiquitinylate itself in vitro in
the presence of purified E1, E2, ubiquitin, and ATP, whereas a CHIP mutant lacking the U-box domain was completely inactive (Fig. 2B), and the point mutation of an invariant histidine in the
U-box domain, corresponding to a conserved RING finger domain histidine residue crucial for E2 binding, greatly reduced the
auto-ubiquitinylation activity (32-34).
To assess if recombinant CHIP could function as a ubiquitin ligase
toward ErbB2, ErbB2 immunopurified from SKBR-3 cells was incubated with
purified CHIP fusion proteins, ubiquitin, and ATP in a rabbit
reticulocyte lysate. Relatively little in vitro ErbB2 ubiquitinylation was observed in the absence of CHIP or ErbB2 (Fig.
2C). Inclusion of recombinant wild type CHIP in the reaction resulted in a marked increase in the level of ubiquitinylated ErbB2
(Fig. 2C, lane 5). In contrast, little or no
increase in ErbB2 ubiquitinylation was observed with the CHIP U-box
domain mutants Overexpression of CHIP Promotes the Down-regulation of
ErbB2--
To assess the effect of CHIP on ErbB2 turnover in
vivo, we cotransfected ErbB2 and Myc-tagged CHIP into 293T cells
and examined the overall levels of ErbB2 with or without 17-AAG
treatment. Coexpression of CHIP led to a slight reduction in the level
of ErbB2 in the absence of 17-AAG treatment (Fig.
3A, lane
3). Furthermore, CHIP overexpression modestly
augmented the reduction in ErbB2 protein levels induced by 17-AAG
treatment (Fig. 3A, lane 4).
Previous analyses have identified the role of Cbl, a RING finger domain
ubiquitin ligase, in ErbB2 down-regulation induced by anti-ErbB2
antibodies or by EGF-induced heterodimerization with EGFR; the
phosphorylated Tyr-1112 in ErbB2 serves as the Cbl-binding site in the
context of antibody-mediated ErbB2 down-regulation (14,
15). Notably, the ability of 17-AAG and/or CHIP to induce the loss of
ErbB2 protein was unaltered by the Y1112F mutation (Fig. 3A,
lanes 7 and 8). That the Y1112F mutation indeed
abrogated the ability of ErbB2 to interact with Cbl was demonstrated by a pull-down assay using lysates of ErbB2-transfected 293T cells stimulated with the humanized anti-ErbB2 antibody
HerceptinTM and the GST-Cbl-N fusion protein, which
incorporates the tyrosine kinase binding domain of Cbl (29). Wild type
ErbB2 bound to GST-Cbl-N and HerceptinTM treatment enhanced
this interaction (Fig. 3B, lane 6); in contrast, the ErbB2 Y1112F mutant failed to bind to GST-Cbl-N with or without HerceptinTM treatment (Fig. 3B, lane
8). Taken together these data indicate that CHIP enhances ErbB2
down-regulation in a Cbl-independent manner.
Given the in vitro ubiquitin ligase activity of CHIP (Fig.
2, B and C) (26, 32, 33, 35) and its ability to
induce the loss of ErbB2 in vivo (Fig. 3A), we
asked if CHIP promotes ErbB2 ubiquitinylation in vivo. For
this purpose, 293T cells transfected with ErbB2 with or without CHIP
were incubated with the proteasome inhibitor MG-132, and the lysates of
these cells were subjected to anti-ErbB2 IP followed by anti-ubiquitin
immunoblotting. Indeed, CHIP overexpression was associated with an
increase in the ubiquitinylation of ErbB2 (Fig. 3C). That
CHIP-dependent enhancement of ErbB2 ubiquitinylation reflected the E3 activity of CHIP was shown by the inability of the
U-box point mutant H260Q to enhance ErbB2 ubiquitinylation in
transfected SKBR-3 cells (Fig. 3D). Furthermore, expression of the H260Q mutant (Fig. 3D, lane 6 versus
lane 2), but not wild type CHIP (lane 4 versus 2), inhibited the 17-AAG-induced
ubiquitinylation of ErbB2, suggesting a role for endogenous CHIP
(and/or CHIP-related E3s) in 17-AAG-induced ErbB2 ubiquitinylation.
CHIP-induced Down-regulation of Cell Surface ErbB2--
Given that
GA targets the Hsp90-associated mature cell surface ErbB2 for
degradation, it was important to assess if CHIP overexpression specifically down-regulates this pool of ErbB2, particularly since previous studies reported CHIP as a ubiquitin ligase toward misfolded Hsp70/Hsp90 client proteins in the endoplasmic reticulum (24, 35).
Therefore, we carried out cell surface biotinylation of vector-
or CHIP-transfected SKBR-3 cells, which express endogenous ErbB2, and
monitored the levels of biotin-labeled cell surface ErbB2 by
streptavidin blotting of anti-ErbB2 IPs and densitometry (Fig.
4A). These analyses showed an
enhancement of the 17-AAG-induced loss of surface ErbB2 in
CHIP-overexpressing SKBR-3 cells as compared with the
vector-transfected cells (Fig. 4A). The reduction in cell
surface (biotin-labeled) ErbB2 closely paralleled the reduction in
total ErbB2 (Fig. 4A, compare streptavidin versus
anti-ErbB2 blots). Although the effect of CHIP was modest, it was
reproducible in additional experiments (data not shown).
CHIP-dependent enhancement of 17-AAG-induced loss of ErbB2
was also observed when the synthesis of new ErbB2 was inhibited with
cycloheximide treatment (Fig. 4B), consistent with
CHIP-induced loss of mature cell surface ErbB2. Although we could not
exclude the possibility that CHIP also targets newly synthesized ErbB2,
the combined results clearly show that CHIP can facilitate the
17-AAG-induced loss of cell surface ErbB2.
CHIP Associates with ErbB2--
To further explore the role of
CHIP in ErbB2 turnover, we examined its in vivo association
with ErbB2 with or without 17-AAG treatment. Because such an
association was predicted to occur via Hsp70/Hsp90 chaperone proteins,
we first demonstrated the coimmunoprecipitation of the endogenous Hsp70
and Hsp90 proteins with Myc-tagged CHIP in the lysates of
CHIP-transfected 293 T cells (Fig.
5A). Next we used
reciprocal IP/Western blotting to show that ErbB2 and CHIP proteins
associate in 293T cells cotransfected with both proteins (Fig.
5B). The level of ErbB2/CHIP coimmunoprecipitation increased
when cells were treated with 17-AAG (Fig. 5B, top
and third panels, lane 5 versus 6).
The 17-AAG-induced CHIP-ErbB2 association correlated with the increased
association of ErbB2 with the Hsp70 chaperone (see Fig.
6). Finally, we used the SKBR-3 cells,
which express moderate levels of CHIP (compared with a number of other
cell lines; data not shown), to demonstrate that both endogenous ErbB2
and CHIP also coimmunoprecipitate (Fig. 5C), indicating
their association under more physiological conditions. Importantly, the
level of the endogenous CHIP-ErbB2 association also increased
substantially upon 17-AAG treatment (Fig. 5C).
Increased ErbB2 Interaction with the CHIP H260Q Mutant--
In
view of the reduced 17-AAG-induced ErbB2 ubiquitinylation upon CHIP
H260Q mutant expression (Fig. 3D), we characterized the
complexes of this mutant with ErbB2 and chaperone proteins using
IP/Western blotting. The wild type and mutant CHIP proteins showed a
similar level of association with Hsp90 or Hsp70, and no changes were
detected in these associations upon 17-AAG treatment (Fig. 6,
left panel). In contrast, substantially more CHIP H260Q mutant coimmunoprecipitated with ErbB2 (Fig. 6, top right
panel; compare lane 5 with lane 3); this
association was further increased by 17-AAG treatment, as with wild
type CHIP (top right panel, lane 3 versus lane 4 and lane 5 versus lane 6). Notably, there was also a
substantial increase in ErbB2-Hsp70 association when CHIP H260Q mutant
was expressed; 17-AAG treatment further enhanced this association (Fig.
6, third right panel). Finally, although 17-AAG reduced the
ErbB2·Hsp90 association in vector- and wild type CHIP-transfected
cells, no 17-AAG-induced dissociation of ErbB2·Hsp90 complex
was observed in CHIP H260Q mutant-transfected cells (Fig. 6,
second right panel). Thus, the ubiquitin ligase activity of
CHIP appears to be crucial in determining the nature of
Hsp70·Hsp90· ErbB2 complexes.
Down-regulation of ErbB2 with anti-ErbB2 antibodies has emerged as
a viable therapeutic strategy for ErbB2-overexpressing breast cancers
and other tumors (37), providing impetus for physiologic and
pharmacologic means to achieve ErbB2 down-regulation. Both
physiological (e.g. via EGF-induced heterodimerization with EGFR) and pharmacological (using anti-ErbB2 antibodies and ansamycin antibiotics such as GA) down-regulation of ErbB2 have been linked to
induction of receptor ubiquitinylation, which apparently targets the
modified receptor for lysosomal and proteasomal degradation (14-21).
Understanding the nature of the ubiquitin ligases recruited to ErbB2 by
these different agents is, therefore, of great interest because such
knowledge could facilitate the development of targeted strategies to
achieve receptor down-regulation as well as combinatorial therapeutic
strategies with agents that recruit distinct ubiquitin ligases.
Recent data indicate that both EGF- and antibody- induced ErbB2
down-regulation are mediated through the recruitment of the ubiquitin
ligase Cbl to ErbB2 autophosphorylated on Tyr-1112 (14, 15). The kinase
activity of ErbB2 is essential for Cbl-dependent ubiquitinylation and down-regulation. In contrast, ErbB2
down-regulation induced by the Hsp90 inhibitor GA required the kinase
domain but not the kinase activity of ErbB2, and the carboxyl-terminal
tail carrying the Cbl-binding site was dispensable (21). Thus, it appeared likely that distinct ubiquitin ligase machinery is recruited to ErbB2 upon GA treatment. Given the recent studies that ansamycins inhibit the ATPase activity of Hsp90 and that ErbB2 constitutively associates with Hsp90, we examined the possibility that the
Hsp70/Hsp90-associated co-chaperone CHIP may function as an
ErbB2-directed ubiquitin ligase. Here we provide evidence that CHIP
functions as a ubiquitin ligase toward ErbB2. Although our results
provide an independent confirmation for similar data published while
this paper was under review (27), the present study provides several
additional insights that are discussed below.
In keeping with a role for CHIP in ErbB2 ubiquitinylation, both our
results and the Xu et al. (27) study establish that ErbB2
functions as a substrate for CHIP-mediated ubiquitinylation in
vitro (Fig. 2, C and D), an activity that
requires the U-box domain, previously shown to be essential for CHIP
ubiquitin ligase activity (26, 32, 33, 35). Furthermore, overexpression of intact CHIP, but not its U-box domain mutant, led to enhanced ubiquitinylation and degradation of ErbB2 in human cells (Fig. 3,
C and D). The ability of the CHIP H260Q mutant to
decrease 17-AAG-induced ErbB2 ubiquitinylation (Fig. 3D) is
suggestive of a dominant negative effect on endogenous CHIP, although
further studies are needed to clarify this possibility. The effects of CHIP on ErbB2 are reminiscent of those observed with cystic fibrosis transmembrane conductance regulator (24), another transmembrane receptor. However, the present study establishes that, in contrast to
CHIP-induced ubiquitinylation of misfolded cystic fibrosis transmembrane conductance regulator in the ER (24), CHIP enhanced the
loss of mature cell surface ErbB2 (Fig. 4, A and
B). The sensitivity of mature cell surface ErbB2 to CHIP is
consistent with the association of Hsp90 with mature ErbB2 and the
requirement of this association for ErbB2 stability, as demonstrated by
the sensitivity of ErbB2 to GA (21).
Our study also shows that CHIP overexpression enhances the
ubiquitinylation and down-regulation of both wild type ErbB2 and a
mutant (Y1112F) that is incapable of interacting with Cbl (Fig. 3B). Thus, our results define CHIP as a novel ErbB2-directed
ubiquitin ligase distinct from the previously reported Cbl ubiquitin ligase.
Both our results (Fig. 5, B and C) and the Xu
et al. (27) study demonstrate that both endogenous
and ectopic CHIP associate with ErbB2, an association likely to be
mediated via the Hsp70/Hsp90 chaperone proteins. Previous studies have
established that CHIP constitutively associates with Hsp70 and Hsp90
through the binding of the amino-terminal tetratricopeptide repeats of
CHIP to the carboxyl terminus of Hsp70 and Hsp90 (22). The peptide
binding region of Hsp90 appears to directly interact with the kinase
domain of ErbB2 (21), strongly suggesting that the CHIP·ErbB2
association is mediated via Hsp70/Hsp90 chaperone proteins. However, it
is not clear at present whether CHIP-induced ErbB2 down-regulation in
the absence of GA is mediated via the Hsp90·CHIP or the Hsp70·CHIP complexes, because both complexes were observed in CHIP-transfected cells, and both Hsp90 and Hsp70 coimmunoprecipitated with ErbB2.
The role of CHIP in GA-induced ErbB2 ubiquitinylation and degradation
is supported by a number of findings presented here and the results of
the Xu et al. (27) study. First, 17-AAG, a potent GA
derivative (as well as GA, data not shown), induced a substantial
increase in ErbB2·CHIP association (Fig. 5, B and C). Notably, this enhancement correlated with an increased
association of Hsp70 with ErbB2 and concomitant Hsp90 dissociation
(Fig. 6), consistent with a primarily Hsp70-mediated CHIP·ErbB2
association (21). Second, the overexpression of CHIP in the context of
17-AAG treatment led to an additive loss of ErbB2 (Figs. 3 and 4).
Finally, the overexpression of a CHIP point mutant (H260Q), which
exhibits reduced ubiquitin ligase activity in vitro (Fig.
2D), reduced the level of ErbB2 ubiquitinylation induced by
17-AAG. However, a substantial level of 17-AAG-induced degradation of
ErbB2 was still observed when the CHIP H260Q mutant was expressed (Fig. 3D). The inability of this mutant to fully block the
17-AAG-induced ErbB2 degradation may be due to several factors. First,
only a proportion of cells was likely to be transfected under the
experimental conditions used. Second, the mutant construct employed,
H260Q, has residual ubiquitin ligase activity (Fig. 2B),
which may itself lead to ErbB2 degradation. We were unable to use a
CHIP U-box-deleted mutant, which exhibits no in vitro
ubiquitin ligase activity (Fig. 2B), for these studies
because it showed markedly reduced association with ErbB2 (data not
shown), potentially confounding the results. Third, CHIP may not be the
sole ubiquitin ligase recruited to ErbB2 upon Hsp90 inhibition. This
possibility is supported by the results of Xu et al. (27)
with CHIP Rather unexpectedly, and distinct from the results of Xu et
al. (27), we observed a dramatically increased association of the
CHIP H260Q mutant with ErbB2 under basal conditions; 17-AAG treatment
further enhanced this association (Fig. 6). Notably, whereas the level
of association of CHIP H260Q with Hsp70 was unaffected compared with
wild type CHIP, dramatically more Hsp70 was associated with ErbB2 both
under basal conditions and upon 17-AAG treatment. However, this mutant
did not show a higher level of association with Hsp90, although it
reduced the GA-induced Hsp90 dissociation from ErbB2 compared with that
seen in untransfected and wild type CHIP-transfected cells. Thus, the
mutant CHIP clearly alters the nature of the chaperone-substrate
complex. These findings strongly suggest that the ubiquitin ligase
activity of CHIP plays a critical role in controlling the chaperone
association/dissociation cycle with client proteins. It is likely that
one function of CHIP may be to facilitate the remodeling of chaperone
complexes. Further studies will be needed to directly address these
issues and to ascertain if the observed effects are specific for ErbB2 or are observed with other CHIP substrates as well. The apparently dichotomous effects of CHIP·H260Q on ErbB2 association with Hsp70 versus Hsp90 may simply reflect the higher basal level of
Hsp90·ErbB2 association compared with Hsp70·ErbB2 association.
Alternatively, these results may reflect a differential functional
impact of CHIP-mediated ubiquitinylation (of substrates or chaperone
proteins themselves) on Hsp70 versus Hsp90 association with
client proteins. In this regard, it is well documented that Hsp90 and
Hsp70 require distinct co-chaperone for association with client
proteins and often engage distinct sets of client proteins (38-41).
Recently CHIP was shown to interact, albeit poorly, with the
anti-apoptotic protein Bag-1, which possesses a ubiquitin-like domain
capable of associating with the 26 S proteasome and additional sequences that interact with Hsp70 (26). These findings have led to a
model that a ternary complex of the substrate (c-Raf in the reported
study) with chaperone and ubiquitin ligase (CHIP) may be directly
targeted to the proteasome for efficient degradation of the target
proteins (26). In preliminary experiments we did not observe an
enhancement of CHIP-induced ErbB2 degradation when Bag-1 was
coexpressed (data not shown). Further studies will be needed to fully
assess the potential role (or lack thereof) of the Bag-1 co-chaperone
in CHIP-dependent ErbB2 degradation.
Our results together with those of Xu et al. (27) lead us to
conclude that CHIP is a novel ErbB2-directed E3 ligase. The independence of this pathway from the previously characterized Cbl
ubiquitin ligase pathway and the recruitment of these pathways by
distinct pharmacologic agents (ansamycins versus anti-ErbB2 antibodies) raise the possibility of combinatorial manipulations of
both pathways for a more effective down-regulation of ErbB2 and better
anti-cancer therapy. Notably, a recent report demonstrated that kinase
inhibitors of ErbB receptors also induce
ubiquitin-dependent receptor degradation and Hsp70
recruitment, and such treatment additively down-regulates the ErbB
receptor levels when combined with anti-receptor antibodies (42). These
findings provide added impetus to delineate the various ErbB2-directed
ubiquitin ligase pathways to devise combinatorial strategies for
treatment of ErbB2-overexpressing cancers.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
tubulin) were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA), monoclonal Ab c-neu Ab-5
(anti-ErbB2) was from Calbiochem, and monoclonal Abs P4G7
(anti-ubiquitin) was from Covance Research Products Inc. (Denver, PA).
Monoclonal Ab 9E10 (anti-Myc) (28) was purified in-house. Purified
humanized anti-ErbB2 antibody HerceptinTM was provided by
Genentech Inc. (South San Francisco, CA). Rabbit polyclonal anti-CHIP
antibody was generated by Covance Research Products Inc. against a
peptide corresponding to human CHIP residues 251-268, and its
reactivity was confirmed by immunoprecipitation and immunoblotting
against transfected Myc-tagged CHIP. Horseradish peroxidase
(HRP)-conjugated protein A and rabbit anti-mouse IgG reagents were from
Zymed Laboratories Inc. (South San Francisco, CA).
U-box (amino acids 1-189) CHIP proteins fused to the carboxyl
terminus of glutathione S-transferase (GST) were also
generated by the PCR. The human ErbB2 expression construct in
pcDNA3 was a kind gift of Dr. Kermit Carraway III (University of
California, Davis, CA). The QuikChange® mutagenesis system
(Invitrogen) was used to generate point mutants of ErbB2 (Y1112F) and
CHIP (H260Q) using mutant primers (sequences available upon request).
All constructs were sequence-verified. The pGEX4T-2 constructs
expressing GST-Cbl-N (Cbl residues 1-357) and GST-Cbl (full-length)
fusion protein have been described previously (29, 30).
80 °C. 5-µg aliquots of
glutathione-Sepharose-immobilized fusion protein were incubated with
purified E1 (20 nM) (Calbiochem), E2 (200 nM)
(UbcH5a; Calbiochem), biotin-labeled ubiquitin (2 µg/ml) (AFFINITI Research Products Ltd., Mamhead, UK), 5 mM ATP, 10 mM dithiothreitol, and 5 mM magnesium chloride
in a 50-µl reaction for 90 min at 30 °C. The beads were washed
twice with RIPA buffer and twice with 50 mM Tris, pH 7.5, 2 mM magnesium chloride and then incubated at 37 °C for 30 min in 50 mM Tris, pH 7.5, 2 mM magnesium
chloride, and 2 mM ATP to remove any bacterial heat shock
proteins and associated proteins (26). The beads were washed three
times with RIPA buffer, and the ubiquitinylated proteins were detected
by immunoblotting with streptavidin-HRP. For in vitro ErbB2
ubiquitinylation, anti-ErbB2 IPs from 500-µg aliquots of SKBR-3 cell
lysate were collected on protein G-Sepharose beads and incubated with 5 µg of the indicated soluble GST fusion proteins, 5 µl of rabbit
reticulocyte lysate (Promega, Madison, WI), 2 µg/ml biotin-labeled
ubiquitin, 10 mM dithiothreitol, and 2 mM ATP
in a 50-µl reaction for 90 min at 30 °C. The beads were washed and
processed for detection of ubiquitinylated proteins as above.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Ubiquitinylation and down-regulation of
endogenous as well as ectopically expressed ErbB2 by 17-AAG.
Endogenous ErbB2-expressing SKBR-3 cells or ErbB2-transfected 293T
cells were treated with vehicle ( ) or 100 nM (for SKBR-3
cells) or 3 µM (for 293T cells) 17-AAG (+) with (+) or
without (
) 50 µM MG-132 for 4 h. MG-132 was added
to the cells 1 h before and continued during the 17-AAG treatment.
One-mg aliquots of lysate were subjected to immunoprecipitation
(IP) with an anti-ErbB2 antibody, washed with RIPA buffer,
resolved by SDS-PAGE, and immunoblotted (IB) with
anti-ubiquitin antibody (top panel). The membrane was
stripped and reprobed with the anti-ErbB2 antibody (second
panel). 25-µg aliquots of whole cell lysate were immunoblotted
directly with the anti-ErbB2 antibody to visualize the levels of
endogenous or transfected ErbB2 protein (bottom panel).
ErbB2-Ub indicates ubiquitinylated ErbB2.
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Fig. 2.
Recombinant CHIP functions as a ubiquitin
ligase toward ErbB2 in vitro. A,
schematic representation of the domain structure of CHIP. The
numbers above correspond to amino acid residues in human
CHIP. The positions of introduced mutations are indicated by
arrows. TPR, tetratricopeptide repeat.
B, auto-ubiquitinylation of CHIP, but not its U-box mutants.
5 µg each of purified GST or GST-CHIP fusion proteins, coated on
glutathione-Sepharose beads, were incubated with purified E1, E2
(UbcH5a), and biotin-labeled ubiquitin (Biotin-Ub) as
indicated. The beads were washed in the presence of ATP to remove any
bacterial heat shock proteins, and the ubiquitinylated CHIP signals
(CHIP-Ub) were detected by immunoblotting (IB)
with streptavidin-HRP. WT, wild type; U-box,
CHIP U-box deletion mutant (residues 1-189); H260Q, U-box point mutant.
NS indicates nonspecific signals. C, in
vitro ErbB2 ubiquitinylation by recombinant CHIP but not its U-box
mutants. Anti-ErbB2 IPs from 500-µg aliquots of SKBR-3 cell lysate
were collected using protein G-Sepharose beads and washed with RIPA
buffer. The bead-bound ErbB2 or anti-ErbB2 antibody was subjected to
in vitro ubiquitinylation in rabbit reticulocyte lysate
(RRL, a source of E1 and E2) and other components as
indicated in A. The beads were further processed as in
A to detect the ubiquitinylated ErbB2 (ErbB2-Ub).
D, CHIP mediates more efficient ErbB2 ubiquitinylation than
Cbl. In vitro ErbB2 ubiquitinylation and detection was
carried out as described in C.
U-box or H260Q (Fig. 2C, lanes
6 and 7). The relative specificity of CHIP-induced
ErbB2 ubiquitinylation was shown by the relatively modest
ubiquitinylation of ErbB2 by GST-Cbl (Fig. 2D), whereas
GST-Cbl efficiently induced the ubiquitinylation of EGFR (data not
shown). These in vitro studies showed that CHIP could
function as a ubiquitin ligase toward ErbB2, suggesting the possibility
that CHIP may regulate ErbB2 turnover in vivo.
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Fig. 3.
Overexpression of CHIP induces ErbB2
ubiquitinylation in vivo and promotes the
down-regulation of ErbB2. A, overexpression of CHIP reduces
the level of wild type (WT) ErbB2 as well as its
Cbl-independent mutant Y1112F. 293T cells were transfected with plasmids encoding wild type ErbB2 or ErbB2 Y1112F
together with a plasmid encoding Myc-tagged CHIP (+) or with control
vector ( ). After 36 h of transfection, the cells were treated
with 3 µM 17-AAG (+) or Me2SO control (
)
for 2 h before cell lysis with lysis buffer. 25-µg aliquots of
whole cell lysate were resolved by SDS-PAGE and serially immunoblotted
(IB) with anti-ErbB2, anti-Myc (CHIP), and anti-tubulin
antibodies. B, failure of ErbB2 Y1112F mutant to interact
with the ErbB2 binding domain of Cbl. 293T cells were transfected with
wild type ErbB2 or its Y1112F mutant. 36 h post-transfection, the
cells were either left unstimulated (
) or stimulated with 10 µg/ml
HerceptinTM (+) for 30 min at 37 °C before lysis. One-mg
aliquots of lysate were used for the pull-down assay with 50 µg of
glutathione-Sepharose-bound GST-Cbl-N fusion proteins. The bead-bound
ErbB2 was detected by anti-ErbB2 immunoblotting. C,
CHIP-induced ErbB2 ubiquitinylation in 293T cells. 293T cells were
transfected with plasmids encoding ErbB2 with (+) or without (-) CHIP.
The cells were treated with 50 µM proteasome inhibitor
MG-132 for 4 h before lysis. Anti-ErbB2 IPs from 1.5-mg aliquots
of lysate were serially immunoblotted with anti-ubiquitin (top
panel) and anti-ErbB2 (middle panel) antibodies.
25-µg aliquots of lysate were directly immunoblotted with anti-Myc
antibody to visualize the transfected CHIP protein (bottom
panel). D, inhibition of 17-AAG-induced ErbB2
ubiquitinylation by CHIP H260Q. The SKBR-3 cells were transfected with
plasmids encoding wild type CHIP or CHIP H260Q or with vector alone
(
). After 36 h of transfection, the cells were pretreated with
50 µM MG-132 (+) for 1 h before a 4-h incubation in
the presence of 100 nM 17-AAG (+) or Me2SO
(
). Anti-ErbB2 IPs from 1-mg aliquots of lysate were serially
immunoblotted with anti-ubiquitin (top panel) and anti-ErbB2
(middle panel) antibodies. 25-µg aliquots of lysate were
directly immunoblotted with anti-Myc antibody to visualize the
transfected CHIP (bottom panel).
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Fig. 4.
CHIP-induced down-regulation of cell surface
ErbB2. A, vector- or CHIP-transfected SKBR-3 cells were
surface-labeled with biotin and treated with 100 nM 17-AAG
or Me2SO control for the indicated time points before
lysis. Anti-ErbB2 IPs from 100-µg aliquots of lysate protein were
immunoblotted (IB) with streptavidin-HRP to visualize the
surface-labeled ErbB2 (top panel) followed by anti-ErbB2
immunoblotting to visualize the total immunoprecipitated ErbB2
(second panel). 25-µg aliquots of lysate were directly
resolved by SDS-PAGE and immunoblotted with anti-Myc antibody to detect
CHIP expression (bottom panel) (representative of two
independent experiments). The bands in the top panel of
A were quantified by densitometry using the Scion Images
software. The data was expressed as a percentage of the signals
obtained with untreated cells (bottom graph). B,
overexpression of CHIP facilitates ErbB2 degradation even after new
protein synthesis is blocked with cycloheximide (CHX)
treatment. 293T cells were cotransfected with ErbB2 and/or CHIP as
described in Fig. 3. The cells were treated with vehicle or 100 µM cycloheximide for 2 h before the addition of 3 µM 17-AAG. 25-µg aliquots of cell lysate were
immunoblotted with the indicated antibodies. ErbB2 signals in the
top panel were quantified by densitometry and plotted as a
percentage of signals in vehicle-treated cells. The results are
representative of three independent experiments.
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Fig. 5.
Association of CHIP with Hsp70 and Hsp90
chaperone proteins and ErbB2. A, association of the
transfected CHIP with Hsp70 and Hsp90 proteins. 293T cells were
transfected with a Myc-CHIP expression plasmid (+) or vector ( ), and lysates were
prepared 36 h after transfection. One-mg aliquots of lysate were
subjected to anti-Myc IPs and serially immunoblotted (IB)
with anti-Hsp70, anti-Hsp90, and anti-Myc antibodies. B,
association of transfected ErbB2 and CHIP proteins and increased
association upon 17-AAG treatment. 293T cells were transfected with
ErbB2 and/or CHIP as indicated. Cells were then treated with 50 µM MG-132 for 1 h followed by a 2-h incubation in
the presence of 3 µM 17-AAG (+) or Me2SO
(
). Anti-ErbB2 or anti-Myc IPs from 1-mg aliquots of lysate were
serially immunoblotted with anti-ErbB2 and anti-Myc (CHIP) antibodies.
C, association of endogenous ErbB2 and CHIP. SKBR-3 cells
were treated with either Me2SO vehicle (
) or MG-132 (+)
for 1 h, and 100 nM 17-AAG (+) or Me2SO
(
) was then added for 1 h. Anti-ErbB2 IPs from 1-mg aliquots of
lysate protein were then serially immunoblotted with anti-CHIP
(top panel) and anti-ErbB2 (second panel)
antibodies. 50-µg aliquots of lysate were directly immunoblotted with
anti-CHIP (third panel) and anti-ErbB2 (bottom
panel) antibodies.
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Fig. 6.
CHIP H260Q-induced enhancement of
ErbB2·Hsp70 association and reduction of 17-AAG-induced Hsp90
dissociation from ErbB2. 293T cells were transfected with
ErbB2 (+) and/or Myc-tagged wild type (WT) CHIP or its H260Q
mutant, as indicated. 36 h post-transfection, the cells were
pretreated with 50 µM MG-132 for 1 h followed by
another 2-h incubation in the presence of 3 µM 17-AAG (+)
or Me2SO ( ). Anti-Myc or anti-ErbB2 IPs from 1-mg
aliquots of lysate were serially immunoblotted (IB) with the
indicated antibodies (in the order of top panel to
bottom panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
fibroblasts, which showed no reduction in
GA-induced ErbB2 degradation (27). Finally, CHIP may exert a more
complex effect on ErbB2 regulation, as Hsp70 itself has been shown to
be a CHIP substrate (33), and this modification may have regulatory
effects on Hsp70-mediated folding and/or degradation of client
proteins. In this regard, it is noteworthy that previous studies have
shown that CHIP inhibits the protein folding function of Hsp70 and
competes for binding of co-chaperones such as p23 (23). Furthermore,
CHIP has been shown to decrease the ATPase activity of Hsp70 and
inhibit its chaperone function in vitro (22). Further
studies of CHIP should help clarify these issues.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Kermit Carraway III for ErbB2 cDNA, Genentech Inc. (South San Francisco, CA) for HerceptinTM and the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis of the National Cancer Institute, National Institutes of Health, CI for 17-AAG. We also thank members of the Band laboratory for helpful suggestions.
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FOOTNOTES |
---|
* This work was supported in part by United States Department of Defense Breast Cancer Program Grant DAMD 17-02-1-0303 and National Institutes of Health Grants CA 87986, CA 75075, and CA 76118 (to H. B.) and CA 81076 and CA 70195 (to V. B.).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.
Scholars of the Massachusetts Department of Public Health Breast
Cancer Research Program.
§ Supported by National Institutes of Health Training Grant T32AR07530.
** To whom correspondence should be addressed: Brigham and Women's Hospital, Smith Bldg., Rm. 538C, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1101; Fax: 617-525-1010; E-mail: hband@rics.bwh.harvard.edu.
Published, JBC Papers in Press, February 6, 2003, DOI 10.1074/jbc.M209640200
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ABBREVIATIONS |
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The abbreviations used are: EGFR, epidermal growth factor (EGF) receptor; CHIP, carboxyl terminus of Hsc70-interacting protein; GA, geldanamycin; 17-AAG, 17-allylamino-geldanamycin; GST, glutathione S-transferase; HRP, horseradish peroxidase; IP, immunoprecipitate; Ab, antibody; E1, ubiquitin-activating enzyme; E2 or Ubc, ubiquitin-conjugating enzyme; RIPA, radioimmune precipitation assay buffer.
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